HPN, a synthetic analogue of bromophenol from red alga Rhodomela confervoides: Synthesis and anti-diabetic effects in C57BL/KsJ-db/db mice

Article abstract of DOI:10.3390/md11020350

3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-(isopropoxymethyl)benzyl)benzene-1, 2-diol (HPN) is a synthetic analogue of 3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6- (ethoxymethyl)benzyl)benzene-1,2-diol (BPN), which is isolated from marine red alga Rhodomela confervoides with potent protein tyrosine phosphatase 1B (PTP1B) inhibition (IC₅₀ = 0.84 μmol/L). The in vitro assay showed that HPN exhibited enhanced inhibitory activity against PTP1B with IC₅₀ 0.63 μmol/L and high selectivity against other PTPs (T cell protein tyrosine phosphatase (TCPTP), leucocyte antigen-related tyrosine phosphatase (LAR), Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2). The results of antihyperglycemic activity using db/db mouse model demonstrated that HPN significantly decreased plasma glucose (P < 0.01) after eight weeks treatment period. HPN lowered serum triglycerides and total cholesterol concentration in a dose-dependent manner. Besides, both of the high and medium dose groups of HPN remarkably decreased HbA1c levels (P < 0.05). HPN in the high dose group markedly lowered the insulin level compared to the model group (P < 0.05), whereas the effects were less potent than the positive drug rosiglitazone. Western blotting results showed that HPN decreased PTP1B levels in pancreatic tissue. Last but not least, the results of an intraperitoneal glucose tolerance test in Sprague-Dawley rats indicate that HPN have a similar antihyperglycemic activity as rosiglitazone. HPN therefore have potential for development as treatments for Type 2 diabetes.

Full text of DOI:10.3390/md11020350

Mar. Drugs 2013, 11, 350-362; doi:10.3390/md11020350
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Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
HPN, a Synthetic Analogue of Bromophenol from Red Alga
Rhodomela confervoides: Synthesis and Anti-Diabetic Effects
in C57BL/KsJ-db/db Mice
Dayong Shi 1,2,*, Shuju Guo 1,2, Bo Jiang 1,2, Chao Guo 1,2, Tao Wang 3, Luyong Zhang 3 and
Jingya Li 4
1
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
E-Mails: guoshuju@qdio.ac.cn (S.G.); beckyjiang0220@163.com (B.J.);
yujuejie0546@126.com (C.G.)
2
Nantong Branch, Institute of Oceanology, Chinese Academy of Sciences, Nantong 226006, China
3
Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, China;
E-Mails: wangtao1331@126.com (T.W.); lyzhang@cpu.edu.cn (L.Z.)
4
National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China;
E-Mail: jyli@mail.shcnc.ac.cn
* Author to whom correspondence should be addressed; E-Mail: shidayong@qdio.ac.cn;
Tel.: +86-0532-8289-8719; Fax: +86-0532-8289-8641.
Received: 14 November 2012; in revised form: 16 January 2013 / Accepted: 21 January 2013 /
Published: 30 January 2013
Abstract: 3,4-Dibromo-5-(2-bromo-3,4-dihydroxy-6-(isopropoxymethyl)benzyl)benzene-
1,2-diol (HPN) is a synthetic analogue of 3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-
(ethoxymethyl)benzyl)benzene-1,2-diol (BPN), which is isolated from marine red alga
Rhodomela confervoides with potent protein tyrosine phosphatase 1B (PTP1B) inhibition
(IC50 = 0.84 μmol/L). The in vitro assay showed that HPN exhibited enhanced inhibitory
activity against PTP1B with IC50 0.63 μmol/L and high selectivity against other PTPs
(T cell protein tyrosine phosphatase (TCPTP), leucocyte antigen-related tyrosine
phosphatase (LAR), Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1)
and SHP-2). The results of antihyperglycemic activity using db/db mouse model
demonstrated that HPN significantly decreased plasma glucose (P < 0.01) after eight weeks
treatment period. HPN lowered serum triglycerides and total cholesterol concentration in a
dose-dependent manner. Besides, both of the high and medium dose groups of HPN
remarkably decreased HbA1c levels (P < 0.05). HPN in the high dose group markedly

