Synthesis of N-substituted 3,5-bis(arylidene)-4-piperidones with high antitumor and antioxidant activity

Article abstract of DOI:10.1021/jm200353f

A series of 3,5-bis(arylidene)-4-piperidone (DAP) compounds are considered as synthetic analogues of curcumin for anticancer properties. We performed structure-activity relationship studies by synthesizing a number of DAPs N-alkylated or acylated with nitroxides or their amine precursors as potent antioxidant moieties. Both subtituents on arylidene rings and on piperidone nitrogen (five- or six-membered, 2- or 3-substituted or 3,4-disubstituted isoindoline nitroxides) were varied. The anticancer efficacy of the new DAP compounds was tested by measuring their cytotoxicity to cancer cell lines A2780 and MCF-7 and to the H9c2 cell line. The results showed that all DAP compounds induced a significant loss of cell viability in the human cancer cell lines tested; however, only pyrroline appended nitroxides (5c (Selvendiran, K.; Tong, L.; Bratasz, A.; Kuppusamy, L. M.; Ahmed, S.; Ravi, Y.; Trigg, N. J.; Rivera, B. K.; Kálai, T.; Hideg, K.; Kuppusamy, P.Mol. Cancer Ther. 2010, 9, 1169-1179), 5e, 7, 9) showed limited toxicity toward noncancerous cell lines. Computer docking simulations support the biological activity tested. These results suggest that antioxidant-conjugated DAPs will be useful as a safe and effective anticancer agent for cancer therapy.

Full text of DOI:10.1021/jm200353f

ARTICLE
pubs.acs.org/jmc
Synthesis of N-Substituted 3,5-Bis(arylidene)-4-piperidones with High
Antitumor and Antioxidant Activity
Tamꢀas Kꢀalai,† M. Lakshmi Kuppusamy,‡ Mꢀaria Balog,† Karuppaiyah Selvendiran,‡ Brian K. Rivera,‡
Periannan Kuppusamy,‡ and Kꢀalmꢀan Hideg*,†
†Institute of Organic and Medicinal Chemistry, University of Pꢀecs, H-7624 Pꢀecs, Szigeti st. 12, Hungary
‡Department of Internal Medicine, Davis Heart and Lung Research Institute, The Ohio State University, Columbus,
Ohio 43210, United States
S Supporting Information
b
ABSTRACT: A series of 3,5-bis(arylidene)-4-piperidone (DAP) compounds are con-
sidered as synthetic analogues of curcumin for anticancer properties. We performed
structureꢀactivity relationship studies by synthesizing a number of DAPs N-alkylated or
acylated with nitroxides or their amine precursors as potent antioxidant moieties. Both
subtituents on arylidene rings and on piperidone nitrogen (five- or six-membered, 2- or
3-substituted or 3,4-disubstituted isoindoline nitroxides) were varied. The anticancer
efficacy of the new DAP compounds was tested by measuring their cytotoxicity to cancer
cell lines A2780 and MCF-7 and to the H9c2 cell line. The results showed that all DAP
compounds induced a significant loss of cell viability in the human cancer cell lines tested; however, only pyrroline appended
nitroxides (5c (Selvendiran, K.; Tong, L.; Bratasz, A.; Kuppusamy, L. M.; Ahmed, S.; Ravi, Y.; Trigg, N. J.; Rivera, B. K.; Kꢀalai, T.;
Hideg, K.; Kuppusamy, P. Mol. Cancer Ther. 2010, 9, 1169ꢀ1179), 5e, 7, 9) showed limited toxicity toward noncancerous cell lines.
Computer docking simulations support the biological activity tested. These results suggest that antioxidant-conjugated DAPs will be
useful as a safe and effective anticancer agent for cancer therapy.
