Ferulic acid amide derivatives as anticancer and antioxidant agents: synthesis, thermal, biological and computational studies

Article abstract of DOI:10.1007/s00044-016-1562-6

Abstract: The design and microwave-assisted synthesis of four series (IIIa–IIIo, Va–Vg, VIIa–VIIg and IXa–IXe) of mono and bis-amide derivatives of ferulic acid have been achieved under solvent-free conditions and, subsequently characterized by spectroscopic techniques. During thermal analysis, all the compounds were found stable up to 100?°C and decomposed through single step at higher temperature. The derivatives were screened for their in vitro cytotoxicity and antioxidant activity, respectively and observed that compound Vb was most active against breast (MCF-7; IC₅₀?=?07.49?μM and MDA-MB-231; IC₅₀?=?07.28?μM), Vd against lung (A549; IC₅₀?=?07.11?μM) and liver (HepG2; IC₅₀?=?08.32?μM), and Ve against cervical (HeLa; IC₅₀?=?07.14?μM) cancer cell lines, while compounds IIIf, IIIl, IIIo, VIIe and IXa–IXe were found to exhibit the strong antioxidant activity with respect to their parent molecule. Previous reports for the biological applications of ferulic acid amides also confirmed the importance of work presented here. The 3D-QSAR studies for anticancer and antioxidant activities were also performed by using CoMFA, and the corresponding contour maps of electrostatic and steric fields have been computed. Statistical analysis between experimental and CoMFA-predicted data for pIC₅₀ have been accomplished by curve fitting analysis which showed the significant correlation. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-016-1562-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1562-6
ORIGINAL RESEARCH
Ferulic acid amide derivatives as anticancer and antioxidant
agents: synthesis, thermal, biological and computational studies
Naresh Kumar1 Sandeep Kumar2 Sheenu Abbat3
•
•
•
Kumar Nikhil1 Sham M. Sondhi2 Prasad V. Bharatam4
Partha Roy1 Vikas Pruthi1
•
•
•
•
Received: 23 November 2015 / Accepted: 19 March 2016
Ó Springer Science+Business Media New York 2016
Abstract The design and microwave-assisted synthesis
of four series (IIIa–IIIo, Va–Vg, VIIa–VIIg and IXa–
IXe) of mono and bis-amide derivatives of ferulic acid
have been achieved under solvent-free conditions and,
subsequently characterized by spectroscopic techniques.
During thermal analysis, all the compounds were found
stable up to 100 °C and decomposed through single step at
higher temperature. The derivatives were screened for their
in vitro cytotoxicity and antioxidant activity, respectively
and observed that compound Vb was most active against
breast (MCF-7; IC50 = 07.49 lM and MDA-MB-231;
IC50 = 07.28 lM), Vd against lung (A549; IC50 = 07.11
lM) and liver (HepG2; IC50 = 08.32 lM), and Ve against
cervical (HeLa; IC50 = 07.14 lM) cancer cell lines, while
compounds IIIf, IIIl, IIIo, VIIe and IXa–IXe were found to
exhibit the strong antioxidant activity with respect to their
parent molecule. Previous reports for the biological appli-
cations of ferulic acid amides also confirmed the importance
of work presented here. The 3D-QSAR studies for anticancer
and antioxidant activities were also performed by using
CoMFA, and the corresponding contour maps of electro-
static and steric fields have been computed. Statistical
analysis between experimental and CoMFA-predicted data
for pIC50 have been accomplished by curve fitting analysis
which showed the significant correlation.
Graphical Abstract
Keywords Ferulic acid Á Amide derivatives Á
Anticancer and antioxidant activity Á 3D-QSAR Á
CoMFA
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-016-1562-6) contains supplementary
material, which is available to authorized users.
Introduction
& Naresh Kumar
nareshkumariitr@gmail.com
Synthesis of heterocyclic molecules, which can be easily
prepared and exhibit good biological activities, is a fasci-
nating area of research (Basoglu et al., 2013). As the limi-
tations of most of the existing anticancer drugs due to various
reasons such as drug resistance, serious side effects, and lack
of efficiency made infectious diseases a vicious cycle,
so there is an urgent need to design improved lead molecules
for the well-being of mankind (Narang et al., 2009). The
dietary antioxidants are considered to play the shielding role
1
Department of Biotechnology, Indian Institute of Technology
Roorkee, Roorkee, Uttarakhand 247667, India
2
Department of Chemistry, Indian Institute of Technology
Roorkee, Roorkee 247667, India
3
Department of Pharmacoinformatics, NIPER, S.A.S. Nagar,
Mohali 160062, India
4
Department of Medicinal Chemistry, NIPER, S.A.S. Nagar,
Mohali 160062, India
123

Med Chem Res
against the development and progressions in the pathological
conditions produced by oxidative stress, and are involved in
many rigorous human diseases mainly including
atherosclerosis, cancer, neurodegenerative disorders, dia-
betes, ageing and many more. Since the last two decades,
polyphenols have drawn the attention of scientific commu-
nity toward their potential antioxidant and other biological
effects (Herrmann, 1989; Clifford, 1990; Sajjadi et al., 2004;
Wiegand et al., 2009; Ferhat et al., 2014). Ferulic acid is a
ubiquitous phenolic derivative of cinnamic acid, exhibits a
wide range of biomedical activities viz., anticancer, antiox-
idant, antimicrobial, anti-inflammatory, antiallergic,
antithrombotic, antiviral, hepatoprotective, increases the
sperm viability, enhances the stability of cytochrome C and
inhibition of apoptosis induced by cytochrome C and
increases the IgE binding to peanut allergens, a b-secretase
modulator with therapeutic potential against Alzheimer’s
disease (Rosazza et al., 1995; Mori et al., 1999; Middleton
et al., 2000; Toshihiro et al., 2000; Ou and Kwok, 2004;
Chung and Champagne, 2011; Mori et al., 2013; Kumar and
Pruthi, 2014). In the past few years, different types of ferulic
acid derivatives have been discovered which exhibit
enhanced biomedical activities as compared to ferulic acid
(Hosoda et al., 2002; Tan and Shahidi, 2011; Teresa et al.,
2011; Li et al., 2012a, b; Piazzon et al., 2012; Sultana, 2012;
Huang et al., 2013; Paiva et al., 2013; Wang et al., 2013;
Kiran et al., 2015). The compounds containing amide moiety
are also associated with the broad spectrum of biomedical
activities (Tamm et al., 2003; Parkesh et al., 2012). Moti-
vated by the wide range of applications shown by ferulic acid
alone, different kinds of ferulic acid derivatives and the
compounds containing the amide moieties, we had devel-
oped a strategy toward the synthesis of different range of
novel amide derivatives of ferulic acid by its structural
modifications comprising the variety of aliphatic, aromatic
and heterocyclic primary amines. Ferulic acid (used in this
study) has been extracted and well characterized by single
crystal X-ray diffraction along with p-coumaric and caffeic
acids by our research group (Kumar and Pruthi, 2015; Ku-
mar et al., 2015). The work presented here might serve to
provide a new therapeutic approach in the field of medicinal
and pharmaceutical chemistry based on the natural sources.
The work will also be useful for the design and synthesis of
solvent-free molecules which leads to a vital drug.
bromide), cell culture grade DMSO, different types of
mono- and bis- amines used in synthesis, 5-fluorouracil (5-
FU), cyclophosphamide and actidione (cycloheximide)
were from HiMedia (Mumbai, India).
Instrumentation
Microwave reactor Anton Paar (monowave 300) and
microwave oven model M197DL (Samsung) were used for
microwave irradiation. The melting point of compounds
were determined by using a JSGW apparatus and are
uncorrected. The FT-IR spectra were taken on a Perk-
inElmer 1600 series FT-IR spectrometer in KBr pellets. All
the NMR experiments (1H and 13C-NMR) were carried out
on a 500 MHz high-resolution NMR spectrometer (Avance
500 Bruker Biospin Intl. AG, Switzerland) in DMSO-d6
with tetramethylsilane (TMS) as internal standard. Perk-
inElmer Clarus 500 gas chromatograph with MS detector
built within was used to record the GC–MS, while APCI
mass was recorded using Finnigan Mat LCQ mass spec-
trometer. The elemental analysis has been carried out on a
Vario EL III Elementar. Thermogravimetry (TG) and
derivative thermogravimetry (DTG) analysis have been
carried out by using a mass of 0.045 g at 10 °C/min under
the nitrogen at 200 ml/min flow rate on a thermogravimetric
analyzer (PerkinElmer’s, California, USA). Absorbance of
the lysates during MTT assay was determined on a Fluostar
optima microplate reader (BMG Labtech, Germany).
General procedure for synthesis of mono and bis
amide derivatives of ferulic acid
(E)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid (ferulic acid)
and corresponding amines (IIa–IIo, IVa–IVg, VIa–VIg and
VIIIa–VIIIe) were mixed together thoroughly in an
equimolar ratio for mono-amide and 2:1 molar ratio for bis-
amide within a Petri dish. The reaction mixture was subjected
to microwave irradiation at 180–450 Watt for 3–7 min. The
progress of the reactions was monitored by thin layer chro-
matography (TLC) on silica gel using ethyl acetate/methanol
(4:1, 3:2 and 7:3) as solvent of elution. TLCs indicated the
absence of starting materials and confirmed the completion of
reactionandformationofproducts. Thecrudeproductsthusso
obtainedwerepurifiedbycrystallizationandre-crystallization
in methanol to give the pure products (IIIa–IIIo, Va–
Vg, VIIa–VIIg and IXa–IXe) in high yield.
Materials and methods
Materials
(E)-N-cyclopropyl-3-(4-hydroxy-3-
methoxyphenyl)acrylamide (IIIa)
All the reagents used in cell culture were taken from
GIBCO (Invitrogen, USA). Penicillin, streptomycin, MTT
(3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H tetrazolium
Yield: 87 %. Amorphous, bright-yellow. mp: 110–112 °C.
FT-IR (KBr, cm-1) mmax: 3435, 2929, 1666, 1606, 1597,
1571. H-NMR (500 MHz, DMSO-d6, ppm) d: 0.37–0.41
1
123

