Evaluation of 18F-labeled benzodioxine piperazine-based dopamine D4 receptor ligands: Lipophilicity as a determinate of nonspecific binding

Article abstract of DOI:10.1021/jm200762g

Derivatization of the putative neuroleptic 1-(2,3-dihydrobenzo[1,4]dioxin- 6-yl)-4-(4-fluorobenzyl)-piperazine (3a) led to a series of new dopamine receptor D₄ ligands displaying high affinity (K_i = 1.1-15 nM) and D₂/D

Full text of DOI:10.1021/jm200762g

Article
pubs.acs.org/jmc
18
Evaluation of F-Labeled Benzodioxine Piperazine-Based Dopamine
D4 Receptor Ligands: Lipophilicity as a Determinate of Nonspecific
Binding
Fabian Kugler,† Wiebke Sihver,† Johannes Ermert,† Harald Hubner,‡ Peter Gmeiner,‡ Olaf Prante,§
̈
̈
and Heinz H. Coenen*,†
†Institute of Neuroscience and Medicine, INM-5, Nuclear Chemistry, Forschungszentrum Julich GmbH, Julich, Germany
̈
̈
‡Department of Chemistry and Pharmacy and §Laboratory of Molecular Imaging, Friedrich-Alexander University, Erlangen, Germany
ABSTRACT: Derivatization of the putative neuroleptic 1-
(2,3-dihydrobenzo[1,4]dioxin-6-yl)-4-(4-fluorobenzyl)-
piperazine (3a) led to a series of new dopamine receptor D4
ligands displaying high affinity (Ki = 1.1−15 nM) and D2/D4
subtype selectivities of about 800−6700. These ligands were
labeled with the short-lived positron emitter fluorine-18 and
analyzed for their potential application for imaging studies by
positron emission tomography (PET). In vitro autoradio-
graphy was used to determine their nonspecific binding
behavior as a result of their structural and thus physicochem-
ical properties. The biodistribution, in vivo stability, and brain uptake of the most promising D4 radioligand candidate were
determined. This proved to be 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-((6-fluoropyridin-3-yl)methyl)piperazine ([18F]3d),
which revealed an excellent binding pattern with a high selectivity and limited nonspecific binding in vitro. This analogue also
exhibited a high stability and an extremely high brain uptake in vivo with specific binding in hippocampus, cortex, colliculus, and
cerebellum as determined by ex vivo autoradiography. Thus, [18F]3d appears as a suitable D4 radioligand for in vivo imaging,
encouraging continued evaluation by PET studies.
methyl)-3-methylbenzamide (PD-168077),16 one of the first
selective D4 agonists developed by Glase in 1997, up to various
D4-selective compounds developed by Gmeiner’s group
INTRODUCTION
■
From the five known subreceptors of the dopaminergic system
the D4 receptor exhibits a specific pharmacological profile,1,2
including 2-[4-(4-chlorophenyl)piperazin-1-ylmethyl]pyrazolo-
and an extensive scope of polymorphism of the human
[1,5-a]pyridine (FAUC 213)17 and the benzoimidazole 2-[4-
(3,4-dimethylphenyl)piperazin-1-ylmethyl]-1H-benzoimidazole
(A-381393),18 the basic structure consists of a piperidine or, in
most cases, a piperazine backbone with two nitrogen-linked
aromatic rings, one of them being an arylamine. The D4
subtype selectivity of compounds under investigation is
strongly dependent on the presence of a methylene linker
that defines the distance between the piperazine ring and the
lipophilic moiety. The relation between structure and binding
behavior was meanwhile also substantiated by CoMFA studies
and integrated in a pharmacophore model.17,19
dopamine receptor D4 (DRD4) gene is known.3,4 It is
attributed to an extremely low density in brain.5,6 Therefore,
there is still ambiguity about physiological and pathophysio-
logical functions as well as exact localization and distribution
density of this subtype,7,8 although the dopamine D4 receptor
was first cloned in 1991.1 Immunohistochemistry and in vitro
hybridization led to contradictory findings and differences
between species but implicated an expected higher expression
of D4 in the prefrontal cortex and the limbic system and also in
the temporal cortex, parts of tectum, and cerebellum.7−11
The D4 receptor became of more interest, since it had
become clear that the atypical neuroleptic clozapine showed a
10-fold higher affinity for D4 than for D2 receptor.1 Therefore,
higher efficacy of clozapine to nonresponders as well as to
therapy of negative symptomatology was discussed as a
consequence of D4 binding. The fact that further studies did
not reproducibly confirm a direct relation between schizo-
phrenia and the D4 receptor density,12,13 which could also be a
result of a lack of true D4 antagonists, indicated that the role in
psychiatric diseases remains questionable. Nevertheless, up to
now various subtype selective ligands were developed for the
D4 receptor.14,15 From N-([4-(2-cyanophenyl)piperazin-1-yl]-
A variety of D4 agonists showed a proerectile effect in animal
experiments,20,21 thereby confirming the role of D4 receptors
on the physiology of sexual functions. It is assumed that
differences in haplotypes of the D4 receptor that show a wide
range of polymorphic tandem repeats of the third cytoplasmic
loop are responsible for libido dysfunctions and for other
neurobehavioral disorders like attention deficit hyperactivity
disorder, novelty seeking, and substance abuse.22 Different
Received: June 14, 2011
Published: October 31, 2011
© 2011 American Chemical Society
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a
Scheme 1. General Synthetic Scheme of Title Structures and Reference Compounds
a
Reaction conditions: CH3OH, CH3COOH, NaBH3CN, 60 °C, 24 h.
binding porperties of D4 ligands with respect to the distinct D4
fluorophenyl)piperazin-1-ylmethyl)indole-5-carbonitrile
(FAUC 316). However, preliminary studies showed that its use
as in vivo radioligand failed because of its high lipophilicity.
