Design and synthesis of heteroaromatic-based benzenesulfonamide derivatives as potent inhibitors of H5N1 influenza A virus

Article abstract of DOI:10.1039/c8md00474a

Influenza A virus is an enveloped negative single-stranded RNA virus that causes febrile respiratory infection and represents a clinically challenging threat to human health and even lives worldwide. Even more alarming is the emergence of highly pathogenic avian influenza (HPAI) strains such as H5N1, which possess much higher mortality rate (60%) than seasonal influenza strains in human infection. In this study, a novel series of heteroaromatic-based benzenesulfonamide derivatives were identified as M2 proton channel inhibitors. A systematic investigation of the structure-activity relationships and a molecular docking study demonstrated that the sulfonamide moiety and 2,5-dimethyl-substituted thiophene as the core structure played significant roles in the anti-influenza activity. Among the derivatives, compound 11k exhibited excellent antiviral activity against H5N1 virus with an EC₅₀ value of 0.47 μM and selectivity index of 119.9, which are comparable to those of the reference drug amantadine.

Full text of DOI:10.1039/c8md00474a

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RESEARCH ARTICLE
Design and synthesis of heteroaromatic-based
benzenesulfonamide derivatives as potent
inhibitors of H5N1 influenza A virus†
ab
Cite this: DOI: 10.1039/c8md00474a
Yongshi Yu,
‡
Tazeem,‡bc Zhichao Xu,b Liaoqi Du,a Mengyu Jin,b
b
a
Chune Dong, b Hai-Bing Zhou
and Shuwen Wu
*
*
Influenza A virus is an enveloped negative single-stranded RNA virus that causes febrile respiratory infection
and represents a clinically challenging threat to human health and even lives worldwide. Even more
alarming is the emergence of highly pathogenic avian influenza (HPAI) strains such as H5N1, which possess
much higher mortality rate (60%) than seasonal influenza strains in human infection. In this study, a novel
series of heteroaromatic-based benzenesulfonamide derivatives were identified as M2 proton channel in-
hibitors. A systematic investigation of the structure–activity relationships and a molecular docking study
demonstrated that the sulfonamide moiety and 2,5-dimethyl-substituted thiophene as the core structure
played significant roles in the anti-influenza activity. Among the derivatives, compound 11k exhibited excel-
lent antiviral activity against H5N1 virus with an EC50 value of 0.47 μM and selectivity index of 119.9, which
are comparable to those of the reference drug amantadine.
Received 23rd September 2018,
Accepted 23rd November 2018
DOI: 10.1039/c8md00474a
rsc.li/medchemcomm
Influenza is a common and serious infectious respiratory
disease caused by influenza viruses belonging to the
Orthomyxoviridae family.1 Despite decades of surveillance and
interventions, seasonal influenza viruses continue to cause
epidemics around the world each year. According to the
World Health Organization, seasonal influenza epidemics
result in 3–5 million flu patients and 290 000 to 650 000
deaths worldwide each year.2 Due to its higher antigenic
mutation, influenza A (flu A) virus is a greater threat to
human health compared to flu B and C strains. Five flu A
pandemics have occurred in the past 100 years: 1918 H1N1
(Spanish flu), 1957 H2N2 (Asian flu), 1968 H3N2 (Hong Kong
flu), 2005 H5N1 (Bird flu) and 2009 H1N1 (Swine flu).3–8
Among them, the 1918 pandemic was the most severe,
leading to a catastrophic 50 million deaths globally.3,9–11
Worrisome, highly pathogenic avian influenza (HPAI) viruses
have recently emerged, including H5N1 (ref. 12–15) and
H7N9.16,17 These viruses possess higher mortality compared
to seasonal influenza viruses.18 HPAI H5N1 flu strains have
been shown to be transmissible among humans after
acquiring only one or few mutations, generating
a
considerable concern among researchers.14,19,20 Although
vaccines and antiviral agents are essential measures for
influenza treatment, anti-influenza agents seem to be more
effective at preventing highly contagious influenza infections
due to the frequent variation of influenza virus through anti-
genic drift and antigenic shift.8 Currently, two classes of anti-
influenza drugs are applied for the prophylaxis and treatment
of influenza infection (Fig. 1): M2 proton channel inhibitors
(amantadine and rimantadine) and neuraminidase inhibitors
(oseltamivir, zanamivir and peramivir). However, the drug re-
sistance of H5N1 viral strains limits the usable clinical range
a State Key Laboratory of Virology, College of Life Sciences, Wuhan University,
Wuhan 430072, China. E-mail: shuwenwu@hotmail.com
b Hubei Province Key Laboratory of Allergy and Immunology, Hubei Province
Engineering and Technology Research Center for Fluorinated Pharmaceuticals,
Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China.
E-mail: zhouhb@whu.edu.cn
c Department of Chemistry, Shia P. G. College (University of Lucknow),
LucknowUttar Pradesh 226020, India
† Electronic supplementary information (ESI) available: NMR spectra of 11–12.
See DOI: 10.1039/c8md00474a
‡ These two authors contributed equally to this work.
Fig. 1 Approved anti-influenza drugs for influenza treatment.
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of these drugs, making the identification of novel anti-
influenza agents imperative.21–24
carboxamide derivatives by shortening the linker length and
transforming the heteroaromatic core structure (Scheme 1).
As a consequence, some small molecules that inhibit in-
fluenza A H5N1 virus have been created through the struc- Results and discussion
tural modification of approved anti-influenza drugs or using
the hit-and-lead discovery strategy (Fig. 2).
Chemistry
The synthesis of the designed heteroaromatic-based benzene-
In 2010, based on high-throughput screening, Severson
and coworkers reported quinazolinone analogues as novel in-
hibitors; compound 1 exhibited an excellent EC50 value of
0.032 μM for both H5N1 and H1N1 viruses.25 In 2011, Liu
and co-workers found that 3-fluoro-N-(2-(piperidin-1-yl)ethyl)-
5-(trifluoromethyl)benzamide hydrochloride (2, Fig. 2) could
inhibit HPAI H5N1 virus infection by blocking viral entry
with an IC50 value of 27.03 μM.26 More recently, polyphenol
(3, Fig. 2) was identified as entry inhibitor against H5N1 virus
with an EC50 value of 1.78 μM, superior to that of the refer-
ence drug amantadine.27 In 2017, Liu's group discovered a se-
ries of oligo-thiophene compounds as HA inhibitors that act
by inhibiting the membrane fusion between the H5N1 virus
and the endosome of the host cell. The most potent inhibitor
(4, Fig. 2) displayed good anti-H5N1 activity with an IC50
value of 0.029 μM.28 In the same year, our group developed
furan–carboxamide derivatives as novel inhibitors of H5N1
influenza A virus. The most potent compound (5, Fig. 2)
showed low-micromolar activity against H5N1 virus with an
EC50 value of 1.25 μM, which was comparable with reference
drug amantadine.29 The systematic investigation of the struc-
ture–activity relationships (SARs) of furan–carboxamide ana-
logues indicated that heteroaromatic groups (e.g., furan and
thiophene moieties) and sulfur atoms played significant roles
in the inhibition of H5N1 virus. Moreover, furan–carboxa-
mide derivatives containing para-substituents on the phenyl
rings showed more potent anti-H5N1 activity than those with
meta- or ortho-substituents on the phenyl rings. A molecular
docking investigation of this series of furan–carboxamide an-
alogues indicated that they cannot efficiently interact with
the M2 protein cavity due to the long linker. Our long-term
aim is the development of antivirals with better inhibitory po-
tency and fewer side effects than currently used drugs;29–33
hence, to develop novel heteroaromatic-based benzene-
sulfonamide compounds as anti-influenza A/H5N1 agents, an
attempt was made to modify the structural features of furan–
sulfon-amide derivatives is shown in Scheme 2. All designed
compounds were synthesized via the sulfonylation reaction of
substituted benzenesulfonyl chloride analogues with diversi-
fied amines. Most amines were formed starting from corre-
sponding carboxylic acid 6, as shown in Scheme 2. First,
heteroaromatic formic acid 6 reacted with thionyl chloride to
produce aromatic formyl chloride (7). Formamide 8 was then
formed via the amination reaction of 7 and ammonium hy-
droxide in the presence of dichloromethane (DCM). Next, the
key amine 9 was synthesized through reduction reaction in
the presence of lithium aluminum hydride followed by the
sulfonylation reaction to generate the final products (11–12)
in 20%–60% yields.
