The potential role of the 5,6-dihydropyridin-2(1: H)-one unit of piperlongumine on the anticancer activity

Article abstract of DOI:10.1039/d0ra08778e

Piperlongumine (PL), a potent anticancer agent from the plant long pepper (Piper longum), contains the 5,6-dihydropyridin-2(1H)-one heterocyclic scaffold and cinnamoyl unit. In this paper, we synthesized a series of PL analogs and evaluated their cytotoxicity against cancer cells for the sake of exploring which pharmacophore plays a more potent role in enhancing the anticancer activities of PL. These results illustrated that the position effect, not the electronic effect, of substituents plays a certain role in the cytotoxicity of PL and its analogs. More important, the 5,6-dihydropyridin-2(1H)-one unit, a potent pharmacophore in enhancing the antiproliferative activities of PL, could react with cysteamine and lead to ROS generation, and then bring about the occurrence of ROS-induced downstream events, followed by cell cycle arrest and apoptosis. This work suggests that introducing a lactam unit containing Michael acceptors may be a potent strategy to enhancing the anticancer activity of drugs. This journal is

Full text of DOI:10.1039/d0ra08778e

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The potential role of the 5,6-dihydropyridin-2(1H)-
one unit of piperlongumine on the anticancer
activity†
Cite this: RSC Adv., 2020, 10, 42128
*
*
Wen-Wen Mu, Peng-Xiao Li, Yue Liu, Jie Yang and Guo-Yun Liu
Piperlongumine (PL), a potent anticancer agent from the plant long pepper (Piper longum), contains the
5,6-dihydropyridin-2(1H)-one heterocyclic scaffold and cinnamoyl unit. In this paper, we synthesized
a series of PL analogs and evaluated their cytotoxicity against cancer cells for the sake of exploring
which pharmacophore plays a more potent role in enhancing the anticancer activities of PL. These
results illustrated that the position effect, not the electronic effect, of substituents plays a certain role in
the cytotoxicity of PL and its analogs. More important, the 5,6-dihydropyridin-2(1H)-one unit, a potent
pharmacophore in enhancing the antiproliferative activities of PL, could react with cysteamine and lead
to ROS generation, and then bring about the occurrence of ROS-induced downstream events, followed
by cell cycle arrest and apoptosis. This work suggests that introducing a lactam unit containing Michael
acceptors may be a potent strategy to enhancing the anticancer activity of drugs.
Received 15th October 2020
Accepted 9th November 2020
DOI: 10.1039/d0ra08778e
rsc.li/rsc-advances
been noted in a number of cancer cell lines and a variety of
animal models.16,17 Moreover, PL has also been found to induce
Introduction
cell death by both caspase-dependent apoptosis and
necrosis.17,18 Additionally, PL could generally inhibit PI3K/Akt/
mTOR signaling pathway18 and glutathione S-transferase p
(GSTp), followed by disrupting the redox homeostasis and
boosting the reactive oxygen species production in cancer
cells.19
a,b-Unsaturated ketones, typically found in PL, curcumi-
noids, cinnamic acids derivatives, could signicantly improve
the pharmacological properties and have been a novel design
concept of covalent drugs.20 a,b-Unsaturated ketones were
usually prepared through the Claisen–Schmidt reaction of
acetone with aldehydes. Zhang et al. developed a more effective
method for synthesizing a,b-unsaturated ketones by using
proline-modied catalyst under very mild and green condi-
tions.21 Moreover, Zhang et al. also reported a gram-scale
protocol for the synthesis of dialkylideneacetones, which was
a very reliable and practical method in laboratory and at the
industrial level.22
However, many efforts have been placed in the synthesizing
PL analogs and exploring their mechanisms against cancer
cells, the two pharmacophore, 5,6-dihydropyridin-2(1H)-one
heterocyclic scaffold and cinnamoyl unit, which plays a more
potent role on enhancing the anticancer activities of PL still need
to be explored. Therefore, we design and synthesized a series of PL
analogs (Scheme 1), and evaluated their cytotoxicity against cancer
cells. Thus we focus on the structure–activity relationship (SAR)
from the perspective of chemistry and explore the apoptotic
mechanism associated with PL analogs.
The lactam ring scaffold, arguably one of the most acknowl-
edged nitrogen-containing heterocycles studied so far, has
attracted much attention in the elds of chemistry, biology and
medicine.1–3 Lactams, containing four-membered heterocy-
cles,4 ve-membered heterocycles,5 six-membered heterocy-
cles6 and so on, have been used extensively as potent
substrates for various types of bioactive molecules. b-Lactam
derivatives, initially focusing on their antibacterial proper-
ties,7 have shown diverse pharmacological activities like
anticancer,4 anti-inammatory8 and anti-HIV activity.9 5,6-
Dihydropyridin-2(1H)-one, an essential six-membered lactam
derivative, is a potent pharmacophore in many naturally
occurring medicines. Drugs containing the 5,6-dihydropyr-
idin-2(1H)-one moiety are of great importance because they
have been proven to show signicant antifungal,10 antidia-
betic11 and anticancer12 activities.