Thus, dihydrobenzo[1,4]dioxine-1-(2,3-dihydrobenzo[1,4]-
dioxin-6-yl)-4-(4-fluorobenzyl)piperazine (3a) was chosen as
alternative lead structure with the goal to develop less lipophilic
derivatives while maintaining the affinity, and three new
derivatives thereof were synthesized and chemically charac-
terized according to Scheme 1.
receptor polymorphism have been attested.23
Noninvasive imaging using selective radioligands for the D4
receptor by positron emission tomography (PET) or single
photon emission computer tomography (SPECT) provides in
principle the opportunity to investigate the D4 receptor
expression in the living human brain with high sensitivity.
From the existing radioligands to determine D4 activity in brain,
tritiated ligands like 2-{4-[(2-[3H]phenyl-1H-imidazol-4-yl)-
methyl]piperazin-1-yl}pyrimidine ([3H]NGD 94-1)24 are of
course not applicable for in vivo imaging, while other suitably
labeled candidates require the blockade of interfering receptor
subtypes because of their insufficient selectivity, as shown by
Boy et al.25 Another major problem is the inadequate ratio of
specific to nonspecific and unspecific binding,26,27 which is
most obtrusive because of the very low D4 receptor density. A
decrease of specific binding to non-D4 receptors (unspecific
binding) could be reached by a better understanding of the
above-mentioned structure−activity relationship (SAR) which
led to ligands with high affinity and selectivity.19,28 However,
the nonspecific binding of D4 radioligand candidates, such as
binding to brain lipids which is hardly reversible and barely
displaceable by nonradioactive ligands, has not yet been
considered adequately by SAR studies and is difficult to
predict. It is anticipated that nonspecific binding correlates with
the lipophilicity of the substance. In fact, such a correlation is
sometimes observed,29−31 and in a recent study on analogues of
β-carboline harmine32 a similar comparison of in vitro data and
lipophilicity was performed.
As a typical D4 ligand lead structure, according to SAR
findings, the fluorinated D4 antagonist 1-(2,3-dihydrobenzo-
[1,4]dioxin-6-yl)-4-(4-fluorobenzyl)piperazine 3a shows an
outstanding subtype selectivity of D2/D4 > 5000.33 The aim
of the present study was to determine the suitability of this
compound to act as a D4 receptor ligand for imaging studies,
when labeled with fluorine-18. Further derivatives of the lead
compound were developed and studied for their dopamine
receptor affinities and functional activity and with special
interest on the determination of specific to nonspecific binding
ratios by means of in vitro autoradiography. As predicted from
the in vitro data, the most suitable radioligand was selected for
determination of its biodistribution, in vivo stability, and brain
uptake. Together with the in vitro and ex vivo radioligand
autoradiography, the results were evaluated in order to assess
the suitability of a new D4 radioligand candidate for in vivo
imaging.
Analogous to the description of Liu et al.,35 the intermediate
1-(1,4-benzodioxine-6-yl)piperazine (1) was synthesized via
cyclization of the commercially available aminodioxine 2,3-
dihydrobenzo[b][1,4]dioxin-6-amine with bis(2-chloroethyl)-
amine hydrochloride.36 Reductive amination with the appro-
priate aldehydes 2a−d led to a series of new 3-substituted 6-(4-
[4-fluorobenzyl]piperazine-1-yl)benzodioxine derivatives ob-
tained in 20−85% yield. They all were used as reference
compounds for chromatographic identification of radioactive
products, for determining their molar activities, and as
competitors for autoradiographic evaluation studies. The 18F-
labeled ligands were obtained in no-carrier-added form by the
same buildup syntheses starting with nucleophilic substitution
with nca [18F]fluoride of analogous benzylaldehydes 2a−c as
precursors, where R2+ represents the trimethylammonium
triflate moiety (-NMe3 TfO−), or chlorine in the case of 2d,
as aromatic leaving group. Optimization of labeling conditions,
comprising variation of temperature, solvent, and anion
activating system, is given elsewhere together with details on
the syntheses of standards, intermediates, and precursors for
radiofluorination.36 The radiochemical yields of the products
[18F]3a−d obtained after both reaction steps, i.e., radiolabeling
and subsequent amination, are summarized in Table 1. In the
first step the nca labeled fluoroaldehydes [18F]2a−d were
obtained with radiochemical yields of 72−80%.
Table 1. Total Radiochemical Yields of [18F]3a−d after
Radiofluorination and Amination in a One-Pot Reaction
[18F]
fluoroaldehyde
final
product
radiochemical yield
(%)
total synthesis time
(min)
[18F]2a
[18F]2b
[18F]2c
[18F]2d
[18F]3a
[18F]3b
[18F]3c
[18F]3d
35 ± 5
20 ± 5
9 ± 4
∼90
∼90
∼120
∼80
15 ± 5
In contrast to the macroscopic synthesis of the ligands, the
reductive amination with the nca intermediate [18F]fluoro-
aldehydes could also be performed in DMSO instead of
methanol. Labeling and amination could therefore be
performed as one-pot reaction. The nonsubstituted and the
methoxy compound ([18F]3a and [18F]3b) were generated in
RESULTS AND DISCUSSION
■
Ligands and 18F-Labeled analogues. Continuing the
efforts on the development of 18F-labeled D4 selective
radioligands for in vivo use34 led to 18F-labeling of 2-(4-(4-
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Table 2. Binding Affinities of the Derivatives 3a−d to the Human Dopamine Receptor Subtypes D2long, D2short, D3, and D4.4, the
Porcine D1 Receptor, and the Porcine 5-HT1A, 5-HT2, and α1 Receptors
a
Ki ± SD (nM)
b
b
b
b
c
d
e
f
compd
R
hD2long
hD2long
hD3
hD4.4
pD1
p5-HT1A
p5-HT2
pα1
3a
3b
3c
3d
H
7600 ± 570
5700 ± 1300
16000 ± 2100
6100 ± 71
3900 ± 71
1.1 ± 0.56
4.2 ± 0.071
7.3 ± 2.6
19000 ± 11000
23000 ± 4200
15000 ± 3600
5200 ± 2200
5600 ± 2100
6200 ± 710
7000 ± 5700
4200 ± 2100
2300 ± 640
4000 ± 780
3300 ± 350
3000 ± 210
11000 ± 710
OMe
OH
17000 ± 1400
9000 ± 1100
29000 ± 2000
5900 ± 640
4200 ± 1300
17000 ± 710
45000 ± 710
15 ± 0.71
35000 ± 4200
21000 ± 2100
5800 ± 2100
a
b
c
d
e
Ki values are the mean values of two independent erxperiments each done in triplicate. [3H]spiperone. [3H]5. [3H]4. [3H]ketanserin.