Structure–activity relationship (SAR) studies
All synthesized novel heteroaromatic-based arylsulfonamide
compounds were tested for anti-influenza activity against
both amantadine-sensitive H5N1 virus and amantadine-
resistant H5N1 virus using plaque reduction assays in MDCK
cells. The reference anti-influenza drugs amantadine (AMD)
and oseltamivir phosphate were used for comparison. The re-
sults of this extensive biological evaluation are described in
Tables 1 and 2. As a global observation, the heteroaromatics
as core structures played significant roles in the activity
against amantadine-sensitive H5N1 virus. Originally, the
designed heteroaromatic-based benzenesulfonamide com-
pound 11a containing furan as a core structure and 4-NO2 as
the R substituent showed good potency against H5N1 virus
(EC50 = 1.06 μM; Table 1, entry 1). To explore the effects of R
substituents on the anti-influenza activity, compounds 11b–e
were synthesized by introducing electron-withdrawing groups
(4-CN, 4-F, 4-Cl and 4-Br) (Table 1, entries 2–5).
Fig. 2 Structures of known small-molecule inhibitors of H5N1 influ-
Scheme 1 Strategy for designing heteroaromatic-based benzene-
enza A virus.
sulfonamide derivatives as novel inhibitors of H5N1 influenza A virus.
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potent analogue 11k exhibited an EC50 value of 0.47 μM,
comparable with those of the reference drugs amantadine
and oseltamivir phosphate (Table 1, entries 11 vs. 15 and 16).
We also explored the influence of other heteroaromatic core
structures on the anti-influenza activity in the presence of
4-NO2 as the R substituent. We found that analogue 11f
showed dramatically decreased inhibition potency when a fu-
ran moiety was the core structure (Table 1, entries 1 vs. 6).
Moreover, introducing a thiophene or 2-chloro-thiophene
moiety as the core structure also resulted in slightly de-
creased anti-influenza activity (Table 1, entries 1 and 12–14).
Meanwhile, we tested the antiviral activity against
amantadine-resistant H5N1 virus in MDCK cells. Unfortu-
nately, these benzenesulfonamide analogues showed no inhi-
bition potency (Table 1, entries 1–14).
Scheme 2 Synthetic route for heteroaromatic-based benzenesulfon-
amide derivatives. Reagents and conditions: (a) SOCl2, toluene, reflux;
(b) NH4OH, DCM, rt; (c) LiAlH4, THF, reflux; (d) Et3N, DCM, rt.
Unfortunately, analogues 11b–e all displayed decreased in-
hibitory potency. Considering that sulfur and oxygen atoms
have similar chemical properties, bioisostere analogues
11g–k were synthesized (Table 1, entries 7–11). Surprisingly,
we found that all compounds displayed superior anti-H5N1
activity with the exception of compound 11i (containing 4-F),
which showed no inhibition potency. Among 11g–k, the most
Next, we further investigated the SAR when using a benzo-
thiophene moiety as the core structure. Initially, we synthe-
sized compound 12a with a C3-substituted benzothiophene
moiety as the core structure and 4-NO2 as the R substituent.
Interestingly, 12a maintained potency against the
amantadine-sensitive H5N1 virus when the benzothiophene
Table 1 Anti-H5N1 activities of furan- or thiophene-based benzenesulfonamide derivatives 11a–n in MDCK cellsa
Core
structure
EC50 (μM)b AMD-sensitive EC50 (μM)c AMD-resistant
Entry Cmpd
R
H5N1
H5N1
CC50 (μM)d
SIe
1
2
3
4
5
6
11a
11b
11c
11d
11e
11f
4-NO2 1.06 0.26
NA
NA
NA
NA
NA
NA
>152.10 40.60 >143.5
4-CN
4-F
4-Cl
4-Br
4.03 1.62
8.08 1.84
1.37 0.27
1.80 0.52
105.39 22.04
138.01 36.00
55.71 16.34
29.63 10.75
>236.30
26.1
17.1
40.7
16.5
>5.0
4-NO2 47.19 17.25
7
8
9
10
11
12
11g
11h
11i
11j
11k
11l
4-NO2 1.29 0.43
NA
NA
NA
NA
NA
NA
>204.36
>217.69
—
52.88 14.56
56.35 14.43
138.77 28.83
>158.4
>351.1
—
75.5
119.9
94.4
4-CN
4-F
0.62 0.098
NAf
g
h
4-Cl
4-Br
0.70 0.16
0.47 0.11
4-NO2 1.47 0.77
13
14
11m
11n
4-NO2 1.91 0.94
NA
NA
133.41 27.82
127.72 28.85
69.8
1.8
4-NO2 70.05 25.87
15
16
AMD
0.43 0.13
0.39 0.10
NA
0.34 0.02
>100.00
>163.26
>235.3
>418.6
Oseltamivir phosphate
a
b
All data were obtained from at least three independent experiments. EC50, concentration that effectively inhibited amantadine-sensitive
c
H5N1 virus plaque formation by 50%. EC50, concentration that effectively inhibited amantadine-resistant H5N1 virus plaque formation by
50%. CC50, concentration that inhibited cell growth by 50% compared with control cultures. Selectivity index (SI) was determined for effec-
tive compounds by dividing CC50 by EC50 (amantadine-sensitive H5N1 virus). NA: no activity (EC50 > 100 μM). —: no detection. —: no se-
d
e
f
g
h
lectivity index.
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Table 2 Anti-H5N1 activities of benzothiophene-based benzenesulfonamide derivatives 12a–s in MDCK cellsa
EC50b (μM) AMD-sensitive EC50c (μM) AMD-resistant
Entry Cmpd
Core structure
R
H5N1
H5N1
CC50d (μM)
>191.45
SIe
1
2
3
12a
12b
12c
4-NO2
4-CN
4-F
1.38 0.29
20.28 8.34
3.42 0.81
1.98 0.68
7.22 1.60
38.56 14.73
10.49 3.78
NAf
NA
NA
NA
NA
NA
NA
NA
NA
>138.7
122.41 32.59 6.0
98.64 29.25
69.56 15.39
87.11 21.71
94.78 14.27
>210.13
28.8
4
5
6
7
12d
12e
12f
12g
4-Cl
4-Br
4-CF3
4-Me
2-F
35.1
12.1
2.5
>20.0
g
h
8
12h
—
—
9
12i
3-F
NA
NA
—
—
10
11
12
13
14
15
16
17
18
19
20
21
12j
12k
12l
12m
12n
12o
12p
12q
4-COCH3 12.04 3.85
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.34 0.02
>193.09
>191.45
—
—
—
>174.47
>179.60
>210.13
—
—
>100.00
>163.26
>16.0
>3.8
—
—
—
>4.3
>4.0
>5.0
—
—
>235.3
>418.6
4-NO2
4-CN
4-F
49.97 14.87
NA
NA
4-Cl
4-Br
4-CF3
4-Me
2-F
NA
40.70 12.11
44.83 16.51
41.96 13.74
NA
12r
12s
AMD
3-F
NA
0.43 0.13
0.39 0.10
Oseltamivir phosphate
a
b
All data were obtained from at least three independent experiments. EC50, concentration that effectively inhibited amantadine-sensitive
c
H5N1 virus plaque formation by 50%. EC50, concentration that effectively inhibited amantadine-resistant H5N1 virus plaque formation by
50%. d CC50, concentration that inhibited cell growth by 50% compared with control cultures. Selectivity index (SI) was determined for the ef-
e
f
g
h
fective compounds by dividing CC50 by EC50 (amantadine-sensitive H5N1 virus). NA: no activity (EC50 > 100 μM). —: no detection. —: no
selectivity index.
moiety was the core structure, indicating that our hypothesis
was rational (Table 1, entries 7, 12–13 vs. Table 2, entry 1).
