2-Phenyl-imidazo[1,2-a]pyridine compounds containing hydrophilic groups as potent and selective ligands for peripheral benzodiazepine receptors: Synthesis, binding affinity and electrophysiological studies

Article abstract of DOI:10.1021/jm8006728

A series of imidazopyridine acetamides were synthesized to evaluate the effects of structural changes at both central (CBRs) and peripheral benzodiazepine receptors (PBRs). These changes include the introduction of polar substituents or ionizable function

Full text of DOI:10.1021/jm8006728

6876
J. Med. Chem. 2008, 51, 6876–6888
2-Phenyl-imidazo[1,2-a]pyridine Compounds Containing Hydrophilic Groups as Potent and
Selective Ligands for Peripheral Benzodiazepine Receptors: Synthesis, Binding Affinity and
Electrophysiological Studies
Nunzio Denora,† Valentino Laquintana,† Maria Giuseppina Pisu,‡,§ Riccardo Dore,‡ Luca Murru,‡ Andrea Latrofa,†
Giuseppe Trapani,*,† and Enrico Sanna*,‡
Dipartimento Farmaco-Chimico, Facolta` di Farmacia, UniVersita` degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy, Dipartimento di
Biologia Sperimentale, Sezione di Neuroscienze, UniVersita` di Cagliari, Cittadella UniVersitaria Monserrato, SS 554 Km 4.5, Monserrato
(Cagliari), Italy, National Research Council (C.N.R.), Institute of Neuroscience, Cagliari, Italy
ReceiVed June 4, 2008
A series of imidazopyridine acetamides were synthesized to evaluate the effects of structural changes at
both central (CBRs) and peripheral benzodiazepine receptors (PBRs). These changes include the introduction
of polar substituents or ionizable functional groups at the 2- and 8-position of the imidazopyridine skeleton.
The results suggest that substituents endowed with hydrogen bonding acceptor and/or donor properties in
the para position of the phenyl ring lead to high affinity for PBR. In electrophysiological studies, it was
found that compounds 9, 12, 13, and 28 markedly enhanced GABA-evoked Cl- currents in Xenopus oocytes
expressing R1ꢀ2γ2 GABAA receptors. The capability of flumazenil to reduce the stimulatory effect exerted
by compound 9 supports the conclusion that the modulatory effects of the examined compounds occur
involving the CBR. The ability of compound 16 to increase GABAA receptor-mediated miniature inhibitory
postsynaptic currents in CA1 pyramidal neurons is indicative of its ability to stimulate the local synthesis
and secretion of neurosteroids.
Introduction
A broad spectrum of functions are associated with the PBR,
including the regulation of cholesterol transport and the synthesis
of steroid hormones, porphyrin transport, and heme synthesis,
apoptosis, cell proliferation, anion transport, regulation of
mitochondrial functions, and immunomodulation.2,3 Among
these different functions, of particular interest are those referring
to the neurosteroid synthesis and the involvement in apoptosis
processes. Thus, it is now well known that steroids synthesized
in the brain modify neuronal activity by modulating GABAA
receptor function. Changes in neurosteroid levels are associated
with various physiological and pathological conditions including
stress, pregnancy, neural development, aging, and postpartum
depression. The neurosteroid levels are also altered by psycho-
active drugs, including ethanol and antidepressants such as
fluoxetine.4 On the other hand, the PBR is component of the
MPTP located at junction sites between the inner and the outer
mitochondrial membranes. MPTP opening induced by some
PBR ligands increases the mitochondrial membrane perme-
ability, allowing the leakage of mitochondrial pro-apoptotic
factors such as caspase-3 and caspase-9.5 Several experimental
evidences also indicate that PBRs are overexpressed in a number
of tumor types, and PBR expression appears to be related to
the tumor malignancy grade.6,7 Therefore, these receptors could
be the target to selectively increase anticancer drug delivery by
using an appropriate PBR ligand-anticancer drug conjugate as
well as appropriate PBR ligands could be used as diagnostic
imaging agents.8,9 On the basis of all these functions, there are
many potential clinical applications of PBR modulation such
as in oncologic, endocrine, neuropsychiatric, and neurodegen-
erative diseases.
During the last decade, the peripheral-type benzodiazepine
receptor (PBRa) has been the object of extensive studies aimed
at defining its biochemical and pharmacological role. In contrast
to central-type benzodiazepine receptors (CBRs), which are
located primarily in neurons in the central nervous system
(CNS), peripheral-type benzodiazepine receptors (PBRs) were
detected in the 1970s as benzodiazepine binding sites mainly
in several peripheral tissues such as steroidogenic organs and
blood cells.
The PBR is a mitochondrial protein of 18 kDa, which can
form a trimeric complex with the voltage dependent anion
channel VDAC (32 kDa) and the adenine nucleotide carrier
ANC (30 kDa). The 18 kDa protein is the minimal functional
unit of PBR and recently has been named “translocator protein”
regardless of its subcellular localization.1
* To whom correspondence should be addressed. For G.T.: phone, (039)
080-5442764; fax, (039) 0805442754; E-mail, trapani@farmchim.uniba.it.
For E.S.: phone, (039) 070-6754139; fax, (039) 07-6754166; E-mail,
esanna@unica.it.
†
Dipartimento Farmaco-Chimico, Facolta` di Farmacia, Universita` degli
Studi di Bari.
‡
Dipartimento di Biologia Sperimentale, Sezione di Neuroscienze,
Universita` di Cagliari, Cittadella Universitaria Monserrato.
§
National Research Council (C.N.R.), Institute of Neuroscience.
a Abbreviations: ACSF, artificial cerebrospinal fluid; Boc, tert-butoxy-
carbonyl; Boc-Gly-OH, N-(tert-butoxycarbonyl)glycine; CBRs, central
benzodiazepine receptors; CDI, N,N′-carbonyldiimidazole; CNS, central
nervous system; DCC, N,N′-dicyclohexylcarbodiimide; DMAP, 4-(dim-
ethylamino)pyridine; DMF, dimethylformamide; DMSO, dimethylsulfoxide;
EEDQ, ethyl 1,2-dihydro-2-ethoxy-1-quinolinecarboxylate; EGTA, ethyl-
englycol-bis-(2-aminoethyl ether)-N,N,N′N′-tetracetic acid; HEPES, 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid; MBS, modified Barth’s
saline; mIPSCs, miniature inhibitory post-synaptic currents; MPTP, mito-
chondrial permeability transition pore; THF, tetrahydrofuran; PBRs,
peripheral benzodiazepine receptors; SAR, structure-activity relationship;
SI, selectivity index; TEA, triethylamine; 3R,5R-TH Prog, 5R-pregnan-3R,-
ol-20-one; TLC, thin layer chromatography.
Nowadays, a wide variety of specific ligands with high affinity
and selectivity for PBR have been identified (Chart 1). For
example, compound 1 (Ro5-4864),10 4′-chlorodiazepam, exhibits
high affinity for the PBR and very low affinity for the CBR.
Compound 2 (PK11195)11 is an isoquinoline derivative, and
10.1021/jm8006728 CCC: $40.75
2008 American Chemical Society
Published on Web 10/04/2008

