All trans 1-(3-arylacryloyl)-3,5-bis (pyridin-4-ylmethylene)piperidin-4-ones as curcumin-inspired antineoplastics

Article abstract of DOI:10.1016/j.ejmech.2014.09.090

A series of eleven N-acryloyl/N-cinnamoyl 3,5-bis(pyridin-4-yl)methylene-4-piperidones were synthesized as curcumin-based candidate antineoplastic agents. The cytostatic potency of these compounds was evaluated against three representative cell lines and all compounds were found to exhibit significant anti-cancer cell activity in vitro. QSAR studies using several physicochemical parameters and 50% inhibitory concentration (IC₅₀) values resulted in certain important correlations which will aid design of more potent analogs. Representative test compounds were investigated in the NCI 60-cell line panel where they were found to display a profound cytotoxicity. These compounds were also potent anti-oxidants and inhibitors of human topoisomerase III±. Representative compounds were well-tolerated by human fibroblasts and by mice during the survival/toxicity studies.

Full text of DOI:10.1016/j.ejmech.2014.09.090

European Journal of Medicinal Chemistry 87 (2014) 461e470
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
All trans 1-(3-arylacryloyl)-3,5-bis(pyridin-4-ylmethylene)piperidin-
-ones as curcumin-inspired antineoplastics
4
a
a
b
b
Nawal K. Paul , Mamta Jha , Khushwant S. Bhullar , H.P. Vasantha Rupasinghe ,
c
a, *
Jan Balzarini , Amitabh Jha
Department of Chemistry, Acadia University, Wolfville, Nova Scotia, Canada B4P 2R6
Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia, Canada B2N 5E3
Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 June 2014
Received in revised form
A series of eleven N-acryloyl/N-cinnamoyl 3,5-bis(pyridin-4-yl)methylene-4-piperidones were synthe-
sized as curcumin-based candidate antineoplastic agents. The cytostatic potency of these compounds
was evaluated against three representative cell lines and all compounds were found to exhibit significant
anti-cancer cell activity in vitro. QSAR studies using several physicochemical parameters and 50%
inhibitory concentration (IC50) values resulted in certain important correlations which will aid design of
more potent analogs. Representative test compounds were investigated in the NCI 60-cell line panel
where they were found to display a profound cytotoxicity. These compounds were also potent anti-
20 September 2014
Accepted 28 September 2014
Available online 30 September 2014
Keywords:
Curcumin
Michael acceptor
Anti-oxidant
oxidants and inhibitors of human topoisomerase II
by human fibroblasts and by mice during the survival/toxicity studies.
© 2014 Elsevier Masson SAS. All rights reserved.
a. Representative compounds were well-tolerated
Cytostatic activity
Topoisomerase II inhibitors
QSAR
1
. Introduction
resulted in an additional site for protein thiolation [16,17]. As ex-
pected, these compounds were found to be more potent than 1, and
on average, a 26-fold increase in potency was observed against
P388 tumor cell screens, when compared to the parent molecule
[17]. Close correlations between electronic properties with respect
to cytotoxic potencies of these molecules were observed, e.g.
placement of electron-withdrawing substituents on the molecule
improved its anti-cancer cell potency, due to an increase in the
electrophilicity of the olefinic bond where thiolation is believed to
occur [17].
The strong cytotoxic activity displayed by N-acryloyl derivatives
of 3,5-bisarylmethylene-4-piperidones made the development of
series 3e6 (Fig. 1) a logical evolution in fine-tuning the cytotoxic
activity of these types of molecules. Replacement of a terminal
acryloyl hydrogen with aryl (series 3) [4] and arylcarbamoyl [5e7]
(series 4 and 5) moieties enabled the electrophilicity of the acryloyl
group to be modulated, as it relates to thiol alkylation and cyto-
toxicity, by modifying the aryl substituents. Series 3 compounds, in
general, were found to be slightly less inhibitory against cancer
cells than their precursor 1 [4]. The arylcarbamoyl-containing E
Curcumin belongs to a well-established therapeutic armamen-
tarium with a strong anticancer activity and remarkable safety
profile [1]. A number of studies have been conducted on a variety of
styryl ketones as curcumin analogs [2e11]. These compounds may
be regarded as Michael acceptors capable of protein thiolation with
little or no capacity for conjugation with nucleic acid(s) nucleo-
philes such as amino or hydroxy groups under physiological con-
ditions [12]. Hence molecules of these types should not be
genotoxic, contrasting several alkylating agents currently used in
cancer chemotherapy [13]. Also, a candidate among these with
drug-like properties will likely be effective against cross-resistant
tumors due to its structural novelty.
3
,5-Bisarylmethylene-4-piperidone derivatives can be regarded
as curcumin analogs as they share an ample structural similarity as
bis-styryl ketones (Fig. 1). The parent 3,5-bisarylmethylene-4-
piperidones
proliferative activity towards leukemia and colon cancer cell lines
14,15]. The conversion of series 1 to N-acryloyl amides 2 (Fig. 1)
1 (Fig. 1) were found to possess strong anti-
[
congeners, 4 (R ¼ H and NO
2
), were found to have comparable
cytotoxic potencies to melphalan [7]. However, similar compounds
with Z geometry (series 5) were generally found to have greater
potency than the parent molecule, 1 (R ¼ H) and were significantly
*
Corresponding author.
E-mail address: ajha@acadiau.ca (A. Jha).
http://dx.doi.org/10.1016/j.ejmech.2014.09.090
223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
0

462
N.K. Paul et al. / European Journal of Medicinal Chemistry 87 (2014) 461e470
Fig. 1. Cytotoxic 3,5-bisarylmethylene-4-piperidones and their structural resemblance to curcumin.
more potent than melphalan [5]. In a related study, an additional
electrophilic site on precursor 1 (R ¼ H) was introduced by N-
acylation using a chalcone-based acid chloride leading to series 6
compounds [4]. Most members of this series were found to be
highly cytotoxic towards murine leukemia P388 cells (IC50 <1 uM
therapeutic effect of these compounds as anti-cancer/oxidant
agents has been explored, along with a toxicity assessment that
may merit the innocuous remedial benefits of these compounds.
2. Results and discussion
for all compounds) than towards the other tumor cell lines tested
þ
(
murine leukemia L1210 and human CD4 T-lymphocyte Molt4/C8
2.1. Chemistry
&
CEM cells) displaying a high degree of selectivity.
In line with our interest in development of nature-inspired
The synthetic scheme followed to prepare the target molecules
6e17 is shown in Scheme 1. The 3,5-bis(4-pyridinylmethylene)-4-
piperidone trihydrochloride 6 was prepared via condensation of
4-piperidone monohydrate hydrochloride with the 4-
pyridinecarboxaldehyde by briskly passing HCl gas in acetic acid
as solvent. Compound 6 was N-acylated with acryloyl or (E)-3-
arylacryloyl groups using corresponding acid chlorides under
anhydrous basic conditions to yield compounds 7e17 (Scheme 1).
All reaction products were purified by column chromatography and
fully characterized based on their spectroscopic data. It was inter-
esting to note that most of the synthesized compounds (8e17)
suffered peak broadening in their NMR spectra due to molecular
dynamics which is common in saturated nitrogen heterocycles
[31e33]. In their C NMR spectra, a couple of peaks, including two
methylenes attached to N were consistently either missing or
broad. To resolve this, we undertook variable temperature NMR
experiments on compound 16 (see Supplementary Data) as a
representative example. Not only peaks for these methylene Cs and
Hs on them were better resolved in the NMR spectra recorded at
temperatures lower (253 K) and higher (313 K) than ambient
temperature, the compound also understandably appear to freeze
in an unsymmetrical conformation at lower temperature (e.g.
anticancer agents [4e9,11,16,18e22], we aimed to design and syn-
thesize potential anticancer agents based on a simplified curcumin
pharmacophore in the present investigation. First, the established
pharmacophore 3,5-bisarylmethylene-4-piperidone was retained
in the newly-designed compounds but the aryl ring was replaced
by a 4-pyridyl group. The rationale behind this change were as
follows: (i) a pyridine ring is known to be electron-deficient [23]
and therefore it will increase the protein-thiolation capacity of
the corresponding styryl ketone functionality and (ii) introduction
of two nitrogen atoms on the molecule will significantly improve
the aqueous solubility (as such and as acid salts) [24] of the
resulting compounds e a highly desirable property for candidate
drugs. Additionally, alike the previous studies [4,5,7,17], it was
decided to introduce an additional site for protein thiolation by
conjugating the substituted cinnamoyl group at the N-1 position of
the 3,5-bis(4-pyridinylmethylene)-4-piperidone pharmacophore.
