Synthesis and biological evaluation of stilbene derivatives coupled to NO donors as potential antidiabetic agents

Article abstract of DOI:10.1080/14756366.2018.1425686

The work is focused on the design of drugs that prevent and treat diabetes and its complications. A novel class of stilbene derivatives were prepared by coupling NO donors of alkyl nitrate and were fully characterised by NMR and other techniques. These compounds were tested in vitro activity, including α-glucosidase inhibitory activity, aldose reductase (AR) inhibitory activity and advanced glycation end products (AGEs) formation inhibitory activity. A class of modified compounds could play a significant effect for treatment of diabetic complications. Target compounds 3e and 7c offered a potential drug design concept for the development of therapeutic or preventive agents for diabetes and its complications.

Full text of DOI:10.1080/14756366.2018.1425686

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of stilbene
derivatives coupled to NO donors as potential
antidiabetic agents
Bing Wang, Teng Liu, Zhongyu Wu, Lei Zhang, Jie Sun & Xiaojing Wang
To cite this article: Bing Wang, Teng Liu, Zhongyu Wu, Lei Zhang, Jie Sun & Xiaojing Wang
(2018) Synthesis and biological evaluation of stilbene derivatives coupled to NO donors as potential
antidiabetic agents, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 416-423, DOI:
10.1080/14756366.2018.1425686
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 416–423
https://doi.org/10.1080/14756366.2018.1425686
RESEARCH PAPER
Synthesis and biological evaluation of stilbene derivatives coupled to NO donors
as potential antidiabetic agents
Bing Wanga,b,c,d, Teng Liua,b,c,d, Zhongyu Wua,b,c,d, Lei Zhanga,b,c,d, Jie Suna,b,c,d
Xiaojing Wanga,b,c,d
and
b
aSchool of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, China; Institute of Materia Medica,
c
d
Shandong Academy of Medical Sciences, Jinan, China; Key Laboratory for Biotech-Drugs Ministry of Health, Jinan, China; Key Laboratory for
Rare & Uncommon Diseases of Shandong Province, Jinan, China
ABSTRACT
ARTICLE HISTORY
Received 4 December 2017
Revised 5 January 2018
Accepted 5 January 2018
The work is focused on the design of drugs that prevent and treat diabetes and its complications. A novel
class of stilbene derivatives were prepared by coupling NO donors of alkyl nitrate and were fully character-
ised by NMR and other techniques. These compounds were tested in vitro activity, including a-glucosidase
inhibitory activity, aldose reductase (AR) inhibitory activity and advanced glycation end products (AGEs)
formation inhibitory activity. A class of modified compounds could play a significant effect for treatment
of diabetic complications. Target compounds 3e and 7c offered a potential drug design concept for the
development of therapeutic or preventive agents for diabetes and its complications.
KEYWORDS
a-Glucosidase inhibitor;
antidiabetic; AGEs formation
inhibitor; stilbene; nitric
oxide donor
1. Introduction
because of its small molecule and lipophilic properties7. Nitric
oxide is synthesised from the amino acid L-arginine in a reaction
mediated by one of several isoforms of the enzyme nitric oxide
synthase (NOS)8, and it plays a very important role in mammalian
physiology and pathophysiology9. The lack of NO relates to many
pathologic processes, thus providing a solid biological basis for
the use of NO replacement therapy. Exogenous NO sources consti-
tute a powerful way to supply NO when the body fails to generate
sufficient NO for normal biological functions. This theory opens up
the possibility of designing new drugs that can deliver NO into tis-
sues and the bloodstream in a sustained and controlled manner.
This objective has been achieved by grafting an organic nitrate
structure onto existing drugs through chemical spacers, such as
aliphatic, aromatic, or a heterocyclic chain. The approach has led
to the synthesis of several new chemical entities whose pharmaco-
logic profile challenges the parent drug, not only on the basis of
new properties, but also raised safety10. Great advances in know-
ledge on biochemical pathways and about pharmacological prop-
erties involving NO have sparked interest in devising hybrid
molecules containing NO-donors that allow synergism of action
between NO and the broad chemical diversity of “native” drugs11.
Diabetes mellitus (DM) is a chronic, incurable metabolic disorder
defined by the dysregulation of glucose homeostasis manifesting
as hyperglycaemia, abnormalities in lipid and protein metabolism,
and the development of both acute and long-term complications
in target organs, including retina, kidney, peripheral nerves, and
the cardiovascular system1. In 2014, the IDF estimated that 8.2%
of adults aged 20–79 (387 million people) were living with dia-
betes, compares with 382 million people in 2013, and this number
was projected to rise beyond 592 million in 20352. Chronic hyper-
glycaemia is the primer of a series of cascade reactions causing
the over production of free radicals and increasing evidences indi-
cate that these contribute to the development of diabetic compli-
cations such as blindness, cardiac and kidney disease. Effective
glycaemic control can lower the incidence of diabetic complica-
tions and reduce their severity.
Stilbene compounds such as rosewood and resveratrol have
been shown to be effective in the treatment of diabetes.
Pterocarpus marsupium Roxb. commonly known as vengisa or
bijasal, is well known for its medicinal properties in Ayurvedic and
Unani systems in the treatment of diabetes. The water extract of
the heartwood and root shows good curative properties for dia-
betes, and this may be due to the presence of pterostilbene3,4
.
2. Results and discussion
Pari and Satheesh5 reported that oral administration of pterostil-
bene decreased glucose levels in streptozotocin (STZ)-diabetic rats
2.1. Chemistry
through long-term trials conducted in 2006. Resveratrol also shows The preparation of the nitrate derivatives following the synthetic
a good candidate as a neutraceutical support for the therapy of routes is summarised in Scheme 1. An alkyl chain with a bromide
obesity and type II diabetes6.
group was introduced to the 40-position of stilbene 1a–b by a
Nitric oxide (NO) is a short-lived free radical whose half-life is selective reaction with an appropriate dibromo alkane, yielding
only a few seconds. It can easily pass through the cell membrane the 40-bromo alkane derivatives 2a–e, which were subsequently
CONTACT Jie Sun
sunjie310@126.com; Xiaojing Wang
xiaojing6@gmail.com
School of Medicine and Life Sciences, University of Jinan-Shandong Academy
of Medical Sciences, Jinan, China
Supplemental data for this article can be accessed here.
