Structure-activity relationship of nonacidic quinazolinone inhibitors of human microsomal prostaglandin synthase 1 (mPGES 1)

Article abstract of DOI:10.1021/jm201687d

Microsomal prostaglandin E synthase 1 (mPGES-1) is a key enzyme of the arachidonic acid cascade. Its product PGE₂ plays an important role in various inflammatory processes, pain, fever, and cancer. Selective inhibition of mPGES-1 might be a promising step to avoid cyclooxygenase-related effects of NSAIDs. We studied a class of quinazolinone derivatives of the lead structure FR20 for their effects on the isolated human and murine enzymes, human HeLa cells, and in various settings of the whole blood assay. Novel compounds with direct enzyme inhibiting activity in the submicromolar range (IC₅₀: 0.13-0.37 μM) were designed using a bioisosteric replacement strategy and proved to be effective in both cells and human whole blood. Furthermore, pharmacological profiling of toxicity and eicosanoid screening with LC/MS-MS was applied to characterize this new class of mPGES-1 inhibitors.

Full text of DOI:10.1021/jm201687d

Article
pubs.acs.org/jmc
Structure−Activity Relationship of Nonacidic Quinazolinone
Inhibitors of Human Microsomal Prostaglandin Synthase 1
(mPGES 1)
Florian Rorsch,*,† Estel.la Buscato,‡ Klaus Deckmann,† Gisbert Schneider,§ Manfred Schubert-Zsilavecz,‡
́
̈
Gerd Geisslinger,† Ewgenij Proschak,‡ and Sabine Grosch†
̈
†Johann Wolfgang Goethe-University, Institute of Clinical Pharmacology, pharmazentrum frankfurt, LiFF/ZAFES, Theodor-Stern-Kai
7, D-60590 Frankfurt/Main, Germany
‡Johann Wolfgang Goethe-University, Institute of Pharmaceutical Chemistry, LiFF/ZAFES, Max-von-Laue-Strasse 9, D-60438
Frankfurt/Main, Germany
§ETH Zurich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland
̈
̈
S
* Supporting Information
ABSTRACT: Microsomal prostaglandin E synthase 1 (mPGES-1) is a key enzyme of the
arachidonic acid cascade. Its product PGE2 plays an important role in various inflammatory processes,
pain, fever, and cancer. Selective inhibition of mPGES-1 might be a promising step to avoid
cyclooxygenase-related effects of NSAIDs. We studied a class of quinazolinone derivatives of the lead
structure FR20 for their effects on the isolated human and murine enzymes, human HeLa cells, and
in various settings of the whole blood assay. Novel compounds with direct enzyme inhibiting activity in the submicromolar range
(IC50: 0.13−0.37 μM) were designed using a bioisosteric replacement strategy and proved to be effective in both cells and human
whole blood. Furthermore, pharmacological profiling of toxicity and eicosanoid screening with LC/MS-MS was applied to
characterize this new class of mPGES-1 inhibitors.
models.15 On the other hand, mPGES-1 activity is directly
INTRODUCTION
■
affected by small-molecule inhibitors. AF3442 2 inhibited
mPGES-1 activity in LPS-stimulated human monocytes and
human whole blood.16 PF-9184 3 exhibits high potency on
recombinant human mPGES-1 with an IC50 of 16.5 nM but
loses its potency on the rat mPGES-1 in vitro (IC50 of 1080 nM
on recombinant rat mPGES-1) and in vivo (no effect in rat air
pouch model).17 A series of phenanthrene imidazole inhibitors
around the lead compound MF63 4 also showed high potency
on the human and guinea pig enzyme and in the human whole
blood assay with selectivity over several other related enzymes
but lacks the ability to inhibit mouse or rat mPGES-1.18
These data illustrate one main problem occurring during the
development of selective human mPGES-1 inhibitors, as they
are inactive regarding the inhibition of the mouse or rat
mPGES-1, thus rendering the application of preclinical animal
models impossible. This species difference is due to several
amino acid exchanges between the human and murine enzyme,
leading to variations in the active site of mPGES-1. Several dual
5-lipoxygenase (5-LO)/mPGES-1 inhibitors extend the phar-
macological profile of anti-inflammatory agents and exhibit
activity in animal models of carrageenan-induced rat pleurisy
and aortic aneurysm.19−22 In addition, natural compounds like
arzanol 5 from Helichrysum italicum have been reported to
affect inflammation and PGE2 levels in carrageenan-induced
pleurisy in rats.23 We recently reported a series of novel
The microsomal prostaglandin E2-synthase 1 (mPGES-1) is an
inducible enzyme of the arachidonic acid cascade and mainly
coupled to the expression of cyclooxygenase-2 (COX-2).1
Proinflammatory stimuli such as lipopolysaccharide (LPS) up-
regulate the protein level of both enzymes. The major product,
prostaglandin E2 (PGE2), plays an important role in the
mediation of pain,2,3 fever,4 the development of various cancer
types,5 and several inflammatory diseases like rheumatoid
arthritis.6 Besides clinically relevant diseases, PGE2 is involved
in gastrointestinal protection7,8 and the onset of wakefulness
and maintenance of the circadian rhythm.9 PGE2 production is
inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs),
specifically selective and nonselective COX-inhibitors. Inhib-
ition of mPGES-1 might improve the pharmacological profile of
current analgesic, anti-inflammatory, and antipyretic medication
due to a reduction of side effects as compared to known
NSAIDs.10,11 Especially patients under continuous long-term
treatment with NSAIDs may suffer from gastrointestinal ulcers
and cardiovascular complications.12,13 Permanent treatment,
however, is indicated for treatment of chronic inflammatory
diseases like rheumatoid arthritis (RA) or arteriosclerosis.
