Exploration of the dihydropyrimidine scaffold for the development of new potential anti-inflammatory agents blocking prostaglandin E2 synthase-1 enzyme (mPGES-1)

Article abstract of DOI:10.1016/j.ejmech.2014.04.061

Agents targeting microsomal prostaglandin E₂ synthase-1 (mPGES-1) would inhibit only PGE₂ production induced by inflammatory stimuli and thus could represent a valuable alternative to non-steroidal anti-inflammatory drugs (NSAIDs) as they should be free from the severe side effects of the classic anti-inflammatory drugs. Although several mPGES-1 inhibitors have been so far identified, none of them is currently in clinical trials, therefore the discovery of new molecular platforms, able to interfere with this interesting target, is urgently required. Here, we report the results of a focused collection of dyhidropyrimidin-2(1H)-one based molecules projected by Virtual Screening computational techniques. The key interactions with the receptor counterpart were introduced as a qualitative filter for the selection of the most promising compounds. The biological data obtained are consistent with the computer-aided suggestions and disclosed two interesting molecules showing in vitro mPGES-1 inhibitory activity in the low μM range.

Full text of DOI:10.1016/j.ejmech.2014.04.061

European Journal of Medicinal Chemistry 80 (2014) 407e415
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Exploration of the dihydropyrimidine scaffold for the development of
new potential anti-inflammatory agents blocking prostaglandin E2
synthase-1 enzyme (mPGES-1)
,
b
Gianluigi Lauro a, Maria Strocchia a, Stefania Terracciano a, Ines Bruno a , Katrin Fischer ,
*
,
Carlo Pergola b, Oliver Werz b, Raffaele Riccio a, Giuseppe Bifulco a
*
a Department of Pharmacy, University of Salerno, via Giovanni Paolo II, 132, 84084 Fisciano, Italy
b Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller University, Jena, Philosophenweg 14, D-07743 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Agents targeting microsomal prostaglandin E2 synthase-1 (mPGES-1) would inhibit only PGE2 produc-
tion induced by inflammatory stimuli and thus could represent a valuable alternative to non-steroidal
anti-inflammatory drugs (NSAIDs) as they should be free from the severe side effects of the classic
anti-inflammatory drugs. Although several mPGES-1 inhibitors have been so far identified, none of them
is currently in clinical trials, therefore the discovery of new molecular platforms, able to interfere with
this interesting target, is urgently required. Here, we report the results of a focused collection of
dyhidropyrimidin-2(1H)-one based molecules projected by Virtual Screening computational techniques.
The key interactions with the receptor counterpart were introduced as a qualitative filter for the se-
lection of the most promising compounds. The biological data obtained are consistent with the
computer-aided suggestions and disclosed two interesting molecules showing in vitro mPGES-1 inhibi-
Received 10 February 2014
Received in revised form
18 April 2014
Accepted 22 April 2014
Available online 24 April 2014
Keywords:
Molecular docking
Inflammation
mPGES-1 inhibitors
Biginelli reaction
tory activity in the low mM range.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
including breast, cervical, colorectal, oral, prostate cancers, and
multiple myeloma [3].
Arachidonic acid (AA) is the substrate of several enzymes
involved in inflammation events. A first processing of AA by both
the constitutively expressed cyclooxygenase (COX)-1 and the
inducible COX-2 leads to the production of prostaglandin PGH2 [1].
PGH2 is a key intermediate that is downstream transformed into a
wide range of inflammatory mediators, including PGE2, which
possess powerful pleiotropic functions and are mediators of several
pathological processes, including chronic inflammation and cancer.
In particular, the correlation between increased levels of PGE2 and
the outbreak of arthritis was highlighted in the past few years, and
the inhibition of PGE2 production and signaling is associated with
reduction of pain and inflammation [2]. Moreover, an over-
production of PGE2 is also related to different types of cancer,
There are three terminal synthases responsible for PGE2
biosynthesis: one cytosolic isoform cPGES, and two membrane
associated enzymes, mPGES-1 [4] and mPGES-2. cPGES and
mPGES-2 are constitutively expressed, while mPGES-1 is an
inducible isoform, and in fact its expression is dramatically
increased in response of a pro-inflammatory stimulus.
mPGES-1 is a member of Membrane-Associated Proteins in
Eicosanoid and Glutathione metabolism (MAPEG) superfamily and
is functionally coupled with COX-2. The therapeutic interventions
in inflammation so far developed include either the inhibition of
COX-1 and COX-2 enzymes by non-steroidal anti-inflammatory
drugs (NSAIDs), or selective COX-2 inhibition by coxibs. Neverthe-
less, they both possess severe gastrointestinal and cardiovascular
side-effects that limit their use in long-term treatments as required
in chronic inflammation processes [5], prompting to explore new
enzymatic targets in this pathway. In this perspective, selective
inhibition of mPGES-1 activity has emerged as an alternative
promising strategy to develop effective and safer agents helpful in
inflammation and in cancer chemoprevention [6]. From a structural
point of view, this enzyme shares 39% sequence identity with
Abbreviations: GSH, glutathione; MAPEG, membrane-associated proteins in
eicosanoid and glutathione metabolism; MGST-1, microsomal glutathione trans-
ferase 1; mPGES-1, microsomal prostaglandin E2 synthase-1; COX, cyclooxygenase;
PG, prostaglandin; ADME, absorption, distribution, metabolism, and excretion.
