Synthesis, modeling studies and evaluation of E-stilbene hydrazides as potent anticancer agents

Article abstract of DOI:10.1016/j.molstruc.2019.07.043

A group of new thermodynamically more stable E-2-styrylbenzohydrazide derivatives (5a-i) were synthesized via palladium catalyzed Mizoroki-Heck reaction conditions. All synthesized compounds were characterized by UV-visible, FT-IR, ¹H NMR,

Full text of DOI:10.1016/j.molstruc.2019.07.043

Accepted Manuscript
Synthesis, modeling studies and evaluation of E-stilbene hydrazides as potent
anticancer agents
Ahsan Iqbal, Zulfiqar Ali Khan, Sohail Anjum Shahzad, Shakeel Ahmad Khan, Syed
Ali Raza Naqvi, Ayesha Bari, Hira Amjad, Muhammad Ihtisham Umar
PII:
S0022-2860(19)30883-X
DOI:
https://doi.org/10.1016/j.molstruc.2019.07.043
MOLSTR 26796
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 May 2019
Revised Date: 7 July 2019
Accepted Date: 9 July 2019
Please cite this article as: A. Iqbal, Z.A. Khan, S. Anjum Shahzad, S. Ahmad Khan, S.A. Raza Naqvi,
A. Bari, H. Amjad, M.I. Umar, Synthesis, modeling studies and evaluation of E-stilbene hydrazides
as potent anticancer agents, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.07.043.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis, Modeling Studies and Evaluation of E-stilbene Hydrazides as
Potent Anticancer Agents
Graphical Abstract
A new series of E-2-styrylbenzohydrazide derivatives (5a-i) have been synthesized,
1
characterized by H NMR, and 13C NMR and screened for their in vitro anticancer activity
against human breast cancer cells (MCF-7). All of the synthesized compounds showed good to
excellent anticancer activity. Kinetic and docking study of the most potent compound 5e was
also performed.

ACCEPTED MANUSCRIPT
Synthesis, Modeling Studies and Evaluation of E-stilbene Hydrazides as Potent Anticancer
Agents
Ahsan Iqbala, Zulfiqar Ali Khana*, Sohail Anjum Shahzadb*, Shakeel Ahmad Khanc, Syed
Ali Raza Naqvia, Ayesha Baria, Hira Amjadc, Muhammad Ihtisham Umard
aDepartment of Chemistry, Government College University Faisalabad-38000, Pakistan
bDepartment of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-
22060, Pakistan
cDepartment of Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon,
China
dDepartment of Pharmacy, COMSATS University Islamabad, Lahore Campus, Defence Road
Lahore-54000, Pakistan
*Correspondence authors at: Department of Chemistry, Faculty of Physical Sciences, Government
College University, Faisalabad 38000, Pakistan (Z.A. Khan); Department of Chemistry, COMSATS
University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan. Tel.: 0092 992 3835916; Fax:
0092 992 383441 (S.A. Shahzad).
E-mail addresses: Zulfiqarchem@gmail.com (Z.A. Khan), sashahzad@cuiatd.edu.pk (S.A. Shahzad).
Abstract:
A group of new thermodynamically more stable E-2-styrylbenzohydrazide derivatives (5a-i)
were synthesized via palladium catalyzed Mizoroki-Heck reaction conditions. All synthesized
1
13
compounds were characterized by UV-visible, FT-IR, H NMR, C NMR, mass spectrometry
and elemental analysis. Anticancer potential of all synthesized E-stilbene analogues (5a-i) were
investigated via cell proliferation assay and apoptosis by Hoechst 33258 staining assay. The most
active compound (5e) showed significant anticancer potential against estrogen dependent human
breast cancer cells (MCF-7). The cell viability and apoptosis of compound (5e) was found 56 ±
0.06% and 80.09 ± 0.07% respectively as compared to standard drug (Doxorubicin) with cell
viability of 62 ± 0.03% and apoptosis 73.69 ± 0.05%. Docking studies revealed higher efficacy
of compound (5e) owing to its better binding affinity and ligand efficiency scores (Ki = 0.000076
nMol). All stilbene hydrazides (5a-i) exhibited significant anticancer activity against human
breast cancer cells (MCF-7). Therefore, stilbene analogues with hydrazide moiety may offer
immense potential for future therapeutic and pharmaceutical applications.
Keywords: Stilbene hydrazides; Mizoroki-Heck reaction; Breast cancer; Docking study

ACCEPTED MANUSCRIPT
1. Introduction
All the complex organ systems are comprised of simple tiny biological entities, the cells that are
the structural and functional units of the human body. Despite of the tremendous functional
differentiation and specialization within tissues, all the cells of an individual share a common
genetic code that governs and monitors complex cellular processes such as proliferation,
differentiation and apoptosis. Each cell duplicates its DNA before cell division through an
extremely controlled process where any defect in base pair sequencing is carefully monitored
and repaired. However, a deviant subsist of such a defect may instigate an array of aberrant
signaling that precipitates into the tumorigenic transition of defected cells [1–2]. Once
transformed, these rebellious cells tend to divide at an uncontrolled pace that is impervious to the
scarcity of oxygen, nutrients and growth factors [3–4]. The hypoxia induced by the anomalous
cell-proliferation further promotes tumor supportive angiogenesis whereas the unavailability of
nutrients is partly managed by recycling of amino acids within the cancer cells through
autophagy (Figure 1). Moreover, the modified tumor microenvironment further supports the
survival of these cells so much so that they leave their origin and dissipate into the surrounding
healthy tissues through a process called metastasis [5–6]. For last many decades, the clinicians
are putting efforts to encounter the perilous habitation of recalcitrant cancerous cells within the
biological system. However, the major impediment in targeting these handful of cells is the fact
that they are camouflaged in billions of normal cells that makes it difficult to completely wipe
them out. Moreover, the cancer cells share the same biochemical building blocks with the normal
cells which makes these multifold normal cells equally susceptible to any cytotoxic treatment
that is aimed to target the relatively fewer mutant cells. Under non-pathological conditions, the
cells with defected DNA undergo apoptosis and hence depleted from the body. However, the
cancer microenvironment keeps on silencing the pro-apoptotic signaling to make these faulty
cells survive. For instance, the upregulation of the anti-apoptotic Bcl-2 protein in chronic
lymphocytic leukemia makes these cancer cells survive within the tumor milieu [7–8]. Hence, a
pro-apoptotic drug could help reverting such silencing signaling in the cancer cells to induce
selective cytotoxic effect without affecting the normal cells.

