Design, Synthesis and Pharmacological Evaluation of Novel Piperlongumine derivatives as Potential Antiplatelet Aggregation Candidate

Article abstract of DOI:10.1111/cbdd.12714

A series of novel piperlongumine derivatives (4a-i, 6a-i) were designed and synthesized. The inhibitory activities of platelet aggregation induced by ADP and AA in vitro have been evaluated by bron turbidimetry and liver microsomal incubated assay. The assay results show that compounds 4e and 6e exhibited remarkable potency to that of the positive control piplartine and aspirin.

Full text of DOI:10.1111/cbdd.12714

Chem Biol Drug Des 2016; 87: 833–840
Research Article
Design, Synthesis and Pharmacological Evaluation of
Novel Piperlongumine derivatives as Potential
Antiplatelet Aggregation Candidate
Yujun Wang1, Jie Wang2,†, Jiaming Li1,*, Yanchun
cell lines was evaluated. It was suggested that side chain
length affects the ability to overcome the MDR cancer
phenotype (7).
Zhang1,*, Weijun Huang1, Jian Zuo1, Huicai Liu1,
Di Xie1 and Panhu Zhu1
1Department of Medicinal Chemistry, Anhui University of
In particular, the antiplatelet aggregation activity of piper-
Chinese Medicine, 103 Meishan Road, Hefei 230031,
China
longumine has attracted global attention. It was reported
that piperlongumine inhibits platelet aggregation as a
thromboxane A2 receptor antagonist (8). In addition,
Park’s team evaluated the anti-atherosclerotic potential of
PL. It was assumed that PL significantly attenuated activa-
tion of NF-jB in response to PDGF-BB stimulation and
inhibited PDGF-BB-induced PDGF receptor beta activation
and suppressed downstream signalling molecules (4).
2Department of Chemistry, Bengbu Medical College,
Bengbu 233030, China
*Corresponding authors: Jiaming Li,
lijiaming2004@sina.com; Yanchun Zhang,
yczhang411@163.com
†Contributed equally to the work.
A series of novel piperlongumine derivatives (4a-i, 6a-i)
were designed and synthesized. The inhibitory activi-
ties of platelet aggregation induced by ADP and AA
in vitro have been evaluated by bron turbidimetry and
liver microsomal incubated assay. The assay results
show that compounds 4e and 6e exhibited remarkable
potency to that of the positive control piplartine and
aspirin.
Thienopyridines, including ticlopidine, clopidogrel, prasug-
rel, (Figure 2) are wildly used in clinical for treating platelet
aggregation by selectively blocking the binding of adenine
diphosphate (ADP)-induced platelet aggregation (9). Cur-
rently, dual treatment with aspirin and clopidogrel is the
ideal treatment of antiplatelet therapy for patients with
acute coronary syndromes (ACS) and prevention of throm-
botic events after percutaneous coronary intervention (PCI)
with stenting (10). However, the bleeding risk, complica-
tions and individual risk still exist (11). Novel safer antipla-
telet agents are urgently needed (12). The thienopyridine
derivatives were designed and synthesized by Zhi’s group
and exhibit potent inhibition to ADP-induced aggregation
than ticlopidine (13).
Key words: antiplatelet aggregation, piperlongumine deriva-
tives, thienopyridine
Received 14 September 2015, revised 30 October 2015 and
accepted for publication 1 December 2015
Correction added on 20 May 2016, after first online
publication: Acknowledgements added.
On careful review at piplartine and the thienopyridine
derivatives, it was noticed that piplartine and ‘grel’ may
share same skeleton. We also predicted that trimethoxy-
phenyl-acryloyl part and thienopyridine may be able to
contribute the activity of antiplatelet aggregation. There-
fore, in the present research, we report the design, syn-
thesis and biological evaluation of the piplartine derivatives
bearing thienopyridine structure (Figure 3; Table 1).
Piper longum L. had been used as a crude drug for the
treatment of the intestinal disorder, asthma and poorly
peripheral blood circulation in Asia for decades. Piper-
longumine (PL, Figure 1), also known as piperaceous
amides or piplartine, is an alkaloid isolated from Piper
longum L, which displays wide range of biological activi-
ties, such as anticancer (1,2), antidiabetic (3), antiplatelet
aggregation (4), antidepression and antifungal activities (5).
Great effort had been paid to the structure modification of
PL to make it more selective and potent to the disease.
By replacing nitrogen atom of lactam in PL with carbon
atom to increase anti-inflammatory activity, Zhou’s group
reach promising anti-inflammatory agent PL-0N (6). 10
amide alkaloid derivatives were synthesized by Lee’s team.
The antiproliferative activity against eight human tumour
Chemistry
Target compounds 4a-i, 6a-i were achieved via the syn-
thetic route outlined in Scheme 1. Different substituted
benzaldehyde was used as starting material. 4a-e and 6a-e
were reached by Knoevenagel, amidation, condensation
reaction and esterification. 6f-i were reached by acylation
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12714
833

Wang et al.
the aqueous phase was acidified with HCl (liq.) to pH = 4.
Then, the separated solid was filtrated and filter was
recrystallized with anhydrous ethanol, to provide the title
compound as
a
white solid (7.92 g, 65.2%),
m.p.124.3~125.7 °C.
Figure 1: Piperlongumine.
(E)-1-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-3-
(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4a)
SOCl2 (15 mL)was added to the solution of 1b (3.00 g,
12.6 mmol) in toluene (45 mL). The reaction mixture was
stirred at 80 °C for 5 h. The reaction mixture was evapo-
rated to give the crude compound 3a.
of 7a-d, which hydroxyl group was protected by acylation,
using Et3N as acid-binding agent, while 4f-i were achieved
using DCC as condensation agent, because of the hydroxyl
group on the benzene ring.
