Bioassay of ferulic acid derivatives as influenza neuraminidase inhibitors

Article abstract of DOI:10.1002/ardp.201900174

Four series of ferulic acid derivatives were designed, synthesized, and evaluated for their neuraminidase (NA) inhibitory activities against influenza virus H1N1 in vitro. The pharmacological results showed that the majority of the target compounds exhibited moderate influenza NA inhibitory activity, which was also better than that of ferulic acid. The two most potent compounds were 1m and 4a with IC₅₀ values of 12.77 ± 0.47 and 12.96 ± 1.34 μg/ml, respectively. On the basis of the biological results, a preliminary structure–activity relationship (SAR) was derived and discussed. Besides, molecular docking was performed to study the possible interactions of compounds 1p, 2d, 3b, and 4a with the active site of NA. It was found that the 4-OH-3-OMe group and the amide group (CON) of ferulic acid amide derivatives were two key pharmacophores for NA inhibitory activity. It is meaningful to further modify the natural product ferulic acid to improve its influenza NA inhibitory activity.

Full text of DOI:10.1002/ardp.201900174

Received: 15 June 2019
Revised: 3 October 2019
Accepted: 4 October 2019
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DOI: 10.1002/ardp.201900174
F U L L P A P E R
Bioassay of ferulic acid derivatives as influenza
neuraminidase inhibitors
1
1
2
1
2
1
Man‐Ying Cui
Ai‐Xi Hu
|
Meng‐Wu Xiao
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Lv‐Jie Xu
|
Yun Chen
|
Ai‐Lin Liu
|
Jiao Ye
|
1
1
College of Chemistry and Chemical
Engineering, Hunan University,
Changsha, China
Abstract
Four series of ferulic acid derivatives were designed, synthesized, and evaluated for
their neuraminidase (NA) inhibitory activities against influenza virus H1N1 in vitro.
The pharmacological results showed that the majority of the target compounds
exhibited moderate influenza NA inhibitory activity, which was also better than that
of ferulic acid. The two most potent compounds were 1m and 4a with IC50 values
of 12.77 ± 0.47 and 12.96 ± 1.34 μg/ml, respectively. On the basis of the biological
results, a preliminary structure–activity relationship (SAR) was derived and discussed.
Besides, molecular docking was performed to study the possible interactions of
compounds 1p, 2d, 3b, and 4a with the active site of NA. It was found that the 4‐OH‐
2
Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking
Union Medical College, Beijing, China
Correspondence
Jiao Ye and Aixi Hu, College of Chemistry and
Chemical Engineering, Hunan University,
410082 Changsha, China.
Email: yejiao@hnu.edu.cn, (J. Y.) and
axhu@hnu.edu.cn (A. H.)
Funding information
Hunan Provincial Natural Science Foundation
of China, Grant/Award Number: 2019JJ40030
3
‐OMe group and the amide group (CON) of ferulic acid amide derivatives were two
key pharmacophores for NA inhibitory activity. It is meaningful to further modify the
natural product ferulic acid to improve its influenza NA inhibitory activity.
K E Y W O R D S
ferulic acid derivatives, molecular docking, neuraminidase inhibitors
[10]
[11]
[12]
1
|
INTRODUCTION
natural product licochalcone,
resveratrol,
kaempferol,
and
so on. So it is a good way to discover new NA inhibitors from natural
products.
Influenza is an acute viral infectious disease of the respiratory tract.
According to statistics from the World Health Organization, seasonal
influenza could cause 2–5 million infections and 250,000–500,000
Ferulic acid, a very common natural product, has a wide range of
pharmacological activities, such as antioxidant, anti‐inflammatory,
[1,2]
[13]
deaths each year worldwide.
With the long‐term research on the
antithrombotic, anticancer, and so on.
In 2016, it was reported
influenza virus, it was found that there are two main glycoproteins—
hemagglutinin (HA) and neuraminidase (NA) on the surface of the
virus, which are responsible for the invasion of virus and the release
that ferulic acid had moderate NA inhibitory activity with an IC50
[
14]
value of 27.16 μg/ml.
However, to date, there are no follow‐up
reports about ferulic acid derivatives as influenza NA inhibitors. In
[15]
[3,4]
of offspring virus, respectively.
Moreover, further research
2017, Enkhtaivan et al.
found that piperazine structural fragment
indicated that over 10 amino acid residues of NA’s active site are
can significantly enhance the NA inhibitory activity of the natural
product berberine (Figure 1a). In our previous research, it was
found that the compounds containing piperazine fragments tend
to exhibit moderate NA inhibitory activity with an IC50 value of
[
5,6]
highly conserved.
Hence, compared with other targets, NA is an
attractive target for anti‐influenza research. However, although there
are two marketed NA inhibitors, oseltamivir and zanamivir, the drug
resistance caused by the high mutability of the influenza virus has
[
16]
20.03 μg/ml
(Figure 1b). Besides, we found that thiazolamide
[7–9]
become increasingly severe.
As to the drug resistance of viruses,
fragments also can act as NA inhibitory active fragments in many
[
17,18]
nonclassical influenza NA inhibitor may be a new research direction.
In recent years, some nonclassical influenza NA inhibitors have been
discovered possessing effective NA inhibitory activity such as the
NA inhibitors
(Figure 1c,d). On the basis of the above research,
we took phenylacrylic acid as the basic fragment to condense with
active piperazine fragments to design and synthesize the target
Arch. Pharm. Chem. Life Sci. 2019;e1900174.
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201900174

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FIGURE 1 Design of the target compounds
compound 1. According to the preliminary activity results, it can be
found that 4‐OH‐3‐OMe groups on the benzene ring of ferulic acid
are the key pharmacophores on NA inhibitory activity. So keeping
active pharmacophore (R = 4‐OH‐3‐OMe), we spliced the ferulic
acid with 2‐aminothiazolyl fragments to design the target com-
pound 2. To further optimize the target compounds 1 and 2 with
better activity and have a discussion on the structure–activity
relationship, we removed the carbon–carbon double bond in target
compounds 1 and 2 to design the target compounds 3 and 4
acid 6 and various piperazine derivatives 7 were condensed using
EDCI/HOBt as the condensing agent to afford cinnamamide
derivatives 1a–q with good yields. Due to the poor reactivity of
2‐aminothiazolyl, it needed to be raised to 115°C to synthesize
compounds 2a–f using EDCI/HOBt as the condensing agent with
moderate yields. Vanillic acid 6i and piperazine derivatives 7 were
condensed to prepare analogues 3a–c, using the same method as
in the synthesis of compound 1 with yields of 53.6%, 88.7%, and
64.3%, respectively. Vanillic acid was subjected to acetylation,
condensation, and hydrolysis in turn to obtain the target
compounds 4a–c. In addition, the Boc‐amine of compound 1q
was deprotected under the action of hydrogen chloride gas in ethyl
acetate to obtain compound 1r. Compound 1f was hydrolyzed by
LiOH aqueous solution to afford the compound 1s. Compound 2h
was synthesized in the same way as in the synthesis of compound
1s. Compound 2e was condensed with methoxyammonium
chloride to afford the compound 2g. All the target compounds
were characterized by NMR spectroscopy.
(
Figure 1).
2
2
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RESULTS AND DISCUSSION
|
Chemistry
.1
The synthetic pathways of ferulic acid derivatives are shown in
Scheme 1. The key intermediate substituted cinnamic acids 6 were
[
19]
synthesized as reported in the literature.
Substituted cinnamic

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SCHEME 1 Synthesis of the target compounds