Mar. Drugs 2013, 11
lowered the insulin level compared to the model group (P < 0.05), whereas the effects were
351
less potent than the positive drug rosiglitazone. Western blotting results showed that HPN
decreased PTP1B levels in pancreatic tissue. Last but not least, the results of an
intraperitoneal glucose tolerance test in Sprague–Dawley rats indicate that HPN have a
similar antihyperglycemic activity as rosiglitazone. HPN therefore have potential for
development as treatments for Type 2 diabetes.
Keywords: bromophenol; protein tyrosine phosphatase 1B inhibitor; db/db mouse model;
anti-diabetes properties
1. Introduction
Type 2 diabetes mellitus (T2DM) is a complex endocrine and metabolic disorder. On the World
Diabetes Day 2011, the International Diabetes Federation’s 5th edition of the Diabetes Atlas indicated
that the number of people living with diabetes was expected to rise from 366 million in 2011 to 552 million
by 2030, if no urgent action is taken. Furthermore, the disease is now being observed with increasing
frequency in young children and adolescents. Current treatment agents for T2DM mainly include
insulin, biguanides, sulfonylureas, α-glucosidase inhibitors and thiazolidinediones (TZDs). However,
adverse effects such as hypoglycemia, weight gain and edema limit their clinical use [1].
Protein tyrosine phosphatase 1B (PTP1B), the first characterized protein tyrosine phosphatase
(PTPases), has become a new target for the treatment of T2DM because of its involvement in the
insulin signaling cascade as a major negative regulator [2]. In addition, molecular biology
investigations have already proven that PTP1B knock-out mice exhibit the phenotypes of increased
insulin sensitivity, improved glucose tolerance, and resistance to diet-induced obesity [3,4]. Based on
the above research results, the inhibition of PTP1B has emerged as a novel therapeutic strategy for the
treatments of Type 2 diabetes mellitus.
In the past decades, there have been a number of reports about the development of synthetic PTP1B
inhibitors [5–7]. However, the highly charged phosphatase active site and the relatively shallow nature
of the surrounding protein surface pose a great challenge to the discovery of cell permeable and orally
bio-available PTP1B inhibitors. At the same time, more and more studies have been focusing on
PTP1B inhibitors isolated from plants, and most of them have demonstrated promising results [8–19].
Bromophenols exists in most marine organisms, such as mollusks, algae, jellyfish and sponges.
Accumulated studies have reported that bromophenols exhibit a wide spectrum of biological and
pharmacological activities including antioxidant activity [20,21], α-glucosidase inhibition [22], and
antitumor effects [23]. Four novel bromophenols from the ethanolic extract of the red alga
Rhodomela confervoides were isolated and screened as PTP1B inhibitors in our lab (Figure 1) [24,25].
In order to obtain new types of PTP1B inhibitors, a series of bromophenol derivatives were
synthesized by using bis-(2,3-dibromo-4,5-dihydroxy-phenyl)-methane (BDDPM) and 3-bromo-4,
5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol (BDB) as lead compounds. Fortunately, two
of the bromophenol derivatives exhibited enhanced PTP1B inhibitory activity and are promising
development prospects [26,27]. In the present study, we selected BPN (3,4-dibromo-5-(2-bromo-3,

