Exploring efficacy of natural-derived acetylphenol scaffold inhibitors for α-glucosidase: Synthesis, in vitro and in vivo biochemical studies

Article abstract of DOI:10.1016/j.bmcl.2020.127528

The discovery of novel α-glucosidase inhibitors and anti-diabetic candidates from natural or natural-derived products represents an attractive therapeutic option. Here, a collection of acetylphenol analogues derived from paeonol and acetophenone were synthesized and evaluated for their α-glucosidase inhibitory activity. Most of derivatives, such as 9a–9e, 9i, 9m–9n and 11d–1e, (IC₅₀ = 0.57 ± 0.01 μM to 8.45 ± 0.57 μM), exhibited higher inhibitory activity than the parent natural products and were by far more potent than the antidiabetic drug acarbose (IC₅₀ = 57.01 ± 0.03 μM). Among these, 9e and 11d showed the most potent activity in a non-competitive manner. The binding processes between the two most potent compounds and α-glucosidase were spontaneous. Hydrophobic interactions were the main forces for the formation and stabilization of the enzyme - acetylphenol scaffold inhibitor complex, and induced the topography image changes and aggregation of α-glucosidase. In addition, everted intestinal sleeves in vitro and the maltose loading test in vivo further demonstrated the α-glucosidase inhibition of the two compounds, and our findings proved that they have significant postprandial hypoglycemic effects.

Full text of DOI:10.1016/j.bmcl.2020.127528

Bioorganic&MedicinalChemistryLetters30(2020)127528
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploring efficacy of natural-derived acetylphenol scaffold inhibitors for α-
glucosidase: Synthesis, in vitro and in vivo biochemical studies
⁎
⁎
Xiao Yua,d, Fan Zhangb,d, Ting Liub,d, Zhigang Liub, Qingjian Dongc, , Ding Lib,
a Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
b Shaanxi Key Laboratory of Natural Products & Chemical Biology, Shaanxi Engineering Center of Bioresource Chemistry & Sustainable Utilization, College of Chemistry &
Pharmacy, Northwest A&F University, Yangling 712100, China
c Department of Nuclear Medicine and PET, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The discovery of novel α-glucosidase inhibitors and anti-diabetic candidates from natural or natural-derived
products represents an attractive therapeutic option. Here, a collection of acetylphenol analogues derived from
paeonol and acetophenone were synthesized and evaluated for their α-glucosidase inhibitory activity. Most of
Acetylphenol
α-Glucosidase
Diabetes
derivatives, such as 9a–9e, 9i, 9m–9n and 11d–1e, (IC50 = 0.57
0.01 μM to 8.45
0.57 μM), exhibited
Structure-activity relationship
Hyperglycemic activity
higher inhibitory activity than the parent natural products and were by far more potent than the antidiabetic
drug acarbose (IC50 = 57.01
0.03 μM). Among these, 9e and 11d showed the most potent activity in a non-
competitive manner. The binding processes between the two most potent compounds and α-glucosidase were
spontaneous. Hydrophobic interactions were the main forces for the formation and stabilization of the enzyme -
acetylphenol scaffold inhibitor complex, and induced the topography image changes and aggregation of α-
glucosidase. In addition, everted intestinal sleeves in vitro and the maltose loading test in vivo further demon-
strated the α-glucosidase inhibition of the two compounds, and our findings proved that they have significant
postprandial hypoglycemic effects.
Diabetes mellitus (DM), often called as diabetes, is characterized as
a group of metabolic disorders with high blood sugar levels over a
prolonged period.1 If left untreated promptly, diabetes can cause a
variety of serious diseases, such as complications, including cardio-
vascular diseases, stroke, blurred vision, diminished renal function or
even death2–4. The incidence of diabetes is rising dramatically all over
the world. An estimated 425 million people worldwide were suffering
from diabetes in 2017,5 and China had about 118 million people with
diabetes.6 The number of diabetic patients is projected to reach 642
million by 2040.5 α-glucosidase, located at the epithelium of the small
intestine, is the most important carbohydrate digestive enzyme.7 One of
the effective methods to suppress postprandial hyperglycemia is to
control the release of glucose by inhibiting α-glucosidase.8 Two α-
glucosidase inhibitors, acarbose and miglitol, have been used clinically
to control postprandial hyperglycemia as the first-line therapy for dia-
betes by the International Diabetes Federation (IDF), but these drugs
usually have many side effects, such as abdominal diarrhea, distention,
flatulence and meteorism9. In addition, due to α-glucosidases playing
an important role in glycosylation of viral envelope proteins, inhibition
of α-glucosidase is a promising strategy in the development of new anti-
HIV agents10. Therefore, the discovery of novel α-glucosidase inhibitors
is an appealing goal in the field of medicinal chemistry.
