A series of 18F-labelled pyridinylphenyl amides as subtype-selective radioligands for the dopamine D3 receptor

Article abstract of DOI:10.1002/cmdc.201000067

Synthesis, biological activity, and structure-selectivity relationship (SSR) studies of a novel series of potential dopamine D3 receptor radioligands as imaging agents for positron emission tomography (PET) are reported. Considering a structurally diverse

Full text of DOI:10.1002/cmdc.201000067

DOI: 10.1002/cmdc.201000067
18
A Series of F-Labelled Pyridinylphenyl Amides as
Subtype-Selective Radioligands for the Dopamine D3
Receptor
Carsten Hocke,*[a] Simone Maschauer,[a] Harald Hꢀbner,[b] Stefan Lçber,[b] Wolfgang Utz,[b]
Torsten Kuwert,[a] Peter Gmeiner,[b] and Olaf Prante[a]
Synthesis, biological activity, and structure–selectivity relation-
ship (SSR) studies of a novel series of potential dopamine D3
receptor radioligands as imaging agents for positron emission
tomography (PET) are reported. Considering a structurally di-
verse library of D3 ligands, SSR studies were performed for a
new series of fluorinated pyridinylphenyl amides using CoMFA
and CoMSIA methods. The in vitro D3 affinities of the predict-
ed series of biphenyl amide ligands 9a–d revealed single-digit
to sub-nanomolar potencies (Ki =0.52–1.6 nm), displaying ex-
cellent D3 selectivity over the D2 subtype of 110- to 210-fold
for the test compounds 9a–c. Radiofluorination by nucleophil-
ic substitution of Br or NO2 by 18F led to radiochemical yields
of 66–92% for [18F]9a–d. However, the specific activities of
[18F]9b and [18F]9d were insufficient, rendering their use for
in vivo studies impossible. Biodistribution studies of [18F]9a
and [18F]9c using rat brain autoradiography revealed accumu-
lation in the ventricles, thus indicating insufficient biokinetic
properties of [18F]9a and [18F]9c for D3 receptor imaging
in vivo.
Introduction
Dopaminergic neurotransmission plays an important role in
various physiological processes. Correspondingly, it is implicat-
ed in the pathogenesis of a variety of diseases including ex-
trapyramidal movement disorders such as Parkinson’s disease
and psychiatric illnesses such as schizophrenia or
drug addiction.[1–3]
BP 897 (1), FAUC 346 (2), CJB 090 (3a), or NGB 2904 (4).[6,22–24]
In 1999, BP 897 received great attention as it was first reported
to be effective in the treatment of cocaine abuse.[6] Compared
to the partial agonist BP 897 (1), FAUC 346 (2) differs by a ben-
Dopamine receptors can be differentiated into the
five subtypes D1–D5. Among these, the D2 subtype
has received most attention owing to its essential
role in the nigrostriatal axis. However, knowledge of
the physiological and pathophysiological roles of the
other subtypes is rapidly evolving. In particular, re-
search has focused on the D3 subtype, as disturban-
ces of its expression and/or activity seem to play a
role in emotional functions associated with reward,
behavioural reinforcement, and craving.[4,5] The D3 re-
ceptor is a therapeutic target for a variety of diseases
including schizophrenia, depression, and Parkinson’s
disease.[6–8] In rats, the D3 receptor is expressed at
high density in the granule cells of the islands of Call-
eja and in the olfactory bulb; moderate density is ob-
served in the nucleus accumbens.[9–14] Selective D3 re-
ceptor partial agonists and antagonists have been in-
vestigated as potential drug abuse therapeutic agents,[15,16] as
nonselective dopaminergic ligands appeared to induce un-
wanted side effects.
[a] Dr. C. Hocke, Dr. S. Maschauer, Prof. T. Kuwert, Dr. O. Prante
Friedrich Alexander University Erlangen–Nꢀrnberg
Clinic of Nuclear Medicine, Friedrich Alexander University
Krankenhausstrasse 12, 91054 Erlangen (Germany)
Fax: (+49)9131-85 39262
In the past few years, a variety of structural analogues of D3
receptor ligands have been developed to improve the D3 re-
ceptor affinity, while simultaneously increasing their D3 selec-
tivity over the interfering D2 subtype.[3,17–21] Most of these
studies were based on D3-selective lead compounds such as
E-mail: carsten.hocke@uk-erlangen.de
[b] Dr. H. Hꢀbner, Dr. S. Lçber, Dr. W. Utz, Prof. P. Gmeiner
Department of Chemistry and Pharmacy
Emil Fischer Center, Friedrich Alexander University
Schuhstrasse 19, 91052 Erlangen (Germany)
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C. Hocke et al.
