Identification of 2-mercaptohexanoic acids as dual inhibitors of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1

Article abstract of DOI:10.1016/j.bmc.2011.04.034

5-Lipoxygenase (5-LO) and microsomal prostaglandin E₂ synthase (mPGES)-1 are key enzymes in the biosynthesis of leukotrienes and prostaglandin (PG)E₂, respectively, and are considered as valuable targets for the treatment of inflamma

Full text of DOI:10.1016/j.bmc.2011.04.034

Bioorganic & Medicinal Chemistry 19 (2011) 3394–3401
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of 2-mercaptohexanoic acids as dual inhibitors
of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1
Christine Greiner a, , Heiko Zettl b, , Andreas Koeberle a, Carlo Pergola c, Hinnak Northoff d,
Manfred Schubert-Zsilavecz b, Oliver Werz c,
⇑
a Department of Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard Karls University Tuebingen, Auf der Morgenstelle 8, D-72076 Tuebingen, Germany
b Institute of Pharmaceutical Chemistry, ZAFES/OSF, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany
c Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, D-07743 Jena, Germany
d Institute for Clinical and Experimental Transfusion Medicine, University Medical Center, Tuebingen, Hoppe-Seyler-Straße 3, 72076 Tuebingen, Germany
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) and microsomal prostaglandin E2 synthase (mPGES)-1 are key enzymes in the bio-
synthesis of leukotrienes and prostaglandin (PG)E2, respectively, and are considered as valuable targets
for the treatment of inflammatory diseases. Here, we present the identification of 2-mercaptohexanoic
acid derivatives as dual inhibitors of 5-LO and mPGES-1. The lead compound 2(4-(3-biphenyloxyprop-
oxy)phenylthio)hexanoic acid (21) inhibits human 5-LO and mPGES-1 in cell-free assays with an
Received 17 February 2011
Accepted 17 April 2011
Available online 22 April 2011
Keywords:
IC50 = 3.5 and 2.2
l
M, respectively, and suppresses 5-LO in intact cells with even a higher potency
M) neither significantly inhibited the related 12- or 15-LOs nor cyclo-
5-Lipoxygenase
Leukotriene
Neutrophils
Inflammation
Prostaglandin
(IC50 = 0.9 M). Compound 21 (10
l
l
oxygenase-1 and -2 or cytosolic phospholipase A2. Based on the selective and potent inhibition of 5-LO
and mPGES-1, further assessment of these 2-mercaptohexanoic acids in preclinical models of inflamma-
tion are warranted.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
addition, the concomitant increase of LT formation observed
during COX inhibition may contribute to the NSAID-related tox-
Prostanoids and leukotrienes (LTs), formed from arachidonic
acid (AA) via the cyclooxygenase (COX)-1/2 and 5-lipoxygenase
(5-LO) pathway, respectively, are powerful lipid mediators that
mediate inflammatory responses, chronic tissue remodelling,
cancer, asthma, and autoimmune disorders, but also possess
homeostatic functions in the gastrointestinal tract, uterus, brain,
kidney, vasculature, and host defence.1 Whether distinct prosta-
noids are beneficial or harmful for the body depends on the tar-
geted organ or tissue as well as on the local concentrations of
the respective prostanoid. For example, prolonged and excessive
formation of prostaglandin (PG)E2 may cause inflammation, pain,
fever and carcinogenesis whereas low amounts of PGE2 in the
stomach or kidney are important for normal physiology of
these organs.2,3 By inhibiting COX-1/2, the non-steroidal anti-
inflammatory drugs (NSAIDs) block the synthesis of all prosta-
noids, including those with protective and beneficial functions
(e.g., PGE2 and PGI2). Based on the resulting lack of these pros-
tanoids, NSAIDs may cause severe side effects including gastro-
intestinal injury, renal irritations, and cardiovascular risks.4 In
icity.5,6 Accordingly, anti-inflammatory agents interfering with
eicosanoid biosynthesis require a well-balanced pharmacological
profile to minimize these on-target side effects. Current anti-
inflammatory drug discovery attempts to identify compounds
that can suppress the massive formation of pro-inflammatory
PGE2 without affecting homeostatic PGE2 and PGI2 synthesis.
To this aim, inhibition of microsomal PGE2 synthase-1
(mPGES-1) activity3,7 may provide efficient anti-inflammatory
drugs with fewer side effects. In fact, among the three isomeric
PGE2 synthases, the inducible isoform mPGES-1 is essentially the
one involved in the overproduction of PGE2 during inflammation.
The concept of selective mPGES-1 inhibition has led to lead
compounds of different structural classes, including indole deriv-
atives,8,9 phenanthrene imidazoles,10 benzamides,11 and pirinixic
acid derivatives.12 A novel pharmacological strategy focuses on
the dual inhibition of mPGES-1 and 5-LO in order to selectively
suppress the synthesis of pro-inflammatory PGE2 and LTs,
respectively.13–15 Besides some natural products including myr-
tucommulone,16 arzanol,17 garcinol,18 and curcumin,19 series of
benzo[g]indole-3-carboxylates,9,20
a-n-alkyl- or a-aryl-substi-
tuted pirinixic acid derivatives,12 and arylpyrrolizines14 were
shown to block both mPGES-1 and 5-LO, and studies of these
compounds in animal models of inflammation revealed high
anti-inflammatory efficacy.17,20,21
⇑
Corresponding author. Tel.: +49 3641 949801; fax: +49 3641 949802.
