Benzonate derivatives of acetophenone as potent α-glucosidase inhibitors: synthesis, structure–activity relationship and mechanism

Article abstract of DOI:10.1080/14756366.2019.1604519

In this article, 23 compounds (6 and 7a–7v) were prepared and evaluated for their in vitro α-glucosidase inhibitory activity. The compounds 7d, 7f, 7i, 7n, 7o, 7r, 7s, 7u, and 7v displayed the α-glucosidase inhibition activity with IC₅₀ values ranging from 1.68 to 7.88 μM. Among all tested compounds, 7u was found to be the most efficient, being 32-fold more active than the standard drug acarbose, which significantly attenuated postprandial blood glucose in mice. In addition, the compound 7u also induced the fluorescence quenching and conformational changes of enzyme, by forming α-glucosidase–7u complex in a mixed inhibition type. The thermodynamic constants recognised the interaction between 7u and α-glucosidase and was an enthalpy-driven spontaneous exothermic reaction. The synchronous fluorescence and CD spectra also indicate that the compound 7u changed the enzyme conformation. The findings identify the binding interactions between new ligands and α-glucosidase and reveal the compound 7u as a potent α-glucosidase inhibitor.

Full text of DOI:10.1080/14756366.2019.1604519

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Benzonate derivatives of acetophenone as
potent α-glucosidase inhibitors: synthesis,
structure–activity relationship and mechanism
Wen-Jia Dan, Qiang Zhang, Fan Zhang, Wei-Wei Wang & Jin-Ming Gao
To cite this article: Wen-Jia Dan, Qiang Zhang, Fan Zhang, Wei-Wei Wang & Jin-Ming Gao
(2019) Benzonate derivatives of acetophenone as potent α-glucosidase inhibitors: synthesis,
structure–activity relationship and mechanism, Journal of Enzyme Inhibition and Medicinal
Chemistry, 34:1, 937-945, DOI: 10.1080/14756366.2019.1604519
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 937–945
https://doi.org/10.1080/14756366.2019.1604519
ARTICLE
Benzonate derivatives of acetophenone as potent a-glucosidase inhibitors:
synthesis, structure–activity relationship and mechanism
Wen-Jia Dan, Qiang Zhang, Fan Zhang, Wei-Wei Wang and Jin-Ming Gao
Shaanxi Key Laboratory of Natural Products and Chemical Biology, College of Chemistry and Pharmacy, Northwest A&F University, Yangling,
Shaanxi, China
ABSTRACT
ARTICLE HISTORY
Received 23 February 2019
Revised 1 April 2019
Accepted 3 April 2019
In this article, 23 compounds (6 and 7a–7v) were prepared and evaluated for their in vitro a-glucosidase
inhibitory activity. The compounds 7d, 7f, 7i, 7n, 7o, 7r, 7s, 7u, and 7v displayed the a-glucosidase
inhibition activity with IC50 values ranging from 1.68 to 7.88 mM. Among all tested compounds, 7u was
found to be the most efficient, being 32-fold more active than the standard drug acarbose, which signifi-
cantly attenuated postprandial blood glucose in mice. In addition, the compound 7u also induced the
fluorescence quenching and conformational changes of enzyme, by forming a-glucosidase–7u complex
in a mixed inhibition type. The thermodynamic constants recognised the interaction between 7u and
a-glucosidase and was an enthalpy-driven spontaneous exothermic reaction. The synchronous fluores-
cence and CD spectra also indicate that the compound 7u changed the enzyme conformation. The find-
ings identify the binding interactions between new ligands and a-glucosidase and reveal the compound
7u as a potent a-glucosidase inhibitor.
KEYWORDS
a-Glucosidase; acetophe-
none derivatives; inhibiting
mechanism; in vitro; in vivo
1. Introduction
the structural characteristics of existing phenolic a-glucosidase
inhibitors and the advantages of natural product resources, in this
research work about 23 derivatives of 2,4-dihydroxy-5-methylace-
tophenone 3 were synthesised. The newly prepared analogs were
also explored for their inhibitory activity and mechanism against
a-glucosidase.
Diabetes mellitus is one of the major chronic diseases1. The inci-
dence and prevalence of diabetes have risen sharply in recent
years, and 642 million people might be suffering from diabetes
until 20402,3. a-Glucosidase is a type of glycoside hydrolase, which
favors in absorption of carbohydrates. By inhibiting its activity, the
absorption of small intestine carbohydrates could be delayed,
which bring on the reduction of postprandial blood glucose4,5
.
