Design and synthesis of cenocladamide analogues and their evaluation against breast cancer cell lines

Article abstract of DOI:10.1039/c6md00577b

This work describes the total synthesis of the alkaloid cenocladamide and a concise library of nine structural analogues aiming at their evaluation against the breast cancer cell line MDA-MB-231. The most promising compound (3; IC₅₀ = 6.6 μM) was also evaluated in a panel of seven breast cancer cell lines and two non-tumorigenic cell lines. We further conducted an initial investigation on the mechanism of action of analogue 3, which lacks the endocyclic double bond when compared to cenocladamide. The present study presents the discovery of a cenocladamide analogue with interesting cytotoxic activity, which could be useful for further optimization towards new chemotherapeutic agents for breast cancer treatment.

Full text of DOI:10.1039/c6md00577b

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RESEARCH ARTICLE
Design and synthesis of cenocladamide analogues
and their evaluation against breast cancer cell
lines†‡
Cite this: DOI: 10.1039/c6md00577b
Carla C. F. Santos,§a Luciana S. Paradela,§b Luiz F. T. Novaes,§a
b
a
*
*
Sandra M. G. Dias and Julio C. Pastre
This work describes the total synthesis of the alkaloid cenocladamide and a concise library of nine struc-
tural analogues aiming at their evaluation against the breast cancer cell line MDA-MB-231. The most prom-
ising compound (3; IC50 = 6.6 μM) was also evaluated in a panel of seven breast cancer cell lines and
two non-tumorigenic cell lines. We further conducted an initial investigation on the mechanism of action
of analogue 3, which lacks the endocyclic double bond when compared to cenocladamide. The present
study presents the discovery of a cenocladamide analogue with interesting cytotoxic activity, which could
be useful for further optimization towards new chemotherapeutic agents for breast cancer treatment.
Received 14th October 2016,
Accepted 21st November 2016
DOI: 10.1039/c6md00577b
www.rsc.org/medchemcomm
inspired by natural compounds.7 When allied with organic
synthesis, the potential to obtain more powerful derivatives
Introduction
Breast cancer is the second most common cancer worldwide
after lung cancer, the fifth most common cause of death re-
lated to cancer, and the leading cause of cancer death in
women. The global burden of breast cancer exceeds all other
types of malignancies and the incidence rates of breast cancer
are increasing.1 Although recent advances have been made in
tumor detection and treatment, breast tumors are still very
challenging in view of their heterogeneity.2,3
Breast cancer treatment includes several strategies from
hormone therapy drugs, to surgery, radio- and chemother-
apy.4 However, resistance of tumor cells to current drugs
available for breast cancer treatment highlights the need for
new chemotherapeutic agents.5
that are not available from natural sources can be leveraged.
Total synthesis still has an import role in the continuing ex-
ploitation of natural products as lead compounds for drug
discovery, as it often represents the most efficient means,
even for complex structures, of providing sufficient quantities
of materials for meaningful biological profile assessments.8
On the other hand, modern and less costly strategies have
been used by organic and medicinal chemists to generate de-
rivatives such as molecular simplification9,10 and diverted to-
tal synthesis, where selected portions of the prototype mole-
cule are modified to understand their importance for the
expression of a certain biological activity of a particular class
of natural products.11,12
In this context, compounds of natural origin play a key
role in the development of novel therapeutics mainly because
of their tremendous structural diversity, serving as unique
and privileged scaffolds for drug discovery.6 Indeed, natural
products have a profound impact in several areas, including
cancer, and it is estimated that ca. 80% of the antitumor
agents introduced in the past decades are of natural origin or
In this study, we employed both strategies to produce a
concise series of analogues of the alkaloid cenocladamide.
Since its isolation in 2000 by Dodson and co-workers from
the leaves of Piper cenocladum,13,14 only studies of its effect
on
certain
herbivores
have
been
conducted.15–17
Cenocladamide is structurally similar to piplartine (also
known as piperlongumine, see Fig. 1), a compound that
selectively kills cancer cells while sparing primary normal
cells, causing an increase in reactive oxygen species (ROS)
a Departamento de Química Orgânica Instituto de Química, Universidade Estadual
de Campinas (Unicamp), CP 6154, CEP 13084-971, Campinas, SP, Brazil.
E-mail: juliopastre@iqm.unicamp.br
b Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em
Energia e Materiais (CNPEM), CEP 13083-100, Campinas, SP, Brazil.
E-mail: sandra.dias@lnbio.cnpem.br
† The authors declare no competing interests.
‡ Electronic supplementary information (ESI) available: Experimental procedures
and NMR spectra. See DOI: 10.1039/c6md00577b
§ These authors contributed equally to this work.
Fig. 1 Chemical structures of cenocladamide and piplartine.
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and decrease in glutathione (GSH).18,19 Despite this structural
similarity, to the best of our knowledge, no study concerning
the evaluation of cenocladamide's potential as a new pharma-
cological agent has been reported so far.
a key pharmacophore, with the exo double bond also playing
a significant role in determining cytotoxicity.21
Results and discussion
Herein, we report the total synthesis of cenocladamide
and a series of analogues aiming at the evaluation of their
effect on several breast tumour cell lines (e.g. MDA-MB-231,
SKBR3, HS578T, HCC38, MDA-MB-468, BT549, T47D and
MCF-7) in comparison with non-tumorigenic breast cell lines
(i.e. MCF10A and iHMEC). Some structural requirements for
the expression of the cytotoxic activity were identified and
preliminary studies were performed in an attempt to unveil
the mechanism responsible for cell death.
Chemistry
Firstly, the 2,3-dihydro-4-pyridone core present in compounds
1–2 and 7–10 was constructed based on the protocols
reported by Brimioulle and Bach.22 Thus, 4-methoxypyridine
(11) was treated with benzyl chloroformate, followed by a re-
duction of the acyl pyridinium intermediate to obtain N-Cbz-
2,3-dihydro-4-pyridone 12 in 68% yield. Next, hydrogenolysis
under standard conditions removed the nitrogen protecting
group to generate 2,3-dihydro-4-pyridone 13 in a quantitative
yield (Scheme 1). Then, 2,3-dihydro-4-pyridone 13 was acyl-
ated with acetic anhydride or acyl chlorides, which were pre-
pared from the corresponding carboxylic acids upon treat-
ment with oxalyl chloride and DMF, furnishing analogues 2,
7–10 and O-acetyl cenocladamide 14. Finally, deprotection of
phenolic acetate with ammonium acetate afforded
cenocladamide (1),23 whose spectroscopic data were identical
to those reported for the natural sample confirming the pro-
posed structure.13
Analogues 5 and 6 were prepared by a coupling reaction
between piperidine (15) and the corresponding carboxylic
acids, employing carbodiimides as carboxyl activators. A simi-
lar procedure was performed using amine 16 to obtain am-
ides 17 and 18, which were then subjected to acidic condi-
tions in order to promote the hydrolysis of the ketal
protecting group thus releasing the ketone functionality pres-
ent in analogues 3 and 4 (Scheme 2).
