Design, synthesis and biological evaluation of uncharged catechol derivatives as selective inhibitors of PTP1B

Article abstract of DOI:10.1016/j.ejmech.2017.05.007

Protein tyrosine phosphatases 1B (PTP1B) is a promising and validated therapeutic target to effectively treat T2DM and obesity. However, the development of charged PTP1B inhibitors was restricted due to their low cell permeability and poor bioavailability. Based on active natural products, two series of uncharged catechol derivatives were identified as PTP1B inhibitors by targeting a secondary aryl phosphate-binding site as well as the catalytic site. The most potent inhibitor 22 showed an IC₅₀ of 0.487?μM against PTP1B and strong selectivity (27-fold) over TCPTP. Kinetic studies were also performed that 22 act as a competitive PTP1B inhibitor. The treatment of C2C12 myotubes with 22 markedly increased the phosphorylation levels of IRβ, Akt and IRS1 phosphorylation. The similarity of its action profiling with that produced by insulin suggested its potential as a new non-insulin-dependent drug candidate.

Full text of DOI:10.1016/j.ejmech.2017.05.007

European Journal of Medicinal Chemistry 136 (2017) 348e359
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of uncharged catechol
derivatives as selective inhibitors of PTP1B
,
b
,
,
b
,
c
,
,
b
,
,
, b
Xiang-Qian Li a 1, Qi Xu a
1, Jiao Luo a c, Li-Jun Wang a b, Bo Jiang a
,
Ren-Shuai Zhang a b, Da-Yong Shi a
,
, b, c, *
a Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
b Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China
c University of Chinese Academy of Sciences, Beijing, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein tyrosine phosphatases 1B (PTP1B) is a promising and validated therapeutic target to effectively
treat T2DM and obesity. However, the development of charged PTP1B inhibitors was restricted due to
their low cell permeability and poor bioavailability. Based on active natural products, two series of un-
Received 19 January 2017
Received in revised form
27 April 2017
Accepted 2 May 2017
Available online 3 May 2017
charged catechol derivatives were identified as PTP1B inhibitors by targeting
phosphate-binding site as well as the catalytic site. The most potent inhibitor 22 showed an IC50 of
0.487 M against PTP1B and strong selectivity (27-fold) over TCPTP. Kinetic studies were also performed
that 22 act as a competitive PTP1B inhibitor. The treatment of C2C12 myotubes with 22 markedly
increased the phosphorylation levels of IR , Akt and IRS1 phosphorylation. The similarity of its action
a secondary aryl
m
Keywords:
b
Type 2 diabetes
PTP1B inhibition
Selectivity
profiling with that produced by insulin suggested its potential as a new non-insulin-dependent drug
candidate.
© 2017 Elsevier Masson SAS. All rights reserved.
Competitive
Cellular activity
1. Introduction
adequate binding affinity. Non-acid-containing ligands targeting
site B are highly desirable for improving the overall druglike
Protein tyrosine phosphatases 1B (PTP1B), a member of PTPs
family, is a key negative regulator in both insulin and leptin
signaling pathways [1,2]. It can down regulate insulin signaling by
dephosphorylating the insulin receptor and several of its down-
stream signaling proteins [3,4], thereby modulates both glucose
and lipid metabolism. PTP1B knockout mice displayed enhanced
insulin sensitivity, lower plasma glucose and insulin levels, without
causing abnormalities in growth or other vital functions [5,6].
Hence, PTP1B is considered as a promising drug target for the
treatment of type 2 diabetes and obesity.
properties.
To increase selectivity versus highly homologous T-cell PTP
(TCPTP) is another great challenge for the design of PTP1B in-
hibitors. T-Cell PTP (TCPTP) is highly homologous to PTP1B, with
74% sequence identity in the catalytic region [14,15]. Unlike the
PTP1B knockout mice that develop normally, the TCPTP knockout
died within 3e5 weeks after birth, because of the defects in he-
matopoiesis and immune function caused by abnormalities in B-
and T-cells [15]. Simultaneous inhibition of PTP1B and TCPTP is
considered unsuitable.
Accordingly, various PTP1B inhibitors, difluoromethylene
phosphonate [7,8] and carboxylic acid pTyr mimetic [9,10], have
been developed extensively over the past decade. However, the
limited cell membrane permeability failed to support them to
progress beyond pre-clinical stage [11e13], owing to the highly
charged pharmacophore that were incorporated to achieve an
Some PTP1B inhibitors with good selectivity over TCPTP (20-
fold) had been reported successively in the last decade
[14,16e18]. However, most of them were acid-containing com-
pounds without cell activity studies and following reports. Non-
acid-containing inhibitors, such as (S)-isothiazolidinone de-
rivatives, showed high affinity for PTP1B (IC50 ¼ 0.055
mM) and low
cellular activity, but exhibited no selectivity over TCPTP [19,20].
Hence, there is an urgent need to develop small molecule PTP1B
inhibitors devoid of any charged moieties and with good selectivity.
In the last few years, we have identified a series of bromophenol
PTP1B inhibitors from the ethanolic extract of the red alga
* Corresponding author. Key Laboratory of Experimental Marine Biology, Institute
of Oceanology, Chinese Academy of Sciences, Qingdao, China.
E-mail address: shidayong@qdio.ac.cn (D.-Y. Shi).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2017.05.007
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

X.-Q. Li et al. / European Journal of Medicinal Chemistry 136 (2017) 348e359
349
rhodomela confervoides (Fig. 1) [21,22]. These natural products
2.2. Structure-based design of PTP1B inhibitors
with an uncharged catechol have shown hypoglycemic effect
mediated by PTP1B inhibition over few in vitro and in vivo diabetic
and obese models. Furthermore, some other groups reported that
many natural PTP1B inhibitors with the catechol pharmacophore
from Broussonetia papyrifera [23], Licorice [24], Tradescantia spa-
thacea Sw. [25], Phellinus igniarius [26], also showed moderate
inhibitory activities against PTP1B (Fig. 1).
In this context, the catechol that these active natural products
have in common was proposed as a lead to guide molecular design
of novel PTP1B inhibitors. Prompted by these findings, two series of
catechol derivatives were prepared and their inhibitory activity
against the PTP1B enzyme was assessed. The results from the
binding assays were also used to re-evaluate the docking results in
order to gain insight into their possible binding modes, allowing
the development of more active compounds in the future.
PTP1B enzyme contains two aryl phosphate-binding sites: a
high affinity catalytic site (site A) and the second phosphotyrosine
binding site (site B). The collective interactions with both sites are
proposed to increase the inhibitory activity for PTP1B and enhance
the selectivity over TCPTP [32,33]. Previous research suggested that
a such inhibitor of PTP1B may require a combination of four moi-
eties: a hydrophilic head, an aromatic center, a linker and a hy-
drophobic tail (Fig. 2) [32]. A hydrophilic head was proposed to
occupy the catalytic site of PTP1B enzyme through hydrogen bonds
with the residues Ser216, Ile219, Gly220 and Arg221, which
embrace the catalytic residue Cys215. The aromatic center was
designed to participate in extensive van der Waals interactions
with Tyr46, Val49, Ile219, and F182 of the flap [19,34,35]. An
appropriate linker could pass through the “gateway” region to ac-
cess the site B from the site A [36]. Hydrophobic substituents of
different sizes were employed to occupy site B, especially the res-
idue Ala27 that is related to selectivity.
3,4-dihydroxybenzaldehyde (1) with
a catechol core was
2. Results and discussion
selected as the hydrophilic head to occupy site A. PTP1B inhibitory
assay of our own laboratory identified that compound 1 exhibited
weak inhibitory activity (29% inhibition at 1 mM, not shown). As
shown in Fig. 3a, the catechol core is accommodated in the catalytic
site of PTP1B enzyme through a network of hydrogen bonds with
the residues Ser216, Ile219, Gly220 and Arg221. Such interactions
do have great influence on the catalytic function of PTP1B, but
inhibitory activity still need to be improved by adding pharmaco-
phore to occupy other binding site. Therefore, the second phos-
photyrosine binding site (site B) was selected to offer opportunities
for both potency and selectivity improvement.
Subsequently, an oxadiazole ring was introduced as an aromatic
center and a corner structure, which could pass through the
“gateway” between site A and site B. Compounds 2e5 were ob-
tained by introducing hydrophobic substitution groups into the
terminal and determined to inhibit PTP1B with IC50 values of
2.1. Chemistry
The designed compounds were synthesized according to the
general method depicted in Scheme 1. The synthesis began with
3,4-dihydroxybenzoic acid to afford the requisite intermediate 4-
(5-mercapto-1,3,4-oxadiazol-2-yl)benzene-1,2-diol through a long
synthetic sequence included esterification, hydrazinolysis and
cyclization reactions [27]. After alkylation with chloracetates or
bromoacetophenones, intermediate 4-(5-mercapto-1,3,4-
oxadiazol-2-yl)benzene-1,2-diol were converted to target com-
pounds 2e18.
