Identification of N-Hydroxycinnamamide analogues and their bio-evaluation against breast cancer cell lines

Article abstract of DOI:10.1016/j.biopha.2018.08.015

The present study demonstrates the identification of N-hydroxycinnamamide derivatives and their anticancer potential against human triple-negative breast cancer cell line MDA-MB?231, MCF-7 and non-malignant origin cell line, HEK-293 (human embryonic kidney). MTT assay was studied with HEK-293 cell line. Anticancer potential of the N-hydroxycinnamamide derivatives were compared with marked drug Tamoxifen through in vitro study. The compound numbers 3b and 3h exhibit most potent activity against antagonistic breast cancer cells (MDA-MB-231) with IC₅₀ 13μM and 5μM respectively. Compound 3h promotes DNA fragmentation and induction of apoptosis. Furthermore, loss of mitochondrial membrane potential induced by compound 3h. The major mechanism of compound 3h for anti-breast cancer activity was probably initiation of reactive oxygen species (ROS) in cancer cells thereby persuading apoptotic cell deaths in cancer cells.

Full text of DOI:10.1016/j.biopha.2018.08.015

Biomedicine&Pharmacotherapy107(2018)475–483
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Identification of N-Hydroxycinnamamide analogues and their bio-
evaluation against breast cancer cell lines
Akhilesh Kumar Shuklaa, Hamidullahb,c,d, Manoj Kumar Shrivasha, Vishwa Deepak Tripathie,
Rituraj Konwarc,d, Jyoti Pandeya,⁎
a
Department of Chemistry, Babasaheb Bhimrao Ambedkar University (A Central University), Lucknow 226025, UP, India
Department of Dermatology, University of Wisconsin Madison, WI 53706, USA
Endocrinology Division, CSIR-Central Drug Research Institute (CDRI), Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India
Academy of Scientific and Innovative Research (AcSIR), CSIR-Central Drug Research Institute Campus, Lucknow 226031, India
b
c
d
e
Department of Chemistry, Maharani Kalyani College Laheriasarai, Lalit Narayan Mithila University, Darbhanga 846001, Bihar, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
The present study demonstrates the identification of N-hydroxycinnamamide derivatives and their anticancer
potential against human triple-negative breast cancer cell line MDA-MB‑231, MCF-7 and non-malignant origin
cell line, HEK-293 (human embryonic kidney). MTT assay was studied with HEK-293 cell line. Anticancer po-
tential of the N-hydroxycinnamamide derivatives were compared with marked drug Tamoxifen through in vitro
study. The compound numbers 3b and 3h exhibit most potent activity against antagonistic breast cancer cells
(MDA-MB-231) with IC50 13μM and 5μM respectively. Compound 3h promotes DNA fragmentation and in-
duction of apoptosis. Furthermore, loss of mitochondrial membrane potential induced by compound 3h. The
major mechanism of compound 3h for anti-breast cancer activity was probably initiation of reactive oxygen
species (ROS) in cancer cells thereby persuading apoptotic cell deaths in cancer cells.
Breast cancer
MDA-MB-231
MCF-7
N-Hydroxycinnamamide
HEK-293
1. Introduction
eCONHOH or eCONHR [5]. Hydroxamic acids are well known as ef-
ficacious molecules in the field of cancer chemotherapy and as a mu-
Cancer is one of the most severe public health issue around the
globe according to the World Health Organization (WHO) [1]. Among
various type of cancers breast cancer is also one of the major causes of
cancer death among women worldwide. Due to its complex cancer
biology, it is necessary to use multiple therapeutic modalities. So far,
the conventional treatments for breast cancer are surgical intervention,
hormonal therapy, radiotherapy and chemotherapy. It is merely re-
sponsible for 20–25% of all cancer cases and 15–18% of cancer deaths
among women [2]. Although the emergence of drugs such as Tamoxifen
and Toremifene makes chemotherapy a viable choice for breast cancer
patients, the development of drug resistance and severe side effects are
unresolved problems in clinical oncology [3]. Therefore, the search for
novel anti-cancer compounds with improved features is needed. In re-
cent oncology research, different breast cancer cell lines have been
applied by investigators for drug discovery purposes and among these
cells estrogen non-dependant MDA-MB-231 is one of the most ex-
tensively used model [4].
tagenic agent. Several hydroxamates based drugs are functioning very
good in clinics for cancer chemotherapy such as SAHA [6,7], PXD-101
(Belinostat,Topotarget) [8] and LBH-589 (panobinostat) [9], which are
approved by the U.S. Food and Drug Administration (FDA) in October
2006, July 2014 and February 2015, respectively (Fig. 1). There are
some other hydroxamate based molecules are in clinical trials, such as
m-carboxycinnamic acid bishydroximic acid (CBHA) [10], SB-939
(phase II) [11] and 4SC-201 (Resminostat, phase II) [12].
Among various derivatives of hydroxamic acid, SAHA
(Suberoylanilide Hydroxamic Acid) is considered as a potent anticancer
agent [13]. These molecules possess very good chelating ability [14].
