Identification of a series of 1,3,4-trisubstituted pyrazoles as novel hepatitis C virus entry inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.09.039

In this report we describe the identification of novel pyrazole analogs as potent hepatitis C virus (HCV) entry inhibitor. The pyrazoles were identified by our phenotypic high-throughput screening using infectious HCV. A series of pyrazole derivatives was synthesized and evaluated for inhibitory activity against HCV in the infectious cell culture system. Through evaluation of selected compounds we observed that the pyrazoles did not interfere with HCV RNA replication but with viral entry as shown by experiments with HCV replicons and HCV pseudo particles, respectively.

Full text of DOI:10.1016/j.bmcl.2013.09.039

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6467–6473
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a series of 1,3,4-trisubstituted pyrazoles as novel
hepatitis C virus entry inhibitors
Jong Yeon Hwang a, Hee-Young Kim b, Dong-Sik Park a, Jihyun Choi a, Sung Min Baek b, Keumhyun Kim b,
Soohyun Kim b, Sikwang Seong c, Inhee Choi a, Hong-gun Lee d, Marc Peter Windisch b, , Jinhwa Lee e,
⇑
⇑
a Medicinal Chemistry Group, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
b Applied Molecular Virology, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
c Department of Applied Chemistry, Kyung Hee University, 1732 Deogyeong-daero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea
d Screening Technology Platforms Group, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
e Late Discovery Program, Institut Pasteur Korea (IP-K), Sampyeong-dong 696, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2013
Revised 20 August 2013
Accepted 12 September 2013
Available online 21 September 2013
In this report we describe the identification of novel pyrazole analogs as potent hepatitis C virus (HCV)
entry inhibitor. The pyrazoles were identified by our phenotypic high-throughput screening using infec-
tious HCV. A series of pyrazole derivatives was synthesized and evaluated for inhibitory activity against
HCV in the infectious cell culture system. Through evaluation of selected compounds we observed that
the pyrazoles did not interfere with HCV RNA replication but with viral entry as shown by experiments
with HCV replicons and HCV pseudo particles, respectively.
Keywords:
Hepatitis C virus
Ó 2013 Elsevier Ltd. All rights reserved.
Phenotypic high-throughput screening
Infectious cell culture system
Pyrazole
Entry inhibitor
Hepatitis C virus (HCV) is a global health concern, affecting
more than 3% of the world’s population by causing chronic liver
disease.1 Viral genome replication is prone to high error rates lead-
ing to a large diversity of HCV genotypes and subtypes2 with differ-
ences in susceptibility to current treatment and outcome of
disease. To date, the standard of care (SOC), a combination of pegy-
respectively. The antiviral activity of hit compounds was deter-
mined by 10-point dose response curve (DRC) analysis. Briefly,
naïve Huh-7 target cells which were plated in 384-well micro-
plates, incubated with serially diluted compounds, and inoculated
with cell culture derived HCV (HCVcc) at a multiplicity of infection
(MOI) 1. At 72 h post-infection live cells were analyzed and the half
maximal effective concentration (EC50) to inhibit viral replication
was determined. In parallel, to rule out that the observed antiviral
effects were due to toxicity induced by compounds, the cytotoxic
concentration (CC50) was calculated by automatically counting
cells of each individual well treated with antivirals.
lated interferon-alpha (PEG-IFNa) and ribavirin (RBV), results fre-
quently in unsatisfactory sustained virologic response rates (SVR)
of only 45–70% for patients infected with HCV genotype (gt) 1
and about 70–80% for those infected with gt 2 or 3.3,4 Unfortu-
nately, this treatment is associated with side effects responsible
for low adherence to therapy.5–8 More recently, two direct-acting
antiviral agents (DAAs) were introduced to the clinics increasing
the SVR rates in HCV gt1 infected patients, but are unfortunately
accompanied by severe side effects.9 This emerging clinical data
encouraged us to develop a high-throughput screening (HTS) assay
to identify novel antiviral targets. By devising screening strategies
using infectious HCV expressing a fluorescent marker protein, we
screened phenotypically the entire viral life cycle.10,11 Identified
inhibitors were triaged in HCV replicon and HCV pseudo particle
(HCVpp) systems to monitor viral RNA replication and viral entry,
In our phenotypic target-free HTS campaign for the discovery of
novel anti-HCV chemical entities we identified among others
R1
R1
N
O
R3
O
N
R1
O
NH
HN
OH
R2
OH
N
R2
3
N
4
N
N
N
N
1
R3
R2
2
3
1
⇑
Corresponding authors.
