Novel tetrahydrobenzo[b]thiophen-2-yl)urea derivatives as novel α-glucosidase inhibitors: Synthesis, kinetics study, molecular docking, and in vivo anti-hyperglycemic evaluation

Article abstract of DOI:10.1016/j.bioorg.2021.105236

α-Glucosidase inhibitors, which can inhibit the digestion of carbohydrates into glucose, are one of important groups of anti-type 2 diabetic drugs. In the present study, we report our effort on the discovery and optimization of α-glucosidase inhibitors with tetrahydrobenzo[b]thiophen-2-yl)urea core. Screening of an in-house library revealed a moderated α-glucosidase inhibitors, 5a, and then the following structural optimization was performed to obtain more efficient derivatives. Most of these derivatives showed increased inhibitory activity against α-glucosidase than the parental compound 5a (IC₅₀ of 26.71 ± 1.80 μM) and the positive control acarbose (IC₅₀ of 258.53 ± 1.27 μM). Among them, compounds 8r (IC₅₀ = 0.59 ± 0.02 μM) and 8s (IC₅₀ = 0.65 ± 0.03 μM) were the most potent inhibitors, and showed selectivity over α-amylase. The direct binding of both compounds with α-glucosidase was confirmed by fluorescence quenching experiments. Kinetics study revealed that these compounds were non-competitive inhibitors, which was consistent with the molecular docking results that compounds 8r and 8s showed high preference to bind to the allosteric site instead of the active site of α-glucosidase. In addition, compounds 8r and 8s were not toxic (IC₅₀ > 100 μM) towards LO2 and HepG2 cells. Finally, the in vivo anti-hyperglycaemic activity assay results indicated that compounds 8r could significantly decrease the level of plasma glucose and improve glucose tolerance in SD rats treated with sucrose. The present study provided the tetrahydrobenzo[b]thiophen-2-yl)urea chemotype for developing novel α-glucosidase inhibitors against type 2 diabetes.

Full text of DOI:10.1016/j.bioorg.2021.105236

Bioorganic Chemistry 115 (2021) 105236
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel tetrahydrobenzo[b]thiophen-2-yl)urea derivatives as novel
α
-glucosidase inhibitors: Synthesis, kinetics study, molecular docking, and
in vivo anti-hyperglycemic evaluation
Hong-Xu Xiea 1, Juan Zhanga 1, Yue Lia, Jin-He Zhanga, Shan-Kui Liua, Jie Zhangb,
,
,
Hua Zhengb, Gui-Zhou Haob *, Kong-Kai Zhua *, Cheng-Shi Jianga
,
,
c,
,*
a School of Biological Science and Technology, University of Jinan, Jinan 250022, China
b Lunan Pharmaceutical Group Co., Ltd., Linyi 273400, China
c Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
α
-Glucosidase inhibitors, which can inhibit the digestion of carbohydrates into glucose, are one of important
groups of anti-type 2 diabetic drugs. In the present study, we report our effort on the discovery and optimization
of -glucosidase inhibitors with tetrahydrobenzo[b]thiophen-2-yl)urea core. Screening of an in-house library
revealed a moderated -glucosidase inhibitors, 5a, and then the following structural optimization was performed
to obtain more efficient derivatives. Most of these derivatives showed increased inhibitory activity against
-glucosidase than the parental compound 5a (IC50 of 26.71 ± 1.80 M) and the positive control acarbose (IC50
of 258.53 ± 1.27 M). Among them, compounds 8r (IC50 = 0.59 ± 0.02 M) and 8s (IC50 = 0.65 ± 0.03 M)
were the most potent inhibitors, and showed selectivity over -amylase. The direct binding of both compounds
Tetrahydrobenzo[b]thiophen-2-yl)urea
α
-Glucosidase inhibitors
α
Non-competitive inhibitor
Molecular docking
α
Anti-hyperglycaemic activity
Type 2 diabetes
α
μ
μ
μ
μ
α
with α-glucosidase was confirmed by fluorescence quenching experiments. Kinetics study revealed that these
compounds were non-competitive inhibitors, which was consistent with the molecular docking results that
compounds 8r and 8s showed high preference to bind to the allosteric site instead of the active site of
α
-glucosidase. In addition, compounds 8r and 8s were not toxic (IC50 > 100 μM) towards LO2 and HepG2 cells.
Finally, the in vivo anti-hyperglycaemic activity assay results indicated that compounds 8r could significantly
decrease the level of plasma glucose and improve glucose tolerance in SD rats treated with sucrose. The present
study provided the tetrahydrobenzo[b]thiophen-2-yl)urea chemotype for developing novel
α-glucosidase in-
hibitors against type 2 diabetes.
1. Introduction
blood glucose [9]. The glucose in the blood comes from the hydrolysis of
carbohydrates catalyzed by enzymes such as -glucosidase and
-amylase [10]. -Glucosidase is a key glycoside hydrolase specifically
hydrolyzing 1,4- -glucopyranosides bond to produce -glucose [11,12].
Inhibition of
α
Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder
characterized by insulin resistance and pancreatic islet β-cell failure [1],
and accounts for 80% to 90% of all diabetic patients [2]. According to
the statistics of the International Diabetes Federation report, about 403
million people were diagnosed with diabetes in 2019 worldwide, and its
prevalence was estimated to be 700 million by 2045 [3]. Diabetes can
increase the risk of various health complications, including stroke [4,5],
peripheral artery disease [6], and cancer [7], and it also affects the
kidneys, heart, and nerves by causing varying degrees of damage [8].
The main strategy for controlling diabetes is to lower the level of
α
α
α
α
α
-glucosidase could retard the absorption of carbohydrates
at the small intestine and reduce the postprandial blood glucose levels
[13,14], while side effects including flatulence are possible as a conse-
quence of bacterial fermentation if simultaneous strong inhibition of
α
-amylases by drugs result in undigested polysaccharides entering the
colon [15,16]. Moreover,
as therapeutic drugs for T2DM. Thus,
one of the most promising targets for the treatment of diabetes [17].
α
-glucosidase inhibitors have been marketed
α-glucosidase enzyme was taken as
* Corresponding authors at: School of Biological Science and Technology, University of Jinan, Jinan 250022, China (C.-S. Jiang).
E-mail addresses: haoguizhouhgz@163.com (G.-Z. Hao), bio_zhukk@ujn.edu.cn (K.-K. Zhu), bio_jiangcs@ujn.edu.cn (C.-S. Jiang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.105236
Received 27 May 2021; Received in revised form 26 July 2021; Accepted 1 August 2021
Available online 8 August 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
Attracted by the important biological properties of
α
-glucosidase, me-
which were then reacted with compound 2f to give the final compounds
8b-8v and 9a-9g, respectively.
dicinal chemists have developed a variety of α-glucosidase inhibitors
[17–30]. Currently, three approved
α
-glucosidase inhibitors are carbo-
As shown in Scheme 3, the synthetic intermediates 2h-2m were
obtained by the reaction of compounds 1h-1m with malononitrile in the
presence of sulfur. 2,4-Dimethoxybenzylamine 3r was reacted with CDI
to produce intermediate 6r, which was then reacted with 2h-2m to
afford the thiophen-urea derivatives 10a-10f. All of the 1H and 13C
NMR, and HRESIMS spectra of target compounds can be found in Sup-
plementary Material.
hydrate mimics, including acarbose, voglibose, and miglitol (Fig. 1) used
in reducing postprandial hyperglycemia [17,31–33]. However, some
undesired effects, including flatulence, diarrhea, and stomach ache,
limited their clinical application [34]. Therefore, there is an urgent need
to discover novel
In our project to identify new
in-house compounds were screened for their activities towards
α
-glucosidase inhibitors to overcome these side effects.
α
-glucosidase inhibitors [35,36], our
α
-glucosidase at an initial concentration of 100 μM, with acarbose as a
2.2.
α
-Glucosidase inhibition assay and SAR study
-glucosidase inhibitory activity of all the target compounds was
positive control. The preliminary results of bioassay revealed that a
compound 5a possessing tetrahydrobenzo[b]thiophen-2-yl)urea core
The
α
showed 87.76% inhibitory activity against α-glucosidase, and its IC50
tested by in vitro enzyme assay, with acarbose as the positive control.
The inhibition rates of tested compounds at 100 M and IC50 values for
compounds with >50% inhibition at 100 M were measured and dis-
value was determined to be 26.71 ± 1.80 μM. To further improve the
μ
α
-glucosidase inhibitory activity of 5a, a systematic structural modifi-
μ
cation was carried out. In this study, a series of thiophen- and thiazole-
urea derivatives were synthesized and evaluated for their activities, and
the structure–activity relationships (SAR) study was discussed. Further,
the direct binding of the most potent compounds 8r and 8s with
played in Table 1. The first round of structural modification towards hit
5a concerned the replacement of cyclohexyl by other ring systems, such
as methyl cyclohexyl (5b), pyrazyl (5c), and 1-methylpiperidinyl (5d),
to explore its effect on the activity. However, the results showed that
α
-glucosidase was confirmed by fluorescence quenching experiments,
none of these three analogues was active towards α-glucosidase.
and their inhibition mechanisms were investigated using molecular
docking and kinetic studies. Finally, the in vitro cytotoxicity and in vivo
anti-hyperglycemic effect of 8r and 8s were also evaluated. Herein, we
report the synthesis, in vitro and in vivo biological evaluations of a series
Then the replacement of carboxylate group with cyano group in the
second round led to the production of 5e-5k possessing different ring
systems fused with thiophen. Interestingly, compared with 5a, a more
potent compound 5f (IC50 of 10.18 ± 0.28 μM) was found, suggesting
of tetrahydrobenzo[b]thiophen-2-yl)urea-based novel
α-glucosidase
that the moiety of 3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophenol core
was favorable for the inhibitory activity, and especially, the cyclohexyl
played a critical role in the activity since other ring systems connected to
thiophenol made the activity of compounds (5e, 5g-5j, and 5k) dimin-
ished. During the third round of optimization, three analogues 5l-5n
were prepared by using thiazole to replace the thiophenol ring. How-
inhibitors.
2. Results and discussion
2.1. Chemical synthesis
ever, these compounds did not exhibit activity towards α-glucosidase,
The synthetic routes of the target thiophen- and thiazole-urea de-
rivatives are outlined in Schemes 1–3, and their structures are displayed
in Tables 1–3. As shown in Scheme 1, the synthetic intermediates 2a-2k
were obtained by the reaction of compounds 1a-1d with cyanoacetate or
the reaction of 1a-1g with malononitrile in the presence of sulfur [37].
p-Methoxybenzyl amine 3a is reacted with N,N’-carbonyldiimidazole
(CDI) to form intermediate 4a [38], which was then reacted with 2a-2k
to afford the thiophen-urea derivatives 5a-5k, while the thiazole-urea
derivatives 5l-5n were obtained by treating 4a with the commercially
available thiazole-2-amines 2l-2n [37].(See Table 4)
with low inhibition rate of 26.43%~2.02% at 100 μM. Therefore, based
on these preliminary results, it was deduced that the 3-cyano-4,5,6,7-
tetrahydrobenzo[b]thiophenol moiety was a key fragment for the
α
-glucosidase inhibitory activity.
In the subsequent round, the 3-cyano-4,5,6,7-tetrahydrobenzo[b]
thiophenol moiety was conserved, and different substituents were
introduced to the benzene ring, resulting into the production of ana-
logues 8b-8v. From the results in Table 2, it was observed that most of
compounds in this series showed improved activity compared to 5f.
Several compounds, such as 8b, and 8p-8s containing the alkoxy group/
s at benzene ring, have lower IC50 values (1.67 ± 0.08 to 0.59 ± 0.02
Compounds 8b-8v and 9a-9g were prepared as shown in Scheme 2.
The starting materials of substituted benzylamines 3 were reacted with
CDI to produce the corresponding intermediates 6b-6v and 7a-7g,
μ
M), indicating that methoxyphenyl and ethoxyphenyl were key phar-
macophores. Whereas the introduction of the bulky phenyl groups (8t-
Fig. 1. Approved
α
-glucosidase inhibitors (A), and the α-glucosidase inhibitors (B) reported in this study.
2

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
Scheme 1. Synthesis of compounds 2a-2k and 5a-5n. Reagents and conditions: (a) S8, Morpholine, EtOH, 60 ℃, 4–7 h; (b) S8, NaAlO2, EtOH, 60 ℃, 4–7 h; (c) CDI,
isopropyl alcohol hydrochloride, DMF, DCM, rt., 3–4 h; (d) NaH, DCM, r.t., 8–10 h.
Scheme 2. Synthesis of compounds 8b-8v and 9a-9g. Reagents and conditions: (a) CDI, isopropyl alcohol hydrochloride, DMF, DCM, r.t., 3–4 h; (b) 2f, NaH, DCM, r.
t., 8–10 h.
Scheme 3. Synthesis of compounds 2l-2q and 10a-10f. Reagents and conditions: (a) S8, Morpholine, EtOH, 60 ℃, 4–7 h; (b) CDI, isopropyl alcohol hydrochloride,
DMF, DCM, r.t., 3–4 h; (c) 2l-2q, NaH, DCM, rt., 8–10 h.
8v) led to the significant decrease or loss of activity, which probably be
attributed to the steric effect of naphthyl. In the meantime, another
series of analogues 9a-9g with substituents at N atom of urea was ob-
tained, however only one compound 9a showed decreased inhibitory
2.3. α-Amylase inhibition assay of compounds 8r and 8s
Next, the inhibitory activities of 8r and 8s against α-amylase were
evaluated with acarbose as a reference. As shown in Fig. 4, the com-
activity with an IC50 value of 4.81 ± 0.09
μM.
parison of their IC50 values indicated that compounds 8r and 8s
In the final round of structural modification, several ring systems
having more structural diversity were fused into the thiophene on basis
of the most potent inhibitor 8r to produce analogues 10a-10f, in which
the meta-dimethoxyphenyl fragment was conserved since it made the
activity improve significantly. However, the activity of all these com-
pounds was decreased or completely lost. The results of bioassay were
shown in Table 3.
exhibited high selectivity towards
α
-glucosidase over
α
-amylase by 51-
-amylase is
important because it could allow the hydrolysis of polysaccharides into
oligosaccharides catalyzed by -amylases to avoid the side effects of
drugs as mentioned above [15,16].
and 86-fold. Selective inhibition against α-glucosidase over α
α
3

