Design, synthesis and biological evaluation of hydrazone derivatives as anti-proliferative agents

Article abstract of DOI:10.1007/s00044-017-1866-1

A series of triaryl-substituted hydrazones as structural acyclic prototypes were synthesized and screened for anti-proliferative activity against breast (Michigan cancer foundation-7 and MD Anderson metastatic breast-231) and uterine cancer (Ishikawa) cel

Full text of DOI:10.1007/s00044-017-1866-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1866-1
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of hydrazone
derivatives as anti-proliferative agents
Ravi Shankar1,2 Ravindra K. Rawal3 Uma S. Singh1 Preeti Chaudhary4
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Rituraj Konwar4 Kanchan Hajela1
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Received: 24 April 2014 / Accepted: 4 March 2017
© Springer Science+Business Media New York 2017
Abstract A series of triaryl-substituted hydrazones as
structural acyclic prototypes were synthesized and screened
for anti-proliferative activity against breast (Michigan can-
cer foundation-7 and MD Anderson metastatic breast-231)
and uterine cancer (Ishikawa) cell lines. Two compounds
were found to be the most active, 5e showed the maximum
inhibition of both functional estrogen receptor containing
Michigan cancer foundation-7 cells (IC50: 7.8 µM) and
Ishikawa cells (IC50: 7.3 µM) whereas, compound 5i was
selectively most active against ER-negative MD Anderson
metastatic breast-231 cells (IC50: 4.7 µM). The inhibitory
effect of 5e in breast cancer and uterine cancer cells was due
to ER antagonistic action, also supported by molecular
docking studies.
Introduction
The discovery and identification of compounds with desired
potency, and selective cytotoxicity against cancer cells that
may lead to the development of new anti-cancer drugs
remains an important objective of medicinal chemists.
Hydrazones represent an important class of compounds
known to possess, antimicrobial, anticonvulsant, analgesic,
anti-inflammatory, anti-platelet, anti-tubercular and anti-
tumor activities (Kumar and Narasimhan 2013). Some
biologically active hydrazones 1–7 are shown in Fig. 1. The
2, 4-dinitrophenyl hydrazones of diaryl ketones have
been reported to possess antiestrogenic and antineoplastic
activities. One of the compound A-007, (4,4'-dihy-
drobenzophenone-2,4-dinitrophenyl-hydrazone (5) is in
phase II clinical trials (Morgan et al. 2003). These hydra-
zones (6) are reported to have pseudo ring structure that
stereo-chemically occupies the estrogenic region of the
receptor thereby inhibiting it from undergoing allosteric
change to prevent transcription of estrogenic information to
the cell nucleus (Pandey et al. 2002; Singh et al. 2008).
Small molecules like guanylhydrazone 7 have been reported
to block estrogen receptor (ER) activity through a non-
traditional mechanism, by directly interfering with coacti-
vator binding to agonist-liganded ER (LaFrate et al. 2008).
From a study of the estrogen-ligand pharmacophore
model (Agatonovic-Kustrin and Turner 2008), subtle
changes in the spatial conformation of the triarylethylene
core lead to some major pharmacological changes. This
hypothesis has led to the development of many different ER
agonists/antagonists, the most promising being the triaryl
pyrazole-based ER antagonists (Stauffer et al. 2001, 2000;
Sun et al. 1999). These observations prompted us to design
and investigate the hydrazone pharmacophore as the acyclic
prototype of triaryl substituted pyrazoles, and to build a
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Keywords Hydrazones Estrogen receptor Antagonists
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Anti-proliferative Anti-cancer
* Kanchan Hajela
kanchan_hajela@cdri.res.in
1
Medicinal & Process Chemistry Division,CSIR-Central Drug
Research Institute, Sector 10,Jankipuram Extension, Sitapur Road,
Lucknow, Uttar Pradesh 226031, India
2
3
4
Medicinal Chemistry Division, CSIR-Indian Institute of
Integrative Medicine, Jammu 180001, India
Department of Pharmaceutical Chemistry, Indo-Soviet College of
Pharmacy, Moga 142001, India
Endocrinology Division, CSIR-Central Drug Research Institute,
Lucknow, Uttar Pradesh 226031, India

Med Chem Res
Fig. 1 Biologically active
hydrazones 1–7
Fig. 2 Structures of tamoxifen,
triphenolic pyrazole and
proposed hydrazone
scaffold with specific alignment of three aryl substituents
suitable for ER affinity and activity. Here, our motivating
factor was to see the flexibility effect of the benzylic group,
and also the effect of distance between A and B rings as in
tamoxifen (7) at 2-position and in pyrazole (8) at 3-position
on the biological activity against ER+ve and ER−ve breast
cancer cell lines. The flexible alignment of an acyclic or
pseudo ring (compound 9) may help in the adoption of a
favorable (antagonist) conformation by the molecules
within the receptor cavity resulting in the enhanced affinity
of the ligand to the receptor amino acids thereby poten-
tiating the biological efficacy of hydrazones (Fig. 2).
silica gel F254 plates. Melting points were taken in open
capillaries on Complab melting point apparatus and are
uncorrected. Infrared spectra were recorded on a Perkin-
1
Elmer AC-1 spectrometer. H nuclear magnetic resonance
(NMR) and 13C NMR spectra were recorded using Bruker
Supercon Magnet DRX-300 spectrometer (operating at 200,
300 MHz for 1H and 50, 75 MHz for 13C) using CDCl3 and
DMSO-d6 as solvents and tetramethylsilane as internal
standard. Chemical shifts are reported in parts per million.
