New Hydroxycinnamic Acid Esters as Novel 5-Lipoxygenase Inhibitors That Affect Leukotriene Biosynthesis

Article abstract of DOI:10.1155/2017/6904634

Leukotrienes are inflammatory mediators that actively participate in the inflammatory response and host defense against pathogens. However, leukotrienes also participate in chronic inflammatory diseases. 5-lipoxygenase is a key enzyme in the biosynthesis

Full text of DOI:10.1155/2017/6904634

Hindawi
Mediators of Inflammation
Volume 2017, Article ID 6904634, 12 pages
https://doi.org/10.1155/2017/6904634
Research Article
New Hydroxycinnamic Acid Esters as Novel 5-Lipoxygenase
Inhibitors That Affect Leukotriene Biosynthesis
Luc H. Boudreau, Grégoire Lassalle-Claux, Marc Cormier, Sébastien Blanchard,
Marco S. Doucet, Marc E. Surette, and Mohamed Touaibia
Department of Chemistry and Biochemistry, Université de Moncton, Moncton, NB, Canada E1A 3E9
Correspondence should be addressed to Mohamed Touaibia; mohamed.touaibia@umoncton.ca
Received 24 January 2017; Revised 3 May 2017; Accepted 8 May 2017; Published 7 June 2017
Academic Editor: Sandra Helena Penha Oliveira
Copyright © 2017 Luc H. Boudreau et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Leukotrienes are inflammatory mediators that actively participate in the inflammatory response and host defense against
pathogens. However, leukotrienes also participate in chronic inflammatory diseases. 5-lipoxygenase is a key enzyme in the
biosynthesis of leukotrienes and is thus a validated therapeutic target. As of today, zileuton remains the only clinically approved
5-lipoxygenase inhibitor; however, its use has been limited due to severe side effects in some patients. Hence, the search for a
better 5-lipoxygenase inhibitor continues. In this study, we investigated structural analogues of caffeic acid phenethyl ester, a
naturally-occurring 5-lipoxygenase inhibitor, in an attempt to enhance the inhibitory activity against 5-lipoxygenase and
determine structure-activity relationships. These compounds were investigated for their ability to attenuate the biosynthesis of
leukotrienes. Compounds 13 and 19, phenpropyl and diphenylethyl esters, exhibited significantly enhanced inhibitory activity
when compared to the reference molecules caffeic acid phenethyl ester and zileuton.
1. Introduction
resins, 30% wax, 10% essential and aromatic oils, 5% pollens,
and 5% of other organic substances (reviewed in [15]). Prop-
olis has been shown to exhibit various beneficial biological
properties, such as anti-inflammatory, antibacterial, antivi-
ral, and anticancer effects [16–18]. Caffeic acid phenethyl
ester (CAPE (2, Figure 1)) is one of the major bioactive com-
ponents of honeybee propolis [19]. We and others have
shown that CAPE (2) and some structural analogues are
potent inhibitors of the 5-LO pathway [20–23].
In this study, CAPE (2) and some structural analogues of
the ester moiety were synthesized in an attempt to enhance
inhibitory activity against 5-LO and determine structure-
activity relationships. As shown in Figure 1, two families of
CAPE analogues were investigated. One is a series of aliphatic
esters and the second is substituted aryl esters. These com-
pounds were investigated for their ability to attenuate the bio-
synthesis of LTs, their specificity to the 5-LO enzyme, and
their potency as inhibitors in a complex environment such
as freshly isolated human blood. In summary, the aryl ester
compounds 13 and 19 exhibited the most potent inhibitory
The 5-lipoxygenase (5-LO) enzyme catalyzes the first two
steps of the conversion of arachidonic acid into leukotrienes
(LTs) [1]. LTs are potent lipid mediators that actively partic-
ipate in numerous inflammatory diseases such as atheroscle-
rosis [2], asthma [3], arthritis [4], and several types of cancers
[5–7]. As the cornerstone of the LT biosynthesis pathway, it
is not surprising that the regulation of 5-LO activity has been
the focal point of many therapeutic approaches in recent
years (reviewed in [8–10]).
Currently, zileuton (1, Figure 1) (Zyflo®) remains the
only clinically used and approved 5-LO inhibitor. It is pri-
marily prescribed to help alleviate chronic asthma symptoms
[11, 12]. However, possible liver toxicity has been associated
with long-term intake of the drug [13]; thus, the search for
better 5-LO inhibitors with less side effects continues.
Amongst the new wave of 5-LO inhibitors are naturally
occurring molecules derived from plant extracts [14]. Honey-
bee propolis is a resinous substance composed of 50% plant

2
Mediators of Inflammation
O
HO
HO
S
O
O
CAPE (2)
N
HO
NH2
Zileuton (1)
HO
HO
O
(10): R = Ph
(3): R = Me
R
(4): R = Et
(11): R = CH2Ph
O
(5): R = n-Pr
(12): R = CH2CH2cyclohexyl
(13): R = CH2CH2CH2Ph
(14): R = CH2CH2PhF(4-F)
(15): R = CH2CH2PhMe(4-Me)
(6): R = I-Pr
(7): R = n-But
(8): R = I-pentyl
(9): R = 3-methylbut-3-enyl
(16): R = CH2CH2PhOMe(4-OMe)
(17): R = CH2CH2Ph(4-NO2)
(18): R = CH2CH2Naph
(19): R = CH2CH(Ph)2
Figure 1: Zileuton (1), CAPE (2), and CAPE analogues investigated in the present study.
activity showing significantly better inhibition when com-
pared to the reference molecules CAPE (2) and zileuton (1).
resuspended at 107 cells/ml in HBSS supplemented with
1.6 mM CaCl2. PRP was processed as previously described
for platelet isolation [30]. Briefly, PRP was centrifuged at
400 g for 2 min at room temperature to remove the remain-
ing erythrocytes. The supernatant was then centrifuged at
1300g for 10min to pellet platelets. Cells were resuspended
in Tyrode Buffer pH 7.4 (134 mM NaCl, 2.9 mM KCl,
0.34 mM Na2HPO4, 12 mM NaHCO3, 20 mM HEPES,
1 mM MgCl2, 5 mM glucose, and 0.5 mg/ml BSA) at
3 × 108 cells/ml in the presence of 5 mM CaCl2.
2. Methods
2.1. CAPE Analog Synthesis. As recently reported [24],
alkyl esters (3–9) were synthesized in one step by Fisher
esterification with selected alcohols and caffeic acid. Aryl
esters (10–19) were synthesized by esterification of (E)-
caffeoyl chloride diacetate with selected alcohols followed
by a de-O-acetylation.
2.4. Biosynthesis of 5-Lipoxygenase Products by PMNL.
Suspended PMNL (107 cells/ml) were incubated at 37°C
with adenosine deaminase (0.3 U/ml, Sigma-Aldrich) and
test compounds at indicated concentrations 5 min before
stimulation. To initiate stimulation, 1 μM of thapsigargin
(Sigma-Aldrich) was added to cells which were then incu-
bated for 15min at 37°C [31]. Two volumes of MeOH:MeCN
(1 :1) containing the internal standard PGB2 (100 ng/ml) was
then added, and samples were processed for RP-HPLC anal-
ysis as described above.
2.2. HEK293 Cell Stimulation and Measurement of 5-LO
Products. HEK293 cells expressing both 5-LO and the 5-
lipoxygenase activating protein (FLAP) were generated as
previously reported [25–27]. For cell stimulation of 5-LO
products, transfected HEK293 cells were collected following
trypsinization and washed and the cell pellet was resus-
pended in Hank’s balanced salt solution (HBSS, Lonza) con-
taining 1.6 mM CaCl2 at a concentration of 5 × 105 cells/ml.
Cells were preincubated with each compound at the indicated
concentration for 5 min at 37°C. Cells were then stimulated
for 15 minutes at 37°C with the addition of 10μM calcium
ionophore A23187 (Sigma-Aldrich) and 10μM arachidonic
acid (Cayman Chemical). Stimulations were stopped by add-
ing 0.5 volume of cold MeOH:MeCN (1 : 1) containing
100 ng/ml of 19-OH prostaglandin B2 (PGB2) as internal
standard. Samples were then processed and 5-LO products
were analyzed by reversed-phase high-performance liquid
chromatography (RP-HPLC) as described previously [20, 28].
2.5. Arachidonic Acid Release. Quantification of arachidonic
acid release from cellular membranes was performed as pre-
viously described [31, 32]. Briefly, freshly isolated PMNL
(107 cells/ml, in HBSS containing 1.6 mM CaCl2 and 0.1%
BSA) were incubated with adenosine deaminase (0.3 U/ml)
and test compounds for 5 min at 37°C. Stimulation was initi-
ated with the addition of 1 μM thapsigargin, followed by
incubation at 37°C for 5 min. The reactions were stopped
with the addition of two volumes of cold MeOH and 300 ng
of arachidonic acid-d8 (Cayman Chemicals) as internal stan-
dard. The samples were stored at −20°C overnight and then
centrifuged the next day at 1000g for 10min. The superna-
tants were diluted with four volumes of acidified water
(0.1%) then processed on an octadecyl (C18) column. Sam-
ples were eluted with the addition of 3 ml of MeOH and dried
under nitrogen. Pentafluorobenzylesters were prepared with
the addition 50μl N,N-diisopropylethylamine (20% in aceto-
nitrile) and 50 μl of 2,3,4,5,6-pentafluorobenzylesters (20% in
acetonitrile). Samples were heated for 40 min at 40°C, then
dried under nitrogen and resuspended in 100 μl of hexane.
2.3. Polymorphonuclear Leukocyte and Platelet Isolation.
Polymorphonuclear leukocytes (PMNL) were isolated as pre-
viously described [20, 29]. Briefly, whole blood was collected
(with anticoagulant citrate dextrose (ACD)) and then centri-
fuged at 500 g for 10 min. Platelet-rich plasma (PRP) was set
aside for platelet isolation (see below), and erythrocytes were
removed by dextran sedimentation. After a centrifugation
step at 900 g ×20 min at room temperature on a lymphocyte
separation medium cushion (density, 1.077g/ml) (Wisent),
PMNL were obtained (>96%) followed by hypotonic lysis
of the remaining erythrocytes. PMNL were counted and

