Synthesis of coumaperine derivatives: Their NF-κB inhibitory effect, inhibition of cell migration and their cytotoxic activity

Article abstract of DOI:10.1016/j.ejmech.2016.10.047

Coumaperine (an amide alkaloid, present in white piper) and its derivatives were synthesized and investigated for their cytotoxicity against L428 and A549 cells and their NF-κB inhibitory activity. It was found that the coumaperine derivatives CP-9 and CP-38 suppress NF-κB subunits p50 and p65 in nuclear fractions by western blot and by NF-κB luciferase reporter gene assay in a dose dependent manner. Confirmation of these results was obtained by confocal microscopy. CP-9, CP-32 and CP-38 also exhibited dose dependent cell cytotoxicity in a L428?cells expressing constitutively active NF-κB and in A549?cells, with an IC50 value of 43.25?μg/ml, 0.39?μg/ml and 16.85?μg/ml respectively against L428?cells and 57.15?μg/ml, 69.1?μg/ml and 63.2?μg/ml respectively against A549?cells. In addition, the coumaperine derivatives show remarkable inhibitory activity on the cancer cell migration assay against A549 lung adenocarcinoma cells at the concentrations of 5?μg/ml, 10?μg/ml, and 5?μg/ml of CP-9, CP-32 and CP-38 respectively. Aromatic substituents and number of olefinic double bond in coumaperine derivatives found to influence the inhibitory activity. In luciferase reporter gene assay, di-olefin conjugated coumaperine derivatives, CP-38, CP-32 and PIP exhibited higher inhibitory activity than their corresponding tri-olefin conjugated coumaperine derivatives, CP-102, CP-146 and PIP-155 respectively. CP-32 with a stronger electron donating group (-N(CH₃)₂) showed better inhibitory activity in luciferase reporter gene assay and in cell proliferation of L428?cells. Simple coumaperine derivative (CP-9, with no substituent) effectively inhibited A549?cells proliferation and migration than the other coumaperine derivatives. CP-9 and CP-38 diminish significantly the NF-κB subunits (p50 and p65) of L428?cells in nuclear fractions at the dosage of 10?μg/ml and 30?μg/ml respectively. Which clearly shows that CP-9 and CP-38 inactivate the NF-κB pathway in?vitro.

Full text of DOI:10.1016/j.ejmech.2016.10.047

Accepted Manuscript
Synthesis of coumaperine derivatives: Their NF-κB inhibitory effect, inhibition of cell
migration and their cytotoxic activity
Natarajan Nandakumar, Subramani Muthuraman, Pushparathinam Gopinath,
Pattusamy Nithya, Jacob Gopas, Rajendran Saravana Kumar
PII:
S0223-5234(16)30909-6
10.1016/j.ejmech.2016.10.047
EJMECH 9012
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 August 2016
Revised Date: 16 October 2016
Accepted Date: 21 October 2016
Please cite this article as: N. Nandakumar, S. Muthuraman, P. Gopinath, P. Nithya, J. Gopas,
R.S. Kumar, Synthesis of coumaperine derivatives: Their NF-κB inhibitory effect, inhibition of cell
migration and their cytotoxic activity, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.10.047.
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Graphical abstract
Coumaperine, a minor amide alkaloid, present in white piper, its analogs (11 derivatives) were
synthesized in less number of synthetic steps and evaluated for NF-ҡB (a key transcription factor
often associated with several inflammatory disorders and different types of cancer) inhibitory
activity. They found to effectively inhibit NF-ҡB proteins in L428 cells in vitro, with a potential
to be used as anti-inflammatory compounds.

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Synthesis of Coumaperine derivatives: their NF-κB inhibitory effect, inhibition of cell
migration and their cytotoxic activity
Natarajan Nandakumara, Subramani Muthuramanb†, Pushparathinam Gopinathc, Pattusamy
Nithyab, Jacob Gopasa* and Rajendran Saravana Kumarb*
aThe Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health
Sciences, and Oncology Laboratory, Soroka University Medical Center, Ben-Gurion University
of the Negev, Beer Sheva, Israel.
bChemistry Division, School of Advanced Sciences, VIT University Chennai Campus, Chennai,
Tamilnadu, India.
CDepartment of Chemistry, Indian Institute of Technology Madras, Chennai, Tamilnadu, India.
†Equally contributed with the first author for this work
*Correspondence to
Dr. Rajendran Saravana Kumar, Ph.D.,
Assistant Professor,
School of Advanced Sciences,
VIT University Chennai Campus,
Vandalore- Kelambakkam Road,
Chennai- 600127 Tamilnadu, India
Email: sar.org@gmail.com
Phone: 91-44-39931479 Fax: NA
Abstract
Coumaperine (an amide alkaloid, present in white piper) and its derivatives were synthesized and
investigated for their cytotoxicity against Hodgkin's lymphoma derived L428 cells and A549
lung adenocarcinoma cells and their NF-ҡB inhibitory activity. It was found that the
coumaperine derivatives CP-9 and CP-38 suppress NF-ҡB subunits p50 and p65 in nuclear
fractions by western blot and by NF-ҡB luciferase reporter gene assay in a dose dependent