A biological response such as anti-cancer activity of therapeutic
agents is due to their interaction with one or several drug targets or
13
receptors. Human topoisomerase IIa has been identified as a po-
tential target of 3,5-bisarylmethylene-4-piperidone derivatives
through some preliminary work in our laboratory [22]. This
enzyme is rich in cysteine residues and is essential for cell prolif-
eration [25e27]. Through transient double-stranded nicks in the
DNA helix, this enzyme is responsible for releasing torsional strain
and facilitating unwinding of the DNA double helix, allowing
replication and transcription processes to occur [25,26,28e30].
Inhibition of these processes causes replication and transcription to
stall, impeding cancer cell proliferation. Alternatively, if topo-
isomerase poisoning occurs, the DNA nicks created by the enzyme
cannot re-legate, resulting in lethal lesions in the DNA frame, which
discrete peaks for each NeCH
2.2. Biological evaluation
2.2.1. Cytostatic activity
2
at 253 K).
To evaluate the cytotoxicity of compounds 6e17 against malig-
nant cell lines, the compounds were evaluated for cytostatic ac-
þ
tivity against murine L1210 leukemia cells, as well as human CD
4
T-
lymphocyte Molt4/C8 and CEM cells, following a literature proce-
dure [34]. The results are presented in Table 1.
then leads to apoptosis [25,26]. Since topoisomerase IIa expression
is enhanced in rapidly proliferating cancerous cells, targeting this
enzyme by Michael-acceptors offers a means of selectivity target-
ing malignant cells to inhibit tumor growth. Furthermore, the
surrender of oxidative stress during carcinogenesis endorses
auxiliary cancer damage; consequently making its attenuation a
crucial strategy in the cancer treatment. Therefore, the dual
The high cytostatic activity of nearly all the compounds tested
against three representative cancer cell lines provides strong evi-
dence that these molecules might be potent antitumour agents as
well as promising lead molecules for further development. The
unsubstituted 3,5-bis(4-pyridinylmethylene)-4-piperidone trihy-
drochloride 6 was found to be equipotent to its phenyl analog 1a

N.K. Paul et al. / European Journal of Medicinal Chemistry 87 (2014) 461e470
463
Scheme 1. Synthetic route to (3E,5E)-3,5-Bis(pyridin-4-ylmethylene)piperidin-4-one (6) and it's amido derivatives 7e17. The R groups are as follows: 7 (R ¼ H), 8 (R ¼ phenyl), 9
R ¼ 4-chlorophenyl), 10 (R ¼ 4-methylphenyl), 11 (R ¼ 4-methoxyphenyl), 12 (R ¼ 4-nitrophenyl), 13 (R ¼ 3,4-dichlorophenyl), 14 (R ¼ 3,4-dimethoxyphenyl), 15 (R ¼ 3,4-
methylenedioxyphenyl), 16 (R ¼ 3,4,5-trimethoxyphenyl), 17 (R ¼ 2-furyl).
(
[
17]. The N-acryloyl analog 7 showed moderate antiproliferative
noted that all test compounds except 7 were more inhibitory than
curcumin [11] in the three representative screens. Encouraged by
these results, compounds 9 and 11 were also evaluated against
NCI's 57 human tumor cell lines (59 cell lines in case of 11) repre-
senting nine different neoplastic conditions, namely leukemia,
melanoma, non-small cell lung, colon, central nervous system,
ovarian, renal, prostate, and breast cancers [36]. The data generated
are summarized in Table 2.
The results shown in Table 2 demonstrate that when all cell lines
are considered, both compounds 9 and 11 are significantly more
cytotoxic than melphalan and curcumin. The GI50 values of the
tested compounds are in the low sub-micromolar range compared
to single/double digit micromolar values for curcumin and
melphalan. An important characteristic of a candidate anticancer
potency (average IC50 34.4 M) against all cell lines tested. This is in
m
contrast to previous observations where inclusion of an additional
electrophilic site in the form of an acroyloyl group, e.g. 2a, led to an
increase (or no change) in the potency [17]. Compared to melphalan
[
35], an established anticancer agent, compounds 6, 8e17 displayed
a significantly greater potency against human Molt4/C8 and CEM T-
lymphocyte cells, and half of the tested compounds possessed
relatively lower potency than melphalan in the L1210 screen.
Among the compounds tested, 11 and 13 were found to be signif-
icantly more selective towards human Molt4/C8 and CEM T-
lymphocyte cells with a selectivity ratio of 26 and 24 respectively.
Compounds 16 and 17, however, were found to be potent inhibitors
against each of the three tumor cell lines screened. It should also be
Table 1
In-vitro cytostatic activities of 3,5-Bis(pyridin-4-ylmethylene)piperidin-4-one (6) and it substituted acryloyl/3-arylacryloyl amides against selected cell lines (7e17).
ID
R
IC50
(
m
M)
SRa
L1210/0
Molt4/C8
CEM/0
1
2
6
7
8
9
1
1
1
1
1
1
1
1
a
a
e
7.0 ± 2.3
8.69 ± 0.73
8.9 ± 0.4
76 ± 17
1.67 ± 0.15
1.42 ± 0.27
1.7 ± 0.1
8.1 ± 3.0
1.8 ± 0.3
0.91 ± 0.17
1.48 ± 0.34
1.7 ± 0.0
19 ± 2
7.7
6.1
5.2
9.4
3.1
3.5
5.8
25.78
5.7
24.2
5.6
b
e
e
H
Phenyl
4.3 ± 4.0
2.7 ± 0.3
2.6 ± 1.0
5.9 ± 3.2
8.0 ± 0.1
5.8 ± 2.8
7.8 ± 1.2
9.7 ± 1.6
0.97 ± 0.54
1.8 ± 1.1
5.8 ± 0.3
22 ± 1
1.4 ± 0.0
0.77 ± 0.54
0.45 ± 0.05
0.86 ± 0.52
1.4 ± 0.4
0.35 ± 0.00
1.4 ± 1.3
2.3 ± 1.1
1.2 ± 0.4
0.32 ± 0.02
2.6 ± 1.2
15 ± 5
4-Chlorophenyl
4-Methylphenyl
4-Methoxyphenyl
4-Nitrophenyl
3,4-Dichlorophenyl
3,4-Dimethoxyphenyl
3,4-Methylenedioxyphenyl
3,4,5-Trimethoxyphenyl
2-Furyl
1.2 ± 0.0
0
1
2
3
4
5
6
7
0.96 ± 0.90
0.23 ± 0.20
1.7 ± 0.0
0.24 ± 0.09
1.7 ± 0.0
2.3 ± 0.6
1.6 ± 0.0
0.38 ± 0.06
3.24 ± 0.79
9.0 ± 1.9
4.2
1.6
5.6
3.9
Melphalan
Curcumin
e
e
2.4
a
Selectivity Ratio, i.e. the ratio of the highest to the lowest IC50 values for each compound.
Reprinted from J. Med. Chem. 44 (2001) 586. Copyright 2001 American Chemical Society.
b

464
N.K. Paul et al. / European Journal of Medicinal Chemistry 87 (2014) 461e470
Table 2
principally existing in a supercoiled state. The results showed that
significant topoisomerase II inhibitory activity by all compounds
was similar to the activity exhibited by cisplatin and sorafenib.
