Design, synthesis and biological activity evaluation of a new class of 2,4-thiazolidinedione compounds as insulin enhancers

Article abstract of DOI:10.1080/14756366.2019.1608197

Diabetes mellitus (DM) is a global disease with a high incidence of type 2 diabetes. Current studies have shown that insulin enhancers play an important role in the treatment of type 2 diabetes and have great importance in the improvement of type 2 diabetes. In this research, Rosiglitazone was taken as the lead compound, and the structure was modified by using the bioisostere principle, and a new class of 2,4-thiazolanedione compound was designed and synthesised. The novel series of compounds were studied for their biological activities in vitro and in vivo. In vitro tests, the biological activities showed that the target compounds have good selective activation of peroxisome-proliferator-activated receptor γ (PPARγ), such as the compounds 6a, 6e, 6f, 6g and 6i, especially the compound 6e to PPARγ was EC₅₀ = 0.03 ± 0.01 μmol/L in vitro. Then, in vivo biological activities’ test results showed that the tendency of increasing in blood sugar had an obvious inhibiting effect, and had a significant insulin hypoglycaemic effect of enhancing and extending the exogenous. In addition, the results of cytotoxicity tests and acute toxicity tests (LD₅₀) showed that these compounds belong to the low toxicity compounds.

Full text of DOI:10.1080/14756366.2019.1608197

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and biological activity evaluation
of a new class of 2,4-thiazolidinedione compounds
as insulin enhancers
Zou Huiying, Chen Guangying & Zhou Shiyang
To cite this article: Zou Huiying, Chen Guangying & Zhou Shiyang (2019) Design, synthesis
and biological activity evaluation of a new class of 2,4-thiazolidinedione compounds as insulin
enhancers, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 981-989, DOI:
10.1080/14756366.2019.1608197
To link to this article: https://doi.org/10.1080/14756366.2019.1608197
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 981–989
https://doi.org/10.1080/14756366.2019.1608197
RESEARCH PAPER
Design, synthesis and biological activity evaluation of a new class of
2,4-thiazolidinedione compounds as insulin enhancers
Zou Huiyinga,b, Chen Guangyinga,b and Zhou Shiyanga,b
b
aCollege of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, China; Key Laboratory of Tropical Medicinal Resource
Chemistry of Ministry of Education, Hainan Normal University, Haikou, China
ABSTRACT
ARTICLE HISTORY
Received 25 February 2019
Revised 28 March 2019
Accepted 8 April 2019
Diabetes mellitus (DM) is a global disease with a high incidence of type 2 diabetes. Current studies have
shown that insulin enhancers play an important role in the treatment of type 2 diabetes and have great
importance in the improvement of type 2 diabetes. In this research, Rosiglitazone was taken as the lead
compound, and the structure was modified by using the bioisostere principle, and a new class of 2,4-thia-
zolanedione compound was designed and synthesised. The novel series of compounds were studied for
their biological activities in vitro and in vivo. In vitro tests, the biological activities showed that the target
compounds have good selective activation of peroxisome-proliferator-activated receptor c (PPARc), such
as the compounds 6a, 6e, 6f, 6g and 6i, especially the compound 6e to PPARc was
EC50 ¼ 0.03 0.01 lmol/L in vitro. Then, in vivo biological activities’ test results showed that the tendency
of increasing in blood sugar had an obvious inhibiting effect, and had a significant insulin hypoglycaemic
effect of enhancing and extending the exogenous. In addition, the results of cytotoxicity tests and acute
toxicity tests (LD50) showed that these compounds belong to the low toxicity compounds.
KEYWORDS
Diabetes mellitus; insulin
enhancers; design;
synthesis;
2,4-thiazolanedione
1. Introduction
was that the binding of insulin antibodies and insulin inhibits the
transport of insulin targets, the insulin receptors on the target
cells were reduced in hyperinsulinaemia, and the acidosis made
the body to become less sensitive to insulin. Therefore, the devel-
opment and use of drugs that could improve patients’ insulin sen-
sitivity and improve the state of insulin resistance were of
great significance for the treatment of diabetes. The insulin
enhancers used clinically include biguanides (Metformin37 and
Phenformin38–42) and thiazolidinediones (Rosiglitazone43–45 and
Pioglitazone46–51) (Figure 2). At present, thiazolidinedione hypogly-
caemic drugs were the main type of insulin enhancers. These
drugs have all characteristics of thiazolidinedione in terms of
chemical structure and could also be regarded as derivatives of
phenylpropionic acid. The mechanism of action of these drugs
could increase the sensitivity of insulin to the target tissue of the
receptor, reduce the production of liver sugars and enhance
the glucose uptake in peripheral tissues. The target of its action
was the peroxidase-proliferator activated receptor (PPAR) of
Diabetes mellitus (DM) is a kind of disease with carbohydrate, pro-
tein and fat metabolism disorder caused by multiple aetiologies,
which has a high incidence, only after those of cardiovascular dis-
ease and tumour1–3. Based on the 16th International Diabetes
Federation (IDF) conference in 1997, diabetes could be divided
into two broad categories: insulin-dependent diabetes mellitus
(IDDM, I diabetes) and non-insulin-dependent diabetes mellitus
(NIDDM, 2 diabetes)4–8. The IDDM patients of pancreatic islet
b-cells in the body were damaged9–14, the level of insulin in the
blood plasma was far lower than the normal value, and most of
the patients were under 30 years old15–19. The disease (1) was
mainly treated with insulin20–22 (Figure 1) and its analogues23–25
.
The NIDDM patients had less insulin resistance and secretion dis-
order, normal or slightly lower insulin level in plasma, and the
internal target tissue of the body was not sensitive to insulin
response26–30. Due to the relative insufficiency of insulin, hypergly-
caemia caused, and most of the patients were over 40years old.
