Synthesis and evaluation of new benzimidazole-based COX inhibitors: A naproxen-like interaction detected by STD-NMR

Article abstract of DOI:10.1039/c5ra04984a

Non-steroidal anti-inflammatory drugs exert their pharmacological activity through inhibition of cyclooxygenase 1 and 2 (COX-1 and COX-2). Recent research suggests that a balanced inhibition of both COX-1 and COX-2 is the key to reduce the side-effects ex

Full text of DOI:10.1039/c5ra04984a

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Synthesis and evaluation of new benzimidazole-
based COX inhibitors: a naproxen-like interaction
detected by STD-NMR†
Cite this: RSC Adv., 2015, 5, 49098
Luısa C. R. Carvalho,a Daniela Ribeiro,b Raquel S. G. R. Seixas,c Artur M. S. Silva,c
´
a
a
d
b
*
Mariana Nave, Ana C. Martins, Stefan Erhardt, Eduarda Fernandes,
e
a
*
*
Eurico J. Cabrita and M. Manuel B. Marques
Non-steroidal anti-inflammatory drugs exert their pharmacological activity through inhibition of
cyclooxygenase 1 and 2 (COX-1 and COX-2). Recent research suggests that a balanced inhibition of both
COX-1 and COX-2 is the key to reduce the side-effects exhibited by COX inhibitors. We developed new
benzimidazole-based compounds that showed a balanced COX inhibition, supported by molecular
docking screening. The human whole blood assays demonstrated that the ester derivatives were potent
inhibitors. Competitive saturation transfer difference (STD)-NMR experiments, in the presence of COX-2,
using naproxen and diclofenac demonstrated that ester derivatives do not compete with diclofenac for
the same binding site, but compete with the allosteric inhibitor naproxen. Combination of NMR
spectroscopy with molecular docking has permitted us to detect a new naproxen-like inhibitor, which
could be used for future drug development.
Received 30th March 2015
Accepted 26th May 2015
DOI: 10.1039/c5ra04984a
www.rsc.org/advances
constitutively expressed in some tissues playing important phys-
iological roles in the brain, kidney or cardiovascular system.3,4
Introduction
The unravelling of COX structure, and the main differences
between COX-1 and COX-2 lead to the development of potent
and highly selective COX-2 inhibitors.5 When compound DuP
697 was reported as the rst selective COX-2 inhibitor in 1990, it
was rapidly perceived that its shape, composed of a 1,2-diaryl
heterocycle template, was an important feature to accommo-
date the drug inside the COX-2 active site.6 This observation
formed the basis of the early work in the eld of selective COX-2
inhibitors (usually known as coxibs). In the late 90s, several
coxibs were released to the market, such as celecoxib, rofecoxib,
valdecoxib and etoricoxib (Fig. 1).7 Coxibs selectively inhibit
COX-2 and hamper the benecial vascular effects of prostacy-
clin (PGI2) without blocking thromboxane A2 (TXA2) formation
produced by COX-1. The loss of the antiplatelet and vasodilatory
effects of PGI2, causes a relative excess of TXA2, promoting
vasoconstriction, platelet aggregation and thrombosis.8 Conse-
quently, some coxibs demonstrated acute cardiovascular side
effects, and some of these drugs, like rofecoxib and valdecoxib,
were removed from the market.9
Non-steroidal anti-inammatory drugs (NSAIDs), such as
ibuprofen, naproxen or diclofenac (Fig. 1),1,2 inhibit cyclo-
oxygenase (COX), an enzyme involved in the inammatory
process, which exists in two isoforms, COX-1 and COX-2.3 This
inhibitory activity is also responsible for the deleterious effects of
NSAIDS that are mainly due to their nonselective character, i.e.
the inhibition of both isoforms.4 COX-1 is constitutively expressed
in all tissues and is involved in the production of prostaglandins
that mediate basic housekeeping functions in the body. On the
other hand, COX-2 is usually recognized as an inducible enzyme,
being expressed in response to inammation. COX-2 is also
a
´
ˆ
LAQV@REQUIMTE, Departamento de Quımica, Faculdade de Ciencias e Tecnologia,
Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal.
E-mail: mmbmarques@fct.unl.pt
b
ˆ
´
´
´
UCIBIO@REQUIMTE, Departamento de Ciencias Quımicas, Laboratorio de Quımica
´
Aplicada, Faculdade de Farmacia, Universidade do Porto, Rua de Jorge Viterbo
Ferreira no. 228, 4050-313 Porto, Portugal
Several studies indicate that prolonged NSAIDs use is also
associated with a small increase in cardiovascular risk and that
myocardial infarctions are comparable between coxibs and the
traditional NSAIDs ibuprofen and diclofenac.10 Surprisingly,
naproxen was not associated with an increase in cardiovascular
events. The divergence between naproxen and other COX inhib-
itors (selective and nonselective) is not clear but may be related to
its longer half-life compared to ibuprofen or diclofenac.11
c
´
QOPNA & Departamento de Quımica, Universidade de Aveiro, 3810-193 Aveiro,
Portugal
dSchool of Life, Sport and Social Sciences, Edinburgh Napier University, Sighthill
Campus, Edinburgh EH11 4BN, UK
eUCIBIO@REQUIMTE, Departamento de Quımica, Faculdade de Ciencias
Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica,
´
ˆ
e
Portugal
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra04984a
49098 | RSC Adv., 2015, 5, 49098–49109
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Fig. 1 Known COXs inhibitors.
Prolonged inhibition of COX-1 would prevent the formation of
The design of new anti-inammatory drugs requires a deep
the prothrombotic TXA2, which might counterbalance the effect understanding of the COX regulation and Ecat/Eallo interplay and
of inhibition of COX-2 that is involved in PGI2 biosynthesis. the inuence of structural features on COX inhibition. The
Therefore and in view of these ndings, some groups support primary source of information towards the rational drug design
that the key to regulate thrombotic events can rely on a balanced of novel COXs inhibitors rely on X-ray crystallography, molec-
inhibition of both COX-1 and COX-2 isoenzymes to ensure a ular modeling and kinetic studies, which are the most used
prostacyclin/thromboxane balance in the body.8a
methods to study the mode of action of COXs inhibitors.
Following our previous studies on COXs inhibition,12 we Recently, we reported an STD-NMR study of known NSAIDs,
aimed to develop a new class of inhibitors that present a such as ibuprofen, diclofenac and ketorolac, to characterize
balanced COX-1/COX-2 inhibition in order to control the severe their binding to both COX-1 and COX-2. The encouraging
side-effects resulting from selective COX inhibition.
results proved that STD-NMR can be a powerful technique to
We decided to investigate the behavior of structures pos- embrace this challenging quest.
sessing both a coxib's shape and an acetic acid chain, which is
To establish the structural determinants for effective
usually associated with non-selective inhibitors. With these interaction with COXs, SAR and molecular docking studies
hybrid structures we aimed to achieve a balanced COX-1/COX- were conducted. The data collected revealed that the target
2 inhibition and elucidate COX-ligand molecular interaction. molecule should contain an arylsulfonamide on a diary-
So far, structural modications of NSAIDs have been per- lheterocyclic scaffold and the presence of a carboxylic moiety.
formed to improve their safety prole, some of these modi- In order to design a library of potent compounds, the best
cations include derivatization of the carboxylate function of results were obtained for the benzimidazole core possessing
NSAIDs.13
two aromatic units at N1 and C2 positions. A heterocyclic core,
Intensive studies have been developed in order to disclose larger than the known coxibs, was chosen on the expectation
COX inhibition mechanism and regulation.14 Despite the efforts that this could be relevant for a balanced COX inhibition. The
on the design of new COX inhibitors and X-ray crystallographic presence of halogen atoms and CF3 group were considered at
studies developed so far, the structural requirements for COX N1-aryl moiety. Additionally, a methylsulfone and a sulfon-
inhibition and selectivity are still not fully understood.
amide bound to the aromatic ring at C2 were also evaluated.
Mechanistically, COXs inhibitors have been classied The existence of an acetic acid chain was investigated as an
according to two major categories: time-dependent and time- important anchor for key interactions with COX active site
independent/rapidly reversible inhibitors.14a
residues. The best results were obtained for a small benz-
Recently, it was proposed that COX-2 acts as a conforma- imidazole library that demonstrated an orientation in COX
tional heterodimer that possesses a catalytic (Ecat) and an allo- active site similar to SC-558 (Fig. 1). Notwithstanding prom-
steric monomer (Eallo).15 Eallo can allosterically inuence the ising results,16 the benzimidazole scaffold has been scarcely
catalytic efficiency of the partner Ecat subunit, i.e. Ecat is regu- explored as COX inhibitor.
lated by Eallo in a manner-related way the ligand is bound to
Eallo. It was also proposed the existence of time-independent STD-NMR experiments of novel benzimidazole derivatives and
inhibitors, such as ibuprofen, that bind both Eallo and Ecat the data rationalization recurring also to the molecular docking
Herein we present the synthesis, biological evaluation and
;
time-dependent inhibitors that bind the COX site of Ecat models. We found novel potent inhibitors and evidence of their
(e.g. celecoxib, diclofenac); and time-dependent substrates (e.g. binding mode.
naproxen, urbiprofen) that function by binding Eallo
.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 49098–49109 | 49099

View Article Online
RSC Advances
Paper
atmosphere in toluene. It is worth mentioning, that the
deliverance of a free amine group can promote the intra-
molecular nucleophilic attack of amine to the methyl ester
group, yielding the corresponding oxindole derivative. Indeed,
despite a preliminary optimization of reaction conditions, the
aminated products were attained, in some cases, with a small
amount of the corresponding oxindole structures. These
compounds were directly carried onto the next step without
purication since both of them presented the same retention
time, impeding their isolation. Benzoylation with sulfa-
moylbenzoyl chloride without the use of any base, promoted
the cyclization to achieve the corresponding benzimidazole
structures 5a–e in good yields. In this step the corresponding
oxindoles were also observed as minor products.
