Development of molecular tools based on the dopamine D3 receptor ligand FAUC 329 showing inhibiting effects on drug and food maintained behavior

Article abstract of DOI:10.1016/j.bmc.2017.04.036

Dopamine D₃ receptor-mediated networks have been associated with a wide range of neuropsychiatric diseases, drug addiction and food maintained behavior, which makes D₃ a highly promising biological target. The previously described do

Full text of DOI:10.1016/j.bmc.2017.04.036

Accepted Manuscript
Development of molecular tools based on the dopamine D3 receptor ligand
FAUC 329 showing inhibiting effects on drug and food maintained behavior
Anne Stö ßel, Regine Brox, Nirupam Purkayastha, Harald Hübner, Carsten
Hocke, Olaf Prante, Peter Gmeiner
PII:
S0968-0896(17)30454-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.036
BMC 13706
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 March 2017
24 April 2017
27 April 2017
Please cite this article as: Stößel, A., Brox, R., Purkayastha, N., Hübner, H., Hocke, C., Prante, O., Gmeiner, P.,
Development of molecular tools based on the dopamine D3 receptor ligand FAUC 329 showing inhibiting effects
on drug and food maintained behavior, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/
j.bmc.2017.04.036
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f
Development of molecular tools based on the dopamine D3
receptor ligand FAUC 329 showing inhibiting effects on drug
and food maintained behavior
Anne Stößela, Regine Broxa, Nirupam Purkayasthaa, Harald Hübnera, Carsten Hockeb,
Olaf Pranteb, Peter Gmeinera*
aDepartment of Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander
University, Schuhstraβe 19, D-91052 Erlangen, Germany
bDepartment of Nuclear Medicine, Ulmenweg 18, D-91054 Erlangen, Germany
*Corresponding Author. Phone: +49-(0)9131-85-24116. Email: peter.gmeiner@fau.de
Keywords
Dopamine D3 receptor; selective D3 antagonist; subtype selectivity; positron emission
tomography (PET); fluorine-18; autoradiography; self-administration; drug maintained
behavior; food maintained behavior

Abstract
Dopamine D3 receptor-mediated networks have been associated with a wide range of
neuropsychiatric diseases, drug addiction and food maintained behavior, which makes
D3 a highly promising biological target. The previously described dopamine D3 receptor
ligand FAUC 329 (1) showed protective effects against dopamine depletion in a MPTP
mouse model of Parkinson`s disease. We used the radioligand [18F]2, a
[18F]fluoroethoxy substituted analog of the lead compound 1 as a molecular tool for
visualization of D3-rich brain regions including the islands of Calleja. Furthermore,
structural modifications are reported leading to the pyrimidylpiperazine derivatives 3 and
9 displaying superior subtype selectivity and preference over serotonergic receptors.
Evaluation of the lead compound 1 on cocaine-seeking behavior in non-human primates
showed a substantial reduction in cocaine self-administration behavior and food intake.
1. Introduction
The neurotransmitter dopamine is synthesized in and released from dopaminergic
neurons to stimulate G-protein coupled receptors, thereby controlling movement,
cognition and emotion.1-3 Dysregulation of the dopaminergic system is implicated in
various pathophysiological conditions including Parkinson`s disease, schizophrenia,
drug abuse and addiction. The dopamine D3 subtype revealed a restricted distribution in
the limbic regions such as nucleus accumbens and the islands of Calleja4-6 implying an
involvement in emotional and cognitive functions and, thus, emerged as an interesting
biological target.7-10 Recently, the D3 antagonist cariprazine received FDA approval for
the treatment of schizophrenia and bipolar disorders (Figure 1).11-13 Moreover, the
regulation of the dopamine system in these mesolimbic brain areas was identified to
play a crucial role in reward-related behavior14 including drug addiction and food
reward-linked obesity.15-20 Recent investigations using [3H]-(+)-7-OH-DPAT for ligand
binding and autoradiographic mapping revealed increased D3 receptor density in the
striatum of human cocaine overdose fatalities.21 Interestingly, the selective D3 receptor
partial agonist BP897 was identified to reduce cocaine cue-controlled seeking behavior

in rats without producing any intrinsic reinforcing effects.22, 23 There is an increasing
evidence that the dopamine D3 receptor may also be involved in seeking behavior of
reinforcing effects of natural rewards like food.24 Support was provided by the finding
that the D3 receptor antagonist SB-277011A produced a significant decrease in food
intake and responses for food in an operant task in Zucker obese and lean rats.25
Further evidence of the involvement of D3 receptors in modulating food-seeking
behavior was demonstrated by a study of Nathan et al.26, which examined the effects of
the D3 receptor antagonist GSK598809 on attentional bias to rewarding food cues in
overweight to obese individuals. Although GSK598809 had only moderating effect on
attentional bias to food cues, individual differences in eating styles impacted on the
effect of GSK598809 on attentional bias, with a stronger drug effect observed in those
with lower restrained eating. However, the involvement of the dopamine receptor
subtypes in reward is more complex and their functional role in drug- and feeding-
related behaviors remains unclear due to the lack of highly selective pharmacological
tools.
We previously described the synthesis of the dopamine receptor partial agonist 1
(FAUC 329), which displays high affinity and selectivity for the D3 receptor.27
Furthermore, we evaluated the effects of 1 in the MPTP (1-methyl-4-phenyl-1,2,3,6-
29
tetrahydropyridine) mouse model of Parkinson`s disease.28,
Compound 1
demonstrated significant neuroprotective effects on dopamine depletion, which was
most pronounced in the nucleus accumbens according to the preferential abundance of
D3 receptors in this region. A preparation of [18F]2 on an analytical scale had been
previously published,30 however, the radiotracer has not yet been investigated for
imaging purposes.
18
In this paper, we describe the evaluation of the F-labeled D3 radioligand [18F]2 by in-
vitro and ex-vivo autoradiography and by in-vivo PET imaging studies. Moreover, in vivo
studies on drug and food maintained behavior are reported and structural variations are
described leading to an enhancement of target selectivity for the pyrimidylpiperazines of
type 3.

