3-O-Arylmethylgalangin, a novel isostere for anti-HCV 1,3-diketoacids (DKAs)

Article abstract of DOI:10.1016/j.bmc.2010.09.021

Through chelation of the metal ions at the enzyme active site, 1,3-diketoacids (DKAs) show potent inhibition of viral enzymes such as HIV integrase and HCV NS5B. In order to optimize the antiviral activity of the DKAs, structural modification of their metal-binding units, keto-enol acids or monoketo acids, have been actively performed. In this study, we proposed 3-O-arylmethylgalangin 3 as an alternative to ortho-substituted aromatic DKA, a potent inhibitor of HCV NS5B. As a proof-of-concept study, a series of 3-O-arylmethylgalangin derivatives (3a-3r) were prepared and their inhibitory activity against HCV NS5B was estimated. Structure-activity relationship of the 3-O-arylmethylgalangin derivatives was in good accordance with that of the ortho-substituted aromatic DKA series. In particular, two galangin ethers (3g and 3i) completely superimposable with the most potent ortho-substituted aromatic DKA analogue (2) in atom-by-atom fashion showed equipotent inhibitory activity to that of 2. Taken together, this result provides convincing evidence that the 3-O-arylmethylgalangin can successfully mimic the chelating function of the DKA pharmacophore to show potent inhibitory activity against the target enzyme, HCV NS5B.

Full text of DOI:10.1016/j.bmc.2010.09.021

Bioorganic & Medicinal Chemistry 18 (2010) 7331–7337
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3-O-Arylmethylgalangin, a novel isostere for anti-HCV 1,3-diketoacids (DKAs)
⇑
Hyo Seon Lee , Kwang-su Park , Bokhui Lee, Dong-Eun Kim, Youhoon Chong
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Through chelation of the metal ions at the enzyme active site, 1,3-diketoacids (DKAs) show potent inhi-
bition of viral enzymes such as HIV integrase and HCV NS5B. In order to optimize the antiviral activity of
the DKAs, structural modification of their metal-binding units, keto-enol acids or monoketo acids, have
been actively performed. In this study, we proposed 3-O-arylmethylgalangin 3 as an alternative to
ortho-substituted aromatic DKA, a potent inhibitor of HCV NS5B. As a proof-of-concept study, a series
of 3-O-arylmethylgalangin derivatives (3a–3r) were prepared and their inhibitory activity against HCV
NS5B was estimated. Structure–activity relationship of the 3-O-arylmethylgalangin derivatives was in
good accordance with that of the ortho-substituted aromatic DKA series. In particular, two galangin
ethers (3g and 3i) completely superimposable with the most potent ortho-substituted aromatic DKA ana-
logue (2) in atom-by-atom fashion showed equipotent inhibitory activity to that of 2. Taken together, this
result provides convincing evidence that the 3-O-arylmethylgalangin can successfully mimic the chelat-
ing function of the DKA pharmacophore to show potent inhibitory activity against the target enzyme,
HCV NS5B.
Received 12 August 2010
Revised 7 September 2010
Accepted 8 September 2010
Available online 16 September 2010
Keywords:
3-O-Arylmethylgalangin
1,3-Diketoacid
Isostere
Hepatitis C Virus (HCV)
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
trast to the active search for novel DKA-based HIV integrase inhib-
itors,4–10 modification of the keto-enol acid or monoketo acid
Originally, through high-throughput screening, 1,3-diketoacids
(DKAs) (1, Fig. 1) were identified as HIV (Human Immunodefi-
ciency Virus) integrase inhibitors.1 Later, the keto-enol acid phar-
macophore of DKA was reported to be responsible for its antiviral
activity by binding to the magnesium ions present in the active site
of the enzyme.2 Unfortunately, the DKAs are known to suffer from
unfavorable physicochemical as well as pharmacokinetical prob-
lems such as chemical instability, irreversible covalent binding to
protein, and poor stability in plasma.3 As these problems can be
mainly attributed to the free carboxylic acid of DKA, there have been
numerous attempts to replace the carboxylic acid with its isosters
such as triazole (S1360, Fig. 1),4 tetrazole (5CITEP, Fig. 1),5 pyridine
(L-870,810, Fig. 1),6–8 and a neutral carbonyl group (Raltegravir,
Fig. 1).9 Recently, the keto-enol acid pharmacophore model was
further refined to identify a monoketo acid as a minimal structural
motif for antiviral activity (Elvitegravir, Fig. 1).10
HCV (Hepatitis C Virus) NS5B RNA-dependent RNA polymerase
has the same feature as HIV integrase of using two metal ions in
the active site as a key component of the catalytic machinery. Even
though these two enzymes catalyze different types of reaction,
they share the common geometry of the active site amino acids
as well as the catalytic metal ions.3 Thus, it is not surprising that
DKAs are also potent inhibitors of HCV NS5B.11 However, in con-
pharmacophore of anti-HCV DKAs has rarely been reported. For
anti-HCV activity, it was reported that an aryl substituent is specif-
ically required in addition to the keto-enol acid pharmacophore of
DKA.11–14 Moreover, substituents at the aromatic ortho-position
were shown to significantly improve antiviral activity of the result-
ing ortho-substituted aromatic DKAs with a cyanopropyl ether
being the optimum functional group (2, Fig. 2a).11 Based on this
information, we reasoned that galangin, a naturally occurring
metal chelator,15–17 might serve as the aforementioned minimal
pharmacophore (monoketo acid), and substitution of its 3-OH
group with arylmethyl functionality (3, Fig. 2a) would successfully
mimic the ortho-substituted aromatic DKA (2). The superimposed
structure of 2 and 3 (Fig. 2b) confirms this idea by showing
atom-by-atom match of the minimal monoketo acid pharmaco-
phore (shown in thick lines) and aromatic alkyloxy substituents.