Piperlongumine (PL), a naturally occurring alkaloid from the
plant long pepper (Piper longum), is chemically characterized by
the presence of the 5,6-dihydropyridin-2(1H)-one heterocyclic
scaffold and cinnamoyl pharmacophore.5 PL has been reported
to possess multiple pharmacological effects including anti-
inammatory,13 anti-platelet,14 anti-bacterial,15 and especially,
anticancer properties.6 The antineoplastic activities of PL have
School of Pharmacy, Liaocheng University, 1 Hunan Street, Liaocheng, Shandong
252000, China. E-mail: yangjie1110@163.com; guoyunliu@126.com; Tel: +86
15063505132
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra08778e
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3.82–3.79 (m, 2H), 2.62–2.59 (m, 2H), 1.90–1.87 (m, 4H); 13C
NMR (125 MHz, CDCl3), d 172.8, 168.5, 161.4, 134.2, 130.3,
128.0, 123.4, 123.3, 122.3, 115.1, 43.6, 33.9, 21.5, 19.6; HRMS m/
z (ES+): [M + Na]+ 270.0852 (theor 270.0906).
(E)-1-(3-(3-Fluorinephenyl)acryloyl)piperidine-2-one (1c). Yield
36.2%, yellow solid, 1HNMR (500 MHz, CDCl3), d 7.64 (d,
J ¼ 16.0 Hz, 1H), 7.43 (d, J ¼ 16.0 Hz, 1H), 7.34–7.32 (m, 2H),
7.26–7.25 (m, 1H), 7.07–7.03 (m, 1H), 3.81–3.79 (m, 2H), 2.63–
2.60 (m, 2H), 1.91–1.88 (m, 4H); 13C NMR (125 MHz, CDCl3),
d 173.9, 169.4, 164.0, 141.5, 137.5, 130.3, 124.2, 123.5, 116.9,
114.5, 44.7, 34.9, 22.5, 20.6; HRMS m/z (ES+): [M + Na]+ 270.0852
(theor 270.0906).
Scheme 1 Molecular structures and synthetics routs of piperlongu-
mine analogs.
(E)-1-(3-(4-Fluorinephenyl)acryloyl)piperidine-2-one (1d). Yield
23.1%, white solid, 1H NMR (500 MHz, CDCl3), d 7.68 (d,
J ¼ 15.5 Hz, 1H), 7.56–7.53 (m, 2H), 7.39 (d, J ¼ 15.5 Hz, 1H),
7.05 (t, J ¼ 8.5 Hz, 2H), 3.81–3.79 (m, 2H), 2.62–2.59 (m, 2H),
1.90–1.87 (m, 4H); 13C NMR (125 MHz, CDCl3), d 173.9, 169.5,
164.8, 141.9, 131.4, 130.1, 121.9, 116.0, 44.6, 34.9, 22.6, 20.6;
HRMS m/z (ES+): [M + Na]+ 270.0856 (theor 270.0906).
(E)-1-(3-(2-Methoxylphenyl)acryloyl)piperidine-2-one (1e). Yield
58.4%, white solid, 1H NMR (500 MHz, CDCl3), d 8.07 (d,
J ¼ 15.5 Hz, 1H), 7.59 (d, J ¼ 7.5 Hz, 1H), 7.51 (d, J ¼ 16.0 Hz,
1H), 7.32–7.30 (m, 1H), 6.94 (t, J ¼ 7.5 Hz, 1H), 6.90 (d,
J ¼ 8.5 Hz, 1H), 3.88 (s, 3H), 3.81–3.79 (m, 2H), 2.61–2.59 (m,
2H), 1.89–1.86 (m, 4H); 13C NMR (125 MHz, CDCl3), d 173.8,
170.1, 158.4, 138.6, 131.2, 128.8, 124.2, 122.4, 120.6, 111.1, 55.5,
44.6, 34.9, 22.6, 20.7; HRMS m/z (ES+): [M + Na]+ 282.1048 (theor
282.1106).
Experimental section
Materials
Roswell Park Memorial Institute (RPMI)-1640 was from
Hyclone. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), rhodamine 123, 20,70-dichlorouorescein diac-
etate, N-acetylcysteine (NAC), GSH and GSSG Assay kit, and
thiobarbituric acid were purchased from Beyotime. Annexin V-
FITC/PI apoptosis detection kit was from BD Biosciences.
Substituted benzaldehyde, malonic acid, 2-piperidone and
piperlongumine were from Energy Chemical. All other chem-
icals were of the highest quality available.
