Synthesis, characterization, molecular docking and cytotoxic activity of novel plumbagin hydrazones against breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2012.03.060

Novel plumbagin hydrazonates were prepared, structurally characterized and evaluated for anti-proliferative activity against estrogen receptor-positive MCF-7 and triple negative MDA-MB-231 and MDA-MB-468 breast cancer cell lines which exhibited superior i

Full text of DOI:10.1016/j.bmcl.2012.03.060

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3104–3108
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, characterization, molecular docking and cytotoxic activity of novel
plumbagin hydrazones against breast cancer cells
Prasad Dandawate a, Ejazuddin Khan a, Subhash Padhye a,b, , Himanshi Gaba c, Swati Sinha c,
⇑
b
Jyoti Deshpande c, K. Venkateswara Swamy c, Madhukar Khetmalas c, Aamir Ahmad b, , Fazlul H. Sarkar
⇑
a ISTRA, Department of Chemistry, MCE Society’s Abeda Inamdar Senior College of Arts, Science and Commerce, University of Pune, Pune 411001, India
b Department of Pathology, Barbara Ann Karmanos Cancer Institute and Wayne State University School of Medicine, 707 HWCRC, 4100 John R St., Detroit, MI 48201, USA
c Dr. D. Y. Patil Institute of Biotechnology and Bioinformatics, Pune 411044, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel plumbagin hydrazonates were prepared, structurally characterized and evaluated for anti-proliferative
activity against estrogen receptor-positive MCF-7 and triple negative MDA-MB-231 and MDA-MB-468
breast cancer cell lines which exhibited superior inhibitory activity than parent plumbagin compound.
Molecular docking studies indicated that hydroxyl groups on plumbagin and hydrazonate side chain
Received 22 February 2012
Revised 13 March 2012
Accepted 15 March 2012
Available online 21 March 2012
favor additional hydrogen bonding interactions with amino acid residues in p50-subunit of NF-
jB
protein and these compounds inhibited NF- B expression which may be responsible for the enhanced
j
Keywords:
Plumbagin hydrazone
Breast cancer
anti-proliferative activity. These compounds were found to be more effective against triple negative
breast cancer cells and might serve as a starting point for building future strategies against triple neg-
ative breast cancers which are known for their increased drug resistance and poor prognosis of breast
cancer patients.
NF-jB
Triple negative breast cancer
Ó 2012 Elsevier Ltd. All rights reserved.
Breast cancer is the most common type of cancer affecting
more than 1 million women and causing high mortality world-
wide. It is estimated that there have been 232,620 new cases
of breast cancer and 39,970 deaths occurring in the USA in
2011.1 A large number of breast cancers express estrogen recep-
tor (ER) as well as progesterone receptor (PR)2 and respond to
hormonal therapy or aromatase inhibitors. However, there is a
group of patients (12–17%) who do not respond to such treat-
ment because of the absence of these two receptors as well as
the receptor HER-2/neu (ErbB2); this group represents a highly
aggressive breast cancer subtype, known as triple-negative
breast cancers (TNBC),3 that is difficult to treat. In recent years
TNBC has gained attention due to its aggressive behavior, poor
prognosis and lack of targeted therapies.
The current therapies for the treatment of TNBC (Fig. 1) include
inhibitors of EGFR (HER1)/tyrosine kinase (Gefitinib), topoisomer-
ase II-a (Etoposide), PARP (Poly-(ADP-ribose)-polymerase) (Olapa-
rib), Src kinase (Dasatinib) and VEGF/VEGFR (Bevacizumab).
Recently, a new approach under a clinical trial has included the
use of microtubule inhibitor, viz. Ixabepilone.4,5 Gene mapping
studies have identified two major breast cancer pre-disposing
genes for TNBC, BRCA1 (chromosome 7q21) and BRCA2 (chromosome
13q12),6,7 which are found to be involved in DNA repair, cell
cycle checkpoint control through regulation of p53 activity and
maintenance of global chromosome stability.8 Some studies have
indicated that breast cancers in patients with BRCA1 germline
mutation are more often negative for ER, PR and HER2 and likely
to be positive for p53 protein, compared with controls.9
PARP-inhibitors10 like Olaparib, Iniparib, Veliparib and platinum-
based drugs like cisplatin11,12 are a few reported therapeutic
agents for BRCA-1 or -2 associated breast cancers.
