Synthesis of 6-hydroxyaurone analogues and evaluation of their α-glucosidase inhibitory and glucose consumption-promoting activity: Development of highly active 5,6-disubstituted derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.06.040

A series of 6-hydroxyaurones and their analogues have been synthesized and evaluated for their in vitro α-glucosidase inhibitory and glucose consumption-promoting activity. These compounds exhibited varying degrees of α-glucosidase inhibitory activity, 11 of them showing higher potency than that of the control standard acarbose (IC₅₀?=?50.30?μM). Surprisingly, analogues devoid of a substituent at C-2 but having an aryl group at C-5 were found to be highly active (e.g., 7f, IC₅₀?=?9.88?μM). Docking analysis substantiated these findings. The kinetic analysis of compound 7f, the most potent α-glucosidase inhibitor of this study, revealed that it inhibited α-glucosidase in an irreversible and mixed competitive mode. In addition, compounds 7f and 10c exhibited significant glucose consumption promoting activity at 1?μM.

Full text of DOI:10.1016/j.bmcl.2017.06.040

Accepted Manuscript
Synthesis of 6-hydroxyaurone analogues and evaluation of their α-glucosidase
inhibitory and glucose consumption-promoting activity: Development of highly
active 5, 6-disubstituted derivatives
Hua Sun, Weina Ding, Xiaotong Song, Dong Wang, Mingzhu Chen, Kaili
Wang, Yazhou Zhang, Peng Yuan, Ying Ma, Runling Wang, Robert H. Dodd,
Yongmin Zhang, Kui Lu, Peng Yu
PII:
S0960-894X(17)30642-X
http://dx.doi.org/10.1016/j.bmcl.2017.06.040
BMCL 25074
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
17 April 2017
12 June 2017
14 June 2017
Please cite this article as: Sun, H., Ding, W., Song, X., Wang, D., Chen, M., Wang, K., Zhang, Y., Yuan, P., Ma,
Y., Wang, R., Dodd, R.H., Zhang, Y., Lu, K., Yu, P., Synthesis of 6-hydroxyaurone analogues and evaluation of
their α-glucosidase inhibitory and glucose consumption-promoting activity: Development of highly active 5, 6-
disubstituted derivatives, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.06.040
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Graphical Abstract
Synthesis of 6-hydroxyaurone analogues and
evaluation of their α-glucosidase inhibitory
and glucose consumption-promoting activity:
Development of highly active 5, 6-
Leave this area blank for abstract info.
disubstituted derivatives
Hua Sun, Weina Ding, Xiaotong Song, Dong Wang, Mingzhu Chen, Kaili Wang, Yazhou Zhang, Peng Yuan,
Ying Ma, Runling Wang, Robert H. Dodd, Yongmin Zhang, Kui Lu and Peng Yu

Bioorganic & Medicinal Chemistry Letters
Synthesis of 6-hydroxyaurone analogues and evaluation of their α-glucosidase
inhibitory and glucose consumption-promoting activity: Development of highly
active 5, 6-disubstituted derivatives
Hua Suna , Weina Dinga , Xiaotong Songa , Dong Wanga , Mingzhu Chena , Kaili Wanga , Yazhou Zhanga ,
Peng Yuana , Ying Mab , Runling Wangb , Robert H. Dodda,c , Yongmin Zhanga,d , Kui Lua,* and Peng Yua, 
aChina International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Tianjin Key Laboratory of Industry
Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457, China.
bTianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical
University, Tianjin 300070, China
c Centre de recherche de Gif-sur-Yvette, Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
dUniversité Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moléculaire UMR CNRS 8232, 4 place Jussieu, 75005, Paris, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of 6-hydroxyaurones and their analogues have been synthesized and evaluated for their
in vitro α-glucosidase inhibitory and glucose consumption-promoting activity. These compounds
exhibited varying degrees of α-glucosidase inhibitory activity, 11 of them showing higher
potency than that of the control standard acarbose (IC50 = 50.30 μM). Surprisingly, analogues
devoid of a substituent at C-2 but having an aryl group at C-5 were found to be highly active
(e.g., 7f, IC50 = 9.88 μM). Docking analysis substantiated these findings. The kinetic analysis of
compound 7f, the most potent α-glucosidase inhibitor of this study, revealed that it inhibited α-
glucosidase in an irreversible and mixed competitive mode. In addition, compounds 7f and 10c
exhibited significant glucose consumption promoting activity at 1 μM.
