Discovery of novel analgesic and anti-inflammatory 3-arylamine-imidazo[1,2-a]pyridine symbiotic prototypes

Article abstract of DOI:10.1016/j.bmc.2008.11.018

We describe herein the design, synthesis and pharmacological evaluation of novel 3-arylamine-imidazo[1,2-a]pyridine derivatives structurally designed as novel symbiotic prototypes presenting analgesic and anti-inflammatory properties. The derivatives obtained were submitted to in vivo assays of nociception, hyperalgesia and inflammation, and to in vitro assays of human PGHS-2 inhibition. These assays allowed the identification of compound LASSBio-1135 (3a) as an anti-inflammatory and analgesic symbiotic prototype. This compound inhibited moderately the human PGHS-2 enzyme activity (IC₅₀ = 18.5 μM) and reverted the capsaicin-induced thermal hyperalgesia (100 μmol/kg, po) similarly to p38 MAPK inhibitor SB-203580 (2). Additionally, LASSBio-1135 (3a) presented activity similar to celecoxib (1) regarding the reduction of the carrageenan-induced rat paw edema (33% of inhibition at 100 μmol/kg, po). We also discovered derivatives LASSBio-1140 (3c) and LASSBio-1141 (3e) as analgesic and anti-inflammatory prototypes, which were able to attenuate the capsaicin-induced thermal hyperalgesia (100 μmol/kg, po) and reduce the carrageenan-induced paw edema (ED₅₀ = 11.5 μmol/kg (3.3 mg/kg) and 14.5 μmol/kg (4.1 mg/kg), respectively), being both more active than celecoxib (1), despite the fact that their effects involve a different mechanism of action. Additionally, derivative LASSBio-1145 (3j) showed remarkable analgesic (ED₅₀ = 22.7 μmol/kg (8.9 mg/kg)) and anti-inflammatory (ED₅₀ = 8.7 μmol/kg (3.4 mg/kg)) profile in vivo (100 μmol/kg; po), in AcOH-induced abdominal constrictions in mice and carrageenan-induced rat paw edema models, respectively, being a novel orally-active anti-inflammatory drug candidate that acts as a selective PGHS-2 inhibitor (IC₅₀ = 2.8 μM).

Full text of DOI:10.1016/j.bmc.2008.11.018

Bioorganic & Medicinal Chemistry 17 (2009) 74–84
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel analgesic and anti-inflammatory
3-arylamine-imidazo[1,2-a]pyridine symbiotic prototypes
Renata B. Lacerda a,b, Cleverton K. F. de Lima a,c, Leandro L. da Silva a,c, Nelilma C. Romeiro a,
Ana Luisa P. Miranda a,c, Eliezer J. Barreiro a,b,c, Carlos A. M. Fraga a,b,c,
*
a Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Faculty of Pharmacy, Federal University of Rio de Janeiro, PO Box 68023, 21941-902, Rio de Janeiro, RJ, Brazil
b Programa de Pós-Graduação em Química, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
c Programa de Pós-Graduação em Farmacologia e Química Medicinal, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe herein the design, synthesis and pharmacological evaluation of novel 3-arylamine-imi-
dazo[1,2-a]pyridine derivatives structurally designed as novel symbiotic prototypes presenting analgesic
and anti-inflammatory properties. The derivatives obtained were submitted to in vivo assays of nocicep-
tion, hyperalgesia and inflammation, and to in vitro assays of human PGHS-2 inhibition. These assays
allowed the identification of compound LASSBio-1135 (3a) as an anti-inflammatory and analgesic sym-
biotic prototype. This compound inhibited moderately the human PGHS-2 enzyme activity
(IC50 = 18.5 lM) and reverted the capsaicin-induced thermal hyperalgesia (100 lmol/kg, po) similarly
to p38 MAPK inhibitor SB-203580 (2). Additionally, LASSBio-1135 (3a) presented activity similar to cele-
coxib (1) regarding the reduction of the carrageenan-induced rat paw edema (33% of inhibition at
Received 13 September 2008
Revised 7 November 2008
Accepted 8 November 2008
Available online 17 November 2008
Keywords:
3-Arylamine-imidazo[1,2-a]pyridines
p38 MAPK
Analgesic
Anti-inflammatory
Symbiotic drugs
100
and anti-inflammatory prototypes, which were able to attenuate the capsaicin-induced thermal hyperal-
gesia (100 mol/kg, po) and reduce the carrageenan-induced paw edema (ED50 = 11.5 mol/kg (3.3 mg/
kg) and 14.5 mol/kg (4.1 mg/kg), respectively), being both more active than celecoxib (1), despite the
fact that their effects involve a different mechanism of action. Additionally, derivative LASSBio-1145
(3j) showed remarkable analgesic (ED50 = 22.7 mol/kg (8.9 mg/kg)) and anti-inflammatory
(ED50 = 8.7 mol/kg (3.4 mg/kg)) profile in vivo (100 mol/kg; po), in AcOH-induced abdominal constric-
tions in mice and carrageenan-induced rat paw edema models, respectively, being a novel orally-active
anti-inflammatory drug candidate that acts as a selective PGHS-2 inhibitor (IC50 = 2.8 M).
lmol/kg, po). We also discovered derivatives LASSBio-1140 (3c) and LASSBio-1141 (3e) as analgesic
l
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l
l
l
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l
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
and preceding the development of an immune response. On the
other hand, the chronic inflammatory process involves the release
of diverse mediators which do not have significant participation in
the acute phase, as interleukins, interferon and tumor necrosis fac-
Inflammation is a complex defensive process that may vary
from a localized to a generalized response characterized by the
accumulation of local fluids and leukocytes with the objective of
eliminating the noxious stimulus. The inflammatory response oc-
curs in three distinct temporal phases, which seems to be mediated
by different physiological and immunological phenomena: the
acute phase, characterized by the transient local vasodilatation
and the increase of the capillary permeability; a delayed sub-acute
phase characterized by the infiltration of leukocytes and other
phagocytising cells; and the proliferative chronic phase, in which
occurs degeneration of the affected tissue and fibrosis.1
tor
a (TNF-a, a cytokine that plays a major role in this kind of
inflammatory process and whose production is associated with
some difficultly treatable diseases, for example, rheumatoid arthri-
tis, Crohn’s disease, among others.2
Although the efficacy of selective PGHS-2 enzyme inhibitors has
been proved, and these drugs together with the classical non-selec-
tive NSAIDs constitute the mainstay of symptomatic treatment of
many inflammatory disorders and soft-tissue injuries, the results
of two major prospective studies—the VIGOR (Vioxx Gastrointesti-
nal Outcomes Research Study) and APPROVe (Adenomatous Polyp
Prevention on Vioxx)—demonstrated an increased risk of serious
cardiovascular events, including heart attacks and strokes in pa-
tients making use of rofecoxib.3 This profile suggests that the ideal
NSAID does not have to be excessively selective for the PGHS-2 en-
zyme, for example, celecoxib (1), and demonstrates the need for
The acute inflammation occurs as the initial response to tissue
injury, being mediated by the release of autacoids, for example,
histamine, serotonin, bradikynin, prostaglandins and leukotrienes,
* Corresponding author. Tel.: +55 21 25626503; fax: +55 21 25626644.
E-mail address: cmfraga@pharma.ufrj.br (C.A.M. Fraga).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.018

R. B. Lacerda et al. / Bioorg. Med. Chem. 17 (2009) 74–84
75
searching new strategies and new targets for efficient and safe
treatment of the chronic inflammatory diseases.4
nase inhibitors. The rational approach exploited in the design of
compounds belonging to series (3) considered the diarylheterocy-
clic subunit as a common structural feature of selective inhibitors
of both targets,10 for example, celecoxib (1) and SB-203580 (2), and
led us to propose the combination of their corresponding pharma-
cophoric groups in a common azahomologue structural pattern, by
applying the molecular hybridization strategy11 (Fig. 1).