Figure 1. Chemical structures of partial D3 agonist cariprazine, D3 antagonist FAUC 329 (1), the
radiolabeled derivative [18F]2 and the highly selective D3 receptor ligand 3.
2. Results and discussion
2.1 Evaluation of the radioligand [18F]2
2.1.1 Optimization of the radiosynthesis
Based on our previous work on the preparation of [18F]2 in an analytical scale,30 it was
our intention here to optimize the radiosynthesis for extended in-vitro and in-vivo PET
imaging studies. To accomplish this goal, we envisaged the synthesis of a suitable
tosylate labeling precursor for an efficient one-step nucleophilic 18F-fluorination that
would provide direct access to [18F]2. Using potassium carbonate as a base, we applied
a three-step synthesis starting with a chemoselective N-alkylation of commercially
available 2-hydroxyphenylpiperazine with 4-bromobutyronitrile to give the respective
nitrile, which was reduced to yield the primary amine 4 (Scheme 1) following a similar
strategy as described previously.31, 32 Subsequently, an amide coupling reaction with
pyrazolo[1,5-a]pyridinyl-2-carboxylic acid27 activated by HATU afforded the
carboxamide 5 in a more straightforward synthesis than described previously30 without
the need of an O-demethylation step.

Scheme 1. Synthesis of radioligand [18F]2. Reagents and conditions: (a) 1. 4-
bromobutyronitrile, K2CO3, KI, acetone, reflux, 16 h; 2. LiAlH4, THF, 0 °C to room temperature,
5 h;32 (b) pyrazolo[1,5-a]pyridine-2-carboxylic acid27, HATU, DIPEA, DMF, room temperature,
16 h; (c) Cs2CO3, CH3CN, 60 °C, 2 h; (d) 1 eq. NBu4OH, DMF, 120 °C, 2 min.
Our initial intention was to synthesize the O-(2-tosyloxy)ethyl derivative of 5 and
subsequently displace the tosyloxy unit by nucleophilic fluorination. However, after
treatment of 5 with ethyleneglycol-1,2-bistosylate under various basic conditions, we
observed intramolecular N-alkylation resulting in the formation of the quaternary
ammonium salt 6 (Scheme 1). Consequently, the O-(2-tosyloxy)ethyl derivative of 5
could not be isolated, therefore a one-step nucleophilic 18F-fluorination on a tosylate
precursor was not achievable. However, the phenol 5 could be used as an intermediate
for both, the preparation of the radioligand [18F]2 via 18F-fluoroalkylation using 1-
[18F]fluoro-2-tosyloxyethane ([18F]7) and for the synthesis of the nonradioactive

reference compound 2 (Supporting Information).
The introduction of the fluorine-18 isotope was performed via nucleophilic substitution of
ethyleneglycol-1,2-bistosylate with [18F]fluoride under classical aminopolyether-assisted
reaction conditions to yield 2-[18F]fluoroethyltosylate [18F]7 as described previously,33, 34
followed by 18F-fluoroethylation of 5 in the presence of N(Bu)4OH to give practical access
to the final product [18F]2 in a preparative scale with a radiochemical yield of 50%
(referred to [18F]Fluoride), a radiochemical purity of >99% and in molar radioactivities of
44 ± 30 GBq/µmol after isolation by radio-HPLC and final formulation of [18F]2 in sterile
saline solution.
2.1.2 In-vitro autoradiography
18
The F-labled pyrazolo[1,5-a]pyridinyl carboxamide radioligand [18F]2 was studied for
its ability to specifically bind to D3 receptors by rat brain autoradiography in vitro. Due to
the subnanomolar D3 affinity (Ki = 0.31 nM) and excellent D3/D2 subtype selectivity (130-
fold for D2Long and 68-fold for D2short) we expected the ability of [18F]2 to specifically label
D3 receptors. The results of the in-vitro autoradiography demonstrated D3-specific
binding of the [18F]2 in rat brain slices, proven by the displacement with the D3 receptor
ligand BP897 (Figure 2). The most pronounced binding was visible in regions that are
known to have high D3 receptor density such as the islands of Calleja, including the
major islands, the lateral septum nuclei as well as striatal and cortical regions.35 In
addition, the distribution pattern of D3 receptor-rich regions in the rat brain was more
differentiated with apparently increased signal-to-noise ratio than for previously reported
diazo derivatives.36

Figure 2. In-vitro autoradiography of coronal rat brain slices. Brain slices were incubated with
the (a) radioligand [18F]2 (b) radioligand [18F]2 in the presence of BP897 (1 µM) and compared
to (c) HE stained slices.
2.1.3 Ex-vivo autoradiography
Ex-vivo rat brain autoradiography of the radioligand [18F]2 qualitatively showed negligible
uptake of the radioligand in the D3-rich brain regions, such as the islands of Calleja or
the nucleus caudatus putamen (striatum). The result are in accordance with those
previously reported for 18F-labeled pyridinylphenyl amides and phenyl
37
azocarboxamides.36,
Our experiments showed a significant accumulation of the
radiotracer in the ventricles, the ependyma cell layer or plexus tissue (Figure 3).
Applying the D3 ligand BP897, only 7-12% (lateral ventricle, LV) and 5-14% (third
ventricle, 3V) of the radioligand was displaced, when the region of interest-to-
background (BG)-ratios in the control slices (without BP897) were compared with the
sections from animals that were co-injected with BP897.

Figure 3. Ex-vivo autoradiography of coronal rat brain sections. Brain sections were obtained
from animals that were injected with (a) radioligand [18F]2 alone and with radioligand containing
the D3 receptor ligand BP897, both at 60 min p.i., (b) HE staining (c) HE stained sections were
overlayed with the autoradiography.
2.1.4 PET studies
PET studies were conducted to further evaluate [18F]2 as a tracer for selective imaging
of dopamine D3 receptors in vivo. The results were in accordance with the ex-vivo
autoradiography and revealed marginal brain uptake of the radioligand in CNS
structures (Figure 4). However, a significant radiotracer uptake in the pituitary gland and
in the brain ventricles was observed. The mean whole pituitary gland uptake of the
radioligand at 40 min p.i. was 0.394 ± 0.016% ID/g and pretreatment with BP897
moderately reduced the uptake of the radioligand in the pituitary gland by 25%. The in
vivo uptake into the ventricular system is not as pronounced as for the azo compound
class previously measured.36 Similar to the previously published data, besides high
uptake of [18F]2 in the pituitary gland, the fourth ventricle also showed uptake as
visualized in the PET scan (Figure 4).

