Synthesis, radiofluorination, and in vitro evaluation of pyrazolo[1,5-a]pyridine-based dopamine D4 receptor ligands: Discovery of an inverse agonist radioligand for PET

Article abstract of DOI:10.1021/jm701375u

A series of fluoro-substituted analogs structurally derived from the aminomethyl-substituted pyrazolo[1,5-a]pyridine lead compounds 9 (FAUC 113) and 10 (FAUC 213) were synthesized and evaluated as high-affinity D₄ receptor (D₄R) ligands (3a-3h, K_i = 1.3-28 nM). The para-fluoroethoxy-substituted derivatives 3f and 3h revealed an outstanding D₄ subtype selectivity of more than 3 orders of magnitude over both congeners D₂ and D₃ combined with inverse agonism at D₄R. The corresponding ¹⁸F-labeled radioligands revealed high serum stability in vitro and log P values of 2-3. In vitro rat brain autoradiography showed specific binding of [¹⁸F]3h in distinct brain regions, including the gyrus dentate of the hippocampus, that were inhibited by both eticlopride (65-80%) and the selective D₄R antagonist 10 (78-93%). The observed binding pattern was mainly consistent with the known D₄R distribution in the rat brain. Thus, [¹⁸F]3h (FAUC F41) represents a potential radioligand for studying the D₄R in vivo by positron emission tomography (PET).

Full text of DOI:10.1021/jm701375u

1800
J. Med. Chem. 2008, 51, 1800–1810
Synthesis, Radiofluorination, and In Vitro Evaluation of Pyrazolo[1,5-a]pyridine-Based
Dopamine D4 Receptor Ligands: Discovery of an Inverse Agonist Radioligand for PET
Olaf Prante,*,† Rainer Tietze,† Carsten Hocke,† Stefan Löber,‡ Harald Hübner,‡ Torsten Kuwert,† and Peter Gmeiner‡
Laboratory of Molecular Imaging, Clinic of Nuclear Medicine, Friedrich-Alexander UniVersity, 91054 Erlangen, Germany, and Department of
Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander UniVersity, 91052 Erlangen, Germany
ReceiVed NoVember 2, 2007
A series of fluoro-substituted analogs structurally derived from the aminomethyl-substituted pyrazolo[1,5-
a]pyridine lead compounds 9 (FAUC 113) and 10 (FAUC 213) were synthesized and evaluated as high-
affinity D4 receptor (D4R) ligands (3a-3h, Ki ) 1.3–28 nM). The para-fluoroethoxy-substituted derivatives
3f and 3h revealed an outstanding D4 subtype selectivity of more than 3 orders of magnitude over both
congeners D2 and D3 combined with inverse agonism at D4R. The corresponding 18F-labeled radioligands
revealed high serum stability in vitro and log P values of 2–3. In vitro rat brain autoradiography showed
specific binding of [18F]3h in distinct brain regions, including the gyrus dentate of the hippocampus, that
were inhibited by both eticlopride (65–80%) and the selective D4R antagonist 10 (78–93%). The observed
binding pattern was mainly consistent with the known D4R distribution in the rat brain. Thus, [18F]3h (FAUC
F41) represents a potential radioligand for studying the D4R in vivo by positron emission tomography (PET).
Introduction
of binding sites defined with [3H]raclopride (D2/D3 antagonist)
were substracted from total binding of [3H]nemonapride.12,13
Therefore, some uncertainties on D4R expression still prevail,
in particular, due to the limited availability of a selective D4
receptor radioligand with high affinity and selectivity over the
other dopamine receptor subtypes.14 Due to the above-described
methodological problems, evidence on the precise distribution
and regulatory mechanisms of the D4R in health and disease is
scarce. It has been implicated in novelty seeking and libidinous
behavior that is influenced by D4R agonists inducing the release
of oxytocin.15–17 Furthermore, the disturbance of neural net-
works involving the D4R has been assumed in various neu-
robehavioral and psychiatric disorders, such as attention-deficit
hyperactivity disorder (ADHDa) and schizophrenia.18–20
The subfamily of D2-like dopamine receptors includes the
D2-, D3-, and D4-receptor subtypes (D2R, D3R, D4R) and
mediates the action of dopamine in the brain by inhibition of
adenylate cyclase activity. Dopamine receptor subtypes differ
with regard to their distribution within the brain and their
density.1 D2Rs are highly expressed in the striatum, whereas
D3Rs are predominantly located in the mesocorticolimbic
system, especially in the nucleus accumbens and the Islands of
Calleja. D3R density is, however, lower than that of the D2Rs.2,3
The D4R was cloned from a genomic library in 1991.4 Some
evidence points to its preferential expression in the prefrontal
cortex and limbic areas such as the hippocampus, the amygdala,
the thalamic reticular nucleus, and the hypothalamus of the rat
brain.5–8 Immunohistological studies using D4 antireceptor
antisera or sequence-specific D4R antibodies have, however,
yielded some discrepant results. Khan et al. found highest
expression in cortical areas and the hippocampus of the rat
brain,8 whereas Ariano et al. found predominant expression of
the rat D4 receptor in the prefrontal cortex.5 Defagot et al.
supplemented these results by reporting highest D4 positive
antibody staining in the prefrontal cortex followed by hippoc-
ampus, cortex, entorhinal cortex, and amygdala.7 Recently,
Noaín et al. reinvestigated the D4R expression pattern in the
brain of bacterial artificial chromosome (BAC) transgenic mice
that express enhanced green fluorescent protein (EGFP) under
the transcriptional control of the mouse D4 receptor gene,
confirming predominant expression in the prefrontal cortex of
such animals.9
Molecular imaging with positron emission tomography (PET)
is in principle suited to gain deeper insight into the physiological
and pathophysiological role of the D4R also in the human.21
However, its use for this purpose has been obviated by the lack
of bioavailable D4 receptor selective radioligands, although
recent progress in the development of dopamine receptor
subtype-selective ligands has been achieved.22,23 Recently,
newly developed compounds with high affinity for D4 receptors
combined with potent serotonin reuptake inhibition, such as Lu
35-13824 and others,25,26 have been described. However, lacking
high specificity toward D4R would prevent these ligands from
serving as lead compounds for the development of PET analogs.
In the past, various attempts have been reported toward the
development of 18F- and 11C-labeled D4 receptor radioligands,
including [11C]SDZ-GLC756,27 methoxybenzamide deriva-
tives,28,29 SB-23575330 or 18F-labeled pyrrolo[2,3-b]pyridines.31,32
However, none of these radioligands has as yet been proven
Moreover, using autoradiography with the D4-selective ra-
dioligand [3H]NGD 94-1,10,11 the apparently low D4 receptor
densities have been reported to range between 9 and 30 fmol/
mg in the entorhinal cortex, septum, and hippocampus of the
rat brain.6 However, this D4 receptor distribution was not
consistent with autoradiography studies, in which the number
a Abbreviations: ADHD, attention-deficit hyperactivity disorder; cLogP,
calculated logarithm of distribution coefficient between octanol and water;
CM, central medial thalamic nucleus; CX, cortex; EOS, end of synthesis;
HC, hippocampus; HRMS, high-resolution mass spectrometry; HT, hypo-
thalamus; MHb, medial habenular nucleus, PBS, phosphate-buffered saline;
PET, positron emission tomography; RCY, radiochemical yield; SD,
standard deviation; s, septum; ST, striatum.
* To whom correspondence should be addressed. Phone: +49 9131
8544440. Fax: +49 9131 8534325. E-mail: olaf.prante@uk-erlangen.de.
†
Laboratory of Molecular Imaging, Clinic of Nuclear Medicine.
Department of Chemistry and Pharmacy.
‡
10.1021/jm701375u CCC: $40.75
2008 American Chemical Society
Published on Web 02/29/2008

D4 Radioligands for PET
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1801
Scheme 2. Syntheses of 3a-3ha
Figure 1. Development of 18F-labeled D4 radioligands.
Scheme 1. Synthesis of (6-Halopyridin-2-yl)piperazines 1a,ba
a Reaction conditions: (a) CH2O, AcOH, CH2Cl2, rt, 24 h; (b)
Na(OAc)3BH, CH2Cl2, rt, 24 h.
Scheme 3. Synthesis of the Labeling Precursor compounds 7a,
7b, 8a, and 8ba
a Reaction conditions: (a) triethyamine, DMF, reflux, 20 h; (b) dioxane/
HCl (1:1), 2 h.
suitable for PET studies due either to low specific binding in
vivo or undesirable pharmacological properties, such as rapid
metabolism.
As a result of our previous drug discovery and structure–ac-
tivity relationship (SAR) studies on selective dopamine D4
receptor–ligands, we developed the pyrazolo[1,5-a]pyridine
derivatives 9 (FAUC 113)33 and 10 (FAUC 213),34 bearing the
(4-chlorophenyl)piperazinylmethyl moiety in 3- and 2-position
of the pyrazolo[1,5-a]pyridine core, respectively, as high-affinity
dopamine D4 receptor–ligands with superior subtype select-
ivity.33–36 Interestingly, 9 has been found to show partial D4R
agonism, whereas 10 revealed complete D4R antagonism in a
cell-based mitogenesis assay using [3H]quinpirole as a refer-
ence.34 Moreover, 10 exhibited atypical antipsychotic properties
in animal models of behavioral neurobiology.36 On the basis
of these previous findings, we chose 9 and 10 as interesting
lead compounds for the development of 18F-labeled PET ligands.