2. Materials and methods
Moreover, the inhibition of a-glucosidase also has positive effects
to treat some diseases such as virosis, cancer, and chronic heart
failure etc6,7. Nowadays, the a-glucosidase inhibitors have been
recognised as an efficient therapy in the treatment of type-II dia-
betes (T2D)8. They inhibit membrane bound a-glucosidase in the
cells lining the small intestine, which in turn decreases the diges-
tion of starch and additional dietary sugars, helping to avoid
hyperglycemia and maintain normal blood sugar levels9. Therefore,
it is significant to discover new classes of a-glucosidase inhibitors
and to investigate the action mechanism of these inhibitors.
Numerous medical and nutritional researches have revealed
that the natural phenols took a key position in prevention and
control of various diseases like diabetes, cancer, and neurodege-
nerative10,11. Various natural products have been reported for their
a-glucosidase inhibition activity. The study of Sun et al.12 and
Zeng et al.13, shows that the natural phenols 30-geranylchalconar-
ingenin 1 and apigenin 2 (Figure 1) both exhibited superior inhib-
ition activity against a-glucosidase than acarbose (a standard
drug). The compound 2,4-dihydroxy-5-methylacetophenone 3 was
isolated from the cultures of Polyporus picipes (Figure 1) by our
group as a good fungicide14–19, which also showed considerable
2.1. General
1H NMR and 13 C NMR spectra were carried out using a 500 MHz
Avance spectrometer (Bruker, USA) or a Varian Mercury 400 MHz
spectrometer. Chemical shifts were measured relative to the
residual solvent peaks of CDCl3 with tetramethylsilane as the
internal standard. High-resolution mass spectra were recorded
on an AB SCIEX Triple TOF 5600þ spectrometer. The in vitro
inhibitory activity and kinetic assay of a-glucosidase were meas-
ured by a microplate reader (Synergy HTX, BioTek Instruments
Inc., Winooski, VT, USA). Fluorescence spectra were conducted on
a spectrofluorometer (LS55, Perkin Elmer Inc., Waltham, MA, USA).
All of the CD spectra were measured at 298 K with a circular spec-
trometer (Chirascan, Applied Photophysics Ltd., UK). Titration
microcalorimetry measurement conducted on a Nano-ITC SV
instrument (TA Instruments Ltd., UK). The surgeries were per-
formed under anesthesia and all efforts were made to minimise
animal suffering. The blood glucose levels were determined via a
glucose detection kit (Robio Co., Ltd., Shanghai, China). The jejuna
for everted sleeve assays were obtained from adult male
inhibitory activity against a-glucosidase in prescreen. Considering Sprague–Dawley rats (SCXK (Shaan) 2017–003, 7 weeks old). The
CONTACT Jin-Ming Gao
jinminggao@nwsuaf.edu.cn
Shaanxi Key Laboratory of Natural Products and Chemical Biology, College of Chemistry and Pharmacy,
Northwest A&F University, Yangling, Shaanxi, China
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

938
W.-J. DAN ET AL.
Figure 1. The structures of 30-geranylchalconaringenin 1, apigenin 2 and 2,4-dihydroxy-5-methylacetophenone 3.
OH
O
OH
OH
O
Br
ZnCl2
Br2
CH3
CH3
CH3COOH
CH3COOH
HO
HO
HO
Br
4
5: 82%
6: 67%
OH
O
Br
O
DMAP
CH3
Acid chloride
R
O
Br
: 78-92%
−
7a 7v
= CH3
= CH
7a:
7b:
7c:
7d:
7e:
7f :
R
R
R
R
R
R
R
R
R
F
F
2CH3
= CH(CH3 2
)
F
F
F
= CH
2CH(CH3)2
7j
7k
7l
7m
7n
7o
7p
= CH
= CH
= CH
= CH
= CH
2CH
2CH(CH3)2
2CH2CH2CH2CH3
Br
N
O2
Cl
Br
2CH2CH2
2CH2CH2CH2
2CH2CH2CH2Cl
N
7g:
7h:
Br
Cl
Br
Br
7s
Cl
7v
7q
7r
7t
7u
7i :
Scheme 1. Synthesis of compounds 7aꢀ7v.
concentrations of glucose in the soaking solution and intestinal benzene to give compound 6 as white crystals, yield (2.06 g, 67%),
sleeve were detected using S-10 biosensor analyzer (Sieman m.p. 174 ꢀ 175 ꢁC. 1H NMR (500 MHz, CDCl3) d 13.31 (s, 1 H), 7.88
Technology Co., Ltd., Shenzhen, China). All reaction progress was (s, 1 H), 2.60 (s, 3 H); 13 C NMR (125 MHz, CDCl3) d 202.0, 160.4,
155.6, 133.4, 115.3, 99.4, 98.9, 26.3. HR-ESI-MS m/z Calcd. for
monitored by thin-layer chromatography (TLC) on silica gel GF254
(Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) with ultra-
violet detection. Reagents were purchased from commercial sour-
ces (Bodi Chemical Co., Ltd., Tianjin, China; Aladdin Industrial Co.,
Ltd., Shanghai, China), and used as received. a-Glucosidase (EC
3.2.1.20, Sigma-Aldrich Chemical, St. Louis, MO, USA); 4-nitro-
phenyl-a-D-glucopyranoside (pNPG) (99%, Macklin Biochemical
Co., Ltd, Shanghai, China).