Insulin resistance, especially in the liver and muscle tissue insulin
resistance of type 3, plays a very important role in the pathogenesis
of diabetes31–33. The NIDDM patients account for about more than
90% of the patients, and the main drugs used for treatment were
promoter to insulin secretion, insulin enhancers and a-glucosidase
inhibitors34–36 (Figure 2). In addition to the main drugs delivery was
injection way of insulin, the other was used oral way for all kinds of
type 2 diabetes drugs, so it was also called oral medications.
nucleus52–58
. The PPAR had three types: PPARa, PPARb and
PPARc (Figure 3). The thiazolidinediones drug could be activated to
PPARc, increase the sensitivity of fat cells, liver cells and skeletal
muscle cells to insulin, and promote the uptake, transport and oxi-
dation of insulin target cells to blood sugar. At the same time, it
can reduce the content of blood sugar and free fatty acids. In add-
ition, Rosiglitazone also increases glucose uptake by glucose trans-
porters 1 and 4. Rosiglitazone could also improve atherosclerosis
and correct lipid disorders. The main adverse effects of
Rosiglitazone were elevated liver transaminase levels, mild oedema
In recent years, the research has shown that the occurrence
and development of insulin resistance in type
2 plays an
extremely important role. The main reason for insulin resistance and anaemia59.
CONTACT Chen guangying
Normal University, Haikou 571158, China
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
chgying123@163.com; Zhou Shiyang
zhoushiyang520@126.com
College of Chemistry and Chemical Engineering, Hainan
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.

982
Z. HUIYING ET AL.
By combining the advantages and disadvantages of thiazoli- the lead compound and modified by the bioisostere principle
dinediones drug in the clinical application, a new type of thia-
zolidinedione structure was designed with Rosiglitazone as
(Figure 4). The newly designed target compounds retain the
basic skeleton structure of Rosiglitazone from a chemical per-
spective, and mainly optimise the structure of benzene ring
and its substituents to achieve the design of compounds
with good biological activity, low toxic and side effects.
These target compounds were synthesised by five steps of
acylchlorination, amidation, condensation, knoevenagel and
addition reaction (Scheme 1). The synthetic route was charac-
terised by simple operation, high total yield and mild reac-
tion conditions.
2. Results and discussion
2.1. The design and synthesis of target compounds
Rosiglitazone was
a thiazolanedione hypoglycaemic agent,
which belongs to insulin enhancers that were used to treat
type 2 diabetes. A recent study43–45 showed that Rosiglitazone
has a mechanism of action of PPARc and increases glucose
uptake by glucose transporters 1 and 4. The Rosiglitazone
could also improve atherosclerosis and correct lipid disorders.
The main adverse effects of Rosiglitazone were elevated liver
transaminase levels, mild oedema and anaemia. It was against
these shortcomings, the Rosiglitazone as the lead compound,
Figure 1. The protein structure of insulin20–22
.
designed and synthesised of
a
new class of 2,4-
Figure 2. The structure of NIDDM drugs.
Figure 3. The protein structure of PPAR52–58
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
983
Figure 4. Design of 2,4-thiazolidinedione compounds.
X1
X1
SOCl2, CH2Cl2
DMF, r.t. 6-10 h
H2NCH2CH2OH
COCl
R1
COOH
R1
CH2Cl2
1a-1f
X1
2a-2f
X2
Cl
CHO
X1
O
C
O
C
R1
NHCH2CH2OH
R1
NHCH2CH2O
CHO
THF, KOH, t.r. 10 h
3a-3f
4a-4l
O
NH
O
X1
O
X2
NH
O
S
O
R1
C NH
O
Acetate/Toluene, r.t. 6 h
S
5a-5l
O
O
X1
X2
NH
Mg/MeOH
r.t. 12 h
O
R1
C NH
O
S
6a-6l
R1
X1
C
C
N
N
C
C
X2
C
N
C
N
C
N
R1
X1
X2
C
N
C
N
C
N
6a:
6b:
6c:
6d:
6e:
6f:
-H
6g:
6h:
6i:
6j:
6k:
6l:
-CH3
-CH3
-OH
-OH
-OH
N
N
C
C
N
N
-H
-H
-H
-CH3
-CH3
-OH
Scheme 1. The synthetic route of 2,4-thiazolidinedione compounds.
thiazolanedione compounds for the treatment of type 2 dia- on the ring selection –H, –CH3 and –OH. Such structural modi-
betes which was very necessary. The novel series of com- fication was designed to change the logP, pKa and spatial
pounds were designed, and retain the basic skeleton structures of the target compounds in order to obtain the tar-
of Rosiglitazone in terms of its chemical structure, namely get compounds with good biological activity and little toxic
2,4-thiazolidinedione and six-membered ring structure (Figure and side effects. In terms of synthetic route, we selected a syn-
4). According to the structure–activity relationship (SAR), the thetic route with simple operation, mild reaction conditions
target compounds should have the same treatment effect as and high total yield. The synthesis of the target compounds
Rosiglitazone or be superior to its effect. In the design of the was of five steps: acylchlorination, amidation, condensation,
target compounds, the six-membered ring and substituents on knoevenagel and addition reaction (Scheme 1). A total of 12
the ring were modified. The specific structure modification was compounds were synthesised and they were analysed by 1H
selection C atom and N atom on the six-membered ring by NMR, 13C NMR, MS and elemental analysis. The yield of the
using the modified bioisostere principle, and the substituents target compounds 6a–6f was 69.8–84.2%.

984
Z. HUIYING ET AL.
Table 1. In vitro the activation of test compounds on PPARa, PPARb and PPARc.
EC50 SDa（lmol/l）
PPARb
Compounds
R1
–H
–H
–H
–H
–CH3
–CH3
–CH3
–CH3
–OH
–OH
–OH
–OH
/
X1
X2
log p
pKa
PPARa
iab
12.90 1.29
13.02 1.56
PPARc
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
C
C
N
N
C
C
N
N
C
C
N
N
/
C
N
C
N
C
N
C
N
C
N
C
N
/
2.68
1.77
1.92
1.38
3.26
2.56
2.83
2.01
2.80
2.11
2.32
1.45
/
10.43
10.38
10.00
9.78
9.77
9.65
9.45
9.41
9.03
9.01
8.66
8.58
/
ia
0.08 0.02
1.29 0.53
0.45 0.21
3.22 0.65
0.03 0.01
0.10 0.10
0.08 0.03
0.28 0.13
0.07 0.02
0.34 0.15
0.21 0.11
2.68 0.46
0.08 0.02
ia
9.18 0.89
ia
ia
ia
ia
ia
12.19 1.21
ia
ia
ia
ia
10.56 1.86
ia
ia
ia
ia
14.53 1.20
ia
10.11 1.12
12.23 1.35
ia
9.34 1.01
9.05 1.15
ia
Rosiglitazone
DMSO
/
/
/
/
/
aData represent the mean values from at least eight independent experiments each in triplicate (n ꢁ 8).
bia ¼ inactive.
