Synthesis and biological evaluation of novel neoflavonoid derivatives as potential antidiabetic agents

Article abstract of DOI:10.1039/c7ra06457h

Various substituted neoflavonoid derivatives were synthesized using sulfated montmorillonite K-10 as a catalyst. This method is environmental friendly, sustainable and economical, convenient in isolation and purification processes, with little byproducts, using earth-abundant catalysts and has relatively high yield. Those neoflavonoid derivatives were screened for antioxidant, a-glucosidase inhibitory, aldose reductase 2 (ALR2) inhibitory and advanced glycation end-product formation inhibitory effects. Most compounds exhibited significant antioxidant and advanced glycation end-product (AGE) formation inhibitory activities. It was interesting to note that out of thirty compounds, 8k and 8l were found to have greater ALR2 inhibitory activity than the standard drug quercetin. The pharmacological studies suggested neoflavonoid with adjacent 7,8-dihydroxy groups were more effective in inhibiting ALR2. Antidiabetic activity studies had shown that compounds 8l and 8m were equipotent to the standard drug glibenclamide in vivo. In summary, the target compound 8l provided a potential drug design concept for the development of therapeutic or prophylactic agents of diabetes and diabetes complications.

Full text of DOI:10.1039/c7ra06457h

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Synthesis and biological evaluation of novel
neoflavonoid derivatives as potential antidiabetic
agents†
Cite this: RSC Adv., 2017, 7, 34448
abcd
abcd
Bing Wang,‡abcd Na Li,‡abcd Teng Liu,abcd Jie Sun
*
*
and Xiaojing Wang
Various substituted neoflavonoid derivatives were synthesized using sulfated montmorillonite K-10 as
a catalyst. This method is environmental friendly, sustainable and economical, convenient in isolation
and purification processes, with little byproducts, using earth-abundant catalysts and has relatively high
yield. Those neoflavonoid derivatives were screened for antioxidant, a-glucosidase inhibitory, aldose
reductase 2 (ALR2) inhibitory and advanced glycation end-product formation inhibitory effects. Most
compounds exhibited significant antioxidant and advanced glycation end-product (AGE) formation
inhibitory activities. It was interesting to note that out of thirty compounds, 8k and 8l were found to have
greater ALR2 inhibitory activity than the standard drug quercetin. The pharmacological studies suggested
neoflavonoid with adjacent 7,8-dihydroxy groups were more effective in inhibiting ALR2. Antidiabetic
activity studies had shown that compounds 8l and 8m were equipotent to the standard drug
glibenclamide in vivo. In summary, the target compound 8l provided a potential drug design concept for
the development of therapeutic or prophylactic agents of diabetes and diabetes complications.
Received 9th June 2017
Accepted 3rd July 2017
DOI: 10.1039/c7ra06457h
rsc.li/rsc-advances
Owing to the potential biological activity and structural
diversity of neoavonoid, they have attracted an appreciable
1. Introduction
amount of attention in the synthetic community in recent
decades.19 There are numerous reports of their synthesis in
literature, but most of them have some disadvantages such as
using expensive raw materials, delivering low regioselectivities
and not economical or environmental friendly.20 Consequently,
researchers are still looking for more environmental benign and
economical synthetic processes. Lee and coworkers21 disclosed
a new approach to the synthesis of neoavonoid, based on
a high yielding montmorillonite K-10 catalyzed lactone ring
forming cyclization process (Fig. 2). In this article, we simplied
and improved Lee's method by employing sulfated montmo-
rillonite K-10 as catalyst (Fig. 2). In contrast to Lee's method, our
substituted phenylpropiolic acids were not converted to acylate
and the nal product could be puried by recrystallization
Derivatives of neoavonoids, also named 4-arylcoumarins, are
widespread in nature and have possessed a broad spectrum of
biological activities1 including antioxidant,2–4 antidiabetic,5
cytotoxic,6–8 antimicrobial,9–11 anti-inammatory,12 anti-
protozoal13–15 and estrogenic16 activities. Korec and co-workers17
reported that oral administration of an extract prepared from
a commercial mixture of the stem barks of H. latiora and E.
caribaeum (CM) of uncertain origin decreased glucose levels in
streptozotocin (STZ)-diabetic mice during short term experi-
ments in 2000. Guerrero-Analco and co-workers18 found that the
extract of H. latiora and several 4-phenylcoumarins isolated
from Rubiaceae were very effective in regulating blood glucose
levels in hyperglycemia in 2007. The structure of the most
effective compound (1,5-O-b-D-glucopyranosyl-7,30,40-trihydroxy-
4-phenylcoumarin) was displayed in Fig. 1.
aSchool of Medicine and Life Sciences, University of Jinan, Shandong Academy of
Medical Sciences, Jinan 250200, Shandong, China. E-mail: sunjie310@126.com;
xiaojing6@gmail.com
bInstitute of Materia Medica, Shandong Academy of Medical Sciences, Jinan 250062,
Shandong, China
cKey Laboratory for Biotech-Drugs, Ministry of Health, Jinan 250062, Shandong, China
dKey Laboratory for Rare & Uncommon Diseases of Shandong Province, Jinan 250062,
Shandong, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra06457h
‡ These authors contributed equally.
Fig. 1 Structure of compound 1.
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nitrobenzene and a lower yield of 66% in the acetic acid
(Table 1). The further studies found that the optimal H2SO4
concentration was 30% to treat montmorillonite K-10. This
paper also examined the reuse of the catalyst on the tandem
esterication alkylation of substituted cinnamic acids and
resorcinol to get dihydrocoumarin 4a. The results showed that
montmorillonite K-10 could be reused without extra treatment
to catalyze the same reaction, and the yield of 4a didn't have
signicant decrease (Table 2). Montmorillonite K-10 also had
such advantages as low cost, non-toxicity, and commercial
availability, rendering the synthetic process more environ-
mental friendly and economical.
Fig. 2 Methods to synthesize neoflavonoid.
8a–8h were synthesized from different substituted cinnamic
acids 2a–2c through four steps as outlined in Scheme 1. The key
intermediates 4-methoxyphenylpropiolic acid (7a), 3,4-dime-
rather than chromatography on silica gel. In addition, we tried
to use acetic acid as the solvent for this reaction, which was in
accord with the concept of modern green chemistry.
thoxyphenylpropiolic
acid
(7b)
and
3,4,5-trimethox-
yphenylpropiolic acid (7c) were prepared according to the
routine reported previously by Sun's group.20 This procedure
mainly included esterication, addition, elimination and acid-
ication reactions. Then the neoavonoids 8a–8h were
successfully obtained via the reaction between key intermedi-
ates 7a–7c and different phenols 3a–3e in the presence of
2. Results and discussion
2.1. Chemistry
The common synthetic strategies for the target compounds 4a–
4p and 8a–8n are summarized in Scheme 1. The synthetic
pathway was started from Knoevenagel condensation reaction
of commercially available material 1a–1e and malonic acid to
afford corresponding 2a–2e.22 Then under the catalysis of
sulfated montmorillonite K-10, 4a–4p can be prepared by the
esterication–cyclization reaction of 2a–2e and different
phenols 3a–3e. In order to conrm the optimal reaction
conditions, 4-methoxyphenylacrylic acid 2a with resorcinol 3a
were chosen as model substrates. This paper screened different
solvents such as nitrobenzene, chlorobenzene and acetic acid. It
was found that this reaction had the highest yield of 79% in the
ꢀ
sulfated montmorillonite K-10 and nitrobenzene at 100 C for
3–10 hours. The crude products were puried by recrystalliza-
tion in pretty good yields (64–75%, Table 3). These neo-
avonoids 8a–8h with 1, 2 or 3 methoxy group at the aryl in C4
could be easily converted to neoavonoids 8i–8n with 1, 2 or 3
hydroxyl group at the aryl in C4 under the catalysis of I2 and Al in
Table 1 The reaction conditions and yield of compound 4a. Reaction
reagents and conditions: 2a (5 mmol), 3a (5.5 mmol), solvents (10 mL)
Concentration Montmorillonite Reaction Yield
No. Solvents
of H2SO4 (%) K-10 (g)
time (h) (%)
1
2
3
4
5
6
7
8
9
Nitrobenzene
Nitrobenzene
0
5
2
2
2
2
2
1
4
6
2
2
27
20
16
8
3
6
3
3
5
10
5
23
41
61
79
65
79
71
45
66
Nitrobenzene 10
Nitrobenzene 20
Nitrobenzene 30
Nitrobenzene 30
Nitrobenzene 30
Nitrobenzene 30
Chlorobenzene 30
10 Acetic acid
30
Table 2 Recovery of montmorillonite K-10 for synthesis of compound
4a and its corresponding yield. Reaction reagents and conditions: 2a (5
mmol), 3a (5.5 mmol), solvents (10 mL), montmorillonite K-10 (2 g)
Recycling times
Reaction time (h)
Yield (%)
0
1
2
3
4
5
3
79
78
74
74
72
71
Scheme 1 General synthetic route to neoflavonoid derivatives 4a–4p
and 8a–8n. Reagents and conditions: (a) malonic acid, piperidine,
pyridine, 90 ꢀC; (b) nitrobenzene or acetic acid, montmorillonite K-10,
100 ꢀC; (c) MeOH, SOCl2, reflux, 4 h; (d) Br2, CH2Cl2, 0 ꢀC, 20 min; (e)
KOH, EtOH, reflux, 6 h; (f) nitrobenzene, montmorillonite K-10, 100 ꢀC;
(g) I2, Al, acetonitrile, reflux, 5 h.
4.5
4.5
4.5
6
4
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Table 3 Compounds 4a–4p and 8a–8n
positive reference substance vitamin C (IC50 ¼ 6.509 ꢁ 0.153 mg
mLꢂ1). These conclusions were similar to ref. 25.
Product
R1
R2
R3
R4–7
Yield (%)
In addition, it can be found from the data that almost all
compounds with two adjacent hydroxy groups exhibit great
hydroxyl radical scavenging activity. Especially, the IC50 values
of compounds 8h, 8k, 8l, 8m and 8n were from 289 ꢁ 0 to 417 ꢁ
21 mg mLꢂ1, which were 2 to 3 folds of the value of vitamin C
(IC50 ¼ 837 ꢁ 24 mg mLꢂ1). The IC50 values, 4f > 8c and 4g > 8d,
indicated that neoavonoid with 3,4-dihydrogen had weaker
hydroxyl radical scavenging activity.
