Discovery of a novel 2,5-dihydroxycinnamic acid-based 5-lipoxygenase inhibitor that induces apoptosis and may impair autophagic flux in RCC4 renal cancer cells

Article abstract of DOI:10.1016/j.ejmech.2019.06.060

The inhibition of 5-lipoxygenase (5-LO), the key enzyme for the biosynthesis of leukotrienes (LTs), has generated increasing enthusiasm as anti-inflammatory and antitumor strategies in recent years. Based on our previous studies, we synthesized a series of dihydroxycinnamic acid-based analogs that might be 5-LO inhibitors. LTs biosynthesis inhibition in HEK293 cells and polymorphonuclear leukocytes (PMNL) was measured and antitumor activities were investigated in Renal Cell Carcinoma (RCC). Results showed that the 2,5-dihydroxycinnamic acid phenethyl ester (10b) was the best 5-LO inhibitor and was 7-fold more potent than Zileuton (1), the only clinically approved 5-LO inhibitor. 2,5-Dihydroxy substitution was more favorable to 5-LO inhibition since compound 10b is twice as active as CAPE (2) which is a 3,4-dihydroxylcinnamic acid ester. Meanwhile, 10b reduced the cell viability of renal cancer cells and was more selective toward RCC4 and 786.0 cells which are deficient for the Von Hippel-Lindau (VHL) tumor suppressor gene. As to the underlying cell-death mechanisms, 10b induced apoptosis in VHL-deficient RCC4 cells. Also, increases in LC3B and p62 expression suggest a blockage of the autophagic flux in RCC in response to 10b.

Full text of DOI:10.1016/j.ejmech.2019.06.060

Accepted Manuscript
Discovery of a novel 2,5-dihydroxycinnamic acid-based 5-lipoxygenase inhibitor that
induces apoptosis and may impair autophagic flux in RCC4 renal cancer cells
Ayyoub Selka, Jérémie A. Doiron, Pierre Lyons, Sonia Dastous, Alison Chiasson,
Marc Cormier, Sandra Turcotte, Marc E. Surette, Mohamed Touaibia
PII:
S0223-5234(19)30587-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.060
EJMECH 11463
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 April 2019
Revised Date: 21 June 2019
Accepted Date: 21 June 2019
Please cite this article as: A. Selka, Jéé.A. Doiron, P. Lyons, S. Dastous, A. Chiasson, M. Cormier,
S. Turcotte, M.E. Surette, M. Touaibia, Discovery of a novel 2,5-dihydroxycinnamic acid-based
5-lipoxygenase inhibitor that induces apoptosis and may impair autophagic flux in RCC4 renal
cancer cells, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.060.
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ACCEPTED MANUSCRIPT
Discovery of a novel 2,5-dihydroxycinnamic acid-based 5-lipoxygenase
inhibitor that induces apoptosis and may impair autophagic flux in
RCC4 renal cancer cells
Ayyoub Selka, Jérémie A. Doiron, Pierre Lyons, Sonia Dastous, Alison Chiasson, Marc
Cormier, Sandra Turcotte, Marc E. Surette, and Mohamed Touaibia*
Department of Chemistry and Biochemistry, Université de Moncton, Moncton, New
Brunswick, Canada.
*
Corresponding author. E-mail address: mohamed.touaibia@umoncton.ca
Graphical Abstract
O
OH
OH
O
HO
HO
Structural
optilization
X
O
X = O, CH2
10b
IC50 on 5-LO: 0.33 (µM)
IC50 on RCC4 (VHL -/-): 8 (µM)
IC50 on RCC4 (VHL +/+): 99 (µM)
S
O
N
HO
NH2
IC50 of the only clinically
approved 5-LO inhibitor:
Zileuton (1): 2.31 (µM)

ACCEPTED MANUSCRIPT
Discovery of a novel 2,5-dihydroxycinnamic acid-based 5-lipoxygenase inhibitor
that induces apoptosis and may impair autophagic flux in RCC4 renal cancer cells
Ayyoub Selka, Jérémie A. Doiron, Pierre Lyons, Sonia Dastous, Alison Chiasson, Marc Cormier,
Sandra Turcotte, Marc E. Surette, and Mohamed Touaibia*
Department of Chemistry and Biochemistry, Université de Moncton, Moncton, New Brunswick,
Canada.
*
Corresponding author. E-mail address: mohamed.touaibia@umoncton.ca
Abstract
The inhibition of 5-lipoxygenase (5-LO), the key enzyme for the biosynthesis of leukotrienes (LTs), has
generated increasing enthusiasm as anti-inflammatory and antitumor strategies in recent years. Based on
our previous studies, we synthesized a series of dihydroxycinnamic acid-based analogs that might be 5-
LO inhibitors. LTs biosynthesis inhibition in HEK293 cells and polymorphonuclear leukocytes (PMNL)
was measured and antitumor activities were investigated in Renal Cell Carcinoma (RCC). Results
showed that the 2,5-dihydroxycinnamic acid phenethyl ester (10b) was the best 5-LO inhibitor and was
7
-fold more potent than Zileuton (1), the only clinically approved 5-LO inhibitor. 2,5-Dihydroxy
substitution was more favorable to 5-LO inhibition since compound 10b is twice as active as CAPE (2)
which is a 3,4-dihydroxylcinnamic acid ester. Meanwhile, 10b reduced the cell viability of renal cancer
cells ꢀand was more selective toward RCC4 and 786.0 cells which are deficient for the Von Hippel-
Lindau (VHL) tumor suppressor gene. As to the underlying cell-death mechanisms, 10b induced
apoptosis in VHL-deficient RCC4 cells. Also, increases in LC3B and p62 expression suggest a blockage
of the autophagic flux in RCC in response to 10b.
Keywords: 5-LO inhibitors, anti-leukotriene therapy, Caffeic acid, Renal cell carcinoma
.

ACCEPTED MANUSCRIPT
Introduction
Several recent studies have demonstrated an increased use of fatty acid substrates as metabolic fuel to
promote inflammatory responses in several diseases [1, 2]. Therapies that investigate these metabolic
pathways may prove to be effective anti-inflammatory approaches. Arachidonic acid (AA) is a
polyunsaturated fatty acid involved in numerous inflammatory diseases through its enzymatic
conversion to potent bioactive metabolites [3]. 5‐Lipoxygenase (5-LO) is the key enzyme in the
conversion of AA to the leukotrienes (LTs), potent mediators of inflammation that are associated with
several inflammatory and allergic disorders [4-8].
Many studies support the association of inflammation with chronic diseases and cancers [9]. Chronic
inflammation is a precursor to many tumors and plays a crucial role in various aspects of tumor
development including proliferation, invasion, metastasis, and angiogenesis [9, 10]. 5-LO is expressed
in several cancer cell types, and its inhibition blocks proliferation and induces apoptosis [11-13]. It was
shown that up-regulation of 5-LO is a common and striking feature of clear cell renal cell carcinoma
(ccRCC) [14]. These highly vascularized tumors are associated with an inactivation of the Von Hippel-
Lindau (VHL) tumor suppressor in most ccRCC cases [15, 16] and overexpression of 5-LO is correlated
with a loss of VHL and high levels of the vascular endothelium growth factor (VEGF) [14].
Zileuton (1) (Figure 1) is the only inhibitor of 5-LO approved for the treatment of inflammatory
diseases. However, its clinical use has been limited because of a poor pharmacokinetic profile and liver
toxicity [17, 18]. Therefore, there is a need for new effective inhibitors of 5-LO and the search for
alternative and potent 5-LO inhibitors with fewer side effects is ongoing. Among the known promising
inhibitors of 5-LO are a variety of polyhydroxylated natural products such as caffeic acid [19] and
flavonoids [20]. Accordingly, our recent results obtained with caffeic acid phenethyl ester (CAPE) 2
(
Figure 1) and several analogs demonstrate an especially effective inhibition of 5-LO [21-24]. Contrary
to the belief that polyphenols inhibit 5-LO exclusively through their antioxidant properties, our results
clearly show that the presence or absence of specific functional groups influence 5-LO inhibition
independent of their impact on antioxidant activity [21-24].
While the effect of CAPE (2) on breast cancer cells has been extensively studied [25, 26], to the best of
our knowledge neither CAPE (2) nor its analogues have ever been tested on renal cancer

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carcinoma (RCC). RCC is the most common type of kidney cancer in adults and is the 10th most
common type of cancer in the world.
N
S
O
O
N
HO
HO
N
HO
O
S
N
NH2
H
Zileuton (1)
CAPE (2)
STF-62247 (3)
Figure 1. Structures of Zileuton (1), CAPE (2) and STF-62247 (3).
The feasibility of targeting the inactivation of VHL using small molecules was previously shown [27-
9]. Besides, it was recently demonstrated that RCC cells depend on iron for growth and survival as a
2
result of VHL inactivation [30]. The chelating effect of iron with polyphenols can be explored as an
option for suppressing the progression of these tumor cells. As part of our continuing interest in phenolic
acid analogs as new 5-LO inhibitors, closely related derivatives of CAPE with the two hydroxyls in
various positions on the phenolic acid moiety were investigated. Thus, this series will compare the
effects of catechols and dihydroxylated analogues of CAPE. Phenpropyl ester analogs as well as the
ketone analog of the best 5-LO inhibitor were also synthesized. The designed and synthesized
compounds were evaluated for their ability to inhibit 5-LO and to induce selective cell death in VHL-
deficient RCC cells (VHL -/-) versus their VHL-positive counterparts (VHL +/+). The designed CAPE
analogs were compared to STF-62247 (3) (Figure 1), as small molecule reported to induce cytotoxicity
and reduce tumor growth of VHL-deficient RCC cells compared to genetically matched cells with wild
type VHL [27].
Results and Discussion
Synthesis of CAPE-based inhibitors.
The 2,4-dihydroxycinnamic acid phenethyl and phenpropyl esters 6 and 7 were prepared to determine
the influence of the position of hydroxyls on biological activity. Following our one pot esterification
procedure [22], commercially available 2,4-dihydroxycinnamic acid 4, 2-phenylethyl bromide 5a or 3-
phenylpropyl bromide 5b gave the corresponding esters 6 and 7 in good yields (Scheme 1). As the 2,3-
dihydroxycinnamic acid was not commercially available, we tried to synthesize it by condensation from
2
,3-dihydroxybenzaldeyde and malonic acid as reported for CAPE synthesis [31]. Unfortunately, these