This chelating property makes them very favourable for enzyme in-
hibition and therefore hydroxamates possess a special place in cancer
drug discovery research. Due to these special properties hydroxamates
are very interesting group for scientists from all over the world. Several
research groups have synthesized different hydroxamic acid moieties
with well-known inhibitors of matrix metalloproteinases (MMPs) [15],
peptidyldeformylases [16], adenylyl cyclases (ACs) [17], inosine
monophosphate dehydrogenase (IMPDH), histone deacetylase (HDAC)
Hydroxamic acids or hydroxamates are carboxylic acids or aldehyde
analogues where eCOOH group or eCHO group has been replaced by
⁎
Corresponding author.
E-mail address: drjyotibbau@gmail.com (J. Pandey).
https://doi.org/10.1016/j.biopha.2018.08.015
Received 11 June 2018; Received in revised form 24 July 2018; Accepted 6 August 2018
0753-3322/©2018ElsevierMassonSAS.Allrightsreserved.

A.K. Shukla et al.
Biomedicine&Pharmacotherapy107(2018)475–483
Fig. 1. Approved and clinical HDACLs with N-hydroxycinnamamide fragment highlighted in red.
[15], carbonic anhydrase [18], tumor necrosis factor converting en-
zyme (TACE, ADAM17) [19] and TNF-α–converting enzyme [20]. One
research group reported antileukemic activity in hydroxamic acids
[21,22]. Azaindolehydroxamic acid derivatives are known to possess
potent anti-HIV activities [23] while sulfonamidohydroxamates are
good anti-osteoarthritis agents [24–26]. In addition to these interesting
properties this moiety is also present in many growth factors [27], food
additives [28], antibiotics [29,30], antitumors [31], antifungals [32],
cell division factors and enzyme inhibitors [33,34]. They have also
shown inhibition against melanogenesis [18]. As many enzymes are
inhibited by hydroxamates, several physiological processes are affected
by this versatile class. Some molecules with hydroxamic acid func-
tionality have also been reported as NO donors [35] and the acetylated
hydroxamates derivatives can act as effective aspirin analogues by
prostaglandin H2 synthase inhibition [36,37].
It is evident from literature survey that hydroxamic acid derivatives
have attracted scientists for their potential as highly efficacious in
combating various etiological factors associated with cancer [38]. They
have also been used as acyl cation equivalents for the preparation of
carbonyl compounds and N-methoxyamides as precursors of N-
methoxy-N-acylnitrenium ions in electrophilic aromatic substitutions
and as precursor for β-lactams synthesis [39,40]. Different N-benzox-
yamides as precursors of hydroxamic acids, are known for different
biological activities including anti-inflammatory [41] antiasthmatic
[42] antimetastatic [43] antibiotic [44] psychotropic [33] insecticidal
[45] acaricidal [46] and nematocidal activity [33]. In this article we
present anticancer property of hydroxamic acid derivatives to show that
this single functional moiety has not only fit in the receptor site but also
create a diversified activity.
aldehyde using HWE reaction and synthesis of 2-O-alkyl benzhy-
droxamic acids.
2. Materials and methods
2.1. Materials
All chemicals, reagents and solvents were purchased from Sigma
Aldrich and Merck. Thin layer chromatography was performed using
silica gel 60 F254 plates with detecting agent iodine vapours, Merck
Silica gel (60–120 mesh) was used for column chromatography. IR
spectra were recorded as thin films or in chloroform soln with a
Perkin–Elmer Spectrum RX-1 (4000–450 cm−1) spectrophotometer. 1H
and 13C NMR spectra were recorded on a Brucker DRX-400 and 101
MHz in DMSO-d6. Chemical shift values are reported in ppm relative to
SiMe4 as internal reference, unless otherwise stated; s (singlet), d
(doublet), t (triplet), m(multiplet); J in hertz. FAB mass spectra were
performed using a mass spectrometer Jeol SX-102 and ESI mass spectra
with Quattro II (Micromass). Melting points were obtained manually by
capillary methods and are uncorrected. Elemental analyses were per-
formed on a Perkin–Elmer 2400 II elemental analyzer. The organic
extracts were dried over anhydrous Na2SO4 and evaporation of the
solvent was carried out on a rotary evaporator under reduced pressure.
2.2. Synthesis of α, β-unsaturated hydroxamic acids
The starting acrylate derivatives (2a-k) were prepared by the
Horner-Wadsworth-Emmons (HWE) olefination of different aromatic
aldehydes (1a-k) with triethylphosphonoacetate in the presence of
LiOH in THF at ambient temperature. The reaction of acrylate
Present work demonstrates synthesis of simple hydroxamic acids by
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A.K. Shukla et al.
Biomedicine&Pharmacotherapy107(2018)475–483
Scheme 1. Synthesis of N-Hydroxycinnamamide derivatives, Reagents and conditions.
