Design, synthesis and structure-activity relationship of novel inhibitors against H5N1 hemagglutinin-mediated membrane fusion

Article abstract of DOI:10.1016/j.ejmech.2012.08.041

We reported previously that a small molecule named CL-385319 could inhibit H5N1 influenza virus infection by targeting hemagglutinin, the envelope protein mediating virus entry. In the present study, a novel series of derivatives focused on the structural

Full text of DOI:10.1016/j.ejmech.2012.08.041

European Journal of Medicinal Chemistry 57 (2012) 211e216
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and structureeactivity relationship of novel inhibitors against
H5N1 hemagglutinin-mediated membrane fusion
,
,
b
,
,
*
Zhibo Zhu a 1, Runming Li a 1, Gaokeng Xiao c, Zhipeng Chen a, Jie Yang a, Qiuhua Zhu a, Shuwen Liu a
a School of Pharmaceutical Sciences, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou 510515, China
b Guangzhou Baidi Biotechnology Co. Ltd, Guangzhou 511495, China
c Institute of Traditional Chinese Medicine and Natural Products, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research,
Jinan University, Guangzhou 510632, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We reported previously that a small molecule named CL-385319 could inhibit H5N1 influenza virus
infection by targeting hemagglutinin, the envelope protein mediating virus entry. In the present study,
a novel series of derivatives focused on the structural variation of CL-385319 were synthesized as specific
inhibitors against the H5 subtype of influenza A viruses. These small molecules inhibited the low pH-
induced conformational change of hemagglutinin, thereby blocking viral entry into host cells.
Received 18 May 2012
Received in revised form
29 August 2012
Accepted 29 August 2012
Available online 7 September 2012
Compound 1l was the most active inhibitor in this series with an IC50 of 0.22 mM. The structureeactivity
relationships analysis of these compounds showed that the 3-fluoro-5-(trifluoromethyl)benzamide
moiety was very important for activity, and the eF group was a better substituent group than eCF3
group in the phenyl ring. The inhibitory activity was sensitive to the benzamide because the oxygen and
hydrogen of the amide served as H-bond acceptor and donor, respectively.
Keywords:
Membrane fusion
Fusion inhibitors
H5N1
Ó 2012 Elsevier Masson SAS. All rights reserved.
Influenza A virus
Hemagglutinin
Structureeactivity relationship
1. Introduction
a trimeric envelope glycoprotein, which can be cleaved into HA1
and HA2 subunits by host proteases [6]. The entry step of virus
The H5N1 avian influenza A virus which caused significant
morbidity and mortality is a serious threat to human health and has
potential human-to-human transmission in the near future.
Currently, only four drugs are available for the prevention and
treatment of influenza virus, which fall into two categories, the M2
ion channel blockers and neuraminidase (NA) inhibitors [1].
However, most of the isolated H5N1 influenza A virus strains are
now resistant to M2 inhibitors, and may rapidly develop resistance
to NA inhibitors [2e5]. Therefore, it is urgently needed to develop
new antiviral drugs, especially those with new mechanisms of
action.
infection is initiated by HA1 binding to sialic acid receptor on the
target cell membrane. The virion is then internalized to the endo-
some by endocytosis. The low endosomal pH leads to an irrevers-
ible conformational change of HA2 protein, resulting in the
membrane fusion. Several small molecules, such as stachyflin [7],
BMY-27709 [8] and CL-385319 [9], can stabilize the conformation
of HA2 at neutral pH or destabilize the HA2 structure at low pH,
leading to the blocking effect of membrane fusion between virus
and endosome. These molecules can be lead compounds to develop
new low molecular influenza virus entry inhibitors.
We reported previously that CL-385319 was effective in inhib-
iting the infection of H5N1 influenza A virus (A/Thailand/Kan353/
An effective antiviral strategy is to block virus entry into the
target cell. Hemagglutinin (HA), the envelope protein of influenza A
virus, is the key protein mediating influenza virus entry and is
a target for developing influenza virus entry inhibitors. HA is
2004) with an IC50 of 27.03 ꢀ 2.54
mM [10]. Mechanism study
showed that CL-385319 could interact with the H5-typed hemag-
glutinin and prevent the low pH-induced conformational change of
the protein, thus blocking the fusion process [10]. In the present
study, a series of derivatives which focused on structural variation of
CL-385319 were synthesized. These novel compounds have potent
activities against the infection of H5N1 pseudovirus which pseu-
dotyped with hemagglutinin from A/Anhui/1/2005(H5N1) strain.
* Corresponding author. Tel.: þ86 20 6164 8538; fax: þ86 20 6164 8655.
E-mail address: liusw@smu.edu.cn (S. Liu).
1
Both authors contributed equally.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.041

212
Z. Zhu et al. / European Journal of Medicinal Chemistry 57 (2012) 211e216
2. Results and discussion
2.2. SAR analysis of the compounds
2.1. Inhibition of the infection of H5N1 pseudovirus
From Table 1, the antiviral activities of compounds 1ke1n were
better than the others, which suggested that the incorporation of 3-
fluoro-5-(trifluoromethyl)benzamide segment to the basic struc-
ture could increase the anti-H5N1 virus activity. Among them,
Previously, we demonstrated that CL-385319 inhibited the
highly pathogenic H5N1 avian influenza A virus by blocking viral
entry [10]. CL-385319 could not inhibit N1-subtype neuraminidase
activity and the absorption of H5-subtype HA to chicken erythro-
cytes. The inhibitory activity of CL-385319 is specific to hemag-
glutinin since it has no inhibitory activity on VSV-G pseudovirus,
which the only difference is that the HA and NA were replaced by
glycoproteins from vesicular stomatitis virus (VSV-G) [10].
It is much more convenient and safer to use the single-cycle
pseudovirus than live H5N1 avian influenza virus to evaluate the
inhibitory activities of modified CL-385319 derivatives against
H5N1 entry. The H5N1 pseudovirus was prepared by cotransfection
of HA plasmid, NA plasmid and an HIV backbone plasmid (pNL4-
3.luc.RꢁEꢁ) to 293T cells.
The compounds designed and synthesized are listed in Table 1.
Compounds 1aet, 2e3 and 4aed were evaluated for the inhibitory
activity against the entry of H5N1 influenza virus. All compounds
possess inhibitory activities against the infection of H5N1 virus
with a potency ranging from moderate to potent. The IC50 values
obtained for these compounds are also summarized in Table 1. The
most active compounds in this series are 1k, 1l, 1m and 1n, with
compound 1l is the most potent inhibitor with an IC50 of 0.22 mM.