2.2. The biological activities
100
2.2.1. The biological activity screening in vitro
98
96
94
92
90
88
86
84
82
80
Drug in vitro experiment was one of the basic contents to evalu-
ate drugs activity, and it was a necessary approach to research
drugs. Drugs’ efficacy was often affected by some physical and
chemical properties of compounds, such as lipid–water partition
coefficient (log p), and dissociation degree (pKa). In this part of
the research, the physical and chemical properties of 2,4-thiazoli-
dinedione compounds were studied that were very necessary to
study the pharmacodynamics (Table 1). In Table 1, we could find
that target compounds were fat-soluble. The fat-soluble com-
pounds were favourable for drugs absorption, and drugs were
easy to enter the cells. This compound the log p was from 1.45 to
3.26. The pKa will affect the absorption and delivery of the drugs,
different environmental (acid and alkalinity) medicine may be dif-
ferent, sometimes exists in the form of ions, sometimes exists in
the form of molecules. Usually in the form of ion drugs has no
effect. Therefore, it was necessary to study the dissociation degree
(pKa) of drugs to study the pharmacodynamics. In Table 1, we
could find that target compounds the pKa were from 8.58 to
10.43, and these compounds could be absorbed in the intestinal
tract. After the physical and chemical properties were studied, the
biological activity was studied. The in vitro biological activity
evaluation was based on the concentration of 50% of maximal
effect (EC50), and PPARa, PPARb and PPARc were selected
(Table 1). In Table 1, we can find that these compounds had a
good biological activity to PPARc, but inactive to PPARa and
PPARb. These data show that these kinds of target compounds
have good selectivity. Especially, as compounds 6a, 6e, 6f, 6g and
f
i
a
e
g
6
6
6
6
ne
o
d
d
d 6
n
n
z
n
u
und
und
u
ta
li
ou
mp
co
o
o
o
o
p
p
p
p
g
m
si
m
o
m
m
o
o
c
co
c
co
R
Figure 5. In vitro activities of PPARc agonists activity.
different R1 (–H, –CH3 and –OH) substituents have little effect on
EC50, which was mainly affected by the two six-membered ring
structures in the target compounds. As could be seen from the
data of in vitro experiments, when both six-membered rings were
benzene rings, the target compounds had a good biological activ-
ity to PPARc. In addition, when the first six-membered ring was
pyridine ring and the second six-membered ring was benzene
ring, it also shows a good biological activity to PPARc.
6i had better biological activity in vitro, which of compound 6e to 2.2.2. The evaluation of biological activity in vivo
PPARc was EC50 ¼ 0.03 0.01 lmol/l. The in vitro biological activity The compounds 6a, 6e, 6f, 6g and 6i were selected from the in
evaluation was based on the concentration of 50% of maximal vivo activity test, and it was used for in vivo activity evaluation. In
effect (EC50), and PPARa, PPARb and PPARc were selected vivo experiments, glucose tolerance test, insulin tolerance test, cell
(Table 1). In Table 1, we can found that these compounds had a survival experiment and acute toxicity were used as the test indi-
good biological activity to PPARc, but inactive to PPARa and cators (Tables 2, 3 and 4). In Table 2, we chose dose 10 mg kgꢀ1
PPARb. These data showed that these kinds of target compounds dꢀ1 and used oral administration. The Rosiglitazone was used as
have good selectivity. Especially, as compounds 6a, 6e, 6f, 6g and the positive reference substance and DMSO was used as the blank
6i had better biological activity in vitro, which of compound 6e to control. The mice were given the drug for 10 d, blood was col-
PPARc was EC50 ¼ 0.03 0.01 lmol/l. After that, the target com- lected from the tail vein of the mice before and after the sugar
pounds 6a, 6e, 6f, 6g and 6i had good biological activity and load for 1 h and 2 h, and the blood glucose level of the mice
were preliminarily screened out for further study on PPARc recep- before and after the sugar load was measured by the glucometer.
tor agonist activity (Figure 5). The experimental results showed The experimental results showed that the compounds 6a, 6e, 6f,
that the five target compounds had good biological activity in 6g and 6i had good resistance activation to glucose. In Table 3,
vitro. According to the structure–activity relationship (SAR), experimental results showed that the compounds 6a, 6e, 6f, 6g

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
985
Table 2. The oral glucose tolerance test.
Blood glucose levelsa (mmol lꢀ1
1 h
)
Compounds
Dose (mg kgꢀ1 dꢀ1
)
0 h
2 h
6a
6e
6f
6g
10
10
10
10
10
10
10
3.376 0.361
3.312 0.301
3.216 0.315
3.112 0.279
3.218 0.317
3.265 0.345
3.471 0.211
6.342 2.011
6.003 1.876
6.651 2.413
6.445 2.220
6.401 2.112
6.351 2.004
8.756 2.102
3.368 1.341
2.953 0.876
4.121 1.922
3.677 1.730
3.265 1.236
3.654 1.232
4.371 1.621
6i
Rosiglitazone
DMSO
aData represent the mean values from at least 10 independent experiments each in triplicate (n ꢁ 10).
Table 3. The effect of compounds on blood glucose under insulin load.
Blood glucose levelsa (mmol lꢀ1
)
Compounds
Dose (mg kgꢀ1 dꢀ1
)
0 h
1 h
2 h
6a
6e
6f
6g
10
10
10
10
10
10
10
5.644 1.035
5.411 1.002
5.714 1.465
5.613 1.246
5.592 1.134
5.613 1.124
5.214 0.761
1.801 0.531
1.762 0.453
1.903 0.611
1.843 0.544
1.792 0.541
1.811 0.497
1.842 0.513
2.498 1.136
2.463 1.211
2.602 1.143
2.566 1.224
2.501 1.025
2.513 1.072
3.024 1.671
6i
Rosiglitazone
DMSO
aData represent the mean values from at least 10 independent experiments each in triplicate (n ꢁ 10).
Table 4. The cytotoxicity and acute toxicity tests.
