N-(4-(4-(2-halogenophenyl)piperazin-1-yl)butyl) substituted cinnamoyl amide derivatives as dopamine D2 and D3 receptor ligands

Article abstract of DOI:10.1002/ardp.200600196

A series of eight substituted N-(4-(4-(2-halogenophenyl)piperazin-1-yl) butyl)-3-phenylacryl amide derivatives have been synthesized and screened for binding affinities at dopamine hD₂ and hD₃ receptors. All compounds have shown high

Full text of DOI:10.1002/ardp.200600196

178
Arch. Pharm. Chem. Life Sci. 2007, 340, 178–184
Full Paper
N-(4-(4-(2-Halogenophenyl)piperazin-1-yl)butyl) Substituted
Cinnamoyl Amide Derivatives as Dopamine D2 and D3 Receptor
Ligands
Oliver Saur1, Anneke E. Hackling1, Sylvie Perachon2, Jean-Charles Schwartz2,3, Pierre Sokoloff3,
and Holger Stark1
1 Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Frankfurt, Germany
2 Laboratoire Bioprojet, Paris, France
3 INSERM, Unitꢀ de Neurobiologie et Pharmacologie Molꢀculaire (U. 573), Centre Paul Broca, Paris, France
A series of eight substituted N-(4-(4-(2-halogenophenyl)piperazin-1-yl)butyl)-3-phenylacryl amide
derivatives have been synthesized and screened for binding affinities at dopamine hD2 and hD3
receptors. All compounds have shown high to remarkable receptor affinities and some have led
to distinct selectivity for D3 receptors. Highest D3-receptor affinity has been observed for 3-(4-ami-
nophenyl)-N-(4-(4-(2-fluorophenyl)piperazin-1-yl)butyl)acryl amide (hD3 Ki 0.9 nM; hD2 Ki 17.4 nM).
Selectivity ratio has been best for 3-(4-chlorophenyl)-N-(4-(4-(2-fluorophenyl)piperazin-1-yl)butyl)-
acryl amide with a 56-fold preference for hD3 versus hD2. A functional activity test has been per-
formed by a mitogenesis test for N-(4-(4-(2-fluorophenyl)piperazin-1-yl)butyl)-3,3-diphenylacryl
amide, which, surprisingly, has shown full agonist properties.
Keywords: Cinnamic acids / D3 receptors / Dopamines / PET / Phenylpiperazines /
Received: November 22, 2006; accepted: February 2, 2007
DOI 10.1002/ardp.200600196
applied to many dopamine receptor ligands: An aryl
Introduction
amide is linked via a tetramethylene spacer to a basic
residue with aryl substituent [7]. So, the partial dopamine
D3 receptor agonist BP 897, one of our lead compounds,
follows these structural analogies [3]. The lead structure
of new compounds based on BP 897 is drawn in bold
(Fig. 1).
BP 897 shows a noteworthy pharmacological profile
with potential for the treatment of Parkinson’s disease
and neuropsychiatric disorders [8, 9]. Replacement of the
naphthylamide residue of BP 897 by a cinnamoyl group
and choosing 1,2,3,4-tetrahydroisoquinoline as basic resi-
due has lead to the full antagonist ST 198 [10, 11]. Antago-
nists also look promising in the treatment of Parkinson's
disease and drug abuse [6, 12, 13]. The 1-(2-methoxyphe-
nyl)piperazine derivatives ST 186 and ST 168 (Table 1) are
partial agonists with even higher affinity at D3 receptors
than that of BP 897 [14]. Gmeiner et al. have reported
about 1-(2,3-dichlorophenyl)piperazine derivatives with
antagonist properties [15]. These dihalogenated phenyl-
piperazine derivatives have shown similar D2 and D3
Dopamine is an essential neurotransmitter in the central
nervous system. Its corresponding receptors are divided
into two receptor families: The D1-like receptors includ-
ing D1 and D5 receptor subtypes activate adenylyl cyclase.