Keywords:
Aurone
α-Glucosidase inhibitor
Inhibitory mechanism
Docking analysis
Glucose consumption
2009 Elsevier Ltd. All rights reserved.
α-Glucosidase catalyzes the cleavage of the 1,4-α-glycosidic bonds of starch and certain disaccharides with concomitant conversion
into glucose. The inhibition of α-glucosidase lowers the rate of glucose absorption by delaying the digestion and absorption of
carbohydrates and extending digestion time, thereby constituting one of the therapeutic approaches for controlling postprandial
hyperglycemia in type 2 diabetes.1 Moreover, α-glucosidase can be related to other diseases, such as cancer,2 viral infections3 and
Pompe disease.4 Thus, α-glucosidase is an attractive target for pharmaceutical studies.
Figure 1. Structures of aurone (1) and 6-hydroxyaurone (2).
Aurones (Fig. 1, 1) chemically, (Z)-2-benzylidene-benzofuran-3(2H)-one, are isomeric compounds of flavanoids widely distributed
in nature. Aurones play an important role in the pigmentation of fruits, vegetables and flowers.5 They also possess a variety of
biological activities such as anti-cancer,6, 7 anti-inflammation,8 antifungal activity,9 and anti-Alzheimer's disease.10 However, aurones
have received little attention in comparison to the structurally similar and widely investigated flavones, isoflavones and chalcones.5 In
———
 Corresponding author. Tel.: +86-22-6091-2592; fax: +86-22-6060-2298; e-mail: yupeng@tust.edu.cn; lukui@tust.edu.cn.

2012, three new geranyl aurones having the 6-hydroxyaurone (2) skeletal structures, were isolated from the leaves of Artocarpus altilis
and exhibited α-glucosidase inhibitory activity in vitro.11 6-Hydroxyaurones with a benzofuran ring are structurally different from other
common representative flavonoids. Our previous studies focused on some natural and synthesized flavones, isoflavones and chalcones
as α-glucosidase inhibitors.12 In the present study, we prepared 6-hydroxyaurone and 6-hydroxybenzofuranone derivatives with the aim
to study structure–activity relationships, and thereby, to potentially provide new candidates possessing enhanced α-glucosidase
inhibitory and cell-based anti-diabetic activities. In addition, the α-glucosidase inhibitory mechanism and cell-based glucose-
consumption inhibitory activity of the most potent compound as well as a molecular docking analysis were explored.
The synthesis of 6-hydroxyaurone and 6-hydroxybenzofuranone derivatives was accomplished as shown in Scheme 1. 6-Hydroxy-3-
coumaranone 3 was first synthesized from resorcinol according to a known procedure.5, 13 The 6-hydroxyaurone derivatives 2, 4a-4e
were then prepared by cross-aldol condensation of 3 with various substituted benzaldehydes. Compounds 4f and 4g were obtained by
deprotection of 4d and 4e using boron tribromide. The double bond at C-2 of 4g was reduced using catalytic hydrogenation to give 4h.
