Anti-anxiety and sedative profile evaluation of imidazo[1,2-a]pyridine derivatives

Article abstract of DOI:10.1002/ddr.20382

(Table Presented) Three imidazo[1,2-a]pyridine-3-nitrosated (L-1, L-2, L-3) and a 3-formyl imidazo[1,2-a]-pyridine thiosemicarbazone (L-4) were synthesized and evaluated for their effects in the elevated plus maze, burying behavior test, rotarod performance, the horizontal wire test, and locomotor activity. L-2 and L-3 increased the percent time spent in the open arms of the plus maze at doses of 1 and 2 mg/kg without modifying the number of total entries. In addition, L-2 and L-3 (1 mg/kg) increased the number of open arm entries indicating anxiolytic-like activity at this dose. In the burying behavioral test, L-1 (2-8mg/kg), L-2 (8mg/kg), and L-3 (4 and 8mg/kg), induced a clear reduction in cumulative burying behavior, without modifying burying behavior latency, thus reducing experimental anxiety. In the rotarod test, L-1 and L-2 impaired rotarod performance only at the highest evaluated dose (64mg/kg) at which reduction of motor activity was observed and thereby no conclusions about myorelaxant effects can be proposed. All compounds showed a clear sedative effect and corresponding ED₅₀ values were obtained. Results indicate that compounds L-1, L-2, and L-3 show a sedative and an anxiolytic profile.

Full text of DOI:10.1002/ddr.20382

DRUG DEVELOPMENT RESEARCH 71:371–381 (2010)
Research Article
Anti-Anxiety and Sedative Profile Evaluation
of Imidazo[1,2-a]Pyridine Derivatives
1
1
Ã
2
´
´
´
´
Margarita Lopez-Martınez, Hector Salgado-Zamora, Eduardo Ramırez San-Juan,
Sergio Zamudio,2 Ofir Picazo,3 Maria Elena Campos,1 and Elia B. Naranjo-Rodriguez4
1Departamento Qu´ımica Orga´nica, Escuela Nacional Ciencias Biolo´gicas, Instituto Polite´cnico
Nacional, Mexico City, Mexico
2Departamento Fisiolog´ıa, Escuela Nacional Ciencias Biolo´gicas, Instituto Polite´cnico Nacional,
Mexico City, Mexico
3Escuela Superior de Medicina, Instituto Polite´cnico Nacional, Mexico City, Mexico
4Facultad de Qu´ımica, Universidad Nacional Auto´noma de Me´xico, Mexico City, Mexico
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Toxicology, Formulation Phases I-III
Technology,
Genomics,
Proteomics
Preclinical
Research
Postmarketing
Phase IV
Drug Delivery,
Regulatory, Quality,
Manufacturing
Pharmacokinetics
ABSTRACT
Three imidazo[1,2-a]pyridine-3-nitrosated (L-1, L-2, L-3) and a 3-formyl imidazo[1,2-a]-
pyridine thiosemicarbazone (L-4) were synthesized and evaluated for their effects in the elevated plus
maze, burying behavior test, rotarod performance, the horizontal wire test, and locomotor activity. L-2 and
L-3 increased the percent time spent in the open arms of the plus maze at doses of 1 and 2 mg/kg without
modifying the number of total entries. In addition, L-2 and L-3 (1 mg/kg) increased the number of open arm
entries indicating anxiolytic-like activity at this dose. In the burying behavioral test, L-1 (2–8mg/kg), L-2
(8mg/kg), and L-3 (4 and 8 mg/kg), induced a clear reduction in cumulative burying behavior, without
modifying burying behavior latency, thus reducing experimental anxiety. In the rotarod test, L-1 and L-2
impaired rotarod performance only at the highest evaluated dose (64 mg/kg) at which reduction of
motor activity was observed and thereby no conclusions about myorelaxant effects can be proposed.
All compounds showed a clear sedative effect and corresponding ED50 values were obtained.
Results indicate that compounds L-1, L-2, and L-3 show a sedative and an anxiolytic profile. Drug Dev
Res 71:371–381, 2010.
r 2010 Wiley-Liss, Inc.
Key words: anxiety; sedation; imidazo[1,2-a]pyridines; behavioral tests
INTRODUCTION
it can result in disturbance of daily life and cause
suffering in patients.
Sleep behavior and anxiety are two major
recurrent problems treated by the general medical
physician. Sleep problems like insomnia are frequently
associated with anxiety disorders [Wittchen and Hoyer,
2001; Ressler and Mayber, 2007] while anxiety is a
common emotional phenomenon in humans. ‘‘Normal’’
anxiety can be defined as occurring as an adaptive
response to various stressors (physiological, psycho-
logical, sociological). When anxiety becomes patho-
Grant sponsor: Consejo Nacional de Ciencia y Tecnolog´ıa
(CONACyT/Me´xico); Grant numbers: 91426; 49937.
Ã
Correspondence to: He´ctor Salgado-Zamora, Escuela
Nacional Ciencias Biolo´gicas, IPN, Mexico City, Mexico.
E-mail: hsalgado47@hotmail.com
Received 19 May 2010; Accepted 12 July 2010
Published online in Wiley Online Library (wileyonlinelibrary.com).
logical, with disproportionate or unjustified reactions, DOI: 10.1002/ddr.20382
ꢀc
2010 Wiley-Liss, Inc.

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´
372
LOPEZ-MARTINEZ ET AL.
