Discovery and biological evaluation of novel 1,4-benzoquinone and related resorcinol derivatives that inhibit 5-lipoxygenase

Article abstract of DOI:10.1016/j.ejmech.2013.06.039

5-Lipoxygenase (5-LO), an enzyme that catalyzes the initial steps in the biosynthesis of pro-inflammatory leukotrienes, is an attractive drug target for the pharmacotherapy of inflammatory and allergic diseases. Here, we present the discovery and biological evaluation of novel series of 1,4-benzoquinones and respective resorcinol derivatives that efficiently inhibit human 5-LO, with little effects on other human lipoxygenases. SAR analysis revealed that the potency of the compounds strongly depends on structural features of the lipophilic residues, where bulky naphthyl or dibenzofuran moieties favor 5-LO inhibition. Among the 1,4-benzoquinones, compound Ig 5-[(2-naphthyl)methyl]-2- hydroxy-2,5-cyclohexadiene-1,4-dione potently blocked 5-LO activity in cell-free assays with IC₅₀ = 0.78 μM, and suppressed 5-LO product synthesis in polymorphonuclear leukocytes with IC₅₀ = 2.3 mM. Molecular docking studies suggest a concrete binding site for Ig in 5-LO where select π-π interactions along with hydrogen bond interactions accomplish binding to the active site of the enzyme. Together, our study reveals novel valuable 5-LO inhibitors with potential for further preclinical assessment as anti-inflammatory compounds.

Full text of DOI:10.1016/j.ejmech.2013.06.039

European Journal of Medicinal Chemistry 67 (2013) 269e279
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery and biological evaluation of novel 1,4-benzoquinone and
related resorcinol derivatives that inhibit 5-lipoxygenase
,
a
,
,
,1
Rosanna Filosa a , Antonella Peduto 1, Polamarasetty Aparoy b 1, Anja M. Schaible c
**
,
Susann Luderer d, Verena Krauth c, Carmen Petronzi e, Antonio Massa e, Mario de Rosa f,
Pallu Reddanna g h, Oliver Werz c
,
,
*
a Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Via Ponte don Melillo, 84084 Fisciano, SA, Italy
b Centre for Computational Biology and Bioinformatics, School of Life Sciences, Central University of Himachal Pradesh, Dharamshala, India
c Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, D-07743 Jena, Germany
d Department of Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8,
D-72076 Tuebingen, Germany
e Department of Chemistry, University of Salerno, Via Ponte don Melillo, 84084 Fisciano, SA, Italy
f Department of Experimental Medicine, University of Naples, Via Costantinopoli, 16, 80138 Naples, Italy
g School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India
h National Institute of Animal Biotechnology, Hyderabad 500046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Lipoxygenase (5-LO), an enzyme that catalyzes the initial steps in the biosynthesis of pro-inflammatory
leukotrienes, is an attractive drug target for the pharmacotherapy of inflammatory and allergic diseases.
Here, we present the discovery and biological evaluation of novel series of 1,4-benzoquinones and
respective resorcinol derivatives that efficiently inhibit human 5-LO, with little effects on other human
lipoxygenases. SAR analysis revealed that the potency of the compounds strongly depends on structural
features of the lipophilic residues, where bulky naphthyl or dibenzofuran moieties favor 5-LO inhibition.
Among the 1,4-benzoquinones, compound Ig 5-[(2-naphthyl)methyl]-2-hydroxy-2,5-cyclohexadiene-
Received 9 February 2013
Received in revised form
14 June 2013
Accepted 15 June 2013
Available online 27 June 2013
Keywords:
5-Lipoxygenase
Leukotriene
Molecular docking
Benzoquinone
Resorcinol
1,4-dione potently blocked 5-LO activity in cell-free assays with IC50 ¼ 0.78
product synthesis in polymorphonuclear leukocytes with IC50 ¼ 2.3 M. Molecular docking studies
suggest a concrete binding site for Ig in 5-LO where select interactions along with hydrogen bond
mM, and suppressed 5-LO
m
pep
interactions accomplish binding to the active site of the enzyme. Together, our study reveals novel
valuable 5-LO inhibitors with potential for further preclinical assessment as anti-inflammatory
compounds.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
is then metabolized by LTA4 hydrolase into the chemotactic LTB4 or
by LTC4 synthase into the cysteinyl (cys)-LTs C4, D4 and E4 which
Leukotrienes (LTs) represent potent lipid mediators that play key
roles in allergic and inflammatory processes and contribute to
cardiovascular diseases (CVD) and various types of cancer [1,2]. Upon
release of arachidonic acid (AA) from phospholipids of cellular
membranes by cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase
(5-LO) converts AA into LTA4 by the aid of the 5-LO-activating
protein (FLAP) that facilitates the transfer of AA toward 5-LO [3]. LTA4
evoke bronchoconstriction and increase vascular permeability [1].
Both, LTB4 and the cys-LTs mediate their biological effects essentially
via selective G protein-coupled receptors (at least two for LTB4 and
more than three for cys-LTs) [4]. Because of the crucial roles of LTs in
pathophysiology, pharmacological strategies have been developed in
order to intervene with LTs. While 5-LO inhibitors and FLAP antag-
onists are used to inhibit the synthesis of LTs, the so-called cys-LT
receptor antagonists prevent the biological actions of cys-LTs [5]. The
5-LO inhibitors are traditionally classified according to their molec-
ular mode of action as redox-type, iron ligand-type, and non redox-
type inhibitors. However, there are currently more and more com-
pounds described that potently inhibit cellular LT synthesis by
different modes of actions such as interference with the regulatory
* Corresponding author. Tel.: þ49 03641 949801; fax: þ49 03641 949802.
** Corresponding author. Tel.: þ39 089969398; fax: þ39 089969602.
E-mail addresses: rfilosa@unisa.it (R. Filosa), oliver.werz@uni-jena.de (O. Werz).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.039

270
R. Filosa et al. / European Journal of Medicinal Chemistry 67 (2013) 269e279
O
H3CO
H3CO
H3CO
H3CO
OCH3
OCH3
H3CO
H3CO
OH
H
i
ii
iii
OCH3
OCH3
1
2
3
Br
R
R
H3CO
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
OH
O
O
iv
v
H3CO
H3CO
Ia-b
6a-b
4
Scheme 1. Synthesis of 2-hydroxy-5-methoxy-1,4-benzoquinones Iaeb. Reagents and conditions: (i) H2O2 30%, H2SO4, MeOH, rt, 2 h; (ii) CH3I, K2CO3, CH3COCH3, 65 ꢀC, 18 h; (iii) H2CO,
HBr 30%, CH3COOH, 55 ꢀC, 6 h; (iv) phenylboronic acid (5a) or naphthalen-2-yl-2-boronic acid (5b), Pd(OAc)2, PPh3, tBuOK, toluene, 110 ꢀC, 18 h; (v) CAN, H2O, CH3CN, rt, 3 h.
