Design and synthesis of a second series of triazole-based compounds as potent dual mPGES-1 and 5-lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.05.014

Microsomal prostaglandin E₂ synthase (mPGES)-1 and 5-lipoxygenase (5-LO) are pivotal enzymes in the biosynthesis of the pro-inflammatory PGE₂ and leukotrienes, respectively. The design and synthesis of a second series of mPGES-1 inhibitors based on a triazole scaffold are described. Our studies allowed us to draw a tentative SAR profile and to optimize this series with the identification of compounds 10, 11 and 14-15 which displayed potent mPGES-1 inhibition in a cell-free assay. In addition, compounds 5, 10, 12 and 14-16 also blocked 5-LO activity in cell-free and cell-based test systems, emerging as very promising candidates for the development of safer and more effective anti-inflammatory drugs.

Full text of DOI:10.1016/j.ejmech.2012.05.014

European Journal of Medicinal Chemistry 54 (2012) 311e323
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of a second series of triazole-based compounds as potent
dual mPGES-1 and 5-lipoxygenase inhibitors
,
a
b
Maria Giovanna Chini a, Rosa De Simone a, Ines Bruno a , Raffaele Riccio , Friederike Dehm ,
*
Christina Weinigel c, Dagmar Barz c, Oliver Werz b, Giuseppe Bifulco a
,
**
a Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
b Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Philosophenweg 14, D-07743 Jena, Germany
c Institute of Transfusion Medicine, University Hospital Jena, 07743 Jena, Germany
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< Design of 2nd series of new triazole-
based compounds as mPGES-1 and
5-LO inhibitors.
< Synthesis of 2e16 performed by
click chemistry and Suzuki cross-
coupling reaction.
< Identification of 10, 11 and 14e15 as
potent mPGES-1 inhibitors.
< 5, 10, 12 and 14e16 emerged as 5-LO
inhibitors.
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 (mPGES)-1 and 5-lipoxygenase (5-LO) are pivotal enzymes in the
biosynthesis of the pro-inflammatory PGE2 and leukotrienes, respectively. The design and synthesis of
a second series of mPGES-1 inhibitors based on a triazole scaffold are described. Our studies allowed us
to draw a tentative SAR profile and to optimize this series with the identification of compounds 10, 11
and 14e15 which displayed potent mPGES-1 inhibition in a cell-free assay. In addition, compounds 5, 10,
12 and 14e16 also blocked 5-LO activity in cell-free and cell-based test systems, emerging as very
promising candidates for the development of safer and more effective anti-inflammatory drugs.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 10 February 2012
Received in revised form
9 May 2012
Accepted 9 May 2012
Available online 18 May 2012
Keywords:
Triazole-based inhibitors
In silico screening
mPGES-1
5-Lipoxygenase
1. Introduction
the prostaglandin (PG) biosynthetic pathway such as PLA2, COX-1
and COX-2; the latter was initially supposed to be a promising
For many years, the main strategy adopted to manage inflam-
matory disorders consisted in the inhibition of the key enzymes of
target for the development of therapeutic interventions owing to
its inducible nature. Unfortunately, the long-term intake of COX-2
inhibitors (coxibs) is associated with severe cardiovascular
toxicity and, thus coxibs are controversially discussed as thera-
peutics [1,2,3]. Lately, mPGES-1 enzyme, which catalyzes the last
step of PGE2 synthesis, has emerged as a valid alternative strategy
in the struggle against inflammation [4]. In the last years, a plethora
* Corresponding author. Tel.: þ39 089 969743; fax: þ39 089 969602.
** Corresponding author. Tel.: þ39 089 969741; fax: þ 39 089 969602.
E-mail addresses: brunoin@unisa.it (I. Bruno), bifulco@unisa.it (G. Bifulco).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.014

312
M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
of data have been gathered highlighting the key role played by this
enzyme in inflammation-related disorders and its ability to affect
PGE2 synthesis only under inflammatory conditions, without sup-
pressing the constitutive prostanoids involved in gastro-protection
and in other crucial physiological functions [5,6,7]. Continuing our
studies on small molecules able to block mPGES-1 activity, we
recently reported the design and synthesis of a small collection of
differently decorated disubstituted triazole derivatives able to
interact with this target at micromolar concentrations. In the
course of our investigations, we identified some interesting mole-
cules able to inhibit mPGES-1 as well as other key enzymes within
the arachidonic acid cascade such as 5-lipoxygenase (5-LO) and 5-
LO-activating protein (FLAP) that are required for leukotrienes (LTs)
formation [8]. The good qualitative coherence found in our
previous work [8] between the biological results and the predic-
tions of molecular docking calculations provided a satisfactory
binding mode model of these triazoles with their respective bio-
logical targets (i.e., mPGES-1, 5-LO, and FLAP), gaining more infor-
mation to envisage further structural optimizations of the basic
template in accordance with the catalytic pocket requirements of
the enzyme. In more detail, the previous investigations have dis-
closed the basic structure 1 [8] (Scheme 1), able to efficiently
interact with key residues in the catalytic site, which needed to be
further optimized for what concerns the substitution pattern on the
biphenyl moiety and in order to provide a better binding affinity
with the target enzyme. In line with these considerations, we have
decided to leave the 2-nitro-4-carboxybenzyl moiety unchanged
(Scheme 1) and to variously decorate the right hand portion of the
molecule, coming to the identification of compound 11, which
retained mPGES-1 inhibition ability, and compound 10, emerging
as potent dual inhibitor of mPGES-1 and 5-LO enzymes. Building on
these results, we have further refined the structure of compound
10, leaving unmodified the right hand portion of the molecule and
introducing some minor modifications on the left hand moiety,
such as the reduction of the nitro group (compound 14), the
elimination of the ortho-substituent on the phenyl ring (compound
15) and the insertion of a sulfonamide function between the phenyl
and the triazole ring (compound 16), reminiscent of the structural
backbone showed by a triazole-based compound previously iden-
tified by us as potent dual inhibitor of mPGES-1 and 5-LO enzymes
[8].
Here we report the computer-aided design, the synthesis and,
the pharmacological profile of this second generation of triazoles
derivatives; our findings provided new interesting hits shedding
further light on the structural requirements needed for an optimal
ligandeenzyme interaction.
2. Results and discussions
2.1. Docking studies
On the basis of the encouraging results regarding the ration-
alization of the inflammation processes related to natural products
[9e13] and the design of new synthetic molecules [8,14,15], here we
N
N
N
O N
2
1
HOOC
N
N
N
N
N
N
N
N
O2N
O2N
O
N
O2N
4
3
N
2
O
NH2
HOOC
O2N
HOOC
HOOC
O2N
OCH3
O
N
N
N
O2N
N
N
N
N
N
N
N
N
COOH
N
6
7
5
HOOC
COOH
HOOC
O2N
HOOC
O
F3C
S
O
N
N
N
N
O2N
H
N
N
N
N
N
Cl
O2N
OCH3
O
OH
8
10
9
OCH3
OCH3
HOOC
HOOC
O2N
HOOC
N
N
N
N
N
O2N
N
N
N
N
O2N
11
S
12
13
HOOC
COOH
OH
HOOC
HOOC
O
F3C
F3C
F3C
N
N
N
H2N
N
N
N
N
Cl
S
N
N
Cl
Cl
O
O
O
O
14
16
15
HOOC
HOOC
HOOC
Scheme 1. Structures of lead compound 1 and its derivatives.

M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
313
report the design and the theoretical evaluation of a new significant
set of molecules obtained by revisiting the lead compound 1 (See
Scheme 1) [8]. In particular, we have substituted the scaffold of 1
with various groups of different size and hydrophobicity with the
aim to improve potency and to trace a tentative SAR profile of this
kind of potential anti-inflammatory drugs. As suggested from our
previous work [8], the 2-nitro-4-carboxybenzyl moiety remarkably
influences the activity of the triazole molecules, so this moiety was
not altered for the molecules 2e13 (Scheme 1) combining it with
small hydrophilic groups (COOH, OH, NH2) at different positions (4,
6e8, 12e13, Scheme 1), or with hydrophobic groups with
increasing size (2e3, 5, 9e11, Scheme 1) to understand their
potential influence on biological profiles. Even if Jegerschöld et al.
[16] have recently elucidated the electron crystallographic struc-
ture of closed inactive conformation of mPGES-1, owing to the lack
of the experimental structure of the mPGES-1 open active form, for
our docking calculations by Autodock4.2 software [17] we used the
microsomal glutathione transferase 1 (MGST-1) [18] as model
receptor. MGST-1 shows 38% sequence identity with the human
mPGES-1 [19] and was successfully used in our previous work [8]
and as template for molecular modeling and dynamics simulation
[20,21]. The sequence alignment of these two MAPEG (membrane-
associated proteins in eicosanoid and glutathione metabolism)
superfamily enzymes [8,20] was used for the in silico screening
analysis. Our results point out two different docking poses for these
triazole-based potential mPGES-1 inhibitors: (a) the first one
includes molecules with ortho- and/or meta-substituted biphenyl
groups (3, 6e8, and 10, Fig. 1A), or with a smaller aromatic ring (11)
as arms at position 4; (b) the second family relates to compounds
containing the biphenyl portion with at least one substituent in the
para position (2, 4e5, 9, 12e13, Fig. 1B). However, all the designed
molecules accommodate in the ligand pocket situated in the region
at the interface of the two mPGES-1 subunits (Fig. 1) [22,23]
interacting with the highly conserved Arg110mPGES-1 (Arg113MGST-
1) [16,24e27] the key catalytic residue Arg126mPGES-1 (Arg129MGST-
1) [16,22,23], and establishing further contacts with Arg38, Leu39,
Val65, Arg70, Tyr130, Thr131, Ser127 [22] (Arg37, Leu38, Val68,
Arg73, Phe133, Phe134 and Gly130 in MGST-1, respectively).