The pharmacotherapy of anxiety and sleep thiosemicarbazone (L-4) were selected from a previous
disorders involves the use of benzodiazepines (BZs) round of preliminary tests and evaluated for their
that have potent anxiolytic, sedative-hypnotic and action on anxiety behavioral tests.
anticonvulsant activity, acting as indirect agonists
of the GABAA (g-aminobutyric acid-A) receptor.
However, a great tendency to produce tolerance,
addiction, ataxia, sleep suppression REM (rapid eye
MATERIALS AND METHODS
Chemistry
movements), and amnesia are factors associated with
BZs [Feldman et al., 1997]. Therefore, treatment for
anxiety and associated pathologies require more
selective drugs with less collateral effects [Kent et al.,
2002; Davidson, 2002].
Imidazo[1,2-a]pyridines, such as zolpidem (sedative-
hypnotic agent) and alpidem (anxiolytic agent), are
chemically non-related to BZs and show more selective
pharmacological action. However, amnesia and insomnia
rebound effects following the use of zolpidem have been
reported while alpidem showed hepatotoxicity [Berson,
2001; Depoortere et al., 1986].
The imidazo[1,2-a]pyridine is an attractive core
that has been evaluated in connection to several
biological activities. In particular 3-nitrosoimida-
zo[1,2-a]pyridines have been evaluated as antiretroviral
[Chaouni-Benabdallah et al., 2001] and anti-parasitic
[Srivastava et al., 1998] agents and have shown
antifungal activity [Yu et al., 2008]. In the present
study, three 2-aryl-3-nitrosoimidazo[1,2-a]pyridines
(L-1-3) and the 3-formyl imidazo[1,2-a]pyridine
Melting points were measured on an Electro-
thermal melting point apparatus and are uncorrected.
1H and 13C nmr spectral data were recorded at 300 and
75 MHz, respectively, using a Bruker DPX 300 MHz
NMR spectrometer. Chemical shifts (d) are given
in parts per million downfield from TMS (d 5 0).
Synthesis of the compounds under investigation
was achieved as depicted in Figure 1. The starting
2-arylimidazo[1,2-a]pyridines were prepared by con-
densation of 2-aminopyridine with the corresponding
phenacyl bromide according to the literature protocol
[Lombardino, 1965]. The nitroso functional group was
introduced by treating the 2-arylimidazopyridine with
nitrous acid generated from sodium nitrite and acetic
acid at low temperature [Chaouni-Benabdallah et al.,
2001]. The carboxaldehyde functional group was
introduced through a Vilsmeier Haack reaction,
using dry N,N-dimethyl formamide and phosphorous
oxychloride, and heating at 801C for 12 h, followed by
treatment with aqueous sodium hydroxide [Fuentes
and Paudler, 1975]. The thiosemicarbazone L-4 was
CF3
N
L-1
L-2
N
NO
8
N
7
N
Ar
Cl
2
N
N
6
3
HONO
5
NO
NO
OMe
OMe
N
L-3
N
N
Ar
N
NO
1
DMF/POCl3
aq. NaOH
N
N
OMe
NH2CSNHNH2
AcOH
Ar
N
N
H
N
N
CHO
NH2
L-4
S
Fig. 1. Synthesis and chemical structures of imidazo[1,2-a]pyridine derivatives.
Drug Dev. Res.

EVALUATION OF IMIDAZO[1,2-a ]PYRIDINES
373
prepared from imidazo[1,2-a]pyridine-3-carboxaldehyde prepared on the experimental day. All drugs were
and thiosemicarbazide in acetic acid [West et al., 1993]. administered intraperitoneally (i.p.) in a volume of
All products were purified and characterized by 10 mL/kg in mouse and 1 mL/kg in rat, 30 min prior to
conventional spectroscopic methods and combustion testing at a single dose.
analysis. Compounds L-1, L-3, and L-4 have not been
Animals
1
previously described in the literature, therefore the H
and 13CNMR data are provided.
Male ICR mice, weighing 18–22 g; male Wistar
rats weighing 180–200 g were purchased from UAM-
2-(30-Trifluoromethylphenyl)-3-Nitroso Imidazo
[1,2-a]Pyridine (L-1)
´
Xochimilco (Mexico City) and Harlan-Mexico (Mexico
City). Mice and rats were housed in groups of five per
cage, allowed access to water and food ad libitum, and
maintained under a constant temperature (23711C)
and humidity (60710%). The rats used for the
Elevated plus-maze (EPM) test were housed under a
normal 12-hour light/dark cycle. In other evaluation
experiments, animals were kept under a 12:12 h
inverted light-dark cycle conditions (lights on
9:00–21:00). Animals were allowed to acclimate for
1–2 weeks prior to use and were transported to the test
room at least 1 h prior to testing. All behavioral
evaluations were carried out between 10 a.m. and 3
p.m. Care and handling of the animals were in
agreement with internationally accepted procedures
and approved by our own institutional Committee
following the recommendation indicated in the Mexican
Technical Specifications for the Production, Care, and
Isolated as a light green solid, in 90% yield, mp
1
124–1251C. H NMR (CDCl3) d H5 9.9 (dd, J5,6 5 8.8,
0
0
0
0
J
5,8 5 1.0, 1H), H5 ,6 8.9 (m, 2 H), H8,2 ,4 7.9–7.2
(m, 3 H), H7 7.7 (t, J 5 7.6, 1 H), H6 7.0 (td, J5,6 5 8.8,
6,7 5 7.7, J6,8 5 2.4, 1 H). 13C NMR d 117.7, 120.0,
J
125.9, 126.4, 127.2, 127.3, 127.8, 127.9, 129.3, 132.4,
134.1, 136.3, 145.5, 153.3. Anal. Calcd. for C14H8N3F3O:
C, 57.73; H, 2.75. Found: C, 57.61; H, 2.90.