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
Lineweaver-Burk plot analysis. As shown in Fig. 3A, the double recip-
rocal plots at different concentrations of substrate had the same
Michaelis constant (Km), while the Vmax decreased with decreasing
concentration of 8r and 8s. This behavior indicated that both com-
Table 1
Inhibitory activity of 5a-5n against
α
-glucosidase.
pounds were non-competitive inhibitors for α-glucosidase [39,40]. In
addition, as shown in Fig. 3B, the plot of the slop against the concen-
tration of 8r and 8s provided the steady-state inhibition constant (Ki),
Compound
R1
Group
Inhibition at 100
87.76
μM (%)
IC50 (μ
M)a
the values of which was estimated to be 8.6
respectively.
μM (8r) and 6.2 μM (8s),
5a
-CO2Et
26.71 ± 1.80
2.6. Homology modelling and molecular docking
b
5b
5c
-CO2Et
-CO2Et
22.71
24.54
-
Homology modelling was used to construct the 3-D structure of
–
α
-glucosidase (Saccharomyces cerevisiae organism, EC:3.2.1.20) with the
online program SWISS MODEL (https://swissmodel.expasy.org/).
Template search result (Table 5) indicated that 3AJ7 was the better
template compared with 3AXH [10]. Therefore, 3AJ7 was selected as
5d
5e
5f
-CO2Et
-CN
4.06
49.04
73.02
–
the template to construct the 3-D structure of α-glucosidase. To validate
–
the stability of 3-D structure of α-glucosidase, 2 ns molecular dynamics
simulation was performed. As illustrated in Fig. S1 (ESI†), the stable
root-mean-square deviation (RMSD) values for backbone atoms of the
-CN
10.18 ± 0.28
α
-glucosidase indicated that the modeled structure was reliable.
Molecular docking simulation was performed to probe the binding
5g
-CN
52.80
–
modes of compounds 8r and 8s with α-glucosidase. As shown in Table 6,
both of the two compounds displayed higher docking scores when they
were docked to the allosteric site compared to the active site. The two
possible binding modes (the allosteric site and the active site) of 8r and
8s were shown in Fig. 4, respectively. These docking results were
consistent with the noncompetitive property of these two compounds
validated in the enzyme kinetic assay. The detailed interactions between
5h
5i
-CN
-CN
39.15
9.46
–
–
the two compounds (8r and 8s) with
shown in Fig. 5. It was seen that the hydrogen bonds, hydrophobic and
the stacking interactions were the main interactions that contributed
α-glucosidase (allosteric site) were
5j
-CN
-CN
ꢀ 0.17
–
π-π
to the binding affinity of inhibitors with the enzyme. The Fig. S2 (ESI†)
of the detailed ligand protein interactions showed that 8r established
two hydrogen bonds with Asn412 and Lys155 residues, and formed
hydrophobic interactions with Phe157, Gly159, Thr234, Ser235,
Leu237, His239, Glu304, Phe311, Arg312, Ile415, Phe420 residues, and
8s formed three hydrogen bonds with Asn241, Trp242, Pro309 residues,
and established hydrophobic interactions with Phe157, Phe231, His239,
Phe310, Phe311, Arg312, His279 residues. From these results of the
docking simulation, it can be found that 8r and 8s bind to allosteric sites
away from the active site (D214, E276 and D349) of the enzyme.
Together, all of the data indicated that compounds 8r and 8s were
5k
67.35
43.53 ± 1.18
5l
5-NO2
26.43
12.10
–
–
5m
5n
2.02
–
Acarbose
258.53 ± 1.27
a
IC50 value was expressed as mean ± SD from three independent experiments.
noncompetitive inhibitor of α-glucosidase.
b
IC50 value was not tested if inhibition rate was <50% at 100 μM.
2.7. Druglikeness property prediction
2.4. Compounds 8r and 8s quenched the intrinsic fluorescence of
α
-glucosidase
Discovery studio 3.0 and online molinspiration were used to predict
the druglikeness properties of 8r and 8s. From the results in Table 7, the
chemical properties of two compounds fell into the Lipinski’s “Rule of
five”, and were predicted to have acceptable solubility and absorption
level. Importantly, both of them may not bind to PPB and CYP2D6,
which possibly indicated fewer side effects of this series of compounds.
The direct interactions of 8r and 8s with
α-glucosidase were
confirmed by performing a fluorescence quenching experiment. As
shown in Fig. 2,
α-glucosidase exhibited an intrinsic fluorescence
emission peak at 335 after being excited at a wavelength of 290 nm.
After treatment with increasing concentrations of compounds 8r
(Fig. 2A) or 8s (Fig. 2B), the intrinsic fluorescence emission of
2.8. In vitro cytotoxicity
α
-glucosidase was gradually quenched in a concentration-dependent
manner, which thus confirmed the interactions of 8r and 8s with
-glucosidase.
Due to that the liver is the primary organ responsible for drug
metabolism, the effects of compounds 8r and 8s on the growth of human
normal hepatocyte (LO2) and human liver cancer (HepG2) cells were
evaluated using the MTT method, with acarbose as negative control and
doxorubicin as positive control [41]. As shown in Fig. 6 and Table 8, the
α
2.5. Kinetic study of 8r and 8s on enzyme inhibition
To further probe the inhibition mechanism of tetrahydrobenzo[b]
cell viability of LO2 cells treated with 100
100.4 ± 4.0%, 94.7 ± 2.4%, and 95.5 ± 1.8%, respectively, and the cell
viability of HepG2 cells treated with 100 M acarbose, 8r, and 8s were
95.2 ± 2.3%, 96.3 ± 2.5%, and 96.0 ± 1.6%, respectively, indicating
μM acarbose, 8r, and 8s were
thiophen-2-yl)urea derivatives on α-glucosidase, an inhibition kinetics
study was performed. The inhibition types of the most potent com-
μ
pounds 8r and 8s, based on their IC50 values, were tested using
4

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
Table 2
Inhibitory activities of 8b-8v and 9a-9g against α-glucosidase.
Compound
X
R1
Inhibition at 100
μM (%)
IC50
(μ
M)a
8b
8c
8d
8e
8f
2-OCH3
3-OCH3
H
H
91.64
79.46
64.59
49.38
68.75
51.34
37.38
89.14
76.54
75.49
70.28
84.54
42.29
71.24
81.37
89.57
88.58
91.12
53.51
36.22
13.62
77.83
21.24
19.28
8.15
1.67 ± 0.08
0.77 ± 0.03
63.11 ± 0.05
H
H
b
2-CH3
3-CH3
4-CH3
4-F
H
-
H
12.61 ± 0.86
8g
8h
8i
H
–
–
H
2-Cl
H
16.53 ± 1.05
8j
3-Cl
H
31.23 ± 1.03
8k
8l
4-Cl
H
34.55 ± 1.48
2-Br
H
9.08 ± 0.09
8m
8n
8o
8p
8q
8r
8s
8t
3-Br
H
16.19 ± 0.87
4-Br
H
–
4-OC2H5
2-OC2H5
3,4-OCH3
2,4-OCH3
3,5-OCH3
H
22.18 ± 1.52
H
0.96 ± 0.04
H
1.41 ± 0.09
H
0.59 ± 0.02
H
0.65 ± 0.03
H
89.67 ± 0.79
8u
8v
9a
9b
9c
9d
9e
9f
H
–
–
4-Ph
H
H
-CH3
-CH3
-CH3
-Et
-i-pr
-Bz
-CH3
4.81 ± 0.09
2-CH3
4-CH3
H
–
–
–
–
–
–
H
14.78
4.51
H
9g
8.88
a
IC50 value was expressed as mean ± SD from three independent experiments.
b
IC50 value was not tested if inhibition rate was <50% at 100 μM.
Table 3
Table 4
Inhibitory activities of 10a-10f against
α-glucosidase.
Inhibitory activities of 8r and 8s against α-amylase.
Compounds
IC50
(
μM)
Indexa
α-glucosidase
α-amylase
8r
0.59 ± 0.02
0.65 ± 0.03
258.53 ± 1.27
30.37 ± 0.58
56.14 ± 0.71
70.82 ± 0.89
51
8s
86
Compound
Group
Inhibition rate at 100
36.20
μM (%)
IC50
(μ
M)a
Acarbose
0.27
b
a
10a
-
IC50 (α-amylase)/IC50 (α-glucosidase).
that both compounds did not affect on the growth of LO2 and HepG2
10b
10c
75.42
9.14
7.17 ± 0.26
cells. The results disclosed that 8r and 8s had CC50 values >100 μM. To
be a potential drug, the ratio of CC50/IC50 (SI) should be equal or great
than 10. In the case of 8r and 8s, their SI values are much >150 indi-
cating that they are non-cytotoxic towards LO2 and HepG2 cell lines and
have selectivity towards the enzyme inhibition [18].
–
10d
10e
26.14
4.80
–
–
2.9. In vivo anti-hyperglycemic effects of 8r and 8s
The antihyperglycemic effects of compounds 8r and 8s on normal
rats were evaluated, with acarbose as a positive control. As shown in
Fig. 7A, the level of the control group increased rapidly after sucrose
treatment (81.00 ± 6.69 mg/dL at 0 min vs 183.45 ± 23.41 mmol/L at
20 min), and the glucose load basically reached the highest level at 30
min after sucrose treatment. In the administration groups, the levels of
blood glucose of rats treated with compounds 8r, 8s or acarbose were
lower than that of control group, and especially it began to decrease
after 45 min. Thus, it could be concluded that they had significant hy-
poglycemic effects. The results in Fig. 7B revealed that the areas under
10f
13.95
–
a
b
IC50 value was expressed as mean ± SD from three independent experiments.
IC50 value was not tested if inhibition rate was<50% at 100 μM.
5

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
Fig. 2. Variation of fluorescence emission spectra of
α
-glucosidase (1U/mL) without or in the presence of 8r (A) and 8s (B). The intrinsic fluorescence emission peak
of α-glucosidase was observed at 335 nm after being excited at 290 nm (left graph), and compounds 8r and 8s quenched the intrinsic fluorescence of enzyme in a
concentration-dependent manner (right graph).
Fig. 3. Kinetic analysis of compounds 8r and 8s interacting with
α-glucosidase. A: Lineweaver-Burk reciprocal plots of initial velocity and increasing substrate
(pNPG) concentration; B: the secondary plot of slopes versus the concentration of inhibitors.
the blood glucose curve (AUC) of the administration groups of 8r and
acarbose significantly decreased in 2 h vs control group, which indicated
that compound 8r could improve the oral sucrose tolerance. All these
results indicated that compound 8r could improve sucrose tolerance in
vivo and suppress the postprandial hyperglycemia by inhibiting
α-glucosidase.
3. Conclusion
In summary, a new tetrahydrobenzo[b]thiophen-2-yl)urea-based
6

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
Fig. 4. The proposed binding modes of compounds 8r, and 8s with
α
-glucosidase (A: allosteric site; B: active site). The
α
-glucosidase and compounds were shown as
surface and sticks, respectively. Residues in the active site (D214, E276 and D349) were also shown as sticks.
study, fluorescence quenching experiments, and molecular docking.
Finally, their in vitro cytotoxicities and in vivo anti-hyperglycaemic ef-
fects were evaluated.
Table 5
Template search results.
Template
Seq Identity
Resolution
Seq Similarity
GMQE
QMEAN
Among all the analogues, most of series of compounds 8, possessing
1-benzyl-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)urea
3AJ7
72.68
72.51
1.30 Å
1.80 Å
0.55
0.55
0.94
0.94
0.08
0.17
3AXH
framework, displayed promising α-glucosidase inhibitory activity, with
compounds 8r and 8s being the most potent inhibitors with IC50 values
of 0.96 ± 0.04 and 0.59 ± 0.02 μM, respectively. The SAR analysis
suggested that ring systems fused with a thiophenyl core could signifi-
cantly affect the activity of compounds, and a cyano group in the thio-
phene and alkoxyl groups one the benzene could improve the
Table 6
Molecular docking score results.
Compounds
Docking score (Allosteric site)
Docking score (Active site)
α
-glucosidase inhibitory activity. In addition, the replacement of thio-
8r
8s
ꢀ 4.665
ꢀ 5.503
ꢀ 4.389
ꢀ 3.832
phene by thiazole resulted in completely loss of activity. Molecular
docking studies revealed that the increase of the activities could be
attributed to hydrogen bonds, hydrophobic and the π-π stacking in-
α
-glucosidase inhibitor 5a was identified from our in-house compound
library, and then a series of its analogues were synthesized and evalu-
ated for their -glucosidase inhibitory activities. In addition, their in-
hibition types and binding with -glucosidase were explored by kinetic
teractions of compounds with the
the most potent compounds 8r and 8s towards
confirmed by fluorescence quenching experiments. Further kinetics
study suggested that both compounds inhibited -glucosidase via a
α
-glucosidase enzyme. The binding of
α
-glucosidase was
α
α
α
Fig. 5. The detailed ligand–protein interactions established between 8s (A)/8s (B) and allosteric site of
α
-glucosidase. The yellow dashed lines represent hydrogen
bonds, and residues involved in the hydrophobic and the π-π stacking interactions were labeled and shown as sticks. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Table 7
Prediction of druglikeness properties of 8r and 8s.
No.
MW
No. HBA
No. HBD
cLogP
No. Rotatable bond
5
Solubility level
Absorption level
PPB
CYP2D6
8r
8s
372
372
6
6
2
2
3.53
2
2
0
0
2.08196
1.18478
FALSE
FALSE
3.732
MW < 500; No. HBA < 10; No. HBD < 5; cLogp < 5; No. Rotatable bond < 10; Solubility Level: (0, Good; 1, Moderate; 2, Poor; 3, Very poor); Absorption Level: (0,
Good; 1, Moderate; 2, Poor; 3, Very poor); PPB: plasma protein binding; CYP2D6: Cytochrome P450 2D6 inhibition.
7

