Structure-activity relationships and effects on neuroactive steroid synthesis in a series of 2-phenylimidazo[1,2-a]pyridineacetamide peripheral benzodiazepine receptors ligands

Article abstract of DOI:10.1021/jm049610q

A series of 36 imidazopyridineacetamides (2-37) were designed and synthesized to evaluate the effects of structural changes on the amide nitrogen at both central (CBRs) and peripheral benzodiazepine receptors (PBRs). These changes include variations in the length and number of the alkyl groups as well as introduction of different aromatic, heteroaromatic, and conformationally constrained groups. The affinities of these compounds for CBRs and PBRs were determined, and the results indicate that bulkiness of the substituents, their branching, and length beyond an optimal value may cause hindrance to the ligand in its interaction with the receptor. The presence of aromatic or conformationally constrained substituents on the carboxamide nitrogen can be conducive to high affinity and selectivity. Furthermore, the ability of a subset of the most active ligands to stimulate synthesis of neuroactive steroids in plasma and brain was evaluated in vivo and in vitro. Compound 3 exhibited very marked effects on the peripheral and central synthesis of neuroactive steroids, while 36 (potent at subnanomolar level) showed a slight ability to affect neuroactive steroid content in the cerebral cortex.

Full text of DOI:10.1021/jm049610q

292
J. Med. Chem. 2005, 48, 292-305
Structure-Activity Relationships and Effects on Neuroactive Steroid Synthesis
in a Series of 2-Phenylimidazo[1,2-a]pyridineacetamide Peripheral
Benzodiazepine Receptors Ligands
Giuseppe Trapani,*,† Valentino Laquintana,† Nunzio Denora,† Adriana Trapani,† Angela Lopedota,†
Andrea Latrofa,† Massimo Franco,† Mariangela Serra,§, Maria Giuseppina Pisu,§, Ivan Floris,§ Enrico Sanna,§
Giovanni Biggio,§, and Gaetano Liso†
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, and C.N.R. Institute of Neuroscience, Unit of Neuropharmacology,
09100 Cagliari, Italy
Received May 27, 2004
A series of 36 imidazopyridineacetamides (2-37) were designed and synthesized to evaluate
the effects of structural changes on the amide nitrogen at both central (CBRs) and peripheral
benzodiazepine receptors (PBRs). These changes include variations in the length and number
of the alkyl groups as well as introduction of different aromatic, heteroaromatic, and
conformationally constrained groups. The affinities of these compounds for CBRs and PBRs
were determined, and the results indicate that bulkiness of the substituents, their branching,
and length beyond an optimal value may cause hindrance to the ligand in its interaction with
the receptor. The presence of aromatic or conformationally constrained substituents on the
carboxamide nitrogen can be conducive to high affinity and selectivity. Furthermore, the ability
of a subset of the most active ligands to stimulate synthesis of neuroactive steroids in plasma
and brain was evaluated in vivo and in vitro. Compound 3 exhibited very marked effects on
the peripheral and central synthesis of neuroactive steroids, while 36 (potent at subnanomolar
level) showed a slight ability to affect neuroactive steroid content in the cerebral cortex.
Introduction
dihydroxy-5R-pregnan-20-one (3R,5R-THDOC), and
dehydroepiandrosterone] are known to modulate
GABAergic and glutamatergic transmissions.4-8 More-
over, it has been proved that PBR expression selectively
increases in brain tumors, and this led to evaluating
appropriate PBR ligands as diagnostic imaging agents9,10
or as receptor-mediated drug carriers to selectively
target anticancer drugs to brain tumors.11-13 The abun-
dance of PBRs in cancers of colon, breast, ovary, and
liver suggests a role of these receptors in tumori-
genesis.14-17 There is also experimental evidence that
PBRs are involved in various neurological diseases,
while PBR-specific ligands have also been shown to
induce apoptosis and cell cycle arrest in cancer cells.18,19
The sequence of primary, secondary, and tertiary
structure of PBRs in several animal species including
human, bovine, rat, and murine has been clarified.20
Furthermore, considerable efforts have been focused on
the identification of new PBR ligands characterized by
high affinity and selectivity. The known PBR-selective
ligands belong to structurally different compounds such
as benzodiazepines ([3H] Ro-54864),21 isoquinolines ([3H]
PK-11195),22 2-aryl-3-indoleacetamides (FGIN-1-27),23
N-phenoxyphenyl-N-isopropoxybenzylacetamides
(DAA1097),24 and some benzothiazepines25 (Chart 1).
Benzodiazepines are used clinically as sedatives,
hypnotics, anxiolytics, muscle relaxants, and anticon-
vulsants. In the brain, two major classes of benzodiaz-
epine receptors have been identified: the “central-type”
and the “peripheral-type” receptors. Central-type ben-
zodiazepine receptors (CBRs) are present exclusively in
the central nervous system (CNS) and mediate the
classical effects of benzodiazepines. Peripheral-type
benzodiazepine receptors (PBRs) are pharmacologically
distinct from CBRs and are present in peripheral tissues
and also in glial cells of the CNS. PBRs are composed
of a multimeric protein complex located on the outer
mitochondrial membrane of astroglial cells.1 Although
the pharmacological role of PBRs in the CNS has not
yet been fully clarified, a wide range of pharmacological
activities, including anticonvulsant, anxiolytic, immu-
nomodulating, and cardiovascular, have been associated
with their activation. In particular, there is growing
experimental evidence suggesting that high-affinity
PBR ligands stimulate neurosteroids synthesis in glial
cells.2,3 In fact, PBR mediates the delivery of cholesterol
to the inner mitochondrial membrane where it is
oxidized by cytochrome P450 scc (side chain cleavage)
to pregnenolone, the parent molecule of endogenous
steroids. Some neurosteroids [pregnenolone sulfate, 3R-
hydroxy-5R-pregnan-20-one (3R,5R-THPROG), 3R,21-
We have recently shown that some 2-phenylimidazo-
[1,2-a]pyridineacetamides are potent and selective ligands
for PBRs and stimulate steroidogenesis in both the brain
and periphery.26-29 Among these ligands, N,N-di-n-
propyl-[2-(4-chlorophenyl)-6,8-dichloroimidazo[1,2-a]-
pyridin-3-yl)]acetamide (denoted as CB 34)28 has been
* To whom correspondence should be addressed. Phone (039) 080-
544-2764. Fax (039) 080-544-2754. E-mail: trapani@farmchim.uniba.it.
† Universita` degli Studi di Bari.
§ Universita` di Cagliari.
C.N.R. Institute of Neuroscience.
10.1021/jm049610q CCC: $30.25 © 2005 American Chemical Society
Published on Web 12/13/2004

Peripheral Benzodiazepine Receptors Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 293
Chart 1
ment; (iii) the presence of an aromatic or heteroaromatic
system with a pendant phenyl ring. Recently, a unique
3D interaction model of endogenous and synthetic PBR
ligands has been described31 which confirmed two
lipophilic regions (L1 and L3), one polar group (H2), and
a lipophilic region modulating the receptor binding (L4)
in which H2 is expressed as essential elements of the
PBR interaction. Modeling of the receptor’s 3D structure
is based on knowledge of the receptor protein’s primary
structure. Receptor-mapping and receptor-fitting ap-
proaches, indeed, led to the suggestion that the amino
acid residues Ser 41, Trp 107, and Trp 161 of the
receptor protein are key elements in the ligand-
receptor interaction. In particular, the amide carbonyl
group of the ligand should be involved in a hydrogen
bonding acceptor interaction with Ser 41, while the
hydrophobic groups attached to C(dO)N should interact
with Trp 107 and Trp 161.30
The series of 2-phenylimidazo[1,2-a]pyridineaceta-
mides 2-37 (Table 1) herein studied was designed
taking into account the chemical structure of compound
1. Thus, the present study probed variations in the
length and number of the alkyl groups on the amide
nitrogen, as well as introduction of different aromatic,
heteroaromatic, and conformationally constrained groups.
This was done with a 2-fold aim: first, in order to find
molecules characterized by high affinity and selectivity
which are also antagonist of neurosteroids synthesis,
and second, to improve our definition of the structure-
activity relationships in this specific region in the
2-phenylimidazopyridineacetamide series of PBR ligands.
shown to be a particularly interesting compound be-
cause of its affinity, selectivity, and stimulation of the
steroidogenesis.26-29 Our data demonstrate that sub-
stitution at the 8-position of the imidazopyridine nucleus
is a key factor for improving affinity and selectivity
toward peripheral binding sites.26-29 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. With the
exception of N,N-di-n-butyl-[2-(4-chlorophenyl)imidazo-
[1,2-a]pyridin-3-yl)]acetamide 1 (Chart 1), all the N,N-
dipropylimidazopyridineacetamides studied markedly
increased the level of neuractive steroids in brain and
therefore can be considered full or partial agonists on
steroidogenesis.27 Compound 1, even though it possess
high affinity for the PBR, showed a low receptor
selectivity and failed to affect the neuractive steroid
content in both plasma and cerebral cortex.27 On the
other hand, to the best of our knowledge, PBR ligands
with high affinity and selectivity which do not stimulate
neurosteroid synthesis (antagonists) have never been
described. Therefore, the identification of PBR ligands
endowed with antagonistic properties on steroidogenesis
remains a worthwhile goal. Such molecules may be the
proper pharmacological tool to increase our understand-
ing of the physiology and pharmacology of PBRs.
On the basis of information from molecular biology
experiments, increasingly accurate PBR ligand-recep-
tor interaction models have been developed.30 These
modeling studies lead to the suggestion that high PBR
affinity requires (i) the presence of a suitably located
and oriented carbonyl C(dO)N; (ii) the presence of
lipophilic substituents attached to the C(dO)N frag-
Chemistry
The imidazopyridineacetamides 2-6 (Table 1) were
synthesized using a previously reported method outlined
in Scheme 1 (Method A). Briefly, compounds 2-6 were
prepared by condensation in DMF at reflux of suitably
substituted 2-aminopyridines 41 with the appropriate
bromoketoamides 40 which, in turn, were prepared by
reaction of 3-benzoylpropionic acids 38 with the ap-
propriate dialkylamines in the presence of ethyl 1,2-
dihydro-2-ethoxy-1-quinolinecarboxylate (EEDQ) as de-
hydrating agent. Next, treatment of the resulting
amides 39 with bromine in carbon tetrachloride gave
the desired compounds 40. As shown in Scheme 2, the
imidazopyridineacetamides 7-9, 12, 13, 17-23, 27-29,
34-36 were prepared by condensation of the imida-
zopyridine-acetic acids 4213 with the appropriate mono-
or dialkylamine hydrochlorides in anhydrous THF and
in the presence of carbonyldiimidazole (CDI) (Method
B). The synthesis of compounds 10, 11, 15, 16, 24-26,
30, and 37 was accomplished by the procedure shown
in Scheme 2 involving the condensation of the imida-
zopyridineacetic acids 4213 with the appropriate mono-
or dialkylamines in anhydrous THF and in the presence
of EEDQ (Method C). An attempt was made to prepare
the tertiary amide N-benzyl-N-methyl-[2-(4-chlorophen-
yl)imidazo[1,2-a]pyridin-3-yl)]acetamide 37 (Table 1) by
N-methylation of the corresponding secondary amide 30
by treatment with NaH in DMF and methyl iodide.
Under these conditions however, the dimethyl-substi-
tuted compound 33 was obtained (Scheme 3, Method
D). Similarly, the dimethyl-substituted compound 32
was obtained by treatment of the secondary amide 28

