Synthesis and evaluation of 18F-labeled dopamine D3 receptor ligands as potential PET imaging agents

Article abstract of DOI:10.1016/j.bmcl.2005.07.037

A series of fluoro substituted aryl carboxamides was synthesized revealing high affinity for the dopamine D3 receptor. In contrast to 2-methoxy substitution, a 2,3-dichloro substitution pattern at the phenylpiperazine moiety induces a 10-fold increase of

Full text of DOI:10.1016/j.bmcl.2005.07.037

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4819–4823
18
Synthesis and evaluation of F-labeled dopamine D3
receptor ligands as potential PET imaging agents
Carsten Hocke,a,* Olaf Prante,a Stefan Lo¨ber,b Harald Hubner,b
¨
Peter Gmeinerb and Torsten Kuwerta
aDepartment of Nuclear Medicine, Krankenhausstrasse 12, D-91054 Erlangen, Germany
bDepartment of Medicinal Chemistry, Emil Fischer Center, Schuhstrasse 19, D-91052 Erlangen,
Friedrich-Alexander University, Germany
Received 11 April 2005; revised 6 July 2005; accepted 14 July 2005
Available online 31 August 2005
Abstract—A series of fluoro substituted aryl carboxamides was synthesized revealing high affinity for the dopamine D3 receptor. In
contrast to 2-methoxy substitution, a 2,3-dichloro substitution pattern at the phenylpiperazine moiety induces a 10-fold increase of
D3 affinity which is expressed by Ki values of 0.53, 1.1, and 9.0 nM for 8b, 8d, and 8f. Applying aromatic 18F-for-Br(Cl) substitution,
high radiochemical yields between 76–82% were obtained for [18F]8c–f. The most promising ligand, [18F]8d, was used as imaging
agent of the D3 receptor in vitro. However, due to the lack of specific binding, further studies should aim at the development of
radioligands with improved D3 receptor selectivity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The dopamine D2-like receptors are involved in numer-
ous physiological processes and are supposed to be key
players in disorders such as schizophrenia, ParkinsonÕs
disease, and cocaine addiction.1–3 This is also illustrated
by the high affinity of antipsychotic drugs, such as hal-
operidol (1), to the D2-like receptors (Fig. 1).
partial agonist with a D2/D3 suptype selectivity of 66-
fold.2
As part of our drug discovery and SAR investigations
on selective dopamine D3 receptor ligands, we devel-
oped the superpotent benzothiophene derivate FAUC
365 (3), displaying a neutral antagonistic behavior and
a 7200-fold selectivity over the D2 subtype.15 Further-
more, we synthesized radioiodinated derivatives of
FAUC365 for non-invasive single-photon emission
tomography (SPET).16 The development of specific
and potent radioligands as positron emission tomogra-
phy (PET) tracers for D3 receptors is an important step
to investigate the role of this receptor subtype in the
pathophysiology of numerous diseases.
The D2-like receptor family comprises of D2, D3, and
D4 receptors. The dopamine D3 receptor was identified
and cloned by Sokoloff et al.,4 and is mainly found in the
mesocorticolimbic system, whereas the D2 subtype is
accumulated in the striatum.5,6 The physiological role
of the D3 receptor is as yet unclear. However, the loca-
tion of this receptor subtype in brain regions implicated
in emotion and cognition makes it an attractive candi-
date for research aimed at elucidating the pathogenesis
of the above-mentioned psychiatric diseases.7–9 To vali-
date this hypothesis, the synthesis of potent and selective
D3 receptor ligands represents an important goal.
Recently, various series of arylpiperazines with high
affinity and selectivity for the D3 receptor were charac-
terized.10–14 These include BP 897 (2), which acts as a
Based on the results of Murray et al.,17 we chose the 4-
bromophenyl carboxamide 4 as an interesting lead com-
pound for the development of 18F-labeled PET tracers.
Recently, this potent D3 receptor ligand (pKi 9.3) was
radioiodinated to allow its application as a SPET
tracer.18
This paper reports the synthesis and in vitro evaluation
of a series of fluoro substituted analogs of 4, represent-
ing our target compounds for the radiosynthesis of 18F-
labeled tracers potentially suitable for PET imaging.
Herein, we prepared a series of respective haloaryl
Keywords: Dopamine; D3 receptor; Radioligand; PET.