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OH
OCH3
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
OH
H
H
7-Hydroxy
79
53
55
52
53
79
83
53
66
72
62
53
64
62
60
70
75
64
67
73
70
65
70
72
93
93
97
90
93
95
OCH3
OCH3
H
OCH3
H
OCH3
OCH3
OH
6-Hydroxy
7,8-Dihydroxy
2.2.2. In vitro a-glucosidase inhibitory activity.26 a-
Glucosidases, enzymes anchored in the brush border of the
small intestine, are responsible for catalyzing the hydrolysis of
carbohydrates.27 Their inhibitors were useful for the treatment
of type II diabetes mellitus.28 Though all the compounds were
tested a-glucosidase inhibitory activity in vitro, as shown in
Table 4, just six compounds (8f, 8h, 8k, 8l, 8m and 8n) pre-
sented moderate to excellent inhibitory activity for a-
glucosidase. Notably, 8l (IC50 ¼ 0.250 ꢁ 0.042 mg mLꢂ1) had
relatively strong activity, which displayed little weaker capacity
than acarbose (IC50 ¼ 0.032 ꢁ 0.002 mg mLꢂ1). None of the
neoavonoid with 3,4-dihydrogen showed good inhibitory
activity, indicating that double bond between C3 and C4 was
necessary for inhibiting a-glucosidase. The results showed that
neoavonoid with adjacent 7,8-dihydroxy groups was weaker
than that with 5,7-dihydroxy groups and 6,8-dihydroxy groups
in meta-position. Aer compound 8h was converted to 8n via
demethylation, its IC50 value decreased from 0.499 ꢁ 0.164 mg
mLꢂ1 to 0.400 ꢁ 0.036 mg mLꢂ1. According to in vitro a-gluco-
sidase inhibition test results, we decided to study in vivo
hypoglycemic activity of compounds 8l, 8m and 8n.
4j
4k
4l
H
5,7-Dihydroxy
OCH3
OCH3
OH
H
OH
OCH3
H
OCH3
H
OCH3
OCH3
H
OCH3
OCH3
OH
4m
4n
4o
4p
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
6,8-Dihydroxy
7-Hydroxy
7,8-Dihydroxy
5,7-Dihydroxy
6,8-Dihydroxy
7-Hydroxy
7,8-Dihydroxy
H
OH
OH
OH
OH
OH
OH
OH
8m
8n
5,7-Dihydroxy
6,8-Dihydroxy
OH
OH
reuxing acetonitrile. Notably, the yield of this demethylating
reaction was more than 90% without complex purication.
2.2.3. In vitro inhibitory activity of AGEs (advanced glyca-
tion end products) formation.29 Advanced glycation end prod-
ucts are a group of complex and heterogeneous compounds,
which are implicated in a number of biochemical abnormalities
associated with diabetes.30 Therefore, the discovery of AGEs
2.2. Biological evaluation
2.2.1. In vitro antioxidant activity. All the synthesized inhibitors would be benecial to the prevention and treatment
compounds were evaluated for their antioxidant activities in the of diabetic or other pathogenic complications. The results listed
way of scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH)23 and in Table 4 displayed that most of the target compounds pre-
hydroxyl radical.24 Vitamin C was used as a reference compound sented strong inhibitory activity to AGEs, even better than
in this assay. As shown in Table 4, most compounds of 4a–4p amino guanidine (AG) (IC50 ¼ 31.265 ꢁ 0.942 mg mLꢂ1). The
and 8a–8n demonstrated moderate to great scavenging activity, IC50 value, 8n < 8k < 8l < 8b < 8c < 8m < 3.0 mg mLꢂ1, which were
among which 8c, 8d and 8h–8n with several hydroxyl groups over 10 folds compared with positive reference substance.
showed excellent activity in the both two aspects.
When the neoavonoids both have the 30,40-dihydroxy groups,
DPPH is a widely used method to evaluate antioxidant those compounds with 6,8-dihydroxy groups were better than
capacities of natural and synthetic products. Although the main those with 7,8-dihydroxy groups and which were stronger than
skeleton for all compounds is same, the slight difference in those with 5,7-dihydroxy groups. The AGEs inhibitory capacities
their inhibitory potential might be due to the different substi- of 8b > 4b, 8c > 4f, 8d > 4g, 8g > 4k and 8m > 4o, indicating that
tution patterns on benzene ring. As shown in Table 4, the absence of 3,4-dihydrogen groups in neoavonoids enhanced
number and position of hydroxy groups were relatively corre- their inhibitory activity to the formation of AGEs. The series of
lated with DPPH radical scavenging activity. Generally, the IC50 values, 8k < 8l < 8c < 8d < 8e showed that neoavonoids
increase of hydroxy groups and the decrease of methoxy groups with 7,8-dihydroxy groups had a strong inhibitory activity of
were positive to this activity, while 3,4-dihydrogen structure was AGEs formation.
adverse to radical scavenging capacity. Moreover, compounds
2.2.4. In vitro ALR2 inhibitory activity.31 The enzyme aldose
with adjacent 7,8-dihydroxy groups or 30,40-dihydroxy groups reductase (ALR2) is a member of the aldo–keto reductase
had great capacity to scavenge DPPH radical, especially superfamily. Diabetic complications including cataracts, reti-
compounds 4i, 4o, 8i, 8k, 8l, 8m and 8n were better than nopathy,
accelerated
atherosclerosis,
and
increased
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Table 4 Biological evaluation in vitro
a-Glucosidase
inhibitory activity
ALR2 inhibitory
activity
Product
DPPH
OH
AGEs inhibitory activity
IC50 value (mg mLꢂ1
)
4a
>1000
>2000
>1000
>1000
N/D
4b
4c
4d
>1000
>2000
>2000
>2000
>1000
>1000
>1000
51.472 ꢁ 1.364
193.967 ꢁ 15.232
>1000
N/D
N/D
N/D
25.146 ꢁ 3.160
>1000
4e
>1000
>2000
>1000
>1000
N/D
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
8a
9.655 ꢁ 0.151
7.828 ꢁ 0.041
6.929 ꢁ 0.016
6.396 ꢁ 0.067
568.079 ꢁ 4.537
459.341 ꢁ 21.791
55.906 ꢁ 1.589
24.462 ꢁ 1.160
250.661 ꢁ 3.690
3.673 ꢁ 0.029
15.732 ꢁ 0.465
>1000
996 ꢁ 39
1061 ꢁ 29
908 ꢁ 32
982 ꢁ 27
>2000
>2000
>2000
>2000
>2000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
74.140 ꢁ 0.420
6.730 ꢁ 0.269
121.279 ꢁ 7.062
29.706 ꢁ 1.808
>1000
7.717 ꢁ 0.241
5.572 ꢁ 0.264
>1000
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
>1000
721 ꢁ 20
4.712 ꢁ 0.383
6.454 ꢁ 0.090
>1000
>2000
>2000
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
>1000
>2000
>1000
>1000
>1000
>1000
1.605 ꢁ 0.038
2.257 ꢁ 0.619
4.356 ꢁ 0.288
6.329 ꢁ 1.713
>1000
5.467 ꢁ 0.463
33.418 ꢁ 0.160
>1000
24.200 ꢁ 1.268
0.617 ꢁ 0.017
1.148 ꢁ 0.125
2.493 ꢁ 0.079
0.468 ꢁ 0.028
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
9.544 ꢁ 0.139
6.961 ꢁ 0.175
24.544 ꢁ 0.275
543.121 ꢁ 23.144
95.245 ꢁ 17.881
10.059 ꢁ 0.204
5.623 ꢁ 0.024
11.450 ꢁ 0.185
3.390 ꢁ 0.006
3.583 ꢁ 0.126
4.014 ꢁ 0.032
3.891 ꢁ 0.002
6.509 ꢁ 0.153
599 ꢁ 16
573 ꢁ 8
938 ꢁ 18
>2000
2.574 ꢁ 0.496
>2000
>1000
410 ꢁ 22
486 ꢁ 20
619 ꢁ 6
341 ꢁ 5
289 ꢁ 0
338 ꢁ 10
417 ꢁ 21
837 ꢁ 24
0.499 ꢁ 0.164
>1000
>1000
1.020 ꢁ 0.062
0.250 ꢁ 0.042
0.469 ꢁ 0.042
0.400 ꢁ 0.036
N/D
0.357 ꢁ 0.015
0.203 ꢁ 0.001
4.97 ꢁ 0.052
4.815 ꢁ 0.189
8n
Vitamin C
Acarbose
AG
0.032 ꢁ 0.002
31.265 ꢁ 0.942
Quercetin
1.119 ꢁ 0.088
cardiovascular risk, which are life threatening risks for diabetic and 8n at concentrations of 10, 100, 1000 mg kgꢂ1 to test their
patients. The development and progression of chronic diabetic oral toxicity in the rst phase, and 1600, 2900 and 5000 mg kgꢂ1
complications are conrmed to be quite related to the activation at the second phase. Results showed that none of the tested
and/or over expression of ALR2 (ref. 32). Therefore, ALR2 compounds signicantly affected mice viability. No death and
inhibitors may play a critical role in preventing or treating these no appetite-suppressant effect were detected in the tested mice
complications. The results listed in Table 4 displayed that the in 14 days. Since no death or damage was observed throughout
target compounds (8k–8n) resulted in high inhibitory activities the experiment, the LD50 was higher than 5000 mg kgꢂ1 for the
on ALR2 (IC50 ¼ 0.203 ꢁ 0.001 to 4.97 ꢁ 0.052 mg mLꢂ1). The three compounds assayed, indicating their innocuousness for
potency of ALR2 inhibition was 0.2–5.5 times of the positive mice.
reference compound quercetin. The target compound 8k and 8l
2.2.6. Acute hypoglycemic assay. The antidiabetic activity
presented strong inhibitory activity to ALR2, showing better was determined by using a standard method.26 As shown in
inhibitory activity than quercetin (IC50 ¼ 1.119 ꢁ 0.088 mg Table 5, the target compounds 8l, 8m and 8n (10 mg kgꢂ1, 30 mg
mLꢂ1). Target compounds 8k and 8l exerted higher ALR2 inhi- kgꢂ1 and 100 mg kgꢂ1 of bw) caused decreases in blood glucose
bition activities than the other serial compounds, indicating levels in STZ-diabetic mice compared with normal mice
that neoavonoid with adjacent 7,8-dihydroxy groups was more (Table 6). Especially, 8l (30 mg kgꢂ1 of bw) caused signicant
effective in inhibiting ALR2. The neoavonoids with 30,40-dihy- decreases in blood glucose levels when compared with vehicle-
droxy groups showed weak effect on enhancing their inhibitory treated groups (p < 0.05). In STZ-diabetic animals, the hypo-
activity to ALR2.