On this basis, the substitutions at ortho, meta and para of the
biphenyl arm with small hydrophilic groups cause a different
pattern of hydrogen bonds with MGST-1 among 4, 6e8, 12e13 (See
Fig. 2A). In particular, even though 6 and 7 have similar docking
COOH of 7 interacts with Arg37 (Arg38mPGES-1) (Fig. 2A). On the
other hand, the para carboxybiphenyl arm of 13 interacts with
aminoacids located at the bottom part of the ligand binding pocket
establishing a
pep interaction with the Phe133 (Tyr130mPGES-1),
and moreover, its 2-nitro-4-carboxybenzyl moiety interacts with
Asn81 and Lys25 (Fig. 2A). The same hydrogen bond pattern is also
observed for the molecules 4, 8 and 12 (See Fig. 2B), where the OH
(12) and/or NH2 (4) group at para position of biphenyl arm do not
allow the hydrogen bonds with Arg37 (Arg38mPGES-1) and/or
Arg129 (Arg126mPGES-1), but allow the
pep interaction with
Phe133 (Tyr130mPGES-1) (See Fig. 2B); whereas for 8, as like for 7, the
hydrogen bond with Arg37 and
and Arg129 are observed.
p-cation interactions with Lys67
Regarding the molecules with bulky groups (2e3, 5, 9, 10e11,
Scheme 1), depicted in Fig. 3, the different steric hindrance of the
arms influences the positioning in the ligand binding site. In
particular, the molecules with para (5) groups, or combined with
meta (2) and/or ortho (9) substituents at biphenyl portion are in
close contact with Gly130, Phe134 and establish
pep interactions
with Phe133 (Tyr130mPGES-1). On the other hand, even though 11
and compounds with bulkier arms (i.e. 3 and 10) have similar
docking poses, the thiophene ring of 11 allows peculiar van-der-
Waals contacts with Ala32 and Val68, and a further
interaction with Lys67; while 10 with respect to 3 establishes
additional -cation interactions with Arg129 (Arg126mPGES-1) and
p-cation
p
Arg37 (Arg38mPGES-1) and hydrophobic contact with Pro125.
From the above considerations, we have identified the differ-
ences between these two families of molecules where the
substituents position on the arms influences the pattern of
hydrophobic interactions and/or the hydrogen bonding. For these
reasons, we decided to undertake the synthesis of the designed
molecules (all within the lowest free energy of binding
(Ebinding) < ꢀ7 kcal/mol) as the starting point for obtaining
preliminary experimental results based on the evaluation of the
bioactivity of this small set of compounds with the aim of a deeper
understanding of the key features of new mPGES-1 triazole-based
inhibitors. On the basis of the biological results, we have decided to
further improve and refine the structure of the most active mole-
cule 10 that was recognized as new lead compound (see Biological
section) for the design of triazole-based mPGES-1 inhibitors. In
particular, we have aimed to improve the biological activity of new
lead compound 10, modifying its 2-nitro-4-carboxybenzyl moiety
(14e16) taking into account our previous considerations [8]. As
shown in Fig. 4, the molecule 15 assumes the same docking pose
with respect to 10, where the elimination of the NO2 group
poses and both molecules are able to establish a p-cation interac-
tion with Arg129 (Arg126mPGES-1), the ortho COOH of 6 forms
a hydrogen bond with Arg129 (Arg126mPGES-1) while the meta
Fig. 1. Two different spatial arrangements of triazole-based compounds 1e13 in the binding site of MGST-1. (A) Superposition of compounds 3 (emerald), 6 (orange), 7 (yellow), 8
(purple), 10 (blue) and 11 (dark green). (B) Superposition of 2 (green), 4 (pink), 5 (gray), 9 (light green), 12 (light pink) and 13 (light brown). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

314
M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
Fig. 2. (A) Three dimensional models of the different hydrogen bond patterns of 6 (orange), 7 (yellow), and 13 (light brown) with MGST-1 binding site. (B) Three dimensional
models of the different hydrogen bond patterns of 8 (purple), 4 (pink) and 12 (light pink) with MGST-1 binding site. In both the figures the crucial aminoacids of MGST-1 receptor
are depicted by stick and balls (by atom type: C, gray; O, red; N, dark blue; and S, yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
influence the strength of the H-bonding with the Arg113
(Arg110mPGES-1), while the -cation interactions with Arg129
(Arg126mPGES-1 and Arg37 (Arg38mPGES-1), and most of the
On this basis, we have synthesized these compounds and
assessed the biological activity to prove our theoretical findings.
p
)
hydrophobic interactions are maintained. On the other hand, the
chemical reduction of the nitro group to NH2 for 14, and the
insertion of a sulfonamide function between the phenyl and the
triazole ring at 1 yielding 16 causes a different positioning of the
compounds compared to 10, and a shifting of the bulky arm at C-4
of the triazole ring in the MGST-1 ligand pocket. In particular, the
NH2 group of 14 establishes two additional hydrogen bonds acting
2.2. Chemistry
For the synthesis of analogs 2e16, the retro-synthetic approach
suggested us to obtain the triazole ring through the copper-
catalyzed 3 þ 2 Huisgen’s cycloaddition between the appropriate
terminal alkynes and azides [27], while to generate the differently
decorated biphenyl systems we relied on the Suzuki cross-coupling
reaction [28] that afforded the desired compounds (Scheme 2).
In more detail, the triazole rings were generated through the
condensation between the appropriate terminal alkynes and the
azides following two different synthetic procedures. Specifically,
when we started from the commercially available azide 17, con-
taining a strong electron-withdrawing group such as the sulfonyl
function that could drive the reaction toward undesired by-
products formation, decreasing the yields of our expected cyclo-
adduct [29], we carried out the reaction with 1-bromo-4-
ethynylbenzene 18 in dry chloroform at 0 ꢁC in presence of 2,6-
lutidine as base (Scheme 3) [30]. On the contrary, when the
1
as H-bond donor with the carbonyl oxygen of Gly223 and Nd of
His75, making also possible the hydrogen bond between the tri-
azole ring with the hydroxyl group of Thr33, and hydrophobic
interactions of the halogenated benzene with Gly130, Pro127 and
Gln126. On the other hand, the sulfonamide function of 16 only
forms a hydrogen bond with the OH moiety of Thr33.
Fig. 3. Three dimensional models of the different hydrogen bond patterns and peculiar
hydrophobic interactions of 2 (green), 3 (emerald), 5 (gray), 9 (light green), 10 (blue),
and 11 (dark green) with the MGST-1 binding site. The crucial aminoacids of MGST-1
are depicted by stick and balls and CPK (by atom type: C, gray; O, red; N, dark blue; and
S, yellow). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Fig. 4. Three dimensional models of the different hydrogen bond patterns and peculiar
hydrophobic interactions of 10 (blue), 14 (light blue), 15 (white), and 16 (cyan) with
MGST-1 binding site. The crucial aminoacids of MGST-1 receptor are depicted by stick
and balls and CPK (colored by atom type: C, gray; O, red; N, dark blue; and S, yellow).
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
315
N
R
N
N
N
N
Br
R
N
OH
OH
R
B
Suzuki
Coupling
1,3-dipolar
cycloaddition
R
R
N3
Br
Scheme 2. Retro-synthetic approach for the synthesis of derivatives 2e16.
azides were not available, the microwave irradiation technique
provided a faster way to obtain the desired triazole intermediates
22e23 (Scheme 3) in a oneepot reaction implicating the in situ
generation of azides starting from the corresponding halides 19e20
and sodium azide (Scheme 3) [31]. The 1,3-dipolar cycloaddition
required the presence of Cu(I) as catalyst for directing the
selective formation of 1,4-disubstituted triazoles [32]. The triazole
intermediates 21e23, obtained from the first step, were further
subjected to the Suzuki cross-coupling reaction with the appro-
priate boronic acid a-l following the experimental conditions
previously optimized by us [33], requiring the use of Pd(dppf)Cl2 as
catalyst and CsF as base in a mixture of tetrahydrofurane/water
(THF/H2O) 1:1, under microwaves irradiation; the desired final
compounds 2e5, 7e12 and 15e16 and the advanced intermediates
24e25 (Scheme 3) were obtained in satisfactory yields.
