Pirinixic acid derivatives as novel dual inhibitors of microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase

Article abstract of DOI:10.1021/jm801085s

Dual inhibition of the prostaglandin (PG) and leukotriene (LT) biosynthetic pathway is supposed to be superior over single interference, both in terms of efficacy and side effects. Here, we present a novel class of dual microsomal PGE₂ synthase

Full text of DOI:10.1021/jm801085s

8068
J. Med. Chem. 2008, 51, 8068–8076
Pirinixic Acid Derivatives as Novel Dual Inhibitors of Microsomal Prostaglandin E2 Synthase-1
and 5-Lipoxygenase
Andreas Koeberle,†,‡ Heiko Zettl,†,§ Christine Greiner,‡ Mario Wurglics,§ Manfred Schubert-Zsilavecz,§ and Oliver Werz*,‡
Department of Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard-Karls-UniVersity Tuebingen, Auf der Morgenstelle 8,
D-72076 Tuebingen, Germany, and Institute of Pharmaceutical Chemistry, ZAFES, LIFF, Goethe-UniVersity Frankfurt, Max-Von-Laue-Strasse
9, D-60438 Frankfurt, Germany
ReceiVed September 1, 2008
Dual inhibition of the prostaglandin (PG) and leukotriene (LT) biosynthetic pathway is supposed to be
superior over single interference, both in terms of efficacy and side effects. Here, we present a novel class
of dual microsomal PGE2 synthase-1/5-lipoxygenase (5-LO) inhibitors based on the structure of pirinixic
acid [PA, 2-(4-chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)acetic acid, compound 1]. Target-
oriented structural modification of 1, particularly R substitution with extended n-alkyl or bulky aryl substituents
and concomitant replacement of the 2,3-dimethylaniline by a biphenyl-4-yl-methane-amino residue, resulted
in potent suppression of mPGES-1 and 5-LO activity, exemplified by 2-(4-(biphenyl-4-ylmethylamino)-6-
chloropyrimidin-2-ylthio)octanoic acid (7b, IC50 ) 1.3 and 1 µM, respectively). Select compounds also
potently reduced PGE2 and 5-LO product formation in intact cells. Importantly, inhibition of cyclooxygenases-
1/2 was significantly less pronounced. Taken together, these pirinixic acid derivatives constitute a novel
class of dual mPGES-1/5-LO inhibitors with a promising pharmacologial profile and a potential for therapeutic
use.
Introduction
mPGES-1 inhibitors have been described thus far. Compound
9 [MK-886, 3-(3-(tert-butylthio)-1-(4-chlorobenzyl)-5-isopropyl-
1H-indol-2-yl)-2,2-dimethylpropanoic acid, IC50 ) 2.4 µM6] and
structural derivatives7 potently inhibit mPGES-1 in Vitro, but
they are afflicted to high plasma protein binding. Recently,
phenanthrene imidazoles have been identified as selective and
orally active mPGES-1 inhibitors (IC50 ) 1.3 µM in whole
blood8) that effectively relieve both pyresis and inflammatory
pain in preclinical models of inflammation.9
Prostaglandins (PGs)a and leukotrienes (LTs) are synthesized
from arachidonic acid (AA) by the initial actions of the key
enzymes cyclooxygenase (COX) and 5-lipoxygenase (5-LO),
respectively.1 Pathological conditions, such as inflammation,
pain, fever, anorexia, atherosclerosis, stroke, and tumorigenesis,
are related to elevated levels of PGE2.2 Accordingly, pharma-
cological inhibitors of COX isoenzymes [i.e., nonsteroidal anti-
inflammatory drugs (NSAIDs) or COX-2-selective coxibs] are
used to treat respective disorders.1 However, other PGs (e.g.,
PGI2 and PGF2R) are needed to maintain homeostasis, and thus,
inhibition of the synthesis of these PGs by NSAIDs is likely
responsible for gastrointestinal and renal side effects.3 Although
coxibs have a lower risk of gastrointestinal damage, they exert
cardiovascular side effects presumably by altering the balance
between PGI2 and thromboxanes.4
PGE2 is synthesized from the COX product PGH2 by three
terminal isoforms of PGE2 synthases (PGES). Increasing
evidence suggests that the microsomal PGE2 synthase-1 (mPGES-
1), which is induced by pro-inflammatory stimuli and function-
ally coupled to COX-2, provides elevated PGE2 levels during
inflammation, fever, and pain.2 Thus, mPGES-1 may constitute
a potential target for therapeutic intervention,5 but only a few
LTs are important mediators of inflammatory and allergic
diseases but also play roles in cardiovascular disease and
cancer.10 Anti-LT therapy is used in asthma therapy and may
possess therapeutic potential also for other LT-related disorders.
It was found that dual inhibition of both the PG and LT
biosynthetic pathway, for example, by 2-(6-(4-chlorophenyl)-
2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl) acetic
acid (licofelone), is superior over single interference, not only
in terms of anti-inflammatory effectiveness but also because of
a lower incidence of gastrointestinal toxicity, typically related
to COX inhibition.11,12 We have recently shown that licofelone
suppresses PGE2 formation primarily by interference with
mPGES-1, whereas COX-2 was hardly affected.13 It is specu-
lated that drugs that selectively suppress PGE2 formation
combined with reduction of LT biosynthesis may lack the
detrimental cardiovascular side effects, implying an alternative
pharmacological strategy for intervention with inflammation.5,14
* To whom correspondence should be addressed: Department of
Pharmaceutical Analytics, Pharmaceutical Institute, Eberhard-Karls-
University Tuebingen, Auf der Morgenstelle 8, D-72076 Tuebingen,
Germany. Telephone: ++49-7071-2978793. Fax: ++49-7071-294565.
E-mail: oliver.werz@uni-tuebingen.de.
Recently, we have shown that aliphatic R substitution of
pirinixic acid [PA, 2-(4-chloro-6-(2,3-dimethylphenylamino)py-
rimidin-2-ylthio)acetic acid, compound 1] enhanced both per-
oxisome proliferator-activated receptor (PPAR)R and PPARγ
agonism15 and enables these derivatives to inhibit cellular LT
formation (IC50 ) 0.6 µM).16 Here, we address the effectiveness
of 1 and its derivatives for inhibition of PGE2 synthesis and
interaction with mPGES-1, 5-LO, and COX-1/2. Although 1
itself failed to suppress PGE2 formation, we present R-substi-
†
Both authors contributed equally to this work.
Eberhard-Karls-University Tuebingen.
Goethe-University Frankfurt.
‡
§
a Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; cPGES,
cytosolic prostaglandin E2 synthase; ELISA, enzyme-linked immunosorbent
assay; FLAP, 5-LO-activating protein; 12-HHT, 12(S)-hydroxy-5-cis-8,10-
trans-heptadecatrienoic acid; IL, interleukin; 5-LO, 5-lipoxygenase; LT,
leukotriene; mPGES, microsomal prostaglandin E2 synthase; NSAID,
nonsteroidal anti-inflammatory drug; PBS, phosphate-buffered saline; PPAR,
peroxisome proliferator-activated receptor; PA, pirinixic acid; PMNL,
polymorphonuclear leukocyte; PG, prostaglandin.
