Synthesis and structure-activity relationships of a new model of arylpiperazines. 5. Study of the physicochemical influence of the pharmacophore on 5-HT1A/α1-adrenergic receptor affinity: Synthesis of a new derivative with mixed 5-HT1A/D2 antagonist properties

Article abstract of DOI:10.1021/jm000929u

In this paper we have designed and synthesized a test series of 32 amide arylpiperazine derivatives VI in order to gain insight into the physicochemical influence of the pharmacophores of 5-HT_1A and α₁-adrenergic receptors. The training set was designed applying a fractional factorial design using six physicochemical descriptors. The amide moiety is a bicyclohydantoin or a diketopiperazine (X = -(CH₂)₃-, -(CH₂)₄-; m = 0, 1), the spacer length is 3 or 4 methylene units, which are the optimum values for both receptors, and the aromatic substituent R occupies the ortho- or meta-position and has been selected from a database of 387 substituents using the EDISFAR program. The 5-HT_1A and α₁-adrenergic receptor binding affinities of synthesized compounds VI (1-32) have been determined. This data set has been used to derive classical quantitative structure-activity relationships (QSAR) and neural networks models for both receptors (following paper). A comparison of these models gives information for the design of the new ligand EF-7412 (46) (5-HT_1A: K_i = 27 nM; α₁: K_i > 1000 nM). This derivative displays affinity for the dopamine D₂ receptor (K_i = 22 nM) and is selective versus all other receptors examined (5-HT_2A, 5-HT₃, 5-HT₄ and Bz; K_i > 1000 nM). EF-7412 (46) acts as an antagonist in vivo in pre- and postsynaptic 5-HT_1A receptor sites and as an antagonist in the dopamine D₂ receptor. Thus, EF-7412 (46) is a derivative with mixed 5-HT_1A/D₂ antagonist properties and this derivative could be useful as a pharmacological tool.

Full text of DOI:10.1021/jm000929u

186
J . Med. Chem. 2001, 44, 186-197
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of a New Mod el of
Ar ylp ip er a zin es. 5.1 Stu d y of th e P h ysicoch em ica l In flu en ce of th e
P h a r m a cop h or e on 5-HT1A/r1-Ad r en er gic Recep tor Affin ity: Syn th esis of a New
Der iva tive w ith Mixed 5-HT1A/D2 An ta gon ist P r op er ties†
Mar´ıa L. Lo´pez-Rodr´ıguez,*,§ M. J ose´ Morcillo,‡ Esther Ferna´ndez,§ Esther Porras,§ Luis Orensanz,
Ma Eugenia Beneytez,# J orge Manzanares,# and J ose Angel Fuentes#
Departamento de Quı´mica Orga´nica I, Facultad de Ciencias Qu´ımicas, and Departamento de Farmacologı´a, Facultad de
Farmacia, Universidad Complutense, 28040 Madrid, Spain, Facultad de Ciencias, Universidad Nacional de Educacio´n a
Distancia, 28040 Madrid, Spain, and Hospital Ramo´n y Cajal, Carretera de Colmenar km. 9, 28034 Madrid, Spain
Received February 23, 2000
In this paper we have designed and synthesized a test series of 32 amide arylpiperazine
derivatives VI in order to gain insight into the physicochemical influence of the pharmacophores
of 5-HT1A and R1-adrenergic receptors. The training set was designed applying a fractional
factorial design using six physicochemical descriptors. The amide moiety is a bicyclohydantoin
or a diketopiperazine (X ) -(CH2)3-, -(CH2)4-; m ) 0, 1), the spacer length is 3 or 4 methylene
units, which are the optimum values for both receptors, and the aromatic substituent R occupies
the ortho- or meta-position and has been selected from a database of 387 substituents using
the EDISFAR program. The 5-HT1A and R1-adrenergic receptor binding affinities of synthesized
compounds VI (1-32) have been determined. This data set has been used to derive classical
quantitative structure-activity relationships (QSAR) and neural networks models for both
receptors (following paper). A comparison of these models gives information for the design of
the new ligand EF-7412 (46) (5-HT1A: Ki ) 27 nM; R1: Ki > 1000 nM). This derivative displays
affinity for the dopamine D2 receptor (Ki ) 22 nM) and is selective versus all other receptors
examined (5-HT2A, 5-HT3, 5-HT4 and Bz; Ki > 1000 nM). EF-7412 (46) acts as an antagonist
in vivo in pre- and postsynaptic 5-HT1A receptor sites and as an antagonist in the dopamine
D2 receptor. Thus, EF-7412 (46) is a derivative with mixed 5-HT1A/D2 antagonist properties
and this derivative could be useful as a pharmacological tool.
In tr od u ction
class have a number of characteristic amino acid pat-
terns in common. In particular, the transmembrane
amino acid sequence of the 5-HT1A subtype is notewor-
thy for its high degree of homology to R1-adrenergic
receptor (approximately 45%).7 So, a great number of
ligands show high 5-HT1A affinity and poor selectivity.
In this way, we have synthesized8 a series of bicyclo-
hydantoin arylpiperazines I, which showed affinity for
Receptors interacting with serotonin (5-HT)2 can be
classified into at least 14 different subtypes on the basis
of operational pharmacology, sequence analysis, and
transduction mechanisms. During the past decade,
5-HT1A receptors have been a major target for neuro-
biological research and drug development.3 Many com-
pounds, belonging to different chemical classes, diplay
high affinity for 5-HT1A receptors. Most of these ligands
are agonists or partial agonists. Full antagonists, devoid
of any agonistic activity at presynaptic or postsynaptic
receptors, are still scarce. Agonists and partial agonists
have been proven to be effective in anxiety and depres-
sion.4 In addition to therapeutic applications in the field
of psychiatry, more recent preclinical studies have
suggested that 5-HT1A receptor agonists have also
pronounced neuroprotective properties.5 Potential thera-
peutic applications for 5-HT1A receptor antagonists are
evaluated, for example, in cognition disorders.6
5-HT1A and R1-adrenergic receptors. Structure-activity
relationship (SAR and 3D-QSAR) studies9 showed that
the length of the alkyl chain is of great importance for
5-HT1A and R1-adrenergic receptor affinities. As a
general trend, maximum affinity for 5-HT1A as well as
R1-adrenergic receptors is reached with n ) 3 or 4, and
reduction of the hydrocarbon chain by 2 and 1 carbon
atoms causes a decrease in affinity in both receptors.
Regarding the aromatic ring substitution it seems that
ortho- and meta-positions are favorable for the affinity
at both receptors, while para-substituted derivatives are
The 5-HT1A receptor belongs to the class of G-protein
coupled receptors (GPCRs), and the members of this
† Dedicated to Professor J ose´ Luis Soto on the occasion of his 70th
birthday.
* To whom correspondence should be addressed. Phone: 34-91-
3944239. Fax: 34-91-3944103. E-mail: mluzlr@eucmax.sim.ucm.es.
§ Departamento de Qu´ımica Orga´nica I, Universidad Complutense.
#
Departamento de Farmacolog´ıa, Universidad Complutense.
‡ Universidad Nacional de Educacio´n a Distancia.
Hospital Ramo´n y Cajal.
10.1021/jm000929u CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/21/2000

Synthesis and SARs of New Arylpiperazines. 5
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2 187
Ta ble 1. Values of the Parameters IA, IB, and In
practically inactive. As part of our ongoing program to
develop new selective compounds, we were interested
in a systematic research based on the no-pharmaco-
phoric and pharmacophoric sites of both receptors, to
gain insight into the structural factors that are respon-
sible for 5-HT1A/R1 selectivity.
parameter
-
+
IA
IB
In
-(CH2)3-
m ) 0
n ) 3
-(CH2)4-
m ) 1
n ) 4
With respect to the no-pharmacophoric sites, in recent
works1,10,11 we have described a series of derivatives II-
IV in order to determine the influence of electronic and
steric factors on the stabilization of receptor-ligand
complex. SAR studies10 on compounds II, which are
devoid of the terminal amide fragment present in
related 5-HT1A ligands but which preserve the steric
requeriments of this moiety, suggest that there is
influence of electronic factors on the no-pharmacophoric
part of the R1-adrenergic receptor site; however, they
have no influence on the stabilization of the 5-HT1A
receptor-ligand complex. In the series1,11 of arylpipera-
zines III and IV, in which we have explored some steric
requirements modifying the size and the shape of the
amide portion, with respect to the bicyclohydantoin I,
we could suggest that the no-pharmacophoric pocket in
the 5-HT1A receptor would have less restriction than the
corresponding pocket in the R1-adrenergic receptor.
These studies allow us to suggest some differences
between the no-pharmacophoric sites of both 5-HT1A and
R1-adrenergic receptors. Regarding the influence of
lipophilic factors, we have synthesized a new series of
compounds V, but no data has been published yet.
Ta ble 2. Marker Point Values Calculated for the Parameter
Space Using Eqs 1-3
parameter
-
0
+
π
σo,m
MR
-0.85
-0.19
8.54
0.25
0.18
16.63
1.34
0.54
24.71
and preliminary pharmacological characterization of
EF-7412 (46), a derivative with mixed 5-HT1A/D2 an-
tagonist properties.
Tr a in in g Set Design
The structural field of compounds VI is defined with
six parameters: three indicator variables (IA, IB and In)
which determine the size of the A and B rings and the
length of the alkyl chain, and the lipophilic (π), elec-
tronic (σo- or σm-), and steric (MR) parameters, which
determine the position and nature of the aromatic
substituent R. Indicator variable IA ) 0 or 1 for X )
-(CH2)3- or -(CH2)4-, IB ) 0 or 1 for m ) 0 (hydantoin)
or m ) 1 (diketopiperazine), and In ) 0 or 1 for n ) 3 or
4.
The application of a factorial experimental design 2n
would imply the synthesis of 64 compounds (26). How-
ever, we have reduced the number of compounds ap-
plying a fractional factorial design: 2n-1 ) 26-1 ) 32
compounds. In this case, the last variable (MR) is a
cross-term of the other five. To apply this technique all
the variables were represented in a binary form ((), and
each experiment was designed so each level combination
occurs only once in all the series. The meaning of the
parameters IA, IB and In is illustrated in Table 1. The
aromatic substituent R was selected using the database
(387 substituents) of the EDISFAR program.12 This
method considers three levels (+, 0, -) to each param-
eter, the marker points (Table 2). The program calcu-
lated the values of the marker points of each parameter
using eqs 1-3:
To gain insight into the physicochemical influence of
5-HT1A and R1 pharmacophores, we have designed a
training set of 32 compounds of general structure VI.