ß 2018 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.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
417
O(CH2)nONO2
O(CH2)nBr
R1
OH
R1
R1
(a)
(b)
R2
R2
R2
3a:R1=R2=OCH3, n=2
3b:R1=R2=OCH3, n=4
3c:R1=R2=OCH3, n=6
3d:R1=R2=OH, n=4
3e:R1=R2=OH, n=6
2a:R1=R2=OCH3, n=2
2b:R1=R2=OCH3, n=4
2c:R1=R2=OCH3, n=6
2d:R1=R2=OH, n=4
2e:R1=R2=OH, n=6
1a:R1=R2=OCH3
1b:R1=R2=OH
OCH2COOH
(e)
OCH2COOC2H5
H3CO
OH
H3CO
H3CO
(c)
(d)
OCH3
5a
OCH3
4a
OCH3
1a
OCH2COO(CH2)nBr
OCH2COO(CH2)nONO2
H3CO
H3CO
(b)
OCH3
6a: n=2
6b: n=4
6c: n=6
OCH3
7a: n=2
7b: n=4
7c: n=6
Scheme 1. General synthetic route to NO-donor compounds 3a–3e and 7a–7c. Reagents and conditions: (a) dibromo alkane, K2CO3, acetone, reflux, 6 h; (b) AgNO3,
acetonitrile, 80 ꢀC, 2 h; (c) ethyl bromoacetate, K2CO3, acetone; (d) KOH, methanol; (e) dibromo alkane, Et3N, acetone, reflux, 24 h.
converted to the nitro esters 3a–e via treatment with AgNO3 in vivo experiments showed that the compounds had no signifi-
(silver nitrate) in anhydrous acetonitrile. Details on the chemical
and spectroscopic characterizations of compounds 3a–e were
described in the Supporting Information.
Stilbene 1a was reacted with bromoacetate to produce
compound 4a. Subsequent hydrolysis of this compound and
reaction with a dibromo alkane produced compounds 6a–c.
These compounds were then reacted with AgNO3, producing
products 7a–c. Details on the chemical and spectroscopic charac-
terizations of compounds 7a–c were described in the Supporting
Information.
cant effect on blood pressure in normal mice (data not shown).
2.2.2. In vitro a-glucosidase inhibitory activity17
a-Glucosidases, enzymes anchored in the brush border of the
small intestine, are responsible for catalysing the hydrolysis of car-
bohydrates. Their inhibitors were useful for the treatment of
type II DM18. As shown in Table 1, just three compounds (3c, 3e,
and 7c) presented moderate inhibitory activity for a-glucosidase.
Notably, 3c (IC50 ¼ 83.12 16.21 mM) had relatively strong activity
which displayed weaker capacity than Acarbose (IC50¼0.05
0.003 mM). None of the linker with 1,2-dibromoethane or 1,4-dibro-
mobutane showed good inhibitory activity, indicating that increas-
ing the length of linker in NO donor compounds was necessary
for inhibiting a-glucosidase. Through comparison of the IC50 val-
ues, 7c>3c, showed that linker of NO donor compounds with car-
bonyl group reduced a-glucosidase inhibitory activity. Through
comparison of the IC50 values, 3d>3e, showed that the length of
linker in NO donor compounds played an important role in
enhancing the a-glucosidase inhibitory activity.
2.2. Biological evaluation
2.2.1. In vitro NO releasing property12
As shown in Table 1, NO appeared to be released from all the stil-
bene derivatives upon incubation with phosphate-buffered saline
solution (PBS at pH 7.4) in the presence of L-cysteine, indicating
that all the stilbene derivatives were exogenous NO donor com-
pounds. The percentage of released NO varied from
0.896 0.004% to 1.391 0.050%, indicating a slow release. The
amount of NO released from sodium nitroprusside (SNP), however,
was substantially higher (6.825 0.187%). At higher concentrations,
particularly under conditions of oxidative stress, NO is highly cyto-
toxic, a feature that is exploited by inflammatory cells in response
to invading pathogens13. Some researchers demonstrated that NO
could stimulate glucose metabolism or transport in adipocytes or
skeletal muscle14–16. Those stilbene derivatives could release NO in
2.2.3. In vitro inhibitory activity of AGEs (advanced glycation end
products) formation19,20
Advanced glycation end products are a group of complex and het-
erogeneous compounds, which are implicated in a number of bio-
chemical abnormalities associated with diabetes21,22. Therefore,
a slow rate. We think this can be useful to treat diabetes. Further the discovery of AGEs inhibitors would be beneficial to the

418
B. WANG ET AL.
Table 1. Biological evaluation in vitro.
IC50 value (lM)
Products
NO releasing activity (%)a
a-Glucosidase inhibitory activityb
>1000
AGEs inhibitory activityc
ALR2 inhibitory activityd
DMSO
Pterostilbene
Resveratrol
3a
3b
3c
3d
3e
7a
7b
7c
SNP
ꢁꢁ
1.99 0.51
0.83 0.21
2.90 0.09
2.39 0.11
1.49 0.20
1.39 0.16
1.05 0.25
1.29 0.04
>1000
17.02 0.31
>1000
ꢁꢁ
ꢁꢁ
241.53 16.98
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
1.029 0.009
1.359 0.034
1.391 0.050
0.896 0.004
0.900 0.006
0.922 0.020
1.000 0.004
1.056 0.023
6.825 0.187
>1000
>1000
83.12 16.21
>1000
178.74 5.76
>1000
>1000
163.40 14.20
ꢁꢁ
>1000
299.25 22.62
10.03 0.15
8.18 0.33
>1000
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
0.97 0.16
>1000
121.13 13.28
ꢁꢁ
ꢁꢁ
ꢁꢁ
0.68 0.07
Acarbose
AG
Quercetin
0.05 0.003
282.81 8.52
3.71 0.29
Each value represents the mean SD (n ¼ 3).