* Corresponding authors.
E-mail addresses: brunoin@unisa.it (I. Bruno), bifulco@unisa.it (G. Bifulco).
http://dx.doi.org/10.1016/j.ejmech.2014.04.061
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

408
G. Lauro et al. / European Journal of Medicinal Chemistry 80 (2014) 407e415
another MAPEG protein, namely microsomal glutathione
transferase-1 (MGST-1). Other related proteins with a less sequence
identity are MGST-2, MGST-3, leukotriene C4 (LTC4) synthase, and
5-lipoxygenase-activating protein (FLAP) [7]. In 2008, Jegerschöld
et al. presented a 3D-electron crystallography structure of mPGES-
1, but the detailed analysis of the protein organization suggested a
closed non-active form [8]. In 2013, Sjögren et al. introduced the
first high resolution X-ray structure of mPGES-1 in the active form
[9], prompting new insights for the structure-based design of new
potent inhibitors. The structural details revealed that the protein is
a membrane homotrimer with three active sites partially occupied
by the cofactor (glutathione, GSH), and recent experimental evi-
dences suggested a 1:3 site reactivity [10]. The asymmetric
monomer is characterized by four-helix, and each active site is
oriented toward the cytoplasmic part of the protein, in particular
between N-terminal parts of helix II and IV of a monomer and the
C-terminal part of helix I and the cytoplasmic domain of the
adjacent monomer (Fig. 1). This protein folding generates a pro-
nounced deep active site occupied by GSH, and in the outer part, an
extended groove between helix I of a monomer and helix IV of the
adjacent monomer is observable. mPGES-1 was also co-crystallized
with a bis-phenyl GSH-derivative (1-(4-phenylphenyl)-2-(S-gluta-
thionyl)-ethanone). Crystallographic data clearly highlighted the
partial replacement of GSH by its derivative; this last showed to
keep the same orientation of GSH in the deep cavity of the active
site, and a perfect accommodation of the bis-phenyl additional
group in the external groove is observed [9].
In recent years, our research group has been deeply involved in
the discovery of potential mPGES-1 inhibitors, as promising anti-
inflammatory and/or anticancer drugs [11e17]. Based on the addi-
tional structural information, we decided to undertake a structure-
based drug design with the aim of identifying new molecular
platforms able to interfere with the target in object. For our pur-
pose, we decided to rely on dihydropyrimidine nucleus that rep-
resents a sort of “privileged structure”, being endowed with several
relevant pharmacological effects, including calcium channel mod-
ulation for the treatment of cardiovascular diseases, a1a-adrenergic
receptor antagonism, useful for benign prostatic hyperplasia and
mitotic kinesin inhibition with anticancer application [18]. In
addition to its interesting biological profile, the dihydropyrimidine
core has received a considerable attention in drug-discovery pro-
cess due to its synthetical accessibility through the easy, cheap and
rapid Biginelli one-pot multicomponent reaction [18].
Following this synthetic strategy, a wide number of differently
decorated dihydropyrimidin-2(1)H-one-based compounds can be
rapidly synthesized combining three synthons: an aldehyde, a 1,3-
dicarbonyl compound and an urea. As first step, we performed a
focused in silico virtual screening on a small set of compounds
synthetically accessible by means of molecular docking (1e36,
Table 1). A qualitative computational filter, based on the respect of
some key interactions with the receptor counterpart, has been
introduced, leading to the identification of a focused set of com-
pounds for the subsequent step of chemical synthesis and biolog-
ical evaluation.
2. Results and discussion
2.1. Docking studies
Molecular docking calculations were performed using the
recently crystallized structure of mPGES-1 (PDB code: 4AL0) [9].
Sjögren et al. have proposed a catalytic process in which GSH
covalently binds PGH2 that subsequently isomerizes to PGE2 thanks
to several amino acid residues close to GSH [9]. A dynamic process
in which PGH2 reaches the binding site and interacts with GSH and
the other residues in the active site can be assumed, and then a
hypothetical mPGES-1 inhibitor can act either as a false substrate
(PGH2) or as a cofactor analog (GSH), or at least it can behave in
both ways [19]. In the latter case, it can be imagined a scenario in
which the inhibitor, in addition to displacing the substrate from its
active site, could also influence the binding mode of GSH inside the
enzyme pocket. In order to simulate the partial or total displace-
ment of GSH, molecular docking calculations were then performed
removing the cofactor from the active site.
We designed new putative dihydropirymidin-2(1H)-one-
featured inhibitors taking into account the synthetic accessibility
of the decorated scaffold. Hence, a first computational study was
focused on a small set of compounds derived from the combination
of the following chemical synthons:
1.Urea, thiourea, or N-methylurea;
2.Ethyl 3-oxobutanoate or ethyl 4-(4-methoxyphenyl)-2,4-
dioxobutanoate as 1,3-dicarbonyl compound;
3.Benzaldehyde.
