Novel cinnamic acid derivatives as antioxidant and anticancer agents: Design, synthesis and modeling studies

Article abstract of DOI:10.3390/molecules19079655

Cinnamic acids have been identified as interesting compounds with antioxidant, anti-inflammatory and cytotoxic properties. In the present study, simple cinnamic acids were synthesized by Knoevenagel condensation reactions and evaluated for the above biological activities. Compound 4ii proved to be the most potent LOX inhibitor. Phenylsubstituted acids showed better inhibitory activity against soybean LOX, and it must be noted that compounds 4i and 3i with higher lipophilicity values resulted less active than compounds 2i and 1i. The compounds have shown very good activity in different antioxidant assays. The antitumor properties of these derivatives have been assessed by their 1/IC50 inhibitory values in the proliferation of HT-29, A-549, OAW-42, MDA-MB-231, HeLa and MRC-5 normal cell lines. The compounds presented low antitumor activity considering the IC₅₀ values attained for the cell lines, with the exception of compound 4ii. Molecular docking studies were carried out on cinnamic acid derivative 4ii and were found to be in accordance with our experimental biological results.

Full text of DOI:10.3390/molecules19079655

Molecules 2014, 19, 9655-9674; doi:10.3390/molecules19079655
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Novel Cinnamic Acid Derivatives as Antioxidant and Anticancer
Agents: Design, Synthesis and Modeling Studies
Eleni Pontiki 1,*, Dimitra Hadjipavlou-Litina 1,*, Konstantinos Litinas 2 and
George Geromichalos 3
1
Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences,
Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
2
Laboratory of Organic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki,
Thessaloniki 54124, Greece
3
Symeonidion Research Center, “Theagenion” Cancer Hospital, Thessaloniki, 54007, Greece
* Authors to whom correspondence should be addressed; E-Mails: epontiki@pharm.auth.gr (E.P.);
hadjipav@pharm.auth.gr (D.H.-L.); Tel.: +30-231-099-7627 (D.H.-L.);
Fax: +30-231-099-7679 (D.H.-L.).
Received: 5 May 2014; in revised form: 13 June 2014 / Accepted: 17 June 2014 /
Published: 7 July 2014
Abstract: Cinnamic acids have been identified as interesting compounds with antioxidant,
anti-inflammatory and cytotoxic properties. In the present study, simple cinnamic acids
were synthesized by Knoevenagel condensation reactions and evaluated for the above
biological activities. Compound 4ii proved to be the most potent LOX inhibitor. Phenyl-
substituted acids showed better inhibitory activity against soybean LOX, and it must be
noted that compounds 4i and 3i with higher lipophilicity values resulted less active than
compounds 2i and 1i. The compounds have shown very good activity in different
antioxidant assays. The antitumor properties of these derivatives have been assessed by their
1/IC50 inhibitory values in the proliferation of HT-29, A-549, OAW-42, MDA-MB-231,
HeLa and MRC-5 normal cell lines. The compounds presented low antitumor activity
considering the IC50 values attained for the cell lines, with the exception of compound 4ii.
Molecular docking studies were carried out on cinnamic acid derivative 4ii and were found
to be in accordance with our experimental biological results.
Keywords: antioxidant activity; inflammation; cancer; lipoxygenase; cinnamic acids;
molecular docking

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Abbreviations
AA: Arachidonic Acid; AAPH: 2,2'-azobis (2-amidinopropane) hydrochloride; ACPYPE:
AnteChamber PYthon Parser interface; clog P: Theoretically calculated lipophilicity; COX:
Cyclooxygenase; DPPH: 2,2-Diphenyl-1-picrylhydrazyl radical; LDL: Low-density lipoprotein; LOX:
Lipoxygenase; NBT: Nitroblue tetrazolium; ND: Neurodegenerative diseases; NDGA:
•
Nordihydroguaretic acid; OH: Hydroxyl radical; O2−·: Superoxide radical; ROS: Reactive oxygen
species; RPTLC: Reverse-phase thin layer chromatography; TCA: Trichloroacetic acid.
1. Introduction
Cancer has become the second major cause of death in developed countries and the clinical
prognosis remains relatively poor. During the last decade extensive research has been done in order to
define the mechanism by which continued oxidative stress could lead to cancer and chronic inflammation.
Although inflammation has long been recognized as a risk and causative factor for several types of
cancer, many of the molecular and cellular mechanisms involved still remain unclear. The functional
relationship between polyunsaturated fatty acid metabolism, inflammation, and carcinogenesis has
been extensively examined in numerous molecular studies, revealing potential novel targets like
arachidonic acid metabolizing enzymes, such as cyclooxygenases (COXs) and lipoxygenases
(LOXs) [1,2]. The formation by these enzymes of various lipid peroxides and bioactive lipids, free
radicals, and aldehydes can induce deleterious gene mutation and post-translational modifications
of key cancer-related proteins, implicated in the regulation of cellular proliferation, apoptosis,
differentiation, and senescence [3–5]. Especially lipoxygenase-catalyzed products may exert their
biological effects in an intracrine manner, through the activation of transcription factors of the
peroxisome proliferator-activated receptor/PPAR family, or may interact with specific trans-membrane
G protein-coupled cell surface receptors in an autocrine or paracrine manner [6,7].
High levels of ROS are able to modify essentially biological molecules, such lipids, proteins and
DNA. It is consistent that rates of ROS production are increased in most diseases [5]. Under “oxidative
stress” conditions, reactive oxygen species (ROS) in the form of superoxide anion, hydroxyl radical
and hydrogen peroxide attack various biological macromolecules (proteins, enzymes, DNA, etc.) or
indirectly may interfere with mechanisms of DNA repair [8]. Oxidative stress can activate signal
transduction pathways, inducing changes in a variety of transcription factors (nuclear factor/NF-κB,
activator proteion/AP-1, hypoxia-inducible factor-1α/HIF-1α, nuclear factor of activated T-cells and
NF-E2 related factor-2/Nrf2) leading to the expression of genes, including those for growth factors,
inflammatory cytokines, chemokines, cell cycle regulatory molecules, and anti-inflammatory molecules
that may induce a high perturbation in the intracellular and intercellular homeostasis, genetic instability,
proliferation, chemoresistance, radioresistance, invasion, angiogenesis, and stem cell survival [9].
It is of increasing interest that the treatment of the above-mentioned pathophysiological conditions
could benefit from the use of drugs that combine the above activities. The urgency and the growing
need for multifunction molecules designed to block multiple targets in cancer cells are well accepted.
During the last decade, natural products bearing the cinnamoyl moiety have attached much attention
due to their broad spectrum of biological activities and low toxicity. Additionally, trans-cinnamic acid

