Hybrid approach for the design of highly affine and selective dopamine D3 receptor ligands using privileged scaffolds of biogenic amine GPCR ligands

Article abstract of DOI:10.1016/j.bmc.2007.08.034

A series of compounds containing privileged scaffolds of the known histamine H₁ receptor antagonists cetirizine, mianserin, ketotifen, loratadine, and bamipine were synthesized for further optimization as ligands for the related biogenic amine binding dopamine D₃ receptor. A pharmacological screening was carried out at dopamine D₂ and D₃ receptors. In the preliminary testing various ligands have shown moderate to high affinities for dopamine D₃receptors, for example, N-(4-{4-[benzyl(phenyl)amino]piperidin-1-yl}butylnaphthalen-2-carboxamide (19a) (hD₃ K_i = 0.3 nM; hD₂ K_i = 703 nM), leading to a selectivity ratio of 2343.

Full text of DOI:10.1016/j.bmc.2007.08.034

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7258–7273
Hybrid approach for the design of highly affine
and selective dopamine D3 receptor ligands using
privileged scaffolds of biogenic amine GPCR ligands
Britta C. Sasse,a Ulrich R. Mach,a Jukka Leppaenen,a,b
Thierry Calmelsc and Holger Starka,*
aInstitute of Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Max-von-Laue-Straße 9, 60438 Frankfurt, Germany
bDepartment of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland
cBioprojet-Biotech, 4 rue du Chesnay Beauregard, BP 96205, 35762 Saint-Gre´goire, France
Received 28 July 2006; revised 31 July 2007; accepted 21 August 2007
Available online 25 August 2007
Abstract—A series of compounds containing privileged scaffolds of the known histamine H1 receptor antagonists cetirizine, mian-
serin, ketotifen, loratadine, and bamipine were synthesized for further optimization as ligands for the related biogenic amine binding
dopamine D3 receptor. A pharmacological screening was carried out at dopamine D2 and D3 receptors. In the preliminary testing
various ligands have shown moderate to high affinities for dopamine D3receptors, for example, N-(4-{4-[benzyl(phenyl)amino]pip-
eridin-1-yl}butylnaphthalen-2-carboxamide (19a) (hD3 Ki = 0.3 nM; hD2 Ki = 703 nM), leading to a selectivity ratio of 2343.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
cognitive symptoms of schizophrenia, additionally pre-
vents from undesirable extrapyramidal side effects,
including tardive dyskinesia, parkinsonism, and dys-
tonic reactions, which are associated with dopamine
D2 receptor antagonism in the caudate putamen.4,9–13
It has been reported that dopamine D3 receptors play
an important role in mediating the reinforcing effects
of psychostimulants, such as cocaine.14 Therefore
selective dopamine D3 receptor antagonists and partial
agonists are under investigation to prove their thera-
peutic potential in the treatment of drug addic-
tion.15,16 Additionally, D3 receptor antagonists might
improve cognitive deficits due to enhancing frontocor-
tical cholinergic transmission.17
All dopamine receptor subtypes belong to class A of G-
protein coupled receptors (GPCRs) and are subdivided
into two subfamilies: D1-like receptors, with its subtypes
D1 (D1a) and D5 (D1b), activating adenylyl cyclase, and
D2-like receptors, including D2, D3, and D4 receptor
subtypes, inhibiting adenylyl cyclase.1–3 Imbalance of
the dopaminergic system is implicated in various neuro-
logical and neuropsychiatric disorders, including Par-
kinson’s disease, schizophrenia, Tourette‘s syndrome,
and drug abuse.1–4
The dopamine D3 receptor subtype has been discov-
ered by Sokoloff and colleagues in 1990.5 Subsequent
identification of its distinct distribution in the limbic
regions of the striatum, predominant in the islands
of Calleja and the nucleus accumbens, allowed to
deduce that it may be a therapeutic target for anti-
psychotic and antiparkinsonian drugs.4,6–8 Selective
antagonism at D3 receptors reduces negative and
Identification of selective dopamine D3 receptor ligands
bearing subnanomolar affinities to lower the risk of mo-
tor extrapyramidal syndrome and to understand the
pharmacological role of dopamine D3 receptors has been
and still is a great topical challenge in drug development.
To date some series of compounds with these require-
ments have been achieved and are actually in ongoing
clinical development as potential therapeutics for the
aforementioned disorders.18,19 Some representative li-
gands showing D3 receptor-preference with antagonist
and partial agonist properties are shown in Figure 1.20–22
Keywords: Privileged structures; Class A GPCR; Dopamine; D3 rece-
ptor; Histamine; H1 receptor; Radioligand competition binding assay.
*
Corresponding author. Tel.: +49 69 798 29302; fax: +49 69 798
29258; e-mail: h.stark@pharmchem.uni-frankfurt.de
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.034

B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
7259
O
O
O
N
S
N
N
N
H
N
Cl
H
H
N
N
OCH3
N
Cl
ST 198
FAUC 365
BP 897
CH3
O
H
O
N
O
O
CH3
O
N
O
S
N
N
H
H
CN
CN
H3C
O
H3C
N
H
S33084
GR218,231
S33138
CF3
N
CH3
CH3
N
H3C
HO
N
S
N
N
A437,203
Figure 1. Dopamine D3 receptor selective antagonists and partial agonists.
Compound BP 897 (Fig. 1) was identified as a selective
dopamine D3 receptor partial agonist with high affinity
binding at human D3 (hD3) (Ki = 0.92 nM) and 70-fold
selectivity over human D2 (hD2) (Ki = 61 nM) behaving
as an antagonist at this subtype.23,24 Further evaluation
of in vitro models showed that accordingly BP 897 dem-
onstrated in many models antagonist D3 receptor profile
in addition to its partial agonist properties.25,26 BP 897
attenuated the behavioral and reinforcing effects of co-
caine, showing a promising property for the treatment
of drug abuse. Regarding the interactions of BP 897 at
multiple classes of monoaminergic receptors, the only
pivotal role of D3 receptors in the mechanism of reduced
cocaine-seeking behavior is not confirmed yet.4 The
treatment of levodopa-induced dyskinesias in patients
with Parkinson’s disease is under investigation.27 To
date BP 897 is ongoing phase II clinical studies.28
(pKi = 9.0) over hD2 (pKi = 7.2).32,33 S33138 (Fig. 1)
and A437,203 (Fig. 1) are promising agents which are
under clinical evaluation. Both compounds behave as
selective dopamine hD3 over hD2 receptor antagonists.
A437,203 has an influence on brain dopamine activity
and has demonstrated antipsychotic properties without
clear extrapyramidale effects.34,35 In vitro data have con-
firmed the former as a highly potent D3 receptor antago-
nist having a Ki value of 2.9 nM.36
Antipsychotic drugs have become first line treatment of
schizophrenia despite their interactions with several neu-
rotransmitter receptors, including histamine H1 recep-
tors, serotonin 5-HT2a receptors, and a1/a2 adrenergic
receptors.37 This multireceptor affinity has been consid-
ered to effect both therapeutic advantages but also ad-
verse effects.38 The antagonist binding profile of
antipsychotics for central histamine H1 receptors has
been well demonstrated by chlorpromazine, a phenothi-
azine derivative, initially developed for its antiallergic
properties by Delay and Deniker in 1952.39 Addition-
ally, histamine H1 receptor antagonists containing tri-
and tetracyclic structures display high affinity for diverse
catecholamine receptors, due to the highly conserved li-
gand–receptor interaction of biogenic amine receptors
by an aspartate (Asp) residue in transmembrane (TM)
domain 3.40–42
Another related development on this lead structure is
FAUC 365 (Fig. 1), an antagonist bearing a heteroatom
substituted bicyclic ring system and a (2,3-dichlorophe-
nyl)piperazine substructure. The compound demon-
strated high affinity for hD3 (Ki = 0.5 nM), while the
affinity for hD2 was dependent on different assay condi-
tions, but provided an impressive high selectivity for
hD3 over hD2 (7200)29 although this could not be con-
firmed by other groups.30 Further development is repre-
sented by the 1,2,3,4-tetrahydroisoquinoline compound
ST 198 (Fig. 1) (Ki = 12 nM (hD3); Ki = 780 nM
(hD2)). It has been reported that this antagonist normal-
izes dopamine D3 receptor function and subsequently
attenuates levodopa-induced dyskinesia.27
Lipophilic/aromatic moieties connected to a basic nitro-
gen atom41 are often claimed as ‘privileged structures’
for GPCRs and are supposed to be important for both
dopamine D2-like receptor and histamine H1 receptor
binding sites. In order to elucidate structure–activity
relationships (SAR) of the D2-like receptor profile show-
ing similar structural requirements than that for H1
receptor binding, we investigated in a novel hybrid
structure development to identify highly affine dopa-
mine D3 receptor selective ligands. An approach was
undertaken by synthesizing hybrids containing privi-
leged scaffolds of histamine H1 receptor antagonists,
The benzopyranopyrrole derivative S33084 (Fig. 1) also
behaves as an antagonist and has displayed high hD3
binding affinity (pKi = 9.5) and >100-fold selectivity ratio
for hD3 receptors.31 Furthermore GR218,231 (Fig. 1), an
aminotetraline derivative with less selectivity for D3
receptors compared to S33084 has been described. This
ligand preferentially interacts as an antagonist at hD3