294 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Table 1. Structure and Physical Properties of Compounds 1-37
Trapani et al.
compd
X
Y
Z
R1
n-C4H9
R2
R3
method
mp (°C)
yield (%)
1
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
H
n-C4H9
n-C4H9
n-C4H9
H
ref 27
2
Cl
Cl
Cl
H
n-C4H9
n-C4H9
n-C6H13
n-C4H9
n-C6H13
n-C4H9
n-C4H9
n-C4H9
n-C4H9
tert-C4H9
n-C3H7
C6H5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
A
180-182
131-133
93-95
142-145
127-129
46-48
55-57
56-58
111-112
238-240
209-211
205-206
15
17
15
19
19
33
20
22
18
15
80
60
3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
A
4
n-C6H13
n-C4H9
n-C6H13
C6H5
A
5
A
6
H
A
7
Cl
Cl
H
B
8
C6H5
C6H5
B
9
B
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CH2C6H5
CH2C6H5
4-NO2-CH2C6H5
H
C
Cl
Cl
Cl
Cl
Cl
H
C
B
B
CH2CHdCH2
CH2CHdCH2
ref 28
-(CH2)4-
C
135-136
191-193
197-198
221-223
158-160
165-167
182-184
131-132
216-218
78-80
34
22
43
36
57
50
26
32
24
37
47
64
82
20
50
90
96
96
97
56
60
62
67
-(CH2)4-
-(CH2)5-
-(CH2)5-
C
H
B
Cl
Cl
Cl
Cl
H
B
-CH2CH(COOC2H5)(CH2)3-
CH2CH(COOC2H5)(CH2)3-
-(CH2)2N(CH2C6H5)(CH2)2-
B
B
B
B
Cl
H
Cl
H
Cl
Cl
H
Cl
Cl
Cl
Cl
Cl
H
B
2-pyridylethyl
CH3
CH3
H
H
C
2-pyridylethyl
4-pyridyl
n-C4H9
H
C
162-164
274 dec
H
C
H
H
B
180-181
240-241
227-228
232-233
136-138
143-146
178-179
176-178
162-164
205-207
195-198
C6H11
C6H11
CH2C6H5
n-C3H7
C6H11
H
H
B
H
H
B
H
H
C
n-C3H7
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
D
E
CH2C6H5
n-C4H9
D
B or F
B or F
B or F
C
n-C4H9
H
Cl
Cl
C6H5
H
CH2C6H5
H
with potassium tert-butoxide in DMF and methyl iodide
(Scheme 3, Method E). It is likely that this double-
methylation reaction is due to the fact that the meth-
ylene group in the starting secondary amides is acidic
enough with a dissociation constant comparable with
that of a CONH moiety. Thus, the methyl-substituted
derivative 31 was prepared starting from the tertiary
amide N,N-di-n-propyl-[2-(4-chlorophenyl)-6,8-dichloro-
imidazo[1,2-a]pyridin-3-yl)]acetamide (i.e., CB 34)26 by
treatment with NaH in DMF and methyl iodide. The
double-methylation reaction was prevented by using
n-BuLi at -45 °C and quenching the resulting anion
with methyl triflate. Under the latter experimental
conditions, the desired monomethyl-substituted tertiary
amides 34 and 35 were obtained starting from the
imidazoacetamide 27 and N-n-butyl-(2-phenyl-6,8-di-
chloroimidazo[1,2-a]pyridin-3-yl)acetamide,27 respec-
tively (Scheme 3, Method F). Similarly, treatment of
compound 13 with n-BuLi and methyl triflate gave the
corresponding monomethyl tertiary amide 36. All com-
spectra, and elemental analyses (Experimental Section).
An interesting feature of the H NMR spectra of these
1
imidazoacetamides concerns the signals associated with
the methylene linked at the carbonyl group and the
methyl and methylene groups characterizing the alkyl
chains of CO-N<R1R2. The CH2CO gives rise to a
singlet in the range 3.7-4.6 δ only when the R1 and R2
groups are of similar size. When these two groups are
very different in size, (e. g., in the case of compounds
10, 12, 22-25, 32-35, and 37) the signals of the CH2-
CO and those of the protons in the R1 and R2 groups
are duplicated due to the partial double bond character
of the amide bond. Physical data for new compounds
are reported in Table 1.
Results and Discussion
Affinities of Imidazopyridine Derivatives for
Peripheral and Central Benzodiazepine Recep-
tors. The affinities of the 36 tested compounds for CBR
and PBR were evaluated by measuring their ability to
displace [3H]-flunitrazepam and [3H]PK 11195 from
1
pounds were fully characterized by IR, H NMR, mass

Peripheral Benzodiazepine Receptors Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 295
Scheme 1a
Scheme 3a
a Method A, Reagents: (a) R1R2NH, EEDQ,THF; (b)Br2/CCl4;
(c) DMF, reflux.
a Method D, Reagents: (a) NaH, room temperature, CH3I, DMF.
Method E, Reagents: (a) Potassium tert-butoxide, CH3I, DMF.
Method F, Reagents: (a) n-BuLi -45°C, methyl triflate, THF.
Scheme 2a
membranes and from brain cerebral cortex cells (n )
35, r2 ) 0.870). This suggests that there are no signifi-
cant differences in the PBR structure in the two tissues
examined, and therefore we limited our SAR and SSR
analysis to biological data from cortex membranes.
Structure-Affinity (SAR) and Structure-Selec-
tivity Relationships (SSR). As mentioned above, even
though compound 1 was characterized by high affinity
for the PBR, it showed poor receptor selectivity and
failed to affect neuroactive steroids content both in
plasma and cerebral cortex. Therefore, our initial efforts
were directed to enhancing the selectivity of compound
1 chosen as template. Thus, compounds 2, 3, 5 as 6- and/
or 8-Cl-substituted analogues of 1 were synthesized,
taking into account that substitution at the 6- and/or
8-position leads to compounds with enhanced affinity
and selectivity for PBR versus CBR. A pairwise com-
parison of binding data showed a marked improvement
in selectivity only for the 8-Cl-substituted compounds
2 and 3 over template 1 (i.e., pIC50 PBR cortex 8.104,
8.284, and 8.230,27 respectively; selectivity index ∆pIC50
3.104, 3.284, and 1.190,27 respectively), whereas com-
pound 5, although endowed with good affinity at the
PBR, showed a modest selectivity (i.e., pIC50 PBR cortex
8.485; selectivity index ∆pIC50 1.434). No significant
improvement in selectivity was caused by increasing the
chain length of the alkyl substituents on the carbox-
amide nitrogen as observed for compounds 4 and 6.
These findings are consistent with suggestions from
QSAR analysis32 on the 2-phenylimidazo[1,2-a]pyridine
derivatives which suggested that a four carbon chain
a Method B, Reagents (a): CDI, THF; (b)+H2NR1R2 Cl-. Method
C, Reagents: (a) R1R2NH, EEDQ,THF.
binding to membrane preparations from the cerebral
cortex and ovary. Their effects were compared with
those of unlabeled PK 11195, a selective ligand for
PBRs22 and with that of CB34.26,29 The measured
binding affinities for CBR and PBR expressed as Ki are
shown in Table 2. For the sake of comparison with
previously reported data, the affinities as well as their
ratios, as a measure of in vitro selectivity, are expressed
also as pIC50. A linear regression analysis on pIC50
values for the whole set of compounds listed in Table 2
showed a good correlation between the data from ovary

296 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Trapani et al.
Table 2. Affinities of Compounds 2-37 for CBR and PBR from Different Tissuesa
Ki (nM) Hill slope pIC50
Ki (nM)
Hill slope
cortex
pIC50
∆pIC50
Ki (nM)
Hill slope
pIC50
∆pIC50
compd
CBR
CBR
CBR PBR cortex
PBR cortex PBR cor-CBR PBR ovary
ovary PBR ovary PBR ov-CBR
1
2
3
4
5
6
7
8
9
7.040
8.230
8.104
8.284
6.424
8.485
8.292
7.939
7.876
8.824
7.616
5.464
7.566
7.701
8.092
6.668
5.907
6.804
8.301
7.454
6.845
4.682
7.412
8.313
5.663
6.046
5.677
6.409
6.640
5.878
6.772
5.920
5.288
5.005
9.347
8.456
9.481
8.623
8.155
1.190
3.104
3.284
1.424
1.434
2.811
2.939
2.876
2.691
2.616
0.464
2.566
2.701
3.092
1.668
0.907
1.804
3.301
0.729
1.845
-0.318
2.412
3.313
0.663
1.046
0.677
1.409
1.640
0.878
1.772
0.920
0.288
0.005
4.347
3.456
4.481
3.623
3.155
7.980
7.646
7.731
5.584
8.172
7.312
8.337
8.319
7.936
7.047
4.740
7.157
7.599
8.252
6.074
5.029
5.837
7.036
6.520
5.597
3.139
6.636
7.449
5.296
5.294
5.387
6.538
5.977
5.884
ND
0.940
2.646
2.731
0.584
1.121
1.831
3.337
3.319
1.803
2.047
-0.260
2.157
2.599
3.252
1.025
0.029
0.837
2.036
-0.205
0.597
-1.861
1.636
2.449
0.296
0.294
0.387
1.538
0.977
0.884
ND
6880
7750
7750
1.02
1.04
1.03
1
0.99
0.98
0.97
0.96
1
1.02
0.88
0.87
0.85
0.93
0.99
0.98
0.97
1
1.02
1
1
1
0.99
0.88
0.93
0.99
0.92
1.01
1.02
1
5.000
5.000
5.000
7.051
5.481
5.000
5.000
6.133
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
6.725
5.000
4.639
2.679
0.88
0.92
0.87
0.93
0.82
0.85
0.9
17.193
13.179
1
1.08
0.88
0.97
0.93
0.98
0.99
1
221.659
1.685
1892.631
4.769
68.90
2560
6880
6880
506
7010
7590
6880
6880
7750
7010
6880
7590
7590
130
6880
6880
6880
6880
7750
7750
7750
6880
7590
7010
7010
7010
7010
7010
6880
6880
6880
6880
3.012
35.314
6.779
3.499
7.841
3.649
0.884
1.1
8.824
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
14.143
2101.357
16.034
11.731
4.769
1.02
0.85
0.93
0.94
0.88
0.9
64.667
0.86
1.02
0.91
1.012
0.91
0.66
0.89
1.03
0.96
0.88
0.9
0.93
0.93
0.88
0.95
1.12
1.16
1.2
12932.951
50.528
18.273
4.061
125.649
757.466
95.905
3.054
19.636
79.773
607.744
6646.968
1033.925
65.375
207.468
1737.374
515896.848
164.149
25.283
3667.789
3685.193
2974.550
170.426
748.974
941.535
0.91
0.85
0.87
1.05
0.93
0.95
0.83
0.97
0.92
0.87
1.025
1.01
1.45
1.1
5.000 12705.88
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
5.000
23.640
2.969
1279.258
529.978
1237.991
169.951
139.889
771.429
98.766
701.299
2992.208
5774.026
0.302
1.89
0.81
1.3
0.95
1.27
1.037
1.2
1931.372
2212.535
8334.674
2.465
0.78
0.87
0.86
0.98
0.97
0.93
5.572
5.513
4.937
8.481
8.638
8.770
ND
0.572
0.513
-0.063
3.481
3.638
3.770
ND
1.03
1
1
0.99
0.88
0.87
1.13
2.157
0.98
0.97
0.93
1.05
1.718
1.268
0.203
1.379
PK 11195 7590
4.269
1.704
1.066
8.620
3.620
a pIC50 values are the negative logarithms of the molar concentrations necessary for 50% inhibition (IC50); data are means of three
separate experiments performed in duplicate which differed by less than 10%; ND, not determined.
is the optimum length for the alkyl substitution on the
carboxamide nitrogen. Replacement of one n-butyl group
in 3 with a phenyl one did not improve affinity and
selectivity (compare 3 with 7, 8, and 9). Introduction of
a benzyl group (compound 10) or a 4-NO2-benzyl group
(compound 12) instead of one n-butyl group in the
parent compound led to a good selectivity for PBR but
a slight decrease in receptor binding. Interestingly, the
low affinity of the ter-butyl-substituted imidazopyridine
11 (pIC50 5.464) in comparison to the corresponding
n-butyl derivative 10 (pIC50 7.616) suggests that in-
creasing the branching of the alkyl substituent on the
carboxamide nitrogen may cause hindrance to the
carboxamide site of the ligand in occupying the cavity
within the receptor. It should be noted that all these
aromatic compounds (i.e., 7-13) were designed in order
to take advantage of a potential hydrophobic interaction
π-π between their aromatic moieties and the amino
acid residues Trp 107 and Trp 161 on the receptor
protein.
We next examined the biological effects of conforma-
tionally constrained analogues. These compounds were
designed taking into consideration that antagonists are
often conformationally restricted analogues of ago-
nists.33 Thus, inspection of the data reported in Table
2 for the dipropenyl- (14), pyrrolidino- (15 and 16), and
piperidino- (17 and 18) derivatives revealed that the
best results in terms of affinity and selectivity were
obtained for compounds 18 and 14. These results
prompted us to synthesize new congeners of 18 with a
polar group such as the ethoxycarbonyl group on the
piperidino moiety (i.e., 19 and 20), the 4′-benzyl-piper-
azino analogue 21, and the benzofused analogues 22 and
23. Interestingly, only the latter two compounds re-
tained a good affinity selectivity (i.e., pIC50 PBR cortex
7.412 and 8.313; selectivity index ∆pIC50 2.412 and
3.313, respectively) (Table 2). These data suggest that
the presence of polar substituents or ionizable groups
on the amide nitrogen seems to be detrimental for high
affinity and selectivity. This was confirmed by introduc-
ing a heteroaryl substituent such as the 2-pyridylethyl
group; the resulting tertiary amides 24 and 25, indeed,
showed a marked decrease in affinity and selectivity
toward PBR.
Monosubstitution on the carboxamide nitrogen led to
a significant decrease in affinity and selectivity for PBR,
as observed for the set of compounds 26-30. Therefore,
these data support the conclusion that a disubstitution
on the carboxamide nitrogen with linear or conforma-
tionally restricted alkyl substituents of optimum length
is of fundamental importance for interaction with the
corresponding complementary site of the receptor. We
next examined the effects on affinity and selectivity of
disubstitution on the carboxamide nitrogen with alkyl
groups of very different lengths and namely, one n-butyl
group or phenyl group or benzyl group, each coupled
with a methyl group. Another reason for designing these
N-methyl congeners 34-37 was to exploit the presence