In this study, we aimed at introducing a (2-fluoroethoxy)phenyl
or a (6-fluoropyridin-2-yl)phenyl group into the positions 2 or
3 of the pyrazolo[1,5-a]pyridine framework, replacing the
4-chlorophenyl moiety of the lead structure, to develop fluoro-
substituted candidates that also should be accessible by efficient
18F-labeling techniques (Figure 1).
a Reaction conditions: (a) C2H4(OTs)2, K2CO3, CH3CN, reflux, 8 h; (b)
dioxane/HCl (1:1); (c) 2a, CH2O, AcOH, CH2Cl2, rt, 24 h; (d) 2b,
Na(OAc)3BH, CH2Cl2, rt, 24 h.
commercially available N-Boc-protected pyridinylpiperazine 4b.
The ortho- and para-(fluoroethoxy)phenyl piperazines 1c and
1d were synthesized as described recently.39
With the arylpiperazines 1a-1d in hand, we prepared a new
series of 6-halopyridin-2-yl-, o-, and p-(2-fluoroethoxy)phenyl-
substituted derivatives of 9 and 10 by employing Mannich
reaction with 2a and reductive amination with pyrazolo[1,5-
a]pyridine-2-carbaldehyde (2b) in the presence of Na(OAc)3BH,
respectively, as depicted in Scheme 2. Following these reaction
pathways, a set of 3-substituted pyrazolo[1,5-a]pyridines (3a,
3b, 3e, and 3f (Scheme 2); 7a, 7b (Scheme 3b) was obtained
in 16–90% yield, and the corresponding series of 2-substituted
target compounds (3c, 3d, 3g, and 3h) was achieved in a yield
of 29–57%. The obtained series of compounds included the
reference compounds of the aspired radiolabeled analogs (3a,
3c, 3e-h), the 18F-labeling precursors for aromatic 18F-for-Br
substitution using ortho-bromo-substituted pyridinyl compounds
3b and 3d, and precursor compounds 7a and 7b suitable for
18F-ethylation using [18F]fluoroethyltosylate.
Extending our recent work on the development of D3- and
D4-selective radioligands,37–40 we herein describe the synthesis,
in vitro characterization, and 18F-labeling of a new series of D4
receptor radioligands derived from the lead compounds 9 and
10. Finally, one promising D4 radioligand with improved D4
subtype selectivity was chosen for in vitro brain autoradiography
to assess its in vivo properties as a D4 PET radioligand
candidate.
Results and Discussions
Chemistry. Our strategy toward the preparation of this new
series of test ligands was based on the availability of the
arylpiperazines 1a-d. Reaction of 2,6-difluoropyridine and
1-Boc-piperazine under basic conditions in DMF and subsequent
cleavage of the t-butyl carbamate (4a) readily afforded (6-
fluoropyridin-2-yl)piperazine (1a; Scheme 1). The corresponding
6-bromo derivative 1b was prepared similarly starting from the
Receptor Binding Experiments. After purification by flash
chromatography and confirmation of chemical purity by LC/
MS, HPLC, and HRMS, the test compounds 3a-h were

1802 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Prante et al.
Table 1. Binding Affinities of the Fluorinated Target Compounds 3a, 3c, and 3e-h to the Human Dopamine Receptor Subtypes D2long, D2short, D3, and
D4, the Porcine D1 Receptor, as Well as the Porcine 5-HT1A, 5-HT2, and R1 Receptors in Comparison with the Labeling Precursors 3b, 3d, 7a, and 7b
and the Reference Compounds 9 and 10
Ki valuesa (nM ( SEM)
[3H]SCH
23990
[3H]8-OH-
DPAT
[3H]
ketanserin
[3H]
prazosin
[3H]spiperone
D2short
cmpd pos.
X
R
D2long
D3
D4.4
D1
5-HT1A
5-HT2
R1
3a
3b
3c
3d
3e
3f
3g
3h
7a
7b
9
3
3
2
2
3
3
2
2
3
3
3
2
N
6-F
6-Br
6-F
330 ( 0
210 ( 30
3400 ( 1300
880 ( 95
150 ( 26
220 ( 65
150 ( 25
1700 ( 790
550 ( 75
180 ( 34
590 ( 60
410 ( 55
8.8 ( 1.0 7300 ( 1300
2.6 ( 0.8 3900 ( 2600
290 ( 85
120 ( 36
5400 ( 250
1200 ( 300
9700 ( 250
4400 ( 650
2900 ( 250
87 ( 9.0
21 ( 1.0
910 ( 200
240 ( 0
N
N
N
3400 ( 600 24 ( 1.5 30000 ( 4500 1200 ( 50
6-Br
930 ( 75
320 ( 27
7.9 ( 0.5 18000 ( 4500
1.6 ( 0.1 2500 ( 87
360 ( 0
120 ( 5.0
CH 2-O(CH2)2F
5.4 ( 0.4
CH 4-O(CH2)2F 66000 ( 13000 79000 ( 18000 8700 ( 2700 28 ( 0.5 33000 ( 5000 13000 ( 0
CH 2-O(CH2)2F
CH 4-O(CH2)2F 30000 ( 1000 50000 ( 5500 15000 ( 1000 13 ( 0.5 29000 ( 2500 1400 ( 200
CH 2-OH
CH 4-OH
CH 4-Cl
CH 4-Cl
14000 ( 1500 4500 ( 380
46 ( 8.5
79 ( 19
940 ( 160 1.3 ( 0.1 4800 ( 100
5700 ( 1200 13000 ( 10000
40 ( 9.5
5500 ( 1600 4100 ( 0
1500 ( 150
4500 ( 450
6100 ( 550
2900 ( 2950
3400 ( 450
4700 ( 1200
87000 ( 1300
2200 ( 670
2200 ( 0
44 ( 15 2800 ( 250
680 ( 430
26 ( 2.5
82 ( 7.0
240 ( 5.0
270 ( 13
3400 ( 250 20 ( 1.5 9900 ( 100 12000 ( 1500 24700 ( 700
4500 ( 360 5.8 ( 0.9 2800 ( 50
1400 ( 130
380 ( 140
900 ( 200
10
6300 ( 1900
5300 ( 720 2.2 ( 0.2 5500 ( 1300 1700 ( 360
a Ki values are expressed as mean ( SEM of 2–8 experiments, each performed in triplicate.
subjected to receptor binding studies to determine their ability
to displace [3H]spiperone from the cloned human dopamine
Table 2. D4 Subtype Selectivities within the Dopamine D2 Receptor
Family of the Fluorinated Target Compounds 3a, 3c, and 3e-h in
Comparison with the Reference Compounds 9 and 10
receptors D2long, D2short,
41 D3,42 and D443 being stably expressed
in Chinese hamster ovary (CHO) cells.44 In addition, the receptor
affinity profile was extended by the related biogenic amine
receptors 5-HT1A, 5-HT2, and R1 that were measured utilizing
porcine cortical membranes and the selective radioligands [3H]8-
OH-DPAT, [3H]ketanserin, and [3H]prazosin, respectively. For
straight comparison of the binding data, the lead compounds 9
and 10 were investigated under identical experimental condi-
tions. The results of the receptor binding studies are presented
in Table 1.
In fact, the dopamine receptor binding profiles of all
compounds indicated poor affinities for the D1, D2, and D3
subtypes and highest affinity for the D4 receptor subtype with
Ki values ranging from 1.3 to 44 nM. The results for the
6-halopyridinyl derivatives 3a-3d showed higher D4 affinities
for the bromo-substituted compounds (3b, 3d) in comparison
with the corresponding fluoro derivatives (3a, 3c; Table 1).
Comparison of the D4 binding data among each test compound
revealed significantly higher D4 receptor affinities in the low
nanomolar range (1–2 nM) for derivatives bearing a fluoro-
ethoxy substituent in ortho-position of the phenylpiperazinyl
moiety (3e, 3g). However, this encouraging result is ac-
companied by an increased affinity to the dopamine D2 receptor
and to the interfering R1 receptor (Table 1, 3e and 3g).
Interestingly, the corresponding para-fluoroethoxy derivatives
3f and 3h displayed a similar receptor binding profile with a Ki
for the D4 receptor of 28 nM and 13 nM, respectively, high
dopamine receptor subtype selectivities (D2/D4 > 2000), and a
substantial low R1 affinity (>4000 nM, Table 1). These findings
are consistent with our recent results on the comparison of ortho-
and para-fluoroethoxyphenyl-substituted 5-cyano indole deriva-
tives.39
D4R subtype selectivitiesa
cmpd pos.
X
R
D
2long/D4.4
D2short/D4.4 D3/D4.4
3a
3e
3f
9
3
3
3
3
2
2
2
2
N
6-F
38
94
2360
500
142
35
25
112
2821
379
71
67
200
311
776
142
CH 2-O(CH2)2F
CH 4-O(CH2)2F
CH 4-Cl
3c
3g
3h
10
N
6-F
CH 2-O(CH2)2F
CH 4-O(CH2)2F
CH 4-Cl
61
723
2308
1546
3846
2864
1154
2409
a D4 receptor subtype selectivities are expressed as the ratios Ki(D2long)/
Ki(D4.4), Ki(D2short)/Ki(D4.4), and Ki(D3)/Ki(D4.4), using the mean Ki values
from Table 1.
Within the series of ortho- and para-fluoroethoxy-substituted
test compounds, there were no significant differences in the D4/
D3 receptor subtype selectivities (Table 2). However, compound
3h displayed a D4/D3 selectivity of >1100, which was compa-
rable to that of the lead compounds 9 (780) and 10 (2400) (Table
2). In general, 3h was characterized as a high affinity D4 receptor
ligand with an outstanding subtype selectivity preferring the D4
receptor, a weak receptor affinity for R1, 5-HT1A, and 5-HT2,
thus illustrating interesting in vitro properties that encouraged
the optimization of the radiosynthesis of the 18F-labeled analog
[18F]3h, aiming at further in vitro studies, such as autoradiog-
raphy using rat brain slices.