2-(40-Chlorophenyl)-3-Nitroso Imidazo[1,2-a]
Pyridine (L-2)
Isolated as a green powder in 75%, mp 223–2241C,
lit mp 222–224 [Salgado-Zamora et al., 2008].
2-(30,40-Dimethoxyphenyl)-3-Nitrosoimidazo
[1,2-a]Pyridine (L-3)
´
Use of Laboratory Animals (Secretarıa de Agricultura,
Ganaderıa, Desarrollo Rural, Pesca y Alimentacion).
Isolated as a dark yellow solid, mp 196–1981C.
`
´
1H NMR (CDCl3) d H5 9.9 (d, J5,6 5 6.0, 1 H), H8 8.4 (d,
J
8,7 5 8.0, 1 H), Ar-H 8.2 (s, 1 H), Ar-H 7.8 (s, 2 H), Ar-H
BEHAVIOURAL TESTING
Elevated Plus-Maze (EPM)
1H7 7.2 (m, 2 H), H6 7.0 (d, J6,7 5 8.0, 1 H), MeO-, 4.0
(s, 3 H), -OMe 3.98 (s, 3 H). 13CNMR 56.0, 56.04, 111.1,
112.8, 117.1, 119.0, 124.3, 125.0, 126.6, 136.2, 146.0,
149.2, 152.5, 153.3, 159.5. Anal. Calcd. for C15H13N3O3:
C, 63.60; H, 4.59. Found: C, 63.74; H, 4.68.
The test apparatus based on that described by
Pellow and colleagues [1985] is widely used for
studying anxiolytic drugs and neurobiological mecha-
nisms of anxiety in rodents due to the natural aversion
of rodents to heights and open spaces. The EPM
consisted of two open arms, 50 ꢁ 10 ꢁ 40 cm, and two
enclosed arms, 50 ꢁ 10 ꢁ 40 cm, with an open top,
arranged so that the arms of the same type were
opposite each other, connected by an open central area
(10 ꢁ 10 cm). The maze was elevated to a height of
50 cm. Thirty minutes after i.p. administration of the
test drug, the rat (N 5 5 and 7) was placed in the center
of the maze, facing one of the enclosed arms. EPM
behavior was videotaped during a 5-min test period.
The following were recorded: the number of entries
and time spent in the open and closed arms, and the
total number of arm entries.
2-(40-Methoxyphenyl)-3-Formylimidazo
[1,2-a]Pyridine Thiosemicarbazone (L-4)
Isolated as a light brown crystalline powder in 98%
1
yield, mp 223–2241C. H NMR (DMSOd6) d H5 9.5
(d, J5,6 5 6.7. 1 H), NH 8.7 (s, 1 H), Ar-H 8.2 (bs, 1 H),
Ar-H 7.8–7.6 (m, 4 H), H7, 7.51 (t, J7,6 5 7.3 1 H), Ar-H
1H6, 7.13–7.06 (m, 3 H), MeO-3.8 (s, 3 H). 13CNMR
55.9, 114.7, 114.9,117.0,126.0, 128.2, 130.2, 131.1, 126.4,
146.6, 150.8, 160.3, 177.3. Anal. Calcd. for C16H15N5SO:
C, 59.07; H, 4.61. Found: C, 59.23; H, 4.26.
Drugs
Diazepam was obtained from Roche and Zolpidem
tartrate from Lab Auylacem. and suspended in 0.3%
Tween 80 (Sigma-Aldrich, St. Louis, MO)/saline
solution. For in vivo pharmacological studies, all
Conditioned Defensive Burying
The test apparatus is based on that described by
compounds were prepared as suspensions in a 10% Pinel and Treit [1978]. The method is useful for testing
aqueous solution of Tween 80 and Polietilenglicol 400 physiological and pharmacological changes in experi-
(Merck-Schuchardt, Hohenbrunn, Germany) freshly mental anxiety [Treit et al.,1980; Treit, 1985]. An
Drug Dev. Res.

´
´
LOPEZ-MARTINEZ ET AL.
374
electrified prod (7 cm long) projecting 2 cm above the traveled, time spent in ambulation, stereotypic move-
floor from one wall of an acrylic cage (27 ꢁ 16 ꢁ 23) ments, and resting by a video-recording system (Video-
was covered with fine sawdust. When the animal mex V. Columbus Instruments, Columbus, OH) during
touched the prod, it received an electric shock of 2 repetitions of 3 min each per animal. After removing
0.3 mA supplied by a constant-current shocker. In this the rat, the open field was cleaned with a solution with
test, the mouse (N 5 6 and 7) was introduced into the damp and dry cloths to remove any residue or odors.