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
comparable anti-hyperglycemic activity with acarbose. As a result, these
compounds may be promising lead compounds for developing novel
α-glucosidase inhibitors used in type 2 diabetes treatment.
4. Materials and methods
4.1. General
The
α
-glucosidase of Saccharomyces cerevisiae (EC 3.2.1.20), porcine
pancreatic
α
-amylase (EC 3.2.1.1), and substrate pNPG (N1377) were
purchased from Sigma-Aldrich. All other commercially available re-
agents were used without further purification. Organic solvents were
evaporated with reduced pressure using a Büchi R-100 evaporator. Re-
actions were monitored by TLC using Yantai Jingyou (China) GF254
silica gel plates. Silica gel column chromatography was performed on
silica gel (200–300 mesh) from Qingdao Hailang (China). NMR spectra
were measured on Bruker Avance III 600 MHz spectrometers. Chemical
shifts were expressed in δ (ppm) and coupling constants (J) in Hz with
solvent signals as internal standards (CDCl3, δH 7.26 ppm and δC 77.2
Fig. 6. Effects of compounds 8r, 8s, acarbose, and doxorubicin at 100 μM on
the cell viability of LO2 and HepG2 cell lines. Data are expressed as mean ± SD
(n = 3), and ****P < 0.0001versus control group.
Table 8
The CC50 values of 8r and 8s towards LO2 and HepG2 cell lines
ppm; DMSO‑d6, δH 2.50 ppm and δ 39.5 ppm). ESI-MS analyses were
C
Compounds
CC50
(μM)
performed on an Agilent 1260–6460 Triple Quard LC-MS instrument
(Agilent, Waldbronn, Germany), and HR-ESI-MS data were acquired on
an Agilent Q-TOF 6520. Medium M199 and bovine serum were obtained
from Gibco Co. (Carlsbad, CA, USA).
LO2
HepG2
8r
>100
>100
>100
>100
8s
>100
Acarbose
Doxorubicin
>100
2.50 ± 0.08
1.32 ± 0.12
4.2. Synthesis of compounds
4.2.1. Synthesis of compounds 2a~2k
General method A:To a solution of cyclic ketone compounds in
anhydrous ethanol (conc. 0.1 M) was added sodium (6 % mmol), sulfur
(1.1 eq) and malononitrile (1 eq) or ethyl cyanoacetate (1 eq) at 60 ◦C.
The reaction mixture was stirred for 4–7 h. After the completion of the
reaction, the reaction solution was concentrated and purified by flash
column chromatography with solution of EtOAc/petroleum ether to
obtain compounds.
4.2.1.1. Ethyl
2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbox-
ylate (2a). Rf = 0.30 (EtOAc/petroleum ether = 1/15), 1H NMR (600
MHz, DMSO‑d6) δ 7.19 (s, 2H), 4.14 (q, J = 7.1 Hz, 2H), 2.59 (t, J = 5.7
Hz, 2H), 2.41 (t, J = 5.7 Hz, 2H), 1.72–1.60 (m, 4H), 1.23 (t, J = 7.1 Hz,
3H). 13C NMR (150 MHz, DMSO‑d6) δ 165.1, 162.9, 131.3, 115.5, 102.6,
58.6, 39.5, 26.5, 23.9, 22.8, 22.4, 14.4.
4.2.1.2. Ethyl 2-amino-6-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate (2b). Rf = 0.30 (EtOAc/petroleum ether = 1/20), 1H NMR
(600 MHz, CDCl3) δ 5.92 (s, 2H), 4.25 (q, J = 7.1 Hz, 2H), 2.91–2.83 (m,
1H), 2.63–2.56 (m, 1H), 2.56–2.52 (m, 1H), 2.13 (m, 1H), 1.90–1.79 (m,
2H), 1.33 (t, J = 7.1 Hz, 4H), 1.04 (d, J = 6.5 Hz, 3H). 13C NMR (150
MHz, CDCl3) δ 166.3, 161.9, 132.2, 117.4, 105.8, 59.5, 32.8, 31.3, 29.6,
26.9, 21.6, 14.6.
4.2.1.3. Ethyl 2-amino-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylate
1
(2c). Rf = 0.30 (EtOAc/petroleum ether = 1/6), H NMR (600 MHz,
Fig. 7. The effects of compounds 8r and 8s on the glucose level in normal rat
model. The SD rats were treated with or without 5 mg/kg tested compounds,
and then 7.5 g/kg sucrose solutions 30 min after oral administration, and the
glucose level was tested at 0–2 h. Data are mean ± SD, n = 12. *P < 0.05 and
**P < 0.001.
CDCl3) δ 6.00 (s, 2H), 4.55 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 3.90 (t, J =
5.6 Hz, 2H), 2.82 (tt, J = 5.6, 2.0 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13
C
NMR (150 MHz, CDCl3) δ 166.0, 162.3, 130.4, 114.9, 105.6, 65.3, 64.7,
59.7, 27.8, 14.6.
4.2.1.4. Ethyl 2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-
3-carboxylate (2d). Rf = 0.32 (EtOAc/petroleum ether = 1/6), 1H NMR
(600 MHz, CDCl3) δ 6.00 (s, 2H), 4.25 (q, J = 7.1 Hz, 2H), 3.36 (q, J =
1.7 Hz, 2H), 2.83 (t, J = 5.9 Hz, 2H), 2.65 (t, J = 5.9 Hz, 2H), 2.43 (s,
3H), 1.32 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 166.1, 162.2,
130.8, 114.7, 105.5, 59.6, 53.4, 52.6, 45.6, 27.5, 14.6.
noncompetitive mechanism, which is consistent with the molecular
docking results that these compounds were more likely to bind to an
allosteric site rather than the active site of α-glucosidase.
The results of in vitro cytotoxicity bioassay showed that compounds
8r and 8s had no significant effect on the growth of HepG2 and LO2 cells
even at 100 μM. Most importantly, compound 8r was demonstrated to
lower glucose levels in SD rats at a dose of 5 mg/kg, and to have a
8

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
4.2.1.5. 2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile
107–108 ℃; Rf = 0.55 (EtOAc/petroleum ether = 1/3), 1H NMR (600
MHz, CDCl3) δ 10.33 (s, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.6
Hz, 2H), 5.38 (s, 1H), 4.42 (d, J = 5.6 Hz, 2H), 4.20 (q, J = 7.1 Hz, 2H),
3.79 (s, 3H), 2.75 (t, J = 5.8 Hz, 2H), 2.60 (t, J = 5.8 Hz, 2H), 1.76 (dd, J
= 5.8, 2.2 Hz, 2H), 1.70 (dd, J = 5.8, 2.2 Hz, 2H)., 1.31 (t, J = 7.1 Hz,
3H). 13C NMR (150 MHz, CDCl3) δ 166.6, 159.2, 155.1, 153.5, 141.0,
141.0, 130.3, 129.2, 114.2, 106.0, 60.3, 55.4, 44.3, 25.1, 23.9, 22.7,
21.7, 14.4. HRMS (ESI+): m/z calcd for C20H24N2O4S+ [M + H]+:
389.4820, found: 389.4863.
1
(2e). Rf = 0.40 (EtOAc/petroleum ether = 1/6), H NMR (600 MHz,
CDCl3) δ 4.64 (s, 2H), 2.78–2.73 (m, 2H), 2.73–2.67 (m, 2H), 2.36 (p, J
= 7.1 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ 165.4, 142.1, 125.3, 115.7,
84.8, 29.4, 28.6, 27.5.
4.2.1.6. 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile
1
(2f). Rf = 0.30 (EtOAc/petroleum ether = 1/6), H NMR (600 MHz,
CDCl3) δ 4.68 (s, 2H), 2.53–2.42 (m, 4H), 1.78 (m, 4H). 13C NMR (150
MHz, CDCl3) δ 160.2, 132.4, 120.6, 115.7, 88.6, 24.6, 24.2, 23.4, 22.2.
4.2.3.2. Ethyl 2-(3-(4-methoxybenzyl)ureido)-6-methyl-4,5,6,7-tetrahy-
drobenzo[b]thiophene-3-carboxylate (5b). Light yellow solid; yield
42.3%; m.p.: 99.5–101 ℃; Rf = 0.60 (EtOAc/petroleum ether = 1/3), 1H
NMR (600 MHz, CDCl3) δ 10.64 (s, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.85 (d,
J = 8.6 Hz, 2H), 5.27 (t, J = 5.0 Hz, 1H), 4.41 (d, J = 5.6 Hz, 2H),
4.26–4.19 (m, 2H), 3.79 (s, 3H), 2.88 (m, 1H), 2.69–2.58 (m, 2H),
2.24–2.19 (m, 1H), 1.89–1.81 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H), 1.25 (d,
J = 2.1 Hz, 1H), 1.05 (d, J = 6.5 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ
167.2, 159.2, 153.6, 151.3, 130.2, 129.3, 124.8, 114.2, 114.1, 109.1,
60.3, 55.4, 53.6, 32.5, 31.4, 29.4, 26.4, 21.6, 14.4. HRMS (ESI+): m/z
calcd for C21H26N2O4+ [M + H]+: 403.1692, found: 403.1713.
4.2.1.7. 2-Amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-car-
bonitrile (2g). Rf = 0.30 (EtOAc/petroleum ether = 1/6), 1H NMR (600
MHz, CDCl3) δ 2.82–2.74 (m, 2H), 2.47–2.43 (m, 2H), 1.74 (dt, J = 9.5,
4.4 Hz, 2H), 1.70–1.66 (m, 2H), 1.57 (dt, J = 5.7, 2.7 Hz, 2H). 13C NMR
(150 MHz, CDCl3) δ 215.4, 111.9, 43.9, 36.3, 30.4, 29.0, 26.2, 24.3.
4.2.1.8. 2-Amino-4-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-car-
bonitrile (2i). Rf = 0.25 (EtOAc/petroleum ether = 1/5),1H NMR (600
MHz, CDCl3) δ 4.67 (s, 2H), 2.80 (m, 1H), 2.52–2.44 (m, 2H), 1.87–1.80
(m, 2H), 1.78–1.74 (m, 1H), 1.59–1.52 (m, 1H), 1.26 (d, J = 7.1 Hz, 3H).
13C NMR (151 MHz, CDCl3) δ 160.5, 137.1, 120.5, 115.9, 86.3, 30.2,
29.4, 24.4, 20.6, 19.9.
4.2.3.3. Ethyl 2-(3-(4-methoxybenzyl)ureido)-4,7-dihydro-5H-thieno[2,3-
c]pyran-3-carboxylate (5c). White solid; yield 32.3%; m.p.: 130.5–131.5
℃; Rf = 0.50 (EtOAc/petroleum ether = 1/2), 1H NMR (600 MHz,
CDCl3) δ 10.60 (s, 1H), 7.24 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H),
5.53 (s, 1H), 4.64 (s, 2H), 4.41 (d, J = 5.6 Hz, 2H), 4.22 (q, J = 7.1 Hz,
2H), 3.91 (t, J = 5.6 Hz, 2H), 3.79 (d, J = 1.0 Hz, 3H), 2.83 (m, 2H), 1.32
(t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 166.7, 159.2, 153.5,
151.8, 129.2, 128.3, 122.6, 114.2, 108.8, 65.2, 64.8, 60.5, 55.4, 44.3,
4.2.1.9. 2-Amino-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carbonitrile
1
(2J). Rf = 0.25 (EtOAc/petroleum ether = 1/4), H NMR (600 MHz,
DMSO‑d6) δ 7.11 (s, 2H), 4.41 (d, J = 2.1 Hz, 2H), 3.82 (t, J = 5.5 Hz,
2H), 2.48–2.35 (m, 2H). 13C NMR (150 MHz, DMSO‑d6) δ 164.0, 129.6,
116.3, 114.9, 83.2, 64.2, 64.1, 25.0.
27.2, 14.4. HRMS (ESI+): m/z calcd for C19H22N2O5 [M + H]+:
+
4.2.1.10. 2-Amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carbonitrile (2k). Rf = 0.50 (Acetone/petroleum ether = 2.5/1), 1H
NMR (600 MHz, DMSO‑d6) δ 7.03 (s, 2H), 3.23 (d, J = 2.1 Hz, 2H), 2.58
(t, J = 5.7 Hz, 2H), 2.42 (td, J = 5.7, 2.8 Hz, 2H), 2.31 (s, 3H). 13C NMR
(150 MHz, DMSO‑d6) δ 163.7, 130.0, 116.5, 114.9, 83.2, 52.7, 51.6,
45.3, 24.8.
391.1328, found: 391.1308.
4.2.3.4. Ethyl 2-(3-(4-methoxybenzyl)ureido)-6-methyl-4,5,6,7-tetrahy-
drothieno[2,3-c]pyridine-3-carboxylate (5d). Yellow solid; yield 34.7%;
m.p.: 111–112 ℃; Rf = 0.40 (MeOH/CH2Cl2 = 5/95), 1H NMR (600
MHz, CDCl3) δ 10.56 (s, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6
Hz, 2H), 5.52 (t, J = 5.6 Hz, 1H), 4.40 (d, J = 5.6 Hz, 2H), 4.22 (q, J =
7.1 Hz, 2H), 3.79 (s, 3H), 3.45 (s, 2H), 2.85 (m, 2H), 2.66 (t, J = 5.9 Hz,
2H), 2.44 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ
166.8, 159.2, 153.5, 151.6, 129.2, 128.7, 122.3, 114.2, 108.7, 60.4,
55.4, 53.4, 52.6, 50.9, 45.7, 44.3, 27.1, 14.4. HRMS (ESI+): m/z calcd
for C20H25N3O4+ [M + H]+: 404.1644, found: 404.1675.
4.2.2. Synthesis of compounds 4a
General method B: To a solution of benzylamine derivatives in
anhydrous DCM (conc. 0.3 M) was slowly added a solution of hydro-
chloric acid in isopropanol with stirring until it becomes thick white
turbid liquid at room temperature. Then CDI (5.6 mmol) in DMF (6 ml)
solution was added dropwise. The reaction mixture was stirred for 3–4 h
at room temperature under argon then was diluted with AcOEt and
brine. The aqueous layer was extracted with AcOEt and the combined
organic extracts were washed with brine, dried over Na2SO4 and
concentrated under vacuum. The precipitate was washed with Et2O to
give the desired N-(4-methoxybenzyl)-1H-imidazole-1-carboxamide
(4a) as a colored solid. White solid; yield 81.2%; Rf = 0.20 (acetone/
petroleum ether = 1/2), 1H NMR (600 MHz, CDCl3) δ 8.07 (s, 1H), 7.39
(t, J = 1.5 Hz, 1H), 7.24 (s, 2H), 6.93 (s, 1H), 6.87 (d, J = 8.6 Hz, 2H),
4.50 (d, J = 5.5 Hz, 2H), 3.79 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
159.5, 149.0, 135.9, 130.1, 129.6, 129.2, 116.4, 114.4, 55.5, 44.6.
4.2.3.5. 1-(3-Cyano-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)-3-(4-
methoxybenzyl)urea (5e). Light yellow solid; yield 73.5%; m.p.: 84–85.5
℃; Rf = 0.25 (EtOAc/petroleum ether = 1/3), 1H NMR (600 MHz,
CDCl3) δ 9.57 (s, 1H), 7.18 (d, J = 8.7 Hz, 2H), 6.79 (s, 1H), 6.78 (d, J =
8.7 Hz, 2H), 4.35 (d, J = 5.5 Hz, 2H), 3.73 (s, 3H), 2.77–2.74 (m, 2H),
2.68–2.65 (m, 2H), 2.35–2.30 (m, 2H). 13C NMR (150 MHz, CDCl3) δ
158.8, 155.9, 154.0, 140.2, 131.5, 130.9, 128.9, 115.6, 113.9, 85.2,
+
55.3, 43.5, 29.3, 28.1, 27.9. HRMS (ESI+): m/z calcd for C17H17N3O2
[M + H]+: 328.1120, found: 328.1118.
4.2.3. Synthesis of compounds 5a-5n
4.2.3.6. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(4-
methoxybenzyl)urea (5f). Light yellow solid; yield 79.1%; m.p.:
158–159 ℃; Rf = 0.30 (acetone/petroleum ether = 1/4),1H NMR (600
MHz, CDCl3) δ 8.69 (s, 1H), 7.22 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz,
2H), 6.04 (t, J = 5.6 Hz, 1H), 4.38 (d, J = 5.6 Hz, 2H), 3.78 (s, 3H), 2.55
(t, J = 5.9 Hz, 2H), 2.40 (t, J = 5.9 Hz, 2H), 1.81–1.76 (m, 4H). 13C NMR
(150 MHz, CDCl3) δ 159.1, 153.7, 151.8, 130.4, 130.2, 129.0, 126.2,
116.2, 114.1, 89.2, 55.4, 43.9, 24.0, 24.0, 23.3, 22.3. HRMS (ESI+): m/z
calcd for C18H19N3O2+ [M + H]+: 342.1276, found: 342.1227.
General method C: To a solution of compound 4 or 6 in DMF was
added compound 2 (1 eq) and NaH (1.2 eq). After stirring for 8–10 h at
room temperature. The reaction mixture was diluted with AcOEt and
brine. The aqueous layer was extracted with AcOEt, and the combined
organic extracts were washed with brine, dried over Na2SO4 and
concentrated under vacuum. The residue was purified by flash column
chromatography to give the desired compound as a solid.
4.2.3.1. Ethyl 2-(3-(4-methoxybenzyl)ureido)-5,6-dihydro-4H-cyclopenta
[b]thiophene-3-carboxylate (5a). White solid; yield 39.2%; m.p.:
9

