Design, synthesis and biological activity of piperlongumine derivatives as selective anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.05.070

In an effort to expand the structure-activity relationship of the natural anticancer compound piperlongumine, we have prepared sixteen novel piperlongumine derivatives with halogen or morpholine substituents at C2 and alkyl substituents at C7. Most of 2-halogenated piperlongumines showed potent in vitro activity against four cancer cells and modest selectivity for lung normal cells. The highly active anticancer compound 11h exhibited obvious ROS elevation and excellent in vivo antitumor potency with suppressed tumor growth by 48.58% at the dose of 2 mg/kg. The results indicated that halogen substituents as electrophilic group at C2 played an important role in increasing cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2014.05.070

Accepted Manuscript
Design, Synthesis and biological activity of piperlongumine derivatives as selective
anticancer agents
Yuelin Wu , Xiao Ming , Chunlin Zhuang , Jing Li , Zhiliang Yu , Guoqiang Dong ,
Jiangzhong Yao , Shengzheng Wang , Yang Liu , Shangchao Wu , Shiping Zhu ,
Chunquan Sheng , Yunyang Wei , Huojun Zhang , Wannian Zhang , Zhenyuan Miao
PII:
S0223-5234(14)00502-9
10.1016/j.ejmech.2014.05.070
EJMECH 7028
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 November 2013
Revised Date: 26 May 2014
Accepted Date: 29 May 2014
Please cite this article as: Y. Wu, X. Ming, C. Zhuang, J. Li, Z. Yu, G. Dong, J. Yao, S. Wang, Y. Liu, S.
Wu, S. Zhu, C. Sheng, Y. Wei, H. Zhang, W. Zhang, Z. Miao, Design, Synthesis and biological activity
of piperlongumine derivatives as selective anticancer agents, European Journal of Medicinal Chemistry
(2014), doi: 10.1016/j.ejmech.2014.05.070.
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*Graphical Abstract (for review)
Click here to download Graphical Abstract (for review): Graphic Abstract.doc
ACCEPTED MANUSCRIPT
Graphic Abstract
Sixteen piperlongumine derivatives with halogen or morpholine at C2 and alkyl
substituents at C7 were prepared as anticancer agents. The highly active anticancer
compound 11h exhibited obvious ROS elevation and excellent in vivo antitumor
potency.

*Highlights (for review)
ACCEPTED MANUSCRIPT
Highlights




Sixteen piperlongumine derivatives were prepared in five or six steps.
2-Halogen piperlongumines showed moderate in vitro activity against four cancer cells.
Most of the compounds showed modest selectivity for lung normal cells.
Halogen substituents at C2 played an important role in increasing cytotoxicity.

*Revised Manuscript
Click here to view linked References
ACCEPTED MANUSCRIPT
Design, Synthesis and biological activity of piperlongumine
derivatives as selective anticancer agents
a
a
a
Yuelin Wu a,b,1, Xiao Ming a,1, Chunlin Zhuang , Jing Li , Zhiliang Yu , Guoqiang
a
Dong a, Jiangzhong Yao , Shengzheng Wang a, Yang Liu a, Shangchao Wu a, Shiping
a
a
a,*
Zhu , Chunquan Sheng , Yunyang Wei b, Huojun Zhang c,*, Wannian Zhang
,
Zhenyuan Miao a,*
a School of Pharmacy, Second Military Medical University, 325 Guohe Road,
Shanghai 200433, People’s Republic of China.
b School of chemical engineering, Nanjing University of Science & Technology, 200
Xiaolingwei, Nanjing 210094, People’s Republic of China.
c Department of Radiation Oncology, Changhai Hospital, Second Military Medical
University, 168 Changhai Road, Shanghai 200433, People’s Republic of China.
*To whom correspondence should be addressed. Mailing address: School of
Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
People’s Republic of China. Phone: 86-21-81871241. Fax: 86-21-81871241. E-mail:
miaozhenyuan@hotmail.com; zhangwnk@hotmail.com; chyyzhj@hotmail.com
1 Both authors contributed equally to this work
1

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Abstract: In an effort to expand the structure-activity relationship of the natural
anticancer compound piperlongumine, we have prepared sixteen novel
piperlongumine derivatives with halogen or morpholine substituents at C2 and alkyl
substituents at C7. Most of 2-halogenated piperlongumines showed potent in vitro
activity against four cancer cells and modest selectivity for lung normal cells. The
highly active anticancer compound 11h exhibited obvious ROS elevation and
excellent in vivo antitumor potency with suppressed tumor growth by 48.58% at the
dose of 2 mg/ kg. The results indicated that halogen substituents as electrophilic group
at C2 played an important role in increasing cytotoxicity.
Keywords：piperlongumine; anticancer agent; selectivity; biological activity
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1. Introduction
Piperlongumine(PL) is an alkaloid isolated from Piper species and displays
anticancer[1-7], anti-diabetic[8], anti-platelet aggregation[9-12], and antifungal
activities[13, 14]. Recently, Raj et al demonstrated selectivity of piperlongumine for
cancer cells. Compared to the normal cell lines, piperlongumine induced cell death in
thirteen cancer lines with an average IC50 value lower than 7 μM[15]. Additionally,
similar effects were observed in vivo in four nude mice models without effect on
normal tissue. Further studies revealed a unique mechanism based on reactive oxygen
species (ROS). Using a phenotypic screen, they found that two proteins:
glutathione-S- transferase-P1 (GSTP1) and carbonyl reductase 1 (CBR1) played a
more important role in the cellular response to ROS. This tumor specific effect of
piperlongumine on cytotoxicity and ROS shows a new strategy for selective targeting
of cancer cells[16-20].