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derivatives, both isolated from plant sources or synthesized, are well known for their antioxidant [10],
antitumor [11], antimicrobial [12] and antimycobacterial properties [13]. Cinnamic acid derivatives,
especially those combining the cinnamoyl moiety with hydroxyl groups, present strong free radical
scavenging properties. Acids, esters, amides, hydrazides and related derivatives of cinnamic acid with
such activities are reported in the literature for their health benefits [14,15]. Especially, p-coumaric
acid or 4-hydroxy-trans-cinnamic acid presents antioxidant activity, involving direct scavenger of
reactive oxygen species (ROS) by minimizing the oxidation of low-density lipoprotein (LDL) [16].
Lipophilic hexylamides and hexylesters of cinnamic and hydrocinnamic acids as well as the
corresponding acid precursors, have been recently studied for their antioxidant profile and found to
play important role in neurodegenerative diseases (ND) due to their ability to cross the blood-brain
barrier [17]. Additionally, 2'-hydroxycinnamaldehyde and the analogue 2'-benzoyloxycinnamaldehyde
induce apoptosis in cancer cells via the induction of cellular reactive oxygen species (ROS) [18].
Recently antitumor activities of various cinnamic acid derivatives were explored by many research
groups [19–22].
In this study we have chosen to synthesize a series of substituted cinnamic acids based on the fact
that the cinnamoyl moiety has been found in a variety of biologically active substances, as already
mentioned. Previous studies of our group have shown that cytotoxic and antiinflammatory effects of
antioxidant cinnamic acids are associated with their pro-oxidant effects [23–26]. The antioxidant,
anti-inflammatory and anticancer properties of cinnamic acids are known to be influenced to a great
extent by the substitutions of the aryl ring and the double bond. In order to enhance the anticancer and
anti-inflammatory activity of these derivatives [25] a series of new cinnamic acids with the appropriate
substituents have been synthesized. These series have been evaluated for their: (a) antioxidant activity
in different assays (b) anticancer activity in different cell lines and (c) ability to inhibit
soybean lipoxygenase. Judging the in vitro results representative derivatives are further subjected to
modeling studies.
2. Results and Discussion
2.1. Chemistry
The synthesis of cinnamic acids was accomplished by a Knoevenagel condensation reaction as
already reported by us [24] and shown in Scheme 1. Acids of series i were derived from the
condensation of the suitable cinnamic aldehyde or not with 3-phenylacetic acid and acetic acid
anhydride in the presence of triethylamine, while 3-substituted-acrylic acids of series ii, were
derived from the condensation of the suitable aldehydes with malonic acid in the presence of pyridine
and piperidine.
Products 1i, 1ii, 2i, 2ii were obtained in satisfactory yields (55%–77%), 4i in 33% yield and
products 3i, 3ii and 4ii only in traces (Table 1). The pure final products were recrystallized from
1
13
ethanol/water. IR, H-NMR, C-NMR and elemental analysis were used for the confirmation of the
synthesized compounds structures (Table 1).

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Scheme 1. Synthesis of cinnamic acids.
Table 1. Chemical structures, physicochemical and reaction data of cinnamic acid
derivatives 1–4.
No
1i
Z
Y
Formula *
Rf
Clog P **
RM # (±SD)
Mp °C
Yield%
C16H11NO2
0.74 a
3.20
−0.895 (0.004) e 155–157
−0.972 (0.032) e 258–260
−0.706 (0.062) e 163–165
−0.868 (0.019) e 170–172
60
67
77
55
12
9
H
C10H7NO2
C17H13NO4
C11H9NO4
C17H14O2
C11H10O2
0.48 a
0.39 b
0.52 b
0.88 b
0.62 b
1.61
3.58
2.00
3.84
2.25
1ii
2i
H
H
2ii
3i
0.987 (0.018) e
−0.893 (0.002) e 163–165
0.566 (0.001) e
145–147
3ii
C17H13BrO2 0.85 c
4.09
135–136
33
3
4i
H
C11H9BrO2
0.89 d
2.51
−0.847 (0.044) e 169–171
4ii
a
b
c
C6H5:C2H5OH (1:1);
CH3COOCH2CH3:CHCl3:petroleum ether (2:1:1);
CHCl3, CH2Cl2:CH3COOC2H5 (2:1);
d CH3COOCH2CH3:CHCl3:petroleum spirit (2:1:1); e CH3OH:H2O:CH3COOH, (77: 23: 0.1); * Elemental analyses for molecular formula
(±0.4%), ** Theoretically calculated clog P values; # RM values are the average of at least 10 measurements.
The syntheses of compounds 1ii [27], 2ii [28] and 3ii [29] have been previously reported.
Compound 1i has been isolated in the cis- form by Chapman et al. [30] whereas no evidence for the
stereochemistry of 3i has been given [31,32].