Figure 4. PET imaging study of [18F]2 in rat, image of frame 30-45 min post radioligand
injection (4V = fourth ventricle).
These findings are in accordance with previously reported in vivo PET recordings of
structurally related benzamide ligands.36, 37 We found that [18F]2 had only low blood-
brain-barrier (BBB) penetration and the detection of specific binding of [18F]2 in CNS
structures of the rat brain by PET imaging was not possible. However, the tracer
revealed significant penetration of the blood-liquor barrier and uptake into brain
ventricles in rats. Moreover, the pituitary gland, lying outside the BBB, showed high
specific binding of [18F]2, a phenomenon that resembles very much the biodistribution of
amisulpiride. It is well known that dopamine receptors are expressed in the pituitary
gland, where they regulate hormone synthesis and secretion through binding of
dopamine. In conclusion, [18F]2 can be used for selectively quantifying the distribution of
D3 receptors in rat brain by in vitro autoradiography, but did not fit for detecting D3
receptors in CNS structures of the living rat brain by PET. The results from our PET
imaging study with [18F]2 suggest that the main mode of action of FAUC 329 in vivo
could be induced by binding to dopamine receptors in the pituitary gland.
2.2 Structural modifications
2.2.1 Synthesis
To further fine-tune the selectivity profile of the investigated D3 ligands, we planned the
synthesis of pyrimidylpiperazines of type 3 and 9, taking advantage of recently
described SAR studies.10, 27, 38 Receptor-ligand interactions that were deduced from the
D3 crystal structure were also helpful for the ligand design.39 Key step of the syntheses
was a HATU and TBTU promoted coupling reaction of the primary amine 8 with

pyrazolo[1,5-a]pyridine-2-carboxylic acid and benzothiophene-2-carboxylic acid,
respectively (Scheme 2). The intermediate 8 was prepared by N-alkylation of the
corresponding pyrimidyl-substituted piperazine with 4-bromobutylonitrile and
subsequent reduction employing LiAlH4.
Scheme 2. Synthesis of pyrimidylpiperazine derivatives 3 and 9. Reagents and conditions:
(a) 4-bromobutyronitrile, K2CO3, KI, MeCN, reflux, 16 h; (b) LiAlH4, THF, 0 °C to room
temperature, 5 h; (c) 8, pyrazolo[1,5-a]pyridine-2-carboxylic acid, HATU, DIPEA, DMF, room
temperature, 16 h; (d) 8, benzo[b]thiophene-2-carboxylic acid, TBTU, DIPEA, DMF, room
temperature, 16 h.
2.2.2 Receptor binding studies
Compound 1 was previously reported as a dopamine D3 receptor ligand with
considerable selectivity over the other dopamine receptor subtypes.27 To analyze
receptor binding and selectivity profiles of the test compounds 3, 6 and 9, radioligand
displacement assays were employed for the subtypes of dopamine receptors, and for
the related serotonergic receptors 5-HT1A and 5-HT2A. The resulting Ki values of the

newly synthesized analogs, compared to the antipsychotic drug cariprazine and the
reference agents were determined in parallel under identical conditions. The data are
listed in Table 1. Compared to the lead compound 1, the fluoro substituted analog 2
displayed a similar binding profile indicating that [18F]2 may act as a valuable imaging
agent, which is able to visualize the CNS activity of compound 1. The spirocyclic-
derivative 6 that emerged as a side product was also investigated with regard to its
receptor binding profile, when moderate D3 affinity was detected (Ki = 200 nM).
Aiming to increase selectivity towards 5-HT1A receptors, modifications of the pyrazolo-
[1,5-a]pyridine moiety and the methoxyphenylpiperazine unit were investigated, when
we followed the findings of previous SAR studies.8, 10, 38, 40-42 The heteroaryl-substituted
analog 3 bearing a pyrimidyl-piperazine scaffold, a moiety that was reported for its drug-
like properties38, showed excellent D3 receptor binding affinity (Ki = 1.2 nM) in the same
range as that of compound 1 (Table 1). Interestingly, a huge increase of D3 selectivity
over 5-HT1A was observed for 3 (Ki (D3/5-HT1A) = 8000). An additional gain in selectivity
could be achieved by the exchange of the pyrazolopyridine moiety by a benzothiophene
ring system. Thus, the target compound 9 showed 1000-20000 fold D3 selectivity over
all biogenic amine receptors investigated.

Table 1. Receptor Binding for D3 Receptor Ligands Employing Human D1, D5, D2long, D2short, D3, D4, 5HT-2A as well as Porcine 5HT-1A Receptors.
Ki values (nM)a
[3H]spiperone
[3H]WAY6001
[3H]SCH 23990
[3H]ketanserin
35
Compd.
hD1
hD5
hD2long
hD2short
hD3
hD4
p5-HT1A
h5-HT2A
22 ± 6
cariprazine
2100 ± 1200 7900 ± 1700
1600 ± 100 3700 ± 2000
0.47 ± 0.13
0.41 ± 0.05
200 ± 40
0.27 ± 0.07
110 ± 10
0.49 ± 0.13
1
(FAUC
270 ± 30
3.5 ± 0.4
150 ± 20
10 ± 4
430 ± 50
329)
2c
3
860 ± 100
3800 ± 1500
39 ± 11
21 ± 4
0.31 ± 0.06
1.2 ± 0.4
200 ± 31
2.4 ± 0.8
160 ± 52
6.5 ± 1.4
520 ± 98
7500 ± 4700 5700 ± 2700
5700 ± 1700b > 50000b
430 ± 55
200 ± 61
4700 ± 400 9800 ± 3200b
2700 ± 1100b
> 10000b
6
6300 ± 2800
4000 ± 900
4500 ± 1100
2700 ± 500
> 10000
> 10000
900 ± 400b
9
> 10000b
> 10000b
> 50000
7100 ± 1900
a Ki values in nM ±SEM are based on the mean of 3-12 experiments each performed in triplicate. b Ki values in nM ±SD based on two experiments.
c previously published in Hocke et al.30

