2,6-Bis-arylmethyloxy-5-hydroxychromones with antiviral activity against both hepatitis C virus (HCV) and SARS-associated coronavirus (SCV)

Article abstract of DOI:10.1016/j.ejmech.2011.09.005

In this study, as a bioisosteric alternative scaffold of the antiviral aryl diketoacids (ADKs), we used 5-hydroxychromone on which two arylmethyloxy substituents were installed. The 5-hydroxychromones (5b-5g) thus prepared showed anti-HCV activity and, de

Full text of DOI:10.1016/j.ejmech.2011.09.005

European Journal of Medicinal Chemistry 46 (2011) 5698e5704
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
2,6-Bis-arylmethyloxy-5-hydroxychromones with antiviral activity against both
hepatitis C virus (HCV) and SARS-associated coronavirus (SCV)
,
1
,
Mi Kyoung Kima , Mi-Sun Yub 1, Hye Ri Parka, Kyung Bo Kima, Chaewoon Leea, Suh Young Choa,
**
Jihoon Kanga, Hyunjun Yoona, Dong-Eun Kima, Hyunah Chooc, Yong-Joo Jeongb , Youhoon Chong
,
a,
*
a Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
b Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, Republic of Korea
c Neuro-Medicine Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seoungbuk-gu, Seoul 136-791, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, as a bioisosteric alternative scaffold of the antiviral aryl diketoacids (ADKs), we used 5-
hydroxychromone on which two arylmethyloxy substituents were installed. The 5-hydroxychromones
(5be5g) thus prepared showed anti-HCV activity and, depending on the aromatic substituents on the
2-arylmethyloxy moiety, some of the derivatives (5be5f) were also active against SCV. In addition, unlike
the ADKs which showed selective inhibition against the helicase activity of the SCV NTPase/helicase, the
5-hydroxychromones (5be5f) were active against both NTPase and helicase activities of the target
enzyme. Among those, 3-iodobenzyloxy-substituted derivative 5e showed the most potent activity
Received 19 May 2011
Received in revised form
8 August 2011
Accepted 3 September 2011
Available online 8 September 2011
Keywords:
Aryl diketoacid (ADK)
5-Hydroxyflavone
5-Hydroxychromone
Hepatitis C
Severe Acute Respiratory Syndrome (SARS)
against HCV (EC50 ¼ 4
m
M) as well as SCV (IC50 ¼ 4
mM for ATPase activity, 11 mM for helicase activity) and
this might be used as a platform structure for future development of the multi-target or broad-spectrum
antivirals.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
scaffold was attempted. Compared with the ADKs, the
5-hydroxyflavone scaffold provides additional advantage because
Aryl diketoacid (ADK) has previously been identified to inhibit
HIV-1 (human immunodeficiency virus) integrase [1] and RNA-
dependent RNA polymerase (RdRp) of hepatitis C virus (HCV)
[2,3]. In our previous communications, we reported the ADKs with
an arylmethyloxy or arylmethylamino substituent attached at the
2-, 3-, or 4-position of an aromatic ring (1, Fig. 1) as inhibitors of the
HCV RdRp activity [4] as well as NTPase/helicase activity of SARS-
associated coronavirus (SARS-CoV, SCV) [5]. Inhibition of the two
different viral enzymes, HCV RdRp and SCV NTPase/helicase, by the
same class of compounds was noteworthy because novel multi-
target or broad-spectrum antivirals might be developed from this
structure. Later, in order to improve unfavorable physicochemical
as well as pharmacokinetic properties associated with the dike-
toacid moiety [6], bioisosteric replacement of pharmacophoric
both sides of the central b-hydroxyketone moiety can be
substituted (R1eR3 in 2, Fig. 1) to allow extensive structureeactivity
relationship study. In our recent proof-of-concept study, the key
pharmacophoric arylmethyloxy group of the antiviral ADKs was
introduced on opposite sides (3- and 7-positions) of the
5-hydroxyflavone core structure to give the corresponding galangin
derivatives, 3-O-arylmethyloxygalangins (3) [7] and 7-O-arylme-
thyloxygalangins (4) [8], and significant anti-HCV activities were
observed from both derivatives. However, unlike the ADKs with
arylmethyloxy substituents (1) which showed inhibitory activity
against both HCV RdRp and SCV NTPase/helicase, the galangin
derivatives (3 and 4) lacked anti-SCV activity [9]. This result
suggests that the HCV RdRp might have a binding site specific for
the 5-hydroxyflavone scaffold around which two hydrophobic sites
with similar shapes are located. It can also be assumed that the SCV
NTPase/helicase might not be able to accommodate a 2-phenyl
group of the galangin scaffold. Thus, in order to confirm this
rationale, we intended to use 5-hydroxychromone instead of
5-hydroxyflavone as a platform structure on which two arylme-
thyloxy substituents were attached at the same time (50, Fig. 1).
However, due to synthetic difficulties associated with synthesis of
50, we flipped a core AC ring system of the chromone scaffold and
diketoacid core structure (thick line in 1, Fig. 1) with
a
b-hydroxyketone moiety (thick line in 2, Fig. 1) of 5-hydroxyflavone
* Corresponding author. Tel.: þ82 2 2049 6100; fax: þ82 2 454 8217.
** Corresponding author. Fax: þ82 2 910 4415.
E-mail addresses: jeongyj@kookmin.ac.kr (Y.-J. Jeong), chongy@konkuk.ac.kr
(Y. Chong).
