Synthesis and pharmacological characterization of benzenesulfonamides as dual species inhibitors of human and murine mPGES-1

Article abstract of DOI:10.1016/j.bmc.2013.10.006

The microsomal prostaglandin E₂ synthase 1 (mPGES-1) became a desirable target in recent years for the research of new anti-inflammatory drugs. Even though many potent inhibitors of human mPGES-1, tested in vitro assay systems, have been synthe

Full text of DOI:10.1016/j.bmc.2013.10.006

Bioorganic & Medicinal Chemistry 21 (2013) 7874–7883
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological characterization
of benzenesulfonamides as dual species inhibitors
of human and murine mPGES-1
Thomas Hanke a, , Florian Rörsch b, , Theresa M. Thieme a, Nerea Ferreiros b, Gisbert Schneider c,
Gerd Geisslinger b, Ewgenij Proschak a, Sabine Grösch b, Manfred Schubert-Zsilavecz a,
⇑
a Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
b Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, LiFF/ZAFES, Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany
c ETH Zürich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 16 October 2013
The microsomal prostaglandin E2 synthase 1 (mPGES-1) became a desirable target in recent years for the
research of new anti-inflammatory drugs. Even though many potent inhibitors of human mPGES-1, tested
in vitro assay systems, have been synthesized, they all failed in preclinical trials in rodent models of
inflammation, due to the lack of activity on rodent enzyme. Within this work we want to present a
new class of mPGES-1 inhibitors derived from a benzenesulfonamide scaffold with inhibitory potency
Keywords:
Human mPGES-1
Murine mPGES-1
Sulfonamides
SAR
Inflammation
Inhibitors
on human and murine mPGES-1. Starting point with an IC50 of 13.8 lM on human mPGES-1 was com-
pound 1 (4-{benzyl[(4-methoxyphenyl)methyl]sulfamoyl}benzoic acid; FR4), which was discovered by
a virtual screening approach. Optimization during a structure–activity relationship (SAR) process leads
to compound 28 (4-[(cyclohexylmethyl)[(4-phenylphenyl)methyl]sulfamoyl]benzoic acid) with an
improved IC50 of 0.8
ical characterization has been carried out to estimate their anti-inflammatory potential.
Ó 2013 Elsevier Ltd. All rights reserved.
lM on human mPGES-1. For the most promising compounds a broad pharmacolog-
1. Introduction
strategies can be distinguished for the interference within the pro-
duction of PGE2.
The microsomal prostaglandin E2 synthase
1
(mPGES-1)
On the one hand small molecules were synthesized which are
belongs to the class of the MAPEG family proteins and catalyzes
the reaction from PGH2 to PGE2. PGE2 is one of the key mediators,
within the prostaglandin pathway, which is responsible for fever,
inflammation and pain.1 Inhibitors of prostaglandin synthesis like
the nonsteroidal anti-inflammatory drugs (NSAIDs) (unselective
or selective COX-1 and COX-2 inhibitors) are one of the most
widespread drugs used in anti-inflammatory therapy. However,
especially in long-term therapy their use is closely related to se-
vere side effects, such as gastrointestinal and renal complications
or increased cardiovascular risk due to the suppression of
physiological relevant prostaglandins. Hence new approaches for
anti-inflammatory therapy are urgently needed. One possibility is
the selective inhibition of the mPGES-1, which has been shown
to be upregulated under inflammatory conditions2 and therefore
gained interest as new anti-inflammatory target. Two different
able to reduce the expression of mPGES-1.3 BTH (Fig. 1) deriving
from the c-hydroxybutenolide series represses mPGES-1 induction
and has even shown activity in vivo in a zymosan-induced mouse
air pouch model of inflammation as well as in a collagen induced
arthritis model.4 On the other hand many compounds were devel-
oped, which suppress the production of PGE2 by direct inhibition of
mPGES-1 activity (for review of mPGES-1 inhibitors see Refs. 5,6).
Even though many companies pursuing the approach of a mPGES-1
inhibitor as a new anti-inflammatory target, but up to now no one
reached clinical trials, albeit various preclinical trials have been
evaluated. MF63 (Fig. 1), a phenanthrene imidazole synthesized
by Merck Frosst, is a highly potent inhibitor of human mPGES-1.7
Cl
Cl
Br
NC
O
S
OH
O
Cl
N
O
N
H
N
H
⇑
Corresponding author. Tel.: +49 69 798 29339; fax: +49 69 798 29332.
E-mail address: schubert-zsilavecz@pharmchem.uni-frankfurt.de (M. Schubert-
Zsilavecz).
HO
BTH
NH
NC
S
O
O
PF-9184
MF63
These authors contributed equally to this work, and are listed in alphabetical
order.