10.1021/jm801085s CCC: $40.75
2008 American Chemical Society
Published on Web 11/19/2008

PA DeriVatiVes Inhibit mPGES-1 and 5-LO
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8069
Scheme 1. General Synthesis of Compounds 5a-7ea
a Reagents and conditions: (i) (Z ) H), thiobarbituric acid, 2-bromoethylester (1.17 equiv), NaOH (1.08 equiv), H2O/EtOH (1:1), 60 °C, 2 h; (Z ) n-Bu),
(Z ) n-Hex), thiobarbituric acid, 2-bromoethylester (1.5 equiv), triethylamine (3.0 equiv), DMF, 80 °C, 1.5-3 h; (ii) POCl3 (18 equiv), N,N-diethylaniline
(1 equiv), RF, 2-3.5 h; (iii) R-NH2 (1.2 equiv), triethylamine (3 equiv), EtOH, RF, 3.5-96 h; (iv) LiOH (3 equiv), EtOH, RT, 24 h; (v) (2-bromoethyl)benzene
(2 equiv), K2CO3 (3 equiv), DMF, 80 °C, 5-9 h.
tuted derivatives as potent dual mPGES-1/5-LO inhibitors in
cellular and cell-free systems.
we pursued our search for improved 5-LO inhibitors with the
core structure of 1 and also addressed their effectiveness for
inhibition of mPGES-1. To initially screen for dual inhibitors
of 5-LO and mPGES-1, compound 1 and its derivatives (at 10
µM) were analyzed for their ability to inhibit the conversion of
PGH2 to PGE2 mediated by mPGES-1 in microsomes of IL-
1ꢀ-treated A549 cells.13 Focus was placed on derivatives that
are substituted in the R position of the carboxyl group. The
representatives 2d, 2e, 2g, and 3a-3d out of this series were
already shown to inhibit 5-LO product synthesis in human
PMNL, and values given in Table 1 are referenced.16 Thus, only
for novel derivatives 2f, 2h, and 4-7e, the 5-LO inhibitory
potential was assessed in the first screening round. The 5-LO
inhibitor 8 [BWA4C, (E)-N-hydroxy-N-(3-(3-phenoxyphenyl)-
allyl)acetamide]19 and the mPGES-1 inhibitor 9 were used as
reference compounds, respectively.
Results and Discussion
Chemistry. The presented compounds were generally syn-
thesized in a four-step reaction, which was modified from the
synthesis of 1 published by D’Atri et al.17 Compounds 2a-4
were synthesized as previously reported.15,18 The synthesis of
compounds 5a-7e is presented in Scheme 1. First, the thiol
group of thiobarbituric acid was etherified with the appropriate
R-bromoethylester (i). The introduction of alkyl chains longer
than four carbons succeeds easier with triethylamine as a
conjugated base in DMF instead of NaOH in H2O/EtOH.17
Aromatic nucleophilic substitution with POCl3 (ii) gave the
chlorinated pyrimidine derivatives. In the following step,
treatment with the appropriate primary amine employing tri-
ethylamine in refluxed EtOH (iii) resulted in monoamination
of the pyrimidine core. These compounds were hydrolyzed with
LiOH in EtOH (iv) to give the desired carboxylic acids. A side
path via a Williamson-like ether synthesis (v) led to sym-
metrically substituted 3,5-bis-ether pyrimidine derivatives, which
were hydrolyzed to the corresponding carboxylic acids as
described (iv).
Screening for Dual 5-LO and mPGES-1 Inhibitors
Derived from Prinixic Acid. We have previously shown that
target-oriented structural modification of 1 led to potent inhibi-
tors of 5-LO product formation in intact polymorphonuclear
leukocytes (PMNL) and to a minor extent also in cell-free
assays.16 In particular, esterification of the carboxylic acid group,
replacement of the 2,3-dimethylaniline by 6-aminoquinoline, and
alkylation in the R position of the carboxylic acid group yielded
compound 3b (IC50 ) 0.6 µM in intact PMNL) as the most
potent derivative out of the structures 1-3d (Table 1 and ref
16). Because dual inhibition of both the LT and the PG
biosynthetic pathway might be advantageous over single inter-
ference,11 potent inhibitors that dually interfere with 5-LO
product and PGE2 formation would be desirable. Accordingly,
In accordance with the literature,6,7,13 compound 9 concentra-
tion-dependently blocked PGE2 formation with an IC50 ) 2.1
µM, and 26 ( 3% activity remained at a concentration of 10
µM. Compound 1 itself did not inhibit mPGES-1 activity.
Introduction of one (2a) or two (2b) methyl groups or a n-butyl
residue (2c) in the R position of the carboxyl group of 1 was
without effect. However, elongation of the n-alkyl chain [n-hexyl
(2d) and n-octyl (2f)] yielded potent inhibitors of mPGES-1
(Table 1). The R-phenyl-substituted 2g was hardly active, but
introduction of a naphthyl moiety (2h) clearly enhanced the
efficacy against mPGES-1, comparable to the n-octyl-substituted
2f. Together, n-hexyl, n-octyl, or naphthyl substitution of 1 in
the R position leads to mPGES-1 inhibition, implying a
requirement of bulky lipophilic substituents. Note that the novel
derivatives 2h and 2f, which are carboxylic acids possessing
bulky R-substituents, also efficiently inhibited 5-LO product
synthesis in PMNL (Table 1), suggesting that similar SARs are
applicable for both mPGES-1 and 5-LO. The effectiveness of
the free carboxylic acids against 5-LO is surprising because,
for interference with 5-LO product formation in PMNL, the
corresponding esters have been found to be generally superior

8070 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Koeberle et al.
Table 1. Inhibition of PGE2 Formation in Microsomal Preparations of A549 Cells and Inhibition of 5-LO Product Formation in PMNL by Test
Compoundsd

PA DeriVatiVes Inhibit mPGES-1 and 5-LO
Table 1. Continued
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8071
a n.i. ) no inhibition. b Published by Werz et al.16 c Inhibition at 1 µM. d Mean values (n ) 3-4) and standard error estimates are given: (/) p < 0.05,
(//) p < 0.01, and (///) p < 0.001.
over the free acids.16 Here, we find that the need for esterifi-
cation was abolished by insertion of long and bulky alkyl/aryl
residues in the R position of the carboxylic group, reflected by
the high efficacy of the R-naphthyl derivative 2h and the R-(n-
octyl)-analogue 2f against 5-LO.
Replacement of the 2,3-dimethylphenyl residue by a quinoline
moiety (3a-3d), regardless of the bridging atom (N or O), was
shown to significantly improve inhibition of 5-LO product
formation.16 Although this derivatization did not enhance the
potency of the R-(n-hexyl)-substituted free acids 3a, 3c, and

8072 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Koeberle et al.
