Synthesis and biological evaluation of piperlongumine derivatives as potent anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2014.10.054

Piperlongumine (PL) and its derivatives were synthesized by the direct reaction between acid chloride of 3,4,5-trimethoxycinnamic acid and various amides/lactams. Later their anti-inflammatory effects were evaluated in lipopolysaccharide (LPS)-induced RAW-264.7 macrophages. Of the piperlogs prepared in this study, the maximum (91%) inhibitory activity was observed with PL (IC₅₀ = 3 μM) but showed cytotoxicity whereas compound 3 (IC₅₀ = 6 μM) which possess α,β-unsaturated γ-butyrolactam moiety offered good level (65%) of activity with no cytotoxicity. This study revealed that amide/lactam moiety connected to cinnamoyl group with minimum 3 carbon chain length and α,β-unsaturation is fruitful to show potent anti-inflammatory activity.

Full text of DOI:10.1016/j.bmcl.2014.10.054

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of piperlongumine derivatives as
potent anti-inflammatory agents
Young Hwa Seo a, Jin-Kyung Kim b, Jong-Gab Jun a,
⇑
a Department of Chemistry and Institute of Applied Chemistry, Hallym University, Chuncheon 200-702, Republic of Korea
b Department of Biomedical Science, College of Natural Science, Catholic University of Daegu, Gyeungsan-Si 700-702, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Piperlongumine (PL) and its derivatives were synthesized by the direct reaction between acid chloride of
3,4,5-trimethoxycinnamic acid and various amides/lactams. Later their anti-inflammatory effects were
evaluated in lipopolysaccharide (LPS)-induced RAW-264.7 macrophages. Of the piperlogs prepared in
Received 22 August 2014
Revised 30 September 2014
Accepted 17 October 2014
Available online xxxx
this study, the maximum (91%) inhibitory activity was observed with PL (IC50 = 3
toxicity whereas compound 3 (IC50 = 6 M) which possess ,b-unsaturated
offered good level (65%) of activity with no cytotoxicity. This study revealed that amide/lactam moiety
lM) but showed cyto-
l
a
c-butyrolactam moiety
Keyword:
connected to cinnamoyl group with minimum 3 carbon chain length and
show potent anti-inflammatory activity.
a,b-unsaturation is fruitful to
3,4,5-Trimethoxycinnamic acid
Piperlongumine
Nitric oxide
Ó 2014 Elsevier Ltd. All rights reserved.
Anti-inflammatory
Inflammation is the body’s natural response to infection, dis-
ease and tissue damage and is an integral part of the immune
response.1 Symptoms of inflammation include swelling, redness
of the area, pain, and sometimes loss of movement or function.2
Inflammation is either acute or chronic and under specific circum-
stances the former could turn into the later one which will become
a causative factor in the pathogenesis. Various molecules involved
in the inflammatory response viz. prostaglandins (PG), leukotri-
enes (LT) and nitric oxide (NO) etc. PGs are produced by cyclooxe-
ganases (COX, which mainly having two forms COX-1 and COX-2)
and LTs are produced by lipoxygenases (LOX). Nitric Oxide (NO) is
Non-steroidal anti-inflammatory drugs (NSAIDs) usage is com-
mon practice in the therapeutic approach to alleviate the symp-
toms associated with both acute and chronic inflammatory
diseases. The activity of NSAIDs is most likely mediated through
their ability to inhibit COX, LOX and iNOS. Although NSAIDs are
effective, there are known risks. They can inhibit the housekeeping
enzyme COX-1 along with COX-2. The problem is that COX-1 inhi-
bition would ‘switch off’ functions like the repair and maintenance
of stomach lining, which results in varying degrees of gastric
health issues, the most significant one is gastrointestinal bleeding.
Therefore, continuous efforts are on to search for more potent and
selective anti-inflammatory drugs with minimal side-effects.
Piperlongumine (PL) 1 is a biologically active dihydropyridone
alkaloid isolated from the various parts of long pepper, Piper lon-
gum L. along with other constituents viz. piperine, sylvatin, ses-
amin and diaeudesmin piperlongumine.4 Long pepper is used to
treat cough, respiratory infections, stomach pains and other dis-
eases in Indian Ayurvedic medicine.5 The chemical structure of
PL is having 3,4,5-trimethoxycinnamoyl moiety, attached to
dihydropyridone and is well characterized (Fig. 1). PL was previ-
ously reported to possess antibacterial and insecticidal proper-
ties.6 In addition PL also showed several noteworthy activities
which include anti-cancer,7 anti-platelet aggregation,8 anti-fun-
gal9 and anti-diabetic.10 In recent studies, PL as a potential
anti-cancer compound demonstrated that it would selectively
kill 13 different human solid tumor cells (including melanoma,
bladder, breast and lung cancer) without affecting their normal
cells.11
a short-lived, lipophilic, free-radical species generated from L-argi-
nine by nitric oxide synthases (NOS). Among the NOS, inducible
isoform of nitric oxide synthase (iNOS) is chiefly responsible for
the large amounts of NO production in inflammatory lesions. NO
is involved physiologically in vasorelaxation, neurotransmission,
inhibition of platelet aggregation, immune defense, and intracellu-
lar signaling. However, NO reacts with superoxide (OÀ2 ) to form
peroxynitrite (ONOOÀ), which is a powerful oxidant. NO bioactivity
is related to the production of many reactive intermediates, but
many of these reactive nitrogen species (RNS) are capable of dam-
aging DNA or hindering DNA repair.3 Therefore, control of the
excess NO production by inhibition of iNOS may exert anti-inflam-
matory effects.
⇑
Corresponding author. Tel.: +82 33 248 2075; fax: +82 33 256 3421.
E-mail address: jgjun@hallym.ac.kr (J.-G. Jun).
http://dx.doi.org/10.1016/j.bmcl.2014.10.054
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Seo, Y. H.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.10.054

