Discovery of novel, potent, selective and cellular active ADC type PTP1B inhibitors via fragment-docking-oriented de novel design

Article abstract of DOI:10.1016/j.bmc.2015.05.032

Fragment-docking-oriented de novel design for both the catalytic site and the C phosphotyrosine binding site led to the discovery of novel scaffold and chemical easy N-(2,5-diethoxy-phenyl)-methanesulfonamide based phosphotyrosine mimetics that when incorporated into ureas are high potent and selective inhibitors of protein tyrosine phosphatase 1B. Among them, compound 15 was shown to be the most potent PTP1B inhibitor with great selectivity over the highly homologous T-cell protein tyrosine phosphatase.

Full text of DOI:10.1016/j.bmc.2015.05.032

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel, potent, selective and cellular active ADC type
PTP1B inhibitors via fragment-docking-oriented de novel design
Yongli Du a, , , Hao Ling a, , Meng zhang c, Jingkang Shen b, Qunyi Li d,
⇑
⇑
a School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology, 3501 Daxue Road, Jinan 250353, China
b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
c Brunswick Laboratories (China), 5 Xing Han Road, Suzhou 215021, China
d Clinical Pharmacy Laboratory, Huashan Hospital, Fudan University, 12 Wu Lu Mu Qi M Road, Shanghai 200040, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Fragment-docking-oriented de novel design for both the catalytic site and the C phosphotyrosine binding
site led to the discovery of novel scaffold and chemical easy N-(2,5-diethoxy-phenyl)-methanesulfon-
amide based phosphotyrosine mimetics that when incorporated into ureas are high potent and selective
inhibitors of protein tyrosine phosphatase 1B. Among them, compound 15 was shown to be the most
potent PTP1B inhibitor with great selectivity over the highly homologous T-cell protein tyrosine
phosphatase.
Received 4 March 2015
Revised 14 May 2015
Accepted 15 May 2015
Available online xxxx
Keywords:
Ó 2015 Published by Elsevier Ltd.
Protein tyrosine phosphatase 1B inhibitor
N-(2,5-Diethoxy-phenyl)-
methanesulfonamide
Type 2 diabetes mellitus
Insulin signaling
1. Introduction
The crystal structure of PTP1B in complex with the IR kinase
activation segment revealed that the large, negatively charged sub-
Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator
of the insulin and leptin receptor pathways.1–3 PTP1B deficient
mice were viable, healthy, and lean and they displayed enhanced
insulin sensitivity and resistance to diet-induced obesity.4,5 This
provides important evidence that PTP1B inhibition would be an
effective diabetes therapy. It has been extensively studied within
academia and the pharmaceutical industry and is regarded by
many as one of the best validated drug targets for intervention in
type 2 diabetes and obesity.
However, there is no drug approved as PTP1B inhibitor until
now, and only two small molecule PTP1B inhibitors entered clini-
cal trials. Nearly all medicinal chemistry efforts have been severely
hindered because the potent phosphotyrosine (pTyr) mimetics,
such as bioisosteric nonhydrolyzable DFMP pTyr mimetics,7,8
carboxylic or dicarboxylic acid pTyr mimetics,10–13 heterocyclic
TDZ and IZD pTyr mimetics,6,14,15 are negatively charged with poor
membrane permeability and low selectivity.4,9
strate interacts with multiple positively charged sites in the pro-
tein (A, B, C, D and E).16 The A site is the catalytic pocket of the
enzyme where phosphotyrosine (Tyr) residues of the IR kinase
activation peptide are dephosphorylated. The A site is 9-Å deep
(from Cys215 to Phe182) and 10-Å wide (from Tyr46 to Gln262).
Its lower half contains the polar phosphate binding loop
(Cys215–Arg221) and the catalytic Cys215. Its upper half contains
hydrophobic residues (Y46, V49, F182, A217, I219, and Q262) that
interact with the aryl ring of pTyr. The D site is a small narrow
pocket, partially shielded from solvent and lined with polar and
charged residues (Tyr46, Glu115, Lys120, Asp181, and Ser216).
The most desirable aspect of building into this site is the possibility
of increasing potency. The C site is highly solvent exposed and
completely flat except for Lys41 and Arg47, and it shares Tyr46
and Asp48 with the A site.17
In our pursuit to discover novel potent and selective PTP1B inhi-
bitors, N-(2,5-diethoxy-phenyl)-methanesulfonamide based pTyr
mimetics were identified through fragment-docking-oriented de
novel design. Here we report the discovery of the design, synthesis
and bioactivity of the novel, potent, selective and cellular active
PTP1B inhibitors which interact with the A, D and C site, namely
ADC type PTP1B inhibitors.
⇑
Corresponding authors. Tel.: +86 531 89631208; fax: +86 0531 89631207
(Y.D.); tel.: +86 21 52889303; fax: +86 21 52889307 (Q.L.).
E-mail addresses: ylduyjs@163.com (Y. Du), qyli1234@163.com (Q. Li).
These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2015.05.032
0968-0896/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Du, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.032

2
Y. Du et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
O
2. Inhibitor design
O
P
HO
HO
HN
Our efforts toward the design of PTP1B inhibitors started with
the analysis of the interactions between PTP1B and its ligand com-
pound 1 (Ki = 1.7 nM, Fig. 1) in the X-ray crystal (PDB code 2CNE).
The DFMP moiety of compound 1 (gray capped sticks, Fig. 2) binds
at the center of the phosphate binding loop (ball and stick) through
multiple hydrogen-bonding interactions with the catalytic residue
in the A site, the C-terminal amide and adjacent amide NHs
hydrogen bond (yellow dashed lines) to the carboxylate of Asp48
at the A–C border, the distal amide CO hydrogen bonds to the
backbone NH of Arg47 and the distal DFMP interacts with Lys41
and Arg47 in the C site.18
NH
O
NH2
F
F
O
F
F
HO
HO
P
O
Compound 1 Ki=1.7nM
Figure 1. Previously reported difluoromethylphosphonic acid (DFMP) derivative.
We envisioned that suitable hydrogen bond acceptors instead of
negatively charged phosphoric acid groups in pTyr mimetics for
hydrogen binding to the catalytic residues in A site, maybe
demonstrate potent affinity and good membrane permeability.
Closer inspection and chemical intuition suggested that neutral
methyl sulfonyl fragment 3 (Fig. 3), probably forming multiple
hydrogen-bonding interactions with the catalytic residues in the
deep A site, maybe effectively mimic the interactions of DFMP 2
(Figs. 2 and 3) containing ligands. Docking simulations of the pro-
posed compound 3 within the ADC site of PTP1B (PDB entry 2CNE)
have been performed using Surflex docking program (Sybyl X 1.0)
with the default setting. Molecular docking result showed that the
two sulfonyloxygens in compound 3 hydrogen bond to Ser216,
Arg221 and Ile219, similar to the phosphate group, in addition to
Gly220 (Fig. 4).