ACCEPTED MANUSCRIPT
Figure 1: Cancer Pathogenesis. The aberrant proliferative signaling such as AkT/mTOR, MAPK
and NF-кB signaling results into the tumorigenic transformation of cells with damaged DNA. On
one side, the hypoxic and ischemic conditions within the cancer microenvironment triggers
pathological angiogenesis and autophagy, the silencing of DNA repair mechanism by the
supportive cancer microenvironment help escaping these cells from the apoptosis.
Over the past decades, a number of synthetic compounds are tested for their cytotoxic and pro-
apoptotic potentials with stilbenes as one of the prominent candidates for future cancer drugs [9–
11]. Some of the potential chemotherapeutic drugs with amide and stilbene moiety are shown in
Figure 2. Resveratrol exhibits its cytotoxic effect by inducing S and G2 phase arrest with
subsequent induction of apoptosis in cancer cells [12]. Likewise, veliparib imparts its anti-tumor
effects by improving DNA repair through poly ADP Ribose polymerase pathway [13].
In continuation of our efforts to develop anticancer agents, various analogues of E-stilbene with
hydrazide moiety are envisioned. In the present work, we have reported the synthesis of nine
novel E-stilbenes bearing hydrazide moiety along with their cytotoxic and pro-apoptotic
evaluation in estrogen dependent MCF-7 cells. The possible binding sites with estrogen receptor

ACCEPTED MANUSCRIPT
proteins, binding energy and inhibition constant (ki) of the tested compounds were assessed by
performing docking studies.
Figure 2: Chemical structures of some anti-breast cancer drugs
2. Experimental
2.1. Chemical reagents
All chemicals and reagents were procured from Sigma/Aldrich and Alfa Aesar and used without
further purification. The solvents were dried by using standard procedures. Melting points were
measured by Büchi 434 melting point apparatus. FT-IR spectra (KBr discs) were recorded on a
Bruker FT-IR IFS48 spectrophotometer. 1H NMR and 13C NMR spectra were recorded in CDCl3
using Bruker AC400 (400 MHz and 100 MHz) spectrometer. Mass spectrometry was carried out
via negative electrospray ionization mode (mass spectrometer ESI Micromass ZMD-2000). The
signals are represented with chemical shifts δ in ppm and splitting pattern is given in s = singlet,
d = doublet, dd = doublet of doublet, t = triplet etc. All reactions were monitored by thin layer
chromatography (TLC). TLC was performed on 2 × 5 cm aluminum sheets preloaded with silica
gel 60F254 to a thickness of 0.25 mm (Merck). The chromatograms were visualized under 365 nm
UV light irradiation.
2.2. Chemistry
In current study, synthesis of E-stilbenes (5a-i) is depicted in Scheme 1. The 2-iodobenzoic acid
(1) was refluxed with absolute ethanol in the presence of sulfuric acid furnished ethyl-2-
iodobenzoate (2) in 75% yields [14]. The resulting ester (2) was subjected to Mizoroki-Heck

ACCEPTED MANUSCRIPT
reaction. The ester (2) was coupled with styrene under Mizoroki-Heck reaction conditions to
give E-stilbene (3) with free ester moiety [15-16]. The ester functionality of compound (3) was
converted to E-stilbene hydrazide (4) by treating with hydrazine hydrate in presence of ethanol
[17]. Finally, compounds (5a-i) were accomplished [18-19] from hydrazide (4) by condensation
with a variety of acid chlorides as outlined in Scheme 1.
2.2.1. Synthesis of Ethyl-2-iodobenzoate (2)
Ethyl-2-iodobenzoate (2) was prepared by a reported procedure [14]. Yield: 75%; IR (υmax, KBr,
cm−1): 3010 (C-H aromatic), 2890 (C-H aliphatic), 1660 (C=O), 1110 (C-O).
2.2.2. Synthesis of Compound (3) via Mizoroki-Heck reaction
Ethyl-2-iodobenzoate (2) (5.1 g, 0.018 mol), styrene (2.59 mL, 0.0225 mol), triethylamine (5
mL), Pd(OAc)2 (0.4 g, 0.0018 mol) and triphenylphosphine (0.94 g, 0.0036 mol) were mixed in a
100 mL round bottom flask. The resulting reaction mixture was stirred at 25 ºC for half an hour.
The reaction mixture was subjected to reflux at 120 ºC for 12–15 hours under argon atmosphere.
After completion of the reaction, the reaction mixture was dissolved in ethyl acetate (10 mL).
The reaction mixture was adsorbed on silica gel and purified by column chromatography using
hexane/ethyl acetate mixture (90:10) as an eluent. The product was obtained as yellowish liquid.
Yield: 80%.
2.2.3. Synthesis of E-2-Styrylbenzohydrazide (4)
Ethanolic (10 mL) solution of E-Ethyl 2-styrylbenzoate (3) (2.52 g, 0.01 mol) and 80%
hydrazine hydrate (2.42 mL, 0.04 mol) were mixed in a 100 mL round bottom flask. The reaction
mixture was heated at 80 °C for appropriate time (monitored by TLC analysis). Reaction
completion was confirmed with thin layer chromatography. The remaining hydrazine hydrate
and ethanol was distilled off under reduced pressure. The obtained crude product was washed
with water and crystallized from 30% aqueous ethanol. Yield: 78%; M.P: 150 ºC; colorless
crystals; IR (υmax, KBr, cm−1): 3340 (N-H), 3010 (C-H aromatic), 2860 (C-H aliphatic), 1660
(C=O), 1595 (C=C) 970 (CH=CH); 1H NMR (CDCl3, 400 MHz) δ: 9.12, (s, 1H, N-H), 7.21-7.71
(m, 9H, Ar-H), 7.15 (d, 1H, HC=CH, J = 16 Hz), 6.82 (d, 1H, HC=CH, J = 16 Hz), 4.23 (broad
singlet, 2H)

