Synthesis and biological evaluation of 3-arylcoumarin derivatives as potential anti-diabetic agents

Article abstract of DOI:10.1080/14756366.2018.1518958

A variety of substituted 3-arylcoumarin derivatives were synthesised through microwave radiation heating. The method has characteristics of environmental friendliness, economy, simple separation, and purification process, less by-products and high reaction yield. Those 3-arylcoumarin derivatives were screened for antioxidant, α-glucosidase inhibitory and advanced glycation end-products (AGEs) formation inhibitory. Most compounds exhibited significant antioxidant and AGEs formation inhibitory activities. Anti-diabetic activity studies showed that compounds 11 and 17 were equipotent to the standard drug glibenclamide in vivo. According to the experimental results, the target compound 35 can be used as a lead compound for the development of new anti-diabetic drugs. The whole experiment showed that anti-diabetic activity is prevalent in 3-arylcoumarins, which added a new natural skeleton to the development of anti-diabetic active drugs.

Full text of DOI:10.1080/14756366.2018.1518958

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 3-
arylcoumarin derivatives as potential anti-diabetic
agents
Yuheng Hu, Bing Wang, Jie Yang, Teng Liu, Jie Sun & Xiaojing Wang
To cite this article: Yuheng Hu, Bing Wang, Jie Yang, Teng Liu, Jie Sun & Xiaojing Wang (2019)
Synthesis and biological evaluation of 3-arylcoumarin derivatives as potential anti-diabetic agents,
Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 15-30
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 34, NO. 1, 15–30
https://doi.org/10.1080/14756366.2018.1518958
RESEARCH PAPER
Synthesis and biological evaluation of 3-arylcoumarin derivatives as potential anti-
diabetic agents
Yuheng Hua,b,c,d, Bing Wanga,b,c,d, Jie Yanga,b,c,d, Teng Liua,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, Shandong, China; Institute of
c
MateriaMedica, Shandong Academy of Medical Sciences, Jinan, Shandong, China; Key Laboratory for Biotech-Drugs Ministry of Health, Jinan,
d
Shandong, China; Key Laboratory for Rare & Uncommon Diseases of Shandong Province, Jinan, Shandong, China
ABSTRACT
ARTICLE HISTORY
Received 10 July 2018
Revised 24 August 2018
Accepted 24 August 2018
A variety of substituted 3-arylcoumarin derivatives were synthesised through microwave radiation heating.
The method has characteristics of environmental friendliness, economy, simple separation, and purification
process, less by-products and high reaction yield. Those 3-arylcoumarin derivatives were screened for anti-
oxidant, a-glucosidase inhibitory and advanced glycation end-products (AGEs) formation inhibitory. Most
compounds exhibited significant antioxidant and AGEs formation inhibitory activities. Anti-diabetic activity
studies showed that compounds 11 and 17 were equipotent to the standard drug glibenclamide in vivo.
According to the experimental results, the target compound 35 can be used as a lead compound for the
development of new anti-diabetic drugs. The whole experiment showed that anti-diabetic activity is
prevalent in 3-arylcoumarins, which added a new natural skeleton to the development of anti-diabetic
active drugs.
KEYWORDS
3-Arylcoumarin; a-glucosi-
dase inhibitor; antidiabetic;
AGEs inhibitor
1. Introduction
significantly decreasing the blood glucose levels of genetically dia-
betic mice. Antioxidant compounds, its free radical scavenger
properties imply a protective effect against oxidative stress in the
early stages of diabetic nephropathy, leading to symptom relief
and improvement in organ function. Antioxidant compounds in
reducing diabetes-related neurotoxicity by the restoration of the
nervous fiber myelination with the normalisation of pain behav-
ior22. These antioxidants fully protected astrocytes from the cas-
pase 3 apoptotic signaling activation induced by toxic substance
and it is well known that glia has a pivotal role in neuropathy
development and astrocytes may be involved in the maintenance
Arylcoumarin is a class of naturally-occurring compounds, the
basic skeleton of which is arylbenzopyranones. It mainly includes
3-arylcoumarin and 4-arylcoumarin. Arylcoumarin was discovered
in abundance existence in some plants. Later on, scientists con-
ducted a lot of syntheses and activity experiments on the basis of
the natural structure of arylcoumarins by splitting and derivatisa-
tion1–3. Among them, the research on 3-arylcoumarins received
great attention. Studies have shown that arylcoumarins exhibit a
variety of activities, such as anti-cancer4, anti-leukemia5, anti-
coagulant, anti-allergic6, anti-inflammatory7, antioxidant8, anti-
of neuropathic pain23,24
.
microbial9–11
,
estrogen receptor affinity12, adenosine receptor
Umbelliferone (UMB), a derivative of coumarin, is a benzopyr-
one in nature, and it is present in the fruits of bitter orange
(Citrus aurantium)25. It has been reported that UMB has a certain
affinity7, and monoamine oxidase B inhibition activity13,14. Rich
pharmacological activity has stimulated interest in the research of
arylcoumarins. These compounds have similarities in chemical
structure with coumarins, flavones, isoflavones, and trans-stilbenes
and they are related to each other in the process of biosynthesis
in plants15. Therefore, they are highly regarded by researchers.
Isoflavones, the most abundant phytoestrogens in soy beans, are
structurally similar to 3-arylcoumarin derivatives. The compounds
have been found effective in the management of diabetes by act-
ing on peroxisome proliferator-activated receptors16. Researchers
found that resveratrol appears a good candidate as a neutraceuti-
cal support for the therapy of obesity and type II diabetes17,18. In
recent years, coumarins and flavonoids have received wide atten-
tion owing to their excellent advanced glycation end products
hypoglycemic activity26. Coumarin may be
a prodrug, and
7-hydroxycoumarin is the pharmacologically active agent27. UMB
is derivatised as 3-arylcoumarin and 4-arylcoumarin (Scheme 1).
Here we focus on the synthesis of 3-arylcoumarin and its pharma-
cological activity. This work has designed and synthesised a series
of compounds, and carried out a series of active screening around
the research of anti-diabetes.
2. Results and discussion
2.1. Chemistry
(AGEs) inhibitory and anti-inflammatory activities19,20
Kuroda et al.21 reported that 3-arylcoumarins have the effect of coumarin derivatives are summarised in (Scheme 2). Substituted
. Minpei The common synthetic strategies for the target compounds 3-aryl-
CONTACT Jie Sun
sunjie310@126.com; Xiaojing Wang
xiaojing6@gmail.com
Institute of MateriaMedica, Shandong Academy of Medical Sciences, Jinan,
Shandong, 250062, 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.

16
Y. HU ET AL.
Scheme 1. Umbelliferone is derivatised as 3-arylcoumarin and 4-arylcoumarin. They have inhibitory activity on a-glucosidase inhibitory activity and AGEs formation
inhibitory activity.
Scheme 2. General synthetic route to 3-arylcoumarin derivatives. (a) sulfur, p-toluene sulfonic acid, morpholine, 120 ꢀC; (b) NaOH (aq), Tetrabutylammonium bromide,
100 ꢀC; (c) Acetic anhydride, Et3N, 112ꢀC; HCl.
phenylacetic acid 2a–2k were synthesised with acetophenone
derivatives 1a–1k. The substituted phenylacetic acid and substi-
tuted salicylaldehyde 3a–3h were used as starting materials to
obtain the corresponding compounds 4–47 by Perkin reaction.
Details on the chemical and spectroscopic characterizations of com-
pounds 4-47 were described in the Supporting Information. In order
to confirm the optimal reaction conditions, 4-hydroxyphenylacetic
acid 2b with 2,4-dihydroxybenzaldehyde 3a were chosen as model
substrates. This paper screened different microwave power and
reaction time. It was found that this reaction had the highest yield
of 96% could be achieved at microwave power of 100 W reaction
time of 70min (Table 1). The previous reaction by heating in an oil
bath required a reaction for 6 h, and now the reaction time was
Table 1. The reaction conditions and yield.
Reaction time (min)
Yield (%)
50
60
70
80
90
Microwave power (W)
60
70
80
90
100
80
83
77
88
85
85
87
79
93
95
86
89
86
93
96
87
90
96
95
93
89
86
86
93
91
product can be obtained by a recrystallisation purification method
with a high yield (Table 2). The results showed that microwave
heating method had such advantages as low cost, low-toxicity, and
good commercial availability, rendering the synthetic process more
shortened to 70min by microwave heating. Some of the crude environmental friendly and economical.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
17
Our group has previously synthesised a series of 4-arylcoumar- (DPPH)28 and hydroxyl radical29. Vitamin C was used as a reference
ins (Scheme 3)2. 4-Arylcoumarin is more difficult to synthesise compound in this assay. As shown in Table 3, most compounds of
than 3-arylcoumarin and has more reaction steps. The synthesis of
4-arylcoumarin has certain guiding significance for the synthesis
of 3-arylcoumarin. In the following experiments and discussion,
we will compare the in vitro activities of 3-arylcoumarin and 4-
arylcoumarin with the same substituents sites.
4–47 demonstrated moderate to great scavenging activity, among
which compound 36 with several hydroxyl groups showed an excel-
lent activity in both aspects.
DPPH is widely used to evaluate antioxidant capacities of nat-
ural and synthetic products. Although the main skeleton of all
compounds is same, the slight difference in their inhibitory poten-
tial might result from the different substitution patterns on ben-
zene ring. As shown in Table 3, the number and position of
hydroxy groups were correlated with DPPH radical scavenging
activity. Generally, the increase of hydroxy groups and the
decrease of methoxygroups were positive to radical scavenging
capacity. Moreover, compounds with adjacent 7,8-dihydroxy
groups or 30,40-dihydroxy groups possessed a great capacity to
scavenge DPPH radical, especially compounds 15, 23, 30, 32–34,
36, and 37, which were better than positive reference substance
Vitamin C (IC50 ¼ 34.94 0.85 mM). These conclusions were similar
to reference [30].
2.2. Biological evaluation
2.2.1. In vitro antioxidant activity
All the synthesised compounds were evaluated for their antioxidant
activities in the way of scavenging 2,2-diphenyl-1-picrylhydrazyl
Table 2. Compounds 4–47.
Product
R1
R2
R3
R4
R5
R6
R7
R8
Yield (%)
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
OH
H
H
OH
F
CH3
CH3
H
H
OH
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
OH
H
H
OH
H
OH
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
OH
H
H
OH
H
H
H
OH
H
H
H
H
78.15
54.2
35.29
49.58
55.55
39.29
37.22
41.21
95.28
57.09
51.18
39.37
44.88
73.23
44.92
51.97
52.26
44.74
40.67
43.28
45.15
46.56
42.16
43.66
29.26
22.22
66.66
45.82
56.62
45.04
48.94
54.25
55.59
72.5
In addition, it can be found from the data that almost all com-
pounds with two adjacent hydroxy groups exhibit hydroxyl radical
scavenging activity. Especially, the IC50 values of compounds 35
and 36 were 245.74 11.87 and 268.68 41.26 mM, respectively,
H
OH
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
CH3
CH3
H
H
H
H
OH
H
H
H
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
H
OH
OH
H
OH
H
OH
H
H
which were threefolds of the value of Vitamin
C
OH
OH
H
H
H
OH
H
(IC50 ¼ 787.50 15.74 mM).
OH
F
F
H
2.2.2. In vitro a-glucosidase inhibitory activity31
a-Glucosidases, enzymes anchored in the brush border of the
small intestine, are responsible for catalyzing the hydrolysis of car-
bohydrates32. Their inhibitors were useful for the treatment of
type II diabetes mellitus33. Though all the compounds were tested
for a-glucosidase inhibitory activity in vitro, as shown in Table 3,
only eight compounds (11, 13, 16, 17, 25, 28, 35, and 39) pre-
sented moderate to excellent inhibitory activity for a-glucosidase.
Notably, 28 (IC50 ¼ 1.37 0.67 mM) had relatively strong activity,
which was a little weaker than acarbose (IC50 ¼ 0.05 0.003 mM).
The 3-arylcoumarin with 7-hydroxyl group showed good inhibitory
activity, indicating that 7-hydroxyl group was very important for
inhibiting a-glucosidase. Comparison of the IC50 values, 4 > 13
and 12 > 28, showed that 3-arylcoumarin with 7-hydroxyl group
was weaker than that with 5,7-dihydroxy groups in meta-position.
Comparison of the IC50 values, 12 > 17 > 28, showed that 3-aryl-
coumarin with 7-hydroxyl group was weaker than that with 6-
hydroxyl group. However, the 3-arylcoumarin with 6-hydroxyl
group was weaker than that of 3-arylcoumarin with 5,7-dihydroxy
groups in meta-positi on in a-glucosidase inhibitory activity.
Comparison of the IC50 values, 13 > 25 > 35 > 28, showed that 3-
arylcoumarin with 40-hydroxy was stronger than that with 40-
methoxy and 40-methyl in a-glucosidase inhibitory activity and the
inhibitory activity of 40-methyl and 40-methoxy group was similar
but stronger than that of hydrogen. After compound 35 was
H
CH3 OH
OCH3
CH3
OCH3
CH3
OH
OH
OH
OH
OH
F
F
H
OCH3
OCH3
OH
H
H
H
H
H
H
H
H
H
H
H
OH
OH
H
H
H
H
H
H
OH
H
H
OH
OH
OH
OCH3
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
OH
CH3 OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
OH
H
OH
H
H
H
H
OH
H
OH
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
H
H
OCH3 OCH3
OCH3 OCH3
H
H
H
H
H
OH
H
H
H
H
H
H
H
32.21
69.82
53.32
26.51
96.18
50.64
56.43
55.52
52.15
54.65
H
H
Br
OH
H
H
OH
H
H
H
OH
H
Et2N
OH
OH
OH
H
OH
OH
Br
Br
Br
Br
H
H
H
H
OH
H
Scheme 3. The main synthesis step of 4-arylcoumarin derivatives. (d) montmorillonite K-10, nitrobenzene, 100 ꢀC; (e) I2, Al, acetonitrile.