In Vitro Functional Assay. Considering the marked neu-
ropharmacological differences of the regioisomers 9 and 10,34
we aimed at extending the data on receptor affinities of our
series of compounds with functional data. Agonist activation
of dopamine receptors is known to increase mitogenesis in
heterologously transfected cell lines that can be determined by
measuring the rate of [3H]thymidine incorporation into growing
cells.45 We studied and compared the intrinsic activities of the
highly D4 selective para-fluoroethoxy-substituted ligands 3f and
3h and the ortho regioisomers 3e and 3g by measuring the rate
Looking at dopamine receptor subtype selectivities in more
detail (Table 2), it is obvious that the pyridinyl compounds 3a
and 3c and the ortho-fluoroethoxyphenyl analogs 3e and 3g
suffer from low subtype selectivities when compared to the lead
structures 9 and 10. Contrary, the corresponding analogs bearing
a para-fluoroethoxyphenyl group (3f and 3h) revealed excellent
dopamine D4/D2 receptor subtype selectivities of >2300 that
were even superior to that of the lead compounds 9 and 10.

D4 Radioligands for PET
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1803
To our knowledge, our finding that 3f and 3h act as inverse
agonists at the D4 receptor represents as yet the first report on
this phenomenon regarding selective ligands for the D4 receptor
subtype. Although intrinsic constitutive receptor activity and
inverse agonism have been frequently observed in vitro for
various GPCRs, the therapeutic relevance of inverse agonist
drugs as yet remains to be elucidated.46,49 However, the para-
(2-fluoroethoxy)phenyl-substituted pyrazolo[1,5-a]pyridines 3f
and 3h could serve as interesting lead compounds for the
development of more potent D4 inverse agonists and for studying
D4 receptor mutants in functional assays.
Clearly, the degree of inverse D4 agonism of potential active
drugs depends on the presence of an adequate degree of
constitutive activity. Moreover, the level of basal activation of
dopamine receptors in vivo is unclear and difficult to measure
due to the presence of endogenous dopamine. However, for
imaging purposes and aiming at the development of D4 PET
ligands, the level of basal activation of D4 receptors in vivo
does not play a key role for being successful because the
application of PET ligands relies on the “tracer concept”, that
is, the use of negligible amounts of radioactive ligands for
binding studies without disturbing the dynamic receptor equi-
librium significantly. One of the most important prerequisites
of a suitable D4 PET ligand is high receptor selectivity, therefore,
inverse agonism could be a useful feature of a suitable D4 PET
radioligand due to the ability of such compounds to preferen-
tially bind to the inactive state of the receptor. Thus, our notion
that 3f and 3h act as inverse agonist D4 ligands might be
beneficial for the development of D4 radioligands with improved
functional selectivity for the inactive D4 receptor conformation.
Figure 2. Intrinsic activities of 3e-h in comparison with the reference
compounds 9 (partial agonist) and 10 (neutral antagonist) derived from
the D4 stimulating effect on cellular [3H]thymidine uptake.
Table 3. Intrinsic Activity of the Pyrazolo[1,5-a]pyridines 3e-h, 9, and
10 Relative to the Reference Compound Quinpirole at the Dopamine D4
Receptor Established by Measuring the Stimulation of Mitogenesis
test compounds
quinpirole 3e
3f
71% -41% 49% -37% 26% <3%
1.0 18 0.8 9.1 12 nd
3g
3h
9c
10c
ligand efficacya
100%
7.1
b
EC50 (nM)
a Rate of incorporation of [3H]thymidine (in %) as a measure of
mitogenetic activity relative to the full agonist effect of quinpirole (100%)
as the mean value of quadruplicates from 10 independent experiments.
b EC50 values are derived from the mean curves of the experiments. c Ref
34; nd ) not determined.
of [3H]thymidine incorporation into growing CHO cells stably
expressing the dopamine D4.2 receptor (Figure 2). When
quinpirole as a full agonist reference of maximum efficacy is
used, the results of the mitogenesis assay for compounds 3e
and 3g clearly revealed partial agonism for D4.2, displaying
intrinsic activities of 70 and 49%, respectively, and substantial
ligand potency, as summarized in Table 3. Interestingly, the
mitogenesis assay demonstrated inverse agonist activity of about
-40% for both para-substituted regioisomers 3f and 3h. The
structurally closely related regioisomers 9 (partial agonist) and
10 (neutral antagonist)34 differ from 3f and 3h by a 4-chlo-
rophenylpiperazine instead of a 4-(2-fluoroethoxy)phenylpi-
perazine core, however, the fluoroethoxy substitution in para-
position obviously results in a shift of the pharmacological
efficacy for this class of pyrazolo[1,5-a]pyridine based D4
ligands toward inverse agonism.
Radiochemistry. As a prerequisite for the radiosynthesis of
the 18F-labeled analogs, we prepared the set of suitable labeling
precursor compounds, as shown in Scheme 3. Starting from
N-Boc-protected hydroxyphenylpiperazine 4c, the tosylethoxy
group was introduced by reaction with ethylene glycol-1,2-
ditosylate in the presence of potassium carbonate to afford 5 in
good yields (63%), employing the protocol of Wang et al.50
Cleavage of the piperazine nitrogen under acidic conditions
provided the free amine (6), which was subsequently reacted
with 2a under Mannich reaction conditions to obtain the desired
labeling precursor 8a bearing a tosylate leaving group in 40%
yield (Scheme 3a). Mannich reactions of 2a with piperazines
4d or 4e, respectively, readily afforded the desired labeling
precursor compounds 7a and 7b (Scheme 3b), each bearing an
aromatic hydroxy group for 18F-fluoroethylation reactions by
the use of [18F]fluoroethyltosylate. Furthermore, the aldehyde
2b and the piperazine 4e in the presence of Na(OAc)3BH
afforded intermediate 7c that was treated with ethylene glycol-
1,2-ditosylate to obtain the desired precursor compound 8b
bearing a tosylate leaving group for direct nucleophilic displace-
ment by [18F]fluoride (Scheme 3b).
In recent years, the increased availability of recombinant and
constitutively active G-protein-coupled receptor (GPCR) systems
led to the discovery of the phenomenon of inverse agonism,
providing evidence for the potential of GPCRs to be active in
absence of agonists.46 Based on the most simple model, GPCRs
exist in a dynamic equilibrium between the active state (R*)
and inactive state (R), when agonists preferentially bind to R*
shifting the equilibrium toward the active conformation, and
neutral antagonists display equal affinity for R* and R. In
contrast, inverse agonists selectively bind to R, thereby causing
a redistribution of receptor species from the active state into
the inactive receptor state, which results in a decrease in
constitutive activity, provided that spontaneous active states of
GPCR had been present in the system.47 Recently, a large series
of typical and atypical antipsychotics was tested for D2-like
receptor activity at cells cotransfected with GR0.48 The majority
of compounds were determined to be D2 and D3 inverse agonists,
whereas, noteably, none was D4 inverse agonist, although many
were potent D4 partial agonists or neutral antagonists.48
With these labeling precursors in hand, we systematically
investigated the 18F-syntheses of [18F]3a, [18F]3c, [18F]3e,
[18F]3f, and [18F]3h leading to the optimized reaction conditions
presented in Table 3. Scheme 4 summerizes the various 18F-
labeling reactions that were performed to obtain the respective
18F ligands. Applying typical reaction conditions for aromatic
18F-for-Br substitution to ortho-bromo-substituted pyridinyl
derivatives (3b or 3d), that is, high reaction temperature (180
°C) and long reaction time (45 min), the 18F-labeled ligands
[18F]3a and [18F]3c were obtained in high radiochemical yields
(RCY) of 69 and 80%, respectively (Scheme 4, Table 4).
Furthermore, the [18F]fluoroethoxy-substituted radioligands
[18F]3e and [18F]3f were achieved by a two-step procedure

1804 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Prante et al.
Scheme 4. Radiosynthesis of 18F-labeled
ing the ability of 10 and 7-iodo-9 to adequately cross the
blood-brain barrier.
Pyrazolo[1,5-a]pyridine Based D4 Radioligandsa
In Vitro Autoradiography. Due to the superior D4R subtype
selectivity together with the convenient and high-yielding
radiosynthesis, we chose the inverse agonist radioligand [18F]3h
to perform in vitro autoradiography studies on native tissue.