To further explore the influence of the R substituent on the
antiviral activity, we designed and synthesized compounds
12b–g and 12j containing electron-withdrawing groups (4-CN,
4-F, 4-Cl, 4-Br, 4-CF3 and 4-COCH3) and an electron-donating
group (4-Me), respectively (Table 2, entries 2–7 and 10). All
compounds displayed inferior activity against amantadine-
sensitive H5N1 virus compared to compound 12a; in particu-
lar, the potency of analogue 12f was approximately 28 times
lower than that of 12a, likely due to the stronger electron-
activity (Table 2, entry 3 vs. 8–9 and entry 13 vs. 18–19), which
is consistent with our prediction. Among benzothiophene-
based benzenesulfonamide derivatives, the C3-substituted
compound 12a displayed potent antiviral activity against
amantadine-sensitive H5N1 virus with an EC50 of 1.38 μM
(Table 2, entry 1). Next, we evaluated the activity against
amantadine-resistant H5N1 virus in MDCK cells. Unfortu-
nately, these benzothiophene-based benzene–sulfonamide de-
rivatives displayed no potency (Table 2, entries 1–19).
Fig. 3 intuitively shows that the 2,5-dimethyl-substituted
thiophene moiety was a more potent core structure than the
2,5-dimethyl-substituted furan moiety or benzo-thiophene
moiety in terms of anti-influenza activity against amantadine-
sensitive H5N1 virus (Fig. 3A and B). Furthermore, the para-
substituent was superior to the meta- or ortho-substituent as
an R group in terms of activity.
The anti-proliferative activities of the synthesized com-
pounds against MDCK cells were also evaluated to monitor
potential cytotoxic effects. As expected, none of the
heteroaromatic-based benzenesulfonamide derivatives obvi-
ously inhibited the growth of MDCK cells at a concentra-
tion of 50 μM, with the exception of compound 11e, con-
solidating the safety of this series of compounds (Tables 1
and 2).
withdrawing
property
of
the
4-CF3
group
(Table 2, entries 1 vs. 6). Meanwhile, analogues 12k–q were
synthesized to study the effect of the C2-substituted benzo-
thiophene moiety as a core structure on the biological activity.
These analogues all exhibited over 30-fold decreased potency
compared to the corresponding analogues 12a–g containing
C3-substituted benzothiophene moiety as the core structure
(Table 2, entries 1–7 and 11–17). We also synthesized ana-
logues 12h–i and 12r–s to study the anti-H5N1 activity of ana-
logues containing ortho- and meta-substituents as R groups.
The biological results indicated no inhibitory activity against
amantadine-sensitive H5N1 virus, showing that
a para-
substituent as the R group is important for the anti-influenza
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Fig. 3 Graphical representation of the effects of the core structure and R substituent of heteroaromatic-based benzenesulfonamide compounds
on activity against amantadine-sensitive H5N1 virus in MDCK cells. A) The effects of 2,5-dimethylfuran or 2,5-dimethylthiophene moiety as core
structure and R substituent of compounds 11a–e and 11g–k on antiviral activity; B) the effects of benzothiophene as core structure and R substitu-
ent of compounds 12a–g and 12k–q on antiviral activity.
Preliminary mechanistic studies based on a time-course
experiment with compound 11k and reference drugs amanta-
dine and favipiravir (T-705) demonstrated that the novel
heteroaromatic-based benzenesulfonamide derivatives prevented
early-stage virus replication (Fig. 4). Similar to the M2 proton
channel inhibitor amantadine, compound 11k likely blocked
channel function to inhibit the uncoating of the viral RNPs.34
To further explore the possible drug target of this series of
novel heteroaromatic-based benzenesulfonamide derivatives,
we performed a yeast growth restoration assay.35–38 The M2
proton channel inhibitor amantadine and neuraminidase
(NA) inhibitor oseltamivir were included as reference drugs.
The yeast toxicities of the heteroaromatic-based benzene-
sulfonamide derivatives were also evaluated to eliminate the
contribution of toxic effects. As shown in Table 3, this series
of compounds exhibited good potency against WT M2 and
displayed little yeast toxicity in the yeast growth restoration as-
say, which was almost consistent with the anti-H5N1 activity re-
sults in MDCK cells (Table 3 vs. Tables 1 and 2). Moreover, com-
pounds 11a, 11h, 11j and 11k showed excellent activity against
WT M2 in comparison to amantadine (Table 3, entries 1, 8, 10–
11 and 34). In contrast, the NA inhibitor oseltamivir showed no
inhibitory potency against WT M2 (Table 3, entry 35). Thus, the
heteroaromatic-based
benzenesulfonamide
analogues
inhibited WT M2 expression in yeast similarly to the M2 inhibi-
tor amantadine, demonstrating that M2 protein was the drug
target of this series of compounds. Unfortunately, these novel
heteroaromatic-based
benzenesulfonamide
derivatives
displayed no potency against mutant S31N/V27A M2 (Table 3).
To further investigate the structure–binding relationships
of these heteroaromatic-based benzenesulfonamide deriva-
tives, we conducted a docking study to explore the binding
mode of 11k with the M2 protein of the H5N1 virus.
Docking experiments were carried out based on the pub-
lished M2 protein (PDB: 2KQT)39 with amantadine.
AutoDockTool 4.2 (ref. 40) was used to study the binding
modes of 11k, and the results indicated a good binding
score (−5.87 kcal mol−1). As shown in Fig. 5A–C, the 2,5-
dimethyl-thiophene moiety of compound 11k enters into
the M2 protein cavity formed by residues Ala-30, Ser-31, Ile-
33, Gly-34 and Ile-35, demonstrating that the 2,5-dimethyl-
thiophene moiety played a significant role in the anti-
influenza activity. Moreover, one oxygen atom of the sulfon-
amide moiety formed a hydrogen bond with the backbone
residue His-37, and the nitrogen atom of the sulfonamide
moiety formed another hydrogen bond with residue Gly-34,
verifying that the sulfonamide moiety is necessary in this
series of compounds (Fig. 5B–D).
Fig. 4 Results of the time-course experiment. Compound 11k (c =
0.68 μg mL−1), AMD (amantadine,
c = ) and T-705
0.26 μg mL−1
(favipiravir, c = 0.63 μg mL−1) were added to MDCK cells that had been
infected with H5N1 virus at an MOI of 2 at the indicated time.