E-mail address: oliver.werz@uni-jena.de (O. Werz).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.034

C. Greiner et al. / Bioorg. Med. Chem. 19 (2011) 3394–3401
3395
We have recently presented the design, synthesis, and the bio-
logical evaluation of novel 2-mercaptohexanoic acid derivatives as
LO, and cPLA2. In contrast to mPGES-1 inhibitors that have just be-
gun to be developed, agents that suppress 5-LO product synthesis
have been the subject of intensive research efforts over the past
30 years and their molecular pharmacology is well characterized.
Thus, the efficient inhibition of 5-LO by the lead compound 21 in
a cell-free and a cell-based assay prompted for more detailed anal-
ysis of the interference with 5-LO and revealed an advantageous
pharmacological profile of this compound.
activators of peroxisome proliferator-activated receptors (PPARs)
a
and c 22
.
Here we present the identification of these 2-mercapto-
hexanoic acid derivatives as dual inhibitors of 5-LO and mPGES-1
that efficiently suppress the formation of LTs and PGE2 without
marked inhibition of the cellular synthesis of other oxygenated
metabolites generated by related enzymes including COX, 12/15-
Ar-OH
(ii)
O
O
O
Ar
SH
+
O
OH
S
S
S
(iv)
(i)
O
O
OH
(iii)
HO
Z
Z
Ar
Z
Ar
Z
HO
O
O
O
O
Z=nBu
Z=H
(ii)
S
(iii)
Ar-residues see Table 1
Ar-OH
O
O
HO
O
Scheme 1. Synthesis of 2-mercaptohexanoic acid derivatives and of the 2-mercaptoacetic acid derivative (1). Reagents and conditions: (i) ethyl-2-bromoacetate (Z = H) or
ethyl-2-bromohexanoate (Z = nBu), Et3N, CHCl3, reflux, 1 h; (ii) 3-bromopropan-1-ol, K2CO3, ACN, reflux, 24–48 h; (iii) DEAD or ADDP, TPP, THF, rt, 4–48 h; (iv) LiOH, MeOH,
H2O, 40 °C, 2 h.
15-H(P)ETE
12-H(P)ETE
5-H(P)ETE
A
B
LPS/fMLP
A23187
500
400
300
160
140
120
100
80
***
LTB
4
200
150
***
100
50
0
60
40
20
0
0
0.3
1
3
10
0
0.3
1
3
10
21 [µM]
21 [µM]
0 µM AA
D
C
20 µM AA
40 µM AA
60 µM AA
120
100
80
60
40
20
0
A23187
NaCl
120
100
80
60
40
20
0
0
0.3
1
3
10
0
0.3
1
3
10
21 [µM]
21 [µM]
Figure 1. Inhibition of 5-LO activity by compound 21 in cell-based assays. (A) Inhibition of 5-LO, 12-LO and 15-LO in human PMNL stimulated with 2.5
20 M AA. (B) Comparison of 5-LO inhibition by 21 between PMNL challenged with 2.5 M A23187 or with 1 g/ml LPS and 100 nM fMLP. (C) Comparison of 5-LO inhibition
by 21 between PMNL challenged with 2.5 M A23187 or 0.3 M NaCl in the presence of 40 M AA, each. (D) Inhibition of 5-LO in PMNL stimulated with 2.5 M A23187
together with exogenous AA at varying concentrations. Data are mean SE, n = 3-4. ⁄⁄⁄p < 0.001 for samples treated with A23187 versus those treated with LPS/fMLP.
lM A23187 plus
l
l
l
l
l
l

3396
C. Greiner et al. / Bioorg. Med. Chem. 19 (2011) 3394–3401
O
O
(ii)
O
O
O
P
Br
O
O
O
O
O
(vi)
(iv)
O
O
OH
O
O
O
O
O
O
(iii)
(i)
O
(v)
OH
O
O
O
OH
(iii)
O
O
O
O
(vi)
(vi)
O
O
OH
OH
O
O
O
O
Scheme 2. Synthesis of carbon analogs 5, 6 and 7. Reagents and conditions: (i) 3-bromopropan-1-ol, K2CO3, ACN, reflux, 24 h; (ii) triethylphosphite, reflux, overnight;
(iii) 4-hydroxybenzaldehyde or ethyl 3-(4-hydroxyphenyl)propanoate, DEAD, TPP, THF, rt, 4 h; (iv) NaH, THF, rt, 24 h; (v) Pd/C, H2, EtOH, rt, 24 h; (vi) LiOH, MeOH, H2O, reflux,
8–20 h.
well-recognized inhibitor of mPGES-1,28 and the selective iron
2. Results and discussion
ligand-type 5-LO inhibitor 25 (BWA4C29) were used as controls.
