Synthesis and evaluation of N-heteroaromatic ring-based analogs of piperlongumine as potent anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2017.06.046

Piperlongumine (PL) selectively targets a wide spectrum of cancer cells and induces their death by triggering various pathways, including apoptosis, necrosis and autophagy. However, the poor solubility is a serious concern for intensive study and clinical application. We synthesized its analogs 1–9 by replacement of the trimethoxyphenyl of PL with an N-heteroaromatic ring and/or not introduction of 2-Cl. These compounds improved aqueous solubility and displayed potent anticancer activity. The most active compound 9 selectively enhanced ROS levels in colon cancer cells and inhibited the cell proliferation but sparing non-tumor colon cells. Importantly, 9 significantly repressed tumor growth in an HCT-116 xenograft mouse model, suggesting that these N-heteroaromatic ring-based analogs of PL warrant further investigation.

Full text of DOI:10.1016/j.ejmech.2017.06.046

Accepted Manuscript
Synthesis and evaluation of N-heteroaromatic ring-based analogs of piperlongumine
as potent anticancer agents
Yu Zou, Chang Yan, Huibin Zhang, Jinyi Xu, Dayong Zhang, Zhangjian Huang, Yihua
Zhang
PII:
S0223-5234(17)30492-0
10.1016/j.ejmech.2017.06.046
EJMECH 9541
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 May 2017
Revised Date: 22 June 2017
Accepted Date: 23 June 2017
Please cite this article as: Y. Zou, C. Yan, H. Zhang, J. Xu, D. Zhang, Z. Huang, Y. Zhang, Synthesis
and evaluation of N-heteroaromatic ring-based analogs of piperlongumine as potent anticancer agents,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.046.
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ACCEPTED MANUSCRIPT
Synthesis and evaluation of N-heteroaromatic ring-based analogs of
piperlongumine as potent anticancer agents
Yu Zou,a,b,# Chang Yan,a,b,# Huibin Zhang,a,b Jinyi Xu,a Dayong Zhang, a,b Zhangjian
Huang,*,a,b and Yihua Zhang *,a,b
aState Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing 210009, P.R. China.
bJiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China
Pharmaceutical University, Nanjing 210009, P.R. China.
# These authors contributed equally to this work.
Graphical Abstract

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Synthesis and evaluation of N-heteroaromatic ring-based analogs of
piperlongumine as potent anticancer agents
Yu Zou,a,b,# Chang Yan,a,b,# Huibin Zhang,a,b Jinyi Xu,a Dayong Zhang, a,b Zhangjian
Huang,*,a,b and Yihua Zhang *,a,b
aState Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing 210009, P.R. China.
bJiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China
Pharmaceutical University, Nanjing 210009, P.R. China.
# These authors contributed equally to this work.
Abstract
Piperlongumine (PL) selectively targets a wide spectrum of cancer cells and induces
their death by triggering various pathways, including apoptosis, necrosis and
autophagy. However, the poor solubility is a serious concern for intensive study and
clinical application. We synthesized its analogs 1−9 by replacement of the
trimethoxyphenyl of PL with an N-heteroaromatic ring and/or not introduction of 2-Cl.
These compounds improved aqueous solubility and displayed potent anticancer
activity. The most active compound 9 selectively enhanced ROS levels in colon
cancer cells and inhibited the cell proliferation but sparing non-tumor colon cells.
Importantly, 9 significantly repressed tumor growth in an HCT-116 xenograft mouse
model, suggesting that these N-heteroaromatic ring-based analogs of PL warrant
further investigation.
Keywords: piperlongumine, N-heteroaromatic ring, solubility, anticancer activity,
ROS.
1. Introduction
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Solubility is one of the key issues for drug candidates. Poor aqueous solubility
often results in poor absorption, even though the permeation ability is great, since the
flux of a drug cross the intestinal membrane is closely related to the concentration
gradient between the intestinal lumen and blood. Additionally, efficacy and risk
evaluation of poorly soluble compounds is challenging because exposure may be hard
to define and the sensitivity of the assays may be diminished. Furthermore, high
concentrations of poorly soluble drugs in some organisms may lead to crystallization
and acute toxicity in cases such as uric acid and gout. As a matter of fact, the poor
solubility has resulted in failures of numerous drug candidate development [1].
Appropriate formulation may be helpful in solving these problems [2], but the
extent of solubility enhancement that can really be achieved is extremely limited.
Additionally, stability and manufacturing problems also have to be in consideration,
since an insoluble drug candidate may require a non or less conventional strategy,
resulting in an increase in cost [3]. Therefore, it would be desirable to generate drug
candidates with an applicable aqueous solubility at the early stage of drug
development. To this regard, structural modification of the molecule is supposed to be
much better than other approaches.