Experimental Section
The solution of 1c in DCM was added at 0 °C slowly to
the mixture of 4,5,6,7-tetrahydrothieno[3,2-c]pyridine
hydrochloride (1.86 g, 13.4 mmol) and Et3N (6 mL) in
CH2Cl2 (50 mL). The reaction mixture was stirred at room
temperature for 2.5 h. The organic phase was washed
with 1 mol/L Na2CO3(liq.), 1% hydrochloric acid and satu-
rated brine, dried over sodium sulphate and evaporated in
vacuo to give the crude product. The crude product was
purified on silica using hexanes/ethyl acetate to provide
the title compound as white solid (2.43 g, 53.3%),
m.p.151.4~152.7 °C; 1H NMR (CDCl3, 300 MHz) d: 7.63
(d, J = 15.6 Hz, 1H, ArCH=), 7.16 (d, J = 5.1 Hz, 1H,
ThH), 6.85–6.80 (m, 2H, -CH= and ThH), 6.76 (s, 2H,
ArH), 4.78 (s, 2H, Py-CH2), 3.98 (t, 2H, J = 4.5 Hz,
Py-CH2), 3.91 (s, 6H, OCH3 9 2), 3.88 (s, 3H, OCH3),
2.96 (br s, 2H, Py-CH2); 13C NMR (CDCl3, 75 MHz)
d:166.0, 153.4, 143.0, 139.6, 132.4, 130.8, 125.2, 124.5,
123.6, 116.6, 105.1, 61.0, 56.2, 46.0, 43.3, 26.1; IR (KBr,
cmꢀ1) υ: 2998.3, 2935.6, 2833.9, 1642.7, 1587.0, 1505.6,
1437.4, 1418.9, 1332.8, 1252.2, 1266.5, 1124.4, 1058.1,
1008.1, 977.9, 826.2, 717.9, 613.2; ESI-Mass for
General
All of the target compounds were purified by column chro-
matography (CC) on silica gel 60 (200–300 mesh). Melting
points were determined using
(RDCSY-I) and are reported directly. NMR spectra were
recorded with a Bruker Avance 300 MHz spectrometer
(Bruker, Fallanden, Switzerland), using TMS as an internal
standard. IR spectra were measured with Shimadzu FTIR-
8400S (Shimadzu,Tokyo, Japan). MS spectra were mea-
sured with a Hewlett-Packard 1100 LC/MSD spectrometer
(Agilent, Waldbronn, Germany).
a capillary apparatus
(E)-3-(3,4,5-trimethoxyphenyl)acrylic acid (2a)
A
solution of 3,4,5-trimethoxybenzaldehyde (10.0 g,
51.0 mmol), malonic acid (6.41 g, 61.5 mmol) in benzene
(150 mL) was treated with pyridine (35 mL), piperidine
(2 mL). The reaction mixture was stirred at reflux for 6 h.
The reaction mixture was cooled to room temperature and
saturated NaHCO3(liq.) (75 mL) was added. After 30 min,
C
19H21NO4S: m/z (M++H) 360.06.
Figure 2: Structure of thienopyridines.
Figure 3: The design of piplartine
derivatives.
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Chem Biol Drug Des 2016; 87: 833–840

Novel Piperlongumine Derivatives
Table 1: The structure of target com-
pounds
Compd
R1
R2
R3
R4
4a
4b
4c
4d
4e
4f
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
OCH3
OCH3
CH3
Cl
NO2
OH
OH
OCH3
OH
OCH3
H
H
H
H
H
H
H
H
4g
4h
4i
OCH3
OH
OCH3
OCH3
OCH3
OCH3
6a
OCH3
O
H
C
3
O
6b
6c
6d
6e
6f
H
OCH3
CH3
Cl
H
H
H
H
H
H
O
H
H
H
H
H
C
C
C
C
C
3
3
3
3
3
O
O
H
O
O
H
O
O
H
NO2
O
O
O
H
H
H
C
3
O
O
O
O
6g
6h
6i
H
C
H
C
H
C
H
C
3
3
3
3
O
OCH3
O
O
H
O
H
C
3
O
O
O
OCH3
OCH3
O
O
H
C
O
3
Compounds 4b-e were synthesized following the same
procedure described above for the preparation of 4a.
129.4, 127.9, 125.2, 124.5, 123.5, 114.9, 114.2, 55.3,
45.9, 43.3, 26.0; IR (KBr, cmꢀ1) υ:3004.3, 2962.5, 2905.6,
1649.8, 1593.0, 1506.3, 1437.5, 1249.2, 1165.5, 1060.8,
1027.9, 980.1, 896.4, 824.6, 716.9; ESI-Mass for
C17H17NO2S: m/z (M++H) 300.13.
(E)-1-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-3-
(4-methoxyphenyl)prop-2-en-1-one (4b)
Off-white solid,yield 61.2%,m.p.131.4~132.7 °C; 1H NMR
(CDCl3, 400 MHz) d: 7.67 (d, J = 15.2 Hz, 1H, ArCH=),
7.50 (d, J = 8.0 Hz, 2H, ArH), 7.15 (d, J = 5.2 Hz, 1H,
ThH), 6.90 (d, J = 8.4 Hz, 2H, ArH), 6.83–6.79 (m, 2H,
ThH and -CH=), 4.76 (s, 2H, Py-CH2), 3.97 (br s, 2H,
Py-CH2), 3.84 (s, 3H, OCH3), 2.94 (br s, 2H, Py-CH2); 13C
NMR (CDCl3, 100 MHz) d:166.3, 160.9, 142.6, 132.3,
(E)-1-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-3-
(4-methylphenyl)prop-2-en-1-one (4c)
White solid,yield 63.7%,m.p.145.8~147.2 °C; 1H NMR
(CDCl3, 400 MHz) d: 7.69 (d, J = 15.6 Hz, 1H, ArCH=),
7.45 (d, J = 7.6 Hz, 2H, ArH), 7.19 (d, J = 8.0 Hz, 2H,
ArH), 7.15 (d, J = 5.2 Hz, 1H, ThH), 6.89 (d, J = 15.6 Hz,
Chem Biol Drug Des 2016; 87: 833–840
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Wang et al.
