Design, synthesis and pharmacobiological evaluation of novel acrylic acid derivatives acting as lipoxygenase and cyclooxygenase-1 inhibitors with antioxidant and anti-inflammatory activities

Article abstract of DOI:10.1016/j.ejmech.2010.10.035

A series of novel acrylic acid derivatives bearing at the 3 position thienyl, furfuryl and 3,5-ditert-butyl-4-hydroxyphenyl substituents have been designed, synthesized and tested as potential dual lipoxygenase/cyclooxygenase-1 (LOX/COX-1) inhibitors and

Full text of DOI:10.1016/j.ejmech.2010.10.035

European Journal of Medicinal Chemistry 46 (2011) 191e200
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and pharmacobiological evaluation of novel acrylic acid
derivatives acting as lipoxygenase and cyclooxygenase-1 inhibitors with
antioxidant and anti-inflammatory activities
,
a
,
b
c
c
Eleni Pontiki a , Dimitra Hadjipavlou-Litina , Konstantinos Litinas , Orazio Nicolotti , Angelo Carotti
*
*
a Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of Thessaloniki, Thessaloniki 54124, Greece
b Laboratory of Organic Chemistry, Department of Chemistry, Aristotelian University of Thessaloniki, Thessaloniki 54124, Greece
c Dipartimento Farmaco-chimico, University of Bari, Via E. Orabona 4, I-70125 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel acrylic acid derivatives bearing at the 3 position thienyl, furfuryl and 3,5-ditert-butyl-4-
hydroxyphenyl substituents have been designed, synthesized and tested as potential dual lipoxygenase/
cyclooxygenase-1 (LOX/COX-1) inhibitors and as antioxidant and anti-inflammatory agents. Some
compounds have shown moderate antioxidant and COX-1 inhibitory activities, very good anti-inflammatory
activity and an inhibition of soybean lipoxygenase (LOX) higher than caffeic acid. In particular, compound
Received 6 May 2010
Received in revised form
14 October 2010
Accepted 29 October 2010
Available online 4 November 2010
4I disclosed a moderate in vitro LOX inhibition with an IC50 ¼ 100
m
M whereas compounds 1I and 2II
exhibited the best, albeit poor, activity as COX-1 inhibition (75% inhibition at 100
mM). Good radical
Keywords:
LOX
COX-1
Antioxidant agents
Anti-inflammatory agents
Lipoxygenase inhibitors
Cyclooxygenase inhibitors
Substituted acrylic acids
scavenging properties were shown by compounds 4I, 3I and 1II. Docking simulations performed on LOX
inhibitor 4I and COX-1 inhibitor 1I indicated that hydrophobic key interactions may govern the enzyme-
inhibitor binding.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
NSAID, is a selective COX-1 inhibitor whereas naproxen and indo-
methacin are non-selective COX inhibitors. In the late 1990s,
Arachidonic acid (AA) is metabolised to eicosanoids by the
cyclooxygenase (COX), lipoxygenase (LOX) and epoxygenase path-
ways [1].
selective COX-2 inhibitors (i.e, coxibes) have been introduced in
clinical trials for the treatment of inflammatory conditions without
causing gastric irritation. However, several coxibes (e.g., rofecoxib
and valdecoxib) have been withdrawn because they have been
associated with myocardial infraction and cardiovascular throm-
botic events [9e11]. COX-2-specific inhibitors, such as celecoxib
(CelebrexÒ) and rofecoxib (VioxxÒ) lack antiplatelet activity
because they do not inhibit the COX-1 isoform and therefore do not
inhibit thromboxane synthesis [12]. These thrombotic side effects
seem to be due to the decreased level of vasodilatory and anti-
aggregatory prostacyclin (PGI2) along with an increased level of the
prothrombotic platelet aggregator TXA2 [3,13e15]. In addition, it
has been pointed out that inhibiting COX pathway could shunt the
metabolism of AA toward the 5-LOX pathway [15], increasing the
formation of leukotrienes leading to inflammation. Unfortunately,
NSAIDs-induced important adverse effects e.g. asthma and
gastrointestinal damages, that limit their use [16e18].
In the COX pathway, the COX-1, COX-2, and COX-3 isoforms
convert AA to the hydroxyendoperoxide PGH2, which is further
metabolized to prostaglandins (PGs), prostacyclin (PGI2) and
thromboxane A2 (TXA2). In contrast, AA is initially converted to
hydroperoxyeicosatetraenoic acids (HPETE), subsequently to
hydroxyeicosatetraenoic acids (HETE) and then to the leukotrienes
(LTs) via the LOX pathway [2,3]. PGs and LTs produced in the COX
and LOX pathways, respectively, have been recognized as pro-
inflammatory mediators in numerous inflammatory diseases,
allergic disorders [4e6] proliferation and neoangiogenesis [7,8].
Many non-steroidal anti-inflammatory drugs (NSAIDs) were
found to inhibit these enzymes. Acetylsalicylic acid, a classical
Compounds that combine COX and LOX inhibition may present
multiple advantages because they synergistically block metabolic
pathways of arachidonic acid cascade and thrombosis and possess
* Corresponding author. Tel.: þ302310997627; fax: þ302310997679.
E-mail addresses: epontiki@pharm.auth.gr (E. Pontiki), hadjipav@pharm.auth.gr
(D. Hadjipavlou-Litina).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.035

192
E. Pontiki et al. / European Journal of Medicinal Chemistry 46 (2011) 191e200
Finally, docking studies were undertaken to gain insight into the
Z
Y
possible binding mode of the most active compounds in the LOX
and COX-1 binding sites.
C
C
H
C
O
X
2. Results and discussion
Y: -Ph, -H Z: different substituents X: -OH, -NHOH, -NHR
2.1. Chemistry
Fig. 1. Substituted acrylic acids and amides, inhibitors of soybean lipoxygenase with
antioxidant and anti-inflammatory activities.