2-Phenyl-imidazo[1,2-a]pyridines Containing Hydrophilic Groups
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6877
Chart 1. Synthetic Ligands for PBR
amino-, hydroxy-, and carboxylic-groups. Consequently, these
polar substituents could be useful in increasing the hydrophilicity
of PBR ligands and, on the other hand, they could be directly
conjugated to anticancer drugs or hydrophilic polymers (e.g.,
polyethylenglicols) with a reversible chemical linkage.
In this paper, we present the synthesis and biological
evaluation of new potent and selective PBR ligands mainly
characterized by hydrophilic substituents at 2- and 8-positions
of the imidazopyridine nucleus. The modulatory action of most
compounds evaluated at human R1ꢀ2γ2 GABAA receptors
expressed in Xenopus oocytes are also discussed.
Chemistry
The imidazopyridine acetamides 9-11, 14, and 16-18
(Table 1) were synthesized using synthetic methods outlined
in Scheme 1. In particular, condensation in DMF at reflux
of suitably substituted 2-aminopyridines 33 with the ap-
propriate bromoketoacids 32 yielded the butyl imidazopyri-
dine acetates 34. Compounds 32, in turn, were prepared by
treatment of the keto-butyl esters 31 with bromine in carbon
tetrachloride. Compounds 34 were hydrolyzed with 1 N
NaOH in n-butanol to give the corresponding acids 35. These
compounds were then converted to the desired compounds
9-11, 14, and 16-18 by condensation of the appropriate
dialkylamines in the presence of EEDQ as dehydrating agent.
In particular, the preparation of the nitroimidazopyridines
14 and 16-18 was readily accomplished by using the H2SO4/
HNO3 mixture. Furthermore, compounds 14, 16, 17, and 18
were smoothly reduced to the corresponding aminoimida-
zopiridines 15, 19, 20, and 21 by using cyclohexene with
10% Pd/C or Sn in HCl.19 As shown in Scheme 2, the
demethylation of the 2-p-methoxyphenylimidazopyridine
acetic acid 35a was accomplished with HBr (48% w/w) at
reflux, giving rise to the 2-p-hydroxyphenylimidazopyridine
acetic acid 36a. The subsequent condensation of this last
compound with the suitable dialkylamine hydrochloride in
anhydrous THF in the presence of CDI gave compounds 6-8.
Treatment of compound 6 with Boc-Gly-OH in the presence
of DCC and the subsequent Boc removal with gaseous HCl
led to compound 12, while treatment of 6 with methyl-4-
chloro-4-oxobutanoate in anhydrous THF gave compound 13.
The synthesis of compound 22 was easily accomplished by
refluxing a solution of 19 in toluene in the presence of
succinic anhydride. The synthesis of compounds 23-27 was
accomplished by the procedures shown in Scheme 3.
Compounds 23-25 were obtained by reaction of the known
N,N-di-n-propyl [2-(4-chlorophenyl)-8-amino-imidazo[1,2-
a]pyridin-3-yl)]acetamide 3815 with the appropriate cyclic
anhydride, while compound 26 was prepared by condensation
of 38 with the N-Boc-glycine and the subsequent selective
deprotection of the amino group with gaseous HCl. Com-
pounds 28 and 29 were prepared by the sequence of reactions
reported in Scheme 4, which involves the synthesis of the
methyl bromoester 43 by a reported procedure,20 followed
by its condensation with 3,5-dichloro-2-amino-pyridine 44
to give, after hydrolysis, the thiazolylimidazopyridine-acetic
acid 46. Condensation of this last compound with the
appropriate dialkyl- or arylalkyl-amine yielded the desired
compounds 28 and 29.
currently it is the most widely used specific probe for peripheral
benzodiazepine receptors. Compound 3 (FGIN-1-27)12 is a
2-aryl-3-indoleacetamide derivative and exhibits high affinity
for the PBR with high selectivity over the CBR. Compound 4
(i.e., Alpidem)13 has an imidazopyridine skeleton in its structure
and binds with high affinity to both PBRs and CBRs.
We have recently reported a series of potent and selective
PBR ligands, designed from Alpidem by introducing several
substituents on the imidazopyridine nucleus (Compounds 5,
Chart 1).14-18 Our structure-activity correlations revealed that
substitution at the 8-position of the imidazopyridine nucleus is
a key factor for improving affinity and selectivity toward
peripheral binding sites. Substitutions at the 8-position with
lipophilic groups and at the para position of the phenyl ring at
C(2) with a chlorine atom are crucial for high affinity and
selectivity.12 Additionally, the substituents on the acetoamide
nitrogen on the 3-position of the imidazopyridine nucleus are
responsible for modulation of affinity.18 Mainly, increasing the
branching of the alkyl substituent on the carboxamide nitrogen
of the potential PBR ligand may cause unfavorable interaction
with the receptor. The presence of aromatic substituents on the
carboxamide nitrogen leads to high affinity and selectivity, while
introduction of polar substituents in this region is detrimental
for binding affinity.
As a continuation of our studies on 2-phenylacetamidoimi-
dazo[1,2-a]pyridines as potent and selective PBR ligands, our
attention has been focused on the 2- and 8-position of the
imidazopyridine nucleus to clarify the effect of substituents in
these specific regions on modulation of affinity to PBR. It should
be noted that, to our knowledge, very few PBR ligands
containing hydrophilic groups have been synthesized so far.
Therefore, the need for a greater aqueous solubility for our PBR
ligands prompted us to introduce polar substituents or ionizable
functional groups. Particularly, the phenyl group on the 2-posi-
tion of the imidazopyridine skeleton was functionalized with
1
All compounds were fully characterized by IR, H NMR,
mass spectra, and elemental analyses (Experimental Section).
As already noted in the previous work,18 an interesting feature
1
of the H NMR spectra of these imidazoacetamides concerns
the signals associated with the methylene linked at the carbonyl

6878 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Table 1. Structure and Physical Properties of Compounds 6-29
Denora et al.
compd
X
Y
R1
R2
R3
R4
yield (%)
mp (°C)
6
7
9
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
H
Cl
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
OH
OH
OCH3
OCH3
OCH3
OCOCH2NH2 ·HCl
OCO(CH2)2COOCH3
NO2
NH2
NH2 HCl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
n-C3H7
CH3
n-C3H7
CH3
n-C3H7
n-C4H9
n-C3H7
n-C4H9
C6H5
43
45
85
83
65
94
95
55
68
95
74
59
68
62
88
54
65
84
77
82
87
97
72
21
22
212-216
200-203
123-125
114-117
201-203
N.D.a
199-201
218-220
200 dec
165 dec
177-180
147-150
155-157
218-220
N.D.a
223-225
217-220
159 dec
230 dec
258 dec
203 dec
N.D.a
253-256
158-163
172-175
10
11
12
13
14
15a
15b
16
17
18
19a
19b
20
21
22
23
24
25
26
27
28
29
CH3
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
CH3
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C4H9
n-C4H9
n-C3H7
n-C3H7
n-C4H9
n-C4H9
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
C6H5
H
H
NO2
NO2
NO2
NH2
NH2 HCl
NH2
NH2
CH3
n-C3H7
n-C3H7
CH3
CH3
NH-CO(CH2)2COOH
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C3H7
CH3
NHCOCH2COOH
NHCO(CH2)2COOH
NHCO(CH2)3COOH
NHCOCH2NH2 ·HCl
NHCO(CH2)2CO2C2H5
Cl
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
a N.D., not determined.
Scheme 1. Preparation of the Imidazopyridine Acetamides 9-11, 14-21a
a Reagents and conditions: (a) n-butanol/H2SO4; (b) Br2/CCl4; (c) n-butanol, ∆; (d) n-butanol/ NaOH 1N; (e) CDI, anhydrous solvent, TEA; (f) EtOH/
HCl, granular tin, ∆; (g) anhydrous THF/HCl(g)
.
group and the methyl and methylene groups characterizing the
alkyl chains of CO-N < R3R4. The -CH2CO- gives rise to a
singlet in the range 3.7-4.6 δ only when the R3 and R4 groups
are of similar size. When these two groups are very different in
size (e.g., in the case of compounds 7, 10, 17, 18, 20, and 21),
the signals of the CH2CO and those of the protons in the R3

2-Phenyl-imidazo[1,2-a]pyridines Containing Hydrophilic Groups
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6879
Scheme 2. Preparation of the Imidazopyridine Acetamides 6-8, 12, 13, 22a
a Reagents and conditions: (a) conc HBr, ∆; (b) CDI, anhydrous THF, TEA; (c) N-Boc-Gly, DCC, anhydrous THF, TEA; (d) 0 °C, CH2Cl2/HCl(g); (e)
methyl-4-chloro-4-oxobutyrate, anhydrous THF; (f) succinic anhydride, toluene, ∆.
and R4 groups are duplicated due to the partial double bond
character of the amide bond. Physical data for new compounds
are reported in Table 1.
H-bonding acceptor moiety (δ1, usually a suitably oriented
amide carbonyl group) plays a key role in the interaction with
PBR.
As above mentioned, to our knowledge, very few PBR ligands
containing hydrophilic groups are known. Therefore, the need
for a greater aqueous solubility for our PBR ligands as well as
the possibility to exploit these hydrophilic groups for a direct
conjugation to drugs, especially anticancer drugs or hydrophilic
polymers with a reversible chemical linkage, prompted us to
introduce polar substituents or ionizable functional groups on
the phenyl at the 2 position of the imidazopyridine skeleton.
Thus, compounds 6-8 as p-OH-substituted 2-phenyl-imida-
zopyridine derivatives were synthesized. Interestingly, it was
noted that introduction of a polar substituent such as the -OH
group in that position leads to compounds with high affinity
for PBR and greater than that of the reference compound 2 (Ki,
1.14 nM); in particular, compound 8 showed an affinity value
in the subnanomolar order. Moreover, the selectivity index (SI)
for PBR versus CBR, expressed as Ki CBR/Ki PBR ratio, for
Results and Discussion
Affinities of Imidazopyridine Derivatives for Peripheral
and Central Benzodiazepine Receptors. The affinities of the
26 tested compounds for PBR and CBR were determined by
measuring their ability to displace compound [3H]-2 and [3H]-
flunitrazepam (5-(2-fluorophenyl)-1-methyl-7-nitro-1,3-dihydro-
2H-1,4-benzodiazepin-2-one) from binding to membrane prepa-
rations obtained from rat cerebral cortex. Their effects were
compared with those of unlabeled 2, a selective ligand for
PBR.11 The measured binding affinity for PBR and CBR
expressed as Ki values are shown in Table 2. The structural
analysis of PBR ligands 6-29 suggests that they may share
the pharmacophore components proposed by Bourguignon21 and
Anzini22 (δ1, LA, PAR, FRA in Figure 1) in which a single

6880 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Denora et al.
Scheme 3. Preparation of the Imidazopyridine Acetamides 23-27a
a Reagents and conditions: (a) Meldrum’s acid, anhydrous THF (23); succinic or glutaric anhydride, toluene, ∆ (24, 25); (b) 50 °C, EtOH/conc H2SO4;
(c) N-Boc-Gly, DCC, anhydrous CH2Cl2, DMAP, TEA; (d) anhydrous CH2Cl2/HCl(g)
.
compounds 6 and 8 was very high (>103). A pairwise
comparison of binding data demonstrated that replacement of
two n-propyl groups on the carboxamide nitrogen in 6 with
methyl/butyl or methyl/phenyl groups (i.e., 7 and 8, respectively)
improved the affinity. Methylation of the p-OH groups of
compounds 6-8 gave the methylethers 9-11, respectively.
Interestingly, the latter compounds retained good affinity and
selectivity for PBR even at subnanomolar level (Table 2). These
results suggest that the presence of substituents endowed with
hydrogen bonding acceptor properties in the para position of
the phenyl ring seems to be favorable for high affinity and
selectivity. Esterification of compound 6 with glycine and
methyl emisuccinate led to compounds 12 and 13, and both of
these compounds showed high affinity and selectivity. These
data suggest that the presence of an ionizable group such as
the amino group of the glycinate 12 on the para-position of the
phenyl ring seems to be favourable for high affinity and
selectivity. To further examine the behavior of an ionizable
group, compounds 15a,b (i.e., p-NH2- and p-NH2 ·HCl sub-
stituted 2-phenyl-imidazopyridine compounds) were synthesized.
Again, high affinity for the PBR was observed even though it
was slightly lower than that of the p-OH derivative 6. However,
compounds 15a,b showed a modest selectivity for this receptor
(SI of 111 and 371, respectively). The high affinity of compound
15b suggests that a substituent endowed with hydrogen bonding
donor properties in the para position of the phenyl ring should
favorably interact with the corresponding complementary site
of the receptor. Therefore, it may be concluded that substitution
with groups characterized by hydrogen bonding acceptor and/
or donor properties in the para position leads to high affinity
for PBR.
We next examined the biological effects of the p-Cl-m-NH2
and p-Cl-m-NH2 ·HCl disubstituted compounds 19a, 20, 21, and
19b, respectively. Interestingly, compounds 19a,b, 20, 21
showed high affinity and, in particular 19a,b, together with the
nitro-compounds 16-18, proved to be among the most active
(at subnanomolar level) compounds in this series. However,
compounds 20 and 21 exhibited a modest selectivity for PBR
with SI of 686 and 187, respectively. In contrast, the carboxylic
acid 22 showed a high selectivity for PBR but an affinity value
(i.e., Ki 14.4 nM) about 20-fold lower than those of compounds
20 and 21. These data support the conclusion that the substitution
with groups possessing hydrogen bonding acceptor and/or donor