The preparation of 2,5-disubstituted oxazole derivatives
(19e27) also required a long synthetic sequence included bromi-
nation, amination and cyclization reactions (Scheme 2). Initially,
variously substituted acetophenones have been synthesized by
copper-catalyzed Ullmann diaryl ether synthesis [28] and were
bromination by copper(II) bromide in refluxing chloroform-ethyl
a-
8.09e35.58
mM respectively (Table 1). As expected, these com-
pounds maintain the binding mode of 1, the additional groups lie
along the tunnel wall and offer a favorable trajectory toward the B
site, as shown in Fig. 3b and c, respectively.
With a purpose to improve the potency of 4 and 5, further
optimization was attempted by introducing different hydrophobic
substituents into their para-position of the terminal phenyl ring.
acetate [29]. Intermediate
with hexamethylenetetramine, and then hydrolyzed by 4 M HCl/
EtOH to afford -aminoacetophenone [30]. The synthesis of target
2,5-disubstituted was achieved in the presence of iodine and TBHP
from -aminoketone and aromatic aldehydes [31].
a-bromoacetophenone were reacted
a
a
Fig. 1. Representative natural PTP1B Inhibitors with the catechol pharmacophore.

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X.-Q. Li et al. / European Journal of Medicinal Chemistry 136 (2017) 348e359
Scheme 1. Synthesis of designed compounds. Reagents and conditions: (a) H2SO4, EtOH, reflux, 4 h; (b) N2H4$H2O (85%, 6.0 equiv), EtOH, reflux, 6 h; (c) CS2 (2.3 equiv), KOH (1.0
equiv), EtOH, reflux, 4 h; (d) KOH (1.0 equiv), Cl-R1 or Br-R1 (1.0 equiv), EtOH, H2O, rt, 1 h.
Scheme 2. Synthesis of designed compounds. Reagents and conditions: (a) CuI (0.1 equiv), K2CO3 (1.2 equiv), DMF, 140 ꢀC, 14 h; (b) CuBr2 (2.0 equiv), CHCl3, EtOAc, reflux, 2 h; (c)
(CH2)6N4 (1.0 equiv), CHCl3, 2 h; (d) HCl (35%, 5.0 equiv), EtOH, rt, 48 h; (e) 3,4-dihydroxybenzaldehyde (1.0 equiv), I2 (0.15 equiv), TBHP (1.0 equiv), DMF, NaHCO3 (1.0 equiv), 70 ꢀC,
10 h.
Fig. 2. Design of catechol derivatives as novel PTP1B inhibitors.

X.-Q. Li et al. / European Journal of Medicinal Chemistry 136 (2017) 348e359
351
Fig. 3. Predicted binding models of PTP1B (1G1H) with (a) compound 1; (b) compound 4; (c) compound 5 (d) compound 13. Carbon, oxygen, nitrogen, bromine, and sulfur atoms
are colored gray 9(light blue), red, blue, green, and yellow, respectively. Hydrogen bonds are depicted as green lines. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
2.3. Entropy-driven optimization of PTP1B inhibitors
Table 1
In vitro inhibitory activity of designed compounds 2e5 to PTP1B.
Ligand binding is a multistep process that involves the des-
olvation and conformational rearrangement of both the ligand and
the binding site [37]. Correspondingly, entropy-driven optimization
by adding lipophilic moieties and applying chain-ring strategies are
successful tools routinely used in medicinal chemistry programs
[37e39]. More lipophilic compounds desolvate more easily,
resulting in a significant reward in desolvation entropy [40].
However, repeated attempts to increase hydrophobic on para-
position of terminal phenyl group (compound 6e18) did not
translate into expected inhibitory activity improvement in our
study. Therefore, entropy-driven optimization by applying chain-
ring became the strategy of further study (compound 19e27,
Fig. 4). Reducing ligand flexibility by limiting its rotational freedom
could decrease the penalty arising from conformational entropy
changes.
Compd
R1
PTP1B
IC50 SD (mM)
2
31.63 2.6
3
4
35.58 4.8
8.09 2.1
Encouraged by the above findings, a series of oxazole com-
pounds (19e27), with rigid structure and lipophilic groups, were
designed and synthesized. The results (shown in Table 3) indicated
that all of these compounds showed potential inhibitory activity
toward PTP1B. The presence of the methyl group give compound 20
5
15.49 4.3
3.09 0.13
Sodium vanadate
improved inhibitory activity against PTP1B (IC50 ¼ 0.809
mM).
Amazingly, when t-butyl was incorporated to para-position,
inhibitory
activity
had
obvious
improvement
(22,
IC50 ¼ 0.487
mM). The molecular modeling (Fig. 5) predicted that
Two series of compounds, 6e8 and 9e18, were synthesized and
showed improved PTP1B inhibitory activity (Table 2). Interestingly,
when introducing methyl group on para-position of phenyl group
compound 20 and 22 maintain the similar binding modes, occu-
pying both site A and site B simultaneously. The additional lipo-
philic groups fill a hydrophobic cavity, which is formed by the
main- and side-chain atoms of Met258, Gly259, Arg254 and Ala27.
(6 and 13) inhibited PTP1B with similar IC50 values, 4.74
3.89 M, respectively.
mM and
m
Docking studies showed that compound 13 do not only form a
network of H-bonds with Ser216, Ile219, Gly220 and Arg221, but
also form an OꢁꢁꢁH-N hydrogen bond with the positive charged
side chain of Arg24 (2.052 Å, Fig. 3d). The terminal p-methyl lies on
a small hydrophobic pocket formed by the main- and side-chain
atoms of Met258, Gly259 and Ala27, which cannot accommodate
a larger group.
2.4. Selectivity over TCPTP
Since Ala27 is not conserved between PTP1B and TCPTP, inter-
action with Ala27 had the potential to impart selectivity to PTP1B
inhibitors [33]. Our molecular modeling predicted that several
potent compounds 7, 13, 22 and 23 interact with Ala27 in an edge-

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X.-Q. Li et al. / European Journal of Medicinal Chemistry 136 (2017) 348e359
Table 2
In vitro inhibitory activity of designed compounds 6e18 to PTP1B.
Compd
Group R2 or R3
PTP1B
Compd
Group R2 or R3
PTP1B
IC50 SD (
m
M)
IC50 SD (mM)
6
7
8
9
10
11
12
R2 ¼ CH3
R2 ¼ OCH3
R2 ¼ OCH2CH3
R3 ¼ Br
4.74 0.9
6.02 1.5
7.94 1.1
115.34 8.6
21.38 3.2
7.79 1.8
9.33 2.2
13
14
15
16
17
18
R3 ¼ OPh-p-CH3
R3 ¼ OPh-p-(i-Pr)
R3 ¼ OPh-p-OEt
R3 ¼ OPh-p-OBn
R3 ¼ OPh-p-(t-Bu)
R3 ¼ OPh-p-EtOMe
3.89 0.8
10.96 3.1
16.98 2.3
21.37 3.7
10.47 1.9
22.75 3.4
3.09 0.13
R3 ¼ CF3
R3 ¼ OBn
R3 ¼ OPh
Sodium vanadate
strong inhibitory activity against PTP1B, kinetic analyses were
performed (Fig. 6). Various concentrations of pNPP (0.5, 1.0, 2.0, 4.0,
and 8.0 mM) were used as a PTP1B substrate in the absence or
presence of compounds. The effects of compounds 12 and 22 on the
kinetic profile of PTP1B-catalyzed pNPP hydrolysis were investi-
gated as described in methods. As the graph showed straight lines
intersected each other on the 1/v axis, compounds 12 and 22
behaved as competitive inhibitors, indicating that they may bind at
the active site of the PTP1B enzyme.
2.6. Cellular activity
Insulin signal transduction initiates when the insulin binds to
the insulin receptor (IR), which stimulates IR intrinsic kinase ac-
tivity and activates a number of downstream molecules including
insulin receptor substrate 1 (IRS1) and Akt by phosphorylation, in
turn, lead to increased influx of glucose into the skeletal muscle
cells [41e43]. Hence, phosphorylation of the signaling molecules
plays a vital role in the activation of insulin signaling pathway.
PTP1B serves as a negative regulator of the insulin activated
signaling pathways, inhibition of which could up-regulate phos-
Fig. 4. Entropy-driven optimization by applying chain-ring strategy.
Table 3
In vitro inhibitory activity of designed compounds 19e27 to PTP1B.
phorylation levels of insulin receptor
substrate 1 (IRS-1) and protein kinase B (Akt).
Given the observed potency of 22 for PTP1B, we next explored
its possible regulatory role in IR , IRS-1 and AKT activation. As
shown in Fig. 7, incubation of C2C12 cells with 22 in 0.5, 1, 2, 4
concentration range increased insulin-mediated IR , IRS-1 and AKT
phosphorylation levels compared to controls. The uptake levels
determined at 2 M concentration were comparable to that pro-
b (IRb), insulin receptor
Compd
Group R4
PTP1B
IC50 SD (mM)
19
20
21
22
23
24
25
26
H
2.63 0.7
b
CH3
OCH3
t-Bu
Ph
i-Pr
t-Pe
OEt
0.807 0.21
6.92 1.3
0.487 1.3
6.25 1.7
7.44 3.9
3.60 1.3
20.82 3.6
19.14 6.7
3.09 0.13
m
M
b
m
duced by insulin (100 nM). These results suggest that 22 possesses
good membrane permeability, and could possibly induce insulin
signaling at the cellular level.
