4,5-Diarylisoxazol-3-carboxylic acids: A new class of leukotriene biosynthesis inhibitors potentially targeting 5-lipoxygenase-activating protein (FLAP)

Article abstract of DOI:10.1016/j.ejmech.2016.02.027

In this article, we report novel leukotriene (LT) biosynthesis inhibitors that may target 5-lipoxygenase-activating protein (FLAP) based on the previously identified isoxazole derivative (8). The design and synthesis was directed towards a subset of 4,5-diaryl-isoxazole-3-carboxylic acid derivatives as LT biosynthesis inhibitors. Biological evaluation disclosed a new skeleton of potential anti-inflammatory agents, exemplified by 39 and 40, which potently inhibit cellular 5-LO product synthesis (IC₅₀ = 0.24 μM, each) seemingly by targeting FLAP with weak inhibition on 5-LO (IC₅₀ ≥ 8 μM). Docking studies and molecular dynamic simulations with 5-LO and FLAP provide valuable insights into potential binding modes of the inhibitors. Together, these diaryl-isoxazol-3-carboxylic acids may possess potential as leads for development of effective anti-inflammatory drugs through inhibition of LT biosynthesis.

Full text of DOI:10.1016/j.ejmech.2016.02.027

European Journal of Medicinal Chemistry 113 (2016) 1e10
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
4,5-Diarylisoxazol-3-carboxylic acids: A new class of leukotriene
biosynthesis inhibitors potentially targeting 5-lipoxygenase-activating
protein (FLAP)
,
*
a
b,
Erden Banoglu a , Ers¸ an Çelikoglu , Susanna Volker c, Abdurrahman Olgaç a,
ꢀ
€
Jana Gerstmeier b, Ulrike Garscha b, Burcu Çalıs¸ kan a, Ulrich S. Schubert c
,
,
d
Andrea Carotti e, Antonio Macchiarulo e, Oliver Werz b
,
c
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Yenimahalle, 06330, Ankara, Turkey
b Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller University Jena, Philosophenweg 14, D-07743, Jena, Germany
c Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743, Jena, Germany
d Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstrasse 10, 07743, Jena, Germany
e
ꢁ
Dipartimento di Scienze Farmaceutiche, Universita di Perugia, Via del Liceo 1, 06123, Perugia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this article, we report novel leukotriene (LT) biosynthesis inhibitors that may target 5-lipoxygenase-
activating protein (FLAP) based on the previously identified isoxazole derivative (8). The design and
synthesis was directed towards a subset of 4,5-diaryl-isoxazole-3-carboxylic acid derivatives as LT
biosynthesis inhibitors. Biological evaluation disclosed a new skeleton of potential anti-inflammatory
agents, exemplified by 39 and 40, which potently inhibit cellular 5-LO product synthesis
Received 26 November 2015
Received in revised form
25 January 2016
Accepted 10 February 2016
Available online 15 February 2016
(IC50 ¼ 0.24
m
M, each) seemingly by targeting FLAP with weak inhibition on 5-LO (IC50 ꢀ 8
mM). Docking
studies and molecular dynamic simulations with 5-LO and FLAP provide valuable insights into potential
binding modes of the inhibitors. Together, these diaryl-isoxazol-3-carboxylic acids may possess potential
as leads for development of effective anti-inflammatory drugs through inhibition of LT biosynthesis.
© 2016 Elsevier Masson SAS. All rights reserved.
Keywords:
Inflammation
Leukotrienes
5-lipoxygenase
5-lipoxygenase-activating protein
Isoxazole
1. Introduction
metabolism [5].
Despite intensive efforts to develop 5-LO/FLAP inhibitors as
Leukotrienes (LTs) are potent lipid mediators that play impor-
tant roles in the pathophysiology of inflammatory, fibrotic and
hyperproliferative diseases such as asthma, chronic obstructive
pulmonary disease (COPD), inflammatory bowel disease (IBD),
arthritis, allergic diseases, autoimmune diseases, cardiovascular
disease (CVD) and certain cancer types [1,2]. In the first step of LT
biosynthesis, 5-lipoxygenase (5-LO) catalyzes the production of the
unstable epoxide LTA4 from arachidonic acid (AA), which is further
metabolized to LTB4 or cysteinyl LTs (cys-LTs) such as LTC4, D4 and
E4 [3,4]. This first step also requires the involvement of 5-LO-acti-
vating protein (FLAP), which acts as a regulatory protein by inter-
action with 5-LO for the transfer of AA to 5-LO for efficient
anti-LTs, zileuton (1), the only 5-LO inhibitor, reached the market
for the treatment of asthma and/or allergic rhinitis, but has expe-
rienced limited use due to its poor pharmacokinetics and observed
idiosyncratic hepatotoxicity [6]. Additionally, the 5-LO inhibitor
setileuton (2, MK-0633) [7e9] and several FLAP inhibitors such as
AM803 (3, GSK2190915), AZD6642 (4) and BI665915 (5) were re-
ported to be in various stages of preclinical and clinical studies for
treatment of asthma and COPD [10e13].
Recent studies have also implicated LTs in CVD such as athero-
sclerosis, myocardial infarction (MI), and stroke [14,15]. 5-LO and
FLAP are considered to be important components of the LT cascade
found in atherosclerotic lesions, thus implicating their involvement
in atherogenesis [16e18]. The 5-LO inhibitor 2 recently completed
phase II clinical trials for atherosclerosis (Clinical trials ID:
NCT00421278) as well as for asthma and COPD [7,19], implying the
value of LT inhibition for CVD besides respiratory disorders.
* Corresponding author. Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Gazi University, 06330, Etiler, Ankara, Turkey.
E-mail address: banoglu@gazi.edu.tr (E. Banoglu).
http://dx.doi.org/10.1016/j.ejmech.2016.02.027
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

2
E. Banoglu et al. / European Journal of Medicinal Chemistry 113 (2016) 1e10
Pharmacological intervention with FLAP has also been reported to
decrease atherosclerotic lesion size in animal models [20e23] and
the FLAP inhibitor veliflapon (6, DG-031/BAY-X1005) has success-
fully passed a phase II human clinical trial for MI, although
participant recruitment in phase III was suspended due to unex-
pected formulation issues. In addition, there is a growing interest in
5-LO or FLAP inhibitors as broad-spectrum LT biosynthesis in-
hibitors in cancer therapy, especially in prostate cancer [24,25] and
certain types of leukemia [26e28].
5-LO conformation represented the first major difficulty to eluci-
date the ligand-active site interactions. Therefore, we referred to
the conserved or unconserved active site interaction models
[33,34]. Grounding on those results, we combined docking studies
with MD simulations (see Supporting information for detail) to
investigate the binding mode of inhibitors taking into account
ligand-induced conformational changes of 5-LO. As a result of the
docking studies, 8 interacts with the binding site engaging residues
Tyr181, Trp599, Phe177, His372, Phe421, Asn425 and His600
(Fig. S1). The 5-LO/8 complex obtained by docking was submitted to
a 10 ns MD study (Fig. 2A). As a result of MD simulations, the o-
hydroxyl group is engaged in a stable hydrogen bonding interaction
with the side-chain of Asn425 (occupancy, 94%, Fig. 2A) orienting
Inspired by the therapeutic potential of LT biosynthesis in-
hibitors, we have recently introduced two promising chemotypes, a
benzimidazole
derivative
1-(2-chlorobenzyl)-2-(1-(4-
isobutylphenyl)ethyl)-1H-benzimidazole (7, BRP-7) and an iso-
xazole derivative 2-[4-(4-chlorophenyl)-3-methyl-1,2-oxazol-5-yl]-
5-[(2-methyl phenyl)methoxy]phenol (8) as anti-LT agents [29,30]
(see Fig. 1). From the SARs [29,31] and detailed pharmacological
studies [32], 7 was revealed as potent inhibitor of LT biosynthesis
the phenyl moiety to engage p-p contacts with Tyr181 (occupancy,
38%), His372 (occupancy, 39%) and Phe421 (occupancy, <30%),
which is in agreement with previously reported data on the cata-
lytic site of 5-LO [35].
targeting FLAP in a cell-based assay (IC50 ¼ 0.15e0.31
affecting isolated 5-LO (IC50 > 10 M), whereas 8 was moderately
effective in a cell-based assay (IC50 ¼ 4.4 M) and directly inhibited
isolated 5-LO (IC50 ¼ 1.9 M), which might be the primary cause
m
M) without
These findings led us to turn our attention into a structure-based
design of new derivatives of 8 involving the isoxazole-3-carboxylic
acid core (32, Fig. 2B), where the carboxyl arm on isoxazole might
influence the pattern of more strong interactions with neighboring
polar amino acids or with amino acid backbones. Indeed, from the
analysis of molecular docking and MD results, both the isoxazol-3-
carboxylic acid analog 32 and 8 disclosed a similar binding mode at
the 5-LO active site (Fig. 2AeB). Compound 32, as already seen for
the compound 8, engaged a strong hydrogen bond reinforced by
charge assistance with Asn425 (occupancy, 99%), as well as main-
taining the key hydrophobic contacts with Tyr181 (occupancy,
<30%), His372 (occupancy, 34%) and Phe421 (occupancy, 32%).