Acetylphenol scaffold has been found in various natural and natural
derived pharmaceuticals. As shown in Fig. 1, Paeonol (2-hydroxy-4-
methoxyacetophenone), a bioactive monomer extracted from the root
bark of Moutan Cortex (Paeonia suffruticosa), is an important phar-
macological ingredient with antiatherosclerotic effects, anti-in-
flammatory and antioxidant activities.11,12 Oxybenzone (benzophe-
none-3) is a common ultraviolet absorbent widely used in sunscreens.13
Acetophenone isolated from the higher fungus Polyporus picipes ex-
hibits antifungal and antibacterial properties.14,15 2′, 4, 4′-trihydrox-
ychalcone is known to stall biosynthesis of mycolic acids by myco-
bacterial FAS-II system.16 Acebutolol is
a cardioselective beta-
adrenergic antagonist for the treatment of hypertension and ventricular
premature beats in adults.17
There are many reports available on α-glucosidase inhibitory ac-
tivity of isolated natural products and natural derived compounds, such
as flavonoids, terpenoids, alkaloids, phenylpropanoids, xanthone and
⁎ Corresponding authors.
E-mail addresses: aiyi_aiyi@163.com (Q. Dong), jbolid@nwsuaf.edu.cn (D. Li).
d These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2020.127528
Received 8 July 2020; Received in revised form 22 August 2020; Accepted 27 August 2020
Availableonline10September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

X. Yu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127528
Fig. 1. Chemical structures of some natural and natural derived pharmaceuticals bearing acetylphenol scaffold.
phenolic compounds.18,19 But inhibitors with the motif of acetylphenol
have been rarely studied. In this study, plenty of acetylphenol analo-
gues derived from paeonol and acetophenone were designed and syn-
thesized in a simple and efficient route. Their α-glucosidase inhibitory
activities were evaluated and the structure-activity relationships were
also discussed. Meanwhile, interaction mechanism of the inhibitors
with α-glucosidase was further explored by using enzyme kinetics,
fluorescence spectroscopy, atomic force microscopy (AFM). Moreover,
inhibitory activity of two representative compounds against α-glucosi-
dase were demonstrated on everted intestinal sleeves in vitro and the
postprandial blood glucose-lowering effect of two representative com-
pounds were investigated in maltose loading test in vivo.
Firstly, commercial available resorcinol and 2, 4-dihydroxy ben-
zaldehyde were used as starting materials. The synthetic route of
compounds 2–5 was outline in Scheme 1. Acylation of resorcinol with
anhydride in boron triflouride diethylether (BF3Et2O) at 90 °C for 16 h
got 2 in excellent yields (85–95%). Introduction of bromine at position
5 of the phenyl ring to afford 4 in yield 75% was achieved by reacting
2f with bromine solution in an acetic acid medium. Compounds 3 (or 5)
were synthesized by the esterification reaction of 2 (or 4) with different
acyl chloride groups in the presence of dichloromethane (DCM) and
triethylamine (TEA) at room temperature. The methyl group at position
5 of the aromatic ring was obtained from the reaction of 2, 4-dihy-
droxybenzaldehyde with sodium cyanoborohydride in THF solution
Scheme 1. The synthetic route for the preparation of compounds 2–5.
2

X. Yu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127528
Scheme 2. The synthetic route for the preparation of compounds 7–11.
Table 1
α-Glucosidase inhibitory activities of compounds 2, 3 and 5.
Comp.
R1
R2
IC50 (μM)a
NA
Comp.