27
EtOMe
Sodium vanadate
3. Conclusion
to-edge fashion. The in vitro selectivity over TCPTP was evaluated
for these four compounds, using pNPP assay and IC50 values in
Table 4. Compounds 7 and 22 showed 16e27-fold selectivity, 13
showed about 2-fold selectivity, while 23 showed no selectivity
over TCPTP enzyme. The introduction of the methoxyl and t-butyl
substituents might force an interaction with Ala27 in PTP1B, which
are less tolerated by the larger replacement (Ser29) at this position
in TCPTP. Ser29 might push the methoxyl and t-butyl substituents
to a different and presumably less favorable conformation.
Two series of uncharged catechol derivatives were synthesized
and identified as competitive PTP1B inhibitors. Among them, the
representative compound 22 had strong inhibitory activity to
PTP1B (IC50 ¼ 0.487
mM) and exhibited good specificity for PTP1B
over TCPTP (27-fold). Further studies on cellular activities revealed
that 22 was membrane-permeable and exerted non-insulin-
dependent cellular effects on the activation of IR
pathway in C2C12 myotubes at a concentration of 2.0
b
, IRS-1 and Akt
M.
m
These results provide further evidence that non-acid-containing
catechol derivatives can be potent inhibitor of PTP1B with high
selectivity over TCPTP and non-insulin-dependent cellular activity.
A hydrophilic head incorporating a hydrophobic tail provide new
opportunity to overcome two challenges for the design of PTP1B
2.5. Kinetic studies
To elucidate the inhibition modes of compounds 20 and 22 with

X.-Q. Li et al. / European Journal of Medicinal Chemistry 136 (2017) 348e359
353
Fig. 5. Predicted binding models of PTP1B (1G1H) with (a) compound 20 and (b) compound 22. Carbon, oxygen, nitrogen, bromine, and sulfur atoms are colored gray (light blue),
red, blue, green, and yellow, respectively. Hydrogen bonds are depicted as green lines. Hydrophobic interaction is labeled by blue line. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
Table 4
Selectivity ratios for the compounds designed to interact with Ala27.
Compounds
7
13
22
23
Terminal groups
OMe
Me
t-Bu
Ph
PTP1B (IC50
,
m
m
M)
M)
6.02 1.3
>100
16.6
3.89 0.67
8.73 2.14
2.24
0.487 0.38
13.31 2.55
27.34
6.25 1.36
5.01 1.34
0.80
TCPTP (IC50
,
Selectivitya
a
TCPTP IC50/PTP1B IC50
.
inhibitors, selectivity and membrane-permeable. These com-
pounds will provide new insights into the design and development
of non-insulin-dependent agents to treat type 2 diabetes.
4. Experimental section
4.1. Chemistry
All commercial reagents were purchased and used without
further purification or distillation unless otherwise stated. Melting
points were measured on a Griffin apparatus and are uncorrected.
NMR (1H, 13C) was obtained with Bruker AV-500 spectrometer with
chemical shifts reported as parts per million (TMS as internal
standard). The following abbreviations are used for multiplicity of
NMR signals: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet,
m ¼ multiplet. Column chromatography was performed on silica
gel 200e300 mesh. Purity of all final products was determined by
analytical HPLC to be >95%. HPLC purity of compounds was
measured with a normal phase HPLC (XBridge C18, 4.6 ꢂ 150 mm,
5 mm) with two diverse wavelength detection systems. Compounds
were eluted using a gradient elution of 40/60 to 0/100H2O/CH3OH
over 30 min at a flow rate of 1.0 mL/min.
4.2. General procedure for the preparation of 4-(5-mercapto-1,3,4-
oxadiazol-2-yl)benzene-1,2-diol
Fig. 6. The effect of 20 and 22 on PTP1B-catalyzed pNPP hydrolysis. (a) 20 concen-
trations were 1, 2, 4, and 8 M, respectively. (b) 22 concentrations were 0, 1, 2, and
M, respectively. The experiment was performed at 37 ꢀC and pH 7.0.
To a solution of 3,4-dihydroxybenzoic acid (15.4 g, 0.1 mol) in
absolute ethanol (200 mL) was added 5 mL concentrated sulfuric
acid. This mixture was heated at reflux for 4 h. After cooling to
ambient temperature, water (100 mL) was slowly introduced over a
period of 10 min. The mixture was extracted with EtOAc, washed
with diluted HCl followed by water, dried with Na2SO4. Then sol-
vent was removed under reduced pressure, the yellow powder as
ethyl 3,4-dihydroxybenzoate was used without further disposal.
Yield 15.8 g, 86.8%.
m
4
m
reaction mixture was diluted with cold water and acidified with
1 M HCl. The precipitated solid was filtered and washed with water
to obtain 3,4-dihydroxybenzohydrazide hydrochloride (12.0 g,
88.9%), which was used in the next step without further
purification.
A
solution of 12.0
g
(0.071 mol) of the 3,4-
The crude product (15.8 g, 0.087 mol) were dissolved in ethanol
(100 mL) and 37.5 g N2H4$H2O were dropwise added. The mixture
was refluxed for 6 h. After cooling to room temperature, the
dihydroxybenzohydrazide and 4.0 g (0.072 mol) KOH in absolute
ethanol (200 mL) was treated to the addition of carbon disulfide

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X.-Q. Li et al. / European Journal of Medicinal Chemistry 136 (2017) 348e359
tR ¼ 4.024 min.
4.3.3. Phenyl 2-((5-(3,4-dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)
thio)acetate (4)
M.p. 119.2e120.6 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.82 (s, 1H),
9.54 (s, 1H), 7.41 (t, J ¼ 7.7 Hz, 2H), 7.33 (s, 1H), 7.26 (overlap, 2H),
7.11 (d, J ¼ 8.2 Hz, 2H), 6.87 (d, J ¼ 8.2 Hz, 1H), 4.50 (s, 2H). 13C NMR
(126 MHz, DMSO-d6)
d 167.39 (s), 166.15 (s), 161.95 (s), 150.80 (s),
149.89 (s), 146.34 (s), 130.11 (s), 126.66 (s), 121.84 (s), 119.15 (s),
116.65 (s), 114.18 (s), 113.75 (s), 34.48 (s). DEPT (135ꢀ)
130.11 (CH),
126.66 (CH),121.84 (CH),119.15 (CH),116.65 (CH),113.75 (CH), 34.47
(negative peak, CH2). DEPT (90ꢀ)
130.11 (CH), 126.66 (CH), 121.84
d
d
(CH), 119.15 (CH), 116.65 (CH), 113.75 (CH). HRMS (ESIþ) m/z:
367.0372 (M þ Na). HPLC purity ¼ 99.883%, tR ¼ 4.25 min.
4.3.4. 2-((5-(3,4-Dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-1-
phenylethan-1-one (5)
M.p. 216.3e218.2 ꢀC.1H NMR (500 MHz, DMSO-d6)
d 9.78 (s, 1H),
9.52 (s, 1H), 8.04 (d, J ¼ 7.5 Hz, 2H), 7.69 (t, J ¼ 7.4 Hz, 1H), 7.56 (t,
J ¼ 7.7 Hz, 2H), 7.30 (s, 1H), 7.21 (d, J ¼ 8.2, 1H), 6.85 (d, J ¼ 8.2 Hz,
1H), 5.11 (s, 2H). 13C NMR (126 MHz, DMSO-d6)
d 193.17 (s), 165.85
Fig. 7. Activation of insulin signaling during exposure of C2C12 myotubes to 22. C2C12
myotubes were treated with 22 in 0.5, 1, 2, 4 mM concentration range or 100 nM insulin
(s), 162.39 (s), 149.78 (s), 146.30 (s), 135.48 (s), 134.42 (s), 129.34 (s),
128.91 (s), 119.08 (s), 116.61 (s), 114.28 (s), 113.70 (s), 40.89 (s). DEPT
for 30 min in serum-free DMEM. Then, phosphorylation levels of IR
were determined by immunoblotting.
b, IRS-1 and Akt
(135ꢀ)
d
134.42 (CH), 129.34 (CH), 128.91 (CH), 119.08 (CH), 116.61
(CH), 113.70 (CH), 40.88 (negative peak, CH2). DEPT (90ꢀ)
d
134.41
(CH),129.34 (CH),128.90 (CH),119.08 (CH),116.60 (CH),113.69 (CH).
HRMS (ESIþ) m/z: 351.0416 (M þ Na). HPLC purity ¼ 98.959%,
tR ¼ 4.375 min.
(12 g, 0.162 mol). This mixture was heated at reflux for 4 h. After
cooling to ambient temperature, concentrated hydrochloric acid
(20 mL) was slowly introduced causing the precipitation of a light
yellow solid, 4-(5-mercapto-1,3,4-oxadiazol-2-yl)benzene-1,2-diol
(10.3 g, 69.1%).