The acid derivatives were obtained by hydrolysis with
LiOH$H2O, a fast and smooth process that yielded the target
1,2-diarylated benzimidazoles 6a–e, quantitatively. All the nal
ester and acid based derivatives were either crystallized or
puried by chromatography. The structures of all intermediates
and target compounds were conrmed and fully characterized
by NMR spectroscopy and mass spectrometry. The compounds
chosen for biological evaluation were all subjected to HPLC
analysis (UV detection at 270 nm) in order to establish their
purity ($95%, if not otherwise denoted). Detailed experimental
conditions and spectral data for all synthesized compounds are
provided in ESI.†
Results and discussion
The classical synthetic methods to attain 1,2-disubstituted
benzimidazoles are usually hampered by regioselectivity issues
and are limited to the available starting materials.17 As a result,
new strategies were developed, such as the metal-catalyzed
arylamination chemistry or the cascade arylamination/
condensation procedures.17 Thus, to attain a more straightfor-
ward route to the benzimidazole assembly, the synthetic route
shown in Scheme 1 was followed.
The introduction of the ester or acid moiety in the position 4
of the benzimidazole ring – which could be a challenging task,
was attained using a commercially available heterocyclic ring.
Thus, despite the not so obvious application, 7-Br isatin 1 was
chosen as starting material. It was anticipated that its reduction
to 7-Br oxindole (not shown) followed by a basic hydrolysis
would give the key synthetic intermediate 2, which contains all
the functional groups necessary for the assembly of the benz-
imidazole core properly substituted at C4. Consequently, the
rst synthetic step involved the reduction of the 7-Br isatin
using the usual Wolff–Kishner conditions. However, it was
envisioned that instead of the 7-Br oxindole, one could obtain
directly compound 2. Since the method involves basic condi-
tions, it was veried that using an excess of KOH (17 equiv.), the
ring opening product 2 was easily obtained in excellent yield.
The amine moiety of compound 2 was readily modied by
oxidation to the nitro group using oxone as the oxidizing
agent. Further esterication with MeOH in aqueous HCl gave
product 3 in 67% yield. Compounds 4a–e were attained in
good yields using a Pd-catalyzed C–N cross-coupling with
several anilines. In this step several catalytic systems were
tested, including the Buchwald conditions.18 However, the
Pd2db3/BINAP system combined with Cs2CO3 in toluene pre-
sented the best results. Subsequently, the reduction of nitro
derivatives 4a–e was performed using Pd/C 10% under H2
The biological evaluation required a thromboxane synthase
inhibitor (TXBSI), (E)-7-phenyl-7-(pyridin-3-yl)hept-6-enoic acid,
that was prepared via a modication of a reported procedure
(see ESI†).19
In order to understand whether the prepared compounds
possess a balanced inhibition between both COX isoforms, the
inhibitory activity of the synthesized compounds was tested
against COX-1 and COX-2 using the human whole blood (HWB)
assay.20 The inhibitory activity studies were performed for the
ester and acid compounds (5a–e and 6a–e, respectively) at
different concentrations, starting at 50 mM and gradually
decreasing the tested concentrations until the compounds
showed inexpressive inhibitory activity. Inhibition of COXs is
expressed as the percent inhibition of control COX-1 or COX-2
activity. The known COX inhibitors indomethacin and cele-
coxib were used as positive controls.
Table 1 Percent inhibition of COX-1 (mean ꢁ SEM)
Compound
50 mM 12.5 mM 5 mM 2.5 mM 1 mM 0.625 mM
5a
5b
5c
5d
5e
6a
6b
6c
98 ꢁ 1 93 ꢁ 2 80 ꢁ 3 60 ꢁ 4
—
—
—
—
—
—
—
—
—
—
15.2 ꢁ 0.4
77 ꢁ 6 61 ꢁ 3 NA
—
—
97 ꢁ 1 81 ꢁ 6 68 ꢁ 6 42 ꢁ 11
32 ꢁ 5
41 ꢁ 14
—
—
—
—
—
—
98 ꢁ 1 94 ꢁ 2 86 ꢁ 5 64 ꢁ 8
74 ꢁ 5 24 ꢁ 2 NA
—
—
—
—
—
—
—
Scheme 1 (i) (1) NH2NH2$H2O, DEG, 80 ꢀC, 1 h; (2) KOH solution (17
equiv.), 120 ꢀC, 2 h, 90%; (ii) oxone, NaHCO3, NaOH, H2O : acetone, rt,
4 h, 65%; (iii) HCl, MeOH, rt, 67%; (iv) arylamine, Pd2dba3 (5 mol%),
BINAP (7.5 mol%), Cs2CO3, toluene, 100 ꢀC, 4 h, 66–89%; (v) H2, Pd/C
10%, toluene, rt, 16 h; (vi) sulfamoylbenzoyl chloride, THF, rt, 4 h, 60–
85% over two steps; (vii) LiOH, THF : H2O, rt, quant.
52 ꢁ 9
25 ꢁ 2
—
—
—
—
—
—
—
NA
NA
NA
NA
NA
—
6d
6e
23 ꢁ 8
—
Indomethacin
—
88 ꢁ 8 55 ꢁ 12
49100 | RSC Adv., 2015, 5, 49098–49109
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Table 2 Percent inhibition of COX-2 (mean ꢁ SEM)
reported, pyrroles containing an acetic ester chain at C3 possess
an increased activity toward COX-2 than the corresponding
acids.21
Compound 50 mM 12.5 mM 5 mM
1.25 mM 0.5 mM 0.125 mM
Our results show that substituents at N1 aryl ring also
inuence the compounds activity. As previously reported, an
halogen at the meta position of the aromatic ring can contribute
to an enhanced activity.22 Indeed, the presence of a uorine or
chlorine atom improved the activity. Compound 5a – which
does not have any substituent in aromatic ring – had similar
values to 5c and 5d, only at higher concentrations. The presence
of a m-CF3 or a m-OAc (5b and 5e), seems to have an unfav-
ourable effect, that can be attributed to the groups bulkiness.
STD-NMR is a useful technique to detect binding of small
molecules to a biological target.23 This technique was used to
investigate the binding mode of the biologically active benz-
5a
5b
5c
5d
5e
6a
6b
6c
88 ꢁ 3
79 ꢁ 1
88 ꢁ 2
84 ꢁ 3
—
—
—
—
81 ꢁ 5 59 ꢁ 4
65 ꢁ 7 31 ꢁ 3
81 ꢁ 2 82 ꢁ 3
81 ꢁ 2 83 ꢁ 3
30 ꢁ 2
NA
—
—
—
42 ꢁ 8
60 ꢁ 8 21 ꢁ 2
83 ꢁ 5 40 ꢁ 4
NA
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
30 ꢁ 3 NA
NA
NA
NA
NA
—
—
—
—
6d
6e
Celecoxib
18 ꢁ 2 NA
NA
NA
—
—
72 ꢁ 10
Tables 1 and 2 show the percentage of inhibition of prosta- imidazoles to COX-2. Due to the poor solubility of esters in the
glandin E2 (PGE2) production via COX-1 and COX-2, for all the buffer solution used in the NMR studies, the experiments were
studied compounds. As observed, the majority of the tested performed only for the ester 5a as well as for all the acids 6a–e
compounds were found to inhibit both COX-1 and COX-2. that were soluble on the studied conditions. The NMR studies
However, it was neatly observed that the ester derivatives 5a–e were undertaken with ovine COX-2 and human COX-2 with
showed higher inhibition values when compared to acid related similar results.
structures 6a–e, inhibiting both COX-1 and COX-2 in a
All compounds analysed showed STD-NMR responses
concentration-dependent manner.
demonstrating interaction with the protein (see spectra in ESI†)
For COX-1 inhibition (Table 1), a clear distinction was and reversible binding (Fig. 2). Nevertheless, when compared
observed for the ester compounds 5a, 5c and 5d when compared with the strong STD-NMR signals of the acid derivatives 6a–e,
to 5b (R ¼ CF3) and 5e (R ¼ OAc). While the rst three the ester compound 5a yielded a poorer STD-NMR spectrum,
compounds presented inhibitory activity above 97 ꢁ 1%, at 50 with a low signal-to-noise ratio, which can be due to its high
mM, compounds 5b and 5e presented 77 ꢁ 6% and 74 ꢁ 5%, affinity towards COX-2.
respectively. For lower concentrations and below 5 mM, 5b and
For both 5a and 6a–e, it is possible to integrate the reso-
5e displayed inhibition values less than 10%, which were nances of the resolved protons signals at the aromatic region
considered not relevant. These facts, allows one to consider 5a, and determine the relative STD intensities. Due to the strong
5c and 5d the most active compounds towards COX-1. Con- signals in the aliphatic region from the buffer and the additives
cerning the acids, only 6a, 6b and 6d presented inhibitory that overlaps with the ligand resonance peaks, it was not
activity at 50 mM. These compounds showed high values of SEM possible to verify the interaction between the acetic acid anchor
making difficult to achieve a concentration-dependent effect.
