Synthesis and biological evaluation of C(5)-substituted derivatives of leukotriene biosynthesis inhibitor BRP-7

Article abstract of DOI:10.1016/j.ejmech.2016.07.004

Pharmacological intervention with 5-lipoxygenase (5-LO) pathway leading to suppression of leukotriene (LT) biosynthesis is a clinically validated strategy for treatment of respiratory and cardiovascular diseases such as asthma and atherosclerosis. Here we describe the synthesis of a series of C(5)-substituted analogues of the previously described 5-LO-activating protein (FLAP) inhibitor BRP-7 (IC₅₀?=?0.31?μM) to explore the effects of substitution at the C(5)-benzimidazole (BI) ring as a strategy to increase the potency against FLAP-mediated 5-LO product formation. Incorporation of polar substituents on the C(5) position of the BI core, exemplified by compound 11 with a C(5)-nitrile substituent, significantly enhances the potency for suppression of 5-LO product synthesis in human neutrophils (IC₅₀?=?0.07?μM) and monocytes (IC₅₀?=?0.026?μM).

Full text of DOI:10.1016/j.ejmech.2016.07.004

European Journal of Medicinal Chemistry 122 (2016) 510e519
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of C(5)-substituted derivatives of
leukotriene biosynthesis inhibitor BRP-7
Serkan Levent a, Jana Gerstmeier b, Abdurrahman Olgaç a, Felix Nikels b, Ulrike Garscha b,
,
**
Andrea Carotti c, Antonio Macchiarulo c, Oliver Werz b, Erden Banoglu a
,
,
*
Burcu Çalıs¸ kan a
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
ꢀ
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:
Pharmacological intervention with 5-lipoxygenase (5-LO) pathway leading to suppression of leukotriene
(LT) biosynthesis is a clinically validated strategy for treatment of respiratory and cardiovascular diseases
such as asthma and atherosclerosis. Here we describe the synthesis of a series of C(5)-substituted an-
Received 28 March 2016
Received in revised form
10 June 2016
Accepted 3 July 2016
Available online 5 July 2016
alogues of the previously described 5-LO-activating protein (FLAP) inhibitor BRP-7 (IC50 ¼ 0.31
mM) to
explore the effects of substitution at the C(5)-benzimidazole (BI) ring as a strategy to increase the po-
tency against FLAP-mediated 5-LO product formation. Incorporation of polar substituents on the C(5)
position of the BI core, exemplified by compound 11 with a C(5)-nitrile substituent, significantly en-
Keywords:
Leukotriene
5-Lipoxygenase-activating protein
FLAP
hances the potency for suppression of 5-LO product synthesis in human neutrophils (IC50 ¼ 0.07
mM) and
monocytes (IC50 ¼ 0.026 M).
m
© 2016 Elsevier Masson SAS. All rights reserved.
Benzimidazole
BRP-7
1. Introduction
edema and mucus secretion.
In intact cells, the 5-LO-mediated formation of LTA4 from AA
also requires a concomitant involvement of the 5-LO-activating
protein (FLAP) that transfers AA to 5-LO for efficient metabolism.
FLAP may function as a regulatory protein for 5-LO at the nuclear
membrane where the LT synthetic complex is assembled [4].
Pharmacological concepts to interfere with LT biosynthesis essen-
tially include direct 5-LO inhibitors or compounds that block the
function of FLAP [2,5]. Although intensive efforts have been made
to develop LT biosynthesis inhibitors, only a single 5-LO inhibitor,
namely zileuton (1), is in clinical use for the treatment of asthma
and/or allergic rhinitis [6]. Meanwhile, 5-LO inhibitor setileuton (2,
MK-0633) and several FLAP inhibitors such as fiboflapon
(GSK2190915, 3), AZD6642 (4) and BI665915 (5) have been re-
ported to be in various phases of preclinical and clinical studies for
treatment of respiratory diseases such as asthma and COPD [7e10]
(Fig. 1). Moreover, beneficial pharmacology of LT biosynthesis in-
hibitors in atherosclerosis was demonstrated, highlighting the
potential value of this therapeutic target for cardiovascular diseases
besides respiratory pathologies [11e15].
Leukotrienes (LTs) are important pro-inflammatory mediators,
which play key roles in the progression of inflammation in various
ailments including asthma, chronic obstructive pulmonary disease
(COPD), arthritis, allergy, cancer, and atherosclerosis [1,2]. LTs are
produced by the 5-lipoxygenase (5-LO) pathway, which is activated
by the release of arachidonic acid (AA) via cytosolic phospholipase
(cPLA2) in response to inflammatory stimuli. Liberated AA is then
converted to the unstable epoxide intermediate LTA4 by 5-LO,
which is a junction point for production of either LTB4 by LTA4
hydrolase or cysteinyl-LTs (LTC4, D4 and E4) by LTC4 synthase [3].
LTB4 acts as ligand for the G protein-coupled receptors (GPCR) BLT1
and BLT2, which promotes the inflammatory response as well as
neutrophil and eosinophil chemotaxis. The cysteinyl-LTs activate
the GPCRs CysLT1 and CysLT2 causing bronchoconstriction, airway
* Corresponding author.
** Corresponding author.
E-mail addresses: banoglu@gazi.edu.tr (E. Banoglu), bcaliskan@gazi.edu.tr
(B. Çalıs¸ kan).
Based on the renowned interest in LT biosynthesis inhibitors
and as a result of our continuing efforts to develop novel
http://dx.doi.org/10.1016/j.ejmech.2016.07.004
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

S. Levent et al. / European Journal of Medicinal Chemistry 122 (2016) 510e519
511
Fig. 1. Chemical structures of LT biosynthesis inhibitors.
compounds along these lines [16e23], we recently identified a non-
acidic benzimidazole derivative (6, BRP-7) bearing an ibuprofen
fingerprint with reasonable potency to suppress 5-LO product
HBr to generate the 5-hydroxy derivatives (6a-b). The 5-
hydroxybenzimidazole derivatives were then alkylated with
appropriate alkyl halides to obtain the target compounds 7a-u. For
the synthesis of 5-cyanobenzimidazole derivative 11 (Scheme 2), N-
2-chlorobenzyl-4-cyano-2-nitroaniline 8 was first prepared from 2-
chloro-5-cyanonitrobenzene by nucleophilic substitution of the
chlorine atom by 2-chlorobenzylamine. Reduction of the nitro
group followed by the standard procedures described in Scheme 2
resulted in the 5-cyanobenzimidazole derivative 11.
All compounds were purified by automated flash chromatog-
raphy and checked for purity with UPLC before being tested in
biological assays (purity was >97%). The structures of the com-
pounds were confirmed by high-resolution mass spectrometry
(HRMS), 1H and 13C NMR spectral data.
synthesis in intact neutrophils (IC50 ¼ 0.31
mM) [16]. Our detailed
pharmacological investigation revealed BRP-7 as a potent inhibitor
of LT biosynthesis targeting FLAP without affecting 5-LO [22,23].
Our preliminary structure-activity relationships (SAR) studies
indicated that (i) the benzyl group in 1-position of the benzimid-
azole (BI) is absolutely essential and ortho substitution increases
the potency, (ii) the isobutyl substitution clearly governs the po-
tency and larger or smaller hydrophobic groups in this position are
not tolerated, (iii) C(5)-BI substitution may favor inhibition of LT
biosynthesis and (iv) the chirality at asymmetric carbon linked at
C(2)-BI was not an important determinant of the biological activity
since a negligible difference in the inhibitory activity of individual
isomers over racemic BRP-7 was previously revealed [16,23]. Since
the number of substitutions was limited to conclusively explain the
contribution of C(5)-BI substitution on pharmacological activity,
here we explored different substituents at C(5)-BI to obtain broad
and non-correlated variation in their lipophilic, electronic and
steric properties. This substitution-focused SAR-study resulted in
the discovery of highly active compounds that might be of impor-
tance to understand the role of C(5)-substitution of the BI in order
to develop efficient inhibitors of LT biosynthesis.
