Developing piperlongumine-directed glutathione S-transferase inhibitors by an electrophilicity-based strategy

Article abstract of DOI:10.1016/j.ejmech.2016.11.034

We report a case of successful design of glutathione S-transferase (GST) inhibitors via a natural product-inspired and electrophilicity-based strategy. Based on this strategy, a novel piperlongumine analog (PL-13) bearing a para-trifluoromethyl group and an α-chlorine on its aromatic and lactam rings, respectively, surfaced as a promising GST inhibitor, thereby overcoming cisplatin resistance in lung cancer A549 cells.

Full text of DOI:10.1016/j.ejmech.2016.11.034

European Journal of Medicinal Chemistry 126 (2017) 517e525
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Developing piperlongumine-directed glutathione S-transferase
inhibitors by an electrophilicity-based strategy
Hai-Bo Wang, Xiao-Ling Jin, Jia-Fang Zheng, Fu Wang, Fang Dai, Bo Zhou*
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, 222 Tianshui Street S., Lanzhou, Gansu 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We report a case of successful design of glutathione S-transferase (GST) inhibitors via a natural product-
inspired and electrophilicity-based strategy. Based on this strategy, a novel piperlongumine analog (PL-
13) bearing a para-trifluoromethyl group and an a-chlorine on its aromatic and lactam rings, respectively,
Received 29 September 2016
Received in revised form
29 October 2016
Accepted 14 November 2016
Available online 15 November 2016
surfaced as a promising GST inhibitor, thereby overcoming cisplatin resistance in lung cancer A549 cells.
© 2016 Elsevier Masson SAS. All rights reserved.
Keywords:
Glutathione S-transferases
Piperlongumine
Electrophilicity
Multiple drug resistance
1. Introduction
substrate, GSH or a previously applied chemosensitizer, ethacrynic
acid [15]. To our knowledge, however, there are few researches
Glutathione S-transferases (GSTs, EC 2.5.1.18), as a superfamily
of phase II detoxification system, catalyze the conjugation of the
reduced glutathione (GSH) to electrophilic centers of cytotoxic
substances, rendering them water soluble to facilitate their elimi-
nation from cells [1,2]. According to their cellular localization, GSTs
can be divided into three family members, that is, the cytosolic,
microsomal and mitochondrial GSTs. In mammals, at least seven
classes cytosolic GSTs have been recognized, namely, alpha (A), mu
(M), pi (P), sigma (S), zeta (Z), theta (T) and omega (O) [3]. Among
the various GSTs, they share the structural similarity of the active
site, which contains a highly specific GSH binding site (G-site) and a
less similar hydrophobic pocket (H-site) [4,5]. GSTs are often
overexpressed in many cancer cells [6,7], and contribute to the
development of resistance by these cells [1] (such as melanoma [8]
and lung cancer [9] cells) towards various anticancer drugs. GST
inhibition has, therefore, been proposed as a potential approach to
overcome multiple drug resistance [10]. For this reason, the past
few decades have witnessed much interest in discovering and
designing various GST inhibitors [9e17] with the aim of developing
them as adjuvants/sensitizers in chemotherapy.
focusing on design of GST inhibitors by a natural product-inspired
and electrophilicity-based strategy. We considered that it might
represent a promising strategy in the development of GST in-
hibitors based on the following reasons: (1) Natural products with
extraordinarily diverse chemical scaffolds provide an important
source of inspiration for developing new class of inhibitors. (2) The
long list of GST inhibitors characterizes a suite of electrophiles
including ethacrynic acid [18] and its analogs [12], curcumin
[18e20], benzyl isothiocyanate [13,21], quinones [11,22e24] and
others. (3) Cysteine residues in GSTs (such as Cys47 and Cys101 of
GSTP1-1 [12,13,25]) are known to be susceptible to covalent
modification by electrophiles which results in inactivation of this
enzyme. (4) For an electrophilic substrate as exemplified by etha-
crynic acid [26,27] and benzyl isothiocyanate [13,21], the inacti-
vation occurs usually by two possible pathways: formation of the
electrophile-GST adduct and the electrophile-GSH conjugate; no
matter what kind of mechanism is employed, electrophilicity
should be an important determinant for the inactivation.
Piperlongumine (PL, Table 1), an alkaloid isolated from Piper
longum L. [28,29], is characterized by the presence of two
a, b-
Most of inhibitors have been designed by mimicking its primary
unsaturated imide functionalities, and has been previously identi-
fied as a promising anticancer molecule by targeting the stress
response to ROS [28], resulting in much effort placed in its chemical
modifications and biological activity exploration [30e34]. This
molecule has been suggested to induce apoptosis by interfering
* Corresponding author.