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manner. Confirmation of these results was obtained by confocal microscopy. CP-9, CP-32 and
CP-38 also exhibited dose dependent cell cytotoxicity in a L428 cells expressing constitutively
active NF-ҡB and in A549 cells, with an IC50 value of 43.25 µg/ml, 0.39 µg/ml and 16.85
µg/ml respectively against L428 cells and 57.15 µg/ml, 69.1 µg/ml and 63.2 µg/ml respectively
against A549 cells. In addition, the coumaperine derivatives show remarkable inhibitory activity
on the cancer cell migration assay against A549 lung adenocarcinoma cells at the concentrations
of 5 µg/ml, 10 µg/ml, and 5 µg/ml of CP-9, CP-32 and CP-38 respectively. Aromatic
substituents and number of olefinic double bond of in coumaperine derivatives found to
influenced the inhibitory activity. In luciferase reporter gene assay, di-olefin conjugated
coumaperine derivatives, CP-38, CP-32 and PIP exhibited higher inhibitory activity than their
corresponding tri-olefin conjugated coumaperine derivatives, CP-102, CP-146 and PIP-155
respectively. CP-32 with a stronger electron donating group (-N(CH3)2) showed better
inhibitionry inhibitory activity in luciferase reporter gene assay and in cell proliferation of L428
cells. Simple coumaperine derivative (CP-9, with no substituent) effectively inhibited A549 cells
proliferation and migration than the other coumaperine derivatives. CP-9 and CP-38 diminish
significantly the NF-ҡB subunits (p50 and p65) of L428 cells in nuclear fractions at the dosage
of 10 µg/ml and 30 µg/ml respectively. Which clearly shows that CP-9 and CP-38 inactivate the
NF-ҡB pathway in vitro.
Keywords
NF-ҡB; L428 cells; A549 cells; coumaperine; piperine; amide alkaloid.
1. Introduction

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Nuclear factor-ҡB (NF-ҡB), a key transcription factor regulates various genes involves in
inflammation, cell proliferation and apoptosis [1-5]. Abnormal activation of NF-κB is often
associated with several cancer cells [5] especially lymphoma cells [7]. Many synthetic and
natural products small molecule inhibitors against NF-κB have been discovered, underscoring it
as a validated drug target [7-10]. Typically, NF-κB exists in its inactive form as a protein
complex with its inhibitor protein IkB in the cytoplasm of resting cells. Activation of NF-κB
occurs through a cascade of pro-inflammatory events, leading to the translocation of NF-κB from
the cytoplasm to nucleus either through the classical or alternative pathway [3, 9]. Dysregulation
of NF-κB is witnessed in several diseased states, hence inhibition of these activation pathway
through various synthetic and natural products have been developed, underscoring it as a
validated drug target [7-10]. Since natural products have been the source of inspiration for
various anticancer agents, it is not surprising that the inhibitors developed against NF-κB are
enriched with several natural products including polyphenols (quercetin, curcumin, epigenin and
resveratrol)
and
terpenoids
(helenalin,
parthenolide,
costunolide
and
thionupharidines/thionuphlutidines) [9, 12]. Nevertheless, lack of any clinical candidate against
this target further demand the identification of new chemical entities, especially natural product
based inhibitors [7, 12]. Piperine, (Figure 1) an amide alkaloid, a major constituent of black
(Piper nigrum Linn) and long (Piper longum Linn) pepper, was identified as a potent NF-κB
inhibitor [13] in lung (adenocarcinoma derived A549 cells [14], and prostate cancer cells
(LNCaP, PC-3 and DU-145) [15]. Furthermore, piperine is known to inhibit LPS-mediated
activation of NF-κB proteins [16]. It is also know to exhibit a wide variety of biological activities
[17-19], including antioxidant [20, 21], anti-inflammatory [22, 23], antimutagenic [24] and
antitumor [25] activity. Coumaperine (Figure 1), a piperine type amide alkaloid present in white