The imidazo[1,2-a]pyridine ring was chosen as the core hetero-
cyclic scaffold of target compounds (3), due to its presence in the
pyridazinone p38 MAPK inhibitor (4)12 and also because it can be
envisioned as a resultant of the fusion of the imidazole and phenyl
rings present in SB-203580 (2). Additionally, the anti-inflamma-
tory and antinociceptive activity of some N-acylhydrazone deriva-
tives belonging to imidazo[1,2-a]pyridine class, for example,
4-bromophenyl derivative (5) was previously described by our
research group,13 corroborating the importance of this azahetero-
cyclic ring for the desired bioprofile (Fig. 1).
The interest in the MAP kinase pathway as a potential target for
the development of novel anti-inflammatory drugs has grown re-
cently. In fact, the identification of p38 MAPK as the target of some
pyridinil-imidazole compounds5 confirmed the role of this intra-
cellular enzyme in the regulation of many physiological and path-
ological states and reinforced the importance of its modulation in
the therapy of some inflammatory diseases. The clinical assays
with the selective p38 inhibitors demonstrate the therapeutic ben-
efits of the blockage of this signaling pathway responsible for the
regulation of cytokines production, for example, IL-1b and TNF-
a 6
. Moreover, the characterization of the pharmacological profile
displayed by the selective p38 inhibitor prototype SB203580 (2),
proved its disease-modifying activity in the adjuvant-induced
arthritis model.6 This set of results strongly suggests that adequate
modulation of production of these cytokines can bring significant
benefits to the therapy of chronic inflammatory diseases,6 for
example, rheumatoid arthritis, psoriasis, asthma, chronicle
obstructive pulmonary disease (COPD) and Crohn’s disease, and
has supported the potential use of selective p38 inhibitors as
monotherapy7 or in set with other drugs able to act synergistically.
The combination of two or more clinically validated therapeutic
targets with mutual participation in specific multifactorial diseases
can represent an interesting strategy to increase the available
pharmacological space for the discovery of new drugs.8 Emerging
from this new Medicinal Chemistry approach, the concept of sym-
biotic drugs appears as an interesting therapeutic innovation in the
search for more efficient and safe drugs for treating chronic dis-
eases. A design of these symbiotic agents should be made to allow
the molecular recognition of two distinct biological targets, in-
volved in the same disease and belonging to different biochemical
pathways, in order to promote the treatment and the prevention of
the harmful stimulus amplification more efficiently.9
The functionalized aromatic rings Ar1 and Ar2, for example,
4-(methylsulfonyl)phenyl, 2-pyridinyl, 4-pyridinyl and other isos-
teric subunits, were elected in order to retain, in just one chemical
entity, the characteristic pharmacophoric groups of selective p38
MAPK and PGHS-2 inhibitors, aiming to favor their simultaneous
blockage (Fig. 1).
2. Results and discussion
2.1. Chemistry
The new functionalized 3-arylamine imidazo[1,2-a]pyridine
derivatives (3a–h) were synthesized by exploring at the key-step,
the three component condensation (3CC) of heterocyclic amidines
with aldehydes and isocyanides described by Groebke14 as a vari-
ation of the classic multi-component condensations originally
developed by Passerini15 and Ugi.16 The synthesis of isocyanides
(6a–d) was performed in two steps, starting with the formylation
of the corresponding aromatic amines (7a–d) by refluxing with
ethyl formate in triethylamine17 to produce the corresponding for-
mamides (8a–d) in 35–95% yield (Scheme 1). The 2-pyridinil-form-
In this context, the present work describes the synthesis and the
investigation of the analgesic and anti-inflammatory properties of
new 3-arylamine-imidazo[1,2-a]pyridine derivatives (3) designed
as symbiotic agent candidates acting as PGHS-2 and p38 MAP ki-
Figure 1. Design concept of new 3-arylamine imidazo[1,2-a]pyridine derivatives (3a–n).

76
R. B. Lacerda et al. / Bioorg. Med. Chem. 17 (2009) 74–84
Scheme 1. Reagents and conditions: (a) ethyl formate, Et3N, reflux, 20–96 h; (b) POCl3, CH2Cl2, Et3N, 0 °C, 2 h; (c) i—HCO2H, Ac2O, 50–60 °C, 2 h, ii—2-aminopyridine, ethyl
ether, rt, 48 h; (d) AcOH, MeOH, rt, 24 h (see Table 1).
amide derivative (8a) obtained in only 35% yield after 96 h could
alternatively be prepared in 78% yield through the formylation of
2-aminopyridine (9) with the more reactive mixed formylacetyl
anhydride generated in situ18 (Scheme 1).
Afterward, the desired isocyanides (6a–d) were obtained in
yields varying from 40% to 90%, exploiting the dehydration of cor-
responding formamides (8a–d) by treatment with POCl3 in trieth-
ylamine (Scheme 1).16,17
matic aldehydes (10–13) and 2-aminopyridine (9) in methanol,
under acidic conditions (Scheme 1 and Table 1).14 Contrary to
our expectations, the 3-arylamino imidazo[1,2-a]pyridine deriva-
tives presenting 4-pyridinyl ring, that is (3c) and (3d), were not
formed when three-component condensation was performed
exploiting 4-pyridinylcarboxaldehyde (11) as the electrophilic spe-
cies. The side-products from these reactions were identified as the
corresponding 4-pyridinyl imines (14) and (15), respectively
(Fig. 2), which are probable resulted from condensation of the het-
eroaromatic aldehyde with the aniline formed due to partial
hydrolysis of isocyanides (6b) and (6d) during the long reaction
The target derivatives 3-arylamine-imidazo[1,2-a]pyridine (3)
were obtained in moderate yields, by applying the multi-compo-
nent condensation of isocyanides (6a–d) with the appropriate aro-
Table 1
Yields and physical properties of the 3-arylamino imidazo[1,2-a]pyridine derivatives (3)
Compound
Ar1
Ar2
Molecular formulaa
MW
Yield (%)
X
Y
R
Z
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3l
3m
3n
N
N
CH
CH
N
N
H
SCH3
H
SCH3
H
H
OCH3
H
H
OCH3
H
SO2CH3
SO2CH3
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
N
CH
CH
C18H14N4
C19H16N4S
C18H14N4
C19H16N4S
C19H15N3
C20H17N3S
C21H19N3OS
C19H16N4S
C20H17N3O2S
C21H19N3O3S
C19H16N4O2S
C19H16N4O2S
C19H16N4O2S
286.33
332.42
286.33
332.42
285.34
331.43
361.46
332.42
363.43
393.46
364.42
364.42
364.42
40
40
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
0 (50)c
0 (60)b
25
CH
C-SCH3
C-SCH3
C-SCH3
C-SO2CH3
C-SO2CH3
C-SO2CH3
CH
50
70
50
60c
60c
80c
60c
85c
CH
N
a
The analytical results for C, H, N, S were within 0.4% of calculated values.
Yields obtained from alternative two step condensation described in Scheme 2.
Obtained by chemoselective oxidation of the corresponding methylsulfide derivatives (3b), (3d) and (3f–h) as described in Scheme 3.
b
c

R. B. Lacerda et al. / Bioorg. Med. Chem. 17 (2009) 74–84
77
and the hydrogen located at C-5 of the azaheterocyclic ring of
derivative (3b), impossible to occur in 2-arylamino isomer (Fig. 3).
All new imidazo[1,2-a]pyridine derivatives described herein
were completely characterized by common spectroscopic methods
(see Section 4) and their analytical results for C, H, and N were
within 0.4% of calculated values.