Figure 1. Published mPGES-1 inhibitors with preclinical results.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.006

T. Hanke et al. / Bioorg. Med. Chem. 21 (2013) 7874–7883
7875
Route A:
Its activity was evaluated in vivo in a preclinical model of
inflammation. Whereas MF63 is able to inhibit human and guinea
pig mPGES-1 highly potent, it was not able to inhibit the mouse or
rat enzyme, and therefore Xu et al. used a knock-in (KI) mouse
expressing human mPGES-1.8 Pfizers compound PF-9184 (Fig. 1),
derived from a series of mPGES-1 inhibitors with an oxicam tem-
plate, is also a highly potent inhibitor of human mPGES-1 with
Step Ia
O
R1
O
S
O
R3
R1 R2
N
R2
NH2
H
H
Cl
Step II
Route B:
O
an IC50 of 0.016
lM in an enzyme based assay and an IC50 of
Step II
M in a cell based assay.9 Even though PF-9184 was highly
O
S
O
OH
O
R3
0.42
l
R1 R2
N
R1
S
H
N
selective on recombinant human (rh) mPGES-1 over rhCOX-1 and
rhCOX-2, it failed to inhibit the mouse or rat mPGES-1 enzymes.10
Pawelzik et al. have identified for the first time key residues which
are responsible for the difference between rat mPGES-1 and human
mPGES-1.11 Three amino acids at position Thr-131, Leu-135 and
Ala-138 in the transmembrane helix four of the human mPGES-1
are exchanged to Val-131, Phe-135 and Phe-138 in the rat enzyme
which functions as a gate keeper for the inhibitor to enter the ac-
tive site. They have shown, that in the rat mPGES-1, these positions
were substituted by larger more bulky aromatic residues. This sub-
stitution pattern occurred only in rat and mouse mPGES-1 but not
in other rodent orthologues.12 This inter-species difference could
be a major drawback for the development of mPGES-1 inhibitors,
which have to be overcome to succeed in preclinical trials in rodent
models of inflammation.13 Recently we presented a series of qui-
nazolinone derivatives identified by virtual screening,14 with high
potency in submicromolar range in a cell based and whole blood
assays on human mPGES-1. However all derivatives of this class
failed to inhibit murine mPGES-1.15 Within this work we want to
present a new class of mPGES-1 inhibitors, developed from a vir-
tual screening with the lead compound FR4 (Compound 1). This
benzenesulfonamide derivative was able to inhibit murine
mPGES-1 (derived from murine RAW and NIH cell lines) in an
about equimolar range in comparison to human mPGES-1.
Cl
O
R2
Route C:
Step II
N
H
O
S
R3
O
Cl
H
O
O
Route D:
OH
Step Ib
OH
N
N
H
O
Scheme 1. Synthesis of benzenesulfonamides and the corresponding tertiary
amine. Reagents and conditions: (step Ia) aldehyde (1 equiv), primary amine
(1.2 equiv), HOAc (2 equiv), sodium triacetoxyborohydride (1.4–2.6 equiv), DCE, rt,
18 h; (step Ib) 4-formylbenzoic acid (2 equiv), dibenzylamine (1 equiv), HOAc
(5 equiv), sodium triacetoxyborohydride (2.6 equiv), THF, rt, 66 h; (step II) second-
ary amine (1 equiv), benzenesulfonyl chloride (1 equiv), TEA (3 equiv), EtOH, from
0 °C to rt, 18 h.
calculated using MOE (Molecular Operating Environment) soft-
ware suite (Chemical Computing Group, Montreal, Canada) and
were subsequently screened using the pharmacophore model
shown in Figure 2. We retrieved FR4 (compound 1) as a hit from
pharmacophore search and purchased it for in vitro screening.
FR4 reduced the mPGES-1 derived PGE2-level in a human in vitro
2. Results and discussion
2.1. Virtual screening
assay by 40% at 5 lM concentration. Comparable results were
obtained with murine mPGES-1 in vitro assays. We therefore
started a structure–activity relationship study with this benzene-
sulfonamide class to proof if this cross-species inhibitory function
is limited to this single compound or a feature of this class. Besides
this we wanted to improve the inhibitory potency and gather infor-
mation on cell toxicity and perform an intensive whole blood
screening to elucidate effects on other enzymes of the arachidonic
acid cascade.
Ligand-based virtual screening is an efficient way to retrieve
novel lead structures based on molecular compounds exhibiting
the desired biological activity.16 In this study a pharmacophore
model was built based on a potent indole-derived inhibitor of
mPGES-1.17
Using this model we screened the Asinex database (version
November 2008, www.asinex.com, Moscow, Russian Federation)
containing 360,169 compounds. Multiple conformers were
2.2. Chemistry
For the preparation of FR4 a linear two step synthesis procedure
was established (Scheme 1). First step was the synthesis of the
secondary amine starting with benzaldehyde and (4-methoxy-
phenyl)methanamine under conditions of a reductive amination.18
The following step was the building of the benzenesulfonamide
(FR4) by coupling of this secondary amine with 4-(chlorosulfo-
nyl)benzoic acid.
These two steps were used for a divergent synthesis of the
corresponding educts to get in a simple manner a broad range of
diverse benzenesulfonamide derivatives. In route A the benzene-
sulfonamides were synthesized in the linear two step synthesis
by alternating the aldehydes and the primary amines in the reduc-
tive amination to get various secondary amines, which were cou-
pled with benzenesulfonyl chloride derivatives. Following route
B, commercially available secondary amines were all coupled with
4-(chlorosulfonyl)benzoic acid to maintain the carboxylic acid
group in position 4 to the benzenesulfonamide. According to route
C dibenzylamine was used as secondary amine with various ben-
zenesulfonyl chlorides to get modifications on the residue three.
Figure 2. Pharmacophore model for the virtual screening of mPGES-1 inhibitors
based on a potent indol inhibitor. Color code: yellow—hydrophobic or aromatic
group, green—hydrophobic group, blue—hydrogen-bond acceptor group.

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T. Hanke et al. / Bioorg. Med. Chem. 21 (2013) 7874–7883
Finally in route D dibenzylamine was used with 4-formylbenzoic
acid in a reductive amination to replace the sulfonamide by a ter-
tiary amine.
position 3 (compound 17) and the replacement of it by a nitrile
group (compound 23) leads just to a slight decline in hmPGES-1
inhibitory potency; whereas the replacement of it by several halo-
gens (compounds 18–22) leads nearly always to a totally loss in
mPGES-1 inhibition. So it seems that an acidic head group in posi-
tion 4 was most favorable.
2.3. Biological assays
2.3.1. Human mPGES-1 activity screening
To investigate the inhibitory potency of these substances on the
human mPGES-1 we compared the inhibition rate of each deriva-
tive at 5 lM on the microsomal fraction of the human mPGES-1
protein (hmPGES-1) in an in vitro activity assay in comparison to
the reference structure compound 1 (FR4).
