Synthesis of novel 4-Boc-piperidone chalcones and evaluation of their cytotoxic activity against highly-metastatic cancer cells

Article abstract of DOI:10.1016/j.bmcl.2019.126760

In this study, six curcuminoids containing a tert-butoxycarbonyl (Boc) piperidone core were successfully synthesized, five of them are novel compounds reported here for the first time. These compounds were prepared through an aldolic condensation by adding tetrahydropyranyl-protected benzaldehydes or substituted benzaldehyde to a reaction mixture containing 4-Boc-piperidone and lithium hydroxide in an alcoholic solvent. A 44–94% yield was obtained supporting the developed methodology as a good strategy for the synthesis of 4-Boc-piperidone chalcones. Cytotoxic activity against LoVo and COLO 205 human colorectal cell lines was observed at GI₅₀ values that range from 0.84 to 34.7 μg/mL, while in PC3 and 22RV1 human prostate cancer cell lines, GI₅₀ values ranging from 17.1 to 22.9 μg/mL were obtained. Results from biochemical assays suggest that the cytotoxicity of the 4-Boc-piperidone chalcones can be linked to their ability to induce apoptosis, decrease the activity of NFκB and cellular proliferation. Our findings strongly support the potential of Boc-piperidone chalcones as novel cytotoxic agents against highly-metastatic cancer cells.

Full text of DOI:10.1016/j.bmcl.2019.126760

Journal Pre-proofs
Synthesis of novel 4-Boc-piperidone chalcones and evaluation of their cytotoxic
activity against highly-metastatic cancer cells
Carlimar Ocasio-Malavé, Metsiel J. Donate, María M. Sánchez, Jesús M. Sosa-
Rivera, Joseph W. Mooney, Tomás A. Pereles-De León, Néstor M. Carballeira,
Beatriz Zayas, Christian E. Vélez-Gerena, Magaly Martínez-Ferrer, David J.
Sanabria-Ríos
PII:
S0960-894X(19)30723-1
DOI:
https://doi.org/10.1016/j.bmcl.2019.126760
BMCL 126760
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
6 August 2019
25 September 2019
15 October 2019
Please cite this article as: Ocasio-Malavé, C., Donate, M.J., Sánchez, M.M., Sosa-Rivera, J.M., Mooney, J.W.,
Pereles-De León, T.A., Carballeira, N.M., Zayas, B., Vélez-Gerena, C.E., Martínez-Ferrer, M., Sanabria-Ríos, D.J.,
Synthesis of novel 4-Boc-piperidone chalcones and evaluation of their cytotoxic activity against highly-metastatic
cancer cells, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126760
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Synthesis of novel 4-Boc-piperidone chalcones and evaluation of their cytotoxic activity
against highly-metastatic cancer cells
a
b
c
b
Carlimar Ocasio-Malavé , Metsiel J. Donate , María M. Sánchez , Jesús M. Sosa-Rivera , Joseph
a
a
d
e
W. Mooney , Tomás A. Pereles-De León , Néstor M. Carballeira , Beatriz Zayas , Christian E.
e
b,c
a1
Vélez-Gerena , Magaly Martínez-Ferrer , David J. Sanabria-Ríos
aFaculty of Science and Technology, Department of Natural Sciences, Inter American University
of Puerto Rico, Metropolitan Campus, PO Box 191293, San Juan, PR 00919, USA
b
School of Pharmacy, Department of Pharmaceutical Sciences, University of Puerto Rico,
Medical Sciences Campus, PO Box 365067, San Juan, PR 00936, USA
University of Puerto Rico Comprehensive Cancer Center, PO Box 363027. San Juan, PR 00936,
c
USA
d
Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, 17 Ave Universidad
STE 1701, San Juan, PR 00925, USA
School of Science, Technology & Environmental, Universidad Metropolitana, PO Box 21150,
e
San Juan, PR 00928, USA
Keywords: Curcumin, curcumin analogs, cytotoxic agents, cancer, apoptosis, cell proliferation
1Corresponding author. Tel.: 787-250-1912 ext. 2332; Fax: 787-250-8736
Email address: dsanabria@intermetro.edu

Abstract
In the present study, six curcuminoids containing a tert-butoxycarbonyl (Boc) piperidone core
were successfully synthesized, five of them for the first time. These compounds were prepared
through an aldolic condensation by adding tetrahydropyranyl-protected benzaldehydes or
substituted benzaldehyde to a reaction mixture containing 4-Boc-piperidone and lithium
hydroxide in an alcoholic solvent. A 44─94% yield was obtained supporting this methodology as
a good strategy for the synthesis of 4-Boc-piperidone chalcones. Cytotoxic activity against LoVo
and COLO 205 human colorectal cell lines was observed at GI values that range from 0.84-34.7
5
0
μg/mL, while in PC3 and 22RV1 human prostate cancer cell lines, GI values ranging from 17.1-
5
0
2
4
2.9 μg/mL were obtained. Results from biochemical assays suggest that the cytotoxicity of the
-Boc-piperidone chalcones can be linked to their ability to induce apoptosis, decrease the
activity of NFκB and cellular proliferation. Our findings strongly support the potential of Boc-
piperidone chalcones as novel cytotoxic agents against highly-metastatic cancer cells.

