Design, synthesis, and in vitro biological evaluation of 1 H -1,2,3-triazole-4-carboxamide derivatives as new anti-influenza A agents targeting virus nucleoprotein

Article abstract of DOI:10.1021/jm2013503

The influenza virus nucleoprotein (NP) is an emerging target for anti-influenza drug development. Nucleozin (1) and its closely related derivatives had been identified as NP inhibitors displaying anti-influenza activity. Utilizing 1 as a lead molecule, we successfully designed and synthesized a series of 1H-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza A agents. One of the most potent compounds, 3b, inhibited the replication of various H3N2 and H1N1 influenza A virus strains with IC ₅₀ values ranging from 0.5 to 4.6 μM. Compound 3b also strongly inhibited the replication of H5N1 (RG14), amantidine-resistant A/WSN/33 (H1N1), and oseltamivir-resistant A/WSN/1933 (H1N1, 274Y) virus strains with IC ₅₀ values in sub-μM ranges. Further computational studies and mechanism investigation suggested that 3b might directly target influenza virus A nucleoprotein to inhibit its nuclear accumulation.

Full text of DOI:10.1021/jm2013503

Article
pubs.acs.org/jmc
Design, Synthesis, and in Vitro Biological Evaluation of 1H-1,2,3-
Triazole-4-carboxamide Derivatives as New Anti-influenza A Agents
Targeting Virus Nucleoprotein
Huimin Cheng,†,⊥ Junting Wan,† Meng-I Lin,‡ Yingxue Liu,† Xiaoyun Lu,† Jinsong Liu,† Yong Xu,†
Jianxin Chen,§ Zhengchao Tu,† Yih-Shyun E. Cheng,‡ and Ke Ding*,†
†Key Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences, 190 Kaiyuan Avenue, Guangzhou 510530, People’s Republic of China
‡Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan, Republic of China
§South China Agricultural University, 483 Wushan Road, Guangzhou 510642, People’s Republic of China
⊥Graduate School of Chinese Academy of Sciences, #19 Yuquan Road, Beijing 100039, China
S
* Supporting Information
ABSTRACT: The influenza virus nucleoprotein (NP) is an emerging target for anti-
influenza drug development. Nucleozin (1) and its closely related derivatives had been
identified as NP inhibitors displaying anti-influenza activity. Utilizing 1 as a lead molecule,
we successfully designed and synthesized a series of 1H-1,2,3-triazole-4-carboxamide
derivatives as new anti-influenza A agents. One of the most potent compounds, 3b, inhibited
the replication of various H3N2 and H1N1 influenza A virus strains with IC50 values ranging
from 0.5 to 4.6 μM. Compound 3b also strongly inhibited the replication of H5N1 (RG14), amantidine-resistant A/WSN/33
(H1N1), and oseltamivir-resistant A/WSN/1933 (H1N1, 274Y) virus strains with IC50 values in sub-μM ranges. Further
computational studies and mechanism investigation suggested that 3b might directly target influenza virus A nucleoprotein to
inhibit its nuclear accumulation.
Most recently, cases of reassortment between swine influenza
and 2009 influenza A (H1N1) in human were reported,18
which highlights the emergent need for developing new anti-
influenza agents with new mechanism of action.
INTRODUCTION
■
Influenza is a seasonally epidemic acute respiratory disease
persistently threatening public health caused by infection of
ribonucleic acid (RNA) influenza viruses of orthomyxoviridae
family. The epidemic and pandemics of influenza have caused
serious impact on worldwide morbidity, mortality, and
economy.1 Three distinct types of influenza viruses are
classified based on their serological subtypes, among which
influenza A viruses are the most virulent human pathogens.
Influenza A viruses possesses eight segmented RNA genomes.
The continuous genetic mutation and reassortment of influenza
A viruses may cause the evasion of primary human immunities
and lead to the resistance against current therapies. More
importantly, the viral genetic shift may eventually cause the
breakthrough of interspecies and interhuman transmission
barriers which will be disastrous to public health.2−5
Currently, two types of small molecular drugs are available
for the treatment of influenza A including M2 ion channel
blockers (i.e., amantidine and rimanitidine) and neuraminidase
inhibitors (i.e., oseltamivir and zanamivir).6−8 However, the
application of M2 ion channel blockers, particularly for
amantidine, has been strictly limited because of the rapid
emergence of drug resistance and the occurrence of central
nervous side effects.9 Oseltamivir resistant viruses have also
been reported since 2005,10,11 and almost all the seasonal
influenza H1N1 viruses circulating in the United States were
resistant to Oseltamivir in the 2008−2009 flu season.12−17
The influenza virus nucleoprotein (NP) is encoded by the
fifth genome segment and abundantly expressed during the
course of infection. Comparing with the viral surface spike
proteins like hemagglutinin and neuraminidase, the influenza
virus nucleoprotein is highly conserved.20−22 During the viral
life cycle, nucleoprotein binds with influenza viral RNA
segments and polymerase subunit proteins (PB1, PB2, and
PA) to form virus ribonucleoprotein (vRNP) complex, which is
transported to host cell nucleus to trigger viral RNA
transcription, replication, and virion assembly.19 Owing to its
indispensable roles in numerous stages of viral multiplication,
influenza virus nucleoprotein has been considered as a novel
target for new anti-influenza drug development. Several
nucleoprotein inhibitors have been reported to display
promising anti-influenza virus activities.23−28
Nucleozin (1)24 is the arguably first small molecular
influenza virus nucleoprotein inhibitor reported by Yuen and
Su independently.24,25 Studies have demonstrated that 1 and its
related derivatives could potently trigger the aggregation of
nucleoprotein and inhibit its nuclear accumulation to display
Received: October 8, 2011
Published: February 14, 2012
© 2012 American Chemical Society
2144
dx.doi.org/10.1021/jm2013503 | J. Med. Chem. 2012, 55, 2144−2153

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Compounds 2, 4, and 5
a
Reagents and conditions: (a) NH2OH·HCl, ethanol aq, 60 °C overnight, 76.4%; (b) NaOH, MeOH:THF:H2O = 1:1:3, overnight, 95.7%; (c)
(COCl)2, dichloromethane, 2.0 h, then 1-(2-chloro-4-nitrophenyl)piperazine, Et3N, dichloromethane, 3.0 h, 60−80%; (d) N2H4·H2O, ethanol, reflux
overnight, 76.4%; (e) BBr3, DCM, −10 °C, 3.0 h, 56.2%.
a
Scheme 2. Synthesis of Compounds 3 and the X-Ray Structure of Intermediate 16a
a
Reagents and conditions: (a) H2O/ethyl acetate, then HCl,NaNO2; (b) NaN3, 0 °C, 2.0 h,59.0%; (c) toluene, reflux, overnight, 80.9% (14 + 15);
(d) LiOH, MeOH:THF:H2O = 1:1:3, overnight, 96%; (e) (COCl)2, dichloromethane, DMF cat., 2.0 h, then 1-(2-chloro-4-nitrophenyl)piperazine,
Et3N, dichloromethane, 3.0 h, 58.0%.
potent anti-influenza virus activities. Further mutational studies
suggested that these inhibitors might directly bind to
nucleoprotein and “induce formation of higher-order nucleo-
protein oligomers” that are unable to migrate into the
nucleus.24−27 However, it remains elusive that how the
inhibitors induced the formation of nucleoprotein oligomers.27
None of the current inhibitors has been approved for clinical
investigation. It is highly desirable to identify new inhibitors for
to further validate nucleoprotein as novel molecular target for
anti-influenza A development. Herein, we report the structural
design, synthesis, and in vitro biological evaluation of 1H-1,2,3-
triazole-4-carboxamide derivatives as new anti-influenza A
agents using 1 as the lead compound.27
condensation/cyclization of ethyl 2-benzoyl-3-oxobutanoate
(6) with hydroxylamine hydrochloride or hydrazine hydrate
yielded ethyl 3-methyl-5-phenylisoxazole-4-carboxylate (7) or
ethyl 5-methyl-3-phenyl-1H-pyrazole- 4-carboxylate (9), re-
spectively. Compound 7 or 9 was hydrolyzed and reacted with
1-(2-chloro-4-nitrophenyl)piperazine to produce the designed
(4-(2-chloro-4-nitrophenyl) piperazin-1-yl)(3-methyl-5-phenyl-
isoxazol-4-yl)methanone (2) or (4-(2-chloro-4-nitrophenyl)-
piperazin-1-yl) (5-methyl-3-phenyl-1H-pyrazol-4-yl) metha-
none (4). (4-(2-Chloro-4-nitrophenyl) piperazin-1-yl)(2′-
biphenyl)methanone (5) was readily prepared by a direct
condensation of 2-phenyl benzoic acid (11) with 1-(2-chloro-4-
nitrophenyl)piperazine.
Compounds 3 were synthesized by using a traditional “3 + 2”
cycloaddition as the key step.29−31 Briefly, the anilines 12 were
diazotized and then reacted with NaN3 to produce the
azidobenzenes 13. Compounds 13 were directly reacted with
CHEMISTRY
■
Compounds 2 and 4 were prepared by using ethyl 2-benzoyl-3-
oxobutanoate as the same starting material (Scheme 1). The
2145
dx.doi.org/10.1021/jm2013503 | J. Med. Chem. 2012, 55, 2144−2153

Journal of Medicinal Chemistry
Article
Figure 1. Design of new anti-influenza A agents using a scaffold-hopping strategy.