aPercentage of NO released relative to a theoretical maximum release of 1 mol NO/mol of test compound; determined by Griess reagent in the pres-
ence of 5 mM L-cysteine, at pH 7.4.
bThe concentration required for a 50% inhibition in the optical density of PNP (p-nitrophenol) at 405 nm relative to DMSO. IC50 values were calculated
from the dose inhibition curve.
cThe concentration required for a 50% inhibition of the fluorescence intensity of AGE relative to 0.1% DMSO, IC50 values were calculated from the
dose inhibition curve.
dThe concentration required for a 50% inhibition of the decrease in the optical density of NADPH at 340 nm relative to DMSO. IC50 values were calcu-
lated from the dose inhibition curve.
ꢁꢁ
p < .01 significantly different ANOVA followed by Dunnett’s t-test for comparison with standard.
prevention and treatment of diabetic or other pathogenic compli- 1000 mg/kg to test their oral toxicity in the first phase, and 1600,
cations23. The results listed in Table 1 displayed that most of the 2900, and 5000 mg/kg at the second phase. Results showed that
none of the tested compounds significantly affected mice’ viability.
No death and no appetite-suppressant effect were detected in the
tested mice in 14 days. Since no death or damage was observed
throughout the experiment, the LD50 was higher than 5000 mg/kg
for the three compounds assayed, indicating their innocuousness
for mice.
target compounds presented strong inhibitory activity to AGEs,
even better than amino guanidine (AG) (IC50 ¼ 282.81 8.52 mM).
Through comparison of the IC50 values, 7c<7 b<3e<7a<
3d<3c<1.5 mM, those compounds were over 100 folds compared
with positive reference AG in inhibitory activity to the formation
of AGEs. The AGEs inhibitory capacities of 3d>3b, 3e>3c, indicate
that absence of 3,5-dihydroxy groups in stilbene compounds
enhanced their inhibitory activity to the formation of AGEs. The
AGEs inhibitory capacities of 7b>3b, 7c>3c, indicate that linker
with carbonyl group in NO donor compounds enhanced their
inhibitory activity to the formation of AGEs.
2.2.6. Acute hypoglycaemic assay
The antidiabetic activity was determined by using a standard
method27. As shown in Table 2, the target compounds 3c, 3e, and
7c (40 mg/kg of bw) caused decreases in blood glucose levels in
STZ-diabetic mice compared with normal mice (Table 2).
Especially, compound 7c caused significant decreases in blood glu-
cose levels when compared with vehicle-treated groups (p < .05).
The target compounds 3c and 3e showed similar antidiabetic
activity throughout the experiment, their hypoglycaemic effect
was weaker than pterostilbene at dose of 40 mg/kg of body
weight. In STZ-diabetic animals, the hypoglycaemic effect of pter-
ostilbene (40 mg/kg of bw) was larger than 50% since 5 h and per-
sisted throughout the experiment. Target compound 7c (40 mg/kg
of bw) caused significant decreases in blood glucose levels com-
pared with pterostilbene (40 mg/kg of bw). This result indicated
that the NO donor was an adjunct to the hypoglycaemic effect in
STZ-diabetic animal. However, glibenclamide (10 mg/kg of bw)
which was used as a positive control, showed the strongest hypo-
glycaemic effect in STZ-induced diabetic mice than all compounds
tested (Table 2). Target compound 7c exerted higher hypogly-
caemic effect than other serial compounds, indicating that linker
with carbonyl group in NO donor compounds and the absence of
3,5-dimethoxy groups in stilbene compounds enhanced their
hypoglycaemic effect (Figure 1).
2.2.4. In vitro ALR2 inhibitory activity24
The enzyme aldose reductase (ALR2) is a member of the aldo–keto
reductase superfamily. The development and progression of
chronic diabetic complications are confirmed to be quite related
to the activation and/or over expression of ALR225. Therefore,
ALR2 inhibitors may play a critical role in preventing or treating
these complications. The results listed in Table 1 displayed that
the target compounds 3c, 3d, 3e, and 7c resulted in high inhibi-
tory activities on ALR2 (IC50 ¼ 8.18–299.25 mM). The target com-
pound 3d and 3e presented strong inhibitory activity to ALR2,
showing little weaker inhibitory activity than quercetin (IC50
¼
3.71 0.29 mM). Target compounds 3d and 3e exerted higher ALR2
inhibition activities than the other serial compounds, indicating
that absence of 3,5-dihydroxy groups in stilbene compounds was
more effective in inhibiting ALR2. Target compounds 3c and 7c
exerted weaker ALR2 inhibition activities, indicating that increasing
the length of linker in NO donor compounds showed weak effect
on enhancing their inhibitory activity to ALR2.
2.2.5. Oral toxicity to mice
3. Conclusions
With reference to Lorke’s method26, Kunming mice were used as
targets to estimate oral toxicity of each compound to mice. We
A novel class of stilbene derivatives was prepared by coupling NO
select compounds 3c, 3e, and 7c at concentrations of 10, 100, and donors of alkyl nitrate. We then studied the pharmacological

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
419
Table 2. Acute effect of compounds 3c, 3e, and 7c on blood glucose levels in STZ-diabetics mice.