2
Y.H. Seo et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
O
O
O
O
O
1
MeO
MeO
2
3
MeO
MeO
8
N
N
N
N
R
6
3, R =
5, R =
7
2, R =
4, R =
5
4
O
OMe
OMe
2-8
Piperlongumine 1
N
H
O
O
O
N
7, R =
6, R =
8, R =
N
H
N
H
Figure 1. Structure of piperlongumine (1) and its derivatives (2–8).
O
O
O
O
O
HN
MeO
MeO
oxalyl chloride
DMF (catalytic)
CH2Cl2, rt, 3 h
98%
MeO
MeO
OH
MeO
MeO
Cl
N
toluene, reflux
18 h, 95%
OMe
9
OMe
10
OMe
2
O
O
O
O
MeO
MeO
MeO
SePh
H2O2
N
PhSeCl, LDA
N
2
-50 - -78 o
C
THF, 0 oC - rt
45 min, 71%
MeO
OMe
5.5 h, 27%
11
OMe
1
Piperlongumine
O
30% H2O2 / BaCO3
HN
H2O, ref lux, 5 h
N
H
30%
12
O
O
O
O
MeO
MeO
MeO
MeO
12
N
N
N
H
n-BuLi, DMF
acrylamide
-78 oC, 1 h
11%
6 (4%)
3
OMe
OMe
THF, reflux
19.5 h
+
O
O
O
O
O
MeO
MeO
HN
MeO
MeO
N
10
toluene, reflux
14.5 h, 64%
7 (73%)
OMe
OMe
4
O
O
O
O
MeO
MeO
MeO
MeO
N
N
H
acetamide
propionamide
toluene, reflux
14.0 h, 33%
H
DMF, reflux
14 h, 41%
OMe
OMe
8
5
Scheme 1. Synthesis of PL and its derivatives.
In continuation of our work12 on the synthesis of bioactive nat-
ural products and their analogues, herein we wish to describe the
synthesis and anti-inflammatory activity evaluation of PL and its
derivatives.
Our approach for the synthesis of PL and its derivatives is out-
lined in Scheme 1. The synthesis was initiated by the conversion
of 3,4,5-trimethoxycinnamic acid (9) to it acid chloride (10)
(Scheme 1). Treatment of compound 9 with oxalyl chloride and
catalytic amount of DMF afforded the acid chloride 10 in 98% yield.
Keeping 10 in hand, we first attempted the PL derivative 2 synthe-
sis by the direct reaction between 10 and piperidin-2-one and the
product was formed in 95% yield. Treatment of compound 2 with
Please cite this article in press as: Seo, Y. H.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.10.054