*
Corresponding author. Tel.: +49 9131 85 33411; fax: +49 9131 85
39262; e-mail: Carsten.Hocke@nuklear.imed.uni-erlangen.de
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.037

4820
C. Hocke et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4819–4823
OCH3
F
N
O
N
N
Cl
N
O
H
OH
(2) BP 897
Cl
(1) Haloperidol
Cl
N
OCH3
N
O
O
N
N
N
H
N
H
S
Br
(3) FAUC 365
(4)
Figure 1. Potent dopamine D3 receptor ligands.
carboxamides and assessed their suitability in 18F-for-Br
and 18F-for-Cl nucleophilic substitution.
acetonitrile) were examined, but the best results were ob-
tained employing the reaction conditions described in
Scheme 2. The RCYs of radiofluorinated nicotinamides
([18F]8c,d) and picolinamides ([18F]8e,f) were about 76–
82%. Significant differences in radiochemical yields
between both heteroaromatic systems could not be ob-
served. The RCYs were not significantly influenced by
the substitution pattern (2-methoxy or 2,3-dichloro) of
the N-phenylpiperazinyl moiety of the molecules. The
nucleophilic aromatic substitution with (n.c.a.)
[18F]fluoride requires aromatic activation in ortho- or
para position. This is usually realized by electronic with-
drawal groups, such as carboxamides and sulfonamides
in para position or ortho halogen-substituted pyri-
dines.22 The radiolabeled compounds ([18F]8c-f) could
be obtained in sufficiently high yield to investigate the
tracer behavior in vivo and in vitro for further studies.
In strong contrast to these results, apparently low or
negligible RCYs of the benzamide and benzenesulfona-
mide derivatives [18F]8a,b and [18F]8g,h, respectively,
were obtained.
The syntheses of the series of compounds are shown in
Scheme 1. The commercially available N-phenylpipera-
zines 5a,b were converted to the respective aminobutyl
derivatives by alkylation with 4-bromobutyronitrile
and subsequent reduction with LiAlH4 in THF affording
the primary amines 6a,b in more than 60% overall yield.
Acylation of 6a,b with 4-bromobenzoyl chloride, 6-chlo-
ronicotinoyl chloride, 6-bromopicolinic acid chloride, or
4-bromobenzenesulfonyl chloride in CH2Cl2 in the pres-
ence of triethylamine afforded the respective amides 7a–
h (53–82% yield) bearing leaving groups (chloride or
bromide) for the nucleophilic substitution with [18F]fluo-
ride.19 Condensation of the amines 6a,b with the
appropriate fluorobenzoic acid chlorides gave the corre-
sponding amides 8a–h in yields of 43–64%.20 These com-
pounds were subjected, after identification21, to receptor
binding studies, as described below to determine the
pharmacological potency of the aspired radiolabeled
analogs.
Radioligand binding assays were employed to investi-
gate the affinity and selectivity of the target compounds
8a–h to the different subtypes of dopamine receptors and
to the related biogenic amine receptors 5-HT1A, 5-HT2,
and a1. Binding affinities at the human dopamine
The radiosynthetic approach and results of radiochemi-
cal yields (RCYs) for the aromatic substitution of 7a–h
with no-carrier-added (n.c.a.) [18F]fluoride are given in
Table 1 and Scheme 2. Alternative solvents (DMF or
Scheme 1. Reagents and conditions: (i) 4-bromobutyronitrile, DMF, 100 ꢁC, 5 h16; (ii) LiAlH4, THF, 0 ꢁC—reflux, 5 h16; (iii) acid chlorides A–H,
CH2Cl2, NEt3, rt (43–82%).

C. Hocke et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4819–4823
4821
Table 1. Radiochemical yields (RCY) [%] of the radiofluorinated compounds [18F]8a–h (500 lL DMSO, 140 ꢁC, n.c.a. [18F]fluoride (20-100 MBq),
Kryptofixꢂ2.2.2. K2CO3, t = 20 min)
Compound
R
R0
Amide
Ar
Leaving group
RCY [%] of [18F]8a–h
LogPa of 8a–h
8a
8b
8c
8d
8e
8f
CH3O
Cl
H
Carboxamide
Carboxamide
Carboxamide
Carboxamide
Carboxamide
Carboxamide
Sulfonamide
Sulfonamide
4-Fluorophenyl
4-Fluorophenyl
Br
Br
Cl
Cl
Br
Br
Br
Br
2
3
2
2
5
4
6
5
3.63
5.27
2.76
4.39
3.11
4.74
3.79
5.43
Cl
H
CH3O
Cl
6-Fluoropyridin-3-yl
6-Fluoropyridin-3-yl
6-Fluoropyridin-2-yl
6-Fluoropyridin-2-yl
4-Fluorophenyl
81
82
76
78
Cl
H
CH3O
Cl
Cl
H
8g
8h
CH3O
Cl
0
Cl
4-Fluorophenyl
0
a Calculated value using the program ClogP; logP of the reference FAUC365 was 5.34.
