Discovery of 5-(3-bromo-2-(2,3-dibromo-4,5-dimethoxybenzyl)-4,5-dimethoxybenzylidene)thiazolidine-2,4-dione as a novel potent protein tyrosine phosphatase 1B inhibitor with antidiabetic properties

Article abstract of DOI:10.1016/j.bioorg.2021.104648

Protein tyrosine phosphatase 1B (PTP1B) is a well-validated target in therapeutic interventions for type 2 diabetes mellitus (T2DM), however, PTP1B inhibitors containing negatively charged nonhydrolyzable pTyr mimetics are difficult to convert to the corr

Full text of DOI:10.1016/j.bioorg.2021.104648

Bioorganic Chemistry 108 (2021) 104648
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of 5-(3-bromo-2-(2,3-dibromo-4,5-dimethoxybenzyl)-4,5-
dimethoxybenzylidene)thiazolidine-2,4-dione as a novel potent protein
tyrosine phosphatase 1B inhibitor with antidiabetic properties
,
b
,
,
,
,b,*
Bo Jianga 1, Jiao Luoa 1, Shuju Guoa b, Lijun Wanga
a Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
b Center for Ocean Mega-Science, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Protein tyrosine phosphatase 1B (PTP1B) is a well-validated target in therapeutic interventions for type 2 dia-
betes mellitus (T2DM), however, PTP1B inhibitors containing negatively charged nonhydrolyzable pTyr mi-
metics are difficult to convert to the corresponding in vivo efficacy owing to poor cell permeability and oral
bioavailability. In this work, molecules bearing less acidic heterocycle 2,4-thiazolidinedione and hydantoin were
designed, synthesized and evaluated for PTP1B inhibitory potency, selectivity and in vivo antidiabetic efficacy.
Type 2 diabetes mellitus
Protein tyrosine phosphatase 1B inhibitors
Thiazolidinedione
Insulin signaling
Anti-diabetic activities
Among them, compound 5a was identified as a potent PTP1B inhibitor (IC50 = 0.86 μM) with 5-fold selectivity
over the highly homologous TCPTP. Long-term oral administration of 5a at a dose of 50 mg/kg not only
significantly reduced blood glucose levels, triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C)
levels but also ameliorated insulin sensitivity in diabetic BKS db mice. Moreover, 5a enhanced the insulin-
stimulated phosphorylation of IRβ, IRS-1 and Akt in C2C12 myotubes. A histopathological evaluation of liver
and pancreas demonstrated that 5a increased liver glycogen storage and improved islet architecture with more
β-cells and fewer
α-cells in diabetic mice. Thus, our work demonstrated that compound 5a could serve as a lead
compound for the discovery of new antidiabetic drugs.
1. Introduction
crucial roles in regulating the intracellular phosphorylation of proteins.
A series of biochemical, cellular and knockout mouse studies have
demonstrated that PTP1B is a key negative regulator of both insulin and
leptin signaling pathways [3–6]. Thus, PTP1B has become an attractive
therapeutic target for T2DM treatment. Additionally, accumulating ev-
idence suggests that PTP1B also plays an increasingly important role in
other human diseases such as breast cancer [7], hepatocellular carci-
noma [8] and nonalcoholic fatty liver disease [9].
Type 2 diabetes mellitus (T2DM) is a complex endocrine and meta-
bolic disease, which is characterized by insulin resistance and progres-
sive β-cell dysfunction. Owing to the sedentary lifestyle and obesity in
the population, T2DM is now a major public health problem worldwide.
According to the latest Diabetes Atlas (9th edition 2019) published by
International Diabetes Federation (IDF), 463 million people (20–79
years) had diabetes in 2019, and this number is estimated to exceed 700
million globally by 2045 [1]. In China, the problem is particularly
overwhelming, with 116 million adults considered to have T2DM in
2019, which ranked first in the world. These staggering data document
the increasing incidence of T2DM and strongly indicate the medical
need for new and better agents to treat this disease.
In light of convincing evidence that PTP1B is related to many human
diseases, the development of small-molecule PTP1B inhibitors has
attracted considerable attention in both fundamental research and
pharmaceutical industries. To date, only two small-molecule PTP1B
inhibitors, ertiprotafib [10] and trodusquemine [11] (Fig. 1), have
reached clinical trials, but the studies were discontinued due to poor
clinical efficacy and undesirable side effects. A first-in-class drug has yet
to be launched; nevertheless, extensive research is underway to develop
a potential drug candidate. Over the past decades, various compounds
Protein tyrosine phosphatase 1B (PTP1B) was the first mammalian
protein tyrosine phosphatase (PTP) to be isolated and characterized
from human placenta [2]. The significance of PTP1B is that it plays
* Corresponding author at: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
E-mail address: wanglijun@qdio.ac.cn (L. Wang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104648
Received 15 June 2020; Received in revised form 15 November 2020; Accepted 6 January 2021
Available online 12 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

B. Jiang et al.
Bioorganic Chemistry 108 (2021) 104648
Fig. 1. Representative examples of PTP1B inhibitors I-VIII and bromophenol compounds IX-XI isolated from red alga.
containing negatively charged non-hydrolyzable pTyr mimetics have
been reported to exhibit excellent potency (at nanomolar range) in vitro
[12,13]; however, the low cell permeability and bioavailability of these
compounds have limited their application for the development of
effective drugs. Therefore, less acidic molecules may provide alterna-
tives in developing less potent PTP1B inhibitors with improved cell
permeability and bioavailability.
Compounds bearing a 2,4-thiazolidinedione (TZD) moiety were
shown to exhibit various biological effects, including anti-inflammatory
[22], antidiabetic [23], antibacterial [24] and anti-Alzheimer’s disease
effects [25]. In particular, TZDs, known as high-affinity PPARγ agonists,
were introduced in the late 1990s as the first agents for the treatment of
type 2 diabetes [26]. Additionally, hydantoin, the bioisostere of TZD,
represent a kind of new and less acidic pTyr mimetic that can be used for
the development of potent PTP1B inhibitors. Our group has long been
engaged in isolation of bromophenol compounds from marine algae
[27]. Fig. 1 shows partial structures of mono-benzyl and di-benzyl
bromophenols and some of them exhibited potent PTP1B inhibition
(compounds IX-XI) [28]. Considering these findings, TZDs and hydan-
toin were introduced on the bromophenols scaffold to obtain potent
PTP1B inhibitors lacking negatively charged pTyr mimetic. The antidi-
abetic activities both in vitro and in vivo as PTP1B inhibitors along with
mechanistic studies were further investigated.