Furthermore, the effects of compounds 13, 15 and 16 were much
more robust than the other compounds, and that of the etoposide
Evaluation of compounds 9, 11 and reference compounds against a panel of human
tumor cell lines.
a
Cell lines
9
11
Melphalanc Curcumind
0.19 24.6 6.7
All cell lines
GI50
SI
(
m
M)a
0.24
148
0.07
b
7080
79
5.6
10
VP-16 and the standard drug at 50 mM concentrations. The gel
Leukemia
GI50
GI50
GI50
GI50
GI50
GI50
GI50
(m
(m
(m
(m
(m
(m
(m
(m
(m
M)
M)
M)
M)
M)
M)
M)
M)
M)
0.14
3.7
9.2
4.7
5.8
5.9
9.1
8.9
11.2
8.1
analysis showed that unlike VP-16 giving a linear DNA band, thus
portraying an interfacial topoisomerase poison, the assayed com-
pounds displayed a catalytic inhibitory activity as they directly
blocked the activity of topoisomerase II directly (no relaxed DNA
but strong supercoiled plasmid, Lanes 1e12, Fig. 2A). To further
substantiate the anticancer activity of active compounds 13, 15 and
Lung cancer
Colon cancer
CNS cancer
Melanoma
Ovarian cancer
Renal cancer
0.46
0.11
0.26
0.18
0.23
0.19
0.30
0.34
0.22 25.6
0.11 48.9
0.17 27.4
0.17 33.0
0.34 41.1
0.20 32.2
0.23 35.5
0.11 33.5
Prostate cancer GI50
Breast cancer
1
6 towards attenuation of topoisomerase II activity, we assessed
their topoisomerase II activity at different concentrations (0.1, 1, 10
and 20 M) and obtained IC50 values. As shown in Fig. 2B, com-
pounds 13 (IC50 1.79 M) and 16 (IC50 1.83 M) emerged as the
strongest inhibitors of topoisomerase II activity in vitro. Interest-
GI50
a
GI50 refers to the compound concentrations required to inhibit the growth of the
m
cells by 50%.
b
m
m
SI refers to the selectivity index. The SI figures for all cell lines were obtained by
dividing the GI50 values of the least and most sensitive cells.
a
c
GI50 values for melphalan were obtained from online NCI database (COMPARE
ingly, at lower concentrations, compound 13 displayed dual prop-
erties of a catalytic inhibitor as well as an interfacial poison (mild
linear bands observed). Overall, the results revealed that all twelve
data vector search, compound ID NSC 8806).
d
GI50 values for curcumin were obtained from online NCI database (COMPARE
data vector search, compound ID NSC 32982).
compounds exhibited tangible influence on topoisomerase II
tivity at low and biologically-relevant concentrations ranging from
.1 to 50 M. From this experiment, we concluded that the com-
a ac-
drug is selective toxicity for certain tumor cell types rather than
their being indiscriminately cytotoxic. The selectivity index (SI)
figures for all tumor cell lines divulge a wide differential sensitivity
of the human tumor cell types to 9 and 11; compound 11 in
0
m
pounds 6e17 demonstrated their anticancer activities through the
restraint of topoisomerase II activity, possibly by blocking the
enzyme activity via excessive supercoiling, which is deleterious to
cancer cells and tumor growth. Similar results have been reported
for curcumin and curcumin-like compounds [39,40] indicating the
ability of such compounds to inhibit the topoisomerase-DNA
cleavable complex.
particular displayed an impressive selectivity (SI 7080).
A
comparative review of the GI50 values with respect to various cell
line classes in Table 2 revealed that both 9 and 11 exerted greater
toxicity against leukemic and colon cancer cell lines than against
those cell lines representing other neoplastic diseases. Melphalan is
used in combination chemotherapy to treat chronic leukemias [37].
The results in Table 2 indicate that 9 as well as 11 are considerably
more potent against leukemic cell lines than melphalan. The novel
findings that the compounds possess significant potencies and
preferential cytotoxicity for certain tumor cells suggests that E,E,E-
2.2.3. Anti-oxidant activity assays
Recent experimental and clinical studies have shown that
oxidative stress and redox imbalance are involved in the pathology
of multiple cancers. The failure or insufficient activity of the
endogenous anti-oxidant defenses (i.e. superoxide dismutase,
glutathione peroxidase, and catalase) during oxidative stress trig-
gers free radicals, which attack DNA, resulting in formation of DNA
adducts [41]. These adducts are implicated in DNA strand breakage
and genetic mutations which may lead to cancer initiation and
progression [42]. Curcumin and its analogs are known to exhibit a
myriad of biological effects (vide supra) via numerous mechanisms,
1
-(3-arylacryloyl)-3,5-bis(pyridin-4-ylmethylene)piperidin-4-ones
(8e17) are candidate anticancer agents with selective toxicity.
2.2.2. Topoisomerase II
a inhibitory activity
DNA topoisomerases are ubiquitous enzymes with a wide
spectrum of cellular functions and their elevated levels in cancer
may make them crucial drug targets. Multiple compounds like
anthracyclines act as topoisomerase inhibitors and cause G1/G2 cell
cycle arrest through DNA breakage. Topoisomerase inhibitors
interfere with topology of DNA by forming cleavage complexes
leading to the inhibition of the cell growth and the cell cycle [38]. As
a series of compounds containing a pharmacophore similar to those
in the compounds under the current investigation were found to
inhibit human topoisomerase II
were undertaken to study their effect on this vital enzyme. Topo-
isomerase II inhibition assays were performed using a commercial
human DNA topoisomerase II inhibition kit, according to the
manufacturer's instructions (Topogen Inc, Columbus, OH). The
topoisomerase II inhibitory activities of compounds 6e17 were
evaluated using an in vitro gel assay to reveal the mechanism
behind the therapeutic action of these compounds. A representa-
tive gel picture displaying the topoisomerase II
tial of the compounds 6e17 is presented in Fig. 2AeB. The results
Table 3
In-vitro antioxidant activities of 3,5-Bis(pyridin-4-ylmethylene)piperidin-4-one (6)
and it substituted acryloyl/3-arylacryloyl amides (7e17).
Compound FRAP (
m
M TE/L) ORAC (
m
M TE/ DPPH (%
inhibition)
ROS (%
inhibition)
L)
a activity [22], appropriate assays
6
7
8
9
28.02 ± 2.7e
206.46 ± 16.5 67.45 ± 1.6bcd
a
59.90 ± 3.6d
fg
cd
bcde
c
cd
7.59 ± 1.4
4.69 ± 0.7
35.56 ± 4.9 65.78 ± 2.7
67.93 ± 4.4
67.12 ± 2.4
65.80 ± 4.1
a
g
de
cd
cd
21.43 ± 4.4 60.22 ± 3.3
de
bcde
bcde
abcd
a
a
ND
22.55 ± 2.3
21.47 ± 2.8
22.87 ± 4.7 66.19 ± 3.3
ef
c
61.25 ± 8.4d
10
45.02 ± 3.2 65.81 ± 5.0
efg
c
cd
cd
1
1
1
1
1
2
3
2
32.04 ± 6.1 68.52 ± 0.8
64.75 ± 3.6
79.11 ± 1.6
77.53 ± 1.9
e
bc
bc
327.90 ± 8.2
4.25 ± 1.6 75.19 ± 1.7
44.48 ± 7.1 63.84 ± 1.0
88.26 ± 6.1d
c
de
373.01 ± 9.2b
c
ab
83.64 ± 9.7b
43.13 ± 2.1 72.38 ± 1.7
ab
cd
abc
cde
bcd
a
bc
a
inhibition poten-
15
449.57 ± 12.4
27.96 ± 1.1 71.30 ± 1.9
78.02 ± 6.6
74.61 ± 3.3
74.48 ± 4.4
bcd
bcd
1
1
6
7
ND
65.55 ± 3.1b 64.76 ± 1.9
71.33 ± 6.3b 67.82 ± 2.2
32.41 ± 2.3e
from this evaluation showed that all the assayed compounds, along
with drugs such as cisplatin and sorafenib (50 mM), exhibited sig-
nificant inhibitory activity towards topoisomerase II in vitro. As
shown in Fig. 2A, compared to the relaxation of a supercoiled
plasmid caused by the topoisomerase II enzyme (i.e. Lane L vs. K),
treatment with compounds 6e17 resulted in the pHOT1 plasmid
a
a
98.7 ± 0.4a
Curcumin 1201.4 ± 0.2
1581.9 ± 28.4 96.8 ± 1.3
Antioxidant capacity measured by FRAP, ORAC, DPPH and ROS radical assays for the
test compounds 6e17 and reference antioxidants, chlorogenic acid and quercetin, at
100 mM concentrations. Means ± standard deviation, followed by the different
superscripted letters (a through g) within column is significantly different, Tukey's
multiple means comparison test, P ꢀ 0.05), ND: Not detected.