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
4.2.3.7. 1-(3-Cyano-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophen-2-yl)-
3-(4-methoxybenzyl)urea (5g). White solid; yield 78.7%; m.p.: 225–226
℃; Rf = 0.65 (EtOAc/petroleum ether = 1/4), 1H NMR (600 MHz,
CDCl3) δ 8.47 (s, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H),
5.89 (t, J = 5.5 Hz, 1H), 4.38 (d, J = 5.5 Hz, 2H), 3.78 (s, 3H), 2.64 (m,
2H), 2.54 (m, 2H), 1.83 (t, J = 5.6 Hz, 2H), 1.61 (d, J = 5.6 Hz, 2H), 1.25
(s, 2H). 13C NMR (150 MHz, CDCl3) δ 159.0, 153.6, 149.5, 134.7, 130.3,
129.7, 128.9, 116.4, 114.0, 91.8, 55.3, 43.8, 32.0, 29.0, 29.0, 28.1, 27.3.
HRMS (ESI+): m/z calcd for C19H21N3O2+ [M + H]+: 356.1433, found:
356.1427.
4.2.3.13. 1-(4-Methoxybenzyl)-3-(4,5,6,7-tetrahydrobenzo[d]thiazol-2-
yl)urea (5m). White solid; yield 51.9%; m.p.: 171–172 ℃; Rf = 0.25
(EtOAc/petroleum ether = 1/1), 1H NMR (600 MHz, CDCl3) δ 7.21 (d, J
= 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 4.40 (d, J = 5.7 Hz, 2H), 3.78 (s,
3H), 2.61 (m, 2H), 2.56 (m, 2H), 1.80 (m, 4H). 13C NMR (150 MHz,
CDCl3) δ 159.3, 158.8, 155.0, 144.1, 130.8, 128.7, 120.2, 113.9, 55.3,
+
43.4, 26.6, 23.3, 22.9, 22.9. HRMS (ESI+): m/z calcd for C16H19N3O2
[M + H]+: 318.1276, found: 318.1293.
4.2.3.14. 1-(4-Methoxybenzyl)-3-(7-oxo-4,5,6,7-tetrahydrobenzo[d]thia-
zol-2-yl)urea (5n). White solid; yield 78.3%; m.p.: 233–234 ℃; Rf =
0.70 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, DMSO‑d6) δ 11.00 (s,
1H), 7.22 (d, J = 8.7 Hz, 2H), 7.09 (d, J = 6.4 Hz, 1H), 6.90 (d, J = 8.7
Hz, 2H), 4.26 (d, J = 5.9 Hz, 2H), 3.72 (s, 3H), 2.78 (t, J = 6.3 Hz, 2H),
2.44 (t, J = 6.4 Hz, 2H), 2.04 (p, J = 6.3 Hz, 2H). 13C NMR (150 MHz,
DMSO‑d6) δ 191.5, 165.0, 164.7, 158.4, 153.2, 131.0, 128.7, 122.5,
113.8, 55.1, 42.5, 37.1, 26.4, 22.6. HRMS (ESI+): m/z calcd for
4.2.3.8. 1-(3-Cyano-6-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-
3-(4-methoxybenzyl)urea (5h). White solid; yield 69.8%; m.p.:
1
218.5–219.5 ℃; Rf = 0.90 (EtOAc/petroleum ether = 1/2), H NMR
(600 MHz, DMSO‑d6) δ 9.93 (s, 1H), 7.22 (d, J = 8.1 Hz, 2H), 7.10 (t, J =
5.8 Hz, 1H), 6.90 (d, J = 8.1 Hz, 2H), 4.25 (d, J = 5.8 Hz, 2H), 3.73 (s,
3H), 2.62 (dd, J = 16.0, 4.9 Hz, 1H), 2.47–2.41 (m, 1H), 2.12 (dd, J =
16.0, 9.6 Hz, 1H), 1.81 (d, J = 12.2 Hz, 2H), 1.34–1.32 (m, 1H), 1.23 (s,
1H), 1.01 (d, J = 6.5 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6) δ 158.8,
153.8, 150.9, 131.6, 129.9, 129.1, 125.0, 115.4, 114.3, 88.9, 55.5, 43.0,
C
16H17N3O3+ [M + H]+: 332.1069, found: 332.1062.
4.2.4. Synthesis of compounds 6b-6v and 7a-7g
+
31.8, 30.4, 29.6, 23.8, 21.6. HRMS (ESI+): m/z calcd for C19H21N3O2
Compounds 6b-6v, 7a-7g were prepared from a series of starting
materials 3 according to the General method B.
[M + H]+: 356.1433, found: 356.1413.
4.2.3.9. 1-(3-Cyano-4-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-
3-(4-methoxybenzyl)urea (5i). Yellow solid; yield 53.1%; m.p.:
114–115.5 ℃; Rf = 0.50 (EtOAc/petroleum ether = 1/4), 1H NMR (600
MHz, DMSO‑d6) δ 10.05 (s, 1H), 7.44 (t, J = 5.7 Hz 1H), 7.22 (d, J = 8.2
Hz, 2H), 6.90 (d, J = 8.2 Hz, 2H), 4.24 (d, J = 5.7 Hz, 2H), 3.72 (s, 3H),
3.15 (d, J = 5.1 Hz, 1H), 2.75 (d, J = 6.3 Hz, 1H), 2.47 (m, 1H), 1.77 (m,
2H), 1.71 (d, J = 4.4 Hz, 1H), 1.54–1.51 (m, 1H), 1.19 (d, J = 6.9 Hz,
3H). 13C NMR (150 MHz, DMSO‑d6) δ 158.1, 153.3, 150.5, 134.3, 130.9,
128.3, 124.5, 114.8, 113.5, 88.0, 54.8, 42.2, 29.4, 27.9, 23.3, 20.3, 18.8.
HRMS (ESI+): m/z calcd for C19H21N3O2+ [M + H]+: 356.1433, found:
356.1405.
4.2.4.1. N-(2-methoxybenzyl)-1H-imidazole-1-carboxamide (6b). Rf =
0.60 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 8.06 (d, J =
1.1 Hz, 1H), 7.35–7.27 (m, 3H), 7.02 (dd, J = 1.6, 0.9 Hz, 1H), 6.94 (td,
J = 7.5, 1.1 Hz, 1H), 6.90 (dd, J = 8.2, 1.0 Hz, 1H), 6.73 (d, J = 6.0 Hz,
1H), 4.57 (d, J = 5.8 Hz, 2H), 3.87 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
157.6, 148.9, 136.0, 130.4, 130.2, 129.7, 125.0, 121.0, 116.0, 110.7,
55.6, 41.2.
4.2.4.2. N-(3-methoxybenzyl)-1H-imidazole-1-carboxamide (6c). Rf
=
0.55 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 8.07 (s, 1H),
7.79 (d, J = 5.8 Hz, 1H), 7.43 (t, J = 1.5 Hz, 1H), 7.24 (t, J = 7.9 Hz, 1H),
6.90–6.80 (m, 4H), 4.51 (d, J = 5.6 Hz, 2H), 3.76 (s, 3H). 13C NMR (150
MHz, CDCl3) δ 160.0, 149.2, 138.8, 135.9, 130.0, 129.9, 120.2, 116.6,
113.9, 113.2, 55.4, 44.9.
4.2.3.10. 1-(3-Cyano-4,7-dihydro-5H-thieno[2,3-c]pyran-2-yl)-3-(4-
methoxybenzyl)urea (5j). Light yellow solid; yield 62.8%; m.p.: 218–219
℃; Rf = 0.65 (EtOAc/petroleum ether = 1/2), 1H NMR (600 MHz,
DMSO‑d6) δ 10.08 (s, 1H), 7.22 (d, J = 8.6 Hz, 2H), 7.17 (t, J = 5.8 Hz,
1H), 6.90 (d, J = 8.6 Hz, 2H), 4.55 (s, 2H), 4.26 (d, J = 5.8 Hz, 2H), 3.85
(t, J = 5.5 Hz, 2H), 3.73 (s, 3H), 2.54 (t, J = 5.5 Hz, 2H). 13C NMR (150
MHz, DMSO‑d6) δ 158.8, 153.8, 151.7, 131.5, 129.1, 128.3, 123.3,
114.9, 114.3, 88.8, 64.2, 64.2, 55.5, 43.0, 24.4. HRMS (ESI+): m/z calcd
for C17H17N3O3+ [M + H]+: 344.1069, found: 344.1060.
4.2.4.3. N-benzyl-1H-imidazole-1-carboxamide (6d). Rf = 0.70 (MeOH/
CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 8.05 (t, J = 1.1 Hz, 1H),
7.92 (t, J = 5.8 Hz, 1H), 7.43 (t, J = 1.5 Hz, 1H), 7.36–7.24 (m, 5H), 6.85
(t, J = 1.2 Hz, 1H), 4.53 (d, J = 5.6 Hz, 2H). 13C NMR (150 MHz, CDCl3)
δ 149.2, 137.3, 135.9, 129.7, 128.9, 128.0, 116.7.
4.2.4.4. N-(2-methylbenzyl)-1H-imidazole-1-carboxamide (6e). Rf
=
0.50 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 7.95 (t, J =
1.1 Hz, 1H), 7.39 (t, J = 1.5 Hz, 1H), 7.25 (d, J = 1.7 Hz, 1H), 7.21–7.14
4.2.3.11. 1-(3-Cyano-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-
yl)-3-(4-methoxybenzyl)urea (5k). Brick red solid; yield 65.4%; m.p.:
201.5–203 ℃; Rf = 0.35 (acetone/petroleum ether = 1/1), 1H NMR
(600 MHz, CDCl3) δ 8.86 (s, 1H), 7.23 (d, J = 8.6 Hz, 2H), 6.84 (d, J =
8.6 Hz, 2H), 6.14 (t, J = 5.7 Hz, 1H), 4.38 (d, J = 5.7 Hz, 2H), 3.77 (s,
3H), 3.34 (d, J = 1.9 Hz, 2H), 2.68 (t, J = 5.8 Hz, 2H), 2.59 (t, J = 5.8 Hz,
2H), 2.42 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 159.0, 153.5, 152.2,
130.4, 129.0, 128.3, 123.2, 115.6, 114.1, 55.3, 52.6, 51.6, 45.3, 43.7,
(m, 3H), 6.89–6.84 (m, 1H), 4.56 (d, J = 5.2 Hz, 2H), 2.34 (s, 3H). 13
C
NMR (150 MHz, CDCl3) δ 148.9, 136.7, 135.9, 134.7, 130.9, 130.0,
128.9, 128.4, 126.5, 116.5, 43.2, 19.2.
4.2.4.5. N-(3-methylbenzyl)-1H-imidazole-1-carboxamide (6f). Rf
=
0.50 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 8.05 (d, J =
1.2 Hz, 1H), 7.71–7.66 (m, 1H), 7.43 (t, J = 1.4 Hz, 1H), 7.21 (t, J = 7.5
Hz, 1H), 7.14–7.07 (m, 3H), 6.90–6.87 (m, 1H), 4.51 (d, J = 5.6 Hz, 2H),
2.32 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 149.1, 138.7, 137.2, 135.9,
129.8, 128.9, 128.8, 128.8, 125.1, 116.6, 45.0, 21.5.
29.7, 24.2. HRMS (ESI+): m/z calcd for C18H20N4O2 [M + H]+:
+
357.1385, found: 357.1370.
4.2.3.12. 1-(4-Methoxybenzyl)-3-(5-nitrothiazol-2-yl)urea
(5l). Light
yellow solid; yield 47.2%; m.p.: 192–193 ℃; Rf = 0.40 (acetone/pe-
1
troleum ether = 1/2), H NMR (600 MHz, DMSO‑d6) δ 11.62 (s, 1H),
4.2.4.6. N-(4-methylbenzyl)-1H-imidazole-1-carboxamide (6g). Rf
=
0.50 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 8.07 (t, J =
1.1 Hz, 1H), 7.48 (d, J = 5.3 Hz, 1H), 7.40 (t, J = 1.5 Hz, 1H), 7.20 (d, J
= 8.1 Hz, 2H), 7.14 (d, J = 7.8 Hz, 2H), 6.90 (t, J = 1.2 Hz, 1H), 4.51 (d,
J = 5.6 Hz, 2H), 2.33 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 149.1, 137.9,
135.9, 134.2, 130.0, 129.6, 128.1, 116.5, 44.8, 21.2.
8.50 (s, 1H), 7.24 (d, J = 8.6 Hz, 3H), 6.90 (d, J = 8.6 Hz, 2H), 4.30 (d, J
= 5.9 Hz, 2H), 3.73 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ 164.8,
158.9, 153.8, 143.9, 131.2, 129.2, 128.8, 114.3, 55.5, 43.1. HRMS
(ESI+): m/z calcd for C12H12N4O4 [M + H]+: 309.0658, found:
309.0663.
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Bioorganic Chemistry 115 (2021) 105236
4.2.4.7. N-(4-fluorobenzyl)-1H-imidazole-1-carboxamide (6h). Rf = 0.50
(MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 8.10 (t, J = 1.1
Hz, 1H), 7.90 (t, J = 5.6 Hz, 1H), 7.43 (t, J = 1.5 Hz, 1H), 7.28 (dd, J =
8.5, 5.4 Hz, 2H), 7.01 (t, J = 8.6 Hz, 2H), 6.87 (t, J = 1.2 Hz, 1H), 4.51
(d, J = 5.7 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ 149.0, 135.8, 129.7,
129.7, 129.6, 116.6, 115.8, 115.6, 44.2.
4.2.4.16. N-(3,4-dimethoxybenzyl)-1H-imidazole-1-carboxamide (6q).
Rf = 0.50 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 8.12 (s,
1H), 7.73–7.67 (m, 1H), 7.46 (s, 1H), 6.92–6.88 (m, 1H), 6.84 (dd, J =
8.1, 2.0 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.77 (d, J = 8.1 Hz, 1H), 4.46
(d, J = 5.5 Hz, 2H), 3.81 (s, 3H), 3.78 (s, 3H). 13C NMR (150 MHz,
CDCl3) δ 149.1, 149.1, 148.7, 136.0, 129.8, 129.8, 120.6, 116.6, 111.5,
111.3, 56.0, 55.9, 44.9.
4.2.4.8. 1,3-bis(2-chlorobenzyl)urea (6i). Rf = 0.45 (MeOH/CH2Cl2
=
5/95), 1H NMR (600 MHz, DMSO‑d6) δ 7.42 (dd, J = 7.8, 1.3 Hz, 2H),
7.37–7.31 (m, 4H), 7.28 (td, J = 7.5, 2.0 Hz, 2H), 6.62 (t, J = 6.1 Hz,
2H), 4.30 (d, J = 6.1 Hz, 4H). 13C NMR (150 MHz, DMSO‑d6) δ 157.8,
137.8, 131.9, 129.0, 128.6, 128.4, 127.1, 40.9.
4.2.4.17. N-(2,4-dimethoxybenzyl)-1H-imidazole-1-carboxamide (6r).
Rf = 0.50 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 8.06 (t,
J = 1.1 Hz, 1H), 7.35 (t, J = 1.5 Hz, 1H), 7.21 (d, J = 8.1 Hz, 1H),
6.99–6.96 (m, 1H), 6.95 (d, J = 5.9 Hz, 1H), 6.46–6.42 (m, 2H), 4.48 (d,
J = 5.6 Hz, 2H), 3.81 (s, 3H), 3.78 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
161.0, 158.6, 148.9, 136.0, 130.9, 130.2, 117.6, 116.1, 104.2, 98.8,
55.6, 55.5, 40.6.
4.2.4.9. N-(3-chlorobenzyl)-1H-imidazole-1-carboxamide (6j). Rf = 0.45
(MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 8.33 (t, J = 5.8
Hz, 1H), 8.19 (t, J = 1.2 Hz, 1H), 7.49 (t, J = 1.5 Hz, 1H), 7.28 (dt, J =
2.6, 1.2 Hz, 1H), 7.24 (dd, J = 4.8, 1.1 Hz, 2H), 7.19 (ddd, J = 5.0, 4.1,
1.7 Hz, 1H), 6.89 (t, J = 1.2 Hz, 1H), 4.50 (d, J = 5.8 Hz, 2H). 13C NMR
(150 MHz, CDCl3) δ 149.2, 139.5, 136.0, 134.7, 130.2, 129.5, 128.1,
128.0, 126.1, 116.9, 44.3.
4.2.4.18. N-(3,5-dimethoxybenzyl)-1H-imidazole-1-carboxamide (6s).
Rf = 0.50 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, CDCl3) δ 8.10 (t,
J = 1.2 Hz, 1H), 7.90 (s, 1H), 7.44 (d, J = 1.6 Hz, 1H), 6.91–6.88 (m,
1H), 6.44 (d, J = 2.3 Hz, 2H), 6.35 (t, J = 2.3 Hz, 1H), 4.46 (d, J = 5.7
Hz, 2H), 3.74 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 161.2, 149.2, 139.6,
135.9, 129.8, 116.7, 106.0, 99.6, 55.5, 45.0.
4.2.4.10. N-(4-chlorobenzyl)-1H-imidazole-1-carboxamide (6k). Rf
=
0.45 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, DMSO‑d6) δ 9.10 (t, J
= 5.9 Hz, 1H), 8.27 (t, J = 1.2 Hz, 1H), 7.70 (t, J = 1.5 Hz, 1H),
7.43–7.39 (m, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.06–7.02 (m, 1H), 4.45 (d,
J = 5.8 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6) δ 149.0, 137.5, 136.0,
131.7, 129.7, 129.2, 128.8, 128.4, 128.1, 116.6, 42.9.
4.2.4.19. N-benzyl-N-methyl-1H-imidazole-1-carboxamide (7a). Rf
=
0.75 (MeOH/CH2Cl2 = 5/95),1H NMR (600 MHz, DMSO‑d6) δ 7.92 (t, J
= 1.1 Hz, 1H), 7.39 (dd, J = 8.1, 6.7 Hz, 2H), 7.35–7.32 (m, 1H), 7.28 (d,
J = 7.5 Hz, 2H), 7.24 (t, J = 1.5 Hz, 1H), 7.06 (t, J = 1.2 Hz, 1H), 4.63 (s,
2H), 3.03 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ 152.2, 137.1, 135.4,
129.8, 129.2, 128.3, 127.6, 118.1, 54.2, 36.5.
4.2.4.11. 1,3-bis(2-bromobenzyl)urea (6l). Rf = 0.45 (MeOH/CH2Cl2
=
5/95), 1H NMR (600 MHz, DMSO‑d6) δ 7.59 (d, J = 7.9 Hz, 2H), 7.35
(dd, J = 18.1, 7.4 Hz, 4H), 7.25–7.15 (m, 2H), 6.66 (t, J = 6.1 Hz, 2H),
4.26 (d, J = 6.0 Hz, 4H). 13C NMR (150 MHz, DMSO‑d6) δ 157.8, 139.3,
132.3, 128.7, 128.7, 127.7, 122.3, 43.4.
4.2.5. Synthesis of compounds 8b-8v, and 9a-9g
Compounds 8b-8v and 9a-9g are correspondingly prepared from
intermediates 6b-6v and 7a-7g, respectively, according to the General
method C.
4.2.4.12. N-(3-bromobenzyl)-1H-imidazole-1-carboxamide (6m). Rf
=
0.45 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, DMSO‑d6) δ 9.10 (t, J
= 5.9 Hz, 1H), 8.28 (t, J = 1.1 Hz, 1H), 7.71 (t, J = 1.5 Hz, 1H), 7.56 (d,
J = 1.8 Hz, 1H), 7.50–7.47 (m, 1H), 7.37 (dt, J = 7.8, 1.4 Hz, 1H), 7.33
4.2.5.1. 1-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(2-
methoxybenzyl)urea (8b). White solid; yield 77.5%; m.p.: 200–201 ℃;
Rf = 0.25 (EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz, CDCl3) δ
8.59 (s, 1H), 7.29 (dd, J = 7.6, 1.7 Hz, 1H), 7.24 (dd, J = 7.6, 1.7 Hz,
1H), 6.89 (t, J = 7.4 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.11 (t, J = 6.0 Hz,
1H), 4.45 (d, J = 6.0 Hz, 2H), 3.84 (s, 3H), 2.55 (m, 2H), 2.46 (m, 2H),
1.81–1.77 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 157.6, 153.7, 151.9,
130.0, 129.5, 128.9, 126.6, 126.2, 120.7, 116.1, 110.3, 55.5, 40.3, 29.8,
24.1, 24.0, 23.3, 22.3. HRMS (ESI+): m/z calcd for C18H19N3O2+ [M +
H]+: 342.1276, found: 342.1283.
(d, J = 7.8 Hz, 1H), 7.05 (t, J = 1.2 Hz, 1H), 4.47 (d, J = 5.8 Hz, 2H). 13
C
NMR (150 MHz, DMSO‑d6) δ 149.0, 141.3, 136.0, 130.7, 130.1, 130.0,
129.7, 126.4, 121.7, 116.6, 43.0.
4.2.4.13. N-(4-bromobenzyl)-1H-imidazole-1-carboxamide (6n). Rf
=
0.45 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, DMSO‑d6) δ 9.10 (t, J
= 5.8 Hz, 1H), 8.27 (d, J = 1.3 Hz, 1H), 7.70 (t, J = 1.4 Hz, 1H),
7.57–7.53 (m, 2H), 7.34–7.29 (m, 2H), 7.04 (t, J = 1.2 Hz, 1H), 4.43 (d,
J = 5.8 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6) δ 149.0, 138.0, 136.0,
131.3, 129.7, 129.6, 120.2 , 116.6, 42.9.
4.2.5.2. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(3-
methoxybenzyl)urea (8c). White solid; yield 75.8%; m.p.: 147–148 ℃;
Rf = 0.25 (EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz, CDCl3) δ
8.74 (s, 1H), 7.22 (t, J = 7.9 Hz, 1H), 6.88 (d, J = 7.9 Hz, 1H), 6.85 (t, J
= 2.0 Hz 1H), 6.78 (dd, J = 8.1, 2.5 Hz, 1H), 6.09 (t, J = 5.7 Hz, 1H),
4.43 (d, J = 5.7 Hz, 2H), 3.77 (s, 3H), 2.55 (t, J = 5.8 Hz, 2H), 2.41 (t, J
= 5.8 Hz, 2H), 1.81–1.76 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 160.0,
153.8, 151.8, 139.9, 130.2, 129.8, 126.3, 119.8, 116.2, 113.1, 112.9,
89.2, 55.3, 44.3, 24.0, 24.0, 23.3, 22.3. HRMS (ESI+): m/z calcd for
4.2.4.14. N-(4-ethoxybenzyl)-1H-imidazole-1-carboxamide (6o). Rf
=
0.60 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, DMSO‑d6) δ 8.07 (s,
1H), 7.63–7.55 (m, 1H), 7.41 (s, 1H), 7.21 (d, J = 8.6 Hz, 2H), 6.89 (s,
1H), 6.84 (d, J = 8.6 Hz, 2H), 4.46 (d, J = 5.5 Hz, 2H), 4.02–3.98 (m,
2H), 1.40 (d, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6) δ 158.8,
149.1, 135.9, 129.9, 129.5, 129.2, 116.5, 114.8, 63.6, 44.5, 15.0.
C
18H19N3O2+ [M + H]+: 342.1276, found: 342.1243.
4.2.4.15. N-(2-ethoxybenzyl)-1H-imidazole-1-carboxamide (6p). Rf
=
0.60 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz, DMSO‑d6) δ
9.08–8.96 (m, 1H), 8.34 (d, J = 1.4 Hz, 1H), 7.79 (q, J = 1.5 Hz, 1H),
7.24 (dd, J = 8.0, 6.4 Hz, 2H), 7.04 (t, J = 1.1 Hz, 1H), 6.98 (d, J = 8.3
Hz, 1H), 6.91 (t, J = 7.3 Hz, 1H), 4.45 (d, J = 5.7 Hz, 2H), 4.06 (q, J =
6.9 Hz, 2H), 1.33 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6) δ
156.0, 148.9, 136.1, 129.5, 128.4, 127.7, 125.9, 120.1, 116.7, 111.5,
63.3, 38.8, 14.7.
4.2.5.3. 1-Benzyl-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)
urea (8d). White solid; yield 64.1%; m.p.: 209.5–210.5 ℃; Rf = 0.35
(EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz, CDCl3) δ 8.68 (s,
1H), 7.33–7.25 (m, 5H), 6.02 (t, J = 5.7 Hz, 1H), 4.47 (d, J = 5.7 Hz,
2H), 2.56 (t, J = 5.9 Hz, 2H), 2.41 (t, J = 5.9 Hz, 2H), 1.81–1.76 (m, 4H).
13C NMR (150 MHz, CDCl3) δ 153.7, 151.8, 138.4, 130.2, 128.8, 127.6,
127.6, 126.3, 116.3, 60.6, 44.4, 24.0, 24.0, 23.3, 22.3. HRMS (ESI+): m/
z calcd for C17H17N3O+ [M + H]+: 312.1171, found: 312.1214.
11