The synthesis of the aryl-acrylic acids was accomplished
according to the Knoevenagel condensation indicated in Scheme 1.
The aryl-acrylic acids of series I were obtained by the condensation
of a suitable aldehyde with phenylacetic acid and acetic anhydride in
the presence of triethylamine, while the 3-substituted-acrylic acids
of series II were obtained by the condensation of the suitable alde-
hyde with malonic acid in the presence of pyridine and piperidine.
All the reactions proceeded smoothly and in good yields
(43e75%), with the only exception of compounds 1I and 4II which
were obtained in lower yields (28 and 27%, respectively). All the
compounds formed were recrystallized from ethanol/water. The
structures of the synthesized compounds, given in Table 1, were
confirmed by IR, 1H NMR, 13C NMR and elemental analysis. All the
acids present the characteristic absorption in the IR (nujol) (nearly
a wide range of anti-inflammatory activities [19]. It has been clearly
shown that dual inhibitors of the COX/LOX enzymatic pathways
offer a better alternative approach in designing new efficacious
drugs with excellent safety profile and least side effects. During the
last few years different groups have synthesized dual COX/LOX
inhibitors [13e15,20e23]. Moreau et al. [14,15] have presented
some aryl-acrylic acids as dual COX/LOX inhibitors while Abdellatif
et al. [24,25] as COX (COX-1/2) inhibitors. Recently, we have
reported a series of substituted acrylic acids and amides having
potent inhibitory activity against soybean lipoxygenase as well as
good antioxidant and anti-inflammatory activities (Fig. 1). These
analogues have been used in the present design and synthesis.
Some of the referred acrylic acids have also been tested as COX-1
inhibitors with promising results [26e29].
Starting from the observation that several pyrazoline, thio-
phene, di-tert-butylphenol, hydrazone, pyrrolidine and pyrazole
derivatives were potent dual 5-LOX and COX inhibitors [30], we
have designed a series of acrylic acid derivatives containing a 5-
membered heterocyclic and the 3,5-ditert-butyl-4-hydroxyphenyl
(DTBHP) substituents as potential dual COX/LOX inhibitor [31e48].
Indeed, DTBHP derivatives have been proved to be good oral anti-
inflammatories devoid of ulcerogenic drawbacks. Some compounds
(IeIV) showing dual COX/5-LOX activity are presented in Fig. 2
[43e48]. Compound S-2474 (III) in which the DTBHP moiety is
linked with a vinyl group to the heterocyclic ring is undergoing
clinical trials as an anti-arthritic drug [43,44]. Other studies have
shown that compounds possessing a propenone moiety, repre-
sented by V in Fig. 2, exhibit also dual COX/LOX inhibition [49].
Our design supported from our previous findings [26e29]
addressed new acrylic acid derivatives combining appropriate
structural and physicochemical features in order to inhibit soybean
lipoxygenase and cyclooxygenase-1 and to present both antioxi-
dant and anti-inflammatory activities. The synthesized compounds
were screened for free radical scavenging ability, in vitro soybean
LOX and COX-1 inhibition and in vivo anti-inflammatory activity.
3200 (OeH), 1720 (C]O), 1625 (C]C), cmꢀ1) and the expected 1
H
NMR signals for the E configuration of the double bond. In some
cases the H of the COOH group was not detectable. Spectral data are
consistent with the proposed structures and are in agreement with
previous findings (226e27, 29).
Compounds 1II [50,51], 2II [50] and 3II [51] were previously
reported, whereas compound 3I has been used as an intermediate
for the synthesis of naphtho[2,1-b]-furanecarboxylic acids by
oxidative photochemical cyclization [52]. Compound 4II was syn-
thesized according to our procedure [28,29], differing from the
Knoevenangel condensation reaction of 3,5-di-tert-butyl-benzalde-
hyde with malonic acid in dioxane reported by Munteanu et al. [53].
2.2. Physicochemical studies
Since lipophilicity is a significant physicochemical property for
the absorption, distribution, metabolism and excretion of drugs
(ADME properties), we measured it experimentally as RM
values
by the RPTLC (reverse-phase thin-layer chromatography) method
and compared it with the corresponding log P values in n-octanol-
buffer estimated by Clog P [54]. RPTLC is considered to be a reli-
able, fast and convenient method for expressing lipophilicity [55].
Apart from the important role of lipophilicity for the ADME
O
O
O
O
t-Bu
HO
t-Bu
HO
S
t-Bu
HO
N
N
S
N
S
OH
t-Bu
t-Bu
S-2474 (III)
NH2
t-Bu
CI-987 (II)
Darbufelone (I)
O
O
t-Bu
HO
X
X
O
(V)
t-Bu
X = O, S, CH=CH, CH=N
KME-4 (IV)
Fig. 2. Dual COX/LOX inhibitors.

E. Pontiki et al. / European Journal of Medicinal Chemistry 46 (2011) 191e200
193
CH COOH
Z
2
C
C
H
C
O
OH
I
Z CH
O
Z
H
)
OH
CO
H (
C
2
2
C
C
H
C
O
OH
II
Pyridine, Piperidine
CH
3
H C
3
CH
3
H C
3
H C
3
H C
O
3
Z:
S
S
HO
3
1
4
2
H C
3
CH
H C
3
3
Scheme 1. Synthesis of aryl-acrylic acid derivatives.
properties, also the radical scavenging property (i.e., in an aqueous
phase) or the chain-breaking antioxidant activity (i.e., in biological
membranes) both taken into account in the present study are
influenced by lipophilicity.