Peripheral Benzodiazepine Receptors Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 297
of a methyl group, to develop a potential positron
emission tomography (PET) tracer that would provide
selective imaging of PBR in vivo. Interestingly, com-
pounds 34-37 proved to be among the most active
compounds in this series. Particularly, compounds 34
and 36 are potent even at subnanomolar level. To
characterize better the role of the carboxamide nitrogen
in these compounds, the propanamides 31-33 were also
evaluated. The observed loss of activity and selectivity
highlights the importance of the presence of a carbonyl
C(dO)N group suitably located and oriented for binding
to PBR [compare 31 and 33 with N,N-di-n-propyl-[2-(4-
chlorophenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)]ac-
etamide (CB 34)26 and 37, respectively]. It is likely that
the presence of the additional methyl group in 31-33
leads to a conformational change with consequent
unfavorable orientation of the carbonyl group for the
interaction with the receptor.
Quantitative Structure-Affinity Relationship
Studies. To gain further insight into the relationships
between the binding affinity to PBRs and physicochem-
ical properties, a QSAR study of the 2-phenylimida-
zopyridines 2-37 was carried out. The data set was
divided into subsets A [characterized by compounds
having open chain alkyl substituents (R1 and R2) on the
carboxamide nitrogen, i.e., compounds 2-11, 13, 14,
27-37] and B which considers the whole data set). Each
compound in subset A was described by 14 parameters.
As a descriptor of molecular lipophilicity, the octanol/
water partition coefficient value calculated using the
CLOGP program34 was used. Bulkiness was param-
etrized by molar volume (mv/1000), calculated with the
ACDLab program,35 molar refractivity (mr/1000)34 and,
for each alkyl substituent, STERIMOL descriptors (i.e.,
the length L, the minimum width B1 and the maximum
width B5 of the alkyl substituents) developed by Verloop
and co-workers.36 The main advantage of using the
STERIMOL parameters over other more common steric
variables is that they allow one to define the dimensions
of a given substituent along fixed directions in a three-
dimensional space and, in this case, they should help
to assess the optimal size of the alkyl groups. A number
of indicator variables were also used to account for the
presence or absence of a chlorine atom in the para
position of the phenyl group (Iz1), the presence or
absence of a chlorine atom both in the para position of
the phenyl group and at the 8-position of the 2-phe-
nylimidazo[1,2-a]pyridine system (Iz2), the presence of
an acetamide or propanamide group (ICH3) and next
whether the carboxamide nitrogen is di- or monosub-
stituted (I) (Table 3). The variable N, denoting the
number of carbon atoms of the alkyl substituents on the
carboxamide nitrogen, was also included. In subset A,
compounds 12, 24-26 were not included because their
STERIMOL parameters are not reported in standard
compilations.
Figure 1. (a) Dendrogram of similarity among variables of
subset A obtained using a hierarchical cluster analysis based
on correlation coefficients. (b) Dendrogram of similarity among
variables of subset B obtained using a hierarchical cluster
analysis based on correlation coefficients.
B1R2. This cluster analysis also proved the presence of
an acetamide group to be somehow related to pIC50.
Thus, we performed a stepwise multiparametric regres-
sion analysis (MRA) using only six predictors (mr, ICH3
,
I, Iz1, LR1, and B1R1), retaining only QSARs with cross-
validated (leave-one-out procedure, loo) q2 > 0.5. MRA
carried out on subset A retained four variables (mr, ICH3
,
I, and B1R1) to generate eq 1, explaining about 80% of
the variance in the PBR binding data:
pIC50 (PBR cortex) ) 12.39 ((4.0) -
491.43 ((342) mr + 2.66 ((0.8) ICH3
+
1.84 ((0.8) I - 1.64 ((1.16) B1R1 (1)
n ) 23, r2 ) 0.799, q2 ) 0.576
where n represents the number of data points, r2 the
squared correlation coefficient, q2 the loo cross-valida-
tion coefficient (an assessment of the ‘internal’ predictive
ability of the QSAR model).
Equation 1 relates PBR cortex affinity mainly to steric
effects parametrized by molar refractivity and the
minimum width B1 of the R1 alkyl substituent. Bulky
substituents on the amide nitrogen are detrimental for
PBR affinity. Furthermore, the significant contribution
to the QSAR model of ICH3 and I suggests the importance
for binding to PBR of effects not already accounted for
by steric constants (mr and B1R1). The positive coef-
ficients of ICH3 and I in eq 1 indicate that the presence
of an acetamide group and disubstitution on carboxa-
mide nitrogen favor the affinities for PBR. These find-
A selection of the pertinent variables was carried out
by considering the intercorrelation among predictors
(physicochemical parameters) and their influence (ex-
plained variance of the biological data) on the regression
model. A cluster analysis based on correlation coef-
ficients among variables (Figure 1a) showed which
parameters contain comparable information, namely
mr, mv, N, B5R2, LR2, and and Clog P as well as I and

