trans-1-[(2-phenylcyclopropyl)methyl]-4-arylpiperazines: Mixed dopamine D2/D4 receptor antagonists as potential antipsychotic agents

Article abstract of DOI:10.1021/jm990562x

The dopaminergic receptor profile of a series of trans-1-[(2-phenylcyclopropyl)methyl]-4-arylpiperazines was examined. Aromatic substitution patterns were varied with the goal of identifying a compound having affinities for the D₂ and D₄ receptors in a ratio similar to that observed for the atypical neuroleptic clozapine. The compounds (1S,2S)-trans-1-[(2-phenylcyclopropyl)methyl]-4-(2,4-dichlorophenyl)piperazin e (5m) and (1S,2S)-trans-1-[(2-phenylcyclopropyl)methyl]-4-(2,4-dimethylphenyl)piperazin e (5t) were selected for functional antagonists at D₂ and D₄ receptors and had a D₂/D₄ ratio approximating that of clozapine; they proved inactive in behavioral tests of antipsychotic activity.

Full text of DOI:10.1021/jm990562x

J . Med. Chem. 2000, 43, 3923-3932
3923
tr a n s-1-[(2-P h en ylcyclop r op yl)m eth yl]-4-a r ylp ip er a zin es: Mixed Dop a m in e
D2/D4 Recep tor An ta gon ists a s P oten tia l An tip sych otic Agen ts
Xiaoyan Zhang, Kevin Hodgetts, Stanislaw Rachwal, He Zhao, J an W. F. Wasley, Kimberly Craven,
Robbin Brodbeck, Andrzej Kieltyka, Diane Hoffman, Maria D. Bacolod, Brian Girard, J ennifer Tran, and
Andrew Thurkauf*
Neurogen Corporation, 35 Northeast Industrial Road, Branford, Connecticut 06405
Received November 9, 1999
The dopaminergic receptor profile of a series of trans-1-[(2-phenylcyclopropyl)methyl]-4-
arylpiperazines was examined. Aromatic substitution patterns were varied with the goal of
identifying a compound having affinities for the D2 and D4 receptors in a ratio similar to that
observed for the atypical neuroleptic clozapine. The compounds (1S,2S)-trans-1-[(2-phenylcy-
clopropyl)methyl]-4-(2,4-dichlorophenyl)piperazine (5m ) and (1S,2S)-trans-1-[(2-phenylcyclo-
propyl)methyl]-4-(2,4-dimethylphenyl)piperazine (5t) were selected for functional antagonists
at D2 and D4 receptors and had a D2/D4 ratio approximating that of clozapine; they proved
inactive in behavioral tests of antipsychotic activity.
In tr od u ction
Schizophrenia is a devastating psychiatric disorder
of unknown etiology whose symptoms may include
hallucinations and social withdrawal. In 1963, Carlsson
and Lindquist first put forward the hypothesis that
schizophrenia was linked to a malfunctioning of dopa-
minergic pathways in the central nervous system (CNS).1
Since that time, many converging lines of scientific
evidence have given support to this idea.2-4 While many
of the prominent antipsychotic agents bind to a wide
array of CNS receptor systems, each has a significant
affinity for the dopamine D2 receptor subtype as part
of its pharmacological profile. In addition, certain
structural classes have been shown to be selective for
dopamine D2-like receptors. Haloperidol (1) and remox-
ipride (2) are representative of two structurally distinct
classes of compounds, the butyrophenones and the
benzamides, respectively, which are both clinically
effective antipsychotic agents and antagonists at dopa-
mine receptor subtype. Haloperidol binds with high
affinity to D2, D3, and D4 receptors, while most repre-
sentatives of the benzamide series are mixed D2/D3
development of extrapyramidal motor side effects and
tardive dyskinesia, while clozapine is free of these
disturbing problems.7 The hypothesis that D4 receptors
are integrally related to schizophrenia was initially
supported by the now controversial8 finding that schizo-
phrenics possessed a 6-fold larger number of D4 recep-
tors than nonschizophrenics.9
After these findings, a dedicated search for D4-
selective agents was carried within many laboratories
resulting in the identification of a wide array of struc-
tural classes having high D4 affinity and selectivity.9-20
Although a number of D4-selective compounds were
advanced to clinical trials, the results of these trials
have, to date, proved disappointing. In a published
report from Merck, schizophrenics treated with L-745,870
(4) demonstrated no significant improvement in either
positive or negative symptomology.21
antagonists.
Strong support for the dopamine hypothesis of Carls-
son and Lindquist is provided by a remarkable correla-
tion between the cerebrospinal fluid concentration of
antipsychotic agents determined at their clinically ef-
fective doses and the affinity of these agents for the
dopamine D2 receptor subtype.5 An important exception
to this correlation is the dibenzo[1,4]diazepine clozapine
(3), whose cerebrospinal fluid concentration correlates
best with its affinity for the D4 receptor subtype.6 This
correlation suggests that, unlike most neuroleptic drugs,
the antipsychotic effects of clozapine are a result of its
interaction with the D4 receptor population rather than
D2 receptor sites. This distinction is important in light
of the observation that the neuroleptics having strong
affinity for D2 receptors also present a high risk for the
These reports lead us to postulate that, while D4 may
play an important role in the actions of clozapine, some
* To whom correspondence should be addressed. Tel: 203-315-3024.
Fax: 203-483-7027. E-mail: arthurkauf@nrgn.com.
10.1021/jm990562x CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/30/2000

3924 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Sch em e 1. Preparation of the Target Moleculesa
Zhang et al.
a
Reagents: Method A: NEt3, CH2Cl2. Method B: EDCl, HOBT, DMAP, CHCl3. Method C: AlH3, THF.
affinity for the D2 receptor subtype may be required for
effective antipsychotic action. Therefore, a compound
having a mix of high D4 and more modest D2 affinity
might better mimic the profile of clozapine than a D4-
selective agent. Thus we set out to obtain a compound
which possessed a D2/D4 binding ratio similar to that
of clozapine. A secondary goal was to minimize R1
binding in order to avert undesirable cardiovascular
effects. In the course of our research into D4-selective
compounds, we identified a number of chemical series
with such receptor pharmacology. Specifically, the
1-phenyl-4-[(trans-2-phenylcyclopropyl)methyl]piper-
azines 5 showed promise in this regard. The parent
compound, trans-1-[(2-phenylcyclopropyl)methyl]-4-phen-
ylpiperazine (5a ), demonstrated an encouraging dopa-
mine receptor profile (D2 ) 37 nM, D4 ) 48 nM) and
served as a starting point for the exploration of the
structure-activity relationships (SARs) within this
series. We set our criteria for compound selection as a
D4 affinity of less than 10 nM and a ratio of D2 to D4
affinities of 2-10. Due to the asymmetric carbon at C-2
of the cyclopropyl, the results of initial receptor screens
would reflect the affinities of a racemic mixture. The
D2 to D4 ratio was therefore set in a wide range around
the observed ratio of approximately 4-7 in the hopes
that, upon resolution, the dopamine receptor profile of
one of the enantiomers would closely resemble that of
clozapine.
eomeric salts.26 However, we found such a procedure
very time-consuming, and therefore an indirect resolu-
tion trans-2-phenyl-1-cyclopropanecarboxylic acid was
investigated as outlined in Scheme 2. Coupling of
racemic trans-2-phenyl-1-cyclopropanecarbonyl chloride
with (R)-R-methylbenzylamine provided diastereomeric
amides 9a ,b, in which 9a could be isolated by crystal-
lization from ethyl acetate. The mother liquid from the
crystallization, containing the mixture of 9a ,b with 9b
as a major component, was hydrolyzed to give a mixture
of 8a ,b with 8b as a major component. The mixed acids
were then coupled with (S)-R-methylbenzylamine fol-
lowed by crystallization from ethyl acetate to provide
9c, the enantiomer of 9a . The amides 9a ,c were
hydrolyzed to provide the chiral acids 8a ,b, respectively.
The absolute configurations of 8a ,b were assigned by
comparison of their optical rotation configurations with
the literature values.26 The absolute configurations of
5l-m ,s,t then followed the assignment of the carboxylic
acid starting materials, and their optical purities were
determined to be >99% by using methanol on a
CHIRACEL OD chiral column. The physical properties
of cyclopropylmethylpiperazines 5a -z and 6a -e are
summarized in Table 1.
Resu lts a n d Discu ssion
Affinity of the compounds listed in Table 1 at D2
receptors was determined via standard competitive
displacement assays using D2 receptors cloned from
human with [3H]YM 09151 as the competitive ligand.13
Affinity at D4 receptors was determined via standard
competitive displacement assays using human D4 recep-
tor clones with [3H]YM 09151 as the competitive ligand.