2.3 Self-Administration Studies
There is a growing body of evidence that dopamine D3 receptors are implicated in the
reinforcing effects of addictive drugs and food.17 We examined the ability of our lead
compound 1 to modify the reinforcing effects of cocaine and food in non-human
primates. Male rhesus monkeys were trained to respond for the delivery of cocaine
and food during daily, 2 hour fixed-ratio (FR30) schedule. This experimental setup
allowed us to investigate the effects of the D3 ligand 1 on cocaine-self administration
as well as food-maintained behavior.
2.3.1 Cocaine self-administration
Cocaine self-administration behavior generally was dose-related and characterized
by an inverted U-shaped function that is characteristic for cocaine-maintained
behavior under fixed-ratio schedules (supplementary data, Figure S1).43
Pretreatment with compound 1 (0.32 mg/kg) produced attenuating effects on the
intake of 0.03 mg/kg per injection i.v. cocaine (Figure 5). Specifically, averaged for
the group of monkeys, compound 1 decreased cocaine-maintained responding to
approximately 20% of control values. Furthermore, pretreatment with various doses
of compound 1 showed dose-related decreases in cocaine-maintained responding
(supplementary data, Figure S2).
2.3.2 Food-maintained behavior
Averaged data also indicate that compound 1 produced decreases in food-
maintained responding prior to the i.v. cocaine self-administration component of the
session (food 1), but did not appreciably alter food-maintained responding following
the i.v. self-administration component (food 2). Pretreatment with the highest dose of
compound 1 (0.32 mg/kg) resulted in an approximately 30% reduction of food-
maintained behavior during food 1 session (Figure 5). The low and intermediate
pretreatment doses of compound 1 (0.1 and 0.18 mg/kg) did not markedly decrease
food 1 and 2 responding (supplementary data, Figure S2). A similar finding was also
reported for buspirone that had inconsistent effects on food-maintained responding,
but produced substantial decreases in cocaine-maintained behavior.44 In conjunction,
the rate-decreasing effects of compound 1 on i.v. cocaine self-administration
behavior and food-maintained responding are not consistent with the idea that its

effects on cocaine-maintained responding are behaviorally selective. In addition, the
limited rate-decreasing effects of compound 1 on food-maintained behavior during
food 2 components in sessions of i.v. saline availability suggest that behaviorally
active doses in the present study had a relatively short (< 2 h) duration of action.
*
5 0
4 0
3 0
2 0
1 0
0
2 0
1 5
1 0
5
*
S A L
0 .3 2 m g /kg 1
0
C o c a in e
F o o d 1
F o o d 2
Figure 5. Mean effects of compound 1 (0.32 mg/kg) on behavior maintained by 0.032 mg/kg
per injection cocaine. Bars represent the number of total cocaine injections that were self-
administered in the 100-min drug component or number of food pellets that were delivered
during Food 1 and Food 2. The results shown for “Sal” (black bars) represent the effects of
saline pretreatment and serve as baseline values for comparison with the effects of
compound 1 (grey bars). Bars represent the mean ± S.E.M. of four individual experiments. *
p < 0.05, significantly different from baseline values as determined by two-tailed t-test.
3. Conclusions
The exact role of the D3 receptor in feeding-related behaviors is still an open
question. An involvement of D3 receptors in the regulation of body weight and body
fat when consuming diets differing in palatability and fat content was suggested by a
study based on the finding that male D3 receptor deficient mice become obese when
fed a high-fat diet.45 Furthermore, a reduced striatal D2/3 receptor availability was
found in obese compared with non-obese humans confirming a role of the
dopaminergic reward system in obesity.46 Our self-administration studies indicate that
our D3 ligand 1 (FAUC 329) exerts inhibitory effects on cocaine and food maintained
behavior of male rhesus monkeys indicating that the compound is a valuable tool for
more specific studies investigating the role of D3 in feeding-related behavior.
A common limitation in clinical and preclinical studies of drug addiction and obesity is

the lack of D3 selective receptor radioligands to evaluate differences in D3 receptor
expression levels. Until now, the development of suitable receptor PET ligands is still
an ongoing challenge because the available agents suffer from selectivity issues and
unfavorable lipophilicity.47, 48 [11C]PHNO is the only radiopharmaceutical in use for
imaging of the D3 receptor, however the selectivity is not very high.49, 50 To overcome
these drawbacks we evaluated the 18F-labeled radioligand [18F]2 as an analog of the
D3 ligand 1 for imaging purposes. The radioligand [18F]2 successfully visualized D3-
rich brain regions including the islands of Calleja by in vitro autoradiography,
However, our PET imaging study revealed that [18F]2 did not significantly accumulate
in CNS structures of the rat brain in vivo. Instead, [18F]2 showed significant uptake in
the brain ventricular system and, more pronounced, in the pituitary gland. Thus,
provided that [18F]2 mimics the biodistribution of 1, it is tempting to speculate that 1
could induce its effects on cocaine-self administration as well as food-maintained
behavior through binding to D3 receptors in the pituitary gland.
To reduce binding affinity towards serotonin 5-HT1A and 5-HT2A receptors in brain
51, 52
tissues,30,
structural modifications were performed resulting in the
pyrimidylpiperazine derivatives 3 and 9 with outstanding selectivity over the
serotonergic congeners. These compounds may serve as leads for further ligand
development.
4. Experimental section
4.1 General
Reagents and dry solvents were of commercial quality and used as purchased. If not
stated otherwise, reactions were carried out under nitrogen atmosphere. MS was run
on a BRUKER ESQUIRE 2000 using ESI ionization. HRMS-ESI was run on an AB
Sciex Triple TOF660 SCiex, source type ESI or at the Chair of Organic Chemistry,
Friedrich Alexander University Erlangen-Nürnberg on a Bruker Daltonik micrOTOF II
focus or Bruker Daltonik maXis 4G, source type ESI or APPI. NMR spectra were
recorded on a Bruker Avance 400 or a Bruker Avance 600 spectrometer at 300 K in
the solvents indicated. Chemical shifts are given in ppm () relative to TMS.
Purification by column chromatography was performed with silica gel 60. TLC
analyses were performed using Merck 60 F254 aluminum plates in combination with