Synthesis of the PL analogs
General procedure for the synthesis of substituted cinnamic
(E)-1-(3-(3-Methoxylphenyl)acryloyl)piperidine-2-one (1f). Yield
acids. The substituted cinnamic acids were synthesized 30.4%, white solid, 1H NMR (500 MHz, CDCl3), d 7.68 (d,
according to the published procedure.5 Briey, the corre- J ¼ 15.5 Hz, 1H), 7.44 (d, J ¼ 15.5 Hz, 1H), 7.29 (d, J ¼ 7.5 Hz,
sponding aldehyde (20 mmol), malonic acid (24 mmol) and 1H), 7.17 (d, J ¼ 7.5 Hz, 1H), 7.08–7.07 (m, 1H), 6.92–6.90 (m,
piperidine (1.2 mL) were dissolved in pyridine (12 mL) and 1H), 3.83 (s, 3H), 3.81–3.79 (m, 2H), 2.62–2.59 (m, 2H), 1.90–1.87
reuxed for 3 h. Aer cooling in an ice bath, the solid were (m, 4H); 13C NMR (125 MHz, CDCl3), d 173.8, 169.6, 159.9, 143.1,
ltered and recrystallized from ethanol to afford the corre- 136.5, 129.7, 122.4, 121.0, 116.0, 113.1, 55.3, 44.6, 34.9, 22.6,
sponding acids.
20.6; HRMS m/z (ES+): [M + Na]+ 282.1055 (theor 282.1106).
(E)-1-(3-(4-Methoxylphenyl)acryloyl)piperidine-2-one (1g). Yield
General procedure for the synthesis of piperlongumine
analogs. The mixture, containing oxalyl chloride (6 mmol), 63.4%, yellow solid, 1H NMR (500 MHz, CDCl3), d 7.70 (d,
a catalytic amount of DMF (0.01 equiv.), the substituted cin- J ¼ 15.5 Hz, 1H), 7.53 (d, J ¼ 9.0 Hz, 2H), 7.37 (d, J ¼ 15.5 Hz,
namic acids (2 mmol) and dry CH2Cl2 (4 mL), was stirred at 1H), 6.89 (d, J ¼ 9.0 Hz, 2H), 3.83 (s, 3H), 3.80–3.78 (m, 2H),
room temperature for 24 h. Aer removing the solvent, the 2.61–2.58 (m, 2H), 1.89–1.86 (m, 4H); 13C NMR (125 MHz,
residue, triethylamine (6 mmol) and lactam (2.4 mmol) was CDCl3), d 173.8, 169.8, 161.2, 143.3, 130.0, 127.9, 119.7, 114.2,
dissolved in dry CH2Cl2 (5 mL), and stirred at room temperature 55.4, 44.5, 35.0, 22.6, 20.6; HRMS m/z (ES+): [M + Na]+ 282.1057
for 24 h before extracting with ethyl acetate. Then the crude (theor 282.1106).
products were puried with a silica gel column.5 Their struc-
(E)-1-(3-(2-Triuoromethylphenyl)acryloyl)piperidine-2-one
(1h). Yield 17.5%, yellow solid, 1H NMR (500 MHz, CDCl3),
1
tures were conrmed by H and 13C NMR spectroscopy.
(E)-1-(3-(3-Phenyl)acryloyl)piperidine-2-one (1a). Yield 33.8%, d 8.04 (d, J ¼ 15.5 Hz, 1H), 7.80 (d, J ¼ 8.0 Hz, 1H), 7.69 (d,
1
white solid, H NMR (500 MHz, CDCl3), d 7.72 (d, J ¼ 15.5 Hz, J ¼ 7.5 Hz, 1H), 7.54 (t, J ¼ 7.5 Hz, 1H), 7.46–7.39 (m, 2H), 3.83–
1H), 7.58–7.56 (m, 2H), 7.46 (d, J ¼ 15.5 Hz, 1H), 7.37–7.36 (m, 3.80 (m, 2H), 2.62–2.59 (m, 2H), 1.91–1.88 (m, 4H); 13C NMR
3H), 3.82–3.79 (m, 2H), 2.62–2.60 (m, 2H), 1.90–1.87 (m, 4H); (125 MHz, CDCl3), d 173.9, 168.9, 137.9, 134.2, 132.0, 129.2,
13C NMR (125 MHz, CDCl3), d 173.9, 169.7, 143.2, 135.2, 130.0, 128.2, 126.3, 126.0, 126.0, 44.6, 34.9, 22.5, 20.6; HRMS m/z (ES+):
128.8, 128.3, 122.1, 44.60, 34.9, 22.6, 20.6; HRMS m/z (ES+): [M + [M + Na]+ 320.0817 (theor 320.0874).
Na]+ 252.0951 (theor 252.1000).
(E)-1-(3-(3-Triuoromethylphenyl)acryloyl) piperidine-2-one (1i).