1
These two authors contributed equally on this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.005

M.K. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 5698e5704
5699
Fig. 1. Structures of ADK with an arylmethyloxy or arylmethylamino substituent (1), 5-hydroxyflavone (2), 3-O-arylmethylgalangins (3), 7-O-arylmethylgalangins (4), 3,7-bis-O-
arylmethyl-5-hydroxychromone (50), and the title compound of this study [2-arylmethyloxy-6-(3-chlorobenzyloxy)-5-hydroxychromone, 5].
introduced the substituents on both 2- and 6-positions. As an
enoleketo arrangement on a flat fused AC ring system forming an
intramolecular hydrogen bond is believed as a key pharmacophoric
element of the flavones as well as the chromones, it was fair to
assume that flipping the AC ring system to a ketoeenol form (CA
ring system) would not affect the pharmacophore of the chromone
scaffold. As shown in Fig. 1, the 2,6-bis-O-arylmethyloxy substitu-
ents attached to the flipped fused ring system in 5 are superim-
posable to those at the 3- and 7-positions of 50. The arylmethyloxy
substituent at 6-position was fixed with a 3-chlorobenzyloxy group
because it provided the 3-O-arylmethyloxygalangin derivatives (3)
with the most potent inhibitory activity against the HCV RdRp
substituted benzyl alcohols to furnish 2,6-bis-arylmethyloxy-5-
hydroxychromone derivatives (10ae10g) in 47e71% yield [12].
Final Lewis acid-mediated demethylation of 10 to 5 necessitated
careful control of the reaction conditions because of concurrent loss
of the arylmethyl moiety. Thus, after optimization of Lewis acid as
well as the reaction conditions, use of less than one equivalent
(w0.89) of AlCl3 in CH2Cl2 was found to be the method of choice to
provide the desired 2-arylmethyloxy-6-(3-chlorobenzyloxy)-5-
hydroxychromones (5ae5g) in moderate (43e57%) yield.
2.2. Biological activity
(IC50 ¼ 0.1
mM) [7]. In contrast, as the anti-HCV activity of the 7-O-
In this study, we combined the pharmacophoric arylmethyloxy
group of 3-O-arylmethylgalangins (3) and 7-O-arylmethylgalangins
(4) on the 5-hydroxychromone scaffold to prepare 7 derivatives of
arylmethylgalangin derivatives (4) was found to be critically
dependent upon size and position of a hydrophobic aromatic
substituent in the arylmethyloxy group [8], the aromatic substit-
uent of the 2-arylmethyloxy group was varied with Cl-, I-, and
CN- at 3-, 4-, and 5-positions of the aromatic ring. Herein we
report synthesis and evaluation of anti-HCV as well as anti-
SCV activity of novel 2-arylmethyloxy-6-(3-chlorobenzyloxy)-5-
hydroxychromones 5 (Fig. 1).
2,6-bis-arylmethyloxy-5-hydroxychromones
(5ae5g).
The
2. Results and discussion
2.1. Chemistry
Synthesis of the title compounds, 2-arylmethyloxy-6-(3-
chlorobenzyloxy)-5-hydroxychromone (5ae5g) is summarized in
Scheme 1.
Following
literature
methods
[10],
2,5-dihydroxy-6-
methoxyacetophenone (6) was prepared from commercially avail-
able 2,5-dihydroxyacetophenone. Due to intramolecular hydrogen
bonding, the 5-OH group of 6 selectively reacted with 3-ClPhCH2Br
in the presence of K2CO3 to provide the alkylated product 7 in 58%
yield. Treatment of 7 with LiHMDS, CS2, and MeI in THF provided the
corresponding ketene dithioacetal, which was then cyclized under
basic conditions to give 2-methylthiochromone 8 in 51% yield [11].
The sulfide 8 was oxidized by mCPBA to 9, whose methylsulfonyl
group was then successfully displaced with sodium salt of variously
Scheme 1. Synthesis of the title compounds (5ae5g).

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M.K. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 5698e5704
synthesized 5-hydroxychromone derivatives were then evaluated
for their antiviral activity against HCV as well as SCV, and the result
is summarized in Table 1.
2.2.2. Anti-SCV activity
Viral helicases couple energy from nucleotide triphosphate
(NTP) hydrolysis with unwinding of duplex viral nucleic acid and,
due to a critical role in viral viability, both NTPase and duplex
unwinding activity have been the subjects of antiviral drug
discovery campaigns [14,15]. In our previous study, we have iden-
tified ADKs (1) as anti-SCV agents with selective inhibition
2.2.1. Anti-HCV activity
While the 5-hydroxychromone derivatives 5be5g with aryl-
methyloxy substituents at both 2- and 6-positions showed
moderate to potent anti-HCV activity, the compound 5a with 2-
benzyloxy substituent (R ¼ H) did not show any anti-HCV
activity. More interestingly, compounds with the same type of
substituent (5b/5c and 5e/5f) showed similar anti-HCV activity
regardless of the position of substitution. In particular, derivatives
(IC50 ¼ 5.4e13.6
mM) against duplex DNA-unwinding helicase
activity of SCV NTPase/helicase without significant impact on the
ATPase activity (1, Table 1) [5]. Unfortunately, presumably due to
the presence of additional 2-phenyl group, neither of the two 5-
hydroxyflavone derivatives (3 and 4) showed anti-SCV activity
(Table 1). Thus, in this study, the 5-hydroxyflavone scaffold was
replaced with a 5-hydroxychromone, on which two arylmethyloxy
substituents were installed. The resulting derivatives 5ae5g
showed interesting structureeactivity relationship. First, in
comparison with the ADKs which showed selective inhibition
against the helicase activity, 5-hydroxy-6-(3-chlorobenzyloxy)-
chromone derivatives with chloro- (5b and 5c) or iodo- (5e and 5f)
benzyloxy substituent inhibited both NTPase and helicase activities
with more potent inhibition against NTPase activity (Table 1).