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concentration of 40 μg/ml in vitro using oseltamivir as positive
control. The results of four series of thiazolylhydrazone derivatives
are summarized in Tables 1–4. Preliminary biological results showed
that many compounds displayed moderate to good inhibitory activity
against NA.
As shown in Table 1, the majority of compound 1 had a moderate
NA inhibitory activity. Especially, most of compounds 1i–r with the
4‐OH‐3‐OMe group on the phenyl ring tended to exhibit better
NA inhibitory activity than compounds 1a–h. Therefore, it can be
speculated that 4‐OH‐3‐OMe on the benzene ring played a decisive
role on the inhibitory activity of NA and the 4‐OH‐3‐OMe group
was a critical pharmacophore.
FIGURE 2 X‐ray crystal structure of compound 2c
Among compounds 1i–r (R = 4‐OH‐3‐OMe), compounds 1i, 1m,
2
.2 Crystal structure
|
1
n, 1o, 1p, and 1r showed the better activity with IC50 values ranging
Compound 2c was determined by single‐crystal X‐ray diffraction.
Yellow crystal of compound 2c was obtained by slow evaporation of
ethanol and water solution at room temperature. The crystal
structure of compound 2c is shown in Figure 2. The space group
from 10 to 20 μg/ml. Especially, compound 1m (R = 4‐OH‐3‐OMe,
1
R = CH ) had the best activity with an IC value of 12.77 ± 0.47 μg/
3
50
1
ml. The activity of compound 1j (4‐OH‐3‐OMe, R = 2‐OMeC H ) and
6
4
1
1q (R = 4‐OH‐3‐OMe, R = Boc) was relatively poor with IC values
5
0
P2
1
/c is as follows: a = 10.43 (3), b = 10.642 (14), c = 17.35 (4) Å,
of 23.56 ± 0.52 and 24.52 ± 1.93 μg/ml. The inhibition rates of
3
α = 90°, β = 90.12 (3)°, γ = 90°, Z = 4, V = 1,926 (7) Å , F(000) = 840,
compound 1k and 1l were even less than 50% at the concentration
−3
−1
1
Mr = 399.47, Dx = 1.377 mg m , μ = 0.20 mm , T = 273 K, Mo Kα
radiation, λ = 0.71073 Å. As depicted in Figure 2, the CH═CH double
bond (C8═C9) of acrylamide is present in the form of geometric E
isomer, which is consistent with the coupling constant (J = 15.6 Hz) of
of 40 μg/ml. The influence on activities of substituents R on the
piperazine ring is as follows: CH > 4‐OHC H > H·HCl ≈ COCH ≈
3
6
4
3
CO Et > C H > 2‐OMeC H > Boc > Ph > 4‐ClC H . On the basis of
2
2
5
6
4
6
4
the above active order, it can be found that the activity of hydrophilic
1
1
E double bond in the H NMR spectrum. The dihedral angle between
substituents on R is better than that of hydrophobic substituents.
phenyl plane and thiazole ring is 18.10°; the dihedral angle between
the thiazole ring and the 1,2,4‐triazole ring is 83.90°. As shown in
Figure 3, the crystal structure is stabilized via intermolecular
hydrogen bond interaction (N1‐HA1…N5). The hydrogen bond length
and angle are 2.956 Å and 164.74°, respectively. The additional
crystal structure and experimental data are available from Support-
ing Information S1 and the Cambridge Crystallographic Data Centre
as CCDC 1890182.
As shown in Table 2, most of compound 2 exhibited moderate NA
2
inhibitory activity. The most potent one was compound 2g (R = Me,
3
R = C(NOCH )CH ) with an IC value of 14.32 ± 0.06 μg/ml. The IC50
3
3
50
values of compounds 2b–e ranged from 10 to 20 μg/ml. The activity of
2
3
compound 2f (R = CH CO Et, R = H) was relatively poor with an IC
50
2
2
value of 24.64 ± 0.93 μg/ml. However, the hydrolyzed compound 2h
2
3
(R = CH COOH, R = H; IC50 = 18.47 ± 0.34 μg/ml) showed better
2
activity than compound 2f. The influence on the activities of substituent
groups at 4‐position and 5‐position of the thiazole ring is as follows:
4
‐Me‐5‐C(NOCH
imidazolyl ≈ 4‐t‐Bu‐5‐1,2,4‐triazolyl > 4‐CH
H > 4‐t‐Bu‐5‐NO2. Preliminary structure–activity relationship shows that
electron‐withdrawing substituent (imidazolyl, 1,2,4‐triazolyl, CO Et,
COCH3, C(NOCH )CH ), which can form a conjugation with the thiazole
ring can contribute to improvement of NA inhibitory activity. Besides,
3
)CH
3
> 4‐Me‐5‐COCH
3
≈ 4‐Me‐5‐CO
2
Et > 4‐t‐Bu‐5‐
2
.3 In vitro inhibitory activity on NA
|
2
COOH > 4‐CH
2
CO Et‐5‐
2
Thirty‐three target compounds were synthesized and evaluated for
the NA inhibitory activity against influenza viral NA (H1N1) at the
2
3
3
2
3
the increasing hydrophilicity of R and R groups can improve NA
inhibitory activity.
To investigate the effect of the length of the carbon chain between
the two rings on the inhibitory activity of NA, compounds 3 and 4 were
designed, synthesized, and evaluated for NA inhibitory activity. The
activity test results indicated that compound 3 possessed poor activities
with an NA inhibition rate lower than 50% at the test concentration of
40 μg/ml (Table 3). Compound 4 had the equivalent activity with ferulic
acid thiazolylamide derivatives 2 (Table 4). The compound 4a (R = 4‐
1
OH‐3‐OMe, R = CH
3
) had better activity than others with an IC50 value
of 12.96 ± 1.34 μg/ml. Comparing the activity, we can speculate that
carbon–carbon double bonds play a key role in the activity of ferulic
acid piperazine amide derivatives 1. However, carbon–carbon double
FIGURE 3 Hydrogen bond diagram of compound 2c

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TABLE 1 Inhibitory activity of compound 1 against neuraminidase in vitro
1
Comp 1
R
R
Inhibition rate (%)
3.78
IC50 (μg/ml)
NA
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
3‐OH
4‐OH
3,4‐(OH)
4‐OMe
4‐OHC
4‐OHC
4‐OHC
4‐OHC
4‐OHC
4‐OHC
4‐OHC
4‐OHC
4‐OHC
6
6
6
6
6
6
6
6
6
H
H
H
H
H
H
H
H
H
4
4
4
4
4
4
4
4
34.69
NA
2
29.00
NA
2.53
NA
4‐NO
4‐CO
2
4.39
NA
2
Me
7.03 ± 1.43
55.25 ± 5.79
38.01 ± 3.18
72.38 ± 0.50
62.99 ± 1.08
29.60
NA
g
h
i
3‐OH‐4‐OMe
4‐OH‐3,5‐ (OMe)
4‐OH‐3‐OMe
4‐OH‐3‐OMe
4‐OH‐3‐OMe
4‐OH‐3‐OMe
4‐OH‐3‐OMe
4‐OH‐3‐OMe
4‐OH‐3‐OMe
4‐OH‐3‐OMe
4‐OH‐3‐OMe
4‐OH‐3‐OMe
4‐COOH
33.94 ± 7.35
NA
2
4
19.06 ± 1.64
23.56 ± 0.52
NA
j
2‐OMeC
6
H
4
k
l
4‐ClC
6
H
4
Ph
40.36
NA
m
n
o
p
q
r
CH
3
80.96 ± 1.70
75.25 ± 1.32
76.23 ± 2.06
74.54 ± 0.25
64.07 ± 1.7
76.62 ± 0.84
1.17
12.77 ± 0.47
16.09 ± 0.96
16.56 ± 0.59
18.36 ± 0.69
24.52 ± 1.93
16.21 ± 0.64
NA
2 5
C H
COCH
CO Et
Boc
3
2
H·HCl
s
6 4
4‐OHC H
bonds have almost no effect on the activity of ferulic acid thiazolylamide
derivatives 2. To sum up, the SAR of the ferulic acid derivatives is
summarized in Figure 4.
According to the docking result in Figures 5a1 and 6a2, it could
be found that the plane of the benzene ring of compound 1p almost
coincides with the plane of the six‐membered ring of oseltamivir.
There are eight hydrogen bonds formed between compound 1p and
the amino acid residues of NA. The stretching direction and angle of
4‐OH‐3‐OMe on the benzene ring of compound 1p are consistent
with that of the acetamido group of oseltamivir. The 4‐OH‐3‐OMe
group on the benzene ring can strongly interact with residues of
Arg152, Trp178 by two hydrogen bonds, which can explain the
reason why the 4‐OH‐3‐OMe group on the benzene ring has a
dominant effect on activity. Besides, the stretching direction and
angle of amide group (CON) of compound 1p are accordant with the
ester group of oseltamivir. Docking result demonstrates that the
amide group (CON) of compound 1p can form five hydrogen bonds
with amino acid residues of Arg371, Try406, and Arg292, which
contribute to the improvement of activity greatly. At last, the ester
2
.4 Docking analysis
|
To understand the interaction between ferulic acid derivatives
and the NA active site, analogs 1p, 2d, 3b, and 4a containing
2
ethoxycarbonyl group (CO Et) were respectively docked into the
active sites of NA (H1N1, PDB entry: 3TI6) using LeDock (http://
[20,21]
www.lephar.com/) with default parameters.
The results were
analyzed and visualized using PyMOL (http://pymol.sourceforge.net/).
LigPlot + v.2.1 was used to depict the two‐dimensional (2D) interac-
[
22]
tion diagrams.
TABLE 2 Inhibitory activity of compound 2 against neuraminidase
2
group (CO Et) of piperazine amide can also form a hydrogen bond
in vitro
with the amino acid residue of Asn294. However, as shown in Figures
5b1 and 6b2, compound 3b cannot bind well to the NA cavity. There
are just two hydrogen bonds with amino acid residues and the amide
Inhibition
2
3
Comp 2
R
R
rate (%)
IC50 (μg/ml)
NA
2
2
2
2
2
2
2
2
a
b
c
d
e
f
t‐Bu
t‐Bu
t‐Bu
Me
NO
2
45.29
imidazol‐1‐yl
72.2 ± 1.28
19.21 ± 1.13
1,2,4‐triazol‐1‐yl 69.34 ± 0.93 19.25 ± 0.14
TABLE 3 Inhibitory activity of compound 3 against neuraminidase
in vitro
CO
2
Et
80.73 ± 1.07 15.93 ± 1.22
78.46 ± 1.19 14.38 ± 0.93
63.83 ± 0.73 24.64 ± 0.93
83.68 ± 1.09 14.32 ± 0.06
75.06 ± 3.64 18.47 ± 0.34
1
Me
COCH
3
Comp 3
3a
R
Inhibition rate (%)
13.76
IC50 (μg/ml)
CH
Me
CH
2
CO
2
Et
H
Boc
NA
NA
NA
g
h
C(NOCH
3
)CH
3
3b
CO
2
Et
17.39
2
COOH
H
3c
4‐OHPh
21.09

