Novel series of benzoquinones with high potency against 5-lipoxygenase in human polymorphonuclear leukocytes

Article abstract of DOI:10.1016/j.ejmech.2015.02.042

5-Lipoxygenase (5-LO) is a potential target for pharmacological intervention with various inflammatory and allergic diseases. Starting from the natural dual 5-LO/microsomal prostaglandin E₂ synthase (mPGES)-1 inhibitor embelin (2,5-dihydroxy-3-

Full text of DOI:10.1016/j.ejmech.2015.02.042

European Journal of Medicinal Chemistry 94 (2015) 132e139
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel series of benzoquinones with high potency against 5-
lipoxygenase in human polymorphonuclear leukocytes
,
,
Rosanna Filosa a *, Antonella Peduto a b, Anja M. Schaible c, Verena Krauth c,
Christina Weinigel d, Dagmar Barz d, Carmen Petronzi a, Ferdinando Bruno a,
Fiorentina Roviezzo e, Giuseppe Spaziano a, Bruno D'Agostino a, Mario De Rosa a,
,
*
Oliver Werz c
a Department of Experimental Medicine, University of Naples, Via Costantinopoli, 16, 80138 Naples, Italy
b Dermofarma, Via Cortenocera, 82030 S. Salvatore Telesino, BN, Italy
c Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Philosophenweg 14, 07743 Jena, Germany
d Institute of Transfusion Medicine, University Hospital Jena, 07743 Jena, Germany
e Department of Experimental Pharmacology, University Federico II of Naples, Naples, Italy
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) is a potential target for pharmacological intervention with various inflammatory
and allergic diseases. Starting from the natural dual 5-LO/microsomal prostaglandin E2 synthase
(mPGES)-1 inhibitor embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone, 2) that suppresses 5-LO ac-
Received 20 December 2014
Received in revised form
18 February 2015
Accepted 21 February 2015
Available online 24 February 2015
tivity in human primary leukocytes with IC50 ¼ 0.8e2
mM, we synthesized 48 systematically modified
derivatives of 2. We modified the 1,4-quinone to 1,2-quinone, mono- or bimethylated the hydroxyl
groups, and varied the C11-n-alkyl residue (C4- to C16-n-alkyl or prenyl) of 2. Biological evaluation yields
potent analogues being superior over 2 and obvious structure-activity relationships (SAR) for inhibition
of 5-LO. Interestingly, conversion to 1,2-benzoquinone and bimethylation of the hydroxyl moieties
strongly improves 5-LO inhibition in polymorphonuclear leukocytes versus 2 up to 60-fold, exemplified
by the C12-n-alkyl derivative 22c (4,5-dimethoxy-3-dodecyl-1,2-benzoquinone) with IC50 ¼ 29 nM.
Regarding inhibition of mPGES-1, none of the novel benzoquinones could outperform the parental
Keywords:
Inflammation
5-Lipoxygenase
Microsomal prostaglandin E2 synthase-1
Arachidonic acid
Benzoquinone
compound 2 (IC50 ¼ 0.21
mM), and only modest suppressive effects on 12- and 15-LOs were evident.
Together, our detailed SAR study reveals 22c as highly potent 5-LO-selective lead compound in intact
cells that warrants further preclinical evaluation as anti-inflammatory agent.
© 2015 Published by Elsevier Masson SAS.
1. Introduction
heme iron-containing dioxygenase, is the key enzyme in the
biosynthesis of LTs that catalyzes the incorporation of molecular
Leukotrienes (LTs) are bioactive lipid mediators produced from
arachidonic acid (AA) with pivotal roles in immune reactions,
inflammation, and allergy [1,2]. 5-Lipoxygenase (5-LO), a non-
oxygen into AA yielding the intermediate 5(S)-hydro-
peroxyeicosatetraenoic acid (5-HPETE), and subsequently de-
hydrates 5-HPETE to LTA4 [3]. The latter is either converted by LTA4
hydrolase to LTB4 or by LTC4 synthases to the glutathione conjugate
LTC4 that is further degraded to LTD4 and LTE4. The biological effects
that are evoked by LTs are mediated by different G protein-coupled
receptors, specific for LTB4 (BLT1/2) or LTC4, D4 and E4 (cysLT1/2), as
well as by additional receptors [4]. While LTB4 causes chemotaxis
towards various types of leukocytes and activates phagocytes, LTC4,
D4 and E4 are potent bronchoconstrictors, cause mucus secretion in
the lung, and increase vasopermeability of postcapillary venules
[1]. Accordingly, LTs are implicated as mediators in a variety of in-
flammatory and allergic diseases including asthma, allergic rhinitis,
Abbreviations: AA, arachidonic acid; CAN, cerium ammonium nitrate; COX,
cyclooxygenase; cPLA2, cytosolic phospholipase A2; FLAP, 5-lipoxygenase-activating
protein; H(P)ETE, hydro(pero)xyeicosatetraenoic acid; 5-LO, 5-lipoxygenase; LT,
leukotriene; mPGES-1, microsomal prostaglandin E2 synthase-1; PBS, phosphate-
buffered saline; PG, prostaglandin; PGC buffer, PBS containing 1 mg/ml glucose plus
1
mM CaCl2; PMNL, polymorphonuclear leukocyte; SAR, structure-activity
relationship.
* Corresponding authors.
E-mail addresses: rosanna.filosa@unina2.it (R. Filosa), oliver.werz@uni-jena.de
(O. Werz).
http://dx.doi.org/10.1016/j.ejmech.2015.02.042
0223-5234/© 2015 Published by Elsevier Masson SAS.