2.2. Biological evaluation and SAR
We previously established that ortho-substituted N-benzyl
group of BRP-7 derivatives governed the potent inhibition of 5-LO
product formation, and lipophilic and electron-withdrawing o-CF3
substitution on N-benzyl slightly improved the activity
(IC50 ¼ 0.25
m
m
M) as compared to N-o-chlorobenzyl derivative BRP-7
M) [16]. In addition, we observed slightly improved
(IC50 ¼ 0.31
potency by substitutions at C(5) of BRP-7, applying a limited
number of substituents such as hydroxyl, methoxy, chlorine and 2-
2. Results and discussion
pyridinylmethyloxy (IC50 ¼ 0.12e0.19
mM) [16]. Based on this initial
screen, we introduced further modifications by varying the sub-
stituent at C(5)-BI basically to obtain compounds with different
physicochemical characteristics (compounds 7aeu in Table 1).
LT biosynthesis is initiated by cPLA2-mediated release of AA,
which is subsequently converted to LTA4 by activated 5-LO with the
aid of FLAP that is essential for cellular LT biosynthesis [4]. Note that
FLAP has no concrete (enzymatic) activity that can be monitored in
a cell-free assay with a select read-out. Therefore, we analyzed the
test compounds in a ‘FLAP-dependent’ cell-based assay for sup-
pression of 5-LO (i.e. LTB4 and its trans isomers and 5-HPETE)
product formation as described before [22]. In this assay, neutro-
phils are challenged with Ca2þ ionophore A23187 that causes
2.1. Chemistry
For the synthesis of compounds 7a-u, we utilized the synthetic
procedure outlined in Scheme 1 by following our published pro-
cedures [16]. All compounds were synthesized as racemates since
we previously showed a negligible difference in the inhibitory
potency of individual enantiomers of BRP-7 [23]. Starting from 2-
nitro-4-methoxyaniline, benzylation and further reduction of the
nitro group generated 2a-b. Following the described conditions
shown in Scheme 1, the 5-methoxybenzimidazole derivatives 5a-b
were obtained. Hydrolysis of the methyl ether was accomplished by

512
S. Levent et al. / European Journal of Medicinal Chemistry 122 (2016) 510e519
Scheme 1. Synthesis of C(5)-substituted analogues of BRP-7. Reagents and conditions: (a) DIEA, DMF; (b) Pd/C, H2 or SnCl2, EtOH; (c) ibuprofen (3), EDC, DMAP, DCM; (d) AcOH, D;
(e) HBr 48%; (f) alkyl halides, K2CO3, AcCN.
Scheme 2. Synthesis of C(5)-nitrile-substituted analogue of BRP-7. Reagents and conditions: (a) 2-chlorobenzylamine, MeOH; (b) SnCl2, EtOH; (c) ibuprofen acyl chloride, DIEA,
DCM; (d) Dioxan, HCl,
D.
substantial formation of 5-LO products as a result of elevated
intracellular Ca2þ concentration where FLAP plays an indispensable
role in facilitating substrate (AA) transfer to 5-LO [3]. The initial
screening of 7a-u for their ability to inhibit 5-LO product formation
in this assay was performed at 1 and 10 mM concentrations of test
compounds. Analyzed 5-LO products include 5-H(P)ETE and all
trans-isomers of LTB4, as well as LTB4 in intact cells.
Among the tested compounds, only compounds carrying 2-
pyridinylmethyloxy (7a), 4- or 2-cyanobenzyloxy (7h and 7l), 2-
allyloxy (7o), 3,3-dimethylallyloxy (7p), 2-pentynoxy (7r), n-buty-
loxy (7s) and methoxycarbonylmethyloxy (7t) substituents showed
beneficial and well tolerated at C(5) position of the BI core for in-
hibition of 5-LO product synthesis (Table 1). Since, 7a, 7o and 7t
were the only derivatives that elicited almost complete inhibition
of 5-LO product formation at 1
response studies were performed in order to calculate the IC50
values, which were determined at 0.15, 0.17 and 0.21 M, respec-
mM, more detailed concentration-
m
tively (Table 2). Suppression of 5-LO product synthesis by 7l (35%)
was more pronounced when N-2-CF3-benzyl was replaced with N-
2-chlorobenzyl (7u, 61%), in which the IC50 value for 7u was
determined at 0.12 mM (Table 2).
On the basis of the contribution of the polar nitrile moiety to the
inhibitory activity, and also considering this moiety as a prevalent
pharmacophoric element contributing to drug-like properties of
many known drug molecules [24], we chose to synthesize the 5-
nitrile derivative of BRP-7 (11, Scheme 2). Interestingly, 5-nitrile
substitution resulted in the highly active derivative (11), which
outperformed BRP-7, as the potency of 11 in the cell-based assay
was improved by 4.4-fold (Table 2). In fact, compound 11
concentration-dependently inhibited the formation of 5-LO-
derived products in A23187-stimulated human neutrophils with an
significant inhibition of 5-LO product synthesis at 1 mM (Table 1).
Interestingly, other p-substituted analogues equipped with
nonpolar or bulky substituents (7be7g) failed to inhibit cellular 5-
LO product synthesis. This suggests that introducing lipophilicity or
increasing the steric bulk at p-phenyl at C(5)-BI (7be7g) impaired
the potency except for 7h (with polar p-CN), indicating that volu-
minous and nonpolar substituents at this position are not well
accommodated. Accordingly, variations of the nonpolar sub-
stituents in o-position of the phenyl ring (i.e., compounds 7ie7k)
abolished the inhibitory activity whereas significant suppression of
IC50 value of 0.07 mM.
5-LO product formation at 1
m
M could be retained with o-CN sub-
Besides neutrophils, monocytes are also a major source of LTs
[25,26]. We thus determined the effects of 11 on 5-LO product
formation in human monocytes stimulated with A23187. Com-
pound 11 potently inhibited the formation of 5-LO products (LTB4
and its trans isomers and 5-H(p)ETE) in A23187-stimulated
stitution (7l) providing an H-bond acceptor. m-Substituted phenyl
analogues (7me7n) were not pursued further as they showed
marked loss of inhibitory activity (Table 1). In addition, multiple
alkoxy substitutions (7oe7s) and ester group (7t) were quite

S. Levent et al. / European Journal of Medicinal Chemistry 122 (2016) 510e519
Table 1 (continued )
513
Table 1
Effects of test compounds on 5-LO product synthesis in a cell-based (intact neu-
trophils) assay.
Cmpd.
R1
R
5-LO product formation in
neutrophils, remaining
activity (% of control) at
Cmpd.
R1
R
5-LO product formation in
neutrophils, remaining
activity (% of control) at
1
m
M
10 mM
7s
7t
CF3
CF3
58.1 24.3
1.9 1.7
n.d.
n.d.
1
m
M
10
mM
7a
7b
CF3
CF3
0.0 0.0
n.d.
7u
Cl
Cl
39.9 14.5
15.6 6.9
92.6 6.3
90.5 14.9
94.3 14.1
80.1 7.5
94.0 9.5
94.4 9.6
n.d.
11
12
0.0 0.0
2.7 1.2
n.d.
n.d.
7c
7d
7e
7f
CF3
CF3
CF3
CF3
CF3
CF3
91.7 9.6
81.6 2.9
94.9 16.9
108.1 10.9
99.9 6.5
71.1 17.6
MK-886
Compounds were tested in neutrophils stimulated with 2.5
given as percentage of control at 1 and 10 M inhibitor concentration (means SE,
n ¼ 4e6). The 100% value of the control (0.3% DMSO, vehicle) corresponds to an
average of 47 ng 5-LO products per 106 neutrophils; n.d., not determined. The FLAP
inhibitor 3-[3-(tert-butylthio)-1-(4-chlorobenzyl)-5-isopropyl-1H-indol-2-yl]-2,2-
dimethylpropanoic acid (12, MK-886, see Fig. S1 for chemical structure) was used
mM A23187. Data are
m
(1 mM) as reference compound.
monocytes with an IC50 ¼ 0.026
that observed for neutrophils (Fig. 2A). In the presence of 10
exogenous AA, 11 still inhibited 5-LO activity with a slightly
reduced potency (IC50 ¼ 0.035 M; Fig. 2B), which was seen with
mM, which was more potent than
mM
7g
7h
m
BRP-7 and other FLAP inhibitors whose potencies are also reduced
by addition of exogenous AA [22,27,28]. In contrast to neutrophils,
monocytes may convert LTA4 to LTC4 by LTC4 synthase, and com-
pound 11 also efficiently suppressed the synthesis of cysteinyl
(cys)-LTs in monocytes stimulated with lipopolysaccharide (LPS)
plus N-formyl-methionyl-leucyl-phenylalanine (fMLP), which was
more physiologically relevant assay condition, with an IC50 of
n.d.