glycemic effect of 8l (30 mg kgꢂ1 of bw) was larger than 50% and
2.2.5. Oral toxicity to mice. With reference to Lorke's persisted throughout the experiment (Table 5). The highest
method,33 Kunming mice were used as targets to estimate oral antihyperglycemic effect was observed at doses of 30 and
toxicity of each compound to mice. We select compounds 8l, 8m 100 mg kgꢂ1 at 7 h (ꢃ77.14 and ꢃ70.56%, respectively) (Table
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Table 5 Acute effect of compounds 8l, 8m and 8n on blood glucose levels in STZ-diabetics mice. Each value is the mean ꢁ SEM for six mice in
each groupa
Blood glucose concentration (mM)
Dose
(per os)
mg kgꢂ1
Test samples
of bw
0 h
1.5 h
3 h
5 h
7 h
9 h
Control
(vehicle)
—
14.7 ꢁ 4.3 14.8 ꢁ 4.8 (0.68)
12.3 ꢁ 6.5 (ꢂ15.65) 11.6 ꢁ 8.4 (ꢂ21.09) 9.2 ꢁ 6.0 (ꢂ31.41) 9.1 ꢁ 4.8 (ꢂ31.41)
Glibenclamide 10
16.1 ꢁ 4.1 7.2 ꢁ 1.2* (ꢂ55.28) 6.9 ꢁ 1.9 (ꢂ57.14) 7.1 ꢁ 1.6 (ꢂ55.90) 6.6 ꢁ 1.4 (ꢂ59.63) 4.7 ꢁ 1.1* (ꢂ70.81)
18.7 ꢁ 7.3 11.1 ꢁ 4.3 (ꢂ40.64) 9.9 ꢁ 5.6 (ꢂ47.06) 8.9 ꢁ 4.4 (ꢂ52.94) 7.9 ꢁ 3.7 (ꢂ57.75) 7.7 ꢁ 3.3 (ꢂ59.36)
17.5 ꢁ 7.5 8.3 ꢁ 2.6* (ꢂ51.43) 5.7 ꢁ 1.1* (ꢂ66.86) 6.1 ꢁ 0.9 (ꢂ65.14) 4.0 ꢁ 1.4* (ꢂ77.14) 5.4 ꢁ 1.9 (ꢂ69.14)
18.0 ꢁ 5.3 9.9 ꢁ 3.2 (ꢂ45.00) 8.7 ꢁ 2.9 (ꢂ51.67) 6.2 ꢁ 1.6 (ꢂ65.56) 5.2 ꢁ 1.5 (ꢂ70.56) 6.5 ꢁ 1.3 (ꢂ63.89)
14.4 ꢁ 5.5 9.6 ꢁ 2.7 (ꢂ33.30) 8.8 ꢁ 3.1 (ꢂ38.90) 8.1 ꢁ 2.2 (ꢂ43.05) 6.5 ꢁ 1.8 (ꢂ54.16) 5.2 ꢁ 1.2* (ꢂ63.19)
15.0 ꢁ 2.1 9.1 ꢁ 1.6 (ꢂ39.33) 8.3 ꢁ 3.0 (ꢂ44.53) 8.5 ꢁ 2.5 (ꢂ43.33) 6.7 ꢁ 2.1 (ꢂ55.20) 5.4 ꢁ 1.3 (ꢂ64.20)
13.6 ꢁ 3.1 9.0 ꢁ 1.9 (ꢂ33.82) 8.3 ꢁ 1.4 (ꢂ38.97) 7.7 ꢁ 1.2 (ꢂ45.59) 6.1 ꢁ 0.7 (ꢂ55.15) 4.9 ꢁ 0.6* (ꢂ63.97)
17.6 ꢁ 5.1 11.7 ꢁ 5.0 (ꢂ33.52) 11.2 ꢁ 3.3 (ꢂ36.36) 10.0 ꢁ 3.0 (ꢂ43.75) 9.4 ꢁ 2.8 (ꢂ46.59) 7.4 ꢁ 2.1 (ꢂ57.39)
18.8 ꢁ 7.4 11.6 ꢁ 4.0 (ꢂ38.30) 10.5 ꢁ 3.7 (ꢂ44.15) 9.1 ꢁ 2.9 (ꢂ51.60) 8.7 ꢁ 4.3 (ꢂ53.72) 7.0 ꢁ 2.9 (ꢂ62.77)
17.5 ꢁ 5.7 11.1 ꢁ 5.9 (ꢂ36.57) 9.9 ꢁ 6.0 (ꢂ43.43) 8.3 ꢁ 1.7 (ꢂ52.57) 8.1 ꢁ 1.8 (ꢂ53.71) 6.9 ꢁ 1.4 (ꢂ60.57)
8l
10
8l
30
8l
100
10
30
100
10
30
8m
8m
8m
8n
8n
8n
100
a
*p < 0.05 signicantly different ANOVA followed by Dunnett's t-test for comparison with respect to initial levels in each group. % variation of
glycemia are in parentheses.
Table 6 Acute effect of compounds 8l, 8m and 8n on blood glucose levels in normal mice. Each value is the mean ꢁ SEM for six mice in each
groupa
Blood glucose concentration (mM)
Dose
(per os)
mg kgꢂ1
Test samples
of bw
0 h
1.5 h
3 h
5 h
7 h
9 h
Control
(vehicle)
—
10.2 ꢁ 1.0 8.1 ꢁ 0.9 (ꢂ20.59)
8.2 ꢁ 1.0 (ꢂ18.82)
8.4 ꢁ 1.5 (ꢂ17.65)
7.1 ꢁ 1.1 (ꢂ29.41) 6.4 ꢁ 0.9 (ꢂ36.27)
Glibenclamide 10
9.0 ꢁ 1.9 6.0 ꢁ 1.0* (ꢂ33.33) 5.5 ꢁ 0.8* (ꢂ38.89) 6.0 ꢁ 1.1* (ꢂ33.33) 5.4 ꢁ 1.1 (ꢂ40.00) 5.5 ꢁ 1.0 (ꢂ38.89)
8l
10
10.3 ꢁ 0.6 8.1 ꢁ 1.0 (ꢂ21.36)
10.5 ꢁ 1.3 6.6 ꢁ 0.8 (ꢂ37.14)
10.4 ꢁ 0.8 8.0 ꢁ 0.9 (ꢂ24.04)
10.7 ꢁ 1.1 9.4 ꢁ 1.2 (ꢂ12.15)
10.8 ꢁ 1.3 9.0 ꢁ 1.4 (ꢂ16.67)
10.6 ꢁ 1.1 9.2 ꢁ 1.1 (ꢂ13.40)
10.7 ꢁ 1.1 9.6 ꢁ 0.7 (ꢂ10.28)
10.7 ꢁ 1.0 9.7 ꢁ 1.1 (ꢂ9.35)
10.6 ꢁ 1.1 9.5 ꢁ 1.0 (ꢂ10.38)
6.5 ꢁ 0.7* (ꢂ36.89) 7.3 ꢁ 1.2 (ꢂ29.13)
6.2 ꢁ 0.9 (ꢂ39.81) 6.6 ꢁ 0.8 (ꢂ35.92)
5.8 ꢁ 1.4 (ꢂ44.76) 7.1 ꢁ 1.5 (ꢂ32.38)
6.4 ꢁ 0.7 (ꢂ39.42) 7.2 ꢁ 0.7 (ꢂ31.73)
8.6 ꢁ 1.4 (ꢂ20.56) 7.8 ꢁ 1.2 (ꢂ27.10)
8.6 ꢁ 1.5 (ꢂ20.37) 7.9 ꢁ 1.2 (ꢂ27.78)
7.7 ꢁ 1.4 (ꢂ27.36) 7.8 ꢁ 1.2 (ꢂ26.42)
8.8 ꢁ 0.9 (ꢂ17.76) 8.2 ꢁ 1.2 (ꢂ23.36)
8.7 ꢁ 1.1 (ꢂ18.69) 7.9 ꢁ 0.9 (ꢂ26.17)
8.6 ꢁ 0.9 (ꢂ18.87) 8.1 ꢁ 1.0 (ꢂ24.53)
8l
30
7.3 ꢁ 0.7 (ꢂ30.48)
7.0 ꢁ 0.8 (ꢂ32.69)
8.7 ꢁ 1.3 (ꢂ18.69)
8.7 ꢁ 1.3 (ꢂ19.44)
8.3 ꢁ 1.0 (ꢂ21.70)
9.3 ꢁ 0.9 (ꢂ13.08)
8.9 ꢁ 1.0 (ꢂ16.82)
8.7 ꢁ 0.9 (ꢂ17.92)
6.5 ꢁ 1.6 (ꢂ38.10)
7.2 ꢁ 0.8 (ꢂ30.77)
9.3 ꢁ 1.0 (ꢂ13.08)
9.3 ꢁ 1.0 (ꢂ13.89)
8.1 ꢁ 1.7 (ꢂ22.64)
8.8 ꢁ 0.7 (ꢂ17.76)
8.9 ꢁ 0.9 (ꢂ16.82)
8.4 ꢁ 1.1 (ꢂ20.75)
8l
100
10
30
100
10
30
8m
8m
8m
8n
8n
8n
100
a
*p < 0.05 signicantly different ANOVA followed by Dunnett's t-test for comparison with respect to initial levels in each group. % variation of
glycemia are in parentheses.
5). In diabetic animals, the highest antihyperglycemic effect of
8m and 8n was observed at doses of 30 mg kgꢂ1 at 9 h (ꢃ64.20
and ꢃ62.77%, respectively) (Table 5). The target compounds 8m
and 8n showed similar antidiabetic activity throughout the
experiment. Glibenclamide (10 mg kgꢂ1 of bw), used as a posi-
tive control, showed maximum hypoglycemic effect at 9 h in
STZ-induced diabetic mice (Table 5). However, the administra-
tion of the target compounds 8l, 8m and 8n (10 mg kgꢂ1, 30 mg
kgꢂ1 and 100 mg kgꢂ1 of bw) did not show signicant decreases
of blood glucose levels in normoglycemic mice (Table 6).
2.2.7. Effects of daily treatment with compounds 8l and 8m
in STZ-induced diabetic mice. The long term antihyperglycemic
effect of the target compounds 8l and 8m was performed by
using a classical chronic experiment with STZ-induced diabetic
mice.34 The results demonstrated that daily oral administration
of the target compound 8l (30 mg kgꢂ1 of bw each time), once
a day, for 16 days, induced pronounced antihyperglycemic effect
in the STZ-diabetic mice (Fig. 3). The target compound 8m was
less efficient in decreasing blood glucose levels in diabetic mice
(Fig. 3). Compounds 8l (30 mg kgꢂ1 of bw each time) restored
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Fig. 3 Long-term effect of the compound 8l and 8m on blood
glucose levels in STZ-diabetic mice. Each value is the mean ꢁ SEM for
six mice in each group. *p < 0.05 significantly different ANOVA fol-
lowed by Dunnett's t-test for comparison with the diabetic control
group at same time.
Fig. 5 Oral glucose tolerance test of compounds 8m and 8l on in
STZ-induced diabetic mice. Each value is the mean ꢁ SEM for six mice
in each group. * p < 0.05 significantly different ANOVA followed by
Dunnett's t-test vs. the diabetic control group at same time.
blood glucose levels to near normal values at the end of the
experiment [Fig. 3 (16 days)] and the effect was equipotent to
that of the glibenclamide (10 mg kgꢂ1 of bw each time) which
was used as a positive control.18 The treatments with the target
compounds 8l (30 mg kgꢂ1 of bw each time) and 8m (30 mg kgꢂ1
of bw each time) also prevented body weight loss in hypergly-
cemic mice (Fig. 4) and the effect was equipotent to that of
glibenclamide (10 mg kgꢂ1 of bw each time). However, 8l was
more efficient than 8m in preventing body weight loss in
hyperglycemic mice (Fig. 4).