N.K. Paul et al. / European Journal of Medicinal Chemistry 87 (2014) 461e470
465
Fig. 2. Topo II (a) and (b) inhibitory activities of 3,5-bis(pyridin-4-ylmethylene)piperidin-4-one (6) and it substituted acryloyl/3-arylacryloyl amides (7e17). (A) The compounds
were examined in a final concentration of 50
m
M, respectively, as designated. Lane K: pHOT1 DNA, supercoiled, Lane L: Marker DNA, Linear pHOT1, Lane E: pHOT1 DNA þ 4 U topo
II þ etoposide, Lane D1: pHOT1 DNA þ 4 U topo II þ Sorafenib, Lane D2: pHOT1 DNA þ 4 U topo II þ Cisplatin, Lanes 1e12: pHOT1 DNA þ 4 U topo II þ test compounds in
designated concentrations. (B) Lane K: pHOT1 DNA, supercoiled, Lane L: Marker DNA, Linear pHOT1, Lane E: pHOT1 DNA þ 4 U topo II þ etoposide, Lanes 1e12: pHOT1 DNA þ 4 U
topo II þ test compounds in described concentrations.
and of particular significance and importance is their compelling
anti-oxidant potential [8]. In order to evaluate compounds 6e17 as
anti-oxidant agents, we conducted in vitro tests using four anti-
oxidant assays, viz. FRAP (ferric-reducing ability of plasma), ORAC
data, as illustrated in Table 3, only a few of the test compounds
showed potent FRAP activity. Among the synthesized compounds,
15 (449.57
mM TE/L) showed the strongest in-vitro activity in
comparison to the other compounds (p ꢀ 0.05). On the contrary,
compounds 9 and 16 exhibited no anti-oxidant activity while the
compounds 12 and 14 closely followed the compound 15 in their
in vitro anti-oxidant potential (p ꢀ 0.05). It was also observed that
anti-oxidant activities of the compounds 6e11 and 17
(
oxygen
diphenylpicrylhydrazyl anti-radical assay) and Cell-ROS assay
cellular reactive oxygen species detection assay) [8,43]. The Cell-
ROS assay employed the use of WI-38 cells (human fibroblasts) as
radical
absorbance
capacity),
DPPH
(2,2-
(
0
0
the cellular substrates together with 2 ,7 -dichlorofluorescein as
(4.69e32.41
mM TE/L) were very weak in comparison to the active
0
the fluorophore and AAPH (2,2 -Azobisisobutyramidinium chlo-
compound 15. For instance, a 96-fold lower anti-oxidant activity
ride) as peroxyl radical generator. Table 3 shows the anti-oxidant
activity data obtained for compounds 6e17 and curcumin (as
was observed in the compound 8 (4.69
compound 15.
mM TE/L) than found for
reference) at 100
m
M concentrations. It should be noted that cur-
The peroxyl radical scavenging activities of these compounds
was assessed by the ORAC assay using fluorescence as the fluo-
rophore, while the relative anti-oxidant potential was estimated by
trolox as the reference index. Interestingly, compound 6, which
exhibited weak activity in FRAP analysis, showed the strongest
ORAC activity in comparison to other compounds in vitro (p ꢀ 0.05).
cumin exhibited the highest antioxidant activity in all the four
antioxidants assays conducted in vitro (p ꢀ 0.05).
The compounds were first screened using the FRAP assay which
determines the anti-oxidant ability of the assayed compounds
3þ
based on the reduction of ferric-tripyridyltriazine (Fe -TPTZ) to its
blue, ferrous form (Fe -TPTZ) resulting in the production of a
stable anti-oxidant radical. According to the FRAP anti-oxidant
2
þ
It was followed by compound 16 (65.55 mM TE/L), which earlier
indicated no anti-oxidant potential in the FRAP analysis. Similarly,
stout anti-oxidant candidates like 12, 14 and 15 displayed much
weaker anti-oxidant potential in ORAC analysis (p ꢀ 0.05); espe-
cially compound 12, which lost about 98% of its anti-oxidant po-
tential in vitro. However, the remaining compounds displayed
similar anti-oxidant potential to FRAP analysis in vitro (p ꢀ 0.05).
Additionally, the in vitro anti-oxidant activity of these com-
pounds was also determined by using the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay, which estimates anti-oxidant activ-
ity by either the direct transfer of H-atom by an anti-oxidant
ꢁ
candidate to DPPH or by monitoring the electron transfer from
ꢁ
the phenoxide anion to DPPH . Crucially, all tested compounds
presented UV absorbance between 250 and 340 nm, thus didn't
interfere with the DPPH absorbance at 517 nm. The results were
ꢁ
calculated as % inhibition of DPPH with respect to an assay control
(no anti-oxidant treatment) (Table 3). The compounds 12, 14 and 15
exhibited the strongest anti-radical scavenging activity, in agree-
ment with the FRAP analysis (p ꢀ 0.05). Likewise, compound 8
ꢁ
displayed the weakest DPPH trapping potential, also in line with
FRAP results in vitro (p ꢀ 0.05). Finally, the interaction of the test
compounds with the cellular ROS was assessed to examine their
radical scavenging ability in a cell model system. The interaction of
the tested compounds with AAPH-induced peroxyl radical was
found to be similar to FRAP and DPPH assays. Among the tested
Fig. 3. In vitro toxicity assessment of 3,5-bis(pyridin-4-ylmethylene)piperidin-4-one
(
6) and it substituted acryloyl/3-arylacryloyl amides (7e17) using LDH release assay
in WI-38 cells (human fibroblasts). Different letters (a through d) on columns desig-
nate a statistical significant difference of P ꢀ 0.05 between the assayed compounds
using a Tukey's test (n ¼ 6), CUR represents curcumin.

466
N.K. Paul et al. / European Journal of Medicinal Chemistry 87 (2014) 461e470
compounds, the strongest peroxyl radical scavenging activity was
displayed by compounds 12e15 that demonstrated superior ac-
tivity than the other compounds in vitro (p ꢀ 0.05). For compounds
Table 4
Correlation constants between IC50 values of compounds under investigation and
their physicochemical parameters.
Correlation coefficient ra
P valueb
6
e11, the ROS scavenging ability, as observed earlier, was frail in
Independent
Dependent
Type
comparison to the other compounds (p ꢀ 0.05). The results were
similar to FRAP and DPPH analysis with the exceptions of com-
pound 9 and 16, which showed no FRAP activity in vitro.
In general, in this study several compounds emerged as potent
anti-oxidants in vitro which may, in part, account for attenuation of
tumor growth and increased oxidative stress, often implicated in
the pathology and progression of carcinogenesis [44].
Charge
Hansch
b
L1210 IC50
CEM IC50
Molt IC50
CEM IC50
CEM IC50
Molt IC50
CEM IC50
CEM IC50
Linear
Linear
Linear
Linear
Semi-log
Linear
Linear
ꢂ0.595
ꢂ0.587
ꢂ0.622
ꢂ0.731
ꢂ0.854
ꢂ0.616
ꢂ0.734
ꢂ0.849
0.091
0.097
0.073
0.025
0.003
0.078
0.024
0.004
p
LogP
Semi-log
a
The magnitude of coefficient r shows the extent (the closer the values to 1, the
2
.2.4. In-vitro toxicology analysis
Pharmacovigilance credence is a crucial strategy for the accep-
better) and the nature (sign positive or negative) of the correlation.
The value of P in the range 0.05e0.1 indicates a trend toward significance, while
values <0.05 suggest significant correlation.
b
tance of a drug candidate as multiple toxicities are associated with
chemotherapy. The in vitro cell culture model can be used to screen
drug candidates for toxicity by evaluating basic parameters of cells
such as the cell viability and membrane damage or rupture. The
in vitro toxicity experiments were performed with the synthesized
N-acryloyl/N-cinnamoyl
piperidones (6e17) in human fibroblasts by quantifying cell
membrane damage following the exposure to the assayed com-
pounds (100 mM). The membrane integrity was evaluated by the
LDH release assay, based on measuring the reduction of tetrazolium
to colored formazan, indicating the membrane damage status. In
order to evaluate whether compounds 6e17 were selectively toxic
towards tumor cells, we conducted viability tests in vitro using the
WI-38 cell line (human fibroblasts). The results showed that the
toxic effect by the assayed compound was negligible towards a
non-cancerous cell line. Fig. 3 shows the LDH release activity for all
compounds and curcumin in comparison to a vehicle control. All
the compounds 6e17, at the assayed concentration showed very
low cytotoxic activity in human fibroblasts, with an average LDH
release of <10% compared to a vehicle control. However, compound
undertaken using certain physicochemical parameters of the
bioactivity. The extent to which the electrophilicity of the N-3-
arylacryloyl systems affected the bioactivity was evaluated by
3,5-bis(pyridin-4-yl)methylene-4-
comparing the electronic charge at the b-position (calculated using
a commercial software) [46] of the test compounds (see structures
for 7e17 in Table 1) with the IC50 values against the three cell lines.
