Quinazolinone derivatives: Synthesis and comparison of inhibitory mechanisms on α-glucosidase

Article abstract of DOI:10.1016/j.bmc.2016.09.042

In this study, eight quinazolinone derivatives were designed and synthesized. Their inhibitory activities on α-glucosidase were assessed in vitro. Two compounds: 2-(4-chlorophenyl)-quinazolin-4(3H)-one (CQ) and 2-(4-bromophenyl)-quinazolin-4(3H)-one (BQ) were found to be potent inhibitors of α-glucosidase with IC₅₀values of 12.5 ± 0.1 μM and 15.6 ± 0.2 μM, respectively. Spectroscopy methods were performed to analyze the inhibitory mechanisms of both compounds on α-glucosidase. The results revealed that they reversibly inhibited α-glucosidase in a non-competitive manner. CQ and BQ could statically quench the fluorescence spectra by formation of an inhibitor-α-glucosidase complex. The interaction between CQ and α-glucosidase depended on hydrogen bonds, electrostatic and hydrophobic force, while the driving force of the binding between BQ and the enzyme was hydrophobic. The docking results showed that BQ was less active than CQ against α-glucosidase because of its weaker interaction with the enzyme. In brief, the quinazolinone derivatives identified in this work were potentially promising candidates for developing as novel anti-diabetic agents.

Full text of DOI:10.1016/j.bmc.2016.09.042

Accepted Manuscript
Quinazolinone derivatives: synthesis and comparison of inhibitory mechanisms
on α-glucosidase
Mankun Wei, Wei-Ming Chai, Rui Wang, Qin Yang, Zhihong Deng, Yiyuan
Peng
PII:
S0968-0896(16)30738-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.042
BMC 13295
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 August 2016
15 September 2016
16 September 2016
Please cite this article as: Wei, M., Chai, W-M., Wang, R., Yang, Q., Deng, Z., Peng, Y., Quinazolinone derivatives:
synthesis and comparison of inhibitory mechanisms on α-glucosidase, Bioorganic & Medicinal Chemistry (2016),
doi: http://dx.doi.org/10.1016/j.bmc.2016.09.042
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Quinazolinone derivatives: synthesis and comparison of inhibitory
mechanisms on α-glucosidase
Mankun Weia, Wei-Ming Chai *,a,b, Rui Wanga, Qin Yanga,b, Zhihong Deng b, Yiyuan
Peng *,a,b
a Key Laboratory of Small Fuctional Organic Molecule, Ministry of Education and
College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi 330022,
China
b Key Laboratory of Green Chemistry, Jiangxi Province Nanchang, Jiangxi 330022,
China
* Corresponding author. E-mail address: yypeng@jxnu.edu.cn; chaiweiming@jxnu.edu.cn

Abstract
In this study, eight quinazolinone derivatives were designed and synthesized.
Their inhibitory activities on ꢀ-glucosidase were assessed in vitro. Two compounds:
2-(4-chlorophenyl)-quinazolin-4(3H)-one (CQ) and
2-(4-bromophenyl)-quinazolin-4(3H)-one (BQ) were found to be potent inhibitors of
ꢀ-glucosidase with IC50 values of 12.5 0.1 ꢁM and 15.6 0.2 ꢁM, respectively.
Spectroscopy methods were performed to analyze the inhibitory mechanisms of both
compounds on ꢀ-glucosidase. The results revealed that they reversibly inhibited
ꢀ-glucosidase in a non-competitive manner. CQ and BQ could statically quench the
fluorescence spectra by formation of an inhibitor-ꢀ-glucosidase complex. The
interaction between CQ and ꢀ-glucosidase depended on hydrogen bonds, electrostatic
and hydrophobic force, while the driving force of the binding between BQ and the
enzyme was hydrophobic. The docking results showed that BQ was less active than
CQ against ꢀ-glucosidase because of its weaker interaction with the enzyme. In brief,
the quinazolinone derivatives identified in this work were potentially promising
candidates for developing as novel anti-diabetic agents.
Kewords: quinazolinone, ꢀ-glucosidase, fluorescence, docking.