R. Filosa et al. / European Journal of Medicinal Chemistry 94 (2015) 132e139
133
O
and autoimmune disorders, as well as cardiovascular diseases [2].
Moreover, due to stimulation of survival, proliferation and migra-
tion of cancer cells by LTs, a role in cancer became apparent [5].
Based on its key function in the initiation of LT-related diseases,
5-LO has long been considered as a promising target for therapeutic
intervention with those disorders [2]. In fact, numerous series of
synthetic agents as well as natural products have been identified as
5-LO inhibitors but most of these compounds exhibited only
limited potency, were rather unspecific for 5-LO with potential side
effects, and/or lacked efficiency in vivo. [6] Moreover, the charac-
terization of the cellular and molecular mechanism of 5-LO inhi-
bition by the inhibitor was often neglected and detailed follow-up
studies were not consequently performed or pursued [7]. For
example, the 1,4-benzoquinone 1 (2,3,5-trimethyl-6-(12-hydroxy-
5,10-dodecadiynyl)-1,4-benzoquinone, AA-861, Fig. 1) was initially
presented in 1982 as one of the first 5-LO inhibitors [8] and many
subsequent pharmacological studies confirmed the efficiency and
anti-inflammatory efficacy in vivo, and 1 even succeeded in a
clinical trial for prevention of seasonal allergic rhinitis [9]. Never-
theless, the precise mechanisms of inhibition of 5-LO has remained
elusive until recently [10].
OH
1
O
O
OH
HO
2
O
O
OH
HO
O
In the course of our previous biological investigations of
simplified derivatives of the marine hydroxyquinone bolinaqui-
none with antitumour activity [11,12], we identified compound 2
(2,5-dihydroxy-3-undecyl-1,4-benzoquinone, embelin, Fig. 1), a
naturally occurring 1,4-benzoquinone from Embelia ribes, that
dually inhibits 5-LO as well as microsomal prostaglandin E2 syn-
thase (mPGES)-1 but does not interfere with 12/15-LOs, cytosolic
phospholipase (PL)A2 or cyclooxygenase (COX) enzymes [13]. In a
parallel study we synthesized and characterized a series of related
2,5-dihydroxylated 1,4-benzoquinones with various lipophilic and
bulky alkyl- or aryl-substituents in 3-position [14], exemplified by
3-((decahydronaphthalen-6-yl)methyl)-2,5-dihydroxycyclohexa-
2,5-diene-1,4-dione (compound 2a, Fig. 1), as 5-LO inhibitors with
anti-inflammatory activity in vivo [15]. The simple structure and
good potency of 2 and 2a with IC50 values in the submicromolar
range stimulated us to systematically modify the structure of 2 and
thus, to improve the inhibitory potential as well as to investigate
SARs. In particular, we aimed at improving the 5-LO inhibitory
potential in intact cells, because the potency of 2 in cell-free assays
2a
Fig. 1. Chemical structures of compound 1, 2 and 2a that inhibit 5-LO.
was reacted with different alkyl halides giving intermediates 5e16
in good yields. Cerium ammonium nitrate (CAN)-mediated oxida-
tive reaction provided a mixture of 2,5-dimethoxy (17a-28a) and 2-
hydroxy-5-methoxy-1,4-benzoquinones (17b-28b) as described for
literature-known compounds 20a, 21a and 20b, 21b (Method A
[17]). A mixture of 2,5-dimethoxy-1,4-benzoquinones (17a-28a)
and 4,5-dimethoxy-1,2-benzoquinones (17c-28c) was achieved
following a modified procedure of the reported method (Method B;
Scheme 2). Treatment of 17a-20a and 22a-28a with 2 M NaOH
allowed to obtain 2,5-dihydroxy-1,4-benzoquinones 17d-20d and
22d-28d in quantitative yields (Scheme 3) [18].
2.2. Biological assays
To study the ability of the test compounds for direct inhibition of
5-LO, we applied a cell-free assay using purified human recombi-
nant 5-LO enzyme [7,19]. To study the inhibitory potency on 5-LO
product formation in intact cells, we used human PMNL that are
a major source for LT biosynthesis [20,21]. PMNL were either
stimulated with the Ca2þ-ionophore A23187 alone or together with
20 mM exogenous AA. Co-addition of AA allows circumventing the
absolute need for endogenous substrate release by cPLA2 and the
AA transfer via the 5-LO-activating protein (FLAP) [7,22]. The 5-LO
(IC50 ¼ 0.06
m
M) was 13- to 33-fold higher than in cell-based test
M) [13].
systems (0.8e2
m
Here we report the synthesis and the biological evaluation of 48
different 3-mono-alkyl-substituted 1,4-benzoquinones (i.e., 17a-
28a, 17b-28b, 17d-20d, 22d-28d) or 1,2-benzoquinones (i.e., 17c-
28c). Whereas the potency of some representatives was clearly
superior over 2 regarding inhibition of 5-LO, inhibition of mPGES-1
was not improved. Our results suggest concrete SARs for 5-LO in-
hibition, that is, (I) methylation of hydroxyl groups at the quinone
core increases the potency especially in intact cells, (II) 1,2-
quinones are more potent than 1,4-quinones, and (III) n-alkyl res-
idues with 10e14 carbon atoms confer highest potency. In fact, the
doubly O-methylated 1,2-benzoquinone 22c with C12-n-alkyl chain
inhibitor
29
(N-(1-benzo[b]thien-2-ylethyl)-N-hydroxyurea;
zileuton) served as reference compound [23].