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attempts failed since the product of the intramolecular esterification with the hydroxyl at the 2-position
is mostly obtained.
OH
O
OH
O
Br
i
OH
+
( )
O
( )n
n
HO
HO
4
5a: n =1
6
: n = 1
5
b: n =2
7: n = 2
Scheme 1. Synthesis of 2,4-dihydroxylated CAPE analogs. Reagents and conditions: (i) Na CO , HMPA, rt, 12 h.
2
3
This esterification is not detected with 3,4-dihydroxycinnamic acid. Acylation of both hydroxyls of 2,3-
dihydroxybenzaldehyde also failed even with excess acetic anhydride. The protection of these hydroxyls
with the allyl group gave the desired product but unfortunately, its reaction with malonic acid gave a
very low yield of condensation product. The deprotection of the two allyl protecting groups was also
problematic since less than 10% of the desired product was obtained.
Always with the same aldehyde, we tried to proceed with Wittig’s condensation to obtain 2,3-
dihydroxylated CAPE analogs 10a and 10d. As shown in Scheme 2, the condensation of the stabilized
ylids 9a-b bearing the phenethyl or phenpropyl ester unit with 1,2-dihydroxybenzaldehydes 8a-c gave
the desired products in good yields. The used ylids were obtained with the esterification of chloroacetic
acid followed by treatment with triphenylphosphine. With the same strategy, and since the
corresponding acid was not available, we obtained the 2,5- (10b, 10e) and 3,5-dihydroxylated (10c)
CAPE analogs (Scheme 2).
R1
O
R1
O
Cl
Ph P
R2
O
R2
R3
O
( )n
( )n
i
+
3
H
O
R3
9
a-b
R4
8
a-c
R4
1
1
1
1
1
0a: R = OH, R = OH, R = H, R = H, n = 1
1 2 3 4
0b: R = OH, R = R = H, R = OH, n = 1
1
2
3
4
0c: R = H, R = OH, R = H, R = OH, n = 1
0d: R = OH, R = OH, R = H, R = H, n = 2
1
2
3
4
1
2
3
4
0e: R = OH, R = R = H, R = OH, n = 2
1
2
3
4
Scheme 2. Synthesis of 2,3-, 2,5-, and 3,5-dihydroxylated CAPE analogs. Reagents and conditions: (i) K CO ,
2
3
DMF, rt, 12h.
Unfortunately, all attempts to synthesize the propyl analogue of 10c as well as 2,6-dihydroxy analogs
failed, and an excess cyclization due to the hydroxyl at the 2-position may explain these failures. As the
2
,5-dihydroxylated CAPE analog 10b surpassed Zileuton (1) and CAPE (2) (see LT biosynthesis
inhibition below), ketone 12 as well as analog 14 lacking the alkene moiety were synthesized to

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investigate the effects of the ester and the alkene of the α,β-carbonyl moieties. As shown in scheme 3,
the 2,5-dihydroxylated ketone analog 12 was obtained following an aldol condensation of 2,5-
dihydroxybenzaldehyde 8b and benzylacetone 11. Following our one pot esterification procedure [22],
commercially available 2,5-dihydroxybenzoic acid 13 and 2-phenylethyl bromide 5a gave the
corresponding ester 14 in good yield (scheme 3).
OH
O
O
OH
O
i
H
+
8b
11
12
OH
OH
OH
O
OH
OH
O
OH
Br
ii
O
+
14
1
3
5a
OH
Scheme 3. Synthesis of 2,5-dihydroxylated ketone 12 and dihydroxybenzoic acid analog 14. Reagents and
conditions: (i) Pyrrolidine (catalytic amount), acetic acid (catalytic amount), THF, reflux, 12h; (ii) Na CO ,
2
3
HMPA, rt, 12 h.
Biological activity
5
-LO products biosynthesis inhibition
Inhibition of LT biosynthesis was tested in HEK293 cells that are stably transfected with human 5-LO.
This model serves as a highly reproducible model of 5-LO product biosynthesis in which compounds
can be preliminarily screened for anti-LTs activity before moving on to more complex systems [32]. All
compounds were assayed at 1ꢀM, including known inhibitors Zileuton (1) and CAPE (2) (Figure 2). As
shown in Figure 2, the 2,3-dihydroxylated catechol analog 10a is more active than Zileuton (1) and as
active as than CAPE (1). While the 2,4- and 3,5-dihydroxylated analogs (6 and 10c) were completely
non-active, the 2,5-dihydroxylated analog 10b was active with an inhibition at around 25% of control,
which clearly surpasses Zileuton (1). Favorable interactions of the 2,3- and 2,5-dihydroxyphenyl moiety
with the enzyme may explain this good activity. To investigate the lipophilic effect, analogs 7, 10d and
1
0e bearing an extra methylene were tested as shown in Figure 2. As previously described with 3,4-
dihydroxy phenpropyl CAPE analog [33], there is no improvement in activity with a slight increase of
lipophilicity, there is rather a decrease in the inhibitory effect of 5-LO. This is also confirmed with all
new analogs as shown in Figure 2. Only 10e, which is an analogue of 10b with an extra methylene,
retains its 5-LO inhibitory activity which can confirm that the 2,5-dihydroxy can be a better substitution.

ACCEPTED MANUSCRIPT
1
25
00
a
a
a
1
75
50
25
0
b
c
c
c
Test compounds (1 M)
Figure 2. Inhibition of 5-LO product biosynthesis by 6, 7, and 10a-e in HEK293 cells. Values are the means ±
SEM of at least three independent experiments, each performed in duplicate; values with different superscripts are
significantly different (p < 0.05) as determined by one-way ANOVA test with subsequent Tukey’s adjustment.
In order to explore the effect of the unsaturated carbonyl moiety and ketone, 2,5-dihydroxy analogs 12
and 14 were tested (Figure 3). The negligible inhibitory effect of the ester analog 14 with the missing
unsaturation clearly demonstrates the importance of this moiety for 5-LO inhibition with this series. On
the other hand, the ketone analogue 12 was equivalent to its ester analog 10b and is probably more
stable as ketones are not hydrolysable like the esters.
Figure 3. Inhibition of 5-LO product biosynthesis by 10b, 12, and 14 in HEK293 cells. Values are the means ±
SEM of at least three independent experiments, each performed in duplicate; values with different superscripts are
significantly different (p < 0.05) as determined by one-way ANOVA test with subsequent Tukey’s adjustment.
To further probe the inhibitory activity of 10a, 10b and 12 selected from preliminary HEK293 cell
screenings (Figures 2 and 3), 5-LO inhibition assays were undertaken in stimulated primary human
immune cells. 5-LO is highly expressed in polymorphonuclear leukocytes (PMNL) and these cells are
4
important physiological producers of LTB [21, 34]. Compound 10b was found to be the best inhibitor

ACCEPTED MANUSCRIPT
exceeding the inhibitory capacity of both Zileuton (1) and CAPE (2) in this model. The presence of the
hydroxyl groups in positions 2 and 5 seems to be an ideal combination for inhibition of 5-LO (Figure 4).
These structural changes may influence the availability, cell permeation or even the stability of
inhibitors in PMNL versus HEK293 cells.
Figure 4. Inhibition of 5-LO product biosynthesis by CAPE (2), 10a, 10b, and 12 in PMNL cells. Values are the
means ± SEM of at least three independent experiments, each performed in duplicate; values with different
superscripts are significantly different (p < 0.05) as determined by one-way ANOVA test with subsequent
Tukey’s adjustment.
The 2,5-dihydroxyphenyl substitution of 10b makes it likely to be less sensitive to sulfation and
glucuronidation than a catechol such as CAPE (2) and 10a. Although less active than the ester analog,
the ketone 12 was equivalent to CAPE (2) and significantly more active than Zileuton (1). Selected
compounds with inhibition of at least 50% at 1µM in PMNL were screened further through
concentration-response assays in PMNL and the superiority of the inhibitory activity of 10b was
confirmed in dose response experiments (Table 1). Compound 10b inhibited 5-LO in a dose-dependent
manner and outperformed both Zileuton (1) and CAPE (2). With an IC50 of 0.33 µM, this 2,5-
dihydroxycinnamic acid phenylethyl ester was 3-fold more potent than CAPE (2) and 7-fold more potent
than Zileuton (1), the only clinically approved 5-LO inhibitor. Compound 10b was 2.7-fold more potent
than the 2,3-dihydroxyl substituted compound 10a which confirms that the 2,5-dihydroxy is a better
substitution. Compound 12, the 2,5-dihydroxy ketone analog, was 1.8-fold more powerful than CAPE
(2) and four times more active than Zileuton (1) (IC50 = 2.3 µM).