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piper (Piper nigrum Linn.) also possess interesting biological activities like hepatocarcinogenesis
in rat models [26, 27]. Unlike piperine, coumpaperine is present in lower concentration [28],
which makes its isolation not only challenging but also became a bottle-neck for various
biological studies. In order to circumvent this situation, we developed an efficient method to
access coumparine and its analogs and study their anticancer properties. Herein, we report the
synthesis and cytotoxic effect of coumparine and its derivatives against L428 and A549 cancer
cell lines and study their NF-ҡB inhibition pathway in vitro.
Figure 1. Structure of amide alkaloids, piperine and coumaperine found in Piper species
2. Materials and Methods
2.1 Materials
All commercially obtained reagents/solvents for the synthesis of coumaperine and its derivatives
were used as received without further purification; chemicals were purchased from
Spectrochem®, SRL®, Acros Organics®, RANKEM®, Fisher Scientific®. and used as received
without further purification. Unless stated otherwise, reactions were conducted in oven-dried
glassware and under normal atmospheric conditions.
Antibodies against p65, Rel B, p50 and p52 were obtained from Santa Cruz Biotechnology, Anti-
mouse and anti-rabbit IgG peroxidase was obtained from Jackson Immuno Research. The anti-β-
actin monoclonal antibody from MP Biomedicals, Alexa flour®-488 conjugated goat anti-mouse

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IgG and CyTM3 -conjugated goat anti-rabbit IgG were obtained from Molecular Probes Inc. and
Jackson Immuno Research Laboratories Inc. respectively and DAPI was obtained from Sigma.
2.2 NMR, IR, High resolution mass spectrometry, and melting point characterization
13
1H NMR and C NMR spectra were recorded on Bruker 500 MHz spectrometer operating with
the 13C resonance frequency of 125 MHz and proton resonance frequency of 500 MHz or Bruker
400 MHz spectrometer operating with the 13C resonance frequency of 100 MHz and proton
resonance frequency of 400 MHz. Data from the 1H NMR spectroscopy are reported as chemical
shift (δ ppm) with the corresponding integration values. Coupling constants (J) are reported in
hertz (Hz). Standard abbreviations indicating multiplicity were used as follows: s (singlet), br
(broad), d (doublet), t (triplet), q (quartet) and m (multiplet). Data from 13C NMR spectra are
reported in terms of chemical shift (δ ppm).
IR spectra were recorded in Thermo Scientific Nicolet Nexus 470 FT-IR spectrometer and band
positions are reported in reciprocal centimeters. Samples were made as pellet with KBr and
recorded.
High-resolution mass spectral data in Electrospray Ionization mode were recorded on Agilant
6520 (Q-TOF) mass spectrometer in positive (ESI+) ion mode.
Melting points were recorded with REMI DDMS 2545. The instrument is calibrated with
benzoic acid before the measurement.
2.3 Cell culture

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Hodgkin’s lymphoma (HL)—derived L428 cells lack IκBα, therefore NF-κB is constitutively
activated and expressed in the nucleus [6, 29]. The cells are maintained in RPMI 1640. The Lung
Adenocarcinoma derived A549 cell lines were maintained and DMEM medium. Media was
supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, and 1% Pen- Strep
(Beit Haemek, Israel). A549 cells were passaged by trypsinization.
2.4 Cell viability by XTT assay
Cell survival was measured by a tetrazolium-formazan XTT assay kit (Beit Haemek) in 96-well
plates. 3 x 104 L428 cells/well in 200 µl medium were treated with different concentrations of
coumaperine derivatives for 48 h at 37 °C. Similarly, 3 x 104 Lung adenocarcinoma A549 cells
were seeded in 96-well plates. After the formation of monolayer (24 h) the cells were treated
with different doses of coumaperine derivatives. XTT solution (40 µl) was added, and the plates
were again incubated for 4 h at 37 °C. Absorbance was read at 450 nm by an ELISA reader.
2.5 Luciferase-NF-κB reporter gene assay
L428 cells stable transfectants with the luciferase NF-κB-Luc reporter gene were generated as
described in Ozer et al., 2009 [12] and were maintained in 500 µg/ml of G418. L428 cells (106
per well) expressing the luciferase-NF-κB reporter gene were incubated in 1 ml of medium
containing the solvent (DMSO) with different concentrations of coumaperine derivatives for 2 h.
Cells were then harvested, lysed and monitored by a luciferase reporter assay kit (Promega)
according to the manufacturer’s instructions. Measurements were carried out using a
luminometer at 300 nm. Data were normalized to the protein concentration in each lysate as
measured by the Bradford method (BioRad).