The amide moiety is a bicyclohydantoin or a diketo-
piperazine (X ) -(CH2)3-, -(CH2)4-; m ) 0, 1), the
spacer length is 3 or 4 methylene units, which are the
optimum values for both receptors, and the aromatic
substituent R occupies the ortho- or meta-position and
has been selected from a database of 387 substituents
using the EDISFAR program.12 In the present paper we
report the design, synthesis and binding profile on
5-HT1A and R1-adrenergic receptors of compounds VI.
This data set will be used to derive classical QSAR and
neural networks models for both receptors (following
paper), which will be useful in the design of the new
ligand EF-7412 (46) (5-HT1A: Ki ) 27 nM; R1: Ki > 1000
nM). We also report here the synthesis, binding assays,
P ) 1/K‚
P
(1)
(2)
(3)
∑
0
i
P- ) P0 - SD
P+ ) P0 + SD
where K is the total number of selected substituents in
the parameter space and SD is the standard deviation
for the corresponding parameter. EDISFAR looked for
the substituents which are nearest, in terms of the

188 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2
Ta ble 3. Selected Substituents for Each Marker Point
Lo´pez-Rodrı´guez et al.
sodium hydride and N,N-dimethylformamide (DMF)
(method A). Treatment of 1-arylpiperazines with 1-bromo-
3-chloropropane, in the presence of potassium carbonate
and DMF, gave the corresponding intermediates 34.
Derivatives 17-32 (n ) 4) were synthesized by treat-
ment of intermediates 35 with the corresponding
1-arylpiperazines in the presence of triethylamine and
in acetonitrile as solvent (method B). The reaction of
compounds 33 with 1,4-dibromobutane gave the key
intermediates 35. When R ) m-NH2 or m-NHCOPri,
compounds VI were prepared by catalytic hydrogenation
of the corresponding nitro compounds and a subsequent
reaction of the amino derivatives with isobutyryl chlo-
ride in pyridine (Scheme 2). Respective hydrochloride
salts were prepared as samples for biological assays. All
new compounds (Table 5) were characterized by IR and
1H and 13C NMR spectroscopy and gave satisfactory
combustion analyses (C, H, N).
marker
points
marker
points
R
R
- - -
+ - -
- + -
+ + -
o-OCH3 or m-NH2
o-CH3
- - +
+ - +
- + +
+ + +
o-CN
m-Br or m-CF3
o-CONHPr
o-COOPr
m-NHCOPri
o-OBu
Ta ble 4. Quality Tests for the Chosen Series: Correlation
Matrix, Its Determinant and Eigen Values for Orthogonality,
and Relative Variance Means for Dissimilarity
π
MR
σ
Eigen % var % cum
π
MR
σ
1
1.375 45.83 45.83
1.0318 34.39 80.23
0.5931 19.77 100
0.3517
-0.168
1
0.0411
1
corr matrix det ) 0.8415 (opt: 1.000)
corr coef mean ) 0.1869 (opt: 0.000)
rel var mean ) 1.2219 (should be >1.000)
Sch em e 1a
Bioch em istr y
The 5-HT1A and R1-adrenergic receptor binding af-
finities of synthesized compounds VI (1-32) were
determined by measurement of the displacement of [3H]-
8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) and
[3H]prazosin binding, respectively, in rat cerebral cortex
membranes. All the compounds were used in form of
hydrochloride salts and were water-soluble. The inhibi-
tion constant Ki was defined from the IC50 by the
Cheng-Prusoff equation.17 The results of these assays
are illustrated in Table 5.
Resu lts a n d Discu ssion
From the binding data, most of compounds VI dem-
onstrated moderate to high affinity for 5-HT1A and R1-
adrenergic receptor binding sites. The results, shown
in Table 5, lead to a series of considerations with regard
to the SAR for compounds of general structure VI. The
wide range of receptor affinities for these compounds,
which is due to a rational choice of substituents, makes
this material suitable for a QSAR investigation. The
results using Hansch analysis and neural networks will
be presented in a subsequent article. However, some
interesting SAR considerations can be made in advance.
(i) Regarding the length of the alkyl chain, although
most of compounds 1-32 show affinity at both receptors,
it seems that ligands 17-32 with a 4-carbon chain in
the spacer display higher affinities at 5-HT1A receptors
than comparably substituted compounds with a 3-car-
bon chain spacer (e.g., 28: Ki(5-HT1A) ) 3.6 nM vs 16:
Ki(5-HT1A) ) 214 nM; 18: Ki(5-HT1A) ) 3.9 nM vs 6:
Ki(5-HT1A) ) 60 nM). With respect to the R1-adrenergic
receptor both series (n ) 3 or 4) show a similar trend
(e.g., 7: Ki(R1) ) 13 nM and 19: Ki(R1) ) 8.0 nM; 11:
Ki(R1) ) 28 nM and 31: Ki(R1) ) 27 nM). This fact is in
agreement with the results previously reported by us1
for compounds III and IV, where the optimum length
of the alkyl chain for 5-HT1A affinity is 4 carbon atoms
and for R1-adrenergic receptors is 3-4 methylene units.
(ii) In general, the size of the amide portion in
compounds 1-32 seems to have no influence on R1-
adrenergic receptor affinity (e.g., 7, 11, 19 and 31: Ki-
(R1) ) 13, 28, 8.0 and 27 nM, respectively). Regarding
the 5-HT1A receptor, there is a little difference in the
affinity between the two series (n ) 3 or 4). For example,
a
Reagents: (a) NaH, DMF, N2, 60 °C, 1 h, then 34, 110 °C,
1-3 h; (b) NaH, DMF, N2, 60 °C, 1 h, then Br(CH2)4Br, 110 °C,
1-3 h; (c) 1-arylpiperazine, NEt3, CH3CN, 60 °C, 20-24 h.
normalized euclidean distance (NED), to the calculated
marker points. So, we selected in each group the
substituent nearest to the marker point or the most
adequate in terms of synthetic feasibility and stability
(Table 3). Once the substituents were selected, the
program tested their quality evaluating the orthogonal-
ity and the dissimilarity of the chosen series, expressed
as the correlation matrix and the relative variances
mean, respectively (Table 4).
Ch em istr y
The synthesis of derivatives VI (1-32) listed in Table
5 is outlined in Scheme 1. Compounds 1-16 (n ) 3)
were obtained by reaction of the hydantoins13,14 or
diketopiperazines15,16 33 with the corresponding 1-aryl-
4-(3-chloropropyl)piperazines 34 in the presence of

Synthesis and SARs of New Arylpiperazines. 5
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2 189
Ki ( SEM (nM)
Ta ble 5. Physical Properties and in Vitro Binding Dataa
compd
X
m
n
R
mp (°C)
formula
5-HT1A [3H]-8-OH-DPAT
R1 [3H]prazosin
1
2
3
4
5
6
7
8
9
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)3-
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
0
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
o-OBu
o-CONHPr
m-NH2
m-Br
o-CH3
187-190
181-183
151-153
205-207
180-182
185-186
55-57
198-201
259-261
69-70
214-215
193-195
149-151
187-188
104-107
276-278
231-233
69-70
185-186
189-192
214-216
85-87
C23H34N4O3‚2HCl
11 ( 2
>1000
30 ( 4
>1000
C23H33N5O3‚2HCl
C19H27N5O2‚2HCl‚2H2O
C19H25BrN4O2‚2HCl
C21H30N4O2‚2HCl
C24H34N4O4‚HCl‚H2O
C21H27N5O2‚2HCl‚1/2H2O
C24H35N5O3‚2HCl‚1/2H2O
C21H30N4O2‚2HCl
C24H34N4O4‚HCl‚H2O
C21H27N5O2‚2HCl
>1000
>1000
33 ( 9
134 ( 2
60 ( 5
29 ( 2
5.3 ( 0.2
41 ( 1
o-COOPr
o-CN
33 ( 12
>1000
13 ( 1
m-NHCOPri
o-CH3
>10000
23 ( 1
27 ( 1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
46
o-COOPr
o-CN
44 ( 3
26 ( 7
28 ( 3
31 ( 5
m-NHCOPri
o-OCH3
o-OBu
C24H35N5O3‚2HCl‚5/2H2O
C22H32N4O3‚2HCl‚2H2O
C25H38N4O3‚2HCl‚H2O
C25H37N5O3‚HCl‚H2O
C22H29F3N4O2‚2HCl‚H2O
C21H30N4O2‚HCl‚3/2H2O
C24H34N4O4‚HCl‚3/2H2O
C21H27N5O2‚HCl
326 ( 72
126 ( 8
110 ( 15
>10000
214 ( 21
16 ( 2
>1000
16 ( 1
79 ( 15
>1000
o-CONHPr
m-CF3
o-CH3
252 ( 3
10 ( 2
51 ( 4
o-COOPr
o-CN
3.9 ( 1.4
3.7 ( 0.8
102 ( 8
1.2 ( 0.1
341 ( 106
80 ( 18
4.0 ( 1.1
8.6 ( 0.7
6.5 ( 1.1
>1000
8.0 ( 1.4
>10000
0.4 ( 0.3
>1000
m-NHCOPri
o-OBu
C24H35N5O3‚2HCl‚H2O
C25H38N4O3‚2HCl
o-CONHPr
m-NH2
m-Br
o-OCH3
o-OBu
o-CONHPr
m-CF3
C25H37N5O3‚HCl‚3/2H2O
C21H31N5O2‚3HCl
167-169
175-176
204-206
188-190
88-90
182-183
266-267
77-78
215 ( 6
5.3 ( 0.3
28 ( 1
C21H29BrN4O2‚HCl‚H2O
C22H32N4O3‚2HCl‚H2O
C25H38N4O3‚2HCl‚2H2O
C25H37N5O3‚2HCl‚H2O
C22H29F3N4O2‚HCl‚H2O
C23H34N4O2‚HCl
C26H38N4O4‚2HCl‚2H2O
C23H31N5O2‚HCl‚H2O
C26H39N5O3‚2HCl‚1/2H2O
C22H33N5O4S‚2HCl‚1/2H2O
18 ( 8
>1000
3.6 ( 0.6
27 ( 8
24 ( 5
14 ( 1
169 ( 64
27 ( 6
95 ( 11
29 ( 2
561 ( 33
27 ( 5
o-CH3
o-COOPr
o-CN
98-100
216-218
187-190
m-NHCOPri
m-NHSO2Et
>10000
>10000
a
All values are means ( SEM of 2-4 experiments performed in triplicate.