Several mPGES-1 inhibitors have been tested in vitro and in
vivo in different disease models (Chart 1).14
For mPGES-1 inhibition, two main approaches have been
pursued. On the one hand, mPGES-1 can be inhibited via
down-regulation of mPGES-1 expression. Benzothiophene γ-
hydroxybutenolide (BTH) 1 significantly reduced PGE2 levels
in the mouse air pouch and collagen-induced arthritis (CIA)
Received: December 14, 2011
Published: March 26, 2012
© 2012 American Chemical Society
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Chart 1. Several Published mPGES-1 Inhibitors
a
Scheme 1. Schematic Synthesis Route for Compounds Based on a Quinazolinone Scaffold
a
Formation of 2,3-disubstituted 3H-quinazolin-4-one is followed by the formation of the final product by a related Goldberg reaction.
nonacidic mPGES-1 inhibitors that were found by a multistep
virtual screening protocol.24 Quinazolinone 7 (FR20) provided
the starting point for an extensive structure−activity relation-
ship study (SAR). Here we present a structure optimization
approach including chemical synthesis of quinazolinone
derivatives, detailed information about activity on human and
murine isolated mPGES-1 enzyme, in vitro nonspecific cellular
toxicity, and impact on different eicosanoids in human whole
blood. As a major outcome of this study, we present a class of
direct human mPGES-1 inhibitors with low nonspecific cellular
toxicity and a broad whole blood eicosanoid profile.
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 derivative at 1 μM
on isolated protein in the mPGES-1 activity assay with the
reference structure 7 (Table 1). Derivatives 7f, 7i, 7k, 7l, 7n,
7o, 7q, and 7r equipotently reduced the PGE2-level 5% or
higher as compared to 7. For those substances, dose−response
curves (exemplary curve in Supporting Information Figure S1)
and IC50 values with 95% confidence intervals were calculated.
IC50 values were between 0.13 μM (7) and 0.37 μM (Table 1).
IC50 values of these derivatives reveal comparable potencies.
Murine mPGES-1 Activity Screening. Because the human
and murine mPGES-1 differ in their amino acid sequences, we
further investigated the influence of 7 and equipotent
derivatives 7f, 7i, 7k, 7l, 7n, 7o, 7q, and 7r on murine
mPGES-1 in vitro. The compounds were tested on the murine
mPGES-1 enzyme in the microsomal fraction of two different
cell types. Mouse macrophage cells (RAW) and mouse
fibroblast cells (NIH-3T3) were stimulated with either LPS
or IL-1β/TNFα to upregulate mPGES-1 protein levels. Protein
extracts were incubated with different concentrations of
quinazolinone series compounds or the mouse mPGES-1
RESULTS
■
Assembly of the Quinazolinone Compound Library
and Development of a Synthesis Route. On the basis of
the reference structure 7, several derivatives (7a−7n, 7r−7x)
were purchased from Asinex (www.asinex.com, Moscow,
Russia). Three novel derivatives (7o, 7p, 7q) were synthesized
(Scheme 1) to extend the chemical diversity and provide a
synthesis route for this scaffold class. All compounds are listed
in Chart 2.
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Chart 2. Twenty-Four Derivatives around the Lead Structure 7 Were Used for Structure−Activity Relationship Studies and
a
Pharmacological Profiling
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Chart 2. continued
a
Three additional compounds (7v−x) with slight variations were added.
inhibitor control substance 6 (YS121).21 Neither RAW- nor
NIH-derived mPGES-1 activity was impaired by quinazolinone
compounds by more than 20% (Table 1), while 6 reduced the
activity by more than 62%.
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Table 1. Measured Activity of the Quinazolinone Series on Isolated Human and Murine (from RAW and NIH Cells) Enzyme at
a
1 μM Final Concentration with Standard Error of the Mean (SEM)
human
murine
mPGES-1
inhibition @ 1
μM [%]
RAW mPGES-1
NIH mPGES-1
COX-1
COX-2
identifier
unit
SEM
[%]
IC50
[μM]
95% confidence inhibition @ 1 μM SEM inhibition @ 1 μM SEM inhibition @ 1 inhibition @ 1
interval [μM]
[%]
[%]
[%]
[%]
μM [%]
μM [%]
7
66.7
32.7
15.3
30.0
14.8
18.8
68.8
15.6
14.7
71.5
61.3
74.2
86.3
41.9
83.6
75.6
61.3
63.1
67.2
15.9
≤0
4.8
6.6
0.13
0.08−0.18
8.4
3.6
10.7
7.2
≤0
6.7
7a
7b
7c
7d
7e
7f
6.0
4.6
5.8
10.1
3.1
0.18
0.21
0.10−0.32
0.13−0.33
12.4
15.3
3.4
4.2
17.1
4.6
2.2
4.6
2.6
≤0
7g
7h
7i
10.7
2.6
3.6
20.1
2.5
7j
8.4
7k
7l
0.9
0.22
0.36
0.13−0.38
0.17−0.77
11.8
9.5
3.5
3.5
20.3
1.9
4.5
8.4
5.8
1.7
−0.8
≤0
≤0
7m
7n
7o
7p
7q
7r
0.8
0.8
0.18
0.37
0.11−0.29
0.26−0.53
15.2
7.5
1.3
6.6
6.0
5.0
16.1
≤0
≤0
1.3
−8.7
10.0
≤0
2.3
3.9
0.26
0.16−0.42
6.0
3.8
2.3
5.1
0.9
1.5
2.8
≤0
≤0
≤0
≤0
b
5.2
nd
17.8
7s
7.2
7t
16.1
13.2
10.7
22.8
7.0
7u
7v
7w
7x
YS121
14.1
37.1
≤0
≤0
66.2 (@ 5 μM)
2.8
62.9 (@ 5 μM)
2.2
a
The 50% inhibitory concentration (IC50) on human enzyme was calculated for each derivative showing larger inhibitory potency at 1 μM than the
lead structure 7. With 95% percent probability the IC50 is in the given range of the confidence interval. Cross-reactivity against COX-1/COX-2
b
enzymes with 1 μM inhibitor concentration was measured in vitro. Data are the mean of n ≥ 3. nd = not determined.