These results suggested the synthesis of a focused library of com-
pounds with decorations on an isoxazole-3-carboxylic acid core
that was designed taking into account both synthetic accessibility,
and the compatibility of new skeleton with the binding re-
quirements of the active pocket to obtain preliminary SAR (Scheme
2). The biological evaluation of this small set of compounds might
be helpful for the comprehension of the key features of new iso-
xazole-3-carboxylic acids as LT biosynthesis inhibitors.
m
m
m
for the decrease of cellular LT formation [29]. However, whether 8
also interferes with FLAP was not explored in those studies.
Therefore, the present work describes the structure-guided design,
synthesis, structureꢁactivity relationship (SAR), and biological
evaluation of novel 4,5-diarylisoxazol-3-carboxylic acids as potent
inhibitors of LT biosynthesis, potentially targeting FLAP.
2. Results and discussion
2.1. Docking studies and molecular dynamic (MD) simulations with
5-LO
Based on the favorable profile of 8, we focused on the prepa-
ration of further derivatives in order to understand SARs for
interference with 5-LO product synthesis. First, we addressed the
necessity of the o-hydroxyl group on C5-phenyl for the bioactivity
of 8. As we previously showed the importance of hydrophobic
contacts over hydrophilic interactions as a general determinant for
many known 5-LO inhibitors [33], our initial hypothesis was the
removal of the o-hydroxyl group to improve the potency. Therefore,
we synthesized two derivatives of 8 by either completely removing
the polar o-hydroxyl moiety (15) or by replacing it by a less polar
methoxy group (16) (Scheme 1). Interestingly, removal of the o-
hydroxyl group from 8 or O-methylation abolished the inhibition of
cellular LT biosynthesis (15e16, Table 1).
2.2. Chemistry
Compounds 15e16 were prepared following the reaction
sequence shown in Scheme 1 using general methods previously
reported [36]. Hence, the
a,b-unsaturated ketone intermediates
(11e12), which were prepared by condensation of 4-
chlorophenylacetone with benzaldehydes (9e10) in the presence
of piperidine, were converted to a,b-unsaturated oximes (13e14).
For rationalizing the inhibitory activity of 8 on 5-LO by means of
molecular docking, we used the recently reported apo form of the
5-LO crystal structure (PDB code 3O8Y). The unavailability of a holo
Subsequent cyclization of the oximes (13e14) generated the 3-
methylisoxazole derivatives (15e16, Scheme 1).
For the synthesis of compounds 32e40, we utilized the
H
O
O
O
O
R
O
H
Br
R
R
R
O
R
O
d
c
a
b
+
+
N
O
O
NOH
OH
Cl
Cl
Cl
Cl
9
R = H
11 R = H
12 R = OCH3
13 R = H
14 R = OCH3
15 R = H
16 R = OCH3
10 R = OCH3
Scheme 1. Synthesis of 4,5-diaryl-3-methylisoxazoles. Reagents and conditions: (a) K2CO3, MeCN, reflux; (b) piperidine, 90 ꢂC; (c) NH2OH.HCl, NaOAc, EtOH, reflux; (d) KI, I2,
NaHCO3, THF, H2O, reflux.

E. Banoglu et al. / European Journal of Medicinal Chemistry 113 (2016) 1e10
3
Table 1
Effects of test compounds on 5-LO product synthesis in a FLAP-dependent cell-based (intact neutrophils) and in a FLAP-independent cell-free (purified 5-LO) assay.
Cmpd
R
R1
5-LO product formation
Cell-based assaya (% remaining activity at 10
m
M)
IC50 [m
M]b
Cell-based
Cell-free
8
OH
H
OCH3
2-MePh
3-FPh
4-MePh
2-MePh
3-FPh
4-MePh
3-CNPh
2-ClPh
2-CF3Ph
pyridin-2-yl
quinolin-2-yl
benzothiazol-2-yl
zileuton
3.0 0.7
101.5 5.4
81.3 8.2
71.9 10.3
55.3 12.1
61.4 12.4
13.1 3.9
19.6 9.5
15.7 4.5
6.9 3.9
5.1 1.5
5.5 2.2
8.4 0.7
2.7 1.4
2.0 0.8
6.7 2.1
4.4 1.1
n.d.
n.d.
n.d.
n.d.
1.9 0.5
n.d.
n.d.
n.d.
n.d.
15
16
23
24
25
32
33
34
35
36
37
38
39
40
1
Et
Et
Et
H
H
H
H
H
H
H
H
H
n.d.
n.d.
1.7 0.5
1.3 0.6
1.4 0.1
4.1 0.3
1.2 0.2
0.77 0.2
4.1 0.1
0.24 0.07
0.24 0.10
0.58 0.11
0.48 0.03
0.38 0.04
5.9 0.4
16.3 5.6
6.1 0.9
6.0 0.7
7.4 2.5
8.0 3.0
>10
0.91 0.25
Compounds were tested for inhibition of 5-LO product formation in human neutrophils stimulated with 2.5 mM A23187 (cell-based assay) and against purified human re-
combinant 5-LO (cell-free assay).
a
Data are given as remaining 5-LO activity as percentage of uninhibited control (100%, containing 0.3% DMSO as vehicle) at 10 mM inhibitor concentration (means SE,
n ¼ 3e4). The 100% value of the control corresponds to an average of 30 6 ng 5-LO products per 106 neutrophils.
b
The IC50 values are given as mean SE of n ¼ 3e4 determinations in duplicates. n.d. not determined.
synthetic procedure outlined in Scheme 2, following published
standard procedures [37]. In brief, the commercially available p-
hydroxyacetophenone was protected with benzyl group (17) prior
to the subsequent reaction to generate a keto-enol ester (18) by
reaction with diethyl oxalate. Formation of the isoxazole ring 19
was done by reaction of 18 with hydroxylamine in ethanol.
Bromination at the 4-position of the isoxazole (20) was achieved
with N-bromosuccinimide (NBS) in the presence of a catalytic
amount of ceric ammonium nitrate (CAN). Compound 20 under-
went a palladium-catalyzed Suzuki cross-coupling reaction with p-
chlorophenylboronic acid to afford 21. Hydrogenation of 21 to
remove benzyl group furnished the intermediate 22, which was
subsequently used to produce desired final compounds 32e40
through first the alkylation of the phenolic hydroxyl and then the
hydrolysis of the ester group. All compounds were purified by
recrystallization or by automated flash chromatography and
checked for purity by TLC and UPLC before being tested in biological
assays (purity was ꢀ95% based on the peak area percentage of UPLC
analysis). The structures of the compounds were confirmed by
high-resolution mass spectrometry (HRMS), elemental analysis,
1H and 13C NMR spectral data.
test compounds in a ‘FLAP-dependent’ cell-based assay for sup-
pression of 5-LO product formation using Ca2þ-ionophore A23817
activated neutrophils, as well as in a ‘FLAP-independent’ cell-free 5-
LO activity assay using human recombinant 5-LO and 20 mM AA as
exogenous substrate. Analyzed 5-LO products include 5-H(P)ETE
and all trans-isomers of LTB4, as well as LTB4 in intact cells. The
direct 5-LO inhibitor zileuton (1) was used as reference drug.