E-mail address: bozhou@lzu.edu.cn (B. Zhou).
http://dx.doi.org/10.1016/j.ejmech.2016.11.034
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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H.-B. Wang et al. / European Journal of Medicinal Chemistry 126 (2017) 517e525
Table 1
GST-inhibitory activity and electrophilicity of PL and its designed analogs.
d
d
Compounda
Inhibition Rate (%)b
30.7 1.2
IC50
(m
M)c
k2 (Mꢀ1sꢀ1
)
Compounda
Inhibition Rate (%)b
30.8 4.6
IC50
(
m
M)c
k2 (Mꢀ1sꢀ1
)
>100
>100
>100
>100
>100
>100
>100
0.46 0.02
0.35 0.02
e
>100
0.99 0.05
0.39 0.01
1.13 0.08
1.35 0.05
0.67 0.01
2.53 0.11
2.64 0.05
15.4 0.6
23.8 4.5
0
31.5 1.0
53.0 8.6
55.5 5.2
40.3 1.0
71.0 6.0
74.7 7.6
>100
50.1 7.5
41.6 5.8
>100
e
2.6 3.6
8.2 2.8
50.5 0.9
0.44 0.01
1.21 0.05
1.15 0.03
10.5 1.0
5.6 0.7
All data listed in the table are the mean SD of triplicate determinations.
a
Molecular structures.
The percent (%) inhibition of compound tested at 100 mM towards equine liver GST.
IC50 value is the concentration of inhibitor to cause 50% inhibition of GST activity.
Reactions were carried out in Tris-HCl (100 mM, pH 7.4) containing EDTA (2 mM)-ethanol (v/v, 2/1) under pseudo-first-order conditions at 25 ꢁC.
b
c
d
e
Not determined due to spectral overlapping.
with redox and ROS homeostatic regulators such as GSTP1 [28], and
used as a GSTP1 inhibitor [35]. Thus, as part of our research project
in designing natural product-inspired anticancer agents [36e40],
we selected this electrophilic molecule as a lead to develop potent
GST inhibitors and to clarify the feasibility of an electrophilicity-
based strategy in designing the inhibitors. In this work, the com-
pounds (PL-1, PL-2, PL-3, PL-4 and PL-5, Table 1) were designed to
explore the structural requirements of PL for its GST-inhibitory
activity, including the two Michael acceptor units, the three
methoxyl groups and the aromatic ring. To moderately improve the
electrophilicity of PL, several structural modifications were applied
(Table 1), including replacement of the nitrogen atom of lactam
entails coupling the corresponding freshly prepared acryloyl chlo-
ride and synthetically accessible lactams (5,6-dihydropyridin-
2(1H)-one (4) or 3-chloro-5,6-dihydropyridin-2(1H)-one (5)) ac-
cording to the published procedures [39,40] (Scheme 1). The syn-
thesis of compounds 4 [39] and 5 [40] has been reported in our
previous papers. Additionally, PL-3 was obtained by catalytic hy-
drogenation of PL-1 over Pd/C.
2.2. Inhibition of equine liver GST
Initial screening was conducted by determining the inhibitory
effects of PL and its analogs on conjugation of 1-chloro-2,4-
dinitrobenzene (CDNB) to GSH [41] and using commercially avail-
able and low-cost equine liver GST, which shares a structure ho-
mology with human GSTA [42]. The results listed in Table 1 allowed
us to identify the following structureeactivity relationships: (1) PL
showed moderate inhibition of equine liver GST with a rate of
30.7%; Either of the two Michael acceptor units is essential for the
GST-inhibitory activity of PL as illustrated by the decreased effi-
ciency of PL-1 and PL-2 along with the inactivity of PL-3; Addi-
tionally, both the three methoxyl groups (PL-4) and the aromatic
ring (PL-5) contribute to the activity of this parent molecule. (2)
Replacement of the nitrogen atom of lactam with a carbon atom
(racemic PL-6) resulted in the increased activity. (3) Introduction of
a meta- or para-trifluoromethyl group (PL-9 and PL-10), rather than
an ortho-one (PL-8), on the aromatic ring tended to increase the
with
withdrawing
a
carbon atom (PL-6), introduction of an electron-
-chlorine on the lactam ring (PL-7), placement of
a
an electron-withdrawing trifluoromethyl group on the aromatic
ring in the ortho (PL-8), meta (PL-9), or para (PL-10) position to the
exocyclic olefin and simultaneous introduction of an
a trifluoromethyl group (PL-11, PL-12 and PL-13).
a-chlorine and
2. Results and discussion
2.1. Chemical synthesis
The compounds (PL, PL-1, PL-2, PL-4, PL-5 and PL-6 [39] as well
as PL-7 [40]) were prepared as reported in our previous papers.