In this way, we obtained a first generation of structurally diverse
molecules (1e6) submitted to docking calculations in order to
verify the presence of some key interactions with the receptor
counterpart:
ꢀ
p
e
p
with Tyr130(A), indicative of a good accommodation
within the GSH binding site;
ꢀ a polar interaction with Ser127(A), a key residue involved in
PGH2 recognition;
ꢀ polar interactions with Thr131(A), Gln134(A), and van der Waals
interactions with Tyr28(B) and Ile32(B), belonging to the
external binding groove.
We noticed that none of these six compounds was able to
occupy the external groove, establishing at the same time the pep
Fig. 1. mPGES-1 structure (secondary structure: chain A blue, chain B red, chain C
orange); dummy atoms define extracellular (above red atoms) and cytoplasmic (below
blue atoms) sides. Glutathione cofactor is depicted in licorice mode (C: green, O red, N,
blue, H light gray), molecular surface focused to the GSH binding site and the external
binding groove is colored in dark gray. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
with Tyr130(A). In particular, compounds 1e3, featuring aliphatic
substituents in 5 and 6 positions, were found not able to interact
with both the sites, showing only a partial placement in the GSH
binding site. However, we found a pose of 4 in which the 4-phenyl
group is partially accommodated in the external groove, while the

G. Lauro et al. / European Journal of Medicinal Chemistry 80 (2014) 407e415
409
Table 1
3,4-dihydropyrimidin-2-one derivatives 1e36.
Entry
R1
R2
R3
R4
X
1
2
3
H
CH3
H
Phenyl
Phenyl
Phenyl
CO2C2H5
CO2C2H5
CO2C2H5
CH3
CH3
CH3
O
O
S
4
5
6
7
8
9
H
CH3
H
H
CH3
H
Phenyl
Phenyl
Phenyl
3-Formylphenyl
3-Formylphenyl
3-Formylphenyl
4-(methoxybenzoyl)
4-(methoxybenzoyl)
4-(methoxybenzoyl)
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CH3
CH3
CH3
O
O
S
O
O
S
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
H
CH3
H
H
CH3
H
H
CH3
H
H
CH3
H
H
CH3
H
H
CH3
H
H
CH3
H
H
CH3
H
3-Formylphenyl
3-Formylphenyl
3-Formylphenyl
3-Ethoxybenzoyl
3-Ethoxybenzoyl
3-Ethoxybenzoyl
3-Ethoxybenzoyl
3-Ethoxybenzoyl
4-(methoxybenzoyl)
4-(methoxybenzoyl)
4-(methoxybenzoyl)
CO2C2H5
CO2C2H5
CO2C2H5
4-(methoxybenzoyl)
4-(methoxybenzoyl)
4-(methoxybenzoyl)
CO2C2H5
CO2C2H5
CO2C2H5
4-(methoxybenzoyl)
4-(methoxybenzoyl)
4-(methoxybenzoyl)
CO2C2H5
CO2C2H5
CO2C2H5
4-(methoxybenzoyl)
4-(methoxybenzoyl)
4-(methoxybenzoyl)
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CO2C2H5
CH3
CH3
CH3
CO2C2H5
CO2C2H5
CO2C2H5
CH3
CH3
CH3
CO2C2H5
CO2C2H5
CO2C2H5
CH3
CH3
CH3
CO2C2H5
CO2C2H5
CO2C2H5
CH3
CH3
CH3
O
O
S
O
O
S
O
O
S
O
O
S
O
O
S
O
O
S
O
O
S
O
O
S
3-Ethoxybenzoyl
(4-cyanophenyl)pyridin-2-yl
(4-cyanophenyl)pyridin-2-yl
(4-cyanophenyl)pyridin-2-yl
(4-cyanophenyl)pyridin-2-yl
(4-cyanophenyl)pyridin-2-yl
(4-cyanophenyl)pyridin-2-yl
6,8-Dibromo-4-oxo-4H-chromen-3-yl
6,8-Dibromo-4-oxo-4H-chromen-3-yl
6,8-Dibromo-4-oxo-4H-chromen-3-yl
6,8-Dibromo-4-oxo-4H-chromen-3-yl
6,8-Dibromo-4-oxo-4H-chromen-3-yl
6,8-Dibromo-4-oxo-4H-chromen-3-yl
5-(3-(trifluoromethyl)phenyl)furan-2-yl
5-(3-(trifluoromethyl)phenyl)furan-2-yl
5-(3-(trifluoromethyl)phenyl)furan-2-yl
5-(3-(trifluoromethyl)phenyl)furan-2-yl
5-(3-(trifluoromethyl)phenyl)furan-2-yl
5-(3-(trifluoromethyl)phenyl)furan-2-yl
H
CH3
H
4(methoxybenzoyl)
4(methoxybenzoyl)
4(methoxybenzoyl)
CO2C2H5
CO2C2H5
CO2C2H5
O
O
S
5-(4-methoxybenzoyl) moiety is properly oriented to establish a
with Tyr130(A) (Fig. 2). A similar binding mode was found for
1-(N-methyl) derivative 5 and for the thio-analog 6.
featuring 5-(3-methoxybenzoyl) and 6-ethylcarboxylate groups.