C2-like domain of 5-LO or by interference with 5-LO-stimulating
factors or mechanisms in the cell other than FLAP [6,7]. Although
much more efforts have been made in order to develop LT synthesis
inhibitors, the LT antagonists dominate on the market and thus far
only zileuton [8], an iron ligand-type inhibitor, has been approved for
anti-LT therapy.
Polyphenols are widely distributed in nature, and many
studies revealed that they constitute a rich source of inhibitors of
5-LO product synthesis [9]. Mechanistically, they may act as
antioxidants thereby keeping the active site iron of 5-LO in the
inactive ferrous state, even though the hydroxy groups might
also contribute to 5-LO inhibition by iron-chelating properties
O
HO
HO
OH
OH
HO
O
O
O
O
O
O
i
ii
+
R
H
OH
10c: R = Cyclohexyl
10d: R = 2-Decahydronaphthalenyl
9
8
7
S
S
R
OH
R
O
O
O
O
O
O
O
O
iii
iv
v
O
12c-d
11c-d
R
R
HO
O
O
O
O
O
O
vi
OH
13c-d
Ic-d
OCH3
O
O
H
H
viii
vii
14
15
10d
Scheme 2. Synthesis of 2,5-dihydroxy-1,4-benzoquinones Iced. Reagents and conditions: (i) Sn, HCl (37%), THF, 100 ꢀC, 1 h; (ii) C4H8O, PPTS, C6H6, 80 ꢀC, 24 h; (iii) n-BuLi, THF,
from ꢁ10 ꢀC to rt, 16 h; (iv) NaHDMS, CS2, CH3I, THF, before ꢁ78 ꢀC, after ꢁ55 ꢀC, then 0 ꢀC, 3.5 h; (v) n-Bu3SnH, AIBN, toluene, from rt to 110 ꢀC, 2.5 h; (vi) HCl 4 M, dioxane, air,
110 ꢀC, 7 h; (vii) MMTPPC, n-BuLi, diethyl ether, from ꢁ40 ꢀC to rt, 30 h; (viii) HClO4 70%, H2O, diethyl ether, reflux, 1 h.

R. Filosa et al. / European Journal of Medicinal Chemistry 67 (2013) 269e279
271
O
OCH3
OH
ii
i
H3CO
H3CO
17
16
O
O
Br
iii
iv
H3CO
H3CO
H3CO
OCH3
18
20
O
O
O
v
HO
HO
HO
OH
O
OH
O
O
21
Ih
Scheme 3. Synthesis of 2-hydroxy-1,4-benzoquinone Ih. Reagents and conditions: (i) DIBALH 1 M, THF, 0 ꢀC, 30 min; (ii) PBr3, C6H6, from 0 ꢀC to rt, 1 h; (iii) 2,4-dimethoxybenzen
boronic acid (19), Pd(OAc)2, PPh3, K3PO4, C7H8, 110 ꢀC, 18 h; (iv) BBr3, DCM, from ꢁ78 ꢀC to rt, 1.5 h; (v) K2NO(SO3)2, Na2CO3 (15%), THF, rt, 3 h.
[10]. Natural compounds and synthetic derivatives possessing a
1,4-benzoquinone moiety are associated with a wide range of
biological properties, including antioxidant, anti-inflammatory
and anti-cancer activities [11]. The anti-inflammatory activity of
1,4-benzoquinones has been associated with suppression of LT
formation [9]. In the cell, the 1,4-benzoquinone moiety can be
reduced to a 1,4-diphenol structure (¼1,4-hydroquinone) due to
the reducing intracellular milieu (e.g. presence of glutathione in
the millimolar range). Such 1,4-diphenols as well as other
polyphenols have been assumed to reduce the active site iron of
5-LO, thereby inhibiting 5-LO activity primarily due to antioxi-
dant properties [12,13]. However, it was also shown that the
potency of 1,4-diphenols and polyphenols does not solely depend
on the reducing properties but instead the structural features
and the degree of lipophilicity are determinants [9,14]. Only few
reports are available that addressed this issue in detail and
respective structureeactivity relationship (SAR) studies are rare.
Therefore, the rational of our study was to provide insights into
SARs with focus on the lipophilic residue at the phenol core.
Here we present the design and synthesis of novel 1,4-
benzoquinones and corresponding resorcinol derivatives that
depending on the overall structure, proved to be potent inhibitors
of human 5-LO in intact cells as well as in cell-free assays, whereas
others inhibited 5-LO surprisingly only in the cell-free test system.
Our data shed light on the SARs related to the lipophilic moieties
connected to the 1,4-benzoquinone or resorcinol backbone,
respectively that determine inhibition of 5-LO without significant
interference with human 12- and 15-LOs. Results from molecular
docking studies that were carried out to investigate their binding
interactions with 5-LO, revealed a correlation between the capacity
to bind and to inhibit 5-LO.
2. Results and discussion
2.1. Chemistry
Compounds
Iaeb
were
synthesized
starting
from
2,4,5-trimethoxyphenol (2) [15] (Scheme 1). The phenol 2 was
methylated to provide 1,2,4,5-tetramethoxybenzene 3, which was
converted into 3-(bromomethyl)-1,2,4,5-tetramethoxybenzene (4)
[16]. Suzuki coupling with commerciallyavailable phenylboronic acid
(5a) and naphthalen-2-yl-2-boronic acid (5b) yielded 6aeb. Oxida-
tion with ammonium cerium nitrate gave desired Iaeb (structures
identified by 1H NMR and confirmed by gCOSY analysis). Compound
H3CO
OCH3
H3CO
OCH3
HO
OH
O
HO
O
i
iii
ii
I
R
R
IIe: R = 4-dibenzofuranyl
IIh:R = 3-benzo[b]thiophenyl
24a: R = 4-dibenzofuranyl
24b: R = 3-benzo[b]thiophenyl
Ii
22
O
Scheme 4. Synthesis of IIe, IIh and Ii. Reagents and conditions: (i) dibenzofuran-4-boronic acid (23a) or benzo[b]thiophen-3-yl-3-boronic acid (23b), K2CO3, PdCl2(dppf), Ethanol,
MW, 25 min; (ii) BBr3, CH2Cl2, ꢁ15 ꢀC to rt, 18 h; (iii) NO(KSO3)2, EtOAceH2O, Na2HPO4, 0 ꢀC to rt, 20 h.