f
[3H]prazosin.
good radiochemical yields of 20−35% after HPLC separation
3b, 3c) led to a minor decrease in D4 affinity of these
compounds. The displacement of the fluorophenyl group by
the pyridinyl substituent 3d induced a minor decrease in D4
affinity by a factor of about 10. However, the D4 subtype
selectivity of >1000 (D4/D3) and >2000 (D4/D2) for 3d was
still adequate and comparable to the values of the previously
described fluoro-substituted pyrazolo[1,5-a]pyridine.34 The
subtype selectivity was high for all derivatives (Table 3) but
highest for 3a, followed by 3b, 3d, and 3c. Remarkably, 3a
displayed a Ki ratio of more than 5000 for D2 (D4/D2) and
3500 for D3 (D4/D3), which is, to our knowledge, one of the
highest selectivities for D4 over D3 reported up to now.
In addition, the data on receptor affinities of the series of
compounds with functional data were extended using our
previously described mitogenesis assay.42 The agonist activation
of dopamine receptors is known to increase mitogenesis in
heterologously transfected cell lines, measuring the rate of
proliferation in growing cells.43 Since agonist ligands
preferentially bind to the active GPCR conformation (high-
affinity state) and antagonists have equal affinity for the active
and inactive state, it is important to determine the intrinsic
activity for a putative radioligand. These data could also help to
interpret differences between agonist and antagonist radio-
ligands in the in vivo binding behavior. Therefore, we
investigated the intrinsic activity of the series of title
compounds by measuring the [3H]thymidine incorporation
into growing CHO cells stably expressing the dopamine D4
receptor.43 In fact a neutral antagonism was determined for all
title compounds under investigation in comparison with the full
agonist quinpirole as a reference.
and with molar activities of about 60 GBq/μmol. Since no
labeling yield could be observed using the trimethylammonium
precursor of the hydroxy compound [18F]2c, the benzyloxy
analogue was used instead. Therefore, the low RCY of only 9%
and a molar activity of only 30 GBq/μmol for [18F]3c were due
to the additional step necessary for cleaving the benzyl
protecting group. [18F]Fluoropyridine ([18F]2d) is sensitive
to degradation; thus, the RCY and molar activity of radioligand
[18F]3d were also rather low (cf. Table 1).
Receptor Binding and Intrinsic Activities. The lead
compound 3a and its derivatives 1-(2,3-dihydrobenzo[1,4]-
dioxin-6-yl)-4-(4-fluoro-3-methoxybenzyl)piperazine (3b), 5-
[4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)piperazin-1-ylmethyl]-2-
fluorophenol (3c), and 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-
yl)-4-((6-fluoropyridin-3-yl)methyl)-piperazine (3d) were sub-
jected to receptor binding studies to test for their ability to
displace [3H]spiperone from the cloned human dopamine
37
receptors D2long, D2short
,
D3,38 and the most common D4
polymorphism D4.4,39 being stably expressed in Chinese
hamster ovary (CHO) cells.40
Additionally, the metabotropic receptors 5-HT1A, 5-HT2A,
and α1, from which it is known that they could reveal
competing binding affinity for putative D4 ligands, were
measured utilizing porcine cortical membranes and the selective
radioligands N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-
N-(2-pyridyl)cyclohexanecarboxamide ([3H]4, [3H]-
WAY100635), [3H]ketanserin, and [3H]prazosin, respec-
tively.41 The highly expressed but structurally and functionally
different dopaminergic receptor D1 was measured using porcine
striatal membranes and the radioligand 7-chloro-3-methyl-1-
phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol ([3H]5, [3H]-
SCH23390). The results of the receptor binding studies are
presented in Table 2.
Because of the promising high D4 receptor affinity and
distinct D4 receptor subtype-selectivity of 3a, 3b, 3c, and 3d,
the radiosyntheses of the corresponding 18F-labeled antagonist
radioligands appeared attractive.
Pharmacology. High selectivity, good uptake and reten-
tion, stability, and low unspecific and nonspecific binding in the
target tissue are key requirements for the suitability of
radioligands in PET studies. While most features can be tested
with the unlabeled compounds, there is little indication for their
nonspecific binding behavior prior to labeling. This may be one
reason why the latter is often a knockout criterion for many
radiotracers that fulfill the former requirements. The radio-
ligand [18F]3a failed in this respect, since it exhibited a
nonspecific binding of >90%. The apparent lipophilicity of 3a
prompted us to increase the hydrophilicity, resulting in the
design of title compounds 3b−d. The results from in vitro
competition experiments of all derivatives directed the selection
of a promising radioligand for further in vivo studies.
All ligands showed high affinity to D4 in the low nanomolar
range and weak affinities to all other tested receptors (Table 2).
Among the series of compounds, the lead compound 3a
showed highest affinity to the D4 receptor combined with an
exceptional D4 subtype selectivity (Table 3). The introduction
of a methoxy or hydroxy group to the fluorophenyl moiety (see
Table 3. D4 Subtype Selectivity within the Family of D2-like
Receptors
ratio Ki(D2 or D3)/Ki(D4.4
)
compd
Ki(hD4.4) (nM)
hD2long/hD4.4
hD2short/hD4.4
hD3/hD4.4
3a
3b
3c
3d
1.1
4.2
7.3
15
6700
4000
1300
2000
5000
3800
830
3600
1400
590
Lipophilicity. As an adequate estimation of this phys-
icochemical property, the MarvinSketch 5.1.4 software44 and
the ALOGPS 2.1 software45 were used to calculate log P values,
3000
1100
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Table 4. Calculated and Experimental log P7.4 of the Derivatives 3a−d
parameter
3a
3b
3c
3d
calcd MarvinSketch
calcd ALOGPS
exptl HPLC
3.35
3.09
2.87
2.49
3.23 ± 0.27
2.71 ± 0.03
nd
3.11 ± 0.45
2.44 ± 0.07
2.33 ± 0.09
2.81 ± 0.37
1.70 ± 0.15
1.78 ± 0.01
2.26 ± 0.32
2.02 ± 0.17
1.81 ± 0.05
exptl octanol/buffer
which were estimated to be between 2.3 and 3.4 (Table 4). The
two different algorithms showed similar results. However,
experimental log P values, ranging from 1.7 to 2.7, were clearly
lower than the theoretical ones. Experimental values were also
obtained by two different methods at pH 7.4, i.e., shake flask
and HPLC method, according to the OECD guidelines for the
testing of chemicals.46 The obtained log P values were
determined to be between 2 and 3, a range that is proposed
for ligands to penetrate the blood−brain barrier, suggesting
sufficient brain uptake of the corresponding radioligands.