We also found that replacement of eH with CH3 in R4 substituent
increases the activity (1e > 1r; 1c > 1t). The structures of 1c and 1l
are very similar, in which 1l has an eF group in the phenyl ring,
while the corresponding position of 1c is a eCF3 group. It is indi-
cated that the eF group is a better substituent group than eCF3
group, and eCF3 is better substituent group than eH in R4
substituent. This finding is also in agreement with the facts that
replacement of eCF3 with eF group or eH with eCF3 enhances the
inhibitory activity (1l > 1c > 1t; 1k > 1b; 1i > 1h; 1n > 1e). The
inhibitory activity is sensitive to benzamide group since sulfon-
amides compounds 2 or 3 are much lower active than 1a. The
weaker activity may be ascribed to the fact that Arg106 from the
HA2 chain of hemagglutinin can form hydrogen bond to the amide
carbonyl oxygen of 1a [9]. Glu105 and Asp109 from HA2 may
participate in chargeecharge interaction with the piperidine
nitrogen and the pyrrole nitrogen. Interestingly, we found that 1g
and 1j were less active although they can also form chargeecharge
interaction with Glu105 and Asp109, which may be due to the 3-
carbon linker between the benzamide and piperazine. Therefore,
a 2-carbon linker is a preference. It was reported that quinolizidine
salicylamides can selectively inhibit the fusion process of the H1-
and H3-subtypes influenza A by stabilizing hemagglutinin and
prevent the essential conformational rearrangement [3e5]. We also
IC50 value lower than 1
mM (Fig. 1). Compounds 1k, 1l and 1m
had no effect on VSV-G pseudovirus infection (Fig. 1), which was
similar to CL-385319, demonstrating that these compounds
specifically interfere with the entry of influenza virus by targeting
hemagglutinin.
Table 1
Inhibitory activities of compounds against H5N1 pseudovirus.
R
NH
R1
O
R1
O
R1
R1
N
O
O
S
R2
R3
R
R2
R3
R
R2
R3
N
H
O
R2
R3
R
N
H
O
R4
1a-1t
R4
3
R4
2
4a-4d
Compounds
R1
R2
R3
R4
R
IC50 (mM)
CL-385319
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
CF3
CF3
CF3
CF3
CF3
Cl
Br
Cl
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
F
2-(Piperidin-1-yl)ethyl
2-(Piperidin-1-yl)ethyl
2-(Pyrrolidin-1-yl)ethyl
2-(Thiophen-2-yl)ethyl
(Pyridine-3-yl)methyl
Phenethyl
1-(Naphthalene-2-yl)ethyl
3-(4-Methylpiperazin-1-yl)propyl
2-Morpholinoethyl
0.37 ꢀ 0.12
2.51 ꢀ 0.16
1.71 ꢀ 0.26
2.44 ꢀ 0.01
2.46 ꢀ 0.08
2.47 ꢀ 0.34
40.77 ꢀ 4.31
66.64 ꢀ 12.4
9.89 ꢀ 2.44
1.22 ꢀ 0.17
84.32 ꢀ 15.06
0.38 ꢀ 0.02
0.22 ꢀ 0.01
0.56 ꢀ 0.01
0.33 ꢀ 0.01
49.00 ꢀ 1.42
59.97 ꢀ 1.57
41.46 ꢀ 1.35
79.96 ꢀ 4.27
7.80 ꢀ 0.01
4.33 ꢀ 0.12
54.87 ꢀ 8.68
61.16 ꢀ 8.61
6.25 ꢀ 0.12
5.13 ꢀ 0.08
9.20 ꢀ 0.50
7.61 ꢀ 0.35
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
F
F
F
F
F
F
F
F
OH
H
H
H
H
F
2-Morpholinoethyl
3-(4-Methylpiperazin-1-yl)propyl
2-(Pyrrolidin-1-yl)ethyl
2-(Thiophen-2-yl)ethyl
(Pyridine-4-yl)methyl
Phenethyl
1-(Naphthalene-2-yl)ethyl
(3-Fluoro-5-(trifluoromethyl)benzamido)ethyl
2-(Pyrrolidin-1-yl)ethyl
Phenethyl
1s
1t
2
3
4a
4b
4c
Cyclohexyl
2-(Thiophen-2-yl)ethyl
2-(Piperidin-1-yl)ethyl
2-(Piperidin-1-yl)ethyl
2-(Piperidin-1-yl)ethyl
2-(Piperidin-1-yl)ethyl
2-(Thiophen-2-yl)ethyl
2-(Thiophen-2-yl)ethyl
e
e
e
4d
e

Z. Zhu et al. / European Journal of Medicinal Chemistry 57 (2012) 211e216
213
3. Conclusions
A
120
100
80
In the present study, a series of derivatives were synthesized
based on the structure of CL-385319. These compounds could
inhibit the infection of pseudoviruses carrying hemagglutinins
from H5N1 strains. 1l is the most active inhibitor in this series with
H5N1
VSV-G
an IC50 of 0.22 mM. The SAR analysis of these compounds showed
60
that the 3-fluoro-5-(trifluoromethyl)benzamide moiety is very
important for activity, while the F- group is a better substituent
group than eCF3 group and the eCF3 group is better than H- group
in the phenyl ring. The inhibitory activity is sensitive to the ben-
zamide because the oxygen of amide is an H-bond acceptor, while
the hydrogen of the amide served as H-bond donor, respectively.
40
20
0
-20
3.13 1.56 0.78 0.39 0.19
0.1
0.05
4. Experimental protocols
Concentration of 1k (
µM)
4.1. Chemistry
B
All reagents and solvents were obtained from commercial
suppliers and were used without further purification. The
benzotriazole-l-yl-oxy-tris-pyrrolidino-phosphonium hexafluoro
phosphate (PyBOP) was obtained from Aladdin (Shanghai, China).
Most of reactions were monitored with a silica gel TLC plate under
UV light followed by visualization with a p-anisaldehyde. 1H NMR
spectra were measured at 400 MHz in CDCl3 unless stated other-
wise and data were reported as follows in ppm (d) from the internal
standard (TMS, 0.0 ppm), and coupling constant in Hz. Melting
points of solid compounds were observed on a Thomas Hoover
capillary melting point apparatus.
