Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase- activating protein (FLAP)

Bioorganic & Medicinal Chemistry 20 (2012) 3728–3741

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Bioorganic & Medicinal Chemistry

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Identiﬁcation of novel benzimidazole derivatives as inhibitors

of leukotriene biosynthesis by virtual screening targeting

5-lipoxygenase-activating protein (FLAP)

Erden Banoglu ^a,, Burcu Çalısßkan ^a, Susann Luderer ^b, Gökçen Eren ^a, Yagmur Özkan ^a,

⇑

Wolfram Altenhofen ^c,, Christina Weinigel ^d, Dagmar Barz ^d, Jana Gerstmeier ^e, Carlo Pergola ^e,

Oliver Werz ^e,

⇑

^aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Etiler, 06330 Yenimahalle, Ankara, Turkey

^bDepartment of Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, D-72076 Tuebingen, Germany

^cChemical Computing Group, AG, Kaiser-Wilhelm-Ring 11, 50676 Koeln, Germany

^dInstitute of Transfusion Medicine, University Hospital Jena, 07743 Jena, Germany

^eChair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, D-07743 Jena, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:

Available online 1 May 2012

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein

(FLAP) inhibitors is a promising strategy to intervene with inﬂammatory, allergic and cardiovascular dis-

eases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore

model led to the identiﬁcation of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole

(7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutro-

Keywords:

5-Lipoxygenase

Leukotriene

Inﬂammation

Neutrophils

phils (IC₅₀= 0.31

causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based

derivatives of 7 were synthesized leading to more potent analogues (70–72, 82) with IC₅₀= 0.12–0.19

lM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP

l

M

in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen ﬁn-

gerprint as a new chemotype for further development of anti-leukotriene agents.

1. Introduction

supply protein for 5-LO at the nuclear membrane.³LTA₄is then rap-

idly converted to either LTB₄by LTA₄hydrolase or to LTC₄by LTC₄

Leukotrienes (LTs) are potent lipid mediators with central roles

in the initiation and ampliﬁcation of the inﬂammatory response

by regulating the recruitment and activation of leukocytes in in-

ﬂamed tissues.^1,2The ﬁrst two steps in LT biosynthesis involve the

key enzyme 5-lipoxygenase (5-LO) that converts arachidonic acid

(AA) into 5-hydroperoxyeicosatetraenoic acid (5-HpETE) followed

by dehydration to the unstable epoxide LTA₄. These initial oxygena-

tion/dehydration steps are aided in the cellular environment by the

5-LO-activating protein (FLAP), which acts as substrate transfer/

synthases. LTC₄is further metabolized to LTD₄and then to LTE₄

which are collectively named cysteinyl(cys)-LTs. LT signals are spe-

ciﬁcally transduced via distinct receptors (BLT1 and BLT2 for LTB₄;

CysLTR1 for LTD_4,CysLTR2 for LTC₄; and CysLT_ER/P2Y12 for LTE₄)

resulting in a number of biological activities including neutrophil

and eosinophil chemotaxis and activation of inﬂammatory re-

sponses by LTB₄, as well as bronchoconstriction, airway edema,

and hypersecretion of mucus by cysLTs.²

There are two major pharmacological strategies pursued in or-

der to intervene with LTs: (I) antagonism of cys-LTs at CysLTR1

and (II) inhibition of 5-LO product (i.e., cys-LTs, LTB₄and

5-H(p)ETE) biosynthesis, where inhibitors of 5-LO product synthe-

sis may be advantageous over CysLTR1 antagonists because of their

interference with all 5-LO-derived products. However, while

CysLTR1 antagonists (e.g., montelukast) are successfully applied

in asthma therapy,⁴the development of LT biosynthesis inhibitors

is less advanced.^5,6So far, the direct 5-LO inhibitor zileuton is the

only marketed LT synthesis inhibitor, but it is not widely prescribed

due to signiﬁcant side-effects, poor pharmacokinetics, and multiple

Abbreviations: AA, arachidonic acid; cPLA₂, cytosolic phospholipase A₂; FLAP, 5-

lipoxygenase-activating protein; 5-HpETE, 5-hydroperoxyeicosatetraenoic acid; 5-

LO, 5-lipoxygenase; LT, leukotriene; PGC buffer, phosphate-buffered saline pH 7.4

containing 1 mg/mL glucose and 1 mM CaCl₂; SAR, structure–activity relationship.

⇑

Corresponding authors. Tel.: +90 312 2023240; fax: +90 312 2235018 (E.B.);

tel.: +49 3641 949801; fax: + 49 3641 949802 (O.W.).

E-mail addresses: banoglu@gazi.edu.tr (E. Banoglu), Oliver.Werz@uni-jena.de

(O. Werz), Oliver.Werz@uni-jena.de (O. Werz).

Present address: Leukocare AG, Am Klopferspitz 19, D-82152 Martinsried,

Germany.

http://dx.doi.org/10.1016/j.bmc.2012.04.048

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

3729

dosing.⁷Recently, a new 5-LO inhibitor (MK-0633, setileuton) was

reported to be under investigation for the treatment of asthma

and atherosclerosis.⁸Besides targeting 5-LO, inhibition of LT bio-

synthesis may also be achieved by targeting FLAP. Early candidates

like MK-886, BAY X1005 and MK-591 that all target FLAP under-

went phase I and II studies and demonstrated clinical beneﬁts in

allergic asthma trials but were not further developed for unpub-

lished reasons.⁹However, the recently developed FLAP inhibitors

such as AM803, AM643, and AM103 (which are deduced from

MK886) were shown to be efﬁcacious in preclinical studies of

inﬂammatory diseases as well as in trials with patients suffering

from asthma and are currently under clinical investigations.^9–13

Though no FLAP inhibitor has yet reached the market, FLAP is cur-

rently considered a promising and clinically relevant target for

pharmacological intervention with LT-related disorders.¹⁴

The aim of this study was the identiﬁcation of new FLAP inhib-

itors by a novel combined structure- and ligand-based virtual

screening approach. Thus, we ﬁrst compiled diverse sets of pub-

lished FLAP inhibitors to be used as queries for a combined ligand-

and structure-based pharmacophore model. We then used this

model to screen libraries of commercially available compounds

for novel chemical scaffolds with potential inhibitory activity on

cellular 5-LO product formation. From a list of 37 candidates, 8

molecules were ‘cherry-picked’ and tested for inhibition of 5-LO

product synthesis in intact cells and the most active derivative

(7) was selected as lead for structure–activity relationship (SAR)

studies. Eventually, this approach led to the identiﬁcation of a

new chemotype bearing a benzimidazole core structure as a potent

inhibitor of LT biosynthesis in intact cells with bioactivities in the

submicromolar range.

2. Results and discussion

2.1. Building of the combined ligand- and structure-based

pharmacophore model for virtual screening

The analysis of the available crystal structure of FLAP¹⁵was

found to be insufﬁcient for a reliable structure-based drug design

due to the low resolution. Therefore, our current work combined

structure- and ligand-based evidences in order to identify novel

chemotypes as potential FLAP inhibitors. The workﬂow of the ap-

plied strategy was: (i) collection of chemically diverse sets of

known FLAP inhibitors; (ii) ﬂexible alignment of their structures

onto the X-ray ligand; (iii) generation of a ligand-based pharmaco-

phore model from the aligned ligands, including an exterior vol-

ume that indicates the shape of the alignment; (iv) use of this

model for the virtual screening of a commercially available com-

pound database; (v) docking of the initial hits from step 4 into

the active site using a pharmacophore-biased placement algo-

rithm, and (vi) selection of the hits on the basis of diversities in

side chain interaction ﬁngerprints among the survived molecules

obtained after post processing of the docking results.

C

B

A

R5

R4

N

R1

R2

O

S

R3

Class 1

R5

O

R1

R4

R2

O

R3

Class 2

R5

R2

R4

R3

R1

O

Class 3

D

S

O

COOH

N

Cl

Figure 1. (A) The FLAP active site identiﬁed by ﬁve subpockets (P1–P5), where P1 and P2 are rather buried and P3–P5 are on the water accessible exterior. (B) Alignment of

literature known active inhibitors from each chemical class (Class 1: 1,2-disubstituted indoles, Class 2: 2,2-bisaryl-bicycloheptanes, Class 3: diaryl-substituted alkanes)

showing P1–P5 exit vectors. (C) The presentation and aligned scaffolds of the chemical classes of literature known active FLAP inhibitors, where R1–5 correspond to the

respective subpockets P1–5. (D) Chemical structure of the well-known FLAP inhibitor 85 (MK-591).

3730

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

classes, that is, 1,2-disubstituted indoles (class 1), 2,2-bisaryl-bicy-

cloheptanes (class 2), and diaryl-substituted alkanes (class 3) where

the available X-ray ligand 85 was a member of the class 1 structural

type (Fig. 1). Alignment scores and pocket side-chain atoms were

used to identify the best matching among the multiple poses per

molecule produced by the algorithm.

Relevant structural features from the aligned training set were

transformed into a consensus pharmacophore model using Ph4

consensus of MOE. Optimizing query simplicity resulted in a six-

featured pharmacophore query which consists of three aromatic

rings and three hydrophobic groups in the shown spatial arrange-

ments as common features of the aligned ligands (Fig. 2). The 3D

pharmacophore query was used to screen a precompiled collection

of 2.8 mio vendor compounds (database available from Chemical

Computing Group, 153 mio conformations) where 1792 com-

pounds matching the desired pharmacophoric features were iden-

tiﬁed. To conﬁrm the hits, they were docked using MOE and

prioritized for biological testing.

2.2. Molecular docking and scoring

FLAP is a homotrimer with three binding sites located between

adjacent monomers.¹⁵We used the FLAP crystal structure in com-

plex with compound 85 (PDB code: 2Q7M) and chains B and C were

selected for docking studies. All hit compounds retrieved from the

pharmacophore-based virtual screening were subjected to molecu-

lar docking to the FLAP binding site located between 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 a result, 1494 of 1792

hits were successfully docked. Then, the docking poses that sur-

vived were ﬁltered according to forceﬁeld reﬁnement and rescoring

function. Thus, 192 virtual hits were identiﬁed and selected as

promising candidates, and the protein–ligand interaction ﬁnger-

prints (PLIFs) application of MOE was used to identify most diverse

interaction pattern. As a result, a subset of 37 hits was selected

based on diversity observed in PLIFs. Finally, 8 candidates (Fig. 3)

were ‘cherry-picked’ from this subset based on chemical intuition

(e.g. compound 7) and literature knowledge (e.g. compound 8)

considering the geometrical and pharmacophoric features as well

as overall lipophilicity of known FLAP inhibitors for evaluation of

Figure 2. Constructed ligand-based pharmacophore model for FLAP inhibitors. The

solid spheres represent the core pattern of aromatic rings. Projected Pi-Ring

features (dotted) constrain the directionality of the interaction. Features shown in

line mode are optional aromatic or hydrophobic features.

The development of the pharmacophore model was initiated by

aligning a set of 202 known active FLAP inhibitors (collected from

published reports^16–21) to the crystallographic data of the complex

of compound 85 (3-[3-(tert-butylthio)-1-(4-chlorobenzyl)-5-(quin-

olin-2-ylmethoxy)-2,3-dihydro-1H-indol-2-yl]-2,2-dimethylpropa-

noic acid, MK-591, Fig. 1) and FLAP (PDB code: 2Q7M) available in

the Brookhaven Protein Data Bank¹⁵by using the software MOE.

The known FLAP inhibitors involved three distinct structural

Cl

N

F₃C

S

O

S

O

S

O

N

O

N

H

HN

Cl

HO

Cl

1

2

3

4

O

Cl

O

Cl

HO

Cl

O

N

O

Cl

N

O

N

H

HO

O

Cl

5

6

7

8

Figure 3. Structures of potentially active FLAP inhibitors identiﬁed by the virtual screening study.

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

3731

inhibition of 5-LO product synthesis in cell-based and cell-free

assays.

reference inhibitor 84 ((E)-N-hydroxy-N-(3-(3-phenoxyphenyl)-

allyl)acetamide, BWA4C) inhibited 5-LO product formation in

these assays (Table 1) as expected.