First optimizations of FR4 occured at position R1 (Table 1) by ex-
change of the benzyl residue with various heterocycles like thio-
Within the knowledge of the SAR at these three positions, sev-
eral modifications were done at once (Table 4). Shortening of the
benzyl by a phenyl residue in position R1, simultaneously by short-
er chains in position R2 with an ethyl (compound 24) or a butyl
(compound 25), results in the complete loss of hmPGES-1 inhibi-
tion. Replacement of the carboxylic acid group in compound 16
with a nitrile group (compound 26) leads also to a drastic reduc-
tion in hmPGES-1 inhibition. However retaining the cyclohexylm-
ethyl scaffold from compound 7 and introduction of the bulky
lipophilic phenoxy group (compound 27) or a biphenylresidue
(compound 28) leads to compounds that inhibit the hmPGES-1
phene (compound 2), furan (compound 3) or
a thiazole
derivative (compound 4), however they all were less active than
FR4. Also the introduction of nitriles on the benzyl residue in com-
pounds 5 and 6 led to a decline of hmPGES-1 inhibition. Only the
replacement of the aromatic system with a cyclohexylmethyl
(compound 7) raised the inhibitory activity from about 40% to
67.9% on the human mPGES-1 enzyme.
Simultaneous to variation at position R1 optimization at R2 were
carried out (Table 2). The abolition of the methoxy group in com-
pound 8 did not have any influence on the inhibition in comparison
to FR4. Nevertheless it appears that shortening of the 4-methoxy-
benzyl to an ethyl (compound 9), a butyl (compound 10) or a
phenyl (compound 11) residue resulted on the human mPGES-1
enzyme in less active derivatives than FR4. Extension of the meth-
ylene spacer in FR4 to an ethylene (compound 12) or a propylene
spacer (compound 13) leads to an improvement of hmPGES-1
inhibition; and even more bulky lipophilic substitution like the
4-(phenylamino)benzyl (compound 14) and the two phenoxy
derivatives (compounds 15 and 16) leads to highly potent hmP-
GES-1 inhibitors with up to 90% inhibition of the hmPGES-1 activ-
up to 80% at a concentration of 5 lM, whereas the p-chlorobenzyl
residue (compound 29) was again less active. Ring closure of R1
and R2 with a 4-phenylpiperazine (compound 30) was slightly
superior to FR4, whereas replacement of the sulfonamide with a
tertiary amine in compound 31 was again drastic less active on
the hmPGES-1 enzyme than compound 8, which emphasize the
necessity of the sulfonamide scaffold.
The five most promising compounds together with the lead
structure FR4, were further evaluated by determining the IC50 val-
ues on hmPGES-1 together with validating the concept of a dual
species inhibitor by testing these compounds on the murine
mPGES-1 as well as their cross-reactivity against the cyclooxygen-
ase COX-1 or COX-2 (Table 5). The lead structure FR4 inhibits the
human mPGES-1 enzyme with an IC50 of 13.8
lM, instead com-
pound 28 inhibits the hmPGES-1 with an IC50 of 0.8
lM which
means that the modifications of compound 28 leads to an increase
in mPGES-1 inhibitory potency of about 17-fold in comparison to
the reference compound FR4. All tested FR4 derivatives also inhibit
the murine mPGES-1 (isolated either from murine macrophages
(RAW) or fibroblasts (NIH)) better than FR4 and have no or only
ity in the in vitro assay at a compound concentration of 5 lM.
Finally we examined modifications at position R3 (Table 3). The
movement of the carboxylic acid from position 4 (compound 8) to
Table 1
In vitro pharmacological characterization of compounds 1–7; variations on benzyl residue
R3
O
SAR of FR4
R1
S
O
N
R2
Compound
R1
R2
R3
hmPGES-1 inhibition [%] SEM @ 5 lM
Modifications at position 1 (R1)
O
1 FR4
–COOH
–COOH
–COOH
–COOH
–COOH
–COOH
–COOH
39.9 8.6
11.9 1.5
14.2 9.8
2.9 6.6
6 0 8.0
6.0 3.7
67.9 3.4
O
O
O
S
2
O
3
S
N
4
NC
O
O
5
6
7
NC
O

T. Hanke et al. / Bioorg. Med. Chem. 21 (2013) 7874–7883
7877
Table 2
In vitro pharmacological characterization of compounds 8–16; variations on 4-methoxybenzyl residue
R3
O
SAR of FR4
R1
S
O
N
R2
Compound
R1
R2
R3
hmPGES-1 inhibition [%] SEM @ 5 lM
Modifications at position 2 (R2)
8
–COOH
–COOH
–COOH
51.4 3.7
60 5.1
9
10
19.1 4.3
11
–COOH
26.4 15.1
12
13
–COOH
–COOH
57.0 2.8
73.0 1.0
14
15
16
–COOH
–COOH
–COOH
78.5 0.8
79.8 2.2
90.0 3.0
N
H
O
O
Table 3
In vitro pharmacological characterization of compounds 17–23; variations of the carboxylic acid (R3)
3
2
1
4
R3
O
SAR of FR4
R1
S
O
N
R2
Compound
R1
R2
R3
hmPGES-1 inhibition [%] SEM @ 5 lM
Modifications at position 3 (R3)
17
18
19
20
21
22
23
3-COOH
4-F
35.8 6.3
60 10.3
60 9.7
4-Cl
3-Cl
6.2 12.2
60 12.3
11.9 1.5
34.1 17.0
2-Cl
4-CF3
4-CN

7878
Table 4
T. Hanke et al. / Bioorg. Med. Chem. 21 (2013) 7874–7883
In vitro pharmacological characterization of compounds 24–31; benzenesulfonamides with various modifications
3
2
1
4
R3
O
SAR of FR4
Compound
R1
S
O
N
R2
R1
R2
R3
hmPGES-1 inhibition [%] SEM @ 5 lM
Benzenesulfonamides with various modifications
24
4-COOH
4-COOH
60 7.9
60 4.6
25
26
4-CN
1.0 7.8
O
O
27
4-COOH
79.2 2.5
28
29
4-COOH
4-COOH
76.0 0.6
48.4 5.4
Cl
O
S
O
O
N
N
30
58.7 2.4
OH
Replacement of the benzenesulfonamide
O
OH
N
31
17.4 5.8
Table 5
In vitro pharmacological characterization of compounds 1, 7, 14, 15, 16, 28 on human and murine mPGES-1 as well as recombinant COX-1 and COX-2
Compound
Human
IC50
Murine
RAW mPGES-1 inhibition NIH mPGES-1 inhibition
[%] SEM @ 5
COX-1 inhibition
COX-2 inhibition
[%] @ 10 lM
[%] @ 10
lM
mPGES-1 inhibition
95% Confidence
[%] SEM@ 5
lM
[lM]
interval [lM]
[%] SEM @ 5
l
M
lM
FR4
7
14
15
16
28
39.9 8.6
67.9 3.4
78.5 0.8
79.8 2.2
90.0 3.0
76.0 0.6
13.8
10.2
7.2
1.7
1.0
3.0–63.5
7.7–13.4
4.3–12.0
1.0–3.0
0.9–1.2
0.3–1.8
29.8 8.3
n.d.