R1
R2
IC50 (μM)a
35.78
2a
/
3f
0.37
0.40
1.68
2b
2c
/
/
NA
3g
3h
14.37
72.49
106.99
0.15
2d
/
81.85
0.98
3i
33.44
0.11
0.68
2e
2f
/
/
74.64
32.95
0.55
0.64
4
/
/
29.01
NA
5a
3a
3b
3c
3d
3e
NA
NA
5b
143.44
14.98
11.35
0.26
5c
0.05
0.32
8.97
NA
0.21
5d
Genistein
Acarbose
7.21
0.41
NA
57.01
0.03
a
Values are the mean
SD. All experiments were performed at least three times; NA: no appreciable inhibitory activity
and HCl at room temperature in yield (82.8%) to give 7. Subsequently,
the preparation of the derivatives 9 and 11 occurred via simple ester-
ification reaction mentioned above as shown in Scheme 2.
activity goes from null to a higher value, as examples of 2a-2e. The 7-
straight chain alkyl group for R1 brings compound 2e with
IC50 = 74.64
0.55 μM. Introduction of the ethyl group with iso-
All synthetic compounds were tested for their abilities to inhibit α-
glucosidase. The result in Table 1 shows compounds 2a and 2b with
short straight chain alkyl group for R1 present no appreciable inhibition
against α-glucosidase. With the increase of the straight chain, the
propyl group for R1 leads to an excellent increase in inhibitory activity
as observed for compound 2f (IC50 = 32.95
0.64 μM), which is
more active than 2e and the standard drug acarbose
(IC50 = 57.01 0.03 μM). It suggests that isopropyl group is favored
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X. Yu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127528
in the mentioned position. So, esterification on the 4-phenyl ring and
introduction of a bromine atom on the 5-phenyl ring of 2f was further
inspected. As shown in Table 1, introduction of 2-iodoacetoxy on the 4-
phenyl ring, as in compound 3g, leads to the most potent activity with
9j). On the contrary, as anticipated, a long straight chain alkyl and
isopropyl group for R1 and iodomethyl group for R2 exert a remarkable
enhancement in potency, leading to the discovery of highly active lead
compounds 9c (IC50
=
3.90
0.66 μM), 9d (IC50 = 1.77
IC50 = 14.37
0.40 μM in this series, while acetoxy and 2-chlor-
0.20 μM), 9e (IC50 = 1.69
0.17 μM) and 9i (IC50 = 4.27
oacetoxy result in a significant deterioration in activity. A similar trend
could be found in the series 5 (compounds 5a-5d). Obviously, 2-io-
doacetoxy would significantly improve the activity, and the compound
3c, compared with 2c, is another compelling example. Furthermore, the
introduction of bromine atoms on the 5-phenyl ring increases the α-
glucosidase inhibitory potency as observed in compound 4, 5b, 5c, and
5d compared to 2f, 3e, 3f and 3g respectively.
0.15 μM). It seems that the longer the straight chain alkyl group for
R2, the higher anti-α-glucosidase activity of the compound. But when
the straight chain of R2 grows to 9 carbon atoms, the activity begins to
decrease. Comparison between results for 9k and 9o showed the
branched chain isomer of R2 has better activity. In the series 9 (com-
pounds 9a–9x), conversion of alkyl group to phenyl group for R2 ex-
hibits moderate effects on the potency. However, it is clear that con-
version of the bromine atom on the 5-phenyl ring to the methyl group
enhances the inhibition activity, such as 9g vs 5b (9h vs 5c and 9i vs
5d).
As Table 2 shows, the substituents of R1 and R2 have substantial
effects on anti-α-glucosidase activity. A short straight chain alkyl group
for R1 and R2 are detrimental to the potency of compounds (8a, 8b, 9f,
Table 2
α-Glucosidase inhibitory activities of compounds 8 and 9.
Comp.
R1
R2
IC50 (μM)a
Comp.
9i
R1
R2
IC50 (μM)a
8a
/
NA
4.27
NA
0.15
8b
8c
8d
8e
8f
/
/
/
/
/
NA
9j
66.46
40.86
35.62
23.70
1.01
9k
9L
9m
9n
43.16
24.69
5.93
8.45
0.87
5.34
0.59
0.12
0.12
0.88
0.57
9a
9b
6.22
5.58
0.01
9o
9p
15.96
14.55
0.19
0.31
0.77
9c
9d
3.90
1.77
0.66
0.20
9q
9r
25.44
27.35
0.20
0.25
9e
9f
1.69
NA
0.17
9s
9t
28.05
10.94
0.56
0.89
9g
139.55
14.87
4.13
9x
26.02
0.09
9h
0.12
a
Values are the mean
SD. All experiments were performed at least three times; NA: no appreciable inhibitory activity.