Therefore, the design of cenocladamide analogues was
planned to evaluate the importance of the structural features
of this natural product related to their biological activity re-
garding cancer cells (Fig. 2). First, we investigated the effect
of replacing a phenolic hydroxyl by a methoxy group (com-
pound 2), to mimic the side chain of piplartine. The influ-
ence of methoxy and hydroxyl groups on the cytotoxic activity
has been extensively investigated and there are several natu-
ral compounds with different degrees of methoxylation and/
or hydroxylation with interesting antitumor properties, such
as resveratrol, combretastatin, curcumin, etoposide, and
others.20 Further, the role of this replacement was also evalu-
ated in compounds 4 and 6, and these groups were removed
in compound 9. Additionally, we explored the modification of
the nitrogenated heterocycle, removing the endocyclic double
bond (compounds 3 and 4), and also removing the double
bond and the ketone group (compounds 5 and 6). Next, a
molecular simplification strategy was employed to verify the
importance of the side chain (compounds 7–8). Thus, the
N-cinnamoyl group was replaced with simple acetyl and ben-
zoyl groups. Finally, removal of the exocyclic double bond
resulted in analogue 10, which could allow the evaluation of
the importance of this electrophilic site for selectivity and cy-
totoxicity. Adams et al. synthesized and tested an array of
piplartine analogues and identified the endo double bond as
Study of the effect of cenocladamide analogues on breast
cancer cell lines
The development of clinical metastasis remains a significant
cause of morbidity and mortality from the disease, and tu-
mor invasion plays a crucial role in this process.24 Therefore,
we began our study using the human metastatic breast
Fig. 2 Design of cenocladamide analogues.
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Scheme 1 Synthesis of cenocladamide (1) and analogues 2, 7–10.
cancer cell line MDA-MB-231,25 which is highly invasive in
comparison with the frequently studied MCF-7 breast cancer
cell line. Cell proliferation/death was evaluated after 3 days of
treatment with cenocladamide and its analogues at a concen-
tration of 20 μM. While most of the compounds did not af-
fect or affected marginally cell proliferation, analogues 9 and
2 inhibited proliferation by ∼50%. Compound 3, on the
other hand, was the most effective compound, killing around
60% of the cells, indicating that the presence of the non-
conjugated ketone and the phenolic group plays a key role in
the cytotoxicity of this family of compounds (Fig. 3A). The
IC50 of 3 in cell proliferation of MDA-MB-231 was 6.6. μM
(Fig. 3B).
From this preliminary screening, it is clear that the exocy-
clic double bond is more important for the cytotoxic effect
displayed by this family of compounds, since compound 2
inhibited cell growth by ∼50% while the saturated derivative
10 did not affect cell proliferation. As indicated for other nat-
ural products and analogues, the α,β-unsaturated scaffold
may act as a Michael acceptor for cysteine residues or other
nucleophiles in biological systems.19,26 On the other hand, re-
moval of the endocyclic double bond resulted in the most cy-
totoxic analogue (3) of this series, which killed ∼60% of the
cells. This effect becomes more evident when we compare
the cytotoxic activity displayed by analogue 3 and the natural
compound cenocladamide (1). This modification may also
change the electronics of the whole molecule since the nitro-
gen lone pair is no longer involved in cross-conjugation and
could affect the strength of the HBA and HBD profile of the
carbonyl and the phenolic hydroxyl groups, respectively. The
importance of the phenolic hydroxyl group as a HBD is also
emphasized if one compares compounds 3 and 4, since
methylation leads to only a marginal effect on cell prolifera-
tion induced by compound 4. Finally, the role of the substitu-
ents at the aromatic ring is less evident: while methylation
ablates the activity of cenocladamide (1), compound 9 lacking
any substituent is able to inhibit cell growth by ∼50% com-
pared to the control. To shed new light on the importance of
the structural requirements for the cytotoxicity of this family
of compounds, new analogues are still needed.
Considering that different cell lines display different sen-
sitivities toward the same cytotoxic agent, we then deter-
mined the overall impact of the treatment with compound 3
over seven other breast cancer cell lines (e.g. SKBR3, HS578T,
HCC38, MDA-MB-468, BT549, T47D and MCF-7), as well as a
non-tumorigenic cell line derived from human fibrocystic
Scheme 2 Synthesis of analogues 3–6: (a) for R = H, DCC, THF, r.t.; (b)
for R = Me, EDCI, Et3N, DMAP, CH2Cl2, r.t.
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Fig. 3 Compound series affected growth/survival of breast cancer cell lines. (A) The compound series was tested against the MDA-MB-231 breast
cancer cell line at 20 μM for 72 hours. (B) Compound 3 dose response in cell proliferation of MDA-MB-231 cells, after 72 hours of treatment. (C)
Eight breast cancer cell lines and 2 non-tumorigenic breast cells were treated with 3 or 5 at 20 μM, or doxorubicin at 1 μM for 72 hours.
mammary tissue (MCF10A)27 and an in-house immortalized
breast epithelial cell line by expression of hTert (iHMEC). For
comparison, we used the chemotherapeutic doxorubicin, con-
sidered the most effective agent in the treatment of breast
cancer patients.28 Compound 3 induced cell death on all
tested cell lines, an effect that was not seen with compound
5, an analogue that lacks the carbonyl group at the heterocy-
clic ring. Compound 3 killed all cell types with an efficacy
varying from 15% to more than 80% of death, while doxoru-
bicin inhibited proliferation from 0.2% to around 50%
amongst the tested cell lines (with the exception of iHMEC
that presented 23.9% of cell death) (Fig. 3C). The observed ef-
fect of doxorubicin on the cell lines is in agreement with the
already documented cytostatic action of this drug.29 Although
compound 3 also caused cell death to the non-tumorigenic
MCF10A and iHMEC cells, they were amongst the least af-
fected cells, pointing out a slight selectivity for tumor cell
lines.
Hoechst and MitoTracker Deep Red, respectively. Compound
3, but not compound 5, increased nuclear fragmentation and
mitochondria staining, which indicates potential increased
mitochondrial biogenesis (Fig. 4A). The effects on nuclei
morphology may indicate pyknosis followed by karyorrhexis,
the irreversible condensation of chromatin and nuclear frag-
mentation, respectively, happening in cells undergoing necro-
sis or apoptosis,30 whereas an increase in MitoTracker inten-
sity may be related to a cytotoxic process involving increased
mitochondrial ROS levels.31 To confirm these findings, we in-
vestigated cell death by flow cytometry using ethidium bro-
mide (EB, which stains necrotic cells) and acridine orange
(AO, a vital dye).32 Both tacrine and compound 3 increased
the cell population stained with EB and formed a second AO
population less stained than the major population found on
control cells (Fig. 4B). Altogether, these data show that 3
imparted severe cell injury that led to cell death.
