Radiosynthesis and evaluation of 18F-labeled dopamine D4-receptor ligands

Article abstract of DOI:10.1016/j.nucmedbio.2020.07.004

Introduction: The dopamine D₄ receptor (D₄R) has attracted considerable attention as potential target for the treatment of a broad range of central nervous system disorders. Although many efforts have been made to improve the performance of putative radioligand candidates, there is still a lack of D₄R selective tracers suitable for in vivo PET imaging. Thus, the objective of this work was to develop a D₄-selective PET ligand for clinical applications. Methods: Four compounds based on previous and new lead structures were prepared and characterized with regard to their D₄R subtype selectivity and predicted lipophilicity. From these, 3-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-1H-pyrrolo[2,3-b]pyridine I and (S)-4-(3-fluoro-4-methoxybenzyl)-2-(phenoxymethyl)morpholine II were selected for labeling with fluorine-18 and subsequent evaluation by in vitro autoradiography to assess their suitability as D₄ radioligand candidates for in vivo imaging. Results: The radiosynthesis of [¹⁸F]I and [¹⁸F]II was successfully achieved by copper-mediated radiofluorination with radiochemical yields of 7% and 66%, respectively. The radioligand [¹⁸F]II showed specific binding in areas where D₄ expression is expected, whereas [¹⁸F]I did not show any uptake in distinct brain regions and exhibited an unacceptable degree of non-specific binding. Conclusions: The compounds studied exhibited high D₄R subtype selectivity and logP values compatible with high brain uptake, but only ligand [¹⁸F]II showed low non-specific binding and is therefore a good candidate for further evaluation. Advances in knowledge: The discovery of new lead structures for high-affinity D₄ ligands opens up new possibilities for the development of suitable PET-radioligands. Implications for patient: PET-imaging of dopamine D₄-receptors could facilitate understanding, diagnosis and treatment of neuropsychiatric and neurodegenerative diseases.

Full text of DOI:10.1016/j.nucmedbio.2020.07.004

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Radiosynthesis and evaluation of 18F-labeled dopamine
D4-receptor ligands
Michael Willmann, Johannes Ermert, Olaf Prante, Harald Hübner,
Peter Gmeiner, Bernd Neumaier
PII:
S0969-8051(20)30195-5
DOI:
https://doi.org/10.1016/j.nucmedbio.2020.07.004
NMB 8160
Reference:
To appear in:
Nuclear Medicine and Biology
Received date:
Accepted date:
29 June 2020
9 July 2020
Please cite this article as: M. Willmann, J. Ermert, O. Prante, et al., Radiosynthesis and
evaluation of 18F-labeled dopamine D4-receptor ligands, Nuclear Medicine and Biology
(2020), https://doi.org/10.1016/j.nucmedbio.2020.07.004
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Radiosynthesis and evaluation of 18F-labeled dopamine D4-receptor ligands
Michael Willmanna, Johannes Ermerta*, Olaf Prantec, Harald Hübnerd, Peter Gmeinerd, Bernd
Neumaiera,b
a
Forschungszentrum Jülich GmbH, Institut für Neurowissenschaften und Medizin, INM-5,
Nuklearchemie, 52425 Jülich, Germany
b
Uniklinik Köln, Institut für Radiochemie und Experimentelle Molekulare Bildgebung, 50937
Köln, Germany
c
Friedrich-Alexander University Erlangen-Nürnberg (FAU), Department of Nuclear Medicine,
Molecular Imaging and Radiochemistry, Translational Research Center, 91054 Erlangen,
Germany
d
Friedrich-Alexander University Erlangen-Nürnberg (FAU), Department Chemistry and
Pharmacy, Medicinal Chemistry, 91058 Erlangen, Germany
* corresponding author: j.ermert@fz-juelich.de
Abbreviated title: 18F-labeled dopamine D4-receptor ligands
Keywords: 18F-labeling, positron emission tomography, autoradiography, dopamine D4-receptor,
schizophrenia, neurological diseases

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A B S T R A C T
Introduction: The dopamine D4 receptor (D4R) has attracted considerable attention as potential
target for the treatment of a broad range of central nervous system disorders. Although many efforts
have been made to improve the performance of putative radioligand candidates, there is still a lack
of D4R selective tracers suitable for in vivo PET imaging. Thus, the objective of this work was to
develop a D4-selective PET ligand for clinical applications.
Methods: Four compounds based on previous and new lead structures were prepared and
characterized with regard to their D4R subtype selectivity and predicted lipophilicity. From these, 3-
((4-(2-fluorophenyl)piperazin-1-yl)methyl)-1H-pyrrolo[2,3-b]pyridine I and (S)-4-(3-fluoro-4-
methoxybenzyl)-2-(phenoxymethyl)morpholine II were selected for labeling with fluorine-18 and
subsequent evaluation by in vitro autoradiography to assess their suitability as D4 radioligand
candidates for in vivo imaging.
Results: The radiosynthesis of [18F]I and [18F]II was successfully achieved by copper-mediated
radiofluorination with radiochemical yields of 7 % and 66 %, respectively. The radioligand [18F]II
showed specific binding in areas where D4 expression is expected, whereas [18F]I did not show any
uptake in distinct brain regions and exhibited an unacceptable degree of nonspecific binding.
Conclusions: The compounds studied exhibited high D4R subtype selectivity and logP values
compatible with high brain uptake, but only ligand [18F]II showed low nonspecific binding and is
therefore a good candidate for further evaluation.
Advances in Knowledge: The discovery of new lead structures for high-affinity D4 ligands opens up
new possibilities for the development of suitable PET-radioligands.
Implications for patient: PET-imaging of dopamine D4-receptors could facilitate understanding,
diagnosis and treatment of neuropsychiatric and neurodegenerative diseases.

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1 Introduction
Dopamine (DA) is an important neurotransmitter involved in cognitive control, reward-related
cognition and motor function that produces its effects by interaction with the DA receptors
belonging to the G-protein-coupled receptor (GPCR) family. DA D2 receptor (D2R) antagonists
have a long history as effective antipsychotic drugs, but their clinical application is limited by
serious extrapyramidal side effects thought to result from DA receptor blockade in the striatum [1].
The DA D4 receptor (D4R), a less abundant member of the D2-like DA receptor subfamily, has
attracted considerable attention as potentially more suitable target for the treatment of central
nervous system disorders such as schizophrenia [2], drug abuse [3] and attention deficit
hyperactivity disorder [4]. First cloned in 1991, D4R was identified as a preferential target of the
atypical antipsychotic agent clozapine [5] showing a 10 fold higher affinity for the D4 than for the
D2 receptor [6, 7] (Fig. 1), which suggested that selective D4R antagonism could be responsible for
the drugs unique clinical efficacy in otherwise treatment-resistant patients [8]. In addition, early
mRNA and immunhistochemical studies indicated that D4R expression is mostly restricted to extra-
striatal (cortical and limbic) brain regions [9, 10], which could explain the very low propensity of
clozapine to induce extrapyramidal side effects [11]. However, further studies could not confirm the
clinical efficiency of D4R antagonists in schizophrenia [12, 13] and a number of inconsistent or
contradictory findings as well as apparent species differences in receptor distribution have
complicated conclusions with regard to the role of D4R in human health and disease [14, 15].
Imaging techniques like positron emission tomography (PET) could greatly aid in deciphering the
physiological and pathophysiological role of D4R, but their application has been hampered by a
number of methodological difficulties. For example, while ligands like L-745,870 (Fig. 2) possess
subnanomolar affinity and >2,000-fold selectivity for D4R over D2R, their aryl piperazine-based
structure is common to many GPCR ligands and associated with high off-target affinity (i.e.
unspecific binding). The latter could be overcome by rationale drug design based on structure-
activity-relationship (SAR) studies, which led to the development of improved ligands with reduced