n.d.
74.7 7.4
71.4 8.3
44.4 7.0
10.6 0.8
48.5 2.6
67.9 1.0
71.2 3.5
65.1 0.7
51.9 4.8
60
10.3
60
12.4
0.3
n.d.
60
18.5
12.9
60
3.1
n.d.
0.8
Cellular PGE2-Inhibition
a weak inhibitory effect on COX-1 or COX-2 activity at 10
concentration.
lM
1 µM
10 µM
100 µM
In addition to the activity on isolated or recombinant enzymes
we investigated the influence of the most active compounds on
higher test systems. Therefore inhibition of cellular produced
PGE2 was tested after incubation of (human) HeLa-cells with FR4
derivatives (Fig. 3). All compounds inhibited the PGE2 production
totally at 100 lM, but inhibition was drastically reduced at lower
concentrations. This effect was not observed for the control com-
pound celecoxib (a COX-2 inhibitor). Nevertheless the derivatives
100
50
0
FR4
Comp. 7
Comp. 10
Comp. 14
Comp. 15
Comp. 16
Celecoxib
7, 14, 15 and 16 showed an inhibitory effect at 10 lM, which could
not be observed for the lead compound FR4.
Figure 3. Inhibition of cellular PGE2 by benzenesulfonamide mPGES-1 inhibitors.
Celecoxib was used as reference substance.

T. Hanke et al. / Bioorg. Med. Chem. 21 (2013) 7874–7883
7879
WST-1
line with the test compounds (also at 1, 10 and 100
lM concentra-
tion) and investigated the cell viability after 24 h (Fig. 4).
All tested compounds did not reduce cell viability up to 10
At higher concentrations (100 lM) a cell toxic effect reducing cell
l
M.
100
50
0
viability to about 50% was observed for all compounds and also
for the reference substance Celecoxib. We therefore conclude that
the cellular PGE2-reduction at lower concentrations is independent
from toxic effects, but may in part be affected at higher concentra-
tions. Compound 28 which was the most active compound in the
previous reported test system (s. Table 5), was regrettably less ac-
tive in human whole blood (s. Fig. 5), maybe due to a strong protein
binding or a reduced cell permeability, so we decided to exclude
this compound from these tests.
FR4
Comp. 7
Comp. 10
Comp. 14
Comp. 15
Comp. 16
Celecoxib
Figure 4. Cell viability of HeLa cells after 24 h of incubation with benzenesulfon-
amide compounds at 1, 10 and 100
reference substance.
lM concentration. Celecoxib was used as
Because tumor cell line based assays are not sufficient to reflect
a higher system with many different cell types, compartments and
changing physicochemical properties we decided to test the
To exclude a potential cell toxic effect, which might also be
responsible for a reduced PGE2 level, we incubated the same cell
Figure 5. Benzenesulfonamides based on the lead compound FR4 were evaluated on their potential to reduce (667% remaining activity) or enhance (P133% remaining
activity) the level of arachidonic acid derived fatty acids in human whole blood.

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T. Hanke et al. / Bioorg. Med. Chem. 21 (2013) 7874–7883
FR4-series inhibitors in human whole blood assays. This test
system allows a setting close to a real life system and is in our
opinion a good choice to study many effects of drugs without the
need of in vivo experiments. Freshly taken venous whole blood
was therefore incubated with the test compound and in parallel
Within this work we present for the first time compounds
which are able to inhibit both species human and murine
mPGES-1 and which might be therefore an interesting starting
point for further in vitro/in vivo pharmacological characterization.
Pawelzik et al. have shown that three amino acids Val-131, Phe-
135 and Phe-138 in the rat enzyme function as gate keeper and
prevent the inhibitors of entering the active site of the mPGES-
1.11 With this benzenesulfonamide scaffold we have identified
obviously a drug class which is better accessible to the active site
of the murine mPGES-1 than other well known mPGES-1 inhibitors
from the literature. However which amino acids in the murine
mPGES-1 are responsible for the gate keeper function remains un-
clear and could maybe be resolved by a site-directed mutagenesis
study.
stimulated with
a proinflammatory substance to upregulate
arachidonic acid derived lipid production. Stimuli are described
in detail in our recent publication.15 We observed a PGE2-reducing
effect at 100
lead structure FR4 and compounds 16 and 28. Lower concentra-
tions (1 and 10 M) did not reduce the level or only slightly (data
lM for compounds 7, 10, 14 and 15 but not for the
l
not shown) which is in accordance with data from the cell based
assay. More interestingly we did not see a reduction of the concur-
rent prostanoids PGF2 and TXB2 and in case of compounds 7, 10
a
and 14 also for PGD2. Prostacyclin (PGI2) was not measurable in
these settings. As expected Celecoxib, which blocks the enzymatic
catalysation of the precursor fatty acid PGH2, reduced the level of
all prostanoids almost completely. Beside prostanoids also leukotr-
Approaches including early screening of related enzymes addi-
tionally may prevent us from late phase unwanted and unexpected
side effects. Our compounds have only negligible cross-reactivities
against COX-1 and COX-2, but two compounds interfered with the
leukotriene/HETE pathways pointing out the importance of meth-
ods monitoring not only the desired target.