Recent studies have reported that small molecules with good enzyme
inhibition and devoid of negatively charged groups (compounds I-III, as
shown in Fig. 1) are highly desirable for the discovery of PTP1B in-
hibitors [14–16]. In addition, natural products are abundant sources of
bioactive molecules. Intriguingly, several natural products have been
found to have PTP1B inhibitory activity through their unique scaffolds,
instead of relying on the functionalities of mimicking pTyr. Fig. 1 dis-
plays some representative PTP1B inhibitors (compounds IV-VIII) iso-
lated from natural plants with IC50 values of moderate to good potency
[17–21].
2

B. Jiang et al.
Bioorganic Chemistry 108 (2021) 104648
2. Chemistry
derivatives 2a-e [29]. Furthermore, the Z-configuration has been
considered thermodynamically more stable than the E-configuration on
the basis of literature data for similar compounds [30,31].
The TZD derivatives 1a-g and hydantoin derivatives 2a-e were
synthesized according to the general procedures depicted in Schemes 1
and 2. Knoevenagel condensation of appropriate benzaldehydes with
TZD or hydantoin in the presence of ammonium acetate generated the
corresponding arylidenes 1a-g and 2a-e in good yields. The compounds
5a-b and 8a-b were synthesized as described inFig. Scheme 3. The key
intermediates 3 and 6 were obtained via well documented procedures
[28]. Oxidation of benzyl alcohol (3) using pyridinium chlorochromate
(PCC) as an oxidant afforded aryl aldehyde 4 in good yield. Alkylation of
4-hydroxy benzaldehyde with benzyl bromide (6) in the presence of
potassium carbonate in acetone gave the corresponding aryl aldehyde 7.
Similarly, the Knoevenagel reaction of 4 or 7 with TZD in acetic acid as
the solvent afforded the target compounds 5a and 8a, respectively.
Finally, the olefinic bond of the 5-arylidene-2,4-thiazolidinediones 5a
and 8a was reduced with hydrogen in methanol using 10% palladium on
carbon as the catalyst to give compounds 5b and 8b, respectively.
The 1H NMR spectra of the 5-arylidene-2,4-thiazolidinediones 1a-g,
5a and 8a showed a singlet for exocyclic olefinic protons at 7.63–7.86
ppm in DMSO‑d6 solution, suggesting that these compounds were
assigned to the Z-configuration. In the case of Knoevenagel condensation
between N1-unsubstituted hydantoin and the corresponding benzalde-
hydes, the Z-configuration was also preferable for the hydantoin
3. Results and discussion
3.1. PTP1B inhibitory activities and structure–activity relationship
The in vitro inhibitory activities of compounds 1a-g, 2a-e, 5a-b and
8a-b were tested against recombinant human PTP1B. First, we measured
the percentage inhibitory rates of all the synthetic compounds at 20 μg/
mL. Compounds with inhibition rates >50% were selected for further
determination of IC50 values. As summarized in Table 1, all the hydan-
toin derivatives exhibited low inhibitory rates, indicating that the
hydantoin was not tolerated. The TZD compounds bearing a mono-aryl
ring offered slightly better activity than the hydantoin ones. Two of
them (1d and 1e) displayed moderate potency (8.34 ± 1.75 and 10.51 ±
2.55 μM, respectively). Although the mono-aryl ring compounds did not
offer acceptable efficacy, it was encouraging to note that the diaryl-
methane scaffold with substitutions of bromines was crucial for better
inhibitory activity against PTP1B (Table 2). Comparing with those of
previously reported diaryl-methane derivatives XII-XIV, compounds 5a-
b and 8a-b showed improved potency with an IC50 range from moderate
to potent (0.86–12.77 μM), thus confirming the favorable role of the
diaryl-methane bearing TZD moiety in enhancing inhibition against the
target enzyme. Furthermore, the number of bromine substitution on the
aryl ring had a certain influence on the inhibitory activity. Especially the
tri-brominated compounds (5a-b and 8a-b) and di-brominated com-
pounds (1d) displayed more significant PTP1B inhibitory activity than
mono-brominated or non-brominated compounds. It is also found that
the reduction of the olefinic bond of 5a was less sensitive to modifica-
tions. On the contrary, the reduction of 8a led to 8b with improved
potency (12.77 ± 6.08 and 2.10 ± 0.23
compound 5a was the most active, with a submicromolar IC50 of 0.86 ±
0.29 M.
μM, respectively). Among them,
μ
3.2. Selectivity, kinetics and docking studies of compound 5a
Although PTP1B has been identified as a validated target for T2DM,
the development of a selective PTP1B inhibitor remains a challenging
task because the closest homologue of PTP1B, TCPTP, shares 74%
sequence identity in the catalytic domain. Therefore, the selectivity
issue for PTP1B is critically important [32]. In addition to good PTP1B
inhibition, compound 5a was selected for testing against a panel of PTPs,
including TCPTP, SHP-1, SHP-2 and LAR. As shown in Fig. 2A, com-
pound 5a showed 5-fold selectivity against TCPTP. Furthermore, 5a
demonstrated excellent selectivity against SHP-1, SHP-2 and LAR (>20-
fold).
Scheme 1. Synthesis of TZD derivatives 1a-g. Reagents and conditions: (i) 3
equiv NH4OAc, AcOH, reflux, 24 h.
Kinetics analysis was conducted to elucidate the mode of inhibition
of compound 5a. A series of concentrations of 5a (1.0, 2.0, 4.0, 8.0 μM)
and the substrate pNPP (0.5, 1.0, 2.0, 4.0 and 8 mM) were used in this
assay. Fig. 2B shows the Lineweaver-Burk plots for inhibition. Various
concentration lines of compound 5a intersected in the second quadrant,
indicating that 5a inhibited PTP1B in a mixed manner. When the in-
hibitor concentration was increased, the increased Km values and
reduced Vmax values confirmed 5a as a mixed inhibition of PTP1B
(Fig. 2C and D).
Molecular docking was conducted to investigate the binding in-
teractions of compound 5a with the PTP1B active sites (PDB code
1G1H). As shown in Fig. 2E and F, the aryl ring bearing TZD moiety
oriented itself to the pocket, which was surrounded by the WPD loop,
the Q loop, and the pTyr loop. The oxygen atom of thiazolidinedione
formed two hydrogen bonds with Arg 221. Another single hydrogen
bond interaction was observed between the oxygen atom on the
methoxy group of 5a and Lys 120. The phenyl ring was sandwiched
between the aromatic side chains of Tyr 46 and Phe 182, which provided
Scheme 2. Synthesis of hydantoin derivatives 2a-e. Reagents and conditions:
(i) 3 equiv NH4OAc, AcOH, reflux, 24 h.
