Discovery of novel bromophenol 3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6- (isobutoxymethyl)benzyl)benzene-1,2-diol as protein tyrosine phosphatase 1B inhibitor and its anti-diabetic properties in C57BL/KsJ-db/db mice

Article abstract of DOI:10.1016/j.ejmech.2013.03.037

In an effort to develop novel small molecule PTP1B inhibitors, a series of bromophenol derivatives were designed, synthesized and evaluated in vitro and in vivo. All of the synthesized compounds displayed weak to potent PTP1B inhibitory activities (5.62-96.25%) at 20 μg/mL. Among these compounds, 3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(isobutoxymethyl)benzyl)benzene-1,2-diol (9) exhibited enhanced PTP1B inhibitory activity (IC₅₀ = 1.50 μM) than the lead compound BDDPM (IC₅₀ = 2.42 μM) and high selectivity against other PTPs (TCPTP, LAR, SHP-1 and SHP-2). Results of anti-diabetic assay using C57BL/KsJ-db/db mouse model demonstrated that compound 9 was effective at lowering blood glucose, total cholesterol and HbA1c (P < 0.01).

Full text of DOI:10.1016/j.ejmech.2013.03.037

European Journal of Medicinal Chemistry 64 (2013) 129e136
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of novel bromophenol 3,4-dibromo-5-(2-bromo-3,4-
dihydroxy-6-(isobutoxymethyl)benzyl)benzene-1,2-diol as protein
tyrosine phosphatase 1B inhibitor and its anti-diabetic properties in
C57BL/KsJ-db/db mice
,
,
,
b
,
a
c
Bo Jiang a b, Shuju Guo a b, Dayong Shi a , Chao Guo , Tao Wang
*
a Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
b Nantong Branch, Institute of Oceanology, Chinese Academy of Sciences, Nantong 226006, China
c Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to develop novel small molecule PTP1B inhibitors, a series of bromophenol derivatives were
Received 12 December 2012
Received in revised form
12 March 2013
Accepted 21 March 2013
Available online 1 April 2013
designed, synthesized and evaluated in vitro and in vivo. All of the synthesized compounds displayed weak
to potent PTP1B inhibitory activities (5.62e96.25%) at 20
(2-bromo-3,4-dihydroxy-6-(isobutoxymethyl)benzyl)benzene-1,2-diol (9) exhibited enhanced PTP1B
M) and high selectivity
mg/mL. Among these compounds, 3,4-dibromo-5-
inhibitory activity (IC50 ¼ 1.50
m
M) than the lead compound BDDPM (IC50 ¼ 2.42
m
against other PTPs (TCPTP, LAR, SHP-1 and SHP-2). Results of anti-diabetic assay using C57BL/KsJ-db/db
mouse model demonstrated that compound 9 was effective at lowering blood glucose, total cholesterol
and HbA1c (P < 0.01).
Keywords:
Type 2 diabetes mellitus
Protein tyrosine phosphatase 1B
Bromophenol
Ó 2013 Elsevier Masson SAS. All rights reserved.
Anti-diabetic activities
1. Introduction
tract infection in patients with T2DM [2]. The “second-generation”
approaches for the treatment of T2DM are including incretin
Type 2 diabetes mellitus (T2DM) is a complex endocrine and
metabolic disorder. Established glucose-lowering medications,
which were called the “first-generation” agents, include insulin,
biguanides, sulfonylureas, and thiazolidinediones (TZDs). However,
the adverse effects such as hypoglycemia, weight increment and
edema limited their clinical use. In addition, recent studies reported
that long-term use of rosiglitazone or pioglitazone was associated
with a significant increase in the risk of myocardial infarction [1] and
a modestly increased risk of any pneumonia or lower respiratory
glucagon-like peptide 1 (GLP-1) analogues, sodium glucose
cotransporter 2 (SGLT2) inhibitors, dipeptidyl peptidase 4 (DPP4)
and protein tyrosine phosphatase 1B (PTP1B) inhibitors [3,4]. Among
them, PTP1B inhibitors have attracted considerable attention.
PTP1B is the first identified and characterized protein tyrosine
phosphatase (PTP) that was purified from human placental tissue
as early as in 1988 [5,6]. Not until 1999 that one research team
announced that PTP1B knockout mice displayed enhanced sensi-
tivity to insulin, lower plasma insulin levels and were resistant to
weight gain [7]. The results were independently confirmed by
another research team a year later [8]. Also, biochemical studies
revealed that treatment of diabetic mice with antisense oligonu-
cleotides (ASOs) resulted in reduced PTP1B expression, and sub-
sequent improvement of insulin sensitivity and glycemic control
[9]. Thus, PTP1B is currently considered to be a promising drug
target for non-insulin dependent diabetes and obesity [10,11].
Furthermore, recent studies have also indicated that PTP1B is
involved in cancer, which makes PTP1B inhibitors to be great po-
tential agents in cancer therapy [12e15].
Abbreviations: PTP1B, protein tyrosine phosphatase 1B; T2DM, type 2 diabetes
mellitus; TZDs, thiazolidinediones; GLP-1, glucagon-like peptide 1; SGLT2, sodium
glucose cotransporter 2; DPP4, dipeptidyl peptidase 4; ASOs, antisense oligonu-
cleotides; SAR, structure activity relationship; TFAA, trifluoroacetic anhydride; TLC,
thin-layer chromatography; HPLC, high-performance liquid chromatography; TMS,
tetramethylsilane; TCPTP, T-cell protein tyrosine phosphatase; SHP-1, Src homology
2-containing protein tyrosine phosphatase-1; SHP-2, Src homology 2-containing
protein tyrosine phosphatase-2; LAR, leucocyte antigen-related tyrosine
phosphatase.
* Corresponding author. Institute of Oceanology, Chinese Academy of Sciences,
Qingdao 266071, China. Tel./fax: þ86 0532 8289 8719.
Given the compelling evidence that PTP1B is implicated in
T2DM, a large number of small molecules have been described over
E-mail address: shidayong@qdio.ac.cn (D. Shi).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.037

130
B. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 129e136
Synthesis of diaryl-methanone compounds was carried out
following the synthetic steps in Scheme 2. Also, FriedeleCrafts
acylation between 1 and corresponding aryl acids 10e12 in the
presence of trifluoroacetic anhydride (TFAA) gave diaryl-
methanones 13e15. Oxidation of ketones 13e15 using KMnO4 as
oxidant afforded aryl acids 16. Further esterification with CH3OH
provided ester 17. The etherificated products 20 and 21 were pre-
pared according to the procedures of 6e9. Interestingly, in order to
prepare the reduction product of keto acid 16 via ZneCu [22] or
NaBH4, we obtained compounds 18 and 19 with more rigid
conformation.