Cell survival ratea (%)
Compounds
10ꢀ6 mol lꢀ1
10ꢀ5 mol lꢀ1
10ꢀ4 mol lꢀ1
10ꢀ3 mol lꢀ1
10ꢀ2 mol lꢀ1
LD50 SDb(mg kgꢀ1
)
6a
6e
6f
6g
100
100
100
100
100
99.9 0.11
100
99.8 0.09
100
100
99.5 0.21
99.3 0.26
100
99.2 0.31
99.8 0.09
99.9 0.02
98.1 0.61
98.1 0.28
99.1 0.08
98.3 0.45
98.6 0.44
98.8 0.29
97.2 0.71
93.1 1.21
94.3 1.02
89.6 1.34
90.5 1.54
92.1 1.43
86.5 2.41
2313.6 1.26
2451.2 1.24
2013.6 1.43
2138.8 1.36
2268.9 1.25
2246.1 1.56
6i
Rosiglitazone
99.8 0.05
aData represent the mean values from at least ten independent experiments each in triplicate (n ꢁ 10).
bData represent the mean values from at least three independent experiments each in triplicate (n ꢁ 3).
and 6i had good resistance activation to insulin. In Table 4, the
results of cell survival experiments show that in the compounds
6a, 6e, 6f, 6g and 6i in the 10ꢀ3–10ꢀ2 mol Lꢀ1, no obvious cyto-
4. Experimental
4.1. Chemistry section
toxic effect was observed. In Table 4, the acute toxicity test results 4.1.1. A general method for synthesis of compounds 2a to 2f
The benzoic acid (1a, 12.03 g, 0.10 mol) was put into 500 ml
round bottom flask, then 200 ml of methylene chloride
(CH2Cl2) was used as the solvent and 10 drops of N,N’-dimethyl
formamide (DMF) used as a catalyst for the reaction were
added. The flask was placed in the ice water bath (ꢂ10 ꢃC),
and a magnetic mixer was used to stir until the reaction liquid
became clarified. The thionyl chloride (SOCl2, 11.25 ml,
0.15 mol) was constantly dropped into the flask under stirring,
and the drop speed rate and the reaction temperature were
controlled (ꢂ10 ꢃC and ꢁ20 min). After the reactants were
added, the reaction lasted for 6–10 h under reflux. The methy-
show the oral administration of the compounds, and it belongs to
low toxicity compounds.
3. Conclusion
We report here the design, synthesis and evaluation of a series of
novel PPARc selective activators containing 2,4-thiazolanedione
compounds. Rosiglitazone is used as the lead compound, a total
of 12 compounds (6a–6l) were designed and synthesised, and
used the bioisostere principle to modify the lead compound. We
selected five steps to synthesis the target compounds, and the lene chloride and excessive thionyl chloride were removed
route with high total yield, mild conditions and simple operation.
under vacuum. The mixture was dried to get the crude product
Structure–activity relationship (SAR) studies led to the identifica- of benzoyl chloride (2a). The crude product was obtained by
tion of PPARc selective activators (the compounds 6a, 6e, 6f, 6g atmospheric distillation, the product was collected at 198 ꢃC,
and 6i) with stronger potency and efficacy to activate PPARc. The and the pure product of benzoyl chloride (2a) as a colourless
compound 6e to PPARc was EC50 ¼ 0.03 0.01 lmol/L in vitro. The transparent liquid was obtained. The general method was used
in vivo biological activity test results showed that the tendency of to synthesise compounds 2c and 2e as all were colourless
increasing the blood sugar had obvious inhibition, and could sig- transparent liquid. And the general method was used to syn-
nificantly enhance and extend the exogenous insulin hypogly- thesise crude compounds 2b, 2d and 2f, the crude products
caemic effect. In addition, the results of cytotoxicity test and acute were recrystallised with hexane, filtered and dried in vacuum
toxicity test showed that these compounds belong to low tox- to give pure products of compounds 2b, 2d and 2f as all were
icity compounds.
of white crystal.

986
Z. HUIYING ET AL.
4.1.2. A general method for synthesis of compounds 3a to 3f
The benzoyl chloride (2a, 14.05 g, 0.10 mol) was put into 500 ml
the filtrate was poured into the ice water. The extract was
extracted three times with ethyl acetate, combined with the
round bottom flask, then 200 ml of methylene chloride (CH2Cl2) superstratum extract, and washed twice with saturated sodium
was added as a solvent for the reaction. The flask was placed in bicarbonate and saturated sodium chloride solution, and dried
the ice water bath (0–10 ꢃC), and a magnetic mixer was used to with anhydrous calcium chloride after washing. Decompression
stir until the reaction liquid became clarified. The ethanolamine
(6.11 g, 0.10 mol) was constantly dropped into the flask under stir-
ring, and the drop speed rate and the reaction temperature were
controlled (0–10 ꢃC and ꢁ20 min). After the reactants were added,
the reaction lasted 6 h under standing. The methylene chloride
(CH2Cl2) was removed under vacuum. The mixture was dried to
get the crude product of N-(2-hydroxyethyl) benzamide (3a). The
crude product was recrystallised with toluene, filtered and dried in
vacuum to give pure product of 4-chloro-N-(hydroxymethyl) ben-
zamide (3a) as a white crystal. The general method was used to
synthesis compound 3b to 3f as all were of white crystal.
distillation and recovery of solvent methanol were done to obtain
the crude product of compound 5a. The crude product was
recrystallized with methanol to give pure product of compound
6a which is of a white powder. The general method was used to
synthesise compounds 6b to 6l as all were of white powder.
N-(2–(4-((2,4-dioxothiazolidin-5-yl) methyl) phenoxy)ethyl) ben-
1
zamide (6a): white powder, yield 84.2%, m.p. 164–166 ꢃC; H NMR
(300 MHz, DMSO) d: 3.32 (2H, d, J ¼ 6.1 Hz, –CH2–), 3.48 (2H, t,
J ¼ 7.2 Hz, –CH2–), 4.14 (2H, t, J ¼ 7.2 Hz, –CH2–), 4.46 (1H, t,
J ¼ 6.1 Hz, –CH–), 6.92 (2H, ddd, J ¼ 8.8, 1.8, 0.5 Hz, Ph-H), 7.11 (2H,
ddd, J ¼ 8.8, 1.0, 0.5 Hz, Ph-H), 7.48 (2H, dddd, J ¼ 8.5, 7.5, 1.4,
0.4 Hz, Ph-H), 7.63 (1H, tt, J ¼ 7.5, 1.5 Hz, Ph-H), 7.85 (2H, dddd,
J ¼ 8.5, 1.6, 1.5, 0.4 Hz, Ph-H); 13C NMR (75 MHz, DMSO) d: 39.4,
39.7, 49.8, 61.2, 115.2, 127.6, 127.9, 128.5, 128.9, 133.7, 1, 158.4,
168.1, 169.1, 175.8; HR-ESI-MS m/z: calcd for C19H18N2O4S｛
(M þ H)þ｝ 370.0986, found 370.4231; Anal. calcd for C19H18N2O4S:
C, 61.61; H, 4.90; N, 7.56; O, 17.28; S, 8.65; found: C, 61.62; H, 4.91;
N, 7.54; O, 17.27; S, 8.66%.
