Targeting inflammation with conjugated cinnamic amides, ethers and esters

Article abstract of DOI:10.2174/1570180816666181129125511

Background: Cinnamic acid is a key intermediate in shikimate and phenylpropanoid pathways. It is found both in free form, and especially in the form of esters in various essential oils, resins and balsams which are very important intermediates in the biosynthetic pathway of several natural products. The cinnamic derivatives play a vital role in the formation of commercially important intermediate molecules which are necessary for the production of different bioactive compounds and drugs. Different substitutions on basic moiety lead to various biological activities. Furthermore, combination of appropriate pharmacophore groups with cinnamic acid derivatives were developed to give hybrids in order to find out promising drug candidates as inhibitors of multiple biological targets associated with inflammation. We found interesting to continue our efforts to design and synthesise three series of novel cinnamic acid-based hybrids: a) nitrooxy esters of cinnamic acid, b) ethers and c) amides of cinnamic acids with arginine, as pleiotropic candidates against multiple targets of inflammation Methods: The synthesis of cinnamic was established by a Knoevenagel-Doebner condensation of the suitable aldehyde either with malonic acid in the presence of pyridine and piperidine, or with phenylacetic acid in the precence of triethylamine in acetic anhydride. The synthesis of the corresponding esters was conducted in two steps. The ethers were synthesized in low yields, with 1,2 – dibromoethane in dry acetone, in the presence of K₂CO₃, to give oily products. The corresponding cinnamic amides were synthesised in a single step. The synthesised hybrids were tested as lipoxygenase (LOX) and cyclooxygenase (COX) inhibitors in vitro. In silico docking was applied to all the novel derivatives. Several molecular properties of the hybrids were calculated in order to evaluate their drug likeness. Results: A number of esters, ethers and amides of selected cinnamic acids, either phenyl substituted or not, has been synthesised and subjected to modelling studies. The compounds were studied in vitro/in vivo for their inhibitory activities on cox and lox, and as antioxidants. Log P values of all the title compounds except of 3a (5.38) were found to be less than 5 and are in agreement to Lipinski’s rule of five, suggesting satisfactory permeability across cell membrane. The molecular modelling study seems to be in accordance with the experimental results for LOX and COX-2. The result of antioxidant activity for amide 3b supports the anti-lox activity. Compound 5d presents the higher in vivo anti-inflammatory. Conclusion: According to the experimental findings compounds 3b and 5d can be used as lead compounds for the design of new molecules to target inflammation.

Full text of DOI:10.2174/1570180816666181129125511

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3
Letters in Drug Design & Discovery, 2020, 17, 3-11
RESEARCH ARTICLE
ISSN: 1570-1808
eISSN: 1875-628X
Targeting Inflammation with Conjugated Cinnamic Amides, Ethers and
Esters
Impact
Factor:
0.953
Ioannis Fotopoulos1, Eleni Pontiki1 and Dimitra Hadjipavlou Litina1,*
1Department of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences, Aristotle University of
Thessaloniki, Thessaloniki 54124, Greece
Abstract: Background: Cinnamic acid is a key intermediate in shikimate and phenylpropanoid
pathways. It is found both in free form, and especially in the form of esters in various essential oils,
resins and balsams which are very important intermediates in the biosynthetic pathway of several
natural products. The cinnamic derivatives play a vital role in the formation of commercially im-
portant intermediate molecules which are necessary for the production of different bioactive com-
pounds and drugs. Different substitutions on basic moiety lead to various biological activities. Fur-
thermore, combination of appropriate pharmacophore groups with cinnamic acid derivatives were
developed to give hybrids in order to find out promising drug candidates as inhibitors of multiple
biological targets associated with inflammation. We found interesting to continue our efforts to de-
sign and synthesise three series of novel cinnamic acid-based hybrids: a) nitrooxy esters of cinnamic
acid, b) ethers and c) amides of cinnamic acids with arginine, as pleiotropic candidates against mul-
tiple targets of inflammation
A R T I C L E H I S T O R Y
Methods: The synthesis of cinnamic was established by a Knoevenagel-Doebner condensation of
the suitable aldehyde either with malonic acid in the presence of pyridine and piperidine, or with
phenylacetic acid in the precence of triethylamine in acetic anhydride. The synthesis of the corre-
Received: September 18, 2018
Revised: November 1, 2018
sponding esters was conducted in two steps. The ethers were synthesized in low yields, with 1,2 –
Accepted: November 26, 2018
dibromoethane in dry acetone, in the presence of K2CO3, to give oily products. The corresponding
DOI:
cinnamic amides were synthesised in a single step. The synthesised hybrids were tested as lipoxy-
10.2174/1570180816666181129125511
ꢀ
genase (LOX) and cyclooxygenase (COX) inhibitors in vitro. In silico docking was applied to all the
novel derivatives. Several molecular properties of the hybrids were calculated in order to evaluate
their drug likeness.
Results: A number of esters, ethers and amides of selected cinnamic acids, either phenyl substituted
or not, has been synthesised and subjected to modelling studies. The compounds were studied in
vitro/in vivo for their inhibitory activities on cox and lox, and as antioxidants. Log P values of all the
title compounds except of 3a (5.38) were found to be less than 5 and are in agreement to Lipinski’s
rule of five, suggesting satisfactory permeability across cell membrane. The molecular modelling
study seems to be in accordance with the experimental results for LOX and COX-2. The result of
antioxidant activity for amide 3b supports the anti-lox activity. Compound 5d presents the higher in
vivo anti-inflammatory.
Conclusion: According to the experimental findings compounds 3b and 5d can be used as lead
compounds for the design of new molecules to target inflammation.