Mediators of Inflammation
3
Samples were quantified by negative ion chemical ionization
gas chromatography/mass spectrometry using TraceGC ultra
column (Thermo) and a Polaris Q mass spectrometer
(Thermo).
2.9. Statistical Analyses. Statistical analysis and graph design
were performed with GraphPad Prism 5 software (GraphPad
Software, San Diego, California). IC50 values were calculated
from a sigmoidal concentration-response curve-fitting
model and are expressed as means with 95% confidence
intervals. All other data are expressed as mean SEM.
One-way ANOVA with Dunnett’s multiple comparison test
(p < 0 05) were performed to determine significant differ-
ence from controls.
2.6. Platelet Activation. Platelets (3 × 108 cells/ml) were pre-
incubated with various test compounds at indicated concen-
trations for 5 min at 37°C. Platelet activation was initiated
with the addition of 10 μM of calcium ionophore A23187
(Sigma-Aldrich) followed by a 5 min incubation time at
37°C. Reactions were stopped with the addition of two
volumes of cold MeOH:MeCN (1 : 1) containing PGB2
(100 ng/ml) as internal standard. Samples were stored over-
night at −20°C and were then centrifuged, the supernatant
was dried under nitrogen, and the samples were resus-
pended in 30% methanol and analyzed by RP-HPLC as
indicated above.
3. Results
3.1. Compound Synthesis. CAPE (2) is a simple molecule for
which several biological activities have been reported. In con-
tinuation of previous work on the development of new 5-LO
inhibitors based on CAPE (2), the synthesis of CAPE ana-
logues and the testing of 5-LO inhibition were undertaken.
With these series of compounds, the importance of the
nature of the ester moiety was explored (Figure 1). Indeed,
this moiety is crucial for the inhibition of 5-LO by CAPE
(2) as demonstrated previously [20].
The first series with the alkyl esters allowed the evalua-
tion of the presence and the nature of an alkyl chain (3–9).
The alkyl chains vary from the simple methyl ester (3), pro-
ceeding to the 3-methyl-3-enyl ester (9) representing a
mimic of CAPE (2) with methyl group and a single double
bond. The second series allowed us to explore the effect of
the presence of an aryl and the effect of a monosubstitution
on this aryl (10–19). The effect of the presence and the length
of a linker between the oxygen and the aryl group was also
investigated (Figure 1). The two series of compounds were
synthesized by two different strategies. The alkyl series was
obtained in a single step following a single esterification of
caffeic acid and an alcohol to give the desired ester. The aryl
esters were synthesized in three steps with the (E)-caffeoyl
chloride diacetate, obtained by the Vilsmeier-Haack adduct
derived from thionyl chloride and N,N-dimethylformamide
as catalyst, and the appropriate alcohol. The structures of
all synthesized esters are summarized in Figure 1.
2.7. Ex Vivo Whole Blood Stimulation. Ex vivo whole blood
stimulation for the biosynthesis of 5-LO products was per-
formed as previously reported [20]. Briefly, blood was col-
lected in tubes containing heparin as anticoagulant. Each
compound tested or its diluent control (DMSO, 0.5%) was
added to 1 ml of heparinized blood and incubated for 5 min
in a water bath at 37°C. Stimulation was initiated with the
addition of 125 μl of 40 mg/ml of opsonized zymosan, sam-
ples were gently vortexed and incubated at 37°C for 30min.
Samples were then centrifuged at 960 g for 10 min at 4°C.
Plasma (350 μl) was removed and added to tubes containing
1.2 ml of CH3OH:CH3CN (1 : 1) containing 100 ng/ml of
PGB2 as internal standard. Samples were stored overnight
at −20°C, then centrifuged at 500 g for 10 min. The superna-
tants were dried under nitrogen, resuspended in 30% MeOH,
and analyzed by RP-HPLC as described above.
2.8. Molecular Docking. Molecular docking was undertaken
with the help of AutoDock 4.0, Autogrid [33], and AutoDock
Tools [34]. The standard AutoDock protocol was followed
unless otherwise noted. Ligands were drawn and then proc-
essed with AutoDock Tools for charge and rotatable bonds
assignment. 5-LO crystal structure chosen for docking is
PDB ID: 3O8Y [34], which is a “stable-5-LOX.” To enable
crystallization, several mutations are present in the noncata-
lytic domain and a small 3 residue sequence in the catalytic
domain is replaced from KKK to ENL. The mutations maybe
affect the structure, but “stable-5-LOX” catalytic activity was
not affected [34]. The protein was prepared with AutoDock
Tools. Water molecules were removed, polar hydrogens
added, and charges assigned. The grid box used a default
spacing of 0.375 with a bounding box of 60, 66, and 60 and
a grid center of −2.24, 25.69, and −0.94, in both cases X, Y,
and Z coordinates. As for the docking settings, 100 runs were
completed per ligand and defaults were kept with exceptions
for the following values: ga_pop_size 5000, ga_num_evals
100,000,000, ga_num_generations 500,000, and sw_max_its
5000. For analysis, AutoDock Tools (Schrödinger Release;
Maestro, version 10.6) and LigPlot+ [35] were used. Results
were clustered with a maximum of 2.00 Å RMSD, and the
largest cluster with the lowest binding energy was chosen.
3.2. Biosynthesis of 5-LO Products. To evaluate CAPE ana-
logues as potential inhibitors of the biosynthesis of 5-LO
products, a first series of experiments was performed in
HEK293 cells expressing both 5-LO and FLAP as previously
described [20, 26, 27, 36]. HEK293 cells were first preincu-
bated with the test compounds (1 μM), then stimulated in
the presence of arachidonic acid and calcium ionophore
A23187 to induce the biosynthesis of 5-LO products
(Figure 2(a)). Compounds that showed similar or better
inhibitory activity than the reference compound CAPE (2),
or decreased the biosynthesis of 5-LO products by more than
50% (when compared to the control), were selected for sub-
sequent testing. Based on these criteria, compounds 9, 13,
14, 15, 16, and 19 were selected for subsequent analyses. Of
importance, zileuton (1), the only 5-LO inhibitor currently
used clinically, was less effective than the selected com-
pounds (Figure 2(a)). It should be noted that many of the
compounds that were not selected for further study are nev-
ertheless inhibitors of 5-LO that may well have IC50
values