4.3.5. p-Tolyl 2-((5-(3,4-dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)
thio)acetate (6)
M.p. 156.9e158.1 ꢀC.1H NMR (500 MHz, DMSO-d6)
d 9.87 (s, 1H),
4.3. General procedure for the preparation of 2e18
9.59 (s, 1H), 7.32 (s, 1H), 7.25 (d, J ¼ 8.2 Hz, 1H), 7.19 (d, J ¼ 8.2 Hz,
2H), 6.97 (d, J ¼ 8.2 Hz, 2H), 6.87 (d, J ¼ 8.2 Hz,1H), 4.46 (s, 2H), 2.26
(s, 3H). 13C NMR (126 MHz, DMSO-d6)
d 167.49 (s), 166.13 (s), 161.99
A mixture of 4-(5-mercapto-1,3,4-oxadiazol-2-yl)benzene-1,2-
diol (105 mg, 0.5 mmol), KOH (20 mg, 0.5 mmol) in 10 mL H2O
was stirred vigorously under nitrogen for 1 h at room temperature.
Under vigorous stirring, chloroacetate or bromoacetophenone
(0.5 mmol) in 20 mL ethanol was slowly added to the mixture. After
1 h, water was slowly added with rapid stirring under ice-water
bath. Crystals were obtained after filtration and purified by silica
gel flash column chromatography (CH2Cl2/acetone ¼ 35:1).
(s), 149.87 (s), 148.58 (s), 146.32 (s), 135.91 (s), 130.42 (s), 121.50 (s),
119.18 (s), 116.64 (s), 114.16 (s), 113.72 (s), 34.42 (s), 20.80 (s). DEPT
(135ꢀ)
d
130.41 (CH), 121.50 (CH), 119.18 (CH), 116.63 (CH), 113.72
(CH), 34.42 (negative peak, CH2), 20.80 (CH3). DEPT (90ꢀ)
130.42
(CH), 121.50 (CH), 119.18 (CH), 116.64 (CH), 113.72 (CH). HRMS
d
(ESIþ) m/z: 381.0538 (M
þ
Na). HPLC purity
¼
99.011%,
tR ¼ 4.388 min.
4.3.1. 4-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)benzene-1,2-diol (2)
4.3.6. 4-Methoxyphenyl 2-((5-(3,4-dihydroxyphenyl)-1,3,4-
M.p.178.8e179.2 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.79 (s, 1H),
oxadiazol-2-yl)thio)acetate (7)
M.p.183.3e183.9 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.82 (s, 1H),
9.52 (s, 1H), 7.43 (d, J ¼ 7.4 Hz, 2H), 7.36e7.29 (m, 3H), 7.26 (d,
J ¼ 7.4 Hz,1H), 7.23 (d, J ¼ 8.2 Hz,1H), 6.86 (d, J ¼ 8.2 Hz,1H), 4.51 (s,
9.54 (s, 1H), 7.32 (s, 1H), 7.25 (d, J ¼ 8.2 Hz, 1H), 7.02 (d, J ¼ 8.9 Hz,
2H). 13C NMR (126 MHz, DMSO-d6)
d 165.95 (s), 162.36 (s), 149.79
2H), 6.94 (d, J ¼ 8.9 Hz, 2H), 6.87 (d, J ¼ 8.2 Hz,1H), 4.47 (s, 2H), 3.72
(s, 3H). 13C NMR (126 MHz, DMSO-d6)
d 167.65 (s), 166.12 (s), 161.96
(s), 146.30 (s), 137.08 (s), 129.43 (s), 129.01 (s), 128.17 (s), 119.09 (s),
116.63 (s), 114.30 (s), 113.71 (s), 36.39 (s). DEPT (135ꢀ)
129.43 (CH),
129.01 (CH), 128.17 (CH), 119.09 (CH), 116.63 (CH), 113.70 (CH),
36.38 (negative peak, CH2). DEPT (90ꢀ)
129.43 (CH), 129.01 (CH),
d
(s), 157.52 (s), 149.88 (s), 146.33 (s), 144.18 (s), 122.65 (s), 119.15 (s),
116.65 (s), 115.00 (s), 114.18 (s), 113.74 (s), 55.87 (s), 34.43 (s). DEPT
(135ꢀ)
d
122.65 (CH), 119.14 (CH), 116.64 (CH), 115.00 (CH), 113.74
(CH), 55.87 (CH3), 34.42 (negative peak, CH2). DEPT (90ꢀ)
122.65
(CH), 119.15 (CH), 116.64 (CH), 115.00 (CH), 113.74 (CH). HRMS
d
128.17 (CH),119.09 (CH),116.63 (CH),113.70 (CH). HRMS (ESIþ) m/z:
d
323.0461 (M þ Na). HPLC purity ¼ 99.019%, tR ¼ 4.725 min.
(ESIþ) m/z: 397.0493 (M
þ
Na). HPLC purity
¼
95.141%,
4.3.2. Methyl 2-((5-(3,4-dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)
tR ¼ 5.251 min.
thio)acetate (3)
M.p.158.2e160.9 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d
9.78 (s,1H),
4.3.7. 4-Ethoxyphenyl 2-((5-(3,4-dihydroxyphenyl)-1,3,4-
9.57 (s, 1H), 7.30 (s, 1H), 7.23 (d, J ¼ 8.2 Hz, 1H), 6.87 (d, J ¼ 8.2 Hz,
oxadiazol-2-yl)thio)acetate (8)
1H), 4.23 (s, 2H), 3.67 (s, 3H). 13C NMR (126 MHz, DMSO-d6)
M.p. 151.2e152.4 ꢀC.1H NMR (500 MHz, DMSO-d6)
d 9.84 (s, 1H),
d
168.76 (s), 165.98 (s), 162.02 (s), 149.84 (s), 146.31 (s), 119.10 (s),
116.65 (s), 114.20 (s), 113.70 (s), 53.17 (s), 34.10 (s). DEPT (135ꢀ)
119.09 (CH), 116.65 (CH), 113.69 (CH), 53.16 (CH3), 34.09 (negative
peak, CH2). DEPT (90ꢀ)
119.10 (CH), 116.65 (CH), 113.70 (CH).
HRMS (ESIþ) m/z: 305.0207 (M þ Na). HPLC purity ¼ 99.255%,
9.56 (s, 1H), 7.34 (s, 1H), 7.25 (d, J ¼ 8.3 Hz, 1H), 7.00 (d, J ¼ 8.9 Hz,
2H), 6.91 (d, J ¼ 8.9 Hz, 2H), 6.88 (d, J ¼ 8.3 Hz,1H), 4.46 (s, 2H), 3.97
(q, J ¼ 6.9 Hz, 2H),1.28 (t, J ¼ 6.9 Hz, 3H). 13C NMR (126 MHz, DMSO-
d
d
d6) d 167.65 (s),166.13 (s),161.97 (s),156.79 (s),149.89 (s),146.34 (s),
144.06 (s), 122.63 (s), 119.15 (s), 116.66 (s), 115.44 (s), 114.18 (s),

X.-Q. Li et al. / European Journal of Medicinal Chemistry 136 (2017) 348e359
355
113.76 (s), 63.83 (s), 34.42 (s), 15.03 (s). DEPT (135ꢀ)
d
122.63 (CH),
4.3.12. 2-((5-(3,4-Dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-1-
119.14 (CH), 116.66 (CH), 115.44 (CH), 113.76 (CH), 63.83 (negative
(4-(p-tolyloxy)phenyl)ethan-1-one (13)
peak, CH2), 34.42 (negative peak, CH2), 15.03 (CH3). DEPT (90ꢀ)
M.p. 195.8e196.3 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 8.04 (d,
d
122.63 (CH), 119.15 (CH), 116.66 (CH), 115.44 (CH), 113.76 (CH).
J ¼ 8.2 Hz, 2H), 7.29 (s, 1H), 7.25 (d, J ¼ 8.3 Hz, 2H), 7.20 (d,
HRMS (ESIþ) m/z: 389.0828 (M þ H). HPLC purity ¼ 95.285%,
J ¼ 8.2 Hz, 1H), 7.02 (overlap, 4H), 6.85 (d, J ¼ 8.2 Hz, 1H), 5.04 (s,
tR ¼ 3.508 min.
2H), 2.30 (s, 3H). 13C NMR (126 MHz, DMSO-d6)
d 191.61 (s), 165.84
(s), 162.79 (s), 162.41 (s), 152.77 (s), 149.78 (s), 146.30 (s), 134.70 (s),
131.57 (s), 131.19 (s), 129.91 (s), 128.91 (s), 120.63 (s), 119.07 (s),
117.23 (s), 116.60 (s), 114.28 (s), 113.70 (s), 40.67 (s), 20.80 (s). DEPT
4.3.8. 1-(4-Bromophenyl)-2-((5-(3,4-dihydroxyphenyl)-1,3,4-
oxadiazol-2-yl)thio)ethan-1-one (9)
(135ꢀ)
d
131.56 (CH), 131.19 (CH), 128.91 (CH), 120.63 (CH), 119.07
(CH), 117.22 (CH), 116.59 (CH), 113.69 (CH), 40.66 (negative peak,
CH2), 20.80 (CH3). DEPT (90ꢀ)
131.57 (CH), 131.19 (CH), 129.34
M.p. 236.6e237.6 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 7.97 (d,
J ¼ 8.5 Hz, 2H), 7.78 (d, J ¼ 8.5 Hz, 2H), 7.29 (s, 1H), 7.20 (d,
J ¼ 8.2 Hz, 1H), 6.85 (d, J ¼ 8.2 Hz, 1H), 5.08 (s, 2H). 13C NMR
d
(CH), 120.63 (CH), 119.07 (CH), 117.23 (CH), 116.60 (CH), 113.69 (CH).
