Pyrrole alkanoic acid derivatives as nuisance inhibitors of microsomal prostaglandin E2 synthase-1

Article abstract of DOI:10.1016/j.ejmech.2011.12.009

Microsomal prostaglandin E₂ synthase-1 (mPGES-1) is an enzyme, which is induced during the inflammatory response. Therefore, inhibitors of this enzyme are considered to be potential anti-inflammatory drugs. We have identified 3-(4-dodecanoyl-1,

Full text of DOI:10.1016/j.ejmech.2011.12.009

European Journal of Medicinal Chemistry 48 (2012) 153e163
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European Journal of Medicinal Chemistry
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Original article
Pyrrole alkanoic acid derivatives as nuisance inhibitors of microsomal
prostaglandin E2 synthase-1
*
Andrea Wiegard, Walburga Hanekamp, Klaus Griessbach, Jörg Fabian, Matthias Lehr
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Hittorfstrasse 58-62, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Microsomal prostaglandin E2 synthase-1 (mPGES-1) is an enzyme, which is induced during the
inflammatory response. Therefore, inhibitors of this enzyme are considered to be potential anti-
inflammatory drugs. We have identified 3-(4-dodecanoyl-1,3,5-trimethylpyrrol-2-yl)propionic acid (12)
as submicromolar inhibitor of mPGES-1. Surprisingly, structural variations made around this lead only
resulted in a relatively small change of enzyme inhibitory potency. Such flat structureeactivity rela-
tionships are reported to be typical for so called nuisance inhibitors, which exert their action not by
directly binding to the enzyme, but by forming colloid-like aggregates at micromolar and sometimes
submicromolar concentrations, which somehow sequester and inhibit enzyme targets without speci-
ficity. Since aggregate-based inhibition is highly sensitive to non-ionic detergents such as Triton X-100,
we investigated some of our compounds for inhibition of human recombinant mPGES-1 also in presence
of this detergent. The pyrrole derivatives 12, 67 and 81, which exhibited IC50 values in absence of Triton
Received 12 September 2011
Received in revised form
29 November 2011
Accepted 4 December 2011
Available online 9 December 2011
Keywords:
Nuisance inhibitor
Microsomal prostaglandin E2 synthase-1
Pyrrole carboxylic acid
Triton X-100
X-100 in the range of 0.1 and 1 mM, were virtually inactive at the highest test concentration of 10 mM
when 0.1% of the detergent was added. In the same way, the published mPGES-1 inhibitor 2-[(4-{[(1,10-
biphenyl)-4-ylmethyl]amino}-6-chloropyrimidin-2-yl)thio]octanoic acid (Cay10589) (6) totally lost its
activity under these conditions. Therefore, these compounds have to be judged as nuisance inhibitors of
the enzyme. In contrast, the known indole derivative 3-[3-(tert-butylthio)-1-(4-chlorobenzyl)-5-
isopropylindol-2-yl]-2,2-dimethylpropionic acid (MK-886) (2) showed a considerable activity (75%
inhibition at 10
m
M) also in the presence of Triton X-100.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
synthase. In endothelic cells PGH2 is converted to prostaglandin I2
(PGI2, prostacyclin) by prostacyclin synthase. In many other cells,
however, the predominant lipid mediator produced is prostaglandin
E2 (PGE2). The isomerization of PGH2 to PGE2 is catalyzed by three
different enzymes [2e5]: the cytosolic prostaglandin E2 synthase
(cPGES), and two membrane bound PGES, namely, the microsomal
prostaglandin synthase-1 and -2 (mPGES-1 and PGES-2). cPGES and
mPGES-2 are constitutively expressed enzymes with cPGES coupling
through COX-1 [2] and mPGES-2 coupling through COX-1 and COX-2
[6]. In contrast, mPGES-1 is an inducible enzyme primarily coupled to
COX-2 [7e10]. Strong inducers of the enzyme are proinflammatory
Prostaglandins and thromboxanes comprise a family of lipid
mediators, which are involved in physiological as well as in patho-
physiological processes [1]. Their synthesis is initiated by a phos-
pholipase A2, which liberates arachidonic acid (AA) from
phospholipid membranes. Arachidonic acid, in turn, is converted to
prostaglandin H2 (PGH2) by cyclooxygenase-1 (COX-1) or
cyclooxygenase-2 (COX-2). PGH2 is an unstable intermediate, which
is further metabolized to different products in dependence of the
downstream enzymes present in the cells. In platelets, its main
reaction product is thromboxane B2 formed via thromboxane
stimuli such as tumor necrosis factor-a (TNF-a) and interleukin-1b
(IL-1 ) leading to a highly elevated PGE2 formation during inflam-
b
mation [9e11]. Mice deficient in mPGES-1 exhibited a reduced
production of inflammatory PGE2 and showed a lower inflammatory
response in the collagen induced arthritis model [12e16]. Further-
more, pain reactions were impaired [14e16] and lipopolysaccharides
were not able to produce fever in such knock-out mice [17]. Gastro-
intestinal toxicity usually elicited by nonsteroidal anti-inflammatory
drugs (NSAIDs) inhibiting COX-1 [13] as well as side effects typically
Abbreviations: mPGES-1, microsomal prostaglandin E2 synthase-1; mPGES-2,
microsomal prostaglandin E2 synthase-2; cPGES, cytosolic prostaglandin E2 syn-
thase; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; PGH2, prostaglandin H2;
PGE2, prostaglandin E2; DMSO, dimethyl sulfoxide; HPLC, high performance liquid
chromatography; UV, ultra violet; MS, mass spectrometry; EI, electron ionization;
ESI, electrospray ionization; APCI, atmospheric pressure chemical ionization.
* Corresponding author. Tel.: þ49 251 83 33331; fax: þ49 251 83 32144.
E-mail address: lehrm@uni-muenster.de (M. Lehr).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.009

154
A. Wiegard et al. / European Journal of Medicinal Chemistry 48 (2012) 153e163
for COX-2 inhibitors such as changes in thrombogenesis and blood
pressure could not be seen in these animals [18,19]. Therefore,
inhibitors of mPGES-1 are expected to be novel analgetic, antipyretic
and anti-inflammatory drugs producing less adverse reactions than
the COX inhibitors presently applied [20e22].
In the past years several inhibitors of mPGES-1 have been
published [21,22]. One of the first compounds reported to possess
inhibitory activities against the enzyme were the sulfonamide
derivative 1 (NS-398) [10] and the indolylpropionic acid 2 (MK-
886) (Fig. 1) [8]. While 1 inhibits mPGES-1 with a relative low
applied as for the preparation of their known corresponding octa-
decanoyl and octadecyl derivatives, respectively [35,36,38,39].
The benzoyl and 2-naphthoyl substituted pyrrolylpropionic acid
derivatives 28 and 30 were synthesized starting from methyl 3-
(1,3,5-trimethylpyrrol-2-yl)propionate according to the procedure
published for the preparation of 12 [35]. The remaining compounds
of this series (17,20,26,27,29,32e33) were prepared from ethyl
1,3,5-trimethylpyrrol-2-carboxylate in an analogous manner as
shown in Scheme 1 for the synthesis of 26.
The pyrrolylbutanoic acid 42 was synthesized as outlined in
Scheme 2. 1,3,5-Trimethylpyrrole-2-carboxylic acid ethylester (21)
was acylated in position 4 with dodecanoyl chloride/AlCl3. Saponifi-
cation of the ester group of obtained intermediate 36by KOH followed
by thermal decarboxylation and formylation by the VilsmeiereHaak
method yielded the carbaldehyde 37, which was converted to the
methoxyvinyl substituted pyrrole 38 by Wittig-reaction with
(methoxymethyl)triphenylphosphonium chloride [40]. From this
compound acetaldehyde 39 was obtained by treatment with chloro-
trimethylsilane/NaI. HornereWadswortheEmmons reaction of 39
with triethyl phosphonoacetate gave the (E)-but-2-enoic ester 40.
Catalytic hydrogenation and final ester hydrolysis with KOH led to the
target compound 42.