Finally, we turned our attention to the position and number of
bromine substitutions on the phenyl ring. As depicted in Scheme 3,
the di-brominated compounds 23, 24 and 25 were formed using
the similar procedures in Scheme 1. Further bromination of com-
pound 28 in the presence of NBS and concd. H2SO4 afforded multi-
brominated compound 29 in moderate yield.
Fig. 1. Structures of bromophenols isolated from red alga Rhodomela confervoides.
the last two decades with the aim of developing potent and se-
lective compounds as drug candidates [16e18]. However, the
highly charged phosphatase active site and the relatively shallow
nature of the surrounding protein surface make great challenge to
medicinal chemists for the discovery of cell permeable and orally
bio-available PTP1B inhibitors.
3. Results and discussion
Our PTP1B small molecule inhibitor program started with the
isolation of some bromophenols (Fig. 1) from the ethanolic extract
of red alga Rhodomela confervoides [19]. Further biological evalua-
tion identified that bromophenol BDDPM was a potent PTP1B in-
3.1. In vitro enzyme-based inhibitory activities assays
The target compounds 3e9, 13e21, 23e25 and 28e29 were
evaluated for their in vitro inhibitory activities against recombinant
PTP1B. We measured the inhibitory rates of all the derivatives at
hibitor (IC50 ¼ 2.42
mM) and selected as a lead compound for
further optimization. In our previous studies, we reported a series
of derivatives which were designed based on symmetric structure
of BDDPM with diverse inhibitory activities against PTP1B [20]. As a
part of our ongoing research, we describe herein the synthesis,
structureeactivity relationship (SAR) and in vivo anti-diabetic
studies of novel bromophenol derivatives as PTP1B inhibitors.
concentration of 20 and 5
mg/mL, and the compounds with good
inhibition rates (>45% at 5
mg/mL) were selected for IC50 assay.
Optimization of BDDPM with varying the substitution on one phenyl
ring was depicted in Table 1. Electron-rich groups were favorable at
this position, especially alkoxyl methylene groups (6e9 and 20e21).
However, replacement of the alkoxyl methylene groups with
carboxyl (16) led to a dramatic decrease in PTP1B inhibitory activity.
Furthermore, the compound with branched-chain in alkoxyl
methylene group (9) exhibited significant PTP1B inhibition (96.25%
2. Chemistry
The synthetic route of diaryl-methane compounds 3e9 was
shown in Scheme 1. The starting materials 1 and 2 were prepared
according to methods described previously [21]. FriedeleCrafts
alkylation between 1 and 2 in the presence of AlCl3 afforded diaryl-
methane 3. Treatment of 3 with NBS under hv condition provided
the corresponding benzyl bromide. Subsequent hydrolysis in the
presence of K2CO3 and 1,4-dioxane afforded benzyl alcohol 5.
Demethylation of compounds 3 and 5 with BBr3 in CH2Cl2 gave
bromophenols in good yields. The bromophenols was treated with
corresponding alcohol in the presence of catalyst H3PO4 to give the
analogues 6e9.
at 20 mg/mL and 48.3% at 5 mg/mL). The diaryl-methane moiety was
selected for further derivatization. As shown in Table 2, replacement
of diaryl-methane with diaryl-methanones moiety (13e17 and 20e
21) was not tolerated. And the compounds containing more rigid
configuration (18 and 19) exhibited significant loss of activity,
indicating that the flexibility of diaryl-methane scaffold appeared to
be optimal. The number of bromine substitution on the phenyl ring
also had a certain influence on the inhibitory activity against PTP1B.
As shown in Table 3, the tri- and tetra-brominated compounds (3e9
and 28e29) displayed more significant PTP1B inhibitory activity
than di-brominated compounds (23e24). A quick examination of
Scheme 1. Synthesis of diaryl-methane compounds 3e9.

B. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 129e136
131
Scheme 2. Synthesis of diaryl-methanone compounds 13e21.
Tables 1e3 revealed that the compounds with free hydroxyl groups
were more potent PTP1B inhibitors than the corresponding
methoxyl compounds (6e9 vs 13e27). Further IC50 determination
showed that compound 9 exhibited enhanced inhibitory activity
mice in treatment group exhibited a downward trend compared to
the model group and no mortality occurred. Besides, compound 9
at different doses reduced the food and water intakes, indicating
that compound 9 could improve the diabetes-related symptoms,
such as polydipsia and polyphagia. The effects of compound 9 on
blood glucose were illustrated in Fig. 2, the positive drug rosigli-
tazone significantly decreased blood glucose from 15.3 to
8.9 mmol/L at the first week of treatment (P < 0.01) and maintained
blood glucose level at approximately 8 mmol/L until the end.
Although slightly weaker than rosiglitazone (48.4%), compound 9
significantly decreased blood glucose (P < 0.01) since the third
week, and obtained 36.4% and 32.7% decrease in the blood glucose
level at high and low dose group, respectively.
against PTP1B with an IC50 of 1.50
mM, which was better than the
lead compound BDDPM (IC50 ¼ 2.42
mM).
In addition to potency improvements, we investigated the
selectivity of compound 9 against other PTPs (TCPTP, LAR, SHP-1
and SHP-2). The results showed that compound 9 had excellent
selectivity against TCPTP, LAR, SHP-1 and SHP-2 (>50-fold).
3.2. In vivo anti-diabetic activities assays
In addition, antidyslipidemic activity of compound 9 was
assessed in the db/db mouse model. As shown in Table 4, the db/db
mice used in this study had significantly elevated triglyceride and
total cholesterol concentrations compared to the db/dm mice
(P < 0.01, P < 0.05). In general, compound 9 at high dose showed
As an interesting new chemical entity, compound 9 was then
selected for anti-diabetic activities evaluation in the db/db mice, a
model of type 2 diabetes. Compound 9 was dosed orally at 25 and
50 mg/kg once a day, and the positive drug rosiglitazone was dosed
at 50 mg/kg. During a 6-week intervention, body weights of db/db
Scheme 3. Synthesis of multi-brominated compounds 23e29.