and protein, e.g. CH2 STD percentages.14b The STD percentages
Similar to COX-1, the same trend was found for COX-2 inhi- observed for the aromatic protons of the heterocyclic core are
bition for acids and esters (Table 2). Within the acids group, 6a above 86%. It is also seen that the aromatic ring containing the
and 6d were the only ones exhibiting inhibitory activities at 50 sulfonamide group has lower STD interaction when compared
mM. The other acid compounds displayed inhibition values lower with the other aromatic protons. This epitope mapping indi-
than 10%, which conducted to high values of SEM thus not being cates that both acids and esters compounds possess a similar
considered. Compounds 5a, 5c and 5d were the most potent, interaction when binding to protein. This observation is in
showing a similar pattern and inhibiting COX-2 by 88 ꢁ 3%, 88 ꢁ agreement with the docking studies that predict the same
2% and 84 ꢁ 3 (50 mM), respectively. At the same concentration, orientation at the active site for both esters and acids (Fig. 3A
compound 5b and 5e exhibited 78.94 ꢁ 0.13% and 83 ꢁ 5%, and B). The ester and acid groups interact with Arg120 in the
respectively. However, below 5 mM, 5e displayed inhibition values same way as non-selective inhibitors such as ibuprofen. The
less than 10%, while for 5b the same was veried below 0.5 mM. STD results also support this binding mode, since as shown in
Therefore the less potent compound was 5e possessing an OAc Fig. 3 protons H6/7 are in close proximity to Leu531 and protons
group at the meta position. Conversely, compound 5d, which has 200/300 are close to Tyr355, Trp387 and Phe518 thus receiving
a chlorine atom, showed the higher potency, inhibiting 81 ꢁ 2% higher STD than the sulphonamide ring which is located in the
at 5 mM. Remarkably this compound has similar inhibitory selective pocket. The docking studies also predict a slightly
potency as celecoxib which showed 72 ꢁ 10% at 5 mM.
stronger binding towards COX-2 for all acid and ester benz-
The biological activity found for the benzimidazole imidazoles (see ESI†). However, the true signicance can only
compounds, suggests that the presence of an ester/carboxylic be established by in vivo and in vitro experiments as the novel
acid side chain at C4 has a strong inuence on COXs inhibi- inhibitors also t inside the COX-1 binding pocket.
tion. Clearly the ester functionality has an important role on the
These ndings are however in contradiction with the activity
inhibition. A different behavior between acid/ester derivatives found on the HWB assay, since under the tested conditions the
was previously observed for pyrrole based structures. As acids 6 were not active. It is important to stress that NMR
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 49098–49109 | 49101

View Article Online
RSC Advances
Paper
Fig. 2 Expansion of the aromatic region of (a) 1H STD-NMR and (b) the reference spectra of: 5a (115 mM) in the presence of hCOX-2 (1.15 mM) and
6a (300 mM) in the presence of oCOX-2 (3 mM), at 600 MHz and 37 ꢀC. The spectra were acquired in 80 mM Tris–HCl buffer in D2O, pH 8, 0.1%
Tween 20 and 300 mM DDC, irradiated at ꢂ300 Hz, using a series of 40 Eburp2.1000 shaped 90ꢀ pulses (50 ms, 1 ms delay between pulses) for a
total saturation time of 2.0 s.
experiments are carried with isolated enzymes while in HWB complete COX inhibition, while naproxen is an allosteric regu-
assays, that recreates physiological conditions, the blood lator that binds to Eallo, causing an incomplete inhibition.
components can affect drug–protein interaction. In fact According to reported IC50 values, diclofenac (IC50 of 38 nM) is a
discrepancies between HWB results and isolated enzymes have more potent inhibitor of COX-2 than naproxen (IC50 ¼ 28 mM).24
already been reported.22 The standard STD-NMR experiment
The initial competitive experiments were performed with
only reports binding events and does not provide information naproxen (protein : ligand, 1 : 50) and 5a at different
about the specicity of the binding or the binding location. naproxen : 5a ratios and differing the order of addition. When
Thus, in order to establish a possible interaction site for these adding 5a to a solution already containing naproxen and for a
compounds, competitive STD-NMR experiments were under- 1 : 0.5 ratio of naproxen : 5a, it was observed that 5a had a
taken. Due to the higher inhibitory activity for esters 5 detected higher STD response than naproxen (Fig. 4A). On the other
in the biological assays, our interest was to verify whether this hand, reversed addition, i.e. adding naproxen to a solution of 5a
compound binds Ecat and/or Eallo. Diclofenac and naproxen until the same 1 : 0.5 ratio, resulted in a clear reduction of 5a
were used as spy molecules to evaluate whether the designed STD response. Two possible outcomes can explain the results
ester compounds have a binding preference for Eallo or Ecat. The obtained; either naproxen expels this compound from its
choice of these drugs is related to their binding mode to the binding site (Eallo) or naproxen modulates Ecat and thus
protein, since these time-dependent inhibitors preferentially decreases 5a affinity to this binding site.
bind to a single monomer.15f Diclofenac binds Ecat and
In order to clarify this behaviour a competitive STD-NMR
competes with AA for the catalytic site which conducts to the experiment was carried with diclofenac (Fig. 4B). When 1
Fig. 3 Docking of compounds in the active site of COX-2: (A) 5a; (B) 6a; and (C) selective inhibitor SC-558 in the active site of COX-2 from X-ray
data (pdb 1CX2).
49102 | RSC Adv., 2015, 5, 49098–49109
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Fig. 4 Expansion of the aromatic region of the reference (left) and 1H STD-NMR spectra (right): (A) (a) naproxen (160 mM) with different 5a
concentrations: (b) 81.5 mM; (c) 160 mM; (B) (a) 5a (160 mM) (b) and with diclofenac 160 mM in the presence of oCOX-2 (3.26 mM), at 600 MHz and
37 ꢀC.
equivalent of diclofenac was added to 5a both compounds Val116, Tyr355 and Leu359. Analogous conclusions have been
showed STD response with similar intensities, suggesting that drawn by others, which proved that the type of ester, e.g.
5a binds to Eallo, without inuence on the binding mode of insertion of isopropyl or butyl moiety, have high inuence in
diclofenac to Ecat
.
the inhibitory activity.21 Unfortunately, it was not possible to
To rationalize the results obtained for the new benzimid- verify the STD response for the acetic acid chain, which could
azole library, the docking data was further analysed (Fig. 3). give valuable information about its interaction with COX-2.
According to the epitope mapping obtained from the STD-NMR,
On the other hand, it was expected that the carboxylate group
one can conclude that both acids 6 and esters derivatives 5 on compounds 6 could particularly interact with Arg120 and
should have a very similar interaction with COX. Additionally, Tyr355 residues, as veried for naproxen or indomethacin and
the lower energy docking structures present an almost identical thus be a key for activity.25 The molecular docking predicted
orientation for both benzimidazoles 5 and 6 (Fig. 3A and B), and such orientation and by STD the epitope mapping observed
very similar to that of the known selective inhibitor SC-558 matched the epitope found for compounds 5, suggesting the
(Fig. 3C). The presence of an arylsulfonamide group has been same orientation within the active site.
pointed as determinant of selectivity of diarylheterocyclic
inhibitors. However, it is known that selectivity prole can be
Experimental
Synthetic procedures
affected by other structural differences on COXs outside the
selective pocket.4a This might explain the lack of selectivity
found for benzimidazole compounds.
The data obtained demonstrates that the presence of the
carboxymethyl ester chain at C4 plays a crucial role on inhibi-
tion. Inspection of the binding mode of the benzimidazoles 5,
reveals that this might be due to an important hydrophobic
interaction between the aliphatic chain and the residues
General. All commercially obtained reagents were used
without further purication unless specied. All the mentioned
solvents used in the reactions were dried by usual methods. All
reactions were performed under argon atmosphere in ame
dried glassware. Flash column chromatography was carried out
using silica gel 60 (220–440 mesh) using the described eluent
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 49098–49109 | 49103

View Article Online
RSC Advances
Paper
for each case. Preparative and analytical TLC was performed (0.25 mL, 0.2 mmol). The reaction mixture was stirred at room
with silica gel 60 plates of 1 mm, 0.5 mm and 0.25 mm, temperature for 1 h. The mixture was evaporated and the crude
respectively. Chromatograms were visualized by UV light and was puried, to give 3 (18 mg, 60%) as a light yellow solid. Rf ¼
stained with adequate staining solution. HPLC was performed 0.7, hexane : EtOAc (3 : 2). mp: 53–56 C; IR (KBr, cmꢂ1): 3075,
ꢀ
using a Merck HITACHI LaChrom equipped with a DAD 2954, 2888, 1737, 1529, 1438, 1368, 1338, 1218, 1162, 1000, 764;
detector L-7450A and a LiChrospher 100 RP-18 (10 mm) 1H NMR (400 MHz, acetone-d6) d: 7.81 (d, J ¼ 7.0 Hz, ArH, 1H),
LiChroCART® 250-4 column; injection volume: 20 mL; (A) water 7.63–7.54 (m, ArH, 2H), 3.80 (s, CH2, 2H), 3.67 (s, CO2CH3, 3H);
(pH 2.5), (B) methanol; gradient elution (time, %A, %B): 0 min, 13C NMR (100 MHz, acetone-d6) d: 170.6 (CO), 152.7 (CqNO2),
50 : 50; 5 min, 30 : 70; 20 min 10 : 90; 30 min 0 : 100; 35 min, 134.3 (CAr), 133.2 (2ꢃ CAr), 130.3 (CqAr), 113.8 (CqAr), 53.0
0 : 100; 38 min 50 : 50. Measured at 270 nm.
Melting points were determined using melting point appa- 295.9529, found 295.9527.
