Design, synthesis of novel azolyl flavonoids and their protein tyrosine Phosphatase-1B inhibitory activities

Article abstract of DOI:10.1016/j.bioorg.2018.06.008

A series of azolyl flavonoids were synthesized and characterized by NMR, IR, MS and HRMS spectra. All the newly prepared compounds were screened for their potential protein tyrosine phosphatase inhibitory activities. Bioactive assay manifested that most of the azolyl flavonoids exhibited good protein phosphatase 1B (PTP1B) inhibitory activities. Especially, triazolyl flavonoid 6a displayed the best inhibitory activity (IC₅₀ = 1.6 μM) with 9.9-fold selectivity for PTP1B over the closely related T-cell protein tyrosine phosphatase (TCPTP). Cell viability assays indicated 6a has lower cytotoxicity. Molecular modeling and dynamics studies revealed the reason of selectivity for PTP1B over TCPTP. Quantum chemical studies were carried out on these compounds to understand the structural features essential for activity.

Full text of DOI:10.1016/j.bioorg.2018.06.008

Accepted Manuscript
Design, Synthesis of Novel Azolyl Flavonoids and Their Protein Tyrosine Phos-
phatase-1B Inhibitory Activities
Ling Zhang, Yu Ge, Hao Ming Song, Qing Ming Wang, Cheng-He Zhou
PII:
S0045-2068(18)30306-7
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.008
YBIOO 2388
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 March 2018
30 May 2018
3 June 2018
Please cite this article as: L. Zhang, Y. Ge, H. Ming Song, Q. Ming Wang, C-H. Zhou, Design, Synthesis of Novel
Azolyl Flavonoids and Their Protein Tyrosine Phosphatase-1B Inhibitory Activities, Bioorganic Chemistry (2018),
doi: https://doi.org/10.1016/j.bioorg.2018.06.008
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Title page
Title:
Design, Synthesis of Novel Azolyl Flavonoids and Their Protein Tyrosine Phosphatase-1B Inhibitory
Activities
Author names and affiliations:
Ling Zhang1,*, Yu Ge1, Hao Ming Song1, Qing Ming Wang1,* and Cheng-He Zhou2
1,* School of Pharmacy, Yancheng Teachers’ University, Yancheng, Jiangsu 224051, People’s Republic of
China
2
Laboratory of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering,
Southwest University, Chongqing, 400715, P.R. China
Tel.: 0515-88258905
E-mail: zling891613@163.com (Ling Zhang), wangqm@yctu.edu.cn (Qing Ming Wang)
Abstract:
A series of azolyl flavonoids were synthesized and characterized by NMR, IR, MS and HRMS spectra. All
the newly prepared compounds were screened for their potential protein tyrosine phosphatase inhibitory
activities. Bioactive assay manifested that most of the azolyl flavonoids exhibited good protein phosphatase
1B (PTP1B) inhibitory activities. Especially, triazolyl flavonoid 6a displayed the best inhibitory activity
(IC50 = 1.6 μM) with 9.9-fold selectivity for PTP1B over the closely related T-cell protein tyrosine
phosphatase (TCPTP). Cell viability assays indicated 6a has lower cytotoxicity. Molecular modeling and
dynamics studies revealed the reason of selectivity for PTP1B over TCPTP. Quantum chemical studies
were carried out on these compounds to understand the structural features essential for activity.
Keywords:
Flavonoid; Azole; Protein tyrosine phosphatase; Selectivity; Cell viability
1

1. Introduction
Protein tyrosine phosphatases (PTPs) play an important role in modulation several cellular signal
transduction pathways and catalysis protein tyrosine dephosphorylation [1]. Dysregulation of PTPs
activities lead to the pathogenesis of many human diseases such as obesity, autoimmune disorders, cancers
and diabetes [2]. Protein tyrosine phosphatases 1B (PTP1B), a key member of the PTP family, could
regulate the insulin sensitivity and act by directly inactivating insulin receptor (IR) through
dephosphorylation tyrosine residues in the regulatory domain. Insulin exerts an important effect in glucose
uptake. Resistance of insulin results in reduced glucose intake and increased hepatic glucose output, which
led to the increase of the blood glucose level [3]. A number of synthetic PTP1B inhibitors have been
discovered [4-6]. Compound 1 was a classic PTP1B inhibitor (IC50 = 32 nM) designed on the basis of
catalytic pocket characteristics and demonstrated modest caco-2 permeability (0.4  10-6 cm/s), but without
selectivity (Figure 1). Compound 2 derived from natural products has also been proved to be active against
PTP1B with micromolar level of activity (IC50 = 2.42 M), however, the selectivity or membrane
permeability was not further reported [7]. PTP inhibitors especially that can inhibit the particular PTP with
good permeability received much attention [8].
Fig. 1 Some reported PTP1B inhibitors
Flavonoids, a type of plant phenolic compounds, are widely found in nature with large potentiality in the
2