Other analogs were constructed by a convergent strategy that

H.-B. Wang et al. / European Journal of Medicinal Chemistry 126 (2017) 517e525
519
Scheme 1. Synthesis of the designed PL analogs. (a) Malonic acid, Pyridine, Piperidine, 95e100 ꢁC, 3 h; (b) SOCl2, CH2Cl2, reflux, 4 h; (c) NaH, THF, 0 ꢁC-rt, 18e24 h; (d) Pd/C, H2, rt,
24 h.
activity. (4) Despite the presence of an
a-chlorine having no
2.4. Theoretical calculation
appreciable effect on the activity of PL, it increased significantly the
activity of trifluoromethyl-containing molecules as exemplified by
PL-11, PL-12 and PL-13. Among the tested compounds, PL-13,
characterized by the presence of a para-trifluoromethyl group and
Interestingly, among all the trifluoromethyl-containing mole-
cules, its position on the aromatic ring is critically important for
improving either the GST-inhibitory activity or the electrophilicity
with the activity order of para (meta) > ortho. To clarify the reason
why the molecules bearing ortho-trifluoromethyl group exhibited
the weaker electrophilicity and GST-inhibitory activity than those
bearing para (meta)-trifluoromethyl groups, we calculated the ge-
ometries of PL-11, PL-12 and PL-13 in the lowest energy confor-
mation via the Gaussian 09 program [44]. As shown in Fig. 2, PL-12
and PL-13 displayed a planar geometry conformation between the
aromatic ring and exocyclic C-C olefin, whereas, PL-11 bearing an
ortho-trifluoromethyl group exhibited a dihedral angle of 28ꢁ. The
significant conformational shift out of planarity might reduce its
reactivity with the active cysteine residues of GST or/and small
molecule thiols due to steric effect.
an
a
-chlorine, was the most active with an inhibition rate of 74.7%
M, at least 18-fold more active than the
and an IC50 value of 5.6
m
parent PL by comparing their IC50 values.
2.3. Evaluation of electrophilicity
The structure-activity data support that the electrophilicity
plays a vital role in the GST-inhibitory activity of PL and its analogs.
To further clarify this point, we evaluated their electrophilicity by
measuring the second-order rate constants (k2) for their reaction
with cysteamine under pseudo-first-order condition according to
the method established by Amslinger et al. [43]. As expected,
among the test compounds, PL-13 demonstrated its strongest
electrophilicity with a k2 value of 2.64 Mꢀ1 sꢀ1 (Table 1), in line with
its most potent GST-inhibitory activity. Furthermore, plotting the k2
values versus the GST-inhibitory rates gave a straight line with a
relatively weak correlation coefficient of 0.59 (Fig. 1A) for all the
test compounds. However, a large deviation from the straight line
was observed in the case of PL-5. Considering that there is a large
structural difference between PL-5 lacking the aromatic ring and
the other molecules, we replotted this figure in the absence of PL-5
and found a strong correlation coefficient of 0.85 (Fig. 1B). The
strong positive correlation indicates that the electrophilicity is an
important determinant for their GST-inhibitory activity, although
other factors also work.
2.5. GST-inhibitory mechanism and Ki determinations
To clarify the mechanism by which PL-13 displayed the equine
liver GST-inhibitory activity, the kinetic inactivation study of the
enzyme was performed according to the procedure reported by
Ang et al. [12] with some modifications. Steady-state kinetic ex-
periments demonstrated that PL-13 was noncompetitive versus 1-
chloro-2,4-dinitrobenzene (CDNB) (KCi DNB ¼ 24.7
mM, Fig. 3A and B)
mM, Fig. 3C and D), indi-
but was competitive for GSH (KGi SH ¼ 7.8
cating that PL-13 is a competitive inhibitor towards GSH and binds
at the G-site, but not the hydrophobic H-site of equine liver GST.
Thus, PL-13 inactivates this enzyme probably by modifying directly
cysteine residues and/or by formation of the PL-13-GSH conjugate.

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H.-B. Wang et al. / European Journal of Medicinal Chemistry 126 (2017) 517e525
Fig. 1. Correlation analysis between the GST-inhibitory activity and the electrophilicity of PL and its designed analogs.
Fig. 2. Geometries of PL-11, PL-12 and PL-13 in the lowest energy conformation.
Fig. 3. Lineweaver-Burk plots. (A) Reciprocal velocity versus reciprocal [CDNB] by varying CDNB concentration at a fixed GSH concentration (1 mM). (B) Determination of the Ki
values by plotting the intercept from A versus [PL-13]. (C) Reciprocal velocity versus reciprocal [GSH] by varying GSH concentration at a fixed CDNB concentration (1 mM). (D)
Determination of the Ki values by plotting the slope from C versus [PL-13]. Data represents the mean of triplicate determinations.