3d toward mPGES-1, compound 3b, the esterified analogue of
3a, was quite efficient and remarkably superior over its 2,3-
dimethylphenyl analogue 2e. Therefore, the presence of a
quinoline residue apparently overcomes the necessity of the free
carboxylic group, whereas all other esters tested were rather
poor inhibitors of mPGES-1. For example, 2e, the ethyl ester
of 2d (that potently inhibited mPGES-1), failed to suppress PGE2
formation, suggesting that the free carboxylic group governs
mPGES-1 inhibition. Why the ester 3b was superior over the
free acid as inhibitor of mPGES-1 is not readily understood
and requires more detailed analysis.
Compound 7b turned out to be the most potent mPGES-1
inhibitor, with an IC50 ) 1.3 µM, being even somewhat superior
over 9 [IC50 ) 2.1 (Table 2) and 2.4 µM6]. Compound 7d was
the most active inhibitor of 5-LO in intact PMNL and purified
5-LO (IC50 ) 0.4 and 1.5 µM, respectively), with similar
potency as 1-(1-(benzo[b]thiophen-2-yl)ethyl)-1-hydroxyurea
(zileuton, Zyflo), the most advanced 5-LO inhibitor under
comparable conditions (IC50 ) 0.5 to 1 µM20). Importantly, the
reported discrepancy between the potency regarding 5-LO
inhibition in cell-based assays (high efficacy) and cell-free
systems (low efficacy) that was originally apparent for the ester
3b and other esterified derivatives of 116 is not evident for the
free acids. Thus, the R-(n-hexyl)-substituted acids bearing a
bulky lipophilic residue at C6 of the pyrimidine ring also
potently inhibit 5-LO in cell-free assays, with IC50 values in
the range of 1.5-6.5 µM, and can thus be designated direct
5-LO inhibitors.
Also, the exchange of the 2,3-dimethylaniline by more bulky
lipophilic substituents at C6 of the pyrimidine, for example, by
3,5-bis(2,2,2-trifluoroethoxy)-aniline (4), led to derivatives that
were similarly active on mPGES-1 as compared to 2d. Of
interest, the carboxylic acid 4 also potently inhibited 5-LO
product synthesis. Hence, bulky lipohilic substituents not only
in the R position but also linked to the pyrimidine ring may
allow the free acid analogues to inhibit cellular 5-LO product
formation. Along these lines, replacement of the 2,3-dimethyl-
aniline moiety and the chlorine at the pyrimidine ring of 1 by
phenylethoxy residues led to compound 5a and its R-butyl
analogue 5b, free acids that moderately (5a) or potently (5b)
blocked 5-LO product synthesis in PMNL, respectively. Both
compounds also inhibited mPGES-1, and again, the correspond-
ing ester of 5b, namely, compound 5c, was not (mPGES-1) or
hardly (5-LO) active.
Ideally, the preferred pharmacological dynamics of a lead
compound for intervention with chronic inflammatory diseases,
CVD, or cancer without typical side effects of NSAIDs and
coxibs could comprise a potent and dually suppressive effect
on mPGES-1 and 5-LO without inhibiting COX isoenzymes.13
Accordingly, selected potent dual mPGES-1/5-LO inhibitors
from the first screening round were investigated for their
pharmacological profile in more detail. Because lipophilic acids,
such as 1 and its derivatives mimicking AA, are likely to act
on COX enzymes and because the active lead compounds inhibit
the COX-related mPGES-1 and 5-LO about equally well, it
appeared reasonable to address if the compounds may also
interfere with COX-1/2. The effects of selected compounds on
the activities of isolated ovine COX-1 and human recombinant
COX-2 [using the COX metabolite 12(S)-hydroxy-5-cis-8,10-
trans-heptadecatrienoic acid (12-HHT) as a biomarker21] were
determined. With the exception of compounds 2h and 4, all
selected derivatives were only moderate inhibitors of COX-1
and COX-2, with IC50 values > 10 µM. In control experiments,
2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]ace-
tic acid (indomethacin, 10 µM) suppressed the activity of COX-1
and 4-(5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl)ben-
zenesulfonamide (celecoxib, 5 µM) inhibited the activity of
COX-2 with 19 ( 2 and 21 ( 3% remaining activity,
respectively. Therefore, we conclude that COX inhibition by
the lead compounds is markedly less pronounced as compared
to interference with 5-LO and mPGES-1.
In view of the SARs regarding mPGES-1 apparent thus far,
bulky lipophilic substituents at C6 of the pyrimidine ring could
basically increase the efficacy. Whereas exchange of the 2,3-
dimethylaniline by phenethylamine (6a) or phenoxyethylamine
(6b or 6c) failed to increase the potency versus 2d, introduction
of a biphenyl-4-amine (7a) or a biphenyl-4-methane amine
moiety (7b) yielded derivatives that potently inhibited mPGES-1
as well as 5-LO. The corresponding esters (exemplified by 7c)
failed to interfere with mPGES-1 and also were hardly active
against 5-LO. Moreover, substitutions of the biphenyl-4-amino
moiety were tolerated, because introduction of a cyano group
in position 4′ (7d) or methyl and methoxy substitutents at the
biphenyl (7e) did not hamper the interference with either
mPGES-1 or 5-LO. Finally, we investigated whether or not the
absolute configuration of the R-C atom next to the carboxyl
group influences the potencies of the compounds. The two
enantiomeric forms of the n-hexyl-substituted derivative 7d,
namely, 7d(R) and 7d(S) were separated and tested for inhibition
of 5-LO and mPGES-1. Neither for inhibition of 5-LO (intact
cells or cell-free assay) nor for mPGES-1 was a marked
discrepancy in the efficacies (mPGES-1, IC50 ) 1.6 and 2.1
µM, respectively, and cell-free 5-LO, IC50 ) 0.8 and 1.5 µM,
respectively) determined, implying that the absolute configu-
ration is essentially negligible. Taken together, two structural
modifications of the parental inactive compound 1, namely, (I)
introduction of an n-hexyl residue in the R position and (II)
replacement of the 2,3-dimethylaniline at the C6 of the
pyrimidine ring by a biphenyl-4-yl-methanamine residue yield
efficient dual mPGES-1/5-LO inhibitors, including 7a, 7b, 7d,
and 7e, with about equal efficacies for both enzymes.
Effectiveness of the PA Derivatives on PGE2 Formation
in Intact A549 Cells. In contrast to the convenient cell-based
assay applied for the evaluation of 5-LO inhibitors, assessment
of mPGES-1 inhibitors in intact cell assays is laborious.13 Hence,
we analyzed only select compounds (i.e., 2d, 4, and 7b) for
inhibition of PGE2 formation in IL-1ꢀ-stimulated A549 cells.