Autoradiography. A high ratio of nonspecific to specific
binding is the main reason for radiotracer failure in vivo. In this
work the nonspecific binding of the radioligands was
determined by using an excess of the corresponding non-
radioactive reference ligand. Since a predominant contribution
to nonspecific binding is the binding to phospholipids, the
degree of nonspecific binding is often related to the lipophilicity
of compounds. The reason why this correlation cannot be
generally applied to all radioligands may be due to possible
structural influences of small molecules on lipid bilayers.47 In
fact, the mechanism of nonspecific binding is still not
completely understood.
the postulated relationship30,31,33 between nonspecific binding
and lipophilicity was found (Figure 1).
The more lipophilic radioligands [18F]3a and [18F]3b
showed unacceptable high nonspecific binding of 96% and
79%, respectively, and seem therefore not suitable as radio-
tracers. In contrast, nonspecific binding of [18F]3c was only
33%, and [18F]3d showed even a lower degree of nonspecific
binding of only 7%. Although [18F]3d exhibited a lower D4
affinity than the other ligands, it lends itself to further ex vivo
studies because of its high selectivity and low nonspecific
binding.
In vitro autoradiography studies of the more hydrophilic
compounds [18F]3c and [18F]3d showed generally a specific
binding throughout the whole rat brain. Both radioligands
displayed a distinct high accumulation in the colliculus and in
the medial nuclei of the cerebellum. As presented in Figure 2a,
there is no marked higher accumulation in hippocampus,
frontal cortex, and striatum. By contrast, in ex vivo auto-
radiography studies with NMRI mice using [18F]3d, high
binding was observed in cortex and hippocampus (Figure 2b).
In comparison with the cresyl violet stained image of the same
slice, the good contrast allows differentiation between the outer
layers of cortex with high binding and lower binding in middle
layers of cortex. In the hippocampus high binding uptake was
observed in dentate gyrus (GD) and cornu ammonis 3 (CA3).
This is in agreement with findings of Prante et al.34 who
described a significant higher uptake in the GD of rat brain
slices with a 18F-labeled pyrazolo[1,5-a]pyridine D4 ligand but
not in the other regions of hippocampus. High uptake in vitro
and ex vivo was observed in regions of the colliculus. This part
of brain, responsible for sorting of optical and acoustical
information, was described as a region where the D4 receptor
should be found7 but often not mentioned as a region of
interest. Otherwise, the high accumulation of [18F]3d in the
cerebellum is contrary to the estimated D4 distribution in brain,
although there is information in the literature on D4R
expressing genes that are also found there.7 However, negligible
uptake of [18F]3d was observed in striatum, which is in
accordance with the low striatal D4 receptor density in rats.6
In order to exclude that different binding of [18F]3d in vitro
and in vivo might be due to differences of species, in vitro
autoradiography of NMRI mouse brain slices was also
performed. Even though a slightly higher binding in cortex
was recognizable here, the contrast of radioligand accumulation
was worse in comparison to the ex vivo autoradiographs.
Possible reasons for that finding could be a metabolisation by a
contingent disposal of enzymes in vitro or by the incubation
buffer used; however, both explanations were rendered false by
experiment. Thus, it was assumed that considerable binding to
intracellular components that are only accessible in vitro may
result in a higher nonspecific background. For a comparison
with an autoradiography from the rare literature showing results
with D4 ligands, sagittal mouse brain slices were chosen and the
ex vivo binding distribution was compared with rat brain slices
of the 131I-labeled derivative of 3-(4-(4-chlorophenyl)-
After determination of suitable affinity and subtype selectivity
to the D4 receptor for all tested ligands, in vitro auto-
radiography studies on horizontal rat brain slices were
performed with the 18F-labeled radioligands to compare the
nonspecific binding values with the lipophilicity (Figure 1). For
this, nonspecific binding was defined as bound 18F-labeled
Figure 1. Relation of experimentally determined log P7.4 values of the
derivatives 3a−d and the nonspecific binding of the 18F-labeled
compounds obtained by blocking studies with the nonradioactive
standards on Wistar rat brain slices in vitro.
compounds in the presence of an excess of the nonradioactive
ligands 3a,b and 3c,d, respectively. No differences in blocking
behavior could be found between blocking with the authentic
reference compound or with one of the other ligands. Thereby,
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Figure 2. (a) In vitro autoradiography of a rat brain (A) with 5 nmol/L [18F]3d and after blocking with 10 μM 3d (B) to show nonspecific binding.
Sagittal slices of in vitro (C) and ex vivo (D) autoradiography studies of mouse brain with [18F]3d. (b) Ex vivo autoradiography (left) of horizontal
planes of a mouse brain 15 min after injection of [18F]3d and the corresponding histological image stained with cresyl violet (right).