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B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
Cl
N
O
N
H3C
O
N
N
HO
N
N
S
N
H3C
O
H3C
O
Cl
O
Cetirizine
Mianserin
Ketotifen
Loratadine
N
N
H3C
Bamipine
Figure 2. Histamine H1 receptor antagonists containing privileged scaffolds.
so-called ‘antihistamines’43 (Fig. 2) and fragments of
dopamine D3 receptor-preferring ligands. The compo-
nents of histamine H1 receptor antagonists comprise
basic substructures of cetirizine ([(4-chlorophenyl)phe-
nylmethyl]piperazine, S1), mianserin (4-(2,3,4,5,10,15-
hexahydro-1H-dibenzo[b:e]pyrazino[2,1-g])azepine, S2),
ketotifen (4-[4-(10-oxo-9,10-dihydro-4H-benzo[4,5]cyclo-
hepta[1,2-b]thiophen)-4-yliden]piperidine, S3), loratadine
(4-[(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-
b]pyridin)-11-yliden]piperidine, S4), and bamipine
(N-benzyl-N-piperidin-4-ylaniline, S5).43 These residues
were connected via a tetramethylene chain to naphta-
len-2-carboxamide, cinnamide, benzo[b]thiophen-2-car-
boxamide and its diminished bioisostere thiophen-2-
carboxamide, or phthalimide moiety, recognized as the
structural elements of BP 897, ST 198, FAUC 365
(Fig. 1), and NAN 190, respectively. NAN (N-(4-[4-(2-
methoxyphenyl)piperazinyl)butyl)isoindolin-1,3-dion)
has demonstrated less binding affinity and selectivity for
dopamine D3 receptors compared to BP 897, ST 198 and
FAUC 365. It has shown high affinity binding at seroto-
nin 5-HT1A receptors and this pharmacological profile
might enhance existing antipsychotic treatment.44 The
phthalimide residue combines two amide functions rig-
idly incorporated into the heteroaromatic moiety and
provides a promising novel scaffold. Moreover, the neces-
sity of an aromatic amide residue has been investigated by
introducing a cyclohexylamide.
and affine dopamine D3 receptor selective ligands and to
elucidate the SAR of dopamine D3 and D2 receptors by
employing privileged scaffolds of histamine H1 receptor
ligands.
2. Chemistry
Benzhydrylpiperazine compounds (3a–d) were prepared
starting from monocarbamate-protected piperazine and
either benzhydryl chlorides (1a, 1b) or benzhydryl alco-
hols (1c, 1d) (Scheme 1).
In the latter case, we utilized a method to couple second-
ary amines and primary, aliphatic alcohols.46 This con-
venient method, which is here applied for the first time
on secondary benzylic alcohols, allowed the preparation
of benzhydrylpiperazines in remarkable yields about
95%. The protective group was subsequently cleaved un-
der alkaline conditions. Comparable to the preparation
of 3a–d, monoacetylated 1,4-diazepane was substituted
with benzhydryl chloride, the acetyl group was cleaved
under strong basic conditions in moderate yields (not
shown).47 Preparation of the final compounds followed
two different routes. A first straightforward approach
involved the alkylation of appropriate secondary amines
with N-(4-bromobutyl)phthalimide (4a–d). Subsequent
hydrazinolysis led to primary amines (5a–c), which on
treatment with different arylcarboxylic acid chlorides re-
sulted in the corresponding amides 6a–8d (Scheme 2).48
For the novel ligands, the calculated clogP values are in
the range of 3.18–6.53 (Table 1) (ChemOffice Ultra 7.0)
which show higher lipiphilicity than of most CNS active
compounds with an logP value of about 2.5 as standard
orientation. The hybrid approach provided compounds
with physicochemical properties allowing them to pene-
trate the blood–brain barrier although they are more
voluminous and more lipophilic than optimal for mar-
keted drugs.45
A more elegant way of preparation was adopted
from the published procedure as mentioned below
(9a–11c) (Scheme 3).46 N-(4-Hydroxybutyl) substituted
arylcarboxamides,
phenylvinylcarboxamides,
and
benzo[b]thiophen-2-carboxamides were coupled to
benzhydrylpiperazines in one-pot procedures. This
method allowed the versatile preparation of numerous
potentially active compounds (10, 11a–c).
The revealed benzhydrylpiperazine derivatives, tri- and
tetracyclic ring system containing compounds and bam-
ipine analogues were preliminary screened for binding
affinities at dopamine hD2 and hD3 receptors in radioli-
gand competition experiment carrying out six-point
measurements (Table 1). The aim was to develop highly
A series of (semi-)rigid analogues closely related to the
benzhydrylpiperazine scaffold 15a, b, 16a–e, 17a, 18a–
b, and 19a–b were prepared, starting from commercially
available compounds, for example, mianserin, ketotifen
or bamipine (Scheme 4).

B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
7261
Table 1. Chemical structures, physical data, and pharmacological screening results for human dopamine D2 and D3 receptor binding affinities
R2
N
R2
O
R
N
N
( )n
R1
N
R1
R
S
N
N
N
N
N
R
(CH2)4
R
(CH2)4
(CH2)4
(CH2)4
(CH2)4
R
Cl
S5
S1
S2
S3
S4
b
e
Compound
Structure
R
R1
R2
n
1
1
Ki (nM)
D2/D3
MW
clogP
4.75
a
a
D2
D3
O
N
4a
4b
S1
S1
H
H
H
598
47.3
28.0
13
453.6
488.1
O
O
N
4-Cl
559
20
5.31
O
O
O
N
N
4c
4d
6a
S1
S1
S1
H
H
H
H
2
2
1
1180
226
133
9
5
467.7
502.1
477.7
4.85
5.41
5.93
O
O
4-Cl
H
42.9
11.1
O
O
457
41
N
H
6b
6c
6d
S1
S1
S1
H
H
H
H
H
H
1
1
1
652
590
621
6.2
30.3
28.4
30.2
105
20
453.7
483.7
433.6
5.28
5.97
4.92
N
H
O
S
N
H
O
S
22
N
H
O
6e
7a
S1
S1
H
H
H
1
1
435
587
14
433.7
512.1
5.01
6.49
N
H
O
O
4-Cl
2.82c 0.4
208
N
H
7b
7c
S1
S1
4-Cl
4-Cl
H
H
1
1
963
2.4d 1.1
401
57
488.1
518.2
5.84
6.53
N
H
O
S
N
1000
17.7
H
O
S
7d
7e
8a
S1
S1
S1
4-Cl
4-Cl
H
H
H
H
1
1
2
160
189
136
38.9
12.3
32.6
4
15
4
468.1
467.1
491.7
5.48
5.57
6.04
N
H
O
N
H
O
N
H
(continued on next page)

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B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
Table 1 (continued)
b
e
Compound
Structure
R
R1
R2
n
Ki (nM)
D2/D3
MW
clogP
a
D2
a
D3
O
8b
8c
S1
S1
H
H
H
H
2
2
161
40.9
36.9
4
467.7
497.8
5.38
6.08
N
H
O
S
N
633
17
H
O
S
8d
10
S1
S1
H
H
H
2
1
663
626
340
2
447.7
513.8
5.02
5.84
N
H
O
S
N
H
2-OCH3
3.60
174
O
11a
11b
11c
S1
S1
S1
2-OCH3
2-OCH3
2-OCH3
OCH3
OCH3
OCH3
1
1
1
313
972
619
15.2
21
64
27
537.8
513.8
543.8
5.68
5.02
5.72
N
H
O
15.2
22.8
N
H
O
S
N
H
O
N
15a
S2
808
500
2
451.6
5.15
O
O
16a
16b
S2
S2
1,324
429
>1000
1
9
475.7
451.6
6.33
5.68
N
H
O
50.2
N
H
O
S
N
16c
16d
S2
S2
4,925
497
>1000
500
5
1
481.7
431.7
6.37
5.32
H
O
S
N
H
O
16e
15b
17a
18a
S2
S3
S3
S4
258
1,149
>1000
252
>1000
0.3
12
431.7
496.7
546.8
581.2
5.41
4.22
5.41
5.57
N
H
O
N
99.1
O
O
O
10.9
92
N
H
6.01
41
N
H
O
S
N
18b
15c
S4
S5
849
128
2.7
314
551.2
467.7
6.46
5.30
H
O
N
H
>1000
0.1
O

B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
7263
Table 1 (continued)
b
e
Compound
Structure
R
R1
R2
n
Ki (nM)
D2/D3
MW
clogP
a
D2
a
D3
O
19a
19b
23a
23b
23c
S5
S5
S5
S5
S5
H
703
0.3
2343
51
6
491.7
497.7
473.6
457.7
535.7
6.49
6.53
3.49
4.14
5.38
N
H
O
S
N
H
1570
31.1
H
O
2-OCH3
2-OCH3
2-OCH3
Acetyl
13,350
>1000
4620
2340
N
H
O
O
Propionyl
Benzoyl
>1000
>1000
1
N
H
5
N
H
O
N
24
S5
S5
S5
2-OCH3
2-OCH3
2-OCH3
H
H
H
2288
163
>1000
2
9
407.5
431.6
407.6
2.65
3.84
3.18
O
O
O
25a
25b
18.4
N
H
>1000
10.8
93
N
H
BP 897
ST 198
52 12
1272 99
0.91 0.2
8.72 0.2
57
146
417.5
334.5
4.44
3.79
a Values (Ki) received by one experiment performed in duplicates and 6 concentrations: 0.1–10,000 nM.
b Ratio is calculated from corresponding Ki values.
c Values (Ki) received by 6 experiments performed in duplicates and 6 concentrations: 0.1–10,000 nM.
d Values (Ki) received by 5 experiments performed in duplicates and 6 concentrations: 0.1–10,000 nM.
e Molecular weights of the analytically tested salt form is given.
O
R2
R2
R2
a
b
O
N
N
X
N
NH
HN
O
N
R1
Y
Y
Y
R1
3a - d
R1
O
1a: X=Cl, Y=R1=R2=H
1b: X=Y=Cl, R1=R2=H
1c: X=OH, R1=OCH3, Y=R2=H
1d: X=OH, R1=R2=OCH3, Y=H
2a: Y=R1=R2=H
2b: Y=Cl, R1=R2=H
2c: R1=OCH3, Y=R2=H
2d: R1=R2=OCH3, Y=H
Scheme 1. Synthesis of N-(diphenylmethyl)piperazine derivatives. Reagents and condition: (a) acetonitrile, K2CO3 for X = Cl; (CH3)3PCH2CN+Iꢀ,
DIPEA, propionitrile for X = OH; (b) KOH, MeOH, H2O, reflux.
The N-methylated compounds 12a, 12b, 12d were con-
verted to the corresponding carbamates 13a, 13b, 13d
employing ethyl chloroformate. The carbamates (includ-
ing loratadine 13c) were cleaved under alkaline condi-
tions to release the secondary amines 14a–d.49,50 These
amine precursors were subsequently coupled with N-
(4-bromobutyl)phthalimide, followed by the cleavage
of the phthalimide and acylation with the activated aryl-
carboxylic acid, or directly coupled with an N-(4-
hydroxybutyl)arylcarboxamide employing cyanometh-
yl(trimethyl)phosphonium iodide to give the final
compounds.43
A third series of compounds involved the preparation of
piperidine analogues of BP 897 (23a–c, 24, 25a, 25b)
(Scheme 5). Starting material was methoxy-substituted
aniline, which was condensed with N-benzylpiperidone
under Dean–Stark-conditions and hydrogenated over
PtO2 (20).51 Debenzylation and alkylation of compound
20 led to compound 24, which is both a final compound
and the starting material for the phthalimide procedure
described above. This deprotection followed by acyla-
tion sequence resulted in compounds 25a, 25b. Alterna-
tively, the acylation of intermediate 20 with different
acylchlorides provided compounds 21a– c, which were