The test compounds were first analysed at a concentration of
2.1. Chemistry
10
lM. IC50 values were determined for those compounds that
suppressed enzyme activity by more than 50%, whereas the
remaining enzyme activity is given for the others (Table 1). Test
concentrations >10 lM in cell-based assays were not considered
The synthesis of the presented compounds 1–24 has been de-
scribed previously.22 The 2-mercaptohexanoic acid derivatives
were synthesized starting with 4-mercaptophenol as shown in
because of inconsistent solubility behavior in the aqueous assay
buffers at higher concentrations, and thus assessment of IC50 val-
ues of poor inhibitors (IC50 >10 lM) was not further explored. As
shown in Table 1, the 2-substituted acetic acid derivative 1 caused
only weak inhibition of both mPGES-1 and of 5-LO, whereas elon-
gation of the alkyl chain to the corresponding hexanoic acid 2
yielded an efficient dual 5-LO/mPGES-1 inhibitor. Of interest, there
was no significant difference in the potencies of the (R)-(3) and the
(S)-(4) enantiomers implying that the absolute configuration of the
Scheme 1. First, nucleophilic substitution with
a-bromoesters
yielded the thioether derivatives. The lipophilic backbone of the
compounds is arranged by two ether syntheses (by coupling with
3-bromopropan-1-ol and by a Mitsunobu reaction with commer-
cially available phenols). Finally, hydrolysis of the ester group
yielded the desired carboxylic acids. Enantioselective resolution
of 2 was achieved by enantioselective preparative HPLC as de-
scribed.23 The carbon analogs (6 and 7) were prepared starting
with the conversion of ethyl 2-bromohexanoate into its phospho-
nate derivative by an Arbuzov reaction (Scheme 2). The product
was coupled by a Wittig–Horner reaction to the aldehyde group
of a precursor derived from 4-hydroxybenzaldehyde. The resulting
ester was directly hydrolyzed (to yield 7) as well as hydrogenated
and afterwards hydrolyzed (to yield 6). Only two steps (Mitsunobu
condensation and hydrolysis) were needed for the preparation of
a
-C atom is negligible. A loss of effectiveness was observed after
replacing the sulfur of 1 or of 2 by carbon yielding 5 and 6, as
well as in the corresponding alkene derivative of 6 ( -(n)-butyl-
a
substituted cinnamonic acid derivative 7). Interestingly, removal
of the two methyl moieties from the phenyl ring of 2 (yielding 8)
was clearly detrimental. Single methylation partially improved
the inhibition for mPGES-1 regardless of the positioning (in ortho
(9), meta (10), para (11)) in the phenyl moiety, though the com-
pounds were less potent than the double (2,3-) methylated 2. Also
insertion of oxygen yielding the 2-methoxy- (12) and the 3-meth-
oxy- (13) derivatives of 8 was essentially without effect. On the
other hand, enlargement of the alkyl substituent such as introduc-
tion of a 2-isopropyl (14) or 3-isopropyl (15) residue significantly
increased the efficiency over the corresponding methyl-substi-
tuted analogs 9 and 10, respectively. Also replacement of the 3-
methyl group in 10 by a trifluoromethyl residue (16) enhanced
both inhibition of 5-LO and of mPGES-1, and the potency to inhibit
5-LO could be further improved by introducing chlorine in 4-posi-
tion (17). Interestingly, the 4-chloro-2-methoxy-phenyl derivative
18 was significantly less efficient. On the other hand, the bicyclic
derivatives 19 and 21 were active, whereas upon replacement by
a quinoline moiety (23), the inhibitory effect was lost. Taken to-
gether, 17 was the most efficient inhibitor for both mPGES-1 and
the a-unsubstituted carbon analog 5.
2.2. Biology
2.2.1. Screening of dual inhibitors of 5-LO and mPGES-1
First, the test compounds were assayed for their ability to inhi-
bit mPGES-1 and 5-LO in cell-free test systems. For analysis of
mPGES-1 inhibition, microsomal preparations of IL-1b-stimulated
A549 cells (5 lg/100 ll) were pre-incubated with the test com-
pounds and assayed for suppression of enzymatic PGE2 formation
from PGH2 at standardized assay conditions (i.e., potassium phos-
phate buffer 0.1 M, pH 7.4, 20 lM PGH2 as substrate, and 2.5 mM
glutathione as co-substrate24), modified according to Jakobsson
et al.25 Since the cyclic peroxide PGH2 is highly reactive and spon-
taneously degrades non-enzymatically to PGF2, PGD2, and PGE2 in
aqueous solutions within a few minutes,26 the enzymatic assay
was performed at 4 °C for 1 min in order to avoid non-enzymatic
conversion of PGH2 to PGE2. For analysis of 5-LO inhibition, human
recombinant 5-LO enzyme was expressed in Escherichia coli and
purified by affinity chromatography, and 5-LO activity was assayed
5-LO with IC50 values of 1.7 and 2 lM, respectively.
