Thiazolidinedione derivatives as PTP1B inhibitors with antihyperglycemic and antiobesity effects

Article abstract of DOI:10.1016/j.bmcl.2009.09.020

Benzylidene-2,4-thiazolidinedione derivatives with substitutions on the phenyl ring at the ortho or para positions of the thiazolidinedione (TZD) group were synthesized as PTP1B inhibitors with IC₅₀ values in a low micromolar range. Compound 3e, the lowest, bore an IC₅₀ of 5.0 μM. In vivo efficacy of 3e as an antiobesity and hypoglycemic agent was evaluated in a mouse model system. Significant improvement of glucose tolerance was observed. This compound also significantly suppressed weight gain and significantly improved blood parameters such as TG, total cholesterol and NEFA. Compound 3e was also found to activate peroxisome proliferator-activated receptors (PPARs) indicating multiple mechanisms of action.

Full text of DOI:10.1016/j.bmcl.2009.09.020

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6161–6165
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thiazolidinedione derivatives as PTP1B inhibitors with antihyperglycemic
and antiobesity effects
Bharat Raj Bhattarai a, ,à, Bhooshan Kafle a, , Ji-Sun Hwang b, Deegendra Khadka a, Sun-Myung Lee c,
a,
Jae-Seung Kang c, Seung Wook Ham d, Inn-Oc Han b, Hwangseo Park e, , Hyeongjin Cho
*
*
a Department of Chemistry, Inha University, Incheon 402-751, Republic of Korea
b Department of Physiology and Biophysics, College of Medicine, Inha University, Incheon 402-751, Republic of Korea
c Department of Microbiology, College of Medicine, Inha University, Incheon 402-751, Republic of Korea
d Department of Chemistry, Chung Ang University, Seoul 156-756, Republic of Korea
e Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzylidene-2,4-thiazolidinedione derivatives with substitutions on the phenyl ring at the ortho or para
Received 25 April 2009
Revised 11 August 2009
Accepted 4 September 2009
Available online 10 September 2009
positions of the thiazolidinedione (TZD) group were synthesized as PTP1B inhibitors with IC50 values in a
low micromolar range. Compound 3e, the lowest, bore an IC50 of 5.0 lM. In vivo efficacy of 3e as an anti-
obesity and hypoglycemic agent was evaluated in a mouse model system. Significant improvement of
glucose tolerance was observed. This compound also significantly suppressed weight gain and signifi-
cantly improved blood parameters such as TG, total cholesterol and NEFA. Compound 3e was also found
to activate peroxisome proliferator-activated receptors (PPARs) indicating multiple mechanisms of
action.
Keywords:
PTP1B inhibitor
Thiazolidinedione
Obesity
Ó 2009 Elsevier Ltd. All rights reserved.
Diabetes
Glitazones
Thiazolidinedione (TZD) derivatives, also called glitazones, were
developed in the early 1980s as drugs for type 2 diabetes. Among
them, troglitazone, rosiglitazone, and pioglitazone were marketed
in the late 1990s (Fig. 1).1 These drugs are known to act by binding
to PPARs.2 Later, troglitazone was withdrawn from the market due
to severe liver toxicity in several patients.3 Rosiglitazone and piog-
litazone are currently in clinical use.