3

B. Jiang et al.
Bioorganic Chemistry 108 (2021) 104648
Scheme 3. Synthesis of TZD derivatives 5a-b and 8a-b. Reagents and conditions: (i) 2 equiv pyridinium chlorochromate, dry CH2Cl2, room temperature, 2 h; (ii) 1.2
equiv TZD, 3 equiv NH4OAc, AcOH, reflux, 24 h; (iii) H2, 10% Pd/C, MeOH, 50 ◦C, 5 h; (iv) 1.2 equiv 4-ydroxybenzaldehyde, K2CO3, acetone, room tempera-
ture, overnight.
a
π
–
π
stacking interaction. The second aryl ring of 5a extended out of the
could affect insulin signaling, we treated C2C12 skeletal muscle cells
pocket.
with a series of concentrations of compound 5a (ranging from 0.01 to 10
μ
M) with/without insulin stimulation. Notably, compound 5a treatment
increased the insulin-stimulated phosphorylation of IRβ Y1158 and
IRS1Y608 (Fig. 3D-E). Moreover, the phosphorylation level of Akt on
Ser473 also increased after compound 5a treatment. Moreover, the
insulin-sensitizing effect of compound 5a was dose-depend at concen-
3.3. Cellular activities of compound 5a
Firstly, the cell permeability of compound 5a was analyzed by HPLC
in C2C12 myotubes. The result showed that the proportion of 5a in the
cell lysate was about 61.43% (Fig. 3A–C), indicating that 5a successfully
crossed the plasma membrane and accumulated intracellularly.
We next examined the cytotoxicity on C2C12 myotubes treated with
various concentrations of compound 5a, and cell viability was measured
trations between 0.1 and 10 μM. Furthermore, the expression level of
PTP1B protein was also examined by immunoblotting, and the results
showed that it was not changed by intervention of compound 5a. These
results demonstrate that compound 5a, a potent PTP1B inhibitor, en-
hances insulin-stimulated signaling without affecting PTP1B protein
levels.
by CCK8 assay. As shown inFig. 3D, up to 25
pound 5a for 24 h did not reduce the survival of C2C12 cells. When
concentration reached to 100 M, the cell viability lose 50%. Accord-
ingly, further in vitro studies on the insulin signaling activation of 5a
μM treatment with com-
μ
3.4. In vivo anti-diabetic activity assays
were conducted in the range of 0.01–10 μM.
The phosphorylation cascade of IRβ, IRS1, and Akt is an indicator of
insulin pathway activation [33]. To determine whether compound 5a
3.4.1. Hypoglycemic effect of compound 5a
The in vivo efficacy of many PTP1B inhibitors is inconsistent with
4

B. Jiang et al.
Bioorganic Chemistry 108 (2021) 104648
effect of compound 5a on glucose tolerance in diabetic mice. As shown
in Fig. 4D, 2 h after of glucose administration, the blood glucose levels of
compound 5a-treated diabetic mice were significantly lower than the
vehicle-treated diabetic mice, suggesting that compound 5a adminis-
tration improved glucose tolerance in diabetic mice.
Table 1
In vitro PTP1B inhibitory activities of compounds 1a-g and 2a-e.
Compds
Inhibition (%) at 20
μg/mL
IC50
(
μ
M)a
1a
13.34
47.51
39.87
95.95
98.82
14.94
35.22
12.08
19.78
28.67
32.20
31.38
NDb
ND
ND
1b
1c
1d
8.34 ± 1.75
3.4.2. Effects of compound 5a on dyslipidemia
1e
10.51 ± 2.55
T2DM is usually accompanied by dyslipidemia, which is character-
ized by elevated total cholesterol (TC) and triglyceride (TG) and reduced
high-density lipoprotein-cholesterol (HDL-C) levels [34]. Thus, we
further examined whether compound 5a could improve serum lipid
dysregulation in diabetic mice. As shown in Fig. 5A and C, compound 5a
significantly reduced TG and LDL-C levels in diabetic BKS db mice
compared with the vehicle treatment group. Compound 5a also effec-
tively suppressed circulating free fatty acid (FFA) levels (Fig. 5E).
However, it had no effect on blood TC and HDL-C levels (Fig. 5B and D).
These results suggested that compound 5a could ameliorate dyslipide-
mia in diabetic mice.
1f
ND
1g
ND
2a
ND
2b
ND
2c
ND
2d
ND
2e
ND
Oleanolic acid
2.04 ± 0.11
a
Values are expressed as means ± standard deviations. All tests were per-
formed in triplicate.
b
ND = not determined, inhibition rate < 50% at 20
μg/mL was not
determined.
3.4.3. Effects of compound 5a on water intake, food intake and body
weight
Table 2
Polyphagia, polyuria and obesity are typical characteristics of T2DM
patients. Next, we tested the effects of compound 5a on water intake,
food intake, and body weight in BKS db mice. At baseline, there was no
difference in water intake, food intake, or body weight among all dia-
betic mice. Compound 5a treatment significantly reduced water intake
in diabetic mice during the intervention compared with vehicle treat-
ment (Fig. 6A). However, both compound 5a and metformin had no
effect on food intake and body weight of diabetic mice (Fig. 6B and C). In
contrast, compound 5a effectively blocked the increased ratio of
abdominal fat (Fig. 6D).
In vitro PTP1B inhibitory activities of compounds 5a-b and 8a-b, in comparison
with previous analogues XII-XIV.
Compds
R1
R2
Inhibition (%) at 20
mL
μg/
IC50
(μ
M)a
5a
CH3
95.72
0.86 ± 0.29
1.84 ± 0.40
3.4.4. Oral administration of compound 5a increased Akt phosphorylation
in skeletal muscle tissues of diabetic mice
5b
8a
CH3
CH3
67.90
93.39
Given that phosphorylation of Akt Ser473 is a key event in glucose
metabolism [35], Akt phosphorylation of skeletal muscle tissues treated
with/without compound 5a was detected. Western blotting results
showed that diabetic mice treated with compound 5a exhibited
increased phosphorylation levels of Akt at serine 473 (Fig. 7).