Table 5. Biodistribution Data of [18F]3d in NMRI Mice (n = 3) after Intravenous Injection
% ID/g tissue
5 min
10 min
15 min
30 min
brain
4.93 ± 1.58
1.80 ± 1.00
12.99 ± 2.53
4.13 ± 1.76
8.23 ± 2.85
4.42 ± 1.62
2.16 ± 0.70
8.36 ± 3.55
2.03 ± 0.51
1.62 ± 0.14
2.85 ± 0.14
0.52 ± 0.04
4.84 ± 1.21
6.78 ± 0.28
5.86 ± 0.62
3.60 ± 0.15
2.68 ± 0.19
11.10 ± 0.37
2.54 ± 0.33
1.29 ± 0.16
2.27 ± 0.17
0.58 ± 0.31
4.79 ± 0.68
5.67 ± 0.30
4.67 ± 0.40
3.19 ± 0.20
2.51 ± 0.39
6.88 ± 1.44
2.70 ± 0.89
1.23 ± 0.33
1.47 ± 0.15
1.13 ± 0.35
2.68 ± 0.80
7.79 ± 0.39
6.03 ± 0.61
3.13 ± 0.18
4.47 ± 0.20
7.43 ± 0.56
0.93 ± 1.05
0.53 ± 0.21
heart
lung
liver
kidney
spleen
intestine
pancreas
bone
blood, final
piperazinylmethyl)pyrazolo(1,5-a)pyridine (FAUC113) (Ki =
2.6 nM).48 In both studies similar binding was found in cortex
and thalamus, which affirms binding in the limbic system. Also,
cerebellar binding of [18F]3d was in accordance with that
previous work.
Biodistribution and in Vivo Stability. For biodistribu-
tion studies on [18F]3d the three NMRI mice were each
sacrificed at 5, 10, 15, and 30 min pi. Because of the low
lipophilicity of the ligand, there was a rapid accumulation in the
kidney. As expected, the hepatic uptake was lower (cf. Table 5).
The high accumulation in the pancreas that even exceeded the
kidney uptake attracted attention. This observation may be
explained by the reported D4 receptor expression in pancreatic
β cells.49 A high penetration of the blood−brain barrier was
observed in spite of a log P of 1.8 with a brain uptake of 4.93%
ID/g tissue after 5 min. After 30 min there was still more than
1% ID/g in the brain. The data showed a consistent decrease
(Figure 3); thus, no compartment-trapping might be
responsible for the high uptake.
In vivo stability of the radioligand [18F]3d in the blood of
mice was checked by thin layer chromatography (TLC) on
aluminum oxide layers. To ensure that all potential metabolites
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Figure 4. In vivo stability of [18F]3d in blood plasma of NMRI mice
measured by radio-TLC. The open circle point shows the result of the
experiment with a higher dilution of analyzed plasma extract from
which was known that all measured activity except from the
radioligand-spot was from true metabolites and was not from artifacts.
Figure 3. Time dependence of uptake of [18F]3d in brain of NMRI
mice after iv injection.
are analyzed, the whole blood and plasma activity were
compared as well as plasma activity and that of plasma extract.
After centrifugation of the blood, about 27% of radioactivity
was found in the cell pellet. After deproteination of the plasma
nearly all radioactivity remained in the serum fraction (>98%).
TLC analysis of this on silica gel plates with the chosen solvent
(ethyl acetate/methanol/diethylamine, 96:2:2) showed only
two fractions, one remaining on the origin indicating the
hydrophilic character of the corresponding compound(s) and
that of the radioligand. Furthermore, it is suggested that
defluorination did not take place to a considerable extent
because of the very low bone uptake at all time points pi (see
Table 5). The half-life of in vivo stability in blood was about 6
min.
Metabolization in brain was measured 10 min after
intravenous injection in the same way but using a higher
dilution with acetonitrile during the extraction step, feasible by
using higher amounts of radioactivity. In mouse brain, no
metabolisation of [18F]3d after 10 min pi was detected.
Analyzing the blood plasma of the same animals and also using
a high dilution during extraction led to metabolite findings
comparable to those before.
Therefore, it is expected that the obtained in vivo stability
curve of [18F]3d (Figure 4) does not contain “pseudo-
metabolites” (artifacts), which may occur by retention of the
radioligand by the remaining dried proteins. It can be assumed
that the different metabolization rate indicated was due to a
small degeneration of the ligand on thin layer plates, which only
occurs under no-carrier-added (nca) conditions and which is
rapid on silica but moderate on Alox plates. This additional
degeneration of the nca product [18F]3d on TLC plates was
determined at contact times of 0, 5, 15, and 30 min (n = 3).
The observed half-lives of degeneration on silica were about 15
min only but about 50 min on aluminum oxide. This is why the
latter was used for all radio-TLC analyses.
series of derivatives were labeled with fluorine-18 and analyzed
for their suitability to act as D4 radioligands, focusing on the
determination of their nonspecific binding. Accordingly, there is
a significant correlation between the log P7.4 values of the
derivatives and the extent of nonspecific binding as determined
by competition experiments in vitro. The determined non-
specific binding of 33% and 7% of the two new derivatives
[18F]3c and [18F]3d, respectively, is within an acceptable range.
The brain distribution of the radioligand [18F]3d in mice
determined ex vivo was essentially in accordance with literature
data on D4 receptor distribution except for an unexpected high
cerebellum uptake. However, it seems to be no decisive
handicap that in vitro autoradiographic studies partially showed
a different binding behavior. Nevertheless, rare and contra-
dictory data on D4 receptor density from literature make it
difficult to compare distribution of [18F]3d with other
radioligands for D4.
The radioligand [18F]3d showed an extremely high brain
uptake in mouse brain, a moderate plasma metabolization rate,
but no metabolites in the brain. All in all it can be assumed that
[18F]3d might be a suitable D4 radioligand for in vivo imaging
studies, which encourages continued evaluation in preclinical
animal models by PET.
MATERIALS AND METHODS
Materials. All chemicals and p.a. solvents were purchased from
Aldrich (Steinheim, Germany) or Fluka (Buchs, Switzerland) and used
without further purification.
■
Sep-Pak C-18 Plus cartridges were purchased from Waters
(Eschborn, Germany). EN cartridges and Li-Chrolut glass columns
(65 mm × 10 mm) were from Merck (Darmstadt, Germany). Thin
layer chromatography (TLC) was run on precoated plates of silica gel
60F254 (Merck) or alumina N. The compounds were detected by UV
at 256 nm. HPLC was performed on the following system from
Dionex (Idstein, Germany): an Ultimate 3000 LPG-3400A HPLC
pump, an Ultimate 3000 VWD-3100 UV/vis detector (272 nm), a
UCI-50 chromatography interface, an injection valve P/N 8215.