272
R. Filosa et al. / European Journal of Medicinal Chemistry 67 (2013) 269e279
H3CO
OCH3
H3CO
OCH3
HO
OH
O
i
ii
B(OH)2
O
25
27
IIi
Scheme 5. Synthesis of IIi. Reagents and conditions: (i) 2-(naphthalen-6-yl)acetic acid (26), ZnCl2, POCl3, 80 ꢀC, 2h; (ii) BBr3, CH2Cl2, ꢁ15 ꢀC to rt, 18 h.
Id was synthesized according to Kim et al. [17] (Scheme 2).
Briefly, diacetonide 9, obtained from treatment of 1,2,4,5-
tetrahydroxybenzene 8 [18] with 2-methoxypropene, was reacted
with appropriate aldehydes 10ced to provide the corresponding al-
cohols 11ced. They were first converted into xanthate derivatives
12ced, according to the BartoneMcCombie reaction [19]; then
treatment with tributyltin hydride and 2,20-azobisisobutyronitrile
afforded compounds 13ced. Final oxidation provided the
2,5-dihydroxy-1,4-benzoquinones Iced. Decahydronaphthalene-2-
carbaldehyde (10d), prepared from octahydronaphthalen-2(1H)-
one (14) as mixture of stereoisomers, was subjected to Wittig
reaction yielding decahydro-2-(methoxymethylene) naphthalene
(15). Treatment with perchloric acid 70% (v/v) in diethyl ether pro-
vided 10d. The 2-hydroxy-1,4-benzoquinones Ieeg and Ilem were
synthesized according to Ref. [20].
The synthesis of compound Ih (Scheme 3) started from dimethyl
naphthalene-2,6-dicarboxylate (16), which was mono reduced to
provide the corresponding alcohol 17 [21] that, after treatment
with PBr3, furnished methyl 6-(bromomethyl)-2-naphthoate (18).
Suzuki coupling with 2,4-dimethoxybenzen boronic acid (19) led to
intermediate 20, which was demethylated and hydrolyzed simul-
taneously with BBr3 and finally oxidized to Ih using Fremy’s salt.
Compounds Ii, IIe and IIh were obtained by Suzuki coupling
between 1-iodo-2,4-dimethoxybenzene (22) and appropriate
boronic acids (23a and 23b), under microwave irradiation. In-
termediates 24a and 24b were O-demethylated to provide resor-
cinol derivatives (IIe and IIh). IIe was finally oxidized with Fremy’s
salt yielding Ii (Scheme 4). The resorcinol derivatives IIbed and
IIfeg were synthesized as previously reported [20]. Compound IIa
was synthesized based on a modified procedure [22] using micro-
wave irradiation for reaction of Suzuki coupling. Compound IIi was
obtained via FriedeleCrafts acylation of 1,3-dimethoxybenzene
boronic acid (25) with 2-(naphthalen-6-yl)acetic acid (26) and
subsequent demethylation with BBr3 (Scheme 5).
2.2. Evaluation of 5-LO activity and structureeactivity relationships
In order to assess the effects of the test compounds on 5-LO
product synthesis, a cell-free assay using isolated human recombi-
nant 5-LO and a cell-based test system using intact human poly-
morphonuclear leukocytes (PMNL) were applied. While the cell-free
assay allows the identification of compounds that directly interfere
with 5-LO catalytic activity (without the need of bioactivation), the
cell-based test system involves various aspects and steps that are
required for 5-LO product synthesis, and as such offers several tar-
gets within the 5-LO activation and reaction cascade (e.g., cPLA2,
FLAP or coactosine-like protein (CLP), 5-LO-activating lipid hydro-
peroxides, protein kinases or Ca2þ mobilization, and 5-LO
Table 1A
2,3,5-Trisubstituted 1,4-benzoquinone derivatives: chemical structures and effects on the activity of 5-LO. Data are given as mean ꢂ S.E.M., n ¼ 3e4.
Cpd
5-LO activity; cell-free
5-LO activity; cell-based
R
R1
Remaining activity at 10
109.6 ꢂ 5.1
m
M (%)
IC50
(
m
M)
Remaining activity at
10 M (%)
IC50
>10
>10
>10
(mM)
m
Ia
Ib
CH3
CH3
> 10
> 10
> 10
103.3 ꢂ 12.2
56.1 ꢂ 7.4
92.3 ꢂ 16.3
Ic
H
H
95.2 ꢂ 15.8
51.0 ꢂ 8.9
90.4 ꢂ 12.5
5.4 ꢂ 2.6
Id
11 ꢂ 5.4
0.58 ꢂ 0.2

R. Filosa et al. / European Journal of Medicinal Chemistry 67 (2013) 269e279
273
translocation/membrane association) for a given compound in order
to suppress 5-LO product generation [23]. Moreover, compounds
that may require bioactivation (as in the case of quinones) in order
to be able to inhibit 5-LO can be identified using the combination of
these two test systems. The reference 5-LO inhibitors 29 ((E)-N-
hydroxy-N-(3-(3-phenoxyphenyl)-allyl)acetamide; BWA4C [24])
and 30 (N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea; zileuton
[8]) were used to control the 5-LO activity assays that blocked 5-LO
By removing one hydroxyl group at the 1,4-benzoquinone core
and shifting the substituent R1 from position 3 to position 2 (Ieem),
we obtained compounds that (except If) directly inhibited 5-LO in
the cell-free assay with IC50 values in the low micromolar or even
submicromolar range (Table 1B). Under the assay conditions of the
cell-free test system, a reduction of the quinone moiety to the hy-
droquinone is rather unlikely. Thus, the unexpected finding that the
quinone form of Ie, gem inhibits 5-LO directly in this cell-free assay
is interesting and might be related to other mechanisms than
simple reduction of the active site iron by the diphenol core as
generally proposed for quinone-type 5-LO inhibitors with antioxi-
dant activity [12e14]. In fact, as shown below, molecular docking
and SAR experiments reveal a correlation between the number
of favorable interactions with 5-LO and inhibitory potency. Com-
pounds Ig and Im, decorated with a naphthylmethyl or a naph-
thoxy residue, turned out to be the most potent inhibitors of 5-LO of
activity with IC50 of 0.22 and 0.56
1.1 M in the cell-based assay, respectively.
As shown in Table 1A, no remarkable inhibition of 5-LO by the 2-
hydroxy-5-methoxy-1,4-benzoquinones (Ia and Ib) in either assay
type was observed (IC50 > 10 M). Also the 2,5-dihydroxy deriva-
mM in the cell-free and 0.3 and
m
m
tive Ic, bearing a lipophilic cyclohexyl ring, remained inactive.