General synthesis of 1aeo, 1ret and 4aed using Method A [11e
13]: A mixture of 1 mmol (1 equiv.) of carboxylic acids, 1.4 mmol
(1.4 equiv.) of amine, 552 mg (1.8 equiv.) of PyBOP and 2 mL of
triethylamine was stirred overnight at room temperature in 40 ml
of a 1:1 mixture of CH2Cl2 and CH3CN. After solvent evaporation,
the crude product was purified by column chromatography on
silica (CHCl3:CH3CH2OH ¼ 9:1) resulting in yellow thick oil that
slowly crystallized.
General synthesis of 1p, 2 and 3 using Method B: To a solution of
benzoyl chloride (1 mmol) in dry dichloromethane (10 ml) was
added amine (1 mmol), and the mixture was stirred for 0.5 h at
room temperature. After solvent evaporation, the crude product
C
120
100
80
60
40
20
0
H5N1
VSV-G
was
purified
by
column
chromatography
on
silica
(CHCl3:CH3CH2OH ¼ 9:1) resulting in yellow thick oil and crystal-
lized from the dichloromethaneepetroleum ether mixture.
Synthesis of 2,4,5-trihydroxy-N-(2-(pyrrolidin-1-yl)rthyl)benza-
mide (1q) [14,15] (Scheme 1): 2,4,5-trimethoxybenzoic acid
(1 mmol) was mixed with PyBOP (1.4 mmol, 1.4 equiv.), 1-(2-
aminoethyl)pyrrolidine (1.8 mmol,1.8 equiv.) in 1:1 mixture of
CH2Cl2 and CH3CN at room temperature. After 2 h, the solvent
(40 ml) was eliminated by normal pressure distillation. Then 20 ml
of fresh CH2Cl2 and 2 ml of BBr3 were added at ꢁ78 ꢂC. After 4 h, 5 ml
of H2O were added to the mixture. After solvent evaporation, the
crude product was purified by column chromatography on silica gel.
-20
6.25 3.13 1.56 0.78 0.39 0.19 0.09 0.04
Concentration of 1m (µM)
Fig. 1. The inhibitory activity of 1k (A), 1l (B) and 1m (C) against A/Anhui/1/2005 H5N1
pseudovirus and VSV-G pseudovirus. The samples were tested in triplicate and the
data were presented in mean ꢀ SD. This experiment was repeated three times with
similar results.
4.1.1. 3,5-Bis(trifluoromethyl)-N-(2-(piperidin-1-yl)ethyl)
benzamide (1a)
The crude product was purified by column chromatography on
silica gel and crystallized from dichloromethaneepetroleum ether,
312 mg of 1a as white solid, 85%. Mp 180e182 ꢂC. CCDC:854218. 1H
found that the inhibitory activities of 1k and 1l can not be improved
by introduction of a eOH group at benzene moiety, such as
compound 1q. Compounds 1f and 1o had lower activities than
compounds 1e and 1n, which suggests that bulky group in R
moiety, could decrease the activity.
NMR (CDCl3, 400 MHz)
d
: 1.87e1.91 (m, 4H, J ¼ 16.0 Hz), 2.23e2.27
(m, 2H, J ¼ 16.0 Hz), 2.73e2.78 (m, 2H, J ¼ 20.0 Hz), 3.31e3.32 (d,
2H, J ¼ 4.0 Hz), 3.66e3.69 (d, 2H, J ¼ 12.0 Hz), 3.95e3.97 (d, 2H,
J ¼ 8.0 Hz), 7.97 (s, 1H), 8.66 (s, 2H), 9.60 (s, 1H);13C NMR (CDCl3,
100 MHz) d: 21.9, 22.5, 34.6, 54.2, 57.4, 121.7, 124.4, 125.1, 128.4,

214
Z. Zhu et al. / European Journal of Medicinal Chemistry 57 (2012) 211e216
O
O
O
O
O
N
PyBOP
N
H
N
OH
H2N
+
CH2Cl2-CH3CN
O
O
O
l 2
C
2
H
OH
OH
O
C
r 3
B
B
N
N
H
HO
1q
Scheme 1.
131.7, 132.0, 135.3, 165.0. The X-ray structure of 1a was showed in
supporting information (Fig. S1).
J ¼ 6.8 Hz), 5.48e5.52 (m, 1H, J ¼ 14.4 Hz), 6.51e6.53 (d, 1H,
J ¼ 7.6 Hz), 7.48e7.52 (m, 3H, J ¼ 16 Hz), 7.82e7.87 (m, 4H,
J ¼ 21.2 Hz), 7.99 (s, 1H), 7.82 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d:
4.1.2. 3,5-Bis(trifluoromethyl)-N-(2-(pyrrolidin-1-yl)ethyl)
benzamide (1b)
21.4, 50.0, 124.6, 125.0, 126.3, 126.5, 127.3, 127.7, 127.9, 128.9, 132.0,
132.4, 133.3, 136.6, 139.5, 163.7; anal. cal. for C21H15F6NO: C 61.32, H
3.68, N 3.41; found C 61.40, H 3.72, N 3.35.
318 mg of 1b as white solid, 90%. Mp 142e144 ꢂC. MS (ESI) m/z:
355.1 (M þ H)þ, 1H NMR (CDCl3, 400 MHz)
d: 2.13e2.25 (d, 4H,
J ¼ 46 Hz), 2.91 (s, 2H), 3.41 (s, 2H), 3.94 (s, 4H), 7.99 (s, 1H), 8.67 (s,
4.1.7. 3,5-Bis(trifluoromethyl)-N-(3-(4-methylpiperazin-1-yl)
propyl)benzamide (1g)
2H), 9.43 (s, 1H);13C NMR (CDCl3, 100 MHz)
121.7, 124.4125.2, 128.4, 128.5, 131.8, 132.1, 135.3, 165.0; anal. cal. for
14H16F4N2O: C 61.74, H 4.21, N 4.50; found C 61.90, H 4.25, N 4.45.
d: 23.3, 36.4, 54.5,55.8,
238 mg of 1g as white solid, 60%. Mp 140e142 ꢂC. MS (ESI) m/z:
C
398.3(M þ H)þ,1H NMR (D2O, 400 MHz)
d: 2.04e2.08 (m, 2H,
J ¼ 1.6 Hz), 2.96 (s, 3H), 3.30e3.31 (m, 2H, J ¼ 16 Hz), 3.45e3.48 (m,
4.1.3. 3,5-Bis(trifluoromethyl)-N-(2-(thiophen-2-yl)ethyl)
benzamide (1c)
2H, J ¼ 12 Hz), 3.64 (s, 8H), 8.19 (s, 1H), 8.22 (s, 2H), 13C NMR (D2O,
100 MHz) d: 23.6, 36.8, 42.8, 48.7, 54.6, 121.7, 124.4, 125.9, 127.8,
The crude product was purified by column chromatography on
silica gel and crystallized from the dichloromethaneepetroleum
ether. 294 mg of 1c as white solid, 85%. Mp 116e117 ꢂC.