2.3. Design of compounds and structure-activity relationship
studies regarding 5-LO
(4,5-dimethoxy-3-dodecyl-1,2-benzoquinone)
reached
IC50
value ¼ 29 nM in intact human polymorphonuclear leukocytes
(PMNL), which is an about 60-fold improvement over 2.
The focus of the compound design was placed on three struc-
tural variations of lead compound 2: (I) the free hydroxyl groups at
the benzoquinone core in comparison to O-methylated analogues,
(II) the length of the n-alkyl residue in 3-position as well as
modification towards prenyl moieties, and (III) alteration of the 1,4-
quinone to the 1,2-quinone core. Starting with the compound 2-
derived 2,5-dihydroxy-1,4-benzoquinone core, we first varied the
length of the n-alkyl residue in position 3 (i.e., C11 in 2). To this aim,
we introduced linear C4-, C6-, C8-, C10-, C12-, C13-, C14-, C15- and
C16-n-alkyl residues or isoprenoid side chains with two or three
2. Results and discussion
2.1. Chemistry
The desired compounds were synthesized starting from 1,2,4,5-
tetramethoxybenzene (3) [16] which was subjected to an
orthoemetalation reaction in the presence of n-BuLi and hexame-
thylphosphoramide (HMPA) (Scheme 1) [11]. The lithium derivative

134
R. Filosa et al. / European Journal of Medicinal Chemistry 94 (2015) 132e139
O
O
O
O
O
O
OH
R
O
O
a
a
R-X
R
O
HO
O
4a-4l
O
R
O
O
O
17d: R= butyl
18d: R= hexyl
19d: R= octyl
20d: R= decyl
22d: R= dodecyl
23d: R= tridecyl
24d: R= tetradecyl
25d: R= pentadecyl
26d: R= hexadecyl
27d: R= geranyl
28d: R= farnesyl
3
17a-28a
4a: iodobutane
4b: iodohexane
4c: bromooctane
4d: iododecane
4e: bromoundecane
4f: iodododecane
4g: bromotridecane
4h: bromotetradecane
4i: bromopentadecane
4j: iodohexadecane
4k: geranyl bromide
4l: farnesyl bromide
5: R= butyl
6: R= hexyl
7: R= octyl
8: R= decyl
9: R= undecyl
10: R= dodecyl
11: R= tridecyl
12: R= tetradecyl
13: R= pentadecyl
14: R= hexadecyl
15: R= geranyl
16: R= farnesyl
Scheme 1. Ortho-metalation of 1, 2, 4, 5-tetramethoxybenzene 3. Reagents and con-
ditions: a) n-BuLi, HMPA, THF, from ꢀ40 ꢁC to room temperature, 12 h.
Scheme 3. Synthesis of 5-dihydroxy-1,4-benzoquinones 17d-20d and 22d-28d. Re-
agents and conditions: a) NaOH 2 M, EtOH, 80 ꢁC, 2 h.
prenyl moieties, respectively, into 3-position, retaining the 2,5-
dihydroxy-1,4-benzoquinone core (“d-series”, Table 1).
Except for the C4-n-alkyl-substituted compound 17d, all de-
rivatives inhibited the activity of isolated 5-LO in the cell-free assay
suppression of LTB4 synthesis in PMNL as compared to one or four
prenyl moieties [24]. Apparently, an extended lipophilic alkyl res-
idue favours 5-LO inhibition which is in agreement with general
findings by others showing that the potency of (poly)phenol-based
5-LO inhibitors is often enhanced due to increasing the lipophility
[7].
Next, we evaluated the inhibitory potential of the test com-
pounds in the 5-LO cell-based assay. In general, all compounds of
this group significantly lost their potency in PMNL. In fact, com-
pounds with C4-, C6-, C8- (i.e., 17d, 18d, 19d) or C16-n-alkyl and
prenyl residues (i.e., 26d, 27d, 28d) only moderately suppressed 5-
with IC50 values ¼ 0.17e4
10 M, inhibited 5-LO by only 43.9%. Note that none of the com-
pounds was superior over parental 2 (IC50 ¼ 0.06 M) [13], in this
mM. Compound 17d, at a concentration of
m
m
respect. Therefore, a relationship between the length of the n-alkyl
chain and the 5-LO inhibitory potential of 3-substituted 2,5-
dihydroxy-1,4-benzoquinones is apparent, where the C11-n-alkyl
residue is seemingly optimal. Compounds 20d, 22d, 24d, and 26d
with C10-, C12-, C14-, and C16-n-alkyl chains, respectively inhibi-
LO product synthesis (IC50 > 10 mM), regardless of the absence or
presence of exogenous AA. However, the C10-, C12-, C13-, C-14- and
C-15-n-alkyl derivatives 20d, 22d, 23d, 24d, and 25d, respectively,
ted 5-LO with low IC50 values between 0.17 and 0.19
the C6-substituted compound 18d is considerably less active
(IC50 ¼ 4 M). The prenyl derivatives showed lower potency with
IC50 values of 1.8 and 2.5 M for compound 27d and 28d, respec-
mM, whereas
m
were able to inhibit 5-LO (IC50 ¼ 2 and 4.6
mM) in PMNL challenged
with A23187; along these lines 2 (with a C11-n-alkyl residue) was
m
tively. Note that two (geranyl) or three (farnesyl) prenyl residues
connected to 1,4-hydroquinone were found before to be superior in
active in PMNL but lost potency (IC50 ¼ 1.7
mM) [13] versus cell-free
5-LO activity as well.