For PGE2 biosynthesis in intact cells, phospholipases A2 liberate
AA as a substrate for COX isoenzymes to produce PGH2 that
is eventually transformed by selective PG or thromboxane
synthases to various structurally related eicosanoids. In IL-1ꢀ-
treated A549 cells, PGH2 is provided by highly expressed
COX-2 and converted by mPGES-1,22 whereas COX-1 could
not be detected.23 After 48 h of treatment with IL-1ꢀ and pre-
incubation (10 min) with test compounds, cells were activated
via massive intracellular Ca2+ influx using 2.5 µM Ca2+
ionophore A23187 [5-methylamino-2-(2S,3R,5R,8S,9S)-3,5,9-
trimethyl-2-(1-oxo-1-(1H-pyrrol-2-yl)propan-2-yl)-1,7-
dioxaspiro(5.5)undecan-8-yl)methyl)benzooxazole-4-carboxyl-
ic acid]. Simultaneously, exogenous AA [1 µM AA and 18.4
kBq [3H]AA] was provided to initiate PGH2 formation and
Detailed Analysis of Selected Test Compounds on mPGES-
1, 5-Lipoxygenase, and Cyclooxygenases. As stated above and
shown in Table 2, there is obviously a strong correlation between
the efficiency against mPGES-1 and 5-LO, and all eight
representatives chosen block both enzymes similarly well with
comparable IC50 values in the range of 1.3-5.6 µM for
mPGES-1 and in the range of 0.4-5 µM for 5-LO in PMNL.

PA DeriVatiVes Inhibit mPGES-1 and 5-LO
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8073
Table 2. IC50 Values for mPGES-1 in Microsomal Preparations of A549 Cells, 5-LO Activity in PMNL, and Purified 5-LO in a Cell-Free Assayc
a n.d. ) not determined. b n.i. ) no significant inhibition at 10 µM. c Inhibition of isolated ovine COX-1 and human recombinant COX-2 by selected
compounds given as remaining activity at 10 µM. Mean values (n ) 3) and standard error estimates are given: (/) p < 0.05, (//) p < 0.01, and (///) p <
0.001.
circumvent the need for release of endogenous AA by phos-
pholipases. 3H-labeled PGE2 was separated by RP-HPLC from
other eicosanoids and quantified by liquid scintillation counting.
As expected, PGE2 formation was almost completely suppressed
by the COX inhibitors indomethacin (10 µM, data not shown)
or celecoxib (5 µM) (Figure 1). Inhibition of mPGES-1 in intact
A549 cells by 9 (33 µM) reduced PGE2 formation by only 63%.
Compounds 2d and 7b concentration-dependently inhibited
PGE2 formation, with apparent IC50 values of 12 and 6 µM,
respectively (Figure 1). However, inhibition of PGE2 formation
already started at effective concentrations g1-3 µM, but neither
2d, 4, or 7b nor the mPGES-1 inhibitor 9 completely inhibited
PGE2 synthesis. Compound 4 was markedly less potent, and
inhibition of PGE2 formation started first at 10 µM, with the
apparent IC50 value being >30 µM (Figure 2).
Conclusions
Here, we present the design of compounds that combine a
potent suppression of PGE2 formation by inhibiting mPGES-1
with a potent 5-LO inhibitory action, leading to reduced levels
of LTs, but lack pronounced interference with both COX-1 and
COX-2. In particular, the biphenylic derivatives of 1 (e.g., 7b)
were most potent, but also 2d and 4 are efficient dual inhibitors
of mPGES-1 and 5-LO in cell-free assay and cell-based systems.
However, in contrast to the COX inhibitors celecoxib and
Figure 1. Chemical structures of selected inhibitors.
indomethacin, PGE2 formation was not completely suppressed
in A549 cells by the derivatives of 1 as well as by 9, with
30-40% PGE2 still remaining, probably related to the action
of other PGE2 synthases not affected by the compounds. Dual
inhibitors of COX/5-LO are superior over compounds interfering

8074 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Koeberle et al.
The synthetic procedure is exemplified by the following descrip-
tion of the synthesis of key compounds 7b and 7d.
Step (i) (Z ) n-Hexyl). 2-Thiobarbituric acid (1 equiv) was
dissolved under heating in DMF/triethylamine (3 equiv), and ethyl-
2-bromooctyl acetate (1.5 equiv) was added dropwise. After 2 h,
the reaction mixture was quenched with 4 parts of water and
extracted with ethyl acetate. The residue obtained from the organic
phase was recrystallized from ethyl acetate/n-hexane to give ethyl
2-(4,6-dihydroxypyrimidin-2-ylthio)octanoate (50%).
Step (ii). To a solution of the thioether resulting from step (i)
(1 equiv) in POCl3 (18 equiv), N,N-diethylaniline (1 equiv) was
added. After refluxing the solution at 110 °C for 3.5 h, the excessive
POCl3 was distilled in Vacuo and the oily residue was poured upon
crushed ice. The aqueous solution was extracted with ethyl acetate,
and combined organic layers were washed with diluted HCl,
saturated NaHCO3 solution, and brine. The organic layer was dried
over MgSO4, filtered, and evaporated under reduced pressure.
Purifying the resulting oil by column chromatography (n-hexane/
ethyl acetate) gave ethyl 2-(4,6-dichloropyrimidin-2-ylthio)octanoate
(85%).
Step (iii). A mixture of the chlorinated pyrimidine (1 equiv)
obtained from step (ii), the appropriate aniline/amine derivate (1.05
equiv), triethylamine (3 equiv), and EtOH was prepared and heated
under reflux (7b-ester, 6 h; 7d-ester, 96 h). The reaction mixture
was evaporated under reduced pressure. The residue was dissolved
in ethyl acetate, and the organic layer was extracted with diluted
HCl, saturated NaHCO3 solution, and brine. After drying over
MgSO4, the solvent was evaporated under reduced pressure and
the residue was purified by column chromatography (n-hexane/
ethyl acetate) to give ethyl 2-(4-(biphenyl-4-ylmethylamino)-6-
chloropyrimidin-2-ylthio)octanoate (7b-ester, 40%) and ethyl
2-(4-chloro-6-(4′-cyanobiphenyl-4-ylamino)pyrimidin-2-ylthio)-
octanoate (7d-ester, 60%), respectively.
Step (iv). The respective ester (1 equiv) from step (iii) was
dissolved in EtOH, and LiOH (3 equiv) was added. After stirring
for 24 h at room temperature, EtOH was removed and the residue
was dissolved in water (under heating, if necessary, low amounts
of MeOH were added). This solution was acidified with diluted
hydrochloric acid, and the precipitate was filtered, washed to
neutrality with water and then with n-hexane. Recrystallization from
n-hexane/ethyl acetate gave the carboxylic acids.