Cancer is an enormous health burden reaching every region of the world but affecting
mostly underdeveloped countries. Today, it accounts for about one in every seven deaths
1
worldwide; more than HIV/AIDS, tuberculosis, and malaria combined. Since many traditional
anticancer therapies are facing numerous problems, such as multidrug resistance, whole body
2
distribution before reaching cancer tissues, and toxic side effects , natural compounds like
curcumin (Curc), the active component of the Asian plant turmeric (Curcuma longa), have been
explored for its broad range of pharmacological effects. The properties of Curc are widely used
by Oriental Medicine for the treatment of various chronic illnesses. Based on its capability to
affect several molecular targets, Curc has the power for the prevention and treatment of
allergies, arthritis, obesity, aging, diabetes, psoriasis, atherosclerosis, autoimmune, and
3
-8
neurodegenerative diseases. Its efficacy as a potent anti-inflammatory has shown promising
9
-13
results as a novel agent to treat different types of carcinoma.
The antimetastatic effect of
Curc has been revealed in human melanoma, carcinoma, colon, liver, pancreatic, bladder,
1
4,15
ovarian and breast cancer.
This is mediated through regulation of various transcription
factors (NFκB, AP-1, STAT), growth factors (VEGF, EGF, FGF), inflammatory cytokines (IL-1, IL-6,
1
6
TNF) and other enzymes (COX-2, LOX, MMP9, MAPK, mTOR, Akt). Apoptosis related proteins
(
BcI-2, DR, Fas, caspases) and the suppression of specific cytochrome P450 enzymes can be
1
7,18
inhibited as well by the chemopreventive effects of Curc.
Although Curc’s low water solubility can lead to poor bioavailability coupled to fast
metabolism and minimum chemical stability, the use of nanotechnology can successfully
enhance Curc’s efficiency for clinical use. Various drug delivery systems have been developed to
overcome this problem. Some of them include polysaccharides, micelles, phospholipid
1
9-21
complexes, liposomes and nanoparticles.
However, the chemical structure of Curc plays a
vital role in its biological action.22-24 Given that Curc is a polyphenol with two carbonyl groups
(
Figure 1), reports revealed that its rapid metabolism and deficient absorption are due to the
2
5-27
presence of two carbonyl functional groups that severely affect its pharmacokinetics.
H
O
H
H
O
O
H
Figur
e 1.
Che
mica
l
struc
tures
H
H
H
H
HO
OH
HO
OH
OCH3
OCH3
OCH3
OCH3
Curcumin (Curc)
C5-Curcumin (C5-Curc)
of Curc and C5-Curc.
In recent years, researchers have devoted their efforts systematically in modifying Curc’s
structure by the deletion of a methylene and the β-diketone moiety group to synthesize C5-
Curcumin (C5-Curc, Figure 1) and other related analogs.28 It was demonstrated that C5-
Curcuminoids display an improved pharmacokinetics profile and increase anti-inflammatory
activity in vitro without noticeable toxicity in vivo when compared to Curc.29,30 To fully
understand the mechanism of action of Curc and related analogs, careful structure activity