UV detection (254 nm) or ninhydrin staining. Analytical HPLC was conducted on an
Agilent 1200 HPLC system employing a DAD detector and a ZORBAX ECLIPSE
XDB-C8 (4.6  150 mm, 5 µm) column with the following binary solvent systems:
System 1: eluent, methanol/0.1% aq formic acid, 10% methanol for 3 min, to 100% in
15 min, 100% for 6 min, to 10% in 3 min, then 10% for 3 min, flow rate 0.5 mL/min, λ
= 210 or 254 nm; System 2: CH3CN/0.1% aq formic acid, 10% CH3CN for 3 min, to
100% in 15 min, 100% for 6 min, to 10% in 3 min, then 10% for 3 min, flow rate
0.5 mL/min, λ = 210 or 254 nm. Preparative HPLC was performed on an Agilent 1100
Preparative Series, using a ZORBAX ECLIPSE XDB-C8 PrepHT (21.5 x 150 mm, 5
μm, flow rate 10 mL/min) column with the solvent systems indicated.
Instruments for analysis and preparative radiosynthesis were used as follows: a
semi-preparative HPLC system (Agilent 1100) equipped with a quaternary pump, a
variable wavelength detector and a radio-HPLC detector D505TR (Canberra
Packard) connected to a PC with HPLC data acquisition software (FLO-One,
Canberra Packard).
4.2 Syntheses.
The synthesis of pyrazolo[1,5-a]pyridine-2-carboxylic as well as FAUC 329 was
conducted as previously described.27 Benzo[b]thiophene-2-carboxylic acid was
purchased
from
Acros
Organics,
2-(tert-butyl)-4-(piperazin-1-yl)-6-
(trifluoromethyl)pyrimidine was provided by Abbott.
4.2.1 2-(4-(4-Aminobutyl)piperazin-1-yl)phenol (4)
The synthesis of 4 was performed as reported by Leopoldo et al.32 with some
modifications. To a suspension of 2-(piperazin-1-yl)phenol (1.00 g, 5.61 mmol),
K2CO3 (1.16 g, 8.42 mmol) and KI (cat.) in dry acetone (30 mL) was added 4-
bromobutyronitrile (0.56 mL, 5.61 mmol) and the reaction mixture was refluxed for 16
h. After cooling to room temperature, solids were filtrated off and the solvent was
removed under reduced pressure. The crude material was purified by flash
chromatography
(CH2Cl2/MeOH/NH3
aq.,
40:1:0.01)
to
give
4-(4-(2-
hydroxyphenyl)piperazin-1-yl)butanenitrile (1.10 g, 80%) as a colorless solid. 1H NMR
(400 MHz, CDCl3) δ 7.17 (dd, J = 7.8, 1.5 Hz, 1H), 7.11 – 7.05 (m, 1H), 6.95 (dd, J =
8.1, 1.4 Hz, 1H), 6.86 (td, J = 7.7, 1.5 Hz, 1H), 2.90 (app t, J = 4.8 Hz, 4H), 2.62 (br s,

4H), 2.55 (t, J = 6.8 Hz, 2H), 2.47 (t, J = 7.1 Hz, 2H), 1.87 (app quint, J = 6.9 Hz, 2H);
13C NMR (100 MHz, CDCl3) δ 151.5, 138.9, 126.5, 121.4, 120.1, 119.7, 114.0, 56.3,
53.8, 52.5, 22.7, 14.9; ESI-MS m/z 246.0 [M+H]+. Lithium aluminum hydride (2.23
mL, 4 M in Et2O) was added dropwise to
a
solution of 4-(4-(2-
hydroxyphenyl)piperazin-1-yl)butanenitrile (728 mg, 2.97 mmol) in anhydrous
tetrahydrofuran (40 mL) at 0 °C. After stirring for 10 min at 0 °C the reaction mixture
was warmed to room temperature and stirred additional 5 h. The reaction was
quenched by adding wet silica gel carefully and portion wise at 0 °C. The solids were
filtered and washed (100 mL, CH2Cl2/MeOH/NH3 aq., 10:1:0.05) and the combined
filtrates were dried (MgSO4). After removal of solvents under reduced pressure the
crude product 4 (571 mg, 77%, colorless foam) was used in the next step without
1
further purification. H NMR (400 MHz, CDCl3) δ 7.16 (dd, J = 7.8, 1.5 Hz, 1H), 7.06
(td, J = 8.0, 1.5 Hz, 1H), 6.93 (dd, J = 8.0, 1.4 Hz, 1H), 6.85 (td, J = 7.7, 1.5 Hz, 1H),
2.91 (app t, J = 4.8 Hz, 4H), 2.73 (t, J = 6.8 Hz, 2H), 2.62 (br s, 4H), 2.48 – 2.37 (m,
13
2H), 1.63 – 1.41 (m, 4H); C NMR (100 MHz, CDCl3) δ 151.5, 139.1, 126.3, 121.4,
120.0, 114.0, 58.5, 53.9, 52.5, 42.1, 31.8, 24.3; ESI-MS m/z 250.1 [M+H]+.
4.2.2
N-(4-(4-(2-hydroxyphenyl)piperazin-1-yl)butyl)pyrazolo[1,5-a]pyridine-2-
carboxamide (5)
Compound 5 was reported previously.30 For this study, 5 was resynthesized using a
modified synthesis as follows: To a solution of pyrazolo[1,5-a]pyridine-2-carboxylic
acid (150 mg, 0.93 mmol) and 4 (230 mg, 0.93 mmol) in anhydrous DMF (5 mL) was
added DIPEA (0.33 mL, 1.86 mmol) and HATU (372 mg, 0.98 mmol). After stirring
the reaction mixture at room temperaturefor 16 h the solvent was removed under
reduced pressure. The crude mixture was suspended in aqueous 5% NaHCO3
solution and extracted with ethyl acetate (3x). The combined organic phases were
washed with water and brine and were dried (MgSO4). After removal of solvents
under reduced pressure the crude material was purified by flash chromatography
1
(CH2Cl2/MeOH/NH3 aq., 20:1:0.01) to give 5 (314 mg, 86%) as a colorless solid. H
NMR (600 MHz, CDCl3) δ 8.38 – 8.34 (m, 1H), 7.61 – 7.51 (m, 1H), 7.29 (br s, J =
5.2 Hz, 1H), 7.18 – 7.12 (m, 2H), 7.09 – 7.04 (m, 2H), 6.94 (dd, J = 8.0, 1.4 Hz, 1H),
6.88 – 6.81 (m, 2H), 3.58 – 3.50 (m, 2H), 2.98 – 2.87 (m, 4H), 2.63 (br s, 4H), 2.51 –
2.45 (m, 2H), 1.76 – 1.63 (m, 4H); 13C NMR (150 MHz, CDCl3) δ 162.2, 151.5, 148.1,
141.4, 139.0, 128.4, 126.4, 123.7, 121.5, 120.0, 119.3, 114.0, 113.6, 98.0, 58.1,