Figure 2. Imine derivatives (14) and (15) evidenced as the major products obtained
from three-component condensation of 4-pyridinylcarboxaldehyde (11), 2-amino-
pyridine (9) and phenylisocyanide (6d) or 4-methylthiophenylisocyanide (6b),
respectively.
2.2. Analgesic and anti-inflammatory activities
First, we evaluated the in vivo analgesic and anti-inflammatory
profile of eight 3-arylamine-imidazo[1,2-a]pyridine derivatives
(3a), (3c), (3e), (3i), (3j), (3l), (3m), (3n) elect, presenting pharma-
cophoric groups for COX-2 and p38 MAPK inhibition, in the acetic
acid induced abdominal constrictions22 in mice and carrageenan-
induced rat paw edema (CIRPE),23 respectively. The new 3-aryl-
amine-imidazo[1,2-a]pyridine compounds and celecoxib (1), used
time of this kinetically unfavorable three-component coupling un-
der acidic conditions.
Given the limitation of classical 3CC methodology to prepare
imidazo[1,2-a]pyridine derivatives (3c) and (3d) we decided to ap-
ply a two step variation of this reaction, where the imine derivative
(16) was initially prepared through the condensation of equimolar
amounts of 4-pyridinylcarboxaldehyde (11) with 2-aminopyridine
(9) in toluene,19 followed by the addition of isocyanide derivative
(6b) or (6d) to furnish the desired compounds (3d) and (3c) in
60% and 50% yield, respectively (Scheme 2 and Table 1).
Finally, the 3-arylamine-imidazo[1,2-a]pyridine derivatives
(3b), (3d) and (3f–h) with the methylsulfide group attached to Ar1
or Ar2 subunits were chemoselectively oxidized to the correspond-
ing sulfones in good yields (60–80%) using OxoneÒ (2KHSO5ÁKH-
SO4ÁK2SO4) as the oxidizing agent (Scheme 3 and Table 1).20
Although Mandair et al.21 have reported the possible formation
of regioisomers during the construction of functionalized imi-
dazo[1,2-a]pyridine ring exploiting the 3CC strategy in a previous
work, the regioselective formation of 3-arylamino derivatives
was confirmed through 2D-NOESY experiment, which showed
the spatial relationship between the hydrogen at the NH group
Figure 3. Characterization of 3-arylamino imidazo[1,2-a]pyridine regioisomer by
NOESY experiment.
Scheme 2. Reagents and conditions: (a) toluene, 50 °C, 30 min; (b) Ph-NC or 4-H3CSPh-NC, NH4Cl, reflux, 30 h (see Table 1).
Scheme 3. Reagents and conditions: (a) OxoneÒ, MeOH, rt, 1 h (see Table 1).

78
R. B. Lacerda et al. / Bioorg. Med. Chem. 17 (2009) 74–84
as standard drug, were administered orally at the dose of
100 mol/kg. The obtained results are disclosed in Tables 2 and
would like to emphasize (3e), which was the most active despite
being a non-functionalized derivative, presenting 64% of inhibi-
l
3, respectively.
tion of constrictions and an ED50 of 14.5
On the other hand, the most active analgesic compound (3j) pre-
sented an ED50 of 8.7 mol/kg (3.4 mg/kg) regarding the anti-
inflammatory effect in CIRPE model, proving to be, once again,
10-fold more potent than celecoxib (1) (ED50 = 87.7 lmol/kg
(33.4 mg/kg)).
Considering that the anti-edematogenic profile observed for
(3j), (3a) and (3e) could depend at least in part on PGHS inhibition,
we decided to perform whole blood in vitro assay25 to investigate
their ability to inhibit isoforms 1 and 2 of the target enzyme. PGHS-
1 activity was measured through TXB2 production in blood sam-
ples drawn from donors that had not taken any NSAID for two
weeks, while LPS-induced PGHS-2 activity was also determined
by TXB2 plasma levels measured by RIA. None of the tested com-
pounds inhibited the production of TXB2 derived from PGHS-1 at
lmol/kg (4.1 mg/kg).
The analysis of these results led us to conclude that the most ac-
tive analgesic compound was the arylsulfone derivative (3j), which
was able to reduce remarkably the AcOH-induced constrictions,
presenting 53.0% of inhibition (Table 2). The expressive reduction
of the analgesic activity evidenced for Ar1 aza-substituted ana-
logues (3m) and (3n), which were both able to reduce the constric-
tions in ca. 33%, haspointed the pharmacophoric character of the
para-methoxyphenyl framework for the antinociceptive profile of
the imidazo[1,2-a]pyridine derivatives of series (3). The ED50 ob-
l
tained for compound (3j), that is, 22.7 lmol/kg (8.9 mg/kg) (Table
1), showed that, as analgesic agent, it is 10-fold more potent than
COX-2 inhibitor celecoxib (1).24
The anti-inflammatory profile of imidazo[1,2-a]pyridine deriva-
tives (3a), (3c), (3e) and (3i–n) was then evaluated in the CIRPE
test,23 as described in Table 3.
the concentration of 100 lM, contrary to the behavior observed
Compounds (3i), (3a), (3c), (3e) and (3j) were able to inhibit
edema formation significantly and the last three were more active
than celecoxib (1), the standard anti-inflammatory drug (30%
for celecoxib (1), which was able to inhibit it in 53.4% at same mo-
lar concentration. The results showed that compounds (3a) and
(3j) were able to inhibit the production of TXB2 from PGHS-2 with
inhibition at 100
l
mol/kg). Among the active derivatives, we
IC50 of 18.5 lM and 2.8 lM, respectively (Table 1). On the other
hand, compound (3e) did not act as PGHS-2 inhibitor, showing that
its great anti-inflammatory activity should be dependent on other
mechanism of action.
Table 2
Analgesic profile of imidazo[1,2-a]pyridine compounds (3a), (3c), (3e) and (3i–n) in
AcOH-induced abdominal constriction test
2.3. Anti-hyperalgesic activity
Compound
Dose
mol/kg)
No. of
Inhibition
(%)
ED50
mol/kg)
The well-known involvement of peripheral p38 MAPK in
hyperalgesic states induced by activation of vanniloid receptors
led to the evaluation of the in vivo antinociceptive profile of 3-
arylamine-imidazo[1,2-a]pyridine compounds in the capsaicin-
induced thermal hyperalgesia model.26 The title compounds
(3), celecoxib (1) and p38 MAPK inhibitor SB203580 (2), used
as reference compounds, were administered orally at the dose
(l
constrictionsa
(l
Vehicle
(1) Celecoxib
(3a)
(3c)
(3e)
(3i)
(3j)
(3l)
(3m)
(3n)
—
59.2 1.33
31.9 4.7
61.2 2.9
51.5 2.3
52.8 2.9
54.8 3.4
27.8 3.7
46.0 1.7
39.2 3.3
39.3 3.3
—
—
100
100
100
100
100
100
100
100
100
46.1*
247.0c
—
À3.0 nsb
13.0 nsb
10.8 nsb
7.4 nsb
53.0*
—
—
—
22.7
—
of 100 lmol/kg. The analysis of the obtained results disclosed
22.2*
in Figure 4 show that only compounds (3j) and (3i) have no ef-
fect on capsaicin-induced thermal hyperalgesia. All the other
compounds tested are effective as anti-hyperalgesic agents.