Docking calculations showed for 4-(3-ethoxybenzyl) derivatives a
binding mode comparable to that found for the previously
considered 4-(3-formylphenyl) derivatives (pose related to 16,
Fig. S2, Supporting Information).
pep
Since the remaining part of the external groove was not occu-
pied on the meta-position of the 4-phenyl group, we decided to
gradually expand the substitutions in this direction. Preserving all
the other substituents, a new set of compounds featuring a new 4-
(3-formyl-phenyl) moiety was evaluated (7e12). In addition, while
a better accommodation of 5-(4-methoxybenzoyl) group was
found, the external groove was still not fully occupied (pose related
to 10 in Fig. S1, Supporting Information).
Also in this case, compounds 7e9, featuring aliphatic sub-
stituents in C5 and C6, were not found compatible with this binding
mode. For these reasons, we decided to progressively increase the
steric hindrance at the C4 position of the dihydropyrimidine scaf-
fold, with the introduction of 3-ethoxybenzyl (13e18), (4-
cyanophenyl)pyridine-2yl (19e24), (6,8 dibromo-4-oxo-4H-chro-
men-3-yl) (25e30), and 5-(3-(trifluoromethyl)phenyl)furan-2-
yl (31e36) substituents, respectively. The selection of hetero-
aromatic rings derived from the aim of increasing the possible polar
interaction with the hydrophilic residues in the external groove.
Furthermore, together with the pep interaction between the 4-
methoxybenzoyl in C5 and Tyr130 (A), only a partial accommoda-
tion of the larger 4-cyanophenyl (pyridine-2yl) and 6,8 dibromo-4-
oxo-4H-chromen-3-yl substituents in the external groove was
detected (poses related to 22 and 28, Figs S3 and S4, Supporting
Information, respectively). Regarding compounds 34e36, docking
analysis showed that the orientation of the 4-(5-(3-(tri-
fluoromethyl)phenyl)furan-2-yl) moiety allows better interactions
with the binding groove counterpart (Fig. 3).
2.2. Chemistry and biological assays
In order to corroborate our computational results, we firstly
decided to synthesize compounds 10, 16, 22, 28, 34 and to submit
them to biological screening. In particular, as in docking simula-
tions we observed a similar behavior for urea, thiourea and N-
methylurea derivatives, we utilized only urea as Biginelli reaction
synthon. With the optimized general conditions reported in
Also in these cases, docking poses satisfying the contemporary
pep
interaction with Tyr130(A) were found only in compounds

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G. Lauro et al. / European Journal of Medicinal Chemistry 80 (2014) 407e415
Fig. 2. a) 3D model of 4 (colored by atom types: C orange, N blue, O red, H light gray) in docking with mPGES-1 (molecular surface represented in white); residues in the active site
represented in licorice (colored by atom types: C white, N blue, O red, H light gray; black captions for residues in chain A and white captions for those in chain B) and related
molecular surfaces depicted in transparent green (for residues able to interact with 4) and transparent red (for residues not able to interact with 4). b) 2D panel representing
interactions between 4 and residues in mPGES-1 binding site. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
confirming our suggestions regarding the appropriate accommo-
dation of a putative inhibitor in the mPGES-1 binding site.
Moreover, maintaining the crucial 4-, 5- and 6- substituents on
the dihydropyrimidine core, we synthesized and tested the bio-
logical activity of the two 34-related compounds, namely the N1-
methyl derivative 35 and the thio-analog 36. The results obtained
showed efficient inhibitory activity for 35 (IC50 ¼ 7.56 ꢁ 0.94
but a weak and incomplete suppression of mPGES-1 activity for 36
(37% inhibition at 10 M, IC50 > 30 M) (Fig. 4 and Table 2). Since
mM),
m
m
the docking model of 36 confirms the respect of the key in-
teractions found for 34 and 35 (Fig. S5, Supporting Information), the
differences in biological activities could be mainly ascribed to the
influence of the chemical properties of sulfur vs. oxygen (such as
dimensions, electronegativity), not properly weighted by the
scoring functions of the docking software.
To further confirm that the presence of both 4-methoxybenzoyl
group at C5, and 5-(3-(trifluoromethyl)phenyl)furan-2-yl group at
C4 is necessary for the activity, compound 32 was synthesized as
negative control. As expected, compound 32, lacking the aromatic
substituent at C5 was found inactive. This confirms our hypothesis of
using a proper qualitative structure-based filter to subsequently
integrate with the docking predicted binding affinities (Table S1,
Supporting Information) for the selection of new mPGES-1 in-
hibitors. In Fig. 5 the docking model related to the active compound
Fig. 3. Shape complementarity between 6-(5-(3-(trifluoromethyl)phenyl)furan-2-yl)
substituent (molecular surface represented in black wireframes) and external binding
groove of mPGES-1 (molecular surface of residues in this site represented in green
wireframes). (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
34 is depicted. The 5-(3-methoxybenzoyl) group establishes a pep
interaction with Tyr130(A), while the bulky 4-(5-(3-(tri-
fluoromethyl)phenyl)furan-2-yl) substituent occupies the external
binding groove, interacting with Thr31(B), Ile32(B), Gln134(A), and
Leu135(A); more specifically, trifluoromethyl terminal group con-
tributes to these interactions, making contacts with Tyr28(B). The 6-
ethylcarboxylate function interacts with a shallow groove directed
toward the cytoplasmic side of the protein, while the urea containing
portions of the molecules establish polar contacts with Ser127(A).