R'
R'
R
N
R
N
i
N
N
N
N
3
3
H
H
O
O
O
O
18F
Br
Cl
18F
N
N
18F] 8a: R = MeO, R' = H
18F] 8b: R = Cl, R' = Cl
[
[
18F] 8c: R = MeO, R' = H
18F] 8d: R = Cl, R' = Cl
7a: R = MeO, R' = H
7b: R = Cl, R' = Cl
7c: R = MeO, R' = H
7d: R = Cl, R' = Cl
[
[
O
O
O
S
O
O
S
O
18F
Br
N
N
18F
Br
18F] 8e: R = MeO, R' = H
18F] 8f: R = Cl, R' = Cl
[
[
18F] 8g: R = MeO, R' = H
18F] 8h: R = Cl, R' = Cl
[
[
7e: R = MeO, R' = H
7f: R = Cl, R' = Cl
7g: R = MeO, R' = H
7h: R = Cl, R' = Cl
Scheme 2. Radiosyntheses of [18F]8a–h starting from the precursors 7a–h; reagents and conditions: (i) n.c.a. [18F]fluoride, phase transfer catalyst
(Kryptofixꢂ2.2.2.), K2CO3, DMSO, 140 ꢁC, 20 min.
23
receptor subtypes D2long, D2short
,
D3,24 and D4.425
D4 with the factor of 10 when the selectivity ratios rise
from 7.9 to 83 (8a,b), 9.2 to 91 (8c,d), and from 0.88
to 11 (8e,f). However, all the developed fluorinated com-
pounds showed a lesser D3 selectivity as reference com-
pound FAUC365.
were measured using membranes of CHO cells stably
expressing these receptors and the radioligand [3H]spip-
erone, as described previously.26 D1 receptor affinities
were determined utilizing porcine striatal membranes
and the D1 selective radioligand [3H]SCH 23390.26
Binding properties to the serotoninergic receptors 5-
HT1A and 5-HT2, and to the adrenergic a1 receptor
were evaluated utilizing porcine cortical membranes
and the selective radioligands [3H]8-OH-DPAT, [3H]ke-
tanserin, and [3H]prazosin, respectively. The results of
the binding experiments listed in Table 2 reveal only
weak affinity to the D1 receptor and a preferred binding
to the receptors of the D2 family. While the benzamide
derivatives 8a–f show a clear binding preference to the
D3 receptor in a low nanomolar range, high affinities
to the D4 receptor were determined for both benzensulf-
onamides 8g and 8h with Ki values of 9.9 and 17 nM,
respectively. A privileged aromatic scaffold is the para
substituted benzamide substructure of the compounds
8a,b and the aza analoges of 8c,d recognizing the D3
receptor in low nanomolar or even subnanomolar con-
centrations (Ki of 8b27 for D3 = 0.53 nM). Looking at
the influence of the phenylpiperazine moiety, the 2,3-
dichloro substitution, when compared to the 2-methoxy
derivatives, induces a 10-fold increase in D3 affinity,
which is expressed by Ki values of 0.53, 1.1, and
9.0 nM for 8b, 8d, and 8f and 4.3, 14, and 86 nM for
the appropriate 2-methoxy derivatives 8a, 8c, and 8e,
respectively. Additionally, the 2,3-dichloro substitution
pattern also improves selectivity of D3 binding against
All test compounds showed good affinity (15–120 nM)
to the serotonin receptor 5-HT1A, but less binding to
the 5-HT2 subtype (84–3700 nM) when the affinity to
this receptor was strongly inferred by substituents of
the phenylpiperazine moiety. The 2-methoxy derivatives
show Ki values only in the micromolar range, whereas
the 2,3-dichloro substitution induces an improved bind-
ing, indicated by an increase of affinity up to 45-fold (for
8c/8d). Interestingly, for all compounds binding affinities
to the a1 receptor were determined with low nanomolar
Ki values when the benzenesulfonamide 8g showed best
binding with 3.9 nM.