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zothiophene ring system instead of the naphthyl moiety. Nota-
bly, FAUC 365, the 2,3-dichlorophenylpiperazinyl analogue of 2,
was described as a D3 antagonist with a 7200-fold selectivity
over the D2 receptor.[22] Furthermore, the aryl amide functional
group also revealed increased D3 selectivity when extended to
a diaryl ring system as reported for CJB 090 (3a) and 3d, repre-
senting less lipophilic analogues of the first-generation D3 an-
tagonist NGB 2904 (4).[17,25]
D3 receptor subtype selectivity, optimal affinity, and de-
creased lipophilicity are some of the most important prerequi-
sites for a successful D3 radioligand suitable for in vivo imag-
ing applications. Positron emission tomography (PET) has
emerged as a highly sensitive imaging method, which would
allow new insight into the role of the D3 receptor subtype in
the pathophysiology of various psychiatric diseases. However,
the lack of bioavailable D3-selective radioligands for PET, which
exclude cross talk with the strongly related subtypes D2 and
D4, thus far hampers the noninvasive investigation of the D3
receptor by PET.
two 18F-substituted pyridinylphenyl amides (3b,c) as analogues
of the lead compound CJB 090, that displayed high affinities at
the D3 receptor [Ki =0.37 nm (3c) and Ki =0.45 nm (3b)], while
having moderate affinity to D2 [Ki =16 nm (3c) and Ki =12 nm
(3b)].[35] In the present work we extended this series of com-
pounds and evaluated the D3 binding specificity of their 18F-la-
belled versions in vivo. We describe the influence of structural
changes on a focused library of fluorinated pyridinylbenza-
mides that were predicted for adequate D3 selectivities and af-
finities by using CoMFA and CoMSIA methods. In this study,
we changed the substitution pattern of the phenylpiperazinyl
moiety introducing the 2-chloro and 3-chloro-2-methoxy sub-
stitution pattern in the phenylpiperazinyl group and varied the
position of the fluorine substituent at the pyridinyl moiety. Fur-
thermore, the predicted D3 selectivities of the new series of
compounds 9a–d were compared with experimental data de-
rived from in vitro binding studies using dopamine receptor
subtype-expressing Chinese hamster ovary cells (CHO). Herein
we report the syntheses of the radioligands [18F]9a–d, their un-
labelled reference compounds 9a–d and the appropriate
bromo or nitro precursors 10a–d for 18F-fluorination. After se-
lection of promising radioligand candidates ([18F]9a and
[18F]9c), their in vivo binding behaviour was assessed by rat
brain autoradiography.
To overcome this drawback, FAUC 346 (2) has been labelled
with the positron emitters carbon-11 and fluorine-18.[26,27] The
11C-labelled candidate has been studied in vivo and revealed
specific uptake in the rat brain, however, the regional brain dis-
tribution has not yet been determined. Despite the promising
results in the rat model, 11C-labelled FAUC 346 failed as a suita-
ble PET radioligand for imaging of D3 receptors in baboon, as
the intravenous injection of BP 897 did not affect the brain
uptake of the radioligand.[26] Furthermore, the 11C-labelled
imidazo[2,1-b]thiazolylpiperazine derivative RGH 1756 also indi-
cated an insufficiently low signal for specific D3 binding
in vivo, possibly due to the presence of competing endoge-
nous dopamine.[28] Recently, the naphthoxazine [11C](+)-PHNO,
a D2/D3 agonist radioligand, has been discovered to act as a
D3 receptor preferring radioligand in vivo, as indicated by its
specific binding in the globus pallidus, thereby reflecting the
D3 receptor distribution more accurately than the nonselective
D2/D3 radioligand [11C]raclopride.[29–32] However, the 18F-la-
belled derivative of PHNO did not show specific binding in D3-
rich brain areas in vivo.[33] In summary, it is tempting to specu-
late that at least some of the D3 radioligand candidates proba-
bly failed because of their high lipophilicity resulting in signifi-
cant nonspecific binding when employed in vivo. Another
reason for the failure of partial agonist radioligands could be
an insufficient D3 receptor affinity preventing efficient compe-
tition with the endogenous agonist dopamine.
Results and Discussion
Prediction of D3-selective PET tracers
In our previous work, we successfully established CoMFA and
CoMSIA models which allowed us a precise prediction of D3 af-
finity and selectivity over both congeners D2long and D4.[35,36]
On this basis, the selectivities of the proposed structures 9a–d
were calculated. The results are listed in Table 1, indicating pKi
differences expressed as Dlog(Ki(D3)/Ki(D2long)) and Dlog(Ki(D3)/
Ki(D4)) values. Employing the CoMFA and CoMSIA models, the
compounds 9a–c were predicted to have high D3 subtype se-
lectivity, as determined by the deviation of theoretical from ex-
perimental values (Table 1).
Chemistry
Following the synthetic strategy that has been proven to be
efficient and reliable in our previous studies,[18,37] we achieved
Moreover, the clearance of promising D3-selective radioli-
gands, such as [11C](+)-PHNO, in distinct brain regions is rela-
tively slow.[32] Therefore, a radioligand with a longer half-life
than C-11 (20 min) would provide a favourable adjustment of
biokinetics and physical half-life of the tracer. The availability
of a suitable 18F-labelled D3 radioligand with the half-life of
110 min would facilitate prolonged PET scanning times in the
clinical setting and would also provide the option to distribute
the radioligand within longer distances for external clinical ap-
plication.
Table 1. Experimental and predicted selectivities of compounds 9a–d.
[b]
[b]
Compd D3/D2 (exp.)[a]
DD3/D2
CoMFA CoMSIA
D3/D4 (exp.)[a]
DD3/D4
CoMFA CoMSIA
9a
9b
9c
9d
2.32
2.07
2.05
1.68
ꢀ0.02
0.21
2.47
2.56
2.05
2.20
0.39 ꢀ0.39
0.23 ꢀ0.20
0.12 ꢀ0.07
ꢀ0.03 ꢀ0.02
ꢀ0.53 ꢀ0.20
ꢀ0.42 ꢀ0.01
ꢀ0.95 ꢀ0.30
[a] Expressed as pKi differences (see also Table 2). [b] D(D3/D2) and D(D3/
D4) are defined as the difference between experimental and calculated
selectivities; the calculated selectivities are expressed as ꢀlog(Ki(D3)/Ki-
(D2long)) and ꢀlog(Ki(D3)/Ki(D4)), respectively.