1. Introduction
Diabetes Mellitus is a health-threatening chronic metabolic disease, arose from
insufficient insulin secretion and characterized by hyperglycemia.1 Enhanced
postprandial glucose which associated with diabetes mellitus type II will increase the
risk of developing atherosclerosis, stroke and other coronary diseases.2 Thus, reduced
the postprandial glucose by inhibiting the digestive enzymes such as ꢀ-glucosidase is
an effective approach for the treatment of diabetes mellitus type II and diabetic
complications.3 ꢀ-Glucosidase (EC 3.2.1.20) is an enzyme which located in the small
intestine epithelium, catalyzing the final step in the hydrolysis of disaccharides and
polysaccharides to glucose. The activity of ꢀ-glucosidase is directly related to the
concentrations of blood glucose, and inhibition of ꢀ-glucosidase is crucial due to the
potential effects of decreased postprandial blood glucose levels. ꢀ-Glucosidase
inhibitors, such as voglibose and acarbose, are clinically utilized for retarding the
rapid generation of blood glucose. However, they often result in some side effects
including diarrhea, abdominal pain and other gastrointestinal disorders in chronic
therapy.4,5 Therefore, the search for efficient and safe ꢀ-glucosidase inhibitors is
desirable for the therapy of postprandial hyperglycemia.
Quinazolinone derivatives endowed with rich pharmacological properties and
biological activities, which have been reported to possess anti-virus,6 anti-bacterial,7,8
anti-inflammatory,9 anti-cancer,10-12 anti-allergic,13 anti-fungal,14 anti-rheumatic,15
anti-convulsant,16-20 and CNS depressant21,22 activities. Hence, quinazolinone
derivatives have been gained a great interest in pharmaceutical chemistry field.23-28

Inspired by previous work,29-31 we designed and synthesized eight quinazolinone
derivatives, and their inhibitory activities on ꢀ-glucosidase were assessed in vitro.
Two
derivatives:
2-(4-chlorophenyl)-quinazolin-4(3H)-one
(CQ)
and
2-(4-bromophenyl)-quinazolin-4(3H)-one (BQ) were identified as potent inhibitors on
ꢀ-glucosidase. Although CQ has been apprised of exhibiting anti-ꢀ-glucosidase
activity,32 the inhibitory mechanisms of these two compounds on ꢀ-glucosidase were
never been covered. The aims of current study were to investigate and compare the
inhibitory mechanisms of quinazolinone derivatives on ꢀ-glucosidase by using kinetic,
fluorescence quenching and molecular docking methods. The findings of this work
would provide a comprehensive understanding about the inhibitory mechanisms of
quinazolinone derivatives on ꢀ-glucosidase and benefit to developing novel
anti-diabetic agents.
2. Experimental
2.1. Chemicals
ꢂ-Glucosidase
(from
Saccharomyces
cerevisiae),
p-nitrophenyl-ꢂ-D-glucopyranoside (pNPG) were obtained from Sigma-Aldrich (St.
Louis, MO, USA). Acarbose was obtained from Aladdin. (Shanghai, China).
Anthranilamide, substituted aldehydes were purchased from 9 Ding chemistry
(shanghai) Co.
2.2. Synthesis and structure characterization of quinazolinone derivatives
Quinazolinone derivatives were synthesized from the condensation of
anthranilamide (0.2 mmol) and substituted aldehydes (0.4 mmol) in the presence of

NaHSO3 (0.4 mmol) in DMAc (Scheme 1). The mixtures were stirred at 180
overnight. Upon completion of the reaction, the mixture was cooled to room
temperature and diluted with ethyl acetate. The resulting mixture was concentrated
under reduced pressure. The residue was purified by column chromatography on silica
gel (EtOAc/petroleum ether) to afford the desired product. The structures of
1
quinazolinone derivatives were characterized by H NMR coupled with FTIR and
ESI-MS analysis.
O
O
O
NH2
NH
DMAC, 180 °C
+
NaHSO3
H
R
NH2
N
R
CQ: R=4-Cl-Ph
BQ: R=4-Br-Ph
FQ: R=4-F-Ph
NQ: R=4-NO2-Ph
DMQ: R=4-(CH3)2N-Ph
2-MQ: R=2-Me-Ph
3-MQ: R=3-Me-Ph
TQ: R=Thiophene-2-yl
Scheme 1 The synthesis of quinazolinone derivatives
2-(4-chlorophenyl)-quinazolin-4(3H)-one (CQ).33
White solid; mp: 299-300 °C
1H NMR (400 MHz, DMSO-d6): ꢃ 12.63 (s, 1H), 8.25 (d, J=8 Hz, 2H), 8.21 (d, J=8
Hz, 1H), 7.90 (t, J=8 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 8 Hz, 2H), 7.59 (t,
J = 8 Hz, 1H).
FTIR (KBr, ꢄ cm-1): 3451.0, 3195.7, 3133.8, 3088.7, 1677.6, 1603.4, 1560.5, 1345.7,
840.9, 763.0.
ESI-MS: m/z [M+H]+ calcd for C14H10ClN2O 257.0482, found 257.0460.