The nucleophilic partners employed in the Suzuki coupling are
all commercially available except for pinacol esters a and l. These
last were obtained, as shown in Scheme 4, starting from the
appropriate boronic acids 26 and 27 which were firstly protected as
pinacol esters 28 and 29, in order to facilitate the purification step
on silica column, and finally were converted in the boronate l
through a reductive amination of 29 in presence of morpholine and
in the amide a subjecting 28 to an amidation with morpholine
using triethylamine (TEA) as base, N-hydroxybenzotriazole (HOBt)
and N,N-dicyclohexylcarbodiimide (DIC) as carboxylate activators
and N,N-dimethylformamide (DMF) as solvent.
Br
SO2N3
N
N
N
SO2
a
COOH
Br
21
COOH
17
18
Br
Br
Br
N
N
N
N
O2N
N
NO2
N
b
Br
Br
HOOC
HOOC
23
COOH
22
COOH
19
20
18
F3C
Cl
N
O
N
N
O
HO
B
B
R
O
O
R1
OH
N
2-5; 7-9; 11-12; 15-16
O
a
N
b
F3C
HO
B
COOH
N
N
OH
O2N
N
Cl
O
S
O
d
O
NH
14
B
HO
HO
d
HOOC
O2N
OH
HO
10
e
B
c
c
O
O
OH
NH2
B
H
O
N
N
OH
B
N
f
O
OH
H
HO
OCH3
OCH3
H3CO
OH
e
e
B
g
6
HOOC
O2N
OH
h
24
B
HO
OH
B
OH
S
i
OH
N
N
N
H
O
OH
B
OCH3
j
OH
13
O
HOOC
B
N
O
O
O
k
25
H
l
Scheme 3. Synthetic protocol employed to generate derivatives 2e16a. a Reagents and conditions: (a) CuI, 2,6-lutidine, CHCl3 dry, 12 h, 0 ꢁC. (b) CuSO4, Cu(0), NaN3, t-BuOH/H2O 1:1,
microwaves, 25 min (c) CsF, Pd(dppf)Cl2, H2O/THF 1:1, microwaves, 25 min (d) SnCl2, EtOH, 80 ꢁC, 30 min (e) KMnO4, Na2HPO4, CH3OH, r.t., 1.5 h.

316
M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
Finally, for the synthesis of compounds 6 and 13, we experi-
activity (45.7% inhibition at 10 mM) but still the IC50 was > 10 mM.
enced a low reactivity of 4- and 2-carboxy-boronic acids in the
Suzuki coupling in the course of our trials, and thus we decided to
subject the intermediates 24e25, appropriately synthesized for the
purpose, to oxidation with KMnO4 and Na2HPO4 in CH3OH as
solvent for converting the aldehyde into the corresponding
carboxylic acid group. On the other hand, to obtain compound 14
we reduced the nitro group of compound 10 to the corresponding
amine employing SnCl2 in ethanol (EtOH) as solvent (Scheme 3).
Before submitting the synthesized compounds 2e16 to the bio-
logical assays, their purities (>95%) were verified by Agilent Tech-
nologies 1200 series high-performance liquid chromatography
(HPLC) with ultraviolet (UV) detection at 280 nm (method: Jupiter
Also the insertion of the bulky naphthyl sulfonamide in 40-position,
yielding compound 5, did not improve the potency. In contrast,
when the bulky, halogenated phenylether was inserted in 20-posi-
tion (compound 10), a significant improvement in the potency was
obtained, with an IC50 value ¼ 1.2
mM. Next, the influence of the 2-
nitro group within the 4-carboxybenzyl moiety on mPGES-1 inhi-
bition was investigated. Omission of the 2-nitro moiety (resulting
in compound 15) slightly improved mPGES-1 inhibition
(IC50 ¼ 1.0
moiety further increased the efficiency and the respective
compound 14 exhibited an IC50 value of 0.68 M, representing the
mM). Replacement of the 2-nitro function by an amino
m
most potent mPGES-1 inhibitor within this study. Exchange of the
methylene bridge next to the triazole of 15 by a sulfone moiety
(compound 16) again abolished the gain of potency.
C-18 column, 250 mm ꢂ 4.60 mm, 5
mm, 300 Å; 1.0 mL/min flow
rate; 5e100% in 30 min of 0.1% TFA/CH3CN-0.1% TFA/H2O).
It was shown in several studies that mPGES-1 inhibitors often
alsoacton relatedenzymes within the arachidonicacid cascade such
as COX or 5-LO enzymes, and it was proposed that dual inhibition of
mPGES-1 and 5-LO might be beneficial for therapeutic use, in
particular because of an expected lower risk of side effects [7,36].
Therefore, and based on the previous observation that compound 1
also efficiently inhibits 5-LO [8], we assessed the potential of the
above-mentioned compounds for inhibition of human 5-LO in well-
established cell-free and a cell-based models [8,34,37]. Interestingly,
inhibition of 5-LO in both the cell-free and the cell-based test
systems essentially correlated with the interference of the
compounds with mPGES-1. Thus, compounds 2e9,12 and 13 that all
failed or hardly inhibited mPGES-1 were also modest 5-LO inhibitors
2.3. Biological activity
In order to assess the ability of the test compounds to interfere
with mPGES-1 activity, a well-established cell-free assay [4,34] was
applied, where microsomes of interleukin-1
cells were used as source for human mPGES-1 enzyme and PGH2
(20 M) as substrate for mPGES-1. The mPGES-1 inhibitor MK-886
(IC50 ¼ 2.4 M) was used a reference compound [35] that inhibited
b-stimulated A549
m
m
the enzymatic reaction by 82 ꢃ 6.4% at a concentration of 10
mM
(not shown). The results of the assessment of the mPGES-1-
inhibitory activities and some SAR comments of compounds 1e16
are given in Table 1. For compound 1, carrying a biphenyl, an IC50
value of 3.2
m
M was obtained, which is in agreement with our
(IC50 > 10
analog 12 of compound 1 that suppressed 5-LO in the cell-free (but
M, respectively.
m
M), except the sulfonamide 5 and the 40-OH biphenyl
previous finding [8]. Bioisosteric replacement of the 4-phenyl
within the biphenyl of 1 by thiophene, yielding 11, was essen-
not in the intact cell) assay with IC50 ¼ 6.1 and 6.8
m
tially tolerated (IC50 ¼ 5.5
m
M). However, substitution of the 4-
On the other hand, the potent mPGES-1 inhibitors 10, 14 and 15
efficiently inhibited 5-LO activity with IC50 values in the range of
phenyl within the biphenyl by polar residues like 30- or 40-OH
(compounds 8 and 12, respectively), 40eNH2e (compound 4) or 20-,
30- or 40-COOH groups (compounds 6, 7 and 13, respectively) was
0.9e2
respectively. One exception was the sulfone 16, which was inactive
for mPGES-1 but a potent 5-LO inhibitor with IC50 ¼1.9 and 1.2 M in
mM in the cell-free and 2.0e5.3 mM in the cell-based assay,
clearly detrimental with IC50 values > 10
substituted analogs 6, 7, and 13 completely failed to significantly
inhibit mPGES-1 up to 10 M. Since we aimed to identify potent
mPGES-1 inhibitors with improved efficiency versus the lead
compound 1 (i.e., IC50 values < 3.2 M), higher concentrations than
10 M were not tested as they appeared not pharmacologically
m
M. In fact, the carboxy-
m
cell-free and cell-based systems, respectively. Obviously, the sulfone
or sulfonamide moieties (in compounds 16 and 5, respectively)
govern 5-LO inhibition but are detrimental for interference with
mPGES-1. Taken together, compound 14 turned out to be the most
potent dual mPGES-1/5-LO inhibitor out of these series with 4- and
7-fold lower IC50 values versus the parental lead compound 1.
To rationalize the inhibitory activity of 5, 10, 12 and 14e16 on 5-
LO by means of molecular docking, we used the crystal structure of
stable 5-LO recently reported by Newcomer and co-workers [38].
We considered the molecules as possible nonredox-type 5-LO
m
m
m
relevant and encouraging. Insertion of 30-morpholino residues
together with a methoxy moiety (compound 2) or bridged via
a keto group (compound 3) also caused strong loss of potency.
Substitution of the 4-phenyl moiety by three methoxy groups in 30-,
40-, and 50-position in compound 9 retained some inhibitory
HO
OH
O
O
H
N
B
B
O
O
OH
N
HOBt, DIC
TEA, DMF
Pinacol
B
O
EtOAc
O
O
O
O
HO
a
26
28
O
H3CO
O
OCH3
H3CO
H
H
N
H
NaCNBH4
AlCl3
CH3OH dry
O
B
Pinacol
EtOAc
N
O
O
B
O
B
O
O
OH
HO
l
27
29
Scheme 4. Synthetic protocol to generate pinacol esters a and l.

M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
317
Table 1
Inhibition of mPGES-1 and 5-LO, and SAR comments on mPGES-1 inhibitory activity of the tested compounds.
N
N
N
X
R
1
R2
HOOC
.