In the present proof-of-concept study, we set out to develop no-
vel HCV NS5B inhibitors by isosteric replacement of the DKA core
structure with galangin. Herein, we report synthesis and biological
evaluation of a series of 3-O-arylmethylgalangin.
2. Results and discussions
2.1. Chemistry
⇑
Corresponding author.
E-mail address: chongy@konkuk.ac.kr (Y. Chong).
These two authors contributed equally to this work.
In our previous report,18 we observed unusual desilylative-
alkylation of the galangin-3-O-silylether (4, Fig. 3) to the
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.021

7332
H. S. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7331–7337
F
Cl
O
OH
O
OH
O
OH
O
O
N
O
N
R
N
N
OH
HN
HN
N
HN
1
S1360
5CITEP
O
OH
N
O
OH
F
O
N
OH
N
Cl
N
N
H
O
H
N
O
S
N
N
F
F
O
OH
NH
O
N
O
N
O
L-870, 810
Raltegravir
Elvitegravir
Figure 1. Structures of the DKA (1) and its isosteric replacements. Keto-enol acid or monoketo acid motif is shown in thick lines.
b
a
CN
R
CN
R
O
O
OH
OH
O
O
O
OH
O
O
OH
5
O
4
O
O
O
3
OH
OH
OH
O
2
7
OH
2
3
Figure 2. (a) Structures of ortho-substituted DKA (2) and 3-O-arylmethylgalangin (3). The pharmacophores are represented in thick lines. (b) Superimposed structures of 2
and 3.
corresponding alkylgalangin (5, Fig. 3), and the nuclear Overhauser
effect (NOE) between the protons in the arylmethyl moiety (H1,
Fig. 3) and 2-phenyl ring (H2, Fig. 3) confirmed the position of
the alkyl substituent.
For synthesis of a series of 3-O-arylmethylgalangin derivatives,
we made use of the desilylative-alkylation of 4 with variously
substituted benzyl bromides (Scheme 1). Preparation of the key
intermediate 4 was started from the commercially available chry-
sin (5, Scheme 1). Treatment of chrysin (5, Scheme 1) with Me2SO4
and K2CO3 in acetone afforded the dimethylchrysin 6 in 95%
yield.19 Hydroxylation at C-3 of this flavone was accomplished in
a highly regioselective manner by treatment with LDA followed
by quenching with B(OMe)3 to afford the dimethylgalangin 7 in
60% yield.20 Protection of the 3-OH group of 7 with TBDMSCl fol-
lowed by Lewis acid-promoted demethylation provided the key
intermediate 4 in 58% combined yield. Arylmethyl bromides with
Ar
OH
O
O
OH
O
O
H1
H1
OTBDMS
O
H2
K2CO3, R-BnBr, acetone
HO
HO
4
3
B
OH2
SiR3
SiR3
OH
O
SiR3
O
OH
O
O
OH
O
O
O
Ar
O
O
O
O
O
A
C
B
Figure 3. Mechanism for the unusual desilylative-alkylation of 4.

H. S. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7331–7337
7333
OH
O
O
OMe O
OMe O
OH
a
b
HO
MeO
O
MeO
O
5
6
7
c
R
OH
O
O
OH
O
OMe O
O
OTBDMS
OTBDMS
e
d
HO
HO
O
MeO
O
3a-3s
4
8
3a
3g
3m
R = 4-Cl
R = 2-Cl
R = 3-Cl
3b R = 2-Br
3c R = 2-CN
3h R = 3-Br
3i R = 3-CN
3n R = 4-Br
3o R = 4-CN
3d R = 2-NO2 3j R = 3-NO2 3p R = 4-NO2
3e R = 2-Me 3k R = 3-Me 3q R = 4-Me
3f R = 2-OMe 3l R = 3-OMe 3r R = 4-OMe
Scheme 1. Synthesis of 3-O-arylmethylgalangin derivatives (3a–3r). Reagents and conditions: (a) Me2SO4, K2CO3, acetone, 60 °C, 95% yield; (b) LDA, B(OMe)3, AcOH, H2O2,
60% yield; (c) DMAP, TBDMSCI, pyridine, 60 °C, 63% yield; (d) BBr3, CH2CI2, 92% yield; (e) R-BnBr, K2CO acetone, 60–80% yield.
various substituents (Cl, Br, CN, NO2, Me and OMe) at 2-, 3-, or 4-
position of the aromatic ring were then reacted with 4 to provide
the desired 3-O-arylmethylgalangin derivatives (3a–3r) in 60–
80% yield.
Even the most potent inhibitors of HCV NS5B (3g and 3i) did not
exert significant effects in inhibiting HCV RNA replication inside
the cell (EC50 = 25 and 36 lM, respectively). Among the series, al-
most similar antiviral activity was observed regardless of position
as well as type of the substituents (EC50, Table 1). This result
2.2. Biological activity
Table 1
The synthesized galangin arylmethyl ethers (3a–3r) were eval-
uated for their ability to inhibit the HCV NS5B enzyme activity
(IC50, Table 1).21 Antiviral activity of the galangin derivatives
(EC50, Table 1) was evaluated in the human hepatoma cell line
Huh-7, carrying the subgenomic HCV genotype 1 replicon with
the luc-ubi-neo (reporter/selective) fusion gene.22–24 The cytostatic
effect of the test compounds (CC50, Table 1) was also evaluated in
the same cell line.