2-(4-Bromophenyl)-quinazolin-4(3H)-one (BQ).34
White solid; mp: 296-297 °C.
1H NMR (400 MHz, DMSO-d6): ꢃ 12.64 (s, 1H), 8.20 (m, 3H), 7.90 (m, 1H), 7.81 (t,
J = 8.0 Hz, 3H), 7.60 (m, 1H).
FTIR (KBr, ꢄ cm-1): 3451.0, 3028.9, 2942.2, 2915.2, 1677.8, 1602.7, 1560.2, 1481.6,
1347.6, 961.0, 798.4.
ESI-MS: m/z [M+H]+ calcd for C14H10BrN2O 300.9976, found 301.0002.
2-(4-Fluorophenyl)-quinazolin-4(3H)-one (FQ).35
White solid; mp: 293-294 °C.
1H NMR (400 MHz, DMSO-d6): ꢃ 12.57 (s, 1H), 8.27 (m, 2H), 8.17 (d, J = 8.0 Hz,
1H), 7.84 (m, 1H), 7.73 (m, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.40 (t, J = 8.0 Hz, 2H).
ESI-MS: m/z [M+H]+ calcd for C14H10FN2O 241.0777, found 241.0773.
2-(4-Nitrophenyl)-quinazolin-4(3H)-one (NQ).36
Brown solid; mp: 363-364 °C.
1H NMR (400 MHz, DMSO-d6): ꢃ 12.82 (s, 1H), 8.41 (m, 4H), 8.19 (m, 1H), 7.87
(m, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.59 (m, 1H).
ESI-MS: m/z [M+H]+ calcd for C14H10N3O3 268.0722, found 268.0728.
2-(4-(Dimethylamino)-phenyl)quinazolin-4 (3H)-one (DMQ).37
White solid; mp: 296-297 °C.
1H NMR (400 MHz, DMSO-d6): ꢃ 12.18 (s, 1H), 8.12 (t, J = 8.0 Hz, 3H), 7.78 (m,
1H), 7.66 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 6.79 (d, J = 8.0 Hz, 2H), 3.02 (s,
6H).
ESI-MS: m/z [M+H]+ calcd for C16H16N3O 266.1293, found 266.1278.
2-(2-Methylphenyl)-quinazolin-4(3H)-one (2-MQ).38
White solid; mp: 214-216 °C.

1H NMR (400 MHz, DMSO-d6): ꢃ 12.45 (s, 1H), 8.19 (m, 1H), 7.84 (m, 1H), 7.70 (d,
J = 8.0 Hz, 1H), 7.55 (m, 2H), 7.44 (m, 1H), 7.35 (m, 2H), 2.40 (s, 3H).
ESI-MS: m/z [M+H]+ calcd for C15H13N2O 237.1028, found 237.1026.
2-(3-Methylphenyl)-quinazolin-4(3H)-one (3-MQ).39
White solid; mp: 210-212 °C.
1H NMR (400 MHz, DMSO-d6): ꢃ 12.47 (s, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.0 (m,
2H), 7.84 (m, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.43 (m, 2H),
2.42 (s, 3H).
ESI-MS: m/z [M+H]+ calcd for C15H13N2O 237.1028, found 237.1006.
2-(Thiophene-2-yl)-quinazolin-4(3H)-one (TQ).33
White solid mp: 275-276 °C.
1H NMR (400 MHz, DMSO-d6): ꢃ 12.66 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.14 (d, J =
8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.81 (m, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.50 (t, J
= 8.0 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H).
ESI-MS: m/z [M+H]+ calcd for C12H9N2OS 229.0436, found 229.0438.
2.3. Enzyme Assay
ꢀ-Glucosidase activity assay was performed on the basis of Ranilla et al.40 with a
slightly modification. The reaction system involved with 50 µL of sample with
different concentrations, 50 µL of ꢀ-glucosidase solution (7U/mL) and 200 µL of
5mM p-nitro-phenyl-ꢀ-D-glucopyranoside solution in 0.1M potassium phosphate
buffer (pH=6.8). The absorbencies were measured every 10s at 405nm in ambient
temperature. The relative enzymatic activity was calculated as follows: The relative
activity (%) = (slope of reaction kinetics equation obtained by reaction with
inhibitor)/(slope of reaction without inhibitor)×100. Acarbose was used as a positive
control. The inhibitory mechanism assay was applied with varying the concentration