Starting from 8 as hit compound, we aimed to explore (i) the
influence of differently positioned substituents on the benzyloxy
arm, (ii) the replacement of benzyloxy by heteroaryl moieties, and
(iii) the role of carboxylic acid or ester groups at position 3 of the
isoxazole for inhibition of 5-LO product synthesis (Fig. 3).
First, we prepared the 3-carboxyl analogue of the inactive 15
yielding 32, which was highly active and even outperformed hit
compound 8, as the potencies of 32 in the cell-free (IC50 ¼ 0.48
and cell-based assay (IC50 ¼ 1.7 M) were improved by 4- and 2.6-
fold, respectively (Table 1). Meanwhile, the corresponding ethyl
ester 23 was found to be inactive (IC50 > 10 M), supporting the
mM)
m
m
notion that the COOH moiety with hydrogen bonding donor feature
was beneficial.
Moving the electron donating methyl residue in ortho position
at the benzyloxy moiety to para position resulted in a steric hin-
drance and caused a loss of bioactivity (34). When the methyl
residue was replaced by an electron-withdrawing chlorine (36) or
trifluoromethyl (37), the potency in cell-based assays slightly
2.3. Biological evaluation and SAR studies
LT biosynthesis is initiated by cytosolic phospholipase A2
(cPLA2)-mediated release of AA, which is subsequently converted to
LTA4 by activated 5-LO with the aid of FLAP that may facilitate ac-
cess of 5-LO to AA [2]. While FLAP is essential for cellular 5-LO
product formation from endogenous AA, it is dispensable for 5-LO
activity in cell-free systems [38]. Therefore, we investigated the
improved (IC50 ¼ 0.77e1.4
m
M) but in the cell-free assay the activity
was impaired (IC50 ¼ 5.9e6.1
mM). Thus, the cell-free data imply
that replacement of an electron donor group in ortho position to an
electron withdrawing group causes loss of activity, most probably
by affecting the
p-p interaction capability of the attached phenyl

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E. Banoglu et al. / European Journal of Medicinal Chemistry 113 (2016) 1e10
Fig. 1. Chemical structures of LT biosynthesis inhibitors.
group with His372. On the other hand, substitution of the benzy-
loxy group with fluorine in meta position (33) was tolerated and
slightly increased the bioactivity in both assays, whereas with
cyano in this position (35), the efficiency clearly dropped (IC50 ¼ 4.1
substituents at the C5 of isoxazole determines the potencies, thus
indicating that substitution pattern of the 4-benzyloxyphenyl
group is crucial for 5-LO inhibitory activity. Apparently, a consis-
tent SAR for direct 5-LO inhibition is not obvious since small
structural differences on the scaffold were not tolerated. Our data
underline the importance of the 4-benzyloxyphenyl moiety for
directly interacting with 5-LO in this structural context. However, it
should be kept in mind that almost all isoxazole-3-carboxylic acid
derivatives were markedly active in the ‘FLAP-dependent’ cell-
based assays (Table 1). Because several derivatives of this com-
pound class (e.g., 34, 36, 37, 39 and 40) potently suppressed cellular
and 16.3 mM in cell-based and cell-free test systems, respectively).
Replacing the benzyloxy moiety by pyridine-2-yl-methyloxy (38)
further reduced the 5-LO inhibitory potency in both assay types,
similar to 3-cyano derivative 35. Again, ester analogues of active 33
and 34 abolished the bioactivity (i.e., 24 and 25), thus highlighting
the free acidic carboxyl group as structural determinant (Table 1).
We also investigated the effects of introducing voluminous
heteroaromatic groups into the molecule. Intriguingly, a substantial
gain of potency in intact cells was obtained when the benzyl was
exchanged by a quinolinyl-methyl (39) or benzothiazolyl-methyl
5-LO product formation (IC50 ¼ 0.24e1.4
m
M) without significantly
affecting 5-LO in the cell-free assay (IC50 ꢀ 5.9
mM), we speculate
that these derivatives primarily act at other targets than 5-LO,
presumably on FLAP.
(40) residues with IC50 ¼ 0.24
pounds 39 and 40 outperformed the reference drug zileuton (1) in
cell-based assay for which the IC50 was determined as 0.91 M in
agreement with the literature [4]. Note that in the cell-free assay 39
M,
mM, each. Therefore, the com-
m
2.4. Docking studies and MD simulations of 39 with FLAP
and 40 were more than 30-fold less potent (IC50 ¼ 8 and > 10
m
To support the idea that 39 and 40 may act as FLAP inhibitor, we
performed docking studies of a library of compounds including
these active molecules, inactive compounds 15, 16, 23e25 and a set
of 1911 decoy compounds as detailed in the Supporting information
(Table S1). Specifically, we wondered whether compounds 39 and
40 could show better binding scores with respect to inactive
compounds (15, 16, 23e25) in a distribution of binding scores
generated with decoy compounds.
respectively), implying that the compounds may target other
components than 5-LO in intact cells leading to reduced 5-LO
product synthesis. In fact, the 5-LO reference inhibitor 1 was also
found equally effective in the cell free assay (IC50 ¼ 0.58
mM,
Table 1).
Taken together, replacing the 3-methyl moiety at the central
isoxazole by a carboxyl group leads to a core structure for potent
inhibitors of cellular 5-LO product synthesis. The nature of
All of the compounds were docked to the site located between

E. Banoglu et al. / European Journal of Medicinal Chemistry 113 (2016) 1e10
5
Fig. 2. Binding mode analysis of 3-methylisoxazole derivative 8 (A), and isoxazole-3-carboxylic acid derivative 32 (B), depicted together with interacting 5-LO (PDB code 3O8Y)
residues. Main interactions are represented schematically with their occupancies calculated in the time window 0e10 ns.
the chains B and C with the key residues B-Phe123, B-Leu120, B-
Ile119, B-Lys116, B-Phe114, B-Ile113, B-Tyr112, B-Thr66, B-Asp63, C-
Val61, C-His28, C-Ala27, C-Phe25 and C-Asn23 as we reported
previously [29]. As a result, we observed that the binding scores
(Emodel score) of 39 and 40 ranked among the top 2.13% and 4.11%
of the distribution, respectively. In contrast, binding scores of the
inactive compounds started ranking after 18% of the distribution.
Hence, docking studies supported the hypothesis that quinoline
derivative 39 and its benzothiazole analogue 40 are indeed FLAP
inhibitors. Taking into account the docking calculations reported
for FLAP before [40e42] and shown in this study, the binding
specificity of 39 was conferred by the H-bond with Lys116 (Fig. S4).
Moreover, our suggested pose for 39 adopts the equivalent spatial
disposition of the co-crystallized inhibitor 41 (MK-591),

6
E. Banoglu et al. / European Journal of Medicinal Chemistry 113 (2016) 1e10
BnO
BnO
HO
BnO
BnO
e
O
c
d
a
b
O
O
N
N
OEt
Br
O
O
O
R
OH
COOEt
COOEt
17
19
18
20
23, 32 R = 2-MePh
24, 33 R = 3-FPh
BnO
HO
O
R
O
25, 34 R = 4-MePh
26, 35 R = 3-CNPh
27, 36 R = 2-ClPh
O
O
O
O
g
f
h
N
N
N
N
28, 37 R = 2-CF3Ph
29, 38 R = Pyridin-2-yl
30, 39 R = Quinolin-2-yl
31, 40 R = Benzothiazol-2-yl
COOEt
COOEt
COOEt
COOH
Cl
Cl
Cl
Cl
22
23 - 31
21
32 -40
Scheme 2. Synthesis of 4,5-diarylisoxazol-3-carboxylic acids. Reagents and conditions: (a) BnBr, K2CO3, MeCN, reflux; (b) (CO2Et)2, NaOEt, EtOH, rt; (c) NH2OH$HCl, EtOH, reflux; (d)
NBS, CAN, AcCN; (e) 4-chlorophenylboronic acid, Pd(PPh3)2Cl2, NaHCO3, H2O, DMF; (f) Pd/C, H2, MeOH:THF (1:1); (g) appropriate benzyl halide, 2-(chloromethyl)pyridine, 2-
(chloromethyl)quinoline or 2-(chloromethyl)benzothiazole, K2CO3, KI, MeCN, reflux; (h) LiOH, THF, H2O.