Med Chem Res
(m, 2H, CH2), 0.56.0.58 (d, 2H, J = 7 Hz, CH2), 2.22–2.24
(m, 1H, CH), 3.81 (s, 3H, OCH3), 5.44 (s, 1H, NH),
6.34–6.37 (d, 1H, J = 16 Hz, Ar), 6.77–6.79 (d, 1H,
J = 8 Hz, Ar), 7.05–7.07 (d, 1H, J = 8 Hz, Ar), 7.26 (s,
1H, Ar), 7.43–7.46 (d, 1H, J = 16 Hz, Ar), 10.27 (s, 1H,
OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d: 13.55,
24.10, 56.09, 112.27, 116.44, 117.28, 120.94, 128.77,
144.61, 145.44, 151.17, 166.09. Anal. Calcd. (%) for
C13H15NO3 (233.26): C, 66.94; H, 6.48; N, 6.00; Found: C,
66.13; H, 6.29; N, 5.57.
1565. 1H-NMR (500 MHz, DMSO-d6, ppm) d: 1.34 (s, 2H,
CH2), 1.46–1.49 (q, 4H, J = 5.5 Hz, J = 5 Hz, 2 9 CH2),
2.26–2.49 (m, 6H, 3 9 CH2), 2.78–2.79 (d, 2H,
J = 5.5 Hz, CH2), 3.78 (s, 3H, OCH3), 5.49 (s, 1H, NH),
6.29–6.33 (d, 1H, J = 16 Hz, Ar), 6.76–6.78 (d, 1H,
J = 8 Hz, Ar), 6.97–6.98 (d, 1H, J = 6 Hz, Ar), 7.16 (s,
1H, Ar), 7.27–7.30 (d, 1H, J = 15.5 Hz, Ar), 10.23 (s, 1H,
OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d: 25.05,
26.53, 36.99, 53.04, 54.73, 57.09, 112.08, 116.15, 117.16,
120.08, 128.21, 144.15, 145.18, 151.16, 166.80. Anal.
Calcd. (%) for C17H24N2O3 (304.38): C, 67.08; H, 7.95; N,
9.20; Found: C, 66.56; H, 7.89; N, 9.01.
(E)-N-cyclohexyl-3-(4-hydroxy-3-
methoxyphenyl)acrylamide (IIIb)
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2,2,6,6-
Yield: 98 %. Crystalline, light-orange. mp: 180–182 °C.
FT-IR (KBr, cm-1) mmax: 3435, 2934, 2857, 1689, 1634,
1594. H-NMR (500 MHz, DMSO-d6, ppm) d: 1.13–1.19
tetramethylpiperidin-4-yl)acrylamide (IIIe)
1
Yield: 84 %. Crystalline, brick color. mp: 118–120 °C. FT-
(q, 1H, J = 30 Hz, J = 13 Hz, CH2), 1.21–1.26 (t, 4H,
J = 25 Hz, J = 13 Hz, 2 9 CH2), 1.54–1.57 (d, 1H,
J = 8 Hz, CH2), 1.67–1.69 (d, 2H, J = 12.5 Hz, CH2),
1.83–1.85 (d, 2H, J = 11 Hz, CH2), 2.76–2.81 (m, 1H,
CH), 3.79 (s, 3H, OCH3), 5.44 (s, 1H, NH), 6.28–6.32 (d,
1H, J = 15.5 Hz, Ar), 6.76–6.77 (d, 1H, J = 8 Hz, Ar),
6.96–6.98 (d, 1H, J = 8.5 Hz, Ar), 7.16 (s, 1H, Ar),
7.238–7.27 (d, 1H, J = 16 Hz, Ar), 10.22 (s, 1H, OH).
13C-NMR (125 MHz, DMSO-d6, ppm) d: 21.01, 28.54,
33.39, 46.78, 57.32, 112.07, 116.27, 117.58, 120.30,
128.41, 144.33, 145.78, 151.12, 166.67. Anal. Calcd. (%)
for C16H21NO3 (275.34): C, 69.79; H, 7.69; N, 5.09;
Found: C, 67.63; H, 7.56; N, 4.89.
1
IR (KBr, cm-1) mmax: 3433, 2961, 1629, 1516. H-NMR
(500 MHz, DMSO-d6, ppm) d: 0.98 (s, 2H, CH2), 1.06 (s,
6H, 2 9 CH3), 1.13 (s, 6H, 2 9 CH3), 1.71–1.73 (d, 2H,
J = 8.5 Hz, CH2), 3.17–3.18 (d, 1H, J = 7 Hz, CH), 3.79
(s, 3H, OCH3), 5.45 (s, 1H, NH), 6.27–6.31 (d, 1H,
J = 16 Hz, Ar), 6.76–6.77 (d, 1H, J = 4 Hz, Ar),
6.95–6.96 (d, 1H, J = 4 Hz, Ar), 7.15 (S, 1H, Ar),
7.21–7.24 (d, 1H, J = 16 Hz, Ar), 10.24 (s, 1H, OH). 13C-
NMR (125 MHz, DMSO-d6, ppm) d: 29.11, 33.13, 46.01,
47.77, 57.09, 112.12, 116.13, 117.09, 120.17, 128.15,
144.52, 145.66, 151.12, 166.20. Anal. Calcd. (%) for
C19H28N2O3 (332.44): C, 68.65; H, 8.49; N, 8.43; Found:
C, 67.82; H, 8.36; N, 8.12.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2-(pyrrolidin-1-
yl)ethyl)acrylamide (IIIc)
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(3-(2-
oxopyrrolidin-1-yl)propyl)acrylamide (IIIf)
Yield: 83 %. Semi-solid, dark-brown. mp: 98–100 °C. FT-
IR (KBr, cm-1) mmax: 3407, 2961, 1638, 1597, 1578, 1557.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 1.67 (s, 4H,
2 9 CH2), 2.36–2.54 (m, 6H, 3 9 CH2), 2.75–2.78 (t, 2H,
J = 12.5 Hz, J = 6.5 Hz, CH2), 3.79 (s, 3H, OCH3), 5.46
(s, 1H, NH), 6.31–6.33 (d, 1H, J = 15.5 Hz, Ar), 6.76–6.78
(d, 1H, J = 8 Hz, Ar), 6.97–6.99 (d, 1H, J = 8 Hz, Ar),
7.17 (s, 1H, Ar), 7.27–7.31 (d, 1H, J = 15.5 Hz, Ar), 10.23
(s, 1H, OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d:
27.10, 37.92, 50.01, 59.01, 112.17, 116.02, 117.18, 120.11,
121.94, 128.23, 129.11, 136.66, 144.21, 145.45, 151.21,
166.36. Anal. Calcd. (%) for C16H22N2O3 (290.36): C,
66.18; H, 7.64; N, 9.65; Found: C, 65.49; H, 7.54; N, 9.17.
Yield: 95 %. Semi-solid, light-brown. mp: 105–107 °C.
FT-IR (KBr, cm-1) mmax: 3415, 3150, 2932, 1678, 1609,
1597, 1555. 1H-NMR (500 MHz, DMSO-d6, ppm) d:
1.53–1.61 (m, 2H, CH2), 1.87–1.94 (m, 2H, CH2),
2.19–2.22 (t, 2H, J = 16 Hz, J = 8 Hz, CH2), 2.54–2.57
(t, 2H, J = 14 Hz, J = 7 Hz, CH2), 3.15–3.19 (t, 2H,
J = 15 Hz, J = 8 Hz, CH2), 3.21–3.25 (t, 2H, J = 13 Hz,
J = 7 Hz, CH2), 3.84 (s, 3H, OCH3), 5.84 (s, 1H, NH),
6.30–6.33 (d, 1H, J = 16 Hz, Ar), 6.76–6.77 (d, 1H,
J = 8 Hz, Ar), 6.99–7.03 (t, 1H, J = 8, J = 7 Hz, Ar),
7.19–7.19 (d, 1H, J = 1 Hz, Ar), 7.29–7.33 (d, 1H,
J = 15.5 Hz, Ar), 10.27 (s, 1H, OH). 13C-NMR (125 MHz,
DMSO-d6, ppm) d: 17.10, 27.94, 32.10, 37.94, 41.39,
43.03, 56.41, 112.17, 116.36, 117.65, 120.05, 129.34,
144.65, 145.12, 151.15, 166.16, 173.32. Anal. Calcd. (%)
for C17H22N2O4 (318.37): C, 64.13; H, 6.97; N, 8.80;
Found: C, 63.74; H, 6.89; N, 8.51.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2-(piperidin-1-
yl)ethyl)acrylamide (IIId)
Yield: 87 %. Semi-solid, brown. mp: 98–100 °C. FT-IR
(KBr, cm-1) mmax: 3394, 3273, 2935, 2838, 1638, 1593,
123