cage and its behavior was recorded for 10 min. During
Locomotor activity in mice was recorded individu-
this period, two parameters were measured: (1) ally in an automatic activity counter (Opto-Varimex;
cumulative burying behavior, i.e., the time the mouse Columbus Instruments) that consists of an acrylic cage
spent burying the prod and (2) burying behavior measuring 51.1 ꢁ 9.5 ꢁ 69.2 cm with two arrays of 15
latency, i.e., the tine elapsed from the first shock to infrared emitting photocells placed perpendicular to each
the defensive behavior display. In this model, cumula- other. The photocells are spaced 2.5 cm apart. The
tive burying has been directly linked to experimental interruption of each infrared beam generated an electric
anxiety levels, while burying behavior latency may impulse, which was processed and presented as a count.
represent an index of the animal coping ability stimulus This test was carried out immediately after evaluating the
response reactivity [Treit et al., 1981; Picazo and animals in the burying behavior paradigm and sedative
´
Fernandez-Guasti, 1995].
effect tests, registering horizontal activity (ambulation)
and vertical activity (rearing) over a 5-min period.
Rotarod Test
Statistical Analysis
The rotating rod (‘‘rotarod’’) test is widely used to
assess ‘‘minimal neurological deficit’’ such as impaired
motor function (i.e., ataxia) and coordination in rodents.
An Ugo Basile accelerating rotarod for rats (model 7750)
was used in order to evaluate possible motor impair-
ment. A modification of the method described by
Dunham and Miya [1956] was adapted. Two hours
before the experiment, rats were selected for their ability
to maintain themselves for at least 2 min on the rod
(diameter, 7 cm) which rotated slowly at a constant speed
(10 r.p.m). Rats satisfying this criterion received i.p.
injections of test compounds (N 5 5 and 7). Then after
30 min, they were retested for their ability to remain on
the rotarod. The number of total falls was recorded.
The one-way variance analysis (ANOVA), fol-
lowed by post-hoc Student-Newman-Keuls was applied
to compare statistical significance between the treated
group and the vehicle control. The Fisher’s exact test
was applied to evaluate statistical significance in the
horizontal Wire test. Po0.05 value was considered
statistically significant. Analysis was done using the
Sigma Stat version 3.5 and Sigma Plot 10 for windows.
RESULTS
Spontaneous Locomotor Activity
The effects of each of the four imidazopyridine
derivatives evaluated at various doses on total activity
during the first hour of testing are depicted in Table 1.
The Horizontal Wire Test
The horizontal wire test was used to assess A significant difference was observed on locomotor
compund effects on muscle tone in mice. The test activity between individual dose groups and the vehicle
apparatus was based on that described by Bonetti control. One-way ANOVA revealed that doses of
[1982]. In this test, the animals were lifted by the tail, 4 and 8 mg/kg decreased both the distance traveled
allowed to grasp a horizontal strung wire (for mice 25 cm (cm) and time ambulatory (sec) of L-1 [F(5,26) 5 28.46;
high, 2 mm diameter, 30 cm long) with their forepaws Po0.001], [F(5,26) 5 11.80; Po0.001], L-2[F(4,26) 5
and then released. The number of animals (N 5 6,7) that 69.653; Po0.001] [F(4,22) 5 18.794; Po0.001], and
were unable to grasp the horizontal wire, with either the L-3[F(5,30) 5 15.644; Po0.001], [F(5,30) 5 11.711;
forepaws, or at least with one hindpaw within 10 sec Po0.001], respectively, while L-4 causes a decrease in
(cut-off: 60 sec) was recorded. In untreated control these parameters, starting at 8 mg/kg [F(4,22) 5 19.519;
animals, this number was consistently zero.
Po0.001] and [F(4,22) 5 4.868; Po0.006]. Diazepam
decreased both the distance traveled [F(4,22) 5 14.151,
Po0.001] and ambulatory time [F(4,22) 5 3.711;
Spontaneous Locomotor Activity Measurement
Exploratory locomotor activity of rats can be P 5 0.019] at 3.5 mg/kg.
evaluated in open field tests. Rats were placed in a
The four imidazopyridine derivatives had signifi-
squared open field (60 ꢁ 60 ꢁ 30 cm). The floor and cant effects on spontaneous locomotor activity in mice
the wall of the compartment were painted in black. on both ambulatory and vertical activity, as seen in
The test was started by placing the rat (one rat per time) Table 2. One-way ANOVA revealed the following: L-1
in the center of the open field to evaluate distance decreased ambulatory [F(11,62) 5 7.841; Po0.001]
Drug Dev. Res.

EVALUATION OF IMIDAZO[1,2-a ]PYRIDINES
375
were noted for closed arm entries [one-way ANOVA
F(4,22) 5 10.236; Po0.001; F(4,22) 5 12.444, Po0.001,
respectively] at 4 and 8 mg/kg while L-2 had significant
increments in open arm entries [F(4,22)5 5.062;
Po0.005] only at 1 mg/kg. As shown in Figure 2, L-3
had significant effects on total arm, open arm, and
closed arm entries [one-way ANOVA F(5,30) 5 4.998,
Po0.002; F(5,30) 5 7.398, Po0.002; F(5,30) 5 6.66;
Po0.001, respectively]. Subsequent post-hoc analyses
revealed that L-3 caused a significant decrease in total
arm (4 and 8 mg/kg) and closed arm entries (1–8 mg/kg).