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B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
O
O
a
Br
N
N
N
( )n
N
HN ( )n
N
Y
Y
O
O
3a
4a
: Y=H; n=1
: Y=H; n=1
3b: Y=Cl; n=1
3e: Y=H; n=2
4b: Y=Cl; n=1
4c: Y=H; n=2
3f
4d
: Y=Cl; n=2
: Y=Cl; n=2
O
b
c
H2N
N
R
S
N
H
N
N
( )n
N
Y
( )n
Y
5a: Y=H; n=1
: Y=Cl; n=1
: Y=H; n=2
6a - e: Y=H; n=1
5b
5c
7a - e
: Y=Cl; n=1
: Y=H; n=2
8a - d
S
R=
6a 8a
-
6b 8b
-
6c 8c
-
6d 8d
-
6e 7e
,
Scheme 2. Synthesis of substituted benzhydrylpiperazine derivatives. Reagents and condition: (a) acetonitrile, K2CO3; (b) N2H4, MeOH, reflux;
(c) ArCOCl, CH2Cl2, K2CO3.
R2
O
R2
O
a
N
R
N
N
R
N
H
H
HN
N
R1
OH
R1
R1=OCH3, R2=H
11a - c: R1=R2=OCH3
3c: R1=OCH3, R2=H
3d: R1=R2=OCH3
10:
9a - c
S
R=
9a, 11a
9b, 11b
9c, 10, 11c
Scheme 3. Alkylation of N-(4-hydroxybutyl)arylcarboxamides with benzhydrylpiperazine derivatives. Reagents and condition: (CH3)3PCH2CN+Iꢀ,
DIPEA; propionitrile; 90 ꢁC, 3 h.
O
R1
N
c
R2
N
O
R1
R2
15a - c
R1
R1
O
O
N
a
b
R2
d, e
HN
R2
H3CN
Cl
O
O
O
14a - d
12a
12b
12d
R1
13a, b, d
R
N
H
f
13c: loratadine
R2
N
16a - e, 17a, 18a - b, 19a - b
R1
N
=
O
N
N
R2
N
S
N
N
N
N
Cl
12a, 13a, 14a,
15a, 16a - e
12b, 13b, 14b,
15b, 17a
13c, 14c,
18a - b
12d, 13d, 14d,
15c, 19a - b
S
S
R =
16a, 17a, 18a, 19a
16b
16c, 18b, 19b
16d
16e
Scheme 4. Synthesis of rigidized analogues of benzhydrylpiperazines. Reagents and conditions: (a) toluene, reflux; (b) KOH, MeOH, reflux; (c) N-(4-
bromobutyl)isoindolin-1,3-dione, K2CO3, acetonitrile; (d) N2H4, MeOH, reflux; (e) ArCOCl, K2CO3, CH2Cl2; (f) N-(4-hydroxybutyl)arylcarbox-
amide, (CH3)3PCH2CN+Iꢀ, DIPEA, propionitrile; 90 ꢁC.

B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
7265
R1
N
R1 OCH3
N
OCH3
OCH3
H
N
O
NH2
a, b
c
d
BnN
BnN
HN
BnN
OCH3
20
21a - c
22a - c
21a: R1 = acetyl-
22a: R1 = acetyl-
d, f
21b: R1 = propionyl-
21c: R1 = benzoyl-
22b: R1 = propionyl-
22c: R1 = benzoyl-
OCH3
O
H
N
N
e
N
O
O
R1 OCH3
N
24
g, h
N
H
N
O
OCH3
H
N
23a, 23b
R
N
23a: R1 = acetyl-
23b: R1 = propionyl-
23c: R1 = benzoyl-
H
N
25a, 25b
25a: R= 2-naphthyl-
25b: R= phenylvinyl-
Scheme 5. Synthesis of piperidine analogues of BP 897. Reagents: (a) toluene, Dean–Stark-conditions; (b) H2/PtO2, MeOH; (c) acylchloride, K2CO3,
CH2Cl2; (d) H2/Pd(OH)2, MeOH; (e) N-(4-oxobutyl)naphthalen-2-carboxamide, NaBH(OAc)3, AcOH, ClCH2CH2Cl; (f) N-(4-bromobutyl)isoin-
dolin-1,3-dione; K2CO3, acetonitrile; (g) N2H4, MeOH; (h) ArCOCl, K2CO3, CH2Cl2.
subsequently debenzylated (22a–c) and reductively
alkylated to the final compounds 23a–c.52
residue (4a) by a naphthalen-2-carboxamide (6a), cinna-
mide (6b), benzo[b]thiophen-2-carboxamide (6c), thio-
phen-2-carboxamide (6d) or cyclohexyl carboxamide
(6e) resulted in similar binding affinities at dopamine
D2 receptors within the series. Alterations led to im-
proved affinity binding data and dopamine D3 recep-
tor-preference for the naphthalen-2-carboxamide 6a
and a distinct enhanced selectivity for the cinnamide
derivative 6b (Ki (D2/D3) = 105). The cinnamide struc-
ture provides an amide in a precise steric space relative
to the inflexible aromatic ring. This exact spacial orien-
tation seems to contribute to the selectivity due to ben-
eficial interactions with the dopamine D3 receptor
binding site. Comparing the affinity profiles of amides
(6) with data received from the imide containing com-
pound 4a led to the assumption that the amide NH func-
tion as a hydrogen bond donor is not essential as a
pharmacophoric element for receptor binding, whereas
the phthalimide moiety neither improves dopamine D3
receptor binding nor selectivity compared to that of
the amides. As demonstrated by the cyclohexyl amide
derivate 6e, an aromatic system in this position is not
necessarily required for affinity binding at both receptor
subtypes.
3. Results and discussion
We investigated in forty-one newly prepared compounds
and determined binding affinities in a competition bind-
ing experiment with six-point measurements in dupli-
cates. Binding assays were carried out using HEK-cells
transfected with human D2S and CHO-cells transfected
with human D3 dopamine receptor cDNAs and
[3H]spiperone (Table 1).53 Data received from one single
experiment can only give preliminary estimates of struc-
ture–activity relationships and further evaluation need
to be carried out to provide variability and reliability
within statistical limits.
The benzhydrylpiperazine residue (S1) based on the
structural element of the antihistaminergic cetirizine
(Fig. 2) has been combined with diverse (hetero)aryl
amides, cinnamides, phthalimides, and cyclohexyl
amides via a butyl spacer (4–11).
Furthermore, the benzhydrylpiperazine element has
been substituted either with a chlorine at 4-position
(4b, 4d, and 7) or methoxy group(s) at 2-position(s) of
the phenyl ring(s) (10 and 11), related to BP 897. For
some compounds, the piperazine ring has been replaced
by 1,4-diazepane (4c, 4d, 8).
The influence of different substitution patterns on the
phenyl rings of the benzhydrylpiperazine system has
been validated in the following series. Analogues of 4a
and 6 have been synthesized by introducing a chloro
atom at 4-position of the phenyl ring in the benzhydryl-
piperazine residue (4b, 7). The chlorine substitution in-
duced inhomogeneous binding profiles at dopamine D2
and D3 receptors when compared to the unsubstituted
series. Binding affinities of the ligands at D3 receptors
have been improved with regard to their chlorine unsub-
stituted analogues with the exception of the thiophen-2-
carboxamide 7d. As for dopamine D2 receptors, affinity
binding data did not change for the phthalimide
(4a ! 4b) and naphthalen-2-carboxamide (6a ! 7a)
derivatives. The modification led to a deterioration of
affinity binding for the cinnamide (6b ! 7b) and
Among the series of benzhydrylpiperazine derivatives,
compounds have revealed moderate nanomolar to low
nanomolar affinities for hD3 receptors and micromolar
to moderate nanomolar binding affinities for hD2
receptors.
The influence of diverse dopamine D3 receptor-prefer-
ring residues on the binding profile at dopamine D3
and D2 receptors has been investigated within com-
pounds 4a and 6. The replacement of the phthalimide