relationship (SAR) analyses in man-made monocarbonyl Curc analogues (MACs) have shown
that (i) strong electron-withdrawing substituents at the 2’ position may increase the molecule’s
bioactivity and the more electronegative is the moiety, the more cytotoxic is the compound; (ii)
a weak electron-donating substituent at the 4’ position is most favorable for the anti-tumor
activity of the compound; and (iii) a strong electron-donating or withdrawing moiety may
3
1
remove or reduce the bioactivity, respectively. Moreover, compounds that are hexa-
substituted can exhibit strong activities, with a 3,4,5-hexasubstitution resulting in the highest
3
2
potency.
Previously, our group reported on a series of symmetrical MACs with cytotoxicity
3
3
against colorectal cancer cells. As a continuation of our study, exploration of new analogs
3
4
were investigated, inspired by the synthetic equivalents made by Gandhy et. al. They reported
several 4-Boc-piperidone containing compounds, RL197 being the most active of the series,
with very potent effects against colon cancer cells by downregulation of several transcription
factors. In addition, they demonstrated that the presence of a tert-butoxycarbonyl (Boc)
protecting group in the nitrogen atom of the chalcone’s six-membered ring core is key for the
cytotoxicity. In a recent study performed by Kálai and colleagues, it was demonstrated that the
removal of N-acyl protecting group 2-fold decrease the anticancer activity of several N-acyl-
3
5
3
,5-bis (4-fluoro-benzylidene) piperidin-4-ones in three different cancer cell lines , which
strongly support those Gandhy’s results that showed that the presence of the Boc-protecting
group is necessary in the design of new MACs containing the piperidone chalcone core. In the
present study, six MACs with the Boc piperidone core were synthesized (five of them for the
first time) with up to a three-step synthetic route. Their cytotoxicity and some mechanistic
aspects were also examined using the highly-metastatic cancer cell lines LoVo, COLO-205, PC3,
and 22RV1.
3
3
In this work, a recently synthetic methodology reported by Sanabria-Ríos et al. was
employed for the preparation of the new 4-Boc-piperidone chalcones (Schemes 1 and 2). In
Scheme 1, tetrahydropyranyl-protected benzaldehydes were added to a reaction mixture
containing 4-Boc-piperidone and lithium hydroxide (LiOH) in ethanol. The resulting
tetrahydropyranyl-protected 4-Boc-piperidone chalcones 3a-c were treated with pyridinium p-
toluene sulfonate (PPTS) in methanol obtaining the desired products 4a-c in 44-65% yield.
O
N
O
O
O
Boc
CHO
R1
CHO
R1
PPTS, CH2Cl2
LiOH H O, EtOH
PPTS, MeOH
rt, 24h
2
R3
R3
R1
N
R1
R3
R3
R1
N
R1
R3
R3
rt, 24h
rt, 24h
Boc
Boc
R2
R2
R2
R2
R2
R2
1a: R1 = -H; R2 = -OH; R3 = -H
1b: R1 = -H;R2 = -OCH3; R3 = -OH
1c: R1 = -H; R2 = -H; R3 = -OH
2a: R1 = -H; R2 = -OTHP; R3 = -H; 97%
3a: R1 = -H; R2 = -OTHP; R3 = -H; 89%
3b: R1 = -H;R2 = -OCH ; R = -OTHP; 66%
4a: R1 = -H; R2 = -OH; R3 = -H; 65%
2b: R1 = -H;R2 = -OCH ; R3 = -OTHP; 64%
3
3
3
4b: R1 = -H;R2 = -OCH ; R3 = -OH; 50%
3
2c: R1 = -H; R2 = -H; R3 = -OTHP; 90%
3c: R1 = -H; R2 = -H; R3 = -OTHP; 56%
4c: R1 = -H; R2 = -H; R3 = -OH; 44%
Scheme 1. Total synthesis of 4-Boc-piperidone chalcones 4a-c.

In Scheme 2, compounds 6a-c were synthesized by reacting a substituted benzaldehyde
and 4-Boc-piperidone with LiOH in either methanol or ethanol. Products 6a and 6b were
obtained in 53 and 67% yield, respectively. In the case of 6c, the compound was obtained in
9
4% yield demonstrating that the use of LiOH as a base was more effective than NaOCH in
3
methanol, an alkaline solution that was previously reported useful for the preparation of this
product.
3
4
O
N
O
Boc
R4
R3
CHO
R1
R4
R3
R4
R3
LiOH H2O, MeOH or EtOH
rt, 24h
R1
N
R1
Boc
R2
R2
R2
5
5
5
a: R1 = -CF3; R2 = -H; R3 = -H; R4 = -H
6a: R1 = -CF ; R2 = -H; R3 = -H; R4 = -H; 53%
3
b: R1 = -H; R2 = -H; R3 = -H; R4 = -H
6b: R1 = -H; R2 = -H; R3 = -H;R4 = -H; 67%
c: R1 = -OCH ; R2 = -H; R3 = -H, R4 = -OCH3
6c: R1 = -OCH ; R2 = -H; R3 = -H; R4 = -OCH ; 94%
3
3
3
Scheme 2. Synthetic approach towards 4-Boc-piperidone chalcones 6a-c.
Both H-NMR and 13C-NMR spectra were recorded on a Brucker Avance 500
1
1
spectrophotometer. H-NMR chemical shifts are reported relative to internal tetramethylsilane
1
3
(
SiMe , 7.26 ppm), while C-NMR chemical shifts were reported in parts per million (ppm)
4
relative to CDCl (77.00 ppm). FT-IR data were generated on a Thermo Scientific Nicolet iS5 FT-
3
IR spectrophotometer. Melting points for 4 and 6 were determined with a Melt-Temp
apparatus and these measurements were uncorrected. Table 1 summarizes the spectroscopic
data obtained from NMR analyses. Detailed chemical characterization of compounds 4 and 6 is
described in the Supporting Information (SI) section.