18
Y. HU ET AL.
Table 3. Biological evaluation In vitro.
Product
DPPH
OH
a-Glucosidaseinhibitory activity
AGEs inhibitory activity
IC50 Value (lM)
>1000
4
5
6
7
76.85 1.30
>1000
>1000
25.67 0.50
280.38 74.79
60.88 0.55
>1000
21.81 3.07
48.36 3.53
>1000
>1000
>1000
>1000
>1000
>1000
8
9
>1000
60.20 1.27
>1000
>1000
>1000
>1000
69.60 5.32
118.29 41.47
>1000
31.86 2.02
>1000
>1000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Vitaminc
Acarbose
AG
564.21 5.20
756.42 8.31
268.98 0.04
167.05 2.01
2.01 0.08
>1000
>1000
>1000
>1000
>1000
16.39 1.23
19.91 3.39
13.46 0.71
212.72 40.35
29.05 2.87
10.16 0.75
11.54 1.30
86.91 1.17
>1000
>1000
1.42 0.04
14.92 0.20
45.24 5.28
47.60 1.93
20.47 0.20
24.68 0.12
20.59 0.39
34.73 1.84
>1000
212.97 4.77
23.06 0.82
75.93 10.75
>1000
45.75 1.27
65.89 1.34
64.55 0.78
2.52 0.41
7.48 0.59
>1000
3.12 0.33
33.49 1.99
2.91 0.11
>1000
5.21 0.07
14.16 1.33
4.23 0.23
22.28 1.34
8.02 0.20
12.99 0.40
>1000
18.31 0.38
25.22 1.72
7.51 0.38
>1000
996.24 8.03
>1000
163.23 7.13
>1000
>1000
>1000
>1000
>1000
>1000
>1000
39.14 3.83
>1000
>1000
>1000
>1000
>1000
15.63 0.11
3.21 0.04
53.92 1.83
603.32 7.42
68.69 13.36
42.01 0.97
41.51 0.18
11.85 0.04
2.18 0.04
595.18 11.29
2.06 0.07
2.98 0.03
3.66 0.03
154.61 10.49
1.33 0.03
173.42 42.58
16.28 0.44
>1000
800.82 41.83
>1000
25.48 0.52
27.42 0.11
11.49 0.37
>1000
70.26 2.95
1.37 0.15
29.89 5.15
19.04 1.11
19.08 0.26
>1000
>1000
>1000
>1000
>1000
981.55 30.04
696.52 19.59
>1000
969.74 70.85
903.16 41.88
>1000
270.14 41.55
244.02 11.78
>1000
>1000
18.80 1.02
10.81 0.63
247.34 36.22
>1000
20.23 0.44
13.09 0.57
>1000
13.43 0.65
39.08 0.76
>1000
>1000
>1000
>1000
>1000
>1000
>1000
5.89 0.06
4.24 0.06
>1000
>1000
886.85 15.82
>1000
595.87 28.77
>1000
688.44 49.70
787.50 15.74
>1000
>1000
>1000
35.71 0.48
487.82 95.05
>1000
2.37 0.06
19.60 1.88
47.75 0.15
298.83 40.15
0.050 0.003
269.87 7.19
Each value represents the mean SD (n ¼ 3).
converted to 28 via demethylation, its IC50 value decreased from 10.00 mM, and the inhibitory effect was over 10 times compared
10.81 0.63 mM to 1.37 0.67 mM. This suggests that the 40-
hydroxyl group is another active site in a-glucosidase inhibitory
with the positive control. When the 3-arylcoumarin both have the
40-hydroxy groups, those compounds with 7-hydroxy group were
better than those with 5,7-dihydroxy groups and which were
stronger than those with 6-hydroxy group. The 40-fluoro group
had substantially no effect on the activity compared with the 40-
hydroxy group, but 40-bromo group decreased the inhibitory activ-
ity. Comparison of the IC50 values, 35 > 28, showed that 3-arylcou-
marin with 40-hydroxy is stronger than that with 40-methoxy and
40-methyl in AGEs formation inhibitory activity.
Our research group has conducted in-depth studies on the syn-
thesis and hypoglycemic activity of 4-arylcoumarin compounds.
Those 4-arylcoumarin compounds were screened for antioxidant,
a-glucosidase inhibitory, aldose reductase 2 inhibitory and AGE
formation inhibitory effects2. The a-glucosidase inhibitory activity
and AGEs formation inhibitory activity of 3-arylcoumarin and 4-
activity. According to in vitro a-glucosidase inhibition test results,
we decided to study in vivo hypoglycemic activity of com-
pounds11, 17, 25, 28, and 35.
2.2.3. In vitro inhibitory activity of AGEs formation34
AGEs are a group of complex and heterogeneous compounds,
which are implicated in a number of biochemical abnormalities
associated with diabetes35. Therefore, the discovery of AGEs
inhibitory activity would be beneficial to the prevention and treat-
ment of diabetic or other pathogenic complications. The results
listed in Table 3 displayed that most of the target compounds
presented better inhibitory activity to AGEs, even better than
amino guanidine (AG) (IC50 ¼ 269.87 7.19 mM). The IC50 values of arylcoumarin substituted at the same position (Table 4). In sum-
compounds 12, 28, 33, 31, 37, 39, and 44 were less than mary, 3-arylcoumarin substituted at the same position is superior

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
19
Table 4. Comparison of in vitro activity of 3-arylcoumarin and 4-arylcoumarin.
3-arylcoumarin 4-arylcoumarin
Activity comparison
IC50 Value(μM)
G a: 27.42 0.11
>
b: 1000
>
A a: 1000
>
b: 1000
G a: 29.89 5.15
>
b: 1000
A a: 7.48 0.59
b: 93.37 4.31
G a: 19.04 1.11
b: > 1000
A
a: > 1000
b: > 1000
G a: 10.81 0.63
b: 16.11 2.10
A a: 5.21 0.07
b: > 1000
G a: 39.08 0.76
b: > 1000
A a: 18.31 0.38
b: 20.50 1.44
a: 3-arylcoumarin; b: 4-arylcoumarin; G: a-glucosidase inhibitory activity; A: AGEs formation inhibitory activity.
to 4-arylcoumarin in terms of a-glucosidase inhibitory activity and 2.2.4. Oral toxicity to mice
With reference to Lorke’s method36, kunming mice were used as
targets to estimate the oral toxicity of each compound to mice.
We select compounds 11, 17, 25, 28, and 35 at concentrations of
10, 100, and 1000 mg/kg to test their oral toxicity in the first
phase, and 1600, 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.
AGEs formation inhibitory activity. Therefore, we have studied the
synthesis and preliminary activity of 3-arylcoumarin with good
practical significance.
The 3-arylcoumarin derivatives are structurally similar to the
trans-stilbene and isoflavone compounds. Therefore, we have
selected synthetic compounds 28 and resveratrol, a representative
compound of trans-stilbene, genistein a representative of isofla-
vone compound, to compare a-glucosidase inhibitory activity and
AGEs formation inhibitory activity (Table 5). The number and loca-
tion of the hydroxyl groups of these three compounds were basic-
ally the same. We found that compound 28 we synthesised is
superior to the other two compounds in both a-glucosidase
inhibitory activity and AGEs formation inhibitory activity. It can be
seen that the closed pyrone ring can enhance a-glucosidase inhib-
ition and AGEs formation inhibitory activity. The 3-arylcoumarin
2.2.5. Acute hypoglycemic assay
The anti-diabetic activity was determined by using a standard
compound having a trans-stilbene structure is superior to the method31. As shown in Table 6, the target compounds 11, 17, 25,
trans-stilbene structured isoflavone.
28, and 35 (10 mg/kg, 30 mg/kg, and 100 mg/kg of body weight

20
Y. HU ET AL.
Table 5. Compound 28 resveratrol and genistein have a comparative effect on a-glucosidase inhibitory activity and AGEs for-
mation inhibitory activity.
α-glucosidase inhibition IC50
Value(μM)
AGEs formation inhibitory
activity IC50 Value(μM)
Compound
structure
Compound 28
19.08 0.26
3.12 0.33
Resveratrol
Genistein
3.64 0.92
> 1000
724.11 39.74
11.04 1.15
Table 6. Acute effect of compounds 11, 17, 25, 28 and 35 on blood glucose levels in STZ-diabetics mice.
Blood glucose concentration(mM)
Test samples
Dose(per os) mg/kg of bw
0h
1.5h
3h
5h
7h
9h
Control (vehicle)
–
24.7 5.8
22.7 5.4
15.9 4.9
(ꢂ35.63)
6.9 1.6
(ꢂ57.14)
15.5 8.5
(ꢂ37.75)
16.5 6.4
(ꢂ34.78)
15.3 3.9
(ꢂ34.89)
22.8 2.3
(ꢂ15.24)
11.6 4.8
(ꢂ50.22)
12.5 4.7
(ꢂ46.58)
14.9 6.5
(ꢂ24.37)
15.8 4.5
(ꢂ36.23)
17.2 2.9
(ꢂ25.36)
11.4 4.2
(ꢂ25.49)
13.5 6.1
(ꢂ25.82)
13.0 6.6
(ꢂ28.57)
10.7 5.7
(ꢂ47.81)
10.5 5.1
(ꢂ51.39)
10.5 4.9
(ꢂ48.28)
15.4 6.5
(ꢂ37.79)
6.7 2.5
(ꢂ55.90)
12.8 10.4
(ꢂ48.59)
11.3 4.0
(ꢂ55.41)
11.1 4.4
(ꢂ52.76)
16.6 5.8
(ꢂ38.29)
8.3 3.8
(ꢂ64.38)
7.6 3.8
(ꢂ67.52)
9.7 3.4
(ꢂ50.76)
8.9 6.3
(ꢂ64.11)
9.9 2.7
(ꢂ56.95)
8.9 2.6
(ꢂ41.83)
12.7 6.7
(ꢂ30.22)
11.0 6.4
(ꢂ39.56)
14.9 7.1
(ꢂ39.88)
6.5 2.2
(ꢂ59.63)
11.4 8.2
(ꢂ54.22)
9.5 3.9
(ꢂ62.40)
8.9 2.8
(ꢂ62.21)
14.8 8.0
(ꢂ44.98)
7.4 6.7
(ꢂ68.24)
6.9 2.6
(ꢂ70.51)
6.9 3.9
(ꢂ64.97)
9.0 4.8
(ꢂ63.71)
8.1 1.6
(ꢂ64.78)
6.4 1.6
(ꢂ58.17)
10.3 5.6
(ꢂ43.41)
11.8 6.0
(ꢂ35.17)
15.8 5.3
(ꢂ8.23)
(ꢂ35.97)
ꢃ
ꢃꢃ
Glibenclamide
10
10
16.9 4.3
24.9 4.1
25.3 6.3
23.5 5.4
26.9 3.8
23.3 3.2
23.4 5.7
19.7 6.1
24.8 4.9
23.0 4.8
15.3 3.8
18.2 6.7
18.2 4.1
20.5 7.1
21.6 4.4
20.3 4.4
7.2 4.0
4.9 2.5
(ꢂ70.81)
11.9 8.5
(ꢂ52.21)
8.1 4.2
(ꢂ68.14)
8.2 2.6
(ꢂ65.27)
14.7 9.5
(ꢂ45.35)
(ꢂ55.28)
20.6 5.9
(ꢂ17.27)
20.0 6.3
(ꢂ20.95)
18.3 3.9
(ꢂ21.88)
24.5 3.0
(ꢂ8.92)
11
11
11
17
17
17
25
25
25
28
28
28
35
35
35
30
100
10
ꢃ
30
17.9 5.7
(ꢂ23.18)
18.8 6.0
(ꢂ19.66)
15.7 6.6
(ꢂ20.30)
21.7 3.9
(ꢂ12.50)
18.3 3.5
(ꢂ20.44)
14.8 5.8
(ꢂ3.27)
6.9 7.9
(ꢂ70.39)
8.0 2.3
(ꢂ65.81)
8.5 2.2
(ꢂ56.85)
8.9 3.8
(ꢂ64.11)
9.1 2.3
(ꢂ60.43)
7.0 2.4
(ꢂ54.25)
7.3 4.5
(ꢂ59.89)
10.2 4.6
(ꢂ43.96)
100
10
30
100
10
30
14.9 6.6
(ꢂ18.13)
17.3 6.7
(ꢂ4.95)
100
10
ꢃ
ꢃ
ꢃ
ꢃ
13.1 6.4
(ꢂ36.10)
5.6 3.1
5.4 3.4
5.7 3.1
(ꢂ72.68)
6.3 4.3
(ꢂ70.83)
7.2 2.6
(ꢂ64.53)
(ꢂ73.66)
(ꢂ72.20)
ꢃ
ꢃ
ꢃ
30
12.0 3.7
(ꢂ44.44)
15.1 5.0
(ꢂ25.62)
5.7 6.7
5.5 5.2
(ꢂ73.61)
7.2 3.9
(ꢂ64.53)
(ꢂ74.54)
ꢃ
100
6.4 2.0
(ꢂ68.47)
Each value is the mean SEM for six mice in each group.
ꢃ
p < 0.05 significantly different ANOVA followed by Dunnett’s t-test for comparison with respect to initial levels in each group.
% Variation of glycemia are in parentheses.
[bw]) caused decreases in blood glucose levels in streptozotocin positive control at 5, 7, and 9 h. In STZ-diabetic animals, the hypo-
(STZ)-diabetic mice compared with normal mice (Table 7). glycemic effect of 17 and 35 (30 mg/kg of bw) was larger than
Especially, 35 (30 mg/kg of bw) caused significant decreases in 50% started from 3 h and persisted throughout the experiment
blood glucose levels when compared with vehicle-treated groups (Table 6). The highest anti-hyperglycemic effect of 35 was
(p < 0.05). The target compound 35 (10 mg/kg of bw) caused sig- observed at doses of 10 and 30 mg/kg at 7 h (ꢁ73.66%) and 9 h
nificant decreases in blood glucose levels even better than (ꢁ74.54%), respectively (Table 6). In diabetic animals, the highest