The results of the in vitro autoradiography using coronal rat
brain slices are presented in Figure 3. All brain areas were
determined by histological counterstaining of each slice and
verification of the brain regions with reference to the rat brain
atlas of Paxinos and Watson.55 In the slices representing total
binding (Figure 3A,F), increased uptake values of [18F]3h were
detected in the brain areas of the gyrus dentate region of the
hippocampus, the hypothalamus (HT), the medial habenular
nucleus (MHb), the central medial thalamic nucleus (CM),
septum (s), and cortex (CX) (Figure 3). Interestingly, total
binding in the area of the striatum (ST) was low in comparison
with the surrounding cortex (Figure 3F). Importantly, the D4-
selective ligand 1034 (Figure 3C,H) and also the nonselective
dopamine receptor antagonist eticlopride (Figure 3D,I) signifi-
cantly inhibited the binding of [18F]3h in the brain regions under
investigation by 78–93% (n ) 9) and 65–80% (n ) 7),
respectively, as determined by integration of regions of interest
(ROI). This result suggested specific binding of [18F]3h in the
above-mentioned areas of the rat brain. Together with low
binding in the striatum, the observed in vitro binding pattern of
[18F]3h excellently reflects the published D4 receptor expression
in rat brain that had been determined by D4R antibody staining
and autoradiography with [3H]NGD 94-1.6–8 However, Primus
et al. observed additional binding sites in the subregions CA1,
CA2, and CA3 of the rat hippocampus by [3H]NGD 94-1
autoradiography,6 whereas we exclusively observed specific
uptake of [18F]3h in the gyrus dentate of the hippocampus and
only background levels of [18F]3h uptake in CA1–3. This
discrepancy could be due to tissue-specific differences in
intrinsic efficacy and receptor content being responsible for
differences in partial agonist ([3H]NGD 94-1)11 and inverse
agonist ([18F]3h) binding potency between the subregions of
the hippocampus.
a Reaction conditions: (a) [18F]fluoride (potassium 222 kryptate), DMSO,
180 °C, 45-60 min; (b) 2-[18F]fluoroethyltosylate, sodium methanolate,
DMF, 120 °C, 5 min; (c) [18F]fluoride (potassium 222 kryptate), CH3CN,
85 °C, 10 min.
consisting of the radiosynthesis of [18F]fluoroethyltosylate51 and
subsequent 18F-ethylation of 7a or 7b in the presence of sodium
methanolate as base (Scheme 4). The final alkylation step of
this laborious labeling procedure revealed high RCYs of 92%
([18F]3e) and 87% ([18F]3f; Table 4), however, the overall
synthesis time was two hours, being a major disadvantage when
considering the short half-life of fluorine-18 (110 min). Utilizing
the tosylate precursors 8a and 8b for a direct kryptate-assisted
nucleophilic 18F-fluorination in acetonitrile afforded the desired
radioligands [18F]3f and [18F]3h in a straightforward reaction
with a yield of 26% (nonoptimized) and 88%, respectively
(Scheme 4). The reaction conditions for the radiosynthesis of
[18F]3f and [18F]3h closely resembled those of the well-known
oncological PET tracer 2-[18F]fluoro-2-deoxyglucose (FDG),52
and the workup procedure consisted of only one HPLC
purification step. Thus, the radiosynthesis of [18F]3h resulted
in a decay-uncorrected RCY of 22% (related to [18F]fluoride)
within a total synthesis time of only 60 min, which is most
favorable when compared with the decay-uncorrected RCY at
end of synthesis of the respective radioligands ([18F]3a-f, Table
4). All radioligands were isolated by semipreparative HPLC and
formulated in phosphate-buffered solution to determine log D7.4
values and to study their in vitro stability in human serum at
37 °C. The radiochemical purity of all radioligands was >98%
and the specific activity was 24–47 GBq/µmol (starting from
relatively low amounts of [18F]fluoride, i.e., 0.6–1.2 GBq).
To provide a “gold standard” of a selective “D4 radioligand”
for comparison with [18F]3h binding, we performed rat brain
autoradiography using [3H]nemonapride as a high affinity
unselective D2, D3, and D4 radioligand supplemented with 1
µM cold raclopride to block D2 and D3 receptors (Figure 3E).
This method has been used previously for quantitative analysis
of the dopamine D4 receptor in the mouse brain.14 As shown in
Figure 3E, specific [3H]nemonapride binding excellently cor-
related with brain areas of high specific binding of [18F]3h (cf.
Figure 3A), thus suggesting accurate localization of D4 receptors
by [18F]3h in the gyrus dentate of the hippocampus, the
hypothalamus (HT), the medial habenular nucleus (MHb), the
central medial thalamic nucleus (CM), and the cortex (CX).
However, in comparison with [18F]3h and similar to [3H]NGD
94-1,6 [3H]nemonapride revealed additional binding sites in
CA1–3 of the hippocampus that did not show specific binding
of [18F]3h. Interfering nondopaminergic and nonserotonergic
binding sites of [3H]nemonapride (1 µM raclopride) in the
striatum of D4R knockout mice have been noticed previously.14
This suggests that [18F]3h could represent a D4 radioligand with
improved selectivity compared to the system [3H]nemonapride/
cold raclopride, possibly due to the inverse agonist properties
of [18F]3h.
Lipophilicity. As a marker of lipophilicity, experimental log
D7.4 values of all tested radioligands ranged from 1.78 to 2.15,
reflecting mainly the influence of the partly protonated pipera-
zine at pH 7.4 (Table 4). However, calculated log P values
obtained by the software cLogP (Biobyte) revealed values of
1.9 for the pyridinyl compounds [18F]3a and [18F]3c, and log
P values of 2.9 for the [18F]fluoroethoxy-substituted radioligands
[18F]3e, [18F]3f, and [18F]3h (Table 4). Due to the fact that
radiotracer uptake in the brain has been determined to be a
function of log P that peaks between log P of 2 and 3,53 the
log P values of our tested 18F-labeled pyrazolo[1,5-a]pyridine-
based D4 radioligands excellently complied with this require-
ment. Moreover, the tested radioligands revealed high stability
in human serum, even after long-term incubation for up to 90
min (Table 4). These in vitro parameters could suggest advanta-
geous in vivo characteristics, such as adequate blood-brain
barrier penetration. This assumption is supported by our previous
in vivo studies on structurally related compounds,36,54 confirm-
In summary, [18F]3h is a promising D4 radioligand candidate
for PET due to its convenient radiosynthesis, superior D4R

D4 Radioligands for PET
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1805
Table 4. Reaction Conditions of the 18F-Syntheses of [18F]3a, [18F]3c, [18F]3e, [18F]3f, and [18F]3h and the Corresponding Radiochemical Yields
(RCY), Experimental log D7.4 Values and In Vitro Stability in Human Seruma
labeling
precursor
18F-labeling conditions
product decay-corrected RCY (%) RCY at EOS (%) serum stabilityb (%) log D7.4 (exp.) cLogPc
3b
3d
7a
7b
8a
8b
[
18F]fluoride, DMSO,
180 °C, 45–60 min
[
[
[
[
[
[
18F]3a
18F]3c
18F]3e
18F]3f
18F]3f
18F]3h
69 ( 8
80 ( 2d
92 ( 7
87 ( 5
26 ( 7e
88 ( 4
13 ( 4
5 ( 1
>98
>99
nd
1.98 ( 0.14
1.78 ( 0.04
2.15 ( 0.03
2.12 ( 0.13
1.9
1.9
2.9
2.9
2-[18F]fluoroethyltosylate,
15 ( 3
12 ( 3
<10e
NaOMe, DMF, 120 °C, 5 min
>96
[
18F]fluoride, CH3CN,
85 °C, 10 min
22 ( 2
>99
1.99 ( 0.06
2.9
a Data are expressed as mean ( SD of 3–9 experiments. RCY ) radiochemical yield. EOS ) end of synthesis. b Intact radioligand after incubation in
human serum at 37 °C for 90 min. c Calculated using the cLogP software (Biobyte Corp.). d Maximum value. e Nonoptimized RCY; nd ) not determined.
Figure 3. In vitro autoradiography of [18F]3h and histological counterstaining of rat brain coronal slices (20 µm; level of hippocampus, B; level
of striatum, G). Total binding (A) in the area of the hippocampus (HC; that is gyrus dentate of HC only), hypothalamus (HT), cortex (CX), medial
habenular nucleus (MHb), and central medial thalamic nucleus (CM), was inhibited by the D4-selective antagonist 10 (1 µM; C) and by the nonselective
dopamine receptor–ligand eticlopride (1 µM; D). In slices at the level of the rat striatum (F-I) specific binding in the cortex (CX) and septum (s),
and negligible binding in the striatum (ST) was detected (F). Again, binding of [18F]3h was inhibited by the D4 selective antagonist 10 (1 µM; G)
and by eticlopride (1 µM; H). (E) Autoradiogram at the level of the hippocampus obtained by incubation with 1 nM [3H]nemonapride in the
presence of 1 µM cold raclopride as a reference (please note that HC staining is positive for CA1, CA2, CA3, and gyrus dentate of HC).
subtype selectivity, high serum stability, relatively low lipo-
philicity, and encouraging in vitro rat brain autoradiography and,
thus, could also be suitable for the observation of a D4 receptor-
specific signal in vivo. However, the in vitro receptor affinity
measured by a cell-based assay was not exceptionally high (13
nM, Table 1), and the specific activity of [18F]3h was relatively
low, suggesting that meaningful imaging of the apparently low
D4 receptor densities in vivo might be problematic. In this
context, it should be noted that data on rat brain D4 receptor
densities are scarce and the target site of inverse D4 agonists
has not yet been explored neither in vitro nor in vivo. Thus,
little is known about tissue-specific differences in intrinsic
efficacy and receptor content that determine the in vivo binding
potential (Bmax/Kd)56 of an inverse agonist D4R radioligand for
PET. Besides high in vivo receptor affinity, parameters such as
blood-brain barrier permeability, metabolic stability, and
“functional selectivity” of inverse agonist candidates, such as
[18F]3h, could additionally influence the in vivo specificity of
a PET D4R radioligand. Further biodistributional studies are
currently in progress to characterize the in vivo specificity of
[18F]3h as a selective inverse agonist PET radioligand for the
D4 receptor.
radioligands had favorable log P values and showed high serum
stability. [18F]3h revealed an outstanding subtype selectivity for
the D4 receptor and could be obtained by a high-yielding
straightforward radiosynthesis that allows easy automation for
further routine radiopharmaceutical production. In vitro auto-
radiography studies using rat brain slices suggest specific binding
of [18F]3h in brain areas that are in agreement with the known
D4R distribution. Thus, [18F]3h (FAUC F41) represents a
potential radioligand for the exploration of D4 receptor densities
in vivo by PET.