To explore the structure and biological activity relationship of piperlongumine, eighty
analogues had been synthesized and screened by Adams et al[21]. Based on the assays
of cellular toxicities, two key pharmacophores: C2-C3 and C7-C8 double bonds had
been identified. Both these Michael acceptors were necessary for PL’s toxicity to
cancer cells. The former was also critical for ROS elevation and glutathione depletion.
However, removal of C7-C8 olefin led to a decrease in cytotoxicity, but did not reduce
the ROS level. Among all the analogues, PL dimer and trimer displayed more potent
activities than the others. SAR indicated that alkynyl substituents can be introduced at
position 2 without effect in cytotoxicity. On the contrary, alkyl and aromatic
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substituents were not tolerated. Although the C7-C8 olefin has been confirmed as one
of two key pharmacophores for cellular toxicity with the example of the inactive
C7-C8 olefin reduction product, we are unaware of the effect of substituents on the
C7-C8 double bond. In order to continue the development of piperlongumine
derivatives as selective anticancer agents, we describe herein the synthesis and
biological activity of a novel series of piperlongumine derivatives on positions 2 and
7. An active piperlongumine derivative was also assayed for its in vivo antitumor
activity in aA549 nude mice model.
2. Chemistry
PL and sixteen derivatives were synthesized according to the modified Raj’s route[15].
As key reaction intermediates, three dihydropyridin-2(1H)-ones 3, 4 and 6 were
prepared in three or four steps from the commercially available piperidin-2-one
1(Scheme 1). Treatment of
1
with PCl5 in chloroform yielded
2,2-dichloropiperidin-2-one 2[22, 23]. Dehydrochloration of 2 at 120°C in the
presence of Li2CO3 provided the corresponding 2-chloro- 5,6-dihydropyridin-
2(1H)-one 3 which was readily converted into 2-morpholino- 5,6-dihydropyridin-
2(1H)-one 4. The third dihydropyridin-2(1H)-one 6 was prepared by the same route as
3 in a 63.1% yield.
With three dihydropyridin-2(1H)-ones in hand, all target piperlongumine derivatives
were synthesized as detailed in Scheme 2. Condensation of 3,4,5-trimethoxy-
benzaldehyde 7 with 2-substituted malonic acids in pyridine yielded 2-substituted
(E)-3-(3,4,5-trimethoxyphenyl)acrylic acids 9a - 9e. Treatment of 9a - 9e with oxalyl
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chloride in anhydrous THF under N2 gave the acyl chlorides 10a - 10e which were
reacted with the three substituted dihydropyridin-2(1H)-ones to afford
piperlongumine derivatives 11a – 11p using anhydrous THF as solvent and TEA as
catalyst.
3. Results and discussion
3.1 In Vitro anticancer screening
The first obtained seven piperlongumine derivatives 11a-11g and PL were evaluated
for in vitro cytotoxicities against four cancer cells (human lung carcinoma A549,
human colorectal carcinoma HCT116, human breast carcinoma ZR-75-30, human
breast carcinoma MDA-MB-231) and human lung normal cells MRC-5 by MTT assay,
respectively. A summary of these results is shown in Table 1. Interestingly,
substituents on position 2 of piperlongumine played an important role in
antiproliferative activities. For example, compounds 11a and 11b without any group
at C2 exhibited potent activities. On the other hand, the 2-chloro derivative 11g also
showed potent activities against all four cells. However, compounds 11c - 11f with
morpholine substituents at C2 were uniformly less active than piperlongumine. In
addition, compound 11g resulted in a nearly two- or three-fold increase in selectivity
for three cancer cell lines HCT116, ZR-75-30, MDA-MB-231 over normal cells
MRC-5.
Encouraged by the preliminary design, a series of piperlongumine derivatives with
halogen substituent at C2 were synthesized to improve antitumor activity and
investigate the SARs. To our delight, most of the 2-halogen substituted
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piperlongumine derivatives showed potent activities against four cancer cell lines
(human lung carcinoma A549, human colorectal carcinoma HCT116, human breast
carcinoma MDA-MB-231, human hepatoma Hep3B) and human fetal lung normal
cells WI38 (Table 2). Remarkably, both 2-chloro substituted piperlongumines 11h –
11k and 2-bromo substituted piperlongumines 11l – 11p showed more potent
activities than piperlongumine. This fact confirmed that halogen substituents as
electrophilic group at C2 increase cytotoxicity. Moreover, selectivity for A549 cells
over normal cells was slightly decreased with the length of carbon chain at C7 except
for compound 11l. For example, 7-methyl-2-chloropiperlongumine 11h with a rate
value of 4.98 showed higher selectivity than 7-propyl-2-chloropiperlongumine 11j in
these assays.
3.2 ROS evaluation
Although the natural product piperlongume, isolated from Piper species has been
recognized as an antitumor agent, its cellular mechanism has not been defined.
Recently a ROS-independent cellular effect has been disclosed as the antitumor
mechanism of piperlongumine[15, 21]. In order to validate the mechanism of the
target compounds, we next determined the effect of piperlongumine derivative 11h on
cellular ROS level in A549 cancer cells by fluorescence microscopy. Treatment with
10 M of 11h for 1h caused an obvious increase in ROS level (Fig. 2). The result
indicated that a halogen substituent at C2 of piperlongumine did not affect the ROS
elevation.