4
Mediators of Inflammation
⁎
⁎
l
)
3
5
o
2
r
t
n
Co
CAPE
Compounds (1 ꢀM)
(a)
125
100
75
50
25
0
0.1
1
10
Compound (ꢀM)
15
16
19
CAPE (2)
9
13
14
(b)
Figure 2: Effect of the test compounds on the biosynthesis of 5-LO products in transfected HEK293 cells (a) and thapsigargin-stimulated
PMNL (b). (a) HEK293 cells expressing both 5-LO and FLAP were incubated in presence of the indicated compounds (1 μM) or their
diluent (control, 0.5% DMSO) for 5 min, then biosynthesis of 5-LO products was initiated with the addition of 10 μM calcium ionophore
A23187 and 10 μM arachidonic acid for 15 min. After stimulation, reactions were stopped and samples were processed for analysis of
5-LO products by RP-HPLC. Total 5-LO products measured represent the sum of LTB4, its trans isomers, and 5-hydroxyeicosatetraenoic
acid. (b) Freshly isolated human PMNL were incubated in presence of test compounds (or their diluent control, DMSO 0.5%) at the
indicated concentrations for 5 min. Biosynthesis of the 5-LO products was initiated with the addition of 1 μM of thapsigargin, followed by
a 15 min incubation period at 37°C. The stimulation was then stopped and samples were processed for RP-HPLC analysis and total 5-LO
products quantification. Total 5-LO products measured represent the sum of LTB4, its trans isomers, 20-COOH- and 20-OH-LTB4 and
5-hydroxyeicosatetraenoic acid. Data are expressed as means SEM of at least 3 independent experiments. ∗Different from control, as
determined by one-way ANOVA with Dunnett’s multiple comparison test (p < 0 05).
inferior to 10μM. If the selected compounds do not per-
form well in future pharmacokinetic studies, these other
compounds may nevertheless be interesting preclinical can-
didates that could be reinvestigated.
Having selected the most potent compounds, a second
series of experiments measuring the dose response of the
inhibition of 5-LO product biosynthesis was conducted using
freshly isolated human PMNL to determine the IC50 values
for each test compound (Figure 2(b) and Table 1). Com-
pounds 13 (IC50 =0.49 μM) and 19 (0.53 μM) had a signifi-
cantly lower IC50 value than CAPE (2) (0.79 μM, Table 1).
Compounds 9 (0.87 μM), 14 (1.01 μM), 15 (1.04 μM), and
16 (1.29 μM) had IC50 values that were similar to CAPE (2)
(Table 1). Of importance, compound 13 (the most potent
inhibitor of 5-LO product biosynthesis) did not induce
early apoptosis of PMNL as indicated by annexin V labelling
(see Supplementary Figure S1 available online at https://
doi.org/10.1155/2017/6904634).
3.3. Molecular Docking. A total of 19 esters were docked to
5-LO in this study. From the obtained docking results, the
binding energy was determined for each ligand as well as

Mediators of Inflammation
5
For both molecules in this subgroup, they still appear to form
the “cage.”
Table 1: Calculated IC50 values of selected compounds for the
inhibition of 5-LO product biosynthesis in human PMNL.
A
second subgroup of the first group contains only
Compound
IC50 (μM) (95% CI)
0.79 (0.65–0.97)
0.87 (0.70–1.11)
0.49 (0.41–0.58)
1.01 (0.69–1.50)
1.04 (0.30–3.65)
1.29 (0.83–2.00)
0.53 (0.41–0.68)
1.9 (1.48–2.42)∗
zileuton (1). It has a smaller size and a different shape than
the other ligands making certain aspects less evident to
compare. When compared with the other groups, zileuton’s
(1) pose overlaps the most with the molecules in the first
group, where its aromatic ring lines up relatively well and
its linear chain generally follows the linker.
The second group is only made of (3), which is positioned
far away from the iron, at the end of the pocket near Leu420
with the linear part of the molecule at the end of the pocket.
This leaves no possibility for (3) to interact directly with the
iron atom without travelling inside the pocket.
CAPE (2)
9
13
14
15
16
19
Zileuton (1)
CI = 95% confidence intervals; ND = IC50 values could not be determine at
tested concentrations used for the analysis; ∗IC50 values taken from [31].
The last group is made up of 8 molecules (4–11), with 4
minor subgroups. The only difference between theses minor
subgroups is a slightly different placement and rotation of
the catechol moiety of the molecule. The ligands are as
follows, with each minor subgroup separated by a semicolon:
(4), (5), (6) and (7); (8) and (9); (10); (11). These compounds
are distant from the iron atom and would need some
movement for the possibility to interact with the iron.
Compared to the other molecules, they are the least stable
with an average binding energy of −7.17kcal/mol versus
the −8.71 kcal/mol of the remaining molecules.
Overall, the most prevalent hydrogen bond is with
Tyr181, present in 8 out of 17 molecules. Hydrogen bonds
with Gln363, His367, and Asn425 are present with 7 mole-
cules, and 6 molecules make hydrogen bonds with Asn407.
Tyr181, Gln363, and Asn425 form bonds with the molecules
that are less stable. Asn407, His367, and to a certain extent
Phe421 seem like the most import residues since the majority
of the better scoring ligands, mostly comprised of molecules
from the first group, form hydrogen bonds with Asn407
and His367. For a portion of the best-ranked ligands, a π-π
interaction is observed with Phe421.
From the images generated by Ligplot+, almost all atoms
are under hydrophobic contact. Under visual inspection,
most of the best scoring ligands occupy a good portion of
the active site cavity and they all seem to block any access
to the iron atom. In the case of (19), the entire cavity is
almost filled. The lesser scored ligands, mostly the last group,
appear to occupy the end of the cavity, near Leu420, and
mostly leave a large opening around the iron atom. This is
especially true for (3).
hydrogen bonds and π-π interactions (Table 2). The most
stable ligand, with the lowest binding energy, was (17)
with a binding energy of −10.91kcal/mol and (18),
−10.61 kcal / mol. The least stable ligand was (4) with a
binding energy of −6.15 kcal/mol.
The pose of each ligand could be classified into 3 larger
groups with a handful of subdivisions. Inside these groups,
the catechol moiety often has only a slight variation between
each molecule. For the monosubstituted ester moiety, the
variation in position is often larger, depending on the charac-
teristics of the substitution.
The first group has all molecules generating double
hydrogen bonds with residue Asn407 and a single hydrogen
bond with His367. This group is formed by (12), (13), (15),
(16), (17), (18), and CAPE (2). To interact with these two
residues, the ligand must complete a “cage” around the iron
atom with the coordinating residues. This cage would block
access to the iron atom of any other molecule. A minimum
of 3 hydrogen bonds are formed, with the possibility of more
being formed depending on the monosubstituted ester moi-
ety. This type of pose would make it hard to displace the
ligand once in place. An advantage of this pose is that
His367 is an iron-coordinating residue [1, 37], and it has
been suggested that His367 may serve as a replaceable iron
ligand [1]. While most atoms in the molecules appear just
beyond the reach of interaction with the iron, this would
make this group perfectly placed to coordinate or interact
with the iron atom after a small shift of the caffeic acid moiety
and possibly replace His367. Figure 3 shows (13) in the active
site as well as all interactions. These 7 ligands are the most
stable of the series with the exception of (19) occupying
the fourth place. The average binding energy for this first
group is −9.23 kcal/mol, compared to −7.30 kcal/mol for the
remaining molecules.
3.4. Arachidonic Acid Liberation from Cells. A limiting factor
in the generation of 5-LO products in PMNL is the bioavail-
ability of its substrate, arachidonic acid, which is acylated in
cell membrane phospholipids and must be released by a phos-
A subgroup of the first group, (14) and (19), occupy a
similar pose to the first group, but they do not form the same
hydrogen bonds. In the case of (19), the molecule is
“inverted” when compared to all other molecules in this
series. Its hydroxyl groups point towards the end of the cavity
versus “pointing” toward the iron atom. This might be due to
the large size of its double phenyls. One of its double phenyls
overlaps with the caffeic acid moiety of the first group of mol-
ecules, while the second phenyl positions itself near Thr364.
pholipase A
2. Since both CAPE (2) and zileuton (1) were pre-
viously shown to inhibit the liberation of cellular arachidonic
acid [31, 38], the test compounds were evaluated to determine
if they would also inhibit arachidonic acid release, thus par-
tially contributing to the decrease of 5-LO products generated
by the cells. As shown in Figure 4, CAPE (2), 9, 13, and 19 sig-
nificantly inhibited cellular arachidonic acid release when
compared to the control (0.5% DMSO, vehicle). On the other
hand, compounds 14, 15, and 16 failed to significantly inhibit