132
B. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 129e136
Table 1
Table 3
In vitro PTP1B inhibitory activities of diaryl-methane compounds 3e9.
In vitro PTP1B inhibitory activities of multi-brominated compounds 23e29.
R1
R2
R3
Inhibition (%)
20 g/mL
IC50 (mM)
Compds
R1
R2
Inhibition (%)
20 g/mL
IC50 (mM)
Compds
m
5 mg/mL
m
5 mg/mL
3
4
5
6
7
8
9
CH3
H
CH3
H
H
H
CH3
CH3
CH2OH
CH2OCH3
CH2O(CH2)2CH3
CH2O(CH2)3CH3
CH2OCH2CH(CH3)2
87.52
53.38
74.86
51.89
86.15
64.56
96.25
43.13a
32.10a
29.07a
25.30a
55.70a
27.17a
48.30a
ND
ND
ND
ND
23
24
25
28
29
CH3
H
H
H
Br
Br
H
H
H
Br
Br
14.46
5.62
50.60
72.64
75.31
ND
ND
ND
ND
ND
ND
ND
CH2OH
CH2OCH2CH3
CH3
17.33a
38.25a
40.13a
2.00b
ND
CH3
ND ¼ not determined.
H
1.50b
a
Values were tested only if its inhibitory ratio greater than 50% at the dose of
mg/mL.
ND ¼ not determined.
20
a
Values were tested only if its inhibitory ratio greater than 45% at the dose of
20
m
g/mL.
b
Values were tested only if its inhibitory ratio greater than 50% at the dose of
mg/mL.
groups. With regard to glycated serum protein (GSP), another
5
important indicator for diabetes control, compound
9 also
remarkably lowered GSP level (P < 0.05) at 50 mg/kg.
better lipid lowering profile (P < 0.05) and decreased the tri-
glycerides and total cholesterol concentrations in a dose-dependent
manner.
4. Conclusions
It is well known that glycated hemoglobin (HbA1c) is an
important criterion for diagnosis of diabetes and the best way to
check if diabetes is under control. As depicted in Table 5, mice in
model group had significantly elevated HbA1c compared to the
db/dm mice (P < 0.01). It is gratifying that compound 9 significantly
decreased HbA1c levels (P < 0.01) at both high and low dose
In summary, we have prepared a series of bromophenol de-
rivatives to search for potent PTP1B inhibitors. The preliminary
structureeactivity relationship acquired showed that alkoxyl
methylene group attached to the phenyl ring and the diaryl-
methane scaffold are favorable to PTP1B inhibitory activity.
Among these derivatives, compound 9 exhibited enhanced inhibi-
tory activity against PTP1B with IC50 of 1.50 mM and high selectivity
against other PTPs. More importantly, compound 9 was also
effective at lowering glucose and HbA1c in db/db mouse model at
25 and 50 mg/kg. Efforts in safety evaluation will be reported in due
course.
Table 2
In vitro PTP1B inhibitory activities of diaryl-methanone compounds 13e21.
5. Experimental
5.1. General
Reagents and all solvents were analytically pure and were used
without further purification. All of the experiments were moni-
tored by analytical thin-layer chromatography (TLC) performed on
silica gel GF254 precoated plates. After elution, the plate was
visualized under UV illumination at 254 nm for UV active
Compds
R1
R2
R3
Inhibition (%)
20 g/mL
IC50 (mM)
m
5 mg/mL
13
14
15
16
17
20
21
H
H
H
CH3
CH3
CH3
COOH
CO2CH3
CH2OCH3
CH2OCH2CH3
28.86
36.68
48.64
29.90
55.34
61.75
73.83
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
23.82a
17.73a
17.63a
Compds
X
Inhibition (%)
20 g/mL
IC50 (mM)
m
5 mg/mL
18
19
O
H
10.57
11.41
ND
ND
ND
ND
ND ¼ not determined.
a
Values were tested only if its inhibitory ratio greater than 50% at the dose of
mg/mL.
Fig. 2. Effects of compound 9 on blood glucose in db/db mouse during 6 weeks
treatment (n ¼ 10) #P < 0.05, ##P < 0.01 vs model.
20

B. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 129e136
133
Table 4
Antidyslipidemic activities of compound 9 in db/db mouse model ðn ¼ 10; x ꢃ sÞ.
Group
Dose (mg/kg)
Triglycerides (mmol/L)
Total cholesterol (mmol/L)
Baseline
2 weeks
6 weeks
Baseline
2 weeks
6 weeks
Control
Model
Compd 9
1.16 ꢃ 0.09
1.30 ꢃ 0.25
1.34 ꢃ 0.18
1.30 ꢃ 0.16
1.31 ꢃ 0.38
1.08 ꢃ 0.07
1.24 ꢃ 0.12**
1.35 ꢃ 0.19
1.16 ꢃ 0.19
1.06 ꢃ 0.19#
1.05 ꢃ 0.11
1.56 ꢃ 0.17**
1.48 ꢃ 0.26
1.41 ꢃ 0.13
1.29 ꢃ 0.14##
3.6 ꢃ 0.2
6.2 ꢃ 1.7**
6.2 ꢃ 1.5
6.2 ꢃ 1.9
6.1 ꢃ 1.7
3.5 ꢃ 0.2
3.3 ꢃ 0.5
9.8 ꢃ 0.7**
7.8 ꢃ 0.9##
7.7 ꢃ 1.1##
6.5 ꢃ 0.4##
9.2 ꢃ 1.2**
9.1 ꢃ 0.9
8.5 ꢃ 0.7
7.7 ꢃ 1.0#
50
25
50
Rosiglitazone
#P < 0.05.
##P < 0.01 vs model.
**P < 0.01 vs control.
materials or coloration by 95% FeCl3eEtOH solution. Column
chromatography was carried out using silica gel (200e300 mesh).