Among the most active imidazo[1,2-a]pyridine derivatives, (3a),
(3l) and (3n) presented bioprofile comparable to selective p38
MAPK inhibitor SB-203580 (2). The first is able to reverse com-
pletely the capsaicin-induced thermal hyperalgesia (Fig. 4A). The
analgesic activity observed for derivative (3a) indicated the impor-
tance of the 2-pyridinil subunit attached to the heterocyclic central
core, as the activity was significantly reduced when this group was
replaced by the isosteric 4-pyridinyl or phenyl rings in compounds
(3e) or (3c), respectively. Moreover, the comparison of the biopro-
file of regioisomers (3m) and (3l) in the exploited hyperalgesy
model led to the conclusion that the connection of 2-pyridinil
group at the amine group instead of C-2 atom of the imi-
dazo[1,2-a]pyridine scaffold is more favorable to the analgesic
activity, once that compound (3m) is more active than compound
(3l) (Fig. 4F vs Fig. 4G). This profile is probably resultant from the
greater conformational flexibility of 2-pyridinil subunit of (3l) in
comparison with (3m), where the nitrogen atom of this heterocy-
clic subunit forms an intramolecular hydrogen bond with the vic-
inal NH group, contributing to the conformational constraint of
this derivative (Fig. 5).
33.7*
—
—
33.6*
Compounds were administered orally; N = 7–10 animals.
a
Results expressed in terms of mean SEM.
Not statistically significant.
Data obtained from Ref. 24.
p < 0.05 (ANOVA one way followed by Dunnet test).
b
c
*
Table 3
Anti-inflammatory activity of imidazo[1,2-a]pyridine derivatives (3a), (3c), (3e) and
(3i–n) in carrageenan-induced rat paw edema (CIRPE) assay and PGHS-2 inhibitory
profile of some elected compounds in human whole blood (HWB) model
Compound
Dose
mol/
D
(
Volumea Inhibition ED50
HWB (PGHS-2)
(
l
l
L)
(%)
(
lmol/
IC50 (l
M)c
kg)
Kg)
Vehicle
(1) Celecoxib
(3a)
(3c)
(3e)
(3i)
(3j)
(3l)
(3m)
(3n)
—
520.8 17.8
—
—
87.7
—
11.5
14.5
—
8.7
—
—
100
100
100
100
100
100
100
100
100
364.1 36.7 30.0*
346.5 51.1 33.4*
260.7 33.0 49.9*
187.8 23.7 63.9*
373.9 41.3 28.2*
303.2 52.7 41.7*
431.3 67.8 17.2 nsb
424.0 35.8 18.5 nsb
511.3 47.5 1.8 nsb
2.9
18.5
—
nsb
—
2.8
—
—
—
—
The results obtained from this pharmacological assay indicate a
possible action towards the p38 MAPK pathway, since inhibitors of
the activity or the activation of the p38 MAPK enzyme can revert
the capsaicin-induced thermal hyperalgesia.
Additionally, these results showed that the selective PGHS-2
inhibitor, celecoxib (1), does not have significant activity in this
model, except for a low activity after 10 min from the beginning
—
Compounds were administered orally; N = 7–10 animals.
a
Results expressed in terms of mean SEM.
Not statistically significant.
b
c
Determined through the measurement of TXB2 production derived from LPS-
induced PGHS-2.
*
p < 0.05 (ANOVA one way followed by Dunnet test).

R. B. Lacerda et al. / Bioorg. Med. Chem. 17 (2009) 74–84
79
Figure 4. Time course effect of compounds (3i) (A), (3a) (B), (3c) (C), (3e) (D), (3j) (E), (3m) (F), (3l) (G), (3n) (H) in the capsaicin-induced thermal hyperalgesia. Compounds
were administered orally. N = 7–10 animals. *p < 0.05 (ANOVA one way followed by Dunnet test).
of the experiment, showing that the reduction of prostanoids bio-
synthesis deriving from the PGHS-2 inhibition does not lead to the
total or partial reversal of capsaicin-induced thermal hyperalgesia.
Thus, we can conclude that the activity shown in this assay for
derivatives (3a), (3c), (3e), (3m), (3l) and (3n) should not be attrib-
uted to an eventual inhibition of PGHS-2.
parison of the interactions performed by SC-558 in the crystal
and the docked structure of (3j) with PGHS-2 (Fig. 6) shows that
the methyl sulfonyl group of (3j) hydrogen bonds to the amino
acid residue His90, similarly to the pharmacophoric sulfonamide
group pertaining to SC-558. Moreover, the methoxyl group hydro-
gen bonds to both Tyr355 and Arg120, in the same region where
the trifluoromethyl group of SC-558 binds, through hydrogen
bonding with Arg120. Additionally, the imidazo[1,2-a]pyridine
ring is not positioned in the same region as the central pyrazolic
core of SC-558. Instead, it is close to the p-Br-phenyl ring of SC-
558 (17), in the aromatic region of the active site lined by aro-
matic amino acid residues such as Phe381, Tyr385 and Trp387,
among others. This result suggests extra interactions of (3j) in
this hydrophobic pocket and indicates an inverted pharmaco-
2.4. Docking studies
The complex generated by docking studies of (3j) with PGHS-2
and superimposition with the structure of the selective inhibitor,
SC-558 (17), co-crystallized with PGHS-2,27 illustrated in Figure 6,
shows that compound (3j) can bind in the active site of this en-
zyme in a similar fashion as the pyrazolic prototype (17). Com-

80
R. B. Lacerda et al. / Bioorg. Med. Chem. 17 (2009) 74–84
Figure 5. Minimum energy conformations obtained for compounds (3m) and (3l) by using the semi-empirical AM1 method.
Figure 6. Representation of the top posing docked complex of (3j) (green carbon atoms) and the PGHS-2 inhibitor SC-558 (17) (yellow carbon atoms) in the active site of
PGHS-2. Hydrogen bonds are shown as green dashed lines. Only polar hydrogens are shown for clarity.
phore compared to what has been described so far for PGHS-2
inhibitors. In short, the described interactions are typical of selec-
tive inhibitors of PGHS-2, confirming the molecular design of the
reported class of analgesic and anti-inflammatory imidazo[1,2-
a]pyridine derivatives (3).
Bio-1140 (3c) showed no relation to PGHS-2 inhibition, which
opens a new possibility for further investigations of the pharmaco-
logical mechanism of action of these compounds.
Finally, it was also demonstrated that imidazo[1,2-a]pyri-
dine derivative LASSBio-1145 (3j) is a novel PGHS-2 inhibitor,
10-fold more potent than celecoxib as an analgesic and an
anti-inflammatory agent in the AcOH-induced abdominal
3. Conclusions
constriction in mice (ED50 = 22.7
lmol/kg (8.9 mg/kg)) and car-
rageenan-induced rat paw edema (ED50 = 8.7
lmol/kg (3.4 mg/
The rational design of new imidazo[1,2-a]pyridine compounds
based on the symbiotic approach, aiming at both p38-MAPK and
PGHS-2 as therapeutic targets, was successful. Compound LASS-
Bio-1135 (3a) was identified as a novel analgesic and anti-inflam-
matory orally-active prototype, acting through the inhibition of
PGHS-2 and probably through the inhibition of the p38-MAPK
pathway, proving to be a symbiotic drug candidate.
In addition, we observed that derivatives LASSBio-1140 (3c) and
LASSBio-1141 (3e) were more active as antiedematogenic agents
than celecoxib at the same molar dose and presented anti-hyperal-
gesic activity in the capsaicin-induced thermal hyperalgesia model.
It is worth considering that the anti-inflammatory effect of LASS-
kg)) models. This result was corroborated by molecular
docking studies with PGHS-2, which showed that this com-
pound presents the pharmacophoric requisites for PGHS-2
inhibition.