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B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
benzo[b]thiophen-2-carboxamide (6c ! 7c) derivatives,
while Ki values of the thiophen-2-carboxamide
(6d ! 7d) and cyclohexyl carboxamide (6e ! 7e) li-
gands demonstrated an improvement of affinities at
dopamine D2 receptors. The chlorine substituent ac-
counts for an additional hydrophobic interaction in
the binding pocket of the dopamine D3 receptor. As pre-
viously mentioned for the unsubstituted ligands 6a and
6b, the naphthalen-2-carboxamide 7a and the cinnamide
7b derivatives possessed the highest affinities for dopa-
mine D3 receptors in this series. A superior selectivity
for the dopamine D3 receptor have been revealed for
7b (Ki (D2/D3) = 401). The affinity and selectivity pro-
files of benzhydrylpiperazine derivatives which are
methoxy disubstituted at 2-positions of the phenyl rings
have been investigated within compounds 11a–11c.
Compared to the chlorine substituted analogues 7a–7c
the modification yielded compounds with slightly de-
clined affinity binding and selectivity for D3 receptors.
In this series, introducing a cinnamide residue (11b)
has been beneficial for selectivity binding for D3 over
D2 receptor as previously demonstrated by the unsubsti-
tuted compound 6b as well as for the chlorine substi-
tuted benzhydrylpiperazine derivative 7b. Replacing
the second methoxy substituent of benzo[b]thiophen-2-
carboxamide derivative 11c resulted in the methoxy
monosubstituted analogue 10 with advanced affinity
and selectivity for the dopamine D3 receptor. In addi-
tion, this modification revealed superior affinity binding
and selectivity for dopamine D3 receptors when com-
pared to its monosubstituted chlorine analogue 7c.
Disubstitution at 2-positions of the phenyl rings causes
a steric restriction of the aromatic diphenyl residues in
relation to the basic nitrogen. By introducing a substitu-
ent in ortho- or para-position, the phenyl rings are more
flexible and capable to adopt a favorable orientation for
dopamine D3 receptor binding.
at dopamine D2 and D3 receptors. A nanomolar binding
result at dopamine D3 receptors has been obtained only
for the cinnamide bearing compound 16b with a modest
D3 receptor-preference. A dopamine D2 receptor-prefer-
ence has been received for the cyclohexyl amide deriva-
tive 16e.
Benzothienylcycloheptadienpiperidine (S3), the sub-
structure of ketotifen (Fig. 2), was incorporated in 15b
and 17a and the modification has resulted in compounds
with nanomolar binding affinity for D3 receptors and
micromolar affinity binding for D2 receptors. Compared
to the phthalimide analogue 15b, the naphthalen-2-car-
boxamide 17a has an improved selectivity ratio for
dopamine D3 over D2 by enhancing the affinity for
dopamine D3 receptors. The loratadine ring system
(S4) was introduced in compounds 18a and 18b and this
has resulted in molecules with moderate nanomolar
affinities for D2 binding. Introducing this bulky cyclic
system has been well tolerated by dopamine D3 recep-
tors affecting low nanomolar binding affinities. High
affinity binding and selectivity for the dopamine
D3 receptor has been revealed for the benzo[b]thio-
phen-2-carboxamide substituted compound 18b (Ki
(D2/D3) = 314).
For a comprehensive series of potentially privileged
structures of H1 receptor antagonists, derivatives con-
taining the phenylaminopiperidine substructure (S5) of
bamipine (Fig. 2) were synthesized (15c, 19a, 19b, 23a–
23c, 24, and 25a–b). In the first series, a benzyl-N-piperi-
din-4-ylaniline has been combined with phthalimide
(15c), naphthalen-2-carboxamide (19a), or benzo[b]thio-
phen-2-carboxamide (19b) connected via a butyl linker.
Introducing the phthalimide residue resulted in a com-
pound with a preference for the dopamine D2 receptor.
The naphthalen-2-carboxamide containing compound
(19a) displayed subnanomolar affinity binding for hD3
(Ki (D3) = 0.3) and moderate affinity binding for hD2
and represents the most selective ligand in this hybrid
approach (Ki (D2/D3) = 2343). The molecule presents a
promising affinity profile regarding the dopamine D3
receptor, but due to the preliminary testing further
investigations for reliability need to be carried out.
The piperazine ring extension of unsubstituted benzhyd-
rylpiperazine derivatives 4a and 6a–6d to a 1,4-diaze-
pane (4c and 8a–8d) resulted in a reduction of affinity
binding for D3 receptors excluding the benzo[b]thio-
phen-2-carboxamide 8c demonstrating similar data as
seen for 6c. Affinity bindings of the naphthalen-2-car-
boxamide 8a and the cinnamide 8b have been enhanced
for D2 receptors, while a reduced binding affinity was
obtained for 4c. The modification in compounds 8c
and 8d did not alter the binding behavior for this recep-
tor subtype. The ring expansion of the chloro substi-
tuted compounds 4b–4d led to improved affinity
binding at D2 receptors but resulted in deterioration of
data for dopamine D3 receptors. Ring extension changes
the position of the basic amine piperazine relative to the
lipophilic aromatic residue. This alteration generally im-
pairs the dopamine D3 receptor–ligand interaction. In
contrast, the interference of dopamine D2 receptor–li-
gand additionally depends on the carboxamide and car-
boximide residue, respectively.
Encouraged by the results of 19a, it was of interest to
validate the influence of an additional hydrogen-bond
acceptor on affinity binding. Consequently, the benzyl
residue has been exchanged by a benzoyl moiety and
the molecule has been sterically restricted by a methoxy
substitution in 2-position of the phenyl ring (23c). Intro-
ducing the oxo functionality and methoxy substituent
led to a compound with micromolar affinities at both
receptor subtypes. By retaining the added oxo hydro-
gen-bond acceptor function, but derogating the benzoyl
residue to an acetyl and propionyl moiety, compounds
23a and 23b were synthesized. As seen for 23c, binding
affinity values were in the micromolar range at both
receptor subtypes and no further improvement concern-
ing the binding profiles has been observed.
Introduction of the hexahydrodibenzopyrazinoazepine
residue (S2), the tetracyclic substructure of mianserin
(Fig. 2), has generated ligands (15a and 16a–e) with
modest nanomolar to micromolar affinity binding values
In the following compounds 24, 25a, 25b, the phenyl-
aminopiperidine moiety with methoxy substitution in

B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
7267
2-position on the phenyl ring has been maintained.
Introducing a phthalimide residue (24) resulted in a
compound with micromolar affinities at dopamine D2
and D3 receptors. The exchange of the imide by a naph-
thalen-2-carboxamide moiety (25a) led to a clear in-
crease in affinity binding at dopamine D2 and D3
receptors. Further variation included the exchange of
the naphthoyl moiety by a cinnamide residue (25b). This
ligand presents a pharmacological binding profile with
low nanomolar binding data for dopamine D3 receptors
and a 93-fold selectivity for dopamine D3 over D2 recep-
tors. The phenylaminopiperidine residue is an analogue
of the 4-(2-methoxyphenyl)piperazino moiety as seen in
BP 897 (Fig. 1) and has been connected via a butyl lin-
ker to the cinnamide substructure of ST 198 (Fig. 1).
This structural combination gives an explanation for
the high affinity and selectivity for the dopamine D3
receptor.
generated ligands with remarkable affinity and selectivity
for dopamine D3 receptors. Incorporation of flexible res-
idues such as phenylaminopiperidine and benzhydrylpip-
erazine, the substructures of histamine H1 receptor
antagonists, has been well-tolerated as demonstrated
by the naphthalen-2-carboxamide containing compound
19a and cinnamide derivative 7b. The former is most
promising compound with a Ki (hD3) = 0.3 nM and a
remarkable selectivity ratio of 2343, while the latter pos-
sesses high affinity for dopamine D3 receptors and a
selectivity ratio of 401 for dopamine D3 over D2 recep-
tors. Most of the rigid and bulky tricyclic or tetracyclic
ring systems have been tolerated by dopamine D2 and
D3 receptors as exemplified by the loratadine derivative
18b with high affinity binding (Ki (D3) = 2.7) and selec-
tivity for the dopamine D3 receptor (Ki (D2/D3) = 314).
The influence of diverse dopamine D3 receptor-prefer-
ring residues on the binding profile at dopamine D3
and D2 receptors has been investigated and the results
indicate an impact on affinity binding for both receptor
subtypes but a clear effect on selectivity ratios.
The existence of highly conserved residues in the ligand
binding pocket of class A family of GPCRs, predomi-
nantly possessing hydrophobic and aromatic properties,
is well established.54 Other regions of the binding site
among this receptor family have variable residues and
strongly influence the selectivity of ligands toward a
receptor subtype.54 In this hybrid approach, we em-
ployed privileged scaffolds of histamine H1 receptor
antagonists expecting the recognition of this structural
element by the highly conserved receptor region in the
related biogenic amine binding dopamine D3 receptor.
It was assumed that the introduction of the dopamine
D3 receptor-preferring substructures has a refined im-
pact on the affinity and selectivity for the dopamine
D3 receptor. The hybrid approach using these privileged
structures provided ligands mostly with nanomolar
affinity and selectivity for dopamine D3 receptors. The
histamine H1 receptor antagonist fragment containing
basic nitrogen connected to an aromatic/lipophilic resi-
due interfered with the binding pocket of D2 and D3
receptors, assumingly interacting with the highly con-
served Asp in TM3.12 The binding site in the pocket
of the dopamine D3 receptor tolerated bulky flexible
as well as bulky rigid aromatic elements. Exchange of
the dopamine D3 receptor-preferring elements influ-
enced binding affinities and selectivity profile.
Although the data have to be confirmed by repetitive
competition binding assays, the preliminary data dem-
onstrate that by the presented hybrid approach we de-
signed potent and selective drugs with an optimized
binding profile for dopamine D3 receptors. A refined
SAR for dopamine D3 and D2 receptors has been dis-
cussed and improves the understanding of ligand–recep-
tor interactions.
5. Experimental
5.1. General experimental
Melting points were determined on a Buchi 510 melting
¨
point apparatus (Buchi, Flawil, Switzerland) and are
¨
1
uncorrected. H NMR spectra were recorded on a Bru-
ker Avance DPX 400 (1H NMR 400 MHz) or Bruker
AC 300 (1H NMR 300 MHz). Chemical shifts are ex-
pressed in parts per million (ppm) downfield from inter-
nal tetramethylsilane (TMS) as reference. The following
abbreviations are used for multiplicity of NMR signals:
br, broad, s, singlet, d, doublet, t, triplet; m, multiplet;
approximate coupling constants in Hertz (Hz); number
of protons. Elemental analyses (C, H, N) were measured
on Elementaranalysator 240C, Vario EL (Perkin-Elmer)
or CHN-Rapid (Heraeus) and were within 0.4% of the
theoretical values for all compounds. Preparative
column chromatography was performed on silica gel
63–200 lM, mobile phase usually dichloromethane/
methanol. Thin-layer chromatography (TLC) was per-
formed on silica gel PF254 plates (Merck). Spectral data
and elemental analyses are shown only for intermediates
and parent compounds, which were obtained by differ-
ent reactions or methods, and additionally for the most
potent compounds (2b, 4, 6–8, 15, 16, 19, 25b). EI-MS
was performed on a Finnigan Varian MATCH/A or a
MAT 171; FAB-MS was performed on a Finnigan
MAT CH5DF (Xe, DMSO, Glycerole, Cu-Target,
Plus-Mode); ESI-MS was performed on a Fisons Instru-
Since the moieties introduced into the novel dopamine
ligands are well-known histamine H1 receptor antago-
nists, a future evaluation on their histamine H1 receptor
binding properties is of absolute necessity. Additionally,
the histamine H1 receptor antagonists have to be phar-
macologically tested for their binding affinities at dopa-
mine D2 and D3 receptors to assess the impact of the
dopamine D3 substructures on the parent ligands.
4. Conclusion
A rational hybrid design of novel ligands has been
sucessfully employed using privileged structures of
ligands for biogenic amine binding GPCRs. The combi-
nation of privileged scaffolds of H1 receptor antagonists
with dopamine D3 receptor-preferring substructures