HRMS (ESIþ) m/z: 457.0857 (M þ Na). HPLC purity ¼ 98.082%,
tR ¼ 4.192 min.
(126 MHz, DMSO-d6)
d 192.56 (s), 165.90 (s), 162.26 (s), 149.83 (s),
146.32 (s), 134.51 (s), 132.42 (s), 130.90 (s), 128.59 (s), 119.08 (s),
116.60 (s), 114.23 (s), 113.69 (s), 40.74 (s). DEPT (135ꢀ)
132.41 (CH),
130.90 (CH), 119.08 (CH), 116.60 (CH), 113.69 (CH), 40.74 (negative
peak, CH2). DEPT (90ꢀ)
132.42 (CH), 130.90 (CH), 119.08 (CH),
d
4.3.13. 2-((5-(3,4-Dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-1-
d
(4-(4-isopropylphenoxy)phenyl)ethan-1-one (14)
116.60 (CH), 113.69 (CH). HRMS (ESIþ) m/z: 328.9518 (M þ Na).
M.p. 170.5e171.9 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 8.05 (d,
HPLC purity ¼ 97.588%, tR ¼ 3.610 min.
J ¼ 8.6 Hz, 2H), 7.32 (s, 1H), 7.31e7.26 (m, 2H), 7.20 (d, J ¼ 8.2, 1H),
7.04 (overlap, 4H), 6.85 (d, J ¼ 8.2 Hz, 1H), 5.04 (s, 2H), 2.91 (m, 1H),
4.3.9. 2-((5-(3,4-Dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-1-
(4-(trifluoromethyl)phenyl)ethan-1-one (10)
1.19 (d, J ¼ 6.9 Hz, 6H). 13C NMR (126 MHz, DMSO-d6)
d 191.60 (s),
165.84 (s), 162.67 (s), 162.42 (s), 153.02 (s), 149.78 (s), 146.30 (s),
145.53 (s), 131.58 (s), 129.97 (s), 128.53 (s), 120.49 (s), 119.07 (s),
117.37 (s), 116.60 (s), 114.28 (s), 113.70 (s), 33.28 (s), 24.37 (s). DEPT
M.p. 237.4e238.3 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.91 (s,
1H), 9.63 (s, 1H), 8.20 (d, J ¼ 8.1 Hz, 2H), 7.91 (d, J ¼ 8.1 Hz, 2H), 7.27
(s, 1H), 7.19 (d, J ¼ 8.2 Hz, 1H), 6.84 (d, J ¼ 8.2 Hz, 1H), 5.09 (s, 2H).
(135ꢀ)
d
131.58 (CH), 128.53 (CH), 120.49 (CH), 119.07 (CH), 117.36
(CH), 116.59 (CH), 113.69 (CH), 40.66 (negative peak, CH2), 33.28
(CH3), 24.37 (CH3). DEPT (90ꢀ)
131.58 (CH), 128.53 (CH), 120.49
13C NMR (126 MHz, DMSO-d6)
d 192.83 (s), 165.94 (s), 162.28 (s),
149.78 (s), 146.24 (s), 138.62 (s), 133.45 (s), 129.71 (s), 126.32 (s),
126.29 (s), 119.16 (s), 116.58 (s), 114.18 (s), 113.66 (s), 40.72 (s). DEPT
d
(CH), 119.07 (CH), 117.36 (CH), 116.60 (CH), 113.69 (CH), 33.28 (CH).
HRMS (ESIþ) m/z: 485.1156 (M þ Na). HPLC purity ¼ 98.905%,
tR ¼ 3.880 min.
(135ꢀ)
d
129.71 (CH), 126.32 (CH), 126.29 (CH), 119.16 (CH), 116.58
(CH), 113.65 (CH), 109.99 (CH), 40.71 (negative peak, CH2). DEPT
(90ꢀ)
129.71 (s), 126.32 (CH), 126.29 (CH), 119.16 (s), 116.58 (s),
d
113.65 (s), 109.99 (CH). HRMS (ESIþ) m/z: 419.0285 (M þ Na). HPLC
4.3.14. 2-((5-(3,4-Dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-1-
purity ¼ 97.648%, tR ¼ 4.525 min.
(4-(4-ethoxyphenoxy)phenyl)ethan-1-one (15)
M.p. 217.3e218.1 ꢀC.1H NMR (500 MHz, DMSO-d6)
d 9.79 (s, 1H),
4.3.10. 1-(4-(Benzyloxy)phenyl)-2-((5-(3,4-dihydroxyphenyl)-
9.51 (s, 1H), 8.03 (d, J ¼ 8.6 Hz, 2H), 7.28 (s, 1H), 7.20 (d, J ¼ 8.2 Hz,
1,3,4-oxadiazol-2-yl)thio)ethan-1-one (11)
1H), 7.06 (d, J ¼ 8.6 Hz, 2H), 7.02e6.94 (m, 4H), 6.85 (d, J ¼ 8.2 Hz,
M.p. 218.7e220.8 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.88 (s,
1H), 5.03 (s, 2H), 4.01 (q, J ¼ 6.9 Hz, 2H), 1.31 (t, J ¼ 6.9 Hz, 3H). 13
C
1H), 9.67 (s, 1H), 7.98 (d, J ¼ 8.6 Hz, 2H), 7.42 (d, J ¼ 7.2 Hz, 2H), 7.36
(t, J ¼ 7.2 Hz, 2H), 7.31 (t, J ¼ 7.1 Hz, 1H), 7.28 (s, 1H), 7.19 (d,
J ¼ 8.2 Hz,1H), 7.12 (d, J ¼ 8.6 Hz, 2H), 6.85 (d, J ¼ 8.2 Hz,1H), 5.17 (s,
NMR (126 MHz, DMSO-d6) d 191.58 (s), 165.83 (s), 163.35 (s), 162.42
(s), 156.20 (s), 149.77 (s), 147.98 (s), 146.29 (s), 131.56 (s), 129.66 (s),
122.17 (s), 119.07 (s), 116.72 (s), 116.59 (s), 116.24 (s), 114.28 (s),
2H), 4.98 (s, 2H). 13C NMR (126 MHz, DMSO-d6)
d 191.45 (s), 165.83
113.69 (s), 63.87 (s), 40.65 (s), 15.11 (s). DEPT (135ꢀ)
d 131.55 (CH),
(s), 163.22 (s), 162.58 (s), 149.75 (s), 146.24 (s), 136.68 (s), 131.34 (s),
128.97 (s), 128.52 (s), 128.38 (s), 128.21 (s), 119.15 (s), 116.59 (s),
115.36 (s), 114.24 (s), 113.66 (s), 70.02 (s), 40.44 (s). DEPT (135ꢀ)
122.17 (CH), 119.07 (CH), 116.72 (CH), 116.59 (CH), 116.24 (CH),
113.69 (CH), 109.99 (CH), 63.87 (negative peak, CH2), 40.65 (nega-
tive peak, CH2), 15.11 (CH3). DEPT (90ꢀ)
d 131.55 (CH), 122.17 (CH),
d
131.34 (CH), 128.96 (CH), 128.52 (CH), 128.21 (CH), 119.15 (CH),
119.07 (CH), 116.72 (CH), 116.59 (CH), 116.24 (CH), 113.69 (CH).
HRMS (ESIþ) m/z: 487.0953 (M þ Na). HPLC purity ¼ 97.334%,
tR ¼ 5.508 min.
116.59 (CH), 115.36 (CH), 113.66 (CH), 70.02 (negative peak, CH2),
40.44 (negative peak, CH2). HRMS (ESIþ) m/z: 457.0834 (M þ Na).
HPLC purity ¼ 96.448%, tR ¼ 3.840 min.