treatment of cancer, car-diovascular disease and neurodegenerative disorders. Recently, these natural
flavones have drawn special attention as they are found to be good PTP1B inhibitors with low toxicity and
high hypoglycemic effects [9]. It has disclosed that they could inhibit PTP1B activity in a non-competitive
way different to the traditional drugs [10]. More and more flavonoids received much attention due to their
unique properties in the inhibition of PTP1B (Figure 1).
Azoles (thiazoles, oxazoles, carbazoles, imidazoles, benz-imidazoles, triazoles, tetrazoles etc) are an
important type of nitrogen heterocycles with aromaticity and electron rich properties. The special structure
enables their derivatives to easily bind with the enzymes and receptors in organisms through noncovalent
interactions such as coordination and hydrogen bonds, thereby giving them various medicinal applications.
Recently, research has established that the introduction of nitrogen-containing heterocyclic moieties into
the flavonoid backbone can improve bioactivity [11-14]. Especially, it reported that presence of azole ring
is beneficial to improve the PTP1B inhibitory activity [15,16]. However, to the best of our knowledge,
azolyl flavonoids have been rarely reported. In view of the above observations herein we incorporated
different azole fragments into the O-7 position of flavonoid to generate a novel class of azolyl flavonoids. It
evidenced that alkyl linkers can modulate physico-chemical properties and thus improve biological potency
[17]. With the aim of better understanding structure–activity relationships and increasing flexibility,
different lengths of alkyl chains were introduced into the target compounds to investigate the influence of
linkers on bioactivities.
The designed structures of this series of novel azolyl flavonoids are shown in Scheme 1. All the newly
synthesized compounds were characterized by spectral analysis and evaluated for their inhibition of PTP1B
in vitro. Molecular modeling, molecular dynamics, energies and plots of HOMO and LUMO, and plots of
MEP were also investigated by quantum chemical calculation to understand the structural features essential
for activity.
2 Results and discussion
2.1. Chemistry
The target azolyl flavonoids were synthesized according to the synthetic route outlined in Scheme 1.
Condensation of 1,3-benzenediol with chloro-acetonitrile in the presence of ZnCl2 in ether was followed by
hydrolysis in water and produced ketone 2 in 86% yield. The resulting 2 was reacted with benzaldehyde in
ethanol with 10% NaOH, was followed by acidification with aqueous HCl to afford intermediate 3 in
3

satisfactory yields [18]. Compound 3 was then further treated with alkyl dibromides in acetone using
potassium carbonate as base to afford bromides 4a-b with yields of 50–76% [19]. The target azolyl
flavonoids 5a–d, 6a–d and 7a–b were conveniently and efficiently obtained in 50–67% yields by the
reaction of bromides 4a–b respectively with 2-methyl-5-nitroimidazole, 4-nitroimidazole, triazole, tetrazole
or imidazole-thiol in acetonitrile at 50℃ in the presence of potassium carbonate as base. The target new
compounds were confirmed by 1H NMR, 13C NMR, IR, MS and HRMS spectra.
Scheme 1 Synthetic routes of azolyl flavonoids. Reagents and conditions: (i) ClCH2CN, EtOEt, HCl (g), 0℃; (ii) 1 mol/L
HCl, H2O, reflux; (iii) benzaldehyde, 10%NaOH, rt, EtOH; (iv) alkyl dibromides, K2CO3, acetone, 50 ºC; (v)
2-methyl-5-nitroimidazole or 4-nitroimidazole, K2CO3, acetonitrile, 50 ºC; (vi) triazole or 5-methyl tetrazole, K2CO3,
acetonitrile, 50 ºC; (vii) imidazole-thiol, K2CO3, acetonitrile, 50 ºC.
2.2. Biological evaluation
2.2.1. Inhibition of phosphatases
The obtained results as depicted in Table 1 revealed that azolyl flavonoids 5a–d, 6a–d and 7a–b could
effectively inhibit the PTP1B activity with IC50 values of 1.6–15.1 μmol/L. Table 1 showed the significant
4

effects of the types of azole rings and alkyl chain lengths on biological activity. Generally, long-chain alkyl
possessed less inhibitory activities than short-chain derivatives. The long alkyl ones with higher
lipophilicity in these compounds might make them unfavourable to be delivered to the binding sites.
Among them, triazolyl functionalized compound 6a gave the best inhibitory activity against PTP1B with
MIC values of 1.6 μmol/L. The substitution of triazole with 2-methyl-5-nitroimidazole, 4-nitroimidazole,
5-methyl tetrazole or thiol-imidazole group which yielded compounds 5a, 5c, 6c and 7a resulted in
relatively lower activity. These results revealed that existence of triazole moiety in this series of azolyl
flavonoids should be of special importance in the PTP1B Inhibition profiles.
In view of above discussion, the anti-PTP1B efficacies should be closely related to azole ring and alkyl
chain lengths to some extent. For this serial compounds, azole moieties contributed to the anti-PTP1B
activities in the order of triazole > imidazole-thiol > 2-methyl-5-nitroimidazole > 4-nitroimidazole >
5-methyl tetrazole derivatives. The triazolyl group was more helpful for increasing anti-PTP1B efficacy in
comparison to other azolyl ones.
Table 1 Inhibitory activities against PTP1B (μmol/L)
Compds
IC50
R
IC50
Compds
R
4.0+1.7
3.6+0.4
5a
5b
5c
6b
6.9+1.3
5.0+1.2
7.0+1.1
1.6+0.9
7.9+0.3
15.1+0.9
3.0+0.04
3.9+0.9
6c
6d
7a
7b
5d
6a
Further, the selectivity of the 6a was determined by measuring their inhibitory activity against a panel of
several phosphatases including TCPTP, PTP-MEG2 and SHP-2. The IC50 values of inhibitor 6a against the
phosphatases, and the selectivity ratios were shown in Table 2. Compound 6a showed no activity against
PTP-MEG2, and only exhibited moderate activity for SHP-2. Compound 6a was selective for PTP1B over
5