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specific binding to non-D4 receptors [16, 17]. However, their use for PET imaging studies is further
complicated by the low abundance of D4R in the brain, as even moderate nonspecific binding of
radioligand candidates can be sufficient to mask specific binding. While difficult to predict based
on SAR studies, nonspecific binding involves partitioning of ligands into phospholipid membranes
[18] and often correlates with their lipophilicity/phospholipicity [19-21]. Here, we report our results
with four candidate D4R ligands (Fig. 3), which were derived from previous and new lead
structures. The potential D4 selective compounds were synthesized and their dopamine receptor
affinities determined. Among these candidates, the two most promising compounds were 18F-
labeled and evaluated by means of in vitro autoradiography to assess their suitability as D4
radioligand candidates for in vivo imaging.
2 Materials and Methods
2.1 Materials
All chemicals and solvents were purchased by Aldrich (Germany), Fluka (Switzerland),
Fluorochem (United Kingdom) and Merck (Germany) and used without any purification. Thin layer
chromatography (TLC) was performed on precoated plates of silica gel 60 F254 (Merck, Darmstadt,
Germany) and the compounds were detected at 254 nm. All reactions sensitive to moisture were
carried out under argon atmosphere in reaction flasks dried overnight at 140 °C prior to use. All
reaction mixtures were magnetically stirred. Organic extracts were dried with anhydrous MgSO4 or
1
Na2SO4. H, 13C, and 19F NMR spectra were recorded at 400.13, 100.61, and 376.49 MHz,
respectively, by means of a Bruker Avance Neo 400 instrument (Bruker Bio Spin GmbH,
Rheinstetten, Germany) in 5 % solution at 25 °C. Chemical shifts (δ) are given in parts per million
(ppm) relative to trace amounts of the residual solvent signals. High-resolution mass spectrometry
(HRMS) analyses were performed using a hybrid linear ion trap FTICR mass spectrometer LTQ-FT
(Thermo Fisher Scientific, Bremen, Germany) equipped with a 7 T superconducting magnet by
infusion. The mass spectrometer was first tuned and calibrated in the positive mode following the

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standard optimization procedure for all voltages and settings. Mass spectra were recorded in full
scan from 200 to 1000 Da with a resolution of 100.000 at m/z 400. All data were processed using
the Xalibur software version 2.1.
2.2 Syntheses of labeling precursors and reference standards
1H-Pyrrolo[2,3-b]pyridine-3-carbaldehyde (1): To a solution of 7-azaindole (10.0 g, 84.7 mmol)
in acetic acid (20 mL) and H2O (40 mL) was added HMTA (10.6 g, 75.3 mmol). The reaction
mixture was stirred at 120 °C for 6 h. It was cooled with an ice bath, and the resulting precipitate
was collected and dried to afford 1 (7.83 g, 71 %) as pale yellow solid. Yield: 7.83 g (71 %); pale
1
yellow solid; Rf 0.34 (80 % EtOAc/PE). H NMR (400 MHz, DMSO-d6) δ 12.69 (s, 1H), 9.93 (s,
1H), 8.47 (s, 1H), 8.40 (dd, J = 7.8, 1.6 Hz, 1H), 8.36 (dd, J = 4.7, 1.7 Hz, 1H), 7.27 (dd, J = 7.8,
4.7 Hz, 1H).13C NMR (101 MHz, DMSO) δ 185.40, 149.39, 144.83, 138.74, 129.21, 118.42,
116.63, 116.47. MS (ESI): m/z [M+H]+ calcd for C8H7N2O+: 147.06; found: 147.13. HRMS (ESI):
m/z [M]+ calcd. for C8H5N2O+: 145.03964; found: 145.04075.
Compounds 2 & 3 were synthesized according to a published procedure [22].
.
3-(1-Phenyl-3-propyl-1H-pyrazol-5-yl)propanal (4): To 3-(1-phenyl-3-propyl-1H-pyrazol-5-
yl)propan-1-ol (2) (1.89 g, 7.74 mmol, 1.0 eq.) in anhydrous DCM (100 mL) at 0 °C (ice bath) was
added NaHCO3 (1.30 g; 15.5 mmol, 2.0 eq.) and Dess-Martin periodinane (DMP) (3.94 g,
9.29 mmol, 1.2 eq.) under argon. The reaction mixture was stirred for 10 min at 0 °C and then
allowed to warm up to ambient temperature. Stirring was continued for 2 h, maintaining the inert
atmosphere. Consumption of the starting material was monitored by TLC. The reaction mixture was
diluted with a 1:1 mixture of 10 % aq. Na2S2O3 and sat. aq. NaHCO3 solution (50 mL) and stirred
for 10 min. Et2O (100 mL) was added and the layers were separated. The organic layer was washed
with above 1:1 mixture of 10 % aq. Na2S2O3 and sat. aq. NaHCO3 solution (50 mL), dried (Na2SO4)
and concentrated in vacuo. The crude material was purified by flash column chromatography