The preparation of the three 1-phenyl-substitued acid deriva-
tives 49, 52 and 54 started from the carboxylic acid ester 46
(Scheme 3). Introduction of the phenyl ring in position 1 of the
pyrrole was accomplished by Cu(II)acetate catalyzed reaction with
phenylboronic acid [41]. FriedeleCrafts acylation of obtained
compound 47 with dodecanoyl chloride/AlCl3 followed by alkaline
ester hydrolysis yielded the test compound 49. For synthesis of the
corresponding acetic and propionic acid derivatives 52 and 54 first
the carboxylic acid group of 49 was removed by heating. The acetic
acid side chain was introduced into the obtained intermediate 50
by copper-assisted coupling with ethyl diazoacetate and subse-
quent ester hydrolysis with KOH. Treatment of 50 with methyl
acrylate/boron trifluoride etherate followed by saponification led to
the propionic acid target compound.
potency (IC50: 10e20 mM), with a published IC50 of 1.6 mM 2 is
a modest inhibitor of the enzyme. Just as well, the IC50 values of the
carboxylic acid derivatives 5 and 6 lie in the lower micromolar
range [23e25]. Structural refinement of 2 led to 3, which exhibited
a nanomolar IC50 [26]. Further inhibitors of mPGES-1 with nano-
molar activity against mPGES-1 are the indole-2-carboxylic acid 4
[27], the imidazole derivatives 7e9 [28e31] and the sulfonamides
10 and 11 [32e34]. Although compounds with high in vitro potency
have been found, actually no mPGES-1 inhibitor is reported
undergoing clinical evaluation.
As shown in Fig. 1, several of the described mPGES-1 inhibitors
are lipophilic carboxylic acid derivatives. For this reason, we started
our search for new inhibitors of the enzyme by testing compounds
with such structural elements already synthesized in our group for
other purposes. During this screening we found that the 4-
acylpyrrol-2-ylpropionic acid 12 (Fig. 1) [35,36] effectively
reduced enzyme activity [37] with an IC50 of 0.80 mM. This paper
reports the effects of the structural variation of this lead compound
on its mPGES-1 inhibitory properties.
2. Results and discussion
2.1. Chemistry
Quite a few of the pyrroles evaluated for mPGES-1 inhibition
have already been published (13e16,18,19,31,34) [35,36,38,39]. For
the synthesis of numerous of the dodecanoyl-substituted target
compounds (35,43e45,55e67,75) similar synthetic routes were
The synthesis of pyrrole propionic acid derivative 74, in which
the propionic acid chain is flanked by a phenyl and a benzyl residue,
Fig. 1. Structures of inhibitors of mPGES-1 activity.

A. Wiegard et al. / European Journal of Medicinal Chemistry 48 (2012) 153e163
155
Scheme 1. Reagents and conditions: (a) 3-heptyloxybenzoyl chloride, AlCl3, CH2Cl2, 0 ꢀC, 30 min, (b) aq. KOH, ethanol, reflux, 2 h, (c) 160e170 ꢀC, 20 min, (d) methyl acrylate,
BF3eEt2O, CH2Cl2, room temp., 5 d, (d) aq. KOH, ethanol, reflux, 30 min.
is outlined in Scheme 4. The nitrile, amide, hydrazide and sulfony-
lamide derivatives of the lead 12 (78e82) were prepared as shown
in Scheme 5.
for the lead compound 12 an IC50 of 0.80
compound 1 (NS-398) used as reference exhibited an IC50 of 20
m
M was determined, while
mM.
The structural modifications of the lead 12 comprised variation
of the dodecanoyl residue, substitution of the pyrrole methyl
groups by hydrogen, phenyl and benzyl, elongation and shortening
of the propionic acid moiety, and bioisosteric replacement of the
carboxylic acid group. The results of the variation of the acyl residue
are shown in Table 1. Increasing as well as decreasing the length of
the dodecanoyl substituent led to a successive reduction of mPGES-
1 inhibitory potency. Constraining the acyl chain by introduction of
an oxyphenyl moiety (20,26,27) did not drastically change activity.
Replacement of these alkoxybenzoyl residues by less bulky benzoyl,
phenylbenzoyl, naphthoyl and 3-phenylpropanoyl substituents
(28e32), respectively, resulted in a loss of mPGES-1 inhibition at
2.2. Biological evaluation
All test assays applied for mPGES-1 inhibitor screening are
basing on a method described by Thorén and Jakobson using
prostaglandin H2 (PGH2) as substrate [10]. One critical factor in
these assays is the chemical instability of PGH2, which degrades in
aqueous solutions non-enzymatically into the enzyme product
PGE2 and into PGD2 with a half life of about 10 min at room
temperature [42]. To minimize non-enzymatic PGE2 formation,
short incubation times (30 se5 min) and low temperatures
(0e20 ꢀC) are generally applied [10,20,22,24e34,37,43e49]. At the
3.3
mM.
Surprisingly, the other structural variations named above did
end of the enzyme reaction unreacted PGH2 is converted to PGF2
a
not lead to a significant alteration of enzyme inhibition. The inhi-
bition values of most of the compounds evaluated lay in the range
of 30e70% at a concentration of 3.3 mM.
by addition of SnCl2 or FeCl2. The enzyme product is quantified by
radioimmunoassay (RIA), enzyme immunoassay (EIA), coupled
spectrophotometric detection, HPLC with UV or radiometric
detection, or homogenous time resolved fluorescence detection
In our opinion, one possible reason for the observed flat struc-
tureeactivity relationships could have been an impaired solubility
of the test compounds at the assay temperature of 0 ꢀC. Since most
of these substances are very lipophilic, the concern existed that
they precipitate in the assay buffer. Therefore, we decided to re-
evaluate the compounds applying an assay temperature of 20 ꢀC.
Since at this time of our investigations microsomal preparations of
human recombinant mPGES-1 were commercially available, we
used this enzyme source for further studies. Applying the altered
(HTRF). As enzyme sources microsomes from IL-1
b stimulated
A549 cells or microsomal preparations of mPGES-1 expressed in
Chinese hamster ovary (CHO) cells, Sf9 insect cells, and Escherichia
coli cells, respectively, are used.
For evaluation of our compounds, the formation of PGE2 from
PGH2 was monitored by online solid phase extraction HPLC with
UV-detection [37]. The enzyme reactions were performed at 0 ꢀC
for 5 min applying A549 cell microsomes, initially. With this assay,
Scheme 2. Reagents and conditions: (a) dodecanoyl chloride, AlCl3, 0 ꢀC, 30 min, (b) 1. aq. KOH, ethanol, reflux, 1 h, 2. 160e170 ꢀC, 20 min, 3. DMF, POCl3, toluene, room temp., 2 h,
(c) (methoxymethyl)triphenylphosphonium chloride, K-tert-butylate, THF, microwave, 80 ꢀC, 60 min, (d) chlorotrimethylsilane, NaI, acetonitrile, room temp., 20 min, (e) triethyl
phosphonoacetate, NaH, THF, room temp., 1 h, (f) H2, Pd/C, THF, ethanol, room temp., 1 h, (g) aq. KOH, ethanol, reflux, 15 min.

156
A. Wiegard et al. / European Journal of Medicinal Chemistry 48 (2012) 153e163
Scheme 3. Reagents and conditions: (a) phenylboronic acid, Cu(II)acetate, triethyl amine, CH2Cl2, room temp., 2 d, (b) dodecanoyl chloride, AlCl3, 0 ꢀC, 2 h, (c) aq. KOH, ethanol,
reflux, 15 min, (d) 160e170 ꢀC, 20 min, (e) ethyl diazoacetate, Cu(o), toluene, 115e120 ꢀC, (f) aq. KOH, ethanol, reflux, 30 min, (g) methyl acrylate, BF3eEt2O, CH2Cl2, room temp., 3 d,
(h) aq. KOH, ethanol, reflux, 30 min.
conditions, for the lead compound 12 an IC50 of 0.67
measured. The published mPGES-1 inhibitors 2 (MK-886) and 6
(pirixinic acid derivative Cay10589) tested in parallel for compar-
m
M was
structural variations made around the lead 12 only resulted in
a small change of activity. In the majority of cases the inhibition
value of the compounds against human recombinant mPGES-1 lay
ison exhibited IC50 values of 1.3
mM and 0.65
mM, respectively.
within 30e70% at 1 mM. The only compounds showing a greater
Because the inhibition values seemed to be slightly higher than in
the first assay, all test compounds here were evaluated at
a concentration of 1 mM.
Comparing the results obtained for the acyl derivatives listed in
Table 1, one striking difference could be noted. In the assay with the
enzyme from A549 cells performed at 0 ꢀC elongation of the
dodecanoyl residue of 12 led to a successive decrease of mPGES-1
inhibitory potency, while in the assay with the recombinant
enzyme carried out at 20 ꢀC such an enlargement of the acyl chain
divergence from this range were the three derivatives, which are
unsubstituted in pyrrole 3 and 5 position (64e66), and two deriv-
atives with a phenyl residue standing vicinal to the acid moiety (49
and 67). Compounds 64e66 were inactive, while 49 and 67 showed
a nearly total inhibition of the enzyme at 1 mM.