6-Alkoxy-3-coumaranones 5a-5b were prepared from 3 using alkyl halides in the presence of K2CO3. Compounds 5a-5b were then
regioselectively brominated with NBS in DMF to form the 5-bromo derivatives 6a-6b in good yield. A Suzuki coupling reaction was
employed to convert 6a-6b to 5-aryl-6-alkoxy aurones 7a-7f. The 2, 5-disubstituted 6-methoxyaurones 8a-8c were synthesized by
cross-aldol condensation of 7d and 7f with benzaldehyde or ethyl glyoxalate as before. The 2-substituted 6-alkoxy aurones 9a-9b were
prepared from intermediate 5a by cross-aldol condensation. 2, 5-Disubstituted 6-hydroxyaurones 10a-10c were prepared from
compounds 7b and 7c by hydrogenolytic removal of the PMB group followed by cross-aldol condensation.
Scheme 1. Synthesis of 6-hydroxyaurone derivatives. Reagents and
conditions: (a) (i) Chloroacetyl chloride, AlCl3, CS2, 0 °C, reflux, 6 h,
71%; (ii) 5% NaOH(aq.), r.t., 4 h, 35%; (b) Benzaldehyde or substituted
benzaldehyde, EtOH, 60% KOH (aq.), r.t., 6 h, 18-36%; (c) BBr3,
anhydrous DCM, 0 °C→r.t., 0.5 h, 33-50%; (d) Pd/C, H2, EtOH, r.t., 1 h,
33%; (e) R1X, K2CO3, DMF, 0 °C→r.t., 6 h, 63-69%; (f) NBS, DMF,
0
°C→r.t., 6 h, 81-96%; (g) Phenylboronic acid or substituted
phenylboronic acid, MeOH, dioxane, Na2CO3(aq.), PdCl2(dppf), 60 °C, 3-
6 h, 35-75%; (h) Benzaldehyde, EtOH, piperidine, r.t., 6 h, or ethyl
glyoxalate solution (50% in toluene), piperidine, 70 °C, 8 h, 153-49%.
The α-glucosidase inhibitory activities of the synthesized compounds were evaluated according to the literature procedure with
minor modifications.14 Acarbose, a clinically used α-glucosidase inhibitor, was chosen as a reference compound for activity comparison.
As can be seen from the data in Table 1, 6-hydroxy aurone (2) showed no appreciable α-glucosidase inhibitory activity. Introducing one
hydroxyl group on the C-ring, as in 2’, 6-dihydroxyaurone (4a), 3’, 6-dihydroxyaurone (4b) and 4’, 6-dihydroxyaurone (4c), led to no
significant increase in inhibition. The trihydroxyaurones 4f and 4g showed a modest improvement in activity compared to 4a-4c.
Methylation of the 2’, 3’-dihydroxy groups of 4g led to the loss of α-glucosidase inhibition (4e). Reduction of the double bond in 4g
resulted in no significant change in activity (4h). Interestingly, etherification of the 6-hydroxy group of compound 2 with the bulky p-
methoxybenzyl (PMB) group (compound 9a) resulted in a significant improvement in activity. This result suggested that α-glucosidase
inhibitory activity might be quite sensitive to structural modifications of the 6-hydroxy group. Replacement of the benzylidene group of
9a by the =CHCOOCH2CH3 group (9b) led to a slight decrease in its activity. Although the absence of the benzylidene group at C-2 (5a)
proved detrimental to the inhibitory activity, the introduction of a phenyl group (7a) or a substituted phenyl group (7b and 7c) all led to
very significant improvements in activity, with compound 7a exhibiting about a 10-fold improvement over 2.
Table 1. α-Glucosidase inhibitory activity of 6-hydroxyaurone and its derivatives.

R1
H
R2
H
H
H
H
H
H
H
H
H
H
H
Ph
R3
IC50 (μM)a
>100
No.