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2.2. Physicochemical Studies
Lipophilicity represents a significant physicochemical property for the absorption, distribution,
metabolism and excretion of drugs (ADME properties). In this study the experimentally lipophilicity
as RM values is measured by the reverse-phase thin layer chromatography (RPTLC), a reliable, fast
and convenient method. The obtained values were compared with the corresponding log P values in
n-octanol-buffer estimated by ClogP [33,34]. It is well known that lipophilicity plays an important role
at the ADME properties. In the present study additionally, it influences the radical scavenging property
(i.e., in an aqueous phase) or the chain-breaking antioxidant activity (i.e., in biological membranes).
An attempt to correlate log P and RM values (Table 1) did not succeed. The different nature of the
hydrophilic and lipophilic phases used in the two systems is the main reason.
2.3. Biological Assays
In this study a series of eight cinnamic acids were synthesized, by application of standard synthetic
methods summarized in Scheme 1, expected to offer inhibition of LOX, protection against radical
attack and cytotoxicity. Antioxidants acting as lipid peroxidation inhibitors could offer for health’s
maintainance and to the compensation of risk factors [5]. Several different antioxidant protocols have
been reported and subjected in critical review [35]. They are all available to be used in order to assess
in vitro antioxidant ability. However, they usually give inconsistent and conflicting responses. Since
the antioxidant capacity of a compound must be evaluated in a variety of milieus, factors such
solubility or steric hindrance must be considered seriously. Also each assay is related to the generation
of a different radical.
The evaluation of the novel acids against soybean lipoxygenase LOX was accomplished by the
UV-based enzyme assay of Pontiki and Hadjipavlou-Litina [23–25]. This assay may be used as a
qualitative or semi-quantitative screen for such activity [36].
Study of LOX IC50 inhibition values demonstrates that compound 4ii is by far the most active
inhibitor, followed by compounds 1i and 2i (Table 2). It seems that the introduction of a phenyl ring at
position 2 favours the inhibitory activity, with the exception of compound 4ii. The rest of the
compounds presented moderate inhibitory activity. In compounds 1i, 2i, 3i and 4i the presence of a
conjugated double bond does not seem to offer to the activity. Thus, compound 1i is more potent than
the conjugated acids 2i, 3i, 4i. Compounds 3i and 4i seem to be equipotent. Thus, the replacement of
an H-atom by a Br-atom in position α does not influence lipoxygenase inhibition. On the contrary a
p-NO2 substituent offers to the inhibition (2i > 3i). Small differences were observed between 2ii and 3ii.
Most of the LOX inhibitors present antioxidant activity or act as free radical scavengers [37] since
lipoxygenation occurs via a carbon centered radical. It has been found that LOX inhibition is
correlated to the reducing ability of the inhibitors of the Fe3+ at the active site to the catalytically
inactive Fe2+ [38,39]. Several LOX inhibitors proved to be excellent ligants for Fe3+.
Lipophilicity is referred [23–25] to as an important physicochemical property for LOX inhibition.
However, herein highly lipophilic compounds 4i and 3i resulted less active than compounds 1i and 2i
(Table 2).

Molecules 2014, 19
Table 2. In vitro lipoxygenase (LOX) inhibitory activity of cinnamic acid derivatives 1–4.
9660
Percent interaction of the cinnamic acids 1–4 with the stable radical 1,1-diphenyl-
picrylhydrazyl (DPPH).
RA % 0.05 mM (±SD) b
RA % 0.1 mM (±SD) b
LOX a
IC50 (μM) (±SD) b
60 ± 0.4
Compd.
20 min
12 ± 0.3
13 ± 0.3
10 ± 0.04
11 ± 0.0.2
4 ± 0.02
11 ± 0.06
7 ± 0.03
17 ± 0.02
85 ± 1.6
60 min
14 ± 0.1
9 ± 0.03
11 ± 0.02
13 ± 0.04
3 ± 0.0
20 min
28 ± 0.6
23 ± 0.5
23 ± 0.7
29 ± 1.0
5 ± 0.0
60 min
20 ± 0.8
21 ± 1.0
23 ± 0.9
24 ± 0.6
6 ± 0.04
16 ± 0.05
5 ± 0.0
1i
1ii
27% ± 0.8 (0.01 mM)
66.5 ± 1.2
2i
2ii
31% ± 0.7 (0.01 mM)
74 ± 1.7
3i
3ii
24% ± 0.4 (0.01 mM)
74 ± 1.8
7 ± 0.0
20 ± 0.2
8 ± 0.03
30 ± 0.9
81 ± 0.8
4i
4 ± 0.01
13 ± 0.05
83 ± 1.1
4ii
10 ± 0.05
29 ± 0.6
NDGA
28 ± 0.4
83 ± 0.7
a Soybean lipoxygenase inhibition expressed as IC50 (μM); b Values are means ± SD of three or four different
determinations. Means within each column differ significantly (p < 0.05).
As already mentioned free radicals are highly implicated in lipoxygenase inhibition, inflammation
and cancer. Compounds possessing these activities in combination with antioxidant activity might be
protective against these diseases and lead to active and useful drugs, so it would be interesting to
evaluate these new acids for their antioxidant activities in comparison to well known antioxidants, i.e.,
nordihydroguaretic acid (NDGA), Trolox and caffeic acid (Tables 2–4).
Table 3. Radical scavenging activity of cinnamic acids derivatives 1–4 of hydroxyl (HO· %)
−·
and superoxide (O2 , %) radicals. Antioxidant activities of cinnamic acids 1–4 in ABTS+ -
decolorization (ABTS+ %) and inhibition of linoleic acid peroxidation (AAPH) assays.
−
ABTS+·(%)
AAPH (%)
·
O2 ·(%)
Compd.
HO (%) 0.1 mM (±SD) a
0.1 mM (±SD) a 0.1 mM (±SD) a 0.1 mM (±SD) a
1i
no
no
75 ± 1.0
26 ± 0.4
47 ± 0.8
26 ± 0.5
35 ± 0.9
26 ± 0.8
31 ± 1.1
24 ± 0.5
26 ± 0.4
88 ± 1.7
-
92 ± 1.2
20 ± 0.1
89 ± 0.9
29 ± 0.7
84 ± 1.4
30 ± 1.0
88 ± 1.8
63 ± 1.1
63 ± 1.3
-
1ii
50 ± 0.5
2i
33 ± 0.8
no
no
2ii
no
3i
81 ± 1.9
81 ± 1.2
no
no
3ii
4i
no
no
4ii
100 ± 1.8
73 ± 1.0
-
no
Trolox
-
Caffeic acid
Ascorbic Acid
46 ± 0.5
-
-
96 ± 0.9
-
No: No action under the used experimental conditions; a Values are means ± SD of three or four different
determinations. Means within each column differ significantly (p < 0.05).
Several assays should be used in order to assess in vitro antioxidant activity. In this way, factors
such as solubility or steric hindrance which may be of overriding importance in one environment but
not in another can be varied. The used methods are associated with the generation of various radicals.