3.3 In vivo anticancer evaluation
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On the basis of the favorable in vitro cellular activity and selectivity for normal cells,
compound 11h was selected for further in vivo antitumor activity studies on human
lung cancer A549 xenograft model. Doxorubicin (DOX), a clinical drug in the
treatment of a wide range of cancers, was chosen as reference. PL was also used as
reference for the effect of the halogen substituent of piperlongumine on inhibition
tumor growth. As showed in Fig. 3, the results revealed that remarkable antitumor
effects were observed in mice treated with compound 11h which was administered
intraperitoneally (ip) for fourteen consecutive days. Furthermore, compound 11h
showed higher in vivo antitumor potency than natural piperlongumine.
At the dose of 2 mg/ kg body weight, compound 11h suppressed tumor growth by
48.58% compared to 38.31% of PL at the same dose. In addition, there is no obvious
weight loss in animals treated with compound 11h. Therefore, the results may suggest
a low toxicity for compound 11h which is worth of further studies as a new potential
antitumor candidate.
4. Conclusions
In this work, a novel series of piperlongumine derivatives with halogen, morpholine
substituents at C2 and alkyl substituents at C7 were synthesized and characterized.
2-Halo-7-alkylpiperlongumines exhibited moderate in vitro activity against all four
cancer cells while the morpholine substituted derivatives at position 7 of
piperlongumine showed diminished toxicities in cells. It was noted that all of the
active compounds revealed modest selectivity for human lung normal cells MRC-5
and human fetal lung normal cells WI38. The highly active anticancer compound 11h
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with selectivity for normal cells exhibited obvious ROS elevation and excellent in
vivo antitumor potency. The present investigation indicated that 2-chloro-7-methyl-
piperlongumine 11h is worthy further of studies as an antitumor candidate.
5. Experimental Section
5.1 Chemistry. General Methods. All starting materials were commercially available
and analytically pure. Melting points were measured on an uncorrected XT4A digital
1
melting point apparatus (Beijing Keyi Instrument Co., Ltd.; China). H NMR spectra
were recorded on a Bruker Avance 300, Bruker Avance 500 and a Bruker Avance 600
spectrometers (Bruker Company, Germany), using TMS as an internal standard and
CDCl3 or DMSO-d6 as solvents. Chemical shifts (δ values) and coupling constants (J
values) are given in ppm and Hz, respectively. The mass spectra were recorded on an
Esquire 3000 LC-MS mass spectrometer. TLC analysis was carried out on silica gel
plates GF254 (Qindao Haiyang Chemical, China). Flash column chromatography was
carried out on silica gel 300 - 400 mesh using a Biotage instrument. Anhydrous
solvents and reagents were all analytically pure and dried by routine protocols.
5.2 3-Chloro-5,6-dihydropyridin-2(1H)-one (3)
A solution of piperidin-2-one (25.0 g, 252.2 mmol) in CH3Cl (250 mL) was treated
with PCl5(158.0 g, 765.5 mmol) at 0-5 °C for 15 min before being refluxed for 3 h.
Then the reaction mixture was cooled down to 25°C and transferred into an ice-water
mixture(500 mL). The aqueous layer was extracted with CH2Cl2 (3 × 50 mL). The
combined organic extracts were washed with saturated saline solution (50 mL), dried
over MgSO4 and concentrated in vacuo to yellow oil 2 which could be used in the
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next step without further purification.
A solution of 2 (4 g, 23.8 mmol) in DMF (12 mL) was treated with Li2CO3 (3.6g, 48.7
mmol) at room temperature. The resulting mixture was then heated to 120 °C for 7 h.
Then the reaction mixture was cooled down to 25°C and transferred into an ice-water
mixture(500 mL). The aqueous layer was extracted with CH2Cl2 (3 × 50 mL). The
combined organic extracts were washed with saturated saline solution (50 mL), dried
over MgSO4, concentrated and purified by flash chromatography (Ethyl
Acetate/Petroleum Ether = 1:1). The product was obtained as a light brown solid 3
(1.1 g, 35.1%).
m.p. 70-72 °C, 1H-NMR (300 MHz, CDCl3)δ: 7.05 (s, 1 H, NHCO), 6.78(t, J = 4.2 Hz,
1 H, =CH), 3.48 (td, J1 = 2.7 Hz, J2 = 7.2 Hz, 2 H, NCH2), 2.44-2.50 (m, 2 H, CH2);
MS (ESI): 132.2 [M+H]+.
5.3 3-morpholino-5,6-dihydropyridin-2(1H)-one (4)
A solution of 3 (1.3 g, 100.0 mmol) and morpholine in DMF (5 mL) was treated with
Li2CO3 (1.5g, 20.3 mmol) at room temperature. The resulting mixture was then heated
at 130 °C for 3 h. Then the reaction mixture was cooled down to 25°C and filtered.
The filtrate was concentrated and the residue was purified by flash chromatography
(Ethyl Acetate/Petroleum Ether = 1:2). The product was obtained as a brown solid 4
(0.6g, 30.2%).
M.p. 123-126 °C, 1H-NMR(CDCl3, 300 MHz)δ: 6.15(s, 1 H, NH), 5.56(t, J = 1.5 Hz,
1 H, =CH), 3.82-3.85 (m, 4 H, 2 CH2), 3.30-3.36 (m, 2 H, CH2), 2.87-2.90 (m, 4 H, 2
CH2), 2.33-2.40 (m, 2 H, CH2); MS (ESI): 183.1 [M+H]+.