PTP-MEG2 and SHP2. Notably, compound 6a showed 9.9-fold selectivity for PTP1B over TCPTP, since
TCPTP was the most closely related to PTP1B in phosphatase.
Table 2 Inhibition of phosphatases by inhibitor 6a
IC50(M)
Compd
PTP1B
1.6
TCPTP
15.8
PTP-MEG2
NA
SHP-2
19.8
Selective
9.9
6a
a NA: no activity
b Selective: IC50(TCPTP)/IC50(PTP1B)
Abbreviations: PTP1B, protein tyrosine phosphatase 1B; TCPTP, T-cell protein tyrosine phosphatase; PTP-MEG2,
megakaryocyte protein tyrosine phosphatase; SHP-2, src homology phosphatase 2.
c
2.2.2. Kinetic analysis of PTP1B inhibition
A kinetic analysis of compound 6a was then conducted using p-NPP as the small-molecule substrate in a
continuously monitored colorimetric assay. Four different concentrations of the substrate p-NPP (1, 2, 4, 8
M) were used in the steady state kinetics assays. Figure 2 shows the Lineweavere Burk double-reciprocal
plot of the kinetics data in the presence of various concentrations of compound 6a. It appears that the lines
converged at an intersection on the y-axis above the x-axis, implying a competitive inhibition mode versus
p-NPP.
700
600
500
8 μM
4 μM
2 μM
1 μM
400
300
200
100
0
-0.5
0.0
0.5
1.0
1.5
2.0
1/[S]
Fig. 2 Compound 6a is a classical competitive inhibitor
2.3. The influence of 6a on cell viability
The effect of 6a on cell viability of HEK293 (human embryonic kidney 293) cells was further tested by
Cell Counting Kit-8 (CCK8) method. As shown in Figure 3, compound 6a showed relative low toxicity to
normal human embryonic kidney cells at high concentration, and the IC50 value was 106 g/mL. The
results demonstrated 6a has lower cytotoxicity.
6

Fig. 3 Relative cell viabilities of compound 6a in HEK293 cells.
2.4. Molecular modeling
To rationalize the observed PTP1B inhibitory activity of the target flavonoids, active site-docking
simulations of the compounds were performed to calculate their lowest-energy conformations (Figure 4 and
Figure 5). The crystal structure data (PTP1B) were obtained from the protein data bank (PDB code: 1G1H).
Target compound 6a was selected to dock with the PTP1B.
According to the docking evaluation, the most active 6a showed high binding energy (E = -8.69 kJ/mol)
with PTP1B (Figure 4). The docking result of compound 6a with PTP1B might rationalize the possible
inhibitory mechanism. The 1-position and 2-position nitrogen atoms of the triazole moiety could form
hydrogen bonds with residue ARG-221 of PTP1B with length of 2.5 and 2.1 Å respectively, which
indicated the necessity of triazole moiety for the increased bioactivity. The oxygen atom on the alkoxy
group could form hydrogen bonds with the residue ARG-221 and SER-216 at the same time, with the
distance of 1.9 and 2.3 Å, respectively. The oxygen atom on the carbonyl could also form hydrogen bond
with the residue LYS-120 at the distance of 2.1 Å. Furthermore, besides hydrogen bonds, the
compound–PTP1B enzyme complex could be stabilized by several hydrophobic interactions (Figure 5).
This cooperative binding might be beneficial to stabilize the compound–PTP1B enzyme complex, which
might be responsible for the good inhibitory efficacy of compound 6a against PTP1B.
7

Fig. 4 Three-dimensional conformation of compound 6a docked onto the active site of PTP1B.
Fig. 5 Two-dimensional conformation of compound 6a docked onto the active site of PTP1B.
In order to explain the selectivity of inhibitor 6a, the binding mode of compound 6a with TCPTP (PDB
code: 1L8K) was performed. Compound 6a showed relatively lower binding energy (E = -7.1 kJ/mol) than
PTP1B. Some main interactions between inhibitor 6a and the receptor can be observed in Figure 6. Only
the 1-position nitrogen atom of the triazole moiety and oxygen atom of this molecule formed hydrogen
bonding with amino acid residues Ser217 and Arg222 with length of 2.6 Å and 2.2 Å respectively, that was
different with the binding pattern of 6a with PTP1B (Figure 4 and 5).
8

Fig. 6 Three-dimensional conformation of compound 6a docked onto the active site of TCPTP.
2.5. Analysis of the Dynamics Studies
A continuous effort was made to explain the selectivity of inhibitor 6a by performing molecular
dynamics (MD) simulations.
The interaction of the protein with ligands is a dynamic process different with static process of molecular
docking. The dynamic process of results in a lower binding energy of the complex and a more stable
complex, and could be displayed by MD simulation. Analysis of the root-mean-squared deviation (RMSD)
values indicates that the RMSDs of both systems reached equilibration and oscillated around an average
value after 20 and 10 ns simulation, respectively. The backbone RMSD value of PTP1B was lower than
that of TCPTP, which suggested that the PTP1B/6a was more stable than TCPTP/6a in 50 ns MD
simulation (Figure 7). All these observations revealed that the interaction between 6a and PTP1B was more
potent and stable than that between 6a and TCPTP. That may be the reason, at least in part, why compound
6a displayed obvious selectivity for PTP1B over TCPTP.
Fig. 7 Time dependence of root-mean-square deviations (RMSD’s) for 6a bound with TCPTP (1L8K, in red) and PTP1B
(1G1H, in black)
9