Figure 1. Cell viability assay with FR20 series compounds on stimulated human HeLa cells. Cell viability is given as percent of DMSO control with
SEM after 24 h of incubation (n ≥ 3).
COX-1 and COX-2 Cross-Reactivity. Cross-reactivity of
these compounds against both COX-isoforms was tested in
vitro. Neither COX-1 nor COX-2 was inhibited by any
compound by more than 20% at 1 μM inhibitor concentration
(Table 1).
Toxicity Assessment. To generate a toxicological profile
for compound 7 and equipotent derivatives 7f, 7i, 7k, 7l, 7n,
7o, 7q, and 7r, we performed a WST-1 cell assay with human
HeLa cells. Cell viability was determined after treatment of cells
with IL-1β and TNFα and simultaneously with increasing
concentrations of the test compounds for 24 h. Stimulation was
applied to modulate an inflammatory setting. All tested
derivatives showed no or only slightly decreased cell viability
at 1 and 10 μM inhibitor concentration (Figure 1). At 100 μM
concentration, the derivatives 7k, 7l, 7n, and 7q as well as the
control substance celecoxib decreased cell viability consid-
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Figure 2. PGE2 levels were measured with LC/MS-MS technique after incubation of stimulated HeLa cells with different concentrations of test
compounds or DMSO. Inhibition is calculated as percent of DMSO control corrected by the background value (unstimulated control) with SEM (n
≥ 3; 7r, n = 2).
erably. The COX-2 inhibitor celecoxib is already known for its
cytotoxic effects in vitro and in vivo and was used to compare
the quinazolinone inhibitors with a reference compound used
in clinical settings.25−27
stable metabolite of LTA4) also enhanced levels of various
hydroxyeicosatetraenoic acids (HETE) were detected after
stimulation.31 The 5-LO-inhibitor zileuton,32 which was used as
control compound, reduced LTB4 and 5(S)-HETE. Derivative
7f of the quinazolinone series enhanced both LTB4 and 5(S)-
HETE levels, while the residual production of 5(S)-HETE was
reduced by 7i. All other tested compounds had no influence on
LTB4 and 5(S)-HETE. 12(S)-HETE, 15(S)-HETE, and ( )11-
HETE levels were not affected at all at 10 μM final
concentration.
Cell-Based mPGES-1 Activity Assay. To determine the
PGE2 reducing effect of the compounds on intact HeLa cells,
the mPGES-1 inhibitors were simultaneously incubated with a
proinflammatory stimulus (IL-1β and TNFα) for 24 h. The
production of PGE2 was determined in the extracellular
supernatant and was reduced dose-dependently with all
inhibitors (Figure 2). We observed a shift in inhibitory potency
in comparison to isolated mPGES-1 enzyme activity of about
28% (7i) to 64% (7n) at 1 μM compound concentration.
Whole Blood Eicosanoid Profiling. To assemble a
preliminary pharmacological profile and to collect first
bioavailability data, we screened compound 7 and derivatives
7f, 7i, 7k, 7l, 7n, 7o, 7q, and 7r in human whole blood assays.
Three different assays were selected to check their influences
on (i) COX-1 mediated prostanoid production (mainly TXA2
and PGD2), (ii) COX-2 coupled prostanoid synthesis (mainly
PGE2 and PGF2α), and (iii) leukotriene/HETE pathways.
Table 2 summarizes the remaining levels of each eicosanoid as
compared to vehicle treated whole blood. COX-1 mediated
prostanoid production was stimulated by platelet clotting of
nonheparinized blood. Instead of the instable COX-1 related
product TXA2,28 the stable metabolite TXB2 was measured. In
this setting, PGD2 is also slightly up-regulated. No compound
had an inhibitory or stimulating effect on the TXB2 and PGD2-
level up to 10 μM final compound concentration (Table 2A).
The selective COX-1 inhibitor SC-560 reduced both TXB2 and
PGD2 as expected. COX-2 related prostaglandin production
was induced by treatment of heparinized whole blood with
LPS, which leads to an increase of mainly COX-2/mPGES-1
coupled PGE2 levels but also to enhanced PGF2α levels. To
exclude effects mediated by COX-1, we mixed all samples with
10 μg/mL acetylsalicylic acid (in this concentration a specific
COX-1 inhibitor29). Except for 7f, all compounds reduced
PGE2 at 100 μM concentration (Table 2B). Celecoxib also
reduced PGF2α levels, while 7f increased its concentration.
To assess the influence of the compounds on the leukotriene
and HETE pathways, we treated heparinized whole blood with
calcium-ionophore to activate the 5-lipoxygenase (5-LO)/5-
lipoxygenase activating protein (FLAP) complex, leading to an
increase of LTA4 levels30 (Table 2C−E). Beside LTB4 (the
DISCUSSION
■
The primary aim of this study was to generate a library of
quinazolinone compounds with different scaffold decorations,
to investigate their influence on the mPGES-1 and other
enzymes of the eicosanoid pathway, and to identify preliminary
structure−activity relationships.