2.2.8. Oral glucose tolerance test of compounds 8m and 8l
on in STZ-induced diabetic mice. The oral glucose tolerance test
was determined by using a standard method.35 Compared with
vehicle treated group (p < 0.05) (Fig. 5), the target compounds 8l
and 8m (30 mg kgꢂ1 of bw) caused a signicant decrease in the
postprandial glycemia peak in both normal (data not shown)
and STZ-diabetic mice. In all cases, the effect of the target
compounds 8l and 8m (30 mg kgꢂ1 of bw) were comparable to
that of the glibenclamide (10 mg kgꢂ1 of bw) used as a positive
control. Compounds 8l and 8m decreased blood glucose levels
to below initial values at the end of the experiment (Fig. 5).
3. Conclusions
Thirty neoavonoid derivatives were synthesized by using
sulfated montmorillonite K-10 as catalyst. Compared with
traditional methods, this method is more environmental
friendly, more sustainable and economical owning to the earth-
abundant catalysts used, more convenient in isolation and
purication processes due to less byproducts, and the yield is
relatively higher than traditional methods. We then studied the
pharmacological activity of the synthesized compounds. In the
respect to its antioxidant activity, compounds 8k, 8l, 8m and 8n
had strong hydroxyl radical scavenging activity. Compounds 8k
and 8l showed signicant effect on treatment of diabetic
complications. Notably, 8l had relatively strong activity, which
displayed little weaker capacity than acarbose. Oral toxicity tests
indicated their innocuousness for mice. We selected
compounds 8l, 8m and 8n to investigate their antidiabetic
activity in vivo. The results showed the effect of the target
compounds 8l and 8m were equipotent to that of the gliben-
clamide used as a positive control in vivo, in all cases. In
conclusion, the target compound 8l offered a potential drug
design concept for the development of therapeutic or preventive
agents for diabetes and complications of diabetes.
4. Experimental
4.1. Synthesis
Fig. 4 Body weight changes of daily treatment with compounds 8l
and 8m in STZ-induced diabetic mice. Each value is the mean ꢁ SEM
for six mice in each group. *p < 0.05 significantly different ANOVA
followed by Dunnett's t-test vs. the diabetic control group at same
time.
4.1.1. Materials and methods. Melting points were deter-
mined using a Thiele tube and were uncorrected. The FT-IR
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spectra were recorded using a Thermo-Nicolet Nexus 670 spec-
4.1.3.3. 7-Hydroxy-4-(3-methoxy-4-hydroxyphenyl)-3,4-
dihydrocoumarin (4c). White solid, yield 55%, mp 194.3–
196.3 ꢀC. IR (KBr, n, cmꢂ1): 3415 (OH); 1724 (C]O); 2994, 1625,
1602, 1518, 1454, 1112, 1028, 841, 809 (Ar); 1259, 1152 (C–O);
2929 (–OCH3). 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.03 (m,
2H), 3.71 (s, 3H), 4.24 (t, J ¼ 6.0 Hz, 1H), 6.44 (d, J ¼ 7.8 Hz, 1H),
6.51 (s, 1H), 6.54 (d, J ¼ 8.4 Hz, 1H), 6.70 (d, J ¼ 7.8 Hz, 1H), 6.78
(s, 1H), 6.84 (d, J ¼ 7.8 Hz, 1H), 8.93 and 9.72 (s, 2H). 13C NMR
(151 MHz, DMSO-d6) d (ppm): 37.35, 38.81, 56.05, 103.69,
112.05, 115.93, 117.23, 119.84, 129.42, 132.94, 146.01, 148.20,
152.37, 157.95 and 168.45. MS: m/z (%): 287.1 [M + 1]+, 162.8.
4.1.3.4. 6-Hydroxy-4-(4-methoxyphenyl)-3,4-dihydrocoumarin
(4d). White solid, yield 52%, mp 167.7–169.7 ꢀC. IR (KBr, n,
cmꢂ1): 3332, (OH); 1728 (C]O); 3000, 1613, 1600, 1502, 1450,
1
trometer with KBr pellets. The H NMR and 13C NMR spectra
were recorded with a Bruker AM-600 spectrometer with TMS as
the internal standard. Chemical shis 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. The absorbance was recorded by a Hitachi U-
3000 UV spectrophotometer. Column chromatography was
performed on silica gel (200–300 mesh). Unless otherwise
noted, all solvents and reagents were commercially available
and used without further purication.
4.1.2. The treatment of montmorillonite K-10. The mont-
morillonite K-10 used in this study was supplied by Aladdin.
This treatment method referred to literature36 with slight
modication. To a 250 mL oven-dried three-neck round-bottom
ask, 13 g montmorillonite K-10 was mixed with 130 mL 30%
H2SO4 solution. This suspension was heated to 95 ꢀC and keep
for 4 hours under magnetic stirring. Then the acid treated clays
were washed thoroughly with distilled water until the pH of the
washings approached 7. The samples were dried in a vacuum
1
1105, 1028, 831, 807 (Ar); 1236, 1146 (C–O), 2829 (–OCH3). H
NMR (600 MHz, DMSO-d6) d (ppm): 3.01 (m, 2H), 3.73 (s, 3H),
4.34 (t, J ¼ 6.3 Hz, 1H), 6.37 (s, 1H), 6.68 (d, J ¼ 8.6 Hz, 1H), 6.92
(d, J ¼ 8.0 Hz, 2H), 6.96 (d, J ¼ 8.6 Hz, 1H), 7.1 (d, J ¼ 8.6 Hz, 2H),
9.34 (s, 1H). 13C NMR (151 MHz, DMSO-d6) d (ppm): 36.8, 39.99,
55.55, 114.68, 115.23, 117.76, 128.05, 129.05, 133.34, 144.43,
154.37, 158.81 and 168.53. MS: m/z (%): 207.7 [M + 1]+, 162.6.
4.1.3.5. 6-Hydroxy-4-(3,4-dimethoxyphenyl)-3,4-dihydrocoumarin
(4e). White solid, yield 53%, mp 166.7–168.5 ꢀC, IR (KBr, n, cmꢂ1):
3259 (OH); 1709 (C]O); 3071, 1593, 1522, 1487, 1451, 1106, 1022,
856, 817 (Ar); 1267, 1142 (C–O); 2790 (–OCH3). 1H NMR (600 MHz,
DMSO-d6) d (ppm): 3.04 (m, 2H), 3.72 and 3.73 (s, 6H), 4.33 (t, J ¼
6.3 Hz, 1H), 6.37 (d, J ¼ 2.6 Hz, 1H), 6.62 (dd, J ¼ 1.6 and 8.2 Hz,
1H), 6.68 (dd, J ¼ 2.7 and 8.7 Hz, 1H), 6.88 (d, J ¼ 1.5 Hz, 1H), 6.91
(d, J ¼ 8.3 Hz, 1H), 6.96 (d, J ¼ 8.7 Hz, 1H), 9.32 (s, 1H). 13C NMR
(151 MHz, DMSO-d6) d (ppm): 36.70, 39.99, 55.90, 111.99, 112.43,
114.67, 115.22, 117.70, 119.88, 128.06, 133.72, 144.42, 148.42,
149.41, 154.35 and 168.59. MS: m/z (%): 301.0 [M + 1]+, 259.0, 162.9.
4.1.3.6. 7,8-Dihydroxy-4-(4-methoxyphenyl)-3,4-dihydrocoumarin
(4f). White solid, yield 79%, mp 168.9–171.1 ꢀC. IR (KBr, n, cmꢂ1):
3330 (OH); 1738 (C]O); 3050, 1634, 1612, 1512, 1458, 1109, 1030,
1002, 830, 802 (Ar); 1236, 1154 (C–O); 2805 (–OCH3). 1H NMR (600
MHz, DMSO-d6) d (ppm): 3.01 (m, 2H), 3.72 (s, 3H), 4.28 (t, J ¼
6.8 Hz, 1H), 6.28 (d, J ¼ 8.0 Hz, 1H), 6.53 (d, J ¼ 8.0 Hz, 1H), 6.88
and 7.06 (d, J ¼ 8.0 Hz, 4H), 8.91 and 9.29 (s, 2H). 13C NMR (151
MHz, DMSO-d6) d (ppm): 37.35, 38.93, 55.53, 111.7, 114.9, 118.5,
119.1, 128.83, 133.74, 134.11, 140.99, 146.31, 158.66 and 168.25.
MS: m/z (%): 286.8 [M + 1]+, 244.6, 178.6, 160.6.
ꢀ
oven at 40 C to get 13.95 g sulfated montmorillonite K-10.
4.1.3. General procedures for the synthesis of 4-aryl-3,4-
dihydrocoumarins 4a–4p. A mixture of 4-methoxyphenylacrylic
acid 2a (5 mmol) and resorcinol 3a (5 mmol) in nitrobenzene
(10 mL) was heated to 100 ꢀC. 2 g of sulfated montmorillonite K-
10 was added to the mixture aer all reagents were dissolved
and the mixture stirred at 100 ꢀC for 3–12 h. The reaction
process was monitored by TLC (CH2Cl2–CH3OH, 10 : 1). The
suspension was directly ltered and a suitable amount of
petroleum ether was added to the ltrate. Then cooled the
mixture and stewing or stirred under low temperature for
a certain length of time to promote crystallization of the
product. The crude product was recrystallized from EtOAc–PE to
afford 7-hydroxy-4-(4-methoxyphenyl)-3,4-dihydrocoumarin 4a.
Compounds 4b–4p were obtained using the same procedures.
4.1.3.1. 7-Hydroxy-4-(4-methoxyphenyl)-3,4-dihydrocoumarin
(4a). White solid, yield: 79%, mp 177.2–178.3 ꢀC, IR (KBr, n,
cmꢂ1): 3332 (OH); 1736 (C]O); 3040, 1614, 1583, 1507, 1450,
1
1105, 1028, 984, 832 (Ar); 1236, 1146 (C–O); 2800 (–OCH3). H
NMR (600 MHz, DMSO-d6) d (ppm): 3.03 (m, 2H), 3.73 (s, 3H),
4.32 (t, J ¼ 6.0 Hz, 1H), 6.53 (dd, J ¼ 2.4 and 8.4 Hz, 1H), 6.55 (d,
J ¼ 2.4 Hz, 1H), 6.84 (d, J ¼ 8.4 Hz, 1H), 6.90 and 7.06 (d, J ¼
1.8 Hz, 4H), 9.74 (s, 1H). 13C NMR (151 MHz, DMSO-d6) d (ppm):
36.76, 37.80, 54.95, 103.17, 111.51, 114.03, 116.47, 128.23,
128.81, 133.53, 151.81, 157.43, 158.10 and 167.73. MS: m/z (%):
207.7 [M + 1]+, 162.6.