The most promising radioligand [18F]8d was used for
preliminary in vitro studies. [18F]8d is structurally relat-
ed to 8b and also revealed a similar D3 receptor affinity
(ca. 1 nM), but significantly higher RCYs were obtained
in the case of [18F]8d, so this radioligand was chosen for
initial receptor autoradiography studies on rat brain
slices. D3 receptor binding was conducted as described
in the literature.28 The results of this initial study could
not reveal a typical D3 receptor distribution, such as in-
creased binding in the brain area of the nucleus accum-
bens. The rat brain slices incubated with [18F]8d showed
a homogeneous distribution and high nonspecific

4822
C. Hocke et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4819–4823
binding, indicating that this ligand may not be a useful
candidate for PET imaging studies. This result is also
in accordance with a recently published report of Tu
et al. describing a C-11-labeled benzamide.29
In summary, we reported the syntheses, binding affini-
ties, 18F-radiosyntheses, and in vitro studies of selective
dopamine D3 receptor ligands. The compounds 8b and
8d were characterized with good affinities to the D3
receptor. Applying aromatic 18F-for-Br(Cl) substitution,
the obtained radiochemical yields of [18F]8c–f were suf-
ficiently high. The most promising nicotinamide deriva-
tive [18F]8d was characterized by an initial in vitro study
indicating undesirable pharmacologic properties. Thus,
future efforts in our laboratory aim at the development
of radioligands with improved D3 receptor selectivity.
Acknowledgments
We thank Dr. J. -C. Schwartz and Dr. P. Sokoloff (IN-
SERM, Paris), Dr. H. H. M. Van Tol (Clarke Institute
of Psychiatry, Toronto), and Dr. J. Shine (The Garvan
Institute of Medical Research, Sydney) for providing
D3, D4.4, and D2 receptor expressing cell lines. Special
thanks are due to Mrs. S. Pachaly for skillful, technical
assistance. We are grateful to Dr. E. Hess and Mr. W.
Sporenberg for providing technical supplies. Last but
not least, we would like to express our gratitude to
Mr. W. Hamkens and the cyclotron staff (Pet Net
GmbH, Erlangen) for the preparation of no-carrier-add-
ed [18F]fluoride. This work was supported by the Deut-
sche Forschungsgemeinschaft (DFG, PR 677/2-1) and
Amersham Healthcare.
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21. 1H NMR (DMSO-d6, 300,18 MHz), 19F NMR (DMSO-
d6, 282,41 MHz), 8a: 1H NMR d (ppm): 1.52 (m, 4H), 2.36
(m, 2H), 2.49 (m, 4H), 2.94 (m, 4H), 3.25 (m, 2H), 3.74 (s,
3H), 6.82–6.87 (m, 4H), 7.2–7.25 (m, 2H), 7.83–7.88 (dd,
2H), 8.37 (t, 1H), 19F NMR d (ppm): ꢀ110.423, 8b: 1H
NMR d (ppm): 1.53 (m, 4H), 2.36 (m, 2H), 2.52 (m, 4H),
2.96 (m, 4H), 3.26 (m, 2H), 7.09 (dd, 1H), 7.23–7.26 (m,
4H), 7.84–7.89 (dd, 2H), 8.37 (t, 1H), 19F NMR d (ppm):
ꢀ110.428, 8c: 1H NMR d (ppm): 1.7 (m, 4H), 2.5 (m, 2H),
2.65 (m, 4H), 3.05 (m, 4H), 3.5 (m, 2H), 3.85 (s, 3H), 6.8–
6.9 (m, 3H), 7.0 (dd, 2H), 7.1 (t, 1H), 8.2 (t, 1H) 8.6 (s,
1H), 8d: 1H NMR d (ppm): 1.7 (m, 4H), 2.5 (m, 2H), 2.55–
2.7 (m, 4H), 2.95–3.1 (m, 4H), 3.5 (m, 2H), 6.9 (m, 2H), 7.0
(m, 1H), 7.15 (dd, 2H), 8.2 (t, 1H), 8.6 (s, 1H), 8e: 1H
NMR d (ppm): 1.48 (m, 2H), 1.54 (m, 2H), 2.34 (m, 2H),
2.48 (m, 4H), 2.94 (m, 4H), 3.29 (dd, 2H), 3.74 (s, 3H),
6.82–6.87 (m, 4H), 7.35 (m, 1H), 7.92 (m, 1H), 8.12 (dd,
1H), 8.6 (t, 1H). 19F NMR d (ppm): ꢀ68.636, 8f: 1H NMR
d (ppm): 1.48 (m, 2H), 1.56 (m, 2H), 2.37 (m, 2H), 2.53 (m,
4H), 2.97 (m, 4H), 3.29 (dd, 2H), 7.09 (m, 1H), 7.23 (m,
2H), 7.36 (m, 1H), 7.93 (m, 1H), 8.12 (dd, 1H), 8.6 (t, 1H),
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19. General procedure for nucleophilic substitution with n.c.a.