O
O
O
O
O
O
a
+
O
R
O
R
O
R
O
O
O
17c-28c
17a-28a
b
17: R= butyl
18: R= hexyl
19: R= octyl
20: R= decyl
21: R= undecyl
22: R= dodecyl
23: R= tridecyl
24: R= tetradecyl
25: R= pentadecyl
26: R= hexadecyl
27: R= geranyl
28: R= farnesyl
O
O
O
O
OH
R
+
O
R
O
O
17b-28b
17a-28a
Scheme 2. Synthesis of 2,5-dimethoxy-1,4-benzoquinones (17a-28a), 2-hydroxy-5-methoxy-1,4-benzoquinones (17b-28b), and 4,5-dimethoxy-1,2-benzoquinones (17c-28c). Re-
agents and conditions: a) Method B. CAN, CH3CN:H2O 7:3, -7 ꢁC, 100; b) Method A. CAN, CH3CN:H2O 7:3, from ꢀ7 ꢁC to room temperature, 2 h.

R. Filosa et al. / European Journal of Medicinal Chemistry 94 (2015) 132e139
135
Table 1
Effects of 2,5-hydroxy-1,4-benzoquinones (“d-series”) on 5-LO activity. Data are expressed as means S.E. of single determinations obtained in three to four independent
experiments.
Cmpd
5-LO activity
IC50 M]
[
m
(remaining activity (%) at 10
mM)
Cell-based
Cell-free
AA
A23187
A23187 þ AA
17d
18d
19d
R ¼ n-butyl
R ¼ n-hexyl
R ¼ n-octyl
>10
(93.5 4.4%)
>10
(133.5 19.0%)
>10
(53.5 4.8%)
2.0 0.3
1.7 0.4
4.6 1.3
2.5 0.4
3.1 0.5
4.3 0.5
>10
(92.8 4.2%)
>10
(79.9 6.6%)
>10
(80.07 8%)
7.1 1.2
3.5 0.5
>10
(65.4 14.2%)
4.0 1.1
0.38 0.04
20d
2 (embelin)
22d
23d
24d
R ¼ n-decyl
0.18 0.01
0.06 0.01
0.17 0.03
0.22 0.09
0.17 0.01
0.23 0.08
R ¼ n-undecyl
R ¼ n-dodecyl
R ¼ n-tridecyl
R ¼ n-tetradecyl
R ¼ n-pentadecyl
3.9
1
4.7 0.2
5.1 0.6
>10
(61.7 2.1%)
>10
(80.5 5.7%)
>10
(81.9 10.8%)
>10
25d
26d
R ¼ n-hexadecyl
R ¼ geranyl
>10
(88.0 16.3%)
>10
(114.3 4.5%)
>10
(85.8 3.9%)
0.9 0.3
0.19 0.04
1.8 0.2
27d
28d
R ¼ farnesyl
2.5 1.4
(66.6 2.8%)
2.5 1.2
29 (zileuton)
0.59 0.10
We assumed that the two polar hydroxyl moieties in 2- and 5-
position of 2 and related compounds of the “d-series”, which
actually are part of two vinylogue acids with dissociable protons,
may confer the compounds high hydrophilicity and thus, hamper
membrane penetration into PMNL to access 5-LO. In fact, 2 was
reported to exist in the mono-deprotonated form at pH ranges
6.0e8.0 with anionic charge [25]. Accordingly, we methylated
either the hydroxyl in 5-position (“b-series”) or both hydroxyl
residues in 2- and 5-position (“a-series”), respectively, which may
improve cellular uptake and thus potency against 5-LO. Compared
to the 2,5-dihydroxy “d”-series, the 5-O-monomethylated ana-
logues (“b-series”) inhibited 5-LO less efficiently in the cell-free
assay (Table 2). For example, compounds 18b, 26b and 27b
for all O,O0-bimethylated analogues (“a-series”, except 26a) over
the monomethylated “b-series”. In particular, bimethylated com-
pounds carrying n-alkyl residues of 6e15 carbons inhibited
A23187-induced 5-LO product synthesis with IC50 ¼ 0.2e1.7
mM,
with a tendency for improved activity in the absence of exogenous
AA (Table 3). Again, also the prenylated derivatives 27a and 28a
exhibited improved potency with IC50
¼
0.3e1.6 mM. Taken
together, O-mono- and even more strikingly O,O0-bimethylation of
2,5-dihydroxy-1,4-benzoquinones carrying a C10- to C15-n-alkyl
chain in 3-position yields highly potent 5-LO inhibitors that effi-
ciently suppress 5-LO product formation in intact cells, with up to
9-fold superiority over parental 2.
Next, we replaced the 2,5-dimethoxy-1,4-benzoquinone core
(Table 2) did not inhibit isolated 5-LO by more than 50% at 10
and the IC50 values of the most active compounds 20d and 2 (0.18
and 0.06 M) shifted to about 22- to 24-fold higher concentrations
(i.e., 4.3 and 3.8 M) for the corresponding 5-methoxy derivatives
20b and 21b, respectively. Interestingly, bimethylation of both hy-
droxyl moieties in 2- and 5-position (“a-series”, Table 3) improved
the efficiency in the cell-free assay, visualized by IC50 values of
m
M
present in compounds 17a-28a by
a
4,5-dimethoxy-1,2-
benzoquinone moiety (“c-series”, Table 4). This modification to-
wards 1,2-quinones further increased the potency against 5-LO in
particular in intact PMNL. In fact, except for the C4-n-alkyl-
substituted 17c, for all other (C6- to C14-n-alkyl and prenyl) de-
rivatives the IC50 values were in the submicromolar range. The C16-
n-alkyl-substituted compound 26c was poorly soluble in the
aqueous assay buffer and thus, could not be tested for 5-LO inhi-
bition. Of particular interest are the C10- to C14-n-alkyl derivatives
20c, 21c, 22c, 23c and 24c which reached IC50 values between 29
and 150 nM in PMNL stimulated with A23187 with or without AA.