5.4 3-bromo-5,6-dihydropyridin-2(1H)-one(6)
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A solution of piperidin-2-one (9.9 g, 100.0 mmol) in CH2Cl2 (200 mL) was treated
with PCl5 (41.6 g, 200.0 mmol) at 0-5 °C for 10 min. Then ZnI2 (1.0 g, 3.0 mmol) was
added under N2 and warmed up to room temperature for 1 h. Then Br2 (32.0 g, 200.0
mmol) in CH2Cl2 (100 mL) was slowly added, the reaction mixture was stirred for 12
h and transferred into an ice-water mixture(500 mL). The aqueous layer was extracted
with CH2Cl2(5 × 30 mL). The combined organic extracts were washed with saturated
saline solution (50 mL), dried over MgSO4, concentrated and purified by flash
chromatography (Ethyl Acetate/Petroleum Ether= 1:4). The product was obtained as a
light brown solid 5 (21.0g, 82%).
A solution of 5 (12.8 g, 50.0 mmol) in DMF (70 mL) was treated with LiCl (4.0 g,
51.0 mmol) and Li2CO3 (7.0 g, 95.0 mmol) at room temperature. The resulting
mixture was then heated at 130 °C for 8 h. Then the reaction mixture was
concentrated and purified by flash chromatography (Ethyl Acetate/Petroleum Ether =
1:2). The product was obtained as a light brown solid 6 (6.8 g, 77%).
M.p. 80-82 °C, 1H-NMR (CDCl3, 300 MHz) δ: 7.05 (t, J = 1.5 Hz, 1 H, =CH), 6.89 (s,
1 H, NH), 3.49 (t, J = 7.2 Hz, 2 H, NCH2), 2.40-2.46 (m, 2 H, CH2); MS (ESI): 176.3
[M+H]+.
5.5 General Procedure for the synthesis of 9a – 9d:
A solution of 3,4,5-trimethoxybenzaldehyde7 (2.0 g, 10.0 mmol), piperidine (0.2 g,
2.0 mmol) and 2-substitued malonic acid (5.9 g, 57.0 mmol) in dry pyridine (20 mL)
was refluxed for 24 h. Then the reaction mixture was concentrated and water (50 mL)
was added slowly. The solution was extracted with ethyl acetate (3 × 20 mL). The
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combined organic extracts were washed with saturated saline solution (50 mL), dried
over MgSO4, concentrated and purified by flash chromatography (Ethyl
Acetate/Petroleum Ether = 1:2) to afford 9a – 9d.
5.5.1 (E)-2-methyl-3-(3,4,5-trimethoxyphenyl)acrylic acid (9a):
White solid, yield 66.1%, m.p. 154-156 °C, 1H-NMR (CDCl3, 300 MHz) δ: 7.77 (s, 1
H, =CH),6.69 (s, 2 H, Ph), 3.90 (s, 9 H, 3 OMe), 2.19 (s, 3 H, Me); MS (ESI): 253.2
[M+H]+.
5.5.2 (E)-2-ethyl-3-(3,4,5-trimethoxyphenyl)acrylic acid (9b):
1
Off-white solid, yield 53.0%, m.p. 120-122 °C, H-NMR (CDCl3, 500 MHz) δ: 7.74
(s, 1 H, =CH), 6.69 (s, 2 H, Ph), 3.91 (s, 3 H, OMe), 3.90 (s, 6 H, 2 OMe), 2.64 (q, J =
7.5 Hz, 2 H, CH2), 1.27 (t, J = 7.5 Hz, 3 H, Me); MS (ESI): 267.1 [M+H]+.
5.5.3 (E)-2-propyl-3-(3,4,5-trimethoxyphenyl)acrylic acid (9c):
1
Off-white solid, yield 36.0%, m.p. 97-99 °C, H-NMR (CDCl3, 500 MHz) δ: 7.73 (s,
1 H, =CH), 6.67 (s, 2 H, Ph), 3.90 (s, 3 H, OMe), 3.88 (s, 6 H, 2 OMe), 2.50-2.58 (m,
2 H, CH2), 1.64-1.69 (m, 2 H, CH2), 1.03 (t, J = 7.5 Hz, 3 H, Me); MS (ESI): 281.4
[M+H]+.
5.5.4 (E)-2-butyl-3-(3,4,5-trimethoxyphenyl)acrylic acid (9d):
1
Off-white solid, yield 33.2%, m.p. 94-96 °C, H-NMR (CDCl3, 300 MHz) δ: 7.72 (s,
1 H, =CH),6.67 (s, 2 H, Ph), 3.89 (s, 3 H, OMe), 3.88 (s, 6 H, 2 OMe), 2.56-2.61 (m,
2 H, CH2, butyl), 1.58-1.66 (m, 2 H, CH2, butyl), 1.40-1.48 (m, 2 H, CH2, butyl ),
0.95 (t, J = 7.5 Hz, 3 H, Me); MS (ESI): 295.2 [M+H]+.
5.6 General Procedure for synthesis of 11a – 11p:
A solution of substitued (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid (9a – 9e) and
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oxalyl chloride (3.89 eq.) in dried THF was refluxed for 4 h under N2. The reaction
was cooled to 0°C and triethylamine (2.00 eq.) was slowly added. The reaction
solution was stirred for 15 min at 0 ºC. Then 3, 4 or 6 (1.50 eq.) in dried THF was
slowly added and stirred at room temperature for 12 h. The mixture was diluted with
CH2Cl2 (30 mL) and washed with saturated ammonium chloride solution (20 mL) and
saturated saline solution (20 mL), dried over MgSO4, concentrated and purified by
flash chromatography (Ethyl Acetate/Petroleum Ether = 1:2) to afford 11a – 11p.