Experimental Section
No-carrier-added (n.c.a.) [18F]fluoride was produced by the
18O(p,n)18F reaction on 18O-enriched (95%) water using a proton
beam of 11 MeV generated by a RDS 111 cyclotron (PET Net
GmbH, Erlangen, Germany). Solid-phase cartridges (Sep-PakPlus
C18 cartridges) were purchased from Waters (Eschborn, Germany).
Melting points (mp) were uncorrected and obtained with a Mel-
TempII apparatus (Laboratory Devices, MA, U.S.A.). Thin layer
chromatography (TLC) was carried out on silica gel-coated
aluminum plates (silica gel/TLC-cards, with fluorescent indicator
254 nm, 0.2 mm, Fluka). Radio-TLC was performed with silica
gel coated plastic sheets (Polygram, Sil G/UV254, Macherey-Nagel).
Radio-HPLC was performed on the following system: Agilent 1100
with a quarternary pump and variable wavelength (VWL) detector
and radio-HPLC-detector (D505TR, Canberra Packard). Each 18F-
labeled compound was identified by retention time (tR) on the radio-
HPLC system and coinjection of the corresponding reference
compound. NMR spectra were recorded on a Bruker Avance 360
Conclusion
We reported the syntheses, binding affinities, functional
characterization, 18F-radiosyntheses, and in vitro studies of
selective pyrazolo[1,5-a]pyridine-based D4 receptor ligands. All

1806 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Prante et al.
or Bruker Avance 600 using TMS as internal standard, and data
are reported as follows: chemical shifts (ppm), multiplicity (s,
singlet; d, doublet; t, triplet; q, quartet; br, broad; m, multiplet),
peak integral. LC-MS analyses were performed on an Agilent 1100
Series analytic HPLC system with a VWL detector coupled to a
Bruker esquire 2000 mass spectrometer with atmospheric pressure
chemical ionization (APCI). High resolution mass spectrometry
(HRMS) was performed on a JEOL JMS-GC Mate II spectrometer.
1-(2-(2-Fluoroethoxy)phenyl)piperazine (1c), 1-(4-(2-fluoroethoxy)-
phenyl)piperazine (1d), and 4-(4-hydroxyphenyl)piperazine-1-car-
boxylic acid t-butyl ester (4c) were synthesized as described
previously.39 Pyrazolo[1,5-a]pyridine (2a) was prepared as reported
by Löber et al.34 4-(6-Bromopyridin-2-yl)piperazine-1-carboxylic
acid t-butyl ester (4b), 1-(2-hydroxyphenyl)piperazine (4d), and
1-(4-hydroxyphenyl)piperazine (4e) were purchased from Sigma-
Aldrich.
3-[4-(2-Hydroxylphenyl)piperazin-1-ylmethyl]pyrazolo[1,5-
a]pyridine (7a). Obtained from 1-(2-hydroxyphenyl)piperazine (4d,
121 mg, 0.68 mmol) as a white solid (145 mg, 0.47 mmol, 69%).
1
Mp 129 °C. TLC (CH2Cl2/methanol, 95:5): Rf ) 0.35. H NMR
(CDCl3, 600 MHz): δ 2.52–2.81 (m, 4H), 2.87–2.94 (m, 4H), 3.79
(s, 2H), 6.76 (dd (t), J ) 6.7 Hz, 1H, 6-H), 6.85 (ddd (dt), J ) 7.7
Hz, J ) 7.7 Hz, J ) 1.4 Hz, 1H), 6.93 (dd, J ) 8.0 Hz, J ) 1.4
Hz, 1H), 7.06 (ddd (dt), J ) 8.0 Hz, J ) 8.0 Hz, J ) 1.5 Hz, 1H),
7.13 (brdd (t), J ) 7.5 Hz, 1H, 5-H), 7.16 (dd, J ) 7.9 Hz, J ) 1.4
Hz, 1H), 7.64 (brd, J ) 8.9 Hz, 1H, 4-H), 7.93 (s, 1H, 2-H), 8.45
(d, J ) 7.0 Hz, 1H, 7-H).
3-[4-(4-Hydroxylphenyl)piperazin-1-ylmethyl]pyrazolo[1,5-
a]pyridine (7b). Obtained from 1-(4-hydroxyphenyl)piperazine (4e,
121 mg, 0.68 mmol) as a white solid (139 mg, 0.45 mmol, 67%).
Mp 173.5 °C. TLC (CH2Cl2/methanol, 95:5): Rf ) 0.26. 1H NMR
(CDCl3, 600 MHz): δ 2.65 (m, 4H), 3.09 (m, 4H), 3.78 (s, 2H),
6.73–6.75 (m, 3H), 6.80–6.85 (m, 2H), 7.11 (brdd (t), J ) 7.6 Hz,
1H, 5-H), 7.64 (brd, J ) 8.7 Hz, 1H, 4-H), 7.92 (s, 1H, 2-H), 8.44
(brd, J ) 7.0 Hz, 1H, 7-H).
General Procedure. Preparation of 3-[4-Arylpiperazin-1-
ylmethyl]pyrazolo[1,5-a]pyridine Derivatives (3a, 3b, 3e, 3f,
7a, 7b, 8a). A solution of 2a (0.3–0.7 mmol, 1 equiv), the
corresponding arylpiperazine (1a-d, 4d, 4e, or 6, 1 equiv), acetic
acid (0.26 mL), and formaldehyde (50 µL, 37% in H2O) in CH2Cl2
(5 mL) was stirred at room temperature (6 h or overnight). The
reaction was quenched by the addition of aqueous saturated
NaHCO3 (10 mL). The solution was extracted with CH2Cl2 (3 ×
10 mL), and the combined organic phases were dried (Na2SO4).
The solvent was evaporated in vacuo and the residue was purified
by silica gel column chromatography (CH2Cl2/methanol, 95:5).
3-[4-(4-(2-Tosylethoxy)phenyl)piperazin-1-ylmethyl]pyrazo-
lo[1,5-a]pyridine (8a). Obtained from 6 as a yellow oil (54 mg,
1
0.11 mmol, 40%). TLC (CH2Cl2/methanol, 95:5): Rf ) 0.28. H
NMR (CDCl3, 360 MHz): δ 2.44 (s, 3H), 2.63–2.68 (m, 4H),
3.07–3.11 (m, 4H), 3.78 (s, 2H), 4.07–4.10 (m, 2H), 4.30–4.34 (m,
2H), 6.71 (d, J ) 9.2 Hz, 2H), 6.75 (ddd (dt), J ) 6.9 Hz, J ) 6.9
Hz, J ) 1.4 Hz, 1H, 6-H), 6.82 (d, J ) 9.2 Hz, 2H), 7.10 (ddd, J
) 8.9 Hz, J ) 6.7 Hz, J ) 1.1 Hz, 1H, 5-H), 7.33 (d, J ) 8.6 Hz,
2H), 7.63 (ddd (dt), J ) 8.9 Hz, J ) 1.2 Hz, J ) 1.2 Hz, 1H, 4-H),
7.81 (d, J ) 8.3 Hz, 2H), 7.91 (s, 1H, 2-H), 8.44 (ddd (dt), J ) 7.0
Hz, J ) 1.0 Hz, J ) 1.0 Hz, 1H, 7-H). LC-MS (APCI): m/z 507.3
[M + H]+, 377.2 [C19H24N2O4 + H]+, 131.0 [C8H7N2 + H]+.
General Procedure. Preparation of 2-[4-Arylpiperazin-1-
ylmethyl]pyrazolo[1,5-a]pyridine Derivatives (3c, 3d, 3g, 3h,
7c). Compound 2b (1 equiv) and the corresponding arylpiperazine
(1a-d, 6, 1 equiv) were dissolved in anhydrous CH2Cl2.
Na(OAc)3BH (3 equiv) was added and the mixture was stirred at
room temperature (24 h). The reaction was quenched by the addition
of saturated NaHCO3 (20 mL) and the solution was extracted with
CH2Cl2 (3 × 10 mL). The organic layer was dried (Na2SO4) and
evaporated to dryness. The crude product was purified by silica
gel column chromatography using CH2Cl2/methanol (95:5) as
eluent.
3-[4-(6-Fluoropyridin-2-yl)piperazin-1-ylmethyl]pyrazolo[1,5-
a]pyridine (3a). Obtained from 1a (79 mg, 0.66 mmol) as a white
solid (45 mg, 0.14 mmol, 22%). Mp 112 °C. TLC (CH2Cl2/
methanol, 95:5): Rf ) 0.37. 1H NMR (CDCl3, 600 MHz): δ
2.54–2.70 (m, 4H), 3.53–3.66 (m, 4H), 3.82 (s, 2H), 6.16 (dd, J )
7.7 Hz, J ) 2.7 Hz, 1H, 5-H (pyridyl)), 6.39 (dd, J ) 8.2 Hz, J )
2.4 Hz, 1H, 3-H (pyridyl)), 6.77 (dd (t), J ) 6.6 Hz, 1H, 6-H),
7.13 (dd (t), J ) 8.0 Hz, 1H, 5-H), 7.51 (ddd (q), J ) 8.2 Hz, 1H,
4-H (pyridyl)), 7.66 (brd, J ) 8.8 Hz, 1H, 4-H), 7.93 (s, 1H, 2-H),
8.45 (d, J ) 7.0 Hz, 1H, 7-H). LC-MS (APCI): m/z 312.4 [M +
H]+.