Multiplicities are reported as follows: singlet (s), doublet
(d), triplet (t), multiplet (m), and broad singlet (brs). FAB
mass spectra were recorded on JEOL SX 102/DA 6000
mass spectrometer using argon/xenon (6 Kv, 10 mA) as the
FAB gas. Pure compounds were obtained by column
chromatography using silica gel (60–120 and 100–200
mesh).
Experimental section
Materials and methods
MTT assay in Breast cancer cell line
All the reagents were purchased commercially from Sigma-
Aldrich and were used without further purification. The
reactions were monitored by thin-layer chromatography on
The MTT assay was used to determine the anti-proliferative
activities of the compounds as described in our earlier

Med Chem Res
communication (Shankar et al. 2009). In short, 3000 cells/
well were seeded in 100 µL Dulbecco's modified eagle
medium media, supplemented with 10% fetal bovine serum
in each well of 96-well cell culture plates and incubated for
24 h at 37 °C in a CO2 incubator so that cells reaches about
70–80% confluency. Test compounds were dissolved in
DMSO and diluted to the desired concentrations in culture
medium so that final DMSO concentration is 0.001% or
less. Cells were treated with 100 µL of each compound of
desired concentration by adding to the wells along with
untreated vehicle control and incubated for 48 h. Post-
treatment period, the media were removed and 100 µL/well
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide) (5 mg/mL) was added and the plates were
further bioincubated for 4 h. Then the supernatant from each
well was carefully removed, formazon crystals were dis-
solved after adding 100 µL of DMSO with shaking under
incubation for 10 min and absorbance at 540 nm wavelength
were recorded with enzyme-linked immunosorbent assay
reader.
the potential for non-polar parts of the receptor, we scaled
van der Waal radii of receptor atoms by 1.00 Å with a
partial atomic charge of 0.25. The ligands were docked with
the active site using the ‘xtra precision’ (XP) Glide algo-
rithm. After successful docking, enzyme-ligand complexes
were subjected to EM to obtain the final binding mode. All
the calculations were performed on Schrödinger Suite 2007
(Schrödinger Inc.) with GB/SA continuum water solvation
model. OPLS_2001 force field was used for ‘XP’ docking
and then the Merck molecular force fields force field was
implemented in MACROMODEL 9.1 for EM.
General procedure for the synthesis of N-benzylated
hydrazones (4a–g)
To a suspension of NaH (60% suspension in oil, 3 mM) in
5 mL of dry N,N-Dimethylformamide (DMF) was added
hydrazone (3a, 20 mM, dissolved in 5 mL dry DMF) at 0 °C
with stirring under nitrogen atmosphere. After 15 min,
substituted benzyl bromide (30 mM, dissolved in 10 mL
DMF) was added dropwise and stirring was continued at
room temperature for 1.5 h. On completion, the reaction
was quenched with cold water to decompose any unreacted
sodium hydride and the solution was extracted with
dichloromethane, washed with water and dried over anhy-
drous calcium chloride. The drying agent was filtered off
and the solvent was removed by distillation. The residue
was purified on a silica gel column using hexane/EtOAc as
eluent to give the corresponding pure hydrazone
derivatives.
Molecular modeling method
Additionally, the comparative docking study of computa-
tionally energy minimized conformation of active molecule
5e and 4-hydroxy tamoxifen (OHT) was performed to
rationalize the activity profile in ER (ERα). The molecular
modeling study was carried out to obtain an insight of the
molecular mechanism of compound 5e by using the
Schrodinger suite (Shiau et al. 1988). The crystal structure
of ER ligand binding domain (LBD) in complex with 4-
hydroxytamoxifen and reference protein coordinates used
for protein preparation were obtained from the Protein Data
Bank (http://www.rcsb.org/pdb/; PDB ID: 3ERT) and fur-
ther modified for Glide docking calculations. The builder
module of Maestro has been used for the structure pre-
paration of ER LBD in complex with 4-hydroxytamoxifen
from the crystal structure (3ERT.pdb). Furthermore, the
manually inspected and corrected structure of ER was
subjected to a single run by multistep Schrödinger’s Protein
preparation wizard to optimize the structure and ensure its
chemical correctness, and to determine the most energeti-
cally favorable conformation. Minimizations were per-
formed until the average root mean square deviation of the
non-hydrogen atoms reached 0.3 Å. The starting con-
formation for the most active compound 5e and 4-hydroxy
tamoxifen were obtained by 5000 steps of Monte Carlo
conformational search under GB/SA continuum water sol-
vation model and Polak-Ribiere Conjugate Gradient method
used for the subsequent energy minimization (EM). After
ensuring that the protein and ligands were in the correct
form for docking, the receptor-grid files were generated
using a grid-receptor generation program of glide. To soften
N-[4-(2-Chloroethoxy)-benzyl]-N-phenyl-N′-(1-phenyl-
ethylidene)-hydrazone (4a) and N-[4-(3-Chloropropoxy)-
benzyl]-N-phenyl-N′-(1-phenyl-ethylidene)-hydrazone
(4b) 4a and 4b were very unstable at room temperature
and were used as such in the next reaction.