Keywords: Cinnamic esters, cinnamic amides, cyclooxygenase, lipoxygenase, drug-likeness, modelling.
1. INTRODUCTION
important intermediates in the biosynthetic pathway of sev-
eral natural products. The cinnamic derivatives play a vital
role in the formation of commercially important intermediate
molecules which are necessary for the production of differ-
ent bioactive compounds and drugs. Cinnamic acid deriva-
tives present a wide range of biological activities: Antituber-
culosis [1], antidiabetic [2], antioxidant [3] antimicrobial [4],
hepatoprotective [5], Central Nervous System Stimulant
(CNS) [6], antidepressant [7], anticholesterolemic [8], anti-
malarial [9], antiviral [10], anxiolytic [11], cytotoxic [12]
Cinnamic acid is a key intermediate in the shikimate and
phenylpropanoid pathways. It is found both in free form, and
especially in the form of esters (ethyl, cinnamyl, benzyl), and
in various essential oils, resins and balsams which are very
*Address correspondence to this author at the Department of Pharmaceuti-
cal Chemistry, School of Pharmacy, Faculty of Health Sciences, Aristotle
University of Thessaloniki, Thessaloniki 54124, Greece;
Tel/Fax: +302310997627; E-mail: hadjipav@pharm.auth.gr
1875-628X/20 $65.00+.00
©2020 Bentham Science Publishers

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Letters in Drug Design & Discovery, 2020, Vol. 17, No. 1
Fotopoulos et al.
O
OH
O
H
N
NH2
Ar
N
H
R
NH
1b - 5b
v)
O
O
O
iii)
ii)
i)
Cl
ONO2
Ar
O
Ar
O
Ar
OH
Ar
O
R
R
R
1a - 5a
1- 5
O
O
O
OH
iii)
OH
iv)
O2NO
OH
O
Br
O
5c
HO
ii)
O
O
ONO2
Cl
iii)
O
O
iv)
O
Cl
O2NO
Br
O
O
O
5d
HO
Ar =
R =
H
HO
Scheme (1). Reagents and Conditions: i) malonic acid, cat. piperidine, pyridine or phenylacetic acid, cat. TEA, Ac2O, ii) 1 chlorethanol,
DCC, cat. DMAP, DCM (dry), iii) NH4NO3, MeCN, iv) 1,2 - dibromoethane, K2CO3, Acetone (dry). v) L – Arginine hydrochloride, BOP,
TEA, DCM (dry).
NH
O
O
OH
O
O
i)
H
N
H2N
N
H
OH
+
NH2
Ar
OH
Ar
N
H
NH3
R
Cl
R
NH
1b - 5b
Scheme (2). Reagents and Conditions: i) BOP, TEA, DCM (dry).
and anti-inflammatory [13]. Furthermore, the combination of
appropriate pharmacophore groups with suitable substituted
cinnamic acids led to conjugates with anti-inflammatory
activities [3]. In recent years, intensive research on cinnam-
ic acid derivatives has been conducted in order to create
new multifunctional drugs [14, 15]. We designed and
synthesised [16-20] several cinnamic acids as potent lipoxy
genase (LOX) and cyclooxygenase (COX-2) inhibitors, anti-
oxidants and anti-inflammatories over the last decade. L-
arginine is characterized as a semi-essential amino acid in-
fluencing the stages of development and health. The infants
cannot synthesize L-arginine thus rendering it an essential ami-
no acid. Furthermore, there is a need for arginine in inflammato-
ry cases such as burns, certain surgeries and sepsis [21].
possesses superoxide scavenging activity and is able to delay
the cell-mediated breakdown of NO as well as to reduce the
oxidation of lipoproteins [23].
Previously reported [17] potent anti-LOX cinnamic acids
e.g. the cinnamic acid, the p-coumaric acid and the naphthyl
substituted acid were used for the synthesis of the esters,
ethers and amides. In this light, we esterified our cinnamic
acids with 2-nitrooxyethanol. Furthermore, we synthesized
some ethers of p-coumaric acid as pleiotropic candidates
against multiple targets of inflammation [24, 25]. These new
derivatives can be divided into three categories: a) nitrooxy
esters of cinnamic acids, b) ethers and c) amides of cinnamic
acids with arginine, and have been evaluated for their: a) anti-
oxidant activity [26] b) in vitro ability to inhibit soybean
lipoxygenase [16], c) in vitro ability to inhibit cyclooxygenase-2
[27] and d) in vivo anti-inflammatory activity using the carra-
geenin mice paw edema [28, 29] (Supplementary material).
2. MATERIALS AND METHODS
L-Arginine is a precursor of endogenous nitric oxide
(NO) [22]. It was also proved that exogenous L-arginine

Targeting Inflammation with Conjugated Cinnamic Amides
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 1
5
We used modelling studies and previous QSAR results
[30] as a tool in our design for LOX inhibitors. The in silico
results supported the synthesis of the compounds.
ues calculated with C-QSAR (as clog P) to the RM values for
the esters (Tables 1 and 2). The following equation describes
this correlation:
The drug-likeness of the derivatives was determined from
the theoretical calculation of various molecular properties
e.g. partition coefficient (log P), Topological Polar Surface
Area (TPSA), hydrogen bond donors and acceptors, rotatable
bonds, number of atoms, molecular weight. The violations of
Lipinski’s rule of five were considered [30].
RM = 0.225 (0.128) clog P - 1.492 (0.511)
N =6, r = 0.926, r2 = 0.857, q2 = 0.627, s = 0.106, F1,4
24.09, α = 0.01
=
We did not succeed to correlate the corresponding RM to
clog P values for the amides. This may be due to the limited
number of compounds (4) and the different nature of the hy-
drophilic and lipophilic phases used within the two systems.