Med Chem Res
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2-
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2-(thiophen-2-
morpholinoethyl)acrylamide (IIIg)
yl)ethyl)acrylamide (IIIj)
Yield: 92 %. Crystalline, yellowish-orange. mp: 121–123 °C.
FT-IR (KBr, cm-1) mmax: 3431, 3219, 2936, 2820, 1638,
1597. 1H-NMR (500 MHz, DMSO-d6, ppm) d: 2.35–2.42 (q,
4H, J = 22 Hz, J = 12 Hz, 2 9 CH2), 2.78–2.80 (t, 2H,
J = 12 Hz, J = 6 Hz, CH2), 3.56–3.57 (t, 6H, J = 8.5 Hz,
J = 4.5 Hz, 3 9 CH2), 3.79 (s, 3H, OCH3), 5.56 (s, 1H, NH),
6.30–6.33 (d, 1H, J = 15.5 Hz, Ar), 6.76–6.78 (d, 1H,
J = 8 Hz, Ar), 6.98–6.99 (d, 1H, J = 6.5 Hz, Ar), 7.18 (s,
1H, Ar), 7.29–7.33 (dd, 1H, J = 7.5 Hz, J = 7 Hz, Ar),
10.28 (s, 1H, OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d:
37.19, 39.44, 52.04, 54.73, 67.09, 112.21, 116.45, 117.09,
120.88, 128.85, 144.15, 145.68, 151.21, 166.04. Anal. Calcd.
(%) for C16H22N2O4 (306.36): C, 62.73; H, 7.24; N, 9.14;
Found: C, 61.84; H, 7.17; N, 8.97.
Yield: 95 %. Amorphous, traditional-brown. mp: 140–142 °C.
FT-IR (KBr, cm-1) mmax: 3431, 2930, 2838, 1634, 1593, 1568.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 2.85–2.87 (t, 2H,
J = 13 Hz, J = 6.5 Hz, CH2), 2.91–2.95 (q, 2H,
J = 12.5 Hz, J = 6.5 Hz, CH2), 3.80 (s, 3H, OCH3), 5.50 (s,
1H, NH), 6.33–6.36 (d, 1H, J = 15.5 Hz, Ar), 6.77–6.78 (d,
1H, J = 8 Hz, Ar), 6.88–6.89 (d, 1H, J = 3 Hz, Ar),
6.94–6.96 (m, 1H, Ar), 7.02–7.04 (dd, 1H, J = 8 Hz,
J = 1.5 Hz, Ar), 7.22–7.23 (s, 1H, Ar), 7.33–7.34 (d, 1H,
J = 4.5 Hz, Ar), 7.389–7.41 (d, 1H, J = 15.5 Hz, Ar), 10.24
(s, 1H, OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d: 28.39,
38.22, 39.44, 50.41, 112.17, 116.05, 117.17, 120.55, 121.94,
128.21, 129.11, 136.36, 144.21, 145.45, 151.15, 166.51. Anal.
Calcd. (%) for C16H17NSO3 (303.38): C, 63.34; H, 5.65; N,
4.62; S, 10.57; Found: C, 62.97; H, 5.53; N, 4.29; S, 10.17.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2-(piperazin-1-
yl)ethyl)acrylamide (IIIh)
(E)-N-(3-(1H-imidazol-1-yl)propyl)-3-(4-hydroxy-3-
methoxyphenyl)acrylamide (IIIk)
Yield: 86 %. Semi-solid, yellowish-brown. mp: 170–172 °C.
FT-IR (KBr, cm-1) mmax: 3401, 2995, 2826, 1632, 1585, 1507.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 2.31–2.34 (d, 6H,
J = 15.5 Hz, 3 9 CH2), 2.71 (s, 6H, 3 9 CH2), 3.78–3.79 (d,
3H, J = 5.5 Hz, OCH3), 5.42 (s, 1H, NH), 6.28–6.31 (d, 1H,
J = 16 Hz, Ar), 6.74–6.77 (t, 1H, J = 15.5 Hz, J = 7.5 Hz,
Ar), 6.96–7.04 (d, 1H, J = 8.5 Hz, Ar), 7.17 (s, 1H, Ar),
7.26–7.29 (d, 2H, J = 15.5 Hz, Ar), 10.24 (s, 1H, OH). 13C-
NMR (125 MHz, DMSO-d6, ppm) d: 37.11, 46.04, 52.39,
54.74, 57.09, 112.28, 116.45, 117.05, 120.88, 128.14, 144.15,
145.57, 151.05, 166.74. Anal. Calcd. (%) for C16H23N3O3
(305.37): C, 62.93; H, 7.59; N, 13.76; Found: C, 61.97; H,
7.52; N, 13.31.
Yield: 86 %. Crystalline, yellowish-orange. mp: 148–150 °C.
FT-IR (KBr, cm-1) mmax: 3413, 3254, 2930, 1636, 1590. 1H-
NMR (500 MHz, DMSO-d6, ppm) d: 1.86–1.89 (q, 2H,
J = 6.5 Hz, J = 6 Hz, CH2), 2.57–2.59 (t, 2H, J = 13 Hz,
J = 6.5 Hz, CH2), 3.79 (s, 3H, OCH3), 4.03–4.05 (q, 2H,
J = 5 Hz, J = 4.5 Hz, CH2), 5.53 (s, 1H, NH), 6.30–6.34 (d,
1H, J = 17.5 Hz, Ar), 6.76–6.78 (d, 1H, J = 8 Hz, Ar), 6.88
(s, 1H, Ar), 6.98–7.02 (t, 1H, J = 19 Hz, J = 5.5 Hz, Ar),
7.17 (s, 2H, Ar), 7.28–7.34 (t, 1H, J = 30 Hz, J = 14.5 Hz,
Ar), 7.63 (s, 1H, Ar), 10.23 (s, 1H, OH). 13C-NMR
(125 MHz, DMSO-d6, ppm) d: 31.03, 38.01, 50.29, 56.57,
112.78, 116.08, 117.08, 120.20, 121.89, 128.01, 128.99,
136.14, 144.21, 145.94, 151.17, 166.15. Anal. Calcd. (%) for
C16H19N3O3 (301.34): C, 63.77; H, 6.36; N, 13.94; Found: C,
62.48; H, 6.24; N, 13.72.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(thiophen-2-
ylmethyl)acrylamide (IIIi)
Yield: 90 %. Amorphous, coffee color. mp: 111–113 °C.
FT-IR (KBr, cm-1) mmax: 3434, 2926, 1685, 1616, 1591,
1564. 1H-NMR (500 MHz, DMSO-d6, ppm) d: 2.93 (s, 2H,
CH2), 3.81 (s, 3H, OCH3), 5.46 (s, 3H, NH), 6.33–6.36 (d,
1H, J = 15.5 Hz, Ar), 6.77–6.78 (d, 1H, J = 8 Hz, Ar),
6.88–6.89 (d, 1H, J = 2 Hz, Ar), 6.94–6.96 (q, 1H,
J = 9.5 Hz, J = 5.5 Hz, Ar), 7.02–7.04 (q, 1H,
J = 10.5 Hz, J = 8.5 Hz, Ar), 7.22–7.23 (d, 1H, J = 2 Hz,
Ar), 7.32–7.34 (d, 1H, J = 4.5 Hz, Ar), 7.37–7.39 (d, 1H,
J = 10.5 Hz, Ar), 10.25 (s, 1H, OH). 13C-NMR (125 MHz,
DMSO-d6, ppm) d: 39.44, 50.41, 56.09, 112.94, 116.12,
117.17, 120.44, 121.61, 128.77, 129.17, 136.11, 144.27,
145.44, 151.28, 166.42. Anal. Calcd. (%) for C15H15-
NSO3 -(289.35): C, 62.26; H, 5.23; N, 4.84; S, 11.08; Found:
C, 61.87; H, 5.12; N, 4.59; S, 10.63.
(E)-N-benzyl-3-(4-hydroxy-3-methoxyphenyl)acrylamide
(IIIl)
Yield: 81 %. Crystalline, yellow. mp: 135–137 °C. FT-IR
(KBr, cm-1) mmax: 3431, 3007, 2933, 1634, 1598, 1504.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 3.79 (s, 2H, CH2),
3.88 (s, 3H, OCH3), 5.56 (s, 1H, NH), 6.32–6.35 (d, 1H,
J = 15.5 Hz, Ar), 6.77–6.79 (d, 1H, J = 8 Hz, Ar),
7.01–7.03 (d, 1H, J = 8 Hz, Ar), 7.21–7.25 (t, 2H,
J = 17.5 Hz, J = 10.5 Hz, Ar), 7.31–7.39 (m, 5H, Ar),
10.27 (s, 1H, OH). 13C-NMR (125 MHz, DMSO-d6, ppm)
d: 45.33, 55.16, 112.14, 116.41, 118.77, 120.14, 126.24,
127.44, 128.17, 129.45, 141.36, 143.58, 145.56, 151.11,
167.24. Anal. Calcd. (%) for C17H17NO3 (283.32): C,
72.07, H, 6.05, N, 4.94; Found: C, 71.26; H, 5.94; N, 4.08.
123

Med Chem Res
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(pyridin-2-
(E)-N-(acridin-9-yl)-3-(4-hydroxy-3-
ylmethyl)acrylamide (IIIm)
methoxyphenyl)acrylamide (Va)
Yield: 98 %. Semi-solid, brown. mp: 120–122 °C. FT-IR
(KBr, cm-1) mmax: 3425, 2895, 1635, 1594, 1557, 1479.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 3.79 (s, 2H, CH2),
3.89 (s, 3H, OCH3), 5.26 (s, 1H, NH), 6.33–6.36 (d, 1H,
J = 16 Hz, Ar), 6.77–6.79 (d, 1H, J = 8.5 Hz, Ar),
7.01–7.02 (d, 1H, J = 9 Hz, Ar), 7.03 (s, 1H, Ar),
7.24–7.27 (t, 1H, J = 8.5 Hz, J = 6.5 Hz, Ar), 7.34–7.38
(d, 1H, J = 14 Hz, Ar), 7.44–7.45 (d, 1H, J = 8 Hz, Ar),
7.75–7.78 (t, 1H, J = 15 Hz, J = 7.5 Hz, Ar), 8.50–8.51
(d, 1H, J = 4 Hz, Ar), 10.25 (s, 1H, OH). 13C-NMR
(125 MHz, DMSO-d6, ppm) d: 48.21, 55.60, 112.78,
116.33, 118.18, 120.03, 121.21, 124.12, 128.28, 137.12,
144.33, 145.13, 147.14, 151.57, 156.15, 167.37. Anal.
Calcd. (%) for C16H16N2O3 (285.32): C, 67.59; H, 5.67; N,
9.85; Found: C, 66.11; H, 5.63; N, 9.18.
Yield: 85 %. Amorphous, aureolin-yellow. mp: 262–264 °C.
FT-IR (KBr, cm-1): 3331, 3172, 1649, 1616, 1589, 1558. 1H-
NMR (500 MHz, DMSO-d6, ppm) d: 3.79 (s, 3H,
1 9 OCH3), 5.44 (s, 1H, 1 9 NH), 6.28–6.31 (d, 1H,
J = 15.5 Hz, Ar), 6.75–6.77 (d, 1H, J = 8 Hz, Ar),
6.96–6.98 (d, 1H, J = 8.5 Hz, Ar), 7.16 (s, 1H, Ar),
7.24–7.27 (d, 1H, J = 11 Hz, Ar), 7.38–7.41 (d, 2H,
J = 15 Hz, J = 7.5 Hz, Ar), 7.47–7.49 (d, 2H, J = 8.5 Hz,
Ar), 7.77–7.80 (q, 2H, J = 15.5 Hz, J = 7.5 Hz, Ar),
8.07–8.09 (d, 2H, J = 9 Hz, Ar), 10.21 (s, 1H, OH). 13C-
NMR (125 MHz, DMSO-d6, ppm) d: 56.14, 112.98, 114.22,
116.17, 118.13, 120.11, 124.22, 126.13, 128.55, 129.11,
129.98, 144.15, 144.99, 148.14, 151.36, 164.55, 167.75.
Anal. Calcd. (%) for C23H18N2O3 (370.40): C, 74.58; H, 4.90;
N, 7.56; Found: C, 73.17; H, 4.79; N, 7.09.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(pyridin-3-
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(4-methylacridin-9-
ylmethyl)acrylamide (IIIn)
yl)acrylamide (Vb)
Yield: 96 %. Semi-solid, red–orange. mp: 128–130 °C.
FT-IR (KBr, cm-1) mmax: 3425, 2907, 1636, 1586, 1511. 1H-
NMR (500 MHz, DMSO-d6, ppm) d: 3.76 (s, 2H, CH2), 3.80
(s, 3H, OCH3), 5.24 (s, 1H, NH), 6.33–6.36 (d, 1H,
J = 16 Hz, Ar), 6.77–6.79 (d, 1H, J = 8 Hz, Ar), 7.04–7.05
(d, 1H, J = 8 Hz, Ar), 7.242 (s, 1H, Ar), 7.32–7.34 (q, 1H,
J = 12.5 Hz, J = 5 Hz, Ar), 7.40–7.44 (d, 1H, J = 16 Hz,
Ar), 7.44–7.46 (d, 1H, J = 7.5 Hz, Ar), 8.42–8.43 (d, 1H,
J = 4.5 Hz, Ar), 8.53 (s, 1H, Ar), 10.27 (s, 1H, 1 9 OH).
13C-NMR (125 MHz, DMSO-d6, ppm) d: 45.01, 55.63,
112.16, 116.33, 118.02, 120.03, 121.24, 124.29, 128.17,
137.55, 144.36, 145.13, 147.75, 151.37, 156.28, 167.45.
Anal. Calcd. (%) for C16H16N2O3 (285.32): C, 67.59; H,
5.67; N, 9.85; Found: C, 66.23; H, 5.62; N, 9.12.
Yield: 88 %. Amorphous, corn-yellow. mp: 271–273 °C.
FT-IR (KBr, cm-1): 3378, 3121, 1646, 1618, 1593, 1562.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 2.28 (s, 3H, CH3),
3.78 (s, 3H, OCH3), 5.49 (s, 1H, NH), 6.28–6.31 (d, 1H,
J = 15 Hz, Ar), 6.75–6.78 (m, 2H, Ar), 6.96–6.98 (d, 1H,
J = 9 Hz, Ar), 7.16–7.18 (q, 2H, J = 10.5 Hz, J = 4.5 Hz,
Ar), 7.27–7.29 (t, 2H, J = 13 Hz, J = 7.5 Hz, Ar),
7.38–7.41 (t, 1H, J = 15 Hz, J = 7.5 Hz, Ar), 7.47–7.49 (d,
1H, J = 8.5 Hz, Ar), 7.77–7.79 (d, 1H, J = 9.5 Hz, Ar),
8.07–8.09 (d, 1H, J = 9 Hz, Ar), 10.24 (s, 1H, OH). 13C-
NMR (125 MHz, DMSO-d6, ppm) d: 22.39, 56.10, 108.55,
112.45, 116.16, 118.38, 119.34, 120.81, 122.22, 126.23,
127.12, 128.22, 128.78, 129.13, 129.97, 130.99, 136.11,
144.14, 145.11, 148.14, 151.36, 155.44, 164.14, 167.15.
Anal. Calcd. (%) for C24H20N2O3 (384.43): C, 74.98; H,
5.24; N, 7.29; Found: C, 74.21; H, 5.13; N, 6.91.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(pyridin-4-
ylmethyl)acrylamide (IIIo)
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(3-methylacridin-9-
Yield: 98 %. Crystalline, yellow. mp: 160–162 °C. FT-IR
(KBr, cm-1) mmax: 3431, 3002, 1636, 1595, 1520, 1459.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 3.77 (s, 2H, CH2),
3.80 (s, 3H, OCH3), 5.43 (s, 1H, NH), 6.34–6.37 (d, 1H,
J = 14.5 Hz, Ar), 6.77–6.78 (d, 1H, J = 8.5 Hz, Ar),
7.04–7.06 (d, 1H, J = 8 Hz, Ar), 7.25 (s, 1H, Ar),
7.35–7.36 (d, 2H, J = 4.5 Hz, Ar), 7.42–7.45 (d, 1H,
J = 14.5 Hz, Ar), 8.47–8.48 (d, 2H, J = 5 Hz, Ar), 10.23
(s, 1H, OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d:
44.27, 55.32, 112.07, 116.02, 118.58, 120.44, 124.41,
128.78, 144.33, 145.78, 147.81, 149.15, 151.82, 167.18.
Anal. Calcd. (%) for C16H16N2O3 (285.32): C, 67.59; H,
5.67; N, 9.85; Found: C, 66.03; H, 5.59; N, 9.09.
yl)acrylamide (Vc)
Yield: 89 %. Amorphous, coyote-brown. mp: 259–261 °C.
FT-IR (KBr, cm-1): 3391, 3211 1641, 1607, 1591, 1555.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 2.27 (s, 3H, CH3),
3.79 (s, 3H, OCH3), 5.49 (s, 1H, NH), 6.28–6.31 (d, 1H,
J = 15 Hz, Ar), 6.76–6.78 (d, 2H, J = 13 Hz, Ar),
6.95–6.98 (m, 2H, Ar), 7.16–7.18 (q, 2H, J = 14.5 Hz,
J = 9.5 Hz, Ar), 7.27–7.30 (q, 2H, J = 15.5 Hz,
J = 10.5 Hz, Ar), 7.38–7.41 (t, 1H, J = 15.5 Hz,
J = 7.5 Hz, Ar), 7.47–7.49 (d, 1H, J = 9.5 Hz, Ar),
7.78–7.79 (d, 1H, J = 9 Hz, Ar), 8.07–8.09 (d, 1H,
J = 10.5 Hz, Ar), 10.22 (s, 1H, OH). 13C-NMR (125 MHz,
123