of the enzyme in the reaction mixture. The inhibition type was then assayed by the
Lineweaver-Burk plot, and the inhibition constant was determined from the secondary
plot.
2.4. The fluorescence quenching analysis
The fluorescence quenching analysis was carried out via a Varian Cary Eclipse
fluorescence spectrophotometer in the range of 300-500 nm, and the excitation
wavelength was set at 280 nm. Briefly, 50 µL of samples with different concentrations
were mixed with 50 µL of ꢀ-glucosidase (0.1 mg/mL) and 900 µL of 0.1 mM sodium
phosphate buffer. All the data was obtained at 25 °C.
The dynamic quenching data were ascertained from the Stern-Volmer equation.41
F0/F = 1 + Kqꢅ0[Q] = 1 + Ksv[Q]……. (1)
In the equation, F0 and F are the fluorescence intensities of ꢀ-glucosidase in the
absence and presence of quinazolinone derivatives. Ksv is the Stern-Volmer quenching
constant. Kq is the quenching velocity constant of biomolecular reaction. ꢅ0 is the
average lifetime of the fluorophore absence of quencher. [Q] is the concentration of
quinazolinone derivatives.
The fluorescence data were further estimated by the modified Stern-Volmer
equation.42
log [(F0 − F)/F] = log K + n log [Q]……(2)
Where K is the binding constant, n represents the number of binding sites.
2.5. Molecular docking
The X-ray structure of ꢂ-glucosidase was downloaded from the RCSB protein
Data Bank. The water molecules in ꢂ-glucosidase were removed, whereas Gasteiger
charges and polar hydrogen atoms were added to the macromolecule by using the
AutoDock tools. The 3D structures of quinazolinone derivatives were generated by

Chem Bio Draw Ultra 8.0. Docking calculations were achieved using the default
parameters. The docked conformation with lowest free energy was selected as the
optimal binding pattern.
3. Results and Discussion
3.1. The effects of quinazolinone derivatives on α-glucosidase activity
The effects of quinazolinone derivatives on the hydrolyzation of
p-nitrophenyl-ꢂ-D-glucopyranoside catalyzed by ꢀ-glucosidase were studied. The
concentrations leading to 50% activity loss (IC50) were listed in Table 1. Substitution
of chlorine at C4’ on phenyl ring in CQ and substitution of bromine at C4’ on phenyl
ring in BQ were found as effective inhibitors on ꢀ-glucosidase with the IC50 values of
12.5 0.1 ꢁM and 15.6 0.2 ꢁM, respectively. When the fluorine group was present
at C4’ (FQ, IC50 >57.2 ꢁM), a significantly decreased in activity on ꢀ-glucosidase was
observed. Whereas nitro group at C4’ phenyl ring (NQ), dimethylamino group at C4’
phenyl ring (DMQ), methyl group at C2’ phenyl ring (2-MQ), methyl group at C3’
phenyl ring (3-MQ) and thiophene group at quinazolinone ring (TQ) completely
diminished the activity on ꢀ-glucosidase. These results indicated that the presence of
chlorine and bromine at C4’ phenyl ring were distinctly responsible for the potent
activity. Compared with the positive control acarbose (IC50=0.475 0.001 mM), CQ
and BQ were prominent ꢀ-glucosidase inhibitors, and with a possibility to adopt as
novel anti-diabetic agents.