’ INTRODUCTION
one or more binding sites as well as variation of hydrophobicity
and hence membrane transportation properties.16
Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)1,6-hepta-
dien-3,5-dion), a natural component of the rhizome of Curcuma
longa, proved to be a powerful chemopreventive and anticancer
agent2ꢀ5 having also anti-inflammatory,6 antibacterial,7 and anti-
oxidant properties.8 However, the clinical use of curcumin has
been limited because of its low anticancer activity and poor
bioabsorption. In the past decade, a novel class of curcumin
analogues, DAPs, has been developed by incorporating a piper-
idone link to the β-diketone structure and fluoro-, methoxy-,
hydroxyl-, chloro-, nitro-, dimethylamino substituents on the
phenyl group.9,10 These curcumin analogues exhibited multi-
drug-resistance reverting11,12 and antimycobacterial13 properties
as well. The idea of evaluation of these compounds as antineo-
plastic agents is based on the assumption that these compounds
may be considered as a Mannich base of dienone and R,
β-unsaturated ketones. These kinds of compound display antic-
ancer properties via a mechanism of action comprising interac-
tions with cellular thiols with little or no affinity for hydroxyl and
amino groups in nucleic acids. The 1,5-diaryl-3-oxo-1,4-penta-
dienyl groups are considered to react at a primary binding site;
however, the bioactivity will be influenced by other structural
units as well. The acylation of piperidone nitrogen increased the
cytotoxic potencies, and increasing the electron-withdrawing
properties of substituents on aromatic ring has an advantageous
effect on cytotoxicity.14ꢀ18 The dimethylene bridge between C2
and C6 atoms in piperidone was accompanied by reduction of
cytotoxic properties exerting a steric impedance to alignment at
In general the DAP compounds were more effective than
curcumin in inhibiting the proliferation of a variety of cancer cell
lines. For example 3,5-bis[2-(fluoro)benzylidene]piperidin-
4-one (EF24) with ortho-fluorinated phenyl group exhibited anti-
cancer activity in vitro when tested using breast cancer, colon
cancer, and ovarian epithelial cancer.19ꢀ21 Its para-fluorinated
derivative 3,5-bis[4-(fluoro)benzylidene]piperidin-4-one (H-4073)
was more potent than 6 (EF24) in inducing cytotoxicity to ovarian
cancer cells.21ꢀ23 DAP compounds have also been shown to be
more readily bioavailable than the parent compound curcumin.24
A nonspecific cytotoxic compound may have side effects
caused by damage to normal cells. Many chemotherapeutic agents
act by producing free radicals, causing oxidative stress in normal
cells.25 It is well-known that nitroxides or their precursors (hydro-
xylamines and sterically hindered amines) scavenge oxygen
radicals in cells that have normal redox status and have beneficial
effect on toxicity and/or efficiency in reactive oxyges species (ROS)
scavengingcomparedtotheoriginaldrug.26,27 Our previousstudies
indicated that nitroxides or their amine precursors play multiple
roles in elimination of ROS formed during doxorubicine meta-
bolism without reducing their anticancer effect.28,29 These results
inspired us to combine anticancer and antioxidant properties
to decrease ROS-promoted damage. The DAPs were ideal
Received: March 27, 2011
Published: June 27, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
Scheme 1a
a Reagents and conditions: (a) compound 2a (1.0 equiv), Et3N (2.0 equiv) CH2Cl2, 0° C f room temp, 1 h, 49%, or compound 2b (1.0 equiv), THF,
reflux, 4 h, 62%; (b) 4aꢀh (1.0 equiv), K2CO3, (1.0 equiv), acetonitrile, reflux, 3 h, 35ꢀ68%; (c) Fe (10.0 equiv), AcOH, 60 °C, 35 min, 56%.
candidates to prove the conception because of the easily variable
nitrogen substituents of the piperidone moiety. This study
presents the synthesis and evaluation of new DAP compounds
with different substituents (F, CF3, OCH3) on the aromatic rings,
as well as variation of nitroxides (saturated, unsaturated, six-
membered, isoindoline, etc.) attached to the piperidone nitrogen.