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4-dihydroxy-6-(ethoxymethyl)benzyl)benzene-1,2-diol) as the lead compound due to its potent PTP1B
inhibition (IC50 = 0.84 μmol/L). Optimization of BPN provided a novel bromophenol analogue HPN
(3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(isopropoxymethyl)benzyl)benzene-1,2-diol) and its structure
is in Figure 1. Herein, we report the synthesis of HPN and use the diabetic mouse strain C57BL/KsJ-db/db
(db/db), which exhibits many of the metabolic disturbances of human Type 2 diabetes including
hyperglycemia, obesity, and early hyperinsulinemia to investigate the pharmacological properties of HPN.
Figure 1. Structures of 3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(isopropoxymethyl)
benzyl)benzene-1,2-diol (HPN) and four bromophenols isolated from red alga
Rhodomela confervoides.
2. Results and Discussion
2.1. Characterization and Identification of HPN
HPN was obtained as a yellowish powder. The electron ionization mass spectroscopy (EIMS)
spectrum showed the tribrominated molecular ion peak cluster at m/z 544/542/540/538 (1:3:3:1). The
molecular formula was determined as C17H16Br3O5 by HREIMS at m/z 536.8525 (calcd for
C17H16Br3O5 536.8548). The 1H NMR spectrum revealed the presence of four phenol hydroxyl protons
(δ 9.72, δ 9.69, δ 9.26, δ 9.11), two phenyl ring protons (δ 6.88, δ 6.05), two methylene protons (δ 4.15,
δ 3.98) and the isopropyl group (δ 3.51, δ 1.02). The 13C NMR spectrum showed the signals
representing twelve phenyl ring carbons (δ 144.9–113.0), one methine carbon (δ 79.1, δ 67.6, δ 38.3)
1
and two methyl carbons (δ 21.7). Detailed spectrum data are: H-NMR (500 MHz, DMSO-d6) δ: 9.72
(s, 1H, OH), 9.69 (s, 1H, OH), 9.26 (s, 1H, OH), 9.11 (s, 1H, OH), 6.88 (s, 1H), 6.05 (s, 1H), 4.15
13
(s, 2H), 3.98 (s, 2H), 3.51 (m, J = 6.43 Hz, 2H), 1.02 (d, J = 6.43 Hz, 6H); C-NMR (125 MHz,
DMSO-d6) δ: 144.9 (C), 144.3 (C), 142.5 (C), 142.4 (C) 130.5 (C), 129.3 (C), 127.5 (C), 115.4 (CH),
114.5 (C), 114.3 (C), 113.9 (CH), 112.9 (C), 79.1 (CH), 67.6 (CH2) 38.3 (CH2), 21.7 (2 × CH3).
EIMS m/z (% relative intensity): 544/542/540/538 [M]+ (1/3/3/1), 484/482/480/478 (2/7/7/2),
467/465/463/461 (2/6/6/2), 403/401/399 (5/10/5), 322/320 (10/9), 82/80 (98/100), 59 (30). HRMS m/z
calcd for C17H16O5Br3 [M − H]−, 536.8548; found, 536.8525.