5.6.1(E)-1-(2-methyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydropyridin-
-2(1H)-one (11a):
1
Light yellow solid, Yield 75.3%, m.p. 91-92 °C, H-NMR (CDCl3, 300 MHz) δ:
6.95-6.98 (m, 1 H, =CH), 6.87 (s, 1 H, =CH), 6.64 (s, 2 H, Ph), 6.02 (d, J = 9.6 Hz, 1
H, =CH), 3.94 (t, J = 6.6 Hz, 3 H, NCH2), 3.88 (s, 9 H, 3 OMe), 2.54-2.59 (m, 2 H,
13
CH2); 2.16 (s, 3 H, CH3); C-NMR (CDCl3, 300 MHz) δ: 168.92, 165.90, 153.37,
145.46, 143.83, 130.66, 125.85, 121.09, 105.47, 60.98, 56.18, 41.66, 29.70, 24.81;
HRMS (ESI) calculated for (C18H21NO5): 331.1447, found 331.1434.
5.6.2(E)-1-(2-ethyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydropyridin-
-2(1H)-one (11b):
Off-white solid, yield 71.6%, m.p. 96-98 °C, 1H-NMR (CDCl3, 600 MHz) δ:
6.91-6.94 (m, 1 H, =CH), 6.70 (s, 1 H, =CH), 6.56 (s, 2 H, Ph), 5.99 (dt, J1 = 9.6 Hz,
J2 = 1.8 Hz, 1 H, =CH), 3.93 (t, J = 6.6 Hz, 2 H, NCH2), 3.85 (s, 9 H, 3 OMe), 2.63 (q,
J = 7.8 Hz, 2 H, CH2), 2.51-2.54 (m, 2 H, CH2), 1.13 (t, J = 7.8 Hz, 3 H, Me);
13C-NMR (CDCl3, 300 MHz) δ: 175.72, 165.22, 152.99, 145.34, 141.23, 137.68,
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131.84, 131.65, 125.38, 106.21, 60.90, 56.08, 42.66, 24.76, 22.44, 13.03; HRMS (ESI)
calculated for (C19H23NO5): 345.1576, found 345.1574.
5.6.3 (E)-morpholino-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydro-
pyridin-2(1H)-one (11c):
Brown oil, yield 45.0%, 1H-NMR (CDCl3, 600 MHz) δ: 7.67 (d, J = 15.0 Hz, 1 H,
=CH), 7.45 (d, J = 15.0 Hz,1 H, =CH), 6.80 (s, 2 H, Ph), 6.18-6.20 (m, 1 H, =CH),
3.94 (t, J = 6.6 Hz , 2 H, NCH2), 3.87-3.89 (m, 13 H, 3 OMe + 2 CH2), 2.94-2.97 (m,
4 H, 2 CH2), 2.45-2.48 (m, 2 H, CH2); 13C-NMR (CDCl3, 300 MHz) δ:169.36, 163.08,
153.31, 143.80, 143.64, 140.03, 130.54, 121.33, 120.76, 105.51, 66.59, 60.88, 56.17,
50.70, 42.16, 23.38； HRMS (ESI) calculated for (C21H26N2O6): 402.1791, found
402.1783.
5.6.4 (E)-morpholino-1-(2-methyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-di-
hydropyridin-2(1H)-one (11d):
Light yellow solid, yield 67.8%, m.p. 127-129 °C, 1H-NMR(CDCl3, 600 MHz) δ:
6.86 (s, 1 H, =CH), 6.63 (s, 2 H, Ph), 5.99-5.60 (m ,1 H, =CH), 3.80-3.90 (m, 15 H,
NCH2 + 3 OMe + 2 CH2), 2.88-2.89 (m, 4 H, 2 CH2), 2.50-2.53 (m, 2 H, CH2), 2.16 (s,
3 H, Me); 13C-NMR (CDCl3, 300 MHz) δ:176.53, 162.61, 152.92, 143.38, 137.97,
134.54, 133.45, 131.59, 120.39, 106.98, 66.61, 60.92, 56.15, 50.70, 43.41, 23.49,
15.85; HRMS (ESI) calculated for (C22H28N2O6): 416.1947, found 416.1938.
5.6.5 (E)-morpholino-1-(2-ethyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydro-
pyridin-2(1H)-one (11e):
Light yellow solid, yield 67.8%, m.p. 149-151°C, 1H-NMR(CDCl3, 600 MHz) δ: 6.70
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(s, 1 H, =CH), 6.58 (s, 2 H, Ph), 5.92 (t, J = 4.8 Hz ,1 H, =CH), 3.84-3.88 (m, 11 H,
NCH2 + 3 OMe), 3.81 (t, J = 4.8 Hz , 4 H, 2 CH2), 2.86 (t, J = 4.8 Hz, 4 H, 2 CH2),
2.65 (q, J = 7.2 Hz, 2 H, CH2), 2.50 (q, J = 6.6 Hz , 2 H, CH2), 1.12 (t, J = 7.2 Hz , 3
H, CH3); 13C-NMR (CDCl3, 300 MHz) δ:176.01, 162.63, 152.97, 143.51, 141.44,
137.81, 131.93, 131.58, 120.23, 106.44, 66.64, 60.93, 56.14, 50.69, 43.13, 23.49,
22.47, 13.00; HRMS (ESI) calculated for (C23H30N2O6): 430.2104, found 430.2107.