1
(E)-1-(3-(2-Fluorinephenyl)acryloyl)piperidine-2-one (1b). Yield Yield 20.0%, white solid, H NMR (500 MHz, CDCl3), d 7.78 (s,
26.5%, white solid, 1HNMR (500 MHz, CDCl3), d 7.84 (d, 1H), 7.73 (d, J ¼ 8.0 Hz, 1H), 7.69 (d, J ¼ 15.5 Hz, 1H), 7.61 (d,
J ¼ 16.0 Hz, 1H), 7.60 (t, J ¼ 7.5 Hz, 1H), 7.50 (d, J ¼ 16.0 Hz, 1H), J ¼ 7.5 Hz, 1H), 7.51–7.45 (m, 2H), 3.82–3.80 (m, 2H), 2.64–2.61
7.35–7.30 (m, 1H), 7.13 (t, J ¼ 7.5 Hz, 1H), 7.09–7.05 (m, 1H), (m, 2H), 1.91–1.88 (m, 4H); 13C NMR (125 MHz, CDCl3), d 173.9,
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169.3, 141.0, 136.0, 131.3, 129.3, 126.3, 124.7, 124.0, 44.7, 34.9,
22.5, 20.6; HRMS m/z (ES+): [M + Na]+ 320.0817 (theor 320.0874).
(E)-1-(3-(4-Triuoromethylphenyl)acryloyl)piperidine-2-one (1j).
Yield 62.5%, white solid, 1H NMR (500 MHz, CDCl3), d 7.69–7.61
(m, 5H), 7.51 (d, J ¼ 16.0 Hz, 1H), 3.83–3.81 (m, 2H), 2.64–2.61
(m, 2H), 1.91–1.89 (m, 4H), 13C NMR (125 MHz, CDCl3), d 173.9,
169.2, 140.8, 138.6, 128.3, 125.7, 124.7, 44.7, 34.9, 22.5, 20.6;
HRMS m/z (ES+): [M + Na]+ 320.0823 (theor 320.0874).
Measurement of lipid peroxidation
A549 cells (3 ꢀ 105 cells per well) were cultured in 6-well plates
and incubated with the vehicle alone or the test compounds for
15 h before harvesting. Then, the cells were lysed and the
supernatant were tested for assessment of the lipid perox-
idation as previously described.24
Measurement of mitochondrial membrane potential
(E)-1-(3-(3,4,5-Trimethoxylphenyl)acryloyl)
piperidine-2-one
(1k). Yield 55.7%, yellow solid, 1H NMR (500 MHz, CDCl3), A549 cells (3 ꢀ 105 cells per well) were plated in 6-well plates
and incubated with the vehicle alone or the test compounds for
d 7.64 (d, J ¼ 15.5 Hz, 1H), 7.37 (d, J ¼ 15.5 Hz, 1H), 6.78 (s, 2H),
15 h. Then, the cells were harvested, and stained with Rhoda-
mine 123 before analyzing by a ow cytometry as previously
reported.23
3.88 (s, 6H), 3.87 (s, 3H), 3.81–3.78 (m, 2H), 2.62–2.59 (m, 2H),
1.89–1.87 (m, 4H); 13C NMR (125 MHz, CDCl3), d 173.9, 169.6,
153.4, 143.5, 140.0, 130.7, 121.3, 105.5, 61.0, 56.2, 44.6, 35.0,
22.6, 20.6; HRMS m/z (ES+): [M + Na]+ 342.1258 (theor 342.1317).
Electrophilicity assessment by a kinetic thiol assay
The reactions of PL and 1k with cysteamine were carried out in
Tris–HCl (100 mM, pH 7.4) and EDTA (2 mM)–ethanol (v/v, 2/1)
under pseudo-rst-order conditions.25 The test compounds (50
In vitro antiproliferative activity
Human lung cancer cells (A549), human ovarian cancer cells
(SK-OV3), human normal liver cells (LO2) and human embry-
onic kidneys cells (293T) were purchased from the Shanghai
Institute of Biochemistry and Cell Biology. The antiproliferative
activities was performed by the MTT assay as reported previ-
ously.23 Briey, A549 (3 ꢀ 103 per well), SK-OV3 (5 ꢀ 103 per
well), LO2 (5 ꢀ 103 per well) and 293T (5 ꢀ 103 per well) were
plated in 96-well plates and incubated with vehicle alone or the
tested compounds for 48 h before measuring.
ꢁ
mM) were mixed with 100-fold cysteamine at 25 C, and moni-
tored the decay of their wavelength maxima by using a TU-1950
UV-vis spectrophotometer. The second-order rate constants (k2)
were obtained by plotting the pseudo-rst-order constants vs.
[cysteamine].
