2-[(4-Phenylpiperazin-1-yl)methyl]imidazo(di)azines as selective D 4-ligands. Induction of penile erection by 2-[4-(2-methoxyphenyl) piperazin-1-ylmethyl]imidazo[1,2-a]pyridine (PIP3EA), a potent and selective D4 partial agonist

Article abstract of DOI:10.1021/jm060166w

A series of novel 2-[(4-phenylpiperazin-1-yl)methyl]imidazoazines and aza-analogues were prepared and screened at selected dopamine, serotonin, and adrenergic receptor subtypes. 2-Substituted imidazopyridines and pyridazines presented high affinities and

Full text of DOI:10.1021/jm060166w

3938
J. Med. Chem. 2006, 49, 3938-3947
2-[(4-Phenylpiperazin-1-yl)methyl]imidazo(di)azines as Selective D4-Ligands. Induction of Penile
Erection by 2-[4-(2-Methoxyphenyl)piperazin-1-ylmethyl]imidazo[1,2-a]pyridine (PIP3EA), a
Potent and Selective D4 Partial Agonist
Ce´cile Enguehard-Gueiffier,† Harald Hu¨bner,§ Ahmed El Hakmaoui,| Hassan Allouchi,‡ Peter Gmeiner,§ Antonio Argiolas,
Maria Rosaria Melis, and Alain Gueiffier*,†
Laboratoire de Chimie The´rapeutique, Laboratoire de Chimie Physique EA 3857, Faculte´ de Pharmacie, 31 aVenue Monge,
F-37200 Tours, France, Department of Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander UniVersity, Schuhstrasse 19,
D-91052 Erlangen, Germany, Department of Chemistry, UniVersity Hassan II Mohammadia, Faculty of Sciences and Techniques,
BP 146 Mohammadia 20800, Morocco, and Bernard B. Brodie Department of Neurosciences, UniVersity of Cagliari,
S.P. Sestu-Monserrato KM 0.700, 09042 Monserrato, Italy
ReceiVed February 14, 2006
A series of novel 2-[(4-phenylpiperazin-1-yl)methyl]imidazoazines and aza-analogues were prepared and
screened at selected dopamine, serotonin, and adrenergic receptor subtypes. 2-Substituted imidazopyridines
and pyridazines presented high affinities and selectivities for D4 dopamine receptors. Whereas functional
experiments indicated neutral antagonists or weak partial agonist effects for most of the target compounds,
the 2-methoxyphenyl substituted 2-piperazinylmethylimidazopyridine 3c (PIP3EA) displayed substantial
agonist efficacy in mitogenesis experiments and GTPγS binding tests, resulting in EC50 values of 3.0 (46%)
and 4.5 nM (57%), respectively. Our D4 agonist 3c induced penile erection in vivo when administered to
rats. This effect was inhibited by L-745,870 a D4 selective antagonist, confirming the mechanistic pathway.
Chart 1. Structure of Dopaminergic Agents Discussed in the
Text
Introduction
Dopamine receptors are divided into D1-like and D2-like
families.1 From the recent progress in molecular cloning
techniques, the existence of subtypes within these families was
described. The D1-like family comprises D1 and D5 subtypes,
whereas the D2-like family consists of D2, D3, and D4 receptors.2
In a recent review, Missale and coworkers reported the structure
and function of these different subtypes in peripheral tissues as
well as in the brain.3 Nevertheless, the D4 receptor remains a
controversial target,4 and the therapeutic potential of D4 ligands
in various psychiatric or neurological disorders, such as
schizophrenia, ADHD (attention deficit hyperactivity disorders),
mood disorders, and Parkinson’s disease, is still under
discussion.5-7 Starting from the atypical antipsychotic clozapine,
which presents a higher affinity for D4 than for D2 receptors,8
several selective dopamine D4 receptor ligands were synthe-
sized.9 SAR studies on D4 antagonists10 and D4 agonists11 have
been recently reviewed indicating an N-arylpiperazinyl group
as a common scaffold.
In recent years, it was demonstrated that D4 agonists, such
as ABT-724,12 PD-168077,13 CP-226,269,14 and A-41299715
(Chart 1), induce penile erection in rats. More recently, using
PD-168077, the potential therapeutic use of D4 agonists in
preventing dermatoses, which are characterized by epidermal
proliferation and epidermal barrier dysfunction, was demon-
strated.16 Unfortunately from these compounds, PD-168077 was
demonstrated to be unstable in acidic solution, excluding oral
administration.17 Furthermore, Moreland and coworkers showed
a poor selectivity of this compound toward the hR1 receptor
(KihR1/KihD4.4 ) 8).18
In our work on selective dopamine ligands, we reported that
azaindoles bearing an arylpiperazinylmethyl moiety at the
3-position of the heterocyclic moiety (e.g., FAUC 113) show
high affinity for dopamine D4 receptors.19 The comparison of
the molecular electrostatic isopotential maps derived by ab initio
molecular orbital calculations at the RFH (restricted Hartree-
* Corresponding author. Tel: +33-247-367-138. Fax: +33-247-367-
288. E-mail: alain.gueiffier@univ-tours.fr
† Laboratoire de Chimie The´rapeutique, Faculte´ de Pharmacie.
‡ Laboratoire de Chimie Physique EA 3857, Faculte´ de Pharmacie.
§ Friedrich-Alexander University.
| University Hassan II Mohammadia.
University of Cagliari.
10.1021/jm060166w CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/27/2006

2-[(4-Phenylpiperazin-1-yl)methyl]imidazo(di)azines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3939
Scheme 2a
Scheme 1a
a Reagents and conditions: (a) piperidine or N-methylpiperazine, reflux
24 h.
we have recently decided to study the application of new
metallo-catalyzed methodologies in this series.25,26 These meth-
ods allow the introduction of various functionalities, and in
regard to FAUC 299, we were first interested in the synthesis
of cyano derivative 8a. Indeed, to the best of our knowledge,
the substitution using sodium or potassium cyanide on 6- or
8-halogenoimidazopyridine was inefficient. Thus, we focused
our interest on a copper-based method described by Buchwald’s
group, starting from the bromo derivative through a one-step
exchange with iodine and followed by cyanation using sodium
cyanide.27 Because iodinated starting material 7d was available,
the reaction was conducted using sodium cyanide with CuI as
the catalyst and N,N′-dimethyl-1,2-diaminoethane as the ligand
in toluene at 112 °C. In these conditions, nitrile 8a was obtained
in 70% yield (Scheme 4).
Lately, we reported that aminations using copper or palladium
catalysts are efficient on 6- or 8-halogenoimidazo[1,2-a]-
pyridine.28,29 In these previously described conditions, 7d was
allowed to react with morpholine under copper catalysis using
ethyleneglycol as the ligand in the presence of potassium
phosphate in 2-propanol, leading to morpholino derivative 8b
in 72% yield.
We then focused on the introduction of an azole group.
Buchwald and coworkers previously reported a methodology
for the N-arylation of indole30 and various nitrogen hetero-
cycles,31 which we extended to the functionalization of the
6-position of the imidazo[1,2-a]pyridine.32 Thus, in the usual
conditions (e.g., CuI, N,N′-dimethyl-1,2-diaminoethane, K3PO4
in toluene), pyrazolyl compound 8c was obtained from 7d in
42% yield (not optimized). Then, to evaluate the effect of the
substitution pattern, the pyrazolyl group was introduced into
position 5. Indeed, it was demonstrated in our laboratory that
the reaction of the 6-halogenoimidazo[1,2-a]pyridine with an
azole in DMF in the presence of cesium carbonate gives the
cine substitution in position 5.32 In these conditions, 7a led to
5-pyrazolyl compound 8d in only 27% yield, mixed with 50%
of the remaining starting material.
a Reagents and conditions: (a) substituted phenylpiperazine (1 equiv),
Na2CO3 (1 equiv), EtOH, reflux 3 h.
Fock) level of theory gave an explanation for the extraordinary
D4 selectivity of the aza-indoles L-745,870 and FAUC 113. Our
very recent studies led us to the 2-substituted indoles FAUC
299 and FAUC 319 and 7a-aza-analogues displaying even higher
affinities and subtype selectivities compared to those of the
respective regioisomers.20 The structural manipulations allowed
us to perform a fine-tuned ligand efficacy.21 In addition, further
3-substituted imidazo[1,2-a]pyridine analogues of type 1 have
been investigated very recently.22 Thus, to further increase D4
ligand efficacy and selectivity toward the adrenergic subtype
R1, suitable structural modifications should be performed on
the imidazoazine and diazine scaffolds. The evaluation of
dopamine D4 receptor affinities followed by binding studies,
functional experiments, and in vivo investigations of the
synthesized compounds should then lead to a final product of
potential interest for the treatment of erectile dysfunction.
Results and Discussion
Chemistry. In the first part of this study, four suitably
functionalized imidazoazine and diazines were obtained with
various phenyl substituents introduced in the imidazo[1,2-a]-
pyridine and [1,2-b]pyridazine series. Thus, the suitable 2-chlo-
romethylimidazoazine and diazines derivatives 2a-d were
refluxed with N-(4-fluoro, 3,4-dichloro, 2-methoxy or 4-chlo-
rophenyl)piperazine in ethanol in the presence of sodium
carbonate to give 3a-j in good yields (Scheme 1).