Similar as the 2,5-dimethoxylated 1,4-benzoquinones most of the
4,5-dimethoxy-1,2-benzoquinones were about equipotent 5-LO
inhibitors in intact cells and in the cell-free assay. Thus, com-
pounds 20c, 21c, 22c, 23c and 24c repressed 5-LO activity with IC50
values in the range of 40e130 nM, being about equipotent to 2
(IC50 ¼ 60 nM). Conclusively, our SAR studies revealed 3-alkyl-
substituted 4,5-dimethoxy-1,2-benzoquinones when equipped
with simple C10- to C14-n-alkyl residues as potent 5-LO inhibitors
with IC50 values in the range of 29e150 nM in PMNL and
40e130 nM for isolated 5-LO.
m
m
0.21e3.3
mM for all derivatives, which are in the range of those for
the unmethylated “d-series”, and even the n-butyl-derivative 17a
was active (IC50 ¼ 3.3
analogue 17d.
mM), in contrast to the unmethylated
Of interest, in the cell-based assay the 5-LO inhibitory capacity
of the O-methylated derivatives was increased, in particular for the
doubly methylated (“a-series”) analogues. The O-monomethylated
compounds carrying C10- to C15-n-alkyl chains were highly active
with IC50 values in the range of 0.3e0.7
mM in PMNL stimulated
with A23187 and 0.5e2.7 M for cells stimulated with A23187 plus
m
AA. Monomethylation conferred the prenyl derivatives 27b and 28b
efficiency versus the inactive unmethylated counterparts 27d and
28d.
The 5-LO inhibitory potency in intact cells was further improved

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R. Filosa et al. / European Journal of Medicinal Chemistry 94 (2015) 132e139
Table 2
Table 4
Effects of 2-hydroxy-5-methoxy-1,4-benzoquinones (“b-series”) on 5-LO activity.
Data are expressed as means S.E. of single determinations obtained in three to four
independent experiments.
Effects of 4,5-methoxy-1,2-benzoquinones (“c-series”) on 5-LO activity. Data are
expressed as means S.E. of single determinations obtained in three to four inde-
pendent experiments.
No.
5-LO activity
IC50 M]
Cmpd
5-LO activity
IC50 M]
[
m
[m
(remaining activity (%) at 10
m
M)
(Remaining activity (%) at 10
mM)
Cell-based
Cell-free
AA
Cell-based
Cell-free
AA
A23187
A23187 þ AA
A23187
A23187 þ AA
17b R ¼ n-butyl
18b R ¼ n-hexyl
19b R ¼ n-octyl
>10
(86.0 6.0%)
>10
(68.9 16.3%) (64.1 8.0%)
>10
(73.5 8.6%)
0.59 0.1
0.68 0.1
0.56 0.04
0.77 0.26
0.27 0.04
0.39 0.14
1.0 0.6
>10
(95.7 7.3%)
>10
>10
(70.5 17.1%)
>10
(71.1 5.3%)
3.0 0.4
17c
R ¼ n-butyl
2.2 0.8
5.1 0.7
>10
(82.9 2.7%)
2.6 0.3
0.33 0.05
0.13 0.01
0.09 0.04
0.13 0.12
0.08 0.01
0.04 0.02
0.62 0.14
18c
19c
20c
21c
22c
23c
24c
25c
R ¼ n-hexyl
0.33 0.10
0.38 0.07
0.04 0.01
0.06 0.01
0.029 0.01
0.12 0.01
0.12 0.03
>10
0.99 0.42
0.82 0.38
0.07 0.01
0.10 0.03
0.15 0.03
0.05 0.03
0.09 0.02
>10
R ¼ n-octyl
>10
R ¼ n-decyl
(72.9 11.7%)
2.7 0.7
1.3 0.4
1.1 0.2
2.3 1.1
0.49 0.1
0.86 0.27
1.1 0.8
R ¼ n-undecyl
R ¼ n-dodecyl
R ¼ n-tridecyl
R ¼ n-tetradecyl
R ¼ n-pentadecyl
20b R ¼ n-decyl
4.3 0.3
3.8 0.5
0.74 0.08
0.92 0.47
0.42 0.01
0.27 0.10
>10
(60.1 8.7%)
>10
(73.6 8.0%)
5.6 0.1
21b R ¼ n-undecyl
22b R ¼ n-dodecyl
23b R ¼ n-tridecyl
24b R ¼ n-tetradecyl
25b R ¼ n-pentadecyl
26b R ¼ n-hexadecyl
(57.6 3.2%)
(66.8 3.5%)
26c
27c
28c
R ¼ n-hexadecyl
R ¼ geranyl
an.d.
an.d.
an.d.
0.60 0.08
0.30 0.07
0.38 0.08
0.15 0.07
0.39 0.10
0.10 0.01
R ¼ farnesyl
27b R ¼ geranyl
28b R ¼ farnesyl
4.1 1.1
1.9 0.1
>10
(65.9 6.0%)
2.4 0.8
a
n.d., not determined.
selective for 5-LO (at least > 66- to 340-fold). The n-hexyl-
substituted compounds 18b and 18c (at 10 M) repressed 12-HETE
m
Table 3
formation in intact cells by 55.5 and 81.4%, respectively, and 18b
inhibited 15-HETE synthesis by 34.2%. The unspecific LO inhibitor
30 (cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, CDC) [27]
inhibited 12- and 15-HETE formation under these conditions, as
expected (Table S1). Moreover, analysis of 22c for inhibition of 12/
15-LO in a cell-free assay (PMNL homogenates) revealed no sig-
Effects of 2,5-methoxy-1,4-benzoquinones (“a-series”) on 5-LO activity. Data are
expressed as means S.E. of single determinations obtained in three to four inde-
pendent experiments.
