7-O-Arylmethylgalangin as a novel scaffold for anti-HCV agents

Article abstract of DOI:10.1016/j.bmcl.2010.08.012

In spite of potent antiviral activity, suboptimal physicochemical properties of aryl diketo acids (ADKs) necessitates modification of the core 1,3-diketo acid functionality into a novel scaffold. As the metal-binding affinity of the diketo acid is the key to the antiviral activity of ADKs, we anticipated 3,5-dihydroxy-4-oxo arrangement of galangin scaffold would serve as an excellent mimic for the diketo acid functionality. In this study, through synthesis and biological evaluation of various galangin derivatives, we have shown that the diketo acid functionality can be successfully replaced with the galangin scaffold by specific combination of the substituents to result in identification of a novel galangin derivative (3s) with anti-HCV activity (EC₅₀ = 0.9 μM) comparable to the ADK counterpart.

Full text of DOI:10.1016/j.bmcl.2010.08.012

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5709–5712
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
7-O-Arylmethylgalangin as a novel scaffold for anti-HCV agents
*
Hyo Seon Lee , Kwang-su Park , Chaewoon Lee, 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:
In spite of potent antiviral activity, suboptimal physicochemical properties of aryl diketo acids (ADKs)
Received 8 July 2010
Revised 2 August 2010
Accepted 3 August 2010
Available online 6 August 2010
necessitates modification of the core 1,3-diketo acid functionality into
a novel scaffold. As the
metal-binding affinity of the diketo acid is the key to the antiviral activity of ADKs, we anticipated 3,5-
dihydroxy-4-oxo arrangement of galangin scaffold would serve as an excellent mimic for the diketo acid
functionality. In this study, through synthesis and biological evaluation of various galangin derivatives,
we have shown that the diketo acid functionality can be successfully replaced with the galangin scaffold
by specific combination of the substituents to result in identification of a novel galangin derivative (3s)
Keywords:
7-O-Arylmethylgalangin
Flavonol
with anti-HCV activity (EC50 = 0.9
l
M) comparable to the ADK counterpart.
Ó 2010 Elsevier Ltd. All rights reserved.
Diketo acid
Hepatitis C virus (HCV)
Infection with hepatitis C virus (HCV) affects more than 170
million people worldwide, and 3–4 million are newly infected each
year. If untreated, more than 80% of HCV infected individuals will
develop chronic hepatitis, which can lead to liver cirrhosis and
eventually to hepatocellular carcinoma.1 No prophylactic vaccine
is available yet and the only approved anti-HCV treatment, combi-
tal-binding unit of the ADK (1). Thus, we reasoned that galangin
derivatives with appropriately substituted arylmethyl group (7-
O-arylmethylgalangin 3, Fig. 1) would serve as an excellent struc-
tural mimic, a bioisostere for ADKs. Herein, we report synthesis
of novel 7-O-arylmethylgalangin derivatives (3) with various aro-
matic substituents (R1 = Cl, Br, CN, NO2, OMe; R2 = H, CN, OMe)
and evaluation of their anti-HCV activities.
nation of pegylated interferon a