Reversed-phase HPLC was carried out using a Gemini 5 mm C18
110A column, for analytical separations with a dimension of 250 mm
× 4.6 mm (flow rate of 1 mL/min) and for semipreparative
applications with a dimension of 250 mm × 10 mm (flow 5 mL/
min), from Phenomenex (Aschaffenburg, Germany). Radio-TLC
CONCLUSION
■
Three derivatives of 1-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-4-(4-
fluorobenzyl)piperazine (3a) were characterized as high-affinity
D4 ligands with substantial D4 selectivity in vitro. It was
observed that ortho-substitution of the carbon-linked aryl
moiety caused no major influence on the binding profile. The
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Journal of Medicinal Chemistry
Article
chromatograms were analyzed by UV detection and on a Packard
instant imager. All labeled ligands [18F]3a−d showed a radionuclidic
and radiochemical purity of >98%. 1H, 13C, and 19F NMR spectra were
recorded on a Bruker DPX Avance 200 spectrometer with samples
dissolved in CDCl3 or DMSO-d6. All shifts are given in γ ppm using
the signals of the appropriate solvent as a reference. Mass spectra were
obtained from a Finnigan Automass Multi mass spectrometer with an
electron beam energy of 70 eV. High resolution electron spray mass
spectra were recorded on an LTQ FT Ultra (Thermo Fischer).
Melting points are uncorrected and were determined on a Mettler FP-
61 apparatus in open capillaries. Elemental analysis (CHN) results
were recorded on a Leco CHNS-932 analyzer. The obtained purity for
all ligands 3a−d was >95%.
Chemistry. General Procedure for Preparation of 6-(4-
Benzylpiperazine-1-yl)benzodioxine Derivatives 3a−d. In a 100
mL two-neck flask 1-(1,4-benzodioxine-6-yl)piperazine (2) (1.816
mmol, 1 equiv) and the corresponding benzaldehyde (2a−d, 2.724
mmol, 1.5 equiv) were dissolved in 20 mL of dry methanol. After an
addition of acetic acid (96%, 5.45 mmol, 3 equiv) sodium
cyanoborohydride (2.724 mmol, 1.5 equiv) was added in small
portions and rinsed with methanol. The mixture was heated for 12−24
h at 60 °C. After the mixture was cooled, the reaction was quenched
by addition of 50 mL of saturated sodium bicarbonate solution, which
was extracted three times with chloroform and washed with brine. The
combined organic layer was dried over sodium sulfate and evaporated
to dryness after filtration. The residue was purified by chromatography
over a silica gel column (n-hexane/ethyl acetate, 1:2).
The corresponding trimethylammonium triflate precursor of the
aldehydes 2a,b (20−25 μmol), the benzyl protected trimethylammo-
nium triflate analogue of 2c (7 μmol) or the chloro derivative of 2d
(35 μmol) in 0.5 mL of anhydrous DMSO was added. Completion of
the 18F-substitution reaction was monitored by radio-HPLC (Gemini
5 μm RP18 A110, 250 mm × 4.6 mm, 1 mL/min, isocratic 60:40:0.1
v/v/v CH3CN/H2O/TEA). In the case of the benzyl protected
derivative, palladium (black) (20 mg) and ammonium formate (250
mg) were added after radiofluorination for cleavage of protection
groups to obtain [18F]2c.
Then a solution of intermediate 1 (8.7 mg, 40 μmol) in 40 μL of
acetic acid and 50 μL of DMSO and a solution of sodium
cyanoborohydride (4 mg, 64 μmol) in 50 μL of DMSO were added
to the radiofluorinated aldehydes. Different from the preparative
amination reaction (cf. Scheme 1) DMSO at a temperature of 110 °C
was used in both steps of the nca labeling process. Subsequently the
reaction mixture was diluted with water and passed through a SepPak
C18 cartridge, washed with water (5 mL), and dried with air. The
cartridge was eluted with 1 mL of acetonitrile and the eluate injected
on a semipreparative HPLC column (Gemini 5 μm RP18 A110, 250
mm × 10 mm, 4 mL/min, isocratic 30:70:0.1 v/v/v CH3CN/H2O/
TFA). The separated fraction was diluted with 15 mL of water and
passed through a second SepPak C18 cartridge. After washing with
water and drying in an argon stream, the cartridge was eluted with 5
mL of diethyl ether which was evaporated in vacuo (800 up to 330
mbar) to yield dry [18F]3a−d. For in vitro studies all derivatives were
formulated in 300 μL of ethanol/saline (5:1) while an amount of 300
μL of saline with 1% Tween 80 was employed for ex vivo studies.
Receptor Binding Experiments. Receptor binding studies were
carried out as previously described.39 In brief, the dopamine D1
receptor assay was done with porcine striatal membranes at a final
protein concentration of 30 μg/assay tube and the radioligand [3H]5
at 0.5 nM (KD = 0.56 nM). Competition experiments with human
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-4-(4-fluorobenzyl)-
piperazine (3a). 3a was obtained as a white solid (315 mg, 0.96
mmol, 60%) from commercially available 4-fluorobenzaldehyde (2a)
(200 mg, 1.61 mmol). Mp 120 °C. TLC (n-hexane/ethyl acetate, 1:2):
Rf = 0.7. 1H NMR (CHCl3, 400 MHz) δ 2.56 (t, J = 4.8 Hz, 4H), 3.06
(t, J = 4.8 Hz, 4H), 3.50 (s, 2H), 4.19 (m, J = 4.5 Hz, 2H), 4.21 (m, J =
4.6 Hz, 2H), 6.43 (m, 2H), 6.75 (m, 1H), 6.98 (m, 1H), 7.29 (m, 2H)
ppm. FT-MS (ESI): m/z 329.166 [M + H]+.
