Synthetic analogue of the natural product piperlongumine as a potent inhibitor of breast cancer cell line migration

Article abstract of DOI:10.21577/0103-5053.20160303

Piperlongumine is a natural amide alkaloid isolated from several species of Piper and is described in the literature as selectively cytotoxic to several cancer cell lines. Inhibiting cell migration has gained considerable interest as an approach for discovering antimetastatic agents because this process is fundamental to metastasis. Piperlongumine, selected from cell-based assay screening of NuBBE Database, inhibited the migration of MDA-MB-231 breast cancer cells with an EC₅₀ of 3.0 ± 1.0 μM by the Boyden chamber assay. A series of five analogous compounds based on the structure of piperlongumine were designed, synthesized and evaluated in cell migration and cytotoxicity assays. The analogue designed by molecular simplification ((E)-N-acryloyl-3-(3,4,5-trimethoxyphenyl)acrylamide) was the most active of the series, with an EC₅₀ of 1.5 ± 1 μM. Additionally, this compound was selectively cytotoxic, with a selectivity index (SI) of 4.4.

Full text of DOI:10.21577/0103-5053.20160303

http://dx.doi.org/10.21577/0103-5053.20160303
J. Braz. Chem. Soc., Vol. 28, No. 3, 475-484, 2017.
Printed in Brazil - ©2017 Sociedade Brasileira de Química
0
103 - 5053 $6.00+0.00
Article
Synthetic Analogue of the Natural Product Piperlongumine as a Potent Inhibitor of
Breast Cancer Cell Line Migration
a
b
b
c
Marilia Valli, Wanessa Altei, Ricardo N. dos Santos, Emilio C. de Lucca Jr., Marco A.
c
a
d
e
e
Dessoy, Renan M. Pioli, Fernando Cotinguiba, Xavier Cachet, Sylvie Michel, Maysa
a
c
b
,a
Furlan, Luiz C. Dias, Adriano D. Andricopulo and Vanderlan S. Bolzani*
a
Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais (NuBBE), Departamento
de Química Orgânica, Instituto de Química, Universidade Estadual Paulista (UNESP),
1
4800-060 Araraquara-SP, Brazil
Laboratório de Química Medicinal e Computacional, Centro de Pesquisa e Inovação em
Biodiversidade e Fármacos, Instituto de Física de São Carlos, Universidade de São Paulo (USP),
3563-120 São Carlos-SP, Brazil
b
1
c
Laboratório de Química Orgânica Sintética, Instituto de Química, Universidade Estadual de
Campinas (UNICAMP), 13083-970 Campinas-SP, Brazil
d
Instituto de Pesquisas de Produtos Naturais (IPPN), Centro de Ciências da Saúde, Universidade
Federal do Rio de Janeiro (UFRJ), 21941-902 Rio de Janeiro-RJ, Brazil
e
Laboratoire de Pharmacognosie, UMR 8638 COMETE CNRS, Faculté de Pharmacie de Paris,
Université Paris Descartes, Sorbonne Paris Cité, Paris, France
Piperlongumine is a natural amide alkaloid isolated from several species of Piper and is
described in the literature as selectively cytotoxic to several cancer cell lines. Inhibiting cell
migration has gained considerable interest as an approach for discovering antimetastatic agents
because this process is fundamental to metastasis. Piperlongumine, selected from cell-based assay
screening of NuBBE Database, inhibited the migration of MDA-MB-231 breast cancer cells with
an EC of 3.0 ± 1.0 µM by the Boyden chamber assay. A series of five analogous compounds based
50
on the structure of piperlongumine were designed, synthesized and evaluated in cell migration
and cytotoxicity assays. The analogue designed by molecular simplification ((E)-N-acryloyl-3-
(
3,4,5-trimethoxyphenyl)acrylamide) was the most active of the series, with an EC of 1.5 ± 1 µM.
50
Additionally, this compound was selectively cytotoxic, with a selectivity index (SI) of 4.4.
Keywords: piperlongumine, piplartine, piperamide, cytotoxicity, cell migration inhibition
Introduction
antimitotic agent that disrupts the cell cycle in cancer cells
by stabilizing microtubules, exhibits marked and selective
7
Metastasis is the process by which undifferentiated
cancer cells migrate to other parts of the body. Suppression
of metastasis is an urgent need in cancer treatment, as most
inhibition of tumor cell migration.