1
13
a,b,c
Table 1. H-NMR and C-NMR characterization of 4-Boc-piperidone chalcones 4 and 6.
O
3
3
R4
R4
1
2
2
R3
R1
N
R1
R3
Boc
R2
R2
13C-NMR chemical shift (ppm)
1H-NMR chemical shift Melting point (C)
(ppm), multiplicity
Compound
C1
C2
C3
H3
4
4
4
a
b
c
192.5
190.8
187.4
186.8
192.2
187.7
141.4
141.4
136.9
137.6
142.5
143.5
142.1
142.1
143.0
143.6
142.5
143.9
7.73, s
7.74, s
7.77, s
7.72, s
7.83, m
7.97, s
151-153
102-104
183-185
90-96
6
6
a
b
146-148
6
c
164-166
a
1
Signals from methyl groups from Boc-protecting groups were observed at 1.26-1.51 ppm in H-NMR and 27.9-
1
3
13
2
8.2 ppm in C-NMR. Tertiary carbons from t-butyl groups were observed at 79.9-80.7 ppm in C-NMR.
Methylene groups from Boc-piperidone six-membered rings showed signals at 3.97-4.74 ppm in H-NMR and 41.1-
5.1 ppm in C-NMR. Carbonyl groups from Boc-protecting groups showed signals at 154.0-156.5 ppm in C-NMR.
1
1
3
13
4
b
1
Signals from methoxy groups in compounds 4b and 6c were observed at 3.49-3.83 ppm in H-NMR and 55.8-56.0
1
3
c
1
ppm in C-NMR. Signals from aromatic groups were observed at 6.70-7.77 ppm in H-NMR and 111.8-137.8 ppm
1
3
in C-NMR.
1
In H-NMR, singlet signals were observed for 4 and 6 in the region of 7.73-7.98 ppm.
These signals are characteristic of compounds containing an α, β-unsaturated carbonyl system36
and demonstrates the success of the synthesis displayed in Schemes 1 and 2. In the case of 6b,
1
a multiplet at 7.83 ppm was also observed in H-NMR, which suggests a long-range coupling
1
3
between H3 and an aromatic hydrogen. In C-NMR, signals that were generated by C1, C2, and
C3 in the regions of 186.8-192.5 ppm (C1) and also, at 140.5-145.1 ppm (C2-C3) demonstrate
the presence of the α, β-unsaturated carbonyl system, which are in accordance with the results
1
from H-NMR displayed in Table 1.
In 2014, Thakur and collaborators reported the total synthesis of novel 3,5-
bis(arylidiene)-4-piperidone based monocarbonylanalogs of curcumin.3 In the latter
publication, the christallographic structure for the (3E,5E)-3,5-Bis-(2,5-difluoro-benzylidene)-1-
7
(
toluene-4-sulfonyl)-piperidin-4-one was reported demostrating that this compound has an E
double bond configuration. Because 6c has a similar structure than the above-mentioned
compound, it was compared the spectroscopic data of 6c with the NMR data reported by
Thakur et al. In that study, it was reported a singlet at 7.66 ppm for the vinylic protons in the
3
7
α, β unsaturated system, while a singlet at 7.97 ppm was observed for 6c. These results are in
3
4,37
agreement with results reported in the literature
and suggest that there is no long-range
coupling between the vinylic protons and the aromatic protons of the aryl substituent as a
2
possible consequence of free rotation of the sigma bond between the sp carbon of the C=C
2
and the sp carbon of the aryl group. Because all vinylic protons showed a singlet in the region

