Novel Aza-resveratrol analogs: Synthesis, characterization and anticancer activity against breast cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2012.12.002

Novel Aza-resveratrol analogs were synthesized, structurally characterized and evaluated for cytotoxic activity against MDA-MB-231 and T47D breast cancer cell lines, which exhibited superior inhibitory activity than parent resveratrol compound. The bindin

Full text of DOI:10.1016/j.bmcl.2012.12.002

Bioorganic & Medicinal Chemistry Letters 23 (2013) 635–640
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel Aza-resveratrol analogs: Synthesis, characterization and
anticancer activity against breast cancer cell lines
c
c
Areej Siddiqui a, Prasad Dandawate b, Rukhsana Rub a, Subhash Padhye b, , Shama Aphale , Alpana Moghe ,
⇑
Amrit Jagyasi d, K. Venkateswara Swamy d, Bhupendra Singh e, Anwesha Chatterjee e, Amruta Ronghe e,
Hari K. Bhat e,
⇑
a ISTRA, MCES’s Allana College of Pharmacy, University of Pune, India
b ISTRA, Department of Chemistry, Abeda Inamdar Senior College, University of Pune, Azam Campus, Pune 411001, India
c Rajiv Gandhi Institute of Information and Biotechnology, BVU, Pune, India
d Dr. D.Y. Patil Institutes of Biotechnology and Bioinformatics, Dr. D.Y. Patil Vidyapeeth, Pune, India
e Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel Aza-resveratrol analogs were synthesized, structurally characterized and evaluated for cytotoxic
activity against MDA-MB-231 and T47D breast cancer cell lines, which exhibited superior inhibitory
activity than parent resveratrol compound. The binding mechanism of these compounds with estrogen
Received 4 September 2012
Revised 18 November 2012
Accepted 3 December 2012
Available online 11 December 2012
receptor-a was rationalized by molecular docking studies which indicated additional hydrogen binding
interactions and tight binding in the protein cavity. Induction of Beclin-1 protein expression in breast
cancer cell lines after treatment with newly synthesized resveratrol analogs indicated inhibition of
growth of these cell lines through autophagy. The study highlighted the advantage of introducing the
imino-linkage in resveratrol motif in enhancing the anticancer potential of resveratrol suggesting that
these analogs can serve as better therapeutic agents against breast cancer and can provide starting point
for building more potent analogs in future.
Keywords:
Resveratrol
Breast cancer
Autophagy
Molecular docking
Ó 2012 Elsevier Ltd. All rights reserved.
In spite of extensive research on cancer and its cellular path-
ways, target identification and drug development, the disease still
remains the major cause of death in economically developed coun-
tries as well as in developing countries.1 Amongst all cancers,
breast cancer is the most common type of cancer affecting more
than one million women and accounting for the highest mortality
worldwide.2,3 It is estimated that there have been 229,060 new
cases diagnosed of breast cancer in USA in 2012 and 39,920 deaths
resulting due to the disease.4 Even though large number of breast
cancers express estrogen receptor (ER) and progesterone receptor
(PR)5 and respond to therapy with hormones or aromatase
inhibitors, there is a group of patients (12–17%) that do not
respond to such treatment due to lack of ER, PR as well as the
Human Epithelial Receptor (Her-2/neu) (ErbB2). These are known
as triple-negative breast cancers (TNBC) which represent a highly
aggressive sub-type of breast cancer that is difficult to treat.6,7
Among the phytochemicals that have been investigated to treat
breast cancer resveratrol stands out as unique because its chemical
space resembles mammalian estrogen 17b-estradiol as shown in
Figure 1 and hence is anticipated to interfere with the functions
of 17b-estradiol based on space considerations.8 It is, therefore,
not surprising to have been implicated in reducing incidence of
hormone-dependant cancers including breast and prostate.9 These
compounds possess ability to modulate the biological responses of
estrogens generally by binding to ER.10 Their structural similarities
with the estrogens include phenolic rings which are crucial for
binding to ER-
a and ER-b receptors, and the hydroxyl groups at
specific positions.11 We have recently summarized the possible
mechanism of actions of these phytoestrogens in prevention of
breast cancer.12
Resveratrol is a naturally occurring stilbene phytoestrogen that
exists in various food and beverages and is known to possess wide
variety of biological properties including antioxidant, anti-
inflammatory and anticancer activities. The compound is known
to modulate several signaling kinases (Raf, Src, MAPK, PKD, PKCd),
transcription factors (p53/p21, IkB kinase/NF-kB), and other targets
(TRAIL/DR4 + 5, Fas/CD95, PI3K/Akt) leading to cancer cell death.13
Being a phytoestrogen, the anticancer activities of resveratrol
against breast cancer have attracted interest of many researchers
including our group. The compound has been shown to inhibit
EGF-mediated migration and expression of MED28, which is a sub-
unit of the mammalian mediator complex for transcription and
matrix metalloproteinase MMP-9 in MDA-MB-231 cells.14 Recently
⇑
Corresponding authors. Tel.: +91 9921529619; fax: +91 20 26446970 (S.P.); tel.:
+1 816 235 5903; fax: +1 816 235 1776 (H.K.B.).