Sch em e 2a
in compounds with n ) 4 has less effect (e.g., ligands
17 (X ) -(CH2)3-, m ) 0, n ) 4, R ) o-CH3: Ki ) 16
nM) and 29 (X ) -(CH2)4-, m ) 1, n ) 4, R ) o-CH3:
Ki ) 27 nM)). This fact can be explained considering
that the optimum length of the alkyl chain for 5-HT1A
affinity is 4 carbon atoms. So, ligands with n ) 3 and
with a no-pharmacophoric part with a bigger volume
(X ) -(CH2)4-, m ) 1) have higher restrictions to reach
the active site of the receptor, while the bicyclohydan-
toin derivatives (X ) -(CH2)3-, m ) 0) allow the
ligand-receptor interaction, probably because part of
the hydantoin portion acts as a spacer. This hypothesis
is in agreement with our previous reports.1
(iii) With respect to the phenylpiperazine substitution,
we observe only a small influence on 5-HT1A/R1 selectiv-
ity. Regarding the ortho-position, electron-donating
substituents (o-CH3, o-OCH3, o-OBu) as well as electron-
withdrawing substituents (o-COOPr, o-CN) give the
highest affinity values at both receptors. An exception
is represented by o-CONHPr, which leads to totally
inactive derivatives. Taking into account that this group
has a similar van der Waals volume as o-COOPr and
that it would not be possible for an unfavorable steric
interaction, we can explain the inactivity of these
derivatives by the smaller lipophilicity of this group
(πo-CONHPr ) -0.09 vs πo-COOPr ) 1.17). Thus, the
5-HT1A/R1 selectivity seems to be difficult to obtain with
aromatic substituents at this position.
a
Reagents: (a) H2, Pd(C), MeOH, rt, 1-3 h; (b) (CH3)2CHCOCl,
pyridine, 0 °C, 1-2 h.
comparing compound 1 (X ) -(CH2)3-, m ) 0, n ) 3, R
) o-OBu: Ki ) 11 nM) with 14 (X ) -(CH2)4-, m ) 1,
n ) 3, R ) o-OBu: Ki ) 110 nM) there is a 10-fold
decrease in 5-HT1A affinity. However, the same change
With regard to the meta-position we can observe that
the increase of volume in this position could lead to
5-HT1A-selective compounds. So, a m-bromo or m-amino
group (VW(Br) ) 17.2 Å3; VW(NH2) ) 11.4 Å3) leads to

190 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2
Lo´pez-Rodrı´guez et al.
Sch em e 3a
F igu r e 1. Dose-response and time-course effects of EF-7412
(46) on rectal temperature. Mouse rectal temperature was
measured 0, 5, 10, 15, 30, 60, and 90 min after sc administra-
tion of either vehicle (water, 4 mL/kg) or doses of EF-7412 (46)
(0, 5, 10, and 20 mg/kg). Values represent the means ( SEM
of rectal temperature in 6-8 mice. A hypothermic response
was considered when decrease of rectal temperature was more
than 1.1 °C.
a
Reagents: (a) EtSO2Cl, pyridine, acetone, N2, 24 h.
compounds with the same affinity at both receptors (e.g.,
derivatives 23 and 24). A trifluoromethyl group (VW
)
24.2 Å3) at this position leads to an increase in the
5-HT1A selectivity (compound 28: 26-fold). But the best
selectivity ratio is reached with the most voluminous
group, a m-NHCOPri (VW ) 70.8 Å3) which leads to the
most selective compounds 20 and 32 (98- and 59-fold,
respectively).
With these previous data, our aim is to gain deeper
insight into the physicochemical factors for 5-HT1A/R1
selectivity. Using classical QSAR (Hansch analysis) and
artificial neural networks (ANNs) studies in both recep-
tors, we have designed the new ligand EF-7412 (46) (X
) -(CH2)3-, m ) 0, n ) 4, R ) m-NHSO2Et) (subse-
quent paper). On this basis, EF-7412 (46) was synthe-
sized by reaction of the amino derivative 44 with
ethylsulfonyl chloride in the presence of pyridine in
anhydrous acetone as solvent (Scheme 3) and the
hydrochloride salt was prepared as a sample for biologi-
cal assays. This compound bound at 5-HT1A sites (Ki )
27 nM) and showed no significant affinity for the R1-
adrenergic receptor (Ki > 1000 nM) (Table 5). Due to
the high 5-HT1A affinity and selectivity over the R1-
adrenergic receptor, compound EF-7412 (46) was se-
lected for further binding and pharmacological studies.
Binding affinities show that compound EF-7412 (46)
exhibits an appreciable affinity for the D2 receptor
subtype (Ki ) 22 nM) and is selective versus all other
receptors examined (5-HT2A, 5-HT3, 5-HT4, and benzo-
diazepine (Bz); Ki > 1000 nM).
F igu r e 2. Time-course effects of EF-7412 (46) on 8-OH-
DPAT-induced hypothermia. Mouse rectal temperature was
measured before the administration of vehicle (water, 4 mL/
kg) or EF-7412 (46) (20 mg/kg). Vehicle or EF-7412 (46) was
given 15, 60, and 120 min before 8-OH-DPAT. Rectal temper-
ature was measured 30 min after 8-OH-DPAT (0.5 mg/kg).
Each bar represents the means ( SEM of rectal temperature
in 6-8 mice. *Values from the 8-OH-DPAT group (line bar)
that are significantly different from the vehicle control group
(white bar). **Values from the EF-7412 (46) groups (black
bars) that are significantly different (P < 0.05) from the 8-OH-
DPAT control group.
P h a r m a cologica l Resu lts. Pharmacological evalu-
ation of the activity of EF-7412 (46) on 5-HT1A receptor
function was assessed by measuring mouse rectal tem-
perature, 5-HT1A agonist-induced behavioral syndrome
in the rat (flat body posture (FBP) and lower lip
retraction (LLR)), corticosterone plasma levels in the
rat, and 5-hydroxyindoleacetic acid (5-HIAA)/5-HT in
mice hypothalamus. Dopamine receptor function was
assesed in mice by measuring 3,4-dihydroxyphenylacetic
acid (DOPAC)/dopamine (DA) ratio in hypothalamus,
and spontaneous locomotor activity in the open field.
Presynaptic 5-HT1A receptor function was assessed by
measuring mouse rectal temperature. Doses ranging
from 5 to 20 mg/kg of EF-7412 (46) did not alter rectal
temperature in mice (Figure 1) but blocked hypothermia
induced by the administration of 8-OH-DPAT (0.5 mg/
kg) (Figure 2). The action of EF-7412 (46) at postsyn-
aptic sites was evaluated by measuring the intensity of
the 5-HT1A agonist-induced behavioral syndrome in the
rat (FBP and LLR) and the increase of corticosterone
secretion in the rat. The administration of EF-7412 (46)
(10 mg/kg) 60 min before behavioral evaluation did not
alter FBP or LLR, but pretreatment with EF-7412 (46)
(1-10 mg) 30 min before the administration of 8-OH-
DPAT (0.5 mg/kg) completely blocked both effects
(Figure 3). Similarly, doses of EF-7412 (46) in the range
0.3-10 mg/kg did not alter corticosterone plasma levels
in the rat, but pretreatment with EF-7412 (46) (10 mg/
kg) 30 min before the administration of 8-OH-DPAT (1
mg/kg) significantly attenuated the enhancement of
corticosterone secretion induced by 8-OH-DPAT (Figure
4). The administration of EF-7412 (46) induced an
increase in 5-HIAA/5-HT and DOPAC/dopamine ratios
in whole hypothalamus of mice. The administration of
8-OH-DPAT blocks the increase in 5-HT neuronal
activity induced by EF-7412 (46) but was without effect
on dopamine neuronal activity (Figure 5A,B). Further

Synthesis and SARs of New Arylpiperazines. 5
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2 191
F igu r e 3. Time-course effects of EF-7412 (46) on FBP and
LLR induced by 8-OH-DPAT. According to an arbitrary scale
going from 0 (no effect) to 3 (maximum effect), FBP and LLR
were evaluated before and 20 min after sc administration of
vehicle (water, 4 mL/kg) or 8-OH-DPAT (1 mg/kg). Vehicle or
EF-7412 (46) (10 mg/kg) was given 30 min before 8-OH-DPAT.
Each bar represents the means ( SEM of a FBP or LLR score
of 8 rats. *Values from the 8-OH-DPAT control group that are
significantly different (P < 0.05) from the vehicle control group.
**Values from the EF-7412 (46)/8-OH-DPAT group that are
significantly different (P < 0.05) from the 8-OH-DPAT control
group. Means score of vehicle-treated rats was 0.
F igu r e 5. Effects of EF-7412 (46) on 5-HIAA/5-HT (A) and
DOPAC/dopamine (B) ratios in mouse whole hypothalamus.
5-HIAA, 5-HT, DOPAC, and dopamine concentrations were
measured 60 min after administration of vehicle (water, 4 mL/
kg sc) or EF-7412 (46) (10 mg/kg). 30 min after EF-7412 (46),
mice received 8-OH-DPAT (0.3 mg/kg). Values represent the
means ( SEM of 5-HIAA/5-HT and DOPAC/dopamine ratios
in vehicle- or drug-treated mice. *Values from EF-7412 (46)
or EF-7412 (46)/8-OH-DPAT-treated mice that are signifi-
cantly different (P < 0.05) from their respective vehicle-treated
mouse control group.
F igu r e 4. EF-7412 (46) effects on plasma corticosterone
increase induced by 8-OH-DPAT in rats. Vehicle (water, 4 mL/
kg) or 8-OH-DPAT (1 mg/kg) was administered 30 min after
administration of vehicle or EF-7412 (46) (10 mg/kg). Values
represent the mean ( SEM of plasma corticosterone levels in
6-8 rats. *Values from the 8-OH-DPAT group that are
significantly different (P < 0.05) from the vehicle control group.
**Values from the EF-7412 (46)/8-OH-DPAT group that are
significantly different (P < 0.05) from the 8-OH-DPAT control
group.
F igu r e 6. Dose-response effects of EF-7412 (46) on locomotor
activity in mice. The total squares crossed by a mouse were
counted, 60 min after administration of either vehicle (water,
4 mL/kg sc) or doses of EF-7412 (46) (0.5, 2.5, 10, 20, and 40
mg/kg). Values represent the means ( SEM of total squares
crossed during 5 min test in vehicle- and drug-treated mice.
*Values from 8-OH-DPAT-treated mice that are significantly
different (P < 0.05) from their respective vehicle-treated group.
evaluation of EF-7412 (46) at the D2 dopamine receptor
was carried out by examining the effects of this com-
pound on spontaneous locomotor activity in the open
field. The results showed that EF-7412 (46) produces a
slight decrease in spontaneous locomotor activity at the
highest dose used (Figure 6). Taken together, these
results suggest that EF-7412 (46) may be acting as an
antagonist at both 5-HT1A and D2 dopamine receptors.