E-mail addresses: mpwindisch@ip-korea.org (M.P. Windisch), jinhwalee@
Figure 1. Structure of 1,3,4-trisubstituted pyrazoles exhibiting anti-HCV activity
(1) and HCV RNA replication inhibitor (2 and 3).
ip-korea.org (J. Lee).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.039

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J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6467–6473
active compounds containing a 1,3,4-trisubstituted pyrazole core
(Fig. 1 and 1). Pyrazoles 1 were counter-screened in the HCV rep-
licon system to filter out viral replication inhibitor. The pyrazole
core is well-known structure with broad biological spectrum in
drug discovery. Two pyrazole structures (212,13 and 314) have been
previously reported to inhibit HCV RNA replication. However,
pyrazoles 1 do not interfere with HCV RNA replication, indicating
a novel mechanism of action (MoA), which led us to pursue exten-
sive medicinal chemistry work to improve anti-HCV potency and
also to characterize the MoA of pyrazoles. Here, we describe the
identification of a series of pyrazole derivatives by conducting
structure-activity relationship (SAR) studies with the pyrazole core
R3
R1
N
R1
N
N
O
O
a
H
N
b
c
R1
N
N
R1
N
R2
R2
R2
7-9,13
6
5
Scheme 1. Reagents and conditions: (a) R2NHNH2, NaOAc, EtOH, microwave, 130 °C, 1 h; (b) POCl3, DMF, 80 °C, 3 h; (c) R3NH, NaBH(OAc)3, DCM, rt.
O
O
O
H2N
O
NH
N
NH
N
O
OH
NC
CO2Et
O
d
N
b
c
a
N
N
F
N
F
F
F
F
F
14
15
16
17
N
O
O
N
O
NH
N
NH
N
N
H
f
NH
N
e
NH
N
F
N
F
N
F
F
F
F
18
19
8o
Ph
O
TfO
HO
N
HN
j
N
N
O
N
i
N
F
h
g
N
F
N
F
F
F
O
F
F
F
24
21
20
22
23
N
Ph
O
N
N
k
f
Ph
NH
N
F
F
N
N
F
F
25
8q
Scheme 2. Reagents and conditions: (a) 2,4-difluorophenyl hydrazine, NaOAc, AcOH, H2O, microwave, 130 °C, 30 min, 86%; (b) 2-MeBnBr, NaH, DMF, rt, 4 h, 69%; (c) LiOH,
H2O/THF (1:3, v/v), rt, overnight, 77%; (d) N,O-dimethylhydroxyamine hydrochloride, EDCÁHCl, HOBt, DMF, rt, overnight, 93%; (e) LAH, THF, À40 to 0 °C, 5 h, 86%; (f) 2-(4-
methylpiperazin-1-yl)ethanamine, NaBH(OAc)3, DCM, rt; (g) 2,4-difluorophenyl hydrazine, NaOAc, AcOH, H2O, microwave, 130 °C, 30 min, 58%; (h) FeCl3, air, DMF, 80 °C, 6 h,
96%; (i) Tf2O, TEA, DCM, rt, 98%; (j) BnZnBr, Pd(OAc)2, X-Phos, THF, 50 °C, overnight, 94%; (k) POCl3, DMF, 80 °C, 3 h, 70%.

J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6467–6473
6469
Table 1
Table 2
Effect of amine on anti-HCV activity
Substituent effect on R1
R3
N
N
N
N
R1
N
NH
N
F
F
N
F
7a-7q
F
No.
NR3
Anti-HCVcc
(EC50, M)
Cytotoxicity
SI
8
l
(CC50
,
lM)
(CC50/EC50)
No.