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TABLE 4 Inhibitory activity of compound 4 against neuraminidase
in vitro
acid derivatives containing the 4‐OH‐3‐OMe group on the benzene
ring always showed much better NA inhibitory activity than ferulic
acid. The most two potent compounds were 1m and 4a with IC50
values of 12.77 ± 0.47 and 12.96 ± 1.34 μg/ml, respectively. Struc-
ture–activity relationship investigation suggested that the 4‐OH‐3‐
OMe group on the benzene ring of ferulic acid derivatives was the
pivotal pharmacophore. Increasing hydrophilicity and polarity of
ferulic acid derivatives were beneficial to improve NA inhibitory
activity. The carbon–carbon double bonds played a key role in the
activity of ferulic acid piperazine amide derivatives 1. However,
carbon–carbon double bonds had almost no effect on the activity of
ferulic acid thiazolylamide derivatives 2. Molecular docking demon-
strated the interaction mode of each series of the compound with the
NA active cavity. Besides, it also preliminarily explained the reason
why each series of compounds possesses different activities.
It was found that the 4‐OH‐3‐OMe group and amide group (CON)
of ferulic acid amide derivatives were two key pharmacophores on
NA inhibitory activity. Ferulic acid has the potential to be further
modified to have better NA inhibitory activity.
2
3
Comp 4
R
R
Inhibition rate (%)
78.93 ± 2.33
IC50 (μg/ml)
12.96 ± 1.34
14.84 ± 2.95
22.00 ± 0.61
4a
4b
4c
Me
CO
COCH
imidazol‐1‐yl
2
Et
Me
3
77.56 ± 4.87
t‐Bu
68.14 ± 2.38
group (CON) cannot interact with NA cavity. This may be the reason
why the activity of compound 3b is so poor.
As to the docking result of compound 2d (Figures 5c1 and 6c2),
the extension direction and hydrogen bond interaction of compound
2e are similar to the docking result of compound 1p. There are also
eight hydrogen bonds formed between compound 2e and the amino
acid residues of NA. The 4‐OH‐3‐OMe group on the benzene ring
always forms two hydrogen bonds with residues of Arg152, Trp178.
In addition, the carbonyl of the amide group (CON) of compound 2e
can interact with residues of Arg371, Try406, and Arg118 by four
hydrogen bonds. The CO Et substituent at the 5‐position on the
2
thiazole ring also formed two hydrogen bonds with Arg292 and
Asn294. As shown in the Figures 5d1 and 6d2, compared with
compound 2d, the molecular structure of compound 4a extends more
flatly without some folding in the molecule. An unfolded molecule
ensures a stronger binding force to the NA cavity, which is very
similar with oseltamivir. So the six hydrogen bonds on the rigid
structure of compound 4a guarantee that compound 4a produces
a stronger interaction with the NA cavity.
4
|
EXPERIMENTS
|
Chemistry
4.1
4
.1.1 | General
Starting materials and solvents were commercially available and
used without further purification. All reactions were monitored by
thin‐layer chromatography (TLC) on 25.4‐ to 6.2‐mm silica gel plates
(GF‐254). Melting points were measured on an X‐4 electrothermal
digital melting point apparatus and uncorrected. Nuclear magnetic
resonance (NMR) was recorded on a Varian INOVA‐400 spectro-
meter using tetramethylsilane (TMS) as an internal standard. The
To sum up, the docking results of compounds 1p, 2d, 3b, and 4a
demonstrate that the 4‐OH‐3‐OMe group on the benzene ring of
ferulic acid amide derivatives is dominant on NA inhibitor activity,
and the amide group (CON) of ferulic acid amide derivatives has a
great contribution to the improvement of activity. These two sites
are just like two fulcrums to make sure that the ferulic acid
derivatives can combine with the NA cavity.
6
solvents for NMR were dimethyl sulfoxide (DMSO)‐d .
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
3
|
CONCLUSIONS
4
.1.2 | General procedure for the synthesis
of compounds 1a–q
Four series of ferulic acid derivatives were designed, synthesized,
and evaluated for their ability to inhibit NA of influenza H1N1 virus.
Compound 2c was confirmed by single‐crystal X‐ray diffraction,
and the C═C double bond was further confirmed as the form of
geometric E isomer. Biological activity results indicated that ferulic
The substituted cinnamic acids were synthesized according
[
23]
to the method reported in reference
with substituted benzal-
dehydes and malonic acid as the raw materials. A mixture of
FIGURE 4 Structure–activity relationship of the ferulic acid derivatives