2.3. Analysis of inhibition of 5-LO product synthesis by the

virtual screening hits

2.4. Chemistry

For 5-LO product biosynthesis in intact cells, the substrate AA is

released from membrane phospholipids by cytosolic phospholi-

pase A₂(cPLA₂), hooked up by FLAP, and then transferred by FLAP

to 5-LO for metabolism.³However, when there is ample supply of

substrate, for example under cell-free assay conditions (i.e., using

isolated 5-LO or cell homogenates), FLAP is dispensable for 5-LO

product synthesis.²Thus, FLAP inhibitors fail to inhibit 5-LO prod-

uct formation in cell-free test systems.⁵In order to differentiate be-

tween putative FLAP inhibitors and direct 5-LO inhibitors, we have

studied the compounds in a ‘FLAP-dependent’, cell-based assay of

5-LO product synthesis using human neutrophils challenged by

Ca²⁺-ionophore A23817, as well as in a ‘FLAP-independent’, cell-

free 5-LO activity assay using human recombinant 5-LO expressed

in Escherichia coli. To evaluate the compounds in neutrophils, the

direct 5-LO products 5-H(p)ETE and all-trans isomers of LTB₄, as

well as LTB₄, were analyzed.

Based on the favorable proﬁle of compound 7 as inhibitor of LT

biosynthesis, we prepared further derivatives in order to perform

SAR studies. For the synthesis of compounds 7 and 13–48, we uti-

lized the synthetic procedure outlined in Scheme 1, following pub-

lished standard procedures. For the synthesis of benzimidazole

derivative 12, we acquired

a two-step procedure whereby

1,2-phenylenediamine 9 was ﬁrst treated with the activated car-

boxylic acid 10 to yield the monoacylated derivative 11 which sub-

sequently converted to the target benzimidazole through

cyclodehydration by reﬂuxing in pure acetic acid.²²For the N-

alkylation of compound 12, the required benzyl/benzoyl halides

were either obtained from commercial sources or prepared ﬁrst

through sodium borohydride reduction of appropriate benzalde-

hydes using PEG400 as a phase transfer catalyst,²³and second,

halogenation of the resulting benzyl alcohol by standard agents

such as PBr₃, SOCl₂and CBr₄. The target compounds 7 and 13–48,

except for 28 and 29, were synthesized by N-benzylation of the

NH-benzimidazole 12 with the obtained benzyl halides using dif-

ferent basic media such as NaH in DMF, KOH in DMSO, and TEA

in THF to obtain the optimal reaction yields. The generation of 28

was accomplished conveniently by directly reacting 12 with 2-

hydroxybenzyl alcohol under neat conditions.²⁴By this method,

2-hydroxybenzyl derivative 28 was successfully synthesized in

moderate yield by taking advantage of the in situ generated o-

methylenequinone as an electrophile. Subsequent acetylation of

28 by acetic acid anhydride in pyridine resulted in 2-acetoxybenzyl

derivative 29.

Compounds 1, 2 and 3 barely inhibited 5-LO product formation

with IC₅₀values >10 lM both in the cell-based and the cell-free

system (Table 1). Compounds 4, 5, and 8 were more potent inhib-

itors as they reduced 5-LO product synthesis in intact neutrophils

(IC₅₀= 4.4–9.1 lM), but had similar effects also in the cell-free as-

say. Also, compound 6 signiﬁcantly inhibited 5-LO activity in the

cell-free assay. From these data we conclude that compounds 4,

5, 6 and 8 primarily inhibit 5-LO leading to reduced cellular 5-LO

product formation. In contrast, compound 7 (a benzimidazole

derivative bearing the isobutylphenylethyl ﬁngerprint of ibupro-

fen) potently suppressed cellular 5-LO product formation with

IC₅₀= 0.31

23% inhibition at 10

5-LO inhibitor but rather affects FLAP, thereby suppressing 5-LO

product synthesis in neutrophils. The FLAP reference inhibitor 83

(3-[3-(tert-butylthio)-1-(4-chlorobenzyl)-5-isopropyl-1H-indol-2-

yl]-2,2-dimethylpropanoic acid, MK-886) and the direct 5-LO

l

M but was much less active on cell-free 5-LO (only

Synthesis of the desmethyl (55) and the geminal dimethyl (56)

analogues of 7 was carried out as outlined in Scheme 2 by using

ibufenac (49) and the geminal dimethyl analogue of ibuprofen

(50) as starting intermediates. Synthesis of 49 was achieved by a

conventional Willgerodt–Kindler synthesis of thiomorpholide from

4-isobutylacetophenone, which was subsequently converted to 49

by hydrolysis.²⁵For the preparation of 50, esteriﬁcation of ibupro-

fen followed by methylation under basic conditions yielded the

geminal dimethyl ester which was readily hydrolyzed to the acid

by using Triton B.²⁶For 63, carrying a methyl group instead of an

isobutyl substituent in compound 7, the starting intermediate 57

was prepared according to general published reports (Scheme 3).

For the synthesis of 57, ethyl p-tolylacetate was methylated with

methyl iodide under standard conditions and the ester was quan-

titatively hydrolyzed to the acid. The commercially available keto-

profen (58) was used instead of 49 for the synthesis of 64 with

benzoyl substituent. The monoacylation, cyclization to benzimid-

azole, and N-benzylation reactions for compounds 49, 50, 57 and

58 were carried out as described for compound 7 (Schemes 2

and 3).

lM, Table 1), suggesting that 7 is not a direct

Table 1

Inhibition of 5-LO product formation by screening hits in cell-free and cell-based

assays

Compd 5-LO product

formation

5-LO product formation

at 10 M, cell-based

IC_50,cell-based

assay (lM)

l

at 10

assay (% of control)

l

M, cell-free

assay (% of control)

1

2

3

4

5

6

7

8

85.3 16.7

116.0 32.3

62.0 13.0

52.8 28.9

58.0 22.7

55.1 14.1

77.1 15.4

16.0 2.0

91.3 29.5

57.9 13.2

80.8 7.3

40.9 9.9

43.6 6.1

78.8 17.4

4.0 1.6

10.0 7.5

2.7 1.2

3.1 2.2

>10

8.8

9.1

>10

0.31

4.4

0.017

0.11

The analogues of compound

7 carrying a substituent at

83

84

87.8 7.3

4.4 0.9

5-position were obtained by the series of reactions given in

Scheme 4. Starting from 4-methoxyaniline (65), N-acetylation fol-

lowed by nitration and hydrolysis afforded 2-nitroaniline deriva-

tive 66 which was benzylated and reduced to generate 68. After

following the standard conditions shown in Scheme 1, the 5-meth-

oxybenzimidazole derivative 70 was obtained. Hydrolysis of the

methyl ether was done by HBr to yield the 5-hydroxy derivative

71. The 5-hydroxybenzimidazole derivative was then alkylated

Values (mean SE, n = 3–4) for 5-LO product formation under cell-free conditions

(40,000Âg supernatants of lysates of E. coli expressing human recombinant 5-LO

incubated with 20 lM AA) and in intact human neutrophils challenged with 2.5 lM

ionophore are given as percentage of control at 10 lM inhibitor concentration. The

100% values of the controls (0.3% DMSO, vehicle) correspond to an average of 44 ng

5-LO products per 10⁶neutrophils and of 310 ng 5-LO products per ml in the cell-

free assay. The FLAP inhibitor 3-[3-(tert-butylthio)-1-(4-chlorobenzyl)-5-isopropyl-

1H-indol-2-yl]-2,2-dimethylpropanoic acid (MK-886, compound 83) and the 5-LO

inhibitor (E)-N-hydroxy-N-(3-(3-phenoxyphenyl)-allyl)acetamide (BWA4C, com-

either with 2-picolylchloride or

a-chloroquinaldine to obtain the

target compounds 72 and 73, respectively.

For the synthesis of 5-chlorobenzimidazole derivatives (Scheme

5), N-benzyl- or N-2-chlorobenzyl-4-chloro-2-nitroanilines 75 and

pound 84) were used (10

values in the cell-based assay were obtained from intact neutrophils challenged

with 2.5 M ionophore.

lM, each) as reference compounds, respectively. IC₅₀

l

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E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

H

NH₂

HO

N

a

+

O

NH₂

9

10

11

b

c

N

H

R₁

7, 13-48

12

R₁:

Cl

CN

O

Cl

14

Br

15

F

I

CF₃

18

7

13

16

17

19

20

21

22

23

F

CN

OH

CF₃

O

F

CN

O

24

25

26

27

28

29

30

31

32

33

O

Cl

N

O

N

O

N

O

Cl

34

35

36

37

38

39

40

41

42

O

Cl

O

OH

O

Cl

43

44

45

46

47

48

Scheme 1. Synthetic strategy for the synthesis of N-benzyl analogues of compound 7 Reagents and conditions: (a) EDC, DMAP, CH₂Cl₂, rt. (b) AcOH, reﬂux; (c) alkylhalide,

KOH/DMSO or NaH/DMF or TEA/THF at rt.

O

R₂

R₁

R₂

R₁

OH

N

H

N

NH₂

b

c

a

R₂

N

R₁

R₂

N

H

R₁

Cl

R₁=R₂= H

55

56

R₁=R₂= H

R₁=R₂= CH₃

53

54

R₁=R₂= H

R₁=R₂= CH₃

R₁=R₂= H

R₁=R₂= CH₃

49

50

51

R₁=R₂= CH₃

52

Scheme 2. Synthesis of achiral analogues of compound 7. Reagents and conditions: (a) o-phenylenediamine, EDC, DMAP, CH₂Cl₂, rt. (b) AcOH, reﬂux.

(c) 2-chlorobenzylbromide, KOH/DMSO, rt.

76 were prepared from 2,5-dichloronitrobenzene by nucleophilic

substitution of the chlorine atom by benzylamine derivatives.

Reduction of the nitro group followed by the standard procedures

described in Scheme 1 resulted in the 5-chlorobenzimidazole

derivatives 81 and 82. All compounds were puriﬁed by automated

ﬂash chromatography and checked for purity with UPLC before

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

3733

R

O

R

N

OH

N

H

NH₂

N

b

a

c

N

H

Cl

R

R= CH₃

61

57

R= CH₃

R= benzoyl 58

59

63

R= CH₃

R= benzoyl 62

R= benzoyl 60

R= benzoyl 64

Scheme 3. Synthesis of 63 and 64. Reagents and conditions: (a) o-phenylenediamine, EDC, DMAP, CH₂Cl₂, rt. (b) AcOH, reﬂux. (c) 2-chlorobenzylbromide, KOH/DMSO, rt.

Cl

H

N

H

NH₂

NO₂

N

a

b

c

H₃CO

NO₂

H₃CO

NH₂

65

66

67

68

d

Cl

H

N

HO

H₃CO

N

e

f

N

H₃CO

NH

O

Cl

70

71

69

g

R=

N

O

N

R

72

73

Cl

N

Scheme 4. Synthesis of C5-substituted analogues of compound 7. Reagents and conditions: (a) (i) Ac₂O, AcOH (ii) HNO₃, AcOH (iii) Claisen’s alkali. (b) 2-

Chlorobenzylbromide, DIPEA, DMF. (c) SnCl₂Á2 H₂O, EtOH (d) ibuprofen, oxalyl chloride, TEA, DMF (e) AcOH, reﬂux. (f) HBr 48% (g) 2-picolylchloride or

a-chloroquinaldine,

Cs₂CO₃, DMF.

Cl

NO₂

Cl

NO₂

Cl

NH₂

a

R

b

R

N

H

N

H

R

75

=-H

R

77

74

=-H

-Cl 76

-Cl 78

c

O

Cl

N

Cl

NH

d

N

H

R

79

R=-H 81

=-H

82

-Cl 80

-Cl

Scheme 5. Synthesis of C5-chloro-substituted analogues of compound 7. Reagents and conditions: (a) benzylamine or 2-chlorobenzylamine, EtOH (b) SnCl₂Á2 H₂O, EtOH (c)

ibuprofen, oxalyl chloride, TEA, DMF (d) AcOH, reﬂux.