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1
(silica: EtOAc/PE) to obtain 1.61 g (86 %) of product 4. Rf 0.51 (33 % EtOAc/PE). H NMR (200
MHz, CDCl3) δ 9.75 (s, 1H), 7.57 – 7.27 (m, 5H), 6.00 (s, 1H), 3.10 – 2.84 (m, 2H), 2.84 – 2.45 (m,
4H), 1.89 – 1.47 (m, 2H), 0.98 (t, J = 7.3 Hz, 3H). 13C NMR (50 MHz, CDCl3) δ 153.78 (s), 142.29
(s), 139.54 (s), 77.79 (s), 77.36 – 76.83 (m), 76.52 (s), 42.67 (s), 30.31 (s), 22.98 (s), 19.01 (s). MS
(ESI): m/z [M+H]+ calcd. for C15H19N2O+: 243.15; found: 243.20.
N-Benzilidene-2-hydroxyethylamine (5): Benzaldehyde (102.04 mL, 1.00 mol), 2-aminoethanol
(60.48 mL, 1.00 mol) and p-toluenesulfonic acid (400 mg, 2 mmol) were added to toluene (400
mL). The solution was refluxed for 4 h in a Dean-Stark apparatus until 18 mL (1.00 mol) of water
were collected in the water trap. The solvent was removed in vacuo, dissolved in EtOAc (400 mL)
and washed with brine (200 mL) and water (200 mL). After drying (MgSO4) and concentration in
vacuo, compound 5 (137.00 g, 92 %) was obtained as orange oil. Yield: 137.00 g (92%); orange oil;
1
Rf 0.68 (17 % EtOAc/PE). H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 7.77 – 7.65 (m, 2H), 7.41
13
(ddd, J = 5.4, 4.3, 2.5 Hz, 3H), 3.93 – 3.87 (m, 2H), 3.79 – 3.71 (m, 2H), 3.45 (s, 1H). C NMR
(101 MHz, CDCl3) δ 163.44, 135.87, 131.03, 128.75, 128.32, 63.41, 62.43. HRMS (ESI): m/z
[M+Na]+ calcd for C9H11NONa+: 172.07383; found: 172.07335.
2-(Benzylamino)ethanol (6): Compound 5 (20 g, 134 mmol) in 200 mL dry MeOH was chilled in
an ice bath and the bottle was flushed with argon. NaCNBH3 (17 g, 270 mmol) was added in
portions over the course of 12 h. The complete consumption of starting material was verified by
TLC. The reaction mixture was concentrated in vacuo and brine (200 mL) was added to the residue,
which was then extracted with DCM (3 x 200 mL). The organic layers were washed with water
(100 mL), dried and concentrated in vacuo to obtain 17.14 g (94 %) of yellow oil of compound 6,
which was purified by acid-base extraction (HCl/NaOH) directly before use. Yield: 17.14 g (94%);
yellow oil; Rf 0.77 (75 % EtOAc/PE). 1H NMR (400 MHz, Chloroform-d) δ 7.28 (tdd, J = 14.4, 9.0,
13
4.8 Hz, 6H), 3.74 (s, 2H), 3.69 – 3.48 (m, 2H), 3.19 (s, 3H), 2.79 – 2.61 (m, 2H). C NMR (101
MHz, CDCl3) δ 139.67, 128.58, 128.31, 127.27, 60.82, 53.55, 50.66, 32.22. MS (ESI): m/z [M+H]+

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calcd for C9H14NO+: 152.11; found: 152.10. HRMS (ESI): m/z [M+H]+ calcd for C9H14NO+:
152.10699; found: 152.10703.
(S)-(4-Benzylmorpholin-2-yl)methanol (7): To compound 6 (9.64 g, 63.8 mmol) in water (5 mL)
and 2-propanol (5 mL) was added (R)-epichlorohydrin (5.9 g, 63.8 mmol) and the solution was
stirred overnight. A 20 wt-% aqueous solution of Et4NOH (61 mL, 83.0 mmol) was added over 5
min. After 4 h, the reaction was adjusted to pH 9 with 1 m HCl (10 mL). Water (50 mL) was added
and the mixture was extracted with DCM (3 × 500 mL). The extracts were combined and
evaporated to an oil, which was purified by flash column chromatography (silica, EtOAc/PE) to
1
afford compound 7. Yield: 4.66 g (35 %); colorless oil; Rf 0.18 (50 % EtOAc/PE). H NMR (400
MHz, CDCl3) δ 7.25 (d, J = 4.4 Hz, 4H), 7.21 – 7.15 (m, 1H), 3.80 (d, J = 1.5 Hz, 1H), 3.64 (dd, J =
12.4, 10.0 Hz, 2H), 3.56 – 3.38 (m, 4H), 2.69 – 2.56 (m, 2H), 2.54 – 2.22 (m, 1H), 2.13 (d, J = 3.4
Hz, 1H), 2.01 – 1.87 (m, 1H). 13C NMR (101 MHz, CDCl3) δ 129.39, 128.44, 127.44, 76.04, 66.61,
+
64.23, 63.40, 54.62, 53.07. MS (ESI): m/z [M+H]+ calcd for C12H18NO2 : 208.13; found: 208.26.
+
HRMS (ESI): m/z [M+H]+ calcd for C12H18NO2 : 208.13321; found: 208.13322.
(S)-Morpholin-2-ylmethanol (8): To a stirred mixture of 7 (2.43 g, 12.0 mmol) and 10 % Pd-C
(2.43 g) in dry MeOH (80 mL) was added NH4CO2H (3.78 g, 60 mmol) in a single portion under
nitrogen. This was refluxed for approx. 30 min until TLC showed complete consumption of the
starting material. The reaction mixture was filtered through a celite pad, which was then washed
with 20 mL CHCl3. Concentration in vacuo afforded 1.40 g of product 8. Yield: 1.40 g (>99 %),
1
pale yellow oil; no UV trace in TLC. H NMR (400 MHz, DMSO) δ 4.64 (s, 2H), 3.75 (dd, J =
11.4, 2.3 Hz, 1H), 3.58 – 3.21 (m, 4H), 3.17 (s, 1H), 2.90 (d, J = 12.1 Hz, 1H), 2.76 (s, 1H), 2.70
(dd, J = 11.6, 3.4 Hz, 1H), 2.44 (dd, J = 12.2, 10.3 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 76.15,
+
65.83, 62.47, 47.08, 44.64, 39.52. MS (ESI): m/z [M+H]+ calcd for C5H12NO2 : 118.09; found:
+
118.15. HRMS (ESI): m/z [M+H]+ calcd for C5H12NO2 : 118.08626; found: 118.08619.
tert-Butyl (S)-2-(hydroxymethyl)morpholine-4-carboxylate (9): 8 (2.88 g, 24.6 mmol) was added
to a stirred solution of guanidine hydrochloride (352 mg, 3.69 mmol) and di-tert-butyl dicarbonate