Second, unlike the anti-HCV activity, inhibitory activity against SCV
NTPase/helicase was dependent upon the position of the aromatic
substituent, and the 3-substituted derivatives (5b and 5e) showed
significantly more potent activity compared with the correspond-
ing 4-substituted congeners (5c and 5f) (Table 1). Third, 3,5-di-
chloro-substituted derivative 5d, which showed the most potent
anti-HCV activity among the series, was not effective in inhibiting
with bulky iodo substituents (5e and 5f, EC50 ¼ 3e4
significantly more active than the chlorine- (5b and 5c,
M) substituted ones. In
mM) were
EC50 ¼ 17e24
m
M) or cyano- (5g, EC50 ¼ 17
m
line with these observations, it is noteworthy that the 3,5-di-
chloro-substituted derivative 5d showed the most potent anti-HCV
activity (EC50 ¼ 1
mM), which is comparable to that of the ADK (1,
EC50 ¼ 0.8 M). Taken together, these results indicate the critical
m
role of the aromatic substituents in binding of the 5-
hydroxychromone derivatives to the viral target protein and
propose a large hydrophobic binding site in the viral target protein
accommodating an aromatic ring with bulky substituents.
As the HCV RdRp was proven to be a viral target enzyme of
ADKs [2e4], it was also assumed as a potential viral target protein
of the bioisosteres of ADKs including flavone as well as
chromone derivatives. Inhibitory activity of the synthesized 5-
hydroxychromone derivatives (5ae5g) against HCV genotype 1b
NS5B
D
c21 enzyme was estimated [13], but the enzyme activity
the SCV NTPase/helicase (IC50 > 50
SCV 5-hydroxychromones, 3-iodo-substituted one (5e) showed
M for ATPase activity, 11 M for
helicase activity). Based on these results, it might be concluded that
the SCV NTPase/helicase has a hydrophobic binding region specific
mM). Fourth, among the anti-
was not affected by addition of the test compounds (data not
shown). Thus, the anti-HCV replicon activity of the 5-
hydroxychromone derivatives could be attributed to a different
mechanism yet to be determined.
the most potent activity (IC50 ¼ 4
m
m
Table 1
Anti-HCV activity, inhibition of the SCV NTPase/helicase, and cytotoxicity of the 2-arylmethyloxy-6-(3-chlorobenzyloxy)-5-hydroxychromones (5ae5g) in comparison with
those of the ADK (1), 3-O-arylmethylgalangin (3), and 7-O-arylmethylgalangin (4).
Compds
R
Antiviral Activity
HCV
Cytotoxicity
M)d
(
m
SCV (IC50
, mM)
(EC50, m
M)a
NTPaseb
Helicasec
5a
5b
5c
5d
5e
5f
5g
1
H
3-Cl
4-Cl
3,5-di-Cl
3-I
4-I
3-CN
3-(4-chlorobenzyl-amino)
3-NO2
3-CN
>50
17
24
1
4
3
17
0.8 [4]
5 [7]
>50
10
28
>50
4
23
>50
40
53
>50
11
31
>50
11 [5]
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
37
>50 [7]
>50
>50
3
4
3 [8]
a
Concentration required to inhibit HCV RNA replication by 50% in HCV replicon cell.
Concentration required to inhibit SCV NTPase activity by 50%.
Concentration required to inhibit duplex DNA-unwinding activity of SCV helicase by 50%.
Concentration required to reduce proliferation of normal human fibroblast cell HS27 by 50%.
b
c
d

M.K. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 5698e5704
5701
for 3-substituted aromatic rings attached to the 6-(3-
chlorobenzyloxy)-5-hydroxychromone core structure.
chromatography on silica gel (Hex:EtOAc ¼ 8:1) to afford 7
(490 mg, 1.60 mmol, 58% yield) as yellow oil: 1H NMR (400 MHz,
DMSO-d6)
J ¼ 9.0 Hz, 1H), 6.58 (d, J ¼ 9.0 Hz, 1H), 5.07 (s, 2H), 3.79 (s, 3H),
2.47 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
201.7, 149.7, 146.7,
143.2, 139.3, 132.6, 129.9, 127.2, 126.7, 125.6, 122.0, 118.1, 110.5,
69.6, 60.6, 31.8; LC/MS (ESI) m/z Found: 307.0245 (M þ H)þ; Calcd
for C16H15ClO4: 306.0659.
d 10.22 (s, 1H), 7.52 (s, 1H), 7.44e7.40 (m, 3H), 7.08 (d,
2.2.3. Cytotoxicity
The 5-hydroxychromone derivatives (5ae5g) showed no cyto-
static effect in human normal fibroblast cell line HS27 up to 50 mM
d
(Table 1).
3. Conclusion
4.1.3. Synthesis of 6-(3-chloro-benzyloxy)-5-methoxy-2-
methylsulfanyl-chromen-4-one (8)
Structureeactivity relationship study of the ADKs and the
5-hydroxyflavones proposed that the 5-hydroxychromone deriva-
tives with two arylmethyloxy substituents would show antiviral
activity against both HCV and SCV. 2-Arylmethyloxy-6-(3-
chlorobenzyloxy)-5-hydroxychromones (5be5g), thus prepared,
showed anti-HCV activity in cell-based assay. As anticipated,
depending on the aromatic substituents on the 2-arylmethyloxy
moiety, some of the derivatives (5be5f) were also active against
SCV. In addition, unlike the ADKs which showed selective inhibition
against the helicase activity of the SCV NTPase/helicase, 2-
arylmethyloxy-6-(3-chlorobenzyloxy)-5-hydroxychromones
The compound 7 (800 mg, 2.61 mmol) obtained above was
dissolved in THF (5 mL) and the resulting solution was added to
a stirred solution of 1M LiHMDS (7.83 mL, 7.83 mmol) in THF
(15 mL) over 10 min at ꢃ78 ꢂC. After 30 min, CS2 (0.24 mL,
3.92 mmol) was added in one portion, and the reaction mixture was
allowed to warm to 0 ꢂC. After 1 h, MeI (0.36 mL, 5.74 mmol) was
added dropwise over 10 min, and stirring was continued for addi-
tional 1 h at room temperature. A solution of aq. 10N KOH (10 mL)
was added and the reaction mixture was stirred at 60 ꢂC for 30 min.