Thus, a compound with dual action such as EF-7412
(46) may contribute to better understanding the role of
simultaneous blockade of both 5-HT1A and D2 receptors
upon serotoninergic and dopaminergic transmission.
Further pharmacological studies are being carried out
in our laboratory in order to determine the potential
antagonist properties of EF-7412 (46) at the dopamine
D2 receptors. The goal of these studies is to examine
the ability of EF-7412 (46) to block behaviors mediated
by D2 receptors such as climbing behavior induced by

192 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2
Lo´pez-Rodrı´guez et al.
1-(3-Ch lor op r op yl)-4-(o-(p r op oxyca r bon yl)p h en yl)p ip -
er a zin e (34d ): yield 2.69 g (69%); oil. Anal. (C17H25ClN2O2)
C, H, N.
1-(3-Ch lor op r op yl)-4-(o-(p r op ylca r ba m oyl)p h en yl)p ip -
er a zin e (34e): yield 2.76 g (71%); oil. Anal. (C17H26ClN3O)
C, H, N.
apomorphine, stereotypy and increased locomotor activ-
ity induced by amphetamine, and the presence or lack
of catalepsy induced by higher doses of this compound.
These results will be included in a pharmacological
manuscript.
1-(3-C h lo r o p r o p y l)-4-(o-c y a n o p h e n y l)p ip e r a zin e
(34f): yield 2.06 g (65%); oil. Anal. (C14H18ClN3) C, H, N.
1-(3-Ch lor op r op yl)-4-(m -(tr iflu or om eth yl)p h en yl)p ip -
er a zin e (34g): yield 2.61 g (71%); oil. Anal. (C14H18ClF3N2)
C, H, N.
1-(3-C h lo r o p r o p y l)-4-(m -n it r o p h e n y l)p ip e r a zin e
(34h ): yield 2.55 g (75%); oil. Anal. (C13H18ClN3O2) C, H, N.
4-(3-C h lo r o p r o p y l)-1-(m -b r o m op h e n yl)p ip e r a zin e
(34i): yield 2.44 g (64%); oil. Anal. (C13H18BrClN2) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
35c,d . To a suspension of the corresponding diketopiperazines
33c,d (26 mmol) in anhydrous N,N-dimethylformamide (30
mL) was added 60% NaH (1.0 g, 26 mmol). After stirring for
1 h at 60 °C under argon, a solution of 1,4-dibromobutane (11.2
g, 52 mmol) in anhydrous N,N-dimethylformamide (25 mL)
was added dropwise. The mixture was refluxed under argon
at 110 °C for 1-3 h. Then, the solvent was evaporated under
reduced pressure, and the residue was resuspended in water
(50 mL) and extracted with methylene chloride (3 × 50 mL).
The combined organic layers were washed with water and
dried over MgSO4. After evaporation of the solvent, the crude
oil was purified by column chromatography and then distil-
lation.
Con clu sion s
We report on the design and synthesis of a training
set of 32 arylpiperazines of general structure VI with
affinity for 5-HT1A and R1-adrenergic receptors. To gain
insight into the physicochemical influence of the phar-
macophore on 5-HT1A/R1 selectivity, classical QSAR and
neural networks models for both receptors are described
in the following paper. A comparison of these models
gives information for the design and synthesis of a new
ligand EF-7412 (46) (5-HT1A: Ki ) 27 nM; R1: Ki > 1000
nM). This compound displays affinity for the dopamine
D2 receptor (Ki ) 22 nM) and is selective versus all other
receptors examined (5-HT2A, 5-HT3, 5-HT4 and Bz).
Pharmacological evaluation of the activity of EF-7412
(46) suggests that it acts as an antagonist in vivo at
pre- and postsynaptic 5-HT1A receptor sites and as an
antagonist at the dopamine D2 receptor.
In this study we describe a derivative (EF-7412, 46)
with mixed 5-HT1A/D2 antagonist properties, and this
derivative could be useful as a pharmacological tool.
2-(4-Br om ob u t yl)-1,4-d ioxop e r h yd r op yr r olo[1,2-a ]-
p yr a zin e (35c): yield 4.14 g (55%); bp 200-220 °C (0.4
mmHg); 1H NMR (CDCl3) δ 1.66-2.16 (m, 7H), 2.36-2.45 (m,
1H), 3.32-3.69 (m, 6H), 3.80 (d, 1H), 4.10 (t, 1H), 4.16 (d, 1H);
13C NMR (CDCl3) δ 22.6, 25.6, 28.9, 29.5, 33.0, 45.1, 45.3, 51.6,
59.0, 163.1, 167.3. Anal. (C11H17BrN2O2) C, H, N.
2-(4-Br om ob u t yl)-1,4-d ioxop e r h yd r op yr id o[1,2-a ]-
p yr a zin e (35d ): yield 4.10 g (52%); bp 145-155 °C (0.2
mmHg). Anal. (C12H19BrN2O2) C, H, N.
Exp er im en ta l Section
Ch em istr y. Melting points (uncorrected) were determined
on a Gallenkamp electrothermal apparatus. Infrared (IR)
spectra were obtained on a Perkin-Elmer 781 infrared spec-
trophotometer. 1H and 13C NMR spectra were recorded on a
Varian VXR-300S or Bruker 250-AM instrument. Chemical
shifts (δ) are expressed in parts per million relative to internal
tetramethylsilane; coupling constants (J ) are in hertz. El-
emental analyses (C, H, N) were determined within 0.4% of
the theoretical values. Thin-layer chromatography (TLC) was
run on Merck silica gel 60 F-254 plates. For normal pressure
and flash chromatography, Merck silica gel type 60 (size 70-
230 and 230-400 mesh, respectively) was used. Unless stated
otherwise, starting materials used were high-grade commercial
products.
The hydantoins 33a ,b and the diketopiperazines 33c,d were
prepared following the procedure reported in refs 13-16.
Noncommercial 1-arylpiperazines (R ) o-OBu, o-COOPr, o-
CONHPr, m-Br, m-NO2) were prepared by reaction of the
corresponding anilines with bis(2-chloroethyl)amine, as previ-
ously described.18,19 1-(o-Cyanophenyl)piperazine18b was ob-
tained by reaction of o-bromobenzonitrile with piperazine. 1-(3-
Chloropropyl)-4-(o-tolyl)piperazine20 (34a ), 1-(3-chloropropyl)-
4-(o-methoxyphenyl)piperazine21 (34b) and compounds 35a ,b8c
were synthesized according to the literature.
1-Ar yl-4-(3-ch lor op r op yl)p ip er a zin es 34c-i. Gen er a l
P r oced u r e. To a stirred suspension of anhydrous potassium
carbonate (1.7 g, 15 mmol), the corresponding 1-arylpiperazine
(12 mmol) in anhydrous N,N-dimethylformamide (12 mL),
under argon at room temperature, was added dropwise
1-bromo-3-chloropropane (1.5 mL, 15 mmol). The reaction
mixture was stirred at room temperature for 16-24 h and was
filtered to eliminate the potassium carbonate. The solvent was
evaporated under reduced pressure and the resultant oil was
purified by column cromatography (eluent: hexane/ethyl
acetate, relative proportions depending upon the compound).
4-(3-C h lo r o p r o p y l)-1-(o-b u t o x y p h e n y l)p ip e r a zin e
(34c): yield 1.98 g (53%); oil; 1H NMR (CDCl3) δ 0.99 (t, 3H),
1.52 (sex, 2H), 1.83 (qt, 2H), 2.00 (qt, 2H), 2.57 (t, 2H), 2.64
(brs, 4H), 3.11 (brs, 4H), 3.63 (t, 2H), 3.99 (t, 2H), 6.84-6.98
(m, 4H); 13C NMR (CDCl3) δ 13.8, 19.3, 29.7, 31.3, 43.1, 50.4,
53.4, 55.4, 67.4, 112.0, 117.9, 120.7, 122.5, 141.1, 151.5. Anal.
(C17H27ClN2O) C, H, N.
Meth od A. P r ep a r a tion of Com p ou n d s 1, 2, 4-7, 9-11,
13-16, a n d 36-38. To a solution of the corresponding
hydantoins or diketopiperazines 33a -d (6 mmol) in anhydrous
N,N-dimethylformamide (3.3 mL) was added slowly 60% NaH
(0.25 g, 6 mmol), and the mixture was warmed under argon
at 60 °C for 1 h. After stirring 1 h, a solution of the
corresponding 1-aryl-4-(3-chloropropyl)piperazine 34 (6 mmol)
in anhydrous N,N-dimethylformamide (3.3 mL) was added
dropwise and the mixture was refluxed at 110 °C under argon
for 1-2 h. Then, the solvent was evaporated under reduced
pressure, the residue was resuspended in water (50 mL) and
extracted with methylene chloride (3 × 50 mL). The combined
organic layers were washed with water and dried over
anhydrous MgSO4. After evaporation of the solvent, the crude
oil was purified by column chromatography (eluents: hexane/
ethyl acetate, ethyl acetate/ethanol or chloroform/methanol,
relative proportions depending upon the compound). Spectral
data of title compounds refer to the free bases, and then
hydrochloride salts were prepared.
2-[3-[4-(o-Bu toxyp h en yl)p ip er a zin -1-yl]p r op yl]-1,3-d i-
oxoper h ydr opyr r olo[1,2-c]im idazole (1): yield 2.19 g (75%);
mp 187-190 °C (methanol/ethyl ether); 1H NMR (CDCl3) δ
0.99 (t, J ) 7.5, 3H, CH3), 1.51 (sex, J ) 7.5, 2H, CH3CH2),
1.65-1.72 (m, 1H, H7), 1.77-1.86 (m, 4H, CH2, CH2CH2O),
2.03-2.10 (m, 2H, 2H6), 2.22-2.26 (m, 1H, H7), 2.45 (t, J )
7.5, 2H, CH2-Npip), 2.62 (brs, 4H, 2CH2-pip), 3.10 (brs, 4H,
2CH2-pip), 3.20-3.28 (m, 1H, H5), 3.55 (td, J ) 7.5, 0.9, 2H,
NCH2), 3.68 (dt, J ) 11.1, 7.8, 1H, H5), 3.98 (t, J ) 6.6, 2H,
OCH2), 4.08 (dd, J ) 9.0, 7.5, 1H, H7a), 6.83-6.97 (m, 4H, ArH);
13C NMR (CDCl3) δ 13.8 (CH3), 19.3 (CH3CH2), 25.0 (CH2), 26.9
(C6), 27.4 (C7), 31.3 (CH2CH2O), 37.3 (NCH2), 45.4 (C5), 50.4
(2CH2-pip), 53.3 (2CH2-pip), 55.7 (CH2-Npip), 63.2 (C7a), 67.4
(OCH2), 111.9 (C6-phenyl), 117.9 (C3-phenyl), 120.7 (C4-phenyl),
122.5 (C5-phenyl), 141.1 (C1-phenyl), 151.5 (C2-phenyl), 160.7
(C3), 173.8 (C1). Anal. (C23H34N4O3‚2HCl) C, H, N.