Table 1. Inhibition of quinazolinone derivatives on ꢀ-glucosidase
Compound
Structure
IC50 (µM)
O
NH
CQ
12.5 0.1
N
Cl
Br
F
O
NH
BQ
15.6 0.2
>57.2
NA
N
O
N
NH
FQ
O
NH
NQ
N
NO2
O
N
NH
DMQ
NA
CH3
N
CH3

O
N
NH
CH3
2-MQ
3-MQ
TQ
NA
NA
NA
O
N
NH
CH3
O
N
NH
S
Values are expressed as mean of triplicate determinations standard deviation
3.2. Inhibitory mechanisms of CQ and BQ on α-glucosidase
To ascertain the mechanisms of CQ and BQ on ꢀ-glucosidase, the plots of the
enzyme activity versus the concentrations of enzyme in the presence of different
amount of compounds were constructed in Fig. 1. The plots showed a good linear
relationship, all the straight lines passing through the origin. Moreover, the slopes of
the lines reduced with raising the concentrations of CQ and BQ, which indicated that
the presence of CQ and BQ did not lessen the amount of ꢀ-glucosidase, but it resulted
in the suppression of enzymatic activity. These results clearly suggested that both CQ
and BQ inhibited ꢀ-glucosidase in a reversible way.

Figure 1 The inhibitory mechanisms of CQ (A) and BQ (B) on ꢀ-glucosidase. The concentrations
of CQ and BQ for curves 0-4 were 0, 8, 12, 16, 24 µM.
3.3. The inhibition modes of BQ and CQ on α-glucosidase
The Lineweaver-Burk reciprocal plots (Fig. 2A, 2B) were applied to analyze the
inhibition modes of CQ and BQ on ꢀ-glucosidase. The vertical axis intercept (1/Vm)
added and the horizontal axis intercept (-1/Km) untouched with mounting the
concentrations of inhibitors, which revealed that CQ and BQ caused a proper

noncompetitive inhibition. The enzyme inhibitor constants (KI) of CQ and BQ were
acquired from the secondary plots (Fig. 2A-1, 2B-1). They were calculated to be 9.1
and 10.6 ꢁM, respectively. The KI value of CQ was smaller than that of BQ, this
manifested that CQ showed a better binding affinity than BQ to ꢀ-glucosidase.
Figure 2 Lineweaver-Burk plots for inhibition of CQ (A) and BQ (B) on ꢀ-glucosidase. The
concentrations of CQ for curves 0-4 were 0, 8, 12, 16, 24 µM; curves 0-3 for the concentrations of
BQ were 0, 8, 12, 16 µM. The inhibition constant KI of CQ and BQ was determined from the
secondary plot A-1 and B-1, respectively.

3.4. Fluorescence quenching of α-glucosidase by CQ and BQ
Figure 3 (A) Fluorescence emission spectra of ꢀ-glucosidase in the presence of CQ at different
concentrations, the concentration of CQ for curves 0-4 were 0, 10, 20, 30, 50 µM, the curves 5 is
fluorescence emission spectra of CQ at the concentration of 50 µM. (B) The stem-volmer plots for
the fluorescence quenching of ꢀ-glucosidase at different concentrations of CQ. (C) Plot of lg [(F0
− F)/F] against lg [Q] for ꢀ-glucosidase various concentrations of CQ. F0 and F are the
fluorescence intensities of ꢀ-glucosidase in the absence and presence of CQ.

Figure 4 (A) Fluorescence emission spectra of ꢀ-glucosidase in the presence of BQ at different
concentrations, the concentration of BQ for curves 0-4 were 0, 10, 20, 30, 60 µM, the curves 5 is
fluorescence emission spectra of BQ at the concentration of 60 µM. (B) The stem-volmer plots for
the fluorescence quenching of ꢀ-glucosidase at different concentrations of BQ. (C) Plot of lg [(F0
− F)/F] versus lg [Q] for ꢀ-glucosidase various concentrations of BQ. F0 and F are the
fluorescence intensities of ꢀ-glucosidase in the absence and presence of BQ.