3. RESULTS AND DISCUSSION
The design of the new multitarget cinnamic derivatives
was based on our previous research [25] combining the moi-
ety of the enoyl-acyl backbone part, the amide or the ester
linkage. (Schemes 1 and 2). Variations were accomplished
by the choice of suitable substituted cinnamic acids which
showed interesting biological activity in our previous work
[25].
The synthesis of cinnamic acids 1, 2, 3, and 4 was estab-
lished by a Knoevenagel-Doebner condensation of the suitable
aldehyde either with malonic acid in the presence of pyridine
and piperidine, or with phenylacetic acid in the presence of
triethylamine in acetic anhydride, as shown in Scheme 1 [25].
The physicochemical and spectroscopic data of the obtained
acids are identical to those given in the literature.
Several molecular properties of the derivatives were the-
oretically calculated in order to evaluate their drug-likeness
[34]. All the compounds have a molecular weight less than
500. Thus, these molecules could be easily transported, dif-
fused and absorbed. Counting the number of hydrogen bond
acceptors (O and N atoms) and the number of hydrogen bond
donors (NH and OH) in the synthesized compounds, it seems
that both properties follow the Lipinski’s rule of five (less
than 10 and 5 respectively). Within the series of the exam-
ined derivatives, compounds 1a-5a and 5c seem to be orally
active in accordance to Lipinski’s rule of five. The hydrogen
bonding of the compounds is highly correlated to the topo-
logical polar surface area. This property is used as a signifi-
cant indicator of the bioavailability of a bioactive molecule.
The TPSA of the derivatives was observed in the range of
81.36-148.53 Å and is well below the limit of 160 Å, indicat-
ing good oral bioavailability. The upper limit for TPSA for a
molecule to penetrate the brain is around 90 Å. The Log P
values of all the title compounds except of 3a (5.38) were
found to be less than 5 and are in agreement to Lipinski’s
rule of five, suggesting satisfactory permeability across the
cell membrane. The in silico predicted values [36], point that
compounds with logBB values more than 0.3 are consid-
ered to be highly absorbed through BBB whereas values
between 0.3 to - 0.1 and less than -0.1 are considered to be
limited transported through BBB. Our findings do not sup-
port the permeability of these hybrids through BBB
(Table 3).
The synthesis of the corresponding esters was conducted
in two steps. The intermediate 2 chlor ethyl ester [31] of the
appropriate acid reacts with ammonium nitrate to give the
corresponding alkyl nitro-ester. The ethers were synthesised
in low yields, with 1,2 - dibromoethane in dry acetone, in the
presence of K2CO3, to give oily products. The corresponding
cinnamic amides were synthesized in one pot by the reaction
of the appropriate acid with
L Arginine HCl and
(Benzotriazol-1-yloxy) tris (dimethylamino) phosphonium
hexafluorophosphate (BOP) as coupling reagent [32] Com-
pound 1b was synthesized for the first time by our group,
while so far in the literature [33] only its isolation from natu-
ral sources has been described. The final amide products
were obtained in medium to good yields (50-92%).
The synthesized derivatives were expected to offer inhi-
bition of lipoxygenase (LOX), cyclooxygenase (COX-2),
protection against radical attack and in vivo anti-
inflammatory activity. The compounds were tested as lipox-
ygenase and cyclooxygenase inhibitors in vitro. Both en-
zymes catalyze the arachidonic acid metabolism giving
chemical mediators of inflammation. Free radicals are highly
implicated with lipoxygenase inhibition and inflammation
Thus, we used the water-soluble AAPH which generates
in vitro free radicals through spontaneous thermal decompo-
sition, in order to study the anti-lipid peroxidation activity of
the derivatives. The experimental conditions employed in
our study significantly resemble the cellular lipid peroxida-
tion due to the activity of the peroxyl radicals. The com-
pounds were tested at 100µM. With the exception of the es-
ters 2a, 5a and 5d as well as of the amides 1b, 4b and 5b, all
the other derivatives showed high anti-lipid peroxidation
activity. The presence of a phenyl substituent increases the
anti-lipid peroxidation activity of ester 3a in comparison to
4a. Ether 5c seems to be more potent than ester 5a whereas
the ether-ester 5d was found to be less potent than both 5c
The crude amides were recrystallized from ethanol and
the esters were subjected to Preparative Thin Layer Chroma-
1
tography (PTLC). IR, H-NMR, 13C-NMR and elemental
analysis were used for the confirmation of the synthesized
compounds’ structures. All the esters and amides presented
the characteristic absorption in the IR (KBr disk) 1720
(C=O), 1625 (C=C), (cm-1) and correspond to the E-isomers
1
(J> 9 Hz). The H-NMR and 13C-NMR data confirmed the
proposed structures and were in agreement with the literature
[16-18]. The LC-MS (ΕSΙ) analysis showed: [Μ+H]+ as well
as [Μ+Νa]+, [Μ+Κ]+, [Μ+Νa+ΜeOH]+, and the peak at m/z
113 for some amides, as mentioned in the literature [33]
(Tables 1-2).
3.1. Physicochemical Studies
We tried to determine the lipophilicity of the synthesized
derivatives experimentally, using the RPTLC method as RM
values [24], since lipophilicity is a significant physicochemi-
cal property determining the ADME properties. We succeed-
ed to correlate the theoretically calculated lipophilicity val-

6
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 1
Fotopoulos et al.
Table 1. Lipophillicity values of cinnamic ester hybrids (clog P and Rm values); % inhibition of lipid peroxidation (% LP); %
in vitro soybean LOX inhibition (%LOX inh.); % in vitro ovine COX – 2 inhibition (% COX-2 inh.); % in vivo inh. of
caraggenin paw edema CPE% ( SD) (0.01 mol/mL/kg body weight).