Convinced that compound 3 was causing cell death, we
wanted to verify if it was also stopping cell proliferation. Our
cell high-content analysis showed that, even at the highest
tested concentration (25 μM), compound 3 did not block cells
entering the S (synthesis) or leaving the M (mitosis) phases
Intrigued by the extreme effect of compound 3 on cell via-
bility, we looked at two morphological features that could of-
fer clues on how this analogue affects cells. MDA-MB-231 cell
nuclei and mitochondria were stained with the fluorophores
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Fig. 4 Analogue 3 affected cell nuclear morphology, mitochondrial staining with MitoTracker and led to increased cell death. (A) High-content
analysis of MDA-MB-231 treated with compound 3, but not compound 5, pointed to increased nuclear fragmentation and, potentially, increased
mitochondrial mass. (B) Flow cytometry of MDA-MB-231 cells treated with compound 3 or tacrine showed increased staining for ethidium bromide
(a dead cell marker) and decreased staining for acridine orange (a viable cell probe), compared to DMSO incubated cells. P1 is the population se-
lected for analysis, and gates 1 and 2 define AO and EB positive cells, respectively.
(Fig. 5A). These data showed that compound 3 does not affect
the cell cycle.
231 cells upon treatment with compound 3. We verified that
compound 3, but not compound 5, increased cell ROS gener-
ation by about 20% after 3 hours of treatment, an effect that
was counteracted by the ROS scavenger N-acetyl-L-cysteine
(NAC). Curiously, the effect of compound 3 was much less
pronounced after 24 hours of treatment (Fig. 5B). These re-
sults indicate that treatment with compound 3 leads to a
Finally, since compound 3 shares a structural similarity
with piplartine, a molecule with the potential of increasing
cellular ROS production,18 and the mitochondria staining as-
say suggested an increase in mitochondrial mass, we tested
whether intracellular ROS were being produced in MDA-MB-
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Fig. 5 Compound 3 did not affect the cell cycle and slightly and transiently raised ROS. (A) Fluorescence microscopy of MDA-MB-231 treated with
compound 3 at increasing doses showed no alteration in EdU incorporation (S phase marker) and pHH3 detection (M phase-specific marker). (B)
Compound 3, but not compound 5, increased by around 20% the intracellular ROS levels only after 3 hours of treatment, an effect that was
counteracted by NAC.
modest ROS increase in cells, which is not persistent and
may not account for the toxicity displayed by this compound
in the panel of breast tumor cell lines used in our study.
with the exocyclic double bond present on the 3,5-dimethoxy-
4-hydroxycinnamoyl (sinapic) side chain. Compound 3 was
prepared in just two steps in 46% overall yield and exhibited
an IC50 equal to 6.6 μM over the proliferation of MDA-MB-
231 cells. This analogue also showed cytotoxicity against
other seven breast cancer cell lines, and less pronounced cy-
totoxicity on two non-tumorigenic cell lines. Our initial mech-
anistic studies indicate that both apoptosis and necrosis are
likely taking place during cell death after treatment with
compound 3, and this compound does not affect the cell cy-
cle. ROS generation by compound 3 was evaluated, but only a
modest increase was observed and may not explain the toxic-
ity mechanism of this compound. The cytotoxicity of com-
pound 3 toward a wide range of breast cancer cell lines de-
serves further attention to validate this compound as a
potential lead compound for the development of new
Conclusions
A concise library of compounds, including the alkaloid
cenocladamide and nine structural analogues, was synthe-
sized and evaluated against the MDA-MB-231 cancer cell line.
The chemical modifications performed on the structure of
cenocladamide allowed the identification of a more potent
analogue (3), providing insights into the structure–activity re-
lationship (SAR) for the series of compounds prepared in this
study. Indeed, two main features seem to be important for
the cytotoxic effect displayed by these compounds: the non-
conjugated ketone at the heterocyclic ring in combination
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candidates for breast cancer treatment. Our work also high-
lights the usefulness of natural products as a source for the
discovery of novel chemical entities with enhanced cytotoxic-
ity for drug development.
(62.9 MHz; CDCl3): δ 35.3 (CH2), 42.3 (CH2), 68.7 (CH2), 107.4
(CH), 128.2 (CH), 128.44 (CH), 128.48 (CH), 134.7 (C0), 143.1
(CH), 152.3 (C0), 192.9 (C0).
2,3-Dihydropyridin-4IJ1H)-one (13). A mixture of benzyl
4-oxo-3,4-dihydropyridine-1IJ2H)-carboxylate (12, 705 mg, 3.0
mmol, 1 eq.) and Pd/C (70 mg, 5% w/w, 0.02 eq.) in EtOH (30
mL) was stirred under an atmosphere of hydrogen at room
temperature for 1.5 h. The product was filtered through a
plug of celite, washed with ethyl acetate and concentrated un-
der reduced pressure. Yield: 296 mg, quant. as a brown oil;
Experimental section
General
Starting materials and reagents were obtained from commer-
cial sources and used as-received unless otherwise specified.
Dichloromethane and triethylamine were treated with cal-
cium hydride and distilled before use. Tetrahydrofuran (THF)
and diethyl ether were treated with metallic sodium and ben-
zophenone and distilled before use. Anhydrous dimethyl-
formamide (DMF) and pyridine were obtained from commer-
cial sources. Anhydrous reactions were carried out with
continuous stirring under an atmosphere of nitrogen. Prog-
ress of the reactions was monitored by thin-layer chromatog-
raphy (TLC) analysis (silica gel 60 F254 on aluminum plates).
1H NMR and 13C NMR were recorded on 250, 400 or 500
MHz equipment, the chemical shifts (δ) were reported in
parts per million (ppm) relative to the deuterated solvent as
the internal standard (CDCl3 7.26 ppm, 77.0 ppm), and cou-
pling constants (J) are in hertz (Hz). Mass spectra were
recorded on a Q-Tof apparatus operating in electrospray
mode (ES). The principal absorptions of infrared spectra with
Fourier transform (FTIR) are listed in cm−1. The purity of all
final compounds was higher than 95% and was determined
by HPLC analysis, using a C18 column (4.6 × 150 mm, parti-
cle size 5 μm) and a mixture of MeOH/H2O or MeCN/H2O as
the eluent. The IUPAC names of the compounds were gener-
ated using ChemBioDraw Ultra 13.0. NMR spectra were
processed using ACD/NMR Processor Academic Edition ver-
sion 12.01.