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Table 3 Growth restoration in yeast expressing WT and mutant M2 by heteroaromatic-based benzenesulfonamide derivatives
Entry
Cmpd
EC50a (μM) WT M2
EC50 (μM) V27A/S31N M2
Yeast toxicityc
1
2
3
4
5
6
7
8
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
1.71 0.64
12.74 5.51
24.00 7.06
3.07 1.00
4.65 0.87
81.48 21.25
2.21 0.92
1.40 0.33
NA
1.08 0.32
1.58 0.56
2.38 0.67
2.01 0.67
NA
3.44 1.15
45.67 15.22
6.22 2.18
5.33 1.77
15.25 5.26
NA
22.68 6.30
NA
NA
27.79 11.58
NA
NA
NA
NA
NA
NA
NAb
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
>215.9
168.8 34.4
>236.5
123.43 16.68
72.6 11.6
>237.4
>205.3
>218.7
9
120.2 26.7
114.1 22.2
133.2 19.4
>224.6
157.5 20.1
>201.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
>192.3
>204.0
133.8 24.9
121.4 20.7
134.1 23.7
>180.4
>211.08
59.1 12.4
>208.5
>194.0
>192.3
>204.0
>208.5
>198.3
>176.2
>180.4
>211.08
>180.5
>180.5
>443.0
>214.5
NA
NA
NA
0.66 0.20
NA
12s
Amantadine
Oseltamivir phosphate
a
b
EC50: concentration that effectively inhibited the expression of M2 protein by 50%. NA: denotes inactive at the highest concentration tested.
Yeast toxicity: concentration that inhibited the proliferation of the control yeast strain by 50%.
c
Conclusions
thin layer chromatography. Column chromatography purifi-
cation was performed using 230–400-mesh silica gel. NMR
spectra were measured on Bruker DRX and DMX spectrome-
In summary, we have designed and synthesized a novel series of
heteroaromatic-based benzenesulfonamide derivatives as M2 pro-
ton channel inhibitors. Several of the synthesized compounds
show sub-micromolar activity against amantadine-sensitive H5N1
virus and low cytotoxicity in MDCK cells. The SAR investigation
and molecular docking results indicated that the 2,5-
dimethylthiophene core structure and sulfonamide moiety
played key roles in the inhibition activity. Among the synthesized
compounds, 11k is the most effective derivative with an EC50
value of 0.47 μM and selectivity index of 119.98, which are com-
parable with those of reference drugs amantadine and oseltamivir
phosphate. A further mechanistic investigation of these potent
M2 inhibitors is underway and will be reported in due course.
1
ters at 400 MHz for H spectra and 100 MHz for 13C spectra.
Spectra were calibrated using the residual solvent signal. All
1
final products were characterized by H NMR, 13C NMR and
MS analyses.
Representative procedure for the synthesis of 2,5-
dimethylfuran-3-carboxamide 8a
Thionyl chloride (0.14 mL, 1.872 mmol) was added to a solu-
tion of 2,5-dimethylfuran-3-carboxylic acid 6a (174.8 mg, 1.248
mmol) in toluene (5 mL), and the mixture was heated under
reflux for 2 h. After the reaction was completed, the solution
was concentrated. Without further purification, the crude
product 2,5-dimethylfuran-3-carbonyl chloride (7a) was imme-
diately used in the next step. Ammonium hydroxide (25%
aqueous solution, 1 mL) was then added dropwise to a solu-
tion of intermediate 7a in anhydrous dichloromethane (5
mL) at 0 °C. The mixture was stirred at room temperature for
Experimental section
Materials and methods
Reagents and materials were obtained from commercial sup-
pliers unless otherwise noted. Reactions were monitored by
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Fig. 5 (A) The solid surface type between 11k with M2 protein (oxygen atoms are shown in red, sulfur atoms are shown in yellowish orange, and
nitrogen atoms are shown in blue). (B) The transparent surface type between 11k with M2 protein (same as above). (C) The hydrophobic
interactions and hydrogen bond interactions formed between 11k with the backbone residues of M2 protein. (D) Computer modeling of the
complex structures of M2 protein with 11k based on the published structure (PDB: 2KQT) of M2 protein with amantadine.
1 h. The mixture was then extracted with ethyl acetate (3 × 30
mL) and brine, dried over anhydrous sodium sulfate, and
concentrated by vacuum pump. The residue was purified by
flash column chromatography and eluted with petroleum
ether/ethyl acetate (2 : 1) to produce the 2,5-dimethylfuran-3-
carboxamide (8a) as a white solid.
addition of 4-nitrobenzene-sulfonyl chloride 10a (275.0 mg,
1.241 mmol). The mixture was stirred at room temperature for
5 h. After concentration under vacuum pump, the residue was
purified by flash column chromatography and eluted with pe-
troleum ether/ethyl acetate (10: 1) to produce N-((2,5-dimethyl-
furan-3-yl)methyl)-4-nitrobenzenesulfonamide 11a in 40% yield
1
as a yellow solid: mp 127–129 °C, H NMR (400 MHz, CDCl3) δ
8.35–8.30 (m, 2H), 8.01–7.96 (m, 2H), 5.61 (s, 1H), 4.95 (t, J =
5.4 Hz, 1H), 3.95 (d, J = 5.6 Hz, 2H), 2.10 (s, 6H). 13C NMR
(100 MHz, CDCl3) δ150.48, 149.92, 147.64, 146.18, 128.29,
124.15, 114.42, 106.18, 38.59, 13.23, 11.34. HRMS (ESI) calcd
for C13H14N2O5S [M + Na]+ 333.0521, found 333.0520.
Representative procedure for the synthesis of N-((2,5-
dimethylfuran-3-yl)methyl)-4-nitrobenzenesulfonamide 11a
Lithium aluminum hydride (128.4 mg, 3.384 mmol) was added
to a solution of 8a (157.0 mg, 1.128 mmol) in anhydrous THF
(5 mL) at 0 °C. The solution was warmed to ambient tempera-
ture and heated under reflux for 4 h. After completion, the re-
action was quenched by the sequential addition of 0.13 mL
H2O, 0.13 mL1 5% NaOH solution, and 0.39 mL H2O. Ethyl ac-
etate was then added to the above mixture followed by stirring
at room temperature for 30 min. Next, the filtrate was dried
over anhydrous sodium sulfate and concentrated by vacuum
pump. Without further purification, the crude (2,5-
dimethylfuran-3-yl)methanamine 9a was directly used in the
next step. Triethylamine (0.19 mL, 1.354 mmol) was added to
a solution of the key amine intermediate 9a (141.2 mg, 1.128
mmol) in anhydrous dichloromethane at 0 °C followed by the
4 - C y a n o - N - ( ( 2 , 5 - d i m e t h y l f u r a n - 3 - y l ) m e t h y l ) -
benzenesulfonamide 11b. 51% yield as a white solid, mp
1
115–117 °C, H NMR (400 MHz, CDCl3) δ 7.95–7.88 (m, 2H),
7.81–7.75 (m, 2H), 5.60 (s, 1H), 4.92 (t, J = 5.5 Hz, 1H), 3.93
(d, J = 5.6 Hz, 2H), 2.14 (s, 3H), 2.11 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 150.41, 147.59, 144.60, 132.74, 127.67, 117.38,
116.13, 114.45, 106.21, 38.53, 13.28, 11.34. HRMS (ESI) calcd
for C14H14N2O3S [M + Na]+ 313.0623, found 313.0617.
N - ( ( 2 , 5 - D i m e t h y l f u r a n - 3 - y l ) m e t h y l ) - 4 - f l u o r o -
benzenesulfonamide 11c. 41% yield as brown solid, mp 81–
1
83 °C. H NMR (400 MHz, CDCl3) δ 7.92–7.73 (m, 2H), 7.27–
7.14 (m, 2H), 5.64 (s, 1H), 4.86 (t, J = 5.3 Hz, 1H), 3.87 (d, J =
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5.6 Hz, 2H), 2.13 (s, 3H), 2.07 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 166.28, 163.75, 150.21, 147.43, 136.18, 129.87,
116.26, 116.03, 114.70, 106.37, 38.41, 13.26, 11.28. HRMS
(ESI) calcd for C13H14FNO3S [M + Na]+ 306.0576, found
306.0573.