1
,2
As extensively reviewed elsewhere, biodiversity in
nature has provided unique chemical scaffolds that have
been used as templates for medicinal chemistry and drug
3
existing drugs only inhibit cell proliferation. Traditionally,
8-13
chemotherapy is based on cytotoxic therapeutic agents
that inhibit proliferation and cause cell death. However,
the strategy of inhibiting cell migration has recently
discovery. The availability of natural compound libraries
is of significant importance for integrating natural products
and medicinal chemistry, especially for the identification of
new bioactive agents and the rational design of compounds
4
gained considerable interest. Several compounds that
1
4
inhibit the process of metastasis have been described to
in drug discovery. Within this context, we selected
the natural product piperlongumine (1), also known as
piplartine, from cell-based assay screening of NuBBE
5
,6
date. Paclitaxel, which was originally discovered as an
15
*e-mail: bolzaniv@iq.unesp.br
Database (NuBBE ) and evaluated its ability to inhibit
DB

4
76
Synthetic Analogue of the Natural Product Piperlongumine as a Potent Inhibitor of Breast Cancer Cell Line Migration
J. Braz. Chem. Soc.
cancer cell migration using biological and target-based
experiments.
activities were further subjected to cytotoxicity assays using
the cancer cell lines MDA-MB-231 and DU-145, as well as
the normal breast cell line (MCF10A), to further evaluate
Piperlongumine (1, Figure 1) is an alkamide isolated
16
31
from several species of Piper (Piperaceae). It is described
their toxicity toward and selectivity for malignant cells.
17
as possessing antifungal properties, cytotoxicity toward
1
8,19
20
several cancer cell lines,
and trypanocidal effects,
Evaluation of piperlongumine activity as an inhibitor of
tumor cell migration
2
1
as well as other biological activities. Furthermore,
piperlongumine selectively induces cell death in cancer
cells but does not reduce viability in normal cells.
2
2
Wound-healing cell assay
This natural compound also demonstrates an interesting
capacity to reduce angiogenesis and inhibit the formation
The wound-healing assay is a simple and effective
in vitro method employed to qualitatively evaluate the
influence of compounds on cell migration. Wounds are
typically created by the removal of a portion of confluent
cells growing in a multiwell plate using a pipette tip; closure
of the uncovered area can then be observed and measured
over time using microscopy or a computer imaging
22,23
of blood vessels in tumor xenografts in mice.
Recent
work suggests that piperlongumine is more effective than
paclitaxel in inhibiting tumor growth and presents excellent
2
2
oral bioavailability. Piperlongumine causes an increase
in reactive oxygen species (ROS) levels in cancer cells
22
29
but not in normal cells, and piperlongumine analogues
system. Positive activity is identified by measuring the
24
also enhanced ROS in PC-3 cells. This activity may be
at least partly due to proteasome inhibition and may be
area of the unclosed wound that remains. Piperlongumine
at 10 µM inhibited 97% of cell migration (Table 1). At
this concentration, its activity was similar to the positive
control colchicine, which inhibited 72% of cell migration
at 1 µM (Table 1).
25,26
responsible for the selective activity of piperlongumine.
Nonetheless, few studies to date have investigated the
27
impact of this compound on cell migration. Accordingly,
we describe an analysis of piperlongumine as an inhibitor
of the migration of breast cancer cell line MDA-MB-231
and an evaluation of its cytotoxicity toward normal and
cancer cell lines. Additionally, we aimed at improving
the biological activity of this compound through the
design, synthesis and biological evaluation of a series of
its derivatives.
Boyden chamber cell assay
Invasion of basement membranes by tumor cells and
the cells’ migration to distant tissues are crucial steps in the
complex multistage process that leads to the formation of a
metastasis. Tumor cells cross basement membranes, invade
the lymphatic or vascular system during dissemination and
then penetrate into a target tissue. The Boyden chamber
assay employs a two-compartment system separated
by a membrane to quantitatively evaluate the effect of
28,30
compounds on the migration of cancer cells in vitro.
Piperlongumine exhibited an EC50 of 3.0 ± 1.0 µM
by the Boyden chamber assay, which is comparable to
that of colchicine, 0.5 ± 0.1 µM, as shown in Table 1.
These results indicate that piperlongumine possesses the
ability to inhibit cell migration with similar potency as
the positive control colchicine (Table 1). Furthermore,
these results are in agreement with those obtained from
wound-healing assays.
Figure 1. Piperlongumine, a natural amide isolated from several species
of Piper (Piperaceae).
Results and Discussion
The study of cell migration and its underlying
mechanisms is of great significance in various fields,
including cancer drug development. Tumor cell migration
is one of the processes necessary for metastasis, and several
approaches have been used to assess the ability of cells to
Table 1. Effects of piperlongumine and colchicine on MDA-MB-231
breast cancer cell migration
a
Wound healing
b
Boyden chamber EC
5
0
Compound
Colchicine
(concentration) /
inhibition
/
µM
28
%
cross tissue barriers. In this work, in vitro cell assays of
2
9
30
a wound-healing and Boyden chamber method were
performed to evaluate the ability of piperlongumine and its
analogues (2-6) to inhibit the migration of MDA-MB-231
cancer cells. Those compounds with clearly observable
70 (1 µM)
0.5 ± 0.1
3.0 ± 1.0
Piperlongumine
97 (10 µM)
a
b
Average of three independent experiments; average of two independent
experiments.