Based on our work on SAR studies guiding the development
of D3-selective lead compounds,[3,34,35] we recently reported
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Dopamine D3 Receptor Ligands
the series of target ligands as shown in Scheme 1. Starting
from commercially available 2-chlorophenylpiperazine (6a) and
3-chloro-2-methoxyphenylpiperazine (6b), which was synthes-
ised by nucleophilic displacement of 1,3-dichloro-2-methoxy-
benzene (5) with piperazine,[38] the nitriles 7a,b were obtained
in good yields by alkylation with 4-bromobutyronitrile. Reduc-
tion of the resulting arylpiperazinylbutyronitriles 7a,b with
LiAlH4 proceeded in good yield to provide 4-[4-(2-chlorophe-
nyl)piperazin-1-yl]butyl-1-amine (8a) and 4-[4-(3-chloro-2-me-
thoxyphenyl)piperazin-1-yl]butyl-
pressed in CHO cells and the radioligand [3H]spiperone.[39–42]
Furthermore, affinities to the serotonin receptors 5-HT1A, 5-HT2,
and the adrenergic a1 receptor were determined using por-
cine cortical membranes and the selective radioligands
[3H]WAY 100635, [3H]ketanserin and [3H]prazosin, respective-
ly.[43]
As displayed in Table 2, all test compounds (9a–d) showed
excellent D3 binding with Ki values in the single-digit or even
sub-nanomolar range. Depending on the substitution pattern
1-amine (8b), respectively. Final-
Table 2. Binding affinities of the fluorinated compounds 9a–d to the human dopamine receptor subtypes
D2long, D2short, D3, and D4, the porcine D1, and the porcine 5-HT1A, 5-HT2, and a1 receptors.
ly, the compounds of interest
were prepared by dicyclohexyl-
carbodiimide (DCC) coupling of
the amines 8a, b with the ap-
propriate fluoropyridinylbenzoic
acids, providing the amides 9a–
d in yields of 43–72%. The pre-
cursors 10a–d were prepared
using the same course of synthe-
sis, by coupling of 8a, b with
the appropriate bromo and ni-
tropyridinylbenzoic acids to give
the amides 10a–d in yields of
52–79%.
Compd
Ki [nm][a]
[3H]WAY 100635 [3H]ketanserin [3H]prazosin
[3H]SCH 23390
D1
[3H]spiperone[b]
D2long D2short
D3
D4.4
5-HT1A
5-HT2
a1
9a
9b
9c
9d
270[c]
340
2400
15000[c]
120[c]
61
180
67
42
22
62
32
0.58[d]
0.52[d]
1.6
170
190
180
220
160
140
270
270
230
170
180
93
8.0
4.8
44
21
1.4
[a] Values reflect the means of two or three experiments, each done in triplicate; SD<35%. [b] D2long and
D2short are two isoforms of the D2 receptor. [c] SD>35% and <50%. [d] SEM<20%.
at the phenylpiperazine moiety, the 3-chloro-2-me-
thoxy substituted analogues 9c, d show Ki values of
1.6 and 1.4 nm, respectively, whereas the 2-chloro-
phenylpiperazinyl derivatives 9a, b bind to the D3 re-
ceptor with sub-nanomolar Ki values of 0.58 and
0.52 nm, respectively. In terms of subtype selectivity
expressed as Ki(D3)/Ki(D2long), compounds 9a–c dem-
onstrated high D3 selectivity of 110- to 210-fold over
D2 (Table 2). This observation could be ascribed to
the variation of the substitution pattern at the pyri-
dinyl moiety, revealing an influence on D2 receptor
recognition. The 6-fluoropyridin-2-yl benzamide sub-
stitution in 9a and 9c decreased the affinity to the
D2 receptor by two to threefold, thereby resulting in
an increased D3 subtype selectivity when compared
with their corresponding structural isomers 9b and
9d, respectively (Table 2). These results indicate that
Scheme 1. Synthesis of 9a–d and 10a–d. Reagents and conditions: a) piperazine, tolu-
ene, Pd(OAc)2, (o-biphenyl)P(tBu)2, NaOtBu, 808C, 21 h (see ref. [38]); b) 4-bromobutyroni-
trile, DMF, 1008C, 5 h (see ref. [18]); c) LiAlH4, THF, 08C!reflux, 5 h; d) 4-(6-bromopyridin-
2-yl)benzoic acid or 4-(6-nitropyridin-3-yl)benzoic acid or 4-(6-fluoropyridin-2-yl)benzoic
acid or 4-(6-fluoropyridin-3-yl)benzoic acid, DCC, DMAP, CH2Cl2, room temperature, over-
night.
the structural modifications successfully induced de-
creased D2 affinity and therefore could be used to
obtain ligands with improved D3 subtype selectivity.
Radiochemistry
The introduction of fluorine-18 via nucleophilic aro-
Receptor binding experiments and data analysis
matic substitution was performed by exchanging bromine or a
nitro group at the pyridinyl benzamide site, following the reac-
tion conditions described previously.[35] The radiosyntheses of
[18F]9a–d are shown in Scheme 2. The nitro precursors 10b, d
and the bromo precursors 10a, c were synthesised as depicted
in Scheme 1. The radiofluorination of the nitro and bromo pre-
cursor (8 mmol) was performed at 1408C in DMF as solvent for
a reaction time of 30 min. The radiolabelled product was ana-
Radioligand displacement experiments were conducted to
evaluate the binding profiles of the fluorinated ligands 9a–d.