In order to analyze whether the compounds penetrate through
the cell membrane and are active also in the cellular context, inter-
ference with 5-LO product formation was assessed in human
using 20 l
M AA as substrate.27 The indole derivative 26 (MK886), a

C. Greiner et al. / Bioorg. Med. Chem. 19 (2011) 3394–3401
3397
Table 1
Inhibition of mPGES-1 and 5-LO in cell-free assays by test compounds and suppression of 5-LO product synthesis in intact cells (expressed as IC50 values, mean of n = 3–5
experiments)
O
R3
X
O
mPGES-1 IC50 in [
(remaining activity at
10 SE [%])
l
M]
5-LO cell-based IC50 in [
(remaining activity at
lM]
5-LO cell-free IC50 in [
(remaining activity at
lM]
Compd
R2
R1
l
M
10
l
M
SE [%])
10
l
M
SE [%])
O
O
X
R1
R2
R3
CH3
CH3
1
2
3
4
5
6
S
H
H
>10 (75.4 0.9)
>10 (69.6 9.8)
>10 (72.4 4.0)
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
S
S
S
Butyl
H
H
H
H
H
2.9
3.0
8.8
(R)-
Butyl
4.2
1.1
10.0
(S)-
Butyl
4.6
2.0
8.2
CH2
CH2
H
>10 (83.9 14.2)
>10 (80.0 2.0)
>10 (81.8 4.4)
>10 (70.8 5.0)
Butyl
7.1
2.0
O
OH
7
>10 (28.3 10.3)
1.6
>10 (88.9 2.7)
O
O
8
9
S
S
Butyl
Butyl
H
H
>10 (81.9 5.6)
8.8
9.5
4.8
>10 (69.4 4.8)
>10 (58.3 6.8)
CH3
CH3
10
S
Butyl
H
>10 (58.0 5.5)
4.8
>10 (79.3 10.6)
H3C
11
12
S
S
Butyl
Butyl
H
H
9.3
5.2
>10 (67.6 11.7)
>10 (79.4 6.4)
OMe
>10 (88.3 3.0)
>10 (57.4 11.9)
OMe
13
14
S
S
Butyl
Butyl
H
H
>10 (59.1 6.5)
4.8
1.6
>10 (67.7 5.8)
CH3
CH3
4.1
7.8
(continued on next page)

3398
C. Greiner et al. / Bioorg. Med. Chem. 19 (2011) 3394–3401
Table 1 (continued)
O
R3
X
O
mPGES-1 IC50 in [
(remaining activity at
l
M]
5-LO cell-based IC50 in [
(remaining activity at
lM]
5-LO cell-free IC50 in [
(remaining activity at
lM]
Compd
R2
R1
10
lM
SE [%])
10
l
M
SE [%])
10
l
M
SE [%])
O
O
X
R1
R2
R3
H3C
CH3
15
16
S
S
Butyl
H
2.7
2.0
1.4
6.0
CF3
Butyl
H
1.6
1.5
9.8
2.0
CF3
Cl
Cl
17
18
S
S
Butyl
Butyl
H
H
1.7
9.4
OMe
>10 (79.3 14.0)
>10 (59.2 7.4)
19
20
S
S
Butyl
Butyl
H
2.2
1.4
4.5
C2H5 nd
>10 (87.4 2.8)
>10 (79.4 28.4)
21
22
S
S
Butyl
Butyl
H
2.2
0.9
3.5
C2H5 nd
>10 (91.5 0.9)
>10 (83.0 38.7)
N
N
23
24
S
S
Butyl
Butyl
H
>10 (ni)
>10 (92.2 3.4)
0.8
>10 (59.0 12.7)
>10 (68.3 12.8)
C2H5 >10 (83.9 3.6)
O
O
25
BWA4C
nd
0.08
0.16
N
OH
S
COOH
26
MK886
N
2.4
0.09
ni at 1 lM
Cl
For compounds with IC50 >10
ni = no inhibition.
lM, the remaining enzymatic activity (in %) is given at 10 lM.
nd = not done.
polymorphonuclear leukocytes (PMNL) stimulated with A23187
analysed by RP-HPLC as described.31 For mPGES-1 inhibitors, a di-
rect test system that allows select analysis of interference with
endogenous mPGES-1 activity in the cell (i.e., the distinct transfor-
mation of PGH2 to PGE2) is not available. We observed that for inhi-
bition of 5-LO product synthesis in intact cells, the compounds
plus exogenous AA (20 lM) which is a suitable and convenient
cell-based model to study 5-LO inhibitors.30 The 5-LO products
(i.e., LTB4 and its trans-isomers and 5(S)-hydro(pero)xy-6-trans-
8,11,14-cis-eicosatetraenoic acid (5-H(P)ETE)) were extracted and

C. Greiner et al. / Bioorg. Med. Chem. 19 (2011) 3394–3401
3399
were consistently more efficient than in cell-free assays, with the
exception of the quinoline-substituted compound 23 that essen-
tially failed in intact cells. For example, the IC50 values of 16 or
inhibitors, non-redox-type 5-LO inhibitors and agents with distinct
modes of 5-LO inhibition.30 The lack of moieties with reducing
properties in the structures of 17, 19 or 21 rather excludes a redox-
or radical scavenging-based 5-LO inhibitory mechanism, and also
typical iron-chelating moieties of prototype iron-ligand inhibitors
are absent. In order to gain more insights into the molecular mode
of action, we performed mechanistic studies with compound 21,
21 were 9.8 and 3.5 lM for isolated 5-LO but 1.6 and 0.9 lM in in-
tact PMNL, respectively. Intriguingly, esterification of 21 and 19
leading to 22 and 20, respectively, caused a strong loss of efficiency
in intact cells as well as in cell-free assays. On the other hand, the
corresponding ethyl ester of the inactive 23, that is, compound 24
turned out to be a potent 5-LO inhibitor in intact cells (but not in
the most attractive 5-LO inhibitor in cell-based (IC50 = 0.9
lM)
and cell-free assays (IC50 = 3.5 M) from our SAR investigations.