We were able to establish a facile synthesis route for the
quinazolinone-based inhibitors of mPGES-1 (Scheme 1). The
presented synthesis route is timesaving, needs small amounts of
material, and enables chemical modifications of at least three
residues around the quinazolinone scaffold. Together with
several other compounds purchased from a commercial
supplier, we investigated the structure−activity relationship of
quinazolinone derivatives of the lead structure 7. The
compound set includes variations at three residue positions
with (halogenated) aromatic and heterocyclic groups (like
thiophene) as well as aliphatic side chains (Chart 2). It turned
out that variations at R1 in most cases considerably reduced the
activity as compared to 7. Only aromatic residues with an ortho-
chloro-substitution (7, 7f) were tolerated, which points to the
importance of a hydrophobic interaction with the ortho-
chlorine atom in contrast to unsubstituted phenyl moiety and
ortho-fluor (7b and 7c with 15% and 30% inhibition at 1 μM).
Additionally, para-chloro substituents led to decreased activity
(7h). Moreover, we observed reduced inhibition when R1 is an
aliphatic residue (7d, 7e); larger substituents like phenethyl
(7a) further impaired activity. Bioisosteric replacement of the
ortho-substituted phenyl-residue R1 by a thiophene (7g)
resulted in a less potent inhibitor than 7.
With 3-chlorbenzyl moiety in the R2 position, the
disubstitution at R1 was investigated; 7o (2-chloro-6-fluoro)
and 7p (2-chloro-6-chloro) exhibit similar inhibitory activity as
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a
Table 2. Human Whole Blood Was Incubated with Quinazolinone Series Compounds
a
Residual production is given in percent of control. Red color indicates a residual production ≤67% (= inhibition), yellow color between 67.1% and
132.9% (= no effect), green color ≥133% (= stimulation) (A) Blood clotting induced mainly COX-1 mediated TXB2 production, but PGD2 was also
detected. (B) Stimulation of COX-2 and mPGES-1 with LPS upregulated PGE2 and PGF2α-levels. (C−E) Calcium ionophore stimulated whole
blood induces LTB4 production via 5-LO/FLAP but also several HETEs.
7, implicating that a substitution at position 6 of the R1-phenyl
is tolerated. Apparently, the R2 residue may be exchanged for a
smaller substituent without losing potency. Simplification of the
[1,1′-biphenyl]-4-ylmethyl- (7) to a benzyl-group (7l), as well
as substitution at the [1,1′-biphenyl]-4-ylmethyl group (7i, 7j)
or at the benzyl-group at different positions (7l, 7n) is also
tolerated, with no detrimental effect on activity. Variations of
the R3 residue were only possible for some derivatives. A
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methyl (7m) and a benzyl residue (7r) yielded comparable
activities, while a replacement with cyclopropyl (7t) abolished
inhibition. We therefore conclude that residue R1 is critical for
the activity of this class of inhibitors, while R2 and R3 play a
minor role. These two residues may serve as a starting point for
further chemical variations.
CONCLUSIONS
■
In this study, we discovered that a ortho-chloro-substitued
phenyl residue at position R1 and a propyl or benzyl residue at
position R3 of the lead structure 7 promote mPGES-1
inhibition. Structural variations at position R2 seem to be less
influential on mPGES-1 inhibition. A detailed whole blood
eicosanoid screening revealed that pharmacological inhibition
of mPGES-1 is not accompanied by a redirection of COX
derived PGH2 to other prostanoids.
In the in vitro single concentration test on isolated human
mPGES-1 protein, 13 out of 25 compounds showed an
inhibition rate higher than 33%, eight of them with an
enhanced potency compared to the lead structure 7 (>66%).
These eight inhibitors were used in all further studies.
Interestingly, no inhibitor has a lower IC50 than 7 (Table 1).
To exclude COX-1/COX-2 related side effects, selectivity of
the compounds against these two enzymes was tested in a
direct inhibitor test. Neither COX-1 nor COX-2 were affected
at 1 μM concentration (inhibition ≪33%). Cellular toxicology
studies exhibit impaired cell viability at 100 μM inhibitor
concentration for four derivatives (Figure 1). All tested
compounds reduced the PGE2 level in a cell-based assay
(Figure 2) between 70% (7k) and 97% (7i) at 10 μM
compound concentration. Tests with a 10-fold higher
concentration (100 μM) did not enhance the inhibitory effect
that was also observed on isolated enzyme. We therefore
conclude that there is no major limitation in cell permeability.
Whole blood eicosanoid screening revealed a distinct profile for
each compound including many fatty acids of the three major
arachidonic acid subcascades: the prostanoids, leukotrienes, and
HETEs. It has been shown in mPGES-1-deficient mice that
PGE2 synthesis may be redirected to PGD2, PGF2α, and 6-keto-
PGF1α (stable metabolite of PGI2).33 This mechanism may
compensate for a loss of PGE2 synthesis in nociceptive
behavior. We therefore determined the level of other
prostanoids and arachidonic acid metabolites. Besides the
desired PGE2 inhibition, no effects on TXB2 and PGD2 levels
were observed. The role of PGD2 in pathological conditions
seems to be diverse: while exogenous PGD2 sustains the
pyrogenic effects of PGE234 in brain tissue, the metabolite 15d-
PGJ2 is involved in anti-inflammatory responses.35 Reduction of
PGD2 might therefore delay the resolution of inflammation.36
PGF2α, which is also not affected, mediates allodynia in the
spinal cord.37 In contrast, PGF2α contributes to resolution of
inflammation in the late phase of carrageenan-induced pleurisy
in rats.38 In addition, leukotrienes and HETEs play a role in
pathological conditions, but their roles are diverse and
compound-dependent, suggesting no common mode of action.