Receptor binding to the dopamine D1 receptor was measured
using membrane preparations from porcine striatum and the
subtype-selective radioligand [3H]SCH 23390.[39] Binding experi-
ments with the human subtypes D2long, D2short, D3, and D4.4
were performed employing the cloned receptors stably ex-
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C. Hocke et al.
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the octanol phase and in water at neutral pH. The lipophilici-
ties of 9a–d were calculated using the software ClogP
(Table 3). The experimental octanol/buffer partition coefficient
(logD7.4; measured with PBS as aqueous phase, pH 7.4) of
[18F]9a–d were experimentally determined to range between
1.77 and 2.88 (Table 3), suggesting an adequate penetration of
the blood–brain-barrier for further in vivo use. The calculated
logP value (ClogP) and logD7.4 differ significantly, most likely
due to the presence of the partly protonated piperazine at
pH 7.4. Moreover, we proved the stability of [18F]9a against
metabolism by serum enzymes. Incubation in human serum
and HPLC analysis showed the excellent stability of [18F]9a
(Figure 1).
Scheme 2. Nucleophilic aromatic substitution: DMF (500 mL), precursor 10a–
d (8 mmol), 1408C, n.c.a.[18F]fluoride (50–70 MBq), PTC: Kryptofix 2.2.2, K2CO3;
t=30 min.
lysed by gradient reversed-phase radio-HPLC. The radiochemi-
cal identities of [18F]9a–d were verified by co-elution of the ra-
dioactive peak with the respective reference compound 9a–d.
The k’ values of the reference fluoro compounds are listed in
Table 3. The radiochemical yields (RCY) of the 18F-labelled com-
pounds [18F]9a–d ranged between 66 and 92%. No significant
differences in the RCY were identified by varying the substitu-
In vivo experiments using rat brain autoradiography
The regional brain distribution of [18F]9a and [18F]9c, respec-
tively, was determined by ex vivo autoradiography of rat brain
slices after intravenous injection
of the tracer into the tail vein of
Table 3. Radiochemical yields (RCY) using the precursor compounds 10a–d and k’ values of the corresponding
radioligands [18F]9a–d and analytical data for [18F]9a–d.
isofluorane-anaesthetised
rats
alone and in combination with
the D3-selective ligand BP 897
(1 mgkgꢀ1), to test for specific
receptor binding. Rats were sac-
rificed by decapitation 60 min
post-injection. In Figure 2 the
autoradiography and histology
of the rat brain slices of [18F]9a
and [18F]9c are shown. Stan-
wood et al. previously described
Precursor
Leaving group
RCY [%][a]
k’ [min][b]
ClogP[c]
logD7.4
10a
10b
10c
10d
Br
NO2
Br
67ꢁ4, [18F]9a
92ꢁ2, [18F]9b
66ꢁ5, [18F]9c
90ꢁ2, [18F]9d
2.43, [18F]9a
2.28, [18F]9b
2.55, [18F]9c
2.40, [18F]9d
4.60
4.39
4.20
3.99
2.88
1.91
2.45
1.77
NO2
[a] 500 mL DMF, 8 mmol precursor, n.c.a.[18F]fluoride (50–70 MBq), Kryptofix 2.2.2, K2CO3, 30 min, 1408C. [b] k’
values from HPLC System A. [c] ClogP calculated by the software ClogP (BioByte Corp.).
tion pattern of the arylpiperazine. By changing the leaving
the regional distribution of the D3 receptor in the rat brain by
quantitative autoradiography using [125I]7-OH-PIPAT.[46] These
authors showed that D3 receptors in the rat brain are located
group from nitro to bromo, a decreased RCY of more than
25% was observed, most likely attributable to the high Ham-
mett constant of the nitro group,[44] indicating that nitro is a
more suitable leaving group for aromatic substitution than
bromine. However, the separation of the nitro precursors 10b
and 10d from the respective 18F-labelled radioligands, [18F]9b
and [18F]9d, by HPLC turned out to be challenging. As a result
we were unable to obtain [18F]9b and [18F]9d with high specif-
ic activity, rendering the use of [18F]9b and [18F]9d for further
in vivo studies impossible.
As 9a and 9c showed favourable selectivity for the D3 sub-
type and excellent sub-nanomolar D3 affinity, we decided to
perform further in vitro and a comparative in vivo experiment
with the 18F-labelled radioligands [18F]9a and [18F]9c. For these
studies, [18F]9a and [18F]9c were isolated by semipreparative
HPLC and solid-phase extraction methods that allowed formu-
lation of each radioligand in an injectable solution.
Lipophilicity and stability in human serum
The lipophilicity of radioligands plays an important role for ad-
equate brain uptake, the distribution of the tracer, and its me-
tabolism and elimination.[45] As a measure of lipophilicity, the
logP value is defined by the octanol/water partition coefficient,
that is, the ratio of the concentration of the test compounds in
Figure 1. Determination of stability of [18F]9a after incubation in human
serum for 60 min at 378C. HPLC analysis of a) treated [18F]9a in comparison
with that of b) pure [18F]9a (HPLC conditions: see Experimental Section).