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FIGURE 5 Specific interactions of compounds 1p (a1), 3b (b1), 2d (c1), and 4a (d1) binding to the active site of neuraminidase (H1N1, PDB:
TI6)
3
different substituted cinnamic acids 6 (1.2 mmol), 1‐hydroxyben-
zotriazole (1.2 mmol), N‐(3‐dimethylaminopropyl)‐N’‐ethylcarbo-
diimide hydrochloride (1.2 mmol) in N,N‐dimethylformamide
(d, J = 15.2 Hz, 1H, CH), 7.46 (d, J = 15.2 Hz, 1H, CH), 7.59
(d, J = 7.6 Hz, 2H, C ), 9.00 (s, 1H, OH), and 10.01 (s, 1H, OH);
C NMR (101 MHz, DMSO‐d ) δ: 45.58, 51.48, 114.72, 115.98,
6 4
H
1
3
6
(
10 ml) was stirred for 30 min, and then piperazine derivatives 7
116.06, 118.89, 126.61, 130.25, 142.39, 144.38, 151.89, 159.53,
(
1 mmol) were added. The reaction mixture was kept stirring at
and 165.30.
room temperature for 8–12 hr confirmed by TLC and then poured
into ice water. The mixture was extracted with ethyl acetate.
Organic phase was washed successively with saturated sodium
bicarbonate, dilute hydrochloric acid solution, and saturated
sodium chloride solution, dried over anhydrous sodium sulfate,
filtered, and evaporated. The crude product was recrystallized
from ethanol to afford the pure compounds 1a–q.
(E)‐3‐(3,4‐Dihydroxyphenyl)‐1‐(4‐(4‐hydroxyphenyl)piperazin‐1‐yl)‐
prop‐2‐en‐1‐one (1c)
1
Light yellow solid. Yield 73.3%; m.p. 233–235°C; H NMR (400 MHz,
DMSO‐d
6.71–7.16 (m, 8H, C
s, 1H, OH), 9.06 (s, 1H, OH), and 9.54 (s, 1H, OH); C NMR
(101 MHz, DMSO‐d ) δ: 114.69, 115.38, 115.97, 116.06, 118.90,
6
) δ: 3.00 (br, 4H, 2 × CH
2 2
), 3.74–3.85 (m, 4H, 2 × CH ),
6 3
H
, C , CH), 7.41 (d, J = 15.2 Hz, 1H, CH), 8.96
6 4
H
13
(
(
E)‐3‐(3‐Hydroxyphenyl)‐1‐(4‐(4‐hydroxyphenyl)piperazin‐1‐yl)‐
prop‐2‐en‐1‐one (1a)
Yellow solid. Yield 61.5%; m.p. 222–225°C; H NMR (400 MHz,
DMSO‐d ) δ: 2.99 (br, 4H, 2 × CH ), 3.72 (br, 2H, CH ), 3.84 (br, 2H,
CH ), 6.70 (d, J = 8.2 Hz, 2H, C ), 6.82–7.26 (m, 7H, C , C
CH), 7.44 (d, J = 15.4 Hz, 1H, CH), 9.00 (s, 1H, OH), and 9.67 (s, 1H,
6
121.12, 127.20, 142.75, 144.42, 145.89, 147.84, 151.82, and 165.28.
1
6
2
2
2
H
6 4
6
H
3
6
H
4
,
(E)‐1‐(4‐(4‐Hydroxyphenyl)piperazin‐1‐yl)‐3‐(4‐methoxyphenyl)‐
prop‐2‐en‐1‐one (1d)
13
1
OH); C NMR (101 MHz, DMSO‐d
6
) δ: 45.62, 51.49, 115.02, 115.97,
17.15, 118.43, 118.92, 119.41, 130.14, 136.89, 142.27, 144.38,
51.87, 158.14, and 164.92.
Yellow solid. Yield 61.2%; m.p. 196–198°C; H NMR (400 MHz,
1
1
DMSO‐d
3.79 (s, 3H, OCH
), 6.83 (d, J = 8.2 Hz, 2H, C
7.16 (d, J = 15.3 Hz, 1H, CH), 7.49 (d, J = 15.3 Hz, 1H, CH),
6
)
δ: 2.96 (br, 4H, 2 × CH
), 3.82 (br, 2H, CH
), 6.97 (d, J = 8.2 Hz, 2H, C
2
), 3.70 (br, 2H, CH
), 6.68 (d, J = 8.2 Hz, 2H,
),
2
),
3
2
C H
6 4
H
6 4
6 4
H
(
E)‐3‐(4‐Hydroxyphenyl)‐1‐(4‐(4‐hydroxyphenyl)piperazin‐1‐yl)‐
prop‐2‐en‐1‐one (1b)
Yellow solid. Yield 56.3%, m.p. 223–226℃; H NMR (400 MHz,
DMSO‐d ) δ: 2.98 (br, 4H, 2 × CH ), 3.71–3.83 (m, 4H, 2 × CH ), 6.70
d, J = 8.0 Hz, 2H, C ), 6.82–6.86 (m, 4H, C , C ), 7.10
1
3
7.69 (d, J = 8.2 Hz, 2H,
NMR (101 MHz, DMSO‐d
C
6
H
4
), and 8.90 (s, 1H, OH);
C
1
6
) δ: 45.57, 51.50, 55.73, 114.65, 115.93,
115.96, 118.92, 128.25, 130.16, 141.92, 144.41, 151.85, 160.88,
and 165.15.
6
2
2
(
H
6 4
6
H
4
6 4
H

8
of 13
CUI ET AL.
|
FIGURE 6 Two‐dimensional binding mode of compounds 1p (a2), 3b (b2), 2d (c2), and 4a (d2) binding to the active site of neuraminidase
(H1N1, PDB: 3TI6)
(
E)‐1‐(4‐(4‐Hydroxyphenyl)piperazin‐1‐yl)‐3‐(4‐nitrophenyl)‐
7.91 (d, J = 8.0 Hz, 2H, C
6
H
4
), 7.99 (d, J = 8.0 Hz, 2H, C
6 4
H ), and 9.00
1
3
prop‐2‐en‐1‐one (1e)
Yellow solid. Yield 79.1%; m.p. 240–242°C; H NMR (400 MHz,
DMSO‐d ) δ: 2.99 (br, J = 5.5 Hz, 4H, 2 × CH ), 3.72 (br, 2H, CH ),
.87 (br, 2H, CH ), 6.68 (d, J = 8.0 Hz, 2H, C ), 6.84 (d, J = 8.0 Hz,
H, C ), 7.55 (d, J = 15.4 Hz, 1H, CH), 7.62 (d, J = 15.4 Hz, 1H, CH),
.03 (d, J = 8.0 Hz, 2H, C ), 8.25 (d, J = 8.0 Hz, 2H, C ), and 8.90
) δ: 45.71, 51.50, 115.97,
18.95, 123.28, 124.31, 129.54, 139.49, 142.24, 144.34, 147.98,
51.89, and 164.26.
(s, 1H, OH); C NMR (101 MHz, DMSO‐d ) δ: 42.30, 45.71, 52.71,
6
1
116.01, 118.92, 121.44, 128.71, 129.97, 130.42, 140.15, 140.62,
144.28, 151.91, 164.60, and 166.35.
6
2
2
3
2
8
2
6 4
H
6 4
H
H
6 4
6
H
4
(E)‐3‐(3‐Hydroxy‐4‐methoxyphenyl)‐1‐(4‐(4‐hydroxyphenyl)‐
piperazin‐1‐yl)prop‐2‐en‐1‐one (1g)
13
(
s, 1H, OH); C NMR (101 MHz, DMSO‐d
6
1
1
1
White solid. Yield 76.0%; m.p. 234–236°C; H NMR (400 MHz,
DMSO‐d
m, 5H, CH
2H, C ), 6.96 (d, J = 8.0 Hz, 1H, C
7.14 (d, J = 8.0 Hz, 1H, C ), 7.21 (s, 1H, C
6
) δ: 2.98 (br, 4H, 2 × CH
, OCH ), 6.70 (d, J = 8.1 Hz, 2H, C
), 7.09 (d, J = 15.2, 1H, CH),
2
), 3.72 (br, 2H, CH
2
), 3.78–3.92
(
2
3
6 4
H ), 6.85 (d, J = 8.0 Hz,
(
E)‐Methyl‐4‐(3‐(4‐(4‐hydroxyphenyl)piperazin‐1‐yl)‐
‐oxoprop‐1‐en‐1‐yl)benzoate (1f)
White solid. Yield 80.3%; m.p. 218–222°C; H NMR (400 MHz,
DMSO‐d ) δ: 3.00 (br, 4H, 2 × CH ), 3.73 (br, 2H, CH ), 3.82–3.94
m, 5H, CH , OCH ), 6.70 (d, J = 8.4 Hz, 2H, C ), 6.85 (d, J = 8.4 Hz,
H, C ), 7.49 (d, J = 15.4 Hz, 1H, CH), 7.58 (d, J = 15.4 Hz, 1H, CH),
6
H
4
6 3
H
3
6
H
3
6
H
3
), 7.42 (d, J = 15.2 Hz,
1
13
1H, CH), 8.93 (s, 1H, OH), and 9.09 (s, 1H, OH); C NMR (101 MHz,
6
2
2
6
DMSO‐d ) δ: 42.23, 45.57, 56.11, 112.34, 114.76, 115.77, 115.97,
(
2
3
H
6 4
118.91, 121.10, 128.57, 142.39, 144.41, 147.00, 149.71, 151.82,
and 165.19.
2
6 4
H