Vol. 28, No. 3, 2017
Valli et al.
477
33
Design and synthesis of compounds using the natural
product piperlongumine as a scaffold
of apoptosis. The designed compounds are presented in
Scheme 1.
A series of synthesized piperlongumine analogues
was recently reported, and it was proposed that the two
reactive electrophilic sites are essential for the toxicity
of these compounds. To enhance the compound’s cell
migration-inhibiting activity, a series of analogues were
designed by taking into account information in the literature
and concepts of molecular simplification and hybridization.
Because the reported substitutions on the aromatic ring of the
The synthesis of piperlongumine using different
3
2,34-37
methods is described in the literature.
One of the
newest and simplest approaches for amide bond formation
consists of the coupling of the NH-lactam (piperidonic
group) with 3,4,5-trimethoxy cinnamoyl chloride in the
3
2
32
presence of a base. Accordingly, the designed compounds
were synthesized in this way from the corresponding
commercially available nitrogen-bearing reagents, as
3
2
3,4,5-trimethoxy cinnamic moiety do not affect the antitumor
described in Scheme 2. The conditions that were able
to enhance the nucleophilicity of the nitrogenous reagent,
including the nature of the solvent, were dependent on
the reactivity of this reagent. A detailed description of the
synthetic methods together with their characterization is
presented in Synthetic methods sub-section.
activity of the compound, we did not modify this part of the
22,32
molecule. Additionally, considering that the double bond
at C12−C13 is a key pharmacophore and that the double
32
bond at C7−C8 plays a significant role in cell toxicity, we
planned modifications of the d-valerolactam ring to study the
effect of this scaffold on cell migration (2-4). Furthermore,
we designed one compound using molecular simplification
by retaining the Michael acceptor and the double bond at
C12−C13, but by removing the lactam (piperidonic)ring (5).
Finally, we used molecular hybridization to design a
hybrid of piperlongumine and chromene (6), the latter of
which is widely known for its cytotoxicity and induction
Biological evaluation of the synthesized compounds
All the synthesized compounds were evaluated by both
the wound-healing and Boyden chamber assays as well as
for their cytotoxicity toward MDA-MB-231, DU-145 and
MCF10A cell lines. The results obtained are presented in
Tables 2 to 4.
Scheme 1. Piperlongumine (1) as the natural product model for compound design.

4
78
Synthetic Analogue of the Natural Product Piperlongumine as a Potent Inhibitor of Breast Cancer Cell Line Migration
J. Braz. Chem. Soc.
Scheme 2. Synthesis of piperlongumine analogues 2-6. Reagents and conditions: (a) (COCl) , DMF (cat.), CH Cl , r.t., 2 h; (b) described in Synthetic
2
2
2
methods sub-section.
Table 2. Effects of piperlongumine analogues (2-6) on the results of
Table 3. Effects of 5 and colchicine on MDA-MB-231 breast cancer
wound-healing (MDA-MB-231 cells) assays
cell migration
a
b
Compound
Inhibition of cell migration at 10 µM / %
Wound healing /
Boyden chamber
EC50 / µM
Compound
Colchicine
(
% inhibition at 10 µM)
2
0
0
70
0.5 ± 0.1
1.5 ± 1.0
3
5
97
4
50
97
0
a
b
Average of three independent experiments; average of two independent
experiments.
5
6
a
Colchicine
70
growth of MDA-MB-231 cells with an EC of 9.0 ± 1.0 µM
50
Piperlongumine
97
and that of normal MCF10A cells with an EC of 40 ± 5 µM.
50
a
Evaluated at 1 µM.
The degree of selectivity can be expressed as the selectivity
index(SI=EC forthenormalbreastcelllineMCF10A/EC
50
50
The analogue designed by molecular simplification (5)
was highly active, inhibiting 97% of MDA-MB-231 cell
migration at 10 µM. Compound 4 presented moderate
activity, with only 50% inhibition of cell migration at
for the cancer cell line MDA-MB-231), which is 4.4 for
compound 5. Piperlongumine has a similar effect, with
an EC50 of 7.0 ± 1.0 µM against MDA-MB-231 breast
cancer cells and an EC of 38 ± 8 µM against MCF10A
50
1
0 µM. The other synthesized compounds presented no
normal breast cells, corresponding to an SI of 5.4 (Table 4).
The cytotoxicity evaluation shows that compound 5 and
piperlongumine have greater cytotoxic effects (EC values)
compared to effects on cell migration, suggesting that their
inhibition of cell migration may not be due to their toxicity.
activity at the concentration range evaluated in this assay.
The most promising designed compound (5) maintains the
biological activity of piperlongumine while possessing a
simpler structure.