6
Mediators of Inflammation
Table 2: Ligand binding energy, interactions, and hydrogen bond lengths.
Molecule
Binding energy (kcal/mol)
Hydrogen bonds
His367, Asn407 × 2
Gln363, His367
Hydrogen bonds length (Å)
π-π Interactions
Phe421
CAPE (2)
Zileuton (1)
(3)
−8.19
−7.18
−6.78
−6.15
−6.63
−6.84
−7.12
−7.22
−7.21
−8.23
−7.96
−8.69
−8.77
−8.04
−8.77
−8.69
−10.91
−10.62
−9.14
2.14, 2.21, 2.26
2.09, 2.11
Phe177
Ala424
1.81
None
(4)
Tyr181, Gln363 × 2, Asn425
Tyr181, Gln363, Asn425
Tyr181, Gln363, Asn425
Tyr181, Gln363, Asn425
Tyr181, Gln363, Asn425
Tyr181, Gln363, Asn425
Tyr181, Gln363, Asn425
Tyr181
2.25, 1.98, 1.94, 2.21
2.02, 1.96,2.01
2.15, 1.94, 1.95
2.48, 1.99, 1.95
1.98, 2.09, 2.00
2.08, 2.02, 1.93
1.99, 2.04, 1.91
2.04
None
(5)
None
(6)
None
(7)
None
(8)
None
(9)
None
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
Tyr181
Tyr181
His367, Asn407 × 2
His367, Asn407 × 2
His367, Ile406
2.01, 2.22, 2.30
2.08, 2.11, 2.27
2.10, 2.26
None
None
Phe421
His367, Asn407 × 2
His367, Asn407 × 2
His367, Asn407 × 2, Ala424
His367, Asn407 × 2
Leu420
2.11, 2.14, 2.45
2.14, 2.21, 2.32
2.50, 2.07, 2.32, 1.99
2.34, 2.07, 2.22
2.61
Tyr181, Phe421
Phe421
Phe177, Phe421
Phe177
Tyr181, Phe421
the release of cellular membrane arachidonic acid, although
compound 14 approached significance (Figure 4).
Although they showed good inhibition of 5-LO, compounds
16 and 19 failed to significantly affect the biosynthesis of 12-
HETE at a concentration of 1 μM although significant inhibi-
tion was measured at a concentration of 3 μM.
3.5. Ex Vivo Biosynthesis of 5-LO Products. Since selected
compounds inhibit the biosynthesis of 5-LO products in
both fresh human PMNL and the HEK293 cell line express-
ing the machinery for leukotriene generation, the ability of
the compounds to inhibit 5-LO product biosynthesis in a
more physiological and complex environment was mea-
sured. Freshly obtained human blood was preincubated in
presence of test compounds (1 μM) and then 5-LO product
biosynthesis was initiated with addition of zymosan. As
shown in Figure 5(a), all compounds that had been selected
after screening in HEK293 cells significantly decreased 5-
LO product biosynthesis in whole blood at 1 μM and 5 μM.
Of importance, in addition to thapsigargin stimulation of
isolated PMNL, the use of zymosan in this series of experi-
ments showed that these compounds inhibit the biosynthesis
of 5-LO products induced by different stimuli, and in a com-
plex matrix.
4. Discussion
Inflammatory lipid mediators, such as LTs generated via the
5-LO pathway, are necessary for the body’s defense against
pathogens. However, the production of LTs has also been
associated with several inflammatory diseases such as asthma
[3], arthritis [4], atherosclerosis [2], and some types of can-
cers [5, 6]. Therefore, physiological and pharmacological reg-
ulation of LT biosynthesis remains a focal point of numerous
research efforts investigating the control of inflammatory
diseases [8, 9, 39]. To this day, zileuton (1) (Zyflo) remains
the only 5-LO inhibitor approved for use by clinicians to help
alleviate symptoms associated with asthma [11, 12]. How-
ever, serious side effects have been observed with the use of
this drug in some patients [13], thus prompting new and
innovative approaches in drug design and development to
regulate the production of LTs.
In this study, we synthesized a new series of structural
analogue of CAPE (2), a natural occurring compound found
in honeybee propolis previously reported as an inhibitor of
lipoxygenase pathways [18, 22, 31]. Several biological proper-
ties of these compounds were evaluated including their activ-
ity as direct 5-LO inhibitors in HEK293 cells, their capacity to
inhibit LT biosynthesis in both human PMNL (important
producers of the potent chemoattractant LTB4) and whole
blood, their effects on the liberation of cellular arachidonic
acid, and finally in their specificity for the 5-LO pathway ver-
sus the 12-LO pathway.
3.6. Inhibition of Platelet-Type 12-Lipoxygenase. Having
identified compounds that inhibit the biosynthesis of lipid
mediators generated via the 5-LO pathway, their ability to
inhibit platelet 12-lipoxygenase (12-LO) was assessed to
determine whether they show specificity. Platelets do not
express 5-LO but express high levels of the 12-LO enzyme
which is responsible for the conversion of arachidonic acid
into 12-hydroxyeicosatetraenoic acid (12-HETE), an inflam-
matory lipid mediator implicated in vascular permeability.
Compounds CAPE (2), 9, 13, 14, and 15 significantly inhib-
ited the biosynthesis of 12-HETE (Figure 5(b)) at a concen-
tration of 1 μM with near-complete inhibition at 3 μM.