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(5.37 g, 24.6 mmol) in EtOH (60 mL) at 40 °C and stirred for 1 h until evolution of CO2 had ceased.
EtOH was evaporated in vacuo and the residue was purified by flash column chromatography
(silica, EtOAc/PE) to afford the pure product 9. Yield: 3.07 g (57 %), colorless solid, no UV trace
in TLC. 1H NMR (400 MHz, CDCl3) δ 3.82 – 3.60 (m, 1H), 2.86 (dd, J = 10.7, 5.8 Hz, 2H), 2.44 (s,
13
3H), 2.03 (s, 3H), 1.24 (d, J = 6.6 Hz, 9H). C NMR (101 MHz, CDCl3) δ 143.84, 83.35, 75.15,
+
62.46, 53.99, 45.63 (s), 29.84, 25.00. MS (ESI): m/z [M+H]+ calcd for C10H20NO4 : 218.14; found:
+
218.23. HRMS (ESI): m/z [M+Na]+ calcd for C10H19NNaO4 : 240.12063; found: 240.12074.
tert-Butyl (S)-2-(phenoxymethyl)morpholine-4-carboxylate (10): To 9 (500 mg, 2.30 mmol) was
added anhydrous THF (1.14 mL), phenol (216 mg, 2.30 mmol) and PPh3 604 mg, 2.30 mmol). The
mixture was sonicated (40 kHz) for several minutes to allow for mixing. More anhydrous THF
(1.26 mL) was added slowly until all compounds were dissolved. Under sonification, DIAD
(0.454 mL, 2.30 mmol) was added dropwise over a course of 2 min. The mixture was sonicated for
further 13 min. Anhydrous MgCl2 (0.44 g, 4.6 mmol) was added and the mixture was refluxed for
2 h, then chilled in an ice bath and filtered to remove the majority of PPh3O byproduct by
complexation. The crude material was purified by flash column chromatography (silica,
hexane/EtOAc) and recrystallized from methanol to obtain 259 mg (77 %) of pure product 10.
Yield: 259 mg (77 %), colorless crystals, Rf 0.60 (33 % EtOAc/PE). 1H NMR (400 MHz, CDCl3) δ
7.26 (s, 2H), 7.07 – 6.77 (m, 3H), 4.08 (ddd, J = 15.3, 12.1, 6.2 Hz, 2H), 3.95 (dd, J = 9.9, 4.9 Hz,
2H), 3.78 (ddd, J = 17.4, 10.1, 7.5 Hz, 1H), 3.65 – 3.54 (m, 1H), 1.47 (s, 9H). 13C NMR (101 MHz,
CDCl3) δ 158.67, 154.90, 129.61, 121.30, 114.73, 80.34, 73.98, 70.69, 68.66, 66.76, 29.85, 28.52.
+
MS (ESI): m/z [M+H]+ calcd for C16H24NO4 : 294.16; found: 294.22. HRMS (ESI): m/z [M+H]+
+
calcd for C16H24NO4 : 294.16998; found: 294.17003.
(S)-(2-Methoxy-5-((2-(phenoxymethyl)morpholino)methyl)phenyl)boronic acid (11): (S)-2-
(Phenoxymethyl)morpholine (455 mg, 2.35 mmol) and (5-formyl-2-methoxyphenyl)boronic acid
(424 mg, 2.35 mmol) in 10 mL MeOH was adjusted to pH 5 with acetic acid. NaCNBH3 (399 mg,
6.36 mmol) was added under argon atmosphere. The mixture was stirred at 60 °C for 24 h. The

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reaction mixture was concentrated in vacuo. To the residue was added water (20 mL), adjusted to
pH 9 with 1 M NaOH and extracted with DCM (3 × 100 mL). The organic layers were dried and
concentrated to obtain crude oil, which was purified by flash column chromatography (silica,
EtOAc/PE) to provide 423 mg (50 %) of pure product 11. Yield: 423 mg (50 %); colorless solid. Rf
1
0.24 (33 % EtOAc/PE). H NMR (400 MHz, CDCl3) δ 7.96 – 7.83 (m, 1H), 7.44 – 7.35 (m, 1H),
7.34 – 7.22 (m, 3H), 6.97 – 6.82 (m, 4H), 4.08 – 3.87 (m, 7H), 3.58 – 3.42 (m, 2H), 2.98 – 2.58 (m,
13
3H), 2.31 – 2.04 (m, 2H). C NMR (101 MHz, CDCl3) δ 163.93, 158.75, 138.33, 133.81, 129.59,
129.39, 128.44, 126.22, 120.93, 114.65, 109.76, 73.90, 69.25, 66.71, 62.70, 55.58, 55.26, 52.85.
+
MS (ESI): m/z [M+H]+ calcd for C19H25NO5 : 358.18; found: 358.20. HRMS (ESI): m/z [M+H]+
+
calcd for C19H25BNO5 : 358.18203; found: 358.18202.
3-[4-(2-Fluorophenyl)piperazin-1-yl]-1H-pyrrolo-[2,3-b]pyridine (I): To 1 (800mg, 5.47mmol)
in methanol (14 mL) was added 1-(2-fluorophenyl)piperazine (986 mg, 5.47 mmol). The reaction
mixture was adjusted with acetic acid to pH 5 and then reacted with NaBH3CN (928 mg,
14.77 mmol) at 80 °C for 15 h. After the solvent has been removed in vacuo, the residue was
diluted with ethyl acetate and neutralized with 10 % NaOH. Product isolation (ethyl acetate)
followed by flash column chromatography (silica: EtOAc/PE) afforded compound I (611 mg, 36 %)
as colorless crystals. Yield: 611 mg (36 %); colorless crystals; Rf 0.10 (50 %; EtOAc/PE). 1H NMR
(400 MHz, DMSO) δ 11.49 (s, 1H), 8.20 (dd, J = 4.7, 1.5 Hz, 1H), 8.06 (d, J = 6.9 Hz, 1H), 7.39 (s,
1H), 7.17 – 6.86 (m, 5H), 3.70 (s, 2H), 3.33 (s, 2H), 2.99 (s, 4H), 2.57 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 156.15, 153.72, 148.73, 142.50, 127.28, 124.79, 122.25, 122.17, 119.77, 119.17, 115.97,
115.76, 115.11, 53.07, 52.41, 50.13. 19F NMR (376 MHz, DMSO) δ -122.79 (s). HRMS (ESI): m/z
+
[M+H]+ calcd for C18H20FN4 : 311.16665; found: 311.16662.
(S)-4-(3-Fluoro-4-methoxybenzyl)-2-(phenoxymethyl)morpholine (II): 10 (116 mg, 0.56 mmol)
was treated with 1 mL trifluoroacetic acid (TFA) in 3 mL DCM and the mixture was stirred for 1 h
at 30 °C. The mixture was then adjusted to pH 9 with 1 m NaOH and extracted with DCM and
concentrated in vacuo to obtain 104 mg of a crude oil. The unprotected amine (104 mg, 0.54 mmol)