Recently, several studies have demonstrated compounds con-
taining scaffolds similar to TZD exhibit inhibitory effects against
PTP1B and other enzymes (Fig. 1). Malamas et al. reported several
azolidinediones, such as A, with low micromolar IC50 values
against PTP1B and with glucose and insulin normalizing effect in
ob/ob and db/db diabetic mouse models.4 Maccari et al. found a
modified thiazolidinedione, B, as a good inhibitor of aldose reduc-
tase, also involved in the complications of diabetes.5 Soon after, the
same group reported a similar class of compounds (e.g., C) having
high binding affinity to the active site of PTP1B.6 Similarly, Combs
et al. reported isothiazolidinone-containing compounds, repre-
sented by D as potent inhibitors of PTP1B.7 Rhodanine derivatives
like E were also found to behave as inhibitors of a low molecular
weight tyrosine phosphatase, PRL-3.8,9
Partial or complete disruption of the PTP1B gene in normal
and diabetic mice resulted in resistance to weight gain and im-
proved insulin responsiveness.10–13 PTP1B inhibition or a reduc-
tion of its cellular abundance in mice resulted in similar
consequences and, as such, provided the rationale for the thera-
peutic strategy for type 2 diabetes and obesity.14 In a recent
study, we identified novel PTP1B inhibitors by means of a com-
puter-aided drug design protocol involving virtual screening.15
One of those inhibitors (6f) was a TZD derivative inhibiting PTP1B
with an IC50 value of 11 lM. In a subsequent experiment, mar-
keted glitazones such as troglitazone, rosiglitazone and pioglitaz-
one were found to behave as medium or low potency inhibitors
of PTP1B with IC50 values of 55–400 lM. Marketed glitazones
are known to exert their antihyperglycemic effects by binding
PPAR. The glitazones are benzylidene-2,4-thiazolidinedione deriv-
atives with substitutions on the phenyl ring at the para position
of the TZD group. Most derivatives reported thus far are those
with the major substituents on the central phenyl ring at the para
position of the TZD group. Therefore, we synthesized TZD deriva-
tives with the major substituent at the ortho (3a–i) as well as
para (6a–h) positions of the TZD group and evaluated their inhib-
itory activity against PTP1B. The in vivo effects of the selected
compound were then tested in a mouse model system.
* Corresponding authors. Tel.: +82 2 3408 3766; fax: +82 2 3408 4334 (H.P.); tel.:
+82 32 860 7683; fax: +82 32 867 5604 (H.C.).
E-mail addresses: hspark@sejong.ac.kr (H. Park), hcho@inha.ac.kr (H. Cho).
These authors contributed equally to this work.
Present address: Faculty of Pharmacy, University of Manitoba, Apotex Centre, 750
à
McDermot Avenue, Winnipeg, Manitoba, Canada R3E 0T5.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.020

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B. R. Bhattarai et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6161–6165
Figure 1. Marketed TZD derivatives and PTP1B inhibitors mentioned in this study.
The TZD derivatives were synthesized by condensation of TZD
strong enough to permit its ionization constant (pKa ꢀ 6.74) to be
directly and accurately determined by conductivity measure-
ments.20 In the calculated PTP1B-3e complex, it is shown that
the two aminocarbonyl oxygens of the inhibitor receive two hydro-
gen bonds from the side chain of Gln266 and backbone amidic
group of Ser216. These hydrogen bonds are expected to play the
role of anchor for binding of the inhibitor in the active site. It is also
noteworthy that the deprotonated TZD moiety of 3e resides in the
vicinity of Cys215 and Arg221 at a distance within 4–5 Å. The prox-
imity to the catalytic residues with the hydrogen-bond stabiliza-
tions indicates that the deprotonated TZD group may serve as an
effective surrogate for the substrate phosphate group. Inhibitor
3e can be further stabilized in the active site by the hydrophobic
interactions of its aromatic rings with the nonpolar residues
including Tyr46, Val49, Phe182, Ala217, and Ile218. Judging from
the structural features in the calculated PTP1B-3e complex, inhib-
itor 3e seems to inhibit the catalytic action of PTP1B by binding the
active site through simultaneous establishment of the multiple
hydrogen bonds and hydrophobic interactions.
and the appropriate benzaldehydes in the presence of a piperidine
catalyst (Scheme 1). Introduction of the phenyl group in 3i, 6g, and
6h was achieved by a Suzuki coupling reaction. The compounds
synthesized in this study inhibited PTP1B more potently compared
to the marketed glitazones; IC50 values of 5–68
lM versus 55–
400 M. Of the two groups of synthesized compounds, 3a–i and
l
6a–h, the 3a–i group with the major substituent ortho to the TZD
group was more potent in PTP1B inhibition. Two compounds, 3e
and 6e, one from each group, were the most potent, with an IC50
value of 5.0 lM (Table 1).