Interacting with Asp48 on the outer edge of the pocket A has
been shown to increase selectivity.18 So the urea group having
1,3-double-NHs was introduced to methyl sulfonyl containing
fragment 3 for interacting with the carboxylate of Asp48. The dock-
ing structure of methyl sulfonyl fragment 3 and DFMP containing
ligand 1 suggested that a one-atom linker off the para position of
the phenyl ring in fragment 3 should be optimal for linking with
the urea group, thus fragment 4 (Fig. 2) was designed. Docking
studies showed that fragment 4 formed a network of H-bonds with
Arg221, Gy220 and Ile219, little different from fragment 3, and
hydrogen-bonding interactions of –NH present at urea group have
also been observed with carboxylate of Asp48 (Fig. 5).
The most desirable aspect of building into D site is the possibil-
ity of increasing potency. The advantage of interacting with
hydrophobic residues (Val49, Phe52, Ile219, and Met258) proximal
to the A site is the possibility of simultaneously increasing affinity
and specificity.17 Docking results suggested that a three-atom
group at 2 and 5 position of the phenyl ring in fragment 4 should
be optimal for interact with the D site and the hydrophobic resi-
dues (Val49, Phe52, Ile219, and Met258). Guided by docking
results, compound 12 (Fig. 2) was designed. Molecular docking
simulation studies of compound 12 displayed the same binding
mode to compound 4 (Fig. 5). As expected, the 2-ethoxy group
pointed to the D site and the 5-ethoxy group pointed to the
hydrophobic residues (Val49, Phe52, Ile219, and Met258). The
scoring results suggested compound 12 was favorable than frag-
ments 3 and 4 in binding energy. We postulated that the neutral
N-(2,5-diethoxy-phenyl)-methanesulfonamide containing com-
pound 12 may provide improved membrane permeability com-
pared to the hard charged phosphonates containing inhibitors.
Thus compound 12 and its analogs were chosen as our initial syn-
thetic targets.
Figure 2. Compound 1 (Gray) was docked into PTP1B crystal structure (2CNE).
Hydrogen bonding interactions are shown with dotted yellow lines. These images
were generated using the Sybyl-x program.
NH
NH
O
O
NH
NH
O
O
F
F
NH
O
NH
NH
O
HO
O
O
S
P
OH
S
S
O
O
O
3
DFMP 2
12
4
Figure 3. Fragments design and growth of PTP1B inhibitor.
Figure 4. Docking model of fragment 3 (blue) on the A site of PTP1B (PDB code
2CNE). Hydrogen bonding interactions are shown with dotted yellow lines. These
images were generated using the Sybylx program.
3. Chemistry
The syntheses of aromatic b-amino-ketone analogs 12–18 were
accomplished as depicted in Scheme 1. Starting from material 5,
intermediate 6 was synthesized through alkylation with bro-
moethane.19 Then intermediate 7 was produced through nitration
Please cite this article in press as: Du, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.032

Y. Du et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
compound 12 was synthesized and identified to posses potent
inhibition activity (IC50 = 537 nM, Table 1) against the PTP1B
enzyme. The potent activity of compound 12 proved the rationality
of our design strategy above. Replacement of the 3-phenyl group
on ureido (12) with a benzyl group in 18 (IC50 = 756 nM, Table 1)
resulted in a dramatic decrease in inhibition activity against the
PTP1B enzyme. The docking structure of PTP1B/18 (Fig. 6) reveals
that the aryl ring of 3-benzyl group on urea does not interact sig-
nificantly with the protein but instead pointing out of the pocket.
Next, N-{2,5-diethoxy-4-[(3-phenyl-ureido)-methyl]-phenyl}-
methanesulfonamide was chosen as general structure and a num-
ber of substituted phenyl-ureido analogs (e.g., 13–17, Table 1)
were synthesized to study the preliminary structure–activity rela-
tionships. 4-methoxy analog (15, IC50 = 203 nM), 4-methyl analog
(13, IC50 = 309 nM) and 4-bromoanalog (14, IC50 = 212 nM) all dis-
played improved PTP1B inhibition activity than compound 12. This
probably because the introduction of hydrophobic group on 4 posi-
tion of phenyl enhanced the hydrophobic interactions between
compounds 13–15 and the residues in C site of PTP1B. However,
replacement of the 4-methoxy group (15) with a 3-methoxy group
in 16 (IC50 = 2733 nM) resulted in a dramatic decrease (10-fold) in
PTP1B inhibition activity. Interestingly, when introducing methoxy
group on both 3 and 4 position of phenyl group (17, IC50 = 204 nM)
still displayed potent inhibition activity, similar to analog 15,
against the PTP1B enzyme.
Figure 5. Superimposition of docking model of fragment 4 (red) and compound 12
(green) on the ADC site of PTP1B (PDB code 2CNE). Hydrogen bonding interactions
are shown with dotted yellow lines. These images were generated using the Sybyl-x
program.
reaction with HNO3 and acetic acid in good yield.20 After bromina-
tion and amination, intermediate 7 was converted to intermediate
10.21,22 Intermediate 11 was obtained in high yield by condensing
benzylamine intermediate 10 with phenyl chloroformate.23,24 The
intermediate 11 effectively reacts with various amines to give
the corresponding N-benzyl urea intermediates 12H–18H in excel-
lent yields.23,24 Intermediates 13I–18I were prepared through the
reduction of nitro intermediates.25 Methanesulfonyl chloride was
condensed with Intermediates 13I–18I separately to give com-
pound 12–18.
Potent active compounds (13, 14, 15) were selected to test
against a panel of PTPs including homologous T-cell protein tyro-
sine phosphatase (TCPTP), SHP1, SHP2 and the receptor-like PTPs,
LAR. As shown in Table 2, all these inhibitors demonstrated excel-
lent PTP1B selectivity over other PTPs. No inhibition of SHP2 and
LAR was observed at 25 lM in the three tested compounds. No
inhibition of SHP1 was observed in compound 13. 10-fold and
40-fold selectivity for PTP1B over SHP1 was observed separately
in compound 14 and 15. Excitingly, compounds 13 (16-fold) and
14 (6-fold) displayed good PTP1B selectivity over TCPTP, and the
most potent compound 15 demonstrated more than 120 times
selectivity for PTP1B over the highly homologous TCPTP.
4. Results and discussion
This is the first attempt to develop neutral N-(2,5-diethoxy-
phenyl)-methanesulfonamide containing compounds as potential
pTyr mimetics to improve PTP1B inhibition, selectivity. Initially,
O N
2
O
O N
2
a
c
O
b
O
OH
HO
O
O
O
Br
7
6
8
5
O
O
O
O
O
O
O N
2
f
d
N
N
O
e
H
O
O N
2
O N
2
O
NH
2
O
9
10
11
O
O
O
O
O
O
O
O
O
R
R
g
R
O
S
N
H
N
H
N
H
N
H
i
h
N
N
H
O
H
N
H N
2
O N
2
H
inter.
comp. 12-18
inter.