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2.6 Cell migration assay
The metastatic potential of the cells depends on other properties such as on their migration
ability. To investigate how coumaperine derivatives affect the cell migration, A549 human lung
carcinoma cells were seeded on 6-well plates without coumaperine derivatives. After the
formation of the monolayer (24 h), coumaperine derivatives and the vehicle were added and the
monolayers were scratched. After 48 h the results were recorded.
2.7 Western blot analysis
Nuclear and cytoplasmic protein lysates (10 x 106 cells per sample) were prepared. Briefly, 10 x
106 cells per samples were incubated for 2 hours with different concentrations of coumaperine
°
derivatives, after incubation, cells were collected by centrifuged at 1000 g for 5 min at 4 C.
Then the cell pellet was washed with 1x PBS for 3 times. Then protease inhibitor 3.2 mg/ml (PI
cocktail tablets, Roche) was added to the cells in Buffer A, containing Tris 1M, NaCl 5M, EDTA
0.5 M and allowed in ice for 15 min. Then the lysates were sheared several times through a
21 gauge needle, followed by centrifugation at 13,000 g for 10 min at 4 ˚C. The supernatant
o
(cytosolic fraction) was collected and stored at -80 C, then remaining cell pellet was washed
again with 1x PBS for 3 times at 2000 g for 5 min at 4 ˚C. Then 40 µl buffer B (Tris 1M, NaCl
5M, MgCl2) was added and kept in ice for 3 min, then 10 µl of 5M NaCl was added, allowed in
ice for 25 min, and centrifuged at 13,000 g for 30 min 4 ˚C. The supernatant was collected
(nuclear fraction). Protein quantification of lysates was determined by the Bradford method
(BioRad). RIPA lysis buffer containing (10 mM Tris pH 8.0, 100 mM NaCl, 5 mM EGTA, 0.1%
SDS, 1% NP-40, 45 mM β-mercaptoethanol, 50 mM NaF) was added to lysates after the
quantification of protein and Protein lysates (40µg) were separated in 10% SDS-polyacrylamide

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gel and blotted onto nitrocellulose membranes. The membranes were incubated with several
primary antibodies and subsequently with peroxidase-linked anti-mouse or anti-rabbit IgG.
Protein bands were detected by chemiluminescence with ECL (Amersham).
2.8 Immunocytochemistry
Cellular localization of NF-κB subunits in treated L428 cells was done in experiments where the
cells were incubated for 2 h at 37 °C with coumaperine derivatives CP-9 and CP-38. After
incubation, the cells were cyto-centrifuged (Shandon Cytospin 4) at 900 rpm for 5 min and fixed
in 8% formalin for 20 min. Samples of these cells were incubated separately with antibodies
against p65 and p50 and then with their respective fluorescent anti-mouse or anti-rabbit IgG-
peroxidase-linked secondary antibody, and the nucleus was stained with DAPI and the signals
were detected using Olympus FV1000-IX81 confocal microscope.
2.9 Statistical analysis
The results are representatives of three independent experiments. For reporter gene assay, each
sample was tested in triplicate.
3. Results
3.1 Synthesis of coumaperine and its derivatives
In order to generate coumaperine derivatives for structure activity relationship studies, it is
necessary to have a robust methodology for the preparation. Methodologies reported in the
literature for the preparation of coumaperine, and its derivatives were associated with several
limitations: A number of synthetic steps to the target, expensive reagents, or tedious synthetic

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procedure [30-33]. In order to have easy access to coumaperine and its derivatives, we modified
the literature procedure [34] to overcome the aforementioned difficulties.
Scheme 1. General reaction scheme for the synthesis of coumaperine and its derivatives.
Coumaperine and its derivatives were prepared from crotonic acid (or sorbic acid), piperidine
and benzaldehyde (or substituted benzaldehyde). At first, crotonic acid (or sorbic acid) is
converted to the corresponding acid chloride by refluxing over thionyl chloride and reacted with
piperidine to form crotonyl piperidine amide (3). It is then reacted with benzaldehyde (or
substituted benzaldehyde) in the presence of a base (KOH or t-BuOK), to achieve the target
molecule (detail synthetic procedure is given in ESI). Following the above described procedure,
coumaperine and its derivatives, shown in figure 2 were synthesized. Attempt to synthesize the
nitro derivative was not successful. Changing the reaction conditions viz performing the reaction
at elevated temperature (90-110 ˚C), with different bases (K2CO3 or NaH) or solvents (DMF,

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toluene or NMP) were not fruitful. Piperine (PIP) was isolated from black pepper by following
the modified literature procedure [35]. A modified literature procedure [35] was adopted for the
extraction. All the compounds were characterized by spectroscopic techniques (1H NMR, 13C
NMR, IR and GC-MS). The unreported compounds, CP-27, CP-50, CP-184 and CP-146 were
additionally characterized by HRMS.
Figure 2. Coumaperine derivatives synthesized.
3.2 Screening of coumaperine derivatives for inhibition of NF-κB in a reporter gene assay.
In order to understand whether coumaperine and its derivatives inhibit the NF-κB in reporter
gene assay, different concentration of coumaperine and its derivatives (10-50 µg/ml) were