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Dopamine D3 Receptor Ligands
tivity for D3 over D2. In vivo studies with [18F]9a and [18F]9c
using rat brain autoradiography did not reveal an intracerebral
distribution of the radioligand in accordance with the known
distribution of the D3 receptor within this species. Besides a
low signal of specific binding in cortical and striatal brain
areas, no specific uptake was observed in the known D3 recep-
tor-rich reference regions. Instead, we observed a significant
accumulation of [18F]9a and [18F]9c within the brain ventricles,
suggesting biological properties for these radioligands such as
binding to interfering receptors or proteins in ventricular com-
partments, that impedes specific binding in D3 receptor-rich
areas for in vivo imaging applications.
Experimental Section
General
Commercial reagents were used as received without additional pu-
rification. [18F]Fluoride was supplied by PET Net GmbH (Erlangen,
Germany). Purifications of experimental compounds were per-
formed by column chromatography using silica gel 60. Radio HPLC
analysis of [18F]9a–d was performed on
a Hewlett Packard
(HP 1100) with a quaternary pump and variable wavelength detec-
tor (HP 1100) and the radiodetector D505TR (Canberra Packard).
Figure 2. Effects of a) [18F]9a and b) [18F]9c. Left columns: haematoxylin/
eosin (HE) staining and rat brain autoradiography obtained from representa-
tive striatal slices and slices containing the brain ventricles (ST=striatum,
CX=cortex, VT=ventricle). Center columns: autoradiography of rat brain
slices 60 min after intravenous injection of [18F]9a (10.4 MBq) or [18F]9c
(7.3 MBq). Right columns: corresponding rat brain slices obtained from ani-
mals co-injected with BP 897 (1 mgkgꢀ1) and [18F]9a (13.9 MBq) or [18F]9c
(6.1 MBq).
System A: (analysis of [18F]9a–d): Chromolith RP18, 100ꢂ4.6 mm
column, eluted with CH3CN/H2O (0.1% TFA), 10:90 (v/v) to 100%
CH3CN (0.1% TFA) over 5 min, flow rate: 4.0 mLminꢀ1
.
System B: (preparative separation of [18F]9a): Kromasil C8, 125ꢂ
8 mm, 5 mm column, eluted with CH3CN/H2O (0.1% TFA), 40:60 (v/
v), flow rate: 4.0 mLminꢀ1
.
1H NMR and 13C NMR spectra were determined on a Bruker AM 360
or a Bruker Avance spectrometer in solution. Proton chemical shifts
(d) are reported in parts per million (ppm) downfield from (CH3)4Si
(0.00 ppm) as an internal standard. Chemical shifts for 13C NMR
spectra are reported as d values relative to the deuterium triplet
signal of the solvent (CDCl3, 77.5 ppm). HR-EIMS were run on a Fin-
nigan MAT TSQ 70 instrument using peak matching (M/DM=
10000).
in the nucleus accumbens, the islands of Calleja, the ventral
pallidum and, to a lower extent, the caudate putamen (stria-
tum). In comparison, our results show that we could detect a
low specific uptake of [18F]9a and [18F]9c in the striatum
(Figure 2), however, a specific signal in other D3 receptor refer-
ence regions was not visible. Besides low striatal binding,
[18F]9a and [18F]9c specifically accumulated in the cortex.
Moreover, our experiments show a significant nonspecific accu-
mulation of both radioligands in the ventricles, the ependyma
cells or plexus tissue (Figure 2). This finding could be due to
rapid and specific transport of [18F]9a and [18F]9c into these
tissue compartments, most likely mediated by transporters,
such as polyamine transporters, that could be capable of spe-
cifically recognising the diaryl amides under investigation. Fur-
ther studies are necessary to characterise this phenomenon in
more detail.
Chemistry
In previous studies, we have described the synthesis and analytical
data of biarylcarboxylic acids using Suzuki coupling, starting from
4-carboxybenzene boronic acid with the corresponding bromo-
substituted pyridine derivatives, in 43–67% yields.[35] The synthesis
and analytical data of 4-[4-(2-chlorophenyl)piperazin-1-yl]butyl-1-
amine (8a) and 4-[4-(3-chloro-2-methoxyphenyl)piperazin-1-yl)bu-
tyl-1-amine (8b) have been described previously.[43,47] In brief, the
N-phenylpiperazines 6a, b were converted into the alkylnitrile de-
rivatives 7a,b by alkylation with 4-bromobutyronitrile and subse-
quent reduction with LiAlH4 in THF affording the primary amines
8a,b in very good yield.
Conclusions
Compounds 9a–d were identified as a new series of fluoro-
substituted diaryl amides derived from 3b,c with substantial
D3 receptor affinity. The SAR established the importance of the
arylpiperazine moiety. Changing the substitution pattern on
the arylpiperazine did not influence the binding affinity to the
D3 receptor, whereas it decreased the affinity for D2 receptors.
Compounds 9a–c had a favourable in vitro binding profile in-
dicated by high affinity to the D3 receptor and superior selec-
General procedure for synthesis of compounds 9a–d and
10a–d
N,N-dimethylpyridine-4-amine (DMAP, 1.3 equiv), dicyclohexylcarbo-
diimide (DCC, 1.3 equiv), and the corresponding carboxylic acids
(1 equiv) were added to a solution of 4-[4-(2-chlorophenyl)pipera-
zin-1-yl]butan-1-amine (8a, 1 equiv) or 4-[4-(3-chloro-2-methoxy-
ChemMedChem 2010, 5, 941 – 948
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MED
phenyl)piperazin-1-yl]butyl-1-amine (8b, 1 equiv) in dry CH2Cl2
(15 mL) under Ar atmosphere. The mixture was stirred at room
temperature overnight. After filtration of the precipitate, the re-
maining solution was washed with H2O (2ꢂ20 mL) and brine
(20 mL). The organic layer was separated, dried over Na2SO4, and
evaporated under reduced pressure to get a residue of crude prod-
uct. Subsequent column chromatography (CH2Cl2/MeOH; 9:1) gave
each compound 9a–d, 10a–d in yields of 43–79%.