Mediators of Inflammation
7
(a)
His600
Asn425
Leu420
Ala603
Ala424
Tyr181
Leu414
Phe421
Trp599
Gln363
Leu607
His372
2.86
CB
N
ND1
CD1
C
CG1
CE1
CG
CA
O
His367
O
CD2
CB
CG2
NE2
C
2.84
Ile406
CA
N
ND2
Leu368
3.19
CG
2.96
OD1
CB
C
Ala410
N
CA
O
Phe177
Asn407
(b)
Figure 3: (13) docking (a); (13) with AutoDock displayed in Maestro and LigPlot (b).

8
Mediators of Inflammation
150
125
100
75
⁎
⁎
⁎
⁎
50
⁎
25
0
1 6
1 9
NA
Compounds (1 ꢀM)
Figure 4: The impact of test compounds on the liberation of arachidonic acid in stimulated PMNL. Human PMNL were incubated with test
compounds (1 μM) or their diluent (control, DMSO 0.5%) for 5 min. Stimulation was then initiated with the addition of 1 μM thapsigargin,
followed by an incubation at 37°C for 5 min. The reactions were stopped with the addition of two volumes of cold MeOH and 300 ng of
arachidonic acid-d8, then samples were stored overnight at −20°C. Free arachidonic acid was first extracted on octadecyl columns, then
pentafluorobenzylesters of arachidonic acid were prepared and samples were measured on GC-MS. Spontaneous release of AA was also
measured in absence of thapsigargin (nonactivated, NA). ∗Different from control as determined by one-way ANOVA with Dunnett’s
multiple comparison test (p < 0 05). Data are expressed as means SEM of at least 3 independent experiments.
100
75
50
25
0
100
75
50
25
0
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
⁎
Compounds
Compounds
1 ꢀM
3 ꢀM
1 ꢀM
5 ꢀM
(a)
(b)
Figure 5: The inhibitory effects of the test compounds of the biosynthesis of 5-LO products in stimulated whole blood (a) and the
biosynthesis of 12-lipoxygenase products in platelets (b). (a) Whole blood was incubated in presence of various compounds (1 μM and
5 μM) or with their diluent (control, DMSO 0.5%) for 5 min, then stimulated with opsonized zymosan (5 mg/ml) for 30 min at 37°C.
Blood was then centrifuged and plasma was collected and added to 3.5 volumes of MeOH:CH3CN (1 : 1). Samples were processed for
analysis by RP-HPLC for total 5-LO products quantification. Total 5-LO products measured represent the sum of LTB4, its trans isomers,
20-COOH and 20-OH-LTB4, and 5-hydroxyeicosatetraenoic acid. (b) Platelets were incubated for 5 min in presence of test compounds
(1 μM and 3 μM) then biosynthesis of 12-HETE was initiated with the addition of 10 μM of calcium ionophore A23187. Reactions
were stopped with the addition of two volumes of MeOH:MeCN (1 : 1 v/v). Samples were processed for analysis by RP-HPLC for
12-hydroxyeicosatetraenoic acid (12-HETE) quantification. ∗Different from control as determined by one-way ANOVA with Dunnett’s
multiple comparison test (p < 0 05). Data are expressed as means SEM of at least 3 independent experiments.

Mediators of Inflammation
9
inhibitory activity, while the only aryl compounds lacking
activity were those devoid of a phenyl moiety suggesting that
the interaction of the phenyl group with Phe421 stabilizes the
compounds in the pocket.
Compound 13 was the only compound that showed sig-
nificantly better inhibition than CAPE (2). The addition of
a methylene group in compound 13 relative to CAPE (2)
appears to be the determinant for the increased activity. This
increase cannot be explained only by an enhancement of the
Molecular docking was undertaken with the help of
AutoDock 4.0, Autogrid [33], and AutoDock Tools [34].
The “stable-5-LOX” (PDB ID: 3O8Y [34]) used for docking
is mutated mostly on the noncatalytic domain with a small
exception of a short 3 residues sequence mutated on the cat-
alytic domain. While these mutations may affect the struc-
ture, test results showed that catalytic fidelity was conserved
as “stable-5-LOX” activity was not affected [34]. While such
docking results can be ambiguous in that they do not always
reflect biological results, more molecular modeling with the
tested molecules and others can nevertheless be helpful for
the design of new analogs.
lipophilic nature of the molecule since an extra CH
2 does not
greatly increase the lipophilicity. Better positioning of the
molecule in the active site of 5-LO as shown with new inter-
actions predicted by in silico modeling likely explains this
increase in inhibitory activity. Conversely, the removal of
methylene groups in 10 and 11 has the opposite effect result-
ing in molecules that are not as effective at inhibiting 5-LO as
CAPE (2), suggesting that placement of the phenyl group in
relation to the caffeoyl moiety is critical for optimal interac-
tions and thus inhibitory activity.
While most esters adopt a cis configuration, the docking
pose of our best compound (13, Figure 3(a)) shows a trans-
ester configuration. The small cavity, which is largely hydro-
phobic, the relatively large substituent (−CH2CH2CH2Ph),
···
and the hydrogen bond completed with His367 (NH O,
2.08 Å) can explain this trans conformation. As the pocket
is relatively small and the substituent is relatively large, this
limits the poses that the ligand can take. Similarly, both oxy-
gens in the ester are in a small polar section of the pocket
enabling the hydrogen bond and diminishing contact with
the hydrophobic residues. For this hydrogen bond and posi-
tioning in the small polar pocket to happen, the ester needs to
adopt a trans conformation to minimize steric blocking by
Gln367 and Phe421. For the ligand to adopt a cis ester, a
major shift would need to happen. Reported crystals of pro-
teins include CAPE (2) (PDB ID: 4QRF), ferulic acid phene-
tyl ester (FAPE) (4PWF), ethyl caffeate (4) (4PWG), and
other derivatives [40]. Of those three molecules, all crystal-
ized in pairs, the pair of FAPE ligands have their ester in a
trans configuration. One of the two ethyl caffeate is in a trans
configuration while the second ethyl caffeate is in a cis config-
uration. As for CAPE (2), both deviate planarity with values
of 38 degrees and 68 degrees. A recently described crystal
structure with a pair of CAPE as ligands, (PDB ID: 5HNT),
again, both deviate from planarity with angles of 53 degrees
and 68 degrees.
In the first part of this study, the objective was to deter-
mine which compounds would potentially act as direct 5-
LO inhibitors. HEK293 cells that expressed the necessary
machinery to convert arachidonic acid into LTs [25, 41] were
stimulated in the presence of exogenous arachidonic acid,
thus bypassing a critical step in the bioavailability of the 5-
LO substrate. In fact, exogenous substrate is required in this
model since stimulated HEK293 cells do not use endogenous
AA as a substrate for 5-LO [42, 43]. This strategy allowed for
the identification of compounds that may affect the 5-LO
pathway by directly inhibiting the enzyme. In addition to
the reference molecules CAPE (2) and zileuton (1), most
compounds significantly inhibited the biosynthesis of 5-LO
products; however, the best compounds tended to be those
from the series of aryl esters, consistent with the observation
that the phenethyl ester moiety of CAPE is required for its
inhibitory activity [31]. Accordingly, compound 9 that was
designed as a mimic of CAPE (2) with a double bond and
methyl group showed the best inhibitory activity amongst
the compounds from the aliphatic series. Consistent with this
model, the simplest aliphatic esters (3, 4) were without
While several compounds, including CAPE (2), appeared
to perform better at inhibiting LT biosynthesis than the refer-
ence molecule zileuton in isolated cell assays, the inhibitory
activity of zileuton (1) was similar to that of other com-
pounds in the whole blood assay. This observation supports
previous studies showing that zileuton (1) was a slightly bet-
ter inhibitor of the biosynthesis of 5-LO products in whole
blood than CAPE (2) [31]. At this time, the reasons for the
better action of zileuton in a complex matrix are not known,
but it could be due to tighter binding of the more lipophilic
CAPE (2) analogues by plasma proteins, rendering the com-
pounds less available for uptake into leukocytes, the target
blood cells that are responsible for LT biosynthesis in this
model [44]. This slightly decreased activity of CAPE and its
analogues may also be attributed to the stability of the
compounds in whole blood due to the presence of plasma
esterases [45, 46]. Nevertheless, CAPE (2) and its analogues
retained significant inhibitory activity in this complex
matrix. Zileuton (1) on the hand, is less susceptible to plasma
esterases as it lacks the ester moiety found in the test com-
pounds. While CAPE’s (2) bioavailability in humans has
never been evaluated, pharmacokinetic studies have been
performed in small rodents with an elimination half-life
ranging from 21min to 92 min in rats following IV adminis-
tration [47, 48]. However, the pharmacokinetic profiles and
bioavailability of polyphenols can vary considerably between
humans and rodents, consequently establishing the bioavail-
ability and pharmacokinetic profiles of our novel compounds
in vivo is an essential step in the development of these potent
5-LO inhibitors.
Most of the compounds that exhibited 5-LO inhibition
also inhibited the platelet 12-LO pathway. Compounds that
display inhibitory properties for more than one inflamma-
tory enzyme have attracted more attention in recent years
[49, 50] due to their ability to simultaneously shut down mul-
tiple inflammatory pathways [9]. All of the compounds
tested for the inhibition of the 12-LO pathway were inhibi-
tory except compounds 16 and 19, suggesting a degree of
specificity for the 5-LO pathway. Therefore, the addition of
an electron-donating group such as OCH3
in 16 does not