The study showed that the DAPs induce preferential toxicity
in cancer cells while sparing noncancereous cells. The results
suggest that the antioxidant (nitroxide) conjugated DAPs will be
useful as safe and effective anticancer agents for cancer therapy.
propose that compounds 1 (H-4073), 6, 8, 10 possess E stereo-
chemistry.10,14,15 The new N-acyl-3,5-bis(4-fluorobenzylidene)-
piperidin-4-ones were prepared by treatment of compound 1 with
freshly prepared paramagnetic acyl chloride 2a30 in the presence of
Et3N in CH2Cl2 or treatment with isocyanate 2b31 generated
in situ by Curtius rearrangement of acyl azide in THF to yield
compounds 3a and 3b. The N-alkyl 3,5-bis(4-fluorobenzylidene)-
piperidin-4-ones were achieved by alkylation of compound 1 with
an equivalent amount of paramagnetic alkyl halides 4a,32 4b,32
4c,33 4d,34 4e,35 4f,36 4g37 in acetonitrile in the presence of K2CO3
to give compounds 5a, 5b, 5c, 5d, 5f, 5g, 5h. Compound 5e
was synthesized by reduction of the nitroxide function of com-
pound 5c with iron powder in glacial acetic acid38 (Scheme 1).
Alkylation of 3,5-bis(2-fluorobenzylidene)piperidin-4-one 6,19
3,5-bis(4-trifluoromethylbenzylidene)piperidin-4-one 8, and
’ CHEMISTRY
A ClaisenꢀSchmidt condensation between 4-piperidone hy-
drochloride and the appropriate aldehyde led to the formation of
DAPs. On the basis of earlier X-ray crystallography data, we
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ARTICLE
Scheme 2a
a Reagents and conditions: (a) compound 4c (1.0 equiv), K2CO3, (1.0 equiv), acetonitrile, reflux, 3 h, 55ꢀ71%.
Scheme 3a
a Reagents and conditions: (a) 4-trifluoromethylbenzaldehyde (2.0 equiv), AcOH saturated with HCl gas, 48 h, room temp, 74%; (b) 2,3,4-
trimethoxybenzaldehyde (2.0 equiv), AcOH saturated with HCl gas, 48 h, room temp, 62%.
3,5-bis(2,3,4-trimethoxybenzylidene)piperidin-4-one 10 with com-
pound 4c as above yielded compounds 7, 9, 11, respectively
(Scheme 2). Compounds 8 and 10 were synthesized by con-
densation of piperidine-4-one HCl (12) salt with 4-trifluoro-
methylbenzaldehyde or 2,3,4-trimethoxybenzaldehyde in AcOH
saturated previously with HCl gas (Scheme 3).
by modifying the DAP compounds with nitroxides by acylation
(3a, 3b) and by alkylation (5aꢀh, 7, 9, 11). In particular 3a and
3b demonstrated a substantial cytotoxic effect against A2780 and
MCF-7 cells. Comparable cytotoxic efficacies were observed with
5c, 5e, 5f, and 9 derivatives. The results further indicated that the
DAPs were more cytotoxic to ovarian cancer cell (A2780) than to
breast cancer cell. Compounds containing 2-substituted pyrroli-
dine nitroxide (5a, 5b), 3,4-disubstituted nitroxide (5d), or
isoindoline-type nitroxides (5g, 5h) exhibited limited toxicity
toward breast cancer cell lines.
We also compared the cytotoxicity of DAPs to a noncancerous
(healthy) cardiac cell line, namely, H9c2, an undifferentiated
neonatal rat cardiomyoblast. Most of the compounds induced a
significant loss of cell viability, although to different extents
(Table 1ꢀ3), the pyrroline-appended DAPs 5c, 5e, and 7 were
significantly less toxic to the healthy cell.
Particularly, the results of 5c seem to suggest a strikingly
differential effect on cancer versus noncancerous cells. Com-
pound 6, which was toxic to breast cancer cell, was toxic to
healthy cells to the same extent. In addition, this differential effect
could stem from the N-hydroxypyrroline function. Overall the
viability results seem to implicate the diarylidenylpiperidone
group in inducing cytotoxicity and N-hydroxypyrroline group
in protecting noncancerous cells.