Table 1. Inhibitory Activities of the Compounds against Influenza A Replication
IC50 (μM)
c
a
b
compd
n
R1
R2
R3
R4
R5
R6
R7
CC50 (μM)
H3N2
H1N1
1
15.9
1.08
6.00
2.62
8.64
2.87
7.61
3.68
5.16
1.97
0.32
4.83
0.71
3.38
0.72
5.24
1.57
3.13
0.68
5.35
5.61
1.20
4.89
4.93
1.58
2.40
3.22
8.42
2a
2b
4a
4b
5a
5b
3a
3b
3c
3d
3e
3f
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
Cl
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
OMe
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
13.77
14.72
5.65
9.38
>50
H
OMe
H
Cl
H
H
Cl
H
H
3g
3h
3i
H
Cl
H
OH
Me
H
7.84
4.92
8.97
16.16
14.80
2.42
>50
H
H
3j
OEt
OiPr
COMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
3k
3l
H
H
H
H
13.72
1.34
8.36
>50
3m
3n
3o
3p
3q
3r
3s
3t
H
H
H
H
Et
H
H
>50
Me
Me
Me
Me
Me
H
H
>50
48.12
16.98
>50
NO2
H
H
H
>50
H
H
H
>50
OMe
Cl
NO2
NO2
H
H
>50
>50
H
H
>50
35.45
>50
d
AMD
>100
2.22
a
b
c
d
Influenza A/HK/8/68 (H3N2) stains. Influenza A/WSN/33 (H1N1) stains. Antiproliferation against MDCK cells. AMD: amantidine. The data
are means of results from at least three independent experiments.
different alkynoates to obtain the key intermediates ethyl 3-
phenyl-3H-1,2,3-triazole-4-carboxylates (14) and the byprod-
ucts ethyl 1-phenyl-1H-1,2,3-triazole-4-carboxylates (15). Com-
pounds 14 and 15 could be separated by silica gel column
chromatography, and the structures of 14 were further
determined by X-ray crystallographic analysis of a representa-
tive compound 16a (Scheme 2).32 With the key intermediates
14 in hands, compounds 3 were readily obtained by hydrolysis
and coupling with substituted piperazines or 1, 4-diazepane
with good yields.
RESULTS AND DISCUSSION
■
Taking compound 1 as the lead compound, we initially
designed isoxazol-4-carboxamide (2a), 1H-1,2,3-triazole-4-
2146
dx.doi.org/10.1021/jm2013503 | J. Med. Chem. 2012, 55, 2144−2153

Journal of Medicinal Chemistry
Article
carboxamide (3a), 1H-pyrazol-4-carboxamide (4a), and 2-
biphenyl carboxamide (5a) as new anti-influenza A agents by
using a scaffold-hopping strategy (Figure 1). The anti-influenza
A virus activities of the compounds were preliminarily evaluated
using Madin−Darby canine kidney (MDCK) cell-based CPE
(cytopathic effect protection) assays. Under the screening
conditions, 1 inhibited the replication of H3N2 (A/HK/8/68)
and H1N1 (A/WSN/33) strains, with IC50 values of 1.01 and
0.32 μM, respectively, which were comparable to the reported
data.24,25 The results also suggested that all the four designed
compounds potently inhibited the replication of H3N2 (A/
HK/8/68) and H1N1 (A/WSN/33) strains, among which 1H-
1,2,3-triazole- 4-carboxamide (3a) displayed the greatest
potency and inhibited the replication of H3N2(A/HK/8/68)
and H1N1 (A/WSN/33) strains, with IC50 values of 5.16 and
3.13 μM, respectively (Table 1). Although compound 3a is
about 5-fold less potent than the original lead compound 1, it
represented a new anti-influenza A agent with a different
chemical scaffold. Therefore, further structural optimization
was conducted to improve its anti-influenza activity. The results
were summarized in Table 1.
toxicity against the MDCK cells with IC50 value of 16 μM.
However, none of the 1H-1,2,3-triazole-4-carboxamide com-
pounds showed obvious cellular growth inhibition against
MDCK cells under 100 μM (Table 1), indicating that the
compounds beard great selectivity indexes (SI = CC50/IC50).
Although 3b was about 2 times less potent than compound
1, it displayed equal potency with amantidine against the
replication of influenza A/HK/8/68 (H3N2) stains with great
selectivity index. Furthermore, 3b also potently inhibited the
replication of A/WSN/33 (H1N1) stains which were resistant
to the clinical M2 blocker amantidine (Table 1), suggesting that
this compound might be used a new lead compound for novel
anti-influenza drug discovery. Therefore, further biological
evaluations were performed to validate its anti-influenza
activity.
Plague counting based viral yield reduction assay is one of
the most direct and reliable methods for evaluating the
inhibitory effects on viral replication. Therefore, the anti-
influenza function of 3b was further validated by using a plaque
reduction assay. As shown in Figure 2, compound 3b dose-
Similar to the previous observation on the derivatives of
compound 1,25,27 a substituted group in phenyl ring A might
have great impact on the anti-influenza activity of the 1H-1,2,3-
triazole-4-carboxamide compounds. For instance, when a
methoxyl group was introduced at R4 position of compound
3a, the anti-influenza activity was obviously improved and the
resulting compound 3b displayed IC50 values of 1.96 and 0.68
μM against the replication of H3N2 (A/HK/8/68) and H1N1
(A/WSN/33) strains, respectively, which was about 3 times
more potent than the original compound 3a. However, when a
methoxyl group was introduced in R5 or R6 position (3b and
3c), the potency was decreased. The R4-Cl− substituted
compound 3e was also more potent than the corresponding R5-
or R6-substituted compound 3f or 3g, which suggested that R4
might be a feasible position for further optimization. Therefore,
a variety of other moieties such as hydroxyl (3h), methyl (3i),
ethoxyl (3j), or acetyl (3k) groups were introduced for further
investigation. However, it was disappointing that none of these
substitutions further improved the potency. The 2′,6′-
dimethoxyl compound 3m (R4, R7-disubstituted compound)
also displayed good potency against the replication of H3N2
(A/HK/8/68) and H1N1 (A/WSN/33) strains. The impact of
R3 was also investigated, and the results indicated that removal
of the methyl group (3n) or replacing it with a slightly bigger
ethyl group (3o) caused dramatic decrease of the potency.
Investigation on the substituted group in phenyl ring B revealed
that both the nitro- (R2) and chloro- (R1) were necessary for
the anti-influenza activity. The potencies were significantly lost
by removal of the nitro or/and chloro groups (3p−3s). It was
also demonstrated that replacement of the piperazine linker
with 1,4-diazenpane (3t) led to about 50-time decrease of the
potency (comparing with 3b).
Figure 2. Compound 3b potently inhibited the plaque formation of
influenza virus infected MDCK cells. Monolayer MDCK cells were
infected with A/WSN/33 (H1N1) at 0.01 MOI (multiplicity of
infection) and overlaid with 1% agar containing various concentrations
of nucleozin (1) (upper panel) and 3b (bottom panel). At 72 h
postinfection, plaques were visualized by stained with crystal violet.
The results shown are representative of at least three independent
experiments.
dependently inhibited the plaque formation of MDCK cells by
A/WSN/33 (H1N1) influenza virus infection. The plaque
formation was completely inhibited by 3b under a concen-
tration of 1.0 μM. Highly consistent with the results from our
CPE assay, compound 1 displayed better plague suppressive
effect than 3b and induced a complete abrogation of viral
plagues at 0.3 μM. The viral yield reduction activities of 1 and
3b were further validated by using microplate counting of
infectious particles (McIP) assay,25 and the deduced IC50 values
were 0.10 and 0.23 μM, respectively.
The anti-influenza activities of 3b were also evaluated against
a panel of other influenza A virus strains, and the results were
summarized in Table 2. It was shown that 3b also potently
inhibited several other H1N1 and H3N2 influenza A virus
strains with varied IC50 values in low μM range, except for the
H3N2 (Br/10/09) strains (IC50 > 50 μM). It was also
noteworthy that 3b potently inhibited the replication of H5N1
(RG14) strains, tamiflu resistant WSN (274Y) A/WSN/1933
(H1N1) strains, and A/Guangdong/1996 Chicken H9N2
influenza virus strains with IC50 values of 0.69, 1.11, and 4.62
μM, respectively.
Utilizing the knowledge of the structure−activity relationship
on compounds 3, methoxyl or chloro substituted derivatives of
compounds 2a, 4a, and 5a were also design and synthesized.
The resulting compounds 2b, 4b, and 5b displayed improved
anti-influenza activities against H3N2 (A/HK/8/68) and
H1N1 (A/WSN/33) strains, with IC50 values comparable to
that of 3b.
Antiproliferative activities of the compounds against MDCK
cells were also evaluated to monitor the potential cytotoxic
effects. Under the assay conditions, 1 displayed moderate
Compound 1 had been reported as a nucleoprotein inhibitor
that triggered the aggregation and inhibited the nuclear
accumulation of influenza virus nucleoproteins.24,25 Given the
structural similarity of the 1H-1,2,3-triazole-4-carboxamide
2147
dx.doi.org/10.1021/jm2013503 | J. Med. Chem. 2012, 55, 2144−2153

Journal of Medicinal Chemistry
Article
nucleoprotein and caused the nucleoproteins to be trapped in
the cytoplasm and scattered randomly in H1N1 (A/WSN/33)
virus infected MDCK cells, indicating nucleoproteins might be
the molecular target of the 1H-1,2,3-triazole-4-carboxamide
analogues.