10
Mediators of Inflammation
seem to be favorable for the inhibition of 12-LO. The exis-
tence of unfavorable interactions with 12-LO may explain
the loss of activity compared to CAPE (2) since both mole-
cules have substantially the same lipophilicity. A significant
increase in the lipophilicity, likely combined with new π-π
interactions following the addition of a second phenyl as in
19 also appears to hinder the ability of this compound to
inhibit the 12-LO.
Conflicts of Interest
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Acknowledgments
Mohamed Touaibia would like to acknowledge the contri-
bution of the Natural Sciences and Engineering Research
Council of Canada (NSERC), the New Brunswick Innovation
Foundation (NBIF), the Canadian Foundation for Innova-
tion (CFI), and Université de Moncton. Sébastien Blanchard
was supported a graduate scholarship from NSERC. Luc
H. Boudreau acknowledges the support of the Canadian
Institutes of Health Research (CIHR), the NBIF and Univer-
sité de Moncton. Marc E. Surette acknowledges the support
of a grant from the CIHR, and funding from the Canada
Research Chairs Program and the New Brunswick Innova-
tion Research Chairs Program.
Since both CAPE (2) and zileuton (1) are known to
interfere with the release of arachidonic acid from cell mem-
branes, likely via a mechanism inhibiting the group IVA
cytosolic phospholipase A2 (cPLA2α) [31, 38], we investi-
gated whether our compounds displayed similar biological
properties. While 9, 13, 19, and CAPE (2) all inhibited the
release of cellular arachidonic acid, compounds 14, 15,
and 16 bearing an extra methyl, fluorine, or a methoxy,
respectively, in the para position did not interfere with AA
liberation. Although the inhibition of AA release contributes
to the inhibition of LT biosynthesis, all compounds never-
theless show inhibition of 5-LO activity as demonstrated
in the experiments in HEK293 cells where exogenous sub-
strate was provided to the cells. Finally, these series of com-
pounds all exhibit antioxidant properties [24], which
contributes to their inhibitory characteristics on enzymes
such as lipoxygenases; however, their ability to inhibit at
the concentrations used in the present study are neverthe-
less dependent on the presence and the nature of the ester
linkage [31].
While leukotrienes are necessary for host defense against
pathogens, they also participate in chronic inflammatory
diseases such as asthma, arthritis, and atherosclerosis. For
years, researchers have targeted 5-LO as a means of regu-
lating the biosynthesis of leukotrienes. To this day, zileu-
ton remains the only clinically used and approved 5-LO
inhibitor but its usage has been linked to severe side
effects in some patients. In this study, we investigated
structural analogues of caffeic acid phenethyl ester, a 5-
LO inhibitor recently described by our team, in an attempt
to enhance the inhibitory activity against 5-LO and deter-
mine structure-activity relationships. These compounds
were investigated for their ability to attenuate the biosyn-
thesis of leukotrienes either as direct 5-LO inhibitors or
preventing its substrate’s release from cellular membranes.
Interestingly, a phenpropyl ester (13) and a diphenylethyl
ester (19) were identified that exhibited potent inhibitory
activity when compared to the reference molecules caffeic
acid phenethyl ester and zileuton. In the search for better
5-LO inhibitors, we provide a better understanding of the
fundamental mechanism by which these molecules exhibit
anti-inflammatory properties, an important step in the
development of novel therapeutic drugs.
References
[1] O. Rådmark, O. Werz, D. Steinhilber, and B. Samuelsson,
“5-Lipoxygenase, a key enzyme for leukotriene biosynthesis
in health and disease,” Biochimica et Biophysica Acta,
vol. 1851, pp. 331–339, 2015.
[2] M. Mehrabian, H. Allayee, J. Wong et al., “Identification of
5-lipoxygenase as a major gene contributing to atherosclero-
sis susceptibility in mice,” Circulation Research, vol. 91,
pp. 120–126, 2002.
[3] J. A. Leff, W. W. Busse, D. Pearlman et al., “Montelukast, a
leukotriene-receptor antagonist, for the treatment of mild
asthma and exercise-induced bronchoconstriction,” The New
England Journal of Medicine, vol. 339, pp. 147–152, 1998.
[4] M. Chen, B. K. Lam, Y. Kanaoka et al., “Neutrophil-derived
leukotriene B4 is required for inflammatory arthritis,” The
Journal of Experimental Medicine, vol. 203, pp. 837–842,
2006, Rockefeller Univ Press.
[5] S. Sarveswaran, D. Chakraborty, D. Chitale, R. Sears, and J.
Ghosh, “Inhibition of 5-lipoxygenase selectively triggers
disruption of c-Myc signaling in prostate cancer cells,” The
Journal of Biological Chemistry, vol. 290, pp. 4994–5006, 2015,
American Society for Biochemistry and Molecular Biology.
[6] K. Wejksza, C. Lee-Chang, M. Bodogai et al., “Cancer-pro-
duced metabolites of 5-lipoxygenase induce tumor-evoked
regulatory B cells via peroxisome proliferator-activated recep-
tor α,” Journal of Immunology, vol. 190, pp. 2575–2584, 2013,
American Association of Immunologists.
[7] S. K. Wculek and I. Malanchi, “Neutrophils support lung col-
onization of metastasis-initiating breast cancer cells,” Nature,
vol. 528, pp. 413–417, 2015, Nature Research.
[8] B. Hofmann and D. Steinhilber, “5-Lipoxygenase inhibitors: a
review of recent patents (2010-2012),” Expert Opinion on
Therapeutic Patents, vol. 23, pp. 895–909, 2013, Taylor &
Francis.
Ethical Approval
[9] C. Pergola and O. Werz, “5-Lipoxygenase inhibitors: a review
of recent developments and patents,” Expert Opinion on
Therapeutic Patents, vol. 20, pp. 355–375, 2010.
[10] D. Steinhilber and B. Hofmann, “Recent advances in the
search for novel 5-lipoxygenase inhibitors,” Basic and Clinical
Pharmacology and Toxicology, vol. 114, pp. 70–77, 2014.
Blood was obtained, with heparin or ACD as anticoagulant,
from healthy consenting volunteers. This research project
was approved by the Comité d’éthique de la recherche avec
les êtres humains at the Université de Moncton.