3-[4-(6-Bromopyridin-2-yl)piperazin-1-ylmethyl]pyrazolo[1,5-
a]pyridine (3b). Obtained from 1b (26.7 mg, 0.11 mmol) as a dark
yellow solid (37 mg, 0.10 mmol, 91%). Mp 98 °C. TLC (CH2Cl2/
MeOH 95:5): Rf ) 0.47. 1H NMR (CDCl3, 360 MHz): δ 2.55–2.60
(m, 4H), 3.53–3.59 (m, 4H), 3.77 (s, 2H), 6.47 (d, J ) 8.3 Hz,
1H), 6.73 (d, J ) 7.5 Hz, 1H), 6.75 (ddd (dt), J ) 6.9 Hz, J ) 1.3
Hz, 1H), 7.11 (ddd, J ) 8.9 Hz, J ) 6.7 Hz, J ) 1.0 Hz, 1H), 7.26
(dd, J ) 8.4 Hz, J ) 7.5 Hz, 1H), 7.63 (brd, J ) 8.9 Hz, 1H), 7.91
(s, 1H, 2-H), 8.44 (ddd (dt), J ) 7.0 Hz, J ) 1.0 Hz, J ) 1.0 Hz,
1H). LC-MS (APCI): m/z 374.0 [M + H]+.
2-[4-(6-Fluoropyridin-2-yl)piperazin-1-ylmethyl]pyrazolo[1,5-
a]pyridine (3c). Obtained from 1a as a brownish solid (59 mg,
0.19 mmol, 57%). Mp 118 °C. TLC (CH2Cl2/methanol, 95:5): Rf
1
) 0.34. H NMR (DMSO-d6, 360 MHz): δ 2.52–2.55 (m, 4H),
3.44–3.49 (m, 4H), 3.71 (s, 2H), 6.24 (dd, J ) 7.7 Hz, J ) 2.8 Hz,
1H, 5-H (pyridyl)), 6.53 (s, 1H, 3-H), 6.65 (dd, J ) 8.2 Hz, J )
2.7 Hz, 1H, 3-H (pyridyl)), 6.82 (ddd (dt), J ) 6.8 Hz, J ) 6.8 Hz,
J ) 1.4 Hz, 1H, 6-H), 7.17 (ddd, J ) 8.9 Hz, J ) 6.7 Hz, J ) 1.1
Hz, 1H, 5-H), 7.59–7.68 (m, 2H, 4-H, 4-H (pyridyl)), 8.59 (dd, J
) 7.0 Hz, J ) 1.0 Hz, 1H, 7-H). LC-MS (APCI): m/z 312.3 [M
+ H]+.
3-[4-(2-(2-Fluoroethoxy)phenyl)piperazin-1-ylmethyl]pyra-
zolo[1,5-a]pyridine (3e). Obtained from 1c (74 mg, 0.33 mmol)
as a stringy yellow oil (19.3 mg, 0.05 mmol, 16%). TLC (CH2Cl2/
1
methanol, 95:5): Rf ) 0.23. H NMR (CDCl3, 600 MHz): δ 2.67
2-[4-(6-Bromopyridin-2-yl)piperazin-1-ylmethyl]pyrazolo[1,5-
a]pyridine (3d). Obtained from 1b as a white solid (159 mg, 0.42
mmol, 35%). Mp 106 °C. TLC (CH2Cl2/methanol, 95:5): Rf ) 0.50.
1H NMR (DMSO-d6, 360 MHz): δ 2.51–2.55 (m, 4H), 3.45–3.50
(m, 4H), 3.70 (s, 2H), 6.53 (s, 1H, 3-H), 6.77 (d, J ) 7.4 Hz, 1H,
pyridyl), 6.78 (d, J ) 8.4 Hz, 1H, pyridyl), 6.82 (ddd (dt), J ) 6.8
Hz, J ) 6.8 Hz, J ) 1.4 Hz, 1H, 6-H), 7.17 (ddd, J ) 8.9 Hz, J )
6.7 Hz, J ) 1.1 Hz, 1H, 5-H), 7.42 (dd, J ) 8.4 Hz, J ) 7.4 Hz,
1H, 4-H (pyridyl)), 7.61 (ddd (dt), J ) 8.9 Hz, J ) 1.2 Hz, J )
1.2 Hz, 1H, 4-H), 8.59 (ddd, J ) 7.0 Hz, J ) 1.0 Hz, J ) 1.0 Hz,
1H, 7-H). LC-MS (APCI): m/z 374.0 [M + H]+, 244.0 [C9H12BrN3
+ H+]).
2-[4-(2-(2-Fluoroethoxy)phenyl)piperazin-1-ylmethyl]pyra-
zolo[1,5-a]pyridine (3g). Obtained from 1c as a pale yellow oil
(22 mg, 0.06 mmol, 29%). TLC (CH2Cl2/methanol, 95:5): Rf )
0.26. 1H NMR (CDCl3, 360 MHz): δ 2.77 (m, 4H), 3.17 (m, 4H),
3.83 (s, 2H), 4.25 (dt, J ) 28.0 Hz, J ) 4.2 Hz, 2H, OCH2), 4.76
(dt, J ) 47.4 Hz, J ) 4.2 Hz, 2H, FCH2), 6.51 (s, 1H), 6.70 (ddd
(m, 4H), 3.12 (m, 4H), 3.77 (s, 2H), 4.25 (dt, J ) 28.1 Hz, J ) 4.1
Hz, 2H, OCH2), 4.76 (dt, J ) 47.5 Hz, J ) 4.2 Hz, 2H, FCH2),
6.74 (ddd (dt), J ) 6.8 Hz, J ) 1.2 Hz, 1H), 6.87–6.83 (m, 1H),
6.98–6.91 (m, 3H), 7.10 (ddd, J ) 8.9 Hz, J ) 6.7 Hz, J ) 0.9 Hz,
1H), 7.64 (brd, J ) 8.9 Hz, 1H), 7.92 (s, 1H, 2-H), 8.43 (d, J )
7.0 Hz, 1H). LC-MS (APCI): m/z 355.1 [M + H]+, 225.1
[C12H17FN2O + H]+, 131.0 [C8H7N2 + H+].
3-[4-(4-(2-Fluoroethoxy)phenyl)piperazin-1-ylmethyl]pyra-
zolo[1,5-a]pyridine (3f). Obtained from 1d (73 mg, 0.33 mmol)
as a white solid after crystallization from methanol (50 mg, 0.14
mmol, 31%). Mp 131 °C. TLC (CH2Cl2/methanol, 95:5): Rf ) 0.28.
1H NMR (CDCl3, 600 MHz): δ 2.61–2.68 (m, 4H), 3.08–3.13 (m,
4H), 3.77 (s, 2H), 4.16 (dt, J ) 27.9 Hz, J ) 4.2 Hz, 2H, OCH2),
4.72 (dt, J ) 47.4 Hz, J ) 4.3 Hz, 2H, FCH2), 6.75 (dd (t), J )
6.4 Hz, 1H), 6.83–6.89 (m, 4H), 7.10 (dd (t), J ) 8.27 Hz, 1H),
7.64 (d, J ) 8.9 Hz, 1H), 7.91 (s, 1H), 8.44 (d, J ) 7.0 Hz, 1H).
LC-MS (APCI): m/z 355.2 [M + H]+, 131.0 [C8H7N2 + H+].

D4 Radioligands for PET
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1807
(dt), J ) 6.9 Hz, J ) 1.4 Hz, 1H), 6.85 (ddd, J ) 7.1, J ) 3.7, J
) 1.4 Hz, 1H), 6.93–6.97 (m, 3H), 7.07 (ddd, J ) 8.90, J ) 6.7
Hz, J ) 1.1 Hz, 1H), 7.47 (ddd, J ) 8.9 Hz, J ) 1.2 Hz, J ) 1.2
Hz, 1H), 8.42 (ddd, J ) 7.1 Hz, J ) 2.0 Hz, J ) 1.0 Hz, 1H).
LC-MS (APCI): m/z 355.2 [M + H]+.
maximum radiochemical yield of [18F]3c was 80 ( 2% after 60
min at 180 °C. The decay-uncorrected radiochemical yield of
[18F]3c averaged 5 ( 1% (n ) 3, based on [18F]fluoride) after a
total synthesis time of 110 min. Radio-HPLC (Lichrosorb RP18,
250 × 4.6 mm, 1 mL/min, linear gradient from 25% to 100%
CH3CN in water (0.1% TFA) over 30 min): tR ) 8.12 min.
3-[4-(2-(2-[18F]Fluoroethoxy)phenyl)piperazin-1-ylmethyl]pyra-
zolo[1,5-a]pyridine ([18F]3e). A solution of 7a (2.3 mg, 7.5 µmol)
in anhydrous DMF (250 µL) and sodium methanolate (50 µL, 0.15
µM in dry methanol) were stirred for 3 min at 120 °C under
nitrogen. 1-[18F]Fluoro-2-tosyloxyethane39,51 (140–250 MBq) in
dry DMF (250 µL) were added, and the progression of the reaction
was analyzed by radio-TLC (CH2Cl2/methanol 95:5). The radio-
chemical yield of [18F]3e was 92 ( 7% after 5 min at 120 °C.
Radio-HPLC (Lichrosorb RP18, 250 × 4.6 mm, 1 mL/min, linear
gradient from 30% to 70% methanol in water (0.1% TFA) over 30
min): tR ) 17.8 min. The reaction was quenched by the addition
of water (4.5 mL) and the solution was passed through a C18-
cartridge (Sep-PakPlus, Waters). The cartridge was washed with
water (10 mL), dried in a stream of nitrogen, and eluted with
CH3CN (1 mL). The solution was subjected to a semipreparative
HPLC column (Lichrolut RP-18, 125 × 8 mm, 4 mL/min, CH3CN/
H2O, 40/60 (0.1% TFA)). The [18F]3e containing fraction was
diluted with water (1:10 v/v) and the product was fixed on a RP18-
cartridge (100 mg, Merck), dried in a nitrogen stream and eluted
with CH3CN (1 mL). The solvent was evaporated and [18F]3e was
formulated in PBS (300 µL). [18F]3e (39–65 MBq) was obtained
in a decay-uncorrected yield of 13 ( 3% (related to [18F]fluoride)
after a total synthesis time of 120 min.