4. Experimental
4.1. Chemistry
Reactions were routinely monitored by thin-layer chromatog-
raphy (TLC) in silica gel (F245 Merck plates) and the products

R. B. Lacerda et al. / Bioorg. Med. Chem. 17 (2009) 74–84
81
visualized with iodine or ultraviolet lamp (254 and 365 nm). 1H
4.1.2. Alternative procedure for preparation of 2-pyridinylfor-
mamide (8a)18
and 13C nuclear magnetic resonance (NMR) spectra were deter-
mined in CDCl3, DMSO-d6 and trifluoroacetic acid-d (TFA) solu-
tions using a Bruker AC-200 spectrometer. Peak positions are
given in parts per million (d) using tetramethylsilane as internal
standard, and coupling constant values (J) are given in Hz. Infra-
red (IR) spectra were obtained using a Nicolet Magna IR 760 spec-
trometer. Samples were examined as potassium bromide (KBr)
disks. Melting points were determined using a Quimis instrument
and are uncorrected. Column chromatography purifications were
performed using silica gel Merck 230–400 mesh. All described
products showed 1H and 13C NMR spectra according to the as-
signed structures.
Formic acid (0.8 ml, 22 mmol) and acetic anhydride (2.0 ml,
22 mmol) were mixed and stirred at 50–60 °C for 2 h. After cooling
this mixture to room temperature, 2-aminopyridine (0.93 g,
10 mmol) was added in portions. Then, ethyl ether (30 ml) was
added and the mixture was allowed to stir for another 48 h at room
temperature. Then, the solvent was concentrated under reduced
pressured, followed by the addition of 10% aq NaHCO3 (30 ml)
solution to the obtained residue and extraction with dichlorometh-
ane (3Â 30 ml). The organic layer was separated, dried over anhy-
drous sodium sulfate, filtered, and concentrated at reduced
pressure to furnish 2-pyridinylformamide (8a) (0.94 g) in 78%
yield. The spectroscopic data of the samples of compound (8a) ob-
tained from this methodology are in agreement with those previ-
ously described.
4.1.1. General procedure for preparation of N-arylformamides
(8a–d)17
A round-bottomed flask fitted with a reflux condenser was fed
with 49.5 mmol of the corresponding arylamines (7a–d) and
23.0 g (25 ml) of ethyl formate. The mixture was stirred and
heated at reflux while 5.5 g (7.6 ml) of triethylamine was added,
and afterward for another 24 h. After cooling at room tempera-
ture the reaction mixture was concentrated, followed by addition
of water (50 ml). The obtained residue was submitted to extrac-
tion with dichloromethane (3Â 40 ml). The organic layer was sep-
arated, dried over anhydrous sodium sulfate, filtered, and
concentrated at reduced pressure to furnish the desired aromatic
formamides (8a–d) as described next. All peaks in the 1H and 13C
NMR spectra of these derivatives were doubled, due to restricted
rotation about the amide bond as previously described by Krein
and Lowary.28
4.1.3. General procedure for preparation of arylisonitriles (6a–
d)16,17
A round-bottomed flask equipped with a thermometer and a
pressure-equalizing dropping funnel bearing a nitrogen inlet was
charged with 40 mmol of the corresponding arylformamide deriv-
ative (8a–d), 13.8 ml of triethylamine and 40 ml of dichlorometh-
ane. After the resulting solution was stirred and cooled to À2°
using an ice-salt bath, 3.7 ml of phosphorus oxychloride was added
dropwise over 15–20 min, while the temperature was kept at 0°.
The mixture became reddish brown as it was stirred and cooled
at 0° for another hour. Then, ice–salt bath was removed and re-
placed by an ice–water bath. Stirring was continued while a 10%
aq Na2CO3 solution (40 ml) was added dropwise at such a rate that
the temperature of the mixture was maintained at 25–30 °C. The
two-phase mixture was stirred for another 30 min, after which
water was added until the volume of the aqueous layer was
brought to 100 ml. The aqueous layer was separated and extracted
with dichloromethane (3Â 80 ml). The organic layers were com-
bined, dried over anhydrous sodium sulfate and evaporated under
reduced pressure to furnish the desired aromatic isonitriles (6a–d)
as a crude oil, which were immediately used in the ‘3CC’ step with-
out further purification.
4.1.1.1. 2-Pyridinylformamide (8a). Derivative (8a) was ob-
tained in 35% yield, as a yellow solid, mp 34–36 °C. 1H NMR
(200 MHz, CDCl3, d): 10.31 (br s, 1H), 10.16 (br s, 1H), 9.31 (d,
1H, J = 8.1 Hz), 8.51 (s, 1H), 8.30–8.28 (m, 2H), 8.26 (d, 1H,
J = 8.1 Hz), 7.80–7.60 (m, 2H), 7.06 (br s, 2H), 6.93 (d, 1H,
J = 8.1 Hz); 13C NMR (50 MHz, CDCl3, d): 163.2, 159.7, 151.1,
151.0, 148.5, 147.2, 139.1, 138.8, 120.2, 119.9, 115.3, 110.7. IR tmax
(KBr): 3197, 2852, 1691, 1595, 1433, 1303, 778, 517 cmÀ1
.
4.1.4. General procedure for preparation of 3-arylamino-
imidazo[1,2-a]pyridines (3a–h) using classical ‘3CC’
methodology14
4.1.1.2. 4-(Methylthio)phenylformamide (8b). Derivative (8b)
was obtained in 90% yield, as a white solid, mp 68–70 °C. 1H
NMR (200 MHz, CDCl3, d): 8.65 (s, 1H), 8.33 (s, 1H), 7.85 (s,
1H), 7.47 (d, 2H, J = 8.1 Hz), 7.23 (m, 4H), 7.03 (d, 2H,
J = 8.1 Hz), 2.46 (s, 6H). 13C NMR (50 MHz, CDCl3, d): 162.7,
159.1, 135.1, 134.3, 134.2, 134.2, 128.3, 127.7, 120.6, 119.5,
16.4; IR tmax (KBr): 3310, 2916, 2866, 1715, 1683, 1527, 1308,
Equimolar amounts of aromatic aldehyde, 2-aminopyridine and
arylisonitrile (1 mmol) were dissolved in 3 ml of methanol, fol-
lowed by addition of 2 mmol of glacial acetic acid. The resulting
mixture was stirred at room temperature for 24 h then acidified
with 1 N HCl until pH 1 and stirred for 30 min to destroy the resid-
ual isonitrile. After concentration of the reaction mixture under
reduced pressure, 10% aq NaHCO3 solution (15 ml) was added to
the obtained residue, followed by extraction with ethyl acetate
(3Â 50 ml). The solvent was evaporated under reduced pressure.
The organic layer was combined, dried over anhydrous sodium sul-
fate, filtered, and concentrated at reduced pressure to furnish the
desired 3-arylamino-imidazo[1,2-a]pyridine derivatives (3a–h) as
described next.
814, 516 cmÀ1
.
4.1.1.3. 4-(Methoxy)phenylformamide (8c). Derivative (8c)
was obtained in 95% yield, as a white solid, mp 62–64 °C. 1H
NMR (200 MHz, CDCl3, d): 8.86 (br s, 1H), 8.50 (br s, 1H), 8.36 (br
s, 1H), 8.25 (br s, 1H), 7.43 (d, 2H, J = 8.5 Hz), 7.02 (d, 2H,
J = 8.5 Hz), 6.83 (m, 4H), 3.77 (s, 3H), 3.74 (s, 3H); 13C NMR
(50 MHz, CDCl3, TMS) d (ppm): 159.1, 157.2, 156.2, 129.8, 129.4,
121.5, 121.1, 114.5, 113.8, 55.1. IR tmax (KBr): 3251, 3057, 2885,
1672, 1497, 1303, 1022, 833, 689, 517 cmÀ1
.