N-(2-((6-((2,4-dioxothiazolidin-5-yl) methyl) pyridin-3-yl) oxy)
ethyl) benzamide (6b): white powder, yield 82.7%, m.p.
171–173 ꢃC; 1H NMR (300 MHz, DMSO) d: 3.14 (2H, d, J ¼ 6.3 Hz,
–CH2–), 3.48 (2H, t, J ¼ 6.5 Hz, –CH2–), 4.15 (2H, t, J ¼ 6.5 Hz,
–CH2–), 4.46 (1H, t, J ¼ 6.3 Hz, –CH–), 7.03 (1H, dd, J ¼ 8.0, 0.5 Hz,
Py-H), 7.32 (1H, dd, J ¼ 8.0, 1.6 Hz, Py-H), 7.46 (2H, dddd, J ¼ 8.5,
7.5, 1.4, 0.4 Hz, Ph-H), 7.65 (1H, tt, J ¼ 7.5, 1.5 Hz, Ph-H), 7.78 (2H,
dddd, J ¼ 8.5, 1.6, 1.5, 0.4 Hz, Ph-H), 8.23 (1H, dd, J ¼ 1.6, 0.5 Hz,
Py-H); 13C NMR (75 MHz, DMSO) d: 30.4, 39.4, 49.8, 61.2, 122.1,
127.8, 128.6, 129.7, 133.7, 142.6, 151.4, 160.4, 168.1, 169.2, 175.7;
HR-ESI-MS m/z: calcd for C18H17N3O4S｛ (M þ H)þ｝ 371.0943,
found 371.4112; Anal. calcd for C18H17N3O4S: C, 58.21; H, 4.61; N,
11.31; O, 17.23; S, 8.63; found: C, 58.22; H, 4.60; N, 11.32; O, 17.21;
S, 8.64%.
4.1.3. A general method for synthesis of compounds 4a to 4l
The compound 3a (16.51 g, 0.10 mol) and the solid potassium
hydroxide (5.61 g, 0.01 mol) were put into 500 ml round bottom
flask, and 100 ml tetrahydrofuran (THF) was added as the reaction
solvent. Under room temperature with magnetic stirring, the 4-
chlorobenzaldehyde (14.05 g, 0.10 mol) was added at a constant
pressure by a drop funnel, and the rate of droplet acceleration
was controlled. When the dripping was completed, under the con-
dition of nitrogen protection, the reaction was refluxed for 10 h.
When the reaction was complete, the reaction solution was
poured into the ice water, then the ethyl acetate extract was
added three times, and added with the superstratum extract. The
extract was washed twice with saturated sodium chloride solution
and dried with anhydrous calcium chloride. Decompression distil-
lation and recovery of ethyl acetate were done to obtain the
crude product of compound 4a. The crude product was recrystal-
lized with toluene filtered, and dried in vacuum to give pure prod-
uct of compound 4a with a white crystal. The general method
was used to synthesise compounds 4b to 4l as all were of
white crystal.
N-(2-(4-((2,4-dioxothiazolidin-5-yl) methyl) phenoxy) ethyl) pico-
linamide (6c): white powder, yield 80.5%, m.p. 174–176 ꢃC; 1H
NMR (300 MHz, DMSO) d: 2.88 (2H, d, J ¼ 6.1 Hz, –CH2–), 3.50 (2H,
t, J ¼ 7.1 Hz, –CH2–), 4.17 (2H, t, J ¼ 7.1 Hz, –CH2–), 4.49 (1H, t,
J ¼ 6.1 Hz, –CH–), 7.01 (2H, ddd, J ¼ 8.8, 1.8, 0.5 Hz, Ph-H), 7.12 (2H,
ddd, J ¼ 8.8, 1.0, 0.5 Hz, Ph-H), 7.56 (1H, ddd, J ¼ 7.6, 5.2, 1.7 Hz,
Py-H), 7.94–8.02 (2H, 8.01 (ddd, J ¼ 8.2, 1.7, 0.5 Hz, Py-H), 7.97
(ddd, J ¼ 8.2, 7.6, 1.9 Hz, Py-H)), 8.71 (1H, ddd, J ¼ 5.2, 1.9, 0.5 Hz,
Py-H); 13C NMR (75 MHz, DMSO) d: 39.2, 39.7, 49.8, 60.8, 115.2,
122.6, 123.4, 127.7, 134.0, 137.2, 148.3, 151.7, 158.4, 164.5, 169.0,
175.6; HR-ESI-MS m/z: calcd for C18H17N3O4S｛ (M þ H)þ｝
371.0942, found 371.4111; Anal. calcd for C18H17N3O4S: C, 58.21; H,
4.61; N, 11.31; O, 17.23; S, 8.63; found: C, 58.22; H, 4.60; N, 11.33;
O, 17.22; S, 8.62%.
N-(2-((6-((2,4-dioxothiazolidin-5-yl) methyl) pyridin-3-yl) oxy)
ethyl) picolinamide (6d): white powder, yield 73.9%, m.p.
189–191 ꢃC; 1H NMR (300 MHz, DMSO) d: 3.12 (2H, d, J ¼ 6.3 Hz,
–CH2–), 3.49 (2H, t, J ¼ 6.9 Hz, –CH2–), 4.18 (2H, t, J ¼ 6.9 Hz,
–CH2–), 4.42 (1H, t, J ¼ 6.3 Hz, –CH–), 7.02 (1H, dd, J ¼ 8.0, 0.5 Hz,
Py-H), 7.33 (1H, dd, J ¼ 8.0, 1.6 Hz, Py-H), 7.57 (1H, ddd, J ¼ 7.6, 5.2,
1.7 Hz, Py-H), 7.92–8.02 (2H, 8.01 (ddd, J ¼ 8.2, 1.7, 0.5 Hz, Py-H),
7.97 (ddd, J ¼ 8.2, 7.6, 1.9 Hz, Py-H)), 8.27 (1H, dd, J ¼ 1.6, 0.5 Hz,
Py-H), 8.70 (1H, ddd, J ¼ 5.2, 1.9, 0.5 Hz, Py-H); 13C NMR (75 MHz,
DMSO) d: 30.4, 39.2, 49.9, 60.8, 122.0, 122.6, 123.5, 129.7, 137.4,
4.1.4. A general method for synthesis of compounds 5a to 5l
The compound 4a (26.93 g, 0.10 mol) and 2,4-thiazolanedione
(11.71 g, 0.10 mol) were put into 500 ml round bottom flask. The
100 ml of glacial acetic acid was added as the reaction solvent.