L-3 significantly increased the total open arm entries
only at 1 mg/kg. Figure 2 shows a significant decrease in
total arm and closed arm entries [one-way ANOVA
F(4,22) 5 5.584, Po0.003; F(4,22)5 11.448, Po0.001]
at 8 mg/kg. No effects of treatment with L-4 were found
for open arm entries [F(4,22) 5 2.429; n.s.]. Diazepam
had significant effects on the number of total arm, open
arm, and closed arm entries [one-way ANOVA,
F(4,22) 58.217, Po0.001; F(4,22) 520.516, Po0.001,
F(4,22) 514.668, Po0.001] as indicated in Figure 2.
Newman-Keuls post-hoc test revealed that diazepam had
a significant increase in open arm and a significant
decrease in closed arm entries at 1 and 2 mg/kg in the
absence of any change in total arm entries while
reduction in total arm and closed arm entries at 3.5 mg/
kg was observed. Zolpidem significantly decreased closed
arm entries [Fig. 3; one-way ANOVA F(3,18) 58.55,
Po0.001] at 0.75 and 1 mg/kg. Furthermore, 1 mg/kg
significantly reduced total arm [F(3,18) 55.806,
Po0.006] and open arm entries [F(3,18) 54.467;
TABLE 1. Average Results Obtained on Spontaneous Locomotor
Activity Parameters in Rat Treated With Imidazopyridine Derivativesa
Locomotor activity
Dose
(mg/kg, ip)
Distance traveled
(cm)
Time ambulatory
(sec)
Compound
Vehicle
Diazepam
0
0.5
1
662.5750.5
609.8726.1
591.9731.4
581.2714.9
31.072.3
31.272.3
30.074.0
30.073.1
2
Ã
ÃÃÃ
3.5
0.5
1
2
4
354.4716.0
18.871.7
L-1
544.8721.5
630.0730.2
573.7722.6
32.671.5
25.673.7
27.474.5
14.372.3
9.570.3
Ã
Ã
Ã
350.3728.7
Ã
8
226.2719.5
L-2
L-3
1
2
4
8
0.5
1
2
4
8
651.7731.9
609.3712.3
28.172.2
27.772.9
Ã
Ã
232.2727.2
68.7736.7
10.871.2
9.673.3
Ã
Ã
683.5737.8
628.6725.6
608.0721.9
28.872.6
31.772.0
31.572.8
Ã
Ã
391.6760.5
16.071.8
Ã
Ã
331.0717.9
15.270.5
L-4
1
2
4
8
595.2713.2
580.2728.3
567.673.9
35.072.7
29.073.8
29.072.8
Ã
ÃÃ
328.8718.6
18.871.6
aThe effects of imidazopyridine derivatives on mean7SEM sponta-
neous locomotor activity during the first hour of testing (Ns 5 5 and
Ã
ÃÃ
ÃÃÃ
7 groups), Po0.001, Po0.006,
Po0.01 versus corresponding
vehicle control.
(120–480 mg/kg) and vertical [F(11,62) 5 10.912; Po0.016].
Po0.001] (4–480 mg/kg) activity. L-2 decreased ambu-
Figure 3 shows the effects of the four imidazo-
latory [F(11,61) 5 9.577; Po0.001](30–480 mg/kg) and pyridine derivatives at various doses on the percent
vertical [F(11,62) 5 9.391; Po0.001] (120–480 mg/kg) of time in the open arms in the elevated plus maze.
activity. L-3 decreased the ambulatory [F(11,61) 5 13; No statistically significant differences with L-1 [one-
Po0.001](120–480 mg/kg) and vertical [F(11,61) 5 6; way ANOVA F(5,26) 5 1.867, n.s] between individual
Po0.001] (120–480 mg/kg) activity. L-4 decreased the dose groups and the vehicle control were found over a
ambulatory [F(11,61) 514.161; Po0.001] (60–480 mg/kg) range of doses (1–8 mg/kg). One-way ANOVA revealed
and vertical [F(11,61) 511.885; Po0.001] (30–480 mg/kg)
a
significant effect of the L-2 derivate
activity.
[F(4,26) 5 19.042; Po0.001] with both 1 and 2 mg/kg
increasing the percent time spent in the open arms. L-3
also had a significant effect on percent time spent in
Elevated Plus-Maze (EPM)
The results for various doses of the four imidazo- the open arms [one-way ANOVA F(5,28) 5 15.767,
pyridine derivates in the EPM are shown in Figure 2. Po0.001]. A Newman-Keuls post hoc test, confirmed
L-1 treatment showed a significant increase in the that L-3 (1 and 2 mg/kg) caused significant increases on
total arm, open arm, and closed arm entries [one- the percent of time spent in the open arms. No effect
way ANOVA F(5,26)5 2.804, Po0.037; F(5,26)5 3, for L-4 [F(4,22) 5 2.052; Po0.122] was found.
Po0.029, F(5,26) 5 2.833; Po0.036, respectively]. Post-
Diazepam produced a significant and selective
hoc analyses revealed that the significant effects were increase on time spent [Fig. 3; one-way ANOVA
attributable to the 4- and 8-mg/kg doses. These reduc- F(4,23) 5 34.33; Po0.001] at 1- and 2-mg/kg dose. No
tions are most likely due to motor effects. Figure 2 reliable differences [one-way ANOVA F(3,18) 5 2.788;
also shows a significant decrease with derivative L-2 on P 5 0.07] between individual dose groups of zolpidem
total and closed arms entries. Significant effects and the vehicle control were found.