12I~18I
12H~18H
No. R:
No. R:
No. R:
No. R:
No. R:
No. R:
15 4-MeOPh-
15H 4-MeOPh-
16H 3-MeOPh-
15I 4-MeOPh-
16I 3-MeOPh-
12H Ph-
12 Ph-
12I Ph-
13H 4-MePh-
13 4-MePh-
16 3-MeOPh-
17 3,4-di-MeOPh-
18 Bn-
13I 4-MePh-
14H 4-BrPh-
17H 3,4-di-MeOPh-
18H Bn-
14 4-BrPh-
17I 3,4-di-MeOPh-
18I Bn-
14I
4-BrPh-
Scheme 1. Reagents and conditions for the synthesis of compounds 12–18: (a) EtBr, K2CO3, DMF, 80 °C, 12 h; (b) HNO3, AcOH, rt, 3 h; (c) NBS, BPO, CCl4, 80 °C,10 h; (d)
phthalimidepotassium, TBAB, MeOH, 80 °C, 0.5 h; (e) N2H4ÁH2O,MeOH, reflux, 4 h; (f) Carbonochloridic acid, phenyl ester, Et3N, DCM; (g) Et3N, dioxane, 80 °C, 8 h; (h)
NiCl2Á6H2O, NaBH4, MeOH, rt, 0.5 h; (i) methanesulfonyl chloride, DCM, N2, pyridine, rt, 8 h.
Please cite this article in press as: Du, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.032

4
Y. Du et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
Table 3
Inhibitory activity of compounds 12–18 against PTP1Ba
The kinetic parameters of PTP1B enzyme in the pNPP hydrolysis in the presence of
different concentrations of the inhibitor 15 LingH5a
O
O
Inhibitor (
l
M)
Km (mM)
Vmax (mOD/min)
R
O
S
N
H
N
H
O
0
1.81
2.96
3.89
4.52
7.32
57.97
58.34
54.79
48.92
52.49
N
0.1
0.2
0.4
0.8
H
O
12-18
Compound
R
IC50(nM) mean SEM
a
Km is the Michaelis constant, Vmax is the maximum rate and were calculated
from the Michaelis–Menten equation.
Control
12
13
14
15
16
17
18
Oleanolic acid
Ph-
2058 202
537 42
309 99
212 57
203 79
2733 23
204 19
756 115
4-MePh-
4-BrPh-
4-MeOPh-
3-MeOPh-
3,4-di-MeO-Ph-
Bn-
a
Values represent triplicate determinations.
Figure 7. Representative competitive inhibitory effect of compound 15 on PTP1B-
catalyzed pNPP hydrolysis. Data were shown by Lineweaver–Burk plot.
Figure 6. Superimposition of docking model of compounds 12 (green) and 18
(pink) on the A, D and C sites of PTP1B (PDB code 2CNE). Hydrogen bonding
interactions are shown with dotted yellow lines. These images were generated
using the Sybyl-x program.
Figure 8. Effect of compound 15 on tyrosine phosphorylation of insulin receptor b
(IRb) in CHO cells transfected with hIRb-expressing plasmid. NC, negative control;
PC, positive control.
The effect of compound 15 on the kinetic profile of PTP1B-cat-
alyzed pNPP hydrolysis was investigated as described in methods.
As shown in Table 3, with the increasing concentrations of com-
pound 15, the Km value of PTP1B increased accordingly. However,
the Vmax value of PTP1B remained almost constant. The kinetic
parameters presented in Table 3 suggested that compound 15
hyperglycaemia and insulin sensitivity in diabetic animal models.
Thus, we evaluated the effects of compound 15 on phosphorylation
level of IRb in CHO cells transfected with hIRbg plasmid. As shown
in Figure 8, compound 15 greatly enhanced insulin-mediated IRb
acted as
a competitive PTP1B inhibitor. Further analysis by
Lineweaver-Burk plot also demonstrated the competitive inhibi-
tory mechanism of 15 on PTP1B (Fig. 7). Kinetic analysis of enzy-
matic reaction suggested 15 was a competitive PTP1B inhibitor.
It has been suggested that the intrinsic tyrosine kinase activity
of the IRb plays a pivotal role in insulin-mediated signaling cas-
cades.26 PTP1B participates in the dephosphorylation and inactiva-
tion of IR, thus attenuating insulin signaling.26 Several studies
indicates that inhibition of PTP1B by specific antisense oligonu-
cleotide27 or inhibitors28,29 results in improvement of
phosphorylation at concentrations of 25 lM and 50 lM.
5. Conclusion
The novel potent and selective PTP1B inhibitors, N-(2,5-di-
ethoxy-phenyl)-methanesulfonamide based phosphotyrosine
mimetics, were discovered through fragment-docking-oriented
de novel design for the A, D and C site of PTP1B. Efficient syntheses
Table 2
a
The inhibitory activity of select compounds 13, 14, 15 as PTP1B inhibitors against a panel of PTPs, IC50
Compd
PTP1B (nM)
1536 267
TCPTP (
l
M)
SHP1 (lM)
SHP2 (
lM)
LAR (lM)
Oleanolic acid
5.46 0.74
Na3VO4
13
14
14.19 2.63
NAb
2.31 0.44
9.08 1.26
11.62 2.84
22.31 4.67
309 99
212 57
203 79
5.19 0.64
1.36 0.27
NA
NA
NA
NA
NA
NA
NA
15
a
The pNPP assay. IC50 values were determined by regression analysis and expressed as means SD of three replications.
NA: Inhibition rate <50% at 25 lM concentration.
b
Please cite this article in press as: Du, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.032

Y. Du et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
were established to provide N-(2,5-diethoxy-phenyl)-methanesul-
fonamide derivatives. The SAR study revealed that compound
15, acting as competitive inhibitor, was the most potent
(IC50 = 203 nM) PTP1B inhibitor with great selectivity (more than
120-fold) over the highly homologous TCPTP. Interestingly,
compound 15 displayed distinct cellular activity in enhancing
insulin-mediated IRb phosphorylation.
These N-(2,5-diethoxy-phenyl)-methanesulfonamide based
ADC type phosphotyrosine mimetics provide novel scaffold and
opportunities for the discovery of such PTP1B inhibitors in the
future. Further optimization of these novel scaffold PTP1B inhibi-
tors will be described in our next reports.
6.1.3. 2-(2,5-Diethoxy-4-nitro-benzyl)-isoindole-1,3-dione (9)
Bromomethyl-2,5-diethoxy-4-nitro-benzene (8, 8.0 g,
a
26.3 mmol), potassium phthalimide (4.87 g, 26.3 mmol) and tetra-
butylammonium bromide (84.7 mg, 0.026 mmol) were mixed in
methanol and heated to 80 °C for 0.5 h. Then the mixture was
cooled to room temperature and the precipitate were collected
by filtration and dried to give the pure product 9 8.5 g (yield
87.4%) of white solid. 1H NMR (400 MHz, CDCl3) d 7.89 (dt,
J = 6.9, 3.5 Hz, 2H), 7.80–7.75 (m, 2H), 7.36 (s, 1H), 7.04 (s, 1H),
4.93 (s, 2H), 4.08 (dq, J = 20.6, 7.0 Hz, 4H), 1.42 (td, J = 7.0, 2.9 Hz,
6H).
6.1.4. N-{4-[3-(4-Bromo-phenyl)-ureidomethyl]-2,5-diethoxy-
phenyl}-methanesulfonamide (14)
6. Experimental section
6.1. Chemistry
2-(2,5-Diethoxy-4-nitro-benzyl)-isoindole-1,3-dione (9, 8.0 g,
21.6 mmol) was dissolved in methanol (120 mL) and the mixture
was heated at 80 °C for 10 min. Then hydrazine hydrate (1.93 g,
30.9 mmol) was added to the solution followed by stirring at
80 °C for 4 h. Then the mixture was cooled to room temperature
and methanol was removed by distillation. The solid residue was
dissolved in dichloromethane (200 mL) and treated with aqueous
20% NaOH (200 mL). The aqueous phase was separated, extracted
with dichloromethane (3 Â 200 mL). The combined organic phases
were dried with Na2SO4, and concentrated under reduced pressure.