1
Rf = 0.13, ethyl acetate; H NMR (250 MHz): δ 2.51 (2H, t, J =
7.6 Hz), 3.60 (2H, t, J = 7.9 Hz), 5.05 (1H, d, J = 7.4 Hz), 5.48
(1H, br. s), 7.20–7.27 (1H, m); 13C NMR (62.9 MHz; CDCl3): δ
35.5 (CH2), 41.3 (CH2), 97.6 (CH), 153.0 (CH), 192.8 (C0).
(E)-1-(3-(3,4,5-Trimethoxyphenyl)acryloyl)-2,3-dihydro-
pyridin-4IJ1H)-one (2). (E)-3-(3,4,5-Trimethoxyphenyl)acrylic
acid (118 mg, 0.49 mmol, 1.6 eq.) was dissolved in
dichloromethane (3 mL) and three drops of dry DMF were
added. Oxalyl chloride (41.8 μL, 0.49 mmol, 1.6 eq.) was
slowly added (gas evolution!) and the reaction mixture was
stirred for 4 h at room temperature. The resulting solution
was added to a solution of 2,3-dihydropyridin-4IJ1H)-one (13,
30 mg, 0.31 mmol, 1 eq.), triethylamine (129 μL, 0.93 mmol,
3 eq.) and DMAP (3.81 mg, 0.03 mmol, 0.1 eq.) in dry
dichloromethane (3 mL), and the resulting mixture was
stirred overnight at room temperature. The reaction was
quenched by washing with a saturated aqueous NH4Cl solu-
tion (10 mL). After separation of the layers, the aqueous layer
was extracted with dichloromethane (3 × 10 mL). The com-
bined organic layers were dried over anhydrous sodium sul-
fate, filtered and concentrated under reduced pressure. The
crude was purified by flash column chromatography, eluting
with hexane/ethyl acetate (30 : 70). Yield: 35 mg, 36% as a
white solid; Rf = 0.25, hexane/ethyl acetate (30 : 70); mp 139–
143 °C; IR (ATR, cm−1): 2945, 2918, 1650, 1611, 1581, 1291,
1155, 1125, 980; 1H NMR (250 MHz): δ 2.63 (2H, t, J = 6.9
Hz), 3.90 (9H, s), 4.18 (2H, t, J = 6.8 Hz), 5.45 (1H, d, J = 8.2
Hz), 6.79 (2H, s), 6.82 (1H, d, J = 15.1 Hz), 7.75 (1H, d, J =
15.1 Hz), 7.95 (1H, d, J = 8.1 Hz); 13C NMR (62.9 MHz;
CDCl3): δ 35.9 (CH2), 42.3 (CH2), 56.2 (CH3), 61.0 (CH3), 105.6
(CH), 108.2 (CH), 113.6 (CH), 129.6 (C0), 140.7 (C0), 142.8
(CH), 147.1 (CH), 153.5 (C0), 165.2 (C0), 193.4 (C0); HRMS
(ESI +) m/z: calcd. for C17H19NO5Na+ [M + Na]+ 340.1155,
found 340.1155.
Synthesis
Benzyl 4-oxo-3,4-dihydropyridine-1IJ2H)-carboxylate (12). A
solution of 4-methoxypyridine (11, 467 μL, 4.44 mmol, 1 eq.)
in dry methanol (6.5 mL) was cooled to −78 °C. Sodium boro-
hydride (252 mg, 6.66 mmol, 1.5 eq.) was added and the mix-
ture was stirred for 15 minutes. Benzyl chloroformate (1000
μL, 6.66 mmol, 1.5 eq.) was dissolved in dry diethyl ether (10
mL) and added dropwise to the reaction mixture. The
resulting suspension was stirred at −78 °C for 3 h. The reac-
tion was quenched by addition of water (12 mL), warmed to
room temperature and stirred for 1 h. The mixture was
extracted with ethyl acetate (3 × 10 mL). The combined or-
ganic layers were washed with brine (10 mL), dried over anhy-
drous sodium sulfate, filtered and concentrated under re-
duced pressure. The crude was purified by flash column
chromatography, eluting with hexane/ethyl acetate (60 : 40).
Yield: 700 mg, 68% as a colorless oil; Rf = 0.50, hexane/ethyl
1-Acetyl-2,3-dihydropyridin-4IJ1H)-one (7). To a solution of
2,3-dihydropyridin-4IJ1H)-one (13, 30.1 mg, 0.31 mmol, 1 eq.)
in dry dichloromethane (3 mL) were added triethylamine
(130 μL, 0.93 mmol, 3 eq.) and DMAP (3.8 mg, 0.03 mmol,
0.1 eq.). Acetic anhydride (44 μL, 0.46 mmol, 1.5 eq.) was
added dropwise to the reaction mixture which was stirred
overnight at room temperature. The reaction was quenched
with a saturated aqueous NH4Cl solution (10 mL). After sepa-
ration of the layers, the aqueous layer was extracted with
dichloromethane (3 × 10 mL). The combined organic layers
were dried over anhydrous sodium sulfate, filtered and con-
centrated under reduced pressure. The crude was purified by
1
acetate (60 : 40); H NMR (250 MHz; CDCl3): δ 2.50 (2H, t, J =
7.6 Hz), 3.99 (2H, t, J = 7.6 Hz), 5.22 (2H, s), 5.29 (1H, d, J =
8.2), 7.32–7.37 (5H, m), 7.81 (1H, d, J = 8.2 Hz); 13C NMR
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flash column chromatography, eluting with hexane/ethyl ace-
tate (50 : 50). Yield: 33.8 mg, 78% as a white solid; Rf = 0.20,
hexane/ethyl acetate (30 : 70); mp 57–60 °C; 1H NMR (250
MHz): δ 2.30 (3H, s), 2.54 (2H, t, J = 7.1 Hz), 4.04 (2H, t, J =
6.8 Hz), 5.32–5.35 (1H, m), 7.45–8.33 (1H, m); 13C NMR (62.9
MHz; CDCl3): δ 21.2 (CH3), 35.6 (CH2), 40.3 (CH2), 107.7
(CH), 143.0 (CH), 168.8 (C0), 193.3 (C0).