(CH3), 37.8 (CH2); HRMS: calcd for C9H8BrNO4 [M + Na]+:
ratus Reichert Thermovar equipped with a Koer plate and are
Compounds 4a–b
uncorrected. IR spectra were recorded using a Perkin-Elmer
General procedure. To a screw-cap sealed tube equipped with
Spectrum 1000 FTIR. NMR spectra were recorded with a a magnetic stir bar, was added Pd2dba3 (5 mol%), BINAP (7.5
Brucker ARX 400 and Brucker Avance 400 spectrometers using mol%) and Cs2CO3 (2 equiv.). The tube was sealed with a suba-
CDCl3, DMSO-d6, acetone-d6 and D2O as solvents using their seal, evacuated and backlled with argon. A solution of the aryl
corresponding CHCl3, DMSO, acetone and water signals as halide (1 equiv.) in dry toluene (0.25 M) was then added via
reference, respectively. Mass spectra were obtained on a syringe, and several cycles vacuum/argon were performed. Then
Micromass AutoSpecQ and a Micromass GTC (MALDI-TOF-MS, was added the aniline (3 equiv.) and the suba-seal was replaced
Matrix: a-cyano-4-hydroxycinnamic acid).
by the teon screw-cap. The reaction mixture was heated at 90
Compound 2. To a 7-bromo-isatin solution (250 mg, 1.11 ꢀC for 4 h. The solution was allowed to cool to room tempera-
mmol) in DEG (2.5 mL) was added hydrazine hydrate 99–100% ture, quenched by the addition of HCl solution (1 M) and
ꢀ
(0.81 mL, 17 mmol). The mixture was heated at 80 C for 1 h diluted with EtOAc and water. Aer extracting with 3 portions of
until it turned strong yellow. The mixture was allowed to cool EtOAc, the combined organic layers were washed with water,
down and a KOH solution (1 g, 1ꢀ8.8 mmol, 17 equiv.) was added. brine, dried over Na2SO4, ltered and concentrated in vacuum.
The mixture was heated at 120 C for additional 2 h. The reac-
Compound 4a. The crude product was puried by pTLC
tion was diluted with water and washed with EtOAc to remove (Et2O : hexane, 1 : 4) to give 4a as red oil (86 mg, 83%). Rf ¼ 0.5,
any unreacted oxindole. The aqueous layer was carefully EtOAc : hexane (1 : 4); IR (NaCl, cmꢂ1): 3381, 2952, 1738, 1592,
1
neutralized with 1 M HCl solution (in an ice bath), and the 1504, 1280, 1175, 1065, 752; H NMR (400 MHz, acetone-d6) d:
product precipitated as a light brown solid. The residue was 8.15 (bs, NH, 1H), 7.41–7.27 (m, ArH, 6H), 7.12 (t, J ¼ 7.1 Hz,
ltrated and washed with cold water to give 2 as a light brown ArH, 1H), 6.91 (d, J ¼ 7.1 Hz, ArH, 1H), 3.93 (s, CH2, 2H), 3.67 (s,
solid (230 mg, 90%). Rf ¼ 0.2, hexane : EtOAc (3 : 2). mp: 206 ꢀC CO2CH3, 3H); 13C NMR (100 MHz, acetone-d6) d: 171.0 (CO),
(decomp.); IR (KBr, cmꢂ1): 3366, 3301, 1695, 1458, 1251, 1229, 141.8 (CqAr), 141.0 (CqAr), 133.5 (CAr), 131.7 (CqAr), 130.4 (2ꢃ
743; 1H NMR (400 MHz, acetone-d6) d: 7.34 (d, J ¼ 8.0 Hz, ArH, CAr), 124.6 (CAr), 124.2 (CAr), 122.7 (2ꢃ CAr), 118.3 (CAr), 52.2
1H), 7.10 (d, J ¼ 7.4 Hz, ArH, 1H), 6.58 (t, J ¼ 7.7 Hz, ArH, 1H), (CO2CH3), 39.6 (CH2); HRMS: calcd for C15H14N2O4 [M + Na]+
4.90 (bs, NH2, 1H), 3.63 (s, CH2, 2H); 13C NMR (100 MHz, 309.0846, found 309.0842.
acetone-d6) d: 172.6 (CO), 132.2 (CAr), 131.3 (CAr), 131.0 (CAr),
Compound 4b. The crude product was puried by pTLC
(Et2O : hexane, 1 : 6) to give 4b as a red solid (80 mg, 95%). Rf ¼
124.8 (CAr), 119.2 (CAr), 38.8 (CH2).
Compound 3. To a NaOH (24 mg, 0.62 mmol) solution at 0 ꢀC 0.5, 2ꢃ (Et2O : hexane, 1 : 4); mp: 69–73 ꢀC; IR (NaCl, cmꢂ1):
1
were added 2 (200 mg, 0.87 mmol) and NaHCO3 (730 mg, 8.7 3379, 2957, 1739, 1593, 1503, 1330, 1166, 1123, 782; H NMR
mmol). The mixture was protected from light and allowed to stir (400 MHz, acetone-d6) d: 8.15 (s, NH, 1H), 7.55–7.33 (m, ArH,
for 10 min. A solution of oxone (1.34 g, 2.18 mmol) in EDTA 6H), 7.07 (d, J ¼ 6.9 Hz, ArH, 1H), 3.92 (s, CH2, 2H), 3.67 (s,
solution (4 ꢃ 10ꢂ4 M, 8 mL) was then added followed by a CO2CH3, 3H); 13C NMR (100 MHz, acetone-d6) d: 170.9 (CO),
solution of H2O : acetone 1 : 1 (8 mL). The mixture was stirred 143.9 (CqAr), 138.9 (CqAr), 133.4 (CAr), 131.3 (CAr), 126.1 (CAr),
for 4 h in an ice/water bath. The mixture was quenched by a 124.2 (CAr), 120.3 (CAr), 119.7 (CAr), 117.4 (CAr), 52.3 (CO2CH3),
saturated NaHSO4 solution until a suspension was formed. 39.1 (CH2); HRMS: calcd for C16H13F3N2O4 [M + Na]+: 377.0720,
Then, was extracted with EtOAc (3 ꢃ 25 mL), washed with water found 377.0725.
and brine, dried over Na2SO4 and evaporated. The yellow
Compound 4c. The crude product was puried by ash
residue was used in the next step without further purication chromatography using a gradient from Et2O : hexane (1 : 6) to
(210 mg, 93%). Rf ¼ 0.2, 2ꢃ (hexane : EtOAc, 3 : 2). IR (KBr, (1 : 5) to give 4c as red solid (79 mg, 89%). Rf ¼ 0.5, 2ꢃ (Et2-
1
cmꢂ1): 3075, 2929, 1719, 1534, 1277, 1229, 942; H NMR (400 O : hexane, 1 : 4); mp: 88–91 ꢀC; IR (NaCl, cmꢂ1): 3379, 2949,
MHz, acetone-d6) d: 7.78 (d, J ¼ 7.9 Hz, ArH, 1H), 7.61 (d, J ¼ 7.5 1742, 1600, 1505, 1357, 1168, 784; 1H NMR (400 MHz, acetone-
Hz, ArH, 1H), 7.53 (t, J ¼ 7.8 Hz, ArH, 1H), 3.77 (s, CH2, 2H); 13
C
d6) d: 8.03 (1H, bs, NH), 7.48–7.32 (3H, ArH, m), 7.03–6.99 (3H,
NMR (100 MHz, acetone-d6) d: 172.3 (CO), 152.7 (CqNO2), 133.9 ArH, m), 6.78 (1H, ArH, m), 3.91 (2H, CH2, s), 3.66 (3H, CO2CH3,
(CAr), 133.3 (CAr), 133.1 (CAr), 131.3 (CqAr), 113.5 (CqAr), 38.4 s); 13C NMR (100 MHz, acetone-d6) d: 170.9 (CO), 164.5 (d, J ¼
(CH2). To the compound (30 mg, 0.078 mmol) solution in DCM 243.1 Hz, CF), 144.8 (CqAr), 142.0 (CqAr), 139.2 (CqAr), 133.4
(1 mL) in an ice bath, was added dropwise diazomethane (CqAr, J ¼ 113.0 Hz), 133.4 (CAr), 131.8 (d, J ¼ 9.7 Hz, CAr), 131.3
49104 | RSC Adv., 2015, 5, 49098–49109
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
(CqAr), 125.8 (CAr), 120.3 (CAr), 116.8 (CAr), 110.0 (d, J ¼ 21.4 (s, CH2, 2H), 3.70 (s, CO2CH3, 3H); 13C NMR (100 MHz, acetone-
Hz, CAr), 107.9 (d, J ¼ 24.7 Hz, CAr), 52.3 (CO2CH3), 39.1 (CH2); d6) d: 172.2 (CO2CH3), 151.3 (C100), 145.8 (C1), 143.2 (C2), 138.5
HRMS: calcd for C15H13FN2O4 [M + Na]+: 327.0752, found (C10), 138.0 (C7), 134.2 (C40), 132.7 (C20), 132.6 (q, J ¼ 32.9 Hz,
327.0748.
C50), 132.2 (C30), 130.8 (C300), 127.4 (C3), 126.9 (C200), 126.6 (d, J
Compound 4d. The crude product was puried by pTLC ¼ 3.8 Hz, C40), 125.5 (d, J ¼ 3.9 Hz, C60), 124.8 (C5) 124.7 (C4),
(CH2Cl2 : hexane : MeOH, 1 : 5 : 0.1) to give 4d as a red oil 124.6 (q, J ¼ 270.7 Hz, CF3), 110.1 (C6), 52.1 (CO2CH3), 36.1
(75 mg, 80%). Rf ¼ 0.5, 2ꢃ (EtOAc : hexane, 1 : 4); IR (NaCl, (CH2); HRMS: m/z calcd for C23H19F3N3O4S [M + Na]+: 490.1043,
1
cmꢂ1): 3383, 2949, 1739, 1587, 1494, 1352, 1284, 1176, 774; H found 490.1051; HPLC purity: 97%, RT ¼ 15.3 min.