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in 2 mL MeOH was added with 3-fluoro-4-methoxybenzaldehyde (83 mg, 054 mmol), the mixture
adjusted to pH 5 with acetic acid and NaBH3CN (92 mg, 2.7 eq.) was added. The mixture was
stirred for 16 h at 60 °C. Aqueous workup and extraction with DCM afforded a crude oil, which
was purified by flash column chromatography (EtOAc/PE) to obtain compound 11. Yield: 183 mg
(99 %); colorless oil. Rf 0.44 (33 % EtOAc/PE). 1H NMR (400 MHz, CDCl3) δ 7.33 – 7.19 (m, 4H),
7.16 – 7.06 (m, 2H), 7.04 – 6.82 (m, 10H), 4.07 – 3.84 (m, 14H), 3.73 (td, J = 11.3, 2.5 Hz, 2H),
3.45 (s, 4H), 2.85 (d, J = 11.1 Hz, 2H), 2.66 (dd, J = 11.4, 1.9 Hz, 2H), 2.21 (td, J = 11.3, 3.3 Hz,
2H), 2.13 – 2.02 (m, 2H), 1.98 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 158.77, 153.65, 151.21,
146.91, 146.80, 130.89, 129.51, 124.77, 124.73, 121.08, 114.70, 113.19, 113.17, 74.20, 69.23,
66.97, 62.41, 56.40, 55.43, 52.93. 19F NMR (376 MHz, CDCl3) δ -135.43. HRMS (ESI): m/z
+
[M+H]+ calcd for C19H23FNO3 : 332.1656; found: 332.1656.
3-((4-(2-Fluorobenzyl)piperazin-1-yl)methyl)-1H-pyrrolo[2,3-b]pyridine (III): 1 (225 mg, 1.54
mmol) was dissolved in 5 mL MeOH and 1-(2-fluorobenzyl)piperazine (299 mg, 1.54 mmol) was
added thereto. The mixture adjusted to pH 5 with acetic acid and NaBH3CN (260 mg, 2.7 eq.) was
added. The mixture was stirred for 16 h at 60 °C. Aqueous workup and extraction with DCM
afforded a crude oil, which was purified by flash column chromatography (silica, EtOAc/PE) to
obtain the pure compound III as yellow oil. Yield: 250 mg (50 %); yellow oil. Rf 0.20 (75 %
1
EtOAc/PE). H NMR (400 MHz, CDCl3) δ 10.69 (s, 2H), 8.29 (d, J = 3.3 Hz, 1H), 8.05 (dd, J =
7.9, 1.5 Hz, 1H), 7.33 (t, J = 7.5 Hz, 1H), 7.28 (s, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.07 (dd, J = 7.3,
13
5.5 Hz, 2H), 7.04 – 6.95 (m, 1H), 3.73 (s, 2H), 3.59 (s, 2H), 3.28 (s, 2H), 2.55 (s, 8H). C NMR
(101 MHz, CDCl3) δ 162.77, 160.32, 148.94, 142.62, 131.84, 128.89, 128.29, 124.71, 124.63,
124.49, 123.95, 123.91, 120.85, 115.71, 115.47, 115.25, 110.57, 55.28, 55.26, 53.53, 52.85, 52.77.
+
19F NMR (376 MHz, CDCl3) δ -117.73. HRMS (ESI): m/z [M+H]+ calcd for C19H12FN4 : 325.1823;
found: 325.1823.
1-(2-Fluorophenyl)-4-(3-(1-phenyl-3-propyl-1H-pyrazol-5-yl)propyl)piperazine
(IV):
4
(220 mg, 0.91 mmol) was dissolved in 10 mL of DCM together with 1-(2-fluorophenyl)piperazine

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(164 mg, 0.91 mmol). To the mixture was added 1.40 g of molecular sieve (4 A). DIPEA (0.16 mL,
0.91 mmol) was slowly added thereto and stirred for 30 min at room temperature. Then,
NaBH(OAc)3 (674 mg, 3.18 mmol) was added and stirred at r.t. for 3 d. Water (5 mL) was added
and the organic layer was separated and concentrated in vacuo to obtain 404 mg of crude oil, which
was purified by flash column chromatography (silica, EtOAc/PE) to obtain 331 mg (90 %) of the
pure product IV as yellow oil. Rf 0.20 (33 % EtOAc/PE). 1H NMR (400 MHz, CDCl3) δ 7.56 – 7.30
(m, 5H), 7.11 – 6.82 (m, 4H), 6.05 (s, 1H), 3.24 – 2.93 (m, 4H), 2.81 – 2.45 (m, 8H), 2.46 – 2.27
(m, 2H), 1.89 – 1.58 (m, 4H), 1.00 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 157.08,
154.63, 153.79, 143.87, 140.30, 140.17, 129.15, 127.62, 125.55, 124.59, 124.55, 122.59, 122.51,
118.99, 116.32, 116.11, 104.46, 77.48, 77.16, 77.16, 76.84, 57.80, 53.32, 50.64, 50.61, 30.51,
19
26.23, 24.21, 23.10, 14.18. F NMR (376 MHz, CDCl3) δ -122.73 (s). HRMS (ESI): m/z [M+H]+
+
calcd for C25H32FN4 : 407.2605; found: 407.2605.
2.3 Receptor binding assay
Radioligand binding studies with the subtypes of the D2 receptor family were performed as
described previously [23]. In brief, competition binding experiments were done using membranes of
CHO cells stably expressing the human D2L, D2S [24], D3 [25] or D4.4 [26] receptor. Radioligand
displacement assays were run in binding buffer (25 mM HEPES, 5 mM MgCl2, 1 mM EDTA,
0.006% BSA at a pH of 7.4) with [3H]spiperone (molar activity = 68 Ci/mmol, PerkinElmer,
Rodgau, Germany). The assays were carried out at a protein concentration of 4 µg/well, Bmax=1900
fmol/µg, KD=0.10 nM and a concentration of the radioligand of 0.2 nM for D2L, of 1 µg/well,
Bmax=500 fmol/µg, KD=0.10 nM and radioligand of 0.2 nM for D2S, of 2 µg/well, Bmax=3500
fmol/µg, KD=0.25 nM and radioligand of 0.3 nM for D3, and of 6 µg/well, Bmax=1800 fmol/µg,
KD=0.40 nM and radioligand of 0.4 nM for D4.4, respectively. Unspecific binding was determined
in the presence of 10 µM haloperidol, protein concentration was established by the method of
Lowry using bovine serum albumin as standard [27]. Competition binding experiments with GPCRs
related to D2-D4 were performed in an analogous manner with membranes from HEK293T cells

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transiently transfected with the corresponding cDNA by using the Mirus TransIT-293 transfection
reagent (PeqLab, Erlangen, Germany) or a solution of polyethylenimine in PBS (PEI, linear 25 kDa,
Polysciences, 1 mg/mL) as transfection reagent at a 3:1 PEI to cDNA ratio [28]. Binding to the
dopamine receptors D1 and D5 was performed with the radioligand [3H]SCH23390 (80 Ci/mmol,
Biotrend, Cologne, Germany) at a concentration of 0.30 nM, KD=0.23 nM, Bmax=3500 fmol/mg and
2 µg protein/well for D1, and of 0.50 nM, KD=0.50 nM, Bmax=1000 fmol/mg and 14 µg protein/well
for D5, respectively. Affinities to serotonin receptors was measured with [3H]WAY100635 (80
Ci/mmol, Biotrend) at 0.20 nM, KD=0.10 nM, Bmax=3000 fmol/mg and 2 µg protein/well for 5-
HT1A, and [3H]ketanserin (47 Ci/mmol, Biotrend) at 0.30 nM, KD=0.35 nM, Bmax=3400 fmol/mg
and 4 µg protein/well for 5-HT1A, respectively. Adrenergic 1A binding was determined applying
[3H]prazosin (84 Ci/mmol, Biotrend) at 0.20 nM, KD=0.095 nM, Bmax=7500 fmol/mg and 2 µg
protein/well.
The resulting competition curves of the receptor binding experiments were analyzed by nonlinear
regression using the algorithms in PRISM 6.0 (GraphPad Software, San Diego, CA). The data were
initially fit 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 [29].
2.4 Radiochemistry
All radiosyntheses were carried out using anhydrous N,N-dimethylacetamide (DMA) (Aldrich).
QMA cartridges (Sep-Pak Accell Plus QMA Carbonate Plus Light, 46 mg sorbent per cartridge)
were obtained from Waters (Waters GmbH, Eschborn, Germany) and preconditioned with 1 mL
H2O directly before use. [18F]Fluoride was produced by the 18O(p,n)18F reaction by bombardment of
enriched [18O]water with 16.5 MeV protons using a BC1710 cyclotron (The Japan Steel Works
Ltd., Shinagawa, Japan) at the INM-5 (Forschungszentrum Jülich). All radiolabeling experiments
were carried out under ambient atmosphere.

