Synthesis, biological evaluation and molecular docking study of N-arylbenzo[d]oxazol-2-amines as potential α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.08.061

A novel series of N-arylbenzo[d]oxazol-2-amines (4a–4m) were synthesized and evaluated for their α-glucosidase inhibitory activity. Compounds 4f–4i, 4k and 4m displayed potent inhibitory activity against α-glucosidase with IC₅₀values in the range of 32.49?±?0.17–120.24?±?0.51?μM as compared to the standard drug acarbose. Among all tested compounds, compound 4g having 4-phenoxy substitution at the phenyl ring was found to be the most active inhibitor of α-glucosidase with an IC₅₀value of 32.49?±?0.17?μM. Analysis of the kinetics of enzyme inhibition indicated that compound 4g is a noncompetitive inhibitor of α-glucosidase with a K_ivalue of 31.33?μM. Binding interaction of compound 4g with α-glucosidase was explored by molecular docking simulation.

Full text of DOI:10.1016/j.bmc.2016.08.061

Accepted Manuscript
Synthesis, biological evaluation and molecular docking study of N-arylben-
zo[d]oxazol-2-amines as potential α-glucosidase inhibitors
Guangcheng Wang, Zhiyun Peng, Jing Wang, Juan Li, Xin Li
PII:
S0968-0896(16)30678-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.08.061
BMC 13244
Reference:
Bioorganic & Medicinal Chemistry
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29 August 2016
Please cite this article as: Wang, G., Peng, Z., Wang, J., Li, J., Li, X., Synthesis, biological evaluation and molecular
docking study of N-arylbenzo[d]oxazol-2-amines as potential α-glucosidase inhibitors, Bioorganic & Medicinal
Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.08.061
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Synthesis, biological evaluation and molecular
docking study of N-arylbenzo[d]oxazol-2-amines as
potential α-glucosidase inhibitors
Guangcheng Wang*, Zhiyun Peng, Jing Wang, Juan Li, Xin Li
College of Chemistry and Chemical Engineering, Jishou University, Jishou 416000,
PR China
*Corresponding Author
Tel.: +86 743 8563911
Fax: +86 743 8563911
E-mail address: wanggch123@163.com
1

Abstract.
A novel series of N-arylbenzo[d]oxazol-2-amines (4a-4m) were synthesized and
evaluated for their α-glucosidase inhibitory activity. Compounds 4f-4i, 4k and 4m
displayed potent inhibitory activity against α-glucosidase with IC50 values in the range
of 32.49±0.17-120.24±0.51 μM as compared to the standard drug acarbose. Among
all tested compounds, compound 4g having 4-phenoxy substitution at the phenyl ring
was found to be the most active inhibitor of α-glucosidase with an IC50 value of
32.49±0.17 μM. Analysis of the kinetics of enzyme inhibition indicated that
compound 4g is a noncompetitive inhibitor of α-glucosidase with a Ki value of 31.33
μM. Binding interaction of compound 4g with α-glucosidase was explored by
molecular docking simulation.
Keywords: Benzoxazole; α-Glucosidase inhibitor; Structure-activity relationship;
Enzyme kinetic study; Molecular docking
2

1. Introduction
α-Glucosidase (EC. 3.2.1.20) is a membrane-bound enzyme that located on the brush
border membrane of the small intestine1. It catalyzes the hydrolysis of the glycosidic
bond at the non-reducing terminal of the sugar, resulting in the release of free glucose
into the digestive tract. Inhibition of α-glucosidase could decrease the rate of
carbohydrates digestion and suppression of postprandial hyperglycemia2. Thus,
α-glucosidase has been regarded as an important target for the discovery and
development of novel anti-diabetic drugs. Currently, there are three α-glucosidase
inhibitors like acarbose, voglibose, and miglitol, which have been used in clinical for
the treatment of type-2 diabetes mellitus3, 4. Furthermore, due to its important role in
carbohydrates digestion and glycoprotein processing, α-glucosidase has also been
recognized as a potential therapeutic target for other carbohydrate mediated diseases
such as HIV5, 6, cancer7 and hepatitis8.
Benzoxazole is an important heterocyclic system, which is found in many biologically
active natural products such as caboxamycin9, UK-110 and nataxazole11 (Figure 1).
Previous studies revealed that benzoxazole derivatives possessed a wide variety of
biological activities including anti-inflammatory12, antimicrobial13, anti-HIV14,
anticancer15, antimycobacterial16, 17, antileishmanial18 activities. Over the last few
years, several compounds based on benzoxazole, like calcimycin and prinaberel19, are
in preclinical or clinical trials (Figure 1). Despite numerous efforts have been
focusing on the research and development of benzoxazole derivatives as potential
drugs20, there have been no reports on their α-glucosidase inhibitory activity.
In our continuing search for biologically active compounds, herein we reported the
synthesis of a novel series of benzoxazole derivatives and tested for their
α-glucosidase inhibitory activity. Furthermore, the structure-activity relationship
(SAR) and molecular docking studies of these compounds were discussed.
3