Piperlongumine (PL, Fig. 1) is a naturally occurring alkaloid isolated from the plant
long pepper, and used traditionally for the treatment of certain diseases, such as
antiplatelet aggregation [4], anti-inflammation [5], and cardiovascular protection [6].
PL has recently been shown to selectively target a wide spectrum of cancer cells [7]
and induce cancer cell death by initiating various pathways, such as apoptosis,
necrosis and autophagy [8,9]. The elevation of reactive oxygen species (ROS),
characteristic of oxidative stress, is an important mechanism by which PL promotes
cancer-selective cell death [7]. However, the poor aqueous solubility of PL is a serious
concern for intensive investigations and clinical application [10,11]. Although a large
variety of derivatives and analogs of PL have been synthesized in order to find agents
with improved properties, few efforts are so far successful [12-15].
The analysis on the structure and biological activity relationship (SAR) of these
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derivatives and analogs reveals that C2-C3 and C7-C8 double bonds are two
pharmacophores as Michael acceptors essential for toxicity of PL to cancer cells (Fig.
1) [16]. The C2-C3 double bond is also critical for ROS elevation and glutathione
depletion. 2-Halogenated PL possesses more potent in vitro anticancer activity than
PL itself owing to its higher reactivity of C2-C3 Michael acceptor [17]. In addition,
removal of C7-C8 olefin decreases cytotoxicity, but does not reduce the ROS levels
[16]. Furthermore, it has been documented that the aromatic ring can be modified to
obtain new analogs with better activity than PL [18].
It is well-known that N-heteroaromatics are generally more water soluble than
corresponding aromatic compounds. For example, the aqueous solubility of pyridine
and quinoline is 162.25 and 7.61 mg/mL [19], 92- and 224-fold greater than
counterparts benzene (1.76 mg/mL) and naphthalene (0.034 mg/mL), respectively
[20]. Accordingly, we hypothesized that replacement of the trimethoxyphenyl of PL
by an N-heteroaromatic ring might improve the aqueous solubility and retain or
increase the anticancer activity. In addition, given that 2-Cl substituted PL is more
potent than PL [17], we therefore designed and synthesized new analogs 1-9 of PL
(Fig. 1), and biologically evaluated their anticancer effects.
Fig. 1. Design of analogs 1-9 by replacement of the trimethoxyphenyl of PL with an
N-heteroaromatic ring and/or not introduction of 2-Cl.
2. Results and discussion
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2.1. Chemistry
The synthetic routes of target compounds 1-9 are shown in Scheme 1. Compounds
1a-9a underwent Wittig-Horner reaction to generate ester 1b-9b. Hydrolysis of 1b-9b
led to free acid 1c-9c, which was converted to acylchloride 1d-9d, without isolation
followed by reacting with 5,6-dihydropyridin-2(1H)-one 13 and 6-chloro-5,6-dihydro-
pyridin-2(1H)-one 15 to provide analogs 1-9, respectively (Scheme 1A). Compound
13 was obtained by treatment of 10 with trimethyl chlorosilane (TMSCl),
phenylselenyl chloride, and hydrogen peroxide, successively. Similarly, reaction of 10
with PCl5 offered 2,2-dichloropiperidin-2-one 14, followed by dehydrochlorination in
the presence of Li2CO3 to provide 6-chloro-5,6-dihydropyridin-2(1H)-one 15
(Scheme 1B) [13].
1
The structures of all target compounds were fully characterized by H-NMR,
13C-NMR, MS, and HRMS, and the C7-C8 olefinic bond in these analogs was
identified as E configuration according to the coupling constant of two corresponding
protons on the double bond. The purity of the compounds was greater than 95%,
determined by high performance liquid chromatography (HPLC) analysis, for the
following biological experiments.
Scheme 1. A. The synthetic route of analogs 1-9. Reagents and conditions: i.
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Wittig-Hornor reaction; ii. KOH, 16 h; iii. anhydrous DCM, pivaloyl chloride, one
drop of DMF; iv. anhydrous THF, TEA, n-butyllithium and compounds 13 or 15. B.
The synthetic route of compounds 13 and 15. Reagents and conditions: i. toluene,
TEA, TMSCl, 0 °C, 4 h; ii. THF, phenylselenyl chloride, LDA, -50 °C; iii. THF,
50%H2O2; iv. PCl5, chloroform, r.t.; v. Li2CO3, LiCl, anhydrous DMF, 130 °C, 7 h.
2.2. Solubility evaluation
The solubility of PL and its analogs 1-8 was determined in pure water by HPLC [21]
as shown in Table1. It was found that all analogs with solubility from 1.95 to 59.83
µg/mL were more soluble in the aqueous solution than PL (1.63 µg/mL). The effects
of nitrogen-containing heteroaromatics are in the order of pyrzine > pyridine >
quinoline. We reasoned that the improvement of the aqueous solubility of the analogs
to various extent should be attributable to the different water-soluble
N-heteroaromatics.