Likewise, the calculated partition coefficient (LogP) and molar
refractivity (MRmol) values of the test compounds were also ob-
tained using a commercial software [46] and utilized as a means of
assessing the effect of the lipophilicity and steric properties of the
compounds with respect to their bioactivity. Physicochemical pa-
rameters of the aromatic substituents [47] such as Hammett
measure of the electronic property of the substituents), Hansch
measure of the lipophilicity of the substituents), and molar
refractivity (MRsub, (a measure of the size of the substituents) were
also used for statistical correlation studies. Linear and semi-
logarithmic correlations were calculated using statistical analysis
s
(a
(a
p
[48] for the IC50 of each tumor cell line and the aforementioned
7
was least innocuous (p ꢀ 0.05) and its toxicity was twice as high
physicochemical parameters. Only the significant correlations ob-
tained are included in Table 4.
as compared to the other compounds exposed to WI-38 cells. The
compounds 6 and 17 were least noxious, as the average LDH release
in their presence was ~5%. Also, these two compounds were non-
toxic and safe in nature as curcumin in human fibroblasts in vitro
No significant correlation (P < 0.01) was observed between
Hammett
aromatic substituents (MRsub) with any of the biological activities.
Multiple significant correlations were obtained for charge
Hansch and LogP with the IC50 values of the three tumor cell lines
s, molar refractivities of the molecule (MRmol) and that of
(
p ꢀ 0.05). The non-toxic presence of these compounds in human
b,
fibroblast cultures in vitro and their strong activity against the
cancer cell lines certainly augments and merits their anticancer
potency.
p
evaluated, but interestingly all of these correlations were negative
in nature. A negative correlation with a trend towards significance
(
P 0.05e0.1) between charge
b
and antiproliferative activity against
increase there is
2
.2.5. In-vivo neurotoxicity studies
Three representative compounds 6,11 and 13 were evaluated for
L1210 and CEM cells indicates that as the charge
b
a decreasing effect (desirable) on the L1210 and CEM IC50 values.
This was expected if the molecules exert their cytostatic effect via
protein thiolation. This result also suggest that analogs with higher
charge
be made and tested to fine-tune the anticancer potential of these
molecules. Negative correlations between the Hansch and LogP
with the IC50 values of the Molt and CEM tumor cell lines indicate
the inverse relationship between lipophilicity of the aryl sub-
stituents (Hansch p) and that of the molecule (LogP). Increase in the
murine toxicity in vivo following a literature procedure [45]. The
compounds were injected intraperitoneally into mice at doses of
3
and 4 h post administration of the compounds. The compounds
were found to be well-tolerated, with 11 and 13 showing no
neurotoxicity. Death was observed in case of compound 6 in one
out of two animals studied at the highest dose administered
0, 100 and 300 mg/kg. The animals were then monitored for 0.5
b than the current compounds (more electrophilic) should
p
(
300 mg/kg) after 4 h. Also, for compound 6, neurological deficit
was displayed at 100 and 300 mg/kg in some of the test animals. No
neurotoxicity was observed for amides 11 and 13 in any of the test
animals after 0.5 h or 4 h at all doses studied. These results indicate
that the amides studied here are well-tolerated and are not general
biocidal agents.
lipophilicity is resulting in a decrease in the IC50 or increase in the
antiproliferative potency. Thus, in future, more lipophilic analogs
should be prepared and investigated.
3. Experimental
2.3. Quantitative structureeactivity relationship studies
All chemicals and reagents were purchased from Aldrich
1
In an attempt to investigate the effect of structural and elec-
tronic parameters on the cytostatic activity of compounds 8e16, a
quantitative structureeactivity relationship (QSAR) study was
Chemical Co. and were used without further purification. H and
13
C NMR were recorded on a Bruker AV300 spectrophotometer at
300 and 75 MHz, respectively in CDCl where residual CHCl
3
3

N.K. Paul et al. / European Journal of Medicinal Chemistry 87 (2014) 461e470
467
1
(
d
¼ 7.27) served as the internal standard for H NMR, and CDCl
3
3.3. General procedure for the synthesis of (3E,5E)-1-((E)-3-
arylacryloyl)-3,5-bis(pyridin-4-ylmethylene)piperidin-4-ones
(8e17)
13
was used as the internal standard (
d
¼ 77.0) for C NMR. Melting
points were recorded on a MEL-TEMP II apparatus and are reported
as uncorrected values. UVeVis and IR spectra were recorded on
Pharmacia Biotech Ultrospec 4000 and Nicolet Avatar 330FT-IR
spectrophotometers, respectively. EIHR-MS spectra were obtained
on a CEC-21 110B (Dupont) spectrometer while ESI-HRMS and
APCI-HRMS spectra were recorded on a microTOF (Bruker Dal-
tonics) spectrometer. Silica 60 F254 aluminum-backed sheets
The mixture of appropriate substituted trans-cinnamic acid
(3 mmol), thionyl chloride (6 mmol) and DMF (1 drop) in dry 1,2-
dichloroethane (10 ml) were refluxed for 4 h under anhydrous
conditions. Volatiles were then removed under reduced pressure at
ꢄ
50 C. The residue (acid chloride) was dissolved in 1,2-
(
Merck, Darmstadt, Germany) and silica gel 60 (SiliaFlash P60,
dichloroethane (5 ml). This solution was added dropwise to an
ice-cold stirred solution of 6 (2 mmol) and triethylamine (9 mmol)
in 1,2-dichloroethane (20 ml) over 10 min under anhydrous con-
ditions. The reaction mixture was stirred for an additional 17 h at
room temperature. Upon completion of the reaction (monitored by
TLC, solvent system 10% MeOH in DCM), the reaction mixture was
Silicycle, Quebec City, Canada) were used for thin layer chroma-
tography (TLC) and flash chromatography, respectively. 2,2-Azobis
(
2-amidinopropane) dihydrochloride (AAPH), 2,2-diphenyl-1-
picrylhydrazyl (DPPH), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ),
nuclease-free agarose, TBE buffer, dimethyl sulfoxide, ferric chlo-
ride, fluorescein, phosphate buffer and trolox were purchased from
SigmaeAldrich (St. Louise, MO, USA). Clear polypropylene 96-well
microplates were obtained from Corning Incorporated (Edison,
NY, USA). Cell culture flasks, DMEM cell growth media, fetal bovine
serum, cell culture grade DMSO, penicillin and streptomycin were
obtained from Cederlane Labs (Burlington, ON, Canada).
washed successively with a saturated NaHCO
water (2 ꢃ 50 ml). The organic layer was dried over anhydrous
Na SO and filtered. The filtrate was rotary-evaporated to dryness
3
solution (50 ml) and
2
4
to yield the crude product which was column-chromatographed on
silica gel (eluent 2% MeOH in DCM) to obtain the pure product.
3.3.1. (3E,5E)-1-((E)-3-Phenylacryloyl)-3,5-bis(pyridin-4-
3
4
.1. Synthesis of (3E,5E)-3, 5-Bis(pyridin-4-ylmethylene)piperidin-
-one trihydrochloride (6)
ylmethylene)piperidin-4-one (8)
Yield: 51%; m.p. 168e170 C. 1H NMR (CDCl
NHeCH
ꢄ
3
): d 4.93 (bs, 4H,
2
), 6.38e6.44 (d, 1H, J ¼ 15.3 Hz, C]CH), 7.13e7.15 (d, 2H,
To an ice-cold stirred mixture of 4-piperidone hydrochloride
J ¼ 5.7 Hz, AreH), 7.32e7.36 (m, 7H, AreH), 7.56e7.61 (d, 1H,
J ¼ 15.3 Hz, C]CH), 7.77 (s, 2H, C]CH), 8.76e8.77 (m, 4H, AreH).
hydrate (30 mmol) and pyridine-4-carboxaldehyde (75 mmol) in
acetic acid (150 ml), dry HCl gas (generated by controlled addition
13
C NMR: 47.12,115.70,124.18,128.01,129.30, 130.62,134.76,135.40,
of conc. H
for 2 h. After stirring the resulting mixture at room temperature for
4 h, dry HCl gas was again passed briskly for 1 h. The stirring was
2
SO
4
to a mixture of NaCl in conc. HCl) was passed briskly
142.13, 145.24, 150.67, 166.20, 186.57. IR (KBr Disc):
n
3015, 2922,
ꢂ1
1653, 1647, 1617, 1591 cm . UV (MeOH):
lmax 204, 284 nm. EI
þ
2
HRMS: calcd. for C26
H
21
N
3
O2, 407.1634 [M ], found: 407.1639.
continued for an additional 48 h. The reaction mixture was cooled
in the refrigerator for 2 h. The yellow precipitate formed was trit-
urated and filtered. The solid precipitate was washed with cold
methanol (2 ꢃ 20 ml) and dried under vacuum.