Table 2. Antiinfluenza Activities of Compound 3b against a
Panel of Influenza A Viruses
IC50 values against
influenza A virus (μM)
a
strains
1
3b
Most recently, the X-ray crystal structure of nucleoprotein
complex with a compound 1 analogue was disclosed. It was
demonstrated that each inhibitor bridged two molecules of NP
(i.e., NP_A and NP_B) protein to form a hexameric complex.27
The interactions of the inhibitor with the Y289 region in NP_A
and the Y52 region in NP_B were critical for binding of the
inhibitor to interface of NP_A and NP_B (Figure 4A,B). The
importance of the Y289 and Y52 regions in nucleoprotein had
also been observed by previous mutational studies.24,25
To understand the potential interaction of 3b with influenza
nucleoproteins, a computational study was performed. The
results suggested that 3b bound to nucleoproteins with a similar
mode to that of the nucleozin analogues (Figure 4). The
carbonyl group of 3b formed an essential hydrogen bond with
the OH group of the S376 residue in NP_B. The 4-nitro-2-
chloro-phenyl moiety might form strong π−π stacking
interaction with the Y289 residue of NP_A, while the 2-
methoxyl phenyl group and the piperazine moiety could
achieve important hydrophobic effect with the Y52 side chain
of NP_B. To further validate the proposed interaction, the anti-
influenza activity of 3b were determined against A/New Jersey/
8/76 strains bearing Y289H mutation and recombinant WSN
viruses harboring either wild-type NP (52Y) or mutated NP
(52H) using 1 as a reference compound. Similar to compound
1, 3b is a potent anti-influenza agent to the WSN virus with
wild-type NP (52Y). However, both Y52H mutated isogenic
WSN virus and Y289H mutated A/New Jersey/8/76 strains
were resistant to 3b (IC50 > 50 μM), highlighting the critical
interaction of 3b with these two residues. These results were
highly consistent to our computational modeling results and
further supported that virus nucleoprotein might be the
molecular target of the 1H-1,2,3-triazole-4-carboxamide anti-
fluenza agents.
b
A/Guangzhou/2009 (H1N1)
0.54
1.59
0.20
0.52
>100
12.4
0.34
0.15
3.87
0.66
0.49
>50
0.52
1.86
0.66
0.90
>100
>50
1.17
0.69
4.62
1.11
1.40
>50
b
A/Brisbane/10/2007 (H1N1)
b
A/Weiss/43 (H1N1)
b
A/Mal/302/54 (H1N1)
b
A/New Jersey/8/76 (H1N1, NP 289H)
b
A/Brisbane/10/2007 (H3N2)
b
A/Udorn/1972 (H3N2)
b
A/RG14 (H5N1)
b
A/Guangdong/96 (H9N2)
c
A/WSN/33 (neuraminidase, 274Y)
c
rWSN (NP, 52Y)
c
rWSN (NP, 52H)
a
A/RG14 (H5N1): the NIBRG14 reasorant strains that harbors the
hemagglutinin and neuraminidase genes from A/VietNam/1194/2004
and other influenza genes from PR8 viruses. H9N2 (GD/96), A/
Chicken/Guangdong/1996 H9N2 strains; rWSN(52Y), rWSN virus
with wild-type NP (52Y); rWSN (52H), rWSN virus with mutated NP
(52H); WSN (274Y), A/WSN/1933 (H1N1) with tamiflu resistant
neuraminidase (274Y). Data obtained by CPE prevention assay.
Data obtained by measurements of NP antigen reduction assay. The
data are means of results from two or three independent experiments.
b
c
derivatives to that of 1, we hypothesized that the new
compounds might also target influenza A nucleoproteins.
Therefore, the effect of 3b on nucleoproteins was inspected
under fluorescence microscopy (Figure 3). Not surprisingly, 3b
potently inhibited the nuclear accumulation of influenza virus A
CONCLUSION
■
In summary, using scaffold-hopping and bioisosteric replace-
ment strategies, we successfully designed and synthesized a
series of 1H-1, 2, 3-triazole-4-carboxamide derivatives as new
anti-influenza A agents based on the chemical structure of
compound 1. One of the most potent compounds 3b potently
inhibited the replication of various H3N2 and H1N1 influenza
A virus strain with IC50 values ranging from 0.5 to 4.6 μM.
Furthermore, 3b also potently inhibited the replication of
H5N1 (RG14), amantidin-resistant A/WSN/33 (H1N1), and
tamiflu-resistant WSN (274Y) A/WSN/1933 (H1N1) with
IC50 values of 0.69, 0.68, and 1.11 μM, respectively. Further
computational study and mechanism investigation suggested
that 3b might directly target influenza virus A nucleoprotein to
inhibit its nuclear accumulation. Our study provided a new
series of lead compounds for anti-influenza drug development
targeting influenza virus nucleoproteins.
Figure 3. Compound 3b blocked nuclear accumulation of Influenza A
nucleoproteins. MDCK cells were infected with A/WSN/33 (H1N1)
viruses at multiplicity of infection (MOI) of 10 in the absence or
presence of 10 μM 1 (middle row) or 3b (bottom row). At 4.0 h post
infection, cells were fixed and stained with DAPI for nucleus (blue),
and FITC-tagged anti-influenza A NP antibodies specific for viral
nucleoprotein (green), respectively. Images were visualized by
confocal microscopy. The illustration is representative of experiment
in triplicate.
EXPERIMENTAL SECTION
■
Reagents and solvents were obtained from commercial suppliers and
used without further purification. Flash chromatography was
performed using silica gel (300−400 mesh). All reactions were
monitored by TLC, and silica gel plates with fluorescence F254 were
used and visualized with UV light. 1H or 13C NMR spectra were
2148
dx.doi.org/10.1021/jm2013503 | J. Med. Chem. 2012, 55, 2144−2153

Journal of Medicinal Chemistry
Article
Figure 4. Compound 3b binds to nucleoprotein with similar mode as that of the nucleozin analogue. (A) nucleozin analogue−NP derivative
complex structure; (B) 3b−NP complex structure predicted by molecular docking. (c) Overlay of 3b and the nucleozin analogue in the
nucleoprotein dimer interface pocket.
recorded on a Bruker AV-400 spectrometer at 400 MHz or Bruker AV-
500 spectrometer at 125 MHz, respectively. Coupling constants (J) are
expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in
parts per million (ppm) units relative to the internal control (TMS).
The low or high resolution of ESIMS was recorded on an Agilent 1200
HPLC-MSD mass spectrometer or Applied Biosystems Q-STAR Elite
ESI-LC-MS/MS mass spectrometer, respectively. The purity of
compounds was determined by reverse-phase HPLC analysis
confirming to be over 95% (>95%). HPLC instrument: DIONEX
SUMMIT HPLC. Column: Diamonsil C18, 5.0 mm, 4.6 mm × 250
mm (Dikma Technologies). PDA-100 photodiode array detector.
Detector: ASI-100 autoinjector. Pump p-680A. Elution, MeOH in
water; flow rate, 1.0 mL/min.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(3-methyl-5-phe-
nylisoxazol-4-yl)methanone 2a. To a stirred solution of 3-methyl-
5-phenylisoxazole-4-carboxylic acid 8 (207.9 mg, 1.02 mmol,
Supporting Information) in dry dichloromethane (DCM, 10 mL)
was added oxalyl chloride (0.26 mL, 3.07 mmol) and two drops of
DMF. The reaction mixture was stirred at room temperature for 2 h,
and the solvent was removed under vacuum. The resulting residue was
diluted with dry DCM (10 mL) and added 164.2 mg (0.68 mmol) 1-
(2-chloro-4-nitrophenyl)piperazine in 2.0 mL dry DCM and followed
by 0.28 mL (2.04 mmol) diethylethanamine in the ice bath. The
reaction mixture was stirred at room temperature for 3 h, and the
organic solvent was removed under vacuum. The resulting residue
diluted with water (30 mL) and extracted with ethyl acetate (2 × 50
mL). The combined organic layer was washed with dilute sodium
hydroxide solution and then with water, dried over anhydrous sodium
sulfate, and concentrated under vacuum to get the crude product
which was purified by column chromatography over silica gel to afford
pure compound 320.4 mg (yield 73.5%). 1H NMR (400 MHz,
CDCl3) δ 8.23 (d, J = 2.8 Hz, 1 H), 8.07 (dd, J = 2.8, 8.8 Hz, 1 H),
7.74−7.71 (m, 2 H), 7.49−7.48 (m, 3 H), 6.93 (d, J = 8.8 Hz, 1 H),
4.02−4.00 (m, 2 H), 3.42 (br, 2 H), 3.23−3.20 (m, 2 H), 2.83 (br, 2
H), 2.37 (s, 3 H). 13C NMR (125 MHz, CDCl3) δ 165.8, 162.5, 159.1,
153.8, 142.9, 131.0, 129.2, 128.0, 126.7(2C), 126.6, 123.4, 119.6,
110.2, 50.7, 50.4, 46.7, 41.8, 10.4. HRMS (ESI) calcd for
C21H20ClN4O4 [M + H]+ 427.1168; found 427.1167; HPLC analysis:
80:20 methanol−water, 9.04 min, 97.5%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(5-(2-methoxy-
1
phenyl)-3-methylisoxazol-4-yl)methanone 2b. Yield 76.5%. H
NMR (400 MHz, CDCl3) δ 8.21 (d, J = 2.4 Hz, 1 H), 8.06 (dd, J =
2.4, 8.8 Hz, 1 H), 7.68 (dd, J = 1.6, 7.6 Hz, 1 H), 7.47 (dt, J = 1.6, 7.8
Hz, 1 H), 7.08 (t, J = 7.6 Hz, 1 H), 6.99 (d, J = 8.4 Hz, 1 H), 6.90 (d, J
= 8.8 Hz, 1 H), 3.91 (br, 2 H), 3.84 (s, 3 H), 3.31 (br, 2 H), 3.15 (br, 2
H), 2.73 (br, 2 H), 2.39 (s, 3 H). 13C NMR (125 MHz, CDCl3) δ
163.5, 163.1, 159.4, 156.4, 153.9, 142.7, 132.5, 129.6, 127.9, 126.5,
123.3, 121.1, 119.5, 116.2, 111.9, 111.3, 55.3, 50.4, 46.6, 41.5, 10.5.