The inhibitory activity of 3e19 was evaluated against a broad
range of PTPs, including TC-PTP, the catalytic domain of SHP-1
(SHP-1cat), the membrane proximal catalytic domain LAR (LAR-
D1), and 2 microbial PTPs, YOP and YPTP1 (Table 2). Compound
3e demonstrated 2.2-, >20-, and 3.7-fold selectivity over TC-PTP,
LAR-D1, and YPTP1, but little selectivity over SHP-1cat and YOP.
To obtain a degree of energetic and structural insight into the
inhibitory mechanism of 3e, its binding mode in the active site of
PTP1B was investigated by docking simulation. The calculated
binding mode of 3e is shown in Figure 2. In the docking simulation,
the amidic nitrogen of the TZD moiety was assumed to be deproto-
nated because the acidity of the TZD proton had been found to be
In vivo efficacy of 3e as an antiobesity and hypoglycemic agent
was evaluated in a mouse model system (C57BL/6J Jms Slc, male).23
Obesity and diabetes were induced in 16 mice by feeding HFD ad
libitum for 8 wk. The HFD-fed mice were then separated into 2
Scheme 1. Reagent and conditions: (a) RPhCH2Cl or RPhCH2Br, K2CO3, DMF or acetone, rt or reflux, 1–3 h; (b) PhB(OH)2, Pd(PPh3)4, toluene, EtOH, Na2CO3, 80 °C; (c) 2,4-TZD,
EtOH, piperidine, reflux, overnight.

B. R. Bhattarai et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6161–6165
6163
Table 1
Inhibitory effect of TZD derivatives against PTP1B
a
Compound
IC50
55
(lM)
Troglitazone
4
Rosiglitazone
Pioglitazone
Ertiprotafib
400 70
220 35
1.4 0.1b
9.0 0.3
8.0 1.0
8.0 1.0
8.0 0.4
5.0 0.1
3a
3b
3c
3d
3e
3f
3g
3h
3i
6a
6b
6c
6d
6e
6f
23
15
3
1
8.0 1.0
16
64
14
68
15
1
3
2
4
1
Figure 2. Calculated binding mode of 3e in the PTP1B active site. Carbon atoms of
the protein and ligand are indicated in green and cyan, respectively. Each dotted
line indicates a hydrogen bond. All hydrogen atoms are omitted for visual clarity.
AUTODOCK program21 was used in docking simulation of 3e in the PTP1B active site.
The 3-D coordinates in the X-ray crystal structure of PTP1B in complex with an N-
phenyloxamate inhibitor (PDB code: 1Q1M)22 were used as the receptor model in
the docking simulations. In the actual docking simulation of the ligand in the active
site of PTP1B, the empirical AUTODOCK scoring function improved by the implemen-
tation of a new solvation model for a compound was employed.15
5.0 0.4
11
1
6g
6h
9.0 0.4
6.0 1.0
a
Values are the mean standard deviations of two or more experiments. PTP1B
assay was performed as previously described using p-nitrophenyl phosphate as the
substrate (2 mM in the reaction mixture).16 IC50 values were determined by mea-
suring the pNPP hydrolase activity in a range of different inhibitor concentrations.
Kinetic data were analyzed using GRAFIT 5.0 program (Erithacus Software).
Data reproduced from our previous publication.17
b
group compared to HFD control (Table 4). Liver, lung, kidney and
white adipose tissue (epididymal and retroperitoneal) were ex-
cised and weighed. The mass of the fat pad of the drug-treated
mice group was significantly lighter than HFD control group (Table
3). These observations are consistent with the decreased body
weight gain. There was no significant difference in liver, lungs,
and kidney weights between the HFD control and 3e-treated mice
groups. However, livers of 3e-treated mice were found to be asso-
ciated with dark-brownish spots not observed in the livers of other
groups, suggesting unwanted side effects of the test compound 3e.
Further derivatization of 3e to increase the inhibitory potency and
to reduce the dose could help to avoid the undesired effects of the
drug.