12.77 ±
6.08
8b
CH3
94.27
2.10 ± 0.23
XII
CH3
CH3
H
CH3
87.52
74.86
86.15
96.25
NDb,c
NDb,c
2.00c
1.50c
3.4.5. Histological analysis of the liver
XIII
XIII
XIV
CH2OH
Impaired glycogen storage is observed in T2DM patients [36]. To
investigate the effect of compound 5a on diabetic mice liver, we per-
formed H&E and PAS staining of liver sections. As shown in Fig. 8, the
liver of vehicle-treated diabetic mice exhibited markedly vacuolated and
swollen hepatocytes, and these vacuolated hepatocytes were signifi-
cantly reduced in the liver of compound 5a-treated mice. PAS staining
was used to detect hepatic glycogen, and severe glycogen depletion was
found in diabetic mice compared with normal BKS mice. However, this
depletion could be reversed by compound 5a treatment, suggesting that
compound 5a promoted liver glycogen storage in diabetic mice.
CH2O(CH2)2CH3
CH2OCH2CH
(CH3)2
H
a
Values are expressed as means ± standard deviations. All tests were per-
formed in triplicate.
b
ND = not determined, inhibition rate <50% at 5 μg/mL was not determined.
c
Ref. [28].
their observed in vitro activities. Thus, we used a spontaneous diabetic
mouse model, BKS db mice, to evaluate the antidiabetic effects of
compound 5a. The first-line T2DM drug, metformin, was used as the
positive control. As shown in Fig. 4A, compound 5a showed a marked
trend towards a decrease in blood glucose levels compared with the
vehicle-treated diabetic mice. 5a significantly lowered the blood glucose
levels of BKS db mice from the 3rd week (p < 0.01) and lasted for the 5th
week. Moreover, the plasma glycosylated serum protein (GSP) levels
were significantly reduced by compound 5a (p < 0.01) (Fig. 4B).
We next examined the effect of compound 5a on insulin sensitivity
using the ITT assay in diabetic BKS db mice. As shown in Fig. 4C,
compound 5a notably suppressed blood glucose levels in diabetic mice
after insulin injection for 30 min (p < 0.05), 60 min (p < 0.01), and 120
min (p < 0.001). These results indicated that insulin sensitivity had been
ameliorated in diabetic mice treated with compound 5a.
3.4.6. Histological analysis of the pancreas
To investigate the beneficial effect of compound 5a on the pancreas,
a series of histological staining analyses were performed. Glucagon-
positive staining showed pancreatic α-cells distributed on the edge of
islets in normal control mice, while islets in diabetic mice contained
more -cells, which were scattered throughout the entire islet (Fig. 9).
Compound 5a could reduce the number of -cells in islets in diabetic
α
α
mice. Insulin-positive cells were markedly decreased compared with
normal BKS mice, indicating a reduced number of β-cells in diabetic
mice. Compound 5a increased the number of insulin-positive cells,
suggesting the presence of more β-cells in the islets of compound 5a-
treated mice. The same results were observed for glucagon-positive and
insulin-positive double-staining.
Furthermore, a simplified OGTT experiment was used to evaluate the
5

B. Jiang et al.
Bioorganic Chemistry 108 (2021) 104648
Fig. 2. Selectivity, kinetic and docking studies of compound 5a. (A) Selectivity of compound 5a against other PTPs. (B) The Lineweaver-Burk plot for inhibition of
PTP1B enzyme reaction by compound 5a. Concentrations of 5a were 1 (×), 2 (▴), 4 (◆) and 8 μM (■), respectively. (C) The plot of apparent Km versus the
concentrations of compound 5a. (D) The plot of Vmax versus the concentrations of compound 5a. (E) Surface representation of PTP1B in Complex with compound 5a.
(F) Binding mode of compound 5a in the active site of PTP1B. Carbon is in green for compound 5a, oxygen is in red, nitrogen is in blue, sulfur is in yellow. Hydrogen
bonds were considered within 2.5 Å and represented as yellow dots. (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
4. Conclusions
gel GF254 precoated plates and visualized with a UV lamp (254 nm) or
95% FeCl3-EtOH solution. Column chromatography was carried out
using silica gel (200–300 mesh). Melting points (mp) were determined
using a Boetius electrothermal capillary melting point apparatus and
were uncorrected. 1H NMR and 13C NMR spectra were recorded on an
Inova (500 MHz) NMR spectrometer for protons and 125 MHz for car-
bon. Deuterated dimethyl sulfoxide (DMSO) was used as a solvent.
Chemical shifts are reported in ppm (δ) relative to tetramethylsilane
(TMS) as an internal standard. Multiplicities are presented as s (singlet),
d (doublet), m (multiplet) and br s (broad signal). Coupling constants (J
values) are given in hertz (Hz). The acidic protons of the amidic –NH in
the thiazolidindione ring and phenolic hydroxyl were not frequently
observed in 1H NMR spectra. High-resolution mass spectral (HRMS) data
were obtained using a Thermo LTQ-Orbitrap mass spectrometer.
In summary, we have prepared a series of TZD and hydantoin de-
rivatives to search for selective PTP1B inhibitors with good cell
permeability and oral bioavailability. The preliminary structure–activity
relationship indicated that the TZD bearing the diaryl-methane scaffold
with substitutions of bromines had favorable PTP1B inhibitory activity.
Among these derivatives, compound 5a exhibited potent inhibitory ac-
tivity against PTP1B and high selectivity against other PTPs. More
importantly, compound 5a effectively lowered glucose, TG and LDL-C
levels in a db/db mouse model at 50 mg/kg. The novel 5-benzylidene-
thiazolidine-2,4-dione reported in this study could provide a possible
lead compound for the development of selective PTP1B inhibitors with
improved pharmacological properties.
5. Experimental
5.1.2. General procedure for the synthesis of TZD derivatives 1a-g
To a stirred suspension of the appropriate benzaldehyde (1 equiv) in
AcOH (20 mL) was sequentially added 2,4-thiazolidinedione (1.2 equiv)
and NH4OAc (3 equiv). After refluxing for 24 h, the reaction mixture was
cooled and then poured into H2O. In the event that a precipitate was
formed, the suspension was filtered, and the residue was washed with
cold water. The washed residue was dried to obtain the desired de-
rivatives of 5-benzylidenethiazolidine-2,4-dione as yellow residues.