1
Determination of the reaction site by H NMR and HRMS
1H NMR spectra of PL (30 mM in d6-DMSO) aer adding
cysteamine (90 mM in d6-DMSO) at set intervals (0, 1 h) were
recorded using a Bruker AV 500 spectrometer. Aer incubation
3 days, the reaction products were monitored by HRMS (Acquity
UPLC I-Class/Xevo G2-XS QTOF).6
Stability assay
The stability of piperlongumine and its analog 1k in PBS buffer
(100 mM) at 25 ꢁC was monitored at their maximum absorbance
for 120 min at 10 min intervals as described previously.24
Statistical analysis
Cell cycle and apoptosis analysis
The data are expressed as the mean ꢂ S.D. of at least three
independent experiments. Data were analyzed by use of SPSS
soware 17.0 (SPSS, Inc., Chicago, IL, USA). The difference
between two groups was analyzed by one-way analysis of vari-
ance (ANOVA) followed by Dunnett test (compare all drug
treatment groups vs. control). The values were considered
signicant at P < 0.05. *: P < 0.05; **: P < 0.01; ***: P < 0.001
compared with control.
A549 cells (3 ꢀ 105) were seeded in 6-well plates and treated
with vehicle alone or the tested compounds for 15 h (for cell
cycle analysis) or 18 h (for cell apoptosis analysis). Then, the
cells were harvested, washed with PBS, labeled with PI or FITC
Annexin-V and PI, and analyzed by using a ow cytometry as
described previously23
Intracellular ROS assay
Results and discussion
Synthesis
A549 (3 ꢀ 105) cells were plated in 6-well plates and incubated
with the vehicle alone or the tested compounds for 6 h. Then,
the cells were collected, stained with DCFH-DA at the dark, and Compounds 1a–k were synthesized according to the synthetic
subsequently analyzed with a ow cytometry as described route illustrated in Scheme 1. Firstly, the appropriate benzal-
previously.23
dehyde was condensed with malonic acid in pyridine to afford
the key intermediate 2. Then compound 2 reacted with oxalyl
chloride in dry CH2Cl2 to afford intermediate 3, followed by
a nucleophilic substitution reaction with 2-piperidone to
Measurement of GSH and GSSG levels
A549 (3 ꢀ 105 cells per well) cells treated with the vehicle alone generate crude products. The title compounds 1a–k were ob-
or the tested compounds for 6 h. Then, the cells were collected, tained with medium yields aer purication by column chro-
lysed and centrifugated, the supernatant was used to determine matography. All compounds were characterized by 1H NMR and
the level of GSH and GSSG using a GSH and GSSG Assay Kit.24 13C NMR.
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Table 1 Cytotoxicity of PL and its analogs against A549 and SKOV3 cellsa
IC50/(mM)
IC50/(mM)
Comps
A549
SKOV3
Comps
A549
SKOV3
161.1 ꢂ 5.4
138.6 ꢂ 9.2
171.8 ꢂ 9.2
178.1 ꢂ 13.2
186.7 ꢂ 11.6
92.5 ꢂ 4.3
72.8 ꢂ 2.3
177.4 ꢂ 7.8
>200
>200
>200
>200
>200
191.0 ꢂ 6.1
132.3 ꢂ 8.2
160.9 ꢂ 1.4
>200
191.0 ꢂ 4.8
83.8 ꢂ 1.9
115.2 ꢂ 1.1
7.9 ꢂ 0.8
>200
143.3 ꢂ 13.8
19.4 ꢂ 1.7
a
The IC50 value is the concentration of a compound tested to cause 50% inhibition of cell viability aer 48 h of treatment, and is expressed as the
mean ꢂ SD for three determinations.
of oleanolic acid–cinnamic acid ester derivatives against MCF-7
cells.28
Cytotoxicity and SAR
The antiproliferative activities of PL and its analogs (1a–k) were
evaluated against A549 and SKOV3 cells by the MTT assay, and
the results were listed in Table 1. In general, the cell-killing
effects of all the PL analogs (1a–k) were much lower than PL.
As a comparision, compounds with electron-donating groups
(–OCH3) exhibited nearly equivalent cytotoxicity to the
compounds with electron-withdrawing groups (–F, –CF3), sug-
gesting that the electronic effect of substituents are not neces-
sary for the antiproliferative activities of PL and its analogs.
Whereas, the compounds with meta-substituents (1c, 1f, 1i)
were more active than their corresponding ortho- and para-
substituted compounds. Compound 1f (meta-methoxyl group, IC50
¼ 143.3 and 83.8 mM in A549 and SKOV3 cells, respectively)
showed stronger cytotoxicity than compounds 1e (ortho-methoxyl
group, IC50 > 200 and ¼ 191.0 mM in A549 and SKOV3 cells,
respectively) and 1g (para-methoxyl group, IC50 > 200 mM in both
A549 and SKOV3 cells). This result indicated that the position
effect of substituents play a certain role on the cytotoxicity of PL
and its analogs. This phenomenon may be attributed to the ster-
eostructures of meta-substituted compounds, which are favour of
binding certain protein in cells, such as thioredoxin reductase
(TrxR), carbonic anhydrase and so on.6
More important, in comparison with the parent PL, all the PL
analogs, lacking of the 5,6-dihydropyridin-2(1H)-one heterocy-
clic scaffold, displayed much lower cytotoxicity (Table 1).