298 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Trapani et al.
Table 3. Physicochemical Parameters Used in the QSAR Study of 2-Phenylimidazo[1,2-a]pyridineacetamides 2-37
compd
Log Pa
mvb
mrb
Iz1
Iz2
ICH3
I
N
LR1
LR2
B1R1
B1R2
B5R1
B5R2
2
6.430
7.140
9.260
6.430
8.540
6.750
7.470
6.750
6.000
6.890
6.460
5.700
5.520
4.600
3.890
4.450
5.160
4.860
5.570
6.470
5.230
5.940
3.860
4.570
5.030
5.280
5.720
5.010
5.720
6.390
6.310
5.970
5.560
4.840
5.880
5.66
0.3575
0.3668
0.4310
0.3575
0.4217
0.3620
0.3712
0.3620
0.3620
0.3909
0.3764
0.3018
0.3347
0.2758
0.2665
0.2826
0.2918
0.3336
0.3428
0.3704
0.3183
0.3275
0.3345
0.3438
0.2904
0.2973
0.3018
0.2925
0.3179
0.3499
0.3383
0.3543
0.3186
0.3090
0.3231
0.3391
0.01213
0.01262
0.01448
0.01213
0.01399
0.01279
0.01328
0.01279
0.01279
0.01374
0.01389
0.01142
0.01164
0.01059
0.01010
0.01056
0.01105
0.01214
0.01263
0.01393
0.01215
0.01264
0.01211
0.01260
0.01121
0.01077
0.01152
0.01103
0.01189
0.01216
0.01245
0.01281
0.01123
0.01074
0.01189
0.01235
1
1
1
1
1
0
1
1
0
1
1
1
1
1
0
0
1
1
1
1
0
1
0
1
0
1
1
0
1
1
1
1
1
0
1
1
1
1
1
0
0
0
1
0
0
1
1
1
1
1
0
0
1
0
1
1
0
1
0
1
0
1
1
0
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
1
1
1
1
1
1
1
8
6.17
6.17
8.22
6.17
8.22
6.17
6.17
6.17
6.17
4.11
4.92
6.28
5.11
-
6.17
6.17
8.22
6.17
8.22
6.28
6.28
6.28
4.62
4.62
-
1.52
1.52
1.52
1.52
1.52
1.52
1.52
1.52
1.52
2.6
1.52
1.71
1.52
-
1.52
4.54
4.54
5.96
4.54
5.96
4.54
4.54
4.54
4.54
3.11
3.49
3.11
3.78
-
4.54
3
8
12
8
1.52
4.54
4
1.52
5.96
5
1.52
4.54
6
12
10
10
10
11
11
10
6
1.52
5.96
7
1.71
3.11
8
1.71
3.11
9
1.71
3.11
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
1.52
6.02
1.52
3.17
-
1
-
1
2.06
5.11
-
6
1.52
3.78
4
-
-
4
-
-
-
-
-
-
5
-
-
-
-
-
-
5
-
-
-
-
-
-
8
-
-
-
-
-
-
8
-
-
-
-
-
-
11
9
-
-
-
-
-
-
-
-
-
-
-
-
9
-
-
-
-
-
-
8
-
-
-
-
-
-
8
-
-
-
-
-
-
5
-
-
-
-
-
-
4
6.17
6.17
6.17
4.62
4.92
6.17
4.62
6.17
6.17
6.28
4.62
2.06
2.06
2.06
2.06
4.92
2.87
2.87
2.87
2.87
2.87
2.87
1.52
1.91
1.91
1.52
1.52
1.91
1.52
1.52
1.52
1.71
1.52
1
4.54
3.49
3.49
6.02
3.49
3.49
6.02
4.54
4.54
3.11
6.02
1
6
1
1
6
1
1
7
1
1
6
1.52
1.52
1.52
1.52
1.52
1.52
1.52
3.49
2.04
2.04
2.04
2.04
2.04
2.04
7
8
5
5
7
8
a Estimated by CLOGP program.34 b Bulkiness was parametrized by molar volume (mv, cm3) calculated by ACDLab program35 and
molar refractivity (mr) estimated by CLOGP software.34 Both calculated molar volumes and molar refractivities were normalized multiplying
them by a factor of 1 × 10-3 The other physicochemical descriptors reported in the Table are so defined: Iz1 presence (value 1) or absence
(value 0) of Cl at Z position; Iz2 presence (value 1) or absence (value 0) of a Cl both at Z and Y positions; ICH3 presence of an acetamido
(value 1) or presence of propanamide group (value 0); I whether the carboxamide nitrogen is bisubstituted (value 1) or monosubstituted
(value 0); N the total number of carbon atoms of the alkylsubstituents on the carboxamide nitrogen; LR1 and LR2 are the L Verloop’s
parameters for R1 and R2, respectively; B1R1 and B1R2 are the B1 Verloop’s parameters for R1 and R2, respectively; B5R1 and B5R2 are the
B5 Verloop’s parameters for R1 and R2, respectively. The Verloop’s parameters were taken from standard compilations.
ings are consistent with previous QSAR analysis32
which, as mentioned above, suggested that a four-carbon
chain is the optimum length for the alkyl substitution
on the carboxamide nitrogen. However, in the present
study, it was found that the minimum width B1 of the
R1 alkyl substituent, rather than its length and the
number of carbon atoms, is a relevant factor for increas-
ing PBR binding affinity.
factors from large sets of correlated descriptors. There-
fore, in this paper, correlation between the eight selected
descriptors and pIC50 values was also established by
PLS. The best computational model was obtained
involving five predictors namely, mr (molar refractivity
mr in quadratic term), Clog P, ICH3, and I with a
maximum of five PLS components explaining more than
75% of the variance in the PBR binding data. This model
gave r2 of 0.752 and q2 of 0.648.
We next explored the best quantitative structure-
activity relationship of the whole data set (subset B).
As molecular descriptors, parameters of lipophilicity
pIC50 (PBR cortex) ) -65.06 ((27.04) +
11300 ((4368) mr - 507.46 ((181.14) mr2 +
2.84 ((0.86) ICH3 + 1.72 ((0.68) I +
1.08 ((0.34) Clog P (2)
(Clog P), bulk (mv, mr), indicator variables (Iz1, Iz2, ICH3
,
I), and N were examined. Again, a cluster analysis based
on correlation coefficients among variables (Figure 1b)
showed which parameters contain comparable informa-
tion. Moreover it was found that mv, mr, and Clog P
are interdependent (r2 > 0.500).
n ) 36, r2 ) 0.752, q2 ) 0.648
The partial least squares (PLS) method is particularly
suited for the extraction of a few highly significant
These relationships suggest that increases in molar
refractivity after some optimum value is reached may

Peripheral Benzodiazepine Receptors Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 299
cause hindrance to the carboxamide site of the ligand
in interacting with the receptor. Again, the positive
coefficient of ICH3 and I in eq 2 suggests that the
presence of an acetamide group and the double substi-
tution on carboxamide nitrogen favor affinity for PBR.
From eq 2 (which considers the whole data set) lipo-
philicity emerges as a physicochemical property which
also correlates with binding affinity. Actually, the
importance of the lipophilicity was clearly demonstrated
by a significant decrease in receptor affinity of com-
pounds 19-21 characterized by hydrophilic substituents
on the amide nitrogen. As a consequence, these results
induced us to the decision to stop the project of further
introduction of hydrophilic substituents in this specific
region.
With the same independent variables in eqs 1 and 2,
a preliminary attempt was made to explore the features
causing selectivity for PBR binding [expressed as ∆pIC50
(PBR cortex)] and statistically poorer relationships were
derived (i.e., n ) 23, r2 ) 0.717, q2 ) 0.495 and n ) 36,
r2 ) 0.621, q2 ) 0.491, respectively) suggesting that
further studies with larger sample size are necessary
to reach a conclusion concerning selectivity require-
ments in this series of ligands.
While the above QSARs highlighted the main phys-
icochemical factors eliciting the binding of 2-phenylimi-
dazo[1,2-a]pyridine derivatives to PBR and could have
value for molecular design, we further explored the
pharmacological profile of the representative compounds
by measuring the effects on steroidogenesis in rats.
Functional Studies. Compounds 2, 3, 7, 14, 18, 34,
35, and 36, as potent and selective examples of the
subsets examined here, were evaluated both for their
effects on human recombinant GABAA receptors, to
determine their capacity to interact with CBRs, and for
their capability to stimulate the synthesis in plasma and
brain of neuroactive steroids in rats.
In Vitro Assay: Electrophysiology in Xenopus
Oocytes. As illustrated in Figure 2, Cl- currents evoked
by GABA at GABAA receptors were not significantly
modified by compounds 2, 3, 14, 18, and 36 tested at
concentrations of 3, 10, and 30 µM. Similarly, PK 11195
failed to produce statistical significant changes in the
amplitude of GABA-evoked currents. By contrast, com-
pound 35 markedly enhanced GABA-evoked currents
(97 ( 6% and 145 ( 10%, at concentrations of 10 and
30 µM, respectively).
In Vivo Assay: Effect of Compounds on Ste-
roidogenesis. As shown in Table 4, intraperitoneal
administration of compounds 2, 3, 7, 14, 34, and 35 (25
mg/kg) resulted in a significant increase in the concen-
tration of neuroactive steroids both in plasma and in
brain. The effects of compound 3 was most marked:
progesterone (PROG): + 121% and +111% in brain and
plasma, respectively; 3R,5R-THPROG: + 52% and +
96% in brain and plasma, respectively; 3R,5R-THDOC:
+ 52% and 131% in the brain and plasma, respectively.
In contrast, compounds 18 and 36 showed little effects;
in particular 36 was able to significantly enhance the
cerebrocortical and plasmatic levels of PROG (+ 39%;
p < 0.05; and + 55%; p < 0.01, respectively) and the
plasma levels of 3R,5R-THPROG (+ 45%, p< 0.01) while
failing to significantly modify brain 3R,5R-THPROG as
well as brain and peripheral levels of 3R,5R-THDOC.
Figure 2. Modulatory action of compounds 2, 3, 14, 18, 35,
36, and PK 11195 at human R1â2γ2L GABAA receptors
expressed in Xenopus oocytes. Values are expressed as per-
centage change induced by the different drugs from the control
response obtained with GABA EC10 (concentration of GABA,
usually ranging from 0.5 to 8 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 four to six different
oocytes) ( SEM. *p < 0.01 vs control response (ANOVA and
Scheffe`’s test).
On the basis of these results, we examined whether 36,
which appears to act as a partial agonist at the PBR,
could antagonize CB 34-induced steroidogenesis.26,37
Prior treatment (-10 min) with 36, at a dose of 25 mg/
kg, significantly decreased the CB 34-induced increases
in the brain (Figure 3) and plasma (data not shown)
concentrations of 3R,5R-THPROG.
Conclusions
Consistent with previous studies,27,32 the results of
this work suggest that bulkiness of the substituents on
the amide nitrogen of the imidazopyridine PBR ligands
is a property mainly responsible for modulation of
affinity. Second, increasing the branching of the alkyl
substituent on the carboxamide nitrogen may cause
hindrance to the ligand in interacting with the receptor
region which should be of restricted size. Third, the
presence of aromatic or conformationally constrained
substituents on the carboxamide nitrogen may favor
high affinity and selectivity, while the presence of polar
substituents or ionizable groups in this region seems
to be detrimental. A QSAR analysis of the measured
binding data indicated that besides the important role
played by disubstitution on the carboxamide nitrogen,
the affinity of compounds 2-37 to PBRs proved to be
linearly correlated with lipophilicity constant and molar
refractivity through a parabolic relationship.
In in vivo studies, compounds 2, 3, 7, 14, and 34 each
exhibited potent and selective effects on the peripheral
and central synthesis of neuroactive steroids, with 3
being the most potent derivatives. The capability of
compound 36 to reduce the stimulatory effect exerted
by CB 34 on the cerebrocortical concentration of 3R,5R-
THPROG is consistent with a partial agonist activity.
We also show that there is no correlation between PBR
affinity and selectivity and steroidogenic activity. The
whole set of compounds examined 2, 3, 7, 14, 18, 34,
35, and 36, indeed, although endowed with high affinity