The reaction mixture was allowed to cool to 0 ꢂC and quenched by
addition of 1N HCl. After phase separation, the aqueous phase was
extracted with CH2Cl2 three times, and the combined organic layers
were dried over MgSO4 and concentrated under reduced pressure.
The crude product was purified by column chromatography on
silica gel (Hex:EtOAc ¼ 2:1) to afford 8 (483 mg, 1.33 mmol, 51%
(5be5f) were active against both NTPase and helicase activities of the
target
chlorobenzyloxy)-5-hydroxychromone (5e) showed the most
potent activity against HCV (EC50 ¼ 4 M) as well as SCV (IC50 ¼ 4
for ATPase activity, 11 M for helicase activity). In the absence of
enzyme.
Among
those,
2-(3-iodobenzyloxy)-6-(3-
m
mM
yield) as white powder: 1H NMR (400 MHz, CDCl3)
d 7.44 (s, 1H),
m
structural information of the SCV NTPase/helicase, this result would
provide valuable information for designing more potent inhibitors.
Also, the antiviral agents active both against HCV and SCV discovered
in this study might be used as a platform structure for future devel-
opment of the multi-target or broad-spectrum antivirals.
7.31e7.29 (m, 3H), 7.23e7.20 (m, 1H), 7.10e7.07 (m, 1H), 6.08 (s, 1H),
5.13 (s, 2H), 3.97 (s, 3H), 2.50 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
175.4, 168.2, 153.1, 149.7, 149.0, 139.1, 134.7, 130.1, 128.4, 127.7,
125.6, 122.3, 119.2, 113.0, 108.3, 72.2, 62.2, 14.0; LC/MS (ESI) m/z
Found 363.0952 (M þ H)þ; Calcd for C18H15ClO4S: 362.0380.
4. Experimental
4.1.4. Synthesis of 6-(3-chloro-benzyloxy)-2-methanesulfonyl-5-
methoxy-chromen-4-one (9)
4.1. Chemistry
To a stirred solution of 8 (500 mg, 1.38 mmol) in CH2Cl2 (20 mL)
was added mCPBA (681 mg, 3.04 mmol) at 0 ꢂC. The reaction
mixture was stirred for 12 h at room temperature, and then washed
with saturated aq. NaHCO3 solution, dried over MgSO4, and
concentrated under reduced pressure. The resulting crude product
was purified by column chromatography on silica gel
(Hex:EtOAc ¼ 2:1) to afford 9 (387 mg, 0.98 mmol, 71% yield) as off-
4.1.1. General remarks
Nuclear magnetic resonance spectra were recorded at 400 MHz
for 1H NMR and at 100 MHz for 13C NMR 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
singlet). Coupling constants are reported in hertz (Hz). The chemical
white powder: 1H NMR (400 MHz, CDCl3)
d 7.44 (s, 1H), 7.37e7.35
shifts are reported as parts per million (
d
) relative to the solvent
(m, 1H), 7.33e7.31 (m, 3H), 7.27 (d, J ¼ 2.2 Hz, 1H), 6.93 (s, 1H), 5.17
peak. All tested compounds were ꢀ95% purity as determined by
reverse phase HPLC. HPLC was performed on equipment with vari-
able wavelength (VW) UV detector and C18 e A 250 ꢁ 4.6 mm
column. Analytical conditions were as follows: Gradient used was
20%e60% acetonitrile in water containing 0.1% formic acid
(0e3 min), 60%e80% acetonitrile in water containing 0.1% formic
acid (3e5 min), 80%e100% acetonitrile in water containing 0.1%
formic acid (5e7 min), 100% acetonitrile in water containing 0.1%
formic acid (7e15 min), 100%e60% acetonitrile in water containing
0.1% formic acid (15e17 min), 60%e20% acetonitrile in water con-
taining 0.1% formic acid (17e20 min). Flow rate was set to 1 mL/min.
UV was detected at two different wavelengths (340 nm and 220 nm).
(s, 2H), 3.99 (s, 3H), 3.22 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
d
176.2, 159.2, 151.3, 149.7, 149.2, 138.3, 134.6, 130.1, 128.5, 127.4,
125.3, 122.9, 119.7, 113.9, 122.2, 71.6, 62.0, 40.8; LC/MS (ESI) m/z
Found: 395.0849 (M þ H)þ; Calcd for C18H15ClO6S: 394.0278.
4.1.5. Synthesis of 2-arylmethyloxy-6-(3-chloro-benzyloxy)-5-
methoxy-chromen-4-one (10)
The procedure described below for the synthesis of 10a is
representative: To a solution of 9 (200 mg, 0.51 mmol) inTHF (12 mL)
was added NaH (41 mg,1.02 mmol) and BnOH (0.06 mL, 0.61 mmol)
at 0 ꢂC, and the mixture was stirred for 6 h at room temperature. The
reaction was quenched with 1N HCl and extracted with CH2Cl2. The
combined organic layers was dried over MgSO4 and concentrated
under reduced pressure. The crude compound was purified by
column chromatography on silica gel (Hex:EtOAc ¼ 3:2) to afford
10a (106 mg, 0.25 mmol, 49% yield) as white powder.