Med Chem Res
DMSO-d6, ppm) d: 26.08, 56.01, 107.11, 112.13, 116.11,
118.38, 119.33, 120.82, 122.11, 127.23, 127.79, 128.22,
128.88, 129.12, 129.98, 131.83, 139.44, 144.11, 145.99,
147.44, 148.33, 151.14, 164.31, 167.32. Anal. Calcd. (%)
for C24H20N2O3 (384.43): C, 74.98; H, 5.24; N, 7.29;
Found: C, 73.67; H, 5.15; N, 6.69.
d: 3.69 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 5.51 (s, 1H,
NH), 6.28–6.31 (d, 1H, J = 14.5 Hz, Ar), 6.75–6.78 (d,
1H, J = 15 Hz, Ar), 6.94–6.98 (m, 2H, Ar), 7.15–7.18 (q,
2H, J = 15.5 Hz, J = 11 Hz, Ar), 7.27–7.31 (q, 2H,
J = 18.5 Hz, J = 10.5 Hz, Ar), 7.38–7.41 (t, 1H,
J = 15.5 Hz, J = 7.5 Hz, Ar), 7.47–7.49 (d, 1H,
J = 9.5 Hz, Ar), 7.77–7.79 (d, 1H, J = 10 Hz, Ar),
8.07–8.09 (d, 1H, J = 11 Hz, Ar), 10.33 (s, 1H, OH). 13C-
NMR (125 MHz, DMSO-d6, ppm) d: 55.73, 56.36, 105.44,
107.29, 112.53, 116.83, 117.29, 118.24, 118.53, 120.16,
122.99, 127.12, 128.08, 128.91, 129.13, 129.82, 144.83,
145.11, 148.92, 150.13, 151.79, 159.52, 164.23, 167.19.
Anal. Calcd. (%) for C24H20N2O4 (400.43): C, 71.99; H,
5.03; N, 7.00; Found: C, 70.33; H, 4.97; N, 6.55.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2-methylacridin-9-
yl)acrylamide (Vd)
Yield: 88 %. Amorphous, buff-yellow. mp: 262–264 °C.
FT-IR (KBr, cm-1): 3331, 3201, 1638, 1603, 1586, 1553.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 2.27 (s, 3H, CH3),
3.66 (s, 3H, OCH3), 5.52 (s, 1H, NH), 6.28–6.32 (d, 1H,
J = 14.5 Hz, Ar), 6.75–6.78 (d, 1H, J = 14.5 Hz, Ar),
6.95–6.98 (m, 2H, Ar), 7.15–7.18 (q, 2H, J = 15 Hz,
J = 9.5 Hz, Ar), 7.27–7.30 (q, 2H, J = 12.5 Hz, J = 7 Hz,
Ar), 7.38–7.41 (t, 1H, J = 16 Hz, J = 7.5 Hz, Ar),
7.47–7.49 (d, 1H, J = 10 Hz, Ar), 7.77–7.79 (d, 1H,
J = 10 Hz, Ar), 8.07–8.09 (d, 1H, J = 10.5 Hz, Ar), 10.09
(s, 1H, OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d: 27.27,
55.33, 107.24, 112.12, 116.26, 118.39, 119.39, 120.27,
121.03, 127.12, 128.02, 128.32, 128.88, 129.24, 130.84,
133.19, 135.42, 144.19, 145.92, 146.43, 148.33, 151.15,
164.31, 167.51. Anal. Calcd. (%) for C24H20N2O3 (384.43):
C, 74.98; H, 5.24; N, 7.29; Found: C, 73.53; H, 5.13; N, 6.51.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2-methoxyacridin-
9-yl)acrylamide (Vg)
Yield: 90 %. Amorphous, yellow. mp: 275–277 °C. FT-IR
(KBr, cm-1): 3407, 3161, 1642, 1611, 1581, 1567. 1H-NMR
(500 MHz, DMSO-d6, ppm) d: 3.67 (s, 3H, OCH3), 3.78 (s,
3H, OCH3), 5.46 (s, 1H, NH), 6.29–6.32 (d, 1H, J = 15 Hz,
Ar), 6.77–6.81 (d, 1H, J = 15 Hz, Ar), 6.94–6.98 (m, 2H,
Ar), 7.16–7.19 (q, 2H, J = 14 Hz, J = 9 Hz, Ar), 7.27–7.30
(q, 2H, J = 16.5 Hz, J = 11.5 Hz, Ar), 7.38–7.41 (t, 1H,
J = 18 Hz, J = 10.5 Hz, Ar), 7.47–7.49 (d, 1H,
J = 8.5 Hz, Ar), 7.78–7.79 (d, 1H, J = 10 Hz, Ar),
8.07–8.09 (d, 1H, J = 10 Hz, Ar), 10.46 (s, 1H, OH). 13C-
NMR (125 MHz, DMSO-d6, ppm) d: 55.51, 56.45, 105.21,
108.11, 112.32, 116.33, 118.31, 120.23, 121.53, 123.86,
127.09, 128.12, 128.77, 129.08, 129.71, 130.13, 143.83,
144.81, 144.92, 147.43, 151.19, 156.52, 163.10, 167.12.
Anal. Calcd. (%) for C24H20N2O4 (400.43): C, 71.99; H,
5.03; N, 7.00; Found: C, 70.21; H, 4.94; N, 6.18.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(4-methoxyacridin-
9-yl)acrylamide (Ve)
Yield: 91 %. Amorphous, yellowish-green. mp: 281–283 °C.
FT-IR (KBr, cm-1): 3403, 3151, 1642, 1609, 1591, 1558. 1H-
NMR (500 MHz, DMSO-d6, ppm) d: 3.78 (s, 3H, OCH3), 3.88
(s, 3H, OCH3), 5.46 (s, 1H, NH), 6.28–6.31 (d, 1H,
J = 15 Hz, Ar), 6.75–6.78 (m, 2H, Ar), 6.96–6.98 (d, 1H,
J = 7.5 Hz, Ar), 7.16–7.18 (q, 2H, J = 12.5 Hz, J = 6.5 Hz,
Ar), 7.27–7.31 (q, 2H, J = 18.5 Hz, J = 11.5 Hz, Ar),
7.38–7.41 (t, 1H, J = 15 Hz, J = 8.5 Hz, Ar), 7.47–7.49 (d,
1H, J = 10 Hz, Ar), 7.75–7.78 (d, 1H, J = 11 Hz, Ar),
8.08–8.10 (d, 1H, J = 10 Hz, Ar), 10.32 (s, 1H, OH). 13C-
NMR (125 MHz, DMSO-d6, ppm) d: 55.21, 56.09, 106.24,
108.10, 112.27, 113.33, 116.34, 118.27, 120.03, 122.13,
125.09, 127.12, 128.08, 128.13, 129.83, 130.14, 139.43,
144.19, 144.92, 148.44, 151.14, 156.15, 164.11, 167.19. Anal.
Calcd. (%) for C24H20N2O4 (400.43): C, 71.99; H, 5.03; N,
7.00; Found: C, 71.16; H, 4.93; N, 6.43.
Synthesis of (E)-3-(4-hydroxy-3-methoxyphenyl)-N-
phenylacrylamide (VIIa)
Yield: 97 %. Crystalline, black-olive. mp: 138–140 °C. FT-
IR (KBr, cm-1): 3434, 3292, 1664, 1614, 1513, 1429, 1322,
1274, 1203, 1173, 1108, 1032. 1H-NMR (500 MHz, DMSO-
d6, ppm) d: 3.78 (s, 3H, OCH3), 6.32–6.35 (d, 1H,
J = 16 Hz, Ar), 6.77–6.78 (d, 1H, J = 8 Hz, Ar), 7.01–7.03
(d, 1H, J = 8 Hz, Ar), 7.21–7.25 (t, 2H, J = 21 Hz,
J = 21 Hz, Ar), 7.30–7.34 (m, 5H, Ar), 8.39 (s, 1H, NH),
10.37 (s, 1H, OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d:
56.32, 112.08, 116.07, 118.44, 120.58, 121.30, 124.41,
128.33, 129.34, 135.78, 144.11, 149.17, 151.45, 166.12.
Anal. Calcd. (%) for C16H15NO3 (269.29): C, 71.36, H, 5.61,
N, 5.20; Found: C, 70.11, H, 5.57, N, 4.93.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(3-methoxyacridin-
9-yl)acrylamide (Vf)
Yield: 93 %. Amorphous, arctic-lime. mp: 278–280 °C.
FT-IR (KBr, cm-1): 1H-NMR (500 MHz, DMSO-d6, ppm)
123