3734

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

Table 2

being tested in biological assays (purity was >97%). The structures

of the compounds were conﬁrmed by high resolution mass spec-

trometry (HRMS), elemental analysis, IR and ¹H NMR spectral data.

Effects of test compounds on 5-LO product synthesis in

neutrophils) assay

a cell-based (intact

Compd

5-LO product formation in intact neutrophils

2.5. Structure–activity relationships (SAR) studies

Remaining activity at 1

l

M (% of control)

IC₅₀(lM)

7

19.4 4.9

47.7 6.2

66.5 11.7

79.7 6.4

65.8 10.7

78.2 3.6

83.9 5.6

100.4 35.2

67.1 8.9

63.0 7.8

83.8 6.6

98.3 7.0

99.8 13.1

18.1 10.0

43.9 7.3

43.5 9.8

84.8 8.3

8.4 3.1

0.31

0.98

4.8

6

3.4

5

4.9

2.3

2.9

3.8

7.2

In order to deduce SARs, we concentrated on four distinct areas

of the lead compound 7. Thus, we approached (i) modiﬁcation of

the N-benzyl group, (ii) removal of the chirality by preparing

desmethyl and geminal dimethyl analogs of 7, (iii) replacement

of the isobutyl moiety of 7 with smaller or larger groups, and (iv)

incorporation of different substituents at the C5-benzimidazole

ring. Together, by installation of distinct chemical functionalities

with the aforementioned modiﬁcations, forty-six benzimidazole

derivatives were synthesized to investigate their inhibitory effect

on 5-LO product formation in the cell-based (intact neutrophils)

assay (Table 2). First, the substitution pattern at the aromatic ring

of the benzyl group was studied as shown by the comparison of the

4-substituted (14–24), 2-substituted (25–31) and 3-substituted

analogues (32–34). Removal of 2-chlorine, resulting in an unsubsti-

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

55

56

63

64

70

71

72

73

81

82

>10 (77.8 10.6)^a

>10

0.5

0.7

0.45

4.5

0.25

0.44

0.25

4.0

n.d.

tuted benzyl (13), decreased the potency (IC₅₀= 0.98

compound 7 (IC₅₀= 0.31 M). Movement of the chlorine substitu-

ent to 4-position (14) also caused a signiﬁcant loss of activity

(IC₅₀= 4.8 M). Substitutions at 4-position other than chlorine,

lM) versus

9.2 4.9

4.89 2.25

71.1 11.9

91.1 18.5

109.2 10.8

24.6 12.5

89.4 2.2

96.1 16.2

120.3 4.7

85.3 11.4

93.6 20.5

74.1 14.1

112.6 13.6

103.5 11.3

88.7 6.0

92.2 18.1

94.4 2.9

86.0 17.5

135.0 23.6

25.6 11.2

33.3 10.3

80.9 13.9

90.2 2.3

7.8 7.0

l

n.d.

0.5

4.4

i.e. electron withdrawing (15–19) and electron donating groups

(20–21), were detrimental resulting in compounds with IC₅₀values

between 2.3 and 6.0 lM. Increasing the steric bulk at 4-position

(22–24) also impaired the potency versus 7, indicating that volu-

minous substituents at this position are not well accommodated.

In contrast, variations of the substituents in 2-position (i.e., com-

pounds 25–31) slightly affected the inhibitory activity (except for

28) and potent, concentration-dependent suppression of 5-LO

>10 (91.5 13)^a

9.4

2.5

2.8

1.95

4.4

>10 (58.8 6.5)^a

10

product formation (IC₅₀= 0.25–0.7

ingly, in this series, a free 2-hydroxyl group caused a signiﬁcant

loss of activity (28, IC₅₀= 4.5 M), but acetylation (29) or methyla-

tion (30) of this residue recovered the activity (IC₅₀= 0.25 and

0.44 M, respectively). Although the reason for this is not clear,

lM) could be retained. Interest-

>10 (58.7 9.5)^a

6

>10 (56.7 16.9)^a

l

>10 (85.5 6.9)^a

0.5

0.7

2.8

3

0.12

0.19

0.18

l

hydrogen bond forming properties or general increased polarity

of the hydroxy-derivative 28 is conceivable. Of interest, volumi-

nous groups are seemingly tolerated at 2-position, deduced from

the almost 2-fold increased potency of the ester 29. 3-Substituted

benzyl analogues (32–34) were not pursued further as they

showed marked loss of inhibitory activity. When the dichloro sub-

stitution pattern was investigated, the 2,4-substitution (35) was

superior over 2,6-substitution (36) with IC₅₀values of 0.5 and

4.5 0.3

7.0 5.2

72.0 11.6

44.9 11.6

18.08 4.27

>10 (53.4 5.44)^a

0.83

0.16

Compounds were tested in neutrophils stimulated with 2.5

given as percentage of control (100%) at 10

lM A23187. Data are

4.4 lM, respectively. Multiple alkoxy substitutions on the phenyl

l

M inhibitor concentration (means SE,

ring (37 and 38) were detrimental, consistent with the result that

voluminous substituents are not well tolerated on the benzyl moi-

ety other than in 2-position. We also investigated the effects of

introducing a heteroaromatic group into the molecule. When the

phenyl was replaced by 2-pyridinyl (39), 3-pyridinyl (40) or

4-pyridinyl (41), the compounds were still active, albeit the po-

n = 4–6) or as IC₅₀values (means). The 100% value of the control (0.3% DMSO,

vehicle) corresponds to an average of 44 ng 5-LO products per 10⁶neutrophils.

a

Remaining activity at 10 lM; n.d., not determined.

carboxylic acid (48) or by its ester (47) was found to be detrimental

suggesting the signiﬁcance of the N-benzyl group for inhibition of

LT biosynthesis. Taken together, 2-substituted N-benzyl groups

clearly govern potent inhibition of LT biosynthesis.

Since all compounds were evaluated as racemates, our second

approach was to investigate the effect of chirality and the impor-

tance of the methyl group on the chiral carbon for the inhibitory

potency of lead compound 7. As seen in Table 2, either deletion

of the methyl group (55) or insertion of a second methyl at the chi-

ral carbon (56) only slightly inﬂuenced the inhibitory potency ver-

tency decreased (IC₅₀= 1.95–2.5 lM). Introducing a larger quino-

line (42) diminished the inhibitory activity which again was in

agreement with the hypothesis that voluminous substituents in

place of the N-benzyl group are detrimental.

We next examined the necessity of the N-benzyl group by

replacing it with a benzoyl substituent. When 43, the derivative

of 7 carrying a 2-chlorobenzoyl moiety instead of N-benzyl, was

analyzed, a loss of activity was clearly apparent (IC₅₀>10 lM).

Similarly, unsubstituted benzoyl (44), and 3-chlorobenzoyl (45)

derivatives appeared to be ineffective. However, incorporation of

a 2-acetoxy-substituent to the benzoyl group partially restored

the inhibitory activity in this series (IC₅₀= 6 lM), supporting the

previous ﬁnding with compound 29 that a 2-acetoxy substituent

sus 7, with IC₅₀values of 0.5 and 0.7 lM, respectively.

Our third goal was to understand the role of the isobutyl ﬁnger-

print of 7 for the potency of compounds and we brieﬂy examined

this by replacing the isobutyl with smaller methyl (63) and also

with bulky benzoyl (64) groups. However, these derivatives also

favors inhibitory activity. Finally, replacement of benzyl by an alkyl

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

3735

Figure 4. Docking of the compounds 7 (A), 70 (B), 71 (C), and 72 (D) at the active site of FLAP. Hydrophobic interactions are presented in red, while hydrogen bonds are in

blue.

showed diminished inhibitory potency indicating that the isobutyl

substituent was favorable for bioactivity.

phenyl moiety positioned at C(2) of the benzimidazole. In addition,

the benzimidazole ring of compounds 7 and 70 is stabilized by

Finally, we evaluated the incorporation of various substituents

at the C(5) of the benzimidazole. At this stage, we aimed to antici-

pate two improvements on 7: (i) the inhibitory potency and (ii) the

probable metabolic stability by blocking the C(5) position at the

benzimidazole, since benzimidazole derivatives generally tend to

be converted to 5-hydroxy derivatives.^27,28Of interest, we found

that electron donating substituents at the C(5) position enhance

the inhibitory potency. Thus, 5-methoxy (70) and 5-hydroxy (71)

analogues are more potent inhibitors than 7 with IC₅₀of 0.12 and

forming CH-p interactions with B-Thr66, while 71 interacts with

the B-Phe114 through its N-chlorobenzyl group. However, the

most potent inhibitors 70 and 71 form an additional H-bond with

the C-Asn57 through their hydroxy or methoxy groups located at

C(5) of the benzimidazole, which was not present in the case of

compound 7. Compound 72 adopts a different binding conforma-

tion versus 70 and 71 in which the 2-pyridinylmethyl group now

occupies the position of the benzimidazole ring in these com-

pounds, however maintaining similar hydrophobic interaction net-

0.19

lM, respectively. Pyridine and quinoline moieties are found in

work such as CH-p interactions with B-Ile119, C-Val21 and C-

a variety of LT biosynthesis inhibitors and also are established phar-

macophores for FLAP inhibitors.^9,11,29Therefore, we sequentially

incorporated a 2-pyridinylmethyl (72) and 2-quinolynylmethyl

(73) into the C(5) position of 7. While the pyridine moiety was well

Ala27. The binding energies and the stability of the formed li-

gand–protein complexes were in good agreement with the ob-

served biological data (Table S1, Supplementary data).

tolerated (IC₅₀= 0.18 lM), the quinoline group caused a complete

3. Conclusions

loss of inhibitory potency. This may indicate that larger substitu-

ents at this position are not tolerated and may hamper the binding

to FLAP. Finally, we synthesized the mono- and dichloro derivatives

81 and 82 of 7 as examples of electron withdrawing substituent

which essentially retained the inhibitory potency (IC₅₀= 0.83 and

There is currently a strong interest in the development of FLAP

inhibitors because of their promising potential as therapeutics in

inﬂammatory and allergic disorders as well as in cardiovascular

disease.^9–13Here, we have implemented for the ﬁrst time a rapid

virtual screening on a large number of compounds from vendor li-

braries based on a combined ligand- and structure-based FLAP

pharmacophore. Though recently discussed as a future opportu-

nity, drug design solely based on the available structure of FLAP

was found to be insufﬁcient due to low resolution. Out of eight

candidates that were tested, compound 7, a benzimidazole deriva-

tive bearing the isobutylphenylethyl ﬁngerprint of ibuprofen, was

identiﬁed as potent suppressor of cellular LT biosynthesis without

0.16 lM, respectively) of 7.

Together, compounds 7, 25–27, 28–31, 35, 55, 56 and the highly

active analogues 70–72 and 82 potently suppress LT biosynthesis

in intact neutrophils. In a ﬁnal step, we performed molecular mod-

eling studies of the most active compounds by using the 3D-struc-

ture of FLAP in complex with the inhibitor 85.¹⁵In our proposed

poses (Fig. 4), compounds 7, 70 and 71 form hydrophobic interac-

tions with the fundamental amino acid B-Ile119 through the

3736

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

inhibiting 5-LO activity directly. SAR investigations revealed that

the 2-substituted N-benzyl group and the isobutyl substituent of

7 clearly govern potent inhibition of LT biosynthesis, and that large

substituents on the C5-benzimidazole are not tolerated. This sug-

gests the benzimidazole scaffold bearing an ibuprofen ﬁngerprint

as a new chemotype for further development of anti-LT agents. A

possible description of the putative FLAP binding mode for the

novel benzimidazole-based compounds was also proposed. In con-

clusion, our results proved these fast synthetically accessible benz-

imidazole-based compounds as a new and innovative scaffold

useful in the rational design of novel developmental candidates

as anti-LT drugs and provide valuable insights into the chemical

decorations functional for the design of further analogues belong-

ing to this class of inhibitors.