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2.2. Enzyme-Based Inhibitory Activities Assays in Vitro
We firstly evaluated the inhibition of HPN against recombinant PTP1B in vitro. HPN exhibited
remarkable inhibitory activity against PTP1B with IC50 0.63 μmol/L, which was more potent than BPN
(IC50 = 0.84 μmol/L). Selectivity is one of the major issues for the development of PTP1B inhibitors as
drugs, hence, we investigated the selectivity of HPN against other PTPs (TCPTP, LAR, SHP-1 and
SHP-2), which share a high degree of structural conservation in the active site. We found that HPN
demonstrated an excellent selectivity against TCPTP, LAR, SHP-1 and SHP-2 (>50-fold).
2.3. Effects of HPN on Body Weight, Food and Water Intake
The typical symptoms of diabetes are polydipsia, polyuria, and polyphagia, so we studied the
effects on body weight, food and water intake after HPN administration for 8 weeks in db/db mice.
Since no small molecule inhibitors of PTP1B are on the market, we selected rosiglitazone as the
positive control, which is an effective antidiabetic drug and works as an insulin sensitizer. During the
8-week period, there were no significant changes in defecation and urination of mice treated with HPN
and rosiglitazone, and no mortality occurred. As shown in Table 1, the initial and final body weights of
the db/db mice were significantly higher than those in db/dm mice. There was no significant difference
in the food and water intake between the model and control groups. The body weights increased
gradually during the treatment period. At the end of the study, the mice in the rosiglitazone treated
group had a significantly higher body weight compared to the HPN treated groups. The mice in the
HPN treated group exhibited greater body weight gain, however, the increase was less than that
observed in the rosiglitazone treated group. In the low-dose treated group, the body weight was
obviously lower than in the model group (P < 0.05). HPN at different doses reduced the food and
water intake in db/db mice, but the differences were not significant. The results indicated that HPN
could reduce diabetes-related symptoms.
Table 1. Effects of HPN on food and water intake, and body weight in db/db mice.
Body weight (g) a
Food intake (g/day) a
initial final
3.42 ± 0.03 3.26 ± 0.40
Water intake (mL/day) a
Group
initial
17.7 ± 0.9
final
initial
final
Control
Model
22.0 ± 2.5
3.46 ± 0.04
7.55 ± 0.17
7.89 ± 0.13
7.96 ± 0.34
7.67 ± 0.04
8.41 ± 0.49
3.67 ± 0.19
13.12 ± 0.95
9.94 ± 0.41
12.00 ± 0.39
12.92 ± 0.36
11.72 ± 0.34
36.6 ± 2.1 ** 53.7 ± 2.6 ** 5.05 ± 0.03 7.67 ± 0.62
HPN (40 mg/kg)
HPN (20 mg/kg)
HPN (10 mg/kg)
Rosiglitazone (20 mg/kg)
35.9 ± 1.6
35.7 ± 1.8
35.7 ± 1.5
36.0 ± 3.1
52.3 ± 3.1
50.5 ± 4.4
48.1 ± 3.0 #
54.8 ± 4.1
4.88 ± 0.07 5.46 ± 0.89
4.85 ± 0.02 6.54 ± 0.70
4.79 ± 0.08 6.65 ± 0.68
5.03 ± 0.06 7.09 ± 0.48
a Data are expressed as means ± SD, n = 10; # P < 0.05; ** P < 0.01 when compared to the control group.
2.4. Effects of HPN on Blood Glucose
Hyperglycemia plays an important role in the development of Type 2 diabetes mellitus, therefore,
the effective control of the blood glucose level is crucial for preventing diabetic complications and
improving the quality of the life in diabetic patients. Firstly, we examined the time-effect relationship
of the compound in a db/db mice model on the fifth day. As shown in Figure 2, the high dose of HPN

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produced a remarkable reduction in plasma glucose at 1 h, 2 h and 4 h during the intervention period
(P < 0.01, P < 0.05), and its greatest effect was 1 h after intragastric administration, lowering plasma
glucose from 18.2 mmol/L to 13.3 mmol/L (P < 0.01). At that time, a notable reduction was observed
in the medium and low dose groups of HPN (P < 0.01, P < 0.05). There was no decline of effect in
plasma glucose prolonged to 8 h and the plasma glucose maintained at 16 mmol/L. The
antihyperglycemic activity results of HPN are illustrated in Table 2, the positive control rosiglitazone
decreased plasma glucose levels from 18.7 mmol/L to 15.5 mmol/L in the first week (P < 0.05).
Similarly, HPN at 40 mg/kg significantly decreased plasma glucose (P < 0.01) and maintained the
plasma glucose level at 15 mmol/L after an 8-week treatment period. In general, the high dose and
medium dose of HPN were more effective in decreasing the blood glucose level compared to the low
dose group of HPN.
Figure 2. The time-effect relationship of HPN on the fifth day in db/db mice model.
# P <0.05, ## P <0.01 vs. model.
Table 2. Effects of HPN on blood glucose level in db/db mice.
Plasma glucose (mmol/L) a
Dose
Group
(mg/kg)
Baseline
6.0 ± 0.6
1 week
6.0 ± 0.4
4 weeks
6.1 ± 0.3
8 weeks
5.8 ± 0.4
Control
Model
18.9 ± 4.5 **
19.8 ± 5.0
18.6 ± 4.5
19.4 ± 4.9
20.0 ± 3.6 **
15.1 ± 3.6 ##
16.6 ± 3.4 #
16.0 ± 3.7 #
20.2 ± 4.6 **
14.7 ± 3.7 ##
15.4 ± 4.7 #
17.5 ± 3.9
18.9 ± 4.1 **
15.8 ± 2.4 #
15.5 ± 3.3 #
16.5 ± 3.5
40
20
10
20
HPN
Rosiglitazone
18.7 ± 3.8
15.5 ± 4.0 #
15.6 ± 3.1 #
13.9 ± 2.9 ##
a
#
##
Data are expressed as means ± SD, n = 10; P < 0.05, P < 0.01 when compared to the model group;
** P < 0.01 when compared to the control group.