As anticipated, the 3-O-arylmethylgalangin derivatives showed
moderate to potent inhibition of the enzyme activity of HCV NS5B
(IC50, Table 1). In general, 2-substituted benzyl ethers (3a–3f) were
not as active as the corresponding regioisomers, 3- or 4-substi-
tuted benzyl ethers (3g–3r). Halogen (Cl, Br) or cyano group substi-
tuted at 3- or 4-position of the benzyl ether motif provided the
corresponding galangin ethers with potent inhibitory activity
against the target enzyme, HCV NS5B. Thus, compounds 3g, 3i,
and 3m–3o showed potent enzyme inhibition (Table 1), and their
HCV NS5B inhibition, anti-HCV activity, and cytostatic effect of 3-O-arylmethylga-
langin derivatives
b,e
Compd
R
Activity
CC50
SIf
M)
(l
M)
(l
Enzyme
Cell-based
inhibitiona,b
antiviral activityb,c,d
(IC50
,
lM)
(EC50, lM)
g
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
2h
2- Cl
Br
>50
10.5
11.0
>50
7.2
38
9
5
>100
12
>100
51
55
53
46
15
21
31
29
28
36
2
—
1.3
g
CN
NO2
Me
—
12
34
28
25
18
36
5
28
22
15
12
10
7
4.3
1.6
1.9
1.8
0.8
0.6
6.2
1.0
0.4
2.4
0.2
4.8
5.7
2.9
5.7
OMe >50
3- Cl
0.1
4.6
0.2
6.4
30.0
Br
CN
NO2
Me
OMe 21.0
4- Cl
0.3
1.4
1.3
>50
45.2
IC50 values (IC50 = 0.1–1.4
anti-HCV DKA 2 (IC50 = 0.35
l
M) were comparable with that of the
l
Br
M).11 It is of particular interest that
CN
NO2
Me
48
40
40
57
compounds 3g and 3i which are completely superimposable to 2
(Fig. 2b) showed the most potent inhibitory activity. In this regard,
it should be pointed out that the compound 2 with a cyanopropyl
group attached to the ortho-position of aromatic DKA (Fig. 2a) was
the most potent inhibitor among the series of the DKA ethers with
different length and terminal functionalities of the ether linkage.11
Therefore, the structure–activity relationship of the 3-O-aryl-
methylgalangin derivatives, in accordance with that of the ortho-
substituted aromatic DKA series, provides convincing evidence
that the galangin ether successfully mimic the chelating function
of the DKA pharmacophore.
14
10
OMe 29.5
0.35
i
i
i
—
—
—
a
b
c
Concentration required to inhibit enzyme activity of HCV NS5B by 50%.
The values obtained as the average of triplicate determinations.
Concentration required to inhibit HCV RNA replication by 50%.
d
Interferon
a-2b was used as a reference compound at 10,000 units/well and
reduced the signal in the viral RNA (luciferase) assay to background levels without
any cytotoxic activity.
e
Concentration required to reduce cell proliferation by 50%.
f
Selectivity index: ratio of CC50 to EC50
Selective antiviral effect.
Ref. 11.
.
g
h
i
Unfortunately, the galangin derivatives showed only moderate
to marginal antiviral activity in cell-based assay (EC50, Table 1).
Not available.

7334
H. S. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7331–7337
indicates that some factors related with the galangin scaffold
might be involved in limiting its biological activity in cell-based as-
say system. Taking the hydrophobic nature of the galangin deriva-
tives into consideration, this finding can probably be explained by
a combination of factors such as poor solubility, poor cell perme-
ability, and high protein binding. Adverse hepatic effect is another
factor limiting development of the keto-enol acid class of com-
pounds into antiviral drugs.25 The galangin derivatives synthesized
in this study also showed significant cytostatic effect against the
hepatoma cell line, Huh-7 (CC50, Table 1). The cytostatic activity
was not significantly different from the antiviral effect to result
in low selectivity index (SI, Table 1). Among the series, bromoben-
zyl ethers (3b, 3h, and 3n) showed significant cytostatic effect irre-
spective of the position of the bromine substituent on the aromatic
ring. With the exception of bromobenzyl ethers, the cytostatic ef-
fect of the galangin derivatives was dependent upon the position
rather than the type of the aromatic substituent with the most
cytostatic derivatives being galangin 3-substituted benzyl ethers
(3g–3l).
chased from Merck. Column chromatography was performed using
either Silica Gel 60 (220–440 mesh) for flash chromatography.
Mass spectrometric data (MS) were obtained by MALDI-TOF-TOF
mass spectrometer (Ultraflex III, Bruker Daltonik).
4.2. Synthesis of the 3-O-arylmethylgalangin derivatives
4.2.1. 5,7-Dimethoxy-2-phenyl-chromen-4-one (6)
Dimethyl sulfate (1.24 g, 9.83 mmol) and K2CO3 (4.35 g,
31.47 mmol) were added to a solution of chrysin 5 (1.00 g,
3.93 mmol) in acetone (20 mL), and the reaction mixture was stir-
red under reflux for 12 h. Volatiles were removed under reduced
pressure and then the residue was taken with CH2Cl2. The organic
layer was washed with water, dried over MgSO4, and concentrated
under reduced pressure. Recrystallization from acetone provided 6
(1.06 g, 3.74 mmol, 95% yield) as pale yellow powder. Data of 1H
NMR spectra were identical to those in the literature:16 1H NMR
(400 MHz, CDCl3) d 7.87–7.85 (m, 2H), 7.49–7.46 (m, 3H), 6.67 (s,
1H), 6.56 (d, J = 2.3 Hz, 1H), 6.37 (d, J = 2.3 Hz, 1H), 3.91 (s, 3H),
3.84, (s, 3H).