While LTB4 and 5(S)-HETE levels were only slightly
affected by the tested quinazolinone substances, 12(S)-HETE,
15(S)-HETE, and ( )11-HETE were not reduced by any
derivative. Compound 7f contrarily enhanced PGE2, PGF2α,
LTB4, and 5(S)-HETE levels, indicating a COX-2/5-LO
mediated proinflammatory effect. Nevertheless, whole blood
assays revealed that inhibition of mPGES-1 with our
compounds is not accompanied by a redirection of COX-
derived PGH2 to other prostanoids and therefore pharmaco-
logical inhibition of mPGES-1 seems to be a promising
approach for the treatment of pain and inflammation. An
approximately 10-fold shift of decreased PGE2 inhibition
potency between activity assays with isolated enzyme and in
whole blood was observed for all compounds. A reduced
unbound fraction of the compounds due to distribution into
cells, membranes, or protein binding may be responsible for
this shift but does not automatically limit in vivo efficacy.39
EXPERIMENTAL SECTION
■
Chemical Methods. Material and Reagents. Compound 7 and
respective derivatives 7a−n and 7r−x were purchased from Asinex
(Asinex, Moscow, Russia) and exhibit ≥95% purity determined by
manufacturer. Compounds 7o−q were synthesized in-house. The
structures of the synthesized compounds were confirmed by H , 13C
1
NMR and mass spectrometry (ESI). The purity of the synthesized
compounds was determined by combustion analysis and was found to
be >95%. Starting materials and solvents were purchased from Sigma-
Aldrich Chemie GmbH (Steinheim, Germany) or Alfa Aesar (Ward
Hill, USA) and were reagent grade and used without further
purification. For the microwave synthesis, a Biotage Initiator 2.0
(300 W) was used and the products were purified using a Varian 971-
1
FP flash purification system on silica gel with 50 μm particle size. H
and 13C NMR spectra were measured in DMSO-d6 or MeOD on a
Bruker AV 250 (250 MHz for H NMR and 63 MHz for the 13C
1
NMR). Chemical shifts are reported in parts per million (ppm) using
tetramethylsilane (TMS) as internal standard. Mass spectra were
obtained on an Electrospray-Ionization Fisons (VG Platform II)
spectrometer measuring in the positive- or negative-ion mode (ESI-
MS system). Combustion analysis was performed by the micro-
analytical laboratory of the Institute of Organic Chemistry and
Chemical Biology, Goethe University Frankfurt, on an Elementar
Vario Micro Tube CHNO rapid elemental analyzer.
Synthesis. The compounds were synthesized in three steps
(Scheme 1) involving the condensation of 5-iodoanthranilic acid
with butyric acid followed by dehydration to form the intermediate
benzoxazinone. Successive addition of 3-chlorbenzylamine provides
2,3-disubstituted-6-iodo-3H-quinazolin-4-one; these reactions were
carried out under microwave conditions.40 The third step comprises
a related Goldberg reaction,41 a copper-catalyzed N-arylation of
amides with a heteroaryl iodide. The reaction is mediated by copper
iodide, N,N′-dimethylethylenediamine, and K3PO4.
Synthesis of 3-(3-Chlorobenzyl)-6-iodo-2-propylquinazolin-
4(3H)-one. To a conical-bottomed microwave vial, a solution of 5-
iodoanthranilic acid (0.500 g, 1.901 mmol, 1 equiv), butyric acid
(0.167 g, 1.901 mmol, 1 equiv), and triphenyl phosphite (0.639 g,
2.091 mmol, 1.1 equiv) in 2 mL of anhydrous pyridine was added. The
mixture was irradiated in a sealed microwave vial for 10 min at 150 °C.
After cooling, 3-chlorbenzylamine (0.269 g, 1.901 mmol, 1 equiv) was
added and irradiated for 5 min at 230 °C. The solvent was removed,
and the residue was resolved in ethyl acetate, washed with saturated
NaHCO3 (2 × 10 mL), 10% HCl (2 × 10 mL), and dried over
MgSO4. The solvent was removed under reduced pressure, and the
crude product was purified by column chromatography on silica gel
with hexane−ethyl acetate (50:50, v/v), obtaining 100 mg (12% yield)
of an off-white solid.
1H NMR (MeOD): δ 8.60 (br, 1H), 8.19 (dd, J = 3.4, 8.6 Hz, 1H),
7.48 (d, J = 8.6 Hz, 1H), 7.33 (m, 3H), 5.46 (s, 2H), 2.87 (t, J = 7.8
Hz, 2H), 1.73 (q, J = 7.5 Hz, 2H), 1.00 (t, J = 7.3 Hz, 3H).
MS (ESI, 70 eV) m/z (%): 439.2 (100%).
Synthesis of 2-Chloro-N-(3-(3-chlorobenzyl)-4-oxo-2-propyl-3,4-
dihydroquinazolin-6-yl)-6-fluorobenzamide (7o). A round-bottomed
flask was charged with CuI (1 mg, 0.005 mmol, 0.01 equiv), 2-chloro-
6-fluorobenzamide (0.114 g, 0.657 mmol, 1.2 equiv), and K3PO4
(0.232 g, 1.094 mmol, 2 equiv), evacuated, and backfilled with argon.