N-[4-(2-Chloroethoxy)-benzyl]-N-phenyl-N′-(1-phenyl-
propylidene)-hydrazone (4c) oil 1H NMR (CDCl3): δ 0.84
(t, 2H), 2.71 (q, 2H), 3.79 (t, 2H), 4.22 (t, 2H), 4.67 (s, 2H),
6.86 (d, 2H, J = 8.4 Hz), 6.92–6.99 (m, 3H, ArH),
7.23–7.28 (m, 2H), 7.32–7.34(m, 2H), 7.41–7.43 (m, 3H)
7.78–7.81 (m, 2H, ArH).
N-[4-(2-Chloroethoxy)-benzyl]-N-phenyl-N′-(1-phenyl-
1
butylidene)-hydrazone (4d) oil, H NMR(CDCl3): δ 0.94
(t, 3H), 1.29 (q, 2H), 2.64 (t, 2H), 3.80 (t, 2H), 4.22 (t, 2H),
4.67 (s, 2H), 6.87 (d, 2H, J = 8.4 Hz), 6.93–6.98 (m, 3H,
ArH), 7.23–7.28 (m, 2H, ArH), 7.33 (d, 2H, J = 8.5 Hz),
7.41–7.43 (m, 3H), 7.75–7.78 (m, 2H); 13C NMR(CDCl3):
δ 12.97, 18.71, 30.31, 40.26, 60.75, 66.65, 113.12, 116.19,
119.66, 125.87, 127.04, 127.59,128.29, 128.53, 130.38,
136.15, 150.14, 155.92, 170.38; ESIMS: 407(M+ + 1).

Med Chem Res
N-[4-(2-Chloroethoxy)-benzyl]-N′-[1-(4-methoxy-phenyl)-
ethylidene]-N-phenyl-hydrazone (4e) oil, 1H NMR
(CDCl3): δ 2.15 (s, 3H, CH3), 3.81 (t, 2H), 3.87 (s, 3H,
OCH3), 4.22 (t, 2H), 4.72 (s, 2H), 6.87 (d, 2H, J = 8.6 Hz),
6.92–6.96 (m, 5H, ArH), 7.24–7.35 (m, 4H, ArH), 7.88 (d,
2H, J = 8.6 Hz); 13C NMR(CDCl3): δ 16.78, 41.99, 55.39,
61.51, 68.13, 113.69, 114.00, 117.08, 120.58, 128.22,
128.95, 129.50, 130.78, 131.94, 150.68, 157.16, 161.12,
166.13; ESIMS: 409(M+ + 1).
7.20–7.29 (m, 4H, ArH), 7.38–7.40 (m, 3H, ArH),
7.82–7.85 (m, 2H); 13C NMR(CDCl3): δ 15.73, 22.93,
24.57, 53.75, 56.66, 61.08, 64.46, 113.00, 116.46, 119.68,
125.32, 126.93, 127.59, 128.11, 128.23, 129.23, 129.80,
137.10, 149.59, 156.48, 163.55; MS(ESI) m/z 428(M+ +
1). Anal. calcd for C28H33N3O: C 78.65, H 7.78, N 9.83%.
Found: C 78.96, H 7.89, N 9.67%.
N-Phenyl-N′-(1-phenyl-ethylidene)-N-[4-(3-pyrrolidin-1-
yl-propoxy)-benzyl]-hydrazone (5c) Oil; yield 65%; 1H
NMR (CDCl3): δ 1.74–1.77 (m, 4H), 1.94–1.98 (m, 2H),
2.09 (s, 3H), 2.45–2.49 (m, 4H), 2.61 (t, 2H), 3.99 (t, 2H),
4.67 (s, 2H), 6.78 (d, 2H, J = 8.4 Hz), 6.83–6.90 (m, 3H,
ArH), 7.16–7.24 (m, 4H, ArH), 7.32–7.36 (m, 3H),
7.76–7.79 (m, 2H); MS(ESI) m/z 428(M+ + 1). Anal. calcd
for C28H33N3O: C 78.65, H 7.78, N 9.83%. Found: C
78.44, H 7.82, N 9.71%.
N-[4-(2-Chloroethoxy)-benzyl]-N-(4-methoxy-phenyl)-N′-
(1-phenyl-ethylidene)-hydrazone (4f) oil, 1H NMR
(CDCl3): δ 2.19 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 3.90 (t,
2H, ClCH2), 4.25 (t, 2H, OCH2), 4.63 (s, 2H), 6.97–6.81
(m, 5H, ArH), 7.41–7.28 (m, 6H, ArH), 7.82–7.79 (m, 2H);
ESIMS: 409(M+ + 1).