The crude was purified by flash column chromatography to give
pure product 10 4.7 g (yield 92.5%) of yellow solid.
2,5-Diethoxy-4-nitro-benzylamine (10, 0.40 g, 1.67 mmol) was
dissolved in dichloromethane. after addition of carbonochloridic
acid phenyl ester (0.287 g, 1.84 mmol) and triethylamine
(0.186 g, 1.84 mmol), the mixture stirring at room temperature
for 0.5 h. Dichloromethane was then removed by distillation. The
residue was dissolved in dichloromethane and extracted with
water (3 Â 100 mL). The combined organic phases were dried with
Na2SO4, and concentrated under reduced pressure. The crude was
purified by flash column chromatography to give pure product
11 0.518 g (yield 86.3%) of yellow solid.
(2,5-Diethoxy-4-nitro-benzyl)-carbamic acid phenyl ester (11,
0.4 g, 1.11 mmol) was dissolved in dioxane after addition of 1-
amino-4-bromobenzene (0.191 g, 1.1 mmol) and triethylamine
(1.12 g, 11.1 mmol). The mixture was heated at 80 °C for 8 h.
Then the mixture was cooled to room temperature and dioxane
was removed by distillation. The residue was dissolved in dichlor-
omethane and extracted with water (3 Â 100 mL). The combined
organic phases were dried with Na2SO4, and concentrated under
reduced pressure. The remaining solid was recrystallized from
methanol to give pure product 1-(4-bromo-phenyl)-3-(2,5-di-
ethoxy-4-nitro-benzyl)-urea (14H) 0.44 g (yield 90.5%), pale yel-
low solid. A mixture of 1-(4-bromo-phenyl)-3-(2,5-diethoxy-4-
nitro-benzyl)-urea (14H, 400 mg, 0.91 mmol), NiCl2Á6H2O
(371 mg, 1.56 mmol), NaBH4 (119 mg, 3.12 mmol), and MeOH
was stirring at room temperature for 0.5 h. The mixture was made
acidic with a 10% HCl solution and extracted with Ethyl acetate, the
General synthetic methods: Unless otherwise noted, all materi-
als were obtained from commercial suppliers and used without
further purification. All reactions were monitored using thin-layer
chromatography (TLC) on silica gel plates. NMR spectra were
determined using a Bruker AVANCE II 400 spectrometers in
CDCl3 or DMSO solution. Elemental analyses were performed on
a Der CHNOS Elementar Analysen-systemeVario EL III.
6.1.1. 1,4-Diethoxy-2-methyl-benzene (6) and 1,4-diethoxy-2-
methyl-5-nitro-benzene (7)
2-Methyl-benzene-1,4-diol (5, 10.0 g, 80.55 mmol) was dis-
solved in dry N,N-dimethyl formamide. After addition of K2CO3
(33.5 g, 242.8 mmol), the solution was heated under nitrogen at
80 °C for 10 min, bromoethane (26.3 g, 241.7 mmol) was added
dropwise to the solution over 30 min, followed by stirring at
80 °C for 12 h. Then the mixture was cooled to room temperature
and N,N-dimethylformamide was removed by distillation. The resi-
due was dissolved in dichloromethane and extracted with water
(3 Â 200 mL). The combined organic phases were dried with
Na2SO4, and concentrated under reduced pressure to give the crude
product 6 8.8 g (yield 60.7%) without further purification.
1,4-Diethoxy-2-methyl-benzene (6, 8.0 g, 44.4 mmol), was dis-
solved in acetic acid (100 mL), and then the HNO3 (11.3 mL,
178.0 mmol), was added to the solution and the mixture was stir-
red at room temperature for 3 h. Acetic acid was then removed by
distillation. The residue was dissolved in dichloromethane and
extracted with water (3 Â 200 mL). The combined organic phases
were dried with Na2SO4 and concentrated under reduced pres-
sure. The remaining solid was recrystallized from methanol to
give pure product 7 8.2 g (yield 82%) of yellow solid. 1H NMR
(400 MHz, CDCl3) d 7.35 (s, 1H), 6.89 (s, 1H), 4.14 (q, J = 7.0 Hz,
2H), 4.03 (q, J = 6.9 Hz, 2H), 2.28 (s, 3H), 1.45 (dd, J = 12.9,
6.8 Hz, 6H).
6.1.2. Bromomethyl-2,5-diethoxy-4-nitro-benzene (8)
1,4-Diethoxy-2-methyl-5-nitro-benzene (7, 7.0 g, 31.1 mmol)
was dissolved in carbon tetrachloride (150 mL). After addition of
benzoyl peroxide (75 mg, 0.3 mmol), the mixture was heated at
80 °C for 10 min, N-bromosuccinimide (5.54 g, 31.1 mmol) was
added to the solution, followed by stirring at 80 °C for 10 h. Then
the mixture was cooled to room temperature and carbon tetrachlo-
ride was removed by distillation. The residue was dissolved in
dichloromethane and extracted with water (3 Â 200 mL). The com-
bined organic phases were dried with Na2SO4, and concentrated
under reduced pressure. The crude product was purified by flash
column chromatography to give pure product 8 8.5 g (yield
89.9%) of yellow solid. 1H NMR (400 MHz, CDCl3) d 7.38 (s, 1H),
7.11 (s, 1H), 4.52 (s, 2H), 4.20–4.09 (m, 4H), 1.48 (dd, J = 13.0,
6.9 Hz, 6H).
organic layer was washed with
a saturated NaCl solution
(3 Â 100 mL), the combined organic phases were dried with
Na2SO4, and concentrated under reduced pressure. The product
was purified by flash column chromatography to give pure product
1-(4-amino-2,5-diethoxy-benzyl)-3-(4-bromo-phenyl)-urea (14I)
0.3 g (yield 80.5%), white solid. 1-(4-amino-2,5-diethoxy-benzyl)-
3-(4-bromo-phenyl)-urea (14I, 260 mg, 0.64 mmol) was dissolved
in dichloromethane after addition of pyridine (56 mg, 0.70 mmol).
The solution was stirring at room temperature for 10 min and
methanesulfonyl chloride (73 mg, 0.64 mmol) was added dropwise
to the solution, followed by stirring at room temperature for 8 h.