Comp. R1
X
R2
mPGES-1 (IC50
remaining activity at 10
[
m
M];
SAR comments on the mPGES-1
mM) inhibitory activity
5-LO (IC50
cell-free
[m
M]; remaining activity at 10
cell-based
mM)
1
2
3
4
NO2 CH2
NO2 CH2
NO2 CH2
NO2 CH2
3.2
Lead compound
6.7
9.2
OCH3
O
> 10 (87.4 ꢃ 9.1%)
> 10 (88.9 ꢃ 4.2%)
> 10 (65.5 ꢃ 5.1%)
30-morpholino residues together with
a methoxy moiety; potency lost
> 10 (84.9 ꢃ 11.1%)
> 10 (62.7 ꢃ 12.2%)
> 10 (63.5 ꢃ 11.3%)
> 10 (111.1 ꢃ 8.4%)
> 10 (125.0 ꢃ 12.0%)
> 10 (100.1 ꢃ 5.3%)
N
O
30-morpholino residues bridged via
keto group; potency lost
N
O
polar residues; potency lost
NH2
O
HN
5
6
NO2 CH2
> 10 (72.5 ꢃ 4.3%)
bulky naphthyl sulfonamide in
6.1
> 10 (96.5 ꢃ 7.3%)
S
O
40-position did not improve the potency
N
COOH
NO2 CH2
> 10 (94.1 ꢃ 8.5%)
polar residues; strong lost of potency
> 10 (75.3 ꢃ 13.1%)
> 10 (65.9 ꢃ 4.7%)
COOH
7
8
NO2 CH2
NO2 CH2
> 10 (82.1 ꢃ 2.5%)
> 10 (55.6 ꢃ 0.8%)
polar residues; strong lost of potency
polar residues, potency lost
> 10 (74.7 ꢃ 9.6%)
> 10 (80.2 ꢃ 2.7%)
> 10 (123.8 ꢃ 5.5%)
> 10 (113.9 ꢃ 10.1%)
OH
9
NO2 CH2
> 10 (54.3 ꢃ 1.5%)
methoxy groups in 30-, 40-, and
50 retained some inhibitory activity
> 10 (64.3 ꢃ 11.8%)
> 10 (90.2 ꢃ 9.3%)
10
NO2 CH2
1.2
bulky, halogenated phenylether is a
preferred substituent; improvement
of potency
2.0
5.3
S
11
12
NO2 CH2
NO2 CH2
5.5
thiophene ring; potency retained
polar residues; potency lost
> 10 (52.2 ꢃ 9.6%)
> 10 (89.7 ꢃ 2.8%)
> 10 (108.3 ꢃ 1.1%)
> 10 (74.2 ꢃ 1.8%)
6.8
OH
13
NO2 CH2
> 10 (93.0 ꢃ 21.2%)
polar residues; strong loss of potency
> 5 (62.7 ꢃ 12.7%) [5
m
M] > 5 (90.9 ꢃ 8.6%) [5
mM]
COOH
(continued on next page)

318
M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
Table 1 (continued )
Comp. R1
X
R2
mPGES-1 (IC50
remaining activity at 10
[
m
M];
SAR comments on the mPGES-1
M) inhibitory activity
5-LO (IC50
cell-free
[m
M]; remaining activity at 10
cell-based
mM)
m
F3C
Cl
14
15
16
NH2 CH2
O
0.68
bulky, halogenated phenylether together 0.9
with 2-amino group is very potent;
highly active. New lead compound
2.1
2.0
1.2
F3C
Cl
H
CH2
1.0
bulky, halogenated phenylether with
omission of the 2-nitro moiety;
1.47
O
slightly less active as compared to 14
F3C
Cl
H
SO2
> 10 (78.6 ꢃ 9.6%)
bulky, halogenated phenylether
together with sulfone bridge;
potency lost
1.96
O
inhibitors [39,40] since it does not show any features of a redox-
active agent or properties of an iron-ligand. Because the 5-LO was
crystallized without ligand [38], we referred to the model interac-
tions described by Wouters et al. [41], which was already success-
fully used by us [8] and other research groups [42,43], and to the
binding mode descriptions recently reported by Banoglu et al. [44]
for nonredox-type 5-LO inhibitors. In our models, in fact, all the
molecules interact with a binding site formed by Arg411, Ile406,
Phe177, Lys409, Tyr181, Leu607, Leu414, Leu420, Trp599, Asn425,
Gln363, Phe421, and Leu368 [41e44] however, the different size of
the second arms influence their binding mode on the protein
molecular surface. As depicted in Fig. 5, for all six potential inhib-
itors, the eCOOH group and the triazole ring of 2-nitro-4-
potency. In fact, in this small group of molecules, all the compounds
presenting a COOH group (6, 7 and 13) completely failed the mPGES-
1 inhibition. In contrast, compound 9, having a three methoxy
phenyl ring at R2 acts as a weak inhibitor. Furthermore, also the
triazole-based compounds showing at R2 a phenyl ring substituted
with bulky groups at meta and/or para position as 2, 3 and 5 are
significantly less potent with respect to the lead compound 1
underlining the negative effect of the steric hindrance in these
positions on the biological activity (Table 1). On the contrary, when
the phenyl ring at R2 is substituted with the bulky halogenated
phenylether (10), the activity increases with respect to the lead
compound 1. Moreover, the presence of the NO2 and/or NH2 may
modulate the activity, in fact both 14 and 15 are new potent mPGES-
1 inhibitors; on the contrarycompound 16, where the substitution of
the methylene bridge with the SO2 group causes a complete loss of
potency, suggesting the critical role of the alkylic linker on the bio-
logical activity. In conclusion, in Scheme 5 a tentative SAR profile of
these triazole-based compounds is reported.
carboxybenzyl form a hydrogen bond with Arg596 and a
p-cation
interaction with Lys243, respectively. On the other hand, the eNH2
group of 14 allows a further hydrogen bond with the carbonyl
oxygen of Gly419 and the carboxylic group in the side chain of
Asp422, while the oxygen of the halogenated arms of 10, 14e16 act
as H-bond acceptors with the NH 3 22 of Gln413, and moreover, they
are able to establish electrostatic interactions with Lys409 and
Lys173 by the chlorine atom. Furthermore, even though 5 shows
a different binding mode compared to the other compounds, it
establishes hydrophobic contacts with Arg411, Asn148, Cys159,
Glu417, Ile415, Lys158, and Trp147.
3. Conclusions
We have designed, synthesized, and performed biological evalu-
ation of a small set of potential anti-inflammatory triazole derivates
(2e16), obtained by modifying the scaffold of lead compound 1
previously reported by us. Our rational design had the aim to increase
the interaction efficiency with the ligand binding site, and it was
helpful for the comprehension of the key features of new mPGES-1
triazole-based compounds to direct the synthesis toward the most
promising mPGES-1 inhibitors. In particular, the different substitu-
tions on the arms at position 4 of the triazole ring caused a great
disparity in activityamong this class of compounds, where only 10,11,
14 and 15 exhibit significant mPGES-1 inhibitoryactivity determining
the critical features necessary for the optimal contact modes with
MGST-1 binding pocket. By mean of molecular docking, in fact,
a satisfactory explanation of the putative binding mode for these
triazole-based mPGES-1 inhibitors was provided. In conclusion, we
have proven the triazole ring with 2-nitro, and/or ammino-2, and/or
carboxybenzyl moiety at position 1 combined with two aromatic
All these interactions provide complexes with an increased
predicted stability that is fully compatible with the results of the
experimental biological assays, giving a good rationalization of the
5-LO inhibitory activity of 5, 10, 12, 14e16.
2.4. SAR profile for the designed molecules 2e16 on the mPGES-1
inhibitory activity
Starting from the lead compound 1, the simple substitution of the
phenyl group with the small aromatic ring as thiophene at R2
(Scheme 5) yielding 11, positively influences the inhibitory activity
on mPGES-1 (Table 1). On the other hand, the triazole compounds
with a phenyl ring at R2 substituted by small polar groups at ortho,
meta and para positions (4, 6e8 and 12e13) show a decrease of

M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
319
600 randomly placed individuals. The maximum number of energy
evaluations and of generations was set up to 5 ꢂ 106 and to 6 ꢂ 106
respectively. Results differing by less than 3.5 Å in positional root-
mean-square deviation (RMSD) were clustered together and rep-
resented by the result with the most favorable free energy of
binding. On the other hand, for the rationalization of the binding
mode of 5, 10, 12, 14e16 with 5LO (pdb code: 3O8Y) [38] we used
Autodock Vina software [45] choosing a grid box size of 30 ꢂ 28 ꢂ 28,
with spacing of 1.000 Å between the grid points, and centered at
ꢀ11.146 (x), 66.57 (y) and 0.523 (z) covering the active site of the
macromolecule. For the docking studies, we used an exhaustiveness
of 8 with maximum energy difference of 3 kcal/mol between the
best binding mode and the worst one displayed. Illustrations of the
3D models were generated using the Python software [46].
4.2. Synthesis of compounds 2e16
4.2.1. Methods and materials
All water and air sensitive reactions were carried out under an
inert atmosphere (N2) in oven- or flame-dried glassware. CH2Cl2,
CHCl3 and THF were distilled from CaH2 immediately prior to use.
Water was degassed under vacuum (10 mbar). All reagents were
used from commercial sources (SigmaeAldrich) without any further
purification. Microwave reactions were performed on a CEM Dis-
coverÒ single mode platform using 10 mL pressurized vials.