Scheme 1: Reagents and conditions: (I) malonic acid, pyridine, benzene, 100 °C; (II) SOCl2 toluene, 80 °C; (III) CH2Cl2, Et3N;
(IV) CH2Cl2, Et3N;
(V) acetic anhydride, acetonitrile, Et3N; (VI) DCC, CH2Cl2; (VII) acetic anhydride,
pyridine; (VIII) acetic anhydride, CH2Cl2, Et3N; (IX) acetonitrille, Et3N; (X) acetic anhydride, acetonitrille, Et3N.
1H, -CH=), 6.83 (d, J = 4.8 Hz, 1H, ThH), 4.77 (s, 2H, Py-
CH2), 3.97 (br s, 2H, Py-CH2), 2.95 (br s, 2H, Py-CH2),
2.37 (s, 3H, CH3); 13C NMR (CDCl3, 100 MHz) d:166.2,
142.9. 139.9, 132.4, 129.5, 127.6, 125.2, 124.5, 123.5,
116.5, 116.2, 45.9, 43.2, 25.9, 21.4; IR (KBr, cmꢀ1) υ:
3076.1, 2923.6, 2833.9, 1643.9, 1596.0, 1518.3, 1455.5,
1452.5, 1323.9, 1219.3, 1054.8, 1013.0, 968.1, 812.6,
737.9; ESI-Mass for C17H17NOS: m/z (M++H) 284.08.
(d, J = 5.2 Hz, 1H, ThH), 6.92 (d, J = 15.6 Hz, 1H, -CH=),
6.83 (d, J = 4.8 Hz, 1H, ThH), 4.76 (s, 2H, Py-CH2), 3.97
(br s, 2H, Py-CH2), 2.95 (br s, 2H, Py-CH2); 13C NMR
(CDCl3, 100 MHz) d:165.7, 141.5, 135.4, 133.7, 132.3,
129.0, 128.9, 125.1, 124.5, 123.6, 118.0, 45.9, 43.3,
25.9; IR (KBr, cmꢀ1) υ:2926.1, 2869.3, 2833.4, 1607.3,
1646.2, 1487.7, 1439.9, 1401.0, 1326.2, 1215.6, 1182.7,
1081.0, 1048.1, 970.4, 805.9, 704.2; ESI-Mass for
C16H14ClNOS: m/z (M++H) 314.17.
(E)-1-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-3-
(4-chlorophenyl)prop-2-en-1-one (4d)
White solid,yield 56.1%,169.5~170.9 °C; 1H NMR (CDCl3,
400 MHz) d: 7.65 (d, J = 15.2 Hz, 1H, ArCH=), 7.47 (d,
J = 8.0 Hz, 2H, ArH), 7.35 (d, J = 8.0 Hz, 2H, ArH), 7.16
(E)-1-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-3-
(4-nitrophenyl)prop-2-en-1-one (4f)
White solid,yield 45.7%,191.3~192.4 °C; 1H NMR (CDCl3,
300 MHz) d: 8.24 (d, J = 8.7 Hz, 2H, ArH), 7.75–7.67 (m,
836
Chem Biol Drug Des 2016; 87: 833–840

Novel Piperlongumine Derivatives
3H, ArCH= and ArH), 7.18 (d, J = 5.1 Hz, 1H, ThH), 7.09 (d,
J = 15.6 Hz, 1H, -CH=), 6.84 (d, J = 3.6 Hz, 1H, ThH), 4.79
(s, 2H, Py-CH2), 3.94 (d, J = 30.3 Hz, 2H, Py-CH2), 2.99 (br
s, 2H, Py-CH2); 13C NMR (CDCl3, 100 MHz) d:165.0, 148.1,
141.5, 140.1, 128.4, 125.2, 124.4, 124.1, 123.8, 120.0,
121.8, 46.1, 44.0, 26.0; IR (KBr, cmꢀ1) υ:3106.0, 2929.6,
2851.8, 1649.8, 1605.0, 1512.3, 1449.5, 1344.9, 1216.3,
1183.4, 1108.6, 1051.8, 962.1, 839.5, 746.8, 708.0; ESI-
Mass for C16H14N2O3S: m/z (M++H) 315.06.
126.6, 125.4, 125.1, 123.6, 122.5, 115.4, 114.6, 111.3; IR
(KBr, cmꢀ1) υ:3100.0, 2929.6, 2842.9, 1766.5, 1640.9,
1581.1, 1524.2, 1452.5, 1362.8, 1297.0, 1243.2, 1162.5,
1120.6, 1057.8, 1030.9, 977.1, 896.4, 833.6, 797.7, 737.9;
ESI-Mass for C17H17NO3S: m/z (M+ꢀH) 314.17.
(E)-1-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-3-
(3-hydroxy-3-methoxyphenyl)prop-2-en-1-one (4h)
White solid,yield 42.1%,m.p.176.3~177.8 °C; 1H NMR
(CDCl3, 300 MHz) d: 7.62 (d, J = 15.3 Hz, 1H, ArCH=),
7.20 (d, J = 2.1 Hz, 1H, ArH), 7.15 (d, J = 5.4 Hz, 1H,
ThH), 7.04 (dd, J1=2.1 Hz, J2=8.4 Hz, 1H, ArH), 6.85–
6.77 (m, 3H, –CH=, ThH and ArH), 4.76 (s, 2H, Py-CH2),
4.02 (m, 1H, Py-CH2), 3.92 (s, 4H, Py-CH2 and OCH3),
2.94(br s, 2H, Py-CH2); 13C NMR (CDCl3, 75 MHz)
d:166.4, 157.0, 148.3, 145.9, 143.0, 128.7, 125.2, 124.5,
123.5, 121.6, 115.2, 112.7, 110.6, 56.0, 45.9, 43.3, 24.9;
IR (KBr, cmꢀ1) υ:3091.0, 2929.6, 2845.9, 1634.9, 1578.1,
1524.3, 1425.6, 1312.0, 1261.1, 1219.3, 1126.6, 1051.8,
1027.9, 977.1, 803.7, 737.9; ESI-Mass for C17H17NO3S:
m/z (M+ꢀH) 314.17.