In conclusion, benzenesulfonamide derivatives seem to be a
good origin for the development of dual species mPGES-1 inhibi-
tors and also for dual target (mPGES-1/FLAP) inhibitors which
might even outperform single target drugs. We would also like to
point out the important impact of the virtual screening. The ligand
based approach was able to successfully select compounds with
potent inhibitory function on mPGES-1, a new scaffold and a
switch from single species inhibitors to dual species inhibitors.
iens and HETEs play
a crucial role during inflammatory
processes.19–22 Particularly LTB4 and 5(S)-HETE should always be
screened during development of mPGES-1 inhibitors because the
5-lipoxygenase-activating protein (FLAP) is also a member of the
MAPEG enzyme family and therefore structurally related to
mPGES-1. A mPGES-1 inhibitor might therefore inhibit FLAP and
hence reduce LTB4 and 5(S)-HETE (and vice versa a flap inhibitor
may inhibit the mPGES-1). Furthermore inhibition of 5-LO could
also be responsible for reduction of LTB4 and 5(S)-HETE, due to
the observation that many mPGES-1 inhibitors are also able to
inhibit 5-LO.5 Indeed we could observe a reduction of these two
fatty acids by compounds 7 and 14. Compound 15 interestingly en-
hanced the production of these fatty acids suggesting another
mode of action. To strengthen the hypothesis of mPGES-1/FLAP
cross-reaction we scanned two additional fatty acids—12(S)-HETE
and 15(S)-HETE. They are not coupled to FLAP and function as a
negative control. No compound decreased or increased the level
of 12(S)- and 15(S)-HETE as expected. Three major outcomes can
be derived from the whole blood experiments. First, as proposed
selective mPGES-1 inhibition does not reduce the level of other
prostanoids; second, the so called ‘shunting effect’ (redirection of
PGH2 to other prostanoids than PGE2) could not be observed but
may nevertheless occur in vivo or with other inhibitors; and third,
mPGES-1 inhibitors should always be screened on FLAP and on
5-LO inhibition, but dual target inhibition might also be desirable
in several inflammatory diseases.
4. Experimental
4.1. Compounds and chemistry
The structures of the presented compounds were verified by 1H,
13C NMR and mass spectrometry (ESI); the purity (>95%) was
determined by combustion analysis. Compounds 1–31 were syn-
thesized as described in Scheme 1. All commercial chemicals and
solvents are of reagent grade and were used without further puri-
fication. 1H and 13C NMR spectra were measured in DMSO-d6 or
CDCl3 on a Bruker AM 250, DPX 250, AV 300 or AV 400 spectrom-
eter. Chemical shifts are reported in parts per million (ppm) using
tetramethylsilane (TMS) as internal standard. Mass spectra were
obtained on a Fisons Instruments VG Platform II Spectrometer
(ESI-MS system) or on a PerSeptive Biosystems Mariner Biospect-
rometry Workstation (nano-spray ESI-MS system) measuring in
the positive- and/or negative-ion mode. The purities of the final
compounds were determined by combustion analysis, which 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. All
compounds described here have a purity of 95% or higher. The syn-
thesis of the presented compounds according to the synthetic
Scheme 1 is exemplified by compound 1 (4-{benzyl[(4-methoxy-
phenyl)methyl]sulfamoyl}benzoic acid, FR4). For more detailed
information and analytical data of the other compounds see the
Supporting information.
3. Conclusions
In this study we discovered that starting from the benzenesul-
fonamide lead compound FR4 replacement of the aromatic system
at position R1 with a cyclohexylmethyl group, or replacement of
the 4-methoxybenzyl group at position R2 with bulky lipophilic
substitution like the 4-(phenylamino)benzyl or phenoxy deriva-
tives leads to highly potent mPGES-1 inhibitors. In contrast
replacement of the carboxylic acid group at position R3 leads to a
decline in mPGES-1 inhibitory potency, indicating the necessity
of this group. Further pharmacological characterization of five
derivatives indicates that especially the replacement of the
4-methoxybenzyl with bulky lipophilic substitutions at position
R2 improves not only the inhibitory potency of the derivatives on
human but also on the mouse mPGES-1. Concomitant dual species
inhibitors allow for the development of drugs against the human
enzyme and in late development phase the testing in animal
models which have an orthologous but not identical enzyme. We
therefore circumvent the expensive and time consuming need of
knock-in (animal) models.
Synthesis
phenyl)methyl]sulfamoyl}benzoic acid.
Step I, Synthesis of 1a (benzyl[(4-methoxyphenyl)methyl]amine):
Benzaldehyde (0.3 g, 2.83 mmol, 1 equiv), (4-methoxy-
of
compound
1
4-{benzyl[(4-methoxy-
phenyl)methanamine (0.78 g, 5.65 mmol, 2 equiv) and glacial ace-
tic acid (0.32 ml, 5.65 mmol, 2 equiv) were dissolved in 20 mL DCE.