4.1.2. Synthesis of 1-[3-(3-chloro-benzyloxy)-6-hydroxy-2-
methoxy-phenyl]-ethanone (7)
To a solution of 6 (500 mg, 2.74 mmol) and K2CO3 (379 mg,
2.74 mmol) in acetone (20 mL) was slowly added (1-
bromomethyl)-3-chlorobenzene (0.32 mL, 2.47 mmol). The reac-
tion mixture was stirred for 2 h at 60 ꢂC and then cooled to room
temperature, filtered with acetone, and concentrated under
reduced pressure. The crude product was purified by column
4.1.6. Synthesis of 2-benzyloxy-6-(3-chloro-benzyloxy)-5-methoxy-
chromen-4-one (10a)
Reaction of BnOH (87 mg, 0.61 mmol) and 9 (200 mg,
0.51 mmol) according to the procedure described above afforded

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M.K. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 5698e5704
10a (106 mg, 0.25 mmol, 49% yield) as white powder: 1H NMR
(400 MHz, CDCl3) 7.44e7.39 (m, 6H), 7.31e7.30 (m, 3H), 7.20 (d,
J ¼ 9.1 Hz, 1H), 7.07 (d, J ¼ 9.1 Hz, 1H), 5.58 (s, 1H), 5.18 (s, 2H), 5.12
(s, 2H), 3.97 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
165.8, 148.8,
61% yield) as white powder: 1H NMR (400 MHz, CDCl3)
d 7.76 (d,
J ¼ 8.2 Hz, 2H), 7.44 (s, 1H), 7.32e7.29 (m, 3H), 7.21 (d, J ¼ 9.1 Hz,
1H), 7.18 (d, J ¼ 8.2 Hz, 2H), 7.07 (d, J ¼ 9.1 Hz, 1H), 5.55 (s, 1H),
5.13 (s, 2H), 5.11 (s, 2H), 3.97 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
d
147.9, 140.0, 136.3, 135.0, 133.6, 130.9, 129.3, 129.2, 129.1, 128.8,
128.3, 127.8, 126.6, 120.5, 117.9, 113.4, 88.8, 71.5, 70.3, 61.7; LC/MS
(ESI) m/z Found 423.1598 (M þ H)þ; Calcd for C24H19ClO5:
422.0921.
d 178.0, 164.8, 149.0, 148.9, 148.5, 138.3, 137.4, 133.9, 132.9, 129.3,
129.0, 127.6, 126.9, 124.8, 121.1, 117.6, 112.1, 94.2, 88.4, 71.3, 69.8,
61.4; LC/MS (ESI) m/z Found 549.0132 (M þ H)þ; Calcd for
C24H18ClIO5: 547.9887.
4.1.7. Synthesis of 2,6-bis-(3-chloro-benzyloxy)-5-methoxy-
chromen-4-one (10b)
4.1.12. Synthesis of 3-[6-(3-chloro-benzyloxy)-5-methoxy-4-oxo-
4H-chromen-2-yloxymethyl]-benzonitrile (10g)
Reaction of (3-chlorophenyl)methanol (87 mg, 0.61 mmol) and
9 (200 mg, 0.51 mmol) according to the procedure described above
for the synthesis of 10a afforded 10b (146 mg, 0.32 mmol, 63%
Reaction of (3-cyanophenyl)methanol (81 mg, 0.61 mmol) and 9
(200 mg, 0.51 mmol) according to the procedure described above
for the synthesis of 10a afforded 10g (107 mg, 0.24 mmol, 47% yield)
yield) as white powder: 1H NMR (400 MHz, CDCl3)
d
7.44 (s, 2H),
as white powder: 1H NMR (400 MHz, CDCl3)
d 7.76 (s,1H), 7.71e7.67
7.37e7.35 (m, 2H), 7.31 (s, 4H), 7.23e7.22 (m,1H), 7.10e7.07 (m,1H),
(m, 2H), 7.58e7.54 (m, 1H), 7.45 (s, 1H), 7.33e7.30 (m, 3H),
7.24e7.22 (m, 1H), 7.11e7.09 (m, 1H), 5.56 (s, 1H), 5.21 (s, 2H), 5.14
5.56 (s, 1H), 5.15 (s, 2H), 5.13 (s, 2H), 3.98 (s, 3H); 13C NMR
(100 MHz, CDCl3)
d
178.6, 165.4, 149.6, 149.1, 138.9, 135.8, 134.9,
(s, 2H), 3.98 (s, 3H); 13C NMR (100 MHz, CDCl3)
d 178.8, 165.4, 149.8,
134.6, 130.2, 129.9, 129.2, 128.3, 127.9, 127.5, 125.8, 125.4, 121.8,
118.2, 112.7, 97.6, 89.0, 72.0, 70.2, 62.1; LC/MS (ESI) m/z Found
457.1011 (M þ H)þ; Calcd for C24H18Cl2O5: 456.0531.
149.7, 149.4, 139.1, 135.8, 134.8, 132.8, 132.1, 131.3, 130.2, 130.0,
128.5, 127.7, 125.7, 122.0, 118.4, 113.5, 113.0, 89.3, 72.1, 69.8, 62.3; LC/
MS (ESI) m/z Found: 448.1598 (M þ H)þ; Calcd for C25H18ClNO5:
447.0874.