(i) LiOH (1.1eq), TEPA (1.1eq), THF, RT (ii) NH2OH.HCl (5 eq), KOH (10 eq), CH3OH, 0 °C- RT.
derivatives (2a-k) with hydroxylamine hydrochloride in the presence of
KOH in methanol at 0–5 °C results in the formation of respective N-
hydroxyacrylamide (3a-k) in moderate to excellent yields with 99.8%
purity. (Scheme 1 ).
and KOH (1.32 g, 23.7 mmol) in methanol at 0–5 °C to room tempera-
ture to gave the corresponding acrylamide 3a in 84% yield (0.85 g) as a
white solid, mp 140–142 °C; IR(νmax,cm−1): 3416, 3039, 1650, 1090,
754, 1H NMR (400 MHz, DMSO-d6); δH 9.57–9.31 (1H, brs, NH)
7.60–7.58 (2H, d, J = 8.3 Hz, Ar-H), 7.46-7.44 (2H, d, J = 8.3 Hz, Ar-
H), 7.41 (1H, brs, CH), 6.49–6.45 (1H, m, J = 15.8 Hz), 2.08 (1H, s,
OH); 13C NMR (101 MHz, DMSO-d6); δC 162.9, 137.5, 134.3, 134.1,
129.6, 129.4, 120.2. HRMS: Calcd. accurate mass for (C9H9ClNO2),
197.0316. Found. [M+H]+ 198.0313.
2.3. Synthesis of 2-O-alkyl benzhydroxamic acids
Compounds methyl 2,4-dihydroxybenzoate (4) on chemoselective
benzylation with benzyl bromide in acetone in the presence of anhy-
drous K2CO3 and catalytic amount of tetrabutylammonium bromide
(TBAB) affords methyl 4-(benzyloxy)-2-hydroxybenzoate (5) in good
yield. Latter (5) on allylation with allyl bromide in refluxing THF in the
(E)-3-(2, 6-dichlorophenyl)-N-hydroxyacrylamide (3b)
presence of anhydrous K2CO3 and
a catalytic amount of tetra-
It was obtained by the reaction of ethyl-3-(2,6-dichlorophenyl) acrylate
2b (0.5 g, 2.03 mmol), hydroxyl amine hydrochloride (0.708 g,
10.15 mmol) and KOH (1.14 g, 20.3 mmol) in methanol at 0–5 °C to
room temperature to gave the corresponding acrylamide 3b in 74%
yield (0.85 g) as a white solid, mp 142–144 °C; IR (νmax, cm−1); 3684,
3411, 1602, 1523, 757, 1H NMR (400 MHz, DMSO-d6); δH 10.31–10.05
(1H, brs, NH), 7.54 (2H, m, Ar-H), 7.49–7.45 (1H, d, J = 16.0 Hz)
7.39–7.35 (1H, t, J = 7.8 Hz) 6.55–6.51 (1H, d, J = 15.9 Hz); 13C NMR
(101 MHz, DMSO-d6); δC 162.0, 134.1, 132.4, 132.0, 130.8, 129.5,
128.1; HRMS: Calcd. accurate mass for (C9H8Cl2NO2); 230.9927.
Found. [M+H]+ 231.9940.
butylammonium bromide (TBAB) resulted in methyl 2-(allyloxy)-4-
(benzyloxy)benzoate (6) [47]. Finally, the methyl benzoate derivative
(6) on reaction with hydroxylamine hydrochloride in the presence of
solid KOH in methanol at 0–5 °C led to the formation of desired 2-al-
loxy-4-benzyloxy N-hydroxybenzamide derivative (7) in 85% yield
(Scheme 2 ).
2.4. General synthesis of hydroxamic acid
To the stirring mixture of desired acrylate (1 mmol) in methanol
added with hydroxylamine hydrochloride (5 mmol) and KOH
(10 mmol) at 0–5 °C, it was stirred at room temperature until con-
sumption of the starting material (according to TLC). After the com-
pletion of reaction, the solvent was evaporated under reduced pressure
to give a crude mass, which was purified by column (SiO2, 60–120
mesh) using a gradient of EtOAc /hexane as eluent to give the pure
compounds 3a-k, 7 in excellent yields.
(E)-3-(pyridin-4-yl)-N-hydroxyacrylamide (3c)
It was obtained by the reaction of ethyl-3-(pyridin-4-yl)acrylate 2c
(0.5 g, 2.82 mmol), hydroxyl amine hydrochloride (0.980 g, 14.1 mmol)
and KOH (1.58 g, 28.2 mmol) in methanol at 0–5 °C to room tempera-
ture to gave the corresponding acrylamide 3c in 70% yield (0.77 g) as a
yellow solid, mp 156–158 °C; IR (νmax, cm−1); 3435, 2855, 1611, 1512,
1400, 756, 669; 1H NMR (400 MHz, DMSO-d6); δH 10.97 (1H, brs, NH),
(E)-3-(4-chlorophenyl)-N-hydroxyacrylamide (3a)
It was obtained by the reaction of ethyl-3-(4-chlorophenyl)acrylate 2a
(0.5 g, 2.37 mmol), hydroxyl amine hydrochloride (0.824 g, 11.8 mmol)
Scheme 2. Synthesis of O-alkyl benzamide
derivative.
Reagents and conditions (i) MeOH, 20%
H2SO4, reflux (ii) Benzyloxybromide, K2CO3,
Acetone, RT, (iii) Allyl bromide, K2CO3, TBAB,
THF, reflux (iv) NH2OH.HCl, KOH, MeOH,
0–30 °C.
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A.K. Shukla et al.