-2a and ribavirin,2 suffers from low
sustained response and numerous side effects.3 As a result, there is
a compelling need to develop safe and more efficacious antiviral
drugs.4
Aryl diketo acids (ADKs) have been reported as inhibitors of
HCV NS5B polymerase with low-micromolar IC50’s.5 In an earlier
paper,6 we reported the design, synthesis, and antiviral evaluation
of a small series of ADK derivatives, identifying some selective HCV
inhibitors. In particular, a 4-chlorophenylmethyloxy or 4-chlor-
ophenylmethylamino substituent provided the resulting ADK
Galangin (2) and 40-substituted galangin derivatives (6a–6c)
were prepared from a literature protocol14–16 using esterification
followed by cyclization of the readily available17 substituted phe-
nol 4 (Scheme 1). Acetylation of 6 with acetic anhydride (Ac2O)
in pyridine afforded the peracetylated galangin derivatives, which
underwent regioselective deacethylation of the 7-OAc group with
PhSH in NMP16,18,19 to provide the corresponding galangin 3,5-
diacetates 7. Alkylation of 7 with variously substituted benzyl bro-
mides followed by deacetylation by treatment with methanolic
ammonia afforded a series of 7-O-arylmethylgalangin 3.
derivative with potent antiviral activity [1, Fig. 1 (EC50 = 0.82 lM)].
However, suboptimal physicochemical properties of ADKs such
as chemical instability, irreversible covalent binding to protein and
poor stability in plasma7 necessitates modification of the core 1,3-
diketo acid functionality into a novel scaffold with more drug-like
properties. Pyrophosphate mimics like ADKs are known to inhibit
HCV NS5B through binding the metal ions at the active site while
the aryl group forms a specific hydrophobic interaction with an
adjacent hydrophobic pocket or surface.5,8–10 In this context,
well-known metal chelating property of naturally occurring flavo-
noids11–13 drew our attention. In particular, 3,5-dihydroxy-4-oxo
arrangement of galangin (2, Fig. 1) is in perfect match with the me-
All synthesized 7-O-arylmethylgalangin derivatives (3a–3u)
were evaluated for their ability to inhibit HCV replication in
Huh-5-2 cells.20,21 INF-
a was included as a positive control, and
the conditions of the luminescence-based assay used to test the
antiviral activity of the compounds were previously described.22,23
The cytostatic effect of the test compounds was also evaluated in
the same cell line.23 Antiviral effect and cytostatic effect are sum-
marized as EC50 and CC50, respectively, in Table 1.
When R1 = H, galangin derivatives (3a–3r) showed moderate to
potent antiviral activity with 3i and 3j (EC50 = 3 and 2 lM, respec-
tively, Table 1) being most active. As was the case in ADKs,6 antivi-
ral activity of the galangin derivatives was critically dependent not
only on the type, but also on the position of the substituent. Thus,
analysis of the anti-HCV activity indicates that the galangin deriv-
atives can be divided into two groups depending upon the R2
* Corresponding author. Tel.: +82 2 2049 6100; fax: +82 2 454 8217.
E-mail address: chongy@konkuk.ac.kr (Y. Chong).
These two authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.012