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Two types of approach have been taken under consideration: (i) the assays in which we have the
scavenging by hydrogen- or electron donation of a preformed free radical as a marker of antioxidant
activity as well as assays (ii) involving the presence of antioxidant system during the generation of
the radical.
Table 4. Anticancer activity of cinnamic acids 1–4. IC50 refers to the concentration of the
compounds (in μM) required for 50% growth inhibition of human cancer cells of several
types. MRC-5 refers to normal cells.
IC50
IC50
IC50
IC50
IC50
IC50
Compd.
HT-29 (μM) A-549 (μM) OAW-42 (μM) MDA-MB-231 (μM) HeLa (μM) MRC-5 (μM)
1i
1ii
2i
>240
>>240
141 ± 1.8
>>240
>240
>240
>>240
>240
>>240
>240
>>240
>240
>>240
222 ± 1.3
>>240
134 ± 1.6
>>240
160 ± 1.1
>>240
199 ± 1.9
>>240
117.5 ± 1.4
240 ± 1.6
>240
96.3 ± 1.1
240 ± 1.0
>240
2ii
3i
>240
>240
>240
3ii
4i
>>240
>240
>>240
>>240
>>240
>>240
240 ± 1.8
92 ± 1.3
24 ± 0.6
210 ± 1.0
173.5 ± 1.4
174 ± 1.8
63.5 ± 0.8
250 ± 1.2
47.5 ± 0.7
80 ± 0.9
30 ± 0.4
4ii
54 ± 1.1
The novel acids were studied for the antioxidant activity by the use of the sTable 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH) at concentrations 0.05 and 0.1 mM after 20 and 60 min (Table 2) [23–25].
In the DPPH assay, the dominant chemical reaction involved is the reduction of the DPPH radical by
single electron transfer SET from the antioxidant. Phenolic compounds eg. NDGA, giving phenoxide
anions are effective antioxidants. The compounds presented reducing abilities which ranged nearly
29% (4ii), small differences were found among the compounds with time and concentration. It has
been found that phenyl-substituted acids present lower activity than the corresponding non-substituted
(2i < 2ii, 3i < 3ii, 4i < 4ii). Due to steric reasons the interaction of DPPH with the tested compounds
is low.
−·
Superoxide (O2 ) anion and hydroxyl radical (.OH) are free radical species of potential importance.
1
OH reactive radical and O2 are responsible for cytotoxicity. During inflammation, ˙OH radicals are
produced and are responsible for tissue damage.
Thus we evaluated our acids for their competition with DMSO for ˙OH radicals was measured,
hydroxyl radicals were produced by the Fe3+/ascorbic acid system and expressed as percent inhibition
of formaldehyde formation at 0.1 mM concentration (Table 3) [23–25]. Compounds 3i, 3ii, 4ii
presented high inhibitory activities of the oxidation of DMSO (33 mM) at 0.1 mM compared with the
reference compound Trolox, while compound 2i showed low inhibitory activity.
−
The superoxide anion radical (O2 ·) is less toxic than the hydroxyl radical, but still being one of the
most known toxic ROS. Two different experimental assays are followed for the determination of
superoxide anion radical scavenging activity: (i) one involves an enzymatic production of superoxide
anions and (ii) the other is supported by a non- enzymatic procedure. Herein the test is performed at
0.1 mM concentration, generating superoxide anion radicals by a non-enzymatically assay (Table 3).
Compound 1i presented the highest activity, followed by compound 1ii (75% and 50%, respectively).