R1
Anti-HCVcc
(EC50, M)
Cytotoxicity
SI
N
H
N
l
(CC50
,
l
M)
(CC50/EC50)
7a
7b
2.2 0.1
2.3 0.1
>20
>9
N
8a
8b
8c
8d
8e
8f
8g
8h
8i
4-Cl–Ph
0.38 0.70
0.36 0.04
0.54 0.03
0.58 0.04
0.81 0.02
1.2 0.1
1.3 0.1
1.6 0.2
1.7 0.2
1.8 0.1
2.1 0. 1
2.8 0.2
3.2 0.2
3.3 0.2
3.8 0.1
4.3 0.3
19
18
20
15
1
1
1
1
50
50
37
26
NH
4-CF3O–Ph
4-CF3–Ph
3-Cl–Ph
4-Me–Ph
Cyclohexyl
4-Me2N–Ph
Ph
3-Me–Ph
4-(Me2NSO2)–Ph
4-MeO–Ph
2-MePh
3-Me2N–Ph
2-Cl–Ph
>20
>20
>8.8
N
H
N
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>25
>17
>15
>13
>12
>11
>9.5
>7.2
>6.3
>6.1
>5.3
>4.7
>1.8
>1.3
7c
2.8 0.1
>7.2
>6.7
N
N
NH
O
7d
3.0 0.1
4.3 0.1
>20
>20
N
N
8j
8k
7c
8l
8m
8n
8o
8p
8q
H
N
7e
7f
>4.6
>3.0
N
H
N
6.7 0.2
7.5 0.6
>20
>20
4-(MeO2C)–Ph
2-MeBnNH
tert-Bu
7g
7h
7i
N
N
>2.7
1
11
16
1
1
Bn
H
N
18
1
17
1
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with
quadruplicate measurements.
OH
N
H
>20
>20
N.A.
H
Table 3
N
7j
>20
>20
N.A.
R2 effect on anti-HCV activity
N
N
7k
7l
N
N
N
>20
>20
>20
>20
N.A.
N.A.
N
N
NH
O
S
O
HN
N
Me
7m
7n
>20
>20
>20
>20
N.A.
N.A.
N
N
R2
N
H
9
N
No.
R2
Anti-HCVcc
(EC50, M)
Cytotoxicity
(CC50 M)
SI
N
l
,
l
(CC50/EC50)
7o
7p
20
>20
>20
>1
HN
9a
9b
9c
9d
9e
9f
9g
9h
9i
4-CF3–Ph
4-NO2–Ph
4-Cl–Ph
4-MeS–Ph
4-CF3O–Ph
3-CF3–Ph
3-Cl–Ph
4-Br–Ph
2,4-Me2–Ph
2,5-F2–Ph
3,4-F2–Ph
2,4-F2–Ph
4-MeO–Ph
4-F–Ph
2-Cl–Ph
Bn
4-NH2–Ph
4-F–Bn
i-Pr
EtOAc
0.46 0.04
0.49 0.05
0.65 0.04
0.67 0.06
0.90 0.75
0.91 0.02
1.0 0.1
1.2 0.1
2.4 0.1
2.5 0.1
2.6 0.1
2.8 0.2
2.8 0.1
3.7 0.2
3.8 0.2
7.5 0.8
10 0.2
11
1
24
>41
26
25
19
20
16
8.0
>8.3
>8.0
>7.7
>7.1
>7.1
>5.4
>5.3
>2.3
>2
>20
17
17
17
18
N
O
>20
N.A.
1
1
1
1
1
HN
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with
quadruplicate measurements. N.A.: not applicable.
16
9.6
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
1
to characterize its anti-HCV activity in the infectious HCV cell cul-
ture system.
9j
9k
6c
9l
9m
9n
9o
9p
9q
9r
To evaluate anti-HCV activity of pyrazoles 1, we explored the ef-
fect of R1, R2, and R3 group. A general procedure for the synthesis of
1,3,4-trisubstituted pyrazoles 1 is depicted in Scheme 1. Commer-
cially available alkyl or aryl ketones were condensed with various
hydrazines under microwave-assistant conditions to afford hydra-
zones 5.15 Subsequent cyclization–formylation of hydrazones 5 un-
der Vilsmeier–Haack conditions gave pyrazole aldehydes 6 in good
yield.16 Reductive amination of the aldehyde 6 with various amines
provided the desired 1,3,4-trisubstituted pyrazole derivatives 7–9
and 13.