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Bioorganic Chemistry 115 (2021) 105236
4.2.5.4. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(2-meth-
ylbenzyl)urea (8e). White solid; yield 78.7%; m.p.: 205.5–206 ℃; Rf =
0.35 (EtOAc/petroleum ether = 1/4), 1H NMR (600 MHz, CDCl3) δ 8.64
(s, 1H), 7.28 (d, J = 6.8 Hz, 1H), 7.17 (m, 3H), 5.90 (t, J = 5.5 Hz, 1H),
4.45 (d, J = 5.5 Hz, 2H), 2.57 (t, J = 5.5 Hz, 2H), 2.37 (t, J = 6.0 Hz, 2H),
2.35 (s, 3H), 1.84–1.76 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 153.5,
152.1, 136.3, 136.1, 130.6, 130.1, 128.2, 127.8, 126.3, 126.3, 116.4,
88.8, 42.5, 24.0, 23.3, 22.3, 19.2. HRMS (ESI+): m/z calcd for
10.03 (s, 1H), 7.40 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 7.25 (t,
J = 5.9 Hz, 1H), 4.32 (d, J = 5.9 Hz, 2H), 2.52 (t, J = 5.2 Hz, 2H), 2.43
(d, J = 5.2 Hz, 2H), 1.77–1.67 (m, 4H). 13C NMR (150 MHz, DMSO‑d6) δ
153.5, 150.2, 138.5, 131.5, 129.8, 129.0, 128.4, 124.9, 114.8, 88.9,
+
42.4, 23.5, 23.3, 22.7, 21.8. HRMS (ESI+): m/z calcd for C17H16ClN3
[M + H]+: 346.0781, found: 346.0790, 348.0756.
4.2.5.11. 1-(2-Bromobenzyl)-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thio-
phen-2-yl)urea (8l). White solid; yield 68.9%; m.p.: 201–202 ℃; Rf =
0.40 (EtOAc/petroleum ether = 1/4), 1H NMR (600 MHz, DMSO‑d6) δ
10.11 (s, 1H), 7.63 (dd, J = 7.9, 1.1 Hz, 1H), 7.41–7.36 (m, 2H), 7.30 (t,
J = 6.0 Hz, 1H), 7.24 (ddd, J = 7.9, 6.5, 2.6 Hz, 1H), 4.37 (d, J = 6.0 Hz,
2H), 2.54–2.51 (m, 2H), 2.46–2.42 (m, 2H), 1.78–1.66 (m, 4H). 13C
NMR (150 MHz, DMSO‑d6) δ 153.4, 150.1, 137.9, 132.5, 129.8, 129.3,
129.2, 127.9, 125.0, 122.5, 114.8, 88.9, 43.6, 23.5, 23.3, 22.7, 21.8.
HRMS (ESI+): m/z calcd for C17H16BrN3+ [M + H]+: 390.0276, found:
390.0288, 392.0271.
C
18H19N3O+ [M + H]+: 326.1327, found: 326.1372.
4.2.5.5. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(3-meth-
ylbenzyl)urea (8f). White solid; yield 81.4%; m.p.: 181–182 ℃; Rf =
0.35 (EtOAc/petroleum ether = 1/4), 1H NMR (600 MHz, CDCl3) δ 8.70
(s, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.13–7.08 (m, 2H), 7.07 (d, J = 7.5 Hz,
1H), 6.03 (t, J = 5.7 Hz, 1H), 4.42 (d, J = 5.7 Hz, 2H), 2.56 (t, J = 5.9 Hz,
2H), 2.40 (t, J = 5.9 Hz, 2H), 2.32 (s, 3H), 1.83–1.76 (m, 4H). 13C NMR
(150 MHz, CDCl3) δ 153.8, 151.8, 138.5, 138.3, 130.2, 128.7, 128.3,
128.3, 126.3, 124.6, 116.3, 89.1, 44.4, 24.0, 24.0, 23.3, 22.3, 21.5.
HRMS (ESI+): m/z calcd for C18H19N3O+ [M + H]+: 326.1327, found:
326.1355.
4.2.5.12. 1-(3-Bromobenzyl)-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thio-
phen-2-yl)urea (8m). White solid; yield 70.5%; m.p.: 213–214 ℃; Rf =
0.40 (EtOAc/petroleum ether = 1/4), 1H NMR (600 MHz, DMSO‑d6) δ
10.05 (s, 1H), 7.49 (s, 1H), 7.45 (dt, J = 6.3, 2.5 Hz, 1H), 7.31 (d, J = 4.1
Hz, 2H), 7.26 (t, J = 6.0 Hz, 1H), 4.33 (d, J = 6.0 Hz, 2H), 2.52 (t, J =
3.2 Hz, 2H), 2.43 (t, J = 3.2 Hz, 2H), 1.72 (m, 4H). 13C NMR (150 MHz,
DMSO‑d6) δ 153.5, 150.1, 142.3, 130.6, 129.8, 129.8, 126.2, 125.0,
121.7, 114.8, 89.0, 42.5, 23.5, 23.3, 22.7, 21.8. HRMS (ESI+): m/z calcd
for C17H16BrN3+ [M + H]+: 390.0276, found: 390.0256, 392.0240.
4.2.5.6. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(4-meth-
ylbenzyl)urea (8g). White solid; yield 80.7%; m.p.: 194–195.5 ℃; Rf =
0.35 (EtOAc/petroleum ether = 1/4), 1H NMR (600 MHz, CDCl3) δ 8.68
(s, 1H), 7.19 (d, J = 7.8 Hz, 2H), 7.11 (d, J = 7.8 Hz, 2H), 6.03 (t, J = 5.6
Hz, 1H), 4.40 (d, J = 5.6 Hz, 2H), 2.56 (t, J = 5.9 Hz, 2H), 2.40 (t, J =
5.9 Hz, 2H), 2.32 (s, 3H), 1.84–1.74 (m, 4H). 13C NMR (150 MHz,
CDCl3) δ 153.7, 151.9, 137.2, 135.3, 130.1, 129.4, 127.6, 126.2, 116.2,
89.1, 44.2, 24.0, 24.0, 23.3, 22.3, 21.2. HRMS (ESI+): m/z calcd for
4.2.5.13. 1-(4-Bromobenzyl)-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thio-
phen-2-yl)urea (8n). White solid; yield 72.6%; m.p.: 224–225.5 ℃; Rf =
0.40 (EtOAc/petroleum ether = 1/4), 1H NMR (600 MHz, DMSO‑d6) δ
10.03 (s, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 3H), 4.30 (d,
J = 5.9 Hz, 2H), 2.55–2.51 (m, 2H), 2.44 (t, J = 5.1 Hz, 2H), 1.76–1.69
(m, 4H). 13C NMR (150 MHz, DMSO‑d6) δ 153.5, 150.2, 138.9, 131.3,
129.8, 129.4, 124.9, 120.0, 114.8, 88.9, 42.4, 23.5, 23.3, 22.7, 21.8.
HRMS (ESI+): m/z calcd for C17H16BrN3+ [M + H]+: 390.0276, found:
390.0273, 392.0256.
C
18H19N3O+ [M + H]+: 326.1327, found: 326.1331.
4.2.5.7. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(4-fluo-
robenzyl)urea (8h). Light yellow solid; yield 47.2%; m.p.: 218–219 ℃;
Rf = 0.30 (EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz, CDCl3) δ
8.67 (s, 1H), 7.27 (dd, J = 8.6, 5.4 Hz, 2H), 7.00 (t, J = 8.6 Hz, 2H), 6.04
(t, J = 5.7 Hz, 1H), 4.42 (d, J = 5.7 Hz, 2H), 2.59–2.53 (m, 2H), 2.40 (t,
J = 5.6 Hz, 2H), 1.79 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 162.3 (d, J
= 245.7 Hz), 153.7, 151.7, 134.2, 134.2, 130.2, 129.4, 129.3, 126.5,
116.3, 115.7, 115.5, 89.2, 43.7, 24.0, 24.0, 23.3, 22.2. HRMS (ESI+): m/
z calcd for C17H16FN3+ [M + H]+: 330.1076, found: 330.1086.
4.2.5.14. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(4-
ethoxybenzyl)urea (8o). Light yellow solid; yield 75.2%; m.p.: 186–187
℃; Rf = 0.30 (EtOAc/petroleum ether = 1/5), 1H NMR (600 MHz,
CDCl3) δ 8.70 (s, 1H), 7.20 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H),
6.04 (t, J = 5.5 Hz, 1H), 4.36 (d, J = 5.5 Hz, 2H), 3.99 (q, J = 7.0 Hz,
2H), 2.55 (t, J = 5.9 Hz, 2H), 2.39 (t, J = 5.9 Hz, 2H), 1.82–1.74 (m, 4H),
1.39 (t, J = 7.0 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 158.4, 153.7,
151.7, 130.2, 130.2, 129.0, 126.2, 116.2, 114.7, 89.2, 63.5, 43.9, 24.0,
24.0, 23.3, 22.3, 15.0. HRMS (ESI+): m/z calcd for C19H21N3O2+ [M +
H]+: 356.1433, found: 356.1456.
4.2.5.8. 1-(2-Chlorobenzyl)-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thio-
phen-2-yl)urea (8i). White solid; yield 80.7%; m.p.: 200.5–202 ℃; Rf =
0.30 (EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz, CDCl3) δ
10.08 (s, 1H), 7.46 (dd, J = 7.5, 1.8 Hz, 1H), 7.39 (dd, J = 7.5, 1.8 Hz,
1H), 7.36–7.31 (m, 2H), 7.29 (t, J = 5.9 Hz, 1H), 4.40 (d, J = 5.9 Hz,
2H), 2.54–2.50 (m, 2H), 2.47–2.40 (m, 2H), 1.77–1.67 (m, 4H). 13C
NMR (150 MHz, CDCl3) δ 153.4, 150.1, 136.4, 132.2, 129.9, 129.4,
129.1, 127.5, 127.3, 125.0, 114.9, 88.9, 41.2, 23.5, 23.3, 22.7, 21.8.
HRMS (ESI+): m/z calcd for C17H16ClN3+ [M + H]+: 346.0781, found:
346.0799, 348.0756.
4.2.5.15. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(2-
ethoxybenzyl)urea (8p). Light yellow solid; yield 45.6%; m.p.:
181.5–182.5 ℃; Rf = 0.30 (EtOAc/petroleum ether = 1/5), 1H NMR
(600 MHz, CDCl3) δ 8.73 (s, 1H), 7.30 (dd, J = 7.5, 1.7 Hz, 1H), 7.21 (td,
J = 7.9, 1.7 Hz, 1H), 6.87 (td, J = 7.5, 1.0 Hz, 1H), 6.82 (d, J = 7.9 Hz,
1H), 6.09 (t, J = 5.9 Hz, 1H), 4.46 (d, J = 5.9 Hz, 2H), 4.05 (q, J = 7.0
Hz, 2H), 2.54 (t, J = 6.0 Hz, 2H), 2.46–2.40 (m, 2H), 1.80–1.74 (m, 4H),
1.42 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 156.9, 153.8,
151.9, 130.0, 129.4, 128.8, 126.7, 126.1, 120.5, 116.2, 111.1, 89.0,
63.6, 40.2, 24.1, 23.9, 23.3, 22.3, 15.1. HRMS (ESI+): m/z calcd for
4.2.5.9. 1-(3-Chlorobenzyl)-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thio-
phen-2-yl)urea (8J). White solid; yield 71.1%; m.p.: 216.5–218 ℃; Rf =
0.30 (EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz, DMSO‑d6) δ
10.04 (s, 1H), 7.40–7.24 (m, 5H), 4.34 (d, J = 6.0 Hz, 2H), 2.52 (d, J =
4.8 Hz, 2H), 2.44 (d, J = 6.0 Hz, 2H), 1.74–1.70 (m, 4H). 13C NMR (150
MHz, DMSO‑d6) δ 153.5, 150.1, 142.1, 133.1, 130.3, 129.8, 126.9,
125.8, 125.0, 114.8, 89.0, 42.5, 23.5, 23.3, 22.7, 21.8. HRMS (ESI+): m/
z calcd for C17H16ClN3 [M + H]+: 346.0781, found: 346.0799,
C
19H21N3O2+ [M + H]+: 356.1433, found: 356.1473.
+
348.0672
4.2.5.16. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(3,4-
dimethoxybenzyl)urea (8q). Light yellow solid; yield 71.3%; m.p.:
160–161 ℃; Rf = 0.45 (EtOAc/petroleum ether = 1/1), 1H NMR (600
MHz, CDCl3) δ 8.68 (s, 1H), 6.84 (d, J = 7.4 Hz, 2H), 6.78 (d, J = 8.0 Hz,
4.2.5.10. 1-(4-Chlorobenzyl)-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thio-
phen-2-yl)urea (8k). White solid; yield 67.1%; m.p.: 210.5–211 ℃; Rf =
0.30 (EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz, DMSO‑d6) δ
12