Table 1. The solubility (µg/mL) of compounds 1-8 and PL
compound
aqueous
1
2
3
4
5
40.37 45.30
46.17
59.83
48.22
solubility
compound
aqueous
PL
1.63
6
7
8
47.91
2.41
1.95
solubility
2.3. Assessment of in vitro anti-proliferative activity
Analogs 1-8 were first tested for their anti-proliferative activity against lung cancer
A549, leukemic K562 and colorectal cancer HCT-116 cell lines by MTT assay. As
shown in Fig. 2, all the analogs showed significant inhibitory activity comparable to
PL. Among them, quinoline-based analog 7 was the most potent against HCT-116
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cells. Since C2-chlorine substituted PL significantly enhanced the anticancer activity
[17], we then introduced chlorine atom into C2 position of 7 in order to further
improve its activity, yielding analog 9, which had a similar solubility in pure water but
an approximately 64-fold greater solubility in PBS aqueous solution (pH = 5) than PL
(1992.7 vs 31.2 µg/ml). Subsequently, we determined IC50 values of compounds 1-9
against HCT-116 as shown in Table 2. Expectedly, 9 (0.47 ± 0.04 µM) exhibited the
strongest cytotoxicity among all the tested compounds, superior to PL (8.13 ± 0.51
µM).
Fig. 2. Inhibitory activity of 1-8 at 10 µM against cancer A549, K562 and HCT-116
cells after incubation for 72 h. Cell proliferation was determined by MTT assay. Data
are means ± SD of the inhibition (%) from three independent experiments.
Table 2. The IC50 (µM) of 1-9 and PL against HCT-116 cells
Compound
HCT-116
4.21 ± 0.26
6.87 ± 0.31
7.05 ± 0.24
3.19 ± 0.12
8.90 ± 0.17
Compound
HCT-116
7.95 ± 0.10
0.93 ± 0.44
8.17 ± 0.31
0.47 ± 0.04
8.13 ± 0.51
1
2
3
4
5
6
7
8
9
PL
Further tests indicated that 9 was also powerful against other two colon cancer cell
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lines with IC50 values of 0.92 ± 0.11 µM and 1.79 ± 0.23 µM, respectively (Table 3).
In sharp contrast, 9 showed much less inhibitory activity on non-tumor colon
CDD-841 cells (IC50 = 50.81 ± 3.42 µM), better than PL (IC50 = 44.32 ± 1.77 µM)
(Table 3), suggesting that 9 may selectively inhibit the colon cancer cells. Thus, 9 was
selected for the following investigations.
Table 3. The IC50 (µM) of 9 against colon cancer cells and non-tumor colon cells a
Cell lines
HT-29
HCT-8
HCT-116
0.47 ± 0.04
8.13 ± 0.11
CCD-841
50.81 ± 3.42
44.32 ± 1.77
0.92 ± 0.11 1.79 ± 0.23
2.65 ± 0.17 4.10 ± 0.20
9
PL
a
Cells were treated in triplicate with tested compounds for 72 h and the cell
viability was determined by MTT assay.
2.4. ROS evaluations
Based on the above exciting results, we examined whether 9 would impact ROS
levels in both colon cancer HCT-116 cells and non-tumor colon CCD-841 cells.
HCT-116 and CCD-841 cells were treated with 9 and PL at 10 µM for 3 h,
respectively, and the levels of intracellular ROS were detected by DCFH-DA probes
using a fluorescent microplate reader to measure the fluorescent signals as shown in
Fig. 3. It is obvious that treatment with 9 rapidly and significantly increased the levels
of intracellular ROS in HCT-116 cells while the same treatment only increased low
levels of ROS in CCD-841 cells. The selectivity of 9 was greater than that of PL,
indicating that 9 more preferably promoted intracellular ROS accumulation relative to
PL in the cancer cells.
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Fig.３. A. The fluorescence microscopy images of HCT-116 or CCD-841 cells treated
with 10 µM PL or 9 for 3 h. B. The fluorescence intensities of the cells. HCT-116 or
CCD-841 cell was treated with 10 µM PL or 9 for 3 h and then detected by
fluorescence microscopy.
2.5. In vivo anti-tumor effects of 9
To evaluate the in vivo anti-colon cancer activity of 9, BALB/c nude mice were
inoculated subcutaneously with colon cancer HCT-116 cells. After the establishment
of solid tumor, two groups of the mice were intraperitoneally treated with 5 mg/kg of
9 and PL daily for 21 consecutive days, respectively (Fig. 4). It could be seen from
Fig. 4A and 4D that in comparison with the vehicle-treated controls, treatment with 9
at 5 mg/kg significantly inhibited the growth of implanted colon cancer cells (2.01 ±
0.34 vs 0.96 ± 0.19 g, p < 0.01), and the tumor weights in the mice were significantly
reduced by 52 %, which was comparable to PL (53 %, w/w).
