Syntheses and radiofluorination of two derivatives of 5-cyano-indole as selective ligands for the dopamine subtype-4 receptor

Article abstract of DOI:10.1002/jlcr.1026

Two fluoroethoxy substituted derivatives, namely 2-[4-(2-(2-fluoroethoxy) phenyl)-piperazin-1-ylmethyl]indole-5-carbonitrile (5a) and 2-[4-(4-(2- fluoroethoxy)-phenyl) piperazin-1-ylmethyl]indole-5-carbonitrile (Sb) were synthesized as analogs of the sele

Full text of DOI:10.1002/jlcr.1026

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 55–70.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1026
Research Article
Syntheses and radiofluorination of two derivatives of
5-cyano-indole as selective ligands for the dopamine
subtype-4 receptor
Rainer Tietze1, Carsten Hocke1, Stefan Lober2, Harald Hubner2, Torsten
¨
¨
Kuwert1, Peter Gmeiner2 and Olaf Prante1,*
1 Laboratory of Molecular Imaging, Clinic of Nuclear Medicine, Friedrich-Alexander
University, Schwabachanlage 6, D-91054 Erlangen, Germany
2 Department of Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander
University, Schuhstrasse 19, D-91052 Erlangen, Germany
Summary
Two fluoroethoxy substituted derivatives, namely 2-[4-(2-(2-fluoroethoxy)phenyl)-
piperazin-1-ylmethyl]indole-5-carbonitrile (5a) and 2-[4-(4-(2-fluoroethoxy)-phenyl)
piperazin-1-ylmethyl]indole-5-carbonitrile (5b) were synthesized as analogs of the
selective D4 receptor ligand 2-[4-(4-fluorophenyl)piperazin-1-ylmethyl]indole-5-carbo-
nitrile (FAUC 316). In vitro characterization using CHO-cells expressing different
dopamine receptor subtypes gave Ki values of 2.1 (5a) and 9.9 nM (5b) for the
dopamine D4 subtype and displayed a 420-fold D4-selectivity over D2 receptors for 5b.
The para-fluoroethoxy substituted candidate 5b revealed substantially reduced a1 and
serotoninergic binding affinities in comparison to the ortho-fluoroethoxy substituted
compound. In order to provide potential positron emission tomography (PET)
imaging probes for the dopamine D4 receptor, 18F-labelling conditions using
[18F]fluoroethyl tosylate were optimized and led to radiochemical yields of 81 ꢀ 5%
([18F]5a) and 47 ꢀ 4% ([18F]5b) (n ¼ 3, decay-corrected and referred to labelling
agent), respectively. Thus, 18F-fluoroethylation favourably at the para position of the
phenylpiperazine moiety of the 5-cyano-indole framework proved to be tolerated by
D4 receptors and could also be applied to alternative scaffolds in order to develop
D4 radioligand candidates for PET with improved D4 receptor affinity and
selectivity. Copyright # 2005 John Wiley & Sons, Ltd.
*Correspondence to: O. Prante, Laboratory of Molecular Imaging, Clinic of Nuclear Medicine, Friedrich-
Alexander University Erlangen-Nurnberg, Schwabachanlage 6, D-91054 Erlangen, Germany.
¨
E-mail: olaf.prante@nuklear.imed.uni-erlangen.de
Contract/grant sponsor: Deutsche Forschungsgemeinschaft; contract/grant number: PR 677/2
Copyright # 2005 John Wiley & Sons, Ltd.
Received 26 August 2005
Revised 24 October 2005
Accepted 25 October 2005

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R. TIETZE ET AL.