(2H, dd, J = 17.6 and 17.6 Hz), 6.18 (1H, d, J = 7.2 Hz), 6.90 (1H,

m), 6.94–7.08 (3H, m), 7.13–7.37 (6H, m), 7.90 (1H, d, J = 8.0 Hz);

HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₇ClN₂403.1941; found

403.1925; Anal. Calcd for C₂₆H₂₇ClN₂: C, 77.50; H, 6.75; N, 6.95.

Found: C, 77.64; H, 6.75; N, 6.92.

4.1.1.4. 1-Benzyl-2-[1-(4-isobutylphenyl)ethyl]-1H-benzimid-

azole (13).

Yield 67%; mp 107.7 °C. ¹H NMR (CDCl₃): d 0.87

(6H, d, J = 6.8 Hz), 1.78 (1H, m) 1.81 (3H, d, J = 6.8 Hz), 2.40 (2H,

d, J = 7.2), 4.18 (1H, q, J = 7.0 Hz), 5.04–5.19 (2H, dd, J = 16.8 and

J = 16.8), 6.87 (2H, m), 7.02 (2H, d, J = 8.0 Hz), 7.09 (2H, d,

J = 8.0 Hz), 7.13–7.28 (6H, m), 7.89 (1H, d, J = 8.0 Hz); HRMS (m/

z): [M+H]⁺calcd for C₂₆H₂₈N₂369.2331; found 369.2324; Anal.

Calcd for C₂₆H₂₈N₂: C, 84.74; H, 7.66; N, 7.60; found: C, 84.67; H,

7.96; N, 7.63.

4. Experimental

4.1.1.5.

benzimidazole (14).

1-(4-Chlorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-1H-

Yield 69%; mp 180.1 °C. ¹H NMR

4.1. Compounds and chemistry

(CDCl₃): d 0.86 (6H, d, J = 6.4 Hz), 1.76 (1H, m), 1.81 (3H, d,

J = 7.2 Hz), 2.39 (2H, d, J = 7.2 Hz), 4.16 (1H, q, J = 7.0 Hz), 5.01–

5.17 (2H, dd, J = 16.8 Hz and J = 16.8 Hz), 6.76 (2H, d, J = 8.8 Hz),

7.01 (2H, d, J = 8.4 Hz), 7.08 (2H, d, J = 8.0 Hz), 7.10–7.27 (5H, m),

7.89 (1H, d, J = 8.4 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₇ClN₂

403.1941. Found 403.1931; Anal. Calcd for C₂₆H₂₇ClN₂: C, 77.50;

H, 6.75; N, 6.95. Found: C, 77.57; H, 6.37; N, 6.98.

¹H NMR spectra were recorded in CDCl₃or DMSO-d₆on a Varian

Mercury 400 MHz High Performance Digital FT-NMR spectrometer

using tetramethylsilane as the internal standard at the NMR facil-

ity of Faculty of Pharmacy, Ankara University. All chemical shifts

were recorded as d (ppm). High resolution mass spectra data

(HRMS) were collected in-house using a Waters LCT Premier XE

Mass Spectrometer (high sensitivity orthogonal acceleration

time-of-ﬂight instrument) operating in either ESI (+) 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

chromatography was performed with a Combiﬂash^ÒRf automated

ﬂash chromatography system with RediSep columns (Teledyne-

Isco, Lincoln, NE, USA) using hexane–EtOAc or DCM–MeOH solvent

gradients. The purity of the ﬁnal compounds was determined to be

>97% by UPLC with UV detector.

4.1.1.6. 1-(4-Bromobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-1H-

benzimidazole (15).

Yield 77%; mp 168.7 °C. ¹H NMR

(CDCl₃): d 0.86 (6H, d, J = 6.4 Hz), 1.76 (1H, m), 1.81 (3H, d,

J = 7.2 Hz), 2.39 (2H, d, J = 7.2 Hz), 4.16 (1H, q, J = 7.0 Hz),

4.99–5.15 (2H, dd, J = 17.2 Hz and J = 16.8 Hz), 6.68 (2H, d,

J = 8.8 Hz), 6.99 (2H, d, J = 8.0 Hz), 7.06 (2H, d, J = 8.0 Hz), 7.07

(1H, m), 7.18–7.30 (4H, m), 7.88 (1H, d, J = 8.4 Hz); HRMS (m/z):

[M+H]⁺calcd for C₂₆H₂₇BrN₂447.1436; found 447.1416; Anal.

Calcd for C₂₆H₂₇ClN₂: C, 69.80; H, 6.08; N, 6.26. Found: C, 69.83;

H, 5.82; N, 6.20.

4.1.1.7.

benzimidazole (16).

1-(4-Fluorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-1H-

Yield 94%; mp 169.4 °C. ¹H NMR

4.1.1. General procedure for the alkylation of benzimidazole

derivatives

(CDCl₃): d 0.86 (6H, d, J = 6.4 Hz), 1.76–1.82 (4H, m), 2.39 (2H, d,

J = 7.2 Hz), 4.19 (1H, q, J = 7.1 Hz), 5.02 (2H, dd, J = 16.4 Hz and

J = 16.8 Hz), 6.78–6.91 (4H, m), 7.01 (2H, d, J = 8.0 Hz), 7.08 (2H,

d, J = 8.4 Hz), 7.11–7.28 (3H, m), 7.88 (1H, d, J = 8.0 Hz); HRMS

(m/z): [M+H]⁺calcd for C₂₆H₂₇FN₂387.2237; found 387.2235;

Anal. Calcd for C₂₆H₂₇FN₂: C, 80.80; H, 7.04; N, 7.25. Found: C,

80.71; H, 7.05; N, 7.17.

4.1.1.1. Method A (for compounds 7, 13–27, 30–42, 47, 48, 55,

56, 63, 64).

To an ice-cooled solution of NaH (60% in oil,

1.5 equiv) in 10 mL DMF or to a solution of KOH (1.5 equiv) in

10 mL DMSO at rt, the appropriate benzimidazole derivative (12,

53, 54, 61 or 62) (1 equiv) was added and stirred for 0.5 h. After

addition of the corresponding alkyl halide (1 equiv), stirring was

continued for 3 h. The reaction mixture was diluted with water, ex-

tracted with EtOAc (30 mL Â 3), dried over anhydrous Na₂SO₄, ﬁl-

tered, and evaporated to dryness. The ﬁnal product was puriﬁed

by automated ﬂash chromatography.

4.1.1.8.

1-(4-Iodobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-1H-

Yield 64%; mp 140.6 °C. ¹H NMR

benzimidazole (17).

(CDCl₃): d 0.86 (6H, d, J = 6.8 Hz), 1.76 (1H, m), 1.82 (3H, d,

J = 7.2 Hz), 2.39 (2H, d, J = 7.2 Hz), 4.16 (1H, q, J = 7.1 Hz), 4.98–

5.14 (2H, dd, J = 16.8 Hz and J = 17.2 Hz), 6.56 (2H, d, J = 8.4 Hz),

7.00–7.16 (5H, m), 7.16–7.29 (2H, m), 7.50 (2H, d, J = 8.8 Hz),

7.89 (1H, d, J = 8.4 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₇IN₂

495.1297; found 495.1313; Anal. Calcd for C₂₆H₂₇IN₂: C, 63.16; H,

5.50; N, 5.67. Found: C, 63.36; H, 5.54; N, 5.49.

4.1.1.2. Method B (for compounds 43–46).

To a solution of

benzimidazole derivative 12 (1 equiv) in anhydrous THF, the

appropriate acyl chloride (1.1 equiv) and TEA (1.5 equiv) was

added. After stirring at rt overnight, the eluent was evaporated in

vacuo and the residue was dissolved in 20 mL DCM. The organic

phase was extracted with 5% NaHCO₃(3 Â 30 mL), brine

(3 Â 30 mL) and the combined organic phases were dried over

anhydrous Na₂SO₄, ﬁltered, and the ﬁltrate was evaporated in va-

cuo to give the product which was subsequently puriﬁed by auto-

mated ﬂash chromatography.

4.1.1.9.

2-[1-(4-Isobutylphenyl)ethyl]-1-[4-(triﬂuoro-

Yield 40%; mp

methyl)benzyl]-1H-benzimidazole (18).

132.3 °C. ¹H NMR (CDCl₃): d 0.84 (6H, d, J = 6.8 Hz), 1.75 (1H, m),

1.83 (3H, d, J = 7.2 Hz), 2.37 (2H, d, J = 7.2 Hz), 4.16 (1H, q,

J = 7.2 Hz), 5.12–5.26 (2H, dd, J = 17.2 Hz and J = 17.2 Hz), 6.89

(2H, d, J = 8.0 Hz), 6.97 (2H, d, J = 8.0 Hz), 7.05–7.07 (3H, m), 7.19

(1H, m), 7.28 (1H, m), 7.44 (2H, d, J = 8.4 Hz), 7.91 (1H, d,

J = 7.6 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₇H₂₇F₃N₂437.2205;

4.1.1.3.

benzimidazole (7).

1-(2-Chlorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-1H-

Yield 82%; mp 105.5 °C. ¹H NMR (CDCl₃):

d 0.83 (6H, dd, J = 6.8 and 2.0 Hz), 1.75 (1H, m), 1.82 (3H, d,

J = 7.2 Hz), 2.35 (2H, d, J = 7.2 Hz), 4.15 (1H, q, J = 7.2 Hz), 5.24

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

3737

found 437.2194; Anal. Calcd for C₂₇H₂₇F₃N₂: C, 74.29; H, 6.23; N,

6.42. Found: C, 74.25; H, 6.22; N, 6.38.

d, J = 7.2 Hz), 4.09 (1H, q, J = 8.6, Hz), 4.98–5.10 (2H, dd, J = 17.6 Hz

and J = 17.2 Hz), 6.25 (1H, d, J = 8.0 Hz), 6.92–7.29 (10H, m), 7.90

(1H, d, J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₇H₃₀N₂

383.2487; found 383.2482; Anal. Calcd for C₂₇H₃₀N₂: C, 84.77; H,

7.90; N, 7.32. Found: C, 84.95; H, 8.15; N, 7.38.

4.1.1.10. 4-({2-[1-(4-Isobutylphenyl)ethyl]-1H-benzimidazol-1-

yl}methyl)benzonitrile (19).

Yield 32%; mp 158.6 °C. ¹H

NMR (CDCl₃): d 0.84 (6H, dd, J = 6.4 Hz and J = 6.4 Hz), 1.74 (1H,

m), 1.82 (3H, d, J = 7.2 Hz), 2.37 (2H, d, J = 7.2 Hz), 4.14 (1H, m),

5.10–5.28 (2H, dd, J = 17.6 Hz and J = 17,6 Hz), 6.86 (2H, d,

J = 8.4 Hz), 6.96 (2H, d, J = 8.0 Hz), 7.02–7.31 (5H, m), 7.45 (2H, d,

J = 6.8 Hz), 7.90 (1H, d, J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd for

4.1.1.17. 1-(2-Fluorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-1H-

benzimidazole (26).

(CDCl₃): d 0.85 (6H, d, J = 6.4 Hz), 1.77 (1H, m), 1.81 (3H, d,

J = 7.2 Hz), 2.38 (2H, d, J = 7.6 Hz), 4.24 (1H, q, J = 6.8 Hz), 5.20

(2H, s), 6.39 (1H, t, J = 7.2 Hz), 6.83 (1H, t, J = 7.6 Hz), 6.98–7.28

(9H, m), 7.88 (1H, d, J = 8.4 Hz); HRMS (m/z): [M+H]⁺calcd for

Yield 69.4%; mp 96.3 °C. ¹H NMR

C₂₇H₂₇N₃394.2283; found 394.2272; Anal. Calcd for C₂₇H₂₇N₃: C,

82.41; H, 6.92; N, 10.68. Found: C, 81.81; H, 6.90; N, 10.38.

C₂₆H₂₇FN₂387.2237; found 387.2224; Anal. Calcd for C₂₆H₂₇FN₂:

4.1.1.11. 2-[1-(4-Isobutylphenyl)ethyl]-1-(4-methylbenzyl)-1H-

benzimidazole (20).