’ RESULTS AND DISCUSSION
The anticancer efficacy of the DAP compounds with various
substituents on aromatic rings and on piperidone nitrogen was
evaluated by measuring the cytotoxicity of the compounds to
well-established cancer cell lines, namely, A2780 and MCF-7
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay. The measurements were performed by
exposing the cells to 10 μM compound for 24 h. The results,
in the form of percent cell viability compared to respective
control, are summarized in Tables 1ꢀ3. The results showed that
all DAP compounds induced a significant loss of cell viability in
both of the human cancer cell lines tested. In particular 3,5-
bis(arylidene)-4-piperidone compounds without nitroxide tag
(1, 6, 8, 10) demomstrated a substantial cytotoxic effect against
A2780 and MCF-7 cells. The electron-withdrawing substituents
(F, CF3) containing derivatives (1, 6, 8) exhibited greater
cytotoxicity than trimethoxy derivative 10, in agreement with
previous findings.14,15 In all caseses the toxicity can be increased
We recently reported the anticancer efficacy of four DAPs,
namely, 1 and 8 without NOH function and 5c and 9 with NOH
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ARTICLE
Table 1. Biological Activity of New N-Acyl-3,5-bis(4-fluoro-
benzylidene)piperidin-4-ones
Table 2. Biological Activity of N-Alkyl-3,5-bis(4-fluorobenzyl-
idene)piperidin-4-ones
function against a number of cancerous (breast, colon, head and
neck, liver, lung, ovarian, and prostate cancer) and noncancerous
(smooth muscle, aortic endothelial, and ovarian surface epithelial
cells) human cell lines. We observed that all four compounds
induced significant loss of cell viability in cancer cells, while 5c
and 9 showed significantly less cytotoxicity in noncancerous
(healthy) cells. Electron paramagnetic resonance (EPR) mea-
surements showed a metabolic conversion of the N-hydroxyla-
mine function to nitroxide with significantly higher levels of the
metaboliteand superoxide radical-scavenging(antioxidant) activity
in noncancerous cells when compared to cancer cells. The anti-
oxidant activity of compounds 5c and 9 in comparison to their
base (non-nitroxide-derivatized) compounds 1 and 8 was re-
ported recently.22 EPR measurements demonstrated substantial
ability of 5c and 9 to scavenge superoxide radicals but not the
base compounds 1 and 8. Among the new compounds 5c
exhibited the best selective toxicity against cancerous cell. For
this reason, this compound was chosen as a new lead compound
for further evaluations. Western blot analysis showed that the
DAP-induced growth arrest and apoptosis in cancer cells were
mediated by inhibition of STAT3 phosphorylation at Tyr705 and
Ser727 residues and induction of apoptotic markers of cleaved
caspase-3 and poly ADP-ribose polymerase (PARP), suggesting
that the antioxidant-conjugated DAPs will be useful as a safe and
effective anticancer agent for cancer therapy.22 In a subsequent
study, we further confirmed the anticancer efficacy of 5c in a
number of established human ovarian cancer cell lines (A2870,
A2780cDDP, OV-4, SKOV3, PA-1, and OVCAR3), as well as in a
murine xenograft tumor (A2780) model.1 Compound 5c de-
monstrated a preferential toxicity toward ovarian cancer cells
while sparing healthy cells. It induced G2/M cell-cycle arrest in
A2780 cells by modulating cell-cycle regulatory molecules p53,
p21, p27, cdk2, and cyclin, and promoted apoptosis by caspase-8
and caspase-3 activation. It also caused an increase in the
expression of functional Fas/CD95 and decreases in STAT3
(Tyr705) and JAK1 phosphorylation. There was a significant
reduction in STAT3 downstream target protein levels including
Bcl-xL, Bcl-2, survivin, and vascular endothelial growth factor
(VEGF), suggesting that 5c exposure disrupted the JAK/
STAT3-signaling pathway. In addition, compound 5c signifi-
cantly inhibited the growth of the ovarian xenografted tumors in
a dosage-dependent manner without any apparent toxicity.