2-Phenyl-imidazo[1,2-a]pyridines Containing Hydrophilic Groups
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6881
Scheme 4. Preparation of the Imidazopyridine Acetamides 28, 29a
a Reagents and conditions: (a) 0 °C; (b) PyH+Br3-, THF; (c) toluene, ∆; (d) dioxane, HCl 1N; (e) EEDQ, THF.
Table 2. Affinities of Compounds 6-29 for CBR and PBR from Rat
Cerebral Cortex and Their Modulatory Action at Human R1ꢀ2γ2 GABAA
Receptors Expressed in Xenopus Oocytes
change ( SEM of
Ki (nM) Ki (nM) GABA induced Cl- GABA induced Cl-
compd CBRa PBRa current (%) at 1 µMb current (%) at 10 µMb
change ( SEM of
6
7
8
9
10
11
12
13
14
>105
2930
5130
>105
>105
1000
>104
>104
>104
1.31
0.87
0.32
0.31
0.73
0.23
1.52
1.69
0.15
2.22
2.76
0.24
0.30
1.16
0.33
0.45
0.78
1.04
-3.9 (5.3
7.9 ( 34.0
-5.9 ( 16.8
8.8 ( 3.5
24.8 ( 14.0
-8.1 ( 4.7
26.6 (6.2
16.6 (4.2
-4.2 ( 10.3
8.1 ( 2.6
29.9 (6.4
14.9 (3.6
-10.2 ( 0.2
12.1 ( 12.2
-6.2 ( 5.4
-2.9 ( 13.7
N.D.
23.8 ( 6.6
-23.6 ( 19.9
6.7 ( 3.2
55.9 (8.4
-16.5 ( 21.0
-8.0 ( 11.0
71.0 ( 15.6
37.5 ( 8.9
-16.6 ( 9.3
23.9 (6.9
-7.4 ( 2.8
9.6 ( 4.0
-9.6 ( 1.3
-20.0 ( 10.9
-38.7 ( 10.5
-2.2 ( 4.8
N.D.
10.7 ( 13.3
10.0 ( 7.8
19.8 ( 14.8
12.3 ( 0.3
-1.9 ( 30.7
N.D.
Figure 1. The suggested four components of the pharmacophore model
for PBR ligands: FRA, freely rotating aromatic ring region; δ1, electron
rich zone; PAR, planar aromatic region; LA, lipophilic area.
at 2-position, were also evaluated. The observed high affinity
and selectivity of these compounds indicates that introduction
of an aromatic ring different from the benzene one is well
tolerated, and this information could have value for molecular
design of further PBR ligands in the imidazopyridine-acetamide
series.
15a 246
15b 1024
16
17
18
>104
>105
19a >104
Among the very few examples known of PBR ligands
containing hydrophilic groups, there is the 8-amino imidazopy-
ridine 38.15 To evaluate whether further polar substituents or
ionizable functional groups could be introduced in this position,
compounds 23-27 were synthesized and examined. Data
suggest that introduction of a -COOH group led to a significant
decrease in affinity for PBR, as observed for the set of
compounds 23-25 (i.e., Ki 193.1, 285.3, and 117.7 nM,
respectively). A pairwise comparison of binding data between
compounds 24 and 27 showed that esterification of carboxylic
acid function led to a significant increase in affinity and
selectivity for PBR (i.e, Ki from 285.3 to 0.86 nM). Finally, in
contrast with that observed for carboxylic acids 23-25, the
presence of the ionizable -NH2 group as in 26 brought about
good affinity (i.e., Ki 14.2 nM) and selectivity for PBR.
Overall, all of these findings could be consistent with the
pharmacophore mentioned above.22 While the SAR studies
highlighted the main physicochemical factors eliciting the
binding of imidazopyridines herein examined to PBR, we further
explored the pharmacological profile of these compounds by
measuring their modulatory effects on the GABAA receptors.
Functional in Vitro Study. a. Electrophysiology in Xeno-
pus Oocytes. To gain further information on the selectivity of
19b >104
20
21
22
23
24
25
26
27
28
29
2
535.0
194.0
>104
>104
>104
>104
>104
>104
>105
>105
>105
9.5 ( 15.6
11.5 ( 11.0
9.6 ( 6.9
1.3 ( 24.0
7.3 ( 25.1
N.D.
14.4
193.1
285.3
117.7
14.2
0.86
N.D.
N.D.
2.07
1.44
1.14
6.3 ( 2.5
-1.2 ( 10.6
4.9 ( 5.3
60.6 ( 12.3
19.4 ( 4.6
9.4 ( 4.8
a Data are means of three separate experiments performed in duplicate
which differed by less than 10%. b Values are expressed as percentage
change induced by the different drugs from the control response obtained
with GABA EC10 (concentration of GABA, usually ranging from 0.5 to 10
µM, which produced a Cl- current the amplitude of which was 10 ( 3%
of the maximal response to 1 mM GABA). Data are means (from five to
ten different oocytes) ( SEM; N.D., not determined.
properties in meta position of the phenyl ring leads to high
affinity binding for the PBR.
To improve our understanding of the structure-activity
relationships (SAR) at the 2-position of the imidazopyridine
skeleton, compounds 28 and 29, characterized by a thiazole ring

6882 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Denora et al.
brain in both glia and neurons, is mediated by the activation of
PBR and such steroids act as allosteric modulators of the
GABAA receptors. Our previous work has demonstrated that
in vitro application of the PBR selective compound N,N-di-n-
propyl-[2-(4-chlorophenyl)-6,8-dichloroimidazo[1,2-a]-pyridin-
3-yl)]acetamide (CB34)15,16 on rat hippocampal slices resulted
in an increased tissue concentration of the neurosteroid 3R,5R-
TH Prog and in a consequent potentiation of the function of
GABAA receptors in CA1 pyramidal neurons.24 We thus applied
a similar experimental protocol in order to test the ability of
these derivatives to modulate GABAA receptor function and the
involvement of neurosteroidogenesis. By using the whole-cell
patch clamp technique, we recorded GABAA receptor-mediated
miniature inhibitory postsynaptic currents (mIPSCs) in CA1
pyramidal neurons present in rat hippocampal slices. Perfusion
of compound 16 (30 µM) for 30 min, which does not directly
modulate GABAA receptor function when tested in oocytes
(Table 2), induced a significant increase in both amplitude and
decay time of mIPSCs (Figure 3). To test whether such effects
were mediated by an increased local secretion of neurosteroids,
we co-applied finasteride (4aR,4bS,6aS,7S,9aS,9bS,11aR)-N-(tert-
butyl)-4a,6a-dimethyl-2-oxo-2,4a,4b,5,6,6a,7,8,9,9a,9b,10,11,11a-
tetradecahydro-1H-indeno[5,4-f]quinoline-7-carboxamide) (1 µM),
an inhibitor of 5R-reductase, together with compound 16.
Finasteride alone did not alter the kinetic characteristics of
GABAA receptor-mediated mIPSCs but abolished the increase
in both amplitude and decay time of these currents induced by
compound 16.
Figure 2. Antagonism by flumazenil of the potentiation of GABA-
evoked Cl- currents by compound 9 at human R1ꢀ2γ2 GABAA receptors
expressed in Xenopus oocytes. Values are expressed as percentage
change of GABA responses induced by compound 9 (10 µM) in the
absence and presence of flumazenil (10 µM). Data are means (from
four different oocytes) ( SEM.
the different compounds to PBR with respect to CBR, we next
examined their capacity to modulate the function of the GABAA
receptor, a pentameric ligand-gated ion channel complex on
which the CBR is located. Thus, we applied the voltage-clamp
technique to measure the modulatory effects of compounds
6-19, 21-25, 28, and 29 on GABA-evoked Cl- currents in
oocytes expressing human R1ꢀ2γ2 receptors. The different
compounds were tested at the concentration of 1 and 10 µM in
comparison with compound 2. Inspection of the data reported
in Table 2 shows that the whole set of compounds examined,
although most endowed with high affinity and selectivity for
PBR did not produce similar modulatory effects on GABAA
receptors. In particular, compounds 6-8, 10-12, 14-18, 19b,
21-25, and 29 did not significantly modify GABA-evoked Cl-
currents, with a percentage change within (30% over the control
response. The reference compound 2 induced a very weak effect
of GABAA receptor function. In contrast, compounds 9, 12, 13,
and 28 markedly enhanced GABA-evoked Cl- currents, par-
ticularly at the concentration of 10 µM. On the other hand,
compound 19a inhibited GABA-evoked Cl- currents by about
40%. To determine whether the modulatory action of these
compounds on GABA-evoked Cl- currents could be mediated
by the central benzodiazepine recognition sites (CBR), we tested
the co-application of the specific benzodiazepine receptor
antagonist flumazenil ((ethyl 8-fluoro-5-methyl-6-oxo-5,6-di-
hydro-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate). At
the concentration of 10 µM, flumazenil completely antagonized
the potentiation of GABA-evoked Cl- currents induced by
compound 9 (10 µM) (Figure 2), supporting the conclusion that
the modulatory effects of the examined compounds occur
involving the CBR. The results clearly show that there is no
correlation between PBR affinity/selectivity and modulatory
activity on GABAA receptors. Data also revealed that, similarly
to what occurs for CBR acting compounds,23 the transition from
active to inactive compounds is consequent to very small
structural changes. Thus, replacement of the two n-propyl groups
on the carboxamide nitrogen of 9 with methyl and n-butyl or
methyl and phenyl groups to give compounds 10 and 11,
respectively, brought about the change from the potentiation of
GABA-evoked Cl- currents occurring in 9 to the inhibition of
such currents observed for 10 and 11. Similarly, substitution of
two n-propyl groups on the carboxamide nitrogen of 28 with
methyl phenyl ones to give compound 29 led to a decrease of
about 40% in potentiation of GABA-evoked Cl- currents at 10
µM.
Conclusions
New potent and selective PBR ligands mainly characterized
by hydrophilic substituents at 2- and 8-positions of the 2-phe-
nylimidazopyridine nucleus have been synthesized. Biological
evaluation of these compounds revealed that, in sharp contrast
to that previously observed with the substitution on the amide
nitrogen, introduction of polar and ionisable substituents on the
para and meta positions of the phenyl ring leads to compounds
with high affinity and selectivity for PBR even at subnanomolar
level and greater than that of the reference compound 2. SAR
analysis indicated that the hydrogen bonding acceptor and/or
donor ability of the substituents in the para/meta positions of
the phenyl ring together with their lipophilic character are the
properties mainly responsible for modulation of affinity. In
addition, bulkiness of the substituents in para/meta positions
seems to be detrimental to the affinities for PBR.
Introduction of hydrophilic substituents such as amido-ester,
amido-amine, and amido-acid groups at the 8-position of the
2-phenylimidazopyridine skeleton leads to compounds endowed
with variable affinity and selectivity ranging from high (i.e.,
amino, amido-ester-, and amido-amine-derivatives) to moderate
(i.e., amido-acid-derivatives) affinity and selectivity.
In electrophysiological studies, while most of the tested
derivatives were devoid of any significant modulatory efficacy
on Xenopus oocytes expressing R1ꢀ2γ2 GABAA receptors,
compounds 9, 12, 13, and 28 markedly enhanced GABA-evoked
Cl- currents, particularly at the concentration of 10 µM. The
capability of flumazenil to reduce the stimulatory effect exerted
by compound 9 supports the conclusion that the modulatory
effects of the examined compounds occur involving the CBR.
This peculiar property found in these compounds may likely
represent a residual characteristic of imidazopyridines such as
compound 4.13 As for the possible reasons for the difference
between the negative results from CBR binding studies (low
affinity) and voltage-clamp experiments (modulation of GABA-
b. Electrophysiology in Rat Hippocampal Slices. As
mentioned above, the neurosteroid synthesis, occurring in the