D2, D3, and D4 receptor subtypes belonging to the D2-like
receptors inhibit adenylyl cyclase [1] Whereas dopamine
D2 receptors are located in highest density in the stria-
tum, D3 receptors are mainly located in the limbic system
[2, 3]. Therefore, the dopamine D3 receptor has become a
promising target for antipsychotic drugs and the treat-
ment of Parkinson's disease [3–5]. Moreover, dopamine
D3 receptors are implicated in the reinforcing effects of
drugs of abuse [6]. A general structural pattern can be
Correspondence: Holger Stark, Institute of Pharmaceutical Chemistry,
Johann Wolfgang Goethe-University, Max-von-Laue-Str. 9, D-60438
Frankfurt, Germany
E-mail: h.stark@pharmchem.uni-frankfurt.de
Fax: +49-69-798-29258
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2007, 340, 178–184
(2-Halogenophenyl)piperazines as DA D2/3 receptor ligands
179
Reagents and conditions: (i) 4-bromobutyronitrile, acetonitrile, or acetone, K2CO3,
reflux, 8 h; (ii) Ra-Ni, NH3 aq., 25 bar H2, 2 d; (iii) malonic acid, pyridine, piperidine,
reflux, 3 h; (iv) oxalyl chloride, reflux, 2 h.
Scheme 1. Synthesis of compounds 1–4.
synthetic approach turned out to be the catalytic hydro-
genation of the nitrile using Raney Nickel [16]. This
method allowed scaling up to amounts of 20 g nitrile as
educt and has been applied for synthesizing 4-(4-(2-fluoro-
phenyl)piperazin-1-yl)butylamine 2b. 4-Iodocinnamic
acid 3 has been prepared by a classic Knoevenagel con-
densation of 4-iodobenzaldehyde with malonic acid in
the presence of piperidine [17]. Decarboxylation of the
product takes place in situ and the cinnamoyl structure
of the product is clearly identified as E-isomer by its
1HNMR spectrum (Scheme 1). The aryl amide moiety (Fig-
ure 1) of all compounds in this series is represented by a
cinnamoyl residue. Cinnamic acid is a structural imita-
tion of naphthalene-2-carboxylic acid of BP 897. The first
connecting aryl ring of the 2-naphthyl residue has been
replaced by a double bound, which provides the amide in
a definite distance to the sterically fixed aryl structure.
Formally, cinnamoyl is a ring-opened and truncated 2-
naphthoyl imitate with slightly altered bond length and
angle. Within this group, the compounds bear a variety
of halide substituents as well as a nitro or amino group,
each in 4-position (5–11). A different substitution pattern
was chosen for compound 12. An additional phenyl ring
in the b-position of the double bond of the cinnamic acid
basic structure has lead to considerably increased lipo-
philicity and bulkiness of this compound. The required
cinnamoyl structure has been generated by a carboxyla-
tion reaction of oxalyl chloride with 1,1-diphenylethy-
lene (Scheme 1) [18, 19]. Advantage has been taken from
the unsubstituted phenyl structure as it disfavours an
unwanted, possibly following additional decarboxyla-
tion reaction. The electronic influence of halogen substi-
tution would have provoked the decarboxylation of the
diphenylacrylic acid. Thus, for a smooth reaction the
Figure 1. General structure pattern of dopamine D3-receptor
selective antagonists and partial agonists.
receptor affinities to their 2-methoxyphenyl analogous
[15]. Therefore, we have created a series of 3-phenylacryl
amide derivatives with (2-halogenophenyl)piperazino
residue. Most compounds of this series are (2-fluorophe-
nyl)piperazine derivatives, which offer an advantage for
the development of potential radioligands for positron
emission tomography (PET) imaging of dopamine D3
receptors. Radioiodination of N-(4-(4-(2-fluorophenyl)pi-
perazin-1-yl)butyl)-3-(4-iodophenyl)acryl amide might
also provide a useful pharmacological tool for in vivo
investigation of D3 receptor distribution using single
photon emission computed tomography (SPECT) ima-
ging techniques. The newly designed compounds have
been prepared and tested for their affinities at hD2 and
hD3 receptors using [125I]iodosulpiride as radioligand in
displacement studies.
Chemistry
Alkylation of piperazine derivatives by 4-bromobutyroni-
trile led to the precursor compound 1a (Scheme 1).
Reduction of nitrile 1a could be achieved by treatment
with lithium aluminium hydride to obtain 4-(4-(2-chloro-
phenyl)piperazin-1-yl)butylamine 2a. A more economic
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180
O. Saur et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 178–184
cinnamoyl derivative ST 186 (Table 1). Therefore, we
firstly had chosen the 4-chloro substituted cinnamoyl
derivative ST 168 for further variations at the phenylpi-
perazino residue. Bioisosteric replacement of the 2-meth-
oxy residue by 2-chloro substitution has lead to a
decrease in D3 receptor affinity and selectivity (Table 1).
