Effects of the aryl linker and the aromatic substituent on the anti-HCV activities of aryl diketoacid (ADK) analogues

Article abstract of DOI:10.1016/j.bmcl.2008.07.008

Based on our pharmacophore model of the aryl diketoacids (ADKs), we designed and prepared a series of novel ADK analogues, which showed potent inhibitory activities against the NS5B polymerase in the submicromolar range. Pharmacophore-guided docking study revealed that the antiviral activities of the ADKs are highly dependent upon the aryl linker as well as the size and position of the aromatic substituent. It is of another importance that, unlike previously reported ADKs, three ADK analogues synthesized in this study effectively blocked Hepatitis C Virus (HCV) replication in the replicon systems.

Full text of DOI:10.1016/j.bmcl.2008.07.008

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4661–4665
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Effects of the aryl linker and the aromatic substituent on the anti-HCV activities
of aryl diketoacid (ADK) analogues
Jinyoung Kim a, , Ki-Sun Kim a, , Hyo Seon Lee a, Kwang-Su Park a, Sun Young Park a, Seock-Yong Kang a,
Soo Jae Lee b, Hyung Soon Park b, Dong-Eun Kim a, Youhoon Chong a,
*
a Department of Bioscience & Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
b Probiond, Co. Ltd., 402 Gwangjin VBS Center, Seoul 143-823, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on our pharmacophore model of the aryl diketoacids (ADKs), we designed and prepared a series of
novel ADK analogues, which showed potent inhibitory activities against the NS5B polymerase in the sub-
micromolar range. Pharmacophore-guided docking study revealed that the antiviral activities of the ADKs
are highly dependent upon the aryl linker as well as the size and position of the aromatic substituent. It is
of another importance that, unlike previously reported ADKs, three ADK analogues synthesized in this
study effectively blocked Hepatitis C Virus (HCV) replication in the replicon systems.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 20 May 2008
Revised 1 July 2008
Accepted 3 July 2008
Available online 9 July 2008
Keywords:
Hepatitis C virus (HCV)
Aryl diketoacid (ADK)
Pharmacophore-guided docking
Aryl linker
Aromatic substituent
From the therapeutic point of view, hepatitis C virus (HCV) of-
fers a number of latent targets for small molecule intervention.1
Initial effort for the development of HCV-directed antiviral agent
has focused on the inhibition of essential virus-encoded enzy-
mes,1b and it is likely that agents able to disrupt function of the
HCV NS5B polymerase will prove effective in the treatment of
HCV infections due to its essential role in viral replication. Ad-
vances in high throughput-screening assays have resulted in the
identification of numerous anti-NS5B compounds broadly catego-
rized as nucleoside (NIs) and non-nucleoside (NNIs) inhibitors.2
Particularly, NNIs represent a chemically diverse family of com-
pounds which act by complex and various mechanisms by binding
near the active site or discrete allosteric sites of NS5B.3 Apart from
allosteric inhibitors, pyrophosphate (1, Fig. 1) mimics like aryl
diketoacids (2, Fig. 1) have been reported to inhibit the HCV
NS5B polymerase through chelation of the divalent cation in the
active site.4–7
Figure 1. Structures of pyrophosphate (1) and aryl diketoacid (2).
provide valuable insights into designing novel NS5B polymerase
inhibitors.
Recently, we proposed a pharmacophore model of ADK ana-
logues which is composed of a metal-binding diketoacid function-
ality, two hydrogen-bonding acceptors, and/or a hydrogen-bond
donor (Fig. 2).10 The pharmacophore model was successfully ap-
plied for the pharmacophore-guided docking followed by 3D-QSAR
study to generate a 3D-QSAR (CoMSIA) model which gave a good
correlation of the biological activities of ADKs with their three-
dimensional structures.11 However, effects of the linker moiety
bridging two aryl groups (XCH2, Fig. 2) as well as the substituent
at the terminal aromatic group (R, Fig. 2) on the antiviral activities
of the ADKs have yet to be evaluated.
Even though the inherent reactivity of the diketoacid moiety
presented a problem for the further development of these com-
pounds as drug candidates,8 ADKs represent a general scaffold
from which an intensive search for chemically and biologically sta-
ble diketoacid replacements has been initiated.9 Thus, information
about the binding mode of the ADKs to the target enzyme might
The docking study revealed that the hinge-like aryl linker which
connects the diketoacid moiety with the terminal aryl group (Fig.
2) is directly hydrogen-bonded to the protein, and, with the me-
tal-binding diketoacid as well as the hinge-like linker bound to
* Corresponding author. Tel.: +82 2 2049 6100; fax: +82 2 454 8217.
E-mail address: chongy@konkuk.ac.kr (Y. Chong).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.008