Key Words: 18F-fluoroethylation; indole; F-18; D4 receptor; positron-emission-
tomography; PET
Introduction
The dopamine D4 receptor subtype belongs to the subfamily of D2-like distinct
dopamine receptors (D2R, D3R and D4R) mediating the action of dopamine
in the brain, but differing in their brain distribution and pharmacological
profiles. Among them, the D4 receptor has been cloned1 and intensively
studied in vitro using knock-out mice or immunohistochemistry with receptor-
specific antibodies.2–5 The precise function and exact distribution of the
dopamine D4 receptor in the CNS are of great interest, as associations are
emerging between D4 receptors and neuropsychiatric disorders, including
schizophrenia, attention-deficit hyperactivity disorder as well as specific
personality traits such as novelty seeking.6–8 Whether the dopamine D4
receptor fulfills distinct functional roles has not yet been satisfactorily
addressed.
Up to now, dopamine D4 receptor concentrations in vitro are usually
determined by indirect binding studies, in which [3H]raclopride binding (D2/
D3 antagonist) is subtracted from total binding measured with [3H]nemona-
pride.9,10 However, this method has yielded conflicting results: whereas
Seeman et al.9 reported an increased D4 receptor density in postmortem brain
tissue of schizophrenic patients, these results could not be confirmed by
others.11,12 Data on the apparently low D4 receptor densities in the brain are
scarce and were reported in analyses using in situ hybridization establishing
receptor expression in the prefrontal cortex and hippocampus.13 Furthermore,
in vitro autoradiography with [3H]NGD 94-1 reflected a distribution unique
among dopamine receptor subtypes and revealed Bmax-values in the low range
from 9 to 30 fmol/mg in distinct human brain regions.14,15 Thus, the selection
criteria for a suitable D4 receptor radioligand for positron emission
tomography (PET) has to consider a high affinity for its target in order to
observe a receptor-specific signal in vivo. Assuming the maximal concentration
of D4 receptors to be equivalent to approximately 3 nM, the in vitro affinity of
a radioligand candidate should be significantly less than Bmax to achieve a high
in vitro binding potential (Bmax/Kd) that could correlate to a suitable target-to-
nontarget ratio in vivo with good contrast for imaging.16 However, the in vivo
distribution of a radioligand at a single time point is likely to be influenced by
various factors besides receptor density and affinity, such as blood flow,
clearance of the radioligand, metabolism and binding to nonspecific sites.
Up to now, the lack of selective D4 receptor radioligands suitable for in vivo
imaging techniques hampers the noninvasive investigation of neurotransmis-
sion by single photon emission tomography (SPET) and positron emission
tomography (PET) as high-performance tools for understanding the
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SYNTHESIS OF 18F-LABELLED D4 RADIOLIGANDS
57
Figure 1. Chemical structure of FAUC 316 (Ki(D4R)=1.0 nM, Ki(D2R)/
Ki(D4R)=19 000)32
neurochemical basis and pathology of neuropsychiatric diseases.17 Several
efforts were reported on the radiosyntheses of 123I-, 18F- and 11C-labelled
ligands for the exploration of D4 receptor density in vivo by SPET and PET,
including [11C]SDZ GLC 756,18 methoxybenzamide derivatives,19,20 SB-
235753,21,22
a
123I-labelled chromeno[3,4-c]pyridinone23 as well as 18F- and
l23I-labelled pyrrolo[2,3-b]pyridines.24–26 However, none of these radioligands
has been proven suitable, due to lack of specificity in vivo or undesirable
pharmacological properties.
As a part of our drug discovery and SAR investigations on selective
dopamine D4 receptor ligands and radiolabelled analogs,27–31 we identified the
indole derivative 2-[4-(4-fluorophenyl)piperazin-1-ylmethyl]indole-5-carboni-
trile(FAUC 316; Figure 1) as a dopamine D4 receptor ligand with high affinity
(KiðD4RÞ ¼ 1:0 nM) and excellent subtype selectivity (KiðD2RÞ=KiðD4RÞ ¼
19 000).32 The aims of the present study were the syntheses of fluoroethoxy
substituted derivatives of the lead compound FAUC 316, the assessment of
their in vitro properties with regard to dopamine receptor affinity and subtype
selectivity and the radiosyntheses of 18F-labelled analogs. We herein report
the effect of fluoroethylation on receptor binding affinities in vitro and the
18F-radiosyntheses of the D4 receptor radioligand candidates 2-[4-(2-(2-[18F]
fluoroethoxy)phenyl)piperazin-1-ylmethyl]indole-5-carbonitrile ([18F]5a) and
2-[4-(4-(2-[18F]fluoroethoxy)phenyl)piperazin-1-ylmethyl]indole-5-carbonitrile
([18F]5b).