(E)-1-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-3-
(4-hydroxyphenyl)prop-2-en-1-one (5d)
NaOH (1.70 g, 42.5 mmol) was added to a solution of
4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride in
dichloromethane (60.0 mL). The reaction was stirred at
room temperature for 3.5 h. The organic phase was
washed with water, dried over sodium sulphate and evap-
orated in vacuo to give the 4,5,6,7-tetrahydrothieno[3,2-c]
pyridine.
To a solution of (E)-3-(4-hydroxyphenyl)acrylic acid (3.50 g,
21.3 mmol), 4,5,6,7-tetrahydrothieno[3,2-c]pyridine (3.00 g,
21.6 mmol) in dichloromethane (60.0 mL) was added DCC
(5.30 g, 25.7 mmol). The reaction was stirred at 0 °C for
6 h. The organic phase was washed with water, dried over
sodium sulphate and evaporated in vacuo to give the
4,5,6,7-tetrahydrothieno[3,2-c]pyridine to give the crude
product. The crude product was purified on silica using
DCM/MeOH to provide the title compound as a white solid
(3.40 g, 54.1%). m.p.209.6~212.1 °C; 1H NMR (DMSO-
d6, 300 MHz) d: 9.86 (s, 1H, OH), 7.57 (d, J = 8.4 Hz, 2H,
ArH), 7.44 (d, J = 15.0 Hz, 1H, ArCH=), 7.34 (d,
J = 4.8 Hz, 1H, ThH), 7.13 (d, J = 15.6 Hz, 1H, -CH=),
6.90 (d, J = 3.0 Hz, 1H, ThH), 6.78 (d, J = 8.4 Hz, 2H,
ArH), 4.79 (s, 1H, Py-CH2), 4.62 (s, 1H, Py-CH2), 3.90 (d,
J = 30.3 Hz, 2H, Py-CH2), 2.83(d, J = 23.1 Hz, 2H, Py-
CH2); 13C NMR (DMSO-d6, 75 MHz) d: 165.5, 159.5,
142.2, 132.7, 129.8, 125.9, 125.4, 125.1, 123.6, 115.7,
114.2, 45.1, 43.1, 25.6; IR (KBr, cmꢀ1) υ: 3120.9, 3016.3,
2935.6, 2842.9, 1640.9, 1578.0, 1518.3, 1449.5, 1273.1,
1219.3, 1192.4, 1162.5, 1063.8, 983.1, 821.6, 764.8;
ESI-Mass for C16H15NO2S: m/z (M++H) 286.15.
(E)-1-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-3-
(4-hydroxy-3,5-dimethoxyphenyl)prop-2-en-1-one
(4i)
Off-white solid,yield 47.8%,m.p.155.7~156.4 °C; 1H NMR
(CDCl3, 300 MHz) d: 7.64 (d, J = 15.0 Hz, 1H, ArCH=),
7.16 (d, J = 5.1 Hz, 1H, ThH), 6.83 (d, J = 5.1 Hz, 1H,
ThH), 6.81~6.76 (m, 3H, -CH= and ArH), 4.77 (s, 2H, Py-
CH2), 3.98 (t, J = 5.4 Hz, 2H, Py-CH2), 3.94 (s, 6H,
OCH3 9 2), 2.96(t, J = 5.1 Hz, 2H, Py-CH2); 13C NMR
(CDCl3, 75 MHz) d:166.3, 157.0, 147.2, 143.5, 136.7,
132.3, 126.7, 125.2, 124.6, 123.6, 115.1, 104.9, 56.4,
46.1, 43.3, 25.0; IR (KBr, cmꢀ1) υ:3100.0, 2929.6, 2839.8,
1643.9, 1605.0, 1512.3, 1455.5, 1428.6, 1335.9, 1264.1,
1210.3, 1150.5, 1108.6, 1057.8, 974.1, 905.3, 824.6,
705.0; ESI-Mass for C18H19NO4S: m/z (M+ꢀH) 344.59.
(E)-5-(3-(3,4,5-trimethoxyphenyl)acryloyl)-5,6,7,7a-
tetrahydrothieno[3,2-c]pyridin-2(4H)-one (5a)
Compound 5a was synthesized following the same proce-
dure described above for the preparation of 4a.
Compounds 4g-i were synthesized following the same
procedure described above for the preparation of 4f.
Yellow solid, 51.6%,m.p.87.4~89.6 °C; 1H NMR (CDCl3,
300 MHz) d: 7.52 (d, J = 15.3 Hz, 1H, ArCH=), 6.69–6.64
(m, 3H, ArH and–CH=), 6.12 (s, 1H, ThH), 4.35–4.29 (m,
1H, Py-CH2), 4.20 (s, 1H, Py-CH), 3.82 (s, 9H,
OCH3 9 3), 3.80(s, 2H, Py-CH2), 3.37 (br s, 1H, Py-CH2),
2.46 (br s, 2H, Py-CH2); 13C NMR (CDCl3, 75 MHz)
d:197.8, 165.9, 153.4, 144.4, 144.2, 139.9, 130.3, 127.3,
115.4, 105.2, 61.0, 56.3, 51.0, 44.5, 42.9, 22.6; IR (KBr,
cm-1) υ:2938.5, 2839.4, 1682.5, 1644.3, 1583.7, 1505.5,
1455.2, 1418.6, 1333.4, 1270.1, 1246.1, 1188.6, 1125.5,
1002.7, 976.8, 825.9, 779.9, 646.9; ESI-Mass for
C19H21NO5S: m/z (M++H) 376.10.
(E)-1-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)-3-
(4-hydroxy-3-methoxyphenyl)prop-2-en-1-one (4g)
White solid,yield 56.4%,m.p.196.2~197.5 °C; 1H NMR
(CDCl3, 400 MHz) d: 7.64 (d, J = 15.2 Hz, 1H, ArCH=),
7.16 (d, J = 5.2 Hz, 1H, ThH), 7.13 (d, J = 8.4 Hz, 1H,
ArH), 7.01 (s, 1H, ArH), 6.93 (d, J = 8.4 Hz, 1H, ArH), 6.83
(d, J = 4.8 Hz, 1H, ThH), 6.78 (d, J = 14.8 Hz, 1H, -CH=),
4.77 (s, 2H, Py-CH2), 4.06–3.99 (m, 1H, Py-CH2), 3.94 (s,
4H, Py-CH2 and OCH3), 2.97(br s, 2H, Py-CH2); 13C NMR
(CDCl3, 100 MHz) d:165.4, 148.5, 147.8, 142.5, 132.7,
Chem Biol Drug Des 2016; 87: 833–840
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Wang et al.