131.1, 131.5, 135.2, 168.1; anal. cal. for C17H21F6N3O: C 51.38, H 5.33,
N 10.57; found C 51.58, H 5.23, N 10.51.
CCDC:826436. 1H NMR (CDCl3, 400 MHz)
d: 3.18e3.26 (m, 2H,
4.1.8. 3,5-Bis(trifluoromethyl)-N-(2-morpholinoethyl)benzamide
(1h)
J ¼ 32.0 Hz), 3.74e3.79 (m, 2H, J ¼ 20.0 Hz), 6.85e6.96 (m, 2H,
J ¼ 44.0 Hz), 6.97e6.98 (m, 1H, J ¼ 4.0 Hz), 7.19e7.20 (d, 1H,
260 mg of 1h as white solid, 70%. Mp 95e97 ꢂC. MS (ESI) m/z:
J ¼ 4.0 Hz), (s, 1H);13C NMR (CDCl3, 100 MHz)
d
: 29.6, 41.7, 121.5,
369.1(M ꢁ H)ꢁ,1H NMR (CDCl3, 400 MHz)
d: 2.54 (s, 4H), 2.64e2.67
(m, 2H, J ¼ 12.0 Hz), 3.58e3.62 (m, 2H, J ¼ 16.8 Hz), 3.73e3.75 (m,
124.2, 125.0, 125.1, 125.6, 127.2, 132.0, 136.6, 140.7, 164.8. The X-ray
structure of 1c was showed in supporting information (Fig. S2).
4H, J ¼ 8.8 Hz), 7.12 (s, 1H), 8.00 (s, 1H), 8.23 (s, 2H);13C NMR (CDCl3,
100 MHz) d: 36.4, 53.3, 55.7, 66.9, 121.6, 124.3, 124.9, 125.0, 127.3,
4.1.4. 3,5-Bis(trifluoromethyl)-N-((pyridine-3-yl)methyl)
benzamide (1d)
127.3, 132.0, 132.3, 136.7, 164.5; anal. cal. for C15H16F6N2O2: C 48.65,
H 4.36, N 7.57; found C 48.80, H 4.26, N 7.51.
296 mg of 1d as white solid, 85%. Mp 152e154 ꢂC. MS (ESI) m/z:
349.1(M þ H)þ,1H NMR (CDCl3, 400 MHz)
d
: 4.68e4.70 (d, 2H,
4.1.9. 3-Fluoro-5-(trifluoromethyl)-N-(2-morpholinoethyl)
benzamide (1i)
J ¼ 6.0 Hz), 7.00 (s, 1H), 7.30e7.33 (m, 1H, J ¼ 12.8 Hz), 7.74e7.77 (d,
1H, J ¼ 11.6 Hz), 8.02 (s, 1H), 8.29 (s, 2H), 8.54e8.55 (m, 2H,
224 mg of 1i as white solid, 70%. Mp 78e79 ꢂC. MS (ESI) m/z:
J ¼ 2.4 Hz);13C NMR (CDCl3, 100 MHz)
d
: 41.9, 123.9, 125.3, 127.4,
321.2(M þ H)þ,1H NMR (CDCl3, 400 MHz)
d: 2.53e2.55 (m, 4H,
127.5, 132.2, 132.5, 133.4, 136.0, 136.2, 149.3, 149.4, 164.6; anal. cal.
for C15H10F6N2O: C 51.73, H 2.89, N 8.04; found C 51.90, H 2.92, N 8.15.
J ¼ 8.4 Hz), 2.63e2.66 (m, 2H, J ¼ 12 Hz), 3.56e3.60 (m, 2H,
J ¼ 16.8 Hz), 3.74e3.76 (m, 4H, J ¼ 8.8 Hz), 6.90 (s, 1H), 7.47e7.49 (d,
1H, J ¼ 8.0 Hz), 7.68e7.71 (d, 1H, J ¼ 8.8 Hz), 7.83 (s, 1H), 13C NMR
4.1.5. 3,5-Bis(trifluoromethyl)-N-phenethylbenzamide (1e)
(CDCl3, 100 MHz) d: 36.3, 53.3, 56.7, 67.0, 115.5, 115.8, 115.8, 117.7,
314 mg of 1e as white solid, 87%. Mp 64e66 ꢂC. MS (ESI) m/z:
117.9, 119.5, 119.6, 138.0, 161.3, 163.7, 164.7; anal. cal. for
C14H16F4N2O2: C 52.50, H 5.04, N 8.75; found C 52.60, H 5.03, N 8.71.
360.1(M ꢁ H)ꢁ,1H NMR (CDCl3, 400 MHz)
d
: 4.68e4.70 (d, 2H,
J ¼ 6.0 Hz), 7.00 (s, 1H), 7.30e7.33 (m, 1H, J ¼ 12.8 Hz), 7.74e7.77 (d,
1H, J ¼ 11.6 Hz), 8.02 (s, 1H), 8.29 (s, 2H), 8.54e8.55 (m, 2H,
4.1.10. 3-Fluoro-5-(trifluoromethyl)-N-(3-(4-methylpiperazin-1-yl)
propyl)benzamide (1j)
J ¼ 2.4 Hz);13C NMR (CDCl3, 100 MHz)
d: 41.9, 123.9, 125.3, 127.4,
127.5, 132.2, 132.5, 133.4, 136.0, 136.2, 149.3, 149.4, 164.6; anal. cal.
for C17H13F6NO: C 51.73, H 2.89, N 8.04; found C 51.90, H 2.92, N 8.15.
261 mg of 1j as white solid, 75%. Mp 177e179 ꢂC. MS (ESI) m/z:
348.2 (M þ H)þ,1H NMR (D2O, 400 MHz)
d: 2.02e2.08 (m, 2H,
J ¼ 24 Hz), 2.96 (s, 3H), 3.29e3.31 (m, 2H, J ¼ 8.0 Hz), 3.42e3.46 (m,
4.1.6. 3,5-Bis(trifluoromethyl)-N-(1-(naphthalene-2-yl)ethyl)
benzamide (1f)
2H, J ¼ 16 Hz), 3.63 (s, 8H), 7.60e7.66 (m, 2H), 7.81 (s, 1H), 13C NMR
(D2O, 100 MHz), d: 23.6, 36.7, 42.8, 54.6, 116.1, 116.4, 117.9, 118.2,
175 mg of 1f as white solid, 84%. Mp 133e135 ꢂC. MS (ESI) m/z:
120.1, 136.4, 136.5, 160.9, 163.4, 168.1; anal. cal. for C16H21F4N3O: C
55.32, H 6.09, N 12.10; found C 55.42, H 6.13, N 12.05.