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Before radiosynthesis, [18F]fluoride was processed as follows. Aqueous [18F]fluoride was loaded
onto an anion-exchange resin (QMA cartridge). It should be noted, that aqueous [18F]fluoride was
loaded onto the cartridge from the male side, whereas flushing, washing and 18F- elution were
carried out from the female side. If MeOH was used for elution, the resin was flushed with MeOH
(2 mL) before use. In order to account for surface-adsorbed [18F]fluoride, reaction vials were
completely emptied after the addition of water, and radioactivity in the aqueous solution and
remaining radioactivity in the reaction vessel was separately determined and quantified. For
radiolabeling, [18F]fluoride was eluted from the QMA cartridge with a solution of Et4NHCO3 (1.0
mg) in nBuOH (500 μL).
General procedure: The borylated precursor (60 μmol) and Cu(OTf)2(py)4 (30 μmol) in DMA (600
μL) were added to the dried [18F]fluoride and the reaction mixture was stirred at 110 °C for 10 min,
then cooled to room temperature, diluted with water (1 mL) and stirred again for 30 s. Thereafter,
RCY was determined by radio-HPLC.
3-((4-(2-[18F]Fluorophenyl)piperazin-1-yl)methyl)-1H-pyrrolo[2,3-b]pyridine
([18F]I):
[18F]Fluoride was eluted from a QMA cartridge with a solution of Et4NHCO3 (1.0 mg) in nBuOH
(500 μL). A solution of tert-butyl 4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)pipera-
zine-1-carboxylate (23 mg, 60 μmol) and Cu(OTf)2(py)4 (20 mg, 30 μmol) in DMA (600 μL) was
added. The reaction mixture was heated at 110 °C for 10 min under air in a capped vial. The solvent
was then removed in vacuo and 1 mL TFA was added. This was heated for 20 min at 110 °C. TFA
was then removed in vacuo and 1 (9 mg, 60 μmol) suspended in 2 mL acetonitrile was added. This
was heated under reduced pressure and with a steam of argon for azeotropic removal of the
ascending water. Then, NaBH3CN (10 mg) in 1 mL methanol was added and the mixture heated at
80 °C for 5 min. The reaction mixture was quenched with water (4 mL) and passed through a C18
cartridge (500 mg), preconditioned with EtOH (1 mL) and water (10 mL). The cartridge was
washed with water (10 mL) and the product was eluted with MeOH (2 mL) and purified by

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preparative HPLC to afford the desired tracer. HPLC conditions for purification: Synergi 4µ Hydro-
RP 150×21.2 mm; eluent: water, 60 % MeCN; flow rate: 6 mL/min; tR = 5.3 min.
(S)-4-(3-[18F]fluoro-4-methoxybenzyl)-2-(phenoxymethyl)morpholine ([18F]II): [18F]Fluoride
was eluted from a QMA cartridge with a solution of Et4NHCO3 (1.0 mg) in nBuOH (500 μL). A
solution of (S)-(2-methoxy-5-((2-(phenoxymethyl)morpholino)methyl)phenyl)boronic acid 11
(21 mg, 60 μmol) and Cu(OTf)2(py)4 (20 mg, 30 μmol) in DMA (600 μL) was added. The reaction
mixture was heated at 110 °C for 10 min under air in a capped vial. The reaction mixture was
quenched with water (4 mL) and passed through a C18 cartridge (500 mg), preconditioned with
EtOH (1 mL) and water (10 mL). The cartridge was washed with water (10 mL) and the product
was eluted with MeOH (2 mL) and purified by preparative HPLC to afford [18F]II. HPLC
conditions for purification: Synergi 4µ Hydro-RP 150×21.2 mm; eluent: water, 60 % MeCN; flow
rate: 6 mL/min; tR = 8.5 min.
2.5 In vitro autoradiography
Wistar rats weighting 250 to 300 g (Charles River Deutschland GmbH) were decapitated, the brains
removed and frozen at −80 °C. For autoradiography, they were warmed to −20 °C, cut into 20 μm
thick horizontal and sagittal sections (CM 3050 or CM 3500 Polycut, Leica AG Microsystems,
Germany), mounted on gelatin-coated object glasses, dried and stored at −80 °C until use.
Autoradiography was performed as described previously [30]. Briefly the frozen brain sections
were thawed, dried in air at room temperature and then preincubated for 5 min at ambient
temperature in 50 mM Tris HCl (pH 7.4). Sections were then incubated for 70 min in Tris HCl
buffer containing a solution of the tracer (2.5 kBq/mL), together or without the ―cold‖ reference
compound at a final concentration of 5 µM. Labeling was terminated by rinsing the sections for 1
min in TrisHCl and dipping them in deionized water. The dried sections were placed on a phosphor
imaging plate (Fuji) together with a tritium standard (Amersham Bioscience). After exposure for 75
minutes, a laser phosphor imager (BAS 5000, Fuji) controlled with software from the vendor
(Version 3.14, Raytest, Germany) was used for image acquisition.

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2.6 HPLC Analysis
Before the determination of radiochemical yield (RCY), reaction mixtures were diluted with 50%
MeOH to dissolve any [18F]fluoride adsorbed onto the reaction vessel walls. HPLC analyses were
carried out on a Dionex Ultimate® 3000 HPLC system and a DAD UV-detector coupled in series
with a Berthold NaI detector. A Chromolith® SpeedROD RP-18e column (Merck, Darmstadt
Germany), 50×4.6 mm was used. UV and radioactivity detectors were connected in series, giving a
18
time delay of 0.1–0.9 min depending on the flow rate. F-Labeled compounds were identified by
co-injection of the unlabeled reference compounds using HPLC. The completeness of the
radioactivity elution was checked by analyzing the same sample amount choosing a column bypass.
Column: Chromolith SpeedROD®, 50×4.6 mm (Merck Millipore); gradient: 0–2 min: 5 % MeCN,
2–2.5 min: 5→20 % MeCN, 2.5–6 min: 20 % MeCN, 6–7 min: 20→70 % MeCN, 7–9 min: 70 %
MeCN, flow rate: 3.0 mL/min.
2.7 Determination of carrier content
The amount of unlabeled carrier was determined from the peak area in UV–HPLC chromatograms
using an UV absorbance/concentration calibration curve ([18F]I: λ = 223 nm; [18F]II: λ = 256 nm).
The solutions of radiolabeled products obtained after HPLC purification were allowed to stand at
ambient temperature for at least 24 h, concentrated under reduced pressure, and the residues were
re-dissolved in the appropriate HPLC eluents (500 μL). The resulting solutions were completely
injected into the HPLC system. The peak area was determined, and the amount of carrier was
calculated according to the calibration curve.
2.8 Estimation of lipophilicity
The software program ChemDraw 16.0 (PerkinElmer) using Crippen's fragmentation method [31]
and the online tools ALOGPS 2.1 [32] and Chemicalize [33] were used for the estimation of LogP
values.
3 Results and discussion