Figure 1. Structures of some biologically active benzoxazole compounds
2. Chemistry
The synthesis of N-arylbenzo[d]oxazol-2-amines 4a-4m was shown in Scheme 1.
The condensation of 2-aminophenol 1 with carbon disulfide (CS2) under basic
conditions in ethanol to give the intermediate benzo[d]oxazole-2-thiol 2, which was
then reacted with SOCl2 in the presence of DMF as catalyst to afford
2-chlorobenzo[d]oxazole 2. Treatment of intermediate 2 with different commercially
available aniline 3a-3m yielded the desired compounds 4a-4m. The structures of all
the new synthesized compounds 4a-4m were characterized by 1H NMR spectra.
Scheme 1. Reagents and conditions: (a) CS2, KOH, EtOH, reflux, 4 h; (b) SOCl2,
DMF, reflux, 2 h; (c) DIEA, DMF, 150 oC, 3 h.
4

3. Results and discussion
3.1. α-Glucosidase inhibition assay
These new synthesized N-arylbenzo[d]oxazol-2-amines 4a-4m were evaluated for
their in vitro α-glucosidase inhibitory activity using acarbose as a positive control
(IC50, 817.38±6.27 μM21, 22). As shown in Table 1, 4f, 4g, 4h, 4k and 4m were more
inhibitive to α-glucosidase than acarbose, especially 4g, with an IC50 value of
32.49±0.17 μM, which was twenty-five folds than the positive control.
The introduction of electron-donating groups such as methyl (4b, 4c, 4d and 4e),
ethoxyl (4l) and methoxyl (4j) into the phenyl ring, results in a significant decrease
the inhibitory activity. It is interesting to point out that 4-phenoxy group at the phenyl
ring of 4g significantly improved the inhibitory activity, thus revealing that the steric
effect of the substitutions at the phenyl ring influences inhibitory activity. Compounds
4f (3-CF3), 4h (4-F), 4i (4-Cl) and 4k (2,4-Cl2) with electron-withdrawing group also
showed good inhibition with IC50 values of 120.24±0.51, 68.37±0.31, 80.39±0.28 and
44.4±0.17 μM, respectively. Out of these compounds, 4k (IC50 = 44.4±0.17 μM) with
strong electron-withdrawing 2,4-dichloro substitution on the phenyl ring, was found
to be the second most active compound. These results indicated the pattern
substitution in the phenyl ring is closely related to the biological activity of this class
of compounds. The binding interaction of these compounds was confirmed through
molecular docking.
Table 1. α-Glucosidase inhibitory activity of N-arylbenzo[d]oxazol-2-amines 4a-4m.
Compound
Structure
IC50 (μM)a
>200
4a
5

>200
4b
>200
>200
>200
4c
4d
4e
120.24±0.51
32.49±0.17
4f
4g
68.37±0.31
80.39±0.28
4h
4i
6

>200
44.4±0.17
>200
4j
4k
4l
64.95±0.25
4m
817.38±6.27
Acarbose
a Acarbose is standard for α-glucosidase inhibition activity
3.2. Homology model
The crystallographic structure of Saccharomyces cerevisiae α-glucosidase enzyme has
not been published yet, a number of homology models of α-glucosidase have been
reported in the literature.21, 23 In order to expose the binding mode between the
compounds and Saccharomyces cerevisiae α-glucosidase at the molecular level, the
3D structure of α-glucosidase was built by means of modeller 9.15 homology
modeling software (http://salilab.org/modeller/). The sequence in FASTA format of
α-glucosidase was retrieved from UniProt (access code P53341). The crystallographic
structure of Saccharomyces cerevisiae isomaltase (PDB ID: 3AJ7) with 72.4% was
selected as the template for modeling.
7