CH3
R
Het
and
O
OH
O
O
O
O
O
N
N
N
CH3
COOR1
COOR1
Cl
Cl
Cl
Hit compound 8
S
N
R = CH3, CF3, F, Cl, CN
R1 = H, C2H5
Het:
N
N
Fig. 3. Major structural modifications of hit compound 8.
maintaining the same interaction network that accounts for a high
affinity binding mode to FLAP (Figs. S3eS4).
of new isoxazole derivatives based on the structure of hit 8 that we
previously identified as an inhibitor of 5-LO product synthesis [29].
We demonstrated that although the activity of all derivatives in
cell-based assays were quite pronounced, the different sub-
stitutions on the benzyloxyphenyl arm at position C5 of the iso-
xazole caused a great discrepancy for inhibition of 5-LO product
synthesis among this class of compounds, where only 32e33
exhibited potent direct 5-LO inhibitory activity (IC50 5-LO ¼ 0.48
Accordingly, to further investigate the binding mode of 39 in
comparison with 41 (MK591) to FLAP taking into account potential
ligand-induced conformational changes, we performed MD simu-
lations using the 3D structure of FLAP (PDB code 2Q7M) in complex
with these docked ligands (see Supporting information, Figs. S3e4)
[39]. Since the available FLAP x-ray structure has a low resolution
(4.2 Å) and does not include membrane coordinates, we analyzed
the binding mode of FLAP/41 and FLAP/39 complexes by means of
MD simulations including membrane insertion (taken from OPM
Database [43]) in the simulation systems (Fig. 4AeB). By inclusion
of the membrane bilayer, it was possible to observe its effect on the
binding in which the adopted techniques have been widely used for
the analysis of other membrane proteins [44]. As a result, the
binding of 39 to FLAP is characterized by several interactions, but
the most stable ones derived from the MD simulation data shows:
(i) the carboxylic acid group makes polar interactions mainly with
B-Lys116 (occupancy, 42%) and C-His28 (occupancy, 36%); (ii) the
and 0.38
inhibitors of cellular 5-LO product synthesis, seemingly by acting
on FLAP (e.g, 34, 36, 37, 39, 40; IC50 cell-based ¼ 0.24e1.4 M). By
mM, respectively), while other derivatives were efficient
m
means of docking studies and MD simulations with 5-LO and FLAP,
satisfactory explanations of the putative binding mode for these
isoxazole-based 5-LO inhibitors were provided, each, demon-
strating the preservation of the important ligand-protein in-
teractions as seen in crystal structures. Based on our results, we can
speculate that the ability of these compounds to interfere with LT
biosynthesis is either caused by 5-LO or FLAP inhibition according
to the superior activity in cell-free or cell-based assays, respectively.
In conclusion, our biological results disclosed two benchmark
compounds 39 and 40 with potent inhibitory activity against
quinoline group engages a
p-p interaction with B-Tyr112 (occu-
pancy, 38%) and makes hydrophobic contacts to B-Thr66 (occu-
pancy, 28%) (Fig. 4B).
cellular 5-LO product synthesis (IC50 ¼ 0.24
due to interference with FLAP but also with weak 5-LO suppression
M). These isoxazole-3-carboxylic acids may be suitable
mM, each) most likely
(IC50 ꢀ 8.0
m
3. Conclusions
for further development of therapeutics in diseases requiring anti-
LT therapy such as respiratory diseases, cardiovascular diseases,
We have designed, synthesized and biologically evaluated a set

E. Banoglu et al. / European Journal of Medicinal Chemistry 113 (2016) 1e10
7
Fig. 4. X-ray binding mode analysis of ligand 41 (MK591) (A) and 39 (B) during interaction with FLAP (PDB code 2Q7M) considering membrane residues. Main interactions are
represented schematically with their occupancies calculated in the time window 0e10 ns.
allergic and autoimmune diseases as well as cancer. An ongoing
comprehensive pharmacological study, beyond the scope of this
work, using various cell-based and cell-free assays combined with
microscopic techniques (e.g. proximity ligation assay), may provide
additional evidence for 39 acting on FLAP in the future.
4. Experimental section
4.1. Chemistry
Extensive experimental information on synthetic procedures
can be found in Supporting information. 1H and 13C NMR spectra
were recorded in CDCl3 or DMSO-d6 on a Varian Mercury 400 MHz
or Bruker Ultrashield 300 MHz High Performance Digital FT-NMR
spectrometer using tetramethylsilane as the internal standard. All

8
E. Banoglu et al. / European Journal of Medicinal Chemistry 113 (2016) 1e10
chemical shifts were recorded as
d
(ppm). High resolution mass
4.1.2.1. 4-(4-Chlorophenyl)-5-{4-[(2-methylbenzyl)oxy]phenyl}iso-
spectra data (HRMS) were collected using a Waters LCT Premier XE
Mass Spectrometer (high sensitivity orthogonal acceleration time-
of-flight instrument) operating in ESI (þ) method, also coupled to
an AQUITY Ultra Performance Liquid Chromatography system
(Waters Corporation, Milford, MA, USA) using a UV detector
xazol-3-carboxylic acid (32). Purified by recrystallization from
methanol. Yield 49.4%; mp 188.7e189.8 ꢂC. 1H NMR (DMSO-d6):
d
2.30 (3H, s), 5.10 (2H, s), 7.10 (2H, d, J ¼ 8.0 Hz), 7.18e7.25 (3H, m),
7.37e7.41 (5H, m), 7.51 (2H, d, J ¼ 8.0 Hz). 13C NMR (DMSO-d6):
d
18.41, 68.11, 113.81, 115.34, 118.84, 125.77, 128.19, 128.23, 128.41,
monitoring at 254 nm with
a
Aquity BEH C18 column
128.58, 128.65, 130.14, 132.08, 133.10, 134.37, 136.65, 155.92, 160.14,
160.87, 166.40. Anal. Calcd for C24H18ClNO4$0.5MeOH C, 67.51; H,
4.62; N, 3.21; found: C, 67.38; H, 4.81; N, 3.37. HRMS (m/z): [MþH]þ
calcd for C24H19ClNO4 420.1003, found 420.0984.
(2.1 ꢃ 100 mm, 1.7 m, 0.3 mL/min flow rate), using a gradient of
m
CH3CN/H2O (1%e90%) containing 1% formic acid. All tested com-
pounds were determined to be ꢀ 95% pure using the analytical
method described above based on the peak area percentage.
Elemental analyses were taken on a Leco 932 CHNS analyzer.
Melting points were determined with an SMP-II Digital Melting
Point Apparatus and are uncorrected (Schorpp Geraetetechnik,
Germany). Flash chromatography was performed with a Combi-
flash® Rf automated flash chromatography system with RediSep
columns (Teledyne-Isco, Lincoln, NE, USA) using n-hexaneeEtOAc
or DCMeMeOH solvent gradient.
4.1.2.2. 4-(4-Chlorophenyl)-5-{4-[(3-fluorobenzyl)oxy]phenyl}iso-
xazol-3-carboxylic acid (33). Purified by recrystallization from iso-
propanol. Yield 65%; mp 190.4e191.1 ꢂC. 1H NMR (DMSO-d6):
d 5.15
(2H, s), 7.09 (2H, d, J ¼ 8.8 Hz), 7.14e7.19 (1H, m), 7.26e7.28 (2H, m),
7.37e7.46 (5H, m), 7.50 (2H, 2H, d, J ¼ 8.8 Hz). 13C NMR (DMSO-d6):
d
68.46, 113.81, 114.25 (d, J ¼ 22.1 Hz), 114.65 (d, J ¼ 20.6 Hz), 115.37,
118.96, 123.53 (d, J ¼ 3.1 Hz), 128.12, 128.38, 128.56, 130.42 (d,
J ¼ 8.4 Hz), 131.99, 133.05, 139.36 (d, J ¼ 7.6 Hz), 155.88, 159.74,
160.78, 162.10 (d,
C
J
¼
242.3 Hz), 166.28. Anal. Calcd for
4.1.1. Synthesis of 3-methylisoxazole derivatives (15e16)
23H15ClFNO4 C, 65.18; H, 3.57; N, 3.30; found: C, 64.79; H, 3.84; N,
A solution of KI (1.8 mmol) and iodine (0.5 mmol) in H2O (4 mL)
was added to a solution of 13e14 (0.5 mmol) and NaHCO3
(2.1 mmol) in THF (6 mL) and water (6 mL) in a reaction flask
covered by aluminum foil. After the reaction mixture was heated at
reflux for 5 h, saturated aqueous sodium bisulfite solution (5 mL)
was added, and this mixture was extracted with EtOAc (2 ꢃ 50 mL).