Med Chem Res
(E)-3-(4-hydroxy-3-methoxyphenyl)-N–o-tolylacrylamide
167.28. Anal. Calcd. (%) for C17H17NO3 (283.32): C,
72.07, H, 6.05, N, 4.94; Found: C, 71.09, H, 5.94, N, 4.05.
(VIIb)
Yield: 87 %. Amorphous, yellow. mp: 162–164 °C. FT-IR
(KBr, cm-1): 3436, 3015, 2968, 1691, 1665, 1619, 1597.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 2.74 (s, 3H, CH3),
3.79 (s, 3H, OCH3), 6.33–6.36 (d, 1H, J = 19 Hz, Ar),
6.77–6.79 (d, 1H, J = 8 Hz, Ar), 7.01–7.02 (d, 1H,
J = 7.5 Hz, Ar), 7.21 (s, 1H, Ar), 7.24–7.27 (t, 1H,
J = 13.5 Hz, J = 8 Hz, Ar), 7.34–7.37 (d, 1H,
J = 15.5 Hz, Ar), 7.43–7.45 (d, 1H, J = 8 Hz, Ar),
7.76–7.78 (d, 1H, J = 9 Hz, Ar), 8.50–8.51 (d, 1H,
J = 2.5 Hz, Ar), 8.95 (s, 1H, NH), 10.24 (s, 1H, OH). 13C-
NMR (125 MHz, DMSO-d6, ppm) d: 16.33, 56.09, 112.11,
116.02, 118.04, 120.83, 121.51, 124.49, 126.49, 128.54,
129.33, 134.80, 135.78, 144.14, 145.49, 151.46, 166.05.
Anal. Calcd. (%) for C17H17NO3 (283.32): C, 72.07, H,
6.05, N, 4.94; Found: C, 71.43, H, 5.97, N, 4.01.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(2-
methoxyphenyl)acrylamide (VIIe)
Yield: 79 %. Crystalline, brown. mp: 174–176 °C. FT-IR
(KBr, cm-1): 3437, 3377, 1691, 1645, 1620, 1588, 1563.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 3.70 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 6.33–6.37 (d, 1H, J = 19 Hz,
Ar), 6.77–6.79 (d, 1H, J = 8 Hz, Ar), 7.01–7.02 (d, 1H,
J = 8 Hz, Ar), 7.21 (s, 1H, Ar), 7.24–7.27 (t, 1H,
J = 13 Hz, J = 6.5 Hz, Ar), 7.34–7.37 (d, 1H, J = 15 Hz,
Ar), 7.43–7.45 (d, 1H, J = 8 Hz, Ar), 7.76–7.78 (t, 1H,
J = 13 Hz, J = 4 Hz, Ar), 8.50–8.51 (d, 1H, J = 3.5 Hz,
Ar), 8.98 (s, 1H, NH), 10.37 (s, 1H, OH). 13C-NMR
(125 MHz, DMSO-d6, ppm) d: 56.01, 56.99, 112.11,
116.01, 118.05, 120.83, 121.34, 124.77, 126.49, 128.57,
129.33, 134.80, 135.78, 144.14, 145.49, 151.42, 167.01.
Anal. Calcd. (%) for C17H17NO4 (299.32): C, 68.22, H,
5.72, N, 4.68; Found: C, 67.37, H, 5.67, N, 4.02.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-m-tolylacrylamide
(VIIc)
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(3-
Yield: 80 %. Crystalline, brass color. mp: 164–166 °C. FT-
IR (KBr, cm-1): 3435, 3189, 3014, 1690, 1665, 1618, 1584,
1564. 1H-NMR (500 MHz, DMSO-d6, ppm) d: 2.86 (s, 3H,
CH3), 3.84 (s, 3H, OCH3), 6.32–6.36 (d, 1H, J = 17 Hz, Ar),
6.77–6.79 (d, 1H, J = 7 Hz, Ar), 7.04–7.06 (d, 1H,
J = 9 Hz, Ar), 7.24 (s, 1H, Ar), 7.31–7.34 (m, 1H, Ar),
7.41–7.44 (d, 1H, J = 14 Hz, Ar), 7.74–7.76 (d, 1H,
J = 8 Hz, Ar), 8.41–8.42 (d, 1H, J = 3.5 Hz, Ar), 8.53 (s,
1H, Ar), 8.87 (s, 1H, NH), 10.24 (s, 1H, OH). 13C-NMR
(125 MHz, DMSO-d6, ppm) d: 26.44, 56.01, 112.11, 116.01,
118.04, 119.21, 120.82, 121.51, 124.49, 128.54, 129.32,
135.80, 138.78, 144.14, 145.49, 151.45, 167.04. Anal. Calcd.
(%) for C17H17NO3 (283.32): C, 72.07, H, 6.05, N, 4.94;
Found: C, 71.21, H, 5.92, N, 4.17.
methoxyphenyl)acrylamide (VIIf)
Yield: 84 %. Crystalline, gray. mp: 128–130 °C. FT-IR
(KBr, cm-1): 3433, 3347, 1689, 1663, 1623, 1596, 1560.
1H-NMR (500 MHz, DMSO-d6, ppm) d: 3.71 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3), 6.33–6.36 (d, 1H, J = 17 Hz,
Ar), 6.77–6.79 (d, 1H, J = 7 Hz, Ar), 7.04–7.05 (d, 1H,
J = 10 Hz, Ar), 7.24 (s, 1H, Ar), 7.31–7.34 (q, 1H,
J = 10 Hz, J = 9 Hz, Ar), 7.41–7.44 (d, 1H, J = 14 Hz,
Ar), 7.74–7.76 (d, 1H, J = 8 Hz, Ar), 8.42–8.43 (d, 1H,
J = 3.5 Hz, Ar), 8.55 (s, 1H, Ar), 8.844 (s, 1H, NH), 10.24
(s, 1H, OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d:
56.02, 56.79, 112.20, 116.08, 118.07, 119.27, 120.52,
121.51, 124.44, 128.54, 129.93, 135.80, 138.78, 144.24,
145.39, 151.35, 167.12. Anal. Calcd. (%) for C17H17NO4
(299.32): C, 68.22, H, 5.72, N, 4.68; Found: C, 67.41, H,
5.63, N, 4.11.
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-p-tolylacrylamide
(VIId)
Yield: 83 %. Crystalline, gray. mp: 158–160 °C. FT-IR
(KBr, cm-1): 3436, 3015, 2978, 1691, 1665, 1619, 1597,
1515, 1464, 1431, 1379, 1273, 1206, 1177, 1113, 1034. 1H-
NMR (500 MHz, DMSO-d6, ppm) d: 2.85 (s, 3H, CH3),
3.83 (s, 3H, OCH3), 6.33–6.36 (d, 1H, J = 14 Hz, Ar),
6.77–6.79 (d, 1H, J = 8.5 Hz, Ar), 7.04–7.06 (d, 1H,
J = 8 Hz, Ar), 7.25 (s, 1H, Ar), 7.35–7.36 (d, 2H,
J = 4.5 Hz, Ar), 7.42–7.45 (d, 1H, J = 15.5 Hz, Ar),
8.47–8.48 (d, 2H, J = 5 Hz, Ar), 8.88 (s, 1H, NH), 10.25
(s, 1H, OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d:
26.83, 56.51, 112.54, 116.01, 118.14, 120.82, 121.45,
127.55, 129.52, 132.80, 134.97, 144.14, 145.49, 151.45,
(E)-3-(4-hydroxy-3-methoxyphenyl)-N-(4-
methoxyphenyl)acrylamide (VIIg)
Yield: 83 %. Crystalline, buff-yellow. mp: 136–138 °C.
FT-IR (KBr, cm-1): 3436, 3359, 1686, 1661, 1620, 1526,
1471, 1429, 1381, 1334, 1271, 1215, 1147, 1034. 1H-NMR
(500 MHz, DMSO-d6, ppm) d: 3.71 (s, 3H, OCH3), 3.80 (s,
3H, OCH3), 6.34–6.37 (d, 1H, J = 15 Hz, Ar), 6.77–6.79
(d, 1H, J = 8 Hz, Ar), 7.05–7.07 (d, 1H, J = 8 Hz, Ar),
7.25 (s, 1H, Ar), 7.36–7.37 (d, 2H, J = 5 Hz, Ar),
7.42–7.45 (d, 1H, J = 15 Hz, Ar), 8.47–8.48 (d, 2H,
J = 5 Hz, Ar), 8.89 (s, 1H, NH), 10.23 (s, 1H, OH). 13C-
123