C8H6Br2O3 [M–H]– 306.8611, found 306.8610.
2.2.2. Synthesis of compounds 7a–7v
A solution of 6 (1 mmol), 4-dimethylaminopyridine (2 mmol) and
acid chloride (1.2 mmol) in dry acetonitrile (15 ml) was stirred at
room temperature. The reaction was monitored by TLC. On com-
pletion, the resulting mixture was directly evaporated. Then the
residue was purified by silica gel column chromatography with
petroleum ether and ethyl acetate (10:1, v/v) to produce 7a–7v
in 78–92% yield.
2.2. Chemistry
The synthesis of natural product 3 has been reported by our
group (Scheme S1)15. Compounds 6 and 7a–7v were carried out
as illustrated in Scheme 1. Details are described below.
2.3. Biological evaluation
2.3.1. a-Glucosidase inhibitory assay
2.2.1. Synthesis of 3,5-dibromo-2,4-dihydroxyacetophenone (6)
To a solution of 2,4-dihydroxyacetophenone 5 (1.52 g, 10 mmol) in
acetic acid (80%, 20 ml), 3.2 g of bromine (1.04 ml, 20 mmol) dis-
The a-glucosidase inhibitory assay was performed according to a
slightly modified method previously reported21,22. In brief, a series
of reaction solutions, including a fixed amount of a-glucosidase
solved in 10 ml acetic acid was added dropwise with continuous (10 unit/mL 3.75 mL), different concentrations of inhibitors (the
stirring20. The mixture was stirred for 15 min. Then the pale red tested compounds or positive controls, 37.5 mL), and sodium phos-
crystals obtained, which were filtered off and recrystallised from phate buffer (PBS, 596.75 mL, 0.1 M, pH 6.8), were incubated at

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
939
37 ꢁC for 10 min. To initiate the reaction, 112.5 mL of pNPG according to the Bradford method27 using bovine serum albu-
(6.0 mM, as a substrate) was added into the pre-incubated mix- min as the standard28
tures, and the final volume of reaction system was kept at 750 mL.
.
The p-nitrophenol released from pNPG substrate was used as the
2.3.5. Fluorescence spectra measurements
target substance to quantify the enzymatic activity. The absorb-
ance of p-nitrophenol was monitored at 405 nm after incubation
at 37 ꢁC for 30 min. All samples were analyzed in triplicate, acar-
bose, and genistein were used as positive controls. The negative
control was prepared by adding PBS instead of a-glucosidase, the
blank was prepared by adding solvent instead of tested com-
pounds, and the inhibition rate was calculated as the following
Equation (1).
The steady state fluorescence emission spectra, were recorded
between 300 and 500 nm at the excitation wavelength of 280 nm
at three different temperatures (25, 31 and 37 ꢁC), and both the
excitation and emission slits were set at 2.5 nm. Compound 7u at
different concentrations (from 0 to 6 mM) were added into the
3.0 ml solution containing a fixed amount of a-glucosidase (2lM).
All of the mixtures were held for 5 min to equilibrate before meas-
urements. The fluorescence spectra of buffer were subtracted as
ðODcontrol ꢀ ODcontrol blankÞꢀðODtest ꢀ ODtest blankÞ
the background fluorescence29–32
.
ꢂ 100%
(1)
ODcontrol ꢀ ODcontrol
blank
The synchronous fluorescence spectra of a-glucosidase in the
absence and presence of compound 7u were scanned by setting
the excitation and emission wavelength intervals (Dk) at 15 and
60 nm, respectively. To eradicate the probability of re-absorption
and inner filter effects in UV absorption, all of the fluorescence
data were corrected for absorption of exciting light and emitted
Equation (1). Inhibition rate calculation formula.