N-[4-(2-Chloroethoxy)-benzyl]-N′-(3,4-dihydro-2H-
naphthalen-1-ylidene)-N-phenyl-hydrazone(4g) oil, 1H NMR
(CDCl3): δ 1.62 (m, 2H), 2.17 (t, 2H),2.65 (t, 2H), 2.66 (t,
2H), 3.88 (t, 2H), 4.43 (s, 2H), 6.72–6.87 (m, 3H, ArH),
7.32–7.47 (m, 6H, ArH), 7.80–7.84 (m, 4H); ESIMS: 404
(M+).
N-Phenyl-N′-(1-phenyl-ethylidene)-N-[4-(3-piperidin-1-yl-
propoxy)-benzyl]-hydrazone (5d) Oil; yield 76%; 1H
NMR (CDCl3): δ 1.24–1.29 (m, 2H), 1.59–1.65 (m, 4H),
2.51–2.52 (m, 4H), 2.76 (t, 2H, NCH2), 4.09 (t, 2H, OCH2),
6.84 (d, 2H, J = 8.4 Hz), 6.89–6.96 (m, 3H), 7.20–7.30 (m,
5H), 7.39–7.41(m, 3H), 7.78–7.79(m, 2H); 13C NMR
(CDCl3): δ 17.03, 24.54, 26.01, 26.91, 54.68, 56.02, 62.35,
66.27, 96.17, 114.19, 117.70, 120.93, 126.60, 128.22,
128.87, 129.34, 129.51, 130.82, 138.38, 150.80, 158.03,
164.84; MS(ESI) m/z 442(M+ + 1). Anal. calcd for
C29H35N3O: C 78.87, H 7.99, N 9.52%. Found: C 78.97, H
7.68, N 9.76%.
General procedure for the synthesis of N-substituted
hydrazones (5a–m)
N-Phenyl-N′-(1-phenyl-ethylidene)-N-[4-(2-pyrrolidin-1-
yl-ethoxy)-benzyl]-hydrazone (5a) A solution of 4a (1.12
mM), pyrrolidine (5 mL) and tetrabutyl ammonium iodide
(10 mg) in dry DMF (15 mL) was heated at 70–75 °C with
stirring for 7 h. After completion, the solution was cooled
and extracted with dichloromethane. The organic phase was
washed with water and dried over anhydrous calcium
chloride. It was filtered, concentrated and the residue pur-
ified on a silica gel column using hexane/EtOAc as eluent to
give the pure compound 5a as colorless oil; yield (69%); 1H
NMR (CDCl3): δ 1.80–1.84 (m, 4H), 2.13 (s, 3H),
2.60–2.63 (m, 4H), 2.89 (t, 2H), 4.08 (t, 2H), 4.71 (s, 2H),
6.84 (d, 2H, J = 8.5 Hz), 6.90–6.95 (m, 3H, ArH),
7.20–7.29 (m, 4H, ArH), 7.38–7.40 (m, 3H), 7.82–7.85 (m,
2H, ArH); 13C NMR(CDCl3): δ 15.73, 22.27, 53.41, 53.79,
61.08, 65.66, 112.98, 116.45, 119.67, 125.32, 126.93,
127.59, 128.10, 128.23, 129.75, 137.11, 149.54, 156.56,
163.56; MS(ESI) m/z 414(M+ + 1). Anal. calcd for
C27H31N3O: C 78.42, H 7.26, N 10.16%. Found: C 78.55%,
H 7.30, N10.02%.
N-Phenyl-N′-(1-phenyl-propylidene)-N-[4-(2-pyrrolidin-1-
yl-ethoxy)-benzyl]-hydrazone (5e) Oil; yield 68%; 1H
NMR (CDCl3): δ 0.82 (t, 3H), 182–1.86 (m, 4H),
2.66–2.90 (m, 4H), 2.92 (t, 2H), 4.13 (t, 2H), 4.68 (s, 2H),
6.90 (d, 2H, J = 8.4 Hz), 6.93–7.01 (m, 3H, ArH),
7.25–7.35 (m, 4H, ArH), 7.42–7.44 (m, 3H, ArH), 7.77 (d,
2H, J = 8.5 Hz); 13C NMR(CDCl3): δ 11.09, 22.83, 23.53,
54.77, 55.16, 61.58, 67.05, 114.36, 117.04, 120.66,
127.26, 128.44, 128.92, 129.72, 130.93, 137.03, 151.28,
157.28, 173.71; MS(ESI) m/z 428(M+ + 1). Anal. calcd for
C28H33N3O: C 78.65, H 7.78, N 9.83%. Found: C 78.46, H
7.92, N 9.68%.