The combined organic extracts were dried over anhydrous MgSO4,
the solvent was removed in vacuo, and the residue was purified by
automated flash column chromatography using hexane-ethyl ace-
tate gradient as eluent to yield 15e16.
3.49. HRMS (m/z): [MþH]þ calcd for C23H16ClFNO4 424.0752, found
424.0734.
4.1.2.3. 4-(4-Chlorophenyl)-5-{4-[(4-methylbenzyl)oxy]phenyl}iso-
xazol-3-carboxylic acid (34). Purified by recrystallization from
methanol. Yield 88%; mp 185.9e186.6 ꢂC. 1H NMR (DMSO-d6):
d
2.29 (3H, s), 5.06 (2H, s), 7.05 (2H, d, J ¼ 8.4 Hz), 7.18 (2H, d,
J ¼ 8.0 Hz), 7.30 (2H, d, J ¼ 8.0 Hz), 7.36e7.39 (4H, m), 7.49 (2H, d,
J ¼ 8.4 Hz). 13C NMR (DMSO-d6):
d 20.75, 69.30, 113.79, 115.40,
118.76, 127.91, 128.22, 128.39, 128.64, 129.99, 132.07, 133.09, 133.43,
137.27, 155.93,160.06,160.88, 166.40. Anal. Calcd for C24H18ClNO4 C,
68.66; H, 4.32; N, 3.34; found: C, 68.64; H, 3.98; N, 3.50. HRMS (m/
z): [MþH]þ calcd for C24H19ClNO4 420.1003, found 420.0982.
4.1.1.1. 4-(4-Chlorophenyl)-5-{4-[(2-methylphenyl)methoxy]phenyl}-
3-methylisoxazole (15). Purified by flash column chromatography
(0% / 30% EtOAc in hexane). Yield 63%; mp 155.8e156.1 ꢂC. 1H
NMR (CDCl3):
d 2.22 (3H, s), 2.36 (3H, s), 5.04 (2H, s), 6.94 (2H, d,
4.1.2.4. 4-(4-Chlorophenyl)-5-{4-[(3-cyanobenzyl)oxy]phenyl}iso-
xazol-3-carboxylic acid (35). Purified by recrystallization from
methanol. Yield 48%; mp 180.5e181.0 ꢂC. 1H NMR (DMSO-d6):
J ¼ 8.8 Hz), 7.22e7.27 (5H, m), 7.37 (1H, m), 7.42 (2H, d, J ¼ 8.8 Hz),
7.46 (2H, d, J ¼ 9.2 Hz). 13C NMR (CDCl3):
d 10.62, 18.89, 68.64,
113.79, 114.96, 120.48, 126.10, 128.44, 128.48, 128.62, 129.37, 129.40,
130.49, 131.26, 134.05, 134.20, 136.70, 159.80, 160.09, 164.59. Anal.
Calcd for C24H20ClNO2: C, 73.94; H, 5.17; N, 3.59; found: C, 73.88; H,
5.11; N, 3.72. HRMS (m/z): [MþH]þ calcd for C24H21ClNO2 391.1339,
found 391.1336.
d
5.20 (2H, s), 7.10 (2H, d, J ¼ 8.8 Hz), 7.38e7.42 (4H, m), 7.50 (2H, d,
J ¼ 8.8 Hz), 7.61 (1H, t, J ¼ 8.0 Hz), 7.77e7.82 (2H, m), 7.91 (1H, s). 13
C
NMR (DMSO-d6):
d 68.13, 111.42, 113.88, 115.38, 118.53, 119.05,
128.12, 128.42, 128.59, 129.70, 131.10, 131.72, 132.01, 132.45, 133.06,
138.17, 155.84, 159.61, 160.79, 166.28. Anal. Calcd for C24H15ClN2O4
C, 66.91; H, 3.51; N, 6.50; found: C, 66.57; H, 3.73; N, 6.36. HRMS (m/
z): [MþH]þ calcd for C24H16ClN2O4 431.0799, found 431.0782.
4.1.1.2. 4-(4-Chlorophenyl)-5-{2-methoxy-4-[(2-methylphenyl)
methoxy]phenyl}-3-methylisoxazole (16). Purified by flash column
chromatography (0% / 30% EtOAc in hexane). Yield 23%; mp
4.1.2.5. 4-(4-Chlorophenyl)-5-{4-[(2-chlorobenzyl)oxy]phenyl}iso-
131.8e132.2 ꢂC. 1H NMR (CDCl3):
d 2.33 (3H, s), 2.39 (3H, s), 3.38
xazol-3-carboxylic acid (36). Purified by recrystallization from
methanol. Yield 93%; mp 128.5 ꢂC. 1H NMR (DMSO-d6):
d
5.18 (2H,
s), 7.13 (2H, d, J ¼ 8.8 Hz), 7.38e7.43 (6H, m), 7.49e7.53 (3H, m),
7.58e7.60 (1H, m). 13C NMR (DMSO-d6):
67.78, 114.61, 116.04,
(3H, s), 5.05 (2H, s), 6.48 (1H, d, J ¼ 2.0 Hz), 6.63 (1H, dd, J ¼ 2.0,
8.8 Hz), 7.12 (2H, d, J ¼ 8.4 Hz), 7.21e7.28 (3H, m), 7.31 (2H, d,
J ¼ 8.4 Hz), 7.38e7.41 (2H, m). 13C NMR (CDCl3):
d 11.25,19.15, 29.93,
d
55.12, 69.04, 99.90, 105.89, 110.34, 116.24, 126.36, 128.77, 128.83,
129.08, 130.19, 130.73, 130.76, 131.82, 133.18, 134.38, 137.06, 158.05,
159.10, 162.18, 163.99. Anal. Calcd for C25H22ClNO3$0.4H2O: C,
70.30; H, 5.38; N, 3.28; found: C, 70.30; H, 5.46; N, 3.18. HRMS (m/
z): [MþH]þ calcd for C25H23ClNO3 420.1366, found 420.1364.
119.82, 128.08, 128.88, 129.19, 129.36, 130.13, 130.77, 131.03, 132.78,
133.48, 133.81, 134.45, 156.64, 160.55, 161.56, 167.04. Anal. Calcd for
C
23H15Cl2NO4 C, 62.74; H, 3.43; N, 3.18; found: C, 62.99; H, 3.53; N,
3.51. HRMS (m/z): [MþH]þ calcd for C24H16Cl2NO4 440.0456, found
440.0446.
4.1.2. Synthesis of title isoxazol-3-carboxylic acid derivatives
(32e40)
4.1.2.6. 4-(4-Chlorophenyl)-5-{4-[(2-trifluoromethylbenzyl)oxy]
phenyl}isoxazol-3-carboxylic acid (37). Purified by recrystallization
Ester intermediates 23e31 (0.003 mol) and lithium hydroxide
(0.008 mol) in THF/water mixture (1:1) were stirred at room
temperature for 4 h. After the reaction is complete, the reaction
mixture was diluted with water, acidified with 2N HCl and the
precipitate was filtered and recrystallized from the appropriate
solvent or purified by automated flash chromatography.
from methanol. Yield 70%; mp 156.3 ꢂC. 1H NMR (CDCl3):
d 5.28 (2H,
s), 6.92e6.94 (2H, d, J ¼ 9.2 Hz), 7.28e7.30 (2H, d, J ¼ 8.4 Hz),
7.39e7.46 (5H, m), 7.55e7.59 (1H, t, J ¼ 7.6 Hz), 7.68e7.71 (2H, t,
J ¼ 7.4 Hz). 13C NMR (DMSO-d6):
d 66.35, 113.89, 115.23, 119.16,
124.16 (q, J ¼ 272.1 Hz), 126.14 (q, J ¼ 5.3 Hz), 126.96 (q, J ¼ 30.4 Hz),
128.10,128.45,128.60,128.88,130.69,132.02,132.78,133.06,134.20,

E. Banoglu et al. / European Journal of Medicinal Chemistry 113 (2016) 1e10
9
155.86, 159.63, 160.79, 166.24. Anal. Calcd for C24H15ClF3NO4$0.4-
CH3OH C, 60.22; H, 3.44; N, 2.88; found: C, 60.16; H, 3.54; N, 3.20.