Unfortunately, the results showed in Table 1 indicated that RM
values did not result any sound correlation with log P and therefore,
in the present case, such parameters were unsuited for modelling
lipophilicity. The lack of correlation between log P and RM might be
due to the different nature of the hydrophilic and lipophilic phases
used in the two experimental systems.
compounds 3I and 4I were found to be more potent than the
corresponding monosubstituted derivatives 3II and 4II (Table 2). A
preliminary multiple regression analysis of anti-inflammatory
activity versus several physicochemical parameters (lipophilicity,
steric and electronic descriptors) of the examined compounds
revealed no significant correlation.
Compounds were evaluated for the inhibition of soybean lip-
oxygenase LOX by the UV-based enzyme assay of Taraborewala
et al. [58]. While one may not extrapolate the quantitative results of
this assay to the inhibition of mammalian 5-LOX, it has been shown
that inhibition of plant LOX activity by NSAIDs is qualitatively quite
similar to the inhibition observed on the rat mast cells LOX and this
assay may be used as a qualitative or semi-quantitative screen for
such activity.
2.3. Biological results and discussion
Protection against inflammation and radical attack, inhibition of
LOX and COX-1, and toxicity were measured as reported in the
Experimental Section.
Examination of LOX IC50 inhibition values shows that compound
4I is the most active inhibitor followed by compound 4II (Table 2).
Not surprisingly, the presence of the DTBHP group favours the
inhibitory activity.
In acute in vivo toxicity experiments, the examined compounds
did not present toxic effects in doses up to 0.5 mmol/kg body weight.
Acute inflammation is due to the release of chemical mediators,
which cause edema as a result of extravasations of fluid and
proteins from the local microvasculature and accumulation of
polymorphonuclear leukocytes at the inflammatory site. The in
vivo anti-inflammatory effects of the tested acrylic acids were
assessed by using the carrageenin-induced rat paw edema (CPE)
model and are presented in Table 2 as percentage of weight
decrease at the right hind paw. The induced edema is a non-
specific inflammation highly sensitive to NSAIDs and it is largely
accepted as a useful tool for studying new anti-inflammatory
agents [56]. It reliably assesses the anti-inflammatory potency of
the NSAIDs and detects during the second phase of inflammation
process that they act as anti-inflammatory agents by inhibiting
Most LOX inhibitors are antioxidants or free radical scavengers
[59], and some studies suggested a relationship between LOX
inhibition and the ability of the inhibitor to reduce the Fe3þ at the
active site to the catalytically inactive Fe2þ [60,61] LOXs contain
a “non-heme” iron per molecule in the enzyme active site as high-
spin Fe2þ in the native state and the high spin in the activated
state Fe3þ. Several LOX inhibitors proved to be excellent ligands
for Fe3þ. It has been demonstrated that their mechanism of action
is presumably related to their coordination with the catalytically
crucial Fe3þ
.
Although lipophilicity is referred to, as an important physico-
chemical property for LOX inhibition [25e28], no clear relationship
between LOX inhibitory activity and log P emerged for our
compounds. Nonetheless, it must be stressed that the highly lipo-
philic compounds 4I and 4II resulted the most active, while the less
lipophilic one, that is compound 3II was the less active inhibitor.
The introduction of a phenyl ring at position 2 led to improved
inhibitory activity with the exception of compound 1I (Table 2).
Compounds 2II and 1II exhibited the highest in vivo anti-
inflammatory activity in CPE model with a 77.5 and 63%, edema
prostaglandin amplification [57]. All the tested acids at 100
mM
concentration induced a significant protection against the CPE
ranging from 10 to 75% edema weight decrease with the exception
of 4II (2%) while the reference drug indomethacin induced a 47%
edema weight decrease at an equivalent dose. The presence of the
phenyl group at position 2 in the 2,3-disubstituted derivatives 3I
and 4I seems to favourably affect the biological response. In fact,

194
E. Pontiki et al. / European Journal of Medicinal Chemistry 46 (2011) 191e200
Table 1
Chemical structures, physicochemical and reaction data of aryl-acrylic acid derivatives 1e4
Z
Y
C
C
H
C
O
OH
.
Compd.
Z
Y
Formula*
Rf
Clog P**
R#M (ꢁSD)
mp ꢂC
yield%
28
CH3
1I
C14H12O2S
0.51a
0.87b
3.73
ꢀ0.563(0.040)d
198e200
S
S
CH3
1II
H
C8H8O2S
2.38
ꢀ0.461(0.038)d
174e175
59
H C
0.76c
0.87b
0.77c
0.83c
3.73
2.38
3.26
1.91
ꢀ0.553(0.022)d
ꢀ0.458(0.027)d
ꢀ0.583(0.014)d
ꢀ0.482(0.013)d
154e155
163e164
144e145
154e155
43
64
46
75
3
2I
C14H12O2S
C8H8O2S
C14H12O3
C8H8O3
S
H C
3
2II
3I
H
S
O
O
H C
3
H C
3
3II
H
CH
3
H C
3
H C
3
HO
4I
C23H28O3
0.71c
6.37
ꢀ0.540(0.011)d
145e147
72
H C
3
H C CH
3
3
CH
3
H C
3
H C
3
4II
HO
H
C17H24O3
0.86c
5.02
ꢀ0.133(0.005)d
95e97
27
H C
3
CH
H C
3
3
*Elemental analyses for molecular formula (ꢁ0.4%).
**Theoretically calculated clog P values.
#RM values are the average of at least 10 measurements.
a
CH3COOC2H5:CHCl3:epetroleum ether 40e65 ꢂC (2:1:1).
CHCl3.
CH2Cl2:CH3COOC2H5 (2:1).
CH3OH:H2O:CH3COOH, (77:23:0.1).
b
c
d
weight reduction respectively (Table 2). These values were even
higher than that observed with an equimolar amount of the
reference compound indomethacin (47%).