410.2(M ꢁ H)ꢁ,1H NMR (CDCl3, 400 MHz)
d: 1.73e1.75 (d, 3H,

Z. Zhu et al. / European Journal of Medicinal Chemistry 57 (2012) 211e216
215
4.1.11. 3-Fluoro-5-(trifluoromethyl)-N-(2-(pyrrolidin-1-yl)ethyl)
4.1.17. 2,4,5-Trihydroxy-N-(2-(pyrrolidin-1-yl)ethyl)benzamide
(1q)
benzamide (1k)
280 mg of 1k as white solid, 92%. Mp 163e164 ꢂC. MS (ESI) m/z:
133 mg of 1q as white solid, 50%. Mp 164e166 ꢂC. MS (ESI) m/z:
304.98 (M þ H)þ,1H NMR (CDCl3, 400 MHz)
d
: 2.19(s, 4H), 3.38e3.41
267.3(M þ H)þ,1H NMR (CDCl3, 400 MHz)
d: 0.45 (s, 2H), 1.50e1.51
(m, 4H, J ¼ 10.8 Hz), 3.90e3.94 (m, 4H, J ¼ 16.0 Hz), 7.42e7.44 (d,
1H, J ¼ 8.0 Hz), 8.09e8.11 (d, H, J ¼ 8.8 Hz), 8.21 (s, 1H), 9.19 (s, 1H),
(d, 2H, J ¼ 4.8 Hz), 1.62e1.66 (m, 2H, J ¼ 16.8 Hz), 2.59e2.62 (m, 2H,
J ¼ 11.6 Hz), 3.51 (s, 2H), 6.56 (s, 1H), 6.58e6.59 (d, 1H, J ¼ 2.8 Hz),
6.60e6.61 (d, 1H, J ¼ 2.4 Hz), 6.64e6.65 (d, 1H, J ¼ 2.4 Hz), 6.67e
6.68 (d, 1H, J ¼ 2.4 Hz), 7.39e7.43 (d, 1H, J ¼ 15.2 Hz), 13C NMR
13C NMR (CDCl3, 100 MHz)
d: 23.3, 36.4, 54.6, 55.8, 115.7, 118.4,
118.6, 120.9, 136.5, 136.6, 161.1, 165.3; anal. cal. for C14H16F4N2O: C
55.26, H 5.30, N 9.21; found C 55.89, H 5.37, N 9.25.
(CDCl3, 100 MHz) d: 24.2, 25.9, 35.7, 54.2, 56.5, 105.0, 105.2, 106.4,
106.6, 127.4, 127.6, 163.8, 163.9, 164.8, 167.3, 169.3; anal. cal. for
C13H18N2O4: C 58.63, H 6.81, N 10.52; found C 58.80, H 6.66, N 10.51.
4.1.12. 3-Fluoro-5-(trifluoromethyl)-N-(2-(thiophen-2-yl)ethyl)
benzamide (1l)
307 mg of 1l as white solid, 97%. Mp 86e88 ꢂC. MS (ESI) m/z:
4.1.18. 3-(Trifluoromethyl)-N-phenethylbenzamide (1r)
316.10 (M ꢁ H)ꢁ,1H NMR (CDCl3, 400 MHz)
d
: 3.16e3.20 (m, 2H,
205 mg of 1r as white solid, 70%. Mp 104e105 ꢂC. MS (ESI) m/z:
J ¼ 13.2 Hz), 3.73e3.77 (m, 2H, J ¼ 18.8 Hz), 6.88e6.89 (m, 1H,
J ¼ 3.2 Hz), 6.97e6.99 (m, 1H, J ¼ 8.8 Hz), 7.19e7.20 (d, H, J ¼ 1.2 Hz),
7.20e7.21 (d, 1H, J ¼ 1.2 Hz), 7.45e7.47 (d, 1H, J ¼ 8.0 Hz), 7.62e7.64
281.1(M þ H)þ,1H NMR (CDCl3, 400 MHz)
d: 2.96e3.00 (m, 2H,
J ¼ 13.6 Hz), 3.74e3.79 (m, 2H, J ¼ 19.2 Hz), 6.22 (s, 1H), 7.26 (s, 1H),
7.30 (s, 1H), 7.35e7.38 (m, 2H, J ¼ 14.4 Hz), 7.55e7.59 (m, 1H,
J ¼ 15.6 Hz), 7.75e7.77 (d, 1H, J ¼ 7.6 Hz), 7.87e7.89 (d, 1H,
(d, 1H, J ¼ 8.4 Hz), 7.75 (s, 1H);13C NMR (CDCl3, 100 MHz)
d: 29.7,
41.6, 117.7, 117.9, 119.4, 119.5, 124.3, 125.6, 127.3, 140.8, 163.7, 164.9;
anal. cal. for C14H11F4NOS: C 52.99, H 3.49, N 4.41, S 10.11; found C
52.89, H 3.47, N 4.45, S 10.18.
J ¼ 7.6 Hz), 7.97 (s, 1H), 13C NMR (CDCl3, 100 MHz)
d: 35.6, 41.3,
123.9, 126.8, 128.0, 128.8, 129.2, 130.1, 135.5, 138.7, 166.1; anal. cal.
for C15H12F3NO: C 64.51, H 4.33, N 5.02; found C 64.60, H 4.23,
N 5.01.