Blood glucose concentration (mM)
Dose (per os)
mg/kg of bw
Test samples
0 h
1.5 h
3 h
5 h
7 h
9 h
Control (vehicle)
Glibenclamide
Pterostilbene
Resveratrol
3c
3c
3c
3e
3e
3e
7c
7c
7c
–
10
40
40
10
40
100
10
40
20.3 10.1
20.2 5.4
17.2 7.0
20.0 2.6
22.6 7.0
21.7 5.7
18.6 1.9
21.8 6.3
20.8 7.6
22.5 5.6
22.0 5.2
20.1 1.9
20.2 2.0
19.0 5.6 (–6.16)
11.6 4.9 (–42.63)
12.1 6.4 (–29.91)
16.3 2.9 (–18.42)
18.1 7.3 (–20.13)
17.2 5.6 (–20.74)
15.9 3.6 (–14.34)
18.8 5.2 (–13.84)
16.9 5.6 (–18.67)
15.7 7.7 (–30.37)
17.0 7.4 (–22.65)
13.2 2.8 (–34.33)
13.7 4.0 (–32.43)
18.2 5.2 (–10.36)
16.4 6.4 (–19.00)
11.5 5.5 (–43.17)
11.5 6.4 (–43.17)
ꢁ
ꢁ
ꢁ
ꢁ
7.6 3.9 (–62.32)
6.1 3.6 (–69.58)
5.7 3.6 (–71.84)
5.6 3.6 (–72.11)
ꢁ
ꢁ
ꢁ
ꢁ
10.2 6.2 (–40.89)
8.4 5.6 (–51.42)
7.7 5.3 (–55.36)
6.9 4.3 (–59.82)
ꢁ
14.0 2.0 (–29.83)
17.0 6.6 (–24.78)
16.3 7.5 (–25.04)
15.2 3.0 (–18.28)
17.8 4.8 (–18.35)
16.5 9.1 (–20.83)
16.0 6.8 (–29.04)
16.9 10.6 (–23.18)
12.4 2.1 (–38.08)
16.8 6.1 (–25.66)
15.7 6.4 (–27.57)
15.1 3.7 (–19.00)
18.6 7.3 (–14.53)
15.0 8.7 (–28.04)
15.4 9.6 (–31.78)
16.1 7.7 (–26.82)
11.2 1.8 (–44.25)
15.7 4.3 (–30.53)
14.3 9.6 (–34.02)
13.4 4.7 (–28.23)
15.6 6.0 (–28.59)
13.3 9.4 (–36.22)
12.9 6.4 (–42.81)
15.7 8.3 (–28.56)
10.7 1.4 (–46.33)
13.2 3.5 (–41.59)
ꢁ
9.9 4.6 (–54.30)
11.1 3.0 (–40.32)
ꢁ
11.5 4.4 (–47.48)
11.0 8.0 (–47.20)
10.9 4.5 (–51.48)
13.3 5.7 (–39.70)
100
10
40
ꢁ
ꢁ
ꢁ
ꢁ
11.0 3.6 (–45.11)
8.7 2.5 (–56.80)
8.2 1.8 (–59.37)
7.1 1.2 (–64.76)
ꢁ
ꢁ
ꢁ
ꢁ
100
11.1 3.0 (–44.88)
9.9 2.5 (–50.83)
8.2 1.9 (–59.49)
6.9 1.4 (–66.01)
Each value is the mean SEM for six mice in each group.
ꢁ
p < .05 significantly different ANOVA followed by Dunnett’s t-test for comparison with respect to initial levels in each group.
% Variation of glycaemia is in parentheses.
25
20
Control
Glibenclamide
Pterostilbene
Resveratrol
3c10mg
15
3c40mg
3c100mg
3e10mg
*
*
*
*
*
10
5
*
*
*
*
3e40mg
*
*
*
*
*
3e100mg
7c10mg
*
*
*
7c40mg
7c100mg
0
0h
1.5h
3h
5h
7h
9h
ꢁ
Figure 1. Acute effect of compounds 3c, 3e, and 7c on blood glucose levels in STZ-diabetics mice. Each value is the mean SEM for six mice in each group. p < .05
significantly different ANOVA followed by Dunnett’s t-test for comparison with respect to initial levels in each group.
activity of the synthesised compounds. In the respect to its NO concept for the development of therapeutic or preventive agents
releasing property, all stilbene derivatives could release NO in a
slow rate. In the respect to anti-diabetic complications, carbonyl
group in the linker and the length of linker showed significant
effect on treatment of diabetic complications. Notably, target com-
pound 3e had strong activity in inhibitory activity of AGEs forma-
tion, which displayed stronger capacity than AG. Compounds 3c,
3e, and 7c showed relatively strong a-glucosidase inhibitory activ-
ity, which displayed weaker capacity than acarbose. Oral toxicity
tests indicated their innocuousness for mice. We selected com-
pounds 3c, 3e, and 7c to investigate their antidiabetic activity in
vivo. The results showed the effect of the target compound 7c
showed the strongest hypoglycaemic effect in STZ-induced dia-
betic mice which was weaker than that of the glibenclamide used
as a positive control in vivo. The result of antidiabetic activity in
vivo indicated that linker with carbonyl group in NO donor com-
pounds enhanced their hypoglycaemic effect. In conclusion, the
for diabetes and complications of diabetes.
4. Experimental
4.1. Synthesis
4.1.1. Materials and methods
Melting points were determined using a Thiele tube and were
uncorrected. The FT-IR spectra were recorded using a Thermo-
1
Nicolet Nexus 670 spectrometer with KBr pellets. The H NMR and
13C NMR spectra were recorded with a Bruker AM-600 spectrom-
eter (Billerica, MA) with TMS as the internal standard. Chemical
shifts were reported at room temperature on a scale (ppm) with
DMSO-d6 as the solvents and J values are given in hertz.
Mass spectra were obtained with an Agilent Trap VL LC/MS
spectrometer (Santa Clara, CA). The absorbance was recorded by a
target compounds 3e and 7c offered a potential drug design Hitachi U-3000 UV spectrophotometer (Tokyo, Japan). Column

420
B. WANG ET AL.
chromatography was performed on silica gel (200–300 mesh). 1574, 1512, 1446, 963, 832 (Ar); 1238, 1146 (C–O). 1H NMR
Unless otherwise noted, all solvents and reagents were commer- (600 MHz, DMSO-d6) d (ppm): 1.43 (m, 4H), 1.70 (m, 4H), 3.97 (t,
cially available and used without further purification.