of 7.72-7.97 (see Table 1) it is believed that the same phenomena described in the literature is
taking place with all the compounds mentioned in Table 1. In addition, we were no able to
1
3
observe the presence of any Z diasteroisomers in C-NMR, specifically, in the region of 136-144
3
4,37
ppm, which is in agreement with those findings reported in the literature.
Once the syntheses of 4 and 6 were carried out, the cytotoxicity of these compounds
against highly-metastatic colorectal cancer cells were also determined by using the CellTiter-
Glo® luminescent cell viability approach. In this methodology, the colorectal cancer cell lines
LoVo and COLO-205 and the normal colorectal cell line CCD18Co were studied. A detailed
description about luminescent cell viability assays is available in the Supplementary Information
(
SI) section. Table 2 shows GI values obtained from CellTiter-Glo® cell viability assays.
5
0
Table 2. Cytotoxic activity of 4-Boc-piperidone chalcones 4 and 6 against highly-metastatic
a
colorectal cancer cell lines.
b
Compounds
GI ± SEM, µg/mL
5
0
LoVo (SInc)
4.1 ± 1.0 (1.6)
9.6 ± 1.0 (1.9)
3.5 ± 1.1 (1.8)
4.6 ± 1.0 (1.7)
6.1 ± 1.1 (2.0)
2.0 ± 0.2 (1.7)
10.0 ± 1.0 (1.5)
4.3 ± 1.0 (1.7)
1.7 ± 0.1 (0.6)
COLO-205 (SIn)
CCD18Co
6.4 ± 1.0
18.3 ± 1.0
6.4 ± 0.3
7.6 ± 1.0
12.4 ± 1.0
1.2 ± 0.1
15.1 ± 1.5
7.2 ± 1.0
1.1 ± 0.1
4
4
4
a
b
c
11.7 ± 1.0 (0.5)
27.4 ± 1.0 (0.7)
20.9 ± 1.1 (0.3)
4.5 ± 1.0 (1.7)
34.7± 1.0 (0.4)
0.84 ± 0.08 (1.4)
19.4 ± 1.9 (0.8)
12.7 ± 1.0 (0.6)
6.8 ± 0.7 (0.2)
6
6
a
b
6
c
d
C5-Curc
Curc
Cisplatin
d
d
a
Experiments were performed in triplicate (N = 3).
b
Results were reported as mean ± SEM from three determinations. GI50 represents the sample concentration that
is required to achieve 50% cell viability and this value was determined from dose-response curves that were
generated by using Graph-Pad Prism® v. 6.01 (GraphPad Software).
c
38
SIn = Selectivity Index. SIn values were calculated as described by Sanabria-Ríos et al.
d
28
C5-Curc was synthesized as described by Liang et al. C5-Curc, Curc, and cisplatin were used as reference
compounds.
It can be noted that compounds 4 and 6 were more cytotoxic against LoVo cells than C5-
Curc suggesting that the presence of the 4-Boc-piperidone core is needed for the cytotoxicity
Table 2). In the case of 4a, 4c, 6a, and 6c, these compounds displayed similar GI values as
(
5
0
Curc or cisplatin, which makes them attractive for further studies in cancer research. In fact,
these results suggest that the presence of an electron donating group, such as -OH, at either
the meta or para position in 4a or 4c, or the presence of an electron withdrawing group, such
as -CF , at the ortho position in 6a, or the presence of methoxy groups at both ortho and para
3
positions in 6c, are needed for their cytotoxicity against LoVo cells. These findings are in
agreement with SAR studies reported in the literature.31
Table 2 also revealed that compounds 4 and 6 were almost 2-fold more selective towards
LoVo cells than towards the normal colorectal cell line CCD18Co. However, the opposite was

observed for COLO-205. Except for compounds 6a and 6c, almost all tested compounds,
including the reference compounds, were more cytotoxic towards CCD18Co than towards
COLO-205 suggesting that COLO-205 is more resistant towards anticancer drugs than LoVo. It
can be appreciated from Table 2 that the presence of a -CF group at the ortho position in 6a or
3
methoxy groups at both ortho and para positions in 6c, are pivotal for their cytotoxicity towards
COLO-205 colorectal cancer cell lines. In general, the cytotoxicity of 4-Boc-piperidone chalcones
against highly-metastatic colorectal cancer cells followed the order: 6c > 6a > 4a > 4c.
To further understand the mechanism of 4-Boc-piperidone chalcones, we employed
Annexin V apoptosis analysis to determine whether 6a or 6c can induce apoptotic effect in
COLO-205 cancer cells. Detailed description of Annexin V assays can be found in the SI section.
Figure 2 shows the apoptotic induction of 6a and 6c on human COLO-205 colorectal
adenocarcinoma cells.
Figure 2. (A) Apoptotic induction comparison of 6a and 6c on human COLO-205 colorectal
adenocarcinoma cells. Annexin V experiments for both 6a and 6c were performed at their
corresponding GI displayed in Table 2. (B) The Annexin V Assay FACS showing the distribution
5
0
of apoptotic COLO-205 cells treated with either 6a or 6c.
Significant apoptotic induction of 6a and 6c was observed compared to the positive control
Camptothecin. The negative control showed an average of 10.7% apoptotic cells, while the
positive Camptothecin control presented an average of 37.3% apoptotic cells. Compound 6a
displayed an average of 35% of apoptotic cells, while 6c presented the highest average with
3
3
6
2% apoptotic cells, considerably higher than previously reported with Curc (27.5%).
The activation of caspases such as caspase 3 and 7 (known as effector caspases), are
3
9
important components of the apoptotic process. These enzymes can be activated by signaling
caspases such casp 8 and 9 and participate in the activation of enzymatic degradation of cell
3
9
structures and content. Therefore, it is possible that compounds 6a and 6c can induce