(E)-5-(3-(3,4,5-trimethoxyphenyl)acryloyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl acetate (6a)
To the solution of 5a (2.00 g, 5.33 mmol), acetic anhy-
dride (1.5 mL) in acetonitrile (40 mL), Et3N (1.5 mL) was
added. The reaction mixture was stirred at room tempera-
ture for 5 h, then evaporated in vacuo. The residue was
solved in DCM and washed with water. The organic phase
was dried over sodium sulphate and evaporated in vacuo
to give the crude product. The crude product was purified
on silica using hexanes/ethyl acetate 20–33% to provide
the title compound as an off- white solid (1.36 g, 61.3%),
m.p.121.8~123.7 °C; 1H NMR (CDCl3, 400 MHz) d: 7.61
(d, J = 15.2 Hz, 1H, ArCH=), 6.81–6.72 (m, 3H, ArH and -
CH=), 6.20 (s, 1H, ThH), 4.66 (s, 2H, Py-CH2), 4.00 (br s,
2H, Py-CH2), 3.91 (s, 6H, OCH3 9 2), 3.88 (s, 3H,
OCH3), 2.85 (br s, 2H, Py-CH2), 2.30 (s, 3H, CH3); 13C
NMR (CDCl3, 100 MHz) d:167.8, 166.0, 153.4, 150.1,
143.2, 139.6, 130.8, 116.8, 116.4, 111.7, 111.4, 105.1,
61.0, 56.2, 45.7, 43.1, 25.4, 20.7; IR (KBr, cm-1)
υ:2938.5, 2836.9, 1763.5, 1643.9, 1587.0, 1500.3,
1422.6, 1335.9, 1273.1, 1186.4, 1126.6, 1057.8, 1004.0,
884.4, 827.6, 722.9; ESI-Mass for C21H23NO6S: m/z
(M++H) 418.01.
1042.9, 983.1, 893.4, 812.6; ESI-Mass for C19H19NO3S:
m/z (M++H) 342.07.
(E)-5-(3-(4-chlorophenyl)acryloyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl acetate (6d)
Off- white solid,yield 62.5%,m.p.115.4~116.2 °C; 1H NMR
(CDCl3, 300 MHz) d: 7.65 (d, J = 15.6 Hz, 1H, ArCH=),
7.47 (d, J = 8.7 Hz, 2H, ArH), 7.35 (d, J = 8.7 Hz, 2H,
ArH), 6.89 (d, J = 15.3 Hz, 1H, -CH=), 6.41 (s, 1H, ThH),
4.65 (s, 2H, Py-CH2), 3.96 (br s, 2H, Py-CH2), 2.84(br s,
2H, Py-CH2), 2.29 (s, 3H, CH3); 13C NMR (CDCl3,
75 MHz) d:167.7, 165.7, 150.1, 141.7, 135.5, 133.7,
129.1, 124.8, 118.1, 117.8, 111.7, 111.3, 45.7, 43.0,
25.4, 20.7; IR (KBr, cmꢀ1) υ:2920.6, 2866.8, 1748.5,
1643.9, 1604.9, 1584.1, 1503.3, 1428.6, 1362.8, 1332.9,
1225.3, 1204.3, 1150.5, 1087.7, 1042.8, 971.1, 893.4,
812.6; ESI-Mass for C18H16ClNO3S: m/z (M++H) 361.96.
(E)-5-(3-(4-nitrophenyl)acryloyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl acetate (6e)
Off- white solid,yield 32.1%,m.p.127.6~128.3 °C; 1H NMR
(CDCl3, 300 MHz) d: 8.24 (d, J = 8.7 Hz, 2H, ArH), 7.74–
7.67 (m, 3H, ArCH= and ArH),7.10–7.05 (m, 1H, -CH=),
6.42 (s, 1H, ThH), 4.67 (s, 2H, Py-CH2), 4.00 (t,
J = 12.0 Hz, 2H, Py-CH2), 2.87 (br s, 2H, Py-CH2), 2.30
(s, 3H, CH3); 13C NMR (CDCl3, 75 MHz) d:167.7, 165.0,
150.3, 148.1, 141.4, 140.2, 128.4, 126,8, 124.1, 122.0,
121.6, 111.7, 45.8, 43.1, 25.4, 20.7; IR (KBr, cmꢀ1) υ:
2930.3, 2895.2, 2840.6, 1754.8, 1645.9, 1607.5, 1508.2,
1440.9, 1203.9, 1104.6, 1059.8, 960.5, 838.8, 755.5;
ESI-Mass for C18H16N2O5S: m/z (M++H) 373.00.
Compounds 6b-e were synthesized following the same
procedure described above for the preparation of 6a.
(E)-5-(3-(4-methoxyphenyl)acryloyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl acetate (6b)
Off- white solid,yield 63.6%,m.p.126.3~127.4 °C;1H NMR
(CDCl3, 300 MHz) d: 7.68 (d, J = 15.3 Hz, 1H, ArCH=),
7.50 (d, J = 8.7 Hz, 2H, ArH), 6.90 (d, J = 8.7 Hz, 2H,
ArH), 6.79 (d, J = 15.3 Hz, 1H, -CH=), 6.41 (s, 1H, ThH),
4.65 (s, 2H, Py-CH2), 3.96 (t, J = 5.4 Hz, 2H, Py-CH2),
3.83 (s, 3H, OCH3), 2.84(t, J = 5.4 Hz, 2H, Py-CH2), 2.29
(s, 3H, CH3); 13C NMR (CDCl3, 75 MHz) d:167.7, 166.3,
160.9, 150.0, 142.8, 129.4, 127.9, 115.0, 114.6, 114.2,
(E)-3-(4-acetoxyphenyl)acrylic acid (7a)
To the solution of (E)-3-(4-hydroxyphenyl)acrylic acid
(6.00 g, 36.6 mmol) in Ac2O (12 mL),pyridine (2 mL) was
added. The reaction mixture was stirred at room tempera-
ture for 3.5 h. Water was added and filtered. The filter was
solved in water. Then saturated Na2CO3 (liq.) was added
to pH = 10. The mixture was filtered. The filtrate was acidi-
fied with con.HCl to pH = 3, then solid appears. The mix-
ture was filtered, and the crude product was obtained.