Western blot analysis of the xenograft tumor tissues showed that
compound 5c inhibited pSTAT3 tyrosine 705 and serine 727
(Tyr705 and Ser727) and JAK1 and increased apoptotic markers
cleaved caspase-3 and PARP. Overall, compound 5c exhibited
significant cytotoxicity toward ovarian cancer cells by inhibition
of the JAK/STAT3-signaling pathway.1
The cellular uptake of 5c was measured in a variety of cancer
cell lines. Compound 5c was taken in cells within 15 min of
exposure, and its uptake was more than 100-fold higher than that
of curcumin. Compound 5c was also retained in cells in an active
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Table 3. Biological Activity of N-(1-Oxyl-2,2,5,5-tetramethyl-
2,5-dihydro-1H-pyrrole-3-ylmethyl)-3,5-bis(4-arylidene)pip-
eridin-4-ones
Table 4. Summary of DAP Compound Docking Simulations
with the Murine STAT3 Dimer (PDB Code 3CWG)
lowest five
binding energies
(kcal/mol)
corresponding constant
of inhibition
simulation
test compd
Ki (nmol)
1
ꢀ8.76a
ꢀ8.76a
ꢀ8.76a
ꢀ8.76a
ꢀ8.75
380.28
379.04
378.52
379.50
386.19
1660
1660
1670
1710
1700
6.75
8
ꢀ7.89a
ꢀ7.89a
ꢀ7.88
ꢀ7.87a
ꢀ7.87a
ꢀ11.15a
ꢀ11.15a
ꢀ11.10
ꢀ11.00
ꢀ10.99
ꢀ10.66
ꢀ10.62
ꢀ10.61
ꢀ10.59a
ꢀ10.59a
5c
6.76
7.24
8.67
8.80
9
15.33
16.32
16.68
17.22
17.34
a The similarity of docked structures is measured by computing the root-
mean-square deviation (rmsd) between the coordinates of the atoms
and creating a clustering of the conformations based on these rmsd
values. In this run, multiple binding conformation clusters were found,
some with equivalent binding energies.
form for 72 h and possibly longer. When administered to rats by
intraperitoneal injection, significantly high levels of 5c were
found in the liver, kidney, stomach, and blood after 3 h. Also,
significant accumulation of 5c was found in murine tumor
xenografts with a dose-dependent inhibition of tumor growth.
The results suggest that 5c has substantially higher bioabsorption
when compared to curcumin.24 The advantageous effect of
nitrogen substitution of DAPs is also demonstrated by results
of Youssef et al. They reported that amino acid conjugates have
better cytostatic activity and bioavailability than DAPs without
N-substitution.39
’ MOLECULAR DOCKING
We performed theoretical modeling calculations using Auto-
Dock (version 4.2) to elucidate the mode of STAT3-inhibition
by these compounds using in silico docking simulations. The
simulation results have provided additional support for the
hypothesis that the mechanism of action of the DAP compounds
is via targeting of the STAT3 pathway. The computations
demonstrate that the DAP compounds have high docking affinity
for the STAT3 dimer (PDB code 3CWG) at the deoxyribonu-
cleic acid (DNA) binding domain of the molecule (Table 4,
Figure 1). This in turn would prevent the activated STAT3
molecule from binding with DNA, thereby inhibiting transcrip-
tion of downstream signaling. One of the more notable results
from the docking simulations is that the DAP compounds with
the N-hydroxypyrroline (nitroxide) moiety, 5c and 9, have
Figure 1. Compound 5c docked to STAT3 dimer. This image was
generated with the free version of PyMol using computational data from
the AutoDock simulations.
substantially lower binding energies than the parent molecules
lacking this functional group (1 and 8). We believe that this
is most likely due to the fact that the molecules bearing the
N-hydroxylamine group are more polar and have a pronounced
asymmetry. Upon examination of the preferred pocket of the
DAP compounds within the DNA-binding domain of the STAT3
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ARTICLE
dimer, it was noted that amino acid residues present include
arginine (Arg), glutamic acid (Glu), Tyr, Ser, aspartic acid (Asp),
asparagine (Asn), and threonine (Thr). All of these amino acids
are classified as polar or as having polar side chains.
3,5-Bis(4-fluorobenzylidene)-1-[(1-oxyl-2,2,5,5-tetramethyl-
2,5-dihydro-1H-pyrrol-3-yl)carbonyl]piperidin-4-one Ra-
dical (3a). To a solution of compound 1 HCl salt (1.73 g, 5.0 mmol)