Since the replacement of the carboxylic acid group of 12 by
bioisosteric residues like nitrile, amide, hydrazide, and sulfonyl-
amide (78e82) also did not lead to a drastical change of activity
(Table 3), we started looking for an explanation for these unordi-
nary results. During these investigations we came across an
excellent publication of Brian K. Shoichet, in which it was described
that flat structureeactivity relationships are unusual with classic,
well behaved drugs but common among so called “nuisance”
inhibitors [50]. These nuisance inhibitors exert their action not by
directly binding to the enzyme, but by forming colloid-like aggre-
gates at micromolar and sometimes submicromolar concentra-
tions, which somehow sequester and inhibit enzyme targets
without specificity. One characteristic of aggregate-based inhibi-
tion is its high sensitivity to non-ionic detergents such as Triton X-
100. Thus, molecules that inhibit an enzyme in the absence and not
length led to
a significant enhancement of activity of the
compounds (Table 1). A reason for this result could lie in solubility
problems of the very lipophilic tetradecanoyl, hexadecanoyl and
octadecanoyl derivatives 17e19 in water at 0 ꢀC.
Table 2 highlights the effects of the substitution of the methyl
substituents in pyrrole 1, 3 and 5 position of 12 and of the variation
of the alkanoic acid chain length on inhibition of human
recombinant mPGES-1. Furthermore, the consequence of the
replacement of the dodecanoyl moiety of the carboxylic acid
derivative 34 by a dodecyl residue (75) is displayed. The inhibition
data obtained with this second assay again demonstrated that most
Scheme 4. Reagents and conditions: (a) N,N-dimethyldodecanamide, POCl3, toluene, reflux, 10 h, (b) aq. KOH, ethanol, reflux, 30 min, (c) 160e170 ꢀC, 20 min, (d) benzyl bromide,
tetrabutylammonium bromide, aq. NaOH (50%), diethyl ether, CH2Cl2, reflux, 3 h, (e) methyl acrylate, BF3eEt2O, CH2Cl2, room temp., 3 d, (f) aq. KOH, ethanol, reflux, 30 min.

A. Wiegard et al. / European Journal of Medicinal Chemistry 48 (2012) 153e163
157
Scheme 5. Reagents and conditions: (a) acrylnitrile, BF3eEt2O, room temp., 1 h, (b) dodecanoyl chloride, AlCl3, CH2Cl2, 0 ꢀCeroom temp., 1.5 h, (c) N,N0-carbonyldiimidazole, NH3
conc., CH2Cl2, 0 ꢀC, 1 h/room temp., 20 min, (c) N,N0-carbonyldiimidazole, hydrazine hydrate, CH2Cl2, room temp., 16 h, (d) methanesulfonamide or benzenesulfonamide, 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimideeHCl (EDCeHCl), 4-dimethylaminopyridine, triethylamine, CH2Cl2, room temp., overnight.
in the presence of Triton X-100 are likely to inhibit the enzyme not
in a “true” manner [50e52]. Therefore, we investigated some of the
compounds described above also in presence of 0.1% Triton X-100.
This detergent was not reported to be used in mPGES-1 screening
assays until now. Only the chemical company Cayman had added
this substance to the assay buffer, when testing the activity of
microsomal preparations of recombinantly produced enzyme [53].
Table 4 shows the inhibition data of the selected compounds ob-
tained without and with the detergent. The pyrrolylpropionic acid
12, the phenylpyrrolylpropionic acid 67, and the methyl-
sulfonylamide derivative 81, which exhibited IC50 values in absence
of Triton X-100 in the range of 0.1 and 1
mM, were virtually inactive
at the highest test concentration of 10 M in presence of 0.1% of the
m
detergent. Just so, the published mPGES-1 inhibitor 6 [25] totally
lost its activity when Triton X-100 was added. Therefore, these
compounds have to be judged as nuisance inhibitors of the enzyme.
In contrast, the indole derivative 2 [8,26] showed a considerable
Table 2
Inhibition of mPGES-1 activity.
Table 1
Inhibition of mPGES-1 activity.
Comp.
R1
R2
R3
n
Inhibition of
human recombinant
Comp.
R
Inhibition of mPGES-1 Inhibition of
from A549 cells at human recombinant
3.3 mPGES-1 at 1
M [%]a M [%]b
mPGES-1 at 1 mM [%]
12
34
35
42
43
44
45
49
52
54
55
56
57
58
59
60
61
62
63
64
65
66
67
74
75
CH3
CH3
CH3
CH3
H
H
H
Phenyl
Phenyl
Phenyl
Benzyl
Benzyl
Benzyl
4-CH3eBenzyl
4-CleBenzyl
4-OCH3eBenzyl
4-OHeBenzyl
4-COOHeBenzyl
4-CONH2eBenzyl
CH3
CH3
CH3
CH3
Benzyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
2
0
1
3
0
1
2
0
1
2
0
1
2
2
2
2
2
2
2
0
1
2
2
2
54 ꢁ 8 (n ¼ 7)
m
m
59
22
70
62
17
38
99
23
65
65
35
62
52
78
72
53
27
28
n.a.
n.a.
n.a.
90
64
37
12
13
14
15
16
17
18
19
20
26
27
28
29
30
31
32
33
C11H23
C3H7
C6H13
C7H15
C9H19
C13H27
C15H31
C17H35
2-Octyloxyphenyl
3-Heptyloxyphenyl 67
4-Hexyloxyphenyl
Phenyl
Naphthalene-1-yl
Naphthalene-2-yl
2-Phenylethyl
4-Phenylphenyl
4-Phenylbenzyl
74 ꢁ 8 (n ¼ 5)
54 ꢁ 8% (n ¼ 7)
n.a.e
n.a.e
n.a.e
n.a.e
91
n.a.c
n.a.c
55
62
63
35
97
97
n.a.c
23
n.a.e
n.a.e
32
64
n.a.c
n.a.c
n.a.c
n.a.c
n.a.c
n.t.d
n.a.e
n.a.e
n.a.e
n.a.e
n.a.e
n.a.e
a
Incubation at 0 ꢀC, values are the means of at least two independent
H
H
H
H
CH3
CH3
determinations.
b
Incubation at 20 ꢀC, values are the means of at least two independent
determinations.
Phenyl
Phenyl
c
n.a.: not active at 3.3
n.t.: not tested.
n.a.: not active at 1
m
M.
d
e
m
M.
Values are the means of at least two independent determinations.

158
A. Wiegard et al. / European Journal of Medicinal Chemistry 48 (2012) 153e163
Table 3
Melting points were determined on a Büchi B-540 apparatus and
are uncorrected. 1H NMR spectra (400 MHz) and 13C NMR spectra
(100 MHz) were recorded on a Varian Mercury Plus 400 spec-
trometer. Electron ionization (EI) mass spectra were obtained on
a Finnigan GCQ apparatus. The high resolution mass spectra were
recorded on a Bruker micrOTOF-Q II spectrometer applying electro
spray chemical ionization (ESI) or atmospheric pressure chemical
ionization (APCI). Purity of the target compound was determined
by reversed phase HPLC on a Phenomenex Aqua RP 18 column
Inhibition of mPGES-1 activity.
Comp.
R
Inhibition of human
recombinant mPGES-1 at 1 mM [%]
(100 ꢂ 4.6 mm, 3
m
) or a Nucleosil 100 RP 18 column (125 ꢂ 3 mm,
12
78
79
80
81
82
COOH
CN
CONH2
CONHNH2
CONHSO2CH3
CONHSO2Phenyl
54 ꢁ 8 (n ¼ 7)
3
m
), eluting with CH3CN/H2O containing 0.1% H3PO4 at a flow rate
22
39
35
52
77
of 0.7 and 0.5 mL/min, respectively. UV-absorbance was measured
at 254 nm. The purity of all target compounds was greater than
95%.
4.1.2. Ethyl 4-(3-heptyloxybenzoyl)-1,3,5-trimethylpyrrole-2-
carboxylate (22)
Values are the means of at least two independent determinations.
A mixture of 3-heptyloxybenzoic acid [54] (141 mg, 0.47 mmol)
and thionyl chloride (5 mL) was heated under reflux for 2 h. The
remaining thionyl chloride was distilled off under reduced pres-
sure. The residue was dissolved in CH2Cl2 (5 mL) and treated at 0 ꢀC
with ethyl 1,3,5-trimethylpyrrole-2-carboxylate (21) [55] (125 mg,
0.69 mmol) followed by anhydrous AlCl3 (118 mg, 0.88 mmol).
After stirring at 0 ꢀC for 30 min, the reaction mixture was diluted
with water and extracted exhaustively with diethyl ether. The
combined organic layers were washed with half-saturated brine
and saturated aqueous NaHCO3 solution, dried (Na2SO4), and
evaporated. Chromatography on silica gel (hexane/ethyl acetate,
activity (75% inhibition at 10 mM) in the presence of Triton X-100,
making it likely that this compound is a true inhibitor of mPGES-1.