Compound 3e shares a common scaffold with glitazones and,
therefore, agonistic effects of 3e to PPARs could be conceived. This
possibility, however, has been ignored to the end of this study, be-
cause 3e suppressed weight gain in this study and glitazones tend
to cause weight gain in previous reports.24 In contradict to these
speculations, in initial experiments, 3e was found to activate PPAR
with potencies comparable to those of troglitazone in cell-based
transactivation assays (data not shown). Part of the biological ef-
fects of 3e observed in this study could be due to PPAR activation,
and this issue is to be further studied. It is also worth to note that,
as a PPAR agonist, 3e is distinct in that the major substituent on the
phenyl ring is at the ortho position of the TZD group. To the best of
our knowledge, all of the benzylidene-2,4-thiazolidinedione
groups. Each group was then given a HFD or HFD plus 3e for 4 wk.
Compound 3e was administered as a mixture with the food (2.0 g
of 3e per kg of diet). The daily uptake of 3e was approximated at
4.8 mg/day/mouse, equivalent to 143 mg/day/kg of mouse weight.
For the lean control group, LFD was fed throughout the test period.
The body weight and food intake were recorded every third day
during the 4 wk of drug feeding period. Compound 3e significantly
suppressed weight gain in diet-induced obese mice (Table 3). Drug
feeding also significantly decreased feed efficiency, weight gain per
calories of food intake, suggesting that the in vivo effect of 3e was
due to the increase in metabolic rate, not the decrease in food in-
take (Table 3). After the 4 wk compound-feeding period and 6 h
of fasting, fasting blood glucose level of all the groups was mea-
sured (Fig. 3 and 0 min) and glucose tolerance test was performed
by intraperitoneal injection of glucose. Tail-blood was taken at
timed intervals up to 120 min. Feeding 3e significantly improved
glucose tolerance, but not fasting glucose levels, in the diet-in-
duced obese/diabetic mice (Fig. 3). At termination of the glucose
tolerance test, three mice groups were maintained on their own
diet for 5 d and the test group on HFD plus 3e. Mice were then
killed after an overnight fast. Mice were anesthetized and blood
samples collected by cardiac puncture for measurement of blood
parameters. Significantly lower levels of total cholesterol, triglycer-
ide, and NEFA were found in the serum of the 3e-treated mice
Table 2
Inhibition of PTP1B and other PTPs by compound 3ea
b
Compound
IC50
(l
M) [Kic,
l
M]
PTP1B
TC-PTP
SHP-1cat
LAR-D1
>100
YOP
YPTP1
3e
5.0 0.1 [2.1]
16 1 [4.5]
5.8 0.4 [2.2]
6.0 0.1
18 1 [7.7]
a
The catalytic domain of SHP-1 (SHP-1cat) and YPTP1 were expressed in E. coli expression systems and purified as previously described.16 LAR-D1 (membrane-proximal
catalytic domain of LAR), TC-PTP and YOP were purchased from New England Biolabs, Inc. (Beverly, MA, USA). The assay condition was the same for all PTPs except the
enzyme concentrations, which were 40 nM for PTP1B, 100 nM for SHP-1cat, 15 nM for YPTP1, 50 units (manufacturer’s definition)/mL for YOP, and 33 units/mL for LAR-D1
and TC-PTP.
b
Values are mean standard deviations of two or more experiments.
c
Ki values were obtained using the relationship: IC50 = Ki(1 + [S]/KM).18

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B. R. Bhattarai et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6161–6165
Table 3
Effect of 3e on body weight and related parametersa
Mice group
Body weight gain (g)
Feed efficiency (wt gain/kcal  100)
Epididymal fat (g)
Retroperitoneal fat (g)
HFD
HFD + 3e
LFD
4.88 0.46
2.16 0.29**
1.22 0.54
1.38 0.11
0.68 0.09**
0.59 0.09
1.87 0.08
1.27 0.17**
0.49 0.04
0.69 0.06
0.42 0.07**
0.11 0.02
a
The obese and lean control groups were fed high fat diet (HFD, D12451, New Brunswick, NJ, USA) or low fat diet (LFD, D12450B), containing 45% and 10% of the calories
from fat, respectively, for 12 wk. The test group (HFD + 3e) was fed a HFD for 8 wk, and then a HFD mixed with 3e for 4 wk. Values are mean standard deviations; n = 8/
group. Significance of the difference between the HFD group and 3e-fed group was calculated by One-way ANOVA, where ** represents p <0.05.