3. Conclusion
4.2.2. 3-Hydroxy-5,7-dimethoxy-2-phenyl-4H-chromen-4-one (7)
The solution of 6 (282 mg, 1 mmol) in anhydrous THF (10 mL)
was added dropwise to the solution containing LDA (2.0 M in
THF; 2 mL, 4 mmol) in THF (10 mL) at À78 °C. The reaction mixture
was stirred for 6 h at À78 °C and then quenched with a solution of
trimethyl borate (0.5 mL) in THF (5 mL). The resulting solution was
stirred for 1 h at À78 °C. The reaction mixture was acidified with
acetic acid (0.4 mL), stirred for 30 min, and then oxidized with
30%-H2O2 (1 mL). The solution was allowed to warm slowly to
room temperature over 12 h. The reaction mixture was neutralized
with saturated aqueous NaHCO3 solution, extracted with CH2Cl2
and dried over MgSO4. After filtration, the filtrate was concentrated
under reduced pressure. The residue was purified by flash chroma-
tography (SiO2, CH2Cl2/EtOAc = 5:1) to afford 7 (179 mg, 0.6 mmol,
60% yield) as dark yellow oil: 1H NMR (400 MHz, CDCl3) d 8.17 (d,
J = 5.5 Hz, 2H), 7.48 (t, J = 7.3 Hz, 2H), 7.40 (t, J = 7.3 Hz, 1H), 6.51 (s,
1H), 6.30 (d, J = 1 Hz, 1H), 3.94 (s, 3H), 3.88 (s, 3H); 13C NMR
DKAs show potent inhibition of viral enzymes such as HIV
integrase and HCV NS5B through chelation of the metal ions at
the active site. There have been many research efforts to optimize
the antiviral activity of the DKA derivatives by structural modifica-
tion of their metal-binding units, keto-enol acids or monoketo
acids. However, bioisosteres of anti-HCV DKAs have received rela-
tively little attention compared with those of HIV integrase inhib-
itors. In this study, in order to obtain a new scaffold as an
alternative to monoketo acids, we designed 3-O-arylmethylgalan-
gin 3 as an inhibitor against HCV NS5B. Among the series of
synthesized galangin arylmethyl ethers (3a–3r), galangin 3-O-
chlorobenzyl ether (3g) and 3-O-cyanobenzyl ether (3i) were the
most potent HCV NS5B inhibitor with equipotent activity to that
of anti-HCV DKA 2. These two galangin analogues, completely
superimposable with 2 in atom-by-atom fashion (Fig. 2b), seem
to fully mimic the chelating function of the DKAs. However, most
of these inhibitors showed a suboptimal activity in the HCV cell-
based assay, which can be mainly attributed to unfavorable phys-
icochemical as well as pharmacokinetic properties such as poor
solubility and high protein binding. In addition, the galangin ethers
failed to overcome the problem of adverse hepatic effect associated
with the DKA analogues.
(100 MHz, CDCl3)
d 171.96, 164.47, 160.50, 158.94, 141.71,
138.14, 131.01, 129.55, 128.45, 127.10, 106.15, 95.68, 92.33,
56.30, 55.75; LC/MS (ESI) m/z Found: 299.3 [M+H]+; calcd for
C17H15O5: 299.1.
4.2.3. 3-(tert-Butyldimethylsilyloxy)-5,7-dimethoxy-2-phenyl-
4H-chromen-4-one (8)
Taken together, the potent inhibition of HCV NS5B by the 3-O-
arylmethylgalangin derivatives demonstrates that the galangin
can replace the metal-binding function of the DKA pharmacophore
but its suboptimal properties need to be tackled by elaborative
modification of the galangin scaffold.
TBDMSCl (1.62 g, 10.73 mmol) was added to a solution of 7
(1.60 g, 5.36 mmol) and DMAP (130 mg, 1.10 mmol) in pyridine
(30 mL). The reaction mixture was warmed to 60 °C and stirred
for 12 h. After reaction, the volatiles were removed under reduced
pressure. The residue was dissolved in CH2Cl2, washed with water,
and dried over MgSO4. The residue was purified by flash column
chromatography (SiO2, CH2Cl2/EtOAc = 4:1) to afford 8 (1.38 g,
3.35 mmol, 63% yield) as yellow solid: 1H NMR (400 MHz, CDCl3)
4. Experimental
4.1. Materials and general methods
d
7.92 (dd, J = 8.2, 1.8 Hz, 2H), 7.46–7.38 (m, 3H), 6.45 (d,
All chemicals including MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetra-zolium bromide)] were purchased from Sigma-Al-
drich. Dulbecco’s Modified Eagle Media (DMEM), penicillin,
streptomycin and fetal bovine serum (FBS) were purchased from
Invitrogen. Nuclear magnetic resonance spectra were recorded on
J = 2.2 Hz, 1H), 6.30 (d, J = 2.2 Hz, 1H), 3.93 (s, 3H), 3.85 (s, 3H),
0.77 (s, 9H), 0.19 (s, 6H); 13C NMR (100 MHz, CDCl3) d 182.00,
163.60, 161.00, 158.72, 138.47, 131.56, 129.63, 128.63, 128.02,
108.20, 95.55, 92.15, 56.53, 55.66, 25.73, 18.75, À3.74; LC/MS
(ESI) m/z Found: 413.4 [M+H]+; calcd for C23H28O5Si: 413.2.
a
bruker 400 AMX spectrometer (Karlsruhe, Germany) at
400 MHz for 1H NMR and at 100 MHz for 13C NMR with tetrameth-
ylsilane as the internal standard. Chemical shift are reported as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br s
(broad singlet). Coupling constants are reported in hertz (Hz).