4.1.3.2. 7-Hydroxy-4-(3,4-dimethoxyphenyl)-3,4-dihydrocoumarin
(4b). White solid, yield 53%, mp 144.8–146.1 ꢀC. IR (KBr, n, cmꢂ1):
3433 (OH); 1767 (C]O); 3030, 1628, 1597, 1514, 1447, 1101, 1025,
848, 810 (Ar); 1267, 1142 (C–O); 2790 (–OCH3). 1H NMR (600 MHz,
DMSO-d6) d (ppm): 3.05 (m, 2H), 3.70 and 3.71 (s, 6H), 4.29 (t, J ¼
6.0 Hz, 1H), 6.52 (s, 1H), 6.53–6.56 (m, 2H), 6.83–6.89 (m, 3H), 9.74
(s, 1H). 13C NMR (151 MHz, DMSO-d6) d (ppm): 37.27, 38.81, 55.93,
55.95, 103.72, 111.76, 112.09, 112.34, 119.54, 129.40, 149.37, 158.00
and 168.37. MS: m/z (%): 300.8 [M + 1]+, 258.7, 190.6, 162.7.
4.1.3.7. 7,8-Dihydroxy-4-(3,4-dimethoxyphenyl)-3,4-dihydro
coumarin (4g). White solid, yield 83%, mp 173.7–174.6 ꢀC. IR
(KBr, n, cmꢂ1): 3423 (OH); 1754 (C]O); 2935, 1610, 1515, 1470,
1
1413, 1025, 807 (Ar); 1240, 1137 (C–O); 2829 (–OCH3). H NMR
(600 MHz, DMSO-d6) d (ppm): 3.04 (m, 2H), 3.70 and 3.71 (s,
6H), 4.27 (t, J ¼ 6.3 Hz, 1H), 6.28, 6.53, 6.56 and 6.87 (d, J ¼
8.2 Hz, 4H), 6.84 (s, 1H), 8.90 and 9.28 (s, 2H). 13C NMR (151
MHz, DMSO-d6) d (ppm): 37.25, 39.98, 55.96, 111.48, 111.87,
112.32, 117.61, 118.26, 119.62, 133.68, 134.56, 140.99, 146.28,
148.26, 149.32 and 168.34. MS: m/z (%): 316.8 [M + 1]+, 178.5.
4.1.3.8. 7,8-Dihydroxy-4-(3-methoxy-4-hydroxyphenyl)-3,4-
dihydrocoumarin (4h). White solid, yield 53%, mp 171.7–
173.1 ꢀC. IR (KBr, n, cmꢂ1): 3373 (OH); 1743 (C]O); 3018, 1612,
1517, 1464, 1033, 812, 693 (Ar); 1274, 1067 (C–O); 2810 (–OCH3).
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1H NMR (600 MHz, DMSO-d6) d (ppm): 3.01 (m, 2H), 3.71 (s, DMSO-d6) d (ppm): 33.55, 37.84, 56.07, 95.08, 99.11, 103.92,
3H), 4.22 (t, J ¼ 6.2 Hz, 1H), 6.29 (d, J ¼ 8.3 Hz, 1H), 6.45, 6.52 112.82, 114.49, 117.61, 135.46, 146.82, 146.85, 153.37, 155.80,
and 6.69 (d, J ¼ 8.1 Hz, 3H), 6.78 (d, J ¼ 1.5 Hz, 1H), 8.88, 8.90 158.20 and 168.47. MS: m/z (%): 303.0 [M + 1]+, 260.8, 178.7,
and 9.26 (s, 3H). 13C NMR (151 MHz, DMSO-d6) d (ppm): 37.34, 150.7, 110.8.
39.98, 56.08, 111.45, 112.19, 115.88, 117.63, 118.45, 119.90,
4.1.3.14. 5,7-Dihydroxy-4-(4-hydroxyphenyl)-3,4-dihydro-
ꢀ
132.95, 133.65, 140.96, 145.97, 146.21, 148.14 and 168.41. MS: coumarin (4n). White solid, yield 62%, mp 269.8–270.6 C.
m/z (%): 303.1 [M + 1]+, 283.7, 260.7, 178.7, 150.6.
IR (KBr, n, cmꢂ1): 3334 (OH); 1724 (C]O); 1616, 1513, 1464,
1
4.1.3.9. 7,8-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-3,4- 1413, 1132, 1062, 829 (Ar); 1288, 1190 (C–O). H NMR (600
dihydrocoumarin (4i). White solid, yield 66%, mp 185.0– MHz, DMSO-d6) d (ppm): 2.76 (dd, J ¼ 15.8, 1.6 Hz, 1H),
187.1 ꢀC. IR (KBr, n, cmꢂ1): 3499, 3420 (OH); 1752 (C]O); 3010, 3.13 (dd, J ¼ 15.8, 6.9 Hz, 1H),4.32 (m, 1H), 6.01 (d, J ¼
1610, 1591, 1518, 1469, 1030, 786, 698 (Ar); 1277, 1068 (C–O); 2.2 Hz, 1H), 6.16 (d, J ¼ 2.3 Hz, 1H), 6.65 (m, 2H), 6.85 (d, J
2810 (–OCH3). 1H NMR (600 MHz, DMSO-d6) d (ppm): 2.97 (m, ¼ 8.5 Hz, 2H), 9.30 (s, 1H), 9.57 (s, 1H), 9.75 (s, 1H). 13C
2H), 3.72 (s, 3H), 4.19 (t, J ¼ 5.4 Hz, 1H), 6.32 (d, J ¼ 7.8 Hz, 1H), NMR (151 MHz, DMSO-d6) d (ppm): 33.37, 37.87, 95.09,
6.51, 6.53 and 6.54 (d, J ¼ 6.0 Hz, 3H), 6.84 (d, J ¼ 7.8 Hz, 1H), 98.63, 104.03, 116.19, 122.46, 128.15, 132.95, 153.36,
8.90, 8.95 and 9.29 (s, 3H). 13C NMR (151 MHz, DMSO-d6) 155.75, 156.51, 158.18, 159.37, and 168.53. MS: m/z (%):
d (ppm): 37.44, 39.11, 56.10, 111.47, 112.85, 114.92, 117.73, 272.7 [M + 1]+, 178.5.
118.25, 133.68, 134.92, 140.96, 146.23, 147.03 and 168.26. MS:
4.1.3.15. 5,7-Dihydroxy-4-(3,4-dihydroxyphenyl)-3,4-dihydro-
m/z (%): 302.9 [M + 1]+, 283.7, 260.7, 178.6, 150.6.
coumarin (4o). White solid, yield 60%, mp 214.7–216.4 ꢀC. IR
4.1.3.10. 5,7-Dihydroxy-4-(4-methoxyphenyl)-3,4-dihydrocoumarin (KBr, n, cmꢂ1): 3272 (OH); 1751 (C]O); 1620, 1521, 1477, 1420,
(4j). White solid, yield 72%, mp 148.4–149.9 C. IR (KBr, n, cmꢂ1): 1190, 1055, 799 (Ar); 1294, 1163 (C–O). 1H NMR (600 MHz,
ꢀ
3510 (OH); 1759 (C]O); 3050, 1635, 1611, 1513, 1457, 1134, 1061, DMSO-d6) d (ppm): 2.72 (dd, J ¼ 15.7, 1.6 Hz, 1H), 3.12 (m, 1H),
1032, 826 (Ar); 1231, 1179 (C–O); 2805 (–OCH3). 1H NMR (600 MHz, 4.25 (m, 1H), 6.00 (d, J ¼ 2.2 Hz, 1H), 6.16 (d, J ¼ 2.3 Hz, 1H),
DMSO-d6) d (ppm): 2.97 (m, 2H), 3.69 (s, 3H), 4.38 (d, J ¼ 6.5 Hz, 1H), 6.34 (dd, J ¼ 8.2, 2.1 Hz, 1H), 6.42 (d, J ¼ 2.1 Hz, 1H), 6.61 (d, J ¼
6.02 (d, J ¼ 2.1, 1H), 6.17 (d, J ¼ 2.2 Hz, 1H), 6.83 and 6.98 (d, J ¼ 8.1 Hz, 1H), 8.75 (s, 1H), 8.84 (s, 1H), 9.55 (s, 1H), 9.72 (s, 1H).
8.6 Hz, 4H), 9.55 and 9.73 (s, 1H). 13C NMR (151 MHz, DMSO-d6) 13C NMR (151 MHz, DMSO-d6) d (ppm): 33.56, 39.48, 94.53,
d (ppm): 33.40, 37.75, 61.07, 94.51, 95.15, 99.18, 103.88, 114.44, 99.17, 104.17, 116.04, 117.85, 133.75, 144.42, 145.54, 153.36,
128.13, 134.74, 153.38, 155.79, 158.28 and 168.41. MS: m/z (%): 286.9 155.79, 158.13, 159.37, and 168.50. MS: m/z (%): 288.9 [M + 1]+,
[M + 1]+, 178.7, 110.8.
4.1.3.11. 5,7-Dihydroxy-4-(3,4-dimethoxyphenyl)-3,4-dihydro-
178.7.
4.1.3.16. 6,8-Dihydroxy-4-(3,4-dimethoxyphenyl)-3,4-dihydro-
coumarin (4k). White solid, yield 62%, mp 185.8–187.7 ꢀC. IR coumarin (4p). White solid, yield 70%, mp 192.8–193.8 ꢀC. IR
(KBr, n, cmꢂ1): 3402, (OH); 1778 (C]O); 1632, 1610, 1500, 1460, (KBr, n, cmꢂ1): 3385 (OH); 1743 (C]O); 3058, 1638, 1608, 1518,
1012, 825 (Ar); 1237, 1148 (C–O). 1H NMR (600 MHz, DMSO-d6) 1450, 1311, 1106, 1024, 867 (Ar); 1267, 1171 (C–O), 2821
1
d (ppm): 3.02 (m, 2H), 3.68 and 3.70 (s, 6H), 4.37 (d, J ¼ 6.6 Hz, (–OCH3). H NMR (500 MHz, DMSO-d6) d (ppm): 2.99 (m, 2H),
1H), 6.01 (s, 1H), 6.17 (s, 1H), 6.40 (d, J ¼ 8.4 Hz, 1H), 6.81 (d, J ¼ 3.72 (d, J ¼ 4.9 Hz, 6H), 4.21 (t, J ¼ 6.7 Hz, 1H), 6.34 (s, 1H), 6.52
9.6 Hz, 2H), 9.56 and 9.74 (s, 2H). 13C NMR (151 MHz, DMSO-d6) (s, 1H), 6.59 (dd, J ¼ 8.2, 1.9 Hz, 1H), 6.84 (d, J ¼ 1.9 Hz, 1H),
d (ppm): 33.76, 37.74, 55.87, 55.95, 95.11, 99.17, 103.74, 111.49, 6.89 (d, J ¼ 8.3 Hz, 1H), 8.86 (s, 1H), 9.19 (s, 1H). 13C NMR (126
112.22, 118.39, 135.29, 148.08, 153.45, 155.81, 158.27 and MHz, DMSO-d6) d (ppm): 37.32, 39.07, 55.98, 55.99, 104.42,
168.47. MS: m/z (%): 316.9 [M + 1]+, 178.7, 164.7, 110.8.
111.92, 112.42, 114.65, 116.68, 119.79, 134.53, 142.44, 144.15,
4.1.3.12. 5,7-Dihydroxy-4-(3-methoxy-4-hydroxyphenyl)-3,4- 145.57, 148.32, 149.38 and 168.69. MS: m/z (%): 317.0 [M + 1]+,
dihydrocoumarin (4l). White solid, yield 53%, mp 173.0– 178.8.