COX-1 plays a role as a “housekeeping enzyme”, for example
maintaining the lining of the stomach and in endothelial cells
contributing to the normal function of the cardiovascular system
via the release of prostacyclin. Thus, inhibition of COX-1 is involved
in the appearance of the unwanted side effects. Based on the above
we found interesting to examine inhibitory activity of compounds
towards COX-1 at 100 M (Table 2). They resulted moderately active
m
and no correlation was observed between the anti-inflammatory
activity and COX-1 inhibition. This is particularly evident for
compound 1I that exhibited the highest inhibition potency but
a weak anti-inflammatory activity in the rat (21% edema weight
reduction) and for compound 2I which had a good anti-inflam-
matory activity (53%) but presented a low COX-1 (12.5%).

E. Pontiki et al. / European Journal of Medicinal Chemistry 46 (2011) 191e200
195
Table 2
Table 4
Radical scavenging activity (%) of hydroxyl (HO,) and superoxide (Oꢀ2 ) radicals by
ꢃ
In vivo anti-inflammatory activity and in vitro lipoxygenase (LOX) and cycloxygenase
(COX-1) inhibitory activities of acrylic acid derivatives 1e4.
acrylic acid derivatives 1e4.
Compd
CPE (%)a
LOXb IC50
(
m
M)
COX-1c
Compd.
HOꢃ (%) 0.01 mM
HOꢃ 0.1 mM
Oꢀ2 (%) 0.1 mM (1 mM)
ꢃ
1I
1II
2I
2II
3I
3II
4I
4II
21.0**
63.0**
53.0*
77.5*
34.0*
10.0*
43.0*
2.0*
310
255
290
280
240
315
100
220
600
75
37.5
12.5
75
nt
nt
nt
nt
1I
1II
2I
2II
3I
3II
4I
4II
100
99.8
98.9
99.3
99.8
98.1
99.8
97.8
88.2
e
99.4
99.8
97.9
97.8
100
99.6
99.5
100
73.4
e
no (100.0)
57.1
no (100.0)
no (66.7)
50.0
14.3
85.7
35.7
e
Caffeic acid
SC-560
Trolox
Caffeic acid
(IC50 ¼ 7.8 nM)
46.5 (86.1)
100%
no: no action under the used experimental conditions.
Indomethacin
NDGA
47*
515
*p < 0.01.
such as the radical ꢃOH. Hydroxyl radicals are among the most
reactive oxygen species and are considered to be in part responsible
for tissue damage occurring in inflammation.
**p < 0.05.
a
% of reduction of the rat paw edema (CPE %) induced by carrageenin at the dose
of 0.01 mmol/Kg/body weight.
b
Soybean lipoxygenase inhibition expressed as IC50
(
m
M).
Therefore, a series of assays were performed to evaluate the
radical scavenging properties of our compounds. Their competition
c
Cyclooxygenase inhibitory activity at 100
Statistical data: student’s t-test.
mM, expressed as % of inhibition.
with DMSO for OH radicals generated by the Fe3þ/ascorbic acid
ꢃ
system expressed as percent inhibition of formaldehyde production
was used for the evaluation of their radical scavenging activity at
0.1 mM concentration (Table 4) [29]. All the compounds strongly
inhibited the oxidation of DMSO (33 mM) at 0.1 mM with an
activity higher than the reference compound Trolox.
An additional assay was performed to evaluate the radical scav-
enging property of our compounds, at 0.1 mM concentration, on
non-enzymatically generated superoxide anion radicals (Table 4).
Compound 4I presented the highest activity, followed by compound
1II (85.7 and 57.1%, respectively).
Mixing heme proteins with H2O2 generates powerful oxidizing
activated heme species and radicals on amino-acids side chains
that can cause lipid peroxidation. As a model of such reactions we
used the peroxidation of arachidonic acid by a mixture of heme and
H2O2 [29]. All the tested compounds significantly inhibited the lipid
peroxidation at 0.1 mM concentration with the exception of
compounds 1II, 2II and 3II (Table 5). The highest activity was
exhibited by compounds 4II bearing the DTBHP group.
It is well known that free radicals play an important role
in inflammatory action [62]. Consequently, compounds with anti-
oxidant properties could be expected to offer protection in
inflammation and lead to potentially effective drugs. In fact, many
non-steroidal anti-inflammatory drugs have been reported to act
either as radical scavengers or as inhibitors of free radical produc-
tion. Thus, we found interesting to test these derivatives for their
antioxidant ability in comparison to well known antioxidants, i.e.,
3,5-di-tert-butyl-4-hydroxy-toluene (DTBHP), nordihydroguaretic
acid (NDGA), trolox and caffeic acid (Tables 2e5) [63].
A number of assays have been used for the measurement of the
antioxidant activity [64e67]. Each method relates to the generation
of different radical, acting through a variety of mechanisms and the
measurement of a range of end points at a fixed time intervals. The
reducing abilities of the examined compounds were determined by
the use of the stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)
at 0.05 and 0.1 mM concentration [29] after 20 and 60 min (Table 3).
The reducing abilities of the tested compounds ranged nearly 37%
with compound 4II and small differences were observed among the
compounds with time and the concentration. As expected, the
DTBHP derivatives (4II and 4I) presented higher reducing ability.
It is well known that rates of reactive oxygen species (ROS)
production are increased in most diseases [68,69]. The cytotoxicity
Generation of the ABTS (2,20-azinobis-(3-ethyl-benzothiazoline-
6-sulfonic acid)) radical cation, formed the basis for a further
determination of the antioxidant activity of our compounds. All
compounds showed remarkable activity with the exception of
compound 1I (18.8%) (Table 5).