2-(4-(Biphenyl-4-ylmethylamino)-6-chloropyrimidin-2-ylth-
io)octanoic Acid (7b). mp ) 110 °C. 1H NMR (300.13 MHz,
(CD3)2SO) δ: 0.77 (t, 3H, CH3-Hex, J ) 6.3 Hz), 1.14-1.28 (m,
8H, CH2-Hex), 1.66-1.84 (m, 2H, CH2-Hex), 4.23 (t, 1H, S-CH,
J ) 7.1 Hz), 4.49 (dd, 1H, CH2-NH, J1 ) 5.4 Hz, J2 ) 15.5 Hz),
4.65 (dd, 1H, CH2B-NH, J1 ) 5.6 Hz, J2 ) 15 Hz), 6.31 (s, 1H,
Pyr-5H), 7.31-7.47 (m, 5H, Ph-H + Ph′-H), 7.61 (d, 2H,
Ph-3H + Ph-5H, J ) 1.8 Hz), 7.64 (d, 2H, Ph′-2H + Ph-6H,
J ) 1.2 Hz), 8.39 (t,1H, -NH, J ) 4.8 Hz). 13C NMR (75.45
MHz, (CD3)2SO) δ: 13.80 (CH3-Hex), 21.89 (CH2-Hex), 26.50
(CH2-Hex), 28.13 (CH2-Hex), 30.95 (CH2-Hex), 31.39
(CH2-Hex), 43.29 (CH2-NH), 47.26 (S-CH), 99.54 (Pyr-C5),
126.50 (2C, Ph-C3 + -C5), 126.67 (2C, Ph′-C2 + -C6), 127.32
(Ph′-C4), 127.89 (2C, Ph-C2 + -C6), 128.85 (2C, Ph′-C3 +
-C5), 138.04 (Ph-C4), 138.91 (Ph′-C1), 139.82 (Ph-C1), 156.57
(Pyr-C6), 162.40 (Pyr-C4), 169.76 (Pyr-C2), 172.72 (COOH).
MS (ESI+): m/e 470.1 [M + H]+. Anal. Calcd (C25H28ClN3O2S):
C, H, N. Yield: 80%.
Figure 2. Effects of test compounds on the formation of PGE2 in intact
A549 cells. A549 cells (4 × 106/mL) were pre-incubated with test
compounds or vehicle (DMSO) for 10 min, and then cellular PGE2
synthesis was elicited by addition of 2.5 µM Ca2+ ionophore A23187
plus 1 µM AA and [3H]AA (18.4 kBq). After 15 min at 37 °C, formed
[3H]PGE2 was analyzed by RP-HPLC and liquid scintillation counting
as described in the Materials and Methods. Data are given as mean (
standard error (SE). n ) 3. (/) p < 0.05, (//) p < 0.01, or (///) p <
0.001 versus vehicle (0.1% DMSO) control. ANOVA + Tukey HSD
posthoc tests. Cele ) celecoxib, 5 µM.
with only COX enzymes (i.e., NSAIDs and coxibs) in terms of
anti-inflammatory effectiveness associated with a lower inci-
dence of gastrointestinal and cardiovascular toxicity.11 This
might be attributable to the accompanied suppression of pro-
inflammatory LTs,24 which significantly contribute to gastric
epithelial injury and atherogenesis.10 For example, licofelone
that blocks mPGES-1, LT formation, and COX-1 without
inhibiting COX-213 exhibits a significantly superior gastric
tolerability and a lower incidence of ulcers compared to (S)-2-
(6-methoxynaphthalen-2-yl)propanoic acid (naproxen) in healthy
humans,25 and in contrast to COX-2-selective inhibitors,
licofelone reduced neointimal formation and inflammation in
an atherosclerotic model.26 To our knowledge, no dual mPGES-
1/5-LO inhibitor has been reported, although MK-886 and
licofeloneinhibitmPGES-16,13 andthe5-LO-activatingprotein,27,28
also leading to suppression of PGE2 and LT formation. Future
experiments aiming to resolve the detailed interference of these
compounds presented here with mPGES-1 and the 5-LO enzyme
as well as studies addressing the effectiveness in ViVo using
animal models of inflammation, fever, pain, cancer, or athero-
sclerosis are necessary and may reveal the therapeutic potential
of PA derivatives for intervention with mPGES-1- and 5-LO-
related diseases.
Materials and Methods
Compounds and Chemistry. Compound 1 was purchased from
Sigma-Aldrich (Deisenhofen, Germany). The structures of previ-
1
ously reported compounds 2a-4 were confirmed by H and 13C
NMR, as well as by mass spectrometry (ESI); the purity (>98%)
was checked by combustion analysis as described.15,18
Compounds 5a-7e were synthesized as described in Scheme 1.
The synthesis is modified from the method reported from D’Atri
et al.17 All commercial chemicals and solvents are of reagent grade
and were used without further purification, unless otherwise
2-(4-Chloro-6-(4′-cyanobiphenyl-4-ylamino)pyrimidin-2-ylth-
io)octanoic Acid (7d). mp ) 111 °C. 1H NMR (300.13 MHz,
(CD3)2SO) δ: 0.77 (t, 3H, CH3-Hex, J ) 6.5 Hz), 1.17-1.36 (m,
8H, CH2-Hex), 1.77-1.94 (m, 2H, CH2-Hex), 4.37 (t, 1H, S-CH,
J ) 7.1 Hz), 6.56 (s, 1H, Pyr-5H), 7.70-7.77 (m, 4H, Ph-H),
7.85-7.92 (m, 4H, Ph′-H), 10.13 (s,1H, -NH). 13C NMR (75.45
MHz, (CD3)2SO) δ: 13.77 (CH3-Hex), 21.87 (CH2-Hex), 26.49
(CH2-Hex), 28.08 (CH2-Hex), 30.93 (CH2-Hex), 31.61
(CH2-Hex), 47.13 (S-CH), 101.44 (Pyr-C5), 109.50 (Ph′-C4),
118.88 (-CN), 120.77 (2C, Ph-C2 + -C6), 126.92 (2C, Ph′-C2
+ -C6), 127.50 (2C, Ph-C3 + -C5), 132.80 (2C, Ph′-C3 + -C5),
139.27 (Ph′-C1), 143.97 (Ph-C1), 157.48 (Pyr-C6), 160.43
1
specified. H and 13C NMR spectra were measured in DMSO-d6
or CDCl3 on a Bruker ARX 300 (1H NMR) and AC 200 E (13C
NMR) spectrometer. Chemical shifts are reported in parts per
million (ppm) using tetramethylsilane (TMS) as an internal standard.
Mass spectra were obtained on a Fissous Instruments VG Platform
2 spectrometer measuring in the positive- or negative-ion mode
(ESI-MS system). Elemental analysis has been performed by the
Microanalytical Laboratory of the Institute of Organic Chemistry
and Chemical Biology, Goethe-University Frankfurt, on a Foss
Heraeus CHN-O-rapid elemental analyzer (for details see the
Supporting Information).

PA DeriVatiVes Inhibit mPGES-1 and 5-LO
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8075
(Pyr-C2), 170.02 (Pyr-C4), 172.38 (COOH). MS (ESI+): m/e
481.0 [M + H]+. Anal. Calcd (C25H25ClN4O2S): C, H, N. Yield:
60%.