C, 80.80; H, 7.04; N, 7.25. Found: C, 80.44; H, 7.12; N, 7.17.

Yield 89%; mp 138.8 °C. ¹H NMR

(CDCl₃): d 0.87 (6H, d, J = 6.8 Hz), 1.79 (4H, m), 2.28 (3H, s), 2.41

(2H, d, J = 7.6 Hz), 4.19 (1H, q, J = 6.8 Hz), 4.99–5.14 (2H, dd,

J = 16.8 Hz and J = 16.8 Hz), 6.77 (2H, d, J = 8.0 Hz), 7.01–7.27 (9H,

m), 7.88 (1H, d, J = 8.4 Hz); HRMS (m/z): [M+H]⁺calcd for

4.1.1.18. 2-({2-[1-(4-Isobutylphenyl)ethyl]-1H-benzimidazol-1-

yl}methyl)benzonitrile (27).

NMR (CDCl₃): 0.83 (6H, m), 1.75 (1H, m), 1.84 (3H, d,

Yield 52%; mp 134.2 °C. ¹H

d

J = 6.8 Hz), 2.31 (2H, d, J = 7.6 Hz), 4.21 (1H, q, J = 7.0 Hz),

5.34–5.48 (2H, dd, J = 18.0 Hz and J = 18.0 Hz), 6.29 (1H, d,

J = 8.0 Hz), 6.90 (2H, d, J = 8.0 Hz), 7.04–7.31 (7H, m), 7.62 (1H, d,

J = 8.0 Hz), 7.91 (1H, d, J = 8.4 Hz); HRMS (m/z): [M+H]⁺calcd for

C₂₇H₂₇N₃394.2283; found 394.2283; Anal. Calcd for C₂₇H₂₇N₃: C,

82.41; H, 6.92; N, 10.68. Found: C, 82.09; H, 6.94; N, 10.50.

C₂₇H₃₀N₂383.2487; found 383.2486; Anal. Calcd for C₂₇H₃₀N₂: C,

84.77; H, 7.90; N, 7.32. Found: C, 84.48; H, 7.54; N, 7.26.

4.1.1.12.

1H-benzimidazole (21).

2-[1-(4-Isobutylphenyl)ethyl]-1-(4-methoxybenzyl)-

Yield 76%; mp 118.4 °C. ¹H NMR

(CDCl₃): d 0.87 (6H, d, J = 6.8 Hz), 1.79–1.81 (4H, m), 2.42 (2H, d,

J = 7.2 Hz), 3.74 (3H, s), 4.21 (1H, q, J = 7.0 Hz), 4.96–5.14 (2H, dd,

J = 16.8 Hz and J = 16.4 Hz), 6.76 (2H, d, J = 8.4 Hz), 6.79 (2H, d,

J = 8.8 Hz), 7.04 (2H, d, J = 7.6 Hz), 7.11 (2H, d, J = 7.6 Hz), 7.13–

7.27 (3H, m), 7.88 (1H, d, J = 8.4 Hz); HRMS (m/z): [M+H]⁺calcd

4.1.1.19. 2-({2-[1-(4-Isobutylphenyl)ethyl]-1H-benzimidazol-1-

yl}methyl)phenol (28).

An equimolar mixture of 12 and 2-

hydroxybenzyl alcohol was heated under neat conditions at

160 °C with intense stirring. The reaction product was solidiﬁed

with petroleum ether and puriﬁed by automated ﬂash chromatog-

raphy. Yield 35%; mp 224.1 °C. ¹H NMR (CDCl₃): d 0.83 (6H, d, J

= 6,8 Hz), 1.75 (1H, m), 1.79 (3H, d, J = 7.2 Hz), 2.36 (2H, d,

J = 7.2 Hz), 4.45 (1H, q, J = 7.2 Hz), 5.21–5.36 (2H, dd, J = 16.8 Hz

and J = 17.2 Hz), 6.49 (1H, d, J = 6.8 Hz), 6.65 (1H, t, J = 7.4 Hz),

6.90 (1H, d, J = 7.6 Hz), 6.96 (2H, d, J = 8.4 Hz), 7.05 (1H, t,

J = 7.7 Hz), 7.14 (2H, d, J = 7.6 Hz), 7.18–7.29 (3H, m), 7.80 (1H, d,

J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₈N₂O 385.2280;

found 385.2274; Anal. Calcd for C₂₆H₂₈N₂O: C, 81.21; H, 7.34; N,

7.29. Found: C, 81.29; H, 7.30; N, 7.26.

for

C₂₇H₃₀N₂O 399.2436; found 399.2429; Anal. Calcd for

C₂₇H₃₀N₂O: C, 81.37; H, 7.59; N, 7.03. Found: C, 80.97; H, 7.98; N,

7.02.

4.1.1.13. 2-[1-(4-Isobutylphenyl)ethyl]-1-(4-isopropylbenzyl)-

1H-benzimidazole (22).

(CDCl₃): 0.87 (6H, d, J = 6.8 Hz), 1.21 (6H, d, J = 7.2 Hz),

Yield 30%; mp 76.5 °C. ¹H NMR

d

1.66–1.84 (4H, m), 2.42 (2H, d, J = 7.2 Hz), 2.84 (1H, m), 4.21 (1H,

q, J = 6.8 Hz), 5.14 (2H, dd, J = 16.8 Hz and J = 16.8 Hz), 6.82 (2H,

d, J = 7.6 Hz), 7.03–7.27 (9H, m), 7.88 (1H, d, J = 8.0 Hz); HRMS

(m/z): [M+H]⁺calcd for C₂₉H₃₄N₂411.2800; found 411.2780; Anal.

Calcd for C₂₉H₃₄N₂: C, 84.83; H, 8.35; N, 6.82. Found: C, 84.45; H,

8.42; N, 6.81.

4.1.1.20.

2-({2-[1-(4-Isobutylphenyl)ethyl]-1H-benzimidazol-

Compound 28 (1 equiv),

1-yl}methyl)phenyl acetate (29).

acetic anhydride (2.5 equiv) and DMAP (0.02 equiv) in pyridine

was stirred for 3 h at room temperature. The reaction mixture

was diluted by ice-cold water, and extracted with EtOAc. The or-

ganic phase dried, ﬁltered and puriﬁed by automated ﬂash chro-

matography. Yield 97%; mp 113.1 °C. ¹H NMR (CDCl₃): d 0.85

(6H, d, J = 5.6 Hz), 1.75 (1H, m), 1.79 (3H, d, J = 7.6 Hz), 2.21 (3H,

s), 2.40 (2H, d, J = 7.2 Hz), 4.01 (1H, q, J = 7.2 Hz), 5.06 (2H, dd,

J = 17.6 Hz and J = 17.6 Hz), 6.39 (1H, d, J = 7.6 Hz), 6.97 (1H, t,

J = 8.0 Hz), 7.01 (2H, d, J = 8.0 Hz), 7.09 (2H, d, J = 8.0 Hz), 7.13–

7.29 (5H, m), 7.89 (1H, d, J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd

4.1.1.14. 1-(4-tert-Butylbenzyl)-2-[1-(4-isobutylphenyl)ethyl]-

1H-benzimidazole (23).

Yield 72%; mp 72 °C. ¹H NMR

(CDCl₃): d 0.88 (6H, d, J = 6.8 Hz), 1.26 (9H, s), 1.82 (4H, m), 2.42

(2H, d, J = 7.2 Hz), 4.22 (1H, q, J = 8.0 Hz), 5.18 (2H, dd, J = 16.8 Hz

and J = 16.4 Hz), 6.82 (2H, d, J = 8.0 Hz), 7.03 (2H, d, J = 7.6 Hz),

7.10 (2H, d, J = 8.0 Hz), 7.16–7.27 (5H, m), 7.87 (1H, d, J = 6.8 Hz);

HRMS (m/z): [M+H]⁺calcd for C₃₀H₃₆N₂425.2957; found

425.2957; Anal. Calcd for C₃₀H₃₆N₂: C, 84.86; H, 8.55; N, 6.60.

Found: C, 84.77; H, 8.53; N, 6.60.

for

C₂₈H₃₀N₂O₂427.2386; found 427.2379; Anal. Calcd for

4.1.1.15. Methyl

zimidazol-1-yl}methyl)benzoate (24).

4-({2-[1-(4-isobutylphenyl)ethyl]-1H-ben-

Yield 74.5%; mp

C₂₈H₃₀N₂O₂: C, 78.84; H, 7.09; N, 6.57. Found: C, 78.96; H, 7.20;

N, 6.10.

127.5 °C. ¹H NMR (CDCl₃): d 0.84 (6H, d, J = 6.4 Hz), 1.76 (1H, m),

1.81 (3H, d, J = 7.2 Hz), 2.37 (2H, d, J = 6.8 Hz), 3.88 (3H, s), 4.15 (1H,

q, J = 7.0 Hz), 5.05–5.26 (2H, dd, J = 17.2 Hz and J = 17.2 Hz), 6.86

(2H, d, J = 8.4 Hz), 6.98 (2H, d, J = 7.6 Hz), 6.98–7.09 (5H, m),

7.18–7.29 (2H, m), 7.87 (1H, m); HRMS (m/z): [M+H]⁺calcd for

4.1.1.21.

1H-benzimidazole (30).

2-[1-(4-Isobutylphenyl)ethyl]-1-(2-methoxybenzyl)-

Yield 78.2%; mp 129.1 °C. ¹H NMR

(CDCl₃): d 0.86 (6H, d, J = 6.4 Hz), 1.78 (1H, m), 1.81 (3H, d,

J = 6.8 Hz), 2.38 (2H, d, J = 6.8 Hz), 3.86 (3H, s, OCH₃), 4.24 (1H, q,

J = 7.0 Hz), 5.09–5.19 (2H, dd, J = 17.6 Hz and J = 17.2 Hz), 6.35

(1H, d, J = 6.8 Hz), 6.68 (1H, t), 6.86 (1H, d, J = 7.6 Hz), 6.98 (2H, d,

J = 7.6 Hz), 7.09–7.25 (6H, m), 7.87 (1H, d, J = 8.0 Hz); HRMS

(m/z): [M+H]⁺calcd for C₂₇H₃₀N₂O 399.2436; found 399.2433;

Anal. Calcd for C₂₇H₃₀N₂O: C, 80.28; H, 7.66; N, 6.88. Found: C,

80.29; H, 7.49; N, 6.85.

C₂₈H₃₀N₂O₂427.2386; found 427.2382; Anal. Calcd for C₂₈H₃₀N₂O₂:

C, 78.84; H, 7.09; N, 6.57. Found: C, 78.90; H, 7.18; N, 6.52.

4.1.1.16. 2-[1-(4-Isobutylphenyl)ethyl]-1-(2-methylbenzyl)-1H-

benzimidazole (25).

d 0.85 (6H, d, J = 6.6 Hz), 1.74–1.80 (4H, m), 2.26 (3H, s), 2.38 (2H,

Yield 43%; mp 93.5 °C. ¹H NMR (CDCl₃):

3738

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

4.1.1.22.

2-[1-(4-Isobutylphenyl)ethyl]-1-[2-(triﬂuoro-

Yield 90%; mp

0.86 (6H, dd, J = 3.6 Hz and

(CDCl₃): d 0.86 (6H, d, J = 6.8 Hz), 1.35 (3H, t, J = 6.8 Hz), 1.41 (3H,

methyl)benzyl]-1H-benzimidazole (31).

t, J = 6.8 Hz), 1.81 (4H, m), 2.40 (2H, d, J = 7.6 Hz), 3.85 (2H, q,

J = 6.8 Hz), 4.02 (2H, q, J = 6.8 Hz), 4.21 (1H, q, J = 6.8 Hz),

4.92–5.15 (2H, dd, J = 16.4 Hz and J = 16.4 Hz), 6.39 (2H, m), 6.71

(1H, d, J = 8.4 Hz), 7.03 (2H, d, J = 7.6 Hz), 7.11 (2H, d, J = 7.6 Hz),

7.15–7.27 (3H, m), 7.86 (1H, d, J = 8.0 Hz); HRMS (m/z): [M+H]⁺

calcd for C₃₀H₃₆N₂O₂457.2855; found 457.2840; Anal. Calcd for

112.5 °C. ¹H NMR (CDCl₃):

d

J = 3.2 Hz), 1.72 (1H, m), 1.82 (3H, d, J = 7.2 Hz), 2.32 (2H, d,

J = 6.8 Hz), 4.14 (1H, q, J = 7.2 Hz), 5.33–5.42 (2H, dd, J = 17.6 Hz

and J = 17.2 Hz), 6.18 (1H, d, J = 7.6 Hz), 6.91 (2H, d, J = 8.0 Hz),

7.03–7.30 (7H, m), 7.66 (1H, d, J = 7.6 Hz), 7.92 (1H, d, J = 8.0 Hz);

HRMS (m/z): [M+H]⁺calcd for C₂₇H₂₇F₃N₂437.2205; found

437.2208; Anal. Calcd for C₂₇H₂₇F₃N₂: C, 74.29; H, 6.23; N, 6.42.