300 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Trapani et al.
Table 4. Effect of ip Administration of Compounds 2, 3, 7, 14, 18, 34, 35, and 36 (25 mg/kg) on Cerebrocortical and Plasmatic Levels
of Neuroactive Steroids in Rats after 30 mina,b
cerebral cortex (ng/g)
plasma (ng/mL)
compounds
progesterone
3R,5R-THPROG
3R,5R-THDOC
progesterone
3R,5R-THPROG
3R,5R-THDOC
vehicle
2
5.2 ( 0.3
10.3 ( 1.7*
11.5 ( 1.1**
5.8 ( 1.0
11.5 ( 1.2**
7.8 ( 0.6**
8.9 ( 1.1*
10.6 ( 0.6**
7.2 ( 0.7*
6.0 ( 0.6
8.8 ( 1.1*
9.1 ( 1.2*
16.3 ( 1.6**
8.6 ( 1.4
7.3 ( 0.6
7.4 ( 1.8
9.2 ( 1.3*
7.4 ( 0.6
2.3 ( 0.3
2.9 ( 0.1
3.5 ( 0.5*
2.6 ( 0.3
3.2( 0.1*
2.9 ( 0.8
3.9 ( 0.4*
4.1 ( 0.5**
2.8 ( 0.3
9.0 ( 1.0
14.0 ( 0.9
23.4 ( 4.0*
27.5 ( 3.7**
21.3 ( 1.9**
18.1 ( 0.2
19.4 ( 2.0*
16.2 ( 1.9
16.5 ( 2.0
20.3 ( 0.2**
7.0 ( 0.9
14.9 ( 0.8**
16.2 ( 2.1**
8.9 ( 1.8
26.0 ( 3.7**
19.0 ( 1.8**
17.3 ( 2.0**
12.2 ( 1.0*
12.2 ( 1.0*
14.9 ( 1.8*
13.6 ( 0.7**
13.9 ( 0.1**
3
7
14
18
34
35
36
9.0 ( 0.8
9.6 ( 0.5*
8.1 ( 1.2
10.4 ( 1.2*
8.3 ( 0.6
b
a Data are expressed as nanograms of steroids per gram of tissue and are the means ( SEM of values from at least eight rats. *p <
0.05; **p < 0.01 vs vehicle-treated animals.
determined on a Varian Mercury 300 MHz instrument.
Chemical shifts are given in d values downfield from Me4Si
as a internal standard. Mass spectra were recorded on an
Agilent 6890-5973 MSDGC-MS spectrometer. Elemental analy-
ses were carried out with a Eurovector EuroEA 3000 C, H, N
analyzer, and results were within (0.40% of theoretical values.
Silica gel 60 (Merck 70-230 mesh) was used for column
chromatography. All the following reactions were performed
under a nitrogen atmosphere.
Materials. The preparation of 2-phenylimidazo[1,2-a]pyri-
dine-3-acetic acids (42) was accomplished following a reported
procedure.13 Ethyl 1,2-dihydro-2-ethoxy-1-quinolinecarboxylate
(EEDQ), N,N′dicyclohexylcarbodiimide (DCC), 1,1′-carbonyl-
diimidazole (CDI), anhydrous tetrahydrofuran, and triethyl-
amine (TEA) were purchased from Sigma-Aldrich (Italy). All
the mono- or dialkylamines were commercially available; the
amine hydrochlorides used in method B, when not com-
mercially available, were prepared as follows: HCl gas was
bubbled into a stirred solution of amine in anydrous CH2Cl2
for 15 min and then excess HCl gas was removed under a
stream of nitrogen. The solvent was evaporated under reduced
pressure to give the hydrochlorides as white solids which were
used immediately.
General Procedure for Preparation of (2-Phenylimi-
dazo[1,2-a]pyridin-3-yl)acetamides 2-6. Method A. To a
solution of the suitably substituted 2-aminopyridine 41 (11
mmol) in DMF (50 mL) was added the appropriate 3-bromo-
3-benzoyl propionamide26,27 (11 mmol). The mixture was
refluxed under stirring and under a nitrogen atmosphere for
7-20 h. The progress of reaction was monitored by TLC. The
solvent was evaporated under reduced pressure, and the
residue was purified by silica gel column chromatography
[light petroleum ether/ethyl acetate 8/2 (v/v) as eluent)] to give
the amide 2-6. Physical data of these compounds are sum-
marized in Table 1.
N,N-Di-n-butyl-[2-(4-chlorophenyl)-8-chloroimidazo-
[1,2-a]pyridin-3-yl]acetamide (2): IR (KBr): 1630 cm-1; 1H
NMR (CDCl3) δ: 0.8-0.9 (m, 6H, CH3), 0.9-1.1 (m, 2H, CH2),
1.1-1.3 (m, 2H, CH2), 1.3-1.5 (m, 4H, CH2), 3.07 (t, 2H,
CH2N), 3.28 (t, 2H, CH2N), 4.05 (s, 2H, CH2CO), 6.7-6.8 (m,
1H, Ar), 7.2-7.3 (m, 1H, Ar), 7.41 (d, J ) 8.4 Hz, 2H, Ar),
7.62 (d, J ) 8.4 Hz, 2H, Ar), 8.20 (d, J ) 7.0 Hz, 1H, Ar); MS
m/z 431 (M+, 19), 275 (base); Anal. (C23H27Cl2N3O) C, H, N.
Figure 3. Effect of pretreatment with compound 36 on the
CB 34-induced increases in the cerebrocortical concentration
of 3R,5R-THPROG. Rats were injected with compound 36 (25
mg/kg ip) 10 min before CB 34 and were killed 30 min after
CB 34. Data are means ( SD from eight rats and are expressed
as a percentage of concentration of vehicle-treated animals.
b
ap < 0.05; p < 0.01 vs vehicle group.
and selectivity, did not produce similar effects on
neurosteroid synthesis. This could suggest that PBR
agonists and antagonists may occupy similar but not
identical binding sites and that, as occurs for other
receptor systems, the transition from agonist to antago-
nist activity is consequent to very small structural
changes. Of interest in this regard is the consideration
that a rank order of intrinsic activity can be envisaged.
Thus, the N,N-di-n-butyl- and the N-butyl,N-methyl-
substituted-imidazopyridines 3 and 35 exhibited high
efficacy in increasing the peripheral and central syn-
thesis of neuroactive steroids (full agonism). Compounds
2, 14, and 34 showed a moderate increase in ste-
roidogenic activity. The effects of congeners 7 and 18
was less marked but significant, while the N-phenyl,N-
methyl derivative 36 (highly potent and selective)
showed little efficacy in increasing steroids content in
the cerebral cortex. This ligand is the first compound
in this series able to produce a small increase in
neurosteroid concentration and to reduce the CB 34-
induced increase in the cerebrocortical levels of 3R,5R-
THPROG.
N,N-Di-n-butyl-[2-(4-chlorophenyl)-6,8-dichloroimida-
zo[1,2-a]pyridin-3-yl]acetamide (3): IR (KBr): 1630 cm-1
;
1H NMR (CDCl3) δ: 0.8-1.0 (m, 6H, CH3), 1.0-1.6 (m, 8H,
CH2), 3.1-3.2 (m, 2H, CH2N), 3.3-3.4 (m, 2H, CH2N), 4.03 (s,
2H, CH2CO), 7.30 (d, J ) 1.7 Hz, 1H, Ar), 7.43 (d, J ) 8.3 Hz,
2H, Ar), 7.60 (d, J ) 8.3 Hz, 2H, Ar), 8.23 (d, J ) 1.7 Hz, 1H,
Ar); MS m/z 465 (M+, 11), 309 (52); Anal. (C23H26Cl3N3O) C,
H, N.
N,N-Di-n-hexyl-[2-(4-chlorophenyl)-6,8-dichloroimida-
Experimental Section
zo[1,2-a]pyridin-3-yl]acetamide (4): IR (KBr): 1645 cm-1
;
Chemistry. Melting points were determined in open capil-
lary tubes with a Bu¨chi apparatus and are uncorrected. IR
spectra were obtained on a Perkin-Elmer IR Fourier transform
spectrophotometer in KBr pellets. 1H NMR spectra were
1H NMR (CDCl3) δ: 0.7-0.9 (m, 6H, CH3), 1.0-1.6 (m, 16H,
CH2), 3.0-3.1 (m, 2H, CH2N), 3.3-3.4 (m, 2H, CH2N), 4.03 (s,
2H, CH2CO), 7.30 (d, J ) 1.4 Hz, 1H, Ar), 7.43 (d, J ) 8.3 Hz,
2H, Ar), 7.59 (d, J ) 8.3 Hz, 2H, Ar), 8.26 (d, J ) 1.4 Hz, 1H,