2-Phenyl-imidazo[1,2-a]pyridines Containing Hydrophilic Groups
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6883
Figure 3. Compound 16 increases GABAA receptor-mediated mIPSCs in rat CA1 pyramidal neurons in a neurosteroid-dependent manner. (A)
Sample traces of mIPSCs recorded before (control), after 30 min of continuous bath application of rat hippocampal slices with compound 16 (30
µM), or in the presence of the GABAA receptor antagonist bicuculline (20 µM). (B) Averaged traces of multiple mIPSCs recorded during a 3 min
period before (control), after 30 min of application of compound 16, and after 30 min of co-application of compound 16 and finasteride (1 µM).
(C) Values represent the mean percent change ( SEM (n ) 6) in mIPSC amplitude induced by compound 16 with or without the application of
finasteride. (D) Averaged traces of multiple mIPSCs showing the increase in decay time induced by compound 16 and the reversal of this effect
by finasteride. (E) Values represent the mean percent change ( SEM (n ) 6) in mIPSC decay time constant induced by compound 16 with or
without the application of finasteride. *p < 0.05 vs control.
evoked Cl- currents) of compounds 9, 12, 13, and 28, it should
be considered that, firstly, the concentration of 10 µM, effective
in the functional assay, might be still compatible with an
estimated affinity for CBR in the high micromolar range, and
secondly, these compounds, as other imidazopyridines such as
zolpidem, may be endowed with selectivity for R1ꢀ2γ2 receptors.
In the binding studies, the membrane preparation that was used
contained a more heterogeneous population of GABAA receptors
and this may result in a reduced binding inhibition compared
to pure R1ꢀ2γ2 receptors if there is lower affinity to other
receptor subtypes. In this respect, it would be also very
interesting to further explore whether the effects of this group
of compounds, as that of imidazopyridines, are influenced by
the subunit composition of the GABA-A receptor.
midal neurons is indicative of its ability to stimulate, through
binding to PBR, local synthesis and secretion of neurosteroids
such as 3R,5R-TH Prog.24 In fact, the co-application of
finasteride, by inhibiting the formation of neurosteroids from
progesterone, prevents this effect. Taken together, these results
indicate that the modulatory effects on the function of GABAA
receptors might be due to release of neurosteroids via the
activation of PBR.
Experimental Section
Chemistry. Melting points were determined in open capillary
tubes with a Bu¨chi apparatus and are uncorrected. IR spectra were
obtained on a Perkin-Elmer IR Fourier transform spectrophotometer
in KBr pellets or in NaCl disks. 1H NMR spectra were determined
on a Varian Mercury 300 MHz instrument. Chemical shifts are
given in δ values referenced to the solvent. Mass spectra were
recorded on a Hewlett-Packard 5995 GC-MS low-resolution
spectrometer or were obtained using an Agilent 1100 LC-MSD trap
system VL instrument using methanol/ammonium formiate 7 mM
9/1 (v/v). Elemental analyses were carried out with a Carlo Erba
model 1106 analyzer, and results were within (0.40% of theoretical
values. Silica gel 60 (Merck 70-230 or 230-400 mesh) was used
However, the results clearly show that there is no correlation
between PBR affinity/selectivity and modulatory effects on the
function of recombinant R1ꢀ2γ2 GABAA receptors. Data also
reveal that the transition from active to inactive compounds is
consequent to very small structural changes.
In the hippocampal slice model, the ability of compound 16
to increase GABAA receptor-mediated mIPSCs in CA1 pyra-

