Synthesis, anti-inflammatory activity and molecular docking studies of 2,5-diarylfuran amino acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.10.018

A series of 2,5-diaryl substituted furans functionalized with several amino acids were synthesized and evaluated as the cyclooxygenases COX-1 and COX-2 enzymes inhibitors. The proline-substituted compound inhibited PGE₂ secretion by LPS-stimula

Full text of DOI:10.1016/j.ejmech.2011.10.018

European Journal of Medicinal Chemistry 47 (2012) 52e58
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, anti-inflammatory activity and molecular docking studies
of 2,5-diarylfuran amino acid derivatives
,
a
b
c
Hélio A. Stefani a , Giancarlo V. Botteselle , Julio Zukerman-Schpector , Ignez Caracelli ,
*
Denis da Silva Corrêa d, Sandra H.P. Farskya, Isabel D. Machado a, José R. Santin a, Cristina B. Hebeda a
a Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil
b Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil
c Departamento de Física, Universidade Federal de São Carlos, SP, Brazil
d Programa de Pós-Graduação em Biotecnologia, Universidade Federal de São Carlos, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,5-diaryl substituted furans functionalized with several amino acids were synthesized and
evaluated as the cyclooxygenases COX-1 and COX-2 enzymes inhibitors. The proline-substituted
compound inhibited PGE2 secretion by LPS-stimulated neutrophils, suggesting selectivity for COX-2.
Molecular docking studies in the binding site of COX-2 were performed.
Received 16 January 2011
Received in revised form
18 August 2011
Accepted 8 October 2011
Available online 20 October 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
2,5-Diarylfuran
Suzuki-Miyaura
Amino acids
COX-1 and COX-2
Docking
1. Introduction
member ring (pyrazole in celecoxib/CelebrexÒ, 3,4-dihydrofuran-
2-one in rofecoxib/VioxxÒ or 1,2-oxazole in valdecoxib/BextraÒ),
Cyclooxygenases (COXs) are expressed in a variety of cells and
are responsible for the metabolism of membrane lipids, generating
prostaglandins (PGs), which are some of the most important
inflammatory mediators [1]. While COX-1 is described as a consti-
tutive enzyme and displays relevant physiological actions, COX-2 is
induced in response to pro-inflammatory stimuli, and its expres-
sion is absent or low in healthy individuals [1,2]. Although COX-2
inhibitors are widely prescribed anti-inflammatory agents,
conversely several important side effects have been associated with
the simultaneous inhibition of COX-1 activity [3e5]. Therefore, the
development of compounds that would inhibit COX-2 almost
exclusively is an important target in order to reduce adverse side
effects during non-steroidal anti-inflammatory treatment, thus
improving therapeutic benefits.
and that several developments were based on that fact using, for
example, tetrahydrofuran [5] or 1H-pyrrole [6], moreover, all these
described compounds are vicinal substituted. Docking studies of
this class of molecules have shown that they accommodate entirely
in the binding site of COX-2 in positions very close to that of SC-558
in the crystal structure of its complex with COX-2 [7]. In view of
this, and as a further extension to this work we decided to
synthesize a series of C2, C5 diaryl substituted furans, instead of
vicinal aryl substituted, to evaluate them as cyclooxygenase
enzyme (COX-1 and COX-2) inhibitors.
2,5-Diarylfuran derivatives have been synthesized via many
different ways, such as Stille coupling [8], SuzukieMiyaura
coupling [9], and PaaleKnoor synthesis [10]. They have also been
obtained from phenacyl bromides [11] as well as from butane or
butyne diones by microwave irradiation [12], and recently from
alkynes by sequential synthesis [13]. Our research group has
expanded the scope of the effective use of potassium organo-
trifluoroborates and organotellurium compounds in cross-coupling
reactions [14], and recently reported the synthesis of 2-aryl and
2,5-diarylfuran and thiophene derivatives by Suzuki-Miyaura
It is interesting to note that most of the COX-2 inhibitors bear
a common structural feature, namely a central heterocycle five-
* Corresponding author. Tel.: þ55 11 3091 3654; fax: þ55 11 3815 4418.