Fluorescence quenching analysis was executed to investigate the intrinsic
interaction mechanisms of CQ and BQ on the ꢀ-glucosidase. As shown in Fig. 3A and
Fig. 4A, ꢀ-glucosidase displayed a strong fluorescence peak at 330nm, which resulted
from the tyrosine residues of ꢀ-glucosidase. While, CQ and BQ had a high emission
peak at 404 and 390nm, respectively. Neverthless, as added different quinazolinone
derivatives, the fluorescence intensity of ꢀ-glucosidase descended gradually with
increasing the concentrations of the test compounds. The relative fluorescence
intensity of enzyme was decreased to 39.3% when the concentration of CQ was
extended to 50 ꢁM, whereas the relative fluorescence intensity of enzyme was only
reduced to 69.3% when the concentration of BQ was rose to 60 ꢁM. These results
showed that CQ was a better fluorescence quencher and easier to combine with the
enzyme. In addition, with increasing the concentrations of CQ and BQ, the highest
emission peak around 330nm no obvious red shift or blue shift, which proved that CQ
and BQ did not affect the conformation of ꢀ-glucosidase. Intriguingly, there were
accompanied increases in the fluorescence emission at 404 and 390nm with the
accretion of CQ and BQ (Fig. 3A, 4A) respectively which caused by the fluorescence
of quinazolinone derivatives.
For further identify the interaction mechanisms of quinazolinone derivatives on
the ꢀ-glucosidase, the fluorescence quenching data were calculated from the plots of
F0/F versus [Q] (Fig. 3B, 4B) based on the previous studies.41 For CQ and BQ, the
values of Kq were determined to be 3.10×1012 L/mol·s and 7.26×1011 L/mol·s,
respectively. The Kq values of BQ and CQ were much greater than the maximum
scatter collision quenching constant 2.0×1010 L/mol·s.43 Undoubtedly, BQ and CQ
evoked quenching were considered as static quenching by formation of an
inhibitor-ꢀ-glucosidase complex.

The binding constants (K) and number of binding sites (n) were obtained through
the plots of lg [(F0 − F)/F] versus lg [Q] (Fig. 3C, 4C ). The results (Table 2) showed
the binding constant (K) was CQ>BQ, which further confirmed that CQ was a better
fluorescence quencher and more preferable to connect with the enzyme.44 The number
of binding sites (n) of BQ and CQ were close to 1.
Table 2. Quenching constants of quinazolinone derivatives on ꢀ-glucosidase
compound
CQ
T( )
25
Ksv (L/mol)
3.10×104
7.26×103
Kq (L/mol.s)
3.10×1012
7.26×1011
K (L/mol)
1.78×104
1.25×104
n
1.13
0.85
BQ
25
3.5. Molecular docking
With the purpose of acquiring a better comprehension on the mutual effects
between quinazolinone derivatives and ꢀ-glucosidase, molecular dockings were
implemented using ActoDock tools. The conformations with lowest free energy was
considered to be the most actively optimal formation. As shown in Fig. 5, hydrogen
bonds were generated between CQ and amino residues: the oxygen atom on the
quinolinone ring and the chlorine atom on the benzene ring could interact with B
chain of residues Ala451 and Ser44, respectively. Besides, there existed an
electrostatic interaction between CQ and A chain of residue Asp441. Furthermore, it
was found that CQ inserted into the hydrophobic region of ꢀ-glucosidase interacting
with A chain residues His348, Ala444 and B chain Arg450, Ala454. These results
vividly displayed that the binding of CQ with ꢀ-glucosidase was driven by hydrogen
bonds, electrostatic and hydrophobic interaction. For compound BQ (Fig. 6), it could
be visible that a hydrophobic pocket constituting A chain of residues His348, Ala444
and B chain of residues Ala454 surrounded and grasped the ligands. The molecular

Figure 5 Molecular docking results of CQ with ꢀ-glucosidase. (A) Hydrogen bonds between CQ
and amino residues. (B) Electrostatic interaction between CQ and residue Asp441. (C)
Hydrophobic interaction of CQ with amino residues.
Figure 6 Hydrophobic interaction of BQ with ꢀ-glucosidase.

docking endorsed and visualized the consequence that the key driving force of the
binding between BQ and the enzyme was hydrophobic interaction. The docking
results clearly showed that CQ and BQ had different interaction with the enzyme. We
speculated that there existed two main reasons lead to the difference between the
chloro group and the bromo group: (1) the electronegativity of chlorine atom is higher
than that of bromine atom, it allowed CQ easier to generate hydrogen bonds and
electrostatic interaction with the amino acid residues; (2) the radius of chlorine atom
is smaller than that of bromine atom, which makes CQ could be better embedded into
the enzyme and interacted with the amino acid residues. Molecular docking analysis
further evidenced that BQ was less active than CQ against ꢀ-glucosidase because of
its weaker interaction with the enzyme.
4. Conclusion
In summary, compound CQ and BQ possessed excellent ꢀ-glucosidase inhibitory
activities comparing with the positive control acarbose. Both of those two compounds
reversibly inhibited the enzyme in a non-competitive manner. And they statically
quenched the fluorescence spectra by formation of an inhibitor-ꢀ-glucosidase
complex. Molecular docking studies revealed that BQ was less active than CQ against
ꢀ-glucosidase because of its weaker interaction with the enzyme. Inhibition the
activity of ꢀ-glucosidase was critical to control postprandial hyperglycemia. Hence,
the quinazolinone derivatives identified in this work were potentially promising
candidates for developing as novel anti-diabetic agents.