The chemical shifts are reported as parts per million (d) relative
to the solvent peak. TLC was performed on Silica Gel 60 F254 pur-
4.2.4. 3-(tert-Butyldimethylsilyloxy)-5,7-dihydroxy-2-phenyl-
4H-chromen-4-one (4)
BBr3 in dichloromethane (1.0 M, 1 mL) was added to a solution
of 8 (100 mg, 0.24 mmol) in dichloromethane (5 mL) dropwise at
room temperature. The reaction mixture was stirred for 6 h at
room temperature and then quenched with saturated aqueous

H. S. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7331–7337
7335
K2CO3 solution (5 mL). The reaction mixture was diluted with
CH2Cl2, washed with water, and dried over MgSO4. The residue
was purified by flash column chromatography (SiO2, CH2Cl2/
EtOAc = 3:1) to afford 4 (85 mg, 0.22 mmol, 92% yield) as yellow so-
lid: 1H NMR (400 MHz, CDCl3) d 12.7 (s, 1H, –OH), 7.92–7.89 (m, 2H),
7.69–7.65 (m, 3H), 6.39 (s, 1H), 6.31 (s, 1H), 5.73 (s, 1H, –OH), 0.81 (s,
9H), 0.11 (s, 6H); 13C NMR (100 MHz, DMSO-d6) d 177.75, 165.72,
162.23, 157.89, 147.19, 138.58, 132. 43, 131.06, 128.84, 104.69,
99.56, 94.96, 27.26, 26.91, À1.75; LC/MS (ESI) m/z; calcd for
4.2.5.6. 5,7-Dihydroxy-3-(2-methoxybenzyloxy)-2-phenyl-4H-
chromen-4-one (3f). 1H NMR (400 MHz, acetone-d6) d 12.80 (s,
1H, –OH), 7.98–8.00 (m, 2H), 7.39–7.52 (m, 3H), 7.34 (d,
J = 7.6 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 6.82–6.86 (m, 2H), 6.51 (s,
1H), 6.30 (s, 1H), 5.22 (s, 2H), 3.64 (s, 3H); 13C NMR (100 MHz,
DMSO-d6)
d 176.19, 162.27, 159.25, 154.85, 154.47, 153.65,
135.12, 128.57, 128.20, 128.03, 127.94, 127.74, 127.58, 126.25,
117.90, 117.83, 102.29, 96.61, 91.56, 66.43, 55.61; LC/MS (ESI)
m/z; calcd for C23H19O6: 391.1, found: 391.1 [M+H]+.
C
21H25rO5Si: 385.1, found: 385.4 [M+H]+.
4.2.5.7. 3-(3-Chlorobenzyloxy)-5,7-dihydroxy-2-phenyl-4H-chro-
men-4-one (3g). 1H NMR (400 MHz, acetone-d6) d 12.7 (s, 1H,
–OH), 8.01 (d, J = 7.0 Hz, 2H), 7.54 (d, J = 6.8 Hz, 3H), 7.32 (s, 3H),
7.41 (s, 1H), 6.52 (s, 1H), 6.31 (s, 1H); 13C NMR (100 MHz, DMSO-
d6) d 179.89, 166.36, 163.31, 158.48, 157.80, 140.73, 138.92,
134.76, 132.58, 131.82, 131.58, 131.44, 130.09, 129.72, 129.63,
128.36, 106.16, 100.34, 95.42, 74.47; LC/MS (ESI) m/z; calcd for
4.2.5. Desilylative-alkylation of 4
Appropriately substituted benzyl bromide (1.1 equiv) was
added dropwise to a solution of 4 (1 equiv) and K2CO3 (1.1 equiv)
in acetone. The reaction mixture was stirred at room temperature
for 48 h. After reaction, the reaction mixture was neutralized with
1N-HCl solution. The reaction mixture was extracted with CH2Cl2
and dried over MgSO4. After concentration under reduced pressure,
the residue was crystallized from CH2Cl2.
C
22H16ClO5: 395.1, found: 395.2 [M+H]+.
4.2.5.8. 3-(3-Bromobenzyloxy)-5,7-dihydroxy-2-phenyl-4H-chro-
men-4-one (3h). 1H NMR (400 MHz, acetone-d6) d 12.7 (s, 1H,
–OH), 8.00–8.02 (m, 2H), 7.52–7.59 (m, 4H), 7.46 (d, J = 7.9 Hz, 1H),
7.37 (d, J = 7.6 Hz, 1H), 7.26 (t, J = 7.8 Hz, 1H), 6.52 (s, 1H), 6.31 (s,
1H), 5.15 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d 179.43, 165.34,
163.55, 158.03, 156.96, 143.66, 137.66, 131.89, 130.56, 129.98,
129.77, 127.89, 126.13, 122.76, 104.69, 96.79, 93.55, 72.66; LC/MS
(ESI) m/z; calcd for C22H16BrO5: 439.1, found: 438.9 [M+H]+.