4.1.13. 3-Fluoro-5-(trifluoromethyl)-N-((pyridine-4-yl)methyl)
benzamide (1m)
4.1.19. N-cyclohexyl-3-(trifluoromethyl)benzamide (1s)
268 mg of 1m as white solid, 90%. Mp 99e101 ꢂC. MS (ESI) m/z:
203 mg of 1s as white solid, 75%. Mp 122e123 ꢂC. MS (ESI) m/z:
299.1(M þ H)þ,1H NMR (CDCl3, 400 MHz)
d
: 4.70e4.71 (d, 2H,
272.2 (M þ H)þ, 1H NMR (CDCl3, 400 MHz)
d: 1.20 (m, 2H,
J ¼ 6.0 Hz), 7.43e7.47 (m, 2H, J ¼ 16.8 Hz), 7.50e7.52 (d, 1H,
J ¼ 8.0 H), 7.78e7.80 (d, 1H, J ¼ 8.8 Hz), 7.87e7.94 (m, 2H,
J ¼ 26.4 Hz), 7.96e7.98 (d, 1H, J ¼ 8.0 Hz), 8.13 (s, 1H), 8.54e8.55 (d,
J ¼ 30.8 Hz), 1.39e1.47 (m, 2H, J ¼ 28.8 Hz), 1.58e1.66 (m, 2H,
J ¼ 31.2 Hz), 1.75e1.81 (m, 2H, J ¼ 20.8 Hz), 2.03e2.07 (m, 2H,
J ¼ 16 Hz), 3.97e4.01 (m, 1H, J ¼ 14.0 Hz), 5.97 (s, 1H), 7.55e7.59 (m,
1H, J ¼ 15.6 Hz), 7.74e7.76 (d, 1H, J ¼ 7.6 Hz), 7.93e7.95 (d, 1H,
1H, J ¼ 4.8 Hz), 8.77 (s, 1H);13C NMR (CDCl3, 100 MHz)
d: 41.5, 118.1,
119.8, 119.9, 124.7, 135.5, 138.9, 145.8, 146.7, 166.2; anal. cal. for
J ¼ 7.6 Hz), 7.99 (s, 1H), 13C NMR (CDCl3,100 MHz)
d: 24.9, 25.6, 33.2,
C
14H10F4N2O: C 56.38, H 3.38, N 9.39; found C 56.41, H 3.35, N 9.40.
49.1, 123.8, 127.9, 129.2, 130.2, 130.9, 135.9, 165.2; anal. cal. for
C14H16F3NO: C 61.98, H 5.94, N 5.16; found C 61.88, H 5.78, N 5.31.
4.1.14. 3-Fluoro-5-(trifluoromethyl)-N-phenethylbenzamide (1n)
288 mg of 1n as white solid, 95%. Mp 163e164 ꢂC. MS (ESI) m/z:
4.1.20. 3-(Trifluoromethyl)-N-(2-(thiophen-2-yl)ethyl)benzamide
(1t)
312.1 (M þ H)þ,1H NMR (CDCl3, 400 MHz)
d: 2.93e2.97 (m, 2H,
J ¼ 16 Hz), 3.70e3.75 (m, 2H, J ¼ 20 Hz), 6.18 (s, 1H), 7.22 (s, 1H),
7.24 (s, 1H), 7.28 (s, 1H), 7.32e7.36 (m, 2H, J ¼ 14.4 Hz), 7.43e7.45 (d,
1H, J ¼ 8.0 Hz), 7.58e7.61 (d, 1H, J ¼ 8.4 Hz), 7.70 (s, 1H); 13C NMR
195 mg of 1t as white solid, 65%. Mp 62e64 ꢂC. MS (ESI) m/z:
322.7(M þ Na), 1H NMR (CDCl3, 400 MHz)
d: 3.16e3.20 (m, 2H,
J ¼ 12.8 Hz), 3.73e3.78 (m, 2H, J ¼ 18.8 Hz), 6.29 (s, 1H), 6.88e6.89
(m, 1H, J ¼ 3.2 Hz), 6.97e6.99 (m, 1H, J ¼ 8.4 Hz), 7.19e7.20 (m, 1H,
J ¼ 6.4 Hz), 7.54e7.58 (m, 1H, J ¼ 15.6 Hz), 7.74e7.76 (d, 1H,
J ¼ 7.6 Hz), 7.88e7.90 (d, 1H, J ¼ 8.0 Hz), 7.99 (s, 1H), 13C NMR
(CDCl3, 100 MHz) d: 35.5, 41.4, 115.5, 115.6, 115.8, 117.7, 117.9, 119.4,
119.5, 128.8, 128.9, 138.0, 138.5, 161.2, 163.7, 164.8; anal. cal. for
14H16F4N2O: C 61.74, H 4.21, N 4.50; found C 61.90, H 4.25, N 4.45.
C
(CDCl3, 100 MHz) d: 29.8, 41.5, 123.9, 124.0, 124.2, 125.6, 127.2, 128.1,
4.1.15. 3-Fluoro-5-(trifluoromethyl)-N-(1-(naphthalene-2-yl)ethyl)
benzamide (1o)
129.2, 130.1, 135.4, 141.0, 166.0; anal. cal. for C13H12F3NOS: C 56.18,
H 4.04, N 4.68, S 10.71; found C 56.32, H 4.13, N 4.61, S 10.61.
343 mg of 1o as white solid, 95%. Mp 183e184 ꢂC. MS (ESI) m/z:
362.4 (M þ H)þ,1H NMR (CDCl3, 400 MHz)
d
: 1.60e1.62 (d, 3H,
4.1.21. 3-(Trifluoromethyl)-N-(2-(piperidin-1-yl)ethyl)
benzensulfonamide (2)
J ¼ 6.8 Hz), 4.56e4.58 (m, 1H, J ¼ 6.8 Hz), 7.55e7.58 (m, 2H,
J ¼ 8.8 Hz), 7.67e7.69 (d, 1H, J ¼ 8.4 Hz), 7.88e7.90 (d, 1H,
J ¼ 4.8 Hz), 7.91e7.96 (m, 2H, J ¼ 18.8 Hz), 7.98 (s, 1H), 8.00 (s, 2H),
238 mg of 3 as white solid, 71%. Mp 171e172 ꢂC. MS (ESI) m/z:
337.0 (M þ H)þ, 1H NMR (CDCl3, 400 MHz)
d: 1.85e1.96 (m, 4H,
8.57 (s, 2H); 13C NMR (CDCl3, 100 MHz)
d: 20.6, 50.1, 124.5, 125.7,
J ¼ 44 Hz), 2.20e2.26 (m, 2H, J ¼ 24 Hz), 2.75e2.82 (m, 2H,
J ¼ 28 Hz), 3.29 (s, 2H), 3.46(s, 2H), 3.62e3.65 (d, 2H, J ¼ 12 Hz),
7.69e7.71 (m, 1H, J ¼ 8.0 Hz), 7.83 (d, 1H), 8.12 (s, 1H), 8.19e8.23 (m,
126.5, 126.6,127.6, 127.8, 128.4, 132.6, 136.7; anal. cal. for
20H15F4NO: C 66.48, H 4.18, N 3.88; found C 66.70, H 4.25, N 3.65.