ꢀ
% LP
inh@
%LOX %COX-2
inh.@ inh. @ ( SD)(0.01 mol / ml/
CPE%
##
Compounds
Ar
R1
R2
Clog P# RM ꢀꢀꢁꢀꢀ
(100μΜ) (100 μΜ) (100 μΜ) kg Body Weight
-0.696ꢀ
H
1a
2a
2.75
86.0
32.0
41.0
no
no
no
36( 0.2)*
n.t.
( 0.044)
-0.482
( 0.04)
4.09
-0.289ꢀꢀ
5.27
3a
4a
74.8
67.4
no
no
no
no
n.t.
( 0.03)
-0.447ꢀ
H
3.92
27.7 ( 0.8) **
( 0.01)
-0.751ꢀ
H
H
5a
5c
2.08
no
86.5
9.6
23.0
9.4
no
no
24( 0.6) **
( 0.015)
-0.954ꢀ
OH
2.44
n.t.
( 0.004)
-0.820ꢀ
62.5/ IC50
=82.5 μΜ
5d
2.95
no
55.5 ( 3.8)**
( 0.053)
H
-
93 (IC50
0.55 μΜ)
=
-
-
-
NDGA
-
-
-
-
-
Trolox
-
-
-
-
-
-
-
-
88
-
-
-
Indomethacin
-
-
95
37.3 ( 1.3)**
##
#clog P: theoretically determined values via C – QSAR program; RM values are the average of at least 5 measurements; SD <10%; no: The compounds did
not show any activity under the reported experimental conditions; nt not tested; *p<0.01, **p<0.1 compared to reference (Student’s T-test).

Targeting Inflammation with Conjugated Cinnamic Amides
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 1
7
Table 2. Lipophillicity values of cinnamic amides hybrids (c log P and Rm values); % inhibition of lipid peroxidation (% LP); %
in vitro soybean LOX inhibition (%LOX inh.); % in vivo inh. of caraggenin paw edema CPE% ( SD) (0.01 mol/mL/kg
body weight).
% LP
inh@
(100μΜ)
%LOX
inh.@ (100
μΜ)
CPE% ( SD) (0.01 mol/mL
Kg Body Weight)
RM** ( SD)
Compounds
Ar
R
Clog P*
28.4( 0.7)*
64.8 ( 3.7)*
H
0.363 ( 0.006)
1b
-1.53
no
33.8
57.4
-0.04 ( 0.001)
3b
0.99
57.9
(IC50
=
51μM)
H
H
0.273 ( 0.006)
0.398 ( 0.001)
4b
5b
-0.36
-2.20
no
no
42( 1.3)*
0.9
no
nt
93 (IC50
0.55 μΜ)
=
-
-
-
-
-
-
-
-
-
NDGA
trolox
-
-
-
-
88
-
-
-
-
indometha-
cin
-
37.3 ( 1.3)**
*clog P: theoretically determined values via C – QSAR programme; ** RM values are the average of at least 5 measurements; SD <10%; no: the compounds did not show any activi-
ty under the reported experimental conditions; *p<0.01, **p<0.1 compared to reference (Student’s T-test).
ꢀ
Table 3. Molecular properties prediction-Lipinski “Rule of five”. Drug likeness of the synthesised compounds.
No OH,
NHd
No rotational
bondse
Compounds
milogPa
TPSAb
No atoms
No O,Nc
No violations
Volumef
MWg
logBBh
1a
2a
3a
4a
5a
5c
1b
3.06
4.40
5.38
4.04
2.59
2.60
-1.05
81.36
81.36
17
23
27
21
18
18
22
6
6
6
6
7
7
7
0
0
0
0
1
1
6
0
0
1
0
0
0
1
7
8
8
7
7
7
9
205.35
276.76
320.75
249.34
213.37
213.37
283.07
237.21
313.30
363.37
287.27
253.21
253.21
304.35
-0.1753
0.0324
81.36
-0.0234
0.1843
81.36
101.59
101.59
128.30
-0.45045
-0.4489
-1.28175
(Table 3) Contd…

8
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 1
Fotopoulos et al.
No OH,
NHd
No rotational
bondse
Compounds
milogPa
TPSAb
No atoms
No O,Nc
No violations
Volumef
MWg
logBBh
3b
4b
5b
5d
1.27
-0.07
-1.53
3.75
128.30
128.30
148.53
145.66
32
26
23
24
7
7
6
6
7
0
1
1
1
1
10
9
398.47
327.06
291.90
280.26
430.51
354.41
320.35
342.26
-0.92215
-1.12985
-1.55845
-0.71135
8
9
11
12
c
aLogarithm of partition coefficient between n-octanol and water (milog P); bTopological Polar Surface Area (TPSA); Number of hydrogen bond acceptors (n-ON); dNumber of hy-
drogen bond donors (n-OHNH); eNumber of rotatable bonds (n-rotb); fMolecular Volume; gMolecular Weight; h Blood Brain Barrier (BBB).
and 5a. Within the amide group, 3b presented higher antiox-
idant activity than 4b in which the phenyl substituent was
absent.
inhibition was measured. The biological results were in ac-
cordance with the in silico study.
Since the inhibition of lipoxygenase occurs via a carbon-
centered radical on a lipid chain and most of the LOX-
inhibitors are antioxidants or free radical scavengers [46], it
is possible that 3b is extended into the hydrophobic domain
of LOX, to prevent the access of the substrate to the active
site, and hence prevent the lipid peroxidation induced by the
enzyme. The result of antioxidant activity for amide 3b sup-
ports the anti-LOX activity.