Ten drops of hexahydropyridine were added as the reaction cata-
lyst, and then 30 ml of toluene was added as the water solvent.
The water divider was connected that divided water for 6 h. When
the water was separated, the reaction liquid was cooled and then
refrigerated for 24 h. The precipitate was precipitated, filtered and
the precipitate was washed with ether for three times, and the
mixture was vacuum dried to get the crude product of compound
5a. The crude product was recrystallised with toluene to give pure
product of compound 5a which is of a white crystal. The general
method was used to synthesise compounds 5b to 5l as all were
of white crystal.
4.1.5. A general method for synthesis of compounds 6a to 6l
The compound 5a (37.04 g, 0.10 mol) was put into 500 ml round
bottom flask, then 200 ml of methanol was added as the solvent.
The magnesium powder (24.00 g, 0.10 mol) was added to a round
bottom flask, and the refluxing reaction was performed for 12 h
with the protection of nitrogen. After the reflux reaction was com- 142.6, 148.4, 151.4, 151.8, 160.4, 164.5, 169.0, 175.7; HR-ESI-MS m/
pleted, it was immediately filtrated, the filtrate was collected, and z: calcd for C17H16N4O4S｛(M þ H)þ｝ 372.0891, found 372.3992;

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
987
Anal. calcd for C17H16N4O4S: C, 54.83; H, 4.33; N, 15.05; O, 17.18; S, 39.2, 39.7, 49.8, 60.8, 115.0, 115.7 127.5, 127.9, 133.0,134.1,
8.61; found: C, 54.81; H, 4.34; N, 15.06; O, 17.17; S, 8.62%.
157.7, 158.6, 168.3,169.0, 175.7; HR-ESI-MS m/z: calcd for
N-(2-(4-((2,4-dioxothiazolidin-5-yl) methyl) phenoxy) ethyl)-4- C19H18N2O5S｛ (M þ H)þ｝ 386.0933, found 386.4221; Anal. calcd
methylbenzamide (6e): white powder, yield 75.1%, m.p. for C19H18N2O5S: C, 59.06; H, 4.70; N, 7.25; O, 20.70; S, 8.30; found:
170–172 ꢃC; 1H NMR (300 MHz, DMSO) d: 2.32 (3H, s, –CH3), 2.88 C, 59.04; H, 4.71; N, 7.26; O, 20.71; S, 8.29%.
N-(2-((6-((2,4-dioxothiazolidin-5-yl) methyl) pyridin-3-yl)oxy)
(2H, d, J ¼ 6.1 Hz, –CH2–), 3.48 (2H, t, J ¼ 7.2 Hz, –CH2–), 4.15(2H, t,
ethyl)-4-hydroxybenzamide (6j): white powder, yield 79.1%, m.p.
J ¼ 7.2 Hz, –CH2–), 4.47 (1H, t, J ¼ 6.1 Hz, –CH–), 7.01 (2H, ddd,
197–199 ꢃC; 1H NMR (300 MHz, DMSO) d: 3.12(2H, d, J ¼ 6.3 Hz,
–CH2–), 3.47 (2H, t, J ¼ 6.5 Hz, –CH2–), 4.18 (2H, t, J ¼ 6.5 Hz,
–CH2–), 4.42 (1H, t, J ¼ 6.3 Hz, –CH–), 6.99 (1H, dd, J ¼ 8.0, 0.5 Hz,
Py-H), 7.01 (2H, ddd, J ¼ 8.6, 1.1, 0.4 Hz, Ph-H), 7.35 (1H, dd, J ¼ 8.0,
1.6 Hz, Py-H), 7.98 (2H, ddd, J ¼ 8.6, 1.7, 0.4 Hz, Ph-H), 8.27 (1H, dd,
J ¼ 1.6, 0.5 Hz, Py-H); 13C NMR (75 MHz, DMSO) d: 30.5, 39.2,
49.8, 60.8, 115.6, 122.0, 127.6, 129.7, 133.0,142.6, 151.6,
157.9, 160.4, 168.3, 169.0, 175,7; HR-ESI-MS m/z: calcd for
C18H17N3O5S｛ (M þ H)þ｝ 387.0887, found 387.4101; Anal. calcd
for C18H17N3O5S: C, 55.81; H, 4.42; N, 10.85; O, 20.65; S, 8.28;
found: C, 55.83; H, 4.41; N, 10.84; O, 20.665; S, 8.27%.
N-(2-(4-((2,4-dioxothiazolidin-5-yl) methyl) phenoxy) ethyl)-5-
hydroxypicolinamide (6k): white powder, yield 77.2%, m.p.
201–203 ꢃC; 1H NMR (300 MHz, DMSO) d: 2.88(2H, d, J ¼ 6.1 Hz,
–CH2–), 3.48 (2H, t, J ¼ 7.1 Hz, –CH2–), 4.17 (2H, t, J ¼ 7.1 Hz,
–CH2–), 4.46 (1H, t, J ¼ 6.1 Hz, –CH–), 7.01 (2H, ddd, J ¼ 8.8, 1.8,
0.5 Hz, Ph-H), 7.10 (2H, ddd, J ¼ 8.8, 1.0, 0.5 Hz, Ph-H), 7.71 (1H, dd,
J ¼ 8.1, 1.8 Hz, Py-H), 7.87 (1H, dd, J ¼ 8.1, 0.5 Hz, Py-H), 8.46 (1H,
dd, J ¼ 1.8, 0.5 Hz, Py-H); 13C NMR (75 MHz, DMSO) d: 39.2,
39.7, 49.8, 60.8, 114.7, 115.1, 122.0, 127.9, 134.0, 135.3, 151.3,
151.9, 158.6, 164.3, 169.0, 175.6; HR-ESI-MS m/z: calcd for
C18H17N3O5S｛ (M þ H)þ｝ 387.0887, found 387.4101; Anal. calcd
for C18H17N3O5S: C, 55.81; H, 4.42; N, 10.85; O, 20.65; S, 8.28;
found: C, 55.83; H, 4.41; N, 10.84; O, 20.665; S, 8.27%.