10
1
>2
N.A.
N.A.
>20
>20
9s
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with
quadruplicate measurements. N.A.: not applicable.

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J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6467–6473
N
N
O
O
N
N
O
H
OH
Cl
Cl
N
H
NH
a
N
N
b
Cl
N
F
N
N
N
N
F
F
F
N
F
F
F
6n
26
11
10
g
O
O
OH
O
H
NH2
N
O
N
N
N
c
e
d
f
N
N
N
F
O
F
F
28
F
F
27
30
29
31
H
N
O
O
N
H
N
N
N
N
i
g
h
j
N
O
Br
N
F
N
N
N
O
O
F
F
F
32
33
12
34
35
Scheme 3. Reagents and conditions: (a) NaClO2, sulfamic acid, H2O, acetone, 0 °C, 3 h, 80%; (b) 2-(4-methylpiperazin-1-yl)ethanamine, EDCÁHCl, HOBt, DMF, rt; (c) methyl
acetoacetate, InBr3, 4 Å molecular sieves, MeOH, rt,18 h, 91%; (d) benzonitrile, Cu(OAc)2, air, 120 °C, microwave, 1 h, 64%; (e) LAH, THF, 0 °C to rt, 3 h, 89%; (g) IBX, DMSO,
80 °C, 2 h, 64%; (g) 2-(4-methylpiperazin-1-yl)ethanamine, NaBH(OAc)3, DCM, rt; (h) NaOAc, EtOH, microwave, 150 °C, 30 min, 62%; (i) PBr3, MeCN, microwave, 150 °C, 77%;
(j) nBuLi/THF, DMF (cat.), À78 °C to rt, 1 h, 79%.
Compound 8o bearing 2-methylbenzylamine moiety at 3-posi-
tion of the pyrazole was synthesized from intermediate 15 which
was prepared by condensation of 2-cyano-3-methoxyacrylate 14
and 2,4-difluorophenylhydrazine under microwave-assistant con-
dition.17 Intermediate 15 was alkylated with 2-methylbenzyl bro-
mide to give pyrazole 16. Subsequent reactions of pyrazole 16
through hydrolysis, Weinreb amide formation, and reduction gave
pyrazole-4-aldehyde 19. The aldehyde 19 was subjected to reduc-
tive amination condition to afford the desired compound 8o.
Compound 8q was synthesized from 3-hydroxypyrazole 22 in
four steps. 3-Hydroxypyrazole 22 was prepared from ethyl acrylate
20 by condensation with hydrazine under microwave assistance,
followed by oxidation with FeCl3 and air.18 Hydroxyl 22 was con-
verted to the corresponding triflate 23 by reaction with triflic
anhydride. Following palladium-catalyzed Negishi coupling condi-
tion19 of pyrazole triflate 23 with benzyl zinc bromide generated
pyrazole 24, which was then subjected to sequential Vilsmeier
formylation and reductive amination step to give the desired prod-
uct 8q (Scheme 2).
Pyrazole-4-carboxamide 10 was prepared by oxidation of car-
boxaldehyde 6n, followed by amide coupling condition. 5-Methyl
substituted pyrazole 11 was generated by following Neumann
et al.20 Enamine 28 prepared from aniline and methyl acetoacetate
with catalytic InBr was coupled with benzene nitrile under copper
acetate, air, and microwave irradiation condition to give carboxylic
ester pyrazole 29. The ester group of pyrazole 29 was reduced by
LAH to give alcohol 30, which was afterwards oxidized by IBX to
afford aldehyde pyrazole 31 as a precursor of compound 11
generated by the following reductive amination. 5-(Amino-
methyl)-pyrazole 12 was prepared from ethyl benzoylacetate 32
in four steps. Benzoylacetate 32 was condensed with hydrazine
22
to give pyrazol-one 33.21 Subsequent bromination with PBr3
and formylation by nBuLi23 generated pyrazole-5-carboaldehyde
35 which was converted to the desired product 12 by the following
reductive amination.