5.1. Chemistry
5.1.1. General
All the reagents and solvents were purchased from commercial
sources and used without further purification. Reactions were moni-
tored by analytical thin layer chromatography (TLC) performed on silica
6

B. Jiang et al.
Bioorganic Chemistry 108 (2021) 104648
Fig. 3. Compound 5a increases insulin signaling activity after insulin stimulation. (A-C) Cell permeability of 5a in C2C12 myotubes. (A) HPLC analysis of DMSO-
treated group (control). (B) HPLC analysis of 5a-treated group. C2C12 myotubes were treated with 5a (10 M) for 8 h in a 10 cm cell plate. (C) Uptake ratio of 5a in
μ
C2C12 myotubes. (D) Cytotoxicity of 5a to C2C12 myotubes. Cells were incubated with 5a for 24 h, and cells viability was determined by CCK8 method. The data was
expressed as the mean ± SEM (n = 6), ***p < 0.001 versus control group. (E) Enhanced insulin signaling in C2C12 myotubes treated with compound 5a. Serum
starved C2C12 myotubes were treated with compound 5a for 8 h and then exposed with/without 100 nM insulin for 5 min. Cells was then lysed and immunoblotting
was performed to detect IRS1/IRβ/Akt phosphorylation and PTP1B protein. (F) Relative ratio of pIRS-1/IRS-1, pIRβ/IRβ and pAkt/Akt. Data are expressed as mean ±
SEM (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001, as indicated.
However, in the absence of precipitation, the mixture was extracted with
EtOAc (2x), washed successively with saturated NaHCO3 solution and
brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The
residue was purified by silica gel column chromatography to afford the
target compounds as a yellowish solid.
DMSO‑d6) δ: 3.82 (s, 3H), 6.93 (d, J = 8.25 Hz, 1H), 7.07 (d, J = 8.25 Hz,
1H), 7.16 (s, 1H), 7.71 (s, 1H), 9.93 (br s, 1H), 12.56 (br s, 1H); 13C NMR
(125 MHz, DMSO‑d6) δ: 55.63, 114.21, 116.13, 119.22, 124.41, 132.56,
147.90, 149.45, 167.38, 167.96; HRESIMS: calc for C11H8NO4S
[Mꢀ H]ꢀ 250.0180, found 250.0178.
5.1.2.1. (Z)-5-(4-hydroxy-3-methoxybenzylidene)thiazolidine-2,4-dione
5.1.2.2. (Z)-5-(3-bromo-4-hydroxy-5-methoxybenzylidene)thiazolidine-
(1a). Yellow solid, Yield 70%, mp: 230–232 ◦C, 1H NMR (500 MHz,
2,4-dione (1b). Yellow solid, Yield 73%, mp: 251–253 ◦C, 1H NMR (500
7

B. Jiang et al.
Bioorganic Chemistry 108 (2021) 104648
Fig. 4. Hypoglycemic effects of compound 5a in diabetic BKS db mice. Mice were treated with vehicle, compound 5a (50 mg/kg/day), or metformin (100 mg/kg/
day) by oral gavage. (A) Blood glucose levels of diabetic mice treated with vehicle, compound 5a, or metformin and of normal BKS mice treated with vehicle after 6 h
fasting. (B) GSP levels of all the groups of mice. (C) Effects of compound 5a on insulin resistance in diabetic mice. (D) Blood glucose levels in vehicle- and compound
5a-treatment group 2 h after glucose administration. Data were shown as means ± SEM (n = 6–8). *p < 0.05, **p < 0.01, and ***p < 0.001, compound 5a-treatment
group versus vehicle-treated diabetic mice, or as indicated.
MHz, DMSO‑d6) δ: 3.88 (s, 3H), 7.17 (s, 1H), 7.35 (s, 1H), 7.69 (s, 1H),
10.42 (br s, 1H), 12.61 (br s, 1H); 13C NMR (125 MHz, DMSO‑d6) δ: 56.3,
109.8, 112.4, 121.2, 125.3, 126.8, 131.1, 146.2, 148.5, 167.2, 167.6;
HRESIMS: calc for C11H7BrNO4S [Mꢀ H]ꢀ 327.9285, found 327.9282.
5.1.2.6. (Z)-5-(2-bromo-3,4-dimethoxybenzylidene)thiazolidine-2,4-dione
(1f). Yellow solid, Yield 91%, mp:198–200 ◦C, 1H NMR (500 MHz,
DMSO‑d6) δ: 3.75 (s, 3H), 3.89 (s, 3H), 7.24 (d, J = 7.5 Hz, 1H), 7.31 (d,
J = 7.5 Hz, 1H), 7.79 (s, 1H); 13C NMR (125 MHz, DMSO‑d6) δ: 56.56,
60.43, 112.91, 121.52, 125.15, 125.45, 126.52, 127.68, 146.93, 154.96,
169.64, 171.21; HRESIMS: calc for C12H9BrNO4S [Mꢀ H]ꢀ 341.9441,
found 341.9445.
5.1.2.3. (Z)-5-(3-bromo-4,5-dimethoxybenzylidene)thiazolidine-2,4-dione
(1c). Yellow solid, Yield 70%, mp: 216–218 ◦C, 1H NMR (500 MHz,
DMSO‑d6) δ: 3.79 (s, 3H), 3.88 (s, 3H), 7.26 (s, 1H), 7.39 (s, 1H), 7.73 (s,
1H), 12.66 (br s, 1H); 13C NMR (125 MHz, DMSO‑d6) δ: 56.81, 60.88,
114.33, 117.84, 124.61, 126.37, 130.94, 131.08, 147.43, 153.95,
167.69, 168.09; HRESIMS: calc for C12H9BrNO4S [Mꢀ H]ꢀ 341.9441,
found 341.9440.
5.1.2.7. (Z)-5-(2,3-dibromo-4-hydroxy-5-methoxybenzylidene)thiazoli-
dine-2,4-dione (1g). Yellow solid, Yield 72%, mp:198–200 ◦C, 1H NMR
(500 MHz, DMSO‑d6) δ: 3.88 (s, 3H), 7.04 (s, 1H), 7.86 (s, 1H), 10.81 (br
s, 1H), 12.70 (br s, 1H); 13C NMR (125 MHz, DMSO‑d6) δ: 56.95, 110.76,
114.85, 120.59, 125.21, 125.61, 131.81, 147.85, 147.91, 167.52,
168.27; HRESIMS: calc for C11H6Br2NO4S [Mꢀ H]ꢀ 405.8390, found
405.8391.