However, replacement of this cyclohexyl by the more hindered
bicyclic decahydronaphthyl moiety led to compound Id with a
moderate potency in the cell-free assay (IC50 ¼ 11
compound potently suppressed 5-LO product synthesis in intact
M. Comparison of Id with inactive Ic dem-
onstrates that structural features and/or size of the lipophilic res-
idue at the 1,4-benzoquinone core strongly determine the
effectiveness for inhibition of 5-LO.
m
M), but this
this particular series with IC50 value of 0.78 and 0.28
tively. Moreover, compound Ig was the most active compound in
M) within this series. Note that Im carries
a methoxy moiety instead of a free hydroxyl group present in the
other derivatives IeeIl, indicating that a free hydroxyl moiety at the
mM, respec-
cells with IC50 ¼ 0.58
m
intact PMNL (IC50 ¼ 2.3
m
quinone core is actually not
a prerequisite for bioactivity.
Table 1B
2,5-Disubstituted 1,4-benzoquinone derivatives: chemical structures and effects on the activity of 5-LO. Data are given as mean ꢂ S.E.M., n ¼ 3e4.
Cpd
5-LO activity; cell-free
5-LO activity; cell-based
R
R1
Remaining activity at 10
m
M (%)
IC50
(
mM)
Remaining activity at 10
mM (%)
IC50 (mM)
Ie
H
8.0 ꢂ 2.5
4.6 ꢂ 1.2
61.3 ꢂ 7.8
>10
If
H
H
74.3 ꢂ 6.1
8.7 ꢂ 3.7
>10
93.3 ꢂ 16.6
5.3 ꢂ 2.4
>10
Ig
0.78 ꢂ 0.1
2.3 ꢂ 0.8
Ih
Ii
H
H
45.2 ꢂ 4.0
9.0 ꢂ 1.0
74.0 ꢂ 5.6
74.1 ꢂ 2.2
>10
>10
30.8 ꢂ 14.9
0.80 ꢂ 0.1
Il
H
14.2 ꢂ 8.6
7.1 ꢂ 3.2
1.4 ꢂ 0.2
15.9 ꢂ 2.7
21.2 ꢂ 8.6
3.0 ꢂ 0.7
6.7 ꢂ 1.0
Im
CH3
0.28 ꢂ 0.1
Zileuton (cmpd 30)
6.6 ꢂ 1.5
0.56 ꢂ 0.1
13.4 ꢂ 5.9
1.1 ꢂ 0.4

274
R. Filosa et al. / European Journal of Medicinal Chemistry 67 (2013) 269e279
Replacement of the naphthylmethyl residue by a benzyl moiety (If)
abrogated 5-LO inhibition, suggesting again that the size and/or
certain structural features of this lipophilic residue are de-
terminants for the interference with 5-LO. Along these lines,
introduction of a 3-nitro-phenyl ring, directly connected to the 5-
hydroxy-1,4-benzoquinone moiety (Ie), restored bioactivity, at
least for inhibition of cell-free 5-LO activity, and also replacement
of the aromatic substituent by an n-hexyl chain (Il) retained the
efficiency versus Ig in the cell-free assay with only a small loss of
potency in intact cells. The insertion of a polar carboxyl group at the
naphthalene ring (Ih) was clearly detrimental versus the parental
Ig, in both read outs, whereas replacement of the naphthylmethyl
group by more hindered heterocycles, like a dibenzofuran ring (Ii),
was tolerated, at least in the cell-free assay (IC50 ¼ 0.80
mM).
Together, for direct interference with 5-LO, the naphthylmethyl or
naphthoxy residues are most favorable.
Unexpectedly, the potency of the active compounds Ieem was
always higher inthe cell-freeversus cell-basedassay, andinparticular
the potent direct 5-LO inhibitors Ii and Im hardly reduced 5-LO
activity in intact cells, which is not readily understood. Possibly, the
cellular uptake of these compounds might be hampered due to the
sterically hindered dibenzofuran and naphthoxy ring, respectively.
To get further insights into the SARs, we next addressed the role
of the 1,4-benzoquinone core and replaced it by resorcinol where
the alkyl and aryl residues from above as well as other related
lipophilic substituents were incorporated in position 4. As can be
Table 2
Resorcinol derivatives: chemical structures and effects on the activity of 5-LO. Data are given as mean ꢂ S.E.M., n ¼ 3e4.
Cpd
5-LO activity; cell-free
5-LO activity; cell-based
R
Remaining activity at 10
m
M (%)
IC50
(
mM)
Remaining activity at 10
m
M (%)
IC50 (mM)
IIa
9.4 ꢂ 2.4
2.8 ꢂ 0.4
22.7 ꢂ 5.3
4.2 ꢂ 1.4
IIb
IIc
84.3 ꢂ 8.2
47.6 ꢂ 4.4
>10
64.1 ꢂ 7.4
60.0 ꢂ 2.2
>10
>10
9.4 ꢂ 1.0
IId
IIe
37.2 ꢂ 4.9
30.7 ꢂ 8.8
7.4 ꢂ 1.5
6.3 ꢂ 0.9
8.9 ꢂ 1.0
2.3 ꢂ 0.5
>10
84.1 ꢂ 12.6
IIf
25.6 ꢂ 7.1
15.4 ꢂ 2.5
4.6 ꢂ 1.8
3.2 ꢂ 0.4
18.6 ꢂ 6.1
10.5 ꢂ 2.5
2.8 ꢂ 2.2
2.1 ꢂ 0.1
IIg
IIh
IIi
100.2 ꢂ 2.1
57.4 ꢂ 3.6
>10
>10
92.8 ꢂ 5.8
42.4 ꢂ 1.8
>10
6.8 ꢂ 0.02

R. Filosa et al. / European Journal of Medicinal Chemistry 67 (2013) 269e279
275
Table 3
seen from Table 2, compounds out of the resorcinol series except
IIb, IIc, IIh and IIi showed good efficiencies for inhibition of 5-LO
with IC50 values in the low micromolar range but they are less
potent than the 1,4-benzoquinones with lowest IC50 values of
Effects of 1,4-benzoquinone and resorcinol derivatives on the activity of 12-LO
and 15-LO in PMNL. Data are given as mean ꢂ S.E.M., n ¼ 3e4.