The 1-(2-fluorophenyl)piperazine derivative 8 has shown
higher D3 receptor affinity than the 2-chloro substituted
compound 5 whereas D2 receptor affinity has not been
significantly different. Compound 8 has shown the best
selectivity for D3 receptors in this series. Methoxy- (of lead
compound BP 897) and fluoro-substituted phenylpipera-
zines may show comparable physicochemical properties
in aqueous environment. But, moreover, fluorine is
expected to show higher metabolic stability and offers
the development of a potential radioligand for PET ima-
ging of dopamine D3 receptors. Therefore, we have pre-
pared a series of 4-substitued cinnamoyl derivatives
(Table 1) containing compounds which have shown
enhanced selectivity ratios and remarkable low to subna-
nomolar binding affinities at dopamine D3 receptors.
Within the 4-halogen substituted cinnamoyl derivatives,
fluoro compound 9 has revealed the highest dopamine
D3 receptor affinity, whereas bromo derivative 7 has dis-
played an improved selectivity for the dopamine D3
receptor. Due to its fluoro substitution of the cinnamoyl
structure and the 2-fluoro-substituted phenylpiperazino
residue, compound 9 offers two possibilities for 18F radio-
labeling. Best results for dopamine D3 receptor selectivity
have been obtained for iodo compound 6 and chloro
compound 8. Both compounds have shown a remarkable
dopamine D3 receptor affinity. Radioiodination of com-
pound 6 might also provide a useful pharmacological
tool for in vivo investigation of D3 receptor distribution
using SPECT imaging techniques. Nitro substitution (10)
has also resulted in improved selectivity for the dopa-
mine D3 receptor, and has, additionally, led to increased
dopamine D3 receptor affinity. The amino-substituted
analogue 11 has displayed a slightly deteriorated selectiv-
ity D2/D3 ratio, but has reached a superior binding affi-
nity for the dopamine D3 receptor. A different substitu-
tion pattern on the cinnamoyl structure has been per-
formed on compound 12. The phenyl substitution in b-
position of the double bond has lead to a 3,3-diphenyl-
acrylamido structure. Here, the lipophilic aryl residue is
considerably increased and bulky. Spatial requirements
are raised due to the voluminous diphenyl structure.
When compared to the above discussed 4-substituted cin-
namoyl derivatives, b-phenyl substitution has resulted in
deteriorated dopamine D3 receptor binding and conse-
quently in deteriorated preference over the dopamine D2
receptor.
Reagents and conditions: (i)a./thionyl chloride, CH2Cl2, Et3N, 08C, 10 min, rt, 1 h; b./
amine, CH2Cl2, Et3N, 08C, 10 min, rt, overnight. (ii) iron powder, FeCl3, H2O, reflux 15
h.
Scheme 2. Synthesis of compounds 5–12.
mixture could be heated to reflux without the product
3,3-diphenylacrylic acid 4 suffering from decomposition.
Different pathways are possible for preparing the ami-
noalkyl spacer with alkanamino residue and aryl substi-
tuent, corresponding to the general structure pattern
(Figure 1).
The cinnamic acid precursors have been converted
into the corresponding acid chlorides by thionyl chloride
and used directly without further purification for the
final acylation of the primary amines 2 (Scheme 2). Yields
have not been optimized on this reaction step. For the
hydrogenation of compound 10 to the corresponding
amino compound 11, iron powder was taken in the pre-
sence of a catalytic amount of FeCl3. Under these condi-
tions, the nitro group is selectively reduced without
affecting the double bound (Scheme 2) [20].
Pharmacological results and discussion
Following a molecular modeling study, which has been
performed on analogous compounds (e.g. ST 168 and
ST 186) E-cinnamide is established as a valuable struc-
tural basis [21]. These findings are confirmed by other
potent E-cinnamoyl derivatives published [22]. Isomeric
Z-derivatives have been reported to possess lower affi-
nities [22]. ST 168 shows a similar D3 receptor affinity but
an increased selectivity compared to the unsubstituted
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Arch. Pharm. Chem. Life Sci. 2007, 340, 178–184
(2-Halogenophenyl)piperazines as DA D2/3 receptor ligands
181
Table 1. Chemical structures, physical data, and pharmacological screening results for human dopamine D2 and D3 receptor binding
affinities.
No.