1-(3-(3,4,5-Trimethoxyphenyl)propanoyl)-2,3-dihydro-
pyridin-4IJ1H)-one (10). 3-(3,4,5-Trimethoxyphenyl)propanoic
acid (79.3 mg, 0.33 mmol, 1.25 eq.) was dissolved in
dichloromethane (3 mL) and three drops of dry DMF were
added. Oxalyl chloride (41.9 μL, 0.33 mmol, 1.25 eq.) was
slowly added (gas evolution!) and the reaction mixture was
stirred for 4 h at room temperature. The resulting solution
was added to a solution of 2,3-dihydropyridin-4IJ1H)-one (25.6
mg, 0.264 mmol, 1 eq.), triethylamine (110 μL, 0.79 mmol, 3
eq.) and DMAP (3.2 mg, 0.02 mmol, 0.1 eq.) in dry
dichloromethane (3 mL), and the resulting mixture was
stirred overnight at room temperature. The reaction was
quenched by washing with a saturated aqueous NH4Cl solu-
tion (10 mL). After separation of the layers, the aqueous layer
was extracted with dichloromethane (3 × 10 mL). The com-
bined organic layers were dried over anhydrous sodium sul-
fate, filtered and concentrated under reduced pressure. The
crude was purified by flash column chromatography, eluting
with hexane/ethyl acetate (30 : 70). Yield: 34 mg, 40% as a col-
orless oil; Rf = 0.23, hexane/ethyl acetate (30 : 70); IR (ATR,
cm−1): 2997, 2942, 2841, 1663, 1590, 1298, 1181, 1124, 1007,
749; 1H NMR (250 MHz; CDCl3): δ 2.53 (2H, t, J = 7.1 Hz),
2.85 (2H, t, J = 6.8 Hz), 2.97 (2H, t, J = 6.8 Hz), 3.81 (3H, s),
3.84 (6H, s), 4.05 (2H, br. s) 5.35 (1H, br. s) 6.43 (2H, s), 8.70–
7.3 (1H, m); 13C NMR (62.9 MHz; CDCl3): δ 31.1 (CH2), 35.4
(CH2), 35.7 (CH2), 41.0 (CH2), 56.1 (CH3), 60.8 (CH3), 105.4
(CH), 108.0 (CH), 136.0 (C0), 136.6 (C0), 142.0 (CH), 153.3
(C0), 170.7 (C0), 193.2 (C0); HRMS (ESI +) m/z: calcd. for
C17H21NO5Na+ [M + Na]+ 342.1312, found 342.1307.
Cenocladamide (1). (E)-3-(4-Acetoxy-3,5-dimethoxyphenyl)-
acrylic acid (91 mg, 0.34 mmol, 1.25 eq.) was dissolved in
dichloromethane (3 mL) and three drops of dry DMF were
added. Oxalyl chloride (27.7 μL, 0.33 mmol, 1.2 eq.) was
slowly added (gas evolution!) and the reaction mixture was
stirred for 4 h at room temperature. The resulting solution
was added to a solution of 2,3-dihydropyridin-4IJ1H)-one (13,
26.5 mg, 0.27 mmol, 1 eq.), triethylamine (114 μL, 0.82
mmol, 3 eq.) and DMAP (3.4 mg, 0.03 mmol, 0.1 eq.) in dry
dichloromethane (3 mL), and the resulting mixture was
stirred overnight at room temperature. The reaction was
quenched by washing with a saturated aqueous NH4Cl solu-
tion (10 mL). After separation of the layers, the aqueous layer
was extracted with dichloromethane (3 × 10 mL). The com-
bined organic layers were dried over anhydrous sodium sul-
fate, filtered and concentrated under reduced pressure. The
crude was purified by flash chromatography, eluting with
hexane/ethyl acetate (30 : 70), and amide 14 was used in the
next reaction. Thus, amide 14 was taken up in a mixture of
MeOH/H2O/THF (4.5 mL, 4 : 1 : 4) and NH4OAc (171 mg, 2,22
mmol, 8.1 eq.) was added in three portions over three days.
After 72 h, the mixture was concentrated in vacuo and
cenocladamide was purified by flash column chromatogra-
phy, eluting with hexane/ethyl acetate (50 : 50). Yield: 15 mg,
18% (2 steps) as a yellowish oil; Rf = 0.20, hexane/ethyl ace-
tate (50 : 50); IR (ATR, cm−1): 2962, 2917, 2850, 1710, 1650,
1510, 1427, 1222, 1120; 1H NMR (250 MHz; CDCl3): δ 2.65
1-Benzoyl-2,3-dihydropyridin-4IJ1H)-one (8). To a solution
of 2,3-dihydropyridin-4IJ1H)-one (13, 30.1 mg, 0.31 mmol, 1
eq.) in dry dichloromethane (3 mL) were added triethylamine
(130 μL, 0.93 mmol, 3 eq.) and DMAP (3.8 mg, 0.03 mmol,
0.1 eq.). Benzoyl chloride (40 μL, 0.34 mmol, 1.1 eq.) was
added dropwise to the reaction mixture and the resulting
mixture was stirred overnight at room temperature. The reac-
tion was quenched by washing with saturated aqueous NH4Cl
solution (10 mL). After separation of the layers, the aqueous
layer was extracted with dichloromethane (3 × 10 mL). The
combined organic layers were dried over anhydrous sodium
sulfate, filtered and concentrated under reduced pressure.
The crude was purified by flash column chromatography,
eluting with hexane/ethyl acetate (30 : 70). Yield: 32 mg, 51%
1
as a colorless oil; Rf = 0.28, hexane/ethyl acetate (60 : 40); H
NMR (250 MHz; CDCl3): δ 2.67 (2H, t, J = 7.1 Hz), 4.17 (2H, t,
J = 7.3 Hz), 5.33 (1H, d, J = 8.2 Hz), 7.46–7.62 (6H, m); 13C
NMR (62.9 MHz; CDCl3): δ 36.0 (CH2), 42.9 (CH2), 107.9 (CH),
128.6 (CH), 128.8 (CH), 131.9 (CH), 132.6 (C0), 144.8 (CH),
170.2 (C0), 193.4 (C0).
1-Cinnamoyl-2,3-dihydropyridin-4IJ1H)-one (9). Cinnamic
acid (68.7 mg, 0.46 mmol, 1.5 eq.) was dissolved in
dichloromethane (3 mL) and three drops of dry DMF were
added. Oxalyl chloride (62.8 mg, 0.49 mmol, 1.6 eq.) was
slowly added (gas evolution!) and the reaction mixture was
stirred for 4 h at room temperature. The resulting solution
was added to a solution of 2,3-dihydropyridin-4IJ1H)-one (30
mg, 0.31 mmol, 1 eq.), triethylamine (129 μL, 0.93 mmol, 3
eq.) and DMAP (3.81 mg, 0.03 mmol, 0.1 eq.) in dry
dichloromethane (3 mL) and the resulting mixture was
stirred overnight at room temperature. The reaction was
quenched by washing with a saturated aqueous NH4Cl solu-
tion (10 mL). After separation of the layers, the aqueous layer
was extracted with dichloromethane (3 × 10 mL). The com-
bined organic layers were dried over anhydrous sodium sul-
fate, filtered and concentrated under reduced pressure. The
crude was purified by flash column chromatography, eluting
with hexane/ethyl acetate (60 : 40). Yield: 29.0 mg, 41% as a
white solid; Rf = 0.32, hexane/ethyl acetate (60 : 40); mp 113–
116 °C; IR (ATR, cm−1): 2950, 2907, 1660, 1625, 1589, 1290,
1
1178, 977, 769; H NMR (250 MHz; CDCl3): δ 2.65 (2H, t, J =
7.6 Hz), 4.19 (2H, t, J = 7.4 Hz), 5.46 (1H, d, J = 8.2 Hz), 6.97
(1H, d, J = 15.3 Hz), 7.43–7.44 (3H, m), 7.57–7.59 (2H, m),
7.86 (1H, d, J = 15.5 Hz), 7.98 (1H, d, J = 7.9 Hz); 13C NMR
(62.9 MHz; CDCl3): δ 35.9 (CH2), 42.3 (CH2), 108.3 (CH),
114.6 (CH), 128.2 (CH), 129.0 (CH), 130.8 (CH), 134.2 (C0),
142.7 (CH), 147.1 (CH), 165.2 (C0), 193.4 (C0); HRMS (ESI +)
+
m/z: calcd. for C14H14NO2 [M
+
H]+ 228.1019, found
228.1009.