3.3. Molecular docking
Molecular docking simulations was carried out to investigate the binding mode of
these compound with Saccharomyces cerevisiae α-glucosidase. The theoretical
binding mode between the second active compound 4k and Saccharomyces cerevisiae
α-glucosidase was shown in Figure 2. Compound 4k adopted a compact
conformation in the pocket of the α-glucosidase. The dichlorophenyl group of 4k bind
at the bottom of the α-glucosidase pocket and made a high density of vander Waals
contacts, whereas the benzoxazole ring of 4k was positioned near the entrance of the
pocket and made only a few contacts. Detailed analysis showed that the
dichlorophenyl group of 4k formed an arene-cation interaction with the residue
Arg-439 and a π-π stacking with the residue Phe-157, respectively. In addition, the
benzoxazole ring of 4k located at the hydrophobic pocket, surrounded by residues
Phe-157, Phe-300 and Val-303. Importantly, a hydrogen bond interaction was
observed between 4k and the residue Asp-349, with the bond length of 3.3 Å. All
these interactions helped 4k to anchor in the binding site of α-glucosidase.
8

Figure 2. Compound 4k was docked into the binding pocket of the Saccharomyces
cerevisiae α-glucosidase.
We further docked the most active compound 4g into the binding site of
Saccharomyces cerevisiae α-glucosidase and the theoretical binding mode was shown
in Figure 3A. Compound 4g adopted a compact conformation in the pocket of the
α-glucosidase. The 4-phenoxyphenyl group of 4g bind at the bottom of the
α-glucosidase pocket and made a high density of vander Waals contacts, whereas the
benzoxazole ring of 4g was positioned near the entrance of the pocket and made only
a few contacts. Detailed analysis showed that the phenyl group in the middle of 4g
formed an arene-cation interaction with the residue Arg-439 and a π-π stacking with
9

the residue Phe-157, respectively. In addition, the benzoxazole ring of 4g located at
the hydrophobic pocket, surrounded by residues Phe-157, Phe-300 and Val-303.
Importantly, a hydrogen bond interaction was observed between 4g and the residue
Asp-349 (bond length: 3.2 Å). Besides, the phenyl group at the terminal of 4g formed
CH-π interactions with the residues Tyr-71 and Phe-177, respectively, which was the
main differences between 4g and 4k, and made 4g more active than 4k (Figure 3B).
In summary, the above molecular simulations give us rational explanation of the
interactions between 4k, 4g and α-glucosidase, which provided valuable information
for further development of α-glucosidase inhibitors.
Figure 3. Compound 4k and 4g were docked into the binding pocket of the
Saccharomyces cerevisiae α-glucosidase.
3.4. Enzyme kinetic analysis of the inhibition of α-glucosidase
To investigate the types of enzyme inhibition of this series of compounds, enzyme
kinetic study of the most active compound 4g was carried out. In the enzyme kinetic
studies, different concentrations of compound 4g (0, 10, 20, 30, 40 and 50 μM) and
substrate (0.25, 0.5, 1 and 2 mM) were used. The Lineweaver-Burk plots showed that
the maximal velocity (Vmax) gradually decreased and Michaelis-Menten constant
10

(Km) remains unchanged with increasing inhibitor concentration indicating a
non-competitive inhibition (Figure 4A). Furthermore, replots of the slope versus
different concentration of compound 4g gave an estimate of the inhibition constant, Ki
of 31.33 μM (Figure 4B). The results indicated that compound 4g is a noncompetitive
inhibitor of α-glucosidase with a Ki value of 31.33 μM.
Figure 4. (A) Lineweaver-Burk plots for inhibition of α-glucosidase in the presence
of 4g. Concentrations of 4g were 0, 10, 20, 30, 40 and 50 μM, respectively. Substrate
p-nitrophenyl α-D-glucopyranoside concentrations were 0.25, 0.5, 1 and 2 mM,
respectively. (B) replots of the slope versus different concentration of compound 4g to
determine inhibition constant.
4. Conclusion
In summary, we synthesized a novel series of N-arylbenzo[d]oxazol-2-amines (4a-4m)
and evaluated for their α-glucosidase inhibitory activity. Among them, compounds 4g
(IC50 = 32.49±0.17 μM) having 4-phenoxy substitution at the phenyl ring displayed
the most potent α-glucosidase inhibitory activity, with twenty-five folds more active
than the standard drug acarbose (IC50 = 817.38±6.27 μM). Docking studies showed
compound 4g have high binding affinity with the α-glucosidase due to the presence of
hydrogen bonds (Asp-349), hydrophobic interactions (Phe-157, Phe-300 and Val-303)
and CH-π interactions (Tyr-71 and Phe-177). This is the first report of benzoxazole
derivatives as inhibitors of α-glucosidase, and the results indicated that this class of
11