5710
H. S. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5709–5712
O
OH
OH
O
OH
O
O
5
OH
3
2
OH
OH
4
O
HO
7
O
O
NH
R1
2'
4'
3'
4"
2"
R2
3"
Cl
1
2
3
(EC50 = 0.82 µM)
Figure 1. Structures of ADK (1), galangin (2), and 7-O-arylmethylgalangin (3) with the numbering system. The common metal-binding unit is represented as thick lines.
BnO
O
O
HO
O
O
AcO
O
O
OBn
BnO
O
OH
OAc
OBn
BnO
a
b, c
d, e
HO
HO
O
BnO
OH
R1
R1
R1
4
5a R1
=
=
=
H
CN
CF3
2
6a
6b
R1
R1
R1
=
=
=
H
CN
CF3
7a R1
=
=
=
=
H
CN
R1
R1
R1
5b
5c
7b
7c
R1
CF3
OMe
5d R1 = OMe
6c R1 = OMe
7d R1
f, g
R1 R2
R1 R2
R1
R2
OH
O
OH
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3m
3n
3o
3p
3q
3r
H
H
H
H
H
H
2"-Cl
2"-Br
2"-CN
2"-NO2
2"-Me
H
H
H
H
H
H
3"-Cl
3"-Br
3"-CN
3"-NO2
3"-Me
H
H
H
H
H
4"-Cl
4"-Br
4"-CN
4"-NO2
4"-Me
4"-OMe
4"-Cl
O
2"
7
O 2
2'
R1
4'
3'
4"
R2
2"-OMe 3l
3"-OMe
H
CN
CF3 4"-Cl
3"
3s
3t
3a 3u
~
3u OMe 4"-Cl
Scheme 1. Synthesis of the 7-O-arylmethylgalangin derivatives (3a–3u). Reagents and conditions: (a) R1COcl, pyridine, rt, 87–89%; (b) K2CO3, Bu4NBr, toluene, 90 °C; (c) 20%
Pd/C, CH2Cl2/MeOH (1:1), 62–65%; (d) Ac2O, pyridine, rt, 77–78%; (e) PhSH, imidazole, NMP, 0 °C, 79–92%; (f) R2PhCH2Br, K2CO3, acetone, rt, 53–76%; (g) NH3/MeOH, rt 71–
92%.
substituents. Three hydrophobic groups of the similar size (Cl, Br,
and Me) substituted at either 200- or 400-position provided the corre-
sponding arylmethylgalangin derivatives (3a, 3b, 3e, 3m, 3n, and
3q) with significant antiviral activity (Table 1, Fig. 2a). On the con-
trary, site-specific antiviral effects were observed when hydrogen
bond acceptors (CN, NO2, or OMe) were substituted at the 300-posi-
tion of the galangin derivatives (3i, 3j, and 3l; Table 1 and Fig. 2a).
In terms of the cytostatic effect, clear structure–activity rela-
tionship was also identified but in different ways. While one group
of the galangin derivatives with halogens (Cl and Br) and OMe-sub-
stituent (3a, 3b, 3f, 3g, 3h, 3l, 3m, 3n, and 3r) was not cytostatic at
the galangin scaffold can work as a diketo acid replacement. How-
ever, 3m and 3n are about 10 times less potent than the ADK ana-
logue 1 (Fig. 1, EC50 = 0.8 l
M),6 and the loss of antiviral activity
upon replacement of diketo acid with a galangin needs to be
overcome.
In this context, it should be reminded that the metal-binding
affinity of the diketo acid is the key to the antiviral activity of ADKs,
and the lower antiviral activity of the galangin derivative may
partly be attributed to its metal-binding capacity different from
that of ADK. For these reasons, it was of our interest to vary the
R1 substituent and monitor its effect on antiviral activity of the
resulting galangin derivative because the metal-binding capacity
of galangin 3-OH can be resonance-controlled by R1. Thus, with
the R2 substituent fixed with that of the most active 3m and 3n
(400-Cl), three functional groups with different electronic property
(CN, CF3, and OMe) were introduced as R1 to provide three more
galangin derivatives (3s, 3t, and 3u, Scheme 1 and Table 1). Elec-
tronic effect of the R1 substituent was manifested in 3s. With elec-
tron withdrawing cyano group conjugated with the 2-phenyl ring,
the galangin derivative 3s showed significantly increased anti-HCV
all up to 100 lM, others with CN, NO2, or Me substituent (3c–3e,
3i–3k, and 3o–3q) showed cytostatic effect regardless of the sub-
stitution position (200, 300, and 400) albeit in different degrees (Table
1, Fig. 2b).
Taken together, the halogen atoms (Cl and Br) substituted at 400-
position of the aromatic ring provided the resulting 7-O-aryl-
methylgalangin derivatives (3m and 3n) with selective (CC50
>100 lM) and potent (EC50 = 8 and 6 lM, respectively) antiviral
activity. These two compounds and the anti-HCV ADK analogue
(1, Fig. 1)6 were found to share the same 4-chlorophenylmethyl
group directly substituted to the metal-binding unit (galangin
and diketo acid, respectively), which provides proof of concept that
activity (EC50 = 0.9
which is comparable to that of the ADK analogue 1 (Fig. 1,
EC50 = 0.8
M).6 Through combination of resonance and inductive
lM, 8.9 times increase compared with 3m),
l