The integrity of the protein was analyzed by plotting Rg values against time. The Rg describes the
overall spread of the molecule and is defined as the root-mean-square distance of the collection of atoms
from their common center of gravity. In the present MD studies, we determined the Rg values of free
PTP1B and PTP1B-compound 6a complex as shown in Figure 8. The Rg values were stabilized at about 35
ns, indicating that the MD simulation achieved equilibrium after 50 ns. Initially, the Rg values of free
PTP1B and PTP1B-compound 6a complex were 1.92 nm. The free PTP1B and PTP1B-compound 6a
complex were stabilized at 1.91±0.01 and 1.89±0.01 respectively. These results suggest that the Rg value
marginally decreased upon binding of compound 6a. The above results emphasize that the compound 6a
bind to PTP1B in MD simulations due to a change in the microenvironment of PTP1B which leads to the
conformational changes in the protein. Thus, the MD simulation data clearly showed that
PTP1B-compound 6a complex was stabilized due to conformational rearrangements.
2.00
PTP1B
PTP1B-compound 6a
1.95
1.90
1.85
1.80
0
10
20
30
40
50
Time (ns)
Fig. 8 Time evolution of the radius of gyration values during 50 ns of MD simulation of PTP1B and PTP1B-compound 6a
complex.
The local protein flexibility was investigated by calculated RMSF values of free PTP1B and
PTP1B-compound 6a complex, which were plotted against residue numbers on a 50 ns trajectory and
distinctly indicate that the flexibility of PTP1B is more than PTP1B-compound 6a complex. The higher
flexibility of PTP1B is mainly because of interactions of compound 6a with the target protein, PTP1B. This
inherent flexibility of PTP1B is likely to play an important role in the ligand binding (Figure 9). The
analysis provides an indication that PTP1B binding site interacts clearly with compound 6a through
remodeling of the compound 6a to the specific sites of PTP1B, which results in structural alteration of the
protein.
10

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
PTP1B-Compound 6a
PTP1B
0
50
100
150
200
250
300
Residue number
Fig. 9 RMSF analysis of complex structure during molecular dynamics simulation
2.6. Molecular orbital calculation
Electronic effects were shown to be related with biological activity of drugs [20]. At the molecular level,
the frontier molecular orbitals (FMO) controlled the reactivity of a molecule, namely the highest occupied
molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) and they exerted
important effect in intermolecular interactions [21]. The extents of these stabilizing interactions in inversely
correlation with the energy gap between the interacting orbitals. Higher HOMO energy and lower LUMO
energy lead to larger stabilizing interactions, favoring for binding with the receptor. Hence, the orbital
energies of both HOMO and LUMO and their gaps were calculated for compounds 6a and 6d as shown in
Table 3. The HOMO-LUMO energy gap value supports the intramolecular charge-transfer interactions
within the molecule. It was noteworthy that the most active compound 6a gave the lowest energy gap (∆ε)
of 10.85 eV.
Table 3 Energies of both HOMO and LUMO and their gaps (in eV) calculated for compounds 6a and 6d.
Compds
6a
εHOMO (eV) εLUMO (eV)
∆ε (eV)
10.85
11.13
-8.80
2.05
1.96
6d
-9.17
The plots of the HOMOs and LUMOs of 6a and 6d were also obtained successfully to further analyze
the main atomic contributions for these orbitals. The results presented in Table 4 demonstrated that the
electron charge cloud is located at the flavonoid ring in highest occupied molecular orbital (HOMO) of 6a
and 6d which indicated that active sites might be at this ring and biological interactions could take place
between positively charged molecules and these sites. It was also found that azole ring and substituents on
the ring did not contribute directly to HOMOs which manifested these groups might be primarily used to
modulate the physicochemical properties. Moreover, the LUMOs of these molecules were also mainly
centered at the flavonoid ring in which nucleophilic attacks might be favorable.
11

Table 4 Plots of the HOMO and LUMO of compounds 6a and 6d
Compds
HOMO
LUMO
6a
6d
2.7. Molecular electrostatic potentials
In order to deeply understand the lower inhibitory activities of compound 6d together with the higher
inhibitory activity of 6a, molecular electrostatic potentials (MEPs) had been proceeded to check the
similarities and differences in electrostatic binding characteristic of the surface of the molecules (Table 5).
Comparison of the electrostatic maps of 6a and 6d revealed that compound 6a had an increased positive
charge regions (blue region) located on the triazole ring probably due to influence of the aromatic
substituent, and 6a also possessed more negative charge regions (red region) on the oxygen atom of
flavonoid ring. It might indicate compound 6a had strong capability of interacting with polar residues of
enzymes or receptors to form hydrogen bond through triazole ring and the oxygen atom of flavonoid ring.
This was consistent with the binding mode obtained from above docking study.
Table 5 Molecular electrostatic potentials (MEPs) of compounds 6a and 6d showing the most positive potential (deepest blue
color), the most negative potential (deepest red color), and the intermediate potential (intermediate shades) regions
Molecular electrostatic potentials
Compds
6a
12

6d
3 Conclusion
In conclusion, a series of azolyl flavonoids were successfully synthesized by a convenient and efficient
1
procedure. Their structures were confirmed by H NMR, 13C NMR, MS, IR and HRMS spectra. The in
vitro protein tyrosine phosphatase inhibition evaluation revealed that most of azolyl flavonoids exhibited
good protein phosphatase 1B (PTP1B) inhibitory activities at the micromolar range. The most active
compound 6a with IC50 values of 1.6 μM displayed 9.9-fold selectivity over TCPTP. Molecular modeling
studies indicated that the triazole ring played an important role in the interaction of inhibitor with PTP1B.
The MD studies revealed that the PTP1B/6a complex was more stable than TCPTP/6a, which was the
potent proof for observed 9.9-fold selectivity of 6a for PTP1B over TCPTP, although TCPTP and PTP1B
shared high degree of homology in catalytic site. These results provided further evidence that azolyl
flavonoids can be potent inhibitors of PTP1B. Further studies on these compounds in cell permeability,
selectivity and cellular activity would be necessary to provide PTP1B inhibitors suitable for in vivo proof of
animal studies in diabetes.
4 Experimental protocols
4.1 General methods
Melting points were recorded on X-6 melting point apparatus and uncorrected. TLC analysis was done
using pre-coated silica gel plates. FT-IR spectra were carried out on Bruker RFS100/S spectrophotometer
(Bio-Rad, Cambridge, MA, USA) using KBr pellets in the 400–4000 cm-1 range. NMR spectra were
recorded on a Bruker AV 300 and 600 spectrometer using TMS as an internal standard. The chemical shifts
were reported in parts per million (ppm), the coupling constants (J) were expressed in hertz (Hz) and
signals were described as singlet (s), doublet (d), triplet (t), as well as multiplet (m). The mass spectra were
recorded on LCMS-2010A and the high-resolution mass spectra (HRMS) were recorded on an IonSpec
FT-ICR mass spectrometer with ESI resource. All other chemicals (Shanghai Titan Technology Co., Ltd) and
solvents were commercially available, and were used without further purification.
13