and Et3N (1.0 g, 10.0 mmol) in CH2Cl2 (35 mL), freshly prepared 2a
(1.01 g, 5.0 mmol) dissolved in CH2Cl2 (10 mL) was added dropwise at
0 °C. The mixture was allowed to warm to room temperature and stirred
for 1 h. The organic phase was washed with brine (20 mL). The organic
phase was separated, dried (MgSO4), filtered, and evaporated. The residue
was purified by flash column chromatography (CHCl3/Et2O) to yield the
title compound as a yellow solid 1.26 g (49%): mp 168ꢀ170 °C, Rf = 0.55
(CHCl3/Et2O, 2:1). MS (EI) m/z (%): 477 (M+, 6), 463 (15), 447 (10),
310 (30), 133 (100). Anal. Calcd for C28H27F2N2O3: C 70.43, H 5.70, N
5.87. Found: C 70.24, H 5.88, N 6.01.
Thus, it stands to reason that DAP variants with modifications
to increase polarity may display even greater binding affinity
(lower binding energy) for the unphosphorylated murine
STAT3 molecule at the targeted binding site on the DNA-
binding domain. We also note that the molecules with the
monofluoro substitution (1 and 5c) on the aromatic rings exhibit
greater binding affinity than the corresponding trifluoromethyl-
substituted compounds (8 and 9). We believe that this is due to
the size (bulkiness) of the trifluoromethyl group, which acts as an
impediment to docking, versus the monofluoro group. Table 4
lists the tested compounds and the five lowest calculated binding
energies and the corresponding constants of inhibition (Ki, nmol).
The DAP compound binding affinity to the STAT3 dimer
determined is ranked as follows: 5c > 9 . 1 > 8. The rankings
also correlate well with the biological activity. Many small
molecules identified as potential drug candidates have reported
binding energies in the ꢀ7 to ꢀ9 kcal/mol range, but few of
these potential drug candidates have Ki < 50 nM. In addition,
many STAT3 inhibitors target the SH2 binding domain of the
molecule, whereas the DAP compounds favor the DNA binding
region. The identification of a previously unreported docking site
for molecular inhibition of STAT3 activity would be of significant
benefit for future drug design.
3,5-Bis(4-fluorobenzylidene)-4-oxo-N-(1-oxyl-2,2,5,5-tet-
ramethyl-2,5-dihydro-1H-pyrrol-3-yl)piperidine-1-carboxa-
mide Radical (3b). To a solution of compound 1 (933 mg, 3.0 mmol)
in anhydrous THF (40 mL), compound 2b (627 mg, 3.0 mmol) was
added. The mixture was heated under reflux for 4 h. After the mixture
was cooled, the THF was evaporated off under reduced pressure. The
residue was partitioned between CH2Cl2 (40 mL) and brine (10 mL).
The organic phase was washed with 10% aqueous K2CO3 (20 mL) and
water (10 mL). The organic phase was separated, dried (MgSO4), filtered,
and evaporated. The residue was purified by flash column chromatogra-
phy (CHCl3/MeOH) to yield the title compound as a yellow solid: 915
3
mg (62%), mp 175ꢀ177 °C, Rf = 0.43 (CHCl /Et2O, 2:1). MS (EI) m/z
(%): 492 (M+, 1), 462 (1), 460 (8), 310 (22), 133 (100). Anal. Calcd for
C28H28F2N3O3: C 68.28, H 5.73, N 8.53. Found: C 68.12, H 5.90, N 8.68.
General Procedure for N-Alkylation of 3,5-Bis(arylidene)-
piperidin-4-one (5a, 5b, 5c, 5d, 5f, 5g, 5h, 7, 9, 11). A mixture of
1 or 6 or 8 or 10 HCl salt (5.0 mmol) and K2CO3 (1.38 g, 10.0 mmol) in
acetonitrile (20 mL) was stirred at room temperature for 30 min. Then
alkyl bromide 4aꢀg (5.0 mmol) was added, dissolved in acetonitrile
(5 mL). The mixture was stirred and refluxed until the consumption of
the starting materials was complete (∼3 h). After the mixture was cooled,
theinorganic saltswere filtered offonsinteredglass filter andwashedwith
CHCl3 (10 mL). The filtrate was evaporated, and the residue was
partitioned between CHCl3 (20 mL) and water (10 mL). The organic
phase was separated, and the aqueous phase was washed with CHCl3
(20 mL). The combined organic phase was dried (MgSO4), filtered, and
evaporated. The residue was purified by flash column chromatography
(hexane/EtOAc) to give the title compounds in 35ꢀ71% yield.