Dichloromethane was then removed by distillation. The solid
residue was dissolved in dichloromethane (50 mL) and washed
with a saturated NaCl solution (3 Â 100 mL). The combined organic
Please cite this article in press as: Du, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.032

6
Y. Du et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
phases were dried with Na2SO4 and concentrated under reduced
pressure. The crude product was purified by flash column chro-
matography to give pure product (14) 0.17 g (yield 54.8%) of white
solid. 1H NMR (400 MHz, DMSO) d 8.85 (s, 1H), 8.73 (s, 1H),
7.44–7.27 (m, 4H), 6.94 (s, 1H), 6.87 (s, 1H), 6.42 (t, J = 6.0 Hz,
1H), 4.21 (d, J = 5.6 Hz, 2H), 3.98 (q, J = 6.8 Hz, 4H), 2.92 (s, 3H),
1.32 (dd, J = 12.0, 6.4 Hz, 6H). 13C NMR (101 MHz, DMSO) d
154.90, 149.65, 145.37, 139.86, 131.30(2C), 125.97, 125.23,
119.55(2C), 113.72, 112.30, 110.55, 64.61, 64.12, 40.20, 38.12,
14.67, 14.61. Anal. (C19H24N3O5SBr): Anal. Calcd for C, 46.92; H,
4.97; N, 8.64; S, 6.59. Found: C, 47.41; H, 4.95; N, 8.74; S, 6.49.
the same procedure described for 14I, 15H (400 mg, 1.03 mmol)
was treated with NiCl2Á6H2O (420 mg, 1.77 mmol) and NaBH4
(134 mg, 3.53 mmol) to afford 290 mg (78.3%) of 1-(4-amino-2,5-
diethoxy-benzyl)-3-(4-methoxy-phenyl)-urea
(15I)
as
a
white solid. According to the same procedure described for 14,
15I (250 mg, 0.70 mmol) was treated with Pyridine (60.5 mg,
0.77 mmol) and methanesulfonyl chloride (80.2 mg, 0.70 mmol)
to afford 145 mg (47.7%) of N-{2,5-diethoxy-4-[3-(4-methoxy-phe-
nyl)-ureidomethyl]-phenyl}-methanesulfonamide (15) as a white
solid. 1H NMR (400 MHz, CDCl3) d 7.15 (d, J = 8.7 Hz, 2H), 7.08 (s,
1H), 6.92–6.81 (m, 3H), 6.75 (s, 1H), 4.35 (s, 2H), 4.03 (q,
J = 6.9 Hz, 2H), 3.96 (q, J = 6.9 Hz, 2H), 3.79 (s, 3H), 2.92 (s, 3H),
1.38 (t, J = 6.9 Hz, 3H), 1.26 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz,
DMSO) d 155.35, 153.96, 149.60, 145.40, 133.55, 126.32, 125.04,
119.42 (2C), 113.88(2C), 113.58, 110.57, 64.56, 64.06, 55.12,
40.10, 38.10. 14.67, 14.62; Anal. (C20H27N3O6S): Anal. Calcd for C,
54.90; H, 6.22; N, 9.60; S, 7.33. Found: C, 56.47; H, 5.87; N, 9.68;
S, 7.34.
6.1.5. N-{2,5-Diethoxy-4-[(3-phenyl-ureido)-methyl]-phenyl}-
methanesulfonamide (12)
According to the same procedure described for 14H, 11
(500 mg, 1.39 mmol) was treated with aniline (129 mg,
1.39 mmol) and triethylamine (1.40 g, 13.9 mmol) to afford
400 mg (yield 80.0%) of 1-(2,5-diethoxy-4-nitro-benzyl)-3-phe-
nyl-urea (12H) as a white solid. According to the same procedure
described for 14I, 12H (360 mg, 1.0 mmol) was treated with
NiCl2Á6H2O (408 mg, 1.7 mmol) and NaBH4 (129 mg, 3.4 mmol) to
afford 285 mg (86.4%) of 1-(4-amino-2,5-diethoxy-benzyl)-3-phe-
nyl-urea (12I) as a white solid. According to the same procedure
described for 14, 12I (260 mg,0.72 mmol) was treated with pyri-
dine (62.9 mg, 0.80 mmol) and methanesulfonyl chloride
(82.4 mg, 0.72 mmol) to afford 180 mg (yield 60.0%) of N-{2,5-di-
ethoxy-4-[(3-phenyl-ureido)-methyl]-phenyl}-methanesulfonamide
(12) as a white solid. 1H NMR (400 MHz, CDCl3) d 7.26 (s, 3H), 7.06
(d, J = 9.1 Hz, 2H), 6.88 (s, 1H), 6.76 (s, 1H), 4.36 (s, 2H), 4.02–3.92
(m, 4H), 2.91 (s, 3H), 1.32 (m, 6H). 13C NMR (101 MHz, DMSO) d
155.11, 149.66, 145.38, 140.43, 128.57 (2C), 126.16, 125.19,
121.02, 117.70(2C), 113.72, 110.56, 64.62, 64.13, 40.21, 38.08,
14.67, 14.61; Anal. (C19H25N3O5S): Anal. Calcd for C, 56.00; H,
6.18; N, 10.31; S, 7.87. Found: C, 56.11; H, 6.03; N, 10.36; S, 8.17.
6.1.8. N-{2,5-Diethoxy-4-[3-(3-methoxy-phenyl)-
ureidomethyl]-phenyl}-methanesulfonamide (16)
According to the same procedure described for 14H, 11
(500 mg, 1.39 mmol) was treated with p-methoxyaniline
(171 mg, 1.39 mmol) and triethylamine (1.40 g, 13.9 mmol) to
afford 445 mg (yield 82.4%) of 1-(2,5-diethoxy-4-nitro-benzyl)-3-
(3-methoxy-phenyl)-urea (16H) as a white solid. According to
the same procedure described for 14I, 16H (400 mg, 1.03 mmol)
was treated with NiCl2Á6H2O (420 mg, 1.77 mmol) and NaBH4
(134 mg, 3.53 mmol) to afford 305 mg (yield 80.9%) of 1-(4-
amino-2,5-diethoxy-benzyl)-3-(3-methoxy-phenyl)-urea (16I) as
a white solid. According to the same procedure described for 14,
16I (260 mg, 0.64 mmol) was treated with pyridine (55.6 mg,
0.70 mmol) and methanesulfonyl chloride (73.3 mg, 0.64 mmol)
to afford 170 mg (yield 54.8%) of N-{2,5-diethoxy-4-[3-(3-meth-
oxy-phenyl)-ureidomethyl]-phenyl}-methanesulfonamide (16) as
a white solid. 1H NMR (400 MHz, CDCl3) d 7.19 (t, J = 8.1 Hz, 1H),
7.10 (s, 1H), 6.94 (s, 1H), 6.90 (s, 1H), 6.77 (d, J = 12.0 Hz, 2H),
6.64 (d, J = 8.2 Hz, 1H), 4.37 (s, 2H), 4.07–3.93 (m, 4H), 3.76 (s,
3H), 2.92 (s, 3H), 1.38 (t, J = 7.0 Hz, 3H), 1.32 (t, J = 6.6 Hz, 3H).
13C NMR (101 MHz, DMSO) d 155.37, 154.01, 149.64, 145.38,
133.58, 126.34, 125.14, 119.48(2C), 113.91(2C), 113.68, 110.55,
64.61, 64.13, 55.15, 40.22, 38.10, 14.67, 14.61; Anal.
(C20H27N3O6S): Anal. Calcd for: C, 54.90; H, 6.22; N, 9.60; S, 7.33.
Found: C, 54.76; H, 6.02; N, 9.66; S, 7.44.