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Table 1. IC50: 5-LO inhibition in human PMNL cells.
Compounds
PMNL stimulation IC50 (µM)
Zileuton (1)
Mean
CI
2.31
1.96 - 2.72
CAPE (2)
Mean
CI
0.97
0.86 - 1.10
1
1
1
0a
0b
2
Mean
CI
0.89
0.69 - 1.1
Mean
CI
0.33
0.30 - 0.36
Mean
CI
0.53
0.44 - 0.65
Values are means from 3 independent experiments, each performed in duplicate.
CI = 95% confidence interval.
Measurement of the anti-radical activity of the most active analogs, as shown in Table 2, indicates that
this ability does not appear to be an important factor for discriminating the degree of 5-LO inhibition.
Indeed, with anti-radical activities similar to those of CAPE (2), 12 and 14, 10b was nevertheless the
most powerful 5-LO inhibitor.
Table 2. Free Radical Scavenging Activity
Compounds
CAPE (2)
IC 50 (µM)
18.20
Mean
CI
15.62 - 21.20
1
1
1
0b
2
Mean
CI
16.41
14.37 - 18.75
Mean
CI
15.04
11.92 - 18.96
4
Mean
CI
17.33
14.66 - 20.50
Vitamin C
Mean
CI
13.94
12.18 - 15.94
Zileuton (1)
>100
Values are means from 3 independent experiments, each performed in triplicate.
CI = 95% confidence interval

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Molecular Docking
Ketone 12 scored the best affinity for 5-LO with -9.1 kcal/mol and a hydrogen bond with His600 (OH
··· N: 3.04 Å) is detected by LigPlot+ (Table 3). His 600 may be necessary for the positioning of
arachidonic acid [35]. Compound 12 also does π-π interactions with His367 and His372, both of which
coordinate the iron atom [36] and His372 may act as a replaceable [37] coordinating ligand for the iron
atom. This would place 12 in a good position to coordinate the iron atom or at a minimum block some
access to the binding site cavity. Analog 10b (-8.8 kcal/mol) makes very similar interactions as 12
hydrogen bond with His600 (OH ··· N: 3.22 Å) and a π-π interaction with His372 (Table 3, Figure 5).
Table 3. Molecular modeling results for modeling affinity, π-π interactions and hydrogen bonds.
Compounds
Affinity
kcal/mol)
π-π
H-Bond
Distance
(Å)
(
1
1
0b
2
-8.8
-9.1
-8.8
His372
His367, His372 His600
His372
Phe421
His600
3.22
3.04
3.07
2.50, 3.15,
CAPE (2)
(
Leu420
Leu420 x 2, Asn425
R) Zileuton (1) -6.6
3
.29
(
S) Zileuton (1) -6.5
His367
Leu420 x 2, Ala424,
Phe421
2.81, 3.09,
3.34, 2.97
This would also positions 10b to be in a considerably good orientation to coordinate the iron atom or at
a minimum block some access to the binding site cavity. CAPE (2) (-8.8 kcal/mol) does a hydrogen
bond with Leu420 (OH ··· O: 3.07 Å) and a π-π interaction with His372.
Figure 5. H-bonds and π-π Interactions predicted by AutoDock Vina docking displayed in LigPlot+ and Maestro
of 10b.
All docked ligands have the same orientation, with the substituted benzene portion pointing towards the
end of the cavity and the non-substituted portion approaching the iron atom. The ligands have a slightly

ACCEPTED MANUSCRIPT
different pose from each other. It seems that the tested ligands place themselves in an opportunistic
position to stop other ligands from entering the active site cavity or at the minimum to interact with key
residues. All docked ligands have a better affinity than Zileuton (1).
Anti-renal cancer activity
We evaluated the effects of CAPE (2), 10b, 12, 14, and STF-62247 (3) on cell viability by XTT assay in
different human kidney cancer cell lines. Clear Cell Renal Cell Carcinoma (ccRCC) RCC4, RCC10 and
7
86.0 VHL -/- and VHL +/+ cells were tested (Table 4).
Treatment of RCC4 cells with 10b significantly decreased cell viability in a dose-dependent manner.
RCC4 cells that lack the VHL tumor suppressor (RCC4 VHL-/-) were sensitive to 10b with an IC50
value of 8 µM and were 12 times more sensitive than their VHL+/+ counterparts. STF-62247 (3) (IC50
.2 M) and CAPE (2) (IC = 14.0 M) were 4.4 and 2.1 times more selective, respectively, toward
RCC4 VHL-/- cells than VHL+/+ cells.
=
1
µ
µ
5
0
Table 4. Cytotoxic effects of 10b, 12, 14, CAPE (2), and STF-62247 (3) on human renal cancer cell lines
determined by Cell Viability Assays. Mean IC values are shown with 95% confidence intervals (CI).
50
RCC4
RCC4
VHL +/+
(µM)
RCC10
VHL -/-
(µM)
RCC10
VHL +/+
(µM)
786.0
VHL -/-
(µM)
786.0
VHL +/+
(µM)
Compounds
VHL -/-
(
µM)
.0
5.9 - 10.8
10b
Mean
8
99.6
5.04
0.96
34.8
>100
-
CI
Mean
CI
29.9 - 257
3.9 - 6.4
0.9 - 1.0
21.4 -55.3
1
2
4
9
.9
12.3
9.2
5.1
20.5
19.0
7.9 - 12.3
10.4 - 14.4 11.9 - 17.4
7.4 - 10.6
15.5 - 27.0 15.6 - 23.2
1
Mean
CI
4
2.8
>100
-
14.8
3.9
>100
-
>100
-
28.2 - 64.8
12.2 - 18.0
2.9 - 5.1
CAPE
2)
STF-
2247
3)
Mean
14.0
29.1
19.8
12.6
26.8
43.8
(
CI
12.4 - 15.7 26.1 - 32.4 15.8 - 24.9 9.3 - 17.1
24.3 - 29.4 35.6 - 53.8
Mean
CI
1.2
5.30
8.6
12.6
5.7
6.9
6
1
.1 - 1.4
4.5 - 6.2
7.8 - 9.5
11.4 - 13.8
5.2 - 6.4
5.6 - 8.4
(
Values are means from 3 independent experiments, each performed in triplicate. CI = 95% confidence interval.
Compared to CAPE (2), compound 10b, having hydroxyls in position 2 and 5, was significantly more
selective towards RCC4 VHL-/- cells, which demonstrates the importance of this di-substitution. On the
other hand, with the same substitution, but in the form of ketone or ester of the corresponding benzoic

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acid, the analogs 12 and 14 were less selective toward RCC4 cells. The VHL-deficient 786.0 cells were
less sensitive to all compounds. These cells were unaffected by 14 while CAPE (IC = 26.8
µ
M) and
5
0
1
0b (IC50 = 34.80 µM) showed similar potency. The test compounds showed varying activity and
selectivity against RCC10 VHL-positive cells. As the aggressive character of the RCCs must be greater
when the VHL tumor suppressor gene is deficient, the next investigations were conducted solely in
RCC4 cells since 10b, as well as STF-62247 (3), were both selective to this VHL-negative cell line. We
also demonstrated that this cell line expresses 5-LO (Figure 6A), consistent with its expression in
primary renal cell carcinomas [14].
In order to better understand the underlying mechanism by which 10b decreased viability in ccRCC, we
examine the effect of this compound on important players in apoptosis, autophagy and DNA damage
(
Figure 6B, 6C). It is well established that the main players for the initiation and execution of the
apoptotic process are caspases [38]. Caspase-3 is one of the most important members of this family and
is activated in apoptotic cells both during extrinsic and intrinsic pathways [39]. The effect of 10b on
caspase-3 was examined. To this end, RCC4 VHL -/- and VHL +/+ cells were treated with 10b and
staurosporine, known to induce apoptosis, and caspase-3 was determined by immunoblotting using an
antibody that binds both the proenzyme and the cleaved caspase-3 (Figure 6B). The results show that
compound 10b stimulates in a dose-dependent manner the cleavage of pro-caspase-3 only in the RCC4
cells which lack the VHL tumor suppressor. No level of cleaved caspase-3 was detected in response to
STF-62247 (3) or doxorubicine, which demonstrates that these compounds have no apoptotic effect.
Although the number of RCC4 cells decreases following treatment with STF-62247 (3) or 10b, the cell
phenotype was different (Figure 6C). STF-62247 has been previously shown to eliminate VHL -/- cells
through autophagy [27]. Altogether, these results suggest that 10b was able to induce apoptosis by
activation of the caspase-3 pathway leading to cell death.
The link between inhibition of 5-LO and potential apoptotic and cytotoxic effects is not clearly
established to date. To the best of our knowledge, this is the first study to investigate polyphenol-based
5
-LO inhibitors on kidney cancer cells. By analyzing primary renal cell carcinomas and their
corresponding normal renal cortex biopsies, Faronato et al. [14] showed that 5-LO protein levels were
significantly increased in the majority of Clear Cell Renal Cell Carcinoma (CC-RCCs), and we confirm
that 5-LO is expressed in RCC4 and RCC4/VHL cells (Figure 6A).

ACCEPTED MANUSCRIPT
A
B
C
Figure 6. Compound 10b inhibits RCC4 VHL -/- cells growth via autophagic- and apoptotic-associated
pathways. (A) 5-LO and pVHL protein expressions in RCC4 and RCC4/VHL cells treated with 10b (5µM). β-
actin was used as loading control. (B) Pro-caspase3, Capase-3, p53, LC3B and p62 protein expressions in 10b-
treated RCC4 were evaluated by western blot analysis. β-actin was used as loading control. Staurosporine was
used as an apoptosis control. Doxorubicin was used as a DNA damage control. (C) Cell images following 24h
treatment with STF-62247 (3) (1.25µM), (10b) (2.5, 5, and 10 µM), and Doxorubicin (0.5µM).