2.3.2. Sucrose–loading test
The carbohydrate loading test was conducted according as Kato
et al.23 and Han et al.24 reported, with slight modifications. Fifteen
male Kunming mice (35–40 g) after an overnight fast were ran-
domly divided into three groups (five mice per group). Sucrose
(2.5 g/kg body weight), as well as the tested compound 7u
(20 mg/kg body weight) and acarbose (20 mg/kg body weight)
were dissolved in 0.5% sodium carboxymethyl cellulose (CMC–Na)
solution, then administered to mice via a gavage method, respect-
ively. A blank control group was loaded with 0.5% CMC–Na solu-
tion only. The blood samples were collected from the mice’s tail
vein at 0, 15, 30, 60, 90, and 120 min, and the blood glucose levels
were determined by a glucose detection kit.
light, based on the Equation S13,13
.
2.3.6. Titration micro-calorimetry measurement
Titration of the compound 7u into the a-glucosidase was per-
formed at 25 ꢁC. All the solutions were prepared in 0.1 M PBS (pH
6.8). The a-glucosidase solution (2.5lM) was placed in the 1.3 ml
sample cell of the calorimeter, and the injection syringe was
loaded with the solution of 7u (30lM, in 0.05% DMSO). The solu-
tion of compound 7u was titrated into the sample cell at 25 ꢁC as
a progression of 25 injections of 10lL. The time delay between
each two injections was 300 s. To ensure comprehensive mixing,
all the sample was stirred at a speed of 300 rpm during experi-
ment. The control experiments included the titration of the com-
2.3.3. Everted sleeve assays
pound 7u solution into 0.1 M PBS (pH 6.8)33–35
.
The inhibitory activity of compound 7u against a-glucosidase was
assayed on everted intestinal sleeves following the methods of
Han et al.24 and Scow et al.25 with little modifications. After a 12-h
fasting, the jejunum of 200–220 g Sprague-Dawley mice was
obtained. Then the proximal jejunum was flushed with pre-cold
isotonic saline (4 ꢁC) and cut into 3 cm-long segments. Each
segment was weighed separately, everted and both ends were
secured by tying knots. Then the sleeves were incubated (90 min
at 37 ꢁC) in mammalian Ringer’s solution containing sucrose
(1.5 mM) and an appropriate concentration of the inhibitor
(0.17 mM). Following the incubation, the concentrations of glucose
in the soaking solution and intestinal sleeves were tested with
biosensor analyzer (S-10). The total glucose concentration was cal-
culated as glucose per gram of gut weight (mmol/g).
2.3.7. Circular dichroism measurement
The CD measurements of a-glucosidase in the presence of com-
pound 7u were conducted in far–UV region (190–250 nm), under
nitrogen environment using a 1.0 mm path length cuvette. The
concentration of a-glucosidase was maintained constant (2 mM)
while the 7u concentration was varied as 0, 1, 2, and 4 mM. All
observed CD spectra were recorded in 0.1 M PBS (pH 6.8)
at 25 ꢁC13,31
.
2.4. Statistical analysis
All data are expressed as mean SD. The statistical analysis was
performed with GraphPad Prism 7.0 (GraphPad, La Jolla, CA, USA),
using one-way ANOVA followed by the Tukey post hoc test (for
comparison among three or more groups). The statistical signifi-
2.3.4. Kinetic analysis
To further explore the inhibitory characteristic of this type of
compounds, kinetic studies of the selected compound 7u were
performed3,13,26. Different final concentrations (0.5, 1, 2, 4, and
8 mM) of the inhibitor 7u around the IC50 values were chosen.
The concentration of a-glucosidase was kept at 1 mM, and the
pNPG concentrations varied from 2.0 to 12.0 mM in the absence
and presence of compound 7u. The type of inhibition was ana-
lyzed on the basis of the Michaelis–Menten constant (Km) and
ꢃ
ꢃꢃ
ꢃꢃꢃ
p < 0.001.
cance was considered at p < 0.05, p < 0.01, and
3. Results and discussion
3.1. Chemistry
The natural product 3 can be easily obtained by reduction and
maximal rate (Vmax) of the enzyme. The parameters were deter- Friedel-Crafts reactions of industrial raw material 2,4-dihydroxy-
mined via Lineweaver–Burk plots, which was obtained by plot- benzaldehyde15. The compound 5, similar to the natural product
ting enzyme reaction velocity (ꢀ) and substrate (pNPG), namely, 3, can be obtained in good yield (82%) from resorcinol 4 with
1/ꢀ versus 1/[pNPG]. The secondary plot slope against [7u] was previously reported catalyst ZnCl2–CH3COOH (Scheme 1). In order
also determined. The protein concentration was determined to increase the metabolic stability36, the Br was used to replace

940
W.-J. DAN ET AL.