HRMS (m/z): [MþH]þ calcd for C24H16ClF3NO4 474.0720, found
474.0730.
(5 ꢃ 106) were resuspended in 1 ml PGC buffer, preincubated for
15 min at 37 ꢂC with test compounds or vehicle (0.1% DMSO) and
Ca2þ-ionophore A23187 (2.5 M) was added. After 10 min at 37 ꢂC
m
the reaction was stopped on ice by addition of 1 ml of methanol.
30 l 1 N HCl and 500 l PBS, and 200 ng prostaglandin (PG)B1 were
m
m
4.1.2.7. 4-(4-Chlorophenyl)-5-[4-(pyridin-2-ylmethoxy)phenyl]iso-
xazol-3-carboxylic acid (38). Purified by flash column chromatog-
raphy (0% / 20% MeOH in DCM) and recrystallized from methanol.
added and the samples were subjected to solid phase extraction on
C18-columns (100 mg, UCT, Bristol, PA, USA). 5-LO products (LTB4
and its trans-isomers, and 5-H(P)ETE) were analyzed by RP-HPLC
and quantities calculated on the basis of the internal standard
PGB1. Cys-LTs C4, D4 and E4 were not detected (amounts were below
detection limit), and oxidation products of LTB4 were not
determined.
Yield 32%; mp 176.8 ꢂC. 1H NMR (DMSO-d6):
d 5.20 (2H, s), 7.10 (2H,
d, J ¼ 8.8 Hz), 7.34e7.52 (8H, m), 7.82e7.86 (1H, m), 8.54 (1H, d,
J ¼ 4.8 Hz). 13C NMR (DMSO-d6):
d 70.39, 113.89, 115.41, 119.04,
121.82, 123.10, 128.19, 128.46, 128.65, 132.08, 133.11, 137.04, 149.14,
155.93, 156.07, 159.85, 160.87, 166.33. Anal. Calcd for
C
22H15ClN2O4$0.3CH3OH C, 64.32; H, 3.92; N, 6.73; found: C, 64.35;
4.2.3. Expression, purification and cell-free activity analysis of
human recombinant 5-LO
Escherichia coli BL21 cells were transformed with pT3-5-LO
plasmid, and recombinant 5-LO protein was expressed at 27 ꢂC as
described [46]. Cells, resuspendend in 50 mM triethanolamine/HCl
H, 3.96; N, 6.67. HRMS (m/z): [MþH]þ calcd for C22H16ClN2O4
407.0799, found 407.0782.
4.1.2.8. 4-(4-Chlorophenyl)-5-[4-(quinolin-2-ylmethoxy)phenyl]iso-
xazol-3-carboxylic acid (39). Purified by recrystallization from iso-
pH 8.0, 5 mM EDTA, soybean trypsin inhibitor (60
phenylmethanesulphonyl fluoride, and lysozyme (500
m
g/ml), 1 mM
propanol. Yield 94%; mp 208.4 ꢂC. 1H NMR (DMSO-d6):
d 5.40 (2H,
mg/ml), were
s), 7.15 (2H, d, J ¼ 8.8 Hz), 7.38e7.42 (4H, m), 7.50 (2H, d, J ¼ 8.8 Hz),
homogenized by sonication (3 ꢃ 15 s). After centrifugation at
40,000 ꢃ g for 20 min at 4 ꢂC, the supernatant was applied to an
ATP-agarose column to partially purify 5-LO as described previ-
ously [46].
7.61e7.67 (2H, m), 7.76e7.80 (1H, m), 7.99 (2H, m), 8.42 (1H, d,
J ¼ 8.0 Hz). 13C NMR (DMSO-d6):
d 70.84, 113.84, 115.42, 119.05,
119.52, 126.59, 127.13, 127.88, 128.09, 128.42, 128.47, 128.58, 129.85,
131.99, 133.03, 137.05, 146.86, 155.84, 156.87, 159.78, 160.77, 166.23.
Anal. Calcd for C26H17ClN2O4$1.0 HOCH(CH3)2 C, 67.93; H, 4.23; N,
5.83; found: C, 67.62; H, 4.26; N, 5.99. HRMS (m/z): [MþH]þ calcd
for C26H18ClN2O4 457.0955, found 457.0954.
Aliquots of semi-purified 5-LO were diluted with ice-cold PBS
containing 1 mM EDTA, and 1 mM ATP was added. Samples were
pre-incubated with the test compounds or vehicle (0.1% DMSO) as
indicated. After 15 min at 4 ꢂC, samples were pre-warmed for
30 s at 37 ꢂC, and 2 mM CaCl2 plus 20
mM AA was added to initiate
5-LO product formation. After 10 min at 37 ꢂC, the reaction was
stopped by addition of 1 ml ice-cold methanol, and the formed
metabolites were analyzed by RP-HPLC as described [47]. 5-LO
products include the all-trans isomers of LTB4 and 5-H(P)ETE.
4.1.2.9. 4-(4-Chlorophenyl)-5-[4-(benzothiazol-2-ylmethoxy)phenyl]
isoxazol-3-carboxylic acid (40). Purified by recrystallization from
methanol. Yield 89%; mp 196.5 ꢂC. 1H NMR (DMSO-d6):
d 5.65 (2H,
s), 7.18 (2H, d, J ¼ 9.2 Hz), 7.38e7.56 (8H, m), 8.02 (1H, d, J ¼ 8.4 Hz),
8.12 (1H, d, J ¼ 7.6 Hz). 13C NMR (DMSO-d6):
d 67.11, 114.02, 115.53,
119.64, 122.35, 122.69, 125.34, 126.30, 128.03, 128.47, 128.58, 131.98,
133.06, 134.34, 152.37, 155.89, 159.02, 160.75, 166.08, 167.51. Anal.
Calcd for C24H15ClN2O4S$0.5CH3OH C, 61.44; H, 3.38; N, 5.85; S, 6.70
found: C, 61.49; H, 3.36; N, 6.20; S, 6.94. HRMS (m/z): [MþH]þ calcd
for C24H16ClN2O4S 463.0519, found 463.0516.
4.3. Statistics
Data are expressed as mean S.E.M. of single determinations
performed in three or four independent experiments at different
days. IC50 values were calculated by nonlinear regression using
SigmaPlot 9.0 (Systat Software Inc., San Jose, USA) one site binding
competition. Statistical evaluation of the data was performed by
one-way ANOVA followed by a Bonferroni or Tukey-Kramer post-
hoc test for multiple comparisons respectively. A p value < 0.05 (*)
was considered significant.
4.2. Biological assays
4.2.1. Cells
Neutrophils were isolated from human blood as reported before
[45]. Briefly, human peripheral blood was obtained from fastened
(12 h) healthy donors with consent that had not taken anti-
inflammatory drugs during the last 10 days, by venipuncture in
heparinized tubes (16 IE heparin/ml blood; University Hospital
Jena, Germany). The blood was centrifuged (4000 ꢃ g for 20 min at
20 ꢂC) for preparation of leukocyte concentrates. Leukocyte con-
centrates were then subjected to dextran sedimentation and
centrifugation on Nycoprep cushions. Contaminating erythrocytes
of pelleted neutrophils were lysed by hypotonic lysis using water.
Neutrophils were washed twice in ice-cold PBS (purity > 96e97%)
and finally resuspended in PBS pH 7.4 containing 1 mg/ml glucose
and 1 mM CaCl2 (PGC buffer).
4.4. Computational methods
All the methodologies and parameters used in this study are
reported in Supplementary information.
Acknowledgments
This study was supported by The Scientific and Technological
Research Council of Turkey (TUBITAK No.108S210). One of the GPUs
used in this study was a donation from NVIDIA Corporation.