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
21
Table 7. Acute effect of compounds 11, 17 and 35 on blood glucose levels in normal mice.
Blood glucose concentration(mM)
Dose (per os)
Test samples
mg/kg of bw
0h
1.5h
3h
5h
7h
9h
Control (vehicle)
–
10.9 1.1
9.2 0.3
8.6 0.5
8.3 0.3
7.8 0.5
7.3 0.4
(ꢂ15.60)
(ꢂ21.10)
(ꢂ24.16)
(ꢂ28.75)
(ꢂ33.33)
ꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
4.1 0.7
Glibenclamide
10
9.8 1.5
10.0 0.7
10.2 1.2
10.4 0.7
10.6 0.4
10.8 0.3
10.4 0.2
10.8 0.6
10.5 0.9
10.6 0.4
6.4 1.4
(ꢂ34.58)
9.1 0.2
(ꢂ15.27)
8.7 1.2
(ꢂ13.62)
7.0 1.1
(ꢂ31.37)
7.9 0.7
(ꢂ23.96)
8.4 1.0
(ꢂ18.39)
7.5 1.2
(ꢂ25.90)
9.2 0.6
(ꢂ14.55)
8.7 1.0
(ꢂ17.72)
8.7 0.5
(ꢂ18.18)
5.8 0.8
(ꢂ41.36)
7.7 0.6
(ꢂ28.35)
6.9 1.1
(ꢂ30.90)
5.0 1.4
(ꢂ49.15)
6.6 0.5
(ꢂ38.01)
4.3 0.6
(ꢂ56.61)
(ꢂ58.31)
ꢃꢃ
ꢃꢃ
11
11
11
17
17
17
35
35
35
10
5.6 0.4
5.5 1.2
(ꢂ48.91)
(ꢂ47.98)
ꢃ
ꢃꢃ
ꢃꢃ
5.1 0.6
30
5.9 1.1
5.2 0.8
(ꢂ40.86)
(ꢂ47.84)
(ꢂ49.17)
ꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
4.3 0.3
100
10
5.9 0.6
(ꢂ42.16)
6.9 0.8
(ꢂ34.19)
6.9 0.9
(ꢂ33.55)
4.8 1.3
4.7 0.4
(ꢂ52.94)
(ꢂ54.25)
(ꢂ58.17)
ꢃꢃ
ꢃꢃ
ꢃꢃ
5.1 0.4
5.7 0.7
5.4 0.5
(ꢂ45.37)
(ꢂ48.56)
(ꢂ51.12)
ꢃꢃ
ꢃꢃ
ꢃꢃ
4.5 0.6
30
5.3 0.7
5.3 1.1
(ꢂ48.71)
(ꢂ48.71)
(ꢂ56.45)
ꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
100
10
6.3 0.9
(ꢂ37.71)
8.2 0.5
(ꢂ24.15)
7.2 0.8
(ꢂ31.65)
5.2 0.5
(ꢂ49.18)
8.0 0.4
(ꢂ25.70)
6.4 1.0
(ꢂ39.24)
4.7 0.4
(ꢂ53.44)
7.4 0.5
(ꢂ31.27)
4.3 0.4
(ꢂ58.03)
7.1 0.3
(ꢂ34.06)
5.9 0.6
(ꢂ44.30)
ꢃꢃ
30
5.8 0.7
(ꢂ44.94)
ꢃ
ꢃ
ꢃꢃ
ꢃꢃ
100
6.3 1.0
(ꢂ40.44)
5.4 0.6
4.8 0.6
4.6 0.7
(ꢂ57.05)
(ꢂ49.22)
(ꢂ55.17)
Each value is the mean SEM for six mice in each group.
ꢃ
p < 0.05 significantly different ANOVA followed by Dunnett’s t-test for comparison with respect to initial levels in each group.
% Variation of glycemia are in parentheses.
anti-hyperglycemic effect of 17 was observed at doses of 30 and caused significant decrease in the postprandial glycemia peak in
both normal (data not shown) and STZ-diabetic mice. The oral
glucose tolerance effect of the target compound 35 (30 mg/kg of
bw) was stronger than that of the glibenclamide (10 mg/kg of bw)
100 mg/kg at 9 h (ꢁ70.39%) and 7 h (ꢁ70.51%), respectively (Table
6). The target compounds 11 and 17 showed similar anti-diabetic
activity throughout the experiment. Glibenclamide (10 mg/kg of
bw), used as a positive control, showed maximum hypoglycemic
effect at 9 h in STZ-induced diabetic mice (Table 6). The adminis-
tration of the target compounds 11, 17, and 35 (10 mg/kg, 30 mg/
kg, and 100 mg/kg of bw) showed significant decreases of blood
glucose levels in normoglycemic mice (Table 7).
used as
a
positive control throughout the experiment.
Compounds 17 and 35 decreased blood glucose levels to below
positive control group the values of the end of the experiment
(Figure 3).
3. Conclusions
2.2.6. Effects of daily treatment with compounds 11, 17, and 35 in
STZ-induced diabetic mice
Forty-four 3-arylcoumarin derivatives were synthesised by using
microwave reactor heating method. Compared with traditional
methods, this method is more environmental friendly, more sus-
tainable and economical, more convenient in isolation and purifi-
cation processes owing to less byproducts, and the yield is
relatively higher than traditional methods. We then studied the
pharmacological activity of the synthesised compounds. All the
synthesised compounds were evaluated for their antioxidant activ-
ities in the way of scavenging DPPH and hydroxyl radical. Most
compounds of 4–47 demonstrated moderate to great scavenging
activity, among which compound 36 with several hydroxyl groups
showed an excellent activity in both aspects.
The long-term anti-hyperglycemic effect of the target compounds
11, 17, and 35 was tested by using a classical chronic experiment
with STZ-induced diabetic mice37. The results demonstrated that
daily oral administration of the target compounds 17 and 35
(30 mg/kg of bw each time), once a day, for 16 days, induced obvi-
ously anti-hyperglycemic effect in the STZ-diabetic mice (Figure 1).
The target compound 11 was less efficient in decreasing blood
glucose levels in diabetic mice (Figure 1). Compounds 35 restored
blood glucose levels to near normal values at the end of the
experiment (Figure 1) (16 days) and the effect was equipotent to
that of the glibenclamide which was used as a positive control38
The treatments with the target compounds 11, 17, and 35 also
.
Compounds 12, 28, and 35 showed significant effect on the
treatment of diabetic complications. Notably, 17, 28, and 35 had
prevented bw loss in hyperglycemic mice (Figure 2) and the effect relatively strong activity, which displayed a little weaker capacity
than acarbose. Oral toxicity tests indicated their innocuousness for
mice. We selected compounds 11, 17, 25, 28, and 35 to investi-
gate their anti-diabetic activity in vivo. June Hyuk Jang et al.40
reported acarbose as a positive control, the effects of scopoletin
on hyperglycemia following a meal were investigated in normal
mice and mice with STZ-induced diabetes. Treatment of mice with
antioxidants viz., Vitamin C and taurine resulted in only a marginal
to moderate protection against STZ-induced elevation in the
plasma glucose levels41. Jeong-Ho Oak et al.42 reported that
was equipotent to that of glibenclamide. Furthermore, 17 and 35
were more efficient than 11 in preventing bw loss in hypergly-
cemic mice (Figure 2).
2.2.7. Oral glucose tolerance test of compounds 11, 17, and 35 on
in STZ-induced diabetic mice
The oral glucose tolerance test was determined by using a stand-
ard method39. Compared with vehicle-treated group (p < 0.05)
(Figure 3), the target compounds 17 and 35 (30 mg/kg of bw) AG (100 mg/kg/day) treatment had no significant effect on

22
Y. HU ET AL.
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
0.00
0
2
4
6
8
10
12
14
16
18
Time(Days)
Nomal Control (vehicle)
Diabetic Control (vehicle)
11 (30mg/kg)
Glibenclamide (10mg/kg)
35 (30mg/kg)
17 (30mg/kg)
Figure 1. Long-term effect of the compounds 11, 17 and 35 on blood glucose levels in STZ-diabetic mice. Each value is the mean SEM for six mice in each group.
ꢃ
p < 0.05 significantly different ANOVA followed by Dunnett’s t-test for comparison with the diabetic control group at same time.
4.5
*
4
3.5
*
*
*
3
2.5
2
*
*
1.5
1
0.5
0
Normal
Diabetes
Glibenclamide
11
17
35
Figure 2. Body weight changes of daily treatment with compounds 11, 17 and 35 in STZ-induced diabetic mice. Each value is the mean SEM for six mice in each
ꢃ
group. p < 0.05 significantly different ANOVA followed by Dunnett’s t-test vs. the diabetic control group at same time.
STZ-induced hyperglycemic mice. The results showed the effect of 13CNMR spectra were recorded with a Bruker AM-600 spectrom-
the target compounds 17 and 35 were equipotent to that of the eter (Billercia, MA) with TMS as the internal standard. Chemical
glibenclamide used as a positive control in vivo. In conclusion, the shifts were reported at room temperature on a scale (ppm) with
target compound 35 offered a potential drug design concept for 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
Hitachi U-3000 UV spectrophotometer (Tokyo, Japan).
Columnchromatography was performed on silica gel (200–300
mesh). Unless otherwise noted, all solvents and reagents were
commercially available and used without further purification.
the development of therapeutic or preventive agents 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
4.1.2. General method for synthesis of compounds 2a-k
uncorrected. The FT-IR spectra were recorded using a Thermo- A solution of sulfur (1.28 g, 40 mmol) and p-toluenesulfonic acid
Nicolet Nexus 670 spectrometer with KBr pellets. The 1HNMR and (0.1 g, 0.6 mmol) in morpholine (6 ml, 60 mmol). Compound 1a