A
phytochemical isolated from Plumbago zeylanica,13 viz.
Plumbagin (1) has been shown to induce apoptosis in hormone
responsive as well as hormone non-responsive TNBC breast
cancer cell lines in our earlier study.14 The compound is one of
the simplest plant secondary metabolites belonging to three
major phylogenic families, viz. Plumbaginaceae, Droseraceae and
Ebenceae, which also exhibits antioxidant,15 anti-inflammatory,16
anticancer,14,17 antibacterial18 and antifungal19 activities. We
have summarized the biological activities of plumbagin and its
derivatives in a recent review.13 The compound is believed to
act via generation of reactive oxygen species (ROS) which subse-
quently damage DNA and enhance cell death.20 Plumbagin inhib-
its the ER-signaling pathway by up-regulating the expression of
46 kDa isoform of ER-a, which is reported to cause inhibition of
HER66-kDa mediated transactivation leading to inhibition of
estrogen-mediated cell growth.21 The hydroxyl group of plumba-
gin is crucial for its activity especially towards inhibition of
⇑
Corresponding authors. Mobile: +91 9921529619; fax: +91 20 26446970 (S.P.);
tel.: +1 313 576 8315; fax: +1 313 576 8389 (A.A.).
E-mail addresses: sbpadhye@hotmail.com, sbpadhye@chem.unipune.ernet.in
(S. Padhye), ahmada@karmanos.org (A. Ahmad).
histone acetyltransferase activity of p300/CBP, which is
a
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.060

P. Dandawate et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3104–3108
3105
Figure 1. Clinically available compounds for treatment of triple negative breast cancer.
of Bcl-2 expression and NF-
work, we have selected NF- B as a target protein for docking stud-
ies of plumbagin hydrazides in p50-subunit of NF- B protein and
j
B activity.14 Hence, in the present
j
j
have evaluated their anti-proliferative activities against hormone
responsive and non-responsive breast cancer cell lines.
The compounds 2–5 were synthesized32 as shown in Scheme 1
by condensation reactions of equimolar quantities of plumbagin
(1) and appropriate hydrazide in absolute methanol in presence
of trifluoroacetic acid as catalyst with continuous stirring at
55 °C for 4 h. The precipitated hydrazones were washed 3 times
with cold methanol, dried under vacuum and purified by column
chromatography using chloroform: methanol as solvent system.
All synthesized compounds were yellow to orange in color
while compositional and spectral data on them is summarized in
Table 1. Infra-red spectra for compounds 2–5 showed medium
intensity band due to azomethine stretch in the region
1589–1604 cmꢀ133 confirming the formation of the hydrazonates.
The broad band in the range of 3109–3269 cmꢀ134 is assigned to
hydroxyl stretch. The compounds exhibited sharp intense bands
at 1640–1647 and 1672–1683 cmꢀ1 due to free quinone carbonyl
and hydrazide carbonyl groups respectively. The aromatic C@C
stretch was observed in the region 1519–1541 cmꢀ1, while the
Scheme 1. Schematic representation of synthesis of plumbagin hydrazides (2–5).
transcriptional activator of ER-a 22
In spite of such promising
.
activities, the compound has remained less explored structurally
for optimization of anticancer activity except for some C-3 substi-
tuted derivatives cyano, chloro, bromo, N-acetyl amino acids moi-
eties23–27 and their metal complexes.28–31
In the present work, we have extended our structural studies on
plumbagin modification of C-4 carbonyl with hydrazide pharmaco-
hydrazinic N–N stretch33 occurred in the range 1040–1058 cmꢀ1
.