N,N′-Dimethylethylenediamine (4 mg, 0.055 mmol, 0.1 equiv), 3-(3-
chlorobenzyl)-6-iodo-2-propylquinazolin-4(3H)-one (0.240 g, 0.547
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mmol, 1 equiv), and tetrahydrofuran (10 mL) were added under
argon. The reaction mixture was stirred at 110 °C for 23 h. The
resulting pale-brown suspension was cooled down and filtered through
a 1 cm pad of silica gel eluting with 20 mL of ethyl acetate. The filtrate
was concentrated, and the residue was purified by column
chromatography on silica gel with hexane−ethyl acetate (50:50, v/v)
provided 189 mg (71% yield) as an off-white solid.
Collection (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 glucose, and
pyruvate and RAW in RPMI medium 1640, containing high glucose,
GlutaMAX. All media contain 10% fetal calf serum (FCS), 100 units/
mL penicillin G, and 100 μg/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 β
(IL-1β) and recombinant human tumor necrosis factor α (TNFα)
were purchased from PeproTech (London, UK).
1H NMR (DMSO-d6): δ 11.19 (br, 1H), 8.68 (d, J = 2.4, 1H), 8.05
(dd, J = 2.4, 8.8 Hz, 1H), 7.73 (d, J = 8.7 Hz, 1H), 7.79−7.43 (m, 6H),
7.17 (d, J = 5.4 Hz, 1H), 5.43 (s, 2H), 2.76 (t, J = 14.8 Hz, 2H), 1.77
(q, J = 7.3 Hz, 2H), 0.96 (t, J = 7.3 Hz, 3H).
mPGES-1 Activity Assay. To investigate the inhibitory activity of
the quinazolinone 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
incubated for 24 h at 37 °C in medium containing 10% FCS. The
medium was removed, and HeLa/NIH cells were stimulated with IL-
1β (1 ng/mL) + TNFα (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 phosphate buffered saline (PBS) and centrifuged at 5000 rpm for 2
min at 4 °C. Cell pellets were frozen in liquid nitrogen. After thawing
the cells, they were resuspended in 800 μL of potassium phosphate
buffer (Kpi buffer, 0.1 M, pH 7.4), containing 1× complete protease
inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), sucrose
(0.25 M), and reduced glutathione (GSH, 1 mM). After sonification
and centrifugation at 45000 rpm for 2 h or 53000 rpm for 1 h at 4 °C,
the microsomal fraction (pellet) was stored at −80 °C. The pellet was
resuspended in 100 μL Kpi buffer (0.1 M, pH 7.4) containing 1×
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.42 Activity of all quinazolinone
derivates was measured at 1 μM final concentration and compared to
the lead compound. The mPGES-1 activity assay was performed on
the basis of Thoren et al.43 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.
13C NMR (DMSO-d6): δ 163.5, 161.7, 160.5, 156.8, 156.1, 143.9,
139.7, 136.6, 133.7, 132.0, 130.9, 127.9, 126.6, 125.8, 124.9, 120.3,
115.7, 114.8, 45.2, 35.8, 19.8, 13.6.
MS (ESI, 70 eV) m/z (%): 484.5 (100%).
Anal. Calcd for C25H20Cl2FN3O2: C, 61.99, H, 4.16, N, 8.68. Found:
C, 61.95, H, 4.12, N, 8.64.
Synthesis of 2,6-Dichloro-N-(3-(3-chlorobenzyl)-4-oxo-2-propyl-
3,4-dihydroquinazolin-6-yl)benzamide (7p). A round-bottomed flask
was charged with CuI (3 mg, 0.015 mmol, 0.05 equiv), 2,6-
dichlorobenzamide (0.09 g, 0.478 mmol, 1.5 equiv), and K3PO4
(0.132 g, 0.638 mmol, 2 equiv), evacuated, and backfilled with
argon. N,N′-Dimethylethylenediamine (3 mg, 0.031 mmol, 0.1 equiv),
3-(3-chlorobenzyl)-6-iodo-2-propylquinazolin-4(3H)-one (0.140 g,
0.319 mmol, 1 equiv), and THF (10 mL) were added under argon.
The reaction mixture was stirred at 110 °C for 23 h. The resulting
pale-brown suspension was cooled down and filtered through a 1 cm
pad of silica gel eluting with 20 mL of ethyl acetate. The filtrate was
concentrated, and the residue was recrystallized from EtOH/water,
obtaining 80 mg (50% yield) as a white solid.
1H NMR (DMSO-d6): δ 11.18 (br, 1H), 8.70 (d, J = 3.3 Hz, 1H),
8.05 (dd, J = 3.3, 9.2 Hz, 1H), 7.76−7.21 (m, 7H), 7.15 (m, 1H), 5.48
(s, 2H), 2.77 (t, J = 8.3 Hz, 2H), 1.78 (q, J = 9.6 Hz, 2H), 0.95 (t, J =
8.2 Hz, 3H).
13C NMR (DMSO-d6): 162.3, 161.3, 155.9, 143.5, 142.6, 139.4,
136.9, 136.0, 133.6, 131.9, 131.1, 128.2, 126.4, 125.3, 119.9, 115.4,
45.4, 35.7, 19.6, 13.3.