In addition, treatment with 9 significantly reduced the volumes and size of colon
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tumors in mice (Fig. 4C). Moreover, there was no obvious abnormality in the liver,
kidney, lung and heart in terms of the size and morphology in both control and
9-treated mice except for a slight, but not significant reduction in body weight as
compared with each other (Fig. 4B).
The reason why 9 showed better inhibitory activity in vitro but similar antitumor
effects in vivo relative to PL is that 9 may have different targets in cancer cells or may
not act on those affected by PL. However, the exact mechanism of action of 9 needs to
be further clarified in future investigations.
A
B
24
control group
PL (5 mg/kg)
9 (5 mg/kg)
22
20
18
16
3
5
7
9
11 13 15 17 19 21
Time post treatment (day)
D
C
2.5
control group
PL (5 mg/kg)
9 (5 mg/kg)
2.0
control group
PL (5 mg/kg)
9 (5 mg/kg)
2.0
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
3
5
7
9
11 13 15 17 19 21
Time post treatment (day)
Fig. 4. Inhibitory effects of 9 on the growth of HCT-116 tumors in vivo. Mice
inoculated with HCT-116 tumors were intraperitoneally treated with PL or 9 per day.
A. Representative images of the tumors were captured. B. The weights of mice were
measured at the indicated time points. C. The volume of tumors was measured at the
indicated time points. D. The weight of tumors was measured at the indicated time
points. Data are shown as means ± SD from each group of mice (n = 8). **P < 0.01 vs
control.
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3. Conclusion
In summary, a series of analogs 1-9 were designed and synthesized by replacement
of the trimethoxyphenyl of PL with an N-heteroaromatic ring and/or not introduction
of 2-Cl. These compounds improved aqueous solubility and showed potent anticancer
activity. The most active compound 9 selectively enhanced ROS levels and inhibited
proliferation in colon cancer HCT-116 cells but sparing the non-tumor colon
CCD-841 cells. Furthermore, 9 significantly inhibited the growth of implanted colon
cancer cells in mice. Collectively, our findings suggest that these N-heteroaromatic
ring-based analogs of PL may deserve intensive investigations.
4. Experimental protocols
4.1. General informations
Nuclear magnetic resonance (NMR) spectra were obtained from a Bruker Avance
300 (1H, 300 MHz; 13C, 75 MHz) or 500 (1H, 500 MHz; 13C, 125 MHz) spectrometer
at 300 K using TMS as an internal standard. Mass spectrometry (MS) spectra were
recorded on a Mariner mass spectrometer. Melting points (mp) were measured by a
Mel-TEMP II apparatus and uncorrected. TLC was performed on silica gel GF/UV
254, and column chromatography was conducted by silica gel (200−300 mesh). The
purities of target compounds were characterized by HPLC analysis (Shimadzu
DGU-20A3R) and HRMS (Agilent Technologies LC/MSD TOF). The target
compounds 1-9 with a purity of >95% were used for subsequent experiments.
Compound 11-15 were prepared as described in reference 13 starting from
commercially available compound 10, and the synthetic procedures of compounds
11-15 were shown on pages S2 and S3 in the Supporting Information.
4.1.1. General procedure for the preparation of 1-9
Compound 1a-9a (1.0 mmol) was dissolved in toluene (70 mL) in a flask which
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was wrapped with tin foil, sodium hydride (1.5 mmol) and triethyl phosphonoacetate
(1.0 mmol) was added respectively. Then the reaction solution was stirred at room
temperature under dark until TLC analysis showed complete conversion. Extracted
with EtOAc, the combined organic phase was washed with saturated brine (50 mL ×
3), and dried over anhydrous sodium sulfate. Concentrated and purified by column
chromatography (PE / EA = 8: 1) to give a pale yellow solid (1b-9b). The synthetic
routes are similar to PL. [7, 12]
Compound 1b-9b (1.0 mmol) was dissolved in a mixed solution of
tetrahydrofuran and water (V(THF) / V(H2O) = 2: 1, 54 mL), then lithium hydroxide
(4.0 mmol) was slowly added, and the mixture was stirred overnight at room
temperature, checking for product formation via TLC (PE / EA = 1: 1). Next, the pH
value of reaction solution was adjusted to 4-5 with 2N hydrochloric acid aqueous
solution. The mixture was then extracted with EtOAc (30 mL × 3), and the combined
organic phase was washed with saturated brine (50 mL × 3), dried over anhydrous
sodium sulfate and concentrated to give a pale yellow solid 1c-9c. The synthetic
routes are similar to PL. [7, 12]
Compound 1c-9c (1.0 mmol) was dissolved in anhydrous tetrahydrofuran (20
mL). To the solution, triethylamine (1.2 mmol) was slowly added and the solution was
cooled to -20 °C, pivaloyl chloride (1.2 mmol) was then added, and the reaction
mixture was stirred for about 45 minutes. Next, the compound 13 or 15 and
n-butyllithium were added to the reaction solution and stirred for 1 h, checked for
product formation via TLC. Then saturated aqueous solution of ammonium chloride
(10 mL) was added to quench the n-butyllithium in the reaction and extracted with
ethyl acetate, the organic phase was washed with saturated brine (30 mL × 3), Dried
over anhydrous sodium sulfate, concentrated and purified by column chromatography
(PE / EA = 3: 1) to afford 1-9.