Cpd
12-LO activity
15-LO activity
2.8 mM (compd. IIa) and 2.3 mM (compd. IId) in the cell-free and
Remaining activity at 10
mM (%)
Remaining activity at 10 mM (%)
cell-based assay, respectively. In contrast to the 1,4-benzoquinones,
the potency of the resorcinol derivatives in the two assays were
rather similar, except for compound IIe that was inactive in intact
cells. While the phenyl-substituted compound IIa exhibits signifi-
Ia
Ib
Ic
Id
Ie
If
Ig
Ih
Ii
84.0 ꢂ 14.0
76.4 ꢂ 26.8
85.2 ꢂ 10.7
60.9 ꢂ 1.6
60.9 ꢂ 6.7
95.4 ꢂ 20.4
71.9 ꢂ 13.3
81.1 ꢂ 7.4
46.2 ꢂ 6.1
89.8 ꢂ 5.8
73.1 ꢂ 16.0
72.3 ꢂ 3.8
90.1 ꢂ 13.5
77.0 ꢂ 1.3
79.2 ꢂ 8.3
93.3 ꢂ 21.8
69.3 ꢂ 8.7
91.4 ꢂ 7.9
115.4 ꢂ 6.5
96.0 ꢂ 10.4
96.4 ꢂ 12.8
89.5 ꢂ 25.9
87.3 ꢂ 5.7
87.9 ꢂ 11.4
116.3 ꢂ 41.8
132.3 ꢂ 32.4
147.2 ꢂ 28.1
80.9 ꢂ 4.34
79.8 ꢂ 10.9
184.6 ꢂ 20.5
47.9 ꢂ 5.3
cant 5-LO inhibitory activity (IC50 ¼ 2.8 and 4.2
mM in cell-free and
cell-based assays, respectively), insertion of a NO2 group in the
phenyl ring (IIb) or the introduction of a methylene spacer (IIc) was
clearly detrimental. Replacement of the phenyl in IIa by a naph-
thylmethyl moiety led to compound IId that was still active in the
cell-free but at least 2.5-fold more potent in intact cells. The elon-
gation of the chain and the insertion of an oxo moiety led to
compound IIi which retained 5-LO inhibitory in intact cells
Il
Im
IIa
IIb
IIc
IId
IIe
IIf
IIg
IIh
IIi
106.5 ꢂ 11.5
107.5 ꢂ 8.1
115.9 ꢂ 10.9
191.1 ꢂ 20.6
181.5 ꢂ 0.7
110.0 ꢂ 15.4
58.3 ꢂ 1.6
(IC50 ¼ 6.8
mM), but was inactive against purified 5-LO. Moreover,
compound IIe, carrying a dibenzofuranyl residue, was about equi-
potent to IId in the cell-free assay, but markedly lost its potency in
the cell-based system. Again, as observed for Ii within the 1,4-
benzoquinone series, the dibenzofuranyl moiety seemingly ham-
pers inhibition of 5-LO in intact cells. When dibenzofuran was
replaced by another hindered heterocycle like thianthrene (IIg) a
significant improvement of potency both in the cell-based and in
the cell-free assay was observed, whereas the smaller benzothio-
phene moiety in IIh did not cause 5-LO inhibition. Finally, exchange
of the phenyl ring of IIa by an n-hexyl (IIf) was tolerated and did not
significantly alter the efficiency in intact cells or cell-free assays.
Together, as observed for the 1,4-benzoquinone series the ability of
resorcinol derivatives to inhibit 5-LO product synthesis strongly
depends on the nature of the lipophilic substituent.
115.1 ꢂ 15.8
108.2 ꢂ 13.3
2.4. Molecular docking studies
To understand the differences in the binding modes of various
types of compounds we performed molecular docking studies.
Individual binding poses of each compound were assessed and
their interactions with potential amino acids in the active site of
the 5-LO protein were analyzed. The best and most energetically
favorable conformation of each compound was selected. The
binding modes of Ib, Ig and IId were obtained by carefully aligning
docked structures of the compounds into the active site of 5-LO
and studied in detail. As shown in Fig. 2, compound Ig that
efficiently inhibits 5-LO is stabilized in the 5-LO active site by
2.3. Evaluation of the effects of the test compounds on other human
LOs and analysis of competition with the substrate of 5-LO
strong hydrogen bond and
p
e
p
interactions. The bulky naphthyl
moiety forms strong
pep interactions with Phe177 and His372.
Besides 5-LO, other related human LOs such as 12-LO or 15-LO
that also abstract a hydrogen bond from AA via a nonheme active
site iron during catalysis were investigated as potential targets of the
test compounds. The activities of 12-LO and 15-LO were analyzed in
cell-based assays in order to assure the availability of “bioactive”
reduced 1,4-diphenol or resorcinol form, using PMNL preparations
that contain 15-LO in eosinophils and 12-LO in adhering platelets. Of
interest, all compounds that efficiently inhibited 5-LO product
The hydroxyl group forms hydrogen bond interactions with
Tyr181, and one of the carbonyl oxygen groups of the benzoqui-
none core forms strong interactions with Gln363 and His367.
Similar interactions were found for the 5-LO inactive Ib, however,
110
AA 2.5 µM
synthesis in the cell-based model with IC50 values ¼ 0.58e6.8
(i.e., Id, Ig, Il, IIa, IId, IIf, IIg, and IIi) failed to markedly affect the
activity of 12- and 15-LO (IC50 > 10 M), and only compound Ii
impaired the activity of 12-LO and compound Im of 15-LO at 10
by somewhat more than 50% (Table 3). These data suggest that the
5-LO inhibitory substances in this study exhibit a certain preference
for 5-LO and are not just unspecific LO inhibitors due to reducing
properties that may simply interfere with the iron-based redox cycle
of any LO.
It appeared reasonable to speculate that the active compounds
(e.g. Ig) inhibit 5-LO activity by competing with AA as substrate for
the enzyme. Therefore, we assessed inhibition of 5-LO product
synthesis by Ig in the cell-free assay, at varying AA concentrations,
mM
100
75
50
25
0
AA 5 µM
AA 10 M
AA 20 µM
AA 40 µM
m
mM
0.1
0.3
1
3
that is, at 2.5, 5, 10, 20 and 40
findings [25], 5-LO product formation continuously raised with
increasing the substrate concentration, was maximal at 20 M AA,
but declined due to substrate inhibition at 40 M AA. As shown in
Fig. 1, variation of the AA concentration caused no clear pattern of
competitive inhibition by Ig and the IC50 were in the close range of
mM. In agreement with previous
compound Ig (µM)
Fig. 1. Inhibition of 5-LO activity by Ig in the cell-free assay at various substrate
concentrations. Purified 5-LO was pre-incubated with Ig or vehicle (DMSO, 0.1%) for
15 min at 4 ꢀC. Samples were warmed up for 30 s at 37 ꢀC and 2 mM CaCl2 plus the
indicated concentrations of AA were added. After 10 min at 37 ꢀC, 5-LO activity was
determined. Data are expressed as percentage of control (100%, uninhibited 5-LO),
means ꢂ S.E.M., n ¼ 3.
m
m
0.31 (at 40 mM AA) and 0.97 mM (at 5 mM AA).