N-Phenyl-N′-(1-phenyl-propylidene)-N-[4-(2-piperidin-1-
yl-ethoxy)-benzyl]-hydrazone (5f) Oil; yield (69%); 1H
NMR (CDCl3): δ 0.80 (t, 3H), 1.42–1.46 (m, 2H),
1.59–1.67 (m, 4H), 2.51–2.53 (m, 4H), 2.67 (q, 2H), 2.78 (t,
2H), 4.11 (t, 2H), 4.66 (s, 2H), 6.87 (d, 2H, J = 8.4 Hz),
6.89–6.99 (m, 3H, ArH), 7.23–7.33 (m, 4H, ArH),
7.42–7.44 (m, 3H, ArH), 7.75–7.78 (m, 2H); 13C NMR
(CDCl3): δ 11.05, 22.82, 24.20, 25.93, 55.08, 57.99,
61.55, 65.91, 114.10, 115.77, 117.01, 120.62, 127.23,
N-Phenyl-N′-(1-phenyl-ethylidene)-N-[4-(2-piperidin-1-yl-
1
ethoxy)-benzyl]-hydrazone (5b) Oil; yield 71%; H NMR
(CDCl3): δ 1.47–1.49 (m, 2H), 1.61–1.68 (m, 4H), 2.13 (s,
3H), 2.52–2.54 (m, 4H), 2.78 (t, 2H), 4.10 (t, 2H), 4.71 (s,
2H), 6.84 (d, 2H, J = 8.5 Hz), 6.90–6.95 (m, 3H, ArH),

Med Chem Res
128.41, 128.89, 129.68, 130.91, 137.01, 151.25, 157.84,
173.71; MS(ESI) m/z 442 (M+ + 1). Anal. calcd for
C29H35N3O: C 78.87, H 7.99, N 9.52%. Found: C 78.76, H
7.82, N 9.44%.
114.47, 116.96, 120.42, 128.17, 128.89, 129.40, 130.88,
131.17, 150.72, 157.76, 161.03, 166.08; MS(ESI) m/z 470
(M+ + 1). Anal. calcd for C29H35N3O: C 76.12, H 7.71, N
9.18%. Found: C 76.30, H 7.76, N 9.32%.
N-Phenyl-N′-(1-phenyl-butylidene)-N-[4-(2-pyrrolidin-1-
yl-ethoxy)-benzyl]-hydrazone (5g) Oil; yield 62%; 1H
NMR (CDCl3): δ 0.73 (t, 3H), 1.21–1.28 (m, 2H),
1.82–1.86 (m, 4H), 2.61–2.66 (m, 6H), 2.92 (t, 2H), 4.13 (t,
2H), 4.67 (s, 2H), 6.89 (d, 2H, J = 8.4 Hz), 6.93–6.99 (m,
3H, ArH), 7.27–7.34 (m, 4H, ArH), 7.41–7.43 (m, 3H),
7.74 (d, 2H, J = 9.4 Hz); 13C NMR(CDCl3): δ 14.18, 20.02,
23.51, 31.71, 54.76, 55.13, 61.59, 67.02, 114.35, 117.06,
120.62, 127.20, 128.40, 128.73, 128.88, 129.66, 130.90,
137.49, 151.25, 157.88, 172.69; MS(ESI) m/z 442(M+ +
1). Anal. calcd for C29H35N3O: C 78.87, H 7.99, N 9.52%.
Found: C 79.11, H 8.05, N 9.34%.
N′-(3,4-Dihydro-2H-naphthalen-1-ylidene)-N-phenyl-N-[4-
(2-pyrrolidin-1-yl-ethoxy)-benzyl]-hydrazone
(5k) Oil;
yield (69%); 1H NMR (CDCl3): δ 1.74–1.82 (m, 2H),
1.83–1.85 (m, 4H), 2.53 (t, 2H), 2.61–2.66 (m, 4H), 2.77 (t,
2H), 2.92 (t, 2H), 4.11 (t, 2H), 4.71 (s, 2H), 6.86 (d, 2H,
J = 8.5 Hz), 6.90–6.95 (m, 3H, ArH), 7.15 (d, 1H, J = 7.5
Hz), 7.21–7.32 (m, 6H, ArH), 8.37 (d, 1H, J = 7.5 Hz); 13
C
NMR(CDCl3): δ 22.24, 23.47, 28.87, 29.77, 54.68, 55.07,
61.83, 66.86, 114.28, 117.35, 120.64, 125.47, 126.26,
128.67, 128.85, 129.32, 129.32, 129.76, 131.19, 140.26,
150.61, 157.73, 164.89; MS(ESI) m/z 440(M+ + 1). Anal.
calcd for C29H33N3O: C 76.73, H 7.51, N 8.95%. Found: C
76.88, H 7.72, N8.52%.
N-Phenyl-N′-(1-phenyl-butylidene)-N-[4-(2-piperidin-1-yl-
1
ethoxy)-benzyl]-hydrazone (5h) Oil; yield 69%; H NMR
N′-(3,4-Dihydro-2H-naphthalen-1-ylidene)-N-phenyl-N-[4-
(CDCl3): δ 0.74 (t, 3H), 0.90–0.92 (m, 2H), 1.21–1.26 (m,
2H), 1.59–1.64 (m, 4H), 2.50–2.52 (m, 4H), 2.62 (t, 2H),
2.76 (t, 2H), 4.08 (t, 2H), 4.63 (s, 2H), 6.84 (d, 2H, J = 8.5
Hz), 6.90–6.96 (m, 3H, ArH), 7.20–7.23 (m, 2H, ArH),
7.26–7.30 (m, 2H, ArH), 7.38–7.41 (m, 3H, ArH),
7.73–7.76 (m, 2H, ArH); 13C NMR(CDCl3): δ 14.24, 19.99,
24.29, 25.97, 31.56, 55.11, 58.01, 61.99, 65.86, 114.24,
117.32, 120.77, 127.14, 128.29, 128.82, 129.50, 129.62,
130.80, 137.46, 151.42, 157.84, 171.73; MS(ESI) m/z 456
(M+ + 1). Anal. calcd for C30H37N3O: C 79.08, H 8.19, N
9.22%. Found: C 79.46, H 8.42, N 9.01%.