Similar binding modes have been observed for the derivatives 35 and
36 (Fig. 6, and S5), while 32 lacked these fundamental interactions.
Scheme 1, the synthesis of these compounds was performed
through a microwave-assisted Biginelli reaction, which provides a
rapid access to substituted dihydropyrimidine derivatives with
good yields and in a short reaction time [20]. Urea or its derivatives
(1.5 equiv), two different 1,3-dicarbonyl compounds (1.0 equiv) and
the appropriate aryl/heteroaryl aldehydes (1.0 equiv), were suc-
cessfully used in a high-speed trimethylsilyl chloride (TMSCl)-
mediated Biginelli condensation, using microwave irradiation.
Synthesized compounds were then purified by reversed-phase
HPLC and characterized by ESI-MS, HRMS and NMR spectra (see
Experimental Section).
3. Materials and methods
Interference of the test compounds with mPGES-1 activity was
investigated in a cell-free assay using the microsomal fraction of
3.1. Computational details
interleukin-1b-stimulated human A549 cells [21]. Inhibition of
mPGES-1 by our compounds was perfectly in line with computa-
tional predictions. In fact, among the tested compounds, an inter-
The chemical structures of investigated compounds were built
with Maestro (version 9.6) [22] Build Panel and then processed
with LigPrep, version 2.8 [22], generating all the possible
esting IC50 value of 4.16 ꢁ 0.47
mM was detected for compound 34,

G. Lauro et al. / European Journal of Medicinal Chemistry 80 (2014) 407e415
411
Scheme 1. General synthetic procedure and chemical structures of synthesized dihydropyrimidine derivatives.
stereoisomers, tautomers, protonation states at a pH of 7.4 ꢁ 1.0,
and finally minimized using OPLS 2005 force field. For the subse-
quent docking calculations, all the structures were converted in the
.pdbqt format using OpenBabel software (version 2.3.2) [23], add-
ing Gasteiger charges.
Protein 3D model was prepared using the Schrödinger Protein
Preparation Wizard [22], starting from the mPGES-1 X-ray struc-
ture in the active form (PDB code: 4AL0). In particular, crystallized
water molecules and GSH cofactor were removed, all hydrogens
were added, and bond orders were assigned. Conformation “A” was
chosen for residues crystallized in two conformations, as reported
in the original PDB file. Protein .pdb file obtained was then pro-
cessed with Autodock Tools 1.5.6 and converted in .pdbqt format,
merging non polar hydrogens and adding Gasteiger charges. Charge
deficit was spread over all atoms of related residues.
chemical shifts are expressed in parts per million (ppm) on the
delta (d
) scale relative to CHCl3 (7.26 ppm for 1H and 77.2 ppm for
13C) as internal reference. Electrospray mass spectrometry (ESI-MS)
was performed on a LCQ DECA TermoQuest (San Josè, California,
USA) mass spectrometer. High resolution mass spectra were ac-
quired on a LTQ Orbitrap XL (Thermo Scientific). Reactions were
monitored on silica gel 60 F254 plates (Merck) and visualized with
cerium sulfate and under UV (
l
¼ 254 nm, 365 nm). Analytical and
semi-preparative reversed-phase HPLC was performed on Agilent
Technologies 1200 Series high performance liquid chromatography
using a Jupiter Proteo C18 reversed-phase column (250 ꢂ 4.60 mm,
ꢀ
ꢀ
4m
, 90 A, flow rate ¼ 1 mL/min; 250 ꢂ 10.00 mm, 10
m, 90 A, flow
rate ¼ 4 mL/min respectively, PhenomenexÒ). The binary solvent
system (A/B) was as follows: 0.1% TFA in water (A) and 0.1% TFA in
CH3CN (B). The absorbance was detected at 280 nm. The purity of
all tested compound (>98%) was determined by HPLC analysis.
Docking calculations were performed using the Autodock-Vina
software [24]. In the configuration file linked to 3D structure of
the protein, we specified coordinates and dimensions along x, y, z
axes of the grid related to the site of presumable pharmacological
interest. In particular, we chose the binding site between A and B
chains, and a grid box size of 24 ꢂ 20 ꢂ 18 and centered at 10.304
3.2.1. Microwave irradiation experiments
All microwave irradiation experiments were carried out in a
dedicated CEM-DiscoverÒ Focused Microwave Synthesis apparatus,
operating with continuous irradiation power from 0 to 300 W
utilizing the standard absorbance level of 300 W maximum power.
The reactions were carried out in 10 mL sealed microwave glass
vials. The DiscoverÔ system also offers controllable ramp time,
hold time (reaction time) and uniform stirring. The temperature
was monitored using the CEM-Discover built-in-vertically-focused
IR temperature sensor. After the irradiation period, the reaction
vessel was cooled rapidly (60e120 s) to ambient temperature by air
jet cooling.
ꢀ
(x), ꢃ11.033 (y), and ꢃ8.384 (z) was set, with spacing of 1.0 A be-
tween the grid points. The exhaustiveness value was set to 512,
saving 30 conformations as maximum number of binding modes.
For all the investigated compounds, all open-chain bonds were
treated as active torsional bonds. Docking results were analyzed
with Autodock Tools 1.5.6. Illustrations of the 3D models were
generated using VMD software [25] and Maestro [22].