276
R. Filosa et al. / European Journal of Medicinal Chemistry 67 (2013) 269e279
it has been observed that none of the carbonyl oxygen groups of
the benzoquinone is involved in interactions with 5-LO. In general,
the presence of non-bonded hydrogen bond acceptor or donor in
the ligand adds a lot of energy to the system, i.e., in the form
of desolvation penalty [26], which may explain the low affinity of
Ig. Previous reports on 5-LO inhibitors clearly suggest that the
presence of hydrogen bond donors is more favorable than
hydrogen bond acceptors for 5-LO inhibition [27,29]. Together, an
obvious correlation between favorable/unfavorable interactions of
the compounds and inhibition of 5-LO activity in the cell-free
assay is evident. In general, the 1,4-benzoquinone series formed
more stable interactions than the resorcinol derivatives which
again correlates to the 5-LO inhibitory potencies.
Ib (IC50 > 10
m
M) in comparison to Ig (IC50 ¼ 0.78
mM). In Ib
the hydroxyl and the methoxy groups form hydrogen bond
interactions, and one of the carbonyl oxygen in Ib occupies a
hydrophobic point in the active site lined by Phe421 and Leu414
(Fig. 2), which is detrimental for the affinity to 5-LO. The strong
p
3. Conclusions
interactions observed in Ig correlate well with the pharmacophore
model and QSAR models of 5-LO reported earlier [27,28]. For the
resorcinol derivative IId, the two hydroxyl groups are involved in
hydrogen bond interactions with Tyr181 and Gln557, and with
His367, respectively. The hydrogen bond donor groups together,
i.e., the two strong hydroxyl moieties, form only three hydrogen
bonds. A stronger hydrogen bond group often implies higher
penalty of desolvation, unless it is involved in prominent hydrogen
bonds with the protein. As the 4-hydroxy moiety is involved in
only one hydrogen bond it does not contribute much to the free
energy, explaining why IId is less active on 5-LO in comparison to
5-LO that catalyzes the first two steps in LT biosynthesis is
considered as drug target for intervention with asthma, allergic
rhinitis, various autoimmune and inflammatory disorders, athero-
sclerosis and various types of cancer [1,5]. Therefore, the develop-
ment of potent and safe 5-LO inhibitors is a major challenge that
has been intensively pursued [7]. We present here novel series of
1,4-benzoquinones and related resorcinol derivatives as potent LT
synthesis inhibitors, with partially submicromolar IC50 values. Our
data provide insights related to SARs for 1,4-benzoquinone (and for
resorcinol) derivatives as 5-LO inhibitors. Thus, the potency of the
1,4-benzoquinones depends on structural features of the lipophilic
residues as demonstrated for instance by comparison of inactive If
with active Ig in cell-free assays or of inactive Ic with active Id in
intact cells. It appears that in particular bulky naphthyl or diben-
zofuran moieties connected to the 1,4-benzoquinone favor 5-LO
inhibition.
A similar pattern was found for the resorcinol
derivatives. As the results from the molecular docking simulations
reflecting binding to 5-LO correlated with the 5-LO inhibitory
potency, we conclude that the compounds mediate their inhibitory
action on the 5-LO enzyme via discrete physical interactions, sup-
ported also by the lack of marked 12- and 15-LO interference.
Together, novel 1,4-benzoquinone derivatives were revealed in this
study as selective and potent inhibitors of 5-LO in cell-free and cell-
based assays. Our results also stimulate for further preclinical in-
vestigations in order to assess the pharmacological properties and
the anti-inflammatory efficacy of selected compounds.
4. Experimental section
4.1. Chemistry
All reagents were analytical grade and purchased from Sigmae
Aldrich (Milano-Italy). Flash chromatography was performed on
Carlo Erba silica gel 60 (230e400 mesh; CarloErba, Milan, Italy). TLC
was carried out using plates coated with silica gel 60 F254 purchased
from Merck (Darmstadt, Germany). Microwave reactions were per-
formed on a CEM DiscoverÒ single mode platform using 10 mL
pressurized vials. Melting points were determined in open capillary
tubes on an Electrothermal 9100 apparatus and are uncorrected.
Reaction yields refer to chromatographically and spectroscopically
pure products. 1H and 13C NMR spectra were registered on a Bruker
AC 300. gCOSY experiments were registered on Bruker AC 500.
Chemical shifts are reported in ppm. All target compounds were
assessed for purity by combustion analysis. All target compounds
were found to be >95% purity. MS spectrometry analysis ESIeMS
was carried out on a Finnigan LCQ Deca ion trap instrument. Mi-
croanalyses were carried out on Carlo Erba 1106 elemental analyzer.
4.2. General procedure for the synthesis of 2-hydroxy-5-methoxy-
2,5-cyclohexadiene-1,4-dione 3-substituted derivatives (Iaeb)
Fig. 2. Interactions of test compounds with 5-LO. (A) Interactions of Ig with 5-LO. The
hydrogen bonds are given as double dotted lines,
p interactions are shown as single
solid lines. (B) Overlay of the conformations of Ib, Ig and IId in the 5-LO active site. The
best and energetically favorable conformations of Ib (blue), Ig (white) and IId (purple)
are superimposed. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
A water solution (8 mL) of ammonium cerium (IV) nitrate (CAN)
(0.486 g, 2.5 equiv.) was added rapidly to the appropriate 1,2,4,5-
tetramethoxybenzene-3-substituted 6aeb (1 equiv.) in acetonitrile

R. Filosa et al. / European Journal of Medicinal Chemistry 67 (2013) 269e279
277
(8 mL) at room temperature. After 2 h the solvent was evaporated.
The mixture was diluted with water, extracted with ethyl acetate,
providing a crude product purified by column chromatography.
(CD3OD, 75 MHz) d 104.1, 109.0,111.6, 112.3,113.0, 119.8,121.0, 121.5,
123.3,124.7, 130.7, 132.9,137.8, 145.8, 156.8,157.1, 158.8. ESIeMS: m/
z 276.87 [Mꢁ]. Anal. Calcd for C18H12O3: C 78.25, H 4.38, O 17.37.
Found: C 78.42, H 4.41, O 17.45.
4.2.1. 3-Benzyl-2-hydroxy-5-methoxy-2,5-cyclohexadiene-1,4-
dione (Ia)
4.4.2. 4-(Benzo[b]thiophen-3-yl)benzene-1,3-diol (IIh)
Elution with hexane/EtOAc (50:50) afforded Ia (0.046 g, 55%) as
Elution with CHCl3/CH3OH (99:1) afforded IIh (48%) as white
yellow powder. 1H NMR (CDCl3, 300 MHz)
d
3.81 (s, 2H), 3.90 (s,
3H), 5.92 (s, 1H), 7.20 (d, J ¼ 8.7 Hz, 1H), 7.25e7.30 (m, 2H), 7.38e
7.40 (m, 2H). 13C NMR (CDCl3, 75 MHz)
26.2, 54.5, 101.2, 124.5,
solid.1H NMR (CD3OD, 300 MHz)
d
6.84 (dd, J ¼ 8.0 e 1.9 Hz,1H), 6.91
(d, J ¼ 1.9 Hz,1H), 7.49e7.59 (m, 3H), 8.04 (d, J ¼ 6.8 Hz,1H), 8.45 (d,
d
J ¼ 6.8 Hz, 1H), 8.70 (d, J ¼ 6.8 Hz, 1H). 13C NMR (CD3OD, 75 MHz)
1268, 128.1, 138.2, 150.2, 159.6, 179.3, 181.8. ESIeMS: m/z 244.9
[Mþ]. Anal. Calcd for C14H12O4: C 68.85, H 4.95, O 26.20. Found: C
68.72, H 4.83O 26.32.
d 103.7,110.2,118.5,122.8,123.2,124.7,141.7,143.8,158.5,159.7. ESIe
MS: m/z 241.13 [Mꢁ]. Anal. Calcd. For C14H10O2S: C, 69.40; H, 4.16; O,
13.21; S, 13.23. Found: C 68.36, H 4.95, O 12.88, S, 12.98.