All final compounds were purified to >95%, purity, as determined
by high-performance liquid chromatography (HPLC). Melting
points were determined using Boetius electrothermal capillary
melting point apparatus and are uncorrected. 1H NMR and 13C
NMR spectra were recorded on an Inova (500 MHz) NMR spec-
trometer for proton and at 125 MHz for carbon. Chemical shifts are
extracts was dried over anhydrous Na2SO4, and evaporated in vacuo
to provide the brownish residue. The residue was purified over
silica gel column chromatography (CHCl3:MeOH,15:1) to afford 4 as
yellowish solid (0.8 g, 90% yield). 1H NMR (500 MHz, DMSO-d6):
d
9.73 (s, 1H), 9.59 (s, 1H), 9.30 (s, 1H), 8.89 (s, 1H), 6.70 (s, 1H), 6.06
(s, 1H), 3.94 (s, 2H), 2.02 (s, 3H); 13C NMR (125 MHz, DMSO-d6):
145.1 (C), 144.5 (C), 142.7 (C), 141.1 (C), 130.1 (C), 128.1 (C), 127.1 (C),
d
116.3 (CH), 114.5 (CH), 114.3 (C), 113.6 (C), 113.1 (C), 48.5 (CH2), 19.3
(CH3). EIMS m/z (% relative intensity): 486/484/482/480 [M]þ (6/20/
20/6). HRMS m/z calcd for C14H10O4Br3 [M ꢁ H]ꢁ, 478.8129; found,
478.8126.
reported in ppm (d) relative to tetramethylsilane (TMS) as an in-
ternal standard. Multiplicities are given as s (singlet), d (doublet),
dd (doubleedoublet), t (triplet), q (quadruplet), m (multiplet) and
br s (broad signal). Mass spectra and high-resolution mass spectral
(HRMS) data were recorded on Autospec Ultima-Tof and Thermo
LTQ-Orbitrap mass spectrometer, respectively.
5.4. (3-Bromo-2-(20,30-dibromo-40,50-dimethoxybenzyl)-4,5-
dimethoxyphenyl)-methanol (5)
5.2. 2,3-Dibromo-1-(20-bromo-30,40-dimethoxy-60-methylbenzyl)-
4,5-dimethoxy benzene (3)
Under hv condition, to a solution of 3 (5.39 g, 0.01 mol) and AIBN
(200 mg, 0.001 mol) in CCl4 (100 mL) was added NBS (1.96 g,
0.011 mol) in three portions with stirring. After 30 min, the mixture
was filtered, and the filtrate was concentrated and purified over
silica gel column chromatography (petroleum ether:ethyl acetate,
8:1) to obtain the benzyl bromide (2.82 g, 45%). The benzyl bromide
was added to the mixture of K2CO3 (4 g, 0.03 mol), H2O (20 mL) and
1,4-dioxane (20 mL). After refluxing for 3 h, the resulting mixture
was cooled to room temperature and extracted with CHCl3. The
organic layer was collected and removed under reduced pressure to
give 4 as white solid (2.27 g, 91% yield). mp: 164e166 ꢀC. 1H NMR
To a stirred solution of 1 (2.31 g, 0.01 mol) and 2 (3.26 g,
0.01 mol) in CH2Cl2 (50 mL) at 0 ꢀC, AlCl3 (1.34 g, 0.01 mol) was
added. After stirring for 30 min at room temperature, the mixture
was poured into ice water and washed with 1 mol/L HCl. The
CH2Cl2 layer was concentrated and the residue was recrystallized
from CH3OH to afford 3 as white solid (4.8 g, 89% yield). mp: 115e
117 ꢀC. 1H NMR (500 MHz, CDCl3):
(s, 2H), 3.89 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H), 3.56 (s, 3H), 2.17 (s,
3H); 13C NMR (125 MHz, CDCl3):
152.5, 152.0, 146.1, 144.9, 135.8,
d 6.76 (s, 1H), 6.15 (s, 1H), 4.19
d
(500 MHz, CDCl3)
(s, 2H), 3.91 (s, 3H), 3.86 (s, 3H), 3.79 (s, 3H), 3.55 (s, 3H); 13C NMR
(125 MHz, CDCl3) : 152.4, 146.1, 145.9, 136.6, 135.8, 128.7, 122.5,
d: 7.09 (s, 1H), 6.14 (s, 1H), 4.52 (s, 2H), 4.22
134.3, 129.6, 122.1, 121.7, 117.6, 113.8, 111.7, 60.4, 56.1, 56.0, 40.5,
20.6. EIMS m/z (% relative intensity): 542/540/538/536 [M]þ (33/
100/100/33).
d
121.7, 117.4, 111.8, 111.4, 63.0, 60.4, 56.0, 39.2. EIMS m/z (% relative
intensity): 556/554/552/550 [M]þ (13/38/38/12).
5.3. 3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-methylbenzyl)
benzene-1,2-diol (4)
5.5. General procedures for the synthesis of compounds 6e9
Compound 3 (1 g, 1.85 mmol) was dissolved in dry CH2Cl2
(15 mL), then BBr3 (15 mL, 1 mol/L in CH2Cl2) was added dropwise
while stirring in ice bath. The reaction mixture stirred for further
4 h at room temperature. Then the solution was poured into
ice-cold water and extracted with ethyl acetate. The organic
Demethylation of 5 via BBr3 was carried out as described for
compound 4. The ethyl acetate layer was collected and concen-
trated, then the residue was used without purification. The residue
and 85% H3PO4 (1 mL) were dissolved in corresponding alcohol
(10 mL). After stirring for 2 h under reflux, the solvent was removed
under reduced pressure. The residue was purified over silica gel
column chromatography (petroleum ether:ethyl acetate, 2:1) to
produce a yellow solid.
Table 5
HbA1c and GSP levels of compound 9 in db/db mouse model ðn ¼ 10; x ꢃ sÞ.
Group
Dose (mg/kg)
HbA1c (%)
GSP (mmol/L)
Control
Model
Compd 9
2.65 ꢃ 0.40
1.62 ꢃ 0.27
4.44 ꢃ 1.42**
3.00 ꢃ 0.62#
3.29 ꢃ 0.50
2.60 ꢃ 0.58##
5.5.1. 3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-(methoxymethyl)
4.90 ꢃ 0.31**
3.03 ꢃ 0.31##
3.24 ꢃ 0.15##
2.56 ꢃ 0.44##
benzyl)benzene-1,2-diol (6)
50
25
50
90% yield. 1H NMR (500 MHz, DMSO-d6)
d
: 9.72 (s, 2H), 9.30 (s,
2H), 6.87 (s, 1H), 6.04 (s, 1H), 4.12 (s, 2H), 3.97 (s, 2H), 3.18 (s, 3H);
13C NMR (125 MHz, DMSO-d6)
Rosiglitazone
d: 144.9 (C), 144.2 (C), 142.6 (2ꢂ C),
#P < 0.05.