ACCEPTED MANUSCRIPT
2.2.4. General procedure (GP-1) for the preparation of aroyl hydrazides (5a-i)
2.2.5. (E)-Nʹ-benzoyl-2-styrylbenzohydrazide (5a)
The compound (5a) was synthesized from E-2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and
benzoyl chloride (0.27 mL, 0.0023 mol) by following general procedure GP-1. Yield: %; light
brown solid; mp: 240 ºC; λmax in nm: 315; IR (υmax, KBr, cm−1): 3215 (N-H), 2990 (C-H
aromatic), 1680 (C=O), 1660 (C=O), 1620 (C=C), 1195 (C-N), 987 (CH=CH); 1H NMR (CDCl3,
400 MHz) δ: (s, 2H, N-H), 7.21-7.78 (m, 14H, Ar-H), 7.11 (d, 1H, HC=CH, J = 16 Hz), 6.94 (d,
1H, HC=CH, J = 16 Hz); 13C NMR (CDCl3, 75 MHz) δ: 169.1, 163.4, 147.8, 141.6, 135.5,
134.7, 132.1, 130.8, 130.3, 129.4, 128.8, 128.6, 128.5, 127.8, 127.3, 127.1, 126.8, 123.5; ESI-
MS: 341.25 [M−H]−
2.2.6. (E)-2-chloro-Nʹ-(2-styrylbenzoyl)benzohydrazide (5b)
The compound (5b) was synthesized from E-2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and
2-chlorobenzoyl chloride (0.4 g, 0.0023 mol) by following general procedure GP-1. Yield: 38%;
brown solid; mp: 226 ºC; λmax in nm: 305; IR (υmax, KBr, cm−1): 3290 (N-H), 2895 (C-H
1
aromatic), 1675 (C=O), 1640 (C=O), 1615 (C=C), 1105 (C-N), 970 (CH=CH), 745 (C-Cl); H
NMR (CDCl3, 400 MHz) δ: 7.09-7.88 (m, 13H, Ar-H), 7.24 (d, 1H, HC=CH, J = 16.5 Hz), 6.92
13
(d, 1H, HC=CH, J = 16.5 Hz), 4.45 (s, 2H, N-H); C NMR (CDCl3, 75 MHz) δ: 170.3, 165.8,
140.5, 138.7, 136.2, 135.4, 135.1, 133.2, 132.5, 130.7, 129.8, 129.4, 128.9, 128.3, 127.6, 127.3,
127.1, 126.9, 126.4, 123.8; ESI-MS: 375.51 [M−H]−.
2.2.7. (E)-N'-(4-chlorobenzoyl)-2-styrylbenzohydrazide (5c)
The compound (5c) was synthesized from E-2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and
4-chlorobenzoyl chloride (0.4 g, 0.0023 mol) by following general procedure GP-1. Yield: 45%;
brown solid; mp: 238 ºC; λmax in nm: 310; IR (υmax, KBr, cm−1): 3310 (N-H), 2955 (C-H
1
aromatic), 1695 (C=O), 1660 (C=O), 1635 (C=C), 1095 (C-N), 997 (CH=CH), 725 (C-Cl); H
NMR (CDCl3, 400 MHz) δ: 7.03-7.95 (m, 13H, Ar-H), 7.29 (d, 1H, HC=CH, J = 16.5 Hz), 6.87
13
(d, 1H, HC=CH, J = 16.5 Hz), 4.53 (s, 2H, N-H); C NMR (CDCl3, 75 MHz) δ: 168.4, 161.7,
136.4.8, 135.6, 134.9, 133.7, 133.2, 131.2, 130.3, 128.4, 128.1, 127.8, 127.7, 127.5, 126.9, 126.4,
126.1, 123.6; ESI-MS: 375.52 [M−H]−.
2.2.8. (E)-2-hydroxy-N'-(2-styrylbenzoyl)benzohydrazide (5d)
The compound (5d) was synthesized from E-2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and
2-hydroxybenzoyl chloride (0.36 g, 0.0023 mol) by following general procedure GP-1. Yield:

ACCEPTED MANUSCRIPT
52%; yellow solid; mp: 264 ºC; λmax in nm: 315; IR (υmax, KBr, cm−1): 3345 (O-H, broad), 3125
(N-H), 2880 (C-H aromatic), 1710 (C=O), 1690 (C=O), 1655 (C=C), 1160 (C-N), 975
1
(CH=CH); H NMR (CDCl3, 400 MHz) δ: 7.21-7.98 (m, 13H, Ar-H), 7.57 (d, 1H, HC=CH, J =
13
16 Hz), 6.95 (d, 1H, HC=CH, J = 16 Hz), 4.82 (s, 2H, N-H), 4.20 (s, 1H, Ar-OH); C NMR
(CDCl3, 75 MHz) δ: 167.9, 164.5, 152.6, 139.3, 137.5, 136.7, 134.9, 134.3, 132.2, 131.5, 130.7,
128.7, 128.6, 127.9, 127.5, 127.2, 127.0, 126.9, 126.4, 125.6; ESI-MS: 357.29 [M−H]−.
2.2.9. (E)-N'-(4-hydroxybenzoyl)-2-styrylbenzohydrazide (5e)
The compound (5e) was synthesized from E-2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and
4-hydroxybenzoyl chloride (0.36 g, 0.0023 mol) by following general procedure GP-1. Yield:
55%; yellow solid; mp: 275 ºC; λmax in nm: 320; IR (υmax, KBr, cm−1): 3352 (O-H, broad), 3102
(N-H), 2795 (C-H aromatic), 1675 (C=O), 1594 (C=O), 1547 (C=C), 1162 (C-N), 1068
1
(CH=CH); H NMR (CDCl3, 400 MHz) δ: 7.22-7.70 (m, 13H, Ar-H), 7.31 (d, 1H, HC=CH, J =
16.7 Hz), 7.01 (d, 1H, HC=CH, J = 16.7 Hz), 5.97 (s, 2H, N-H), 4.21 (s, 1H, Ar-OH); 13C NMR
(CDCl3, 75 MHz) δ: 175.4, 170.0, 140.7, 139.9, 136.3, 133.1, 133.0, 129.0, 128.9, 128.1, 128.0,
127.8, 126.7, 126.6, 125.4, 124.4, 123.7, 122.9; ESI-MS: 357.29 [M−H]−.
2.2.10. (E)-2-nitro-N'-(2-styrylbenzoyl)benzohydrazide (5f)
The compound (5f) was synthesized from E-2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and 2-
nitrobenzoyl chloride (0.43 g, 0.0023 mol) by following general procedure GP-1. Yield: 35%;
light brown solid; mp: 218 ºC; λmax in nm: 330; IR (υmax, KBr, cm−1): 3305 (N-H), 2890 (C-H
aromatic), 1670 (C=O), 1620 (C=O), 1595 (C=C), 1345 (Ar-NO2), 1205 (C-N), 990 (CH=CH);
1H NMR (CDCl3, 400 MHz) δ: 7.30-8.18 (m, 13H, Ar-H), 7.41 (d, 1H, HC=CH, J = 16.5 Hz),
13
7.11 (d, 1H, HC=CH, J = 16.5 Hz), 5.13 (s, 2H, N-H); C NMR (CDCl3, 75 MHz) δ: 171.2,
162.5, 148.3, 138.5, 137.8, 137.7, 136.6, 134.8, 134.1, 131.2, 130.7, 129.4, 128.6, 128.3, 127.6,
127.1, 126.6, 125.9, 124.8, 124.3; ESI-MS: 386.27 [M−H]−.
2.2.11. (E)-N'-(4-nitrobenzoyl)-2-styrylbenzohydrazide (5g)
The compound (5g) was synthesized from E-2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and
4-nitrobenzoyl chloride (0.43 g, 0.0023 mol) by following general procedure GP-1. Yield: 40%;
dark brown solid; mp: 204 ºC; λmax in nm: 340; IR (υmax, KBr, cm−1): 3195 (N-H), 2905 (C-H
aromatic), 1680 (C=O), 1660 (C=O), 1625 (C=C), 1330 (Ar-NO2), 1180 (C-N), 980 (CH=CH);
1H NMR (CDCl3, 400 MHz) δ: 7.35-8.14 (m, 13H, Ar-H), 7.28 (d, 1H, HC=CH, J = 16.5 Hz),
13
6.85 (d, 1H, HC=CH, J = 16.5 Hz), 5.42 (s, 2H, N-H); C NMR (CDCl3, 75 MHz) δ: 174.5,