7i
7j
CF3
CF3
97.1 13.7
90.9 8.3
89.7 4.5
n.d.
0.012 mM,.
Because there is no enzymatic function for FLAP (an integral
membrane protein) has yet been discovered and since FLAP is
operative only in a complex cellular environment, proof of direct
functional interference of a test compound with this protein is
laborious. To confirm that compound 11 acts on FLAP and not on 5-
LO to suppress cellular 5-LO product formation, we assayed 11 in a
cell-free 5-LO activity assay using isolated human recombinant 5-
LO as the enzyme source. As shown in Fig. 3A, 11 failed to sub-
7k
7l
CF3
CF3
CF3
92.2 15.7
65.2 19.4
98.2 2.9
92.2 15.5
n.d.
7m
n.d.
stantially inhibit 5-LO activity up to 10
mM, while the direct 5-LO
inhibitor zileuton (3 M) blocked enzyme activity by > 80% (not
m
shown) as expected and as reported in previous studies by us
[17,29]. Moreover, we analyzed 11 in a cell-based model suitable for
studying FLAP functionality in 5-LO product biosynthesis [30].
Thus, HEK293 cells stably expressing solely 5-LO (HEK_5-LO) and
HEK cells expressing both 5-LO and FLAP (HEK_5-LO/FLAP) were
investigated; the FLAP inhibitor MK-886 was used as reference
control [31]. Compound 11 failed to suppress A23187-induced 5-LO
product synthesis in HEK_5-LO (devoid of FLAP) but efficiently
reduced 5-LO product biosynthesis in HEK cells expressing both 5-
LO and FLAP; the same pattern was also found for MK-886 (Fig. 3B).
Together, our data suggest that the efficient inhibition of cellular 5-
7n
CF3
92.0 9.8
85.9 8.3
7o
7p
CF3
CF3
7.5 2.4
0.6 0.5
n.d.
60.2 9.3
7r
CF3
55.6 19.7
n.d.

514
S. Levent et al. / European Journal of Medicinal Chemistry 122 (2016) 510e519
Table 2
His28, C-Glu31, B-Val61, B-Ala63, B-Tyr112, B-Ile113, B-Lys116, B-
Phe114, B-Lys116, B-Ile119, B-Leu120, B-Phe123, in agreement with
our previous reports [16,17]. These starting complexes have then
been submitted to 10 ns MD study (Fig. S3). As a result, the binding
orientations of the stereoisomers of 11 to FLAP are characterized by
several interactions (Fig. 4AeB). For the purpose of clarity, we
report the most conserved interactions derived from the MD sim-
Inhibition of 5-LO product formation by
selected compounds in cell-based assay.a
Cmpd.
IC50 mM
7a
7o
7t
7u
11
0.15
0.17
0.21
0.12
0.07
ulations (occupancy ꢁ 30%). The BI is engaged in a strong catione
p
interaction with Lys116 (occupancy, 56% for (S)-stereoisomer and
31% for (R)-stereoisomer) orienting the o-Cl-benzyl moiety to form
a
IC50 values in the cell-based assay were
obtained from intact neutrophils challenged
pep contacts with Phe123 (occupancy, 30% for (S)-stereoisomer
with 2.5 mM ionophore.
and 70% for (R)-stereoisomer), in agreement with our previously
reported data on the arachidonate-binding site of FLAP (Fig. 4AeB)
[17]. After analyzing the water molecules with VMD 1.9.2 [33]
during MD simulations, we also realized that there exist several
bridging water molecules maintaining similar contacts between
C(5)-nitrile of both stereoisomers and Lys116, polar head of POPC
membrane and His28.
LO product formation by 11 is due to interference with FLAP.
2.3. Docking studies and molecular dynamic simulations of 11 with
FLAP
Although biological assays were carried out for racemates, both
stereoisomers (S and R) were considered for rationalizing the
inhibitory activity of 11 by means of molecular modeling. Thus, we
combined docking studies of (S)- and (R)-11 with molecular dy-
namic simulations (MDs) using the FLAP crystal structure (PDB
code: 2Q7M) [32]. This computational strategy allows to investigate
the binding mode of inhibitors taking into account ligand-induced
conformational changes of FLAP. Docking studies focused on the
binding site located between the chains B and C to predict the top
scored pose (Glide SP scores are ꢀ6.74 for (S)-11 and ꢀ6.96 for (R)-
11) showing C(5)-nitrile analogue of BRP-7 (11) interacting with
residues C-Val20, C-Val21, C-Asn23, C-Gly24, C-Phe25, C-Ala27, C-
3. Conclusion
FLAP inhibition is a promising strategy for the pharmacological
therapy of respiratory and cardiovascular disorders, and several
FLAP inhibitors have proceeded to human clinical trials, however,
no drug molecule has yet reached the market, despite the prom-
ising potential of these molecules [34]. We recently presented BRP-
7 as a novel FLAP inhibitor chemotype, which lacks typical phar-
macophoric moieties of other published FLAP inhibitors [16,22,34].
In the present study, modifications of the C(5)-position of the BI
ring in BRP-7 with differently substituted benzyloxy or alkyloxy
Fig. 2. Inhibition of 5-LO product formation by 5-CN-BRP-7 (11) in human monocytes. Effect of compound 11 on LTB4 and 5-H(P)ETE synthesis in monocytes stimulated with
A23187 (2.5 M) in the absence (A) or presence (B) of exogenous AA (10 M).
m
m
B
A
+ 0.3 µM comp.11
+ 0.3 µM MK886
100
75
50
25
0
100
75
50
25
0
- - - - - - - - - - - - - -
- - - - - - - - - - - - - -
100
75
50
25
0
0.1
1
10
HEK_
5-LO
HEK_
5-LO
HEK_
5-LO/FLAP
HEK_
5-LO/FLAP
comp. 11 [µM]
Fig. 3. Effects of compound 11 on 5-LO activity in a cell-free assay and in HEK293 cells. (A) Isolated human recombinant 5-LO was preincubated with 11 and 5-LO activity was
induced by addition of 20 M AA. (B) HEK293 cells, transfected with 5-LO alone (HEK_5-LO) or with 5-LO and FLAP (HEK_5-LO/FLAP), were pre-incubated with 11 or MK-886 (or
vehicle control), and activated with 2.5 M A23187 plus 3 M AA. 5-LO products (trans-isomers of LTB4 and 5-HETE) were determined by HPLC. Data, given as % residual activity of
uninhibited vehicle control (¼ 100%), are means S.E.M., n ¼ 3.
m
m
m

S. Levent et al. / European Journal of Medicinal Chemistry 122 (2016) 510e519
515
not seem to form this H-bond interaction because they cannot be
positioned in an effective way as active derivatives in the vicinity of
Lys116 (unpublished observations). In conclusion, substitution of
the C(5)-position of the BI ring in BRP-7 might be a valuable means
to develop new BRP-7 derivatives with greater potency. Further
exploration of this position is under investigation in our laboratory.
4. Experimental section
4.1. Chemistry
1H and 13C NMR spectra were recorded in CDCl3 or DMSO-d6 on
a Varian Mercury 400 MHz High Performance Digital FT-NMR
spectrometer using tetramethylsilane as the internal standard. All
chemical shifts were recorded as
d (ppm). High resolution mass
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). Melting points were
determined with an SMP-II Digital Melting Point Apparatus and are
uncorrected (Schorpp Geraetetechnik, Germany). Flash chroma-
tography was performed with a Combiflash®Rf automated flash
chromatography system with RediSep columns (Teledyne-Isco,
Lincoln, NE, USA) using hexaneeEtOAc or DCMeMeOH solvent
gradients. The purity of the final compounds was determined to be
>97% by UPLC with UV detector.
4.1.1. General procedure for the alkylation of 5-hydroxy
benzimidazole derivatives 6a-b
To a solution of compound 6a-b (1 equiv) in acetonitrile was
added K2CO3 (1.7 equiv) followed by corresponding benzyl halides
(1.5 equiv). The mixture was refluxed for 5 h then filtered and
evaporated in vacuo. Purification of the crude product by auto-
mated flash chromatography provided the title compound.