Biomedicine&Pharmacotherapy107(2018)475–483
8.60–8.65 (2H, d, J = 5.9 Hz, Ar-H), 7.53–7.51 (2H, d, J = 6.0 Hz, Ar-
It was obtained by the reaction of (E)-ethyl 3-(4-(benzyloxy)phenyl)
acrylate 2g (1.0 g, 3.35 mmol), hydroxyl amine hydrochloride (1.16 g,
16.75 mmol) and KOH (1.87 g, 3.35 mmol) in methanol at 0–5 °C to
room temperature to gave the corresponding acrylamide 3g in 75%
yield (0.75 g) as a brown solid, mp 123–125 °C; IR (νmax, cm−1): 3409,
3104, 1669, 1537, 1491, 1232, 747, 641; 1H NMR (400 MHz, DMSO-
d6); δH 10.55 (1H, brs, NH), 7.52–7.50 (2H, d, J = 8.1 Hz, Ar-H),
7.46–7.38 (5H, m, Ar-H) 7.35–7.33 (2H, d, J = 8.0 Hz, Ar-H),
6.41–6.37 (1H, d, J = 16.56 Hz, CH), 6.35–6.32 (1H, d, J = 15.68 Hz,
CH), 5.14 (2H, s, OCH2), 13C NMR (101 MHz, DMSO-d6); δC 159.8,
143.9, 138.4, 137.2, 128.9, 128.3, 128.0, 117,1, 115.6, 69.7; HRMS:
Calcd. accurate mass for (C16H16NO3), 269.1125. Found. [M+H]+
270.1116.
H), 7.44–7.40 (1H, d, J = 15.8 Hz), 6.70–6.67 (1H, d, J = 15.8 Hz); 13
C
NMR (101 MHz, DMSO-d6); δC 161.7, 150.2, 141.9, 135.8, 123.6, 121.5
HRMS: Calcd. accurate mass for (C8H9N2O2), 165.0659. Found. [M
+H]+ 166.0672.
(E)-3-(4-(1H-imidazol-1-yl) phenyl)-N-hydroxyacrylamide (3d)
(E)-N-hydroxy-3-(3,4,5-trimethoxyphenyl)acrylamide (3h)
It was obtained by the reaction of (E)-ethyl 3-(4-(1H-imidazol-1-yl)
phenyl)acrylate 2d (1.0 g, 4.12 mmol), hydroxyl amine hydrochloride
(1.434 g, 20.63 mmol) KOH (2.31 g, 40.27 mmol) in methanol at 0–5 °C
to room temperature to gave the corresponding acrylamide 3d in 85%
yield (0.85 g) as a light brown solid, mp 158–160 °C; IR (νmax, cm−1):
3435, 2855, 1611, 1512, 1400, 756, 669; 1H NMR (400 MHz, DMSO-
d6); δH 10.21–9.64(1H, brs, NH), 8.31 (1H, m, Ar-H), 7.78 (1H, m, Ar-
H),7.70 (4H, m, Ar-H) 7.50–7.46 (1H, d, J = 15.9 Hz, CH), 7.12 (1H, s,
Ar-H), 6.53–6.49 (1H, d, J = 16.0, CH), 13C NMR (101 MHz, DMSO-d6);
It was obtained by the reaction of (E)-ethyl 3-(3,4,5-trimethoxyphenyl)
acrylate 2h (1.0 g, 3.35 mmol), hydroxyl amine hydrochloride (1.16 g,
16.75 mmol) and KOH (1.87 g, 3.35 mmol) in methanol at 0–5 °C to
room temperature to gave the corresponding acrylamide 3 h in 85%
yield (0.85 g) as a brown solid, mp 120–122 °C, IR(νmax,cm−1): 3392,
2919, 1673, 1507, 1290, 703; 1H NMR (400 MHz, DMSO-d6) δ 10.57
(1H, brs, NH) 7.42–7.39 (2H, d, J = 9.56 Hz, Ar-H), 6.99–6.96 (1H, d,
J = 15.72 Hz, CH), 6.45–6.41 (1H, d, J = 15.88 Hz, CH), 3.81 (s, 6 H),
3.68 (s, 3 H).13C NMR (101 MHz, DMSO-d6) δ 153.5, 139.1, 138.9,
130.9, 118.8, 105.4, 60.5, 56.3. HRMS: Calcd. accurate mass for
(C12H16NO5), 253.1023. Found: [M+H]+ 254.1032.
δ
C 163.0, 137.7, 137.4, 135.9, 133.8, 130.5, 129.4, 120.8, 120.0, 118.2;
HRMS: Calcd. accurate mass for (C12H12N3O2), 229.0924. Found: [M
+H]+ 230.0924
(2E,2E')-3,3'-(1,3-phenylene)bis(N-hydroxyacrylamide) (3e)
(E)-N-hydroxy-3-(4-(2-(hydroxyamino)-2-oxoethoxy)phenyl)acryla-
mide (3i)
It was obtained by the reaction of diethyl-1,3-(3-phenyl)acrylate 2e
(1.0 g, 3.55 mmol), hydroxyl amine hydrochloride (1.20 g, 18.75 mmol)
and KOH (1.92 g, 3.45 mmol) in methanol at 0–5 °C to room tempera-
ture to gave the corresponding acrylamide 3e in 75% yield (0.75 g) as a
brown solid, mp 123–125 °C; IR (νmax, cm−1): 3415, 2865, 1655, 1570,
1295, 858, 755, 659; 1H NMR (400 MHz, DMSO-d6) δH 10.04 (2H, brs,
NH), 7.84 (1H, s, Ar-H), 7.57-7.55 (2H, d, J = 7.4 Hz, Ar-H), 7.48–7.44
(2H, d, J = 16.0 Hz, CH), 7.42 (1H, s, Ar-H), 6.58–6.54 (2H, d,
J = 15.76 Hz, CH), 13C NMR (101 MHz, DMSO-d6) δ 163.0, 138.0,
135.9, 128.5, 127.3, 120.4, 39.2. HRMS: Calcd. accurate mass for
(C12H13N2O4), 248.0870. Found. [M+H]+ 249.0872.