Molecules 2014, 19
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The ABTS•+ is derived from the oxidation of ABTS by potassium persulfate. It is a decolorization
assay. The addition of electron-donating antioxidants leads to ABTS•+ reduction. The chemistry taking
place involves the direct generation of the ABTS•+ with no involvement of an intermediary radical.
The cation radical is formed prior to the addition of the antioxidant and does not take place continually
in the presence of the antioxidant. All compounds presented moderate activity with the exception of
compound 1ii (47.4%) (Table 3). For the above mentioned test the results taken do not define the role
of lipophilicity.
For the in vitro study of the free radical production, azo compounds generating free radicals through
spontaneous thermal decomposition are used. The water soluble 2,2'-azobis(2-amidinopropane)
hydrochloride (AAPH) is recommended as appropriate for measuring radical-scavenging activity
in vitro. The activity of the peroxyl radicals produced by the action of AAPH greatly resembles to
cellular activities such as lipid peroxidation. The phenyl substituted acids presented remarkable
activity at 0.1 mM concentration (84%–92%) compared to the non-substituted derivatives (20%–63%).
Compounds 1i, 2i, 3i, 4i with higher lipophilicity showed higher anti-lipid peroxidation response.
Epidemiological studies revealed the link between reactive oxygen species, inflammation and high
cancer risk. In order to diminish the pro-cancerous mechanisms a key treatment strategy is to reduce
the free radical load and consequently prevent potential damage to cellular compounds such as DNA,
proteins and lipids. Moreover, it has been found that LOX metabolism as well as arachidonic acid
metabolites play an important role in tumor progression and survival [40] and are implicated in the
etiology of mammary carcinogenesis [41]. Studies have demonstrated that levels of several eicosanoids
are increased in breast cancer in comparison to benign breast tumours [42,43]. Thus, LOX inhibitors
may have chemopreventive activity in lung carcinogenesis [44,45].
Thus, we conducted in vitro studies in two colon cancer cell lines (HT-29 and HCT-15) reported
time and dose-dependent stimulation of cell proliferation by LTB4 and 12-HETE [46]. The synthesized
acids have been tested in five different human tumour cell lines: HT-29 (colon), A-549 (lung), OAW-
42 (ovarian), MDA-MB-231 (breast), HeLa (immortal cells from cervical cancer) and MRC-5 normal
cells. On the basis of the in vitro testing results, most of these compounds with different substitution
patterns exhibited significant anticancer activity as depicted in Table 4.
For the HT-29 (colon cancer) compound 4ii presented remarkable anticancer activity, followed by
compound 2i. For the A-549 (lung) cell lines, compound 2i showed very good activity, followed by
compounds 4ii and 4i. For the OAW-42 (ovary cancer) compounds 4ii, 2i and 4ii possessed the better
inhibitory anticancer activity. For the MDA-MB-231 (breast) cells compound 4ii presented remarkable
anticancer activity, followed by compounds 2i and 4ii. For the HeLa cervical cancer cells compounds
4ii and 4i showed the higher activities, followed by compounds 2i and 2ii. For the rest of the
compounds the derived results were not satisfactory (>240 or >>240 μM ).
Normal lung MRC-5 cells found to be more resistant against the tested compounds (higher IC50
values). In all the cell lines 4-bromo-5-phenylpenta-2,4-dienoic acid (4ii) presents the best
anticancer activity.

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2.4. Computational Studies
2.4.1. Computational Methods, Docking Simulations
All the molecules were constructed with the ChemDraw program [47] and converted into
3D-Structures with the OpenBabel program [48], by using MMFF94 force field. Protein setup was
performed using the UCSF Chimera software [49,50]. Τhe AnteChamber PYthon Parser interfacE
(ACPYPE) tool [51] was employed to generate the topologies of the ligands. ACPYPE tool is written
in python to use Antechamber [52,53] to generate topologies for chemical compounds was used for the
parameterization of the ligands. Energy minimizations where carried out with the molecular simulation
toolkit GROMACS [54] using the AMBER99SB-ILDN force field [55].
Docking calculations were performed with the software Autodock Vina [56]. The PyRx
program [57] was employed to generate the docking input files and to analyze the docking results. The
proteins were considered rigid. Performing a blind docking the protein 1RRH soybean LOX, 4ii is
properly aligned in the binding site of LOX. The single bonds were considered as active torsional
bonds. An exhaustiveness value of 64 was used for the docking studies with a maximum output of 100
binding modes. The conformation results of ligands in the binding site of 1RRH are identical with the
ones in 1IK3. The docking results, as a set of solutions, are ranked according to their scoring function
values, defined by the 3D coordinates of its atoms and expressed as a PDB file.
2.4.2. Molecular Docking Studies on Lipoxygenase
The lack of structural data for human LOX, lead us to model human LOX using soybean enzyme
because of its availability and its well characterized structure [58]. For the docking studies we selected
4ii which presents a good combination of in vitro anti-LOX result. The possible mechanism of action,
as well as the differences in activity toward soybean LOX compared to the other derivatives could be
explained by docking calculations.
For the docking studies of LOX we have used the 1RRH (soybean lipoxygenase) accessible from
the Protein Data Bank (PDB) presenting a resolution of 2 Å [59]. We used the 1RRH from PDB with
Fe+3 running blind docking. Two soybean LOX models were derived from 1RRH. The first one has the
ligand taken from 1IK3 (ligand ID: 9OH) into the catalytic site while the other one for the blind
docking simulation has no ligand. The metal center in both models has charge q = +3 with no bond
restraint between the iron and the ligands. Lennard-Jones parameters for Fe (III) force field can be
resumed as: σvdw = 2.138157e-01 nm, εvdw = 2.092e-1 kJ/mol. The confirmation of the docking study
was accomplished with the crystallisation of the ligands in the protein of complex 1RRH. The results
are in accordance with the structure of the molecules in the active site of the protein.
The docking study was performed for all the compounds. or the visualization of the docking results
two compounds have been selected, 4ii presenting the best anti-LOX activity and 3ii, the simplest one
of the series, presenting the lowest activity. The docking orientations of compound 4ii are given in
Figure 1 and of compound 3ii in Figure 2. Compound 4ii presents significant high binding energy to
the protein (−6.8 kcal/mol) while compound 3ii presents (−6.2 kcal/mol). A number of H-bonds were
developed. These H-bonds were observed for compound 4ii between: (a) oxygen of the carbonyl group
with THR792 and (b) the oxygen of the hydroxyl group with SER793. For compound 3ii H-bonds

Molecules 2014, 19
9664
were developed between the oxygen of the carbonyl group with ARG535. Weak hydrophobic
interactions were also observed, supporting an even stronger binding of the compounds to the LOX cavity.
Comparing the G-score of the series to the in vitro results it is concluded that the docking cannot
really predict the in vitro activity under the reported experimental conditions, however it gives an idea
for the interactions between the compounds and the active site.
Figure 1. Docked poses of conjugate 4ii (Magenta) in the LOX binding site. Ball and stick
models are used to render the side-chains of relevant binding site residues. The iron ion is
depicted as an orange sphere.
Figure 2. Docked poses of conjugate 3ii (Purple) in the LOX binding site. Ball and stick
models are used to render the side-chains of relevant binding site residues. The iron ion is
depicted as an orange sphere.
3. Experimental
3.1. Materials and Instruments
All chemicals, solvents, chemical and biochemical reagents were of analytical grade and purchased
from commercial sources. Soybean lipoxygenase, linoleic acid sodium salt, arachidonic acid (AA)
were obtained from Sigma Chemical, Co. (St. Louis, MO, USA), 1,1-diphenyl-2-picrylhydrazyl
(DPPH), nordihydroguairetic acid (NDGA) are purchased from the Aldrich Chemical Co. (Milwaukee,
WI, USA).
Melting points (uncorrected) were determined on a MEL-Temp II (Lab. Devices, Holliston, MA,
USA). UV-Vis spectra were obtained on a Perkin-Elmer 554 double beam spectrophotometer and on a