3.3.2. (3E,5E)-1-((E)-3-(4-Chlorophenyl)acryloyl)-3,5-bis(pyridin-
4-ylmethylene)piperidin-4-one (9)
Yield: 20%; m.p. 164e166 C. 1H NMR (CDCl
ꢄ
d
4.92 (bs, 4H,
3
):
NHeCH
2
), 6.33e6.38 (d, 1H, J ¼ 15.6 Hz, C]CH), 7.03e7.06 (d, 2H,
3
.1.1. (3E,5E)-3, 5-Bis(pyridin-4-ylmethylene)piperidin-4-one
trihydrochloride (6)
Yield 55%; m.p. 212e214 C. H NMR (CDCl
.00 (bs, 4H, NHeCH ), 7.43e7.46 (m, 4H, AreH), 7.51 (s, 2H, C]
CH), 8.64e8.66 (d, 4H, J ¼ 6.6 Hz, AreH). C NMR (CDCl
24.43, 133.72, 138.22, 142.64, 150.58, 187.35. EI HRMS: calcd. for
J ¼ 8.4 Hz, AreH), 7.30e7.34 (m, 6H, AreH), 7.50e7.55 (d, 1H,
1
3
J ¼ 15.3 Hz, C]CH), 7.77 (s, 2H, C]CH), 8.76e8.78 (m, 4H, AreH).
C
ꢄ
1
3
):
d
2.82 (bs, 1H, NH),
NMR: 48.00, 116.21, 124.18, 129.15, 129.57, 133.22, 135.34, 136.53,
d
4
2
142.12, 143.41, 150.89, 165.87, 186.40. IR (KBr Disc.):
n
3069, 2926,
13
ꢂ1
3
):
d
48.18,
1646,1606,1119,1094, 715, 696 cm . UV (MeOH):
lmax 206, 286 nm.
þ
1
3
EI HRMS: calcd. for C26H20Cl N O2, 441.1244 [M ], found: 441.1249.
þ
C
17
H
15
N
3
O, 277.1215 [M ], found: 277.1221.
3.3.3. (3E,5E)-1-((E)-3-(4-Methylphenyl)acryloyl)-3,5-bis(pyridin-
3
.2. Synthesis of (3E,5E)-1-acryloyl-3,5-bis(pyridin-4-ylmethylene)
4-ylmethylene)piperidin-4-one (10)
ꢄ
1
piperidin-4-one, (7)
Yield: 58%; m.p. 175e177 C. H NMR (CDCl
3
): d 2.36 (s, 3H,Ar-
CH
3
), 4.92 (bs, 4H, NHeCH
2
), 6.32e6.37 (d, 1H, J ¼ 15.3 Hz, C]CH),
To an ice-cold stirred solution of 6 (2 mmol) and triethylamine
7.01e7.04 (d, 2H, J ¼ 8.7 Hz, AreH), 7.12e7.14 (d, 2H, J ¼ 7.8 Hz,
AreH), 7.30e7.34 (m, 4H, AreH), 7.54e7.59 (d, 1H, J ¼ 15.3 Hz, C]
(
9 mmol) in dry 1,2-dichloroethane (20 ml), solution of acryloyl
13
chloride (3 mmol) in dry 1,2-dichloroethane (5 ml) was added
dropwise over 10 min under anhydrous conditions. The reaction
mixture was stirred for additional 8 h at room temperature. Upon
completion of the reaction (monitored by TLC, solvent system 10%
MeOH in DCM), the reaction mixture was washed successively with
CH), 7.76 (s, 2H, C]CH), 8.76e8.78 (m, 4H, AreH). C NMR: d 21.83,
44.64, 114.49, 124.19, 128.02, 130.0, 132.01, 135.46, 141.11, 142.10,
144.88, 150.97, 166.34, 184.50. IR (KBr Disc):
n 2926, 1645, 1590,
ꢂ1
1441, 1384 cm . UV (MeOH):
lmax 224, 290 nm. EI HRMS: calcd. for
þ
27 23
C H N
3
O
2
, 421.1790 [M ], found: 421.1773.
saturated NaHCO
organic layer was dried over anhydrous Na
3
solution (50 ml) and water (2 ꢃ 50 ml). The
2
SO and filtered. The
4
3.3.4. (3E,5E)-1-((E)-3-(4-Methoxyphenyl)acryloyl)-3,5-
filtrate was rotary-evaporated to dryness to yield the crude product
which was column-chromatographed on silica gel (eluent 2%
MeOH in DCM) to obtain the pure product. Yield: 48%; m.p.
bis(pyridin-4-ylmethylene)piperidin-4-one (11)
ꢄ
1
Yield: 57%; m.p. 181e184 C. H NMR (CDCl
OCH ), 4.87 (bs, 4H, NHeCH
3
): d 3.79 (s, 3H, Ar-
3
2
), 6.20e6.26 (d, 1H, J ¼ 15.3, C]CH),
ꢄ
1
6
8e172 C (D). H NMR (CDCl
3
):
d
4.83 (bs, 4H, NHeCH
2
), 5.83e5.96
6.78e6.81 (d, 2H, J ¼ 9.0 Hz, AreH), 7.04e7.07 (d, 2H, J ¼ 9, AreH),
(
dd, 1H, J ¼ 8.4, 3.6 Hz, HC]CH
2
), 6.22 (m, 1H, C]CH), 7.285 (s, 4H,
7.29e7.39 (m, 4H, AreH), 7.49e7.55 (d, 1H, J ¼ 15.3 Hz, ¼CH), 7.70
13
13
AreH), 7.71 (s, 2H, C]CH), 8.69e8.71 (d, J ¼ 3.9 Hz, 4H, AreH).
C
(s, 2H, C]CH), 8.72e8.73 (m, 4H, AreH). C NMR:
d
45.10, 55.78,
NMR: 47.18, 125.2, 129.4, 134.4, 136.7, 142.0, 151.5, 155.4, 185.4. IR
114.69, 121.73, 124.21, 127.41, 129.70, 135.52, 142.09, 144.54, 145.09,
ꢂ1
(
KBr Disc):
n
3087, 2923, 1643, 1627, 1600, 1459, 1376 cm . APCI
150.73, 161.68, 166.38, 186.55. IR (KBr Disc):
n
3055, 2930, 1683,
max 205, 231,
ꢂ1
HRMS: calcd. for C20
18
H N
3
O
2
, 332.1399 [MþH], found: 332.1391.
1674, 1598, 1574, 1252, and 1174 cm . UV (MeOH):
l

468
N.K. Paul et al. / European Journal of Medicinal Chemistry 87 (2014) 461e470
þ
2
4
83 nm. EI HRMS: calcd. for C27
37.1742.
23
H N
3
O
3
, 437.1739 [M ], found:
1H,J ¼ 9 Hz, AreH), 7.33e7.48 (m, 7H, AreH), 7.78 (s, 2H, AreH),
13
8.77e8.79 (m, 4H, AreH). C NMR:
d 47.29, 112.78, 113.05, 115.29,
1
24.17, 131.44, 135.34, 144.90, 142.08, 150.93, 151.34, 165.97, 186.53.
ꢂ1
3.3.5. (3E,5E)-1-((E)-3-(4-Nitrophenyl)acryloyl)-3,5-bis(pyridin-4-
IR (Nujol):
n
3170, 2953, 2851, 1656, 1600, 1460, 1376, 1154 cm . UV
ylmethylene)piperidin-4-one (12)
(MeOH):
l
max 272 nm. APCI HRMS: calcd. for C24 , 398.1505
19 3 3
H N O
Yield: 41%; m.p. 91e94 C. 1H NMR(CDCl
ꢄ
): d 4.94 (bs, 4H,
[MþH], found: 398.1501.