H.-B. Wang et al. / European Journal of Medicinal Chemistry 126 (2017) 517e525
521
2.6. Cytotoxicity assay and combination index
at a low-toxic concentration (5
mM) according to the method as
described previously [9]. As shown in Fig. 4, 5
mM PL-13 improved
With these results in hand, we next wondered if PL-13 could
serve as an intracellular GST inhibitor to sensitize cancer cells
resistant to chemotherapeutic drugs. Accordingly, we chose lung
cancer A549 cells (resistant to cisplatin partially by GSTM1 [9]) as a
model to evaluate impact of PL-13 on cisplatin-induced cell death
obviously the efficacy of cisplatin (2.5, 5,10 and 20
m
M) and induced
synergistically death of A549 cells (combination index < 1),
whereas the parent PL failed to exhibit this effect. The synergistic
interaction shows clearly that PL-13 can sensitize A549 cells
resistant to cisplatin. Furthermore, the synergistic interaction was
completely reversed by addition of dithiothreitol (DTT), a thiol-
containing compound as an effective competitor for intracellular
nucleophiles, highlighting that the Michael acceptor units (elec-
trophilicity) of PL-13 are crucial for its sensitization.
2.7. GST activity in living A549 cells
Lastly, to elucidate whether PL-13 achieved the above sensiti-
zation by targeting intracellular GSTs, we employed a GST fluo-
rescent probe (DNs-CV) developed by Zhang et al. [45] to image the
GST activity in living A549 cells. When the cells were incubated
with DNs-CV for 0.5 h, a strong red fluorescence appeared (Fig. 5A).
However, the fluorescence signal was inhibited significantly by
1.5 h of pretreatment with PL-13 (Fig. 5D). In contrast, the parent PL
has no appreciable effect on the signal under the same conditions
(Fig. 5C). Noticeably, the PL-13-induced signal attenuation was
largely abrogated by pretreating the cells with DTT (Fig. 5F). The
above experiments provides crucial evidence that PL-13 indeed
targets intracellular GSTs, thereby overcoming cisplatin resistance
in lung cancer A549 cells, and further emphasizes that PL-13 is a
more effective GST inhibitor than the parent PL in terms of its
increased moderately electrophilicity.
3. Conclusions
In summary, this work emphasizes a natural product-inspired
and electrophilicity-based strategy to develop GST inhibitors. This
strategy is exemplified by PL and its designed analogs. Among
which, a novel molecule, PL-13 surfaced as a promising GST in-
hibitor to effectively overcome cisplatin resistance in lung cancer
A549 cells (Scheme 2).
4. Experimental section
4.1. General information
Lyophilized GST from equine liver, Roswell Park Memorial
Institute (RPMI)-1640, cisplatin,
amine were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
L-glutathione (GSH) and cyste-
(MTT) was purchased from Amresco (Solon, OH, USA). Human lung
cancer A549 cells were obtained from the Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences
(Shanghai, China). GST fluorescent probe (DNs-CV) was synthesized
as previously reported [45]. Unless otherwise stated, all materials
were purchased from commercial sources and used without further
purification.
1H NMR and 13C NMR spectra were recorded with a Bruker
AVIII-400 MHz spectrometer (Bruker Daltonic Inc., Bremen, Ger-
many). The chemical shifts were reported in parts per million
(ppm) relative to the solvent peak. The electron spray ionization
mass spectra (ESI-MS) and high resolution mass spectra (HRMS)
were recorded with Bruker micrOTOF II mass spectrometer (Bruker
Daltonic Inc., Bremen, Germany) and Orbitrap Elite (Thermo Sci-
entific), respectively. The melting points were determined with X-
4B melting point apparatus and are uncorrected. The purity of the
target molecules was further checked by HPLC analysis using a
Waters liquid chromatograph system equipped with a 1525 binary
Fig. 4. Effect of 5
mM PL (A) or PL-13 (B) on the death of A549 cells induced by
different concentrations of cisplatin (2.5, 5, 10 and 20 mM), and the related combination
index analysis (C) using CalcuSyn software. A549 cells (3 ꢂ 104 cells/mL) were pre-
treated with or without DTT (250
mM) for 1 h, and then treated with PL or PL-13
(5
mM) for 2 h, followed by incubation with various concentration of cisplatin for
another 48 h. Each experiment was performed in triplicate. *P < 0.05, ***P < 0.001.