compound could be used as lead compound for the development of novel
α-glucosidase inhibitors.
5. Experimental section.
5.1. Chemistry.
All starting materials and reagents were purchased from commercial suppliers. TLC
was performed on 0.20 mm Silica Gel 60 F254 plates (Qingdao Ocean Chemical
Factory, Shandong, China). Nuclear magnetic resonance spectra (NMR) were
recorded on a Bruker spectrometer (400 MHz) with TMS as an external reference and
reported in parts per million.
5.1.1. Benzo[d]oxazole-2-thiol (1)
To a solution of o-amino phenol (10 mmol, 1.09 g) and KOH (10 mmol, 0.56 g) in
o
ethanol (50 mL) was added CS2 (40 mmol, 3.05g). The mixture was stirred at 50 C
for 4 h and monitored by TLC. After completion of reaction, the solvent was removed
under reduced pressure. The residue was poured into ice cold water and 10 % HCl
was added to adjust pH = 6-7. The precipitated was filtered, washed with water, dried
and purified by silica gel column chromatography (petroleum ether/EtOAc, 8:1).
White solid, yield 54.2 %, m.p. 191-192 °C.
5.1.2. 2-Chlorobenzo[d]oxazole (2)
To a suspension of 1 (10 mmol, 1.51 g) in SOCl2 (50 mL) was added few drops of
DMF, and the mixture heated at reflux for 5 h. After the solvent was evaporated, the
crude product was purified by silica gel column chromatography (petroleum
ether/EtOAc, 10:1). Colorless oil, yield 69.7 %.
12

5.1.3. General procedures for the synthesis of N-arylbenzo[d]oxazol-2-amines
(4a-4m)
A mixture of 2 (151 mg, 1 mmol), different substituted aniline 3a-3m (2 mmol) and
diisopropylethylamine (2 mmol) in DMF (20 mL) was stirred at 150 °C for 3 h. After
the completion of the reaction, the mixture was poured into 100 mL of water and
extracted with ethyl acetate (100 mL×3). The combined organic layers were dried
over Na2SO4 and concentrated under vacuum. The residue was purified by silica gel
column chromatography (petroleum ether/EtOAc, 10:1 to 4:1) to give the title product
4a-4m.
5.1.3.1. N-Phenylbenzo[d]oxazol-2-amine (4a)
o
1
Yellow solid, yield 56.2 %, m.p. 170-172 C, H NMR (CDCl3, 400 MHz) δ:
7.10-7.15 (m, 2H, Ar-H), 7.21-7.26 (m, 1H, Benzoxazole-H), 7.34 (d, 1H, J = 8.0 Hz,
Benzoxazole-H), 7.38-7.42 (m, 2H, Ar-H), 7.48 (d, 1H, J = 8.0 Hz, Benzoxazole-H),
13
7.60-7.63 (m, 2H, Ar-H), 7.93 (s, 1H, NH); C NMR (CDCl3, 100 MHz) δ: 109.2,
117.0, 118.6, 121.8, 123.4, 124.3, 129.4, 138.0, 142.1, 147.9, 158.7; MS (ESI, m/z):
211.01 [M+H]+.
5.1.3.2. N-(p-Tolyl)benzo[d]oxazol-2-amine (4b)
o
1
White solid, yield 46.9 %, m.p. 176-178 C, H NMR (CDCl3, 400 MHz) δ: 2.35 (s,
3H, CH3), 7.11-7.25 (m, 4H, Ar-H), 7.32-7.34 (m, 1H, Ar-H), 7.47-7.49 (m, 3H, Ar-H),
13
7.92 (s, 1H, NH); C NMR (CDCl3, 100 MHz) δ: 20.8, 109.2, 116.8, 119.0, 121.6,
124.3, 129.9, 133.1, 135.4, 142.2, 148.0, 159.1; MS (ESI, m/z): 225.02 [M+H]+.
5.1.3.3. N-(m-Tolyl)benzo[d]oxazol-2-amine (4c)
o
1
White solid, yield 32.1 %, m.p. 137-140 C, H NMR (CDCl3, 400 MHz) δ: 2.40 (s,
3H, CH3), 6.92 (d, 1H, J = 7.2 Hz, Ar-H), 7.11-7.15 (m, 1H, Benzoxazole-H),
7.22-7.26 (m, 1H, Benzoxazole-H), 7.26-7.30 (m, 1H, Ar-H), 7.34 (d, 1H, J = 8.0 Hz,
Benzoxazole-H), 7.41-7.44 (m, 2H, Ar-H), 7.48 (d, 1H, J = 8.0 Hz, Benzoxazole-H),
13
7.90 (s, 1H, NH); C NMR (CDCl3, 100 MHz) δ: 21.6. 109.3, 115.9, 116.7, 119.5,
121.6, 124.3, 124.3, 129.2, 138.0, 139.3, 142.0, 148.0, 159.1; MS (ESI, m/z): 225.03
13