NMR (400 MHz, acetone-d6) d: 8.03 (s, NH, 1H), 7.48–7.02 (m,
Compounds 5c. The residue was puried by pTLC
ArH, 7H), 3.91 (s, CH2, 2H), 3.66 (s, CO2CH3, 3H); 13C NMR (100 [EtOAc : hexane (2 : 3)] to give 5c as a white solid (36 mg, 59%);
MHz, acetone-d6) d: 171.0 (CO), 144.3 (CqAr), 141.8 (CqAr), 139.2 Rf ¼ 0.4, hexane : EtOAc (2 : 3); mp: 179–181 ꢀC; IR (KBr, cmꢂ1):
(CqAr), 135.4 (CqAr), 133.4 (CAr), 131.7 (CAr), 131.4 (CqAr), 3368, 1732, 1596, 1492, 1338, 1166; 1H NMR (400 MHz, acetone-
125.8 (CAr), 123.4 (CAr), 121.0 (CAr), 120.2 (CAr), 119.5 (CAr), d6) d: 7.89 (d, J ¼ 8.6 Hz, H200, 2H), 7.78 (d, J ¼ 8.6 Hz, H300, 2H),
52.3 (CO2CH3), 39.2 (CH2); HRMS: calcd for C15H13ClN2O4 [M + 7.67 (dd, J ¼ 14.5, 8.1 Hz, H30, 1H), 7.43–7.29 (m, H20, H40, H60,
Na]+: 343.0456, found 343.0453.
3H), 7.30–7.29 (m, H4, H5, 2H), 7.22–7.20 (m, H6, 1H), 6.72 (bs,
Compounds 4e. The crude product was puried by pTLC NH2, 2H), 4.17 (s, CH2, 2H), 3.70 (s, CO2CH3, 3H); 13C NMR (100
(Et2O : hexane, 1 : 3 to 1 : 2) to give 4e as a red oil (105 mg, 66%). MHz, acetone-d6) d: 172.2 (CO2CH3), 164.0 (d, J ¼ 247.3 Hz, C50),
Rf ¼ 0.5, EtOAc : hexane (2 : 3); IR (NaCl, cmꢂ1): 2954, 1745, 151.2 (C100), 145.8 (C1), 143.1 (C2), 139.2 (d, J ¼ 10.1 Hz, C10),
1
1538, 1368, 1239, 1173, 849; H NMR (400 MHz, acetone-d6) d: 138.1 (C7), 134.3 (C400), 132.7 (d, J ¼ 9.3 Hz C30), 130.7 (C300),
8.08 (bs, 1H), 7.45–7.33 (m, ArH, 3H), 7.12 (dd, J ¼ 7.9, 1.7 Hz, 127.4 (C3), 126.9 (C200), 124.8 (d, J ¼ 3.3 Hz, C20), 124.8 (C5),
ArH, 1H), 7.02–6.99 (m, ArH, 2H), 6.82 (dd, J ¼ 8.1, 1.5 Hz, ArH, 124.7 (C4), 116.91 (d, J ¼ 21.1 Hz, C60), 116.01 (d, J ¼ 23.7 Hz,
1H), 3.91 (s, CH2, 2H), 3.66 (s, CO2CH3, 3H), 2.24 (s, OCH3, 3H); C40), 110.2 (C6), 52.1 (CO2CH3), 36.1 (CH2); HRMS: m/z calcd for
13C NMR (100 MHz, acetone-d6) d: 171.0 (CO), 169.7 (COCH3),
C
22H18N3O4FS [M + Na]+: 439.1002, found: 439.1004. HPLC
153.0 (CqAr), 143.4 (CqAr), 140.0 (CqAr), 133.5 (CAr), 131.6 purity: 96%, RT ¼ 12.9 min.
(CqAr), 131.0 (CAr), 125.2 (CAr), 119.4 (CAr), 119.0 (CAr), 117.4
(CAr), 115.3 (CAr), 52.3 (CO2CH3), 39.4 (CH2), 21.1 (COCH3).
Compounds 5a–e
Compounds 5d. The residue was puried by pTLC [2ꢃ
EtOAc : hexane (2 : 3)] to give 5d as a white solid (85 mg, 84%).
Rf ¼ 0.2, hexane : EtOAc (3 : 2); mp: 197–199 ꢀC; IR (KBr, cmꢂ1):
General procedure. The corresponding 4a–e (1 equiv.) solution 3368, 2949, 1734, 1590, 1339, 1165, 757; 1H NMR (400 MHz,
in THF (0.5 M) was added dropwise to a sulfamoylbenzyl chlo- acetone-d6) d: 7.90 (d, J ¼ 8.7 Hz, H200, 2H), 7.80 (d, J ¼ 8.7 Hz,
ride (2.5 equiv.) suspension in dry THF (0.5 M) placed in an ice H300, 2H), 7.66–7.64 (m, H20, H30, H60, 3H), 7.49–7.47 (m, H40,
ꢀ
ꢀ
bath. The mixture was stirred for 1 h at 0 C and then at 50 C 1H), 7.30–7.28 (m, H4, H5, 2H), 7.20–7.18 (m, H6, 1H), 6.73 (bs,
13
overnight. The reaction was quenched with water (15 mL), NH2, 2H), 4.17 (s, CH2, 2H), 3.69 (s, CO2CH3, 3H); C NMR
extracted with EtOAc and washed with brine. The combined (100 MHz, acetone-d6) d: 172.2 (CO2CH3), 151.2 (C100), 145.8
organic layer was dried over Na2SO4 and concentrated in (C1), 143.2 (C2), 139.1 (C10), 138.1 (C7), 135.8 (C50), 134.3 (C400),
vacuum.
132.4 (C30), 130.7 (C300), 130.1 (C60), 128.6 (C20), 127.4 (C40, C3),
Compound 5a. The residue was puried by pTLC 126.9 (C200), 124.8 (C5), 124.7 (C4), 110.2 (C6), 52.0 (CO2CH3),
[EtOAc : hexane (2 : 3)] to give 5a as a light pink solid (51 mg, 36.1 (CH2); HRMS: m/z calcd for C22H18N3O4ClS [M + Na]+:
ꢀ
89%). Rf ¼ 0.2, 2ꢃ (EtOAc : hexane 2 : 3); mp: 199–200 C; IR 455.0707, found: 455.0717. HPLC purity: 96%, RT ¼ 14.5 min.
1
(KBr, cmꢂ1): 3383, 3270, 1724, 1500, 1329, 1170, 767; H NMR
Compound 5e. The residue was puried by pTLC [(2ꢃ
(400 MHz, acetone-d6) d: 7.87 (d, J ¼ 8.0 Hz, H200, 2H), 7.76 (d, EtOAc : hexane (2 : 3)] to give 5e as a white solid (95 mg, 73%).
J ¼ 8.0 Hz, H300, 2H), 7.64–7.61 (m, H30, H40, 3H), 7.52–7.49 (m, Rf ¼ 0.25, hexane : EtOAc (3 : 2); mp: 167–169 ꢀC; IR (KBr,
H20, 2H), 7.29–7.27 (m, H4, H5, 2H), 7.16 (m, H6, 1H), 6.69 (bs, cmꢂ1): 3317, 2956, 1768, 1718, 1596, 1342, 1165, 758; H NMR
1
NH2, 2H), 4.18 (s, CH2, 2H), 3.70 (s, CO2CH3, 3H); 13C NMR (100 (400 MHz, acetone-d6) d: 7.88 (d, J ¼ 8.4 Hz, H200, 2H), 7.80 (d,
MHz, acetone-d6) d: 172.3 (COCH3), 151.3 (C100), 145.3 (C1), J ¼ 8.5 Hz, H300, 1H), 7.64 (t, J ¼ 8.1 Hz, H30, 1H), 7.41–7.33 (m,
143.2 (C2), 138.4 (C10), 137.8 (C7), 134.4 (C400), 131.1 (C30), 130.7 H20, H40, H60, 3H), 7.30–7.27 (m, H4, H5, 2H), 7.20–7.18 (m, H6,
(C300), 130.0 (C40), 128.6 (C20), 127.3 (C3), 126.9 (C200), 124.6 (C5), 1H), 6.72 (bs, NH2, 1H), 4.17 (s, CH2, 2H), 3.69 (s, CO2CH3, 3H),
124.5 (C4), 110.3 (C6), 52.1 (CO2CH3), 36.2 (CH2); HRMS m/z 2.28 (s, COCH3, 3H); 13C NMR (151 MHz, acetone-d6) d: 172.2
calcd for C22H20N3O4S [M + H]+: 422.1169, found 422.1162; (CO2CH3), 169.5 (COCH3), 152.8 (C50), 151.1 (C100), 145.6 (C1),
HPLC purity: 98%, RT ¼ 12.8 min.
143.1 (C2), 138.4 (C10), 138.2 (C7), 134.3 (C400), 131.7 (C30), 130.6
Compound 5b. The residue was puried by pTLC (2ꢃ (C300), 127.3 (C3), 126.8 (C200), 125.8 (C5), 124.7 (C4), 124.6 (C40),
EtOAc : hexane 1 : 2) and recrystallized from ethanol to give 5b 123.4 (C20), 122.4 (C60), 110.2 (C6), 52.0 (CO2CH3), 36.1 (CH2),
as a white solid (65 mg, 62%). Rf ¼ 0.2, EtOAc : hexane (1 : 1); 20.9 (COCH3); HRMS: m/z calcd for C24H21N3O6S [M + Na]+:
mp: 259–261 C; IR (KBr, cmꢂ1): 3310, 1716, 1345, 1317, 1172, 479.1151, found: 479.1154. HPLC purity: 94%, RT ¼ 11.1 min.
ꢀ
1
1130, 845; H NMR (400 MHz, acetone-d6) d: 7.98 (s, H60, 1H),
7.95 (dd, J ¼ 7.9 Hz, H40, 1H), 7.89–7.87 (m, H30, H200, 3H), 7.82
Compounds 6a–e
General procedure. To the 5a–e (1 equiv.) solution in a diox-
(dd, J ¼ 8.0 Hz, H20, 1H), 7.77 (d, J ¼ 8.5 Hz, H300, 2H), 7.31–7.30 ane : THF : water mixture (1 : 1 : 1, 0.5 M), was added
(m, H4, H6, 2H), 7.20–7.18 (m, H5, 1H), 6.72 (bs, NH2, 2H), 4.17 LiOH$H2O (5 equiv.). The mixture was stirred at room
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 49098–49109 | 49105

View Article Online
RSC Advances
Paper
temperature until total conversion veried by TLC (about 1 h). H40, 1H), 7.34–7.28 (m, H4, H5, 2H), 7.20 (dd, J ¼ 7.3, 1.5 Hz, H6,
1
The mixture was diluted with EtOAc and washed with water and 1H), 6.71 (bs, NH2, 2H), 4.16 (s, CH2, 2H); H NMR (600 MHz,
brine. The combined organic layer was dried over Na2SO4 and D2O) d: 8.01 (d, J ¼ 5.9 Hz, H200, 2H), 7.87 (d, J ¼ 8.3 Hz, H300,
concentrated in vacuum.