D
2long, D2short,
37 D3,38 and D4.440 receptors were run with preparations
of membranes from CHO cells stably expressing the corresponding
receptor and [3H]spiperone at a final concentration of 0.2 nM. The
assays were carried out at a protein concentration of 2−6 μg/assay
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-4-(4-fluoro-3-
methoxybenzyl)piperazine (3b). The methoxy derivative was
obtained from the commercially available 4-fluoro-3-methoxybenzal-
dehyde (2b) (420 mg, 2.72 mmol) as a pale yellow crystalline solid
(551 mg, 1.54 mmol, 85%). Mp 100 °C. TLC (n-hexane/ethyl acetate,
tube and KD values of 0.091, 0.090, 0.11, and 0.15 nM for the D2long
,
D
2short, D3, and D4.4 receptors, respectively. 5-HT and α1 receptor
binding experiments were performed with homogenates prepared from
porcine cerebral cortex as described.41 Assays were run with
membranes at a protein concentration per each assay tube of 80,
110, and 40 μg/mL for 5-HT1A, 5-HT2, and α1 receptor, respectively,
and radioligand concentrations of 0.2 nM for [3H]prazosin, 0.5 nM for
[3H]ketanserin, and 0.3 nM for [3H]4 with KD values of 0.30 nM for
5-HT1A, 0.71 nM for 5-HT2, and 0.16 nM for the α1 receptor. Protein
concentration was established by the method of Lowry using bovine
serum albumin as standard.52
Mitogenesis Assay. Determination of the intrinsic activity of the
representative compound at the dopamine D4.2 receptor was carried
out by measuring the incorporation of [3H]thymidine into growing
cells after stimulation with the test compound as described in the
literature.42,53 For this assay D4.2 expressing CHO10001 cells have
been incubated with 0.02 μCi [3H]thymidine per well (specific activity
of 25 μCi/mmol, Biotrend, Cologne, Germany). The data of four dose
response experiments for each compound have been normalized and
pooled to get mean values for each concentration tested. These data
showed a neutral antagonist effect for each compound compared to
the effects of the full agonist quinpirole.
Data Analysis. The resulting competition curves of the receptor
binding experiments were analyzed by nonlinear regression using the
algorithms in PRISM 3.0 (GraphPad Software, San Diego, CA). The
data were fitted using a sigmoid model to provide an IC50 value,
representing the concentration corresponding to 50% of maximal
inhibition. IC50 values were transformed to Ki values according to the
equation of Cheng and Prusoff.54
As a reference, binding curves resulting from the mitogenesis assay
with the full agonist quinpirole were analyzed by nonlinear regression.
All data of the reference and of the test compounds were normalized
(basal effect of 0%; maximum effect of the full agonist quinpirole of
1
1:2): Rf = 0.44. H NMR (CHCl3, 400 MHz) δ 2.56 (t, J = 4.8 Hz,
4H), 3.06 (t, J = 4.8 Hz, 4H), 3.48 (s, 2H), 3.88 (s, 3H), 4.18 (m, J =
1.6 Hz, 2H), 4.20 (m, J = 1.6 Hz, 2H), 6.44 (m, 1H), 6.45 (m, 1H),
6.75 (m, 1H), 6.82 (m, 1H), 6.98 (m, 2H) ppm. FT-MS (ESI): m/z
359.18 [M + H]+.
5-((4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)piperazin-1-yl)-
methyl)-2-fluorophenol (3c). The hydroxyl derivative was obtained
from 4-fluoro-3-hydroxybenzaldehyde (2c) (70 mg, 0.5 mmol) as a
white crystalline solid (112 mg, 0.35 mmol, 66%). Mp 186−187 °C.
1
TLC (n-hexane/ethyl acetate, 1:2): Rf = 0.35. H NMR (CHCl3, 400
MHz) δ 2.57 (m, 4H), 3.06 (m, 4H), 3.45 (s, 2H), 4.18 (m, 2H), 4.20
(m, 2H), 6.44 (m, 2H), 6.74 (m, 1H), 6.79 (m, 1H), 6.98 (m, 2H),
9.84 (br, 1H) ppm. FT-MS (ESI): m/z 345.19 (100) [M + H]+.
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-4-((6-fluoropyridin-3-yl)-
methyl)piperazine (3d). Method A. 3d was obtained from 6-
fluoronicotinaldehyde (2d) (100 mg, 0.8 mmol) as a white crystalline
solid (35 mg, 0.11 mmol, 13.7%). Mp 94 °C. TLC: (chloroform/
methanol, 8:1) Rf = 0.63; (ethyl acetate/methanol/diethylamine,
96:2:2): Rf = 0.78 . 1H NMR (CHCl3, 400 MHz) δ 2.52 (br, 4H), 3.00
(br, 4H), 3.47 (s, 2H), 4.14 (m, 2H), 4.16 (m, 2H), 6.38 (m, 2H), 6.71
(m, 1H), 6.84 (dd, J = 2.8 Hz/8.4 Hz, 1H), 7.74 (ddd, J = 2.3 Hz/8.0
Hz/8.1 Hz, 1H), 8.09 (d, J = 1.9 Hz, 1H) ppm. FT-MS (ESI): m/z
330.16 [M + H]+.
General Procedure for the Syntheses of No-Carrier-Added
Labeled 6-(4-[18F]Fluorobenzylpiperazine-1-yl)benzodioxine Deriv-
atives [18F]3a−d. The nca [18F]fluoride was produced via the
18O(p,n)18F nuclear reaction with 17 MeV protons at the JSW
cyclotron BC 1710 of Forschungszentrum Juelich50 and transferred
into a dry Kryptofix 2.2.2 potassium carbonate complex by standard
procedures.51
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Journal of Medicinal Chemistry
Article
100%) and then combined to get mean values. If an agonist effect was
detectable, nonlinear regression analysis of the resulting curve
provided the EC50 value representing the concentration corresponding
to 50% of maximal stimulation as a measure of potency.
In Vivo Stability of 1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-
4-((6-[18F]fluoropyridin-3-yl)methyl)piperazine ([18F]3d). In
order to determine the stability of [18F]3d in mouse serum, removed
blood was centrifuged for separation of the plasma which was
deproteinated with 2 or 9 volumes of acetonitrile. Samples of 2.5 μL
were taken from the extract and analyzed via radio-TLC using ethyl
acetate/methanol/diethylamine (96:2:2). In order to estimate the
recovery of [18F]3d in the extracted solution and to exclude loss of
original ligand or possible metabolites, samples of 2.5 μL of origin
blood and plasma were submitted to TLC analysis (ethyl acetate/
methanol/diethylamine, 96:2:2): Rf = 0.82 ([18F]3d); Rf = 0−0.2
([18F]metabolites).