[M+H]+.
5.1.3.4. N-(o-Tolyl)benzo[d]oxazol-2-amine (4d)
o
1
Yellow solid, yield 47.1 %, m.p. 98-100 C, H NMR (CDCl3, 400 MHz) δ: 2.36 (s,
3H, CH3), 7.06-7.13 (m, 2H, Ar-H), 7.20-7.25 (m, 2H, Ar-H), 7.29-7.33 (m, 2H,
13
Ar-H), 7.45 (d, 1H, J = 8.0 Hz, Benzoxazole-H), 8.06 (d, 1H, J = 8.0 Hz, Ar-H); C
NMR (CDCl3, 100 MHz) δ: 17.8, 109.1, 117.1, 121.1, 121.7, 124.2, 124.5, 127.2,
128.2, 130.8, 136.0, 142.2, 148.0, 159.1; MS (ESI, m/z): 225.04 [M+H]+.
5.1.3.5. N-(2,4-Dimethylphenyl)benzo[d]oxazol-2-amine (4e)
Brown solid, yield 48.1 %, m.p. 95-97 oC, 1H NMR (CDCl3, 400 MHz) δ: 2.32 (s, 3H,
CH3), 2.33 (s, 3H, CH3), 7.05-7.11 (m, 3H, Ar-H), 7.20 (t, 1H, J = 6.8 Hz,
Benzoxazole-H), 7.30 (d, 1H, J = 7.6 Hz, Benzoxazole-H), 7.43 (d, 1H, J = 7.6 Hz,
Benzoxazole-H), 7.82 (d, 1H, J = 8.0 Hz, Ar-H); 13C NMR (CDCl3, 100 MHz) δ: 17.8,
20.9, 109.0, 116.8, 121.5, 122.2, 124.1, 127.6, 129.3, 131.5, 133.3, 134.6, 142.5,
148.1, 159.7; MS (ESI, m/z): 239.02 [M+H]+.
5.1.3.6. N-(3-(Trifluoromethyl)phenyl)benzo[d]oxazol-2-amine (4f)
White solid, yield 66.8 %, m.p. 190-193 oC, 1H NMR (CDCl3, 400 MHz) δ: 7.15-7.19
(m, 1H, Benzoxazole-H), 7.26-7.29 (m, 1H, Ar-H), 7.35-7.40 (m, 2H, Ar-H),
7.50-7.54 (m, 2H, Ar-H), 7.88-7.89 (m, 2H, Ar-H), 8.03 (s, 1H, NH); 13C NMR
((DMSO-d6, 100 MHz) δ: 113.6, 119.1, 121.9, 123.1, 125.9, 126.7, 128.8, 134.3,
135.4, 144.4, 147.3, 152.2, 162.7; MS (ESI, m/z): 278.97 [M+H]+.
5.1.3.7. N-(4-Phenoxyphenyl)benzo[d]oxazol-2-amine (4g)
o
1
Brown solid, yield 52.0 %, m.p. 140-143 C, H NMR (CDCl3, 400 MHz) δ:
7.00-7.02 (m, 2H, Ar-H), 7.06-7.10(m, 3H, Ar-H), 7.11-7.14 (m, 1H, Benzoxazole-H),
7.21-7.26 (m, 1H, Benzoxazole-H), 7.31-7.35 (m, 3H, Ar-H), 7.46 (d, 1H, J = 8.0 Hz,
Benzoxazole-H), 7.57 (d, 2H, J = 8.0 Hz, Ar-H); 13C NMR (CDCl3, 100 MHz) δ:
109.2, 116.8, 118.4, 120.1, 120.5, 121.7, 123.1, 124.4, 129.8, 133.6, 142.0, 147.9,
153.0, 157.7, 158.9; MS (ESI, m/z): 302.99 [M+H]+.
5.1.3.8. N-(4-Fluorophenyl)benzo[d]oxazol-2-amine (4h)
14