Azo compounds generating free radicals through spontaneous
thermal decomposition are useful for in vitro studies of free radical
production. The water soluble 2,20-azobis (2-amidinopropane)
hydrochloride (AAPH) has been extensively used as a clean and
controllable source of thermally produced alkylperoxyl free
of Oꢀ2 and H2O2 in living organisms is mainly due to their trans-
ꢃ
formation into ꢃOH reactive radical and 1O2. During the inflam-
matory process, phagocytes generate the superoxide anion radical
at the inflamed site and this is connected to other oxidizing species
Table 5
Antioxidant activities of acrylic acid derivatives 1e4 in the heme dependent lipid
Table 3
þꢃ
peroxidation (LP %), ABTSþ - decolorization (ABTS %) and inhibition of linoleic acid
ꢃ
Reducing ability (RA %) of acrylic acid derivatives 1e4.
peroxidation (AAPH) assays.
Compd.
RA % 0.05 mM
RA % 0.1 mM
RA % 0.2 mM
ABTSþ % 0.1 mM
AAPH% 0.1 mM
ꢃ
Compd
LP% 1 mM
20 min
60 min
20 min
60 min
20 min
60 min
1I
1II
2I
2II
3I
3II
4I
4II
35.5
1.2
55.8
1.2
49.2
7.9
39.3
54.1
nt
18.8
41.7
41.1
44.2
65.6
37.1
60.3
53.9
88.0
95.8
20.1
64.9
37.5
73.4
32.6
74.9
60.1
74.8
63.0
nt
1I
1II
2I
2II
3I
3II
4I
1.7
3.2
3.6
3.5
3.2
4.8
2.8
28.1
84.8
no
no
5.9
3.2
6.7
4.4
3.6
8.6
7.6
31.6
81.0
2.8
2.5
4.9
4.3
4.8
3.4
9.7
9.3
6.4
10.0
9.6
17.3
47.7
90.3
7.2
7.2
7.3
2.6
6.5
2.9
1.0
1.4
3.0
5.3
26.9
83.1
7.6
6.7
12.2
36.9
82.6
66.2
26.5
94.6
4II
NDGA
Trolox
Ascorbic Acid
nt
no: no activity under the used experimental conditions.
nt: not tested.

196
E. Pontiki et al. / European Journal of Medicinal Chemistry 46 (2011) 191e200
radicals. Also this assay was performed to better characterize the
antioxidant activity of our compounds. All compounds presented
a good activity at 0.1 mM concentration (20.1e74.9%).
2.4. Docking studies
Even if the observed LOX and COX-1 inhibitory affinities of the
analyzed acrylic acids were quite low, we decided to perform some
docking simulations on the most active compounds to get some
clues on the nature and type of interactions governing the inhibitor
binding [70,71].
Simulations were carried out with GOLD, a software based on
a genetic algorithm to explore the ligand conformational space [72].
Docking poses were obtained by applying both Chemscore and
Goldscore, fitness functions available for scoring. As easily inter-
pretable results were obtained on the basis of a recently published
work [73], all the results reported in the present paper are referred
to the Chemscore fitness function. The coordinates for the reference
protein were obtained from 1IK3 (soybean lipoxygenase) and 1Q4G
(ovine cyclooxygenase-1) available from the Protein Data Bank
(PDB) with a resolution of 2 Å [74]. These complexes were prepared
for docking studies by adding hydrogen atoms, removing water and
co-crystallized inhibitors and refined by using the Protein Prepa-
ration tool implemented in Maestro 7.5 [75]. Enzyme-inhibitor
interactions within a radius equal to 12 Å centered on reported
bound inhibitors were taken into account.
The docking simulation of the most active compound 4I toward
LOX showed that the enzyme-inhibitor complex was stabilized by
hydrophobic interactions occurring between the aromatic moieties
of the ligand and lipophilic residues of the binding site. In parti-
cular the 3,5-di-tert-butyl-4-hydroxy-phenyl group was oriented
towards the hydrophobic region lined by Leu565, Ile572, Val566,
Leu515 and Trp519 while the 2-phenyl moiety protruded into
a hydrophobic cavity formed by residuesLeu277, Ile772, Thr274,
Leu273, Ile857 and Ile557. In addition, the carboxylic functional
Fig. 4. Representation of the top-scored docking pose of compound 1I, rendered as
a stick model, into the COX-1 binding site (PDB code 1Q4G). Side-chains of relevant
binding site residues are rendered as ball and stick models. A possible HB is indicated
with a dashed line.
group was supposed to coordinate the iron ion as it showed
a similar structural arrangement compared to the peroxide group
of the co-crystallized ligand (Fig. 3).
The docking top-score binding pose indicated the availability of
some room in the para position of the 3,5-di-tert-butyl-4-hydroxy-
phenyl ring but the removal of the OH group might eliminate the
good antioxidant activity of this compound. However, room for
medium-sized substituents could be found at position 2 or on the
2-phenyl ring of the acrylic acid derivatives.
As far as the COX-1 inhibition is concerned, the most active
inhibitor 1I was docked into the enzymatic binding site. It could
establish many hydrophobic interactions with its 2-phenyl ring
leaned towards Phe381 and Tyr385 and the 3-methylthiophen ring
pointing towards Leu531, Val116 and Leu359. Finally the carboxylic
group could establish an H-bond interaction with the NH group of
the backbone of Ser353. In this case, structural modifications
aiming at the improvement of binding affinity could be made at
position 3 where larger room may be available for hydrophobic
substituents (Fig. 4).
3. Conclusions
The present study showed that certain novel acrylic acid
derivatives possess a high anti-inflammatory activity. Moreover,
ꢃ
most of them proved to be potent OH scavengers, inhibited lipid
peroxidation and in vitro soybean LOX, and exhibited a good anti-
oxidant activities in a variety of tests. The high antiradical in vitro
activity of the tested compounds supported, at least in part, the
good in vivo anti-inflammatory activity.
The most active compounds docked successfully into the active
site of the inhibited enzymes. Inhibitory activity of the most potent
compounds was explained mostly by hydrophobic interactions.