Because in all screening assays for mPGES-1 inhibitors micro-
somal preparations were used, it is imaginable that the nuisance
compounds not only lead to a reduced mPGES-1 activity by forming
aggregates that physically interact with the enzyme, but also by
disturbing the structure of the microsomes. Beside the prevention
of aggregate formation, therefore, the effect of Triton X-100 here
could also result from a stabilization of the microsomes against the
test compounds leading to a preservation of enzyme activity.
As a result of the present investigation it appears to be recom-
mendable that the activity of published mPGES-1 inhibitors should
be re-evaluated with Triton X-100 present in the incubation buffer,
particularly in those cases, in which flat structureeactivity rela-
tionships have been found.
9:1) yielded 22 as an oil (144 mg, 46%). 1H NMR (CDCl3):
d 0.89 (t,
J ¼ 6.7 Hz, 3H), 1.24e1.40 (m, 9H), 1.40e1.52 (m, 2H), 1.73e1.85 (m,
2H), 2.17 (s, 3H), 2.20 (s, 3H), 3.82 (s, 3H), 3.99 (t, J ¼ 6.6 Hz, 2H),
4.31 (q, J ¼ 7.1 Hz, 2H), 7.03e7.11 (m, 1H), 7.27e7.36 ppm (m, 3H),
MS (EI) m/z (%): 399 (100) Mþ, 284 (89).
4.1.3. 4-(3-heptyloxybenzoyl)-1,3,5-Trimethylpyrrole-2-carboxylic
acid (23)
3. Conclusion
A solution of 22 (128 mg, 0.32 mmol) in ethanol (5 mL) was
treated with 10% aqueous KOH (15 mL). The resulting mixture was
refluxed for 2 h, cooled, diluted with water, acidified with dilute
HCl, and extracted exhaustively with diethyl ether. The combined
organic phases were washed with dilute HCl, dried (Na2SO4) and
evaporated to yield 23 as an oil (112 mg, 94%). 1H NMR (CDCl3):
The in vitro assays commonly used for the evaluation of mPGES-
1 inhibitors can lead to false positive results, especially when the
test compounds are lipophilic carboxylic acid derivatives. These
pitfalls can be avoided by addition of the detergent Triton X-100 to
the assay composition.
d
0.89 (t, J ¼ 6.8 Hz, 3H), 1.16e1.40 (m, 6H), 1.40e1.54 (m, 2H),
4. Experimental section
1.71e1.86 (m, 2H), 2.19e2.27 (m, 6H), 3.84 (s, 3H), 3.95e4.04 (m,
2H), 7.06e7.11 (m, 1H), 7.22e7.35 ppm (m, 3H), MS (EI) m/z (%): 371
(24) Mþ, 326 (100).
4.1. Chemistry
4.1.1. General
4.1.4. (3-Heptyloxyphenyl)(1,2,4-trimethylpyrrol-3-yl)methanone
(24)
Column chromatography was performed on silica gel 60,
particle size 0.040e0.063 mm, from Merck or MachereyeNagel.
Compound 23 (103 mg, 0.28 mmol) was heated at 160e170 ꢀC
for 20 min. Chromatography on silica gel (hexane/ethyl acetate,
9:1) yielded 24 as an oil (65 mg, 71%). 1H NMR (CDCl3):
d 0.89 (t,
Table 4
J ¼ 6.8 Hz, 3H), 1.22e1.40 (m, 6H), 1.40e1.50 (m, 2H), 1.71e1.86 (m,
2H), 1.92 (s, 3H), 2.17 (s, 3H), 3.48 (s, 3H), 3.96 (t, J ¼ 6.6 Hz, 2H),
6.32 (s, 1H), 7.00e7.05 (m, 1H), 7.21e7.32 ppm (m, 3H), MS (EI) m/z
(%): 327 (93) Mþ, 326 (100).
Inhibition of human recombinant mPGES-1 activity in absence and in presence of
Triton X-100.
Comp.
Inhibition of human
recombinant
Inhibition of human
recombinant mPGES-1
in presence of 0.1%
mPGES-1 IC50 [m
M]a
Triton X-100 at 10 mM
4.1.5. Methyl 3-[4-(3-heptyloxybenzoyl)-1,3,5-trimethylpyrrol-2-yl]
propionate (25)
12
67
81
0.67 ꢁ 0.34
0.14 ꢁ 0.03
0.60 ꢁ 0.26
1.3 ꢁ 0.42
n.a.b
n.a.b
A solution of 24 (62 mg, 0.19 mmol) in dry CH2Cl2 (5 mL) was
n.a.b
treated with methyl acrylate (17
mL, 0.19 mmol) and boron tri-
2 (MK-866)
6 (Cay10589)
75 ꢁ 13c
fluoride diethyl etherate (12 L). After being stirred at room
m
0.65 ꢁ 0.20
n.a.b
temperature for 5 d, the mixture was diluted with water and
extracted exhaustively with diethyl ether. The combined organic
layers were dried (Na2SO4) and evaporated. The residue was
a
Mean ꢁ standard deviation, n ¼ 4 (in case of 12: n ¼ 5).
n.a.: not active, n ¼ 3.
b
c
Mean ꢁ standard deviation, n ¼ 5.

A. Wiegard et al. / European Journal of Medicinal Chemistry 48 (2012) 153e163
159
chromatographed on silica gel (hexane/ethyl acetate, 9:1) to yield
25 as a solid (33 mg, 42%), mp 54e55 ꢀC. 1H NMR (CDCl3):
0.89
4.1.9. (E)- and (Z)-1-[5-(2-Methoxyvinyl)-1,2,4-trimethylpyrrol-3-
yl]dodecan-1-one (38)
d
(t, J ¼ 6.9 Hz, 3H), 1.20e1.39 (m, 6H), 1.39e1.50 (m, 2H), 1.71e1.83
(m, 2H), 1.92 (s, 3H) 2.15 (s, 3H), 2.44e2.51 (m, 2H), 2.86e2.94
(m, 2H), 3.44 (s, 3H), 3.67 (s, 3H), 3.96 (t, J ¼ 6.6 Hz, 2H),
7.00e7.05 (m, 1H), 7.22e7.31 ppm (m, 3H), MS (EI) m/z (%): 413
(32) Mþ, 340 (100).
In a microwave tube (10 mL), a solution of 37 (423 mg,
1.38 mmol) in dry THF (1 mL) was treated with (methoxymethyl)
triphenylphosphonium chloride (497 mg, 1.45 mmol) and potas-
sium-tert-butylate (170 mg, 1.52 mmol). The reaction mixture was
then stirred and irradiated in a microwave oven at 90 W and 80 ꢀC
for 60 min. After cooling and addition of water, the reaction
mixture was exhaustively extracted with ethyl acetate. The organic
phases were dried (Na2SO4) and concentrated. Silica gel chroma-
tography (hexane/ethyl acetate, 9:1) gave an oily product, which
was a mixture of the E and Z-isomers in a ratio of 6:4 (372 mg, 78%).
4.1.6. 3-[4-(3-Heptyloxybenzoyl)-1,3,5-trimethylpyrrol-2-yl]
propionic acid (26)
A solution of 25 (31 mg, 0.08 mmol) in ethanol (4 mL) was
treated with 10% aqueous KOH (1.3 mL). The resulting mixture was
refluxed for 30 min, cooled, diluted with water, acidified with dilute
HCl, and extracted exhaustively with diethyl ether. The combined
organic phases were washed with dilute HCl, dried (Na2SO4) and
evaporated to a few mL. By addition of petroleum ether, 26
1H NMR (CDCl3):
d
0.88 (t, J ¼ 6.8 Hz, 3H), 1.17e1.41 (m, 16H),
1.58e1.74 (m, 2H), 2.17 (s, 1.2H), 2.25 (s, 1.8H), 2.47 (s, 3H),
2.68e2.75 (m, 2H), 3.36e3.41 (m, 3H), 3.68e3.73 (m, 3H), 5.06 (d,
J ¼ 6.7 Hz, 0.4H, Z-isomer), 5.43 (d, J ¼ 12.8 Hz, 0.6H, E-isomer), 6.17
(d, J ¼ 6.7 Hz, 0.4H, Z-isomer), 6.52 ppm (d, J ¼ 12.8 Hz, 0.6H, E-
isomer), MS (EI) m/z (%): 347 (49) Mþ, 192 (100).
precipitated (26 mg, 88%), mp 105 ꢀC. 1H NMR (CDCl3):
d 0.87 (t,
J ¼ 6.8 Hz, 3H), 1.17e1.38 (m, 6H), 1.38e1.50 (m, 2H), 1.69e1.81 (m,
2H),1.91 (s, 3H) 2.13 (s, 3H), 2.51 (t, J ¼ 7.9 Hz, 2H), 2.90 (t, J ¼ 7.9 Hz,
2H), 3.43 (s, 3H), 3.96 (t, J ¼ 6.6 Hz, 2H), 6.99e7.04 (m, 1H),
4.1.10. 2-(4-Dodecanoyl-1,3,5-trimethylpyrrol-2-yl)acetaldehyde
(39)
7.18e7.30 ppm (m, 3H), 13C NMR (CDCl3):
d 10.9, 12.3, 14.1, 19.4,
22.6, 29.0, 31.8, 26.0, 29.2, 30.4, 33.9, 68.2, 114.1, 116.0, 118.4, 120.7,
121.9, 127.3, 129.0, 134.1, 142.6, 159.1, 177.7, 194.1 ppm, MS (EI) m/z
(%): 399 (75) Mþ, 340 (100), HRMS-ESI m/z [M þ Na]þ calcd for
C24H33NO4: 422.2302, found: 422.2301.