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2.5. Effects of HPN on Serum Lipids
It is well documented that there is an elevation of serum lipid concentration in diabetics. As
illustrated in Table 3, 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). The serum triglyceride
and total cholesterol levels of the rosiglitazone treated group were much lower than in the model group
(P < 0.01, P < 0.05). The HPN lowered the triglycerides and total cholesterol concentration in a
dose-dependent manner in the HPN-treated group, and the high-dose group showed a better lipid
lowering profile (P < 0.05) than other dose groups.
Table 3. Effects of HPN on serum lipid in db/db mice.
Triglycerides (mmol/L) a
Total cholesterol (mmol/L) a
Dose
Group
(mg/kg)
Baseline
4 weeks
8 weeks
1.19 ± 0.24
Baseline
4 weeks
8 weeks
2.99 ± 0.72
Control
Model
1.19 ± 0.25
1.30 ± 0.26
3.01 ± 0.83
3.32 ± 0.80
1.47 ± 0.30 * 1.69 ± 0.39 * 1.74 ± 0.40 ** 4.20 ± 1.17 * 7.22 ± 2.91 ** 7.18 ± 2.18 **
40
20
10
20
1.36 ± 0.35
1.44 ± 0.42
1.44 ± 0.28
1.46 ± 0.23
1.32 ± 0.21 #
1.32 ± 0.27 #
1.45 ± 0.30
1.32 ± 0.34 #
1.36 ± 0.31 #
1.47 ± 0.32
1.36 ± 0.23 #
3.89 ± 0.93
3.86 ± 1.42
4.72 ± 0.91
4.19 ± 1.04
4.67 ± 1.55 #
5.01 ± 1.45
5.54 ± 1.59
4.67 ± 1.42 #
5.01 ± 1.64 #
5.12 ± 2.31
5.79 ± 1.69
4.44 ± 1.60 ##
HPN
Rosiglitazone
1.33 ± 0.27 #
a
#
##
Data are expressed as means ± SD, n = 10; P < 0.05, P < 0.01 when compared to the model group; ** P < 0.01
when compared to the control group.
2.6. Effects of HPN on Glycated Hemoglobin and Glycated Serum Protein
Glycated hemoglobin (HbA1c) and glycated serum proteins (GSP) are well known parameters of
glycemic control. Hence, we were interested to investigate the effect of HPN on HbA1c and GSP
levels. As depicted in Table 4, the blood HbA1c and GSP concentrations in the rosiglitazone group
were significantly lower than in the model group (P < 0.01). Also, HPN of high and medium dose
groups remarkably decreased HbA1c levels (P < 0.05). On the other hand, all dose groups of HPN
reduced GSP levels in a dose-dependent manner.
Table 4. Effects of HPN on glycated hemoglobin and glycated serum protein.
Group
Control
Model
Dose (mg/kg)
HbA1c a (%)
1.1 ± 0.5
GSP a (mmol/L)
1.3 ± 0.2
4.5 ± 1.3 **
3.2 ± 0.8 #
3.5 ± 0.9 #
3.7 ± 1.2
2.8 ± 0.9 ##
##
4.8 ± 1.0 **
3.2 ± 0.9 ##
3.3 ± 1.4 #
3.7 ± 1.3 #
3.1 ± 1.0 ##
40
20
10
HPN
Rosiglitazone
20
a
#
Data are expressed as means ± SD, n = 10; P < 0.05, P < 0.01 when compared to the model group;
** P < 0.01 when compared to the control group.