4.2.2. 3-[(2-Naphthyl)methyl]-2-hydroxy-5-methoxy-2,5-
cyclohexadiene-1,4-dione (Ib)
4.4.3. 1-(2,4-Dihydroxyphenyl)-2-(naphthalen-3-yl)ethanone (IIi)
Elution with CHCl3 afforded IIi (68%) as beige solid. 1H NMR
Elution with CHCl3 afforded Ib (0.065 g, 63%) as dark yellow
(CD3OD, 300 MHz)
d
4.70 (s, 2H), 6.29 (d, J ¼ 2.4 Hz, 1H), 6.39 (dd,
solid. 1H NMR (CDCl3, 300 MHz)
d
3.85 (s, 3H), 3.97 (s, 2H), 5.86 (s,
1H), 7.41 (m, 1H), 7.44 (m, 2H), 7.48 (dd, J ¼ 6.6 e 1.5 Hz, 1H), 7.74 (s,
1H), 7.76e7.80 (m, 3H). 13C NMR (CDCl3, 75 MHz)
27.9, 54.5, 100.1,
J ¼ 6.0 and 2.4 Hz, 1H), 7.33e7.48 (m, 4H), 7.78 (d, J ¼ 9.0 Hz, 1H),
7.83e7.88 (m, 2H), 7.93 (d, J ¼ 9.0 Hz,1H). 13C NMR (CD3OD, 75 MHz)
d
d 43.6, 103.2, 107.9, 115.2, 125.1, 125.9, 126.8, 128.2, 131.5, 132.8, 136.7,
123.8, 124.3, 125.8, 126.2, 127.1, 150.3, 158.8, 179.8, 180.7. ESIeMS:
m/z 295.60 [Mþ]. Anal. Calcd for C18H14O4: C 73.46, H 4.79, O 21.75.
Found: C 73.68, H 4.96,O 21.95.
158.7, 160.2, 205.1. ESIeMS: m/z 277.54 [Mꢁ]. Anal. Calcd. For
18H14O3: C, 77.68; H, 5.07; O, 17.25. Found: C 77.82, H 5.12, O 17.42.
C
4.5. General procedure for the synthesis of p-benzoquinones Ih and Ii
4.3. General procedure for the synthesis of 2,5-dihydroxy-2,5-
cyclohexadiene-1,4-dione 5-substituted derivatives (Iced)
To a solution of the dihydroxyl derivatives 21 and IIe (1.0 equiv.)
in tetrahydrofuran (10 mL) at room temperature, a solution of
Fremy’s salt (2.5 equiv.) in 5.7 mL of 15% Na2CO3, (1.6 equiv.) was
added. The solution was stirred vigorously for 8 h, until reaction
was completed and reaction mixture, after acidification with H2SO4
2 N, was extracted with EtOAc (3 ꢃ 25 mL). Desired compounds
were obtained after purification by flash chromatography.
To a solution of bisacetonide 13ced (1.0 equiv.) in 1,4-dioxane
(3.0 mL) was added an aqueous solution of HCl 4 N (4.58 mL) at
room temperature. The reaction mixture was stirred for 7 h under
reflux conditions and an aqueous solution of NaOH 2 N (5 mL) was
added. The resulting mixture was extracted with CHCl3. The organic
layers were combined, dried over Na2SO4, filtered and concentrated
in vacuo yielding a crude product, purified by flash chromatography.
4.5.1. 6-((4-Hydroxy-3,6-dioxocyclohexa-1,4-dienyl)methyl)
naphthalene-2-carboxylic acid (Ih)
4.3.1. 3-(Cyclohexylmethyl)-2,5-dihydroxycyclohexa-2,5-diene-1,4-
dione (Ic)
Elution with a mixture of CH2Cl2/MEOH/CH3COOH (80:15:05)
afforded quinone Ih as brown powder (0.040 g, 45%). 1H NMR
Elution with EtOAc/hexane (70:30) afforded Ic (0.047 g, 69%) as
yellow solid. Spectroscopical data are reported by Kim et al. [17].
(CD3OD, 300 MHz)
d
4.77 (dd, J ¼ 21.8 e 8.1 Hz, 2H), 5.36 (d, J ¼ 8.1 Hz,
1H), 5.96(d, J ¼ 8.1 Hz, 1H), 6.17 (s, 1H), 6.36e6.28 (m, 3H), 6.46 (d,
J ¼ 8.1 Hz, 1H), 7.03 (s, 1H). 13C NMR (CD3OD, 75 MHz)
d 35.2, 106.0,
4.3.2. 3-((Decahydronapththalen-3-yl)methyl)-2,5-dihydroxy-2,5-
cyclohexadiene-1,4-dione (Id)
109.4, 118.6, 120.2, 126.8, 127.6, 128.1, 128.4, 131.1, 132.1, 134.3, 150.4,
159.8, 160.0, 181.9, 182.1. ESIeMS: m/z 309.10 [Mþ]. Anal. Calcd. For
Elution with EtOAc/hexane (70:30) afforded Id as an orange
C18H12O5: C 70.13, H 3.92, O 25.95. Found: C 70.50, H 4.10, O 26.30.
solid (0.055 g, 57%). 1H NMR (CDCl3, 300 MHz)
d
0.72e1.08 (m, 3H),
1.27e1.39 (m, 4H), 1.32e1.42 (m, 2H), 1.56e1.72 (m, 8H), 2.25e2.42
(m, 2H), 6.03 (s, 1H), 7.70 (br, 2H). 13C NMR (CDCl3, 75 MHz)
21.2,
4.5.2. 5-(4-Dibenzofuranyl)-2-hydroxy-2,5-cyclohexadiene-1,4-
dione (Ii)
d
26.3, 28.3, 29.2 31.5, 33.3, 38.4, 42.5, 42.8, 112.3, 119.8, 150.3, 152.4,
174.6, 179.8. ESIeMS: m/z 292.33 [Mþ]. Anal. Calcd for C17H22O4: C
70.32, H 7.64, O 22.04. Found: C 70.21, H 7.75O 22.54.