4.1.2.1 (E)-1-(3-(Pyridin-2-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (1). Mp:
77-80 ; 1H NMR (300 MHz, CDCl3): δ 8.61 (s, 1H), 7.83-7.65 (m, 3H), 7.46 (d, J =
9.0 Hz, 1H), 7.16-7.11 (m, 1H), 6.94-6.91 (m, 1H), 6.02 (d, J = 9.0 Hz, 1H), 4.08-4.00
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(m, 2H), 2.46 (s, 2H); 13C NMR (75 MHz, CDCl3): δ 169.0, 165.8, 153.9, 150.3,
145.8, 142.0, 136.7, 126.1, 125.8, 124.1, 41.8, 25.0; HRMS (ESI +) m/z: Calcd. for
C13H12N2O2Na [M+Na] + 251. 0796, found 251.0801, ppm error 0.5.
4.1.2.2 (E)-1-(3-(Pyridin-3-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (2). Mp:
1
99-102 ; H NMR (300 MHz, CDCl3): δ 8.74 (s, 1H), 8.55 (s, 1H), 7.92-7.80 (m,
1H), 7.65-7.49 (m, 2H), 7.49-7.40 (m, 1H), 6.93 (s, 1H), 6.02 (d, J = 9.0 Hz, 1H),
4.02 (t, J = 6 Hz, 2H), 2.46 (s, 2H); 13C NMR (75 MHz, CDCl3): δ 168.2, 165.7,
153.8, 148.2, 145.7, 141.9, 136.7, 126.1, 125.8, 124.0, 41.8, 24.9; HRMS (ESI +) m/z:
Calcd. for C13H12N2O2Na [M+Na] + 251.0796, found 251.0803, ppm error 0.7.
4.1.2.3 (E)-1-(3-(Pyridin-4-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (3). Mp:
90-94 ; 1H NMR (300 MHz, CDCl3): δ 8.59 (s, 2H), 7.74-7.37 (m, 4H), 6.94 (d, J =
6 Hz, 1H), 6.02 (d, J = 9.0 Hz, 1H), 4.08-4.00 (m, 2H), 2.56-2.34 (m, 2H); 13C NMR
(75 MHz, CDCl3): δ169.0, 164.7, 153.2, 148.0, 145.7, 141.9, 136.5, 126.1, 125.2,
124.1, 41.0, 24.9; HRMS (ESI +) m/z: Calcd. for C13H12N2O2Na [M+Na] + 251.0796,
found 251.0802, ppm error 0.6.
4.1.2.4 (E)-1-(3-(Pyrazin-2-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (4). Mp:
1
110-115 ; H NMR (300 MHz, CDCl3): δ 8.67 (s, 1H), 8.58 (s, 1H), 8.48 (s, 1H),
7.90 (d, J = 15.0 Hz, 1H), 7.63 (d, J = 15.0 Hz, 1H), 6.97-6.92 (m, 1H), 6.02 (d, J =
9.0 Hz, 1H), 4.02 (t, J = 12.0 Hz, 2H), 2.50-2.45 (m, 2H); 13C NMR (75 MHz, CDCl3):
δ 168.5, 149.5, 146.1, 145.1, 145.0, 144.9, 137.7, 131.1, 128.4, 125.7, 41.8, 25.0;
+
HRMS (ESI +) m/z: Calcd. for C12H11N3O2Na [M+Na] 252.0749, found 252.0753,
ppm error 0.4.
4.1.2.5 (E)-1-(3-(3-Methylpyrazin-2-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (5).