Affinity at R1 receptors was determined via standard
competitive displacement assays using rat brain homo-
genate with [3H]prazosin as the competitive ligand. The
results of these assays are displayed in Table 2. The
reference compound remoxipride (2) was prepared by
condensation of commercially available 3-bromo-2,6-
dimethoxybenzoic acid with (-)-(S)-2-aminomethyl-1-
ethylpyrrolidine.27
Our initial synthetic efforts in this series focused on
the phenyl attached to the piperazine. To obtain a
general sense of how the affinities for D2, D4, and R1
were effected by the electronic character of this phenyl
group, a representative electron-withdrawing group
(chlorine) and electron-donating group (methoxy) were
introduced as a single substituent at each of the three
available positions (compounds 5b-g). Examination of
Ch em istr y
The basic synthetic route to the novel cyclopropyl-
methylpiperazines 5a -z and 6a -e prepared in this
work is summarized in Scheme 1 and Table 1. Coupling
of either trans-2-phenyl-1-cyclopropanecarbonyl chloride
or trans-2-aryl-1-cyclopropanecarboxylic acids 8a -d
with various arylpiperazines 7a -z provided key inter-
mediates, the tertiary amides 10a -z and 11a -e. The
amides were reduced by alane to elaborate the corre-
sponding cyclopropylmethylpiperazines 5a -z and 6a -
e. Several of the N-arylpiperazines used in this study
were not commercially available and were prepared by
several different methods according to the literature
reports.22-24 The trans-2-aryl-1-cyclopropanecarboxylic
acids 8c-d were prepared by cyclopropanation of the
appropriate cinnamic acids, using the method of
Friedrich.25
It has been reported that the chiral trans-2-phenyl-
1-cyclopropanecarboxylic acids 8a ,b could be obtained
by repeated fractional recrystallization of their diaster-

trans-1-[(2-Phenylcyclopropyl)methyl]-4-arylpiperazines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3925
Sch em e 2. Indirect Resolution of Chiral Acid 8a ,ba
a
Reagents: (a) Method A; (b) recrystallization in EtOAc; (c) hydrazine monohydrate, KOH, ethylene glycol, 160 °C; (d) Method B.
the data indicates that, relative to the parent compound
5a , dopamine D4 binding was essentially unchanged for
5c,f,g, while significant improvement was seen for the
ortho-substituted compounds 5b,e and the p-chloro
derivative 5d . The binding of the compounds to R1
showed a somewhat greater sensitivity to the placement
of the substituents with somewhat higher affinities
observed for the ortho substituents and lower affinities
observed for the meta and para substituents. The
finding that both electron-withdrawing and electron-
donating groups at the meta and para positions lose
affinity in this assay indicates that a steric interaction
may be responsible for these results. Highest D2 affinity
was observed for both ortho-substituted derivatives
5b,e.
derivative 5k displayed an receptor profile near enough
to the target profile to warrant further examination of
its enantiomers.
Compounds 5n -p represent various substitution
patterns which were examined. The receptor profiles of
these examples were found to be largely uninteresting
with the exception of the 2,4-dimethyl derivative 5r
which met the desired affinity criteria.
The pyridyl derivatives 5w -y and the 2-pyrimidinyl
5z were examined as examples of electron-deficient
hetero aromatics. Although compounds containing the
fragments 2-pyridyl- and 2-pyrimidinylpiperazine have
previously been identified as D4 antagonists,19 their
presence within the cyclopropyl series was detrimental
to dopamine binding.
Compounds 5h -k represent various dichlorinated
substitution patterns of the 1-phenylpiperazine. The 3,5-
dichloro derivative 5i showed a significant loss of D4
affinity relative to D2, while the 2,3-dichloro and 3,4-
dichloro derivatives 5h ,j displayed unacceptably high
affinities for D2 and R1, respectively. The 2,4-dichloro
At this point, an examination of the effect of substitu-
tion at the para position of the phenyl attached to the
cyclopropyl was undertaken. An examination of the
receptor affinities for compounds 6a -e indicates that
while the receptors would tolerate the presence of a para
substituent, it would not adversely effect R1 binding

3926 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Zhang et al.
Ta ble 1. Physical Data for Cyclopropylmethylpiperazines 5a -z and 6a -e
compd
X
R
config
method
formula
mp (°C)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
1R,2R
A
A
A
A
A
A
A
A
A
A
A
B
B
A
A
A
A
A
B
B
A
A
A
A
A
A
B
b
C
C
C
C
20H24N2
79-80
2-Cl
3-Cl
4-Cl
2-OCH3
3-OCH3
4-OCH3
2,3-diCl
3,5-diCl
3,4-diCl
20H23N2Cl‚HBr
201-202
195-196
229a
20H23N2Cl‚2HBr
20H23N2Cl‚2HBr‚0.5H2O
C21H26N2O‚HBr‚H2O
C21H26N2O‚HBr‚H2O‚0.75H2O
C21H26N2O‚2HBr
C
C
C
C
C
C
C21H25N2OCl‚HBr
C21H25N2OF‚2HBr
C21H25N2Cl‚HBr
C21H25N2F‚HBr
C22H28N2‚HBr
C22H28N2‚2HBr
C22H28N2‚2HBr
C21H25N2OCl‚HBr
C21H24N2OCl2‚HBr
C
C
129-130
220a
218-219
209-210
243-244
157-158
206-207
197-198
142-143
206-207
208-209
206-207
204-205
209-210
215-217
224-225
209-210
233-234
110
20H22N2Cl2‚HBr
20H22N2Cl2‚HBr
20H22N2Cl2‚HBr‚0.5H2O
20H22N2Cl2‚HBr
2,4-diCl
2,4-diCl
2,4-diCl
20H22N2Cl2‚2HBr
20H22N2Cl2‚HBr
1S,2S
2-OCH3-4-Cl
2-OCH3-4-F
2-CH3-4-Cl
2-CH3-4-F
2,4-diCH3
2,4-diCH3
2,4-diCH3
2-OCH3-5-Cl
3,5-diCl-4-OCH3
2-pyridyl
3-pyridyl
4-pyridyl
2-pyrimidinyl
2,4-diCl
racemate
racemate
racemate
racemate
racemate
1R,2R
5s
5t
1S,2S
5u
5v
5w
5x
5y
5z
6a
6b
6c
6d
6e
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
19H23N3‚2HCl‚0.5H2O
19H23N3‚2HBr
238
225
172
C19H23N3‚2HBr
C
C21H24N2OCl2‚HBr
C
C
C
18H21N4‚2HCl
OCH3
OH
F
F
F
223-224
210-211
203
2,4-diCl
2-Cl
3-Cl
4-Cl
20H22N2Cl2O‚HBr
20H22FClN2‚2HBr‚0.5H2O
20H22FClN2‚2HBr‚0.5H2O
B
B
B
215a
203
C20H22FClN2‚2HBr‚0.5H2O
a
b
Decomposed. Prepared from 6a .
relative to the corresponding compound lacking the
substituent.
this case dopamine, will induce the binding of GTP by
the G-protein. When present, the nonhydrolyzing ra-
dioligand GTP[γ-35S] will also bind under these agonist
stimulated conditions and thus can provide a functional
measure of G-protein activation. In CHO cells stably
expressing human D2 receptors, compounds 5m ,t an-
tagonized dopamine-stimulated GTP[γ-35S] binding with
Ki values of 181 and 61 nM, respectively. As a control,
haloperidol displayed a Ki of 7 nM. Similarly, in CHO
cells stably expressing human D4 receptors, compounds
5m ,t antagonized dopamine-stimulated GTP[γ-35S] bind-
ing with Ki values of 24 and 13 nM, respectively. In the
D4 experiment, haloperidol displayed a Ki of 2 nM.
From the data obtained from this study, the racemic
compounds 5k ,r met the initial criteria for D2, D4, and
R1 binding and were selected for resolution. The resolu-
tions were carried out as described in the Experimental
Section, and the absolute configurations were assigned
by comparison of the optical rotation configurations of
the carboxylic acid starting materials with literature
values. Compounds 5l,m represent the 1R,2R and 1S,2S
enantiomers of the 2,4-dichloro derivative 5k . Similarly,
5s,t represent the 1R,2R and 1S,2S enantiomers of the
2,4-dimethyl derivative 5r .
In both cases, significantly higher D4 affinity resided
within the 1S,2S enantiomers while somewhat higher
D2 affinity was observed for the 1R,2R enantiomers.
Similar R1 affinity was observed for each of the enan-
tiomers within the racemic pair. The D2/D4 ratio for
5m ,t were thus approximately 18 and 9, respectively.
Although these represented a D2/D4 ratio somewhat
higher than that found in clozapine, 5m ,t were exam-
ined in behavioral tests of both antipsychotic activity
and extrapyramidal symptoms.
Functional assays to determine antagonist properties
of 5m ,t at the dopamine receptors was carried out using
a modification of the method of Wieland and J akobs.28
Both D2 and D4 are G-protein coupled receptors (GPCR).