E-mail address: hstefani@usp.br (H.A. Stefani).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.018

H.A. Stefani et al. / European Journal of Medicinal Chemistry 47 (2012) 52e58
53
boronic acid 3 using 1,10-bis-(diphenylphosphino)ferrocene (dppf)
as the ligand in the presence of Ag2O as an additive and methanol as
a solvent at room temperature (Scheme 1). The 3-AFA 4 was ob-
tained in 65% yield.
1) n-BuLi/THF, -78 °C, 3 h
n-BuTe
O
O
2
2) Te°, -40 °C, 3 h
3) n-BuBr, -10 °C, 1 h
rt, 2 h
OMe
OMe
1
75%
It is worth pointing out that the reaction shown above is time-
dependent. Attempts to synthesize 3-AFA 4 after stirring for 2
and 3 h gave the product in low yields of 25% and 35%, respectively.
When the reaction mixture was stirred for 6 h, the product was
obtained in moderate yield of 65%. For the formation of the inter-
mediates 2-(4-methoxy)furyl lithium and 2-(4-methoxy)furyl tel-
lurolate a period of 3 h of stirring is required.
Pd(OAc)2 (10mol%),
dppf(20mol%), Ag2O (2.0 eq.)
MeOH, rt, 6 h
H2N
O
B(OH)2
OMe
4
65%
NH2
3
Scheme 1. Synthesis of 2,5-diarylfuran 3-AFA.
The optimal reaction conditions for promoting the coupling
between the 3-AFA 4 and the Boc-glycine 5a were examined. In the
first approach diethylcianophosphonate (DEPC) was used as the
catalyst, Et3N as the base, and DMF as the solvent. The product 6a
was formed in low yield together with a few side products. This
reaction condition was also used with other amino acids (Boc-
alanine 5b, Boc-leucine 5d and Boc-phenylalanine 5f), and in all
cases with low yield and also unwanted side products were formed.
Then the peptide coupling was performed under the usual
conditions [19], that is, using a two molar excess of Boc-glycine 5a,
diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole
(HOBt) in dichloromethane (DCM) at room temperature for 20 h.
The desired product Boc-Gly 6a was obtained in an excellent yield
of 81% and no side products were observed in this reaction
(Scheme 2).
cross-coupling reactions between potassium aryltrifluoroborates
and furanyl tellurides catalyzed by palladium [15]. Taking advan-
tage of this well-established synthesis route, and that compounds
functionalized with amino or carboxyl groups are commonly used
for the conjugation of biomolecules [16], a series of C2, C5 diaryl
substituted furans, instead of 3,4-diaryl substituted [17], and
functionalized with amino acids were obtained, evaluated as
cyclooxygenase enzymes (COX-1 and COX-2) inhibitors. Docking
studies were also performed.
2. Methods
The reaction proved to be efficient as the conjugation with
several other natural amino acids 5bel, with the appropriate side
chain protection was performed and the corresponding
compounds, 6ael, were obtained in moderate to excellent yields, as
shown in Table 1.
The COX inhibitor Screening Assay directly measures PGF2 by
a
SnCl2 reduction of COX-derived PGH2 produced in the COX reac-
tion. The prostanoid product is quantified via enzyme immuno-
assay (EIA) using a broadly specific antiserum that binds to all the
major PG compounds. This assay includes COX-1 and COX-2
enzymes, allowing the screenning of isoenzyme-specific inhibi-
tors (Cayman, catalog number 560131). In brief, amino acid-sub-
3.2. COX inhibition activity
stitued compounds (10 nM; 20 mL) or celecoxib (10 nM; 20 mL) were
incubated with COX-1 or COX-2 enzyme during 10 min, 37 ꢀC.
Following this period of incubation, arachidonic acid was added
As stated before, several COX-2 inhibitors, namely the coxibs,
have as a common feature vicinal diaryl substituted heterocycles
and as the compounds synthesized in our lab resemble these COX
inhibitors, with the difference of being 2,5-diaryl substituted, their
biological activity and the influence of different amino acids were
investigated quantifying prostaglandin E2 (PGE2) levels in neutro-
phil culture supernatants.