o
1
Yellow solid, yield 34.1 %, m.p. 166-168 C, H NMR (CDCl3, 400 MHz) δ:
7.07-7.15 (m, 3H, Ar-H), 7.22-7.26 (m, 1H, Benzoxazole-H), 7.33 (d, 1H, J = 8.0 Hz,
Benzoxazole-H), 7.45 (d, 1H, J = 8.0 Hz, Benzoxazole-H), 7.56-7.59 (m, 2H, Ar-H);
13C NMR (CDCl3, 100 MHz) δ: 109.2, 115.9, 116.1, 116.8, 120.5, 120.6, 121.9, 124.4,
134.0, 141.9, 147.9, 157.9, 158.7, 160.3; MS (ESI, m/z): 229.00 [M+H]+.
5.1.3.9. N-(4-Chlorophenyl)benzo[d]oxazol-2-amine (4i)
o
1
Yellow solid, yield 41.0 %, m.p. 169-172 C, H NMR (CDCl3, 400 MHz) δ:
7.13-7.17 (m, 1H, Benzoxazole-H), 7.23-7.27 (m, 1H, Benzoxazole-H), 7.35-7.37 (m,
3H, Ar-H), 7.48 (d, 1H, J = 7.2 Hz, Benzoxazole-H), 7.57-7.60 (m, 2H, Ar-H), 7.67 (s,
1H, NH); 13C NMR ((DMSO-d6, 100 MHz) δ: 113.5, 121.7, 124.1, 126.4, 128.7,
131.6, 133.5, 147.4, 152.2, 163.0; MS (ESI, m/z): 244.98 [M+H]+.
5.1.3.10. N-(4-Methoxyphenyl)benzo[d]oxazol-2-amine (4j)
o
1
Brown solid, yield 21.1 %, m.p. 132-135 C, H NMR (CDCl3, 400 MHz) δ: 3.82 (s,
3H, OCH3), 6.93-6.95 (m, 2H, Ar-H), 7.08-7.12 (m, 1H, Benzoxazole-H), 7.19-7.23
(m, 1H, Benzoxazole-H), 7.31 (d, 1H, J = 8.0 Hz, Benzoxazole-H), 7.43 (d, 1H, J =
13
8.0 Hz, Benzoxazole-H), 7.49-7.51 (m, 2H, Ar-H); C NMR (CDCl3, 100 MHz) δ:
55.6, 109.1, 114.6, 116.6, 121.1, 121.4, 124.3, 131.1, 142.2, 148.0, 156.2, 159.5; MS
(ESI, m/z): 241.03 [M+H]+.
5.1.3.11. N-(2,4-Dichlorophenyl)benzo[d]oxazol-2-amine (4k)
o
1
White solid, yield 45.7 %, m.p. 90-93 C, H NMR (CDCl3, 400 MHz) δ: 7.16-7.20
(m, 1H, Benzoxazole-H), 7.25-7.29 (m, 1H, Benzoxazole-H), 7.34-7.38 (m, 2H,
Ar-H), 7.42 (d, 1H, J = 2.0 Hz, Ar-H), 7.47 (s, 1H, NH), 7.53-7.55 (m, 1H,
Benzoxazole-H), 8.57 (d, 1H, J = 8.8 Hz, Ar-H); 13C NMR (CDCl3, 100 MHz) δ:
109.2, 117.9, 119.6, 121.9, 122.7, 124.4, 127.8, 128.2, 128.8, 133.3, 142.1, 147.7,
156.6; MS (ESI, m/z): 278.90 [M+H]+.
5.1.3.12. N-(4-Ethoxyphenyl)benzo[d]oxazol-2-amine (4l)
Brown solid, yield 26.2 %, m.p. 123-125 oC, 1H NMR (CDCl3, 400 MHz) δ: 1.41 (m,
3H, J = 6.8 Hz, OCH2CH3), 4.03 (m, 2H, J = 6.8 Hz, OCH2CH3), 6.82-6.97 (m, 2H,
15