Results and discussion
Our previous biological investigation of a representative library of 13 indole
derivatives showed highly potent and selective dopamine D4 receptor binding
profiles, when positions 2 and 5 proved highly suitable attachment positions
for the aminomethyl and cyano groups, respectively.32 Consequently, this
substitution pattern at the indole core unit was maintained for the syntheses of
fluoro substituted analogs presented herein. Starting from the commercially
available 2- and 4-hydroxyphenylpiperazines, the corresponding fluoroethoxy
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R. TIETZE ET AL.
substituted phenylpiperazines 4a and 4b were obtained by a three step
procedure including classical N-protection of the piperazine nitrogen as t-
butyl carbamate (Scheme 1). First, introduction of a t-butyloxycarbonyl
(BOC) protecting group was accomplished using BOC anhydride in N,N-
dimethyl formamide (DMF) in the presence of triethylamine to obtain
compounds 2a,b. Subsequently, fluoroethylation of the aromatic hydroxyl
group was realized by reaction of 2a,b with l-fluoro-2-tosyloxyethane
(fluoroethyl tosylate) in the presence of tetrabutylammonium hydroxide
(N(Bu)4OH) to afford 3a and 3b, respectively, in good yields (72 and 86%)
employing the protocol of Wilson et al.33 Deprotection of the piperazine
nitrogen under acidic conditions provided the desired fluoroethoxy substituted
phenylpiperazines 4a and 4b in yields of 60 and 50%, respectively (Scheme 1).
With the 2- and 4-(2-fluoroethoxy)phenyl substituted piperazines (4a,b) in
hand, we needed access to the appropriate 5-cyano-indole-2-aldehyde (1) in
order to allow reductive amination leading to target compounds 5a-6b
(Scheme 2). Starting from 2-(hydroxymethyl)indole-5-carbonitrile,34 we easily
gained the corresponding aldehyde 1 by oxidation using manganese(IV) oxide
at ambient temperature. Thus, the fluoroethoxy substituted reference
compounds 5a and 5b as well as the labelling precursor compounds 6a and
6b were obtained by the same experimental procedure using Na(OAc)3BH as
reducing agent in the coupling of 1 with 4a, 4b, or 2-/4-hydroxyphenylpiper-
azine, respectively (Scheme 2).
After purification by flash chromatography on silica gel and confirmation of
chemical purity by LC/MS, the novel fluoroethoxyphenyl substituted 5-cyano-
Scheme 1. (i) (BOC)2O, DMF; (ii) 1-fluoro-2-tosyloxyethane, N(Bu)4OH,
DMF; (iii) conc. HCl
Scheme 2. Syntheses of the target compounds 5a,b as well as the corresponding
labelling precursors 6a,b: (i) MnO2, CH2Cl2; (ii) Na(OAc)3BH, CH2Cl2
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SYNTHESIS OF 18F-LABELLED D4 RADIOLIGANDS
59
indoles (5a, 5b) and the corresponding hydroxyphenyl substituted derivatives
(6a, 6b) were characterized in vitro for their ability to displace [3H]spiperone
D336 and D347
35
from the cloned human dopamine receptors D2long, D2short
,
being stably expressed in chinese hamster ovary (CHO) cells.38 D1 affinity was
determined by employing porcine striatal membrane preparations and the D1
selective antagonist [3H]SCH 23390. In addition, receptor affinities to the
related biogenic amine receptors 5-HT1A, 5-HT2 and a1 were evaluated
utilizing porcine cortical membranes and the selective radioligands [3H]8-OH-
DPAT, [3H]ketanserin and [3H]prazosin, respectively. For comparison of the
binding data, the reference compound FAUC 316 was investigated under the
same conditions (Table 1).