The flask was evacuated and refilled with argon and stirred for
3.5 h at room temperature. After imine formation sodium triacet-
oxyborhydride (0.84 g, 3.95 mmol, 1.4 equiv) was added and the
solution stirred at room temperature for 25 h. The reaction was

T. Hanke et al. / Bioorg. Med. Chem. 21 (2013) 7874–7883
7881
quenched by addition of 20 mL aqueous NaHCO3 and the product
was extracted by ethyl acetate. The organic layer was dried over
MgSO4 and solvent of the organic phase was evaporated under
reduced pressure. The crude product was further purified by col-
umn chromatography on silica gel (n-hexane–ethyl acetate) to ob-
bated for 24 h at 37 °C in medium containing 10% FCS. The med-
ium was removed, and HeLa/NIH cells were stimulated with IL-1b
(1 ng/mL) + TNF
a (5 ng/mL) for 16 h, RAW cells were stimulated
with 10 mg/mL LPS for 16 h. Cells were scraped in 2 mL of phos-
phate buffered saline (PBS) and centrifuged at 5000 rpm for 2 min
at 4 °C. Cell pellets were frozen in liquid nitrogen. After thawing
tain benzyl[(4-methoxyphenyl)methyl]amine as
a yellow oil;
yield: 29% (0.19 g). 1H NMR (250.13 MHz, (CD3)2SO) d: 3.60 (s,
2H, N–CH2–), 3.65 (s, 2H, N–CH2–), 3.73 (s, 3H, O–CH3), 6.87 (d,
the cells, they were resuspended in 800 lL of potassium phos-
phate buffer (Kpi buffer, 0.1 M, pH 7.4), containing 1ꢁ complete
protease inhibitor cocktail (Roche Diagnostics, Mannheim,
Germany), sucrose (0.25 M), and reduced glutathione (GSH,
1 mM). After sonification and centrifugation at 45.000 rpm for
2 h or 53.000 rpm for 1 h at 4 °C the microsomal fraction (pellet)
2H, OMe–Ph-H3/5, J = 8.57 Hz), 7.19–7.35 (m, 7H, OMe–Ph-H2/6
,
Ph-H2/3/4/5/6). 13C NMR (62.90 MHz, (CD3)2SO) d: 51.57 (NH–CH2),
52.04 (NH–CH2), 54.97 (O–CH3), 113.50 (Ph-C3+C5), 126.42 (Ph0-
C4), 127.85 (Ph0-C2+C6), 128.04 (Ph0-C3+C5), 129.01 (Ph-C2+C6),
132.73 (Ph-C1), 140.89 (Ph0-C1), 158.00 (Ph-C4). MS (ESI+): m/
e = 228.21 [M+H]+.
was stored at ꢀ80 °C. The pellet was resuspended in 100
lL Kpi
buffer (0.1 M, pH 7.4) containing 1ꢁ Roche complete and reduced
GSH (2.5 mM). To homogenize the solution a sonification step
was applied and total protein content was measured using the
Bradford method. Activity of all benzenesulfonamide derivatives
Step II, Synthesis of 1 4-{benzyl[(4-methoxyphenyl)methyl]sulfa-
moyl}benzoic acid: Precursor 1a (0.16 g, 0.70 mmol, 1 equiv) and
triethylamine (0.29 mL, 2.11 mmol, 3 equiv) were dissolved in
20 mL EtOH at room temperature. After cooling at 0 °C 4-(chloro-
sulfonyl)benzoic acid was slowly added and the reaction mixture
was allowed to come to room temperature. After 17 h the reaction
was stopped and the solvent was evaporated under reduced pres-
sure. The residue was first dissolved in 1 N NaOH and finally the
product was precipitated by the addition of 2 N HCl. The precipita-
tion was extracted by dichloromethane and the organic layer was
dried over MgSO4. The crude product was finally recrystallized
by dichloromethane/n-hexane to obtain 4-{benzyl[(4-methoxy-
phenyl)methyl]sulfamoyl}benzoic acid as a white solid; yield:
47% (0.14 g). 1H NMR (250.13 MHz, (CD3)2SO) d: 3.70 (s, 3H, –O–
CH3), 4.27 (s, 2H, N–CH2), 4.32 (s, 2H, N–CH2), 6.78 (d, 2H, Ph0-
H3/H5, J = 5.00 Hz), 7.00 (d, 2H, Ph0-H2/H6, J = 5.00 Hz), 7.11 (m,
2H, Ph00-H2/H6), 7.23–7.25 (m, 3H, Ph00-H3/H4/H5), 7.97 (d, 2H, Ph-
H3/H5, J = 7.50 Hz), 8.12 (d, 2H, Ph-H2/H6, J = 7.50 Hz), 13.52 (s,
1H, –COOH). 13C NMR (100.61 MHz, (CD3)2SO) d: 50.55 (NH–
CH2), 50.76 (NH–CH2), 55.03 (O–CH3), 113.67 (Ph0-C3+C5), 127.18
(Ph-C3+C5), 127.39 (Ph00-C4), 127.50 (Ph0-C1), 128.07 (Ph00-C2+C6),
128.23 (Ph00-C3+C5), 129.71 (Ph0-C2+C6), 130.17 (Ph-C2+C6), 134.40
(Ph-C1), 136.04 (Ph00-C1), 143.33 (Ph-C4), 158.66 (Ph0-C4), 166.15
(–COOH). MS (ESIꢀ): m/e = 410.6 [MꢀH]ꢀ Anal. Calcd C22H21NO5S
C, H, N, S: Calcd C 64.22, H 5.14, N 3.40, S 7.79; found C 63.98, H
5.07, N 3.23, S 7.95; diff. C ꢀ0.24, H ꢀ0.07, N ꢀ0.17, S +0.16.