Two compounds, that are 1II and 2II, were found to present
a promising anti-inflammatory profile with a high antiedematous
activity, significant inhibitory activity on LOX and COX-1 and good
hydroxyl radical scavenging activity. However, the inhibition of
Fig. 3. Representation of the top-scored docking pose of compound 4I, rendered as
a stick model, into the soybean LOX binding site (PDB code 1IK3). Side-chains of
relevant binding site residues are rendered as ball and stick models. The iron ion is
rendered as a gray sphere.

E. Pontiki et al. / European Journal of Medicinal Chemistry 46 (2011) 191e200
197
both COX-1 and LOX enzymes was generally very low. Since inhi-
bition of COX-1 is involved in the appearance of the unwanted side
effects, this dual inhibition might produce a protective effect as
well as a reduction in inflammation. Some of the synthesized
compounds might constitute initial leads for the design of new
more potent therapeutics against inflammation. This design could
be guided by the insights gained by our preliminary docking studies
on LOX and COX active sites and by our previous results [26e29].
Starting from these data, further investigations will be carried out
to gain insight into the mechanism of antiphlogistic activity of the
examined compounds.
12.4, 122.5, 125.9,126.0, 128.6, 129.1,129.6, 130.2, 133.5,133.6,134.8,
138.5, 139.0, 168.9; Anal. C, H, N. Calcd %: (C14H12O2S) C: 68.83, H:
4.95; Found %: C: 68.98, H: 4.77.
4.1.1.2. (E)-3-(5-Methyl-thiophen-2-yl)-2-phenylacrylic acid (2I). IR
(Nujol) (cmꢀ1): 3175, 2961e2876, 1730e1713, 1615, 1385; 1H NMR
(CDCl3): d, (ppm) 2.33 (3H, s, CH3), 6.62e7.62 (8H, ms), 11.28 (1H, s,
COOH); Anal. C, H, N. Calcd %: (C14H12O2S) C: 68.83, H: 4.95; Found
%: C: 68.82, H: 4.73.
4.1.1.3. (E)-3-(5-Methyl-furan-2-yl)-2-phenylacrylic acid (3
I). IR
(Nujol) (cm-1): 3175, 2961e2897, 1700e1679, 1662, 1388; 1H NMR
4. Experimental section
(CDCl3): d, (ppm) 2.21 (3H, s, CH3), 5.77e5.89 (2H, m), 7.26e7.75
(6H, ms); 13C NMR (CDCl3): 13.7, 109.1, 117.3, 126.0, 127.9, 128.6,
129.2, 129.3, 130.0, 135.0, 139.0, 149.0, 155.0, 168.0; Anal. C, H, N.
Expected %: (C14H12O3) C: 73.67, H: 5.30; Found %: C: 73.94, H: 4.98.
Materials: All chemicals, solvents and reagents were of analy-
tical grade and purchased from commercial sources. Soybean Lip-
oxygenase and porcine heme were obtained from Sigma Chemical,
Co. (St. Louis, MO, USA). The kit for COX activity assay was
purchased by Cayman. For the in vivo experiments, male and female
Fischer-344 rats (180e240 g) were used.
4.1.1.4. (E)-3-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-phenylacrylic
acid (4
NMR (CDCl3):
I
). IR (Nujol) (cm-1): 3196e2940, 2870, 1730, 1653, 1380; 1H
d
, (ppm) 1.08e1.37 (18H, m, 6xCH3), 7.06e7.61 (8H,
Synthesis: All starting materials were obtained from commercial
sources and used without further purification. Melting Points
(uncorrected) were determined on a MEL-Temp II (Lab. Devices,
Holliston, MA, USA). UVeVis spectra were obtained on a Per-
kineElmer 554 double beam spectrophotometer and on a Hitachi
U-2001 spectrophotometer. Infrared spectra (film as Nujol mulls)
were recorded with PerkineElmer 597 spectrophotometer (Per-
kineElmer Corporation Ltd., Lane Beaconsfield, Bucks, England)
and a Shimadzu FTIR-8101M. The 1H Nucleic Magnetic Resonance
(NMR) spectra were recorded at 300 MHz on a Bruker AM 300
spectrometer (Bruker Analytische Messtechnik GmbH, Rheinstet-
ten, 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. Representative 13C NMR spectra are given.
m), 7.93 (1H, s, OH); Anal. C, H, N. Calcd %: (C23H28O3) C: 78.38,
H: 8.01; Found %: C: 78.06, H: 8.25.
4.2. General procedure for the synthesis of 3-heteroaryl-acrylic
acids 1e4II [26e29]
Title compounds were prepared, as illustrated in Scheme 1, by
a Knoevenangel condensation of the suitable heteroarylaldehyde
with malonic acid. Malonic acid (0.01 mol) was dissolved in 1.12 mL
of pyridine and the heteroaldehyde (0.01 mol) and piperidine
(0.01 mol) were added. The mixture was refluxed until the emission
of CO2 stopped. Then the solution was poured into 2 N HCl and then
on ice. The formed precipitate was collected by filtration and
recrystallized from water or from 3:1 water/ethanol mixture. If no
precipitate was formed an extraction with 3 ꢄ 100 mL CHCl3 or
CH2Cl2 was made and the organic phase was collected, dried over
MgSO4, and evaporated to dryness affording a residue that was
recrystallized from aqueous ethanol.
Chemical shifts are expressed in d (ppm) and coupling constants J in
Hz. Mass spectra were determined on a VG-250 spectrometer (VG-
Labs., Tritech, England) with ionization energy maintained at 70 eV.
Elemental analyses for C and H gave values acceptably close to the
theoretical values (ꢁ0.4%) in a PerkineElmer 240B CHN analyzer.
Reactions were monitored by thin-layer chromatography (TLC), on
aluminum cards precoated with 0.2 mm of silica gel and a fluores-
cent indicator.