Specially, compound 1k exhibited the less antiproliferative
activities against A549 and SKOV3 cells with the IC50 values of
115.2 and greater than 200 mM, respectively. Additionally, the
stability of PL and its analog 1k in PBS buffer were determined.
As shown in Fig. 1, no apparent changes were observed in the
case of PL and 1k. The above results clearly indicated that the
5,6-dihydropyridin-2(1H)-one pharmacophore play a potent role
on the antiproliferative activities of PL and its analogs. As far as
the 5,6-dihydropyridin-2(1H)-one pharmacophore is concerned,
it is a potent strategy to increase the activity by introducing the
pharmacophore into drug molecules. Moreover, some previous
studies have shown that PL selectively kill cancer cells without
damaging certain normal cells, such as astrocyte, breast
epithelial (76N), keratinocytes (HKC) cells and so on.29,30 Unex-
pectedly, compared with cancer cells, PL and 1k displayed no
obvious selectivity of cytotoxicity against normal cells (LO2 and
In addition, the antiproliferative activity of 1c against SKOV3
was much higher than that of compound 1k. This result hinted
that the cytotoxicity of PL analogs may be partly attributed to the
cinnamoyl pharmacophore, and the rational modication of
cinnamoyl unit might be conduce to increase their anticancer
activity. For example, the cinnamic acid–progenone hybrids,
with 3-OH or 4-OH cinnamoyl unit, exhibited more activity
towards A549 and HL-60 cells than corresponding methoxyl-
hybrids.26 Gaikwad et al. deduced that cinnamamides with
electron-withdrawing groups at the para position were impor-
tant for their anticancer activity.27 The 4-methylcinnamic acid
unit has the ability to remarkably improve the inhibition activity
Fig. 1 Stability assessment on PL (50 mM) and its analog 1k (50 mM) in
PBS buffer (100 mM) at 25 ꢁC by monitoring the decrease in their
maximum absorbance.
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Table 2 Cytotoxicity of PL and its analogs against LO2 and 293T cells
ROS accumulation and ROS-induced downstream events
ROS generation has been implicated as an upstream signal that
could bring about signaling transduction culminating in
apoptosis.31 Based on the above results, we initially deem that
the ROS production should be a potent mechanism underlying
the antiproliferative activities of PL. Therefore, we next deter-
mined the ROS accumulation using DCFH-DA, a widely used
oxidation-sensitive probe to measure intracellular ROS levels.32
As shown in Fig. 3A, 6 h treatment with PL induced an apparent
intracellular ROS accumulation in a dose-dependent manner.
The uorescence intensity measured in cells treated with 30 mM
PL were increased by approximately 1.7-fold compared with the
vehicle control. In contrast, 100 mM 1k exhibited almost no ROS-
generating ability under the same conditions. As expected,
pretreatment with NAC, an effective antioxidant, the ROS
induced by 30 mM PL were almost completely abolished
(Fig. 3A), in line with the results obtained by the cell cycle and
apoptosis analysis. This result also suggested that the ROS
generation induced by PL may be related with its cytotoxicity,
cell cycle arrest and apoptotic activities.
The ratio of GSH/GSSG, a characteristic of intracellular redox
balance, could be vulnerable to change by the sustained ux of
ROS.33 Therefore, we determined the redox balance in A549 cells
to ascertain whether the ROS accumulation induced by PL
resulted in an imbalance of intracellular redox state. As shown
in Fig. 3B, the ratios of GSH/GSSG were sharply decreased and
exhibited an excellent dose-dependent fashion. The redox
imbalance induced by 100 mM 1k was equal to that induced by
20 mM PL. Expectly, pretreatment with NAC, the redox imbal-
ance induced by 30 mM PL was effectively prevent.
IC50/(mM)
LO2
IC50/(mM)
Comp.
293T
Comp.
LO2
293T
182.6 ꢂ 1.8
PL (1l)
10.4 ꢂ 0.3
7.5 ꢂ 0.4
1k
>200
293T cells) (Table 2). As listed in Table 2, PL exhibited the
antiproliferative activities against normal LO2 and 293T cells
with the IC50 values of 10.4 and 7.5 mM, respectively.
Cell cycle and apoptosis study by ow cytometry
To investigate the possible mechanisms underlying the anti-
proliferative activities, we further determined the effects of PL
and 1k on cell cycle arrest and apoptosis by ow cytometry. As
shown in Fig. 2A, 15 h of treatment with PL arrested cell cycle at
G2/M phase in a dose-dependent manner. Increasing concen-
tration from 10 to 30 mM led to a successive increase of G2/M
phase cell population from 20.0% (control) to 33.8% (30 mM).