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B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
mentsVG Platform II. Data are listed as mass number (m/
z) and relative intensity (%). Descriptions for general pro-
cedure for the preparation have been exemplified with the
first compound mentioned if not stated otherwise.
by column chromatography (CH2Cl2/MeOH (9:1)) and
crystallized as the oxalate from ethanol/diethylether.
5.7. General procedure for the preparation of 6–8, 16–19,
and 25. Method C2
5.2. General procedure for the preparation of 2a, 2b.
Method A1
The purified compound (6.9 mmol) was dissolved in
dry methanol (30 mL) and hydrazine hydrate (0.6 g,
12 mmol) was added and refluxed for 3 h. In cases where
the reaction was not complete, more hydrazine hydrate
(0.3 g, 6 mmol) was added and again refluxed for 2 h.
After evaporation of hydrazine and methanol under
vacuum, ether was added and filtrated. The filtrate was
concentrated under vacuum; the crude product was used
in the next reaction without further purification. To an
ice-cooled suspension of the amine component (5 mmol)
and K2CO3 (0.69 g, 5 mmol) in dry dichloromethane
(10 mL) was added a solution of the arylcarboxylic acid
chloride (5 mmol) in dry dichloromethane (5 mL) in a
dropwise manner. After the addition was complete, the
solution was stirred for 2–6 h at room temperature.
The mixture was washed with saturated NaHCO3
(10 mL), water (5 mL) and brine (5 mL). The organic
phase was dried (MgSO4), freed from volatiles under
vacuum and purified by column chromatography and
crystallized from ethanol/diethylether as the oxalate salt.
A
suspension of the monoprotected piperazine
derivative (12 mmol), the appropriately substituted
chloro(diphenyl)methane (9 mmol) and K2CO3 (1.4 g,
10 mmol) and dry acetonitrile (40 mL) was stirred for 24 h
at rt. After filtration, the solvent was evaporated under vac-
uum. The residue was purified by column chromatography.
5.3. General procedure for the preparation of 2c, 2d.
Method A2
Cyanomethyl(trimethyl)phosphonium iodide (1.8 g,
7.5 mmol) was added to a mixture of the appropriate
benzhydryl alcohol (6.6 mmol), ethyl(piperazino)formi-
ate (0.98 g, 6.2 mmol), diisopropylethylamine (DIPEA)
(1.01 g, 7.8 mmol), and propionitrile (10 mL), and the
mixture was stirred at 90 ꢁC for 3 h under argon atmo-
sphere. The mixture was allowed to cool to room tem-
perature and a solution of potassium carbonate (3 g)
in water (30 mL) was added, and the product was
extracted with dichloromethane (3· 30 mL). The com-
bined organic layers were dried with sodium sulfate,
and concentrated under vacuum to yield 2.51 g of a
brown oil. Purification by column chromatography
(CH2Cl2/MeOH (99:1) yielded 1.18 g (46%) of the prod-
uct as a white solid.
5.8. General procedure for the preparation of 10, 11,
and 16–19. Method C3
Cyanomethyl(trimethyl)phosphonium iodide (1.8 g,
7.5 mmol) was added to a mixture of the appropriate
secondary amine (0.98 g, 6.2 mmol), N-(4-hydroxybu-
tyl)arylcarboxamide (6.6 mmol), DIPEA (1.01 g, 7.8
mmol), and propionitrile (10mL), and the mixture was
stirred at 90 ꢁC for 3 h under argon atmosphere. The
mixture was allowed to cool to room temperature and
a solution of potassium carbonate (3 g) in water
(30 mL) was added, and the product was extracted with
dichloromethane (3· 30 mL). The combined organic
layers were dried with sodium sulfate, and concentrated
under vacuum to yield 2.51 g of brown oil. The crude
product was purified by column chromatography
(CH2Cl2/MeOH (9:1)) and precipitated with oxalic acid
from ethanol/diethylether.
5.4. General procedure for the preparation of 13a,
13b, 13d. Method A3
To a suspension the N-methylated compound (7.1 mmol)
in dry toluene (60 mL) was added ethyl chloroformiate
(16 g, 147 mmol). The mixture was refluxed for 5 h. After
completion of the reaction monitored by TLC, the excess
of the ethyl chloroformiate and the solvent were removed
under reduced pressure. The product was used in the next
step without further purification.
5.5. General procedure for the preparation of 3a–d.
Method B
5.9. General procedure for the preparation of 23.
Method C4
The N-ethoxycarbonyl protected compound (2.9 mmol)
was refluxed in methanol (20 mL) and water (7 mL) in
the presence of KOH (2.5 g) for 24 h. Methanol was evap-
orated and to the residue was added water (30 mL). The
mixture was extracted with dichloromethane and purified
by flash chromatography (CH2Cl2/MeOH (95:5)).
5.9.1. N-(4-Oxobutyl)naphthalen-2-carboxamide. A solu-
tion of N-(4,4-diethoxybutyl)naphthalen-2-carboxamide
(0.5 g, 1.6 mmol), acetic acid (2 mL) and HCl (1 N,
1 mL) in ethanol (5 mL) was stirred for 16 h at room
temperature. After completion of the reaction was con-
firmed by TLC, the solvent was evaporated under vac-
uum (T < 40 ꢁC). The residue was dissolved in
dichloromethane (10 mL) and washed with saturated
NaHCO3 solution (5 mL). The organic phase was dried
with Na2SO4. The solvent was evaporated under vac-
uum. The crude product was dissolved in 1,2-dichloro-
ethane (9 mL). The secondary amine (0.6 mmol), acetic
acid (0.3 mL) and NaHB(OAc)3 (0.6 g, 3 mmol) were
added. The suspension was stirred under exclusion of
5.6. General procedure for the preparation of 4 and 24.
Method C1
A suspension of the secondary amine (10 mmol), N-
(bromobutyl)isoindoline-1,3-dione (2.8 g, 10 mmol)
and K2CO3 (1.4 g, 10 mmol) and dry acetonitrile
(40 mL) was stirred for 24 h. After filtration, the solvent
was evaporated under vacuum. The residue was purified