Found: C, 74.41; H, 6.03; N, 6.61.

C₃₀H₃₆N₂O₂: C, 78.91; H, 7.95; N, 6.13. Found: C, 78.67; H, 8.06;

N, 6.14.

4.1.1.29. 2-[1-(4-Isobutylphenyl)ethyl]-1-(2,3,4-trimethoxyben-

4.1.1.23. 1-(3-Fluorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-1H-

benzimidazole (32).

zyl)-1H-benzimidazole (38).

Yield 51%; mp 152.4 °C. ¹H NMR

Yield 40%; mp 112.5 °C. ¹H NMR

(CDCl₃): d 0.86 (6H, d, J = 6.8 Hz), 1.78 (1H, m), 1.82 (3H, d,

J = 7.2 Hz), 2.39 (2H, d, J = 7.6 Hz), 3.64 (6H, s), 3.79 (3H, s) 4.24

(1H, q, J = 7.2 Hz), 4.98–5.16 (2H, dd, J = 16.8 Hz and J = 16.8 Hz),

6.06 (2H, s), 7.09 (2H, d, J = 8.0 Hz), 7.13–7.28 (5H, m), 7.89 (1H,

d, J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₉H₃₄N₂O₃

459.2648; found 459.2644; Anal. Calcd for C₂₉H₃₄N₂O₃: C, 75.95;

H, 7.47; N, 6.11. Found: C, 75.81; H, 7.53; N, 6.20.

(CDCl₃): d 0.86 (6H, d, J = 6.4 Hz), 1.75–1.83 (4H, m), 2.40 (2H, d,

J = 7.2 Hz), 4.18 (1H, q, J = 7.2 Hz), 5.18 (2H, dd, J = 17.2 Hz and

J = 16.8 Hz), 6.51 (1H, d, J = 9.6 Hz), 6.63 (1H, d, J = 8.0 Hz), 6.89

(1H, m), 7.01 (2H, d, J = 8.4 Hz), 7.08 (2H, d, J = 8.4 Hz), 7.12–7.29

(4H, m), 7.88 (1H, d, J = 7.6 Hz); HRMS (m/z): [M+H]⁺calcd for

C₂₆H₂₇FN₂387.2237; found 387.2244; Anal. Calcd for C₂₆H₂₇FN₂:

C, 80.80; H, 7.04; N, 7.25. Found: C, 80.91; H, 7.25; N, 7.17.

4.1.1.30. 2-[1-(4-Isobutylphenyl)ethyl]-1-(pyridin-2-ylmethyl)-

4.1.1.24. 3-({2-[1-(4-Isobutylphenyl)ethyl]-1H-benzimidazol-1-

yl}methyl)benzonitrile (33).

1H-benzimidazole (39).

Yield 54%; mp 100.2 °C. ¹H NMR

Yield 41.5%; mp 142.6 °C. ¹H

(CDCl₃): d 0.84 (6H, d, J = 6.8 Hz), 1.73 (1H, m), 1.83 (3H, d,

J = 6.8 Hz), 2.54 (2H, d, J = 7.2 Hz), 4.29 (1H, q, J = 7.2 Hz), 5.31

(2H, dd, J = 17.2 Hz and J = 17.6 Hz), 6.31 (1H, d, J = 7.6 Hz), 6.95

(2H, d, J = 8.4 Hz), 7.08–7.34 (7H, m), 7.88 (1H, d, J = 8.0 Hz), 8.53

(1H, d, J = 4.8 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₅H₂₇N₃

370.2283. found 370.2297; Anal. Calcd for C₂₅H₂₇N₃: C, 81.26; H,

7.37; N, 11.37. Found: C, 81.25; H, 7.25; N, 11.27.

NMR (CDCl₃): d 0.84 (6H, dd, J = 6.4 Hz and J = 6.4 Hz), 1.77 (1H,

m), 1.84 (3H, d, J = 6.8 Hz), 2.37 (2H, d, J = 6.8 Hz), 4.17 (1H, q,

J = 7.2 Hz), 5.08–5.25 (2H, dd, J = 17.2 Hz and J = 17.2 Hz), 6.97

(2H, d, J = 8.4 Hz), 7.03–7.31 (8H, m), 7.46 (1H, d, J = 8.0 Hz), 7.90

(1H, d, J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₇H₂₇N₃

394.2283; found 394.2293; Anal. Calcd for C₂₇H₂₇N₃: C, 82.41; H,

6.92; N, 10.68. Found: C, 82.55; H, 7.23; N, 10.66.

4.1.1.31. 2-[1-(4-Isobutylphenyl)ethyl]-1-(pyridin-3-ylmethyl)-

4.1.1.25. Methyl

zimidazol-1-yl}methyl)benzoate (34).

3-({2-[1-(4-isobutylphenyl)ethyl]-1H-ben-

Yield 81.1%; mp

1H-benzimidazole (40).

Yield 45%; mp 89.6 °C. ¹H NMR

(CDCl₃): d 0.85 (6H, d, J = 6.4 Hz), 1.78 (1H, m), 1.82 (3H, d,

J = 7.2 Hz), 2.38 (2H, d, J = 6.8 Hz), 4.14 (1H, q, J = 7.2 Hz),

5.04–5.26 (2H, dd, J = 16.8 Hz and J = 16.8 Hz), 6.87–7.30 (9H, m),

7.89 (1H, d, J = 8.0 Hz), 8.36–8.45 (2H, m); HRMS (m/z): [M+H]⁺

calcd for C₂₅H₂₇N₃370.2283. found 370.2265; Anal. Calcd for

87.6 °C. ¹H NMR (CDCl₃): d 0.85 (6H, dd, J = 6.4 Hz and J = 6.4 Hz),

1.76 (1H, m,), 1.82 (3H, d, J = 7.2 Hz), 2.37 (2H, d, J = 6.8 Hz), 3.89

(3H, s), 4.19 (1H, q, J = 6.8 Hz), 5.08–5.25 (2H, dd, J = 16.8 Hz and

J = 17.2 Hz), 6.85 (1H, d, J = 8.0 Hz), 6.99 (2H, d, J = 7.6 Hz), 7.06–

7.29 (7H, m), 7.72 (1H, s), 7.89 (1H, m); HRMS (m/z): [M+H]⁺calcd

C₂₅H₂₇N₃: C, 81.26; H, 7.37; N, 11.37. Found: C, 81.43; H, 7.32; N,

for

C₂₈H₃₀N₂O₂427.2386; found 427.2401; Anal. Calcd for

11.21.

C₂₈H₃₀N₂O₂: C, 78.84; H, 7.09; N, 6.57. Found: C, 78.92; H, 7.18;

N, 6.49.

4.1.1.32. 2-[1-(4-Isobutylphenyl)ethyl]-1-(pyridin-4-ylmethyl)-

1H-benzimidazole (41).

Yield 77.6%; mp 157.5 °C. ¹H NMR

4.1.1.26. 1-(2,4-Dichlorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-

1H-benzimidazole (35).

(CDCl₃): d 0.84 (6H, d, J = 6,4 Hz), 1.75 (1H, m), 1.83 (3H, d,

J = 7.2 Hz), 2.37 (2H, d, J = 6.8 Hz), 4.14 (1H, q, J = 7.2 Hz),

5.01–5.24 (2H, dd, J = 17.6 Hz and J = 17,6 Hz), 6.71 (2H, d,

J = 5.2 Hz), 6.98 (2H, d, J = 7.6 Hz), 7.05–7.31 (5H, m), 7.91 (1H, d,

J = 8.0 Hz), 8.41 (2H, d, J = 6.0 Hz); HRMS (m/z): [M+H]⁺calcd for

Yield 70%; mp 115.1 °C. ¹H NMR

(CDCl₃): d 0.83 (6H, d, J = 6.8 Hz), 1.72 (1H, m), 1.84 (3H, d,

J = 7.2 Hz), 2.34 (2H, d, J = 7.6 Hz), 4.16 (1H, q, J = 7.2 Hz), 5.17–

5.22 (2H, dd, J = 18.0 Hz and J = 18.0 Hz), 6.00 (1H, d, J = 8.4 Hz),

6.82 (1H, dd, J = 8.4 Hz and J = 0.2 Hz), 6.91 (2H, d, J = 8.0 Hz),

7.04 (3H, m), 7.20 (1H, t, J = 8.0 Hz and J = 7.2 Hz), 7.29 (1H, t,

J = 8.0 Hz and J = 7.6 Hz), 7.35 (1H, d, J = 0.2 Hz), 7.90 (1H, d,

J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₆Cl₂N₂437.1551;

found 437.1533; Anal. Calcd for C₂₆H₂₆Cl₂N₂: C, 71.39; H, 5.99; N,

6.40. Found: C, 71.14; H, 5.85; N, 6.54

C₂₅H₂₇N₃370.2283. found 370.2274; Anal. Calcd for C₂₅H₂₇N₃: C,

81.26; H, 7.37; N, 11.37. Found: C, 81.56; H, 7.55; N, 11.08.

4.1.1.33. 2-({2-[1-(4-Isobutylphenyl)ethyl]-1H-benzimidazol-1-

yl}methyl)quinoline (42).

Yield 44.3%; mp 108.4 °C. ¹H NMR

(CDCl₃): d 0.78 (6H, dd, J = 6.8 Hz and J = 6.8 Hz), 1.68 (1H, m), 1.85

(3H, d, J = 7.2 Hz), 2.98 (2H, d, J = 7.6 Hz), 4.36 (1H, q, J = 8.8 Hz),

5.49 (2H, dd, J = 17.2 Hz and J = 17.2 Hz), 6.40 (1H, d, J = 8.4 Hz),

6.89 (2H, d, J = 8.0 Hz), 7.12 (2H, d, J = 8.0 Hz), 7.15–7.29 (3H, m),

7.53 (1H, t, J = 7.2 Hz), 7.70–7.79 (3H, m), 7.90 (1H, d, J = 8.0 Hz),

8.05 (1H, d, J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₉H₂₉N₃

420.2440. found 420.2458; Anal. Calcd for C₂₉H₂₉N₃: C, 83.02; H,

6.97; N, 10.02. Found: C, 83.26; H, 7.16; N, 9.99.

4.1.1.27. 1-(2,6-Dichlorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-

1H-benzimidazole (36).

Yield 47%; mp 143.1 °C. ¹H NMR

(CDCl₃): d 0.88 (6H, d, J = 6.8 Hz), 1.81 (1H, m), 1.89 (3H, d,

J = 6.8 Hz), 2.42 (2H, d, J = 6.8 Hz), 4.58 (1H, q, J = 7.2 Hz), 5.28

(2H, dd, J = 15.6 Hz and J = 15.2 Hz), 6.81 (1H, d, J = 8.0 Hz), 7.01–

7.09 (5H, m), 7.15–7.21 (2H, m), 7.28–7.31 (2H, m), 7.82 (1H, d,

J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₆Cl₂N₂437.1551;

found 437.1548; Anal. Calcd for C₂₆H₂₆Cl₂N₂Á0.15H₂O: C, 70.95;

H, 6.02; N, 6.37. Found: C, 70.86; H, 5.90; N, 6.39.