For analysis of acute cytotoxicity of the compounds during pre-
incubation periods (30 min at 37 ꢂC), cellular integrity of neutro-
phils was analyzed by trypan blue exclusion with a Vi-cell counter
(Beckmann Coulter GmbH, Krefeld). None of the compounds
caused significant loss of neutrophil viability within 30 min (data
not shown).
Appendix A. Supplementary information
Supplementary information related to this article can be found
at http://dx.doi.org/10.1016/j.ejmech.2016.02.027.
References
4.2.2. Determination of 5-LO products in intact cells
[1] J.Z. Haeggstrom, C.D. Funk, Lipoxygenase and leukotriene pathways:
biochemistry, biology, and roles in disease, Chem. Rev. 111 (2011)
For determination of LO products in intact cells, neutrophils

10
E. Banoglu et al. / European Journal of Medicinal Chemistry 113 (2016) 1e10
5866e5898.
[24] S. Sarveswaran, C.E. Myers, J. Ghosh, MK591, a leukotriene biosynthesis in-
hibitor, induces apoptosis in prostate cancer cells: synergistic action with
LY294002, an inhibitor of phosphatidylinositol 30-kinase, Cancer Lett. 291
(2010) 167e176.
[2] O. Radmark, B. Samuelsson, Regulation of the activity of 5-lipoxygenase, a key
enzyme in leukotriene biosynthesis, Biochem. Biophys. Res. Commun. 396
(2010) 105e110.
[3] O. Radmark, O. Werz, D. Steinhilber, B. Samuelsson, 5-lipoxygenase, a key
enzyme for leukotriene biosynthesis in health and disease, Biochim. Biophys.
Acta 1851 (2015) 331e339.
[4] O. Werz, 5-lipoxygenase: cellular biology and molecular pharmacology, Curr.
Drug Targets Inflamm. Allergy 1 (2002) 23e44.
[5] J. Gerstmeier, C. Weinigel, S. Rummler, O. Radmark, O. Werz, U. Garscha, Time-
resolved in situ assembly of the leukotriene-synthetic 5-lipoxygenase/5-
lipoxygenase-activating protein complex in blood leukocytes, FASEB J.
(2015), http://dx.doi.org/10.1096/fj.15-278010. Epub.
[25] S. Sarveswaran, V. Thamilselvan, C. Brodie, J. Ghosh, Inhibition of 5-
lipoxygenase triggers apoptosis in prostate cancer cells via down-regulation
of protein kinase C-epsilon, Biochim. Biophys. Acta 1813 (2011) 2108e2117.
[26] X. Chen, S. Sood, C.S. Yang, N. Li, Z. Sun, Five-lipoxygenase pathway of
arachidonic acid metabolism in carcino-genesis and cancer chemoprevention,
Curr. Cancer Drug Targets 6 (2006) 613e622.
[27] Y. Chen, D. Li, S. Li, The Alox5 gene is a novel therapeutic target in cancer stem
cells of chronic myeloid leukemia, Cell Cycle 8 (2009) 3488e3492.
[28] T.J. Kennedy, C.Y. Chan, X.Z. Ding, T.E. Adrian, Lipoxygenase inhibitors for the
[6] S.E. Wenzel, A.K. Kamada, Zileuton: the first 5-lipoxygenase inhibitor for the
treatment of asthma, Ann. Pharmacother. 30 (1996) 858e864.
[7] J.A. Bernstein, N. Liu, B.A. Knorr, S.S. Smugar, W.D. Hanley, T.F. Reiss,
treatment of pancreatic cancer, Expert Rev. Anticancer Ther. 3 (2003)
525e536.
[29] E. Banoglu, B. Caliskan, S. Luderer, G. Eren, Y. Ozkan, W. Altenhofen,
C. Weinigel, D. Barz, J. Gerstmeier, C. Pergola, O. Werz, Identification of novel
benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual
screening targeting 5-lipoxygenase-activating protein (FLAP), Bioorg. Med.
Chem. 20 (2012) 3728e3741.
S. Greenberg, MK-0633,
a potent 5-lipoxygenase inhibitor, in chronic
obstructive pulmonary disease, Respir. Med. 105 (2011) 392e401.
[8] Y. Ducharme, M. Blouin, C. Brideau, A. Chateauneuf, Y. Gareau, E.L. Grimm,
H. Juteau, S. Laliberte, B. MacKay, F. Masse, M. Ouellet, M. Salem, A. Styhler,
R.W. Friesen, The discovery of setileuton,
a
potent and selective 5-
[30] B. Caliskan, S. Luderer, Y. Ozkan, O. Werz, E. Banoglu, Pyrazol-3-propanoic acid
derivatives as novel inhibitors of leukotriene biosynthesis in human neutro-
phils, Eur. J. Med. Chem. 46 (2011) 5021e5033.
[31] R. Sardella, S. Levent, F. Ianni, B. Caliskan, J. Gerstmeier, C. Pergola, O. Werz,
E. Banoglu, B. Natalini, Chromatographic separation and biological evaluation
of benzimidazole derivative enantiomers as inhibitors of leukotriene
biosynthesis, J. Pharm. Biomed. Anal. 89 (2014) 88e92.
[32] C. Pergola, J. Gerstmeier, B. Monch, B. Caliskan, S. Luderer, C. Weinigel, D. Barz,
J. Maczewsky, S. Pace, A. Rossi, L. Sautebin, E. Banoglu, O. Werz, The novel
benzimidazole derivative BRP-7 inhibits leukotriene biosynthesis in vitro and
in vivo by targeting 5-lipoxygenase-activating protein (FLAP), Br. J. Pharmacol.
171 (2014) 3051e3064.
lipoxygenase inhibitor, ACS Med. Chem. Lett. 1 (2010) 170e174.
[9] Y.S. Wasfi, C. Villaran, B. de Tilleghem Cle, S.S. Smugar, W.D. Hanley, T.F. Reiss,
B.A. Knorr, The efficacy and tolerability of MK-0633, a 5-lipoxygenase inhib-
itor, in chronic asthma, Respir. Med. 106 (2012) 34e46.
[10] R. Chaudhuri, V. Norris, K. Kelly, C.Q. Zhu, C. Ambery, J. Lafferty, E. Cameron,
N.C. Thomson, Effects of a FLAP inhibitor, GSK2190915, in asthmatics with
high sputum neutrophils, Pulm. Pharmacol. Ther. 27 (2014) 62e69.
[11] R.M. Follows, N.G. Snowise, S.Y. Ho, C.L. Ambery, K. Smart, B.A. McQuade,
Efficacy, safety and tolerability of GSK2190915, a 5-lipoxygenase activating
protein inhibitor, in adults and adolescents with persistent asthma: a rand-
omised dose-ranging study, Respir. Res. 14 (2013) 54.
[12] M. Lemurell, J. Ulander, S. Winiwarter, A. Dahlen, O. Davidsson, H. Emtenas,
J. Broddefalk, M. Swanson, D. Hovdal, A.T. Plowright, A. Pettersen, M. Ryden-
Landergren, J. Barlind, A. Llinas, M. Herslof, T. Drmota, K. Sigfridsson, S. Moses,
C. Whatling, Discovery of AZD6642, an inhibitor of 5-lipoxygenase activating
protein (FLAP) for the treatment of inflammatory diseases, J. Med. Chem. 58
(2015) 897e911.
[13] H. Takahashi, D. Riether, A. Bartolozzi, T. Bosanac, V. Berger, R. Binetti,
J. Broadwater, Z. Chen, R. Crux, S. De Lombaert, R. Dave, J.A. Dines, T. Fadra-
Khan, A. Flegg, M. Garrigou, M.H. Hao, J. Huber, J.M. Hutzler, S. Kerr, A. Kotey,
W. Liu, H.Y. Lo, P.L. Loke, P.E. Mahaney, T.M. Morwick, S. Napier, A. Olague,
E. Pack, A.K. Padyana, D.S. Thomson, H. Tye, L. Wu, R.M. Zindell,
A. Abeywardane, T. Simpson, Synthesis, SAR, and series evolution of novel
oxadiazole-containing 5-lipoxygenase activating protein inhibitors: discovery
of 2-[4-(3-{(r)-1-[4-(2-amino-pyrimidin-5-yl)-phenyl]-1-cyclopropyl-ethyl}-
[1,2,4]oxadiazol-5-yl)-pyrazol-1-yl]-N,N-dimethyl-acetamide (BI 665915),
J. Med. Chem. 58 (2015) 1669e1690.
[14] C. Whatling, W. McPheat, M. Herslof, The potential link between atheroscle-
rosis and the 5-lipoxygenase pathway: investigational agents with new im-
plications for the cardiovascular field, Expert. Opin. Investig. Drugs 16 (2007)
1879e1893.