ACCEPTED MANUSCRIPT
169.3, 140.9, 135.7, 134.9, 133.1, 132.9, 132.8, 129.5, 128.7, 128.5, 128.4, 127.8, 127.6, 126.7,
126.4, 126.2, 124.5; ESI-MS: 386.25 [M−H]−.
2.2.12. (E)-N'-(4-aminobenzoyl)-2-styrylbenzohydrazide (5h)
The compound (5h) was synthesized from E-2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and
4-aminobenzoyl chloride (0.38 g, 0.0023 mol) by following general procedure GP-1. Yield:
49%; brown solid; mp: 281 ºC; λmax in nm: 350; IR (υmax, KBr, cm−1): 3315 (N-H), 2970 (C-H
aromatic), 1715 (C=O), 1675 (C=O), 1630 (C=C), 1205 (C-N), 995 (CH=CH); 1H NMR (CDCl3,
400 MHz) δ: 8.96, (s, 2H, NH2), 7.21-7.70 (m, 13H, Ar-H), 7.31 (d, 1H, HC=CH, J = 16 Hz),
6.97 (d, 1H, HC=CH, J = 16 Hz), 4.52 (s, 2H, N-H); 13C NMR (CDCl3, 75 MHz) δ: 175.4, 169.9,
140.0, 136.9, 134.1, 133.9, 133.4, 132.9, 132.3, 130.5, 129.3, 128.8, 128.3, 127.7, 127.6, 127.2,
126.5, 124.8; ESI-MS: 356.32 [M−H]−.
2.2.13. (E)-2-iodo-N'-(2-styrylbenzoyl)benzohydrazide (5i)
The compound (5i) was synthesized from E-2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and 2-
iodobenzoyl chloride (0.61 g, 0.0023 mol) by following general procedure GP-1. Yield: 34%; off
white solid mp: 292 ºC; λmax in nm: 360; IR (υmax, KBr, cm−1): 3257 (N-H), 2950 (C-H
1
aromatic), 1695 (C=O), 1680 (C=O), 1645 (C=C), 1180 (C-N), 1005 (CH=CH); H NMR
(CDCl3, 400 MHz) δ: 7.04-7.92 (m, 13H, Ar-H), 7.36 (d, 1H, HC=CH, J = 16 Hz), 6.95 (d, 1H,
13
HC=CH, J = 16 Hz), 5.02 (s, 2H, N-H); C NMR (CDCl3, 75 MHz) δ: 169.2, 160.7, 139.4,
136.7, 135.6, 135.0, 133.7, 131.4, 130.2, 128.7, 128.6, 128.4, 128.3, 127.9, 127.5, 126.5, 126.2,
125.9, 125.8, 105.8; ESI-MS: 467.12 [M−H]−.
Scheme 1: Schematic representations for the synthesis of analogues of stilbene hydrazides (5a-i)

ACCEPTED MANUSCRIPT
2.3. In vitro anticancer activity
2.3.1 Cell proliferation assay
The cytotoxic potential of compounds (5a-i) was evaluated by using cell proliferation assay via
standard method described by Fakhroueian et al. [20]. Cell Titer 96 aqueous one solution cell
proliferation assay kit (Promega, USA) was used to perform cell proliferation assay according to
the method advised by the manufacturer. Briefly, the human breast carcinoma cells MCF-7 (1 ×
105) were seeded in 96-well microplate in 100 µL media. The cells were incubated for 72 hours
at 37 ˚C and 5% CO2 and further treated with 50 µmol L–1 compounds (5a-i) in triplicate. The 50
µM concentration of doxorubicin was employed as positive control. Cell Titer 96 aqueous
solutions (20 µL) were introduced after exposing for 72 hours. The plate was kept in incubator
for 1 hour at 37 °C and 5% CO2 under a humidified atmosphere. The absorbance measurement of
the formazan product was carried out at 490 nm with a micro plate reader. The blank control was
also measured by recording the absorbance at 490 nm with wells carrying medium combined
with Cell Titer 96 aqueous solution but no cells. Values are stated as percent of the control
(without compound set at 100%). The cell survival was measured in percent as, OD value of
cells treated with the test compound - OD value of culture medium / (OD value of control cell -
OD value of culture medium) x 100%.
2.3.2. Apoptosis by Hoechst 33258 staining assay
Since the compounds (5a-i) exhibited cytotoxicity on MCF-7 cells, therefore their pro-apoptotic
effect was determined through Hoechst staining assay. For this purpose, MCF-7 cells (1×105)
were seeded into 96-well plates in 2.0 mL of medium for 48 hours. Then, 50 µmol L–1
concentration of compounds (5a-i) and standard drug in each plate was incubated for 48 hours.
The cells were washed with phosphate-buffered saline (PBS) and stained thereafter for 10
minutes at room temperature in PBS containing 40% paraformaldehyde and 10 mg mL–1 Hoechst
33258 dye. The Hoechst stained MCF-7 cells were cultured on sterilized coverslips and treated
as specified by literature [21]. Shortly, PBS washed cells fixed with 3.7% formaldehyde in PBS
were further stained with 0.4 mg mL–1 Hoechst dye (Molecular Probes, Eugene, USA) in PBS
for 15 min. After washing twice with PBS and once with water, coverslips were air-dried and
mounted with Slow Fade (Molecular Probes) mounting media. Afterward, evaluations of
morphological changes in nuclear condensation and fragmentation were carried out instantly at
550 nm of emission using a fluorescent microscope (Olympus, Japan).