The reaction was initiated with 20 μM PGH2 (Larodan, Malmo,
̈
Sweden) and terminated after 1 min by adding a stop solution
containing 40 mM iron chloride (FeCl2) and 80 mM citric acid. For
the solid phase extraction procedure, 100 μL of reaction solution was
mixed for 3 min with 700 μL of ultrapure water, 100 μL of 0.15 M
EDTA, 20 μL of MeOH, and 20 μL of internal standard (25 ng/mL
PGE2-d4, 50 ng/mL PGD2-d4), all from Cayman Chemical Company
(Ann Arbor, USA), centrifuged at 1200 rpm for 3 min, and passed
through a 30 mg of Bond Elut NEXUS 96 round-well plate (Agilent
MS (ESI, 70 eV) m/z (%): 500.3 (100%).
Anal. Calcd for C25H20Cl3N3O2: C, 59.96, H, 4.03, N, 8.39. Found:
C, 60.19, H, 4.21, N, 8.36.
Synthesis of N-(3-(3-Chlorobenzyl)-4-oxo-2-propyl-3,4-dihydro-
quinazolin-6-yl)-2-methylbenzamide (7q). A round-bottomed flask
was charged with CuI (43 mg, 0.228 mmol, 0.2 equiv), 2-
methylbenzamide (0.185 g, 1.368 mmol, 1.2 equiv), and K3PO4
(0.484 g, 2.280 mmol, 2 equiv), evacuated, and backfilled with
argon. N,N′-Dimethylethylenediamine (40 mg, 0.456 mmol, 0.4
equiv), 3-(3-chlorobenzyl)-6-iodo-2-propylquinazolin-4(3H)-one (0.5
g, 1.140 mmol, 1 equiv), and THF (10 mL) were added under argon.
The reaction mixture was stirred at 110 °C for 23 h. The resulting
pale-brown suspension was cooled down and filtered through a 1 cm
pad of silica gel eluting with 20 mL of ethyl acetate. The filtrate was
concentrated, and the residue was purified by flash chromatography on
silica gel (hexane−ethyl acetate, 0→ 80% ethyl acetate, 4%/min)
provided 179 mg (35% yield) as an off-white solid.
Technologies GmbH, Boblingen, Germany) preconditioned with 1
̈
mL of MeOH, followed by 1 mL of ultrapure water. The cartridge was
washed with 1 mL of 30% MeOH. Prostaglandins were eluted with 1
mL of hexane−ethylacetate−isopropranol (30:65:5, v/v/v). After
evaporating the solvent under nitrogen atmosphere, the residue was
reconstituted in 100 μL of acetonitrile−H2O−formic acid
(20:80:0.0025 v/v/v). Samples were measured by LC-MS/MS
technique (LC unit: Agilent 1200 series, Agilent Technologies
GmbH, Boblingen, Germany. MS/MS unit: AB SCIEX QTRAP
̈
5500, Applied Biosystems, Foster City, USA) as described
previously.44 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.
1H NMR (DMSO-d6): δ 10.69 (br, 1H), 8.74 (d, J = 2.1 Hz, 1H),
8.13 (dd, J = 2.3, 8.8 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 7.0
Hz, 1H), 7.49−7.33 (m, 6H), 7.07 (m, 1H), 5.45 (s, 2H), 2.76 (t, J =
7.4 Hz, 2H), 2.48 (s, 3H), 1.76 (q, J = 7.3 Hz, 2H), 0.96 (t, J = 7.3 Hz,
3H).
13C NMR (DMSO-d6): δ 168.2, 161.4, 155.8, 143.0, 139.4, 136.9,
135.6, 133.4, 130.7, 129.9, 129.1, 126.9, 125.8, 124.7, 120.3, 115.6,
45.3, 36.0, 19.4, 19.1, 13.6.
MS (ESI, 70 eV) m/z (%): 446.5 (100%).
Anal. Calcd for C26H24ClN3O2: C, 70.03, H, 5.42, N, 9.42. Found:
C, 69.94, H, 5.33, N, 9.21.
Cell Biological Methods. Cells and Reagents. HeLa (human cervix
carcinoma) and NIH-3T3 (Swiss mouse fibroblast) cells were
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, MI, USA), according to the
manufacturer’s protocol. SC-560, a selective COX-1 inhibitor, and
celecoxib, a selective COX-2 inhibitor, were used as positive controls.
The COX assay is based on the determination of PGE2, PGD2, and
purchased from Deutsche Sammlung fur Mikroorganismen and
̈
Zellkulturen (DSMZ, Braunschweig, Germany). RAW 264.7 (mouse
macrophage) cells were purchased from American Type Culture
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PGF2α amounts produced by SnCl2 reduction of COX-derived PGH2.
Then 100 μL of reaction solution was diluted with 200 μL of MeOH.
Finally, 70 μL of diluted reaction solution was mixed for 3 min with
700 μL of ultrapure water, 100 μL of 0.15 M EDTA, 20 μL of MeOH,
and 20 μL of internal standard (25 ng/mL PGE2-d4, 25 ng/mL PGD2-
d4, 25 ng/mL TXB2-d4, 50 ng/mL PGF2α-d4, 37.5 ng/mL 6-keto-
PGFlα-d4). The amount of prostaglandins was quantified by LC-MS/
MS analysis after solid phase extraction as described above (see
mPGES-1 activity assay).
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 density of 3 × 103 cells in 100 μL of culture medium
containing 10% FCS into 96-well microplates and incubated for 24 h
at 37 °C. Medium was removed, and HeLa cells were stimulated with
IL-1β (1 ng/mL) + TNFα (5 ng/mL) and simultaneously treated with
increasing concentrations of the compounds (1, 10, and 100 μM) or
DMSO. After 24 h, 10 μL of WST-1 reagent was added to each well
and the cells were incubated for further 90−150 min. The formation 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).
with test compounds in DMSO. Finally, 10 μg/mL acetylsalicylic acid
was added to inhibit COX-1. After 15 min incubation at 37 °C, 500
μg/mL (10 μg/mL final concentration) lipopolysaccharide (LPS) in
10 μL autologous plasma was added to stimulate COX-2/mPGES-1.