4.1.1.34. 1-(2-Chlorobenzoyl)-2-[1-(4-isobutylphenyl)ethyl]-1H-

1H-benzimidazole (43).

Yield 52%; mp 93.1 °C. ¹H NMR

(CDCl₃): d 0.83 (6H, d, J = 6.8 Hz), 1.77 (1H, m), 1.84 (3H, d,

J = 7.2 Hz), 2.38 (2H, d, J = 7.2 Hz), 4.95 (1H, m), 6.37 (1H, m),

6.99–7.09 (5H, m), 7.25–7.29 (3H, m), 7.45–7.51 (2H, m), 7.81

4.1.1.28. 1-(3,4-Diethoxybenzyl)-2-[1-(4-isobutylphenyl)ethyl]-

1H-benzimidazole (37).

Yield 48.2%; mp 115.5 °C. ¹H NMR

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

3739

(1H, d, J = 7.6 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₅ClN₂O

417.1734. found 417.1723; Anal. Calcd for C₂₆H₂₅ClN₂O: C, 74.90;

H, 6.04; N, 6.72. Found: C, 74.64; H, 6.17; N, 6.74.

2.37 (2H, d, J = 6.8 Hz), 4.90 (2H, s), 6.33 (1H, d, J = 8.0 Hz), 6.91

(1H, d, J = 8.4 Hz), 6.98 (3H, m), 7.08 (2H, d, J = 8.4 Hz), 7.13–7.30

(4H, m), 7.90 (1H, d, J = 7.6 Hz); HRMS (m/z): [M+H]⁺calcd for

C₂₇H₂₉N₂Cl 417.2098. found 417.2080; Anal. Calcd for C₂₇H₂₉N₂Cl:

4.1.1.35. 1-Benzoyl-2-[1-(4-isobutylphenyl)ethyl]-1H-benzimid-

azole (44).

C, 77.77; H, 7.01; N, 6.72. Found: C, 77.90; H, 7.34; N, 6.64.

Yield 75%; oil. ¹H NMR (CDCl₃): d 0.72 (6H, dd,

J = 6.4 Hz and J = 6.4 Hz), 1.66 (1H, m), 1.86 (3H, d, J = 7.2 Hz),

2.54 (2H, d, J = 7.2 Hz), 4.85 (1H, q, J = 7.2 Hz), 6.52 (1H, d,

J = 8.0 Hz), 6.91 (2H, d, J = 8.4 Hz), 7.01–7.05 (3H, m), 7.25–7.41

(5H, m), 7.57 (1H, m), 7.84 (1H, d, J = 8.4 Hz); HRMS (m/z):

[M+H]⁺calcd for C₂₆H₂₆N₂O 383.2123. found 383.2142; Anal. Calcd

for C₂₆H₂₆N₂O: C, 81.64; H, 6.85; N, 7.32. Found: C, 81.11; H, 6.92;

N, 7.42.

4.1.1.42. 1-(2-Chlorobenzyl)-2-[1-(4-methylphenyl)ethyl]-1H-

benzimidazole (63).

Yield 64%; mp 132.2 °C. ¹H NMR (CDCl₃):

d 1.81 (3H, d, J = 7.2 Hz), 2.23 (3H, s), 4.13 (1H, q, J = 7.0 Hz), 5.22

(2H, m), 6.18 (1H, d, J = 7.4 Hz), 6.92 (1H, t, J = 8.0 Hz), 6.97 (2H, d,

J = 7.6 Hz), 7.06 (2H, d, J = 8.0 Hz), 7.09–7.37 (5H, m), 7.90 (1H, d,

J = 7.8 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₃H₂₁N₂Cl 361.1472.

found 361.1472; Anal. Calcd for C₂₃H₂₁N₂Cl: C, 76.55; H, 5.87; N,

7.76. Found: C, 76.73; H, 5.79; N, 7.68.

4.1.1.36. 1-(3-Chlorobenzoyl)-2-[1-(4-isobutylphenyl)ethyl]-1H-

benzimidazole (45).

Yield 85%; oil. ¹H NMR (CDCl₃): d 0.73

4.1.1.43.

(4-{1-[1-(2-Chlorobenzyl)-1H-benzimidazol-2-

Yield 56%; mp

(6H, dd, J = 6.4 Hz and J = 6.4 Hz), 1.68 (1H, m), 1.86 (3H, d,

J = 6.8 Hz), 2.33 (2H, d, J = 7.6 Hz), 4.82 (1H, q, J = 7.0 Hz), 6.55

(1H, d, J = 8.4 Hz), 6.93 (2H, d, J = 8.0 Hz), 7.01 (2H, d, J = 8.4 Hz),

7.07 (1H, m), 7.22–7.30 (4H, m), 7.53 (1H, m), 7.84 (1H, d,

J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₅ClN₂O 417.1734.

found 417.1739; Anal. Calcd for C₂₆H₂₅ClN₂Oꢀ1.0H₂O: C, 71.79;

H, 6.25; N, 6.44. Found: C, 71.82; H, 6.20; N, 6.52.

yl]ethyl}phenyl)(phenyl)methanone (64).

147.3 °C. ¹H NMR (CDCl₃): d 1.86 (3H, d, J = 7.2 Hz), 4.27 (1H, q,

J = 7.2 Hz), 5.20–5.36 (2H, dd, J = 18.0 Hz and J = 18.0 Hz), 6.14

(1H, d, J = 8.0 Hz), 6.88 (1H, t, J = 7.6 Hz), 7.11–7.66 (14H, m),

7.89 (1H, d, J = 8.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₉H₂₃N₂OCl

451.1577. found 451.1565; Anal. Calcd for C₂₉H₂₃N₂OCl: C, 77.24;

H, 5.14; N, 6.21. Found: C, 77.04; H, 5.04; N, 6.08.

4.1.1.37. 2-({2-[1-(4-Isobutylphenyl)ethyl]-1H-benzimidazol-1-

yl}carbonyl)phenyl acetate (46).

4.1.1.44.

1-(2-Chlorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-5-

Compound 69 (1 equiv)

Yield 91%; oil. ¹H NMR

methoxy-1H-benzimidazole (70).

(CDCl₃): d 0.78 (6H, d, J = 6.8 Hz), 1.72 (1H, m), 1.83 (3H, d,

J = 7.2 Hz), 2.35 (2H, d, J = 7.6 Hz), 4.85 (1H, q, J = 7.0 Hz), 6.66

(1H, d, J = 8.0 Hz), 6.96 (2H, d, J = 8.0 Hz), 7.03–7.27 (6H, m), 7.57

(1H, t, J = 7.6 Hz), 7.80 (1H, d, J = 8.4 Hz); HRMS (m/z): [M+H]⁺calcd

was reﬂuxed in 25 mL acetic acid for 4 h. The reaction mixture

was evaporated in vacuo, and dissolved in 50 mL DCM. The organic

phase was extracted with 5% NaHCO₃(50 mL Â 2) and brine

(50 mL x 2). Combined organic phases were dried over anhydrous

Na₂SO₄, ﬁltered and evaporated to dryness. The oily residue was

puriﬁed by automated ﬂash chromatography. Yield 77%; oil. ¹H

NMR (CDCl₃): d 0.83 (6H, d, J = 6.8 Hz), 1.74 (1H, m), 1.83 (3H, d,

J = 6.8 Hz), 2.35 (2H, d, J = 6.8 Hz), 3.87 (3H, s), 4.13 (1H, q,

J = 8.8 Hz), 5.15–5.25 (2H, dd, J = 18.0 Hz and J = 18.0 Hz), 6.17

(1H, d, J = 8.0 Hz), 6.82–7.15 (8H, m), 7.35 (1H, m), 7.38 (1H, d,

J = 2.4 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₇H₂₉N₂OCl 433.2047;

found 433.2049; Anal. Calcd for C₂₇H₂₉N₂OClꢀ0.4H₂O: C, 73.67;

H, 6.82; N, 6.36. Found: C, 73.57; H, 7.12; N, 6.40.

for

C₂₈H₂₈N₂O₃441.2178. found 441.2173; Anal. Calcd for

C₂₈H₂₈N₂O₃: C, 76.34; H, 6.41; N, 6.36. Found: C, 76.19; H, 6.65;

N, 6.32.

4.1.1.38. Methyl 2-[1-(4-isobutylphenyl)ethyl]-1H-benzimid-

azole-1-carboxylate (47).

Yield 33%; mp 103.5 °C. ¹H NMR

(CDCl₃): d 0.87 (6H, d, J = 6.6 Hz), 1.81 (1H, m), 1.85 (3H, d,

J = 6.8 Hz), 2.41 (2H, d, J = 7.2 Hz), 3.56 (3H, s), 4.24 (1H, q,

J = 7.2 Hz), 4.65 (2H, dd, J = 18.0 Hz and J = 18.0 Hz), 7.04 (2H, d,

J = 8.4 Hz), 7.09 (2H, d, J = 8.4 Hz), 7.14–7.30 (3H, m), 7.86 (1H,

m); HRMS (m/z): [M+H]⁺calcd for C₂₂H₂₆N₂O₂351.2073. found

351.2063; Anal. Calcd for C₂₂H₂₆N₂O₂: C, 75.40; H, 7.48; N, 7.99.

Found: C, 75.19; H, 7.62; N, 7.74.

4.1.1.45. 1-(2-Chlorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-1H-

benzimidazol-5-ol (71).

Compound 70 (1 equiv), 48% HBr

(66 equiv) in 2 mL acetic acid was reﬂuxed for 2 h. The reaction

mixture was diluted by distilled water, extracted with EtOAc

(60 mL Â 2). The organic phase was dried over Na₂SO₄, ﬁltered,

evaporated in vacuo, and the ﬁnal product was puriﬁed by

automated ﬂash chromatography. Yield 60.3%; mp 213.2 °C. ¹H

NMR (CDCl₃): d 0.83 (6H, dd, J = 6.8 Hz and J = 6.4 Hz), 1.74 (1H,

m), 1.80 (3H, d, J = 6.8 Hz), 2.23 (2H, d, J = 6.8 Hz), 4.13 (1H, m),

5.19 (2H, m), 6.20 (1H, d, J = 7.6 Hz), 6.89 (1H, m), 6.90 (2H, d,

J = 8.4 Hz), 6.94 (2H, d, J = 8.0 Hz), 7.08 (2H, d, J = 8.4 Hz), 7.14 (1H,

m), 7.34–7.39 (2H, m); HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₇ClN₂O

419.1890. Found 419.1878; Anal. Calcd for C₂₆H₂₇ClN₂O: C, 74.54;

H, 6.50; N, 6.69. Found: C, 74.01; H, 6.58; N, 6.66.

4.1.1.39.

carboxylic acid (48).

2-[1-(4-Isobutylphenyl)ethyl]-1H-benzimidazole-1-

Yield 58.2%; mp 173.8 °C. ¹H NMR

(CDCl₃): d 0.83 (6H, d, J = 6.8 Hz), 1.61 (3H, d, J = 6.8 Hz), 1.76

(1H, m), 2.34 (2H, d, J = 7.2 Hz), 4.39 (1H, q, J = 6.8 Hz), 4.63 (2H,

dd, J = 17.6 Hz and J = 17.2 Hz), 6.98 (2H, d, J = 8.0 Hz), 7.05 (2H,

d, J = 8.0 Hz), 7.21–7.29 (3H, m), 7.73 (1H, m); HRMS (m/z):

[M+H]⁺calcd for C₂₁H₂₄N₂O₂337.1916. found 337.1916; Anal.

Calcd for C₂₁H₂₄N₂O₂: C, 74.97; H, 7.19; N, 8.33. Found: C, 74.90;

H, 7.04; N, 8.13.

4.1.1.40. 1-(2-Chlorobenzyl)-2-(4-isobutylbenzyl)-1H-benzimid-

azole (55).

Yield 34%; mp 100.1 °C. ¹H NMR (CDCl₃): d 0.84

4.1.1.46.