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
23
100
80
60
40
20
0
0
0.5
1
1.5
2
2.5
3
3.5
−20
−40
−60
Time (h)
Diabetic Control (vehicle)
Glibenclamide (10 mg/kg)
11 (30 mg/kg)
17 (30 mg/kg)
35 (30 mg/kg)
Figure 3. Oral glucose tolerance test of compounds 11, 17, and 35 on in STZ-induced diabetic mice. Each value is the mean SEM for six mice in each group.
ꢃ
p < 0.05 significantly different ANOVA followed by Dunnett’s t-test vs. the diabetic control group at same time.
(20 mmol) was added and the mixture was heated in an oil bath J ¼ 2.2 Hz, 2H) and 6.37 (t, J ¼ 2.2 Hz, 1H). 13CNMR (150 MHz,
at 120 ꢀC for 8–10 h. Methanol (30 ml) and activated carbon (0.1g) DMSO-d6) d (ppm): 161.11, 157.85, 140.08, 129.43, 128.43, 128.40,
were added and the mixture was heated in an oil bath for 5 min 125.62, 116.03, 104.52, 99.75. MS: m/z(%): 238.9[M þ 1]þ, 182.9
8-Hydroxy-3-arylcoumarin (5). White solid, yield 54%, m.p.
199.8–200.1 ꢀC. IR (KBr, ꢀ, cmꢂ1):3201(OH); 1681(C ¼ O); 1607, 1572,
1470, 1136, 958(Ar).1HNMR (600 MHz, DMSO-d6) d(ppm): 10.24 (s,
1H), 8.20 (s, 1H), 7.73 (d, J ¼ 7.7 Hz, 2H), 7.46 (t, J ¼ 7.4 Hz, 2H),
7.42 (d, J ¼ 7.2 Hz, 1H), 7.18 (dt, J ¼ 15.4, 7.6 Hz, 2H), 7.10 (d,
J ¼ 7.7 Hz, 1H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 160.11,
144.80, 142.14, 141.52, 135.22, 128.99, 128.68, 127.18, 125.03,
120.90, 119.10, 118.48. MS: m/z(%): 238.9[M þ 1]þ, 210.8,
182.8, 164.7.
then allowed to cool to room temperature. Ethanol (30 ml, 70%)
and NaOH solution (3 ml, 50%) were added to the mixture. After
being stirred and heated to reflux for 4 h, the resulting mixture
was quenched by the addition of 100 ml water, later the solution
was acidified to pH 3–4 with concentrated HCl and precipitated
crystals. The precipitate was filtrated and the filter cake was
washed with water to give phenylacetic acid 2a (1.99 g, 73%
yield).
Compounds
2d–k
were
obtained
using
the
same procedures.
3-(4-Hydroxyphenyl)-coumarin (6). White solid, yield 35%,
m.p.202.3–203.3 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3211(OH); 1688(C ¼ O); 3065,
1610, 1517, 1453, 1118, 957, 814 (Ar).1HNMR (600 MHz, DMSO-d6)
d(ppm): 9.76 (s, 1H), 8.14 (s, 1H), 7.75 (dd, J ¼ 7.7, 1.5 Hz, 1H), 7.60
(m, 2H), 7.58 (m, 1H), 7.41 (d, J ¼ 8.3 Hz, 1H), 7.36 (td, J ¼ 7.6,
1.0 Hz, 1H), 6.84 (m, 2H). 13CNMR (150 MHz, DMSO-d6) d (ppm):
160.37, 158.47, 153.09, 138.97, 131.59, 130.31, 128.79, 127.19,
125.69, 124.98, 120.18, 116.21, 115.53. MS: m/z(%): 239.0
[M þ 1]þ, 182.8.
4.1.3. General method for the synthesis of 3-arylcoumarin 4–47
Compound 2a (1.36 g, 10 mmol) and compound 3a (1.54 g,
10 mmol) were added to triethylamine (5.56 g, 55 mmol) and acetic
anhydride (6.12 g, 60 mmol) and then the mixture was heated by
microwave reactor and refluxed for 70 min. The resulting mixture
was quenched by the addition of water, and a large amount of
solid was precipitated. The resulting mixture was quenched by the
addition of water, and a large amount of solid was precipitated.
The filtrate was vacuum filtered and the resulting solid was
washed three times with water .The obtained solid was dissolved
in 10 ml of absolute ethanol, and then reacted by adding hydro-
chloric acid (40 ml, 20%) at 80 ꢀC for 3 h (TLC monitoring). The
reaction solution was poured into 50 ml of ice water and stirred to
precipitate a large amount of light yellow solid. After standing
and filtration, the solid was washed with water until the pH of the
wash solution was nearly neutral. The solid was recrystallised from
3-(4-Fluorophenyl)-coumarin (7). White solid, yield 50%,
m.p.196.7–197.7 ꢀC. IR (KBr, ꢀ, cmꢂ1): 1696(C ¼ O); 3058, 1604,
1513, 1455, 1128, 951, 824, 815 (Ar); 1093(C-F).1HNMR (600 MHz,
DMSO-d6) d(ppm): 8.27 (s, 1H), 7.79 (m, 3H), 7.64 (t, J ¼ 7.8 Hz, 1H),
7.45 (d, J ¼ 8.3 Hz, 1H), 7.39 (t, J ¼ 7.5 Hz, 1H), 7.32 (t, J ¼ 8.8 Hz,
2H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 163.51, 161.88, 160.20,
153.43, 141.05, 132.24, 131.52, 131.50, 131.24, 131.18, 129.13,
126.33, 125.12, 119.92, 116.36, 115.69, 115.55. MS: m/z(%):
241.1[M þ 1]þ, 184.9, 164.8.
ethanol/water to obtain compound
4 (1.86 g, 73% yield).
8-Hydroxy-3-(4-methylphenyl)-coumarin (8). White solid, yield
56%, m.p.211.7–212.5 ꢀC, IR (KBr, ꢀ, cmꢂ1): 3220(OH); 1686(C ¼ O);
1607, 1576, 1514, 1470, 1132, 950, 834 (Ar).1HNMR (600 MHz,
Compounds 5–47 were obtained using the same procedures.
7-Hydroxy-3-arylcoumarin (4). White solid, yield: 78%, m.p.
201.2–203.3 ꢀC, IR (KBr, ꢀ, cmꢂ1): 3225(OH); 1716(C ¼ O); 3057, DMSO-d6) d(ppm): 10.22 (s, 1H), 8.17 (s, 1H), 7.64 (d, J ¼ 8.0 Hz,
1610, 1580, 1446, 988, 842 (Ar).1HNMR (600 MHz, DMSO-d6) 2H), 7.27 (d, J ¼ 8.0 Hz, 2H), 7.18 (m, 2H), 7.10 (m, 1H), 2.36 (s, 3H).
d(ppm): 9.59 (s, 1H), 7.42 (d, J ¼ 8.6 Hz, 2H), 7.16 (d, J ¼ 16.4 Hz, 13CNMR (150 MHz, DMSO-d6) d (ppm): 160.15, 144.77, 142.03,
1H), 6.94 (d, J ¼ 16.4 Hz, 1H), 6.77 (d, J ¼ 8.6 Hz, 2H), 6.72 (d, 140.82, 138.51, 132.30, 129.25, 128.83, 127.04, 124.98, 120.96,

24
Y. HU ET AL.
119.00, 118.32, 21.31. MS: m/z(%): 253.0[M þ 1]þ, 224.8, 113.27, 113.26. MS: m/z (%): 254.8[M þ 1]þ, 226.7, 198.7,
196.8, 178.8.
184.7, 132.7.
7-Hydroxy-3-(4-methylphenyl)-coumarin (9). White solid, yield
7-Hydroxy-3-(2-hydroxyphenyl)-coumarin (16). White solid,
39%, m.p.242.9–244.1 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3236(OH); 1692(C ¼ O); yield 45%, m.p.200.3–202.3 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3202(OH);
1591, 1512, 1451, 1123, 943, 819 (Ar).1HNMR (600 MHz, DMSO-d6) 1676(C ¼ O); 1648, 1582, 1518, 1449, 1136, 959, 852(Ar). 1HNMR
d (ppm): 10.59 (s, 1H), 8.12 (s, 1H), 7.60 (m, 3H), 7.25 (d, J ¼ 7.9 Hz, (600 MHz, DMSO-d6) d (ppm): 10.53 (s, 1H), 9.49 (s, 1H), 7.90 (s,
2H), 6.82 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.76 (d, J ¼ 2.3 Hz, 1H), 2.34 (s, 1H), 7.56 (d, J ¼ 8.5 Hz, 1H), 7.24 (d, J ¼ 7.3 Hz, 1H), 7.20 (t,
3H).13CNMR (150 MHz, DMSO-d6) d (ppm): 161.56, 160.59, 155.27, J ¼ 7.7 Hz, 1H), 6.90 (d, J ¼ 8.1 Hz, 1H), 6.84 (t, J ¼ 7.4 Hz, 1H), 6.81
140.94, 137.91, 132.66, 130.35, 129.22, 128.57, 122.57, 113.82, (dd, J ¼ 8.5, 2.1 Hz, 1H), 6.76 (d, J ¼ 1.9 Hz, 1H). 13CNMR (150 MHz,
112.52, 102.16, 21.28. MS: m/z(%): 253.0[M þ 1]þ, 224.8, DMSO-d6) d (ppm): 161.36, 160.24, 155.54, 155.44, 142.86, 131.35,
196.8, 181.8.
130.07, 129.76, 123.07, 121.71, 119.13, 116.11, 113.58, 112.24,
8-Hydroxy-3-(2-methylphenyl)-coumarin (10). White solid, 102.26. MS: m/z (%): 255.0[M þ 1]þ, 226.9, 198.8, 164.7, 132.7.
yield 37%, m.p.212.7–213.0 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3205(OH);
6-Hydroxy-3-(4-hydroxyphenyl)-coumarin (17). White solid,
1677(C ¼ O); 3058, 1612, 1471, 1142, 1060, 959 (Ar).1HNMR yield 73%, m.p.274.8–275.6 ꢀC. IR (KBr, ꢀ, cmꢂ1):3265(OH);
(600 MHz, DMSO-d6) d (ppm): 10.25 (s, 1H), 7.98 (s, 1H), 7.30 (m, 1686(C ¼ O); 1606, 1514, 1460, 1132, 975, 812 (Ar).1HNMR
4H), 7.18 (t, J ¼ 6.8 Hz, 2H), 7.13 (dd, J ¼ 6.9, 2.4 Hz, 1H), 2.22 (s, (600 MHz, DMSO-d6) d (ppm): 9.72 (d, J ¼ 4.7 Hz, 2H), 8.05 (s, 1H),
3H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 159.84, 144.93, 143.20, 7.58 (m, 2H), 7.25 (d, J ¼ 8.9 Hz, 1H), 7.07 (d, J ¼ 2.8 Hz, 1H), 7.00
142.44, 136.87, 135.56, 130.42, 130.39, 128.92, 128.71, 126.18, (dd, J ¼ 8.9, 2.9 Hz, 1H), 6.84 (m, 2H). 13CNMR (150 MHz, DMSO-d6)
125.03, 120.58, 118.96, 118.50, 19.97. MS: m/z(%): 253.0[M þ 1]þ, d (ppm): 160.61, 158.38, 154.24, 146.50, 138.99, 130.33, 127.18,
224.9, 196.8, 181.7.
125.87, 120.71, 119.61, 117.06, 115.47, 112.75. MS: m/z (%):
7-Hydroxy-3-(2-methylphenyl)-coumarin (11). White solid, 254.6[M þ 1]þ, 226.5, 198.5, 164.5, 132.6.
yield 41%, m.p.214.1–214.6 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3204 (OH);
7-Hydroxy-3-(4-fluorophenyl)-coumarin (18). White solid, yield
1677(C ¼ O); 1612, 1471, 1060, 959(Ar). 1HNMR (600 MHz, DMSO- 45%, m.p.245.7–247.4 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3202(OH); 1679(C ¼ O);
d6) d (ppm): 10.60 (s, 1H), 7.90 (s, 1H), 7.57 (d, J ¼ 8.5 Hz, 1H), 7.27 1624, 1508, 1453, 1122, 959, 831 (Ar).1HNMR (600 MHz, DMSO-d6)
(m, 4H), 6.83 (dd, J ¼ 8.5, 2.1 Hz, 1H), 6.79 (d, J ¼ 2.0 Hz, 1H), 2.20 d (ppm): 10.63 (s, 1H), 8.16 (s, 1H), 7.75 (m, 2H), 7.60 (d, J ¼ 8.5 Hz,
(s, 3H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 161.65, 160.26, 1H), 7.28 (m, 2H), 6.83 (dd, J ¼ 8.5, 2.3 Hz, 1H), 6.76 (d, J ¼ 2.2 Hz,
155.69, 143.20, 136.96, 135.87, 130.53, 130.35, 130.29, 128.66, 1H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 163.15, 161.75, 161.52,
126.15, 124.17, 113.78, 112.10, 102.35, 20.02. MS: m/z(%): 160.56, 155.39, 141.58, 131.96, 131.94, 130.89, 130.83, 130.47,
253.1[M þ 1]þ, 224.7, 196.7, 181.6, 146.5.
121.63, 115.58, 115.44, 113.91, 112.41, 102.20. MS: m/z (%):
7-Hydroxy-3-(4-hydroxyphenyl)-coumarin (12). White solid, 256.8[M þ 1]þ, 236.7, 200.8, 164.7.
yield 95%, m.p.327.1–328.0 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3386(OH);
8-Hydroxy-3-(4-fluorophenyl)-coumarin (19). White solid, yield
1675(C ¼ O); 1607, 1515, 1446, 1121, 951, 835(Ar). 1HNMR 52%, m.p.201.3–203.1 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3233(OH); 1686(C ¼ O);
(600 MHz, DMSO-d6) d (ppm): 10.52 (s, 1H), 9.64 (s, 1H), 8.03 (s, 1605, 1509, 1472, 1134, 957, 840 (Ar); 1083(C-F).1HNMR (600 MHz,
1H), 7.55 (dd, J ¼ 13.6, 8.6 Hz, 3H), 6.82 (m, 3H), 6.74 (d, J ¼ 2.1 Hz, DMSO-d6) d (ppm): 10.25 (s, 1H), 8.21 (s, 1H), 7.80 (m, 2H), 7.31 (m,
1H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 161.20, 160.74, 157.95, 2H), 7.19 (m, 2H), 7.11 (dd, J ¼ 7.2, 2.2 Hz, 1H). 13CNMR (150 MHz,
155.00, 139.64, 130.06, 129.99, 126.16, 122.70, 115.45, 113.72, DMSO-d6) d (ppm): 163.47, 161.84, 160.13, 144.81, 142.11, 141.48,
112.65, 102.12. MS: m/z(%): 255.0[M þ 1]þ, 226.9, 198.9, 184.8.
131.60, 131.58, 131.23, 131.17, 126.15, 125.06, 120.85, 119.09,
5,7-Dihydroxy-3-arylcoumarin (13). White solid, yield 57%, 118.51, 115.65, 115.51. MS: m/z (%): 257.0[M þ 1]þ, 228.9, 200.9,
m.p.259.4–260.9 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3259(OH); 1677(C ¼ O); 1609, 182.9, 153.9.
1519, 1447, 1124, 1013, 956, 840(Ar). 1HNMR (600 MHz, DMSO-d6)
7-Hydroxy-3-(2,5-dimethylphenyl)-coumarin (20). White solid,
d (ppm): 10.75 (s, 1H), 10.43 (s, 1H), 8.07 (s, 1H), 7.66 (m, 2H), 7.43 yield 52%, m.p.189.1–190.6 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3218(OH);
(dd, J ¼ 10.5, 4.7 Hz, 2H), 7.36 (dd, J ¼ 8.2, 6.5 Hz, 1H), 6.30 (d, 11681(C ¼ O); 1593, 1455, 1116, 1013, 951, 815 (Ar).1HNMR
J ¼ 2.1 Hz, 1H), 6.24 (d, J ¼ 2.0 Hz, 1H). 13CNMR (150 MHz, DMSO- (600 MHz, DMSO-d6) d (ppm): 10.25 (s, 1H), 8.21 (s, 1H), 7.80 (m,
d6) d (ppm):162.59, 160.75, 156.68, 156.28, 136.36, 135.92, 128.67, 2H), 7.31 (m, 2H), 7.18 (m, 2H), 7.11 (dd, J ¼ 7.2, 2.2 Hz, 1H).
128.59, 128.22, 120.13, 102.81, 98.83, 94.15. MS: m/z(%): 13CNMR (150 MHz, DMSO-d6) d (ppm): 161.60, 160.23, 155.66,
255.0[M þ 1]þ, 226.9, 198.8, 148.8.
143.07, 135.70, 135.04, 133.78, 131.04, 130.27, 130.24, 129.23,
8-Hydroxy-3-(4-hydroxyphenyl)-coumarin (14). White solid, 124.32, 113.76, 112.11, 102.34, 20.91, 19.55. MS: m/z (%):
yield 51%,m.p.237.8–238.7 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3387, 3280(OH); 267.2[M þ 1]þ, 238.9, 210.9, 192.8, 146.6.
1703(C ¼ O); 1610, 1513, 1473, 1122, 1060, 952, 837 (Ar).1HNMR
8-Hydroxy-3-(2,5-dimethylphenyl)-coumarin (21). White solid,
(600 MHz, DMSO-d6) d (ppm): 10.18 (s, 1H), 9.73 (s, 1H), 8.08 (s, yield 45%, m.p.183.2–187.4 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3243(OH);
1H), 7.59 (m, 2H), 7.16 (m, 2H), 7.07 (dd, J ¼ 7.3, 2.1 Hz, 1H), 6.85 1676(C ¼ O); 1611, 1500, 1470, 1060, 947, 816 (Ar).1HNMR
(m, 2H).13CNMR (150 MHz, DMSO-d6) d (ppm): 160.30, 158.41, (600 MHz, DMSO-d6) d (ppm): 10.24 (s, 1H), 7.96 (s, 1H), 7.18 (m,
3H), 7.13 (m, 2H), 7.10 (s, 1H), 2.30 (s, 3H), 2.16 (s, 3H). 13CNMR
144.71, 141.79, 139.41, 130.30, 127.02, 125.77, 124.92, 121.12,
118.79, 117.94, 115.50. MS: m/z(%): 254.9[M þ 1]þ, 226.8, 198.8, (150 MHz, DMSO-d6) d (ppm): 159.81, 144.92, 143.08, 142.42,
164.8, 132.8.
135.40, 135.10, 133.70, 130.91, 130.32, 129.49, 128.86, 125.02,
7,8-Dihydroxy-3-arylcoumarin (15). White solid, yield 39%, 120.59, 118.92, 118.46, 20.91, 19.50. MS: m/z (%): 267.1[M þ 1]þ,
m.p.254.0–255.0 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3508(OH); 1724(C ¼ O); 3052, 223.9, 210.8, 180.8, 146.6.
8-Hydroxy-3-(4-methoxyphenyl)-coumarin (22). White solid,
1621, 1591, 1514, 1445, 1104, 1022, 955, 814(Ar).
1HNMR (600 MHz, DMSO-d6) d (ppm): 10.15 (s, 1H), 9.42 (s, 1H), yield 41%, m.p.223.5–225.8 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3394(OH);
8.12 (s, 1H), 7.71 (dd, J ¼ 5.2, 3.3 Hz, 2H), 7.45 (dd, J ¼ 10.3, 4.7 Hz, 1705(C ¼ O); 1607, 1513, 1470, 1125, 1030, 926, 831 (Ar); 1289,
2H), 7.38 (m, 1H), 7.11 (d, J ¼ 8.5 Hz, 1H), 6.84 (d, J ¼ 8.4 Hz, 1H). 1195(C-O), 2834(-OCH3).1HNMR (600 MHz, DMSO-d6)
d (ppm):
13CNMR (150 MHz, DMSO-d6) d (ppm): 160.52, 150.08, 143.67, 10.20 (s, 1H), 8.14 (s, 1H), 7.71 (m, 2H), 7.16 (m, 2H), 7.05 (ddd,
142.18, 135.68, 132.30, 128.78, 128.63, 128.43, 122.41, 119.68, J ¼ 7.2, 1.9 Hz, 4H), 3.81 (s, 3H). 13CNMR (150 MHz, DMSO-d6) d