4.3.15. 1-(4-(4-(Benzyloxy)phenoxy)phenyl)-2-((5-(3,4-
4.3.11. 2-((5-(3,4-Dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-1-
dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)thio)ethan-1-one (16)
(4-phenoxyphenyl)ethan-1-one (12)
M.p. 226.6e228.3 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.80 (s,
M.p. 189.6e191.2 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d
9.77 (s, 1H),
1H), 9.52 (s, 1H), 8.03 (d, J ¼ 8.7 Hz, 2H), 7.45 (d, J ¼ 7.4 Hz, 2H), 7.38
(t, J ¼ 7.4 Hz, 2H), 7.32 (t, J ¼ 7.2 Hz, 1H), 7.29 (s, 1H), 7.20 (d, J ¼ 8.2,
1H), 7.08 (overlap, 4H), 6.99 (d, J ¼ 8.7 Hz, 2H), 6.86 (d, J ¼ 8.2 Hz,
1H), 5.09 (s, 2H), 5.03 (s, 2H). 13C NMR (126 MHz, DMSO-d6)
9.53 (s, 1H), 8.06 (d, J ¼ 8.7 Hz, 2H), 7.45 (t, J ¼ 7.8 Hz, 2H), 7.29 (s,
1H), 7.25 (t, J ¼ 7.4 Hz, 1H), 7.20 (d, J ¼ 8.2 Hz, 1H), 7.13 (d, J ¼ 7.8 Hz,
2H), 7.06 (d, J ¼ 8.7 Hz, 2H), 6.85 (d, J ¼ 8.2 Hz, 1H), 5.05 (s, 2H). 13
C
NMR (126 MHz, DMSO-d6)
d
191.65 (s), 165.85 (s), 162.41 (s), 162.35
d 191.58 (s), 165.84 (s), 163.28 (s), 162.42 (s), 156.03 (s), 149.78 (s),
(s), 155.20 (s), 149.79 (s), 146.30 (s), 131.60 (s), 130.84 (s), 130.19 (s),
125.40 (s), 120.57 (s), 119.08 (s), 117.65 (s), 116.60 (s), 114.27 (s),
148.29 (s), 146.30 (s), 137.37 (s), 131.56 (s), 129.70 (s), 128.89 (s),
128.33 (s), 128.20 (s), 122.18 (s), 119.08 (s), 116.78 (s), 116.68 (s),
116.60 (s), 114.29 (s), 113.70 (s), 70.11 (s), 40.66 (s). DEPT (135ꢀ)
113.69 (s), 40.66 (s). DEPT (135ꢀ)
d
131.60 (CH), 130.84 (CH), 125.40
(CH), 120.57 (CH), 119.08 (CH), 117.65 (CH), 116.60 (CH), 113.69 (CH),
109.99 (CH), 40.66 (negative peak, CH2). DEPT (90ꢀ)
131.60 (CH),
d
131.56 (CH), 128.89 (CH), 128.33 (CH), 128.20 (CH), 122.18 (CH),
d
119.08 (CH), 116.78 (CH), 116.68 (CH), 116.60 (CH), 113.70 (CH), 70.11
130.84 (CH), 125.40 (CH), 120.57 (CH), 119.08 (CH), 117.65 (CH),
116.59 (CH), 113.69 (CH), 109.99 (CH). HRMS (ESIþ) m/z: 343.0683
(M þ Na). HPLC purity ¼ 95.787%, tR ¼ 3.302 min.
(negative peak, CH2), 40.66 (negative peak, CH2). DEPT (90ꢀ)
d
131.56 (CH), 128.89 (CH), 128.33 (CH), 128.20 (CH), 122.18 (CH),
119.08 (CH), 116.78 (CH), 116.68 (CH), 116.60 (CH), 113.70 (CH).

356
X.-Q. Li et al. / European Journal of Medicinal Chemistry 136 (2017) 348e359
HRMS (ESIþ) m/z: 549.1107 (M þ Na). HPLC purity ¼ 96.918%,
tR ¼ 6.443 min.
iodine (0.6 mmol), TBHP (300 mL), NaHCO3 (2 mmol). After the
reaction mixture was stirred for 10 h at 70 ꢀC, the reaction mixture
was extracted with EtOAc, washed with diluted HCl followed by
water, dried with Na2SO4. Then solvent was removed under
reduced pressure and purified by silica gel column chromatography
(CH2Cl2/MeOH ¼ 40:1) to afford the desired product.
4.3.16. 1-(4-(4-(Tert-butyl)phenoxy)phenyl)-2-((5-(3,4-
dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)thio)ethan-1-one (17)
M.p. 150.6e151.7 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.79 (s, 1H),
9.51 (s, 1H), 8.05 (d, J ¼ 8.6 Hz, 2H), 7.46 (d, J ¼ 8.6 Hz, 2H), 7.29 (s,
1H), 7.20 (d, J ¼ 8.2 Hz, 1H), 7.04 (overlap, 4H), 6.85 (d, J ¼ 8.2 Hz,
4.4.1. 4-(5-(4-Phenoxyphenyl)oxazol-2-yl)benzene-1,2-diol (19)
1H), 5.04 (s, 2H),1.28 (s, 9H). 13C NMR (126 MHz, DMSO-d6)
d
191.62
M.p. 209.1e210.6 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.54 (s,1H),
(s), 165.84 (s), 162.60 (s), 152.77 (s), 149.78 (s), 147.78 (s), 146.30 (s),
131.59 (s), 130.01 (s), 127.50 (s), 120.10 (s), 119.07 (s), 117.42 (s),
116.60 (s), 114.28 (s), 113.70 (s), 40.66 (s), 34.65 (s), 31.66 (s). DEPT
9.36 (s, 1H), 7.76 (d, J ¼ 8.3 Hz, 2H), 7.62 (s, 1H), 7.48e7.37 (overlap,
3H), 7.36 (d, J ¼ 8.2 Hz, 1H), 7.17 (t, J ¼ 7.3 Hz, 1H), 7.09 (d, J ¼ 8.3 Hz,
2H), 7.06 (d, J ¼ 8.1 Hz, 2H), 6.85 (d, J ¼ 8.2 Hz, 1H). 13C NMR
(135ꢀ)
d
131.59 (CH), 127.50 (CH), 120.10 (CH), 119.07 (CH), 117.42
(CH), 116.60 (CH), 113.69 (CH), 40.66 (negative peak, CH2), 31.66
(CH3). DEPT (90ꢀ)
131.59 (CH), 127.50 (CH), 120.10 (CH), 119.08
(126 MHz, DMSO-d6) d 161.04 (s), 157.13 (s), 156.63 (s), 149.85 (s),
148.59 (s), 146.09 (s), 130.61 (s), 126.11 (s), 124.31 (s), 123.64 (s),
d
123.47 (s), 119.42 (d), 118.57 (d), 116.46 (s), 113.58 (s). DEPT (135ꢀ)
(CH),117.43 (CH),113.70 (CH). HRMS (ESIþ) m/z: 499.1312 (M þ Na).
d
130.61 (CH), 126.11 (CH), 124.31 (CH), 123.64 (CH), 119.45 (CH),
119.38 (CH), 118.45 (CH), 116.46 (CH), 113.57 (CH). DEPT (90ꢀ)
130.61 (CH), 126.10 (CH), 124.31 (CH), 123.64 (CH), 119.45 (CH),
HPLC purity ¼ 95.853%, tR ¼ 3.592 min.
d
4.3.17. 2-((5-(3,4-Dihydroxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-1-
119.38 (CH),118.45 (CH),116.46 (CH),113.57 (CH). HRMS (ESIþ) m/z:
(4-(4-(2-methoxyethyl)phenoxy)phenyl)ethan-1-one (18)
346.1073 (M þ H). HPLC purity ¼ 98.152%, tR ¼ 6.027 min.
M.p.179.8e220.8 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.79 (s,1H),
9.51 (s, 1H), 8.05 (d, J ¼ 8.7 Hz, 2H), 7.30 (overlap, 3H), 7.20 (d,
4.4.2. 4-(5-(4-(p-tolyloxy)phenyl)oxazol-2-yl)benzene-1,2-diol
J ¼ 8.2 Hz, 1H), 7.04 (overlap, 4H), 6.85 (d, J ¼ 8.2 Hz, 1H), 5.04 (s,
(20)
2H), 3.53 (t, J ¼ 6.7 Hz, 2H), 3.23 (s, 3H), 2.81 (t, J ¼ 6.7 Hz, 2H). 13
C
M.p. 205.8e207.0 ꢀC. 1H NMR (600 MHz, DMSO-d6)
d 9.58 (s,
NMR (126 MHz, DMSO-d6)
d
191.61 (s), 165.84 (s), 162.61 (s), 162.42
1H), 9.40 (s, 1H), 7.79e7.73 (m, 2H), 7.62 (s, 1H), 7.44 (s, 1H), 7.38 (d,
J ¼ 8.2 Hz, 1H), 7.23 (d, J ¼ 8.4 Hz, 2H), 7.08e7.05 (overlap, 2H), 6.98
(d, J ¼ 8.4 Hz, 2H), 6.87 (d, J ¼ 8.2 Hz, 1H), 2.30 (s, 3H). 13C NMR
(s), 153.34 (s), 149.78 (s), 146.30 (s), 136.37 (s), 131.58 (s), 131.08 (s),
130.01 (s), 120.44 (s), 119.08 (s), 117.40 (s), 116.60 (s), 114.28 (s),
113.70 (s), 73.06 (s), 58.27 (s), 40.66 (s), 35.04 (s). DEPT (135ꢀ)
(151 MHz, DMSO-d6)
d 157.85 (s), 154.24 (s), 150.04 (s), 148.71 (s),
d
131.58 (CH), 131.08 (CH), 120.44 (CH), 119.08 (CH), 117.40 (CH),
146.22 (s), 133.71 (s), 131.13 (s), 131.11 (s), 126.18 (s), 123.21 (s),
119.77 (s), 119.01 (s), 118.82 (s), 118.57 (s), 116.58 (s), 20.85 (s). DEPT
116.60 (CH), 113.70 (CH), 73.06 (negative peak, CH2), 58.27 (CH3),
40.67 (negative peak, CH2), 35.04 (negative peak, CH2). DEPT (90ꢀ)
(135ꢀ)
d
131.12 (CH), 126.18 (CH), 123.21 (CH), 119.77 (CH), 119.01
(CH), 118.82 (CH), 118.57 (CH), 116.58 (CH), 20.85 (CH3). DEPT (90ꢀ)
131.11 (CH), 126.18 (CH), 123.21 (CH), 119.77 (CH), 119.01 (CH),
d
131.58 (CH), 131.08 (CH), 120.44 (CH), 119.08 (CH), 117.40 (CH),
116.60 (CH),113.69 (CH). HRMS (ESIþ) m/z: 501.1113 (M þ Na). HPLC
d
purity ¼ 95.371%, tR ¼ 4.973 min.