4.1.1.1. 2-(1-(4-Isobutylphenyl)ethyl)-5-(pyridin-2-ylmethoxy)-1-(2-
(trifluoromethyl) benzyl)-1H-benzo[d]imidazole (7a). Obtained by
the reaction of picolylchloride with 6b and purified by flash column
chromatography (0% / 40% EtOAc in hexane). The oily product in
ether was solidified by addition of 1e2 eq of conc. HCl. Yield 57.5%;
Fig. 4. Binding mode analysis of A) (S)- and B) (R)-stereoisomers of 11 during inter-
action with FLAP (PDB code: 2Q7M). Ligand protein interactions during 10 ns long MD
simulations are represented by their occupancy values. Water molecules are mapped
volumetrically around C(5)-nitrile by calculating occupancies and combining with
average values with VolMap tool VMD 1.9.2.
mp 135.0e136.0 ꢂC. 1H NMR (CDCl3):
d
0.81 (3H, d, J ¼ 6.4 Hz), 0.83
(3H, d, J ¼ 6.4 Hz), 1.71e1.75 (1H, m), 2.12 (3H, d, J ¼ 7.2 Hz), 2.35
(2H, d, J ¼ 6.8 Hz), 4.49 (1H, q, J ¼ 7.2 Hz), 5.60 (2H, s), 5.72 (2H, s),
6.11 (1H, d, J ¼ 7.6 Hz), 7.00 (2H, d, J ¼ 8.0 Hz), 7.11e7.23 (4H, m),
7.39 (1H, t, J ¼ 7.8 Hz), 7.54 (1H, d, J ¼ 8.0 Hz), 7.75 (1H, d, J ¼ 7.6 Hz),
7.86e7.89 (1H, m), 8.03 (1H, s), 8.18 (1H, d, J ¼ 7.2 Hz), 8.47 (1H, t,
groups are described. The various nonpolar or bulky substituents
on the benzyl group were not tolerated and resulted in mostly
inactive (7b-g, i-k, m, n) or weak inhibitors, such as 7h, l and u,
which are equipped with polar nitrile at p- and o-positions. Mod-
ifications of C(5)-position with smaller alkoxy analogues were also
beneficial (7o-s) leading to improved inhibitors. Finally, the direct
addition of a nitrile group at C(5)-BI of BRP-7 (yielding 11)
improved the potency to inhibit cellular 5-LO product synthesis
J ¼ 7.2), 8.88 (1H, d, J ¼ 5.2 Hz). 13C NMR (CDCl3):
d 20.48, 22.20,
22.24, 29.99, 38.16, 44.76, 45.15, 66.63,100.78,112.49,117.75,123.99
(q, 1JC-F ¼ 275 Hz), 125.37, 125.79, 125.91, 126.67 (q, 3JC-F ¼ 6.0 Hz),
2
127.05, 127.09 (q, JC-F ¼ 30.5 Hz), 127.15, 128.63, 130.28, 130.39,
with IC50 values in the range of 0.012e0.07 mM. These results can be
131.82, 132.75, 134.86, 142.07, 142.28, 145.28, 151.67, 155.33, 156.54.
HRMS (m/z) [MþH]þ calcd for C33H33F3N3O: 544.2576; found,
544.2570.
explained by molecular modeling studies, which indicate the for-
mation of additional hydrogen bonds when 11 is bound in the
arachidonate-binding site of FLAP. The nitrile group of 11 is able to
interact with polar amino acids (i.e. Lys116 and His28) and polar
membrane heads through water-mediated hydrogen bond in-
teractions. This may explain why a C(5)-nitrile substitution (Com-
4.1.1.2. 2-(1-(4-Isobutylphenyl)ethyl)-5-((4-methylbenzyl)oxy)-1-(2-
(trifluoromethyl) benzyl)-1H-benzo[d]imidazole (7b). Obtained by
the reaction of 4-methylbenzyl bromide with 6b and purified by
flash column chromatography (0% / 20% EtOAc in hexane). Yield
pound 11) led to a more effective molecule (IC50 ¼ 0.07
mM) as
M). The modeling
compared to the parent BRP-7 (IC50 ¼ 0.31
m
60.0%; mp 137.6e138.3 ꢂC. 1H NMR (CDCl3):
d 0.82 (3H, d,
studies may also help to understand why the o-nitrile substituted
(7u), allyloxy (7o) and ester (7t) derivatives are highly effective as
J ¼ 6.4 Hz), 0.84 (3H, d, J ¼ 6.4 Hz), 1.70e1.76 (1H, m), 1.80 (3H, d,
J ¼ 6.8 Hz), 2.33 (2H, d, J ¼ 6.8 Hz), 2.36 (3H, s), 4.15 (1H, q,
J ¼ 6.8 Hz), 5.09 (2H, s), 5.32 (1H, d, J ¼ 18.4 Hz), 5.39 (1H, d,
J ¼ 18.4 Hz), 6.19 (1H, d, J ¼ 7.6 Hz), 6.88e6.93 (4H, m), 7.05 (2H, d,
J ¼ 8.0 Hz), 7.13 (1H, t, J ¼ 7.6 Hz), 7.19 (2H, d, J ¼ 7.2 Hz), 7.24e7.28
well (IC50 ¼ 0.12e0.21
mM), since they all form similar H-bond in-
teractions with Lys116 at the membrane opening due to effective
positioning of C(5)-substituents (Fig. S2). The inactive analogues do

516
S. Levent et al. / European Journal of Medicinal Chemistry 122 (2016) 510e519
(1H, m), 7.36 (2H, d, J ¼ 7.6 Hz), 7.48 (1H, s), 7.65 (1H, d, J ¼ 7.6 Hz).
HRMS (m/z): [MþH]þ calcd for C35H36F3N2O: 557.2780; found,
557.2798.
(CDCl3):
d
0.82 (3H, d, J ¼ 6.8 Hz), 0.84 (3H, d, J ¼ 6.8 Hz), 1.69e1.76
(1H, m), 1.80 (3H, d, J ¼ 6.8 Hz), 2.33 (2H, d, J ¼ 7.2 Hz), 4.11 (1H, d,
J ¼ 6.8 Hz), 5.13 (2H, s), 5.33 (1H, d, J ¼ 18.0 Hz), 5.39 (1H, d,
J ¼ 18.0 Hz), 6.19 (1H, d, J ¼ 7.6 Hz), 6.88e6.95 (4H, m), 7.05 (2H, d,
J ¼ 8.0 Hz), 7.11e7.15 (1H, m), 7.23e7.29 (3H, m), 7.45 (1H, d,
J ¼ 2.0 Hz), 7.50 (2H, d, J ¼ 8.4 Hz), 7.65 (1H, d, J ¼ 7.6 Hz). HRMS (m/
z) [MþH]þ calcd for C35H33F6N2O2: 627.2446; found: 627.2468.
4.1.1.3. 2-(1-(4-Isobutylphenyl)ethyl)-1-(2-(trifluoromethyl)benzyl)-
5-((4-(trifluoromethyl)benzyl)oxy)-1H-benzo[d]imidazole
(7c).