B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
7269
light for 24 h at room temperature. The organic phase
was washed with saturated NaHCO3 solution (10 mL)
and water (15 mL), dried (MgSO4) and freed from sol-
vents under vacuum. The crude product was purified
by column chromatography. The pure compound was
precipitated with oxalic acid from ethanol/diethylether.
lid; mp 208 ꢁC; 1H NMR (300 MHz, DMSO-d6) d 1.52–
1.68 (m, 4H), 2.35–2.65 (m, 4H), 2.84–3.26 (m, 6H),
3.5–3.65 (s, br, 2H), 4.40 (s, 1H), 7.13–7.48 (m, 10H),
7.78–7.90 (m, 4H). Anal. (C29H31N3O2ÆC2H2O ) calcd:
4
C, 68.49; H, 6.12; N, 7.73; found: C, 68.63; H, 6.02;
N, 7.86. ESI-MS: 454 (M+H+, 100), 228(M+2H+, 15).
5.10. Preparation and analytical data of intermediate and
final compounds
5.10.7. N-(4-{4-[(4-Chlorophenyl)phenylmethyl]piperazin-
1-yl}butyl)isoindolin-1,3-dione (4b). Method A3, method
B, method C1. Yield (all steps) 61%; white solid; mp
189 ꢁC; H NMR (300 MHz, DMSO-d6) d 1.6 (s, br,
4H), 2.50 (s, br, 4H), 2.96 (s, br, 2H), 3.08 (s, br, 4H),
3.57 (s, br, 2H), 4.38 (s, br, 1H), 7.12–7.53 (m, 9H),
7.78–7.93 (m, 4H). Anal. (C29H30ClN3O2ÆC2H2O4)
calcd: C, 64.41; H, 5.58; N, 7.27; found: C, 64.46; H,
5.51; N, 7.46. ESI-MS: 489 (M+H+, 100).
1
5.10.1. Ethyl {4-[(4-chlorophenyl)phenylmethyl]piperazin-
1-yl}carboxylate (2b). Method A1 with ethyl(piperazi-
no)formate and chloro-(4-chlorophenyl)(phenyl)meth-
ane. Yield 96%. White solid, mp 78 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 1.30 (t, J = 7.1 Hz, 3H), 2.44–
2.56 (m, 4H), 3.27–3.55 (m, 4H), 4.10–4.34 (m, 2H),
4.39 (s, 1H), 7.11–7.50 (m, 9H).
5.10.8. N-{4-[4-(Diphenylmethyl)-1,4-diazepan-1-yl]butyl}iso-
indolin-1,3-dione (4c). Method A3, method B, method
C1. Yield (all steps) 37%; white solid; mp 102 ꢁC; H
NMR (300 MHz, DMSO-d6) d 1.50–1.85 (m, 4H),
1.85–2.08 (m, 2H), 2.52–2.65 (m, 2H), 2.68–2.93 (m,
2H), 2.95–3.63 (m, 8H), 4.71 (s, 1H), 7.05–7.65 (m,
10H), 7.7–7.98 (m, 4H). Anal. (C30H33N3O2Æ C2H2O4Æ
2 H2O) calcd: C, 64.74; H, 6.62; N, 7.08; found: C,
64.79; H, 6.47; N, 7.18. ESI-MS: 467 (M+H+, 100).
5.10.2. [4-(Diphenylmethyl)-1,4-diazepan-1-yl]ethanone.
Method A1 with 1-(1,4-diazepan-1-yl)ethanone and
chloro (diphenyl)methane. Yield 71%. Yellowish solid,
mp 149 C. 1H NMR (300 MHz, DMSO-d6) d 1.78–
2.04 (m, 5H), 3.09–3.58 (m, 8H), 4.71 (s, 1H), 7.01–
7.61 (m, 10 H).
1
5.10.3. N-{4-[4-(N0-Benzyl-N0-phenylamino)piperidin-1-
yl]butyl}isoindolin-1,3-dione (15c). Method A3, method
B, method C1. Yield (all steps) 54%; white solid; mp
145 ꢁC; Rf 0.3 (CH2Cl2/MeOH (9:1)). 1H NMR
(300 MHz, DMSO-d6) d 1.55–1.75 (m, 4H), 1.78–2.05
(m, 4H), 2.89–3.14 (m, 4H), 3.32–3.50 (m, 2H), 3.53–
3.64 (s, br, 2H), 4.09–4.22 (m, 1H), 4.42 (s, 2H), 6.57–
6.78 (m, 3H), 7.07–7.37 (m, 7H), 7.78–7.95 (m, 4H).
Anal. (C30H32N3O2Æ0.8 C2H2O4ÆH2O) calcd: C, 68.06;
H, 6.62; N, 7.54; found: C, 67.76; H, 6.88; N, 7.27.
ESI-MS: 467 (M+H+, 100).
5.10.9. N-(4-{4-[(4-Chlorophenyl)phenylmethyl]-1,4-dia-
zepan-1-yl}butyl)isoindolin-1,3-dione (4d). Method A3,
method B, method C1. Yield (all steps) 20%; white solid;
mp 104 ꢁC; 1H NMR (300 MHz, DMSO-d6) d 1.47–1.82
(m, 4H), 1.87–2.12 (m, 2H), 2.52–2.65 (m, 2H), 2.67–
2.90 (m, 2H), 2.91–3.67 (m, 8H, H-9), 4.71 (s, 1H),
7.05–7.67 (m, 9H), 7.7–7.98 (m, 4H). Anal.
(C30H32ClN3O2ÆC2H2O4ÆH2O) calcd: C, 63.00; H, 5.95;
N, 6.89; found: C, 62.89; H, 5.68; N, 6.62. ESI-MS:
503 (M+H+, 100).
5.10.4. N-[4-(2,3,4,5,10,15-Hexahydro-1H-dibenzo[b: e]pyr-
azino[2,1-g]azepin-1-yl)butyl]isoindolin-1,3-dione (15a).
Method A3, method B, method C1. Yield (all steps)
62%; white solid; mp 238 ꢁC; 1H NMR (300 MHz,
DMSO-d6) d 1.66 (s, br, 4H), 2.84–3.15 (m, 4H), 3.25–
3.53 (m, 5H), 3.53–3.69 (m, 2H), 4.25 (d, J = 10.1 Hz,
1H), 4.64 (d, J = 12.4 Hz, 1H), 6.87 (t, J = 7.3Hz, 1H),
6.95–7.32 (m, 7H), 7.82–7.93 (m, 4H). Anal.
(C29H29N3O2ÆC2H2O 4) calcd: C, 68.75; H, 5.77; N,
7.76; found: C, 68.51; H, 5.71; N, 7.88. ESI-MS: 452
(M+H+, 100).
5.10.10. N-(4-{4-[Benzyl(phenyl)amino]piperidin-1-yl}buty-
lnaphthalen-2-carboxamide (19a). Starting with the free
base of 15c, method C2. Yield 80%; yellowish solid;
mp 166 ꢁC; 1H NMR (300 MHz, DMSO-d6) d 1.52–
1.88 (m, 4H), 1.89–2.07 (m, 4H), 2.93–3.12 (m, 4H),
3.27–3.52 (m, 4H), 4.11–4.24 (m, 1H), 4.41 (s, 2H),
6.58–6.64 (m, 1H), 6.70–6.74 (m, 2H), 7.07–7.36 (m,
7H), 7.55–7.65 (m, 2H), 7.87–8.05 (m, 4H), 8.44 (s,
1H), 8.70 (t, J = 5.5 Hz, 1H). Anal. (C33H37N3OÆ
C2H2O4) calcd: C, 72.27; H, 6.76; N, 7.22; found: C,
72.18; H, 6.75; N, 7.45.
5.10.5. N-{4-[4-(10-Oxo-9,10-dihydro-4H-benzo[4,5]cyclo-
hepta[1,2-b] thiophen)-4-ylidenpiperidin-1-yl]butyl}isoin-
dolin-1,3-dione (15b). Method A3, method B, method
C1. Yield (all steps) 9.5%; yellowish solid; mp 204 ꢁC;
1H NMR (300 MHz, DMSO-d6) d 1.63 (s, br, 4H),
2.33–2.45 (m, 2H), 2.45–3.1 (m, 6H), 3.17–3.42 (m,
2H), 3.57–3.78 (m, 3H), 4.38 (d, J = 13.6 Hz, 1H),
7.12–7.43 (m, 5H), 7.80–7.93 (m, 4H), 7.98 (d, J = 5.0
Hz, 1H). Anal. (C30H28N2O3ÆC2H2O4) calcd: C, 65.52;
H, 5.15; N, 4.78; found: C, 65.22; H, 5.20; N, 4.98.
ESI-MS: 497 (M+H+, 100).
5.10.11. N-[4-(2,3,4,5,10,15-Hexahydro-1H-dibenzo[b:e]pyr-
azino[2,1-g]azepin-1-yl)butyl] naphthalen-2-carboxamide
(16a). Starting with the free base of 15a, method C2. Yield
79%; mp 134 ꢁC; 1H NMR (300 MHz, DMSO-d6) d 1.40–
1.57 (m, 2H), 1.63–1.78 (m, 2H), 2.88–3.13 (m, 4H), 3.13–
3.55 (m, 7H), 4.25 (d, J = 10.3 Hz, 1H), 4.64 (d, J = 12,3
Hz, 1H), 6.82–6.88 (m, 1H), 6.94–7.23 (m, 7H), 7.56 (m,
2H), 7.86–8.05 (m, 4H), 8.35 (s, 1H). Anal.
(C32H33N3OÆ0.5C2H2O4ÆH2O) calcd: C, 73.58; H, 6.74;
N, 7.80; found: C, 73.65; H, 6.79; N, 8.00.
5.10.6. N-{4-[4-(Diphenylmethyl)piperazin-1-yl]butyl}iso-
indolin-1,3-dione (4a). Method C1. Yield 75%. White so-
5.10.12. N-{4-[4-(Diphenylmethyl)piperazin-1-yl]butyl}naph-
thalen-2-carboxamide (6a). Starting with the free base