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incubated with L428 cells constitutively expressing NF-κB and the luciferase- NF-κB reporter
gene for 2 h. A dose dependent inhibition of NF-κB was observed (Table 1).
Table 1. Effect of coumaperine derivatives on NF-κB luciferase reporter gene assay. Triplicate
wells of L428 cells expressing the luciferase-NF-κB reporter gene were incubated with
Coumaperine derivatives or with solvent and a positive control, Nuphar extract [12] for 2 h. The
cells were then harvested and lysed; cell extracts were monitored by a luciferase reporter assay
kit. NF-κB-luciferase activity in control cells was taken as 100%. The average and standard
deviation of three independent experiments are shown. CP-9, CP-38 and CP-32 showed the
better inhibition at lower doses.
% NF-κB inhibition–Luciferase activity
Compound
Structure
Concentration of coumaperine derivatives in µg/mL
10µg/mL 20µg/mL 30µg/mL 40µg/mL 50µg/mL
code
71.9%
68.4%
73.9%
79.6%
82.2%
89.8%
85.9%
91.5%
88.2%
92.2%
CP-9
CP
69.7%
57.6%
76.3%
61.7%
77.2%
63.2%
86.9%
69.7%
89.9%
72.2%
CP-38
CP-184
95.5%
96.6%
97.3%
97.7%
97.9%
CP-32

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CP-10
CP-50
43.4%
39.2%
67.5%
52.9%
73.92%
59.2%
78.9%
59.8%
80.1%
62.9%
CP-27
34.1%
70.3%
44.6%
71.4%
53.1%
75.3%
56.9%
78.1%
59.8%
80.1%
CP-158
PIP
67.3%
34.0%
26.4%
67.5%
56.6%
34.0%
85.1%
65.8%
44.8%
86.6%
71.0%
50.7%
90.2%
71.2%
51.8%
PIP-155
CP-102
CP-146
37.2%
56.2%
56.8%
57.2%
59.9%
3.3 Cytotoxic effect of coumaperine derivatives CP-9, CP-32, CP-38 on cell proliferation
To investigate the potential inhibition of coumaperine derivatives on cancer cell proliferation,
they were incubated with L428 and A549 cells in triplicate for 48 h and the treatment was
evaluated using the XTT assay. Control cells represent 100% survival. The average and standard
deviation of three independent experiments are shown (in Figures 3a-b). Coumaperine
derivatives CP-9, CP-32 and CP-38 markedly inhibited cell proliferation in a dose dependent

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manner against L428 cells with the IC50 value of 43.25 µg/ml, 0.39 µg/ml and 16.85 µg/ml
respectively. The IC50 values against A549 cells were found to be at the concentrations of 57.15
µg/ml, 69.1 µg/ml and 63.2 µg/ml respectively (Figures 4a-b).

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Figure 3. Cytotoxic effect of coumaperine derivatives a) CP-9 and 38; b) CP-32 against L428
cell proliferation. L428 cells were incubated for 48 h in triplicate with different concentrations of
coumaperine derivatives. Cell survival was measured by a tetrazolium-formazan XTT assay kit.
Control cells represent 100% survival. The average and standard deviation of three independent
experiments are shown.

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Figure 4. Cytotoxic effect of coumaperine derivatives a) CP-9 and 38; b) CP-32 on A549 cell
proliferation. A549 cells were seeded and incubated for 24 h to form a monolayer. Then the cells
were incubated for 48 hrs in triplicate with different concentrations of coumaperine derivatives.
Cell survival was measured by a tetrazolium-formazan XTT assay kit. Control cells represent
100% survival. The average and standard deviation of three independent experiments are shown.
3.4 Effect of Coumaperine derivatives CP-9, CP-32 and CP-38 on the inhibition of A549 cell
migration
In the cell migration assay, the vehicle (DMSO) treated cells mostly closed the wound after 48 h
incubation, in contrast to coumaperine derivatives treated cells, where wound closure was
prevented (Figure 5a-c) at the concentration of 5µg/mL, 10µg/mL and 5µg/mL of CP-9, CP-32,

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and CP-38 respectively which clearly shows that these compounds inhibited A549 cell migration
in culture.
Figure 5. Wound-scratch assay: Effect of coumaperine derivatives a) CP-9, b) CP-32 and c) CP-
38 on the A549 cell migration. A549 cell monolayers were "scratched" and were grown in the
absence (control) or presence of coumaperine derivatives (2-10 µg/ml) respectively for 48 h.
Coumaperine derivatives prevented the cells from closing the wound (Representative of three
independent experiments).