ACCEPTED MANUSCRIPT
2.4. Computational approach
2.4.1. Repossession of protein and Ligands
The receptor protein as human estrogen receptor is found to be involved in the breast cancer.
Therefore, the three dimensional structure was retrieved from protein data band with PDB ID:
2IOK and resolution of 2.4 Å [22]. It was observed that water molecules and other ligands were
also embedded in the protein structure. Subsequently, they were removed using Accelrys
discovery studio visualizer [23]. The protein structure had to dock with different ligands using
molecular docking. The ligands were drawn in PubChem Sketcher V2.4 after observing their
effects in lab work. The main compound taken was compound (5a) while the other compounds
were drawn on the basis of addition of different groups like nitro group, amine group, hydroxy
group and halogens at ortho and para position which was then analyzed computationally. The
compounds drawn in sketcher was downloaded in form of sdf file which was latter converted
into .pdb form using “Biovia discovery studio visualizer 2017”. The force field was also applied
after geometrical optimization.
2.4.2. Determination of binding sites
The active sites or the binding sites are very crucial in the determination of computational
activity of any compound with protein which not only reveals its structural activity but also its
bioactivity. The human estrogen receptor for breast cancer was analyzed using 3D ligand site and
equated with the previous literature [24].
2.4.3. Docking
The docking was applied on different compounds (5a-i)) to find its placement in the desired
protein. Further, docking score of different compounds were compared along with their
combining residue sets by using “Biovia discovery studio visualizer 2017”. The ligands were
docked using autodock, vina autodock and viewed by discovery studio resulting in 2D and 3D
representation. The kollman charges were added in the receptor protein while gasteiger partial
charges were added in ligands using autodock. The grid was designed after conversion the files
into pdbqt format using autodock which was later used in script writing in vina autodock [25-27].

ACCEPTED MANUSCRIPT
2.4.4. Statistical analysis
Data analysis was performed using IBM statistical package for social sciences (SPSS) software
Version 23.0. All computational calculations were carried out in triplicate and obtained values
were stated as mean ± SEM (n=3). Antimicrobial and antioxidant assays were calculated with
appropriate dilutions for each sample. Statistical analysis was performed by one-way analysis of
variance (ANOVA) followed by post-hoc Duncan multiple range test. The difference between
groups was considered significant at p <0.05.
3. Results and discussion
3.1. Chemistry
1
The structures of new compounds were deduced by probing the UV-visible, FT-IR, H NMR,
13C NMR, and mass spectral results. The UV-visible spectroscopy was used to characterize the
compounds (5a-i) by using CHCl3 as a solvent. The maximum absorption bands (λmax) of
compounds (5a-i) were appeared in the UV-visible spectra ranging from value 305-360 nm. The
literature revealed that λmax values of related stilbene compounds were in the range of 290-360
nm [28].
The FT-IR spectra showed a strong band in the range of 960-1070 cm−1 attributed to the out of
1
plane bending vibration of C-H bond of E-ethylene bridge of stilbene skeleton [28]. H NMR
spectra also confirmed the required number of protons in the title compounds. The chemicals
shift (δ) values (from 6.85-8.18 ppm) were assigned to aromatic and olefinic protons in structure
of designed compounds [29-30]. The trans-geometry of olefinic protons in the stilbene structure
was confirmed by the appearance of two doublets in spectra with large coupling constants (J =
16-17 Hz). FT-IR, 1H NMR and 13C NMR of most active compound 5e are shown in Figures 3–
5.

ACCEPTED MANUSCRIPT
Figure 3: FT-IR spectrum of compound 5e
Figure 4: 1H NMR spectrum of compound 5e

ACCEPTED MANUSCRIPT
Figure 5: 13C NMR spectra of compound 5e
3.2. Biological Studies
3.2.1. In vitro anticancer activity
Chemotherapy is the major approach for both localized and metastasized cancer. Stilbene
analogues with antioxidant and antiproliferative effect has been shown to inhibit a variety of
primary tumors [31]. Therefore, the synthesized compounds together with standard drug
(doxorubicin) were screened for their in vitro cytotoxicity and growth inhibitory activities
against estrogen dependent human breast carcinoma (MCF-7). Most of the synthesized aroyl
hydrazides were significantly cytotoxic against breast cancer cells MCF-7. The cell viability of
synthesized compounds was found in the range of 56–95% as shown in Figure 6. The standard
drug showed cell survival rate of 62 ± 0.03%.
The SAR study revealed that the compound 5a with non-substituted phenyl moiety showed
activity of 73 ± 0.01%, while introduction of electron donating hydroxyl group such as 4-
hydroxyphenyl moiety in compound 5e increases activity and lessen the cell viability of survival
up to 56 ± 0.06%. Whereas the introduction of strong electron withdrawing nitro group such as
4-nitrophenyl in case of compound 5g decreases the activity and enhances the cell viability of

ACCEPTED MANUSCRIPT
survival up to 95 ± 0.02% as compared to 5a (with non-substituted phenyl). Similar decrease in
activity was also observed in case of chloro- and iodo-functionalities present in compounds 5b,
5c and 5i with cell survival rate of 86 ± 0.02%, 78 ± 0.04% and 84 ± 0.04% respectively.
Figure 6: Anticancer activity of doxorubicin and synthesized stilbene hydrazides (5a-i) against
breast cancer cells
3.2.2. Apoptosis by Hoechst 33258 staining assay
The apoptosis induced by compounds (5a-i) was verified on MCF-7 cells stained with Hoechst
33258 for 48 hours in comparison with standard drug as shown in Figure 7. The typical
cytological changes appeared in the cancer cells were investigated using fluorescence
microscope. Several signifcant changes in cancer cells are appeared for instance activation of
caspases, phosphatidylserine, externalization, plasma membrane blebbing, bi-nucleation,
cytoplasmic vacuolation, cell shrinkage, nuclear fragmentation, chromatin condensation and
chromosomal apoptotic bodies and DNA fragmentation (Figure 7). Moreover, the number of
rounded-up cells with plasma membrane blebs increases with an increase in incubation time. As
shown in Figures 7e and 7f where morphological findings indicated that the compounds 5d and
5e reduced cell viability of survival and induced apoptosis (Table 1) in MCF–7. These
cytological changes indicate that the cells were committed to a specific mode of cell death
resulting in apoptosis.