4.2 Synthesis of azolyl flavonoids
4.2.1. Synthesis of 7-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)-2-phenyl-4H-chromen-4-one (5a)
A mixture of 2-methyl-5-nitro-1H-imidazole (0.76 g, 6 mmol) and potassium carbonate (0.83 g, 6 mmol) in
CH3CN (20 mL) was stirred for 1 h at room temperature, and then compound 4a (1.73 g, 5 mmol) was added.
The resulting mixture was stirred at 50 ℃ for 16 h (monitored by TLC, eluent, petroleum ether/ethylacetate, 3/1,
V/V). The solvents were evaporated under reduced pressure, and the residue was treated with water (50 mL) and
extracted with chloroform (350 mL). The organic layers were combined, dried with anhydrous Na2SO4, and
concentrated under reduced pressure. The crude product was purified via silica gel column chromatography
(eluent, petroleum ether/ethylacetate, 5/1, V/V) to afford compound 5a (1.25 g) as white power. Yield: 64%; mp:
199–200 ℃; IR (KBr, cm-1) ν: 3052 (Ar–H), 2965 (CH3), 1811 (aromatic frame), 1700 (C=O), 1619, 1578
(aromatic frame); 1H NMR (400 MHz, DMSO-d6) δ: 8.31 (s, 1H, imidazole 4-H), 8.01–7.82 (m, 2H, Ph-2,6-2H),
7.54 (d, J = 7.5 Hz, 1H, flavone-5-H), 7.50–7.42 (m, 3H, Ph-3,4,5-3H), 7.22 (d, J = 2.3 Hz, 1H, flavone-3-H),
6.83 (s, 1H, flavone-6-H), 6.72 (dd, J = 7.5, 2.0 Hz, 1H, flavone-8-H), 4.22 (q, J = 4.1 Hz, 2H, N-CH2), 4.00 (dd,
J = 10.2, 5.1 Hz, 2H, O-CH2), 2.33 (s, 3H, CH3) ppm; 13C NMR (101 MHz, DMSO-d6) δ: 181.1, 167.2, 165.3,
155.1, 147.8, 145.5, 132.2, 131.6, 131.0, 130.4, 129.6, 128.2, 126.6, 115.3, 112.8, 99.5, 66.5, 49.4, 27.1, 20.1
ppm; MS (m/z): 392 [M+H]+; HRMS (TOF) calcd for C21H17N3O5: [M+H]+, 392.1246; found, 392.1248.
4.2.2. Synthesis of 7-(4-(2-methyl-5-nitro-1H-imidazol-1-yl)butoxy)-2-phenyl-4H-chromen-4-one (5b)
Compound 5b was prepared according to the experimental procedure for compound 5a. White solid; Yield:
62%; mp: 202–203 ℃; IR (KBr, cm-1) ν: 3049 (Ar–H), 2988 (CH3), 1800 (aromatic frame), 1726 (C=O), 1625,
1538, 1462 (aromatic frame); 1H NMR (400 MHz, DMSO-d6) δ: 8.23 (s, 1H, imidazole 4-H), 8.22–7.95 (m, 2H,
Ph-2,6-2H), 7.68 (d, J = 8.1 Hz, 1H, flavone-5-H), 7.52–7.41 (m, 3H, Ph-3,4,5-3H), 7.19 (d, J = 2.0 Hz, 1H,
flavone-3-H), 6.89 (s, 1H, flavone-6-H), 6.79 (dd, J = 8.2, 3.0 Hz, 1H, flavone-8-H), 5.11–5.00 (m, 2H, N-CH2),
4.77–4.64 (m, 2H, O-CH2), 2.40 (s, 3H, CH3), 1.65 (dt, 4H, J = 14.2, 7.1 Hz, CH2CH2) ppm; 13C NMR (101
MHz, DMSO-d6) δ: 182.5, 169.5–168.1, 167.0, 164.1–163.9, 148.5, 133.7–132.2, 131.3, 128.5, 126.2, 113.8,
111.6, 98.3, 66.6, 52.2, 10.6 ppm; MS (m/z): 420 [M+H]+; HRMS (TOF) calcd for C23H21N3O5: [M+H]+,
420.1559; found, 420.1557.
4.2.3. Synthesis of 7-(2-(4-nitro-1H-imidazol-1-yl)ethoxy)-2-phenyl-4H-chromen-4-one (5c)
Compound 5c was prepared according to the experimental procedure for compound 5a. White power. Yield:
69%; mp: 190–191 ℃; IR (KBr, cm-1) ν: 3053 (Ar–H), 1811 (aromatic frame), 1726 (C=O), 1648, 1574
(aromatic frame); 1H NMR (400 MHz, DMSO-d6) δ: 8.07 (s, 1H, imidazole 2-H), 7.82 (s, 1H, imidazole 5-H),
14