C
2H, J ¼ 16 Hz), 13C NMR (CDCl3, 100 MHz)
d: 21.8, 22.6, 54.3, 57.5,
4.1.16. 2-(3-Fluoro-5-(trifluoromethyl)benzamido)ethyl 3-fluoro-5-
(trifluoromethyl) benzoate (1p)
121.9, 124.1, 124.1, 124.6, 129.4, 130.6, 140.9; anal. cal. for
C14H19F3N2O2S: C 49.99, H 5.69, N 8.33,S 9.53; found C 49.86,
344 mg of 1p as white solid,78%. Mp 193e184 ꢂC. MS (ESI) m/z:
H 5.63, N 8.31,S 9.43.
464.6(M þ Na),1H NMR (CDCl3, 400 MHz)
d: 3.88e3.90 (m, 2H,
J ¼ 5.2 Hz), 4.60e4.61 (m, 2H, J ¼ 4.8 Hz), 6.87 (s, 1H), 7.44e7.46 (d,
1H, J ¼ 7.2 Hz), 7.52e7.54 (d, 1H, J ¼ 7.2 Hz), 7.66e7.68 (d, 1H,
4.1.22. 2-(Piperidin-1-yl)ethyl 3-fluoro-5-(trifluoromethyl)
benzoate (3)
J ¼ 7.6 Hz), 7.79(s, 1H), 7.89e7.91 (d, 1H, J ¼ 8.0 Hz), 8.09 (s, 1H); 13
C
207 mg of 2 as white solid, 65%. Mp 179e181 ꢂC. MS (ESI) m/z:
NMR (CDCl3, 100 MHz)
d
: 39.9, 64.4, 105.0, 115.8, 117.5, 117.7, 118.0,
320.0 (M þ H)þ, 1H NMR (CDCl3, 400 MHz)
d: 1.89e2.05 (m, 4H,
119.5, 120.0, 120.20, 122.3, 132.8, 137.3, 161.7, 164.6, 165.1; anal. cal.
for C18H11F8NO3: C 48.99, H 2.51, N 3.17; found C 48.90, H 2.54,
N 3.15.
J ¼ 40 Hz), 2.29e2.32 (d, 2H, J ¼ 12.0 Hz), 2.77e2.85 (m, 2H,
J ¼ 32 Hz), 3.44e3.45 (d, 2H, J ¼ 4.0 Hz), 3.66e3.69 (d, 2H,
J ¼ 12 Hz), 4.99 (s, 2H), 7.57e7.58 (d, 1H, J ¼ 4.0 Hz), 7.96e7.9 8(d,

216
Z. Zhu et al. / European Journal of Medicinal Chemistry 57 (2012) 211e216
1H, J ¼ 8.0 Hz), 8.09 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d
: 21.8, 22.6,
and the NA plasmid from the N1 subtype strain A/Thailand/Kan353/
2004. Briefly, 293T cells (60e70% confluent) were co-transfected
with 2 mg HA plasmid, 2 mg NA plasmid and 3 mg HIV backbone
53.9, 55.5, 59.9, 117.8, 120.2, 120.4, 122.4, 132.2, 161.2, 163.5, 163.7;
anal. cal. for C15H17F4NO2: C 56.42, H 5.37, N 4.39; found C 56.32, H
5.23, N 4.31.
plasmid (pNL4-3.luc.RꢁEꢁ) into six-well plate using the calcium
phosphate precipitation method [10]. Forty-eight hours after
transfection, the culture supernatants were harvested and centri-
fuged at 2000 rpm for 5 min. Aliquots were stored at ꢁ70 ꢂC. The
amount of pseudotyped particles was quantitated using the HIV-1
p24 ELISA kit from Retro-Tek (Buffalo, NY).
4.1.23. 5-Chloro-N-(2-(piperidin-1-yl)ethyl)pyridine-3-
carboxamide (4a)
192 mg of 4a as white solid, 72%. Mp 102e103 ꢂC. MS (ESI) m/z:
268.9 (M þ H)þ, 1H NMR (DMSO-d6, 400 MHz)
d: 1.44 (s, 2H), 1.56e
1.57 (m, 4H, J ¼ 4.4 Hz), 2.41 (s, 4H), 2.53e2.54 (d, 2H, J ¼ 4.8 Hz),
For measuring the inhibitory activities of test compounds,
MDCK cells (1 ꢃ104/well) were seeded in 96-well plates and grown
overnight. Tested compounds at indicated concentrations were
incubated with pseudotyped particles (1 ng p24/well) for 30 min at
37 ꢂC. Subsequently, the virusecompound mixture was transferred
to the cells and incubated for an additional 48 h. Cells were washed
with phosphate buffer saline (PBS) and lysed with the lysing
reagent included in the luciferase kit (Promega, Madison, WI).
Aliquots of cell lysates were transferred to 96-well flat bottom
luminometer plates (Costar), followed by the addition of luciferase
substrate (Promega). The luciferase activity was measured in
a microplate luminometer. As a negative control, VSV-G pseudo-
typed particles were incubated with the tested compound instead
of H5N1 pseudovirus.
3.49e3.50 (d, 2H, J ¼ 4.4 Hz), 7.28 (s, 2H), 8.11 (s, 1H), 8.62e8.64 (d,
1H, J ¼ 5.2 Hz), 8.81(s, 1H), 13C NMR (DMSO-d6, 100 MHz)
d: 24.3,
26.0, 36.6, 54.2, 56.7, 131.4, 135.0, 145.5, 150.9, 164.0; anal. cal. for
C
13H18ClN3O: C 58.31, H 6.78, N 15.69; found C 58.32, H 6.73, N
15.61.
4.1.24. 5-Bromo-N-(2-(piperidin-1-yl)ethyl)pyridine-3-
carboxamide (4b)
218 mg of 4b as white solid, 70%. Mp 96e97 ꢂC. MS(ESI) m/z:
312.8, 314.8 (M þ H)þ, 1H NMR (CDCl3, 400 MHz)
d: 1.44 (s, 2H),
1.57e1.59 (d, 4H, J ¼ 5.2 Hz), 2.41 (s, 4H), 2.53e2.56 (m, 2H,
J ¼ 12 Hz), 3.49e3.53 (m, 2H, J ¼ 16 Hz), 7.28 (s, 2H), 8.23e7.27 (m,
1H, J ¼ 8.4 Hz), 8.71e8.75 (d, 1H, J ¼ 12.4 Hz), 8.85 (s, 1H), 13C NMR
(CDCl3, 100 MHz) d: 24.3, 26.0, 36.6, 54.2, 56.7, 56.7, 121.0, 131.8,
137.9, 145.8, 145.9, 153.0, 153.1, 164.0; anal. cal. for C13H18BrN3O: C
50.01, H 5.81, N 13.46; found C 50.12, H 5.76, N 13.61.