(Sigma A2767, Deisenhofen, Germany), and the column was eluted
as described previously.33 Partially purified 5-LO was immediately
used for in Vitro activity assays.
Assay Systems. Materials. DMEM/high glucose (4.5 g/L)
medium, penicillin, streptomycin, and trypsin/EDTA solution were
obtained from PAA (Coelbe, Germany). PGH2 was obtained from
Larodan (Malmo¨, Sweden). 11ꢀ-PGE2, PGB1, MK-886, human
recombinant COX-2, and ovine isolated COX-1 were obtained from
Cayman Chemical (Ann Arbor, MI). [5,6,8,9,11,12,14,15-3H] AA
([3H]AA) was obtained from BioTrend Chemicals GmbH (Cologne,
Germany). Ultima Gold XR was obtained from Perkin-Elmer
(Boston, MA). All other chemicals were obtained from Sigma-
Aldrich (Deisenhofen, Germany), unless stated otherwise.
Cells and Cell Viability Assay. Blood cells were freshly isolated
from leukocyte concentrates obtained at the Blood Center of the
University Hospital Tuebingen (Germany) as described.29 In brief,
venous blood was taken from healthy adult donors that did not take
any medication for at least 7 days, and leukocyte concentrates were
prepared by centrifugation (4000g, 20 min, 20 °C). Cells were
immediately isolated by dextran sedimentation and centrifugation
on Nycoprep cushions (PAA, Coelbe, Germany). For incubations
with solubilized compounds, methanol or DMSO was used as
vehicle, never exceeding 1% (v/v). PMNL were immediately
isolated from the pellet after centrifugation on Nycoprep cushions,
and hypotonic lysis of erythrocytes was performed as described.30
Cells were finally resuspended in phosphate-buffered saline (PBS)
at pH 7.4, containing 1 mg/mL glucose and 1 mM CaCl2 (PGC
buffer) (purity > 96-97%).
Determination of 5-LO Activity of Semi-purified 5-LO.
Partially purified 5-LO was added to 1 mL of a 5-LO reaction mix
(PBS at pH 7.4, 1 mM EDTA, and 1 mM ATP). Samples were
pre-incubated with the test compounds for 10 min at 4 °C and
prewarmed for 30 s at 37 °C, and 2 mM CaCl2 and 20 µM AA
were added to start 5-LO product formation. The reaction was
stopped after 10 min at 37 °C by the addition of 1 mL ice-cold
methanol, and the formed metabolites were analyzed as described
for intact cells.
Induction of mPGES-1 in A549 Cells and Isolation of
Microsomes. Preparation of A549 cells was performed as de-
scribed.22 In brief, cells (2 × 106 cells) in 20 mL of DMEM/high
glucose (4.5 g/L) medium containing FCS (2%, v/v) were incubated
for 16 h at 37 °C and 5% CO2. Subsequently, the culture medium
was replaced by fresh medium; IL-1ꢀ (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 homogenization buffer (0.1 M potassium phos-
phate buffer at pH 7.4, 1 mM PMSF, 60 µg/mL soybean trypsin
inhibitor, 1 µg/mL leupeptin, 2.5 mM glutathione, and 250 mM
sucrose) was added, and after 15 min, cells were resuspended and
sonicated on ice (3 × 20 s). The homogenate was subjected to
differential centrifugation at 10000g for 10 min and 174000g for
1 h at 4 °C. The pellet (microsomal fraction) was resuspended in
1 mL of homogenization buffer, and the protein concentration was
determined by the Coomassie protein assay.
A549 cells were cultured in DMEM/high glucose (4.5 g/L)
medium supplemented with heat-inactivated fetal calf serum (10%,
v/v), penicillin (100 units/mL), and streptomycin (100 µg/mL) at
37 °C and 5% CO2. After 3 days, confluent cells were detached
using 1× trypsin/EDTA solution and reseeded at 2 × 106 cells in
20 mL of medium. Cell viability was measured using the colori-
metric thiazolyl blue tetrazolium bromide (MTT) dye reduction
assay as described.31 A549 cells (4 × 104 cells/100 µL of medium)
were plated into a 96-well microplate and incubated at 37 °C and
5% CO2 for 16 h. Then, test compounds or solvent (DMSO) were
added, and the samples were incubated for another 5 h. MTT (20
µL, 5 mg/mL) was added, and the incubations were continued for
4 h. The formazan product was solubilized with sodium dodecyl-
sulfate [10% (w/v), in 20 mM HCl], and the absorbance of each
sample was measured at 595 nm relative to that of vehicle (DMSO)-
treated control cells using a multiwell scanning spectrophotometer
(Victor3 plate reader, Perkin-Elmer, Rodgau-Juegesheim, Germany).
Compounds 1, 2d, 4, and 7b did not significantly reduce cell
viability within 5 h at 10 µM (data not shown), excluding possible
acute cytotoxic effects of the compound in the cellular assays.
Determination of 5-LO Product Formation in Intact Cells.
For assays of intact cells, 5 × 106 freshly isolated PMNL were
resuspended in 1 mL of PGC buffer. After pre-incubation with the
test compounds (15 min, RT), 5-LO product formation was started
by addition of 2.5 µM Ca2+-ionophore A23187 plus 20 µM AA.
After 10 min at 37 °C, the reaction was stopped with 1 mL of
methanol and 30 µL of 1 N HCl, 200 ng of prostaglandin B1, and
500 µL of PBS were added. Formed 5-LO metabolites were
extracted and analyzed by HPLC as described.30 5-LO product
formation is expressed as nanograms of 5-LO products per 106 cells,
which includes LTB4 and its all-trans isomers, and 5(S)-hydro(p-
ero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid. Cysteinyl LTs C4,
D4, and E4 were not detected, and oxidation products of LTB4 were
not determined.
Determination of PGE2 Synthase Activity in Microsomes of
A549 Cells. Microsomal membranes of A549 cells were diluted in
potassium phosphate buffer (0.1 M, pH 7.4) containing 2.5 mM
glutathione (100 µL total volume), and test compounds or vehicle
(DMSO) was added. After 15 min, PGE2 formation was initiated
by the addition of PGH2 (20 µM, final concentration). After 1 min
at 4 °C, the reaction was terminated with 100 µL of stop solution
(40 mM FeCl2, 80 mM citric acid, and 10 µM of 11ꢀ-PGE2), and
PGE2 was separated by solid-phase extraction on reversed-phase
(RP) C18 material using acetonitrile (200 µL) as an eluent and
analyzed by RP-HPLC [30% acetonitrile aqueous + 0.007% TFA
(v/v), Nova-Pak C18 column, 5 × 100 mm, 4 µm particle size,
flow rate of 1 mL/min], with UV detection at 195 nm. 11ꢀ-PGE2
was used as an internal standard to quantify PGE2 product formation
by integration of the area under the peaks.