1-(2-Chlorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-5-

To a solu-

(6H, d, J = 6.8 Hz), 1.76 (1H, m), 2.36 (2H, d, J = 7.6 Hz), 4.21 (2H,

s), 5.31 (2H, s), 6.24 (1H, d, J = 7.2 Hz), 6.92–7.26 (9H, m), 7.37

(1H, d, J = 8.0 Hz), 7.88 (1H, d, J = 8.0 Hz); HRMS (m/z): [M+H]⁺

calcd for C₂₅H₂₅N₂Cl 389.1785. found 389.1797; Anal. Calcd for

(pyridin-2-ylmethoxy)-1H-benzimidazole (72).

tion of compound 71 (1 equiv) in DMF was added Cs₂CO₃(4 equiv)

followed by 2-picolylchloride (1 equiv) and TBAI (0.06 equiv). The

mixture was heated overnight under N₂then diluted with water

and extracted with EtOAc (3 Â 50 ml). The organic phase was dried

(Na₂SO₄) and concentrated. Puriﬁcation of the residue by ﬂash

chromatography provided the title compound. Yield 60%; mp

128.6 °C. ¹H NMR (CDCl₃): d 0.83 (6H, d, J = 6.6 Hz), 1.74 (1H, m),

1.79 (3H, d, J = 7.2 Hz), 2.34 (2H, d, J = 6.8 Hz), 4.12 (1H, q,

J = 6.8 Hz), 5.20 (2H, m), 5.27 (2H, s), 6.19 (1H, d, J = 7.4 Hz),

C₂₅H₂₅N₂Cl: C, 77.20; H, 6.48; N, 7.20. Found: C, 77.57; H, 6.59;

N, 7.15.

4.1.1.41. 1-(2-Chlorobenzyl)-2-[1-(4-isobutylphenyl)-1-methyl-

ethyl]-1H-benzimidazole (56).

NMR (CDCl₃): d 0.84 (6H, d, J = 6.4 Hz), 1.76 (1H, m), 1.78 (6H, s),

Yield 70%; mp 164.2 °C. ¹H

3740

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

6.89–6.99 (5H, m), 7.07 (2H, d, J = 8.0 Hz), 7.13–7.22 (2H, m), 7.35

(1H, d, J = 7.6 Hz), 7.45 (1H, d, J = 8.0 Hz), 7.57 (1H, d, J = 7.6 Hz),

7.71 (1H, m), 8.62 (1H, d, J = 5.6 Hz); HRMS (m/z): [M+H]⁺calcd

queries, the Ph4 consensus module of MOE, which calculates a con-

sensus query consisting of most conserved features of a given

alignment, was used. The ﬁnal query was adjusted manually to

optimize the hit rate of true positives. A volume constraint was

added to further constrain the search.

for

C₃₂H₃₂ClN₃O 510.2312; found 510.2307; Anal. Calcd for

C₂₆H₂₇ClN₂O: C, 75.35; H, 6.32; N, 8.24. Found: C, 75.35; H, 6.65;

N, 8.09.

4.2.3. Virtual screening

4.1.1.47. 2-[({1-(2-Chlorobenzyl)-2-[1-(4-isobutylphenyl)ethyl]-

A presampled collection of 2.8 mio vendor compounds (153 mio

conformations) was obtained from CCG libraries. The 3D search

with MOE was done using the pharmacophore model described

above. Only compounds that matched all the features of the query

were retained as hits.

1H-benzimidazol-5-yl}oxy)methyl]quinoline (73).

The title

compound was synthesized by the same procedure for 72 starting

from 2-(chloromethyl)quinoline. Yield 57%; mp 86.7 °C. ¹H NMR

(CDCl₃): d 0.83 (6H, dd, J = 6.8 Hz and J = 6.8 Hz), 1.74–1.79 (4H,

m), 2.34 (2H, d, J = 7.2 Hz), 4.11 (1H, q, J = 7.0 Hz), 5.20 (2H, m),

5.45 (2H, s), 6.19 (1H, d, J = 7.2 Hz), 6.89–6.7.15 (8H, m), 7.35

(1H, d, J = 7.6 Hz), 7.48–7.55 (2H, m), 7.74 (2H, m), 7.82 (1H, d,

J = 8.4 Hz), 8.11 (1H, d, J = 8.4 Hz), 8.20 (1H, d, J = 8.4 Hz); HRMS

(m/z): [M+H]⁺calcd for C₃₆H₃₄ClN₃O 560.2469. Found 560.2476;

Anal. Calcd for C₂₆H₂₇ClN₂Oꢀ0.7H₂O: C, 75.49; H, 6.22; N, 7.33.

Found: C, 75.32; H, 6.07; N, 7.15.

4.2.4. Postprocessing ﬁltering

The hits retrieved from the chemical databases were ﬁltered

using molecular docking and protein-ligand interaction ﬁnger-

prints (PLIFs) using MOE. The FLAP crystal structure in complex

with compound 85 (PDB code: 2Q7M) was used and the chains B

and C were selected for docking studies. For the ﬁrst docking step,

pharmacophore placement (six-point matching while the excluded

volume is ignored) and Alpha HB were used as scoring functions.

Then, the raw poses obtained from the ﬁrst step were submitted

to the second step in which FF reﬁnement (receptor ﬂexible, tether

weight = 6) and rescoring function Afﬁnity dG were used.

GOLD 5.0.1 was also used to conﬁrm binding modes of the syn-

thesized compounds. The active site was deﬁned with 10 Å radius

around the bound inhibitor 85. Top 10 scored conformation of

every ligand was allowed to be saved at the end of the calculation.

The early termination option was used to skip the genetic optimi-

zation calculation when any ﬁve conformations of a particular

compound predicted within the RMSD value of 1.5 Å. Gold score

was tested as ﬁtness function. The docking poses for each com-

pound were stored in a MOE database and further processed.

The interaction ﬁngerprint of a protein-ligand complex was cal-

culated by the presence or absence of six types of intermolecular

interactions: hydrogen bond with side chain donor, hydrogen bond

with side chain acceptor, hydrogen bond with backbone donor,

hydrogen bond with backbone acceptor, ionic attraction, and sur-

face contact. The resulting ﬁngerprints were extracted from MOE

for the analysis.

4.1.1.48. 1-Benzyl-5-chloro-2-[1-(4-isobutylphenyl)ethyl]-1H-

benzimidazole (81).

The title compound was synthesized

by the same procedure for 70 starting from compound 79. Yield

52%; oil. ¹H NMR (CDCl₃): d 0.86 (6H, d, J = 6.8 Hz), 1.78–1.81

(4H, m), 2.40 (2H, d, J = 7.6 Hz), 4.17 (1H, q, J = 7.6 Hz), 4.99–5.20

(2H, dd, J = 16.8 Hz and J = 16.8 Hz), 6.82–7.26 (11H, m), 7.84

(1H, d, J = 2.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₇N₂Cl

403.1941. found 403.1937; Anal. Calcd for C₂₆H₂₇N₂Cl: C, 77.50;

H, 6.75; N, 6.95. Found: C, 77.54; H, 6.76; N, 6.86.

4.1.1.49.

5-Chloro-1-(2-chlorobenzyl)-2-[1-(4-isobutyl-

The title compound

phenyl)ethyl]-1H-benzimidazole (82).

was synthesized by the same procedure for 70 starting from com-

pound 80. Yield 35%; mp 107.5 °C. ¹H NMR (CDCl₃): d 0.86 (6H, dd,

J = 2.0 Hz and J = 2.4 Hz), 1.74 (1H, m), 1.79 (3H, d, J = 6.8 Hz), 2.34

(2H, d, J = 7.2 Hz), 4.15 (1H, q, J = 6.8 Hz), 5.21 (2H, m), 6.12 (1H, d,

J = 8.0 Hz), 6.89–7.15 (8H, m), 7.35 (1H, d, J = 8.0 Hz), 7.85 (1H, d,

J = 2.0 Hz); HRMS (m/z): [M+H]⁺calcd for C₂₆H₂₆N₂Cl₂437.1551;

found 437.1562; Anal. Calcd for C₂₆H₂₆N₂Cl₂: C, 71.39; H, 5.99; N,

6.40. Found: C, 77.71; H, 6.03; N, 6.59.

4.2. Molecular modelling studies

4.3. Assay systems

4.2.1. Computational approach

4.3.1. Materials

The energy calculation, pharmacophore search and docking

studies for virtual screening were performed using the MOE

2010.10 suite (Chemical Computing Group, CCG). The 3D struc-

tures were generated in the protonated state assuming physiolog-

ical conditions and energy minimized using MOE to a RMSD

gradient of 0.05 kcal/mol Å with MMFF94Â forceﬁeld. Partial

charges were automatically assigned. The molecular docking pro-

gram GOLD 5.0.1 (Cambridge Crystallographic Data Centre, CCDC)

was also used to conﬁrm the binding modes of the synthesized

compounds. Ligand receptor interactions were analyzed using the

Ligand Interaction module available in MOE and visualizations

were performed using Pymol (DeLano Scientiﬁc).

Arachidonic acid, Ca²⁺ionophore A23187, and all other ﬁne

chemicals were from Sigma (Deisenhofen, Germany), unless stated

otherwise. HPLC solvents were from Merck (Darmstadt, Germany).

4.3.2. Cells

Human neutrophils were freshly isolated from leukocyte con-

centrates obtained at the Blood Center of the University Hospital

Tuebingen (Germany) or 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 cen-

trifugation at 4,000Âg for 20 min at 20 °C. Neutrophils were imme-

diately isolated by dextran sedimentation, centrifugation on

Nycoprep cushions (PAA Laboratories, Linz, Austria), and hypotonic

lysis of erythrocytes as described previously.³⁰Cells were ﬁnally

resuspended in phosphate-buffered saline pH 7.4 (PBS) containing

1 mg/mL glucose and 1 mM CaCl₂(PGC buffer).

4.2.2. Pharmacophore model

The alignment was produced using a slightly relaxed represen-

tation of the X-ray ligand with MOE Align. For those known FLAP

inhibitors containing chiral centers with undetermined stereo-

chemistry, all possible isomers were enumerated and the collec-

tion of the lowest energy conformers aligned onto X-ray ligand

pose. The minimization protocol applied decreasing ﬂat bottom

constraints to retain as much of the original geometry as possible

while removing artiﬁcial strain. To generate the pharmacophore

4.3.3. Determination of 5-LO product formation in cell-based

assays

For assays of intact cells, 5 Â 10⁶freshly isolated neutrophils

were resuspended in 1 mL PGC buffer. After preincubation with

E. Banoglu et al. / Bioorg. Med. Chem. 20 (2012) 3728–3741

3741

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started by addition of 2.5 M A23187. Treatment with 2.5

A23187 for 10 min appeared not toxic for neutrophils as deter-

mined by trypan blue staining and light microscopy. After 10 min

at 37 °C, the reaction was stopped with 1 mL of methanol and

l

lM

30 lL of 1 N HCl, 200 ng prostaglandin B₁and 500 lL of PBS were

added. Formed 5-LO metabolites were extracted and analyzed by

HPLC as described.³⁰5-LO product formation is expressed as ng

of 5-LO products per 10⁶cells, which includes LTB₄and its all-trans

isomers, and 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetrae-

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from E. coli and determination of 5-LO activity in cell-free

systems

E. coli BL21 was transformed with pT3–5LO plasmid, recombi-

nant 5-LO protein was expressed at 37 °C, and 40,000Âg superna-

tants were prepared as described.³¹For determination of 5-LO

activity in neutrophil homogenates, 5 Â 10⁶freshly isolated neu-

trophils were resuspended in 1 mL of PBS containing 1 mM EDTA

and sonicated (3 Â 10 s). Aliquots of the E. coli lysate supernatants

(corresponding to 2 mL E. coli cell culture) were diluted with PBS/

EDTA plus 1 mM ATP to 1 mL and pre-incubated with the test com-

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This study was supported by The Scientiﬁc and Technological

Research Council of Turkey (TÜBITAK No. 108S210). We want to

thank Bianca Jazzar for expert technical assistance.

_

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Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.048.

References and notes

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Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase- activating protein (FLAP)

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