Drug Dev. Res.

´
´
LOPEZ-MARTINEZ ET AL.
376
Drug Dev. Res.

EVALUATION OF IMIDAZO[1,2-a ]PYRIDINES
377
18
16
14
12
10
8
18
18
16
14
12
10
8
A
B
C
16
14
12
*
*
*
10
*
*
*
8
*
*
*
*
*
6
6
4
2
0
6
*
*
*
*
*
4
4
*
*
2
2
0
0
0
0.5
1
2
4
8
0
0.5
1
2
4
8
0
1
2
4
8
L-1 (mg/kg)
L-3 (mg/kg)
L-2 (mg/kg)
Total
Open
Close
18
16
14
12
10
8
18
16
14
12
10
8
18
16
14
12
10
8
D
E
F
*
*
*
*
*
6
6
6
*
*
*
*
*
*
4
4
4
*
2
2
2
0
0
0
0
1
2
4
8
0
0.5
1
2
3.5
0
0.5
0.75
1
L-4 (mg/kg)
Diazepam (mg/kg)
Zolpidem (mg/kg)
Fig. 2. The effects of various doses of the four imidazopyridine derivatives (A, B, C, D), diazepam (E) and zolpidem (F) on the means7SEM of
Ã
total, open and closed arms entries of the plus maze (Ns 5 5 and 7 groups). Pr0.05 versus vehicle control.
Conditioned Defensive Burying
cumulative burying behavior and burying behavior
The burying behavior latency and the cumulative latency [Fig. 4B, one-way ANOVA F(6,37) 55.893;
burying behavior of the four imidazopyridines at various Po0.001, F(6,37) 53.771; Po0.005]. A Newman-Keuls
doses (Fig. 4A–D), diazepam (Fig. 4E), and zolpidem post-hoc test confirmed that L-2 at 8 and 15 mg/kg
(Fig. 4F) on male mice are shown in Figure 4. In suppresses conditioned burying while it caused an
Figure 4A, conditioned defensive burying was signifi- increase on burying behavior latency at 15 mg/kg. L-3
cantly suppressed [F(6,36)5 7.347, Po0.001] by L-1 caused significant differences in cumulative burying
over a dose range of 2–15 mg/kg, whereas significantly behavior [F(6,36) 5 6.307, Po0.001] with no change in
increased effects [F(6,36) 5 4.443, Po0.002] on the burying behavior latency [F(6,36) 52.237; n.s.] observed
burying behavior latency at the highest dose of 15 mg/kg (Fig. 4C). No change in burying behavior latency was
was observed, an effect likely due to motor effects. found [Fig. 4D, one-way ANOVA [F(6,37) 51.762, n.s.]
Treatment with L-2 also had an increased effect on for L-4 at any doses evaluated while a decreased effect
Drug Dev. Res.

´
´
378
LOPEZ-MARTINEZ ET AL.
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
*
A
B
C
*
*
*
0
0.5
1
2
4
8
0
0.5
1
2
4
8
0
1
2
4
8
L-3 (mg/kg)
L-1 (mg/kg)
L-2 (mg/kg)
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
*
D
F
E
*
0
0.5
0.75
1
0
1
2
4
8
0
0.5
1
2
3.5
Zolpidem (mg/kg)
L-4 (mg/kg)
Diazepam (mg/kg)
Fig. 3. The effects of various doses of the four derivates of imidazopyridines (A, B, C, D), diazepam (E), and zolpidem (F) on the mean7SEM
Ã
percent of time spent in the open arms of the plus maze Ns 5 5 and 7 groups). Po0.05 versus vehicle control.
[F(6,37) 53.648, Po0.006] on cumulative burying [F(6,35)5 2.929; Po0.02] and L-2 [F(6,35) 5 6.436;
behavior was found.
Po0.001] only at the highest dose (64 mg/kg) effects,
Diazepam (Fig. 4E) had a significant effect most likely due to motor effects. The imidazopyridines
[F(6,38) 5 12.024; Po0.001] on cumulative burying had no effect on grasping reflex in the horizontal wire
behavior. Student-Newman-Keuls post-hoc compari- test at any doses evaluated while diazepam impaired
sons demonstrated a predictable decrease in the time rotarod performance at 2.75mg/kg and prevented
of cumulative burying behavior with diazepam at doses grasping reflex in 71% of the mice at 1.5 mg/kg (Table 3).
of 0.75–1.5 mg/kg, with an increase in burying behavior
latency occurring at 1.5 mg/kg [F(6,38) 5 8.424;
DISCUSSION
Po0.0.001; Fig. 4E]. Zolpidem decreased cumulative
In this study a series of imidazo[1,2-a]pyridines
burying behavior [F(4,26) 5 19.69; Po0.001] at 3 and
were synthesized and evaluated for behavioral responses
in comparison to zolpidem and the BZ diazepam based
on structural analogy with the former and a qualitatively
similar performance with the latter.
10 mg/kg and burying behavior latency increased at the
same doses [F(4,26) 5 41.351; Po0.001; Fig. 4F].
Motor Coordination (Rotarod and the
Horizontal Wire) Tests
The results obtained in the rotarod test determined
The effects of each of the four imidazopyridines that the imidazo[1,2-a]pyridine derivatives L-1 and L-2
in rats on total numbers of falls on the rotarod impaired rotarod performance only at the highest
(2–64 mg/kg) and the percent of mice with impaired evaluated dose (64 mg/kg) at which reduction of motor
grasping reflex in the horizontal wire test (4–60 mg/kg) activity was observed (the results are not shown) and
was determined.