Stimulation of a GPCR by an appropriate agonist, in
When male Sprague-Dawley rats were injected sub-
cutaneously with clozapine, haloperidol, and remox-
ipride 30 min prior to receiving amphetamine (0.5 mg/
kg), the agents produced a significant and dose-
dependent decline in stimulated locomotor activity (P
< 0.05). The minimum effective doses (defined as the
lowest dose tested that produced a significant effect
relative to the amphetamine alone control group using
a Fisher’s LSD post hoc test, P < 0.05) were 0.5, 0.06,
and 0.5 mg/kg for clozapine, haloperidol, and remox-
ipride, respectively. Neither 5m nor 5t caused a sig-
nificant reduction in amphetamine-induced locomotor
activity at the doses tested (4-16 mg/kg). In the
catalepsy experiments, haloperidol and remoxipride
displayed minimum effective doses of 0.5 and 64 mg/

trans-1-[(2-Phenylcyclopropyl)methyl]-4-arylpiperazines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3927
Ta ble 2. Binding Affinities of Cyclopropylmethylpiperazines 5a -z and 6a -e
IC50 (nM ( SEM)
compd
X
R
config
D2
D4
R1
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
1R,2R
37 ( 2
5 ( 1
146 ( 13
246 ( 20
5 ( 2
48 ( 4
8 ( 2
22 ( 3
10 ( 2
2-Cl
3-Cl
4-Cl
2-OCH3
3-OCH3
4-OCH3
2,3-diCl
3,5-diCl
3,4-diCl
85 ( 17
5 ( 2
70 ( 6
81 ( 5
5 ( 2
324 ( 22
633 ( 41
329 ( 15
1341 ( 64
45 ( 9
311 ( 18
348 ( 39
322 ( 17
29 ( 4
14 ( 3
307 ( 86*
31 ( 3
3 ( 1
171 ( 16
752 ( 81
2 ( 1
37 ( 4
73 ( 14
9 ( 3
32 ( 8
164 ( 28
10 ( 3
9 ( 1
133 ( 21
6 ( 2
3 ( 1
6 ( 1
31 ( 11*
68 ( 3
7 ( 1
17 ( 3
8 ( 2
9 ( 2
5j
5k
5l
208 ( 20
65 ( 4
2,4-diCl
2,4-diCl
2,4-diCl
64 ( 2
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
5z
6a
6b
6c
6d
6e
1
1S,2S
93 ( 7
2-OCH3-4-Cl
2-OCH3-4-F
2-CH3-4-Cl
2-CH3-4-F
2,4-diCH3
2,4-diCH3
2,4-diCH3
2-OCH3-5-Cl
3,5-diCl-4-OCH3
2-pyridyl
3-pyridyl
4-pyridyl
2-pyrimidinyl
2,4-diCl
racemate
racemate
racemate
racemate
racemate
1R,2R
17 ( 1
10 ( 2
128 ( 17
68 ( 14
60 ( 2
144 ( 11
137 ( 21
131 ( 14
8 ( 3
45 ( 5
1S,2S
73 ( 2
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
racemate
5 ( 2
228 ( 23
263 ( 15
16 ( 6
96 ( 5
>10000
>10000
567 ( 51*
45 ( 4
43 ( 5
10 ( 2
29 ( 2
30 ( 6
5 ( 1
193 ( 16
55 ( 12
1246 ( 296
>10000
>10000
>10000
766 ( 59*
168 ( 9
61 ( 4
74 ( 8
9 ( 2
56 ( 8
61 ( 4
7 ( 2
OCH3
OH
F
F
F
7 ( 1
12 ( 2
2,4-diCl
2-Cl
3-Cl
4-Cl
13 ( 4
56 ( 10
202 ( 11
4 ( 2
(haloperidol)
(remoxipride)
(clozapine)
2
3
880 ( 17
113 ( 9
3872
17 ( 3
>4000
4 ( 1
kg, respectively. Clozapine and 5m ,t showed no signifi-
cant cataleptic effects over the dose range tested (1-40
mg/kg).
microsomal fractions obtained from human and rat liver
homogenates. Upon exposure to human liver mi-
crosomes the half-lives (t1/2) of 5m ,t were determined
to be 19 and 16 min, respectively. Much shorter half-
lives of 2 and 3 min, respectively, were observed upon
exposure to rat liver microsomes. The short half-lives
of the compounds in rat may, in part, explain the lack
of observable D2 behavioral effects, although clozapine
also shows a fairly short (5 min) half-life in rat.
Alternatively, the D2/D4 hypothesis may be flawed.
Proper testing of the hypothesis will require a series of
D2/D4 antagonists having a more favorable pharmaco-
kinetic profile.
In conclusion, a series of trans-1-[(2-phenylcyclopro-
pyl)methyl]-4-arylpiperazines were prepared and tested
for their ability to bind to adrenergic R1 and dopamine
D2 and D4 receptor subtypes in order to identify candi-
dates with both D2/D4 ratios similar to that of clozapine
and a low propensity to produce untoward cardiac
responses. Two compounds having the 1S,2S absolute
configuration and a 2,4-dimethyl or 2,4-dichloro substi-
tution pattern on the 4-aryl were selected. While the
compounds had D2/D4 ratios approximating that of
clozapine and, in functional assays, were found to be
antagonists at the dopamine subtypes, behavioral effects
observed in rats for both typical and atypical antipsy-
In our examination of the 2-phenylcyclopropylami-
nomethylpiperazines as potential antipsychotic agents,
we have attempted to approximate the D2/D4 ratio of
clozapine on the theory that this ratio might be a key
to its atypical nature. A practical limitation to this
approach may be found in the suggestion by other
researchers that the primary behavioral assays of
antipsychotic efficacy are largely D2-mediated. With this
in mind, the inability of 5m ,t to display significant
effects in amphetamine-timulated locomotor activity as
a test of antipsychotic efficacy is somewhat puzzling in
light of the fact that the control compounds of haloperi-
dol and remoxipride are effective in this assay while
having greater and lesser D2 affinity, respectively, than
5m ,t. It has been demonstrated that the aromatic
6-methoxy substituent of remoxipride is metabolically
cleaved in rat to the corresponding 6-hydroxy. The
resulting 2-hydroxybenzamide, FLA 797, displays a
significantly greater D2 affinity (12 nM)29 than does
remoxipride and thus may account for the observed
behavioral profile of remoxipride.
The time course for P450 enzymatic degradation of
5m ,t was determined by exposure of the compounds to

3928 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Zhang et al.
tr a n s-1-[(2-P h en ylcyclopr opyl)m eth yl]-4-(4-ch lor oph en -
yl)p ip er a zin e Dih yd r obr om id e (5d ). 37% yield from trans-
2-phenyl-1-cyclopropanecarbonyl chloride and 1-(4-chlorophen-
yl)piperazine: mp 229 °C dec; 1H NMR (400 MHz, DMSO-d6)
δ 1.05-1.16 (m, 2H), 1.40-1.42 (m, 1H), 2.00-2.04 (m, 1H),
3.04 (t, J ) 11.6 Hz, 2H), 3.13-3.22 (m, 2H), 3.26 (d, J ) 6.5
Hz, 2H), 3.56 (d, J ) 11.6 Hz, 2H), 3.91 (bs, 2H), 7.01 (d, J )
8.4 Hz, 2H), 7.13 (d, J ) 7.6 Hz, 2H), 7.26-7.30 (m, 5H); MS
(LC-MS) m/e 327 (free base, MH+). Anal. (C20H23N2Cl‚2HBr‚
0.5H2O) C, H, N.
chotic agents were absent. Thus, while these or similar
compounds might prove to be effective antipsychotics
in humans, the short t1/2 in rat leaves the hypothesis
that a compound with the proper D2/D4 ratio will display
an atypical behavioral profile untested at this time.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Thomas-
Hoover capillary melting-point apparatus and are uncorrected.
Elemental analyses were obtained for all compounds tested
for binding. Elemental analysis were performed at Robertson
Microlabs, Madison, NJ , and the results were within (0.4%
tr a n s-1-[(2-P h en ylcyclop r op yl)m eth yl]-4-(2-m eth oxy-
p h en yl)p ip er a zin e Hyd r obr om id e (5e). 91% yield from
trans-2-phenyl-1-cyclopropanecarbonyl chloride and 1-(2-meth-
1
oxyphenyl)piperazine: mp 129-130 °C; H NMR (400 MHz,
1
of the theoretical values unless otherwise indicated. H NMR
DMSO-d6) δ 1.05-1.15 (m, 2H), 1.39-1.41 (m, 1H), 2.01-2.06
(m, 1H), 2.93 (t, J ) 11.6 Hz, 2H), 3.20-3.29 (m, 4H), 3.50-
3.59 (m, 4H), 3.77 (s, 3H, OCH3), 6.87-6.99 (m, 4H), 7.13-
7.18 (m, 3H), 7.27 (t, J ) 7.6 Hz, 2H); MS (LC-MS) m/e 323
(free base, MH+). Anal. (C21H26N2O‚HBr‚H2O) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m eth yl]-4-(3-m eth oxy-
p h en yl)p ip er a zin e Hyd r obr om id e (5f). 91% yield from
trans-2-phenyl-1-cyclopropanecarbonyl chloride and 1-(3-meth-
oxyphenyl)piperazine: mp 220 °C dec; 1H NMR (400 MHz,
DMSO-d6) δ 1.05-1.15 (m, 2H), 1.41-1.44 (m, 1H), 2.01-2.06
(m, 1H), 3.01 (t, J ) 11.6 Hz, 2H), 3.14-3.30 (m, 4H), 3.55-
3.66 (m, 2H), 3.71 (s, 3H, OCH3), 3.81-3.86 (m, 2H), 6.43 (dd,
J ) 2.4, 8.0 Hz, 1H), 6.52 (t, J ) 2.4 Hz, 1H), 6.56 (dd, J )
2.4, 8.2 Hz, 1H), 7.13-7.18 (m, 4H), 7.25-7.29 (m, 2H); MS
(LC-MS) m/e 323 (free base, MH+). Anal. (C21H26N2O‚HBr‚
H2O) C, H, N.
spectra were recorded in CDCl3 (unless otherwise noted) with
tetramethylsilane (TMS) as the internal standard on a Varian
Unity 400 spectrometer. Electron ionization mass spectra (MS)
were recorded on a Hewlett-Packard 5890 mass spectrometer.