3.4. Biological evaluations
3.4.1. Cytotoxicity evaluations
3
NHeCH
2
), 6.48e6.53 (d, 1H, J ¼ 15.2 Hz, C]CH), 7.24e7.27 (d, 2H,
J ¼ 8.7 Hz, AreH), 7.29e7.34 (m, 4H, AreH), 7.56e7.61 (d, 1H,
J ¼ 15.6 Hz, AreH), 7.79 (s, 2H, C]CH), 8.16e8.19 (d, 2H, J ¼ 8.7 Hz,
1
3
AreH), 8.77e8.79 (m, 4H, AreH). C NMR:
d 47.65, 120.04, 124.15,
1
1
1
24.58, 128.56, 135.09, 140.83, 141.87, 141.99, 148.78, 150.96, 165.14,
86.17. IR (KBr Disc): 2925, 1734, 1684, 1675, 1576, 1521, 1429,
384, 1343 cm . UV (MeOH): max 204, 299 nm. ESI-HRMS: calcd.
for C26
, 453.1596 [MþH], found: 453.1565.
The Molt4/C8, CEM and L1210 assays were conducted using a
literature methodology [34] and the resulting data are reported in
Table 1. The human tumor cell line screen at the National Cancer
Institute (NCI) was undertaken by a reported procedure [36] using
n
ꢂ1
l
21 4 4
H N O
57 or 59 tumor cell lines. The data are summarized in Table 2.
3.3.6. (3E,5E)-1-((E)-3-(3,4-Dichlorophenyl)acryloyl)-3,5-
bis(pyridin-4-ylmethylene) piperidin-4-one (13)
3.4.2. Culture of human fibroblasts
ꢄ
1
Human fibroblasts or WI-38 cells (ATCC® CCL-75™) were ob-
tained from Cederlane Labs (Burlington, ON, Canada) and cultured
according to the supplier's instructions. The cells were maintained
Yield: 42%; m.p. 184e186 C. H NMR(CDCl
NHeCH
), 6.36e6.51 (m, 4H, AreH, ¼CH), 7.34e7.41 (m, 5H, AreH,
C]CH), 7.76 (s, 2H, C]CH), 8.75e8.77 (m, 4H, AreH). C NMR:
44.91, 117.50, 124.17, 126.83, 129.69, 131.30, 133.67, 134.77, 135.17,
3
): d 4.91 (bs, 4H,
2
13
2
ꢄ
d
in 75 cm culture flasks at a temperature of 37 C in a humidified
1
42.17, 151.02, 165.49, 186.28. IR (KBr Disc):
n
3050, 2925, 1645,
incubator (VWR International, Mississauga, ON) supplied with 95%
air and 5% CO . WI-38 cells were supplied with DMEM medium,
2
ꢂ1
1619, 1600, 1545, 1416, 1384, 823, 759, 597, 522 cm . UV (MeOH):
l
max 204, 300 nm. ESI HRMS: calcd. for C26
H19Cl
2
N
3
O
2, 476.0933
supplemented with 10% FBS and 100 mg/L of penicillin and strep-
tomycin. The growth medium of cultured cells was changed every
þ
[M ], found: 476.0910 and 478.0908.
4
8 h.
3.3.7. (3E,5E)-1-((E)-3-(3,4-Dimethoxyphenyl)acryloyl)-3,5-
bis(pyridin-4-ylmethylene) piperidin-4-one (14)
3.4.3. Topoisomerase assays
ꢄ
1
Yield: 67%; m.p. 82e86 C. H NMR(CDCl
.74 (s, 3H, OCH ), 4.95 (bs, 4H, NHeCH
AreH, ¼CH), 7.29e7.34 (m, 5H, AreH, C]CH), 7.78 (s, 2H, C]CH),
3
):
d
3.60 (s, 3H, OCH
3
),
All test compounds were assayed at a concentration of 50 mM
3
3
2
), 6.75e6.86 (m, 4H,
using a commercial kit, (Topogen Inc, Columbus, OH). The com-
pounds were challenged against 4 units of topoisomerase enzyme
using pHOT1 DNA as the substrate. The assay is based upon eval-
uating the formation of DNA cleavage products, primarily linearized
DNA (interfacial poisons) and/or supercoiled product (catalytic
inhibitory compounds). All the preparatory and analysis steps were
followed according to the manufacturer's instructions and all
samples were loaded directly onto a 2% agarose gel in 1x TBE (Tris/
Borate/EDTA) running buffer. Gels were run until the dye front was
about 80% down the gel and then stained using Gel red nucleic acid
dye (VWR, ON, Canada). In addition to VP16 (etoposide), sorafenib
and cisplatin were also used as drug standards in the analysis. The
DNA bands were visualized by placing gels on a UV sample tray,
followed by exposure to UV light using gel Doc™ EZ System (Biorad,
ON, Canada). After general screening of all compounds, the active
compounds were run at different concentrations to obtain IC50
values for their topoisomerase II inhibition potential. The band-
wise topoisomerase inhibition was calculated using Gel Doc™
EZ's software SYBR safe by measuring the relative front of each
band with an exposure time of 0.25 s through a UV trans-
illumination mode.
1
3
8
1
1
1
.73e8.75 (m, 4H, AreH). C NMR:
15.50, 116.69, 117.26, 124.25, 124.33, 135.35, 140.83, 142.21, 150.80,
53.30, 153.80, 166.84, 186.25. IR (Nujol): 3025, 2955, 2922, 1658,
637, 1591, 1579, 1460, 1376 cm . EI-HRMS: calcd. for C28
d 48.02, 56.11, 56.15, 112.61,
n
ꢂ1
25 3 4
H N O ,
þ
4
67.1845 [M ], found: 467.1847.
3.3.8. (3E,5E)-1-((E)-3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)-3,5-
bis(pyridin-4-ylmethylene) piperidin-4-one (15)
ꢄ
1
Yield: 45%; m.p. 177e180 C. H NMR(CDCl
3
): d 4.90 (bs, 4H,
NHeCH ), 6.02 (s, 2H, OeCH
2
2
eO), 6.18e6.23 (d, 1H, J ¼ 15.3 Hz, C]
CH), 6.54 (s, 1H, AreH), 6.74e6.77 (m, 2H, AreH), 7.29e7.34 (m, 4H,
AreH), 7.48e7.53 (d, 1H, J ¼ 15 Hz, C]CH), 7.76 (s, 2H, C]CH),
13
8
.76e8.78 (m, 4H, AreH). C NMR: d 47.40, 102.04, 106.38, 108.96,
1
1
1
13.44, 124.49, 129.15, 135.46, 140.10, 144.77, 141.99, 148.71, 149.97,
50.98, 165.30, 186.60. IR (KBr Disc): 3070, 3049, 2970, 1678, 1647,
593,1260,1245, 839, 698, 509 cm . UV (MeOH): max 205, 235, 292.
, 451.1537 [M ], found: 451.1532.
n
ꢂ1
l
þ
21 3 4
EI-HRMS: calcd. for C27H N O
3.3.9. (3E,5E)-1-((E)-3-(3,4,5-Trimethoxyphenyl)acryloyl)-3,5-
bis(pyridin-4-ylmethylene) piperidin-4-one (16)
ꢄ
1
Yield: 62%; m.p. 198e200 C. H NMR(CDCl
OCH ), 3.86 (s, 3H, OCH ), 4.95 (bs, 4H, NHeCH ), 6.33e6.38 (d, 1H,
5 Hz, C]CH), 6.46 (s, 2H, AreH), 7.32e7.35 (m, 4H, AreH),
.51e7.56 (d, 1H, J ¼ 15 Hz, C]CH), 7.79 (s, 2H, AreH), 8.75e8.77
3
):
d
3.78 (s, 6H,
3.4.4. Antioxidant activity assays
3
3
2
3.4.4.1. Ferric reducing ability of a plasma (FRAP) assay. The FRAP
assay was performed according to the method described earlier [8].
Briefly, the FRAP working reagent was prepared by adding 300 mM
acetate buffer (pH 3.6), 10 mM TPTZ solution and 20 mM ferric
1
7
(
13
m, 4H, AreH). C NMR:
23.21, 129.42, 134.53, 140.25, 141.33, 144.09, 149.96, 153.06, 165.19,
85.23. IR (KBr Disc): 3025, 2945, 2922, 1683, 1634, 1591, 1504,
455, 1124 cm . ESI HRMS: calcd. for C29
, 498.2029 [MþH]
d 44.66, 55.81, 60.38, 105.00, 113.89,
1
chloride in the ratio of 10:1:1. The test compounds (20
mL) and FRAP
1
n
working reagent (180 L) were added to 96-well microplates and
m
ꢂ1
1
H
27
N
3
O
5
absorbance was read at 590 nm using the FLUOstar OPTIMA plate
reader (BMG Labtech, Durham, NC, USA). The anti-oxidant capacity
þ
,
found: 498.2012.