Reactions were monitored on silica gel 60 F254 (Merck) plates and
visualized with potassium permanganate or ninhydrin and under UV
Fig. 5. Three dimensional models of the 5 (gray), 10 (blue), 12 (light pink), 14 (light
blue), 15 (white), and 16 (cyan) with 5-LOX. The 5-LOX is represented by molecular
surface (A) (colored by atom type: C, gray; O, red; N, dark blue; and S, yellow) and
purple ribbon (B). The crucial aminoacids of 5-LOX are depicted by stick and balls and
CPK (colored by atom type: C, gray; O, red; N, dark blue; and S, yellow). (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
rings at position 4, and/or with an ortho-substituted biphenyl with
bulky, halogenated phenylether as new scaffold utilizable in the
rational design of innovative mPGES-1 inhibitors. Moreover, the
ability of the compounds to interfere with mPGES-1 significantly
correlates with inhibition of 5-LO; in this regard we were able to
rationalize the inhibitoryactivityof 5,10,12, and 14e16 on 5-LOat the
molecularlevel. As a major result, we present compound 14 as a novel
potent dual mPGES-1/5-LO inhibitor with IC50 values in the sub-
micromolar range. Whether or not these in-vitro properties of
compound 14 translate into anti-inflammatory activity in vivo is
a challenging question and currently under investigation along with
detailed analysis of its molecular pharmacology.
detection (
l
¼ 254 nm, 365 nm). Flash column chromatography was
performed using Merck 60/230e400 mesh silica gel. Analytical and
semi-preparative reverse-phase HPLC purifications were performed
on an Agilent Technologies 1200 series using Jupiter C-18 column
(250 ꢂ 4.60 mm, 5
m
m, 300 Å; 250 ꢂ 10.00 mm, 10
mm, 300 Å,
respectively). Purity grade of final products was determined on
a Agilent Technologies 1200 series HPLC using analytical reverse-
phase columns (Jupiter C-18, 250 ꢂ 4.60 mm, 5
mm, 300 Å).
Reaction yields refer to chromatographically and spectroscopi-
cally pure products. Proton-detected (1H, HMBC, HSQC) and carbon-
detected NMR spectra were recorded on Bruker instruments of
Avance series operating at 300 and 500 MHz and 75 and 125 MHz,
respectively. Chemical shifts are expressed in parts per million
4. Experimental section
(ppm) on the delta (d) scale. The solvent peak was used as internal
4.1. Computational details
reference: for 1H-NMRCDCl3 ¼7.26 ppm and CD3OD ¼ 3.34 ppm; for
13C NMR: CDCl3 ¼ 77.0 ppm and CD3OD ¼ 47.7 ppm. Multiplicities
are reported as follows: s, singlet; d, doublet; t, triplet; m, multiplet.
Mass spectra (MS) were recorded on a ThermoQuest GCeMS
equipped with a ZebronTM Z5-ms column (capillary in cage
column 30 m ꢂ 0.25 mm ꢂ 0.25 mm) using an electron impact ion
source (EIeMS).
We performed molecular docking calculations by Autodock4.2
software [17] on 4x AMD Opteron SixCore at 2.4 GHz. For all the
docking studies on MGST1 (pdb code: 2H8A) [18] a grid box size of
98 ꢂ 98 ꢂ 126 with spacing of 0.375 Å between the grid points, and
centered at ꢀ43.667 (x), 33.493 (y) and 2.656 (z) was used covering
the active site of the target. For all the docked structures, all bonds
were treated as active torsional bonds. To achieve a representative
conformational space during the docking studies and for taking into
account the variable number of active torsions, 10 calculations
consisting of 256 runs were performed, obtaining 2560 structures
for each ligand. The Lamarckian genetic algorithm (LGA) was
employed for docking calculations, choosing an initial population of
The purity of all tested compounds (>95%) were determined by
Agilent Technologies 1200 series high-performance liquid chro-
matography (HPLC) with UV detection at 280 nm (Method: Jupiter
C-18 column, 250 ꢂ 4.60 mm, 5
mm, 300 Å; 1.0 mL/min flow rate;
5e100% in 30 min of 0.1% TFA/CH3CN-0.1% TFA/H2O).
4.2.2. Esterification of boronic acids 26 and 27
The boronic acids 26 and 27 (0.667 mmol) were dissolved in 6 mL
of ethyl acetate and, stirring the solution, pinacol (0.667 mmol) was
added. After 4 h the reaction was stopped adding anhydrous Na2SO4
(1 g) and CaCl2 (1 g). The mixture was filtered and concentrated in
vacuo (Yields: 91% of 28 and 89% of 29).
methylene bridge essential
for the inhibitory activity
NO2 and /or NH2
not critical for
the inhibitory
activity
N
N
N
Aromatic ring
preferably substituted
with bulky halogenated
X
R1
phenylether at 2' position
4.2.3. Synthesis of morpholin-4-yl-[3-(4,4,5,5-tetramethyl-[1,3,2]
dioxa-borolan-2-yl)-phenyl]-methanone a
R2
HOOC
The pinacol ester 28 (1 equiv.) and morpholine (2 equiv.) were
dissolved in DMF. TEA, HOBt and DIC (2 equiv. of each one) were
Scheme 5. SAR summary profile for the designed compounds 2e16.

320
M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
added. The mixture was leaved at room temperature for 48 h under
stirring. When TLC showed the consumption of the pinacol ester 28,
the reactionwas stopped adding HCl 1 N (10 mL). The aqueous phase
was extracted with ethyl acetate (3 ꢂ 10 mL) and the organic phase
was washed firstly with a saturate solution of NaHCO3 and thenwith
brine. The organics were dried over Na2SO4, filtered and concen-
trated in vacuo. The crude was purified by flash chromatography
(10% diethyl ether/n-hexane to 50% diethyl ether/n-hexane). Yield:
under inert atmosphere of nitrogen. When TLC analysis indicated
complete consumption of the reactants, the reaction mixture was
diluted with 10 mL of water. The aqueous layer was extracted with
ethyl acetate (3 ꢂ 10 mL). The organics were dried over Na2SO4,
filtered and concentrated in vacuo. The crude was purified by flash
chromatography (10% diethyl ether/n-hexane to 80% diethyl ether/
n-hexane) and furnished 57% of pure desired triazole 21 as a white
powder.1H NMR
d (300 MHz; CD3OD): 8.48 (1H, s), 8.23e8.16 (4H,
85%: 1H-NMR
d
(300 MHz; CDCl3): 7.99 (1H, s), 7.83 (1H, d), 7.45 (1H,
m), 7.66 (2H, d), 7.53 (2H, d); HRMS calcd. for C15H11BrN3O4S:
d), 7.37 (1H, t), 3.85 (2H, t), 3.70 (4H, m), 3.52 (2H, t),1.34 (12H, s). ES-
[M þ H]þ 407.96 and 409.96 (1:1); found 407.8 and 409.8 (1:1).
MS calcd. for C17H25BNO4: [M þ H]þ 318.18; found 318.3.
4.2.9. Suzuki coupling to generate compounds 2e5; 7e12; 15e16
and 24e25: general procedure
4.2.4. Synthesis of 4-[2-methoxy-5-(4,4,5,5-tetramethyl-[1,3,2]
dioxaborolan-2-yl)-benzyl]-morpholine l
In a CEM DiscoverÒ vial, each of the intermediate 21e23 (1 equiv.),
the appropriate boronic acid ael (1.5 equiv.), Pd(dppf)Cl2 (0.05 equiv.)
Under inert atmosphere (N2), 1 equiv. of boronic ester 29 was
dissolved in anhydrous CH3OH (1 mL/0.18 mmol of ester). The
mixture was kept under stirring at room temperature; anhydrous
morpholin (4 equiv.), ZnCl2 (0.5 equiv.) and NaCNBH3 (1 equiv.) were
added. After 4 h, when the reagents disappeared, the reaction was
stopped and 10 mL of an aqueous solution of NaOH 0.1 M was added.
After concentration of CH3OH in vacuo, the aqueous phase was
extracted with ethyl acetate (3 ꢂ10 mL) and the organics were dried
overNa2SO4, filtered and concentrated invacuo. The obtainedoil was
purified on silica gel by flash chromatography (100% n-hexane to
and CsF (4 equiv.) were placed. Water (500 mL) and THF (500 mL) were
added under N2 atmosphere. The mixture was irradiated for
20e30 min, setting the power at 200 W, the temperature at 120 ꢁC,
the pressure at 250 psi and the Power Max ON. At completion of the
reaction, the vial was cooled to 50 ꢁC by gas jet cooling before it was
opened. After diluting with 10 mL of aqueous solution of HCl 1 N, the
aqueous layer was extracted with ethyl acetate (3 ꢂ 10 mL). The
organics were then dried over Na2SO4, filtered and concentrated in
vacuo. The crude was purified by flash chromatography (10% diethyl
ether/n-hexane to 40% diethyl ether/n-hexane).