The dopamine receptor binding profiles of the test compounds clearly
indicate poor affinities for the D1, D2 and D3 subtypes, with the exception of
compound 5a indicating a distinct influence when introducing a fluoroethoxy
substituent at the ortho position of the phenylpiperazin moiety. All test
compounds revealed a Ki value in the low nanomolar range for the D4 receptor
(Table 1). Comparison of the D4 binding data among each other showed
significantly higher D4 receptor affinities for the derivatives with substituents
in ortho position of the phenylpiperazinyl group (5a: 2.1 nM, 6a: 1.6 nM). The
corresponding derivatives substituted in para position (5b: 9.9 nM and 6b:
3.1 nM) showed decreased D4 binding affinity, whereas dopamine receptor
subtype selectivities were superior in comparison with 5a and 6a, respectively.
Interestingly, a significant effect on a1 binding was observed when comparing
derivatives substituted at 2-position to those substituted at 4-position. This
effect was especially illustrated by a 70-fold loss of a1 binding of 5b when
comparing to a1 affinity of 5a. In general, 5b was characterized as a high
affinity D4 receptor ligand with good dopamine subtype receptor selectivity,
weak receptor affinity for a1, 5-HT1A and 5-HT2, and thus illustrating superior
in vitro properties in comparison with 5a. When comparing 5b to the reference
lead compound FAUC 316, displacement of the para-fluorine substituent by a
para-fluoroethoxy group led to reduced binding properties in vitro (Table 1).
It is tempting to speculate whether 18F-labelled indoles [18F]5a and [18F]5b
could be suitable radioligands for the observation of a receptor-specific signal
with PET, due to the low D4 receptor concentration in the brain. However, the
in vitro binding profile of 5a demonstrated a Ki value for the D4 receptor in the
low nanomolar range (2.1 nM), which could be acceptable for an adequate
binding potential (Bmax/Kd) when assuming an existing D4 receptor
concentration of roughly 3 nM in the brain. Moreover, [18F]5b is characterized
by an improved D4 receptor selectivity including less binding affinity to
adrenergic and serotoninergic sites, which could be beneficial when performing
in vitro autoradiography studies on native tissue. Due to the high D4 receptor
affinity of 5a and distinct D4 receptor selectivity of 5b, we aimed at optimizing
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R. TIETZE ET AL.
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS OF 18F-LABELLED D4 RADIOLIGANDS
61
the radiosyntheses of the corresponding 18F-labelled analogs, in order to
provide putative D4 receptor radioligands that could be further investigated by
in vitro assays or used to assess the pharmacokinetics of these 5-cyano-indole
derivatives in vivo.
6a and 6b were used for the following 18F-radiolabelling studies with
[18F]fluoroethyl tosylate under varied reaction parameters. Compounds 5a and
5b served as authentic reference compounds in analytical radio-HPLC to
confirm chemical identity of [18F]5a and [18F]5b. The radiosyntheses of [18F]5a
and [18F]5b are depicted in Scheme 3. Based on the nucleophilic 18F-for-OTos
substitution on bistosyloxyethane as described by Block and coworkers,39 we
isolated [18F]fluoroethyl tosylate by semipreparative reversed-phase HPLC
followed by solid phase extraction. Slight modifications of the procedure
described by Block et al.39 were introduced concerning reaction time and
reaction temperature. In order to improve the radiochemical yield (RCY) of
the prosthetic group [18F]fluoroethyl tosylate, the reaction temperature was
increased to 908C, while the reaction time was shortened to 3 min. These
reaction conditions provided limited amounts of an 18F-labelled hydrophilic
by-product (55%; probably 2-[18F]fluoroethanol occurring under basic
reaction conditions) and an improved radiochemical yield of 84% as
determined by radio-HPLC (Scheme 3, step 1). The reaction parameters for
the 18F-fluoroethylation key step were investigated with respect to the choice
of reaction solvent, base and reaction time as summarized in Table 2.