174.0 ꢀC. IR (KBr, n, cmꢂ1): 3404, (OH); 1767 (C]O); 3030, 1628,
4.1.4. General procedures for the synthesis of 4-arylcou-
1597, 1514, 1447, 1101, 1025, 848, 810 (Ar); 1267, 1142 (C–O), marins 8a–8h. A mixture of 4-methoxybenzaldehyde 1a (200
2790 (–OCH3). 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.01 (m, mmol), malonic acid (240 mmol) and piperidine (2 mL) in
2H), 3.70 (s, 3H), 4.33 (d, J ¼ 6.6 Hz, 1H), 6.00 (s, 1H), 6.16 (s, pyridine (50 mL) was heated to reux for 8 h at 90 ꢀC. Aer the
1H), 6.30 (d, J ¼ 7.8 Hz, 1H), 6.62 (d, J ¼ 8.4 Hz, 1H), 6.75 (s, 1H), reaction is completed, hydrochloric acid solution (150 mL,
8.91, 9.54 and 9.72 (s, 3H). 13C NMR (151 MHz, DMSO-d6) 3 mol Lꢂ1) was added. Filtrate to obtain a white crude product
d (ppm): 33.73, 37.86, 56.00, 95.09, 99.16, 103.95, 111.78, 115.72, 24 h later. The crude product was recrystallized from absolute
118.78, 133.68, 145.78, 148.05, 153.42, 155.78, 158.19 and ethanol to afford 4-methoxyphenylacrylic acid 2a.
168.55. MS: m/z (%): 303.1 [M + 1]+, 261.1, 179.1, 151.1, 111.2.
SOCl2 (60 mmol) was added to a solution of 2a (40 mmol) in
4.1.3.13. 5,7-Dihydroxy-4-(3-hydroxy-4-methoxyphenyl)-3,4- anhydrous methanol (100 mL). The solution was then
dihydrocoumarin (4m). White solid, yield 64%, mp 228.3– mechanically stirred and reuxed for 2 h. Stop the reaction and
230.3 ꢀC. IR (KBr, n, cmꢂ1): 3332 (OH); 1751 (C]O); 3065, 1638, cool for 10 minutes to concentrate, then ice water (50 mL) was
1589, 1514, 1463, 1137, 1026, 841, 797 (Ar); 1285, 1166 (C–O), added and followed by removal of the solvent in vacuo to obtain
2840 (–OCH3). 1H NMR (600 MHz, DMSO-d6) d (ppm): 2.98 (m, the crude product 4-methoxyphenyl ethyl acrylate 5a.
2H), 3.70 (s, 3H), 4.29 (d, J ¼ 6.4 Hz, 1H), 6.01 (d, J ¼ 2.2 Hz, 1H),
To a solution of 4-methoxyphenyl ethyl acrylate 5a in CH2Cl2
6.16 (d, J ¼ 2.2 Hz, 1H), 6.55–6.39 (m, 2H), 6.80 (d, J ¼ 8.3 Hz, (80 mL) was introduced Br2 (2.1 mL, 41 mmol) dropwise into the
1H), 8.93 (s, 1H), 9.57 (s, 1H), 9.74 (s, 1H). 13C NMR (151 MHz, solution at 0 ꢀC for 20 min until the reaction solution does not
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fade. Continue stirring for one hour, the reaction solution was
washed with 10% aqueous solution of sodium bisulte to
remove excess bromine and washed again with water. The
organic layer was concentrated in vacuo and afforded the crude
product 2,3-dibromo-4-methoxyphenylpropionate 6a. A mixture
of 6a and KOH (6.8 g, 121 mmol) in ethyl alcohol (100 mL) was
heated to reux for 10 h. Aer the reaction stops, add 20 mL
water to dissolve the salt, with concentrated hydrochloric acid
to adjust the pH to strong acid, with dichloromethane to extract
the reaction solution, then concentrating in vacuo to obtain the
crude product. The crude product was subjected to column
chromatography (silica, EtOAc–PE, 1 : 2) to obtain puried
compound 4-methoxybenzene propionic acid 7a (4.6 g, in 65%
yield).
4.1.4.4. 7,8-Dihydroxy-4-(3,4-dimethoxyphenyl)-coumarin
(8d). Pink solid, yield: 73%, mp 126.1–127.3 ꢀC, IR (KBr, n,
cmꢂ1): 3322 (OH); 1700 (C]O); 2954, 1604, 1519, 1505, 1440,
1
1020, 817 (Ar); 1257, 1142 (C–O); 2834 (–OCH3). H NMR (600
MHz, DMSO-d6) d (ppm): 3.82 (s, 3H), 3.84 (s, 3H), 6.15 (s, 1H),
6.80 (d, J ¼ 8.7 Hz, 1H), 6.93 (d, J ¼ 8.7 Hz, 1H), 7.06 (d, J ¼
8.2 Hz, 1H), 7.10 (dd, J ¼ 8.8, 4.8 Hz, 2H), 9.38 (s, 1H),10.17 (s,
1H). 13C NMR (151 MHz, DMSO-d6) d (ppm): 39.56, 56.08,
110.23, 112.05, 112.11, 112.61, 112.73, 117.97, 121.64, 128.23,
133.06, 144.45, 149.20, 149.97, 150.31, 156.27 and 160.72. MS:
m/z (%): 314.9 [M + 1]+, 299.8, 164.6.
4.1.4.5. 7,8-Dihydroxy-4-(3,4,5-trimethoxyphenyl)-coumarin
(8e). Pink solid, yield: 70%, mp 281.3–281.5 ꢀC, IR (KBr, n,
cmꢂ1): 3266 (OH); 1702 (C]O); 2944, 1624, 1519, 1504, 1446,
1
To a solution of 4-methoxyphenylpropionic acid 7a (1.76 g,
10 mmol) and catechol (1.2 g, 11 mmol), in nitrobenzene (20
1037, 867 (Ar); 1255, 1171 (C–O); 2847 (–OCH3). H NMR (600
MHz, DMSO-d6) d (ppm): 3.74 (s, 3H), 3.83 (s, 6H), 6.21 (s, 1H),
6.81 (d, J ¼ 10.3 Hz, 3H), 6.95 (d, J ¼ 8.7 Hz, 1H), 9.43 (s, 1H),
10.24 (s, 1H). 13C NMR (151 MHz, DMSO-d6) d (ppm): 39.49,
49.07, 56.49, 60.54, 106.42, 110.54, 111.91, 112.83, 118.02,
131.35, 133.03, 138.60, 144.34, 150.03, 153.39, 156.41 and
160.68. MS: m/z (%): 345.0 [M + 1]+, 318.9, 299.0.
ꢀ
mL, anhyd) and the solution was stirred and heated to 100 C.
Then, 4 g acidied montmorillonite K-10 was added and the
reaction was monitored by TLC (CH2Cl2 : CH3OH, 10 : 1) and
the reaction was completed aer 5 hours. Hot lter, add 30 mL
of petroleum ether in the ltrate and place the natural crystal-
lization overnight. The solvent was removed by vacuum suction
ltration, washed with 20 mL of petroleum ether and dried to
obtain pure product 4-(40-methoxyphenyl)-7-hydroxycoumarin
8a (2.0 g, in 75% yield). Compounds 8b–8h were obtained by
using the same procedures.
4.1.4.6. 5,7-Dihydroxy-4-(4-methoxyphenyl)-coumarin
(8f).
Red solid, yield: 65%, mp 256.6–258.2 ꢀC, IR (KBr, n, cmꢂ1):
3208 (OH); 1696 (C]O); 1635, 1514, 1468, 1023, 825 (Ar); 1267,
1182 (C–O); 2868 (–OCH3). 1H NMR (600 MHz, DMSO-d6)
d (ppm): 3.79 (s, 3H), 5.74 (s, 1H), 6.22 (dd, J ¼ 48.3, 2.3 Hz, 2H),
6.93 (m, 2H), 7.29 (m, 2H), 10.15 (s, 1H), 10.42 (s, 1H). 13C NMR
(151 MHz, DMSO-d6) d (ppm): 49.07, 55.57, 95.10, 99.61, 101.11,
110.45, 111.64, 113.10, 129.52, 130.96, 140.06, 156.29, 157.60,
159.64, 160.46 and 162.03. MS: m/z (%): 285.0 [M + 1]+, 252.8,
152.6.
4.1.4.1. 7-Hydroxy-4-(4-methoxyphenyl)-coumarin (8a). Pink
solid, yield: 75%, mp 265.1–266.3 ꢀC, IR (KBr, n, cmꢂ1): 3206
(OH); 1702 (C]O); 3006, 1617, 1608, 1511, 1443, 1122, 1036,
1
1002, 828 (Ar); 1251, 1184 (C–O); 2841 (–OCH3). H NMR (600
MHz, DMSO-d6) d (ppm): 3.84 (s, 3H), 6.11 (s, 1H), 6.79 (d, J ¼
8.7 Hz, 2H), 7.11 (d, J ¼ 7.6 Hz, 2H), 7.36 (d, J ¼ 8.1 Hz, 1H), 7.48
(d, J ¼ 7.6 Hz, 2H), 10.65 (s, 1H). 13C NMR (151 MHz, DMSO-d6)
d (ppm): 39.57, 55.81, 60.23, 103.16, 110.26, 111.25, 113.58,
114.75, 127.78, 128.66, 130.47, 155.57, 156.07, 160.71, 160.82
and 161.78. MS: m/z (%): 268.9 [M + 1]+, 162.7.
4.1.4.7. 5,7-Dihydroxy-4-(3,4-methoxyphenyl)-coumarin (8g).