##P < 0.01 vs model.
**P < 0.01 vs control.
130.5 (C), 128.6 (C), 127.5 (C), 115.4 (CH), 114.5 (C), 114.3 (C), 113.8
(CH), 113.0 (C), 72.0 (CH2), 57.3 (CH3), 38.2 (CH2).

134
B. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 129e136
5.5.2. 3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-(propoxymethyl)
2.22 (s, 3H); 13C NMR (125 MHz, CDCl3)
d: 194.2 (C), 154.0 (C), 152.0
benzyl)benzene-1,2-diol (7)
(C), 150.8 (C), 144.6 (C), 135.7 (C), 133.7 (C), 133.0 (C), 124.3 (C), 116.3
(C), 115.5 (C), 115.1 (C), 113.8 (C), 60.6 (C), 60.5 (C), 56.3 (C), 56.0 (C),
20.0 (C). EIMS m/z (% relative intensity): 556/554/552/550 [M]þ
(20/52/52/20).
65% yield. 1H NMR (500 MHz, DMSO-d6)
d: 9.75 (s, 1H), 9.69 (s,
1H), 9.27 (s,1H), 9.14 (s,1H), 6.88 (s,1H), 6.05 (s,1H), 4.17 (s, 2H), 3.99
(s, 2H), 3.26 (t, J ¼ 6.52, 2H), 1.14 (m, 2H), 0.79 (t, J ¼ 7.36, 3H); 13C
NMR (125 MHz, DMSO-d6)
d: 144.9 (C), 144.3 (C), 142.5 (2ꢂ C), 130.5
(C),129.0 (C),127.5 (C),115.4 (CH),114.6 (C),114.3 (CH),113.9 (C),112.9
(C), 71.2 (CH2), 70.3 (CH2), 38.3 (CH2), 22.2 (CH2),10.4 (CH3). EIMS m/z
(% relative intensity): 544/542/540/538 [M]þ (1/3/3/1). HRMS m/z
calcd for C17H16O5Br3 [M ꢁ H]ꢁ, 536.8548; found, 536.8570.
5.7. 3-Bromo-2-(2,3-dibromo-4,5-dimethoxybenzoyl)-4,5-
dimethoxybenzoic acid (16)
Aryl acid 15 (1 g, 1.8 mmol) was dissolved in mixture of t-BuOH
and H2O (1:1, 20 mL). The mixture was heated to 80 ꢀC, then KMnO4
(0.57 g, 3.6 mmol) was added over 1 h and TLC was used to monitor
the reaction. After the reaction, MnO2 was filtered and the filtrate
was acidified to pH ¼ 2 by adding 10% HCl. The precipitate was
collected to afford 17 as white solid (0.64 g, 61% yield). mp: 167e
5.5.3. 3,4-Dibromo-5-(2-bromo-6-(butoxymethyl)-3,4-dihydroxy-
benzyl)benzene-1,2-diol (8)
58% yield. 1H NMR (500 MHz, DMSO-d6)
d: 9.75 (s, 1H), 9.69 (s,
1H), 9.27 (s, 1H), 9.15 (s, 1H), 6.87 (s, 1H), 6.04 (s, 1H), 4.16 (s, 2H),
3.98 (s, 2H), 3.29 (t, J ¼ 6.44, 2H), 1.36 (m, 2H), 1.22 (m, 2H), 0.80 (t,
170 ꢀC. 1H NMR (500 MHz, DMSO-d6)
3.94 (s, 3H), 3.82 (s, 6H), 3.74 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
: 191.6 (CO), 166.2 (COOH), 153.4, 152.0, 150.4, 149.3, 135.8, 134.0,
d: 7.59 (s, 1H), 7.31 (s, 1H),
J ¼ 7.35, 3H); 13C NMR (125 MHz, DMSO-d6)
d: 144.9 (C), 144.2 (C),
142.5 (2ꢂ C), 130.5 (C), 129.0 (C), 127.5 (C), 115.4 (CH), 114.6 (C),
114.3 (CH), 113.9 (C), 112.9 (C), 70.4 (CH2), 69.2 (CH2), 38.3 (CH2),
31.1 (CH2), 18.7 (CH2), 13.6 (CH3). EIMS m/z (% relative intensity):
558/556/554/552 [M]þ (1/3/3/1). HRMS m/z calcd for C18H18O5Br3
[M ꢁ H]ꢁ, 550.8704; found, 550.8688.
d
127.3, 124.2, 116.4, 116.1, 115.5, 114.2, 60.9 (CH3), 60.8 (CH3), 56.8
(2ꢂ CH3). EIMS m/z (% relative intensity): 586/584/582/580 [M]þ
(3/10/10/3).
5.8. Methyl 3-bromo-2-(2,3-dibromo-4,5-dimethoxybenzoyl)-4,5-
5.5.4. 3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-(isobutoxymethyl)
dimethoxybenzoate (17)
benzyl)benzene-1,2-diol (9)
54% yield.1H NMR (500 MHz, DMSO-d6)
d: 9.75(s,1H), 9.69(s,1H),
To the solution of 16 (0.58 g,1 mmol) in CH3OH (10 mL) was added
concd. H2SO4 (0.5 mL). The mixture was heated to reflux for
further12 h. The mixture was removed under reduced pressure. The
residue was recrystallized from CH3OH and H2O to give 17 as white
9.27 (s, 1H), 9.15 (s, 1H), 6.88 (s, 1H), 6.04 (s, 1H), 4.16 (s, 2H), 3.98 (s,
2H), 3.08 (d, J ¼ 6.48, 2H),1.68 (m,1H), 0.78 (d, J ¼ 6.68, 6H); 13C NMR
(125 MHz, DMSO-d6)
d
: 144.9 (C), 144.2 (C), 142.5 (2ꢂ C), 130.5 (C),
129.0 (C),127.5 (C),115.4 (CH),114.6 (C),114.3 (CH),113.9 (C),113.0 (C),
76.4 (CH2), 70.6 (CH2), 38.3 (CH2), 27.8 (CH), 19.1 (2ꢂ CH3). EIMS m/z
(% relative intensity): 558/556/554/552 [M]þ (1/3/3/1). HRMS m/z
calcd for C18H18O5Br3 [M ꢁ H]ꢁ, 550.8704; found, 550.8680.