4.1.8. Synthesis of 6-(3-chloro-benzyloxy)-2-(4-chloro-benzyloxy)-
5-methoxy-chromen-4-one (10c)
4.1.13. Synthesis of 2-arylmethyloxy-6-(3-chloro-benzyloxy)-5-
hydroxy-chromen-4-one (5)
Reaction of (4-chlorophenyl)methanol (87 mg, 0.61 mmol) and
9 (200 mg, 0.51 mmol) according to the procedure described above
for the synthesis of 10a afforded 10c (151 mg, 0.33 mmol, 65% yield)
The procedure described below for the synthesis of 5a is
representative: To a solution of 10a (118 mg, 0.28 mmol) in CH2Cl2
(20 mL) was added AlCl3 (33 mg, 0.25 mmol) at 0 ꢂC and the
mixture was stirred for 4 h at room temperature. The reaction was
quenched with 1N HCl and extracted with EtOAc, dried over MgSO4
and concentrated under reduced pressure. The crude compound
was purified by column chromatography on silica gel
(Hex:EtOAc:MC ¼ 2:1:1) to afford 5a.
as white powder: 1H NMR (400 MHz, CDCl3)
d 7.44 (s,1H), 7.41e7.37
(m, 4H), 7.33e7.30 (m, 3H), 7.21 (d, J ¼ 9.2 Hz, 1H), 7.08 (d,
J ¼ 9.2 Hz, 1H), 5.59 (s, 1H), 5.15 (s, 2H), 5.13 (s, 2H), 3.98 (s, 3H); 13C
NMR (100 MHz, DMSO-d6)
d 177.2, 165.2, 148.5, 148.3, 139.5, 133.6,
133.4, 133.1, 130.4, 130.2, 128.7, 127.9, 127.3, 126.1, 120.0, 117.4, 112.9,
88.3, 70.0, 69.8, 61.2; LC/MS (ESI) m/z Found 457.1121 (M þ H)þ;
Calcd for C24H18Cl2O5: 456.0531.
4.1.14. Synthesis of 2-benzyloxy-6-(3-chloro-benzyloxy)-5-
hydroxy-chromen-4-one (5a)
4.1.9. Synthesis of 6-(3-chloro-benzyloxy)-2-(3,5-dichloro-
benzyloxy)-5-methoxy-chromen-4-one (10d)
The desired compound 5a (65 mg, 0.16 mmol, 57% yield) was
Reaction of (3,5-dichlorophenyl)methanol (108 mg, 0.61 mmol)
and 9 (200 mg, 0.51 mmol) according to the procedure described
above for the synthesis of 10a afforded 10d (177 mg, 0.36 mmol,
obtained as white powder: 1H NMR (400 MHz, CDCl3)
d 13.02 (s,
1H), 7.46e7.40 (m, 5H), 7.37e7.28 (m, 4H), 7.12 (d, J ¼ 9.0 Hz, 1H),
6.74 (d, J ¼ 9.0 Hz, 1H), 5.58 (s, 1H), 5.26 (s, 2H), 5.16 (s, 2H); 13C
71% yield) as white powder: 1H NMR (400 MHz, CDCl3)
d
7.44 (s,
NMR (100 MHz, CDCl3) d 185.0, 167.3, 150.9, 148.2, 142.3, 139.0,
1H), 7.38 (t, J ¼ 1.8 HZ, 1H), 7.33e7.29 (m, 5H), 7.22 (d, J ¼ 9.1 Hz,
134.5, 133.4, 130.2, 129.3, 129.0, 128.2, 128.1, 127.6, 125.6, 122.1,
109.6, 105.2, 86.9, 71.7, 71.5 LC/MS (ESI) m/z Found: 409.1545
(M þ H)þ; Calcd for C23H17ClO5: 408.0765; HPLC retention time of
10.77 min, >97% pure at 340 nm and >95% pure at 220 nm.
1H), 7.08 (d, J ¼ 9.1 Hz, 1H), 5.54 (s, 1H), 5.13 (s, 2H), 5.12 (s, 2H); 13C
NMR (100 MHz, CDCl3)
d 177.7, 164.2, 148.7, 148.6, 148.3, 138.0,
136.3,134.7,133.7,129.1,128.2,127.4,126.6,125.0,124.5,120.8,117.3,
111.9, 88.2, 71.0, 68.4, 61.2; LC/MS (ESI) m/z Found: 491.0715
(M þ H)þ; Calcd for C24H17Cl3O5: 490.0142.
4.1.15. Synthesis of 2,6-bis-(3-chloro-benzyloxy)-5-hydroxy-
chromen-4-one (5b)
4.1.10. Synthesis of 6-(3-chloro-benzyloxy)-2-(3-iodo-benzyloxy)-
5-methoxy-chromen-4-one (10e)
Reaction of 10b (60 mg, 0.13 mmol) with AlCl3 (15 mg,
0.12 mmol) according to the procedure described above for the
synthesis of 5a afforded 5b (32 mg, 0.07 mmol, 54% yield) as: 1H
Reaction of (3-iodophenyl)methanol (143 mg, 0.61 mmol) and 9
(200 mg, 0.51 mmol) according to the procedure described above
for the synthesis of 10a afforded 10e (165 mg, 0.30 mmol, 59% yield)
NMR (400 MHz, CDCl3)
d 12.97 (s, 1H), 7.46 (s, 1H), 7.44 (s, 1H),
7.40e7.29 (m, 6H), 7.13 (d, J ¼ 9.0 Hz, 1H), 6.75 (d, J ¼ 9.0 Hz, 1H),
as white powder: 1H NMR (400 MHz, CDCl3)
d
7.80e7.72 (m, 2H),
7.44e7.08 (m, 8H), 5.56 (s, 1H), 5.13 (s, 2H), 5.11 (s, 2H), 3.98 (s, 3H);
13C NMR (100 MHz, CDCl3)
179.4, 166.1, 150.4, 150.3, 149.9, 139.7,
5.57 (s, 1H), 5.23 (s, 2H), 5.16 (s, 2H); 13C NMR (100 MHz, DMSO-d6)
d
183.2, 166.0, 148.0, 145.8, 140.6, 138.3, 135.3, 132.0, 131.8, 129.3,
d
129.1, 127.5, 126.9, 126.5, 126.1, 125.7, 124.9, 120.0, 107.4, 104.3, 85.0,
69.3, 68.7; LC/MS (ESI) m/z Found: 443.0911 (M þ H)þ; Calcd for
C23H16Cl2O5: 442.0375; HPLC retention time of 11.20 min, >97%
pure at 340 nm and >95% pure at 220 nm.