Additionally, aer 18 h treatment, the number of apoptotic
A549 cells was strikingly increased with increasing concentra-
tions (10, 20 and 30 mM) of PL (Fig. 2B). For example, 30 mM PL
caused 22.7% apoptosis of the total cell count. In contrast, 100
mM 1k displayed no obvious effect on both the cell cycle arrest
and induction of apoptosis (Fig. 2A and B). Moreover,
pretreatment with N-acetylcysteine (NAC), a potent antioxidant,
noticeably reversed the cell cycle arrest and apoptosis induced
by PL (Fig. 2A and B), preliminarily suggesting the involvement
of reactive oxygen species.
Additionally, the sustained ux of ROS could be susceptible
to attack plasma membrane and result in lipid peroxidation.32
Thus, we measured the amount of malondialdehyde (MDA), the
biomarker of lipid peroxidation, by using thiobarbituric acid-
reactive substance. Aer exposed to PL for 15 h, a dose-
dependent increase for the MDA levels in cells was observed
(Fig. 3C). Treatment with 30 mM PL, the level of MDA were
increased by about 3-fold compared with the vehicle control. In
Fig. 2 The effect of PL and its analog 1k on the cell cycle arrest (A) and
apoptosis (B) in A549 cells. Cells were treated with PL and 1k at the
indicated concentrations for 15 h (in cell cycle analysis) and 18 h (in cell Fig. 3 The effects of PL on the ROS accumulation (A) and ROS-
apoptosis analysis) in the absence or presence of NAC. Data are induced downstream events (B) the redox imbalance; (C) lipid per-
representative of three independent experiments.
oxidation; (D) the loss of MMP.
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contrast, 100 mM 1k was less active in changing the MDA levels and cysteamine for 1h, the doublet integral of the olenic
(Fig. 3C). And pretreatment with NAC also abolished the lipid proton at d 5.965 (H-4) decreased from 1.00 to 0.12, and
peroxidation induced by 30 mM PL, supporting the lipid perox- a weaker doublet appeared at d 5.917 with the integral
idation was associated with ROS production.
increasing from 0.00 to 0.03 (Table 4). This result illustrated
The excess ROS could damage the functions of mitochon- that cysteamine was mainly added to the 5,6-dihydropyridin-
drial, which playing a dominant role in the regulation of cell 2(1H)-one heterocyclic unit, and suggested that the a,b-unsat-
survival.34 We subsequently determined the loss of mitochon- urated ketone of 5,6-dihydropyridin-2(1H)-one heterocyclic unit
drial membrane potential (MMP) using rhodamine 123 by ow was more vulnerable to attack by cysteamine than that of cin-
cytometry. As shown in Fig. 3D, we found that PL triggered an namoyl pharmacophore. Additionally, the HRMS analysis
obvious decrease in the collapse of MMP in a dose-dependent showed that the 1 : 1 and 1 : 2 adducts (m/z: 417.1466 and
fashion. Moreover, 30 mM PL caused an 80% decrease of MMP 494.1611, respectively) were formed between PL and cysteamine
in A549 cells compared with the vehicle control, and the aer 3 days of incubation (Fig. 4B).
decrease was signicantly blocked by pretreatment with NAC.
Based on the above results, we found that PL could cause the
The anticancer mechanism of PL and its analogs against A549
cells
generation of ROS, a signal molecule to modulate various bio-
logical processes,32 and lead to the occurence of ROS-induced
ROS, a class of unstable chemically reactive, are ubiquitous
downstream events, such as redox imbalance, lipide perox-
common by-products in the process of life such as mitochon-
drial metabolism and protein folding.35 ROS also can be
produced by NADPH oxidases, a family of enzymes that transfer
electrons from NADPH to O2 to generated ROS, not as by-
products.36 Cancer cells, which display increased ROS to
idation and the loss of MMP. In addition, the antioxidant of
NAC could effectively block the damages induced by PL in A549
cells.
maintain their phenotypes, are more rely on the “redox adap-
tation” mechanism compared with normal cells. Stimulating
intracellular ROS to a toxicity threshold to induce cell death is
one of the important strategy for treatment cancer.6 A large
number of chemotherapeutic drugs containing Michael
acceptor, such as curcumin, PL, quinones and so on, are widely
used for the treatment of cancer.37 The Michael acceptor of
chemotherapeutic agents can irreversibly inhibit TrxR and
trigger intracellular ROS accumulation.6 As shown in Table 3,
the reactive activity of PL with cysteamine was more than that of
compound 1k. Moreover, the Michael acceptor of lactam of PL
analogs was the major reaction site of cysteamine (Fig. 4A).
These results also hinted that the ROS generation induced by PL
and 1k (Fig. 3A) was major attributed to the electrophilicity of
Michael acceptor of lactam of PL.