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
1H), 6.05 (t, J = 5.6 Hz, 1H), 4.38 (d, J = 5.6 Hz, 2H), 3.84 (s, 6H), 2.55
(t, J = 5.9 Hz, 2H), 2.42–2.37 (m, 2H), 1.80–1.73 (m, 4H).13C NMR (150
MHz, CDCl3) δ 153.7, 151.6, 149.2, 148.5, 130.9, 130.2, 126.3, 119.9,
116.1, 111.2, 111.0, 89.3, 56.0, 56.0, 44.3, 24.0, 24.0, 23.3, 22.3. HRMS
7.53–7.48 (m, 1H), 7.36 (d, J = 7.4 Hz, 2H), 7.30 (t, J = 8.3 Hz, 3H),
4.59 (s, 2H), 3.07 (s, 3H), 2.59 (t, J = 5.9 Hz, 2H), 2.52 (t, J = 5.9 Hz,
2H), 1.82–1.76 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 149.7, 136.4,
130.4, 129.1, 128.0, 127.5, 127.1, 114.9, 91.7, 52.7, 34.9, 23.9, 23.9,
23.2, 22.2. HRMS (ESI+): m/z calcd for C18H19N3O+ [M + H]+:
326.1327, found: 326.1399.
(ESI+): m/z calcd for C19H21N3O3 [M + H]+: 372.1382, found:
+
372.1418.
4.2.5.17. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(2,4-
dimethoxybenzyl)urea (8r). Light yellow solid; yield 68.4%; m.p.:
186–187.5 ℃; Rf = 0.45 (EtOAc/petroleum ether = 1/1), 1H NMR (600
MHz, CDCl3) δ 8.65 (s, 1H), 7.18 (d, J = 8.2 Hz, 1H), 6.41 (d, J = 2.4 Hz,
1H), 6.38 (dd, J = 8.2, 2.4 Hz, 1H), 6.15 (t, J = 5.9 Hz, 1H), 4.37 (d, J =
5.9 Hz, 2H), 3.79 (s, 3H), 3.77 (s, 3H), 2.53 (t, J = 5.7 Hz, 2H), 2.45 (d, J
= 5.7 Hz, 2H), 1.82–1.75 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 160.6,
158.6, 153.7, 130.3, 130.0, 126.0, 119.1, 116.0, 103.9, 98.6, 55.5, 55.5,
4.2.5.23. 3-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-1-methyl-
1-(2-methylbenzyl)urea (9b). Yellow solid; yield 51.1%; m.p.:
119.5–121.5 ℃; Rf = 0.55 (EtOAc/petroleum ether = 1/4), 1H NMR
(600 MHz, CDCl3) δ 7.45 (s, 1H), 7.25–7.19 (m, 3H), 7.17 (dd, J = 7.6,
1.9 Hz, 1H), 4.61 (s, 2H), 3.04 (s, 3H), 2.59 (t, J = 5.6 Hz, 2H), 2.53 (t, J
= 5.6 Hz, 2H), 2.32 (s, 3H), 1.83–1.76 (m, 4H).13C NMR (150 MHz,
CDCl3) δ 153.4, 149.7, 136.4, 133.9, 131.1, 130.5, 128.1, 127.4, 127.2,
126.6, 114.9, 91.9, 50.6, 34.6, 24.1, 24.0, 23.3, 22.3, 19.3. HRMS
(ESI+): m/z calcd for C19H21N3O+ [M + H]+: 340.1484, found:
340.1489.
+
39.9, 24.1, 23.9, 23.3, 22.3. HRMS (ESI+): m/z calcd for C19H21N3O3
[M + H]+: 372.1382, found: 372.1408.
4.2.5.18. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(3,5-
dimethoxybenzyl)urea (8s). ellow solid; yield 71.1%; m.p.: 193–194 ℃;
Rf = 0.45 (EtOAc/petroleum ether = 1/1), 1H NMR (600 MHz, CDCl3) δ
8.74 (s, 1H), 6.45 (d, J = 2.2 Hz, 2H), 6.33 (t, J = 2.2 Hz, 1H), 6.10 (t, J
= 5.7 Hz, 1H), 4.39 (d, J = 5.7 Hz, 2H), 3.75 (s, 6H), 2.55 (t, J = 6.1 Hz,
2H), 2.42 (t, J = 6.1 Hz, 2H), 1.83–1.73 (m, 4H).13C NMR (150 MHz,
CDCl3) δ 161.1, 153.8, 151.7, 140.7, 130.3, 126.3, 116.2, 105.4, 99.3,
89.3, 55.4, 44.4, 24.0, 23.3, 22.3. HRMS (ESI+): m/z calcd for
4.2.5.24. 3-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-1-methyl-
1-(4-methylbenzyl)urea (9c). Yellow solid; yield 47.1%; m.p.: 103–104
℃; Rf = 0.55 (EtOAc/petroleum ether = 1/4), 1H NMR (600 MHz,
CDCl3) δ 7.46 (s, 1H), 7.22–7.15 (m, 4H), 4.54 (s, 2H), 3.07 (s, 3H), 2.59
(t, J = 6.1 Hz, 2H), 2.52 (t, J = 6.1 Hz, 2H), 2.34 (s, 3H), 1.85–1.75 (m,
4H).13C NMR (150 MHz, CDCl3) δ 153.4, 149.8, 137.9, 133.3, 130.5,
129.9, 127.6, 127.1, 115.0, 91.7, 52.6, 24.1, 24.0, 23.3, 22.3, 21.2.
HRMS (ESI+): m/z calcd for C19H21N3O+ [M + H]+: 340.1484, found:
340.1513.
C
19H21N3O3+ [M + H]+: 372.1382, found: 372.1388.
4.2.5.19. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(naph-
thalen-2-ylmethyl)urea (8t). Yellow solid; yield 65.3%; m.p.: 209–210
℃; Rf = 0.30 (EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz,
DMSO‑d6) δ 10.05 (s, 1H), 7.92–7.87 (m, 3H), 7.79 (s, 1H), 7.53–7.44
(m, 3H), 7.35 (t, J = 5.9 Hz, 1H), 4.51 (d, J = 5.8 Hz, 2H), 2.52 (t, J =
5.4 Hz, 2H), 2.44 (t, J = 5.4 Hz, 2H), 1.76–1.68 (m, 4H).13C NMR (150
MHz, DMSO‑d6) δ 153.6, 150.3, 136.9, 132.9, 132.2, 129.8, 128.1,
127.6, 126.3, 125.8, 125.2, 124.9, 114.9, 88.8, 43.2, 23.5, 23.3, 22.7,
21.8. HRMS (ESI+): m/z calcd for C21H19N3O+ [M + H]+: 362.1327,
found: 362.1364.
4.2.5.25. 1-Benzyl-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-
1-ethylurea (9d). Yellow solid; yield 68.8%; m.p.: 109–110 ℃; Rf = 0.40
(EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz, CDCl3) δ 7.42–7.36
(m, 3H), 7.33 (dd, J = 7.8, 2.7 Hz, 3H), 4.57 (s, 2H), 3.57–3.45 (m, 2H),
2.58 (t, J = 6.2 Hz, 2H), 2.51 (t, J = 6.2 Hz, 2H), 1.83–1.74 (m, 4H), 1.26
(t, J = 7.2 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 153.1, 149.7, 136.5,
130.4, 129.3, 128.3, 127.4, 127.0, 114.8, 91.7, 50.7, 43.1, 24.0, 23.9,
23.3, 22.3, 13.5. HRMS (ESI+): m/z calcd for C19H21N3O+ [M + H]+:
340.1484, found: 340.1517.
4.2.5.26. 1-Benzyl-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-
4.2.5.20. 1-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-3-(naph-
thalen-1-ylmethyl)urea (8u). Yellow solid; yield 53.4%; m.p.: 190–191
℃; Rf = 0.30 (EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz,
CDCl3) δ 8.65 (s, 1H), 8.00–7.97 (m, 1H), 7.82 (dd, J = 7.9, 1.6 Hz, 1H),
7.76 (d, J = 8.2 Hz, 1H), 7.50 (dd, J = 6.7, 1.5 Hz, 1H), 7.47–7.42 (m,
2H), 7.38 (dd, J = 8.2, 7.0 Hz, 1H), 6.11 (t, J = 5.4 Hz, 1H), 4.86 (d, J =
5.4 Hz, 2H), 2.52 (t, J = 5.8 Hz, 2H), 2.17 (t, J = 5.8 Hz, 2H), 1.80–1.76
(m, 2H), 1.74–1.71 (m, 2H).13C NMR (150 MHz, CDCl3) δ 153.5, 151.6,
133.9, 133.5, 131.4, 130.1, 128.8, 128.6, 126.7, 126.4, 126.0, 125.5,
125.5, 123.4, 116.0, 89.1, 42.4, 23.9, 23.8, 23.3, 22.3. HRMS (ESI+): m/
z calcd for C21H19N3O+ [M + H]+: 362.1327, found: 362.1363.
1-isopropylurea (9e). Yellow solid; yield 66.4%; m.p.: 118.5–120 ℃; Rf
1
= 0.45 (EtOAc/petroleum ether = 1/6), H NMR (600 MHz, CDCl3) δ
7.42 (t, J = 7.6 Hz, 2H), 7.39–7.36 (m, 2H), 7.34 (t, J = 7.3 Hz, 1H), 7.28
(s, 1H), 4.78–4.70 (m, 1H), 4.46 (s, 2H), 2.55 (t, J = 6.1 Hz, 2H), 2.46 (t,
J = 6.1 Hz, 2H), 1.80–1.72 (m, 4H), 1.28 (d, J = 6.8 Hz, 6H).13C NMR
(150 MHz, CDCl3) δ 153.7, 149.5, 136.6, 130.5, 129.7, 128.5, 126.9,
126.8, 114.5, 91.6, 47.4, 45.8, 24.0, 23.9, 23.3, 22.3, 20.8. HRMS
(ESI+): m/z calcd for C20H23N3O+ [M + H]+: 354.1640, found:
354.1684.
4.2.5.27. 1,1-Dibenzyl-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
yl)urea (9f). Yellow solid; yield 56.4%; m.p.: 167–168 ℃; Rf = 0.25
(EtOAc/petroleum ether = 1/6), 1H NMR (600 MHz, CDCl3) δ 7.45 (d, J
= 3.4 Hz, 1H), 7.43–7.31 (m, 10H), 4.73–4.59 (m, 4H), 2.60 (d, J = 6.0
Hz, 2H), 2.50 (d, J = 6.0 Hz, 2H), 1.81–1.78 (m, 4H).13C NMR (150
MHz, CDCl3) δ 153.8, 149.4, 136.2, 130.6, 129.4, 128.4, 127.7, 127.1,
114.6, 92.0, 51.5, 24.0, 23.9, 23.3, 22.3. HRMS (ESI+): m/z calcd for
4.2.5.21. 1-([1,1′-Biphenyl]-4-ylmethyl)-3-(3-cyano-4,5,6,7-tetrahy-
drobenzo[b]thiophen-2-yl)urea (8v). Yellow solid; yield 48.9%; m.p.:
211–211.5 ℃; Rf = 0.45 (EtOAc/petroleum ether = 1/6), 1H NMR (600
MHz, CDCl3) δ 8.65 (s, 1H), 7.57–7.53 (m, 4H), 7.42 (t, J = 7.7 Hz, 2H),
7.38 (d, J = 8.1 Hz, 2H), 7.36–7.32 (m, 1H), 6.07 (t, J = 5.6 Hz, 1H),
4.51 (d, J = 5.6 Hz, 2H), 2.54 (t, J = 5.6 Hz, 2H), 2.40 (t, J = 5.6 Hz, 2H),
1.76 (m, 2H), 1.69 (m, 2H).13C NMR (150 MHz, CDCl3) δ 153.7, 151.8,
140.8, 140.5, 137.4, 130.2, 128.9, 128.1, 127.5, 127.5, 127.1, 126.4,
116.3, 89.3, 44.2, 24.0, 24.0, 23.2, 22.2. HRMS (ESI+): m/z calcd for
C
24H23N3O+ [M + H]+: 388.1505, found: 388.1520.
4.2.5.28. 3-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-1-methyl-
1-(naphthalen-1-ylmethyl)urea (9g). Yellow solid; yield 51.2%; m.p.:
151–152 ℃; Rf = 0.30 (EtOAc/petroleum ether = 1/6), 1H NMR (600
MHz, CDCl3) δ 8.04 (d, J = 8.1 Hz, 1H), 7.88 (dd, J = 7.8, 1.7 Hz, 1H),
7.84 (d, J = 8.2 Hz, 1H), 7.55–7.50 (m, 3H), 7.46 (dd, J = 8.2, 7.0 Hz,
1H), 7.39 (dd, J = 7.0, 1.2 Hz, 1H), 5.08 (s, 2H), 2.99 (s, 3H), 2.62 (t, J =
5.6 Hz, 2H), 2.53 (t, J = 5.6 Hz, 2H), 1.84–1.79 (m, 4H).13C NMR (150
C
23H21N3O+ [M + H]+: 388.1484, found: 388.1453.
4.2.5.22. 1-Benzyl-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-
1-methylurea (9a). Yellow solid; yield 67.2%; m.p.: 115–116.5 ℃; Rf =
0.30 (EtOAc/petroleum ether = 1/5), 1H NMR (600 MHz, CDCl3) δ
13