5.1.2.4. (Z)-5-(2,3-dibromo-4,5-dimethoxybenzylidene)thiazolidine-2,4-
dione (1d). Yellow solid, Yield 75%, mp: 283–285 ◦C, 1H NMR (500
MHz, DMSO‑d6) δ: 3.81 (s, 3H), 3.89 (s, 3H), 7.18 (s, 1H), 7.81 (s, 1H),
12.60 (br s, 1H); 13C NMR (125 MHz, DMSO‑d6) δ: 56.44, 60.40, 111.96,
118.29, 121.97, 127.88, 130.53, 130.58, 148.17, 152.40, 166.68,
167.47; HRESIMS: calc for C12H8BrNO4S [Mꢀ H]ꢀ 421.8526, found
421.8522.
5.1.3. General procedure for the synthesis of hydantoin derivatives 2a-e
As described in procedure 5.1.2.
5.1.3.1. (Z)-5-(3-bromo-4,5-dimethoxybenzylidene)imidazolidine-2,4-
dione (2a). Yellow solid, Yield 86%, mp: 280–282 ◦C, 1H NMR (500
MHz, DMSO‑d6) δ: 3.75 (s, 3H), 3.89 (s, 3H), 6.35 (s, 1H), 7.20 (s, 1H),
7.46 (s, 1H), 10.76 (br s, 1H), 11.28 (br s, 1H); 13C NMR (125 MHz,
DMSO‑d6) δ: 56.82, 60.71, 107.15, 114.10, 117.55, 125.59, 128.26,
130.95, 145.91, 153.94, 156.64, 166.24; HRESIMS: calc for
C12H10BrN2O4 [Mꢀ H]ꢀ 324.9829, found 324.9823.
5.1.2.5. (Z)-5-(3-bromo-4,5-dihydroxybenzylidene)thiazolidine-2,4-dione
(1e). Yellow solid, Yield 72%, mp: 276–278 ◦C, 1H NMR (500 MHz,
DMSO‑d6) δ: 7.01 (s, 1H), 7.28 (s, 1H), 7.59 (s, 1H); 13C NMR (125 MHz,
DMSO‑d6) δ:110.64, 114.93, 121.25, 125.72, 127.39, 131.75, 146.27,
147.07, 168.03, 168.50; HRESIMS: calc for C10H5BrNO4S [Mꢀ H]ꢀ
313.9128, found 313.9132.
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Bioorganic Chemistry 108 (2021) 104648
Fig. 5. Effects of compound 5a on dyslipidemia in diabetic mice. Serum TG (A), TC (B), LDL-C (C), HDL-C (D), and circulating FFA (E) levels in normal BKS mice
treated with vehicle, and in diabetic mice treated with vehicle, compound 5a, and metformin. Data were shown as means ± SEM (n = 6–8). *p < 0.05, **p < 0.01,
and ***p < 0.001 as indicated. n.s., not significant.
5.1.3.2. (Z)-5-(2-bromo-3,4-dimethoxybenzylidene)imidazolidine-2,4-
dione (2b). Yellow solid, Yield 68%, mp: 288–290 ◦C, 1H NMR (500
MHz, DMSO‑d6) δ: 3.74 (s, 3H), 3.88 (s, 3H), 6.52 (s, 1H), 7.11 (d, J =
8.5 Hz, 1H), 7.45 (d, J = 8.5 Hz, 1H), 10.55 (br s, 1H), 11.27 (br s,
1H)13C NMR (125 MHz, DMSO‑d6) δ: 56.81, 60.48, 106.78, 112.65,
120.34, 125.99, 126.22, 129.26, 146.65, 153.83, 156.17, 165.86;
HRESIMS: calc for C12H10BrN2O4 [Mꢀ H]ꢀ 324.9830, found 324.9829.
5.1.3.3. (Z)-5-(4-hydroxy-3-methoxybenzylidene)imidazolidine-2,4-dione
(2c). Yellow solid, Yield 85%, mp: 216–218 ◦C, 1H NMR (500 MHz,
DMSO‑d6) δ: 3.83 (s, 3H), 6.36 (s, 1H), 6.79 (d, J = 7 Hz, 1H), 7.07 (d, J
= 7 Hz, 1H), 7.11 (s, 1H), 9.46 (br s, 1H), 10.42 (br s, 1H), 11.14 (br s,
1H); 13C NMR (125 MHz, DMSO‑d6) δ: 56.36, 110.34, 113.73, 116.28,
124.04, 124.87, 125.98, 148.14, 148.30, 156.29, 166.23; HRESIMS: calc
for C11H9N2O4 [Mꢀ H]ꢀ 233.0633, found 233.0723.
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Bioorganic Chemistry 108 (2021) 104648
Fig. 6. Effects of compound 5a on water intake, food intake and body weight in diabetic mice. Water intake (A), food intake (B), and body weight (C) of BKS db mice
fed a normal chow diet and treated with/without compound 5a. (D) Abdominal fat ratio of mice treated with compound 5a. Data were shown as means ± SEM (n =
6–8). *p < 0.05, **p < 0.01, and ***p < 0.001, compound 5a-treatment group versus vehicle-treated diabetic mice, or as indicated.
Fig. 7. Effect of compound 5a on Akt phosphorylation in skeletal muscle tissues of diabetic mice. (A) Immunoblotting analysis of Akt phosphorylation. Skeletal
muscle tissues were isolated from BKS mice treated with vehicle, and BKS db mice treated with vehicle or compound 5a. Western blotting was used to detect Akt
phosphorylation. (B) Ratio of Akt phosphorylation to total Akt. Data are expressed as means ± SEM (n = 3). *p < 0.05 and ***p < 0.001 as indicated.
5.1.3.4. (Z)-5-(3-bromo-4-hydroxy-5-methoxybenzylidene)imidazolidine-
2,4-dione (2d). Yellow solid, Yield 68%, mp: 220–222 ◦C, 1H NMR (500
MHz, DMSO‑d6) δ: 3.87 (s, 3H), 6.32 (s, 1H), 7.10 (s, 1H), 7.39 (s, 1H);
13C NMR (125 MHz, DMSO‑d6) δ: 56.90, 109.01, 110.64, 112.60,
126.47, 126.62, 146.31, 149.17, 156.26, 166.13; HRESIMS: calc for
C11H8BrN2O4 [Mꢀ H]ꢀ 310.9673, found 310.9763.
388.8786.