(2-piperidin-1-yl-ethoxy)-benzyl]-hydrazone
(5l) Oil;
yield (69%); 1H NMR (CDCl3): δ 1.45–1.47 (m, 2H),
1.58–1.66 (m, 4H), 1.69–1.78 (m, 2H), 2.51–2.55 (m. 6H),
2.75–2.79 (m, 4H), 4.09 (t, 2H), 4.70 (s, 2H), 6.83 (d, 2H, J
= 8.5 Hz), 6.87–6.95 (m, 3H, ArH), 7.14 (d, 1H, J = 7.5
Hz), 7.29–7.32 (m, 3H, ArH), 8.35 (d, 1H, J = 7.5 Hz); MS
(ESI) m/z 454(M+ + 1). Anal. calcd for C30H35N3O:
C 76.96, H 7.71, N 8.69%. Found: C 76.74, H 7.58,
N8.42%.
N-(4-Methoxy-phenyl)-N′-(1-phenyl-ethylidene)-N-[4-(2-
piperidin-1-yl-ethoxy)-benzyl]-hydrazone (5m) Oil; yield
1
N′-[1-(4-Methoxy-phenyl)-ethylidene]-N-phenyl-N-[4-(2-
pyrrolidin-1-yl-ethoxy)-benzyl]-hydrazone (5i) Oil; yield
69%; 1H NMR (CDCl3): δ 1.80–1.85 (m, 4H), 2.12 (s, 3H),
2.61–2.64 (m, 4H), 2.91 (t, 2H), 3.86 (s, 3H, OCH3), 4.11
(t, 2H), 4.70 (s, 2H), 6.87 (d, 2H, J = 8.6 Hz), 6.90–6.95
(m, 5H, ArH), 7.22–7.31 (m, 4H, ArH), 7.81 (d, 2H, J =
8.6 Hz); 13C NMR(CDCl3): δ 16.76, 23.50, 54.72, 55.11,
55.37, 61.51, 66.92, 113.65, 114.32, 116.97, 120.43,
128.17, 128.88, 129.41, 130.88, 131.19, 150.72, 157.77,
161.03, 166.10; MS(ESI) m/z 456(M+ + 1). Anal. calcd for
C28H33N3O: C 75.81, H 7.50, N 9.47%. Found: C 75.99, H
7.76, N 9.32%.
64%; H NMR (CDCl3): δ 1.47–1.51 (m, 6H), 2.42–2.45
(m, 4H), 2.76 (t, 2H), 3.88 (s, 3H, OCH3), 4.08 (t, 2H,
OCH2), 4.68 (s, 2H), 6.84 (d, 2H, J = 8.5 Hz), 6.90–6.93
(m, 5H, ArH), 7.25–7.29 (m, 4H, ArH), 7.79 (d, 2H, J =
8.5 Hz); MS(ESI) m/z 458(M+ + 1). Anal. calcd for
C29H35N3O2: C 76.12, H 7.71, N 9.18%. Found: C 76.45, H
7.98, N 9.06%.
Result and Discussion
Chemistry
N′-[1-(4-Methoxy-phenyl)-ethylidene]-N-phenyl-N-[4-(2-
piperidin-1-yl-ethoxy)-benzyl]-hydrazone (5j) Oil; yield
(68%); H NMR (CDCl3): δ 1.43–1.48 (m, 2H), 1.59–1.64
(m, 4H), 2.12 (s, 3H), 2.49–2.51 (m, 4H), 2.78 (t, 2H), 3.86
(s, 3H, OCH3), 4.10 (t, 2H), 4.70 (s, 2H), 6.86 (d, 2H, J =
8.5 Hz), 6.90–6.95 (m, 5H, ArH), 7.22–7.31 (m, 4H, ArH),
7.81 (d, 2H, J = 8.5 Hz); 13C NMR(CDCl3): δ 16.76, 24.19,
25.92, 55.05, 55.37, 57.98, 61.52, 65.88, 113.65, 114.32,
The proposed compounds were synthesized as outlined in
the Scheme 1. Phenyl hydrazones (3a–f) were prepared by
heating a mixture of phenyl hydrazines (1a and b) with
acyclic or cyclic ketones (2a–e) under acid catalyzed con-
ditions in excellent yields. Since the hydrazones were
unstable and decomposed rapidly, they were used in situ for
N-alkylation with 4-(ω-chloroalkyl) benzyl bromides in the
presence of NaH/DMF at 0 °C under nitrogen atmosphere to
1

Med Chem Res
Scheme 1 Reagents and
conditions: a CH3COOH/EtOH,
b NaH/DMF, c TBAI, DMF
pyrrolidine/piperidine
give the desired triaryl substituted hydrazone framework
(4a–g). To convert these hydrazones into potential estrogen
antagonists or ER modulators, tert.amino groups, the most
essential determinant known to impart mixed agonist/
antagonist activity were introduced by substituting the
chloride group with secondary amines such as piperidine or
pyrrolidine in the presence of the catalytic quantity of tet-
rabutylammonium iodide (TBAI) forming the target mole-
cules in good yields (5a–m).