138.9, 137.5, 136.8, 135.3, 131.3, 130.7, 129.0, 128.3, 127.8, 126.2,
122.5, 119.0, 113.5, 95.4, 89.8, 72.7, 70.8, 62.9; LC/MS (ESI) m/z
Found: 549.1215 (M þ H)þ; Calcd for C24H18ClIO5: 547.9887.
4.1.11. Synthesis of 6-(3-chloro-benzyloxy)-2-(4-iodo-benzyloxy)-
5-methoxy-chromen-4-one (10f)
4.1.16. Synthesis of 6-(3-chloro-benzyloxy)-2-(4-chloro-
benzyloxy)-5-hydroxy-chromen-4-one (5c)
Reaction of (4-iodophenyl)methanol (143 mg, 0.61 mmol) and
9 (200 mg, 0.51 mmol) according to the procedure described
above for the synthesis of 10a afforded 10f (170 mg, 0.31 mmol,
Reaction of 10c (280 mg, 0.61 mmol) with AlCl3 (72 mg,
0.54 mmol) according to the procedure described above for the
synthesis of 5a afforded 5c (126 mg, 0.28 mmol, 46% yield) as white

M.K. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 5698e5704
5703
powder: 1H NMR (400 MHz, CDCl3)
d
12.97 (s, 1H), 7.45 (s, 1H),
HPLC: retention time of 10.24 min, >97% pure at 340 nm and >95%
7.42e7.36 (m, 4H), 7.34e7.32 (m, 1H), 7.30e7.28 (m, 2H), 7.13 (d,
J ¼ 9.0 Hz, 1H), 6.73 (d, J ¼ 9.0 Hz, 1H), 5.56 (s, 1H), 5.22 (s, 2H), 5.16
pure at 220 nm.
(s, 2H); 13C NMR (100 MHz, CDCl3)
d 184.0, 166.1, 150.0, 147.2, 141.5,
4.2. Biological assay
138.1,134.4, 133.6,131.0,129.0,128.5,128.3,127.3,126.7,124.7,121.3,
108.7, 104.3, 86.0, 70.7, 69.8; LC/MS (ESI) m/z Found: 443.0949
(M þ H)þ; Calcd for C23H16Cl2O5: 442.0375; HPLC retention time of
11.16 min, >97% pure at 340 nm and >95% pure at 220 nm.
4.2.1. 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
(University of Heidelberg, Heidelberg, Germany) [16]. Huh-5-2 cells
were seeded at a density of 5 ꢁ 103 per well in a tissue culture-
treated white 96-well view plate in complete DMEM supple-
4.1.17. Synthesis of 6-(3-chloro-benzyloxy)-2-(3,5-dichloro-
benzyloxy)-5-hydroxy-chromen-4-one (5d)
Reaction of 10d (72 mg, 0.15 mmol) with AlCl3 (18 mg,
0.13 mmol) according to the procedure described above for the
synthesis of 5a afforded 5d (34 mg, 0.07 mmol, 47% yield) as white
mented with 500 m
g/mL G418. After incubation for 24 h at 37 ꢂC (5%
CO2), medium was refreshed (with G418) and DMSO stock of test
compounds was added. After 4 days of incubation at 37 ꢂC, cell
culture medium was removed and luciferase activity was deter-
mined using the Steady-Glo luciferase assay system (Promega, Lei-
den, The Netherlands).
powder: 1H NMR (400 MHz, CDCl3)
d 12.93 (s, 1H), 7.46 (s, 1H), 7.40
(s, 1H), 7.33e7.29 (m, 5H), 7.15 (d, J ¼ 9.0 Hz, 1H), 6.75 (d, J ¼ 9.0 Hz,
1H), 5.56 (s,1H), 5.20 (s, 2H), 5.17 (s,1H); 13C NMR (100 MHz, CDCl3)
d
184.9, 166.7, 160.5, 150.9, 148.0, 142.5, 138.9, 136.7, 135.7, 129.9,
129.3, 128.2, 127.6, 126.0, 125.6, 122.2, 113.8, 105.3, 87.0, 71.6, 69.6;
LC/MS (ESI) m/z Found: 477.0615 (M þ H)þ; Calcd for C23H15Cl3O5:
475.9985; HPLC retention time of 11.89 min, >97% pure at 340 nm
and >95% pure at 220 nm.
4.2.2. Anti-SCV assay
Carboxytetramethylrhodamine (TAMRA)-modified 45-base-
oligomer and fluorescein-modified 25-base-oligomer were
purchased from Integrated DNA Technologies: 50-20T25Tam (50-
TTTTTTTTTTTTTTTTTTTTGAGCGGATTACTATACTACATTAGA (TAMR
A)-30) and 30-0T25Flu (50-(Fluorescein)TCTAATGTAGTATAGT
AATCCGCTC-30). The helicase substrate was prepared by annealing
the two oligomers, which resulted in 25 base pairs of dsDNA with
4.1.18. Synthesis of 6-(3-chloro-benzyloxy)-5-hydroxy-2-(3-iodo-
benzyloxy)-chromen-4-one (5e)
Reaction of 10e (375 mg, 0.68 mmol) with AlCl3 (80 mg,
0.61 mmol) according to the procedure described above for the
synthesis of 5a afforded 5e (198 mg, 0.37 mmol, 54% yield) as white
single-stranded 20 dT of 50-overhang. A 80
helicase (150 nM) in 20 mM HEPES (pH 7.4) buffer was added to
each well of the 96-well assay plate which already contained 1 l of
various concentrations of chemical compounds. After 5 min incu-
bation at rt, the FRET based dsDNA-unwinding assay was started by
ml solution of SCV
powder: 1H NMR (400 MHz, CDCl3)
d 13.02 (s, 1H), 7.80 (s, 1H), 7.74
(d, J ¼ 7.9 Hz, 1H), 7.46 (s, 1H), 7.40 (d, J ¼ 7.9 Hz, 1H), 7.33e7.29 (m,
m
3H), 7.19e7.12 (m, 2H), 6.74 (d, J ¼ 8.9 Hz, 1H), 5.56 (s, 1H), 5.19 (s,
2H), 5.16 (s, 2H); 13C NMR (100 MHz, CDCl3)
d 185.0, 167.1, 151.0,
148.2, 142.5, 139.1, 138.4, 136.9, 135.8, 134.6, 130.7, 130.0, 128.3,
127.7, 127.2, 125.7, 122.3, 109.7, 105.4, 94.7, 87.0, 71.7, 70.5; LC/MS
(ESI) m/z Found: 535.1316 (M þ H)þ; Calcd for C23H16ClIO5:
533.9731; HPLC retention time of 11.56 min, >97% pure at 340 nm
and >95% pure at 220 nm.