50
According to the Boyden chamber assay, compound 5
has an EC of 1.5 ± 1.0 µM, which is comparable to the
Tubulin polymerization assay
5
0
EC of colchicine (0.5 ± 0.1 µM), as presented in Table 3.
Microtubules are essential for the development and
maintenance of cell shape, division and mitosis and
are also involved in cell signalling and the transport of
vesicles, mitochondria and other components inside
5
0
Interestingly, compound 5 displays increased inhibition
capacity compared to the original parent compound
piperlongumine.
Piperlongumine and 5 display selective toxicity toward
the normal breast cell line MCF10A. Compound 5 inhibits the
3
8-44
cells.
Microtubules are formed via a dynamic
process of polymerization and depolymerization of
Table 4. Cytotoxicities of piperlongumine and 5 against MDA-MB-231, DU-145 and MCF10A cell lines
Compound
Doxorubicin
Piperlongumine
5
EC50 MDA-MB-231 / µM
3.0 ± 1.0
EC50 DU-145 / µM
5.5 ± 0.5
EC50 MCF10A / µM
30 ± 8
Selectivity index (SI)
10
5.4
4.4
7.0 ± 1.0
11.0 ± 3.0
38 ± 8
9.0 ± 1.0
12.0 ± 1.0
40 ± 5

Vol. 28, No. 3, 2017
Valli et al.
479
4
2,44,45
heterodimers formed by α- and β-tubulin proteins.
significant structural variability in natural products
and the usefulness of this variability in drug discovery.
Piperlongumine, which was selected among many
natural compounds already identified and available in
Due to their essential role during cellular division processes,
microtubules are among the most common and effective
targets in cancer cells.
Recent studies have reported the ability of aryl-
substituted piperlongumine analogues to inhibit microtubule
NuBBE , inhibited migration of the breast cancer cell line
DB
MDA-MB-231 with an EC of 3 ± 1 µM by the Boyden
50
4
6
polymerization in vitro. However, other independent
studies of piperlongumine and similar derivatives have
demonstrated substantial anti-inflammatory activity for
this class of compounds. Based on these facts and the
interesting potential of microtubules as an anticancer
target, we performed fluorescence-based in vitro studies
to evaluate the ability of piperlongumine to inhibit
microtubule formation and to characterize this system as
a possible molecular mechanism explaining the activity of
this molecule and its derivatives.
chamber assay. Additionally, we designed and synthesized
five analogues of piperlongumine through molecular
simplification. Among these compounds, 5 was the most
active, with an EC of 1.5 ± 1 µM against breast cancer
50
cell MDA-MB-231 migration by the Boyden chamber
assay. Additionally, we evaluated the cytotoxicity of these
compounds, and both piperlongumine and 5 presented
high selectivity for tumor cells, with a selectivity index
of 4.44 and 5.42, respectively. Furthermore, taking into
account the reported activities of piperlongumine as an anti-
inflammatory agent and inhibitor of microtubule activity,
we evaluated the ability of this compound to modulate
tubulin polymerization in vitro. The results suggest that
piperlongumine does not interact with microtubules and
that its activity is likely related to another molecular
mechanism.
The evaluation of microtubule polymerization in the
presence of different concentrations of piperlongumine is
shown in Figure 2.
Experimental
Isolation of piperlongumine (1)
The fruits of Piper tuberculatum were harvested from
a specimen growing under greenhouse conditions at the
Institute of Chemistry (UNESP, Araraquara, São Paulo
State, Brazil) in January of 2012. The botanical material
was properly identified by Dr Guillermo E. D. Paredes
from the University Pedro Ruiz Gallo in Peru. A voucher
specimen (K-167) was deposited in the herbarium of the
Institute of Botany of São Paulo in São Paulo, Brazil. The
dried fruits (803 g) were extracted by maceration with
dichloromethane:methanol (2:1, 3 × 1200 mL) under
ultrasonication. The residue was concentrated under
vacuum to obtain 48.90 g of extract (6.1%). The whole
extract was first fractioned under low pressure by normal-
phase column chromatography with silica gel (70 mesh) as
the stationary phase (63 µM, 300 × 60 mm). The solvent
system was a mixture of (A) n-hexane and (B) ethyl
acetate in a gradient from 5 to 50% B, which afforded
11 fractions. Fraction 3 formed a precipitate, which was
Figure 2. Fluorescence-based microtubule polymerization assays in
the presence of different concentrations of piperlongumine (from 1 to
200 µM). DMSO and colchicine at 10 µM were used as negative and
positive controls, respectively, for polymerization inhibition. The data
correspond to the average value of three independent replicates.