J=8.4 Hz, 2H), 7.88 (d, J=8.4 Hz, 2H), 7.92–7.98 (m, 1H), 8.41 ppm
(d, J=2.5 Hz, 1H); 13C NMR (CDCl3) d=24.36, 27.41, 39.99, 49.98,
53.67, 58.03, 58.95, 109.51, 109.75, 116.93, 123.30, 124.56, 127.08,
127.83, 128.73, 134.62, 139.46, 139.70, 139.75, 145.85, 145.95,
146.45, 148.60, 162.59, 167.06 ppm; HRMS calcd for C27H30ClFN4O2
[M+]: 496.2041, found: 496.2042.
N-[4-[4-(2-Chlorophenyl)piperazin-1-yl]butyl]-4-(6-bromopyridin-
2-yl)-benzamide (10a). Compound 10a was prepared from 4-[4-
(2-chlorophenyl)piperazin-1-yl]butyl-1-amine (8a) and 4-(6-bromo-
pyridin-2-yl)benzoic acid according to the general procedure. Yield:
N-[4-[4-(2-Chlorophenyl)piperazin-1-yl]butyl]-4-(6-fluoropyridin-
2-yl)benzamide (9a). Compound 9a was prepared by coupling of
4-[4-(2-chlorophenyl)piperazin-1-yl]butyl-1-amine (8a) and 4-(6-flu-
oropyridin-2-yl)benzoic acid according to the general procedure.
Yield: 72%; 1H NMR (360 MHz, CDCl3): d=1.49–1.55 (m, 2H), 1.59
(td, J=13.7, 6.7 Hz, 2H), 2.27–2.37 (m, 2H), 2.49 (s, 4H), 2.85 (d, J=
57.53 Hz, 4H), 3.37 (dd, J=12.4, 6.7 Hz, 2H), 6.76 (dd, J=8.1,
2.7 Hz, 1H), 6.79–6.87 (m, 2H), 7.01–7.07 (m, 1H), 7.21 (dd, J=7.9,
1.5 Hz, 1H), 7.26 (q, J=5.6 Hz, 1H), 7.49 (dd, J=7.5, 2.1 Hz, 1H),
7.67–7.73 (m, 1H), 7.75–7.79 (m, 2H), 7.87–7.93 ppm (m, 2H);
13C NMR (CDCl3): d=24.24, 27.28, 39.86, 50.73, 53.08, 57.86, 108.09,
108.34, 117.49, 120.14, 123.47, 126.78, 127.35, 128.47, 130.38,
135.63, 139.76, 141.66, 141.71, 148.94, 154.70, 154.79, 162.37,
163.95, 167.13 ppm; HRMS calcd for C26H28ClFN4O [M+]: 466.1936,
found: 466.1936.
1
79%: H NMR (600 MHz, CDCl3): d=1.57–1.74 (m, 4H), 2.43 (t, J=
6.9 Hz, 2H), 2.60 (s, 4H), 3.01 (s, 4H), 3.40–3.52 (m, 2H), 6.89–6.97
(m, 2H), 7.12–7.19 (m, 1H), 7.29–7.35 (m, 2H), 7.37–7.42 (m, 1H),
7.52–7.58 (m, 1H), 7.63 (dd, J=7.7, 0.9 Hz, 1H), 7.82–7.88 (m, 2H),
7.95–8.01 ppm (m, 2H); 13C NMR (CDCl3): d=24.13, 27.22, 39.84,
50.74, 53.09, 57.79, 119.08, 120.13, 123.45, 126.73, 126.74, 127.38,
127.39, 128.42, 130.34, 135.58, 138.95, 139.78, 142.00, 148.87,
156.95, 167.15 ppm; HRMS calcd for C26H28BrClN4O [M+]: 526.1135,
found: 526.1135.
N-[4-[4-(2-Chlorophenyl)piperazin-1-yl]butyl]-4-(6-nitropyridin-3-
yl)-benzamide (10b). Compound 10b was prepared from 4-[4-(2-
chlorophenyl)piperazin-1-yl]butyl-1-amine (8a) and 4-(6-nitropyri-
din-3-yl)benzoic acid according to the general procedure. Yield:
N-[4-[4-(2-Chlorophenyl)piperazin-1-yl]butyl]-4-(6-fluoropyridin-
3-yl)benzamide (9b). Compound 9b was prepared from 4-[4-(2-
chlorophenyl)piperazin-1-yl]butyl-1-amine (8a) and 4-(6-fluoropyri-
din-3-yl)benzoic acid according to the general procedure. Yield:
1
54%: H NMR (360 MHz, CDCl3): d=1.61–1.79 (m, 4H), 2.50 (t, J=
6.9 Hz, 2H), 2.65 (s, 4H), 3.04 (s, 4H), 3.46–3.58 (m, 2H), 6.91–6.99
(m, 2H), 7.07 (t, J=5.4 Hz, 1H), 7.13–7.21 (m, 1H), 7.28–7.36 (m,
1H), 7.66–7.72 (m, 2H), 7.93–7.99 (m, 2H), 8.16–8.22 (m, 1H), 8.33
(td, J=3.0, 1.5 Hz, 1H), 8.79–8.83 ppm (m, 1H); 13C NMR (CDCl3):
d=24.27, 27.34, 40.02, 50.86, 53.23, 57.89, 118.14, 120.12, 123.63,
127.45, 127.51, 128.12, 128.60, 130.53, 135.97, 137.82, 137.84,
141.18, 147.04, 148.96, 155.84, 166.62 ppm; HRMS calcd for
C26H28ClN5O4 [M+]: 493.1881, found: 493.1881.