HRMS (ESI) calcd for 457.1273 [M + H]+; found 457.1276. HPLC
analysis: 80:20 methanol−water, 8.84 min, 95.3% in purity.
Synthesis of 1H-1,2,3-Triazole-4-carboxamide Derivatives 3.
Method A: The procedure was similar to that of 2.
Method B: To a stirred solution of 1-phenyl-1H-1,2,3-triazole-5-
carboxylic acids 16 (1 mmol, Supporting Information) and aryl
poperazine (1.1 mmol) in dry DCM (10 mL) was added 1-ethyl-3-(3-
dimethyllaminopropyl) carbodiimide hydrochloride (EDC·HCl) (1.5
mmol), 1-hydroxybenzotriazole anhydrous (HOBt) (1.2 mmol), and
triethylamine (3 mmol). The resulting mixture was stirred at room
temperature for 3 h, and the organic solvent was removed from the
reaction mixture under vacuum. The resulting residue was diluted with
water and extracted with ethyl acetate (2 × 50 mL).The combined
organic layer was washed with dilute sodium hydroxide solution and
then with water, dried over anhydrous sodium sulfate, and
concentrated under vacuum to get the crude product which was
purified by column chromatography over silica gel to afford pure
compound (yield 58−96%).
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(4-methyl-1-phe-
nyl-1H-1,2,3-triazol-5-yl)methanone 3a. Method A, yield 58.1%.
1H NMR (400 MHz, CDCl3) δ 8.23−8.21 (m, 1 H), 8.09−8.06 (m, 1
H), 7.60−7.50 (m, 5 H), 6.87(d, J = 8.8 Hz, 1 H), 3.89 (br, 2 H), 3.24
(br, 6 H), 2.45 (s, 3 H). 13C NMR (125 MHz, CDCl3) δ 160.0, 153.6,
143.0 (2C), 136.4, 129.8, 129.7, 128.2 (2C), 126.6, 123.4, 123.3, 119.6,
50.4, 50.2, 46.4, 42.0, 10.5. HRMS (ESI) calcd for C20H20ClN6O3 [M
+ H]+ 427.1280; found 427.1278. HPLC analysis: 75:25 methanol−
water, 8.01 min, 95.3%.
2149
dx.doi.org/10.1021/jm2013503 | J. Med. Chem. 2012, 55, 2144−2153

Journal of Medicinal Chemistry
Article
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(2-methoxy-
159.3, 154.1, 150.4, 141.9, 140.1, 130.6, 130.0 126.7, 126.4, 126.0,
124.1, 123.8, 120.7, 119.3, 116.5, 50.0, 49.7, 46.6, 41.4, 10.5. HRMS
(ESI) calcd for C20H20ClN6O4 [M + H]+ 443.1229; found 443.1224.
HPLC analysis: 70:30 methanol−water, 9.53 min, 96.0%.
phenyl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3b. Method
1
A, yield 82.5%. H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 2.0 Hz, 1
H), 8.10 (dd, J = 2.4, 8.8 Hz, 1 H), 7.53 (d, J = 7.6 Hz, 1 H) 7.48 (t, J
= 8.0 Hz, 1 H), 7.12 (t, J = 7.6 Hz, 1 H), 7.06 (d, J = 8.4 Hz, 1 H),
6.97 (d, J = 9.2 Hz, 1 H), 3.81 (br, 5 H), 3.47 (br, 2 H), 3.13 (br, 2 H),
2.93 (br, 2 H), 2.46 (s, 3 H). 13C NMR (125 MHz, CDCl3) δ 160.2,
153.8, 152.6, 142.9, 141.9, 131.3, 129.8, 128.1, 127.6, 126.6, 125.6,
123.4, 121.4, 119.6, 112.2, 56.1, 50.7, 50.3, 46.8, 42.0, 11.0. HRMS
(ESI) calcd for C21H22ClN6O4 [M + H]+ 457.1386; found 457.1384.
HPLC analysis: 75:25 methanol−water, 7.60 min, 97.1%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(4-methyl-1-o-
tolyl-1H-1,2,3-triazol-5-yl)methanone 3i. Method A, yield 92.8%.
1H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 2.8 Hz, 1 H), 8.09 (dd, J =
2.8, 8.8 Hz, 1 H), 7.43−7.37 (m, 2 H), 7.34−7.30 (m, 1 H), 7.26 (d, J
= 8.4 Hz, 1 H), 6.93 (d, J = 8.8 Hz, 1 H), 3.82 (br, 2 H), 3.44 (br, 2
H), 3.06 (br, 2 H), 2.86 (br, 2 H), 2.47 (s, 3 H), 2.22 (s, 3 H). 13C
NMR (125 MHz, CDCl3) δ 159.6, 153.6, 143.0, 141.9, 135.3, 135.0,
131.6, 130.4, 129.6, 128.2, 126.7, 126.6, 126.4, 123.4, 119.7, 50.8, 50.3,
46.6, 42.0, 17.8, 10.8. HRMS (ESI) calcd for C21H22ClN6O3 [M + H]+
441.1437; found 441.1428. HPLC analysis: 75:25 methanol−water,
9.24 min, 95.1%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(3-methoxy-
phenyl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3c. Method
1
A, yield 66.8%. H NMR (400 MHz, CDCl3) δ 8.26−8.20 (m, 1 H),
8.11−8.04 (m, 1 H), 7.40 (t, J = 8.0 Hz, 1 H), 7.15 (s, 1 H), 7.11 (d, J
= 4.0 Hz, 1 H), 7.00 (d, J = 8.4 Hz, 1 H), 6.89 (d, J = 8.8 Hz, 1 H),
3.91 (br, 2 H), 3.84 (s, 3 H), 3.24 (br, 4 H), 2.43 (s, 3 H). 13C NMR
(125 MHz, CDCl3) δ 160.5, 160.0, 153.6, 143.0, 142.9, 137.3, 130.5,
128.1 (2C), 126.6, 123.4, 119.6, 115.5, 115.0, 108.9, 55.7, 50.4, 50.2,
46.4, 42.0, 10.5. HRMS (ESI) calcd for C21H22ClN6O4 [M + H]+
457.1386; found 457.1379. HPLC analysis: 80:20 methanol−water,
6.89 min, 97.8%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(2-ethoxyphen-
yl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3j. Method B,
1
yield 95.9%. H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 2.4 Hz, 1
H), 8.10 (dd, J = 2.4, 8.8 Hz, 1 H), 7.50−7.44 (m, 2 H), 7..11 (t, J =
7.6 Hz, 1 H), 7.06 (d, J = 8.0 Hz, 1 H), 6.92 (d, J = 9.2 Hz, 1 H), 4.08
(q, J = 7.2 Hz, 2 H), 3.79 (br, 2 H), 3.37 (br, 2 H), 3.08 (br, 2 H), 2.79
(br, 2 H), 2.48 (s, 3 H), 1.30 (t, J = 7.2 Hz, 3 H). 13C NMR (125
MHz, CDCl3), δ 160.1, 153.8, 152.6, 142.9, 142.4, 131.4, 129.9, 128.1,
127.9, 126.6, 125.9, 123.4, 121.1, 119.6, 113.4, 65.0, 50.6, 50.3, 46.7,
42.0, 14.5, 10.9. HRMS (ESI) calcd for C22H24ClN6O4 [M + H]+
471.1542; found 471.1534. HPLC analysis: 75:25 methanol−water,
9.23 min, 96.7%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(4-methoxy-
phenyl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3d. Method
1
A, yield 88.2%. H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 2.8 Hz, 1
H), 8.08 (dd, J = 2.8, 8.8 Hz, 1 H), 7.49 (d, J = 8.8 Hz, 2 H), 7.02 (d, J
= 8.8 Hz, 2 H), 6.91 (d, J = 8.8 Hz, 1 H), 3.90 (br, 2 H), 3.85 (s, 3 H),
3.26 (br, 2 H), 3.11(br, 2 H), 2.75 (br, 2 H), 2.44 (s, 3 H). 13C NMR
(125 MHz, CDCl3) δ 160.4, 160.0, 153.6, 142.9, 142.6, 129.4, 128.2,
128.1, 126.5, 124.7, 123.3, 119.6, 114.7, 55.6, 50.5, 50.2, 46.4, 41.9,
10.5. HRMS (ESI) calcd for C21H22ClN6O4 [M + H]+ 457.1386;
found 457.1386. HPLC analysis: 75:25 methanol−water, 9.07 min,
97.2%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(2-isopropoxy-
phenyl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3k. Method
1
B, yield 93.4%. H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 2.8 Hz, 1
H), 8.09 (dd, J = 2.4, 8.8 Hz, 1 H), 7.48−7.43 (m, 2 H), 7.11−7.06
(m, 2 H), 6.90 (d, J = 9.2 Hz, 1 H), 4.57−4.48 (m, 1 H), 3.79 (br, 2
H), 3.35 (br, 2 H), 3.08 (br, 2 H), 2.77 (br, 2 H), 2.48 (s, 3 H), 1.24
(s, 3 H), 1.22 (s, 3 H). 13C NMR (125 MHz, CDCl3), δ 159.9, 153.8,
152.1, 142.9, 142.5, 131.3, 130.0, 128.1(2C), 126.9, 126.6, 123.4,
121.1, 119.5, 115.3, 72.5, 50.7, 50.3, 46.7, 42.0, 21.9, 10.9. HRMS
(ESI) calcd for C23H26ClN6O4 [M + H]+ 485.1699; found 485.1695.