4.1.4.1. 2-Phenyl-N-(2-pyridinyl)imidazo[1,2-a]pyridin-3-amine
4.1.1.4. Phenylformamide (8d). Derivative (8d) was obtained in
80% yield, as a yellow solid, mp 34–36 °C. 1H NMR (200 MHz,
CDCl3, d): 9.13 (br s, 1H), 8.78 (br s, 1H), 8.38 (br s, 2H), 7.60–
7.57 (m, 2H), 7.42–7.31 (m, 4H), 7.26–7.05 (m, 4H); 13C NMR
(50 MHz, CDCl3, d): 162.7, 159.3, 136.7, 136.5, 129.3, 128.7,
124.9, 124.4, 119.8; IR tmax (KBr): 3264, 3057, 2874, 1695, 1597,
(3a).
Derivative (3a) was obtained in 40% yield, by condensa-
tion of 2-aminopyridine (9), phenylisonitrile (6d) and 2-pyridinyl-
carboxaldehyde (10), as a yellow solid, mp 235–237 °C. 1H NMR
(200 MHz, CDCl3, d): 8.55 (d, 1H, J = 4.4 Hz), 8.29 (s, 1H), 8.18 (d,
1H, J = 8.0 Hz), 7.75 (td, 1H, J = 7.8 and 1.6 Hz), 7.69–7.60 (m, 2H),
7.26–7.10 (m, 4H), 6.91 (t, 1H, J = 7.3 Hz), 6.75 (t, 1H, J = 7.3 Hz),
6.65 (d, 2H, J = 7.8 Hz); 13C NMR (50 MHz, CDCl3, d): 154.3, 148.7,
1442, 1311, 753, 541 cmÀ1
.

82
R. B. Lacerda et al. / Bioorg. Med. Chem. 17 (2009) 74–84
143.4, 141.3, 136.5, 131.8, 129.3, 125.2, 123.9, 123.8, 121.4, 120.8,
4.1.4.7. Alternative two-step ‘3CC’ procedure for preparation of
3-arylamino-imidazo[1,2-a]pyridine derivatives (3c) and
120.7, 117.8, 116.6, 111.8; IR tmax (KBr): 3207, 2951, 1601, 1560,
1387, 1237, 833, 750, 698 cmÀ1
.
(3d). 19 A solution of 2-aminopyridine (1 mmol) and 4-pyridine-
carboxaldehyde (1 mmol) in toluene (15 ml) was heated at 50 °C
for 30 min. Then, after addition of solid ammonium chloride
(2 mmol) and the respective arylisonitrile derivative (1 mmol),
the reaction mixture was refluxed for another 30 h. While still
hot, the resulting dark brown solution was washed with warm
water (50 ml), cooled down to room temperature and diluted with
n-hexane (50 ml). The solvent was concentrated under reduced
pressure and the desired product was obtained through acid–base
extraction. The solid obtained was recrystalized from hexane–ethyl
acetate (1:1) to furnish imidazo[1,2-a]pyridine derivatives as de-
scribed next.
4.1.4.2. 2-(4-(Methylthio)phenyl)-N-(2-pyridinyl)imidazo[1,2-
a]pyridin-3-amine (3b). Derivative (3b) was obtained in 40%
yield, by condensation of 2-aminopyridine (9), 4-(methyl-
thio)phenylisonitrile (6b) and 2-pyridinylcarboxaldehyde (10), as
a yellow solid, mp 154–156 °C. 1H NMR (200 MHz, CDCl3, d):
8.53 (d, 1H, J = 4.8 Hz), 8.27 (s, 1H), 8.18 (d, 1H, J = 8 Hz), 7.73
(td, 1H, J = 7.8 and 1.7 Hz), 7.68–7.55 (m, 2H), 7.26–7.08 (m,
4H), 6.73 (t, 1H, J = 6.7 Hz), 6.59 (d, 2H, J = 8.6 Hz), 2.66 (s, 3H);
13C NMR (50 MHz, CDCl3, d): 154.6, 149.0, 142.0, 136.9, 132.2,
130.1, 129.4, 125.3, 124.2, 124.0, 121.8, 121.0, 118.2, 117.6,
112.2, 18.0; IR tmax (KBr): 3306, 3020, 1597, 1493, 1387, 1237,
750, 689 cmÀ1
.
4.1.4.8. 2-(4-Pyridinyl)-N-phenyl-imidazo[1,2-a]pyridin-3-amine
(3c). Derivative (3c) was obtained in 50% yield, by condensation
of 2-aminopyridine (9), phenylisonitrile (6d) and 4-pyridinylcar-
boxaldehyde (11), as a brown solid, mp 208–210 °C. 1H NMR
(200 MHz, CDCl3, d): 8.49 (d, 2H, J = 5.4 Hz), 8.03 (d, 2H,
J = 5.6 Hz), 7.94 (d, 1H, J = 6.8 Hz), 7.68 (d, 1H, J = 9.0 Hz), 7.38–
7.27 (m, 3H), 7.23 (d, 1H, J = 9.0 Hz), 6.98–6.82 (m, 2H), 6.74–
6.65 (m, 3H); 13C NMR (50 MHz, CDCl3, d): 149.5, 144.0, 142.9,
141.1, 136.1, 129.9, 125.9, 121.0, 120.2, 120.2, 117.9, 113.4,
112.8; IR tmax (KBr): 3246, 3029, 1601, 1493, 1391, 1240, 829,
4.1.4.3. N,2-Diphenyl-imidazo[1,2-a]pyridin-3-amine (3e). Deriv-
ative (3e) was obtained in 25% yield, by condensation of 2-amino-
pyridine (9), phenylisonitrile (6d) and benzaldehyde (12), as a
white–green solid, mp 232–234 °C. 1H NMR (200 MHz, DMSO-d6,
d): 8.21 (s, 1H), 8.04 (d, 2H, J = 7.2 Hz), 7.92 (d, 1H, J = 6.7 Hz),
7.61 (d, 1H, J = 9.0 Hz), 7.41–7.26 (m, 4H), 7.12 (t, 2H, J = 7.6 Hz),
6.91 (t, 1H, J = 6.7 Hz), 6.71 (t, 1H, J = 7.2 Hz), 6.49 (d, 2H,
J = 7.8 Hz); 13C NMR (50 MHz, DMSO-d6, d): 145.1, 141.4, 137.0,
133.2, 129.1, 128.0, 127.1, 126.0, 124.8, 122.6, 118.5, 118.2,
116.6, 112.5, 111.9; IR tmax (KBr): 3179, 2983, 2910, 1599, 1562,
801, 756 cmÀ1
.
1495,1387, 774, 751, 689 cmÀ1
.
4.1.4.9. 2-(4-Pyridinyl)-N-(4-(methylthio)phenyl)-imidazo[1,2-
a]piridin-3-amine (3d). Derivative (3d) was obtained in 60%
yield, by condensation of 2-aminopyridine (9), 4-(methylthio)phe-
nylisonitrile (6b) and 4-pyridinylcarboxaldehyde (11), as a brown
solid, mp 189–190 °C. 1H NMR (200 MHz, DMSO-d6, d): 8.58 (d,
2H, J = 4.5 Hz), 8.42 (br s, 1H), 8.05–7.85 (m, 3H), 7.67 (d, 1H,
J = 9.0 Hz), 7.36 (t, 1H, J = 7.8 Hz), 7.15 (d, 2H, J = 8 Hz), 6.96 (t,
1H, J = 6.5 Hz), 6.51 (d, 2H, J = 8.2 Hz), 2.35 (s, 3H); 13C NMR
(50 MHz, DMSO-d6, d): 149.4, 142.9, 141.6, 140.1, 134.1, 129.4,
125.6, 122.7, 120.4, 120.0, 117.0, 113.4, 112.3, 16.4; IR tmax
(KBr): 3207, 2951, 1632, 1601, 1513,1387, 1231, 828, 734,
4.1.4.4. 2-(4-(Methylthio)phenyl)-N-phenyl-imidazo[1,2-a]pyri-
din-3-amine (3f). Derivative (3f) was obtained in 50% yield,
by condensation of 2-aminopyridine (9), phenylisonitrile (6d)
and 4-(methylthio)benzaldehyde (13), as a white solid, mp 232–
234 °C. 1H NMR (200 MHz, TFA, d): 8.91 (d, 1H, J = 6.2 Hz), 8.51–
8.49 (m, 2H), 8.26 (d, 2H, J = 8 Hz), 7.99 (br s, 1H), 7.90–7.79 (m,
4H), 7.52–7.21 (m, 3H), 3.02 (s, 3H); 13C NMR (50 MHz, TFA, d):
145.6, 145.4, 140.0, 137.3, 132.4, 132.38, 129.6, 129.0, 127.6,
125.5, 123.5, 120.3, 119.8, 114.2, 108.0, 15.6; IR tmax (KBr): 3156,
2983, 2910, 1597, 1487, 1387, 1354, 1254, 1097, 828, 751, 691,
508 cmÀ1
.