All synthesized compounds were evaluated for their ability to
inhibit the HCV life cycle and cytotoxicity as described earlier. First
we evaluated anti-HCV activity depending on NR3 group with var-
ious amines (Table 1). Several compounds (7a–7g) showed a sin-
gle-digit micromolar (
without cytotoxicity in the testing range (CC50 >20
them compounds 7a and 7b were the most potent molecules with
l
M) antiviral activity (EC50 = 2.2À7.5
lM)
M). Among
l
2.2 and 2.3 lM EC50 value, respectively. The anti-HCV activity of
this series appeared to be significantly influenced by NR3 substitu-
tions. With the exception of compound 7n bearing a pyridine moi-
ety, only amines that have two or three basic nitrogen atoms and at
least three carbon units between two terminal nitrogen atoms
showed antiviral activity.
We further investigated the effect of R1 group on pyrazoles;
therefore, we used a 2,4-difluorophenyl for the R2 group and a
(4-methylpiperazino)ethylamine for the NR3 group (Table 2). In
this series, most of compounds (7c and 8a–8o) showed good activ-
ities with EC50 values below 10 lM, except the tert-Bu and Bn
substituent (8p and 8q). In addition, electron-withdrawing groups
(EWG) at para-position in phenyl ring are more favorable than
electron-donating groups (EDG) (4-Cl, 4-CF3O, 4-CF3 > 4-Me >

J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6467–6473
6471
Table 4
4-MeO). Compound 8j and 8n slightly reduced antiviral activities
although having a strong EWG at para-position. Cyclohexyl com-
pound 8f was almost equipotent with phenyl compound 8h, indi-
cating that the cyclic system is favorable in comparison of tert-
butyl 8p, but the pi–pi interaction is not essential. Among this ser-
ies tested, compounds 8a and 8b exhibited good inhibitory activity
against HCV with acceptable selectivity index values (SI = 50)
Additionally, further substitution effects of R2 on HCV inhibitory
activities were explored and the data were summarized in Table 3.
In this series, we fixed 2-methylphenyl for R1 group and a (4-meth-
ylpiperazino)ethylamine for the NR3 group. A set of pyrazoles in
this series showed a broad activity range depending on substitu-
ents of R2 group. Among mono-substituents, no clear correlation
Anti-HCV activity of compounds 10–12
No.
Structure
Anti-HCVcc
(EC50, M)
Cytotoxicity
(CC50 M)
l
, l
N
N
O
NH
Cl
N
10
>20
>20
N
F
between antiviral activity and electronic effect (
r value) or hydro-
F
phobicity effect (
p
value) by substituents was observed.24 How-
N
ever, the electron-withdrawing group is likely to be more
favorable than the electron-donating group for antiviral activity.
Among halides at 4-position in phenyl ring, the anti-HCV activity
are Cl > Br > F in order. Benzyl substituted pyrazoles 9o and 9q re-
duced antiviral activity and no inhibitory activities were observed
in aliphatic alkyl pyrazoles 9r–9s (i-Pr and ethylacetate). Reducing
nitro group 9b to amine 9p decreased anti-HCV activity by 20-fold.
With this preliminary SAR data in hand, we designed and syn-
thesized additional pyrazole derivatives to elucidate essential ele-
ments in the pyrazole to be active against HCV (Scheme 3 and
Table 4). To address the importance of methylene amine moiety
(–CH2NR3) in pyrazoles, pyrazole-amide 10 was evaluated, and it
N
NH
Cl
11
8.5 0.8
>20
N
N
F
H
N
N
N
was inactive in the testing range (EC50 >20
tuted compound 11 at 5-postion of pyrazole slightly reduced
anti-HCV activity (EC50 = 8.5 M) compared to compound 6c. Com-
pound 12 bearing a methylene amine moiety at 5-position of the
lM). Methyl substi-
N
N
12
13
1
>20
l
pyrazole also showed reduced antiviral activity with 13
value.
lM EC50
F
Table 5
SAR of 1,3,4-trisubstituted pyrazoles
R1
N
R3
NH
∗
R3
=
N
N
∗
N
Amine-1
Amine-2
N
N
∗
N
Amine-3
R2
No.