5.6.6 (E)-morpholino-1-(2-propyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-di-
hydropyridin-2(1H)-one (11f):
Yellow solid, yield 59.7%, m.p. 124-126 °C, 1H-NMR (CDCl3, 600 MHz) δ: 6.74 (s, 1
H, =CH), 6.58 (s, 2 H, Ph), 5.90 (t , J = 4.8 Hz ,1 H, =CH), 3.80-3.90 (m, 15 H, NCH2
+ 3 OMe + 2 CH2), 2.85 (t , J = 4.2 Hz ,4 H, 2 CH2), 2.60 (t , J = 4.2 Hz, 2H, CH2),
2.50 (q, J = 6.0 Hz , 2 H, CH2), 1.54-1.58 (m, 2 H, CH2), 0.94 (t, J = 7.2 Hz , 3 H,
CH3); 13C-NMR (CDCl3, 300 MHz) δ: 176.30, 162.58, 152.97, 143.56, 140.23, 137.83,
132.63, 131.60, 120.13, 106.48, 66.63, 60.94, 56.12, 50.69, 43.25, 31.45, 23.49, 22.10,
14.36; HRMS (ESI) calculated for (C24H32N2O6): 444.2260, found 444.2251.
5.6.7 (E)-3-Cloro-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydro-pyridin-
-2(1H)-one (11g):
Yellow solid, yield 56.0%, m.p. 140-144 °C, 1H-NMR (CDCl3, 600 MHz) δ: 7.73 (d,
J = 15.6 Hz, 1 H, =CH),7.45 (d, J = 15.6 Hz, 1 H, =CH), 7.11 (t, J = 4.8 Hz, 1 H,
=CH), 6.83 (s, 2 H, Ph), 4.12 (t, J = 6.6 Hz, 2 H, NCH2), 3.90-3.92 (m, 9 H, 3 OMe),
2.57-2.60 (m, 2 H, CH2); 13C-NMR (CDCl3, 300 MHz) δ: 168.52, 161.42, 153.40,
145.02, 141.18, 140.24, 130.37, 128.21, 120.23, 105.64, 60.98, 56.23, 41.80, 25.30;
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HRMS (ESI) calculated for (C17H18ClNO5): 351.0874, found 351.0863.
5.6.8 (E)-3-Cloro-1-(2-methyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydro-
pyridin-2(1H)-one (11h):
Light yellow solid, yield 53.3%, m.p. 105-108 °C, 1H-NMR (CDCl3, 300 MHz) δ:
7.10 (t, J = 4.5 Hz, 1 H, =CH), 6.90 (s, 1H, =CH), 6.63 (s, 2 H, Ph), 3.96 (t, J = 6.3
Hz, 2 H, NCH2), 3.87 (s, 9 H, 3 OMe), 2.64 (q, J = 6.3 Hz,2 H, CH2), 2.15 (s, 3 H,
CH3); 13C-NMR (CDCl3, 300 MHz) δ: 175.71, 160.80, 152.98, 140.88, 138.06,
134.26, 133.87, 131.37, 127.84, 106.88, 60.93, 56.17, 43.16, 25.33, 15.62; HRMS
(ESI) calculated for (C18H20ClNO5): 365.1030, found 365.1015.
5.6.9 (E)-3-Cloro-1-(2-ethyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydro-
pyridin-2(1H)-one (11i):
Off white solid, yield 49.7%, m.p. 127-129 °C, 1H-NMR (CDCl3, 600 MHz) δ: 7.07 (t,
J = 4.8 Hz, 1 H, =CH), 6.74 (s, 1 H, =CH), 6.56 (s, 2 H, Ph), 3.96 (t, J = 6.6 Hz, 2 H,
NCH2), 3.86 (s, 3 H, OMe), 3.85 (s, 6 H, 2 OMe), 2.61-2.68 (m, 4 H, 2 CH2), 1.14 (t,
J = 7.2 Hz, 3 H, CH3); 13C-NMR (CDCl3, 300 MHz) δ: 175.42, 160.92, 153.03,
140.78, 140.60, 137.92, 132.93, 131.33, 127.96, 106.31, 60.93, 56.15, 43.00, 25.34,
22.37, 13.05; HRMS (ESI) calculated for (C19H22ClNO5): 379.1187, found
379.1187.
5.6.10 (E)-3-Cloro-1-(2-propyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydro-
pyridin-2(1H)-one (11j):
Off white solid, yield 41.6%, m.p. 121-123 °C, 1H-NMR (CDCl3, 300 MHz) δ: 7.08 (t,
J = 4.8 Hz, 1 H, =CH), 6.78 (s, 1 H, =CH), 6.57 (s, 2 H, Ph) , 3.96 (t, J = 6.3 Hz, 2 H,
15

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NCH2), 3.86 (s, 3 H, OMe), 3.85 (s, 6 H, 2 OMe), 2.56-2.66 (m, 4 H, 2 CH2),
1.56-1.61 (m, 2 H, CH2), 0.96 (t, J = 7.2 Hz, 3 H, CH3); 13C-NMR (CDCl3, 300 MHz)
δ: 175.59, 160.88, 153.00, 140.81, 139.31, 137.95, 133.53, 131.33, 127.88, 106.36,
60.88, 56.09, 43.06, 31.24，25.30, 22.09, 14.31; HRMS (ESI) calculated for
(C20H24ClNO5): 393.1343, found 393.1354.