6884 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Denora et al.
for column chromatography. All the following reactions were
performed under a nitrogen atmosphere, and the progress of the
reactions was monitored by thin-layer chromatography (TLC) on
using Kieselgel 60 F254 (Merck) plates.
1H, Ar), 9.70 (bs 1H, OH). MS m/z 439 (M+, 19), 305 (base).
Anal. (C23H19Cl2N3O2) C, H, N.
2-(6,8-Dichloro-2-(4-nitrophenyl)imidazo[1,2-a]pyridin-3-yl)-
N,N-dipropylacetamide (14). IR (KBr): 1644, 1523, 1342 cm-1
.
1H NMR (CDCl3) δ: 0.8-1.0 (m, 6H, CH3), 1.5-1.7 (m, 4H, CH2),
3.22 (t, J ) 7.7 Hz, 2H, CH2N), 3.34 (t, J ) 7.7 Hz, 2H, CH2N),
4.07 (s, 2H, CH2CO), 7.34 (d, J ) 1.6 Hz, 1H, Ar), 7.85 (d, J )
8.8 Hz, 2H, Ar), 8.12 (d, J ) 1.6 Hz, 1H, Ar), 8.31 (d, J ) 8.8 Hz,
2H, Ar). MS m/z 448 (M+, 10), 320 (28). Anal. (C21H22Cl2N4O3)
C, H, N.
Materials. 4-(4-Nitrophenyl)-4-oxobutanoic acid (30b) and 4-(4-
chloro-3-nitrophenyl)-4-oxobutanoic acid (30c) were prepared fol-
lowing a literature procedure.20 The general procedure for prepa-
ration of 2-phenylimidazo[1,2-a]pyridine 3-acetic acids (35a,
35c-d) was accomplished following a reported procedure.8 Com-
pound [6,8-dichloro-2-(1,3-thiazol-2-yl)imidazo[1,2-a]pyridin-3-
yl]acetic acid 46 was synthesized using a previously reported
method.25 The starting 2-amino-pyridine compounds (33), N,N′-
dialkylamines, DCC, CDI, EEDQ, DMAP, 3-(4-methoxybenzoyl)-
propionic acid, hydrobromic acid 48%, TEA, 2,2-dimethyl-1,3-
dioxane-4,6-dione (Meldrum’s acid), succinic anhydride, glutaric
anhydride, methyl 4-chloro-4-oxobutyrate, Boc-Gly-OH, and an-
hydrous THF were purchased from Sigma-Aldrich (Italy). Com-
mercial reagent grade chemicals and solvents were used without
further purification.
2-(6,8-Dichloro-2-(4-chloro-3-nitrophenyl)imidazo[1,2-a]pyridin-
3-yl)-N,N-dipropylacetamide (16). IR (KBr): 1645, 1540, 1363
cm-1 1H NMR (CDCl3) δ: 0.8-1.0 (m, 6H, CH3), 1.5-1.7 (m,
.
4H, CH2), 3.2-3.4 (m, 4H, CH2N), 4.05 (s, 2H, CH2CO), 7.34 (m,
1H, Ar), 7.64 (d, J ) 8.5 Hz, 1H, Ar), 7.8-8.0 (m, 2H, Ar), 8.0-8.2
(m, 2H, Ar). MS m/z 452 (M+, 27), 324 (17). Anal. (C21H21Cl3-
N4O3) C, H, N.
N-Butyl-2-(6,8-dichloro-2-(4-chloro-3-nitrophenyl)imidazo[1,2-
a]pyridin-3-yl)-N-methylacetamide (17). IR (KBr): 1651, 1536,
1357 cm-1. 1H NMR (CDCl3) δ: 0.9-1.0 (m, 3H, CH3), 1.2-1.4
(m, 2H, CH2), 1.5-1.7 (m, 2H, CH2), 2.99 (s, 1.5H, CH3N), 3.11
(s, 1.5H, CH3N), 3.34 (t, J ) 7.7 Hz, 1H, CH2N), 3.44 (t, J ) 7.7
Hz, 1H, CH2N), 4.04 (s, 1H, CH2CO), 4.06 (s, 1H, CH2CO), 7.34
(s, 1H, Ar), 7.63 (d, J ) 8.2 Hz, 2H, Ar), 7.8-8.0 (m, 1H, Ar),
8.0-8.2(m,1H,Ar).MSm/z468(M+,14),356(15);.Anal.(C20H19Cl3-
N4O3) C, H, N.
General Procedure for Preparation of (2-Phenyl-imidazo[1,2-
a]pyridine-3-yl)acetamides 9-11, 14, 16-18. To a stirred solution
of the suitable 2-amino-pyridine 33 (13 mmol) in n-BuOH (50 mL)
the butyl-bromoketo ester 32a-d (10 mmol) was added and the
resulting mixture was refluxed for 48 h. The reaction was monitored
by TLC and, when it was completed, the solvent was evaporated
under reduced pressure. The residue was dissolved in CHCl3 (20
mL), washed with 0.1N HCl, and dried (Na2SO4). Evaporation of
the solvent gave a residue that was purified by silica gel chroma-
tography (light petroleum ether/ ethyl acetate 8/2 (v/v) as eluent)
to give the desired butyl (2-phenyl-imidazo[1,2-a]pyridin-3-yl)ac-
etates 34a-d. Next, to a solution of 34 (5 mmol) in n-butanol (20
mL) NaOH 1 N (2 mL) was added dropwise. The mixture was
stirred at room temperature for 12 h. Then, the solvent was
evaporated under reduced pressure and the residue was taken up
with water (25 mL) and extracted with CHCl3 (3 × 20 mL). The
cooled water phase was acidified to pH 4 with 0.1 N HCl, and the
resulting precipitate of the pure acid was collected in good yield
by filtration and dried under vacuum. A solution of the prepared
acid 35a-d (1 mmol) and CDI (1.3 mmol) in anhydrous THF or
DMF (20 mL) was stirred at room temperature. Then, the suitable
amine 33 (1.3 mmol) and TEA (1.5 mmol) were added and the
mixture was stirred for 8-12 h. Solvent was removed under reduced
pressure and the residue was taken up with water, extracted with
CHCl3 (3 × 30 mL), and dried over Na2SO4. Solvent was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (light petroleum ether/ethyl
acetate 6/4(v/v) as eluent)) to give the required compounds 9-11,
14, and 16-18.
N-Butyl-2-(8-chloro-2-(4-chloro-3-nitrophenyl)imidazo[1,2-a]py-
ridin-3-yl)-N-methylacetamide (18). IR (KBr): 1623 cm-1. 1H NMR
(CDCl3) δ: 0.9-1.0 (m, 3H, CH3), 1.2-1.4 (m, 2H, CH2), 1.5-1.7
(m, 2H, CH2), 2.92 (s, 1.5H, CH3N), 2.98 (s, 1.5H, CH3N), 3.2-3.5
(m, 2H, CH2N), 4.08 (s, 1H, CH2CO), 4.10 (s, 1H, CH2CO), 6.8-
7.0 (m, 2H, Ar), 7.3-7.7 (m, 3H, Ar), 7.9-8.2 (m, 1H, Ar). MS
(ESI) m/z 432.9 [M - H]-. Anal. (C20H20Cl2N4O3) C, H, N.
General Procedure for Conversion of 14 and 16-18 to Com-
pounds 15a, 15b, 19a, 19b, 20, and 21. To a stirred solution of the
nitro compounds 14 and 16-18 (0.67 mmol) in ethanol (50 mL)
and conc HCl (20 mL), granular tin (13.4 mmol) was added. The
mixture was refluxed for 2 h and then diluted with 50 mL of water
and filtered through a pad of celite. The solvent was evaporated
under reduced pressure, and the residue was taken up with 25 mL
of 20% NaHCO3, extracted with ethyl acetate (3 × 20 mL), and
dried (Na2SO4). Evaporation of the solvent gave a residue that was
purified by silica gel chromatography (light petroleum ether/ ethyl
acetate 1/1 (v/v) as eluent). Compounds 15b and 19b were prepared
by bubbling gaseous HCl into a solution of the amine 15a and 19a,
respectively, in anydrous THF for 15 min. The excess of HCl was
removed under a stream of nitrogen, and the solvent was evaporated
under reduced pressure to give the corresponding hydrochlorides
as white solids.
2-(6,8-Dichloro-2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)-
2-(2-(4-Aminophenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)-
N,N-dipropylacetamide (9). IR (KBr): 1628 cm-1 1H NMR
.
N,N-dipropylacetamide (15a). IR (KBr): 3442, 3346, 1634 cm-1
.
(CDCl3) δ: 0.71 (t, J ) 7.4 Hz, 3H, CH3), 0.85 (t, J ) 7.4 Hz, 3H,
CH3), 1.4-1.6 (m, 4H, CH2), 3.07 (t, J ) 7.4 Hz, 2H, CH2N),
3.28 (t, J ) 7.4 Hz, 2H, CH2N), 3.85 (s, 3H, CH3O), 4.06 (s, 2H,
CH2CO), 7.00 (d, J ) 8.5 Hz, 2H, Ar), 7.29 (s, 1H, Ar), 7.58 (d,
J ) 8.5 Hz, 2H, Ar), 8.31 (s, 1H, Ar). MS m/z 433 (M+, 15), 305
(base). Anal. (C22H25Cl2N3O2) C, H, N.
1H NMR (CDCl3) δ: 0.71 (t, J ) 7.3 Hz, 3H, CH3), 0.83 (t, J )
7.3 Hz, 3H, CH3), 1.4-1.6 (m, 4H, CH2), 3.26 (t, J ) 7.7 Hz, 2H,
CH2N), 3.29 (t, J ) 7.7 Hz, 2H, CH2N), 3.80 (br, 2H, NH2), 4.08
(s, 2H, CH2CO), 6.75 (d, J ) 8.8 Hz, 2H, Ar), 7.2-7.3 (m, 1H,
Ar), 744 (d, J ) 8.8 Hz, 2H, Ar), 8.29 (d, J ) 1.8 Hz, 1H, Ar).MS
m/z 418 (M+, 18), 290 (base). Anal. (C21H24Cl2N4O) C, H, N.
2-(2-(3-Amino-4-chlorophenyl)-6,8-dichloroimidazo[1,2-a]pyri-
din-3-yl)-N,N-dipropylacetamide (19a). IR (KBr): 3447, 3354, 1633
cm-1. 1H NMR (CDCl3) δ: 0.77 (t, J ) 7.4 Hz, 3H, CH3), 0.85 (t,
J ) 7.4 Hz, 3H, CH3), 1.4-1.6 (m, 4H, CH2), 3.11 (t, J ) 7.4 Hz,
2H, CH2N), 3.28 (t, J ) 7.4 Hz, 2H, CH2N), 4.05 (s, 2H, CH2CO),
4.14 (br, 2H, NH2), 6.84 (m, 1H, Ar), 7.15 (m, 1H, Ar), 7.2-7.4
(m, 1H, Ar), 8.25 (m, 1H, Ar). MS m/z 452 (M+, 27), 324 (93).
Anal. (C21H23Cl3N4O) C, H, N.
N-Butyl-2-(6,8-dichloro-2-(4-methoxyphenyl)imidazo[1,2-a]py-
ridin-3-yl)-N-methylacetamide (10). IR (KBr): 1624 cm-1. 1H NMR
(CDCl3) δ: 0.7-0.9 (m, 3H, CH3), 1.0-1.6 (m, 4H, CH2), 2.89 (s,
1.5H, CH3N), 2.92 (s, 1.5H, CH3N), 3.13 (t, J ) 7.4 Hz, 1H, CH2N),
3.38 (t, J ) 7.4 Hz, 1H, CH2N), 3.85 (m, 3H, CH3O), 4.04 (s, 1H,
CH2CO), 4.07 (s, 1H, CH2CO), 6.99 (d, J ) 8.0 Hz, 2H, Ar),
7.2-7.4 (m, 1H, Ar), 7.5-7.7 (m, 2H, Ar), 8.2-8.4 (m, 1H, Ar).
MS m/z 419 (M+, 16), 305 (base). Anal. (C21H23Cl2N3O2) C,
H, N.
2-(2-(3-Amino-4-chlorophenyl)-6,8-dichloroimidazo[1,2-a]pyri-
din-3-yl)-N-butyl-N-methylacetamide (20). IR (KBr): 3340, 3355,
1633 cm-1. 1H NMR (CDCl3) δ: 0.9-1.0 (m, 3H, CH3), 1.2-1.4
(m, 2H, CH2), 1.5-1.7 (m, 2H, CH2), 3.01 (s, 1.5 H, CH3N), 3.11
(s, 1.5 H, CH3N), 3.34 (t, J ) 7.7 Hz, 1H, CH2N), 3.44 (t, J ) 7.7
Hz, 1H, CH2N), 4.04 (s, 1H, CH2CO), 4.04 (br, 2H, NH2), 4.06 (s,
2-(6,8-Dichloro-2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)-
N-methyl-N-phenylacetamide (11). IR (KBr): 1651 cm-1. 1H NMR
(CDCl3) δ: 3.31 (s, 3H, CH3N), 3.84 (s, 3H, CH3O), 3.85 (s, 2H,
CH2CO), 6.90 (d, J ) 8.5 Hz, 2H, Ar), 7.17 (d, J ) 6.8 Hz, 1H,
Ar), 7.2-7.4 (m, 5H, Ar), 7.51 (d, J ) 8.5 Hz, 2H, Ar), 8.24 (m,