Acknowledgments
Financial support from National Natural Science Foundation of China (No. 21162012
and 31501414), the open project programs of CAS Key Laboratory of Molecular
Recognition and Function, Chinese Academy of Sciences and key laboratory of
Functional Small Organic Molecule, Ministry of Education, Jiangxi Normal
University (NO.KLFS-KF-201408 and KLFS-KF-201419) is gratefully
acknowledged.
References
1. Fatmawati, S.; Shimizu, K.; Kondo, R.; Phytomedicine 2011, 18, 1053.
2. Rother, K. I.; New Engl. J. Med. 2007, 356, 1499.
3. Casirola, D. M.; Ferraris, R. P.; Metab. 2006, 55, 832.
4. Chiasson, J. L.; Josse, R. G.; Gomis, R.; Hanefeld, M.; Karasik, A.; Laakso, M.; Group, S. N. T.
R.; J. Am. Med. Assoc. 2003, 290, 486.
5. Kawamori, R.; Tajima, N.; Iwamoto, Y.; Kashiwagi, A.; Shimamoto, K.; Kaku, K.; Group, V. P.
S.; Lancet. 2009, 373, 1607.
6. Li, H. Y.; Huang, R. Q.; Qiu, D. W.; Yang, Z.; Liu, X.; Ma, J. A.; Ma, Z. H.; Prog. Nat. Sci.
1998, 8, 359.
7. Bedi, P. M.; Kumar, V.; Mahajan, M. P.; Bioorg. Med. Chem. Let. 2004, 14, 5211.
8. Kung, P. P.; Casper, M. D.; Cook, K. L.; Wilson-Lingardo, L.; Risen, L. M.; Vickers, T. A.;
Ecker, D. J.; J. Med. Chem. 1999, 42, 4705.
9. Chandrika, P. M.; Yakaiah, T.; Rao, A. R. R.; Narsaiah, B.; Reddy, N. C.; Sridhar, V.; Rao, J. V.;
Eur. J. Med. Chem. 2008, 43, 846.

10. Li, Y.; Lu, W.; Saini, S. K.; Moukha-Chafiq, O.; Pathak, V.; Ananthan, S.; Oncotarget 2016, 7,
11263.
11. Isanbor, C.; O’Hagan, D.; J. Fluorine Chem. 2006, 127, 303.
12. Qazi, A. K.; Hussain, A.; Khan, S.; Aga, M. A.; Behl, A.; Ali, S.; Mondhe, D. M.; Cancer Lett.
2015, 359, 47.
13. Chandrika, P. M.; Ram Rao, A. R.; Narsaiah, B.; Raju, M. B.; Int. J. Chem. Sci. 2008, 6, 1119.
14. Mohamed, M. S.; Kamel, M. M.; Kassem, E. M.; Abotaleb, N.; El-moez, S. I. A.; Ahmed, M.
F.; Eur. J. Med. Chem. 2010, 45, 3311.
15. Ohta, Y.; Fukuda, S.; Baba, A.; Nagai, H.; Tsukuda, R.; Sohda, T.; Makino, H.;
Immunopharmacology 1996, 34, 17.
16. Srivastava, A. V. K.; Kumar, A.; Eur. J. Med. Chem. 2002, 37, 873.
17. Chapleo, C. B.; Mayer, M.; Myer, P. L.; Saville, J. F.; Smith, A. C. B.; Stilling, M. R.;
Tulloch, I. F.; Walter, D. S.; Welbourn, A. P.; J. Med. Chem. 1986, 29, 2273.
18. Srivastava, A. V. K.; Kumar, A.; Bioorg. Med. Chem. Lett. 2004, 12, 1257.
19. Aziza, M. A.; Ibrahim, M. K.; El-Helpy, A. G.; Al-Azhar J. Pharm. Sci. 1994, 14, 193.
20. Bekhit, A. A.; Khalil, M. A.; Pharmazie 1998, 53, 539.
21. Ergenc, N.; Buyuktimkin, S.; Capan, G.; Baktir, G.; Rollas, S.; Pharmazie 1991, 46, 290.
22. Kashaw, S. K.; Kashaw, V.; Mishra, P.; Jain, N. K.; Arkivoc, 2008, 14, 17.
23. Khan, I.; Zaib, S.; Batool, S.; Abbas, N.; Ashraf, Z.; Iqbal, J.; Saeed, A.; Bioorg. Med. Chem.
2016, 24, 2361.
24. Khan, I.; Ibrar, A.; Ahmed, W.; Saeed, A.; Eur. J. Med. Chem. 2015, 90, 124.
25. Yasuda, H.; Kobayashi, S.; Costa, D. B.; Lancet Oncol. 2012, 13, e23.