solid (0.49 g, 82% yield). mp: 176e178 ꢀC.1H NMR (500 MHz, CDCl3)
d:
7.56 (s, 1H), 7.50 (s, 1H), 3.97 (s, 3H), 3.92 (s, 6H), 3.83 (s, 3H), 3.76 (s,
3H); 13C NMR (125 MHz, CDCl3)
d: 191.7 (CO), 165.1 (COO), 153.1 (C),
151.9 (C), 151.0 (C), 150.1 (C), 136.9 (C), 133.5 (C), 125.5 (C), 124.5 (C),
117.3 (C), 116.0 (C), 115.7 (C), 113.6 (C), 60.8 (OCH3), 60.6 (OCH3), 56.3
(2ꢂ OCH3), 52.7 (C). EIMS m/z (% relative intensity): 600/598/596/594
[M]þ (8/23/23/8).
5.6. General procedures for the synthesis of compounds 13e15
To the suspension of corresponding aryl acids 10e12 (7 mmol)
in TFAA (15 mL) were added 85% H3PO4 (0.5 mL) and 1 (7 mmol)
under ice-bath, and the mixture was heated to reflux and stirred for
further 2 h. The mixture was poured into ice-cold water and
extracted with CH2Cl2. The combined extracts were dried over
anhydrous Na2SO4, and evaporated in vacuo. The residue was
recrystallized in ethyl alcohol to give 13e15 as white solid.
5.9. 4-Bromo-3-(2,3-dibromo-4,5-dimethoxyphenyl)-5,6-dimetho-
xyisobenzofuran-1(3H)-one (18)
16 (1 g, 1.7 mmol) was suspended into 10% KOH solution
(40 mL), then ZneCu complex (6 g) was added, the mixture was
heated to refluxed for 10 h. The complex was filtered. After acidized
by 10% HCl, the filtrate was extracted with CH2Cl2. The CH2Cl2 layer
was concentrated and the residue was recrystallized in C2H5OHe
CH3COCH3 (1:1) to give 18 as white solid (0.52 g, 53% yield). mp:
5.6.1. (2-Bromo-3,4-dimethoxy-6-methylphenyl)(3,4-dimethoxy-
phenyl)methanone (13)
81% yield. 1H NMR (500 MHz, CDCl3)
d: 7.59 (s, 1H), 7.18 (d,
188e190 ꢀC. 1H NMR (500 MHz, CDCl3)
J ¼ 7.5, 2H), 6.70 (s,1H), 6.65 (s,1H), 6.23 (s,1H), 3.95 (s, 3H), 3.91 (s,
3H), 3.90 (s, 3H), 3.82 (s, 3H). 13C NMR (125 MHz, CDCl3)
: 170.9
d: 7.33 (s, 1H), 6.86 (d,
J ¼ 8.14, 1H), 6.81 (d, J ¼ 8.14, 1H), 6.75 (s, 1H), 3.94 (s, 3H), 3.92 (s,
3H), 3.90 (s, 3H), 3.84 (s, 3H), 2.14 (s, 3H); 13C NMR (125 MHz,
d
CDCl3)
d
: 195.1 (CO), 154.1, 153.6, 149.5, 144.5, 133.6, 132.0, 129.7,
(CO), 154.9 (C), 150.6 (C), 149.9 (C), 149.3 (C), 144.1 (C), 128.7 (C),
120.2 (CH), 117.7 (C), 111.0 (CH), 109.9 (CH), 105.7 (CH), 104.0 (CH),
82.4 (CH), 56.3 (2ꢂ CH3), 55.9 (2ꢂ CH3).
125.6, 114.8,113.6,110.6,110.2, 60.6 (CH3), 56.1 (CH3), 56.0 (2ꢂ CH3),
19.5 (CH3). EIMS m/z (% relative intensity): 396/394 [M]þ (58/58).
5.6.2. (2-Bromo-3,4-dimethoxy-6-methylphenyl)(3-bromo-4,5-
5.10. 7-Bromo-1-(2,3-dibromo-4,5-dimethoxyphenyl)-5,6-dime-
dimethoxyphenyl)methanone (14)
thoxy-1,3-dihydroisobenzofuran (19)
80% yield. 1H NMR (500 MHz, CDCl3)
d
: 7.48 (s, 1H), 7.34 (s, 1H),
6.75 (s, 1H), 3.90 (s, 3H), 3.85 (s, 6H), 3.80 (s, 3H), 2.10 (s, 3H); 13C
NMR (125 MHz, CDCl3) : 194.2, 154.0, 152.0, 150.8, 144.6, 135.7,
Compound 16 (0.5 g, 0.9 mmol), NaBH4 (0.35 g, 9 mmol) and
AlCl3 (1.1 g, 8 mmol) were dissolved in THF (20 mL) and the mixture
was heated to reflux for 10 h. Then the mixture was poured into ice-
water, and extracted with ethyl ether. The ethyl ether layer was
collected and removed under reduced pressure. The residue was
purified over silica gel column chromatography (petroleum
ether:ethyl acetate, 3:1) to produce 19 as white solid (0.2 g, 42%
d
133.7, 133.0, 124.3, 116.3, 115.5, 115.1, 113.8, 60.6, 60.5, 56.3, 56.0,
20.0. EIMS m/z (% relative intensity): 476/474/472 [M]þ (47/95/47).
5.6.3. (2-Bromo-3,4-dimethoxy-6-methylphenyl)(2,3-dibromo-4,5-
dimethoxyphenyl)methanone (15)
90% yield, mp: 86e88 ꢀC. 1H NMR (500 MHz, CDCl3)
d: 7.16 (s,
yield). 1H NMR (500 MHz, CDCl3)
1H), 5.26 (d, J ¼ 12, 1H), 5.15 (d, J ¼ 12, 1H), 3.93 (s, 3H), 3.84 (s, 6H),
d: 6.83 (s, 1H), 6.61 (s, 1H), 6.39 (s,
1H), 6.74 (s, 1H), 3.91 (s, 3H), 3.90 (s, 3H), 3.81 (s, 3H), 3.79 (s, 3H),

B. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 129e136
135
3.66 (s, 3H). 13C NMR (125 MHz, CDCl3)
d
: 154.2, 152.6, 147.8, 146.3,
NMR (125 MHz, acetone-d6)
d
: 146.1, 144.2, 142.9, 141.4, 133.1, 130.5,
136.6, 136.3, 132.3, 122.4, 118.6, 112.8, 112.2, 103.9, 87.7, 73.6, 60.7
(CH3), 60.5 (CH3), 56.3 (CH3), 56.1 (CH3). MS: m/z 556/554/552/550
[M]þ (15/46/47/17).