Obtained by the reaction of 4-trifluoromethylbenzyl bromide with
6b and purified by flash column chromatography (0% / 40% EtOAc
in hexane). The oily product in ether was solidified by addition of
1e2 eq of conc. HCl. Yield 70.8%; mp 98.0e99.0 ꢂC. 1H NMR (CDCl3):
4.1.1.8. 4-(((2-(1-(4-Isobutylphenyl)ethyl)-1-(2-(trifluoromethyl)
benzyl)-1H-benzo[d]imidazol-5-yl)oxy)methyl)benzonitrile
(7h).
d
0.82 (3H, d, J ¼ 6.8 Hz), 0.83 (3H, d, J ¼ 6.8 Hz), 1.72e1.76 (1H, m),
Obtained by the reaction of -bromo-p-tolunitrile with 6b and
a
2.12 (3H, s), 2.36 (2H, d, J ¼ 7.2 Hz), 4.45 (1H, s), 5.24 (2H, s), 5.57
(2H, s), 6.10 (1H, d, J ¼ 6.8 Hz), 7.01 (2H, d, J ¼ 8.0 Hz), 7.06e7.21 (5H,
m), 7.39 (1H, t, J ¼ 7.4 Hz), 7.60 (2H, d, J ¼ 8.0 Hz), 7.67 (2H, d,
J ¼ 8.0 Hz), 7.75 (1H, d, J ¼ 8.0 Hz), 7.92 (1H, s). HRMS (m/z):
[MþH]þ calcd for C35H33F6N2O: 611.2497; found, 611.2517.
purified by flash column chromatography (0% / 40% EtOAc in
hexane). The oily product in ether was solidified by addition of 1e2
eq of conc. HCl. Yield 78.5%; mp 107.2e108.1 ꢂC. 1H NMR (CDCl3):
d
0.82 (3H, d, J ¼ 6.8 Hz), 0.83 (3H, d, J ¼ 6.8 Hz), 1.71e1.76 (1H, m),
2.13 (3H, s), 2.36 (2H, d, J ¼ 7.6 Hz), 4.54 (1H, s), 5.26 (2H, s), 5.58
(2H, s), 6.09 (1H, m), 7.01 (2H, d, J ¼ 7.2 Hz), 7.08e7.20 (5H, m), 7.40
(1H, t, J ¼ 7.6 Hz), 7.61 (2H, d, J ¼ 7.6 Hz), 7.71 (2H, d, J ¼ 8.0 Hz), 7.75
4.1.1.4. 2-(1-(4-Isobutylphenyl)ethyl)-5-((4-methoxybenzyl)oxy)-1-
(2-(trifluoromethyl)
Obtained by the reaction of 4-meyhoxybenzyl bromide with 6b and
purified by flash column chromatography (0% / 40% EtOAc in
hexane). The oily product in ether was solidified by addition of 1e2
eq of conc. HCl. Yield 67.0%; mp 231.3e232.1 ꢂC. 1H NMR (CDCl3):
benzyl)-1H-benzo[d]imidazole
(7d).
(1H, d, J ¼ 8.0 Hz), 7.97 (1H, s). 13C NMR (CDCl3):
d 20.69, 22.21,
22.25, 30.02, 38.19, 44.79, 69.96, 99.39,111.92,112.05,117.94,118.60,
123.99 (q, 1JC-F ¼ 272 Hz), 125.71, 126.55, 126.76 (q, 3JC-F ¼ 5.3 Hz),
2
127.13, 127.16 (q, JC-F ¼ 30.3 Hz), 127.89, 128.65, 130.29, 130.68,
132.51, 132.75, 135.05, 141.33, 142.09, 154.82, 157.47. HRMS (m/z):
[MþH]þ calcd for C35H33F3N3O: 568.2576; found: 568.2579.
d
0.82 (3H, d, J ¼ 6.8 Hz), 0.83 (3H, d, J ¼ 6.8 Hz), 1.70e1.76 (1H, m),
2.13 (3H, d, J ¼ 4.8 Hz), 2.35 (2H, d, J ¼ 7.2 Hz), 3.84 (3H, s), 4.48 (1H,
s), 5.15 (2H, s), 5.57 (2H, s), 6.10 (1H, d, J ¼ 6.8 Hz), 6.87e6.90 (1H,
m), 6.99e7.12 (7H, m), 7.20 (2H, d, J ¼ 7.6 Hz), 7.32 (1H, t, J ¼ 8.0 Hz),
7.38 (1H, t, J ¼ 7.6 Hz), 7.74 (1H, d, J ¼ 8.0 Hz), 7.91 (1H, s); 13C NMR
4.1.1.9. 2-(1-(4-Isobutylphenyl)ethyl)-5-((2-methylbenzyl)oxy)-1-(2-
(trifluoromethyl) benzyl)-1H-benzo[d]imidazole (7i). Obtained by
the reaction of 2-methylbenzyl bromide with 6b and purified by
flash column chromatography (0% / 20% EtOAc in hexane). Yield
(CDCl3): d 20.47, 22.21, 22.25, 30.01, 38.08, 44.79, 55.31, 70.79, 99.21,
1
111.71, 112.99, 113.95, 118.24, 119.88, 124.00 (q, JC-F ¼ 272 Hz),
71.6%; mp 143.3e144.4 ꢂC. 1H NMR (CDCl3):
d 0.82 (3H, d,
3
2
125.65, 126.11, 126.70 (q, JC-F ¼ 6.1 Hz), 127.07, 127.11, (q, JC-
J ¼ 6.8 Hz), 0.83 (3H, d, J ¼ 6.8 Hz), 1.70e1.77 (1H, m), 1.81 (3H, d,
J ¼ 6.8 Hz), 2.34 (2H, d, J ¼ 6.8 Hz), 2.39 (3H, s), 4.12 (1H, q,
J ¼ 6.8 Hz), 5.11 (2H, s), 5.33 (1H, d, J ¼ 18.0 Hz), 5.40 (1H, d,
J ¼ 18.0 Hz), 6.21 (1H, d, J ¼ 8.0 Hz), 6.92e6.94 (4H, m), 7.06 (2H, d,
J ¼ 8.0 Hz), 7.14 (1H, t, J ¼ 7.2 Hz), 7.21e7.29 (4H, m), 7.45e7.47 (1H,
m), 7.52 (1H, s), 7.66 (1H, d, J ¼ 7.2 Hz). HRMS (m/z) [MþH]þ calcd
for C35H36F3N2O: 557.2780; found: 557.2779.
¼ 31.7 Hz), 128.62, 129.75, 130.27, 130.66, 132.75, 134.96, 137.40,
F
142.04, 154.45, 158.15, 159.84. HRMS (m/z): [MþH]þ calcd for
C
35H36F3N2O2: 573.2729; found, 573.2725.
4.1.1.5. 5-((4-(tert-Butyl)benzyl)oxy)-2-(1-(4-isobutylphenyl)ethyl)-
1-(2-(trifluoromethyl) benzyl)-1H-benzo[d]imidazole (7e).
Obtained by the reaction of 4-t-butylbenzyl bromide with 6b and
purified by flash column chromatography (0% / 4% MeOH in
4.1.1.10. 2-(1-(4-Isobutylphenyl)ethyl)-1-(2-(trifluoromethyl)
benzyl)-5-((2-(trifluoromethyl)benzyl)oxy)-1H-benzo[d]imidazole
(7j). Obtained by the reaction of 2-trifluoromethylbenzyl bromide
with 6b and purified by flash column chromatography (0% / 40%
EtOAc in hexane). The oily product in ether was solidified by
addition of 1e2 eq of conc. HCl. Yield 73.5%. mp 127.0e128.0 ꢂC. 1H
DCM). Yield 49.8%; mp 72.4e73.6 ꢂC. 1H NMR (CDCl3):
d 0.79 (3H, d,
J ¼ 6.4 Hz), 0.81 (3H, d, J ¼ 6.4 Hz), 1.31 (9H, s), 1.68e1.75 (1H, m),
1.80 (3H, d, J ¼ 6.4 Hz), 2.31 (2H, d, J ¼ 7.2 Hz), 4.11 (1H, m), 5.08 (2H,
s), 5.32 (1H, d, J ¼ 18.0 Hz), 5.39 (1H, d, J ¼ 18.0 Hz), 6.17 (1H, d,
J ¼ 8.4 Hz), 6.90 (4H, s), 7.04 (2H, d, J ¼ 7.6 Hz), 7.11 (1H, t, J ¼ 7.4 Hz),
7.24e7.39 (5H, m), 7.49 (1H, s), 7.63 (1H, d, J ¼ 7.6 Hz). HRMS (m/z):
[MþH]þ calcd for C38H42F3N2O: 599.3249; found: 599.3248.
NMR (CDCl3):
d
0.82 (3H, d, J ¼ 6.8 Hz), 0.83 (3H, d, J ¼ 6.8 Hz),
1.72e1.76 (1H, m), 2.13 (3H, d, J ¼ 4.4 Hz), 2.36 (2H, d, J ¼ 7.2 Hz),
4.50 (1H, m), 5.31 (2H, s), 5.57 (2H, s), 6.11 (1H, d, J ¼ 7.2 Hz),
7.00e7.32 (4H, m), 7.21e7.23 (3H, m), 7.37e7.50 (2H, m), 7.61 (1H, t,
J ¼ 7.2 Hz), 7.69e7.76 (3H, m), 7.93 (1H, s). HRMS (m/z): [MþH]þ
calcd for C35H33F6N2O: 611.2497; found: 611.2498.