E-mail addresses: sbpadhye@hotmail.com (S. Padhye), bhath@umkc.edu (H.K.
Bhat).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.002

636
A. Siddiqui et al. / Bioorg. Med. Chem. Lett. 23 (2013) 635–640
OH
Resveratrol has been very effective as an antiproliferative agent
in vitro but its poor bioavailability and rapid metabolism limits its
use in vivo studies as the preferred anticancer agent. In humans, it
is readily metabolized in the liver by phase II drug metabolizing
enzymes to water soluble glucuronide and sulfate metabolites,
accounting for its urine excretion.27 Resveratrol has a plasma
half-life of 8–14 min in humans.27 In rodents, only 1.5% of resvera-
trol reaches to the plasma compartment.28 Resveratrol can also
bind to DNA and in presence of Cu2+ ions and can induce DNA
strand breaks. In complex with Cu2+, resveratrol reduces Cu2+ to
Cu+ while the emerging oxidized resveratrol products further en-
hance the genotoxicity.29 Gao et al. have observed strong antipro-
liferative effect of resveratrol in vitro on 32Dp210 leukemia cells
but when they inoculated these leukemia cells into mice and trea-
ted with 8 mg/kg body weight of resveratrol, no anti-leukemic ef-
fect was detected. Even higher doses of resveratrol were able to
protect only a negligible number of mice from leukemia.30 This
week in vivo response is thought to be due to poor bioavailability
and rapid metabolism of the compound.
These problems can be addressed adequately by synthesis of
resveratrol analogs. and few analogs of resveratrol have been syn-
thesized by substitutions of hydroxy-,31 methoxy-,32 fluoro-,33 ace-
tate-,34 methyl ether-35 groups either on ‘A’ or ‘B’ ring. Some of
these have been found to exert potent anticancer activity than res-
veratrol. Recently, Roberti and group have reported a potent and
apoptosis inducing analog of resveratrol bearing 3,4-dihydroxy
substitution against HL-60 leukemia cell line,31 while Murias
et al. have shown that resveratrol bearing trihydroxyl pharmaco-
phores exhibited COX-2 inhibition at nanomolar range.36 Cai
et al. have shown that the 3,4-hydroxy substitution in ‘A’ ring of
resveratrol significantly enhance the apoptosis inducing ability of
resveratrol36a These studies seem to indicate that incorporation
of the polyhydroxyl substitution on resveratrol may help in
enhancing its cytotoxic and apoptosis inducing ability. Hence, in
the present work we have synthesized some novel analogs of res-
veratrol (Fig. 1) by maintaining 3,4-dihydroxy substitution on the
A ring while varying the substituents at C-4 position on the B ring
along with inclusion of Aza functionality in the conjugated system.
The compounds 3a–f were synthesized following the scheme
shown in Figure 2 by condensing equimolar amount of 3,4-dihy-
droxy benzaldehyde (1) with respective 4-substituted anilines
(2a–f) in absolute ethanol in presence of catalytic amount of concd.
HCl at 60 °C. The resulting reaction product was washed with cold
ethanol and purified by Silica gel column chromatography. All syn-
thesized compounds are shades of pale yellow to reddish brown.