[18F]fluoride on aromatics: [18F]Fluoride was produced by
the 18O(p,n)18F reaction using a RDS 111 cyclotron (CTI)
at PET Net GmbH (Erlangen, Germany). A QMA-
cardridge with [18F]fluoride (20–100 MBq) was eluted
with a solution of 15 mg Kryptofix 2.2.2.ꢂ/15 lL of 1 N
potassium carbonate stock solution in 1 mL acetonitrile/
water (8:2). The solvent was evaporated under a stream of
argon at 80 ꢁC and the azeotropic drying step was
repeated two times using 500 lL acetonitrile. The dry
residue was resolubilized with a solution of 10–20 lmol
precursor 7a–e in 500 lL dry DMSO. The solution was
heated to 140 ꢁC for 20 min. Samples of the solution
(25 lL) were isolated in periods of 2, 5, 10, 20, and 30 min.
These samples were used for determination of radiochem-
ical yields by reversed-phase HPLC. The identification of
radiofluorinated compounds [18F]8a–h was performed by
gradient reversed-phase radio HPLC (RP 18 Select B5
column (250 · 4 mm)) eluted with acetonitrile/water (20/80
to 70/30 v/v, 0.1% TFA, 1 mL/min) using the UV
absorbance at 254 nm of standard compounds 8a–h as a
reference signal. Analytical HPLC was performed on the
following system: HPLC Hewlett Packard (HP 1100) with
a quaternary pump and variable wavelength detector (HP
1100) connected to a radio-HPLC detector D505TR
(Canberra Packard). Computer analysis of the HPLC
data was performed using FLO-One software (Canberra
1
19F NMR d (ppm): ꢀ68.634, 8g: H NMR d (ppm): 1.38
(m, 4H), 2.23 (m, 2H), 2.43 (m, 4H), 2.76 (m, 2H), 2.91 (m,
4H), 3.74 (s, 3H), 6.82–6.88 (m, 4H), 7.38 (m, 2H), 7.59 (t,
1
1H), 7.8 (dd, 2H), 19F NMR d (ppm): ꢀ107.845, 8h: H
NMR d (ppm): 1.39 (m, 4H), 2.25 (m, 2H), 2.46 (m, 4H),
2.77 (m, 2H), 2.93 (m, 4H), 7.1 (dd, 1H), 7.24 (m, 2H),
7.38 (m, 2H), 7.58 (t, 1H), 7.81 (m, 2H), 19F NMR d
(ppm): ꢀ107.808.
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28. In-vitro autoradiography was performed as described
by: Zhang, K.; Weiss, N. T.; Tarazi, F. I.; Kula, N. S.;
Baldessarini, R. J. Brain Res. 1999, 847, 32. Briefly,
20 lm frontal brain slices were preincubated for 30 min
Packard). Electronic autoradiography (InstantImagerTM
,
Canberra Packard) was used to analyze radio-TLC data.
20. To a solution of 6a,b (0.5 mmol) and triethylamine
(1 mmol) in CH2Cl2 (10 mL) was added the appropriate
fluoro substituted benzoic acid chloride or sulfonic acid
chloride (1.1 equiv) at room temperature. The mixture was
stirred at room temperature overnight. After the addition
of aqueous NaHCO3 solution, the mixture was stirred for
5 min and the organic layer was separated. The organic
phase was dried (Na2SO4), filtered, and concentrated.
Product isolation was followed by column chromatogra-
phy on silica gel (CH2Cl2/methanol 95/5) to give 8a–h (43–
64% yield).
in a buffer containing 50 mM Tris–HCl (pH 7.4),
40 mM NaCl, and 0.3 mM GTP. Subsequently, the
brain slices were incubated in fresh buffer containing 5–
10 MBq [18F]8d and 5lM DTG (to block sigma receptor
sites) for 60 min. Nonspecific binding was determined
with 1 lM S(ꢀ)eticlopride. After washing with binding
buffer and cold water, the distribution of [18F]8d in
brain slices were measured with a Micro-Imagerꢂ system
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