The performed molecular modelling study provided a
useful interpretation of the experimental results [35-43]. The
preferred docking pose for the most potent derivative 3b is
shown in Fig. (1). The binding of 3b to soybean LOX (PDB
code: 3PZW) has a higher AutoDockVina score (-10.1)
compared to the other docked derivatives. From the docking
results, it seems that the designed derivatives present allo-
steric interactions with the enzyme. Furthermore, 3b is able
to accommodate the extensively hydrophobic cavity close to
the active site with possible hydrophobic interactions (π-π
stacking).
In silico docking for COX-2 was applied to all the novel
derivatives. Compound 5d was found to be the most potent
among the group presenting an AutoDock Vina score (-7.1).
It seems that 5d interacts with the enzyme in an allosteric
manner. It is worth to be mentioned that SC-558, a selective
COX-2 inhibitor, accommodates in the protein in the same
way as 5d (Fig. 2) with hydrophobic interactions in an allo-
steric mode. Considering the in vitro assay, only compound
5d - the nitrooxy ester-ether, presents IC50 =82.5µM for
COX. This result is according to the modelling findings.
Fig. (1). Docking orientation of 3b (depicted in turquoise) bound to
soybean lipoxygenase (LOX-1).
Fig. (2). Docking poses of SC-558 (selective COX-2 inhibitor,
depicted in grey) and 5d (depicted in magenta) bound to COX-2.
The evaluation of the novel acids against soybean lipox-
ygenase LOX was accomplished by the UV-based enzyme
assay of Pontiki, E. & Hadjipavlou-Litina, D. [17, 18, 44].
This assay may be used as a qualitative or semi-quantitative
screening for such activity [45].
The esters 1a-5a, as well as the nitrooxy ester-ether 5d
and the ether 5c, did not present any LOX inhibition at
100µM. On the contrary, the amide of naphthyl cinnamic
acid 3b presented a value of IC50 = 51μM. In the case of the
derivative 4b in which the phenyl substituent was absent, no
We decided to study the in vivo anti-inflammatory effect
of representative compounds using the carrageenin hind paw
edema model. Carrageenin has been accepted as a useful
phlogistic agent, releases prostaglandins, and is preferred for

Targeting Inflammation with Conjugated Cinnamic Amides
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 1
9
seeking new anti-inflammatories, inhibitors of prostaglandin
synthesis and COX [28]. Therefore, we considered to study
the in vivo anti-inflammatory activity of the most potent
COX inhibitor, the ether-ester 5d. Simultaneously, in order
to delineate the role of substituent Ar we tested esters 1a, 4a
and 5a in order to delineate the role of substituent Ar. Com-
pound 5d was found to present the highest in vivo anti-
inflammatory activity within the ester group- followed by 1a,
4a and 5a,- which is correlated to its anti-COX activity. The
presented activity is higher than the standard drug indometh-
acin. Lipophilicity does not seem to play any role to this in
vivo activity. However, the volume of substituent Ar seems
to decrease the activity (4a, naphthyl). The 3b was found to
be the most potent amide and simultaneously the most active
molecule among the tested compounds. The amides 4b and
1b follow. The naphthyl amides are more potent anti-
inflammatories than the phenyl derivatives e.g. 4b>1b. The
presence of the naphthyl moiety seems to be crucial for the
in vivo response. The perusal of the in vitro/in vivo results
points that the 3b and 5d can be used as leads for the design
of new molecules to target inflammation.
ΝΟ
=
Nitric Oxide
QSAR
TMPD
=
Quantitative Structure-activity Relationships
N, N, N’,N’-tetramethyl-p-phenylenediamine
=
ETHICS
PARTICIPATE
APPROVAL
AND
CONSENT
TO
The experimental protocols were approved by the Animal
Ethics Committee of the Prefecture of Central Macedonia
(no. 270079/2500), Greece.
HUMAN AND ANIMAL RIGHTS
No humans are used in this study. Our studies were in
accordance with recognised guidelines on animal experimen-
tation. (Guidelines for the care and use of laboratory animals
published by the Greek Government 160/1991, based on EU
regulations 86/609). Balb/c mice (25-35g, 3-4 months old)
were kept in the Centre of the School of Veterinary Medicine
(EL54 BIO42), Aristotle University of Thessaloniki, which
is registered by the official state veterinary authorities (pres-
idential degree 56/2013, in harmonization with the European
Directive 2010/63/EEC).
Further experiments are in progress to investigate the role
of the L-arginine on the activity.
CONCLUSION
CONSENT FOR PUBLICATION
A number of esters, ethers and amides of cinnamic, p-
coumaric and naphthyl acetic acids, phenyl or not phenyl
substituted, a number has been synthesized and subjected to
modelling studies. The compounds were studied as inhibitors
of COX, LOX, as antioxidants in vitro and as anti-
inflammatories in vivo. The result of antioxidant activity for
amide 3b supports the anti-LOX activity. Compounds 3b and
5d were found to be potent anti-inflammatories in vivo and
could be used as leads for the design of new pleiotropic mol-
ecules to target inflammation.
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
The authors confirm that the data supporting the findings
of this research are available within the manuscript and its
supplementary material.
FUNDING
None.