the hydrogen atoms by conventional halogenation reaction and genistein was 54.74 and 22.64 lM, respectively. These results indi-
the moderate yield (67%) was acquired20. The structure of com-
cated that those compounds were more effective against a-gluco-
sidase than the standard drug acarbose.
pound 6 was also verified by X-ray single crystal diffraction (CCDC
1869203). With this compound 6 in hand, the main focus was on
modifying phenolic hydroxyl at OH-40, which is more reactive than
OH-20 due to presence of intramolecular hydrogen bond. That’s
why a series of ester derivatives 7a–7v (78–92% yields) was syn-
thesised, with various chain alkanes and substituted phenyl
groups, which in turn varied in chain length, electron-inducing
ability and substitution position. All these compounds were thor-
oughly characterised (see Supporting Information for details,
Figures S2–S24). This diversity of product may help to explore the
interactions between small molecules and biotarget.
The SARs of these derivatives were summarised briefly as fol-
lowing: (1) longer straight-chain alkyl at ester substitution sites
could increase the level of a-glucosidase inhibition, as IC50 of com-
pounds 7a and 7f were >20 and 6.73 mM, respectively; (2) the
introduction of branched alkyl chain are favorable, as inhibiting
activity of compounds 7c (IC50 ¼ 19.35 mM) was superior than 7a;
(3) the haloalkyl chain might improve the inhibition, such as com-
pound 7i (IC50 ¼ 3.27 mM). However, compound 7 h was a special
case which lost the activity; (4) the aromatic ring substituted
groups were beneficial for improving the activity, except for the
compounds 7k, 7 m, 7q, and 7t. Especially, the large steric and
electron-withdrawing groups on the aromatic rings were essential
for enhancing the a-glucosidase inhibitory activity (the IC50 of 7u
was 1.68 mM). Compound 7u was found as the most high active
against a-glucosidase among all newly synthesised compounds.
We thus furtherly explored its mechanism on a-glucosidase inhib-
ition by several methods.
3.2. Biological evaluation
3.2.1. In vitro a-glucosidase inhibitory activity and SARs
As presented in Table 1, all the compounds were tested for their
inhibition rate under 20 mM. Then the active compounds whose
inhibition rates were over 70% were evaluated for their IC50 val-
ues. The IC50 values were found between 1.68 and 7.88 lM for 7d,
7f, 7i, 7n, 7o, 7r, 7s, 7u, and 7v, while that of acarbose and
3.2.2. Sucrose-loading test
The sucrose loading assay was performed to confirm whether
compound 7u kept reduction effects on postprandial blood glu-
cose levels in vivo. The blood glucose levels were measured as
scheduled time, by the glucose detection kit. As shown in Figure
2(A), the blood glucose level of fasted mice was rapidly increased
from 5.40 to 9.50 mM after administration of sucrose (2.5 g/kg
body weight) for 15 min. In comparison, the blood glucose level of
Table 1. a-Glucosidase inhibition of high active compounds in vitro.
Compd.
IC50 (mM)
Compd.
7l
IC50 (mM)
3
16.22 0.41
17.38 0.55
19.35 0.46
6.08 0.20
10.89 0.07
6.73 0.81
12.43 0.27
3.27 0.39
17.49 0.53
12.07 0.22
3.46 0.26
1.97 0.28
4.62 0.24
7.88 0.17
6
7n
7o
7r
7s
7u
7v
Genistein
Acarbose
7c
7d
7e
7f
7g
7i
7j
1.68 0.05 blank group exhibited almost no variations through the test time
1.85 0.27
22.64 3.03
54.74 0.16
course. Meanwhile, an obvious repressive effect on blood glucose
concentrations at peaked 15 min was observed for treated group
(compound 7u), which were similar to acarbose. Interestingly,
Figure 2. (A) Effects of compound 7u and acarbose on postprandial blood glucose levels. (B) Inhibitory activity of compound 7u (0.17 mM) and acarbose (0.17 mM) on
mice intestinal a-glucosidase. Total concentration of glucose (lmol/g); (C) and (D) Plots of the concentrations of glucose (lmol/g) in and out of the intestinal sleeves,
respectively.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
941
both blood glucose concentrations of 7u treated group and 7u was more efficient to inhibit the transformation of sucrose to
acarbose treated group were at lower level than the model group, glucose. Accordingly, compound 7u was taken as a significant
and exhibited no significant difference up to 90 min. It can be inhibitor of a-glucosidase. The inhibitory activity of a-glucosidase
inferred that the hyperglycemic inhibition by 7u was probably
associated with its inhibitory activity on the carbohydrate-digestive
enzymes. For example, a-glucosidase, or the glucose transporters,
including sodium-dependent glucose cotransporters (SGLTs) and
the facilitative glucose transporters (GLUTs)24.
could effectively reduce postprandial blood glucose, which agreed
with the result of sucrose-loading test. Hence, the major concen-
tration was exploring the inhibitory mechanism of compound 7u
against a-glucosidase.