3.2. Chemistry
3.2.2. General procedure for microwave-assisted Biginelli reaction
A mixture of appropriate aldehyde (1.0 mmol), urea or its de-
rivatives (thiourea and N-methylurea) (1.5 mmol), 1,3-dicarbonyl
compound (1.0 mmol) in acetonitrile (1.5 mL) were placed in a
10 mL microwave glass vial equipped with a small magnetic stirring
bar. TMSCl (1.0 mmol) was added and the mixture was then stirred
under microwave irradiation at 120 ꢄC for 15e20 min. After irra-
diation, the reaction mixture was cooled to ambient temperature
by air jet cooling, cold water was added and the vial was poured
All commercially available starting materials were purchased
from SigmaeAldrich and were used as received. All solvents used
for the synthesis were of HPLC grade; they were purchased from
SigmaeAldrich and Carlo Erba Reagenti. All NMR spectra (1H,
HMBC, HSQC) were recorded on a Bruker Avance 600 MHz in-
strument at T ¼ 298 K (1H at 600 MHz, 13C at 150 MHz). All com-
pounds were dissolved in 0.5 mL of 99.95% CDCl3 (Carlo Erba, 99.95
Atom % D). Coupling constants (J) are reported in Hertz, and

412
G. Lauro et al. / European Journal of Medicinal Chemistry 80 (2014) 407e415
reversed-phase HPLC (on
a
Jupiter Proteo C18 column:
ꢀ
250 ꢂ 10.00 mm, 10
m, 90 A, flow rate ¼ 4 mL/min) using the
gradient conditions reported below for each compound. The final
products were obtained with high purity> 98% detected by HPLC
analysis and were fully characterized by ESI-MS, HRMS and NMR
spectra.
3.2.2.1. Compound 10. Compound 10 was obtained by following
the general procedure as a yellow gelatinous solid (120.9 mg, 85%
yield). RP-HPLC tR ¼ 24.7 min, gradient condition: from 5% B to 25%
B in 10 min, increased to 100% B in 45 min, flow rate of 4 mL/min,
l
¼ 280 nm. 1H NMR (500 MHz, CDCl3):
d
¼ 0.85 (t, J ¼ 7.1 Hz, 3H),
3.81 (s, 3H), 3.88e4.01 (m, 4H), 5.59 (s, 1H), 6.77 (d, J ¼ 8.9 Hz, 2H),
7.44 (t, J ¼ 7.8 Hz,1H), 7.53e7.61 (m, 3H), 7.76 (br s, 2H), 9.92 (s,1H);
13C NMR (125 MHz, CDCl3):
d
¼ 13.7, 54.6, 57.8, 61.9, 112.7, 113.3,
128.4, 128.8, 130.7, 131.6, 132.0, 133.2, 190.7. ESMS, calcd for
C
C
22H20N2O6 408.13; found m/z ¼ 409.1 [M þ H]þ. HRMS, calcd for
22H21N2O6 [M þ H]þ 409.1400, found 409.1388.
3.2.2.2. Compound 16. Compound 16 was obtained by following
the general procedure as a brown gelatinous solid (132.5 mg, 78%
yield). RP-HPLC tR ¼ 22.9 min, gradient condition: from 5% B to 30%
B in 10 min, increased to 100% B in 40 min, flow rate of 4 mL/min,
Fig. 4. Concentration-response analysis of compounds 34, 35, and 36 for inhibition of
mPGES-1 in a cell-free assay. Data are given as mean ꢁ SEM, n ¼ 3.
l
¼ 280 nm. 1H NMR (500 MHz, CDCl3):
d
¼ 0.85 (t, J ¼ 7.1 Hz, 3H),
1.33 (t, J ¼ 7.1 Hz, 3H), 3.79 (s, 3H), 3.85e3.98 (m, 4H), 5.46 (s, 1H),
6.72e6.82 (m, 4H), 7.13 (t, J ¼ 7.8 Hz, 1H), 7.35 (br s, 1H), 7.57 (d,
Table 2
mPGES-1 inhibition by tested compounds 10, 16, 22, 28, 32, 34,
35, 36.
J ¼ 8.8 Hz, 2H); 13C NMR (125 MHz, CDCl3):
¼ 13.2, 14.2, 56.1, 55.9,
d
56.1, 59.6, 113.4, 113.9, 114.2, 119.6, 119.8, 130.7, 131.4, 131.7. ESMS,
calcd for C23H24N2O6 424.16; found m/z ¼ 425.2 [M þ H]þ. HRMS
calcd for C23H25N2O6 [M þ H]þ 425.1713, found 425.1692.
Compound ID
IC50 þ SEM (
m
M)a
10
16
22
28
32
34
35
36
n.a.
n.a
n.a
n.a
3.2.2.3. Compound 22. Compound 22 was obtained by following
the general procedure as a yellow powder (144 mg, 79%); RP-
HPLC tR ¼ 20.8 min, gradient condition: from 20% B to 100% B
n.a
4.16 ꢁ 0.47
7.56 ꢁ 0.94
>30
in 45 min, flow rate of 4 mL/min,
CDCl3):
l
¼ 280 nm. 1H NMR (500 MHz,
d
¼ 0.83 (t, J ¼ 7.1 Hz, 3H), 3.74 (s, 3H), 3.84e3.99 (m, 2H),
a
n.a: no activities at concentration lower than 10 mM found.