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
MHz, CDCl3) δ 153.4, 149.7, 134.1, 131.7, 131.5, 130.6, 129.0, 129.0,
127.3, 126.9, 126.5, 126.3, 125.4, 123.4, 115.0, 92.1, 50.4, 33.8, 24.1,
24.0, 23.3, 22.3. HRMS (ESI+): m/z calcd for C22H21N3O+ [M + H]+:
376.1484, found: 376.1527.
153.3, 150.9, 129.4, 129.2, 122.7, 118.8, 114.9, 104.3, 98.4, 88.1, 65.2,
55.5, 55.2, 38.4, 32.4, 30.1, 21.4. HRMS (ESI+): m/z calcd for
C19H21N3O4+ [M + H]+: 388.1331, found: 388.1334.
4.2.6.9. 1-(3-Cyano-9-methyl-5,6,7,8-tetrahydro-4H-5,8-epi-
4.2.6. Synthesis of compounds2l-2q, and 10a-10f
minocyclohepta[b]thiophen-2-yl)-3-(2,4-dimethoxybenzyl)urea
(10c).
Following the General method A, the intermediate 2l-2q was pre-
pared from compound 1h-1m and malononitrile. Then, compounds 10a-
10f were obtained by treating the intermediate 2l-2q with compound 6r
according to the General method B.
Brown solid; yield 51.5%; m.p.: 176–178 ℃; Rf = 0.70 (MeOH/CH2Cl2
= 15/85), 1H NMR (600 MHz, CDCl3) δ 8.63 (s, 1H), 7.18 (d, J = 8.2 Hz,
1H), 6.43 (d, J = 2.4 Hz, 1H), 6.40 (dd, J = 8.2, 2.4 Hz, 1H), 6.08 (t, J =
5.9 Hz, 1H), 4.37 (d, J = 5.9 Hz, 2H), 3.81 (s, 3H), 3.78 (s, 3H), 3.77 (s,
1H), 3.45 (dd, J = 7.4, 4.4 Hz, 1H), 2.87 (dd, J = 16.6, 4.4 Hz, 1H), 2.32
(s, 3H), 2.26–2.14 (m, 2H), 2.09 (d, J = 16.6 Hz, 1H), 1.89–1.83 (m, 1H),
1.51 (ddd, J = 11.4, 8.8, 5.4 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ
160.6, 158.5, 153.6, 151.7, 130.3, 128.6, 125.5, 119.0, 115.7, 103.8,
98.6, 58.9, 56.7, 55.5, 55.5, 39.9, 35.9, 35.2, 29.6, 28.8. HRMS (ESI+):
m/z calcd for C21H24N4O3+ [M + H]+: 413.1647, found: 413.1648.
4.2.6.1. tert-butyl
2-amino-3-cyano-4,7-dihydrothieno[2,3-c]pyridine-6
(5H)-carboxylate (2l). Rf = 0.20 (EtOAc/petroleum ether = 1/4), 1H
NMR (600 MHz, CDCl3) δ 4.35 (s, 2H), 3.64 (s, 2H), 2.57 (s, 2H), 1.46 (s,
9H). ESI-MS: m/z 280 [M + H]+.
4.2.6.2. 2-Amino-6-hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophene-3-car-
bonitrile (2m). Rf = 0.75 (EtOAc/petroleum ether = 3/2), 1H NMR (600
MHz, DMSO‑d6) δ 4.15 (t, J = 1.7 Hz, 2H), 3.36 (t, J = 6.1 Hz, 2H), 2.76
(td, J = 5.4, 4.8, 2.9 Hz, 2H). ESI-MS: m/z 195.1 [M + H]+.
4.2.6.10. 1-(3-Cyano-5,6,7,8-tetrahydro-4H-5,8-epiminocyclohepta[b]thi-
ophen-2-yl)-3-(2,4-dimethoxybenzyl)urea (10d). Brown solid; yield
1
43.2%; m.p.: 123–124 ℃; Rf = 0.20 (MeOH/CH2Cl2 = 1/9), H NMR
(600 MHz, DMSO‑d6) δ 7.11 (d, J = 8.3 Hz, 1H), 7.06 (t, J = 5.8 Hz, 1H),
6.57 (d, J = 2.4 Hz, 1H), 6.48 (dd, J = 8.3, 2.4 Hz, 1H), 4.25 (d, J = 5.3
Hz, 1H), 4.19 (d, J = 5.8 Hz, 2H), 3.86 (dd, J = 7.8, 4.6 Hz, 1H), 3.81 (s,
3H), 3.74 (s, 3H), 2.84 (dd, J = 16.3, 4.6 Hz, 1H), 2.30 (dd, J = 16.3, 1.2
Hz, 1H), 1.99 (s, 1H), 1.91 (dp, J = 11.2, 5.9 Hz, 1H), 1.83 (ddd, J =
11.6, 8.9, 2.5 Hz, 1H), 1.54–1.48 (m, 1H). 13C NMR (150 MHz,
DMSO‑d6) δ 160.1, 158.0, 153.3, 150.4, 129.8, 129.4, 126.2, 118.8,
114.7, 104.3, 98.4, 88.6, 55.5, 55.3, 52.7, 51.9, 38.4, 36.3, 33.3, 28.5.
HRMS (ESI+): m/z calcd for C20H22N4O3+ [M + H]+: 399.1491, found:
399.1486.
4.2.6.3. 2-Amino-9-methyl-5,6,7,8-tetrahydro-4H-5,8-epiminocyclohepta
1
[b]thiophene-3-carbonitrile (2n). Rf = 0.25 (MeOH/CH2Cl2 = 1/9), H
NMR (600 MHz, CDCl3) δ 4.67 (d, J = 12.4 Hz, 2H), 3.74 (d, J = 5.2 Hz,
1H), 3.49 (s, 2H), 2.93 (dd, J = 16.6, 4.5 Hz, 1H), 2.39 (s, 3H), 1.94–1.88
(m, 1H), 1.59 (m, 1H), 0.86 (dt, J = 16.6, 7.9 Hz, 1H). ESI-MS: m/z 220
[M + H]+.
4.2.6.4. 2-Amino-5,6,7,8-tetrahydro-4H-5,8-epiminocyclohepta[b]thio-
phene-3-carbonitrile (2o). Rf = 0.25 (MeOH/CH2Cl2 = 1/9, +0.5%
TEA), 1H NMR (600 MHz, DMSO‑d6) δ 6.91 (s, 2H), 3.90 (d, J = 4.8 Hz,
1H), 3.69 (dd, J = 7.7, 4.4 Hz, 1H), 2.70 (dd, J = 16.1, 4.4 Hz, 1H), 2.09
(d, J = 16.1 Hz, 1H), 1.93 (m, 1H), 1.79 (m, 2H), 1.47–1.42 (m, 1H). ESI-
MS: m/z 206 [M + H]+.
4.2.6.11. 1-(6-Acetyl-3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-
yl)-3-(2,4-dimethoxybenzyl)urea (10e). White solid; yield 62.3%; m.p.:
1
205–206 ℃; Rf = 0.50 (MeOH/CH2Cl2 = 5/95), H NMR (600 MHz,
DMSO‑d6) δ 10.08 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.06 (t, J = 5.6 Hz,
1H), 6.57 (d, J = 2.2 Hz, 1H), 6.48 (dt, J = 8.3, 1.8 Hz, 1H), 4.49 (dt, J =
27.7, 1.8 Hz, 2H), 4.19 (dd, J = 5.8, 3.6 Hz, 2H), 3.82 (s, 3H), 3.74 (s,
3H), 3.68 (dt, J = 18.0, 5.8 Hz, 2H), 2.61 (td, J = 5.5, 2.8 Hz, 1H),
2.49–2.46 (m, 1H), 2.06 (d, J = 24.8 Hz, 3H). 13C NMR (150 MHz,
DMSO‑d6) δ 168.9, 168.7, 160.1, 158.0, 153.2, 153.2, 151.4, 129.5,
129.4, 128.8, 121.7, 121.4, 118.7, 114.6, 104.4, 98.4, 87.9, 55.6, 55.3,
43.9, 42.8, 38.5, 37.9, 24.3, 23.4, 21.8, 21.3. HRMS (ESI+): m/z calcd
for C20H22N4O4+ [M + H]+: 415.1440, found: 415.1450.
4.2.6.5. 6-Acetyl-2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-car-
bonitrile (2p). Rf = 0.15 (MeOH/CH2Cl2 = 5/95), 1H NMR (600 MHz,
DMSO‑d6) δ 7.30 (d, J = 10.9 Hz, 2H), 4.48 (d, J = 22.6 Hz, 2H), 3.78
(dt, J = 18.4, 5.8 Hz, 2H), 2.63 (tt, J = 3.5, 2.2 Hz, 2H), 2.17 (d, J = 28.8
Hz, 3H). ESI-MS: m/z 222 [M + H]+.
4.2.6.6. 2-amino-5,5,7,7-tetramethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyri-
dine-3-carbonitrile (2q). Rf = 0.35 (MeOH/CH2Cl2 = 1/9), 1H NMR
(600 MHz, DMSO‑d6) δ 7.03 (s, 2H), 2.18 (s, 2H), 1.24 (s, 6H), 1.08 (s,
6H). ESI-MS: m/z 236 [M + H]+.
4.2.6.12. 1-(3-Cyano-5,5,7,7-tetramethyl-4,5,6,7-tetrahydrothieno[2,3-c]
pyridin-2-yl)-3-(2,4-dimethoxybenzyl)urea (10f). Brown solid; yield
60.1%; m.p.: 92–93 ℃; Rf = 0.15 (MeOH/CH2Cl2 = 5/95), 1H NMR
(600 MHz, DMSO‑d6) δ 10.03 (s, 1H), 7.11 (d, J = 8.3 Hz, 1H), 7.07 (t, J
= 5.9 Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.48 (dd, J = 8.3, 2.4 Hz, 1H),
4.18 (d, J = 5.8 Hz, 2H), 3.82 (s, 3H), 3.74 (s, 3H), 2.28 (s, 2H), 1.31 (s,
6H), 1.09 (s, 6H). 13C NMR (150 MHz, DMSO‑d6) δ 160.1, 158.0, 153.3,
150.8, 132.5, 129.4, 127.6, 118.9, 115.1, 104.3, 98.4, 88.4, 55.6, 55.3,
51.7, 49.8, 38.4, 36.7, 33.6, 29.6. HRMS (ESI+): m/z calcd for
4.2.6.7. Tert-butyl 3-cyano-2-(3-(2,4-dimethoxybenzyl)ureido)-4,7-dihy-
drothieno[2,3-c]pyridine-6(5H)-carboxylate (10a). White solid; yield
49.9%; m.p.: 173–174.5 ℃; Rf = 0.15 (EtOAc/petroleum ether = 1/2),
1H NMR (600 MHz, CDCl3) δ 8.74 (s, 1H), 7.17 (d, J = 8.2 Hz, 1H),
6.43–6.36 (m, 2H), 6.18 (s, 1H), 4.43–4.35 (m, 4H), 3.77 (d, J = 7.6 Hz,
6H), 3.65 (s, 2H), 2.53 (s, 2H), 1.47 (s, 9H). 13C NMR (150 MHz,
DMSO‑d6) δ 160.1, 158.0, 153.9, 153.2, 151.4, 129.4, 128.8, 118.7,
114.5, 104.3, 98.4, 88.0, 79.4, 55.5, 55.2, 38.4, 28.0. HRMS (ESI+): m/z
calcd for C23H28N4O5+ [M + H]+: 473.1859, found: 473.1859.
C
22H28N4O3+ [M + H]+: 429.1960, found: 429.1966.
4.3.
α-Glucosidase inhibition bioassay
4.2.6.8. 1-(3-Cyano-6-hydroxy-4,5,6,7-tetrahydrobenzo[b]thiophen-2-
yl)-3-(2,4-dimethoxybenzyl)urea (10b). White solid; yield 62.7%; m.p.:
231–233 ℃; Rf = 0.25 (EtOAc/petroleum ether = 3/2), 1H NMR (600
MHz, DMSO‑d6) δ 9.94 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.03 (t, J = 5.8
Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 6.48 (dd, J = 8.3, 2.4 Hz, 1H), 4.91 (d,
J = 4.1 Hz, 1H), 4.18 (d, J = 5.7 Hz, 2H), 3.96–3.89 (m, 1H), 3.82 (s,
3H), 3.74 (s, 3H), 2.76 (dd, J = 15.9, 4.9 Hz, 1H), 2.58–2.52 (m, 1H),
2.44 (ddd, J = 15.9, 7.8, 5.8 Hz, 1H), 2.41–2.35 (m, 1H), 1.88–1.81 (m,
1H), 1.69–1.62 (m, 1H).13C NMR (150 MHz, DMSO‑d6) δ 160.1, 158.0,
The inhibitory activity against
α
-glucosidase of tested compounds
was performed according to the reported methods [10,36]. Acarbose
was used as a positive control. The tested compounds and acarbose were
respectively dissolved in DMSO, while the enzyme and the substrate
were both dissolved in PBS (pH = 6.8). After pre-incubation of tested
compounds with α μL substrate
-glucosidase in PSB (37 ◦C, 15 min), 25
buffers were added to the system and the incubation was continued at
37 ◦C for another 15 min. Finally, the reaction was terminated by the
14