Ar-H), 7.05-7.13 (m, 1H, Ar-H), 7.19-7.31 (m, 2H, Ar-H), 7.39-7.53 (m, 3H, Ar-H),
13
7.63 (s, 1H, NH); C NMR (CDCl3, 100 MHz) δ: 14.9, 63.9, 109.1, 115.2, 116.6,
121.0, 121.4, 124.2, 131.0, 142.2, 148.0, 155.5, 159.5; MS (ESI, m/z): 255.01
[M+H]+.
5.1.3.13. N-(2-Ethoxyphenyl)benzo[d]oxazol-2-amine (4m)
Brown solid, yield 40.7 %, m.p. 40-43 oC, 1H NMR (CDCl3, 400 MHz) δ: 1.49 (t, 3H,
J = 6.8 Hz, OCH2CH3), 4.03 (m, 2H, J = 6.8 Hz, OCH2CH3), 6.88-6.90 (m, 1H, Ar-H),
6.98-7.05 (m, 2H, Ar-H), 7.10-7.14 (m, 1H, Benzoxazole-H), 7.21-7.25 (m, 1H,
Benzoxazole-H), 7.33 (d, 1H, J = 8.0 Hz, Benzoxazole-H), 7.50 (d, 1H, J = 8.0 Hz,
Benzoxazole-H), 7.70 (s, 1H, NH), 8.39 (d, 1H, J = 7.6 Hz, Ar-H); 13C NMR (CDCl3,
100 MHz) δ: 14.9, 64.3, 108.9, 110.9, 117.4, 121.2, 121.9, 122.6, 124.1, 127.5, 142.7,
146.6, 147.8, 157.7; MS (ESI, m/z): 255.01 [M+H]+.
5.2. In vitro assay of α-glucosidase inhibitory activity
The α-glucosidase inhibition assay had been carried out using baker’s yeast
α-glucosidase (EC 3.2.1.20) and p-nitrophenyl α-D-glucopyranoside. The test
compounds were dissolved in DMSO to prepare the required distributing
concentration. α-Glucosidase inhibitory activity was assayed in 0.1 M phosphate
o
buffer (pH 6.8) at 37 C. The enzyme (0.1 U/mL) in phosphate buffer saline was
incubated with various concentrations of test compounds (DMSO, final concentration
o
5% v/v) at 37 C for 15 min. Then 1.25 mM p-nitrophenyl α-D-glucopyranoside was
o
added to the mixture as a substrate. After further incubation at 37 C for 30 min. The
absorbance was measured spectrophotometrically at 405 nm. The sample solution was
replaced by DMSO as a control. Acarbose was used as a positive control. All
experiments were carried out in triplicates. The% inhibition has been obtained using
16

the formula: Inhibition (%) = (1-∆Asample/∆Acontrol) * 100%. IC50 value is defined
as a concentration of samples inhibiting 50% of α-glucosidase activity under the
stated assay conditions.
5.3. Molecular docking
Molecular docking studies were performed to investigate the binding mode between
the compounds 7k, 7g and α-glucosidase using Autodock vina 1.1.2. The 3D
structures of 7k and 7g were obtained by ChemBioDraw Ultra 14.0 and ChemBio3D
Ultra 14.0 softwares. The AutoDockTools 1.5.6 package was employed to generate
the docking input files. The search grid of α-glucosidase was identified as center_x:
-19.676, center_y: -7.243, and center_z: -21.469 with dimensions size_x: 15, size_y:
15, and size_z: 15. The value of exhaustiveness was set to 20. For Vina docking, the
default parameters were used if it was not mentioned. The best-scoring poses as
judged by the Vina docking score were chosen and visually analyzed using PyMOL
1.7.6 software (http://www.pymol.org/).
Conflict of Interest
The authors confirm that this article content has no conflict of interest.
Acknowledgments
This work was supported by Hunan Provincial Natural Science Foundation of China
(2015JJ3099), Scientific Research Fund of Hunan Provincial Education Department
(15B194), Jishou University for talent introduction and the college students' research
learning and innovative experiment plan project of Jishou University and Hunan
17

Province (Jing Wang, [2016]283).
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