Synthesis and SARs of New Arylpiperazines. 5
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2 193
2-[3-[4-(o-(P r opylcar bam oyl)ph en yl)piper azin -1-yl]pr o-
p yl]-1,3-d ioxop er h yd r op yr r olo[1,2-c]im id a zole (2): yield
1.95 g (65%); mp 181-183 °C (methanol/ethyl ether). Anal.
(C23H33N5O3‚2HCl) C, H, N.
2-[3-[4-(m -Br om op h en yl)p ip er a zin -1-yl]p r op yl]-1,3-d i-
oxoper h ydr opyr r olo[1,2-c]im idazole (4): yield 2.28 g (77%);
mp 205-207 °C (methanol/ethyl ether). Anal. (C19H25BrN4O2‚
2HCl) C, H, N.
Npip), 2.52 (td, J ) 13.0, 2.5, 1H, H6ax), 2.64 (brs, 4H, 2CH2-
pip), 3.09 (brs, 4H, 2CH2-pip), 3.44 (t, J ) 7.2, 2H, NCH2), 3.83
(d, J ) 12.0, 1H, H9a), 3.86 (s, 3H, OCH3), 4.02 (s, 2H, 2H3),
4.67 (dm, J ) 13.2, 1H, H6ec), 6.84-7.02 (m, 4H, ArH); 13C
NMR (CDCl3) δ 23.6 (CH2), 24.2, 24.4 (C7,C8), 31.2 (C9), 42.3
(C6), 44.5 (NCH2), 49.4 (C3), 50.5 (2CH2-pip), 53.3 (2CH2-pip),
55.2 (OCH3), 55.4 (CH2-Npip), 59.1 (C9a), 111.0 (C6-phenyl),
118.1 (C3-phenyl), 120.8 (C4-phenyl), 122.8 (C5-phenyl), 141.1
(C1-phenyl), 152.1 (C2-phenyl), 161.6 (C4), 165.1 (C1). Anal.
(C22H32N4O3‚2HCl‚2H2O) C, H, N.
2-[3-[4-(m -Nit r op h en yl)p ip er a zin -1-yl]p r op yl]-1,3-d i-
oxop er h yd r op yr r olo[1,2-c]im id a zole (36): yield 1.93 g
2-[3-[4-(o-Bu toxyp h en yl)p ip er a zin -1-yl]p r op yl]-1,4-d i-
oxoper h ydr opyr ido[1,2-a ]pyr azin e (14): yield 1.66 g (52%);
mp 187-188 °C (acetone). Anal. (C25H38N4O3‚2HCl‚H2O) C, H,
N.
2-[3-[4-(o-(P r opylcar bam oyl)ph en yl)piper azin -1-yl]pr o-
p yl]-1,4-d ioxop er h yd r op yr id o[1,2-a ]p yr a zin e (15): yield
2.11 g (69%); mp 104-107 °C (methanol/ethyl ether). Anal.
(C25H37N5O3‚HCl‚H2O) C, H, N.
2-[3-[4-(m-(Tr iflu or om eth yl)p h en yl)pip er a zin -1-yl]p r o-
p yl]-1,4-d ioxop er h yd r op yr id o[1,2-a ]p yr a zin e (16): yield
2.32 g (73%); mp 276-278 °C (methanol/ethyl ether). Anal.
(C22H29F3N4O2‚2HCl‚H2O) C, H, N.
Meth od B. P r ep a r a tion of Der iva tives 17-19, 21, 22,
24-31, a n d 39-41. To a suspension of the corresponding
derivatives 35a -d (9 mmol) and the arylpiperazine (15 mmol)
in acetonitrile (19 mL) was added 2.0 mL of triethylamine (1.5
g, 14.6 mmol). The mixture was refluxed for 20-24 h. Then,
the solvent was evaporated under reduced pressure and the
residue was resuspended in water and extracted with dichlo-
romethane (3 × 100 mL). The combined organic layers were
washed with water and dried over MgSO4. After evaporation
of the solvent the crude oil was purified by column chroma-
tography (eluents: hexane/ethyl acetate, ethyl acetate/ethanol
or chloroform/methanol, relative proportions depending upon
the compound). Spectral data refer to the free base and then
hydrochloride salts were prepared.
(76%); mp 223-226 °C (methanol/ethyl ether). Anal. (C19H25
-
N5O4‚HCl) C, H, N.
2-[3-[4-(o-Tolyl)p ip er a zin -1-yl]p r op yl]-1,3-d ioxop er h y-
d r oim id a zo[1,5-a ]p yr id in e (5): yield 2.15 g (81%); mp 180-
1
182 °C (methanol/ethyl ether); H NMR (CDCl3) δ 1.24-1.50
(m, 3H, H6ax, H7ax, H8ax), 1.73 (d, J ) 9.6, 1H, H6ec), 1.83 (qt, J
) 7.5, 2H, CH2), 1.97 (d, J ) 12.9, 1H, H7ec), 2.20 (dd, J )
12.6, 3.0, 1H, H8ec), 2.27 (s, 3H, CH3), 2.44 (t, J ) 7.5, 2H,
CH2-Npip), 2.57 (brs, 4H, 2CH2-pip), 2.82 (td, J ) 13.2, 3.3,
1H, H5ax), 2.90 (t, J ) 4.8, 4H, 2CH2-pip), 3.57 (t, J ) 7.5, 2H,
NCH2), 3.73 (dd, J ) 11.7, 3.9, 1H, H8a), 4.16 (dd, J ) 12.9,
4.2, 1H, H5ec), 6.92-7.01 (m, 2H, H4- and H6-phenyl), 7.12-
7.16 (m, 2H, H3- and H5-phenyl); 13C NMR (CDCl3) δ 17.8
(CH3), 22.7 (C7), 24.9 (C6), 25.3 (CH2), 27.7 (C8), 37.1 (NCH2),
39.2 (C5), 51.6 (2CH2-pip), 53.6 (2CH2-pip), 55.8 (CH2-Npip),
57.3 (C8a), 118.8 (C6-phenyl), 122.9 (C4-phenyl), 126.4 (C5-
phenyl), 130.9 (C3-phenyl), 132.4 (C2-phenyl), 151.4 (C1-phen-
yl), 154.5 (C3), 173.1 (C1). Anal. (C21H30N4O2‚2HCl) C, H, N.
2-[3-[4-(o-(P r opoxycar bon yl)ph en yl)piper azin -1-yl]pr o-
p yl]-1,3-d ioxop er h yd r oim id a zo[1,5-a ]p yr id in e (6): yield
2.21 g (74%); mp 185-186 °C (methanol/ethyl ether). Anal.
(C24H34N4O4‚HCl‚H2O) C, H, N.
2-[3-[4-(o-Cya n op h en yl)p ip er a zin -1-yl]p r op yl]-1,3-d i-
oxoper h ydr oim idazo[1,5-a ]pyr idin e (7): yield 2.34 g (84%);
mp 55-57 °C (methanol/ethyl ether). Anal. (C21H27N5O2‚2HCl‚
1/2H2O) C, H, N.
2-[4-[4-(o-Tolyl)p ip er a zin -1-yl]b u t yl]-1,3-d ioxop er h y-
d r op yr r olo[1,2-c]im id a zole (17): yield 2.81 g (72%); mp
231-233 °C (methanol/ethyl ether); 1H NMR (CDCl3) δ 1.49-
1.74 (m, 5H, -(CH2)2-, H7), 1.98-2.11 (m, 2H, 2H6), 2.19-2.23
(m, 1H, H7), 2.29 (s, 3H, CH3), 2.42 (t, J ) 7.5, 2H, CH2-Npip),
2.59 (brs, 4H, 2CH2-pip), 2.93 (t, J ) 5.1, 4H, 2CH2-pip), 3.20-
3.26 (m, 1H, H5), 3.50 (t, J ) 7.5, 2H, NCH2), 3.67 (dt, J )
11.1, 6.9, 1H, H5), 4.06 (dd, J ) 9.3, 7.2, 1H, H7a), 6.96 (td, J
) 8.4, 1.2, 1H, H4-phenyl), 7.01 (dd, J ) 6.9, 1.5, 1H,
H6-phenyl), 7.13-7.17 (m, 2H, H3- and H5-phenyl); 13C NMR
(CDCl3) δ 17.7 (CH3), 23.9, 25.9 (-(CH2)2-), 26.8 (C6), 27.4 (C7),
38.6 (NCH2), 45.3 (C5), 51.5 (2CH2-pip), 53.5 (2CH2-pip), 57.9
(CH2-Npip), 63.1 (C7a), 118.7 (C6-phenyl), 122.8 (C4-phenyl),
126.3 (C5-phenyl), 130.8 (C3-phenyl), 132.3 (C2-phenyl), 151.3
(C1-phenyl), 160.6 (C3), 173.8 (C1). Anal. (C21H30N4O2‚HCl‚3/
2H2O) C, H, N.
2-[3-[4-(m -Nit r op h en yl)p ip er a zin -1-yl]p r op yl]-1,3-d i-
oxop er h yd r oim id a zo[1,5-a ]p yr id in e (37): yield 1.60 g
(61%); mp 228-231 °C (acetone). Anal. (C20H27N5O4‚HCl) C,
H, N.
2-[3-[4-(o-Tolyl)p ip er a zin -1-yl]p r op yl]-1,4-d ioxop er h y-
d r op yr r olo[1,2-a ]p yr a zin e (9): yield 2.26 g (85%); mp 259-
1
261 °C (methanol/ethyl ether); H NMR (CDCl3) δ 1.79 (qt, J
) 7.2, 2H, CH2), 1.92-2.08 (m, 3H, 2H7, H8), 2.29 (s, 3H, CH3),
2.35-2.39 (m, 1H, H8), 2.43 (t, J ) 6.9, 2H, CH2-Npip), 2.59
(brs, 4H, 2CH2-pip), 2.93 (t, J ) 4.8, 4H, 2CH2-pip), 3.39-3.46
(m, 1H, 1NCH2), 3.52-3.64 (m, 3H, 1NCH2, 2H6), 3.87 (d, J )
16.5, 1H, H3b), 4.08 (t, J ) 7.2, 1H, H8a), 4.19 (d, J ) 16.5, 1H,
H
3a), 6.97 (td, J ) 6.6, 1.2, 1H, H4-phenyl), 7.02 (d, J ) 7.2,
1H, H6-phenyl), 7.14-7.18 (m, 2H, H3- and H5-phenyl); 13C
NMR (CDCl3) δ 17.7 (CH3), 22.5 (C7), 24.3 (CH2), 28.6 (C8),
44.7 (NCH2), 45.0 (C6), 51.5 (2CH2-pip), 52.1 (C3), 53.5 (2CH2-
pip), 55.3 (CH2-Npip), 58.9 (C8a), 118.7 (C6-phenyl), 122.9 (C4-
phenyl), 126.3 (C5-phenyl), 130.8 (C3-phenyl), 132.3 (C2-
phenyl), 151.2 (C1-phenyl), 163.2 (C4), 167.1 (C1). Anal.