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
addition of 50
μ
L 0.2 M sodium carbonate solution. The optical density
per-residue model quality has been assessed using the QMEAN scoring
function [47]. The built model was selected for the following molecular
docking simulation.
(OD) was measured at an absorbance wavelength of 405 nm using a
Microplate Reader (Tecan, Switzerland). The calculation formula of the
inhibition rate is: % inhibition rate = [(A1 - A0)/A0] × 100%, where A1
and A0 are the absorbance of the test compound with or without. The
IC50 value is determined from the graph of the relationship between the
inhibition rate and the different concentration of the test compound. The
experiment was performed in triplicate.
Molecular docking simulation was performed with Glide (grid-based
ligand docking with energetics) program. Firstly, the ligands were pre-
pared by the LigPrep module (LigPrep, version 3.4) in Schrodinger
software (Schrodinger, LLC: New York, NY, 2015). Next, the coordinates
of modeled
α-glucosidase protein structure were optimized by the
module of Protein Preparation Wizard module in the Maestro program
(Maestro, version 10). Then the protein grid files were generated with
the Receptor Grid Generation module. Finally, the prepared ligands
were docked to the protein grid files with the XP precision mode. The
top-ranking pose was selected for binding mode analysis.
4.4.
α
-Amylase inhibition bioassay
The
α-amylase activity of compounds was assessed based on a pre-
viously reported protocolwith slight modification [42]. The tested
compounds and acarbose were respectively dissolved in DMSO, and
porcine pancreatic
α-amylase (EC 3.2.1.1) was dissolved in 0.1 M
4.8. Molecular dynamics (MD) simulation
phosphate buffer at pH 6.8. After 10 min of incubation of 75 µL of the
enzyme solution and 75 µL of tested compound solution at 25 ◦C, 75 µL
of the starch solution (0.1% p/v in 0.1 M phosphate buffer at pH 6.8)
was added to start the reaction and the mixture were incubated for 10
min at 25 ◦C. Then the reaction was stopped by the addition of 62.5 µL
dinitrosalicylic (DNS) reagent. The optical density (OD) was measured
at an absorbance wavelength of 580 nm using a Tecan Microplate
Reader. The absorbance was recorded at 580 nm using a microplate
reader. The calculation formula of the inhibition rate is: % inhibition
rate = [(A0 – A1)/A0] × 100%, where A0 was the absorbance without
tested compound, A1 was the absorbance of the tested compound at
different concentrations.
MD simulation was performed to verify stability of constructed ho-
mology model. H++ program [48] was used to predict the protonation
states of all ionizable residues. A periodic box of transferable intermo-
lecular potential 3P water molecules that extended 10.0 Å from the
protein atoms was used. Counter-ions (Na+) were added to neutralize
the simulation system. AMBER 14.0 package [49] was used to perform
MD simulations. Isothermal-isobaric (NPT) ensemble and periodic
boundary conditions were used. The force field for this simulation was
Amber14SB. SHAKE algorithm [50] and particle-mesh Ewald method
[51] were respectively used to constrain small bonds involving
hydrogen atoms and to calculate the electrostatic interactions. The non-
bonded pairs were updated every 25 steps while the non-bonded cutoff
was set to 10 Å. 2 ns MD simulation was coupled to a 300 K thermal bath
at 1 bar pressure by using the algorithm of Berendsen et al. [52].
4.5. Fluorescence quenching experiment
The fluorescence quenching experiment was referred to a reported
protocol [43]. Briefly,
α
-glucosidase (1U/mL) was pre-treated with
4.9. Prediction of druglikeness properties
certain concentrations of inhibitors for 30 min at 37 ◦C in the fluores-
cence spectrophotometer (Agilent Cary Eclipse). Then 100
μL of the
The pharmacokinetic properties of 8r and 8s were predicted by the
“ADMET Descriptors” module implemented in DS3.0 [53] and online
molinspiration [54].
above solution (pH 6.8) was added accurately to the quartz cell. The
blank was used for buffer spectrum values. The excitation wavelength
was 290 nm, and the emission spectrum was recorded from 300 to 420
nm at 37 ◦C.
4.10. Cell viability assay
4.6. Kinetics of
α
-glucosidase inhibitors
The HepG2 and LO2 cell lines were cultured in a proper medium
supplemented with 10% fetal bovine serum in a humidified atmosphere
of 5% CO2 at 37 ◦C. Cell suspensions were plated in 96-well plates at a
density of 2 × 104 cells/cm3. After incubation for 24 h, the cells were
treated with various concentrations of tested substances for 48 h and
Inhibition type of inhibitors towards
α
-glucosidase was studied ac-
cording to a described method [44]. Increasing concentrations of pNPG
were used as substrates in the absence or presence of tested compounds
at four different concentrations around the IC50 values. The inhibitory
then incubated with 100 μ
L of MTT at 37 ◦C for 2 h. The formazan dye
kinetics of the investigated compounds on
α
-glucosidase was analyzed
product was measured by the absorbance at 490 nm on a Tecan Spark
multimode microplate reader (Switzerland).
using the Lineweaver-Burk plot of the substrate concentration and
velocity.
4.11. Hypoglycemic activity assay
4.7. Molecular modeling
Experiments were performed in 190 ~ 220 g SD rats (Jinan Pengyue
Experimental Animal Breeding Co. Ltd, Jinan, Shandong, China) housed
in groups of 6 on a 12 h/12 h light–dark schedule cycle. All in vivo ex-
periments followed the ARRIVE guidelines [55]. After 3 days of stabi-
lization, in each experimental group, 12 SD rats were used. Each tested
group received a dose of acarbose and tested compound (5 mg/kg), and
then 7.5 g/kg sucrose solution was given 30 min after oral administra-
tion; the control group was only received the sucrose solution. Blood
samples were collected from the tail vein before and after sucrose
administration at 10, 20, 30, 45, 60, 90, and 120 min. The plasma
glucose was recorded using glucometer (Roche, Accu-chek performa).
The online program SWISS MODEL (https://swissmodel.expasy.
org/) was used to perform homology modelling. First, the primary
amino acid sequence of α-glucosidase (Saccharomyces cerevisiae organ-
ism, EC:3.2.1.20) was uploaded to the program, then the SWISS-MODEL
template library (STML, last update: 2021-03-31, last included PDB
release: 2021-03-26) was searched with BLAST [45] and HHBlits [46]
for evolutionary related structures matching the uploaded sequence
(target sequence) when the Search For Templates was performed. Next,
according to the reported result, the template was chosen for homology
modelling of α-glucosidase crystal structure. Model was built based on
the target-template alignment using ProMod3. Coordinates which are
conserved between the target and the template are copied from the
template to the model. Insertions and deletions are remodeled using a
fragment library. Side chains are then rebuilt. Finally, the geometry of
the resulting model is regularized by using a force field. The global and
4.12. Data analysis
Data are presented as mean ± SD, and at least three independent
replications were performed. Images were processed by use of GraphPad
15

H.-X. Xie et al.
Bioorganic Chemistry 115 (2021) 105236
improvement revised consensus statement for endovascular therapy of acute
ischemic stroke, Int. J. Stroke. 13 (2018) 612–632.
Prism 5 (GraphPad Software, La Jolla, CA, USA). One-way ANOVA
combined with Bonferroni post hoc tests were used to determine the
significance between different groups. P < 0.05 was considered statis-
tically significant. For in vivo experiments, the level of blood glucose was
expressed in mmol/L, and the areas under the curve (AUC) for 120 min
were established.
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105236.
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