breast and uterine cancer cells. Triaryl substituted hydra-
zones (4a–i) without the basic amino functionality were
found to be inactive against both the cancer cell lines,
however, subsequent replacement of terminal halogen with
basic amino groups like piperidine or pyrrolidine rendered
these hydrazones (5a–m) significantly active, showing anti-
proliferative activity against the cancer cell lines. Of all the
hydrazones tested, anti-proliferative activity against ER-
positive breast cancer cell line michigan cancer foundation-
7 (MCF-7) was highest for 5e (IC50: 7.8 μM) followed by 5i
(IC50: 8.4 μM) > 5d (IC50: 9 μM) > 5f (IC50: 11 μM) > 5m
(IC50: 11.8 μM) > 5a (IC50: 16 μM), rest of the compounds
were either inactive or moderately active (Table 1).
In-vitro anti-proliferative activity
Antagonistic action on ER in breast cancer cells results in
the inhibition of its estrogen-induced proliferation. There-
fore, in order to evaluate our hypothesis for synthesizing
novel hydrazones as ER-targeting anti-cancer agents, the
molecules were screened against functional ER bearing
When screened against ER−ve MD Anderson metastatic
breast-231 (MDA-MB-231) cancer cell line, compound
5i was found to be the most active (IC50: 4.7 μM).
Other compounds showing significant activity were

Med Chem Res
Table 1 Physical characteristics and anti-proliferative activity of N-substituted hydrazones 5a–m
Compd.
No
Molecular
formula
Molecular
Mass
Structure
IC50 (μM)
MCF-7
16
MDA-MB-231
16
Ishikawa
NA
HEK-231
16
5a
5b
C
27H31N3O
413.25
427.26
C28H33N3O
NA
NA
NA
NA
NA
NA
NA
NA
5c
C
28H33N3O
427.26
5d
5e
C29H35N3O
C28H33N3O
C29H35N3O
441.28
427.26
9
8
14.7
7.3
28.9
23.3
7.8
6.2
5f
441.28
441.28
11
13
18
24.7
14.7
5g
C
29H35N3O
NA
NA
NA
5h
5i
C30H37N3O
455.29
443.26
457.27
NA
8.4
NA
NA
4.7
8.8
NA
NA
17
C
C
28H33N3O2
6
5j
29H35N3O2
19
NA

Med Chem Res
Table 1 continued
Compd.
No
Molecular
formula
Molecular
Mass
Structure
IC50 (μM)
MCF-7
MDA-MB-231
Ishikawa
HEK-231
5k
C29H33N3O
439.26
11.8
9.3
NA
14
5l
C30H35N3O
453.28
457.27
NA
NA
6
7
NA
11
15
NA
27
15
NA
13
5m
C29H35N3O2
Tamoxifen
NA not active up to 100 µM
5e (IC50: 6.2 μM) > 5k (IC50:7 μM) > 5d (IC50: 8 μM) > 5j
(IC50: 8.8 μM) > 5m (IC50: 9.3 μM) > 5f (IC50:13 μM) and
5a (IC50: 16 μM). Since MDA-MB-231 is an ER-negative
cell line, significant activity of 5i and e indicated the pos-
sibility of these compounds exerting the inhibitory effect
through some target other than ER. Further, screening of
compounds in uterine cancer Ishikawa cell line, 5i again
showed considerable activity (IC50: 6 μM) followed by
compounds, 5e (IC50: 7.3 μM) > 5d (IC50: 14.7) > 5k (IC50:
15) > 5f (IC50: 18) > 5j (IC50:19). In order to confirm if the
cytotoxic effects of the compounds were specific to cancer
cells only, they were also tested for their cytotoxicity in
normal non-cancer specific human kidney epithelial cell line
HEK-293, wherein, compounds, 5d (IC50: 18.9 μM), 5k
(IC50: 14 μM), 5g (IC50: 14.7 μM), 5l (IC50: 15 μM) and 5a
(IC50: 16 μM) were found to be cytotoxic to HEK-293 cells,
whereas, 5e (IC50: 23.3 μM) and 5i (IC50: 27 μM) did not
exhibit any significant cytotoxicity towards normal cell line.