Then, the nature of the six-membered ring substitution was
investigated. It is well established that chlorine in position 6 of
the imidazo[1,2-b]pyridazine series easily gives nucleophilic
substitutions.23,24 In our case, the chlorine displacement was
performed on 3f using piperidine or 4-methylpiperazine, leading
to the attempted compounds 4a,b in good yields (Scheme 2).
In the same way, the six-membered ring substitution was
investigated in the imidazo[1,2-a]pyridine series. To this end,
the 6- or 8-halogenated starting materials 6a-d were prepared
by the condensation of 2-amino-3 or 5-halopyridines 5a-d with
1,3-dichloroacetone. Then, the reaction with N-(2-methoxyphe-
nyl)piperazine led to the attempted compounds 7a-d. Nucleo-
philic substitution on 6a using N-(3,4-dichlorophenyl)piperazine
gave 7e (Scheme 3).
Finally, we have recently reported that the Suzuki-type cross
coupling reaction occurs on 8-halogenoimidazo[1,2-a]pyridine;33
therefore, the reaction of 7c with phenylboronic acid gave
compound 8e in 54% yield (not optimized) using tetrakis-
(triphenylphosphine)palladium as the catalyst and sodium
hydroxide as the base in DME/water. The crystal data for 3a
could be obtained.
Biological Activity. Receptor binding profiles of the syn-
thesized compounds were determined in vitro by measuring their
ability to compete with [3H]spiperone for the cloned human
dopamine receptor subtypes D2long, D2short, D3, and D4.4 stably
expressed in Chinese hamster ovary cells (CHO).34-38 D1
receptor affinity was measured by employing porcine striatal
membranes and the D1 selective radioligand [3H]SCH 23390.39
The investigation of serotonin 5-HT1A and 5-HT2 affinities was
performed as described in the literature.40
Because nucleophilic substitution reactions are usually unsuc-
cessful in positions 6 and 8 of the imidazo[1,2-a]pyridine series,

3940 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Enguehard-Gueiffier et al.
Scheme 3a
a Reagents and conditions: (a) 1,3-dichloroacetone, EtOH, reflux; (b) substituted arylpiperazine (1 equiv), Na2CO3 (1 or 2 equiv for chlorhydrates), EtOH,
reflux 3h.
Scheme 4a
a Reagents and conditions: (a) KCN (1.2 equiv), CuI (10 mol %), N,N′-dimethyl-1,2-diaminoethane (1 equiv), toluene, 112 °C; (b) morpholine (1.1
equiv), CuI (15 mol %), ethyleneglycol (2 equiv), K3PO4 (2 equiv), iPrOH, 85 °C; (c) pyrazole (1 equiv), CuI (5 mol %), N,N′-dimethyl-1,2-diaminoethane
(15 mol %), K3PO4 (2.2 equiv), toluene, 112 °C; (d) pyrazole (1 equiv), Cs2CO3 (2.1 equiv), DMF, 112 °C; (e) phenylboronic acid (1 equiv), Pd(PPh3)4 (5
mol %), NaOH (2 equiv), DME/H2O.
The Ki values of the test compounds are listed in Table 1 in
comparison to those for FAUC 113, FAUC 299, and clozapine.
The first general point that emerged from the data is a low D1
affinity for all compounds investigated. A comparison of the
4-fluorophenyl derivatives 3a, 3e, 3i, and 3j shows that the
number and position of nitrogen atoms within the heteroarene
moiety strongly influences D4.4 receptor recognition. Heteroatom
exchange in positions 7 or 8 of the bicyclic heterocycle is
detrimental for D4.4 receptor affinity as previously observed for
3-substituted derivatives.19 The highest affinities for the D4
binding site were determined for the imidazo[1,2-a]pyridine and
imidazo[1,2-b]pyridazine series with Ki values in the range of
1.2 to 7.0 nM for test compounds 3b-h, although they present
micromolar affinities at D3. With respect to the phenyl sub-
stituent, a chlorine atom appeared to be superior to a fluorine
atom in the para position as indicated by Ki values of 7 and 43
nM in the imidazo[1,2-a]pyridine series and 2 and 4 nM in the
imidazo[1,2-b]pyridazine series. A second chlorine atom in the
meta position even increased the D4.4 affinity with Ki values of
1.2-1.3 nM in both series (compounds 3b and 3f). A 2-methoxy

2-[(4-Phenylpiperazin-1-yl)methyl]imidazo(di)azines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3941
Table 1. Receptor Binding Data of Different 2-[4-(Phenylpiperazin-1-yl)methyl]imidazoazines and Diazines Derivatives Employing Bovine D1 and
Human D2, D3, and D4.4 Receptorsa
[3H]SCH23390
D1
[3H]spiperone
D2 short
[3H]8-OH-DPAT [3H]ketanserin [3H]prazosin
compd
3a
3b
3c
3d
3e
3f
D2 Long
D3
D4.4
5-HT1A
5-HT2
R1
8400 ( 1100
2300 ( 50
21000 ( 4000 19000 ( 3500 3100 ( 750
44 ( 1.5
3600 ( 650
2300 ( 500
2200 ( 1000
1700 ( 730
nd
860 ( 25
260 ( 40
3200 ( 730
990 ( 120
2700 ( 650
930 ( 170
1700 ( 170
1.4 ( 0.14
2.8 ( 1.1
7.0 ( 1.3
4.0 ( 0.85
1.3 ( 0.51
1.6 ( 0.49
2.0 ( 0.74
1200 ( 100
61 ( 7.3
20000 ( 0
3900 ( 2300
5900 ( 1400
98 ( 4.5
6700 ( 1100
7000 ( 0
15000 ( 2500 16000 ( 3500 8800 ( 2300
1200 ( 100
60 ( 6.5
9900 ( 1200
1100 ( 100
96 ( 4.5
12000 ( 3400 5600 (3400
670 ( 55
79 ( 3.0
ndb
nd
1500 ( 250
7700 ( 550
5500 ( 350
15000 ( 3000
1500 ( 150
1400 ( 100
7700 ( 950
360 ( 97
24 ( 0.33
390 ( 90
nd
1200 ( 540
190 ( 23
3g
3h
3i
3400 ( 100
64 ( 3.6
12000 ( 1000 8200 ( 150
64000 ( 3000 68000 ( 6000 42000 ( 3000 500 ( 75
910 ( 90
nd
380 ( 55
nd
3j
47000 ( 22000 55000 ( 5500 49000 ( 5000 60000 ( 2300 2800 (220
2200 ( 150
13000 ( 11000 8000 ( 800
nd
nd
nd
4a
4b
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
1100 ( 210
710 ( 65
7800 ( 1100
140 ( 15
72 ( 6.0
150 ( 25
97 ( 14
850 ( 130
880 (120
800 ( 20
120 ( 20
1900 ( 640
nd
nd
nd
nd
nd
nd
7100 ( 100
6400 ( 800
5600 ( 1900
5900 ( 200
2500 ( 0.0
130 ( 10
73 ( 9.0
240 ( 25
88 ( 12
0.25 ( 0.040 nd
nd
nd
nd
nd
nd
nd
600 ( 25
0.33 ( 0.040 nd
670 ( 110
470 ( 55
0.99 ( 0.21
nd
0.53 ( 0.050 nd
nd
nd
nd
nd
nd
nd
nd
nd
3500 ( 100
3700 ( 450
2700 ( 150
100 ( 17
280 ( 5.0
280 ( 5.0
28 ( 5.0
1000 ( 180
3500 ( 1200
2100 ( 500
2400 ( 200
1000 ( 200
390 ( 110
960 ( 45
0.99 ( 0.12
1.3 ( 0.27
2.3 ( 0.23
1.6 ( 0.32
nd
nd
nd
nd
40000 ( 10000 4200 ( 750
6400 ( 1200
8700 ( 50
27000 ( 0.0
2900 ( 100
210 ( 20
530 ( 10
590 ( 60
49 ( 10
41 ( 1.5
3200 ( 400
nd
nd
0.94 ( 0.040 nd
nd
nd
nd
nd
0.84 ( 0.13
16 ( 0.50
3.6 ( 0.87
nd
clozapine 420 ( 50
28 ( 0.50
4300 ( 650
nd
nd
nd
FAUC
113
12000 ( 500
5000 ( 650
1400 ( 130
380 ( 140
240 ( 5.0
FAUC
299
FAUC
319
13000 ( 500
8600 ( 2400
3100 ( 550
290 ( 30
1700 ( 220
0.52 ( 0.050 nd
nd
nd
nd
nd
28000 ( 6500 19000 ( 4000 15000 ( 2000 1.0 ( 0.057 nd
a The Ki values (nM) are the means of 2-5 independent experiments each carried out in triplicate. b nd: values not determined.
group is also well tolerated with Ki values of 2.8 nM for 3c
and 1.6 nM for 3g. In terms of selectivity, with the exception
of 3e (KihD3/KihD4.4 ) 72), the 4-fluoro, 4-chloro, and 3,4-
dichloro compounds presented high selectivities for D4R toward
D1, D2L, D2S, and D3 receptors (KiD/KihD4.4 >440). Changing
to the 2-methoxy substitution gave a slightly decreased selectiv-
ity for imidazopyridine 3c toward D2R (KihD2/KihD4.4 ) 332
for D2S and 353 for D2L) and an even more significant effect
for 3g (KihD2/KihD4.4 ) 49 for D2S and 60 for D2L). Finally, a
cyclic amine (piperidine 4a and N-methylpiperazine 4b) in the
6-position of the imidazo[1,2-b]pyridazine dramatically in-
creased the Ki values toward D4.4 receptors.