White solid, 6.17 g,yield 81.9%,m.p.216.4~218.7.
111.8, 111.3, 55.4, 45.6, 43.0, 25.4, 20.7; IR (KBr, cmꢀ1
)
υ:2938.5, 2839.9, 1757.5, 1643.9, 1599.0, 1503.3,
1440.5, 1371.8, 1303.0, 1252.2, 1207.3, 1144.5, 1030.9,
977.1, 926.3, 890.4, 818.6, 728.9; ESI-Mass for
C19H19NO4S: m/z (M++H) 357.98.
(E)-5-(3-(4-methylphenyl)acryloyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridin-2-yl acetate (6c)
Off- white solid,yield 57.2%,m.p.117.2~118.6 °C; 1H
NMR (CDCl3, 400 MHz) d: 7.68 (d, J = 15.2 Hz, 1H,
ArCH=), 7.44 (d, J = 8.0 Hz, 2H, ArH), 7.19 (d,
J = 7.6 Hz, 2H, ArH), 6.87 (d, J = 15.6 Hz, 1H, -CH=),
6.41 (s, 1H, ThH), 4.66 (s, 2H, Py-CH2), 3.96 (br s, 2H,
Py-CH2), 2.84(br s, 2H, Py-CH2), 2.37 (s, 3H, CH3), 2.29
(s, 3H, CH3); 13C NMR (CDCl3, 100 MHz) d:167.7,
166.2, 150.1, 143.1, 140.0, 132.4, 129.5, 127.8, 116.4,
116.1, 111.7, 111.3, 45.6, 43.0, 25.4, 21.4, 20.7; IR
(KBr, cmꢀ1) υ:2935.6, 2896.7, 2848.8, 1757.5, 1646.8,
1596.0, 1494.4, 1434.6, 1365.8, 1219.3, 1144.5,
(E)-4-(3-(2-acetoxy-6,7-dihydrothieno[3,2-c]pyridin-
5(4H)-yl)-3-oxoprop-1-en-1-yl)phenyl acetate (7b)
Off-white solid, yield 47.6%,m.p.103.6~106.2 °C; 1H NMR
(CDCl3, 300 MHz) d: 7.68 (d, J = 15.6 Hz, 1H, ArCH=),
7.55 (d, J = 8.7 Hz, 2H, ArH), 7.11 (d, J = 8.7 Hz, 2H,
ArH), 6.87 (d, J = 15.3 Hz, 1H, -CH=), 6.41 (s, 1H, ThH),
4.65 (s, 2H, Py-CH2), 3.95 (br s, 2H, Py-CH2), 2.84(br s,
2H, Py-CH2), 2.31 (s, 3H, CH3), 2.29 (s, 3H, CH3); 13C
NMR (CDCl3, 75 MHz) d:169.2, 167.7, 165.9, 151.6,
150.1, 142.0, 133.0, 128.9, 122.0, 117.8, 117.4, 111.8,
111.3, 45.7, 43.0, 25.4, 21.2, 20.7; IR (KBr, cmꢀ1
)
838
Chem Biol Drug Des 2016; 87: 833–840

Novel Piperlongumine Derivatives
υ:3068.0, 2927.1, 2839.9, 1751.6, 1645.9, 1613.9,
1585.1, 1424.9, 1370.5, 1047.0, 909.2, 896.4, 848.4;
ESI-Mass for C20H19NO5S: m/z (M++H) 386.00.
Pharmacology
Each chemical compound was dissolved in 20% dimethyl
sulfoxide (DMSO) in water. The artery blood sample col-
lected from Male New Zealand rabbit was mixed with just
enough sodium citrate with a concentration 3.8% to prevent
clotting, and centrifuged at room temperature for 5 min at
800 rpm to give platelet-rich plasma (PRP). The remaining
blood was further centrifuged for 15 min at 599.4 g to give
platelet-poor plasma (PPP). Aspirin and Piplartine were used
as reference. The tested target compounds were given at
0.8, 4, 20, 100, 500 lg/mL separately. A 20 lL of blank sol-
vent or test compounds was added to 170 lL of PRP, and
after 5 min, the aggregation was initiated by adding 10 lL
of ADP (4 lmol/L), in each well of 96-well plates, respec-
tively. Optical density (OD) at 630 nm was measured with
Microplate Reader (BioTek ELX800) after 1, 2, 4, 6 min of
the addition. All the experiments were performed in tripli-
cate. The platelet aggregation rate was expressed as PAR
(%) and was calculated as follows:
Compounds 6g-i were synthesized following the same
procedure described above for the preparation of 6f.