4.2.1. (E)-3-(3-Methylthiophen-2-yl)acrylic acid (1II
)
IR (Nujol) (cmꢀ1): 3100e2900,1720,1650,1380; 1H NMR (CDCl3):
, (ppm) 2.58 (3H, s, CH3), 6.18 (1H, d, J ¼ 15 Hz, CH]CH), 6.89 (1H, d,
d
J ¼ 6 Hz), 7.31 (1H, d, J ¼ 6 Hz), 7.96 (1H, d, J ¼ 15 Hz); 13C NMR (CDCl3):
14.4,114.9,128.0,131.4,133.6,137.9,142.3,172.3; Anal. C, H, N. Calcd %:
(C8H8O2S) C: 57.12, H: 4.79; Found %: C: 57.31, H: 4.89.
4.1. General procedure
4.2.2. (E)-3-(5-Methylthiophen-2-yl)acrylic acid (2II
IR (Nujol) (cmꢀ1): 3100, 2961e2876, 1773e1753, 1655, 1387; 1H
NMR (CDCl3):
CH), 6.74 (1H, d, J ¼ 3 Hz), 7.09 (1H, d, J ¼ 3 Hz), 7.80 (1H, d, J ¼ 15 Hz,
CH]CH); 13C NMR (CDCl3): 15.9, 119.9, 126.7, 127.9, 132.4, 139.7,
144.0, 171.0; Anal. C, H, N. Calcd %: (C8H8O2S) C: 57.12, H: 4.79;
Found %: C: 57.51, H: 4.89.
)
4.1.1. Synthesis of substituted 2-phenyl-3-heteroaryl-acrylic acids
1Ie4I
d
, (ppm) 2.58 (3H, s, CH3), 6.09 (1H, d, J ¼ 15 Hz, CH]
Title compounds were prepared by a Knoevenangel reaction, as
illustrated in Scheme 1, according to literature methods [26e29]. A
suitable aldehyde (0.015 mol) was condensed with phenylacrylic
acid(0.015 mol) andacrylic acidanhydride (10 mL) inthe presence of
triethylamine (5 mL). The reaction mixture was refluxed for 5 h. The
solution was poured into 2 N HCl, then on ice and the formed
precipitate was collected by filtration and recrystallized from 50%
aqueous ethanol. In case that no precipitate was formed an extrac-
tion with 3 ꢄ 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.
4.2.3. (E)-3-(5-Methylfuran-2-yl)acrylic acid (3II
IR (Nujol) (cmꢀ1): 3100, 2900e2950, 1750e1700; 1H NMR
(DMSO-d6, CDCl3):
)
d
, (ppm) 2.36 (3H, s, CH3), 6.10 (1H, d, J ¼ 3 Hz),
6.23 (1H, d, J ¼ 15 Hz, CH]CH), 6.56 (1H, d, J ¼ 3 Hz), 7.45 (1H, d,
J ¼ 15 Hz, CH]CH), 8.27 (1H, s, COOH); 13C NMR (CDCl3): 13.9,
109.0, 112.7, 117.5, 133.0, 141.0, 149.3, 171.6; Anal. C, H, N. Calcd %:
(C8H8O3) C: 63.15, H: 5.30; Found %: C: 63.14, H: 5.43.
4.1.1.1. (E)-3-(3-Methyl-thiophen-2-yl)-2-phenylacrylic acid (1I). IR
(Nujol) (cmꢀ1): 3150, 2918e2705, 1720, 1666, 1606, 1384; 1H NMR
4.2.4. (E)-3-(3,5-Di-tert-butyl-4-hydroxyphenyl)acrylic acid (4II
IR (Nujol) (cmꢀ1): 3175, 2940e2854, 1740, 1687e1615; 1H NMR
(CDCl3): , (ppm) 1.37e1.43 (18H, m, 6xCH3), 6.51 (1H, s, OH),
)
(CDCl3 ):
d
, (ppm) 2.40 (3H, s, CH3), 6.80 (1H, d, J ¼ 6 Hz), 7.16 (1H, d,
J ¼ 6 Hz), 7.28e7.47 (6H, ms), 11.00 (1H, s, COOH); 13C NMR (CDCl3 ):
d

198
E. Pontiki et al. / European Journal of Medicinal Chemistry 46 (2011) 191e200
7.09e7.15 (1H, d, J ¼ 18 Hz, CH]CH), 7.22e7.84 (3H, m); Anal. C, H,
absorbance at 611 nm. The absorption due to the spontaneous
oxidation of TMPD was subtracted from the initial rate of oxidation
observed in presence of AA. The inhibition of the compounds at
0.1 mM concentration was determined after preincubation for
6 min with the enzyme in the presence of heme and TMPD and the
reaction was started by adding AA. SC-560 has been used as
a reference COX-1 inhibitor (Table 2).
N. Calcd %: (C17H24O3) C: 73.88, H: 8.75; Found %: C: 73.85, H: 8.42.
4.3. Physicochemical studies
4.3.1. Determination of RM values
Reversed phase TLC (RPTLC) was performed on silica gel plates
impregnated with 55% (v/v) liquid paraffin in light petroleum ether.
The mobile phase was a methanol/water mixture (77/23, v/v) con-
taining 0.1% of acetic acid. The plates were developed in closed
chromatography tanks saturated with the mobile phase at 24 ꢂC.
Spots were detected under UV light or by iodine vapours. RM values
were determined from the corresponding Rf values (from ten indi-
vidual measurements) using the equation RM ¼ log [(1/Rf)ꢀ1] [29].
CF3
Cl
N
N
OCH3
4.3.2. Estimation of lipophilicity as Clog P
Lipophilicity was theoretically calculated as Clog P values in n-
octanol-buffer by CLOGP Programme of Biobyte Corp [76].