LIST OF ABBREVIATIONS
AAPH
Arg
=
=
=
2,2’-azobis-2-methyl-propanimidamide
Arginine
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
BOP
(Benzotriazol-1-yloxy) tris (dimethylmino)
Phosphonium Hexafluorophosphate
ACKNOWLEDGEMENTS
clogP
COX
CPE
=
=
=
=
=
=
=
=
=
Theoretically Calculated Molar Lipophilicity
Cycloxygenase
The authors would like to thank E.E. Vlachou, Ph.D.
candidate at Department of Chemistry, Aristotle University
of Thessaloniki for providing the MS spectra, and Dr. Fani
Tsitouroudi, Research Associate, Imperial College London,
for the language refinement. We are thankful to Biobyte and
Dr. A. Leo for free use of C-QSAR and support. Dr. E. Pon-
tiki would like to thank Mr. A. Patsilinakos from the De-
partment of Chemistry and Drug Technologies, “Sapienza”
University of Rome, Italy, for his help during the Molecular
Docking studies and the visualization of the results.
Carrageenin-induced Paw Edema
N, N’-dicyclohexylcarbodiimide
Linoleic Acid
DCC
LLA
LOX
MS
Lipoxygenase
Mass Spectrometry
SUPPLEMENTARY MATERIAL
NDGA
NMR
Nordihydroguaiaretic Acid
Nuclear Magnetic Resonance
Supplementary material is available on the publisher’s
web site along with the published article.

10 Letters in Drug Design & Discovery, 2020, Vol. 17, No. 1
Fotopoulos et al.
[17]
[18]
Pontiki, E.; Hadjipavlou-Litina, D. Synthesis and pharmacochemi-
cal evaluation of novel aryl-acetic acid inhibitors of lipoxygenase,
antioxidants, and anti-inflammatory agents. Bioorg. Med. Chem.,
2007, 15(17), 5819-5827.
REFERENCES
[1]
Rastogi, N.; Goh, K.S.; Wright, E.L.; Barrow, W.W. Potential drug
targets for Mycobacterium avium defined by radiometric drug-
inhibitor combination techniques. Antimicrob. Agents Chemother.,
1994, 38(10), 2287-2295.
[http://dx.doi.org/10.1016/j.bmc.2007.06.001] [PMID: 17604175]
Pontiki, E.; Hadjipavlou-Litina, D.; Geromichalos, G.; Papageor-
giou, A. Anticancer activity and quantitative-structure activity rela-
[http://dx.doi.org/10.1128/AAC.38.10.2287] [PMID: 7840559]
Adisakwattana, S.; Moonsan, P.; Yibchok-Anun, S. Insulin-
releasing properties of a series of cinnamic acid derivatives in vitro
and in vivo. J. Agric. Food Chem., 2008, 56(17), 7838-7844.
[http://dx.doi.org/10.1021/jf801208t] [PMID: 18651742]
Chung, H.S.; Shin, J.C. Characterization of antioxidant alkaloids
and phenolic acids from anthocyanin-pigmented rice (Oryza sativa
cv. Heugjinjubyeo). Food Chem., 2007, 104(4), 1670-1677.
[http://dx.doi.org/10.1016/j.foodchem.2007.03.020]
Naz, S.; Ahmad, S.; Ajaz Rasool, S.; Asad Sayeed, S.; Siddiqi, R.
Antibacterial activity directed isolation of compounds from Onos-
ma hispidum. Microbiol. Res., 2006, 161(1), 43-48.
[http://dx.doi.org/10.1016/j.micres.2005.05.001]
16338589]
[2]
[3]
[4]
tionship (QSAR) studies of
a series of antioxidant/anti-
inflammatory aryl-acetic and hydroxamic acids. Chem. Biol. Drug
Des., 2009, 74(3), 266-275.
[http://dx.doi.org/10.1111/j.1747-0285.2009.00864.x]
19703028]
[PMID:
[19]
Pontiki, E.; Hadjipavlou-Litina, D.; Litinas, K.; Nicolotti, O.; Ca-
rotti, A. Design, synthesis and pharmacobiological evaluation of
novel acrylic acid derivatives acting as lipoxygenase and cyclooxy-
genase-1 inhibitors with antioxidant and anti-inflammatory activi-
ties. Eur. J. Med. Chem., 2011, 46(1), 191-200.
[http://dx.doi.org/10.1016/j.ejmech.2010.10.035]
21106277]
[PMID:
[PMID:
[20]
[21]
[22]
Pontiki, E.; Hadjipavlou-Litina, D.; Litinas, K.; Geromichalos, G.
Novel cinnamic acid derivatives as antioxidant and anticancer
agents: Design, synthesis and modeling studies. Molecules, 2014,
19(7), 9655-9674.
[5]
[6]
[7]
Pérez-Alvarez, V.; Bobadilla, R.A.; Muriel, P. Structure-
hepatoprotective activity relationship of 3,4-dihydroxycinnamic ac-
id (caffeic acid) derivatives. J. Appl. Toxicol., 2001, 21(6), 527-
531.
[http://dx.doi.org/10.1002/jat.806] [PMID: 11746202]
Gunia-Krzyżak, A.; Pańczyk, K.; Waszkielewicz, A.M.; Marona,
H. Cinnamamide derivatives for central and peripheral nervous sys-
tem disorders-A review of structure-activity relationships. Chem.
Med. Chem., 2015, 10(8), 1302-1325.
[http://dx.doi.org/10.1002/cmdc.201500153] [PMID: 26083325]
Fajemiroye, J.O.; Prabhakar, P.R.; Cunha, L.C.; Costa, E.A.;
Zjawiony, J.K. 22-azidosalvinorin A exhibits antidepressant-like
effect in mice. Eur. J. Pharmacol., 2017, 800, 96-106.
[http://dx.doi.org/10.1016/j.ejphar.2017.02.031]
28219707]
[http://dx.doi.org/10.3390/molecules19079655] [PMID: 25004073]
Wu, G.; Jaeger, L.A.; Bazer, F.W.; Rhoads, J.M. Arginine defi-
ciency in preterm infants: Biochemical mechanisms and nutritional
implications. J. Nutr. Biochem., 2004, 15(8), 442-451.