Determination of PGE2 Formation in Intact A549 Cells. A549
cells (2 × 106), treated with IL-1ꢀ for 72 h as described above,
were resuspended in 0.5 mL of PBS containing 1 mM CaCl2 and
pre-incubated with the indicated compounds at 37 °C for 10 min,
and PGE2 formation was started by the addition of Ca2+-ionophore
A23187 (2.5 µM), AA (1 µM), and [3H]AA (18.4 kBq). The
reaction was stopped after 15 min at 37 °C, and the samples were
put on ice. After centrifugation (800g, 5 min, 4 °C), the supernatant
was acidified to pH 3 by the addition of citric acid (20 µL, 2 M)
and the internal standard 11ꢀ-PGE2 (2 nmol) was added. PGE2 was
separated by solid-phase extraction and RP-HPLC as described
above. The amount of 11ꢀ-PGE2 was quantified by integration of
the area under the eluted peaks. For quantification of radiolabeled
PGE2, fractions (0.5 mL) were collected and mixed with Ultima
Gold XR (2 mL) for liquid scintillation counting in a LKB Wallac
1209 Rackbeta liquid scintillation counter.
Activity Assays of Isolated COX-1 and COX-2. Inhibition of
the activities of isolated ovine COX-1 and human COX-2 was
performed as described.21,34 Although the purified COX-1 is not
of human origin, ovine COX-1 is generally used for inhibitor studies
when examining the effectiveness of compounds on the activity of
isolated COX-1 enzyme.34 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 at pH 8, 5 mM
glutathione, 5 µM hemoglobin, and 100 µM EDTA at 4 °C and
pre-incubated with the test compounds for 5 min. Samples were
prewarmed for 60 s at 37 °C, and AA (5 µM for COX-1 and 2 µM
Expression and Purification of Human Recombinant 5-LO
from Escherichia coli. E. coli MV1190 was transformed with pT3-
5LO plasmid, and recombinant 5-LO protein was expressed at 27
°C as described.32 Cells were lysed by incubation in 50 mM
triethanolamine/HCl at pH 8.0, 5 mM EDTA, soybean trypsin
inhibitor (60 µg/mL), 1 mM phenylmethylsulfonyl fluoride (PMSF),
and lysozyme (500 µg/mL), homogenized by sonication (3 × 15 s),
and centrifuged at 100000g for 70 min at 4 °C. The resulting
100000g supernatants were applied to an ATP-agarose column

8076 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Koeberle et al.
of the peroxisome proliferator activated receptor R and γ. Arch. Pharm.
(Weinheim, Ger.) 2008, 341, 191–195.
(16) Werz, O.; Greiner, C.; Koeberle, A.; Hoernig, C.; George, S.; Popescu,
L.; Syha, I.; Schubert-Zsilavecz, M.; Steinhilber, D. Novel and potent
inhibitors of 5-lipoxygenase product synthesis based on the structure
of pirinixic acid. J. Med. Chem. 2008, 51, 5449–5453.
(17) D’Atri, G.; Gomarasca, P.; Resnati, G.; Tronconi, G.; Scolastico, C.;
Sirtori, C. R. Novel pyrimidine and 1,3,5-triazine hypolipidemic agents.
J. Med. Chem. 1984, 27, 1621–1629.
(18) Popescu, L.; Rau, O.; Bottcher, J.; Syha, Y.; Schubert-Zsilavecz, M.
Quinoline-based derivatives of pirinixic acid as dual PPAR R/γ
agonists. Arch. Pharm. (Weinheim, Ger.) 2007, 340, 367–371.
(19) Tateson, J. E.; Randall, R. W.; Reynolds, C. H.; Jackson, W. P.;
Bhattacherjee, P.; Salmon, J. A.; Garland, L. G. Selective inhibition
of arachidonate 5-lipoxygenase by novel acetohydroxamic acids:
Biochemical assessment in vitro and ex vivo. Br. J. Pharmacol. 1988,
94, 528–539.
for COX-2) was added to start the reaction. After 5 min at 37 °C,
the COX product 12-HHT was extracted and then analyzed by
HPLC as described.29
Statistics. Data are expressed as mean ( SE. The IC50 values were
analyzed using GraphPad Prism. The program Graphpad Instat (Graphpad
Software, Inc., San Diego, CA) was used for statistical comparisons.
Statistical evaluation of the data was performed by one-way ANOVAs
for independent or correlated samples followed by Tukey HSD posthoc
tests. A p value of <0.05 (/) was considered significant.
Acknowledgment. We thank Gertrud Kleefeld and Bianca
Jazzar for expert technical assistance. This work was supported
by the LOEWE Lipid Signaling Forschungszentrum Frankfurt
(LiFF).
(20) Carter, G. W.; Young, P. R.; Albert, D. H.; Bouska, J.; Dyer, R.; Bell,
R. L.; Summers, J. B.; Brooks, D. W. 5-Lipoxygenase inhibitory
activity of zileuton. J. Pharmacol. Exp. Ther. 1991, 256, 929–937.
(21) Capdevila, J. H.; Morrow, J. D.; Belosludtsev, Y. Y.; Beauchamp,
D. R.; DuBois, R. N.; Falck, J. R. The catalytic outcomes of the
constitutive and the mitogen inducible isoforms of prostaglandin H2
synthase are markedly affected by glutathione and glutathione per-
oxidase(s). Biochemistry 1995, 34, 3325–3337.
(22) Jakobsson, P. J.; Thoren, S.; Morgenstern, R.; Samuelsson, B.
Identification of human prostaglandin E synthase: A microsomal,
glutathione-dependent, inducible enzyme, constituting a potential novel
drug target. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7220–7225.
(23) Asano, K.; Lilly, C. M.; Drazen, J. M. Prostaglandin G/H synthase-2
is the constitutive and dominant isoform in cultured human lung
epithelial cells. Am. J. Physiol. 1996, 271, L126–L131.
(24) Celotti, F.; Laufer, S. Anti-inflammatory drugs: New multitarget
compounds to face an old problem. The dual inhibition concept.
Pharmacol. Res. 2001, 43, 429–436.
(25) Bias, P.; Buchner, A.; Klesser, B.; Laufer, S. The gastrointestinal
tolerability of the LOX/COX inhibitor, licofelone, is similar to placebo
and superior to naproxen therapy in healthy volunteers: Results from
a randomized, controlled trial. Am. J. Gastroenterol. 2004, 99, 611–
618.
Supporting Information Available: Chemical synthesis and
routine analytics by 1H and 13C NMR, mass spectrometry (ESI-),
and elemental analysis. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) Funk, C. D. Prostaglandins and leukotrienes: Advances in eicosanoid
biology. Science 2001, 294, 1871–1875.
(2) Samuelsson, B.; Morgenstern, R.; Jakobsson, P. J. Membrane pros-
taglandin E synthase-1: A novel therapeutic target. Pharmacol. ReV.
2007, 59, 207–224.
(3) Rainsford, K. D. Anti-inflammatory drugs in the 21st century. Subcell.
Biochem. 2007, 42, 3–27.