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3.5 Western Blot Analysis: Monitoring the expression of NF-κB subunits (p50, p65 and RelB) on
coumaperine derivatives (CP-9, CP-32, CP-38) treated cytosolic and nuclear fraction.
The degree of expression of proteins such as p50, p65 and RelB, both in cytosolic and nuclear
extracts of coumaperine derivatives treated and untreated L428 cells were determined by western
blot. The results show that the NF-κB subunits p50 and p65 were diminished mainly in the
nucleus upon coumaperine derivatives CP-9 and CP-38 treatment and the dose response was
observed starting from 2.5 µg/ml to 10 µg/ml (Figures 6a, c) whereas CP-32 did not show
prominent reduction of these proteins (Figure 6b).

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Figure 6. Western-blot analysis monitors the expression of NF-κB proteins, p50, p65 and RelB,
both in cytosolic and nuclear extracts: Coumaperine derivatives a) CP-9, b) CP-32 and c) CP-38
treatment decreases the quantity of nuclear NF-κB subunits. Western blot of the NF-κB subunits
in L428 cells that were incubated with coumaperine derivatives at different concentrations or
with vehicle (DMSO; Control) for 2 h. Cytosolic and Nuclear extracts were prepared and
western blots were run with antibodies against p50, p65 and Rel B. Anti-β-actin and Lamin B
was used as loading control (Representative of at least three independent experiments).
The active compounds inhibited only the classical NF-κB pathway but not the alternative one
(RelB)

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3.6 Confocal Microscopic Studies: Nuclear localization of NF-κB subunits (p50 and p65) in
L428 cells treated with CP-9 and CP-38
Further support to the western blot results comes from nuclear localization studies, the immuno-
cytochemical comparison of treated and untreated L428 cells with CP-9 and CP-38 was carried
out. Interestingly, the coumaperine derivative CP-9 and CP-38 treated cells shows the depletion
of constitutive NF-κB subunits from the nuclei of most treated cells in a dose dependent manner.
The depletion of NF-κB subunits was found to be at the concentrations of 10 µg/ml and 30µg/ml
of CP-9 and CP-38 respectively and was comparable to that of control cells (Figure 7a-b).
Figure 7. Confocal microscopic image: Coumaperine derivatives CP-9 and CP-38 treatment
depletes the nuclear NF-κB subunits a) p50 and b) p65. L-428 cells treated with 10 µg/ml of CP-
9 and 30 µg/mL of CP-38 were cyto-centrifuged (Shandon Cytospin 4) at 900 rpm for 5 min and
fixed in 8% formalin for 20 minutes. Then the cells were incubated with antibodies against p65
and p50, followed by their respective fluorescent secondary antibodies, and the nucleus were
stained with DAPI and the signals were detected using Olympus FV1000-IX81 confocal
microscope.

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Discussion
With the synthesized compounds in hand, we decided to study their cytotoxic effect against
cancer cell lines—Hodgkin’s lymphoma derived L428 cells and lung adenocarcinoma derived
A549 cell and their NF-κB inhibition pathway. Piperine is known to exhibit myriad biological
activities including, anti-inflammatory and anti-tumor activities [14], arises in part due to its
antioxidant activity, correlated to the presence of phenolic moiety, Michael acceptor unit and
conjugated double bonds. Piperlongumine, an another important pepper alkaloid present in Piper
longum L., is found to be selectively toxic to cancer cells in vitro and in vivo [36], also possess
phenolic moiety, Michael acceptor unit (two) and conjugated double bonds. Hence, it is
anticipated that the coumaperine and its derivatives possessing the aforementioned structural
features may also show anti-inflammatory and anti-cancer activity.
Several pepper alkaloids are shown to be a potent NF-κB inhibitor. Piperlongumine and several
of its analogs have been found to effectively inhibited (IC50 = 1.76 µM (0.56 µg/ml), 1.89-6.06
µM (0.6–1.92 µg/ml) respectively) constitutive expression of NF-kB in A549 cells [37]. Further,
piperlongumine effectively inhibited DNA binding activity of NF-κB in A549 cells in
concentration dependent manner with maximum inhibitory activity at 25 µM (7.92 µg/ml) [37].
There are plethora reports in which piperine is shown to inhibit NF-κB activation in several cell
lines. In RAW 264.7 cells, piperine (at 100 µg/ml), significantly reduced the LPS-mediated NF-
κB activation (by ~75%) in a luciferase reporter gene assay [16]. It also significantly inhibited (at
100 µg/ml) IL-β1 induced NF-κB activation (by ~90%) in human OA chondrocytes [22].
The synthesized compounds were initially screened for their NF-κB inhibitory activity using
luciferase reporter gene assay with various concentrations (results summarized in Table 1). The