4.1.1.6. 5-((4-Bromobenzyl)oxy)-2-(1-(4-isobutylphenyl)ethyl)-1-(2-
(trifluoromethyl) benzyl)-1H-benzo[d]imidazole (7f). Obtained by
the reaction of 4-bromobenzyl bromide with 6b and purified by
flash column chromatography (0% / 40% EtOAc in hexane). Yield
72.8%; mp 143.8e144.5 ꢂC. 1H NMR (CDCl3):
d
0.82 (3H, d,
4.1.1.11. 5-((2-Chlorobenzyl)oxy)-2-(1-(4-isobutylphenyl)ethyl)-1-(2-
(trifluoromethyl) benzyl)-1H-benzo[d]imidazole (7k). Obtained by
the reaction of 2-chlorobenzyl bromide with 6b and purified by
flash column chromatography (0% / 20% EtOAc in hexane). Yield
J ¼ 6.8 Hz), 0.83 (3H, d, J ¼ 6.8 Hz), 1.70e1.76 (1H, m), 1.80 (3H, d,
J ¼ 6.8 Hz), 2.34 (2H, d, J ¼ 6.8 Hz), 4.11 (1H, q, J ¼ 6.8 Hz), 5.08 (2H,
s), 5.33 (1H, d, J ¼ 18.0 Hz), 5.39 (1H, d, J ¼ 18.0 Hz), 6.19 (1H, d,
J ¼ 7.6 Hz), 6.86e6.93 (4H, m), 7.04 (2H, d, J ¼ 8.4 Hz), 7.13 (1H, t,
J ¼ 7.6 Hz), 7.24e7.38 (1H, m), 7.34 (2H, d, J ¼ 8.4 Hz), 7.44 (1H, d,
J ¼ 2.0 Hz), 7.51 (2H, d, J ¼ 8.4 Hz), 7.65 (1H, d, J ¼ 7.6 Hz). HRMS (m/
z): [MþH]þ calcd for C34H33BrF3N2O: 621.1728; found: 621.1727.
85.0%; mp 99.2e100.2 ꢂC. 1H NMR (CDCl3):
d
0.81 (3H, d, J ¼ 6.4 Hz),
0.82 (3H, d, J ¼ 6.4 Hz), 1.69e1.76 (1H, m), 1.80 (3H, d, J ¼ 6.4 Hz),
2.32 (2H, d, J ¼ 6.8 Hz), 4.11 (1H, m), 5.23 (2H, s), 5.34 (1H, d,
J ¼ 18.8 Hz), 5.40 (1H, d, J ¼ 18.8 Hz), 6.19 (1H, d, J ¼ 7.6 Hz),
6.91e6.93 (4H, m), 7.05 (2H, d, J ¼ 7.6 Hz), 7.13 (1H, t, J ¼ 7.6 Hz),
4.1.1.7. 2-(1-(4-Isobutylphenyl)ethyl)-5-((4-(trifluoromethoxy)
benzyl)oxy)-1-(2-(trifluoromethyl)benzyl)-1H-benzo[d]imidazole
(7g). Obtained by the reaction of 4-trifluoromethoxybenzyl bro-
mide with 6b and purified by flash column chromatography (0% /
40% EtOAc in hexane). Yield 79.6%; mp 171.0e172.0 ꢂC. 1H NMR
7.25e7.30 (3H, m), 7.39e7.60 (3H, m), 7.65 (1H, d, J ¼ 7.6 Hz). 13
C
NMR (CDCl3): d 21.78, 22.27, 22.33, 30.07, 38.49, 43.33, 44.88, 68.04,
1
3
103.77, 109.76, 113.28, 124.01 (q, JC-F ¼ 272 Hz), 125.99 (q, JC-
¼ 5.8 Hz), 126.36, 126.83 (q, 2JC-F ¼ 30.2 Hz), 126.91, 126.95, 127.35,
F
128.70,128.83,129.35,129.49,130.31,132.28,132.60,134.23,134.99,

S. Levent et al. / European Journal of Medicinal Chemistry 122 (2016) 510e519
517
2
137.45,139.44,140.33,155.18,157.76. HRMS (m/z): [MþH]þ calcd for
126.83 (q, JC-F ¼ 31.0 Hz), 126.95, 127.31, 129.46, 130.13, 132.26,
133.56, 134.29, 139.52, 140.29, 143.09, 155.19, 157.66. HRMS (m/z)
[MþH]þ calcd for C30H32F3N2O: 493.2467; found: 493.2474.
C
34H33ClF3N2O: 577.2234; found: 577.2236.
4.1.1.12. 2-(((2-(1-(4-Isobutylphenyl)ethyl)-1-(2-(trifluoromethyl)
benzyl)-1H-benzo[d]imidazol-5-yl)oxy)methyl)benzonitrile (7l).
Obtained by the reaction of -bromo-o-tolunitrile with 6b and
4.1.1.16. 2-(1-(4-Isobutylphenyl)ethyl)-5-((3-methylbut-2-en-1-yl)
oxy)-1-(2-(trifluoromethyl)benzyl)-1H-benzo[d]imidazole
Obtained by the reaction of 3,3-dimethylallyl bromide with 6b and
purified by flash column chromatography (0% / 40% EtOAc in
hexane). The oily product in ether was solidified by addition of 1e2
eq of conc. HCl. Yield 68.6%, mp 159.0e160.0 ꢂC. 1H NMR (CDCl3):
a
(7p).
purified by flash column chromatography (0% / 40% EtOAc in
hexane). The oily product in ether was solidified by addition of 1e2
eq of conc. HCl. Yield 84.3%, mp 150.3e150.7 ꢂC. 1H NMR (CDCl3):
d
0.82 (3H, d, J ¼ 6.8 Hz), 0.83 (3H, d, J ¼ 6.8 Hz), 1.72e1.76 (1H, m),
2.14 (3H, s), 2.36 (2H, d, J ¼ 7.6 Hz), 4.49 (1H, s), 5.33 (2H, s), 5.58
(2H, s), 6.12 (1H, s), 7.01 (2H, d, J ¼ 6.8 Hz), 7.08e7.22 (5H, m),
7.40e7.50 (2H, m), 7.67e7.76 (4H, m), 7.80 (1H, s). 13C NMR (CDCl3):
d
0.76 (3H, d, J ¼ 6.0 Hz), 0.78 (3H, d, J ¼ 6.0 Hz), 1.65e1.74 (1H, m),
1.76 (6H, s), 1.80 (3H, d, J ¼ 6.8 Hz), 2.31 (2H, d, J ¼ 6.8 Hz), 4.64 (2H,
d, J ¼ 6.4 Hz), 4.78 (1H, m), 5.45 (t, 1H, J ¼ 6.4 Hz), 5.70 (1H, d,
J ¼ 18.0 Hz), 5.84 (1H, d, J ¼ 18.0 Hz), 6.20 (1H, d, J ¼ 7.6 Hz), 6.96
(2H, d, J ¼ 8.0 Hz), 7.06e7.24 (4H, m), 7.34e7.42 (3H, m), 7.76 (1H, d,
d
20.65, 22.22, 22.25, 30.02, 38.20, 44.79, 44.92, 69.37, 100.12,
1
111.98, 112.45, 117.10, 117.84, 124.01 (q, JC-F ¼ 272 Hz), 125.80,
3
2
126.70 (q, JC-F ¼ 5.3 Hz), 127.11 (q, JC-F ¼ 31.2 Hz), 127.12, 128.65,
129.08, 129.86, 130.30, 130.61, 132.85, 133.15, 133.36, 134.94, 139.14,
142.07, 154.83, 157.75. HRMS (m/z): [MþH]þ calcd for C35H33F3N3O:
568.2576; found: 568.2573.