The newly synthesized analogs (3a–f) of resveratrol were char-
acterized by UV, IR, NMR and Mass Spectroscopy (Table 1). The
infra-red spectra of compounds 3a–f exhibited a characteristic
broad band in the range of 3400–3462 cmÀ1 due to hydroxyl
OH
H
OH
OH
H
H
17Β-estradiol
OH
HO
N
Resveratrol
HO
HO
Compounds 3a-f
OH
OH
OH
H
C
B
B
N
C
H
C
OH
A
A
H
HO
HO
Resveratrol
Aza Linkage
Resveratrol
Ethene Linkage
Figure 1. Structural similarities between 17b-estradiol, Aza-resveratrol and com-
pounds 3a–f.
it was reported to inhibit EGF-induced epithelial to mesenchymal
cell transformation in MCF-7 cells.15 The compound has been
found to induce apoptosis mediated by activation of MAP kinase
pathways,16 modulation of phosphorylated Akt and caspase-9 in
MCF-7 cells,17 upregulation of ERK1/2 and suppression of Bcl-2
expression in MDA-MB-231 cells18, and activation of the p53 in
T47D breast cancer cell lines.19 Further, it has been suggested that
resveratrol-induced caspase-3 activation is required for PARP deg-
radation and induction of apoptosis in MDA-MB-231 cells.20 It has
been shown that resveratrol can induce accumulation of COX-2
which facilitates apoptosis in MCF-7 and MDA-MB-231 breast can-
cer cells.21 The compound is found to exert its anticancer effects in
animal models of breast cancer-induced by 7,12-dimethylbenz-
(a)-anthracene,22 as well as N-methyl-N-nitrosourea.23 It has also
been demonstrated that resveratrol-induced activation of Sirt1
and inhibition of survivin expression elicits a more profound inhib-
itory effect on BRCA-1 (breast cancer type-1 susceptibility protein)
mutant cells than on Brca-1-wild-type cancer cells both in vitro
and in vivo.24 The combination of resveratrol with quercetin, and
catechin has been found to reduce primary tumor growth of breast
cancer xenografts in a nude mouse model,25 while significant low-
ering of tumor growth, decreased angiogenesis, and increased
apoptotic index in MDA-MB-231 (ER
a
À, ERb+) tumors has been
found in resveratrol-treated nude mice.26
R
H
O
HO
HO
HCl, 60°C, stirr
absolute ethanol
H2N
HO
N
H
+
R
HO
3a-e
2a-e
1
a R= OCH3, b R= CH3,
c R= F, d R= Cl, e R= OH
H
N
H
O
H2N
HO
HO
HCl, 60°C, stirr
absolute ethanol
N
H
+
N
H
HO
HO
1
3f
2f
Figure 2. Synthetic scheme for Aza-resveratrol analogs.

A. Siddiqui et al. / Bioorg. Med. Chem. Lett. 23 (2013) 635–640
637
Table 1
Compositional and spectral data on Aza-resveratrol analogs
Code
Mol. formula
Electronic spectra (nm)
IR frequencies (cmÀ1
C–H
)
Mass spectra
Calcd Found
O–H
C@N
C@C
C–X
3a
3b
3c
3d
3e
3f
C
C
C
C
C
C
14H13NO3
14H13NO2
13H10 FNO2
14H10 ClNO2
13H11NO3
15H12N2O2
235, 279, 315
235, 280, 312
235, 281, 315
237, 281, 312
236, 278, 312
237, 280, 310
3284.88
3535.64
3350.49
3302.24
3533.71
3342.75
1587.47
1600.90
1585.54
1589.40
1600.90
1533.46
3090.07
3041.84
3026.41
3047.63
3043.77
3005.20
1518.03
1518.03
1510.31
1518.03
1518.03
1487.17
—
—
243.25
227.25
231.22
247.67
229.23
252.26
243
228
230
246
229
251
1020.38
704
—
—
Figure 3. Binding of Aza-resveratrol analogs into the active site of estrogen receptor-a, as assessed by computer modeling studies.