1
Mp: 121-133 ; H NMR (300 MHz, CDCl3): δ 8.39 (d, J = 9.0 Hz, 2H), 7.94-7.79
(m, 2H), 6.96-6.90 (m, 1H), 6.02 (d, J = 12.0 Hz, 1H), 4.02 (t, J = 6.0 Hz, 2H), 2.67 (s,
3H), 2.49-2.44 (m, 2H); 13C NMR (75 MHz, CDCl3): δ 168.8, 165.7, 153.1, 147.5,
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145.9, 144.1, 142.5, 136.2, 128.9, 125.7, 41.8, 27.8, 21.8; HRMS (ESI +) m/z: Calcd.
for C13H13N3O2Na [M+Na] + 266.0905, found 266.0911, ppm error 0.6.
4.1.2.6 (E)-1-(3-(5-Methylpyrazin-2-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (6).
Mp: 175-180 ; 1H NMR (300 MHz, CDCl3): δ 8.55 (s, 1H), 8.45 (s, 1H), 7.85 (d, J
= 15.0 Hz, 1H), 7.65 (d, J = 15.0 Hz, 1H), 6.97-6.91 (m, 1H), 6.03 (d, J = 12.0 Hz,
13
1H), 4.02 (t, J = 6.0 Hz, 2H), 2.57 (s, 3H), 2.50-2.45 (m, 2H); C NMR (75 MHz,
CDCl3): δ 168.8, 165.6, 157.5, 154.2, 146.7, 144.6, 142.3, 136.2, 128.1, 125.6, 41.8,
+
27.4, 20.9; HRMS (ESI +) m/z: Calcd. for C13H13N3O2Na [M+Na] 266.0905, found
266.0911, ppm error 0.6.
4.1.2.7 (E)-1-(3-(Quinolin-2-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (7). Mp:
84-89 ; 1H NMR (300 MHz, CDCl3): δ 8.15-8.13 (m, 2H), 7.91 (s, 2H), 7.81 (d, J =
9.0 Hz, 1H), 7.69-7.66 (m, 2H), 7.55-7.50 (m, 1H), 6.98-6.92 (m, 1H), 6.05 (d, J = 9.0
13
Hz, 1H), 4.05 (t, J = 6.0 Hz, 2H), 2.54-2.46 (m, 2H); C NMR (75 MHz, CDCl3):
δ168.4, 162.8, 153.9, 148.5, 146.7, 143.9, 141.5, 130.3, 130.1, 128.3, 127.4, 126.6,
+
126.2, 120.4, 41.9, 27.8; HRMS (ESI +) m/z: Calcd. for C17H14N2O2Na [M+Na]
301.0953, found 301.0960, ppm error 0.7.
4.1.2.8 (E)-1-(3-(Quinoline-3-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (8). Mp:
86-91 ; 1H NMR (300 MHz, CDCl3): δ 8.48 (s, 1H), 8.12 (dd, J = 15.0 and 6.0 Hz,
2H), 7.93 (s, 2H), 7.77 (d, J = 15.0 Hz, 1H), 7.69-7.72 (m, 2H), 7.54 (t, J = 7.5 Hz,
13
1H), 6.07 (d, J = 9.0 Hz, 1H), 4.07 (t, J = 6.0 Hz, 2H), 2.52-2.45 (m, 2H); C NMR
(75 MHz, CDCl3): δ 168.4, 162.8, 149.3, 148.5, 146.7, 143.9, 141.5, 130.1, 129.7,
128.3, 127.6, 127.4, 127.0, 126.2, 41.9, 27.8; HRMS (ESI +) m/z: Calcd. for
C17H14N2O2Na [M+Na] + 301.0953, found 301.0960, ppm error 0.7.
4.1.2.9 (E)-3-Chloro-1-(3-(quinoline-2-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (9).
1
Mp: 93-96 ; H NMR (300 MHz, CDCl3): δ 8.08 (dd, J = 15.0 and 6.0 Hz, 2H),
7.91-7.76 (m, 2H), 7.74 (d, J = 12.0 Hz, 1H), 7.70-7.67 (m, 2H), 7.52 (t, J = 6.0 Hz),
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13
7.10-7.03 (m, 1H), 4.09 (t, J = 6.0 Hz, 2H), 2.56 (d, J = 6.0 Hz, 2H) ; C NMR (75
MHz, CDCl3): δ 168.4, 161.6, 153.9, 148.5, 143.9, 141.5, 136.8, 130.2, 130.1, 127.7,
127.5, 127.4, 126.6, 120.5, 42.0, 25.5; HRMS (ESI +) m/z: Calcd. for
C17H13ClN2O2Na [M+Na] + 335.0563, found 336.0571, ppm error 0.8.
4.2 Solubility test
Compounds 1-8 and PL were added to the indicated solvent. After shaking and
centrifuging, the supernatant was taken to determine the concentration of the
compounds for calculation of the corresponding solubility. The samples (20 µL each)
were analyzed by HPLC (Shimadzu DGU-20A3R) on Shimadzu-GL WondaSil
C18-WR column. The mobile phase was acetonitrile–water (60/40, v/v) at a flow rate
of 1.0 mL/min with the detection wavelength at 254 nm. This experiment was
repeated in triplicates.