4

X. Yu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127528
Table 3
280 nm. By the increasing the concentration of compound 9e (0.33 to
3.66 μM), or 11d (0.16 to 1.83 μM) in the mixture system, the fluor-
escence intensity of α-glucosidase was quenched gradually without any
distinct shift at maximum peak wavelength. It was suggested that the
two compounds may interact with α-glucosidase.
α-Glucosidase inhibitory activities of compounds 11.
Compound
R2
IC50 (μM)a
10
/
20.98
0.79
5.81
11a
121.05
To gain insights into the nature of the interactions between the
compounds and the enzyme, the fluorescence quenching plots of 9e and
11b
11c
11d
11e
30.85
11.91
0.57
1.86
0.54
0.85
0.01
0.01
11d were modeled with the well-known Stern-Volmer equation20,22
,
and inserted into upper right corner of the Fig. 3A and B respectively.
The Ksv values of the compound 9e and 11d to α-glucosidase decreased
gradually with rising temperature (Table 4), suggesting that a static
quenching happened by forming an enzyme-inhibitor complex20. The
Kq values of 9e (2.27 × 1012,1.95 × 1012, 1.64 × 1012 L mol−1 s−1 at
11f
15.65
24.89
25.35
0.41
0.16
0.04
298, 304 and 310 K, respectively) and 11d (2.98 × 1012, 2.59 × 1012
,
11g
11h
2.24 × 1012 L mol−1 s−1 at 298, 304 and 310 K, respectively) binding
to α-glucosidase were two order of magnitude greater than the maximal
scatter collision quenching constant (2.0 × 1010 L mol−1 s−1), in-
dicating that the fluorescence of α-glucosidase was quenched by 9e and
11d through a static quenching mechanism. At the same temperatures,
the Ksv values of 11d were slightly higher than those of 9e, indicating
that the former had stronger impact on the fluorescence quenching of
α-glucosidase.
11i
25.18
27.61
0.06
0.21
Additionally, the quenching process was further analyzed using the
double-logarithm equation20. As show in Table 4, the values of Ka were
in the order of 104 Lmol−1, indicating a moderate binding affinity of
compound 9e or 11d with α-glucosidase. Furthermore, the value of Ka
was inversely correlated with temperature, suggesting that the stability
of the compound-α-glucosidase complex decreased at a higher tem-
perature. In addition, the value of n (approximately 1) further con-
firmed the existence of only one binding site for compound 9e or 11d
on α-glucosidase.23
11k
11L
20.55
0.38
a
Values are the mean
SD. All experiments were performed at least three
times.
In order to further clarify the binding forces between the inhibitor
and α-glucosidase, the thermodynamic parameters were determined
using the van’t Hoff equations:20 As revealed in Table 4, the negative
values for the change of free energy ( G0) indicated that the interaction
between α-glucosidase and 9e or 11d occurred spontaneously. The
negative H0 suggested that the binding of α-glucosidase with 9e or
11d were exothermic process. In addition, the positive values of S0
were typical proof for the system becoming more disordered during the
formation of the 9e or 11d - α-glucosidase complex. The values of
negative H0 and positive S0 also suggested that hydrophobic inter-
actions and hydrogen bonds contributed dominantly to the formation
and stabilization of the enzyme - inhibitor complex.24
In the series 10 and 11 (compounds 11a-11L) as shown in Table 3,
most compounds examined possess good to excellent anti-α-glucosidase
activity, wherein compound 11d is by far the most potent one with
IC50 = 0.57
0.01 μM. The results clearly indicate that iodomethyl
group for R2 is a breakthrough in this SAR study. Also, the introduction
of the bromine atom on the 3-phenyl and the methyl group on the 5-
phenyl ring are found to be beneficial to increase the potency.