A
statistically reliable effect thereby no conclusions about myorelaxant effects can be
was observed on total numbers of falls for L-1 proposed. In contrast, derivates did not exhibit effects on
Drug Dev. Res.

EVALUATION OF IMIDAZO[1,2-a ]PYRIDINES
379
600
500
400
300
200
100
0
600
500
400
300
200
100
0
600
A
B
C
500
400
*
300
*
200
*
100
0
*
*
*
*
*
*
*
0
0.5
1
2
4
8
15
0
0.5
1
2
4
8
15
0
0.5
1
2
4
8
15
L-1 (mg/kg)
L-2 (mg/kg)
L-3 (mg/kg)
Burying behavior latency
Cumulative burying behavior
*
600
500
400
300
200
100
0
600
500
400
300
200
100
0
600
500
400
300
200
100
0
F
E
D
*
*
*
*
*
*
*
*
*
0
0.5
1
2
4
8
15
0
0.3
1
3
10
0
0.25 0.5 0.75
1
1.25 1.5
L-4 (mg/kg)
Zolpidem (mg/kg)
Diazepam (mg/kg)
Fig. 4. The effects of the four imidazopyridine derivatives (A, B, C, D), diazepam (E) and zolpidem (F) at various doses on the means7SEM of
Ã
cumulative burying behavior and burying behavior latency of male mice (Ns 5 6 and 7 groups). Po0.05 versus vehicle control.
the horizontal wire test even at the highest evaluated obtained in locomotor activity measured on the open field
doses (Table 3). Diazepam impaired the motor ability test. As expected, Diazepam significantly increased the
at minimum doses of 2.75 mg/kg, similar to previous open arms entries and percent of time spent in the open
reports [Soderpalm et al., 1988].
arms of the plus maze at doses of 1 and 2 mg/kg without
Anxiolytic-like behavior was determined for modifying the number of total entries [Soderpalm et al.,
derivatives L-2 and L-3 based on the following facts: 1988; Frankowska et al., 2007].
Treatment with L-2 or L-3 significantly increased the
The most widely used behavioral tests of anxiolytic
percent of time spent in the open arms of the plus properties of drugs are conflict models. Since the drug
maze at doses of 1 and 2 mg/kg without modifying the effects in conflict models (the Conditioned Defensive
number of total entries. In addition, L-2 and L-3 at Burying test) are positively correlated with BZ receptor
1 mg/kg increased the number of entries to open arms. binding [Lippa et al., 1978], the effects produced by the
It must be emphasized that the previous results are not imidazopyridines during performance on the plus maze
mediated by motor actions (Table 1).
test were compared to those generated from treatment
The total number of arm entries is directly related with aolpidem and diazepam on Conditioned Defensive
to locomotor activity [Pellow et al., 1986], although this Burying test. Derivatives L-1 (2–15 mg/kg), L-2 (8 and
parameter cannot be considered independently of the 15 mg/kg), L-3 (4–15 mg/kg), and L-4 (15 mg/kg)
anxiety state. In agreement with the previous statement, induced a clear reduction in cumulative burying behavior.
L-1, L-2, and L-3 at higher doses (4 and 8 mg/kg) As a consequence, an effect in the reduction in experi-
decreased the number of total entries and the number of mental anxiety was obtained [Treit et al., 1980; Treit,
entries to open and closed arms. L-4 showed a similar 1985]. L-1 and L-2 modified the burying behavior latency
effect at a dose of 8 mg/kg. Results also agree with those only at a dose of 15 mg/kg. This would indicate that the
Drug Dev. Res.

´
´
LOPEZ-MARTINEZ ET AL.
380
TABLE 3. Average Results Obtained With Imidazopiridine Treatment on Rotarod (Rat) and Horizontal Wire (Mice) Tests to Evaluated
Myorelaxant Effecta
Rotarod
Total falls (5 min)
Horizontal Wire test
Compound
Dose (mg/Kg)
Dose (mg/Kg)
Total falls (1 min)
Mice with impaired grasping reflex (%)
0
Tween 80 (10%)
Diazepam
L-1
0
2.75
2
4
8
16
32
64
2
4
8
16
32
64
2
4
8
16
32
64
2
4
8
1.870.3
5.4370.61
2.070.6
1.370.4
1.370.2
2.370.6
2.670.5
0
1.5
0.3370.21
Ã
Ã
Ã
6.070.4
71
4
8
15
30
60
0.570.3
0.370.3
2.271.4
1.670.5
2.670.9
0
0
33
33
33
ÃÃ
4.070.9
1.270.4
2.070.3
2.070.4
1.770.4
2.070.4
2.270.7
2.770.5
2.070.6
2.370.6
2.070.6
2.370.5
3.070.6
1.370.3
1.570.4
1.570.4
2.670.5
3.270.6
4.270.3
L-2
L-3
L-4
4
8
15
30
60
0.770.4
1.770.4
1.370.7
1.670.6
1.970.6
0
17
17
17
50
4
8
15
30
60
0.870.4
1.070.4
2.170.7
2.370.6
2.270.6
0
0
50
50
50
4
8
15
30
60
0.370.2
0.870.4
0.370.2
0.470.4
1.870.6
0
0
0
0
20
16
32
64
Ã
aThe effects of imidazopyridine derivatives on mean7SEM myorelaxant activity in rat (N 5 6 and 7 groups) and mice (Ns 5 6,7/group),
ÃÃ
Ã
Po0.001, Po0.02 versus vehicle control.