ACCEPTED MANUSCRIPT
Figure 7: Hoechst 33258 stained morphology of control (a), and MCF-7 cell line treated with
Componds 6a (b), Componds 6b (c), Componds 6c (d), Componds 6d (e), Componds 6e (f),
Componds 6f (g), Componds 6g (h), Componds 6h (i) and Doxorubicin (j).

ACCEPTED MANUSCRIPT
Table 1. Effect of E-stilbene analogues (5a-i) on apoptosis of MCF-7 cells by Hoechst dye
staining method.
Sample
Apoptosis %
7.30 ± 0.03
Control
Compund 5a
Compund 5b
Compund 5c
Compund 5d
Compund 5e
Compund 5f
Compund 5g
Compund 5h
Compund 5i
Doxorubicin
60.98 ± 0.07
45.21 ± 0.06
55.98 ± 0.09
75.09 ± 0.07
80.09 ± 0.07
25.09 ± 0.07
30.09 ± 0.07
69.09 ± 0.07
48.31 ± 0.09
73.69 ± 0.05
Values are mean ± SD triplicate assays
3.3 Computational Approach
3.3.1. Binding sites
The binding sites were analyzed using 3D ligand that revealed a number of binding residues to
be present at active site for binding with the ligands. The residues were Met343, Leu346,
Thr347, Ala350, Glu353, Leu384, Leu387, Met388, Leu391, Arg394, Phe404, Val418, Met421,
Ile424, Leu428, His524 and Leu525. The residues from receptor protein were the same as those
already found out in the literature review [32-34].
3.3.2. Docking
The docking was performed with a number of different compounds (5a-i). The interaction of
receptor protein and compounds 5a revealed the binding of important residue leucine, which is
considered important in treating cancer. The bonding formed includes number of different bonds
like hydrogen bond, pi-acceptor and pi-donor bond owing to the presence of benzoyl group and

ACCEPTED MANUSCRIPT
its electron delocalization property [35–36]. All the ligands with different group bound with the
same binding pocket and almost with the same residues with slight difference of number of
residues and bond type. The docking score indicated the strength of bond formed while it also
helped us to find the inhibitor constant (Ki). The inhibitor constant is of vital importance in the
drug designing as it indicates the inhibitor potency to produce half concentration after inhibition.
The Ki has an inevitable effect on IC50 effecting the mode of action of a drug. For better IC50
value, the Ki value must be the least one, which further plays its role in better drug activity [37].
The Table 2 provided the results with docking score and inhibitor constant values. The binding
poses of compounds (5d, 5e and 5h) at their maximum bound cavity is showed in Figure 8–13.
Table 2. The results of compounds (5a-i) with docking score and inhibitor constant values.
Ligands
Compound 5a
Compound 5b
Compound 5c
Compound 5d
Compound 5e
Compound 5f
Compound 5g
Compound 5h
Binding affinity
Ki (nMol)
0.000210
0.000150
0.000150
0.000090
0.000076
0.001138
0.000150
0.000105
0.001138
–9.1
–9.3
–9.3
–9.6
–9.7
–8.1
–9.3
–9.5
–8.1
Compound 5i
Values are mean ± SD triplicate assays
The docking score revealed that the compound 5a shows a small value of binding affinity
leading to more binding energy. The para substituted compounds showed better activity towards
the protein rather than the ortho one. The substitution of –Cl group at ortho and para position in
compound 5b and 5c respectively produced the same results which were obviously due to their
deactivating nature. Whereas the substitution of –NO2 group at ortho and para position in
compound 5f and 5g respectively produced distinct results. The –NO2 group at para position
produced about same results as that of –OH group at para but the –NO2 group at ortho position
further increased the binding affinity value leading to low activity than the reference compound.
Similarly the –NH2 group in compound 5h on para position showed comparable results with the
results of compounds 5d and 5e due to electron donating nature. The substitution of –OH group
at ortho position in compound 5d and at para position in compound 5e provided the best and
distinctive results along with its hydrogen bonding. Here the value at para position was
somehow better but the both results were almost comparable yet much better than the reference

ACCEPTED MANUSCRIPT
compound revealing that the presence of strong electron activating or electron donating groups
(+R) play important role in protein ligand binding and better activity towards the breast cancer
[38]. Hence, the close agreement was observed between the experimental results of anticancer
activity and the docking results of the synthesized compounds.
Figure 8: 2D Plot, binding affinity of compound 5d at highly bound cavity
Figure 9: Binding of compound 5d into protein active site, as assessed by computer modeling
studies

ACCEPTED MANUSCRIPT
Figure 10: 2D Plot, binding affinity of compound 5e at highly bound cavity
Figure 11: Binding of compound 5e into protein active site, as assessed by computer modeling
studies
Figure 12. 2D Plot, binding affinity of compound 5e at highly bound cavity

ACCEPTED MANUSCRIPT
Figure 13. Binding of compound 5h into protein active site, as assessed by computer modeling
studies
4. Conclusion
A small library of stilbene hydrazide based nine compounds (5a-i) were synthesized via
Mizoroki-Heck reaction as a key step. The synthesized compounds were evaluated for potent
anticancer activity by cell proliferation assay and apoptosis by Hoechst 33258 staining assay.
The results of this study indicated that the synthesized compounds 5d, 5e and 5h having
hydroxyl and amino substituent possess potent anticancer properties. Docking process revealed
the binding, active site, their mode of action and bioactivity.
Acknowledgements
The authors (Dr. S. A. Shahzad & Dr. Z. A. Khan) is very grateful to the Higher Education
Commission (HEC) of Pakistan for providing financial support vide No.
5290/Federal/NRPU/R&D/HEC/2016.
Conflict of Interest
No conflict of interest is associated with this work.