J ¼ 6.2 Hz, 2H), 4.52 (t, J ¼ 6.5 Hz, 2H), 6.14 (s, 1H), 6.41 (s, 2H),
6.93 (m, 4H), 7.49 (d, J ¼ 7.8 Hz, 2H), 9.21 (s, 2H). 13C NMR
(150 MHz, DMSO-d6) d (ppm): 25.32, 25.57, 26.44, 28.94, 31.15,
67.79, 74.27, 102.39, 104.86, 115.07, 127.05, 127.95, 128.22, 130.01,
139.56, 158.77 and 158.98. MS: m/z (%):346.0 [M þ 1]þ, 282.0,
240.8.
4.1.2. General method for synthesis of compounds 2a–e
Anhydrous potassium carbonate (0.69 g, 5.0 mmol) was added to a
solution of 1a (2.56 g, 10 mmol) in 50 ml of anhydrous acetone, fol-
lowed by refluxing until the solution became clear. Then, 1,2-
dibromo ethylene (9.40 g, 50 mmol) was added dropwise, followed
by refluxing for 24 h and vacuum filtration. This procedure yielded
concentrated filter liquor, from which a white solid was obtained.
The solids were collected, washed with petroleum, 1% NaOH and
water, respectively, then dried. Compounds 2d–2e were obtained
using the same procedures.
4.1.3.5. O40-Nitrooxyethyl resveratrol (3e). White solid, yield: 69%,
mp 134.2–135.9 ꢀC, IR (KBr, ꢀ, cmꢂ1): 3491 (OH); 1606 (–ONO2);
1573, 1512, 964, 834 (Ar); 1255, 1149 (C–O). 1H NMR (600 MHz,
DMSO-d6) d (ppm): 1.43 (m, 4H), 1.70 (m, 4H), 3.97 (t, J ¼ 6.5 Hz,
2H), 4.52 (t, J ¼ 6.6 Hz, 2H), 6.13 (t, J ¼ 2.1 Hz, 1H), 6.40 (d,
J ¼ 2.1 Hz, 2H), 6.92 (m, 4H), 7.49 (d, J ¼ 8.8 Hz, 2H), 9.20 (s, 2H).
13C NMR (150 MHz, DMSO-d6) d (ppm): 25.33, 25.57, 26.45, 28.94,
67.79, 74.28, 102.38, 104.86, 115.07, 127.05, 127.95, 128.23, 130.01,
139.56, 158.77 and 158.98. MS: m/z (%):374.2 [M þ 1]þ, 328.0,
204.8.
4.1.3. General method for synthesis of compounds 3a–e
Compound 2a (3.63 g, 10 mmol) was dissolved in 50 ml of anhyd-
rous acetonitrile, followed by heating to 50 ꢀC. AgNO3 (1.70 g,
10 mmol) and acetonitrile (20 ml) were added. The mixture was
stirred by heating to 80 ꢀC in the dark for 2 h. The precipitate was
filtered out, and filtrate was concentrated. When cooling at room
temperature, white crystals were precipitated. The filtrate was vac-
uum filtered and the resulting solid was washed three times with
water then dried to obtain white crystal 3a. Compounds 3b–3e
were obtained using the same procedures.
4.1.4. General method for synthesis of compounds 6a–c
A mixture of 4-methoxybenzaldehyde 1a (200 mmol), malonic acid
(240 mmol) and piperidine (2 ml) in pyridine (50 ml) was heated to
reflux for 8 h at 90 ꢀC. After the reaction is completed, hydrochloric
acid solution (150 ml, 3 mol/l) was added. Then filtered to obtain a
white crude product 24 h later. The crude product was recrystal-
lised from absolute ethanol to afford 4-methoxyphenylacrylic acid
2a.
Anhydrous potassium carbonate (0.69 g, 5.0 mmol) was added
to a solution of 1a (2.56 g, 10 mmol) in 50 ml of anhydrous acet-
one, followed by refluxing until the solution became clear. Then,
ethyl bromoacetate (2.3 ml, 20 mmol) was added dropwise, fol-
lowed by refluxing for 24 h and vacuum filtration. This procedure
yielded concentrated filter liquor, from which a white solid was
obtained. The solids were collected, washed with petroleum, 1%
NaOH and water, respectively, then dried to obtain white solid 4a
(3.16 g, in 92% yield).
4.1.3.1. O40-Nitrooxyethyl pterostilbene (3a). White solid, yield:
60%, mp 142.2–143.2 ꢀC, IR (KBr, ꢀ, cmꢂ1): 2966 (–CH3); 1624
(–ONO2); 1592, 1513, 1455, 1040, 960, 829 (Ar); 1238, 1149 (C–O).
1H NMR (600 MHz, DMSO-d6) d (ppm): 3.78 (s, 6H), 4.34 (m, 2H),
4.89 (m, 2H), 6.40 (t, J ¼ 2.2 Hz, 1H), 6.75 (d, J ¼ 2.2 Hz, 2H), 6.98
(d, J ¼ 8.7 Hz, 2H), 7.05 (d, J ¼ 16.4 Hz, 1H), 7.22 (d, J ¼ 16.4 Hz,
1H), 7.55 (d, J ¼ 8.7 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) d
(ppm): 55.65, 64.51, 72.50, 100.01, 104.70, 115.25, 127.00, 128.36,
128.89, 130.62, 139.84, 158.04 and 161.12. MS: m/z (%): 346.0
[M þ 1]þ, 269.8, 240.8.
Potassium hydroxide (3.36 g, 60 mmol) was added to a solution
of 4a (3.42 g, 10 mmol) in anhydrous methanol (150 ml). The solu-
tion was then mechanically stirred and heat refluxed for 3 h until a
white solid that does not dissolve in methanol was obtained. The
solid dissolved in water. Then, the pH was adjusted to the desired
acidity with hydrochloric acid, a white solid that does not dissolve
in water was obtained. The solution underwent vacuum filtration,
washed with water, then dried to obtain white solid 5a (2.78 g, in
89% yield).