groups, while the formation of imino functionality was indicated
by the presence of
(C@N) stretch at 1570–1600 cmÀ137 The
medium to weak bands at 3057–3061 cmÀ1 can be attributed to
aromatic (C@C) stretch of the aromatic
(C–H) stretches.38 The
m
m
m

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A. Siddiqui et al. / Bioorg. Med. Chem. Lett. 23 (2013) 635–640
Table 2
broad signal appearing at 9.500 ppm in case of compound 3e is
due to para-hydroxyl in ‘B’ ring.41 The methylinic proton exhibited
a singlet at 8.350–8.479 ppm.42 The additional singlet seen at 3.507
and 2.088 ppm in case of compounds 3a and 3b were due to
4-methoxy41 and 4-methyl groups on the ‘B’ ring.43 The protons
of the aromatic rings are seen as sharp signals at 6.389–
7.412 ppm.41 The 13C NMR spectra of compounds 3a–f exhibited
aromatic carbon signals in the range of 108.434–152.654 ppm,41
while peak of methylinic carbon was found in the range of
159.693–162.429 ppm. The peaks appeared at 55.920 and
21.044 ppm in compounds 3a and 3b were ascribed to 4-meth-
oxy41 and 4-methyl groups on the ‘B’ ring. The 3,4-hydroxyl
carbons of ‘A’ ring have exhibited their peaks in the region
146.382–146.777 and 151.375–152.654 ppm, respectively.41
In order to act as antioxidants, resveratrol and its analogs have
to undergo oxidation. In a scavenging reaction the hydrogen atom
is donated to the radical present in the biological system thereby
neutralizing it. The hydrogen atoms are supplied by dissociation
of the hydroxyl groups. The resveratrol molecule contains both res-
orcinol and phenol moieties with a conjugated double bond be-
tween the two. Cyclic voltammogram of trans-resveratrol in
DMSO solvent shows the presence of two oxidation peaks at
+0.65 and +0.92 V associated with resorcinol and phenol moieties,
respectively which have no counterparts on the reduction side.44
On the other hand resveratrol analogs described in the present
work contain catechol and phenol moieties. Among the newly syn-
thesized analogs, 3e shows a broad oxidation peak at +0.64 V with-
out reducing counterpart. When the phenolic hydroxyl is replaced
by the methyl group as in 3b, the peak is shifted to +0.31 V. The
reduction peak corresponding to imino functionality can be ob-
served at À0.64 and À0.67 V in both the analogs, respectively,
(Supplementary data, Fig. 1S).
Docking results and consensus scores of Aza-resveratrol analogs
Code Binding energy Hydrogen
Bond
distance
(Å)
No. of
hydrogen
bonds
LogP
(Kcal/mole)
bonding
residues
Res
3a
À8.6
À6.8
ARG394-NH2
HIS524-ND1
GLY521-O
ARG394-NH2
HIS524-ND1
HIS524-ND1
GLY521-O
GLU353-OE1
HIS524-ND1
HIS524-ND1
HIS524-ND1
HIS524-ND1
ARG394-NH2
GLU353-OE1
GLY521-O
3.0
2.5
2.2
2.4
2.5
2.4
2.7
1.8
2.0
2.9
2.5
2.2
3.0
2.6
2.3
2.9
2.8
2.5
1
3
2.73
3.0
3b
À7.6
3
3.61
3c
3d
À8.1
À7.4
1
2
3.28
3.68
3e
3f
À8.2
4
2.73
À7.1
4
2.66
ARG394-NH2
ARG394-NH2
LEU387-O
Res = Resveratrol.
ring was found at 1515–1520 cmÀ1 in all compounds. In haloge-
nated compounds, C–Cl and C–F bands were observed at 704 and
1020 cmÀ1
,
respectively.39 The mass spectroscopic analysis
showed molecular ion peaks for compounds 3a–f corresponding
to M+ within the expected range (228–251) confirming the forma-
tion of expected imino-Schiff base species. The electronic absorp-
tion Spectra exhibited mainly three intense absorptions where
bands at 220–245 and 280 nm were ascribed to
p ?
p⁄ transition
of the aromatic ring and imino functionality, respectively. The
band at longer wavelength (312–320 nm) originates from the
intra-molecular charge transfer (CT) transition.40 1H NMR
spectra of all compounds exhibited two singlets in the range
9.493–9.687 ppm due to hydroxyl groups on the ‘A’ ring, while
ER-a is a critical growth regulatory gene in breast cancer and its
expression in breast cancer cells is critical for tumor progression.45
Its expression in tumor tissues has been considered as a favorable
predictor of prognosis in endocrine treatment.46 Hence, we have
Figure 4. Cytotoxic activity of Aza-resveratrol analogs against (A) MDA-MB-231, (B) T47D breast cancer cells, (C) MCF-10A non-neoplastic breast epithelia cells.