4-Chloro-N-((2,5-dimethylfuran-3-yl)methyl)benzenesulfon-
amide 11d. 48% yield as grey solid, mp 85–87 °C. 1H NMR
(400 MHz, CDCl3) δ 7.75 (d, J = 8.6 Hz, 2H), 7.45 (d, J = 8.7
Hz, 2H), 5.62 (s, 1H), 4.81 (t, J = 5.5 Hz, 1H), 3.86 (d, J = 5.6
Hz, 2H), 2.12 (s, 3H), 2.08 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 150.27, 147.46, 139.03, 138.66, 129.22, 128.57, 114.64,
106.33, 38.45, 13.27, 11.31. HRMS (ESI) calcd for
C13H14ClNO3S [M + Na]+ 322.0281, found 322.0277.
4-Bromo-N-((2,5-dimethylfuran-3-yl)methyl)benzenesulfon-
amide 11e. 51% yield as a brown solid, mp 103–105 °C. 1H
NMR (400 MHz, CDCl3) δ 7.68 (d, J = 8.6 Hz, 2H), 7.62 (d, J =
8.6 Hz, 2H), 5.62 (s, 1H), 4.83 (t, J = 5.5 Hz, 1H), 3.87 (d, J =
5.7 HZ, 2H), 2.13 (s, 3H), 2.09 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 150.28, 147.46, 139.20, 132.21, 128.65, 127.48,
114.62, 106.32, 38.45, 13.29, 11.32. HRMS (ESI) calcd for
C13H14BrNO3S [M + Na]+ 365.9775, found 365.9773.
100 °C. H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.6 Hz, 2H),
7.43 (d, J = 8.6 Hz, 2H), 6.29 (s, 1H), 4.97 (t, J = 5.6 Hz, 1H),
3.97 (d, J = 5.7 Hz, 2H), 2.27 (s, 3H), 2.21 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 138.97, 138.60, 136.38, 134.31, 131.04,
129.20, 128.48, 126.01, 40.23, 14.94, 12.63. HRMS (ESI) calcd
for C13H14ClNO2S2 [M + Na]+ 338.0052, found 338.0048.
N-[(2,5-Dimethylthiophen-3-yl)methyl]-4-bromo-
benzenesulfon-amide 11k. 53% yield as a white solid, mp
1
119–121 °C. H NMR (400 MHz, CDCl3) δ 7.72–7.54 (m, 4H),
6.29 (s, 1H), 4.71 (t, J = 5.4 Hz, 1H), 3.98 (d, J = 5.6 Hz, 2H),
2.29 (s, 3H), 2.22 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
139.15, 136.48, 134.38, 132.21, 130.96, 128.58, 127.47, 125.94,
40.26, 14.97, 12.64. HRMS (ESI) calcd for C13H14BrNO2S2 [M +
Na]+ 381.9547, found 381.9543.
4-Nitro-N-(thiophen-3-ylmethyl)benzenesulfonamide 11l.
52% yield as a white solid, mp 131–133 °C. 1H NMR (400
MHz, CDCl3) δ 8.32 (d, J = 5.3 Hz, 2H), 7.99 (d, J = 4.9 Hz,
2H), 7.23 (d, J = 2.0 Hz, 1H), 7.09 (s, 1H), 6.87 (d, J = 3.7 Hz,
1H), 5.12 (t, J = 5.9 Hz, 1H), 4.28 (d, J = 5.7 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 149.99, 146.05, 136.43, 128.26, 127.00,
126.87, 124.29, 123.46, 42.48. HRMS (ESI) calcd for
C11H10N2O4S2 [M + Na]+ 320.9980, found 320.9975.
4-Nitro-N-(thiophen-2-ylmethyl)benzenesulfonamide 11 m.
36% yield as a white solid, mp 154–156 °C. 1H NMR (400
MHz, CDCl3) δ 8.32 (d, J = 8.8 Hz, 2H), 8.00 (d, J = 8.9 Hz,
2H), 7.20–7.13 (m, 2H), 6.91–6.82 (m, 2H), 5.09 (t, J = 5.6 Hz,
1H), 4.47 (d, J = 5.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ
150.00, 146.05, 138.20, 128.34, 127.14, 127.00, 126.38, 124.32,
N-(Furan-3-ylmethyl)-4-nitrobenzenesulfonamide 11f. 28%
1
yield as a white solid, mp 120–122 °C. H NMR (400 MHz, Ac-
etone-d6) δ 8.41 (d, J = 8.8 Hz, 2H), 8.15–8.05 (m, 2H), 7.46–
7.36 (m, 2H), 7.21 (s, 1H), 6.32 (s, 1H), 4.12 (d, J = 6.1 Hz,
2H). 13C NMR (100 MHz, Acetone-d6) δ 149.97, 146.89,
143.51, 140.55, 128.40, 124.23, 121.46, 110.11, 37.92. HRMS
(ESI) calcd for C11H10N2O5S [M + Na]+ 305.0208, found
305.0205.
42.17. HRMS (ESI) calcd for C11H10N2O4S2 [M
320.9980, found 320.9980.
+
Na]+
N-[(2,5-Dimethylthiophen-3-yl)methyl]-4-nitro-
benzenesulfon-amide 11g. 41% yield as a yellow solid, mp
N - [ ( 5 - C h l o r o t h i o p h e n - 2 - y l ) m e t h y l ] - 4 - n i t r o -
benzenesulfonamide 11n. 60% yield as a white solid, mp
118–120 °C. 1H NMR (400 MHz, Acetone-d6) δ 8.48–8.29 (m,
2H), 8.10 (dd, J = 8.8, 2.1 Hz, 2H), 7.64–7.45 (m, 1H), 7.31
(dd, J = 5.1, 1.1 Hz, 1H), 6.89 (m, 1H), 6.80 (s, 1H), 4.46 (d, J
= 6.1 Hz, 1H), 4.41 (d, J = 6.2 Hz, 1H). 13C NMR (100 MHz, Ac-
etone-d6) δ 146.93, 139.87, 128.40, 128.35, 126.63, 126.33,
126.07, 125.73, 124.29, 41.68. HRMS (ESI) calcd for
C11H10N2O4S2 [M + Na]+ 320.9980, found 320.9980.
N - ( 1 - B e n z o t h i o p h e n - 3 - y l m e t h y l ) - 4 - n i t r o -
benzenesulfonamide 12a. 35% yield as a white solid, mp
114–116 °C. 1H NMR (400 MHz, Acetone-d6) δ 8.32–8.28 (m,
2H), 8.12–8.06 (m, 2H), 7.80 (d, J = 7.2 Hz, 1H), 7.70 (d, J =
7.1 Hz, 1H), 7.64 (s, 1H), 7.34–7.25 (m, 2H), 7.20 (d, J = 0.6
Hz, 1H), 4.56 (d, J = 5.8 Hz, 2H). 13C NMR (100 MHz, Ace-
tone-d6) δ 146.44, 140.31, 137.56, 131.50, 128.03, 125.65,
124.07, 123.76, 122.51, 121.87, 40.86. HRMS (ESI) calcd for
C15H12N2O4S2 [M + Na]+ 371.0136, found 371.0131.