118.82 (CH), 118.57 (CH), 116.58 (CH). HRMS (ESIþ) m/z: 360.1212
(M þ H). HPLC purity ¼ 99.000%, tR ¼ 4.994 min.
4.4. General procedure for the preparation of 19e27
4.4.3. 4-(5-(4-(4-Methoxyphenoxy)phenyl)oxazol-2-yl)benzene-
Copper(II) bromide (0.050 mol) and acetophenone (0.025 mol)
to be brominated (0.03 mol) were placed in a flak fitted with a
reflux condenser. Ethyl acetate (25 mL) and chloroform (25 mL)
were added. The resulting reaction mixture was refluxed with
vigorous stirring to ensure complete exposure of the copper(II)
bromide to the reaction medium until the reaction was complete as
judged by a color change of the solution from green to amber,
disappearance of all black solid, and cessation of hydrogen bromide
evolution. The copper(I) bromide was collected by filtration and
washed well with ethyl acetate. The solvents were removed from
the filtrate under reduced pressure. The resulting product, 2-
bromoacetophenone, was purified by column chromatography on
silica gel with petroleum ether and ethyl acetate (25:1) as the
mobile phase.
A solution of 2-bromoacetophenone (0.0628 mol) and hexa-
methylenetetramine (9.67 g, 0.07 mmol) in CHCl3 (50 mL) was
stirred at room temperature for 2 h. The precipitated solid was
filtered and washed with CHCl3 to obtain quaternary ammonium
compounds, which was used in the next step without further
purification.
1,2-diol (21)
M.p. 236.9e238.1 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.54 (s,
1H), 9.38 (s,1H), 7.71 (d, J ¼ 8.6 Hz, 2H), 7.58 (s,1H), 7.43 (s,1H), 7.36
(d, J ¼ 8.2 Hz, 1H), 7.03 (d, J ¼ 9.0 Hz, 2H), 7.00 (d, J ¼ 8.6 Hz, 2H),
6.97 (d, J ¼ 9.0 Hz, 2H), 6.85 (d, J ¼ 8.2 Hz, 1H), 3.74 (s, 3H). 13C NMR
(126 MHz, DMSO-d6)
d 160.93 (s), 158.48 (s), 156.32 (s), 149.95 (s),
149.33 (s), 148.55 (s), 146.08 (s), 126.01 (s), 123.34 (s), 122.70 (s),
121.40 (s), 118.72 (s), 118.43 (s), 118.13 (s), 116.46 (s), 115.61 (s),
113.56 (s), 55.86 (s). DEPT (135Ί)
(CH), 118.43 (CH), 118.13 (CH), 116.46 (CH), 115.61 (CH), 113.56 (CH),
55.86 (CH3). DEPT (90ꢀ)
126.01 (CH), 123.34 (CH), 121.40 (CH),
118.43 (CH), 118.12 (CH), 116.46 (CH), 115.61 (CH), 113.55 (CH).
HRMS (ESIþ) m/z: 376.1164 (M þ H). HPLC purity ¼ 98.996%,
tR ¼ 5.978 min.
d 126.01 (CH), 123.34 (CH), 121.40
d
4.4.4. 4-(5-(4-(4-(tert-butyl)phenoxy)phenyl)oxazol-2-yl)benzene-
1,2-diol (22)
M.p. 202.4e204.5 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.54 (s,
1H), 9.35 (s, 1H), 7.74 (d, J ¼ 8.4 Hz, 2H), 7.60 (s, 1H), 7.45e7.37
(overlap, 3H), 7.35 (d, J ¼ 8.1 Hz, 1H), 7.07 (d, J ¼ 8.4 Hz, 2H), 6.97 (d,
J ¼ 8.4 Hz, 2H), 6.85 (d, J ¼ 8.1 Hz, 1H), 1.27 (s, 9H). 13C NMR
Concentrated HCl (30 mL) was dropped into the solution of
quaternary ammonium in ethanol (100 mL). The reaction mixture
was stirred at room temperature for 48 h. The white solid was
removed by filtration and the filtrate was concentrated in vacuo.
Recrystallization of the crude product yielded the desired product
2-aminoacetophenone as a white needle-like crystal.
(126 MHz, DMSO-d6)
d 161.00 (s), 157.41 (s), 154.18 (s), 149.89 (s),
148.57 (s), 146.65 (s), 146.08 (s), 127.24 (s), 126.06 (s), 123.54 (s),
123.24 (s), 119.20 (s), 118.95 (s), 118.69 (s), 118.43 (s), 116.45 (s),
113.56 (s), 34.54 (s), 31.69 (s). DEPT (135ꢀ)
d
127.24 (CH), 126.07
(CH), 123.54 (CH), 119.20 (CH), 118.95 (CH), 118.43 (CH), 116.45 (CH),
113.56 (CH), 31.69 (CH3). DEPT (90ꢀ)
127.24 (CH), 126.06 (CH),
123.54 (CH), 119.19 (CH), 118.95 (CH), 118.43 (CH), 116.45 (CH),
To a DMF (10 mL) solution of aldehydes (2 mmol) were suc-
cessively added 2-aminoacetophenone hydrochloride (4 mmol),
d

X.-Q. Li et al. / European Journal of Medicinal Chemistry 136 (2017) 348e359
357
113.56 (CH). HRMS (ESIþ) m/z: 402.1687 (M
purity ¼ 96.681%, tR ¼ 7.809 min.
þ
H). HPLC
113.54 (CH), 63.83 (negative peak, CH2), 15.14 (CH3). DEPT (90ꢀ)
126.01 (CH), 123.35 (CH), 121.38 (CH), 118.41 (CH), 118.13 (CH),
d
116.44 (CH), 116.10 (CH), 113.54 (CH). HRMS (ESIþ) m/z: 390.1329
4.4.5. 4-(5-(4-([1,10-Biphenyl]-4-yloxy)phenyl)oxazol-2-yl)
benzene-1,2-diol (23)
(M þ H). HPLC purity ¼ 99.181%, tR ¼ 6.547 min.
M.p. 263.2e234.2 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d
9.56 (s,
4.4.9. 4-(5-(4-(4-(2-Methoxyethyl)phenoxy)phenyl)oxazol-2-yl)
1H), 9.37 (s, 1H), 7.78 (d, J ¼ 8.6 Hz, 2H), 7.68 (d, J ¼ 8.6 Hz, 2H), 7.63
(overlap, 3H), 7.45 (s, 1H), 7.43 (t, J ¼ 8.3 Hz, 2H), 7.38 (d, J ¼ 8.2 Hz,
1H), 7.32 (t, J ¼ 8.3 Hz,1H), 7.15 (d, J ¼ 8.7 Hz, 2H), 7.12 (d, J ¼ 8.7 Hz,
benzene-1,2-diol (27)
M.p.142.7e144.0 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.53 (s,1H),
9.35 (s, 1H), 7.74 (d, J ¼ 8.7 Hz, 2H), 7.60 (s, 1H), 7.42 (s, 1H), 7.35 (d,
J ¼ 8.2 Hz, 1H), 7.25 (d, J ¼ 8.5 Hz, 2H), 7.06 (d, J ¼ 8.7 Hz, 2H), 6.97
(d, J ¼ 8.5 Hz, 2H), 6.84 (d, J ¼ 8.2 Hz, 1H), 3.52 (d, J ¼ 6.8 Hz, 2H),
3.23 (s, 3H), 2.78 (t, J ¼ 6.8 Hz, 2H). 13C NMR (126 MHz, DMSO-d6)
2H), 6.86 (d, J ¼ 8.2 Hz, 1H). 13C NMR (126 MHz, DMSO-d6)
d 161.09
(s), 156.93 (s), 156.36 (s), 149.84 (s), 148.61 (s), 146.11 (s), 139.83 (s),
136.16 (s), 129.37 (s), 128.81 (s), 127.67 (s), 126.91 (s), 126.14 (s),
123.72 (s), 123.68 (s), 119.71 (s), 119.63 (s), 118.70 (s), 118.48 (s),
d
161.00 (s), 157.45 (s), 154.75 (s), 149.88 (s), 148.57 (s), 146.08 (s),
116.48 (s), 113.60 (s). DEPT (135ꢀ)
d
129.37 (CH), 128.81 (CH), 127.66
(CH), 126.91 (CH), 126.14 (CH), 123.71 (CH), 119.71 (CH), 119.63 (CH),
118.48 (CH), 116.47 (CH), 113.60 (CH). DEPT (90ꢀ)
129.37 (CH),
135.20 (s), 130.86 (s), 126.07 (s), 123.55 (s), 123.24 (s), 119.34 (s),
119.15 (s), 118.68 (s), 118.43 (s), 116.45 (s), 113.56 (s), 73.16 (s), 58.26
d
(s), 35.01 (s). DEPT (135ꢀ)
d 130.86 (CH), 126.06 (CH), 123.55 (CH),
128.81 (CH), 127.67 (CH), 126.91 (CH), 126.14 (CH), 123.72 (CH),
119.71 (CH), 119.63 (CH), 118.48 (CH), 116.47 (CH), 113.60 (CH).