After 24 h incubation time at 37 °C, samples were chilled on ice for 5
min and plasma was collected and prostaglandins extracted and
measured by LC-MS/MS (see Whole Blood COX-1 Assay).
Whole Blood Leukotriene/HETE Assay. To assess the influence of
quinazolinone derivatives on different enzymes in the leukotriene and
HETE pathway, 500 μL of human heparinized whole blood was mixed
with test compounds and incubated for 15 min at 37 °C. Afterward,
the whole blood was stimulated with 20 μM calcium ionophore
(A23187) in autologous plasma for a 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 20 μL of EtOH and 20 μL of internal standard containing 25 ng/
mL 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. Then 600 μL of
ethyl acetate was added and samples were mixed and centrifuged at
13000 rpm for 3 min. The organic upper phase was extracted.
Extraction of leukotrienes and HETEs was repeated once. Solvent was
evaporated under nitrogen atmosphere, and the residue was
reconstituted in 50 μL of 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 AB SCIEX QTRAP 5500 (Applied
Biosystems, Foster City, CA, USA) operating in multiple reaction
monitoring (MRM).45 Chromatographic separation was performed on
a Gemini-NX 5u C18 110A column (150 mm × 2 mm inner diameter,
5 μm particle size, Phenomenex, Aschaffenburg, Germany).
Cell-Based mPGES-1 Activity Assay. first, 2 × 104 HeLa cells were
incubated in 24-well microplates for 24 h at 37 °C. Then the medium
was replaced with fresh medium containing 1 ng/mL IL-1β + 5 ng/mL
TNFα (stimulated control) or an equal amount of PBS (unstimulated
control) and test compound (1, 10, or 100 μM) or DMSO. After
incubation for 24 h at 37 °C, cell supernatant was collected. Then
400−500 μL of supernatant was mixed with 400 μL of 45 mM H3PO4,
100 μL of 0.15 M EDTA, 20 μL of MeOH, and 20 μL of internal
standard (25 ng/mL PGE2-d4, 25 ng/mL PGD2-d4, 25 ng/mL TXB2-
d4, 50 ng/mL PGF2α-d4, 37.5 ng/mL 6-keto-PGFlα-d4). The amount of
prostaglandins was quantified by LC-MS/MS analysis after solid phase
extraction as described above (see mPGES-1 activity assay).
Whole Blood Eicosanoid Screening. To determine possible effects
of quinazolinone derivatives on different eicosanoids levels in human
whole blood, a eicosanoid profile was created for each compound.
Therefore, three different experimental settings were used to stimulate
the production of eicosanoid synthesis.
A stimulating effect of a compound was assumed when the relative
level of an eicosanoid exceeded +33% compared to control, an
inhibitory effect when levels were reduced by −33% or more, as
compared to control.
Whole Blood COX-1 Assay. Because 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
ASSOCIATED CONTENT
■
S
* Supporting Information
IC50 dose−response curve of compound 7. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49 69 6301 6086. Fax: +49 69 6301 7636. E-mail:
Roersch@em.uni-frankfurt.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by Merz GmbH & Co. KGaA, the
LOEWE Lipid Signaling Forschungszentrum Frankfurt (LiFF),
the Oncogenic Signaling Frankurt (OSF), the European
Graduate School “Roles of Eicosanoids in Biology and
(SARSTEDT AG & Co, Numbrecht, Germany) from healthy donors
̈
who had not taken any NSAIDs for at least one week. Then 500 μL of
blood per sample was directly mixed with rising concentrations of test
compounds. Negative controls were immediately chilled on ice to
minimize thrombocyte aggregation. All other samples were incubated
at 37 °C for 1 h, and blood was allowed to clot.
́
Medicine” (DFG GRK 757/1), the DAAD/Fundacion Obra
Social “la Caixa”, and the Fonds der Chemischen Industrie.
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 the extraction procedure, plasma was mixed for
3 min with 600 μL of 45 mM H3PO4, 100 μL of 0.15 M EDTA, 10 μL
of 2 mg/mL butylated hydroxytoluene, 20 μL of MeOH, and 20 μL of
internal standard (25 ng/mL PGE2-d4, 25 ng/mL PGD2-d4, 25 ng/mL
TXB2-d4, 50 ng/mL PGF2α-d4, 37.5 ng/mL 6-keto-PGF1α-d4).
TXB2 (instead of the instable TXA2) was measured together with
PGE2, PGD2, 6-keto-PGF1α, and PGF2a after solid phase extraction as
described above (see mPGES-1 activity assay).
ABBREVIATIONS USED
■
5-LO, 5-lipoxygenase; COX, cyclooxygenase; HETE, hydrox-
yeicosatetranoid acid; IC50, half-maximal inhibitory concen-
tration; LC-MS/MS, liquid chromatography with combined
tandem mass spectrometry; LPS, lipopolysaccharide; LTB4,
leukotrien B4; mPGES-1, microsomal prostaglandin E synthase
1; NSAIDs, nonsteroidal anti-inflammatory drugs; PGE2,
prostaglandin E2; SAR, structure−activity relationship
Whole Blood COX-2 Assay. The mPGES-1 expression is mainly
coupled to COX-2 and therefore 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
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̈
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