(4) McGettigan, P.; Henry, D. Cardiovascular risk and inhibition of
cyclooxygenase: a systematic review of the observational studies of
selective and nonselective inhibitors of cyclooxygenase 2. J. Am. Med.
Assoc. 2006, 296, 1633–1644.
(5) Friesen, R. W.; Mancini, J. A. Microsomal prostaglandin E2 synthase-1
(mPGES-1): A novel anti-inflammatory therapeutic target. J. Med.
Chem. 2008, 51, 4059–4067.
(6) Claveau, D.; Sirinyan, M.; Guay, J.; Gordon, R.; Chan, C. C.; Bureau,
Y.; Riendeau, D.; Mancini, J. A. Microsomal prostaglandin E
synthase-1 is a major terminal synthase that is selectively up-regulated
during cyclooxygenase-2-dependent prostaglandin E2 production in
the rat adjuvant-induced arthritis model. J. Immunol. 2003, 170, 4738–
4744.
(7) Riendeau, D.; Aspiotis, R.; Ethier, D.; Gareau, Y.; Grimm, E. L.; Guay,
J.; Guiral, S.; Juteau, H.; Mancini, J. A.; Methot, N.; Rubin, J.; Friesen,
R. W. Inhibitors of the inducible microsomal prostaglandin E2 synthase
(mPGES-1) derived from MK-886. Bioorg. Med. Chem. Lett. 2005,
15, 3352–3355.
(26) Vidal, C.; Gomez-Hernandez, A.; Sanchez-Galan, E.; Gonzalez, A.;
Ortega, L.; Gomez-Gerique, J. A.; Tunon, J.; Egido, J. Licofelone, a
balanced inhibitor of cyclooxygenase and 5-lipoxygenase, reduces
inflammation in a rabbit model of atherosclerosis. J. Pharmacol. Exp.
Ther. 2007, 320, 108–116.
(27) Dixon, R. A.; Diehl, R. E.; Opas, E.; Rands, E.; Vickers, P. J.; Evans,
J. F.; Gillard, J. W.; Miller, D. K. Requirement of a 5-lipoxygenase-
activating protein for leukotriene synthesis. Nature 1990, 343, 282–
284.
(28) Fischer, L.; Hornig, M.; Pergola, C.; Meindl, N.; Franke, L.; Tanrikulu,
Y.; Dodt, G.; Schneider, G.; Steinhilber, D.; Werz, O. The molecular
mechanism of the inhibition by licofelone of the biosynthesis of
5-lipoxygenase products. Br. J. Pharmacol. 2007, 152, 471–480.
(29) Albert, D.; Zundorf, I.; Dingermann, T.; Muller, W. E.; Steinhilber,
D.; Werz, O. Hyperforin is a dual inhibitor of cyclooxygenase-1 and
5-lipoxygenase. Biochem. Pharmacol. 2002, 64, 1767–1775.
(30) Werz, O.; Burkert, E.; Samuelsson, B.; Rådmark, O.; Steinhilber, D.
Activation of 5-lipoxygenase by cell stress is calcium independent in
human polymorphonuclear leukocytes. Blood 2002, 99, 1044–1052.
(31) Tretiakova, I.; Blaesius, D.; Maxia, L.; Wesselborg, S.; Schulze-
Osthoff, K.; Cinatl, J., Jr.; Michaelis, M.; Werz, O. Myrtucommulone
from Myrtus communis induces apoptosis in cancer cells via the
mitochondrial pathway involving caspase-9. Apoptosis 2008, 13, 119–
131.
(32) Fischer, L.; Szellas, D.; Rådmark, O.; Steinhilber, D.; Werz, O.
Phosphorylation- and stimulus-dependent inhibition of cellular 5-li-
poxygenase activity by nonredox-type inhibitors. FASEB J. 2003, 17,
949–951.
(33) Brungs, M.; Rådmark, O.; Samuelsson, B.; Steinhilber, D. Sequential
induction of 5-lipoxygenase gene expression and activity in Mono Mac
6 cells by transforming growth factor ꢀ and 1,25-dihydroxyvitamin
D3. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 107–111.
(8) Cote, B.; Boulet, L.; Brideau, C.; Claveau, D.; Ethier, D.; Frenette,
R.; Gagnon, M.; Giroux, A.; Guay, J.; Guiral, S.; Mancini, J.; Martins,
E.; Masse, F.; Methot, N.; Riendeau, D.; Rubin, J.; Xu, D.; Yu, H.;
Ducharme, Y.; Friesen, R. W. Substituted phenanthrene imidazoles
as potent, selective, and orally active mPGES-1 inhibitors. Bioorg.
Med. Chem. Lett. 2007, 17, 6816–6820.
(9) Xu, D.; Rowland, S. E.; Clark, P.; Giroux, A.; Cote, B.; Guiral, S.;
Salem, M.; Ducharme, Y.; Friesen, R. W.; Methot, N.; Mancini, J.;
Audoly, L.; Riendeau, D. MF63 {2-(6-chloro-1H-phenanthro[9,10-
d]imidazol-2-yl)isophthalonitrile}, a selective microsomal prostaglan-
din E synthase 1 inhibitor, relieves pyresis and pain in preclinical
models of inflammation. J. Pharmacol. Exp. Ther. 2008, 326, 754–
763.
(10) Peters-Golden, M.; Henderson, W. R., Jr. Leukotrienes. N. Engl.
J. Med. 2007, 357, 1841–1854.
(11) Fiorucci, S.; Meli, R.; Bucci, M.; Cirino, G. Dual inhibitors of
cyclooxygenase and 5-lipoxygenase. A new avenue in anti-inflam-
matory therapy. Biochem. Pharmacol. 2001, 62, 1433–1438.
(12) Kulkarni, S. K.; Singh, V. P. LicofelonesA novel analgesic and anti-
inflammatory agent. Curr. Top. Med. Chem. 2007, 7, 251–263.
(13) Koeberle, A.; Siemoneit, U.; Buehring, U.; Northoff, H.; Laufer, S.;
Albrecht, W.; Werz, O. Licofelone suppresses prostaglandin E2
formation by interference with the inducible microsomal prostaglandin
E2 synthase-1. J. Pharmacol. Exp. Ther. 2008, 326, 975–982.
(14) Wang, M.; Song, W. L.; Cheng, Y.; Fitzgerald, G. A. Microsomal
prostaglandin E synthase-1 inhibition in cardiovascular inflammatory
disease. J. Intern. Med. 2008, 263, 500–505.
(34) Mitchell, J. A.; Akarasereenont, P.; Thiemermann, C.; Flower, R. J.;
Vane, J. R. Selectivity of nonsteroidal antiinflammatory drugs as
inhibitors of constitutive and inducible cyclooxygenase. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 11693–11697.
(15) Rau, O.; Syha, Y.; Zettl, H.; Kock, M.; Bock, A.; Schubert-Zsilavecz,
M. R-Alkyl substituted pirinixic acid derivatives as potent dual agonists
JM801085S