Med Chem Res
NMR (125 MHz, DMSO-d6, ppm) d: 56.08, 56.58, 112.45,
116.02, 118.26, 120.81, 121.40, 127.74, 129.52, 132.80,
134.87, 144.45, 145.49, 151.67, 167.38. Anal. Calcd. (%)
for C17H17NO4 (299.32): C, 68.22, H, 5.72, N, 4.68;
Found: C, 67.07, H, 5.61, N, 3.99.
Ar), 7.44–7.46 (d, 1H, J = 8 Hz, Ar), 7.56–7.88 (t, 2H,
J = 13 Hz, J = 7.5 Hz, Ar), 8.49–8.51 (d, 1H, J = 5 Hz,
Ar), 8.98 (s, 2H, 2 9 NH), 10.31 (s, 2H, 2 9 OH). 13C-
NMR (125 MHz, DMSO-d6, ppm) d: 17.99, 46.09, 47.95,
56.31, 112.09, 116.83, 118.58, 120.69, 127.89, 143.11,
144.23, 151.25, 166.44. Anal. Calcd. (%) for C23H26N2O6
(426.46): C, 64.78; H, 6.15; N, 6.57; Found: C, 64.14; H,
6.11; N, 6.01.
Synthesis of N,N’-ethylen-Bis[(E)-3-(4-hydroxy-3-
methoxyphenyl)-acrylamide] (IXa)
Yield: 93 %. Crystalline, yellowish-brown. mp:
146–148 °C. FT-IR (cm-1): 3431, 3146, 2904, 1641, 1599,
1556. 1H-NMR (500 MHz, DMSO-d6, ppm) d: 3.47–3.50
(t, 4H, J = 15 Hz, J = 7.5 Hz, 2 9 CH2), 3.79 (s, 6H,
2 9 OCH3), 5.48 (s, 2H, 2 9 NH), 6.28–6.31 (d, 2H,
J = 15 Hz, Ar), 6.75–6.77 (d, 2H, J = 10 Hz, Ar),
6.95–6.97 (d, 2H, J = 8 Hz, Ar), 7.19 (s, 2H, Ar),
7.25–7.28 (d, 2H, J = 13 Hz, Ar), 10.22 (s, 2H, 2 9 OH).
13C-NMR (125 MHz, DMSO-d6, ppm) d: 39.002, 56.320,
112.070, 116.302, 118.444, 120.585, 128.445, 143.135,
144.412, 151.447, 166.298. Anal. Calcd. (%) for
C22H24N2O6 (412.44): C, 64.07; H, 5.87; N, 6.79; Found:
C, 63.48; H, 5.79; N, 6.37.
N,N’-butylen-Bis[(E)-3-(4-hydroxy-3-methoxyphenyl)-
acrylamide] (IXd)
Yield: 95 %. Crystalline, brown. mp: 136–138 °C. FT-IR
(cm-1): 3401, 3188, 2935, 1642, 1617, 1589, 1561. H-
1
NMR (500 MHz, DMSO-d6, ppm) d: 1.94–1.97 (t, 4H,
J = 15 Hz, J = 12.5 Hz, 2 9 CH2), 2.93–2.96 (t, 4H,
J = 15 Hz, J = 7.5 Hz, 2 9 CH2), 3.75 (s, 6H,
2 9 OCH3), 5.43 (s, 2H, 2 9 NH), 6.29–6.32 (d, 2H,
J = 15 Hz, Ar), 6.75–6.78 (d, 2H, J = 10 Hz, Ar),
6.95–6.97 (d, 2H, J = 9 Hz, Ar), 7.19 (s, 2H, Ar),
7.25–7.27 (d, 2H, J = 13 Hz, Ar), 10.27 (s, 2H, 2 9 OH).
13C-NMR (125 MHz, DMSO-d6, ppm) d: 27.42, 41.95,
56.34, 112.09, 116.33, 118.21, 120.18, 127.99, 143.51,
144.43, 151.46, 166.17. Anal. Calcd. (%) for C24H28N2O6
(440.49): C, 65.44; H, 6.41; N, 6.36; Found: C, 64.89; H,
6.33; N, 5.97.
N,N’-(trimethylene)-Bis[(E)-3-(4-hydroxy-3-
methoxyphenyl)-acrylamide] (IXb)
Yield: 97 %. Crystalline, antique-white. mp: 114–116 °C.
FT-IR (cm-1): 3534, 3436, 3013, 2916, 1639, 1592, 1566,
N,N’-3-(4-(3-aminopropyl)piperazin-1-yl)propylen)-
1
1551. H-NMR (500 MHz, DMSO-d6, ppm) d: 1.95–1.98
Bis[(E)-3-(4-hydroxy-3-methoxyphenyl)-acrylamide] (IXe)
(m, 2H, CH2), 2.92–2.95 (m, 4H, 2 9 CH2), 3.78 (s, 6H,
2 9 OCH3), 5.48 (s, 2H, 2 9 NH), 6.29–6.32 (d, 2H,
J = 15 Hz, Ar), 6.75–6.77 (d, 2H, J = 10 Hz, Ar),
6.96–6.98 (d, 2H, J = 9 Hz, Ar), 7.19 (s, 2H, Ar),
7.25–7.28 (d, 2H, J = 13 Hz, Ar), 10.22 (s, 2H, 2 9 OH).
13C-NMR (125 MHz, DMSO-d6, ppm) d: 28.41, 39.01,
56.74, 112.07, 116.31, 118.42, 120.66, 128.04, 143.11,
144.90, 151.46, 166.89. Anal. Calcd. (%) for C23H26N2O6
(426.46): C, 64.78; H, 6.15; N, 6.57; Found: C, 64.01; H,
6.08; N, 6.29.
Yield: 89 %. Crystalline, brown. mp: 130–132 °C. FT-IR
(cm-1): 3371, 3163, 1647, 1612, 1589, 1572. 1H-NMR
(500 MHz, DMSO-d6, ppm) d: 1.89–1.92 (t, 4H,
J = 15 Hz, J = 7.5 Hz, 2 9 CH2), 2.82–2.85 (t, 12H,
J = 15 Hz, J = 7.5 Hz, 6 9 CH2), 3.88 (s, 6H,
2 9 OCH3), 3.95–3.98 (m, 4H, 2 9 CH2), 5.42 (s, 2H,
2 9 NH), 6.31–6.34 (d, 2H, J = 15 Hz, Ar), 6.75–6.77 (d,
2H, J = 10 Hz, Ar), 6.97–6.99 (d, 2H, J = 9 Hz, Ar), 7.11
(s, 2H, Ar), 7.24–7.27 (d, 2H, J = 15 Hz, Ar), 10.24 (s,
2H, 2 9 OH). 13C-NMR (125 MHz, DMSO-d6, ppm) d:
27.11, 38.01, 51.51, 53.52, 56.45, 112.33, 116.83, 118.21,
120.23, 127.86, 143.13, 144.83, 151.11, 166.12. Anal.
Calcd. (%) for C30H40N4O6 (552.66): C, 65.20; H, 7.30; N,
10.14; Found: C, 64.89; H, 7.21; N, 9.93.
N,N’-propylen-Bis[(E)-3-(4-hydroxy-3-methoxyphenyl)-
acrylamide] (IXc)
Yield: 93 %. Crystalline, apricot color. mp: 94–96 °C. FT-
IR (cm-1): 3436, 3197, 2935, 1596, 1591, 1582, 1566. 1H-
NMR (500 MHz, DMSO-d6, ppm) d: 1.40–1.43 (d, 3H,
J = 14 Hz, CH3), 2.76–2.78 (q, 1H, J = 7 Hz, J = 4 Hz,
CH), 3.70 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 4.77–4.80
(d, 2H, J = 15 Hz, CH2), 6.33–6.37 (d, 1H, J = 19 Hz,
Ar), 6.77–6.79 (d, 1H, J = 8 Hz, Ar), 7.01–7.03 (d, 1H,
J = 10.5 Hz, Ar), 7.21 (s, 1H, Ar), 7.24–7.27 (t, 1H,
J = 12.5 Hz, J = 7 Hz, Ar), 7.35–7.38 (d, 1H, J = 16 Hz,
Cytotoxicity assay
A variety of human cancer cell lines (breast: MCF-7 and
MDA-MB-231, lung: A549, Cervical: HeLa and liver:
HepG2) and one normal stem cell line (BM-MSC) were
acquired from National Centre for Cell Sciences, Pune,
India, for primary screening of synthesized molecules as
123

Med Chem Res
strong anticancer agents. The cells were grown in the tissue
culture flasks at 37 °C within the carbon dioxide incubator
(5 % CO2, 90 % RH) by using growth medium which
contained the RPMI-1640 medium (with 2 mM glutamine),
supplemented with 10 % fetal bovine serum, 100 lg/mL
streptomycin and 100 units/mL penicillin at pH 7.4. In
brief, 5 9 103 cells in 200 ml of medium were seeded in
96-well Elisa plates. Serial dilutions of tested synthesized
derivatives ranging from 0 to 100 mM in DMSO have been
putted to the monolayer, and 0.1 % DMSO was used as
vehicle control during MTT assay. After 24 h of cell cul-
ture, 50 ml (5 mg/ml) of MTT was added in the culture
plates and incubated at 37 °C for 4 h in CO2 incubator
under dark. MTT mixed culture plates were aspirated;
25 ml of Sorensen glycine buffer (0.1 M glycine and 0.1 M
NaCl, pH 10.5) with 200 ml of DMSO have been added
into the plates to lyse the cells and for the solubilization of
the water insoluble formazones (Sondhi et al., 2012;
Kumar et al., 2014). Finally, the absorbance of all the
lysates were taken at 570 nm with the help of Fluostar
optima microplate reader (BMG Labtech, Germany). The
IC50 values were calculated by using the software Graph-
Pad Prism 5.02, and the percentage inhibition was calcu-
lated with the help of mathematical formula which is given
below:
method reported earlier (Li et al., 2012a, b). The scav-
enging capability of test compounds was calculated using
the following equation:
Percentage DPPH radical scavenging activity
k517 À S
ꢀ
ꢁ
¼
1 À
 100
k517 À C
where k517-C is absorbance of a control with no radical
scavenger and k517-S is absorbance of the remaining DPPH
in the presence of scavenger.
3D-QSAR modeling
Five CoMFA models based on the anticancer activities of
21 compounds and one CoMFA model based on the
antioxidant activity of 34 compounds have been generated.
The pIC50 values exhibited by the synthesized derivatives
were used as the dependent variable for the generation of
CoMFA model. SYBYL7.1 (Silicon Graphics Octane2
workstation containing IRIX6.5 operating system) was
used for all the calculations performed during the molec-
ular modeling exercise. The PM3 method was applied for
the energy minimization step with 0.005 kcal/mol energy
gradient convergence criterion, and the Gasteiger-Huckel
charges were assigned to all the compounds during their
Mean OD of vehicle treated cells ðnegative controlÞ À Mean OD of treated cells  100
Mean OD of vehicle treated cells (negative control)
DPPH assay
modeling (Gasteiger and Marsili, 1980). The random
selections have been made to choose the training and test
set compounds for anticancer (16 and 5 compounds) and
antioxidant model (26 and 8 compounds). The database
alignment method was applied for the alignment of mole-
cules and selected the lowest energy conformer of the most
active compound as a template (Fig. S35). The Compara-
tive Molecular Field Analysis (CoMFA) method was exe-
cuted for the evaluation of steric and electrostatic fields and
partial least square (PLS) analysis was also carried out to
correlate the CoMFA fields with experimentally exhibited
biological activities by amide derivatives (Cramer et al.,
1988a, b; Wold, 1991). The leave-one-out (LOO) method
has been used for the cross-validation (rc2v) of data, and the
formula given below was used for the calculation of pre-
dictive correlation (rp2red):
The earlier reported methods for antioxidant assays with
modifications were used here to determine the spectral
values of ferulic acid amide derivatives (Blois, 1958;
Sharma and Bhat, 2009; Vashisth et al., 2015). The cur-
rently used method of microplate assay has its own bene-
fits. It is environmental friendly and also reduces the time
and sample as compared to the commonly used in vitro
antioxidant methods (labor, time, sample and reagent
consuming) for testing of antioxidant activity of the com-
pounds. Briefly, the methanolic DPPH solution (50,
100 lg/mL) was added into each sample of different con-
centration (200 lL, 12.5–100 lg/mL). The solutions
(sample with DPPH) were mixed gently and incubated for
30 min in the dark at room temperature, and absorbance
was recorded at 517 nm. Different concentrations of
methanolic DPPH (5–50 lg/mL) have been taken in the
formation of standard curve. The concentration of DPPH in
the reaction mixture was calculated according to the
rp2red ¼ ðSD À PRESSÞ=SD
where SD is the sum of the squared deviations between the
test set biological activities and mean activities of the
123

Med Chem Res
O
O
H3CO
HO
OCH3
NH R4
IXa-e
N
H
OH
R4
R4
d
e
CH2CH2CH2CH2
a
b
c
CH2CH2
CH2CH2CH2
MWI
300 W / 110oC
7 min
(CH2)3
NH2R4NH2
VIIIa-e
N
N (CH2)3
CH2CH
CH3
R3 R2
R2
N
R3
R1
NH2
R5
H2N
N
R1
O
O
O
R5
VIa-g
H3CO
HO
OCH3
OH
H3CO
HO
N
H
N
H
OH
IVa-g
MWI
MWI
450 W / 145oC
5 min
300 W / 125oC
5 min
I
VIIa-g
Va-g
R5
R5
MWI
180 W / 85oC
3 min
RNH2
IIa-o
e
f
g
OCH3(O)
OCH3(m)
OCH3(p)
a
b
c
d
H
R1
R2
H
R3
CH3(O)
CH3(m)
CH3(p)
a
b
c
d
e
f
H
H
CH3
H
H
H
CH3
H
H
OCH3
H
H
O
H
CH3
H
OCH3
H
H3CO
HO
H
N
R
H
g
H
OCH3
IIIa-o
where R is same for IIa-o and IIIa-o
R
R
R
f
k
l
N CH2CH2CH2
O
a
N
N CH2CH2CH2
H2
C
b
c
g
O
N CH2CH2
H2
C
N
N CH2CH2
HN N CH2CH2
H2
m
n
h
i
N
d
e
N CH2CH2
CH3
CH2
CH2
C
S
H3C
HN
H3C
o
N
j
CH2CH2
S
CH3
Scheme 1 Schematic representation of synthesis of amide derivatives of ferulic acid, i.e., IIIa–IIIo, Va–Vg, VIIa–VIIg and IXa–IXe
training set molecules and PRESS is the sum of the squared
deviations between the actual and predicted activities of the
test compounds (Cramer et al., 1988a, b).
mathematical validation by using MATLAB R2013a tool-
box. The correlation coefficient (R) value, which measures
the potency and direction of a linear association between
two variables, was calculated between simulated and
experimental data (Kumar and Bhalla, 2011; Kumar and
Garg, 2014; Goel and Singh, 2013; Goel and Kumar,
2014). All the experiments were carried out in triplicate for
reproducing the results, and the mean values were taken for
the comparison of data.
Statistical analysis
The values of pIC50 for anticancer and antioxidant activi-
ties of all the derivatives obtained experimentally and
theoretically have been tested statistically for their
123