129.7, 123.2, 115.8, 114.7, 114.5, 109.6, 70.9, 65.7, 36.1, 15.1. EIMS m/z
(% relative intensity): 450/448/446 [M]þ (1.5/3/1.5).
5.15. 2,3,6-Tribromo-4,5-dimethoxybenzaldehyde (26)
5.11. General procedures for the synthesis of compounds 20e21
To the suspension of 1 (2.45 g, 0.01 mol) in concd. H2SO4 (20 mL)
was added NBS (3.9 g, 0.02 mmol) under ice-bath, and the mixture
was stirred for further 2 h at room temperature. The mixture was
poured into ice-cold water and extracted with CHCl3. The combined
extracts were washed with Na2CO3 and dried over anhydrous
Na2SO4, and evaporated in vacuo to provide the brownish residue.
The residue was purified by silica gel column chromatography
(petroleum ether:ethyl acetate, 1:1) to give white solid (3.2 g, 79%
The title compounds were prepared from 15 using the proce-
dure previously described for compounds 6e9 and were purified by
silica gel column chromatography (petroleum ether:ethyl acetate,
3:1) to give yellowish slurry.
5.11.1. (2-Bromo-3,4-dimethoxy-6-(methoxymethyl)phenyl)(2,3-
dibromo-4,5-dimethoxyphenyl)methanone (20)
59% yield. 1H NMR (500 MHz, CDCl3)
d
: 7.17 (s, 1H), 7.04 (s, 1H),
4.44 (s, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 3.82 (s, 3H), 3.76 (s, 3H), 3.35
(s, 3H); 13C NMR (125 MHz, CDCl3)
: 193.7 (C), 154.2 (C), 151.8 (C),
yield). mp: 160e162 ꢀC. 1H NMR (500 MHz, CDCl3)
d: 10.10 (s, 1H),
4.00 (s, 3H), 3.93 (s, 3H).
d
150.5 (C), 145.7 (C), 135.7 (C), 135.1 (C), 132.1 (C), 124.2 (C), 116.5 (C),
116.1 (C), 115.5 (C), 111.7 (C), 69.9 (C), 63.5 (C), 60.7 (C), 60.6 (C), 56.3
(C), 56.1 (C). EIMS m/z (% relative intensity): 586/584/582/580 [M]þ
(3/9/9/3).
5.16. (2,3,6-Tribromo-4,5-dimethoxyphenyl)methanol (27)
To a solution of compound 26 (3.89 g, 10 mmol) in CH3OH
(20 mL) was added NaBH4 (0.2 g, 5 mmol) over 10 min. After the
reaction, the mixture was acidified with 10% HCl and poured into
water. The mixture was extracted with CH2Cl2. The organic phase
was combined and dried over anhydrous Na2SO4 and concentrated
in vacuo to give 27 as white solid (3.64 g, 90% yield).
5.11.2. (2-Bromo-6-(ethoxymethyl)-3,4-dimethoxyphenyl)(2,3-
dibromo-4,5-dimethoxyphenyl)methanone (21)
56% yield. 1H NMR (500 MHz, CDCl3)
d: 7.17 (s, 1H), 7.04 (s, 1H),
4.44 (s, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 3.82 (s, 3H), 3.76 (s, 3H), 3.43
(q, J ¼ 7 Hz, 2H), 1.08 (t, J ¼ 7 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d:
5.17. 1,2,4-Tribromo-3-(2-bromo-3,4-dimethoxy-6-methylbenzyl)-
5,6-dimethoxybenzene (28)
193.7 (C), 154.2 (C), 151.8 (C), 150.5 (C), 145.7 (C), 135.7 (C), 135.1 (C),
132.1 (C), 124.2 (C), 116.5 (C), 116.1 (C), 115.5 (C), 111.7 (C), 69.9 (C),
66.5 (C), 60.7 (C), 60.6 (C), 56.3 (C), 56.1 (C), 14.9 (C). EIMS m/z (%
relative intensity): 600/598/596/594 [M]þ (2/7/7/2).
The title compound was prepared from 1 and 27 using the
procedure previously described for compound 3 and was purified
by recrystallization from CH3OH to give white solid. 74% yield. 1H
5.12. 3-Bromo-2-(3-bromo-4,5-dimethoxybenzyl)-4,5-dimethoxy-
1-methylbenzene (23)
NMR (500 MHz, CDCl3)
(s, 3H), 3.83 (s, 3H), 3.81 (s, 3H), 1.98 (s, 3H); 13C NMR (125 MHz,
CDCl3) : 151.2, 150.6, 150.4, 137.4, 133.5, 129.0, 126.5, 123.2, 122.8,
d: 6.58 (s, 1H), 4.68 (s, 2H), 3.89 (s, 3H), 3.86
d
The title compound was prepared from 1 and 22 using the
procedure previously described for compound 3 and was purified
by recrystallization from CH3OH to give white solid. 79% yield. mp:
122.0, 121.8, 114.2, 60.5, 60.4, 56.0, 55.9, 43.3, 21.3.
5.18. 1,2,4-Tribromo-3-(2,5-dibromo-3,4-dimethoxy-6-methylbenzyl)-
5,6-dimethoxybenzene (29)
116e118 ꢀC. 1H NMR (500 MHz, CDCl3)
d
: 6.76 (d, J ¼ 1.83 Hz, 1H),
6.72 (s, 1H), 6.60 (d, J ¼ 1.83 Hz,1H), 4.11 (s, 2H), 3.87 (s, 3H), 3.84 (s,
3H), 3.81 (s, 3H), 3.77 (s, 3H), 2.23 (s, 3H); 13C NMR (125 MHz,
The title compound was prepared from 28 using the procedure
previously described for compound 26 to give white solid. 53%
CDCl3) d: 153.5, 151.8, 144.8, 144.7, 136.5, 133.9, 129.9, 123.8, 121.9,
yield. 1H NMR (500 MHz, CDCl3)
d
: 4.78 (s, 2H), 3.90 (s, 3H), 3.89 (s,
117.5, 113.9, 111.8, 60.5, 60.4, 56.1, 56.0, 37.8, 20.8. EIMS m/z (%
relative intensity): 462/460/458 [M]þ (45/95/50).