Gen er a l P r oced u r e for th e P r ep a r a tion of tr a n s-1-[(2-
P h en ylcyclopr opyl)m eth yl]-4-ar ylpiper azin es fr om tr a n s-
2-P h en yl-1-cyclop r op a n eca r bon yl Ch lor id e (Meth od A).
tr a n s-1-[(2-P h en ylcyclopr opyl)m eth yl]-4-(2-ch lor oph en yl)-
p ip er a zin e Hyd r obr om id e. (5b) To a solution of 1-(2-
chlorophenyl)piperazine hydrochloride (0.59 g, 3.0 mmol) in
10 mL of dichloromethane was added triethylamine (0.61 g,
6.0 mmol) followed by the addition of trans-2-phenyl-1-cyclo-
propanecarbonyl chloride (0.54 g 3.0 mmol) at 0 °C. The
mixture was stirred at room temperature for 3 h and then
quenched by the addition of 10 mL of water. The aqueous layer
was extracted with chloroform (10 mL × 2) and the combined
organics were washed with brine, dried over Na2SO4 and
evaporated to dryness to give 1.0 g (98%) of 10b as a light
yellow oil: 1H NMR (400 MHz, CDCl3) δ 1.31-1.34 (m, 1H),
1.67-1.73 (m, 1H), 1.99-2.05 (m, 1H), 2.48-2.55 (m, 1H),
3.03-3.06 (m, 4H), 3.81-3.85 (m, 4H), 6.98-7.03 (m, 2H),
7.12-7.18 (m, 2H), 7.20-7.29 (m, 4H), 7.31-7.39 (m, 1H); MS
(LC-MS) m/e 341 (MH+). This crude product was used in the
next step without further purification.
To a 10-mL solution of alane (AlH3; 0.5 M in THF) was
added a solution of 10b in 5 mL of THF at 0 °C. The mixture
was stirred at room temperature for 3 h, then carefully
quenched with NaOH (10%). The aqueous layer then was
extracted with EtOAc (30 mL × 2). The combined organics
were washed with brine, dried over Na2SO4 and concentrated.
The free base was dissolved in 2-propanol and then treated
with HBr (48%). Crystallization of the salt from i-PrOH/i-Pr2O
provided the desired product (1.0 g, 82%) as white crystals:
mp 201-202 °C; 1H NMR (400 MHz, CD3OD) δ 1.14-1.28 (m,
2H), 1.45-1.50 (m, 1H), 2.07-2.10 (m, 1H), 3.18-3.29 (bs, 4H),
3.39-3.71 (bd, 6H), 7.08-7.28 (m, 5H), 7.30-7.34 (m, 3H), 7.41
(dd, J ) 1.37, 7.96 Hz, 1H); MS (LC-MS) m/e 327 (free base,
MH+). Anal. (C20H23N2Cl‚HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m eth yl]-4-(4-m eth oxy-
p h en yl)p ip er a zin e Dih yd r obr om id e (5g). 90% yield from
trans-2-phenyl-1-cyclopropanecarbonyl chloride and 1-(4-meth-
1
oxyphenyl)piperazine: mp 218-219 °C; H NMR (400 MHz,
DMSO-d6) δ 1.06-1.15 (m, 2H), 1.42-1.44 (m, 1H), 2.01-2.05
(m, 1H), 2.92 (t, J ) 11.6 Hz, 2H), 3.14-3.28 (m, 4H), 3.55-
3.66 (m, 4H), 3.68 (s, 3H, OCH3), 6.83 (d, J ) 9.2 Hz, 2H),
6.94 (d, J ) 9.2 Hz, 2H), 7.13-7.18 (m, 3H), 7.25-7.29 (m,
2H); MS (LC-MS) m/e 323 (free base, MH+). Anal. (C21H26N2O‚
2HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(2,3-d ich lo-
r op h en yl)p ip er a zin e Hyd r obr om id e (5h ). 28% yield from
trans-2-phenyl-1-cyclopropanecarbonyl chloride and 1-(2,3-
dichlorophenyl)piperazine: mp 209-210 °C; 1H NMR (400
MHz, DMSO-d6) δ 1.01-1.16 (m, 2H), 1.40-1.43 (m, 1H),
2.02-2.07 (m, 1H), 3.07 (t, J ) 12.4 Hz, 2H), 3.23-3.29 (m,
4H), 3.46 (bd, J ) 13.6 Hz, 2H), 3.61 (bd, J ) 11.2 Hz, 2H),
7.13-7.18 (m, 3H), 7.21-7.29 (m, 3H), 7.32-7.39 (m, 2H); MS
(LC-MS) m/e 361 (free base, MH+). Anal. (C20H22N2Cl2‚HBr)
C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(3,5-d ich lo-
r op h en yl)p ip er a zin e Hyd r obr om id e (5i). 21% yield from
trans-2-phenyl-1-cyclopropanecarbonyl chloride and 1-(3,5-
dichlorophenyl)piperazine: mp 243-244 °C; 1H NMR (400
MHz, DMSO-d6) δ 1.04-1.10 (m, 1H), 1.12-1.17 (m, 1H),
1.39-1.42 (m, 1H), 1.99-2.02 (m, 1H), 3.09-3.16 (m, 2H),
3.25-3.29 (m, 4H), 3.53-3.55 (m, 2H), 3.99-4.02 (m, 2H), 6.95
(s, 1H), 7.04 (s, 2H), 7.12-7.18 (m, 3H), 7.25-7.29 (m, 2H);
MS (LC-MS) m/e 361 (free base, MH+). Anal. (C20H22N2Cl2‚
HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(3,4-d ich lo-
r op h en yl)p ip er a zin e Hyd r obr om id e (5j). 33% yield from
trans-2-phenyl-1-cyclopropanecarbonyl chloride and 3,4-dichlo-
rophenyl piperazine: mp 157-158 °C; 1H NMR (free base, 400
MHz, CDCl3) δ 0.84-0.89 (m, 1H), 0.98-1.02 (m, 1H), 1.25-
1.29 (m, 1H), 1.68-1.73 (m, 1H), 2.36-2.41 (m, 1H), 2.59-
2.64 (m, 1H), 2.66-2.71 (m, 4H), 3.17-3.20 (m, 4H), 6.73 (dd,
J ) 2.8, 9.0 Hz, 1H), 6.95 (d, J ) 3.2 Hz, 1H), 7.05-7.07 (m,
2H), 7.14-7.18 (m, 1H), 7.25-7.29 (m, 3H); MS (LC-MS) m/e
361 (free base, MH+). Anal. (C20H22N2Cl2‚HBr‚0.5H2O) C, H,
N.
Compounds 5a -k ,n -r ,u -z were prepared according to
Method A.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-p h en ylp ip -
er a zin e (5a ). 50% yield from trans-2-phenyl-1-cyclopropan-
ecarbonyl chloride and 1-phenylpiperazine: mp 79-80 °C; 1H
NMR (400 MHz, CDCl3) δ 0.84-0.89 (m, 1H), 0.97-1.01 (m,
1H), 1.26-1.31 (m, 1H), 1.68-1.73 (m, 1H), 2.38-2.43 (m, 1H),
2.59-2.67 (m, 1H), 2.70-2.75 (m, 4H), 3.18-3.23 (m, 4H), 6.86
(t, J ) 7.2 Hz, 1H), 6.92 (d, J ) 8.0 Hz, 2H), 7.06 (d, J ) 7.2
Hz, 2H), 7.15 (t, J ) 8.0 Hz, 1H), 7.24-7.28 (m, 4H); MS (LC-
MS) m/e 293 (free base, MH+). Anal. (C20H24N2) C, H, N.
tr a n s-1-[(2-P h en ylcyclopr opyl)m eth yl]-4-(3-ch lor oph en -
yl)p ip er a zin e Dih yd r obr om id e (5c). 38% yield from trans-
2-phenyl-1-cyclopropanecarbonyl chloride and 1-(3-chlorophen-
1
yl)piperazine: mp 195-196 °C; H NMR (400 MHz, DMSO-
d6) δ 1.11-1.16 (m, 2H), 1.40-1.42 (m, 1H), 2.00-2.04 (m, 1H),
3.04 (t, J ) 11.6 Hz, 2H), 3.13-3.22 (m, 2H), 3.26 (d, J ) 6.5
Hz, 2H), 3.56 (d, J ) 11.6 Hz, 2H), 3.91 (bs, 2H), 6.86 (d, J )
8 Hz, 1H), 6.95-6.97 (m, 1H), 7.05 (s, 1H), 7.12-7.18 (m, 4H),
7.23-7.29 (m, 2H); MS (LC-MS) m/e 327 (free base, MH+).