4.2.5.1. 3-(2-Chlorobenzyloxy)-5,7-dihydroxy-2-phenyl-4H-chro-
men-4-one (3a). 1H NMR (400 MHz, acetone-d6) d 12.70 (s, 1H),
7.98 (d, J = 7.1 Hz, 2H), 7.59–7.61 (m, 1H), 7.47–7.52 (m, 3H),
7.27–7.35 (m, 3H), 6.53 (s, 1H), 6.31 (s, 1H), 5.27 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) d 178.41, 164.83, 161.72, 157.03, 156.53,
137.41, 134.29, 132.94, 131.23, 131.07, 130.25, 129.47, 128.78,
128.69, 127.98, 127.55, 104.85, 99.14, 94.19, 70.91; LC/MS (ESI)
m/z; calcd for C22H16ClO5: 395.1, found: 394.2 [M+H]+.
4.2.5.9.
3-[(5,7-Dihydroxy-4-oxo-2-phenyl-4H-chromen-3-
4.2.5.2. 3-(2-Bromobenzyloxy)-5,7-dihydroxy-2-phenyl-4H-chro-
men-4-one (3b). 1H NMR (400 MHz, acetone-d6) d 12.7 (s, 1H,
–OH), 7.98–8.01 (m, 2H), 7.62 (d, J = 7.5 Hz, 1H), 7.47–7.53 (m,
4H), 7.34 (t, J = 7.6 Hz, 1H), 7.22 (t, J = 7.7 Hz, 1H), 6.53 (d,
J = 1.3 Hz, 1H), 6.31 (d, J = 1.1 Hz, 1H), 5.25 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) d 178.25, 166.42, 161.83, 158.85, 157.94,
138.86, 137.98, 132.10, 130.33, 129.81, 129.54, 128.63, 127.95,
127.93, 126.78, 104.58, 96.83, 94.35, 66.78; LC/MS (ESI) m/z Found:
440.1 [M+H]+; calcd for C22H16BrO5: 439.1.
yloxy)methyl]benzonitrile (3i). 1H NMR (400 MHz, acetone-d6)
d 12.6 (s, 1H, –OH), 8.01 (dd, J = 7.4, 1.4 Hz, 2H), 7.56–7.51 (m,
3H), 7.37 (s, 1H), 7.34–7.30 (m, 3H), 6.51 (d, J = 1.8 Hz, 1H), 6.30
(d, J = 1.8 Hz, 1H), 5.14 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d
179.59, 165.22, 163.31, 158.17, 157.64, 139.57, 138.45, 132.84,
132.71, 132.53, 131.88, 131.44, 130.23, 129.51, 129.39, 119.12,
113.10, 106.14, 99.68, 94.85, 73.68; LC/MS (ESI) m/z; calcd for
C
23H16NO5: 386.1, found: 386.2 [M+H]+.
4.2.5.10. 5,7-Dihydroxy-3-(3-nitrobenzyloxy)-2-phenyl-4H-chro-
men-4-one (3j). 1H NMR (400 MHz, acetone-d6) d 12.7 (s, 1H,
–OH), 8.26 (s, 1H), 8.15 (d, J = 8.3 Hz, 1H), 7.98–8.00 (m, 2H), 7.83
(d, J = 7.3 Hz, 1H), 7.61 (t, J = 7.9 Hz, 1H), 7.51–7.53 (m, 3H), 6.52
(s, 1H), 6.31 (s, 1H), 5.29 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d
178.24, 164.86, 161.67, 156.93, 156.43, 147.82, 138.94, 137.20,
134.73, 131.05, 130.15, 129.76, 128.56, 122.96, 122.93, 104.63,
98.97, 93.99, 72.58; LC/MS (ESI) m/z; calcd for C22H16NO7: 406.1,
found: 406.2 [M+H]+.
4.2.5.3. 2-[(5,7-Dihydroxy-4-oxo-2-phenyl-4H-chromen-3-yloxy)-
methyl]benzonitrile (3c). 1H NMR (400 MHz, acetone-d6) d 12.66
(s, 1H, –OH), 7.94–7.96 (m, 2H), 7.61–7.71 (m, 3H), 7.46–7.54 (m,
4H), 6.52 (d, J = 1.9 Hz, 1H), 6.31 (d, J = 2.0 Hz, 1H), 5.35 (s, 2H); 13C
NMR (100 MHz, DMSO-d6)
d 178.27, 164.94, 161.70, 157.05,
156.67, 139.59, 137.10, 133.42, 133.06, 130.16, 129.57, 129.45,
129.42, 128.87, 128.73, 117.42, 111.78, 104.84, 98.49, 94.24, 66.39;
LC/MS (ESI) m/z; calcd for C23H16NO5: 386.1, found: 386.1 [M+H]+.
4.2.5.4. 5,7-Dihydroxy-3-(2-nitrobenzyloxy)-2-phenyl-4H-chro-
men-4-one (3d). 1H NMR (400 MHz, acetone-d6) d 12.6 (s, 1H,
–OH), 7.99–8.09 (m, 4H), 7.75–7.79 (m, 1H), 7.58–7.61 (m, 1H),
7.49–7.54 (m, 3H), 6.54 (s, 1H), 6.32 (s, 1H), 5.53 (s, 2H); 13C
NMR (100 MHz, DMSO-d6) d 178.42, 164.85, 161.65, 157.54,
156.66, 147.45, 137.98, 134.66, 134.34, 130.33, 129.56, 128.99,
128.34, 127.9, 124.98, 104.55, 97.39, 94.05, 66.55; LC/MS (ESI)
m/z; calcd for C22H16NO7: 406.1, found: 406.1 [M+H]+.