was measured at 5 lM final concentration and compared to the
lead compound FR4. The mPGES-1 activity assay was performed
on the basis of Thoren et al.23 Briefly, 0.15 mg/mL of human HeLa
derived or murine NIH derived protein or 0.3 mg/mL of murine
RAW derived protein was incubated with each compound for
30 min on ice. The reaction was initiated with 20 lM PGH2
(Larodan, Malmö, Sweden) and terminated after 1 min by adding
a stop solution containing 40 mM iron chloride (FeCl2) and
80 mM citric acid. For solid phase extraction procedure 100
reaction solution was mixed for 3 min with 700 L of ultrapure
water, 100 L of 0.15 M EDTA, 20 L MeOH, 20 L internal stan-
lL
l
l
l
l
dard (25 ng/mL PGE2-d4, 25 ng/mL PGD2-d4, 25 ng/mL TXB2-d4,
50 ng/mL PGF2 -d4, 37.5 ng/mL 6-keto-PGFl -d4), all from Cay-
a
a
man Chemical Company (Ann Arbor, USA). After centrifugation
at 1200 rpm for 3 min, the samples were extracted using a
30 mg Bond Elut NEXUS 96 round-well plate (Agilent Technolo-
gies GmbH, Böblingen, Germany). The plate was preconditioned
with 1 mL MeOH, followed by 1 mL ultrapure water. After this,
it was washed with 1 mL 30% MeOH and dried for 7 min at max-
imum vacuum. Prostaglandins were eluted with 1 mL hexane–
ethylacetate–isopropranol (30:65:5, v/v/v). The elution solvent
was evaporated under nitrogen atmosphere at 45 °C and the res-
idue was reconstituted in 100 lL acetonitrile–H2O–formic acid
(20:80:0.0025 v/v/v). Samples were measured by LC–MS/MS
technique (LC unit: Agilent 1200 series, Waldbronn, Germany,
MS/MS unit: AB SCIEX QTRAP 5500, AB Sciex, Darmstadt,
Germany) as described previously.24 Compounds with improved
inhibitory effect as compared to the lead structure were used
for further experiments. For IC50 calculation the mPGES-1 assay
was performed with increasing compound concentrations. The
IC50 was calculated using GraphPad Prism 5 software (GraphPad
Software, Inc., San Diego, CA 92130, USA) by fitting the four
parameter logistic curve. With 95% probability the estimated
IC50 is in the range of the given confidence interval.
4.2. Cell biological methods
4.2.1. Cells and reagents
HeLa (human cervix carcinoma) and NIH-3T3 (Swiss mouse
fibroblast) cells were purchased from Deutsche Sammlung für
Mikroorganismen und Zellkulturen (DSMZ, Braunschweig,
Germany). RAW 264.7 (Mouse leukaemic monocyte macrophage
cell line) cells were purchased from American Type Culture Col-
lection (ATCC, Manassas, USA). HeLa were incubated in RPMI
medium 1640, containing high glucose,
L-glutamine and
25 mM HEPES, NIH in Dulbecco’s MEM containing 4.5 g/L glu-
cose and pyruvate and RAW in RPMI medium 1640, containing
high glucose, GlutaMAX. All media contain 10% fetal calf serum
4.2.3. COX-inhibitor screening assay
To distinguish between mPGES-1 and COX-1 or COX-2 derived
inhibition of PGE2 production direct inhibition of the COX-1 (ovine)
and COX-2 (human recombinant) enzyme was measured using a
COX inhibitor screening assay kit (Cayman Chemicals, Ann Arbor,
Mich., USA), according to the manufacturer’s protocol. SC-560, a
selective COX-1 inhibitor, and celecoxib, a selective COX-2 inhibi-
tor, were used as positive controls. The COX assay is based on
(FCS), 100 units/mL penicillin G and 100 lg/mL streptomycin
which were purchased from Invitrogen (Darmstadt, Germany).
Cells were cultured at 37 °C in an atmosphere containing 5%
CO2. Recombinant human IL-1 beta (IL-1b) and recombinant hu-
man tumor necrosis factor alpha (TNF
PeproTech (London, UK).
a) were purchased from
the determination of PGE2, PGD2 and PGF2 amounts produced
a
4.2.2. mPGES-1 activity assay
by SnCl2 reduction of COX-derived PGH2. 100
tion was diluted with 200 L MeOH. 70 L of diluted reaction solu-
tion was mixed for 3 min with 700 L of ultrapure Water, 100 L of
0.15 M EDTA, 20 L MeOH and 20 L internal standard (25 ng/mL
PGE2-d4, 25 ng/mL PGD2-d4, 25 ng/mL TXB2-d4, 50 ng/mL
lL of reaction solu-
To investigate the inhibitory activity of the benzenesulfon-
amide derived compounds on the mPGES-1 enzymes in vitro,
the microsomal fraction of human HeLa, murine RAW and murine
NIH cells were prepared. Approximately 3 ꢁ 106 cells were incu-
l
l
l
l
l
l

7882
T. Hanke et al. / Bioorg. Med. Chem. 21 (2013) 7874–7883
PGF2 -d4, 37.5 ng/mL 6-keto-PGFl -d4). The amount of prostaglan-
4.2.8. Whole blood COX-2 assay
a
a
dins was quantified by LC–MS/MS analysis after solid phase extrac-
tion as described above (see Section 4.2.2).
The mPGES-1 expression is mainly coupled to COX-2 and there-
fore many proinflammatory stimuli, activating the COX-2, also
upregulate the protein level of mPGES-1. For the COX-2 assay
blood was collected in NH4-heparin containing monovettes (SAR-
STEDT AG & Co, Nümbrecht, Germany) which prevent blood clot-
4.2.4. WST-1 cell viability assay
The water soluble tetrazolium-1 salt (Roche Diagnostics,
Mannheim, Germany) was used to determine the cell viability after
treatment of cells with the compounds. HeLa cells were seeded at a
ting. 500
in DMSO. 10
1. After 15 min incubation at 37 °C, 10
(LPS) in 10 L autologous plasma was added to stimulate COX-2/
l
l heparinized blood was mixed with test compounds
g/ L acetylsalicylic acid was added to inhibit COX-
g/ L lipopolysaccharide
l
l
density of 3 ꢁ 103 cells in 100
l
L culture medium containing 10%
l l
FCS into 96-well microplates and incubated for 24 h at 37 °C.
l
Medium was removed and HeLa cells were stimulated with IL-1b
mPGES-1. After 24 h incubation time at 37 °C samples were chilled
on ice for 5 min and plasma was collected, prostaglandins ex-
tracted and measured by LC–MS/MS (see Section 4.2.7).