689 cmÀ1
.
4.1.4.5. 2-(4-(Methylthio)phenyl)-N-(4-methoxyphenyl)-imi-
dazo[1,2-a]pyridin-3-amine (3g). Derivative (3g) was ob-
4.1.5. General procedure for preparation of arylsulfones (3i–n)20
A solution of 11.8 g of oxoneÒ in 25 ml of water was added
dropwise to a solution of 6.4 mmol of the corresponding arylsul-
fide derivative (3b), (3d) or (3f–h) in 25 ml of MeOH. The
obtained mixture was stirred at room temperature for one hour
when the TLC analysis indicated the end of the reaction, and
then poured into an ice–water mixture. The resulting precipitate
was filtered out, washed with water (15 ml) and air dried to
furnish the desired arylsulfone derivatives (3i–n) as described
next.
tained in 68% yield, by condensation of 2-aminopyridine (9), 4-
methoxyphenylisonitrile (6c) and 4-(methylthio)benzaldehyde
(13), as a white solid, mp 184–186 °C. 1H NMR (200 MHz, DMSO-
d6, d): 8.01 (d, 2H, J = 8.1 Hz), 7.92 (br s, 2H), 7.59 (d, 1H,
J = 8.7 Hz), 7.27 (d, 3H, J = 7.8 Hz), 6.89 (t, 1H, J = 6.7 Hz), 6.75 (d,
2H, J = 8.7 Hz), 6.43 (d, 2H, J = 8.7 Hz), 3.62 (s, 3H), 2.46 (s, 3H).
13C NMR (50 MHz, DMSO-d6, d): 152.1, 141.4, 138.9, 137.1, 136.7,
130.1, 126.6, 125.5, 124.7, 122.7, 119.2, 116.7, 114.8, 113.5,
111.8, 54.9, 14.3. IR tmax (KBr): 3207, 2983, 1513, 1387, 1231,
1035, 828, 759, 508 cmÀ1
.
4.1.5.1. 2-(4-(Methylsulfonyl)phenyl)-N-phenyl-imidazo[1,2-a]-
pyridin-3-amine (3i). Oxidation of arylsulfide derivative (3f)
followed by purification on silica gel column using a mixture
of hexane/AcOEt mixture as eluent furnished the compound
(3i) in 60% yield, as white solid, mp >250 °C. 1H NMR
(200 MHz, DMSO-d6, d): 8.37 (s, 1H), 8.30 (d, 2H, J = 8.2 Hz),
7.99–7.93 (m, 3H), 7.66 (d, 1 H, J = 8.9 Hz), 7.35 (t, 1H,
J = 8.2 Hz), 7.14 (t, 2H, J = 7.6 Hz), 6.95 (t, 1H, J = 6.6 Hz), 6.73
(t, 1H, J = 7.2 Hz), 6.51 (d, 2H, J = 7.8 Hz), 3.21 (s, 3H); 13C NMR
(50 MHz, DMSO-d6, d): 145.0, 142.0, 139.1, 138.5, 135.5, 129.5,
127.1, 126.7, 125.8, 123.2, 120.5, 118.8, 117.3, 112.9, 112.6,
43.4; IR tmax (KBr): 3164, 3095, 2918, 2857, 1597, 1487, 1311,
4.1.4.6. 2-(4-(Methylthio)phenyl)-N-(2-pyridinyl)-imidazo[1,2-
a]pyridin-3-amine (3h). Derivative (3h) was obtained in 50%
yield, by condensation of 2-aminopyridine (9), 2-pyridinylisonitrile
(6a) and 4-(methylthio)benzaldehyde (13), as a light yellow solid,
mp 233–235 °C. 1H NMR (200 MHz, CDCl3, d): 8.88 (s, 1H), 7.99–
7.94 (m, 3H), 7.87 (d, 1H, J = 6.8 Hz), 7.62–7.50 (m, 2H), 7.31–
7.25 (m, 3H), 6.87 (t, 1H, J = 6.7 Hz), 6.71 (t, 1H, J = 6.2 Hz), 6.57
(d, 1H, J = 8.2 Hz), 2.46 (s, 3H); 13C NMR (50 MHz, CDCl3, d):
156.8, 148.0, 141.6, 137.9, 137.2, 136.8, 130.3, 126.8, 125.5,
124.7, 123.1, 117.8, 116.7, 114.5, 111.8, 107.8, 14.5. IR tmax
(KBr): 3179, 2983, 2910, 1601, 1495, 1444, 1387, 1231, 1097,
828, 751, 698, 508 cmÀ1
.
1150, 958, 762, 541 cmÀ1
.

R. B. Lacerda et al. / Bioorg. Med. Chem. 17 (2009) 74–84
83
4.1.5.2. 2-(4-(Methylsulfonyl)phenyl)-N-(4-methoxyphenyl)-imi-
dazo[1,2-a]pyridin-3-amine (3j). Oxidation of arylsulfide deriv-
ative (3g) followed by purification on silica gel column using a
dichloromethane/MeOH mixture as eluent furnished the com-
pound (3j) in 60% yield, as white–green solid, mp 180–182 °C. 1H
NMR (200 MHz, CDCl3, d): 8.36 (d, 2H, J = 8.4 Hz), 8.25 (s, 1H),
7.99 (d, 1H, J = 6.7 Hz), 7.80–7.55 (m, 4H), 7.13 (t, 1H, J = 6.7 Hz),
6.76 (d, 2H, J = 8.9 Hz), 6.60 (d, 2H, J = 8.9 Hz), 3.72 (s, 3H), 2.95
(s, 3H). 13C NMR (50 MHz, CDCl3, d): 158.7, 150.0, 141.7, 137.5,
129.1, 129.5, 127.4, 125.5, 125.0, 122.6, 116.7, 115.9, 113.8,
112.1, 54.8, 44.9. IR tmax (KBr): 3240, 2925, 2857, 1597, 1512,
pared with the vehicle control group. Results are expressed as
the mean SEM of n animals per group.
4.2.2. Carragenaan-induced rat paw edema assay23
Fasted Wistar rats of both sexes (150–200 g) were used. The
anti-inflammatory activity was determined in vivo using the car-
rageenan induced rat paw edema test (CIRPE). Test compounds
(3) and celecoxib (1) were orally administered at a dose of
100 lmol/kg, as a suspension in 5% arabic gum in saline (vehi-
cle). The control animals received an equal volume of vehicle.
After 1 h, animals were then injected subplantarly with either
0.1 ml of 1% carrageenan solution in saline (0.1 mg/paw) into
the right hind paws and sterile saline (NaCl 0.9%) into the left
hind paws. Three hours after the subplantar injection, the paw
volumes were measured using a glass plethysmometer coupled
to a peristaltic pump. The edema was calculated as the volume
difference between the carrageenan and saline-treated paw.