HPLC analysis: 80:20 methanol−water, 7.70 min, 97.1%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(2-chlorophen-
yl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3e. Method A,
1
yield 72.3%. H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 2.4 Hz, 1
H), 8.11 (dd, J = 2.4, 8.8 Hz, 1 H), 7.58−7.48 (m, 4 H), 7.01 (d, J =
9.2 Hz, 1 H), 3.85 (br, 2 H), 3.66 (br, 2 H), 3.14 (br, 4 H), 2.48 (s, 3
H). 13C NMR (125 MHz, CDCl3) δ 159.3, 153.7, 143.0, 141.4, 134.2,
131.5, 130.3, 130.1, 129.9, 129.3, 128.1, 128.0, 126.6, 123.4, 119.7,
50.9, 50.3, 47.0, 42.0, 11.2. HRMS (ESI) calcd for C20H19Cl2N6O3 [M
+ H]+ 461.0890; found 461.0878. HPLC analysis: 80:20 methanol−
water, 6.75 min, 96.0%.
1-(2-(5-(4-(2-Chloro-4-nitrophenyl)piperazine-1-carbonyl)-4-
methyl-1H-1,2,3-triazol-1-yl)phenyl)ethanone 3l. Method A,
1
yield 64.1%. H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 2.8 Hz, 1
H), 8.07 (dd, J = 2.4, 8.8 Hz, 1 H), 7.76 (dd, J = 1.2, 6.8 Hz, 1 H),
7.65−7.61 (m, 2 H), 7.55 (dd, J = 1.6, 7.6 Hz, 1 H), 6.90 (d, J = 4.8
Hz, 1 H), 3.85 (br, 2 H), 3.66 (br, 2 H), 3.06 (br, 2 H), 2.88 (br, 2 H),
2.53 (s, 3 H), 2.45 (s, 3 H). 13C NMR (125 MHz, CDCl3) δ 199.6,
159.6, 153.8, 142.8, 141.6, 136.7, 133.6, 131.8, 130.4, 130.1, 128.4,
128.1, 126.6, 123.3, 119.5, 51.0, 50.3, 46.7, 42.0, 29.4, 10.8. HRMS
(ESI) calcd for C22H22ClN6O4 [M + H]+ 469.1386; found 469.1383.
HPLC analysis: 75:25 methanol−water, 7.57 min, 98.4%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(2,6-dimethox-
yphenyl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3m. Meth-
od A, yield 85.7%. 1H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 2.4 Hz,
1 H), 8.10 (dd, J = 2.4, 8.8 Hz, 1 H), 7.41 (t, J = 8.8 Hz, 1 H), 6.96 (d,
J = 8.8 Hz, 1 H), 6.68 (d, J = 8.4 Hz, 2 H), 3.78 (s, 10 H), 3.51 (br, 2
H), 3.08 (br, 2 H), 2.96 (br, 2 H), 2.47 (s, 3 H). 13C NMR (125 MHz,
CDCl3) δ 159.9, 155.9, 153.9, 142.9, 141.3, 131.8, 130.4, 128.1, 126.6,
123.4, 119.6, 114.5, 104.6, 56.4, 50.9, 50.5, 46.7, 41.9, 11.1. HRMS
(ESI) calcd for C22H24ClN6O5 [M + H]+ 487.1491; found 487.1492.
HPLC analysis: 75:25 methanol−water, 7.13 min, 97.2%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(3-chlorophen-
yl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3f. Method A,
1
yield 82.0%. H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 2.8 Hz, 1
H), 8.10 (dd, J = 2.4, 8.8 Hz, 1H), 7.63 (s, 1 H), 7.52−7.46 (m, 3 H),
6.94 (d, J = 9.2 Hz, 1 H), 3.94 (br, 2 H), 3.33 (br, 2 H), 3.16 (br, 2 H),
2.83 (br, 2 H), 2.45 (s, 3 H). 13C NMR (125 MHz, CDCl3) δ 159.7,
153.5, 143.0, 137.2, 135.5, 130.8, 129.7, 128.2, 128.1, 126.6, 123.4,
121.2, 119.7, 50.6, 50.2, 46.5, 42.1, 10.5. HRMS (ESI) calcd for
C20H19Cl2N6O3 [M + H]+ 461.0890; found 461.0874. HPLC analysis:
80:20 methanol−water, 8.42 min, 97.5%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(4-chlorophen-
yl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3g. Method A,
1
yield 63.9%. H NMR (400 MHz, CDCl3) δ 8.25 (s, 1 H), 8.10 (d,
J = 8.8 Hz, 1 H), 7.56−7.50 (m, 4 H), 6.94 (d, J = 9.2 Hz, 1 H), 3.92
(br, 2 H), 3.30 (br, 2 H), 3.14 (br, 2 H), 2.83 (br, 2H), 2.44 (s, 3H).
13C NMR (125 MHz, CDCl3) δ 159.8, 153.5, 143.1, 142.9, 135.7,
134.8, 130.0, 128.2(2C), 126.6, 124.5, 123.4, 119.7, 50.7, 50.3, 46.5,
42.0, 10.6. HRMS (ESI) calcd for C20H19Cl2N6O3 [M + H]+
461.0890; found 461.0878. HPLC analysis: 80:20 methanol−water,
8.25 min, 97.3%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(2-methoxy-
phenyl)-1H-1,2,3-triazol-5-yl)methanone 3n. Method B, yield
86.5%. 1H NMR (400 MHz, CDCl3), δ 8.27 (d, J = 2.4 Hz, 1 H), 8.12
(dd, J = 2.4, 8.8 Hz, 1 H), 7.88 (s, 1 H), 7.59 (d, J = 7.6 Hz, 1 H), 7.49
(t, J = 7.6 Hz, 1 H), 7.15 (t, J = 7.6 Hz, 1 H), 7.06 (d, J = 8.4 Hz, 1 H),
7.01 (d, J = 8.8 Hz, 1 H), 3.85 (br, 2 H), 3.81 (s, 3 H), 3.69 (br, 2 H),
3.18 (br, 2 H), 3.06 (br, 2 H). 13C NMR (125 MHz, CDCl3), δ 159.1,
153.8, 152.3, 143.0, 132.9, 132.3, 131.3, 128.1, 127.4, 126.7, 125.5,
123.5, 121.4, 119.7, 112.1, 56.0, 50.7, 50.3, 47.1, 42.0. HRMS (ESI)
calcd for C20H20ClN6O4 [M + H]+ 443.1229; found 443.1232. HPLC
analysis: 70:30 methanol−water, 9.82 min, 95.9%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(1-(2-hydroxy-
phenyl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3h. Method
1
A, yield 32.5%. HNMR (400 MHz, DMSO-d6) δ 10.51 (br, 1 H),
8.27 (d, J = 2.8 Hz, 1 H), 8.20 (dd, J = 2.8, 8.8 Hz, 1 H), 7.43 (dd, J =
2.4, 8.0 Hz, 1 H), 7.36−7.32 (m, 1 H), 7.29 (d, J = 8.8 Hz, 1 H), 7.02
(d, J = 8.0 Hz, 1 H), 6.99 (t, J = 8.0 Hz, 1 H), 3.70 (br, 2 H), 3.55 (br,
2 H), 3.20 (br, 4 H), 2.35 (s, 3 H). 13CNMR (125 MHz, DMSO-d6) δ
2150
dx.doi.org/10.1021/jm2013503 | J. Med. Chem. 2012, 55, 2144−2153

Journal of Medicinal Chemistry
Article
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(4-ethyl-1-(2-me-
thoxyphenyl)-1H-1,2,3-triazol-5-yl)methanone 3o. Method B,
yield 96.2%. 1H NMR (400 MHz, DMSO-d6) δ 8.25 (d, J = 2.4 Hz, 1
H), 8.17 (dd, J = 2.8, 8.8 Hz, 1 H), 7.55 (t, J = 8.0 Hz, 1 H), 7.49 (d, J
= 7.2 Hz, 1 H), 7.27 (d, J = 8.0 Hz, 1 H), 7.14 (t, J = 7.6 Hz, 1 H),
3.78 (s, 3 H), 3.66 (br, 2 H), 3.48 (br, 2 H), 3.17 (br, 4 H), 2.75 (q, J
= 7.6 Hz, 2 H), 1.28 (t, J = 7.6 Hz, 3H). 13C NMR (125 MHz, DMSO-
d6) δ 159.5, 154.4, 153.0, 146.5, 142.4, 131.9, 130.0, 127.9, 126.9,
126.4, 125.4, 124.2, 121.3, 121.2, 113.2, 56.5, 50.6, 50.3, 46.8, 41.9,
18.6, 13.6. HRMS (ESI) calcd for C22H24ClN6O4 [M + H]+ 471.1542;
found 471.1545. HPLC analysis: 75:25 methanol−water, 9.17 min,
97.8%.