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(2H, t, J = 7.3 Hz), 3.96 (6H, s), 4.20 (2H, t, 7.6 Hz), 5.46 (1H,
d, J = 8.4 Hz), 5.87 (1H, br. s), 6.78 (1H, d, J = 15.3 Hz), 6.82
(2H, s), 7.77 (1H, d, J = 15.2 Hz), 7.99 (1H, d, J = 8.2 Hz); 13C
NMR (62.9 MHz; CDCl3): δ 36.0 (CH2), 42.4 (CH2), 56.4 (CH3),
105.4 (CH), 108.1 (CH), 112.0 (CH), 125.7 (C0), 137.8 (C0),
142.9 (CH), 147.3 (C0), 147.6 (CH), 165.4 (C0), 193.5 (C0);
HRMS (ESI +) m/z: calcd. for C16H17NO5Na+ [M + Na]+
326.0999, found 326.0995.
(E)-3-(4-Hydroxy-3,5-dimethoxyphenyl)-1-(piperidin-1-yl)-
prop-2-en-1-one (5). To a solution of piperidine (15, 21.6 μL,
0.219 mmol, 1 eq.) in anhydrous THF (5 mL) were added
sinapic acid (50.0 mg; 0.219 mmol, 1 eq.) and DCC (45.6 mg;
0.219 mmol, 1 eq.). The mixture was stirred at room tempera-
ture for 24 h. The solvent was removed and the crude was pu-
rified by flash column chromatography, eluting with hexane/
ethyl acetate (60 : 40). Yield: 37 mg, 58% as a white solid; Rf =
0.58, hexane/ethyl acetate (60 : 40); mp 126–128 °C; IR (ATR,
cm−1): 2929, 2849, 1639, 1582, 1462, 1337, 1113; 1H NMR
(500 MHz; CDCl3): δ 1.61–1.68 (6H, m), 3.60–3.68 (4H, m),
3.92 (6H, s), 5.82 (1H, br. s), 6.72 (1H, d, J = 15.4 Hz), 6.75
(2H, s), 7.56 (1H, d, J = 15.4 Hz); 13C NMR (62.9 MHz; CDCl3):
δ 24.6 (CH2), 25.6 (CH2), 27.0 (CH2), 43.3 (CH2), 46.9 (CH2),
56.3 (CH3), 104.7 (CH), 115.3 (CH), 126.9 (C0), 136.4 (C0),
142.6 (CH), 147.1 (C0), 165.4 (C0).
ethyl acetate (30 : 70); IR (ATR, cm−1) 2964, 2876, 2841, 1639,
1601, 1515, 1334, 1223, 1116, 985; 1H NMR (250 MHz,
CDCl3): δ 1.68–1.73 (4H, m), 3.73 (4H, br. s), 3.85 (6H, s), 3.94
(4H, s), 6.71 (2H, s), 6.72 (1H, d, J = 15.2 Hz), 7.54 (1H, d, J =
15.3 Hz); 13C NMR (62.9 MHz; CDCl3): δ 34.8 (CH2), 35.3
(CH2), 40.1 (CH2), 43.5 (CH2), 56.2 (CH3), 64.3 (CH2), 104.7
(CH), 106.8 (C0), 114.5 (CH), 126.4 (C0), 136.5 (C0), 143.2
(CH), 147.1 (C0), 165.5 (C0); HRMS (ESI +) m/z: calcd. for
C18H24NO6+ [M + H]+ 350.1598, found 350.1586.
(E)-1-(1,4-Dioxa-8-azaspiroij4.5]decan-8-yl)-3-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (18). To a solution of (E)-3-
(3,4,5-trimethoxyphenyl)acrylic acid (120 mg, 0.504 mmol, 1
eq.) in dry dichloromethane (5 mL) were added EDCI (115
mg, 0.6 mmol, 1.2 eq.), DMAP (74.0 mg; 0.6 mmol, 1.19 eq.)
and triethylamine (83.6 μL, 0.6 mmol, 1.2 eq.). The mixture
was stirred at room temperature for 30 minutes. Next, 1,4-
dioxa-8-azaspiroij4.5]decane (16) was added (78.5 μL; 0.6
mmol, 1.2 eq.) and the reaction mixture was stirred at room
temperature for 24 h. Then, dichloromethane (10 mL) was
added and the reaction mixture was washed with an aqueous
HCl solution (1 M, 30 mL), a saturated aqueous NaHCO3 so-
lution (30 mL) and brine (30 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered and concentrated
under reduced pressure. The crude was purified by flash
column chromatography, eluting with hexane/ethyl acetate
(50 : 50). Yield: 57 mg, 31% as a white solid; Rf = 0.27,
hexane/ethyl acetate (30 : 70); mp 148–149 °C; IR (ATR, cm−1):
(E)-1-(Piperidin-1-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-
one (6). To a solution of (E)-3-(3,4,5-trimethoxyphenyl)acrylic
acid (120 mg, 0.504 mmol, 1 eq.) in dry dichloromethane (5
mL) were added EDCI (115 mg, 0.6 mmol, 1.2 eq.), DMAP
(74.0 mg; 0.6 mmol, 1.2 eq.) and triethylamine (84 μL, 0.6
mmol, 1.2 eq.). The mixture was stirred at room temperature
for 30 minutes. Then, piperidine (15) was added (51.1 mg;
0.6 mmol, 1.2 eq.) and the reaction mixture was stirred at
room temperature for 24 h. Dichloromethane (10 mL) was
added and the reaction mixture was washed with an aqueous
HCl solution (1 M, 30 mL), a saturated aqueous NaHCO3 so-
lution (30 mL) and brine (30 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered and concentrated un-
der reduced pressure. The crude was purified by flash col-
umn chromatography, eluting with hexane/ethyl acetate (50 :
50). Yield: 81.4 mg, 53% as an yellow oil; Rf = 0.19, hexane/
1
2972, 2942, 2873, 1641, 1585, 1334, 1126; H NMR (250 MHz;
CDCl3): δ 1.68–1.72 (4H, m), 3.72 (4H, br. s), 3.82 (3H, s), 3.85
(6H, s), 3.95 (4H, s), 6.70 (2H, s), 6.76 (1H, d, J = 15.3), 7.52
(1H, d, J = 15.3); 13C NMR (62.9 MHz; CDCl3): δ 34.6 (CH2),
35.7 (CH2), 40.2 (CH2), 43.8 (CH2), 56.0 (CH3), 60.7 (CH3),
64.3 (CH2), 104.9 (CH), 106.7 (C0), 116.3 (CH), 130.7 (C0),
139.4 (C0), 142.6 (CH), 153.2 (C0), 165.2 (C0); HRMS (ESI +)
m/z: calcd. for C19H25NO6Na+ [M + Na]+ 386.1574, found
386.1558.