53.9, 52.6, 39.1, 27.6, 24.4; ESI-MS m/z 394.2 [M+H]+.
4.2.3
4'-(4-(Pyrazolo[1,5-a]pyridine-2-carboxamido)butyl)-2,3-
dihydrospiro[benzo[b][1,4]oxazine-4,1'-piperazin]-4-ium
4-
methylbenzenesulfonate (6)
A solution of 5 (21.0 mg, 53 µmol) in anhydrous acetonitrile (1.5 mL) was treated with
Cs2CO3 (26.0 mg, 80 µmol), heated to 60 °C and a solution of bistosyloxyethan (59.0
mg, 159 µmol) in anhydrous acetonitrile (1.5 mL) was added dropwise. The reaction
mixture was stirred at 60 °C for 2 h. After removal of the solvent the crude product
was purified by short flash chromatography (CH2Cl2/MeOH/25% aq. NH4OH; 5:1:0.1)
to give 6 (19.0 mg, 85%) as a colorless solid. 1H NMR (600 MHz, CDCl3) δ 8.36 (d, J
= 7.0, 1H, pyrazolopyridine-H7), 7.73 (d, J = 8.1 Hz, 2H, tosylate-H2/H2’), 7.62 (d, J =
8.2 Hz, 1H, H20), 7.56 (d, J = 8.9 Hz, 1H, pyrazolopyridine-H4), 7.46 (br t, J = 5.5 Hz,
1H, NH), 7.38 – 7.34 (m, 1H, H18), 7.15 – 7.05 (m, 6H, pyrazolopyridine-H5, tosylate-
H3/H3’, pyrazolopyridine-H3, H17, H19), 6.83 (td, J = 6.9, 1.2 Hz, 1H,
pyrazolopyridine-H6), 4.71 – 4.63 (m, 2H, CH2O), 4.52 – 4.46 (m, 2H, CH2N+), 4.20 –
4.11 (m, 2H, 2 x N+CHax), 3.91 – 3.82 (m, 2H, 2 x N+CHeq), 3.52 (q, J = 6.6 Hz, 2H,
CH2NH), 3.26 – 3.15 (m, 2H, 2 x NCHax), 3.04 – 2.96 (m, 2H, 2 x NCHeq), 2.62 (t, J =
6.9 Hz, 2H, CH2N), 2.31 (s, 3H, CH3), 1.74 – 1.66 (m, 2H, CH2CH2N), 1.64 – 1.55 (m,
13
2H, CH2CH2NH); C NMR (150 MHz, CDCl3) δ 162.3 (CO), 149.5 (Cq), 148.0 (Cq),
143.7 (Cq), 141.3 (Cq), 139.2 (Cq), 131.8 (CH, C18), 129.4 (Cq), 128.6 (2 x CH),
128.5 (CH), 125.9 (2 x CH), 123.8 (CH), 122.6 (CH), 121.1 (CH), 120.6 (CH), 119.2
(CH), 113.7 (CH), 97.9 (CH), 65.0 (CH2, C14/C14’), 60.6 (CH2, C16), 56.6 (CH2,
C12), 54.8 (CH2, C15), 46.5 (CH2, C13/C13’), 38.9 (CH2, C9), 27.4 (CH2, C11), 23.6
(CH2, C10), 21.3 (CH3); ESI-MS m/z 420.3 [M]+; HRMS-ESI (m/z): [M]+: calcd. for
C24H30N5O2: 420.2394, found: 420.2394; HPLC: System 1: tR = 13.5 min, purity 96%,
System 2: tR = 11.2 min, purity 97%.
4.2.4 4-(4-(2-(tert-Butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butan-1-
amine (8)
Compound 8 was reported previously.53 For this study, 8 was prepared by a modified
synthesis as follows: To a suspension of 4-(tert-butyl)-6-(piperazin-1-yl)-2-
(trifluoromethyl)pyrimidine (1.0 g, 3.46 mmol), K2CO3 (0.96 g, 6.92 mmol) and KI