apoptosis through the inhibition of effector caspases. For this reason, it was carried out in vitro
assays using the fluorescent inhibitor probe FAM-DEVD-FMK to label active caspases 3 and 7
enzymes in
COLO-205. Results from these assays are displayed in Figure 3.
Figure 3. (A) Histograms showing the activation effect of compounds 6a and 6c on caspases 3 &
7
. Annexin V experiments for both 6a and 6c were performed at their corresponding GI50
displayed in Table 2. Experiments were carried out in triplicate (N = 3). (B) FACS plot showing
distribution of apoptotic COLO-205 cells treated with either 6a and 6c.
Results in Figure 3 reveal that the activation of caspases 3 & 7 is statistically significant (P
<
0.05) when compared to the negative vehicle control. The negative control (no treatment)
presented an average of 15.3% of cells undergoing apoptosis, while the positive control
Camptothecin) presented an average of 59.3%. Compounds 6a and 6c presented an average of
1.5% and 35.7%, respectively (Figure 3). The results above are in accordance with results in
(
3
Figure 2, which revealed significant amounts of cells undergoing apoptosis as caspase 3 & 7
activation is closely associated with apoptotic cell death.
Previously, Sanabria-Ríos and colleagues synthesized C5-curcumin-fatty acid (C5-Curc-FA)
3
3
conjugates to establish the structure-activity relationship (SAR) of their anticancer activity.
Interestingly, they found that one of the conjugates, C5-Curc-decanoic acid (DA), inhibits the
3
3
activity of NFκB at an GI of 18.2 µg/mL. Moreover, they showed that C5-Curc-DA causes an
5
0
apoptotic effect at a GI of 46.0 µg/mL in the colorectal adenocarcinoma cell line COLO-205,
5
0
which is likely due to the observed significant mitochondrial membrane permeabilization, and
caspase 3 and 7 activation, that occurs following C5-Curc-DA treatment. Based on these
findings, it is hypothesized that the 4-Boc-piperidone chalcones will also inhibit NFκB activity.
To test this hypothesis, we carried out NFκB luciferase reporter assays aimed at determining
the inhibitory effects of 6a on the activity of NFκB. Further description of NFκB luciferace
reporter assays is available in the SI.

Results displayed in Figure 4 show the inhibitory effect of 6a on the reporter activity of
NFκB. It can be observed that the luminescence properties, after treatment with 6a, showed a
sigmoidal response associated with the logarithm of the concentration of the chalcone. This
4
0-43
behavior was also observed for Curc, a well-known inhibitor of NFκB.
The fact that Curc is
3
.5-fold higher than 6a suggests that the presence of a monocarbonyl moiety and the presence
of -CF groups are influencing the inhibition of the reporter activity of NFκB. The results above
3
are important because the NFκB pathway controls the genes that are linked to apoptosis and
cell proliferation such as c-IAP1, c-IAP2, and IXAP (antiapoptotic genes), the TNF receptor-
4
4
associated factors (TRAF1 and TRAF2), the Bcl-2 homologue A1/Bfl-1, and IEX-IL. Apoptosis
results displayed in Figure 2, in combination with the ones of Figure 4, do not discard the
hypothesis that the inhibition of NFκB pathway can be also involved in the apoptosis effect of 4-
Boc-piperidone chalcones in COLO-205.
Figure 4. Drug-response curves showing the inhibitory effect of both Curc and 6a on the NFκB
Reporter-HEK293 cells. Experiments were performed in triplicate (N=3). Results were reported
as mean ± SEM from three determinations. Non-linear regression analysis was performed with
GraphPad Prism v. 6.01 (GraphPad Software). The IC50 values were determined by the
concentration causing a half-maximal percent activity.
Intended to further explore the potential of 4-Boc-piperidone chalcones as cytotoxic
agents against highly-metastatic cancer cell lines, the MTS approach was employed for
determining the cytotoxicity of these compounds on PC3 and 22RV1 cells. Compound 4c was
tried first because it displayed significant cytotoxicity against LoVo cells (Table 2).
Figure 5 shows the cytotoxic effect of 4c and related analogs on the cell viability of PC3
and 22RV1. It was observed that C5-Curc significantly decreased cell viability in PC3 and 22RV1
cells, when compared to the control (DMSO). At similar ranges of concentrations than C5-Curc,
4
(
c showed a significant decrease (P < 0.05) in the cell viability when compared with the control
DMSO) in 22RV1 cells, with GI50 lower than PC3 cells, suggesting that 22RV1 is more
susceptible towards 4c. In the case of Curc, this compound significantly decreased the cell