(E)-1-(3-(4-Hydroxy-3,5-dimethoxyphenyl)acryloyl)piperidin-
4-one (3). A solution of (E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-
1-(1,4-dioxa-8-azaspiroij4.5]decan-8-yl)prop-2-en-1-one (17, 56
mg, 0.16 mmol, 1 eq.) and p-toluenesulfonic acid mono-
hydrate (2.4 mg, 0.013 mmol, 0.08 eq.) in a water–ethanol
mixture (3 mL, 1 : 2) was heated at reflux for 3 h. The solvent
was removed in vacuo and the residue was added to a satu-
rated aqueous NaHCO3 solution (20 mL), then extracted with
ethyl acetate (20 mL) and washed with brine (20 mL). The or-
ganic layer was dried over anhydrous sodium sulfate, filtered
and concentrated under reduced pressure. The crude was
purified by flash column chromatography, eluting with hex-
ane/ethyl acetate (30 : 70). Yield: 30 mg, 63% as a yellowish
solid; Rf = 0.30, hexane/ethyl acetate (30 : 70); mp 192–203 °C;
IR (ATR, cm−1): 2961, 2918, 2849, 1712, 1647, 1511, 1427,
1
ethyl acetate (50 : 50); H NMR (250 MHz; CDCl3): δ 1.57–1.60
(6H, m), 3.58 (4H, br. s), 3.82 (3H, s), 3.85 (6H, s), 6.70 (2H,
s), 6.75 (1H, d, J = 15.5 Hz), 7.50 (1H, d, J = 15.5 Hz); 13C
NMR (62.9 MHz; CDCl3): δ 24.4 (CH2), 43.2 (CH2), 46.9 (CH2),
56.0 (CH3), 60.7 (CH3), 104.7 (CH), 116.8 (CH), 130.9 (C0),
139.2 (C0), 142.0 (CH), 153.2 (C0), 165.1IJC0).
(E)-3-(4-Hydroxy-3,5-dimethoxyphenyl)-1-(1,4-dioxa-8-
azaspiroij4.5]decan-8-yl)prop-2-en-1-one (17). To a solution of
(E)-1-(1,4-dioxa-8-azaspiroij4.5]decan-8-yl)-3-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (16, 57.6 μL, 0.440 mmol,
1 eq.) in anhydrous THF (10 mL) were added sinapic acid
(101 mg, 0.440 mmol, 1 eq.) and DCC (91.7 mg, 0.440 mmol,
1 eq.). The mixture was stirred at room temperature for 24 h.
The solvent was removed and the crude was purified by flash
column chromatography, eluting with hexane/ethyl acetate
(30 : 70). Yield: 113 mg, 73% as a yellow oil; Rf = 0.28, hexane/
1
1225, 1123; H NMR (250 MHz, CDCl3): δ 2.54 (4H, t, J = 6.1
Hz), 3.92 (6H, s), 3.97 (4H, t, J = 6 Hz), 5.86 (1H, br. s), 6.77
(2H, s), 6.78 (1H, d, J = 15.3 Hz), 7.65 (1H, d, J = 15.1 Hz); 13C
NMR (62.9 MHz; CDCl3): δ 41.1 (CH2), 44.1 (CH2), 56.4 (CH3),
105.0 (CH), 113.8 (CH), 126.3 (C0), 136.9 (C0), 144.4 (CH),
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147.2 (C0), 166.0 (C0), 206.8 (C0); HRMS (ESI +) m/z: calcd. for
C16H19NO5Na+ [M + Na]+ 328.1155, found 328.1151.
clei was quantified using the plate-reader fluorescence micro-
scope Operetta (PerkinElmer) and the software Columbus
(Perkin Elmer). The percentage of proliferation (when the
number of cells at time 72 h of treatment > time 24 h) was
calculated with the equation 100 × [(Compound72h/Media24h)/
(DMSO72h/Media24h)], while the percentage of cell loss (when
the number of cells at time 72 h of treatment < time 24 h)
(E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)piperidin-4-one
(4). A solution of (E)-1-(1,4-dioxa-8-azaspiroij4.5]decan-8-yl)-3-
(3,4,5-trimethoxyphenyl)prop-2-en-1-one (18, 57.0 mg; 0.16
mmol, 1 eq.) and p-toluenesulfonic acid monohydrate (2.4
mg, 0.013 mmol, 0.08 eq.) in a water–ethanol mixture (3 mL,
1 : 2) was heated at reflux for 3 h. The solvent was removed in
vacuo and the residue was added to a saturated aqueous
NaHCO3 solution (20 mL), extracted with ethyl acetate (20
mL), and the organic phase was washed with brine (20 mL),
dried over anhydrous sodium sulfate, filtered and concen-
trated under reduced pressure to furnish the product. Yield:
37 mg, 74% as a white solid; Rf = 0.24, hexane/ethyl acetate
(30 : 70); mp 100–106 °C; IR (ATR, cm−1): 2991, 2969, 1716,
was calculated with the equation 100 × [1 − (Compound72h
/
Media24h)].
Nuclear fragmentation and mitochondrial fluorescence
microscopy assays. Nuclei and mitochondria were stained
with 1 μg mL−1 Hoechst 33342 and 300 nM MitoTracker Deep
Red (Life Technologies), respectively, in RPMI and 10% fetal
bovine serum for 60 minutes and imaged with the plate-
reader fluorescence microscope Operetta (PerkinElmer). The
software Columbus was used to identify the nuclear and cyto-
plasmic compartments and the average of intensity of
MitoTracker staining was determined at the cytoplasm com-
partment and defined as the mitochondrial mass. For
nuclear fragmentation index analysis, we used the Harmony
(Perkin Elmer) “Apoptosis-1” module imported to the Colum-
bus from the Ready-Made Solution (RMS) collection.