2H), 7.73–7.67 (m, H20, H30, 2H), 7.62 (s, H60, 1H), 7.51–7.49 (m,
Compound 6a. The compound was obtained as a light pink H40, H5, 2H), 7.44 (d, J ¼ 7.2 Hz, H4, 1H), 7.37 (d, J ¼ 8.2 Hz, H6,
solid (quant.). Rf ¼ 0.1, EtOAc : hexane (3 : 2); mp: 275–278 ꢀC; 1H), 4.06 (s, CH2, 2H); 13C NMR (100 MHz, acetone-d6) d: 172.5
IR (KBr, cmꢂ1): 3418, 3331, 1668, 1338, 1160; 1H NMR (400 MHz, (CO2H), 151.1 (C100), 145.8 (C1), 143.0 (C2), 139.0 (C10), 138.0
acetone-d6) d: 7.87 (d, J ¼ 8.3 Hz, H200, 2H), 7.75 (d, J ¼ 8.3 Hz, (C7), 135.8 (C50), 134.2 (C400), 132.4 (C30), 130.7 (C300), 130.1 (C60),
H300, 2H), 7.63–7.48 (m, H10, H20, H30, 5H), 7.28–7.12 (m, H4, 128.6 (C20), 127.6 (C40), 127.4 (C3), 126.9 (C200), 124.8 (C5), 124.8
H5, H6, 3H), 7.03 (bs, NH2, 2H), 4.11 (s, CH2, 2H); 1H NMR (600 (C4), 110.1 (C6), 36.5 (CH2); HRMS: m/z calcd for C21H16N3O4ClS
MHz, D2O) d: 7.99 (d, J ¼ 8.3 Hz, H200, 2H), 7.86 (d, J ¼ 8.4 Hz, [M + H]+: 441.0550, found: 441.0564. HPLC purity: 92%, RT ¼
H300, 2H), 7.70–7.79 (m, H30, H40, H50, 3H), 7.53–7.35 (m, H10, 12.11 min.
H60, H4, H5, H6, 5H), 4.06 (s, CH2, 2H); 13C NMR (100 MHz,
Compound 6e. The compound was obtained as a white solid
acetone-d6) d: 173.1 (CO2H), 151.1 (C100), 145.9 (C1), 143.1 (C2), (8 mg, quant.). Rf ¼ 0, EtOAc : hexane (3 : 2); mp: 245–247 ꢀC; IR
138.2 (C10), 137.7 (C7), 134.2 (C400), 130.9 (C30), 130.4 (C300), (KBr, cmꢂ1): 3368, 1596, 1338, 1161, 757; H NMR (400 MHz,
1
129.8 (C40), 128.4 (C20), 127.8 (C3), 126.7 (C200), 124.4 (C4, C5), acetone-d6) d: 7.89 (d, J ¼ 8.5 Hz, H200, 2H), 7.82 (d, J ¼ 8.5 Hz,
109.9 (C6), 36.5 (CH2); HRMS: m/z calcd for C21H17N3O4S [M + H300, 2H), 7.44 (t, J ¼ 8.3 Hz, H30, 1H), 7.30–7.29 (m, H4, H5, 2H),
Na]+: 407.0940, found: 407.0922. HPLC purity: 97%, RT ¼ 9.60 7.20 (dd, J ¼ 6.8, 2.3 Hz, H6, 1H), 7.06 (dd, J ¼ 7.9 Hz, H20, 1H),
min.
6.96–6.94 (m, H40, H60, 2H), 6.71 (bs, NH2, 1H), 4.16 (s, CH2,
1
Compounds 6b. The compound was obtained as a white 2H); H NMR (600 MHz, D2O) d: 7.98 (d, J ¼ 8.2 Hz, H200, 2H),
solid (quant.). Rf ¼ 0.1, EtOAc : hexane (3 : 2); mp: 265–268 ꢀC; 7.88 (d, J ¼ 8.3 Hz, H300, 2H), 7.45–7.36 (m, H30, H4, H5, H6, 4H),
IR (KBr, cmꢂ1): 3416, 3343, 3078, 1671, 1458, 1338, 1162, 1128, 6.93 (d, J ¼ 9.4 Hz, H20, 1H), 6.78 (s, H60, 1H), 6.73 (d, J ¼ 8.2 Hz,
756; H NMR (400 MHz, acetone-d6) d: 7.99 (s, H60, 1H), 7.95– H40, 1H), 4.04 (s, CH2, 2H); 13C NMR (100 MHz, acetone-d6) d:
1
7.85 (m, H200, H30, H40, 4H), 7.82–7.75 (m, H300, H20, 3H), 7.33– 172.4 (CO2H), 159.7 (C50), 151.0 (C100), 145.6 (C1), 142.9 (C2),
7.28 (m, H4, H5, 2H), 7.17 (dd, J ¼ 7.3, 1.8 Hz, H6, 1H), 7.11 (bs, 138.6 (C10), 138.2 (C7), 134.4 (C400), 131.8 (C30), 130.5 (C300),
1
NH2, 2H), 4.11 (s, CH2, 2H); H NMR (600 MHz, D2O) d: 8.08– 127.3 (C3), 126.8 (C200), 124.6 (C5), 124.5 (C4), 119.3 (C40), 117.1
7.95 (m, H200, H60, 3H), 7.92–7.81 (m, H300, H30, H40, H20, 5H), (C20), 115.3 (C60), 110.4 (C6), 36.7 (CH2); HRMS: m/z calcd for
7.50–7.35 (m, H4, H5, H6, 3H), 4.06 (s, CH2, 2H); 13C NMR (100
C
21H17N3O5S [M + H+]+: 423.0889, found: 423.0887. HPLC
MHz, acetone-d6) d: 173.1 (CO2H), 151.2 (C100), 146.1 (C1), 143.1 purity: 95%, RT ¼ 7.5 min.
(C2), 138.4 (C10), 137.9 (C7), 133.9 (C40), 132.7 (C20), 132.5 (q, J ¼
32.8 Hz, C50), 132.2 (C30), 130.6 (C300), 128.0 (C3), 126.8 (C200),
Human whole blood assays for COX-2 and COX-1
126.5 (d, J ¼ 3.8 Hz, C40), 125.4 (d, J ¼ 3.8 Hz, C60), 124.8 (C5),
124.7 (C4), 109.8 (C6), 36.4 (CH2); HRMS: m/z calcd for The blood was collected from healthy human volunteers,
C
22H17N3F3O4S [M + Na]+: 476.0888, found: 476.0886. HPLC following informed consent. Venous blood was collected by
Purity: 98%, RT ¼ 13.25 min.
antecubital venipuncture, into heparin-Li+ vacuum tubes.
Compound 6c. The compound was obtained as a white solid
Reagents. The following reagents were purchased from
(quant.). Rf ¼ 0.1, EtOAc : hexane (3 : 2); mp: 256–258 ꢀC; IR Sigma-Aldrich Co. LLC (St. Louis, USA): dimethylsulfoxide
(KBr, cmꢂ1): 3420, 3337, 3084, 1676, 1594, 1341, 1162, 754; H (DMSO), acetylsalicylic acid, gentamicin sulfate, cremophor®
1
NMR (400 MHz, acetone-d6) d: 7.90 (d, J ¼ 8.5 Hz, H200, 2H), 7.79 EL, lipopolysaccharides from Escherichia coli 026:B6 (LPS),
(d, J ¼ 8.5 Hz, H300, 2H), 7.68 (dd, J ¼ 14.5, 8.1 Hz, H30, 1H), 7.45– calcium ionophore (A23187), Dulbecco's phosphate-buffered
7.29 (m, H20, H40, H60, H4, H5, 5H), 7.22 (dd, J ¼ 7.3, 1.8 Hz, H6, saline (DPBS). For the synthesis of thromboxane synthase
1
1H), 6.70 (bs, NH2, 2H), 4.16 (s, CH2, 2H); H NMR (600 MHz, inhibitor (TXBSI) see ESI.†19 The “PGE2 Enzyme Immunoassay
D2O) d: 8.01 (d, J ¼ 8.4 Hz, H200, 2H), 7.87 (d, J ¼ 8.4 Hz, H300, (EIA) Kit” was obtained from Enzo Life Sciences (Lausen,
2H), 7.71 (dd, J ¼ 15.0, 8.4 Hz, H30, 1H), 7.52–7.37 (m, H20, H40, Switzerland).