l
cell-free assays) with an IC50 = 0.8
l
M, which is not readily under-
Preparations of PMNL, isolated from human peripheral blood,
generate not only 5-LO products but also transform AA via plate-
let-type 12-LO (present in PMNL adherent platelets) and 15-LO
(in eosinophilic granulocytes) to 12(S)-hydro(pero)xy-5,8-cis-10-
trans-14-cis-eicosatetraenoic acid (12(S)-H(P)ETE) and 15(S)-hy-
dro(pero)xy-5,8,11-cis-13-trans-eicosatetraenoic acid (15(S)-H(P)
ETE). As shown from Figure 1A, compound 21 caused no significant
suppression of 12(S)-H(P)ETE and 15(S)-H(P)ETE in PMNL up to
stood. Together, 2-mercaptohexanoic acids with large and lipo-
philic aryloxy residues exemplified by the leads 17, 19 and 21
represent potent dual inhibitors of 5-LO and mPGES-1.
In view of their structure, the active 2-mercaptohexanoic acids
may resemble long chain fatty acids such as AA. Since 5-LO binds
AA as substrate in the active site and AA is an inhibitor of
mPGES-1 (IC50 = 0.3 lM), it appeared reasonable that these 2-
mercaptohexanoic acids act as AA mimetics and bind into the ac-
tive sites of mPGES-1 and of 5-LO. In view of such considerations,
besides 5-LO and mPGES-1, other enzymes within the AA cascade
could be affected by the 2-mercaptohexanoic acids. Hence, we
investigated the effects of the lead compounds 17, 19 and 21 on
the activities of isolated COX-1 and -2, and (at least for 21) on iso-
lated cPLA2. Reference inhibitors were indomethacin for COX-1,
10
l
M, again supporting selectivity for 5-LO/mPGES-1.
The structural properties led us to hypothesize that 21 may act
as non-redox-type 5-LO inhibitor. Because the potency of non-
redox-type 5-LO inhibitors can vary depending on the stimulus
and signalling pathway inducing 5-LO product synthesis,32 we ana-
lysed the potency of 21 on 5-LO product formation in PMNL stim-
ulated with A23187 and in cells challenged with fMLP upon
celecoxib for COX-2 and the cPLA2 inhibitor N-{(2S,4R)-4-(biphe-
priming with LPS. Whereas A23187 activates 5-LO via a Ca2+
-
a
nyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-ben-
zoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylide-
nemethyl)-phenyl]acrylamide that blocked the respective enzyme
activities as expected (not shown). The results presented in
Table 2 show that none of the 2-mercaptohexanoic acid derivatives
possess a significant inhibitory effect on any of these enzymes,
suggesting selectivity for 5-LO and mPGES-1.
dependent mechanism, LPS/fMLP may partially act by Ca2+ but also
via induction of 5-LO phosphorylation by mitogen-activated pro-
tein kinases (MAPK).33 In fact, non-redox-type 5-LO inhibitors
were shown to be less potent when 5-LO is activated by phosphor-
ylation.32 As shown in Figure 1B, the potency of 21 was somewhat
decreased upon stimulation with LPS/fMLP as compared to
A23187. Moreover, we studied the efficiency of 21 in PMNL ex-
posed to 300 mM NaCl that causes cell stress leading to 5-LO acti-
vation by phosphorylation independent of Ca2+. However, no
striking differences in the potency of 21 were evident (Fig. 1C).
Next, we tested whether the substrate concentration may influ-
ence the 5-LO inhibitory effect of 21, again a phenomenon ex-
pected for competitive, non-redox-type 5-LO inhibitors.34 The
potency of 21 is only slightly reduced when exogenous AA was
provided in excess (up to 40), suggesting that 21 inhibits 5-LO
essentially independent of the substrate concentration (Fig. 1D).