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3.1 Synthesis of reference compounds I-IV and labeling precursor 12
Based on azaindoles like L-745.870 [34, 35], FAUC 113/213 [36-38], ABT-124 [39, 40] or PIP3EA
[41] as D4R selective lead structures, compounds I and III were prepared by reductive amination of
1H-Pyrrolo[2,3-b]pyridine-3-carbaldehyde 1 and 1-(2-fluorophenyl)piperazine (2-FPP) (for I, see
scheme 1) or 1-(2-fluorobenzyl)piperazine (for III, see scheme 2) using NaBH3CN as reductant.
To obtain 1H-pyrrolo[2,3-b]pyridine-3-carbaldehyde 1, a formylation of 7-azaindol was carried out
using either the Vilsmeier-Haack reaction [42] or the Duff reaction [43], with the Duff reaction
performing better in terms of yield (71%) and ease of work-up.
Reference compound IV was chosen based on D4R selective piperazinyl-propyl-pyrazole
derivatives as alternative lead structures and synthesized according to a known procedure modified
only by the use of the less toxic oxidant Dess-Martin periodinane (DMP) in the oxidation step
(Scheme 3) [22].
The synthesis of the chiral alkoxy morpholines II and 12 was achieved according to a procedure
from HENEGAR [44] (Scheme 4), starting with the condensation of benzaldehyde and 2-
aminoethanol under Dean-Stark conditions, yielding the benzyl-protected ethanolamine 6 (94 %)
after reduction of the aldimine 5 by NaBH4 (92 %). Chirality was introduced by the reaction of 6
with enantiomerically pure (R)-epichlorohydrin, yielding an intermediate chlorohydrin (Scheme 4).
Without isolation of the intermediate, the cyclization reaction forming the epoxide was initiated by
addition of Et4NOH. Under inversion of the configuration (Walden inversion), the epoxide
underwent further cyclization to form the morpholine oxide 7 in a yield of 35 %. Subsequent
Mitsunobu reaction [45] with the phenol of either the N-benzyl or the N-Boc protected morpholine
proceeded under sonification (40 kHz) in 15 minutes with comparable yields, but reductive
cleavage (HCO2NH4, 10 % Pd-C) of the benzyl group, which was used as a chromophore for
reaction monitoring, gave significantly better yields (77 %) when it was performed prior to
Mitsunobu coupling. Since the Mitsunobu reaction failed with the unprotected morpholine, it was
again protected with the easily removable N-Boc group in a yield of 57 %. Reductive amination

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(NaBH3CN) with either 3-fluoro-4-methoxybenzaldehyde or (5-formyl-2-methoxyphenyl)boronic
acid yielded the reference compound II (99 %) or the radiolabeling precursor 12 (50 %)
respectively. Enantiomeric purity (>99 %) was conserved for both compounds throughout the
synthesis and verified by chiral HPLC.
3.2 Receptor binding studies
The target compounds I-IV were subjected tabto in vitro receptor binding assays to test their ability
to displace [3H]SCH 23990 from the cloned human D1-like DA receptors D1R and D5R, or
[3H]spiperone from the cloned human D2-like DA receptors D2Rlong, D2Rshort, D3R and the most
common D4R polymorphism D4.4R, which were stably expressed in Chinese hamster ovary (CHO)
cells. In addition, binding to the cloned human serotonin receptors 5-HT1AR and 5-HT2AR as well as
the cloned human alpha adrenergic receptor α1A transiently transfected in human embryonic kidney
(HEK293T) cells were examined by displacement experiments with the selective radioligands
[3H]WAY600135, [3H]ketanserin and [3H]prazosin respectively. The results of these experiments
are summarized in Tab. 1. All compounds exhibited high affinity for the D4 receptor subtype with
Ki values ranging from 4.1 to 18 nM (Tab. 1) and, except for compound IV, reduced affinity for all
other receptors tested (Tab. 2). Based on its very high affinity for the human α1A receptor (Ki=1.2
nM, Tab. 1 & 2) combined with a high predicted lipophilicity (see below), compound IV was
excluded from further studies. In comparison with reported D4-subtype selective ligands, such as
FAUC 213 with a D4-over-D3 selectivity of 2400-fold [37], compound III showed very similar D4
subtype selectivity (2000-fold, Tab. 2), but its D4 affinity (Ki=18 nM, Tab. 1) was almost ten-fold
lower than that of FAUC 213 (2 nM). However, the outstanding subtype selectivity and very low
affinity for 5-HT receptors suggested that this compound might still be worthwhile for radiolabeling
and future in vivo studies. The highest affinity for the D4 receptor was found for the 2-
fluorophenylpiperazinyl 7-azaindole (I) (Ki=4.1 nM), which exhibited moderate to good selectivity
over 5-HT (140 to 3400-fold) and D2 (230 to 340-fold) receptors (Tab. 2). The morpholine
derivative (II) showed a less variable D4-over-5-HT selectivity (570 to 610-fold) but only low D4-

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subtype selectivity (35 to 36-fold) (Tab. 2). Thus, taken together, ligand I and II both revealed
adequate D4 affinity combined with variable affinities to 5-HT (I > II) and non-D4 DA (I < II)
receptors.
3.3 Lipophilicity
A number of previous studies indicate that blood-brain-barrier (BBB) penetration and nonspecific
binding are often correlated with the lipophilicity of a ligand. To obtain a rough measure of this
physiochemical property, various tools were used to calculate logP and logD7.4 values for
compounds I-IV. As illustrated in Tab. 3, the different algorithms showed similar results, with
values determined to be between 2 and 3 for compounds I-III, a range considered optimal for
ligands to penetrate the blood-brain-barrier without excessive nonspecific binding. In contrast and
as already noted above, values between 5 and 6 were obtained for compound IV, suggesting that
nonspecific binding of this ligand could be high. Therefore, IV was not selected for radiosynthesis
and excluded from further evaluation.
3.4 Radiosynthesis
Due to their promising D4 selectivities, compounds I & II were selected for radiolabeling and
subsequent in vitro autoradiographic studies. The synthesis of radiotracer [18F]I was performed by a
three-step one-pot reaction without the need for isolation of any intermediates (Scheme 5). Attempts
for late stage radiofluorination were not successful since the corresponding ortho-borylated
precursor for [18F]I was challenging to synthesize and degraded upon purification by column
chromatography.
The [18F]fluoride used for radiofluorination was trapped on a QMA cartridge and eluted with
tetraethylammonium bicarbonate (TEAHC) in n-butanol with an elution efficacy of 90 ± 5 %.
A Cu(II)-mediated radiofluorination protocol as described by ZISCHLER et al. [46], with
commercially
available
tert-butyl
4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)piperazine-1-carboxylate as the precursor was performed at 110 °C for 10 min, which led