2-[4-(4-(2-Fluoroethoxy)phenyl)piperazin-1-ylmethyl]pyra-
zolo[1,5-a]pyridine (3 h). Obtained from 1d as a pale yellow oil
(30.6 mg, 0.09 mmol, 57%). TLC (CH2Cl2/methanol, 95:5): Rf )
1
0.21. H NMR (CDCl3, 360 Hz): δ 3.40 (brs, 8H), 4.16 (dt, J )
27.8 Hz, J ) 4.2 Hz, 2H, OCH2), 4.51 (s, 2H), 4.72 (dt, J ) 47.4
Hz, J ) 4.2 Hz, 2H, FCH2), 6.76 (s, 1H, 3-H), 6.83–6.93 (m, 5H),
7.19 (dd, J ) 8.9 Hz, J ) 6.8 Hz, 1H, 5-H), 7.58 (brd, J ) 8.8 Hz,
1H, 4-H), 8.44 (brd, J ) 6.6 Hz, 1H, 7-H). LC-MS (APCI): m/z
355.3 [M + H]+.
2-[4-(4-Hydroxyphenyl)piperazin-1-ylmethyl]pyrazolo[1,5-
a]pyridine (7c). Obtained from 1-(4-hydroxyphenyl)piperazine (4e)
as a white solid (77.5 mg, 0.25 mmol, 52%). TLC (CH2Cl2/
1
methanol, 95:5): Rf ) 0.35. H NMR (CDCl3, 600 MHz): δ 2.75
(brs, 4H), 3.13 (brs, 4H), 3.84 (brs, 2H), 6.50 (s, 1H, 3-H), 6.72
(dd (t), J ) 7.0 Hz, 1H, 6-H), 6.75 (d, J ) 9.0 Hz, 2H), 6.84 (d,
J ) 8.9 Hz, 2H), 7.08 (dd (t), J ) 7.7 Hz, 1H, 5-H), 7.47 (brd, J
) 8.9 Hz, 1H, 4-H), 8.42 (dd, J ) 7.0 Hz, J ) 0.9 Hz, 1H, 7-H).
LC-MS (APCI): m/z 309.2 [M + H]+.
2-[4-(4-(2-Tosylethoxy)phenyl)piperazin-1-ylmethyl]pyrazo-
lo[1,5-a]pyridine (8b). Compound 8b was prepared following the
synthetic protocol for 5. Starting from 7c (95 mg, 0.31 mmol),
ethylene glycol-1,2-ditosylate (342 mg, 0.92 mmol) and potassium
carbonate (25 mg, 0.18 mmol) gave 8b as a pale yellow oil (58
mg, 0.12 mmol, 37%). TLC (CH2Cl2/methanol, 95:5): Rf ) 0.29.
1H NMR (CDCl3, 360 MHz): δ 2.44 (s, 3H), 2.69–2.82 (m, 4H),
3.01–3.15 (m, 4H), 3.82 (s, 2H), 4.06–4.10 (m, 2H), 4.31–4.36 (m,
2H), 6.49 (brs, 1H, 3-H), 6.70 (ddd (dt), J ) 6.9 Hz, J ) 6.9 Hz,
J ) 1.4 Hz, 1H, 6-H), 6.71 (d, J ) 9.2 Hz, 2H), 6.83 (d, J ) 9.2
Hz, 2H), 7.07 (ddd, J ) 8.9 Hz, J ) 6.7 Hz, J ) 1.1 Hz, 1H, 5-H),
7.30–7.35 (m, 2H), 7.47 (brd, J ) 8.9 Hz, 1H, 4-H), 7.78–7.83
(m, 2H), 8.42 (ddd, J ) 7.0 Hz, J ) 2.1 Hz, J ) 1.1 Hz, 1H, 7-H).
LC-MS (APCI): m/z 507.5 [M + H]+.
3-[4-(4-(2-[18F]Fluoroethoxy)phenyl)piperazin-1-ylmethyl]pyra-
zolo[1,5-a]pyridine ([18F]3f). Variation A: [18F]3f was synthesized
by 18F-fluoroethylation as described for [18F]3e using 7b (2.5 mg,
16 µmol) as the labeling precursor and 1-[18F]fluoro-2-tosyloxy-
ethane in the presence of sodium methanolate in DMF/methanol
(550 µL, 9:1 v/v). The radiochemical yield of [18F]3f was 87 (
5% after 5 min at 120 °C as determined by radio-HPLC (Lichrosorb
RP18, 250 × 4.6 mm, 1 mL/min, linear gradient from 30 to 70%
methanol in water (0.1% TFA) over 30 min): tR ) 17.0 min). The
workup procedure, including semipreparative HPLC, was performed
as described for [18F]3e. [18F]3f was obtained in a decay-
uncorrected yield of 12 ( 3% after a total synthesis time of 120
min.
Radiochemistry. 3-[4-(6-[18F]Fluoropyridin-2-yl)piperazin-
1-ylmethyl]pyrazolo[1,5-a]pyridine ([18F]3a). No-carrier-added
[18F]fluoride (400–800 MBq) was eluted from a QMA-cartridge
with a solution of Kryptofix 2.2.2 (15 mg) and K2CO3 (15 µL, 1
M) in acetonitrile/water (8:2, 1 mL). The solution was evaporated
using a stream of nitrogen at 85 °C and coevaporated to dryness
with CH3CN (2 × 200 µL). A solution of 3b (5.6 mg, 15 µmol) in
anhydrous DMSO (0.5 mL) was added at 180 °C. The progression
of the reaction was analyzed by radio-HPLC. The radiochemical
yield of [18F]3a was 69 ( 8% after 45 min, as determined by radio-
HPLC from a sample withdrawn from the reaction mixture
(Lichrosorb RP18, 250 × 4.6 mm, 1 mL/min, linear gradient from
Variation B: [18F]3f was synthesized by direct nucleophilic
substitition with no-carrier-added [18F]fluoride in a Kryptofix 2.2.2-
promoted reaction as described above (see [18F]3a). A solution of
8a (6 mg, 12 µmol) in anhydrous CH3CN (0.5 mL) was added to
a reaction vessel containing the dried [K⊂222]+[18F]F- complex
and the reaction mixture was stirred at 85 °C. The progression of
the reaction was analyzed by radio-HPLC. The radiochemical yield
of [18F]3f was 26 ( 7% (n ) 4) after 10 min as determined by
radio-HPLC from a sample withdrawn from the reaction mixture
(Kromasil C8, 250 × 4.6 mm, 1 mL/min, linear gradient from 30
to 100% CH3CN in water (0.1% TFA) over 30 min): tR ) 9.13
min). The workup procedure including semipreparative HPLC was
performed as described for [18F]3h. Starting from [18F]fluoride
(300–800 MBq), [18F]3f was obtained in a decay-uncorrected yield
of <10% after a total synthesis time of about 60 min. Variation B
was used to obtain PBS-formulated [18F]3f for the determination
of serum stability.
25% to 100% CH3CN in water (0.1% TFA) over 30 min; tR
)
7.38 min). The reaction mixture was diluted with water (0.5 mL)
and passed through a C18-cartridge (Sep-PakPlus, Waters), washed
with water (10 mL), and eluted with acetonitrile (1.5 mL) in a
reaction vessel. The solvent was evaporated and the residue was
dissolved in acetonitrile/water (2:3 v/v, 0.5 mL) for injection on a
semipreparative HPLC system. [18F]3a was isolated by semi-
preparartive reversed-phase HPLC (Lichrosorb RP18, 125 × 8 mm,
4 mL/min, CH3CN/H2O (30/70; 0.1% TFA)). The fraction contain-
ing [18F]3a was diluted with water (1:10) and the radiolabeled
product was fixed on a RP18-cartridge (100 mg, Merck). The
cartridge was dried in a nitrogen stream and eluted with acetonitrile
(1 mL). The solvent was evaporated in vacuo and [18F]3a (52–104
MBq) was formulated in phosphate-buffered saline (PBS, 0.3 mL)
for further experimental use. Starting from [18F]fluoride (400–800
MBq), the decay-uncorrected radiochemical yield of [18F]3a was
13 ( 4% (n ) 3, based on [18F]fluoride) after a total synthesis
time of 95 min.
2-[4-(2-(2-[18F]Fluoroethoxy)phenyl)piperazin-1-ylmethyl]pyra-
zolo[1,5-a]pyridine ([18F]3h). No-carrier-added [18F]fluoride (600–
1200 MBq) was eluted from a QMA-cartridge and the drying
process was performed as described above. A solution of 8b (6.1
mg, 12 µmol) in anhydrous CH3CN (0.5 mL) was added to the
dried kryptate ([K⊂222]+[18F]F-) and the reaction mixture was
stirred at 85 °C. The progression of the reaction was analyzed by
radio-HPLC. The radiochemical yield of [18F]3h was 88 ( 4% (n
) 3) after 10 min as determined by radio-HPLC from a sample
withdrawn from the reaction mixture (Kromasil C8, 250 × 4.6 mm,
1 mL/min, linear gradient of 30 to 100% CH3CN in water (0.1%
TFA) over 30 min): tR ) 9.63 min). The reaction was quenched
2-[4-(6-[18F]Fluoropyridin-2-yl)piperazin-1-ylmethyl]pyra-
zolo[1,5-a]pyridine ([18F]3c). The radiosynthesis of [18F]3c was
accomplished in a manner identical to [18F]3a, using precursor 3d
(5.6 mg, 15 µmol) dissolved in anhydrous DMSO (0.5 mL). The

1808 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Prante et al.
by the addition of water (10 mL) and the solution was passed
through a C18-cartridge (Sep-PakPlus, Waters), washed with water
(5 mL), and eluted with CH3CN (1.5 mL). [18F]3h was purified
with HPLC on a Kromasil C-8 column (125 × 8 mm, 4 mL/min,
CH3CN/H2O, 30/70 (0.1% TFA)). [18F]3h (tR ) 4.85 min) was
collected, concentrated by solid-phase extraction (RP18-cartridge,
100 mg, Merck), and eluted with ethanol (1 mL). The solvent was
removed by evaporation and [18F]3h (130–270 MBq) was formu-
lated in PBS (300 µL). The decay-uncorrected radiochemical yield
of [18F]3h averaged 22 ( 2% (n ) 9, based on [18F]fluoride) after
a total synthesis time of 60 min and the specific activity was >40
GBq/µmol as determined by radio-HPLC with monitoring UV
absorbance at 254 nm using authentic 3h as a reference.