We evaluated the 5-HT1A, 5-HT2, and R1 recognition for
compounds 3a-d and 3f-h with the aid of [3H]-8-OH-DPAT,
[3H]ketanserin, and [3H]prazosin labeled porcine brain homo-
genate, respectively. With the exception of 3g, which showed
a good affinity for the 5-HT1A receptor (Ki ) 24 nM, Kip5-
HT1A/KihD4.4 ) 15), a high selectivity was observed for all target
compounds. Nevertheless, there is clear evidence for a loss of
selectivity toward 5-HT1A in the imidazo[1,2-b]pyridazine series.
Interestingly, only moderate R1 affinities (Ki ) 60-260 nM)
were observed. Thus, the very potent D4 ligands turned out to
also display also substantial selectivity over R1.
From 7a-d, iodine or bromine appeared to be well tolerated
in positions 6 and 8 giving derivatives displaying the highest
affinities (Ki < 1 nM). A comparison between 7a and 7e,
confirmed a decrease of the selectivity toward D2 receptors
induced by the presence of 2-methoxy substitution. Introducing
a cyano in position 8 gave compound 8a with high affinity (Ki
) 1.3 nM) and an interestingly recovered selectivity toward
D2L (KiD2L/KiD4.4 ) 3230) and D2S (KiD2S/KiD4.4 ) 2076).
Changing to a phenyl group enhanced the affinity when a loss
of specificity toward D2 was noted (KiD2S/KiD4 ) 33). The same
observation was made when introducing a morpholine or a
pyrazol-1-yl group in position 8. When the pyrazolyl group was
introduced in position 5, the affinity was conserved (Ki ) 0.94
nM), and a good selectivity toward hD2S was noted (KihD2S/
KihD4 ) 300).
Ligand efficacy of selected compounds was determined by
mitogenesis assays, measuring the rate of [3H]thymidine incor-
20
poration into growing CHO10001 cells stably expressing D4.2
.
Intrinsic activity was quantified by the determination of the
effective concentration (EC50) of a test compound and by
comparing the maximal effect to that of a full agonist (Table
2).39,40 The tested compounds (3b,d, 3f-h, 7a,b, and 7d,e) were
shown to be complete antagonists, whereas 3c appeared to be
a partial agonist with an EC50 value of 3.0 nM and an intrinsic
effect of 46% compared to that of the nonselective full agonist
quinpirole. To corroborate the functional activity of the test
compounds using a second functional experiment, a [35S]GTPγS
binding assay was realized. Employing membrane preparations
from human D4.4 receptor expressing CHO cells, the dose
dependent stimulation of [35S]GTPγS binding was determined
to distinguish between agonist and antagonist properties and to
measure the potency and efficacy at the D4 receptor.41,42 Using
this assay, agonist efficacy of target compound 3c was confirmed
revealing an EC50 value of 4.5 nM and an intrinsic activity of
57% when 3d appeared to be a weak agonist (18%).
In addition, we investigated the affinity of the most interesting
compound 3c to the agonist binding site of the D2 receptor.
Using the agonist radioligand [3H]-7-OH-DPAT for competition
experiments with human D2long and native porcine D2 receptor-
containing membranes, the Ki values for 3c of 110 and 400 nM,
respectively, were measured. Although these data may indicate
the weak agonist effect of 3c at D2 receptors, a detailed analysis
of the binding curves derived from competition experiments with
the antagonist [3H]spiperone did not give any evidence for a
biphasic curve shape, which should be expected when measuring

3942 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Enguehard-Gueiffier et al.
Table 2. Intrinsic Activities of 3b-d, 3f-h, 7a, b, d, and e as Well as
Reference Compound Quinpirole Derived from the Stimulating Effect on
the Mitogenesis of D4 Receptor-Expressing Cells and the D4 Receptor
Mediated Binding of [35S]GTPγS
[3H]thymidine
[35S]GTP
uptake (mitogenesis)a
S bindingb
EC50
intrinsic effect
(% quinpirole)d
EC50
intrinsic effect
entry
(nM)c
(nM)e
(% quinpirole)f
3b
3c
3d
3f
3g
3h
7a
7b
7d
7e
0
46
6
0
0
0
0
10
0
0
ndg
4.5
2.6
nd
nd
nd
nd
nd
nd
nd
49
nd
57
18
nd
nd
nd
nd
nd
nd
nd
100
3.0
1.8
Figure 1. Effect of compound 3c, administered by s.c. or icv injections,
on penile erection in male rats: comparison with PD-186077. Com-
pound 3c and PD 168077 were dissolved in ethanol and diluted with
distilled water to a final 0.3% ethanol concentration and injected s.cly
in a volume of 0.2 mL/rat and icvly in a volume of 10 µL/rat. After
treatment, the rats were individually placed in Plexiglas cages (30 ×
30 × 30 cm3) and observed for 60 min to count penile erection episodes.
The values are means ( SEM of 6 rats per group. *P < 0.001 with
respect to vehicle-treated rats (compound 3c or PD 168077 ) 0).
0.52
1.4
quinpirole
100
a Determined with CHO10001 cells stably expressing the human D4.2
receptor. b Determined with CHO K1 cells stably expressing the human
D
an agonist compound. To prove these findings, we performed
a functional experiment by the determination of the agonist
stimulated [35S]GTPγS binding at the D2short receptor; 3c was
not able to stimulate the receptor up to a concentration of 10
µM.
4.4 receptor. c The EC50 values were derived from a mean curve of 2-11
independent experiments. d The rate of incorporation of [3H]thymidine as
evidence for mitogenesis activity relative to the maximal effect of the full
agonist quinpirole ()100%) used as a reference. e The EC50 values were
derived from a mean curve of three or four independent experiments.
f Maximum binding of [35S]GTPγS induced by receptor activation at 1 µM
relative to the effect of the reference agonist quinpirole ()100%) (at 10
µM) derived from six independent experiments. g nd: values not determined.
Because of the interest in recent studies on D4 agonists in
the treatment of erectile dysfunction,12,13,15,43 compound 3c was
then tested in rats to study its ability to promote erection after
systemic (subcutaneous, s.c.) or intracerebroventricular (icv)
administration. When given subcutaneously (s.cly), 3c induced
penile erection with an inverted U dose-response curve (Figure
1). The minimal effective dose was 5 µg/kg of body weight,
which induced penile erection in 60% of the treated rats and
increased penile erection episodes from 0.3 ( 0.02 to 1.02 (
0.05. The maximal effect was obtained with 20 µg/kg, which
induced penile erection in all treated rats and increased penile
erection episodes to 2.69 ( 0.43. A much lower response was
seen with doses of 30 µg/kg or higher. Similar results were
found with an icv injection of compound 3c. The minimal
effective dose was 0.5 µg/rat, which increased penile erection
episodes from 0.14 ( 0.02 to 1.12 ( 0.32, whereas the maximal
response (2.52 ( 0.61) was found with the dose of 1 µg/rat. A
much lower response was found with the dose of 5 µg/rat or
higher (Figure 1). A comparison of the dose-response curves
of 3c with those of PD-168077, another D4 agonist that induces
penile erection in rats,13,15 given s.cly or intracerebroventricularly
(icvly) revealed that 3c was more effective than PD-168077
because compound 3c induces a higher number of episodes of
penile erections/rat, and higher doses of PD-168077 were
necessary to induce penile erection when given either s.cly or
icvly (Figure 1). Perhaps more important for this work, the pro-
erectile effect of compound 3c given s.cly (20 µg/kg) or icvly
(1 µg/rat) was reduced by more than 80%, not only by clozapine
and haloperidol, which block all dopamine receptor subtypes,
but also by L-745,870, a selective D4 antagonist,10 all given
intraperitoneally (i.p.) at the dose of 1 mg/kg, 15 min before
compound 3c (Table 3).
Table 3. Effect of L-745,870, Haloperidol, and Clozapine on
Compound 3c-induced Penile Erectiona
treatment
penile erections/rat
i.p. saline (1 mL) + s.c. vehicle (0.2 mL)
i.p. saline (1 mL)+ s.c. 3c (20 µg/kg)
i.p. L-745,870 (1 mg/kg) + s.c. 3c (20 µg/kg)
i.p. haloperidol (1 mg/kg) + s.c. 3c (20 µg/kg)
i.p. clozapine (1 mg/kg) + s.c. 3c (20 µg/kg)
i.p. saline (1 mL) + icv vehicle (10 µl)
i.p. saline (1 mL) + icv 3c (1 µg)
i.p. L-745,870 (1 mg/kg) + icv 3c (1 µg))
i.p. haloperidol (1 mg/kg) + icv 3c (1 µg))
i.p. clozapine (1 mg/kg) + icv 3c (1 µg))
0.31 ( 0.06
2.42 ( 0.51b
0.53 ( 0.12c
0.49 ( 0.12c
0.50 ( 0.12c
0.26 ( 0.05
2.32 ( 0.32b
0.67 ( 0.09c
0.59 ( 0.09c
0.54 ( 0.09c
a Compound 3c was dissolved in ethanol and then diluted with water to
a final concentration of 0.3% of ethanol (vehicle). Haloperidol, clozapine
or L-745,870 was dissolved in saline and administered by i.p. injection 15
min before compound 3c was administered. After treatment, the rats were
observed individually for 60 min to count penile erection episodes. The
values are means ( SEM of 6 rats/group. b P < 0.001 with respect to saline
c
+ vehicle treated rats. P < 0.001 with respect to compound 3c-treated
rats.
especially 2-methoxy derivative 3g. With regard to phenyl
substitution and its influence on D4 affinity, all of the substit-
uents tested (fluorine, chlorine, and methoxy) were well
tolerated. Nevertheless, the introduction of a 2-methoxy group
led to a decrease in selectivity for D4 receptors toward D2
subtypes.