Besides cell-based analysis, studies with 21 were performed in
cell-free assays. Wash-out experiments (dilution from 10 to
2.2.2. Analysis of the mechanism of 5-LO inhibition by
compound 21
Based on their molecular mode of action, 5-LO inhibitors can be
categorized in general as redox-type inhibitors, iron ligand-type
Table 2
Inhibition of COX-1 and COX-2 and of cPLA2 by the compounds 17, 19 and 21 at a
concentration of 10 lM, n = 3
Compd COX-1 (remaining
activity at
COX-2 (remaining
activity at
cPLA2 (remaining
activity at
10
l
M
SE [%])
10
lM
SE [%])
10
lM
SE [%])
1 lM) demonstrated that the inhibitory effect of 21 on isolated
17
19
21
71.3 2.0
ni
79.2 8.8
ni
ni
ni
nd
nd
5-LO can be (at least partially) reverted (Fig. 2A), implying that
the compound does not irreversibly inactivate 5-LO or acts by
covalent binding to it. The potency of non-redox-type 5-LO inhib-
itors is typically impaired at elevated hydroperoxide levels.35,36
105.5 19.0
ni = no inhibition.
nd = not done.
120
A
B
120
100
80
60
40
20
0
w/o
100
80
60
40
20
0
+ 13(S)-HpODE
1
10 1 (10)
0
1
3
10
30
21 [µM]
21 [µM]
Figure 2. Inhibition of 5-LO activity by compound 21 in cell-free assays. (A) Inhibition of isolated human recombinant 5-LO by 21 is partially reversible upon 10-fold dilution.
(B) Impact of 13(S)-HpODE on the potency of 21 to inhibit isolated 5-LO. Data are mean SE, n = 4–5.

3400
C. Greiner et al. / Bioorg. Med. Chem. 19 (2011) 3394–3401
Hence, we analyzed inhibition of 5-LO by 21 in the absence and
presence of 1 M 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid
(13(S)-HpODE). As can be seen from Figure 2B, the potency of 21
was not affected by 13(S)-HpODE. Thus, 21 does not exhibit typical
properties of non-redox-type 5-LO inhibitors such as ZM230487 or
CJ-13610.35,36
3.2.2. Cells and cell viability assay
l
Human PMNL were freshly isolated from leukocyte concen-
trates obtained at the Blood Center of the University Hospital Tueb-
ingen (Germany). In brief, venous blood was taken from healthy
adult donors and leukocyte concentrates were prepared by centri-
fugation at 4000Âg for 20 min at 20 °C. PMNL were immediately
isolated by dextran sedimentation, centrifugation on Nycoprep
cushions (PAA Laboratories, Linz, Austria), and hypotonic lysis of
erythrocytes as described previously.38 Cells were finally resus-
pended in phosphate-buffered saline pH 7.4 (PBS) containing
1 mg/ml glucose and 1 mM CaCl2 (PGC buffer).
2.3. Discussion
One current pharmacological strategy aiming to overcome
NSAID-related toxicity pursues the dual interference with
mPGES-1 and 5-LO. In contrast to COX inhibition, interference with
mPGES-1 is thought to selectively suppress the pathological PGE2
generation thereby maintaining the house-keeping functions of
other prostanoids.13 Moreover, experimental evidence from inves-
tigations of NSAIDs in vitro and in vivo suggests that the elevated
LT formation due to target-related inhibition of prostanoid biosyn-
thesis contributes to the toxicity of NSAIDs.5,6 Here, we identified
simple 2-mercaptohexanoic acid derivatives as dual mPGES-1/5-
LO inhibitors with IC50 values in the low micromolar or even sub-
micromolar range. The lead compounds (i.e., 17, 19 and 21), elon-
gated with lipophilic bulky aryloxy moieties, have a well-balanced
activity on both enzymes and, at least concerning 5-LO, are active
also in intact cells with even improved efficiency. Elucidation of
the molecular mode of action of 5-LO inhibition revealed no
congruent mechanism with other typical 5-LO inhibitors such as
redox-active agents, iron-ligands or non-redox-type/competitive
5-LO inhibitors. Importantly, the lead 21 showed advantageous
features in terms of 5-LO inhibition such as no marked loss of po-
tency at elevated peroxide tone and at increased substrate concen-
trations, and no strong dependency on the stimulus. Moreover, 21
does not inhibit COX-1 and -2, cPLA2 and other human LOs (i.e.,
12- and 15-LO). Only few synthetic dual 5-LO/mPGES-1 inhibitors
have been described thus far13,15 and data related to gastrointesti-
nal, renal or cardiovascular toxicity of these substances in animal
models are still missing. At least, two recent studies of dual
mPGES-1/5-LO inhibitors (indole-3-carboxylates9 and pirinixic
acid derivatives37 in vivo provided encouraging results in terms
of anti-inflammatory efficacy. Together, the novel lead structures
presented in this study may serve as potential candidates for
intervention with inflammatory diseases and deserve further
exploration in preclinical studies.