ACCEPTED MANUSCRIPT
References
[1]
[2]
M. Usman, E.V. Volpi, DNA damage in obesity: Initiator, promoter and predictor of cancer,
Mutat. Res. 778 (2018) 23–37.
E. Bonazzoli, E. Cocco, S. Lopeza, S. Bellonea, L. Zammataro, A. Bianchi, A. Manzano, G.
Yadav, P. Manara, E. Perrone, K. Haines, M. Espinal, K. Dugan, G. Menderes, G. Altwerger, C.
Han, B. Zeybek, B. Litkouhi, E. Ratner, D.-A. Silasi, G.S. Huang, M. Azodi, P. E. Schwartz,
PI3K oncogenic mutations mediate resistance to afatinib in HER2/neu overexpressing
gynecological cancers, Gynecol. Oncol. 153 (2019) 158–164.
[3]
[4]
[5]
[6]
[7]
M. Archetti, K.J. Pienta, Cooperation among cancer cells: applying game theory to cancer, Nat.
Rev. Cancer 19 (2019) 110–117.
M.V. Liberti, J.W. Locasale, The Warburg effect: How does it benefit cancer cells? Trends
Biochem. Sci. 41 (2016) 211–218.
M. Pein, T. Oskarsson, Microenvironment in metastasis: roadblocks and supportive niches, Am.
J. Physiol. Cell Physiol. 309 (2015) C627–638.
D.F. Quail, J.A. Joyce, Microenvironmental regulation of tumor progression and metastasis, Nat.
Med. 19 (2013) 1423–1437.
M. Dancescu, M. Rubio-Trujillo, G. Biron, D. Bron, G. Delespesse, M. Sarfati, Interleukin 4
protects chronic lymphocytic leukemic B cells from death by apoptosis and upregulates Bcl-2
expression, J. Exp. Med. 176 (1992) 1319–1326.
[8]
L.1. Sanz, J.A. Garcia-Marco, B. Casanova, M.T. de La Fuente, M. García-Gila, A. Garcia-Pardo,
A. Silva, Bcl-2 family gene modulation during spontaneous apoptosis of B-chronic lymphocytic
leukemia cells, Biochem. Biophys. Res. Commun. 315 (2004) 562–567.
R. Csuk, S. Albert, B. Siewert, S. Schwarz, Synthesis and biological evaluation of novel (E)
stilbene-based antitumor agents, Eur. J. Med. Chem. 54 (2012) 669–678.
[9]
[10]
R. Mikstacka, M. Zielińska-Przyjemska, Z. Dutkiewicz, M. Cichocki, T. Stefański, M.
Kaczmarek, W. Baer-Dubowska, Cytotoxic, tubulin-interfering and proapoptotic activities of 4'-
methylthio-trans-stilbene derivatives, analogues of trans-resveratrol, Cytotechnology 70 (2018)
1349–1362.
[11]
M.N. Azmi, M.F.M. Din, C.H. Kee, M. Suhaimi, A.K. Ping, K. Ahmad, M.A. Nafiah, N.F.
Thomas, K. Mohamad, L. K. Hoong, K. Awang, Design, synthesis and cytotoxic evaluation of o-
carboxamido stilbene analogues, Int. J. Mol. Sci. 14 (2013) 23369–23389.
T.-C. Hsieh, J.M. Wu, Differential effects on growth, cell cycle arrest, and induction of apoptosis
by resveratrol in human prostate cancer cell lines, Exp Cell Res. 249 (1999) 109–115.
L.M. Wagner, Profile of veliparib and its potential in the treatment of solid tumors, Onco. Targets
Ther. 8 (2015) 1931–1939.
Z.A. Khan, T. Wirth, Synthesis of indene derivatives via electrophilic cyclization, Org. Lett. 11
(2009) 229-231.
Z. A. Khan, I. Michio, T. Wirth, Novel cyclization cascades to functionalized indanes and
tetrahydronaphthalenes, Tetrahedron 66 (2010) 6639-6646.
[12]
[13]
[14]
[15]
[16]
[17]
H.A. Dick, R.F. Heck, Organophosphinepalladium complexes as catalysts for vinylic hydrogen
substitution reactions, J. Am. Chem. Soc. 96 (1974) 1133-1136.
M. Yar, M. Bajda, R. A. Mehmood, L. Sidra, N. Ullah; L. Shahzadi, M. Ashraf, T. Ismail, S.A.
Shahzad, Z.A Khan et al. Design and synthesis of new dual binding site cholinesterase inhibitors:
In vitro inhibition studies with in silico docking, Lett. Drug Des. & Dis. 11 (2014) 331-338.
A.P. Dwivedi, S. Kumar, V. Varshney, A.B. Singh, A.K. Srivastava, D.P. Sahu, Synthesis and
antihyperglycemic activity of novel N-acyl-2-arylethylamines and N-acyl-3-coumarylamines,
Bioorg. Med. Chem. Lett. 18 (2008) 2301-2305.
[18]