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Hitachi U-2001 spectrophotometer. Infrared spectra (film as Nujol mulls) were recorded with
Perkin-Elmer 597 spectrophotometer (Perkin-Elmer Corporation Ltd., Lane Beaconsfield, Bucks,
1
England) and a Shimadzu FTIR-8101M. The H-Nucleic Magnetic Resonance (NMR) spectra were
recorded at 300 MHz on a Bruker AM 300 spectrometer (Bruker Analytische Messtechnik GmbH,
Rheinstetten, Germany) in CDCl3 or DMSO using tetramethylsilane as an internal standard unless
otherwise stated. 13C-NMR spectra were obtained at 75.5 MHz on a Bruker AM 300 spectrometer
in CDCl3 or DMSO solutions with tetramethylsilane as internal reference unless otherwise stated.
Chemical shifts are expressed in δ (ppm) and coupling constants J in Hz. Elemental analyses for C and
H gave values acceptably close to the theoretical values (±0.4%) in a Perkin-Elmer 240B CHN
analyzer. Reactions were monitored by thin-layer chromatography (TLC), on aluminum cards precoated
with 0.2 mm of silica gel and fluorescent indicator.
3.2. Chemistry General Procedure
3.2.1. Synthesis of Phenyl Substituted Cinnamic Acids 1i-4i [23–25]
Title compounds were prepared by a Knoevenangel reaction, as shown in Scheme 1. according to
literature methods [23–25]. A suitable aldehyde (0.015 mol) was condensed with phenylacetic acid
(0.015 mol) and acrylic acid anhydride (10 mL) in the presence of triethylamine (5 mL). The reaction
mixture was refluxed for 5 h. The solution was poured into 2 N HCl, and ice and the formed
precipitate was collected by filtration and recrystallized from 50% aqueous ethanol. In case that no
precipitate was formed an extraction with 3 × 100 mL CHCl3 was made and the organic phase was
collected, dried over MgSO4 and evaporated to dryness affording a residue that was recrystallized from
50% aqueous ethanol.
1
3-(4-Cyanophenyl)-2-phenylacrylic acid (1i) [30]. H-NMR (CDCl3): δ, (ppm) 7.39–7.46 (m, 7H), 7.51
(d, 1H, J = 9 Hz), 7.56 (s, 1H), 7.65 (d, 1H, J = 9 Hz) Anal. C, H, N. Calcd %: (C16H11NO2) C: 77.10, H:
4.45, N: 5.62 Found %: C: 76.96, H: 4.39, N: 5.68.
5-(4-Nitrophenyl)-2-phenylpenta-2,4-dienoic acid (2i). IR (Nujol) (cm−1): 3050–2940,1720, 1640
1H-NMR (CDCl3): δ, (ppm) 6.95–7.06 (m, 1H), 7.31–7.33 (m, 2H), 7.45–7.50 (m, 7H), 7.73 (d, 1H,
13
J = 9 Hz, phenyl), 8.16 (d, 1H, J = 9 Hz, Ph), 10.6 (s, 1H, COOH). C-NMR (CDCl3): 124.1 127.7,
128.2, 128.4, 128.7, 128.9, 129.9, 131.4, 134.2, 136.6, 140.1, 153.6, 163.4 Anal. C, H, N. Calcd %:
(C17H13NO4) C: 69.15, H: 4.44, N: 4.74, Found %: C: 69.2, H: 4.61, N: 4.35.
2,5-Diphenylpenta-2,4-dienoic acid (3i) [31,32]. 1H-NMR (CDCl3): δ, (ppm) 7.33–7.41 (m, 11H), 7.82
(d, 1H, J = 9 Hz, Ph), 7.94 (d, 1H, J = 9 Hz, Ph). Anal. C, H. Calcd %: (C17H14O2) C: 81.58, H: 5.64,
Found %: C: 81.46, H: 5.25.
4-Bromo-2,5-diphenylpenta-2,4-dienoic acid (4i). IR (Nujol) (cm−1): 3000–2900, 1750, 1630, 1480.
1H-NMR (CDCl3): δ, (ppm) 7.34–7.45 (m, 7H), 7.61–7.62 (b, 1H), 7.81–7.84 (m, 2H), 7.93–7.96 (m, 2H).
13C-NMR (CDCl3): 117.3, 126.2, 126.4, 127.8, 128.7, 128.8, 129.2, 129.9, 132.3, 133.3, 135.3, 137.2,
165.4. Anal. C, H. Calcd %: (C17H13BrO2) C: 62.03, H: 3.98, Found %: C: 62.05, H: 3.08.