6.1.6. N-[2,5-Diethoxy-4-(3-p-tolyl-ureidomethyl)-phenyl]-
methanesulfonamide (13)
According to the same procedure described for 14H, 11
(500 mg, 1.39 mmol) was treated with 4-methylaniline (149 mg,
1.39 mmol) and triethylamine (1.40 g, 13.9 mmol) to afford
430 mg (yield 83.0%) of 1-(2,5-diethoxy-4-nitro-benzyl)-3-p-
tolyl-urea (13H) as a white solid. According to the same procedure
described for 14I, 13H (350 mg, 0.94 mmol) was treated with
NiCl2Á6H2O (384 mg, 1.6 mmol) and NaBH4 (122 mg, 3.2 mmol) to
afford 275 mg (yield 85.5%) of 1-(4-amino-2,5-diethoxy-benzyl)-
3-p-tolyl-urea (13I) as a white solid. According to the same proce-
dure described for 14, 13I (260 mg, 0.76 mmol) was treated with
pyridine (65.9 mg, 0.83 mmol) and methanesulfonyl chloride
(87.0 mg, 0.76 mmol) to afford 160 mg (yield 50.2%) of N-[2,5-di-
ethoxy-4-(3-p-tolyl-ureidomethyl)-phenyl]-methanesulfonamide
(13) as a white solid. 1H NMR (400 MHz, CDCl3) d 7.10 (d,
J = 10.8 Hz, 4H), 6.90 (d, J = 6.8 Hz, 1H), 6.75 (s, 1H), 4.36 (d,
J = 5.6 Hz, 2H), 3.99 (m, 4H), 2.92 (s, 3H), 2.32 (s, 3H), 1.38 (t,
J = 6.8 Hz, 3H), 1.30–1.26 (m, 3H). 13C NMR (101 MHz, DMSO) d
155.19, 149.66, 145.38, 137.87, 128.97, 128.57, 126.25, 125.17,
121.02, 117.81, 117.67, 113.71, 110.55, 64.61, 64.13, 40.21, 38.08,
20.21, 14.67, 14.61; Anal. (C20H27N3O5S): Anal. Calcd for C, 56.99;
H, 6.46; N, 9.97; S, 7.61. Found: C, 56.95; H, 6.10; N, 9.94; S, 7.68.
6.1.9. N-{4-[3-(3,4-Dimethoxy-phenyl)-ureidomethyl]-2,5-
diethoxy-phenyl}-methanesulfonamide (17)
According to the same procedure described for 14H, 11
(500 mg, 1.39 mmol) was treated with 3,4-dimethoxyaniline
(213 mg, 1.39 mmol) and triethylamine (1.40 g, 13.9 mmol) to
afford 440 mg (yield 75.6%) of 1-(2,5-diethoxy-4-nitro-benzyl)-3-
(3,4-dimethoxy-phenyl)-urea (17H) as a white solid. According to
the same procedure described for 14I, 17H (400 mg, 0.95 mmol)
was treated with NiCl2Á6H2O (388 mg, 1.63 mmol) and NaBH4
(124 mg, 3.26 mmol) to afford 300 mg (yield 80.9%) of 1-(4-
amino-2,5-diethoxy-benzyl)-3-(3,4-dimethoxy-phenyl)-urea (17I)
as a white solid. According to the same procedure described for
14, 17I (260 mg, 0.64 mmol) was treated with pyridine (55.6 mg,
0.70 mmol) and methanesulfonyl chloride (73.3 mg, 0.64 mmol)
to afford 170 mg (yield 54.8%) of N-{4-[3-(3,4-dimethoxy-phe-
nyl)-ureidomethyl]-2,5-diethoxy-phenyl}-methanesulfonamide (17)
as a white solid. 1H NMR (400 MHz, CDCl3) d 7.08 (s, 1H), 6.94 (s,
1H), 6.89 (s, 1H), 6.82–6.73 (m, 2H), 6.70 (d, J = 8.1 Hz, 1H), 6.56
(s, 1H), 4.36 (s, 2H), 3.99 (ddd, J = 25.1, 13.6, 6.7 Hz, 4H), 3.84 (d,
J = 12.4 Hz, 6H), 2.92 (s, 3H), 1.38 (t, J = 6.7 Hz, 3H), 1.27 (t,
J = 5.6 Hz, 3H); 13C NMR (101 MHz, DMSO) d 155.28, 149.65,
6.1.7. N-{2,5-Diethoxy-4-[3-(4-methoxy-phenyl)-
ureidomethyl]-phenyl}-methanesulfonamide (15)
According to the same procedure described for 14H, 11
(500 mg, 1.39 mmol) was treated with m-methoxyaniline
(171 mg, 1.39 mmol) and triethylamine (1.40 g, 13.9 mmol) to
afford 450 mg (yield 83.3%) of 1-(2,5-diethoxy-4-nitro-benzyl)-3-
(4-methoxy-phenyl)-urea (15H) as a white solid. According to
Please cite this article in press as: Du, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.032

Y. Du et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
148.89, 145.38, 143.63, 134.30, 126.32, 125.15, 113.67, 112.89,
110.54, 109.69, 103.80, 64.63, 64.12, 56.03, 55.37, 40.22, 38.10,
14.67, 14.61; Anal. (C21H29N3O7S): Anal. Calcd for C, 53.95; H,
6.26; N, 8.99; S, 6.86. Found: C, 53.69; H, 6.25; N, 8.87; S, 7.17.
(6 Â 105 cells per dish). 2
l
g of the human IR plasmid was intro-
duced into cells by using FuGENE 6 transfection reagent (Roche)
according to the manufacturer’s protocol. Cells were transfected
for 8 h, and harvested with 0.05% trypsin and 0.02% EDTA prior
to reseeding onto a 6-well plate. After 24 h with 5% CO2 at 37 °C,
CHO cells serum free starved for 2 h were incubated with com-
pounds for 4 h, then 10 nM insulin was added stimulation for
10 min before harvested.
6.1.10. N-[4-(3-Benzyl-ureidomethyl)-2,5-diethoxy-phenyl]-
methanesulfonamide (18)
According to the same procedure described for 14H, 11 (500 mg,
1.39 mmol) was treated with benzylamine (149 mg, 1.39 mmol)
and triethylamine (1.40 g, 13.9 mmol) to afford 420 mg (81.1%) of
1-benzyl-3-(2,5-diethoxy-4-nitro-benzyl)-urea (18H) as a white
solid. According to the same procedure described for 14I, 18H
(400 mg, 1.07 mmol) was treated with NiCl2Á6H2O (438 mg,
1.84 mmol) and NaBH4 (140 mg, 3.68 mmol) to afford 300 mg (yield
81.5%) of 1-(4-amino-2,5-diethoxy-benzyl)-3-benzyl-urea (18I) as a
white solid. According to the same procedure described for 14, 18I
(250 mg, 0.73 mmol) was treated withpyridine (63.3 mg, 0.80 mmol)
and methanesulfonyl chloride (83.6 mg, 0.73 mmol) to afford 170 mg
(yield 55.9%) of N-[4-(3-benzyl-ureidomethyl)-2,5-diethoxy-phe-
nyl]-methanesulfonamide (18) as a white solid. 1H NMR (400 MHz,
DMSO) d 8.80 (s, 1H), 7.34 –7.16 (m, 5H), 6.87 (s, 1H), 6.84 (s, 1H),
6.51 (t, J = 6.0 Hz, 1H), 6.23 (t, J = 5.6 Hz, 1H), 4.20 (t, J = 7.5 Hz, 2H),
4.16 (d, J = 5.9 Hz, 2H), 3.94 (m, 6.9 Hz, 4H), 2.92 (s, 3H), 1.31 (m
J = 6.9, 3.7 Hz, 6H). 13C NMR (101 MHz, DMSO) d 155.19, 149.66,
145.38, 137.87, 128.97, 128.57, 126.25, 125.17, 121.02 (s), 117.81,
117.67, 113.71, 110.55, 64.61, 64.13, 40.21, 38.08, 20.21, 14.67,
14.61; Anal. (C20H27N3O5S): Anal. Calcd for C, 56.99; H, 6.40; N,
9.97; S, 7.61. Found: C, 56.73; H, 6.46; N, 10.04; S, 8.02.