127.9, 127.4, 126.6, 123.4, 119.5, 112.1, 50.2, 46.9, 41.8, 10.7. HRMS
(ESI) calcd for C21H21ClN5O3 [M + H]+ 426.1328; found 426.1327.
HPLC analysis: 75:25 methanol−water, 10.73 min, 98.1%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(3-(2-chlorophen-
yl)-5-methyl-1H-pyrazol-4-yl)methanone 4b. Method B, yield
45.6%. 1H NMR (400 MHz, DMSO-d6) δ 13.10 (s, 1 H), 8.22 (d, J =
2.0 Hz, 1 H), 8.15 (d, J = 8.8 Hz, 1 H), 7.60−7.42 (m, 4 H), 7.14 (d, J
= 8.8 Hz, 1 H), 3.48 (br, 4 H), 2.78 (br, 4 H), 2.32 (s, 3 H). 13C NMR
(125 MHz, DMSO-d6) δ 164.5, 154.1, 147.3, 141.8, 139.3, 132.8,
131.9, 129.5, 127.1, 126.3, 125.9, 123.8, 120.4, 113.0, 49.9(4C), 9.9.
HRMS (ESI) calcd for 460.0938 [M + H]+; found 460.0941. HPLC
analysis: 80:20 methanol−water, 8.09 min, 95.7% in purity.
Biphenyl-2-yl(4-(2-chloro-4-nitrophenyl)piperazin-1-yl)-
(4-(2-Chlorophenyl)piperazin-1-yl)(1-(2-methoxyphenyl)-4-
1
methanone 5a. Followed procedure for 2, yield 80.6%. H NMR
methyl-1H-1,2,3-triazol-5-yl)methanone 3p. Method B, yield
1
(400 MHz, CDCl3) δ 8.19 (d, J = 2.8 Hz, 1 H), 8.04 (dd, J = 2.4, 8.8
Hz, 1 H), 7.53−7.34 (m, 9 H), 6.76 (d, J = 9.2 Hz, 1 H), 4.05−4.01
(m, 1 H), 3.58−3.52 (m, 1 H), 3.18−3.12 (m, 2 H), 3.00−2.96 (m, 1
H), 2.79−2.76 (m, 1 H), 2.67−2.61 (m, 1 H), 1.82−1.76 (m, 1 H).
13C NMR (125 MHz, CDCl3) δ 170.0, 154.2, 142.6, 139.8, 138.3,
134.8, 129.7, 129.3, 128.9, 128.7, 128.0, 127.9, 127.8, 126.5, 123.3,
119.4, 49.9, 46.3, 41.3. HRMS (ESI) calcd for C23H21ClN3O3 [M +
H]+ 422.1266; found 422.1266. HPLC analysis: 80:20 methanol−
water, 13.98 min, 98.6%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(2′-methoxybi-
phenyl-2-yl)methanone 5b. Yield 90.9%. 1H NMR (400 MHz,
CDCl3) δ 8.20 (d, J = 2.4 Hz, 1 H), 8.06 (dd, J = 2.2, 8.8 Hz, 1 H),
7.48−7.31 (m, 7 H), 7.04−6.96 (m, 2 H), 6.82 (d, J = 8.8 Hz, 1 H),
3.87−3.81 (m, 1 H), 3.78 (s, 3 H), 3.62−3.60 (m, 1 H), 3.28−3.23
(m, 1 H), 3.13−3.11 (m, 2 H), 2.87−2.84 (m, 1 H), 2.69−2.65 (m, 1
H), 2.10−2.04 (m, 1 H). 13C NMR (125 MHz, CDCl3) δ 170.1, 156.4,
154.2, 142.5, 135.4, 135.2, 131.5, 131.1, 129.4, 128.9, 128.5,
127.8(2C), 127.7, 126.5, 123.3, 120.7, 119.4, 110.8, 55.4, 50.3, 50.2,
46.4, 41.3. HRMS (ESI) calcd for 452.1372 [M + H]+; found
452.1378. HPLC analysis: 80:20 methanol−water, 14.26 min, 99.2% in
purity.
MDCK Cell-Based Anti-influenza Assay (CPE). Cell-based anti-
influenza virus inhibitor screening was based on the principle of
cytopathic effect (CPE) protection assay as described in Supporting
Information.33
Cytotoxicity Assay. Procedure is similar to the CPE assay, but no
virus was added.
NP Antigen Reduction Assay. Subconfluent MDCK cells were
infected with 500 PFU of WSN virus in 96-well black clear bottom
plates and treated with varied testing compounds. After a 24 h
incubation, the cells were fixed and treated with 0.5% triton X-100
followed by NP antigen staining using anti-NP antibody (Millipore
MAB8251) and FITC conjugated second antibody for fluorescence
measurement (ex480, em535).
Plaque Reduction Assay. The plaque reduction assay was
performed in 6-well tissue culture plates. The MDCK cells were
seeded at 6 × 105 cells/well in DMEM (Invitrogen) with 1 mg/mL
TPCK and 0.3% BSA and incubated overnight at 37 °C in a
humidified 5% CO2 incubator. The confluent monolayers of MDCK
cells were then washed twice with 2 mL of Hanks and inoculated with
0.01MOI A/WSN/33 (H1N1) virus. After 1 h of virus adsorption at
37C, the viral inoculums were removed and the cells were washed
twice with PBS. The cell monolayers were then overlaid with MEM
medium containing 1% agar and serial compounds dilutions in the
presence of 2 mg/mL trypsin and 0.3% BSA. Three days after
infection, the monolayers were fixed and stained with 0.1% crystal
violet solution. The number of virus-induced plaques was counted.
McIP Assay. The plaque reduction was also determined in a high
throughput manner using the microplate counting of infectious
particles (McIP) assay.25 Briefly, the viral yields in the harvested
conditional media after anti-influenza treatments were measured as the
number of infection centers determined by NP staining after overnight
infection of MDCK cells in microwells.
91.2%. H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 8.0 Hz, 1 H),
7.46 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1 H), 7.22 (t, J = 7.6 Hz, 1
H), 7.11 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1 H), 7.01 (t, J = 7.2
Hz, 1 H), 6.92 (d, J = 8.0 Hz, 1 H), 3.81 (br, 5 H), 3.45 (br, 2 H), 2.98
(br, 2 H), 2.80 (br, 2 H), 2.47 (s, 3 H). 13C NMR (125 MHz, CDCl3)
δ 160.1, 152.7, 148.2, 141.7, 131.2, 130.7, 130.1, 128.9, 127.7, 127.6,
125.6, 124.5, 121.2, 120.3, 112.1, 56.0, 51.3, 50.8, 47.2, 42.3, 11.0.
HRMS (ESI) calcd for C21H23ClN5O2 [M + H]+ 412.1535; found
412.1537. HPLC analysis: 80:20 methanol−water, 6.21 min, 99.6%.
(1-(2-Methoxyphenyl)-4-methyl-1H-1,2,3-triazol-5-yl)(4-(4-
nitrophenyl)piperazin-1-yl)methanone 3q. Method B, yield
1
94.0%. H NMR (400 MHz, DMSO-d6) δ 8.08 (d, J = 9.6 Hz, 2
H), 7.54−7.48 (m, 2 H), 7.24 (d, J = 8.4 Hz, 1 H), 7.13 (t, J = 7.6 Hz,
1 H), 7.02 (d, J = 9.6 Hz, 2 H), 3.76 (s, 3 H), 3.63 (br, 2 H), 3.51 (br,
2 H), 3.50 (br, 2 H), 3.37 (br, 2 H), 2.30 (s, 3 H). 13CNMR (125
MHz, DMSO-d6) δ 159.4, 154.7, 152.8, 141.2, 137.8, 131.7, 130.5,
127.7, 126.1, 125.5, 121.3, 113.3, 113.1, 56.5, 46.8, 46.1, 45.9, 41.5,
11.0. HRMS (ESI) calcd for C21H23N6O4 [M + H]+ 423.1776; found
423.1773. HPLC analysis: 70:30 methanol−water, 5.58 min, 96.4%.
(1-(2-Methoxyphenyl)-4-methyl-1H-1,2,3-triazol-5-yl)(4-phe-
nylpiperazin-1-yl)methanone 3r. Method B, yield 86.2%. 1H NMR
(400 MHz, CDCl3) δ 7.53 (d, J = 7.2 Hz, 1 H), 7.45 (t, J = 7.6 Hz, 1
H), 7.28 (t, J = 7.2 Hz, 2 H), 7.11 (t, J = 7.6 Hz, 1 H), 7.02 (d, J = 8.0
Hz, 1 H), 6.94−6.87 (m, 3 H), 3.79 (br, 5 H), 3.44 (br, 2 H), 3.13 (br,
2 H), 2.94 (br, 2 H), 2.46 (s, 3 H). 13C NMR (125 MHz, CDCl3) δ
160.0, 152.6, 150.6, 141.7, 131.2, 130.0, 129.3, 127.6, 125.6, 121.2,
120.9, 116.8, 112.1, 56.0, 49.7, 49.4, 46.8, 42.0, 11.0. HRMS (ESI)
calcd for C21H24N5O2 [M + H]+ 378.1925; found 378.1921. HPLC
analysis: 70:30 methanol−water, 6.88 min, 98.3%.