Receptor Binding Experiments. Receptor binding experiments
were performed, as described previously.44 Briefly, the dopamine
D1 receptor assay was done with porcine striatal membranes (40
µg/assay tube) and at 0.3 nM [3H]SCH23390 (Kd ) 0.5 nM).
Competition experiments with the human D2long, D2short, D3, and
D4.4 receptors were performed using preparations of membranes
from CHO cells stably expressing the corresponding receptor (6–30
µg/assay tube) and [3H]spiperone at a final concentration of 0.5
nM. The obtained Kd values for [3H]spiperone were of 0.10 nM
for D2long, D2short, and D3 and 0.10–0.13 nM for D4.4. Serotonin
5-HT1A, 5-HT2, and adrenergic R1 binding were measured utilizing
porcine cortical membranes and the selective radioligands [3H]8-
OH-DPAT, [3H]ketanserin, and [3H]prazosin, respectively, each at
a final concentration of 0.5 nM. The resulting competition curves
were analyzed by nonlinear regression using the algorithm in
PRISM (GraphPad Software, San Diego, U.S.A.). The data were
fitted using the sigmoid model to provide IC50 values, which were
transformed to Ki values according to the equation of Cheng and
Prusoff.57
centrifugation for 3 min. The organic layer was analyzed by radio-
TLC using CH2Cl2/methanol (95:5). In some cases, an aliquot of
the organic layer (10 µL) was evaporated to dryness under reduced
pressure and the residue was dissolved in water/CH3CN (1:1 v/v,
50 µL) for radio-HPLC analysis.
In Vitro Autoradiography of Rat Brain Slices. Rat brain slices
were prepared from Sprague–Dawley rats according to protocols
approved by the local Animal Protection Authorities (Regierung
Mittelfranken, Germany, No. 54-2531.31-28/06). Female Spra-
gue–Dawley rats (200–240 g) were housed in groups of 2–3 in
individual cages in a temperature- and air-controlled room with
free access to food and water (Franz-Penzoldt Center, Erlangen).
Animals were maintained on a 12 h light/dark cycle before
experimental use. Autoradiography of rat brain slices was performed
following the procedure of Zhang et al.59 with slight modifications.
Sprague–Dawley rats were sacrificed by decapitation and brains
were removed and frozen in cooled hexane at -70 °C. Coronal
brain sections (20 µm) were cut on a cryostat microtome (HM550,
Microm, Germany) and thaw-mounted on covered glass slides
(Histobond). The brain slices were carefully dried at room tem-
perature and preincubated for 2 × 5 min (50 mM Tris-HCl, 120
mM NaCl, 1 mM EDTA, 5 mM MgCl2, pH 7.4). The sections were
incubated at room temperature for 60 min in assay buffer (50 mM
Tris-HCl, 120 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 0.5 µM
1,3-di-o-tolylguanidine (DTG, sigma receptor agonist), 0.1 µM
pindolol (serotonin receptor antagonist), pH 7.4) containing 110
kBq/mL [18F]3h in the presence and absence of eticlopride (1 µM)
or 10 (1 µM), respectively. Coronal brain sections of the same level
of the hippocampus were preincubated, as described, and incubation
with 1 nM [3H]nemonapride (D2, D3, D4 radioligand, obtained from
Perkin-Elmer, specific activity 82.7 Ci/mmol) in the presence of 1
µM raclopride, to block D2 and D3 receptors, was performed using
the assay buffer, as described above. Nonspecific binding was
measured in the presence of 60 µM sulpiride. Subsequently, slices
were washed two times for 5 min in fresh cold assay buffer and
dipped briefly in ice-cold distilled water. The slices were dried under
a slight stream of air. The brain slices were then covered by a solid
scintillator sheet for real-time autoradiography (µ-Imager, Biospace).
18F radioactivity measurements were started within 2 h after
incubation with a duration of 15 min for each slice. The duration
of 3H radioactivity measurements was 48 h. After 18F autoradiog-
raphy, conventional HE staining of the same sections was per-
formed, allowing definition of regions of interest (ROIs) referring
to the rat brain atlas of Paxinos and Watson.55 The ROIs were then
transferred to the autoradiograms for determination of intensities
(cpm/mm2) within each brain region using the software provided
by the manufacturer (Beta-Vision Software, Biospace).
Mitogenesis Assay. The mitogenesis experiments were per-
formed with a CHO10001A cell line stably transfected with the
rat dopamine D4.2 receptor.58 In brief, cells were grown in MEM
R-medium supplemented with fetal calf serum (FCS), L-glutamine,
penicillin G, streptomycin, and hygromycin B at 37 °C under a
humidified atmosphere of 5% CO2 at a density of 10000 cells/well.
After 72 h, the growth medium was removed and the cells were
rinsed twice with serum-free medium. Incubation was started by
the addition of seven different concentrations of each test compound
(with a final concentration of 0.01–10000 nM) diluted in sterile
water (10 µL) to each well containing serum-free medium (90 µL).
Eight wells of every plate contained 100 µL serum-free medium
or medium supplemented with 10% FCS to control stimulation of
growth. After incubation for 20 h, [3H]thymidine (0.02 µCi, specific
activity ) 25 Ci/mmol) in serum-free medium (10 µL) was added
to each well and incubation was proceeded for 2 h at 37 °C. Finally,
cells were trypsinized and harvested onto GF/C filters using an
automated cell harvester. Filters were washed four times with ice-
cold PBS buffer and counted in a microplate scintillation counter.
Determination of Partition Coefficient (log D7.4). The lipo-
philicity of 18F-labeled radioligands was assessed by determination
of the water-octanol partition coefficient following the procedure
described previously.54 1-Octanol (0.5 mL) were added to a solution
of approximately 25 kBq of the 18F-labeled compound in PBS (0.5
mL, pH 7.4) and the layers were vigorously mixed for 3 min at
room temperature. The tubes were centrifuged (3000 rpm, 10 min)
and three samples of 100 µL of each layer were counted in a gamma
counter (Wallac Wizard). The partition coefficient was determined
by calculating the ratio cpm (octanol)/cpm (PBS) and expressed
as log D7.4 ) log(cpmoctanol/cpmbuffer). For each test ligand, 2–3
independent experiments were performed in triplicate and data were
provided as mean values ( standard deviation.
Acknowledgment. The authors thank Professor Dr. Ingmar
Blümcke (Institute of Neuropathology, Erlangen) for his expert
support in the anatomical determination of rat brain regions and
Mrs. Bianca Weigel for technical assistance. We thank Dr. J.-
C. Schwartz and Dr. P. Sokoloff (INSERM, Paris), Dr. J. Shine
(The Garvan Institute of Medical Research, Sydney) and the
late Dr. H. H. M. Van Tol (Clarke Institute of Psychiatry,
Toronto) for providing D3, D2, and D4.4 receptor expressing cell
lines, respectively, and Dr. R. M. Huff (Pfizer, Kalamazoo, MI)
for providing D4.2 receptor expressing cells. We gratefully
acknowledge Wilhelm Hamkens, Marco Pschierer, and Jan
Kubin (PET Net GmbH, Erlangen) for producing and delivering
[18F]fluoride and for excellent collaboration. This work was
supported by the Deutsche Forschungsgemeinschaft (DFG, PR
677/2-1 and PR 677/2-2).
Stability in Human Serum. The serum stability of 18F-labeled
compounds was evaluated by incubation in human serum at 37 °C
for up to 90 min. An aliquot of the PBS-formulated 18F-labeled
compound (20 µL, 250 MBq/mL) was added to a sample of human
serum (200 µL), and the mixture was incubated at 37 °C. Samples
of 10 µL each were taken after designated periods (10–90 min)
and quenched in methanol/CH2Cl2 (1:1 v/v, 100 µL) followed by
Supporting Information Available: Experimental procedures
and analytical data for compounds 1a,b, 2b, 4a, 5, and 6, and
tabulated HPLC and HRMS data for compounds 3a-3h, 7a, 7b,
and 8b. This material is available free of charge via the Internet at
http://pubs.acs.org.

D4 Radioligands for PET
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1809
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Larsen, A. K.; Christoffersen, C. T.; Ramirez, T.; Marcus, M. M.;
Svensson, T. H.; Di Matteo, V.; Esposito, E.; Bang-Andersen, B.; Arnt,
J. Lu 35-138 ((+)-(S)-3-{1-[2-(1-acetyl-2,3-dihydro-1H-indol-3-yl)
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D4 receptor antagonist and serotonin reuptake inhibitor: Characteri-
sation of its in vitro profile and pre-clinical antipsychotic potential.
Eur. J. Pharmacol. 2007, 573, 148–160.
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