4.3 Biological assays
4.3.1 MTT assay
Human colon, colorectal and non-tumor colon epithelial cells (1 × 106
cells/well) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum
(FBS) in 96-well plates overnight and treated with vehicle alone or the tested
compounds at the indicated concentrations for 24, 48, or 72 h. During the last 4-h
culture, the cells were exposed to 20 µL of MTT (5 mg/mL, in PBS). The generated
formazan in individual wells was dissolved in DMSO (150 µL), and measured for the
absorbance at 570 nm using a microplate reader. The inhibition rates were calculated
by the formula of (1-OD (experimental cells)/OD (control cells)) x 100%.
4.3.2 Measurement of ROS generation
The tested cells (1 × 106 cells/well) were treated in triplicate with vehicle DMSO
(0.1%, v/v) alone, PL or 9 at the indicated concentrations for 3 h, and the cells were
stained with dihydroethidium (DHE, Beyotime). The levels of intracellular ROS were
measured as the fluorescent signals using a fluorescence microplate reader (300 and
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610 nm).
4.3.3 Evaluation of in vivo antitumor activity
All animal experimental protocols were approved by the Administration Committee of
Experimental Animals in Jiangsu Province and the Ethics Committee of China
Pharmaceutical University. Male BALB/c nude mice 3–5 weeks of age (Silaike
Experiment Animal Co., Ltd, Shanghai, China) were inoculated subcutaneously with
1 × 107 HCT-116 cells. After the formation of a solid tumor with a volume of about 75
mm3, the tumor-bearing mice were randomly divided into four groups with eight mice
in each group. The groups with compound 9 and PL treatment received a dosage (5
mg/kg) every day. At the end of the experiment, the mice were sacrificed, their tumors
were dissected, and the tumor size and weight were measured. The tumor inhibitory
ratio was calculated by the following formula: tumor inhibitory ratio (%) =
[(Wcontrol − Wtreated)/Wcontrol] × 100%. Wtreated and Wcontrol were the average
tumor weights of the treated and control mice, respectively. The tumor diameters were
measured with calipers, and the tumor volume was calculated by the formula V (mm3)
= d2 × D/2, where D is the largest diameter and d the smallest diameter.
Acknowledgments
This study was supported by grants from the National Natural Science Foundation of
China (No. 21372261 and No. 81673305) and Jiangsu Province Funds for
Distinguished Young Scientists (BK20160033). Part of the work was supported by the
Program for New Century Excellent Talents in University (NCET-13-1033) and the
Jiangsu Shuang Chuang team.
References
[1] Y.W. Alelyunas, J. R. Empfield, D. McCarthy, R.C. Spreen, K. Bui, L.
Pelosi-Kilby, C. Shen, Experimental solubility profiling of marketed CNS drugs,
exploring solubility limit of CNS discovery candidate, Bioorg. Med. Chem. Lett. 20
(2010) 7312-7316.
15

ACCEPTED MANUSCRIPT
[2] M. Palucki, J.D. Higgins, E. Kwong, A.C. Templeton, Strategies at the interface of
drug discovery and development: Early optimization of the solid state phase and
preclinical toxicology formulation for potential drug candidates, J. Med. Chem. 53
(2010) 5897-5905.
[3] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in drug discovery
and development settings, Adv. Drug. Delivery Rev. 46 (2001) 3-26.
[4] B.S. Park, D.J. Son, W.S. Choi, G.R. Takeoka, S.O. Han, T.W. Kim, S.E. Lee,
Antiplatelet activities of newly synthesized derivatives of piperlongumine, Phytother.
Res. 22 (2008) 1195-1199.
[5] Y.H. Seo, J.K. Kim, J.G. Jun, Synthesis and biological evaluation of
piperlongumine derivatives as potent anti-inflammatory agents, Bioorg. Med. Chem.
Lett. 24 (2014) 5727-5730.
[6] S.K. Ku, J.A. Kim, J.S. Bae, Vascular barrier protective effects of piperlongumine
in vitro and in vivo, Inflamm. Res. 63 (2014) 369-379.
[7] L. Raj, T. Ide1, A.U. Gurkar, M. Foley, M. Schenone, X. Li, N.J. Tolliday, T.R.
Golub, S.A. Carr, A.F. Shamji, A.M. Stern, A. Mandinoval1, S.L. Schreiber, S.W. Lee,
Selective killing of cancer cells by a small molecule targeting the stress response to
ROS, Nature 475 (2011) 231-234.
[8] E.H. Kong, Y.J. Kim, H.J. Cho, S.N. Yu, K.Y. Kim, J.H. Chang, S.C. Ahn,
Piplartine induces caspase-mediated apoptosis in PC-3 human prostate cancer cells,
Oncol. Rep. 20 (2008) 785-792.