A. Siddiqui et al. / Bioorg. Med. Chem. Lett. 23 (2013) 635–640
639
Expression of Beclin-1 is known to be suppressed in breast cancers
and its overexpression is reported to inhibit breast cancer.48 Thus,
induction of Beclin-1 by resveratrol analogs may be of therapeutic
advantage in prevention against breast cancer. The ability of resve-
ratrol to induce autophagy leading cell death has been noted only
in case of ovarian, colon and chronic myelogenous leukemia
cells.51–53
Present work has thus indicated that substitution of appropriate
groups with favorable stereochemistry on the resveratrol motif
along with imino-functionality between the two rings may prove
advantageous for tailoring these molecules for use as therapeutic
agents against breast cancer.
Figure 5. Induction of Beclin-1 protein expression by Aza-resveratrol analogs 3b
and 3f. Representative Western blots to show the protein expression of Beclin-1 in
MDA-MB-231 and T47D breast cancer cells treated with resveratrol (50
lM), 3b
(50 M) or 3e (50 M) for 12 h. Thirty microgram total protein from cell lines was
l
l
size fractionated on a 12% SDS–polyacrylamide gel and transferred onto a PVDF
membrane under standard conditions. Beclin-1 primary antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) was used for immunodetection. The same mem-
Supplementary data
brane was stripped and incubated with
luminescent detection was performed using the BM Chemiluminescence Detection
kit (Roche, Indianapolis, IN).
a-tubulin antibody (Santa Cruz). Chemi-
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
12.002.
References and notes
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a
protein cavity in order compare their binding profiles towards
this biomarker. Compounds 3a–f were found to dock into the ac-
tive site of ER- with a good fit indicating that substitutions em-
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allowing additional hydrogen bonding interactions (Fig. 3, Table 2).
Compounds 3a–f exhibited binding energies in the range of À6.8–
À8.2 Kcal/mol compared to resveratrol, which suggest tight bind-
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a protein cavity. The compound 3e
has shown the highest stable binding energy where vicinal hydro-
xyl groups on ring A undergo hydrogen bonding interaction with
HIS524 (2.5 and 2.2 Å) residues while methyl group interacted
with ARG394 (3.0 Å) and GLU354 (2.6 Å) residues, respectively.
Resveratrol exhibits only one hydrogen bonding interaction with
ARG394 (3.0 Å) via para-hydroxyl group on B ring. It was apparent
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the ER-
protein cavity.
a protein leading to stabilization of these ligands into the
The newly synthesized compounds were tested and compared
with resveratrol for their cytotoxic effects against triple negative
MDA-MB-231 and progesterone receptor rich T47D human breast
cancer cell lines as well as MCF-10A human normal breast cell line
using MTT assay. All compounds exhibited cytotoxic effects against
MDA-MB-231 (Fig. 4A) and T47D (Fig. 4B) breast cancer cell lines at
50 lM concentration. Compounds 3b and 3e exhibited most potent
activity against MDA-MB-231(with 65–75% cytotoxicity) and T47D
cells (with 40–60% cytotoxicity), while resveratrol induced only
40% cytotoxicity against both cell lines tested. The compounds 3b
and 3e exhibited IC50 value of 21 and 29
231, and 32 and 44 M against T47D breast cancer cell lines,
respectively, as compared to resveratrol with IC50 value of 66 and
76 M, respectively. The compound 3f exhibited 40% cytotoxicity
against T47D cells at 50 M, but was ineffective to induce cytotox-
lM against MDA-MB-
l
l
l
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trol analogs exhibited any cytotoxic effects against MCF-10A cells
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at 50 lM concentration (Fig. 4C) suggesting its safety towards nor-
mal breast cells and selectivity to cancer cells. Resveratrol is
known to induce apoptosis but our newly synthesized resveratrol
analogs 3b and 3e seem to inhibit cell growth through autophagy-
mediated cell death.26 The protein isolates from MDA-MB-231
and T47D cell lines were analyzed for the protein expression of
Beclin-1 by standard Western blotting techniques.49,50 Induction
of Beclin-1 protein (an indicator for autophagy)47 in MDA-MB-231
and T47D cells after treatment with 3b or 3e indicated
autophagy-mediated inhibition of growth in these cells (Fig. 5).

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