CUI ET AL.
9 of 13
|
(
E)‐3‐(4‐Hydroxy‐3,5‐dimethoxyphenyl)‐1‐(4‐(4‐hydroxyphenyl)‐
piperazin‐1‐yl)prop‐2‐en‐1‐one (1h)
Yellow solid. Yield 42.5%; m.p. 229–231°C; H NMR (400 MHz,
DMSO‐d ) δ: 3.00 (br, 4H, 2 × CH ), 3.73 (br, 2H, CH ), 3.80–3.92
m, 8H, CH 2 × OCH ), 6.71 (d, J = 8.0 Hz, 2H, ), 6.86
d, J = 8.0 Hz, 2H, C ), 7.06 (s, 2H, C ), 7.18 (d, J = 15.2 Hz,
H, CH), 7.48 (d, J = 15.2 Hz, 1H, CH), 8.85 (s, 1H, OH), and 8.97
(E)‐3‐(4‐Hydroxy‐3‐methoxyphenyl)‐1‐(4‐methylpiperazin‐1‐yl)‐
prop‐2‐en‐1‐one (1m)
1
1
Yellow solid. Yield 72.5%; m.p. 65–66°C; H NMR (400 MHz, DMSO‐
6
2
2
d
6
) δ: 2.22 (s, 3H, NCH
2 × CH ), 3.86 (s, 3H, OCH
(m, 2H, C , CH), 7.35 (s, 1H, C
and 9.46 (s, 1H, OH).
3
), 2.34 (br, 4H, 2 × CH
), 6.80 (d, J = 8.0 Hz, 1H, C
), 7.43 (d, J = 15.2 Hz, 1H, CH),
2
), 3.58–3.71 (m, 4H,
(
(
2
,
3
C
H
6 4
2
3
6 3
H ), 7.06–7.12
H
6 4
6
H
2
H
6 3
6 3
H
1
13
(
s, 1H, OH); C NMR (101 MHz, DMSO‐d
16.04, 119.02, 125.94, 137.80, 143.36, 144.38, 148.40, 151.65,
and 165.68.
6
) δ: 56.57, 106.21, 115.15,
1
(E)‐1‐(4‐Ethylpiperazin‐1‐yl)‐3‐(4‐hydroxy‐3‐methoxyphenyl)‐
prop‐2‐en‐1‐one (1n)
1
Yellow solid. Yield 66.2%; m.p. 55–58°C; H NMR (400 MHz, DMSO‐
(
E)‐3‐(4‐Hydroxy‐3‐methoxyphenyl)‐1‐(4‐(4‐hydroxyphenyl)‐
piperazin‐1‐yl)prop‐2‐en‐1‐one (1i)
Brownish yellow solid. Yield 56.9%; m.p. 225–227°C; H NMR
400 MHz, DMSO‐d ) δ: 2.97 (br, 4H, 2 × CH ), 3.71 (br, 4H, 2 × CH ),
), 6.67 (d, J = 8.0 Hz, 2H, C ), 6.78 (d, J = 8.0 Hz,
H, C6H3), 6.8 (d, J = 8.0 Hz, 2H, C ), 7.08–7.12 (m, 2H, C , CH),
.34 (s, 1H, C ), 7.43 (d, J = 15.2 Hz, 1H, CH), 8.89 (s, 1H, OH),
d
6
) δ: 1.04 (t, J = 7.2 Hz, 3H, CH
3.58 (br, 4H, 2 × CH ), 3.72 (br, 4H, 2 × CH
(d, J = 8.0 Hz, 1H, C ), 7.06–7.11 (m, 2H, C
), 7.42 (d, J = 15.2 Hz, 1H, CH), and 9.68 (s, 1H, OH).
3
), 2.37–2.38 (m, 6H, 2 × CH
), 3.84 (s, 3H, OCH
, CH), 7.34 (s, 1H,
2
, NCH
2
),
2
2
3
), 6.83
1
6
H
3
6 3
H
(
6
2
2
6 3
C H
3
1
7
.83 (s, 3H, OCH
3
6 4
H
H
6 4
6
H
3
(E)‐1‐(4‐Acetylpiperazin‐1‐yl)‐3‐(4‐hydroxy‐3‐methoxyphenyl)‐
prop‐2‐en‐1‐one (1o)
6 3
H
13
1
and 9.42 (s, 1H, OH); C NMR (101 MHz, DMSO‐d
6
) δ: 45.48, 50.93,
6.26, 111.60, 114.89, 115.90, 115.98, 118.95, 123.04, 127.15,
42.85, 148.31, 148.92, 151.84, and 165.39.
White solid. Yield 58.9%; m.p. 168–171°C; H NMR (400 MHz,
5
DMSO‐d
2 × CH ), 3.83 (s, 3H, OCH
m, 2H, C , CH), 7.33 (s, 1H, C
and 9.47 (s, 1H, OH); C NMR (101 MHz, DMSO‐d
6
) δ: 2.04 (s, 3H, COCH
), 6.78 (d, J = 7.9 Hz, 1H, C
), 7.44 (d, J = 15.2 Hz, 1H, CH),
) δ: 21.72, 56.26,
3
), 3.48 (br, 4H, 2 × CH
2
), 3.67 (br, 4H,
1
2
3
6 3
H ), 7.05–7.12
(
6
H
3
6 3
H
1
3
(
E)‐3‐(4‐Hydroxy‐3‐methoxyphenyl)‐1‐(4‐(2‐methoxyphenyl)‐
piperazin‐1‐yl)prop‐2‐en‐1‐one (1j)
White solid. Yield 65.2%; m.p. 123–125°C; H NMR (400 MHz,
DMSO‐d ) δ: 2.97 (br, 4H, 2 × CH ), 3.72 (br, 2H CH ), 3.80 (s, 3H,
OCH ), 3.84 (br, 5H, OCH , CH ), 6.78 (d, J = 8.0 Hz, 1H, C ), 6.90
m, 2H, C ), 6.97 (m, 2H, C ), 7.10–7.13 (m, 2H, C , CH), 7.36
s, 1H, C ), 7.45 (d, J = 15.1 Hz, 1H, CH), and 9.46 (s, 1H, OH);
) δ: 45.76, 51.35, 55.83, 56.26, 111.56,
12.40, 114.92, 115.90, 118.79, 121.31, 123.07, 123.32, 127.18,
41.27, 142.78, 148.33, 148.95, 152.51, and 165.31.
6
111.69, 114.80, 115.90, 123.04, 127.08, 142.99, 148.29, 149.00,
165.52, and 168.95.
1
6
2
2
3
3
2
6
H
3
(E)‐Ethyl‐4‐(3‐(4‐hydroxy‐3‐methoxyphenyl)acryloyl)‐
piperazine‐1‐carboxylate (1p)
(
(
6
H
4
H
6 4
6 3
H
1
3
1
H
6 3
C
White solid. Yield 96.7%, m.p. 140–142°C; H NMR (400 MHz,
NMR (101 MHz, DMSO‐d
6
DMSO‐d
(br, 2H, CH
(q, J = 6.8 Hz, 2H, OCH
m, 2H, C , CH), 7.33 (s, 1H, C
and 9.46 (s, 1H, OH); C NMR (101 MHz, DMSO‐d
6
) δ: 1.20 (t, J = 6.7 Hz, 3H, CH
), 3.71 (br, 2H, CH ), 3.83 (s, 3H, OCH
), 6.78 (d, J = 8.0 Hz, 1H, C ), 7.03–7.11
), 7.44 (d, J = 15.2 Hz, 1H, CH),
) δ: 15.01, 56.26,
3
), 3.41 (br, 4H, 2 × CH
2
), 3.57
1
1
2
2
3
), 4.07
2
6 3
H
(
6
H
3
6 3
H
1
3
(
E)‐1‐(4‐(4‐Chlorophenyl)piperazin‐1‐yl)‐3‐(4‐hydroxy‐3‐
methoxyphenyl)prop‐2‐en‐1‐one (1k)
Brownish yellow solid. Yield 87.9%; m.p. 136–138°C; H NMR
400 MHz, DMSO‐d ) δ: 3.19 (br, 4H, 2 × CH ), 3.74 (br, 2H, CH ),
.87 (m, 5H, CH , OCH ), 6.81 (d, J = 8.0 Hz, 1H, C ), 7.02
), 7.13–7.16 (m, 2H, + CH), 7.28
), 7.38 (s, 1H, C ), 7.48 (d, J = 15.2 Hz, 1H,
) δ: 45.14,
9.27, 56.28, 111.71, 114.83, 115.91, 117.73, 123.07, 123.25,
27.14, 129.14, 142.91, 148.31, 148.98, 150.09, and 165.3.
6
61.43, 111.68, 114.76, 115.92, 123.03, 127.09, 143.06, 148.32,
149.00, 155.13, and 165.57.
1
(
6
2
2
3
2
3
6
H
3
(E)‐tert‐Butyl‐4‐(3‐(4‐hydroxy‐3‐methoxyphenyl)acryloyl)‐
piperazine‐1‐carboxylate (1q)
(
d, J = 8.4 Hz, 2H,
d, J = 8.4 Hz, 2H, C
C
6
H
4
6 3
C H
1
(
6
H
4
H
6 3
White solid. Yield 94.3%, m.p. 191–193°C; H NMR (400 MHz,
1
3
CH), and 9.50 (s, 1H, OH); C NMR (101 MHz, DMSO‐d
6
CDCl
2 × CH
(d, J = 8.2 Hz, 1H, C
), and 7.63 (d, J = 15.2 Hz, 1H, CH).
CDCl ‐d ) δ: 28.39, 56.01, 80.38, 109.95, 113.97, 114.84, 122.07,
127.60, 143.61, 146.82, 147.62, 154.62, and 166.01.
3
) δ: 1.48 (s, 9H, 3 × CH
), 3.93 (s, 3H, OCH ), 6.70 (d, J = 15.2 Hz, 1H, COCH), 6.92
), 6.99 (s, 1H, C ), 7.09 (d, J = 8.2 Hz, 1H,
3 2
), 3.49 (br, 4H, 2 × CH ), 3.67 (br, 4H,
4
1
2
3
6
H
3
6 3
H
1
3
C
H
6 3
C NMR (101 MHz,
(
E)‐3‐(4‐Hydroxy‐3‐methoxyphenyl)‐1‐(4‐phenylpiperazin‐1‐yl)‐
prop‐2‐en‐1‐one (1l)
Yellow solid. Yield 95.0%; m.p. 158–160°C; H NMR (400 MHz,
DMSO‐d ) δ: 3.19 (br, 4H, 2 × CH ), 3.75 (br, 2H, CH ), 3.87 (s + br,
H, CH , OCH ), 6.81–7.28 (m, 8H, C , C , CH), 7.39 (s, 1H,
), 7.48 (d, J = 15.2 Hz, 1H, CH), and 9.50 (s, 1H, OH); C NMR
101 MHz, DMSO‐d ) δ: 45.28, 49.53, 56.28, 111.68, 114.88, 115.91,
16.34, 119.78, 123.07, 127.16, 129.45, 142.87, 148.32, 148.97,
51.31, and 165.37.
3
6
1
6
2
2
4
.1.3 | Synthesis of (E)‐3‐(4‐hydroxy‐3‐
5
2
3
H
6 5
6 3
H
methoxyphenyl)‐1‐(piperazin‐1‐yl)prop‐2‐en‐1‐one
hydrochloride (1r)
1
3
6 3
C H
(
6
1
1
A solution of compound 1q (1 mmol) in dry ethyl acetate (10 ml) was
passed into dry hydrogen chloride gas at room temperature and