5.1.4. Procedure for the synthesis of TZD derivatives 5a-b
To the solution of benzyl alcohol 9 (2.0 g, 3.6 mmol) in dry CH2Cl2
(20 mL) was added PCC (1.6 g, 0.73 mmol). The mixture was stirred at
room temperature for 2 h. The suspension was filtered, and the filtrate
was washed with H2O (2x). The organic layer was dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by silica gel
column chromatography using petroleum ether/ethyl acetate (5:1, v/v)
to afford the aryl aldehyde 4 as a white solid with a yield of 78.9%.
The TZD derivative 5a was prepared as described in 5.1.2.
5.1.3.5. (Z)-5-(2,3-dibromo-4-hydroxy-5-methoxybenzylidene)imidazoli-
dine-2,4-dione (2e). Yellow solid, Yield 72%, mp: >300 ◦C, 1H NMR
(500 MHz, DMSO‑d6) δ: 3.93 (s, 3H), 6.48 (s, 1H), 7.09 (s, 1H), 10.64 (s,
1H), 11.31 (s, 1H); 13C NMR (125 MHz, DMSO‑d6) δ: 56.81, 108.45,
112.29, 113.89, 118.34, 125.39, 129.66, 146.25, 147.69, 156.21,
165.75; HRESIMS: calc for C11H7Br2N2O4 [Mꢀ H]ꢀ 388.8778, found
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Bioorganic Chemistry 108 (2021) 104648
Fig. 8. Effect of compound 5a on liver glycogen storage in diabetic mice. H&E staining and PAS staining of liver sections in normal BKS mice treated with vehicle,
and diabetic BKS db mice treated with vehicle or compound 5a (scale bar, 100 m).
μ
Fig. 9. Effect of compound 5a on pancreas. H&E staining, insulin immunostaining, glucagon immunostaining, and insulin and glucagon double-immunostaining of
pancreas from BKS mice treated with vehicle, and diabetic BKS db mice treated with vehicle or compound 5a (scale bar, 100 m).
μ
5.1.4.1. (Z)-5-(3-bromo-2-(2,3-dibromo-4,5-dimethoxybenzyl)-4,5-dime-
thoxybenzylidene) thiazolidine-2,4-dione (5a). Yellow solid, Yield 90%,
mp: 194–195 ◦C, 1H NMR (500 MHz, DMSO‑d6) δ: 3.54 (s, 3H), 3.69 (s,
3H), 3.80 (s, 3H), 3.89 (s, 3H), 4.26 (s, 2H), 6.19 (s, 1H), 7.14 (s, 1H),
7.63 (s, 1H), 12.58 (br s, 1H); 13C NMR (125 MHz, DMSO‑d6) δ: 31.25,
56.45, 56.81, 60.55, 60.82, 110.04, 111.86, 112.74, 116.95, 121.78,
122.87, 130.60, 131.28, 131.34, 136.21, 146.09, 147.64,152.64,
152.69, 168.45; HRESIMS: calc for C21H17Br3NO6S [Mꢀ H]ꢀ
647.8332, found 647.8328.
A solution of 5a (1.3 g, 2.0 mmol) and Pd/C (50 mg, 10% palladium
on carbon) was suspended in dry methanol (20 mL) and stirred at 50 ◦C
under a 1.5 MPa hydrogen atmosphere for 5 h. After completion, the Pd/
C was removed via filtration through a Celite pad. The filtrate was
evaporated to dryness under reduced pressure. The resulting oil residue
was chromatographed on silica gel in 20% ethyl acetate in hexane with
0.1% glacial acid to give 5b.
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Bioorganic Chemistry 108 (2021) 104648
5.1.4.2. 5-(3-bromo-2-(2,3-dibromo-4,5-dimethoxybenzyl)-4,5-dimethox-
ybenzyl)thiazolidine-2,4-dione (5b). Yellow solid, Yield 86%, mp:
92–93 ◦C, 1H NMR (500 MHz, DMSO‑d6) δ: 2.97–3.21 (m, 2H), 3.49 (s,
3H), 3.69 (s, 3H), 3.71 (s, 3H), 3.84 (s, 3H), 4.23 (s, 2H), 4.81–4.84 (m,
1H), 6.04 (s, 1H), 7.09 (s, 1H), 12.07 (br s, 1H); 13C NMR (125 MHz,
DMSO‑d6) δ: 31.25, 36.04, 52.63, 56.40, 56.66, 60.55, 60.64, 112.25,
114.32, 117.06, 121.67, 122.12, 129.35, 134.54, 136.38, 145.76,
146.02, 152.48, 152.72, 172.07, 176.17; HRESIMS: calc for
C21H19Br3NO6S [Mꢀ H]ꢀ 649.8489, found 649.8495.
5.3. Biological assays
5.3.1. Enzymatic activity assay and kinetics study
PTP1B inhibitory activities of synthetic compounds were measured
using recombinant human PTP1B1-321 proteins. Enzymatic activity
methods including selectivity against other PTPs were performed as
previously described [27]. In the kinetics study, a series of concentra-
tions of pNPP (0.5, 1.0, 2.0, 4.0 and 8.0 mM) were used as PTP1B sub-
strate in the presence of compound 5a (1.0, 2.0, 4.0 and 8.0
μM).
Lineweaver-Burk plotting of the enzymatic reaction was used to identify
5.1.5. Procedure for the synthesis of TZD derivatives 8a-b
the inhibition type of compound 5a.
The suspension of benzyl bromide 6 (1.0 g, 1.6 mmol), 4-ydroxyben-
zaldehyde (0.4 g, 3.2 mmol) and K2CO3 (0.3 g, 2.4 mmol) in acetone (10
mL) was stirred at room temperature overnight. The mixture was
filtered, and the filtrate was evaporated in vacuo. The residue was pu-
rified by silica gel column chromatography using petroleum ether/ethyl
acetate (6:1, v/v) as the eluent to afford 7.
5.3.2. Cell treatment and western blotting
C2C12 cells (a murine myoblast cell line) were cultured in DMEM
containing 10% FBS and seeded in a six-well cell plate at 4 × 105 cells/
well. When the cells had reached 80–90% confluence, the medium was
replaced with DMEM supplemented with 10% horse serum to induce cell
differentiation for an additional 4 days. After overnight starvation, the
cells were treated for 8 h with different concentrations of compound 5a
The TZD derivative 8a was prepared as described in 5.1.2.
(0, 0.01, 0.1, 1 and 10
stimulation for 5 min.