2-Phenyl-imidazo[1,2-a]pyridines Containing Hydrophilic Groups
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6885
1H, CH2CO), 6.84 (m, 1H, Ar), 7.15 (m, 1H, Ar), 7.2-7.4 (m,
1H, Ar), 8.25 (m, 1H, Ar). MS m/z 440 (M+, 20), 321 (base). Anal.
(C20H21Cl3N4O) C, H, N.
washed with 5% NaHCO3, and dried (Na2SO4). Solvent was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (CH2Cl2/ acetone 9/1 (v/v)
as eluent) to give the Boc-protected product 37. Next, to the stirred
and ice-cooled solution of 37 (200 mg, 0.35 mmol) in anhydrous
CH2Cl2 (20 mL), gaseous HCl was bubbled for 30 min. Evaporation
of the solvent under reduced pressure gave the corresponding Boc-
deprotected compound 12 as hydrochloride salt in good yield.
4-(6,8-Dichloro-3-(2-(dipropylamino)-2-oxoethyl)imidazo[1,2-
a]pyridin-2-yl)phenyl 2-Aminoacetate (12). IR (KBr): 3406, 1640,
1629 cm-1. 1H NMR (D2O) δ: 0.6-0.8 (m, 6H, CH3), 1.4-1.6 (m,
4H, CH2), 3.1-3.3 (m, 4H, CH2N), 4.15 (s, 2H, CH2CO), 4.17 (s,
2H, NCH2COO), 7.29 (d, J ) 8.8 Hz, 2H, Ar), 7.51 (d, J ) 8.8
Hz, 2H, Ar), 7.76 (d, J ) 1.6 Hz, 1H, Ar), 8.31 (d, J ) 1.6 Hz,
1H, Ar). MS (ESI) m/z 477.2 [M + H]+. Anal. (C23H26Cl2N4O3)
C, H, N.
4-(6,8-Dichloro-3-(2-(dipropylamino)-2-oxoethyl)imidazo[1,2-
a]pyridin-2-yl)phenyl Methyl Succinate (13). To a stirred solution
of 6 (200 mg, 0.48 mmol) in anhydrous THF (20 mL) methyl
4-chloro-4-oxobutyrate (152 µL, 0.95 mmol) was added dropwise
at room temperature. Then after 1 h, the solvent was evaporated
under reduced pressure and the residue was dissolved in EtOAc
(20 mL), washed with 5% NaHCO3, and dried (Na2SO4). Evapora-
tion of the solvent gave a residue that was purified by silica gel
column chromatography (light petroleum ether/ethyl acetate 1/1 (v/
v) as eluent). IR (KBr): 1757, 1744, 1626 cm-1. 1H NMR (CDCl3)
δ: 0.73 (t, J ) 7.4 Hz, 3H, CH3), 0.81 (t, J ) 7.4 Hz, 3H, CH3),
1.4-1.6 (m, 4H, CH2), 2.5-2.9 (m, 4H, OCOCH2CH2COO), 3.21
(t, J ) 7.1 Hz, 2H, CH2N), 3.29 (t, J ) 7.1 Hz, 2H, CH2N), 3.98
(s, 3H, CH3OCO), 4.03 (s, 2H, CH2CO), 7.16 (d, J ) 8.8 Hz, 2H,
Ar), 7.2-7.3 (m, 1H, Ar), 7.60 (d, J ) 8.8 Hz, 2H, Ar), 8.19 (d, J
) 1.6 Hz, 1H, Ar). MS (ESI) m/z 534.2 [M + H]+. Anal.
(C26H29Cl2N3O5) C, H, N.
2-(2-(3-Amino-4-chlorophenyl)-8-chloroimidazo[1,2-a]pyridin-
3-yl)-N-butyl-N-methylacetamide (21). IR (KBr): 3451, 3365, 1646
1
cm-1. H NMR (CDCl3) δ: 0.9-1.0 (m, 3H, CH3), 1.2-1.4 (m,
2H, CH2), 1.5-1.7 (m, 2H, CH2), 2.85 (s, 1.5H, CH3N), 2.89 (s,
1.5H, CH3N), 3.12 (t, J ) 7.7 Hz, 1H, CH2N), 3.34 (t, J ) 7.7 Hz,
1H, CH2N), 4.08 (s, 1H, CH2CO), 4.10 (s, 1H, CH2CO), 4.2 (br,
2H, NH2), 6.76 (t, J ) 6.9 Hz, 1H, Ar), 6.8-6.9 (m, 1H, Ar),
7.2-7.4 (m, 3H, Ar), 8.1-8.2 (m, 1H, Ar). (ESI) m/z 405 [M +
H]+. Anal. (C20H22Cl2N4O) C, H, N.
Synthesis of the Emisuccinamide 22. A mixture of 19 (1 g, 1.8
mmol) and succinic anhydride (324 mg, 3.2 mmol) was refluxed
in toluene (50 mL) overnight. Then the reaction mixture was
allowed to cool at room temperature and the resulting precipitate,
corresponding to essentially pure acid, was recovered by filtration,
washed with toluene, and then dried under vacuum.
4-(2-Chloro-5-(6,8-dichloro-3-(2-(dipropylamino)-2-oxoethyl)imi-
dazo[1,2-a]pyridin-2-yl)phenylamino)-4-oxobutanoic acid (22). IR
1
(KBr): 3339, 1686, 1617 cm-1. H NMR (DMSO-d6) δ: 0.7-0.9
(m, 6H, CH3), 1.4-1.6 (m, 4H, CH2), 2.4-2.7 (m, 4H,
CH2CH2COO), 3.20 (t, J ) 7.4 Hz, 2H, CH2N), 3.33 (t, J ) 7.4
Hz, 2H, CH2N), 4.26 (s, 2H, CH2CO), 7.39 (d, J ) 8.5 Hz, 1H,
Ar), 7.55 (d, J ) 8.5 Hz, 1H, Ar), 7.66 (s, 1H, Ar), 7.97 (s, 1H,
Ar), 8.57 (s, 1H, Ar), 9.65 (s, 1H, NH), 12.16 (br, 1H, COOH).
MS (ESI) m/z 551.2 [M - H]-. Anal. (C25H27Cl3N4O4) C, H, N.
General Procedure for Preparation of (2-Phenyl-imidazo[1,2-
a]pyridine-3-yl)acetamides 6-8. A stirred solution of compound
35a (1g, 2.8 mmol) in conc HBr (50 mL) was refluxed for 4 h.
The reaction mixture was then cooled and poured in water (100
mL). The resulting precipitate 36a was collected by filtration,
washed several times with water, and dried under vacuum. Next a
solution of the acid 36a (500 mg, 1.49 mmol) and CDI (313 mg,
1.93 mmol) in anhydrous THF (25 mL) was stirred at room
temperature. After 30 min, the suitable dialkylamine (1.9 mmol)
and TEA (2.2 mmol) were added and the stirring was prolonged
for 12 h. Then the reaction mixture was taken up with water,
extracted with CHCl3, and dried over Na2SO4. Solvent was
evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography (light petroleum ether/ ethyl
acetate 1/1 (v/v) as eluent) to give the desired compounds.
2-(6,8-Dichloro-2-(4-hydroxyphenyl)imidazo[1,2-a]pyridin-3-yl)-
Preparation of (2-Phenyl-imidazo[1,2-a]pyridine-3-yl)acetamides
23-25. A solution of 38 (500 mg, 1.3 mmol) and 2,2-dimethyl-
1,3-dioxane-4,6-dione (Meldrum’s acid, 281 mg, 1.95 mmol) in
anhydrous THF (25 mL) was stirred at room temperature for 48 h.
Solvent was evaporated under reduced pressure, and the residue
was dissolved in ethyl acetate (20 mL), washed with 0.1 N HCl,
and dried (Na2SO4). The organic solvent was evaporated under
reduced pressure, and the residue was purified by silica gel column
chromatography (CHCl3/CH3OH 9/1 (v/v) as eluent) to give the
acid 23. Compounds 24 and 25 were prepared as follows: A mixture
of 38 (1 g, 2.6 mmol) and succinic or glutaric anhydride (5.2 mmol)
was refluxed in toluene (50 mL) overnight. Then, the reaction
mixture was allowed to cool at room temperature and the resulting
precipitate, corresponding to essentially pure acid, was recovered
by filtration, washed with toluene, and then dried under vacuum.
3-(2-(4-Chlorophenyl)-3-(2-(dipropylamino)-2-oxoethyl)imida-
zo[1,2-a]pyridin-8-ylamino)-3-oxopropanoic acid (23). IR (KBr):
N,N-dipropylacetamide (6). IR (KBr): 1638, cm-1 1H-NMR
.
(DMSO-d6) δ: 0.7-0.9 (m, 6H, CH3), 1.4-1.7 (m, 4H, CH2),
3.2-3.3 (m, 4H, CH2NCO), 4.20 (s, 2H, CH2CON), 6.83 (d, J )
8.5 Hz, 2H, Ar), 7.41(d, J ) 8.5 Hz, 2H, Ar), 7.60 (d, J ) 1.6 Hz,
1H, Ar), 8.55 (d, J ) 1.6 Hz, 1H, Ar), 9.65 (s, 1H, OH). MS (ESI)
m/z 420.2 [M + H]+. Anal. (C21H23Cl2N3O2) C, H, N.
2-(6,8-Dichloro-2-(4-hydroxyphenyl)imidazo[1,2-a]pyridin-3-yl)-
N-buthyl-N-methylacetamide (7). IR (KBr): 1631 cm-1. 1H NMR
(CDCl3) δ: 0.7-1.0 (m, 3H, CH3), 1.0-1.6 (m, 4H, CH2), 2.89 (s,
1.5H, CH3N), 2.92 (s, 1.5H, CH3N), 3.14 (t, J ) 7.4 Hz, 1H, CH2N),
3.38 (t, J ) 7.4 Hz, 1H, CH2N), 4.01 (s, 1H, CH2CO), 4.04 (s, 1H,
CH2CO), 6.7-6.9 (m, 2H, Ar), 7.3-7.5 (m, 3H, Ar), 8.2-8.4 (m,
1H, Ar), 9.69 (br 1H, OH). MS m/z 405 (M+, 2), 291 (base). Anal.
(C20H21Cl2N3O2) C, H, N.
1
1722, 1607, 1591 cm-1. H NMR (DMSO-d6) δ: 0.77-0.85 (m,
6H, CH3), 1.4-1.6 (m, 4H, CH2), 3.1-3.5 (m, 6H, CH2N +
COCH2COO), 4.20 (s, 2H, CH2CO), 6.84 (t, J ) 7.0 Hz, 1H, Ar),
7.51 (d, J ) 8.4 Hz, 2H, Ar), 7.69 (d, J ) 8.4 Hz, 2H, Ar), 7.85
(m, 1H, Ar), 8.08 (m, 1H, Ar), 10.0 (s, 1H, NH), 12.8 (br, 1H,
COOH). MS (ESI) m/z 469.0 [M - H]-. Anal. (C24H27ClN4O4) C,
H, N.
2-(6,8-Dichloro-2-(4-hydroxyphenyl)imidazo[1,2-a]pyridin-3-yl)-
N-methyl-N-phenylacetamide (8). IR (KBr): 1648 cm-1. 1H NMR
(CDCl3) δ: 3.31 (s, 3H, CH3N), 3.83 (s, 2H, CH2CO), 6.76 (d, J )
8.5 Hz, 2H, Ar), 7.17 (d, J ) 6.9 Hz, 2H, Ar), 7.2-7.5 (m, 6H,
Ar), 8.23 (d, J ) 1.4 Hz, 1H, Ar), 9.65 (br 1H, OH). MS (ESI) m/z
425.9 [M + H]+. Anal. (C22H17Cl2N3O2) C, H, N.
Preparation of (2-Phenyl-imidazo[1,2-a]pyridine-3-yl)acetamides
12, 13. Compound 12 was prepared as follows: A solution of Boc-
Gly-OH (248 mg, 1.42 mmol) and DCC (177 mg, 0.85 mmol) in
anhydrous THF (25 mL) was stirred for 30 min at 0 °C by using
an ice bath. After this time, the compound 6 (300 mg, 0.71 mmol)
and TEA (0.15 mL, 1.06 mmol) were added and the mixture was
stirred overnight at room temperature. Solvent was evaporated under
reduced pressure, and the residue was dissolved in CHCl3 (20 mL),
4-(2-(4-Chlorophenyl)-3-(2-(dipropylamino)-2-oxoethyl)imida-
zo[1,2-a]pyridin-8-ylamino)-4-oxobutanoic Acid (24). IR (KBr):
1720, 1640 cm-1. 1H NMR (DMSO-d6) δ: 0.7-0.9 (m, 6H, CH3),
1.4-1.6 (m, 4H, CH2), 2.42 (t, J ) 6.6 Hz, 2H, CH2CONH), 2.48
(t, J ) 6.6 Hz, 2H, CH2COO), 3.20 (t, J ) 7.7 Hz, 2H, CH2N),
3.30 (t, J ) 7.7 Hz, 2H, CH2N), 4.18 (s, 2H, CH2CO), 6.8-6.9 (m,
1H, Ar), 7.49 (d, J ) 8.5 Hz, 2H, Ar), 7.6-8.0 (m, 3H, Ar), 8.0-8.4
(m, 1H, Ar), 9.89 (s, 1H, NH), 12.0 (br, 1H, COOH). MS (ESI)
m/z 483.2 [M - H]-. Anal. (C25H29ClN4O4) C, H, N.
5-(2-(4-Chlorophenyl)-3-(2-(dipropylamino)-2-oxoethyl)imida-
zo[1,2-a]pyridin-8-ylamino)-5-oxopentanoic Acid (25). IR (KBr):
3287, 1696, 1645, cm-1. 1H NMR (DMSO-d6) δ: 0.7-0.9 (m, 6H,
CH3), 1.4-1.6 (m, 4H, CH2), 1.8-1.9 (m, 2H, CH2), 2.41 (t, J )
6.6 Hz, 2H, CH2CONH), 2.57 (t, J ) 6.6 Hz, 2H, CH2COO), 3.20