For total protein extraction, CHO cells were lysed in the ice-cold
buffer A (1% Triton X-100, 100 mM sodium pyrophosphate, 2 mM
phenylmethylsulfonyl fluoride (PMSF), 50 mM HEPES, pH7.4) and
centrifuged at 15,000g for 20 min at 4 °C. Supernatants were col-
lected and stored at À80 °C. For subcellular fractionation, cells
were lysed in buffer B (0.25 M sucrose, 2 mM EDTA, 1 mM phenyl-
methylsulfonyl fluoride (PMSF) and 10 mM Tris-HCl, pH 7.4). The
lysates were centrifuged at 750g for 15 min, and the supernatants
were centrifuged at 12,000g for 20 min to isolate the crude plasma
membrane fraction as the pellets. Equal amount of protein from
each sample were denatured and subjected to SDS-PAGE and blot-
ted on PVDF membrane, which was incubated for 2 h at room tem-
perature with blocking buffer (5% non-fat milk, 0.1% Tween 20, in
TBS, pH 7.6) and then probed with primary antibodies (PY-20
and b-actin) overnight at 4 °C. After incubation with the appropri-
ate secondary antibodies, the immunoreactive band was detected
by an ECL Western blotting detection system (GE Healthcare).34
6.5. Molecular docking
The crystal structure of the progesterone binding domain of
PTP1B in complex with compound 1 was retrieved from the RCSB
Protein Data Bank (PDB entry 2CNE). All the docking simulations
of the studied compounds within the binding pocket of PTP1B have
been performed using Surflex docking program (Sybyl X 1.0) with
the default settings.
6.2. PTP1B and related PTPs biological assay
PTP1Binhibitoryactivities were measuredaspreviously described
with minor revision.30,31 Briefly, PTP1B hydrolyzes pNPP to pNP that
can be detected at 405 nm. Test compounds were predispensed in 96-
well microplates as 1.0
lL aliquots per well in 100% DMSO. The PTP1B
enzymatic assay was carried out in a total volume of 100
lL per well
Acknowledgment
in assay plates with 30 nM recombinant PTP1B protein, 2 mM p-ni-
trophenylphosphonic acid (pNPP), 1 mM dithiothreitol and 1 mM
EDTA (pH 6.5). After 30 min incubation at 37 °C, the reaction was ter-
minated by addition of 2.5 M NaOH. The amount of hydrolysis pro-
duct, pNP, was monitored by detection of the absorbance at
405 nm. IC50 values were calculated by Graphpad Prism 5.0 software
(Logit method). To determine cross-reactivity of test compounds, the
same protocol was followed for inhibition assay with TCPTP.
To study the inhibition on the other PTPs family members,
SHP1, SHP2 and LAR assays were performed according to proce-
dures described previously.32
This work was supported by a Grant from the National Natural
Science Foundation of China (81202389 and 81302853).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.05.032.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
6.3. Characterization of the inhibitor on enzyme kinetics
1. Asante-Appiah, E.; Kennedy, B. P. Am. J. Physiol. Endocrinol. Metab. 2003, 284,
E663.
2. Cook, W. S.; Unger, R. H. Dev. Cell. 2002, 2, 385.
To characterize the inhibitor of PTP1B, the assay was carried out
in a 100 lL system containing 50 mM MOPS, pH 6.5, 30 nM PTP1B,
pNPP in 2-fold dilution from 64 mM, and different concentrations of
the inhibitor.33 In the presence of the competitive inhibitor, the
Michaelis–Menten equation is described as 1/v = (Km/[Vmax[S]])
(1 + [I]/Ki) + 1/Vmax, where Km is the Michaelis constant, v is the ini-
tial rate, Vmax is the maximum rate, and [S] is the substrate concen-
tration. The Ki value was obtained by the linear replot of apparent
Km/Vmax (slope) from the primary reciprocal plot versus the inhibi-
tor concentration [I] according to the equation Km/Vmax = 1 + [I]/Ki.
3. Byon, J. C.; Kusari, A. B.; Kusari, J. Mol. Cell. Biochem. 1998, 182, 101.
4. Combs, A. P. J. Med. Chem. 2010, 53, 2333.
5. Szczepankiewicz, B. G.; Liu, G.; Hajduk, P. J.; Abad-Zapatero, C.; Pei, Z.; Xin, Z.;
Lubben, T. H.; Trevillyan, J. M.; Stashko, M. A.; Ballaron, S. J.; Liang, H.; Huang,
F.; Hutchins, C. W.; Fesik, S. W.; Jirousek, M. R. J. Am. Chem. Soc. 2003, 125, 4087.
6. Combs, A. P.; Yue, E. W.; Bower, M.; Ala, P. J.; Wayland, B.; Douty, B.; Takvorian,
A.; Polam, P.; Wasserman, Z.; Zhu, W.; Crawley, M. L.; Pruitt, J.; Sparks, R.;
Glass, B.; Modi, D.; McLaughlin, E.; Bostrom, L.; Li, M.; Galya, L.; Blom, K.;
Hillman, M.; Gonneville, L.; Reid, B. G.; Wei, M.; Becker-Pasha, M.; Klabe, R.;
Huber, R.; Li, Y.; Hollis, G.; Burn, T. C.; Wynn, R.; Liu, P.; Metcalf, B. J. Med. Chem.
2005, 48, 6544.
7. Puius, Y. A.; Zhao, Y.; Sullivan, M.; Lawrence, D. S.; Almo, S. C.; Zhang, Z.-Y. Proc.
Natl. Acid. Sci. U.S.A. 1997, 94, 13420.
8. Lau, C. K.; Bayly, C. I.; Gauthier, J. Y.; Li, C. S.; Therien, M.; Asante-Appiah, E.;
Cromlish, W.; Boie, Y.; Forghani, F.; Desmarais, S.; Wang, Q.; Skorey, K.;
Waddleton, D.; Payette, P.; Ramachandran, C.; Kennedy, B. P.; Scapin, G. Bioorg.
Med. Chem. Lett. 2004, 14, 1043.
6.4. Effect of PTP1B inhibitors on the phosphorylation level of IR
in transfected CHO cells
CHO cells were cultured in the presence of F12 supplemented
with 10% FBS and seeded 24 h before transfection in 6 cm dish
9. Low, J.-L.; Chai, C. L. L.; Yao, S. Q. Antioxid. Redox. Signal. 2014, 20, 2225.
Please cite this article in press as: Du, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.032

8
Y. Du et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
10. Liu, G.; Xin, Z.; Liang, H.; Abad-Zapatero, C.; Hajduk, P. J.; Janowick, D. A.;
Szczepankiewicz, B. G.; Pei, Z.; Hutchins, W. C.; Ballaron, S. J.; Stashko, M. A.;
Lubben, T. H.; Berg, C. E.; Rondinone, C. M.; Trevillyan, J. M.; Jirousek, M. R. J.