1
47%; H NMR (360 MHz, CDCl3): d=1.67 (ddd, J=10.5, 9.2, 4.1 Hz,
2H), 1.70–1.76 (m, 2H), 2.48 (t, J=7.1 Hz, 2H), 2.64 (s, 4H), 3.03 (s,
4H), 3.51 (dd, J=12.3, 6.6 Hz, 2H), 6.95 (dd, J=3.0, 1.6 Hz, 1H),
6.96 (dd, J=3.6, 1.4 Hz, 1H), 7.01 (dd, J=8.5, 2.5 Hz, 1H), 7.17
(ddd, J=8.0, 7.4, 1.5 Hz, 2H), 7.31–7.34 (m, 1H), 7.54–7.58 (m, 2H),
7.88–7.92 (m, 2H), 7.95 (ddd, J=8.4, 7.6, 2.6 Hz, 1H), 8.39 ppm (d,
J=2.62 Hz, 1H); 13C NMR (CDCl3): d=24.28, 27.34, 39.94, 50.84,
53.19, 57.92, 109.42, 109.67, 120.12, 123.59, 126.92, 127.42, 127.82,
128.59, 130.49, 133.65, 134.57, 139.25, 139.61, 139.66, 145.72,
145.81, 148.95, 162.46, 164.06, 166.98 ppm; HRMS calcd for
C26H28ClFN4O [M+]: 466.1936, found: 466.1936.
N-[4-[4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl]butyl]-4-(6-
bromopyridin-2-yl)-benzamide (10c). Compound 10c was pre-
pared from 4-[4-(3-chloro-2-methoxyphenyl)piperazin-1-yl]butyl-1-
amine (8b) and 4-(6-bromopyridin-2-yl)benzoic acid according to
1
the general procedure. Yield: 65%: H NMR (360 MHz, CDCl3): d=
1.58–1.77 (m, 4H), 2.45 (t, J=6.9 Hz, 2H), 2.59 (s, 4H), 3.08 (s, 4H),
3.40–3.54 (m, 2H), 3.84 (t sext., J=6.6, 5.0 Hz, 3H), 6.74 (dd, J=8.0,
1.6 Hz, 1H), 6.91 (t, J=8.0 Hz, 1H), 6.95–7.00 (m, 1H), 7.09 (t, J=
5.4 Hz, 1H), 7.43 (dd, J=7.7, 0.9 Hz, 1H), 7.59 (t, J=7.7 Hz, 1H),
7.67 (dd, J=7.7, 0.9 Hz, 1H), 7.81–7.89 (m, 2H), 7.98–8.05 ppm (m,
2H); 13C NMR (CDCl3): d=24.26, 27.32, 39.93, 49.91, 53.56, 57.94,
58.86, 116.97, 119.18, 123.12, 124.51, 126.87, 126.90, 127.42, 128.54,
135.71, 139.02, 139.99, 142.15, 146.39, 148.50, 157.12, 167.21 ppm;
HRMS calcd for C27H30BrClN4O2 [M+]: 556.1241, found: 556.1240.
N-[4-[4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl]butyl]-4-(6-
fluoropyridin-2-yl)benzamide (9c). Compound 9c was prepared
from 4-[4-(3-chloro-2-methoxyphenyl)piperazin-1-yl]butyl-1-amine
(8b) and 4-(6-fluoropyridin-2-yl)benzoic acid according to the gen-
1
eral procedure. Yield: 69%; H NMR (360 MHz, CDCl3): d=1.62–1.68
(m, 2H), 1.69–1.74 (m, 2H), 2.45 (dd, J=16.3, 9.3 Hz, 2H), 2.58 (s,
4H), 3.08 (s, 4H), 3.50 (dd, J=12.3, 6.7 Hz, 2H), 3.82–3.86 (m, 3H),
6.70–6.76 (m, 1H), 6.87–6.93 (m, 2H), 6.97 (dt, J=8.0, 2.1 Hz, 1H),
7.03 (t, J=5.4 Hz, 1H), 7.62 (dd, J=7.5, 2.1 Hz, 1H), 7.83–7.87 (m,
3H), 8.02–8.03 (m, 1H), 8.03–8.05 ppm (m, 1H); 13C NMR (CDCl3):
d=24.41, 27.39, 40.00, 50.00, 53.64, 58.02, 58.89, 108.26, 108.51,
117.00, 117.59, 123.15, 124.53, 126.92, 127.44, 128.60, 135.75,
141.74, 141.79, 146.47, 148.55, 154.87, 154.96, 162.53, 164.11,
167.19 ppm; HRMS calcd for C27H30ClFN4O2 [M+]: 496.2041, found:
496.2041.