10 of 13
CUI ET AL.
|
stirred for 2 hr confirmed by TLC. Then the precipitate was filtered
(E)‐N‐(4‐(tert‐Butyl)‐5‐(1H‐imidazol‐1‐yl)thiazol‐2‐yl)‐
and washed with ethyl acetate to afford the pure compound 1r.
3‐(4‐hydroxy‐3‐methoxyphenyl)acrylamide (2b)
1
1
White solid. Yield 95.9%; m.p. 196–198°C; H NMR (400 MHz,
Light yellow solid. Yield 38.2%, m.p. 244–246°C; H NMR (400 MHz,
DMSO‐d
6
) δ: 3.12 (br, 4H, 2 × CH
), 6.82 (d, J = 7.6 Hz, 1H, C
.34 (s, 1H, C ), 7.48 (d, J = 15.2 Hz, 1H, CH), 9.56 (s, 1H, OH), and
.60 (s, 2H, NH
11.85, 114.27, 115.96, 123.18, 126.94, 143.59, 148.33, 149.22,
2
), 3.83 (s, 3H, OCH
3
), 3.95 (br, 3H,
DMSO‐d
(d, J = 15.6 Hz, 1H, CH), 6.88 (d, J = 7.6 Hz, 1H, C
(m, 2H, N , C ), 7.21 (s, 1H, C ), 7.47 (s, 1H, N
(d, J = 15.6 Hz, 1H, CH), 7.94 (s, 1H, N ), 9.71 (s, 1H, OH), and
12.40 (s, 1H, NH); C NMR (101 MHz, DMSO‐d ) δ: 29.89, 35.76,
56.04, 112.18, 116.01, 116.34, 119.71, 122.62, 124.70, 126.17,
29.19, 143.91, 148.34, 149.86, 153.73, 153.89, and 164.57.
6
) δ: 1.15 (s, 9H, 3 × CH
3
), 3.85 (s, 3H, OCH
), 7.12–7.14
), 7.67
3
), 6.77
2
7
9
1
× CH
2
6
H
3
), 7.06–7.13 (m, 2H, C
6
3
H , CH),
6 3
H
6
H
3
C H
2 3 3
6
H
3
6
H
3
2 3 3
C H
13
2
); C NMR (101 MHz, CDCl
3
‐d
6
) δ: 43.25, 56.33,
2 3 3
C H
1
3
6
and 165.66.
1
(
E)‐N‐(4‐(tert‐Butyl)‐5‐(1H‐1,2,4‐triazol‐1‐yl)thiazol‐2‐yl)‐
4
.1.4 | Synthesis of (E)‐4‐(3‐(4‐(4‐hydroxyphenyl)‐
3
‐(4‐hydroxy‐3‐methoxyphenyl)acrylamide (2c)
piperazin‐1‐yl)‐3‐oxoprop‐1‐en‐1‐yl)benzoic acid (1s)
1
Light yellow solid. Yield 40.1%, m.p. 126–128°C; H NMR (400 MHz,
DMSO‐d ) δ: 1.14 (s, 9H, 3 × CH ), 3.85 (s, 3H, OCH ), 6.78
d, J = 15.6 Hz, 1H, CH), 6.88 (d, J = 8.0 Hz, 1H, ), 7.14
d, J = 8.0 Hz, 1H, C ), 7.22 (s, 1H, C ), 7.69 (d, J = 15.6 Hz, 1H,
), 8.98 (s, 1H, N ), 9.74 (s, 1H, OH),
and 12.53 (s, 1H, NH); C NMR (101 MHz, DMSO‐d ) δ: 29.74,
5.83, 56.05, 112.23, 115.91, 116.35, 119.03, 122.66, 126.15,
44.09, 148.33, 149.11, 149.90, 152.70, 154.73, 155.34, and 164.70.
To a solution of compound 1p (1 mmol) in ethanol (10 ml) was
added 2 N lithium hydroxide solution (2 ml) and stirred for 8 hr
confirmed by TLC. Then, dilute hydrochloric acid was added into
the reaction solution to adjust pH to 4–5. The precipitate was
filtered and washed with water to afford the pure product 1s.
6
3
3
(
(
6 3
C H
6
H
3
6 3
H
CH), 8.28 (s, 1H, N
3
C
2
H
2
3 2 2
C H
13
6
1
White solid. Yield 70.0%; m.p. 285–288°C; H NMR (400 MHz,
3
1
DMSO‐d
CH ), 6.68 (d, J = 8.3 Hz, 2H, C
.45 (d, J = 15.4 Hz, 1H, CH), 7.57 (d, J = 15.4 Hz, 1H, CH), 7.86 (d,
J = 7.9 Hz, 2H, C ), 7.96 (d, J = 7.9 Hz, 2H, C ), 8.94 (s, 1H,
OH), and 13.00 (s, 1H, COOH); C NMR (101 MHz, DMSO‐d ) δ:
5.59, 51.71, 116.01, 119.14, 121.10, 128.56, 130.10, 131.69,
39.79, 140.81, 164.57, and 167.36.
6
) δ: 2.98 (br, 4H, 2 × CH
2
), 3.72 (br, 2H, CH
2
), 3.86 (br, 2H,
2
6
H
4
), 6.84 (d, J = 8.3 Hz, 2H, C
6 4
H
),
7
(
E)‐Ethyl‐2‐(3‐(4‐hydroxy‐3‐methoxyphenyl)acrylamido)‐
‐methylthiazolyl‐5‐carboxylate (2d)
Light yellow solid. Yield 40.2%, m.p. 185–187°C; H NMR (400 MHz,
DMSO‐d ) δ: 1.29 (t, J = 7.0 Hz, 3H, CH ), 2.56 (s, 3H, CH ), 3.83
s, 3H, OCH ), 4.25 (q, J = 7.0 Hz, 2H, OCH ), 6.71 (d, J = 15.6 Hz, 1H,
CH), 6.85 (d, J = 8.0 Hz, 1H, C ), 7.12 (d, J = 8.0 Hz, 1H, C ), 7.21
s, 1H, C ), 7.67 (d, J = 15.6 Hz, 1H, CH), 9.70 (s, 1H, OH), and
12.54 (s, 1H, NH); C NMR (101 MHz, DMSO‐d
56.06, 61.23, 112.00, 115.86, 116.32, 116.67, 123.77, 125.92,
29.18, 148.44, 150.65, 153.49, 162.87, 167.69, and 191.54.
6
H
4
6 4
H
4
1
3
6
1
4
1
6
3
3
(
3
2
6
H
3
6 3
H
(
6 3
H
1
3
4
.1.5 | General procedure for the synthesis
6
) δ: 14.60, 17.48,
of compounds 2a–f
1
A mixture of ferulic acid derivative 3 (1.2 mmol), 1‐hydroxybenzo-
triazole (1.2 mmol), N‐(3‐dimethylaminopropyl)‐N’‐ethylcarbodiimide
hydrochloride (1.2 mmol) in N,N‐dimethylformamide (10 ml) was
stirred for 30 min and then 2‐aminothiazolyl derivative 4 (1 mmol)
was added. The mixture was stirred at 115°C for 8–16 hr confirmed
by TLC, and cooled at room temperature. The reaction solution was
poured into ice water. The precipitate was filtered and washed
successively with saturated sodium bicarbonate, dilute hydrochloric
acid solution, and water, recrystallized from ethanol to afford the
pure product 2a–f.
(
E)‐N‐(5‐Acetyl‐4‐methylthiazol‐2‐yl)‐3‐(4‐hydroxy‐3‐
methoxyphenyl)acrylamide (2e)
Yellow solid. Yield 34.6%, m.p. 256–258°C; H NMR (400 MHz,
DMSO‐d ) δ: 2.43 (s, 3H, CH ), 2.45 (s, 3H, CH ), 3.77 (s, 3H, OCH ),
.66 (d, J = 16.0 Hz, 1H, CH), 6.80 (d, J = 8.0 Hz, 1H, C ), 7.06
d, J = 8.0 Hz, 1H, C ), 7.15 (s, 1H, C ), 7.61 (d, J = 16.0 Hz,
H, CH), 9.69 (s, 1H, OH), and 12.49 (s, 1H, NH); C NMR (101 MHz,
1
6
3
3
3
6
6 3
H
(
6
H
3
6 3
H
1
3
1
6
DMSO‐d ) δ: 18.59, 30.50, 56.07, 112.04, 115.90, 116.28, 122.95,
126.02, 126.09, 144.39, 148.34, 149.95, and 154.99.
(
E)‐N‐(4‐(tert‐Butyl)‐5‐nitrothiazol‐2‐yl)‐3‐(4‐hydroxy‐3‐
methoxyphenyl)acrylamide (2a)
Red solid. Yield 36.8%, m.p. 262–263°C; H NMR (400 MHz, DMSO‐
) δ: 1.50 (s, 9H, 3 × CH ), 3.88 (s, 3H, CH ), 6.82 (d, J = 15.6 Hz, 1H,
CH), 6.91 (d, J = 8.0 Hz, 1H, C ), 7.19 (d, J = 8.0 Hz, 1H, C ), 7.26
s, 1H, C ), 7.79 (d, J = 15.6 Hz, 1H, CH), 9.81 (s, 1H, OH), and
(E)‐Ethyl‐2‐(2‐(3‐(4‐Hydroxy‐3‐methoxyphenyl)acrylamido)‐
thiazol‐4‐yl)acetate (2f)
1
1
Light yellow solid. Yield 32.1%, m.p. 181–183°C; H NMR (400 MHz,
d
6
3
3
DMSO‐d
(s, 3H, OCH
CH), 6.85 (d, J = 8.0 Hz, 1H, C
(d, J = 8.0 Hz, 1H, C ), 7.18 (s, 1H, C
CH), 9.64 (s, 1H, OH), and 12.24 (s, 1H, NH); C NMR (101 MHz,
DMSO‐d ) δ: 14.57, 37.19, 56.07, 60.79, 111.03, 112.05, 116.32,
6
) δ: 1.19 (t, J = 7.0 Hz, 3H, CH
), 4.09 (q, J = 7.1 Hz, 2H, OCH
), 6.99 (s, 1H, SNC
), 7.62 (d, J = 15.6 Hz, 1H,
3
), 3.70 (s, 2H, CH
), 6.69 (d, J = 15.6 Hz, 1H,
H), 7.09
2
), 3.83
H
6 3
6
H
3
3
2
(
6
H
3
6
H
3
3
13
1
5
1
3.01 (s, 1H, NH); C NMR (101 MHz, DMSO‐d
6
) δ: 28.48, 36.95,
6
H
3
6 3
H
1
3
6.03, 112.28, 115.09, 116.36, 123.13, 125.94, 137.86, 145.55,
48.37, 150.31, 158.34, 163.32, and 165.55.
6