Using DMF and sodium methoxide (NaOMe) as a base to generate the
phenoxide from precursor 6a did not lead to a rapid progression of the 18F-
fluoroethylation in ortho position. By increasing the reaction temperature
from 100 to 1408C, the RCY of [18F]5a decreased to 13%. At 1008C the
remaining radioactivity at the end of reaction was present as [18F]fluoroethyl
tosylate (50 ꢀ 3%, t ¼ 5 min), while at 1408C an accelerated degradation of
[18F]fluoroethyl tosylate was observed probably due to the formation of
Scheme 3. Two-step procedure for the radiosynthesis of [18F]5a and [18F]5b: (i)
[Kꢁ222]+[18F]Fꢂ, bistosyloxyethane, CH3CN, T=908C, 3 min; (ii) for [18F]5a:
6a, NaOMe, DMSO, T=1208C, 5 min; for [18F]5b: 6b, N(Bu)4OH, DMF,
T=1408C, 5 min
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R. TIETZE ET AL.
Table 2. Radiochemical yields (RCY) for the 18F-fluoroethylation of 6a and 6b using
1-[18F]fluoro-2-tosyloxyethane
Product
Solvent/base
T (8C)
t (min)
RCY (%)a
[18F]5a
DMF/NaOMe
100
1
5
15
1
5
10
1
3
10
1
2
12; 13 (n ¼ 2)
37; 39 (n ¼ 2)
67 ꢀ 5
14 ꢀ 5
34 ꢀ 3
47 ꢀ 1
7; 11 (n ¼ 2)
8; 16 (n ¼ 2)
7; 20 (n ¼ 2)
31; 45 (n ¼ 2)
57; 67 (n ¼ 2)
70 ꢀ 5
80 ꢀ 2
81 ꢀ 5
70 ꢀ 5
0b
120
140
120
DMSO/NaOMe
3
5
10
15
1–15
1–15
1–15
1
[18F]5b
DMF/NaOMe
DMSO/NaOMe
DMF/NaH
120
140
120
100
0b
0b
DMF/N(Bu)4OH
9c
3
5
10c
7c
120
1
5
10
15
1
24 ꢀ 5
37 ꢀ 8
31 ꢀ 4
34 ꢀ 5
41 ꢀ 10
47 ꢀ 4
36 ꢀ 5
0b
140
140
5
15
1–15
DMSO/N(Bu)4OH
aRCY were determined by radio-HPLC and related to 1-[18F]fluoro-2-tosyloxyethane. Values are expressed
as mean ꢀ standard deviation (SD) of three independent experiments (n ¼ 3). Independent experimental
values are expressed for n ¼ 2 (10.8 mmol precursor, V ¼ 350 ml).
bDetermined by radio-TLC.
cData of a single experiment.
2-[18F]fluoroethanol (ꢃ70%, t ¼ 10 min). The low RCY of [18F]5a using the
solvent system DMF/NaOMe could be due to limited solubility of the sodium
phenoxide in DMF and thus decreased nucleophilicity. Thus, we examined the
influence of the solvent on the RCY under identical experimental conditions.
We used dimethyl sulfoxide (DMSO) instead of DMF, since this solvent
provides excellent properties for dissolving sodium salts as already has been
exploited in numerous 18F-fluoroethylation procedures, such as the radio-
synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine and others.40–42 As shown in
Table 1, the use of DMSO revealed an accelerated formation of the desired
product [18F]5a, obtaining a RCY of about 80% (decay-corrected and related
to [18F]fluoroethyl tosylate) within 5–10 min at 1208C.