μM) following insulin (100 nM) stimulation or no
5.1.5.1. (Z)-5-(4-(3-bromo-2-(2,3-dibromo-4,5-dimethoxybenzyl)-4,5-
dimethoxyphenoxy)benzylidene)thiazolidine-2,4-dione (8a). Light yellow
solid, Yield 92%, mp: 207–208 ◦C, 1H NMR (500 MHz, DMSO‑d6) δ: 3.55
(s, 3H, OCH3), 3.69 (s, 3H), 3.76 (s, 3H), 3.90 (s, 3H), 4.16 (s, 2H), 5.13
(s, 2H), 6.06 (s, 1H), 6.85 (d, J = 7.5 Hz, 2H), 7.43 (s, 1H), 7.45 (d, J =
7.5 Hz, 2H), 7.70 (s, 1H), 12.58 (br s, 1H); 13C NMR (125 MHz,
DMSO‑d6) δ: 31.25, 56.15, 56.75, 60.61, 69.22, 112.64, 115.25, 116.91,
121.07, 121.31, 122.52, 126.29, 130.31, 132.24, 132.44, 133.24,
136.66, 145.80, 146.70, 152.40, 152.44, 160.09, 168.05, 168.56;
HRESIMS: calc for C28H23Br3NO7S [Mꢀ H]ꢀ 753.8751, found
753.8752.
Whole cell lysates were extracted using ice-cold RIPA buffer, and
protein concentrations were measured using a BCA Protein Assay Kit.
Proteins were separated on a polyacrylamide gel and transferred to and
detected on PVDF membranes with specific primary antibodies. Relative
band densities were detected with Western Blotting Substrate (Thermo
Scientific, Waltham, MA, USA).
5.3.3. Cell permeability assay
The permeability of compound 5a was carried out in C2C12 myo-
tubes. The differentiated C2C12 cells were treated with 5a (0.1 µM) and
incubated for 8 h at 37 ◦C. The medium was then removed and the cells
were washed with PBS. Then, methanol was added to fix the cells. The
mixture was treated with ultrasound and the solution was collected by
centrifugation. The equal amount of the internal standard (0.1 µM) was
added into the solution. The resulting solution was evaporated under
reduced pressure and the residue was dissolved in methanol (1 mL) for
A solution of 8a (0.76 g, 1.0 mmol) and Pd/C (30 mg, 10% palladium
on carbon) was suspended in dry dichloromethane (15 mL) and stirred at
50 ◦C under a 1.5 MPa hydrogen atmosphere for 5 h. After completion,
the Pd/C was removed via filtration through a Celite pad. The filtrate
was evaporated to dryness under reduced pressure. The resulting oil
residue was subjected to chromatography on silica gel in 20% ethyl
acetate in hexane with 0.1% glacial acid to give 8b.
subsequent HPLC analysis (π-Nap, mobile phase: methanol/H2O +
0.05% TFA). The gradient elution was 80% methanol to 90% methanol
in 15 min followed by 90% methanol in next 15 min and 80% methanol
in the next 10 min at a flow rate of 1 mL/min.
5.1.5.2. 5-(4-(3-bromo-2-(2,3-dibromo-4,5-dimethoxybenzyl)-4,5-dime-
thoxyphenoxy)benzyl)thiazolidine-2,4-dione (8b). Light yellow solid,
Yield 81%, mp: 102–104 ◦C, 1H NMR (500 MHz, DMSO‑d6) δ: 2.99–3.29
(m, 2H), 3.56 (s, 3H), 3.69 (s, 3H), 3.75 (s, 3H), 3.87 (s, 3H), 4.16 (s,
2H), 4.83 (m, 1H), 5.00 (s, 2H), 6.10 (s, 1H), 6.69 (d, J = 8.5 Hz, 2H),
7.08 (d, J = 8.5 Hz, 2H), 7.38 (s, 1H), 12.03 (br s, 1H); 13C NMR (125
MHz, DMSO‑d6) δ: 31.12, 36.69, 53.43, 56.09, 56.59, 60.49, 68.71,
112.59, 114.81, 116.82, 121.12, 122.23, 129.43, 130.01, 130.64,
133.65, 136.58, 145.68, 146.41, 152.24, 152.33, 157.39, 172.15,
176.16; HRESIMS: calc for C28H25Br3NO7S [Mꢀ H]ꢀ 755.8907, found
755.8925.
5.3.4. Cell viability test
C2C12 cells were first seeded in a 96-well plate, and after overnight
incubation cells treated with compound 5a (0.01–100 µM) for 24 h.
Then cells were incubated with 10 uL CCK8 solution each well for 1 h in
the 37 ◦C cell incubator. Finally, the absorbance was determined using a
multi-well microplate reader at the wavelength of 450 nm. Cells exposed
to DMSO were used as control.
5.3.5. Animal study
5.2. Molecular docking
Animal studies were performed according to approved regulatory
standards under protocol HAIFAJIZI-2013–3 (approval date: 2013-12-
09; Animal Care and Use Committee of Institute of Oceanology, Chi-
nese Academy of Sciences). 4–6 weeks male BKS.Cg-Dock7m+/
+Leprdb/J mice and wild control (BKS mice) were purchased from the
Model Animal Research Centre (MARC) of Nanjing University. All mice
were fed ad libitum a normal chow diet and water. After 1 week of
acclimatization, qualified diabetic mice were randomly divided into
three different treatment groups (n = 8): vehicle treatment group (BKS
db, 1.5% CMC-Na), metformin treatment group (metformin, 100
mg•kgꢀ 1 body wt•dayꢀ 1), and compound 5a treatment group (5a, 50
mg•kgꢀ 1 body wt•dayꢀ 1). Age-matched male BKS mice (n = 8) were
treated with vehicle (1.5% CMC-Na) and used as a normal control group.
Metformin and compound 5a (in 1.5% CMC-Na) were orally
administered once/daily between 08:00 and 09:00 using a cannula.
The molecular docking study was performed using the 1.5.6 version
of AutoDock combined with PyMol software. The 3D structure of com-
pound 5a was constructed using ChemDraw 12.0, and then the MM2
energy was minimized. The crystal structure of PTP1B was obtained
from the RCSB Protein Data Bank (PDB code: 1G1H) by removing water
molecules and bound ligands and adding hydrogens and Kollman
charges. The domain sphere was defined by the binding site of 5a in the
crystal pose in 1G1H. Each docking experiment was performed 300
times, resulting in 300 docking conformations. All other parameters
were default values of the system. The best model was selected based on
the best stable energy.
12

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Bioorganic Chemistry 108 (2021) 104648
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Declaration of Competing Interest
The authors have declared no conflict of interest.
Acknowledgements
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