[15] S. Xu, L. Tang, Y. Mi, J. Jiang, M. Zhu, B. Chen, B. Wang, T. Li, C. Xu, J. Wang,
X. Lu, W. Ge, L. Wang, J. Huang, Clinical significance of leukotriene b4 and
extracellular matrix metalloproteinase inducer in acute coronary syndrome,
Clin. Investig. Med. 36 (2013) E282eE289.
[33] G. Eren, A. Macchiarulo, E. Banoglu, From molecular docking to 3D-quanti-
tative structure-activity relationships (3D-QSAR): insights into the binding
mode of 5-lipoxygenase inhibitors, Mol. Inf. 31 (2012) 123e134.
[34] M.G. Chini, R. De Simone, I. Bruno, R. Riccio, F. Dehm, C. Weinigel, D. Barz,
O. Werz, G. Bifulco, Design and synthesis of a second series of triazole-based
compounds as potent dual mPGES-1 and 5-lipoxygenase inhibitors, Eur. J.
Med. Chem. 54 (2012) 311e323.
[35] 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)
217e219.
[36] A.G. Habeeb, P.N. Praveen Rao, E.E. Knaus, Design and synthesis of 4,5-
diphenyl-4-isoxazolines: novel inhibitors of cyclooxygenase-2 with anal-
gesic and antiinflammatory activity, J. Med. Chem. 44 (2001) 2921e2927.
[37] P.A. Brough, W. Aherne, X. Barril, J. Borgognoni, K. Boxall, J.E. Cansfield,
K.M. Cheung, I. Collins, N.G. Davies, M.J. Drysdale, B. Dymock, S.A. Eccles,
H. Finch, A. Fink, A. Hayes, R. Howes, R.E. Hubbard, K. James, A.M. Jordan,
A. Lockie, V. Martins, A. Massey, T.P. Matthews, E. McDonald, C.J. Northfield,
L.H. Pearl, C. Prodromou, S. Ray, F.I. Raynaud, S.D. Roughley, S.Y. Sharp,
A. Surgenor, D.L. Walmsley, P. Webb, M. Wood, P. Workman, L. Wright, 4,5-
diarylisoxazole Hsp90 chaperone inhibitors: potential therapeutic agents for
the treatment of cancer, J. Med. Chem. 51 (2008) 196e218.
[38] O. Werz, D. Steinhilber, Therapeutic options for 5-lipoxygenase inhibitors,
Pharmacol. Ther. 112 (2006) 701e718.
[39] A.D. Ferguson, B.M. McKeever, S. Xu, D. Wisniewski, D.K. Miller, T.T. Yamin,
R.H. Spencer, L. Chu, F. Ujjainwalla, B.R. Cunningham, J.F. Evans, J.W. Becker,
Crystal structure of inhibitor-bound human 5-lipoxygenase-activating pro-
tein, Science 317 (2007) 510e512.
[40] R. De Simone, M.G. Chini, I. Bruno, R. Riccio, D. Mueller, O. Werz, G. Bifulco,
Structure-based discovery of inhibitors of microsomal prostaglandin E2
synthase-1, 5-lipoxygenase and 5-lipoxygenase-activating protein: promising
hits for the development of new anti-inflammatory agents, J. Med. Chem. 54
(2011) 1565e1575.
[41] A.D. Ferguson, Structure-based drug design on membrane protein targets:
human integral membrane protein 5-lipoxygenase-activating protein,
Methods Mol. Biol. 841 (2012) 267e290.
[16] M. Back, Inflammatory signaling through leukotriene receptors in athero-
sclerosis, Curr. Atheroscler. Rep. 10 (2008) 244e251.
[17] R. De Caterina, A. Zampolli, From asthma to atherosclerosise5-lipoxygenase,
leukotrienes, and inflammation, N. Engl. J. Med. 350 (2004) 4e7.
[18] R. Spanbroek, R. Grabner, K. Lotzer, M. Hildner, A. Urbach, K. Ruhling,
M.P. Moos, B. Kaiser, T.U. Cohnert, T. Wahlers, A. Zieske, G. Plenz, H. Robenek,
P. Salbach, H. Kuhn, O. Radmark, B. Samuelsson, A.J. Habenicht, Expanding
expression of the 5-lipoxygenase pathway within the arterial wall during
human atherogenesis, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 1238e1243.
[19] J. Roos, C. Oancea, M. Heinssmann, D. Khan, H. Held, A.S. Kahnt, R. Capelo, E. la
Buscato, E. Proschak, E. Puccetti, D. Steinhilber, I. Fleming, T.J. Maier,
M. Ruthardt, 5-lipoxygenase is a candidate target for therapeutic manage-
ment of stem cell-like cells in acute myeloid leukemia, Cancer Res. 74 (2014)
5244e5255.
[42] J.F. Evans, A.D. Ferguson, R.T. Mosley, J.H. Hutchinson, What's all the FLAP
about?: 5-lipoxygenase-activating protein inhibitors for inflammatory dis-
eases, Trends Pharmacol. Sci. 29 (2008) 72e78.
[20] M. Back, Leukotriene signaling in atherosclerosis and ischemia, Cardiovasc.
Drugs Ther. 23 (2009) 41e48.
[43] M.A. Lomize, A.L. Lomize, I.D. Pogozheva, H.I. Mosberg, OPM: orientations of
proteins in membranes database, Bioinformatics 22 (2006) 623e625.
[44] C. Kandt, W.L. Ash, D.P. Tieleman, Setting up and running molecular dynamics
simulations of membrane proteins, Methods 41 (2007) 475e488.
[45] A.M. Schaible, H. Traber, V. Temml, S.M. Noha, R. Filosa, A. Peduto, C. Weinigel,
D. Barz, D. Schuster, O. Werz, Potent inhibition of human 5-lipoxygenase and
microsomal prostaglandin E synthase-1 by the anti-carcinogenic and anti-
inflammatory agent embelin, Biochem. Pharmacol. 30 (1) (2016) 276e285.
[46] L. Fischer, D. Szellas, O. Radmark, D. Steinhilber, O. Werz, Phosphorylation-
and stimulus-dependent inhibition of cellular 5-lipoxygenase activity by
nonredox-type inhibitors, FASEB J. 17 (2003) 949e951.
[21] M. Back, A. Sultan, O. Ovchinnikova, G.K. Hansson, 5-Lipoxygenase-activating
protein: a potential link between innate and adaptive immunity in athero-
sclerosis and adipose tissue inflammation, Circ. Res. 100 (2007) 946e949.
[22] J. Jawien, M. Gajda, M. Rudling, L. Mateuszuk, R. Olszanecki, T.J. Guzik,
T. Cichocki, S. Chlopicki, R. Korbut, Inhibition of five lipoxygenase activating
protein (FLAP) by MK-886 decreases atherosclerosis in apoE/LDLR-double
knockout mice, Eur. J. Clin. Investig. 36 (2006) 141e146.
[23] H. Qiu, A. Gabrielsen, H.E. Agardh, M. Wan, A. Wetterholm, C.H. Wong,
U. Hedin, J. Swedenborg, G.K. Hansson, B. Samuelsson, G. Paulsson-Berne,
J.Z. Haeggstrom, Expression of 5-lipoxygenase and leukotriene A4 hydrolase
in human atherosclerotic lesions correlates with symptoms of plaque insta-
bility, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 8161e8166.
[47] O. Werz, E. Burkert, B. Samuelsson, O. Radmark, D. Steinhilber, Activation of 5-
lipoxygenase by cell stress is calcium independent in human poly-
morphonuclear leukocytes, Blood 99 (2002) 1044e1052.