N-(2-((6-((2,4-dioxothiazolidin-5-yl) methyl) pyridin-3-yl) oxy)
ethyl)-5-hydroxypicolinamide (6l): white powder, yield 70.6%, m.p.
211–213 ꢃC; 1H NMR (300 MHz, DMSO) d: 3.11 (2H, d, J ¼ 6.3 Hz,
–CH2–), 3.47 (2H, t, J ¼ 6.9 Hz, –CH2–), 4.18 (2H, t, J ¼ 6.9 Hz,
–CH2–), 4.42 (1H, t, J ¼ 6.3 Hz, –CH–), 7.00 (1H, dd, J ¼ 8.0, 0.5 Hz,
Py-H), 7.35 (1H, dd, J ¼ 8.0, 1.6 Hz, Py-H), 7.70 (1H, dd, J ¼ 8.1,
1.8 Hz, Py-H), 7.88 (1H, dd, J ¼ 8.1, 0.5 Hz, Py-H), 8.27 (1H, dd,
J ¼ 1.6, 0.5 Hz, Py-H), 8.46 (1H, dd, J ¼ 1.8, 0.5 Hz, Py-H); 13C NMR
(75 MHz, DMSO) d: 30.4, 39.2, 49.8, 60.8, 114.8, 119.8,122.1, 129.0,
135.2, 142.6, 151.3, 151.5, 151.9, 160.2, 164.3, 169.0,175.6; HR-ESI-
MS m/z: calcd for C17H16N4O5S｛ (M þ H)þ｝ 387.0887, found
387.4101; Anal. calcd for C17H16N4O5S: C, 52.57; H, 4.15; N, 14.43;
O, 20.60; S, 8.25; found: C, 52.59; H, 4.15; N, 14.45; O, 20.61;
S, 8.24%.
J ¼ 8.8, 1.8, 0.5 Hz, Ph-H), 7.06–7.20 (4H, 7.16 (ddd, J ¼ 8.5, 1.2,
0.5 Hz, Ph-H), 7.11 (ddd, J ¼ 8.8, 1.0, 0.5 Hz, Ph-H)), 7.86 (2H,
ddd, J ¼ 8.5, 1.7, 0.5 Hz, Ph-H); 13C NMR (75 MHz, DMSO) d: 21.4,
39.2, 39.7, 49.8, 60.8, 115.2, 127.5, 127.9, 128.6, 131.0,
139.7, 158.5, 168.3, 169.0, 175.6; HR-ESI-MS m/z: calcd for
C20H20N2O4S｛ (M þ H)þ｝ 384.1142, found 384.4503; Anal. calcd
for C20H20N2O4S: C, 62.48; H, 5.24; N, 7.29; O, 16.65; S, 8.34; found:
C, 62.46; H, 5.25; N, 7.28; O, 16.66; S, 8.35%.
N-(2-((6-((2,4-dioxothiazolidin-5-yl) methyl) pyridin-3-yl) oxy)
ethyl)-4-methylbenzamide (6f): white powder, yield 74.2%, m.p.
177–179 ꢃC; 1H NMR (300 MHz, DMSO) d: 2.32 (3H, s, –CH3), 3.12
(2H, d, J ¼ 6.3 Hz, –CH2–), 3.48 (2H, t, J ¼ 6.5 Hz, –CH2–), 4.18 (2H, t,
J ¼ 6.5 Hz, –CH2–), 4.42 (1H, t, J ¼ 6.3 Hz, –CH–), 7.01 (1H, dd,
J ¼ 8.0, 0.5 Hz, Py-H), 7.16 (2H, ddd, J ¼ 8.5, 1.2, 0.5 Hz, Ph-H), 7.35
(1H, dd, J ¼ 8.0, 1.6 Hz, Py-H), 7.86 (2H, ddd, J ¼ 8.5, 1.7, 0.5 Hz, Ph-
H), 8.26 (1H, dd, J ¼ 1.6, 0.5 Hz, Py-H); 13C NMR (75 MHz, DMSO) d:
21.2, 30.6, 39.4, 49.8, 60.8, 122.0, 127.5, 128.6, 129.4, 131.0, 139.7,
142.6, 151.5, 160.2, 168.1, 169.0, 175.6; HR-ESI-MS m/z: calcd for
C19H19N3O4S｛(M þ H)þ｝ 385.1094, found 385.4381; Anal. calcd
for C19H19N3O4S: C, 59.21; H, 4.97; N, 10.90; O, 16.60; S, 8.32;
found: C, 59.23; H, 4.96; N, 10.91; O, 16.60; S, 8.30%.
N-(2-(4-((2,4-dioxothiazolidin-5-yl) methyl) phenoxy) ethyl)-5-
methylpicolinamide (6g): white powder, yield 73.1%, m.p.
180–182 ꢃC; 1H NMR (300 MHz, DMSO) d: 2.32 (3H, s, –CH3), 3.12
(2H, d, J ¼ 6.3 Hz, –CH2–), 3.48 (2H, t, J ¼ 6.5 Hz, –CH2–), 4.18(2H, t,
J ¼ 6.5 Hz, –CH2–), 4.42 (1H, t, J ¼ 6.3 Hz, –CH–), 7.01 (1H, dd,
J ¼ 8.0, 0.5 Hz, Py-H), 7.16 (2H, ddd, J ¼ 8.5, 1.2, 0.5 Hz, Ph-H), 7.35
(1H, dd, J ¼ 8.0, 1.6 Hz, Py-H), 7.86 (2H, ddd, J ¼ 8.5, 1.7, 0.5 Hz, Ph-
H), 8.26 (1H, dd, J ¼ 1.6, 0.5 Hz, Py-H); 13C NMR (75 MHz, DMSO) d:
17.6, 39.2, 39.7, 49.8, 60.8, 115.0, 122.0, 127.8, 132.5, 134.0, 137.3,
152.0,152.8, 158.6, 164.3, 169.0, 175.6; HR-ESI-MS m/z: calcd for
C19H19N3O4S｛ (M þ H)þ｝ 385.1095, found 385.4382; Anal. calcd
for C19H19N3O4S: C, 59.21; H, 4.97; N, 10.90; O, 16.60; S, 8.32;
found: C, 59.23; H, 4.96; N, 10.90; O, 16.60; S, 8.31%.