H60, H4, H5, H6, 6H), 4.06 (s, CH2, 2H); 13C NMR (100 MHz,
COX-1 and COX-2 assays. The human whole blood assays to
acetone-d6) d: 172.4 (CO2H), 164.0 (d, J ¼ 247.3 Hz, C50), 151.1 assess the COXs inhibition were performed as previously
(C100), 145.8 (C1), 143.0 (C2), 139.2 (d, J ¼ 10.2 Hz C10), 138.0 reported.26
(C7), 134.2 (C400), 132.6 (d, J ¼ 9.4 Hz, C30), 130.7 (C300), 127.5
For the COX-2 assay, the samples of fresh heparinized blood
(C3), 126.9 (C200), 124.8 (d, J ¼ 3.2 Hz, C20), 116.90 (d, J ¼ 21.1 were aliquoted to 800 mL, mixed with 10 mL of TXBSI (nal
Hz, C60), 116.0 (d, J ¼ 23.7 Hz, C40), 110.2 (C6), 36.5 (CH2); concentration ¼ 1 mM) and 50 mL acetylsalicylic acid (nal
HRMS: m/z calcd for C21H16N3O4FS [M + Na]+: 425.0846, found: concentration ¼ 10 mg mLꢂ1). The use of TXBSI reduces the
425.0843; HPLC purity: 96%, RT ¼ 9.95 min.
amount of LPS needed and its incubation period, and the ace-
Compound 6d. The compound was obtained as a white solid tylsalicylic acid exclude any contribution of COX-1. Aer adding
(quant). Rf ¼ 0.1, EtOAc : hexane (3 : 2); mp: 269–270 ꢀC; IR 100 mL of the tested compound (0.125–50 mM) in DMSO/
1
(KBr, cmꢂ1): 3338, 3068, 1671, 1591, 1338, 1160; H NMR (400 cremophor/ethanol 1% (1 : 10), samples were equilibrated in a
MHz, acetone-d6) d: 7.90 (d, J ¼ 8.4 Hz, H200, 2H), 7.80 (d, J ¼ 8.4 humidied incubator (37 C, 5% CO2). Aer 15 min, 50 mL of
ꢀ
Hz, H300, 2H), 7.68–7.63 (m, H20, H30, H60, 3H), 7.51–7.48 (m, LPS (nal concentration ¼ 10 mg mLꢂ1) were added into all caps
49106 | RSC Adv., 2015, 5, 49098–49109
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
(except those for basal data; 50 mL DPBS-Gentamicin were to suppress the water proton signals. A spin lock lter (T1r) with
added instead) to induce COX-2 expression and samples were a 2 kHz eld and a length of 20 ms was applied to suppress
incubated for another 5 h. The reactions were stopped by add- protein background. Selective saturation of protein resonances
ing an equal volume (1000 mL) of ice cold DPBS-Gentamicin (on resonance spectrum) was performed by irradiating at ꢂ300
buffer into the aliquots and further cooling on ice for 10 min. Hz using a series of 40 Eburp2.1000 shaped 90ꢀ pulses (50 ms, 1
The samples were centrifuged (1000ꢃg, 4 ꢀC, 15 min) and ms delay between pulses) for a total saturation time of 2.0 s. For
the plasma was removed and stored at ꢂ20 ꢀC until further the reference spectrum (off resonance) the samples were irra-
examination.
diated at 20 000 Hz. Proper control experiments were performed
For the COX-1 assay, the samples of fresh heparinized blood with the reference samples in order to optimize the frequency
were aliquoted to 500 mL, mixed and incubated with 5 mL of for protein saturation (ꢂ0.5 ppm) and off-resonance irradiation,
TXBSI (nal concentration ¼ 1 mM) and 5 mL of the tested to ensure that the ligand signals were not affected. The STD
compound (0.625–50 mM) in DMSO, in a water bath at 37 ꢀC for effect was calculated using (I0 ꢂ ISTD)/I0, in which (I0 ꢂ ISTD) is
15 min. TXBSI, reduces the amount of A23187 needed, which the peak intensity in the STD spectrum and I0 is the peak
results in a higher prostaglandin production. During this intensity in the off-resonance spectrum. The STD intensity of
incubation period, the TXBSI and the tested compounds the largest STD effect was set to 100% as a reference, and the
interact with all the cells and the other components of the relative intensities were determined.
blood. Subsequently, 2.5 mL of A23187 (nal concentration ¼
12.2 mg mLꢂ1) was added into all microtubes (except those for
Molecular docking
basal data where 2.5 mL of DPBS were added instead) and the
The docking investigations were carried with the docking
mixture was incubated for 1 min, to trigger COX-1 activity. The
program AutoDock 4.2 (Release 4.2.5.1). Marvin 2012 (ref 27)
reaction was stopped by cooling the samples at 0 ꢀC for 5 min.
was used to generate the 3D structure for the new inhibitors.
The samples were centrifuged (1000ꢃg, 4 ꢀC, 20 min) and
The following X-ray structure were used for COX1 2AYL28 and for
plasma was removed and stored at ꢂ20 ꢀC until further
COX2 3PGH29 in the docking study. The box centre was built
examination.
around the C(b) of the residue 523, which is isoleucine in COX-1
Determination of PGE2 production. PGE2 concentrations in
˚
and valine for COX-2. The box size was 100 A and a total of 100
runs were performed for each docking calculation.
thawed plasma supernatants were determined using the above
mentioned commercial EIA kit, as an indicator of COXs activity,
according to the manufacturer's instructions. COXs activity is
dened as the production of PGE2 in the vehicle-treated and
LPS or A23187-treated blood over that of background levels in
Conclusions
unstimulated blood at time zero. Results are expressed as the Recent research on inammatory related disease suggests that a
percent inhibition of control PGE2 production. Each study balanced inhibition of both COX-1 and COX-2 isoenzymes is the
corresponds to at least three experiments, with different key to reduce the side-effects exhibited by COX inhibitors.
donors.
Intensive research efforts have been devoted to avoid the gastric
damaging and cardiovascular effects of these anti-inammatory
drugs.
STD-NMR experiments
To address this problem we have designed and developed a
COX-2 from sheep placenta and COX-2 human recombinant new library of hybrid COX inhibitors based on the benzimid-
were purchased from Cayman Chemical (Ann Arbor, MI, U.S.). azole core with motifs from both selective and non-selective
The protein was supplied in 80 mM Tris–HCl, pH 8.0, 0.1% known-inhibitors. The HBW assay revealed that the ester
Tween 20, and 300 mM diethyldithiocarbamate (DDC) and was derivatives 5 were indeed strong inhibitors of both COXs iso-
used as such. Naproxen was purchased from Sigma, and forms, and slightly selective for COX-2 at low concentrations.
diclofenac was purchased from Merck and used as such. For the The inhibitory activity found prompted us to explore its binding
STD-NMR experiments, naproxen and diclofenac stock solu- interaction to COX-2 by employing STD-NMR experiments. The
tions (20 mM) were prepared in [d6]DMSO. From these, an structural requirements that rule activity and binding to COX
amount of 15 mL was added to the COX solution directly in the were investigated. The STD-NMR experiments revealed an
NMR tube. Then 80 mM Tris–HCl buffer at pH 8.0 was used to interaction with COX-2. Furthermore, the epitope mapping
adjust the volume to 200 mL. Final concentrations of COX-2 and obtained clearly highlighted that arylsulfonamide received less
inhibitor were 3 and 300 mM, respectively. For the competition saturation and thus the hydrophobic interaction may rule the
binding experiments, the ratio of inhibitors/protein was kept to binding. Additionally competitive STD-NMR was carried for 5a
100 : 1 and the solutions were prepared as above.
with known drugs, suggesting that this compound has a
All STD-NMR experiments were acquired at 37 ꢀC in a Bruker naproxen-like behaviour binding to the allosteric monomer.
Avance III spectrometer operating at 600 MHz, with a 5 mm Molecular docking supported the compounds design and data
triple resonance cryogenic probe head. The STD-NMR spectra rationalization.
were acquired with 1024 transients in a matrix with 32k data
We have experimentally demonstrated that the presence of
points in t2 in a spectral window of 12 019.23 Hz centered at an ester moiety in cooperation with a coxib shaped molecule
2814.60 Hz. Excitation sculpting with gradients was employed containing an aryl sulphonamide provides a balanced COX-1/
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 49098–49109 | 49107

View Article Online
RSC Advances
Paper
COX-2 inhibition. Moreover we demonstrated that a properly
functionalized benzimidazole core can result in potent COX
inhibitors.
These results are important for further investigations on the
drug design of novel inhibitors and on the disclosure of allo-
steric COX regulation.
A. W. Veenhuizen, Y. Y. Zhang and P. C. Isakson, J. Med.
Chem., 1997, 40, 1347; (b) J. L. Hillson and D. E. Furst,
Expert Opin. Pharmacother., 2000, 1, 1053; (c) J. J. Talley,
D. L. Brown, J. S. Carter, M. J. Graneto, C. M. Koboldt,
J. L. Masferrer, W. E. Perkins, R. S. Rogers, A. F. Shaffer,
Y. Y. Zhang, B. S. Zweifel and K. Seibert, J. Med. Chem.,
2000, 43, 775; (d) D. Riendeau, M. D. Percival, C. Brideau,
´
S. Charleson, D. Dube, D. Ethier, J.-P. Falgueyret,
Acknowledgements
R. W. Friesen, R. Gordon, G. Greig, J. Guay, J. Mancini,
M. Ouellet, E. Wong, L. Xu, S. Boyce, D. Visco, Y. Girard,
P. Prasit, R. Zamboni, I. W. Rodger, M. Gresser,
A. W. Ford-Hutchinson, R. N. Young and C.-C. Chan, J.
Pharmacol. Exp. Ther., 2001, 296, 558; (e) D. J. Cochrane,
B. Jarvis and G. M. Keating, Drugs, 2002, 62, 2637.
8 (a) C. D. Funk and G. A. FitzGerald, J. Cardiovasc. Pharmacol.,
2007, 50, 470; (b) Y. Yu, E. Ricciotti, R. Scalia, S. Y. Tang,
G. Grant, Z. Yu, G. Landesberg, I. Crichton, W. Wu,
E. Pure, C. D. Funk and G. A. FitzGerald, Sci. Transl. Med.,
2012, 4, 132.