Anti-inflammatory activity was expressed as percentage of inhi-
bition of the edema when compared to the vehicle group con-
trol. Results are expressed as the mean SEM of n animals per
group.
1308, 1237, 1150, 958, 762, 541 cmÀ1
.
4.1.5.3. 2-(4-(Methylsulfonyl)phenyl)-N-(2-pyridinyl)-imidazo[1,2-
a]pyridin-3-amine (3l). Oxidation of arylsulfide derivative (3h)
followed by purification on silica gel column using a dichlorometh-
ane/MeOH mixture as eluent furnished the compound (3l) in 80%
yield, as white solid, mp >250 °C. 1H NMR (200 MHz, DMSO-d6,
d): 9.04 (s, 1H), 8.26 (d, 2H, J = 8.5 Hz), 7.94 (d, 4H, J = 8.5 Hz),
7.67–7.54 (m, 2H), 7.34 (td, 1H, J = 0.8 and 6.7 Hz), 6.92 (t, 1H,
J = 6.7 Hz), 6.67 (d, 1H, J = 8.3 Hz), 6.74 (m, 1H), 3.21 (s, 3H); 13C
NMR (50 MHz, DMSO-d6, d): 156.1, 147.6, 141.5, 138.6, 138.2,
137.6, 134.8, 126.7, 126.3, 125.1, 123.1, 119.3, 116.7, 114.4,
111.9, 107.7, 43.0; IR tmax (KBr): 3142, 2920, 1700, 1601, 1476,
4.2.3. Capsaicin-induced hyperalgesia26
The anti-hyperalgesic activity was investigated using the
capsaicin-induced thermal hyperalgesia test adapted from
Mizushima et al.26 Animals (fasted Wistar rats of both sexes
(150–200 g)) were placed in a hot plate apparatus (Ugo Basile,
Model-DS 37) at a temperature of 52 0.1 °C to record the accli-
matization and the basal latency of the withdrawal response.
Then, test compounds (3), celecoxib (1) and SB203580 (2) were
1310, 1147, 772, 536 cmÀ1
.
4.1.5.4. 2-(2-Pyridinyl)-N-(4-(methylsulfonyl)phenyl)-imidazo[1,2-
a]pyridin-3-amine (3m). Oxidation of arylsulfide derivative (3b)
followed by purification on silica gel column using a dichlorometh-
ane/MeOH mixture as eluent furnished the compound (3m) in 60%
yield, as yellow solid, mp 227–230 °C. 1H NMR (200 MHz, DMSO-
d6, d): 9.0 (s, 1H), 8.52 (d, 1H, J = 3.9 Hz), 8.13 (d, 1H, J = 7.9 Hz),
7.93 (d, 1H, J = 6.8 Hz), 7.86 (td, 1H, J = 1.7 and 7.7 Hz), 7.70–
7.54 (m, 3H), 7.36–7.24 (m, 1H), 6.97 (t, 1H, J = 6.7 Hz), 6.63 (d,
2H, J = 8.6 Hz), 3.06 (s, 3H); 13C NMR (50 MHz, DMSO-d6, d):
151.3, 148.7, 147.6, 140.0, 135.0, 134.7, 127.8, 127.3, 123.9,
121.7, 120.7, 119.7, 118.2, 116.0, 111.5, 111.2, 42.5; IR tmax
(KBr): 3133, 2900, 2800, 1591, 1505, 1400, 1288, 1145, 772, 543,
administered at a dose of 100
lmol/kg. After 1 h, 0.1 ml of 5%
capsaicin solution in DMSO (5
lg/paw) was injected into
animals’ the right hind paws. The withdrawal latency of the
right hind paw was determined at 2, 5, 10, 30 and 60 min
post-challenge with a chronometer. The basal latencies were
found to be 10–16 s. A cut-off time of 21 s was established to
prevent any injury in the paw.
4.2.4. Determination of the inhibition of PGHS25
530 cmÀ1
.
To evaluate the action of the compounds onto PGHS-1, fresh
blood was collected into tubes containing no anticoagulant from
normal volunteers who had not ingested any drugs within the pre-
vious two weeks. Blood aliquots were incubated with the com-
pounds or vehicle (DMSO) at 37 °C for 1 h under constant
agitation. At the end of incubation, the serum was obtained by cen-
trifugation (6500 rpm for 10 min) and was tested for thromboxane
B2 production using an enzyme immunoassay kit (Amersham Bio-
sciences) according to the manufacturer’s instructions.
To evaluate the action of the compound onto PGHS-2, fresh
blood was collected in heparinized tubes from normal volunteers
as previously described. Aliquots of blood were incubated with
the compounds or vehicle (DMSO) for 15 min at 37 °C under
constant agitation. After that, the blood was incubated with
4.1.5.5. 2-(4-Pyridinyl)-N-(4-(methylsulfonyl)phenyl)-imidazo[1,2-
a]pyridin-3-amine (3n). Oxidation of arylsulfide derivative (3d)
followed by purification on silica gel column using a dichlorometh-
ane/MeOH mixture as eluent furnished the compound (3n) in 85%
yield, as brown crystals, mp 194–196 °C. 1H NMR (200 MHz, DMSO-
d6, d): 9.12 (s, 1H), 8.59 (d, 2H, J = 6.4 Hz), 8.03 (d, 1H, J = 6.4 Hz),
7.93 (s, 2H), 7.70 (d, 3H, J = 7.9 Hz), 7.39 (t, 1H, J = 6.8 Hz), 6.99 (t,
1H, J = 6.2 Hz), 6.70 (d, 2H, J = 7.2 Hz), 3.09 (s, 3H); 13C NMR
(50 MHz, DMSO-d6, d): 146.9, 149.6, 142.3, 140.3, 134.8, 130.2,
129.2, 126.1, 123.2, 120.3, 119.2, 117.5, 113.1, 112.8, 44.0; IR tmax
(KBr): 3201, 3034, 2962, 2918, 1594, 1503, 1415, 1310, 1296, 1145,
958, 831, 750, 525 cmÀ1
.
20
l
L of lipopolysaccharide (LPS) solution at a final concentra-
g/ml (SIGMA serotype 055:B5, diluted in phosphate
4.2. Pharmacology
tion of 10
l
buffer saline (PBS), pH 7.0) under constant agitation for 6 h at
37 °C in order to induce PGHS-2 expression. After incubation,
the tubes were centrifuged at 6500 rpm for 10 min to obtain
plasma that was frozen until the day of the assay. Plasma was
tested for tromboxane B2 production using an enzyme immuno-
assay kit (Amersham Biosciences) according to the manufac-
turer’s instructions.
4.2.1. Acetic acid-induced abdominal constriction in mice22
The antinociceptive activity was determined in vivo using swiss
mice (18–23 g) abdominal constriction test induced by acetic acid
0.6% (0.1 ml/10 g; ip). Test compounds (3) and celecoxib (1) were
administered orally at a dose of 100 lmol/kg, as a suspension in
5% arabic gum in saline (vehicle). After 1 h, acetic acid was injected
as previously described and 10 min after, the number of constric-
tions per animal was recorded for 20 min. The control animal
received an equal volume of vehicle. Analgesic activity was
expressed as percentage of inhibition of constrictions when com-
4.2.5. Docking studies
The molecular docking studies with PGHS-2 were performed
using the flexible molecular docking software FlexE.29

84
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Acknowledgments
The authors acknowledge financial support and fellowships (to
R.B.L., C.K.F.L., L.L.S., N.C.R., A.L.P.M., C.A.M.F., E.J.B.) from CAPES
(BR), CNPq (BR), FAPERJ (BR), PRONEX (BR), and IM-INOFAR (BR,
#420.015/05-1).
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