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H.-B. Wang et al. / European Journal of Medicinal Chemistry 126 (2017) 517e525
Fig. 5. Fluorescence images for the GST activity in living A549 cells in the presence of (A) DNs-CV, (B) DNs-CV and DTT, (C) DNs-CV and PL, (D) DNs-CV and PL-13, (E) DNs-CV, PL
and DTT, (F) DNs-CV, PL-13 and DTT. A549 cells (2 ꢂ 105 cell/mL) were pretreated with or without DTT (500
mM) for 1 h, and then treated with or without PL or PL-13 (25 mM) for
1.5 h followed by incubation with DNs-CV (2
mM) for 0.5 h. Scale bar ¼ 50
mm.
Scheme 2. Proposed mechanisms for the PL-13-induced sensitization in cisplatin-resistant A549 cells.
HPLC pump, a 2998 photodiode array detector and a reverse phase
route described in Scheme 1 and the details are described as below.
C-18 column (Sun Fire™, 4.6 mm ꢂ 150 mm).
4.2.1. Synthesis of substituted cinnamic acids (2a-2c) (step a in
Scheme 1) [39]
4.2. Synthesis of the PL and its analogs
To a mixture of the corresponding aldehyde (10 mmol), pyridine
(6 mL) and piperidine (0.6 mL) was added malonic acid (1.25 g,
12 mmol). The mixture was stirred for 3 h at 90e100 ꢁC, then
cooling to room temperature. The reaction mixture was neutralized
Synthesis of PL and its analogs including PL, PL-1, PL-2, PL-4,
PL-5 and PL-6 [39] as well as PL-7 [40] was reported in our pre-
vious papers, and other analogs were synthesized according to the

H.-B. Wang et al. / European Journal of Medicinal Chemistry 126 (2017) 517e525
523
with hydrochloric acid solution (10 mol/L, 80 mL) at 0 ꢁC. The white
solid was filtered, washed with cold water, and dried to afford the
corresponding acid.
3.3 mmol, 1.1 equiv) according to the general procedure. The re-
action mixture was stirred for 20 h. The crude product was purified
by flash column chromatography (petroleum ether/ethyl acetate, 4/
1) to give PL-10 (312 mg).
4.2.2. Synthesis of substituted cinnamic acid chlorides (3a-3c) (step
b in Scheme 1) [39]
(E)-1-(3-(4-(Trifluoromethyl)phenyl)acryloyl)-5,6-
dihydropyridin-2(1H)-one (PL-10). White solid, yield 35.2%; M.p.
To a solution of the corresponding acid (3 mmol) in dry CH2Cl2
(6 mL) was added thionyl chloride (0.65 mL, 9 mmol). The mixture
was heated to reflux for 4 h under dry condition. The solvent was
removed under reduced pressure to give the corresponding acyl
chloride. The crude product was used to the next step without
further purification.
107e109 ꢁC; Purity 99.22% (H2O/MeOH ¼ 40/60, Rt ¼ 17 min); 1H
NMR (400 MHz, CDCl3)
d
7.72 (d, J ¼ 15.6 Hz,1H), 7.69e7.62 (m, 4H),
7.56 (d, J ¼ 15.6 Hz, 1H), 6.97 (dt, J ¼ 10.0, 4.0 Hz, 1H), 6.05 (dt,
J ¼ 10.0, 2.0 Hz, 1H), 4.06 (t, J ¼ 6.4 Hz, 2H), 2.52e2.48 (m, 2H); 13
C
NMR (100 MHz, CDCl3)
d 168.4, 165.8, 145.8, 141.2, 138.5, 131.4,
128.3, 125.7, 124.4, 123.9, 41.6, 24.7; HRMS (ESI): m/z calculated for
[M þ Na]þ: 318.0712, found: 318.0708, error ¼ 1.3 ppm.
4.2.3. Synthesis of the PL analogs (PL-8, PL-9, PL-10, PL-11, PL-12
and PL-13) (step c in Scheme 1)
4.2.3.4. Synthesis of PL-11. Compound PL-11 was synthesized from
(E)-3-(2-(trifluoromethyl)phenyl)acryloyl chloride (3a) (702 mg,
3 mmol, 1 equiv) and 3-chloro-5,6-dihydropyridin-2(1H)-one (5)
(432 mg, 3.3 mmol, 1.1 equiv) according to the general procedure.
The reaction mixture was stirred for 18 h. The crude product was
purified by flash column chromatography (petroleum ether/ethyl
acetate, 4/1) to give PL-11 (188 mg).