Cmpd
5-LO activity
IC50 M]
[
m
(Remaining activity (%) at 10
mM)
Cell-based
Cell-free
nificant inhibition up to 10 mM, while 5-LO activity was abolished in
the same incubations (not shown).
A23187
A23187 þ AA AA
Since 2 was also active on human mPGES-1 (IC50 ¼ 0.21
mM) [13]
17a
18a
19a
20a
21a
22a
23a
24a
25a
26a
R ¼ n-butyl
3.4 1.4
7.9 1.0
3.3 0.9
we tested the novel compounds for their ability to inhibit mPGES-1
activity. For analysis of mPGES-1 inhibition, the well-recognized
R ¼ n-hexyl
0.32 0.21
0.32 0.01
0.60 0.38
0.20 0.02
0.63 0.41
0.23 0.04
0.29 0.09
0.39 0.14
>10
1.4 0.4
2.6 1.2
R ¼ n-octyl
0.57 0.17
1.7 0.9
1.2 0.3
1.8 0.7
cell-free assay using microsomes from interleukin-1
A549 cells and PGH2 (20 M) as substrate was utilized [28]. The
mPGES-1 inhibitor 31 (3-[3-(tert-butylsulfanyl)-1-[(4-
b-stimulated
R ¼ n-decyl
m
R ¼ n-undecyl
R ¼ n-dodecyl
R ¼ n-tridecyl
R ¼ n-tetradecyl
R ¼ n-pentadecyl
R ¼ n-hexadecyl
0.59 0.11
0.49 0.10
0.31 0.05
1.4 0.8
0.86 0.27
3.5 0.9
0.93 0.13
0.61 0.08
0.26 0.01
0.21 0.03
0.27 0.08
1.6 0.2
chlorophenyl)methyl]-5-(propan-2-yl)-1H-indol-2-yl]-2,2-
dimethylpropanoic acid, MK886) served as reference compound
[28]. Inhibition of mPGES-1 clearly depended on both, the
(unmethylated) free hydroxyl groups and the length of the 3-
substituted alkyl chain, with active compounds (i.e.,
(80.1 9.1%)
0.45 0.2
0.31 0.1
27a
28a
R ¼ geranyl
R ¼ farnesyl
1.6 0.2
1.2 0.2
3.3 0.7
1.7 0.7
IC50 < 10 mM) carrying 8 to 15 carbons. Of interest, none of the
synthetic derivatives could outperform the parental compound 2.
Mono- or bimethylation of the hydroxyl groups (“a-, b- and c-se-
ries”) clearly decreased mPGES-1-inhibitory potency (Table S1) and
only the 2,5-dihydroxy derivatives 19d, 20d, and 23d with C8-, C10-
2.4. Effects of benzoquinones on the 5-LO-related enzymes 12-LO,
15-LO-1 and on mPGES-1
and C12-n-alkyl chains reached IC50 values ꢂ1
derivative 19d (IC50 ¼ 0.8 M), carrying a C8-n-alkyl residue, was
still about 4-fold less potent than 2. Thus, 2 with unmethylated 2,5-
dihydroxy moieties and C11-n-alkyl residue and IC50
M [13] exhibits optimal structural properties as
mM. The most potent
Besides 5-LO, other human LOs such as 12-LO and 12/15-LO (also
termed 15-LO-1) catalyze the incorporation of molecular oxygen
into AA (at C12 and C15, respectively) as substrate [26]. In order to
determine the selectivity of the investigated benzoquinones, we
analyzed their effects on the formation of 12(S)-hydroxy-6-trans-
8,11,14-cis-eicosatetraenoic acid (12-HETE) and 15(S)-hydroxy-
5,8,11-cis-,13-trans-eicosatetraenoic acid (15-HETE) that are pro-
duced by 12- and 12/15-LOs in PMNL incubations as well. Except for
compounds 19c and 23a as well as for the C6-n-alkyl derivatives
18b and 18c, none of the test compounds at a concentration of
m
a
value ¼ 0.21
m
mPGES-1 inhibitor out of all these alkyl-substituted benzoquinone
derivatives.
3. Conclusions
10 mM significantly suppressed the synthesis of 12-HETE or of 15-
HETE in PMNL incubations (Table S1), indicating that in particular
the most active compounds (e.g., 20c, 21c, 22c, and 23c) are highly
Among the multitude of 5-LO inhibitors discovered thus far,
especially from natural sources, many compounds exhibit disad-
vantageous structural features that impair the 5-LO inhibitory

R. Filosa et al. / European Journal of Medicinal Chemistry 94 (2015) 132e139
137
effectiveness inside the cell, apparently due to inadequate uptake
or intracellular inactivation. This is the case also for the natural
4.1.2. General procedure for the synthesis of compounds 17d-20d,
22d-28d
product 2 that potently inhibits 5-LO in cell-free assays with an
Aqueous sodium hydroxide (2 M; 1 ml) was added to a solution
of 17a-20a or 22a-28a (0.045 mmol) in ethanol (2 ml). The mixture
was heated to 70 ꢁC for a period of 2 h and allowed to cool to room
temperature. The reaction mixture was diluted with hydrochloric
acid (2 M; 5 ml) then extracted with ethyl acetate (3 ꢃ 5 ml). The
combined organic phases were dried over MgSO4 and the solvent
was evaporated to give the title compounds as orange solids.