To a solution of 38 (361 mg, 1.04 mmol) in acetonitrile (29 mL)
were added NaI (183 mg, 1.22 mmol) and chlorotrimethylsilane
(160 mL, 0.75 mmol). The mixture was stirred at room temperature
for 20 min, diluted with 0.5 M aqueous Na2S2O3 solution (114 mL)
and extracted exhaustively with ethyl acetate. The combined
organic layers were dried (Na2SO4) and evaporated. Chromatog-
raphy on silica gel (hexane/ethyl acetate, 8:2) yielded 39 as solid
4.1.7. Ethyl 4-dodecanoyl-1,3,5-trimethylpyrrole-2-carboxylate
(36)
A solution of ethyl 1,3,5-trimethylpyrrole-2-carboxylate (21)
[55] (1.05 g, 5.79 mmol) in CH2Cl2 (30 mL) was treated with
dodecanoyl chloride (1.5 mL, 6.5 mmol) and cooled to 0 ꢀC. Then
anhydrous AlCl3 (865 mg, 6.48 mmol) was added in portions and
the mixture was stirred at 0 ꢀC for 30 min. After addition of half-
saturated brine, the reaction mixture was exhaustively extracted
with diethyl ether. The combined organic layers were washed with
saturated aqueous NaHCO3 solution, dried (Na2SO4) and concen-
trated. The residue was purified by silica gel chromatography
(hexane/ethyl acetate, 9:1) to give 36 as a solid (1.49 g, 71%), mp
(116 mg, 33%), mp 73e74 ꢀC. 1H NMR (CDCl3):
d
0.88 (t, J ¼ 6.8 Hz,
3H), 1.18e1.38 (m, 16H), 1.61e1.72 (m, 2H), 2.23 (s, 3H), 2.47 (s, 3H),
2.67e2.75 (m, 2H), 3.36 (s, 3H), 3.67 (d, J ¼ 2.3 Hz, 2H), 9.58 ppm (t,
J ¼ 2.3 Hz, 1H), MS (EI) m/z (%): 334 (5) Mþ, 304 (100).
4.1.11. (E)-Ethyl 4-(4-dodecanoyl-1,3,5-trimethylpyrrol-2-yl)but-2-
enoate (40)
A suspension of sodium hydride (60% in mineral oil) (16 mg,
0.39 mmol) in dry THF (1 mL) was treated at 0 ꢀC with a solution of
triethyl phosphonoacetate (85 mL, 0.43 mmol) in dry THF (1 mL).
37e39 ꢀC. 1H NMR (CDCl3):
19H), 1.59e1.71 (m, 2H), 2.40 (s, 3H), 2.48 (s, 3H), 2.69e2.75 (m,
2H), 3.74 (s, 3H), 4.28e4.35 ppm (m, 2H), MS (EI) m/z (%): 363 (20)
Mþ, 208 (100).
d
0.87 (t, J ¼ 6.9 Hz, 3H), 1.18e1.41 (m,
The mixture was stirred at 0 ꢀC for 30 min. Then a solution of 39
(111 mg, 0.33 mmol) in dry THF (1 mL) was added at this
temperature and stirring was continued at room temperature for
1 h. After addition of water, the reaction mixture was extracted
exhaustively with diethyl ether. The combined organic phases
were washed with brine, dried (Na2SO4) and evaporated. Chro-
matography on silica gel (hexane/ethyl acetate, 9:1) yielded 40 as
4.1.8. 4-Dodecanoyl-1,3,5-trimethylpyrrole-2-carbaldehyde (37)
A mixture of 36 (508 mg, 1.51 mmol), ethanol (20 mL) and 10%
aqueous KOH (20 mL) was heated under reflux for 1 h, cooled,
diluted with water, acidified with 10% aqueous HCl and extracted
exhaustively with diethyl ether. The combined organic phases were
washed with dilute aqueous HCl, dried (Na2SO4) and concentrated.
The dry residue was heated at 160e170 ꢀC for 20 min. The reaction
was performed a second time. A solution of obtained intermediate
(694 mg, 2.38 mmol) in toluene (4 mL) was added dropwise to
a solid (56 mg, 42%), mp 47e48 ꢀC. 1H NMR (CDCl3):
d 0.87 (t,
J ¼ 6.8 Hz, 3H), 1.19e1.39 (m, 19H), 1.62e1.73 (m, 2H), 2.17 (s, 3H),
2.47 (s, 3H), 2.67e2.74 (m, 2H), 3.34 (s, 3H), 3.45 (dd, J ¼ 5.4 Hz and
1.7 Hz, 2H), 4.17 (q, J ¼ 7.1 Hz, 2H), 5.61 (dt, J ¼ 15.6 Hz and 1.8 Hz,
1H), 6.97 ppm (dt, J ¼ 15.6 Hz and 5.5 Hz, 1H), MS (EI) m/z (%): 404
(14) Mþ, 220 (100).
a mixture of dry N,N-dimethylformamide (553
m
L, 7.14 mmol) and
4.1.12. Ethyl 4-(4-dodecanoyl-1,3,5-trimethylpyrrol-2-yl)butanoate
(41)
POCl3 (222
m
L, 2.38 mmol) that had been stirred before at 0 ꢀC for
30 min. After stirring at room temperature for 2 h, a solution of
sodium acetate (1 g) in water (4 mL) was added and the mixture
was heated under reflux for 15 min. Then the reaction mixture was
cooled, diluted with water, and extracted exhaustively with diethyl
ether/CH2Cl2 (3:1). The combined organic phases were dried
(Na2SO4) and evaporated. The residue was chromatographed on
silica gel (CH2Cl2/ethyl acetate, 9:1) to yield 37 as a solid (452 mg,
A solution of 40 (56 mg, 0.14 mmol) in THF/ethanol (1:1) (5 mL)
was treated with a small amount of palladium (10%) on charcoal
and stirred under a balloon filled with H2 at room temperature for
1 h. The reaction mixture was filtered through Celite. The residue
was washed with THF and the combined filtrates were evaporated
to dryness. Silica gel chromatography (hexane/ethyl acetate, 9:1)
yielded 41 as an oil (45 mg, 80%). 1H NMR (CDCl3):
d 0.88 (t,
47%), mp 60e62 ꢀC. 1H NMR (CDCl3):
d
0.87 (t, J ¼ 6.8 Hz, 3H),
J ¼ 6.8 Hz, 3H), 1.19e1.39 (m, 19H), 1.62e1.72 (m, 2H), 1.72e1.82 (m,
2H), 2.19 (s, 3H), 2.33 (t, J ¼ 7.1 Hz, 2H), 2.46 (s, 3H), 2.55e2.62 (m,
2H), 2.67e2.73 (m, 2H), 3.43 (s, 3H), 4.12 ppm (q, J ¼ 7.1 Hz, 2H), MS
(EI) m/z (%): 406 (14) Mþ, 250 (100).
1.16e1.44 (m, 16H), 1.62e1.75 (m, 2H), 2.47 (s, 3H), 2.52 (s, 3H),
2.68e2.76 (m, 2H), 3.87 (s, 3H), 9.78 ppm (s, 1H), MS (EI) m/z (%):
319 (28) Mþ, 164 (100).

160
A. Wiegard et al. / European Journal of Medicinal Chemistry 48 (2012) 153e163
4.1.13. 4-(4-Dodecanoyl-1,3,5-trimethylpyrrol-2-yl)butanoic acid
(42)
(d, J ¼ 1.0 Hz, 3H), 2.39 (s, 3H), 2.73e2.79 (m, 2H), 6.48e6.50 (m,
1H), 7.22e7.27 (m, 2H), 7.36e7.49 ppm (m, 3H), MS (EI) m/z (%): 354
(4) Mþ, 198 (100).