(C21H30N4O2‚2HCl) C, H, N.
2-[4-[4-(o-(P r op oxyca r bon yl)p h en yl)p ip er a zin -1-yl]bu -
tyl]-1,3-d ioxop er h yd r op yr r olo[1,2-c]im id a zole (18): yield
2.28 g (50%); mp 69-70 °C (acetone). Anal. (C24H34N4O4‚HCl‚
3/2H2O) C, H, N.
2-[4-[4-(o-Cya n op h en yl)pip er a zin -1-yl]bu tyl]-1,3-d ioxo-
p er h yd r op yr r olo[1,2-c]im id a zole (19): yield 2.41 g (64%);
mp 185-186 °C (methanol/ethyl ether). Anal. (C21H27N5O2‚
HCl) C, H, N.
2-[3-[4-(o-(P r opoxycar bon yl)ph en yl)piper azin -1-yl]pr o-
p yl]-1,4-d ioxop er h yd r op yr r olo[1,2-a ]p yr a zin e (10): yield
1.94 g (65%); mp 69-70 °C (acetone). Anal. (C24H34N4O4‚HCl‚
H2O) C, H, N.
2-[4-[4-(m -Nitr op h en yl)p ip er a zin -1-yl]bu tyl]-1,3-d ioxo-
p er h yd r op yr r olo[1,2-c]im id a zole (39): yield 2.29 g (58%);
mp 197-198 °C (methanol/ethyl ether). Anal. (C20H27N5O4‚
HCl) C, H, N.
2-[3-[4-(o-Cya n op h en yl)p ip er a zin -1-yl]p r op yl]-1,4-d i-
oxop er h yd r op yr r olo[1,2-a ]p yr a zin e (11): yield 2.45 g
(90%); mp 214-215 °C (acetone). Anal. (C21H27N5O2‚2HCl) C,
H, N.
2-[4-[4-(o-Bu toxyph en yl)piper azin -1-yl]bu tyl]-1,3-dioxo-
p er h yd r oim id a zo[1,5-a ]p yr id in e (21): yield 2.92 g (63%);
mp 214-216 °C (methanol/ethyl ether). Anal. (C25H38N4O3‚
2HCl) C, H, N.
2-[3-[4-(m -Nit r op h en yl)p ip er a zin -1-yl]p r op yl]-1,4-d i-
oxop er h yd r op yr r olo[1,2-a ]p yr a zin e (38): yield 0.97 g
(37%);mp 247-249 °C (methanol/ethylether).Anal.(C20H27N5O4‚
HCl) C, H, N.
2-[4-[4-(o-(P r op ylca r ba m oyl)p h en yl)p ip er a zin -1-yl]bu -
tyl]-1,3-d ioxop er h yd r oim id a zo[1,5-a ]p yr id in e (22): yield
2.29 g (49%); mp 85-87 °C (methanol/ethyl ether). Anal.
(C25H37N5O3‚HCl‚3/2H2O) C, H, N.
2-[4-[4-(m -Br om op h en yl)p ip er a zin -1-yl]b u t yl]-1,3-d i-
oxop er h yd r oim id a zo[1,5-a ]p yr id in e (24): yield 2.95 g
2-[3-[4-(o-Met h oxyp h en yl)p ip er a zin -1-yl]p r op yl]-1,4-
d ioxop er h yd r op yr id o[1,2-a ]p yr a zin e (13): yield 1.80 g
(59%); mp 149-151 °C (methanol/ethyl ether); 1H NMR
(CDCl3) δ 1.39-1.61 (m, 3H, H7ax, H8ax, H9ax), 1.70-1.76 (m,
1H, H7ec), 1.82 (qt, J ) 7.1, 2H, CH2), 1.99 (d, J ) 12.5, 1H,
H
8ec), 2.36 (dm, J ) 13.3, 1H, H9ec), 2.44 (t, J ) 6.9, 2H, CH2-

194 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2
Lo´pez-Rodrı´guez et al.
(65%); mp 175-176 °C (methanol/ethyl ether). Anal. (C21H29
BrN4O2‚HCl‚H2O) C, H, N.
2-[4-[4-(m -Nitr op h en yl)p ip er a zin -1-yl]bu tyl]-1,3-d ioxo-
p er h yd r oim id a zo[1,5-a ]p yr id in e (40): yield 2.80 g (65%);
mp 65-67 °C (methanol/ethyl ether). Anal. (C21H29N5O4‚HCl‚
3/2H2O) C, H, N.
2-[4-[4-(o-Meth oxyp h en yl)p ip er a zin -1-yl]bu tyl]-1,4-d i-
oxop er h yd r op yr r olo[1,2-a ]p yr a zin e (25): yield 2.30 g
(52%);mp 204-206 °C (methanol/ethylether).Anal.(C22H32N4O3‚
2HCl‚H2O) C, H, N.
2-[4-[4-(o-Bu toxyph en yl)piper azin -1-yl]bu tyl]-1,4-dioxo-
p er h yd r op yr r olo[1,2-a ]p yr a zin e (26): yield 2.43 g (49%);
mp 188-190 °C (acetone). Anal. (C25H38N4O3‚2HCl‚2H2O) C,
H, N.
2-[4-[4-(o-(P r op ylca r ba m oyl)p h en yl)p ip er a zin -1-yl]bu -
tyl]-1,4-d ioxop er h yd r op yr r olo[1,2-a ]p yr a zin e (27): yield
2.46 g (50%); mp 88-90 °C (acetone). Anal. (C25H37N5O3‚2HCl‚
H2O) C, H, N.
2-[4-[4-(m -(Tr iflu or om eth yl)p h en yl)p ip er a zin -1-yl]bu -
tyl]-1,4-d ioxop er h yd r op yr r olo[1,2-a ]p yr a zin e (28): yield
3.19 g (72%); mp 182-183 °C (acetone). Anal. (C22H29F3N4O2‚
HCl‚H2O) C, H, N.
2-[4-[4-(o-Tolyl)p ip er a zin -1-yl]b u t yl]-1,4-d ioxop er h y-
d r op yr id o[1,2-a ]p yr a zin e (29): yield 2.43 g (62%); mp 266-
267 °C (acetone/ethyl ether). Anal. (C23H34N4O2‚HCl) C, H, N.
2-[4-[4-(o-(P r op oxyca r bon yl)p h en yl)p ip er a zin -1-yl]bu -
tyl]-1,4-d ioxop er h yd r op yr id o[1,2-a ]p yr a zin e (30): yield
2.45 g (47%); mp 77-78 °C (methanol/ethyl ether). Anal.
(C26H38N4O4‚2HCl‚2H2O) C, H, N.
2-[4-[4-(o-Cya n op h en yl)p ip er a zin -1-yl]bu tyl]-1,4-d ioxo-
p er h yd r op yr id o[1,2-a ]p yr a zin e (31): yield 2.42 g (58%);
mp 98-100 °C (methanol/ethyl ether). Anal. (C23H31N5O2‚HCl‚
H2O) C, H, N.
-
(brine). The organic layer was dried (Na2SO4) and the solvent
evaporated under reduced pressure to afford the desired
compounds which were converted to their hydrochloride salts.
2-[3-[4-[m -(2-Met h ylp r op a n a m id o)p h en yl]p ip er a zin -
1-yl]p r op yl]-1,3-d ioxop er h yd r oim id a zo[1,5-a ]p yr id in e
(8): yield 1.44 g (69%); mp 198-201 °C (methanol/ethyl ether).
Anal. (C24H35N5O3‚2HCl‚1/2H2O) C, H, N.
2-[3-[4-[m -(2-Met h ylp r op a n a m id o)p h en yl]p ip er a zin -
1-yl]p r op yl]-1,4-d ioxop er h yd r op yr r olo[1,2-a ]p yr a zin e
(12): yield 1.23 g (55%); mp 193-195 °C (methanol/ethyl
ether). Anal. (C24H35N5O3‚2HCl‚5/2H2O) C, H, N.
2-[4-[4-[m -(2-Met h ylp r op a n a m id o)p h en yl]p ip er a zin -
1-yl]b u t yl]-1,3-d ioxop e r h yd r op yr r olo[1,2-c]im id a zole
(20): yield 1.32 g (62%); mp 189-192 °C (methanol/ethyl
ether). Anal. (C24H35N5O3‚2HCl‚H2O) C, H, N.
2-[4-[4-[m -(2-Meth ylp r op a n a m id o)p h en yl]p ip er a zin -1-
yl]bu t yl]-1,4-d ioxop er h yd r op yr id o[1,2-a ]p yr a zin e (32):
yield 0.90 g (41%); mp 216-218 °C (methanol/ethyl ether).
Anal. (C26H39N5O3‚2HCl‚1/2H2O) C, H, N.