(4-(2-Methoxy-4-nitrophenyl)piperazin-1-yl)(1-(2-methoxy-
phenyl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3s. Method
1
B, yield 93.6%. H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 7.2 Hz, 1
H), 7.71(s, 1 H), 7.52 (d, J = 7.6 Hz, 1 H), 7.46 (t, J = 8.0 Hz, 1 H),
7.11 (t, J = 7.6 Hz, 1 H), 7.04 (d, J = 8.0 Hz, 1 H), 6.80 (d, J = 8.8 Hz,
1 H), 3.95 (s, 3 H), 3.80 (br, 5 H), 3.46 (br, 2 H), 3.15 (br, 2 H), 2.97
(br, 2 H), 2.46 (s, 3 H). 13C NMR (125 MHz, CDCl3) δ 160.1, 152.6,
151.3, 146.2, 142.8, 141.8, 131.2, 129.9, 127.6, 125.6, 121.3, 117.6,
116.9, 112.1, 106.6, 56.0(2C), 50.0, 49.6, 46.8, 42.0, 11.0. HRMS
(ESI) calcd for C22H25N6O5 [M + H]+ 453.1881; found 453.1881.
HPLC analysis: 74:26 methanol−water, 5.69 min, 96.6%.
(4-(2-Chloro-4-nitrophenyl)-1,4-diazepan-1-yl)(1-(2-methox-
yphenyl)-4-methyl-1H-1,2,3-triazol-5-yl)methanone 3t. Method
1
A, yield 74.5%. H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 2.4 Hz, 1
H), 8.03−7.99 (m, 1 H), 7.47−7.41 (m, 2 H), 7.08−6.94 (m, 3 H),
3.86−3.84 (m, 1 H), 3.76 (br, 4 H), 3.51−3.49 (m, 1 H), 3.45−3.42
(m, 1 H), 3.36 (br, 1 H), 3.29 (br, 1 H), 3.19 (br, 2 H), 2.43 (s, 3 H),
2.05 (br, 1 H), 1.94 (br, 1 H), 1.82 (br, 1 H). HRMS (ESI) calcd for
C22H24ClN6O4 [M + H]+ 471.1542; found 471.1545. HPLC analysis:
80:20 methanol−water, 5.61 min, 99.8%.
(4-(2-Chloro-4-nitrophenyl)piperazin-1-yl)(5-methyl-3-phe-
nyl-1H-pyrazol-4-yl)methanone 4a. Followed the procedure for 3,
1
method B, yield 60.2%. H NMR (400 MHz, CDCl3) δ 11.49 (br, 1
H), 8.21 (d, J = 2.4 Hz, 1 H), 8.05 (dd, J = 2.4, 8.8 Hz, 1 H), 7.56 (d, J
= 6.8 Hz, 2 H), 7.39−7.35 (m, 3 H), 6.82 (d, J = 9.2 Hz, 1 H), 3.92
(br, 2 H), 3.29 (br, 2 H), 3.11 (br, 2 H), 2.52 (br, 2 H), 2.29 (s, 3 H).
13C NMR (125 MHz, CDCl3) δ 165.7, 154.1, 142.7, 128.9, 128.8,
Immunofluorescence Microscopy. MDCK cells were seed to 35
mm culture plate or 6-well plates with coverslips and grow to 70−80%
confluence, and then were infected with A/WSN/33 (H1N1) viruses
2151
dx.doi.org/10.1021/jm2013503 | J. Med. Chem. 2012, 55, 2144−2153

Journal of Medicinal Chemistry
Article
at MOI of 10 in the presence or absence of 10 μM compounds. At 2.0,
4.0, and 6.0 h postinfection, the inoculums were aspirated and the cells
were fixed by 4% paraformaldehyde at 4 C for 1 h. After fixation, the
cells were washed and incubated with blocking buffer for 1 h and then
incubated with FITC-tagged NP primary antibody (Abcam, MA, USA)
in antibody dilution buffer (dilution 1: 20) for 2.0 h. The coverslips
were then washed and counterstained with 4′,6-diamidino-2-phenyl-
indole, dihydrochloride (DAPI) (Invitrogen) for nucleus localization
and mounted on slides using antifade mounting medium. The
immunofluorescence images were photographed using a Leica laser
scanning confocal microscope.
Computational Study. The structure of influenza virus
nucleoprotein oligomers was retrieved from the Protein Data Bank
(PDB code: 3ROS). The protein was processed using protein
preparation wizard, which assigns bond orders and adds hydrogens
and missing atoms. Compound 3b was prepared in LigPrep (LigPrep,
Estoepangestie, A. T.; Chaisingh, A.; Auewarakul, P.; Long, H. T.;
Hanh, N. T.; Webby, R. J.; Poon, L. L.; Chen, H.; Shortridge, K. F.;
Yuen, K. Y.; Webster, R. G.; Peiris, J. S. Genesis of a highly pathogenic
and potentially pandemic H5N1 influenza virus in eastern Asia. Nature
2004, 430, 209−213.
(4) Webby, R. J.; Swenson, S. L.; Krauss, S. L.; Gerrish, P. J.; Goyal,
S. M.; Webster, R. G. Evolution of swine H3N2 influenza viruses in the
United States. J. Virol. 2000, 74, 8243−8251.
(5) Zhou, N. N.; Senne, D. A.; Landgraf, J. S.; Swenson, S. L.;
Erickson, G.; Rossow, K.; Liu, L.; Yoon, K.; Krauss, S.; Webster, R. G.
Genetic reassortment of avian, swine, and human influenza A viruses in
American pigs. J. Virol. 1999, 73, 8851−8856.
(6) Davies, W. L.; Hoffmann, C. E.; Paulshock, M.; Wood, T. R.;
Haff, R. F.; Grunert, R. R.; Watts, J. C.; Hermann, E. C.; Neumayer, E.
M.; Mcgahen, J. W. Antiviral activity of 1-adamantanamine
(Amantadine). Science 1964, 144, 862−863.
(7) Vonitzstein, M.; Wu, W. Y.; Kok, G. B.; Pegg, M. S.; Dyason, J.
C.; Jin, B.; Phan, T. V.; Smythe, M. L.; White, H. F.; Oliver, S. W.;
Colman, P. M.; Varghese, J. N.; Ryan, D. M.; Woods, J. M.; Bethell, R.
C.; Hotham, V. J.; Cameron, J. M.; Penn, C. R. Rational design of
potent sialidase-based inhibitors of influenza-virus replication. Nature
1993, 363, 418−423.
(8) Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H. T.; Zhang, L. J.;
Swaminathan, S.; Bischofberger, N.; Chen, M. S.; Mendel, D. B.; Tai,
C. Y.; Laver, W. G.; Stevens, R. C. Influenza neuraminidase inhibitors
possessing a novel hydrophobic interaction in the enzyme active site:
design, synthesis, and structural analysis of carbocyclic sialic acid
analogues with potent anti-influenza activity. J. Am. Chem. Soc. 1997,
119, 681−690.
(9) De Clercq, E. Antiviral agents active against influenza A viruses.
Nature Rev. Drug Discovery 2006, 5, 1015−1025.
(10) de Jong, M. D.; Thanh, T. T.; Khanh, T. H.; Hien, V. M.; Smith,
G. J. D.; Chau, N. V.; Cam, B. V.; Qui, P. T.; Ha, D. Q.; Guan, Y.;
Peiris, J. S. M.; Hien, T. T.; Farrar, J. Oseltamivir resistance during
treatment of influenza A (H5N1) infection. New Engl. J. Med. 2005,
353, 2667−2672.
(11) Lackenby A, H. O.; Dudman, S. G.; Meijer, A.; Paget, W. J.;
Hay, A. J.; Zambon, M. C. Emergence of resistance to oseltamivir
among influenza A(H1N1) viruses in Europe. Eurosurveillance 2008,
13, 1−2.
(12) Regoes, R. R.; Bonhoeffer, S. Emergence of drug-resistant
influenza virus: population dynamical considerations. Science 2006,
312, 389−391.
(13) Webster, R. G.; Govorkova, E. A. H5N1 influenzacontinuing
evolution and spread. New Engl. J. Med. 2006, 355, 2174−2177.
(14) Lackenby, A.; Thompson, C. I.; Democratis, J. The potential
impact of neuraminidase inhibitor resistant influenza. Curr. Opin.
Infect. Dis. 2008, 21, 626−638.