8.05–7.96 (m, 2H, Ph-2,6-2H), 7.65 (d, J = 8.5 Hz, 1H, flavone-5-H), 7.52–7.41 (m, 3H, Ph-3,4,5-3H), 7.24 (d, J
= 2.6 Hz, 1H, flavone-3-H), 6.87 (s, 1H, flavone-6-H), 6.80 (dd, J = 8.3, 2.6 Hz, 1H, flavone-8-H), 4.52 (q, J =
4.2 Hz, 2H, N-CH2), 4.46 (dd, J = 10.1, 5.1 Hz, 2H, O-CH2) ppm; 13C NMR (101 MHz, DMSO-d6) δ: 181.7,
167.2, 165.4, 153.2, 148.4, 146.3, 133.5, 132.2, 131.1, 130.2, 129.3, 128.2, 126.0, 115.5, 114.5, 112.2, 98.6, 63.1,
41.2 ppm; MS (m/z): 378 [M+H]+; HRMS (TOF) calcd for C20H15N3O5: [M+H]+, 378.1090; found, 378.1096.
4.2.4. Synthesis of 7-(3-(4-nitro-1H-imidazol-1-yl)propoxy)-2-phenyl-4H-chromen-4-one (5d)
Compound 5d was prepared according to the experimental procedure for compound 5a. White solid; Yield:
60%; mp: 201–202 ℃; IR (KBr, cm-1) ν: 3036 (Ar–H), 1824 (aromatic frame), 1744 (C=O), 1624, 1535, 1461
(aromatic frame); 1H NMR (400 MHz, DMSO-d6) δ: 8.24–8.19 (m, 2H, Ph-2,6-2H), 8.11 (s, 1H, imidazole 2-H),
8.02 (s, 1H, imidazole 5-H), 7.58 (d, J = 7.4 Hz, 1H, flavone-5-H), 7.50–7.40 (m, 3H, Ph-3,4,5-3H), 7.22 (d, J =
2.5 Hz, 1H, flavone-3-H), 6.91 (s, 1H, flavone-6-H), 6.76 (dd, J = 8.3, 3.5 Hz, 1H, flavone-8-H), 5.13–5.01 (m,
2H, N-CH2), 4.82–4.68 (m, 2H, O-CH2), 1.791.76 (m, 2H, CH2CH2), 1.721.68 (m, 2H, CH2CH2) ppm; 13C
NMR (101 MHz, DMSO-d6) δ: 180.6, 168.9, 166.5, 165.2, 164.2, 148.5, 134.7, 132.3, 129.4, 125.3, 115.6, 112.7,
98.4, 67.1, 53.2, 20.6 ppm; MS (m/z): 392 [M+H]+; HRMS (TOF) calcd for C21H17N3O5: [M+H]+, 392.1246;
found, 392.1240.
4.2.5. Synthesis of 7-(2-(1H-1,2,4-triazol-1-yl)ethoxy)-2-phenyl-4H-chromen-4-one (6a)
Compound 6a was prepared according to the experimental procedure for compound 5a. White power. Yield:
67%; mp: 185–186 ℃; IR (KBr, cm-1) ν: 3061 (Ar–H), 1804 (aromatic frame), 1718 (C=O), 1617, 1571
1
(aromatic frame); H NMR (400 MHz, DMSO-d6) δ: 8.62 (s, 1H, triazole-5-H), 8.02 (s, 1H, triazole-3-H),
8.00–7.95 (m, 2H, Ph-2,6-2H), 7.69 (d, J = 8.6 Hz, 1H, flavone-5-H), 7.55–7.43 (m, 3H, Ph-3,4,5-3H), 7.21 (d, J
= 2.1 Hz, 1H, flavone-3-H), 6.86 (s, 1H, flavone-6-H), 6.82 (dd, J = 8.6, 2.1 Hz, 1H, flavone-8-H), 4.66 (q, J =
4.6 Hz, 2H, N-CH2), 4.55 (dd, J = 10.3, 5.3 Hz, 2H, O-CH2) ppm; 13C NMR (101 MHz, DMSO-d6) δ: 182.1,
168.3, 166.3, 152.0, 147.6, 145.2, 132.4, 131.6, 131.2, 130.4, 129.5, 128.8, 126.1, 114.6, 113.4, 111.6, 98.4, 67.2,
48.4 ppm; MS (m/z): 334 [M+H]+; HRMS (TOF) calcd for C19H15N3O3: [M+H]+, 334.1192; found, 334.1197.
4.2.6. Synthesis of 7-(4-(1H-1,2,4-triazol-1-yl)butoxy)-2-phenyl-4H-chromen-4-one (6b)
Compound 6b was prepared according to the experimental procedure for compound 5a. White power. Yield:
64%; mp: 190–191 ℃; IR (KBr, cm-1) ν: 3034 (Ar–H), 1901 (aromatic frame), 1763 (C=O), 1625, 1598, 1575
1
(aromatic frame); H NMR (400 MHz, DMSO-d6) δ: 8.53 (s, 1H, triazole-5-H), 7.93 (s, 1H, triazole-3-H),
8.02–7.94 (m, 2H, Ph-2,6-2H), 7.53 (d, J = 8.5 Hz, 1H, flavone-5-H), 7.50–7.44 (m, 3H, Ph-3,4,5-3H), 7.24 (d, J
= 2.3 Hz, 1H, flavone-3-H), 6.85 (s, 1H, flavone-6-H), 6.79 (dd, J = 8.5, 2.2 Hz, 1H, flavone-8-H), 4.66 (q, J =
15