3H), 3.86 (s, 6H), 2.14 (s, 3H); 13C NMR (125 MHz, CDCl3)
d
: 150.6,
150.5, 149.6, 149.0, 136.8, 134.2, 134.0, 123.0, 122.2, 122.1, 121.4,
121.1, 60.9, 60.7, 44.2, 21.1.
5.13. (3-Bromo-2-(3-bromo-4,5-dimethoxybenzyl)-4,5-dimethoxy-
phenyl)methanol (24)
5.19. Enzymatic activity assay in vitro
The title compound was prepared from 23 using the procedure
previously described for compound 5 to afford white solid. 32%
Recombinant human GST-PTP1B (hGST-PTP1B) protein was
used to measure the PTP1B inhibitory activities of synthetic com-
pounds. The assay was conducted as described by Zhang et al. [23]
with minor modifications. HPN was dissolved in DMSO and
distributed to 96-well clear polystyrene plate. DMSO was distrib-
uted as the full enzyme activity. After adding an assay mixture,
hGST-PTP1B was added to initiate the reaction. The enzymatic ac-
tivity assay was carried out in a mixture containing 50 mM MOPS,
pH 6.5, 2 mM pNPP, 30 nM PTP1B and 2% DMSO, and the catalysis of
pNPP was continuously monitored at 405 nm for 2 min at 30 ꢀC.
Inhibitory rate was calculated according to the formula:
yield. mp: 156e158 ꢀC. 1H NMR (500 MHz, CDCl3)
d
: 7.05 (s, 1H),
6.74 (s,1H), 6.60 (s,1H), 4.59 (s, 2H), 4.17 (s, 2H), 3.89 (s, 3H), 3.86 (s,
3H), 3.79 (s, 3H), 3.76 (s, 3H); 13C NMR (125 MHz, CDCl3)
: 153.6,
d
152.2, 145.9, 144.8, 136.5, 136.4, 129.2, 123.7, 122.3, 117.6, 111.7, 111.6,
63.3, 60.5, 60.4, 56.1, 56.0, 36.5.
5.14. 3-Bromo-4-(3-bromo-4,5-dihydroxybenzyl)-5-(ethoxymethyl)-
benzene-1,2-diol (25)
The title compound was prepared from 24 using the procedure
previously described for compound 6e9 and was purified by silica
gel column chromatography (petroleum ether:ethyl acetate, 1:1) to
ꢀ
ꢁ.
% inhibition ¼ 100 ꢂ VDMSO ꢁ Vsample VDMSO
:
give yellowish solid. 90% yield. 1H NMR (500 MHz, acetone-d6)
d:
6.97 (s, 1H), 6.73 (d, J ¼ 2.4 Hz, 1H), 6.49 (d, J ¼ 2.4 Hz, 1H), 4.32 (s,
IC50 value was determined with 30 nM PTP1B, 2 mM pNPP in
2H), 4.08 (s, 2H), 3.43 (q, J ¼ 7.2 Hz, 2H), 1.10 (t, J ¼ 7.2 Hz, 3H); 13C
50 mM MOPS at pH 6.5 and the inhibitors diluted around the

136
B. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 129e136
estimated IC50 values. IC50 was calculated from the non-linear curve
fitting of percent inhibition (% inhibition) vs inhibitor concentration
[I] by using the following equation:
Foundation (Grant No. AS2011013), the Qingdao Municipal Natural
Science Foundation (Grant No. 10-3-4-8-2-JCH) and Shangdong
Excellent Young Scientists Award Fund (Grant No. BS2009YY011).
% inhibition ¼ 100=f1 þ ðIC50=½IꢄÞkg;
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where k is the Hill coefficient.
To study the inhibition selectivity on other PTP family members,
TCPTP, SHP-1, SHP-2 and LAR were prepared as described previ-
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5.20. Anti-diabetic activity assays in db/db mice
C57BL/KsJ db/db mice (6e8 weeks age) and their non-diabetic
controls (db/dm mice, 6e8 weeks age) were purchased from
Experimental Animal Center of Military Medical Sciences (Beijing,
China). The mice were housed in a room controlled for temperature
(24 ꢃ 2.0 ꢀC), relative humidity (60e80%) and 12/12 h light/dark
cycle (lights on at 6.00 a.m.). All the mice were allowed free access
to fresh water and laboratory chow. After one week adaptation
period, the mice were divided into five groups (n ¼ 10 in each
group), the control group (db/dm mice), the model (0.5% CMC-Na
containing 2% tween-80) group, rosiglitazone group (50 mg/kg),
compound 9 high-dose group (50 mg/kg) and low-dose group
(25 mg/kg). Rosiglitazone was dissolved in distilled water and
compound 9 was suspended in 0.5% CMC-Na containing 2% tween-
80 and administered orally for six weeks (approximately 0.2 mL/
20 g body weight). Body weights and food intakes were measured
weekly. Blood glucose concentrations were checked with ONE
TOUCH UltraÒ glucometer (LifeScan, USA) weekly. Serum triglyc-
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method (Whitman Biotech Co., Ltd., Nanjing, China) every two
weeks. At the end of 6-week treatment period, the mice were fasted
overnight. 1 h after administration, mice were excised eyeballs for
blood sampling. Blood samples were collected into heparin-coated
tubes. HbA1c concentration was measured by HbA1c reagent
(Whitman Biotech Co., Ltd., Nanjing, China). After centrifugation at
3500 g for 15 min at 4 ꢀC, the plasma was carefully removed from
the sample. The GSP levels were determined by fructosamine
method using GSP assay kit (Whitman Biotech Co., Ltd., Nanjing,
China).
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Conflict of interest
The authors have declared no conflict of interest.
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Acknowledgments
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (Grant No. 41276167 and
41206066), the Natural Science Foundation of Jiangsu Province
(Grant No. BK 2012223), the Nantong Municipal Natural Science