2-[4-[4-(m -(Eth ylsu lfon a m id o)p h en yl)p ip er a zin -1-yl]-
b u t yl]-1,3-d ioxop er h yd r op yr r olo[1,2-c]im id a zole (E F -
7412, 46). To a suspension of 44 (2.40 g, 6 mmol) and dry
pyridine (0.8 mL) in anhydrous acetone (5.7 mL) at room
temperature under argon, was added ethylsulfonyl chloride
(0.6 mL, 0.8 g, 6 mmol). After stirring overnight at room
temperature, the mixture was diluted with water and the
solvents were evaporated under reduced pressure. The solid
residue was purified by column chromatography (ethyl acetate/
ethanol 9:1), to afford 2.28 g (82%) of EF-7412 (46), which was
converted to its hydrochloride salt: mp 187-190 °C (methanol/
ethyl ether); 1H NMR (CDCl3) δ 1.35 (t, J ) 7.5, 3H, CH3),
1.50-1.72 (m, 5H, -(CH2)2-, H7), 2.05-2.13 (m, 2H, 2H6), 2.23-
2.27 (m, 1H, H7), 2.41 (t, J ) 7.5, 2H, CH2-Npip), 2.57 (t, J )
4.8, 4H, 2CH2-pip), 3.12 (q, J ) 7.5, 2H, SO2CH2), 3.20 (t, J )
5.4, 4H, 2CH2-pip), 3.23-3.29 (m, 1H, H5), 3.50 (t, J ) 7.2,
2H, NCH2), 3.69 (dt, J ) 11.1, 7.2, 1H, H5), 4.09 (dd, J ) 9.0,
7.5, 1H, H7a), 6.67-6.71 (m, 2H, H4- and H6-phenyl), 6.82 (t, J
) 2.1, 1H, H2-phenyl), 7.18 (t, J ) 8.4, 1H, H5-phenyl); 13C
NMR (CDCl3) δ 8.2 (CH3), 23.6, 25.9 (-(CH2)2-), 26.9 (C6), 27.5
(C7), 38.6 (NCH2), 45.4 (C5, SO2CH2), 48.4 (2CH2-pip), 52.9
(2CH2-pip), 57.7 (CH2-Npip), 63.3 (C7a), 107.5 (C2-phenyl),
111.0, 112.1 (C4- and C6-phenyl), 130.0 (C5-phenyl), 137.9 (C3-
phenyl), 152.2 (C1-phenyl), 160.8 (C3), 174.0 (C1). Anal.
(C22H33N5O4S‚2HCl‚1/2H2O) C, H, N.
2-[4-[4-(m -Nitr op h en yl)p ip er a zin -1-yl]bu tyl]-1,4-d ioxo-
p er h yd r op yr id o[1,2-a ]p yr a zin e (41): yield 2.62 g (59%);
mp 192-193 °C (methanol/ethyl ether). Anal. (C22H31N5O4‚
HCl‚3/2H2O) C, H, N.
Gen er a l P r oced u r e. Syn th esis of Com p ou n d s 3, 23,
a n d 42-45. To a solution of 36-41 (5 mmol) in methanol (18
mL) was added 0.1 g of 10% Pd(C). The mixture was hydro-
genated (35 psi) at room temperature for 1-3 h. The reaction
mixture was filtered over Celite and evaporated to dryness to
afford the pure amines. Spectral data refer to the free bases,
and then hydrochloride salts were prepared.
2-[3-[4-(m -Am in op h en yl)p ip er a zin -1-yl]p r op yl]-1,3-d i-
oxoper h ydr opyr r olo[1,2-c]im idazole (3): yield 2.05 g (88%);
mp 151-153 °C (methanol/ethyl ether). Anal. (C19H27N5O2‚
2HCl‚2H2O) C, H, N.
Ra d ioliga n d Bin d in g Assa ys. For all receptor-binding
assays, male Sprague-Dawley rats (Rattus norvegicus albi-
nus), weighing 180-200 g, were killed by decapitation and the
brains rapidly removed and dissected.
2-[3-[4-(m -Am in op h en yl)p ip er a zin -1-yl]p r op yl]-1,3-d i-
oxop er h yd r oim id a zo[1,5-a ]p yr id in e (42): yield 2.40 g
(83%);mp 138-139 °C (methanol/ethylether).Anal.(C20H29N5O2‚
3HCl) C, H, N.
2-[3-[4-(m -Am in op h en yl)p ip er a zin -1-yl]p r op yl]-1,4-d i-
oxop er h yd r op yr r olo[1,2-a ]p yr a zin e (43): yield 2.64 g
(97%);mp 165-167 °C (methanol/ethylether).Anal.(C20H29N5O2‚
3HCl‚7/2H2O) C, H, N.
2-[4-[4-(m-Am in oph en yl)piper azin -1-yl]bu tyl]-1,3-dioxo-
p er h yd r op yr r olo[1,2-c]im id a zole (44): yield 2.23 g (93%);
mp 92-94 °C (methanol/ethyl ether). Anal. (C20H29N5O2‚3HCl)
C, H, N.
2-[4-[4-(m-Am in oph en yl)piper azin -1-yl]bu tyl]-1,3-dioxo-
p er h yd r oim id a zo[1,5-a ]p yr id in e (23): yield 1.98 g (80%);
mp 167-169 °C (methanol/ethyl ether). Anal. (C21H31N5O2‚
3HCl) C, H, N.
2-[4-[4-(m-Am in oph en yl)piper azin -1-yl]bu tyl]-1,4-dioxo-
p er h yd r op yr id o[1,2-a ]p yr a zin e (45): yield 2.60 g (97%);
mp 147-150 °C (d) (methanol/ethyl ether). Anal. (C22H33N5O2‚
3HCl‚3/2H2O) C, H, N.
Gen er a l P r oced u r e. P r ep a r a tion of Der iva tives 8, 12,
20, a n d 32. To a solution of 42-45 (4 mmol) in pyridine (50
mL) at 0 °C was added dropwise 0.5 mL of isobutyryl chloride
(0.50 g, 4 mmol). After stirring at room temperature for 1.5 h,
the mixture was washed with a saturated aqueous solution of
CuSO4, water and a saturated aqueous solution of NaCl
5-HT1A R ecep t or . The receptor binding studies were
performed by a modification of a previously described proce-
dure.22 The cerebral cortex was homogenized in 10 volumes of
ice-cold Tris buffer (50 mM Tris-HCl, pH 7.7 at 25 °C) and
centrifuged at 28000g for 15 min. The membrane pellet was
washed twice by resuspension and centrifugation. After the
second wash the resuspended pellet was incubated at 37 °C
for 10 min. Membranes were then collected by centrifugation
and the final pellet was resuspended in 50 mM Tris-HCl, 5
mM MgSO4, and 0.5 mM EDTA buffer (pH 7.4 at 37 °C).
Fractions of the final membrane suspension (about 1 mg of
protein) were incubated at 37 °C for 15 min with 0.6 nM [3H]-
8-OH-DPAT (133 Ci/mmol), in the presence or absence of
several concentrations of the competing drug, in a final volume
of 1.1 mL of assay buffer (50 mM Tris-HCl, 10 nM clonidine,
30 nM prazosin, pH 7.4 at 37 °C). Nonspecific binding was
determined with 10 µM 5-HT.
5-HT2A Recep tor . The receptor binding assays were per-
formed by a modification of a previously described procedure.23
The frontal cortex was homogenized in 60 volumes of ice-cold
buffer (50 mM Tris-HCl, 0.5 mM Na2EDTA, 10 mM MgSO4,
pH 7.4 at 37 °C), and centrifuged at 30000g for 15 min. The
membrane pellet was resuspended in buffer and incubated at
37 °C for 15 min. After centrifuging at 30000g for 15 min, the
membranes were washed twice by resuspension and centrifu-
gation, and the final pellet was resuspended in assay buffer

Synthesis and SARs of New Arylpiperazines. 5
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2 195
(50 mM Tris-HCl, 0.5 mM Na2EDTA, 10 mM MgSO4, 0.1%
ascorbic acid, 10 µM pargyline, pH 7.4 at 37 °C). Fractions of
the final membrane suspension (about 0.5 mg of protein) were
incubated at 37 °C for 15 min with 0.4 nM [3H]ketanserin (77
Ci/mmol), in the presence or absence of several concentrations
of the competing drug, in a final volume of 2 mL of assay
buffer. Nonspecific binding was determined with 1 µM cin-
anserin.
For all binding assays, competing drug, nonspecific, total
and radioligand bindings were defined in triplicate. Incubation
was terminated by rapid vacuum filtration through Whatman
GF/B filters, presoaked in 0.05% poly(ethylenimine), using a
Brandel cell harvester. The filters were then washed with the
assay buffer and dried. The filters were placed in poly-
(ethylene) vials to which was added 4 mL of a scintillation
cocktail (Aquasol), and the radioactivity bound to the filters
was measured by liquid scintillation spectrometry. The data
were analyzed by an iterative curve-fitting procedure (program
Prism, Graph Pad), which provided IC50, Ki, and r2 values for
test compounds, Ki values being calculated from the Cheng
and Prusoff equation.17 The protein concentrations of the rat
cerebral cortex and the rat striatum were determined by the
method of Lowry,29 using bovine serum albumin as the
standard.
5-HT3 Recep tor . Binding assays were performed according
to the procedure previously described in the literature.24 The
cerebral cortex was homogenized in 9 volumes of 0.32 M
sucrose and centrifuged at 1000g for 10 min. The supernatant
was centrifuged at 17000g for 20 min. The membrane pellet
was washed twice by resuspension in 60 volumes of 50 mM
Tris-HCl buffer (pH 7.4 at 25 °C) and centrifugation at 48000g
for 10 min. After the second wash the resuspended pellet was
incubated at 37 °C for 10 min, and centrifuged at 48000g for
10 min. Membranes were resuspended in 2.75 volumes of assay
buffer (50 mM Tris-HCl, 10 µM pargyline, 0.6 mM ascorbic
acid, and 5 mM CaCl2, pH 7.4 at 25 °C). Fractions of 100 µL
of the final membrane suspension (about 2 mg/mL of protein)
were incubated at 25 °C for 30 min with 0.7 nM [3H]LY 278584
(83 Ci/mmol), in the presence or absence of six concentrations
of the competing drug, in a final volume of 2 mL of assay
buffer. Nonspecific binding was determined with 10 µM 5-HT
and represented less than 30% of the total binding.
P h a r m a cologica l Meth od s. An im a ls. Male Swiss albino
mice (20-25 g, BW) and male Sprague-Dawley rats (200-
250 g, BW) were obtained from Interfauna Ibe´rica (Sant Feliu´
de Codines, Barcelona, Spain) and maintained in a tempera-
ture and light (25 ( 1 °C, light on between 8.00 a.m. and 8.00
p.m.) controlled environment. Food and tab water were
provided ad libitum. All experiments were performed between
9.00 a.m. and 1.00 p.m.
Hyp oth er m ia . After removal of mice from their home
cages, basal rectal temperature was measured with a lubri-
cated digital thermistoprobe which was inserted into the
rectum 1.5 cm for 40 s. Rectal temperature was determined
again after the appropriate treatments with 8-OH-DPAT, EF-
7412 (46) or vehicle. The difference between the temperature
measured before and after the administration represents an
index of hypothermia. A decrease of more than 1.1 °C from
basal rectal temperature was considered a hypothermic re-
sponse.
5-HT4 Recep tor . Binding assays were performed according
to the procedure previously described in the literature.25 The
striatum was homogenized in 15 volumes of ice-cold 50 mM
HEPES buffer (pH 7.4 at 4 °C) and centrifuged at 48000g for
10 min. The pellet was resuspended in 4.5 mL of assay buffer
(50 mM HEPES, pH 7.4 at 4 °C). Fractions of 100 µL of the
final membrane suspension were incubated at 37 °C for 30
min with 0.1 nM [2H]GR 113808 (85 Ci/mmol), in the presence
or absence of six concentrations of the competing drug, in a
final volume of 1 mL of assay buffer. Nonspecific binding was
determined with 30 µM 5-HT and represented less than 40%
of the total binding.