Although colchicine completely inhibited microtubule
formation at a concentration of 10 µM, piperlongumine did
not affect the formation of microtubules in vitro over a range
of 1 to 200 µM. These results indicate that piperlongumine
does not interact with tubulin and is unable to modulate
microtubule polymerization. Accordingly, the findings
suggest a different mechanism underlying the anticancer
activity of piperlongumine and its derivatives, which may
filtered and washed with MeOH in an ice bath to furnish
47,48
1
be related to anti-inflammatory effects.
piperlongumine (1, 1.19 g). H NMR (600 MHz, CDCl )
3
d (ppm) 2.43 (2H, m, J 6.3, 4.3, 1.8 Hz), 3.82 (3H, s), 3.84
(6H, s), 3.99 (2H, t, J 6.5 Hz), 5.99 (1H, dt, J 9.7, 1.7 Hz),
Conclusions
6
.75 (2H, s), 6.89 (1H, dt, J 9.4, 4.1 Hz), 7.37 (1H, d,
This work represents an interesting example of the
J 15.5 Hz), 7.62 (1H, d, J 15.5 Hz).

4
80
Synthetic Analogue of the Natural Product Piperlongumine as a Potent Inhibitor of Breast Cancer Cell Line Migration
J. Braz. Chem. Soc.
Cell assays
Cell cultures
in the insert was removed along with the cells that had not
migrated to the bottom of the membrane. The cells that
had migrated to the lower portion of the membrane were
fixed and stained with 1% aqueous toluidine blue in borax
for 5 min. The membranes were cut using a surgical lancet
and placed on a slide, and the migrated cells were counted
using a Nikon Eclipse E600 optical microscope (Nikon
Instruments Inc., Melville, NY, USA) and NIS-Elements
Image processing software. The assays were performed in
duplicate using colchicine as a positive control and DMSO
as a negative control. The EC was determined from the
curve of the log concentration versus the percent inhibition.
The curves were plotted using data obtained for six different
concentrations of the compounds tested in duplicate. The
curves were fitted using the software Sigmaplot 11.0 by the
method of nonlinear regression - dynamic fitting.
MDA-MB-231 human breast cancer cells were cultured
at 37 °C and 5% CO in Leibovitz’s medium, and MCF-7
human breast cancer cells were cultured in DMEM
2
(
Dulbecco’s modified Eagle’s medium, Cultilab) at 37 °C
and 5% CO . DU-145 human prostate tumor cells were
2
cultured under the same conditions using RPMI (Roswell
Park Memorial Institute medium). The cells were stored in
liquid nitrogen in culture medium containing 5% DMSO
and 20% foetal bovine serum (FBS, sterile, Cultilab).
50
Wound-healing assay
A wound-healing assay was performed to qualitatively
assess the potential of the compounds to inhibit cell
29
5
migration. Cells were cultured in 24-well plates (1 × 10 cells
per well) containing 1 mL of culture medium and 10% FBS.
After the cells reached confluence, the culture medium was
removed, and a wound was generated in the cell monolayer
with the aid of a sterile pipette tip. The test compounds
were dissolved in DMSO, diluted in the culture medium
and added to each well (final DMSO concentration = 0.1%).
The system was photographed using a Nikon Eclipse TS100
inverted microscope (4× magnification, Nikon Instruments
Inc., Melville, NY, USA); images were taken at 0 h and
Cytotoxicity assay
A cytotoxicity evaluation was performed to test the
toxicity of the compounds toward the cancer cell lines
MDA-MB- 231 and DU-145. The assay was performed
3
in 96-well plates to which approximately 5 × 10 cells
per well were added. After checking cell adhesion (at
approximately 24 hours), piplartine was dissolved in
DMSO in a concentration of 100 mM (stock solution).
For the assay, compounds were added into wells in serial
dilutions (100-0.04 µM). The plate was incubated for 24 h,
after which 20 µL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) was added to each well. After 4 h of incubation, the
absorbance was measured at 490 nm using a SpectraMax Plus
384 - Absorbance Microplate Reader (Molecular Devices,
Sunnyvale, USA). The plate was maintained at 24 °C, and
the assays were performed in triplicate using doxorubicin
after 22 h of incubation at 37 °C and 5% CO . The percent
2
inhibition shown by the test compound was determined by
the difference in the wound area at 0 and 22 h. The extent
of the area was measured using NIS - Elements software.
The assays were performed in triplicate, with colchicine as
29
a positive control and DMSO as a negative control.
Boyden chamber assay
31
The Boyden chamber assay was performed to
quantitatively evaluate the effect of the compounds on cell
as a positive control and DMSO as a negative control.
2
8,30
migration.
This assay utilizes a hollow plastic chamber
Tubulin polymerization assay (fluorescence based)
that is sealed at one end with a porous membrane. This
chamber/insert is suspended over a larger well that contains
medium and a chemoattractant. Cells are placed inside the
insert and allowed to migrate through the pores from one
side of the membrane to the other. Migratory cells are then
stained and counted. The assays were performed in 24-well
A fluorescence-based assay was applied (Cytoskeleton
Inc., Denver, CO, USA) to determine the EC of effects
50
49-52
on tubulin polymerization.