ACCEPTED MANUSCRIPT
[19]
[20]
X. Li, J. Shen, L. Tan, Z. Zhang, D. Gao, J. Luo, H. Cheng, X. Zhou, J. Ma, K. Ding, X. Lu,
Design and synthesis of N-(4-aminopyridin-2-yl)amides as B-Raf(V600E) inhibitors, Bioorg.
Med. Chem. Lett. 26 (2016) 2760-2763.
Z. Fakhroueian, A.M. Dehshiri, F. Katouzian, P. Esmaeilzadeh, In vitro cytotoxic effects of
modified zinc oxide quantum dots on breast cancer cell lines (MCF7), colon cancer cell lines
(HT29) and various fungi. J. Nanopart. Res. 16 (2014) 2483.
[21]
[22]
D. Denoyer, S. Masaldan, S. La Fontaine, M.A. Cater, Targeting copper in cancer therapy:
'Copper That Cancer', Metallomics 7 (2015) 1459-1476.
K.D. Dykstra, L. Guo, E.T. Birzin, W. Chan, Y.T. Yang, E.C. Hayes, C.A. DaSilva, L.Y. Pai,
R.T. Mosley, B. Kraker, P.M. Fitzgerald, F. DiNinno, S.P. Rohrer, J.M. Schaeffer, M.L.
Hammond. Estrogen receptor ligands. Part 16: 2-Aryl indoles as highly subtype selective ligands
for ERalpha, Bioorg. Med. Chem. Lett. 17 (2007) 2322–2328.
[23]
[24]
[25]
Dassault Systèmes BIOVIA, Biovia Discovery Studio Visualizer, v16.1.0, San Diego: Dassault
Systèmes, 2016.
M.N. Wass, L.A. Kelley, M.J. Sternberg, 3DLigandSite: predicting ligand-binding sites using
similar structures, Nucleic Acids Res. 38 (2010) W469-73.
G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, A.J. Olson, AutoDock4 and
AutoDockTools4: Automated docking with selective receptor flexibility, J. Comput. Chem. 30
(2009) 2785–2791.
[26]
K. Rizwan, N. Rasool, R. Rehman, T. Mahmood, K. Ayub, T. Rasheed, G. Ahmad, A. Malik,
S.A. Khan, M.N. Akhtar, N.B. Alitheen. Facile synthesis of N-(4-bromophenyl)-1-(3-
bromothiophen-2-yl) methanimine derivatives via Suzuki cross-coupling reaction: their
characterization and DFT studies, Chem. Cent. J. 12 (2018) 1-9.
[27]
[28]
O. Trott, A.J. Olson, AutoDock Vina: Improving the speed and accuracy of docking with a new
scoring function: Efficient optimization and multithreading, J. Comput. Chem. 31 (2010) 455–
461.
E. Wyrzykiewicz, M. Wendzonka, B. Kedzia, Synthesis and antimicrobial activity of new (E)-4-
[piperidino (4'-methylpiperidino-, morpholino-) N-alkoxy]stilbenes, Eur. J. Med. Chem. 41
(2006) 519-525.
[29]
[30]
E. Wyrzykiewicz, A. Blaszczyk, B. Kedzia, Synthesis and antimicrobial activity of (E)-
acetoxystilbenes and α,α′-dibromoacetoxybibenzyls, IL Farmaco 55 (2000) 151-157.
W. Jian, D. He, P. Xi, X. Li, Synthesis and biological evaluation of novel fluorine-containing
stilbene derivatives as fungicidal agents against phytopathogenic Fungi, J. Agri. Food Chem. 63
(2015) 9963-9969.
[31]
[32]
[33]
[34]
M.N. Azmi, M.F.M. Din, C.H. Kee, M. Suhaimi, A.K. Ping, K. Ahmad, M.A. Nafiah, N.F.
Thomas, K. Mohamad, L.K. Hoong, K. Awang, Design, synthesis and cytotoxic evaluation of o-
carboxamido stilbene analogues, Int. J. Mol. Sci. 14 (2013) 23369-23389.
W. Ahmad, S.A. Khan, K.S. Munawar, A. Khalid, S. Kawanl Synthesis, characterization and
pharmacological evaluation of mixed ligand-metal complexes containing omeprazole and 8-
hydroxyquinoline, Trop. J. Pharm. Res. 16 (2017) 1137-1146.
S.A. Khan, W. Ahmad, K.S. Munawar, S. Kanwal, Synthesis, spectroscopic characterization and
biological evaluation of Ni (II), Cu (II) and Zn (II) complexes of diphenyldithiocarbamate, Indian
J. Pharm. Sci. 80 (2018) 480-488.
S.A. Khan, S. Shahid, S. Kanwal, G. Hussain, Synthesis characterization and antibacterial activity
of Cr (III), Co (III), Fe (II), Cu (II), Ni (III) complexes of 4-(2-(((2-hydroxy-5-nitrophenyl)
diazenyl)(phenyl) methylene) hydrazinyl) benzene sulfonic acid based formazan dyes and their
applications on leather, Dyes Pigments 148 (2018), 31-43.
[35]
S.A. Khan, F. Noreen, S. Kanwal, A. Iqbal, G. Hussain, Green synthesis of ZnO and Cu-doped
ZnO nanoparticles from leaf extracts of Abutilon indicum, Clerodendrum infortunatum,
Clerodendrum inerme and investigation of their biological and photocatalytic activities, Mater.
Sci. Eng. C 82 (2018) 46-59.

ACCEPTED MANUSCRIPT
[36]
S.A. Khan, S. Shahid, S. Kanwal, K. Rizwan, T. Mahmood, K. Ayub, Synthesis of novel metal
complexes of 2-((phenyl (2-(4-sulfophenyl) hydrazono) methyl) diazenyl) benzoic acid formazan
dyes: Characterization, antimicrobial and optical properties studies on leather, J. Mol. Struct.
1175 (2019) 73-89.
[37]
[38]
K.A. Bachmann, J.D. Lewis, Predicting inhibitory drug—drug interactions and evaluating drug
interaction reports using inhibition constants, Ann. Pharmacother. 39 (2005) 1064-1072.
C.W. Mai, M. Yaeghoobi, N. Abd-Rahman, Y.B. Kang, M.R. Pichika, Chalcones with electron-
withdrawing and electron-donating substituents: Anticancer activity against TRAIL resistant
cancer cells, structure–activity relationship analysis and regulation of apoptotic proteins, Eur. J.
Med. Chem. 77 (2014) 378-387.

ACCEPTED MANUSCRIPT
Figure 1: Cancer Pathogenesis. The aberrant proliferative signaling such as AkT/mTOR, MAPK
and NF-кB signaling results into the tumorigenic transformation of cells with damaged DNA. On
one side, the hypoxic and ischemic conditions within the cancer microenvironment triggers
pathological angiogenesis and autophagy, the silencing of DNA repair mechanism by the
supportive cancer microenvironment help escaping these cells from the apoptosis.
Figure 2: Chemical structures of some anti-breast cancer drugs

ACCEPTED MANUSCRIPT
O
I
O
O
OEt
NH2NH2 • H2O
EtOH/H
65%
OH
OEt
Pd(II)acetate,
I
EtOH, 80%
PPh3, Et3N, 60%
3
2
1
O
O
H
H
N
O
N
O
Cl
N
NH2
R
R
H
4
5a-i
5a: R = H; 5b: R = 2-Cl; 5c: R = 4-Cl; 5d: R = 2-OH; 5e: R = 4-OH; 5f: R = 2-NO2
5g: 5h: 5i:
R = 4-NO2;
R = 4-NH2;
R = 2-I
Scheme 1: Schematic representations for the synthesis of analogues of stilbene hydrazides (5a-i)
Figure 3: FT-IR spectrum of compound 5e

ACCEPTED MANUSCRIPT
Figure 4: 1H NMR spectrum of compound 5e

ACCEPTED MANUSCRIPT
Figure 5: 13C NMR spectrum of compound 5e
Figure 6: Anticancer activity of doxorubicin and synthesized derivatives (5a-i) against breast
cancer cells

ACCEPTED MANUSCRIPT
Figure 7: Hoechst 33258 stained morphology of control (a), and MCF-7 cell line treated with
Componds 6a (b), Componds 6b (c), Componds 6c (d), Componds 6d (e), Componds 6e (f),
Componds 6f (g), Componds 6g (h), Componds 6h (i) and Doxorubicin (j).