Triethylamine (4.15 ml, 30 mmol) was added to a solution of 5a
(3.14 g, 10 mmol) in acetone (100 ml). The mixture was then
refluxed for 30 min. 1,2-Dibromoethane (9.40 g, 50 mmol) was
dribbled into the mixture, followed by refluxing for 8 h and filtra-
tion to remove precipitates, then concentrating in vacuo to obtain
the crude product. The crude product was subjected to column
chromatography (silica, EtOAc–PE, 1:4) to obtain purified com-
pound 6a (2.05 g, in 49% yield). Compounds 6b–6c were obtained
using the same procedures.
4.1.3.2. O40-Nitrooxyethyl pterostilbene (3b). White solid, yield:
70%, mp 110.9–111.3 ꢀC, IR (KBr, ꢀ, cmꢂ1): 2972 (–CH3); 1620
(–ONO2); 1593, 1514, 1455, 1024, 965, 832 (Ar); 1257, 1148 (C–O).
1H NMR (600 MHz, DMSO-d6) d (ppm): 1.82 (m, 4H), 3.77 (s, 6H),
4.03 (t, J ¼ 5.9 Hz, 2H), 4.60 (t, J ¼ 6.2 Hz, 2H), 6.39 (t, J ¼ 2.2 Hz,
1H), 6.74 (d, J ¼ 2.2 Hz, 2H), 6.94 (d, J ¼ 8.7 Hz, 2H), 7.02 (d,
J ¼ 16.4 Hz, 1H), 7.21 (d, J ¼ 16.4 Hz, 1H), 7.52 (d, J ¼ 8.7 Hz, 2H).
13C NMR (150 MHz, DMSO-d6) d (ppm): 23.47, 25.42, 55.65, 67.33,
74.01, 99.95, 104.64, 115.17, 126.63, 128.31, 129.03, 130.04, 139.90,
158.77 and 161.12. MS: m/z (%):374.1 [M þ 1]þ, 298.0, 268.8, 253.9.
4.1.3.3. O40-Nitrooxyethyl pterostilbene (3c). White solid, yield:
71%, mp 110.9–111.1 ꢀC, IR (KBr, ꢀ, cmꢂ1): 2943 (–CH3); 1617
(–ONO2); 1592, 1513, 1458, 1038, 971, 831 (Ar); 1258, 1148 (C–O).
1H NMR (600 MHz, DMSO-d6) d (ppm): 1.43 (dd, J ¼ 15.2, 9.9 Hz,
4H), 1.70 (m, 4H), 3.77 (s, 6H), 3.98 (t, J ¼ 5.6 Hz, 2H), 4.53 (t,
J ¼ 6.1 Hz, 2H), 6.39 (s, 1H), 6.74 (s, 2H), 6.93 (d, J ¼ 8.0 Hz, 2H),
7.01 (d, J ¼ 16.4 Hz, 1H), 7.20 (d, J ¼ 16.3 Hz, 1H), 7.51 (d,
J ¼ 7.9 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) d (ppm): 25.32,
25.57, 26.45, 28.94, 55.65, 67.81, 74.28, 99.93, 104.64, 115.14,
126.55, 128.30, 129.06, 129.90, 139.92, 158.93 and 161.11. MS: m/z
(%):402.3 [M þ 1]þ, 356.0, 326.0, 268.9, 240.8, 204.8.
4.1.5. General method for synthesis of compounds 7a–c
Compound 6a (2.11 g, 5 mmol) was dissolved in 50 ml of anhyd-
rous acetonitrile, followed by heating to 50 ꢀC. AgNO3 (0.85 g,
5 mmol) and acetonitrile (20 ml) were added. The mixture was
4.1.3.4. O40-Nitrooxyethyl resveratrol (3d). White solid, yield: 46%, stirred by heating to 80 ꢀC in the dark for 2 h. The precipitate was
mp 140.4–141.3 ꢀC, IR (KBr, ꢀ, cmꢂ1): 3386 (OH); 1625 (–ONO2); filtered out, and filtrate was concentrated. When cooling at room

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
421
Table 3. The amount and order of each reactant of a-glucosidase inhibition
test.
their care were performed in strict accordance with the National
Care and Use of Laboratory Animals by the National Animal
Research Authority (China) and guidelines of Animal Care and Use
issued by University of Jinan Institutional Animal Care and Use
Committee. The experiments were approved by the Institutional
Animal Care and Use Committee of the School of Medicine and
Life Sciences, University of Jinan. All efforts were made to minim-
ise animal’s suffering and to reduce the number of animals used.
Volume (lL)
Blank
group
Control
group
Sample blank
group
Sample
group
Reagents
PBS
20
0
20
10
10
0
20
10
20
10
20
0
10
10
20
0
Compounds/Inhibitors
PNPG
Water
Mix well and incubate at 37 ꢀC for 10 minutes
a-Glucosidase
Mix well and react at 37 ꢀC for 20 minutes
Na2CO3 70
0
10
0
10
70
4.2.2. Detection of nitrite
A solution of the appropriate compound (80 mL) in dimethyl sulph-
oxide (DMSO) was added to 8 ml of 1:1 v/v mixture of 50 mM PBS
70
70
(pH 7.4) with MeOH, containing 5 ꢃ 10ꢂ4
M L-cysteine. The final
concentration of target compounds was 10ꢂ4 M. After 1 h at 37 ꢀC
in dark, the reaction mixture was treated with 2 ml of the Griess
reagent [sulphanilamide (1 g), N-naphthylethylenediamine dihydro-
chloride (0.1 g), 85% phosphoric acid (2.5 ml) in distilled water
(final volume: 100 ml)]. After 10 min at 37 ꢀC in dark, the absorb-
ance was measured at 540 nm. Sodium nitrite standard solutions
(1–80 mmol/ml) were used to construct the calibration curve. The
results were expressed as the percentage of NO released (n ¼ 3)
relative to a theoretical maximum release of 1 mol NO/mol of test
compound.
temperature, white crystals were precipitated. The filtrate was vac-
uum filtered and the resulting solid was washed three times with
water then dried to obtain white solid 7a. Compounds 7b–7c
were obtained using the same procedures.