Microtubule-associated, protein-rich tubulin protein
-1
(2 mg mL , porcine, cytoskeleton) in a buffer containing
80 mM piperazine-N,N’-bis[2-ethanesulfonic acid]
sequisodium salt (PIPES) at pH 6.9, 2.0 mM magnesium
chloride, 0.5 mM ethylene glycol-bis(β-amino-ethyl ether)
N,N,N’,N’-tetra-acetic acid (EGTA), 1.0 mM guanosine
triphosphate (GTP) and 10% glycerol was placed in
each well of a 96-well plate (OptiPlate F -1536 - Black;
PerkinElmer Inc., Waltham, MA, USA). For each assay,
45 µL of the tubulin solution was incubated with 5 µL of
the compound at a 10-fold concentration and 10% dimethyl
TM
plates (BD BioCoat Migration/Invasion Chambers) with
inserts of 6.5 mm diameter and 8 µM pore size. To the inner
4
part of the insert (upper chamber), 4 × 10 cells/300 µL
culture medium (without FBS) and the test compound were
added; to the lower compartment (well of the plate), 700 µL
of culture medium supplemented with 10% FBS and the
test compound were added. This system was incubated for
6
h at 37 °C and 5% CO . After incubation, the medium
2

Vol. 28, No. 3, 2017
Valli et al.
481
sulfoxide (DMSO), resulting in a final solution with the
(200 mg, 0.84 mmol) in CH Cl (8.0 mL) under a nitrogen
2
2
4
9
desired concentration of 1% DMSO. All assays were
performed in triplicate, with 1% DMSO as the negative
control and 10 µM colchicine as the positive control.
Using an excitation wavelength of 360 nm, the rate of
polymerization was monitored at 37 °C by measuring the
fluorescence emission at 440 nm with a Victor 3 fluorimeter
plate reader and Wallac 1420 Multilabel Plate Counter
software (PerkinElmer Inc., Waltham, MA, USA).
atmosphere was added oxalyl chloride (358 µL, 4.2 mmol)
and a catalytic amount of dimethylformamide (DMF). The
reaction was stirred at room temperature for 2 hours. The
residue, compound 7, a yellow solid, was dried and used
32
in the next step without further purification.
1-((E)-3-(3,4,5-Trimethoxyphenyl)acryloyl)-1H-pyrrole-2,5-
dione (2)
CH Cl (8 mL), triethylamine (352 µL, 2.52 mmol), and
2
2
Materials and instruments
maleimide (96 mg, 1 mmol) were added to 7 (0.84 mmol).
The reaction mixture was stirred at room temperature
overnight and then quenched by addition of a saturated
aqueous ammonium chloride solution. The product was
extracted with CH Cl ; the combined extracts were dried
Reagents were purchased from Sigma-Aldrich,
Acros Organics, or Fluka and were used without further
purification; solvents were purified according to standard
2
2
53
methods. Reaction progress was monitored by thin layer
chromatography (TLC) on aluminium plates pre-coated
with silica gel F254 layers (0.2 mm thickness; Whatman)
and visualized under UV light (254 and 366 nm). Column
chromatography was carried out over silica gel (particle
over MgSO and filtered, and the solvent was removed
4
3
2
under reduced pressure. The product was purified by
column chromatography using cyclohexane:ethyl acetate
(7:3) for elution to afford compound 2 (121 mg, 45%
1
yield) as a pure reddish-brown solid. H NMR (400 MHz,
1
size of 35-70 µM; Acros Organics). H NMR spectra were
CDCl ) d (ppm) 3.94 (s, 3H), 3.95 (s, 6H), 6.88 (s,
3
recorded using Bruker AC 300 (300 MHz), Bruker Avance
2H), 6.92 (s, 2H), 7.23 (d, 1H, J 15.5 Hz), 7.91 (d, 1H,
1
3
4
00 (400 MHz) or Bruker Avance III 600 (600 MHz)
J 15.5 Hz); C NMR (75 MHz, CDCl ) d 56.2, 61.0,
3
spectrometers; spectra were referenced against the internal
106.0, 117.9, 129.6, 135.4, 141.0*, 148.4, 153.5, 162.5*,
167.9; Figures S1-S3 in Supplementary Information;
standard TMS (dTMS 0.00) or against the residual solvent
1
+
resonances of CDCl (d 7.26 H); the multiplicity (s,
HRMS (ESI-TOF) m/z: 318.0977 [M + H] (calcd. for
3
+
singlet; d, doublet; t, triplet; q, quartet; dd, doublet of
C H NO , 318.0972), 340.0796 [M + Na] (calcd. for
1
6
16
6
13
doublet) and coupling constant (Hz) are noted. C NMR
spectra were recorded using Bruker AC 300 (75 MHz) or
C H NNaO , 340.0792).