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2.7. Effects of HPN on Plasma Insulin Level
In general, the db/db mice exhibit an initial phase of hyperinsulinemia. In this study, the plasma
insulin levels in db/db mice were significantly higher than in the control group at 4 weeks and 8 weeks
(P < 0.01). As shown in Table 5, the rosiglitazone treated group had a significantly lower insulin level
at 20 mg/kg (P < 0.01). As for HPN treated group, the effects were less marked than for the
rosiglitazone group. The insulin levels increased during the treatment period from 4 weeks to 8 weeks.
Only the high dose of HPN markedly lowered plasma insulin levels compared to the model
group (P < 0.05).
Table 5. Effects of HPN on plasma insulin level in db/db mice.
Insulin (ng/mL) a
Group
Dose (mg/kg)
4 weeks
8 weeks
Control
Model
1.18 ± 0.37
1.36 ± 0.42
22.56 ± 6.61 **
15.30 ± 6.26 #
15.94 ± 7.02
15.55 ± 3.79 #
27.94 ± 4.78 **
20.80 ± 8.31 #
22.22 ± 6.10 #
23.40 ± 6.50
10.80 ± 3.43 ##
40
20
10
20
HPN
Rosiglitazone
10.60 ± 3.71 ##
a
#
##
Data are expressed as means ± SD, n = 10; P < 0.05, P < 0.01 when compared to the model group;
** P < 0.01 when compared to the control group.
2.8. PTP1B Expression in Pancreatic Tissue (Western Blotting)
A Western blotting assay was conducted to verify the expression of PTP1B protein in pancreatic
tissue and to determine the inhibition effect of HPN against PTP1B. As illustrated in Figure 3, PTP1B
expression in pancreatic tissue of db/db mice is much higher than in that of db/dm mice. It is apparent
that HPN decreases PTP1B levels at high, medium and low doses, whereas the effects are not as potent
as with rosiglitazone.
Figure 3. Western blotting assay of HPN.
2.9. Intraperitoneal Glucose Tolerance Test in Sprague–Dawley Rats
An intraperitoneal glucose tolerance test (IPGTT) in Sprague–Dawley (SD) rats was performed to
investigate the effects of HPN on glucose tolerance during the treatment period and explore the
possible mechanism of antihyperglycemic activity of HPN. As illustrated in Table 6, there are no
differences of plasma glucose levels among the control, rosiglitazone and HPN groups prior to glucose
load. After glucose administration, the plasma glucose levels significantly elevated at 0.5 and 1 h

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(from 4 mmol/L to 10.7 mmol/L) and decreased to 7.3 mmol/L at 2 h. Similar trends were observed in
the rosiglitazone and HPN groups. Furthermore, we examined the insulin levels. As shown in Figure 4,
insulin levels in the rosiglitazone and HPN groups were higher than in the control group, but the
difference was not statistically significant. In general, as the findings of plasma glucose and insulin
levels in the HPN group are similar to those of the rosiglitazone group, it is then implied that HPN
conducts its antihyperglycemic activity by enhancing insulin sensitivity.
Table 6. Effects of HPN on blood glucose in intraperitoneal glucose tolerance test
(IPGTT) in Sprague–Dawley (SD) rats.
Plasma glucose (mmol/L) a
Dose
Group
(mg/kg)
0 h
0.5 h
1 h
2 h
Control
HPN
4.0 ± 0.4
4.0 ± 0.8
4.0 ± 0.4
4.2 ± 0.5
3.9 ± 0.3
10.7 ± 3.5
10.4 ± 2.2
10.7 ± 3.1
10.9 ± 2.9
9.7 ± 1.7
10.7 ± 2.8
10.3 ± 2.1
10.3 ± 2.1
10.8 ± 2.7
10.4 ± 2.5
7.3 ± 1.4
7.4 ± 0.9
7.8 ± 1.0
8.3 ± 1.1
7.4 ± 1.1
40
20
10
20
Rosiglitazone
a Data are expressed as means ± SD, n = 10.
Figure 4. Effects of HPN on insulin level in IPGTT in SD rats.
3. Experimental Section
3.1. General Experimental Procedures
Reagents and all solvents were analytically pure and were used without further purification. All of
the experiments were monitored 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 materials or coloration by 95% FeCl3-EtOH solution. Column chromatography was
carried out using silica gel (200–300 mesh). All final compounds were purified to >95% purity, as
1
determined by high-performance liquid chromatography (HPLC). H-NMR and 13C-NMR spectra