CH-Ph
2
4a
93.00±5.61
>100
H
CH-o-OH-Ph
H
CH-m-OH-Ph
4b
4c
76.71±5.05
H
CH-p-OH-Ph
H
CH-m, p-diOCH3-Ph
>100
4e
79.25±0.45
67.60±7.07
63.39±6.79
19.83±3.75
33.44±1.43
H
CH-o, p-diOH-Ph
4f
H
CH-m, p-diOH-Ph
4g
H
CH2-m, p-diOH-Ph
4h
9a
PMB
PMB
PMB
PMB
CH-Ph
CHCOOCH2CH3
9b
5a
H
>100
10.57±1.19
10.94±2.06
24.87±3.56
20.12±3.45
21.24±2.93
9.88±0.18
>100
H
7a
PMB p-CF3-Ph
H
7b
7c
PMB
CH3
CH3
CH3
CH3
CH3
CH3
H
p-F-Ph
Ph
H
H
H
7d
7e
p-CF3-Ph
p-F-Ph
Ph
H
7f
CH-Ph
8a
p-F-Ph
p-F-Ph
p-F-Ph
p-CF3-Ph
p-CF3-Ph
CH-Ph
>100
8b
8c
26.60±4.58
11.08±0.23
14.30±1.37
30.94±2.71
50.30±2.75
CHCOOCH2CH3
CHCOOCH2CH3
CHCOOCH2CH3
CH-Ph
10a
10b
10c
Acarb
H
H
a Results are the average of three independent experiments, each performed in duplicate. Standard deviations were below ±20%. bAcar (Acarbose) was used as
the reference compound.
These data all suggest that at least two substituents on the 6-hydroxybenzofuran-3(2H)-one structure are crucial for good inhibition
of α-glucosidase and these should be amenable to further modification. A logical option was the replacement of the PMB group in 7a-
7c by a methyl group (7d-7f). Of these, 7f (IC50 = 9.88 μM) was found to have the highest activity, being even more active than the
market drug acarbose (IC50 = 50.30 μM). However, introduction of the benzylidene group at C-2 in compounds 7d and 7f resulted in
significant decrease in inhibitory activity (8a and 8b). The low activity of compounds 8a and 8b now suggested that the presence of
three substituents on the 6-hydroxybenzofuran-3(2H)-one nucleus was unfavorable for activity. We hypothesize that the substituents at
the C-2, C-5 and 6-OH positions of the 6-hydroxybenzofuran-3(2H)-one directly impact on the steric environment, causing vastly
different α-glucosidase inhibitory activity. This is confirmed by replacement of the benzylidene group at C-2 of 8b to the more flexible
=CHCOOCH2CH3 (8c) which led to improvement in activity, albeit modest.
To determine the mechanisms of α-glucosidase inhibition by the most potent compound 7f, kinetic analysis was performed. As
shown in Figure 2A, the value of 1/V increased with increasing concentrations of compound 7f, but the intercept on the X-axis and Y-
axis remained constant. This result suggests a mixed competitive inhibition by compound 7f. The plots of the velocity versus enzyme
concentrations in the presence of different concentrations of compound 7f gave a family of parallel straight lines (Fig. 2B). Increasing
the inhibitor concentrations did not affect the slopes of the lines, indicating that compound 7f is an irreversible inhibitor. We further
evaluated whether the inhibitory effect of compound 7f is irreversible by a dialysis experiment. The results are presented in Fig. 2C.
After dialysis, the activity of α-glucosidase preincubated with 7f showed relatively less activity, which was similar to that of the treated
α-glucosidase without further dialysis, indicating that the enzymatic activity was not recovered after dialysis. These results provide
further evidence that the inhibition of α-glucosidase by 7f is irreversible.
A
B
2500
2000
1500
1000
500
0.008
0.006
0.004
0.002
0.000
0 μM
0 μM
15 μM
20 μM
10 μM
20 μM
-6
-4
-2
0
2
4
6
8
10
0
2
4
6
8
1/(S, mM)
Concentration (mU)
C
120
Enzyme Dialyzed
100
80
Enzyme/ 7f Dialyzed
Enzyme/ 7f
60
40
**
**

Figure 2. Inhibitory kinetics of compound 7f on α-glucosidase. (A)
Lineweaver–Burk plots for inhibition of compound 7f (●, 0 μM; ▲, 15
μM; ■, 20 μM) on α-glucosidase. (B) The interactions of compound 7f
and α-glucosidase (●, 0 μM; ■, 10 μM; ▲, 20 μM). (C) α-Glucosidase
activity after dialysis or non-dialysis in presence of DMSO or compound
7f. **P<0.01.