addition of 20
m
l 5ꢁ reaction solution [5 mM MgCl2, 45 mM ATP,
25 mM DTT, and 100 nM dsDNA substrate in 20 mM HEPES (pH
7.4)]. The reaction mixture was further incubated for 2 min at 37 ꢂC
and stopped with 100
ml of termination solution [0.1 M EDTA and
0.4
m
M trap DNA (unmodified 25 bases 30-0T25 oligomer) in 20 mM
HEPES (pH 7.4)]. The sample was excited at 485 nm and the fluo-
rescence was measured at 535 nm.
4.1.19. Synthesis of 6-(3-chloro-benzyloxy)-5-hydroxy-2-(4-iodo-
benzyloxy)-chromen-4-one (5f)
Reaction of 10f (318 mg, 0.58 mmol) with AlCl3 (68 mg,
0.52 mmol) according to the procedure described above for the
synthesis of 5a afforded 5f (132 mg, 0.25 mmol, 43% yield) as white
4.2.3. Cell viability assay
Normal human fibroblast cells (HS27) were seeded
(5 ꢁ 103 cells/well) in tissue-cultured COSTAR clear bottom 96-well
plate in complete DMEM (Dulbecco’s Modified Eagle Media) and
incubated for 1 day (37 ꢂC, 5% CO2). Prepared test compounds
dissolved in DMSO were diluted into 6 different concentrations (0.1,
0.5, 1, 5, 10, 50 mM) and added to the media. After 24 h, cell viability
was estimated by MTT assay. Each experiment was performed in
triplicates and repeated three times.
powder: 1H NMR (400 MHz, CDCl3)
d 12.97 (s, 1H), 7.77 (d,
J ¼ 8.2 Hz, 2H), 7.46 (s, 1H), 7.35e7.29 (m, 3H), 7.18 (d, J ¼ 8.2 Hz,
2H), 7.13 (d, J ¼ 8.9 Hz, 1H), 6.73 (d, J ¼ 8.9 Hz, 1H), 5.56 (s, 1H), 5.20
(s, 2H), 5.16 (s, 2H); 13C NMR (100 MHz, CDCl3)
d 186.7, 168.8, 152.7,
149.9, 144.2, 140.8, 139.9, 136.3, 134.9, 131.7, 131.5, 130.0, 129.4,
127.4, 124.0, 111.4, 107.0, 96.9, 88.7, 73.4, 72.6; LC/MS (ESI) m/z
Found 535.0123 (M þ H)þ; Calcd for C23H16ClIO5: 533.731 HPLC:
retention time of 11.52 min, >97% pure at 340 nm and >95% pure at
220 nm.
Acknowledgments
4.1.20. Synthesis of 3-[6-(3-chloro-benzyloxy)-5-hydroxy-4-oxo-
4H-chromen-2-yloxymethyl]-benzonitrile (5g)
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (2010-
0008260), by Priority Research Centers Program through the
National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (2009-0093824), by
a grant from ORP 11-28-66 (NIAS), and by a grant from Next
Generation Biogreen 21 program PJ007982 (Korea Rural Develop-
ment Administration). Y.-J. Jeong was supported by a grant of the
Korea Healthcare technology R&D Project, Ministry for Health,
Welfare & Family Affairs, Republic of Korea (A090410), and the
research program 2010 of Kookmin University in Korea.
Reaction of 10g (300 mg, 0.67 mmol) with AlCl3 (79 mg,
0.60 mmol) according to the procedure described above for the
synthesis of 5a afforded 5g (146 mg, 0.34 mmol, 51% yield) as white
powder: 1H NMR (400 MHz, CDCl3)
d 12.91 (s, 1H), 7.76 (s, 1H),
7.72e7.67 (m, 2H), 7.59e7.55 (m, 1H), 7.46 (s, 1H), 7.39e7.30 (m,
3H), 7.14 (d, J ¼ 9.0 Hz, 1H), 6.75 (d, J ¼ 9.0 Hz, 1H), 5.57 (s, 1H), 5.29
(s, 2H), 5.17 (s, 2H); 13C NMR (100 MHz, CDCl3)
d 186.8, 168.6, 152.8,
149.9,144.4,140.8,137.1,136.4,134.6,133.8,133.0,131.8,130.1,129.5,
127.5, 124.1, 120.0, 115.2, 111.5, 107.2, 88.9, 73.5, 71.8; LC/MS (ESI) m/
z Found: 434.1511 (M þ H)þ; Calcd for C24H16ClNO5: 433.0717;

5704
M.K. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 5698e5704
[7] H.S. Lee, K.-S. Park, B. Lee, D.-E. Kim, Y. Chong, Bioorg. Med. Chem. 18 (2010)
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