[http://dx.doi.org/10.1016/j.jnutbio.2003.11.010]
15302078]
[PMID:
de Nigris, F.; Lerman, L.O.; Ignarro, S.W.; Sica, G.; Lerman, A.;
Palinski, W.; Ignarro, L.J.; Napoli, C. Beneficial effects of antioxi-
dants and L-arginine on oxidation-sensitive gene expression and
endothelial NO synthase activity at sites of disturbed shear stress.
Proc. Natl. Acad. Sci. USA, 2003, 100(3), 1420-1425.
[PMID:
[http://dx.doi.org/10.1073/pnas.0237367100] [PMID: 12525696]
Wallner, S.; Hermetter, A.; Mayer, B.; Wascher, T.C. The alpha-
amino group of L-arginine mediates its antioxidant effect. Eur. J.
Clin. Invest., 2001, 31(2), 98-102.
[8]
[9]
Sharma, P. Cinnamic acid derivatives: A new chapter of various
pharmacological activities. J. Chem. Pharm. Res., 2011, 3(2), 403-
423.
[23]
Gayam, V.; Ravi, S. Cinnamoylated chloroquine analogues: A new
structural class of antimalarial agents. Eur. J. Med. Chem., 2017,
135, 382-391.
[http://dx.doi.org/10.1046/j.1365-2362.2001.00771.x]
11168445]
[PMID:
[24]
[25]
Peperidou, A.; Pontiki, E.; Hadjipavlou-Litina, D.; Voulgari, E.;
Avgoustakis, K. Multifunctional cinnamic acid derivatives. Mole-
cules, 2017, 22(8), E1247.
[http://dx.doi.org/10.1016/j.ejmech.2017.04.063]
28460312]
[PMID:
[10]
Gravina, H.D.; Tafuri, N.F.; Silva Júnior, A.; Fietto, J.L.R.;
Oliveira, T.T.; Diaz, M.A.N.; Almeida, M.R. In vitro assessment of
the antiviral potential of trans-cinnamic acid, quercetin and morin
against equid herpesvirus 1. Res. Vet. Sci., 2011, 91(3), e158-e162.
[http://dx.doi.org/10.1016/j.rvsc.2010.11.010] [PMID: 21159355]
Yoon, B.H.; Jung, J.W.; Lee, J-J.; Cho, Y-W.; Jang, C-G.; Jin, C.;
Oh, T.H.; Ryu, J.H. Anxiolytic-like effects of sinapic acid in mice.
Life Sci., 2007, 81(3), 234-240.
[http://dx.doi.org/10.3390/molecules22081247] [PMID: 28757554]
Peperidou, A.; Kapoukranidou, D.; Kontogiorgis, C.; Hadjipavlou-
Litina, D. Multitarget molecular hybrids of cinnamic acids. Mole-
cules, 2014, 19(12), 20197-20226.
[http://dx.doi.org/10.3390/molecules191220197]
25474291]
[PMID:
[11]
[12]
[26]
Liégeois, C.; Lermusieau, G.; Collin, S. Measuring antioxidant
efficiency of wort, malt, and hops against the 2,2′-azobis(2-
amidinopropane) dihydrochloride-induced oxidation of an aqueous
dispersion of linoleic acid. J. Agric. Food Chem., 2000, 48(4),
1129-1134.
[http://dx.doi.org/10.1016/j.lfs.2007.05.007] [PMID: 17570441]
Hedvati, L.; Nudelman, A.; Falb, E.; Kraiz, B.; Zhuk, R.; Sprecher,
M. Cinnamic acid derived oxazolinium ions as novel cytotoxic
agents. Eur. J. Med. Chem., 2002, 37(7), 607-616.
[http://dx.doi.org/10.1016/S0223-5234(02)01375-2]
12126779]
[http://dx.doi.org/10.1021/jf9911242] [PMID: 10775361]
Kulmacz, R.J.; Lands, W.E.M. Requirements for hydroperoxide by
the cyclooxygenase and peroxidase activities of prostaglandin H
synthase. Prostaglandins, 1983, 25(4), 531-540.
[PMID:
[27]
[28]
[13]
[14]
[15]
Natella, F.; Nardini, M.; Di Felice, M.; Scaccini, C. Benzoic and
cinnamic acid derivatives as antioxidants: Structure-activity rela-
tion. J. Agric. Food Chem., 1999, 47(4), 1453-1459.
[http://dx.doi.org/10.1016/0090-6980(83)90025-4]
6410459]
[PMID:
[http://dx.doi.org/10.1021/jf980737w] [PMID: 10563998]
Sova, M. Antioxidant and antimicrobial activities of cinnamic acid
derivatives. Mini Rev. Med. Chem., 2012, 12(8), 749-767.
Winter, C.A.; Risley, E.A.; Nuss, G.W. Carrageenin-induced ede-
ma in hind paw of the rat as an assay for antiiflammatory drugs.
Proc. Soc. Exp. Biol. Med., 1962, 111(3), 544-547.
[http://dx.doi.org/10.2174/138955712801264792]
22512578]
[PMID:
[http://dx.doi.org/10.3181/00379727-111-27849]
14001233]
[PMID:
Bernini, R.; Mincione, E.; Barontini, M.; Provenzano, G.; Setti, L.
Obtaining 4-vinylphenols by decarboxylation of natural 4-
hydroxycinnamic acids under microwave irradiation. Tetrahedron,
2007, 63(39), 9663-9667.
[29]
[30]
Chi, S-C.; Jun, H.W. Anti-inflammatory activity of ketoprofen gel
on carrageenan-induced paw edema in rats. J. Pharm. Sci., 1990,
79(11), 974-977.