5.6.11 (E)-3-Cloro-1-(2-butyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydro-
pyridin-2(1H)-one (11k):
Off white solid, yield 47.3%, m.p. 83-85°C, 1H-NMR (CDCl3, 300 MHz) δ: 7.08 (t, J
= 4.5 Hz, 1 H, =CH), 6.77 (s, 1 H, =CH), 6.57 (s, 2 H, Ph), 3.97 (t, J = 6.3 Hz,2 H,
NCH2), 3.86 (s, 9 H, 3 OMe), 2.58-2.66 (m, 4 H, 2 CH2), 1.53-1.62 (m, 2 H, CH2),
1.32-1.40 (m, 2 H, CH2), 0.89 (t, J =7.2Hz, 3H, Me); 13C-NMR (CDCl3, 300 MHz) δ:
175.67, 160.91, 153.03, 140.72, 139.46, 137.94, 133.43, 131.36, 127.99, 106.33,
60.93, 56.12, 43.08, 30.91, 29.02, 25.35, 23.01, 13.95; HRMS (ESI) calculated for
(C21H26ClNO5): 407.1500, found 407.1490.
5.6.12 (E)-3-Bromo-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydropyridin-
-2(1H)-one (11l):
Yellow solid, yield 73.2%, m.p. 138-144 °C, 1H-NMR (CDCl3, 300 MHz) δ: 7.71 (d,
1 H, J = 15.3 Hz, =CH), 7.41 (d, 1 H, J = 15.3 Hz, =CH),7.36 (t, J=4.5Hz, 1 H, =CH),
6.81 (s, 2 H, Ph) , 4.11 (t, J = 6.3 Hz , 2 H, NCH2), 3.90 (s, 6 H, 2 OMe), 3.89 (s, 3 H,
OMe), 2.50-2.56 (m, 2 H, CH2); 13C-NMR (CDCl3, 300 MHz) δ: 168.63, 161.12,
153.41, 145.95, 145.04, 140.26, 130.39, 120.25, 119.14, 105.68, 60.99, 56.26, 41.91,
26.72； HRMS (ESI) calculated for (C17H18BrNO5): 395.0368, found 395.0371.
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5.6.13 (E)-3-Bromo-1-(2-methyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-di-
hydropyridin-2(1H)-one (11m):
Light yellow solid, yield 65.3%, m.p. 99-102 °C, 1H NMR (CDCl3, 300 MHz) δ: 7.36
(t, J = 4.5 Hz, 1 H, =CH), 6.89 (s, 1H, =CH), 6.63 (s, 2 H, Ph), 3.97 (t, J = 6.3 Hz, 2
H, NCH2), 3.87 (s, 9 H, 3 OMe), 2.57-2.63 (m, 2 H, CH2), 2.15 (s, 3 H, Me);
13C-NMR (CDCl3, 300 MHz) δ: 175.84, 160.54, 152.98, 145.64, 138.09, 134.23,
131.38, 133.89, 118.68, 106.93, 60.93, 56.19, 43.24, 26.73, 15.65; HRMS (ESI)
calculated for (C18H20BrNO5): 409.0525, found 409.0510.
5.6.14 (E)-3-Bromo-1-(2-ethyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydro-
pyridin-2(1H)-one (11n):
Yellow solid, yield 67.1%, m.p 85-87 °C, 1H NMR: (CDCl3, 300 MHz) δ: 7.36 (t, J =
4.5 Hz, 1 H, =CH), 6.76 (s, 1 H, =CH), 6.58 (s, 2 H, Ph), 3.99 (t, J = 6.3 Hz,2 H,
NCH2), 3.88 (s, 9 H, 3 OMe), 2.57-2.69 (m, 4 H, 2 CH2), 1.16 (t, J = 7.5 Hz, 3 H, Me);
13C-NMR (CDCl3, 300 MHz) δ: 175.53, 160.65, 153.02, 145.57, 140.58, 137.92,
132.95, 131.33, 118.80, 106.33, 60.93, 56.14, 43.08, 26.73, 22.36, 13.05; HRMS (ESI)
calculated for (C19H22BrNO5): 423.0681, found 423.0688.
5.6.15 (E)-3-Bromo-1-(2-propyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydro-
pyridin-2(1H)-one (11o):
Off white solid, yield 56.4%, m.p 80-82°C, 1H NMR: (CDCl3, 300 MHz) δ: 7.35 (t, J
= 4.5 Hz, 1 H, =CH), 6.77 (d, J = 3.3 Hz, 1 H, =CH), 6.57 (s, 2 H, Ph), 3.97 (t, J = 6.3
Hz, 2 H, NCH2), 3.87 (s, 3 H, OMe), 3.86(s, 6 H, 2 OMe), 2.56-2.62 (m, 4 H, 2 CH2),
1.56-1.61 (m, 2 H, CH2), 0.87-0.97 (m, 3 H, Me); 13C-NMR (CDCl3, 300 MHz) δ:
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175.75, 160.63, 153.00, 145.56, 139.36, 137.91, 133.57, 133.42, 118.79, 106.33,
60.93, 56.11, 43.16, 29.02, 26.72, 23.01, 13.93;HRMS (ESI) calculated for
(C20H24BrNO5): 437.0838, found 437.0821.
5.6.16 (E)-3-Bromo-1-(2-butyl-3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6-dihydro-
pyridin-2(1H)-one (11p):
Off white solid, yield 49.7%, m.p.109-112 °C, 1H NMR: (CDCl3, 300 MHz) δ: 7.35 (t,
J = 4.5 Hz, 1 H, =CH), 6.77 (s, 1 H, =CH), 6.58 (s, 2 H, Ph), 3.98 (t, J = 6.3 Hz, 2 H,
NCH2), 3.89 (s, 9 H, 3 OMe), 2.56-2.63 (m, 4 H, 2 CH2), 1.54-1.59 (m, 2 H, CH2),
1.29-1.40 (m, 2 H, CH2), 0.90 (t, J = 7.2 Hz, 3 H, Me); 13C-NMR (CDCl3, 300 MHz)
δ: 175.78, 160.66, 153.00, 145.55, 139.44, 138.31, 133.44, 131.36, 118.80, 106.33,
60.94, 56.11, 43.17, 30.90, 29.03, 26.74, 23.02, 13.94; HRMS (ESI) calculated for
(C21H26BrNO5): 451.0994, found 451.0980.