4.2.5.11.
5,7-Dihydroxy-3-(3-methylbenzyloxy)-2-phenyl-4H-
chromen-4-one (3k). 1H NMR (400 MHz, acetone-d6) d 12.8 (s,
1H, –OH), 8.01–8.03 (m, 2H), 7.51–7.58 (m, 3H), 7.08–7.18 (m,
4H), 6.51 (d, J = 2.0 Hz, 1H), 6.30 (d, J = 2.0 Hz, 1H), 5.12 (s, 2H),
2.86 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 179.78, 165.38,
163.29, 158.14, 157.40, 138.66, 138.60, 137.64, 131.71, 131.69,
130.32, 129.68, 129.64, 129.30, 128.96, 126.64, 106.04, 99.66,
94.66, 74.93, 21.36; LC/MS (ESI) m/z Found: 375.2 [M+H]+; calcd
for C23H19O5: 375.1.
4.2.5.5. 5,7-Dihydroxy-3-(2-methylbenzyloxy)-2-phenyl-4H-chro-
men-4-one (3e). 1H NMR (400 MHz, acetone-d6) d 12.77 (s, 1H,
–OH), 7.96–7.98 (m, 2H), 7.48–7.59 (m, 3H), 7.11–7.33 (m, 4H), 6.51
(d, J = 1.9 Hz, 1H), 6.31 (d, J = 1.9 Hz, 1H), 5.15 (s, 2H), 2.29 (s, 3H);
13C NMR (100 MHz, DMSO-d6): d 178.63, 164.80, 161.73, 157.01,
156.56, 137.66, 137.23, 134.84, 131.27, 130.41, 130.33, 130.25,
129.72, 128.79, 128.73, 125.91, 104.86, 99.16, 94.30, 72.12, 18.63;
LC/MS (ESI) m/z; calcd for C23H19O5: 375.1, found: 375.1 [M+H]+.
4.2.5.12. 5,7-Dihydroxy-3-(3-methoxybenzyloxy)-2-phenyl-4H-
chromen-4-one (3l). 1H NMR (400 MHz, acetone-d6) d 12.7 (s,
1H, –OH), 8.03–8.05 (m, 2H), 7.53–7.55 (m, 3H), 7.20 (t,
J = 7.8 Hz, 1H), 6.82–6.93 (m, 3H), 6.51 (d, J = 1.4 Hz, 1H), 6.30 (d,
J = 1.1 Hz, 1H), 5.15 (s, 2H), 3.70 (s, 3H); 13C NMR (100 MHz,
DMSO-d6)
d 178.59, 164.58, 161.31, 159.16, 156.58, 155.82,
137.55, 136.58, 130.78, 130.34, 130.59, 128.97 (br), 128.63,

7336
H. S. Lee et al. / Bioorg. Med. Chem. 18 (2010) 7331–7337
119.45, 113.94, 113.84, 104.86, 98.35, 94.10, 73.50, 55.59; LC/MS
MgCl2, 20 mM NaCl, 1 mM dithiothreitol, 0.05% Tween 20, 1% glyc-
erol, 5 pmol of biotin-dG12 (primer), 0.5 pmol of poly(rC)300 (tem-
(ESI) m/z; calcd for C23H19O6: 391.1, found: 391.1 [M+H]+.
plate),
(0.15
1
l
M
GTP, 0.1–0.3
lCi of [a-
32P]GTP, and 2.5 pmol
4.2.5.13.
3-(4-Chlorobenzyloxy)-5,7-dihydroxy-2-phenyl-4H-
l
g) of HCV polymerase protein in a final volume of 75 lL.
chromen-4-one (3m). 1H NMR (400 MHz, acetone-d6) d 12.7 (s,
1H, –OH), 9.94 (s, 1H), 8.01 (d, J = 7.2 Hz, 2H), 7.58–7.51 (m, 3H),
7.39 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 8.2 Hz, 2H), 6.52 (s, 1H), 6.31
(s, 1H), 5.13 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d 178.44,
164.81, 161.68, 157.02, 156.39, 137.47, 133.09, 131.31, 130.35,
129.70, 129.63, 129.37, 128.01, 127.70, 104.78, 95.00, 93.30,
73.00; LC/MS (ESI) m/z; calcd for C22H16ClO5: 395.1, found: 395.2
[M+H]+.
Reactions were initiated by addition of enzyme and incubated for
30 min at 30 °C. Reactions were stopped by addition of 33 mM
EDTA, and polynucleotide products were collected by filtration
through DEAE Filtermat papers (Wallac); unincorporated triphos-
phate was removed by washing the filters with 5% dibasic sodium
phosphate. The filters were counted in a Packard Tri-Lux Microbeta
scintillation counter. Compounds to be tested were added at vari-
ous concentrations from stocks in 10% dimethyl sulfoxide (DMSO)–
water (final DMSO concentration = 1% of the reaction mixture).
Fifty percent inhibitory concentration (IC50) values were estimated
from the primary cpm data (collected in triplicate).
4.2.5.14.