According to mitogenesis and GTPγS assays, the 2-substi-
tuted imidazo[1,2-a]pyridine 3c (PIP3EA) showed substantial
agonist efficacy. In line with the above in vitro results,
compound 3c was shown to be capable of inducing penile
erection in rats after s.c. and icv injections with an efficacy
higher than that of PD-168077, a D4 agonist previously shown
to be capable of inducing penile erection in rats.13,15 The reason
PIP3EA, which presents an intrinsic activity of only 50%
compared to that of full agonists in the above tests, shows such
a good efficacy in the rat model of penile erection is unknown
at present. However, similar low intrinsic activity was also found
with other partial D4 agonists that induce penile erection (e.g.,
ABT 724, intrinsic activity 60%). 13,15 Because the pro-erectile
Conclusions
Herein we have presented a novel series of 2-(4-arylpiperazin-
1-ylmethyl)imidazoazines and diazines. From the synthesized
compounds, the imidazo[1,2-a]pyridine and imidazo[1,2-b]-
pyridazine derivatives were found to present high affinity and
selectivity for D4R over D1, D2L, D2S, and D3 as well as 5-HT2
and R1 receptors. A good selectivity toward the 5-HT1A receptors
was also noticed with the imidazo[1,2-a]pyridine series, but it
was decreased with the imidazo[1,2-b]pyridazine compounds,

2-[(4-Phenylpiperazin-1-yl)methyl]imidazo(di)azines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3943
1H, H-3), 7.58 (d, 1H, J ) 9 Hz, H-8), 8.08 (d, 1H, J ) 6.8 Hz,
H-5). 13C NMR (50 MHz, CDCl3) δ 49.46 (pip), 53.53 (pip), 56.97
(CH2), 111.43 (C-3), 112.59 (C-6), 117.56 (Cl-Ph-2,6), 117.87 (C-
8), 124.69 (Cl-Ph-4), 124.73 (C-7), 125.91 (C-5), 129.30 (Cl-Ph-
3,5), 144.00 (C-8a*), 145.54 (C-2*), 150.40 (Cl-Ph-1).
6-Chloro-2-[4-(3,4-dichlorophenyl)piperazin-1-ylmethyl]imi-
dazo[1,2-b]pyridazine (3f). Mp 118-120 °C. 1H NMR (200 MHz,
CDCl3) δ 2.71 (m, 4H, pip), 3.21 (m, 4H, pip), 3.80 (s, 2H, CH2),
6.72 (dd, 1H, J ) 8.9-2.8 Hz, diCl-Ph-6), 6.94 (d, 1H, J ) 2.8
Hz, diCl-Ph-2), 7.05 (d, 1H, J ) 9.4 Hz, H-7), 7.25 (d, 1H, J )
8.9 Hz, diCl-Ph-5), 7.87 (d, 1H, J ) 9.4 Hz, H-8), 7.90 (s, 1H,
H-3). 13C NMR (50 MHz, CDCl3) δ 48.91 (pip), 53.28 (pip), 56.92
(CH2), 115.66 (diCl-Ph-6), 116.38 (C-3), 117.50 (diCl-Ph-2), 119.22
(C-7), 122.39 (diCl-Ph-4), 126.90 (C-8), 130.75 (diCl-Ph-5), 133.10
(diCl-Ph-3), 137.78 (diCl-Ph-1), 145.19 (C-8a*), 147.03 (C-2*),
151.01 (C-6).
effect of compound 3c is reduced not only by haloperidol and
clozapine, which block all dopamine receptor subtypes, but also
by the selective D4 antagonist L-745,870, these findings provide
further support for the role of central D4 receptors in the control
of penile erection and the possible therapeutic use of D4 agonists
in the treatment of erectile dysfunctions.
Experimental Section
Chemistry Methods. All solvents were anhydrous reagents from
commercial sources. Unless otherwise noted, all chemicals and
reagents were obtained commercially and used without purification.
NMR spectra were recorded at 200 MHz (1H) or 50 MHz (13C) on
a Bruker DPX instrument or at 360 MHz (1H) or 90 MHz (13C) on
a Bruker AM 360. The chemical shifts are reported in parts per
million (ppm, δ) relative to residual deuterated solvent peaks. The
possible inversion of two values in the NMR spectra is expressed
by an asterisk. Known compounds were prepared according to a
literature procedure: 2-chloromethylimidazo[1,2-a]pyridine (2a),
6-chloro-2-chloromethylimidazo[1,2-b]pyridazine (2b), 2-chloro-
methylimidazo[1,2-a]pyrazine (2c), and 2-chloromethylimidazo[1,2-
a]pyrimidine (2d).44-47
6-Chloro-2-[4-(2-methoxyphenyl)piperazin-1-ylmethyl]imidazo-
[1,2-b]pyridazine (3g). The title compound was obtained as an
oil.1H NMR (200 MHz, CDCl3) δ 2.82 (m, 4H, pip), 3.17 (m, 4H,
pip), 3.86 (s, 2H, CH2), 3.89 (s, 3H, CH3), 6.87-7.06 (m, 4H,
CH3O-Ph), 7.07 (d, 1H, J ) 9.4 Hz, H-7), 7.89 (d, 1H, J ) 9.4
Hz, H-8), 7.95 (s, 1H, H-3).
2-[4-(4-Fluorophenyl)piperazin-1-ylmethyl]imidazo[1,2-a]py-
ridine (3a). Typical procedure: a mixture of 2-chloromethylimi-
dazoazine or diazine (5 mmol), 1-(substituted phenyl)piperazine
(5 mmol), and sodium carbonate (5 mmol) in dry ethanol (20 mL)
was refluxed for 3 h. After cooling, ethanol was removed in vacuo,
and the residue was dissolved in water and extracted with
dichloromethane. The organic layer was dried (CaCl2), evaporated,
and the residue purified by chromatography (neutral alumina-
6-Chloro-2-[4-(4-chlorophenyl)piperazin-1-ylmethyl]imidazo-
[1,2-b]pyridazine (3h). Mp 149-151 °C. H NMR (200 MHz,
1
CDCl3) δ 2.75 (m, 4H, pip), 3.23 (m, 4H, pip), 3.83 (s, 2H, CH2),
6.86 (d, 2H, J ) 8.7 Hz, Cl-Ph-2,6), 7.07 (d, 1H, J ) 9.4 Hz,
H-7), 7.22 (d, 2H, J ) 8.7 Hz, Cl-Ph-3,5), 7.89 (d, 1H, J ) 9.4
Hz, H-8), 7.93 (s, 1H, H-3). 13C NMR (50 MHz, CDCl3) δ 49.49
(pip), 53.54 (pip), 57.06 (CH2), 116.40 (C-3), 117.66 (Cl-Ph-2,6),
119.17 (C-7), 124.90 (Cl-Ph-4), 126.93 (C-8), 129.34 (Cl-Ph-3,5),
137.82 (Cl-Ph-1), 145.43 (C-8a), 147.02 (C-2), 150.32 (C-6).
2-[4-(3,4-Dichlorophenyl)piperazin-1-ylmethyl]-6-(piperidin-
1-yl)imidazo[1,2-b]pyridazine (4a). Typical procedure: a solution
of 3f (0.5 g, 1.26 mmol) in the suitable amine (5 mL) was refluxed
for 24 h. After cooling, the mixture was diluted with water and
extracted with dichloromethane. The organic layer was dried
(CaCl2), evaporated, and the residue purified by chromatography
1
dichloromethane). Mp 124-126 °C. H NMR (200 MHz, CDCl3)
δ 2.73 (m, 4H, pip), 3.13 (m, 4H, pip), 3.76 (s, 2H, CH2), 6.72 (td,
1H, J ) 6.8-1.1 Hz, H-6), 6.80-6.97 (m, 4H, F-Ph), 7.11 (ddd,
1H, J ) 8.9-6.8-1.1 Hz, H-7), 7.52 (s, 1H, H-3), 7.54 (bd, 1H,
J ) 8.9 Hz, H-8), 8.04 (dt, 1H, J ) 6.8-1.1 Hz, H-5).