Med Chem Res
Fig. 1 Simultaneous TG-DTG thermogram of IIIa, Va, VIIa and IXa under nitrogen
Thermal analysis
Results and discussions
Chemistry
Thermal behavior of a molecule is exciting predicting
their stability, shelf lives and suitable storage conditions
(Shamsipur et al., 2013). Thermokinetic data could be
calculated by thermal analysis, which makes the possible
determination of thermodynamic parameters for a drug
candidate (Lever and Papadaki, 2004). Here, all
the derivatives, like their parent molecule (ferulic acid),
were found stable up to 100 °C and decomposed through
one step during thermal studies. After 100 °C, the TG-
DTG curves showed irregular pattern which confirmed the
maximum decomposition. The graphical representation of
thermal analysis curves for the first compound from each
series is shown in Fig. 1, and the details of results are
provided in Table S1. The compound IIIa was decom-
posed immediately after melting, and it continued up to
364 °C with 98.1 % weight loss (DTG peak at 205 °C),
which corresponds to endotherm at 292 °C in DTA. In
Va, the weight loss (97.6 %) was observed in the tem-
perature range of 264–378 °C (DTG peak at 341 °C). The
thermal decomposition of VIIa occurred in the tempera-
ture range of 174–402 °C, which showed the weight loss
of 97.9 % with the DTG peak at 257 °C, while IXa
decomposed in the 148–367 °C temperature range with
Microwave-assisted synthesis of four series of mono and bis-
amide derivatives of ferulic acid has been achieved at room
temperature by simple grinding of the mixture of ferulic acid
and corresponding amines (IIa–IIo, IVa–IVg, VIa–VIg and
VIIIa–VIIIe; Scheme 1) in 1:1 and 1:2 molar ratio within
3–7 min. All the molecules were well characterized by
highly sophisticated techniques including FT-IR, FT-NMR,
mass spectroscopy, TGA-DTG thermal analysis as well as
elemental analysis for C, H and N. The structural data
obtained from spectral (Fig. S1–Fig. S34) and elemental
studies for the compounds reported in the experimental
sectionhave fully supported the chemical structures assigned
to them. The 1H-NMR spectra for all the compounds did not
show any change after keeping them for 10 days at room
temperature; similarly the same spectra were also collected
after 6 months when compared with the previous data and
found negligible changes in the results with respect to the
starting spectroscopic data. Commencing on the results from
above studies, we can conclude that the currently synthesized
amide derivatives are stable in solid as well liquid phase even
after a long interval of time.
123

Med Chem Res
Normal (stem)
Table 1 In vitro anti-proliferative activity (% growth inhibition) of ferulic acid amide derivatives (10 lM concentration)
Compound name
Breast (MCF-7)
Breast (MDA-MB-231)
Lung (A549)
Liver (HepG2)
Cervical (HeLa)
IIIa
29.19
27.95
23.51
32.30
27.72
26.85
20.28
25.35
59.09
62.55
43.36
51.92
58.22
55.88
50.81
57.24
75.53
66.64
68.97
66.26
79.11
78.04
51.67
48.62
49.68
51.63
39.07
42.68
48.63
24.01
33.63
27.57
28.96
26.91
23.56
15.54
13.17
15.22
16.98
30.18
21.77
24.22
21.87
27.22
29.29
30.55
51.48
56.83
47.77
56.55
55.09
57.68
58.33
60.82
77.25
74.10
78.11
62.84
61.12
69.51
51.91
43.88
47.82
55.14
40.78
49.83
55.66
19.09
25.02
22.24
19.95
19.37
20.18
20.22
15.84
17.96
26.17
19.07
27.28
33.89
35.52
31.13
17.81
31.12
54.04
56.81
52.27
56.12
58.34
53.42
56.49
69.95
72.64
75.10
79.17
78.56
80.63
83.71
60.26
61.03
53.34
65.88
58.42
55.49
60.65
27.18
31.76
31.64
27.51
27.19
20.66
30.17
11.18
14.11
27.69
34.94
20.65
25.14
29.69
29.92
24.93
29.75
53.55
50.63
51.39
47.68
44.72
51.09
41.62
59.36
61.95
63.35
66.46
63.51
66.37
68.32
45.35
48.35
52.33
55.91
46.58
50.46
56.68
25.77
20.96
23.76
22.67
22.59
21.09
32.72
19.50
22.13
22.73
31.88
25.68
29.22
32.17
30.24
23.74
27.93
65.62
61.91
55.71
58.42
55.43
53.14
54.11
78.11
71.39
69.19
82.95
81.01
70.97
73.82
63.41
59.42
55.84
60.69
54.48
57.28
63.98
29.09
32.95
38.01
23.59
16.92
21.91
23.84
20.16
22.37
NA
IIIb
NA
IIIc
NA
IIId
NA
IIIe
NA
IIIf
NA
IIIg
06.63
NA
IIIh
IIIi
11.36
13.63
07.68
18.37
14.21
15.34
08.44
17.24
16.47
12.49
06.63
09.76
17.05
19.13
08.44
12.69
08.96
10.27
08.01
06.25
03.68
NA
IIIj
IIIk
IIIl
IIIm
IIIn
IIIo
Va
Vb
Vc
Vd
Ve
Vf
Vg
VIIa
VIIb
VIIc
VIId
VIIe
VIIf
VIIg
IXa
IXb
NA
IXc
NA
IXd
05.59
08.61
NA
IXe
Ferulic acid
5 Fluorouracil
Cyclophosphamide
Cycloheximide
25.21
19.27
23.98
NA not applicable
mass loss of 98.9 % (DTG peak at 198 °C). Other com-
pounds of all the series also exhibited the same stability
behavior.
against the five different human cancer cell lines, i.e.,
breast (MDA-MB-231 and MCF-7), cervical (HeLa), lung
(A549) and liver (HepG2) along with one normal (non-
cancerous) cell line at a concentration of 10 lM. The
results obtained from these screenings are summarized in
Table 1, which confirmed that twenty-one compounds
(IIIi–IIIo, Va–Vg, and VIIa–VIIg) exhibited the notice-
able anti-proliferative activity against all tested cancer cell
lines and they also showed very less or negligible effect on
In vitro cytotoxicity
The evaluation of in vitro cytotoxicity of synthesized
derivatives (IIIa–IIIo, Va–Vg, VIIa–VIIg and IXa–IXe)
have been calculated by MTT assay as primary screening
123

Med Chem Res
Table 2 IC50 (lM) values of in vitro anti-proliferative activity of active ferulic acid derivatives
Compound name
Breast (MCF-7)
Breast (MDA-MB-231)
Lung (A549)
Liver (HepG2)
Cervical (HeLa)
IIIi
12.88 3.96
09.87 2.97
17.29 1.96
11.98 3.12
11.42 3.77
13.09 4.38
13.44 2.76
09.17 3.47
07.49 3.22
09.23 3.69
08.38 3.64
09.74 3.09
08.06 3.09
08.12 3.88
13.05 3.72
15.81 3.65
15.28 3.37
12.78 3.88
15.27 3.33
16.55 3.19
16.17 3.22
14.08 2.59
12.97 3.41
16.91 2.76
12.28 3.88
13.06 4.22
14.32 3.72
14.91 3.23
08.99 3.52
07.29 2.72
07.66 3.36
07.48 3.01
09.89 3.71
10.34 3.19
08.99 3.13
12.67 3.72
17.64 3.81
16.01 3.22
12.49 2.82
17.04 2.98
13.14 3.64
12.74 3.86
13.45 3.35
13.44 4.02
12.09 3.72
12.18 4.12
12.22 5.03
12.55 3.09
13.77 3.54
07.84 2.89
08.13 3.11
07.21 3.53
07.11 3.48
07.48 4.52
08.11 3.72
08.04 3.92
09.77 3.72
09.19 3.53
09.16 3.21
08.98 3.56
11.67 4.01
10.84 3.09
09.54 2.64
15.91 3.98
16.14 3.88
14.61 3.19
15.17 3.21
16.82 4.05
15.49 3.13
18.28 3.34
10.59 4.27
09.88 4.67
10.21 4.37
09.31 4.05
10.41 3.86
11.08 4.12
10.37 3.79
15.92 4.29
13.54 4.78
12.13 4.09
11.05 3.82
14.36 2.98
13.02 3.44
13.11 3.73
10.97 3.79
12.07 4.01
12.26 3.81
12.05 4.04
13.71 3.51
12.83 2.92
15.82 4.43
07.51 3.84
08.75 3.72
08.06 4.19
07.29 3.55
07.14 2.68
08.98 3.18
09.65 4.27
09.89 3.72
10.89 3.72
09.74 3.98
09.53 3.09
10.62 3.76
09.87 2.97
09.17 4.12
IIIj
IIIk
IIIl
IIIm
IIIn
IIIo
Va
Vb
Vc
Vd
Ve
Vf
Vg
VIIa
VIIb
VIIc
VIId
VIIe
VIIf
VIIg
the normal stem cells while compounds IIIa–IIIh and
IXa–IXe were found to exhibit very low level of activity.
In comparison with their parent molecule, these amide
derivatives showed an increased level of anticancer activ-
ity. Further, these twenty-one compounds were studied for
the calculation of their half maximal inhibitory concen-
tration (IC50) values by serial dilution method, and the
results are given in Table 2. On seeing this table, we can
conclude that the range of IC50 values was laid between
07.11 to 18.28 lM in different kinds of studied cancer cell
lines. The amide derivatives which contain the moieties of
aliphatic and aromatic amines showed significant anti-
proliferative activity and a very low range of inhibitory
concentration but the compounds Va–Vg (containing
acridine moiety) showed the best activity as minimum
value of IC50 (07.11–11.08 lM) against all the studied cell
lines. The most promising compound against breast, lungs,
liver and cervical cancer cell lines was Vb (IC50 val-
ues = 7.49 3.22 and 7.29 2.72 lM), Vd (IC50 val-
ues = 7.11 3.48 & 8.31 4.05 lM) and Ve (IC50
values = 7.14 2.68 lM), respectively.
scavenging activity of a molecule. It possesses an unpaired
electron and produces a stable violet color in methanol
solution at 517 nm. The DPPH free radical is reduced into
DPPH-H in the presence of hydrogen donating molecule.
All the synthesized derivatives have been tested for their
antioxidant activity by DPPH assay, and their inhibition
constant (EC50) were calculated. The results of in vitro free
radical scavenging activities assay given in Table 3
revealed that nine compounds (IIIf, IIIl, IIIo, VIIe and
IXa–IXe) showed superior activity with EC50 ranges from
18.37 to 25.44 lM, as compared to the standard ascorbic
acid (EC50 = 20.14 lM) and isolated ferulic acid
(EC50 = 34.16 lM). Thirteen derivatives were found to
possess poor antioxidant activity having their EC50 values
C35 lM, while twelve compounds exhibited moderate
activity with the EC50 value between 26.89 and 34.81 lM.
The experiments were repeated for three times for repro-
ducing the obtained results and their statistical analyses.
Among all synthesized derivatives, IXa was found to be
the most active molecule during the DPPH assay with EC50
value of 18.37 2.74 lM. From the above results, it is
clear that the amide derivatives, like their parent molecule,
also showed the antioxidant activity. This is due to the
presence of phenoxy radical and resonance stabilization
which are also present in the ferulic acid. Compounds from
bis-amide series (IXa–IXe) have two phenolic OH in their
In vitro antioxidant activity
1,1-Diphenyl-2-Picrylhydrazyl radical (DPPH) is a well-
known chemical for the evaluation of free radical
123