6886 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Denora et al.
(t, J ) 7.7 Hz, 2H, CH2N), 3.30 (t, J ) 7.7 Hz, 2H, CH2N), 4.07
(s, 2H, CH2CO), 6.8-7.0 (m, 1H, Ar), 7.51 (d, J ) 8.5 Hz, 2H,
Ar), 7.6-8.0 (m, 3H, Ar), 8.0-8.4 (m, 1H, Ar), 9.89 (s, 1H, NH),
12.0 (br, 1H, COOH). MS (ESI) m/z 497.2 [M - H]-. Anal.
(C26H31ClN4O4) C, H, N.
the European Communities Council Directive of 24 November 1986
(86/609/EEC).
In Vitro Receptor Binding Assays. [3H]Flunitrazepam Bind-
ing. Cerebral cortex was homogenized with a Polytron PT 10 in
50 volumes of ice-cold 50 mM Tris-HCl (pH 7.4) buffer, and the
homogenate was centrifuged twice at 20000g for 10 min. The final
pellet was reconstituted in 50 volumes of Tris-HCl buffer and used
for the binding assay. [3H]Flunitrazepam binding was determined
in a final volume of 1000 µL, comprising 400 µL of membrane
suspension (0.4-0.5 mg of protein), 400 µL of Tris-HCl buffer,
100 µL of [3H]Flunitrazepam (74 Ci/mmol; New England Nuclear,
final concentration 0.5 nM), and 100 µL of drug solution or solvent.
Incubations were performed for 60 min at 0 °C and were terminated
by rapid filtration through glass-fiber filter strips (Whatman GF/
B). The filters were then rinsed with ice-cold Tris-HCl buffer, and
filter-bound radioactivity was quantified by liquid scintillation
spectrometry. Nonspecific binding was determined as binding in
the presence of 5 µM diazepam and represented about 10% of total
binding.
Preparation of 2-(8-(2-Aminoacetamido)-2-(4-chlorophenyl)imi-
dazo[1,2-a]pyridin-3-yl)-N,N-dipropylacetamide (26). A solution of
Boc-Gly-OH (455 mg, 2.6 mmol) and DCC (400 mg, 1.94 mmol)
in anhydrous CH2Cl2 (25 mL) was stirred for 30 min at 0 °C by
using an ice bath. After this time, the compound 38 (500 mg, 1.3
mmol), DMAP (15 mg, 0.13 mmol), and TEA (0.27 mL, 1.94
mmol) were added and the stirring was prolonged overnight at room
temperature. Solvent was evaporated under reduced pressure, and
the residue was dissolved in CHCl3 (20 mL), washed with 5%
NaHCO3, dried (Na2SO4), and evaporated. The residue was purified
by silica gel column chromatography (CH2Cl2/ethyl acetate 7/3 (v/
v) as eluent) to give the Boc-protected product 39. Next, in a stirred
and ice-cooled solution of the compound 39 (200 mg, 0.37 mmol)
in anhydrous CH2Cl2 (20 mL), gaseous HCl was bubbled for 30
min. Evaporation of the solvent under reduced pressure gave the
corresponding Boc-deprotected compound 26 as hydrochloride salt
[3H-]-2 Binding. After killing, the brain was rapidly removed
from rats, the cerebral cortex was dissected, and all tissue were
stored at -80 °C until assayed. The tissues were thawed and
homogenized in 50 volumes of Dulbecco’s phosphate-buffered
saline (PBS), pH 7.4, at 4°C with a Polytron PT 10 (setting 5, for
20 s). The homogenate was centrifuged at 40000g for 30 min, and
the resulting pellet was resuspended in 50 volumes of PBS and
recentrifuged. The new pellet was resuspended in 10 volumes of
PBS and used for the assay. [3H]-2 binding was determined in a
final volume of 500 µL, comprising 50 µL of membrane suspension
(0.15-0.20 mg protein), 50 µL of [3H]-2 (85.5 Ci/mmol, New
England Nuclear; final assay concentration 1 nM), 350 µL of PBS
buffer, and 50 µL of drug solution or solvent. Incubations were
initiated by the addition of membranes and were terminated 90 min
later by rapid filtration through glass-fiber filter strips (Whatman
GF/B) that had been presoaked with 0.3% polyethyleneimine and
placed in a Cell Harvester manifold (Brandel). The filters were
rinsed five times with 4 mL of ice-cold PBS buffer, after which
filter-bound radioactivity was quantified by liquid scintillation
spectrometry. Nonspecific binding was defined as the binding in
the presence of 10 µM of unlabelled 2 (Sigma).
Functional in Vitro Studies. a. Electrophysiological Record-
ings Using Xenopus Oocytes. Complementary DNAs encoding the
human R1, ꢀ2, and γ2L GABAA receptor subunits were subcloned
into the pCDM8 expression vector (Invitrogen, San Diego, CA).
The cDNAs were purified with the Promega Wizard Plus
Miniprep DNA purification system (Madison, WI) and then
resuspended in distilled water, divided into portions, and stored
at -20°C until used for injection. Stage V and VI oocytes were
manually isolated form sections of Xenopus laeVis ovary, placed
in MBS containing 66 mM NaCl, 1 mM KCl, 10 mM HEPES-
NaOH (pH 7.5), 0.82 mM MgSO4, 2.4 mM NaHCO3, 0.91 mM
CaCl2, and 0.33 mM Ca(NO3)2 and treated with 0.5 mg/mL
collagenase type IA (Sigma) in collagenase buffer (83 mM NaCl,
2 mM KCl, 1 mM MgCl2, 5 mM HEPES-NaOH (pH 7.5) for 10
min at room temperature to remove the follicular layer. A mixture
of GABAA receptor R1, ꢀ2, and γ2L subunit cDNAs (total of 1.5
ng of cDNA in 30 nL in a 1:1:1 ratio) was injected into the
oocytes nucleus using a 10 µL glass micropipette (10-15 µm
tip diameter). The injected oocytes were cultured at 19 °C in
sterile MBS supplemented with streptomycin (10 µg/mL),
penicillin (10 U/mL), gentamicin (50 µg/mL), 0.5 M theophyl-
line, and 2 mM sodium pyruvate. Electrophysiological recordings
began approximately 24 h following cDNA injection. Oocytes
were placed in a 100 µL rectangular chamber and continuously
perfused with MBS solution at a flow rate of 2 mL/min at room
temperature. The animal pole of oocytes was impaled with two
glass microelectrodes (resistance between 0.5 and 3 MΩ) filled
with filtered 3 M KCl, and the membrane voltage was clamped
at -70 mV with an Axoclamp 2-A amplifier (Axon Instruments,
Burlingame, CA). Currents were continuously recorded on a
strip-chart recorder. Resting membrane potential usually varied
1
in good yield. IR (KBr): 3421, 1635, cm-1. H NMR (CDCl3) δ:
0.6-0.8 (m, 6H, CH3), 1.4-1.6 (m, 4H, CH2), 3.15 (t, J ) 6.9 Hz,
2H, CH2N), 3.23 (t, J ) 6.9 Hz, 2H, CH2N), 4.06 (s, 2H, CH2CO),
4.23 (s, 2H, NCH2CO), 7.25 (t, J ) 7.1 Hz, 1H, Ar), 7.43 (d, J )
8.5 Hz, 2H, Ar), 7.49 (d, J ) 8.5 Hz, 2H, Ar), 7.80 (d, J ) 7.7 Hz,
1H, Ar), 8.17 (d, J ) 7.7 Hz, 1H, Ar). MS (ESI) m/z 442.2 [M +
H]+. Anal. (C23H28ClN5O2) C, H, N.
Preparation of 4-(2-(4-Chlorophenyl)-3-(2-(dipropylamino)-2-
oxoethyl)imidazo[1,2-a]pyridin-8-ylamino)-4-oxobutanoate (27). A
solution of the acid 24 (400 mg, 0.83 mmol) and concentrated
H2SO4 (2 mL) in ethanol (20 mL) was stirred at 50 °C for 12 h.
Then, the solvent was evaporated under reduced pressure and the
residue was taken up with 5% NaHCO3 (25 mL), extracted with
EtOAc (3 × 25 mL), and dried (Na2SO4). Evaporation of the solvent
gave a residue that was purified by silica gel column chromatog-
raphy (CHCl3/ethyl acetate 95/5 (v/v) as eluent). IR (KBr): 1733,
1
1698, 1639 cm-1. H NMR (CDCl3) δ: 0.68 (t, J ) 7.4 Hz, 3H,
CH3), 0.73 (t, J ) 7.4 Hz, 3H, CH3), 1.26 (t, J ) 7.4 Hz, 3H,
CH3), 1.4-1.6 (m, 4H, CH2), 2.7-2.9 (m, 4H, OCOCH2CH2CON),
3.10 (t, J ) 7.4 Hz, 2H, CH2N), 3.27 (t, J ) 7.4 Hz, 2H, CH2N),
4.06 (s, 2H, CH2CO), 4.15 (q, J ) 7.4 Hz, 2H, CH2OCO), 6.8-7.0
(m, 1H, Ar), 7.49 (d, J ) 8.5 Hz, 2H, Ar), 7.62 (d, J ) 8.5 Hz,
2H, Ar), 7.7-7.8 (m, 1H, Ar), 7.9-8.0 (m, 1H, Ar), 8.80 (s, 1H,
NHCO). MS (ESI) m/z 535.2 [M + Na]+. Anal. (C27H33ClN4O4)
C, H, N.
Preparation of (2-Thiazolyl-imidazo[1,2-a]pyridine-3-yl)aceta-
mide 29. Compound 29 was prepared following the previously
reported method for 28.25
2-(6,8-Dichloro-2-(thiazol-2-yl)imidazo[1,2-a]pyridin-3-yl)-N-
methyl-N-phenylacetamide (29). To a stirred solution of 46 (0.25
g, 0.76 mmol) in anhydrous THF (20 mL) at room temperature
were added in the order EEDQ (0.28 g, 1.14 mmol), the appropriate
dialkylamine (0.92 mmol), and, after 10 min, TEA dropwise (0.16
mL, 1.14 mmol). Stirring was continued for additional 24 h at room
temperature and then the solvent was evaporated under reduced
pressure. The resulting residue was purified by column chroma-
tography on silica gel (petroleum ether/ethyl acetate 7:3 v/v as
1
eluent). IR (KBr):1650 cm-1. H NMR (CDCl3) δ: 3.28 (s, 3Η,
CH3N), 4.44 (s, 2H, -CH2CON), 7.2-7.5 (m, 7H, Ar), 7.68 (d, J
) 3.0 Hz, 1H, Ar), 8.28 (d, J ) 1.6 Hz, 1H, Ar). MS (ESI): m/z
417.0 [M + H]+. Anal. (C19H14Cl2N4OS) C, H, N.
Biological Methods. Materials. Male Sprague-Dawley CD rats
(Charles River, Como, Italy) at 6-8 weeks of age were used for
binding experiments. They were housed for at least 7 days before
experiments and were maintained under an artificial 12 h light/12
h-dark cycle (light on 0800 to 2000 hours) at a constant temperature
of 23 ( 2 °C and 65% humidity. Food and water were freely
available until the time of experiment. Animal care and handling
throughout the experimental procedures were in accordance with