CUI ET AL.
11 of 13
|
1
1
16.56, 122.59, 126.29, 143.30, 144.31, 148.35, 149.71, 158.36,
64.11, and 170.53.
56.13, 112.19, 115.36, 115.94, 118.93, 120.92, 126.87, 144.40,
147.74, 148.50, 151.82, and 169.62.
4
.1.6 | Synthesis of (2E)‐3‐(4‐hydroxy‐3‐
methoxyphenyl)‐N‐(5‐(1‐(methoxyimino)‐ethyl)‐
‐methylthiazol‐2‐yl)acrylamide (2g)
Ethyl 4‐(4‐hydroxy‐3‐methoxybenzoyl)piperazine‐1‐carboxylate (3b)
1
White solid. Yield 88.7%, m.p. 137–139°C; H NMR (400 MHz,
4
DMSO‐d
6
) δ: 1.21 (t, J = 7.0 Hz, 3H, CH
br, 4H, 2 × CH ), 3.81 (s, 3H, OCH ), 4.08 (q, J = 7.0 Hz, 2H, OCH
.83 (d, J = 8.0 Hz, 1H, C ), 6.89 (d, J = 8.0, 1H, C ), 6.99 (s, 1H,
), and 9.49 (s, 1H, OH); C NMR (101 MHz, DMSO‐d ) δ: 15.02,
3
), 3.44 (br, 4H, 2 × CH
2
), 3.51
(
2
3
2
),
To a solution of compound 2e (0.5 mmol) in N,N‐dimethylformamide
5 ml) was added the mixed aqueous solution (2 ml) of methoxyam-
6
6
H
3
6 3
H
(
1
3
C H
6 3
6
monium chloride (1 mmol) and sodium acetate (1 mmol). Then the
mixture was stirred at 80°C for 10 hr confirmed by TLC and cooled at
room temperature. The reaction solution was poured into ice water.
The precipitate was filtered and washed successively with saturated
4
1
3.77, 56.12, 61.37, 112.22, 115.38, 120.99, 126.62, 147.72, 148.61,
55.08, and 169.87.
sodium bicarbonate and water to afford the pure product 2g. White
tert‐Butyl 4‐(4‐hydroxy‐3‐methoxybenzoyl)‐
piperazine‐1‐carboxylate (3c)
1
solid. Yield 60.6%, m.p. 133–135℃; H NMR (400 MHz, DMSO‐d
6
)
δ: 2.23 (s, 2H, C
3
NSCH
, Z‐type), 2.45 (s, 2H, CH
s, 3H, NOCH ), 6.72 (d, J = 15.6 Hz, 1H, CH), 6.87 (d, J = 8.0 Hz, 1H,
), 7.13 (d, J = 8.0 Hz, 1H, C ), 7.22 (s, 1H, C ), 7.65
d, J = 15.6 Hz, 1H, CH), 9.70 (s, 1H, OH), and 12.24 (s, 1H, NH).
3
, E‐type), 2.43 (s, 1H, C
3
NSCH
3
, Z‐type), 2.26
1
White solid. Yield 64.3%, m.p. 56–58°C; H NMR (400 MHz, DMSO‐
(
(
s, 1H, CH
3
3
, E‐type), 3.85 (s, 3H, OCH
3
), 3.90
d
6
) δ: 1.41 (s, 9H, 3 × CH
× CH ), 3.79 (s, 3H, OCH
d, J = 8.0 Hz, 1H, C ), 6.96 (s, 1H, C
NMR (101 MHz, DMSO‐d ) δ: 28.49, 44.14, 56.11, 79.63, 112.18,
15.37, 120.98, 126.66, 147.70, 148.57, 154.30, and 169.86.
3
), 3.36 (br, 4H, 2 × CH
), 6.80 (d, J = 8.0 Hz, 1H, C
), and 9.44 (s, 1H, OH);
2
), 3.43 (br, 4H,
3
2
2
3
6 3
H ), 6.86
6
C H
3
H
6 3
6 3
H
13
(
6
H
3
6
H
3
C
(
6
1
4
.1.7 | Synthesis of (E)‐2‐(2‐(3‐(4‐hydroxy‐3‐
methoxyphenyl)acrylamido)‐thiazol‐4‐yl)acetic
acid (2h)
4
.1.9 | General procedure for the synthesis
To a solution of compound 2f (1 mmol) in ethanol (10 ml) was added
of compounds 4a–c
2
N lithium hydroxide solution (2 ml) and stirred for 8 hr confirmed
Acetyl vanillic acid is synthesized according to the method reported
by TLC. Then dilute hydrochloric acid was added into the reaction
[24]
in reference.
White solid. Yield 76.1%, m.p. 135–136°C. Com-
solution to adjust pH to 4–5. The precipitate was filtered and washed
pounds 10a–c were synthesized by the same method as for the
synthesis of compound 2. Compounds 10a–c were hydrolyzed by
the same method as for the synthesis of compound 2h to produce the
target compounds 4a–c.
with water to afford the pure product 2h. White solid. Yield 65.9%,
1
m.p. 210–212°C; H NMR (400 MHz, DMSO‐d
6
) δ: 3.62 (s, 2H, CH
), 6.69 (d, J = 15.6 Hz, 1H, CH), 6.85 (d, J = 8.0 Hz,
), 6.96 (s, 1H, SNC H), 7.10 (d, J = 8.0 Hz, 1H, C ), 7.19
), 7.63 (d, J = 15.6 Hz, 1H, CH), 9.63 (s, 1H, OH), 12.24
2
),
3.83 (s, 3H, OCH
3
1H, C
6
H
3
3
6 3
H
(s, 1H, C
6
H
3
1
3
(
s, 1H, NH), and 12.33 (s, 1H, COOH); C NMR (101 MHz, DMSO‐d
6
)
Ethyl 2‐(4‐hydroxy‐3‐methoxybenzamido)‐4‐methylthiazolyl‐
δ: 37.38, 56.07, 110.76, 112.02, 116.30, 116.58, 122.58, 126.28,
43.23, 144.88, 148.33, 149.67, 158.15, 164.06, and 172.05.
5
‐carboxylate (4a)
White solid. Yield 95.0%, m.p. 238–241°C; H NMR (400 MHz,
DMSO‐d ) δ: 1.32 (t, J = 7.2 Hz, 3H, CH ), 2.61 (s, 3H, CH ), 3.89
s, 3H, OCH ), 4.28 (q, J = 7.1 Hz, 2H, CH ), 6.93 (d, J = 8.4 Hz,
H, C ), 7.67 (d, J = 8.4, 2.0 Hz, 1H, C ), 7.77 (s, 1H, C ), 10.07
s, 1H, OH), and 12.79 (s, 1H, NH); C NMR (101 MHz, DMSO‐d
δ: 14.68, 17.43, 56.22, 60.93, 112.28, 114.51, 115.72, 122.43, 123.12,
47.81, 151.81, 156.51, 161.11, 162.70, and 165.44.
1
1
6
3
3
(
3
2
4
.1.8 | General procedure for the synthesis
1
H
6 3
6
H
3
6 3
H
of compounds 3a–c)
1
3
(
6
)
The same method as for the synthesis of compounds 1a–o was
carried out with vanillic acid and piperazine derivatives as raw
materials and dichloromethane as the solvent to afford the
products 3a–c.
1
N‐(5‐Acetyl‐4‐methylthiazol‐2‐yl)‐4‐hydroxy‐3‐
methoxybenzamide (4b)
(
4‐Hydroxy‐3‐methoxyphenyl)(4‐(4‐hydroxyphenyl)piperazin‐1‐yl)‐
methanone (3a)
Light yellow solid. Yield 53.6%, m.p. 148–150°C; H NMR (400 MHz,
DMSO‐d ) δ: 2.98 (br, 4H, 2 × CH ), 3.64 (br, 4H, 2 × CH ), 3.81 (s, 3H,
OCH ), 6.69 (d, J = 8.0 Hz, 2H, C ), 6.83–6.85 (m, 3H, C
), 6.90 (d, J = 7.2 Hz, 1H, C ), 7.01 (s, 1H, C ), 8.93 (s, 1H,
OH), and 9.48 (s, 1H, OH); C NMR (101 MHz, DMSO‐d ) δ: 50.95,
1
Brownish yellow solid. Yield 84.6%, m.p. 230–232°C; H NMR
(400 MHz, DMSO‐d ) δ: 2.47 (s, 3H, CH ), 2.59 (s, 3H, CH ), 3.86
(s, 3H, OCH ), 6.89 (d, J = 8.4 Hz, 1H, C ), 7.64 (d, J = 8.4 Hz, 1H,
), and 7.75 (s, 1H, C ); C NMR (101 MHz, DMSO‐d ) δ:
1
6
3
3
6
2
2
3
6 3
H
1
3
3
6
H
4
H
6 3
+
C H
6 3
H
6 3
6
C
6
H
4
6
H
3
6
H
3
18.58, 30.55, 56.17, 112.33, 115.62, 123.02, 123.57, 125.03, 151.51,
154.97, 162.45, 166.21, and 190.90.
1
3
6