viability in both PC3 cells and 22RV1 cells when compared to the control (DMSO) at similar
concentration range of 4c. Significant cytotoxicity was observed as well when both PC3 and
2
2RV1 were treated with Dxtl (P < 0.05). In both prostate cancer cell lines, the GI values were
5
0
remarkably lower than 4c and other analogs.
B)
A)
Figure 5. Inhibitory effect of 4c and related analogs on the cell viability of both PC3 (A) and
2RV1 (B). PC3 and 22RV1 cells were treated with 4c at concentrations ranging from 5 to 30
µg/mL. C5-Curc, Curcumin, and Docetaxel (Dxtl) were used as reference compounds. PC3 and
2RV1 were treated with C5-Curc at concentrations ranging from 5 to 30 µg/mL, Curcumin at
concentrations ranging from 0.001 to 30 µg/mL and Dxtl at concentrations that range from
.002 to 0.01 µg/mL. Cell viability was determined after 48h of incubation with the MTS reagent
see SI section). Experiments were performed in triplicates (N=3). Statistical analysis was
2
2
0
(
performed using One-way ANOVA, followed by Dunnett’s test. Mean ± SEM with P<0.05 was
statistically significant. Graphs and statistical analyses were performed with GraphPad Prism v.
6
.01 (GraphPad Software).
Cytotoxic activity of organic compounds can be explained by several mechanisms, one
of them being cellular proliferation. Cellular proliferation is key in cancer development and
4
5,46
progression, particularly in prostatic diseases.
This mechanism can be analyzed by using
Ki67 antigen, a marker that works against nuclear antigens in specific phases in the cell
4
7,48
cycle.
In the present study, immunofluorescence assays were performed to determine
whether 4c is able to decrease cell proliferation in both PC3 and 22RV1 after 48h of exposition.
Results in Figure 6 show that 4c decreased cell proliferation in both PC3 and 22RV1. In
PC3, 4c was the only compound that significantly inhibited cell proliferation when compared to
control (DMSO), Curc, C5-Curc, and Dxtl. In 22RV1, all compounds tested (i.e. 4c, Curc, C5-Curc,
and Dxtl) inhibited the Ki67 protein expression. However, Dxtl was the compound that better
inhibited the Ki67 protein expression (Figure 6B). These results clearly demonstrate the
potential of 4-Boc-piperidone chalcones as cytotoxic agents against highly metastatic prostate

cancer cells, and are similar to previous studies addressing the synthesis of Curc analogs to
4
9
inhibit the proliferation of prostate cancer cells.
Figure 6. Inhibitory effect on the proliferation activity (Ki67 antibody) in treated PC3 (A) or
2RV1 (B) cells with 4c after 48h of incubation. Immunofluorescence assay was performed to
determine Ki67 activity. PC3 cells were treated with 4c (22.9 µg/mL), C5-curc (24.8 µg/mL), Curc
2
(
13.8 µg/mL) and Dxtl (9 ng/mL); while 22RV1 cells were treated with 4c (17.1 µg/mL), C5-Curc
(22.7 µg/mL), Curc (14.3 µg/mL), and Dxtl (6 ng/mL). Representative images of Ki67 protein
staining PC3 or 22RV1 cells treated with 4c, Curc, C5-Curc, and Dxtl. Nuclei are stained with blue
DAPI (left panels in Figures 5A and 5B) and Ki67 protein is stained in red (right panels in Figures
6
A and 6B). Statistical analysis shows that Ki67 activity was decreased with 4c when compared
to control (DMSO), Curc, and C5-Curc. Dxtl decreased Ki67 protein expression more efficiently
when compared to control (DMSO), 4c, C5-Curc, and Curc. Statistical analysis was performed

using One-way ANOVA, followed by Dunnett’s test. Mean ± SEM (*P<0.05). Experiments were
performed in triplicates (N=3).
Since, the ability of 6a, a synthetic analog of 4c, in inhibiting the reporter activity of
NFκB in HEK293 cells (Figure 4) was demonstrated, we hypothesized that the cytotoxic activity
of 4c against PC3 and 22RV1 can be linked to the inhibition of NFκB. To test this hypothesis,
immunofluorescence assays were carried out to determine whether 4c inhibits the
translocation of NFκB in the nucleus of both PC3 and 22RV1. For these assays, primary
antibodies against NFκB were used, as well as secondary anti-mouse, and DAPI for staining the
nucleus of both PC3 and 22RV1 prostate cancer cell lines (see SI section for further details).
Our results show that 4c significantly decrease the activity of NFκB in PC3 cells and
2RV1 cells (Figure 7) when compared to control (DMSO), Curc, and C5-Curc (compounds that
2
most significantly inhibited the activity of NFκB). Curc only showed significant difference when
compared to control (DMSO) in PC3 cells (Figure 7A). In the case of Dxtl, this compound
significantly inhibited the activity of NFκB in both prostate cancer cells when compared to
control (DMSO). Results in Figure 7 agree with findings from NFκB reporter activity assays
displayed in Figure 4. In addition, these results strongly support the hypothesis that the
inhibition of NFκB can be involved in the cytotoxicity activity of 4c in either PC3 or 22RV1
prostate cancer cells.

Figure 7. Inhibitory effect on the activity of NFκB in both PC3 (A) and 22RV1(B) cells after
treatment with 4c, C5-Curc, Curc, and Dxtl at 48h of incubation. Immunofluorescence assay was
performed to determine NFκB inhibitory activity. PC3 cells were treated with 4c (22.9 µg/mL),
C5-curc (24.8 µg/mL), Curc (13.8 µg/mL) and Dxtl (9 ng/mL); while 22RV1 cells were treated
with 4c (17.1 µg/mL), C5-Curc (22.7 µg/mL), Curc (14.3 µg/mL), and Dxtl (6 ng/mL).
Representative images of NFκB staining PC3 and 22RV1 treated with 4c, C5-Curc, Curc and Dxtl.
Nuclei were stained with blue DAPI (left panels in Figures 7A and 7B) and NFκB is stained in
green (right panels in Figures 7A and 7B). Statistical analysis showed that NFκB activity was
decreased with 4c treatment when compared to control (DMSO) and Curc and C5-Curc.
Statistical analysis was performed using One-way ANOVA, followed by Dunnett’s test. Mean ±
SEM (*P<0.05). Experiments were performed in triplicates (N=3).