Mediators of Inflammation
11
[11] H. Nelson, J. Kemp, W. Berger et al., “Efficacy of zileuton
controlled-release tablets administered twice daily in the treat-
ment of moderate persistent asthma: a 3-month randomized
controlled study,” Annals of Allergy, Asthma & Immunology,
vol. 99, pp. 178–184, 2007.
[12] E. Israel, P. Rubin, J. P. Kemp et al., “The effect of inhibition of
5-lipoxygenase by Zileuton in mild-to-moderate asthma,”
Annals of Internal Medicine, vol. 119, pp. 1059–1066, 1993,
American College of Physicians.
[25] L. H. Boudreau, J. Bertin, P. P. Robichaud et al., “Novel
5-lipoxygenase isoforms affect the biosynthesis of 5-
lipoxygenase products,” The FASEB Journal, vol. 25,
pp. 1097–1105, 2011.
[26] J. Doiron, L. H. Boudreau, N. Picot, B. Villebonet, M. E.
Surette, and M. Touaibia, “Synthesis and 5-lipoxygenase
inhibitory activity of new cinnamoyl and caffeoyl clusters,”
Bioorganic
&
Medicinal Chemistry Letters, vol. 19,
pp. 1118–1121, 2009.
[13] M. C. Liu, L. M. Dubé, and J. Lancaster, “Acute and chronic
effects of a 5-lipoxygenase inhibitor in asthma: a 6-month ran-
domized multicenter trial. Zileuton Study Group,” Journal of
Allergy and Clinical Immunology, vol. 98, pp. 859–871, 1996.
[14] O. Werz, “Inhibition of 5-lipoxygenase product synthesis by
natural compounds of plant origin,” Planta Medica, vol. 73,
pp. 1331–1357, 2007.
[15] S. Huang, C.-P. Zhang, K. Wang, G. Q. Li, and F.-L. Hu,
“Recent advances in the chemical composition of propolis,”
Molecules, vol. 19, pp. 19610–19632, 2014, Multidisciplinary
Digital Publishing Institute.
[16] A. P. Tiveron, P. L. Rosalen, M. Franchin et al., “Chemical
characterization and antioxidant, antimicrobial, and anti-
inflammatory activities of south Brazilian organic propolis,”
PLoS One, vol. 11, article e0165588, 2016, Public Library of
Science. Agbor G, editor.
[17] I. A. Freires, S. M. de Alencar, and P. L. Rosalen, “A pharma-
cological perspective on the use of Brazilian red propolis and
its isolated compounds against human diseases,” European
Journal of Medicinal Chemistry, vol. 110, pp. 267–279, 2016.
[18] O. K. Mirzoeva and P. C. Calder, “The effect of propolis and its
components on eicosanoid production during the inflamma-
tory response. Prostaglandins, leukotrienes and essential fatty
acids,” Churchill Livingstone, vol. 55, pp. 441–449, 1996.
[19] M. F. Tolba, S. S. Azab, A. E. Khalifa, S. Z. Abdel-Rahman, and
A. B. Abdel-Naim, “Caffeic acid phenethyl ester, a promising
component of propolis with a plethora of biological activities:
a review on its anti-inflammatory, neuroprotective, hepato-
protective, and cardioprotective effects,” IUBMB Life, vol. 65,
pp. 699–709, 2013.
[20] L. H. Boudreau, J. Maillet, L. M. LeBlanc et al., “Caffeic acid
phenethyl ester and its amide analogue are potent inhibitors
of leukotriene biosynthesis in human polymorphonuclear leu-
kocytes,” PLoS One, vol. 7, article e31833, 2012, Public Library
of Science. Proost P, editor.
[27] E. P. Allain, L. H. Boudreau, N. Flamand, and M. E. Surette,
“The intracellular localisation and phosphorylation profile of
the human 5-lipoxygenase Δ13 isoform differs from that of
its full length counterpart,” PLoS One, vol. 10, article
e0132607, 2015, Public Library of Science. Samant R, editor.
[28] P. P. Robichaud, S. J. Poirier, L. H. Boudreau et al., “On the
cellular metabolism of the click chemistry probe 19-alkyne
arachidonic acid,” Journal of Lipid Research, 2016, American
Society for Biochemistry and Molecular Biology.
[29] A. Böyum, “Isolation of mononuclear cells and granulocytes
from human blood. Isolation of monuclear cells by one cen-
trifugation, and of granulocytes by combining centrifugation
and sedimentation at 1 g,” Scandinavian Journal of Clinical
and Laboratory Investigation. Supplementum, vol. 97,
pp. 77–89, 1968.
[30] N. Cloutier, A. Pare, R. W. Farndaleetal., “Plateletscanenhance
vascular permeability,” Blood, vol. 120, pp. 1334–1343, 2012.
[31] L. H. Boudreau, J. Maillet, L. B. LM et al., “Caffeic acid phe-
nethyl ester and its amide analogue are potent inhibitors of
leukotriene biosynthesis in human polymorphonuclear leuko-
cytes,” PloS One, vol. 7, article e31833-8, 2012, Proost P, editor.
[32] M. E. Surette, J. D. Winkler, A. N. Fonteh, and F. H. Chilton,
“Relationship between arachidonate—phospholipid remodel-
ing and apoptosis,” Biochemistry, vol. 35, pp. 9187–9196,
1996, American Chemical Society.
[33] G. M. Morris, R. Huey, W. Lindstrom et al., “AutoDock4 and
AutoDockTools4: automated docking with selective receptor
flexibility,” Journal of Computational Chemistry, vol. 30,
pp. 2785–2791, 2009, Wiley Subscription Services, Inc., A
Wiley Company.
[34] G. M. Morris, D. S. Goodsell, M. E. Pique et al., AutoDock
Version 4.2; Updated for Version 4.2.6, s.l.: The Scripps
Research Institute, Molecular Graphics Laboratory, La Jolla,
CA, USA, 2009.
[35] R. A. Laskowski and M. B. Swindells, “LigPlot+: multiple
ligand–protein interaction diagrams for drug discovery,”
Journal of Chemical Information and Modeling, vol. 51,
pp. 2778–2786, 2011, American Chemical Society.
[21] J. A. Doiron, B. Métayer, R. R. Richard et al., “Clicked
cinnamic/caffeic esters and amides as radical scavengers and
5-lipoxygenase inhibitors,” International Journal of Medicinal
Chemistry, vol. 2014, Article ID 931756, p. 12, 2014, Hindawi
Publishing Corporation.
[22] G. F. Sud'ina, O. K. Mirzoeva, M. A. Pushkareva, G. A. Korshu-
nova, N. V. Sumbatyan, and S. D. Varfolomeev, “Caffeic acid
phenethyl ester as a lipoxygenase inhibitor with antioxidant
properties,” FEBS Letters, vol. 329, pp. 21–24, 1993.
[23] J. A. Doiron, L. M. LeBlanc, M. J. Hébert et al., “Structure-
activity relationship of caffeic acid phenethyl ester analogs as
new 5-lipoxygenase inhibitors,” Chemical Biology & Drug
Design, vol. 89, pp. 514–528, 2017.
[24] J. T. Sanderson, H. Clabault, C. Patton et al., “Antiproliferative,
antiandrogenic and cytotoxic effects of novel caffeic acid deriv-
atives in LNCaP human androgen-dependent prostate cancer
cells,” Bioorganic & Medicinal Chemistry, vol. 21, pp. 7182–
7193, 2013.
[36] L. H. Boudreau, N. Picot, J. Doiron et al., “Caffeoyl and cinna-
moyl clusters with anti-inflammatory and anti-cancer effects.
Synthesis and structure–activity relationship,” New Journal of
Chemistry, vol. 33, pp. 1932–1940, 2009.
[37] N. C. Gilbert, S. G. Bartlett, M. T. Waightet al., “Thestructure of
human 5-lipoxygenase,” Science, vol. 331, pp. 217–219, 2011,
American Association for the Advancement of Science.
[38] A. Rossi, C. Pergola, A. Koeberle et al., “The 5-lipoxygenase
inhibitor, zileuton, suppresses prostaglandin biosynthesis by
inhibition of arachidonic acid release in macrophages,” British
Journal of Pharmacology, vol. 161, pp. 555–570, 2010,
Blackwell Publishing Ltd.
[39] M. Peters-Golden and W. R. Henderson, “Leukotrienes,” New
England Journal of Medicine, vol. 357, pp. 1841–1854, 2007.