In Vitro Cytotoxicity Assay. Cells were plated in 96-well microtiter plates at a
density of 5 × 103/well and incubated in a humidified atmosphere with 5% CO2 at
37 °C for 24 h. Test compounds were added to triplicate wells with different
concentrations and 0.1% DMSO for control. After incubation for 72 h, 20 μL of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 mg/mL)
was added to each well and the plate was incubated for an additional 4 h. The
formazan was dissolved in 100 μL of DMSO. The absorbance (OD) was read on a
WellscanMK-2 microplate reader (Labsystems) at 570 nm. The concentration causing
50% inhibition of cell growth (IC50) was determined by the Logit method.
ROS assay. Cells were treated with compound 11h and DMSO for 1 hour and ROS
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generation was detected with 2'-,7'-dichlorofluorescein diacetate (DCFH-DA)
(Invitrogen, Carlsbad, CA, USA). Cells were incubated with 10 M of DCFH-DA for
30 min at 37°C, washed twice with PBS and immediately analyzed by a fluorescence
microscope.
In vivo antitumor activity. The compound was dissolved in normal saline using
tween-80 as solubilizer. BALB/C nude male mice (Certificate SCXK-2007-0005,
weighing 18 g to 20 g) were obtained from Shanghai Experimental Animal Center,
Chinese Academy of Sciences. A549 lung cancer cell suspensions were implanted
subcutaneously into the right axilla region of mice. Treatment was begun when
implanted tumors had reached a volume of about 100-300 mm3 (after 17 days). The
animals were randomized into appropriate groups (six animals/treatment and eight
animals for the control group) and administered by ip injection for fourteen
consecutive days after implantation of cells. Tumor volumes were monitored by
caliper measurement of the length and width and calculated using the formula of TV =
1/2 × a × b2, where a is the tumor length and b is the width. Tumor volumes and body
weights were monitored every 4 days over the course of treatment. Mice were
sacrificed on day 30 to 33 after implantation of cells and tumors were removed and
recorded for analysis.
ACKNOWLEDGEMENTS
This work was supported in part by the National Natural Science Foundation of China
(Grant 81373331), and the Bio-Pharmaceutical Project of Science and Technology of
Shanghai (No. 13431900302), Major Special Project for the Creation of New Drugs
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(Grant Nos. 2014ZX09101-004-002). We gratefully acknowledge technical support
from Prof. Jing An (Shanghai University, P. R. China) for part of biological activity
assays.
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Figure Captions
Figure 1. Structure of piperlongumine (PL)
Figure 2. Piperlongumine derivative 11h (10 M) induces ROS elevation in A549 cells for 1h. A,
control; B, compound 11h; C, mean fluorescence of control and 11h
Figure 3. Growth of sc implanted human xenograft in nude mice treated with vehicle,
PL (ip×14, 2 mg/Kg), 11h (ip×14, 2 mg/Kg) and DOX (iv×1, 10 mg/Kg) in A549
lung cancer xenograft model. Mean ± SE for group of 6 mice (P<0.05).
Table 1. In vitro antitumor activity of 2 and 7-substituted piperlongumine derivatives
Table 2. In vitro antitumor activity of 2-halogen substituted piperlongumine
derivatives
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Fig. 1. Structure of piperlongumine (PL)
23

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Fig. 2. Piperlongumine derivative 11h (10 M) induces ROS elevation in A549 cells for 1h. A,
control; B, compound 11h; C, mean fluorescence of control and 11h
24

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Fig. 3. Growth of sc implanted human xenograft in nude mice treated with vehicle,
PL (ip×14, 2 mg/Kg), 11h (ip×14, 2 mg/Kg) and DOX (iv×1, 10 mg/Kg) in A549
lung cancer xenograft model. Mean ± SE for group of 6 mice (P<0.05).
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Table 1. In vitro antitumor activity of 2 and 7-substituted piperlongumine derivatives
IC50 a (M)
ZR-75-30
Compds
A549
HCT116
MDA-MB-231
MRC-5
PL
11a
11b
11c
11d
11e
11f
22.85
23.95
17.95
>100
>100
>100
>100
19.83
6.04
10.34
12.34
47.20
>100
>100
>100
4.28
5.86
3.32
8.46
11.71
23.67
>100
>100
>100
>100
6.71
35.04
19.37
13.53
56.05
>100
>100
52.53
14.30
3.16
52.64
>100
>100
>100
4.49
11g
a Values were measured with MTT method.
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Table 2. In vitro antitumor activity of 2-halogen substituted piperlongumine
derivatives
IC50 a (M)
MDA-MB-231
Compds
A549
HCT116
Hep3B
WI38
PL
11h
11i
5.90
3.94
4.06
3.81
3.78
4.79
2.67
2.18
2.63
2.56
21.80
9.85
9.17
8.08
7.53
7.12
6.20
3.02
2.14
2.59
19.53
6.07
7.27
8.13
6.55
2.73
2.47
2.12
2.36
2.44
69.46
16.69
19.03
22.36
14.75
8.09
26.78
19.60
5.48
11j
10.42
7.93
11k
11l
20.65
7.16
11m
11n
11o
11p
3.10
8.12
7.94
5.54
5.02
14.85
9.21
a Values were measured with MTT method.
27