It was obtained by the reaction of (E)-ethyl 3-(4-(2-ethoxy-2-ox-
oethoxy)phenyl)acrylate 2i (1.0 g, 3.35 mmol), hydroxyl amine hydro-
chloride (1.16 g, 16.75 mmol) and KOH (1.87 g, 3.35 mmol) in me-
thanol at 0–5 °C to room temperature to gave the corresponding
acrylamide 3i in 72% yield (0.72 g) as a brown solid, mp 122–124 °C; IR
(νmax,cm−1): 3402, 3019, 1680, 1290, 767, 658; 1H NMR (400 MHz,
DMSO-d6); δH 10.71 (1H, brs, NH), 9.06 (1H, brs, NH) 7.52–7.50 (2H, d,
J = 6.64 Hz, Ar-H), 7.43–7.39 (1H, d, J = 15.72 Hz, CH), 6.91 (2H, m,
Ar-H), 6.36–6.32 (1H, d, J = 1 Hz, CH), 4.50 (2H, s, OCH2), 13C NMR
(101 MHz, DMSO-d6); δC164.5, 163.5, 159.2, 138.3, 129.4, 128.5,
117.3, 115.5, 66.2, 40.5, 40.3, 40.1, 39.9, 39.7, 39.5, 39.3. HRMS:
Calcd. accurate mass for (C13H15N2O5), 278.0975. Found. [M+H]+
279.0972.
(2E,2E')-3,3'-(1,4-phenylene)bis(N-hydroxyacrylamide) (3f)
(E)-N-hydroxy-3-(1-methyl-1H-indol-3-yl) acrylamide (3 j)
It was obtained by the reaction of diethyl-1,4-(3-phenyl)acrylate 2f
(1.0 g, 3.52 mmol), hydroxyl amine hydrochloride (1.16 g, 17.65 mmol)
and KOH (1.87 g, 3.55 mmol) in methanol at 0–5 °C to room tempera-
ture to gave the corresponding acrylamide 3f in 79% yield (0.79 g) as a
brown solid, mp 124–126 °C;IR (νmax, cm−1); 3515, 2955, 1661, 1580,
1305, 765, 679; 1H NMR (400 MHz, DMSO-d6); δH 9.92 (2H, brs, NH),
7.66–7.54 (4H, m, Ar-H), 7.43–7.41 (2H, d, J = 10.2 Hz, CH),
6.55–6.52 (2H, d, J = 11.0 Hz, CH), 13C NMR (101 MHz, DMSO-d6); δC
163.0, 138.1, 138.0, 136.2, 134.3, 131.6, 129.5, 128.50, 127.1, 120.2,
39.8. HRMS: Calcd. accurate mass for (C12H13N2O4), 248.0870. Found.
[M+H]+ 249.0867.
It was obtained by the reaction of (E)-ethyl 3-(1-methyl-1H-indol-3-yl)
acrylate 2j (1.0 g, 3.35 mmol), hydroxyl amine hydrochloride (1.16 g,
16.75 mmol) and KOH (1.87 g, 3.35 mmol) in methanol at 0–5 °C to
room temperature to gave the corresponding acrylamide 3j in 80%
yield (0.80 g) as a brown solid, mp 125–127 °C; IR (νmax, cm−1): 3449,
3129, 2830, 1664, 1507, 1215; 1H NMR (400 MHz, DMSO-d6) δ 11.90
(s, 1H, NH), 7.53–7.51 (2H, d, J = 8.12 Hz, Ar-H), 7.30–7.26 (1H, d,
J = 16.2 Hz, CH), 7.23 (1H, s, Ar-H), 7.21–7.19 (2H, d, J = 7.92 Hz, Ar-
H), 6.32–6.28 (1H, d, J = 15.92 Hz, CH), 3.82 (3H, s, CH3).13C NMR
(101 MHz, DMSO-d6) δ 169.0, 138.3, 138.2, 135.2, 125.9, 122.9, 121.5,
(E)-3-(4-(benzyloxyphenyl)-N-hydroxyacrylamide (3g)
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120.3, 112.6, 111.1, 40.6, 33.3. HRMS: Calcd. accurate mass for
(C12H13N2O2), 216.0972. Found. [M+H]+ 217.0978
(E)-3-(furan-2-yl)-N-hydroxyacrylamide (3k)
3.3. Cell cycle analysis
Effect of compound 3h of cell cycle phase distribution was mea-
sured with flow cytometry technique using propidium iodide (PI)
staining method. For this, 1 × 106 MDA-MB-231 cells were seeded in
six well plates and allowed to grow for 24 h. Next day, cells were
treated with compound 3h for another 24 h. At the end of that in-
cubation peroid, cells were harvested, washed with PBS and fixed with
70% chilled ethanol. Fixed cells were washed with PBS and stained with
PI (30 μg/mL) in PBS containing 10 μg/mL RNase A for 30 min at room
temperature in dark condition. DNA content of the cells was measured
using a FACS Calibur flow cytometer using FACScan (Becton Dickinson)
and percentages of cells in each phases of cell cycle were calculated.