26. Roskoski, R.; Pharmacol. Res. 2014, 79, 34.
27. Alaa, A. M.; Abou-Zeid, L. A.; ElTahir, K. E. H.; Mohamed, M. A.; El-Enin, M. A. A.;
El-Azab, A. S.; Bioorg. Med. Chem. 2016, 24, 3818.
28. Cortot, A. B.; Repellin, C. E.; Shimamura, T.; Capelletti, M.; Zejnullahu, K.; Ercan, D.; Jänne,
P. A.; Cancer Res. 2013, 73, 834.
29. Pan, Z. Z.; Li, H. L.; Yu, X. J.; Zuo, Q. X.; Zheng, G. X.; Shi, Y.; Chen, Q. X.;
J. Agri. Food Chem. 2011, 59, 6645.
30. Chen, L. H.; Hu, Y. H.; Song, W.; Song, K. K.; Liu, X.; Jia, Y. L.; Chen, Q. X.;
J. Agri. Food Chem. 2012, 60, 1542.
31. Pan, Z. Z.; Zhu, Y. J.; Yu, X. J.; Lin, Q. F.; Xiao, R. F.; Tang, J. Y.; Chen, Q. X.;
Liu, B.; J. Agri. Food Chem. 2012, 60, 10784.
32. Javaid, K.; Saad, S. M.; Rasheed, S.; Moin, S. T.; Syed, N.; Fatima, I.; Choudhary, M. I.;
Bioorg. Med. Chem. 2015, 23, 7417.
33. Xu, W.; Jin, Y.; Liu, H.; Jiang, Y.; Fu, H.; Org. Lett. 2011, 13, 1274.
34. Chen, J.; Wu, D.; He, F.; Liu, M.; Wu, H.; Ding, J.; Su, W.; Tetrahedron Lett. 2008, 49, 3814.
35. Wang, R.; Chai, W. M.; Yang, Q.; Wei, M. K.; Peng, Y. Y. Bioorg. Med. Chem. 2016, 24, 4620.
36. Bakavoli, M.; Shiri, A.; Ebrahimpour, Z.; Rahimizadeh, M.; Chin. Chem. Lett. 2008, 19, 1403.
37. Chen, J.; Wu, D.; He, F.; Liu, M.; Wu, H.; Ding, J.; Su, W.; Tetrahedron Lett. 2008, 49, 3814.
38. Hikawa, H.; Ino, Y.; Suzuki, H.; Yokoyama, Y.; J. Org. Chem. 2012, 77, 7046.
39. Heravi, M. M.; Montazeri, N.; Rahimzadeh, M.; Bakavoli, M.; Ghassemzadeh, M. Pol. J.
Chem. 2004, 78, 2101.
40. Ranilla, L. G.; Kwon, Y. I.; Apostolidis, E.; Shetty, K.; Biores. Technol. 2010, 101, 4676

41. Eftink, M. R.; Ghiron, C. A.; Anal. Biochem. 1981, 114, 199.
42. Zhao, Y.; Cao, Y.; Han, F. M.; Chen, Y.; Spectral Anal. (Beijing, China) 2008, 28, 904.
43. Lakowicz, J. R. (Ed.).; Springer Science Business Media, 2013.
44. He, H.; Lu, Y. H.; J. Agri. Food chem. 2013, 61, 8110.
Graphical Abstract