Neutrophils are cells that are activated, secreting microbicidal
compounds, when microorganisms made their way into tissues,
thus killing the injurious agent [20]. They are also the most
important cells in the initial stage of sepsis [21]. In an exacerbated
inflammatory response, neutrophils activities cause huge tissue
damage [20], and therefore, inhibitions of neutrophils activities are
a target for developing anti-inflammatory drugs.
(10 mM; 10
stopped by adding 50
m
L) and incubated for 2 min at 37 ꢀC. The reaction was
m
l of HCl (1 M) and afterwards 100 ml of SnCl2
saturated solution was added. The solution was incubated during
5 min, at room temperature. The PGE2 formation was quantified in
EIA procedure (420 nm). The PGE2 formation was calculated on
basis on a standard curve and percentage of inhibition was based on
100% initial activity sample.
3. Results and discussion
3.1. Synthesis of 2,5-diarylfuran amino acid derivatives
By applying the technique described by Botteselle et al. [15]. The
synthesis of the desired 3-AFA 4 was accomplished starting from 2-
(4-methoxyphenyl)furan 1 as depicted in Scheme 1. The resulting
arylfuran 1 was converted into the butyltelluride 2 by treatment
with n-BuLi in THF followed by the addition of elemental tellurium
to generate the intermediate tellurolate, which was alkylated with
n-BuBr. This method was previously described in the literature [18].
The 2-(butyltellanyl)-5-(4-methoxyphenyl)furan 2 was subjected
to palladium(II) acetate-catalyzed SuzukieMiyaura coupling to
The effect on cell viability was measured on neutrophils treated
with 1, 10, 100, and 1000 nM of the amino acid-substituted
compounds. Concentrations greater than 100 nM reduced neutro-
phil viability, compared to control cells (data not shown). Incuba-
tion of neutrophils with 10 nM of the different amino acid-
substituted compounds did not modify the cell viability (Fig. 1).
PGE2 levels in the supernatant of cultured neutrophils incubated
with celecoxib and compounds with proline, valine, cysteine, and
phenylalanine at a final concentration of 10 nM, in absence or
Boc
H
N
NH2
N
H
DIC, HOBt
DCM, r.t., 20 h
O
+
N-Boc-Gly
O
O
MeO
MeO
3-A
FA 4
6a
5a
81 %
Scheme 2. Peptide coupling conditions used for the conjugation of 3-AFA 4 with N-Boc-Gly 5a.

54
H.A. Stefani et al. / European Journal of Medicinal Chemistry 47 (2012) 52e58
Table 1
Conjugation of several natural protected amino acids 5a-l with a free probe 3-AFA 4.
H
N
NH2
DIC
AA
AA
5a-l
O
+
O
HOBt, DCM
MeO
MeO
3-A
FA
4
6a-l
Entry
1
Product
Yield (%)a
81
Boc
Boc
H
N
N
H
O
O
6a
M eO
H
N
N
H
2
3
63
86
O
O
6b
M eO
M eO
Boc
H
N
N
H
O
O
6c
Boc
H
N
4
79
N
H
O
O
6d
M eO
M eO
H
N
N
5
6
91
73
Boc
O
O
6e
Ph
Boc
Tos
H
N
N
H
O
O
6f
M eO
H
N
HN
NH
7
55
Boc
H
N
N
H
O
O
MeO
M eO
SBn
6g
Cbz
H
N
N
H
8
9
50
89
O
O
6h
M eS
Boc
H
N
N
H
O
6i
O
M eO
M eO
O Bz
Boc
H
N
H
10
85
N
O
6j
O

H.A. Stefani et al. / European Journal of Medicinal Chemistry 47 (2012) 52e58
55
Table 1 (continued )
Entry
Product
Yield (%)a
O
t-Bu
O
11
43
Fm oc
H
N
N
H
O
6l
O
M eO
a
Isolated yields.
Table 2
Binding energy for ligands in the active site of COX-2.