6-Chloro-2-[4-(4-fluorophenyl)piperazin-1-ylmethyl]imidazo-
1
[1,2-b]pyridazine (3e). Mp 111-113 °C. H NMR (200 MHz,
1
CDCl3) δ 2.75 (m, 4H, pip), 3.16 (m, 4H, pip), 3.82 (s, 2H, CH2),
6.84-7.01 (m, 4H, F-Ph), 7.05 (d, J ) 9.4 Hz, H-7), 7.87 (d, J )
9.4 Hz, H-8), 7.92 (s, 1H, H-3).
(neutral alumina-dichloromethane). Mp 135-137 °C. H NMR
(200 MHz, CDCl3) δ 1.71 (m, 6H, piperidine), 2.73 (m, 4H, pip),
3.22 (m, 4H, pip), 3.50 (m, 4H, piperidine), 3.75 (s, 2H, CH2),
6.75 (dd, 1H, J ) 8.9-2.9 Hz, diCl-Ph-6), 6.83 (d, 1H, J ) 9.9
Hz, H-7), 6.96 (d, 1H, J ) 2.9 Hz, diCl-Ph-2), 7.28 (d, 1H, J )
8.9 Hz, diCl-Ph-5), 7.64 (d, 1H, J ) 9.9 Hz, H-8), 7.65 (s, 1H,
H-3).13C NMR (50 MHz, CDCl3) δ 24.84 (piperidine), 25.79
(piperidine), 47.89 (piperidine), 48.99 (pip), 53.17 (pip), 57.18
(CH2), 110.83 (C-7), 115.64 (diCl-Ph-6), 115.83 (C-3), 117.50
(diCl-Ph-2), 122.26 (diCl-Ph-4), 125.71 (C-8), 130.77 (diCl-Ph-
5), 133.11 (diCl-Ph-3), 136.44 (diCl-Ph-1), 141.58 (C-8a*), 151.17
(C-2*), 155.58 (C-6).
2-[4-(4-Fluorophenyl)piperazin-1-ylmethyl]imidazo[1,2-a]py-
razine (3i). Mp 127-129 °C. 1H NMR (200 MHz, CDCl3) δ 2.72
(m, 4H, pip), 3.12 (m, 4H, pip), 3.81 (s, 2H, CH2), 6.82-6.97 (m,
4H, F-Ph), 7.66 (s, 1H, H-3), 7.83 (d, 1H, J ) 4.5 Hz, H-5), 8.02
(dd, 1H, J ) 4.5-1.5 Hz, H-6), 9.03 (m, 1H, H-8).
2-[4-(4-Fluorophenyl)piperazin-1-ylmethyl]imidazo[1,2-a]py-
1
rimidine (3j). Mp 170-172 °C. H NMR (200 MHz, CDCl3) δ
2.77 (m, 4H, pip), 3.13 (m, 4H, pip), 3.83 (s, 2H, CH2), 6.82-6.98
(m, 5H, F-Ph, H-6), 7.55 (s, 1H, H-3), 8.44 (dd, 1H, J ) 6.7-2.1
Hz, H-5), 8.49 (dd, 1H, J ) 4.1-2.1 Hz, H-7).
2-[4-(3,4-Dichlorophenyl)piperazin-1-ylmethyl]-6-(4-meth-
ylpiperazin-1-yl)imidazo[1,2-b]pyridazine (4b). Mp 178-180 °C.
1H NMR (200 MHz, CDCl3) δ 2.39 (s, 3H, CH3), 2.58 (m, 4H,
6-pip), 2.72 (m, 4H, 2-pip), 3.22 (m, 4H, 2-pip), 3.53 (m, 4H, 6-pip),
3.76 (s, 2H, CH2), 6.75 (dd, 1H, J ) 8.9-2.9 Hz, diCl-Ph-6), 6.83
(d, 1H, J ) 9.9 Hz, H-7), 6.97 (d, 1H, J ) 2.9 Hz, diCl-Ph-2),
7.28 (d, 1H, J ) 8.9 Hz, diCl-Ph-5), 7.67 (s, 1H, H-3), 7.69 (d,
1H, J ) 9.9 Hz, H-8). 13C NMR (50 MHz,CDCl3) δ 46.59 (CH3),
46.67 (6-pip), 49.00 (2-pip), 53.19 (2-pip), 55.01 (6-pip), 57.15
(CH2), 110.29 (C-7), 115.65 (diCl-Ph-6), 115.88 (C-3), 117.51
(diCl-Ph-2), 122.29 (diCl-Ph-4), 125.94 (C-8), 130.78 (diCl-Ph-
5), 133.12 (diCl-Ph-3), 136.55 (diCl-Ph-1), 141.95 (C-8a*), 151.16
(C-2*), 155.31 (C-6).
6-Bromo-2-(chloromethyl)imidazo[1,2-a]pyridine (6a). Typical
procedure: a mixture of the conveniently substituted 2-aminopy-
ridine (50 mmol), 1,3-dichloroacetone (50 mmol), and dimethoxy-
ethane (30 mL) was stirred overnight at room-temperature. After
which time, the resulting solid was collected and washed with
several milliliters of dimethoxyethane. The solid was then dissolved
in 150 mL of ethanol and refluxed for 2 h. The solvent was then
removed under reduced pressure, and the residue was suspended
in H2O, made alkaline with Na2CO3, and extracted with CH2Cl2.
2-[4-(3,4-Dichlorophenyl)piperazin-1-ylmethyl]imidazo[1,2-
a]pyridine (3b). Mp 103-105 °C. 1H NMR (200 MHz, CDCl3) δ
2.74 (m, 4H, pip), 3.21 (m, 4H, pip), 3.78 (s, 2H, CH2), 6.73 (dd,
1H, J ) 8.9-2.9 Hz, diCl-Ph-6), 6.79 (td, 1H, J ) 6.8-1.2 Hz,
H-6), 6.95 (d, 1H, J ) 2.9 Hz, diCl-Ph-2), 7.16 (ddd, 1H, J )
9-6.8-1.2 Hz, H-7), 7.26 (d, 1H, J ) 8.9 Hz, diCl-Ph-5), 7.56 (s,
1H, H-3), 7.58 (m, 1H, H-8), 8.09 (dt, 1H, J ) 6.8-1.2 Hz, H-5).
Anal. Calcd for C18H18Cl2N4: C, 59.84; H, 5.02; N, 15.51. Found:
C, 59.81; H, 5.13; N, 15.43.
2-[4-(2-Methoxyphenyl)piperazin-1-ylmethyl]imidazo[1,2-a]-
pyridine (3c). Mp 142-144 °C.1H NMR (200 MHz, CDCl3) δ
2.81 (m, 4H, pip), 3.13 (m, 4H, pip), 3.83 (s, 3H, CH3O), 3.86 (s,
2H, CH2), 6.76 (td, 1H, J ) 6.6-1.2 Hz, H-6), 6.86 (m, 1H, CH3O-
Ph-3), 6.92-7.02 (m, 3H, CH3O-Ph-4,5,6), 7.15 (ddd, 1H, J )
9-6.6-1.2 Hz, H-7), 7.56 (s, 1H, H-3), 7.59 (m, 1H, H-8), 8.08
(dt, 1H, J ) 6.6-1.2 Hz, H-5).
2-[4-(4-Chlorophenyl)piperazin-1-ylmethyl]imidazo[1,2-a]py-
ridine (3d). Mp 141-143 °C. 1H NMR (200 MHz, CDCl3) δ 2.75
(m, 4H, pip), 3.20 (m, 4H, pip), 3.79 (s, 2H, CH2), 6.77 (t, 1H, J
) 6.8 Hz, H-6), 6.84 (d, 2H, J ) 8.8 Hz, Cl-Ph-2,6), 7.17 (dd, 1H,
J ) 9-6.8 Hz, H-7), 7.20 (d, 2H, J ) 8.8 Hz, Cl-Ph-3,5), 7.55 (s,

3944 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Enguehard-Gueiffier et al.
After drying (CaCl2), the organic layers were evaporated to dryness.
The residue was chromatographed on neutral alumina eluting with
CH2Cl2; 53% yield (not optimized). Mp 125 °C. H NMR (200
MHz, CDCl3) δ 4.77 (s, 2H, CH2), 7.28 (dd, 1H, J ) 9.6-1.8 Hz,
H-7), 7.50 (d, 1H, J ) 9.6 Hz, H-8), 7.61 (s, 1H, H-3), 8.26 (m,
1H, H-5).
(C-7), 141.78 (CH3O-Ph-1), 142.39 (C-8a*), 144.93 (C-2*), 152.67
(CH3O-Ph-2).
1
8-Iodo-2-[4-(2-methoxyphenyl)piperazin-1-ylmethyl]-6-meth-
ylimidazo[1,2-a]pyridine (7d). The title compound was prepared,
following the procedure used for 7b, in 81% yield. Mp 143 °C. 1H
NMR (360 MHz, CDCl3) δ 2.28 (s, 3H, CH3), 2.81 (m, 4H, pip),
3.14 (m, 4H, pip), 3.86 (s, 5H, CH2, CH3O), 6.86 (dd, 1H, J )
7.8-1 Hz, CH3O-Ph-6), 6.89-7.02 (m, 3H, CH3O-Ph-3,4,5), 7.51
(m, 1H, H-7), 7.62 (s, 1H, H-3), 7.84 (m, 1H, H-5). 13C NMR (50
MHz, CDCl3) δ 17.43 (CH3), 50.53 (pip), 53.37 (pip), 55.22 (CH2),
56.63 (CH3O), 83.00 (C-8), 111.07 (CH3O-Ph-6), 112.39 (C-3),
118.05 (CH3O-Ph-3), 120.83 (CH3O-Ph-4), 122.44 (C-6), 122.67
(C-5), 123.39 (CH3O-Ph-5), 136.39 (C-7), 141.29 (CH3O-Ph-1),
143.34 (C-8a*), 144.35 (C-2*), 152.15 (CH3O-Ph-2).