A549 cells were cultured in DMEM/High glucose (4.5 g/l) med-
ium supplemented with heat-inactivated fetal calf serum (10%, v/
v), penicillin (100 U/ml), and streptomycin (100 lg/ml) at 37 °C
in a 5% CO2 incubator. After 3 days, confluent cells were detached
using 1 Â trypsin/EDTA solution and reseeded at 2 Â 106 cells in
20 ml medium in 175 cm2 flasks. Cell viability was measured using
the colorimetric MTT dye reduction assay. A549 cells (4 Â 104
cells/100 ll medium) were plated into a 96-well microplate and
incubated at 37 °C and 5% CO2 for 16 h. Then, test compounds
(10 M, each) or solvent (DMSO, never exceeding a final concentra-
tion of 0.3%) were added, and the samples were incubated for an-
other 5 h. MTT (20 l, 5 mg/ml) was added and the incubations
l
l
were continued for 4 h. The formazan product was solubilized with
SDS (10%, m/v in 20 mM HCl) and the absorbance of each sample
was measured at 595 nm relative to the absorbance of vehicle
(DMSO)-treated control cells using a multiwell scanning spectro-
photometer (Victor3 plate reader, PerkinElmer, Rodgau-Jueges-
heim, Germany). None of the compounds significantly reduced
cell viability at a concentration of 10 lM (data not shown), exclud-
ing possible acute cytotoxic effects of the compounds in the cellu-
lar assays.
3.2.3. Determination of product formation by 5-LO, 15-LO and
12-LO in cell-based assays
For assays of intact cells stimulated with ionophore A23187
or NaCl, 5 Â 106 freshly isolated PMNL were resuspended in
1 ml PGC buffer. After preincubation with the compounds for
15 min at 37 °C, 5-LO product formation was started by addition
of 2.5 lM A23187 plus exogenous AA (at the indicated concen-
trations) or 0.3 M NaCl was supplemented 3 min before addition
of AA. After 10 min at 37 °C, the reaction was stopped with 1 ml
of methanol and 30
ll of 1 N HCl, 200 ng prostaglandin B1 and
500 l of PBS were added. Formed 5-LO metabolites were ex-
l
tracted and analyzed by HPLC as described.39 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)-di-hy-
droxy-6,10-trans-8,14-cis-eicosatetraenoic acid (5(S),12(S)-DiHE-
TE), and 5-H(P)ETE. Cysteinyl LTsC4, D4 and E4 were not
detected, and oxidation products of LTB4 were not determined.
15-H(P)ETE was analyzed as product of 15-LO and 12-H(P)ETE
as product of 12-LO. To assess 5-LO product formation in cells
stimulated with fMLP, 2 Â 107 PMNL were resuspended in 1 ml
3. Experimental
3.1. Compounds and chemistry
Compounds 1–24 were synthesized as previously reported.22
All structures were confirmed by 1H and 13C NMR, as well as by
mass spectrometry (ESI). The purity (>98%) was checked by com-
bustion analysis as described.22
3.2. Assay systems
PBS/glucose, primed with 1 lg/ml LPS for 10 min at 37 °C and
0.3 U/ml adenosine deaminase was added. After another
10 min, cells were treated with the compounds for 10 min and
3.2.1. Materials
The cPLA2
inhibitor N-{(2S,4R)-4-(biphenyl-2-ylmethyl-
1 lM fMLP was added. The reaction was stopped on ice after
a
isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-
2-yl methyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl]
acrylamide was from Calbiochem (Bad Soden, Germany). DMEM/
High Glucose (4.5 g/l) medium, penicillin, streptomycin, trypsin/
EDTA solution, PGH2, Larodan (Malmö, Sweden); 11b-PGE2, PGB1,
MK886, [5,6,8,9,11,12,14,15-3H] arachidonic acid ([3H]AA),
BioTrend Chemicals GmbH (Cologne, Germany); Ultima Gold™ XR,
Perkin Elmer (Boston, MA). All other chemicals were obtained from
Sigma–Aldrich (Deisenhofen, Germany) unless stated otherwise.
5 min and cells were centrifuged (800Âg, 10 min). Formed LTB4
was determined by ELISA according to the manufacturer’s proto-
col (Assay Designs, Ann Arbor, MI).
3.2.4. Expression and purification of human recombinant 5-LO
from E. coli, and determination of 5-LO activity in cell-free
systems
E. coli BL21 was transformed with pT3-5LO plasmid, and recom-
binant 5-LO protein was expressed at 37 °C, and 5-LO was purified

C. Greiner et al. / Bioorg. Med. Chem. 19 (2011) 3394–3401
3401
as described.32 Purified 5-LO was immediately used for 5-LO activ-
ity assays. Aliquots of purified 5-LO (0.5 g) were added to 1 ml
PBS/EDTA plus 1 mM ATP and pre-incubated with the test com-
pounds. After 5–10 min at 4 °C, samples were pre-warmed for
30 s at 37 °C, and 2 mM CaCl2, and the indicated amounts of AA
t test for paired and correlated samples was applied. A p value of
<0.05 (⁄) was considered significant.
l
Acknowledgments
(routinely 20
l
M AA) with or without 1
l
M 13(S)-HpODE were
We thank Gertrud Kleefeld and Daniela Müller for expert tech-
nical assistance. C.P. received a Carl-Zeiss stipend.
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 metabolites were analyzed by HPLC as described for in-
tact cells.