ACCEPTED MANUSCRIPT
Further studies are required to evaluate whether 5-LO-targeted therapy using 5-LO inhibitors might
prove effective against RCCs. While the involvement of leukotrienes in apoptosis is not fully elucidated,
a recent epidemiological study showed asthmatic patients taking montelukast and zafirlukast, two
widely used leukotriene receptor antagonists used for asthmatic prevention and control, have lower risk
for several types of cancers compared to non-users [40]. Accordingly, in vitro studies have reported
montelukast-induced apoptosis in renal cell carcinoma, prostate cancer, testicular cancer, colon cancer,
lung cancer and neuroblastoma [41-46]. To examine whether 10b induces DNA damage, Western
blotting was employed to detect the changes of p53, a DNA damage marker which can be activated in
response to DNA damage. As shown in Figure 6B, RCC4 VHL -/- and RCC4 VHL +/+ cells exposed to
1
0b or STF-62247 (3) showed no detectable p53, which implies that DNA damage was not triggered in
the cells exposed to these two compounds (Figure 6B).
Doxorubicin was used as a positive DNA damage control and clearly showed an elevation of p53
expression. Characterization of STF-62247 (3) on the autophagic flux recently showed that this
compound blocks late stages of autophagy [47]. Inhibition of the early stage of autophagy rendered
VHL -/- cells insensitive to STF-62247 demonstrating the importance of this process in selective cell
death. To determine the effects of 10b on autophagy, western blots were employed to detect the
expression of autophagy-related proteins (Figure 6B). LC3B, a marker of autophagy, exists in two
forms: LC3B-I and LC3B-II. Briefly, the activation of autophagic pathways leads to the lipidation of
LC3B-I, converting it to its active form, LC3B-II. Once active, LC3B-II will incorporate itself in the
autophagosome membranes [48]. As expected, STF-62247 increased LC3B-I conversion to LC3B-II
following 24h treatment and this was more apparent in VHL-/- cells. Interestingly, a dose-dependent
increase of both LC3B-I and LC3B-II protein expression was observed in response to 10b which was
higher in RCC4 VHL -/- versus RCC4 VHL +/+ cells. Compared to STF-62247 (
3), 10b seems to
convert less LC3B-I to LC3B-II (Figure 6A).
The p62 protein (SQSTM1) is commonly used to study the autophagic flux because it serves as a
scaffold in autophagosomes and can then be degraded by autophagy [49]. Increased p62 expression is
often associated with inhibited autophagic flux due to lack of its degradation. As expected, increased
p62 expression was observed in STF-62247 (
3)-treated cell lines (Figure 6A). Following treatment with
1
0b, increased protein expression of p62 is observed in both RCC4 VHL-/- and RCC4 VHL +/+ cells
although the increase is stronger in RCC4 VHL +/+ cells. These results indicate that compound 10b may
block the autophagic flux in both cell lines but additional investigations would be required to further

ACCEPTED MANUSCRIPT
determine whether autophagy is important in 10b cytotoxicity, which appears to be mainly related to
apoptosis.
Conclusion
In conclusion, we synthesized a series of closely related derivatives of CAPE (2), where the two
hydroxyl positions on the phenolic acid moiety were investigated. Our results revealed that the 2,5-
dihydroxycinnamic acid phenethyl ester 10b was the best 5-LO inhibitor. The presence of the
unsaturated carbonyl moiety appears to be crucial for the 5-LO inhibition, while the ester function
affords slightly better inhibition than the ketone. Furthermore, compound 10b decreased cell viability,
induced apoptosis and may block autophagic flux in VHL-negative renal cell carcinoma (RCC4).
Therefore, we have developed a more efficient 5-LO inhibitor than CAPE (2) that can be used to
investigate the molecular mechanisms of 5-LO inhibition, apoptosis, autophagy regulation, and can
potentially be applied for future anti-inflammatory and renal cancer therapies.
Experimental section
Chemistry
All chemicals used were purchased from Aldrich (CA). Purification of compounds was carried out by
flash chromatography (CombiFlash®, Separation System SG 100C, ISCO). TLC was run on silica gel
coated aluminum sheets (SiliaPlate TLC, Silicycle®) with detection by UV light (254 nm, UVS-11,
Mineralight® shortwave UV lamp). Melting points were obtained using a MELTEMP (model 1001D)
melting point apparatus. NMR spectra were recorded on Bruker Avance III 400 MHz spectrometer.
High-resolution mass measurements were performed on Bruker® Doltonics’ micrOTOF instrument in
positive or negative electrospray
.
General procedure for the esterification of phenolic acids
To a vigorously stirred solution of Na CO (1.2 eq) in 6 ml of HMPA, 500 mg of the appropriate
2
3
phenolic acid (1 eq) is added after which the reaction vessel is put under argon atmosphere. After 30 min
of stirring, the appropriate bromide (1.1 eq) is dissolved in 1 ml of HMPA and added dropwise to the
reaction mixture over a period of 30 min. A small amount of KI is added, and the reaction vessel is
thoroughly flushed with argon gas and sealed under balloon pressure. The reaction mixture is stirred in
an ice bath for 72 hr under argon atmosphere. The resulting solution is quenched with 30 ml of ice water
and stirred for 30 min. The aqueous phase is extracted three times with AcOEt (30 ml). The organic
4
fractions are then combined, washed with brine, dried over MgSO , and concentrated. The resulting oil
is purified by flash chromatography

ACCEPTED MANUSCRIPT
Phenethyl (
Following our general procedure for the esterification of phenolic acids with 2,4-dihydroxycinnamic
acid ( ) (500 mg, 3.05 mmol, 1 eq), Na CO (384 mg, 3.62 mmol, 1.2 eq), and (2-bromoethyl)benzene
E)-3-(2,4-dihydroxyphenyl)acrylate (6)
4
2
3
(0.47 ml, 3.45 mmol, 1.1 eq), compound
6
was obtained as a white solid after flash chromatography
1
(
0%–30% AcOEt/Hex), yield = 78%, Mp = 123-125°C, Rf = 0.43 (30% AcOEt/Hex). H NMR (400
MHz, MeOD, 25°C),
δ (ppm) = 7.92-7.88 (d, 1H, J = 16 Hz, ArCH=CH-), 7.34-7.21 (m, 5H, H ), 6.41-
ar
6
3
1
3
3
.37 (d, 1H, J = 16 Hz, ArCH=CH-), 6.35-6.33 (m 2H, Har), 4.36 (t, 2H, J = 6.8 Hz, -COOCH
2
CH
2
Ar),
1
3
.01 (t, 2H, J = 6.8 Hz, -COOCH CH Ar). C NMR (100 MHz, MeOD, 25°C),
δ (ppm) = 168.80,
2
2
60.93, 158.68, 141.41, 138.08, 130.18, 128.60, 1228.09, 126.10, 113.43, 112.84, 107.44, 102.11, 64.64,
+
4.84. HRMS m/z calc. for C H O + (H ): 285.1121; found: 285.1121.
1
7
16
4
-Phenylpropyl (E)-3-(2,4-dihydroxyphenyl)acrylate (7)
Following our general procedure for the esterification of phenolic acids with 2,4-dihydroxycinnamic
acid (
4
) (500 mg, 2.77 mmol, 1 eq), Na CO (440 mg, 4.16 mmol, 1.2 eq), and (2-bromopropyl)benzene
2
3
(0.506 ml, 3.33 mmol, 1.1 eq), compound
7
was obtained as a white solid after flash chromatography
1
(
0%–30% AcOEt/Hex), yield = 68%, Mp = 119-120°C, Rf = 0.28 (30% AcOEt/Hex). H NMR (400
MHz, DMSO, 25°C),
δ (ppm) = 10.14 (s, 1H, OH), 9.87 (s, 1H, OH), 7.79 (d, 1H, J = 16 Hz,
ArCH=CH-), 7.43 (d, 1H, J = 8.6 Hz, Har), 7.31-7.17 (m, 5H, Har), 6.42-6.38 (d, 1H, J= 16 Hz,
ArCH=CH-), 6.37 (d, 1H, J = 2.5 Hz, H ), 6.28 (dd, 1H, J = 2.5, 8.5 Hz, H ), 4.10 (t, 2H, J = 6.6 Hz, -
ar
ar
COOCH
2
CH
2
CH
2
Ar), 2.68 (t, 2H, J= 7.9 Hz, -COOCH
2
CH
2
CH
2
Ar), 1.93 (quint, 2H, J = 6.64 Hz, -
1
3
COOCH CH CH Ar). C NMR (100 MHz, DMSO, 25°C),
δ (ppm) = 167.69, 161.42, 158.99, 141.68,
2
2
2
1
40.97, 130.88, 128.81, 128.76, 126.33, 113.38, 113.05, 108.27, 102.93, 63.32, 31.95, 30.43. HRMS
+
m/z calc. for C18
H
18
O
4
+ (H ): 299.1278; found: 299.1283.
General procedure for the synthesis of the 2-chloroacetate esters
Under inert atmosphere, to a solution of 2-phenylethanol or 3-phenylpropanol and pyridine (2.2 eq) in
dry dichloromethane (20 mL) was added drop wise at 0 °C 2-chloroacetyl chloride (2.2 eq), after the
complete addition (30 min) the mixture was then stirred at room temperature for 3 h. TLC indicated the
complete consumption of alcohol. The formed pyridinium was filtered and the mixture was then washed
with 5% hydrochloride solution (3 × 50 mL), water (2 × 50 mL) and brine (50 mL), dried over MgSO4