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1
of 4a, method C2. Yield 90%; mp 118 ꢁC; H NMR
(300 MHz, DMSO-d6) d 1.52–1.78 (m, 4H), 2.45–2.71
(m, 4H), 3.01 (s, br, 2H), 3.13 (s, br, 4H), 3.34 (s, br,
2H), 4.40 (s, 1H), 7.16–7.37 (m, 6H), 7.40–7.50 (m,
4H), 7.53–7.65 (m, 2H), 7.78–8.08 (m, 4H), 8.45 (s,
1H), 8.71 (s, br, 1H). Anal. (C32H35N3OÆC2H2O4) calcd:
C, 71.94; H, 6.57; N, 7.40; found: C, 71.78; H, 6.47; N,
7.50. ESI-MS: 478 (M+H+, 100).
J = 9.93 Hz, 1H), 4.65 (d, J = 12.24 Hz, 1H), 6.89 (t,
J = 7.29 Hz, 1H), 7.02–7.36 (m, 8H), 7.80 (d, J = 4.38
Hz, 2H), 8.56 (t, J = 5.51 Hz, 1H). Anal. (C26H29N3O-
SÆ0.8C2H2O4Æ1.8 H2O) calcd: C, 60.70; H, 6.29; N,
7.58; found: C, 60.73; H, 6.10; N, 7.29. ESI-MS: 432
(M+H+, 100).
5.10.18. N-[4-(2,3,4,5,10,15-Hexahydro-1H-dibenzo[b:e]-
pyrazino[2,1-g]azepin-1-yl)butyl]benzo[b]thiophen-2-car-
boxamide (16c). Starting with the free base of 15a, meth-
5.10.13. N-(4-{4-[(4-Chlorophenyl)phenylmethyl]pipera-
zin-1-yl}butyl)naphthalen-2-carboxamide (7a). Starting
with the free base of 4b, method C2. Yield 85%; mp
118 ꢁC; 1H NMR (300 MHz, DMSO-d6) d 1.54–1.82
(m, 4H), 2.43–2.81 (m, 4H), 3.02 (s, br, 2H), 3.14 (s,
br, 4H), 3.34 (s, br, 2H), 4.41 (s, 1H), 7.13–7.67 (m,
11H), 7.78–8.08 (m, 4H), 8.45 (s, 1H), 8.71 (s, br, 1H).
Anal. (C32H34ClN3OÆC2H2O4) calcd: C, 67.82; H, 6.03;
N, 6.98; found: C, 67.57; H, 5.95; N, 7.00. ESI-MS:
497 (M+H+, 100).
1
od C2. Yield 93%; mp 171 ꢁC; H NMR (300 MHz,
DMSO-d6) d 1.48–1.82 (m, 4H), 2.76–3.12 (m, 4H),
3.18–3.55 (m, 7H), 4.20 (d, J = 10.1 Hz, 1H), 4.64 (d,
J = 12.4 Hz, 1H), 6.88 (t, J = 7.29 Hz, 1H), 6.96–7.30
(m, 7H), 7.26–7.54 (m, 2H), 7.88–8.03 (m, 3H), 8.83 (t,
J = 5.4 Hz, 1H). Anal. (C30H31N3OSÆC2H2O4) calcd:
C, 67.23; H, 5.78; N, 7.35; found: C, 67.02; H, 5.78;
N, 7.32. ESI-MS: 482 (M+H+, 100).
5.10.19. N-[4-(2,3,4,5,10,15-Hexahydro-1H-dibenzo[b:e]-
pyrazino[2,1-g]azepin-1-yl)butyl]cyclohexancarboxamide
(16e). Starting with the free base of 15a, method C2.
Yield 86%; mp 142 ꢁC; H NMR (300 MHz, DMSO-
d6) d 1.05–1.51 (m, 8H), 1.51–1.82 (m, 6H), 2.02–
2.15 (m, 1H) 2.87–3.12 (m, 4H), 3.18–3.55 (m,
7H), 4.20 (d, J = 10.1 Hz, 1H), 4.64 (d, J =
12.4 Hz, 1H), 6.88 (t, J = 7.29 Hz, 1H), 6.96–7.30
(m, 7H), 7.72 (t, J = 5.4 Hz, 1H). Anal.
(C28H37N3OÆC2H2O4Æ0.25 H2O) calcd: C, 68.48; H,
7.57; N, 7.99; found: C, 68.25; H, 7.77; N, 8.20.
ESI-MS: 432 (M+H+, 100).
5.10.14. N-{4-[4-(Diphenylmethyl)-1,4-diazepan-1-yl]butyl}-
naphthalen-2-carboxamide (8a). Starting with the free
base of 4c, method C2. Yield 70%; mp 76 ꢁC; 1H
NMR (300 MHz, DMSO-d6) d 1.55–1.80 (m, 4H),
1.92 (s, br, 2H), 2.55–2.79 (m, 2H), 2.75 (s, br, 2H),
3.10–3.28 (m, 4H), 3.28–3.45 (m, 4H), 4.68 (s, 1H),
7.18 (t, J = 7.3 Hz, 2H), 7.29 (t, J = 7.5 Hz, 4H),
7.6 (d, J = 7.3 Hz, 4H), 7.58–7.64 (m, 2H), 7.91–8.05
(m, 4H), 8.45 (s, 1H), 8.73 (s, br, 1H). Anal.
(C33H37N3OÆC2H2O4ÆH2O) calcd: C, 70.10; H, 6.89;
N, 7.01; found: C, 69.98; H, 6.74; N, 7.17. ESI-MS:
492 (M+H+, 100).
1
5.10.20. N-[4-(4-Diphenylmethylpiperazin-1-yl)butyl]cin-
namide (6b). Starting with the free base of 4a, method
C2. Yield 89%; mp 182 ꢁC; 1H NMR (300 MHz,
DMSO-d6) d 1.39–1.74 (m, 4H), 2.50–2.72 (m, 4H),
2.88–3.33 (m, 8H), 4.40 (s, 1H), 6.64 (d, J = 15.8 Hz,
1H), 7.12 (t, J = 4.3 Hz, 1 H), 7.21–7.51 (m, 13H),
7.51–7.62 (m, 2H), 8.53–8.64 (m, 1H). Anal.
(C30H35N3OÆC2H2O4) calcd: C, 70.70; H, 6.86; N, 7.73;
found: C, 70.53; H, 6.70; N, 7.83.
5.10.15. N-(4-{4-[Benzyl(phenyl)amino]piperidin-1-yl}butyl)-
benzo[b]thiophen-2-carboxamide (19b). Starting with the
free base of 15c, method C2. Yield 78%; mp 182 ꢁC; 1H
NMR (300 MHz, DMSO-d6) d 1.49–1.90 (m, 4H), 1.91–
2.13 (m, 4H), 2.90–3.09 (m, 4H), 3.21–3.52 (m, 4H),
4.13–4.27 (m, 1H), 4.43 (s, 2H), 6.53–6.68 (m, 1H),
6.73–6.77 (m, 2H), 7.02–7.33 (m, 7H), 7.38–7.45 (m,
2H), 7.80–7.98 (m, 2H), 8.32 (s, 1H), 8.87 (s, br, 1H).
Anal. (C31H35N3OSÆC2H2O4) calcd: C, 67.44; H, 6.35;
N, 7.15; found: C, 67.18; H, 6.42; N, 7.23. ESI-MS:
498 (M+H+, 100).
5.10.21. N-[4-(4-Diphenylmethylpiperazin-1-yl)butyl]thio-
phen-2-carboxamide (6d). Starting with the free base of
4a, method C2. Yield 89%; mp 182 ꢁC; 1H NMR
(300 MHz, DMSO-d6) d 1.45–1.72 (m, 4H), 2.50–2.72
(m, 4H), 2.93–3.33 (m, 8H), 4.40 (s, 1H), 7.12 (t,
J = 4.3 Hz, 1H), 7.16–7.25 (m, 2H), 7.31 (t, J = 7.1
Hz, 4H), 7.38–7.51 (m, 4H), 7.69–7.81 (m, 2H), 8.53–
8.64 (m, 1H). Anal. (C26H31N3OSÆC2H2O4) calcd: C,
64.22; H, 6.35; N, 8.02; found: C, 64.06; H, 6.39; N,
7.96.
5.10.16. N-[4-(2,3,4,5,10,15-Hexahydro-1H-dibenzo[b:e]-
pyrazino[2,1-g]azepin-1-yl)butyl]cinnamide (16b). Start-
ing with the free base of 15a, method C2. Yield
87%; mp 138 ꢁC; 1H NMR (300 MHz, DMSO-d6) d
1.41–1.58 (m, 2H), 1.58–1.79 (m, 2H), 2.85–3.55 (m,
11H), 4.25 (d, J = 10.31 Hz, 1H), 4.65 (d, J = 12.34
Hz, 1H), 6.63 (d, J = 15.82 Hz, 1H), 6.88 (t, J = 7.31
Hz, 1H), 6.97–7.28 (m, 7H), 7.32–7.47 (m, 4H),
7.48–7.60 (m, 2H), 8.22 (t, J = 5.47 Hz, 1H). Anal.
(C30H33N3OÆC2H2O4) calcd: C, 70.96; H, 6.51; N,
7.76; found: C, 70.75; H, 6.55; N, 7.69. ESI-MS: 452
(M+H+, 100).
5.10.22. N-[4-(4-Diphenylmethylpiperazin-1-yl)butyl]benzo-
[b]thiophen-2-carboxamide (6c). Starting with the free
base of 4a, method C2. Yield 87%; mp 144 ꢁC; 1H
NMR (300 MHz, DMSO-d6) d 1.47–1.78 (m, 4H),
2.50–2.72 (m, 4H), 2.91–3.42 (m, 8H), 4.39 (s, 1H),
7.14–7.23 (m, 2H), 7.30 (t, J = 7.4 Hz, 4H), 7.35–7.51
(m, 6H), 7.88–8.06 (m, 2H), 8.11 (s, 1H), 8.80–8.92 (m,
1H). Anal. (C30H33N3OSÆC2H2O4) calcd: C, 66.99; H,
6.15; N, 7.32; found: C, 67.03; H, 6.14; N, 7.35. ESI-
MS: 484 (M+H+, 100).
5.10.17. N-[4-(2,3,4,5,10,15-Hexahydro-1H-dibenzo[b:e]-
pyrazino[2,1-g]azepin-1-yl)butyl]thiophen-2-carboxamide
(16d). Starting with the free base of 15a, method C2.
1
Yield 92%; mp 186 ꢁC; H NMR (300 MHz, DMSO-
d6) d 1.45–1.86 (m, 4 H), 2.92–3.63 (m, 11H), 4.26 (d,