ꢂ1
results were expressed as
mM TE L of solution.
3.3.10. (3E,5E)-1-((E)-3-(Furan-2-yl)acryloyl)-3,5-bis(pyridin-4-
ylmethylene)piperidin-4-one (17)
3.4.4.2. Oxygen radical absorbance capacity (ORAC) assay. The
ORAC assay was performed according to the method described
earlier [8]. Briefly, 35 mL of trolox standard or sample, 130 mL of
Yield: 40%; m.p. 93e96 C. 1H NMR(CDCl
NHeCH ), 6.35e6.40 (d, 1H, 15 Hz, C]CH), 6.91e6.94 (d,
ꢄ
):
d
4.91 (bs, 4H,
3
2

N.K. Paul et al. / European Journal of Medicinal Chemistry 87 (2014) 461e470
469
fluorescein (0.957
the black 96-well plates and pre-incubated at 37 C for 10 min. The
m
M) and 35
m
L of AAPH (150 mM) were added to
300). One out of two mice died at a 300 mg/kg dose during the 4 h
test. Mice were fed, handled and housed in accordance with the
procedures outlined in the National Research Council publication
“Guide for the Care and Use of Laboratory Animals”. The animals
were euthanized using the guidelines of the Institute of Laboratory
Resources.
ꢄ
assay controls included a positive control with 140
0.96 M) and 60 L of phosphate buffer (75 mM, pH 7) along a
buffer control with 200 L of phosphate buffer. A negative control
comprised of 130 L fluorescein, 35 L of AAPH and 35 L of
mL fluorescein
(
m
m
m
m
m
m
phosphate buffer. The fluorescence signal was read at an excitation
and emission wavelength of 485 nm and 510 nm using the FLUOstar
3
.5. Statistical analyses using electronic and physicochemical
OPTIMA plate reader (BMG Labtech, Durham, NC, USA). The anti-
parameters
ꢂ1
oxidant capacity results were expressed as
m
M TE L of solution.
Electronic and physicochemical properties such as charge
partition coefficient (LogP), and molar refractivity of the molecule
were calculated using a commercial software [46]. The values for
Hammett s, Hansch p, and molar refractivity (MRsub) of the sub-
stituents were taken from the literature [47]. The MRsub value of the
hydrogen atom is 1.03 and not 0.00. Since the compound with most
substitution is trisubstituted (16), the MRsub figure for the unsub-
stituted compound 8 was 3.09. The correlations between these
molecular descriptors and the IC50 values of the three cell lines
b,
3
.4.4.3. The 2,2-diphenylpicrylhydrazyl (DPPH) assay. The DPPH
assay was performed according to the method described earlier [8].
Briefly, the test compounds were challenged against 0.2 mM DPPH
solutions in a 1:1 ratio in 96-well plates. Upon addition of both
compounds and DPPH solution, the plates were incubated at 25 C
for 10 min. The DPPH percentage inhibition was calculated from the
following equation after absorbance was measured using
FLUOstar OPTIMA plate reader (BMG Labtech, Durham, NC, USA) at
5
ꢄ
a
20 nm % inhibition ¼ 100(Ablank ꢂ Asample)/Ablank
(
[
Table 1) were generated using a commercial software package
48]. All significant correlations are reported in Table 4.
3
.4.4.4. Cellular reactive oxygen species (ROS) detection assay.
ROS were analyzed using a methodology outlined previously [43].
4
. Conclusions
Briefly, WI-38 cells were seeded in a 24-well sterile plate at the
5
density of 2 ꢃ 10 cells per well. Cells were allowed to adhere for
In conclusion, the series of all trans 1-(3-arylacryloyl)-3,5-bis(-
2
4 h and then incubated with the test compounds for 12 h prior to
pyridin-4-ylmethylene)piperidin-4-ones were found to possess
potent cytostatic activity against representative cancer cell lines.
They were found to be significantly more potent and selective than
the reference drugs melphalan and curcumin against human CD
lymphocyte Molt C4/8 and CEM tumor cell proliferation. QSAR re-
sults indicate that more lipophilic analogs with a more electrophilic
oxidative insult using AAPH. After incubation, cell growth media
containing the test compounds were removed and cells were
washed twice with PBS. Cellular growth media were changed to
þ
4
T-
FBS-free medium and 5
mM of fluorescein was added to the cells,
and the plate was again placed in the incubator for 5 min. After
5
min, 30 mM AAPH was added to the wells of a 96-well plate at
3
-arylacryloyl group may further afford bioactivity. As these com-
ꢄ
temperature of 37 C. The 96-well plates were immediately placed
pounds have been shown to inhibit topoisomerase II, and since they
are structurally divergent from known topoisomerase II inhibitors,
they may be of value in treating drug-resistant tumors. The in vitro
lung and in vivo neurotoxicity experiments revealed that the final
test compounds (amides) were well-tolerated by human fibroblasts
in the fluorescence plate reader at an excitation wavelength of
4
90 nm and an emission wavelength of 510 nm. Fluorescence was
measured at intervals of 60 min (initial reading at 0 min and final
reading at 60 min) and ROS were estimated as percentage inhibi-
tion of flourescein with respect to an assay control.
as well as by mice at concentrations up to 100 mM and 300 mg/kg
body weight, respectively. Strong anti-oxidant activity was noted
for selective derivatives, with some members exhibiting greater
anti-oxidant activity than the parent compound. These results
indicate that the investigations of all trans 1-(3-arylacryloyl)-3,5-
bis(pyridin-4-ylmethylene)piperidin-4-ones is a promising area of
study to fine-tune the anticancer potential of these molecules.
3
.4.5. In-vitro toxicology analysis
In vitro toxicity analysis was conducted using the CytoTox-
ONE™ homogeneous membrane integrity assay (Promega Corpo-
4
ration, Madison, WI). Briefly, 2 ꢃ 10 WI-38 cells were seeded using
1
00
mL media in a 96-well tissue culture plate. Cells were pretreated
ꢄ
with the test compounds for 24 h and placed at the 37 C in a
humidified incubator (VWR International, Mississauga, ON) for
ꢄ
Acknowledgments
2
4 h. Plates were removed from 37 C incubator and placed at room
temperature for 30 min. All the remaining steps for membrane
damage assessment assay were performed according to the man-
ufacturer's instructions. The absorbance value was read using a
BioTek, Power wave XS2 spectrophotometer (BioTek, Winooski, VT)
at the wavelength of 490 nm. The percentage membrane damage
was expressed as the release of the enzyme lactate dehydrogenase
from cells with respect to the positive control cells.
The authors thank the following agencies for financial support,
namely the Nova Scotia Health Research Foundation (A.J.), Natural
Sciences and Engineering Research Council of Canada (A. J.), Fonds
voor Wetenschappelijk Onderzoek-Vlaanderen (J. B.). Appreciation
is extended to Mrs. Lizette van Berckelaer for conducting the Molt4/
C8, CEM and L1210 assays, the National Cancer Institute, U.S.A.,
which provided the data using the panel of human tumor cell lines
and the National Institute of Neurological Disorders and Stroke,
U.S.A. for undertaking the short-term toxicity studies in mice.
H.P.V.R. gratefully acknowledges funding from the Canada Research
Chair program and the Discovery Grant program of the Natural
Sciences and Engineering Research Council (NSERC) of Canada.
3.4.6. In-vivo toxicity evaluations
Compounds 6, 11 and 13 were examined for short-term survival
and neurotoxicity by the National Institute of Neurological Disor-
ders and Stroke according to their protocols [45]. In brief, mice were
injected intraperitoneally with doses of 30, 100 and 300 mg/kg for
each compound and the animals were observed at the end of 0.5
and 4 h. Only compound 6 demonstrated neurotoxicity in the
rotarod method [49] (number of animals demonstrating neuro-
toxicity/total number of animals tested, time of test in h, dose of
compound in mg/kg): (1/8, 0.5, 100; 1/4, 0.5, 300; 3/4, 4, 100; 1/2, 4,
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.090.

470
N.K. Paul et al. / European Journal of Medicinal Chemistry 87 (2014) 461e470
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