To a solution of 5,6-dihydropyridin-2(1H)-one [39] or 3-chloro-
5,6-dihydropyridin-2(1H)-one (3.3 mmol) [40] at 0 ꢁC in tetrahy-
drofuran (15 mL) was added the corresponding acyl chloride
(3 mmol). NaH (3.3 mmol) was slowly added, and the reaction
mixture was stirred for 2 h at 0 ꢁC. After 2 h, the stirring was
continued at room temperature for 18e24 h under dry air. The
reaction mixture was poured into appropriate ice water, stirred for
a few minutes, and then extracted with ethyl acetate. The combined
organic phases were dried (Na2SO4), and the solvent was evapo-
rated under reduced pressure. The residue was purified by flash
column chromatography (petroleum ether/ethyl acetate) to give
the target molecules.
(E)-3-Chloro-1-(3-(2-(trifluoromethyl)phenyl)acryloyl)-5,6-
dihydropyridin-2(1H)-one (PL-11). White solid, yield 19%; M.p.
118e119 ꢁC; Purity 99.43% (H2O/MeOH ¼ 35/65, Rt ¼ 11 min); 1H
NMR (400 MHz, CDCl3)
d
8.11 (dq, J ¼ 15.6, 2.0 Hz, 1H), 7.84 (d,
J ¼ 8.0 Hz, 1H), 7.70 (d, J ¼ 8.0 Hz, 1H), 7.56 (t, J ¼ 7.6 Hz, 1H),
7.49e7.44 (m, 2H), 7.11 (t, J ¼ 4.4 Hz, 1H), 4.11 (t, J ¼ 6.4 Hz, 2H),
2.62e2.57 (m, 2H); 13C NMR (100 MHz, CDCl3)
d 167.8, 161.4, 141.3,
4.2.3.1. Synthesis of PL-8. Compound PL-8 was synthesized from
(E)-3-(2-(trifluoromethyl)phenyl)acryloyl chloride (3a) (702 mg,
3 mmol, 1 equiv) and 5,6-dihydropyridin-2(1H)-one (4) (320 mg,
3.3 mmol, 1.1 equiv) according to the general procedure. The re-
action mixture was stirred for 22 h. The crude product was purified
by flash column chromatography (petroleum ether/ethyl acetate, 4/
1) to give PL-8 (514 mg).
139.4, 133.8, 132.0, 129.5, 128.9, 128.3, 128.1, 126.0, 125.3, 124.0, 41.7,
25.3; HRMS (ESI): m/z calculated for [M þ Na]þ: 352.0323, found:
352.0318, error ¼ 1.4 ppm.
4.2.3.5. Synthesis of PL-12. Compound PL-12 was synthesized from
(E)-3-(3-(trifluoromethyl)phenyl)acryloyl chloride (3b) (702 mg,
3 mmol, 1 equiv) and 3-chloro-5,6-dihydropyridin-2(1H)-one (5)
(432 mg, 3.3 mmol, 1.1 equiv) according to the general procedure.
The reaction mixture was stirred for 18 h. The crude product was
purified by flash column chromatography (petroleum ether/ethyl
acetate, 4/1) to give PL-12 (532 mg).
(E)
-1-(3-(2-(Trifluoromethyl)phenyl)acryloyl)-5,6-
dihydropyridin-2(1H)-one (PL-8). White solid, yield 58.1%; M.p.
99e101 ꢁC; Purity 95.13% (H2O/MeOH ¼ 40/60, Rt ¼ 12 min); 1H
NMR (400 MHz, CDCl3)
d
8.08 (dq, J ¼ 15.6, 2.0 Hz, 1H), 7.81 (d,
J ¼ 8.0 Hz, 1H), 7.69 (d, J ¼ 7.6 Hz, 1H), 7.55 (t, J ¼ 7.6 Hz, 1H), 7.48 (d,
J ¼ 15.6 Hz, 1H), 7.46 (t, J ¼ 7.46 Hz, 1H), 6.95 (dt, J ¼ 10.0, 4.0 Hz,
1H), 6.05 (dt, J ¼ 10.0, 1.6 Hz, 1H), 4.06 (t, J ¼ 6.4 Hz, 2H), 2.52e2.47
(E)-3-Chloro-1-(3-(3-(trifluoromethyl)phenyl)acryloyl)-5,6-
dihydropyridin-2(1H)-one (PL-12). White solid, yield 53.9%; M.p.
130e131 ꢁC; Purity 95.29% (H2O/MeOH ¼ 30/70, Rt ¼ 8 min); 1H
(m, 2H); 13C NMR (100 MHz, CDCl3)
d
168.2, 165.7, 145.7, 138.4,
NMR (400 MHz, CDCl3) d 7.81 (s, 1H), 7.78e7.74 (m, 2H), 7.63 (d,
134.1, 132.0, 129.1, 128.7, 128.2, 126.0, 125.9, 125.8, 124.0, 41.6, 24.7;
J ¼ 7.6 Hz, 1H), 7.54 (d, J ¼ 15.6 Hz, 1H), 7.51 (t, J ¼ 7.6 Hz, 1H), 7.12 (t,
MS (ESI): (m/z) ¼ 318.0337 [M þ Na]þ.