Anal. (C20H23N2Cl‚2HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(2,4-d ich lo-
r op h en yl)p ip er a zin e Hyd r obr om id e (5k ). 75% yield from
trans-2-phenyl-1-cyclopropanecarbonyl chloride and 2,4-dichlo-

trans-1-[(2-Phenylcyclopropyl)methyl]-4-arylpiperazines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3929
rophenylpiperazine: mp 206-207 °C; 1H NMR (400 MHz,
DMSO-d6) δ 1.06-1.15 (m, 2H), 1.43-1.45 (m, 1H), 2.02-2.07
(m, 1H), 3.05 (t, J ) 12.0 Hz, 2H), 3.21-3.27 (m, 4H), 3.42-
3.45 (m, 2H), 3.60-3.62 (m, 2H), 7.13-7.18 (m, 3H), 7.23-
7.29 (m, 2H), 7.40 (dd, J ) 2.4, 8.0 Hz, 2H), 7.60 (d, J ) 2.4
Hz, 1H). Anal. (C20H22N2Cl2‚HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(4-ch lor o-2-
m eth oxyph en yl)piper azin e Hydr obr om ide (5n ). 63% yield
from trans-2-phenyl-1-cyclopropanecarbonyl chloride and 1-(4-
chloro-2-methoxyphenyl)piperazine: mp 206-207 °C; 1H NMR
(400 MHz, DMSO-d6) δ 1.00-1.15 (m, 2H), 1.40-1.42 (m, 1H),
2.01-2.04 (m, 1H), 2.93 (t, J ) 12.4 Hz, 2H), 3.22-3.27 (m,
4H), 3.47-3.55 (m, 4H), 3.79 (s, 3H, OCH3), 6.94 (s, 2H), 7.02
(s, 1H), 7.12-7.18 (m, 3H), 7.25-7.29 (m, 2H); MS (LC-MS)
m/e 357 (free base, MH+). Anal. (C21H25N2OCl‚HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(4-flu or o-2-
m eth oxyp h en yl)p ip er a zin e Dih yd r obr om id e (5o). 29%
yield from trans-2-phenyl-1-cyclopropanecarbonyl chloride and
1-(4-fluoro-2-methoxyphenyl)piperazine: mp 208-209 °C; 1H
NMR (400 MHz, DMSO-d6) δ 1.06-1.15 (m, 2H), 1.40-1.43
(m, 1H), 2.00-2.03 (m, 1H), 2.90 (t, J ) 11.2 Hz, 2H), 3.22-
3.28 (m, 2H), 3.43 (12.8, 2H), 3.56 (d, J ) 10.4 Hz, 2H), 3.79
(s, 3H, OCH3), 6.68-6.73 (m, 1H), 6.89-6.97 (m, 2H), 7.13-
7.18 (m, 3H), 7.25-7.29 (m, 2H); MS (LC-MS) m/e 341 (free
base, MH+). Anal. (C21H25N2OF‚2HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(4-ch lor o-2-
m eth ylp h en yl)p ip er a zin e Hyd r obr om id e (5p ). 90% yield
from trans-2-phenyl-1-cyclopropanecarbonyl chloride and 1-(4-
chloro-2-methylphenyl)piperazine: mp 206-207 °C; 1H NMR
(400 MHz, DMSO-d6) δ 1.08-1.17 (m, 2H), 1.40-1.43 (m, 1H),
2.03-2.05 (m, 1H), 2.25 (s, 3H, CH3), 2.96 (t, J ) 10.2 Hz,
2H), 3.20-3.30 (m, 6H), 3.55-3.57 (m, 2H), 7.07 (d, J ) 8.8
Hz, 2H), 7.14-7.24 (m, 4H), 7.26-7.30 (m, 2H); MS (LC-MS)
m/e 341 (free base, MH+). Anal. (C21H25N2Cl‚HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(p yr id in -2-
yl)p ip er a zin e Dih yd r och lor id e (5w ). 25% yield from trans-
2-phenyl-1-cyclopropanecarbonyl chloride and 1-(pyridin-2-
yl)piperazine: mp 110 °C; 1H NMR (free base, 400 Hz, CDCl3)
δ 0.86 (dt, J ) 8.4 and 5.6 Hz, 1H), 1.00 (dt, J ) 8.4 and 4.8
Hz, 1H), 1.27 (m, 1H), 1.70 (dt, J ) 8.4 and 4.8 Hz, 1H), 2.40
(dd, J ) 12.8 and 6.8 Hz, 1H), 2.60 (dd, J ) 12.4 and 6.0 Hz,
1H), 2.66 (m, 4H), 3.57 (m, 4H), 6.62 (dd, J ) 7.6 and 5.2 Hz,
1H), 6.64 (d, J ) 8.4 Hz, 1H), 7.07 (m, 2H), 7.16 (tt, J ) 7.6
and 1.2 Hz, 1H), 7.26 (t, J ) 7.2 Hz, 2H), 7.47 (ddd, J ) 9.2,
7.6 and 2.0 Hz, 1H), 8.19 (dd, J ) 4.8 and 2.0 Hz, 1H). Anal.
(C19H23N3‚2HCl‚0.5H2O) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(p yr id in -3-
yl)p ip er a zin e Dih yd r obr om id e (5x). 63% yield from trans-
2-phenyl-1-cyclopropanecarbonyl chloride and 1-(pyridin-3-
yl)piperazine: mp 238 °C; 1H NMR (free base, 400 Hz, CDCl3)
δ 0.86 (dt, J ) 8.8 and 5.2 Hz, 1H), 1.00 (dt, J ) 8.8 and 4.8
Hz, 1H), 1.27 (m, 1H), 1.71 (dt, J ) 8.4 and 4.8 Hz, 1H), 2.40
(dd, J ) 12.4 and 6.8 Hz, 1H), 2.61 (dd, J ) 12.4 and 5.6 Hz,
1H), 2.70 (m, 4H), 3.24 (t, J ) 5.2 Hz, 4H), 7.06 (d, J ) 7.6
Hz, 2H), 7.16 (m, 3 H), 7.26 (t, J ) 8.0 Hz, 2 H), 8.08 (dd, J )
3.6 and 1.6 Hz, 1H), 8.30 (d, J ) 1.6 Hz, 1H). Anal. (C19H23N3‚
2HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(p yr id in -4-
yl)p ip er a zin e Dih yd r obr om id e (5y). 75% yield from trans-
2-phenyl-1-cyclopropanecarbonyl chloride and 1-(pyridin-4-
yl)piperazine: mp 225 °C; 1H NMR (free base, 400 Hz, CDCl3)
δ 0.86 (dt, J ) 9.2 and 4.8 Hz, 1H), 1.00 (dt, J ) 8.8 and 4.8
Hz, 1H), 1.26 (m, 1H), 1.70 (dt, J ) 9.2 and 4.0 Hz, 1H), 2.37
(dd, J ) 12.4 and 6.8 Hz, 1H), 2.60 (dd, J ) 12.0 and 5.6 Hz,
1H), 2.64 (m, 4H), 3.34 (m, 4H), 6.64 (dd, J ) 4.8 and 2.0 Hz,
2H), 7.05 (d, J ) 8.0 Hz, 2H), 7.16 (m, 1H), 7.27 (t, J ) 7.6
Hz, 2H), 8.26 (dd, J ) 5.6 and 1.2 Hz, 2H). Anal. (C19H23N3‚
2HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(p yr im id in -
2-yl)p ip er a zin e Dih yd r och lor id e (5z). 54% yield from
trans-2-phenyl-1-cyclopropanecarbonyl chloride and 1-(pyri-
midin-2-yl)piperazine: mp 172 °C; 1H NMR (free base, 400 Hz,
CDCl3) δ 0.85 (dt, J ) 8.8 and 5.2 Hz, 1H), 1.00 (dt, J ) 8.4
and 4.8 Hz, 1H), 1.27 (m, 1H), 1.69 (dt, J ) 9.2 and 4.8 Hz,
1H), 2.40 (dd, J ) 12.4 and 6.8 Hz, 1H), 2.58 (dd, J ) 11.6
and 6.4 Hz, 1H), 2.60 (m, 4H), 3.85 (m, 4H), 6.47 (t, J ) 4.8
Hz, 1H), 7.05 (dd, J ) 6.8 and 1.6 Hz, 2H), 7.15 (t, J ) 7.6 Hz,
1H), 7.26 (t, J ) 8.0 Hz, 2H), 8.29 (d, J ) 4.4 Hz, 2H). Anal.