Med Chem Res
Analysis of 3D-QSAR results
Table 3 EC50 (lg/ml) values of free radical scavenging activity of
active ferulic acid derivatives
Based on the results of in vitro biological activities, we
have developed a total of six (6) CoMFA models to find out
the structure activity relationship among the synthesized
amide derivatives of ferulic acid. The pictorial represen-
tation of each model has been shown in Fig. 2. These
models were further studied on the basis of contour maps
and mathematical factors such as steric-electrostatic con-
tributions, cross-validation coefficient (rc2v), non-cross-
validation coefficient (rn2cv), predictive correlation coeffi-
cient (rp2red) and the standard error of estimation (SEE). The
data used as input in the generation of CoMFA models are
provided as Table S2 and Fig. S36. The final values of
studied factors are summarized in Table 4, which was
generated after the statistical analysis of data. In the
CoMFA models for breast (Fig. 2a, b), lung (Fig. 2c) and
liver (Fig. 2d) cancer cells and DPPH assay (Fig. 2f)
showed significantly high steric contributions as compared
to electrostatic one, while in case of cervical cancer cells
(Fig. 2e) the electrostatic contribution is slightly higher
than steric interactions indicating their roles in the variation
of biological activities of synthesized derivatives. These
results are also confirmed by the statistical validation of
data (Table 4). In the CoMFA model for MCF-7 cells,
green contours were observed close to o-position and yel-
low contours around rest of the acridine ring indicated that
the increasing and decreasing steric bulk at these position,
respectively, would results in the enhanced bioactivities.
This is an established fact that compounds with higher
pIC50 values have bulky substituents at o-position of acri-
dine ring and no bulky group was noticed near other
positions of acridine ring. The two small red contours near
the central and un-substituted acridine rings positions were
observed, while blue contour was observed at substituted
bulky group of acridine ring. The red contour signifies the
presence of electron-rich regions, whereas the blue contour
corresponds to the regions where an increased positive
charge would result in boost up of bioactivity. In the MDA-
MB-231 CoMFA model, green contour was observed near
the o-position of acridine ring while a slight disagreement
was noticed for the yellow colored contours as compared to
MCF-7 cells. The number of bulky disfavored regions is
less for the MDA-MB-231 model. More importantly in
MCF-7 model, the m- and p-positions of the substituted
acridine ring are demonstrated with yellow contour indi-
cating it as sterically disfavored while the same site has
been demonstrated with green contours in MDA-MB-231
indicating that at these regions the steric bulk is favored for
increasing the biological activity. The similar region in the
models of A549 and HeLa cells was also demonstrated by
green contours (Fig. 2c, e). Unlike the breast cancer
models, these models disfavor steric bulk at the o-position
Compound
EC50
IIIa
IIIb
IIIc
46.83 3.48
31.54 2.74
38.85 3.49
36.28 2.94
47.29 3.27
23.39 3.84
28.11 2.72
34.28 2.46
28.65 2.79
26.94 3.26
42.83 3.62
19.87 3.32
29.78 3.71
28.19 3.63
25.44 3.67
36.55 3.62
45.58 4.28
48.41 3.54
49.95 4.34
35.24 3.72
41.17 3.82
37.96 3.68
27.14 3.57
37.73 2.97
33.12 2.83
34.81 2.79
23.64 3.73
32.78 2.96
27.43 3.37
18.37 2.74
20.09 2.77
21.81 2.56
20.89 3.52
24.68 2.69
34.16 2.52
16.64 1.51
IIId
IIIe
IIIf
IIIg
IIIh
IIIi
IIIj
IIIk
IIIl
IIIm
IIIn
IIIo
Va
Vb
Vc
Vd
Ve
Vf
Vg
VIIa
VIIb
VIIc
VIId
VIIe
VIIf
VIIg
IXa
IXb
IXc
IXd
IXe
Ferulic acid
Ascorbic acid
structures, so they can donate 2 protons with DPPH; thus,
these molecules were found better antioxidant agents
among all derivatives. These molecules were also found to
exhibit improved antioxidant activity as compared to cur-
cumin (EC50 = 29.99 0.39), a natural antioxidant also
having two phenolic OH (Shang et al., 2010). We have also
found a positive correlation between antioxidant and anti-
cancer activity of synthesized amides. Compounds (IIIf,
IIIl, IIIo, and VIIe) showed a noticeable increase in its
biological activities as compared to ferulic acid.
123

Med Chem Res
Fig. 2 STDDEV*COEFF plots of the CoMFA steric and electrostatic
contour maps for a MCF-7 b MDA-MB-231 c A549 d HepG2 e HeLa
f antioxidant models. (The most active molecule is displayed in the
background. Green region sterically favored, yellow region sterically
disfavored, red region negatively charged favored, and blue region
positively charged favored) (Color figure online)
of acridine ring. Moreover, positively charged substituents
are favored at the unsubstituted acridine ring (indicated by
blue contours) in both the models, the effect being more
pronounced in case of A549 cells. Positively charged
substituents are also favored near o-position (toward ferulic
acid) of unsubstituted acridine ring. In the HepG2 model,
steric contribution was found to be relatively higher than
electrostatic contribution and the same was also noticed in
contour maps (Fig. 2d). In this model, steric bulk is highly
disfavored around the acridine ring (as can be seen from
the cloud of yellow contour around the acridine ring).
Small blue contour was found around the o-position of
unsubstituted acridine ring, very similar to A549 and HeLa
cells CoMFA models, suggesting the importance of the
positively charged group. In the antioxidant model
(Fig. 2f), green and yellow contours were observed around
terminal ring and linker region, while blue contour around
–NH group of linker chain and red contour near carbonyl
group suggest the importance of positively and negatively
charged substitutions at these positions. On a comparative
scale, the negative charge substituents contributes more in
the breast cancer cell lines, while in all other cases very
small red contours were noticed.
Analysis of statistical results
The statistical analysis has been done by curve fitting
analysis to validate the experimental and theoretical data
obtained from biological and computational studies,
123

Med Chem Res
Antioxidant
˚
Table 4 CoMFA analysis for anticancer and antioxidant activities of ferulic acid derivatives (Grid spacing = 2.0 A)
Parameters
Breast (MCF-7)
Breast (MDA-MB-231)
Lung (A549)
Liver (HepG2)
Cervical (HeLa)
rc2v
rn2cv
0.712
0.966
0.025
4
0.711
0.949
0.031
4
0.858
0.991
0.011
4
0.767
0.934
0.25
0.816
0.859
0.039
5
0.583
0.903
0.044
4
SEE
ONC
rp2red
3
0.915
78.878
0.822
51.329
0.999
81.318
0.884
56.338
0.0814
85.620
0.792
48.960
F value
Field contributions
Steric
0.653
0.347
0.686
0.314
0.524
0.476
0.595
0.405
0.484
0.516
0.592
0.408
Electrostatic
rc2v cross-validation correlation coefficient, rn2cv non-cross-validation correlation coefficient, SEE standard error of estimation, ONC optimum
number of components, rp2red predictive correlation coefficient, F value F test value
respectively. All the experiments performed during anti-
cancer and antioxidant activity evaluations of synthesized
molecules have been repeated three times for the mini-
mization of error and statistical validation of data
(Table S3). Summary of statistical analysis showed that the
values of correlation coefficient (R) for all the studied
cases lied between 0.8699–0.9872 and 0.8554–0.9764 in
training and test sets, respectively. The plots for curve
fitting analysis are given in Fig. S36. Commenting on the
statistical results, we inferred that simulated values of
pIC50 statistically are in line with the experimental results
and outcome in all the cases is worthy. The reasonable
correlation between the experimental and predicted values
of biological activities of the training and test set com-
pounds would be used as a guide in designing of new
molecules as anticancer and antioxidant agents based on
ferulic acid.
in vitro free radical scavenging activity; nine compounds
were found to exhibit increased activity as compared to
ferulic acid. As it is a proven fact that oxidative stress leads
to many dreadful human diseases like cancer, we have
designed such kind of molecules which are acting both as
antioxidant and anticancer agent. It is envisaged that there is
a probable relationship between the observed anticancer
and antioxidant activity of the amide derivatives. These
compounds may serve as a lead molecule in quest of a vital
anticancer drug, which will also minimize the oxidative
stress started due to various physiological conditions in
cells. To further establish the correlation among experi-
mental results, the QSAR analyses were performed, and six
different CoMFA models have been generated and ana-
lyzed. The statistical analysis, contour maps analysis, and
comparison between steric and electrostatic field contribu-
tion were carried out to understand the correlation between
their chemical structure and biological activities. The
results of contour maps analyses aligned with the statistical
validation proves the accuracy of these models and statis-
tically significant correlation in all aforesaid discussed
cases. Also these outcomes could be used as a base model in
the designing of novel amide derivatives of natural pheno-
lics that will lead to a high-quality drug which acts both as
anticancer and antioxidant agent. Therefore, they would
lead as a possible pharmaceutical supplement.
Conclusions
Ferulic acid is an imperative natural lead molecule for
designing the compounds of biological interest. In this
work, thirty four amide derivatives of ferulic acid have been
synthesized under solvent-free conditions by microwave-
assisted condensation reaction, and fully characterized by
spectroscopic techniques. The stability of these amide
derivatives have been confirmed by thermal and spectro-
scopic results. After the successful structural and thermal
characterization, the derivatives have been screened for
their different biological applications. Twenty-one deriva-
tives (IIIi–IIIo, Va–Vg, and VIIa–VIIg) were found to
exhibit noticeable in vitro anticancer activity against all
studied cancer cell lines, while all the derivatives showed
negligible effect on normal cells. They were also tested for
Supplementary material
1
All the additional Figures (Figs. S1–S36) containing H-
NMR, 13C-NMR, mass spectra, Mass fragmentation pat-
tern, alignment of all molecules during 3D-QSAR, with
additional Tables (Tables S1–S3) are available in PDF
formats.
123

Med Chem Res
Acknowledgments Naresh Kumar gratefully acknowledges CSIR,
New Delhi, India, for financial assistance, Indian Institute of Tech-
nology Roorkee and NIPER Mohali for instrumentation facility.
Kumar N, Pruthi V (2014) Potential applications of ferulic acid from
natural sources. Biotechnol Rep 4:86–93
Kumar N, Pruthi V (2015) Structural elucidation and molecular
docking of ferulic acid from Parthenium hysterophorus possess-
ing COX-2 inhibition activity. 3. Biotech 5(4):541–551
Kumar S, Kumar N, Roy P, Sondhi SM (2014) Efficient synthesis of
heterocyclic compounds derived from 2,6-dioxopiperazine
derivatives and their evaluation for anti-inflammatory and
anticancer activities. Med Chem Res 23(9):3953–3969
Kumar N, Pruthi V, Goel N (2015) Structural, thermal and quantum
chemical studies of p-coumaric and caffeic acids. J Mol Struct
1085:242–248
Compliance with ethical standards
Conflict of interest Authors declare no conflict of interest.
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