Eva lu a tion of 5-HT1A Recep tor -Med ia ted Beh a vior in
th e Ra t. Male Sprague-Dawley rats were acclimatized to the
housing environment and handled every day for 1 week before
testing. Rats were housed in groups of 3-4/cage. Lower lip
retraction (LLR) and flat body posture (FBP) were used to
evaluate the 5-HT1A receptor-mediated behavior. Both re-
sponses were measured 5, 10, 20 and 30 min after drug
administration by using a 0-3 scale as previously described.30
r1-Ad r en er gic Recep tor . The radioligand receptor binding
studies were performed according to a previously described
procedure.26 The cerebral cortex was homogenized in 20
volumes of ice-cold buffer (50 mM Tris-HCl, 10 mM MgCl2,
pH 7.4 at 25 °C) and centrifuged at 30000g for 15 min. Pellets
were washed twice by resuspension and centrifugation. Final
pellets were resuspended in the same buffer. Fractions of the
final membrane suspension (about 250 µg of protein) were
incubated at 25 °C for 30 min with 0.2 nM [3H]prazosin (23
Ci/mmol) in the presence or absence of several concentrations
of the competing drug, in a final volume of 2 mL of buffer.
Nonspecific binding was determined with 10 µM phentolamine.
D2-Dop a m in er gic Recep tor . The receptor binding studies
were performed according to a previously described proce-
dure.27 The corpus striatum was homogenized in 50 mM Tris-
HCl buffer (pH 7.7 at 25 °C) and centrifuged at 48000g for 10
min. The pellet was resuspended and centrifuged as before.
The final pellet was resuspended in 50 mM Tris-HCl buffer
(pH 7.4 at 25 °C) containing 120 mM NaCl, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2 and 0.1% ascorbic acid. Fractions of the
final membrane suspension (125-150 µg of protein) were
incubated at 25 °C for 60 min with 0.8 nM [3H]raclopride (77
Ci/mmol), in the presence or absence of the competing drug,
in a final volume of 1.1 mL of the assay buffer (pH 7.4 at 25
°C). Nonspecific binding was determined with 1 µM (+)-
butaclamol.
Cor ticoster on e Ra d ioim m u n oa ssa y. Male Sprague-
Dawley rats were acclimatized to the housing environment and
handled every day for 1 week before testing. Rats were housed
in groups of 3-4/cage. After the appropiate treatments with
the vehicle, EF-7412 (46) or EF-7412 (46)/8-OH-DPAT, rats
were decapited and trunk blood was collected. The samples
were centrifugated and plasma samples were stored frozen at
-80 °C before analysis. Corticosterone was extracted following
a previously reported method.31 Briefly, corticosterone binding
protein transcortin was hydrolyzed by incubation with trypsin
(Boehringuer, Mannheim, Madrid, Spain) for 60 min at room
temperature. Trypsin was inactivated by trypsin inhibitor
treatment (Boehringuer, Mannheim, Madrid, Spain) for 30 min
at room temperature. Corticosterone (Sigma Chemicals Co.,
Madrid, Spain) was used as the assay standard. Radioimmu-
noassays were performed adding to this mixture corticosterone
antiserum (Bioclin Clinical Services Ltd., Cardiff, U.K.) and
[3H]corticosterone (Amersham, Madrid, Spain) at specific
activity of 88 Ci/mmol (8000 cpm/tube). After incubation at 4
°C for 16-24 h, the antigen-antibody complex was separated
from unbound antigen with 1% charcoal and centrifugated at
4 °C, 3000 rpm, for 10 min. Finally, the supernatant was
decanted and mixed with liquid scintillation cocktail before
counting. The lower limit of sensitivity of assay was 2 ng/mL.
The intra- and interassay variability was 4.76% and 5.84%,
respectively.
Bz Recep tor . The receptor binding assays were performed
according to a previously described procedure.28 The cerebral
cortex was homogenized in 25 mM potassium phospate (KPi)
buffer (pH 7.4). Homogenate fractions (about 100 µg of protein)
were incubated at 0-4 °C for 90 min with 0.25 nM [3H]-
flunitrazepam (37 Ci/mmol), in the presence or absence of
several concentrations of the competing drug, in a final volume
of 1 mL of 25 mM KPi (pH 7.4). Nonspecific binding was
determined with 2 µM diazepam.
Neu r och em ica l Activity: 5-HIAA/5-HT a n d DOP AC/
DA Ra tios. Following appropriate treatments, mice were
decapitated and brains were removed from the skull. Hypho-
talami were dissected on ice and inmediately frozen over dry
ice. Tissues samples were placed in 200 µL of 0.1 M phosphate-

196 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 2
Lo´pez-Rodrı´guez et al.
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Wijngaarden, I., Soudijn, W., Eds.; Elsevier: The Netherlands,
1997. (b) Olivier, B.; Soudijn, W.; van Wijngaarden, I. The 5-HT1A
Receptor and its Ligands: Structure and Function. Prog. Drug
Res. 1999, 52, 103-165.
citrate buffer (pH 2.5) containing 15% methanol and stored
at -80 °C until assayed.
On the day of the assay tissue samples were thawed,
sonicated for 3 s (Vibra Cell, model VC-501, Sonics and
Materials Inc., Danbury, CT) and centrifuged for 60 s in a
Microfuge (IEC, model Centra-MP4R, Needham, MA). 5-HIAA,
5-HT, DOPAC and DA concentrations in hypothalamus tissue
extracts were measured by high-performance liquid chroma-
tography (HPLC) with electrochemical detection. 20 µL of the
supernatant were injected onto a C18 reverse-phase analytical
column (5-µm spheres, 250 × 4.6 mm; Nucleosi, Scharlau,
Barcelona, Spain) which was protected by a precolumn car-
tridge filter (5-µm spheres, 30 × 4.6 mm). The HPLC column
was coupled to a single coulometric electrode conditioning cell
in series with dual electrode analytical cells (Coulochem II,
ESA, Bedford, MA). The conditioning electrode potential was
set at 100 nA and the analytical electrodes were set at +0.12
and -0.31 V relative to internal Ag reference electrodes. The
HPLC mobile phase consisted of 1.0 M phosphate/citrate buffer
(pH 2.8) containing 0.1 mM ethylenediaminetetraacetic acid
(EDTA), 0.050% sodium octylsulfate and 25% methanol.
5-HIAA, 5-HT, DOPAC and dopamine contents of each sample
were quantitated by comparing peak heights with those peaks
of standards assayed the same day as determined by Shimadzu
intregrator (Shimadzu, model C-R4AX-CHROMATOPAC, Ky-
oto, J apan). The lower limit of sensitivity of this assay for these
compounds was 2-8 pg/sample. Tissue pellets were dissolved
in 1.0 N NaOH and assayed for protein.29
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Lines of Rats Differ in Social Interaction and Hippocampal
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Locom otor Activity. Open field locomotor activity was
measured in male Swiss albino mice, after the appropriate
treatments with EF-7412 (46) and 8-OH-DPAT, in an open
field. The apparatus consisted in a square box (30 × 30 cm)
with 10-cm high walls. The floor was a white plastic sheet
divided into 25 (5 × 5) equally sized squares painted in fine
black. Mice were maintained in the animal house at least 5
days before the experiment. On the day of the experiment
drugs were dissolved in distilled water and sc injected. Mice
were individually tested in 5-min sessions in the apparatus
described above. The floor of each box was cleaned between
sessions. Each mouse was tested by placing it in one corner of
the square field. Its behavior was recorded on a videotape and
locomotor activity analysis was subsequently performed from
the recording. The number of total squares were counted
during a 5-min test. A mouse whose four paws were in a new
square was considered as having crossed a square.
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Rosado, M. L.; Benhamu´, B.; Sanz, A. M. 2-[4-(o-Methoxyphenyl)-
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Synthesis and Structure-Activity Relationships of a New Model
of Arylpiperazines. 1. 2-[[4-(o-Methoxyphenyl)piperazin-1-yl]-
methyl]-1,3-dioxoperhydroimidazo[1,5-a]pyridine: A Selective
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M. J .; Ferna´ndez, E.; Schaper, K.-J . Synthesis and Structure-
Activity Relationships of a New Model of Arylpiperazines. 2.
Three-Dimensional Quantitative Structure-Activity Relation-
ships of Hydantoin-Phenylpiperazine Derivatives with Affinity
for 5-HT1A and R1 Receptors. A Comparison of CoMFA Models.
J . Med. Chem. 1997, 40, 1648-1656.
(10) Lo´pez-Rodr´ıguez, M. L.; Morcillo, M. J .; Ferna´ndez, E.; Porras,
E.; Murcia, M.; Sanz, A. M.; Orensanz, L. Synthesis and
Structure-Activity Relationships of a New Model of Arylpip-
erazines. 3. 2-[ω-(4-Arylpiperazin-1-yl)alkyl]perhydropyrrolo[1,2-
c]imidazoles and -perhydroimidazo[1,5-a]pyridines: Study of the
Influence of the Terminal Amide Fragment on 5-HT1A Affinity/
Selectivity. J . Med. Chem. 1997, 40, 2653-2656.
(11) Lo´pez-Rodr´ıguez, M. L.; Morcillo, M. J .; Rovat, T. K.; Ferna´ndez,
E.; Sanz, A. M.; Orensanz, L. 1-[ω-(4-Arylpiperazin-1-yl)alkyl]-
3-diphenylmethylene-2,5-pyrrolidinediones as 5-HT1A Receptor
Ligands: Study of the Steric Requirements of the Terminal
Amide Fragment on 5-HT1A Affinity/Selectivity. Bioorg. Med.
Chem. Lett. 1998, 8, 581-586.
Sta tistica l An a lyses. Statistical analyses were performed
using analysis of varriance followed by the Student-Neeew-
man-Keul’s test. Differences were considered significant if the
probability of error was less than 5%.
Ack n ow led gm en t. This work was supported by the
DGESIC (PB97-0282), CICYT 960360, and Comunidad
de Madrid (08.5/000466.1/98). The authors are grateful
to UNED for a predoctoral grant to E.F. and E.P.
Su p p or tin g In for m a tion Ava ila ble: Characterization
data for new compounds 2-4, 6-8, 10-12, 14-16, 18-33,
34d -i, 35d , and 36-45. This information is available free of
charge via the Internet at http://pubs.acs.org.
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