Formula
R1
R2
R3
MW
MP
(8C)
Yield
(%)
Ki [nM]a)
hD2
hD2/
b)
hD3
hD3
5
6
7
8
9
10
11
12
C23H27Cl2N3O6C2H2O46H2O Cl
H
H
H
H
H
H
H
Cl
F
F
F
F
F
F
F
540.4
507.4
464.9
420.4
489.5
426.5
396.5
547.6
105.5c) 19
167.5d)
162.7d) 10
18 l 5
3.2 l 0.6
7.9
3.4
1.7 € 0.4
2.4
0.9
21.8
0.33 l 0.1
0.44 l 0.09
0.92 l 0.2
420 l 240
166.1 l 27.6
170
190
25.6 € 1.0
57
17.4
211
13.54 l 1.2
22.6 l 1.0
61 l 0.2
23
52
22
56
15
24
19
10
41
51
66
C23H27FIN3O
I
9
C23H27BrFN3O60.25H2O
C23H27ClFN3O60.25H2O
C23H27F2N3O6C2H2O4
C23H27FN4O3
C23H29FN4O
C29H32FN3O6C2H2O4
Br
Cl
F
NO2
NH2
H
158.7d)
6
174.8e) 10
164.8d) 21
151.0d) 19
Phenyl
H
H
164.8f)
9
ST 186g)
ST 168g)
BP 897h)
H
Cl
OCH3
OCH3
a)
Values (Ki) are mean of at least two independent determinations (lSEM of at least three independent determinations performed
in triplicate).
b)
c)
d)
e)
f)
Ratio is calculated from corresponding Ki values.
Crystallized from ethanol/diethylether.
Crystallized from 2-propanol.
Crystallized from methanol/diethylether.
Crystallized from 2-propanol/diethylether.
Ref. 14.
g)
h)
Ref. 8.
In this study, compound 12 with its untypical struc-
Experimental
ture has been functionally tested in a mitogenesis assay
on [3H]thymidine incorporation in NG 108-15 cells [23,
24]. Agonist-induced incorporation of [3H]thymidine into
growing cells has been used as basic principle. The intrin-
sic activity is quantified by determination of the effective
concentration (EC50) of the test compound and by com-
paring the maximum effect to that of the full agonist
dopamine [25]. Results have surprisingly designated a
full dopamine D3 receptor agonist with an intrinsic activ-
ity of l1 and an EC50 value of l10 nM. 2,3-Chloro-disub-
stitution of the 4-phenylpiperazino residue would have
lead to a typical antagonist structure [6]. As no typical
agonist elements are given, the lack of antagonist proper-
ties complies with anticipation. The replacement of a 2-
methoxy substituent, which is supposed to be indicative
of partial agonism [6], by a 2-fluoro substituent in the 4-
phenylpiperazino residue may have caused the change to
full agonism. Since the intrinsic activity of this outstand-
ing structure 12 could be attributed to its basic residue,
no further mitogenesis tests have been performed within
this series of compounds presented here.
Chemistry
Melting points were determined on an Electrothermal IA 9000
digital or a Bꢀchi (Germany) 512 melting point apparatus and
are uncorrected. 1H NMR spectra were recorded on a Bruker (Ger-
1
many) Avance DPX 400 (400 MHz) spectrometer. H NMR data
are reported in the following order: chemical shift (d) in ppm
downfield from tetramethylsilane as internal reference; multi-
plicity (br, broad; s, singlet; d, doublet; dd, double doublet; t, tri-
plet; quint, quintet; m, multiplet); approximate coupling con-
stants (J) in Hertz (Hz); number and assignment of protons; *,
exchangeable by D2O; Pipera, piperazinyl. EI-MS was performed
on a Finnigan (USA) Varian MAT CH7A or a MAT 212 Varian,
FAB+-MS on a Finnigan (USA) FAB MAT CH5DF (80 eV,) with
xenon, DMSO as solvent, and a matrix of glycerol. Data are listed
as mass number (m/z) and relative intensity (%). Elemental ana-
lyses (C, H, N) were measured on Perkin Elmer (USA) 240 B, Per-
kin Elmer (USA) 240 C, Vario-EL (Perkin Elmer, USA) or CHN-
Rapid (Heraeus, Germany) and were within l 0.4% of the theore-
tical values for all compounds. Hydrogenations were carried out
on an autoclave model IV, 500 mL (Roth, Germany). Preparative,
centrifugally accelerated, rotatory chromatography was per-
formed using a Chromatotron 8924 (Harrison Research, USA)
and glass rotors with 4 mm layers of silica gel 60 PF254 containing
gypsum (Merck, Germany). Analytical thin layer chromatogra-
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182
O. Saur et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 178–184
phy (TLC) was performed on silica gel 60 F254 plates (Merck, Ger-
many). The spots were visualized with fast blue salt B, ninhydrin
or by UV absorption at 254 nm. Chemical procedures are exemp-
lary described for the fluoro compounds.