50% ethyl acetate/n-hexane). Yield: 75%: 1H-NMR
d (300 MHz;
CDCl3): 7.98 (1H, d), 7.78 (1H, s), 7.24 (1H, d), 4.53 (2H, s), 4.10 (2H, t),
3.93 (3H, s), 3.76 (2H, t), 3.47 (4H, m),1.35 (12H, s); ESeMS calcd. for
C18H29BNO4: [M þ H]þ 334.21; found 334.1.
4.2.10. 4-[4-(40-methoxy-30-morpholin-4-ylmethyl-biphenyl-4-yl)-
[1,2,3]triazol-1-ylmethyl]-3-nitro-benzoic acid 2
Yield: 54%; 1H NMR
d (300 MHz; CD3OD): 8.79 (1H, s), 8.56 (1H,
4.2.5. Synthesis of triazoles 22e23 from halides 19e20: general
procedure
s), 8.35 (1H, d, J ¼ 7.89 Hz), 8.02 (2H, d, J ¼ 8.11 Hz), 7.88 (1H, d,
J ¼ 8.11 Hz), 7.79 (2H, d, J ¼ 7.45 Hz), 7.75 (1H, d, J ¼ 7.89 Hz), 7.33
(1H, d, J ¼ 8.11 Hz), 6.23 (1H, s), 4.12 (2H, t, J ¼ 7.31 Hz), 4.5 (2H, s),
4.01 (3H, s), 3.82 (2H, t, J ¼ 6.37 Hz), 3.45 (4H, m); ES-MS calcd. for
C28H28N5O6: [M þ H]þ 530.20; found 530.2.
The appropriate halide 19e20 (1.1 mmol), 1-bromo-4-
ethynylbenzene 18 (1.0 mmol) and sodium azide (1.3 mmol) were
suspended in a 1:1 mixture of water and t-BuOH (1.5 mL each) in
a 10 mL crimp-sealed thick-walled glass tube equipped with a small
magnetic stirring bar. Copper wire (0.80 mmol) and copper sulfate
4.2.11. 4-{4-[30-(Morpholine-4-carbonyl)-biphenyl-4-yl]-[1,2,3]
solution (1 N, 200
m
L) were added to the mixture. Then the mixture
triazol-1-ylmethyl}-3-nitro-benzoic acid 3
was irradiated for 30 min setting the power at 200 W, the
temperature at 120 ꢁC, the pressure at 250 psi and the Power Max
ON. After completion of the reaction, the vial was cooled to 50 ꢁC by
gas jet cooling before it was opened. The mixture was then diluted
with water (20 mL) and filtered. The residue was washed with cold
water (20 mL), 0.25 N HCl (20 mL) and finally with petroleum ether
(20 mL) to furnish the desired triazoles 22e23.
Yield: 56%; 1H NMR
d (300 MHz; CD3OD): 8.79 (1H, s), 8.58 (1H,
s), 8.35 (1H, d, J ¼ 7.89 Hz), 8.02 (2H, d, J ¼ 8.11 Hz), 7.88 (1H, d,
J ¼ 8.11 Hz), 7.79 (2H, d, J ¼ 7.89 Hz), 7.75 (1H, s), 7.65 (1H, t,
J ¼ 6.37 Hz), 7.45 (1H, d, J ¼ 8.33 Hz), 7.33 (1H, d, J ¼ 8.33 Hz), 6.23
(2H, s), 4.16 (2H, t, J ¼ 6.37 Hz), 3.88 (2H, t, J ¼ 6.37 Hz), 3.48 (4H,
m); 13C NMR
d (75 MHz; CD3OD): 170.1, 165.4, 147.1, 146.2, 140.5,
139.5, 135.6, 134.4, 133.5, 131.6, 130.9, 130.2, 129.9, 128.5, 127.6,
126.8, 126.3, 125.9, 124.3, 121.8, 67.1, 66.2, 50.8, 49.1, 44.4. ES-MS
calcd. for C27H24N5O6: [M þ H]þ 514.16; found 514.2.
4.2.6. 4-[4-(4-Bromo-phenyl)-[1,2,3]triazol-1-ylmethyl]-benzoic
acid 22
Yield: 76%; 1H NMR
d
(300 MHz; CD3OD): 8.07 (2H, d), 7.65 (1H,
4.2.12. 4-[4-(40-amino-biphenyl-4-yl)-[1,2,3]triazol-1-ylmethyl]-3-
s), 7.58 (2H, d), 7.45 (2H, d), 7.42 (2H, d), 5.23 (2H, s); HRMS calcd.
for C16H13BrN3O2: [M þ H]þ 358.01 and 360.01 (1:1); found and
358.2 and 360.2 (1:1).
nitro-benzoic acid 4
Yield: 49%;1HNMR
d (300 MHz;CD3OD):8.77 (1H, s), 8.53 (1H, s),
8.26 (1H, d, J ¼ 8.11 Hz), 7.88 (2H, d, J ¼ 7.67 Hz), 7.70 (2H, d,
J ¼ 8.33 Hz), 7.49 (2H, d, J ¼ 7.33 Hz), 7.24 (1H, d, J ¼ 8.11 Hz), 6.81 (2H,
4.2.7. 4-[4-(4-Bromo-phenyl)-[1,2,3]triazol-1-ylmethyl]-3-nitro-
d, J ¼ 8.11 Hz), 6.22 (2H, s); 13C NMR
d (75 MHz; CD3OD): 165.6,147.8,
benzoic acid 23
146.8,139.6,137.9,136.9,134.6,131.9,131.5,130.3,130.0,127.5,126.5,
126.1, 126.1, 122.0, 116.6, 50.9. ESeMS calcd. for C22H18N5O4:
[M þ H]þ 416.13; found 416.1.
Yield: 73%; 1H NMR
d (300 MHz; CD3OD): 8.72 (1H, s), 8.35 (1H,
s), 8.24 (1H, d), 7.70 (2H, d), 7.56 (2H, d), 7.18 (1H, d), 6.08 (2H, s);
HRMS calcd. for C16H12BrN4O4: [M þ H]þ 403.00 and 404.99 (1:1);
found 403.3 and 405.3 (1:1).
4.2.13. 4-{4-[40-(6-dimethylamino-naphthalene-2-sulfonylamino)-
biphenyl-4-yl]-[1,2,3]triazol-1-ylmethyl}-3-nitro-benzoic acid 5
4.2.8. Synthesis of 4-[4-(4-bromo-phenyl)-[1,2,3]triazole-1-
sulfonyl]-benzoic acid 21
1-Bromo-4-ethynylbenzene 18 (0.60 mmol) and 4-
carboxybenzene-sulfonazide 17 (0.50 mmol) were dissolved in
1.00 mL of dry chloroform. 2,6-lutidine (0.60 mmol) and CuI
(0.05 mmol) were added and the solution was stirred for 12 h at 0 ꢁC
Yield: 51%; 1H NMR
d (300 MHz; CD3OD): 8.77 (1H, s), 8.55 (1H,
s), 8.24 (2H, d, J ¼ 7.67 Hz), 7.89 (1H, d, J ¼ 7.67 Hz), 7.71 (2H, d,
J ¼ 7.89 Hz), 7.55 (2H, d, J ¼ 7.67 Hz), 7.50e7.46 (3H, m), 7.32 (2H, d,
J ¼ 7.67 Hz), 7.13e7.09 (8H, m), 6.95 (1H, d, J ¼ 8.11 Hz), 6.85 (1H, d,
J ¼ 8.11 Hz), 6.19 (2H, s); 13C NMR
d (75 MHz; CD3OD): 167.4, 160.7,
153.5, 149.8, 148.4, 142.6, 141.2, 140.5, 139.1, 138.9, 136.8, 133.8,

M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
321
132.4, 132.2, 132.0, 131.1, 130.0, 129.2, 129.0, 128.1, 126.0, 125.8,
4.2.20. 4-{4-[20-(2-chloro-5-trifluoromethyl-phenoxymethyl)-
biphenyl-4-yl]-[1,2,3]triazol-1-ylmethyl}-benzoic acid 15
121.0, 119.0, 117.7, 112.8, 111.1, 52.5, 47.3. ESeMS calcd. for
C34H29N6O6S: [M þ H]þ 649.18; found 649.1.
Yield: 65%; 1H-NMR
d (300 MHz; CD3OD): 8.41 (1H, s), 8.18 (2H,
d, J ¼ 7.67 Hz), 7.86 (2H, d, J ¼ 7.89 Hz), 7.62 (1H, d, J ¼ 7.67 Hz),
4.2.14. 40-[1-(4-carboxy-2-nitro-benzyl)-1H-[1,2,3]triazol-4-yl]-
7.54e7.42 (8H, m), 7.18 (1H, d, J ¼ 8.77 Hz), 7.07 (1H, s), 5.73 (2H,
s), 5.11 (2H, s); 13C NMR
d (75 MHz; CD3OD): 167.1, 154.4, 146.9,
biphenyl-3-carboxylic acid 7
Yield: 45%; 1H NMR
d (500 MHz; CD3OD): 8.77 (1H, s), 8.63 (1H, s),
142.2, 141.3, 139.9, 133.7, 131.5, 130.8, 130.4, 130.1, 128.4, 126.0,
125.8, 125.6, 122.6, 119.0, 118.9, 112.3, 111.7, 111.3, 110.0, 108.6,
105.8, 69.8, 53.2. ESeMS calcd. for C30H22ClF3N3O3: [M þ H]þ
564.12; found 564.2.