3.2.4. Kinetic analysis of inhibitory type
3.2.3. Everted sleeve assays
To explore the type of inhibition characterised by 7u against a-glu-
cosidase, the kinetic assay was carried out by Lineweaver–Burk
equation and Michaelis–Menten equation (Equations S2–S4).
As shown in Lineweaver–Burk double-reciprocal plot (Figure 3(A)),
the lines intersected in second quadrant, while, Vmax and Km
changed with varying the concentration of compound 7u. These
results indicated that 7u induced a mixed-type inhibition. The sec-
ondary plots (inset of Figure 3(A)) showed a good linear relation-
ship, suggesting that the inhibitor (7u) bound mainly in a single
kind of inhibition sites on a-glucosidase. The kinetic parameters
(Table 2) were also obtained by the nonlinear regression of the
Michaelis–Menten plots (Supplementary Figure S1) and the Ki
value of this mixed-type inhibition was calculated as 2.28 lM.
The a-glucosidase is one of the most important carbohydrate
digestive enzymes which are found on the surface membrane of
the intestine. In order to explore the inhibitory activity of com-
pound 7u against intestine a-glucosidase, the inhibitory activity of
a-glucosidase on everted intestinal sleeves was tested. As shown
in Figure 2(B), both 7u (0.17 mM) and acarbose (0.17 mM) led to
decreasing the concentration of total glucose from the first
(5 min) to the last (90 min) time-point, and the concentrations
of glucose were found much lower than that of the blank.
Additionally, the inhibitory activity of compound 7u was better
than that of acarbose. The glucose concentrations of compound
7u and acarbose-treated groups in the intestinal sleeves were
lower than that of blank (Figure 2(C)), which indicated both 7u
and acarbose could inhibit the activity of glucose transporters,
such as SGLT1 and GLUT2, and help reducing the glucose absorp-
3.2.5. Fluorescence quenching mechanism and binding
tion from the intestinal tract to the blood25. The compound 7u characterisations
also decreased the glucose concentrations in the solution present The endogenous fluorescence property of a-glucosidase is mainly
outside the intestinal sleeves (Figure 2(D)), which suggested that derived from aromatic amino acid residues, such as tryptophan
Figure 3. (A) Lineweaver–Burk plots. c(7u) ¼ 0.5, 1, 2, 4 and 8 lM for curves a ! e, respectively. The inset was the secondary plots of slope (the upper left) and Y-
intercept (the lower left) versus [7u]. (B) Fluorescence spectra of a-glucosidase in the presence of 7 u at various concentrations (pH 6.8, T ¼ 25ꢁC, kex ¼ 280 nm, kem
¼ 346nm). c(a-glucosidase) ¼ 2 mM, and c(7u) ¼ 0, 0.5, 1, 2, 3, 4, 5 and 6 mM for curves a ! h, respectively. Curve i shows the emission spectrum of 7 u at the con-
centration of 6 mM. The Stern–Volmer plots for the fluorescence quenching of a-glucosidase by 7 u at different temperatures were inserted. (C) T ¼ 31 ꢁC. (D) T ¼ 37 ꢁC.

942
W.-J. DAN ET AL.
Table 2. Kinetic parameters of a-glucosidase in the presence of 7u.
Table 4. Thermodynamic parameters for the 7u ꢀ a-glucosidase system.
Concentrations
of 7u (mM)
T (oC)
DGo (kJ·molꢀ1
)
DHo (kJ·molꢀ1
ꢀ75.82 0.11
)
DSo (J·molꢀ1·Kꢀ1
ꢀ146.32 0.02
)
Vmax (mMꢄminꢀ1
)
Km (mM)
Ki (mM)
25
31
37
ꢀ32.21 0.06
ꢀ31.33 0.03
ꢀ30.46 0.02
0
1
2
4
8
1.60 0.03
1.39 0.02
1.24 0.01
1.04 0.01
0.84 0.02
5.19 0.67
6.18 0.59
8.17 0.37
9.08 0.53
10.83 0.96
2.28 0.46
the dominant contribution not only in formation but also in sta-
bilisation of the a-glucosidase–7u complex. The reaction also
characterised an enthalpy-driven process due to |DHꢁ| > |TDSꢁ|.
Table 3. Quenching constants (Ksv), binding constants (Ka) and number of bind-
ing sites (n) for the 7u ꢀ a-glucosidase system at different temperatures.