Molecules 2014, 19
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3.2.2. General Procedure of the Synthesis of Cinnamic Acids 1ii–4ii [23–25]
The synthesis of the title compounds (Scheme 1) follows a Knoevenangel condensation of the
suitable heteroarylaldehyde (0.01 mol) with malonic acid (0.01 mol) dissolved in 1.12 mL of pyridine
and piperidine (0.01 mol). The mixture was refluxed until the emission of CO2 stopped. The reaction
solution was poured into 2N HCl and ice and the formed precipitate was collected by filtration and
recrystallized from water or from 3:1 water/ethanol. In the case that no precipitate was formed the
water phase was extracted with 3 × 100 mL CHCl3 or CH2Cl2 and the organic phase was collected,
dried over Mg2SO4, and evaporated to dryness affording a residue that was recrystallized from
aqueous ethanol.
1
3-(4-Cyanophenyl)acrylic acid (1ii) [27]. H-NMR (DMSO): δ, (ppm) 6.53 (d, 1H, J = 15 Hz,
CH=CH), 7.09 (d, 1H, J = 6 Hz), 7.43 (d, 1H, J = 6 Hz), 7.57–7.61 (s, 2H), 7.66 (d, 1H, J = 15 Hz,
CH=CH). Anal. C, H, N. Calcd %: (C10H7NO2) C: 69.36, H: 4.07, N: 8.09, Found %: C: 69.49, H:
4.23, N: 8.00.
1
5-(4-Nitrophenyl)penta-2,4-dienoic acid (2ii) [28]. H-NMR (DMSO): δ, (ppm) 6.09 (d, 1H, J = 12 Hz,
CH=CH=CH=CH), 6.93–7.14 (m, 2H, CH=CH=CH=CH), 7.42 (d, 1H, J =12 Hz, CH=CH=CH=CH),
7.66–8.21 (m, 4H, Ph). Anal. C, H, N. Calcd %: (C11H9NO4) C: 60.27, H: 4.14, N: 6.39, Found %: C:
59.95, H: 4.53, N: 6.79.
1
5-Phenylpenta-2,4-dienoic acid (3ii) [29]. H-NMR (CDCl3): δ, (ppm) 6.00 (d, 1H, J = 15 Hz,
CH=CH), 6.86–6.99 (m, 1H), 732–7.40 (m, 4H), 7.47–7.50 (m, 3H). Anal. C, H. Calcd %: (C11H10O2)
C: 75.84, H: 5.79, Found %: C: 76.08, H: 5.42.
1
4-Bromo-5-phenylpenta-2,4-dienoic acid (4ii). IR (Nujol) (cm−1): 3020–2900, 1750, 1650. H-NMR
(CDCl3): 7.34–7.66 (m, 4H), 7.81–7.90 (m, 2H), 7.99–8.02 (m, 2H), 9.33 (s, 1H). 13C-NMR (CDCl3):
117.2, 118.3, 127.9, 128.1, 128.6, 136.8, 143.8, 170.4. Anal. C, H, N. Calcd %: (C11H9BrO2) C: 52.20,
H: 3.58 Found %: C: 52.35, H: 3.62.
3.3. Physicochemical Studies
3.3.1. Determination of RM Values
Reversed phase TLC (RPTLC) was studied using silica gel plates saturated with 55% (v/v) liquid
paraffin in light petroleum ether and methanol/water mixture (77/23, v/v) containing 0.1% of acetic
acid was used as a mobile phase. Spots were detected under UV light or by iodine vapours. RM values
were determined by the corresponding Rf values (from ten individual measurements) using the
equation RM = log [(1/Rf) − 1] [23–25].
3.3.2. Estimation of Lipophilicity as Clog P
Bioloom of Biobyte Corp was used for the theoretical calculation of lipophilicity as Clog P values [34].

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3.4. Biological Experiments
3.4.1. Experiments in vitro
The in vitro assays were repeated at least in triplicate and the standard deviation of absorbance was
less than 10% of the mean.
3.4.1.1. Soybean Lipoxygenase Inhibition Study in vitro [23–25]
In vitro inhibitory LOX assay is accomplished as described previously. The novel derivatives for
testing (stock solutions 10 mM in DMSO) were incubated at room temperature with sodium linoleate
(0.1 mM) and 0.2 mL of LOX enzyme solution (1/9 × 10−4 w/v in saline). The conversion of sodium
linoleate to 13-hydroperoxylinoleic acid was measured at 234 nm and compared with the reference
inhibitor. Several concentrations were used for the IC50 determination (Table 2).
3.4.1.2. Interaction of the New Acrylic Acids with the Stable Radical 1,1-diphenyl-picrylhydrazyl
(DPPH) [23–25]
To a solution of DPPH in absolute ethanol the appropriate volume of the compounds (0.05 and
0.1 mM final concentrations) dissolved in DMSO was added. The absorbance was recorded at 517 nm
after 20 and 60 min at room temperature (Table 3).
3.4.1.3. Hydroxyl Radicals Scavenging Activity [23–25]
3+
The hydroxyl radicals were produced by the Fe 3+/ascorbic acid system. EDTA (0.1 mM), Fe
(167 μM), DMSO (33 mM) in phosphate buffer (50 mM, pH 7.4), the tested compounds (0.1 mM) and
ascorbic acid (10 mM) were mixed in test tubes. The solutions were incubated at 37 °C for 30 min.
The reaction was stopped by CCl3COOH (17% w/v) (Table 4) and the % scavenging activity of the
tested compounds for hydroxyl radicals was given.
3.4.1.4. Superoxide Radical Scavenging Activity [23–25]
Superoxide radicals were produced non-enzymatically by mixing PMS, NADH and air–oxygen.
The nitroblue tetrazolium method was used for the estimation of the produced radicals. The reaction
mixture containing compounds (0.1 mM), 3 μM PMS, 78 μM NADH, and 25 μM NBT in 19 μM
phosphate buffer pH 7.4 was incubated for 2 min at room temperature. The acrylic acids were
preincubated for 2 min before adding NADH (Table 4) and the absorption was recorded at 560 nm.
3.4.1.5. Inhibition of Linoleic Acid Peroxidation [25]
For initiating the free radical, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) is used. The
final solution in the UV cuvette consisted of ten microliters of the 16 mM linoleate sodium dispersion
0.93 mL of 0.05 M phosphate buffer, pH 7.4, thermostatted at 37 °C. 50 μL of 40 mM AAPH solution
was added as a free radical initiator at 37 °C under air and 10 μL of the tested compounds. The