(C18H21N4‚2HCl) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of tr a n s-1-
[(2-P h en ylcyclop r op yl)m eth yl]-4-a r ylp ip er a zin es fr om
tr a n s-2-Ar yl-1-cyclop r op a n eca r boxylic Acid s (Meth od
B). (1S,2S)-tr a n s-1-[(2-P h en ylcyclop r op yl)m eth yl]-4-(2,4-
d ich lor op h en yl)p ip er a zin e Hyd r obr om id e (5m ). (Di-
methylamino)pyridine (DMAP; 0.73 g, 6 mmol), 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI; 1.15
g, 6.0 mmol) and 1-hydroxybenzotriazole (HOBT; 0.81 g, 6.0
mmol) were successively added to a solution of (1S,2S)-trans-
2-phenyl-1-cyclopropanecarboxylic acid (8b; 0.95 g, 5.8 mmol)
and 1-(2,4-dichlorophenyl)piperazine (1.35 g, 5.8 mmol) in 40
mL of CH2Cl2 at 0 °C with stirring. After being stirred
overnight, the reaction was quenched by the addition of 50
mL of water. The aqueous layer was extracted with chloroform
(40 mL × 2) and the combined organic layers were washed
with brine, dried over Na2SO4 and evaporated to dryness to
give 1.2 g (55%) of 10m as a light yellow oil: 1H NMR (400
MHz, CDCl3) δ 1.29-1.34 (m, 1H), 1.69-1.71 (m, 1H), 1.99-
2.03 (m, 1H), 2.49-2.54 (m, 1H), 3.01-3.12 (m, 4H), 3.80-
3.83 (m, 4H), 6.92 (d, J ) 8.4 Hz, 1H), 7.12 (d, J ) 7.6 Hz,
2H), 7.19-7.22 (m, 2H), 7.28-7.31 (m, 3H); MS (LC-MS) m/e
375 (MH+). The crude product was used in the second step
without further purification.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(4-flu or o-2-
m eth ylp h en yl)p ip er a zin e Hyd r obr om id e (5q). 44% yield
from trans-2-phenyl-1-cyclopropanecarbonyl chloride and 1-(4-
fluoro-2-methoxyphenyl)piperazine: mp 204-205 °C; 1H NMR
(400 MHz, DMSO-d6) δ 1.06-1.15 (m, 2H), 1.42-1.44 (m, 1H),
2.02-2.06 (m, 1H), 2.25 (s, 3H, CH3), 2.97 (t, J ) 12.0 Hz,
2H), 3.14-3.26 (m, 6H), 3.54-3.58 (m, 2H), 6.96-7.00 (m, 1H),
7.03-7.18 (m, 5H), 7.25-7.29 (m, 2H); MS (LC-MS) m/e 325
(free base, MH+). Anal. (C21H25N2F‚HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m eth yl]-4-(2,4-d im eth -
ylp h en yl)p ip er a zin e Hyd r obr om id e (5r ). 77% yield from
trans-2-phenyl-1-cyclopropanecarbonyl chloride and 2,4-di-
methylphenylpiperazine: mp 209-210 °C; 1H NMR (free base,
400 MHz, CDCl3) δ 0.86-0.90 (m, 1H), 0.94-1.01 (m, 1H),
1.28-1.31 (m, 1H), 1.69-1.72 (m, 1H), 2.26 (s, J ) 6 Hz, 6H,
2CH3), 2.43-2.47 (m, 1H), 2.57-2.62 (m, 1H), 2.71 (bs, 4H),
2.91-2.94 (m, 4H), 6.92-7.00 (m, 3H), 7.06-7.08 (m, 2H),
7.15-7.16 (m, 1H), 7.24-7.27 (m, 2H); MS (LC-MS) m/e 321
(free base, MH+). Anal. (C22H28N2‚HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m et h yl]-4-(5-ch lor o-2-
m eth oxyph en yl)piper azin e Hydr obr om ide (5u ). 51% yield
from trans-2-phenyl-1-cyclopropanecarbonyl chloride and 1-(4-
chloro-2-methylphenyl)piperazine: mp 209-210 °C; 1H NMR
(400 MHz, DMSO-d6) δ 1.06-1.14 (m, 2H), 1.40-1.42 (m, 1H),
2.01-2.03 (m, 1H), 2.93 (t, J ) 12.0 Hz, 2H), 3.22-3.29 (m,
6H), 3.56 (bd, J ) 10.4 Hz, 2H), 3.77 (s, 3H, OCH3), 6.94-
6.99 (m, 2H), 7.04 (dd, J ) 2.4, 8.0 Hz, 1H), 7.12-7.18 (m,
3H), 7.25-7.29 (m, 2H); MS (LC-MS) m/e 357 (free base,
MH+). Anal. (C21H25N2OCl‚HBr) C, H, N.
tr a n s-1-[(2-P h en ylcyclop r op yl)m eth yl]-4-(3,5-d ich lor o-
4-m eth oxyp h en yl)p ip er a zin e Hyd r obr om id e (5v). 81%
yield from trans-2-phenyl-1-cyclopropanecarbonyl chloride and
1-(3,5-dichloro-4-methoxyphenyl)piperazine: mp 233-234 °C;
1H NMR (400 MHz, DMSO-d6) δ 1.04-1.07 (m, 1H), 1.13-
1.14 (m, 1H), 1.39-1.41 (m, 1H), 1.99-2.03 (m, 1H), 3.03 (t, J
) 12.0 Hz, 2H), 3.10-3.20 (m, 2H), 3.24-3.25 (m, 2H), 3.54
(bd, J ) 11.6 Hz, 2H), 3.72 (s, 3H, OCH3), 3.88-3.90 (m, 2H),
7.12-7.19 (m, 5H), 7.25-7.29 (m, 2H); MS (LC-MS) m/e 391
(free base, MH+). Anal. (C21H24N2OCl2‚HBr) C, H, N.
To a 20-mL solution of AlH3 (0.5 M in THF) was added a
solution of 10m in 10 mL of THF at 0 °C. The mixture was
stirred at room temperature for 3 h, then carefully quenched
with NaOH (10%). The aqueous layer was then extracted with
EtOAc (30 mL × 2). The combined organics were washed with
brine, dried over Na2SO4 and concentrated. The free base was

3930 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Zhang et al.
dissolved in 2-propanol and then treated with HBr (48%) to
adjust the pH to 3. Crystallization of the salt in i-PrOH/i-Pr2O
provided the desired product (1.25 g, 88%) as white crystals:
and 1-(3-chlorophenyl)piperazine: mp 215 °C dec; 1H NMR
(free base, 400 Hz, CDCl3) δ 0.88 (dt, J ) 5.0, 8.5 Hz, 1H),
0.97 (dt, J ) 5.0, 8.5 Hz, 1H), 1.21-1.26 (m, 1H), 1.68 (dt, J )
5.0, 9.0 Hz, 1H), 2.39 (dd, J ) 7.0, 13.0 Hz, 1H), 2.59 (dd, J )
7.0, 13.0 Hz, 1H), 2.63-2.68 (m, 4H), 3.12-3.22 (m, 4H), 6.76-
6.81 (m, 2H), 6.87 (s, 1H), 6.97-7.02 (m, 2H), 7.15 (t, J ) 8.5
Hz, 1H), 7.21-7.30 (m, 2H). Anal. (C20H22FClN2‚2HBr‚0.5H2O)
C, H, N.
1
mp 142-143 °C; H NMR (400 MHz, DMSO-d6) δ 1.06-1.15
(m, 2H), 1.43-1.45 (m, 1H), 2.02-2.07 (m, 1H), 3.05 (t, J )
12.0 Hz, 2H), 3.21-3.27 (m, 4H), 3.42-3.45 (m, 2H), 3.60-
3.62 (m, 2H), 7.13-7.18 (m, 3H), 7.23-7.29 (m, 2H), 7.40 (dd,
J ) 2.4, 8.0 Hz, 2H), 7.60 (d, J ) 2.4 Hz, 1H); MS (LC-MS)
20
m/e 361 (free base, MH+); [R]D +94.0° (free base, c ) 0.436,
tr a n s-1-[(2-(4-F lu or op h en yl)cyclop r op yl)m eth yl]-4-(4-
ch lor op h en yl)p ip er a zin e Dih yd r obr om id e (6e). 79% yield
from trans-2-(4-fluorophenyl)-1-cyclopropanecarboxylic acid
CH3Cl). Anal. (C20H22N2Cl2‚HBr) C, H, N. The optical purity
was shown to be >99% by HPLC analysis (CHIRACEL OD,
1.0 mL/min, methanol). The approximate retention time was
6.45 min.
1
and 1-(4-chlorophenyl)piperazine: mp 203 °C; H NMR (free
base, 400 Hz, CDCl3) δ 0.85 (dt, J ) 5.0, 8.5 Hz, 1H), 0.94 (dt,
J ) 5.0, 8.5 Hz, 1H), 1.21-1.23 (m, 1H), 1.70 (dt, J ) 5.0, 9.0
Hz, 1H), 2.41 (dd, J ) 7.5, 13.0 Hz, 1H), 2.60 (dd, J ) 7.5,
13.0 Hz, 1H), 2.68-2.70 (m, 4H), 3.16-3.20 (m, 4H), 6.82-
6.85 (m, 2H), 6.94 (t, J ) 8.8 Hz, 1H), 7.00-7.04 (m, 3H), 7.18-
7.21 (m, 2H). Anal. (C20H22FClN2‚2HBr‚0.5H2O) C, H, N.
Compounds 5l,m ,s,t and 6a -e were prepared according to
Method B.
(1R,2R)-tr a n s-1-[(2-P h en ylcyclop r op yl)m eth yl]-4-(2,4-
dich lor oph en yl)piper azin e Dih ydr obr om ide (5l). mp 197-
198 °C; 1H NMR (free base, 400 MHz, CDCl3) δ 0.85-0.89 (m,
1H), 0.97-1.01 (m, 1H), 1.25-1.31 (m, 1H), 1.69-1.74 (m, 1H),
2.42-2.47 (m, 1H), 2.58-2.63 (m, 1H), 2.74 (bs, 4H), 3.06 (bs,
4H), 6.96 (d, J ) 8.4 Hz, 1H), 7.06-7.08 (m, 2H), 7.15-7.20
(m, 2H), 7.28-7.31 (m, 3H); MS (LC-MS) m/e 361 (free base,
tr a n s-1-[(2-(4-Hyd r oxyp h en yl)cyclop r op yl)m eth yl]-4-
(2,4-d ich lor op h en yl)p ip er a zin e Hyd r obr om id e (6b). To
a solution of 6a (free base, 0.5 g 1.3 mmol) in 12 mL of CH2Cl2
was added 5 mL of BBr3 solution (1.0 M in CH2Cl2) at -78 °C.
The mixture was warmed to room temperature and then
quenched with 15 mL of CH3OH. The volatile was removed
under reduced pressure. The residue was partitioned between
CHCl3 (30 mL) and saturated NaHCO3 solution and the pH
of the aqueous was carefully adjusted to 10 by 10% NaOH.