Y.H. Seo et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
phenylselenyl chloride gave the
subsequently subjected to oxidation with hydrogen peroxide to
furnish piperlongumine 1 in 71%.
a
-phenylseleno imide 11 which
Anti-inflammatory activities of piperlogs 1–8
Compound
NO production (% inhibition)
Next, we prepared 1H-pyrrol-2(5H)-one 12 from pyrrole follow-
ing the literature procedure13 which was further used in the PL
derivative 3 preparation. Treatment of compound 12 with n-BuLi
at À78 °C followed by slow addition of 10 resulted 3. PL derivatives
4, 5 and 8 were obtained by direct reaction of 10 with the commer-
cially available pyrrolidin-2-one, propionamide and acetamide in
64%, 33% and 41%, respectively. Finally, reaction of 10 with acryl-
amide offered PL derivative 7 as a major product along with com-
pound 6. The structures of all the products were settled from their
spectral (1H and 13C NMR and MS) data.
1
2
l
M
10 lM
Medium (MED)
1
2
3
4
5
6
7
1 (98)⁄⁄⁄
2
9
1 (98)⁄⁄⁄
1 (91)⁄⁄⁄
89 2 (11)⁄⁄⁄
99 2 (1)
54 11 (46)⁄⁄⁄
35 1 (65)⁄⁄⁄
72 1 (28)⁄⁄⁄
84 6 (16)⁄⁄⁄
59 1 (41)
95 (5)⁄⁄
99 1 (1)
101 3 (À1)
97 2 (3)
99 1 (1)
78 1 (22)⁄⁄⁄
84 2 (16)⁄⁄
100 1 (0)
8
LPS
101 2 (À1.0)
100 1 (0)
In order to evaluate the anti-inflammatory effects of the pre-
pared piperlongumine (1) and its derivatives (2–8), we measured
the amount of nitric oxide (NO) which is one of the essential medi-
ators on inflammation, in lipopolysaccharide (LPS)-stimulated
RAW264.7 macrophages.14
The results are reported as mean value SEM for n = 3. Statistical significance is
based on the difference when compared with LPS-treated groups (⁄⁄P <0.01, ⁄⁄⁄P
<0.001).
% Inhibition is based on LPS as shown in parenthesis.
Anti-inflammatory activity: Effect of PL (1) and its derivatives (2–
8) on NO generation by induced macrophages was monitored
(Table 1). Lipopolysaccharide (LPS) treated RAW 264.7 has been
used to stimulate the production of NO through the activation of
iNOS. Of the piperlogs prepared in the present study, only four
compounds (i.e., PL, compound 2, 3 and 6) showed significant
Table 2
Proliferation effect of piperlogs 1–8
Compound
Proliferation
1
l
M
10
100
77 1⁄⁄
l
M
Medium (MED)
100
115
107
101
105
97
103
97
104
4
3
4
3
2
7
1
5
6
4
1
2
3
4
5
6
7
8
activities at 10
itory activity was observed with PL (91%) followed by compound 3
(65%), 2 (46%) and 6 (41%). The cell viability assay at 10 M con-
lM. Among the 8 compounds, the maximum inhib-
101
113
102
103
2
2
8
6
l
centration was not affected by compounds 2, 3, 4, 5 and 8 indicat-
ing no cytotoxicity as shown in Table 2. IC50 values of these
compounds (1–8) were evaluated by using GraphPad Prism 4.0
software and showed 3, 14, 6, 80, 64, 17, 64 and 341 lM, respec-
tively, (Fig. 2). From the aforementioned pharmacological results,
83 2⁄
92
8
104
3
The results are reported as mean value SEM for n = 3. Statistical significance is
based on the difference when compared with Medium groups (⁄P <0.05, ⁄⁄P <0.01).
we can conclude that amide/lactam moiety connected to cinnam-
oyl group with minimum 3 carbon chain length and a,b-unsatura-
tion is fruitful as the compound 3 was found to show potent anti-
inflammatory activity with no cytotoxicity (Fig. 3).
In summary, we have described the synthesis of piperlongu-
mine (1) and its derivatives (2–8) by the direct reaction between
the acid chloride of 3,4,5-trimethoxycinnamic acid and various
amides/lactams. Later, the anti-inflammatory effects of these com-
pounds were evaluated in lipopolysaccharide (LPS) stimulated
RAW-264.7 macrophages. Of the piperlogs prepared in this study,
the maximum inhibitory activity was observed with PL (91%) but
showed cytotoxicity whereas compound 3 which possess
a,b-
unsaturated -butyrolactam moiety offered good level (65%) of
c
activity with no cytotoxicity. This study revealed that amide/lac-
tam moiety connected to cinnamoyl group with minimum 3 car-
bon chain length and
a,b-unsaturation is fruitful to show potent
IC50 Values 1: 3 µM, 2: 14 µM, 3: 6 µM, 4: 80 µM, 5: 64 µM, 6: 17 µM, 7: 64
µM, 8: 341 µM
anti-inflammatory activity. Gaining boost from this study, further
investigation about the mechanism of action is under progress
and will be disclosed in due course.
Figure 2. IC50 values for NO production..
O
O
O
O
O
O
O
O
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
N
N
N
N
H
OMe
1 (Piperlongumine)
OMe
OMe
OMe
2
3
6
IC50 Values
Cytotoxicity at 10
3
μΜ
6 μΜ
14 μΜ
non-toxic
17 μΜ
toxic
non-toxic
toxic
μΜ
Figure 3. Comparison of IC50 values with cytotoxicity of active compounds.
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Y.H. Seo et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.10.
054.
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