IC50 ¼ 0.06
m
M but showed reduced potency in leukocytes (up to
33-fold; IC50 ¼ 0.8e2
mM) [13], seemingly related to the polar acidic
hydroxyl groups that may hamper membrane permeability and/or
might be subject to rapid oxidation in the cell. From the systematic
structural modifications of 2, aiming to obtain analogues with
improved efficiency in intact cells, bimethylation of the hydroxyl
moieties was revealed as successful mean to significantly improve
cellular 5-LO inhibition. Conversion of the 1,4- to
a
1,2-
4.2. Biological evaluation and assay systems
benzoquinone core further governs 5-LO inhibitory potency, and
when such 4,5-dimethoxy-1,2-benzoquinones are equipped with
simple C10- to C14-n-alkyl residues highly potent 5-LO inhibitors
with IC50 values in the range of 29e150 nM in leukocytes and
40e130 nM for isolated 5-LO are obtained. From our SAR analysis of
the 48 novel derivatives, compound 22c turned out to be of
particular pharmacological value, as it most potently inhibited
cellular 5-LO product synthesis. Regarding inhibition of mPGES-1,
none of the novel benzoquinones could outperform the parental
4.2.1. Materials
The 5-LO inhibitor 29 (zileuton) was from Sequoia Research
Products (Oxford, UK), and the mPGES-1 inhibitor 30 (MK886) and
the LO inhibitor 31 (CDC) were from Cayman Chemical (Ann Arbor,
€
MI). PGH2 was from Larodan (Malmo, Sweden). DMEM/High
Glucose (4.5 g/l) medium, Nycoprep, penicillin, streptomycin, and
trypsin/EDTA solution were from PAA Laboratories (Linz, Austria).
HPLC solvents were from VWR (Darmstadt, Germany). All other
chemicals were purchased from SigmaeAldrich (Deisenhofen,
Germany), unless stated otherwise.
compound 2 (IC50 ¼ 0.21
m
M). Importantly, 22c failed to strongly
inhibit mPGES-1 (IC50 > 10
mM) and did not affect the related 12-
and 15-LO, thus implying specific interference with 5-LO rather
than unspecific formation of radical intermediates. Only very few
other selective 5-LO inhibitors have been reported with higher
potency than 22c in intact cells. Together, our systematic natural
product-based synthesis of novel benzoquinones identified privi-
leged, simple structures that are selective and highly potent 5-LO
inhibitors with excellent cellular activity that warrant further
pharmacological characterization and preclinical analysis.
4.2.2. Cells and cell isolation
PMNL were isolated from human blood as reported before [13].
In brief, human peripheral blood was obtained from fastened (12 h)
healthy donors with consent that had not taken any anti-
inflammatory drugs during the last 10 days, with venipuncture in
heparinized tubes (16 IE heparin/ml blood) (University Hospital
Jena, Germany). The blood was centrifuged at 4000ꢃ g for 20 min at
20 ꢁC for preparation of leukocyte concentrates. Leukocyte con-
centrates were then subjected to dextran sedimentation and
centrifugation on Nycoprep cushions. Contaminating erythrocytes
of pelleted polymorphonuclear leukocytes (PMNL) were lysed by
hypotonic lysis. PMNL were washed twice in ice-cold PBS
(purity > 96e97%) and finally resuspended in PBS pH 7.4 containing
1 mg/ml glucose and 1 mM CaCl2 (PGC buffer) as indicated.
For analysis of acute cytotoxicity of the compounds during pre-
incubation periods (30 min at 37 ꢁC), cellular integrity of PMNL was
analyzed by trypan blue exclusion with a Vi-cell counter (Beck-
mann Coulter GmbH, Krefeld). None of the compounds caused
significant loss of PMNL viability within 30 min (data not shown).
4. Experimental section
4.1. Compounds and chemistry
All reagents were analytical grade and purchased from Sigma-
eAldrich (Milano, Italy). Flash chromatography was performed on
Carlo Erba silica gel 60 (230÷400 mesh; Carlo Erba, Milan, Italy). TLC
was carried out using plates coated with silica gel 60 F254 nm
purchased from Merck (Darmstadt, Germany). 1H and 13C NMR
spectra were registered on a Bruker AC 300. Chemical shifts are
reported in ppm. The purity (95% or higher) of all final products that
were evaluated for bioactivity was assessed by combustion analysis
(using a Carlo Erba 1106 elemental analyser) and is reported in the
Supporting Information. The test compounds were dissolved in
DMSO and stored in the dark at ꢀ20 ꢁC; freezing/thawing cycles
were kept to a minimum.
4.2.3. Expression and purification of human recombinant 5-LO
E.coli BL21 was transformed with pT3-5-LO plasmid, and re-
combinant 5-LO protein was expressed at 27 ꢁC as described [29].
Cells were lysed in 50 mM triethanolamine/HCl pH 8.0, 5 mM EDTA,
soybean trypsin inhibitor (60
methanesulphonyl fluoride, and lysozyme (500
m
g/ml),
1
mM phenyl-
mg/ml), homoge-
4.1.1. General procedure for synthesis of compounds 17a-28a, 17b-
28b, 17c-28c
nized by sonication (3 ꢃ 15 s), and centrifuged at 40,000ꢃ g for
20 min at 4 ꢁC. The 40,000ꢃ g supernatant (S40) was applied to an
ATP-agarose column to partially purify 5-LO as described previ-
ously [29]. Semi-purified 5-LO was immediately used for activity
assays.