General procedure for preparation of 3-phenylacryl
amides 5-10, 12
Thionyl chloride (30 mmol) was added to 2 mL of dry CH2Cl2 and
a catalytic amount of triethylamine. This mixture was protected
from light, cooled with ice, and stirred under argon. 3.0 mmol
of the carboxylic acid was added carefully. The mixture was stir-
red for 10 min under ice and for 1 h without cooling. Then, the
solvent and the excessive thionyl chloride were removed under
reduced pressure. The crude acid chloride obtained was dis-
solved in 4 mL of dry CH2Cl2 and given to a mixture of amine 2
(2.7 mmol), triethylamine (8.1 mmol), and 7 mL of dry CH2Cl2,
which had been stirred under argon and ice cooling for 10 min.
Stirring continued under additional light protection overnight,
until no more amine could be detected by TLC control. The sol-
vent was removed under reduced pressure and the mixture stir-
red with water to give a light brown, solid product. Water was
decanted and the product recrystallized from 2-propanol.
Further purification using a chromatotron (PE, CHCl3 1:1, atm
of NH3) afforded the product.
4-(4-(2-Fluorophenyl)piperazin-1-yl)butyronitrile 1b
A mixture of 1-(2-fluorophenyl)piperazine (40 mmol), 4-bromo-
butyrontrile (44 mmol), K2CO3 (60 mmol), and
a catalytic
amount of KI in 100 mL of acetone was heated to reflux for 8 h
and then stirred at ambient temperature overnight. The suspen-
sion was filtrated and the residue washed with acetone. The fil-
trate was evaporated to dryness and dried under reduced pres-
sure. The product suited well for the next reaction step without
further purification. Yield 84%. C14H18FN3 (247.32), colorless oil.
1H NMR (400 MHz, [D6]DMSO): d = 7.13–7.08 (m, 2H, F-Ph-3H, -
5H), 7.03–6.93 (m, 2H, F-Ph-4H, -6H), 3.01 (t, J = 4.7, 4H, Pipera-
2H, -6H), 2.53 (t, J = 4.6, 4H, Pipera-3H, -5H), 2.49 (partially cov-
ered by DMSO peak, m, 2H, NC-CH2), 2.42 (t, J = 6.9, 2H, CH2-
Pipera), 1.75 (2H, NC-CH2-CH2). EI-MS: 247 (M+, 72), 207 (52), 193
(100).
3-(4-Chlorophenyl)-N-(4-(4-(2-fluorophenyl)piperazin-1-
yl)butyl)acryl amide 5
4-(4-(2-Fluorophenyl)piperazin-1-yl)butylamine 2b
1H NMR (400 MHz, [D6]DMSO): d = 8.17 (m*, 1H, NH), 7.59 (d, J =
8.1, 4-Cl-Ph-2H, -6H), 7.44 (m, 4H, 4-Cl-Ph-3H, -5H, 4-Cl-Ph-CH, 2-Cl-
Ph-3H), 7.31 (m, 1H, 2-Cl-Ph-4H), 7.18 (m, 1H, 2-Cl-Ph-6H), 7.09 (m,
1H, 2-Cl-Ph-5H), 6.73 (d, J = 15.8, 1H, CO-CH), 3.39-2.66 (partially
covered by H2O peak, 12H, CONH-CH2, 8-Pipera-H, 1-Pipera-CH2 ),
1.76-1.52 (m, 4H, CONH-CH2-CH2-CH2). EI-MS: 430 (M+, 7), 209
(100), 194 (20), 165 (88), 154 (27), 139 (28), 125 (13), 70 (91), 56
(31), 44 (23). Anal. calcd: C, 55.6; H, 5.78; N, 7.78; found: C, 55.2;
H, 5.48; N, 7.38.
Reduction of the nitrile 1b (30 mmol) was accomplished with
freshly prepared Raney nickel (from 90 mmol aluminum-nickel-
alloy) in 150 mL of aqueous ammonia 25% and 25 bar H2 pres-
sure at room temperature for 2 d. Cautious filtration and eva-
poration to dryness delivered an oily product, which crystallized
as hydrogen dioxalate from 2-propanol. Yield 74%.
1
C14H22FN362C2H2O4 (431.42), m.p. 90-1038C. H NMR (400 MHz,
[D6]DMSO): d = 7.99 (s*, br, 1H, H2N), 7.19–7.00 (m, 4H, F-Ph), 3.22
(brs, 4H, Pipera-2H, -6H), 3.11 (brs, 4H, Pipera-3H, -5H), 2.91 (t, J =
7.0, 2H, H2N-CH2), 2.82 (t, J = 7.2, 2H, CH2-Pipera), 1.69–1.67 (m,
2H, H2N-CH2-CH2), 1.63–1.58 (m, 2H, CH2-CH2-Pipera). FAB+-MS:
252 ([M+H]+, 92), 72 (100).