Red solid, yield: 70%, mp 183.6–184.8 ꢀC, IR (KBr, n, cmꢂ1):
3468 (OH); 1675 (C]O); 3051, 1595, 1516, 1458, 1079, 840 (Ar);
1246, 1171 (C–O); 2841 (–OCH3). 1H NMR (600 MHz, DMSO-d6)
d (ppm): 3.75 (s, 3H), 3.79 (s, 3H), 5.79 (s, 1H),6.19 (d, J ¼ 2.3 Hz,
1H), 6.26 (d, J ¼ 2.3 Hz, 1H), 6.88 (dd, J ¼ 8.2, 2.0 Hz, 1H), 6.95
(dd, J ¼ 5.1, 3.1 Hz, 2H), 10.14 (s, 1H), 10.42 (s, 1H). 13C NMR
(151 MHz, DMSO-d6) d (ppm): 39.5, 55.95, 95.09, 99.69, 101.15,
110.49, 111.01, 112.40, 120.45, 132.32, 147.94, 149.18, 156.36,
157.30, 157.61, 160.49 and 162.02. MS: m/z (%): 315.0 [M + 1]+,
164.7.
4.1.4.8. 6,8-Dihydroxy-4-(3,4-dimethoxyphenyl)-coumarin
(8h). Red solid, yield: 72%, mp 311.6–313.7 ꢀC. IR (KBr, n, cmꢂ1):
3230 (OH); 1669 (C]O); 3003, 1598, 1516, 1464, 1023, 847 (Ar);
1262, 1170 (C–O); 2831 (–OCH3). 1H NMR (600 MHz, DMSO-d6)
d (ppm): 3.84 (d, J ¼ 7.3 Hz, 6H), 6.14 (s, 1H), 6.82 (s, 1H), 6.97 (s,
1H), 7.10 (m, 3H), 9.48 (s, 1H), 10.25 (s, 1H). 13C NMR (151 MHz,
DMSO-d6) d (ppm): 39.50, 56.09, 103.64, 110.41, 110.68, 111.38,
112.11, 112.45, 121.47, 128.23, 143.27, 148.98, 149.16, 150.24,
150.84, 155.57 and 162.02. MS: m/z (%): 315.0 [M + 1]+, 164.7.
4.1.5. General procedures for the synthesis of 4-arylcou-
marins 8i–8n. To a solution of iodine (9.6 g, 37.6 mmol) and
aluminum (1.0 g, 37 mmol) in acetonitrile (150 mL) and the
solution was stirred and heated to reux for 3 h. Then, cooled to
room temperature and compound 8a (0.54 g, 2 mmol) was
4.1.4.2. 7-Hydroxy-4-(3,4-dimethoxyphenyl)-coumarin
(8b).
Pink solid, yield: 64%, mp 239.4–240.5 ꢀC, IR (KBr, n, cmꢂ1):
3355 (OH); 1732 (C]O); 3069, 1622, 1558, 1519, 1450, 1139,
1
1014, 860, 820 (Ar); 1253, 1178 (C–O); 2843 (–OCH3). H NMR
(600 MHz, DMSO-d6) d (ppm): 3.83 (d, J ¼ 12.1 Hz, 6H), 6.16 (s,
1H), 6.79 (dd, J ¼ 6.3, 2.3 Hz, 2H), 7.09 (ddd, J ¼ 11.2, 10.1,
5.1 Hz, 3H), 7.41–7.47 (m, 1H), 10.62 (s, 1H). 13C NMR (151
MHz, DMSO-d6) d (ppm): 39.57, 49.07, 56.09, 56.09, 103.12,
110.32, 111.26, 112.18, 112.56, 113.60, 121.62, 127.94, 128.82,
149.28, 150.39, 155.75, 156.08, 160.75 and 161.75. MS: m/z (%):
299.0 [M + 1]+, 282.9, 256.8, 164.7.
4.1.4.3. 7,8-Dihydroxy-4-(4-methoxyphenyl)-coumarin
(8c).
Red solid, yield: 67%, mp 108–110 C, IR (KBr, n, cmꢂ1): 3480
(OH); 1685 (C]O); 3165, 1601, 1560, 1508, 1442, 1177, 1017,
856, 819 (Ar); 1297, 1177 (C–O); 2841 (–OCH3). 1H NMR (600
MHz, DMSO-d6) d (ppm): 3.84 (s, 3H), 6.09 (s, 1H), 6.82 (dd, J ¼
29.0, 8.7 Hz, 2H), 7.10 (d, J ¼ 8.7 Hz, 2H), 7.47 (d, J ¼ 8.6 Hz, 2H),
9.42 (s, 1H), 10.16 (s, 1H). 13C NMR (151 MHz, DMSO-d6)
d (ppm): 39.56, 39.70, 55.79, 110.16, 112.06, 112.72, 114.65,
117.83, 128.07, 130.48, 133.08, 144.42, 150.00, 156.11, 160.67
and 161.73. MS: m/z (%): 284.8 [M + 1]+, 256.7, 152.6.
ꢀ
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added. Continue to reux and the reaction was monitored by 119.26, 130.93, 144.50, 146.03, 156.85, 157.65, 160.55 and
TLC (CH2Cl2 : CH3OH, 6 : 1) until the starting material dis- 161.90. MS: m/z (%): 286.9 [M + 1]+, 152.6.
appeared completely. The reaction solution was concentrated,
4.1.5.6. 6,8-Dihydroxy-4-(3,4-dihydroxyphenyl)-coumarin
and 5% sodium bisulte was added to remove excess iodine. (8n). Light yellow solid, yield: 95%, mp 301.5–303.5 ꢀC, IR (KBr,
Aer the hydrochloric acid was acidied, the mixture was n, cmꢂ1): 3365 (OH); 1683 (C]O); 1607, 1520, 1446, 1395, 861
extracted with ethyl acetate. The organic layer was concentrated (Ar); 1276 (C–O). 1H NMR (600 MHz, DMSO-d6) d (ppm): 5.99 (s,
to obtain 4-(40-hydroxyphenyl)-7-hydroxycoumarin 8i (0.46 g, in 1H), 6.80 (dd, J ¼ 7.9, 2.3 Hz, 2H), 6.88 (d, J ¼ 2.1 Hz, 1H), 6.90
92% yield). Compounds 8j–8n were obtained by using the same (d, J ¼ 8.1 Hz, 1H), 6.98 (s, 1H), 9.31 (s, 1H), 9.40 (s, 1H), 9.44 (s,
procedures.
1H), 10.20 (s, 1H). 13C NMR (151 MHz, DMSO-d6) d (ppm): 56.19,
4.1.5.1. 7-Hydroxy-4-(3,4-dihydroxyphenyl)-coumarin
(8i). 103.61, 109.82, 110.78, 111.58, 116.30, 120.27, 126.95, 143.27,
Yellow solid, yield: 92%, mp 265.5–267.9 C, IR (KBr, n, cmꢂ1): 145.84, 147.39, 148.97, 150.85, 155.98 and 161.15. MS: m/z (%):
ꢀ
3430 (OH); 1667 (C]O); 1627, 1542, 1513, 1444, 1116, 1007, 839 287.1 [M + 1]+, 152.8.
1
(Ar); 1263, 1194 (C–O). H NMR (600 MHz, DMSO-d6) d (ppm):
6.04 (s, 1H), 6.81 (dt, J ¼ 10.3, 1.9 Hz, 3H), 6.90 (dd, J ¼ 5.1, 4.2. Biological activity
2.9 Hz, 2H), 7.46 (d, J ¼ 8.5 Hz, 1H), 9.37 (s, 1H), 9.52 (s, 1H),
4.2.1. Animals. Normoglycemic Kunming mice, weight 18–
22 g, were obtained from Jinan PengYue Experimental Animal
Co., Ltd. (License number: SCXK (Lu) 2014–0007). The animals
were housed under standard laboratory conditions and main-
10.66 (s, 1H). 13C NMR (151 MHz, DMSO-d6) d (ppm): 49.07,
103.10, 109.61, 111.24, 113.47, 116.31, 120.45, 126.55, 128.87,
145.89, 147.55, 156.06, 160.82 and 161.67. MS: m/z (%): 270.9
[M + 1]+, 242.2.
tained on a standard pellet diet and water ad libitum. All
4.1.5.2. 7,8-Dihydroxy-4-(4-hydroxyphenyl)-coumarin
(8j).
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
minimize animal's suffering and to reduce the number of
animals used.
Yellow solid, yield: 93%, mp 272.0–273.1 C, IR (KBr, n, cmꢂ1):
3352 (OH); 1685 (C]O); 2959, 1610, 1586, 1514, 1454, 1111,
1042, 838 (Ar); 1229, 1066 (C–O). 1H NMR (600 MHz, DMSO-d6)
d (ppm): 6.06 (s, 1H), 6.80 (d, J ¼ 8.8 Hz, 1H), 6.90 (dd, J ¼ 18.2,
8.6 Hz, 3H), 7.36 (d, J ¼ 8.5 Hz, 2H), 9.93 (s, 1H), 9.37 (s, 1H),
10.14 (s, 1H). 13C NMR (151 MHz, DMSO-d6) d (ppm): 39.56,
60.23, 109.76, 112.09, 112.66, 115.98, 117.93, 126.43, 130.54,
133.04, 144.45, 149.91, 156.46, 159.25 and 160.74. MS: m/z (%):
270.8 [M + 1]+, 242.7, 152.5.
ꢀ
4.1.5.3. 7,8-Dihydroxy-4-(3,4-dihydroxyphenyl)-coumarin
(8k). Yellow solid, yield: 97%, mp 182.0–183.1 ꢀC, IR (KBr, n,
cmꢂ1): 3265 (OH); 1666 (C]O); 2918, 1597, 1531, 1449, 1343,
1119, 1043, 847 (Ar); 1243, 1043 (C–O). 1H NMR (600 MHz,
DMSO-d6) d (ppm): 6.02 (s, 1H), 6.80 (s, 1H), 6.81 (d, J ¼ 2.0 Hz,
1H), 6.89 (m, 2H), 6.95 (d, J ¼ 8.8 Hz, 1H), 9.35 (s, 1H), 9.41 (s,
1H), 9.48 (s, 1H), 10.20 (s, 1H). 13C NMR (151 MHz, DMSO-d6)
d (ppm): 49.06, 109.54, 112.60, 116.31, 118.02, 120.45, 126.88,
133.01, 144.44, 145.79, 147.43, 149.87, 156.62, 160.77 and
172.51. MS: m/z (%): 286.9 [M + 1]+, 240.8, 152.6.
4.1.5.4. 7,8-Dihydroxy-4-(3,4,5-trihydroxyphenyl)-coumarin
(8l). Light yellow solid, yield: 90%, mp 312.5–313.5 ꢀC, IR (KBr,
n, cmꢂ1): 3386 (OH); 1704 (C]O); 2851, 1600, 1539, 1452, 1317,
1038, 833 (Ar); 1226, 1077 (C–O). 1H NMR (600 MHz, DMSO-d6)
d (ppm): 5.98 (s, 1H), 6.42 (s, 2H), 6.80 (d, J ¼ 8.8 Hz, 1H), 6.99
(d, J ¼ 8.8 Hz, 1H), 8.60 (s, 1H), 9.22 (s, 2H), 9.36 (s, 1H), 10.13 (s,
1H). 13C NMR (151 MHz, DMSO-d6) d (ppm): 56.54, 60.23,
4.2.2. 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 and coworkers24
with slight modications. In test tube 1 mL H2O2 solution
(0.03%), 1 mL FeSO4 solution (2 mmol Lꢂ1), 1 mL salicylic acid
solution (10 mmol Lꢂ1) and 1 mL of 4-arylcoumarins and
standard water solution with different concentration (0.4–
2.0 mg mLꢂ1) were mixed. Finally adds 1 mL H2O2 solution
(0.03%) to this mixture to start the reaction. Solutions were kept
at 37 ꢀC. Aer 30 minutes, 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 compounds and vitamin C was calculated
using the following equation:
$OH scavenging effect (%) ¼ [A0 ꢂ (A1 ꢂ A2)]/A0 ꢄ 100%
107.99, 109.35, 112.06, 112.55, 118.11, 125.98, 132.98, 135.19, where A0 is the absorbance of 1.0 mL distilled water + 1.0 mL
144.43, 146.60, 149.86, 156.95 and 160.76. MS: m/z (%): 302.9 [M H2O2 + 1.0 mL FeSO4 + 1.0 mL salicylic acid + 1.0 mL H2O2; A1 is
+ 1]+, 256.8, 152.7.