4.1.5.1. O40-Nitrooxyethyl pterostilbene (7a). White solid, yield:
51%, mp 95.2–96.3 ꢀC, IR (KBr, ꢀ, cmꢂ1): 2941 (–CH3); 1743 (C¼O);
1613 (–ONO2); 1591, 1510, 1456, 977, 836 (Ar); 1257, 1149 (C–O).
1H NMR (600 MHz, DMSO-d6) d (ppm): 3.77 (s, 6H), 4.47 (m, 2H),
4.77 (m, 2H), 4.86 (s, 2H), 6.39 (t, J ¼ 2.1 Hz, 1H), 6.75 (d,
J ¼ 2.1 Hz, 2H), 6.96 (d, J ¼ 8.7 Hz, 2H), 7.04 (d, J ¼ 16.4 Hz, 1H),
7.22 (d, J ¼ 16.4 Hz, 1H), 7.53 (d, J ¼ 8.7 Hz, 2H). 13C NMR
(150 MHz, DMSO-d6) d (ppm): 54.59, 60.01, 63.93, 70.86, 98.98,
4.2.3. Influence on blood pressure
Influence on blood pressure was analysed in adult normotensive
103.63, 114.24, 126.01, 127.17, 127.77, 129.75, 138.75, 156.65, mice (25–30 g). After one week of acclimation, mean arterial pres-
160.05 and 168.01. MS: m/z (%):404.0 [M þ 1]þ, 327.9, 268.8, 253.8.
sure values were measured using the tail-cuff method with a
blood pressure monitor (BP-2010, Softron Beijing Biotechnology
Co., Ltd., Beijing, China) from 0 h to 8 h after administration of the
standard and test compounds28. The test compounds (40 mg/kg
and100 mg/kg) and standard (losartan, 20 mg/kg) were
administered.
4.1.5.2. O40-Nitrooxyethyl pterostilbene (7b). White solid, yield:
76%, mp 124.3–124.9 ꢀC, IR (KBr, ꢀ, cmꢂ1): 2947 (–CH3); 1747
(C¼O); 1628 (–ONO2); 1593, 1512, 1450, 975, 841 (Ar); 1239, 1148
(C–O). 1H NMR (600 MHz, DMSO-d6) d (ppm): 1.69 (m, 4H), 3.77 (s,
6H), 4.16 (t, J ¼ 5.8 Hz, 2H), 4.52 (t, J ¼ 6.0 Hz, 2H), 4.83 (s, 2H),
6.39 (t, J ¼ 2.1 Hz, 1H), 6.74 (d, J ¼ 2.1 Hz, 2H), 6.95 (d, J ¼ 8.7 Hz,
2H), 7.04 (d, J ¼ 16.4 Hz, 1H), 7.21 (d, J ¼ 16.4 Hz, 1H), 7.53 (d,
J ¼ 8.7 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) d (ppm): 23.17,
24.89, 31.16, 55.65, 64.41, 65.09, 73.77, 100.03, 104.69, 115.24,
127.05, 128.25, 128.84, 130.75, 139.83, 157.81, 161.12 and 169.23.
MS: m/z (%): 432.5 [M þ 1]þ, 356.4, 241.3.
4.2.4. In vitro a-glucosidase inhibitory activity
a-Glucosidase (G0660-750UN, Sigma Aldrich, St. Louis, MO) and 4-
nitrophenyl a-D-glucopyranoside (PNPG, Macklin) were dissolved in
phosphate buffer (pH 6.8, 100 mM), and the test compounds were
dissolved in DMSO solution. The experiment was divided into
blank group, control group, sample blank group and sample
group. The reagents were loaded in 96-well plates at the dose of
the table (Table 3). The solution was bathed in 37 ꢀC water for
10 min, after the end, enzyme solution was added. After reaction
at 37 ꢀC for 20 min, 70 mL Na2CO3 solution (0.2 mM) was added to
stop the reaction. All experiments were run in triplicate. Acarbose
4.1.5.3. O40-Nitrooxyethyl pterostilbene (7c). White solid, yield:
79%, mp 98.0–99.1 ꢀC, IR (KBr, ꢀ, cmꢂ1): 2900 (–CH2); 1765 (C¼O);
1639 (–ONO2); 1591, 1513, 1456, 973, 841 (Ar); 1205, 1155 (C–O).
1H NMR (600 MHz, DMSO-d6) d (ppm): 1.31 (m, 4H), 1.60 (m, 4H),
3.77 (s, 6H), 4.12 (t, J ¼ 6.5 Hz, 2H), 4.48 (t, J ¼ 6.6 Hz, 2H), 4.82 (s,
2H), 6.39 (t, J ¼ 1.9 Hz, 1H), 6.74 (d, J ¼ 2.1 Hz, 2H), 6.94 (d,
J ¼ 8.7 Hz, 2H), 7.04 (d, J ¼ 16.4 Hz, 1H), 7.21 (d, J ¼ 16.4 Hz, 1H),
7.53 (d, J ¼ 8.7 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) d (ppm):
25.13, 25.27, 26.38, 28.32, 55.65, 64.85, 65.11, 74.20, 100.04, 104.67,
115.23, 127.03, 128.24, 128.82, 130.73, 139.82, 157.82, 161.11 and
169.27. MS: m/z (%):460.8 [M þ 1]þ, 414.4, 384.5.
(Sigma Aldrich, St. Louis, MO) was used as a standard inhibitor29
.