N-(4-(4-(2-Fluorophenyl)piperazin-1-yl)butyl)-3-(4-
iodophenyl)acryl amide 6
1H NMR (400 MHz, [D6]DMSO): d = 8.12 (t, J = 5.8, 1H, NH), 7.77 (d, J
= 8.2, 2H, I-Ph-3H, -5H), 7.36 (d, J = 15.1, 1H, I-Ph-CH, d, J = 8.6, 2H,
I-Ph-2H, -6H), 7.13-7.07 (m, 2H, F-Ph-3H, -5H), 7.03–6.93 (m, 2H, F-
Ph-4H, -6H), 6.66 (d, J = 15.8, 1H, CH-CO), 3.20 (d, br, J = 5.5, 2H,
HN-CH2), 3.00 (t, J = 4.3, 4H, Pipera-2H, -6H), 2.51-2.50 (partially
covered by DMSO peak, m, 4H, Pipera-3H, -5H), 2.34 (brs, 2H, CH2-
Pipera), 1.49 (brs, 4H, HN-CH2-CH2-CH2). EI-MS: 507 (M+., 5), 57 (16),
193 (100) FAB+-MS: 508 ([M+H]+, 100). Anal. calcd: C, 54.45; H,
5.36; N, 8.28; found: C, 54.24; H, 5.29; N, 8.23.
4-Iodocinnamic acid 3
Malonic acid (10 mmol) was dissolved in 50 mL of pyridine and
0.5 mL of piperidine. The mixture was protected from light and
stirred under argon. Then, 4-iodobenzaldehyde (8 mmol) was
added and the mixture heated to reflux. After 3 h no more alde-
hyde was detected by TLC. The mixture was cooled down to
ambient temperature and the acid liberated with 10 N HCl and
ice under vigorous stirring. The solid product obtained was
washed with water several times and dried extensively under
1
reduced pressure. Yield 86%. C9H7IO2 (274.05), m.p. 127.08C. H
NMR (400 MHz, [D6]DMSO): d = 13.99 (brs*, 1H, COOH), 7.79 (d, J =
8.2, 2H, I-Ph-3H, -5H), 7.54 (d, J = 16.1, 1H, I-Ph-CH), 7.49 (d, J = 8.3,
2H, I-Ph-2H, -6H), 6.57 (d, J = 16.0, 2H, CH-COOH). EI-MS: 274 (M+,
100).
3-(4-Bromophenyl)-N-{4-[4-(2-fluorophenyl)piperazin-1-
yl)butyl)acryl amide 7
1H NMR (400 MHz, [D6]DMSO): d = 8.12 (t, J = 5.4, 1H, NH), 7.61 (d, J
= 8.4, 2H, Br-Ph-3H, -5H), 7.51 (d, J = 8.4, 2H, Br-Ph-2H, -6H), 7.39
(d, J = 15.8, 1H, Br-Ph-CH), 7.13-7.07 (m, 2H, F-Ph-3H, -5H), 7.03–
6.93 (m, 2H, F-Ph-4H, -6H), 6.65 (d, J = 15.8, 1H, CH-CO), 3.21–3.19
(m, 2H, HN-CH2), 3.00–2.99 (m, 4H, Pipera-2H, -6H), 2.51 (partially
covered by DMSO peak, brs, 4H, Pipera-3H, -5H), 2.34 (brs, 2H,
CH2-Pipera), 1.49 (brs, 4H, HN-CH2-CH2-CH2). FAB+-MS: 463 (2), 462
(6), 461 (2), 460 ([M+H]+, 6), 209 (4), 193 (6). Anal. calcd: C, 59.42;
H, 5.96; N, 9.04; found: C, 59.42; H, 6.11; N, 8.85.
3,3-Diphenylacrylic acid 4
Compound was prepared according to known methods [19].
1
Yield 44%. C15H12O2 (224.26), m.p. 161.58C (m.p. 1678C [19]). H
NMR (400 MHz, [D6]DMSO): d = 12.44 (s*, 1H, COOH), 7.41-7.36 (m,
3H, Z-Ph-3H, 4-H, 5-H, 3H, E-Ph-3H, -4H, -5H), 7.27-7.25 (m, 2H, Z-
Ph-2H, -6H), 7.16-7.14 (m, 2H, E-Ph-2H, -6H). EI-MS: 224 (M+, 100).