To analyze the type of α-glucosidase inhibition exerted by the
acetylphenol derivatives, the enzyme kinetic studies of compounds 9e
and 11d were performed by using Lineweaver-Burk plots study. As
display in Fig. 2, compounds 9e and 11d inhibit α-glucosidase in the
non-competitive manner obviously, as all the lines cross the negative
direction of the horizontal axis and the value of Vmax decreases,
whereas −1/Km (horizontal axis intercept) is constant with increasing
concentrations of the inhibitors. And the replots of slope and Y-inter-
cept versus [9e] (or [11d]) were linear. These results indicated that the
acetylphenol derivatives bound to one or a class of inhibition sites
around the substrate site of α-glucosidase20,21. Moreover, the inhibitory
constant (Ki) values of compounds 9e and 11d for α-glucosidase were
The synchronous fluorescence was introduced to reflect the change
of the polarity microenvironment around the tyrosine (Tyr) and tryp-
tophan (Trp) residues when the D-value (Δλ) between excitation and
emission wavelength was stabilized at 15 or 60 nm, respectively.22 The
fluorescence intensities of Trp decreased more obviously than that of
Tyr after addition of compound 9e or 11d (Fig. 4), implying that the
two inhibitors may quench α-glucosidase fluorescence mainly by
quenching the fluorescence of Trp residue. The maximum emission
wavelength of Tyr or Trp residue did not show any shift (Fig. 4A and B),
suggesting that the microenvironment around the Tyr and Trp residues
displayed no discernable change during the binding process of 9e. In
contrast, the maximum emission wavelength of Tyr had a red shift from
290 to 294 nm as the concentration of 11d increased (Fig. 4C), while
that of Trp exhibited a blue shift from 286 to 282 nm (Fig. 4D). The blue
shift at the fluorescence emission peaks indicated that the hydro-
phobicity around the Trp residue increased and thus Try residue was
less exposed to the solvent.25
calculated as 1.62
0.31 and 0.65
0.15 μM, respectively. The
inhibition ability of the two compounds against α-glucosidase de-
termined by the values of Ki was consistent with the results of the en-
zyme activity assay.
As presented in Fig. 3, the compounds 9e and 11d show very weak
fluorescence, while α-glucosidase exhibits a strong intrinsic fluores-
cence emission peak at 337 nm after being excited at a wavelength of
5

X. Yu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127528
Fig. 2. Linweave-Burk plots of compounds 9e and 11d. (A) The concentrations of compound 9e were 0.0 μM , 0.625 μM, 1.25 μM and 2.5 μM, (B) The concentrations
of compound 11d were 0.0 μM, 0.3 μM, 0.46 μM and 0.92 μM with different concentrations of pNPG and c(α-glucosidase) = 6.10−7 M. The secondary plots of slope
(the upper left) and Y-intercept (the lower left) vs. [9e] (or [11d]) were in the inset.
Fig. 3. Fluorescence spectra of α-glucosidase in the presence of 9e (A) and 11d (B) at various concentrations (pH 6.8, T = 298 K, λex = 280 nm, λem = 337 nm). c
(α-glucosidase) = 6 × 10−7 mol L−1, c(9e) = 0, 0.33, 0.66, 1.0, 1.33, 1.66, 2.0, 2.32, 2.66, 3.0, 3.32 and 3.66 × 10−6 mol L−1 and c(11d) = 0, 0.16, 0.33, 0.50,
0.66, 0.83, 1.0, 1.16, 1.33, 1.5, 1.66 and 1.83 × 10−6 mol L−1 for curves a-m, respectively. Curve n shows the emission spectrum of 9e at concentrations of
3.0 × 10−5 mol L−1, or 11d of 1.50 × 10−5 mol L−1
.
Table 4
Quenching constant (KSV), binding constant (Ka), and relative thermodynamic parameters for the interaction of 9e or 11d with α-glucosidase at different tem-
peratures.
Comp.
Ksv (x104 Lmol−1
)
Ra
Ka (×104 Lmol−1
)
n
Rb
H0
(kJmol−1
)
G0 (kJmol−1
)
S0
(Jmol−1K−1
)
T(K)
9e
298
304
310
298
304
310
2.27
1.95
1.64
2.98
2.59
2.24
0.03
0.04
0.03
0.04
0.03
0.03
0.9955
0.9953
0.9838
0.9931
0.9863
0.9812
1.39
1.34
1.30
1.56
1.45
1.36
0.04
0.04
0.03
0.03
0.04
0.04
1.17
1.15
1.14
1.00
0.97
0.96
0.01
0.03
0.03
0.02
0.03
0.02
0.9709
0.9883
0.9952
0.9914
0.9903
0.9794
−17.69
−17.96
−18.23
−36.51
−36.85
−37.19
0.4
0.3
0.4
0.3
0.2
0.3
−4.28
0.3
45.01
57.02
0.1
0.1
11d
−19.52
0.2
a
R is the correlation coefficient for the Ksv values. bR is the correlation coefficient for the Ka values.