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were recorded on an Inova (500 MHz) NMR spectrometer for proton and at 125 MHz for carbon. Mass
spectra and high-resolution mass spectral (HRMS) data were recorded on Autospec Ultima-Tof and
Thermo LTQ-Orbitrap mass spectrometer, respectively.
3.2. Preparation of HPN
The synthetic route of HPN is shown in Scheme 1. The starting Materials 1 and 2 were prepared
according to our previous study [28]. Friedel–Crafts alkylation between 1 and 2 in the presence of
AlCl3 gave diaryl-methane 3. Treatment 3 with NBS under hv condition provided the benzyl bromide,
which was subsequently hydrolysed in the presence of K2CO3 and 1,4-dioxane gave benzyl alcohol 5.
Demethylation of Compounds 3 and 5 with BBr3 in CH2Cl2 cleanly gave bromophenol in good yields.
The bromophenol was treated with isopropyl alcohol in the presence of catalyst H3PO4 afforded HPN.
Scheme 1. Synthetic route of HPN.
Reagents and conditions: (i) AlCl3, CH2Cl2, r.t.; (ii) NBS, AIBN, CCl4, hv; (iii) K2CO3,
1,4-dioxane, H2O, 90 °C; (iv) BBr3, CH2Cl2, 0 °C—r.t.; (v) isopropyl alcohol, cat. H3PO4, r.t.
3.3. Enzymatic Activity Assay in Vitro
Recombinant human GST-PTP1B (hGST-PTP1B) protein was used to measure the inhibition of
HPN on PTP1B activity, which was determined by monitoring the hydrolysis of pNPP (para-nitrophenyl
phosphate). Dephosphorylation of pNPP generates the product pNP, which can be monitored at
405 nm. HPN was solubilized in dimethyl sulfoxide (DMSO) and distributed to a 96-well clear
polystyrene plate. DMSO was distributed as the full enzyme activity. After adding an assay mixture,
GST-PTP1B was added to initiate the reaction. The high-throughput screening was carried out in a
mixture containing MOPS, pNPP, PTP1B and DMSO, and the catalysis of pNPP was continuously
monitored at 405 nm for 2 min at 30 °C. The inhibitory rate was calculated according to the formula:
% inhibition = 100 × (VDMSO − Vsample)/VDMSO
All the assays were performed in triplicate.
(1)
IC50 value was calculated using GraphPad Prism 4.00 (GraphPad Software Inc., USA). To study the
inhibition selectivity on other PTP family members, 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) and LAR (leucocyte antigen-related tyrosine phosphatase) were
prepared as described previously [29]. Assays for these PTPs were performed at the optimal pH for