Peripheral Benzodiazepine Receptors Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 301
Ar); MS m/z 309 [M+- (C6H13) 2NCO, 53]; Anal. (C27H34Cl3N3O)
C, H, N.
7.7 (m, 2H, Ar), 8.33 (d, J ) 1.5 Hz, 1H, Ar); MS m/z 387 (M+,
15), 275 (base); Anal. (C20H19Cl2N3O) C, H, N.
N,N-Di-n-butyl-[2-(4-chlorophenyl)-6-chloroimidazo[1,2-
a]pyridin-3-yl]acetamide (5): IR (KBr): 1630 cm-1; 1H NMR
(CDCl3) δ: 0.8-1.0 (m, 6H, CH3), 1.0-1.6 (m, 8H, CH2), 3.1-
3.2 (m, 2H, CH2N), 3.3-3.4 (m, 2H, CH2N), 4.02 (s, 2H, CH2-
CO), 7.14 (dd, J ) 9.4 and 1.9 Hz, 1H, Ar), 7.40 (d, J ) 8.5
Hz, 2H, Ar), 7.5-7.6 (m, 3H, Ar), 8.2-8.3 (m, 1H, Ar); MS m/z
431 (M+, 15), 275 (base); Anal. (C23H27Cl2N3O) C, H, N.
N,N-Di-n-hexyl-[2-(4-chlorophenyl)-6-chloroimidazo[1,2-
a]pyridin-3-yl]acetamide (6): IR (KBr): 1640 cm-1; 1H NMR
(CDCl3) δ: 0.8-0.9 (m, 6H, CH3), 1.0-1.6 (m, 16H, CH2), 3.0-
3.2 (m, 2H, CH2N), 3.3-3.4 (m, 2H, CH2N), 4.05 (s, 2H, CH2-
CO), 7.16 (dd, J ) 9.4 and 1.9 Hz, 1H, Ar), 7.43 (d, J ) 8.5
Hz, 2H, Ar), 7.5-7.7 (m, 3H, Ar), 8.28 (d, J ) 1.2 Hz, 1H, Ar);
MS m/z 487 (M+, 15), 275 (base); Anal. (C27H35Cl2N3O) C, H,
N.
General Procedure for Preparation of (2-Phenylimi-
dazo[1,2-a]pyridin-3-yl)acetamides 7-9, 12, 13, 17-23,
27-29, 34-36. Method B. A solution of the appropriate acid
42 (1 mmol) and CDI (1.3 mmol) in anhydrous THF (20 mL)
was stirred at room temperature. Then, the suitable amine
hydrochloride (HNR1R2‚HCl) (1.5 mmol) was added and the
mixture stirred at room temperature or under a gentle heating
for 4-12 h. The reaction mixture was washed 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 8/2 (v/v) as eluent)] to give
the required compounds 7-9, 12, 13, 17-23, 27-29, 34-36.
Physical data of these compounds are summarized in Table
1.
2-(4-Chlorophenyl)-3-[(2-oxo-2-piperidin-1-ylethyl)]-
6,8-dichloroimidazo[1,2-a]pyridine (18): IR (KBr): 1640
1
cm-1; H NMR (CDCl3) δ: 1.3-1.4 (m, 2H, CH2), 1.5-1.7 (m,
4H, CH2), 3.3-3.4 (m, 2H, CH2), 3.5-3.6 (m, 2H, CH2), 4.06
(s, 2H, CH2CO), 7.35 (d, J ) 1.5 Hz, 1H, Ar), 7.42 (d, J ) 8.4
Hz, 2H, Ar), 7.61 (d, J ) 8.4 Hz, 2H, Ar), 8.25 (d, J ) 1.5 Hz,
1H, Ar); MS m/z 421 (M+, 18), 309 (base); Anal. (C20H18Cl3N3O)
C, H, N.
Ethyl 1-[2-(4-chlorophenyl)-6-chloroimidazo[1,2-a]py-
ridin-3-yl]acetyl)piperidin-3-yl]acetate (19): IR (KBr):
1
1724, 1634 cm-1; H NMR (CDCl3) δ: 1.1-1.4 (m, 3H, CH3),
1.5-1.8 (m, 2H, CH2), 1.8-2.1 (m, 2H, CH2), 2.4-2.6 (m, 1H,
CH), 3.1-3.5 (m, 2H, CH2), 3.7-3.9 (m, 1H, CH2), 3.9-4.4 (m,
5H, CH2CO, CH2O and CHCO), 7.1-7.2 (m, 1H, Ar), 7.4-7.6
(m, 5H, Ar), 8.2-8.3 (m, 1H, Ar); MS m/z 459 (M+, 17), 275
(base); Anal. (C23H23N3Cl2O3) C, H, N.
Ethyl 1-[2-(4-chlorophenyl)-6,8-dichloroimidazo[1,2-a]-
pyridin-3-yl]acetyl)piperidine-3-yl]acetate (20):
IR
(KBr): 1727, 1644 cm-1; 1H NMR (CDCl3) δ: 1.2-1.3 (m, 3H,
CH3), 1.5-1.8 (m, 2H, CH2), 1.8-2.1 (m, 2H, CH2), 2.4-2.6
(m, 1H, CH), 3.1-3.5 (m, 2H, CH2), 3.7-3.9 (m, 1H, CH2), 3.9-
4.4 (m, 5H, CH2CO, CH2O and CHCO), 7.2-7.3 (m, 1H, Ar),
7.4-7.7 (m, 4H, Ar), 8.2-8.3 (m, 1H, Ar); MS m/z 493 (M+,
21), 309 (base); Anal. (C23H22N3Cl3O3) C, H, N.
2-(4-Chlorophenyl)-3-[(4-benzylpiperazin-1-yl)-2-oxo-
ethyl]-6,8-dichloroimidazo[1,2-a]pyridine (21): IR (KBr):
1640 cm-1; 1H NMR (CDCl3) δ: 2.3-2.4 (m, 4H, CH2N), 3.4-
3.8 (m, 6H, CH2N and CH2-Ar), 4.0 (s, 2H, CH2CO), 7.2-7.8
(m, 10H, Ar), 8.17 (s, 1H, Ar); MS m/z 512 (M+, 5), 309 (30);
Anal. (C26H23Cl3N4O) C, H, N.
N-n-Butyl-N-phenyl-(2-phenyl-6,8-dichloroimidazo[1,2-
a]pyridin-3-yl)acetamide (7): IR (KBr): 1630 cm-1; 1H NMR
(CDCl3) δ: 0.86 (t, J ) 7.3 Hz, 3H, CH3), 1.2-1.4 (m, 2H, CH2),
1.4-1.6 (m, 2H, CH2), 3.60-3.70 (m, 2H, CH2N), 3.77 (s, 2H,
CH2CO), 7.0-7.2 (m, 2H, Ar), 7.2-7.4 (m, 7H, Ar), 7.4-7.6
(m, 2H, Ar), 8.15 (d, J ) 1.9 Hz, 1H, Ar); MS m/z 451 (M+,
28), 275 (base); Anal. (C25H23Cl2N3O) C, H, N.
2-Phenyl-3-[(6,8-dichloroimidazo[1,2-a]pyridin-3-yl)-
acetyl]-1,2,3,4-tetrahydroisoquinoline (22): IR (KBr):1620
cm-1; 1H NMR (CDCl3) δ: 2.65 (t, J ) 6.5 Hz, 1,1H, CH2), 2.85
(t, J ) 6.5 Hz, 0.9H, CH2), 3.45 (t, J ) 6.5 Hz, 1.1H, CH2),
3.80 (t, J ) 6.5 Hz, 0.9H, CH2), 4.17 (s, 1.1H, CH2CO), 4.20 (s,
0.9H, CH2CO), 4.35 (s, 0.9H, CH2), 4.71 (s, 1.1H, CH2), 6.5-
7.8 (m, 10H, Ar), 8.31 (d, J ) 6.7 Hz, 0.55H, Ar), 8.38 (d, J )
1.8 Hz, 0.45H, Ar); MS m/z 435 (M+, 7), 275 (base); Anal.
(C24H19Cl2N3O) C, H, N.
2-(4-Chlorophenyl)-3-[(6,8-dichloroimidazo[1,2-a]pyri-
din-3-yl)acetyl]-1,2,3,4-tetrahydroisoquinoline (23): IR
(KBr):1620 cm-1; 1H NMR (CDCl3) δ: 2.70 (t, J ) 6.5 Hz, 1.1H,
CH2), 2.85 (t, J ) 6.5 Hz, 0.9H, CH2), 3.56 (t, J ) 6.5 Hz, 1.1H,
CH2), 3.80 (t, J ) 6.5 Hz, 0.9H, CH2), 4.12 (s, 1.1H, CH2CO),
4.14 (s, 0.9H, CH2CO), 4.44 (s, 0.9H, CH2), 4.72 (s, 1.1H, CH2),
6.7-7.8 (m, 9H, Ar), 8.22 (d, J ) 1.8 Hz, 0.55H, Ar), 8.26 (d,
J ) 1.8 Hz, 0.45H, Ar); MS m/z 469 (M+, 4), 309 (60); Anal.
(C24H18Cl3N3O) C, H, N.
N-n-Butyl-N-phenyl-[2-(4-chlorophenyl)-6,8-di-
chloroimidazo[1,2-a]pyridin-3-yl]acetamide (8):
IR
(KBr): 1640 cm-1 1H NMR (CDCl3) δ: 0.88 (t, J ) 7.3 Hz,
;
3H, CH3), 1.2-1.4 (m, 2H, CH2), 1.4-1.6 (m, 2H, CH2), 3.6-
3.7 (m, 2H, CH2N), 3.79 (s, 2H, CH2CO), 7.0-7.2 (m, 2H, Ar),
7.2-7.4 (m, 6H, Ar), 7.46 (d, J ) 8.5 Hz, 2H, Ar), 8.19 (d, J )
1.9 Hz, 1H, Ar); MS m/z 485 (M+, 29), 309 (base); Anal. (C25H22-
Cl3N3O) C, H, N.
N-n-Butyl-N-phenyl-[2-(4-chlorophenyl)-6-chloroimi-
dazo[1,2-a]pyridin-3-yl]acetamide (9): IR (KBr): 1640
cm-1; 1H NMR (CDCl3) δ: 0.84 (t, J ) 7.4 Hz, 3H, CH3), 1.2-
1.4 (m, 2H, CH2), 1.4-1.6 (m, 2H, CH2), 3.67 (t, J ) 7.6 Hz,
2H, CH2N), 3.77 (s, 2H, CH2CO), 7.0-7.2 (m, 3H, Ar), 7.2-
7.4 (m, 5H, Ar), 7.4-7.6 (m, 3H, Ar), 8.1-8.2 (m, 1H, Ar); MS
m/z 451 (M+, 22), 275 (base); Anal. (C25H23Cl2N3O) C, H, N.
N-n-Butyl-N-4-nitrobenzyl-[2-(4-chlorophenyl)-6,8-
dichloroimidazo[1,2-a]pyridin-3-yl]acetamide (12): IR
(KBr):1645 cm-1; 1H NMR (CDCl3) δ: 0.73 (t, 2.1H, CH3), 0.84
(t, 0.9H, CH3), 1.4-1.6 (m, 2H, CH2), 3.12 (t, J ) 6.5 Hz, 1,-
4H, CH2N), 3.34 (t, J ) 6.5 Hz, 0,6H, CH2N), 4.02 (s, 0,6H,
CH2CO), 4.16 (s, 1,4H, CH2CO), 4.40 (s, 0,6H, CH2-Ar), 4.64
(s, 1,4H, CH2-Ar), 6.9-7.7 (m, 8H, Ar), 8.1-8.4 (m, 2H, Ar);
MS m/z 530 (M+, 8), 309 (base); Anal. (C25H21Cl3N4O3) C, H,
N.
N-n-Butyl-[2-(4-chlorophenyl)-6,8-dichloroimidazo[1,2-
a]pyridin-3-yl]acetamide (27): IR (KBr): 3295, 1643 cm-1
;
1H NMR (CDCl3) δ: 0.88 (t, 3H, CH3), 1.2-1.6 (m, 4H, CH2),
3.25 (q, 2H, CH2NH), 3.92 (s, 2H, CH2CO), 7.3-7.4 (m, 1H,
Ar), 7.43 (d, J ) 8.4 Hz, 2H, Ar), 7.70 (d, J ) 8.4 Hz, 2H, Ar),
8.1-8.2 (m, 1H, Ar); MS m/z 409 (M+, 14), 309 (base); Anal.
(C19H18Cl3N3O) C, H, N.
N-Cyclohexyl-[2-(4-chlorophenyl)-6,8-dichloroimida-
zo[1,2-a]pyridin-3-yl]acetamide (28): IR (KBr): 3295, 1630
1
cm-1; H NMR (CDCl3) δ: 0.9-1.2 (m, 2H, CH2), 1.2-1.4 (m,
2H, CH2), 1.5-1.9 (m, 6H, CH2), 3.7-3.8 (m, 1H, CH), 3.87 (s,
2H, CH2CO), 5.52 (d, J ) 7.0 Hz, 1H, NH), 7.33 (d, J ) 1.6
Hz, 1H, Ar), 7.43 (d, J ) 8.5 Hz, 2H, Ar), 7.65 (d, J ) 8.5 Hz,
2H, Ar), 8.13 (d, J ) 1.6 Hz, 1H, Ar); MS m/z 435 (M+, 15),
309 (base); Anal. (C21H20Cl3N3O) C, H, N.
N-Phenyl-[2-(4-chlorophenyl)-6,8-dichloroimidazo[1,2-
a]pyridin-3-yl]acetamide (13): IR (KBr):3265, 1650 cm-1
;
1H NMR (CDCl3) δ: 4.08 (s, 2H, CH2CO), 7.0-7.2 (m, 2H, Ar),
7.2-7.5 (m, 5H, Ar), 7.6-7.7 (m, 3H, Ar), 8.16 (d, J ) 6.5 Hz,
1H, Ar); MS m/z 429 (M+, 16), 309 (base); Anal. (C21H14Cl3N3O)
C, H, N.
N-Cyclohexyl-(2-phenyl-6,8-dichloroimidazo[1,2-a]-
pyridin-3-yl)acetamide (29): IR (KBr): 3290, 1635 cm-1; 1H
NMR (CDCl3) δ: 0.9-1.2 (m, 2H, CH2), 1.2-1.5 (m, 2H, CH2),
1.5-1.9 (m, 6H, CH2), 3.8-3.9 (m, 1H, CH), 3.90 (s, 2H, CH2-
CO), 5.70 (br s, 1H, NH), 7.2-7.6 (m, 4H, Ar), 7.6-7.8 (m, 2H,
Ar), 8.0-8.3 (m, 1H, Ar); MS m/z 401 (M+, 15), 275 (base);
Anal. (C21H21N3Cl2O) C, H, N.
2-Phenyl-3-[(2-oxo-2-piperidin-1-ylethyl)]-6,8-di-
chloroimidazo[1,2-a]pyridine (17): IR (KBr):1627 cm-1; 1H
NMR (CDCl3) δ: 1.3-1.4 (m, 2H, CH2), 1.5-1.7 (m, 4H, CH2),
3.2-3.3 (m, 2H, CH2), 3.5-3.6 (m, 2H, CH2), 4.09 (s, 2H, CH2-
CO), 7.34 (d, J ) 1.5 Hz, 1H, Ar), 7.4-7.5 (m, 3H, Ar), 7.6-
N-n-Butyl,N-methyl-[2-(4-chlorophenyl)-6,8-dichloro-
imidazo[1,2-a]pyridin-3-yl]acetamide (34): IR (KBr): 1640