8.38(1H, s), 8.37(1H,d,J¼ 8.11Hz),8.08(1H,d,J¼ 7.89Hz),8.04(1H,d,
J ¼ 7.89Hz), 8.09(2H, d, J ¼ 8.11 Hz), 7.89 (2H, d, J ¼ 8.11 Hz), 7.70 (1H, t,
J ¼ 7.67 Hz), 7.35 (1H, d, J ¼ 8.11 Hz), 6.22 (2H, s); 13C NMR
d (125 MHz;
CD3OD): 167.3, 165.4, 147.2, 146.4, 139.9, 138.9, 134.0, 133.5, 132.8,
132.3, 130.0, 129.6, 128.8, 128.2, 127.9, 127.4, 126.5, 125.8, 125.4, 121.8,
50.4. ESeMS calcd. for C23H17N4O6: [M þ H]þ 445.11; found 445.1.
4.2.21. 4-{4-[20-(2-chloro-5-trifluoromethyl-phenoxymethyl)-
biphenyl-4-yl]-[1,2,3]triazole-1-sulfonyl}-benzoic acid 16
Yield: 63%; 1H-NMR
d (300 MHz; CD3OD): 8.18 (1H, s), 7.86 (2H,
4.2.15. 4-[4-(30-hydroxy-biphenyl-4-yl)-[1,2,3]triazol-1-ylmethyl]-
d, J ¼ 8.11 Hz), 7.63 (1H, d, J ¼ 7.67 Hz), 7.55 (2H, d, J ¼ 8.11 Hz), 7.45
(1H, t, J ¼ 5.92 Hz), 7.43e7.36 (7H, m), 7.18 (1H, d, J ¼ 8.11 Hz), 7.09
3-nitro-benzoic acid 8
(1H, s), 5.12 (2H, s). 13C NMR
d (75 MHz; CD3OD): 167.1, 159.9, 143.6,
Yield: 42%; 1H NMR
d (300 MHz; CD3OD): 8.76 (1H, s), 8.23 (1H,
142.2, 140.0, 135.9, 135.1, 131.3, 131.1, 133.2, 130.9, 130.6, 130.1, 129.8,
129.7, 128.5, 127.5, 126.8, 125.5, 124.5, 123.1, 118.1, 113.2, 108.3, 69.3.
ESeMS calcd. for C29H20ClF3N3O5S: [M þ H]þ 614.07; found 614.1.
d, J ¼ 7.89 Hz) 8.12 (1H, s), 7.83 (2H, d, J ¼ 8.11 Hz), 7.62 (2H, d,
J ¼ 7.89 Hz), 7.41 (1H, s), 7.23 (1H, t, J ¼ 7.67 Hz), 6.77e6.72 (3H, m),
6.05 (2H, s); 13C NMR
d (75 MHz; CD3OD): 165.1, 157.5, 147.3, 146.1,
140.3, 139.2, 134.5, 132.1, 130.9, 130.2, 128.4, 128.0, 126.1, 125.9,
125.2, 121.7, 118.3, 116.2, 115.5, 50.8. ESeMS calcd. for C22H17N4O5:
[M þ H]þ 417.11; found 417.1.
4.2.22. Reduction of nitro-derivative 10 to amine 14: general
procedure
A solution of compound 10 and stannous chloride dihydrate
(5 equiv.) in ethanol was stirred at 80 ꢁC for about 30 min. The
solvent was removed in vacuo. Ethyl-acetate (10 mL) was added to
the residue and washed with saturated sodium bicarbonate
(2 ꢂ 10 mL). The organic layer was dried over Na2SO4 and
concentrated. The obtained compound 14 was directly purified on
4.2.16. 4-[4-(30,40,50-trimethoxy-biphenyl-4-yl)-[1,2,3]triazol-1-
ylmethyl]-3-nitro-benzoic acid 9
Yield: 71%; 1H NMR
d (300 MHz; CD3OD): 8.79 (1H, s), 8.56 (1H,
s), 8.34 (1H, d, J ¼ 7.45 Hz), 8.00 (2H, d, J ¼ 8.33 Hz), 7.81 (2H, d,
J ¼ 8.55 Hz), 7.31 (1H, d, J ¼ 8.11 Hz), 7.01 (2H, s), 6.20 (2H, s), 4.01
semi-preparative HPLC (yield: 83%). 1H-NMR
d (300 MHz; CD3OD):
(6H, s), 3.80 (3H, s); 13C NMR
d (75 MHz; CD3OD): 165.4,153.7,147.9,
7.68 (1H, s), 7.18 (2H, d, J ¼ 8.33 Hz), 6.97 (1H, d, J ¼ 7.67 Hz),
6.89e6.78 (6H, m), 6.72 (2H, d, J ¼ 7.89 Hz), 6.54e6.51 (2H, m), 6.42
(1H, s), 4.96 (2H, s), 4.45 (2H, s). ESeMS calcd. for C30H23ClF3N4O3:
[M þ H]þ 579.13; found 579.0.
146.1, 141.1, 137.7, 136.6, 134.5, 131.1, 130.3, 129.3, 129.0, 127.6, 126.3,
125.9, 122.2, 104.2, 60.0, 55.5, 50.6. ESeMS calcd. for C25H23N4O7:
[M þ H]þ 491.15; found 491.2.
4.2.17. 4-{4-[20-(2-chloro-5-trifluoromethyl-phenoxymethyl)-
biphenyl-4-yl]-[1,2,3]triazol-1-ylmethyl}-3-nitro-benzoic acid 10
4.2.23. Oxydation of aldehyde-group to carboxylic acid: general
procedure
Yield: 62%; 1H NMR
d (300 MHz; CD3OD): 8.78 (1H, s), 8.48 (1H, s),
The advanced intermediates 24 and 25 (1 equiv.) were dis-
solved in CH3OH (1 mL) and 0.5 mL of a solution 0.5 M of
Na2HPO4 in water was added. Finally 0.5 mL of a solution 0.5 M of
KMnO4 in water was slowly added dropwise over a period of 1 h.
After 1.5 h, the reaction mixture was quenched with 20 mL of HCl
8.29 (1H, d, J ¼ 8.33 Hz), 7.90 (2H, d, J ¼ 8.11 Hz), 7.65 (1H, d,
J ¼ 7.67 Hz), 7.50 (2H, d, J ¼ 7.89 Hz), 7.45e7.37 (6H, m), 7.18 (1H, d,
J ¼ 7.89 Hz), 6.18 (2H, s), 5.15 (2H, s); 13C NMR
d (75 MHz; CD3OD):
165.7, 154.4, 148.1, 145.8, 142.2, 140.0, 135.9, 135.1, 133.8, 133.2, 131.5,
131.2, 130.8, 130.5, 130.1, 130.0, 129.9, 129.6, 128.5, 126.2, 125.7, 123.2,
119.0, 118.9, 111.3, 110.1, 69.8, 50.8. ESeMS calcd. for C30H21ClF3N4O5:
[M þ H]þ 609.11; found 609.1.
1
M. The aqueous layer was extracted with ethyl acetate
(3 ꢂ 30 mL) and the organics were dried with Na2SO4, filtered
and concentrated in vacuo.
4.2.24. 40-[1-(4-carboxy-2-nitro-benzyl)-1H-[1,2,3]triazol-4-yl]-
4.2.18. 3-Nitro-4-[4-(4-thiophen-2-yl-phenyl)-[1,2,3]triazol-1-
ylmethyl]-benzoic acid 11
biphenyl-2-carboxylic acid 6
Yield: 54%; 1H NMR
d (300 MHz; CD3OD): 8.75 (1H, s), 8.55 (1H,
Yield: 89%; 1H-NMR
d (300 MHz; CD3OD): 8.08 (1H, s), 7.82 (1H,
s), 8.34 (1H, d, J ¼ 8.33 Hz), 7.92 (2H, d, J ¼ 8.11 Hz), 7.75 (2H, d,
J ¼ 7.89 Hz), 7.51 (1H, d, J ¼ 8.11 Hz), 7.45 (1H, d, J ¼ 7.89 Hz), 7.27
(1H, d, J ¼ 8.11 Hz), 7.12 (1H, t, J ¼ 7.31 Hz), 6.18 (2H, s); 13C NMR
s), 7.64 (1H, d, J ¼ 7.89 Hz), 7.23 (2H, d, J ¼ 8.11 Hz), 7.17 (1H, d,
J ¼ 7.45 Hz), 6.92 (1H, t, J ¼ 6.79 Hz), 6.83e6.76 (4H, m), 6.58 (1H, d,
J ¼ 7.45 Hz), 5.50 (2H, s). 13C NMR
d (75 MHz; CD3OD): 169.1, 165.5,
d
(75 MHz; CD3OD): 165.3, 147.9, 146.1, 141.1, 135.6, 134.3, 133.9,
147.2, 146.0, 140.6, 139.8, 138.8, 133.2, 132.5, 132.1, 130.3, 130.1,
128.5, 128.1, 126.9, 126.7, 125.8, 125.4, 121.9, 50.8. ESeMS calcd. for
C23H17N4O6: [M þ H]þ 445.11; found 445.1.