T (oC)
KSV (ꢂ 104 Lꢄmolꢀ1
)
Ra
Ka (ꢂ 105 Lꢄmolꢀ1
)
n
Rb
3.2.6. Isothermal titration calorimetry binding assays
25
31
37
5.00 0.04
4.74 0.03
4.31 0.02
0.9937
0.9905
0.9964
4.35 0.05
2.50 0.04
1.33 0.03
1.23
1.19
1.15
0.9986
0.9990
0.9993
Isothermal titration calorimetry (ITC) was carried out to study the
interactions between compound 7u and a-glucosidase, which
could give direct measurement of the binding constant, the stoi-
chiometry, the heat of reaction, as well as the indirect access to
other thermodynamic parameters such as entropic binding contri-
bution and Gibbs free energy. As shown in Figure 4, the green
curve corresponded to a binding model with a 1:1 stoichiometry,
which also fitted with the change of enthalpy (DH, ꢀ100.00 kJ·
molꢀ1), entropy (DS, –204.50 J· molꢀ1· Kꢀ1) and free energy (DG,
–39.04 kJ· molꢀ1). These data indicated that the binding was
enthalpy-driven and spontaneous reaction which conformed to
the fluorescence result. The value of binding constant Ka was
6.91 ꢂ 106 L·molꢀ1, demonstrating a good binding affinity of com-
pound 7u for a-glucosidase. Fluorescence technology and ITC are
common methods for studying the interactions between small
molecular ligands and protein35,37,38. In this work, the thermo-
dynamic parameters obtained by these two methods showed the
same trend. However, there are some difference in their values,
which could be attributed to factors such as the tested solvent
environment, the macromolecular state, and the electrostatic
repulsions between the molecules that bear the same charge35.
aThe correlation coefficient for the KSV value.
bThe correlation coefficient for the Ka value.
and tyrosine3. So the fluorescence quenching experiments could
be used to characterise the binding constant (Ka), binding site (n)
and mechanism of binding between 7u and the enzyme. As
shown in Figure 3(B)–(D), a-glucosidase possessed a strong fluor-
escence-emission peak at 350 nm, which quenched regularly with
the addition of increasing the sample (7u) concentrations. The
data suggested the change of a-glucosidase structure, at the
meantime, reflecting the formation of a-glucosidase–7u complex.
To shed light on the interaction mechanism, the Stern–Volmer
equation (Equations S5) was used to analyze the fluorescence
data. The Stern–Volmer plots (the inset in Figure 3(B)) suggested
that the quenching course was a single mode. Generally, there are
two types of quenching i.e. dynamic quenching and static
quenching. Usually, static quenching is characterised by decreas-
ing quenching constant with increasing temperature29. As listed in
Table 3, the values of KSV have a negative correlation with tem-
perature, specifying a static quenching mechanism. Furthermore,
the values of Kq (1012 L·molꢀ1·sꢀ1) far exceeded than maximum
diffusion collision quenching constant (2.0 ꢂ 1010 L· molꢀ1· sꢀ1),
Similar situations have also been reported in the literatures35,37
The accurate thermodynamic parameters need to be further
studied in combination with other techniques, such as surface
.
which also suggested a static quenching process. The intrinsic plasmon resonance (SPR)38,39 and thermal shift assays (TSA)40,41
binding constant Ka and the number of binding sites n values of
.
a-glucosidase with 7u could be calculated by the double loga-
3.2.7. Synchronous fluorescence
rithm equation (Equations S6). The Table 3 presented the values
of Ka which were in the order of 105 L·molꢀ1, indicating a moder-
ate to good binding affinity of compound 7u for the a-glucosi-
dase. In addition, the values of Ka were also inversely correlated
with temperature that evoked the stability of the
compound–enzyme complex decreased at a higher temperature.
Furthermore, the value of n was approximate to 1, which indi-
cated the presence of a single binding site for understudy com-
pound (7u) on a-glucosidase.
The microenvironment changes of fluorophores in a-glucosidase
were monitored by synchronous fluorescence spectroscopy, which
was done by measuring the possible shift of spectra in maximum
emission wavelength before and after binding with ligand. By set-
ting the Dk (Dk ¼ kem – kex) at 15 or 60 nm, the information
about the microenvironment changes of Tyr and Trp residues
were obtained, respectively29. As shown in Figure 5, the synchron-
ous fluorescence intensity of both Tyr and Trp residues decreased
with the concentration of 7u increased. No evident shift was
To further characterise the intermolecular forces between
a-glucosidase and 7u, the thermodynamic parameters were also observed at the maximum emission wavelength of Tyr residues
calculated by using van’t Hoff equation (Equation S7 and S8),
which determined the main forces contributing to the ligand–pro-
tein stability.