It was obtained by the reaction of (E)-ethyl 3-(furan-2-yl)acrylate 2k
(0.5 g, 2.82 mmol), hydroxyl amine hydrochloride (0.980 g, 14.1 mmol)
and KOH (1.58 g, 28.2 mmol) in methanol at 0–5 °C to room tempera-
ture to gave the corresponding acrylamide 3k in 70% yield (0.77 g) as a
yellow solid, mp 156–158 °C, IR(νmax,cm−1): 3412, 3129, 1683, 1510,
652; 1H NMR (400 MHz, DMSO-d6) δ 9.86 (1H, brs, NH), 7.75 (s, 1 H),
7.28–7.25 (1H, d, J = 15.6 Hz, CH), 6.75 (s, 1 H), 6.56 (s, 1 H),
6.28–6.24 (1H, d, J = 15.6 Hz, CH),13C NMR (101 MHz, DMSO-d6) δ
163.0, 151.3, 145.1, 126.1, 116.8, 114.1, 112.8. HRMS: Calcd. accurate
mass for (C7H8NO3), 153.0499. Found: [M+H]+ 154.0489.
3.4. Hoechst staining
2-(allyloxy)-4-(benzyloxy)-N-hydroxybenzamide (7)
To determine effect of compound 3h on nuclear morphology,
fluorescent microscopy was carried out using Hoechst staining. 1 × 106
MDA-MB-231 cells were seeded in 6-well culture plates for 24 h. After
another 24 h of treatment with compound 3h, cells were washed with
PBS, fixed with 4% paraformaldehyde for 15 min and subsequently cells
were permeabilized with 3% paraformaldehyde containing 0.1%
tritonX-100 for 10 min. Finally, cells were stained with 1 μg/ml of
Hoechst-33358 (Sigma, USA) for 30 min at room temperature, washed
with PBS and photographed using fluorescent microscopy.
It was obtained by the reaction of methyl-2-allyloxy-4-benzylox-
ybenzoate 6 (1.0 g, 3.35 mmol), hydroxyl amine hydrochloride (1.16 g,
16.75 mmol) and KOH (1.87 g, 3.35 mmol) in methanol at 0–5 °C to
room temperature to gave the corresponding acrylamide 7 in 85% yield
(0.85 g) as a brown solid, mp 124–126 °C; IR (νmax, cm−1): 3399, 3019,
1641, 1607, 1491, 1421, 1215, 1182, 757, 669; 1H NMR (400 MHz,
DMSO-d6); δH 9.08 (1H, brs, NH), 7.60–7.57 (1H, m, Ar-H) 7.45–7.43
(2H, d, J = 7.1 Hz, Ar-H), 7.41–7.37 (2H, t, J = 7.0 Hz, Ar-H),
7.35–7.31 (1H, m, Ar-H), 6.68–6.67 (2H, m, Ar-H), 6.08-5.99 (1H, m,
CH=CH2), 5.41–5.36 (1H, dd, J1 = 17.2 Hz, J2 = 1.1 Hz, CH=CH2),
3.5. Apoptosis assay
Effect of compound 3h on cell death was carried out using flow
cytometry based Annexin V-FITC/PI apoptosis kit (Sigma, USA).
1 × 106 MDA-MB-231 cells were seeded in 6-well culture plates for
24 h, followed by treatment with compound 3h for another 24 h. After
24 h of treatment, cells were harvested, washed with PBS and stained
with Annexin V-FITC/PI kit for 10 min. Finally, samples were analyzed
by flow cytometry (FACScan, Becton Dickinson).
5.28–5.25 (1H, d,
J
=
10.5 Hz, CH=CH2), 5.13 (2H, s,
OCH2),4.67–4.66 (2H, d, J = 4.9 Hz, OCH2); 13C NMR (101 MHz,
DMSO-d6); δC 163.6, 161.7, 157.3, 137.0, 133.6, 128.9 (2C), 128.4,
128.2 (2C), 118.4, 115.3, 106.9, 100.9, 69.9, 69.3. HRMS: Calcd. ac-
curate mass for (C17H18NO4), 299.1230. Found: [M+H]+ 300.1221.
3. Bioassay
3.6. Mitochondrial membrane potential analysis
3.1. Cell culture
To analyze effect of compound 3h on mitochondrial membrane
potential, 1 × 106 MDA-MB-231 cells were seeded in 6-well culture
plates and grown for 24 h. Grown cell monolayers were treated with
compound 3h for another 24 h. At the end of treatment, cells were
harvested, washed with PBS and incubated with 5 μg of JC-1 dye for
30 min at room temperature, followed by washing and re-suspension in
300 μl PBS. Finally, JC-1 stained MDA-MB-231 cells were analyzed by
flow cytometry using FACScan (Becton Dickinson).