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SYNTHESIS OF 18F-LABELLED D4 RADIOLIGANDS
63
Surprisingly, these reaction conditions were not transferable to the
18F-fluoroethylation of the para substituted precursor 6b to give [18F]5b
(Table 2). More than 90% of the remaining radioactivity at end of reaction
was detected as unreacted [18F]fluoroethyl tosylate when using the solvent/
base systems DMF/NaOMe or DMSO/NaOMe. Changing the base from
NaOMe to equimolar amounts of sodium hydride also did not reveal any
reaction or degradation of [18F]fluoroethyl tosylate. However, following the
protocol of Wilson et al.33 we used tetrabutylammonium hydroxide as base
promotor to generate the para phenoxide of 6b in DMF. Under these
conditions, addition of [18F]fluoroethyl tosylate lead to satisfactory radio-
chemical yields of [18F]5b at 1408C (40–50%, decay-corrected and related to
[18F]fluoroethyl tosylate; Table 2). In comparison with the radiosynthesis of
the [18F]fluoroethoxy substituted derivative [18F]5a, 18F-fluoroethylation in the
para position of the phenylpiperazine moiety using the solvent system DMF/
N(Bu)4OH allowed the use of a higher reaction temperature (1408C) and
proceeded more rapidly reaching a maximum RCY of [18F]5b within a shorter
reaction time of 1–5 min. At later time points, the RCY of [18F]5b slightly
decreased, suggesting degradation of the final product under basic reaction
conditions. As the major by-product we detected an unknown hydrophilic 18F-
labelled compound (Rt ¼ 2:9 min, radio-HPLC), probably due to the
formation of 2-[18F]fluoroethanol as also observed for the reaction of 6a with
[18F]fluoroethyl tosylate at 1408C (see above). Efforts to improve the RCY of
the para substituted product [18F]5b by using DMSO/N(Bu)4OH failed (Table
2), so that the solvent system DMF/N(Bu)4OH at 1408C turned out to be the
optimal reaction medium for the radiosynthesis of [18F]5b.
This optimization study for the syntheses of the 18F-labelled radioligands
[18F]5a (DMSO/NaOMe, 1208C, 5 min) and [18F]5b (DMF/N(Bu)4OH, 1408C,
5 min) led to adequate radiochemical yields of 80 and 47%, respectively,
permitting further in vitro and in vivo studies.
Experimental
General
All chemicals and reagents were of analytical grade and obtained from
commercial sources. [18F]Fluoride was produced by the 18O(p, n)18F reaction
on 18O-enriched (95%) water using a 11 MeV proton beam generated by a
RDS 111 cyclotron (PET Net GmbH, Erlangen, Germany). Solid phase
cartridges (Sep-Pak1Plus C18 cartridges) were purchased from Waters
(Eschborn, Germany). Thin layer chromatography (TLC) was carried out
on silica gel-coated aluminium plates (silica gel/TLC-cards, with fluorescent
indicator 254 nm, layer thickness 0.2 mm, Fluka); for radio-TLC plastic sheets
(Polygram1, Sil G/UV254, Macherey-Nagel) were used. Compounds were
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R. TIETZE ET AL.
visualized by UV light (254 nm). Analytical radio-HPLC was performed on the
following system: HPLC Hewlett Packard (HP 1100) with a quarternary pump
and variable wavelength detector (HP 1100) and radio-HPLC-detector
D505TR (Canberra Packard). Computer analysis of the HPLC data was
performed using FLO-One software (Canberra Packard). NMR spectra were
recorded on a Bruker Avance 360 or Bruker Avance 600 using TMS as
internal standard (all data were expressed in parts per million (ppm)). LC-MS
analyses were performed on an Agilent 1100 Series analytic HPLC system with
a VWL detector, coupled to a Bruker esquire 2000 mass spectrometer with
atmospheric pressure chemical ionization (APCI). Melting points (m.p.) were
uncorrected and obtained with a Buchi apparatus. 1-fluoro-2-tosyloxyethane
¨
was synthesized as previously described by Block et al.39 and identity was
1
confirmed by TLC (ethyl acetate/hexane 7:3, Rf ¼ 0:89), H-NMR (CDCl3: d
2.46 (s, 3H), 4.24–4.32 (dt, 2H, OCH2), 4.45–4.63 (dt, 2H, FCH2), 7.34–7.38
(m, 2H) 7.79–7.83 (m, 2H)) and HPLC (Lichrosorb RP18, 250 ꢄ 4:6 mm
1 ml/min, 30–80% CH3CN in water (0.1% TFA) in 25 min: Rt ¼ 13:40 min).