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to [18F]13 in 60-80 % radiochemical yield (RCY). Deprotection was achieved with excess
trifluoroacetic acid (TFA) at 110 °C for 20 min. Under less harsh conditions, deprotection did not
proceed quantitatively. Subsequently, the D4R-radioligand [18F]I was obtained by reductive
amination with azaindole 3-carbaldehyde 1, using an excess of NaBH3CN as reductant, for 5 min at
80 °C. The crude solution was passed through a C18 cartridge to trap the product, washed with
water and eluted with methanol. [18F]I was then isolated by semi-preparative HPLC with a total
RCY of 7 %.
18
Precursor 11 (Scheme 6) was used for aromatic nucleophilic F-substitution with the same Cu(II)-
mediated radiofluorination protocol by ZISCHLER et al. as used above [46]. [18F]Fluoride was
trapped on a QMA cartridge and eluted with tetraethylammonium bicarbonate (TEAHC) into a
solution of 11 and [Cu(OTf)2(py)4] in N,N-dimethylacetamide (DMA) and reacted at 110 °C for
10 min to obtain [18F]II. The crude solution was passed through a C18 cartridge to trap the product,
washed with water and eluted with methanol. Finally, the radiotracer [18F]II was (partially) isolated
by semi-preparative HPLC with a RCY of 66% (n=5).
3.5. Autoradiography
The main reason for failure of D4R selective radioligand candidates in in vivo experiments is a high
ratio of nonspecific to specific binding. Here, we used in vitro autoradiography in competition with
the non-labeled reference compounds to determine nonspecific binding of the radiolabeled
compounds [18F]I and [18F]II. To this end, rat brain slices were incubated either with the labeled
compounds alone (total binding) or with the labeled compounds and an excess of the corresponding
non-labeled reference compound (non-specific binding).
In three independent experiments with molar activities of up to 90 GBq/µmol, the D4 receptor
ligand [18F]I did not show any uptake in distinct brain regions and exhibited an unacceptably high
degree of non-specific binding that could not be displaced by the non-radioactive reference

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compound (Fig. 4). Thus, [18F]I is presumably unsuitable for in vivo PET imaging applications and
was therefore excluded from further studies.
For comparison, Fig. 5 shows horizontal and sagittal rat brain slices incubated with the chiral
radioligand [18F]II at a molar activity of 80 GBq/µmol (starting activity: 2450 MBq). Due to the
relatively low molar activity that was achieved by the manual radiosynthesis, the signal-to-noise
ratio of the autoradiography images could most likely be further improved by the use of an
automated synthesis module.
In any case, [18F]II appeared to show specific binding throughout the brain, with no uptake in the
striatum but distinct high accumulation in the colliculi and medial nuclei of the cerebellum (Fig. 5),
which is in line with previous results obtained using in vitro autoradiography with [18F]FPPB by
KÜGLER et al. [21]. On the other hand, little binding was observed in prefrontal cortex (PFC) and
hippocampus (HC), even though mRNA [9], immunhistochemical [15, 47, 48] and autoradiographic
[21, 49, 50] findings indicate predominant expression of D4R in these regions of the rat brain.
Interestingly, lack of binding in PFC and HC was also observed in previous in vitro
autoradiography experiments with [18F]FPPB, while ex vivo autoradiography with the same
radioligand revealed clear binding in these regions [21]. With this in mind, our findings encourage
further evaluation of [18F]II brain uptake and distribution in preclinical animal models by PET.
4. Conclusion
In summary, four compounds that appeared suitable for the development of a D4-selective
radioligand for PET imaging were first examined for their D4R subtype selectivity. Based on the
properties found, two of the compounds were 18F-labeled and examined by in vitro
autoradiography.
Of
these,
only
ligand
(S)-4-(3-[18F]fluoro-4-methoxybenzyl)-2-
(phenoxymethyl)morpholine [18F]II showed low non-specific binding and accumulation in distinct,

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extra-striatal brain regions. Further work is in progress to study the brain uptake and metabolism of
[18F]II in preclinical animal models.
5. Acknowledgement
The authors would like to thank Annette Schulze, Dr. Dirk Bier and Dr. Marcus Holschbach (all
INM-5), and Dr. Sabine Willbold (Zentralinstitut für Engineering, Elektronik und Analytik, ZEA-
3), all Forschungszentrum Jülich, for recording the spectroscopic data and conducting the biological
studies.

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Figures Capture:
Figure 1. The first atypical antipsychotic clozapine in comparison with typical antipsychotics.
Figure 2. D4 selective receptor ligands ABT-724 and L-745,870 with binding affinities and
calculated LogP values (ChemDraw 16).
Figure 3. Compounds selected for binding studies
Figure 4. In vitro autoradiography of horizontal rat brain slices with 2.31 kBq/mL [18F]I alone
(Total binding) or after blocking with 5 µM non-labeled I (Non-specific binding). i) Total binding
profile. ii) Competition with non-radioactive [19F]I, displaying non-specific binding.
Figure 5. In vitro autoradiography of horizontal (top) and sagittal (bottom) rat brain slices with
2.31 kBq/mL [18F]II alone (Total binding) or after blocking with 5 µM non-labeled II (Nonspecific
binding). cbl: cerebellum, co: colliculi, c: cortex).
Scheme 1. Synthesis of reference compound I a) Hexamethylenetetramine (HMTA, 120 °C, 6 h). b)
1-(2-fluorophenyl)piperazine, NaBH3CN, pH 5 (AcOH), 80 °C, 15 h.

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Scheme 2. Synthesis of reference compound III a) 1-(2-fluorobenzyl)piperazine, NaBH3CN, pH 5
(AcOH), 80 °C, 15 h.
Scheme 3. Synthesis of reference compound IV a) NaOMe, benzene, 30-50 °C. b) TEA 3 eq.,
MeOH, r.t., 4 h. c) DMP, DCM, r.t., 12 h. d) NaBH3CN, MeOH, 80 °C, 12 h.
Scheme 4. Synthesis of labeling precursor 22 and reference compound II a) 2-aminoethanol,
toluene b) NaBH3CN, r.t., 12 h. c) (R)-epichlorhydrine d&e) 20 wt-% aq. Et4NOH,
2-PrOH/H2O (1:1) f) 10 % Pd-C, NH4CO2H, MeOH g) guanidine*HCl, di-tert-butyl dicarbonate,
EtOH h) phenol, PPh3, diisopropyl azodicarboxylate (DIAD), THF, 40 kHz, 15 min i) HCl,
NaBH3CN, MeOH, 60 °C, 12 h.
Scheme 5. Synthesis of compound [18F]I a) Elution of 18F- with Et4NHCO3 in nBuOH;
[Cu(OTf)2(py)4], DMA, 110 °C, 10 min. b) TFA, 110 °C, 20 min. c) 3, NaBH3CN, 80 °C, 5 min.
(RCY determined by radio-HPLC).
Scheme 6. Synthesis of compound [18F]II a) Elution of 18F- with Et4NHCO3 in nBuOH;
[Cu(OTf)2(py)4], DMA, 110 °C, 10 min.