Breast Cancer cell lines, MDA-MB-231 and MCF-7 were originally
obtained from American type of cell culture collection (ATCC), USA and
stock was maintained in laboratory. Non-malignant origin cell line,
HEK-293 (human embryonic kidney) was obtained from institutional
cell repository of animal tissue culture facility. Cells were cultured in
CO2 incubator at 37 °C with 5% CO2 and 95% humidity in RPMI-
1640 growth medium supplemented with 10% FBS and 1% of antibiotic
and antimycotic solution.
3.7. Reactive oxygen species (ROS) analysis
3.2. Cell viability assay
ROS was measured in terms of reactive oxygen induced fluorescence
of DCF-DA, basically a non-fluorescent cell staining dye. For this,
1 × 106 MDA-MB-231 cells were seeded in 6-well plates for 24 h, fol-
lowed by treatment with compound 3h for 24 h. At the end of in-
cubation, cells were harvested, washed with PBS and then fixed with
chilled absolute methanol. Cells were stained with 10 μg/mL DCFH-DA
and incubated at room temperature in dark condition for 30 min. After
incubation, cells were centrifuged and re-suspended in 300 μl PBS and
analyzed by flow cytometry (FACScan, Becton Dickinson, USA).
MTT (3-(4,5-dimethylthiazol-2 yl)-2,5-diphenyl tetrazolium bro-
mide) assay was performed to determine cell viability upon compound
exposure as per method described earlier. In brief, cells @ 1 × 104/well
were seeded in 96 well microculture plates in 100 μl RPMI-1640
medium and incubated for 24 h at 37 °C in a CO2 incubator. Compounds
were diluted to required concentration in phenol red free RPMI-1640
medium supplemented with 2% FBS. After 24 h of incubation, cells
were treated with desired concentration of compounds and respective
vehicle alone control for 24 h. At the end of incubation, 10 μL (5 mg/
mL) of MTT (Sigma, USA) was added and incubated for another 3 h.
Finally, supernatant was carefully discarded and 200 μl of DMSO was
added to dissolve formazan crystals under gentle shaking in plate
shaker (Biosan, USA) and absorbance at 570 nm wavelength was re-
corded in a microplate reader (Microquant, BioTek).
3.8. Evaluation of biological activity
All the synthesized compounds were screened for anti-cancer ac-
tivity in both MCF-7 (ER + ve) and MDA-MB-231 (ER-ve) cells and
HEK-293 cells using MTT assay (Table 1).
479

A.K. Shukla et al.
Biomedicine&Pharmacotherapy107(2018)475–483
Table 1
Table 2
Anti-proliferative effect of synthesized compounds with cell line MCF-7, MDA-
MB-231 and HEK-293.
Analysis of the interaction between breast cancer target and synthesized mo-
lecules.
Structure
Code
MCF-7
IC50 μM
MDA-MB-231
IC50 μM
HEK-293
IC50 μM
Ligands
Docking energies (Kcal/mol)
Ligands
Docking energies
(Kcal/mol)
3a
3b
> 50
> 50
> 50
3a
3b
3c
3d
3e
3f
−6.0
−6.1
−5.5
−6.7
−7.0
−7.1
3 g
3 h
3i
3 j
3k
7
−7.4
−6.7
−6.9
−6.9
−5.3
−7.7
30
2.26
13
2.62
> 50
3c
3d
> 50
> 50
45
39
1.43
3.45
> 50
> 50
ligands and EGFR kinase. The analysis shows that amino acids (Leu 844
,
Lys745, Val726, Met793, and Cys775) in EGFR protein is the potential
target site for binding of compound3b by formation of hydrogen bond
with chloro and hydroxamic acid groups of compound3b (Fig. 2). The
compound3e interacted with amino acids (Leu718, Val726 and Ala743),
3e
> 50
42
2.37
> 50
compound3f interacted with amino acids (Ala 743, Val 726, Leu
Met766 and Thr854), compound3g interacted with amino acids (Leu844
Leu792, Leu 718, Ala743, Thr854 and Met793), compound3h interacted
with amino acids (Thr854, Ala743, Leu718, Val726 and Leu844) (Table 2),
,
,
844
3f
> 50
34.3
41
35
3.12
3.45
> 50
> 50
3 g
1.54
and compound7 interacted with amino acids (Leu792, Leu718, Val726
Ala743 and Lys745) (Fig. 3).
,
3 h
31
3.24
5
2.16
> 50
3i
> 50
> 50
41
43
2.54
3.43
> 50
> 50
4. Results and discussions
Among all the synthesized compounds, 3b and 3h shows potent
anticancer activity against breast cancer cell line. MCF-7 and MDA-MB-
231 but their activities were more pronounced in aggressive breast
cancer model (MDA-MB-231 cells) with IC50 13μM and 5μM respec-
tively. Out of the series compound 3h was the most active and potent
analogue with lowest IC50 against MDA-MB-231 cells. Interestingly,
compound 3h shows no cytotoxicity against HEK-293 cells suggesting
that compound 3h was probably the safest compound of this series
with specific activity against cancer cells.