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
25
(ppm): 160.26, 160.02, 144.75, 141.89, 140.04, 130.29, 127.40, 1H), 7.99 (s, 1H), 7.57 (d, J ¼ 8.5 Hz, 1H), 7.18 (d, J ¼ 2.2 Hz, 1H),
126.71, 124.96, 121.05, 118.89, 118.12, 114.18, 114.13, 55.70. MS: 6.99 (dd, J ¼ 8.2, 2.2 Hz, 1H), 6.78 (ddd, J ¼ 34.2, 17.2, 2.2 Hz, 3H).
m/z (%): 269.0[M þ 1]þ.
13CNMR (150 MHz, DMSO-d6) d (ppm): 161.16, 160.65, 154.94,
7,8-Dihydroxy-3-(4-methylphenyl)-coumarin (23). White solid, 146.13, 145.23, 139.55, 130.07, 126.59, 122.78, 119.96, 116.26,
yield 43%, m.p.226.8–227.5 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3155(OH);
115.78, 113.70, 112.65, 102.07. MS: m/z(%): 270.8[M þ 1]þ, 252.7,
1682(C ¼ O); 1619, 1517, 1315, 1125, 1029, 910, 810 (Ar).1HNMR
214.6, 180.6, 162.6.
(600 MHz, DMSO-d6) d (ppm): 10.13 (s, 1H), 9.40 (s, 1H), 8.08 (s,
7,8-Dihydroxy-3-(4-hydroxyphenyl)-coumarin
(30).
White
1H), 7.61 (m, 2H), 7.25 (d, J ¼ 7.9 Hz, 2H), 7.10 (d, J ¼ 8.5 Hz, 1H), solid, yield 67%, m.p.287.5–288.9 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3448(OH);
6.84 (d, J ¼ 8.4 Hz, 1H), 2.34 (s, 3H). 13CNMR (150 MHz, DMSO-d6) d 1682(C ¼ O); 3087, 1609, 1519, 1439, 1321, 1013, 821 (Ar).1HNMR
(ppm): 160.56, 149.92, 143.56, 141.51, 137.84, 132.75, 132.27, (600 MHz, DMSO-d6) d (ppm): 10.05 (s, 1H), 9.63 (s, 1H), 9.37 (s,
129.20, 128.61, 122.32, 119.54, 113.30, 113.23, 21.28. MS: m/z (%): 1H), 7.99 (s, 1H), 7.55 (m, 1H), 7.07 (d, J ¼ 8.5 Hz, 1H), 6.82 (dt,
269.1[M þ 1]þ, 222.8, 212.9, 194.9, 166.9.
J ¼ 4.3, 2.6 Hz, 1H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 160.71,
7-Hydroxy-3-(4-methoxyphenyl)-coumarin (24). White solid, 157.91, 149.55, 143.35, 140.22, 132.23, 130.02, 126.25, 122.45,
yield 45%, m.p.227.8–228.8 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3226(OH);
119.24, 115.43, 113.44, 113.17. MS: m/z(%): 270.9[M þ 1]þ, 242.8,
1697(C ¼ O); 1611, 1513, 1464, 1300, 1030, 947, 827 (Ar); 1247, 214.8, 196.8, 168.9.
1181(C-O), 2834(-OCH3).1HNMR (600 MHz, DMSO-d6)
d
(ppm):
5,7-Dihydroxy-3-(4-fluorophenyl)-coumarin (31). White solid,
10.56 (s, 1H), 8.08 (s, 1H), 7.66 (d, J ¼ 8.8 Hz, 2H), 7.58 (d, J ¼ 8.5 Hz, yield 46%, m.p.267.0–267.6 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3232(OH);
1H), 7.00 (d, J ¼ 8.8 Hz, 2H), 6.82 (dd, J ¼ 8.5, 2.2 Hz, 1H), 6.75 (d, 1676(C ¼ O); 1612, 1307, 1008, 951, 826 (Ar); 1071(C-F).1HNMR
J ¼ 2.1 Hz, 1H), 3.80 (s, 3H). 13CNMR (150 MHz, DMSO-d6) d (ppm): (600 MHz, DMSO-d6) d (ppm): 10.80 (s, 1H), 10.49 (s, 1H), 8.10 (d,
161.38, 160.70, 159.62, 155.12, 140.23, 130.20, 129.98, 127.78, J ¼ 1.8 Hz, 1H), 7.63 (m, 4H), 6.27 (d, J ¼ 36.4 Hz, 2H). 13CNMR
122.32, 114.09, 113.79, 112.59, 102.15, 55.66. MS: m/z (%): (150 MHz, DMSO-d6) d (ppm): 162.87, 160.54, 156.82, 156.34,
269.0[M þ 1]þ, 240.9, 212.9, 122.9, 106.9.
136.65, 135.11, 131.58, 130.64, 121.42, 118.77, 102.78, 98.87, 94.20.
5,7-Dihydroxy-3-(4-methylphenyl)-coumarin (25). White solid, MS: m/z(%): 272.9[M þ 1]þ, 226.8, 216.9, 198.9, 186.8, 170.8.
yield 47%, m.p.220.1–222.0 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3382(OH);
1675(C ¼ O); 1589, 1508, 1470, 1013, 831 (Ar).
7,8-Dihydroxy-3-(4-fluorophenyl)-coumarin (32). White solid,
yield 57%, m.p.259.8–260.7 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3172(OH);
1HNMR (600 MHz, DMSO-d6) d (ppm): 10.72 (s, 1H), 10.40 (s, 1677(C ¼ O); 1612, 1511, 1470, 1156, 1026, 835 (Ar); 1089(C-F).
1H), 8.03 (s, 1H), 7.56 (d, J ¼ 8.1 Hz, 2H), 7.23 (d, J ¼ 8.0 Hz, 2H), 1HNMR (600 MHz, DMSO-d6) d (ppm): 10.17 (s, 1H), 9.43 (s, 1H),
6.30 (d, J ¼ 2.0 Hz, 1H), 6.23 (d, J ¼ 1.9 Hz, 1H), 2.34 (s, 3H). 8.12 (s, 1H), 7.76 (m, 2H), 7.27 (dd, J ¼ 14.8, 5.9 Hz, 2H), 7.10 (d,
13CNMR (150 MHz, DMSO-d6) d (ppm): 162.41, 160.78, 156.55, J ¼ 8.4 Hz, 1H), 6.85 (d, J ¼ 8.4 Hz, 1H). 13CNMR (150 MHz, DMSO-
156.16, 137.59, 135.71, 133.00, 129.25, 128.42, 120.07, 102.82, d6) d (ppm): 163.12, 161.50, 160.54, 150.11, 143.64, 142.15, 132.31,
98.80, 94.12, 21.25. MS: m/z(%): 269.0[M þ 1]þ, 240.9, 212.9, 148.6.
7-Methoxy-3-(4-hydroxyphenyl)-coumarin (26). White solid,
yield 42%, m.p.209.0–209.8 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3336(OH);
132.06, 132.03, 130.93, 130.87, 121.39, 119.67, 115.56, 115.42,
113.31, 113.19. MS: m/z(%): 272.9[M þ 1]þ, 252.8, 216.9, 198.9.
7,8-Dihydroxy-3-(2,5-dimethylphenyl)-coumarin (33). White
1685(C ¼ O); 1606, 1506, 1436, 1127, 1023, 947, 821 (Ar); 1269, solid, yield 45%, m.p.190.3–190.5 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3193(OH);
1172(C-O).1HNMR (600 MHz, DMSO-d6) d (ppm): 9.68 (s, 1H), 8.08 1679(C ¼ O); 1612, 1511, 1465, 1340, 1040, 935, 843 (Ar). 1HNMR
(s, 1H), 7.66 (m, 1H), 7.56 (m, 2H), 6.97 (m, 2H), 6.83 (m, 2H), 3.86 (600 MHz, DMSO-d6) d (ppm): 10.14 (s, 1H), 9.41 (s, 1H), 7.84 (s,
(d, J ¼ 1.6 Hz, 3H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 162.40, 1H), 7.15 (d, J ¼ 7.7 Hz, 1H), 7.11 (dd, J ¼ 7.8, 1.2 Hz, 1H), 7.06 (m,
160.62, 158.10, 154.90, 139.31, 130.05, 129.80, 125.97, 123.65, 2H), 6.84 (d, J ¼ 8.4 Hz, 1H), 2.29 (s, 3H), 2.14 (s, 3H). 13CNMR
115.48, 113.71, 112.98, 100.63, 56.38. MS: m/z(%): 268.9[M þ 1]þ, (150 MHz, DMSO-d6) d (ppm): 160.20, 149.94, 143.93, 143.61,
240.8, 212.8, 162.7.
135.82, 135.04, 133.79, 132.41, 131.05, 130.25, 129.19, 124.11,
119.38, 113.18, 112.88, 20.91, 19.56. MS: m/z(%): 282.9[M þ 1]þ.
7,8-Dihydroxy-3-(4-methoxyphenyl)-coumarin (34). White
6-Methoxy-3-(4-hydroxyphenyl)-coumarin (27). White solid,
yield 44%, m.p.195.6–197.2 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3333(OH);
1683(C ¼ O); 1606, 1515, 1465, 1131, 1036, 972, 837 (Ar); 1265, solid, yield 49%, m.p.234.4–235.4 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3299(OH);
1176(C-O).1HNMR (600 MHz, DMSO-d6) d (ppm): 9.75 (s, 1H), 8.09 1697(C ¼ O); 1605, 1517, 1470, 1308, 1130, 1012, 830 (Ar). 1HNMR
(s, 1H), 7.59 (d, J ¼ 8.6 Hz, 2H), 7.35 (d, J ¼ 9.0 Hz, 1H), 7.30 (d, (600 MHz, DMSO-d6) d (ppm): 10.09 (s, 1H), 9.39 (s, 1H), 8.05 (s,
J ¼ 2.9 Hz, 1H), 7.17 (dd, J ¼ 9.0, 3.0 Hz, 1H), 6.85 (d, J ¼ 8.6 Hz, 2H), 1H), 7.67 (m, 2H), 7.08 (d, J ¼ 8.5 Hz, 1H), 7.00 (m, 2H), 6.83 (d,
3.82 (d, J ¼ 5.9 Hz, 3H). 13CNMR (150 MHz, DMSO-d6) d (ppm): J ¼ 8.4 Hz, 1H), 3.80 (s, 3H). 13CNMR (151 MHz, DMSO-d6) d (ppm):
160.48, 158.49, 156.09, 147.50, 138.83, 130.31, 127.46, 125.75, 160.67, 159.59, 149.73, 143.44, 140.80, 132.26, 130.01, 127.88,
120.64, 119.04, 117.28, 115.54, 110.95, 56.13. MS: m/z(%): 122.07, 119.38, 114.07, 113.38, 113.21, 55.66. MS: m/z(%):
268.5[M þ 1]þ, 240.4, 212.4, 184.3, 132.4.
285.0[M þ 1]þ, 256.9, 238.9, 228.9.
5,7-Dihydroxy-3-(4-hydroxyphenyl)-coumarin
(28).
White
5,7-Dihydroxy-3-(4-methoxyphenyl)-coumarin (35). White
solid, yield 29%, m.p.298.1–299.6 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3248(OH); solid, yield 54%, m.p.259.4–260.8 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3279(OH);
1666(C ¼ O); 1608, 1513, 1444, 1116, 1007, 987, 838 (Ar).1HNMR 1681(C ¼ O); 1610, 1509, 1474, 1121, 1009, 955, 810 (Ar); 1299,
(600MHZ, DMSO-d6) d (ppm): 10.85 (s, 1H), 10.51 (s, 1H), 9.71 (s, 1181(C-O).1HNMR (600 MHz, DMSO-d6) d (ppm): 10.69 (s, 1H),
1H), 7.96 (s, 1H), 7.50 (d, J ¼ 8.6 Hz, 3H), 6.82 (d, J ¼ 8.6 Hz, 3H), 10.37 (s, 1H), 8.00 (s, 1H), 7.62 (d, J ¼ 8.6 Hz, 2H), 6.99 (d, J ¼ 8.7 Hz,
6.40 (d, J ¼ 2.0 Hz, 1H), 6.24 (d, J ¼ 1.9 Hz, 1H). 13CNMR (150 MHz, 2H), 6.29 (d, J ¼ 0.8 Hz, 1H), 6.23 (s, 1H), 3.79 (s, 3H). 13CNMR
DMSO-d6) d (ppm): 162.16, 161.00, 157.78, 156.41, 155.84, 134.61, (151 MHz, DMSO-d6) d (ppm): 162.20, 160.90, 159.42, 156.43,
129.75, 126.49, 120.09, 115.50, 102.84, 98.94, 94.00. MS: m/z(%): 156.02, 135.05, 129.81, 128.16, 119.88, 114.11, 102.86, 98.80, 94.10,
270.9[M þ 1]þ, 242.8, 214.7, 186.6, 158.6.
55.63. MS: m/z(%): 285.1[M þ 1]þ, 270.0, 256.9, 228.9, 122.6.
7,8-Dihydroxy-3-(3,4-dihydroxyphenyl)-coumarin (36). White
7-Hydroxy-3-(3,4-dihydroxyphenyl)-coumarin
(29).
White
solid, yield 22%, m.p.306.0–307.8 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3294(OH); solid, yield 56%, m.p.294.1–295.5 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3313(OH);
1685(C ¼ O); 1609, 1513, 1454, 1148, 1042, 837 (Ar).1HNMR 1686(C ¼ O); 1610, 1529, 1467, 1306, 1104, 1051, 959, 871
(600 MHz, DMSO-d6) d (ppm): 10.51 (s, 1H), 9.13 (s, 1H), 9.02 (s, (Ar).1HNMR (600 MHz, DMSO-d6) d (ppm): 10.05 (s, 1H), 9.36