2-(Chloromethyl)-6-iodoimidazo[1,2-a]pyridine (6b). 48% yield
1
(not optimized). Mp 161 °C. H NMR (200 MHz, CDCl3) δ 4.76
(s, 2H, CH2), 7.37 (bs, 2H, H-8, H-7), 7.58 (s, 1H, H-3), 8.36 (m,
1H, H-5).
8-Bromo-2-(chloromethyl)-6-methylimidazo[1,2-a]pyridine (6c).
The compound was obtained in 36% yield (not optimized). Mp
1
>250 °C. H NMR (200 MHz, CDCl3) δ 2.34 (s, 3H, CH3), 4.82
(s, 2H, CH2), 7.36 (d, 1H, J ) 1.2 Hz, H-7), 7.66 (s, 1H, H-3),
7.87 (m, 1H, H-5).
6-Bromo-2-[4-(3,4-dichlorophenyl)piperazin-1-ylmethyl]imi-
dazo[1,2-a]pyridine (7e). The title compound was prepared fol-
lowing the procedure used for 7a using 1-(3,4-dichlorophenyl)-
piperazine in 87% yield. Mp 58-59 °C. 1H NMR (360 MHz,
CDCl3) δ 2.70 (m, 4H, pip), 3.19 (m, 4H, pip), 3.75 (s, 2H, CH2),
6.71 (dd, 1H, J ) 8.9-2.8 Hz, diCl-Ph-6), 6.93 (d, 1H, J ) 2.8
Hz, diCl-Ph-2), 7.20 (dd, 1H, J ) 9.6-1.8 Hz, H-7), 7.24 (dd, 1H,
J ) 8.9 Hz, diCl-Ph-5), 7.46 (d, 1H, J ) 9.6 Hz, H-8), 7.51 (s,
1H, H-3), 8.22 (m, 1H, H-5). 13C NMR (50 MHz, CDCl3) δ 48.83
(pip), 53.24 (pip), 56.66 (CH2), 107.33 (C-6), 111.91 (C-3), 115.70
(diCl-Ph-6), 117.56 (diCl-Ph-2), 118.44 (C-8), 122.43 (diCl-Ph-
4), 125.99 (C-5), 128.35 (C-7), 130.80 (diCl-Ph-5), 133.10 (diCl-
Ph-3), 143.97 (C-8a*), 144.44 (C-2*), 150.98 (diCl-Ph-1).
8-Cyano-2-[4-(2-methoxyphenyl)piperazin-1-ylmethyl]-6-me-
thylimidazo[1,2-a]pyridine (8a). A screw-cap test tube was
charged with 0.23 g (0.5 mmol) of 7d, 29.4 mg (0.6 mmol) of
sodium cyanide, and 9.52 mg (10 mol %) of copper(I)iodide. A
Teflon septum was attached, and the tube was evacuated and back-
filled with argon. The evacuated/backfield sequence was repeated
an additional time. Then, 44 mg (0.5 mmol, 53.2µL) of N,N′-
dimethyl-1,2-diaminoethane and toluene (1 mL) were added by
syringe under argon. The screw-cap test tube was sealed with a
cap, and the reaction mixture was stirred magnetically at 112 °C
for 24 h. After cooling, the suspension was diluted with dichlo-
romethane and then washed with sodium carbonate solution
followed by water. The organic layer was dried over calcium
chloride and evaporated to dryness. The residue was chromato-
graphed on neutral alumina eluted with ethyl acetate-hexane (50:
2-(Chloromethyl)-8-iodo-6-methylimidazo[1,2-a]pyridine (6d).
The compound was obtained in 43% yield (not optimized). Mp
146 °C. 1H NMR (200 MHz, CDCl3) δ 2.31 (s, 3H, CH3), 4.82 (s,
2H, CH2), 7.58 (d, 1H, J ) 1.2 Hz, H-7), 7.70 (s, 1H, H-3), 7.87
(m, 1H, H-5).
6-Bromo-2-[4-(2-methoxyphenyl)piperazin-1-ylmethyl]imidazo-
[1,2-a]pyridine (7a). To a solution of 1.23 g (5 mmol) of 6-bromo-
2-chloromethylimidazo[1,2-a]pyridine (6a) and 0.97 g (5 mmol)
of 1-(2-methoxyphenyl)piperazine in anhydrous ethanol (25 mL)
was added 0.55 g (5 mmol) of sodium carbonate. The suspension
was refluxed for 3 h. After cooling, the ethanol was removed in
vacuo, the residue dissolved in water, and then extracted with
dichloromethane. The organic layer was dried over calcium chloride,
filtered, and evaporated to dryness. The residue was chromato-
graphed on silica gel, eluting with dichloromethane to give 1.62 g
(81%) of 7a. Mp 108-109 °C. 1H NMR (200 MHz, CDCl3) δ 2.85
(m, 4H, pip), 3.15 (m, 4H, pip), 3.84 (s, 2H, CH2), 3.88 (s, 3H,
CH3O), 6.86-7.04 (m, 4H, CH3O-Ph), 7.23 (dd, 1H, J ) 9.5-1.8
Hz, H-7), 7.50 (d, 1H, J ) 9.5 Hz, H-8), 7.58 (s, 1H, H-3), 8.26
(m, 1H, H-5). 13C NMR (50 MHz, CDCl3) δ 50.85 (pip), 53.84
(pip), 55.74 (CH2), 56.86 (CH3O), 107.19 (C-6), 111.52 (CH3O-
Ph-6), 111.90 (C-3), 118.51 (C-8), 118.63 (CH3O-Ph-3), 121.38
(CH3O-Ph-4), 123.33 (CH3O-Ph-5), 125.93 (C-5), 128.12 (C-7),
141.67 (CH3O-Ph-1), 143.94 (C-8a*), 144.86 (C-2*), 152.64
(CH3O-Ph-2).
6-Iodo-2-[4-(2-methoxyphenyl)piperazin-1-ylmethyl]imidazo-
[1,2-a]pyridine (7b). To a solution of 2 g (6.83 mmol) of 6-iodo-
2-chloromethylimidazo[1,2-a]pyridine (6b) and 1.56 g (6.83 mmol)
of 1-(2-methoxyphenyl)piperazine hydrochloride in anhydrous
ethanol (25 mL) was added 1.45 g (13.66 mmol) of sodium
carbonate. The suspension was refluxed for 3 h. After cooling, the
ethanol was removed in vacuo and the residue dissolved in water,
and then extracted with dichloromethane. The organic layer was
dried over calcium chloride, filtered, and evaporated to dryness.
The residue was chromatographed on silica gel, eluting with
1
50 v/v) to give 126 mg (70%) of 8a. Mp 149-150 °C. H NMR
(360 MHz, CDCl3) δ 2.36 (s, 3Η, CΗ3), 2.80 (m, 4H, pip), 3.12
(m, 4H, pip), 3.85 (s, 3H, CH3O), 3.86 (s, 2H, CH2), 6.84-7.01
(m, 4H, CH3O-Ph), 7.44 (d, 1H, J ) 1.4 Hz, H-7), 7.60 (s, 1H,
H-3), 8.07 (m, 1H, H-5). 13C NMR (50 MHz, CDCl3) δ 17.72
(CH3), 50.58 (pip), 53.47 (pip), 55.32 (CH2), 56.44 (CH3O), 101.69
(C-8), 111.19 (CH3O-Ph-6), 112.05 (C-3), 115.11 (CN*), 118.16
(CH3O-Ph-3), 120.94 (CH3O-Ph-4), 120.99 (C-6*), 122.85 (CH3O-
Ph-5), 127.35 (C-5), 133.79 (C-7), 141.34 (CH3O-Ph-1*), 141.47
(C-8a*), 145.92 (C-2*), 152.27 (CH3O-Ph-2).
1
dichloromethane to give 2.23 g (73%) of 7b. Mp 78-80 °C. H
NMR (360 MHz, CDCl3) δ 2.79 (m, 4H, pip), 3.14 (m, 4H, pip),
3.80 (s, 2H, CH2), 3.86 (s, 3H, CH3O), 6.86 (dd, 1H, J ) 8.2-1.4
Hz, CH3O-Ph-6), 6.90-7.02 (m, 3H, CH3O-Ph-3,4,5), 7.31 (dd,
1H, J ) 9.6-1.8 Hz, H-7), 7.38 (d, 1H, J ) 9.6 Hz, H-8), 7.51 (s,
1H, H-3), 8.34 (m, 1H, H-5). 13C NMR (50 MHz, CDCl3) δ 50.48
(pip), 53.41 (pip), 55.24 (CH2), 56.42 (CH3O), 74.71 (C-6), 110.68
(C-3), 111.11 (CH3O-Ph-6), 118.10 (C-8), 118.40 (CH3O-Ph-3),
120.89 (CH3O-Ph-4), 122.71 (CH3O-Ph-5), 130.28 (C-5), 131.96
(C-7), 141.34 (CH3O-Ph-1), 143.52 (C-8a*), 144.49 (C-2*), 152.18
(CH3O-Ph-2).