[http://dx.doi.org/10.1002/jps.2600791106] [PMID: 2292773]
Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Exper-
imental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv.
Drug Deliv. Rev., 2001, 46(1-3), 3-26.
[http://dx.doi.org/10.1016/j.tet.2007.07.035]
[16]
Pontiki, E.; Hadjipavlou-Litina, D. Antioxidant and anti-
inflammatory activity of aryl-acetic and hydroxamic acids as novel
lipoxygenase inhibitors. Med. Chem., 2006, 2(3), 251-264.
[http://dx.doi.org/10.2174/157340606776930763]
16948471]
[PMID:
[http://dx.doi.org/10.1016/S0169-409X(00)00129-0]
11259830]
[PMID:

Targeting Inflammation with Conjugated Cinnamic Amides
Letters in Drug Design & Discovery, 2020, Vol. 17, No. 1 11
[31]
Detterbeck, R.; Hesse, M. Synthesis and structure elucidation of
open-chained putrescine-bisamides from Aglaia species. Tetrahe-
dron, 2002, 58(34), 6887-6893.
[40]
[41]
Wang, J.; Wang, W.; Kollman, P.A.; Case, D.A. Automatic atom
type and bond type perception in molecular mechanical calcula-
tions. J. Mol. Graph. Model., 2006, 25(2), 247-260.
[http://dx.doi.org/10.1016/j.jmgm.2005.12.005] [PMID: 16458552]
Lindorff-Larsen, K.; Piana, S.; Palmo, K.; Maragakis, P.; Klepeis,
J.L.; Dror, R.O.; Shaw, D.E. Improved side-chain torsion potentials
for the Amber ff99SB protein force field. Proteins, 2010, 78(8),
1950-1958.
[http://dx.doi.org/10.1016/S0040-4020(02)00751-2]
[32]
[33]
Prakash, S.M.D.; Samanta, S.; Sinha, R.K. Design, synthesis and
antidiabetic, cardiomyopathy studies of cinnamic acid-amino acid
hybrid analogs. Med. Chem., 2014, 4(2), 345-350.
Sahakitpichan, P.; Disadee, W.; Ruchirawat, S.; Kanchanapoom, T.
L-(-)-(N-trans-cinnamoyl)-arginine, an acylamino acid from Glinus
oppositifolius (L.) Aug. DC. Molecules, 2010, 15(9), 6186-6192.
[http://dx.doi.org/10.3390/molecules15096186] [PMID: 20877215]
MolInspiration Cheminformatics. www.molinspiration.com
Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Green-
blatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF Chimera-a visualiza-
tion system for exploratory research and analysis. J. Comput.
Chem., 2004, 25(13), 1605-1612.
[http://dx.doi.org/10.1002/prot.22711] [PMID: 20408171]
Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS
4:ꢀAlgorithms for highly efficient, load-balanced, and scalable mo-
lecular simulation. J. Chem. Theory Comput., 2008, 4(3), 435-447.
[http://dx.doi.org/10.1021/ct700301q] [PMID: 26620784]
Trott, O.; Olson, A.J. AutoDock Vina: Improving the speed and
accuracy of docking with a new scoring function, efficient optimi-
zation, and multithreading. J. Comput. Chem., 2010, 31(2), 455-
461.
[42]
[43]
[34]
[35]
[http://dx.doi.org/10.1002/jcc.20084] [PMID: 15264254]
Fiser, A. Šali, A. Modeller: Generation and refinement of homolo-
gy-based protein structure models. Meth. Enzymol., 2003, 374, 461-
491.
O’Boyle, N.M.; Banck, M.; James, C.A.; Morley, C.; Vander-
meersch, T.; Hutchison, G.R. Open Babel: An open chemical
toolbox. J. Cheminform., 2011, 3(1), 33.
[36]
[37]
[PMID: 19499576]
[44]
[45]
Pontiki, E.; Hadjipavlou-Litina, D. Lipoxygenase inhibitors: A
comparative QSAR study review and evaluation of new QSARs.
Med. Res. Rev., 2008, 28(1), 39-117.
[http://dx.doi.org/10.1002/med.20099] [PMID: 17191217]
Taraporewala, I.B.; Kauffman, J.M. Synthesis and structure-
activity relationships of anti-inflammatory 9,10-dihydro-9-oxo-2-
acridine-alkanoic acids and 4-(2-carboxyphenyl)aminobenzenealkanoic
acids. J. Pharm. Sci., 1990, 79(2), 173-178.
[http://dx.doi.org/10.1186/1758-2946-3-33] [PMID: 21982300]
Halgren, T.A. Merck molecular force field. I. Basis, form, scope,
parameterization, and performance of MMFF94. J. Comput. Chem.,
1996, 17(5ꢁ6), 490-519.
[38]
[39]
[http://dx.doi.org/10.1002/jps.2600790219] [PMID: 2109057]
Müller, K. 5-Lipoxygenase and 12-lipoxygenase: Attractive targets
for the development of novel antipsoriatic drugs. Arch. Pharm.
(Weinheim), 1994, 327(1), 3-19.
[http://dx.doi.org/10.1002/(SICI)1096-
[46]
987X(199604)17:5/6<490::AID-JCC1>3.0.CO;2-P]
Sousa da Silva, A.W.; Vranken, W.F. ACPYPE - AnteChamber
python parser interface. BMC Res. Notes, 2012, 5(1), 367.
[http://dx.doi.org/10.1186/1756-0500-5-367] [PMID: 22824207]
[http://dx.doi.org/10.1002/ardp.19943270103] [PMID: 8117187]