26
Y. HU ET AL.
(s, 1H), 9.12 (s, 1H), 9.02 (s, 1H), 7.95 (s, 1H), 7.19 (d, J ¼ 2.1 Hz, 111.94, 102.83, 98.82, 94.09, 56.03, 56.00. MS: m/z(%):
1H), 7.07 (d, J ¼ 8.4 Hz, 1H), 7.00 (dd, J ¼ 8.2, 2.1 Hz, 1H), 6.80 (dd, 315.0[M þ 1]þ, 298.9, 270.8, 244.9.
7,8-Dihydroxy-3-(3,4-dimethoxyphenyl)-coumarin (43). White
J ¼ 17.7, 8.3 Hz, 2H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 160.62,
solid, yield 51%, m.p.290.5–291.9 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3402(OH);
149.51, 146.09, 145.21, 143.29, 140.13, 132.20, 126.69, 122.54,
1685(C ¼ O); 1611, 1519, 1113, 1022, 847 (Ar); 1262(C-O).1HNMR
119.99, 119.25, 116.31, 115.79, 113.44, 113.14. MS: m/z(%):
287.0[M þ 1]þ, 268.8, 240.8, 212.8, 184.7.
(600 MHz, DMSO-d6) d (ppm): 10.10 (s, 1H), 9.38 (s, 1H), 8.09 (s,
1H), 7.31 (m, 2H), 7.09 (d, J ¼ 8.5 Hz, 1H), 7.01 (d, J ¼ 8.2 Hz, 1H),
7-Hydroxy-3-(3,4-dimethoxyphenyl)-coumarin (37). White
solid, yield 73%, m.p.220.8–222.1 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3355(OH);
6.83 (d, J ¼ 8.4 Hz, 1H), 3.80 (d, J ¼ 8.6 Hz, 7H). 13CNMR (150 MHz,
DMSO-d6) d (ppm): 160.59, 149.74, 149.30, 148.74, 143.43, 140.99,
1731(C ¼ O); 3068, 1622, 1605, 1519, 1449, 1313, 1013, 860 (Ar);
132.25, 128.10, 122.13, 121.43, 119.39, 113.36, 113.22, 112.55,
111.87, 56.07, 56.03. MS: m/z(%): 315.0[M þ 1]þ, 299.9, 287.0,
271.9, 259.0.
1276, 1177(C-O), 2842(-OCH3).1HNMR (600 MHz, DMSO-d6)
d
(ppm): 10.56 (s, 1H), 8.13 (s, 1H), 7.58 (d, J ¼ 8.5 Hz, 1H), 7.31 (dd,
J ¼ 7.7, 5.1 Hz, 2H), 7.01 (d, J ¼ 8.3 Hz, 1H), 6.79 (dd, J ¼ 40.3, 5.3 Hz,
2H), 3.80 (m, 6H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 159.48,
153.28, 148.73, 147.73, 145.58, 138.78, 126.66, 125.90, 120.78,
119.61, 118.85, 116.12, 111.80, 111.71, 110.82, 55.09, 55.03. MS: m/
z(%): 299.0[M þ 1]þ, 270.9, 237.9, 182.9, 136.9.
7-Hydroxy-3-(4-brominephenyl)-coumarin (44). White solid,
yield 56%, m.p.270.5–271.6 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3231(OH);
1677(C ¼ O); 1589, 1449, 1122, 943, 856 (Ar); 528(C-Br).1HNMR
(600 MHz, DMSO-d6) d (ppm): 10.66 (s, 1H), 8.21 (s, 1H), 7.68 (d,
J ¼ 8.6 Hz, 2H), 7.64 (d, J ¼ 8.6 Hz, 2H), 7.61 (d, J ¼ 8.5 Hz, 1H), 6.84
(dd, J ¼ 8.5, 2.0 Hz, 1H), 6.76 (d, J ¼ 1.9 Hz, 1H). 13CNMR (150 MHz,
DMSO-d6) d (ppm): 161.96, 160.34, 155.49, 141.89, 134.78, 131.58,
130.78, 130.62, 121.76, 121.35, 113.99, 112.38, 102.22. MS: m/z(%):
318.2[M þ 1]þ, 237.8, 209.8.
8-Hydroxy-3-(3,4-dimethoxyphenyl)-coumarin (38). White
solid, yield 32%, m.p.162.8–163.8 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3233(OH);
1685(C ¼ O); 1605, 1516, 1466, 1133, 1024, 868 (Ar); 1294, 1171(C-
O), 2834(-OCH3).1HNMR (600 MHz, DMSO-d6) d (ppm): 10.21 (s,
1H), 8.18 (s, 1H), 7.36 (dd, J ¼ 4.4, 2.4 Hz, 2H), 7.18 (m, 2H), 7.09
(dd, J ¼ 7.4, 2.1 Hz, 1H), 7.04 (d, J ¼ 9.0 Hz, 1H), 3.82 (d, J ¼ 7.2 Hz,
6H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 160.18, 149.75, 148.76,
144.75, 141.87, 140.22, 127.57, 126.75, 124.97, 121.77, 121.03,
118.88, 118.13, 112.66, 111.84, 56.10, 56.04. MS: m/z(%):
298.9[M þ 1]þ, 283.9, 270.9, 237.9, 210.9.
8-Hydroxy-3-(4-brominephenyl)-coumarin (45). White solid,
yield 56%, m.p.235.5–236.9 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3233(OH);
1686(C ¼ O); 1608, 1469, 1131, 1012, 839 (Ar); 524(C-Br).1HNMR
(600 MHz, DMSO-d6) d (ppm): 10.26 (s, 1H), 8.25 (s, 1H), 7.71 (m,
2H), 7.67 (m, 2H), 7.19 (m, 2H), 7.12 (dd, J ¼ 7.5, 2.0 Hz, 1H).
13CNMR (150 MHz, DMSO-d6) d (ppm): 159.91, 144.82, 142.17,
141.81, 134.41, 131.63, 131.07, 125.97, 125.11, 122.38, 120.79,
119.19, 118.70. MS: m/z(%): 318.4[M þ 1]þ, 237.5, 209.6.
6-Hydroxy-3-(3,4-dimethoxyphenyl)-coumarin (39). White
solid, yield 70%, m.p.216.1–217.9 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3284(OH);
1663(C ¼ O); 1519, 1451, 1129, 1023, 873 (Ar); 1264, 1173(C-
O).1HNMR (600 MHz, DMSO-d6) d (ppm): 9.74 (s, 1H), 8.15 (s, 1H),
7.34 (m, 2H), 7.27 (d, J ¼ 8.9 Hz, 1H), 7.09 (d, J ¼ 2.7 Hz, 1H), 7.03
(m, 2H), 3.81 (d, J ¼ 5.9 Hz, 6H). 13CNMR (150 MHz, DMSO-d6) d
(ppm): 160.48, 154.28, 149.73, 148.73, 146.58, 139.78, 127.66,
126.90, 121.78, 120.61, 119.85, 117.12, 112.80, 112.71, 111.82,
56.09, 56.03. MS: m/z(%): 299.1[M þ 1]þ, 284.0, 271.0, 255.8, 237.8.
3-(4-brominephenyl)-coumarin (40). White solid, yield 53%,
m.p.192.8–193.8 ꢀC. IR (KBr, ꢀ, cmꢂ1): 1714(C ¼ O); 3048, 1607,
1453, 1300, 1115, 1005, 955, 836 (Ar); 513(C-Br).1HNMR (600 MHz,
DMSO-d6) d (ppm): 8.32 (s, 1H), 7.79 (dd, J ¼ 7.7, 1.4 Hz, 1H), 7.71
(m, 2H), 7.68 (m, 2H), 7.64 (ddd, J ¼ 8.7, 7.5, 1.6 Hz, 1H), 7.45 (d,
J ¼ 8.3 Hz, 1H), 7.40 (td, J ¼ 7.6, 0.9 Hz, 1H). 13CNMR (150 MHz,
DMSO-d6) d (ppm): 159.98, 153.49, 141.38, 134.33, 132.44, 131.67,
131.07, 129.24, 126.16, 125.17, 122.47, 119.86, 116.40. MS: m/z(%):
301.8[M þ 1]þ, 221.9.
5,7-Dihydroxy-3-(4-brominephenyl)-coumarin
(46).
White
solid, yield 52%, m.p.288.8–289.8 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3249(OH);
1678(C ¼ O); 1615, 1509, 1471, 1009, 830 (Ar); 564(C-Br).1HNMR
(600 MHz, DMSO-d6) d (ppm): 10.75 (s, 1H), 10.44 (s, 1H), 8.05 (s,
1H), 7.71 (m, 2H), 7.25 (t, J ¼ 8.9 Hz, 2H), 6.29 (d, J ¼ 2.1 Hz, 1H),
6.23 (d, J ¼ 2.0 Hz, 1H). 13CNMR (150 MHz, DMSO-d6) d (ppm):
162.62, 160.76, 156.68, 156.25, 136.35, 132.29, 131.07, 130.72,
130.67, 119.11, 115.57, 115.43, 102.76, 98.84, 94.15. MS: m/
z(%): 333.9[M þ 1]þ.
7,8-Dihydroxy-3-(4-brominephenyl)-coumarin (47). White solid,
yield 55%, m.p.275.1–276.8 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3274(OH);
1692(C ¼ O); 1619, 1510, 1465, 1316, 1129, 1010, 825 (Ar); 524(C-
Br).1HNMR (600MHZ, DMSO-d6) d (ppm): 10.22 (s, 1H), 9.45 (s, 1H),
8.17 (s, 1H), 7.69 (m, 2H), 7.64 (m, 2H), 7.11 (d, J¼ 8.5 Hz, 1H), 6.85
(d, J ¼ 8.4 Hz, 1H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 160.31,
150.33, 143.71, 142.47, 134.88, 132.32, 131.56, 130.82, 121.69, 121.09,
119.85, 113.37, 113.15. MS: m/z(%): 334.5[M þ 1]þ, 253.5, 225.5.
7-Diethylamino-3-(4-hydroxyphenyl)-coumarin (41). White
solid, yield 27%, m.p.190.2–191.8 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3280(OH);
1686(C ¼ O); 1613, 1519, 1455, 1310, 1135, 1013, 831 (Ar).1HNMR
(600 MHz, DMSO-d6) d (ppm): 9.57 (s, 1H), 7.94 (s, 1H), 7.53 (d,
J ¼ 8.3 Hz, 2H), 7.48 (d, J ¼ 8.8 Hz, 1H), 6.80 (d, J ¼ 8.3 Hz, 2H), 6.71
(d, J ¼ 8.7 Hz, 1H), 6.54 (s, 1H), 3.44 (q, J ¼ 6.9 Hz, 4H), 1.14 (t,
J ¼ 7.0 Hz, 6H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 161.15,
4.2. Biological activity
4.2.1. Animals
Normoglycemic Kunming mice, weight 18–20 g, were obtained
157.51, 155.89, 150.49, 139.84, 132.00, 129.75, 129.70, 129.14, from Jinan PengYue Experimental Animal Co., Ltd. Jinan, China,
126.69, 119.62, 115.39, 109.50, 109.06, 96.60, 46.17, 44.53, 12.82. (License number: SCXK (Lu) 2014–0007). The animals were housed
MS: m/z(%): 310.3[M þ 1]þ, 281.1, 254.1.
under standard laboratory conditions and maintained on a stand-
ard pellet diet and water ad libitum. All experiments involving liv-
ing animals and 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
5,7-Dihydroxy-3-(3,4-dimethoxyphenyl)-coumarin (42). White
solid, yield 96%, m.p.322.0–324.1 ꢀC. IR (KBr, ꢀ, cmꢂ1): 3468(OH);
1674(C ¼ O); 3050, 1515, 1457, 1320, 1007, 840 (Ar); 1246, 1140(C-
O), 2841(-OCH3).1HNMR (600 MHz, DMSO-d6) d (ppm): 10.70 (s,
1H), 10.39 (s, 1H), 8.03 (s, 1H), 7.24 (m, 2H), 7.00 (d, J ¼ 8.5 Hz, 1H),
6.30 (d, J ¼ 2.1 Hz, 1H), 6.23 (d, J ¼ 1.9 Hz, 1H), 3.80 (dd, J ¼ 8.5, Animal Care and Use Committee. The experiments were approved
6H). 13CNMR (150 MHz, DMSO-d6) d (ppm): 162.24, 160.85, 156.46, by the Institutional Animal Care and Use Committee of the School
156.00, 149.13, 148.72, 135.28, 128.43, 121.19, 120.00, 112.42, of Medicine and Life Sciences, University of Jinan. All efforts were