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Bioorganic&MedicinalChemistryLetters30(2020)127528
Fig. 4. Synchronous fluorescence spectra of α-glucosidase in the absence and presence of 9e (A, B) or 11d (C, D) with Δλ = 15 nm (A, C) and Δλ = 60 nm (B, D) at
pH 6.8, T = 298 K. c(α-glucosidase) = 6 × 10−7 mol L−1, c(9e) = 0, 0.33, 0.66, 1.0, 1.33, 1.66, 2.0, 2.32, 2.66, 3.0 and 3.32 × 10−6 mol L−1 and c(11d) = 0.16,
0.33, 0.50, 0.66, 0.83, 1.0, 1.16, 1.33, 1.5 and 1.66) × 10−6 mol L−1 for curves a-j , respectively.
Atomic Force Microscopy (AFM) is a useful tool imaging technique
in visualizing the interaction between small molecules and protein
owing to its high resolution.7,25 As shown in Fig. 5A, free α-glucosidase
was uniformly distributed on the mica surfaces, and its topography
image can be clearly observed. Cross-sectional of free α-glucosidase
molecules showed the average width and height of the individual α-
glucosidase molecules reached 28.35 nm and 0.87 nm, respectively.
Upon complexation with 9e or 11d, α-glucosidase molecules appeared
to be more swollen and disorder on the mica substrate (Fig. 5B and C,
respectively). The mean width and height of the individual α-glucosi-
dase molecule reached 47.5 and 0.75 nm for 9e, and 29.62 and 3.97 nm
for 11d. The increase in the size indicated that both compounds
interacted with α-glucosidase and that an enzyme - inhibitor complex
was formed. After binding with the compounds, the dispersion of the
protein aggregation became more evident in the AFM photograph,
which suggests that the formation of the enzyme - inhibitor complex
may cause a major destabilization of the native conformation of α-
glucosidase. From the three-dimensional graph of free α-glucosidase
(Fig 4D) and α-glucosidase − 9e or 11d complex (Fig. 5E, F), respec-
tively, it was clearly observed that the interaction between the in-
hibitors and α-glucosidase induced the topography image changes of α-
glucosidase and aggregation, which may be due to the changes of mi-
croenvironment around α-glucosidase molecule and the enzyme mole-
cule exposing to a more hydrophobic environment after interaction
7

X. Yu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127528
Fig. 5. AFM topography image of free α-glucosidase (A), 9e -α-glucosidase (B) and 11d-α-glucosidase (C) complex adsorbed onto mica with tapping mode in air, the
scan size of the image is 2.0 μm × 2.0 μm, cross-sectional images were in the inset, respectively . Panels D, E and F are the three-dimensional graphs for panels A, B
and C, respectively. c(α-glucosidase) = 6 × 10−7 mol L−1, c(9e) = c(11d) = 2.5 × 10−6 mol L−1.
with the inhibitors. To minimize the detrimental factors to the form a
stable structure, the molecules of α-glucosidase reduced the surface
area in contact with water by the aggregation together. The results
suggested that hydrophobic interactions existed between α-glucosidase
with 9e and 11d, which was consistent with that of above thermo-
dynamic analysis. Comparison between the results for 9e and 11d, it
was showed the different influence of 9e and 11d on the conformation
of α-glucosidase, which may be the reason for the different performance
of them in synchronous fluorescence study.
sleeves from the first (5 min) to the last (90 min) time-point. And the
AUC values showed clearly the decrease of blood glucose in another
way. Meanwhile, the efficacy of the two synthetic inhibitors is better
than that of acarbose. The results indicated that 9e, 11d could effec-
tively inhibit the intestinal α-glucosidase and delay the hydrolysis of
disaccharide into glucose.