N - ( 1 - B e n z o t h i o p h e n - 3 - y l m e t h y l ) - 4 - c y a n o -
benzenesulfonamide 12b. 28% yield, semi-solid. 1H NMR
(400 MHz, Acetone-d6) δ 7.94 (d, J = 8.5 Hz, 2H), 7.89–7.83
(m, 4H), 7.49 (s, 1H), 7.39–7.35 (m, 3H), 4.50 (d, J = 6.1 Hz,
2H). 13C NMR (100 MHz, Acetone-d6) δ 144.96, 140.38,
137.66, 132.69, 131.55, 127.45, 125.55, 124.54, 124.11, 122.64,
121.92, 117.39, 115.33, 40.91. HRMS (ESI) calcd for
C16H12N2O2S2 [M + Na]+ 351.0238, found 351.0234.
1
134–136 °C. H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 8.7 Hz,
2H), 7.93 (d, J = 8.7 Hz, 2H), 6.28 (s, 1H), 5.18 (t, J = 5.4 Hz,
1H), 4.06 (d, J = 5.6 Hz, 2H), 2.23 (d, J = 2.2 Hz, 6H). 13C
NMR (100 MHz, CDCl3) δ149.83, 146.08, 136.62, 134.62,
130.77, 128.16, 125.91, 124.07, 40.30, 14.89, 12.65. HRMS
(ESI) calcd for C13H14N2O4S2 [M + Na]+ 349.0293, found
349.0285.
N-[(2,5-Dimethylthiophen-3-yl)methyl]-4-cyano-
benzenesulfon-amide 11h. 60% yield as a white solid, mp
1
108–110 °C. H NMR (400 MHz, CDCl3) δ 7.90–7.80 (m, 2H),
7.75–7.68 (m, 2H), 6.26 (s, 1H), 5.39 (t, J = 5.7 Hz, 1H), 4.02
(d, J = 5.7 Hz, 2H), 2.25 (s, 3H), 2.21 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 144.54, 136.65, 134.61, 132.68, 130.72, 127.56,
125.84, 117.37, 116.08, 40.30, 14.94, 12.65. HRMS (ESI) calcd
for C14H14N2O2S2 [M + Na]+ 329.0394, found 329.0389.
N-[(2,5-Dimethylthiophen-3-yl)methyl]-4-fluoro-
benzenesulfon-amide 11i. 43% yield as a grey solid, mp 87–
1
89 °C. H NMR (400 MHz, CDCl3) δ 7.92–7.73 (m, 2H), 7.22–
7.09 (m, 2H), 6.32 (s, 1H), 4.76 (t, J = 5.5 Hz, 1H), 3.98 (d, J =
5.7 Hz, 2H), 2.29 (s, 3H), 2.21 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 66.27, 163.74, 136.42, 136.09, 134.32, 131.05, 129.82,
125.99, 116.26, 116.04, 40.24, 14.94, 12.60. HRMS (ESI) calcd
for C13H14FNO2S2 [M + Na]+ 322.0348, found 322.0344.
N-[(2,5-Dimethylthiophen-3-yl)methyl]-4-chloro-
benzenesulfon-amide 11j. 41% yield as a grey solid, mp 98–
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N - ( 1 - B e n z o t h i o p h e n - 3 - y l m e t h y l ) - 4 - f l u o r o -
benzenesulfonamide 12c. 36% yield, semi-solid. 1H NMR
(400 MHz, CDCl3) δ 7.88–7.75 (m, 3H), 7.70–7.63 (m, 1 H),
7.36–7.34 (m, 2H), 7.24 (s, 1H), 7.11–7.07 (m, 2H), 4.97 (t, J =
5.5 Hz, 1H), 4.39 (d, J = 5.6 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 166.27, 163.74, 140.53, 137.21, 130.34, 129.81,
129.72, 125.40, 124.81, 124.43, 122.92, 121.56, 116.34, 116.11,
106 °C. H NMR (400 MHz, CDCl3) δ 7.81–7.76 (m, 1H), 7.68–
7.64 (m, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.50 (d, J = 8.1 Hz, 1H),
7.37–7.32 (m, 3H), 7.22–7.18 (m, 2H), 5.15 (s, 1H), 4.39 (d, J =
5.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 163.65, 161.15,
141.85, 139.88, 139.41, 139.15, 130.98, 124.68, 123.59, 123.30,
122.91, 122.36, 120.16, 114.69, 42.97. HRMS (ESI) calcd for
C15H12FNO2S2 [M + Na]+ 344.0191, found 344.0190.
41.36. HRMS (ESI) calcd for C15H12FNO2S2 [M
344.0191, found 344.0187.
+
Na]+
4 - A c e t y l - N - ( 1 - b e n z o t h i o p h e n - 3 - y l m e t h y l ) -
benzenesulfonamide 12j. 35% yield as a white solid, mp 174–
1
N - ( 1 - B e n z o t h i o p h e n - 3 - y l m e t h y l ) - 4 - c h l o r o -
benzenesulfonamide 12d. 29% yield as a yellow solid, mp
176 °C. H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 8.3 Hz, 2H),
7.87 (d, J = 8.3 Hz, 2H), 7.79–7.77 (m, 1H), 7.68–7.64 (m, 1H),
7.35–7.32 (m, 2H), 7.24 (s, 1H), 5.10 (t, J = 5.5 Hz, 1H), 4.43
(d, J = 5.8 Hz, 2H), 2.63 (s, 2H). 13C NMR (100 MHz, CDCl3) δ
196.83, 143.51, 140.52, 139.79, 137.15, 130.24, 128.76, 127.27,
125.62, 124.81, 124.44, 122.87, 121.52, 41.42, 26.93. HRMS
(ESI) calcd for C17H15NO3S2 [M + Na]+ 368.0391, found
368.0388.
N - ( 1 - B e n z o t h i o p h e n - 2 - y l m e t h y l ) - 4 - n i t r o -
benzenesulfonamide 12k. 52% yield as a white solid, mp
165–166 °C. H NMR (400 MHz, Acetone-d6) δ 8.08 (d, J = 8.2
Hz, 2H), 7.86 (d, J = 8.3 Hz, 2H), 7.81 (d, J = 7.2 Hz, 1H), 7.72
(dd, J = 6.7, 2.0 Hz, 1H), 7.52 (m, 1H), 7.32–7.27 (m, 2H), 7.21
(s, 1H), 4.54 (d, J = 6.0 Hz, 2H). 13C NMR (100 MHz, Acetone-
d6) δ 141.87, 140.78, 140.32, 130.60, 129.29, 125.33, 124.98,
124.34, 124.15, 123.07, 43.40. HRMS (ESI) calcd for
C15H12N2O4S2 [M + Na]+ 371.0136, found 371.0136.
1
130–132 °C. H NMR (400 MHz, CDCl3) δ 7.74–7.71 (m, 1H),
7.66–7.62 (m, 2H), 7.60–7.56 (m, 1H), 7.32–7.30 (m, 1H),
7.29–7.26 (m, 3H), 7.16 (s, 1H), 4.91 (t, J = 5.8 Hz, 1H), 4.32
(d, J = 5.3 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 140.50,
139.15, 138.07, 137.14, 130.23, 129.24, 128.44, 125.46, 124.80,
124.41, 122.92, 121.50, 41.36. HRMS (ESI) calcd for
C15H12ClNO2S2 [M + Na]+ 359.9896, found 359.9891.
N-(1-Benzothiophen-3-ylmethyl)-4-brormobenzene-
sulfonamide 12e. 28% yield as a yellow solid, mp 138–140 °C.