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Arch. Pharm. Chem. Life Sci. 2007, 340, 178–184
(2-Halogenophenyl)piperazines as DA D2/3 receptor ligands
183
6H), 6.55 (d, J = 8.5, 2H, H2N-Ph-3H, -5H), 6.28 (d, J = 15.7, 1H, CH-
CO), 5.51 (s*, 2H, H2N), 3.17 (d, J = 5.5, 2H, HN-CH2), 3.00–2.99 (m,
4H, Pipera-2H, -6H), 2.51 (partially covered by DMSO peak, s, 4H,
Pipera-3H, -5H), 2.33 (brs, 2H, CH2-Pipera), 1.47 (brs, 4H, HN-CH2-
CH2-CH2). EI-MS: 396 (M+9, 5), 193 (36), 146 (100). Anal. calcd: C,
69.67; H, 7.37; N, 14.13; found: C, 69.67; H, 7.56; N, 13.96.
3-(4-Chlorophenyl)-N-(4-(4-(2-fluorophenyl)piperazin-1-
yl)butyl)acryl amide 8
1H NMR (400 MHz, [D6]DMSO): d = 8.12 (t, J = 5.5, 1H, NH), 7.58 (d,
J = 8.5, 2H, Cl-Ph-2H, -6H), 7.47 (d, J = 8.5, 2H, Cl-Ph-3H, -5H), 7.40
(d, J = 15.8, 1H, Cl-Ph-CH), 7.13–7.07 (m, 2H, F-Ph-3H, -5H), 7.03-
6.93 (m, 2H, F-Ph-4H, -6H), 6.63 (d, J = 15.8, 1H, CH-CO), 3.20–3.19
(m, 2H, HN-CH2), 3.00–2.99 (m, 4H, Pipera-2H, -6H), 2.51 (partially
covered by DMSO peak, brs, 4H, Pipera-3H, -5H), 2.34 (brs, 2H,
CH2-Pipera), 1.49 (brs, 4H, HN-CH2-CH2-CH2). Anal. calcd: C, 65.71;
H, 6.59; N, 9.99; found: C, 65.58; H, 6.71; N, 9.80.
Pharmcological assays
Binding studies
For binding assays, Chinese hamster ovary (CHO) cells were sta-
bly transfected with cDNA of human D2L and D3 receptors and
cloned [26, 27]. Binding to dopamine D2 and D3 receptors was
evaluated by displacement studies with [125I]iodosulpiride [28]. Ki
values are calculated from the IC50 values according to the
Cheng–Prusoff equation [29]. These cell lines were cultured in
Dulbecco's Modified Eagle Medium, which was supplemented in
10% fetal calf serum, in an atmosphere of 5% CO2. Cells were har-
vested from culture dishes in the presence of 0.2% trypsin, were
centrifuged at 2000 g for 5 min and then homogenized in
10 mM Tris-HCl, pH 7.4, containing 5 mM MgCl2, using a Poly-
tron. The homogenate was centrifuged at 20000 g for 15 min at
48C and the pellet was resuspended by sonication in 50 mM of
Tris-HCl, pH 7.4, containing: NaCl, 120 mM; KCl, 5 mM; CaCl2,
2 mM; and MgCl2, 8 mM (incubation buffer). Membranes were
used either immediately or after storage at –708C. A membrane
volume of 200 lL, diluted in incubation buffer supplemented
with 0.2% bovine serum albumin was added to polystyrene tubes
containing (in 100 lL) 0.1 nM [125I]iodosulpiride and drug,
diluted in 100 lL of incubation buffer. Nonspecific binding was
determined in the presence of 1 lM nemonapride. Incubations
were run at 308C for 30 min. Reactions were stopped by vacuum
filtration through Whatman (Great Britain) GF/B glass-fiber fil-
ters coated in 0.3% polyethylenimine with automated cell har-
vester (Brandel-Beckman, Gaithersburg, MD, USA). Filters were
rinsed three times with 5 mL of ice-cold incubation buffer and
counted by liquid scintillation in 5 mL of ACS II (Amersham,
USA).
3-(4-Fluorophenyl)-N-(4-(4-(2-fluorophenyl)piperazin-1-
yl)butyl)acryl amide 9
1H NMR (400 MHz, [D6]DMSO): d = 8.18 (t, J = 5.4, 1H, NH), 7.63
(dd, J = 5.6, 2H, F-Ph), 7.42 (d, J = 15.8, 1H, 4-F-Ph-CH), 7.25 (t, J =
8.8, 2H, F-Ph), 7.19–7.00 (m, 4H, F-Ph), 6.59 (d, J = 15.8, 1H, CH-
CO), 3.22–3.21 (m, 2H, HN-CH2, 4H, Pipera-2H, -6H), 3.15 (brs, 4H,
Pipera-3H, -5H), 2.95 (t, J = 7.7, 2H, CH2-Pipera), 1.66–1.62 (m, 2H,
HN-CH2-CH2), 1.54-1.49 (m, 2H, CH2-CH2-Pipera). FAB+-MS: 400
([M+H]+, 2), 149 (3). Anal. calcd: C, 61.34; H, 5.97; N, 8.58; found:
C, 60.97; H, 5.92; N, 8.25.