Elution with hexane/EtOAc (60:40) afforded Ii (0.056 g, 54%) as
light brown solid. 1H NMR (DMSO-D6, 300 MHz)
d
6.11 (s, 1H), 6.99
(s, 1H), 7.30e7.36 (m, 3H), 7.46 (d, J ¼ 7.4 Hz, 1H), 7.8e8.0 (m, 3H),
11.0 (br; 1H). 13C NMR (DMSO-D6, 75 MHz)
110.1, 111.5, 111.6,
d
4.4. General procedures for synthesis of resorcinol derivatives (IIe,
IIh, IIi)
113.0, 119.1, 121.0, 121.7, 123.3, 124.7, 129.6, 132.7, 134.0, 145.7, 145.8,
156.3, 169.0, 181.3, 187.0. ESIeMS: m/z 291.01[Mþ]. Anal. Calcd for
C18H10O4: C 74.48, H 3.47, O 22.05. Found: C 74.56, H 3.53, O 22.16.
The appropriate 2,4-dimethoxybenzene aryl derivatives 24a, 24b
and 27 (1.0 equiv.) in dry dichloromethane (12 mL), cooled to ꢁ15 ꢀC,
were reacted in the presence of boron tribromide (8.8 equiv.). The
mixture was stirred for 18 h then deionized water was added, fol-
lowed by dichloromethane. The combined organic layer was dried
over Na2SO4, filtered, and purified to obtain desired compounds.
4.6. Docking protocol
Molecular docking was performed to predict the binding mode
of the ligands in the binding site of 5-LO; the recently reported
crystal structure of 5-LO (PDB: 3O8Y) was used [30]. The structures
of all compounds in this study were sketched and minimized using
Cerius2. GOLD (Genetic Optimization of Ligand Docking), a docking
program based on genetic algorithm was used to dock the in-
hibitors [31]. During docking, the default algorithm speed was
selected. The number of poses for each inhibitor was set to 100, and
early termination was allowed if the top three bound
4.4.1. 1-(4-Dibenzofuranyl)-2,4-benzendiol (IIe)
Elution with hexane/EtOAc (90:10e60:40) afforded IIe (82%) as
white solid. 1H NMR (CD3OD, 300 MHz)
d 6.85 (s, 1H), 6.87 (d,
J ¼ 8.1 Hz, 1H), 7.22e7.30 (m, 3H), 7.68 (t, J ¼ 7.2 Hz, 1H), 7.79 (d,
J ¼ 8.5 Hz, 1H), 7.96 (m, 2H), 8.13 (d, J ¼ 7.2 Hz, 1H). 13C NMR

278
R. Filosa et al. / European Journal of Medicinal Chemistry 67 (2013) 269e279
ꢀ
conformations of a ligand were within 1.5 A RMSD. As it is well
known, in LOs the substrate/inhibitor binding site is near to the
conserved iron binding site [29]. Thus, the active site was centered
on the ligand’s binding site oxyterminal of Ile. The interactions and
binding modes were analyzed in Accelrys Discover Visualizer 2.5.
SAR and prominent differences in the interactions of various de-
rivatives with 5-LO have been illustrated.
Acknowledgments
The authors thank Daniela Müller and Bianca Jazzar for expert
technical assistance and Aureliasan GmbH (Tuebingen, Germany)
for financial support.
Appendix A. Supplementary data
4.7. Biological evaluation and assay systems
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.039.
4.7.1. Cells and isolation
PMNL were isolated from buffy coats obtained from the Blood
Centre, University Hospital Tuebingen (Tuebingen, Germany) as
described [32]. In brief, human fresh blood was collected in hepa-
rinized tubes (16 I.E. heparin/mL blood) by venipuncture from
fasted (12 h) adult healthy volunteers, with consent, and leukocyte
concentrates were prepared by centrifugation (4000 ꢃ g, 20 min,
20 ꢀC). The subjects had no apparent inflammatory conditions and
had not taken anti-inflammatory drugs for at least ten days prior to
blood collection. Cells were immediately isolated by dextran sedi-
mentation and centrifugation on Nycoprep cushions (PAA Labora-
tories, Linz, Austria) and hypotonic lysis of erythrocytes was
performed as described [32]. Cells were finally resuspended in PBS
pH 7.4 containing 1 mg/mL glucose and 1 mM CaCl2 (PGC buffer)
(purity > 96e97%).
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4.7.2. Determination of 5-LO, 12-LO, and 15-LO product formation
in cell-based assays
For assays of 5-LO, 12-LO and 15-LO, 5 ꢃ 106 freshly isolated
PMNL were resuspended in 1 mL PGC buffer. After pre-incubation
with the compounds for 15 min at 37 ꢀC, 5-LO product formation
was started by addition of 2.5
10 min at 37 ꢀC, the reactionwas stopped with 1 mL of methanol and
30 L of 1 N HCl, 200 ng PGB1 and 500 L of PBS were added. Formed
mM A23187 and 20 mM AA. After
m
m
5-LO, 12-LO and 15-LO metabolites were extracted and analyzed by
HPLC as described [33]. 5-LO product formation is expressed as ng of
5-LO products per 106 cells, which includes LTB4 and its all-trans
isomers, 5(S),12(S)-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic
acid (5(S),12(S)-DiHETE), and 5(S)-hydro(pero)xy-6-trans-8,11,14-
cis-eicosatetraenoic acid (5-H(p)ETE). Cysteinyl LTs C4, D4 and E4
were not detected, and oxidation products of LTB4 were not deter-
mined. The formation of the 12-LO product 12(S)-hydro(pero)xy-
5,8-cis-10-trans-14-cis-eicosatetraenoic acid (12-H(P)ETE) and of
the 15-LO product 15(S)-hydro(pero)xy-5,8,11-cis-13-trans-eicosa-
tetraenoic acid (15-H(P)ETE) is expressed as ng per 106 cells.
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4.7.3. Expression and purification of human recombinant 5-LO from
Escherichia coli, and determination of 5-LO activity in cell-free
systems
E. coli BL21 was transformed with pT3-5LO plasmid, human re-
combinant 5-LO protein was expressed at 37 ꢀC and purified as
described [34]. Purified 5-LO was immediately used for 5-LO activity
assays. 0.5
incubated with the test compounds. After 5e10 min at 4 ꢀC, sam-
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mg purified 5-LO were diluted with PBS/EDTA and pre-
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product formation. The reactionwas stopped after 10 min at 37 ꢀC by
addition of1 mL ice-cold methanolandthe formed metabolites were
analyzed by HPLC as described for intact cells.
4.8. Statistics
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