A phenolic C–O vibration could be observed at 1229 cmꢀ1 for free
plumbagin ligand which was shifted to higher frequency region
1274–1284 cmꢀ1 in the hydrazonate derivatives.31 The mass
spectroscopic analysis showed molecular ion peaks corresponding
to M+ and M++H ions at 308.10, 308.10, 322.09 and 307.12,
phore which has been known to induce inhibition of NF-jB
activation with concomitant down-regulation of Bcl-2 expression.
In our earlier study, we found that plumbagin efficiently induced
apoptosis in breast cancer cells with concomitant down-regulation
Table 1
Compositional and spectral data on plumbagin hydrazides (2–5)
Code
Mol. formula
Electronic spectra (nm)
IR frequencies (cmꢀ1
)
Mass spectra
Calcd
O–H
C@O
C@N
C–O
N–N
Found
1
2
3
4
5
C
C
C
C
C
11H8O3
394.0
344.5
351.5
373.5
346.5
3445
1663, 1645
1672, 1640
1680, 1647
1683, 1641
1680, 1645
—
1604
1589
1604
1599
1229
1282
1274
1280
1284
—
—
—
17H13N3O3
17H13N3O3
18H14N2O4
18H14N2O3
3213–3109
3194–3157
3269–3234
3195–3157
1058
1040
1055
1051
307.30
307.30
322.32
306.32
308.10
308.10
322.09
307.12

3106
P. Dandawate et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3104–3108
Figure 2. Anticancer activity of plumbagin hydrazides against (A) MCF-7 and (B) MDA-MB-231 breast cancer cell lines. Cells were treated for 48 h with the compounds before
analysis through MTT assay.
respectively, confirming the formation of hydrazonate species pro-
posed for compounds 2–5. The electronic spectra showed strong
Table 2
IC50 values
determined by MTT assays
(l
M
S.E.) of compounds 1–5 against breast cancer cell lines, as
intense intra-ligand absorptions in the region 344–373 nm ascrib-
able to
p–
p⁄ and n–p⁄ transitions. A signal observed in the 1H NMR
Cell line
spectra of the compounds at 2.10–2.12 ppm is due to the methyl
group.30 The two peaks observed at 12.31–12.52 ppm and 12.87–
12.98 ppm disappeared upon D2O exchange confirming the
presence of –NH and hydroxyl groups. The hydroxyl proton of
plumbagin showed an up-field shift upon formation of hydrazo-
nates, while methylidene proton (C-3) exhibited a down-field shift
appearing at 7.89–8.22 ppm. The protons of the aromatic ring of
Code
MCF-7
MDA-MB-231
MDA-MB-468
1
2
3
4
5
5.5 0.54
6.2 0.45
2.7 0.32
5.3 0.41
2.8 0.26
3.5 0.34
3.3 0.46
1.9 0.28
3.8 0.42
2.1 0.34
2.5 0.33
3.3 0.42
1.9 0.25
2.9 0.32
2.0 0.31
Figure 3. Binding of plumbagin hydrazides into the active site of p50 subunit of NF-B, as assessed by computer modeling studies.

P. Dandawate et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3104–3108
3107
Table 3
Docking results and consensus scores of synthesized plumbagin hydrazides
Code
Docking energy
Binding energy
Hydrogen bonding residues
Bond distance (Å)
No. of hydrogen bonds
LogP
1
2
3
4
5
ꢀ7.71
ꢀ7.78
ꢀ7.52
ꢀ7.69
ꢀ7.93
ꢀ7.68
ꢀ7.77
ꢀ7.43
ꢀ7.66
ꢀ7.88
LYS114-OA, ASN136-HA, HIS115-OA
GLY66-OA, GLY66-HA
GLY66-HA, GLY66-OA
GLY66-HA, GLY66-OA, HIS64-OA
GLY66-HA, SER78-OA
2.125, 1.823, 1.836
1.788, 1.782
1.811, 1.835
1.784, 1.832, 2.191
1.782, 1.991
3
2
2
3
2
0.89
2.86
3.13
4.17
4.00
adducts with Cys-62 residues of p50. Hence, we evaluated NF-
inhibitory potential of compounds 1–5 by performing molecular
docking studies on them in the p50-subunit of NF- B protein cav-
ity.39 Compounds 2–5 were found to dock into the active site of
p50-subunit of NF- B with good fit confirming that hydrazide sub-
jB
j
j
stitution does not introduce any major steric change in the parent
plumbagin moiety except allowing additional hydrogen bonding
interactions (Fig. 3, Table 3). The higher logP values observed for
the hydrazonate derivatives indicate that they can easily internal-
ize through cell membrane. Their higher binding energies were in
the range of ꢀ7.43 to ꢀ7.88 kcal/mol, which are higher than those
observed for the parent plumbagin compound, indicate tight bind-
ing in the active site of p50-subunit of NF-jB protein promoted
through hydrogen bonding interaction with GLY66 (1.784 Å),
GLY66 (1.832 Å), HIS64 (2.191 Å) residues, respectively.