1
1649, 1585, 1416, 1268, 1125, 1002, 829; H NMR (400 MHz;
CDCl3): δ 2.52 (4H, t, J = 6.4 Hz), 3.85 (3H, s); 3.87 (6H, s),
3.95 (4H, t, J = 6.0 Hz), 6.73 (2H, s); 6.80 (1H, d, J = 15.3 Hz);
7.63 (1H, d, J = 15.3 Hz); 13C NMR (101 MHz; CDCl3): δ 41.5
(CH2), 44.5 (CH2), 56.4 (CH3), 61.2 (CH3), 105.4 (CH), 115.7
(CH), 130.7 (C0), 140.1 (C0), 144.2 (CH), 153.6 (C0), 166.0 (C0),
+
207.0 (C0); HRMS (ESI +) m/z: calcd. for C17H22NO5 [M + H]+
320,1492, found 320.1479.
Cell cycle fluorescence microscopy assay. After 72 h of 0.2–
25 μM compound 3 treatment, MDA-MB-231 cells were la-
belled with 30 μM EdU for 30 minutes. After that, cells were
fixed with 3.7% paraformaldehyde in PBS at room tempera-
ture for 20 minutes. Cells were then washed twice in PBS and
permeabilized with 0.1% Triton X100 in PBS for 15 minutes
at room temperature. The incorporated EdU was labeled with
AF488 using the Click-iT EdU labeling kit (Life Technologies)
as guided by the fabricant. Following blocking for 30 minutes
with 3% BSA in PBS, cells were incubated with 1 : 250 anti-
pHH3 (S10) primary antibody (Cell Signaling Technologies)
diluted in TBS at room temperature for 60 minutes. Cells
were washed with PBS and then incubated with 1 : 300 AF647
Biology
Cell culture. The breast cancer cell lines MDA-MB-231,
MDA-MD-468, BT549, HCC38, HS578T, MCF7, SKBR3, T47D
and MCF-10A were purchased from the American Type Cul-
ture Collection (ATCC). The human mammary primary cells
(HMEC) were purchased from Lonza and immortalized by
stable transduction with the retroviral vector pBABE-hygro-
hTERT (Telomere Reverse Transcriptase). pBABE-hygro-
hTERT was a gift from Bob Weinberg (Addgene plasmid #
1773).33 All tumor cell lines were cultivated in RPMI 1640 me-
dia (Sigma) supplemented with 10% fetal bovine serum
(Vitrocell). MCFA-10A cells were cultivated in F12/DMEM
supplemented with 5% horse serum, 20 ng mL−1 epithelial
growth factor (EGF), 0.1 μg mL−1 cholera toxin, 10 μg mL−1
bovine insulin, and 0.5 μg mL−1 hydrocortisone. iHMEC cells
were grown in serum-free mammary epithelial growth me-
dium (MEGM) supplemented with a proprietary combination
of hormones (BulletKit) from Lonza. All cells were grown at
37 °C in a 5% CO2 humidified incubator. Cells used for the
in vitro assays were 90–100% viable as assessed by Trypan
blue (Sigma) staining.
Proliferation assay. Three thousand cells were plated in a
96-well plate format and allowed to adhere overnight. In the
following day, cells were treated in a single dose (20 μM) or
in a serial dilution (from 1.5–50 μM) of the compounds for
72 hours. The number of cells at 24 hours was measured and
considered as plated cells (Media24h). Media was replaced
once after 48 hours. DMSO (0.5%) or 1 μM doxorubicin was
used as negative and positive control, respectively. Cells were
fixed with 3.7% formaldehyde and stained with 0.4 μg mL−1
DAPI (Sigma-Aldrich). The total number of DAPI-stained nu-
secondary antibody (LifeTechnologies) and
1
μg mL−1
Hoechst 33342 in TBS at room temperature for 60 minutes.
Cells were finally washed twice with 0.05% Tween20 in PBS
and left in PBS until microscope analysis. The Harmony
(Perkin Elmer) RMS “Cell Cycle” module was used for the
analysis.
Intracellular ROS fluorescence microscopy assay. MDA-
MB-231 cells were treated with 6 μM compound 3 or 20 μM
compound 5 for 3 and 24 hours, with and without 3 mM of
the antioxidant N-acetyl-L-cysteine (NAC, Sigma-Aldrich).
Intracellular ROS was measured using 10 μM of the
membrane permeable dye 2′,7′-dichlorodihydrofluorescein
diacetate (H2DCFDA, Life Technologies) for 30 minutes at
37 °C. Cells were washed with PBS and the fluorescence
intensity was measured with the Operetta fluorescence
microscopy and analyzed with the software Columbus.
Flow cytometry toxicity assay. One million MDA-MB-231
cells were seeded in 100 mm dishes and, after 24 h, treated
for 72 h with compound 3 (40 μM) or compound 5 (20 μM)
or for 24 h with 2 μM tacrine (Sigma-Aldrich). To quantify live
and dead cell populations, the DNA-binding dyes acridine
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orange (AO, Sigma-Aldrich) and ethidium bromide (EB,
Sigma-Aldrich) were used. AO is a cell-permeant nucleic acid
binding dye that emits green fluorescence when bound to
dsDNA and red fluorescence when bound to ssDNA or RNA.
EB, on the other hand, selectively and covalently labels DNA
in dead cells since it is relatively impermeant to live cells.
Floating and adherent cells were collected by trypsinization,
pelleted by centrifugation, washed twice with PBS and
resuspended in 300 μL PBS containing 0.2 μM AO and 0.5
μM EB. All samples were stained and analyzed immediately
at room temperature using a FACS Count II instrument (BD
Biosciences) and the FlowJo software. AO and EB were excited
with a blue laser (488 nm). Emission was detected with a
525/20 nm filter (for AO) and a 635/20 nm filter (for EB).
Four-parameter list mode data were acquired for analysis.
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14 For more information about amides from the genus Piper,
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Model Genus0020for Studies of Phytochemistry, Ecology, and
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18 L. Raj, T. Ide, A. U. Gurkar, M. Foley, M. Schenone, X. Li,
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19 R. C. Barcelos, J. C. Pastre, D. B. Vendramini-Costa, V.
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Acknowledgements
The authors gratefully acknowledge financial support from
the São Paulo Research Foundation (FAPESP, awards No.
2014/26378-2, 2014/25770-6, 2014/15968-3 and 2015/08199-6),
CNPq (award No. 453862/2014-4) and FAEPEX-UNICAMP
(award No. 0877/14). Prof. Dr. Marcos N. Eberlin and Renan
S. Galaverna are acknowledged for HRMS analysis.
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