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simplest coumaperine derivative, CP-9 without any substituted substituent on the aromatic ring
(CP-9), exhibited 71.9% inhibition at 10 µg/ml, introduction of –OH or -OMe group at the para
position (CP and CP-38) lead to marginal decrease in the activity, 68.4% and 69.7% inhibitions
respectively at 10 µg/ml. However, replacing –OMe with –Ocyclopentyl group decreasesd the
activity significantly (69.7 to 57.6%), suggesting a bulkier hydrophobic group is not acceptable
at this position. Incorporation of more than one -OMe group with respect to para methoxy group
viz dimethoxy in the case of CP-10 and trimethoxy groups, as in the case of CP-50 and CP-27
decreases the activity, suggesting, unsubstitution (CP-9) or monosubstitution (CP-38) is optimal
for the desired activity. Replacing electron donating methyl group in CP-38 (-OMe) with
electron withdrawing acetyl (–Ac) group as in CP-158 (-OAc) leads to marginal improvement in
the activity (69.7 to 71.4%). Further, the effect of electron donating substituent was studied
using
-N(CH3)2 group. To our surprise, we observed excellent inhibitory activity (95.5% at 10 µg/ml),
suggesting, a stronger electron donating substituent brings beneficial activity.
Next, we turned our attention to the influence of number of olefinic double bonds, and
incorporated an additional olefinic double bond to CP-38, CP-32 and PIP, which respectively
gave the compounds CP-102, CP-146 and PIP-155. All the three compounds, CP-102, 146 and
PIP-155 (tri-olefin conjugated) showed lesser activity than their di-olefin conjugated counter
parts (Table 1). Thus, increasing the number of olefinic double bond from di to tri-olefin
conjugation decreases the activity. Taken together, unsubstitution/monosubstitution (at para
position) and di-olefin conjugated coumaperine derivatives showed better inhibition of NF-ҡB in
a luciferase reporter gene assay. Based on the above inhibition study, the compounds, CP-9, CP-

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32 and CP-38 were chosen for further biological studies including, inhibition of cell proliferation
(L428 and A549 cells) and cell migration (A549 cells) and NF-ҡB inhibition pathway.
In order to study the effect of coumaperine derivatives on cell proliferation CP-9, CP-32 and CP-
38 were incubated with cancer cell lines L428 and highly resistant A549. All the compounds
inhibited cell proliferation markedly in a dose dependent manner (Figure 3 and 4). Against
L428 cells, CP-32 (IC50: 0.39 µg/ml) exhibited better inhibition of cell proliferation than CP-38
(IC50: 16.85µg/ml) and CP-9 (IC50: 43.25µg/ml). The difference in inhibitory activity may be
correlated to electron donating ability of substituent on the aromatic moiety. CP-32 with stronger
electron donating group –N(CH3)2 showed better inhibitory activity than CP-38 and CP-9 with
relatively weaker electron donating groups, -OCH3 and –H respectively (-N(CH3)2 > -OMe > H).
Against A549 cells, the simple coumaperine derivative, CP-9 (with no substituent on the
aromatic moiety) exhibited better inhibition (IC50: 57.15 µg/ml) of cell proliferation than CP-32
(IC50: 69.1µg/ml) and CP-38 (IC50: 63.2µg/ml) with electron donating substituents -N(CH3)2
and –OMe respectively. Thus, inhibitory activity of coumaperine derivatives, CP-9, CP-32 and
CP-38 against A549 cells is just reverse to L428 cells. In other words, inhibitory activity of
coumaperine derivatives with a weaker electron donating substituent (on the aromatic moiety)
showed better inhibitory activity than coumaperine derivatives with a stronger electron donating
substituent (-H > -OCH3 > -N(CH3)2).
CP-9, CP-32 and CP-38 were then evaluated for inhibition of A549 cell migration. The
coumaperine derivatives were added to the monolayer of A549 Human Lung carcinoma cells and
scratched to study their ability to inhibit cell migration, CP-9 and CP-38 exhibited better
inhibition of cell migration at lower concentration (5 µg/ml) than CP-32 (10 µg/ml) (Figure 5).