J ¼ 4.4 Hz, 1H), 4.11 (t, J ¼ 6.4 Hz, 2H), 2.62e2.57 (m, 2H); 13C NMR
(100 MHz, CDCl3)
d 168.1, 161.4, 142.4, 141.3, 135.6, 131.4, 131.3,
4.2.3.2. Synthesis of PL-9. Compound PL-9 was synthesized from
(E)-3-(3-(trifluoromethyl)phenyl)acryloyl chloride (3b) (702 mg,
3 mmol, 1 equiv) and 5,6-dihydropyridin-2(1H)-one (4) (320 mg,
3.3 mmol, 1.1 equiv) according to the general procedure. The re-
action mixture was stirred for 24 h. The crude product was purified
by flash column chromatography (petroleum ether/ethyl acetate, 4/
1) to give PL-9 (630 mg).
129.4, 128.2, 126.6, 124.9, 123.9, 123.1, 41.8, 25.3; HRMS (ESI): m/z
calculated for [M
error ¼ 2.0 ppm.
þ
Na]þ: 352.0323, found: 352.0316,
4.2.3.6. Synthesis of PL-13. Compound PL-13 was synthesized from
(E)-3-(4-(trifluoromethyl)phenyl)acryloyl chloride (3c) (702 mg,
3 mmol, 1 equiv) and 3-chloro-5,6-dihydropyridin-2(1H)-one (5)
(432 mg, 3.3 mmol, 1.1 equiv) according to the general procedure.
The reaction mixture was stirred for 18 h. The crude product was
purified by flash column chromatography (petroleum ether/ethyl
acetate, 4/1) to give PL-13 (325 mg).
(E)-1-(3-(3-(Trifluoromethyl)phenyl)acryloyl)-5,6-
dihydropyridin-2(1H)-one (PL-9). White solid, yield 71.2%; M.p.
69e70 ꢁC; Purity 97.35% (H2O/MeOH ¼ 40/60, Rt ¼ 15 min); 1H
NMR (400 MHz, CDCl3)
d
7.80e7.71 (m, 3H), 7.61 (d, J ¼ 7.6 Hz, 1H),
7.56e7.49 (m, 2H), 6.97 (dt, J ¼ 10.0, 4.0 Hz, 1H), 6.06 (d, J ¼ 10.0 Hz,
(E)-3-Chloro-1-(3-(4-(trifluoromethyl)phenyl)acryloyl)-5,6-
dihydropyridin-2(1H)-one (PL-13). White solid, yield 33%; M.p.
105e109 ꢁC; Purity 99.75% (H2O/MeOH ¼ 35/65, Rt ¼ 14 min); 1H
1H), 4.05 (t, J ¼ 6.4 Hz, 2H), 2.52e2.48 (m, 2H); 13C NMR (100 MHz,
CDCl3)
d 168.5, 165.8, 145.8, 141.4, 135.9, 131.3, 129.3, 126.3, 125.6,
124.7, 123.8, 41.6, 24.7; MS (ESI): (m/z) ¼ 318.0356 [M þ Na]þ.
NMR (400 MHz, CDCl3)
d
7.75 (d, J ¼ 15.6 Hz, 1H), 7.69 (d, J ¼ 8.4 Hz,
2H), 7.64 (d, J ¼ 8.4 Hz, 2H), 7.55 (d, J ¼ 15.6 Hz, 1H), 7.12 (t,
4.2.3.3. Synthesis of PL-10. Compound PL-10 was synthesized from
(E)-3-(4-(trifluoromethyl)phenyl)acryloyl chloride (3c) (702 mg,
3 mmol, 1 equiv) and 5,6-dihydropyridin-2(1H)-one (4) (320 mg,
J ¼ 4.4 Hz, 1H), 4.11 (t, J ¼ 6.4 Hz, 2H), 2.62e2.58 (m, 2H); 13C NMR
(100 MHz, CDCl3)
d 168.1, 161.5, 142.3, 141.4, 138.2, 131.6, 128.4,
128.1, 125.7, 123.8, 123.6, 41.7, 25.3; HRMS (ESI): m/z calculated for

524
H.-B. Wang et al. / European Journal of Medicinal Chemistry 126 (2017) 517e525
[M þ Na]þ: 352.0323, found: 352.0319, error ¼ 1.1 ppm.