132.2, 130.4, 130.1, 129.1, 128.8, 126.6, 124.9, 124.2, 121.9, 120.2, 50.7.
ESeMS calcd. for C20H15N4O4S: [M þ H]þ 407.07; found 407.0.
4.2.19. 4-[4-(40-hydroxy-biphenyl-4-yl)-[1,2,3]triazol-1-ylmethyl]-
4.2.25. 40-[1-(4-carboxy-2-nitro-benzyl)-1H-[1,2,3]triazol-4-yl]-
3-nitro-benzoic acid 12
biphenyl-4-carboxylic acid 13
Yield: 47%; 1H NMR
d (300 MHz; CD3OD): 8.75 (1H, s), 8.53 (1H,
Yield: 86%; 1H-NMR
d (300 MHz; CD3OD): 8.08 (1H, s), 7.86 (1H,
s), 8.25 (1H, d, J ¼ 7.67 Hz), 7.89 (2H, d, J ¼ 8.11 Hz), 7.71 (2H, d,
s), 7.64 (1H, d, J ¼ 7.67 Hz), 7.48 (2H, d, J ¼ 8.11 Hz), 7.33 (2H, d,
J ¼ 8.11 Hz), 7.49 (2H, d, J ¼ 8.11 Hz), 7.24 (1H, d, J ¼ 8.11 Hz), 6.81
J ¼ 8.33 Hz), 7.17 (1H, d, J ¼ 8.11 Hz), 6.95 (2H, d, J ¼ 8.11 Hz), 6.59
(2H, d, J ¼ 8.11 Hz), 6.19 (2H, s); 13C NMR
d (75 MHz; CD3OD): 165.0,
(2H, d, J ¼ 8.11 Hz), 5.50 (2H, s). 13C NMR
d (75 MHz; CD3OD): 167.1,
157.3, 147.9, 166.0, 141.3, 135.0, 134.5, 131.7, 131.1, 130.2, 129.6, 128.2,
126.6, 126.0, 125.9, 121.9, 115.5, 50.7. ESeMS calcd. for C22H17N4O5:
[M þ H]þ 417.11; found 417.1.
165.5, 147.2, 146.1, 140.5, 139.9, 138.9, 134.1, 133.8, 132.6, 130.2,
130.0, 128.2, 128.0, 126.9, 125.9, 121.9, 50.7. ESeMS calcd. for
C23H17N4O6: [M þ H]þ 445.11; found 445.0.

322
M.G. Chini et al. / European Journal of Medicinal Chemistry 54 (2012) 311e323
4.3. Bioactivity assays
pre-incubated with the test compounds as indicated. After 10 min
at 4 ꢁC, samples were pre-warmed for 30 s at 37 ꢁC, and 2 mM CaCl2
plus 20 M AA was added to start 5-LO product formation. The
4.3.1. Assay systems and materials
m
Dulbecco’sModifiedEagleMedium(DMEM)/highglucose(4.5g/L)
medium, penicillin, streptomycin, trypsin/ethylenediaminetetraace-
tate (EDTA) solution, and LSM 1077 lymphocyte separation medium
reaction was stopped after 10 min at 37 ꢁC by addition of 1 mL ice-
cold methanol, and the formed metabolites were analyzed by
RP-HPLC as described [48]. 5-LO products include the all-trans
isomers of LTB4 and 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosa-
tetraenoic acid.
were obtained from PAA (Pasching, Austria). IL-1b was obtained from
ReproTech (Hamburg, Germany). Fetal calf serum (FCS), phenyl-
methylsulfonylfluoride (PMSF), leupeptin, soybean trypsin inhibitor
(STI), glutathione (reduced), PGB1, lysozyme, Ca2þ-ionophore A23187,
and arachidonic acid were obtained from SigmaeAldrich (Deisen-
4.3.6. Isolation of neutrophils and determination of 5-LO activity in
neutrophils
hofen, Germany). MK886 and 11
Chemical (Ann Arbor, MI). PGH2, adenosine triphosphate (ATP), iso-
propyl- -1-thiogalactopyranoside (IPTG), and dextrane were
obtained from Larodan (Malmoe, Sweden), Roche Diagnostics
(Mannheim, Germany), AppliChem (Darmstadt, Germany), and Fluka
(Neu-Ulm, Germany), respectively. A549 cells were provided by the
Karolinska Institute (Stockholm, Sweden). Leukocyte concentrates
from human healthy volunteers were provided by Institute of
Transfusion Medicine, University Hospital Jena, Germany.
b
-PGE2 were obtained from Cayman
Neutrophils were freshly isolated from leukocyte concentrates
obtained at the Institute of Transfusion Medicine, University
Hospital Jena, Germany, as described [49]. In brief, venous blood
was taken from healthy adult donors that did not take any medi-
cation for at least 7 days, and leukocyte concentrates were prepared
by centrifugation (4000 ꢂ g, 20 min, 20 ꢁC). Neutrophils were
immediately isolated from the pellet after centrifugation on
Nycoprep cushions, and hypotonic lysis of erythrocytes was per-
formed. Neutrophils were finally resuspended in PBS pH 7.4 (PBS)
containing 1 mg/mL glucose and 1 mM CaCl2 (PGC buffer)
(purity > 96e97%).
b-D
4.3.2. Cell culture
A549 cells were grown in DMEM/high glucose (4.5 g/mL)
medium supplemented with heat-inactivated FCS (10%, v/v),
Freshly isolated neutrophils (1 ꢂ 107/mL PGC buffer) were pre-
incubated with the test compounds for 15 min at 37 ꢁC and 5-LO
penicillin (100 U/mL) and streptomycin (100
m
g/mL). After three
product formation was started by addition of 2.5
A23187 plus 20
M AA. After 10 min at 37 ꢁC, the reaction was
stopped with 1 mL of methanol and 30 l of 1 N HCl, and then,
200 ng PGB1 and 500 l PBS were added. Formed 5-LO metabolites
mM ionophore
days, confluent cells were detached using 1x trypsin/EDTA and
m
reseeded with a density of 1 ꢂ 105 cells/ml medium.
m
m
4.3.3. Preparation of crude mPGES-1 in microsomes of A549 cells
and determination of mPGE2 synthase activity
Preparation of A549 cells and determination of mPGES-1
activity was performed as described previously [34]. In brief,
were extracted and analyzed by HPLC as described [8]. 5-LO
products include LTB4 and its all-trans isomers, and 5(S)-hydro(-
pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid. Cysteinyl-LTs C4,
D4, and E4 were not detected, and oxidation products of LTB4 were
not determined.
A549 cells were treated with 1 ng/mL interleukin-1b for 48 h at
37 ꢁC and 5% CO2. After sonification, the homogenate was subjected
to differential centrifugation at 10,000 ꢂ g for 10 min and
174.000 ꢂ g for 1 h at 4 ꢁC. The pellet (microsomal fraction) was
resuspended in 1 mL homogenization buffer (0.1 M potassium
phosphate buffer pH 7.4, 1 mM phenylmethanesulphonyl fluoride,
4.3.7. Statistics
Data are expressed as mean ꢃ SE. IC50 values were graphically
calculated from measurements at 4-5 different concentrations of
the compounds using SigmaPlot 9.0 (Systat Software Inc., San Jose,
USA). The program Graphpad Instat (Graphpad Software Inc., San
Diego, CA) was used for statistical comparisons. Statistical evalua-
tion of the data was performed by one-way ANOVAs for indepen-
dent or correlated samples followed by Tukey HSD post-hoc tests.
Where appropriate, Student’s t test for paired and correlated
samples was applied. A P value of <0.05 was considered significant.
60
mg/mL soybean trypsin inhibitor, 1 mg/mL leupeptin, 2.5 mM
glutathione, and 250 mM sucrose), and the total protein concen-
tration was determined. Microsomal membranes were diluted in
potassium phosphate buffer (0.1 M, pH 7.4) containing 2.5 mM
glutathione. Test compounds or vehicle were added, and after
15 min at 4 ꢁC, the reaction (100
addition of PGH2 (20
wise). After 1 min at 4 ꢁC, the reaction was terminated using stop
solution (100 l; 40 mM FeCl2, 80 mM citric acid, and 10 M of 11b-
ml total volume) was initiated by
mM, final concentration, unless stated other-
Acknowledgments
m
m
PGE2 as internal standard). PGE2 was separated by solid phase
extraction and analyzed by RP-HPLC as described [34].
Financial support by the University of Salerno and by Ministero
dell’Istruzione, dell’Università e della Ricerca (MIUR), PRIN 2009
“Design, conformational and configurational analysis of novel
molecular platforms”. Both the institutions are gratefully
acknowledged.
4.3.4. Expression and purification of human 5-LO from Escherichia
coli (E. coli)
E.coli MV1190 was transformed with pT3-5-LO plasmid, and
recombinant 5-LO protein was expressed at 27 ꢁC as described [47].
Cells were lysed in 50 mM triethanolamine/HCl pH 8.0, 5 mM EDTA,
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