R1
R2
R3
Anti-HCVcc (EC50
,
l
M)
Cytotoxicity (CC50
,
l
M)
SI (CC50/EC50)
12m
12n
12b
12o
12a
12f
12c
12r
12h
12d
12e
12q
12g
12l
CF3
CF3
Cl
CF3
Cl
Cl
Cl
CF3
CF3O
Cl
NO2
NO2
NO2
NO2
NO2
CF3
NO2
CF3
NO2
CF3
CF3
CF3
NO2
CF3
CF3
Amine 1
Amine 2
Amine 2
Amine 3
Amine 1
Amine 3
Amine 3
Amine 3
Amine 2
Amine 1
Amine 2
Amine 2
Amine 1
Amine 3
Amine 1
Amine 1
Amine 3
Amine 2
0.11 0.02
0.12 0.03
0.13 0.02
0.15 0.02
0.29 0.05
0.29 0.04
0.30 0.01
0.33 0.06
0.34 0.04
0.36 0.03
0.36 0.03
0.38 0.07
0.4 0.04
3.2 0.1
4.9 0.1
3.7 0.1
2.3 0.1
5.0 0.1
1.9 0.1
11 1
1.2 0.6
1.6 0.3
3.0 0.1
2.5 0.1
2.0 0.1
4.4 0.2
2.2 0.1
3.0 0.4
1.5 0.1
18 1
29
41
28
15
17
6.7
37
3.6
4.6
8.4
6.9
5.3
11
5.3
6.9
3.1
30
Cl
CF3
CF3O
CF3O
CF3O
CF3
CF3O
CF3O
0.41 0.05
0.44 0.07
0.49 0.05
0.60 0.06
0.75 0.03
12j
12p
12i
CF3
NO2
CF3
12k
2.3 0.1
3.1
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate measurements. Compounds were sorted by EC50 values.

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J. Y. Hwang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6467–6473
Table 6
Determination of antiviral activity of compound 7c and 8a in the HCVpp system
No.
Structure
Anti-HCVpp gt-1 (EC50,
l
M)
Cytotoxicity (CC50
,
lM)
VSVg-pp
Inactive
F
F
F
O
O-
N+
EI-1
0.040
>25
N
N
O
N
H
N
N
H
N
N
F
N
H
7f
6.1
1.9
>25
9.1
Inactive
Inactive
N
F
N
N
N
H
F
N
8a
N
F
Cl
EC50 and CC50 values were determined by 5-point DRC analysis with duplicate measurements.
On the basis of the evaluation of the initial series of pyrazoles,
we further designed and synthesized a limited set of pyrazole com-
pounds to improve anti-HCV activity and selectivity index. This
study focused on a substitution pattern containing (i) 4-Cl or
4-CF3 for R1 group, (ii) 4-NO2 or 4-CF3 for R2 group, and (iii) three
selected amines as shown in Table 5. This set of compounds was
synthesized as described in Scheme 1 and evaluated for antiviral
activity and summarized in Table 5.
antiviral efficacy. Furthermore, based on HCVpp experiments, this
pyrazole series appears to inhibit early entry steps in the viral life
cycle as demonstrated by HCVpp experiments. Although anti-HCV
potency of this series might be potentially further improved, the
1,3,4-trisubstituted pyrazole scaffold can be utilized as a novel en-
try inhibitor to elucidate the HCV life cycle.
Acknowledgments
All compound synthesized in this series demonstrated signifi-
cantly improved anti-HCV potency with sub-micromolar ranges.
Among them compound 12m was the most potent compound with
In memoriam Dr. Junwon Kim and Keumhyun Kim. This work
was supported by the National Research foundation of Korea
(NRF) grant funded by the Korea Government (MEST) (No. 2012-
00011), Gyeonggi-do and KISTI.
0.11 lM EC50. However, the majority of compounds in this series
was accompanied by increased cytotoxicity, thereby providing
poor selectivity index values. The exceptions were compound
12n and 12c, showing moderate selectivity index values of 41
and 37, respectively.
By using the infectious HCV cell culture system during primary
screening, facilitating to target the entire viral life cycle, the MoA
and the targets of hit compounds are initially unknown. In order
to identify which step of the viral life cycle is inhibited, we devised
strategies to elucidate the MoA of the 1,3,4-trisubstituted pyrazole
scaffold. We demonstrated that these pyrazoles do not interfere
with HCV RNA replication by counter-screening in HCV replicon
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