SC-560
4.4. Biological experiments
4.4.2.3. Determination of the reducing activity of the stable radical
1,1-diphenyl-picrylhydrazyl (DPPH) [27,29]. To a solution of DPPH in
absolute ethanol an equal volume of the compounds dissolved in
DMSO was added. A stock solution (10 mM) for the compounds was
used, the concentrations of the final solutions of the compounds
were 0.05, 0.1 and 0.2 mM. After 20 and 60 min at room temper-
ature the absorbance was recorded at 517 nm (Table 3).
4.4.1. Experiments in vivo
4.4.1.1. Inhibition of the carrageenin-induced edema [27,29]. Edema
was induced in the right hind paw of Fisher 344 rats (150e200 g) by
the intradermal injection of 0.1 mL 2% carrageenin in water. Both
sexes were used. Females pregnant were excluded. Each group was
composed of 6e15 animals. The animals, which have been bred in
our laboratory, were housed under standard conditions and
received a diet of commercial food pellets and water ad libitum
during the maintenance but they were entirely fasted during the
experiment period. Our studies were carried out in accordance with
recognised guidelines on animal experimentation.
4.4.2.4. Competition of the tested compounds with DMSO for
hydroxyl radicals [27,29]. The hydroxyl radicals generated by the
Fe3þ/ascorbic acid system, were detected by the determination of
formaldehyde produced from the oxidation of DMSO. The reaction
mixture contained EDTA (0.1 mM), Fe3þ (167
mM), DMSO (33 mM)
The tested compounds 0.01 mmol/kg body weight, were sus-
pended in water, with few drops of Tween 80 and ground in
a mortar before use and were given intraperitoneally simulta-
neously with the carrageenin injection. The rats were euthanized
3.5 h after carrageenin injection. The difference between the
weight of the injected and not-injected paws was calculated for
each animal. The change in paw weight was compared with that in
control animals (treated with water) and expressed as a percent
inhibition of the edema (CPE % values in Table 2). Indomethacin
0.01 mmol/kg was used as reference compound. CPE % are the mean
values from two different experiments with a standard error of the
mean less than 10%.
in phosphate buffer (50 mM, pH 7.4), the tested compounds and
ascorbic acid (10 mM). After 30 min of incubation at 37ꢂ C the
reaction was stopped with CCl3COOH (17% w/v) and the % compe-
tition of the tested compounds with DMSO for hydroxyl radicals
was determined (Table 4).
4.4.2.5. Non enzymatic assay of superoxide radical scavenging
activity [27,29]. The superoxide producing system was set up by
mixing PMS, NADH and aireoxygen. The production of superoxide
was estimated by the nitroblue tetrazolium method. The reaction
mixture containing compounds, 3
mM PMS, 78 mM NADH, and
25 M NBT in 19 M phosphate buffer pH 7.4 was incubated for
m
m
2 min at room temperature and the absorption measured at 560 nm
against a blank containing PMS. The tested compounds were pre-
incubated for 2 min before adding NADH and the % superoxide
radical scavenging activity was determined (Table 4).
4.4.2. Experiments in vitro
In the in vitro assays each experiment was performed at least in
triplicate and the standard deviation of absorbance was less than
10% of the mean.
4.4.2.6. Heme protein-dependent lipid degradation [27,29]. 50
mM
4.4.2.1. Soybean lipoxygenase inhibition study in vitro [27,29]. In
vitro study was evaluated as reported previously. The tested
compounds dissolved in ethanol were incubated at room temper-
ature with sodium linoleate (0.1 mM) and 0.2 mL of enzyme solu-
tion (1/9 ꢄ 104 w/v in saline). The conversion of sodium linoleate to
13-hydroperoxylinoleic acid at 234 nm was recorded and compared
with the appropriate standard inhibitor (Table 2).
heme, arachidonic acid (0.4 mM) the compounds at the various
concentrations tested, H2O2 (0.5 mM) were incubated together for
10 min at 37 ꢂC in KH2PO4eKOH buffer (50 mM, pH 7.4). The
product of peroxidation was detected using the TBA test. The
compounds were added in DMSO solution, which has no effect on
the assay (Table 5) and % heme protein-dependent lipid degrada-
tion was determined.
4.4.2.2. In vitro cyclooxygenase-1 (COX-1) inhibition study [27].
Cyclooxygenase (COX) activity was determined by using arach-
idonic acid (AA) as substrate and N,N,N,N-tetramethylphenylene-
diamine (TMPD) as co-substrate. The reaction mixture (1 mL)
4.4.2.7. ABTSþ$ - decolorization assay for antioxidant activity [77].
ABTS was dissolved in water to a 7 mM concentration. ABTS radical
cation (ABTSþ$) was produced by reacting ABTS stock solution with
2.45 mM potassium persulfate and allowing the mixture to stand in
the dark at room temperature for 12e16 h before use. For the
present study, the ABTSþ$ solution was diluted with ethanol to an
absorbance of 0.70 at 734 nm and equilibrated at 30 ꢂC. Stock
contained 0.75
mM heme, 128 mM TMPD, 80 mM AA and 1.5 mg
enzyme, in 0.1 M Tris/HCl (pH 8.5). The oxidation of substrate was
measured, at room temperature by monitoring the increase of

E. Pontiki et al. / European Journal of Medicinal Chemistry 46 (2011) 191e200
199
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solutions of the tested compounds in DMSO were diluted so that,
after introduction of a 10 mL aliquot of each dilution into the assay,
they produced between 20% and 80% inhibition of the blank
absorbance. After addition of 1.0 mL of diluted ABTSþ$ solution
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l
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m
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initiator. This assay can be used to follow oxidative changes and to
determine the inhibition of linoleic acid peroxidation induced by
each tested compound.
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Pontiki Eleni is grateful to the “Foundation for Education and
European Culture” for financial support and to the Research
Committee of the Aristotle University of Thessaloniki for a Schol-
arship. We also wish to thank Dr C. Hansch and Biobyte Corp. 201
West 4th Str., Suite 204, Claremont CA California, 91711, USA for free
access to the C-QSAR program
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