To further verify the potency of compounds 9e and 11d to reduction
of postprandial blood glucose in vivo, the maltose loading test was
performed by using the reported method7,26,27 The results were pre-
sented in Fig. 7. It was showed that the concentration of blood glucose
of the blank group was not significant changes throughout the whole
process; however, it increased acutely and peaked at 15 min after the
intragastric administration of maltose in the vehicle group. In com-
parison, treatment with 9e or 11d could obviously attenuate the in-
crease of blood glucose levels in a dose-dependent manner from 15 min
In order to prove that this kind of inhibitors also has expectant in-
hibitory effects on animal α-glucosidase, the inhibitory activity of
compounds 9e and 11d against α-glucosidase on sections of the everted
intestine was studied. As shown in Fig. 6, 9e (0.173 mM), 11d
(0.173 mM), and acarbose (0.5 mM, the positive drug) significantly
reduced glucose concentrations both within and outside the intestinal
8

X. Yu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127528
Fig. 6. Inhibitory activity of 9e and 11d (1.73 × 10−5 mol L−1) on rat intestinal α-glucosidase. A is the total concentration of glucose; C and E are the concentrations
of glucose in and out of the intestinal sleeves, respectively. Blood glucose AUC values (0–90 min) of the total concentration of glucose (B), the concentrations of
glucose in (D) and out (F) of the intestinal sleeves, respectively are also calculated. The values are represented as the mean
standard deviation. The data was
analyzed using Dunnett's multiple comparison test, *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the vehicle group.
to 120 min, and the hyperglycemic activity of 11d was as prominent as
in the acarbose-treated group. Moreover, it could be found that the AUC
of compounds 9e, 11d and acarbose were significantly lower than the
vehicle group, which meant that they may improve the oral maltose
tolerance in mice.
non-competitive manner. The binding processes between the two most
potent compounds and α-glucosidase were spontaneous, and induced
the topography image changes and aggregation of α-glucosidase.
Hydrophobic interactions and hydrogen bonds were the main forces for
the formation and stabilization of the enzyme – acetylphenol scaffold
inhibitor complex. Inhibitory activity of the two representative com-
pounds against α-glucosidase was further demonstrated on everted in-
testinal sleeves in vitro and the results showed the inhibitory efficacy of
the two synthetic inhibitors is better than that of acarbose. Most im-
portantly, the maltose loading test in vivo proved they have prominent
In summary, we here report a collection of acetylphenol analogues
derived from paeonol and acetophenone. Two of the derivatives, 9e and
11d are the most potent compounds found in this study, with IC50
values of 1.69
0.17 μM and 0.57
0.01 μM, respectively, against
α-glucosidase. Both of them inhibit the activity of α-glucosidase in a
9

X. Yu, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127528
Fig. 7. Postprandial blood glucose lowering effect of 9e and 11d (A) and blood glucose AUC (B) after intragastric administration of maltose on Kunming mice. The
values are represented as the mean
standard deviation. The data was analyzed using Dunnett's multiple comparison test, *p < 0.05, **p < 0.01, and
***p < 0.001 compared with the vehicle group.
postprandial hypoglycemic effects. These compounds may serve as lead
for the future research in order to identify a potent and safer therapy for
the treatment of diabetes mellitus.
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Author Contributions
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of atherosclerosis. Oxid Med Cell Longevity. 2018;2018.
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
12. Li X-J, Shi X-W, Shuai Q, Gao J-M, Zhang A-L. Bioactive metabolites from bio-
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Declaration of Competing Interest
14. Li D, Tuong Thi Mai L, Dan W-J, et al. Natural products as sources of new fungicides
(IV): synthesis and biological evaluation of isobutyrophenone analogs as potential
inhibitors of class-II fructose-1,6-bisphosphate aldolase. Bioorg Med Chem.
2018;26(2):386–393.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
15. Tuong Thi Mai L, Wang W-W, Zhang F, et al. Structure-antifungal relationships and
preventive effects of 1-(2,4-dihydroxyphenyl)-2-methylpropan-1-one derivatives as
potential inhibitors of class-II fructose-1,6-bisphosphate aldolase. Pestic Biochem
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Acknowledgments
This work was supported by the grants from the National Natural
Science Foundation of China (No. 21502152), the Natural Science
Foundation of Shaanxi Province (No. 2019JM-334), Jinlei Research
Fund for the growth of pediatric endocrinologists (PEGRF201708001).
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