(E)-5-(3-(4-acetoxy-3-methoxyphenyl)acryloyl)-4,5,
6,7-tetrahydrothieno[3,2-c]pyridin-2-yl acetate (6g)
Off-white solid, yield 51.2%,m.p.130.1~130.9 °C; 1H NMR
(CDCl3, 300 MHz) d: 7.65 (d, J = 15.6 Hz, 1H, ArCH=), 7.17
(dd, J1 = 1.5 Hz, J2 = 8.1 Hz, 1H, ArH), 7.09–7.03 (m, 2H,
ArH), 6.84 (d, J = 15.3 Hz, 1H, -CH=), 6.42 (s, 1H, ThH),
4.65 (s, 2H, Py-CH2), 3.96 (br s, 2H, Py-CH2), 3.88 (s, 3H,
OCH3), 2.85(br s, 2H, Py-CH2), 2.33 (s, 3H, CH3), 2.29 (s,
3H, CH3); 13C NMR (CDCl3, 75 MHz) d:168.8, 167.7, 165.8,
151.3, 150.1, 142.3, 140.9, 134.2, 128.4, 124.8, 123.1,
120.4, 117.9, 117.6, 111.6, 55.9, 45.7, 43.0, 25.4, 20.7,
20.6; IR (KBr, cmꢀ1) υ:3068.0, 3010.3, 2930.3, 2869.4,
1767.6, 1649.1, 1613.9, 1591.5, 1511.4, 1456.9, 1399.3,
1300.0, 1255.2, 1123.8, 1047.0, 973.3, 890.0, 822.8,
745.9; ESI-Mass for C21H21NO6S: m/z (M++H) 416.00.
PAR ð%Þ ¼ ð1 ꢀ OD1/OD0Þ ꢁ 100%:
Here, OD0 and OD1 indicated the optical density before
the ADP was added and after the ADP was added,
respectively. The following equation was used to calculate
the inhibition rate of platelet aggregation (IR).
(E)-5-(3-(3-acetoxy-4-methoxyphenyl)acryloyl)-4,5,
6,7-tetrahydrothieno[3,2-c]pyridin-2-yl acetate (6h)
Off-white solid, yield 37.1%,m.p.140.6~142.2 °C; 1H NMR
(CDCl3, 300 MHz) d: 7.62 (d, J = 15.3 Hz, 1H, ArCH=),
7.38 (dd, J1 = 2.1 Hz, J2 = 8.4 Hz, 1H, ArH), 7.27 (s, 1H,
ArH), 7.96 (d, J = 8.4 Hz, 1H, ArH), 6.77 (d, J = 14.4 Hz,
1H, -CH=), 6.41 (s, 1H, ThH), 4.65 (s, 2H, Py-CH2), 3.99 (br
s, 2H, Py-CH2), 3.86 (s, 3H, OCH3), 2.84 (br s, 2H, Py-CH2),
2.34 (s, 3H, CH3), 2.29 (s, 3H, CH3); 13C NMR (CDCl3,
75 MHz) d:168.9, 167.7, 165.9, 151.3, 150.1, 142.4, 140.9,
134.2, 128.4, 123.2, 120.4, 117.9, 117.5, 111.6, 56.0,
45.7, 43.0, 25.4, 20.7; IR (KBr, cmꢀ1) υ: 3093.6, 3055.2,
2923.8, 2843.8, 1767.6, 1649.1, 1604.3, 1511.4, 1434.5,
1364.1, 1300.0, 1268.0, 1127.1, 1053.4, 1027.8, 886.9,
822.8; ESI-Mass for C21H21NO6S: m/z (M++H) 416.00.
IR ð%Þ ¼ ð1 ꢀ PAR test/PAR blankÞ ꢁ 100%:
Here, PAR test and PAR blank represented the PAR of the
tested compounds and the blank solvent, respectively.
Table 2: The inhibitory activities of 4a-4i and 6a-6i against plate-
let aggregation induced by AA or ADP (n = 4) in vitro
IC50/mM
Compd
AA
20.751 ꢂ 1.450
ADP
6.710 ꢂ 0.697
4a
4b
7.794 ꢂ 0.390
259.381 ꢂ 16.148
13.234 ꢂ 1.130
0.130 ꢂ 0.023
21.520 ꢂ 1.323
2.138 ꢂ 0.243
0.403 ꢂ 0.066
0.332 ꢂ 0.076
21.556 ꢂ 1.313
21.962 ꢂ 1.192
10.127 ꢂ 0.703
4.671 ꢂ 0.323
0.108 ꢂ 0.014
16.005 ꢂ 1.110
26.092 ꢂ 1.832
85.418 ꢂ 3.718
13.246 ꢂ 1.184
5.489 ꢂ 0.348
4.431 ꢂ 0.319
13.085 ꢂ 1.943
41.483 ꢂ 2.610
8.428 ꢂ 0.733
0.403 ꢂ 0.080
17.602 ꢂ 1.443
0.310 ꢂ 0.070
7.012 ꢂ 0.374
2.806 ꢂ 0.778
4.484 ꢂ 0.567
14.366 ꢂ 1.316
0.241 ꢂ 0.077
0.466 ꢂ 0.071
3.145 ꢂ 0.720
3.185 ꢂ 0.453
2.309 ꢂ 0.730
1.710 ꢂ 0.479
0.077 ꢂ 0.013
11.121 ꢂ 0.790
12.588 ꢂ 1.313
4c
4d
4e
4f
4g
(E)-5-(3-(4-acetoxy-3,5-dimethoxyphenyl)acryloyl)-
4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl acetate
(6i)
4h
4i
6a
White solid, yield 41.7%,m.p.136.8~137.7 °C; 1H NMR
(CDCl3, 300 MHz) d: 7.61 (d, J = 15.3 Hz, 1H, ArCH=),
6.82 (d, J = 15.3 Hz, 1H, -CH=), 6.76 (s, 2H, ArH), 6.43
(s, 1H, ThH), 4.66 (s, 2H, Py-CH2), 3.97 (br s, 2H, Py-
CH2), 3.86 (s, 6H, OCH392), 2.85(br s, 2H, Py-CH2), 2.35
(s, 3H, CH3), 2.30 (s, 3H, CH3); 13C NMR (CDCl3,
75 MHz) d: 168.6, 167.7, 165.8, 152.3, 150.1, 142.9,
133.6, 129.9, 118.0, 117.5, 111.7, 111.3, 104.5, 56.2,
45.7, 43.0, 24.2, 20.7, 20.5; IR (KBr, cmꢀ1) υ: 3074.4,
3007.1, 2978.3, 2943.1, 2847.0, 1761.2, 1642.7, 1604.3,
1505.0, 1460.1, 1421.7, 1370.5, 1338.4, 1194.3, 1130.3,
1063.0, 1011.7, 883.6, 832.4, 794.7; ESI-Mass for
C22H23NO7S: m/z (M++H) 446.00.