Acknowledgments
This work was supported by the Natural Science Foundation of
China (30772602, 81102792), the Guangdong Pearl River Scholar
Funded Scheme, and the foundation from Key Laboratory of
Prevention and Control of Emerging Infectious Diseases of Guang-
dong Higher Education Institutes (KLB09007).
4.1.25. 5-Chloro-N-(2-(thiophen-2-yl)ethyl)pyridine-3-
carboxamide (4c)
200 mg of 4c as white solid, 75%. Mp 124e125 ꢂC. MS (ESI) m/z:
267.9 (M þ H)þ, 1H NMR (DMSO-d6, 400 MHz)
d: 3.09e3.12 (m, 2H,
J ¼ 14.4 Hz), 3.55e3.56 (m, 2H, J ¼ 5.6 Hz), 6.94e6.98 (m, 2H,
J ¼ 15.2 Hz), 7.34e7.36 (d, 1H, J ¼ 5.2 Hz), 8.28e8.29 (m, 1H,
J ¼ 4.0 Hz), 8.78e8.79 (d, 1H, J ¼ 2.0 Hz), 8.96e8.97 (d, 2H,
Appendix A. Supporting information
J ¼ 1.6 Hz), 13C NMR (DMSO-d6, 100 MHz)
d: 29.0, 41.0, 124.1, 125.3,
Supporting information related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.08.041.
126.9, 131.0, 131.1, 134.5, 141.2, 146.7, 150.3, 163.4; anal. cal. for
C
12H11ClN2OS: C 54.03, H 4.16, N 10.50,S 12.02; found C 54.12, H
4.13, N 10.61, S 12.08.
References
4.1.26. 5-Bromo-N-(2-(thiophen-2-yl)ethyl)pyridine-3-
carboxamide (4d)
[1] H.P. Hsieh, J.T. Hsu, Curr. Pharm. Des. 13 (2007) 3531e3542.
[2] K.E. Doucette, F.Y. Aoki, Expert Opin. Pharmacother. 2 (2001) 1671e1683.
[3] R.K. Gupta, J.S. Nguyen-Van-Tam, N. Engl. J. Med. 355 (2006) 1419e1431.
[4] Q.M. Le, M. Kiso, K. Someya, Y.T. Sakai, T.H. Nguyen, K.H. Nguyen, N.D. Pham,
H.H. Ngyen, S. Yamada, Y. Muramoto, T. Horimoto, A. Takada, H. Goto,
T. Suzuki, Y. Suzuki, Y. Kawaoka, Nature 437 (2005) 1108.
239 mg of 4d as white solid, 77%. Mp 114e115 ꢂC. MS (ESI) m/z:
312.2 (M þ H)þ, 1H NMR (CDCl3, 400 MHz)
d: 3.13e3.16 (m, 2H,
J ¼ 13.2 Hz), 3.68e3.73 (m, 2H, J ¼ 19.2 Hz), 6.84e6.85 (d, 2H,
J ¼ 3.2 Hz), 6.93e6.95 (m, 1H, J ¼ 8.4 Hz), 7.03 (s, 1H), 7.15e7.16 (d,
1H, J ¼ 5.2 Hz), 8.20 (s, 1H), 8.68e8.69 (d, 1H, J ¼ 2.0 Hz), 8.77e8.78
[5] A. Moscona, N. Engl. J. Med. 360 (2009) 953.
[6] J. Skehel, Biologicals 37 (2009) 177e178.
(d, 1H, J ¼ 1.6 Hz), 13C NMR (CDCl3, 400 MHz)
d: 29.7, 41.6, 121.0,
[7] J. Yoshimoto, M. Kakui, H. Iwasaki, H. Sugimoto, T. Fujiwara, N. Hattori,
Microbiol. Immunol. 44 (2000) 677e685.
[8] G. Luo, A. Torri, W.E. Harte, S. Danetz, C. Cianci, L. Tiley, S. Day, D. Mullaney,
K.L. Yu, C. Ouellet, P. Dextraze, N. Meanwell, R. Colonno, M. Krystal, J. Virol. 71
(1997) 4062e4070.
124.2, 125.6, 127.2, 131.6, 137.9, 140.8, 145.9, 153.2, 164.4; anal. cal.
for C12H11BrN2OS: C 46.31, H 3.56, N 9.00,S 10.30; found C 46.22, H
3.58, N 9.02, S 10.28.
[9] S.J. Plotch, B. O’Hara, J. Morin, O. Palant, J. LaRocque, J.D. Bloom Jr.,
S.A. Lang, M.J. DiGrandi, M. Bradley, R. Nilakantan, Y. Gluzman, J. Virol. 73
(1999) 140e151.
[10] S.W. Liu, R.M. Li, R.T. Zhang, C. Chan, B.M. Xi, Z.B. Zhu, J. Yang, K.M.P. Vincent,
J. Zhou, M. Chen, M. Jan, K. Frank, P. Stephan, H. Thomas, D. Ursula, P. Pan,
L. Du, S.B. Jiang, B. Zheng, Eur. J. Pharmacol. 660 (2011) 460e467.
[11] L. Maton, D. Taziaux, J.P. Soumillion, J.H. Jiwan, J. Mater. Chem. 15 (2005)
2928e2937.
[12] C.A.G.N. Montalbetti, V. Falque, Tetrahedron 61 (2005) 10827e10852.
[13] W. Wang, J.S. McMurray, Tetrahedron Lett. 40 (1999) 2501e2504.
[14] I. Ryu, H. Matsubara, S. Yasuda, H. Nakamura, D.P. Curran, J. Am. Chem. Soc.
124 (2002) 12946e12947.
4.2. Measurement of the inhibitory activity against H5N1
pseudovirus
MDCK cells and 293T cells were obtained from the American
Type Culture Collection (ATCC). Cells were grown in Dulbecco’s
Modified Eagle Medium (DMEM, Gibco) containing glutamine,
supplemented with 10% fetal calf serum (FCS).
The H5N1 pseudoviruses were prepared by transfecting HA
plasmid from the H5 subtype strain A/Anhui/1/2005(H5N1) strain
[15] A.K. Chakraborti, L.A. Sharma, M.K. Nayak, J. Org. Chem. 67 (2002) 6407e6414.