4.7. Cytotoxicity assay and combination index
The inhibitory effect of the compounds on cell viability was
evaluated by the well-known MTT assay. Briefly, A549 cells
(3 ꢂ 104 cells/mL) were seeded in 96-well plate and cultured for
4.2.4. Synthesis of PL-3 (step d in Scheme 1) [46]
To a solution of PL-1 (960 mg, 3 mmol) in ethanol (40 mL) was
added 10% Pd/C (0.2 g). The reaction mixture was stirred for 24 h at
room temperature under hydrogen. The catalyst was filtered away
and then the solvent was evaporated. The crude product was
further purified by flash column chromatography (petroleum
ether/ethyl acetate, 15/1) to give PL-3 (900 mg).
24 h. Then, the cells were pretreated with or without DTT (250
m
M)
M) for 2 h, followed
by incubation with various concentration of cisplatin for another
48 h. Thereafter, the medium was removed, 100 L RPMI-1640
for 1 h, and then treated with PL or PL-13 (5
m
m
medium with MTT solution was supplemented and cells were
1-(3-(3,4,5-Trimethoxyphenyl)propanoyl)piperidin-2-one (PL-3).
incubated for 4 h at 37 ꢁC in the dark. The medium was then
White solid, yield 93.5%; M.p. 83e84 ꢁC; Purity 95.43% (H2O/
MeOH ¼ 50/50, Rt ¼ 11 min); 1H NMR (400 MHz, CDCl3)
d
6.45 (s,
removed and 100 mL DMSO was added into each well. The optical
density was measured at 570 nm using a Bio-Rad M680 microplate
reader. The percentage of cell viability was calculated by comparing
with control cells. Combination Index was calculated using Calcu-
Syn software (Biosoft, Ferguson, MO, USA).
2H), 3.84 (s, 6H), 3.81 (s, 3H), 3.71 (t, J ¼ 6.0 Hz, 2H), 3.22 (t,
J ¼ 7.6 Hz, 2H), 2.90 (t, J ¼ 7.6 Hz, 2H), 2.55e2.51 (m, 2H), 1.82e1.80
(m, 4H); 13C NMR (100 MHz, CDCl3)
d 176.1, 173.4, 153.1, 137.0, 136.2,
105.4, 60.8, 56.0, 44.0, 41.3, 34.9, 31.5, 22.4, 20.2; MS (ESI): (m/
z) ¼ 344.1092 [M þ Na]þ.
4.8. Living cell imaging
4.3. Inhibition of equine liver GST and Ki determination
A549 cells (2 ꢂ 105 cells/mL) were plated in 6-well plate and
cultured for 24 h. The cells were pretreated with or without DTT
Inhibition rates, IC50 values and Ki values were measured by
using the procedure reported by Ang et al. [12] with some modi-
fications, which was adapted for use in 96-well plates. Briefly, for
(500
(25
mM) for 1 h, and then treated with or without PL or PL-13
m
M) for 1.5 h, followed by incubation with DNs-CV (2 mM) for
0.5 h at 37 ꢁC in dark. The fluorescent images were obtained by
the inhibition rate experiments, equine liver GST (2.25
mg) was
Leica DM 4000B microscope.
incubated with inhibitor (100 M) in 100 mM potassium phosphate
m
buffer (pH 6.5) containing 1 mM GSH at 37 ꢁC for 30 min (final
volume 0.3 mL). The reaction was initiated with 1-chloro-2,4-
dinitrobenzene (CDNB, final concentration 1 mM) and detected at
340 nm for 3 min by TECAN Infinite M200 multimode microplate
reader (25 ꢁC). The IC50 values were determined by fitting the in-
hibition rates against the corresponding inhibitor concentrations
with sigmoidal analysis. Ki values were assayed by fixing the GSH
concentration at 1 mM and varying the CDNB concentrations (from
0.1 to 1 mM) and vice versa, at a PL-13 concentration (0, 12.5, 25 or
4.9. Statistical analysis
Data are the mean SD. Statistical significance was performed
using a one-way ANOVA followed by LSD analysis. P < 0.05 was
considered as significant difference.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant No. 21372109) and Gansu Key Tech-
nologies R & D Program (143NKDA028).
50
mM) and a fixed equine liver GST concentration (15 mg/mL).
Lineweaver-Burk plots were plotted with reciprocal velocity versus
reciprocal GSH/CDNB concentration from which KCi DNB and KiGSH
values were calculated.
Appendix A. Supplementary data
4.4. Determination of k2 values
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.11.034.
Reactions were investigated by fixing individual compound
concentration (40
mM) and varying cysteamine concentration
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wavelength maxima by using a Varian Cary 300 Spectrophotom-
eter. The second-order rate constants (k2) were obtained by plot-
ting the pseudo-first-order constants versus the corresponding
cysteamine concentration [43].
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