(cat.) in anhydrous acetonitril (30 mL) was added 4-bromobutyronitrile (3.46 mmol,
0.35 mL) and the reaction mixture was refluxed for 16 h. After cooling to room
temperature it was quenched by the addition of water and the aqueous phase was
extracted with DCM (3x). The combined organic layers were dried (Na2SO4) and
solvents were removed under reduced pressure. The crude material was purified by
flash chromatography (EtOAc/Cyclohexane, 2:8) to give 4-(4-(6-(tert-butyl)-2-
(trifluoromethyl)pyrimidin-4-yl)piperazin-1-yl)butanenitrile (1.26 g, 97%) as a colorless
solid. 1H NMR (600 MHz, CDCl3) δ 6.59 (s, 1H), 3.71 ( br s, 4H), 2.55 – 2.50 (m, 6H),
2.48 (t, J = 7.0 Hz, 2H), 1.87 (app p, J = 6.8 Hz, 2H), 1.34 (s, 9H). 13C NMR (151
MHz, CDCl3) δ 178.0, 162.3, 155.1 (q, J = 33.7 Hz), 121.5 (q, J = 274.7 Hz), 119.8,
95.8 (q, J = 3.2 Hz), 56.4, 52.8, 44.0, 39.7, 29.6, 22.9, 15.1; ESI-MS m/z 355.1
[M+H]+. Lithium aluminum hydride (6.33 mL, 1 M in THF) was added dropwise to a
solution
of
4-(4-(6-(tert-butyl)-2-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-
yl)butanenitrile (1.50 g, 4.22 mmol) in anhydrous tetrahydrofuran (100 mL) at 0 °C.
After stirring for 10 min at 0 °C the reaction mixture was warmed to room temperature
and stirred additional 5 h. The reaction was quenched by dropwise addition of sat.
NaHCO3 at 0 °C. The solids were filtered of and the solution was dried (MgSO4).
After removal of solvents under reduced pressure the crude product 8 (1.21 mg,
80%, colorless oil) was used in the next step without further purification. ESI-MS m/z
360.2 [M+H]+.
4.2.5
N-(4-(4-(2-(tert-Butyl)-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-
yl)butyl)pyrazolo[1,5-a]pyridine-2-carboxamide x HCOOH (3)
To a solution of pyrazolo[1,5-a]pyridine-2-carboxylic acid (2.40 mg, 14.8 µmol) and 8
(6.00 mg, 14.8 µmol) in anhydrous DMF (1 mL) was added DIPEA (5.00 µL, 29.6
µmol) and HATU (5.90 mg, 15.5 µmol). After stirring the reaction mixture at room
temperature for 16 h the solvent was removed under reduced pressure. The crude
mixture was suspended in aqueous 5% NaHCO3 solution and extracted with ethyl
acetate (3x). The combined organic phases were washed with water and brine and
were dried (MgSO4). After removal of solvents under reduced pressure the crude
material was purified by preparative HPLC (acetonitrile in 0.1% aqueous HCOOH,
5% to 95%) to give 3 (2.25 mg, 28%) as a colorless solid. 1H NMR (600 MHz, CDCl3)
δ 8.38 – 8.34 (m, 1H), 7.61 – 7.57 (m, 1H), 7.31 (br t, J = 5.2 Hz, 1H), 7.16 (ddd, J =
8.9, 6.7, 1.0 Hz, 1H), 7.07 (d, J = 0.6 Hz, 1H), 6.87 (td, J = 6.9, 1.3 Hz, 1H), 6.60 (s,

1H), 3.75 (br s, 4H), 3.55 (app q, J = 6.6 Hz, 2H), 2.55 (br s, 4H), 2.48 (br s, 2H),
13
1.77 – 1.61 (m, 4H), 1.36 (s, 9H); C NMR (150 MHz, CDCl3) δ 177.7, 162.2, 154.9
(q, JCF = 33.7 Hz), 148.0, 141.4, 128.3, 123.7, 121.3 (q, JCF = 275 Hz), 119.3, 113.6,
98.0, 95.6 (q, JCF = 3.4 Hz), 57.9, 52.7, 43.8, 39.5, 39.1, 29.7, 29.4, 27.6, 24.2; ESI-
MS m/z 504.3 [M+H]+; HRMS-ESI (m/z): [M+H]+: calcd. for C25H33F3N7O: 504.2693,
found: 504.2688; HPLC: System 1: tR = 16.3 min, purity 99%, System 2: tR = 16.2
min, purity 99%.
4.2.6
N-(4-(4-(2-tert-butyl-6-(trifluoromethyl)pyrimidin-4-yl)piperazin-1-
yl)butyl)benzo[b]thiophene-2-carboxamide (9)
To a solution of benzothiophene-2-carboxylic acid (50.0 mg, 0.28 mmol) and 8
(120 mg, 0.33 mmol) in anhydrous DMF (5 mL) was added DIPEA (0.10 mL,
0.56 mmol) and TBTU (90.0 mg, 0.28 mmol). After stirring the reaction mixture at
room temperature for 16 h the solvent was removed under reduced pressure. The
crude mixture was suspended in aqueous 5% NaHCO3 solution and extracted with
ethyl acetate (3x). The combined organic phases were washed with water and brine
and were dried (MgSO4). After removal of solvents under reduced pressure the crude
material was purified by flash chromatography (CH2Cl2/MeOH, 20:1) to give 9
1
(97.0 mg, 67%) as a colorless solid. H NMR (600 MHz, CDCl3) δ 7.86 (s, 1H), 7.86-
7.80 (m, 2H), 7.43 – 7.37 (m, 2H), 6.99 (br s, 1H), 6.60 (s, 1H), 3.87 (br s, 4H), 3.52
(app q, J = 6.2 Hz, 2H), 2.86 (app t, J = 5.0 Hz, 4H), 2.78 – 2.70 (m, 2H), 1.82 – 1.67
(m, 4H), 1.34 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 178.2, 166.4, 162.6, 162.0, 155.3
(q, JCF = 34.1 Hz), 140.8, 139.2, 138.6, 126.3, 125.3, 125.0, 124.9, 121.1 (q, JCF
=
275 Hz), 95.7 (q, JCF = 3.0 Hz), 57.1, 51.7, 42.16, 39.5, 39.2, 29.3, 26.8, 22.3; ESI-
MS m/z 520.3 [M+H]+; HRMS-ESI (m/z): [M+H]+: calcd. for C26H33F3N5OS: 520.2352,
found: 520.2341; HPLC: System 1: tR = 19.6 min, purity 98%, System 2: tR = 16.8
min, purity 99%.
4.2.7
N-(4-(4-(2-(2-Fluoroethoxy)phenyl)piperazin-1-yl)butyl)pyrazolo[1,5-
a]pyridine-2-carboxamide x HCOOH (2)
Compound 2 was synthesized in accordance to Hocke et al.30 with slight
modifications: To a suspension of 5 (10.0 mg, 25 µmol), Cs2CO3 (20 mg, 62.5 µmol)
in dry acetonitrile (2 mL) was added 2-fluoroethyl-4-methylbenzenesulfonate 7 (8.30
mg, 38 µmol) and the reaction mixture was refluxed for 14 h. After cooling to room