The aqueous layer was extracted with CHCl3 (20 mL × 2) and
the combined organics were washed with brine, dried over Na2-
SO4 and concentrated. The crude product was purified by
chromatography to provide 0.35 g (73%) of colorless oil, which
was then converted to hydrobromide salt: mp 210-211 °C;
1H NMR (400 MHz, DMSO-d6) δ 0.96-1.02 (m, 2H), 1.27-
1.28 (m, 1H), 1.91-1.97 (m, 1H), 3.03 (t, J ) 11.2 Hz, 2H),
3.22-3.29 (m, 4H), 3.43 (bd, J ) 12.0 Hz, 2H), 3.59 (bd, J )
10.8 Hz, 2H), 6.66 (d, J ) 8.4 Hz, 2H), 6.93 (d, J ) 8.4 Hz,
2H), 7.24 (d, J ) 9.2 Hz, 1H), 7.40 (dd, J ) 9.2, 2.8 Hz, 1H),
7.60 (d, J ) 2.8 Hz, 1H); MS (LC-MS) m/e 377 (free base,
MH+). Anal. (C20H22N2Cl2O‚HBr) C, H, N.
In d ir ect Resolu tion of tr a n s-2-P h en yl-1-cyclop r op a n -
eca r boxylic Acid (8a ,b). To a solution of (R)-R-methylben-
zylamine (17.6 g, 145 mmol) in 200 mL of dichloromethane
was added 20 mL of triethylamine followed by the addition of
trans-2-phenyl-1-cyclopropanecarbonyl chloride (25.0 g 138.4
mmol) at 0 °C. The mixture was stirred at room temperature
for 3 h and then quenched by the addition of aqueous HCl (5%,
100 mL). The aqueous layer was extracted with chloroform
(100 mL × 2) and the combined organic layers was washed
with brine, dried over Na2SO4 and evaporated to dryness to
give 36 g of crude product as an off-white solid. The crude
product was dissolved in 350 mL of EtOAc at reflux and then
cooled to room temperature. After filtration, 9.0 g of crystals
were obtained and it was recrystallized from 180 mL of EtOAc
to provide the amide 9a (5.5 g, 15%) as white needles: mp
180-181 °C; 1H NMR (400 MHz, CDCl3) δ 1.21-1.28 (m, 1H),
1.51 (d, J ) 9.2 Hz, 3H, CH3), 1.59-1.67 (m, 2H), 2.45-2.52
(m, 1H), 5.11-5.21 (m, 1H, CH), 5.83 (bd, J ) 9.6 Hz, 1H, NH),
7.05-7.08 (m, 2H), 7.15-7.20 (m, 1H), 7.25-7.31 (m, 4H),
7.34-7.37 (m, 3H); MS (LC-MS) m/e 265 (MH+); [R]D20 -207°
(c ) 0.50, CHCl3). The mother liquid of the crystallization was
concentrated to give 16.0 g of a mixture of 9a ,b (9b major
component), which was utilized in the next step.
MH+); [R]D -89.4° (free base, c ) 0.4675, CHCl3). Anal.
20
(C20H22N2Cl2‚2HBr) C, H, N. The optical purity was shown to
be >99% by HPLC analysis (CHIRACEL OD, 1.0 mL/min,
methanol). The approximate retention time was 7.38 min.
(1R,2R)-tr a n s-1-[(2-P h en ylcyclop r op yl)m eth yl]-4-(2,4-
d im et h ylp h en yl)p ip er a zin e Dih yd r ob r om id e (5s). 19%
yield from (1R,2R)-trans-2-phenyl-1-cyclopropanecarboxylic
acid (8a ) and 1-(2,4-dimethylphenyl)piperazine: mp 215-217
1
°C; H NMR (400 MHz, CD3OD) δ 1.14-1.19 (m, 1H), 1.22-
1.28 (m, 1H), 1.16-1.49 (m, 1H), 2.06-2.09 (m, 1H), 2.25 (s,
3H, CH3), 2.28 (s, 3H, CH3), 3.02-3.10 (m, 2H), 3.22-3.30 (m,
6H), 3.67-3.72 (m, 2H), 6.98 (d, J ) 1.1 Hz, 2H), 7.03 (s, 1H),
7.14-7.20 (m, 3H), 7.26-7.30 (m, 2H); MS (LC-MS) m/e 321
(MH+); [R]D -58.5° (c ) 0.582, CH3OH). Anal. (C22H28N2‚
20
2HBr) C, H, N. The optical purity was shown to be >99% by
HPLC analysis (CHIRACEL OD, 1.0 mL/min, methanol).
(1S,2S)-tr a n s-1-[(2-P h en ylcyclop r op yl)m eth yl]-4-(2,4-
d im eth ylp h en yl)p ip er a zin e Dih yd r obr om id e (5t). 29%
yield from (1S,2S)-trans-2-phenyl-1-cyclopropanecarboxylic
acid (8b) and 1-(2,4-dimethylphenyl)piperazine: mp 224-225
1
°C; H NMR (400 MHz, CD3OD) δ 1.14-1.19 (m, 1H), 1.22-
1.28 (m, 1H), 1.16-1.49 (m, 1H), 2.06-2.09 (m, 1H), 2.25 (s,
3H, CH3), 2.28 (s, 3H, CH3), 3.02-3.10 (m, 2H), 3.22-3.30 (m,
6H), 3.67-3.72 (m, 2H), 6.98 (d, J ) 1.1 Hz, 2H), 7.03 (s, 1H),
7.14-7.20 (m, 3H), 7.26-7.30 (m, 2H); MS (LC-MS) m/e 321
(MH+); [R]D +60.7° (c ) 0.594, CH3OH). Anal. (C22H28N2‚
20
2HBr) C, H, N. The optical purity was shown to be >99% by
HPLC analysis (CHIRACEL OD, 1.0 mL/min, methanol).
tr a n s-1-[(2-(4-Meth oxyp h en yl)cyclop r op yl)m eth yl]-4-
(2,4-d ich lor op h en yl)p ip er a zin e Hyd r obr om id e (6a ). 60%
yield from trans-2-(4-methoxyphenyl)-1-cyclopropanecarboxylic
acid28 and 1-(2,4-dichlorophenyl)piperazine: mp 223-224 °C;
1H NMR (400 MHz, DMSO-d6) δ 1.00-1.08 (m, 2H), 1.33-
1.34 (m, 1H), 1.97-1.99 (m, 1H), 3.01-3.07 (m, 3H), 3.20-
3.29 (m, 3H), 3.44 (d, J ) 12.8 Hz, 2H), 3.60 (d, J ) 9.6 Hz,
2H), 3.70 (s, 3H, OCH3), 6.83 (d, J ) 8.4 Hz, 2H), 7.06 (d, J )
8.4 Hz, 2H), 7.24 (d, J ) 8.8 Hz, 1H), 7.40 (dd, J ) 2.4, 8.6 Hz,
1H), 7.60 (2.4, 1H); MS (LC-MS) m/e 391 (free base, MH+).
Anal. (C21H24N2OCl2‚HBr) C, H, N.
tr a n s-1-[(2-(4-F lu or op h en yl)cyclop r op yl)m eth yl]-4-(2-
ch lor op h en yl)p ip er a zin e Dih yd r obr om id e (6c). 67% yield
from trans-2-(4-fluorophenyl)-1-cyclopropanecarboxylic acid29
The mixed amide 9a ,b (16.0 g, 60 mmol) was suspended in
300 mL of ethylene glycol and mixed with 15 g of KOH and 9
mL (180 mL) of hydrazine monohydrate. The mixture was
stirred at 160 °C for 30 h and then quenched by the addition
of 200 mL of water at room temperature. The aqueous layer
was extracted with Et2O (200 mL) and the ether layer was
discarded. The aqueous layer was acidified carefully with
concentrated HCl to pH ∼ 1, then extracted with EtOAc (300
mL × 3). The extracts were washed with brine, dried over Na2-
SO4 and concentrated to give 9.5 g (98%) of gummy oil, which
is a mixture of trans-2-phenyl-1-cyclopropanecarboxylic acid
(8a ,b) (8b major component). This acid was coupled with (S)-
1
and 1-(2-chlorophenyl)piperazine: mp 203 °C; H NMR (free
base, 400 Hz, CDCl3) δ 0.86 (dt, J ) 5.0, 8.5 Hz, 1H), 0.96 (dt,
J ) 5.0, 8.5 Hz, 1H), 1.20-1.26 (m, 1H), 1.71 (dt, J ) 5.0, 9.0
Hz, 1H), 2.42 (dd, J ) 7.0, 12.5 Hz, 1H), 2.61 (dd, J ) 7.0,
12.5 Hz, 1H), 2.69-2.72 (m, 4H), 3.22-3.25 (m, 4H), 6.93-
7.06 (m, 6H), 7.20 (t, J ) 8.0 Hz, 1H), 7.35 (dd, J ) 1.0 and
8.0 Hz). Anal. (C20H22FClN2‚2HBr‚0.5H2O) C, H, N.
tr a n s-1-[(2-(4-F lu or op h en yl)cyclop r op yl)m eth yl]-4-(3-
ch lor op h en yl)p ip er a zin e Dih yd r obr om id e (6d ). 74% yield
from trans-2-(4-fluorophenyl)-1-cyclopropanecarboxylic acid

trans-1-[(2-Phenylcyclopropyl)methyl]-4-arylpiperazines
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21 3931
R-methylbenzylamine (7.10 g, 59 mmol) with EDCI (11.31 g,
59 mmol), HOBT (7.97 g, 59 mmol) and 10 mL of trimethyl-
amine in 100 mL of CH2Cl2 at room temperature. The crude
product was crystallized twice in EtOAc to provide the amide
4. Da t a An a lysis. Binding data were analyzed with the
nonlinear curve-fitting program RS/1 (BBN Software Products,
Cambridge, MA). Calculated IC50 values were then converted
to Ki values using the Cheng-Prusoff correction30 with the
following equation: Ki ) IC50/(1 + [L]/Kd), where [L] is the
radioligand concentration and Kd is the previously determined
dissociation constant for [3H]YM 09151 at the cloned human
D2 receptor (0.070 nM) and cloned human D4 receptor (0.37
nM).