Compounds 3 and 5 were especially potent against TNBC cancer
cell line MDA-MB-231 (IC50 values 1.9 and 2.1 lM, respectively)
Figure 4. Anticancer activity of plumbagin hydrazides against another TNBC cell
line, MDA-MB-468. Cells were treated for 48 h with the compounds before analysis
through MTT assay.
(Fig. 2 and Table 2) which prompted us to check the activity of
compounds in yet another TNBC cell line, MDA-MB-468. We found
that the compounds were active against this TNBC cell line as well
(Fig. 4). The degree of anti-proliferative effect of the compounds
matched their effect against another TNBC cell line, MDA-MB-
231 as shown above in Figure 2 and the IC50 values of the most
effective compounds 3 and 5 was almost similar against MDA-
MB-231 and MDA-MB-468 cells (Table 2). This further supports
our observation that the compounds are particularly active against
the hormone-insensitive breast cancer cells. Our previous work
Figure 5. Representative Western blot analysis showing the effects of compounds
1–5 on NF-
jB p65 subunit in MDA-MB-231 breast cancer cells. Cells were treated
established a dose-dependent inhibition of NF-
plumbagin14 and results presented above (Fig. 3) suggested an
effective docking of compounds in NF- B. Therefore, we assessed
the direct effect of compounds on NF- B p65-subunit in MDA-
MB-231 cells. Western blot analysis clearly demonstrated an inhi-
bition of NF- B by the compounds (Fig. 5). While plumbagin (1)
inhibited NF-
B as expected,14 other derivatives were also ob-
served to inhibit NF- B to varying extents. In particular, com-
pounds 3 and 5 were found to be more effective than the parent
compound in inhibiting NF- B expression. In summary, present
work indicates that plumbagin hydrazonates might serve as a
starting point for building future strategies against hormone non-
responsive triple negative breast cancers, which are well known
for their drug resistant characteristics and for poor prognosis of
breast cancer patients.
jB activity by
with 1.5 M compounds for 48 h. C: vehicle-treated control cells. b-Actin protein
l
was used as protein loading control.
j
j
plumbagin and hydrazides showed sharp peaks at 7.23–7.61 and
7.06–9.15 ppm, respectively. 13C NMR spectra of all plumbagin
hydrazides showed disappearance of the signal at 190.3 ppm due
to C-4 carbonyl of plumbagin30 while exhibiting a new signal at
157.3–157.8 ppm ascribed to imino carbon confirming the forma-
tion of hydrazonates at C-4 position. The C-5 hydroxyl group exhi-
bit shifted from 161.2 to 163.2–165.2 region31 whereas the
aromatic carbon atoms appeared in the range of 115.9–
152.8 ppm. The methylidenic carbon (C-3) was located between
139.4 and 141.7 ppm.30
The anti-proliferative activity of plumbagin and its hydrazo-
nates was evaluated against MCF-7 (ER-positive) and MDA-
MB-231 (ER-negative, TNBC) breast cancer cell lines35,36 (Fig. 2).