reactivity of the animal is decreased. However, it is signs were variably present beginning at dose levels
important to note that at such a dose, two out of six above 8 mg/kg for L-1 and 15 mg/kg for L-2, L-3, L-4,
animals were sedated. As expected, diazepam reduced with larger standard errors often seen at these border-
conditioned defensive burying at experimental doses line doses. Diazepam and zolpiden begin a sedative
0.75–1.5 mg/kg and increased significantly the burying effect at minimum doses of 1 and 3 mg/kg, respectively.
behavior latency but at sedative doses higher than
It has been suggested that imidazopyridines
1.5 mg/kg. On its own, zolpidem (3 mg/kg) showed such as zolpidem and alpidem have preferential affinity
significant reduction on both parameters but at the same for the type o-1 GABAAA/BZ receptor complex
dose reduced motor activity and, therefore, no conclusions (a structure that contains a1 subunits) [Langer et al.,
about reactivity or anxiety-like effects can be proposed.
1985; Depoortere et al., 1986; Benavides et al., 1988],
To confirm sedative effects of the drugs, the which may suggest that the tested imidazopyridines
motor activity was evaluated. The ED50 values in mice exert their effect through a gabaergic mechanism.
were calculated using a probit analysis (Table 2).
Different reports [Rudolph et al., 1999; McKernan
The sedative effect consists of significant reduction et al., 2000] indicate that benzodiazepine receptor-active
in ambulatory and vertical activity. The ED50 doses agents produce sedation through interactions with
found were: 7.43 mg/kg for L-1, 19.07 mg/kg for L-2, a1-containing GABAA BZ receptor complex, and that
30.88 mg/kg for L-3, and 17.70 mg/kg for L-4.
those compounds without this activity may act as
All compounds decreased motor activity in anxyolytic agents (possibly by complexation to other
mice and rats; thus, the evaluated imidazopyridines a-subunits of the receptor) with diminished sedative
had a sedative effect at higher doses than diazepam and motor-impairing side effects. In light of the present
(0.95 mg/kg) and zolpidem (3 mg/kg). At high doses, findings, further investigation of the specificity of
mice moved very slowly and showed long pauses with imidazopyridines evaluated for GABAA benzodiazepine
minimum movement and partially closed eyes. These receptor subtypes is warranted.
Drug Dev. Res.

EVALUATION OF IMIDAZO[1,2-a ]PYRIDINES
381
It was concluded that the tested imidazopyridines
showed sedative properties at minimum doses of 8 and
15 mg/kg (mice) and 4 and 8 mg/kg (rats). In addition,
L-1, L-2, and L-3 produced anxiolytic-like behavior in
tested animals. Ongoing efforts are focused on whether
the differential anxiolytic and sedative potencies of the
four imidazopyridines observed in this study could be
attributed to partial agonist actions.
pharmacological profile of SL 80.0570-23N, a new short acting
hyonotic. J Neurochem 44:179.
Lippa AS, Klepner CA, Yunger L, Sano MC, Smith WV, Beer B. 1978.
Relationship between benzodiazepine receptors and experimental
anxiety in rats. Pharmacol Biochem Behav 9:853–856.
Lombardino JG. 1965. Preparation and new reactions of imidazo
[1m2-apytidines. J Otg Chem 30:2403–2407.
McKernan RM, Rosal TW, Reynols DS, Sur C, Waffor KA,
Atack JR, Farrar S, Myers J, Cook G, Ferris P, Garrent L,
Bristo L, Marshall G, Macaulay A, Brown N, Howell O. 2000.
Sedative but no anxiolytic properties of benzodiazepines are mediated
by the GABA(A) receptor alpha 1 subtype. Nat Neurosci 3:587–592.
ACKNOWLEDGMENTS
We thank Dr. Atonatiu Gomez Martinez bioterio
UNAM for useful assistance in the management of animals;
Carlos Gomez Castro and Patricia Mendoza Hernandez for
Pellow S, File SE. 1986. Anxiolytic and anxiogenic drug effects on
exploratory activity in an elevated plus-maze: a novel test of
anxiety in rat. Pharmacol Biochem Behavior 24:525–529.
´
´
technical assistance; Nadia A. Lopez-Martınez for her
Pellow S, Chopin P, File SE, Briley Mike. 1985. Validation of open:
closed arm entries in an elevated plus-maze as a measure of
anxiety in he rat. J Neurosci Methods 14:149–167.
´
careful assistance with the experiments. M. Lopez
´
Martılnez thanks Conacyt for a doctoral studies grant
202056. Financial support from the ‘‘Consejo Nacional de
´
Picazo O, Fernandez-Guasti A. 1995. Anti-anxiety effects of
´
´
Ciencia y tecnologıa’’ (CONACyT/Mexico) through pro-
jects 91426 and 49937 is gratefully acknowledged.
progesterone and some of its reduced metabolites: an evaluation
using the burying behavior test. Brain Res 680:135–141.
Pinel JP, Treit D. 1978. Buying as a defensive response in rats.
J Comp Physiol Psychol 92:708–712.
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