HRMS (ESIþ) m/z: 444.1187 (M þ Na). HPLC purity ¼ 98.458%,
tR ¼ 5.957 min.
119.34 (CH), 119.15 (CH), 118.43 (CH), 116.45 (CH), 113.55 (CH), 73.16
(negative peak, CH2), 58.26 (CH3), 35.00 (negative peak, CH2). DEPT
(90ꢀ)
d 130.86 (CH), 126.07 (CH), 123.55 (CH), 119.34 (CH), 119.15
(CH), 118.43 (CH), 116.45 (CH), 113.56 (CH). HRMS (ESIþ) m/z:
426.1305 (M þ Na). HPLC purity ¼ 99.033%, tR ¼ 3.197 min.
4.4.6. 4-(5-(4-(4-Isopropylphenoxy)phenyl)oxazol-2-yl)benzene-
1,2-diol (24)
4.5. Enzyme inhibition assays
M.p. 196.1e196.8 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.53 (s, 1H),
9.35 (s, 1H), 7.74 (d, J ¼ 8.7 Hz, 2H), 7.60 (s, 1H), 7.42 (s, 1H), 7.35 (d,
J ¼ 8.2 Hz, 1H), 7.26 (d, J ¼ 8.5 Hz, 2H), 7.06 (d, J ¼ 8.7 Hz, 2H), 6.98
(d, J ¼ 8.5 Hz, 2H), 6.84 (d, J ¼ 8.2 Hz, 1H), 2.87 (m, 1H), 1.19 (d,
The PTP1B enzyme reaction was carried out under the condi-
tions described previously [44]. The effect of each compound on the
PTP1B-catalyzed pNPP hydrolysis was determined at 37 ꢀC and pH
J ¼ 6.9 Hz, 6H). 13C NMR (126 MHz, DMSO-d6)
d
160.99 (s), 157.50
7.5 in a 100
contained 1
0.01 M) and 99
m
m
L reaction system in a 96-well plate. Each reaction
L compound in DMSO (final concentration from 100 to
L assay buffer (10 mM Tris-HCl, 25 mM NaCl,
(s), 154.43 (s), 149.89 (s), 148.57 (s), 146.08 (s), 144.42 (s), 128.29 (s),
126.07 (s), 123.53 (s), 123.20 (s), 119.38 (s), 119.13 (s), 118.69 (s),
118.43 (s), 116.45 (s), 113.56 (s), 33.22 (s), 24.43 (s). DEPT (135ꢀ)
m
m
1 mM EDTA and 2 mM DTT) containing 4 mM pNPP and 60 nM
PTP1B. The PTP1B-catalyzed reaction was started by addition of the
PTP1B and terminated by the addition of 1 N NaOH after 30 min.
The amount of product p-nitrophenol was determined from the
absorbance at 405 nm detected by a SpectraMAX Plus 384 micro-
plate pectrophotometer. The IC50 value was obtained by plotting
relative PTP1B activity versus inhibitor concentration and fitting to
equation %Inhibition ¼ Bottom þ (Top-Bottom)/(1 þ 10(lgIC50ꢃX)*h).
d
128.29 (CH), 126.06 (CH), 123.53 (CH), 119.38 (CH), 119.12 (CH),
118.43 (CH), 116.45 (CH), 113.56 (CH), 33.21 (CH), 24.42 (CH3). DEPT
(90ꢀ)
128.29 (CH), 126.07 (CH), 123.53 (CH), 119.38 (CH), 119.13
d
(CH), 118.43 (CH), 116.45 (CH), 113.56 (CH), 33.22 (CH). HRMS (ESIþ)
m/z: 410.1349 (M þ Na). HPLC purity ¼ 97.981%, tR ¼ 7.449 min.
4.4.7. 4-(5-(4-(4-(tert-pentyl)phenoxy)phenyl)oxazol-2-yl)
benzene-1,2-diol (25)
M.p. 218.4e219.6 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d
9.45
4.6. Docking studies
(overlap, 2H), 7.74 (d, J ¼ 8.7 Hz, 2H), 7.60 (s, 1H), 7.42 (s, 1H), 7.35
(overlap, 3H), 7.06 (d, J ¼ 8.6 Hz, 2H), 6.98 (d, J ¼ 8.6 Hz, 2H), 6.84 (d,
J ¼ 8.2 Hz, 1H), 1.58 (t, J ¼ 7.4 Hz, 2H), 1.23 (s, 6H), 0.62 (t, J ¼ 7.4 Hz,
The crystal structure of the PTP1B (PDB ID: 1G1H) was obtained
from the protein bank in the RCSB. The 3D structures of the in-
hibitors were generated using Chembio3D Ultra 11.0 followed by
energy minimization. AutoDock 4.0 program equipped with ADT
was used to perform the automated molecular docking. Grid maps
covering residues in the catalytic site were defined for all inhibitors
in the AutoDock calculations using a grid spacing of 60 Å. The GA-LS
algorithm was adopted using default settings. For each docking job,
200 hybrid GA-LS runs were carried-out. A total of 200 possible
binding conformations were generated and grouped into clusters
based on a 1.0 Å cluster tolerance. The docking models were
analyzed and represented using ADT.
3H). 13C NMR (126 MHz, DMSO-d6)
d 161.00 (s), 157.42 (s), 154.11 (s),
149.89 (s), 148.58 (s), 146.09 (s), 144.91 (s), 127.87 (s), 126.07 (s),
123.54 (s), 123.24 (s), 119.20 (s), 118.91 (s), 118.68 (s), 118.43 (s),
116.45 (s), 113.57 (s), 37.70 (s), 36.69 (s), 28.77 (s), 9.51 (s). DEPT
(135ꢀ)
d
127.87 (CH), 126.07 (CH), 123.54 (CH), 119.20 (CH), 118.90
(CH), 118.43 (CH), 116.45 (CH), 113.56 (CH), 36.68 (CH), 28.76
(negative peak, CH2), 9.51 (CH3). DEPT (90ꢀ)
127.87 (CH), 126.07
d
(CH), 123.54 (CH), 119.20 (CH), 118.90 (CH), 118.43 (CH), 116.45 (CH),
113.56 (CH), 40.23 (CH), 36.69 (CH). HRMS (ESIþ) m/z: 416.1850
(M þ H). HPLC purity ¼ 99.160%, tR ¼ 9.472 min.
4.4.8. 4-(5-(4-(4-Ethoxyphenoxy)phenyl)oxazol-2-yl)benzene-1,2-
4.7. Cell culture and differentiation
diol (26)
M.p. 225.9e227.1 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d
9.53 (s,1H),
A mouse muscle myoblasts cell line (C2C12) was obtained from
American Type Culture Collection (Rockville, MD) and was cultured
in DMEM supplemented with 10% fetal calf serum and 1%
penicillin-streptomycin and incubated in an atmosphere of 95% O2
and 5% CO2 in air. After confluence, the culture medium was
replaced with DMEM containing 10% horse serum to initiate
myogenic differentiation [45]. After differentiation, myotubes were
subjected to various treatment described below.
9.35 (s, 1H), 7.71 (d, J ¼ 8.8 Hz, 2H), 7.58 (s, 1H), 7.41 (s, 1H), 7.35 (d,
J ¼ 8.2 Hz, 1H), 7.06e6.97 (overlap, 4H), 6.95 (d, J ¼ 8.2 Hz, 2H), 6.84
(d, J ¼ 8.2 Hz, 1H), 4.00 (q, J ¼ 7.0 Hz, 2H), 1.31 (t, J ¼ 7.0 Hz, 3H). 13
C
NMR (126 MHz, DMSO-d6) d 160.92 (s), 158.48 (s), 155.61 (s), 149.94
(s), 149.22 (s), 148.54 (s), 146.07 (s), 126.01 (s), 123.35 (s), 122.69 (s),
121.38 (s), 118.70 (s), 118.41 (s), 118.14 (s), 116.45 (s), 116.10 (s),
113.54 (s), 63.83 (s), 15.14 (s). DEPT (135ꢀ)
d
126.01 (CH), 123.35
(CH), 121.38 (CH), 118.41 (CH), 118.13 (CH), 116.45 (CH), 116.10 (CH),
C2C12 myotubes were starved in serum-free DMEM for 24 h,

358
X.-Q. Li et al. / European Journal of Medicinal Chemistry 136 (2017) 348e359
after exchanged with fresh serum-freemedium, then they were
exposed to each concentration of compound 22. Insulin resistance
was induced in C2C12 myotubes by treating them with palmitic
acid (PA) (0.75 mM) for 16 h. PA was prepared by conjugating it
with bovine serum albumin as previously reported and bovine
serum albumin was included in the cell culture medium for every
condition tested as a control. PA were added when C2C12 myotubes
had been pre-incubated with compound 22 for 1 h.
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Acknowledgment
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2016ZDJS07A13,
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Resources Service Network Programme of Chinese Academy of
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.05.007.
ABBREVIATIONS
PTP1B Protein tyrosine phosphatase 1B
TCPTP
TBHP
pNPP
T-cell protein tyrosine phosphatase
tert-Butyl hydroperoxide
disodium 4-nitrophenyl phosphate
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