All compounds exhibited growth inhibitory activity against both
the cell lines at significantly lower concentrations which are
achievable at tissue level. The data from MTT assays was used to
j
j
j
j
Acknowledgments
P.D. acknowledges Human Resource Development Group, Coun-
cil of Scientific & Industrial Research, New Delhi, India, for provid-
ing Senior Research Fellowship. Mr. Ankit Rochani and Professor
Utpal Tatu of Department of Biochemistry, Indian Institute of Sci-
ence, Bangalore, INDIA, are thanked for providing NMR and Mass
spectroscopic analysis.
calculate IC50 values of the compounds (Table 2). NF-
the major targets of plumbagin contributing to its anticancer prop-
erty.14 The mammalian NF-
B constitutes of five polypeptide
sub-units, p50, p52, p65 (RelA), RelB and c-Rel with commonly
observed homodimers p50 and heterodimer p50/p65, respec-
tively.34 Direct binding of the inhibitor to the p50-subunit of target
jB is one of
j
References and notes
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benzohydrazide), yield: 66%, UV–vis (DMSO): k nm: (373.5). IR (KBr disk), m,
cmꢀ1: 3269–3234 (–OH), 1683, 1641 (–C@O), 1604 (C@N), 1280 (C–O), 1055 (N–
N). 1H NMR (500 MHz, DMSO-d6) (d, ppm) 2.12 (s, 3H, –CH3), 6.99 (dd, J = 7.5 Hz,
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157.61, 157.80, 164.37, 184.64. ESIMS (m/z): Calcd 322.32, found 322.09 (M+), in
accordance with MF C18H14N2O4.
5
((11E)-N0-(5-hydroxy-2-methyl-1-oxonaphthalen-4(1H)-ylidene)benzohyd-
razide), yield: 58%, UV–vis (DMSO): k nm:(346.5). IR (KBr disk),
, cmꢀ1: 3195–
m
3157 (–OH), 1680, 1645 (–C@O), 1599 (C@N), 1284 (C–O), 1051 (N–N). 1H NMR
(500 MHz, DMSO-d6) (d, ppm) 2.12 (s, 3H, –CH3), 7.24 (d, J = 8.0 Hz, 1H), 7.49 (t,
J = 7.5 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.67 (t, J = 7.5 Hz, 3H), 8.01 (d, J = 7.0 Hz,
2H), 8.22 (s, 1H),12.33 (s, 1H, D2O exchangeable), 12.98 (s, 1H, D2O
exchangeable). 13C NMR (125 MHz, DMSO-d6) (d, ppm) 16.90, 116.70, 118.16,
122.51, 125.45, 128.98, 131.44, 132.71, 133.02, 141.27, 144.68, 157.86, 165.22,
184.72. ESIMS (m/z): Calcd 306.32, found 307.12 (M++H), in accordance with MF
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36. Cell Growth Inhibition Studies by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium Bromide (MTT) Assay: Cells (3 ꢁ 103 /well) were seeded in 96-well
culture plates. Each treatment had eight replicate wells and, moreover, each
experiment was repeated at least three times. Test compounds were dissolved
in DMSO and added to cells 24 h after seeding. At the end of treatment, MTT
(0.5 mg/mL) was added and plates incubated at 37C for 2 h followed by
replacement of media with DMSO at room temperature for 30 min. Ultra
Multifunctional Microplate Reader (TECAN) was used to record the absorbance.
2012.
37. Xia, Y. F.; Ye, B. Q.; Li, Y. D.; Wang, J. G.; He, X. J.; Lin, X.; Yao, X.; Ma, D.;
Slungaard, A.; Hebbel, R. P.; Key, N. S.; Geng, J. G. J. Immunol. 2004, 173, 4207.
38. Chen, K. M.; Spratt, T. E.; Stanley, B. A.; De Cotiis, D. A.; Bewley, M. C.; Flanagan,
J. M.; Desai, D.; Das, A.; Fiala, E. S.; Amin, S.; El-Bayoumy, K. Cancer Res. 2007,
67, 10475.