ACCEPTED MANUSCRIPT
and concentrated on a rotary evaporator. The residue was purified by column chromatography eluting
with hexane to gradually increase the polarity of ethyl acetate to obtain the desired ester.
Phenethyl 2-chloroacetate
Following our general procedure for the synthesis of the 2-chloro acetate ester with 2-phenylethanol (2
g, 8.18 mmol) and pyridine (1.45 mL, 18 mmol) and 2-chloroacetyl chloride (1.43 mL, 18 mmol),
phenethyl 2-chloroacetate was obtained as a colorless liquid after flash chromatography, yield = 70%, Rf
1
=
0.45 (10% AcOEt/Hex). H NMR (400 MHz, CDCl )
δ
(ppm) = 7.30 (m, 5H, H ), 4.43 (t, 2H, J = 7.2
3
ar
1
3
Hz, CH
2
-O), 4.06 (s, 2H, CH
2 2 3
-Cl), 3.01 (t, 2H, J = 7.2 Hz, CH -Ph). C NMR (101 MHz, CDCl ), δ
(
(
ppm) = 167.25, 137.18, 128.91, 128.61, 126.78, 66.58, 40.86, 34.92. HRMS m/z calc. for C H ClO +
1
1
13
2
+
4
NH ): 216.0786; found: 216.0788.
Phenylpropyl 2-chloroacetate
Following our general procedure for the synthesis of the 2-chloro acetate ester with 3-phenylpropanol (1
g, 7.34 mmol) and pyridine (0.65 mL, 8.1 mmol) and 2-chloroacetyl chloride (0.64 mL, 8.1 mmol),
phenylpropyl 2-chloroacetate was obtained as a colorless liquid after flash chromatography, yield =
1
5
2
2%, Rf = 0.78 (50% AcOEt/Hex). H NMR (400 MHz, CDCl
3
)
δ
(ppm) = 7.27 (m, 5H, HAr), 4.24 (t,
H, J = 6.4 Hz, CH -O), 4.06 (s, 2H, CH -Cl), 2.73 (t, 2H, J = 7.6 Hz, CH -Ph), 2.04 (m, 2H, CH -CH -
2
2
2
2
2
13
CH ). C NMR (101 MHz, CDCl ),
δ
(ppm) = 167.34, 140.84, 128.51, 128.40, 126.14, 65.54, 40.87,
2
3
+
3
2.02, 29.96. HRMS m/z calc. for C11
H
13ClO
2
+ (NH
4
): 230.0942; found: 230.0935.
General procedure for the synthesis the phosphonium ylids
Under inert atmosphere, to a solution of triphenylphosphine in benzene (5 mL) was added phenylethyl
or phenpropyl 2-chloroacetate (1.1 eq). The reaction mixture was stirred for one week at room
temperature and then washed with anhydrous diethyl ether to afford the corresponding phosphonium
chloride
Phosphonium, [2-oxo-2-(2-phenylethoxy)ethyl]triphenyl chloride (9a
, which was used in the next step without purification.
)
Following our procedure for the synthesis of the phosphonium chlorides with phenylethyl 2-
chloroacetate (1.5 g, 7.55 mmol) and triphenylphosphine (1.8 g, 6.86 mmol) compound 9a was obtained
1
as a white solid, mp: 174 °C, yield = 74%. H NMR (400 MHz, CDCl )
δ (ppm) = 7.76 (td, 3H, J = 7.2
3
Hz, 1.5 Hz, Har), 7.52 (m, 13H, Har), 7.14 (d, 2H, J = 2.7 Hz, Har), 6.86 (dd, 2H, J = 2.0, 3.6 Hz, Har
)
1
3
4
.70 (s, 2H, CH -P), 4.18 (t, 2H, J = 5.8 Hz, CH -O), 2.62 (t, 2H, J = 5.9 Hz, CH -Ph). C NMR (101
2
2
2
MHz, CDCl
3
),
δ
(ppm) = 164.91, 164.87, 137.12, 134.95, 134.92, 134.07, 133.96, 130.25, 130.12,

ACCEPTED MANUSCRIPT
1
2
28.85, 128.58, 126.65, 118.73, 117.84, 67.10, 34.52, 33.05, 32.490. HRMS m/z calc. for C28H26ClO P
+
+
(+K - H O): 446.1196; found: 445.1214.
2
Phosphonium [2-oxo-2-(2-phenylpropoxy)ethyl]triphenyl chloride (9b
)
Following our procedure for the synthesis of the phosphonium chlorides with phenylpropyl 2-
chloroacetate (0.75 g, 3.52 mmol) and triphenylphosphine (0.84 g, 3.2 mmol) compound 9b was
1
obtained as a white solid, yield = 48%. H NMR (400 MHz, CDCl )
δ (ppm) = 7.94 (dd, 6H, J = 7.6,
3
1
3.2 Hz, H ), 7.71 (m, 9H, H ), 7.22 (m, 3H, H ), 7.05 (d, 2H, J =7.2 Hz, H ), 5.82 (d, 2H, J = 14 Hz,
ar
ar
ar
ar
CH
2
-P), 3.99 (t, 2H, J = 6.4 Hz, CH
2
2 2 2 2
-O), 2.49 (t, 2H, J = 7.2 Hz, CH -Ph), 1.76 (m, 2H, CH -CH -CH ).
1
3
C NMR (101 MHz, CDCl3),
δ (ppm) = 164.94, 164.90, 140.64, 135.05, 135.02, 134.09, 133.98,
1
30.29, 130.16, 128.43, 128.34, 126.06, 118.71, 117.83, 66.02, 33.12, 32.56, 31.77, 29.61. HRMS m/z
+
calc. for C H ClO P + (H ): 440.1880; found: 440.1900.
2
9
28
2
General Procedure for Witting coupling
Under inert atmosphere, to a solution of the appropriate phosphonium chloride (1 eq) and potassium
carbonate (4.5 eq) in dry DMF (5 mL) was added the appropriate dihydroxybenzaldehyde (1.1 eq). The
mixture was then stirred at room temperature overnight. TLC indicated the complete consumption of the
phosphonium salt. The mixture was then diluted with 5% hydrochloride in brine solution (50 mL),
extracted with ethyl acetate (3×50 mL), washed successively with 5% hydrochloride in brine solution
(
50 mL), water (2×50 mL) and brine (50 mL), dried over MgSO
evaporator. The residue was purified by column chromatography eluting with hexane to gradually
increase the polarity of ethyl acetate. The coupling constants of double bonds ( = 16 Hz) confirmed the
geometry of 10a and 12
)-phenethyl 3-(2,3-dihydroxyphenyl)acrylate (10a
4
and concentrated on a rotary
J
E
-
e
.
(E
)
Following our general procedure for the Wittig couplig with phosphonium [2-oxo-2-(2-phenylethoxy)
ethyl]triphenyl chloride (0.4 g ,0.86 mmol), potassium carbonate (0.53 g, 3.87 mmol 4.5 eq) and 2,3-
dihydroxybenzaldehyde (0.13 g, 0.94 mmol) compound 10a was obtained as a yellow oil after flash
1
chromatography, yield = 58%, Rf = 0.12 (20% AcOEt/Hex). H NMR (400 MHz, CDCl )
δ (ppm) =
3
7
.98 (d, 1H, J = 16.1 Hz, CH=CH-C=O), 7.36-7.24 (m, 5H, H ), 7.06 (dd, 1H, J = 1.1, 7.8 Hz, H ), 6.89
ar ar
(
dd, 1H, J = 1.1, 7.8 Hz, Har), 6.79 (t, 1H, J= 7.8 Hz, Har), 6.60 (d, 1H, J = 16.1 Hz, CH=CH-C =O), 6.15
s br, 1H, OH), 5.95 (s br, 1H, OH), 4.45 (t, 2H, J = 7.1 Hz, CH -O), 3.04 (t, 2H, J= 7.1 Hz, CH -
(
2
2
1
3
3
Ph). C NMR (101 MHz, CDCl ), δ (ppm) = 167.95, 144.32, 143.37, 140.13, 137.84, 128.97, 128.54,

ACCEPTED MANUSCRIPT
+
1
3
26.59, 121.75, 121.06, 120.16, 118.83, 116.70, 65.23, 35.20. HRMS m/z calc. for C17
07.0941; found: 307.0940.
16 4
H O + (Na ):
(E)-Phenethyl 3-(2,5-dihydroxyphenyl)acrylate (10b)
Following our general procedure for the Wittig couplig with phosphonium [2-oxo-2-(2-phenylethoxy)
ethyl]triphenyl chloride (0.4 g ,0.86 mmol), potassium carbonate (0.53 g, 3.87 mmol 4.5 eq) and 2,5-
dihydroxybenzaldehyde (0.13 g, 0.94 mmol) compound 10b was obtained as a yellow oil after flash
1
chromatography, yield = 75%, Rf = 0.32 (40% AcOEt/Hex). H NMR (400 MHz, CDCl )
δ (ppm) =
3
9
.54 (s br, 1H, OH), 8.90 (s br, 1H, OH) 7.79 (d, 1H, J= 16 Hz, CH=CH-C =O), 7.27 (m, 5H, H ), 6.90
ar
(
d, 1H, J = 2.8 Hz, H ), 6.71 (m, 2H, H ), 6.41 (d, 1H, J = 16 Hz, CH=CH-C =O), 4.33 (t, 2H, J = 6.8
ar ar
1
3
Hz, CH -O), 2.96 (t, 2H, J = 6.8 Hz, CH -Ph). C NMR (101 MHz, DMSOd ),
δ (ppm) = 167.04,
2
2
6
1
3
50.37, 150.19, 140.79, 138.55, 129.35, 128.83, 126.83, 121.23, 119.86, 117.45, 116.99, 113.93, 64.86,
+
4.94. HRMS m/z calc. for C H O + (H ): 285.1121; found: 285.1113.
1
7
16
4
(E)-Phenethyl 3-(3,5-dihydroxyphenyl)acrylate (10c)
Following our general procedure for the Wittig couplig with phosphonium, [2-oxo-2-(2-phenylethoxy)
ethyl]triphenyl chloride (400 mg ,0.86 mmol), potassium carbonate (0.53 g, 3.87 mmol 4.5 eq) and 3,5-
dihydroxybenzaldehyde (132 mg, 0.94 mmol) compound 10c was obtained as a white solid after flash
1
chromatography, yield = 69%, Mp = 140-142°C, Rf = 0.38 (40% AcOEt/Hex). H NMR (400MHz,
CDCl , 25°C),
δ
(ppm) = 9.45 (s, 2H, OH), 7.41(d, 1H, J = 16 Hz, ArCH=CH-), 7.34-7.23 (m, 5H, Har),
3
6
.48 (m, 2H, H ), 6.36 (m, 1H, ArCH=CH-), 6.32-6.29 (m, 1H, H ), 4.33 (t, 2H, J = 6.9 Hz, -
ar
ar
1
3
COCH CH -), 2.95 (t, 2H, J = 6.9 Hz, -COCH CH -). C NMR (100MHz, CDCl , 25°C),
δ (ppm) =
2
2
2
2
3
1
66.53, 159.16, 145.60, 138.50, 136.00, 129.37, 128.84, 126.85, 117.83, 106.74, 105.40, 65.02, 34.89.
+
HRMS m/z calc. for C H O + (H ): 285.1121; found: 285.1109.
1
7
16
3
(E)-3-Phenylpropyl 3-(2,3-dihydroxyphenyl)acrylate (10d)
Following our general procedure for the Wittig coupling with phosphonium, [2-oxo-2-(2-
phenylpropoxy)ethyl]triphenyl-, chloride (0.35 g, 0.73 mmol), potassium carbonate (0.45 g, 3.28 mmol)
and 2,3-dihydroxybenzaldehyde (0.11 g, 0.8 mmol) compound 10d was obtained as a yellow oil after
1
flash chromatography, yield = 67%, Rf = 0.43 (40% AcOEt/Hex). H NMR (400 MHz, CDCl )
δ (ppm)
3
=
8.01 (d, 1H, J =16.4 Hz, CH=CH-C=O), 7.22 (m, 5H, H ), 7.01 (dd, 1H, J = 0.8, 8 Hz, H ), 6.83 (dd,
Ar ar
1
2
H, J = 1.4, 7.6 Hz, H ), 6.70 (t, 1H, J = 8.0 Hz, H ), 6.58 (d, 1H, J = 16.4 Hz, CH=CH-C =O), 4.19 (t,
ar ar
13
H, J = 6.4 Hz, CH -O), 2.75 (t, 2H, J = 7.2 Hz, CH -Ph), 2.02 (m, 2H, CH -CH -CH ). C NMR (101
2
2
2
2
2