To a solution of compounds 5e16 (1 mmol) in CH3CN (10 ml),
was added a solution of CAN (2.5 mmol) in CH3CNeH2O (10 mL,
7:3) dropwise at ꢀ7 ꢁC (salt-ice bath).
Method A. The reaction was stirred for 10 min at ꢀ7 ꢁC and
diluted with diethyl ether (50 ml). The organic layer was washed
two times with distilled water (20 ml), brine (20 ml), dried over
Na2SO4, and concentrated in vacuo. The residue was purified over
silica gel using hexane:EtOAc (8:2) as eluent to give compounds
17a-28a and 17c-28c.
4.2.4. Determination of 5-LO activity in the cell-free assay
Aliquots of semi-purified 5-LO were diluted with ice-cold PBS
containing 1 mM EDTA, and 1 mM ATP was added. Samples were
pre-incubated with the test compounds or vehicle (0.1% DMSO) as
indicated. After 10 min at 4 ꢁC, samples were pre-warmed for 30 s
Method B. The reaction was allowed to stir at rt for 2 h, and
diluted with ether (50 ml). The organic layer was washed with
distilled water (20 ml), brine (20 ml), dried over Na2SO4, and
concentrated in vacuo. The residue was purified over silica gel using
hexane:EtOAc (8:2e7:3) as eluent to give compounds 17a-28a and
17b-28b.
at 37 ꢁC, and 2 mM CaCl2 plus 20
mM AA was added to start 5-LO
product formation. The reaction was stopped after 10 min at
37 ꢁC by addition of 1 ml ice-cold methanol, and the formed me-
tabolites were analyzed by RP-HPLC as described [30]. 5-LO prod-
ucts include the all-trans isomers of LTB4 and 5(S)-hydro(pero)xy-

138
R. Filosa et al. / European Journal of Medicinal Chemistry 94 (2015) 132e139
Pharmacol. Ther. 112 (2006) 701e718.
6-trans-8,11,14-cis-eicosatetraenoic acid.
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For determination of LO products in intact cells, PMNL (5 ꢃ 106)
were resuspended in 1 ml PGC buffer, preincubated for 15 min at
37 ꢁC with test compounds or vehicle (0.1% DMSO), and incubated
for 10 min at 37 ꢁC with the indicated stimuli. Ca2þ ionophore
A23187 (2.5
the reaction was stopped on ice by addition of 1 ml of methanol.
30 l 1 N HCL and 500 l PBS, and 200 ng prostaglandin (PG)B1
were added and the samples were subjected to solid phase
extraction on C18-columns (100 mg, UCT, Bristol, PA, USA). 5-LO
products (LTB4, trans-isomers, 5-H(P)ETE), and the 12- and 15-LO
products 12-HETE and 15-HETE, respectively, were analyzed by
RP-HPLC and quantities calculated on the basis of the internal
standard PGB1. Cys-LTsC4, D4 and E4 were not detected (amounts
were below detection limit), and oxidation products of LTB4 were
not determined.
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Preparations of A549 cells and determination of mPGES-1 ac-
tivity was performed as described previously [28]. In brief, A549
cells were treated with 1 ng/ml Il-1b
for 48 h at 37 ꢁC and 5% CO2.
Cells were harvested, sonicated and the homogenate was subjected
to differential centrifugation at 10,000ꢃ g for 10 min and 174,000ꢃ
g for 1 h at 4 ꢁC. The pellet (microsomal fraction) was resuspended
in 1 ml homogenization buffer (0.1 M potassium phosphate buffer,
pH 7.4, 1 mM phenylmethanesulfonyl fluoride, 60
mg/ml soybean
trypsin inhibitor, 1 g/ml leupeptin, 2.5 mM glutathione, and
m
250 mM sucrose). Microsomal membranes were diluted in potas-
sium phosphate buffer (0.1 M, pH 7.4) containing 2.5 mM gluta-
thione. Test compounds or vehicle were added, and after 15 min at
4
ꢁC reaction was initiated by addition of PGH2 at the indicated
concentration. After 1 min at 4 ꢁC, the reaction was terminated
using stop solution (40 mM FeCl2, 80 mM citric acid, and 10 M 11b-
m
PGE2 as internal standard. PGE2 was separated by solid-phase
extraction and analyzed by RP-HPLC as described, previously [28].
4.2.7. Statistics
Data obtained are expressed as mean
S.E. of single de-
terminations performed in three or four independent experiments
at different days. IC50 values were graphically calculated from
averaged measurements at 4-5 different concentrations of the
compounds using SigmaPlot 12.0 (Systat Software Inc., San Jose,
USA). Statistical evaluation of the data was performed by one-way
ANOVA followed by a Bonferroni or TukeyeKramer post-hoc test
for multiple comparisons respectively. A p value <0.05 (*) was
considered significant.
Acknowledgement
The authors thank Katrin Fischer, Monika Listing, Marius Melzer,
and Heidi Traber for expert technical assistance.
Appendix A. Supplementary data
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J. Maczewsky, S. Pace, A. Rossi, L. Sautebin, E. Banoglu, O. Werz, The novel
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in vivo by targeting 5-lipoxygenase-activating protein (FLAP), Br. J. Pharmacol.
171 (2014) 3051e3064.
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D.W. Brooks, 5-Lipoxygenase inhibitory activity of zileuton, J. Pharmacol. Exp.
Ther. 256 (1991) 929e937.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.02.042.
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