B. C. Sasse et al. / Bioorg. Med. Chem. 15 (2007) 7258–7273
7271
5.10.23. N-[4-(4-Diphenylmethylpiperazin-1-yl)butyl]cyclo-
hexancarboxamide (6e). Starting with the free base of
4a, method C2. Yield 90%; mp 128 ꢁC; H NMR (300
MHz, DMSO-d6) d 1.05–1.45 (m, 6H), 1.45–1.73 (m,
8H), 2.04 (t, J = 5.6 Hz, 1H), 2.51 (s, br, 4H), 2.87–
3.22 (m, 8H), 4.41 (s, 1H), 7.21 (t, J = 7.3 Hz, 2H),
7.28–7.36 (m, 4H), 7.36–7.45 (m, 4H), 7.70 (t,
J = 5.5 Hz, 1H). Anal. (C28H39N3OÆC2H2O4Æ0.75 H2O)
calcd: C, 67.08; H, 7.97; N, 7.82; found: C, 67.02; H,
7.74; N, 8.03. ESI-MS: 434 (M+H+, 100).
MHz, DMSO-d6) d 1.45–1.61 (m, 2H), 1.63–1.83 (m,
2H), 1.87–2.1 (m, 2H), 2.53–2.67 (m, 2H), 2.74–2.89
(m, 2H), 3.05–3.52 (m, 8H), 4.76 (s, 1H), 7.07–7.47 (m,
7H), 7.48–7.58 (d, J = 7.32 Hz, 4H), 7.70–7.86 (m,
2H), 8.64 (t, J = 5.52 Hz, 1H). Anal. (C27H33N3OSÆ1.5-
C2H2O4Æ1.5 H2O) calcd: C, 58.92; H, 6.46; N, 6.87;
found: C, 58.75; H, 6.64; N, 6.80. ESI-MS: 448
(M+H+, 100).
1
5.10.30. N-{4-[4-(Diphenylmethyl)-1,4-diazepan-1- yl]butyl}-
benzo[b]thiophen-2-carboxamide (8c). Starting with the
free base of 4c, method C2. Yield 71%; mp 148 ꢁC;
1H NMR (300 MHz, DMSO-d6) d 1.48–1.64 (m, 2H),
1.65–1.81 (m, 2H), 1.84–1.98 (m, 2H), 2.50–2.60 (m,
2H), 2.67–2.83 (m, 2H), 3.06–3.46 (m, 8H), 4.67 (s,
1H), 7.12–7.38 (m, 6H), 7.39–7.53 (m, 6H), 7.87–8.05
(m, 2H), 8.09 (s, 1H), 8.86 (t, J = 5.40 Hz, 1H). Anal.
(C31H35N3OSÆC2H2O4ÆH2O) calcd: C, 65.43; H, 6.49;
N, 6.94; found: C, 65.59; H, 6.23; N, 7.02.
5.10.24. N-(4-{4-[(4-Chlorophenyl)phenylmethyl]pipera-
zin-1-yl}butyl)cinnamide (7b). Starting with the free base
1
of 4b, method C2. Yield 84%; mp 158 ꢁC; H NMR
(300 MHz, DMSO-d6) d 1.35–1.73 (m, 4H), 2.48 (s, br,
4H), 2.90–3.35 (m, 8H), 4.42 (s, 1H), 6.64 (d, J = 15.6
Hz, 1H), 7.13–7.65 (m, 14H), 8.25 (s, br, 1H). Anal.
(C30H34ClN3OÆC2H2O4) calcd: C, 66.48; H, 6.28; N,
7.27; found: C, 66.24; H, 6.31; N, 7.13.
5.10.25. N-(4-{4-[(4-Chlorophenyl)phenylmethyl]pipera-
zin-1-yl}butyl)thiophen-2-carboxamide (7d). Starting with
the free base of 4b, method C2. Yield 87%; mp 159 ꢁC;
1H NMR (300 MHz, DMSO-d6) d 1.35–1.73 (m, 4H),
2.48 (s, br, 4H), 2.90–3.35 (m, 8H), 4.42 (s, 1H), 7.05–
7.59 (m, 10H), 7.59–7.80 (m, 2H), 8.57 (s, br, 1H). Anal.
(C26H30ClN3OSÆC2H2O4) calcd: C, 60.26; H, 5.78; N,
7.53; found: C, 60.02; H, 5.79; N, 7.53.
5.10.31. N-(4-{4-[(2-Methoxyphenyl)amino]piperidin-1-
yl}butyl)cinnamide (25b). Starting with the free base of
24, method C2. Yield 65%; mp 82 ꢁC; H NMR (300
MHz, DMSO-d6) d 1.45–1.80 (m, 6H), 2.03–2.13 (m,
2H), 2.96–3.06 (m, 4H), 3.18–3.27 (m, 2H), 3.31–3.58
(m, 2H), 3.73 (s, 3H), 6.5–6.86 (m, 5H), 7.32–7.45 (m,
4H), 7.55–7.60 (m, 2H), 8.19 (s, br, 1H). Anal.
(C25H33N3O2ÆC2H2O4) calcd: C, 65.17; H, 7.09; N,
8.44; found: C, 64.92; H, 7.00; N, 8.47. ESI-MS: 408
(M+H+, 100).
1
5.10.26. N-(4-{4-[(4-Chlorophenyl)phenylmethyl)piperazin-
1-yl]butyl}benzo[b]thiophen-2-carboxamide (7c). Starting
with the free base of 4b, method C2. Yield 63%; mp
128 ꢁC 1H NMR (300 MHz, DMSO-d6) d 1.35–1.73
(m, 4H), 2.48 (s, br, 4H), 2.90–3.35 (m, 8H), 4.42 (s,
1H), 7.05–7.59 (m, 11H), 7.90–8.05 (m, 2H), 8.09 (s,
1H), 8.57 (s, br, 1H). Anal. (C28H32ClN3OSÆC2H2O4)
calcd: C, 63.20; H, 5.63; N, 6.91; found: C, 63.18; H,
5.66; N, 6.84. ESI-MS: 495 (M+H+, 100).
5.11. Pharmacology experiments
5.11.1. Dopamine receptors binding studies. Cell culture
was carried out using standard procedures. Human
D2S and D3 receptors were expressed in stably transfec-
ted Human Embryonic Kidney (HEK) and Chinese
hamster ovary (CHO) cells, respectively. The CHO-D3
cells were cultured at 37 ꢁC in Dulbecco’s Modified Ea-
gle Medium (DMEM, Cambrex Bio Sciences, Rockland
Inc) supplemented with 10% dialysed fetal calf serum
(Invitrogen,Co, Carlsbad, CA), 100 Units/ml penicil-
lin-steptomycin (Cambrex, Bio Sciences, Rockland
Inc), Hepes 20 mM, pH 7.4 and 2 mM glutamine
(Cambrex, Bio Sciences, Rockland Inc) in an atmo-
sphere of 5% CO2.53 The HEK-D2S cell line was ob-
tained following transfection by pCDNA3.1-D2S
expressing vector. HEK-D2S were cultured at 37 ꢁC in
Dulbecco’s Modified Eagle’s Medium (DMEM-
NUT.F-12, Cambrex, Bio Sciences, Rockland Inc)
supplemented with 10% fetal calf serum (Cambrex, Bio
Sciences, Rockland Inc), 100 U/ml penicillin-steptomy-
cin (Cambrex, Bio Sciences, Rockland Inc), Hepes
20 mM, pH 7.4, 400 lg/ml geneticin (Cambrex, Bio
Sciences, Rockland Inc) and 2 mM glutamine (Camb-
rex, Bio Sciences, Rockland Inc) in an atmosphere of
5% CO2.
5.10.27. N-(4-{4-[(4-Chlorophenyl)phenylmethyl]pipera-
zin-1-yl}butyl)cyclohexancarboxamide (7e). Starting with
the free base of 4b, method C2. Yield 79%; mp 109 ꢁC;
1H NMR (300 MHz, DMSO-d6) d 1.05–1.73 (m, 14H),
1.95–2.1 (m, 1H), 2.48 (s, br, 4H), 2.90–3.35 (m, 8H),
4.42 (s, 1H), 7.05–7.59 (m, 9H), 7.73 (s, br, 1H). Anal.
(C26H38ClN3OÆC2H2O4Æ0.5 H2O) calcd: C, 63.54; H,
7.29; N, 7.41; found: C, 63.71; H, 7.31; N, 7.35. ESI-
MS: 445 (M+H+, 100).
5.10.28. N-{4-[4-(Diphenylmethyl)-1,4-diazepan-1-yl]butyl}-
cinnamide (8b). Starting with the free base of 4c, method
C2. Yield 79%; mp 89 ꢁC; 1H NMR (300 MHz, DMSO-
d6) d 1.42–1.57 (m, 2H), 1.60–1.79 (m, 2H), 1.85–2.01
(m, 2H), 2.51–2.61 (m, 2H), 2.71–2.83 (m, 2H), 3.04–
3.46 (m, 8H), 4.68 (s, 1H), 6.64 (d, J = 15.81 Hz, 1H),
7.12–7.24 (m, 2H), 7.24–7.63 (m, 14H), 8.25 (t,
J = 5.47 Hz, 1H). Anal. (C31H37N3OÆ1.2C2H2O4Æ2
H2O) calcd: C, 65.58; H, 7.15; N, 6.87; found: C,
65.79; H, 6.91; N, 7.18. ESI-MS: 468 (M+H+, 100).
Incubations containing 2 nM [3H] spiperone (specific
activity 15 Ci/mmol, Perkin-Elmer Life Sciences, Bos-
ton, MA) were run in duplicate in 0.1% polyethylen-
imine (PEI) (Sigma–Aldrich, Inc, St. Louis, MI)
coated multiscreen GF/B 96 wells microplates (Milli-
5.10.29. N-{4-[4-(Diphenylmethyl)-1,4-diazepan-1-yl]butyl}-
thiophen-2-carboxamide (8d). Starting with the free base
1
of 4c, method C2. Yield 65%; mp 84 ꢁC; H NMR (300

7272
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adding per well 250 ll membrane suspension diluted
to 10 lg protein/ml. Two microliters of tested
compounds diluted in dimethyl sulfoxide (Sigma–Al-
drich, Inc, St. Louis, MI) were added in increasing
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the presence of 5 lM haloperidol (Sigma–Aldrich,
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14.50 microbeta Trilux counter (Wallac-Perkin Elmer,
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nonlinear regression using the Origin 6.0 software
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Acknowledgment
We thank Dr. P. Sokoloff (INSERM, Paris) for provid-
ing cell lines expressing dopamine D3 and D2s receptors.
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