1H NMR (400 MHz, CDCl3) δ 7.81–7.79 (m, 1H), 7.68–7.61 (m,
3H), 7.53 (d, J = 8.6 Hz, 2H), 7.37–7.33 (m, 2H), 7.23 (s, 1H),
5.00 (t, J = 5.7 Hz, 1H), 4.39 (d, J = 5.8 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 140.53, 138.64, 137.17, 132.21, 130.28, 128.51,
127.63, 125.51, 124.81, 124.43, 122.95, 121.53, 41.36. HRMS
(ESI) calcd for C15H12BrNO2S2 [M + Na]+ 403.9391, found
403.9388.
N-(1-Benzothiophen-3-ylmethyl)-4-(trifluoromethyl)-
benzene-sulfonamide 12f. 36% yield, semi-solid. 1H NMR
(400 MHz, CDCl3) δ 7.84 (d, J = 8.2 Hz, 2H), 7.80–7.70 (m,
1H), 7.67–7.55 (m, 3H), 7.36–7.30 (m, 2H), 7.25 (d, J = 7.4 Hz,
1H), 5.18 (t, J = 5.5 Hz, 1H), 4.44 (d, J = 5.7 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 143.16, 140.48, 137.08, 130.09, 127.38,
125.94, 125.91, 125.82, 124.83, 124.42, 122.93, 121.41, 41.39.
HRMS (ESI) calcd for C16H12F3NO2S2 [M + Na]+ 394.0159,
found 394.0157.
N - ( 1 - B e n z o t h i o p h e n - 3 - y l m e t h y l ) - 4 - m e t h y l -
benzenesulfonamide 12g. 29% yield as a white solid, mp
158–160 °C. 1H NMR (400 MHz, Acetone-d6) δ 7.82–7.78 (m,
1H), 7.73 (d, J = 8.2 Hz, 2H), 7.68–7.66 (m, 1H), 7.35–7.32 (m,
2H), 7.26–7.21 (m, 4H), 4.93 (t, J = 5.9 Hz, 1H), 4.35 (d, J = 6.0
Hz, 2H), 2.41 (s, 3H). 13C (100 MHz, Acetone-d6) δ 143.64,
140.54, 137.32, 136.48, 130.64, 129.74, 127.18, 125.11, 124.73,
124.38, 122.86, 121.70, 41.40, 21.58. HRMS (ESI) calcd for
C16H15NO2S2 [M + Na]+ 340.0442, found 340.0438.
1
N - ( 1 - B e n z o t h i o p h e n - 2 - y l m e t h y l ) - 4 - c y a n o -
benzenesulfonamide 12l. 42% yield as a white solid, mp 144–
1
146 °C. H NMR (400 MHz, Acetone-d6) δ 8.04 (d, J = 8.1 Hz,
2H), 7.91 (d, J = 8.7 Hz, 2H), 7.83 (d, J = 7.1 Hz, 1H), 7.73 (d,
J = 7.0 Hz, 1H), 7.59 (s, 1H), 7.38–7.29 (m, 2H), 7.21 (s, 1H),
4.55 (d, J = 5.6 Hz, 2H). 13C NMR (100 MHz, Acetone-d6) δ
146.11, 142.02, 140.79, 140.36, 133.80, 128.58, 125.34, 125.31,
124.35, 124.01, 123.09, 118.23, 116.50, 43.38. HRMS (ESI)
calcd for C16H12N2O2S2 [M + Na]+ 351.0238, found 351.0233.
N - ( 1 - B e n z o t h i o p h e n - 2 - y l m e t h y l ) - 4 - f l u o r o -
benzenesulfonamide 12m. 50% yield as a white solid, mp
1
149–151 °C. H NMR (400 MHz, CDCl3) δ 7.80–7.75 (m, 2H),
7.75–7.71 (m, 1H), 7.67–7.63 (m, 1H), 7.43–7.37 (m, 2H),
7.36–7.27 (m, 2H), 7.07 (s, 1H), 5.11 (t, J = 6.0 Hz, 1H), 4.44
(d, J = 6.0 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 166.42,
139.87, 139.55, 139.16, 129.97, 129.37, 128.60, 124.69, 124.59,
123.56, 123.23, 122.36, 116.45, 116.22, 42.91. HRMS (ESI)
calcd for C15H12FNO2S2 [M + Na]+ 344.0191, found 344.0187.
N - ( 1 - B e n z o t h i o p h e n - 2 - y l m e t h y l ) - 4 - c h l o r o -
benzenesulfonamide 12n. 46% yield as white solid, mp 171–
N - ( 1 - B e n z o t h i o p h e n - 3 - y l m e t h y l ) - 2 - f l u o r o -
benzenesulfonamide 12h. 33% yield as a grey solid, mp 197–
199 °C. 1H NMR (400 MHz, CDCl3) δ 7.82–7.77 (m, 1H), 7.70–7.65
(m, 1H), 7.61–7.58 (m, 1H), 7.54–7.50 (m, 1H), 7.45–7.39 (m, 1H),
7.37–7.31 (m, 2H), 7.25–7.19 (m, 2H), 4.99 (t, J = 5.7 Hz, 1H),
4.45–4.39 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 163.60, 161.10,
141.73, 140.54, 137.20, 130.89, 130.29, 125.49, 124.82, 124.45,
122.90, 121.52, 120.02, 114.58, 41.40. HRMS (ESI) calcd for
C15H12FNO2S2 [M + Na]+ 344.0191, found 344.0188.
1
173 °C. H NMR (400 MHz, CDCl3) δ 7.88–7.83 (m, 1H), 7.78–
7.72 (m, 2H), 7.68–7.61 (m, 1H), 7.39 (d, J = 8.6 Hz, 1H),
7.35–7.27 (m, 2H), 7.12–7.06 (m, 2H), 5.19–5.08 (m, 1H), 4.44
(d, J = 6.0 Hz, 2H). 13C NMR (400 MHz, CDCl3) δ 139.87,
139.60, 139.17, 129.95, 129.86, 129.36, 128.59, 124.67, 124.57,
123.56, 123.22, 122.35, 116.43, 116.21, 42.89. HRMS (ESI)
calcd for C15H12ClNO2S2 [M + Na]+ 359.9896, found 359.9892.
N - ( 1 - B e n z o t h i o p h e n - 2 - y l m e t h y l ) - 4 - b r o m o -
benzenesulfonamide 12o. 53% yield as a white solid, mp
194–196 °C. 1H NMR (400 MHz, Acetone-d6) δ 7.86–7.79 (m,
N - ( 1 - B e n z o t h i o p h e n - 3 - y l m e t h y l ) - 3 - f l u o r o -
benzenesulfonamide 12i. 27% yield as white solid, mp 104–
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3H), 7.73–7.71 (m, 3H), 7.37–7.28 (m, 3H), 7.22 (s, 1H), 4.47
(d, J = 5.6, 2H). 13C NMR (100 MHz, Acetone-d6) δ 142.36,
141.26, 140.79, 140.46, 133.08, 129.75, 127.43, 125.25, 125.23,
124.31, 123.68, 123.10, 43.41. HRMS (ESI) calcd for
C15H12BrNO2S2 [M + Na]+ 403.9391, found 403.9388.
MDCK (Madin–Darby canine kidney) cells were purchased
from the American Type Culture Collection. A confluent
monolayer of MDCK cells was prepared in six-well plates. The
cells were infected with amantadine-sensitive H5N1 virus [A/
chicken/Hubei/327/2004 (H5N1)] and amantadine-resistant
H5N1 virus at 100 PFU per well in DMEM supplemented with
0.001% DEAE-dextran, 2 mg mL−1 TPCK-treated trypsin, and
0.5% agarose in the presence of various concentrations of
test compounds for 40 min at 37 °C. Medium containing
0.001% DEAE-dextran, 2 mg mL−1 TPCK-treated trypsin, 0.5%
agarose and test compounds at the same concentrations was
then added. After 2–3 days of incubation at 37 °C in a CO2 in-
cubator, cell monolayers were fixed with 3% formaldehyde
and stained with 0.5% crystal violet, and the EC50 values were
determined. Amantadine was included in all experiments as
a reference compound.