302 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Trapani et al.
1
cm-1; H NMR (CDCl3) δ: 0.8-1.0 (m, 3H, CH3), 1.1-1.6 (m,
2-[6,8-Dichloro-2-(4-chlorophenyl)imidazo[1,2-a]pyri-
din-3-yl]-N-methyl-N-(2-pyridin-3-ylethyl)acetamide
4H, CH2), 2.9-3.0 (m, 3H, CH3N), 3.1-3.3 (m, 1H, CH2N), 3.3-
3.5 (m, 1H, CH2N), 4.02 (s, 1H, CH2CO), 4.05 (s, 1H, CH2CO),
7.3-7.4 (m, 1H, Ar), 7.43 (d, J ) 8.2 Hz, 2H, Ar), 7.5-7.7 (m,
2H, Ar), 8.1-8.2 (m, 0.5H, Ar), 8.2-8.3 (m, 0.5H, Ar); MS m/z
423 (M+, 14), 309 (base); Anal. (C20H20Cl3N3O) C, H, N.
N-n-Butyl,N-methyl-(2-phenyl-6,8-dichloroimidazo[1,2-
1
(25): IR (KBr):1645 cm-1; H NMR (CDCl3) δ: 2.88 (s, 1.5H,
CH3), 2.98 (s, 1.5H, CH3), 3.03 (t, 2H, CH2), 3.7-3.8 (m, 2H,
CH2), 3.92 (s, 1H, CH2CO), 3.95 (s, 1H, CH2CO), 7.0-7.6 (m,
8H, Ar), 7.6-7.7 (m, 0.5H, Ar), 8.11 (d, J ) 1.6 Hz, 0.5H, Ar),
8.4-8.6 (m, 1H, Ar); MS m/z 472 (M+, 17), 309 (37); Anal.
(C23H19Cl3N4O) C, H, N.
a]pyridin-3-yl)acetamide (35): IR (KBr): 1642 cm-1 1H
;
NMR (CDCl3) δ: 0.8-1.0 (m, 3H, CH3), 1.0-1.4 (m, 2H, CH2),
1.4-1.6 (m, 2H, CH2), 2.89 (s, 1.5H, CH3N), 2.92 (s, 1.5H,
CH3N), 3.1-3.2 (m, 1H, CH2N), 3.3-3.5 (m, 1H, CH2N), 4.06
(s, 1H, CH2CO), 4.09 (s, 1H, CH2CO), 7.29 (d, J ) 1.9 Hz, 1H,
Ar), 7.4-7.5 (m, 3H, Ar), 7.6-7.7 (m, 2H, Ar), 8.23 (d, J ) 1.9
Hz, 0.5H, Ar), 8.31 (d, J ) 1.9 Hz, 0.5H, Ar); MS m/z 389 (M+,
12), 275 (base); Anal. (C20H21Cl2N3O) C, H, N.
N-Methyl-N-phenyl-[2-(4-chlorophenyl)-6,8-dichloroim-
idazo[1,2-a]pyridin-3-yl]acetamide (36): IR (KBr): 1650
cm-1; 1H NMR (CDCl3) δ: 3.30 (s, 3H, CH3N), 3.85 (s, 2H, CH2-
CO), 7.1-7.2 (m, 2H, Ar), 7.3-7.4 (m, 6H, Ar), 7.4-7.6 (m,
2H, Ar), 8.2-8.3 (m, 1H, Ar); MS m/z 443 (M+, 18), 309 (base);
Anal. (C22H16Cl3N3O) C, H, N.
2-(6,8-dichloro-2-phenylimidazo[1,2-a]pyridin-3-yl)-N-
pyridin-4-ylacetamide (26): IR (KBr): 3240, 1704 cm-1; 1H
NMR (DMSO-d6) δ: 4.35 (s, 2H, CH2CO), 7.3-7.6 (m, 5H, Ar),
7.6-7.8 (m, 3H, Ar), 8.42 (d, J ) 6.3 Hz, 2H, Ar), 8.85 (d, J )
1.6 Hz, 1H, Ar); MS m/z 396 (M+, 11), 275 (base); Anal. (C20H14-
Cl2N4O) C, H, N.
N-Benzyl-[2-(4-chlorophenyl)-6,8-dichloroimidazo[1,2-
a]pyridin-3-yl]acetamide (30): IR (KBr): 3300, 1630 cm-1
;
1H NMR (DMSO-d6) δ: 3.90 (s, 2H, CH2CO), 4.32 (d, J ) 6.5
Hz, 2H, CH2-Ar), 7.1-7.4 (m, 8H, Ar), 7.76 (d, J ) 8.2 Hz,
2H, Ar), 8.37 (d, J ) 6.5 Hz, 1H, Ar), 8.5-8.6 (m, 1H, NH);
MS m/z 443 (M+, 10), 309 (base); Anal. (C22H16Cl3N3O) C, H,
N.
General Procedure for Preparation of (2-phenylimi-
dazo[1,2-a]pyridin-3-yl)acetamides 10, 11, 15, 16, 24-26,
30, and 37. Method C. To a stirred solution of the required
imidazo[1,2-a]pyridine-3-acetic acid 42 (1 mmol) in anhydrous
THF (20 mL) were added EEDQ (1.2 mmol) and the appropri-
ate amine and then TEA (1.3 mmol) dropwise. Stirring was
prolonged at room temperature for 6-12 h and then the
mixture poured into 20 mL of water and extracted with CHCl3
(3 × 30 mL). The organic layer was separated, washed with
water and brine, and dried over Na2SO4. Solvent was evapo-
rated under reduced pressure, and the resulting residue was
purified by silica gel column chromatography [light petroleum
ether/ethyl acetate 8/2 (v/v) as eluent)] to give the required
compounds. 10, 11, 15, 16, 24-26, 30, and 37. Physical data
of these compounds are summarized in Table 1.
N-Benzyl-N-n-butyl-(2-phenyl-6,8-dichloroimidazo[1,2-
a]pyridin-3-yl)acetamide (10): IR (KBr):1645 cm-1; 1H NMR
(CDCl3) δ: 0.79 (t, 1.35 H, CH3), 0.91 (t, 1.65H, CH3), 0.95-
1.15 (m, 0.9 H, CH2), 1.2-1.3 (m, 1.10 H, CH2), 1.4-1.6 (m,
2H, CH2), 3.08 (t, J ) 6.5 Hz, 1.1H, CH2), 3.43 (t, J ) 6.5 Hz,
0.9H, CH2), 4.01 (s, 0.9H, CH2CO), 4.13 (s, 1.1H, CH2CO), 4.41
(s, 0.9H, CH2-Ar), 4.58 (s, 1.1H, CH2-Ar), 6.9-9.0 (m, 12H,
Ar); MS m/z 465 (M+, 15), 275 (base); Anal. (C26H25N3Cl2O) C,
H, N.
N-n-Benzyl-N-methyl-(2-(4-chlorophenyl-6,8-dichloro-
imidazo[1,2-a]pyridin-3-yl)acetamide (37): IR (KBr): 1633
cm-1; 1H NMR (CDCl3) δ: 2.89 (s, 1.5H, CH3N), 3.05 (s, 1.5H,
CH3N), 4.05 (s, 1H, CH2CO), 4.08 (s, 1H, CH2CO), 4.45 (s, 1H,
CH2), 4.59 (s, 1H, CH2), 6.9-7.0 (m, 1H, Ar), 7.2-7.6 (m, 9H,
Ar), 8.13 (d, J ) 1.9 Hz, 0.5H, Ar), 8.18 (d, J ) 1.9 Hz, 0.5H,
Ar); MS m/z 457 (M+, 15), 309 (base); Anal. (C23H18Cl3 N3O)
C, H, N.
Preparation of N,N-Di-n-propyl-2-[2-(4-chlorophenyl)-
6,8-dichloroimidazo[1,2-a]pyridin-3-yl)]propanamide (31).
Method D. To a stirred solution of N,N-di-n-propyl-[2-(4-
chlorophenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)]aceta-
mide26 (0.2 g, 0.46 mmol) in DMF (10 mL) and cooled at 0 °C
(ice bath) was added NaH (20 mg, 0.83 mmol). After 10 min,
CH3I (0.1 g, 0.69 mmol) was dropwise added and stirring was
prolonged for further 30 min at room temperature. Then, the
reaction mixture was treated with 10 mL of water and the
organic phase extracted with CHCl3 (3 × 5 mL) and dried over
Na2SO4. Evaporation of the solvent gave a residue which
resulted the essentially pure amide 31: IR (KBr):1630 cm-1
;
1H NMR (CDCl3) δ: 0.31 (t, 3H, J ) 6.7 Hz, CH3), 0.71 (t, 3H,
J ) 6.7 Hz, CH3), 1.0-1.2 (m, 2H, CH2), 1.3-1.4 (m, 2H, CH2),
1.69 (d, J ) 7.0 Hz, 3H, CH3CH), 2.4-2.6 (m, 1H, CH2N), 2.6-
2.8 (m, 1H, CH2N), 2.9-3.0 (m, 1H, CH2N), 3.3-3.5 (m, 1H,
CH2N), 4.48 (q, J ) 7.0 Hz, 1H, CHCO), 7.28 (d, J ) 7.0 Hz,
1H, Ar), 7.46 (d, J ) 7.0 Hz, 2H, Ar), 7.64 (d, J ) 7.0 Hz, 2H,
Ar), 8.78 (d, J ) 7.0 Hz, 1H, Ar); MS m/z 451 (M+, 7), 323
(base); Anal. (C22H24Cl3N3O) C, H, N.
N-Benzyl-N-tert-butyl-[2-(4-chlorophenyl)-6,8-di-
chloroimidazo[1,2-a]pyridin-3-yl]acetamide (11): IR
1
(KBr): 1620 cm-1; H NMR (CDCl3) δ: 1.45 (s, 9H, C(CH3)3),
4.02 (s, 2H, CH2CO), 4.56 (s, 2H, CH2N), 7.05 (d, J ) 6.0 Hz,
2H, Ar), 7.2-7.6 (m, 6H, Ar), 7.46 (d, J ) 8.0 Hz, 2H, Ar),
8.19 (s, 1H, Ar); MS m/z 499 (M+, 4), 309 (70); Anal. (C26H24-
Cl3N3O) C, H, N.
In a similar way was prepared compound N-benzyl,N-
methyl-2-[2-(4-chlorophenyl-6,8-dichloroimidazo[1,2-a]-
pyridin-3-yl)]propanamide (33) starting from N-benzyl,N-
methyl-[2-(4-chlorophenyl)-6,8-dichloroimidazo[1,2-a]pyridin-
2-(4-Chlorophenyl)-6,8-dichloro-3-(2-oxo-2-pyrrolidin-
1-ylethyl)imidazo[1,2-a]pyridine (15): IR (KBr):1625 cm-1
;
3-yl)]acetamide (30): IR (KBr):1640 cm-1 1H NMR (CDCl3)
;
1H NMR (CDCl3) δ: 1.8-2.0 (m, 4H, CH2), 3.34 (t, J ) 6.5 Hz,
2H, CH2N), 3.49 (t, J ) 6.5 Hz, 2H, CH2N), 4.00 (s, 2H, CH2-
CO), 7.33 (d, J ) 1.8 Hz, 1H, Ar), 7.44 (d, J ) 8.3 Hz, 2H, Ar),
7.62 (d, J ) 8.3 Hz, 2H, Ar), 8.26 (d, J ) 1.8 Hz, 1H, Ar);
MS m/z 407 (M+, 19), 309 (base); Anal. (C19H16Cl3N3O) C, H,
N.
δ: 1.66 (d, J ) 7.1 Hz, 1.5H, CH3), 1.73 (d, J ) 7.1 Hz, 1.5H,
CH3), 2.40 (s, 1.5H, CH3N), 2.93 (s, 1.5H, CH3N), 4.00 (q, J )
17.6 Hz 1H, CHCO), 4.3-4.7(m, 2H, CH2), 6.3-6.4 (m, 1H,
Ar), 6.9-7.7 (m, 9H, Ar), 8.68 (d, J ) 1.8 Hz 0.5H, Ar), 8.75
(d, J ) 1.8 Hz 0.5H, Ar); MS m/z 471 (M+, 5), 323 (base); Anal.
(C24H20Cl3N3O) C, H, N.
2-Phenyl-6,8-dichloro-3-(2-oxo-2-pyrrolidin-1-ylethyl)-
Preparation of N-Cyclohexyl,N-methyl-2-[2-(4-chloro-
phenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)]propanamide
(32). Method E. A solution of N-cyclohexyl-[2-(4-chlorophen-
yl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)]acetamide 28 (0.7 g,
1.6 mmol) in DMF (5 mL) maintained at room temperature
and under stirring was treated with a solution of potassium
tert-butoxide (0.36 g, 2 mmol), and after 10 min to the resulting
mixture was dropwise added CH3I (0.3 g, 2.1 mmol). Stirring
was prolonged for 30 min and then the reaction mixture poured
with caution into 10 mL of water. The organic phase was
extracted with CHCl3 (3 × 5 mL), dried (Na2SO4), and
evaporated under reduced pressure. The resulting residue was
1
imidazo[1,2-a]pyridine (16): IR (KBr):1628 cm-1; H NMR
(CDCl3) δ: 1.8-2.0 (m, 4H, CH2), 3.29 (t, J ) 6.5 Hz, 2H,
CH2N), 3.48 (t, J ) 6.5 Hz, 2H, CH2N), 4.03 (s, 2H, CH2CO),
7.31 (d, J ) 1.8 Hz, 1H, Ar), 7.4-7.5 (m, 3H, Ar), 7.66 (d, J )
6.7 Hz, 2H, Ar), 8.31 (d, J ) 1.8 Hz, 1H, Ar); MS m/z 373 (M+,
17), 275 (base); Anal. (C19H17Cl2N3O) C, H, N.
2-[6,8-Dichloro-2-phenylimidazo[1,2-a]pyridin-3-yl]-N-
methyl-N-(2-pyridin-3-ylethyl)acetamide (24): IR (KBr):
1
1645 cm-1; H NMR (CDCl3) δ: 2.81 (s, 1.5H, CH3), 2.94 (s,
1.5H, CH3), 2.9-3.0 (m, 2H, CH2), 3.7-3.8 (m, 2H, CH2), 3.91
(s, 1H, CH2CO), 3.96 (s, 1H, CH2CO), 7.0-7.7 (m, 9H, Ar), 7.69
(s, 0.5H, Ar), 8.13 (s, 0.5H, Ar), 8.3-8.6 (m, 1H, Ar); MS m/z
438 (M+, 26), 275 (42); Anal. (C23H20Cl2N4O) C, H, N.
the essentially pure compound 32: IR (KBr):1635 cm-1
;
1H
NMR (CDCl3) δ: 1.1-1.6 (m, 10H, CH2), 1.6-1.8 (m, 3H, CH3),