~
ˆ
The authors acknowledge to Funda¸cao para a Ciencia e
Tecnologia (FCT) for funding the projects PTDC/QUI-QUI/
104056/2008 and PTDC/QUI/65187/2006. Luısa Carvalho
acknowledges FCT the nancial support for the PhD grant
(SFRH/BD/63407/2009). Daniela Ribeiro acknowledges FCT the
nancial support for the PhD grant (SFRH/BD/72966/2010),
within the ambit of “QREN – POPH – Tipologia 4.1 – For-
´
˜
maçao Avançada”, co-sponsored by FSE and by national funds
of MCTES. The NMR spectrometers are part of The National
NMR Facility, supported by Fundaçao para a Ciencia e a Tec-
˜
ˆ
nologia (RECI/BBB-BQB/0230/2012).
9 (a) C. Roubille, J. Martel-Pelletier, J. M. Davy, B. Haraoui and
J. P. Pelletier, Anti-Inammatory Anti-Allergy Agents Med.
Chem., 2013, 12, 55; (b) T. Grosser, S. Fries and
G. A. FitzGerald, J. Clin. Invest., 2006, 116, 4.
Notes and references
1 (a) J. R. Vane, Nature (London), New Biol., 1971, 231, 232; (b) 10 W. B. White, G. Faich, A. Whelton, C. Maurath, N. J. Ridge,
J. B. Smith and A. L. Willis, Nature (London), New Biol., 1971,
231, 235; (c) S. H. Ferreira, S. Moncada and J. R. Vane, Nature
(London), New Biol., 1971, 231, 237.
K. M. Verburg, G. S. Geis and J. B. Leowith, Am. J.
Cardiol., 2002, 89, 425.
11 V. Strand, Lancet, 2007, 370, 2138.
˜
2 (a) K. D. Rainsford, Int. J. Clin. Pract., 2013, 67, 9; (b) 12 M. S. Estevao, L. C. Carvalho, M. Freitas, A. Gomes, A. Viegas,
F. D. Hart and P. L. Boardman, Br. Med. J., 1963, 2, 965; (c)
A. R. Sallmann, Am. J. Med., 1986, 80, 29; (d) K. C. Duggan,
J. Manso, E. Fernandes, E. J. Cabrita, S. Erhardt and
M. M. B. Marques, Eur. J. Med. Chem., 2012, 54, 823.
M. J. Walters, J. Musee, J. M. Harp, J. R. Kiefer, J. A. Oates 13 T. Narsinghani and R. Sharma, Chem. Biol. Drug Des., 2014,
and L. J. Marnett, J. Biol. Chem., 2010, 285, 34950. 84, 1.
3 (a) J. L. Masferrer, K. Seibert, B. Zweifel and P. Needleman, 14 (a) W. L. Smith, Y. Urade and P.-J. Jakobsson, Chem. Rev.,
Proc. Natl. Acad. Sci. U. S. A., 1992, 89, 3917; (b)
M. K. O'Banion, H. B. Sadowski, V. Winn and D. A. Young,
J. Biol. Chem., 1991, 266, 23261; (c) W. L. Xie,
J. G. Chipman, D. L. Robertson, R. L. Erikson and
D. L. Simmons, Proc. Natl. Acad. Sci. U. S. A., 1991, 88,
2692; (d) L. J. Marnett, S. W. Rowlinson, D. C. Godwin,
A. S. Kalgutkar and C. A. Lanzo, J. Biol. Chem., 1999, 274,
22903; (e) W. L. Smith, R. M. Garavito and D. L. DeWitt, J.
Biol. Chem., 1996, 271, 33157.
4 (a) R. G. Kurumbail, A. M. Stevens, J. K. Gierse,
J. J. McDonald, R. A. Stegeman, J. Y. Pak, D. Gildehaus,
J. M. Miyashiro, T. D. Penning, K. Seibert, P. C. Isakson
and W. C. Stallings, Nature, 1996, 384, 644; (b)
H. E. Vonkeman and M. A. van de Laar, Semin. Arthritis
Rheum., 2010, 39, 294.
2011, 111, 5821; (b) A. Viegas, J. Manso, M. C. Corvo,
M. M. B. Marques and E. J. Cabrita, J. Med. Chem., 2011,
54, 8555; (c) V. Limongelli, M. Bonomi, L. Marinelli,
F. L. Gervasio, A. Cavalli, E. Novellino and M. Parrinello,
Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 5411; (d)
V. Limongelli, M. Bonomi and M. Parrinello, Proc. Natl.
Acad. Sci. U. S. A., 2013, 110, 6358; (e) A. L. Blobaum and
L. J. Marnett, J. Med. Chem., 2007, 50, 1425.
15 (a) C. Yuan, R. S. Sidhu, D. V. Kuklev, Y. Kado, M. Wada,
I. Song and W. L. Smith, J. Biol. Chem., 2009, 284, 10046;
(b) N. P. Sharma, L. Dong, C. Yuan, K. R. Noon and
W. L. Smith, Mol. Pharmacol., 2010, 77, 979; (c) G. Rimon,
R. S. Sidhu, D. A. Lauver, J. Y. Lee, N. P. Sharma, C. Yuan,
R. A. Frieler, R. C. Trievel, B. R. Lucchesi and W. L. Smith,
Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 28; (d) L. Dong,
A. J. Vecchio, N. P. Sharma, B. J. Jurban, M. G. Malkowski
and W. L. Smith, J. Biol. Chem., 2011, 286, 19035; (e)
H. Zou, C. Yuan, L. Dong, R. S. Sidhu, Y. H. Hong,
D. V. Kuklev and W. L. Smith, J. Lipid Res., 2012, 53, 1336;
(f) L. Dong, N. P. Sharma, B. J. Jurban and W. L. Smith, J.
Biol. Chem., 2013, 288, 28641.
5 D. Picot, P. J. Loll and R. M. Garavito, Nature, 1994, 367, 243.
6 K. R. Gans, W. Galbraith, R. J. Roman, S. B. Haber, J. S. Kerr,
W. K. Schmidt, C. Smith, W. E. Hewes and N. R. Ackerman, J.
Pharmacol. Exp. Ther., 1990, 254, 180.
7 (a) T. D. Penning, J. J. Talley, S. R. Bertenshaw, J. S. Carter,
P. W. Collins, S. Docter, M. J. Graneto, L. F. Lee,
J. W. Malecha, J. M. Miyashiro, R. S. Rogers, D. J. Rogier, 16 (a) L. Franke, E. Byvatov, O. Werz, D. Steinhilber,
S. S. Yu, G. D. Anderson, E. G. Burton, J. N. Cogburn,
S. A. Gregory, C. M. Koboldt, W. E. Perkins, K. Seibert,
P. Schneider and G. Schneider, J. Med. Chem., 2005, 48,
6997; (b) Y. Bansal and O. Silakari, Bioorg. Med. Chem.,
49108 | RSC Adv., 2015, 5, 49098–49109
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
2012, 20, 6208; (c) A. Rathore, M. U. Rahman, A. A. Siddiqui,
A. Ali and M. Shaharyar, Arch. Pharm., 2014, 347, 923; (d)
P. Anzellotti, P. Patrignani and M. Anzini, J. Med. Chem.,
2007, 50, 5403.
M. Gaba, S. Singh and C. Mohan, Eur. J. Med. Chem., 2014, 23 (a) B. Meyer and T. Peters, Angew. Chem., Int. Ed., 2003, 42,
76, 494.
864; (b) Y. S. Wang, D. Liu and D. F. Wyss, Magn. Reson.
Chem., 2004, 42, 485.
24 T. D. Warner, F. Giuliano, I. Vojnovic, A. Bukasa,
J. A. Mitchell and J. R. Vane, Proc. Natl. Acad. Sci. U. S. A.,
1999, 96, 7563.
17 L. C. R. Carvalho, E. Fernandes and M. M. B. Marques,
Chem.–Eur. J., 2011, 17, 12544.
18 N. Zheng, K. W. Anderson, X. H. Huang, H. N. Nguyen and
S. L. Buchwald, Angew. Chem., Int. Ed., 2007, 46, 7509.
19 K. Kato, S. Ohkawa, S. Terao, Z.-I. Terashita and 25 K. C. Duggan, M. J. Walters, J. Musee, J. M. Harp, J. R. Kiefer,
K. Nishikawa, J. Med. Chem., 1985, 28, 287. J. A. Oates and L. J. Marnett, J. Biol. Chem., 2010, 285, 34950.
20 (a) S. Laufer and S. Luik, Methods Mol. Biol., 2010, 644, 91; (b) 26 D. Ribeiro, M. Freitas, S. M. Tome, A. M. S. Silva, S. Laufer,
´
S. Laufer, C. Greim, S. Luik, S. S. Ayoub and F. Dehner,
Inammopharmacology, 2008, 16, 155.
J. L. F. C. Lima and E. Fernandes, Inammation, 2015,
38(2), 858.
21 M. Biava, G. C. Porretta, G. Poce, C. Battilocchio, F. Manetti, 27 ChemAxon (http://www.chemaxon.com).
M. Botta, S. Forli, L. Sautebin, A. Rossi, C. Pergola, 28 K. Gupta, B. S. Selinsky and P. J. Loll, Acta Crystallogr., Sect.
C. Ghelardini, N. Galeotti, F. Makovec, A. Giordani,
D: Biol. Crystallogr., 2006, 62, 151.
P. Anzellotti, P. Patrignani and M. Anzini, J. Med. Chem., 29 R. G. Kurumbail, A. M. Stevens, J. K. Gierse, J. J. McDonald,
2010, 53, 723.
R. A. Stegeman, J. Y. Pak, D. Gildehaus, J. M. Miyashiro,
T. D. Penning, K. Seibert, P. C. Isakson and
W. C. Stallings, Nature, 1996, 384, 644.
22 M. Biava, G. C. Porretta, G. Poce, S. Supino, S. Forli,
M. Rovini, A. Cappelli, F. Manetti, M. Botta, L. Sautebin,
A. Rossi, C. Pergola, C. Ghelardini, E. Vivoli, F. Makovec,
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 49098–49109 | 49109