Ligand
Goldscore
DGbinding (kcal/mol)
Redocking
Pro-R
Pro-S
Cys-R
Cys-S
Phe-R
Phe-S
Val-R
78.41
67.13
65.39
50.06
83.08
70.44
66.71
74.02
72.41
ꢁ10.70
ꢁ9.48
ꢁ9.30
ꢁ7.65
ꢁ11.20
ꢁ9.84
ꢁ9.44
ꢁ10.22
ꢁ10.05
ꢁ9.65
Val-S
Fig. 1. Effects of substituted amino acid groups on neutrophils viability. Cells were
obtained from male Wistar rats 4 h after intraperitoneal injection of oyster glycogen
(1%, 10 mL). Neutrophils were incubated with 10 nM of each compound in the
absence or presence of LPS (5 mg/mL) and for 18 h. Control cells were incubated
with cell medium (RPMI1640 þ 10% FBS). Neutrophil viability was determined by
trypan blue staining.
stimulation (in comparison to basal values). The valine, cysteine,
and phenylalanine-substituted compounds did not modify the
elevated concentration of PGE2; on the other hand, the proline-
substituted compound and celecoxib, a selective COX-2 inhibitor,
decreased LPS-induced PGE2 secretion (Fig. 2).
It has been shown that 18 h of culture rat leukocytes were
found to express COX-1 and there were no detectable levels of
COX-2, being the latter highly expressed after LPS stimulation
[22]. Thus, by measuring the PGE2 levels in non-stimulated and
LPS-stimulated neutrophils, it is shown that all amino acid-
substituted compounds did not affect COX-1 activity, as basal
values of PGE2 were not altered, but the proline-substituted
Table 3
Main interactions of Pro ligand in COX-2.
Ligand
Ligand
atom
Pro
Distance (Å)
Common part of the molecule
on the active site of COX-2
O1
O1
O1
O1
O2
O2
O3
O3
H1
H4
H8
H9
H10
H10
H14
H14
H18
H19
H19
H25
H25
H26
H26
H26
H27
H30
ARG120:HH11
TYR355:HH
ALA527:HB2
ALA527:HB3
HIS90:HE2
SER353:HA
THR94:HG22
THR94:HG23
LEU352:O
TYR355:OH
LEU352:O
LEU352:O
GLN192:OE1
SER353:O
GLN192:OE1
ASP515:O
PRO191:O
PRO191:O
ASP515:O
THR94:O
TYR355:O
THR94:O
2.88
3.00
3.18
3.30
2.02
2.80
2.66
3.48
2.87
2.99
2.76
3.17
2.13
3.02
2.68
3.36
3.24
3.39
2.98
3.38
2.50
3.15
3.13
2.50
2.78
3.15
Fig. 2. PGE2 levels were quantified in neutrophil culture supernatant by EIA. The
dotted line represents the basal value. Results are expressed as the mean ꢂ s.e.m. of
cells collected from four animals. *P < 0.001 vs. control.
presence of LPS, are shown in Fig. 2. In absence of stimulation,
similar levels of PGE2 were found in all cases as shown by the
dotted line in Fig. 2. LPS induced the secretion of PGE2, and
increased levels of the mediator were detected in control cells after
COX-1
COX-2
25
20
15
10
5
***
***
**
Part of the ligand exposed
to the solvent
0
Control
Proline
Valine
Cysteine Phenilalanine Celecoxib
10 nM
GLY354:O
TYR355:O
THR94:O
Fig. 3. Inhibition on COX-1 and COX-2 activities were measured using the EIA COX
inhibitors screening assay. Results are expressed as the mean ꢂ s.e.m. of % of inhibition.
***P < 0.001 and **P < 0.01 vs. control.
GLY354:O

56
H.A. Stefani et al. / European Journal of Medicinal Chemistry 47 (2012) 52e58
Fig. 4. (a) The ligands inside the active site. COX-2 is represented in ribbon form. In cyan is shown the part of the protein that penetrates the single leaflet of the lipid bilayer. (b) The
Pro compound (the common part of the molecules is in red), docked together with celecoxib and SC-558 from the crystal structure (PDB code: 1CX2). The figure was drawn with DS-
Visualizer [30].(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. The Pro-ligand/p interactions. For the sake of clarity, not all the H-atoms are shown. The figure was drawn with DS-Visualizer [30].