16 15 6
13
Bruker Avance III 600 (150 MHz) spectrometers. C NMR
2-((E)-3-(3,4,5-Trimethoxyphenyl)acryloyl)isoindoline-1,3-
dione (3)
chemical shifts are reported in parts per million relative to
13
the solvent. All C NMR spectra were determined with
broadband decoupling. When necessary, the structures of
the compounds were verified and the signals unambiguously
CH Cl (8 mL), triethylamine (352 µL, 2.52 mmol),
2
2
and phthalimide (147 mg, 1 mmol) were added to 7
(0.84 mmol). The reaction mixture was stirred at room
temperature overnight and then quenched by addition
of a saturated aqueous ammonium chloride solution.
The product was extracted with CH Cl ; the combined
1
1
assigned by 2D NMR techniques: H- H COSY (correlation
1
1
spectroscopy), H- H NOESY (nuclear Overhauser effect
1
3
1
spectroscopy), C- H HMQC (heteronuclear multiple
2
2
13
1
quantum correlation), and C- H HMBC (heteronuclear
extracts were dried over MgSO and filtered, and the
4
13
32
multiple bond correlation). Some C values were assigned
using evidence from HMBC and are marked with an asterisk
in Synthetic methods sub-section. Electrospray ionization
time-of-flight (ESI-TOF) high-resolution mass spectra
solvent was removed under reduced pressure. The
residue was purified by column chromatography using
cyclohexane:ethyl acetate (7:3) for elution to afford
compound 3 (121 mg, 40% yield) as a pure light yellow
1
(
(
HRMS)weremeasuredusingaMicrOTOFQIIspectrometer
Bruker Daltonics, Billerica, MA, USA) operating in positive
ion mode. ChemDraw Ultra 8.0 was used to calculate the
solid. H NMR (600 MHz, CDCl ) d 3.90 (s, 3H), 3.92
3
(s, 6H), 6.86 (s, 2H), 7.28 (d, 1H, J 15.5 Hz), 7.86 (dd,
2H, J 5.5, 3.0 Hz), 7.90 (d, 1H, J 15.5 Hz), 8.00 (dd, 2H,
+
+
13
values of the pseudo-molecular ions [M + H] and [M + Na] .
J 5.5, 3.0 Hz); C NMR (150 MHz, CDCl ) d 56.2, 61.0,
3
1
06.0, 118.7, 124.4, 129.7, 131.4, 135.4, 141.0, 147.9,
Synthetic methods
153.5, 163.7, 165.7; Figures S4-S6 in Supplementary
+
Information; HRMS (ESI-TOF) m/z: 368.1132 [M + H]
+
(E)-3-(3,4,5-Trimethoxyphenyl)acryloyl chloride (7)
(calcd. for C H NO , 368.1129), 390.0948 [M + Na]
2
0
18
6
To a solution of (E)-3,4,5-trimethoxycinnamic acid
(calcd. for C H NNaO , 390.0948).
2
0
17
6

4
82
Synthetic Analogue of the Natural Product Piperlongumine as a Potent Inhibitor of Breast Cancer Cell Line Migration
J. Braz. Chem. Soc.
(
E)-1-(1H-Indol-1-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-
314.0999). The synthesis of this compound by a similar
method was recently described.
54
1
-one (4)
CH Cl (4 mL), sodium hydride (33 mg, 0.84 mmol),
2
2
and indole (59 mg, 0.5 mmol) were added to 7 (0.42 mmol).