temperature the solvent was removed under reduced pressure and the crude mixture
was suspended in aqueous 5% NaHCO3 solution and extracted with ethyl acetate
(3x). The combined organic phases were washed with brine and were dried (MgSO4).
After removal of solvents under reduced pressure the crude material was purified by
preparative HPLC (acetonitrile in 0.1% aqueous HCOOH, 5% to 95%) to give 2 (6.50
mg, 54%) as a colorless solid. 1H NMR (600 MHz, MeOD) δ 8.56 (d, J = 7.0 Hz, 1H),
7.70 (d, J = 8.9 Hz, 1H), 7.25 (dd, J = 8.9, 6.7 Hz, 1H), 7.03 – 6.92 (m, 6H), 4.80 –
4.69 (m, 2H), 4.29 – 4.21 (m, 2H), 3.49 (t, J = 6.6 Hz, 2H), 3.32 – 3.28 (m, 2H), 3.23
13
(br s, 4H), 3.08 (br s, 2H), 2.94 – 2.86 (m, 2H), 1.81 – 1.69 (m, 4H); C NMR (150
MHz, MeOD) δ 151.3, 147.3, 140.6, 128.3, 124.0, 123.4, 121.6, 118.8, 118.3, 113.8,
113.7, 97.0, 81.9 (d, JCF = 169 Hz), 67.8 (d, JCF = 19.6 Hz), 57.1, 52.5, 48.7, 38.3,
26.7, 22.1, 2C were not observed; ESI-MS m/z 440.2 [M+H]+; HRMS-ESI (m/z):
[M+H]+: calcd. for C25H30FN5O2: 440.2465, found: 440.2459; HPLC: System 1: tR =
16.2 min, purity 95%, System 2: tR = 13.6 min, purity 95%.
4.3 Radiosynthesis of [18F]2
After evaporation of the solvent from the reaction vessel containing [18F]fluoride, the
K2CO3 base and the aminopolyether 2.2.2, residual traces of water were removed by
azeotropic drying after the addition of acetonitrile at 80 °C. The labeling precursor
(bistosyloxyethan, 10 µmol) was dissolved in anhydrous acetonitrile (0.4 ml) and
added to the vial containing [18F]fluoride. The reaction mixture was heated at 85 °C
for 3 min. [18F]Fluoroethyltosylate [18F]7 was obtained in a radiochemical yield (RCY)
of 65-80% and isolated by HPLC (isocratic conditions, 65:35 acetonitrile / 0.1 M
ammonium acetate, C-18 column, 4 mL/min, Rt = 7.0 min). The product fraction was
collected, diluted with water (10 mL) and passed through a C18 cartridge (Sep-Pak,
Waters). After washing the cartridge with water (10 mL), the Sep-Pak cartridge was
dried in a He-gas stream. [18F]Fluoroethyltosylate [18F]7 was eluted with 300 µL DMF
for the direct use in the second reaction step with phenol 5 (10 µmol in 200 µL DMF)
in the presence of tetrabutyammonium hydroxide (10 µmol, 10 µL, 1N). The 18F-
fluoroalkylation was achieved within 2 min at 120 °C. The reaction mixture was
diluted with 10 mL of water and passed through a C18 cartridge. The cartridge was
eluted with 500 μL of acetonitrile and the eluate diluted with 500 μL of water and
subjected to the 1 mL sample loop of the HPLC instrument. The preparative

separation was carried out on the same column as described above with isocratic
conditions of 25:75 acetonitrile / 0.1 M ammonium acetate, 4 mL / min, providing the
final product [18F]2 in 50% RCY. After HPLC isolation and collection of the product-
containing fractions by solid-phase extraction with C18 cartridge (Sep-Pak, Waters),
the ethanol solution was evaporated on a rotary evaporator in a glass flask and 0.9%
sterile saline solution (2 mL) was added to redissolve the product. N-[4-[4-(2-
[18F]fluoroethoxyphenyl)-piperazin-1-yl]-butyl]-pyrazolo-[1,5-a]pyridin-2-carboxamide
[18F]2 was identified by analytical radio-HPLC (isocratic conditions, 25:75 acetonitrile
/ 0.1 M ammonium acetate, Luna C-18 column 150 × 4.6 mm, 1 mL/min, Rt = 14.5
min). [18F]2 was isolated in a radiochemical purity of >99% and in molar
radioactivities of 44 ± 30 GBq/µmol. The final solution was used for PET imaging
studies on rats and ex-vivo autoradiography.
Acknowledgements
The authors gratefully acknowledge NIDA/DTMC/CPB, National Institutes of Health
(Dr. Ming Shih) and the contractors Dr. Jack Bergman, Dr. Nancy K. Mello, Dr. S.
Barak Caine of the Harvard Medical School - McLean Hospital for conducting the
self-administration experiments. We thank the German Research Foundation (DFG)
for financial support (GRK 1910). The project was supported by the US National of
Health grant GM106990.
Supplementary data
Supplementary data includes effects of compound 1 on cocaine dose effect function
as well as dose-ranging studies, experimental procedures for known derivatives and
analytical data for newly synthesized compounds and descriptions of the assays.
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Development of molecular tools based on the dopamine D3
receptor ligand FAUC 329 showing inhibiting effects on
drug and food maintained behavior
Anne Stößela, Regine Broxa, Nirupam Purkayasthaa, Harald Hübnera, Carsten
Hockeb, Olaf Pranteb, Peter Gmeinera*
aDepartment of Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander
University, Schuhstraβe 19, D-91052 Erlangen, Germany
bDepartment of Nuclear Medicine, Ulmenweg 18, D-91054 Erlangen, Germany
*
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