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of microsomes, and determination of PGE2 synthase activity
Preparation of A549 cells and determination of mPGES-1 activ-
ity was performed as described previously.24 In brief, A549 cells
(2 Â 106 cells in 20 ml medium) were plated in 175 cm2 flasks
and incubated for 16 h at 37 °C and 5% CO2. Subsequently, the cul-
ture medium was replaced by fresh DMEM/High glucose (4.5 g/l)
medium containing fetal calf serum (2%, v/v). In order to induce
mPGES-1 expression, IL-1b (1 ng/ml) was added, and cells were
incubated for another 72 h. Thereafter, cells were detached with
trypsin/EDTA, washed with PBS, and frozen in liquid nitrogen.
Ice-cold homogenisation buffer (0.1 M potassium phosphate buffer
pH 7.4, 1 mM phenylmethanesulfonylfluoride, 60
lg/ml soybean
trypsin inhibitor, 1 g/ml leupeptin, 2.5 mM glutathione, and
l
250 mM sucrose) was added, and after 15 min, cells were resus-
pended and sonicated on ice (3 Â 20 s). The homogenate was sub-
jected to differential centrifugation at 10,000Âg for 10 min and
174,000Âg for 1 h at 4 °C. The pellet (microsomal fraction) was
resuspended in 1 ml homogenization buffer, and the total protein
concentration was determined by Coomassie protein assay. Micro-
somal membrane fractions were stored at À80 °C for several
weeks. Microsomal membranes were diluted in potassium phos-
phate buffer (0.1 M, pH 7.4) containing 2.5 mM glutathione to give
13. Koeberle, A.; Werz, O. Curr. Med. Chem. 2009, 16, 4274.
14. Liedtke, A. J.; Keck, P. R.; Lehmann, F.; Koeberle, A.; Werz, O.; Laufer, S. A. J. Med.
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L.; Troschutz, R.; Werz, O. J. Med. Chem. 2009, 52, 3474.
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Pergola, C.; Koeberle, A.; Werz, O.; Sautebin, L.; Cuzzocrea, S. J. Pharmacol. Exp.
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22. Zettl, H.; Steri, R.; Lammerhofer, M.; Schubert-Zsilavecz, M. Bioorg. Med. Chem.
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a final concentration of 50
(DMSO at a final concentration of 1%) were added, and after
15 min at 4 °C, the reaction (100 l total volume) was initiated
by addition of PGH2 (20 M, final concentration). After 1 min at
4 °C, the reaction was terminated using stop solution (100 l;
40 mM FeCl2, 80 mM citric acid, and 10 M of 11b-PGE2). PGE2
lg/ml. Test compounds or vehicle
l
l
l
23. Lammerhofer, M.; Pell, R.; Mahut, M.; Richter, M.; Schiesel, S.; Zettl, H.;
Dittrich, M.; Schubert-Zsilavecz, M.; Lindner, W. J. Chromatogr., A 2010, 1217,
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24. Koeberle, A.; Siemoneit, U.; Buehring, U.; Northoff, H.; Laufer, S.; Albrecht, W.;
Werz, O. J. Pharmacol. Exp. Ther. 2008, 326, 982.
l
was separated by solid phase extraction on reversed phase (RP)-
C18 material as previously described.24 11b-PGE2 was used as
internal standard to quantify PGE2 product formation by integra-
tion of the area under the peaks.
25. Jakobsson, P. J.; Thoren, S.; Morgenstern, R.; Samuelsson, B. Proc. Natl. Acad. Sci.
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3.2.6. Activity assays of isolated COX-1 and -2
Inhibition of the activities of isolated COX-1 and COX-2 was
performed as described.24 Briefly, purified COX-1 (ovine, 50 units)
or COX-2 (human recombinant, 20 units) were diluted in 1 ml
reaction mixture containing 100 mM Tris buffer pH 8, 5 mM
glutathione, 5
pre-incubated with the test compounds for 5 min. Samples were
pre-warmed for 60 s at 37 °C, and AA (5 M for COX-1, 2 M for
lM haemoglobin, and 100 lM EDTA at 4 °C and
l
l
COX-2) was added to start the reaction. After 5 min at 37 °C,
12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid was extracted
and then analyzed by HPLC.
33. Radmark, O.; Werz, O.; Steinhilber, D.; Samuelsson, B. Trends Biochem. Sci.
2007, 32, 332.
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3.2.7. Statistics
Data are expressed as mean SE. IC50 values were graphically
calculated from averaged measurements at five different concen-
trations of the compounds (routinely 0.1–10 lM) using SigmaPlot
9.0 (Systat Software Inc., San Jose, USA). The program Graphpad In-
stat (Graphpad Software Inc., San Diego, CA) was used for statisti-
cal comparisons. Statistical evaluation of the data was performed
by one-way ANOVAs for independent or correlated samples fol-
lowed by Tukey HSD post-hoc tests. Where appropriate, Student’s
39. Werz, O.; Burkert, E.; Samuelsson, B.; Radmark, O.; Steinhilber, D. Blood 2002,
99, 1044.