J ¼ 7.2 Hz). 13C NMR (CDCl3):
d 18.33, 20.45, 22.21, 22.25, 25.86,
30.01, 38.05, 44.79, 65.89, 98.62,111.60,118.38,118.49,124.02 (q, 1JC-
¼ 272 Hz), 125.66, 125.79, 126.69 (q, 3JC-F ¼ 5.3 Hz), 127.08, 127.10,
F
2
(q, JC-F ¼ 31.3 Hz), 128.59, 130.25, 130.71, 132.74, 134.99, 139.55,
141.99, 154.26, 158.43. HRMS (m/z) [MþH]þ calcd for C32H36F3N2O:
4.1.1.13. 2-(1-(4-Isobutylphenyl)ethyl)-5-((3-methylbenzyl)oxy)-1-
521.2780; found: 521.2775.
(2-(trifluoromethyl)
benzyl)-1H-benzo[d]imidazole
(7m).
Obtained by the reaction of 3-methylbenzyl bromide with 6b and
purified by flash column chromatography (0% / 20% EtOAc in
4.1.1.17. 2-(1-(4-Isobutylphenyl)ethyl)-5-(pent-2-yn-1-yloxy)-1-(2-
(trifluoromethyl) benzyl)-1H-benzo[d]imidazole (7r). Obtained by
the reaction of 1-bromopent-3-yne with 6b and purified by flash
column chromatography (0% / 40% EtOAc in hexane). The oily
product in ether was solidified by addition of 1e2 eq of conc. HCl.
hexane). Yield 54.0%, mp 129.6e130.7 ꢂC. 1H NMR (CDCl3):
d 0.81
(3H, d, J ¼ 6.4 Hz), 0.82 (3H, d, J ¼ 6.4 Hz), 1.68e1.75 (1H, m), 1.80
(3H, d, J ¼ 6.8 Hz), 2.33 (2H, d, J ¼ 6.8 Hz), 2.37 (3H, s), 4.11 (1H, q,
J ¼ 6.8 Hz), 5.09 (2H, s), 5.32 (1H, d, J ¼ 18.0 Hz), 5.39 (1H, d,
J ¼ 18.0 Hz), 6.20 (1H, d, J ¼ 7.6 Hz), 6.91e6.93 (4H, m), 7.05 (2H, d,
J ¼ 8.0 Hz), 7.11e7.14 (2H, m), 7.24e7.29 (4H, m), 7.48 (1H, s), 7.65
Yield 76.7%, mp 118.0e119.0 ꢂC. 1H NMR (CDCl3):
d 0.84 (6H, d,
J ¼ 6.4 Hz), 1.18 (3H, t, J ¼ 7.0 Hz) 1.62e1.77 (1H, m), 2.15 (3H, d,
J ¼ 6.8 Hz), 2.28 (2H, q, J ¼ 7.0 Hz), 2.37 (2H, d, J ¼ 7.0 Hz), 4.55 (1H,
d, J ¼ 6.8 Hz), 4.80 (2H, s), 5.58 (2H, s), 6.13 (1H, s), 7.04e7.41 (8H,
(1H, d, J ¼ 7.6 Hz). 13C NMR (CDCl3):
d 20.41, 21.40, 22.22, 22.26,
30.02, 38.05, 44.74, 44.80, 71.04, 99.10, 111.70, 118.30, 124.01 (q, 1JC-
¼ 272 Hz), 124.91, 125.63, 126.03, 126.72 (q, 3JC-F ¼ 5.8 Hz), 127.06,
m), 7.75 (1H, d, J ¼ 7.6 Hz), 7.85 (1H, s). 13C NMR (CDCl3):
d 12.55,
F
2
127.10 (q, JC-F ¼ 31.2 Hz), 128.55, 128.58, 128.62, 129.08, 130.28,
13.53, 20.60, 22.22, 22.25, 30.02, 38.18, 44.80, 44.96, 57.71, 73.11,
1
130.66, 132.40, 132.75, 134.94, 135.73, 138.41, 142.05, 154.41, 158.31.
HRMS (m/z) [MþH]þ calcd for C35H36F3N2O: 557.2780; found:
557.2777.
90.54, 99.56, 111.73, 118.20, 124.00 (q, JC-F ¼ 271.2 Hz), 125.82,
126.34, 126.73 (q, 3JC-F ¼ 5.3 Hz), 126.98 (q, 2JC-F ¼ 30.3 Hz), 127.15,
127.58, 130.27, 130.67, 132.35, 132.83, 134.92, 142.03, 154.70, 157.34.
HRMS (m/z): [MþH]þ calcd for C32H34F3N2O: 519.2623; found:
519.2632.
4.1.1.14. 5-((3-Fluorobenzyl)oxy)-2-(1-(4-isobutylphenyl)ethyl)-1-
(2-(trifluoromethyl)
benzyl)-1H-benzo[d]imidazole
(7n).
Obtained by the reaction of 3-fluorobenzyl bromide with 6b and
purified by flash column chromatography (0% / 4% DCM in
MeOH). The oily product was triturated by cold isopropanol. Yield
4.1.1.18. 5-Butoxy-2-(1-(4-isobutylphenyl)ethyl)-1-(2-(tri-
fluoromethyl)benzyl)-1H-benzo[d]imidazole (7s). Obtained by the
reaction of 1-bromobutane with 6b and purified by flash column
chromatography (0% / 40% EtOAc in hexane). The oily product in
ether was solidified by addition of 1e2 eq of conc. HCl. Yield 69.4%,
92.5%, mp 84.3e84.7 ꢂC. 1H NMR (CDCl3):
d
0.81 (3H, d, J ¼ 6.4 Hz),
0.82 (3H, d, J ¼ 6.4 Hz), 1.66e1.76 (1H, m), 1.81 (3H, d, J ¼ 7.2 Hz),
2.32 (2H, d, J ¼ 7.2 Hz), 4.12 (1H, q, J ¼ 7.2 Hz), 5.12 (2H, s), 5.33 (1H,
d, J ¼ 18.0 Hz), 5.39 (1H, d, J ¼ 18.0 Hz), 6.18 (1H, d, J ¼ 8.0 Hz)
6.91e6.94 (4H, m), 6.97e7.05 (3H, m), 7.10e7.20 (4H, m), 7.31e7.36
(1H, m), 7.45 (1H, s), 7.64 (1H, d, J ¼ 7.6 Hz). HRMS (m/z): [MþH]þ
calcd for C34H33F4N2O: 561.2529; found: 561.2526.
mp 109.0e110.0 ꢂC. 1H NMR (CDCl3):
d
0.82 (3H, d, J ¼ 6.8 Hz), 0.83
(3H, d, J ¼ 6.8 Hz), 0.98 (3H, t, J ¼ 7.2 Hz) 1.47e1.53 (2H, m),
1.73e1.82 (3H, m), 2.12 (3H, bs), 2.35 (2H, d, J ¼ 7.6 Hz), 4.07 (2H, t,
J ¼ 6.8 Hz), 4.46 (1H, bs), 5.56 (2H, s), 6.10 (1H, m), 7.00e7.03 (4H,
m), 7.20e7.40 (4H, m), 7.74e7.77 (2H, m). HRMS (m/z): [MþH]þ
calcd for C31H36F3N2O: 507.2780; found: 507.2778.
4.1.1.15. 5-(Allyloxy)-2-(1-(4-isobutylphenyl)ethyl)-1-(2-(tri-
fluoromethyl)benzyl)-1H-benzo[d]imidazole (7o). Obtained by the
reaction of allyl bromide with 6b and purified by flash column
chromatography (0% / 30% EtOAc in Hexane). The oily product
was triturated by acetone-petroleum ether mixture. Yield 51.1%, mp
4.1.1.19. Methyl
fluoromethyl)benzyl)-1H-benzo[d]imidazol-5-yl)oxy)acetate
2-((2-(1-(4-isobutylphenyl)ethyl)-1-(2-(tri-
(7t).