The NFkB immune-fluorescence activity results in Figure 7 were confirmed by using the
PathScan® Total NFkB p65 Sandwich ELISA kit (Cell Signaling Technology, Danvers, MA). This
methodology consists of a solid phase sandwich enzyme-linked immunosorbent assay (ELISA)
that allows the detection and quantification of endogenous levels of total NFkB p65 protein in
either PC3 or 22RV1. Figure 8 shows the inhibitory activity of 4c, C5-Curc, Curc, and Dxtl on the
expression of NFkB in both PC3 and 22RV1. It can be observed in Figure 8A, that both 4c and
Curc (positive control) displayed a significant inhibitory effect on the transcriptional activity of
NFkB when compared to the control (P <0.0001). Also, it is appreciated that Dxtl displayed
decreased expression of NFkB than Dxtl in PC3. Moreover, we observed that 4c, C5-Curc, and
Dxtl displayed inhibitory activity on the expression of NFkB in 22RV1 (Figure 8B), which are
comparable with those findings displayed in Figure 7.
Figure 8. Histograms showing inhibitory effect of 4c, C5-Curc, Curc, and Dxtl on the
transcriptional activity of NFkB p65 in both PC3 (A) and 22RV1 (B). Both prostate cancer cell
lines were exposed to the above-mentioned treatments at their corresponding GI ’s reported
5
0
in Figure 5. Experiments were performed in triplicate (N=3) using protocols that were provided
by the manufacturer. Statistical analysis was performed using One-way ANOVA followed by the
Dunnett's multiple comparisons test. Results were reported as mean ± SEM and experimental
means were compared with the control. (**) = P<0.01, (***) = P < 0.001, (****) = P < 0.0001.
In summary, our results clearly demonstrated that 4 and 6 are promising for further
investigations addressing the development of valuable cytotoxic agents against highly-
metastatic cancer cells. In the case of 6a and 6c, these compounds displayed potent cytotoxicity
as cisplatin towards LoVo and COLO-205 cells but less toxicity towards the normal colorectal
CCD18Co cells. In addition, the cytotoxicity of 6a and 6c can be linked to their ability to induce
apoptosis or inhibit the activity of NFκB. Moreover, we gathered preliminary results that
suggest that 4c displayed cytotoxicity towards highly-metastatic PC3 and 22RV1 prostate cancer
cell lines by decreasing cell proliferation and the activity of NFκB. To better understand the

cytotoxicity of 4 and 6 in both colorectal and prostate cancer cells, several biological studies are
already being pursued.

Acknowledgements
This project was supported by the National Center for Research Resources and the
National Institute of General Medical Sciences of the National Institutes of Health through
Grant Number P20GM103475-16. Dr. D.J. Sanabria-Ríos would like to thank the IAUPR-MC Seed
Money for sponsoring part of this project. Special thanks to the Materials Characterization
Center (MCC) for the use of NMR and to Dr. Christian Morales, Ms. Denisse Alequin-Torres, and
Ms. Ashley Rivera for assisting in the chemical characterization in this study. Mr. Joseph
Mooney and Mr. Tomás Pereles-De León acknowledge the support of the PR-INBRE program for
the undergraduate research fellowship provided. Dr. M. Martínez-Ferrer thanks to the financial
support provided by the University of Puerto Rico Comprehensive Cancer Center to perform
part of this work involving highly-metastatic prostate cancer cells (PC3 and 22RV1). Finally, we
want to acknowledge Dr. Denniss Phillips and the Proteomics and Mass Spectrometry Core
Facility at University of Georgia for carrying out ESI-HRMS experiments. Also, we acknowledge
Crown Bioscience, Inc., and BPS Bioscience, Inc. for their assistance in performing cytotoxicity
assays (involving LoVo, COLO-205, and CCD18Co cells), and conducting NFκB activity studies,
respectively.

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Conflict of Interests Declaration
The authors declare no conflict of interests.
Graphical abstract

Highlights

4-Boc piperidone chalcones are prepared, for the first time, to investigate their cytotoxic
activity against colorectal and prostate cancer cells.
1
13


4-Boc piperidone chalcones are characterized by H-NMR, C-NMR, and melting point.
Significant cytotoxic activity is observed when LoVo, COLO-205, PC3, and 22RV1 are
treated with 4-Boc piperidone chalcones.


4-Boc-piperidone chalcones induce apoptosis in COLO-205 possibly through the
inhibition of NFKB.
4-Boc-piperidone chalcones decrease cellular proliferation and nuclear NFkB activity in
both PC3 and 22RV1 cells.