2-Phenyl-imidazo[1,2-a]pyridines Containing Hydrophilic Groups
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6887
between -25 and -45 mV. GABA (dissolved in MBS) and drugs
(dissolved in dimethyl sulfoxide and then diluted in MBS) were
perfused for 20 s (7-10 s were required to reach equilibrium in
the recording chamber). Maximal percentage of dimethyl su-
foxide in the final dilution of the compounds was 0.1%, which,
alone, did not influence GABA-evoked Cl- currents. Intervals
of 5-10 min were allowed between drug applications. Modula-
tion of GABA-evoked Cl- current amplitude by drugs is
expressed as percentage change, [(I′/I) - 1] · 100%], where I is
the average of control amplitude obtained before drug application
and after drug washout and I′ is the average of the agonist-
induced current amplitude obtained in the same cell in the
presence of drug.
data for compounds 31a,c, 32a,c, 34a,c, 35a,b,c, 36a, 37, and 39.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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A number of 5-10 different oocytes were used for testing each
of the two concentrations of the different compounds, and oocytes
were obtained from different frog donors.
b. Patch Clamp Recording in Rat Hippocampal Slices. Male
Sprague-Dawley rats were anesthetized by intraperitoneal injection
of ketamine (250 mg per kilogram of body mass), and the brain
was rapidly removed into ice-cold cutting solution (220 mM
sucrose, 2 mM KCl, 1.3 mM NaH2PO4, 12 mM MgSO4, 0.2 mM
CaCl2, 10 mM glucose, 2.6 mM NaHCO3 (pH 7.3) equilibrated
with 95% O2 and 5% CO2). Coronal slices (thickness, 300 µm) of
the hippocampus were cut with a Vibratome 1000 plus (Vibratome,
St. Louis, MO) and then ACSF containing 126 mM NaCl, 3 mM
KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 10 mM
glucose, and 26 mM NaHCO3 (pH 7.3), equilibrated with 95% O2
and 5% CO2, first for 40 min at 34°C and then for 30 min at room
temperature before beginning experiments.
Tissue slices were transferred to a chamber perfused with
ACSF at a rate of ∼2 mL/min at room temperature. Whole-cell
patch clamp electrophysiological recordings from CA1 pyramidal
neurons were performed with an Axopatch 200-B amplifier
(Axon Instruments, Union City, CA) and an infrared-differential
interference contrast microscope. Patch microelectrodes (boro-
silicate capillaries with a filament; outer diameter, 1.5 µm) (Sutter
Instruments, Novato, CA) were prepared with a two-step vertical
puller (Sutter Instruments) and had a resistance of 4-to 6 MΩ.
mIPSCs were recorded at a holding potential of -65 mV with
an internal solution containing 140 mM CsCl, 2 mM MgCl2, 1
mM CaCl2, 10 mM EGTA, 10 mM Hepes-CsOH (pH 7.3), 2
mM adenosine triphosphate (disodium salt), and 5 mM QX-314
(lidocaine N-ethyl bromide). Access resistance varied between
20 and 40 MΩ; if it changed by >20% during an experiment,
the recording was discarded. Currents through the patch clamp
amplifier were filtered at 2 kHz and digitized at 5.5 kHz with
commercial software (pClamp 8.2; Axon Instruments). The
external solution (ACSF) contained 500 µM lidocaine and 3 mM
kynurenic acid. The mIPSCs were analyzed with Mini Analysis
5.4.17 software (Synaptosoft, Decatur, GA). Each event identified
was confirmed by visual inspection for each experiment. We
evaluated the effects of the various drugs on the amplitude and
decay time of mIPSCs in individual neurons by cumulative
probability analysis, with statistical significance determined with
the Kolmogorov-Smirnov nonparametric two-sample test.
Statistical Analysis. Statistical comparisons of pooled data were
performed by one-way analysis of variance followed by Scheffe’s
post hoc test. In all cases, a P value of < 0.05 was considered
statistically significant.
Acknowledgment. This work was supported by a grant from
Ministero dell’Universita` e della Ricerca (PRIN 2006 MiUR
for G.T. and E.S.) as well as from Fondi d’Ateneo (Universita`
degli Studi di Bari). We thank Giovanni Dipinto and Antonio
Palermo for their skillful technical assistance in recording mass
and NMR spectra, respectively.
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