6-Methyl-2-[4-(2-methoxyphenyl)piperazin-1-ylmethyl]-8-N-
morpholinoimidazo[1,2-a]pyridine (8b). A screw-cap test tube
was charged with 231 mg (0.5 mmol) of 7d, 14.3 mg (15 mol %)
of copper(I)iodide, and 212.3 mg (1 mmol) potassium phosphate.
A Teflon septum was attached, and the tube was evacuated and
back-filled with argon. The evacuated/backfield sequence was
repeated two additional times. Then 47.9 mg (0.55 mmol, 48 µL)
of morpholine and 62 mg (1 mmol, 55 µL) of ethyleneglycol and
2-propanol (1 mL) were added by syringe under argon. The screw-
cap test tube was sealed with a cap, and the reaction mixture was
stirred magnetically at 85 °C for 48 h. After cooling, the suspension
diluted with dichloromethane and washed with sodium carbonate
solution followed by water. The organic layer dried over calcium
chloride and evaporated to dryness. The residue was chromato-
graphed on neutral alumina eluted with ethyl acetate-hexane (50:
8-Bromo-2-[4-(2-methoxyphenyl)piperazin-1-ylmethyl]-6-me-
thylimidazo[1,2-a]pyridine (7c). The title compound was prepared,
following the procedure used for 7b, in 83% yield. Mp 149-150
°C. 1H NMR (200 MHz, CDCl3) δ 2.32 (s, 3H, CH3), 2.82 (m, 4H,
pip), 3.15 (m, 4H, pip), 3.88 (s, 3H, CH3O), 3.89 (s, 2H, CH2),
6.86-7.05 (m, 4H, CH3O-Ph), 7.30 (s, 1H, H-7), 7.60 (s, 1H, H-3),
7.86 (m, 1H, H-5).13C NMR (50 MHz, CDCl3) δ 18.25 (CH3), 51.04
(pip), 53.89 (pip), 55.75 (CH2), 57.15 (CH3O), 111.00 (C-8), 111.55
(CH3O-Ph-6), 112.79 (C-3), 118.58 (CH3O-Ph-3), 121.35 (CH3O-
Ph-4), 122.43 (C-6), 123.08 (C-5), 123.25 (CH3O-Ph-5), 130.00
1
50 v/v) to give 151 mg (72%) of 8b. oil. H NMR (360 MHz,
CDCl3) δ 2.58 (s, 3Η, CΗ3), 2.81 (m, 4H, pip), 3.13 (m, 4H, pip),
3.51 (m, 4H, mor), 3.81 (s, 2H, CH2), 3.85 (s, 3H, CH3O), 3.97
(m, 4H, mor), 6.21 (s, 1H, H-7), 6.84-7.01 (m, 4H, CH3O-Ph),

2-[(4-Phenylpiperazin-1-yl)methyl]imidazo(di)azines
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3945
7.41 (s, 1H, H-5), 7.53 (m, 1H, H-3). 13C NMR (50 MHz, CDCl3)
δ 18.44 (CH3), 49.88 (mor), 50.60 (pip), 53.38 (pip), 55.35 (CH2),
56.71 (CH3O), 66.86 (mor), 109.23 (C-3), 111.20 (CH3O-Ph-6,
C-7), 116.79 (C-5), 118.25 (CH3O-Ph-3), 120.98 (CH3O-Ph-4),
121.70 (C-6), 122.83 (CH3O-Ph-5), 139.48 (C-8a*), 139.98 (C-
2*), 141.39 (CH3O-Ph-1*), 152.30 (CH3O-Ph-2), C-8 not found.
2-[4-(2-Methoxyphenyl)piperazin-1-ylmethyl]-6-methyl-8-
(pyrazol-1-yl)imidazo[1,2-a]pyridine (8c). A screw-cap test tube
was charged with 231 mg (0.5 mmol) of 7d, 34 mg (0.5 mmol) of
pyrazole, 5 mg (5 mol %) of copper(I)iodide, and 223 mg (1.1
mmol) potassium phosphate. A Teflon septum was attached, and
the tube was evacuated and back-filled with argon, and the
evacuated/backfield sequence was repeated two additional times.
Then, 6.61 mg (15 mol %, 8 µL) of N,N′-dimethyl-1,2-diamino-
ethane and toluene (1 mL) were added by syringe under argon.
The screw-cap test tube was sealed with a cap, and the reaction
mixture was stirred magnetically at 112 °C for 24 h. After cooling,
the suspension was diluted with dichloromethane and washed with
sodium carbonate solution followed by water. The organic layer
was dried over calcium chloride and evaporated to dryness. The
residue was chromatographed on neutral alumina, eluting with ethyl
acetate-hexane (70:30 v/v) to give 84 mg (42%) of 8c. Mp 152
δ 2.38 (s, 3H, CH3), 2.88 (m, 4H, pip), 3.18 (m, 4H, pip), 3.89 (s,
5H, CH3, CH2), 6.89 (d, 1H, J ) 7.7 Hz, CH3O-Ph-6), 6.94-7.08
(m, 3H, CH3O-Ph-3,4,5), 7.14 (d, 1H, J ) 1.4 Hz, H-7), 7.38-
7.56 (m, 3H, Ph-3,4,5), 7.58 (s, 1H, H-3), 7.88 (H-5), 8.04 (m,
2H, Ph-2,6). 13C NMR (50 MHz, CDCl3) δ 18.61 (CH3), 51.02
(pip), 53.81 (pip), 55.77 (CH2), 57.15 (CH3O), 111.56 (CH3O-Ph-
6), 111.67 (C-3), 118.66 (CH3O-Ph-3), 121.39 (CH3O-Ph-4), 122.06
(C-6), 122.78 (C-5), 123.29 (CH3O-Ph-5), 126.35 (C-7), 128.59
(C-8), 128.92 (Ph-3,5), 129.30 (Ph-2,6), 129.73 (Ph-4), 137.03 (Ph-
1), 141.78 (CH3O-Ph-1), 143.24 (C-8a*), 143.73 (C-2*), 152.69
(CH3O-Ph-2).
Receptor Binding Experiments. Receptor binding experiments
were carried out as described in the literature.37 In brief, the
dopamine D1 receptor assay was done with porcine striatal
membranes at a final protein concentration of 40 µg/assay tube
and the radioligand [3H]SCH 23390 at 0.3 nM (Kd ) 0.62-1.1
nM). Competition experiments with human D2long, D2short, D3, and
D4.4 receptors were run with preparations of membranes from CHO
cells expressing the corresponding receptor and [3H]spiperone at a
final concentration of 0.1 nM. The assays were carried out with a
protein concentration of 3-20 µg/assay and Kd values of 0.06-
0.21 nM for D2long, 0.07-0.15 nM for D2short, 0.10-0.35 nM for
D3, and 0.10-0.50 nM for D4.4. The determination of the binding
affinity to the agonist binding site of the D2 receptor was done
with the radioligand [3H]7-OH-DPAT at 0.5 nM (Kd ) 2.5 nM for
1
°C. H NMR (360 MHz, CDCl3) δ 2.38 (s, 3Η, CΗ3), 2.84 (m,
4H, pip), 3.14 (m, 4H, pip), 3.85 (s, 5H, CH2, CH3O), 6.50 (dd,
1H, J ) 2.5-1.8 Hz, Pyr-4), 6.84-7.02 (m, 4H, CH3O-Ph), 7.57
(s, 1H, H-3), 7.76 (d, 1H, J ) 1.8 Hz, Pyr-3*), 7.76 (s, 1H, H-7),
7.80 (m, 1H, H-5), 9.51 (d, 1H, J ) 2.5 Hz, Pyr-5*). 13C NMR (50
MHz, CDCl3) δ 18.66 (CH3), 51.01 (pip), 53.84 (pip), 55.77 (CH2),
56.99 (CH3O), 107.57 (Pyr-4), 111.52 (CH3O-Ph-6), 112.40 (C-
3), 116.13 (C-7), 118.64 (CH3O-Ph-3), 121.07 (C-5), 121.38 (CH3O-
Ph-4), 122.24 (C-6), 123.35 (CH3O-Ph-5), 128.38 (C-8), 132.76
(Pyr-5*), 137.57 (C-8a), 141.54 (CH3O-Ph-1*), 141.71 (Pyr-3*),
143.58 (C-2), 152.67 (CH3O-Ph-2).
D
2long; Kd ) 1.6 nM for porcine D2) and a protein concentration of
10 µg/assay and 100 µg/assay for D2long and porcine D2, respec-
tively.