Since PNPG can produce glucose and p-nitrophenol (PNP) under
the action of a-glucosidase, PNP has the greatest absorption at
405 nm. The absorbance was determined by the microplate reader,
and the inhibition rate of a-glucosidase and the IC50 value of each
sample were calculated according to the formula.
È
Inhibition rate % ¼ ½ðAC ꢂ ABÞ ꢂ ðAS ꢂ ASBÞꢄ=ðAC ꢂ ABÞg ꢃ 100%
where AC is the absorbance of control group; AB is the absorbance
of blank group; AS is the absorbance of sample group; ASB is the
absorbance of sample blank group.
4.2. Biological activity
4.2.1. Animals
Normoglycemic Kunming mice, weight 18–20 g, were obtained
from Jinan PengYue Experimental Animal Co., Ltd. (License num-
ber: SCXK (Lu) 2014-0007). The animals were housed under stand- 4.2.5. In vitro inhibitory activity of AGEs formation
ard laboratory conditions and maintained on a standard pellet diet To prepare the AGE reaction solution, 10 mg/ml of bovine serum
and water ad libitum. All experiments involving living animals and albumin in 50 mM PBS (pH 7.4) was added to 0.2 M glucose, and

422
B. WANG ET AL.
0.02% sodium azide was added to prevent bacterial growth. The experimentation but allowed free access to tap water throughout.
reaction mixture (3 ml) was then mixed with various concentra- The compounds (at the doses of 10, 40, and 100 mg/kg of bw)
tions (0.5–1000 mg/ml) of the target compounds (1 ml) dissolved in were suspended in 0.05% Tween-80 in saline solution.
DMSO. After incubating at 37 ꢀC for 14 d, the fluorescence inten-
Glibenclamide (10 mg/kg of bw) was suspended in the same
sity of AGE was determined by a fluorospectrophotometer (PE,
Cincinnati, OH) with excitation and emission wavelengths at
350 nm and 420 nm30, respectively. All experiments were run in
triplicate. Aminoguanidine hydrochloride was used as a reference
compound. The inhibition rate of AGEs formation and the IC50
value of each sample were calculated according to the formula.
vehicle. The target compounds were freshly prepared immediately
before experimentation and administered by the intragastrical
route at the doses of 10 ml/kg of bw. Control mice received only
the vehicle (0.05% Tween-80 in saline solution) by the same route.
Blood glucose levels were measured at 0, 1.5, 3, 5, 7, and 9 h after
drugs administration33.
È
Inhibition rate % ¼ ½ðAC ꢂ ABÞ ꢂ ðAS ꢂ ASBÞꢄ=ðAC ꢂ ABÞg ꢃ 100%
4.2.9. Statistical analysis
where AC is the absorbance of control group (1.0 ml glucose
þ1.0 ml bovine serum albumin þ1.0 ml sodium azide þ1.0 ml
DMSO); AB is the absorbance of blank group (1.0 ml PBS þ1.0 ml
bovine serum albumin þ1.0 ml sodium azide þ1.0 ml DMSO); AS is
the absorbance of sample group (1.0 ml glucose þ1.0 ml bovine
serum albumin þ1.0 ml sodium azide þ1.0 ml target compound
solution or aminoguanidine hydrochloride solution); ASB is the
absorbance of sample blank group (1.0 ml PBS þ1.0 ml bovine
serum albumin þ1.0 ml sodium azide þ1.0 ml target compound
solution or aminoguanidine hydrochloride solution).
Data were shown as mean SD. Differences between individual
groups were analysed by using ANOVA followed by Dunett’s test.
A difference with a p value of < .05 was considered to be
significant.
Ethical statement
All experiments involving living animals and their care were per-
formed in strict accordance with the National Care and Use of
Laboratory Animals by the National Animal Research Authority
(China) and guidelines of Animal Care and Use issued by
University of Jinan Institutional Animal Care and Use Committee.
The experiments were approved by the Institutional Animal Care
and Use Committee of the School of Medicine and Life Sciences,
University of Jinan. All efforts were made to minimise animal’s suf-
fering and to reduce the number of animals used.
4.2.6. In vitro ALR2 inhibitory activity
After homogenisation and centrifugation, the crude ALR2 from
mice was obtained31. The inhibitory activity of the compounds on
ALR2 was carried out using crude enzyme and different concentra-
tions of the compounds (1–1000 mg/ml) in 200 mM PBS (pH 6.2)
containing 0.10 mM NADPH. The reaction was initiated by addition
of glyceraldehyde and the decrease in the optical density of
NADPH at 340 nm was recorded for 3 min. All experiments were
run in triplicate. IC50 of the compounds was calculated. The flavon-
oid quercetin was used as a reference in the ALR2 assay.
Disclosure statement
The authors declare that they have no competing interests.
Funding
4.2.7. Oral toxicity to mice
Experiments were performed on Kunming mice (male and female
half, body weight range, 25–30 g). Mice were housed in a climate
and light controlled room with a 12 h light/dark cycle. Twelve
hours before experiments, food was withheld, but animals had
free access to drinking water. The compounds were suspended in
vehicle (Tween-80, 0.2% in saline). The concentrations were
adjusted to orally administrate 0.2 ml/10 g of bw (body weight).
Mice were treated in two phases. In the first, intragastric doses of
10, 100 and 1000 mg/kg of bw of compounds were administered.
On the second, the doses were adjusted to 1600, 2900 and
5000 mg/kg of bw of compounds. In both phases, mice were
observed daily in a period of 14 days for mortality, toxic effects
and/or changes in behavioural pattern. At the end of the experi-
ments, the mice were sacrificed in a CO2 chamber.
The authors are grateful to support from the Project of Shandong
Province Higher Educational Science and Technology Program
(J14M02), the Science and Technology Research Program of
Shandong Academy of Medical Sciences (2014–4), Shandong
Provincial Natural Science Foundation (ZR2015YL041), and the
Innovation Project of Shandong Academy of Medical Sciences.
ORCID
Jie Sun
http://orcid.org/0000-0003-0552-9061
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