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oxidation of linoleic acid sodium salt results a conjugated diene hydroperoxide. The reaction is
monitored at 234 nm (Table 3).
3.4.1.6. ABTS+-Decolorization Assay [25]
In this assay, ethanol is used for the dilution of the ABTS+• solution. ABTS is dissolved in water to
a 7 mM and reacts with 2.45 mM potassium phosphate to produce the ABTS radical cation (ABTS+•)
by allowing the mixture to stand in the dark at room temperature for 12–16 h before use. Ten μL of
diluted ABTS+• solution (734 nm) is added to 10 μL of antioxidant compounds or Trolox standards
(final concentration 0–0.1 mM) in ethanol and the absorbance is measured at 30 °C exactly 1 min after
the initial mixing (Table 3).
3.4.1.7. Cytotoxic Activity
Cell Lines and Culture Maintenance
Human cancer cell lines used as targets were HT-29 (colon), A-549 (lung), OAW-42 (ovarian),
MDA-MB-231 (breast), HeLa (immortal cells from cervical cancer). All cells were obtained from the
American Type Culture Collection (ATCC). Cells were routinely grown as monolayer cell cultures in
T-75 flasks (Costar) in an atmosphere containing 5% CO2 in air and 100% relative humidity at 37 °C
and subcultured twice a week, restricting the total number of cell passages below 20. The culture
medium used was Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) [60]
supplemented with 10% fetal bovine serum (Gibco), 2 mM glutamine (Sigma), 100 μg/mL streptomycin
and 100 IU/mL penicillin.
Trypan Blue Exclusion
The loss of membrane integrity, as a morphological characteristic for cell death, was assayed by
Trypan Blue exclusion [61]. The number of cells that were alive was estimated through a
haematocytometer and phase-contrast microscopy. Each result represented the mean of four
independent measurements and used for the inoculation of cells in the microplates.
Cell Inoculation–Drug Exposure–SRB Cytotoxicity Assay
Cell passages were carried out by detaching adherent, logarithmically growing cells after addition
of 2–3 mL of a mixture of 0.05% solution of trypsin (Gibco, 1:250) in phosphate-buffered saline
(PBS) with 0.02% EDTA and incubation for 3–5 min at 37 °C. For the experiments, cells were plated
(100 μL containing 10,000 cells/well) in 96-well flat-bottom microplates microplates (Costar-Corning,
Acton, MA 01720, USA) [62] so that untreated cells were in exponential growth phase at the time of
cytotoxicity evaluation. Cells were left for 24 h at 37 °C to resume exponential growth and stabilization
and afterwards exposed to tested agents for 48 h by the addition of an equal volume (100 μL) of either
complete culture medium (control wells), or twice the final drug concentrations diluted in complete
culture medium (test wells). Drug cytotoxicity was measured by means of the SRB colorimetric assay
estimating the survival fractions (SF) as the percent of control (untreated cells) absorbance. The SRB
assay was carried out as previously described [63] and modified by our group [64]. In brief, culture

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medium was aspirated prior to fixation using a microplate-multiwash device (Tri-Continent Scientific,
Inc., Grass Valley, CA, USA) and 50 μL of 10% cold (4 °C) trichloroacetic acid (TCA) were gently
added to the wells. Microplates were left for 30 min at 4 °C, washed five times with deionized water
and left to dry at room temperature for at least 24 h. Subsequently, 70 μL of 0.4% (w/v)
sulforhodamine B (Sigma) in 1% acetic acid solution were added to each well and left at room
temperature for 20 min. SRB was removed and the plates were washed five times with 1% acetic acid
before air drying. Bound SRB was solubilized with 200 μL of 10 mM unbuffered Tris-base solution
and plates were left on a plate shaker for at least 10 min. Absorbance was read in a 96-well plate
reader at 492 nm subtracting the background measurement at 620 nm. The test optical density (OD)
value was defined as the absorbance of each individual well, minus the blank value (“blank” is the
mean optical density of the background control wells, n = 8). Mean values and the coefficient of
variation (CV) from six replicate wells were calculated automatically. Results were expressed as the
“survival fraction” (SF), as shown below.
Calculation of Results
For each tested compound a dose-effect curve was produced. Sextuplicate determinations gave a
CV (Standard Deviation/mean %) of much less than 10%, resulting in standard error (SE) which was
very low in all cases. The data showing inhibition of cellular growth are expressed as the fraction of
cells that remains unaffected (fu) (survival fraction, SF), which is derived from the following equation:
fu = ODx/ODc
where ODx and ODc represent the test and the control optical density, respectively. Drug potency was
expressed in terms of IC50 values (50% inhibitory concentration) calculated from the plotted dose-
effect curves (through least-square regression analysis) (Table 4).
4. Conclusions
The present study shows that the synthesized cinnamic acids constitute a promising class of
antioxidant anticancer compounds. Docking studies support the in vitro anti-LOX activity indicating
the entrance of 4ii into the cavity of LOX. Compound 4ii presented a promising anti-cancer activity,
high LOX inhibitory activity as well as hydroxyl scavenging high and anti-LPO activities. Thus, acid
4ii might be used as a lead compound for the design of new multifunction agents.
Acknowledgements
We would like to thank A. Leo and Biobyte Corp. 201 West 4th Str., Suite 204, Claremont CA
California, 91711, USA for free access to the C-QSAR program. E. Pontiki would like to thank A.
Patsilinakos from the Department of Chemistry and Drug Technologies, “Sapienza” University of
Rome, Italy, and C. Kontogiorgis from the School of Pharmacy of Aristotle University of Thessaloniki
for their help on Molecular Docking studies and the visualization of the results. E. Pontiki is grateful to
the “Foundation for Education and European Culture” and Aristotle University of Thessaloniki for
financial support.

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Author Contributions
D.H.-L and E.P., are equally contributed in the writing, design, synthesis, biological evaluation and
analysis of the data. K.L. is involved in the NMR research whereas G.G. in the cytotoxicity assays. All
authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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