12
Mediators of Inflammation
[40] T. Yokoyama, Y. Kosaka, and M. Mizuguchi, “Inhibitory activ-
ities of propolis and its promising component, caffeic acid phe-
nethyl ester, against amyloidogenesis of human transthyretin,”
Journal of Medicinal Chemistry, vol. 57, pp. 8928–8935, 2014,
American Chemical Society.
[41] O. Rådmark, O. Werz, D. Steinhilber, and B. Samuelsson, “5-
Lipoxygenase: regulation of expression and enzyme activity,”
Trends in Biochemical Sciences, vol. 32, pp. 332–341, 2007.
[42] P. P. Robichaud, S. J. Poirier, L. H. Boudreau et al., “On the
cellular metabolism of the click chemistry probe 19-alkyne
arachidonic acid,” Journal of Lipid Research, vol. 57,
pp. 1821–1830, 2016.
[43] J. Gerstmeier, C. Weinigel, D. Barz, O. Werz, and U. Garscha,
“An experimental cell-based model for studying the cell
biology and molecular pharmacology of 5-lipoxygenase-
activating protein in leukotriene biosynthesis,” Biochimica et
Biophysica Acta, vol. 1840, pp. 2961–2969, 2014.
[44] M. E. Surette, A. Odeimat, R. Palmantier, S. Marleau, P. E.
Poubelle, and P. Borgeat, “Reverse-phase high-performance
liquid chromatography analysis of arachidonic acid metabo-
lites in plasma after stimulation of whole blood ex vivo,”
Analytical Biochemistry, vol. 216, pp. 392–400, 1994.
[45] N. Celli, L. K. Dragani, S. Murzilli, A. Tommaso Pagliani, and
A. Poggi, “In vitro and in vivo stability of caffeic acid phenethyl
ester, abioactivecompoundofpropolis,”JournalofAgricultural
and Food Chemistry, vol. 55, pp. 3398–3407, 2007, American
Chemical Society.
[46] T. Satoh, P. Taylor, W. F. Bosron, S. P. Sanghani, M.
Hosokawa, and B. N. La Du, “Current progress on esterases:
from molecular structure to function,” Drug Metabolism and
Disposition, vol. 30, pp. 488–493, 2002, American Society for
Pharmacology and Experimental Therapeutics.
[47] X. Wang, J. Pang, J. A. Maffucci et al., “Pharmacokinetics of
caffeic acid phenethyl ester and its catechol-ring fluorinated
derivative following intravenous administration to rats,”
Biopharmaceutics & Drug Disposition, vol. 30, pp. 221–228,
2009, John Wiley & Sons, Ltd.
[48] J. Yang, P. D. Bowman, S. M. Kerwin, and S. Stavchansky,
“Development and validation of an LCMS method to deter-
mine the pharmacokinetic profiles of caffeic acid phenethyl
amide and caffeic acid phenethyl ester in male Sprague–
Dawley rats,” Biomedical Chromatography, vol. 28, pp. 241–
246, 2014.
[49] T. Hanke, F. Dehm, S. Liening et al., “Aminothiazole-featured
Pirinixic acid derivatives as dual 5-lipoxygenase and micro-
somal prostaglandin E2 synthase-1 inhibitors with improved
potency and efficiency in vivo,” Journal of Medicinal Chemis-
try, vol. 56, pp. 9031–9044, 2013, American Chemical Society.
[50] D. Altavilla, L. Minutoli, F. Polito et al., “Effects of flavocoxid, a
dual inhibitor of COX and 5-lipoxygenase enzymes, on benign
prostatic hyperplasia,” British Journal of Pharmacology,
vol. 167, pp. 95–108, 2012, Blackwell Publishing Ltd.

MEDIATORS
of
INFLAMMATION
The Scientific
Journal of
Gastroenterology
World Journal
Research and Practice
Diabetes Research
Disease Markers
Hindawi Publishing Corporation
http://www.hindawi.com
Hindawi Publishing Corporation
Hindawi Publishing Corporation
http://www.hindawi.com
Hindawi Publishing Corporation
Hindawi Publishing Corporation
http://www.hindawi.com
Volume 2014
http://www.hindawi.com
Volume 2014
Volume 2014
http://www.hindawi.com
Volume 2014
Volume 2014
International Journal of
Journal of
Immunology Research
Endocrinology
Hindawi Publishing Corporation
http://www.hindawi.com
Hindawi Publishing Corporation
http://www.hindawi.com
Volume 2014
Volume 2014
Submit your manuscripts at
https://www.hindawi.com
BioMed
PPAR Research
Research International
Hindawi Publishing Corporation
http://www.hindawi.com
Hindawi Publishing Corporation
http://www.hindawi.com Volume 2014
Volume 2014
Journal of
Obesity
Evidence-Based
Journal of
Journal of
Stem Cells
Complementary and
Ophthalmology
International
Alternative Medicine
Oncology
Hindawi Publishing Corporation
Hindawi Publishing Corporation
http://www.hindawi.com
Hindawi Publishing Corporation
Hindawi Publishing Corporation
http://www.hindawi.com
Hindawi Publishing Corporation
http://www.hindawi.com
http://www.hindawi.com
Volume 2014
Volume 2014
http://www.hindawi.com
Volume 2014
Volume 2014
Volume 2014
Parkinson’s
Disease
Computational and
Behavioural
Neurology
Mathematical Methods
Oxidative Medicine and
AIDS
in Medicine
Research and Treatment
Cellular Longevity
Hindawi Publishing Corporation
Hindawi Publishing Corporation
http://www.hindawi.com
Hindawi Publishing Corporation
http://www.hindawi.com
Hindawi Publishing Corporation
Hindawi Publishing Corporation
http://www.hindawi.com
http://www.hindawi.com
Volume 2014
Volume 2014
Volume 2014
http://www.hindawi.com
Volume 2014
Volume 2014