4.6 Hz, 2H, N-CH2), 4.55 (dd, J = 10.3, 5.3 Hz, 2H, O-CH2), 2.01 (m, 2H, CH2), 1.81 (m, 2H, CH2) ppm; 13C
NMR (101 MHz, DMSO-d6) δ: 182.1, 168.3, 166.3, 152.0, 147.6, 145.2, 132.4, 131.8, 131.4, 130.2, 129.1, 128.2,
125.4, 115.5, 113.2, 110.4, 97.2, 63.1, 44.1, 13.6, 12.1 ppm; MS (m/z): 362 [M+H]+; HRMS (TOF) calcd for
C21H19N3O3: [M+H]+, 362.1505; found, 362.1509.
4.2.7. Synthesis of 7-(2-(5-methyl-1H-tetrazol-1-yl)ethoxy)-2-phenyl-4H-chromen-4-one (6c)
Compound 6c was prepared according to the experimental procedure for compound 5a. White solid; Yield:
64%; mp: 201–202 ℃; IR (KBr, cm-1) ν: 3050 (Ar–H), 1805 (aromatic frame), 1716 (C=O), 1615, 1548, 1482
1
(aromatic frame); H NMR (400 MHz, DMSO-d6) δ: 8.01–7.95 (m, 2H, Ph-2,6-2H), 7.68 (d, J = 8.5 Hz, 1H,
flavone-5-H), 7.54–7.46 (m, 3H, Ph-3,4,5-3H), 7.22 (d, J = 2.0 Hz, 1H, flavone-3-H), 6.87 (s, 1H, flavone-6-H),
6.79 (dd, J = 8.5, 2.1 Hz, 1H, flavone-8-H), 5.13–5.06 (m, 2H, N-CH2), 4.76–4.69 (m, 2H, O-CH2), 2.47 (s, 3H,
CH3) ppm; 13C NMR (101 MHz, DMSO-d6) δ: 182.3, 168.3–168.1, 166.0, 163.1–162.9, 147.6, 132.7–132.5,
131.5, 129.4, 126.1, 114.8, 111.5, 98.4, 66.7, 52.1, 10.9 ppm; MS (m/z): 349 [M+H]+; HRMS (TOF) calcd for
C19H16N4O3: [M+H]+, 349.1301; found, 349.1308.
4.2.8. Synthesis of 7-(4-(5-methyl-1H-tetrazol-1-yl)butoxy)-2-phenyl-4H-chromen-4-one (6d)
Compound 6d was prepared according to the experimental procedure for compound 5a. White solid; Yield:
61%; mp: 204–205 ℃; IR (KBr, cm-1) ν: 3044 (Ar–H), 1815 (aromatic frame), 1725 (C=O), 1634, 1557, 1455
1
(aromatic frame); H NMR (400 MHz, DMSO-d6) δ: 8.10–7.99 (m, 2H, Ph-2,6-2H), 7.59 (d, J = 8.5 Hz, 1H,
flavone-5-H), 7.51–7.45 (m, 3H, Ph-3,4,5-3H), 7.19 (d, J = 3.2 Hz, 1H, flavone-3-H), 6.75 (s, 1H, flavone-6-H),
6.71 (dd, J = 8.3, 2.0 Hz, 1H, flavone-8-H), 5.16–5.08 (m, 2H, N-CH2), 4.68–4.62 (m, 2H, O-CH2), 2.31 (s, 3H,
CH3), 2.11 (m, 2H, CH2), 1.93 (m, 2H, CH2) ppm; 13C NMR (101 MHz, DMSO-d6) δ: 183.1, 169.0, 167.2, 164.9,
148.3, 133.6, 131.2, 129.5, 125.3, 114.6, 112.3, 98.5, 66.6, 53.1, 13.2, 10.9 ppm; MS (m/z): 376 [M+H]+; HRMS
(TOF) calcd for C21H20N4O3: [M+H]+, 376.4160; found, 376.4162.
4.2.9. Synthesis of 7-(2-((1H-imidazol-2-yl)thio)ethoxy)-2-phenyl-4H-chromen-4-one (7a)
Compound 7a was prepared according to the experimental procedure for compound 5a. Yellow solid; Yield:
62%; mp: 191192 ℃; IR (KBr, cm-1) ν: 3458 (NH), 3060 (Ar–H), 1805 (aromatic frame), 1717 (C=O), 1614,
1546, 1481 (aromatic frame); 1H NMR (400 MHz, DMSO-d6) δ: 8.01–7.96 (m, 2H, Ph-2,6-2H), 7.69 (d, J = 8.6
Hz, 1H, flavone-5-H), 7.54–7.45 (m, 3H, Ph-3,4,5-3H), 7.28 (d, J = 1.1 Hz, 1H, flavone-3-H), 7.22 (d, J = 2.0
Hz, 1H, imidazole-5-H), 6.98 (d, J = 1.2 Hz, 1H, imidazole-4-H), 6.86 (s, 1H, flavone-6-H), 6.83 (dd, J = 8.6,
2.1 Hz, 1H, flavone-8-H), 4.44–4.36 (m, 2H, N-CH2), 3.43 (t, J = 6.4 Hz, 2H, O-CH2) ppm; 13C NMR (101
16