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
27
Table 8. The amount and order of each reactant of a-glucosidase inhibition test.
Volume (lL)
Sample blank group
Reagents
Blank group
Control group
Sample group
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 ten minutes
a-Glucosidase
Mix well and react at 37 ꢀC for 20 minutes
Na2CO3 70
0
10
70
0
10
70
70
made to minimise animal’s suffering and to reduce the number of 3-arylcoumarins solution or standard; A2 is the absorbance of
animals used.
2.0 ml 3-arylcoumarins solution or standard þ2.0 ml abso-
lute ethanol.
4.2.2. Influence on blood pressure
Influence on blood pressure was analyzed in adult normotensive 4.2.4. In vitro a-glucosidaseinhibitory activity
mice (25–30 g). After 1 week of acclimation, mean arterial pressure a-Glucosidase (G0660-750UN, Sigma Aldrich) and 4-Nitrophenyl
values were measured using the tail-cuff method with a blood a-D-glucopyranoside (PNPG, Macklin) were dissolved in phosphate
pressure monitor (BP-2010, Softron Beijing Biotechnology Co., Ltd.) buffer (pH 6.8, 100 mM), and the test compounds were dissolved
from 0 to 8 h after administration of the standard and test com-
pounds. The test compounds (40 mg/kg and 100mg/kg) and
standard (Losartan, 20 mg/kg) were administered.
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 8). The solution was bathed in 37 ꢀC water for 10 min, and
then enzyme solution was added. After reaction at 37 ꢀC for
20 min, 70 lL Na2CO3 solution (0.2 mM) was added to stop the
reaction. All experiments were run in triplicate. Acarbose (Sigma
Aldrich) was used as a standard inhibitor. Since PNPG can produce
glucose and p-Nitrophenol (PNP) under the action of a-glucosi-
dase, 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 calcu-
lated according to the formula.
4.2.3. In vitro antioxidant activity
The ability of the target compounds and vitamin C to scavenge the
hydroxyl radical was evaluated with method described by Ismaili
et al.29 with slight modifications. In test tube, 1.0 ml H2O2 solution
(0.03%), 1.0 ml FeSO4 solution (2 mmol.Lꢂ1), 1.0 ml salicylic acid
solution (10 mmol.Lꢂ1) and 1.0 ml of 3-arylcoumarins and standard
water solution with different concentration (0.4–2.0 mg/mL) were
mixed. Finally, 1.0ml H2O2 solution (0.03%) was added to this mix-
ture to start the reaction. Solutions were kept at 37ꢀC. After
30min, the absorbance was measured at 510 nm against the
reagent blank, where the distilled water was substituted for the
H2O2 solution. The percentage of OH scavenging by target com-
pounds and vitamin C was calculated the following equation:
È
É
Inhibition rate % ¼ ½ðAC ꢂ ABÞ ꢂ ðAS ꢂ ASBÞꢄ=ðAC ꢂ ABÞ ꢅ 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.
ð
Þ
OH scavenging effect % ¼ ½A0ꢂðA1ꢂA2Þꢄ=A0 ꢅ 100%
where A0 is the absorbance of 1.0 ml distilled water þ1.0 ml
H2O2 þ 1.0 ml FeSO4 þ 1.0 ml salicylic acid þ1.0 ml H2O2; A1 is the
absorbance of 1.0 ml 3-arylcoumarins or standard þ1.0 ml
H2O2 þ 1.0 ml FeSO4 þ 1.0 ml salicylic acid þ1.0 ml H2O2; A2 is the
absorbance of 1.0 ml 3-arylcoumarins or standard þ1.0 ml
4.2.5. In vitro inhibitory activity of AGEs formation
To prepare the AGEs reaction solution, 10 mg/mL of bovine serum
albumin in 50 mM PBS (pH 7.4) was added to 0.2 M glucose, and
0.02% sodium azide was added to prevent bacterial growth. The
reaction mixture (3.0 ml) was then mixed with various concentra-
tions (0.5–1000 lg/mL) of the target compounds (1.0 ml) dissolved
in DMSO. After incubating at 37 ꢀC for 14 d, the fluorescence
intensity of AGE was determined by a fluorospectrophotometer
(PE, USA) with excitation and emission wavelengths at 350 nm
and 420 nm, respectively. All experiments were run in triplicate.
Aminoguanidine hydrochloride was used as a reference com-
pound. The inhibition rate of AGEs formation and the IC50 value
of each sample were calculated according to the formula.
H2O2 þ 1.0 ml
þ1.0 ml H2O2.
FeSO4 þ 1.0 ml
absolute
absolute
ethanol
The effect of target compounds and vitamin C to scavenge the
DPPH free radical was evaluated with the method described by
Villano et al.43 with slight modifications.
Two millilitres of different concentrations of the sample solu-
tion was added to 2.0 ml of DPPH solution (0.04 mg/mL), shaken
and allowed to react at room temperature for 30 min. The absorb-
ance value was measured at 517 nm with an ultraviolet-visible
spectrophotometer. The blank reference cuvette contained abso-
lute ethanol. All measurements were performed in triplicate. IC50
values were calculated. The percentage of DPPH radical scaveng-
ing rate of the target compound is calculated as follows:
È
É
Inhibition rate % ¼ ½ðAC ꢂ ABÞ ꢂ ðAS ꢂ ASBÞꢄ=ðAC ꢂ ABÞ ꢅ 100%
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
ð
Þ
DPPH free radical scavenging effect % ¼ ½A0 ꢂ ðA1 ꢂ A2Þꢄ=A0 ꢅ 100%
where A0 is the absorbance of 2.0 ml DPPH solution þ2.0 ml abso- serum albumin þ1.0 ml sodium azide þ1.0 ml target compound
lute ethanol; A1 is the absorbance of 2.0 ml DPPH solution þ2.0 ml solution or Aminoguanidine hydrochloride solution); ASB is the