To further study the anti-diabetic activity of compound 7f, the glucose consumption in HepG2 cells was evaluated and compared to
7a and 10c, as well as metformin which was used as positive control (Fig. 3). Compounds 7f and 10c were able to promote glucose
consumption in HepG2 cells at 1μM, but 7a showed no such activity.
4.0
*
3.5
*
3.0
*
2.5
2.0
1.5
0.5
0.0
k
an
M
M
M
l
1 μ
1 μ
1m
n
B
7f
i
7a 1 μM
10c
m
or
f
t
e
M
Figure 3. Compounds 7a, 7f and 10c promoted glucose consumption in
HepG2 cells. Data are the average of three independent experiments, each
performed in duplicate. *P < 0.05 vs. Blank.
Molecular docking was performed with compounds 7f and 8b to identify binding modes. As the crystal structure of α-glucosidase
from Saccharomyces cerevisiae is not available, a docking study was conducted using the crystal structure of isomaltase (PDB No.
3A4A) from S. cerevisiae that was reported to be a more suitable template because its sequence is 71% identical and 84% similar to that
of S. cerevisiae α-glucosidase. The main interactions of acarbose with S. cerevisiae α-glucosidase and S. cerevisiae isomaltase are quite
similar.15 Compounds 7f and 8b were docked in the binding pocket of isomaltase and compared to acarbose (Fig. 4). Three hydrogen
bonds are established between the fluorine and carbonyl group of compound 8b and the side chains of Arg 315, Glu 411, and Arg 442
(Fig. 4A). Formation of six H-bonds by 7f with the key residues Glu 277, Asp 352, Arg 315, Glu 411, Asn 415 and Arg 442 was
observed in the docking study (Fig. 4B). In addition, the formation of van der Waals and electrostatic interactions by 7f provides further
stabilization for 7f and makes it fit very tightly in the enzyme’s active pocket (Fig. 4C). Calculated docking scores of 7f (45 kcal/mol)
were higher than for 8b (33 kcal/mol) and acarbose (38 kcal/mol), consistent with their potency (Table 1).
A
B
C
A
2500
2000
1500
1000
500
0 μM
15 μM
20 μM
Figure 4. Binding modes of 7f, 8b, and acarbose in the enzyme active
site. Docking of 2D int0erac2tion4dia6grams of compounds 8b (A), and 7f
-6
-4
-2
8
10
(B). (C) Compound 7f (gray) and acarbose (yellow) superimposed in the
1/(S, mM)
active pocket. Blue and green dotted lines indicate hydrogen bonds.

In conclusion, twenty-three natural and unnatural analogues of 6-hydroxyaurones and 6-hydroxybenzofuran-3(2H)-ones were
synthesized and evaluated for their α-glucosidase inhibitory and glucose consumption promoting activity. Compound 7f was identified
as a new α-glucosidase inhibitor with an IC50 value of 9.88 μM acting in a mixed competitive and irreversible inhibitory mode.
Molecular docking studies indicated that hydrogen bonding, van der Waals, and electrostatic interactions, together with a good shape
match to the active pocket, could explain the high affinity of compound 7f for α-glucosidase. In addition, compound 7f showed glucose
consumption-promoting activity in HepG2 cells at 1 μM, comparable to metformin at a concentration of 1 mM. In view of these
significant results, compound 7f presents considerable potential as a lead compound for the treatment of diabetes and other diseases
involving α-glucosidase.
Acknowledgments
The authors are sincerely thankful for financial support from the Chinese National Natural Science Foundation (21502138).
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