Peripheral Benzodiazepine Receptors Ligands
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1 303
2.31 (s, 1.2H, CH3N), 2.70 (s, 1.8H, CH3N), 2.9-3.1 (m, 0.6H,
CHN), 4.3-4.4 (m, 0.4H, CHN), 4.45 (q, J ) 7.1 Hz 0.4H,
CHCO), 4.60 (q, J ) 7.1 Hz, 0,6H, CHCO), 7.2-7.3 (m, 1H,
Ar), 7.4-7.8 (m, 4H, Ar), 8.68 (d, J ) 1.6 Hz 0.4H, Ar), 8.76
(d, J ) 1.6 Hz 0.6H, Ar); MS m/z 463 (M+, 7), 323 (base); Anal.
(C23H24Cl3N3O) C, H, N.
µL, comprising 50 µL of membrane suspension (0.15-0.20 mg
protein), 50 µL of [3H]PK 11195 (85.5 Ci/mmol, New England
Nuclear; final assay concentration 1M), and 400 µL of PBS
buffer (pH 7.4 at 25 °C). Incubation (25 °C) was initiated by
the addition of membranes and were terminated 90 min later
by rapid filtration through glass-fiber filter strips (Wathaman
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 unlabeled PK 11195
(Sigma).
Preparation of N-n-Butyl,N-methyl-[2-(4-chlorophen-
yl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)]acetamide (34).
Method F. To a solution of N-n-butyl-[2-(4-chlorophenyl)-6,8-
dichloroimidazo[1,2-a]pyridin-3-yl)]acetamide 27 (0.2 g, 0.49
mmol) in anydrous THF (3 mL), cooled at -45 °C with a liquid
nitrogen bath, was dropwise added a solution of n-BuLi 2.5 M
in hexane (200 µL, 0.5 mmol), and the mixture was stirred for
10 min.. Then, methyl triflate (0.1 g, 1.4 mmol) was slowly
added, and stirring was prolonged for 30 min. The progress of
reaction was monitored by HPLC analysis. Then, to the
mixture was added water (5 mL), and the organic phase was
extracted with CHCl3 (3 × 10 mL). The organic layer was
separated and dried over Na2SO4. Solvent was evaporated
under reduced pressure, and the resulting residue was purified
by silica gel column chromatography [CHCl3/acetone 97/3
(v/v) as eluent)] to give the required compound 34.
High-performance liquid chromatography (HPLC) analyses
were performed with a Waters (Waters Corp., Milford, MA)
600 pump equipped with a Waters 990 variable wavelength
UV detector. HPLC mobile phase was prepared from HPLC-
grade methanol. For analysis, a reversed phase Simmetry C18
(25 cm × 4.6 mm; 5 µm particles) column in conjunction with
a precolumn module was eluted with methanol:deionized water
(75:25) (injection volume 20 µL) in isocratic mode. The flow
rate of 1 mL/min was maintained, and the column effluent
was monitored continuously at 254 nm.
Functional in Vitro Studies. Electrophysiological Stud-
ies Using Xenopus Oocytes. Complementary DNAs encoding
the human R1, â2, 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 sterile distilled water, divided into
portions, and stored at -20 °C until used for injection. Stage
V and VI oocytes were manually isolated from sections of
Xenopus laevis ovary, placed in modified Barth’s saline (MBS)
containing 88 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 collage-
nase 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, â2, 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 micropipet (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 record-
ings 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 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 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 to 10 s were required
to reach equilibrium in the recording chamber). Intervals of
5-10 min were allowed between drug applications. Modulation
of GABA-evoked Cl- currents by drugs is expressed as
percentage change, [(I′/I) - 1] × 100%, where I is the average
of control responses obtained before drug application and after
drug washout, and I′ is the average of the agonist-induced
responses obtained from the same cell in the presence of drug.
Functional in Vivo Studies. Effect of Compounds 2,
3, 7, 14, 18, 34, and 35 on Plasma and Brain Concentra-
tion of Neuroactive Steroids. Drug Administration.
Compounds 2, 3, 7, 14, 18, 34, and 35 were dissolved by adding
three drops of Tween 80 per 5 mL of saline and were
administered intraperitoneally in a volume of 0.3 mL per 100
g of body mass.
Extraction and Assay of Steroids. Rats were killed either
with a guillotine (for measurement of plasma steroids) or by
focused microwave irradiation (70 W/cm2 for 4 s) of the head
(for measurement of brain steroids). This latter procedure
results in a virtually instantaneous inactivation of brain
enzymes,38 thus minimizing postmortem steroid metabolism.
The brain was rapidly (<1 min) removed from the skull, and
the cerebral cortices were dissected and then frozen at -20
°C until steroid extraction. Steroids were extracted and
purified as previously described.39 In brief, steroids present
in tissue homogenates [600 mg of cortex in 4 mL of phosphate-
buffered saline (pH 7.0)] were extracted three times with ethyl
In a similar way were prepared compounds 35 and 36.
Biological Methods. Materials. Male Sprague-Dawley
CD rats at 30 days of age, immediately after weaning, were
housed for 30 days either in groups of eight per cage. They
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 the experiment. Animal care and
handling throughout the experimental procedures were in
accordance with the European Communities Council Directive
of 24 November 1986 (86/609/EEC). All experiments were
performed between 10:00 a.m. 1:00 p.m. CB 34 was dissolved
by adding one drop of Tween 80 per 5 mL of physiological
saline and was administered intraperitoneally in a volume of
0.3 mL per 100 g of body mass.
In Vitro Receptor Binding Assays. [3H]Flunitrazepam
Binding. Cerebral cortex was homogenized with a Polytron
PT 10 in 50 volumes of ice-cold 50 mM Tris-HCl (pH 7.4), and
the homogenate was centrifuged twice at 20 000g 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, compris-
ing 400 µL of membrane suspension (0.4 to 0.5 mg of protein),
400 µL of Tris-HCl buffer, 100 µL of [3H]-flunitrazepam (74
Ci mmol-1; New England Nuclear), 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 quan-
titated by liquid scintillation spectrometry. Nonspecific binding
was determined as binding in the presence of 5 µM diazepam
and represented about 10% of total binding.
[3H]PK 11195 Binding. After killing, the brain was rapidly
removed from rats, the cerebral cortex was dissected and all
tissues were stored at -80 °C until assayed. The tissues were
thawed and homogenized in 50 volumes of Dulbecco’s phos-
phate-buffered saline (PBS) pH 7.4 at 4 °C with a Polytron
PT 10 (setting 5, for 20 s). The homogenate was centrifuged
at 40 000g for 30 min, and the resulting pellet was resus-
pended in 50 volumes of PBS and recentrifuged. The new pellet
was resuspended in 10 volumes of PBS and used for the assay.
[3H]PK 11195 binding was determined in a final volume of 500

304 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 1
Trapani et al.
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acetate, and the combined organic phases were dried under
vacuum. The resulting residue was dissolved in 5 mL of
n-hexane and applied to a SepPak silica cartridge (Waters),
and components were eluted with n-hexane and 2-propanol
(7:3, vol/vol). Steroids were separated and further purified by
HPLC on a 5-µm Lichrosorb-diol column (250 by 4 mm;
Phenomenex) with a discontinuous gradient of 2-propanol (0
to 30%) in n-hexane. Progesterone, which coelutes with
cholesterol, was further purified by washing the corresponding
dried HPLC fractions first with 200 µL of dimethyl sulfoxide
and then with 400 µL of water. Progesterone was extracted
from the aqueous phase twice with 1.5 mL volumes of
n-hexane. The recovery (70 to 80%) of steroids through the
extraction and purification procedures was monitored by
adding a trace amount (6000 to 8000 cpm; 20 to 80 Ci/mmol)
of tritiated standard to the brain homogenate.
Blood was collected from the trunk of killed rats into
heparinized tubes and centrifuged at 900g for 20 min at room
temperature. The resulting plasma was frozen (-80 °C) until
assayed for steroids. Steroids were extracted from plasma
three times with 1.5 mL of ethyl acetate.
Steroids from both brain tissue and plasma were quantified
by radioimmunoassay as previously described39,40 with specific
antibodies to progesterone (ICN, Costa Mesa, CA), 3R,5R-
THDOC, and 3R,5R-THPROG (generated in rabbits and sheep,
respectively, and characterized as previously described).40
Statistics. Data are presented as means ( SEM and were
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Acknowledgment. This work was supported by a
grant from Ministero dell′Universita` e della Ricerca
Scientifica e Tecnologica (COFIN 2002 MIUR). We
thank Mr Giovanni Dipinto for skilful technical as-
sistance in recording mass spectra and determining
microanalytical data. Thanks are also due to Dr. Kunal
Roy (Jadavpur University, Calcutta, India) for providing
us a reprint of their QSAR work on imidazopyridines
(ref 32).
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Supporting Information Available: Microanalytical data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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