5.62 (s, 1H), 6.71 (d, J ¼ 9.0 Hz, 2H), 7.24e7.37 (m, 1H), 7.60e7.63
(m, 4H), 7.74e7.88 (m, 4H); 13C NMR (125 MHz, CDCl3):
d
¼ 13.4,
into crushed ice and then at 4 ꢄC overnight. The resulting precipi-
tate was filtered and washed with a cold mixture of ethanol/water
(1:1) (3 ꢂ 3 mL), to give the desired product in good yields (75e
90%). HPLC purification was performed by semi-preparative
55.6, 59.7, 62.9, 113.5, 114.0, 120.1, 120.3, 127.3, 127.7, 129.8, 131.4,
141.4. ESMS, calcd for C27H22N4O5 482.16; found m/z ¼ 483.1
[M þ H]þ. HRMS calcd for C27H23N4O5 [M þ H]þ 483.1668, found
483.1636.
Fig. 5. a) 3D model of identified inhibitor 34 (colored by atom types: C sky blue, N blue, O red, H light gray, F pink) in docking with mPGES-1 (molecular surface represented in
white); residues in the active site represented in licorice (colored by atom types: C white, N blue, O red, H light gray; black captions for residues in chain A and white captions for
those in chain B) and related molecular surfaces depicted in transparent green. b) 2D panel representing interactions between 34 and residues in mPGES-1 binding site (the legend
related to the 2D panel is reported in the lower part of the figure). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)

G. Lauro et al. / European Journal of Medicinal Chemistry 80 (2014) 407e415
413
Fig. 6. a) 3D model of compound 35 (colored by atom types: C blue, N blue, O red, H light gray) in docking with mPGES-1 (molecular surface represented in white); residues in the
active site represented in licorice (colored by atom types: C white, N blue, O red, H light gray; black captions for residues in chain A and white captions for those in chain B) and
related molecular surfaces depicted in transparent green. b) 2D panel representing interactions between 35 and residues in mPGES-1 binding site. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
3.2.2.4. Compound 28. Compound 28 was obtained by following
the general procedure as a yellow powder (68.7 mg, 75% yield); RP-
HPLC tR ¼ 26.4 min, gradient condition: from 5% B to 30% B in 5 min,
(br s, 2H), 7.66e7.73 (m, 4H); 13C NMR (125 MHz, CDCl3):
d
¼ 13.7,
32.6, 50.8, 56.0, 63.5, 108.0, 110.8, 114.6, 120.7, 121.2, 121.7127.4,
128.6, 130.3, 133.5. ESMS, calcd for C27H23F3N2O6 528.15; found m/
z ¼ 529.1 [M þ H]þ. HRMS, calcd for C27H24F3N2O6 529.1586, found
529.1564.
increased to 100% B in 45 min, flow rate of 4 mL/min,
l
¼ 280 nm. 1H
NMR (500 MHz, CDCl3):
d
¼ 0.98 (t, J ¼ 7.1 Hz, 3H), 3.88 (s, 3H),
4.02e4.12 (m, 2H), 5.42 (s, 1H), 6.98 (d, J ¼ 8.7 Hz, 2H), 7.10 (s, 1H),
7.92 (d, J ¼ 8.7 Hz, 2H), 8.03 (br s, 1H), 8.25 (br s, 1H); 13C NMR
3.2.2.8. Compound 36. Compound 36 was obtained by following
the general procedure as a pale brown solid (122.7 mg, 90% yield);
RP-HPLC tR ¼ 32.3 min, gradient condition: from 5% B to 35% B in
10 min, increased to 100% B in 40 min, flow rate of 4 mL/min,
(125 MHz, CDCl3):
d
¼ 13.0, 61.7, 62.7, 75.3, 112.5, 127.4, 130.7, 135.3,
136.3, 148.5, 157.4. ESMS, calcd for C24H18Br2N2O7 606.22; found m/
z ¼ 606.8 [M þ H]þ. HRMS, calcd for C24H19Br2N2O7 [M þ H]þ
604.9559, found 604.9533.
l
¼ 280 nm. 1H NMR (500 MHz, CDCl3):
d
¼ 0.85 (t, J ¼ 7.1 Hz, 3H),
3.75 (s, 3H), 3.85e4.00 (m, 2H), 5.58 (s, 1H), 6.42 (d, J ¼ 3.3 Hz, 1H),
6.58 (d, J ¼ 3.3 Hz, 1H), 6.78 (d, J ¼ 8.7 Hz, 2H), 7.43e7.51 (m, 2H),
7.60 (br s, 2H), 7.74 (d, J ¼ 8.7 Hz, 2H); 13C NMR (125 MHz, CDCl3):
3.2.2.5. Compound 32. Compound 32 was obtained by following
the general procedure as a red solid (67.3 mg, 75% yield); RP-HPLC
tR ¼ 26.7 min, gradient condition: from 5% B to 40% B in 10 min,
d
¼ 13.7, 52.2, 55.1, 62.8, 107.4, 110.7, 113.6, 113.8, 120.4, 124.0, 126.8,
increased to 100% B in 40 min, flow rate of 4 mL/min,
l
¼ 280 nm. 1H
128.9, 129.2, 131.1. ESMS, calcd for C26H21F3N2O5S 530.11; found m/
z ¼ 531.1 [M þ H]þ. HRMS, calcd for C26H22F3N2O5S [M þ H]þ
531.1202, found 531.1172.