the absorbance of 1.0 mL 4-arylcoumarins or standard + 1.0 mL
4.1.5.5. 5,7-Dihydroxy-4-(3,4-dihydroxyphenyl)-coumarin H2O2 + 1.0 mL FeSO4 + 1.0 mL salicylic acid + 1.0 mL H2O2; A2 is
(8m). Light yellow solid, yield: 93%, mp 261.5–262.5 ꢀC, IR (KBr, the absorbance of 1.0 mL 4-arylcoumarins or standard + 1.0 mL
n, cmꢂ1): 3305 (OH); 1698 (C]O); 1597, 1522, 1442, 1349, 1033, H2O2 + 1.0 mL FeSO4 + 1.0 mL absolute ethanol + 1.0 mL H2O2.
845 (Ar); 1265, 1083 (C–O). 1H NMR (600 MHz, DMSO-d6)
The effect of target compounds and vitamin C to scavenge
d (ppm): 5.69 (s, 1H), 6.18 (d, J ¼ 2.4 Hz, 1H), 6.25 (d, J ¼ 2.3 Hz, the DPPH free radical was evaluated with method described by
1H), 6.62 (dd, J ¼ 8.1, 2.1 Hz, 1H), 6.72 (m, 2H), 9.01 (s, 1H), 9.11 Villano and coworkers37 with slight modications.
˜
(s, 1H), 10.11 (s, 1H), 10.40 (s, 1H). 13C NMR (151 MHz, DMSO-
2.0 mL of different concentrations of the sample solution
d6) d (ppm): 49.07, 60.26, 95.02, 99.61, 101.19, 110.04, 115.94, was added to 2.0 mL of DPPH solution (0.04 mg mLꢂ1), shaken
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and allowed to react at room temperature for 30 min. The solution, 10 mg mLꢂ1 of bovine serum albumin in 50 mM PBS
absorbance value was measured at 517 nm with an ultraviolet- (pH 7.4) was added to 0.2 M glucose, and 0.02% sodium azide
visible spectrophotometer. The blank reference cuvette con- was added to prevent bacterial growth. The reaction mixture (3
tained absolute ethanol. All measurements were performed in mL) was then mixed with various concentrations (0.5–1000 mg
triplicate. IC50 values were calculated. The percentage DPPH mLꢂ1) of the target compounds (1 mL) dissolved in DMSO. Aer
ꢀ
free radical scavenging rate was determined as
incubating at 37 C for 14 d, the uorescence intensity of AGE
was determined by a uorospectrophotometer (PE, USA) with
excitation and emission wavelengths at 350 nm and 420 nm,
respectively. All experiments were run in triplicate. Amino-
guanidine 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.
DPPH free radical scavenging effect (%) ¼ [A0 ꢂ (A1 ꢂ A2)]/A0
ꢄ 100%
where A0 is the absorbance of 2.0 mL DPPH solution + 2.0 mL
absolute ethanol; A1 is the absorbance of 2.0 mL DPPH solution
+ 2.0 mL 4-arylcoumarins solution or standard; A2 is the
absorbance of 2.0 mL 4-arylcoumarins solution or standard +
2.0 mL absolute ethanol.
Inhibition rate% ¼ {[(AC ꢂ AB) ꢂ (AS ꢂ ASB)]/(AC ꢂ AB)}
ꢄ 100%
4.2.3. In vitro a-glucosidase inhibitory activity. a-Glucosi-
dase (G0660-750UN, Sigma Aldrich) and 4-nitrophenyl a-D-glu- where AC is the absorbance of control group (1.0 mL glucose +
copyranoside (PNPG, Macklin) were dissolved in phosphate 1.0 mL bovine serum albumin + 1.0 mL sodium azide + 1.0 mL
buffer (pH 6.8, 100 mM), and the test compounds were dis- DMSO); AB is the absorbance of blank group (1.0 mL PBS + 1.0
solved in DMSO solution. The experiment was divided into mL bovine serum albumin + 1.0 mL sodium azide + 1.0 mL
blank group, control group, sample blank group and sample DMSO); AS is the absorbance of sample group (1.0 mL glucose +
group. The reagents were loaded in 96-well plates at the dose of 1.0 mL bovine serum albumin + 1.0 mL sodium azide + 1.0 mL
the Table 7. The solution was bathed in 37 ꢀC water for 10 min, target compound solution or aminoguanidine hydrochloride
aer the end, enzyme solution was added. Aer reaction at solution); ASB is the absorbance of sample blank group (1.0 mL
37 ꢀC for 20 min, 70 mL Na2CO3 solution (0.2 mM) was added to PBS + 1.0 mL bovine serum albumin + 1.0 mL sodium azide +
stop the reaction. All experiments were run in triplicate. Acar- 1.0 mL target compound solution or aminoguanidine hydro-
bose (Sigma Aldrich) was used as a standard inhibitor. Since chloride solution).
PNPG can produce glucose and p-nitrophenol (PNP) under the
4.2.5. In vitro ALR2 inhibitory activity. Aer homogeniza-
action of a-glucosidase, PNP has the greatest absorption at tion and centrifugation, the crude ALR2 from mice was ob-
405 nm. The absorbance was determined by the microplate tained.38 The inhibitory activity of the compounds on ALR2 was
reader, and the inhibition rate of a-glucosidase and the IC50 carried out using crude enzyme and different concentrations of
value of each sample were calculated according to the formula. the compounds (1–1000 mg mLꢂ1) in 200 mM PBS (pH 6.2)
containing 0.10 mM NADPH. The reaction was initiated by
Inhibition rate% ¼ {[(AC ꢂ AB) ꢂ (AS ꢂ ASB)]/(AC ꢂ AB)}
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 avonoid quercetin was used as a reference in
ꢄ 100%
where AC is the absorbance of control group; AB is the absor-
bance of blank group; AS is the absorbance of sample group; ASB
is the absorbance of sample blank group.
4.2.4. In vitro inhibitory activity of AGEs (advanced glyca-
tion end products) formation. To prepare the AGE reaction
the ALR2 assay.
4.2.6. 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 experi-
ments, food was withheld, but animals had free access to
drinking water. The compounds were suspended in vehicle
Table 7 The amount and order of each reactant of a-glucosidase
(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 rst, intragastric doses of 10, 100
and 1000 mg kgꢂ1 of bw of compounds were administered. On
the second, the doses were adjusted to 1600, 2900 and 5000 mg
kgꢂ1 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 behavioral pattern. At the end of the experiments
the mice were sacriced in a CO2 chamber.
4.2.7. Acute hypoglycemic assay. Type II diabetes mellitus
was induced in mice by a single intraperitoneal injection of
freshly prepared STZ (Sigma Aldrich) dissolved in 0.1 M citrate
buffer, pH 4.5, in a volume of 110 mg kgꢂ1 of bw. Aer 7 days of
inhibition test
Volume (mL)
Blank
Control
Sample blank Sample
Reagents
group group
group
group
PBS
20
0
10
0
20
10
20
0
10
10
20
0
Compounds/inhibitors
PNPG
Water
20
10
20
10
Mix well and incubate at 37 ꢀC for 10 minutes
a-Glucosidase
Mix well and react at 37 ꢀC for 20 minutes
Na2CO3 70 70
0
10
0
10
70
70
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STZ administration, blood glucose levels of each mouse were
determined. Mice with blood glucose levels higher than 11 mM
were considered diabetic and were included in the study.
STZ-Induced diabetic mice and normal mice were placed in
single cages with wire-net oors 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
kgꢂ1 of bw) were suspended in 0.05% Tween-80 in saline solu-
tion. Glibenclamide (10 mg kgꢂ1 of bw) was suspended in the
same vehicle. The target compounds were freshly prepared
immediately before experimentation and administered by the
intragastrical route at the doses of 10 mL kgꢂ1 of bw. Control
mice received only the vehicle (0.05% Tween-80 in saline solu-
tion) by the same route. Blood glucose levels were measured at
0, 1.5, 3, 5, 7, 9 h aer drugs administration.
4.2.10. Statistical analysis. Data were shown as mean ꢁ
S.D. differences between individual groups were analyzed by
using ANOVA followed by Dunnett's test. A difference with a P
value of <0.05 was considered to be signicant.
Conflict of interest
There are no conicts of interest to declare.
Acknowledgements
The authors are grateful to support from the Project of Shan-
dong Province Higher Educational Science and Technology
Program (J14M02), the Science and Technology Research
Program of Shandong Academy of Medical Sciences (2014-4),
4.2.8. Effects of daily treatment with compounds 8l and 8m
in STZ-induced diabetic mice. Type II diabetes mellitus was
induced in 24 mice by the same method showed in the section of
the induction of experimental diabetes. Thereaer, the target
compounds 8l, 8m and glibenclamide were administered once (9
a.m.) on a daily basis, for a period of 16 days, to STZ-induced
diabetic mice which were divided into four groups (A–D). A ve
group of normal mice (group E) was used as a control. Groups D
(diabetic control) and E received vehicle (0.05% Tween-80 in saline
solution) per os during 16 days. Group A received glibenclamide
(10 mg kgꢂ1 of bw daily). Groups B and groups C received different
treatments: (B) compound 8l (30 mg kgꢂ1 of bw daily); (C)
compound 8m (30 mg kgꢂ1 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 condi-
tions and maintained with free access to water and food during all
the experiment. Blood glucose concentration on animals of dia-
betic (groups A–D) and control group (group E) was estimated at
days 1, 4, 7, 10, 13 and 16. The body weights of the animals were
measured concomitantly to the blood glucose analyses. All exper-
iments were carried out using six animals per group.
Shandong
Provincial
Natural
Science
Foundation
(ZR2015YL041) and the Innovation Project of Shandong
Academy of Medical Sciences.
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34460 | RSC Adv., 2017, 7, 34448–34460
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