39. Docking studies: All the docking calculations were carried out with AutoDock
4.0 software to analyze ligand interactions with the crystal structure binding
site of p50-NFêB obtained from PDB ID (1NFK). AutoDock calculates a rapid
energy evaluation through pre-calculated grids of affinity potentials with a
variety of search algorithms to find appropriate binding positions. The active
site of the enzyme was defined to include residues 59–71. The 3-D grid box has
been generated with a grid centre co-ordinates comprising of grid spacing
0.375 Å and 60 ꢁ 60 ꢁ 60 point size considering active site residues included
within it. Stable docking conformation of compounds achieved by
implementing energy minimization parameter AMBER force field until the
gradient convergence value of 0.05 Kcal/mol was reached with distance-
dependent dielectric function (€=4r). New designed all plumbagin analogs, as
well as PL were placed in grid box of p50 for docking process. Customized
docking parameters were set in AutoDock for best results for understanding
interaction studies with new designed compounds. Parameter settings were
set to 1500 iterations, 50 population sizes, 100.0 kcal/mol of energy threshold
for pose generation, 300 simplex evolution steps and 1.0 neighbor distance
factor. For preparing the AutoDock docking parameter file we used default
settings (genetic algorithm parameters: population size = 150, number of
energy evaluations = 2.5 ꢁ 107, rate of gene mutation = 0.02, rate of
crossover = 0.8, maximum number of generations = 27000, number of GA
runs = 10, initial dihedrals were randomly specified, elitism value was set to 1).
Prior to docking, total Kollman and Gasteiger charges were added to the
protein and the ligand. For each compound, the most stable docking model was
selected based upon confirmation of best scored predicted by the AutoDock
scoring function.
31. Chen, Z. F.; Tan, M. X.; Liu, Y. C.; Peng, Y.; Wang, H. H.; Liu, H. G.; Liang, H. J.
Inorg. Biochem. 2011, 105, 426.
32. Synthesis of Plumbagin Hydrazides: The ligands (2–5) was synthesized by
condensation of equimolar quantities of plumbagin and isonicotyl, nicotyl,
salicyl and benzoyl hydrazide respectively in methanol in presence of trifluoro
acetic acid as catalyst with continuous stirring at 60 °C for 4 h. The precipitated
compounds were washed 2–3 times with cold methanol, dried under vacuum
and purified by column chromatography using chloroform: methanol (9:1) as
solvent system.
2
((11E)-N0-(5-hydroxy-2-methyl-1-oxonaphthalen-4(1H)-ylidene)isonicotin-
ohydrazide), yield: 68%, UV–vis (DMSO): k nm:(344.5). IR (KBr disk),
m
, cmꢀ1
:
H
3213–3109 (–OH), 1672, 1640 (–C@O), 1604 (C@N), 1282 (C–O), 1058 (N–N). 1
NMR (500 MHz, DMSO-d6) (d, ppm) 2.13 (s, 3H, –CH3), 7.27 (d, J = 10.0 Hz, 1H),
7.52 (t, J = 9.5 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 6.0 Hz, 2H), 8.18 (s, 1H),
8.83 (d, 7.0 Hz, 2H), 12.52 (s, 1H, D2O exchangeable), 12.88 (s, 1H, D2O
exchangeable). 13C NMR (125 MHz, DMSO-d6) (d, ppm) 16.43, 115.91, 117.83,
122.16, 124.84, 130.98, 131.23, 139.43, 141.25, 150.21, 157.42, 163.44, 184.13.
ESIMS (m/z): Calcd 307.30, found 308.10 (M++H), in accordance with MF
C17H13N3O3.
3((11E)-N0-(5-hydroxy-2-methyl-1-oxonaphthalen-4(1H)-ylidene)nicotinohy-
drazide), yield: 64%, UV–vis (DMSO): k nm:(351.5). IR (KBr disk), m
, cmꢀ1: 3194–
3157 (–OH), 1680, 1647 (–C@O), 1589 (C@N), 1274 (C–O), 1040 (N–N). 1H NMR
(500 MHz, DMSO-d6) (d, ppm) 2.10 (s, 3H, –CH3), 7.23 (d, J = 8.0 Hz, 1H), 7.48 (t,