Obtained by the reaction of methyl bromoacetate with 6b and
purified by flash column chromatography (0% / 40% EtOAc in
hexane). The oily product in ether was solidified by addition of 1e2
eq of conc. HCl. Yield 83.1%, mp 158.0e159.0 ꢂC. 1H NMR (CDCl3):
93.5e94.5 ꢂC. 1H NMR (CDCl3):
d
0.81 (3H, d, J ¼ 6.4 Hz), 0.83 (3H, d,
J ¼ 6.4 Hz), 1.70e1.77 (1H, m), 1.81 (3H, d, J ¼ 6.8 Hz), 2.34 (2H, d,
J ¼ 6.8 Hz), 4.11 (1H, q, J ¼ 6.8 Hz), 4.60 (2H, d, J ¼ 5.2 Hz), 5.28e5.31
(2H, m), 5.38 (1H, d, J ¼ 18 Hz), 5.45 (1H, dd, J ¼ 17.2, 1.6 Hz),
6.06e6.16 (1H, m), 6.20 (1H, d, J ¼ 8.0 Hz), 6.84e6.93 (4H, m), 7.05
(2H, d, J ¼ 8.4 Hz), 7.13 (1H, t, J ¼ 7.8 Hz), 7.25e7.28 (1H, m), 7.42
d
0.82 (3H, d, J ¼ 6.8 Hz), 0.83 (3H, d, J ¼ 6.8 Hz), 1.72e1.76 (1H, m),
2.12 (3H, d, J ¼ 7.2 Hz), 2.36 (2H, d, J ¼ 7.2 Hz), 3.85 (3H, s), 4.49 (1H,
q, J ¼ 7.2 Hz), 4.78 (2H, s), 5.57 (2H, s), 6.10 (1H, d, J ¼ 8.0 Hz), 7.01
(2H, q, J ¼ 8.2 Hz), 7.07e7.23 (5H, m), 7.40 (1H, t, J ¼ 8.0 Hz),
7.73e7.76 (2H, m). HRMS (m/z) [MþH]þ calcd for C30H32F3N2O3:
525.2365; found: 525.2363.
(1H, d, J ¼ 2.4 Hz), 7.65 (1H, d, J ¼ 7.6 Hz). 13C NMR (CDCl3):
d 21.80,
22.27, 22.33, 30.07, 38.49, 43.30, 44.88, 69.53,103.47, 109.66, 113.28,
117.64, 124.01 (q, 1JC-F ¼ 271 Hz), 125.96 (q, JC-F ¼ 5.7 Hz), 126.37,
3

518
S. Levent et al. / European Journal of Medicinal Chemistry 122 (2016) 510e519
4.1.1.20. 2-(((1-(2-Chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-
Germany) was used.
benzo[d]imidazol-5-yl)oxy)methyl)benzonitrile (7u). Obtained by
the reaction of
a
-bromo-o-tolunitrile with 5b and purified by flash
4.2.3. Determination of the activity of isolated 5-LO in cell-free
assays
5-LO activity in cell-free assays was assessed using recombinant
5-LO expressed in E. coli BL21 cells (transformed with pT3-5-LO
plasmid) and purified on an ATP-agarose column as described
column chromatography (0% / 40% EtOAc in hexane). The oily
product in ether was solidified by addition of 1e2 eq of conc. HCl.
Yield 61.5%, mp 179.0e180.0 ꢂC. 1H NMR (CDCl3):
d 0.84 (6H, d,
J ¼ 6.4 Hz), 1.71e1.80 (1H, m), 2.12 (3H, bs), 2.37 (2H, d, J ¼ 7.2 Hz),
4.50 (1H, bs), 5.31 (2H, s), 5.42 (2H, s), 6.20 (1H, d, J ¼ 7.6 Hz),
7.00e7.05 (3H, m), 7.14 (2H, s), 7.22e7.26 (3H, m), 7.43e7.50 (2H,
before [36]. Semi-purified 5-LO (0.5 mg/ml) were diluted with ice-
cold PBS containing 1 mM EDTA, and 1 mM ATP was added. Sam-
ples were pre-incubated with the test compounds or vehicle (0.1%
DMSO) as indicated. After 15 min at 4 ꢂC, samples were pre-
m), 7.66e7.75 (3H, m), 7.97 (1H, s). 13C NMR (CDCl3):
d 20.56, 22.38,
22.31, 30.06, 38.19, 44.83, 46.02, 69.22, 99.95, 112.11, 112.39, 117.15,
117.61, 126.54, 127.10, 127.67, 129.07, 129.81, 129.92, 130.02, 130.11,
130.32, 132.19, 133.15, 133.37, 135.16, 139.18, 142.03, 154.60, 157.58.
HRMS (m/z): [MþH]þ calcd for C34H33ClN3O: 534.2312; found:
534.2311.
warmed for 30 s at 37 ꢂC, and 2 mM CaCl2 plus 20
mM arach-
idonic acid 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 [35]. 5-LO products include the all-trans iso-
mers of LTB4 and 5-H(P)ETE.
4.1.1.21. 1-[(2-Chlorophenyl)methyl]-2-{1-[4-(2-methylpropyl)
phenyl]ethyl}-1H-benzimidazol-5-carbonitrile (11). Compound 10
(1 equiv) was refluxed in 5 ml dioxane and 1.5 ml conc. HCl for 1.5 h.
The reaction mixture was evaporated in vacuo, and dissolved in
20 ml EtOAc. The organic phase was extracted with 5% NaHCO3
(30 ml ꢃ 2). Combined organic phases were dried over anhydrous
Na2SO4, filtered and evaporated to dryness. The crude was purified
by automated flash chromatography. Yield 71%, mp 114.0e114.5 ꢂC.
4.2.4. Determination of 5-LO product synthesis in HEK293 cells
Transfection of HEK293 cells with pcDNA3.1/neom (þ)_5-LO
and pcDNA3.1/Hygro (ꢀ)_FLAP and verification of protein expres-
sion was performed as described before [30]. HEK293 cells were
harvested by trypsinization and centrifugation (1000 ꢃ g; 10 min;
4
ꢂC). 5-LO product synthesis in intact cells was determined as
1H NMR (CDCl3):
d
0.84 (3H, d, J ¼ 6.8 Hz), 0.86 (3H, d, J ¼ 6.8 Hz),
previously described [30]. Briefly, 1 ꢃ 106 cells in 1 ml PGC buffer
1.73e1.78 (1H, m), 1.82 (3H, d, J ¼ 6.8 Hz), 2.36 (2H, d, J ¼ 7.6 Hz),
4.18 (1H, q, J ¼ 6.8 Hz), 5.27 (2H, s), 6.11 (1H, d, J ¼ 7.6 Hz),
6.92e6.98 (3H, m), 7.06 (2H, d, J ¼ 8.0 Hz), 7.13e7.19 (2H, m), 7.39
(1H, dd, J ¼ 7.6, 0.8 Hz) 7.44 (1H, dd, J ¼ 8.4, 1.6 Hz), 8.20 (1H, d,
were stimulated with 2.5 mM A23187 plus 3 mM exogenous AA for
10 min at 37 ꢂC. The reaction was stopped by addition of 1 ml ice-
cold methanol and 5-LO products (all-trans isomers of LTB4 and 5-
H(P)ETE) were extracted and analyzed as described above.
J ¼ 0.8 Hz). 13C NMR (CDCl3):
d 21.69, 22.31, 22.35, 30.07, 38.69,
44.81, 44.90, 105.49, 110.46, 119.91, 124.75, 126.27, 126.45, 126.94,
127.24, 129.06, 129.67, 129.72, 132.04, 132.28, 138.47, 138.79, 140.73,
142.17, 160.12; HRMS (m/z): [MþH]þ calcd for C27H27ClN3 428.1894;
found 428.1893.
4.3. Computational methods
Docking and MD simulation protocols are reported in
Supporting Information.
4.2. Biological studies
Acknowledgments
4.2.1. Cells
We greatly acknowledge The Scientific and Technological
Research Council of Turkey (TÜBITAK) for financial support (Grant
No. 112S596). One of the GPUs used in this study was a donation
from NVIDIA Corporation.
_
Human neutrophils were freshly isolated from leukocyte con-
centrates obtained at the Institute of Transfusion Medicine Uni-
versity Hospital Jena (Germany). In brief, venous blood was taken
from healthy adult donors that did not take any medication for at
least 7 days and leukocyte concentrates were prepared by centri-
fugation at 4000 g for 20 min at 20 ꢂC. Neutrophils were immedi-
ately isolated by dextran sedimentation, centrifugation on
Nycoprep cushions (PAA Laboratories, Linz, Austria), and hypotonic
lysis of erythrocytes as described previously [22]. Cells were finally
resuspended in phosphate-buffered saline pH 7.4 (PBS) containing
1 mg/ml glucose and 1 mM CaCl2 (PGC buffer).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.07.004.
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