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J. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4661–4665
Figure 4. ADK analogues studied.
followed by hydrolysis with sodium hydroxide in a mixture of
methanol and water provided the desired ADKs (5–12) in 70–88%
yields.
Figure 2. Pharmacophore model of the ADKs: metal-binding diketoacid, two
hydrogen-bond acceptors, and/or a hydrogen-bond donor.
All compounds synthesized were assayed against HCV genotype
the protein, the terminal aryl group has only limited chance to
interact with the enzyme. Moreover, the pharmacophore-guided
docking study suggested that, in addition to the pharmacophore
model described above (Fig. 2), there are two separate aryl-binding
sites (hydrophobic pocket and groove, Fig. 3) which can accommo-
date the terminal aryl groups of the ADKs. In particular, both aryl-
binding domains have additional hydrophobic holes which are in
perfect size and shape for accommodating a chlorine substituent
(Fig. 3). Taken together, we envisaged that, upon binding of the
ADKs to the enzyme active site, the aryl linker determines the loca-
tion of the terminal aryl group of which hydrophobic interaction
with the aryl-binding site governs the antiviral activities of the
ADKs.
This paper describes a proof-of-concept effort designed to verify
the effects of the aryl linker (X@O or NH, Fig. 4) and the terminal
aryl substituents (Y = H, or Cl, Fig. 4) of the ADKs on their antiviral
activities.
The general procedure for syntheses of the ADK analogues was
adapted from the previous structure–activity relationship study by
Summa et al.4 (Scheme 1). Thus, O-benzylacetopheonones and N-
benzylacetopheonoes (4) were prepared by benzylation of the
appropriate acetophenones (3) with benzyl bromide or p-chloro
benzyl bromide in DMF in the presence of K2CO3 at 60 °C for
24 h (Scheme 1). Reaction of the acetophenones (4) thus obtained
with dimethyl oxalate in the presence of sodium methoxide in THF,
1b NS5B
Dc21 enzyme to assess their inhibitory activity (IC50,
Table 1).12 Also, all analogues were evaluated in a cell-based assay,
in which the HCV subgenomic replicon RNA-harboring NS5B gene
was transfected and expressed in the Huh-7 hepatoma cell lines,
and EC50 and CC50 values are reported (Table 1).13
The inhibitory activities of two known compounds (7 and 11)
against the target enzyme (IC50’s: 4.6 and 28 lM, respectively)
were comparable to the previously reported data (8.0 and 17 lM,
respectively).7,14 In general, the unsubstituted ADKs (5, 9 and 11)
showed only marginal anti-HCV activities (IC50’s: 8.3, 30, and
28
12) were clearly more potent inhibitors of the HCV NS5B polymer-
ase (IC50’s: 1.5, 2.0, and 0.96 M, respectively). Unlike other para-
lM), but the corresponding para-chloro analogues (6, 10 and
l
chloro analogues, compound 8 showed decreased antiviral activity
compared with its unsubstituted counterpart 7 (Table 1) whereas
the N-benzyl analogue of 8 (12) showed very potent enzyme inhib-
itory activity (IC50: 0.96 lM, Table 1). It is of another interest that,
even though the regioisomers 6 and 10 have different configura-
tions and thereby different spatial orientations of the terminal aryl
groups with respect to the diketoacid moieties, they show almost
equipotent antiviral activities.
Binding modes of the ADK analogues provided by the pharma-
cophore-guided docking were in fairly good agreement with the
structure–activity discussion described above (Fig. 5). First, com-
pounds with IC50 values less than 10 lM (5, 6, 10, 11, and 12) gave
docking poses at the active site but others (7, 8, and 9) failed to
dock. In particular, the meta configurations of 7 and 8 proved un-
able to locate the terminal aryl groups at either of the terminal
aryl-binding sites, and, as a result, the para-chloro substitution
has no effect on the anti-HCV effect of 8. Second, due to the differ-
ent locations of the aryl linkers as well as the terminal aryl groups
between 6 and 10 with respect to the diketoacid moieties, the
regioisomers 6 and 10 gave completely different docking poses
(Fig. 5a and b) of which terminal aromatic groups were bound to
the two different aryl-binding sites: the hydrophobic pocket and
the hydrophobic groove, respectively. Most importantly, even
though 7 and 8 failed to produce binding poses due to the lack of
the hydrophobic interactions with the aryl-binding sites, their N-
benzyl analogue 12 successfully located its terminal aryl group at
the hydrophobic pocket (Fig. 5c). The N-benzyl linker of 12 is
hydrogen-bonded to the carbonyl oxygen of Leu159 (Fig. 5c) as a
hydrogen-bond donor whereas the oxygen atom in the O-benzyl
linker is hydrogen-bonded to the amide NH of Leu159 (Fig. 5a) as
a hydrogen-bond acceptor. Thus, the subtle change in the hydrogen
bonding pattern of 12 induced by the presence of an amino group
as a hydrogen-bond donor instead of an acceptor enabled 12 to lo-
cate its terminal aromatic group at the hydrophobic pocket (Fig.
5c) leading to the remarkably improved IC50 value. Analysis of
the binding modes of 6 (Fig. 5a) and 12 (Fig. 5c) also reveals that
the interaction of the chlorine substituent with the hydrophobic
Figure 3. Two binding modes of the ADK analogues at the HCV NS5B polymerase:
Red and yellow sticks indicate ADK molecules 12 and 10 (Scheme 1) docked at the
hydrophobic pocket (red dotted circle) and groove (yellow dotted circle), respec-
tively. Hydrophobic holes inside the aryl-binding domains are denoted as blocked
arrows.

J. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4661–4665
4663
Scheme 1. Syntheses of the ADK analogues. Reagents and conditions: K2CO3, PhCH2Br or 4-CIPhCH2Br, acetone, rt; (b) dimethyl oxalate, NaOMe, THF, rt; (c) NaOH, MeOH/
H20, rt.
Table 1
Biological activity of the ADK analogues
Compound
IC50
(lM)
EC50
(l
M)
CC50 (lM)
5
6
7
8
8.3
1.5
0.66
3.1
30
> 50
40
0.54
28
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
4.6 (8.0a)
28
9
30
2.0
10
11
12
28 (17a)
0.96
0.82
a
IC50 values from the literature Refs. 7 and 14.
hole (Fig. 3) inside the pocket controls the inhibitory activities of
the ADKs: the para-chloro substituent of 12 is well oriented to
snuggle into the hole deep inside the hydrophobic pocket, which
explains lower IC50 value of 12 (Fig. 5c) than that of 6 (Fig. 5a).
To our knowledge, no data of antiviral activity in the cell-based
assay for the pyrophosphate mimics including diketoacids are
available presumably due to the high ionic nature and instability
of these compounds. Thus, it is of particular interest that the inhib-
itory activities of 10 and 12 against the NS5B enzyme were main-
tained in the HCV replicon assay. It is also noteworthy that 5, 10,
and 12 showed more potent antiviral activities in the cell-based as-
say (EC50 values of 0.66, 0.54 and 0.82
enzyme inhibition assay (IC50 values of 8.3, 2.02 and 0.96
respectively), which warrants further investigation of these ADKs
regarding their mode of actions in the cell-based assay. No ADK
l
M, respectively) than in the
lM,
analogues synthesized showed cytotoxicity up to 100 lM.
In summary, due to the chemical and enzymatic instability of
ADK analogues, many ADK derivatives including 5,6-dihydroxypy-
rimidine-4-carboxylic acids9 are under development. However, as
these derivatives are only different variations of the diketoacid
functionality, understanding the binding mode of the ADK would
provide valuable insights into designing novel inhibitors of the

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J. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4661–4665
Figure 5. Pharmacophore-guided docking poses of (a) 6, (b) 10, and (c) 12. Connolly surface of the enzyme, ADK analogues, and chlorine atoms are represented in gray
surface, capped sticks, and space fill model, respectively. Colored dishes indicate specific interactions around the pharmacophore with the enzyme residues: electrostatic
interaction with divalent metal ions (purple), H-bond acceptors (blue), and H-bond donors (red).
NS5B polymerase. Thus, based on our pharmacophore model of the
ADK analogues, we designed and prepared a series of novel ADK
analogues and evaluated their antiviral activities. We showed that
ADKs with potent antiviral activities are highly dependent upon
the aryl linkers as well as the size and position of the aromatic sub-
stituents. More interestingly, the hereto unreported potent antivi-
ral activities of the pyrophosphate mimics in the subgenomic
replicon assay were produced by the newly synthesized ADK
analogues.
Acknowledgments
This work was supported by Grant No. KRF-2007-313-C00476
from the Korea Research Foundation, Republic of Korea (MOEHRD,

J. Kim et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4661–4665
4665
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Basic Research Promotion Fund) and by grants from Biogreen 21
(Korea Ministry of Agriculture and Forestry), and the second Brain
Korea 21 (Korea Ministry of Education). J.K. and H.S.L. are sup-
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7. Di Santo, R.; Fermeglia, M.; Ferrone, M.; Paneni, M. S.; Costi, R.; Artico, M.;
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