N-(4-(4-(2-Fluorophenyl)piperazin-1-yl)butyl)-3-(4-
nitrophenyl)acryl amide 10
1H NMR (400 MHz, [D6]DMSO): d = 8.26 (d, J = 8.6, 1H, NH, 2H, O2N-
Ph-3H, -5H), 7.83 (d, J = 8.7, 2H, O2N-Ph-2H, -6H), 7.53 (d, J = 15.8,
1H, O2N-Ph-CH), 7.13-7.07 (m, 2H, F-Ph-3H, -5H), 7.03–6.93 (m,
2H, F-Ph-4H, -6H), 6.82 (d, J = 15.8, 1H, CH-CO), 3.23-3.22 (m, 2H,
HN-CH2), 3.00–2.99 (m, 4H, Pipera-2H, -6H), 2.51 (partially cov-
ered by DMSO peak, brs, 4H, Pipera-3H, -5H), 2.34 (brs, 2H, CH2-
Pipera), 1.50 (brs, 4H, HN-CH2-CH2-CH2). FAB+-MS: 427 ([M+H]+, 80),
193 (89). Anal. calcd: C, 64.77; H, 6.38; N, 13.14; found: C, 64.56;
H, 6.49; N, 13.16.
N-(4-(4-(2-Fluorophenyl)piperazin-1-yl)butyl)-3,3-
diphenylacryl amide 12
1H NMR (400 MHz, [D6]DMSO): d = 7.95 (t, J = 5.6, 1H, NH), 7.37–
7.31 (m, 6H, Ph), 7.23–7.21 (m, 2H, Ph), 7.19-7.00 (m, 2H, Ph, 4H,
F-Ph), 6.44 (s, 1H, CH-CO), 3.21 (brs, 4H, Pipera-2H, -6H), 3.10 (brs,
4H, Pipera-3H, -5H), 3.03 (dd, J = 6.6, 2H, HN-CH2), 2.88 (t, J = 7.7,
2H, CH2-Pipera), 1.56–1.49 (m, 2H, HN-CH2-CH2), 1.39–1.32 (m,
2H, CH2-CH2-Pipera). FAB+-MS: 459 (18), 458 ([M+H]+, 52), 207 (100).
Anal. calcd: C, 67.99; H, 6.26; N, 7.67; found: C, 67.69; H, 6.05; N,
7.37.
Functional receptor tests
A mitogenesis test was performed on NG 108-15 cells expressing
the dopamine D3 receptor, measuring [3H]thymidine incorpora-
tion [28]. NG 108-15 cells expressing the human D3 receptor were
cultured in Dulbecco's Modified Eagle Medium supplemented in
10% fetal calf serum in an atmosphere of 5% CO2 and were plated
in collagen-coated 96-well plates. After a 24 h culture, cells were
washed twice with culture medium without feral calf serum
and incubated for 16 h with 1 lM forskoline and quinpirole in
increasing concentrations, in the absence or presence of com-
pounds at 1.5, 3, 30, or 300 nM. Then, [3H]thymidine (1 lCi/well)
was added for 2 h and cells were harvested by vacuum filtration
through Whatman GF/C glass-fiber filters using an automated
cell harvester. The filters were rinsed 15 times with 200 lL of
phosphate buffered saline. Radioactivity was counted by liquid
scintigraphy in 5 mL of ACS II.
3-(4-Aminophenyl)-N-(4-(4-(2-fluorophenyl)piperazin-1-
yl)butyl)acryl amide 11
Iron powder (2.1 mmol), a catalytic amount of FeCl3, and com-
pound 10 (2 mmol) were suspended in 10 mL of H2O. The mix-
ture was heated to reflux for 15 h. During this time, catalytic
amounts of iron powder (0.2 mmol) and FeCl3 were added twice.
Cooled down to ambient temperature, the mixture was
quenched with 2 N NaOH and filtrated. The filtrate was
extracted with CH2Cl2 three times. The combined organic layers
were treated with brine and evaporated to dryness. The yellow
oil obtained was purified using a chromatotron (CHCl3, atm of
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