N-[4-[4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl]butyl]-4-(6-ni-
tropyridin-3-yl)-benzamide (10d). Compound 10d was prepared
from 4-[4-(3-chloro-2-methoxyphenyl)piperazin-1-yl]butyl-1-amine
(8b) and 4-(6-nitropyridin-3-yl)benzoic acid according to the gener-
al procedure. Yield: 52%: 1H NMR (360 MHz, CDCl3): d=1.59–1.81
(m, 4H), 2.48 (t, J=6.8 Hz, 2H), 2.62 (s, 4H), 3.10 (s, 4H), 3.53 (q,
J=6.4 Hz, 2H), 3.84 (d, J=4.6 Hz, 3H), 6.74 (dd, J=8.0, 1.6 Hz, 1H),
6.87–7.02 (m, 3H), 7.65–7.73 (m, 2H), 7.90–7.98 (m, 2H), 8.18 (dd,
J=8.4, 2.4 Hz, 1H), 8.35 (dd, J=8.4, 0.7 Hz, 1H), 8.83 ppm (dd, J=
2.3, 0.7 Hz, 1H); 13C NMR (CDCl3): d=24.32, 27.36, 40.04, 49.98,
53.66, 57.97, 58.94, 116.88, 118.19, 123.29, 124.54, 127.60, 128.14,
128.72, 136.00, 137.90, 137.94, 141.24, 146.41, 147.12, 148.59,
155.93, 166.74 ppm; HRMS calcd for C27H30ClN5O4 [M+]: 523.1986,
found: 523.1986.
N-[4-[4-(3-Chloro-2-methoxyphenyl)piperazin-1-yl]butyl]-4-(6-
fluoropyridin-3-yl)benzamide (9d). Compound 9d was prepared
from 4-[4-(3-chloro-2-methoxyphenyl)piperazin-1-yl]butyl-1-amine
(8b) and 4-(6-fluoropyridin-3-yl)benzoic acid according to the gen-
1
eral procedure. Yield: 43%; H NMR (360 MHz, CDCl3) d=1.64–1.76
(m, 4H), 2.43–2.51 (m, 2H), 2.62 (s, 4H), 3.10 (s, 4H), 3.52 (dd, J=
12.2, 6.5 Hz, 2H), 3.83 (d, J=20.0 Hz, 3H), 6.74 (dd, J=8.1, 1.4 Hz,
1H), 6.87–6.94 (m, 2H), 7.01 (ddd, J=9.5, 8.3, 2.14 Hz, 2H), 7.58 (d,
946
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 941 – 948

Dopamine D3 Receptor Ligands
cold hexane (ꢀ508C). Brain slices (20 mm) were prepared on a cryo-
stat microtome (HM550, Microm) and thaw-mounted on glass
slides (Histobond). The slides were placed on a phosphor imaging
screen (Kodak K-screen, Biorad) and measured overnight. The
screen was analysed by a Fluor-S MultiImager using the software
Quantity One (version 4.6.3; Bio-Rad Laboratories). For histological
analyses, the slices were counter-stained with haematoxylin and
eosin (HE) following standard procedures. Histological images were
obtained by a digital camera equipped to an Olympus light micro-
scope.
Radiochemistry
No-carrier-added aqueous [18F]fluoride ion was produced in a RDS
111 cyclotron (PET Net GmbH, Erlangen, Germany) by irradiation of
enriched [18O]H2O via the 18O(p,n)18F nuclear reaction. The
[18F]fluoride ions were transferred on a QMA cartridge (Waters). Elu-
tion of [18F]fluoride from the cartridge was performed using an
aqueous solution of Kryptofix 2.2.2 (10 mg) and potassium carbon-
ate stock solution (13 mL, 1m) in CH3CN/H2O (4:1). The solvent was
evaporated under a stream of Ar at 808C and the azeotropic
drying step was repeated twice using 500 mL CH3CN. The syntheses
of [18F]9a–d were performed by nucleophilic fluorination under the
reaction conditions indicated in Table 3, following the previously
described experiments.[35]
Acknowledgements
Preparation and isolation of [18F]9a and [18F]9c: After synthesis of
[18F]9a or [18F]9c via nucleophilic fluorination of 10a or 10c with
[18F]fluoride, the reaction mixtures were cooled and then diluted
with 20 mL H2O. The resulting suspensions were transferred on a
RP18 cartridge (300 mg, Sep-Pak, Waters). After drying, the car-
tridge was eluted with CH3CN (2.5 mL). For isolation of [18F]9a or
[18F]9c, this solution was fractionated by semipreparative HPLC
(System B). The product fractions were diluted with H2O (20 mL),
concentrated by solid-phase extraction (Lichrosorb-RP18 cartridge,
100 mg, Merck), and eluted from the cartridge with EtOH (1 mL).
After evaporation of the solvent, [18F]9a or [18F]9c were redissolved
as appropriate for further experimental use.
We gratefully acknowledge Mrs. Bianca Weigel for skillful techni-
cal assistance and Mr. Wilhelm Hamkens at PET Net GmbH for
producing and delivering [18F]fluoride (RDS 111/CTI, PET Net
GmbH, Erlangen). This work was supported by a grant from the
Deutsche Forschungsgemeinschaft (DFG, PR 677/2-3).
Keywords: autoradiography · D3 receptors · dopamine ·
positron emission tomography · QSAR
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Received: February 16, 2010
Revised: March 15, 2010
Published online on April 20, 2010
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ChemMedChem 2010, 5, 941 – 948