’ CONCLUSIONS
The present study demonstrated that the earlier DAP com-
pounds can be N-alkylated or acylated with nitroxides or their
amine precursors as potent antioxidant moieties. Measurement
of the cytotoxicity of the new compounds to cancer cell lines
A2780 and MCF-7 and to H9c2 noncancerous (healthy) cardiac
cell line has shown that the modified compounds are more
effective as anticancer compounds but at the same time was less
toxic to noncancereous (healthy) cells. Computer docking simu-
lations support the empirical data collected. Among the com-
pounds tested 5c was chosen as the lead compound for further
studies. These results support the earlier findings that nitroxides
and their precursors do not compromise the anticancer effect of
the modifed molecules, but they have a beneficial effect on the
original activity.
3,5-Bis(4-fluorobenzylidene)-1-[(2,2,5,5-tetramethyl-2,5-
dihydro-1H-pyrrol-3-yl)methyl]piperidin-4-one (5e). To a so-
lution of nitroxide 5c (2.31 g, 5.0 mmol) in AcOH (25 mL), iron powder
(2.8 g, 50.0 mmol) was added. The mixture was stirred at 60 °C for 30
min. After cooling, the reaction mixture was diluted with water (40 mL)
and filtered. The filtrate was basified with solid K2CO3 to pH 8
(intensive foaming). The aqueous phase was extracted with CHCl3
containing 10% MeOH (2 ꢁ 30 mL), and the combined organic phase
was dried (MgSO4), filtered, and evaporated. The residue was purified
by flash column chromatography (CHCl3/MeOH) to give the title
compound as a yellow solid: 1.25 g (56%), mp 158ꢀ160 °C, Rf = 0.31
(CHCl3/MeOH, 9:1). MS (EI) m/z (%): 448 (M+ <1), 433 (12), 324
(13), 133 (56), 124 (100). Anal. Calcd for C28H30F2N2O: C 74.98, H
6.54, N 5.87. Found: C 75.01, H 6.68, N 5.70. 1H NMR (CD3OD): 7.73
(s, 2H); 7.46 (q, J = 15.4 Hz, ArH, 4H); 7.17 (t, J = 8.7 Hz, ArH, 4H);
5.38 (s, CH, 1H); 3.82, (s, N(CH2)2, 4H); 3.18 (s, CH2, 2H); 1.12
(s, CH3, 6H); 0.99 (s, CH3, 6H).
’ EXPERIMENTAL SECTION
Melting points were determined with a Boetius micro melting point
apparatus and are uncorrected. Elemental analyses (C, H, N, S) were
performed on a Fisons EA 1110 CHNS elemental analyzer. Mass spectra
were recorded on a Thermoquest Automass Multi and VG TRIO-2
instruments and in the EI mode. 1H NMR spectra were recorded with a
Varian UNITY INOVA 400 WB spectrometer. Chemical shifts are
referenced to Me4Si. Measurements were run at 298 K probe tempera-
ture in CDCl3 solution. ESR spectra were taken on Miniscope MS 200 in
10ꢀ4 M CHCl3 solution, and monoradicals gave a triplet line. Flash
column chromatography was performed on Merck Kieselgel 60
(0.040ꢀ0.063 mm). Qualitative TLC was carried out on commercially
prepared plates (20 cm ꢁ 20 cm ꢁ 0.02 cm) coated with Merck
Kieselgel GF254. All chemicals were purchased from Aldrich. Com-
pounds 1,10 4a,32 4b,32 4c,33 4d,34 4e,35 4f,36 4g,37 5c,1 and 619 were
prepared as described earlier. All compounds were more than 95% pure.
In Silico Docking Simulations. Previously published work by our
group1,22 has demonstrated that these compounds act upon the signal
transducer and activator of transcription 3 (STAT3) pathway. This
phenomenon was investigated in more detail through in silico molecular
docking simulations using the freely available program AutoDock
(version 4.2).40,41 The target macromolecule used in these studies was
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a nontransformed murine STAT3 dimer downloaded from the RCSB
Protein Data Bank (PDB code 3CWG).42 Energy-minimized 3D mol-
ecular topographies of the DAP compounds were obtained using the
Dundee PRODRG2 server.43 To identify the site on the STAT3 dimer
with the highest binding affinity for the DAP compounds, blind docking
was accomplished using 0.625 Å grid spacing with 128 points in each of
the X, Y, and Z directions. This grid covered the majority of the
previously defined 3CWG molecular structure, including the entire
SH2, linker, and DNA-binding domains, with partial coverage of the
coilꢀcoil domain.42 Specific docking at the preferential site identified
by blind docking was accomplished using 0.375 Å grid spacing with 100
points in each of the X, Y, and Z directions. Dockings were automatically
ranked by AutoDock according to the lowest calculated binding energies
(kcal/mol).