12 of 13
CUI ET AL.
|
N‐(4‐(tert‐Butyl)‐5‐(1H‐imidazol‐1‐yl)thiazol‐2‐yl)‐4‐hydroxy‐
‐methoxybenzamide (4c)
White solid. Yield 88.9%, m.p. 165–167°C; H NMR (400 MHz,
DMSO‐d ) δ: 1.18 (s, 9H, 3 × CH ), 3.90 (s, 3H, OCH ), 6.93
d, J = 8.2 Hz, 1H, C ), 7.09 (s, 1H, N ), 7.47 (s, 1H, N ),
.67 (dd, J = 8.2, 1.8 Hz, 1H, C ), 7.78 (d, J = 1.8 Hz, 1H, C ), 7.94
), 10.10 (s, 1H, OH), and 12.53 (s, 1H, NH); C NMR
101 MHz, DMSO‐d ) δ: 29.87, 35.79, 56.27, 112.33, 115.63, 119.64,
22.55, 123.07, 124.69, 129.23, 141.34, 147.76, 151.68, 153.75,
54.53, and 165.38.
ACKNOWLEDGMENTS
3
This study was financially supported by the Hunan Provincial
Natural Science Foundation of China (No. 2019JJ40030).
The Institute of the Chinese Academy of Medical Sciences and
Peking Union Medical College provided the biological activity
assays.
1
6
3
3
(
6
H
3
2
C
H
3 3
2 3 3
C H
7
6
H
3
6 3
H
3
1
(
(
2 3 3
s, 1H, N C H
6
CONFLICT OF INTEREST
1
1
The authors declare no conflict of interest.
ORCID
4
.2 NA inhibition assay
|
Ai‐Xi Hu
http://orcid.org/0000-0001-8676-6020
The biological test method is according to the methods reported in
[
25]
the literature.
All the target compounds were tested for their
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assays based on the method reported by Zhang et al.
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The compound 2′‐(4‐methylumbellifer‐yl)‐α‐D‐acetyl neuraminic acid
MUNANA) was purchased from Sigma‐Aldrich as the substrate of
The
[28]
(
NA; and cleavage of this substrate by NA produces a fluorescent
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[29]
excitation wavelength of 360 nm.
The intensity of fluorescence
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can reflect the activity of NA sensitively.
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morpholino)ethanesulfonic acid; pH 6.5) was added to a 96‐well
microtiter plate. Meanwhile, blank, enzyme, and positive drug
oseltamivir were set. The reaction was started by the addition of
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the substrate containing 10 μl of 4 mmol/l CaCl
2
,
20 μl of
MUNANA and 30 μl water to the plate. After
incubation for 60 min, the reaction was terminated by adding 150 μl
4 mmol/l NaOH in 83% ethanol. The resulting fluorescence was
−
1 −1
2
0 μg·mmol ·l
1
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quantified at an excitation wavelength of 360 nm and an emission
wavelength of 450 nm. The data are expressed as the mean of
three independent experiments. The IC50 was calculated by plotting
the percent of inhibition of NA activity versus the inhibitor
concentration.
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4
.3 Docking study
|
1
0877.
[
[
14] M. Hariono, N. Abdullah, K. V. Damodaran, E. E. Kamarulzaman,
The crystal structure data of H1N1 NA–oseltamivir complex (PDB
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docked compounds 1p, 2d, 3b, and 4a were energy‐minimized using
the MM2 force‐field. Then, LeDock was used to have the docking
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Z
max = 30.2). The results were analyzed and visualized by PyMOL.
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106317049 B 2018.
[22]
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2
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Additional supporting information may be found online in the
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Bioassay of ferulic acid derivatives as influenza neuraminidase
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