(15) Dharan, N. J.; Gubareva, L. V.; Meyer, J. J.; Okomo-Adhiambo,
M.; McClinton, R. C.; Marshall, S. A.; St. George, K.; Epperson, S.;
Brammer, L.; Klimov, A. I.; Bresee, J. S.; Fry, A. M. Infections with
oseltamivir-resistant influenza A(H1N1) virus in the United States.
JAMA, J. Am. Med. Assoc. 2009, 301, 1034−1041.
(16) Layne, S. P.; Monto, A. S.; Taubenberger, J. K. Pandemic
influenza: an inconvenient mutation. Science 2009, 323, 1560−1561.
(17) Moscona, A. Global Transmission of oseltamivir-resistant
influenza. New Engl. J. Med. 2009, 360, 953−956.
(18) Nalluswami, K.; Nambiar, A.; Perrianne; Lurie; Moll, M.; Lute,
J.; Simwale, O. Swine-origin influenza A (H3N2) virus infection in two
childrenIndiana and Pennsylvania, July−August 2011. MMWR,
Morbidity Mortality Wkly. Rep. 2011, 60, 1213−1215.
version 2.5, Schrodinger, LLC, New York, NY, 2011) module using
̈
OPLS-2005 force field. Molecular docking was performed in Glide
module (Glide, version 5.7, Schrodinger, LLC, New York, NY, 2011).
̈
Compound 3b was docked into the pocket at the dimer interface with
extra precision scoring function.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures for key chemical intermediates, detail
1
procedure for CPE assays, HNMR, 13C NMR, and HPLC
spectrum for compound 2a, 2b, 3a−3t, 4a, 4b, 5a, and 5b. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (8620)32015276. Fax: (8620)32015299. E-mail:
ding_ke@gibh.ac.cn.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank National Basic Research Program of China (grant
2010CB529706, 2009CB940904), the 100-Talent program of
Chinese Academy of Sciences (CAS), and key innovative drug
discovery project of Guangzhou city (grant 2009Z1-E911,
2010J-E551) for their financial support. We also thank the
scientists from Roche Pharma Research and Early Development
(Shanghai) for the assistance to determine the anti-influenza
activities of compound 1 and 3b against A/New Jersey/8/76,
A/Weiss/43, and A/Mal/302/54 strains.
ABBREVIATIONS USED
■
NP, nucleoprotein; RNA, ribonucleic acid; vRNP, virus
ribonucleoprotein complex; CPE, cytopathic effect protection
assays; MDCK, Madin-Darby canine kidney cells; AMD,
amantidine; McIP, microplate counting of infectious particles
assay; MOI, multiplicity of infection
REFERENCES
■
(1) Monto, A. S. The risk of seasonal and pandemic influenza:
prospects for control. Clin. Infect. Dis. 2009, 48, S20−S25.
(2) Karasin, A. I.; Schutten, M. M.; Cooper, L. A.; Smith, C. B.;
Subbarao, K.; Anderson, G. A.; Carman, S.; Olsen, C. W. Genetic
characterization of H3N2 influenza viruses isolated from pigs in North
America, 1977−1999: evidence for wholly human and reassortant virus
genotypes. Virus Res. 2000, 68, 71−85.
(19) Portela, A.; Digard, P. The influenza virus nucleoprotein: a
multifunctional RNA-binding protein pivotal to virus replication. J.
Gen. Virol. 2002, 83, 723−734.
(20) Shu, L. L.; Bean, W. J.; Webster, R. G. Analysis of the evolution
and variation of the human influenza A virus nucleoprotein gene from
1933 to 1990. J. Virol. 1993, 67, 2723−2729.
(3) Li, K. S.; Guan, Y.; Wang, J.; Smith, G. J.; Xu, K. M.; Duan, L.;
Rahardjo, A. P.; Puthavathana, P.; Buranathai, C.; Nguyen, T. D.;
2152
dx.doi.org/10.1021/jm2013503 | J. Med. Chem. 2012, 55, 2144−2153

Journal of Medicinal Chemistry
Article
(21) Ng, A. K.; Wang, J. H.; Shaw, P. C. Structure and sequence
analysis of influenza A virus nucleoprotein. Sci. China, Ser. C: Life Sci.
2009, 52, 439−449.
(22) Li, Z.; Watanabe, T.; Hatta, M.; Watanabe, S.; Nanbo, A.;
Ozawa, M.; Kakugawa, S.; Shimojima, M.; Yamada, S.; Neumann, G.;
Kawaoka, Y. Mutational analysis of conserved amino acids in the
influenza A virus nucleoprotein. J. Virol. 2009, 83, 4153−4162.
(23) Tao, Y. Z. J.; Ye, Q. Z.; Krug, R. M. The mechanism by which
influenza A virus nucleoprotein forms oligomers and binds RNA.
Nature 2006, 444, 1078−1082.
(24) Kao, R. Y.; Yang, D.; Lau, L.-S.; Tsui, W. H. W.; Hu, L.; Dai, J.;
Chan, M.-P.; Chan, C.-M.; Wang, P.; Zheng, B.-J.; Sun, J.; Huang, J.-
D.; Madar, J.; Chen, G.; Chen, H.; Guan, Y.; Yuen, K.-Y. Identification
of influenza A nucleoprotein as an antiviral target. Nature Biotechnol.
2010, 28, 600−605.
(25) Su, C.-Y.; Cheng, T.-J. R.; Lin, M.-I.; Wang, S.-Y.; Huang, W.-I.;
Lin-Chu, S.-Y.; Chen, Y.-H.; Wu, C.-Y.; Lai, M. M. C.; Cheng, W.-C.;
Wu, Y.-T.; Tsai, M.-D.; Cheng, Y.-S. E.; Wong, C.-H. High-throughput
identification of compounds targeting influenza RNA-dependent RNA
polymerase activity. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 19151−
19156.
(26) Fedichev, P.; Timakhov, R.; Pyrkov, T.; Getmantsev, E.; Vinnik,
A. Structure-based drug design of a new chemical class of small
molecules active against influenza A nucleoprotein in vitro and in vivo.
PLoS Currents 2011, 3, RRN1253.
(27) During preraration of this manuscript, Gerritz et al. reported 5-
phenyl-3H-1,2,3-triazol-4-carboxyl amides as new NP inhibitors
Gerritz, S. W.; Cianci, C.; Kim, S.; Pearce, B. C.; Deminie, C.;
Discotto, L.; McAuliffe, B.; Minassian, B. F.; Shi, S.; Zhu, S.; Zhai, W.;
Pendri, A.; Li, G.; Poss, M. A.; Edavettal, S.; McDonnell, P. A.; Lewis,
H. A.; Maskos, K.; Mortl, M.; Kiefersauer, R.; Steinbacher, S.; Baldwin,
̈
E. T.; Metzler, W.; Bryson, J.; Healy, M. D.; Philip, T.; Zoeckler, M.;
Schartman, R.; Sinz, M.; Leyva-Grado, V. H.; Hoffmann, H.-H.;
Langley, D. R.; Meanwell, N. A.; Krystal, M. Inhibition of influenza
virus replication via small molecules that induce the formation of
higher-order nucleoprotein oligomers. Proc. Natl. Acad. Sci. U.S.A.
2011, 108, 15366−15371.
(28) Shen, Y.-F.; Chen, Y.-H.; Chu, S.-Y.; Lin, M.-I.; Hsu, H.-T.; Wu,
P.-Y.; Wu, C.-J.; Liu, H.-W.; Lin, F.-Y.; Lin, G.; Hsu, P.-H.; Yang, A.-S.;
Cheng, Y.-S. E.; Wu, Y.-T.; Wong, C.-H.; Tsai, M.-D. E339...R416 salt
bridge of nucleoprotein as a feasible target for influenza virus
inhibitors. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 16515−16520.
(29) Gerritz, S. Aminoacetamide acyl guanidines as beta-secretase
inhibitors. U.S. Patent Application 20060287287, 2006.
(30) Bell, M. G.; Doti, R. A.; Dowling, M. S.; Genin, M. J.; Lander, P.
A.; Ma, T.; Mantlo, N. B.; Ochoada, J. M.; Stelzer, L. S.; Stites, R. E.;
Warshawsky, A. M. Compounds and methods for modulating FXR.
Patent WO 2007/140174 A2, 2007.
(31) Zhao, B. X.; Zhang, L.; Zhu, X. S.; Wan, M. S.; Zhao, J.; Zhang,
Y.; Zhang, S. L.; Miao, J. Y. Synthesis and discovery of a novel pyrazole
derivative as an inhibitor of apoptosis through modulating integrin
beta 4, ROS, and p53 levels in vascular endothelial cells. Bioorg. Med.
Chem. 2008, 16, 5171−5180.
(32) The crystal data was deposited at the CambridgeCrystallo-
graphic Data Centre. Deposted numember is CCDC 862140.
(33) Tang, G.; Lin, X.; Qiu, Z.; Li, W.; Zhu, L.; Wang, L.; Li, S.; Li,
H.; Lin, W.; Yang, M.; Guo, T.; Chen, L.; Lee, D.; Wu, J. Z.; Yang, W.
Design and synthesis of benzenesulfonamide derivatives as potent anti-
influenza hemagglutinin inhibitors. ACS Med. Chem. Lett. 2011, 2,
603−607.
2153
dx.doi.org/10.1021/jm2013503 | J. Med. Chem. 2012, 55, 2144−2153