3-(4-Bromobenzyloxy)-5,7-dihydroxy-2-phenyl-4H-
chromen-4-one (3n). 1H NMR (400 MHz, acetone-d6) d 12.7 (s,
1H, –OH), 7.99–8.01 (m, 2H), 7.53–7.61 (m, 3H), 7.46 (d,
J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 6.52 (s, 1H), 6.30 (s, 1H),
5.12 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d 176.50, 162.91,
159.75, 154.92, 154.47, 135.54, 134.26, 129.60, 129.48, 128.95,
128.82, 128.42, 126.84, 119.57, 102.87, 97.25, 92.08, 71.24; LC/
MS (ESI) m/z; calcd for C22H16BrO5: 439.1, found: 440.8 [M+H]+.
4.4. Anti-HCV assay
The human hepatoma cell line Huh-7, carrying the subgenomic
HCV genotype 1 replicon with the luc-ubi-neo (reporter/selective)
fusion gene, was kindly provided by Dr. Ralf Bartenschlager (Uni-
versity of Heidelberg, Heidelberg, Germany).19–21 Huh-5-2 cells
were seeded at a density of 5 Â 103 per well in a tissue culture-
treated white 96-well view plate in complete DMEM supple-
4.2.5.15. 4-[(5,7-Dihydroxy-4-oxo-2-phenyl-4H-chromen-3-yloxy)-
methyl]benzonitrile (3o). 1H NMR (400 MHz, acetone-d6) d 12.6 (s,
1H, –OH), 7.99–8.02 (m, 2H), 7.75 (d, J = 8.1 Hz, 2H), 7.71 (d,
J = 8.2 Hz, 2H), 7.51–7.62 (m, 3H), 6.53 (d, J = 1.8 Hz, 1H), 6.31 (d,
J = 1.9 Hz, 1H), 5.24 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d
178.31, 164.88, 161.69, 157.03, 156.43, 142.52, 137.53, 132.49,
131.47, 130.27, 128.93, 128.79, 119.06, 111.03, 104.83, 94.24, 93.03,
73.07; LC/MS (ESI) m/z; calcd for C23H16NO5: 386.1, found: 386.3
[M+H]+.
mented with 500 lg/mL G418. After incubation for 24 h at 37 °C
(5% CO2), medium was refreshed (with G418) and DMSO stock of
test compounds were added. After 4 days of incubation at 37 °C,
cell culture medium was removed and luciferase activity was
determined using the Steady-Glo luciferase assay system (Prome-
ga, Leiden, The Netherlands).
4.5. Cytostatic effect
4.2.5.16. 5,7-Dihydroxy-3-(4-nitrobenzyloxy)-2-phenyl-4H-chro-
men-4-one (3p). 1H NMR (400 MHz, acetone-d6) d 12.6 (s, 1H,
–OH), 8.18 (d, J = 8.7 Hz, 2H), 8.00–8.02 (m, 2H), 7.69 (d, J = 8.7 Hz,
2H), 7.52–7.57 (m, 3H), 6.53 (d, J = 2.0 Hz, 1H), 6.32 (d, J = 2.0 Hz,
1H), 5.30 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d 177.70, 164.29,
161.07, 156.45, 155.91, 146.82, 144.02, 136.91, 130.87, 129.64,
128.62, 128.35, 128.20, 123.07, 104.22, 98.64, 93.70, 72.18; LC/MS
(ESI) m/z; calcd for C22H16NO7: 406.1, found: 406.1 [M+H]+.
Huh-5-2 cells were seeded at a density of 5 Â 103 per well of a
96-well plate in complete DMEM with the appropriate concentra-
tions of G418. Serial dilutions of the test compounds in complete
DMEM without G418 were added 24 h after seeding. Cells were al-
lowed to proliferate for 3 days at 37 °C, after which the cell number
was determined by WST-1 assay.
Acknowledgments
4.2.5.17.
5,7-Dihydroxy-3-(4-methylbenzyloxy)-2-phenyl-4H-
chromen-4-one (3q). 1H NMR (400 MHz, acetone-d6) d 12.8 (s,
1H, –OH), 8.02 (d, J = 7.3 Hz, 2H), 7.52–7.54 (m, 3H), 7.24 (d,
J = 7.8 Hz, 2H), 7.09 (d, J = 7.7 Hz, 2H), 6.51 (s, 1H), 6.30 (s, 1H),
5.11 (s, 2H), 2.29 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d 178.59,
164.79, 161.73, 156.98, 156.27, 137.78, 137.58, 133.71, 131.25,
130.46, 129.19, 129.12, 128.83, 128.78, 104.79, 99.14, 94.15,
73.86, 21.12; LC/MS (ESI) m/z; calcd for C23H19O5: 375.1, found:
375.1 [M+H]+.
This work was supported by a grant of the Korea Healthcare
technology R&D Project, Ministry for Health, Welfare & Family Af-
fairs, Republic of Korea (A08-4628-AA2023-08N1-00010A), Prior-
ity Research Centers Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology (2009-0093824), and Biogreen 21 (Korea
Ministry of Agriculture and Forestry).
References and notes
4.2.5.18. 5,7-Dihydroxy-3-(4-methoxybenzyloxy)-2-phenyl-4H-
chromen-4-one (3r). 1H NMR (400 MHz, acetone-d6) d 12.8 (s,
1H, –OH), 8.00–8.03 (m, 2H), 7.50–7.55 (m, 3H), 7.26 (d,
J = 8.5 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 6.51 (d, J = 1.7 Hz, 1H),
6.30 (d, J = 1.7 Hz, 1H), 5.09 (s, 2H), 3.77 (s, 3H); 13C NMR
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