1
9c (3.96 g, 26%) as long-needle cystals: mp 175-176 °C; H
NMR (400 MHz, CDCl3) δ 1.21-1.28 (m, 1H), 1.51 (d, J ) 9.2
Hz, 3H, CH3), 1.59-1.67 (m, 2H), 2.45-2.52 (m, 1H), 5.11-
5.21 (m, 1H, CH), 5.83 (bd, J ) 9.6 Hz, 1H, NH), 7.05-7.08
(m, 2H), 7.15-7.20 (m, 1H), 7.25-7.31 (m, 4H), 7.34-7.37 (m,
3H); MS (LC-MS) m/e 265 (MH+); [R]D +216° (c ) 0.73,
20
5. Dop a m in e F u n ction a l Assa ys. CHO cells stably ex-
pressing either human D4.2 or D2 receptor were grown to
confluency in Ham’s media supplemented with 10% fetal calf
serum, harvested, and then stored as pellets at -80 °C.
Thawed cells were homogenized using a Polytron (30 s, setting
5) in 50 mM Tris pH 7.4, 10 mM MgCl2 and 2 mM EGTA.
Membrane homogenates were centrifuged at 14000g for 10 min
and the pellet washed one time in cold PBS. The final pellet
was resuspended in homogenization buffer and stored at -80
°C. On the day of the assay, thawed membrane homogenates
were resuspended in assay buffer (50 mM Tris pH 7.4, 120
mM NaCl, 10 mM MgCl2, 2 mM EGTA, 0.1% BSA, 0.1 mM
bacitracin, 100 KIU/mL aprotinin, 5 µM GDP) and added to
reaction tubes at a concentration of 25 µg/0.200 mL. Reactions
were initiated by the addition of 100 pM GTP[γ-35S], dopamine
(0.01 nM-10 µM) and individual compounds ranging in
concentration from 0.1 nM to 10 µM. Following a 30-min
incubation at 22 °C, the reaction was terminated by vacuum
filtration over GF/C filters with ice-cold wash buffer (50 mM
Tris pH 7.4, 5 mM MgCl2). Bound GTP[γ-35S] was determined
by liquid scintillation spectrometry. Nonspecific binding was
defined by 10 µM GTP[γ-35S] and represented less than 10%
of total binding.
6. Beh a vior a l Assa ys. Male Sprague-Dawley rats weigh-
ing 200-350 g served as subjects. The animals were housed
in groups of three in a temperature-controlled (21 ( 1 °C)
animal facility on a 12-h light-dark cycle (lights on at 0630 h)
and had free access to food and water.
Locomotor activity was measured in eight computerized
Digiscan-16 animal activity monitors (Omnitech Electronics,
Columbus, OH) equipped with 48 infrared photocell emittors
and detectors (2.5 cm between sensors). Each box (41 × 41 ×
30 cm3) was constructed of Plexiglass sides and floor.
Rats were pretreated with clozapine (0.125-8.0 mg/kgsc),
haloperidol (0.0075-0.25 mg/kg sc), 5m (4-16 mg/kg sc) or
5t (4-16 mg/kg sc) 30 min prior to an injection of 0.5 mg/kg
d-amphetamine (ip). Immediately following the amphetamine
administration, the rats were placed into the Omnitech activity
boxes where horizontal (total distance traveled) and vertical
activity (rearing) were measured for 1 h.
7. Micr osom a l Meta bolism Assa ys. Into a 5-mL deep well
microtiter plate was placed a microsomal reaction preparation
containing the following: microsomal protein (50 µL, having
a P450 content of ∼0.5 nmol/mg protein), test compound
solution (10 µL of 10 mmol solution in DMSO), 0.1 M
phosphate buffer (798 µL). The plate was preincubated at 39°
C for 10 min. A cofactor mixture was prepared by dissolving
NADP (16.2 mg) and glucose-6-phosphate dehydrogenase (45.4
mg) in 4 mL of 100 mM MgCl2 solution. The enzyme reaction
was then initiated by the addition of 142 µL of the cofactor
mixture to the microsomal reaction preparation followed by
shaking. At each time point (0, 1, 3, 5 and 10 min), 175 µL of
the reaction mixture was transferred via pipet to be quenched
with 175 µL of ice-cold acetonitrile. After the final sample was
collected, the samples were centrifuged at 6000 rpm for 10 min.
HPLC analysis of the samples was then carried out with UV
detection. Concentrations were determined using a standard
concentration curve.
CHCl3).
(1R,2R)-tr a n s-2-P h en yl-1-cyclopr opan ecar boxylic Acid
(8a ). 93% yield from 9a following the procedure described
above: 1H NMR (400 MHz, CDCl3) δ 1.40-1.45 (m, 1H), 1.64-
1.70 (m, 1H), 1.88-1.94 (m, 1H), 2.58-2.64 (m, 1H), 7.10-
20
7.19 (m, 2H), 7.27-7.32 (m, 3H); [R]D -425° (c ) 0.725,
CHCl3) [lit.26 [R]D -410° (c ) 1.0, CHCl3)].
20
(1S,2S)-tr a n s-2-P h en yl-1-cyclop r op a n eca r boxylic Acid
(8b). 99% yield hydrolyzed from 9c following the same
procedure as described above: 1H NMR (400 MHz, CDCl3) δ
1.40-1.45 (m, 1H), 1.64-1.70 (m, 1H), 1.88-1.94 (m, 1H),
2.58-2.64 (m, 1H), 7.10-7.19 (m, 2H), 7.27-7.32 (m, 3H);
[R]D +453° (c ) 0.9, CHCl3) [lit.26 [R]D +405° (c ) 1.0,
20
20
CHCl3)].
Biologica l Meth od s. 1. Exp r ession of Recom bin a n t
Dop a m in e Recep tor s. The recombinant human D4.2 receptor
was prepared from the D4.2 minigene expression construct by
replacement of the NotI-KasI fragment containing two introns
with a synthetic DNA fragment encoding the intron-deleted
sequence (Genbank #HSD4DOP). Stable clones expressing
each receptor were isolated under G418 selection after calcium
phosphate transfection of CHO-K1 cells (the human D4.2
plasmid was cotransfected with pSV2Neo; Clontech). The
membranes prepared from cell pellets were stored at -80 °C.
2. Mem br a n e P r ep a r a tion . Pellets containing selected
cloned dopamine receptor membrane were thawed on ice and
resuspended in ice cooled 50 mM Tris buffer (pH 7.4 at 25 °C)
containing 120 mM NaCl, 1 mM EDTA and 5 mM MgCl2. All
subsequent work was performed on ice. The membranes were
homogenized on a Brinkmann polytron (10 s, setting 5). The
homogenate was centrifuged at 48000g and 4 °C for 10 min
(DuPont Sorvall RC5B). The pellet was resuspended in fresh
buffer and centrifugation was repeated. The pellet was again
resuspended in fresh buffer and centrifuged a final time at
48000g and 4 °C for 10 min. The pellet was resuspended to a
final concentration of 100 mg protein/mL with 50 mM Tris
buffer (pH 7.4 at 25 °C) containing 120 mM NaCl, just prior
to addition to the assay tubes. The protein content was
determined using the Bio-Rad assay (Hercules, CA), with
bovine plasma γ-globulin as a standard.
3. Bin d in g Assa y. For D2 and D4 binding, each sample was
tested in triplicate in a final volume of 0.5 mL in polypropylene
microtube strips containing 0.1 nM [3H]YM 09151 (81.4 Ci/
mmol; NEN DuPont) and CHO cell homogenate (40 µg protein)
in 50 mM Tris buffer (pH 7.4 at 25 °C) containing 120 mM
NaCl. After a 2-h incubation at 25 °C at room temperature
the samples were rapidly filtered through 1% PEI-treated
GF/C filters using a Tomtec harvester 96. The filters were
rinsed with two washes of assay buffer. After air-drying, bound
radioactivity was then quantitated via the BetaPlate scintil-
lation counter at an efficiency of 65%. Nonspecific binding was
defined with 1 mM spiperone.
For R1 binding, each sample was tested in triplicate in a
final volume of 0.5 mL in polypropylene microtube strips
containing 0.2 nM [3H]prazosin and rat cortex homogenate in
50 mM Tris buffer (pH 7.4 at 25 °C) containing 0.01%
Refer en ces
ascorbate. After
a 30-min incubation at 25 °C at room
temperature the samples were rapidly filtered through un-
treated GF/C filters using a Tomtec harvester 96. The filters
were rinsed with two washes of assay buffer. After air-drying,
bound radioactivity was then quantitated via the BetaPlate
scintillation counter at an efficiency of 65%. Nonspecific
binding was defined with 1 µM phentolamine.
(1) Carlsson, A.; Lindquist, M. Effect of Chlorpromazine or Halo-
peridol on Formation of 3-Methoxytyramine and Normetaneph-
rine in Mouse Brain. Acta Pharmacol. Toxicol. 1963, 20, 140-
144.
(2) Snyder, S. H. The Dopamine Hypothesis of Schizophrenia: Focus
on the Dopamine Receptor. Am. J . Psychiatry 1976, 133, 197-
202.

3932 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 21
Zhang et al.
(3) Seeman, P. Dopamine Receptors and the Dopamine Hypothesis
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