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The above results indicate that the coumaperine derivative with a weaker electron donating
substituent (CP-9, -H and CP-38, -OMe), showed better inhibition against A549 cell migration
than a stronger electron donating substituent (CP-32, -N(CH3)2).
Coumaperine derivatives were then studied for inhibition of NF-ҡB proteins of L428 cells. CP-9,
CP-32 and CP-38 were treated with L428 cells and the nuclear and cytoplasmic protein lysates
were prepared and expressions of NF-ҡB subunits were analyzed by western blot. CP-9 and CP-
38 suppress significantly the p50 and p65 subunits both in cytosolic and nuclear fractions at the
dosage of 10 µg/ml, 30 µg/ml respectively (Figure 6a, c). But, CP-32 did not show prominent
reduction of these proteins (Figure 6b). Suggesting that coumaperine derivatives with a weaker
electron donor (-H > -OCH3 > -N(CH3)2) may beneficial for inhibition of NF-ҡB subunits (p50
and p65). In addition to western blot analysis, the inhibition of NF-ҡB subunits (p50 and p65) by
coumaperine derivatives (CP-9, CP-32 and CP-38) was confirmed by nuclear localization using
confocal microscopyic images. The immuno-cytochemical comparison of treated and untreated
L428 cells shows, depletion of NF-κB subunits such as p50 and p65 from the nuclei of most
treated cells in a dose dependent manner (10 µg/ml and 30 µg/ml of CP-9 and CP-38
respectively) and was comparable to that of control cells (confocal microscopic images, Figure
7). Which clearly indicates C-P9 and CP-38 inactivates the NF-ҡB pathway in vitro.
4. Conclusion
Understanding inhibition pathway of NF-ҡB, a key transcription factor often associated with
several diseased states including cancer, by small molecules is crucial for design and
development of efficient inhibitors for biological targets. Coumaperine and its derivatives were
synthesized and investigated for inhibition of NF-ҡB pathway and evaluated for cytotoxicity

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against cancer cell lines, Hodgkin’s lymphoma derived L428 cells and lung adeno-carcinoma
derived A549 cells. Initial Screening against inhibition of NF-ҡB reporter gene (of L428 cells)
showsed, coumaperine derivatives inhibited constitutively expressing NF-ҡB by a reporter gene
assay in a dose dependent manner. The Substituent on the aromatic moiety and number of
olefinic double bond influenced the inhibitory activity. CP-32 with a stronger electron donating
group (-N(CH3)2) showed highest inhibitory activity (at 10 µg/ml) among the coumaperine
derivatives tested. The Introduction of more than one methoxy group (compare CP-10 with 27
and 50) to the aromatic ring and increasing the number of olefinic double bond (from two to
three, compare CP-32, 38 and PIP with CP-146, 102, and PIP-15 respectively) decrease the
inhibitory activity. Based on luciferase assay study, CP-9, 32 and 38 were further evaluated for
inhibition of L428 and A549 cell proliferation cancer cells (L428 and A549) proliferation and
A549 cell migration (A549). They exhibited dose dependent activity. In the Cell proliferation
study shows that coumaperine derivatives exhibited complementary inhibitory pattern against
L428 and A549 cells. CP-32 with a stronger electron donating showed better inhibition (IC50:
0.39 µg/ml) than simple coumaperine derivative CP-9 (with no substituent) (IC50: 43.25 µg/ml)
against L428 cell proliferation. Against A549 cells proliferation, simple coumaperine derivative,
CP-9 showed better inhibition (IC50: 57.15 µg/ml) than CP-32 (IC50: 69.1 µg/ml). The cell
migration study showed revealed that CP-9 and 38 inhibited A549 cell migration at lower
concentration (5 µg/ml) than CP-32 (10 µg/ml). The effect of coumaperine derivatives, CP-9,
32, and 38 on inhibition of NF-ҡB inhibition pathway was studied via western blot and confocal
microscopy. CP-9 and CP-38 suppress NF-ҡB subunits p50 and p65 both in cytosolic and
nuclear fractions in a concentration dependent manner, whereas CP-32 did not show prominent
reduction of these proteins. CP-9 and CP-38 diminish p50 and p65 proteins significantly at the

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dosage of 10 µg/ml and 30 µg/ml respectively. We conclude that coumaperine derivatives, CP-9
and CP-38 strongly inactivate the NF-ҡB pathway in vitro, with a potential to be used as anti-
inflammatory compounds.
Acknowledgements
MR and Dr. RSK gratefully acknowledge Chemistry Division and VIT University Chennai,
India, for financial support and infrastructure facility to perform this work. Thanks extended to
SAIF, VIT University, Vellore for recording GC-MS and NMR for the samples. The PBC
research fellowship extended by the Planning and Budgeting Committee (PBC) of the Council
for Higher Education in Israel, to Dr. NK is gratefully acknowledged.
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