The investigation of 5-HT1A and 5-HT2 binding was performed
as described in the literature.40 In brief, porcine cortical membranes
were subjected to the binding assay at a concentration of 80-110
µg/assay tube for the determination of 5-HT1A and 5-HT2 binding
using [3H]8-OH-DPAT and [3H]ketanserin, each at a final concen-
tration of 0.5 nM with Kd values of 1.1-4.1 nM (for 5-HT1A) and
1.9-4.8 nM (for 5-HT2). The protein concentration was established
by the Lowry method using bovine serum albumin as the standard.48
Data analysis of the resulting competition curves was ac-
complished by nonlinear regression analysis using the algorithms
in PRISM (GraphPad Software, San Diego, CA). The Ki values
were derived from the corresponding EC50 data using the equation
of Cheng and Prusoff.49
2-[4-(2-Methoxyphenyl)piperazin-1-ylmethyl]-5-(pyrazol-1-
yl)imidazo[1,2-a]pyridine (8d). A screw-cap test tube was charged
with 0.2 g (0.5 mmol) of 7a, 34 mg (0.5 mmol) of pyrazole, and
342 mg (1.05 mmol) of cesium carbonate. A Teflon septum was
attached, and the tube was evacuated and back-filled with argon,
and the evacuated/backfield sequence was repeated an additional
time. N,N-Dimethylformamide (1 mL) was added by syringe under
argon. The screw-cap test tube was sealed with a cap, and the
reaction mixture was stirred magnetically at 112 °C for 24 h. After
cooling, the suspension was taken up in water, extracted with
dichloromethane. The organic layer was washed three times with
water, dried over calcium chloride, and evaporated to dryness. The
residue was chromatographed on neutral alumina eluted with ethyl
acetate-hexane (60:40 v/v) and then ethyl acetate to give the
remaining 100 mg (50%) of starting material 7a and 52 mg (27%)
Mitogenesis Experiments. The determination of the stimulating
effects of the test compounds on mitogenesis as a functional assay
was done with the CHO 10001 cell line stably expressing the human
dopamine D4.2 receptor as referred to in the literature.38,40 In detail,
5000 cells/well were grown for 72 h in a 96 well plate in the
appropriate medium supplemented with serum. After washing and
adding a serum free medium, the cells were incubated for 20 h
with the test compound at 8 different concentrations in the range
of 0.01-10 000 nM as hexaduplicates. After the addition of 0.02
µCi/well of [3H]thymidine (specific activity 25 Ci/mmol, Amersham
Biosciences), the incubation of the D4 expressing cells was
continued for 2 h. Finally, the cells were harvested, and the
incorporated radioactivity was measured with a microplate scintil-
lation counter. Experimental data resulting from the mitogenesis
assay were each normalized and then combined to get a mean curve.
A nonlinear regression analysis of this curve provided the EC50
values as a measure of potency. The top value of the curve
represented the maximal intrinsic activity, which was correlated to
the effect of the full agonist quinpirole (100%).
1
of 8d. Mp 160-161 °C. H NMR (360 MHz, CDCl3) δ 2.86 (m,
4H, pip), 3.17 (m, 4H, pip), 3.87 (s, 5H, CH3O, CH2), 6.62 (dd,
1H, J ) 2.4-2.1 Hz, Pyr-4), 6.87 (d, 1H, J ) 7.3 Hz, H-6), 6.85-
7.05 (m, 4H, CH3O-Ph-3,4,5,6), 7.29 (dd, 1H, J ) 9-7.3 Hz, H-7),
7.69 (d, 1H, J ) 9 Hz, H-8), 7.90-7.95 (m, 3H, Pyr-3,5, H-3). 13
C
NMR (50 MHz, CDCl3) δ 50.85 (pip), 53.78 (pip), 55.70 (CH2),
56.87 (CH3O), 106.81 (C-6), 108.34 (Pyr-4), 110.31 (C-3), 111.45
(CH3O-Ph-6), 117.31 (C-8), 118.60 (CH3O-Ph-3), 121.34 (CH3O-
Ph-4), 123.24 (CH3O-Ph-5), 124.22 (C-7), 131.45 (Pyr-5*), 133.41
(C-5), 141.75 (CH3O-Ph-1), 143.01 (Pyr-3*), 144.38 (C-2*), 146.65
(C-8a*), 152.61 (CH3O-Ph-2).
2-[4-(2-Methoxyphenyl)piperazin-1-ylmethyl]-6-methyl-8-
phenylimidazo[1,2-a]pyridine (8e). A mixture of 207.5 mg (0.5
mmol) of 7c, 67 mg (0.5 mmol) of phenylboronic acid, 29 mg (5
mol %) of tetrakis(triphenylphosphine)palladium, and 40 mg (1
mmol) of sodium hydroxide was suspended in 1,2-dimethoxyethane
(4 mL) and water (2 mL) and heated at 85 °C for 3 h. After cooling
and stirring at room temperature for 17 h, dichloromethane was
added, and the resulting mixture was washed with water. The
organic layer was dried over calcium chloride and evaporated to
dryness. The residue was chromatographed on neutral alumina
eluted with ethyl acetate-hexane (70:30 v/v) to give 112 mg (54%
not optimized) of 8e. Mp 59-60 °C. 1H NMR (360 MHz, CDCl3)
Determination of [35S]GTPγS Binding. The [35S]GTPγS bind-
ing assay was established on the basis of literature.41 The preparation
of membranes from CHO cells stably expressing the human D4.4
and the human D2short receptors, with a receptor density of Bmax
)
2.25 and 6.23 pmol/mg protein, were diluted in HEPES buffer (20
nM HEPES, 10 nM MgCl2, 100 nM NaCl; pH 7.4) and incubated
at 37 °C with 1 µM GDP in HEPES buffer and the test compounds
(in HEPES buffer supplemented with 0.1 nM dithiothreitol) in eight
different concentrations (0.01-10 000 nM) as hexaduplicates at a
final volume of 200 µL in 96-well microplates. After 30 min, 0.1
nM [35S]GTPγS (specific activity 1250 Ci/mmol, Perkin-Elmer)

3946 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Enguehard-Gueiffier et al.
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was added, and incubation continued for a further 30 min. The
experiment was terminated by rapid filtration through GF/B filters
using an automated cell harvester (Brandel), and the filters were
washed five times with ice-cold washing buffer (140 mM NaCl,
10 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4; pH 7.4) and dried
at 60 °C for 3 h, and the trapped radioactivity was counted in a
microplate scintillation counter (MicroBeta Trilux, Perkin-Elmer).
To confirm the efficacy derived from the dose-response curves,
the experiments were repeated with representative concentrations
of the test compounds (0.01 and 1000 nM) in comparison with the
effect of 10 000 nM quinpirole. For all experiments, the maximum
stimulation induced with 10 000 nM quinpirole was about 35% and
150% over basal activity for the D4 and D2short receptors, respec-
tively. Data analysis was done in the same manner described for
the mitogenesis experiments (see above) comparing the derived
EC50 values and efficacy to the effect of the reference compound
quinpirole.
Penile Erection Experiments. The effect of 3c on penile erection
was studied in male rats as described in the literature.50,51 Sprague-
Dowley male rats (225-250 g, Harlan Nossan, Italy) were housed
at 24 °C and 60% humidity, with water and standard laboratory
food ad libitum. Compound 3c and PD-168077 were dissolved in
ethanol and diluted with distilled water (0.3% ethanol concentration)
and injected s.cly in a volume of 0.2 mL/rat or icvly in a volume
of 10 µL/rat. For icv injections, a chronic stainless guide cannula
(28 gauge) was implanted stereotaxically in the skull of the animals
(coordinates A 1, L 1, and V 5 52) under chloral hydrate anaesthesia
three days before the experiments. The day of the experiments, the
male rats were individually placed in Plexiglas cages (30 × 30 ×
30 cm3) for 30 min and injected s.cly or icvly and observed for 60
min to count penile erection episodes. When haloperidol, clozapine,
or L-745,870 was used, the compound was dissolved in saline and
administered by i.p. injections 15 min before the D4 agonists were
administered. Penile erection was scored when the penis emerged
from the penile sheath, which was usually accompanied by penile
grooming and hip flexion. At the end of the experiments, the rats
were sacrificed by decapitation, and the brains were removed and
stored in 2% aqueous formaldehyde for 12-15 days. To localize
the position of the tip of the injection cannula, 50 µm transverse
brain sections were prepared by means of a freezing microtome
stained with Neutral Red and inspected on a phase contrast
microscope. The site of the probe tip was localized by following
the probe tract through a series of brain sections. Only those animals
found to have the probe tip correctly positioned icvly were
considered for the statistical evaluation of the results (Student’s t
test).
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Acknowledgment. We thank Dr. H. H. M. Van Tol (Clarke
Institute of Psychiatry, Toronto), Dr. J.-C. Schwartz, and Dr.
P. Sokoloff (INSERM, Paris) as well as Dr. J Shine (The Garvan
Institute of Medical Research, Sydney) for providing dopamine
D4, D3, and D2 receptor expressing cell lines, respectively. Dr.
R. Huff (Pharmacia & Upjohn, Inc. Kalamazoo, MI) is
acknowledged for providing D4 expressing cell lines employed
for mitogenesis. Thanks are also due to Mrs H. Szczepanek,
Mrs P. Schmitt, and Mrs P. Hu¨bner for skillfull technical
assistance. This work was supported by the Deutsche Fos-
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Industrie. C.E.-G. and A.G. would like to thank Ms. Margaret
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Supporting Information Available: X-ray crystallographic data
for compound 3a and purity data of compounds 3a-i, 4a-b, 7a-
e, and 8a-e. This material is available free of charge via the Internet
at http://pubs.acs.org.
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