(Figure 5(A)). However, Trp residues had a noticeable red shift
(from 277.5 to 286.5 nm) as shown in Figure 5(B). The result indi-
cated that 7u did not alter the polarity around Tyr residues signifi-
cantly, while the polarity around the Trp residues enhanced and
the hydrophobicity decreased. In addition, the gradually declined
fluorescence intensity with increased concentration of 7u further
supported the occurrence of fluorescence quenching.
The values of DHꢁ and DSꢁ were obtained from the linearly
fitted plot of logKa against 1/T. The values of Ka rose with the
temperature enhancement, which directed that the binding
ability of 7u to a-glucosidase became stronger with increasing
temperature, while the binding reaction was exothermic. The
negative values of DGꢁ showed that the binding process was
thermodynamically favorable and occurred spontaneously. As
the calculated paramꢀet1ers listed in Table 4, the negative values
of DHꢁ (–75.82 kJ·mol ) and DSꢁ (–146.32 J·molꢀ1·Kꢀ1) indicated
3.2.8. Circular dichroism spectroscopy
The CD spectroscopy has considered as a reliable and sensitive
method for monitoring the structural changes of bio-macromole-
the action of van der Waals force and hydrogen bonds offered cules in interaction with small molecules. As shown in Figure 6,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
943
Figure 4. Calorimetric titration of the a-glucosidase with compound 7 u at 25 ꢁC. Heat flow as a function of time (purple). The green curve corresponds to the theoret-
ical independent model. The thermodynamic constants are presented in the pane.
Figure 5. Synchronous fluorescence spectra of a-glucosidase in the presence of increasing concentrations of 7 u (A) Dk ¼ 15 nm, (B) Dk ¼ 60 nm (pH 6.8, T ¼ 25 ꢁC).
c(a-glucosidase) ¼ 2 mM. c(7u) ¼ 0, 0.5, 1, 2, 3, 4, 5 and 6 mM for curves a ! h, respectively.
the CD spectra of a-glucosidase exhibited a pair of negative bands
at 209 and 220 nm, which were the characteristic of the a-helical
ꢃ
ꢃ
structure of protein, originated from the n!p and p!p elec-
tron transfer for the peptides bonds of the a-helix3,42. On adding
various concentrations of ligand (7u), both intensity of the nega-
tive bands decreased without any significant changes in the peak
position and shape. The results revealed that binding of 7u to
a-glucosidase encouraged the structural transformation of poly-
peptides, resulting in the inhibition of a-glucosidase activity.
4. Conclusions
Figure 6. CD spectra of a-glucosidase in the presence of 7 u. c(a-glucosidase) ¼
2 mM. The molar ratios of 7 u to a-glucosidase were 0:1, 1:1, 2:1 and 4:1 for
curves a ! d, respectively.
This work demonstrated the a-glucosidase inhibitory potential
of natural 2,4-dihydroxy-5-methylacetophenone scaffold and its

944
W.-J. DAN ET AL.
derivatives. The in vitro study revealed that compounds 7d, 7f, 7i,
7n, 7o, 7r, 7s, 7u, and 7v were efficient a-glucosidase inhibitors
with IC50 values of 1.68–7.88 lM, which were much stronger than
that of acarbose and genistein. The most promising derivative
7u effectively reduced the level of postprandial blood glucose in
vivo mainly through inhibition of the activity of a-glucosidase.
Furthermore, this research displayed that compound 7u inhibited
the activity of a-glucosidase in a mixed-type manner, with its Ki
value of 2.28 mM. As an enthalpy-driven spontaneous process, the
compound 7u bound to a-glucosidase to form a complex with
one affinity binding site. Overall, this research could enrich the
types of candidate a-glucosidase inhibitors and provide more
options for efficient chemotherapies in the treatment of Type-
II diabetes.
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Ethical statement
The animal experimental procedures performed in this study were
approved by the Animal Ethics Committee of Xi’an Jiaotong
University, and the protocols were in accordance with the
Guidelines for Care and Use of Laboratory Animals: 8th Edition,
ISBN-10: 0–309-15396–4. All surgeries were performed under anes-
thesia and all efforts were made to minimise animal suffering.
Acknowledgements
We thank Dr. Jiang-Kun Dai for fluorescence and ITC data analysis.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by the Natural Science Basic
Research Plan in Shaanxi Province of China (2014JZ2-001), the
Program of Unified Planning Innovation Engineering of Science &
Technology in Shaanxi Province (No.2015KTCQ02-14).
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