Determination of Partition Coefficients (log P7.4). By use of
the HPLC method corresponding to the OECD guideline for the
testing of chemicals,46 the lipophilicity of compounds was determined
using a LiChrospher 100 RP-8 (5 mm) column (Merck). As eluent,
Soerensen buffer was used (methanol/phosphate buffer, 75:25 (v/v),
at a pH of 7.4). The retention times of a number of reference
compounds (ascorbic acid, benzaldehyde, anisole, toluene, 4-
bromoanisole, 4-iodoanisole) with known log P values (ranging from
−1.67 to 3.24) were determined and the capacity factors k′ calculated.
Plotting log k′ against log P gave the reference curve used to determine
the log P values of 3a−d by their respective retention times.
By use of the shake flask method corresponding to the OECD
guideline for the testing of chemicals,46 the lipophilicity of ligands was
determined by analyzing the partitioning of the 18F-labeled products
between a phosphate buffer (pH 7.4) and an n-octanol phase. Then
4.5 μL of the pure 18F-labeled substance in absolute ethanol was added
to a mixture of 1.5 mL of phosphate buffer and 150 μL of n-octanol.
The system was shaken for 3 min and afterward centrifuged for 5 min
at 130 00 rpm. On a silica TLC plate, 2.5 μL of the organic phase and
5 μL of the buffer phase were stippled four times, respectively.
Partitioning of radioactivity in both phases was determined via an
instant imager.
For the determination of possible cerebral metabolites, mouse brain
(n = 3) was homogenized with a Potter Elvehjem homogenizer and
treated, as described above, with 9 volumes of acetonitrile.
Staining of Brain Slices by Cresyl Violet. After total decay of
the fluorine-18 in the brain sections, cresyl violet staining of cell nuclei
of these sections was performed to identify the different brain regions.
After fixation with neutral buffered formalin for 30 min at 4 °C the
sections were rinsed twice with water and thereafter incubated in cresyl
violet solution (40 mM sodium acetate in 10% glacial acetic acid) for
30 min at 60 °C. After a short water rinse the sections were
dehydrated by 70%, 80%, 90%, and 100% of ethanol followed by a
xylol bath, each step for 5 min. The staining was sealed with DPX
Mountant for histology (Fluka).
AUTHOR INFORMATION
Corresponding Author
*Phone: +49 2461 614141. Fax: +49 2461 612535. E-mail: h.h.
coenen@fz-juelich.de.
■
Animals. The 4−6 month old Wistar rats (230−250 g body
weight) and 2−6 month old female NMRI mice were purchased from
Charles River Laboratories (Wilmington, MA). All animals were kept
under a natural light/dark cycle and had access to water and food ad
libitum. The local government approved all procedures according to
the German Law on the Protection of Animals (sections 7−9
TierSchG). Animal experiments were also approved by the Animal
Research Committee of the Scientific and Technical Advisory Board of
ACKNOWLEDGMENTS
■
The authors thank Dr. M. Holschbach and Dr. D. Bier, both at
INM-5, for performing NMR and MS spectra and for excellent
help and discussions on synthetic and pharmacological
concerns, Dr. S. Wilbold and Dr. J. Bachhausen, both of
Central Institute of Chemical Analysis, Forschungszentrum
the Research Center Julich, Germany. For conducting of ex vivo
̈
experiments with living animals, the number for approval was AZ
9.93.2.10.35.07.244.
In Vitro Autoradiography of Rat and Mouse Brain Slices.
After the animals were sacrificed, whole brains were rapidly removed
and immediately frozen at −80 °C until use. Rat brain sections were
prepared in a cryostat microtome (CM 3050, Leica, section thickness
20 μm) at −20 °C, thaw-mounted onto silica-coated object slides dried
on silica gel overnight at 4 °C, and stored at −80 °C until use.
Incubation conditions for in vitro autoradiography of all tested
substances were similar to those previously described by Zhang et al.55
All incubations were performed at 22 °C in Tris-HCl buffer (50
mmol/L, pH 7.4). After preincubation in buffer for 10 min rat brain
slices were incubated in 5 nmol/L [18F]3a or [18F]3b or [18F]3d or 10
nmol/L [18F]3c and mouse brain slices in 20 nmol/L [18F]3d for 30
min either with 10 μmol/L displacer (spiperone or cold standard) or
with the same amount of DMSO, respectively. They were washed
twice for 5 min in ice-cold buffer, rapidly rinsed in ice-cold distilled
water, and placed under a stream of dry air for rapid drying. Object
slides were exposed to a γ-sensitive film for 15−30 min and laser
scanned by a phosphor imager BAS 5000 (Fuji). The evaluation of
receptor autoradiography was processed according to standard image
Julich, Germany, for performing NMR spectra and elemental
̈
analysis, respectively. This work was supported by the Deutsche
Forschungsgemeinschaft (DFG, Grant PR 677/2-3).
ABBREVIATIONS USED
■
5-HT, 5-hydroxytryptamine; CA, cornu ammonis; CHO,
Chinese hamster ovary; CoMFA, comparative molecular field
analysis; DR, dopamine receptor; GD, gyrus dentatus; GPCR,
G-protein-coupled receptor; HPLC, high performance liquid
chromatography; ID, injection dose; iv, intravenous; MS, mass
spectrometry; nca, no-carrier-added; NMR, nuclear magnetic
resonance; NMRI, Navy Medical Research Institute; OECD,
Organization for Economic Cooperation and Development;
PET, positron emission tomography; pi, postinjection; RCY,
radiochemical yield; SAR, structure−activity-relationship;
SPECT, single photon emission computer tomography; TLC,
thin layer chromatography
analysis software (AIDA 2.31; Raytest Isotopenmeßgerate, Germany).
̈
Nonspecific binding was defined as the residual activity in the presence
of cold standard. Specific binding was calculated as the difference
between total and nonspecific binding.
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