6b
6c
6d
6e
6f
6g
6h
6i
Piperlongumine
Aspirin
Bold indicates compounds 4e and 6e exhibit best platelet aggre-
gation inhibit rate.
Chem Biol Drug Des 2016; 87: 833–840
839

Wang et al.
The ability of the tested compounds to inhibit platelet
aggregation was evaluated by IC50, calculated from the
inhibition ratio linearity regression equation, which was fit-
ted based on the inhibition ratio.
4. Son D.J., Kim S.Y., Han S.S., Kim C.W., Kumar S.,
Park B.S., Lee S.E., Yun Y.P., Jo H., Park Y.H. (2012)
Piperlongumine inhibits atherosclerotic plaque forma-
tion and vascular smooth muscle cell proliferation by
suppressing PDGF receptor signaling. Biochem Bio-
phys Res Commun;427:349–354.
Results and Discussion
5. da Silva R.V., Navickiene H.M.D., Kato M.J., Bolzania
V.D.S., Meda C.I., Young M.C.M., Furlan M. (2002)
Antifungal amides from Piper arboreum and Piper
tuberculatum. Phytochemistry;59:521–527.
6. Sun L.D., Wang F., Dai F., Wang Y.H., Lin D., Zhou B.
(2015) Development and mechanism investigation of a
new piperlongumine derivative as a potent anti-inflam-
matory agent. Biochem Pharmacol;95:156–169.
7. Wang Y.H., Wang L.T., Hsieh K.Y., Natschke S.L.,
Tang G.H., Long C.L., Lee K.H. (2014) Multidrug resis-
tance-selective antiproliferative activity of Piperamide
alkaloids and synthetic analogues. Bioorg Med Chem
Lett;24:4818–4821.
The synthesized piperlongumine derivatives were evaluated
for their inhibitory effect on AA/ADP-induced platelet
aggregation by Born’s Method (14) and liver microsomal
incubated assay in vitro (15), while aspirin and piperlongu-
mine were used as a positive control. The assay results
are summarized in Table 2. The results demonstrate that
compared with positive control, all the target compounds
had moderate IC50 value. Compound 6a-e showed better
inhibition effect on platelet aggregation induced by AA. It
was speculated that these may be due to the elctro-with-
draw effect of the substituent group. Especially, com-
pounds 4e, 6e display obviously superior inhibition effect
on platelet aggregation induced by AA/ADP.
8. Iwashita M., Oka M., Ohkubo S., Saito M., Nakahata
N. (2007) Piperlongumine,
a constituent of Piper
longum L., inhibits rabbit platelet aggregation as a
thromboxane A2 receptor antagonist. Eur J Pharma-
col;570:38–42.
Conclusion
9. Aalla S., Gilla G., Metil D.S., Anumula D.R., Vummen-
thala P.R., Padi P.R. (2012) Process improvements of
prasugrel hydrochloride: an adenosine diphosphate
receptor antagonist. Org Process Res Dev;16:240–
243.
In summary, 18 piperlongumine derivatives were synthe-
sized and evaluated for their inhibitory effect on AA/ADP-
induced platelet aggregation. The assay results indicated
that 4e, 6e exhibited comparable potency with that of
aspirin and piperlongumine.
€
10. Varenhorst C., Jensevik K., Jernberg T., Sundstrom
A., Hasvold P., Held C., Lagerqvist B., James S.
(2014) Duration of dual antiplatelet treatment with
clopidogrel and aspirin in patients with acute coronary
syndrome. Eur Heart J;35:969–978.
Acknowledgements
We gratefully acknowledge support from the: Anhui natural
science research in colleges and universities important
project: KJ2016A414.
11. Shan J.Q., Zhang B.Y., Zhu Y.Q., Jiao B., Zheng
W.Y., Qi X.W., Gong Y.C., Yuan F., Lv F.S., Sun H.B.
(2012) Overcoming clopidogrel resistance: discovery of
vicagrel as a highly potent and orally bioavailable anti-
platelet agent. J Med Chem;55:3342–3352.
References
12. Zhu Y.Q., Zhou J., Jiao B. (2013) Deuterated clopido-
grel analogues as a new generation of antiplatelet
agents. ACS Med Chem Lett;4:349–352.
1. Duh C.Y., Wu Y.C., Wang S.K. (1990) Cytotoxic pyri-
done alkaloids from the leaves of piper-aborescens.
J Nat Prod;53:1575–1577.
13. Zhi S., Zheng G., Liu B., Wang J.Y., Liu D.K. (2012)
Synthesis
of
N-substituted-2-{4,5,6,7-tetrahy-
2. Han S.S., Tompkins V.S., Son D.J., Kamberos N.L.,
Stunz L.L., Halwani A., Bishop G.A., Janz S. (2013)
Piperlongumine inhibits LMP1/MYC-dependent mouse
Blymphoma cells. Biochem Biophys Res Commun;
436:660–665.
3. Rao V.R., Muthenna P., Shankaraiah G., Akileshwari
C., Babu K.H., Suresh G., Babu K.S., Kumar R.S.C.,
Prasad K.R., Yadav P.A., Petrash J.M., Reddy G.B.,
Rao J.M. (2012) Synthesis and biological evaluation of
new piplartine analogues as potent aldose reductase
inhibitors (ARIs). Eur J Med Chem;57:344–361.
drothieno[3,2-c]pyridine-5-yl}acetamides and their
anti-platelet aggregation activities. Chin
J Synth
Chem;20:312–315.
14. Born G. (1962) Aggregation of blood platelets by ade-
nosine diphosphate and its reversal. Nature;194:927–
929.
15. Mauriz L.M., Eduardo A.S., Gouvea D.R., Vessecchi
R., Pupo M.T., Lopes N.P., Kato M.J., Oliveira A.R.
(2014) In vitro metabolism of the alkaloid piplartine by
rat liver microsomes. J Pharm Biomed Anal;95:113–
120.
840
Chem Biol Drug Des 2016; 87: 833–840