A solution of 41 (45 mg, 0.11 mmol) in ethanol (5 mL) was
treated with 10% aqueous KOH (1.7 mL). The resulting mixture was
refluxed for 15 min, cooled, diluted with water, acidified with dilute
HCl, and extracted exhaustively with diethyl ether. The combined
organic phases were washed with dilute HCl, dried (Na2SO4) and
evaporated to yield 42 as an oil (36 mg, 91%). 1H NMR (CDCl3):
4.1.18. Ethyl 2-(4-dodecanoyl-3,5-dimethyl-1-phenylpyrrol-2-yl)
acetate (51)
A solution of 50 (164 mg, 0.46 mmol) in dry toluene (5 mL) was
treated with a solution of ethyl diazoacetate (63 mg, 0.55 mmol)
and some copper powder and heated at 115e120 ꢀC until the
evolution of nitrogen had ceased. After cooling, the mixture was
directly chromatographed on silica gel (hexane/ethyl acetate, a)
9.75:0.25, b) 9.5:0.5) to yield 51 as an oil (57 mg, 28%). 1H NMR
d
0.87 (t, J ¼ 6.8 Hz, 3H), 1.18e1.40 (m, 16H), 1.59e1.72 (m, 2H),
1.74e1.85 (m, 2H), 2.19 (s, 3H), 2.39 (t, J ¼ 7.0 Hz, 2H), 2.47 (s, 3H),
2.61 (t, J ¼ 7.5 Hz, 2H), 2.67e2.75 (m, 2H), 3.42 ppm (s, 3H), 13C NMR
(CDCl3): d 12.3, 12.6, 14.1, 22.7, 29.3, 29.6, 31.9, 23.2, 24.5, 25.1, 30.4,
32.7, 42.2, 115.1, 120.6, 128.5, 135.9, 178.2, 199.2 ppm, MS (EI) m/z
(%): 378 (8) Mþ, 164 (100), HRMS-ESI m/z [M þ Na]þ calcd for
C23H39NO3: 400.2822, found: 400.2825.
(CDCl3):
d
0.88 (t, J ¼ 6.8 Hz, 3H), 1.16 (t, J ¼ 7.1 Hz, 3H), 1.18e1.42
(m, 16H), 1.63e1.76 (m, 2H), 2.23 (s, 3H), 2.26 (s, 3H), 2.73e2.80 (m,
2H), 3.34 (s, 2H), 4.01 (q, J ¼ 7.1 Hz, 2H), 7.09e7.23 (m, 2H),
7.42e7.51 ppm (m, 3H), MS (EI) m/z (%): 440 (17) Mþ, 284 (100).
4.1.14. Ethyl 3,5-dimethyl-1-phenylpyrrole-2-carboxylate (47)
To a solution of ethyl 3,5-dimethylpyrrole-2-carboxylate (46)
(899 mg, 5.37 mmol) in dry CH2Cl2 (5 mL) was added phenyl-
boronic acid (1.42 g, 11.7 mmol), Cu(II)acetate (1.53 g, 8.41 mmol)
and triethylamine (1.5 mL, 11 mmol). The mixture was stirred at
room temperature for 2 d, filtered through Celite, dried (Na2SO4)
and evaporated. The residue was chromatographed on silica gel
(hexane/ethyl acetate, a) 9.5:0.5, b) 9:1) to yield 47 as a solid
4.1.19. 2-(4-Dodecanoyl-3,5-dimethyl-1-phenylpyrrol-2-yl)acetic
acid (52)
A mixture of 51 (50 mg, 0.11 mmol), ethanol (5 mL) and 10%
aqueous KOH (2 mL) was heated under reflux for 30 min, cooled,
diluted with water, acidified with dilute HCl and extracted
exhaustively with diethyl ether. The combined organic phases were
washed with dilute aqueous HCl, dried (Na2SO4) and concentrated.
The product was purified by RP-HPLC (acetonitrile/H2O/formic acid,
9:1:0.04). The eluates were concentrated under reduced pressure
until most of the acetonitrile was distilled off. The remaining
solvent was removed by freeze drying yielding 52 as a solid (27 mg,
(274 mg, 21%), mp 41 ꢀC. 1H NMR (CDCl3):
d
1.05 (t, J ¼ 7.1 Hz, 3H),
1.96 (s, 3H), 2.38 (s, 3H), 4.04 (q, J ¼ 7.1 Hz, 2H), 5.91 (s, 1H),
7.14e7.20 (m, 2H), 7.35e7.46 ppm (m, 3H), MS (EI) m/z (%): 243
(100) Mþ, 170 (63).
64%), mp 87 ꢀC. 1H NMR (CDCl3):
d
0.88 (t, J ¼ 6.8 Hz, 3H), 1.18e1.46
4.1.15. Ethyl 4-dodecanoyl-3,5-dimethyl-1-phenylpyrrole-2-
carboxylate (48)
(m, 16H), 1.61e1.78 (m, 2H), 2.23 (s, 3H), 2.28 (s, 3H), 2.76 (t,
J ¼ 7.4 Hz, 2H), 3.39 (s, 2H), 7.13e7.21 (m, 2H), 7.43e7.53 ppm (m,
A solution of 47 (631 mg, 2.59 mmol) in dry CH2Cl2 (20 mL) was
treated with dodecanoyl chloride (0.69 mL, 2.93 mmol) and cooled
to 0 ꢀC. Then anhydrous AlCl3 (438 mg, 3.28 mmol) was added in
portions and the mixture was stirred at 0 ꢀC for 2 h. After addition
of half-saturated brine, the reaction mixture was exhaustively
extracted with CH2Cl2/diethyl ether (3:1). The combined organic
layers were washed with saturated aqueous NaHCO3 solution, dried
(Na2SO4) and concentrated. The residue was purified by silica gel
chromatography (hexane/ethyl acetate, 9.5:0.5) to give 48 as a solid
3H), 13C NMR (CDCl3):
d 12.3, 13.7, 14.1, 22.7, 29.3, 29.6, 29.7, 31.9,
24.4, 30.2, 42.8, 117.8, 121.8, 122.2, 128.6, 129.0, 129.5, 135.9, 137.0,
175.7, 198.7 ppm, MS (EI) m/z (%): 412 (8) Mþ, 226 (100), HRMS-ESI
m/z [M þ Na]þ calcd for C26H37NO3: 434.2666, found: 434.2664.
4.1.20. Methyl 3-(4-dodecanoyl-3,5-dimethyl-1-phenylpyrrol-2-yl)
propionate (53)
A solution of 50 (175 mg, 0.49 mmol) in dry CH2Cl2 (5 mL) was
treated with methyl acrylate (46
mL, 0.50 mmol) and boron tri-
(481 mg, 44%), mp 53e54 ꢀC. 1H NMR (CDCl3):
d
0.88 (t, J ¼ 6.8 Hz,
fluoride diethyl etherate (63 L). After being stirred at room
m
3H), 0.96 (t, J ¼ 7.1 Hz, 3H), 1.18e1.44 (m, 16H), 1.66e1.75 (m, 2H),
2.18 (s, 3H), 2.60 (s, 3H), 2.75e2.81 (m, 2H), 4.01 (q, J ¼ 7.1 Hz, 2H),
7.12e7.17 (m, 2H), 7.41e7.48 ppm (m, 3H), MS (EI) m/z (%): 426 (7)
Mþ, 285 (100).
temperature for 3 d, the mixture was diluted with water and
extracted exhaustively with diethyl ether. The combined organic
layers were dried (Na2SO4) and evaporated. The residue was
chromatographed on silica gel (hexane/ethyl acetate, 9.5:0.5) to
yield 53 as an oil (68 mg, 31%). 1H NMR (CDCl3):
3H), 1.18e1.43 (m, 16H), 1.62e1.78 (m, 2H), 2.14e2.22 (m, 5H), 2.28
(s, 3H), 2.66e2.78 (m, 4H), 3.58 (s, 3H), 7.15e7.20 (m, 2H),
7.43e7.53 ppm (m, 3H), MS (EI) m/z (%): 440 (19) Mþ, 284 (100).
d
0.88 (t, J ¼ 6.8 Hz,
4.1.16. 4-Dodecanoyl-3,5-dimethyl-1-phenylpyrrole-2-carboxylic
acid (49)
A solution of 48 (430 mg, 1.01 mmol) in ethanol (5 mL) was
treated with 20% aqueous KOH (2 mL). The resulting mixture was
refluxed for 15 min, cooled, diluted with water, acidified with dilute
HCl, and extracted exhaustively with diethyl ether. The combined
organic phases were washed with dilute HCl, dried (Na2SO4) and
evaporated to yield 49 as a solid (282 mg, 70%), mp 95e96 ꢀC. 1H
4.1.21. 3-(4-Dodecanoyl-3,5-dimethyl-1-phenylpyrrol-2-yl)
propionic acid (54)
A mixture of 53 (53 mg, 0.12 mmol), ethanol (7.5 mL) and 10%
aqueous KOH (2.5 mL) was heated under reflux for 30 min, cooled,
diluted with water, acidified with dilute HCl and extracted
exhaustively with diethyl ether. The combined organic phases were
washed with dilute aqueous HCl, dried (Na2SO4), and concentrated
to a few mL. By addition of petroleum ether, 54 precipitated (50 mg,
NMR (CDCl3):
d
0.87 (t, J ¼ 6.8 Hz, 3H),1.16e1.44 (m,16H),1.62e1.77
(m, 2H), 2.15 (s, 3H), 2.58 (s, 3H), 2.77 (t, J ¼ 7.4 Hz, 2H), 7.08e7.16
(m, 2H), 7.38e7.46 ppm (m, 3H), MS (EI) m/z (%): 397 (2) Mþ, 198
(100), HRMS-ESI m/z [M þ Na]þ calcd for C25H35NO3: 420.2509,
found: 420.2519.