H.A. Stefani et al. / European Journal of Medicinal Chemistry 47 (2012) 52e58
57
Table 4
Amino acid hydrophobicities.
For the studied ligands the most hydrophilic moiety is proline,
so it would be expected that, as this part of the ligand is exposed to
the solvent, it should be the most active, moreover, the lowest
activity should be expected for valine, which is the most hydro-
phobic. As this is precisely what was observed in the inhibition
experiment, it can be rationalized that, in this case, inhibition
capacity was related to the part of the ligand that was outside the
active site, thus, exposed to the solvent.
Amino Acid
Hydropathy index
(highly hydrophilic)
Proline
ꢁ1.6
Cysteine
Phenylalanine
Valine
2.5
2.8
4.2
(highly hydrophobic)
4. Conclusion
compound markedly reduced LPS-induced PG2 secretion, which
may indicate a selective action on COX-2 activity. The effect of
proline-substituted on COX-2 activity was corroborated using
COX inhibitors screening assay [23,24]. As shown in Fig. 3, only
proline-substituted compound exhibited the same inhibitory
potency of celecoxib on COX-2 activity. Although, valine or
cistein-substituted compounds did not affect PGE2 levels on LPS-
stimulated neutrophils supernatant, their direct incubation with
the enzymes inhibited COX-2 action. The activities were lower
than those found in proline-substituted compound and celecoxib.
This difference on obtained data may be due experimental
conditions, involving different biological systems. While, PGE2
levels were quantified in cultured cells, which depend on cell
metabolism, COX activity was measured directly on enzyme
system, without cell participation. No significant inhibition on
COX-1 activity was detected in all of compounds studied. As
previously reported, the adverse effects of COX inhibitors are
mainly linked to inhibition of the production of COX-1 generated
products, which exert physiological actions [1].
We described herein the synthesis of a novel 2,5-diarylfuran
scaffold and its conjugation with different amino acids (3-AFA).
We showed that the proline-substituted compound inhibited PGE2
secretion by LPS-stimulated neutrophils and inhibited COX-2
activity similarly to celecoxib, showing selectivity for COX-2
activity.
The analysis of docking results allowed us to postulate that the
inhibition activity of the studied compounds was modulated by the
hydrophilicity of the amino acid that was attached to them.
Finally, we expect that these results will contribute to the
development of newer anti-inflammatory molecules with fewer
adverse side effects.
Acknowledgments
We thank the Brazilian Agencies FAPESP (07/59404-2 to H.A.S.),
CNPq (300613/2007 to H. A. S., 306532/2009-3 to J. Z.-S., 308116/
2010-0 to I.C.) and CAPES (Rede Nanobiotec-Brasil 808/2009 to J. Z.-
S. and I. C.) for financial support.
3.3. Docking studies
Appendix. Supplementary data
In order to rationalize the different biological results shown in
Fig. 2, molecular docking studies of the S and R-enantiomers for
compounds with Pro, Cys, Phe and Val, modeled with MarvinSketch
[25], and celecoxib were performed. The GOLD program (version
4.1.1) with GOLDScore was used [26e28]. The obtained GOLDScores
and corresponding binding energies are shown in Table 2.
The docking studies showed that, in all cases, the common part
of the molecules, namely the 2-(4-methoxyphenyl)-5-phenylfuran
moiety, fits in the long narrow hydrophobic channel of the COX-2
active site in the same fashion, whether the substituent amino
acids are positioned at the entrance of the channel that opens to the
active site, thus this entrance is in some instance “closed” by the
amino acid moiety which is, then, exposed to the solvent (Fig. 3a).
In Fig. 3b it is shown the superposition of the ligand SC-558, as it is
in the X-ray crystal structure [7] (PDB code: 1CX2 [29]) to the
docking results of celecoxib and the Pro compound, as represen-
tative of the poses of the compounds studied here. It can be seen
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.10.018.
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