The reaction mixture was stirred at room temperature
overnight and then quenched by addition of a saturated
aqueous ammonium chloride solution. The product was
extracted with CH Cl ; the combined extracts were dried
(2E)-3-(3,4,5-Trimethoxyphenyl)-N-(2-oxo-2H-chromen-6-
yl)acrylamide (6)
CH Cl (1 mL), triethylamine (352 µL, 2.52 mmol), and
2
2
6-amino-chromen-2-one (161 mg, 1 mmol) were added to
7 (0.84 mmol). The reaction mixture was stirred at room
temperature for 2 hours and then quenched by addition of a
saturated aqueous ammonium chloride solution. The product
was extracted with ethyl acetate; the combined extracts were
2
2
over MgSO and filtered, and the solvent was removed
4
3
2
under reduced pressure. The residue was purified by
column chromatography using cyclohexane:ethyl acetate
(
7:3) for elution to afford compound 4 (58 mg, 41%
dried over MgSO and filtered, and the solvent removed
under reduced pressure. The residue was purified by column
4
1
32
yield) as a pure reddish-brown solid. H NMR (400 MHz,
CDCl ) d 3.96 (s, 3H), 3.99 (s, 6H), 6.74 (d, 1H, J 3.7 Hz),
chromatography using an n-hexane:ethyl acetate gradient
from 7:3 to 0:1 for elution to afford compound 6 (80 mg,
21% yield) as a pure light yellow solid. H NMR (600 MHz,
3
6
7
7
1
1
1
.89 (s, 2H), 7.16 (d, 1H, J 15.3 Hz), 7.32 (dd, 1H, J 8.0,
.8 Hz), 7.41 (dd, 1H, J 8.2, 8.0 Hz), 7.63 (d, 1H, J 7.8 Hz),
.69 (d, 1H, J 3.7 Hz), 7.94 (d, 1H, J 15.3 Hz), 8.57 (d,
32 1
CDCl ) d 3.89 (s, 3H), 3.91 (s, 6H), 6.47 (d, 1H, J 9.5 Hz),
3
1
3
H, J 8.2 Hz); C NMR (75 MHz, CDCl ) d 56.3, 61.0,
6.54 (d, 1H, J 15.3 Hz), 6.78 (s, 2H), 7.30 (d, 1H, J 8.9 Hz),
7.51 (dd, 1H, J 8.9, 2.5 Hz), 7.71 (d, 1H, J 15.3 Hz), 7.73
(d, 1H, J 9.5 Hz), 7.81 (s, 1H), 8.18 (bs, 1H; obs.: broad
3
05.6, 109.2, 116.4, 116.9, 120.9, 123.8, 124.6, 125.1,
29.9*, 130.5*, 135.8*, 140.5*, 146.7, 153.5, 164.2*;
13
Figures S7-S9 in Supplementary Information; HRMS
signal); C NMR (150 MHz, CDCl ) d 56.2, 61.0, 105.3,
3
+
(
3
3
ESI-TOF) m/z: 338.1386 [M + H] (calcd. for C H NO ,
117.2, 117.3, 118.5, 119.1, 119.6, 123.5, 129.9, 134.6, 140.1,
142.9, 143.5, 150.5, 153.5, 160.8, 164.1; Figures S13-S15
in Supplementary Information; HRMS (ESI-TOF)
20
20
4
+
38.1387), 360.1219 [M + Na] (calcd. for C H NNaO ,
20 19 4
60.1206).
+
m/z: 382.1285 [M + H] (calcd. for C H NO , 382.1285),
21
20
6
+
(E)-N-Acryloyl-3-(3,4,5-trimethoxyphenyl)acrylamide (5)
404.1097 [M + Na] (calcd. for C H NNaO , 404.1105).
21 19 6
To a solution of acrylamide (710 mg, 10 mmol) in THF
(
50 mL) at −78 °C, n-BuLi (4.8 mL, 12 mmol; 2.5 M in
Supplementary Information
n-hexane) was added dropwise. The reaction mixture was
warmed to room temperature, and the reaction proceeded
for 7 hours. The reaction mixture was cooled to −78 °C,
and a solution of 7 (20 mmol) in THF (28 mL) was added
dropwise. The reaction was warmed to room temperature,
allowed to proceed for 15 hours and then quenched by
addition of a saturated aqueous ammonium chloride
solution. The reaction mixture was extracted with diethyl
ether; the combined organic extracts were washed with a
saturated solution of sodium chloride, dried over MgSO4,
filtered and evaporated to dryness under vacuum. The
residue was purified by successive recrystallization from
ethyl ether (30 mL) and subsequently with ethanol (70 mL)
to afford compound 5 (920 mg, 30% yield) as a pure yellow
Supplementary information (NMR spectra) is available
free of charge at http://jbcs.sbq.org.br as PDF file.
Acknowledgments
This work was supported by the State of São Paulo
Research Foundation (FAPESP, Fundação de Amparo à
Pesquisa do Estado de São Paulo), grant No. 2013/07600-3;
Biota-FAPESP, grant No. 2003/02176-7; SISBIOTA-
CNPq-FAPESP, grant No. 2010/52327-5; Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
and Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq). The researchers also acknowledge
scholarships, grant No. 2010/17329-7, No. 2010/06741-4
and No. 2007/56140-4 from the São Paulo Research
Foundation (FAPESP).
37 1
solid. H NMR (600 MHz, CDCl ) d 3.89 (s, 3H), 3.90 (s,
3
6
1
H), 5.94 (dd, 1H, J 10.5, 1.2 Hz), 6.56 (dd, 1H, J, 16.6,
.2 Hz), 6.77 (dd, 1H, J 16.6, 10.5 Hz), 6.83 (s, 2H), 7.31
(
d, 1H, J 15.6 Hz), 7.79 (d, 1H, J 15.6 Hz), 9.17 (s, 1H);
1
3
C NMR (150 MHz, CDCl ) d 56.2, 61.0, 105.8, 118.6,
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Submitted: October 4, 2016
Published online: November 17, 2016