28
Y. HU ET AL.
absorbance of sample blank group (1.0 ml PBS þ1.0 ml bovine 4.2.9. Oral glucose tolerance test of compounds 11, 17, and 35 in
serum albumin þ1.0 ml sodium azide þ1.0 ml target compound
STZ-induced diabetic mice
solution or Aminoguanidine hydrochloride solution).
The target compounds 11, 17, and 35 (30 mg/kg of bw) and con-
trol drugs (glibenclamide 10 mg/kg of bw) were dissolved in the
vehicle (Tween-80, NaCl 0.9%). Glucose (2 g/kg of bw) was used
for the glucose tolerance tests. The control mice group received
only the vehicle. All cases were treated orally. Blood samples were
collected from caudal vein by means of a small incision in the end
of the tail. Blood glucose levels were estimated by a commercial
glucometer (Bayer Contour TS). For the anti-hyperglycemic tests,
blood samples were obtained 0.5, 1, 1.5, 2, and 3 h after the glu-
cose load. Percentage variation of glycemia for each group was
calculated with respect to initial (0 h) level according to:
4.2.6. Oral toxicity to mice
Experiments were performed on Kunming mice (male and female
half, bw 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. 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 behav-
ioural pattern. At the end of the experiments, the mice were sacri-
ficed in a CO2 chamber.
Â
Ã
% Variation of glycemia ¼ ðGi ꢂ GtÞ=Gi ꢅ 100%
where Gi is initial glycemia values and Gt is the glycemia value
after samples administration. For the anti-hyperglycemic tests,
data were represented as % variation of glycemia versus time.
Studies were performed in normoglycemic and STZ-induced dia-
betic mice.
4.2.7. Acute hypoglycemic assay
Type II diabetes mellitus was induced in mice by a single intraperi-
toneal injection of freshly prepared STZ (Sigma Aldrich) dissolved in
0.1M citrate buffer, pH 4.5, in a volume of 110 mg/kg of bw. After
7 days of STZ administration, blood glucose levels of each mouse
were determined. Mice with blood glucose levels higher than
11mM were considered diabetic and were included in the study.
STZ-induced diabetic mice and normal mice were placed in sin-
gle cages with wire-net floors and deprived of food for 12 h
before experimentation but allowed free access to tap water
throughout. The compounds (at the doses of 10, 30, and
100 mg/kg of bw) were suspended in 0.05% Tween-80 in saline
solution. Glibenclamide (10 mg/kg of bw) was suspended in the
same vehicle. The target compounds were freshly prepared imme-
diately before experimentation and administered by the intragas-
trical route at the doses of 10 ml/kg of bw. Control mice received
4.2.10. Statistical analysis
Data were shown as mean SD Differences between individual
groups were analyzed by using ANOVA followed by Dunett’s test.
A difference with a p value of <0.05 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
only the vehicle (0.05% Tween-80 in saline solution) by the same and Use Committee of the School of Medicine and Life Sciences,
route. Blood glucose levels were measured at 0, 1.5, 3, 5, 7, and
9 h after drugs administration.
University of Jinan. All efforts were made to minimise animal’s suf-
fering and to reduce the number of animals used.
4.2.8. Effects of daily treatment with compounds 11, 17, and 35 in
STZ-induced diabetic mice
Disclosure statement
The authors declare that they have no competing interests.
Type II diabetes mellitus was induced in 24 mice by the same
method showed in the section of the induction of experimental
diabetes. Thereafter, the target compounds 11, 17, 35, and gliben-
clamide were administered once (9 a.m.) on a daily basis, for a
period of 16 days, to STZ-induced diabetic mice which were div-
ided into five groups (A-E). A six group of normal mice (Group F)
was used as a control. Groups E (diabetic control) and F received
vehicle (0.05% Tween-80 in saline solution) per os for 16 days.
Group A received glibenclamide (10 mg/kg of bw daily). Groups
B–D received different treatments: B, compound 11 (30 mg/kg of
bw daily); C, compound 17 (30 mg/kg of bw daily); D, compound
35 (30 mg/kg of bw daily). All tested materials were suspended in
the same vehicle and administered per os daily for 16 days. The
animals were housed under standard laboratory conditions and
maintained with free access to water and food during all the
experiment. Blood glucose concentration on animals of diabetic
(Groups A–E) and control group (Group F) was estimated at days
1, 4, 7, 10, 13, and 16. The bw of the animals were measured con-
comitantly to the blood glucose analyses. All experiments were
carried out using six animals per group.
Funding
The authors are grateful to support from the Science and
Technology Research Program of Shandong Academy of Medical
Sciences (2017–16), Shandong Provincial Natural Science
Foundation [ZR2018LH021] and the Innovation Project of
Shandong Academy of Medical Sciences.
ORCID
Jie Sun
http://orcid.org/0000-0003-0552-9061
References
1. Kabeya LM, de Marchi AA, Kanashiro A, et al. Inhibition
of horseradish peroxidase catalytic activity by new

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
29
3-phenylcoumarin derivatives: synthesis and structure-activ- 20. Reis J, Gaspar A, Milhazes N, et al. Chromone as a privileged
ity relationships. Bioorg Med Chem. 2007;15:1516–24.
2. Wang B, Li N, Liu T, et al. Synthesis and biological evaluation
of novel neoflavonoid derivatives as potential antidiabetic
agents. RSC Adv. 2017;7:34448–60.
scaffold in drug discovery: recent advances. J Med Chem.
2017;60:7941–57.
21. Mimaki Y, Al EAE. Phenolics with PPARjLigand〣 inding
Activity Obtained from Licorice (Glycyrrhiza uralensis
Roots) and Ameliorative Effects of Glycyrin on
ꢀ
3. Keresztury G, Holly S, Komlosi V, et al. Analysis of the vibra-
tional spectra of new OH-containing E-4-arylmethylene-3-
isochromanones and 3-arylcoumarins. J Biochem Biophys
Methods. 2006;69:163–77.
Genetically Diabetic KK〢
35(14):4267–72.
22. Di CML, Zanardelli M, Bartolucci G, et al. In vitro evidence
for the use of astragali radix extracts as adjuvant against
oxaliplatin-induced neurotoxicity. Planta Medica. 2015;81:
1045–55.
y Mice. Cheminform, 2004;
4. Zhao H, Yan B, Peterson LB, et al. 3-Arylcoumarin derivatives
manifest anti-proliferative activity through Hsp90 inhibition.
ACS Med Chem Lett. 2012;3:327–31.
5. Billard C, Menasria F, Quiney C, et al. 4-Arylcoumarin ana-
logues of combretastatins stimulate apoptosis of leukemic
cells from chronic lymphocytic leukemia patients. Exp
Hematol. 2008;36:1625–33.
6. de Souza Santos M, Freire de Morais Del Lama MP,
Deliberto LA, et al. In situ screening of 3-arylcoumarin deriv-
atives reveals new inhibitors of mast cell degranulation.
Arch Pharm Res. 2013;36:731–8.
7. Matos MJ, Hogger V, Gaspar A, et al. Synthesis and adeno-
sine receptors binding affinities of a series of 3-arylcoumar-
ins. J Pharm Pharmacol. 2013;65:1590–7.
8. Limper C, Wang Y, Ruhl S, et al. Compounds isolated from
Psoralea corylifolia seeds inhibit protein kinase activity and
induce apoptotic cell death in mammalian cells. J Pharm
Pharmacol. 2013;65:1393–408.
9. Yu K, Fu YJ, Zu YG, et al. Cajanuslactone, a new coumarin
with anti-bacterial activity from pigeon pea [Cajanus cajan
(L.) Millsp.] leaves. Food Chem. 2010;121:1150–5.
10. ElSeedi HR. Antimicrobial arylcoumarins from Asphodelus
microcarpus. J Nat Prod. 2007;70:118–20.
11. Canning C, Sun S, Ji X, et al. Antibacterial and cytotoxic
activity of isoprenylated coumarin mammea A/AA isolated
from Mammea africana. J Ethnopharmacol. 2013;147:259–62.
12. Kirkiacharian S, Lormier AT, Chidiack H, et al. Synthesis and
binding affinity to human alpha and beta estrogen recep-
tors of various 7-hydroxycoumarins substituted at 4- and
3,4- positions. IL Farmaco. 2005;36:981–6.
23. Di CML, Zanardelli M, Failli P, et al. Oxaliplatin-induced oxi-
dative stress in nervous system-derived cellular models:
could it correlate with in vivo neuropathy?. Free Radic Biol
Med. 2013;61:143–50.
24. Mannelli LDC, Zanardelli M, Landini I, et al. Effect of the
SOD mimetic MnL4 on in vitro and in vivo oxaliplatin tox-
icity: possible aid in chemotherapy induced neuropathy.
Free Radic Biol Med. 2016;93:67–76.
25. Ramesh B, Pugalendi KV. Antioxidant role of Umbelliferone
in STZ-diabetic rats. Life Sci. 2006;79:306–10.
26. Ramesh B, Pugalendi KV. Antihyperglycemic effect of umbel-
liferone in streptozotocin-diabetic rats. J Med Food. 2006;9:
562–6.
27. Ramesh B, Viswanathan P, Pugalendi KV. Protective effect of
Umbelliferone on membranous fatty acid composition in
streptozotocin-induced diabetic rats. Eur J Pharmacol. 2007;
566:231–9.
ꢂ
ꢀ
ꢀ
28. Veselinovic JB, Veselinovic AM, Vitnik ZJ, et al. Antioxidant
properties of selected 4-phenyl hydroxycoumarins: inte-
grated in vitro and computational studies. Chem Biol
Interact. 2014;214:49–56.
29. Ismaili L, Nadaradjane A, Nicod L, et al. Synthesis and
antioxidant activity evaluation of new hexahydropyrimido
[5,4-c]quinoline-2,5-diones
and
2-thioxohexahydropyri-
mido[5,4-c]quinoline-5-ones obtained by Biginelli reaction in
two steps. Eur J Med Chem. 2008;39:1270–5.
30. Zhang K, Ding W, Jie S, et al. Antioxidant and antitumor
activities of 4-arylcoumarins and 4-aryl-3,4-dihydrocoumar-
ins. Biochimie. 2014;48:16–22.
ꢀ
13. Matos MJ, Vilar S, Garcıa-Morales V, et al. Insight into the
functional and structural properties of 3-arylcoumarin as an
interesting scaffold in monoamine oxidase B inhibition.
ChemMedChem. 2014;9:1488–500.
ꢀ
31. Mata R, Cristians S, Escandon-Rivera S, et al. Mexican antidia-
betic herbs: valuable sources of inhibitors of a-glucosidases.
J Nat Prod. 2013;76:468–83.
14. Ferino G, Cadoni E, Matos MJ, et al. MAO inhibitory activity
of 2-arylbenzofurans versus 3-arylcoumarins: synthesis, in
vitro study, and docking calculations. ChemMedChem. 2013;
8:956–66.
15. Xiao CF, Tao LY, Sun HY, et al. Design, synthesis and antitu-
mor activity of a series of novel coumarin-stilbenes hybrids,
the 3-arylcoumarins. Chin Chem Lett. 2010;21:1295–8.
16. Kalaiselvan V, Kalaivani M, Vijayakumar A, et al. Current know-
ledge and future direction of research on soy isoflavones as a
therapeutic agents. Pharmacogn Rev. 2010;4:111–7.
17. Norouzzadeh M, Amiri F, Saboor-Yaraghi AA, et al. Does
resveratrol improve insulin signalling in HepG2 cells?. Can J
Diabetes. 2017;41:211–6.
ꢀ
ꢀ
32. Rivera-Chavez J, Figueroa M, Gonzalez MDC, et al.
a-Glucosidase inhibitors from a xylaria feejeensis associated
with Hintonia latiflora. J Nat Prod. 2015;78:730–6.
33. Liu D, He W, Wang Z, et al. Design, synthesis and biological
evaluation of 30-benzylated analogs of 30-epi-neoponkoranol
as potent a-glucosidase inhibitors. Eur J Med Chem. 2016;
110:224–36.
34. Ni Z, Zhuge Z, Li W, et al. Inhibitory effects of hydroxysafflor
yellow A on the formation of advanced glycation end prod-
ucts in vitro. Biol Pharm Bull. 2012;35:2050–3.
35. Zou XQ, Peng SM, Hu CP, et al. Synthesis, characterization
and vasculoprotective effects of nitric oxide-donating deriva-
tives of chrysin. Bioorg Med Chem. 2010;18:3020–5.
ꢀ ꢀ
ꢀ
ꢀ
18. Seree E, Burcelin R, Savouret JF. Le resveratrol en comple-
ꢀ
ꢀ
ment therapeutique: une piste pour la prevention du dia-
ꢀ
36. Deciga-Campos M, Rivero-Cruz I, Arriaga-Alba M, et al. Acute
ꢁ
ꢀ
ꢀ
bete de type 2?. Medecine Des Maladies Metaboliques.
2009;3:486–90.
toxicity and mutagenic activity of Mexican plants used in
traditional medicine. J Ethnopharmacol. 2007;110:334–42.
37. Cristians S, Bye R, Navarrete A, Mata R. Gastroprotective
effect of Hintonia latiflora, and Hintonia standleyana,
19. Emami S, Ghanbarimasir Z. Recent advances of chroman-4-
one derivatives: synthetic approaches and bioactivities. Eur J
Med Chem. 2015;93:539–63.

30
Y. HU ET AL.
aqueous extracts and compounds. J Ethnopharmacol. 2012; 41. Muralidhara SB. Early oxidative stress in testis and epididy-
145:530–5.
mal sperm in streptozotocin-induced diabetic mice: its pro-
gression and genotoxic consequences. Reprod Toxicol. 2017;
23:578–87.
38. Guerrero-Analco J, Medina-Campos O, Brindis F, et al.
Antidiabetic properties of selected Mexican copalchis of the
Rubiaceae family. Phytochemistry. 2007;68:2087–95.
39. Rhodes CN, Brown DR. Catalytic activity of acid-treated
montmorillonite in polar and non-polar reaction media.
Catal Lett. 1994;24:285–91.
42. Oak JH, Youn JY, Cai H. Aminoguanidine inhibits aortic
hydrogen peroxide production, VSMC NOX activity and
hypercontractility in diabetic mice. Cardiovasc Diabetol.
2009;8:65–71.
~
ꢀ
ꢀ
ꢀ
40. Jang JH, Park JE, Han JS, et al. Scopoletin inhibits a-glucosi- 43. Villano D, Fernandez-Pachon MS, Moya ML, et al. Radical
dase in vitro and alleviates postprandial hyperglycemia in
scavenging ability of polyphenolic compounds towards
mice with diabetes. Eur J Pharmacol. 2018;834:152–6.
DPPH free radical. Talanta. 2007;71:230–5.