NMR (500 MHz, CDCl3):
d
¼ 1.29 (t, J ¼ 7.1 Hz, 3H), 2.55 (s, 3H), 3.26
(s, 3H), 4.10e4.20 (m, 2H), 5.52 (s, 1H), 6.21 (d, J ¼ 3.3 Hz, 1H), 6.62
(d, J ¼ 3.3 Hz, 1H), 7.45e7.52 (m, 3H), 7.81 (br s, 1H); 13C NMR
(125 MHz, CDCl3):
d
¼ 13.7, 14.8, 32.6, 56.0, 60.2, 108.0, 110.8, 127.4,
3.3. Bioactivity assays
128.6, 130.3, 133.5. ESMS, calcd for C20H19F3N2O4 408.13.; found m/
z ¼ 409.1 [M þ H]þ. HRMS, calcd for C20H20F3N2O4 [M þ H]þ
409.1375, found 409.1354.
3.3.1. Assay systems and materials
Dulbecco’s Modified Eagle Medium (DMEM)/high glucose
(4.5 g/L) medium, penicillin, streptomycin, trypsin/ethylenedi-
aminetetraacetate (EDTA) solution, and LSM 1077 lymphocyte
separation medium were obtained from PAA (Pasching, Austria). IL-
3.2.2.6. Compound 34. Compound 34 was obtained by following
the general procedure as a red gelatinous solid (149.2 mg, 88%
yield); RP-HPLC tR ¼ 34.2 min, gradient condition: from 5% B to 35%
B in 15 min, increased to 100% B in 40 min, flow rate of 4 mL/min,
1b was obtained from ReproTech (Hamburg, Germany). Fetal calf
serum (FCS), phenylmethylsulfonylfluoride (PMSF), leupeptin,
soybean trypsin inhibitor (STI), glutathione (reduced), PGB1, lyso-
zyme, Ca2þ-ionophore A23187, and arachidonic acid were obtained
from SigmaeAldrich (Deisenhofen, Germany). MK886 and
l
¼ 280 nm. 1H NMR (500 MHz, CDCl3):
d
¼ 0.85 (t, J ¼ 7.1 Hz, 3H),
3.74 (s, 3H), 3.85e4.00 (m, 2H), 5.59 (s, 1H), 6.37 (d, J ¼ 3.3 Hz, 1H),
6.57 (d, J ¼ 3.3 Hz, 1H), 6.75 (d, J ¼ 8.4 Hz, 2H), 7.41e7.47 (m, 2H),
7.60 (br s, 2H), 7.73 (d, J ¼ 8.4 Hz, 2H); 13C NMR (125 MHz, CDCl3):
11
b-PGE2 were obtained from Cayman Chemical (Ann Arbor, MI).
d
¼ 13.7, 52.7, 55.4, 63.0, 107.5, 109.7, 111.6, 116.8, 121.7, 124.4, 127.0,
PGH2,
adenosine triphosphate (ATP), isopropyl- -d-1-
b
129.5, 130.8, 131.7. ESMS, calcd for C26H21F3N2O6 514.14; found m/
z ¼ 515.1 [M þ H]þ. HRMS, calcd for C26H22F3N2O6 [M þ H]þ
515.1430, found 515.1403.
thiogalactopyranoside (IPTG), and dextrane were obtained from
Larodan (Malmoe, Sweden), Roche Diagnostics (Mannheim, Ger-
many), AppliChem (Darmstadt, Germany), and Fluka (Neu-Ulm,
Germany), respectively. A549 cells were provided by the Karolinska
Institute (Stockholm, Sweden). Leukocyte concentrates from hu-
man healthy volunteers were provided by Institute of Transfusion
Medicine, University Hospital Jena, Germany.
3.2.2.7. Compound 35. Compound 35 was obtained by following
the general procedure as a red gelatinous solid (84.3 mg, 76% yield);
RP-HPLC tR ¼ 30.3 min, gradient condition: from 5% B to 40% B in
10 min, increased to 100% B in 40 min, flow rate of 4 mL/min,
l
¼ 280 nm. 1H NMR (500 MHz, CDCl3):
d
¼ 1.05 (t, J ¼ 7.1 Hz, 3H),
3.3.2. Cell culture
A549 cells were grown in DMEM/high glucose (4.5 g/mL) me-
dium supplemented with heat-inactivated FCS (10%, v/v), penicillin
3.20 (s, 3H), 3.82 (s, 3H), 3.76e3.88 (m, 2H), 5.54 (s, 1H), 6.40 (d,
J ¼ 3.3 Hz, 1H), 6.60 (d, J ¼ 3.3 Hz, 1H), 6.87 (d, J ¼ 8.7 Hz, 2H), 7.48

414
G. Lauro et al. / European Journal of Medicinal Chemistry 80 (2014) 407e415
(100 U/mL) and streptomycin (100
mg/mL). After three days,
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In conclusion, we have reported the structure-based design of a
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a