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each individual enzyme activity. These enzymes and HPN were pre-incubated for 3 min at 4 °C, and
the assays were initiated by adding substrates. Assays performed for SHP-1, SHP-2 and LAR were
done using 3-O-methylfluorescein phosphate (OMFP) as a substrate.
3.4. Animals
C57BL/KsJ db/db mice (6–8 weeks of age) and their non-diabetic controls (db/dm mice, 6–8 weeks
of age) were purchased from Experimental Animal Center of Military Medical Sciences (Beijing,
China). The Sprague–Dawley (SD) rats (180–220 g) were provided by Shanghai SIPPR/BK Lab
Animal Ltd. All the animals were left to acclimatize for 1 week before the experimental period. The
animals were housed in a room controlled for temperature (24 ± 2.0 °C), relative humidity
(60%–80%), and 12/12 h light/dark cycle (lights on at 6:00 am). All the animals were allowed free
access to fresh water and laboratory chow.
3.5. Experiment Design
The db/db mice were randomly divided into five groups matched for blood glucose levels, each
group consisting of ten mice, as follows:
Model group: db/db mice given 0.5% CMC-Na and 2% Tween-80.
Positive drug group: db/db mice given rosiglitazone (20 mg/kg).
HPN high-dose group: db/db mice given HPN (40 mg/kg).
HPN medium-dose group: db/db mice given HPN (20 mg/kg).
HPN low-dose group: db/db mice given HPN (10 mg/kg).
The db/dm mice were used as control group. Rosiglitazone was dissolved in distilled water and
HPN were suspended in 0.5% CMC-Na containing 2% Tween-80 and administered orally for eight
weeks (approximately 0.2 mL/20 g body weight). Body weights and food intake were measured
weekly. Blood glucose concentrations were checked with ONE TOUCH Ultra® glucometer (LifeScan,
USA) weekly. Serum triglyceride and total cholesterol levels were measured by an enzymatic method
(Whitman Biotech Co., Ltd., Nanjing, China) every two weeks. At the end of the 8-week treatment
period, the mice were fasted overnight. One hour after administration, the mice’s eyeballs were
excised 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 the GSP assay kit (Whitman Biotech Co.,
Ltd., Nanjing, China). The plasma insulin levels were determined using an ELISA kit (ExCell Biology
Inc., Shanghai, China).
3.6. Western Blotting Assay
Western blotting was performed for the analysis of PTP1B expression in pancreatic tissue using
standard protocols. Fourty micrograms of protein were subjected to 10% (w/v) SDS-polyacrylamide
gels and transferred to a nitrocellulose membrane. The membrane was blocked for 1 h in tris-buffered
saline with tween (TBST) (PBS, 0.05% Tween-20) containing 3% nonfat dry milk and incubated with

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antibodies for PTP1B (Santa Cruz Biochemicals, Dallas, FL, USA). After they were washed in TBST,
the corresponding secondary antibodies were incubated at room temperature for 1 h. The
antibody-reactive bands were revealed by the enhanced chemiluminescence kit (Amersham, UK) and
were exposed using ChemiDoc XRS Gel Imager (Bio-rad, USA).
3.7. Intraperitoneal Glucose Tolerance Test (IPGTT) in SD Rats
The rats were divided into five groups, each group consisting of ten rats, as follows:
Control group: SD rats given 0.5% CMC-Na.
Positive drug group: SD rats given rosiglitazone (20 mg/kg).
HPN high-dose group: SD rats given HPN (40 mg/kg).
HPN medium-dose group: SD rats given HPN (20 mg/kg).
HPN low-dose group: SD rats given HPN (10 mg/kg).
The IPGTT was conducted at the end of 7-day treatment. SD rats were fasted overnight (16–18 h)
and then given vehicle, positive drugs and HPN as mentioned above. Five hours after dosing, the
fasting blood glucose level was measured from tail-tip blood using the ONE TOUCH Ultra®
glucometer (LifeScan, USA). Then the mice were injected intraperitoneally with glucose (2.5 g/kg
body weight). Blood glucose was measured again after 0.5, 1 and 2 h for the IPGTT.
3.8. Statistical Analysis
Data are presented as mean ± SD. Statistical analysis of the data for multiple comparisons was
performed by analysis if variance (ANOVA). For single comparison, the significance of differences
between means was determined by t-test. Values of P < 0.05 were considered statistically significant,
and a value of P < 0.001 was considered statistically most significant.
4. Conclusions
In summary, we have discovered a novel synthetic analogue of bromophenol HPN and evaluated its
anti-diabetic activities in db/db mice. Both the results of anti-diabetic activities in vitro and in vivo are
inspiring. Hence, the chemical entity reported in this study could provide a possible opportunity for
developing a novel PTP1B inhibitor with promising pharmacological properties. The acute toxicity
assay of HPN is in progress and will be reported in due course.
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. BK2012223), the Nantong Municipal Natural Science 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).

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