ACCEPTED MANUSCRIPT
MHz, DMSOd
6
),
δ
(ppm) = 168.19, 145.60, 145.31, 141.26, 140.90, 128.05, 125.59, 121.33, 119.03,
1
16.87, 116.16, 63.43, 48.25, 48.04, 47.83, 47.61, 47.40, 47.19, 46.97, 31.79, 30.22. HRMS m/z calc.
+
for C H O + (H ): 299.1278; found: 299.1279.
1
8
18
4
(E)-3-Phenylpropyl 3-(2,5-dihydroxyphenyl)acrylate (10e)
Following our general procedure for the Wittig coupling with phosphonium, [2-oxo-2-(2-
phenylpropoxy)ethyl]triphenyl, chloride (0.35 g, 0.73 mmol), potassium carbonate (0.45 g, 3.28 mmol)
and 2,5-dihydroxybenzaldehyde (0.11 g, 0.8 mmol) compound 10e was obtained as a yellow oil after
1
flash chromatography, yield = 53%, Rf = 0.35 (40% AcOEt/Hex). H NMR (400 MHz, CDCl )
δ (ppm)
3
=
7.95 (d, 1H, J =16 Hz, CH=CH-C =O), 7.23 (m, 5H, Har), 6.93 (s br, 1H, Har), 6.72 (s br, 1H, Har),
6
.50 (d, 1H, J = 16 Hz, CH=CH-C =O), 4.19 (t, 2H, J = 6.4 Hz, CH -O), 2.75 (t, 2H, J = 7.2 Hz, CH -
2
2
1
3
Ph), 2.03 (m, 2H, CH
2
-CH
2
-CH
2
). C NMR (101 MHz, DMSOd
6
),
δ
(ppm) = 168.11, 150.12, 149.91,
1
4
41.26, 140.71, 128.08, 125.59, 121.47, 118.82, 116.55, 113.27, 63.45, 53.39, 48.24, 47.81, 47.60,
+
7.38, 47.17, 46.96, 31.79, 30.21. HRMS m/z calc. for C H O + (H ): 299.1278; found: 299.1277.
1
8
18
4
(E)-1-(2,5-Dihydroxyphenyl)-5-phenylpent-1-en-3-one (12)
Pyrrolidine (catalytic amount) and acetic acid (catalytic amount) were added to a solution of 4-
phenylbutan-2-one 11 (3 mmol) and 2, 5-dihydroxy benzaldehyde (3.2 mmol) and in THF (20 mL). The
reaction mixture was concentrated in vacuo, diluted with H O (30 mL) and extracted with ethyl acetate
2
(
3 X 30 mL). The combined organic fractions were washed with brine, dried over MgSO , and
4
concentrated under reduced pressure. Compound 12 was obtained as a yellow solid after flash
chromatography (0%–30% AcOEt/Hex), yield = 73%, Mp = 160-162°C, Rf = 0.43 (30% AcOEt/Hex).
1
H NMR (400MHz, DMSO-d
6
, 25°C), δ (ppm) = 9.70 (s, 1H, OH), 8.90 (s, 1H, OH), 7.75(dd, 1H, J =
2
, 16 Hz, ArCH=CH-), 7.29-7.24 (m, 4H, H ), 7.19-7.15 (m, 1H, H ), 6.94 (s, 1H, m, 1H, H ), 6.77 (d,
ar
ar
ar
J = 16 Hz, ArCH=CH-), 6.72-6.69 (m, 2H, H ), 3.00-2.96 (m, 2H, -COCH CH -), 2.89-2.85 (m, 2H, -
ar
2
2
1
3
COCH
2
CH2-). C NMR (100MHz, DMSO-d
6
, 25°C),
δ
(ppm) = 150.43, 150.39, 141.81, 137.96,
1
28.79, 128.73, 126.29, 125.61, 121.60, 119.92, 117.49, 113.52, 42.11, 30.03. HRMS m/z calc. for C H
17
+
1
0
O
3
4
+ (NH ): 280.0968; found: 280.0981.
Phenethyl 2,5-dihydroxybenzoate (14
)
Following our general procedure for the esterification of phenolic acids with 2,5-dihydroxybenzoic acid
2 3
(13) (500 mg, 3.24 mmol, 1 eq), Na CO (415 mg, 3.91 mmol, 1.2 eq), and (2-bromoethyl)benzene 5a
(0.5 ml, 3.8 mmol, 1.1 eq), compound 14 was obtained as a white solid after flash chromatography (0%–
1
2
0% AcOEt/Hex), yield = 68%, Mp = 85-88°C, Rf = 0.25 (30% AcOEt/Hex). H NMR (400 MHz,

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CDCl
3
, 25°C),
δ
(ppm) = 10.34 ( s, 1H, OH), 7.37-7.34 (m, 2H, Har), 7.30-7.27 (m, 3H, Har), 7.26-7.25
(
m, 1H, H ), 7.03-7.00 (m, 1H, H ), 6.91-6.88 (m, 1H, H ), 4.57 (t, 2H, J = 7Hz, -COOCH CH -), 4.55
ar
ar
ar
2
2
1
3
(
s, 1H, OH), 3.10 (t, 2H, J = 7Hz, -COOCH CH ). C NMR (100 MHz, CDCl , 25°C),
δ
(ppm) =
2
2-
3
1
69.59, 155.99, 147.60, 137.39, 128.89, 128.68, 126.80, 124.06, 118.55, 114.70, 112.27, 65.87, 35.05.
+
HRMS m/z calc. for C H O + (Na ): 281.0784; found: 281.0779.
1
5
14
4
Biological evaluation
-LO product biosynthesis assays in HEK293 cells
5
HEK293 cells were stably co-transfected with a pcDNA3.1 vector expressing 5-LO and a pBUDCE4.1
vector expressing 5-LO activating protein (FLAP) as previously reported [32, [21]. The resulting stable
double transfectants were propagated in culture and aliquots were frozen. Once thawed for a series of
experiments, each aliquot of cells is cultured for a maximum of 6 weeks before being discarded. For cell
stimulation of 5-LO products, transfected HEK293 cells were collected following trypsinization, washed
and the cell pellet was resuspended in Hank’s balanced salt solution (HBSS) (Lonza, Walkerville, MD)
5
-1
containing 1.6 mM CaCl at a concentration of 5 x 10 cells mL . Cells were pre-incubated with each
2
o
compound or their diluent at the indicated concentration for 5 min at 37 C. Cells were then stimulated
o
for 15 minutes at 37 C with the addition of 10 µM calcium ionophore A23187 (Sigma–Aldrich,
Oakville, ON, Canada) and 10 µM arachidonic acid (Cayman Chemical, Ann Arbor, MI). Stimulation
was stopped and processed for RP-HPLC analysis as described previously [21].
5
-LO products biosynthesis assays in human PMNL cells
Human PMNL prepared from peripheral blood of healthy consenting volunteers as described [21] were
7
suspended in HBSS containing 1.6 mM CaCl
2
(10 cells/ml) and pre-incubated with compounds or their
o
diluent for 5 min at 37 C in the presence of 0.4 U/ml of adenosine deaminase (Sigma-Aldrich,
o
Oakville, On, Canada). Cells were then stimulated for 15 min at 37 C with 1 µM thapsigargin (Sigma-
3
Aldrich) [21]. Reactions were stopped by the addition of 0.5 mL of cold MeOH:CH CN (1:1) and 50 ng
o
of PGB as internal standard and samples were stored at -20 C until processing for RP-HPLC analysis
2
as indicated above.
Antiradical activity
In a serial dilution, 200, 100, 50, 25, 12.5, 6.25, 3.125
0b 12, and 14 were plated in a 96-well micro plate. Afterwards, 200
well, incubated for 30 minutes in the dark with slight agitation. The absorbance was measured at
17 nm. Antiradical activity (% of DPPH inhibition) was calculated using the following formula*:
µ
M of Vitamin C (positive control), CAPE (
2),
1
,
µ
M of DPPH was added to each
5