N-(2-((6-((2,4-dioxothiazolidin-5-yl) methyl) pyridin-3-yl)oxy)
ethyl)-5-methylpicolinamide (6h): white powder, yield 73.1%, m.p.
189–191 ꢃC; 1H NMR (300 MHz, DMSO) d: 2.26 (3H, s, –CH3), 3.12
(2H, d, J ¼ 6.3 Hz, –CH2–), 3.49 (2H, t, J ¼ 6.9 Hz, –CH2–), 4.18 (2H, t,
J ¼ 6.9 Hz, –CH2–), 4.42 (1H, t, J ¼ 6.3 Hz, –CH–), 7.01 (1H, dd,
J ¼ 8.0, 0.5 Hz, Py-H), 7.35 (1H, dd, J ¼ 8.0, 1.6 Hz, Py-H), 7.86–7.94
(2H, 7.92 (dd, J ¼ 8.2, 0.6 Hz, Py-H), 7.91 (dd, J ¼ 8.2, 2.0 Hz, Py-H)),
8.26 (1H, dd, J ¼ 1.6, 0.5 Hz, Py-H), 8.46 (1H, dd, J ¼ 2.0, 0.6 Hz,
Py-H); 13C NMR (75 MHz, DMSO) d: 17.6, 30.4, 39.2, 49.8,
60.8, 122.0, 122.2, 129.7, 132.5, 137.2, 142.6, 151.4, 152.0,
152.6 160.4, 164.5, 169.0, 175.6; HR-ESI-MS m/z: calcd for
C18H18N4O4S｛ (M þ H)þ｝ 386.1048, found 386.4261; Anal. calcd
for C18H18N4O4S: C, 55.95; H, 4.70; N, 14.50; O, 16.56; S, 8.30;
found: C, 55.93; H, 4.71; N, 14.51; O, 16.55; S, 8.31%.
4.2. Biological section
4.2.1. Screening of biological activity of compounds in vitro
According to the literature60, the cells containing PPAR (a, b
and c) were obtained and transfected into 96-well plates the day
before for cells culture. Transfection was performed according to
the instructions of human PPAR enzyme-linked immunoassay.
Various compounds (6a–6l) were dissolved in DMSO and were
added after the cells adhered to the wall. The Rosiglitazone was
used as the positive control group and DMSO was used as the
blank control group during the experiment. After 24 h, the drugs
were added, and 20 ll of MTT was added to each well for another
6 h, and the cell liquid was collected by removing the culture
medium, and 150 ll of DMSO was added to each hole. And the
absorbance value (A) of each hole was measured under the wave-
N-(2-(4-((2,4-dioxothiazolidin-5-yl) methyl) phenoxy) ethyl)-4-
hydroxybenzamide (6i): white powder, yield 80.3%, m.p.
192–194 ꢃC; 1H NMR (300 MHz, DMSO) d: 2.87(2H, d, J ¼ 6.1 Hz,
–CH2–), 3.47 (2H, t, J ¼ 7.2 Hz, –CH2–), 4.12 (2H, t, J ¼ 7.2 Hz,
–CH2–), 4.45 (1H, t, J ¼ 6.1 Hz, –CH–), 6.97–7.03 (4H, 6.99
(ddd, J ¼ 8.8, 1.8, 0.5 Hz, Ph-H), 7.01 (ddd, J ¼ 8.6, 1.1, 0.4 Hz,
Ph-H)), 7.12 (2H, ddd, J ¼ 8.8, 1.0, 0.5 Hz, Ph-H), 7.98 (2H, length of 460 nm. The concentration for 50% of maximal effect
ddd, J ¼ 8.6, 1.7, 0.4 Hz, Ph-H); 13C NMR (75 MHz, DMSO) d: (EC50, the concentration that can cause the maximum effect of

988
Z. HUIYING ET AL.
50%) as an indicator of in vitro screening. EC50 was one of the
drugs’ safety indicators, and, in general, the EC50 values were
higher and the drug was safer.
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4.2.2. In vivo animal study
(1) Glucose tolerance test: The healthy mice (20 2 g) were selected.
After 1 week of acclimation, mice with no body surface damage,
good general condition and flexible reaction were selected for the
experiment. The test compounds were dissolved with DMSO, and
were given a dose volume of 0.2 ml 10 gꢀ1, and a dose of 10mg
kgꢀ1, and given by gavage (oral) once a day. The selected mice were
randomly divided into 10, each male and female, with Rosiglitazone
as the positive control group and DMSO as the blank group. The
mice were given the drug for 10d, and after the last dose, the mice
fasted for 12 h. The next day, blood was collected from the tail vein
of the mice before and after the sugar load for 1 h and 2 h, and the
blood glucose level of the mice before and after the sugar load was
measured by the glucometer. (2) Insulin tolerance test: The healthy
mice were randomly divided into 10, each male and female, with
Rosiglitazone as the positive control group and DMSO as the blank
group, given the drug continuously for 15 d. The 12 h of fasting after
the last administration, blood was taken from the orbital venous
plexus (used to measure the blood glucose level after fasting). Each
group was immediately given a subcutaneous injection of 0.2 lg kgꢀ1
of insulin. After 1 h and 2 h of injection, blood was taken again. (3)
Cell survival experiment: The HUVEC cells were inoculated on the 96-
well plate at the initial concentration of 3ꢄ 103 cells/holes, and 100 ll
RPMI 1640 medium were added to each hole. At 5% CO2 and 37 ꢃC
cell cultivation in 24 h, every pore solution in different concentrations.
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Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The project was sponsored by Innovation Team Project of
Universities in Chongqing (No. CXTDX201601018). The project was
also sponsored by Chongqing University Students’ Training Project
of Innovation and Undertaking (201510637085), Doctoral Program
of Chongqing Normal University (No. 12XLB006), Outstanding
Achievements Transformation Project in Chongqing Normal
University (No. 15XZH08), the National Natural Science Foundation
(21662012 and 41866005), the Postgraduate Research and
Innovation Project of Hainan Normal University (Hsyx2018-8) and
Open Foundation Project of Key Laboratory of Tropical Medicinal
Resource Chemistry of Ministry of Education (RDZH2019002).
ORCID
Zhou Shiyang
http://orcid.org/0000-0002-1100-8863
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