H. S. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5709–5712
5711
Table 1
Anti-HCV activity and cytostatic effect of 7-O-arylmethylgalangin derivativesa
b,c
c,d
Compd
R1
R2
EC50
(
lM)
CC50
(lM)
SIe
Position
200
Substituent
f
3a
3b
3c
3d
3e
3f
H
H
H
H
H
H
Cl
Br
CN
NO2
Me
OMe
30
10
39
25
7
>100
>100
22
32
17
—
—
f
0.6
1.3
2.4
f
53
>100
—
f
3g
3h
3i
3j
3k
3l
H
H
H
H
H
H
300
400
400
Cl
Br
CN
NO2
Me
OMe
22
37
3
2
27
15
>100
>100
72
76
35
—
—
f
24
38
1.3
f
>100
—
f
3m
3n
3o
3p
3q
3r
H
H
H
H
H
H
Cl
Br
CN
NO2
Me
OMe
8
6
19
10
6
>100
>100
94
93
88
—
—
f
4.9
9.3
14.7
f
38
>100
—
3s
3t
3u
CN
CF3
OMe
Cl
0.9
3
6
67
73
64
74.4
24.3
10.7
a
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.
b
Concentration required to inhibit HCV RNA replication by 50%.
The values obtained as the average of triplicate determinations.
Concentration required to reduce cell proliferation by 50%.
c
d
e
Selectivity index: ratio of CC50 to EC50
Select antiviral effect.
.
f
Figure 2. Correlation of the (a) anti-HCV activity (EC50) and (b) cytostatic effect (CC50) with the position of the arylmethyl substituent (R2) (Cl: ꢀ, Br: j, CN: N, NO2: ꢀ, OMe: *,
Me: d).
effect, the cyano group must have dramatically affected the elec-
tronic state of the galangin 3-OH and thereby its metal-binding
capacity. In comparison, with the lack of resonance effect, substitu-
tion with a CF3 group resulted in only 2.6 times increase in antivi-
the metal-binding unit, and the anti-HCV activity of 3s is compara-
ble to that of the ADK analogue 1. Taken together, the galangin
scaffold successfully replaced the diketo acid structure to show po-
tent antiviral activity by specific combination of the aromatic sub-
stituents, which warrants extensive structure–activity relationship
study around the galangin scaffold.
ral activity of 3t (EC50 = 3
with electron-donating OMe-substituent (3u) showed no signifi-
cant change (EC50 = 6 M, Table 1).
lM, Table 1). The galangin derivative
l
In summary, as a replacement of the unstable diketo acid core
structure, we proposed galangin as a novel anti-HCV scaffold. Syn-
thesis and biological evaluation of the substituted galangin deriva-
tives revealed interesting structure–activity relationship and
identified a specific anti-HCV compound 7-O-(400-chlorophenyl-
methyl)galangin (3m) of which antiviral activity was further opti-
mized by introduction of an electron-withdrawing cyano group
conjugated with 2-phenyl group (3s). The most active galangin
derivative 3s has the same pattern of aromatic substitution with
the ADK analogue 1 at the arylmethyl group directly attached to
Acknowledgments
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), Priority
Research Centers Program through the National Research Founda-
tion of Korea (NRF) funded by the Ministry of Education, Science
and Technology (2009-0093824), and Biogreen 21 (Korea Ministry
of Agriculture and Forestry).

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H. S. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5709–5712
14. Caldwell, S. T.; Petersson, H. M.; Farrugia, L. J.; Mullen, W.; Crozier, A.; Hartley,
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supplemented 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 (Promega, Leiden, The Netherlands). In order to estimate the
cytostatic effects, Huh-5-2 cells were seeded at a density of 5 ꢀ 103 per well of a
96-well plate in complete DMEM with the appropriate concentrations of G418.
Serial dilutions of the test compounds in complete DMEM without G418 were
added 24 h after seeding. Cells were allowed to proliferate for 3 days at 37 °C,
after which the cell number was determined by WST-1 assay.