Apoptosis, programmed cell death, can be initiated by an
intrinsic pathway or an extrinsic pathway. ROS are usually
involved in both intrinsic and extrinsic pathways of apoptosis.38
Extensive research has proved the potential of ROS in inducing
apoptosis of cancer cells. PL substantially induced A549 cells
apoptosis in a dose-dependent manner. Chemotherapeutic
agents can inhibit the antioxidant defense systems or increase
ROS production, which mediated activation of c-Jun N-terminal
Electrophilicity assessment and determination of the reaction
site of PL
Drugs, containing Michael acceptor, could irreversibly inhibit
the TrxR and trigger ROS generation. Piperlongumine (PL)
contains two Michael acceptor units, 5,6-dihydropyridin-2(1H)-
one heterocyclic scaffold and cinnamoyl pharmacophore. In
order to ascertain which pharmacophore plays a more potent
role on enhancing the anticancer activities of PL, we assessed
the electrophilicity of PL and its analog 1k by measuring their
second-order rate constants with cysteamine. On the basis of
the k2 values (Table 3), the electrophilicity of PL (k2 ¼ 2.26 Mꢃ1
sꢃ1) was much higher than its analog 1k (k2 ¼ 1.80 Mꢃ1 sꢃ1). The
result indeed accord with their cytotoxicity and proapoptotic
activities. The results of electrophilicity and cytotoxicity also
hinted that the cinnamoyl unit of PL showed a certain chemical
reactivity toward cysteamine, and the 5,6-dihydropyridin-2(1H)-
one heterocyclic scaffold was crucial to the cytotoxicity, proap-
optotic activities, ROS accumulation and ROS-induced down-
stream events.
To identify the specic site for the reaction of PL with
cysteamine, the 1H NMR spectroscopy changes of PL in the
presence of three equivalents of cysteamine were also moni-
tored in d6-DMSO. The doublet at d 6.974 (H-1 and H-10) was the
protons of the aromatic nucleus (Fig. 4A), and the integral was
calibrated to 2.00 hydrogen. Aer incubating a mixture of PL
Table 3 Second-order rate constants (k2) for the reaction of PL and its
analog 1k with cysteamine at 25 ꢁCa
Comp.
k2 (Mꢃ1 sꢃ1
)
Comp.
k2 (Mꢃ1 sꢃ1
)
PL (1l)
2.26 ꢂ 0.04
1k
1.80 ꢂ 0.08
Fig. 4 Reaction of PL with cysteamine: (A) 1H NMR spectra of PL in d6-
DMSO before and after adding cysteamine; (B) HRMS spectrum
recorded 3 days after mixing PL with 3 eq. of cysteamine.
a
Reactions were carried out in Tris–HCl (100 mM, pH 7.4) and EDTA (2
mM)–ethanol (v/v, 2/1) under pseudo-rst-order conditions. Data are
expressed as the mean ꢂ SD for three determinations.
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Table 4 Quantification of the analytes after the addition of cysteamine
Coupling constants
Signals assigned
Chemical shi (ppm)
Coupled splitting
(J/Hz)
Time (h)
Integral
Sum of integral
H-1
H-10
H-4
6.974
6.974–6.890
5.965
s
—
d
d/d
—
—
9.5
9.5/9.5
0
1
0
1
2.00
2.00
1.00
0.12/0.03
2.00
2.00
1.00
0.15
5.965/5.917
Conflicts of interest
The authors declare that they have no conict of interest.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant No. 81901420), Shandong Provin-
cial Natural Science Foundation (Grant No. ZR2018LH022), the
Open Project of Shandong Collaborative Innovation Centre for
Antibody Drugs (Grant No. CIC-AD1822 and CIC-AD1824).
Scheme 2 The 5,6-dihydropyridin-2-(1H)-one unit is crucial for the
anticancer activity.
kinases (JNK) pathway, inhibition of NF-kB activation and
induction of mitochondrial dysfunction to promote cancer cell
apoptosis. As shown in Fig. 3B, PL remarkably decrease the ratio
of GSH/GSSG may be due to the irreversible inhibition of TrxR,
and the redox imbalance induced by 1k may be attributed to the
GSH depletion. In addition, ROS accumulation could lead the
oxidation of intracellular lipids and proteins. As illustrated in
Fig. 3C, PL obviously induced lipid peroxidation in a dose-
dependent fashion. PL also disrupted the MMP in a dose-
dependent manner as illustrated in Fig. 3D. All of the above
results, ROS accumulation, redox imbalance, lipid perox-
idation, the loss of MMP and apoptosis, induced by PL can be
reversed by pretreatment with NAC, a well known antioxidant.
This phenomenon indicated that PL induced apoptosis of A549
cells through ROS-mediated pathway.
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In conclusion, we synthesized a panel of PL analogs and eval-
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