3 j
3k
7
42
45
2.32
3.34
> 50
37
> 50
> 50
3.12
2.04
Tamoxifen
10
1.32
15
50
2.18
Compound 3h arrest progression of cells at G0/G1 check point
Compound 3h dose dependently inhibited progression of cell cycle
at G0/G1 check point (Fig. 4). Furthermore, dose dependent increase of
sub-diploid populations were also increased which indicate possible
DNA fragmentation and apoptosis of MDA-MB-231 cells due to com-
pound 3h treatment.
3.9. Docking study on EGFR protein target of breast cancer in silico
molecular docking analysis
We have analyzed the synthesized compounds through docking
study using Autodock 4.2 to exploit the possible interactions between
Fig. 2. Interaction between EGFR target and 3b, 3e–3h and 7.
480

A.K. Shukla et al.
Biomedicine&Pharmacotherapy107(2018)475–483
Fig. 3. 3D Structure of docking image of 3b, 3e–3h and 7 with EGFR target protein.
Fig. 4. Effect of compound 3h on cell cycle progression in MDA-MB-231 cells. Cells were treated with compound 3h for 24 h washed with PBS, stained with PI and
analyzed by flow cytometry. Quantification of flow cytometry data has been shown as percent of total cells.
Fig. 5. Effect of compound 3h on nuclear morphology in MDA-MB-231 cells. Cells were treated with compound 3h for 24 h washed with PBS, fixed with paraf-
ormaldehyde, stained with Hoechst-33358 and analyzed by fluorescent microscopy.
Compound 3h induces DNA damage and apoptosis in MDA-MB-231
cells
If any compound treatment causes DNA damage and fragmentation,
altered nuclear morphology of MDA-MB-231 cells can be monitored
with DNA binding fluorescent Hoechst stain under florescence
microscopy. In this case, compound 3h induced dose-dependent DNA
fragmentations in treated group as compared to non-treated vehicle
control clearly indicating cells were undergoing apoptosis due to
compound exposure (Fig. 5). This data was further confirmed by flow
cytometry using Annexin V-FITC/PI dual staining technique. Results
481

A.K. Shukla et al.
Biomedicine&Pharmacotherapy107(2018)475–483
Fig. 6. Effect of compound 3h on apoptosis in MDA-MB-231 cells. Cells were treated with compound 3h for 24 h washed with PBS, stained with Annexin V-FITC/PI
and analyzed by flow cytometry. Quantification of flow cytometry data has been shown as percent of total cells.
Fig. 7. Effect of compound 3h on MMP in MDA-MB-231 cells. Cells were treated with compound 3h for 24 h, washed with PBS, stained with JC-1 and analyzed by
flow cytometry. Quantification of flow cytometry data has been shown as percent of total cells.
(Fig. 7). This clearly indicates significant loss of MMP in MDA-MB-231
cells due to compound 3h treatment.
Compound 3h induce ROS generation in MDA-MB-231 cells
Reactive oxygen species (ROS) is a well-known mediator of apop-
tosis and majority of chemotherapeutics detrimental to cancer cells due
to their ROS inducing capacity [48,49]. As compound 3h induces
apoptosis, it is possible that it might induce ROS in MDA-MB-231 cells.
Therefore, effect of compound 3h on ROS generation in MDA-MB-231
cells was evaluated by flow cytometry using DCFH-DA dye. Flow cy-
tometric data revealed that treatment of compound 3h significantly
induced ROS generation compared to non-treated vehicle control MDA-
MB-231 cells (Fig. 8).
5. Conclusions
Fig. 8. Effect of compound 3h on ROS generation in MDA-MB-231 cells. Cells
were treated with compound 3h for 24 h washed with PBS, stained with DCFH-
DA and analyzed by flow cytometry.
The present study investigates the anticancer effects of N-
Hydroxycinnamamide derivatives in human breast cancer cells.
Molecular docking results showed that 3b, 3e–3h and 7 could act on
the active sites of pro-apoptotic proteins EGFR to induce apoptotic
death of cancer cells. Out of twelve compounds screened, compound
3b and compound 3h exhibits most potent activity against aggressive
breast cancer cells (MDA-MB-231) with IC50 13 μM and 5 μM respec-
tively. Compound 3h inhibits cell cycle progression by accumulating
cells at G0/G1 check points. Moreover, compound 3h promotes DNA
fragmentation and induction of apoptosis. Furthermore, loss of mi-
tochondrial membrane were also potentially induced by compound 3h.
One of the major mechanisms of action of compound 3h for its anti-
cancer activity is probably induction of ROS in cancer cells thereby
showed that compound 3h induces apoptosis dose dependently in
MDA-MB-231 cells as compared to non-treated vehicle control cells
(Fig. 6).
Compound 3h induce MMP loss in MDA-MB-231 cells
Further, effect of compound 3h on mitochondria as alterations in
MMP was carried out using membrane potential sensitive dye JC-1. Any
increase in intensity of green fluorescence from red fluorescence of JC-1
dye stained cells indicates mitochondrial depolarization. Flow cyto-
metric data indicate increase of green fluorescence in MDA-MB-231
cells with treatment of increasing concentration of compound 3h
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Biomedicine&Pharmacotherapy107(2018)475–483
inducing apoptotic cell deaths in cancer cells. Our findings suggest that
compounds3b and 3h might be considered as potential candidates for
novel anticancer drug in the future.
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Acknowledgement
The authors thank the UGC fellowship for financial support of this
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2018.08.015.
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