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Figure 3. Glucose Tolerance Test. HFD + 3e group were maintained on a HFD for 8
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19. Spectral data for compound 3e: mp: 204–206 °C; EI-MS: m/z 526 [M+]. IR (KBr,
cmÀ1): 3447, 3190, 3071, 1754, 1697, 1487, 1363, 1276, 1173, 1137. 1H NMR
(DMSO-d6): d 12.70 (br s, 1H, NH), 8.19 (s, 2H), 8.11 (s, 1H), 7.93 (s, 1H), 7.66
(dd, J = 8.8 and 2.6 Hz, 1H), 7.48 (d, J = 2.4 Hz, 1H), 7.23 (d, J = 9.2 Hz, 1H), 5.43
(s, 2H); 13C NMR (DMSO-d6): d 167.40, 166.94, 155.69, 139.77, 134.34, 130.63,
130.54, 130.31, 128.20, 125.87, 124.61, 124.45, 121.89, 115.61, 113.02, 68.77.
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**
fed group was calculated by One-way ANOVA, where represents p <0.05.
Table 4
Effect of 3e on blood parametersa
Mice group Total cholesterol (mg/dL) Triglyceride (mg/dL) NEFA (mM)
HFD
HFD + 3e
LFD
152
125 7**
107
6
124 16
78 10**
0.71 0.22
0.22 0.02**
0.18 0.01
5
65
5
a
The levels of serum triglyceride and total cholesterol were measured using
diagnostic kits TG E and T-Cho E (Wako Pure chemical Industries, Ltd Osaka, Japan),
respectively. Serum concentration of non-esterified free fatty acids (NEFA) was
determined using free fatty acids half-micro test kits (Roche Diagnostics Gmbh,
Penzberg, Germany) following manufacturer’s protocol with minor modifications.
Values are mean standard deviations; n = 8/group. Significance of the difference
between HFD group and 3e-fed group was calculated by One-way ANOVA, where
** represents p <0.05.
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Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
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Trevillyan, J. M.; Jirousek, M. R. J. Med. Chem. 2003, 46, 4232.
derivatives previously reported have a major substituent at the
para position.
23. Mouse experiment: Twenty-four mice (4-wk old, 17–19 g, Japan SLC, Haruno
Breeding branch, Hamamatsu, Japan) were individually housed and
maintained in a 12 h dark-light cycle at 22 2 °C. They were fed ad libitum.
After acclimatization for 1 wk by feeding a LFD, the mice were divided into two
groups; LFD (8 mice) and HFD (16 mice) groups. The LFD-fed lean control group
was maintained on a LFD throughout the study. The HFD group of mice was
provided with a HFD for the first 8 wk of the study for development of obesity/
diabetes. Then, they were separated into two groups with the same mean body
weight. Each group was then given a HFD or HFD plus 3e for 4 wk. Compound
3e was administered as a mixture with the food (2.0 g of 3e per kg of diet). For
this, 3e (400 mg) was dissolved in 0.5 mL DMSO and homogenized with 1%
aqueous solution of polyglycerol fatty acid ester (19.5 mL, Mitsubishi-kagaku
foods, Tokyo, Japan). The homogeneous suspension was mixed thoroughly in a
Acknowledgements
Support for this work provided by Inha University. B. Kafle, D.
Khadka and B.R. Bhattarai were recipients of a BK21 fellowship.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.09.020.

B. R. Bhattarai et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6161–6165
6165
powder form of HFD (200 g) and kneaded into a dough. Body weight and food
intake were recorded every third day during the drug feeding period. At the
end of the drug feeding period, glucose tolerance tests were performed. After a
5 d recovery period on their own diet (the test group on HFD plus 3e), mice
were fasted overnight and blood collected by cardiac puncture under
secobarbital anesthesia. Liver, lung, kidney, and white adipose tissue were
excised and weighed.
24. Mudaliar, S.; Henry, R. R. In Ellenberg s Diabetes Mellitus; Porte, D., Sherwin, R.
S., Baron, A., Eds., 6th ed.; Theory and Practice; McGraw-Hill: New York, 2002;
pp 531–565.