Med. Chem. 2003, 46, 3437.
19. Zhao, Y.; Shirai, Y.; Slepkov, A. D.; Cheng, L.; Alemany, L. B.; Sasaki, T.;
Hegmann, F. A.; Tour, J. M. Chem. Eur. J. 2005, 11, 3643.
20. Shchekotikhin, A. E.; Makarov, I. G.; Buyanov, V. N.; Preobrazhenskaya, M. N.
Chem. Heterocycl. Compd. 2005, 41, 914.
11. Xin, Z.; Liu, G.; Abad-Zapatero, C.; Pei, Z.; Szczepankiewicz, B. G.; Li, X.; Zhang,
T.; Hutchins, C. W.; Hajduk, P. J.; Ballaron, S. J.; Stashko, M. A.; Lubben, T. H.;
Trevillyan, J. M.; Jirousek, M. R. Bioorg. Med. Chem. Lett. 2003, 13, 3947.
12. Liu, G.; Xin, Z.; Pei, Z.; Hajduk, P. J.; Abad-Zapatero, C. W.; Hutchins, C.; Zhao,
H.; Lubben, T. H.; Ballaron, S. J.; Haasch, D. L.; Kaszubska, W.; Rondinone, C. M.;
Trevillyan, J. M.; Jirousek, M. R. J. Med. Chem. 2003, 46, 4232.
13. Wilson, D. P.; Wan, Z.-K.; Xu, W.-X.; Kirincich, S. J.; Follows, B. C.; Joseph-
McCarthy, D.; Foreman, K.; Moretto, A.; Wu, J.; Zhu, M.; Binnun, E.; Zhang, Y.-L.;
Tam, M.; Erbe, D. V.; Tobin, J.; Xu, X.; Leung, L.; Shilling, A.; Tam, S. Y.; Mansour,
T. S.; Lee, J. J. Med. Chem. 2007, 50, 4681.
14. Combs, A. P.; Zhu, W.; Crawley, M. L.; Glass, B.; Polam, P.; Sparks, R. B.; Modi,
D.; Takvorian, A.; McLaughlin, E.; Yue, E. W.; Wasserman, Z.; Bower, M.; Wei,
M.; Rupar, M.; Ala, P. J.; Reid, B. M.; Ellis, D.; Gonneville, L.; Emm, T.; Taylor, N.;
Yeleswaram, S.; Li, Y.; Wynn, R.; Burn, T. C.; Hollis, G.; Liu, P. C.; Metcalf, B. J.
Med. Chem. 2006, 49, 3774.
21. Makhija, Mahindra T.; Kasliwal, Rajesh T.; Kulkarnia, Vithal M.; Neamati, Nouri
Bioorg. Med. Chem. 2004, 12, 2317.
22. Weingarth, Markus; Raouafi, Noureddine; Jouvelet, Benjamin; Duma, Luminita
Chem. Commun. 2008, 5981.
23. Liu, Qi; Luedtke, Nathan W.; Tor, Yitzhak Tetrahedron Lett. 2001, 42, 1445.
24. Diss, Maria L.; Kennan, Alan J. Biopolymers 2007, 86, 276.
25. Takeuchi, Yasuo; Oda, Toshiaki; Chang, Ming-rong; Okamoto, YoKo; Ono,
Junko, et al. Chem. Pharm. Bull. 1997, 45, 406.
26. Panzhinskiy, E.; Ren, J.; Nair, S. Curr. Med. Chem. 2013, 20, 2609.
27. Zinker, B. A.; Rondinone, C. M.; Trevillyan, J. M.; Gum, R. J.; Clampit, J. E.;
Waring, J. F.; Xie, N.; Wilcox, D.; Jacobson, P.; Frost, L.; Kroeger, P. E.; Reilly, R.
M.; Koterski, S.; Opgenorth, T. J.; Ulrich, R. G.; Crosby, S.; Butler, M.; Murray, S.
F.; McKay, R. A.; Bhanot, S.; Monia, B. P.; Jirousek, M. R. Proc. Natl. Acid. Sci.
U.S.A. 2002, 99, 11357.
28. Fukuda, S.; Ohta, T.; Sakata, S.; Morinaga, H.; Ito, M.; Nakagawa, Y.; Tanaka, M.;
Matsushita, M. Diabetes Obes. Metab. 2010, 12, 299.
29. Maeda, A.; Kai, K.; Ishii, M.; Ishii, T.; Safranal, Akagawa M. Mol. Nutr. Food Res.
2014, 58, 1177.
15. Douty, B.; Wayland, B.; Ala, P. J.; Bower, M. J.; Pruitt, J.; Bostrom, L.; Wei, M.;
Klabe, R.; Gonneville, L.; Wynn, R.; Burn, T. C.; Liu, P. C.; Combs, A. P.; Yue, E. W.
Bioorg. Med. Chem. Lett. 2008, 18, 66.
16. Salmeen, A.; Andersen, J. N.; Myers, M. P.; Tonks, N. K.; Barford, D. Mol. Cell.
2000, 6, 1401.
17. Iversen, L. F.; Andersen, H. S.; Branner, S.; Mortensen, S. B.; Peters, G. H.; Norris,
K.; Olsen, O. H.; Jeppesen, C. B.; Lundt, B. F.; Ripka, W.; Moller, K. B.; Moller, N.
P. J. Biol. Chem. 2000, 275, 10300.
18. Ala, P. J.; Gonneville, L.; Hillman, M.; Becker-Pasha, M.; Yue, E. W.; Douty, B.;
Wayland, B.; Polam, P.; Crawley, M. L.; McLaughlin, E.; Sparks, R. B.; Glass, B.;
Takvorian, A.; Combs, A. P.; Burn, T. C.; Hollis, G. F.; Wynn, R. J. Biol. Chem. 2006,
281, 38013.
30. Otake, K.; Azukizawa, S.; Fukui, M.; Kunishiro, K.; Kamemoto, H.; Kanda, M.;
Miike, T.; Kasai, M.; Shirahase, H. Bioorg. Med. Chem. 2012, 20, 1060.
31. Lakshminarayana, N.; Prasad, Y. R.; Gharat, L.; Thomas, A.; Narayanan, S.;
Raghuram, A.; Srinivasan, C. V.; Gopalan, B. Eur. J. Med. Chem. 2010, 45, 3709.
32. Shi, L.; Yu, H. P.; Zhou, Y. Y.; Du, J. Q.; Shen, Q.; Li, J. Y.; Li, J. Acta Pharmacol. Sin.
2008, 29.
33. Zhi, Y.; Gao, L. X.; Jin, Y.; Tang, C. L.; Li, J. Y.; Li, J.; Long, Y. Q. Bioorg. Med. Chem.
2014, 22, 3670.
34. Chen, L.; Li, Q. Y.; Shi, X. J.; Mao, S. L.; Du, Y. L. J. Agric. Food Chem. 2013, 61,
10714.
Please cite this article in press as: Du, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.032