99%), mp 74e76 ꢀC. 1H NMR (CDCl3):
d
0.87 (t, J ¼ 6.8 Hz, 3H),
1.18e1.43 (m, 16H), 1.61e1.76 (m, 2H), 2.15e2.24 (m, 5H), 2.26 (s,
3H), 2.64e2.78 (m, 4H), 7.14e7.20 (m, 2H), 7.43e7.53 ppm (m, 3H),
4.1.17. 1-(2,4-Dimethyl-1-phenylpyrrol-3-yl)dodecan-1-one (50)
Compound 49 (258 mg, 0.68 mmol) was heated at 160e170 ꢀC
for 20 min to yield 50 as an oil (175 mg, 74%). The product was used
for the further reactions without purification. 1H NMR (CDCl3):
13C NMR (CDCl3):
d 12.1, 13.5, 14.1, 19.5, 22.7, 29.3, 29.6, 29.7, 31.9,
24.5, 33.9, 42.8, 115.6, 121.7, 128.0, 128.4, 128.9, 129.5, 135.3, 137.5,
177.5, 199.1 ppm, MS (EI) m/z (%): 426 (14) Mþ, 270 (100), HRMS-ESI
m/z [M þ Na]þ calcd for C27H39NO3: 448.2822, found: 448.2821.
d
0.88 (t, J ¼ 6.8 Hz, 3H), 1.17e1.42 (m, 16H), 1.67e1.76 (m, 2H), 2.32

A. Wiegard et al. / European Journal of Medicinal Chemistry 48 (2012) 153e163
161
4.1.22. Ethyl 4-dodecanoyl-5-methyl-3-phenylpyrrole-2-
carboxylate (69)
A solution of ethyl 5-methyl-3-phenylpyrrole-2-carboxylate
[56] (68) (900 mg, 3.93 mmol) in toluene (15 mL) was added
dropwise to a refluxing solution of N,N-dimethyldodecanamide
as a solid (55 mg, 65%), mp 121e123 ꢀC. 1H NMR (CDCl3):
d
0.88
(t, J ¼ 6.9 Hz, 3H), 0.91e1.34 (m, 16H), 1.34e1.46 (m, 2H),
2.03e2.12 (m, 2H), 2.12e2.22 (m, 2H), 2.42 (s, 3H), 2.66e2.74
(m, 2H), 5.13 (s, 2H), 6.93 (d, J ¼ 7.2 Hz, 2H), 7.22e7.39 ppm (m,
8H), 13C NMR (CDCl3):
d 11.8, 14.1, 19.6, 22.7, 29.2, 29.3, 29.4,
(955
m
L, 4.2 mmol) and POCl3 (416
m
L, 4.53 mmol) in toluene
29.6, 31.9, 24.8, 34.3, 42.5, 46.8, 121.9, 123.1, 125.5, 127.0, 127.6,
127.8, 128.3, 129.0, 130.3, 134.5, 136.6, 136.9, 177.4, 200.6 ppm,
HRMS-ESI m/z [M þ Na]þ calcd for C33H43NO3: 524.3135, found:
524.3137.
(20 mL). After stirring under reflux for 10 h, a solution of sodium
acetate (6 g) in water (24 mL) was added and the mixture was
further heated under reflux for 15 min. Then the reaction mixture
was cooled, diluted with water, and extracted exhaustively with
diethyl ether and CH2Cl2. The combined organic phases were dried
(Na2SO4) and evaporated. The residue was chromatographed on
silica gel (hexane/ethyl acetate, 9:1) to yield 69 as a solid (299 mg,
4.1.28. 3-(1,3,5-Trimethylpyrrol-2-yl)propanenitrile (77)
A mixture of 1,2,4-trimethylpyrrole (76) (2.0 g, 18.3 mmol),
acrylnitrile (2 mL), and boron trifluoride diethyl etherate (1 mL)
was stirred at room temperature for 1 h. After addition of diethyl
ether, the organic phase was washed with water, dried (Na2SO4)
and evaporated. The residue was chromatographed on silica gel to
19%), mp 56e58 ꢀC. 1H NMR (CDCl3):
0.91e1.44 (m, 21H), 1.96e2.09 (m, 2H), 2.55 (s, 3H), 4.09 (q,
J ¼ 7.1 Hz, 2H), 7.27e7.41 (m, 5H), 9.34 ppm (s, broad, 1H), MS (EI)
m/z (%): 411 (11) Mþ, 271 (100).
d 0.80e0.91 (m, 3H),
yield 77 as a solid (747 mg, 25%), mp 56 ꢀC. 1H NMR (CDCl3):
d 2.02
(s, 3H), 2.17 (s, 3H), 2.45 (t, J ¼ 7 Hz, 2H), 2.93 (t, J ¼ 7 Hz, 2H), 3.41
(s, 3H), 5.69 ppm (s, 1H), MS (CI) m/z (%): 163 (100) (M þ 1)þ, 122
(22).
4.1.23. 4-Dodecanoyl-5-methyl-3-phenylpyrrole-2-carboxylic acid
(70)
Compound 69 (457 mg, 1.11 mmol) was saponified according to
the procedure described for the synthesis of 54. Recrystallization
from petroleum ether yielded 70 (292 mg, 69%), mp 130e131 ꢀC. 1H
4.1.29. 3-(4-Dodecanoyl-1,3,5-trimethylpyrrol-2-yl)propanenitrile
(78)
NMR (CDCl3):
d
0.88 (t, J ¼ 6.8 Hz, 3H), 0.91e1.45 (m, 18H),
Compound 78 was prepared from 77 (700 mg, 5.54 mmol) in
a similar manner as described for the synthesis of 36. Chromatog-
raphy on silica gel yielded 79 as a solid (507 mg, 27%), mp 75e76 ꢀC.
1.93e2.03 (m, 2H), 2.52 (s, 3H), 7.27e7.44 ppm (m, 5H), MS (EI) m/z
(%): 384 (3) Mþ, 243 (100).
1H NMR (CDCl3):
d
0.89 (t, J ¼ 7 Hz, 3H), 1.14e1.51 (m, 16H), 1.67
4.1.24. 1-(2-Methyl-4-phenylpyrrol-3-yl)dodecan-1-one (71)
Compound 70 (263 mg, 0.68 mmol) was heated at 160e170 ꢀC
for 20 min. Chromatography on silica gel (hexane/ethyl acetate,
9:1) yielded 71 as a solid (178 mg, 76%), mp 57 ꢀC. 1H NMR (CDCl3):
(quint, J ¼ 7 Hz, 2H), 2.19 (s, 3H), 2.45 (s, 3H), 2.45 (t, J ¼ 7 Hz, 2H),
2.69 (t, J ¼ 7 Hz, 2H), 2.96 (t, J ¼ 7 Hz, 2H), 3.41 ppm (s, 3H). MS (EI)
m/z (%): 344 (27) Mþ, 189 (100).
d
0.88 (t, J ¼ 6.8 Hz, 3H), 0.94e1.37 (m, 16H), 1.37e1.55 (m, 2H),
4.1.30. 3-(4-Dodecanoyl-1,3,5-trimethylpyrrol-2-yl)propanamide
2.22e2.34 (m, 2H), 2.50 (s, 3H), 6.55 (s, 1H), 7.26e7.41 (m, 5H),
8.25 ppm (s, broad, 1H), MS (EI) m/z (%): 339 (21) Mþ, 184 (100).