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(
ꢁ0) − (ꢁ1)
ꢁꢂꢃꢄꢅꢄꢃꢆꢇ% =
ꢇꢈꢇ100
(
ꢁ0)
*
A0 : DPPH and ethanol, A1 : DPPH + compound
Mammalian cell culture, treatment and imaging
The human ccRCC cell lines (RCC4, RCC10 and 786.0) and their isogenic counterparts stably
expressing VHL were kindly provided by Amato J. Giaccia (Stanford University, CA, USA). Cells were
maintained in Dulbeco’s Modified Eagle Medium (DMEM) high glucose medium, supplemented with
1
0% fetal bovine serum (FBS) (Wisent BioScience, QC, Canada), 2 mM L-glutamine and 1 mM sodium
pyruvate (GE Health Life Sciences, ON, Canada). All cell lines were tested for mycoplasma and
authentication were performed by short tandem repeat (STR) DNA profile at Genetica DNA
Laboratories (Burlington, NC, USA). Cells were seeded in 96-well microplates and treated for 24 hours
with STF-62247 (3) (Cayman Chemical co, MI, USA) as well as CAPE (2) and the best 5-LO inhibitors:
1
0b 12, and 14 Staurosporine and Doxorubicine were used as controls in specified experiments. All cell
,
.
imaging was taken with a Nikon TMS-F inverted microscope.
Cell viability assays (XTT)
3
3
3
Cell lines were seeded at 5x10 , 5x10 and 3x10 cells per 96 wells for RCC4, RCC10 and 786.0 cells,
respectively, and were treated with increasing concentrations of STF-62247 ( ), CAPE ( ), 10b 12, and
. After 4 days incubation, a solution consisting of 0.3 mg/ml of XTT (Invitrogen, X6493), phenol red-
free media (Wisent, 319-051-CL), 20% FBS and 2.65 g/ml of N-methyl dibenzopyrazine methyl
3
2
,
1
4
µ
sulfate (PMS) (Sigma Aldrich, ON, Canada) was added to the cells and incubated for 1h at 37°C. The
absorbance was read at 450 nm on a Spectramax Plus spectrophotometer (Molecular Devices, USA).
IC50 values were calculated using a non-linear regression with GraphPad Prism.
ꢍꢎꢏꢊꢃꢏꢐ − ꢑꢒꢓꢔ
ꢉꢄꢊꢋꢄꢌꢄꢃꢆꢇ =
ꢇꢈꢇ100
ꢕꢖꢗꢃꢎꢖꢌ − ꢑꢒꢓꢔ
Western blot assay
Total proteins were extracted using M-PER lysis buffer (50 mM Tris (HCl) pH 7.5, 200 mM NaCl,
.25% Triton X-100, 10% Glycerol) containing 1X protease and phosphatase inhibitor cocktails. Cell
lysates were centrifuged at 12,000 x g for 10 min at 4˚C and quantified by BCA protein assay kit
Thermo Fisher scientific) using the SpectraMax Plus 384 microplate reader. Proteins (30µg) were
separated on 10-15% SDS/PAGE gel and transferred onto a 0.45 micron PVDF transfer membrane
0
(

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(
Immobilon-P, Millipore, IE). Membranes were blocked using 5% skim milk diluted in a solution of
0
.075% PBS-Tween (PBS-T) and incubated overnight in 3% BSA with specific primary antibodies: 5-
LO (Cell Signaling, 3289), pVHL (Cell Signaling, 68547), Caspase-3 (Abcam, 32351), p53 (Cell
Signaling, 2527T), p62 (SQSTM1) (Novus, NBP1-48320SS), LC3B (Cell Signaling, 3868), -Actin
Santa Cruz, E3013). Immunoblots were washed and incubated with HRP-conjugated secondary
β
(
antibodies diluted in 5% skim milk PBS-T (Jackson Immunoresearch Laboratories, PA, USA) and
visualized using the Amersham ECL Primer Western Blotting Detection reagent (GE Healthcare Life
Sciences, ON, Canada) on a Chemidoc MP Imager (BIO-RAD, ON, Canada).
Statistical analysis
Statistical analyses and graph design were performed using GraphPad Prism 5 software (GraphPad
Software, San Diego, California).
Molecular docking
Molecular docking was undertaken with AutoDock Vina software [50]. The protocol established by De
Lucia et al [51] was adapted to complete the molecular docking study. This is the same protocol
followed in a previous study [22]. In short, the chosen structure was PDB: 3O8Y [36]. It is currently the
best available structure. The crystal does have 2 minor issues, but it is still judged usable [51]. A minor
number of mutations have been done to give the protein some stability to enable crystallization. These
are not situated near the active site and should not have any impact on the results [36]. Similarly, the
crystal structure is in apo. To help diminish the impact of this, the structure was minimized according to
the protocol reference in De Lucia et al [51]. Ligands were drawn and minimized before modeling.
Settings were taken from the established protocol kept to the default values. The best generated pose
+
was selected for analysis. Results were analyzed with Maestro (Maestro, 2016) and LigPlot (Laskowski
and Swindells, 2011; “LigPlot+ v.1.4 - multiple ligand-protein interaction diagrams for drug discovery,”
n.d.) Along with binding affinity generated by AutoDock Vina.
Acknowledgments
M.T. would like to acknowledge the contribution of the Natural Sciences and Engineering Research
Council of Canada (NSERC) and Université de Moncton. MT and M.E.S. Acknowledge the support of
the New Brunswick Innovation Foundation (NBIF) and the Canadian Foundation for Innovation (CFI),
M.E.S. was supported by the NBIF Innovation Research Chairs program. S.T., P.L., and S.D would like

ACCEPTED MANUSCRIPT
to acknowledge the support of NBIF through the research technician initiative. S.T is supported by a
Research Chair of the Canadian Cancer Society.
References:
[
1] E. Gatza, D.R. Wahl, A.W. Opipari, T.B. Sundberg, P. Reddy, C. Liu, G.D. Glick, J.L. Ferrara, Manipulating the
bioenergetics of alloreactive T cells causes their selective apoptosis and arrests graft-versus-host disease, Sci.
Transl. Med., 3 (2011) 67ra68-67ra68.
[
2] F. Caspary, G. Elliott, B. Navé, P. Verzaal, M. Rohrbach, P. Das, L. Nagelkerken, J. Nieland, A new therapeutic
approach to treat psoriasis by inhibition of fatty acid oxidation by Etomoxir, Br. J. Dermatol., 153 (2005) 937-
44.
9
[
[
[
3] E.A. Dennis, P.C. Norris, Eicosanoid storm in infection and inflammation, Nat. Rev. Immunol., 15 (2015) 511.
4] M. Peters-Golden, W.R. Henderson Jr, Leukotrienes, N. Engl. J. Med., 357 (2007) 1841-1854.
5] M. Romano, J. Clària, Cyclooxygenase-2 and 5-lipoxygenase converging functions on cell proliferation and
tumor angiogenesis: implications for cancer therapy, FASEB J., 17 (2003) 1986-1995.
6] P. Praveen Rao, Q.-H. Chen, E.E. Knaus, Synthesis and structure− acꢀvity relaꢀonship studies of 1, 3-
diarylprop-2-yn-1-ones: dual inhibitors of cyclooxygenases and lipoxygenases, J. Med. Chem., 49 (2006) 1668-
683.
7] J.A. Baron, R.S. Sandler, R.S. Bresalier, A. Lanas, D.G. Morton, R. Riddell, E.R. Iverson, D.L. DeMets,
Cardiovascular events associated with rofecoxib: final analysis of the APPROVe trial, Lancet, 372 (2008) 1756-
764.
8] J.Z. Haeggstrom, C.D. Funk, Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in
disease, Chem. Rev., 111 (2011) 5866-5898.
[
1
[
1
[
[
[
[
9] A. Mantovani, P. Allavena, A. Sica, F. Balkwill, Cancer-related inflammation, Nature, 454 (2008) 436.
10] L.M. Coussens, Z. Werb, Inflammation and cancer, Nature, 420 (2002) 860.
11] I.M. Avis, M. Jett, T. Boyle, M.D. Vos, T. Moody, A.M. Treston, A. Martínez, J.L. Mulshine, Growth control of
lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling, J. Clin. Invest., 97 (1996) 806-
13.
12] M.-T. Huang, W. Ma, P. Yen, J.-G. Xie, J. Han, K. Frenkel, D. Grunberger, A.H. Conney, Inhibitory effects of
8
[
caffeic acid phenethyl ester (CAPE) on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in
mouse skin and the synthesis of DNA, RNA and protein in HeLa cells, (1996).
[
13] N. Merchant, L.V.K.S. Bhaskar, S. Momin, P. Sujatha, A.B. Reddy, G.P. Nagaraju, 5-Lipoxygenase: its
involvement in gastrointestinal malignancies, Critical reviews in oncology/hematology, (2018).
14] M. Faronato, G. Muzzonigro, G. Milanese, C. Menna, A. Bonfigli, A. Catalano, A. Procopio, Increased
expression of 5-lipoxygenase is common in clear cell renal cell carcinoma, Histol. Histopathol., 22 (2007) 1109-
118.
15] M. Gerlinger, S. Horswell, J. Larkin, A.J. Rowan, M.P. Salm, I. Varela, R. Fisher, N. McGranahan, N. Matthews,
[
1
[
C.R. Santos, Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion
sequencing, Nat. Genet., 46 (2014) 225.
[
16] J.-J. Patard, N. Rioux-Leclercq, D. Masson, S. Zerrouki, F. Jouan, N. Collet, C. Dubourg, B. Lobel, M. Denis, P.
Fergelot, Absence of VHL gene alteration and high VEGF expression are associated with tumour aggressiveness
and poor survival of renal-cell carcinoma, Br. J. Cancer, 101 (2009) 1417.