[9] X.X. Xiong, J.M. Liu, X.Y. Qiu, F. Pan, S.B. Yu, X.Q. Chen, Piperlongumine
induces apoptotic and autophagic death of the primary myeloid leukemia cells from
patients via activation of ROS-p38/JNK pathways, Acta. Pharmacol. Sin. 36 (2015)
362-374.
[10]Fofaria, N. M.; Qhattal, H. S. S.; Liu, X.; Srivastava, S. K. Nanoemulsion
formulations for anti-cancer agent piplartine – characterization, toxicological,
pharmacokinetics and efficacy studies. Int. J. Pharm. 2015, 498, 12-22.
[11]Alhassan, A.; Aaron, P.; Kelly, K.; Myrdal, P. B. Preformulation Studies on
Piperlongumine. Plos. One. 2016, 11, e0151707.
[12]Adams, D. J.; Dai, M.; Pellegrino, G.; Wagner, B. K.; Stern, A. M.; Shamji, A. F.;
Schreiber, S. L. Synthesis, cellular evaluation, and mechanism of action of
piperlongumine analogs. PNAS. 2012, 109, 15115-15120.
[13]Wu, Y. L.; Min, X.; Zhuang, C. L.; Li, J.; Yu, Z. L.; Dong, G. Q.; Yao, J. Z.; Wang,
S. Z.; Liu, Y.; Wu, S. C.; Zhu, S. P.; Sheng, C. Q.; Wei, Y. Y.; Zhang, H. J.; Zhang, W.
N.; Miao, Z. Y. Design, synthesis and biological activity of piperlongumine
derivatives as selective anticancer agents. Eur. J. Med. Chem. 2014, 82, 545-551.
[14]Punganuru, S. R.; Madala, H. R.; Venugopal, S. N.; Samala, R.; Mikelis, C.;
Srivenugopal, K. S. Design and synthesis of a C7-aryl piperlongumine derivative with
potent antimicrotubule and mutant p53-reactivating properties. Eur. J. Med. Chem.
2016, 107, 233-244.
[15]Zhang, Y.; Ma, H.; Wu, Y.; Wu, Z.; Yao, Z.; Zhang, W.; Zhuang, C.; Miao, Z.
Novel non-trimethoxylphenyl piperlongumine derivatives selectively kill cancer cells.
16

ACCEPTED MANUSCRIPT
Bioorg. Med. Chem. Lett. 2017, available online.
[16] D.J. Adamsa, M. Daia, G. Pellegrinoa, B.K. Wagnera, A.M. Stema, A.F. Shamjia,
S.L. Schreibera, Synthesis, cellular evaluation and mechanism of action of
piperlongumine analogs, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 15115-15120.
[17] W.J. Yan, Q. Wang, C.H. Yuan, F. Wang, Y. Ji, F. Dai, X.L. Jin, B. Zhou,
Designing piperlongumine-directed anticancer agents by an electrophilicity-based
prooxidant strategy: A mechanistic investigation, Free Radical Bio. Med. 97 (2016)
109-123.
[18] Y. Liao, X.J. Niu, B.L. Chen, H. Edwards, L.P. Xu, C.Z. Xie, H. Lin, L. Polin,
J.W. Taub, Y.B. Ge, Z.H. Qin, Synthesis and antileukemic activities of
piperlongumine and HDAC inhibitor hybrids against acute myeloid leukemia cells, J.
Med. Chem. 59 (2016) 7974-7990.
[19] B. Yang, Z.J. Li, L.C. Lei, F.F. Sun, J.K. Zhu, Prediction of setschenow constants
of N-heteroaromatics in NaCl solution based on the partial charge on the heterocyclic
nitrogen atom, Environ. Sci. Pollut. Res. 23 (2016) 3399-3405.
[20] W.H. Xie, W.Y. Shiu, D. Mackay, A review of the effect of salts on the solubility
of organic compounds in seawater, Mar. Environ. Res. 44 (1997) 429-444.
[21] G. Gross, J. Tardio, O. Kuhlmann, Solubility and stability of dalcetrapib in
vehicles and biological media, Int. J. Pharm. 437 (2012) 103-109.
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Highlights
ò 9 was 64-fold greater soluble in PBS aqueous solution (pH = 5) than
piperlongumine (PL).
ò 9 was more cytotoxic (IC50 = 0.47-1.79 µM) than PL (IC50 = 2.65-8.13 µM)
against three colon cancer cell lines but sparing non-tumor colon cells.
ò 9 more preferably promoted ROS accumulation than PL in colon cells.
ò 9 significantly suppressed tumor growth in vivo comparable to PL.