N-(1-Benzothiophen-2-ylmethyl)-4-(trifluoromethyl)-
benzene-sulfonamide 12p. 35% yield as a white solid, mp
1
178–180 °C. H NMR (400 MHz, Acetone-d6) δ 8.08 (d, J = 8.2
Hz, 2H), 7.87 (d, J = 8.3 Hz, 2H), 7.83–7.79 (m, 1H), 7.72 (dd,
J = 6.7, 2.1 Hz, 1H), 7.53 (t, J = 6.1 Hz, 1H), 7.37–7.27 (m,
2H), 7.22 (s, 1H), 4.64–4.45 (m, 1H). 13C NMR (100 MHz, Ace-
tone-d6) δ 141.23, 139.88, 139.48, 127.78, 126.10, 126.06,
124.35, 123.40, 122.99, 122.14, 42.50. HRMS (ESI) calcd for
C16H12F3NO2S2 [M + Na]+ 394.0159, found 394.0156.
N - ( 1 - B e n z o t h i o p h e n - 2 - y l m e t h y l ) - 4 - m e t h y l -
benzenesulfonamide 12q. 40% yield as a white solid, mp
174–176 °C. 1H NMR (400 MHz, Acetone-d6) δ 7.78–7.73 (m,
3H), 7.65 (dd, J = 6.7, 2.0 Hz, 1H), 7.34–7.27 (m, 3H), 7.07 (s,
1H), 4.80 (t, J = 5.8 Hz, 1H), 4.43 (d, J = 5.8 Hz, 2H), 2.41 (s,
3H). 13C NMR (100 MHz, Acetone-d6) δ 139.76, 129.77,
127.26, 124.55, 124.49, 123.52, 123.03, 122.36, 42.97, 21.56.
HRMS (ESI) calcd for C16H15NO2S2 [M + Na]+ 340.0442, found
340.0438.
Cytotoxicity assay
The cytotoxicities of compounds in MDCK cells were tested
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) method. Briefly, cells were seeded at a density
of 2 × 104 per well into 96-well plates. The next day, the cell
medium was removed, and fresh media containing DMEM
plus test compounds at various concentrations were added to
each well. After 24 h, 5 mg mL−1 MTT solution was added to
each well, and the plates were incubated for 4 h at 37 °C in a
CO2 incubator. Successively, a solubilization solution was
added to lyse the cells. After 3 h of incubation at 37 °C, ab-
sorbance was measured at 575 nm using an ELISA plate
reader (Tecan Sunrise).
N - ( 1 - B e n z o t h i o p h e n - 2 - y l m e t h y l ) - 2 - f l u o r o -
benzenesulfonamide 12r. 49% yield as a white solid, mp 120–
1
122 °C. H NMR (400 MHz, CDCl3) δ 8.31 (d, J = 8.8 Hz, 2H),
7.98 (d, J = 8.8 Hz, 2H), 7.23–7.16 (m, 1H), 7.08 (d, J = 1.9 Hz,
1H), 6.86–6.80 (m, 1H), 5.10 (s, 1H), 4.26 (d, J = 5.7 Hz, 2H).
13C NMR (100 MHz, CDCl3) δ 157.55, 139.92, 139.35, 139.22,
135.07, 130.24, 124.60, 124.49, 124.33, 123.55, 123.30, 122.31,
117.00, 116.79, 42.97. HRMS (ESI) calcd for C15H12FNO2S2 [M
+ Na]+ 344.0191, found 344.0187.
Time-of-addition assay
N - ( 1 - B e n z o t h i o p h e n - 2 - y l m e t h y l ) - 3 - f l u o r o -
benzenesulfonamide 12s. 36% yield as a white solid, mp
MDCK cells were seeded in 48-well plates and infected with
amantadine-sensitive H5N1 virus at an MOI of 2. Compound
11k and AMD were added at the time of infection (0 h) and
at 1, 2, 3, 4, 6 and 8 h after infection. Viral titers were deter-
mined at 12 h after infection by plaque assay as described
above. Compound 11k was added at a concentration of 1.9
μM. T-705 (4.0 μM) and AMD (1.7 μM) were used as control
treatments.
1
106–108 °C. H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 7.4 Hz,
1H), 7.66 (d, J = 7.1 Hz, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.47–
7.42 (m, 1H), 7.37–7.21 (m, 3H), 7.10 (s, 1H), 5.01 (t, J = 5.6,
1H), 4.47 (d, J = 6.1 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ
163.65, 161.15, 141.85, 139.88, 139.41, 139.15, 130.98, 124.68,
123.59, 123.30, 122.91, 122.36, 120.16, 114.69, 114.45, 42.97.
HRMS (ESI) calcd for C15H12FNO2S2 [M + Na]+ 344.0191,
found 344.0189.
Determination of M2 inhibitory activity by yeast growth
restoration assay
Plaque reduction assay
The coding sequences of the wild-type, V27A/S31N-mutated
M2 gene from the H5N1 strain [A/chicken/Hubei/327/
2004IJH5N1)] were introduced downstream of the GAL1 pro-
moter by PCR amplification followed by recombination clon-
ing in yeast.35 Plasmids were rescued from the yeast strains,
and the coding sequences were verified. The yeast strains
were propagated in synthetic complete (SC) medium (pH 6.5)
lacking leucine and containing glucose to repress M2 gene
expression. After harvesting the yeast by centrifugation, the
pellets were washed with water to remove traces of glucose
and then suspended to A600 = 0.01 in liquid SC medium (pH
6.5) lacking leucine and containing 2% galactose. Samples
The sequence of the M2 protein of amantadine-sensitive H5N1 vi-
rus [A/chicken/Hubei/327/2004 (H5N1)] is MSLLTEVETPTRNEWEC
RCSDSSDPLVVAASIIGILHLILWILDRLFFKCIYRRLKYGLKRGPSTAG-
VPESMREEYRQEQQSAVDVDDGHFVNIELE. Amantadine-resistant
H5N1 virus was obtained by screening amantadine-resistant H5N1
flu strains based on amantadine-sensitive H5N1 virus [A/chicken/
Hubei/327/2004 (H5N1)]. We confirmed that the mutation site of
the M2 protein of amantadine-resistant H5N1 virus was M2-S31 N
by sequencing M2 protein. M2-S31N is the most common
amantadine-resistant mutation site among isolated influenza A vi-
ruses (H5N1, H1N1, etc.).
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(100 μL) were distributed into 96-well plates. The compounds
were transferred to the yeast plates at different concentra-
tions, and the plates were incubated at 30 °C in a humidified
incubator. After 46–48 h, the cells were suspended by
vortexing, and A600 was measured. The percentage of growth
restoration was calculated as described previously.36 Com-
pounds leading to growth restoration were retested in tripli-
cate at different concentrations against WT M2, V27A/S31N
M2 and empty plasmid strains to confirm their activity and
determine EC50.
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There are no conflicts to declare.
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Acknowledgements
We are grateful to the NSFC (81772236, 81773557 and
81573279), Major Project of Technology Innovation Program
of Hubei Province (2016ACA126, 2018ACA123), NSFHP
(2017CFA024), the Fundamental Research Funds for the Cen-
tral Universities of China (2042017kf0288), and Open Re-
search Fund Program of the Hubei Province Engineering and
Technology Research Center for Fluorinated Pharmaceuticals
for support of this research.
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