ACCEPTED MANUSCRIPT
[
17] W. Berger, M. De Chandt, C. Cairns, Zileuton: clinical implications of 5-Lipoxygenase inhibition in severe
airway disease, Int. J. Clin. Pract., 61 (2007) 663-676.
18] L. García-Marcos, A. Schuster, E.G. Pérez-Yarza, Benefit-risk assessment of antileukotrienes in the
management of asthma, Drug Saf., 26 (2003) 483-518.
19] K. Yasuko, N. Tomohiro, M. Sei-Itsu, L. Ai-Na, F. Yasuo, T. Takashi, Caffeic acid is a selective inhibitor for
leukotriene biosynthesis, Biochim. Biophys. Acta, Lipids Lipid Metab., 792 (1984) 92-97.
20] J. Baumann, F.v. Bruchhausen, G. Wurm, Flavonoids and related compounds as inhibitors of arachidonic
acid peroxidation, Prostaglandins, 20 (1980) 627-639.
21] L.H. Boudreau, J. Maillet, L.M. LeBlanc, J. Jean-François, M. Touaibia, N. Flamand, M.E. Surette, Caffeic acid
[
[
[
[
phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human
polymorphonuclear leukocytes, PLoS One, 7 (2012) e31833.
[
22] M. Touaibia, M.J. Hébert, N.A. Levesque, J.A. Doiron, M.S. Doucet, J. Jean-François, M. Cormier, L.H.
Boudreau, M.E. Surette, Sinapic acid phenethyl ester as a potent selective 5-lipoxygenase inhibitor: Synthesis
and structure–activity relationship, Chem. Biol. Drug Des., 92 (2018) 1876-1887.
[
23] J.A. Doiron, L.M. Leblanc, M.J. Hébert, N.A. Levesque, A.F. Paré, J. Jean-François, M. Cormier, M.E. Surette,
M. Touaibia, Structure–activity relationship of caffeic acid phenethyl ester analogs as new 5-lipoxygenase
inhibitors, Chem. Biol. Drug Des., 89 (2017) 514-528.
[
24] J. Doiron, L.H. Boudreau, N. Picot, B. Villebonet, M.E. Surette, M. Touaibia, Synthesis and 5-lipoxygenase
inhibitory activity of new cinnamoyl and caffeoylclusters, Bioorg. Med. Chem. Lett., 19 (2009) 1118-1121.
25] S. Torki, A. Soltani, H. Shirzad, N. Esmaeil, M. Ghatrehsamani, Synergistic antitumor effect of NVP-BEZ235
and CAPE on MDA-MB-231 breast cancer cells, Biomed. Pharmacother., 92 (2017) 39-45.
26] A.-P. Beauregard, J. Harquail, G. Lassalle-Claux, M. Belbraouet, J. Jean-Francois, M. Touaibia, G.A.
Robichaud, CAPE analogs induce growth arrest and apoptosis in breast cancer cells, Molecules, 20 (2015) 12576-
2589.
27] S. Turcotte, D.A. Chan, P.D. Sutphin, M.P. Hay, W.A. Denny, A.J. Giaccia, A molecule targeting VHL-deficient
renal cell carcinoma that induces autophagy, Cancer Cell, 14 (2008) 90-102.
28] P.D. Sutphin, D.A. Chan, J.M. Li, S. Turcotte, A.J. Krieg, A.J. Giaccia, Targeting the Loss of the <em>von
Hippel-Lindau</em> Tumor Suppressor Gene in Renal Cell Carcinoma Cells, Cancer Res., 67 (2007) 5896-5905.
29] D.A. Chan, P.D. Sutphin, P. Nguyen, S. Turcotte, E.W. Lai, A. Banh, G.E. Reynolds, J.-T. Chi, J. Wu, D.E. Solow-
[
[
1
[
[
[
Cordero, M. Bonnet, J.U. Flanagan, D.M. Bouley, E.E. Graves, W.A. Denny, M.P. Hay, A.J. Giaccia, Targeting
GLUT1 and the Warburg Effect in Renal Cell Carcinoma by Chemical Synthetic Lethality, Sci. Transl. Med., 3
(2011) 94ra70-94ra70.
[
30] C.J. Greene, N.J. Sharma, P.N. Fiorica, E. Forrester, G.J. Smith, K.W. Gross, E.C. Kauffman, Suppressive
effects of iron chelation in clear cell renal cell carcinoma and their dependency on VHL inactivation, Free
Radicals Biol. Med., (2018).
[
31] A. Saeed, P.A. Mahesar, S. Zaib, M.S. Khan, A. Matin, M. Shahid, J. Iqbal, Synthesis, cytotoxicity and
molecular modelling studies of new phenylcinnamide derivatives as potent inhibitors of cholinesterases, Eur. J.
Med. Chem., 78 (2014) 43-53.
[
32] E.P. Allain, L.H. Boudreau, N. Flamand, 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, 10
(2015) e0132607.
[
33] L.H. Boudreau, G. Lassalle-Claux, M. Cormier, S. Blanchard, M.S. Doucet, M.E. Surette, M. Touaibia, New
hydroxycinnamic acid esters as novel 5-lipoxygenase inhibitors that affect leukotriene biosynthesis, Mediators
Inflammation, 2017 (2017).
[
34] P. Borgeat, B. Samuelsson, Metabolism of arachidonic acid in polymorphonuclear leukocytes. Structural
analysis of novel hydroxylated compounds, J. Biol. Chem., 254 (1979) 7865-7869.
35] S. Mitra, S.G. Bartlett, M.E. Newcomer, Identification of the substrate access portal of 5-lipoxygenase,
Biochemistry, 54 (2015) 6333-6342.
[

ACCEPTED MANUSCRIPT
[
36] N.C. Gilbert, S.G. Bartlett, M.T. Waight, D.B. Neau, W.E. Boeglin, A.R. Brash, M.E. Newcomer, The structure
of human 5-lipoxygenase, Science, 331 (2011) 217-219.
37] O. Rådmark, O. Werz, D. Steinhilber, B. Samuelsson, 5-Lipoxygenase, a key enzyme for leukotriene
biosynthesis in health and disease, Biochim. Biophys. Acta, Mol. Cell Biol. Lipids, 1851 (2015) 331-339.
38] C. Pop, G.S. Salvesen, Human caspases: activation, specificity, and regulation, J. Biol. Chem., 284 (2009)
1777-21781.
[
[
2
[
[
39] N.A. Thornberry, Caspases: key mediators of apoptosis, Chem. Biol., 5 (1998) R97-R103.
40] M.-J. Tsai, P.-H. Wu, C.-C. Sheu, Y.-L. Hsu, W.-A. Chang, J.-Y. Hung, C.-J. Yang, Y.-H. Yang, P.-L. Kuo, M.-S.
Huang, Cysteinyl leukotriene receptor antagonists decrease cancer risk in asthma patients, Sci. Rep., 6 (2016)
3979.
41] K. Funao, M. Matsuyama, T. Naganuma, Y. Kawahito, H. Sano, T. Nakatani, R. Yoshimura, The cysteinylLT1
receptor in human renal cell carcinoma, Mol. Med. Rep., 1 (2008) 185-189.
42] M. Matsuyama, K. Funao, Y. Kawahito, H. Sano, J. Chargui, J.-L. Touraine, T. Nakatani, R. Yoshimura,
2
[
[
Expression of cysteinylLT1 receptor in human testicular cancer and growth reduction by its antagonist through
apoptosis, Mol. Med. Rep., 2 (2009) 163-167.
[
43] M. Matsuyama, T. Hayama, K. Funao, Y. Kawahito, H. Sano, Y. Takemoto, T. Nakatani, R. Yoshimura,
Overexpression of cysteinyl LT1 receptor in prostate cancer and CysLT1R antagonist inhibits prostate cancer cell
growth through apoptosis, Oncol. Rep., 18 (2007) 99-104.
[
44] S. Savari, M. Liu, Y. Zhang, W. Sime, A. Sjölander, CysLT1R antagonists inhibit tumor growth in a xenograft
model of colon cancer, PLoS One, 8 (2013) e73466.
45] B. Sveinbjörnsson, A. Rasmuson, N. Baryawno, M. Wan, I. Pettersen, F. Ponthan, A. Orrego, J.Z. Haeggstrom,
[
J.I. Johnsen, P. Kogner, Expression of enzymes and receptors of the leukotriene pathway in human
neuroblastoma promotes tumor survival and provides a target for therapy, FASEB J., 22 (2008) 3525-3536.
[
46] M.-J. Tsai, W.-A. Chang, P.-H. Tsai, C.-Y. Wu, Y.-W. Ho, M.-C. Yen, Y.-S. Lin, P.-L. Kuo, Y.-L. Hsu, Montelukast
induces apoptosis-inducing factor-mediated cell death of lung cancer cells, Int. J. Mol. Sci., 18 (2017) 1353.
47] N. Bouhamdani, D. Comeau, K. Cormier, S. Turcotte, STF-62247 accumulates in lysosomes and blocks late
stages of autophagy to selectively target VHL-inactivated cells, Am. J. Physiol., 0 null.
[
[
[
48] N. Mizushima, T. Yoshimori, How to Interpret LC3 Immunoblotting, Autophagy, 3 (2007) 542-545.
49] T. Utaipan, A. Athipornchai, A. Suksamrarn, S. Chunsrivirot, W. Chunglok, Isomahanine induces endoplasmic
reticulum stress and simultaneously triggers p38 MAPK-mediated apoptosis and autophagy in multidrug-
resistant human oral squamous cell carcinoma cells, Oncol. Rep., 37 (2017) 1243-1252.
[
50] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring
function, efficient optimization, and multithreading, J. Comput. Chem., 31 (2010) 455-461.
51] D. De Lucia, O.M. Lucio, B. Musio, A. Bender, M. Listing, S. Dennhardt, A. Koeberle, U. Garscha, R. Rizzo, S.
[
Manfredini, Design, synthesis and evaluation of semi-synthetic triazole-containing caffeic acid analogues as 5-
lipoxygenase inhibitors, Eur. J. Med. Chem., 101 (2015) 573-583.

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Research highlight
►
►
►
►
►
New dihydroxycinnamic acid-based analogs were screened for their 5-LO inhibition
2,5-Dihydroxycinnamic acid phenethyl ester was the best 5-LO inhibitor
2,5-Dihydroxycinnamic acid phenethyl was 7-fold more potent than Zileuton
2,5-Dihydroxycinnamic acid phenethyl ester decreased cell viability of RCC4
2,5-Dihydroxycinnamic acid phenethyl ester induced apoptosis in of RCC4 cells