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’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis and characterization
b
of all new compounds and details of biological assay. This material
is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 36-72-536-220. Fax: 36-72-536-219. E-mail: kalman.hideg@
aok.pte.hu.
(11) Das, U.; Molnꢀar, J.; Barꢀath, Z.; Bata, Z.; Dimmock, J. R. 1-[4-(2-
Aminoethoxy)phenylcarbonyl]-3,5-bis-(benyzlidene)-4-oxopiperidines:
a novel series of highly potent revertrands of P-glycoprotein associated
multidrug resistance. Bioorg. Med. Chem. Lett. 2008, 18, 3484–3487.
(12) Das, S.; Das, U.; Selvakumar, P.; Sharma, R. K.; Balzarini, J.; De
Clercq, E.; Molnꢀar, J.; Serly, J.; Barꢀath, Z.; Schatte, G.; Bandy, B.; Gorecki,
D. K. J.; Dimmock, J. R. 3,5-Bis(benzylidene)-4-oxo-1-phosphonopiper-
idines and related diethyl esters: potent cytotoxins with multi-drug-
resistance reverting properties. ChemMedChem 2009, 4, 1831–1840.
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N-Aroyl-3,5-bis(benzylidene)-4-piperidones: a novel class of antimyco-
bacterial agents. Bioorg. Med. Chem. 2008, 16, 3602–3607.
’ ACKNOWLEDGMENT
This work was supported by a grant from Hungarian National
Research Fund (Grants OTKA K 81123 and OTKA-NKTH
K67597). The authors thank Krisztina Kish for elemental analysis
and Zoltꢀan Berente (Department of Biochemistry and Medicinal
Chemistry, University of Pꢀecs, Hungary) for NMR measurements.
(14) Dinets, I. L.; Makarov, M. V.; Artyushin, O. I.; Ribalkina, E. Y.;
Lyssenko, K. A.; Timofeeva, T. V.; Antipin, M. Y. Phosphoryl substituted
3,5-bis(arilydene)-4-piperidones possessing high antitumor activity.
Phosphorus, Sulfur Silicon Relat. Elem. 2008, 183, 619–620.
(15) Makarov, M. V.; Rybalkina, E. Y.; Roschenthaler, G. V.; Short,
K. W.; Timofeeva, T. V.; Odinets, I. L. Design, cytotoxic and fluorescent
properties of novel N-phosphorylalkyl substituted E,E-3,5-bis(arylidene)-
piperid-4-ones. Eur. J. Med. Chem. 2009, 44, 2135–2144.
(16) Pati, H. N.; Das, U.; Das, S.; Bandy, B.; De Clercq, E.; Balzarini,
J.; Kawase, M.; Sakagami, H.; Quail, J. W.; Stables, J. P.; Dimmock, J. R.
The cytotoxic properties and preferential toxicity to tumour cells
displayed by some 2,4-bis(benzylidene)-8-methyl-8-azabicyclo[3.2.1]-
octan-3-ones and 3,5-bis(benzylidene)-1-methyl-4-piperidones. Eur. J.
Med. Chem. 2009, 44, 54–62.
’ ABBREVIATIONS USED
A2780, human epithelial cancer cell line; MCF-7, human breast
cancer cell line; H9c2, undifferentiated neonatal rat cardiomyo-
blasts; DAP, 3,5-bis(arylidene)-4-piperidones; ROS, reactive oxygen
species; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide; STAT3, signal transducer and activator of tran-
scription 3; PARP, poly ADP-ribose polymerase; JAK, tyrosine
kinase; VEGF, vascular endothelial growth factor; DNA,
deoxyribonucleic acid; EPR, electron paramagnetic resonance
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