Further, it is also interesting to note that of the two active
compounds, 5e is antagonistic on both the cell types, MCF-
7 cells (IC50: 7.8 µM) and Ishikawa cells (IC50: 7.3 µM)
whereas, compound 5i was selectively most active against
ER-negative MDA-MB 231 cells (IC50: 4.7 µM). Following
the similarity of triaryl substituted hydrazone pharmaco-
phore as structural acyclic proptotype of triaryl pyrazole and
tamoxifen, we decided to focus our initial structure–activity
relationship exploration efforts around the triaryl hydrazone
moiety with differently substituted phenyl rings. A change
in the structure of the alkyl group at position-1 from phenyl-
ethylidene to phenyl-propylidene to phenyl-butylidene and
to 3,4-dihydro-2H-napthalen-1-ylidene resulted in minor
change in the anti-proliferative activity. Increasing the
length of carbon chain from methyl to ethyl or propyl
caused an increase in the activity of ER +ve MCF cells
(IC50: 7.8 μM) and Ishikawa cells (IC50: 7.3 μM), however,
a decrease in the activity was observed for ER-negative
MDA-MB (231) cells (5i, IC50: 4.7 μM). Replacing the 5-
membered pyrrolidine ring to 6-membered piperidine ring
in the basic side chain (5e to 5f) led to a decrease in the
anti-proliferative activity against both the cell lines. An
exploration of the structure activity relationship study
indicated that oxyethyl-pyrrolidine substituted hydrazones
(5e and i) showed better inhibitory effect as compared to the
oxyethyl-piperidine group (5d and f, Table 1).

Med Chem Res
Fig. 3 Docking pose of most active compound 5e (a) and comparative docking orientation and receptor interactions of 5e (gray),
4-hydroxytamoxifen (OHT; green) (b) and ERα (PDB Code: 3ERT). Yellow dotted lines are hydrogen bonding interactions (<2.5 Å)
Molecular docking studies
orientation of pyrrolidino ethyl side chain of 5e is compar-
able to that of the bulky dimethylaminoethyl side chain of
OHT and occupies the nearly the same space within the
cavity of ERα, though it is slightly bent outwards due to an
additional benzylic (CH2) group causing an increase in the
length. Outward deviation of the C-aryl ring and unfavorable
orientation of amino chain of 5e from that of tamoxifen
(Fig. 3b) may be responsible for weak binding interaction of
the ligand with the salient amino acids essential for receptor
binding affinity. The ring B of compound 5e makes contacts
with the residues (Met-343, Leu-346, Met-421, Ile-424, Gly-
521, His-524 and Leu-525), most of which also interact with
the ring B of OHT. These favorable interactions and the
similarity in orientation of aryl rings A, B and C and the
basic amino chains of 4-OHT over that of 5e at the aryl ring
C, conform for the antagonistic profile shown in MCF-7,
MDA-MB-231 and Ishikawa cell lines.
To rationalize the activity profile in terms of interaction with
ER (ERα), the molecular modeling study was carried out to
obtain an insight into the molecular mechanism of compound
5e by comparing docking of OHT and computationally
energy minimized conformation of active molecule 5e using
the Schrodinger suite. The crystal structure of ERLBD in
complex with 4-hydroxytamoxifen and reference protein
coordinates used for protein preparation were obtained from
the Protein Data Bank (http://www.rcsb.org/pdb/; PDB ID:
3ERT) and further modified for Glide docking calculations.
Figure. 3a, b, shows the orientation and interaction of the
active molecule 5e within the ER cavity in comparison to 4-
hydroxy tamoxifen.
As is evident from Fig. 3a, the compound 5e with its three
aryl rings A, B and C (Fig. 2) occupies the same pocket that
recognizes OHT, also the orientation of the aryl ring A of 5e
is in the same location as the ring A of OHT with its phenolic
hydroxyl forming hydrogen bond with the side chains of
Glu-353 and Arg-394. However, absence of any hydroxyl
group or a hydrogen bond donor/acceptor group at the 4th
position of the aryl ring A of 5e results in the loss of this
crucial H-bond interaction with Glu-353 and Arg-394, which
is reflected in weak binding interaction with ERα as seen in
(Fig.3b). Further, it was also observed that the binding
Conclusion
The biological activity results show, that compound 5e is
comparable in its antagonistic activity to tamoxifen against
MCF-7, MDA-MB-231 and Ishikawa cell lines, which is
reflected in the docking studies also, but a slight increase in
length due to an additional methylene group causes

Med Chem Res
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Hooper CL, Fan D, Jursic BS (2003) Design, synthesis, and
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diaryl and triaryl hydrazone derivatives as possible estrogen
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unfavorable twisting and weak binding interactions with
salient amino acids. Structural modification of the active
compounds with insertion of groups and optimization of
chain length and its orientation for specific binding inter-
actions within the receptor cavity is in progress.
Shankar R, Chakravarti B, Singh US, Ansari MI, Deshpande S,
Dwivedi SKD, Bid HK, Konwar R, Kharkwal G, Chandra V,
Dwivedi A, Hajela K (2009) Synthesis and biological evaluation
of 3,4,6-triaryl-2-pyranones as a potential new class of anti-breast
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Greene GL (1988) The structural basis of estrogen receptor/
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tamoxifen. Cell 95:927–937
Acknowledgements The authors, RS and USS are grateful to CSIR
for Senior research fellowships and to the Ministry of Health & Family
Welfare for financial assistance for the biological screening of com-
pounds. All the authors are thankful to Sophisticated Analytical
Instrument Facility for spectroscopic analyses of the pure compounds.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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(2000) Pyrazole ligands: structure-affinity/activity relationships
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