MHz, DMSO-d6) δ: 182.1, 168.4, 166.6, 147.7, 140.1, 132.4, 131.6, 130.3, 129.5, 129.0, 126.0, 123.8, 113.5,
111.4, 68.1, 33.3, 32.4 ppm; MS (m/z): 365 [M+H]+; HRMS (TOF) calcd for C20H16N2O3S: [M+H]+, 365.0960;
found, 365.0962.
4.2.10. Synthesis of 7-(4-((1H-imidazol-2-yl)thio)butoxy)-2-phenyl-4H-chromen-4-one (7b)
Compound 7b was prepared according to the experimental procedure for compound 5a. Yellow solid; Yield:
57%; mp: 199201 ℃; IR (KBr, cm-1) ν: 3455 (NH), 3061 (Ar–H), 1815 (aromatic frame), 1750 (C=O), 1624,
1555, 1482 (aromatic frame); 1H NMR (400 MHz, DMSO-d6) δ: 8.02–7.95(m, 2H, Ph-2,6-2H), 7.77 (d, J = 8.2
Hz, 1H, flavone-5-H), 7.58–7.47 (m, 3H, Ph-3,4,5-3H), 7.25 (d, J = 1.6 Hz, 1H, flavone-3-H), 7.20 (d, J = 2.4
Hz, 1H, imidazole-5-H), 6.96 (d, J = 1.6 Hz, 1H, imidazole-4-H), 6.85 (s, 1H, flavone-6-H), 6.65 (dd, J = 8.4,
2.5 Hz, 1H, flavone-8-H), 4.45–4.31 (m, 2H, N-CH2), 3.45 (t, J = 6.2 Hz, 2H, O-CH2), 2.05 (m, 2H, CH2), 1.98
(m, 2H, CH2) ppm; 13C NMR (101 MHz, DMSO-d6) δ: 183.4, 169.3, 166.4, 147.6, 141.2, 134.3, 132.4, 131.4,
129.8, 129.0, 128.1, 123.6, 114.6, 112.3, 69.7, 33.4, 31.3, 14.3, 11.7 ppm; MS (m/z): 393 [M+H]+; HRMS (TOF)
calcd for C22H20N2O3S: [M+H]+, 393.1273; found, 393.1275.
4.3. PTPs inhibition assay
The PTPs were obtained from Prof. Zhang of Shangxi University and expressed and purified as
described previously [22]. The inhibitory effects and kinetics analysis of the azoyl flavonoids against PTPs
were measured, using p-nitrophenol phosphate (p-NPP) as the substrate [23]. Briefly, the assays were
performed on a 96-well plate in 20 mM MOPS buffer, pH 7.2, containing 50 mM NaCl. 10 L of the
compound at various concentrations was mixed with PTPs solution (82 L) for 35 min at 37℃. Then 2 L
of p-NPP (0.1 M) substrate was added. After incubation for about 30 min, the assays were terminated by
the addition of 6 L of 2 M NaOH. The released p-nitrophenolate ion was determined by measuring the
absorbance at 405 nm using microplate reader. The results of non-enzymatic hydrolysis of 2 L p-NPP
were compensated by measuring the control without enzyme addition. IC50 values were obtained by fitting
the concentration-dependent inhibition curves using the Origin program (Origin Lab, Northampton, MA).
The inhibiting kinetic analysis was carried out according to the Lineweaver-Burk plot analysis.
Phosphatase activities were measured at a fixed enzyme (PTP1B) concentration (0.2 mol/L) while
concentrations of the substrate (p-NPP) and the inhibitor 6a were varied. The data were fitted using Origin
program to generate the Lineweaver-Burk plot.
4.4. The influence of 6a on cell viability
17

The influence of 6a on cell viability toward HEK293 cells (which were gifts from Prof. Yang, college of
pharmacy, ChongQing Medical University, ChongQing, P. R. China) was determined by using Cell
Counting
Kit-8
(CCK8,
Yeasen
Company)
based
on
WST-8
(4-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-
tetraz-ol-3-ium-5-yl)benzene-1,3-disulfonate)
reduction assay following literature procedures [24]. The HEK293 cells (5000 cells per well) were seeded
into 96-well plates. The cells were then incubated in a culture medium containing compound 6a with a
particular concentration for 24 h. After that, 10 mL of CCK8 was added to each well. After 4 h, the
unreacted dye was removed by aspiration. The OD values were spectrophotometrically measured in an
ELISA plate reader (model 550, Bio-Rad) at a wavelength of 450 nm. The cell survival was expressed as
follows: cell viability = (OD treated/OD control)100%.
Acknowledgments
This work was partially supported by Natural Science Foundation of the Jiangsu Higher Education
Institutions of China (No. 17KJB150041), the practice inovation trainng program projects for the Jiangsu
College students (No. 201710324004X, 201710324020Z, 201710324126C).
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21

► Prepared azolyl flavonoids gave anti-PTP1B activity at the micromolar range.
► Compound 6a (IC50 = 1.6 μM) displayed 9.9-fold selectivity for PTP1B over TCPTP.
► Cell viability assays indicate 6a has lower cytotoxicity.
► Molecular modeling and dynamics studies reveal the reason of selectivity.
22

Design, Synthesis of Novel Azolyl Flavonoids and Their Protein Tyrosine Phosphatase-1B Inhibitory Activities
Ling Zhang1,*, Yu Ge1, Hao Ming Song1, Qing Ming Wang1,* and Cheng-He Zhou2
1* School of Pharmacy, Yancheng Teachers’ University, Yancheng, Jiangsu 224051, People’s Republic of China.
2 Laboratory of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing,
400715, P.R. China
Triazolyl flavonoid 6a displayed the best inhibitory activity (IC50 = 1.6 μM) with good selectivity for PTP1B
over TCPTP. Cell viability assays indicate 6a is cell permeable with lower cytotoxicity. Molecular modeling and
dynamics studies reveal the reason of selectivity for PTP1B over TCPTP.
IC50 = 1.6 μmol/L
23