(1 ng/mL) + TNF
increasing concentrations of the compounds (1, 10 and 100
or DMSO. After 24 h, 10 L of WST-1 reagent was added to each
a
(5 ng/mL) and simultaneously treated with
l
M)
l
well and the cells were incubated for further 90–150 min. The for-
mation of the dye was measured at 450 nm against a reference
wavelength of 620 nm using a 96-well spectrophotometric plate
reader (SpectraFluor Plus, Tecan, Crailsheim, Germany).
4.2.9. Whole blood leukotriene/HETE assay
To assess the influence of benzenesulfonamide derivatives on
different enzymes in the leukotriene- and HETE-pathway 500 lL
human heparinized whole blood was mixed with test compounds
and incubated for 15 min at 37 °C. Afterwards the whole blood
4.2.5. Cell based mPGES-1 activity assay
was stimulated with 20 lM calcium ionophore (A23187) in autol-
2 ꢁ 104 HeLa cells were incubated in 24-well microplates for
ogous plasma for further 15 min at 37 °C. Blood samples were
chilled on ice for 5 min, plasma collected (centrifugation at 4 °C
with 2000 rpm for 20 min) and leukotrienes and HETEs extracted
by liquid–liquid extraction. Therefore, plasma was mixed with
24 h at 37 °C. The medium was replaced with fresh medium con-
taining 1 ng/mL IL-1b + 5 ng/mL TNF
amount of PBS (unstimulated control) and test compound (1, 10 or
100 M) or DMSO. After incubation for 24 h at 37 °C, cell superna-
tant was collected. 400–500 L supernatant was mixed with
400 L of 45 mM H3PO4, 100 L of 0.15 M EDTA, 20 L MeOH
and 20 internal standard (25 ng/mL PGE2-d4, 25 ng/mL
a (stimulated control) or equal
l
20
LTB4-d4, 25 ng/mL 5(S)-HETE-d8, 25 ng/mL 12(S)-HETE-d8, 25 ng/
mL 15(S)-HETE-d8 and 25 ng/mL 20-HETE-d6. 600 L ethyl acetate
lL EtOH and 20 lL internal standard containing 25 ng/mL
l
l
l
l
l
l
L
was added and samples were mixed and centrifuged at 13.000 rpm
PGD2-d4, 25 ng/mL TXB2-d4, 50 ng/mL PGF2 -d4, 37.5 ng/mL
for 3 min. Organic upper phase was collected and the aqueous
a
6-keto-PGFl -d4). The amount of prostaglandins was quantified
by LC–MS/MS analysis after solid phase extraction as described
above (see Section 4.2.2).
phase was extracted again with 600
ing the organic phases they were evaporated under a gentle stream
of nitrogen at 45 °C and the residue was reconstituted in 50
lL ethyl acetate. After combin-
a
lL
MeOH–H2O (50:50 v/v). LTB4, 5(S)-HETE, 12(S)-HETE, 15(S)-HETE
and 20-HETE were analyzed by LC–MS/MS technique on a tandem
mass spectrometer QTRAP 5500 (AB Sciex, Darmstadt, Germany)
operating in multiple reaction monitoring (MRM).25 Chromato-
graphic separation was performed on a Gemini-NX C18 column
4.2.6. Whole blood eicosanoid screening
To determine possible effects of benzenesulfonamide deriva-
tives on different eicosanoids levels in human whole blood, a eicos-
anoid profile was created for each compound. Therefore three
different experimental settings were used to stimulate the produc-
tion of eicosanoid synthesis.
(150 ꢁ 2 mm inner diameter, 5
lm particle size, Phenomenex,
Aschaffenburg, Germany).
A stimulating effect of a compound was assumed when the rel-
ative level of an eicosanoid exceeded +33% compared to control, an
inhibitory effect when levels were reduced by ꢀ33% or more, as
compared to control.
4.2.10. Statistics
Whole blood remaining activity (as compared to vehicel treated
control) is presented as means SEM (standard error of the mean).
The data were analyzed using unpaired two-tailed t test with 95%
confidence interval. Graph Pad Prism software (GraphPad Soft-
ware, Inc., La Jolla, CA 92037, USA, version 5.00) was used for sta-
tistical analysis.
4.2.7. Whole blood COX-1 assay
Since TXA2 is the major product of the COX-1/thromboxane-A
synthase-pathway (which is activated during blood clotting) this
setting was used to investigate the effect of mPGES-1 inhibitors
on these two enzymes. Human venous whole blood was collected
in neutral monovettes without any additives (SARSTEDT AG & Co,
Nümbrecht, Germany) from healthy donors who had not taken
Acknowledgments
This research was supported by Merz GmbH & Co.KGaA, the
LOEWE Lipid Signaling Forschungszentrum Frankfurt (LiFF), Deut-
sche Forschungsgesellschaft DFG (Sachbeihilfe PR 1405/2-1 and
SFB 1039 project A07), the Oncogenic Signaling Frankurt (OSF),
the European Graduate School ‘Roles of Eicosanoids in Biology
and Medicine’ (DFG GRK 757/1), and the Fonds der Chemischen
Industrie.
any NSAIDs for at least one week. 500 lL blood per sample was
directly mixed with rising concentrations of test compounds. Neg-
ative controls were immediately chilled on ice to minimize throm-
bocyte aggregation. All other samples were incubated at 37 °C for
1 h and blood was allowed to clot.
Samples were chilled on ice for 5 min and plasma was extracted
by centrifugation at 4 °C with 2000 rpm for 20 min. Plasma samples
were stored at ꢀ80 °C. For extraction procedure plasma was mixed
References and notes
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10
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TXB2-d4, 50 ng/mL PGF2 -d4, 37.5 ng/mL 6-keto-PGF1 -d4).
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