(79)
A
solution of 12 (150 mg, 0.41 mmol) and N,N0-carbon-
yldiimidazole (200 mg) in dry CH2Cl2 (8 mL) was stirred at room
temperature for 1 h. After cooling in an ice bath, conc. NH3 (8 mL)
was added and the mixture was stirred with ice cooling for 1 h and
at room temperature for 20 min. Then water and NaCl were added,
and the reaction mixture was extracted with diethyl ether and with
CH2Cl2. The combined organic layers were washed with dilute
aqueous NaOH, dried (Na2SO4) and evaporated. Chromatography
on silica gel (CHCl3/THF, 3:1) yielded 79 as a solid (26 mg, 17%), mp
4.1.25. 1-(1-Benzyl-2-methyl-4-phenylpyrrol-3-yl)dodecan-1-one
(72)
A mixture of 71 (171 mg, 0.50 mmol), benzyl bromide (94 mg,
0.55 mmol), tetrabutylammonium bromide (81 mg, 0.25 mmol),
diethyl ether (5 mL), CH2Cl2 (2.5 mL), and 50% aqueous NaOH
(2.5 mL) was refluxed for 3 h with vigorous stirring. The reaction
mixture was cooled and the organic phase separated. The aqueous
layer was extracted exhaustively with diethyl ether/CH2Cl2 (3:1),
and the combined organic phases were dried (Na2SO4) and evap-
orated. The residue was chromatographed on silica gel (hexane/
ethyl acetate, 9:1) to yield 72 as an oil (184 mg, 86%). 1H NMR
136e137 ꢀC. 1H NMR (CDCl3):
d
0.88 (t, J ¼ 7 Hz, 3H), 1.19e1.43 (m,
16H), 1.65 (quint, J ¼ 7 Hz, 2H), 2.21 (s, 3H), 2.34 (t, J ¼ 8 Hz, 2H),
2.46 (s, 3H), 2.70 (t, J ¼ 7 Hz, 2H), 2.93 (t, J ¼ 8 Hz, 2H), 3.43 (s, 3H),
5.25e5.35 ppm (m, broad, 2H). MS (EI) m/z (%): 362 (2) Mþ, 304
(100).
(CDCl3):
d
0.88 (t, J ¼ 6.8 Hz, 3H), 0.95e1.36 (m, 16H), 1.39e1.53 (m,
2H), 2.26e2.34 (m, 2H), 2.40 (s, 3H), 5.06 (s, 2H), 6.54 (s, 1H),
7.04e7.12 (m, 2H), 7.23e7.40 ppm (m, 8H), MS (EI) m/z (%): 430 (20)
Mþ, 289 (100).
4.1.31. 3-(4-Dodecanoyl-1,3,5-trimethylpyrrol-2-yl)propanehydraz-
ide (80)
A
solution of 12 (150 mg, 0.41 mmol) and N,N0-carbon-
4.1.26. Methyl 3-(1-benzyl-4-dodecanoyl-5-methyl-3-phenylpyrrol-
2-yl)propionate (73)
yldiimidazole (200 mg) in dry CH2Cl2 (8 mL) was stirred at room
temperature for 1 h. After addition of hydrazine hydrate (0.25 mL),
the mixture was stirred at room temperature for 16 h. Then water
and NaCl were added and the mixture was extracted exhaustively
with diethyl ether. The combined organic layers were dried
(Na2SO4) and evaporated. The product was precipitated from THF/
petroleum ether and subsequently dissolved again in diethyl ether/
CH2Cl2 (5:1). The organic solution was washed with dilute aqueous
HCl, dried (Na2SO4), and concentrated to a few mL. After addition of
petroleum ether, 80 precipitated (96 mg, 62%), mp 96e98 ꢀC. 1H
Compound 72 (173 mg, 0.40 mmol) was reacted with methyl
acrylate as described for the preparation of 53. Chromatography on
silica gel (hexane/ethyl acetate, 9:1) yielded 73 as an oil (88 mg,
42%). 1H NMR (CDCl3):
d
0.88 (t, J ¼ 6.8 Hz, 3H), 0.92e1.33 (m, 16H),
1.33e1.49 (m, 2H), 2.00e2.12 (m, 2H), 2.13e2.24 (m, 2H), 2.42 (s,
3H), 2.66e2.78 (m, 2H), 3.53 (s, 3H), 5.15 (s, 2H), 6.94 (d, J ¼ 7.2 Hz,
2H), 7.20e7.44 ppm (m, 8H), MS (ESI) m/z [M þ Na]þ 538.4.
4.1.27. 3-(1-Benzyl-4-dodecanoyl-5-methyl-3-phenylpyrrol-2-yl)
propionic acid (74)
Compound 73 (87 mg, 0.17 mmol) was saponified according
to the procedure described for the synthesis of 54 to yield 74
NMR (CDCl3):
d
0.88 (t, J ¼ 7 Hz, 3H), 1.19e1.43 (m, 16H), 1.65 (quint,
J ¼ 7 Hz, 2H), 2.19 (s, 3H), 2.23 (t, J ¼ 8 Hz, 2H), 2.45 (s, 3H), 2.69 (t,
J ¼ 7 Hz, 2H), 2.93 (t, J ¼ 8 Hz, 2H), 3.43 (s, 3H), 6.64 ppm (s, 1H). MS
(EI) m/z (%): 377 (23) Mþ, 304 (100).

162
A. Wiegard et al. / European Journal of Medicinal Chemistry 48 (2012) 153e163
4.1.32. 3-(4-Dodecanoyl-1,3,5-trimethylpyrrol-2-yl)-N-
(methanesulfonyl)propanamide (81)
of the internal standard PGE1. The ratio of enzymatic to non-
enzymatic production of PGE2 usually was about 2.5e3.
A mixture of 12 (100 mg, 0.27 mmol), methanesulfonyl chloride
(67 mg, 0.70 mmol), 4-dimethylaminopyridine (10 mg, 0.08 mmol),
3-(3-dimethylaminopropyl)-1-ethyl carbodiimideehydrochloride
(EDCeHCl) (48 mg), triethylamine (0.07 mL) and CH2Cl2 (15 mL)
was stirred at room temperature overnight. After addition of
CH2Cl2, the organic solution was washed with water, dried (Na2SO4)
and evaporated. Chromatography on silica gel with ethyl acetate
followed by reprecipitation from CH2Cl2/petroleum ether (1:5)
yielded 81 as a solid (58 mg, 49%), mp 133 ꢀC. 1H NMR (CDCl3):
For calculation of enzyme inhibition the peak ratio of the
enzyme product PGE2 formed enzymatically and the internal
standard PGE1 obtained in presence of a test compound was
compared with the mean level of this peak ratio determined in
absence of test compounds (¼control tests). The IC50 values were
calculated with the aid of Probit transformation.
The
chlorobenzyl)-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid,
MK-886) and
(2-[(4-{[(1,10-biphenyl)-4-ylmethyl]amino}-6-
reference
inhibitors
2
(3-[3-(tert-butylthio)-1-(4-
6
d
0.89 (t, J ¼ 7 Hz, 3H), 1.22e1.43 (m, 12H), 1.56e1.67 (m, 6H), 2.20
chloropyrimidin-2-yl)thio]octanoic
purchased from Cayman Chemicals.
acid,
Cay10589)
were
(s, 3H), 2.46 (s, 3H), 2.47 (t, J ¼ 7 Hz, 2H), 2.69 (t, J ¼ 7 Hz, 2H), 2.96
(t, J ¼ 7 Hz, 2H), 3.28 (s, 3H), 3.44 ppm (s, 3H). MS (EI) m/z (%): 440
(10) Mþ, 304 (100).
4.2.2. Test assay with Triton X-100
The assay was carried out in the same way applying 186
mL Tris/
4.1.33. 3-(4-Dodecanoyl-1,3,5-trimethylpyrrol-2-yl)-N-
(benzenesulfonyl)propanamide (82)
Compound 12 (364 mg, 1 mmol) was reacted with benzene-
sulfonamide according to the procedure described for the synthesis
of 81. Chromatography on silica gel (petroleum ether/ethyl acetate,
7:3) yielded 82 as a solid (99 mg, 20%), mp 56e57 ꢀC. 1H NMR
glutathione-buffer, which contained 0.108% Triton X-100 (w/v). The
final concentration of Triton X-100 was 0.10% (w/v). For the enzy-
matic reactions the commercial available enzyme solution was
diluted only 1:100 with phosphate buffer. The peak areas of PGE2
formed in the incubations with the enzyme in absence of test
compounds averaged 40e50% of the area of the internal standard
PGE1. The ratio of enzymatic to non-enzymatic production of PGE2
here was about 1.
(CDCl3):
d
0.88 (t, J ¼ 7 Hz, 3H), 1.15e1.33 (m, 16H), 1.66 (quint,
J ¼ 7 Hz, 2H), 2.08 (s, 3H), 2.40 (t, J ¼ 7 Hz, 2H), 2.43 (s, 3H), 2.68 (t,
J ¼ 7 Hz, 2H), 2.85 (t, J ¼ 7 Hz, 2H), 3.32 (s, 3H), 4.90 (s, broad, 1H),
7.55e7.63 (m, 2H), 7.68 (t, J ¼ 8 Hz, 1H), 8.05 ppm (d, J ¼ 8 Hz, 2H).
MS (EI) m/z (%): 502 (2) Mþ, 77 (100).
Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2011.12.009.
4.2. Inhibition assays for mPGES-1 applying recombinant human
enzyme
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4.2.1. Test assay without Triton X-100
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