2-(Hydroxymethyl)indole-5-carbonitrile (LC-MS (APCI): m/z 173.0
½M þ Hꢅþ) was available as a product of a former research study (Department
of Medicinal Chemistry).34 Each 18F-labelled compound was identified by
retention time (Rt) on the radio-HPLC system and co-injection of the
corresponding reference compound.
Chemistry
5-Cyano-indole-2-aldehyde (1). 1 was synthesized by oxidation of 2-(hydro-
xymethyl)indole-5-carbonitrile (1.4 g, 8.14 mmol) using manganese(IV) oxide
(7.08 g, 80.28 mmol) in 30 ml CH2Cl2. The suspension was stirred at room
temperature for 24 h. The reaction mixture was filtered through cellulose and
the precipitate was washed with ethyl acetate. The organic layer was removed
under reduced pressure to give the product as a pale yellow solid (1.09 g,
6.41 mmol, 79%), which was used for subsequent reactions without further
1
purification. TLC (CH2Cl2/MeOH 95:5): Rf ¼ 0:43. H-NMR (DMSO): d
7.53 (s, 1H), 7.64 (m, 2H), 8.36 (s, 1H), 9.96 (s, 1H, CHO), 12.48 (s, 1H, NH).
4-(2-Hydroxyphenyl)piperazine-1-carboxylic acid t-butyl ester (2a). Anhy-
drous di-t-butyldicarbonate (720 mg, 3.3 mmol) was dissolved in 10 ml DMF
and slowly added to a solution of 1-(2-hydroxyphenyl)piperazine (534 mg,
3.0 mmol) and triethylamine (334 mg, 3.3 mmol) in 20 ml DMF. After stirring
at room temperature for 1 h, the reaction was stopped by adding 30 ml of a
saturated NaHCO3 solution. The solution was extracted with ethyl acetate
(3 ꢄ 20 ml) and the organic solvent was removed under reduced pressure. The
crude product was purified by silica gel chromatography using ethyl acetate/
hexane (3:7) as eluant. 2a was isolated in 58% yield (511 mg, 1.74 mmol) as a
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SYNTHESIS OF 18F-LABELLED D4 RADIOLIGANDS
1
65
yellow oil. TLC (ethyl acetate/hexane 3:7): Rf ¼ 0:60. H-NMR (CDCl3):
d 1.48 (s, 9H, t-butyl), 2.85 (t, 4H), 3.59 (t, 4H), 6.87–7.15 (m, 4H).
4-(4-Hydroxyphenyl)piperazine-1-carboxylic acid t-butyl ester (2b). 2b was
synthesized as described for 2a. Starting from 178 mg (1.0 mmol) 1-(4-
hydroxyphenyl)piperazine, 2b was obtained as a yellow oil in a yield of 64%
1
(188 mg, 0.64 mmol). TLC (ethyl acetate/hexane 3:7): Rf ¼ 0:30. H-NMR
(CDCl3): d 1.50 (s, 9H, t-butyl), 2.91 (t, 4H), 3.56 (t, 4H), 6.76 (m, 2H), 6.90
(m, 2H).
4-(2-(2-Fluoroethoxy)phenyl)piperazine-1-carboxylic acid t-butyl ester
(3a). Following a protocol reported by Wilson et al.,33 a mixture of 2a
(149 mg, 0.51 mmol), 1-fluoro-2-tosyloxyethane (171 mg, 0.79 mmol) and a
solution of tetrabutylammonium hydroxide (0.51 ml, 1.4 N in dry MeOH)
were dissolved in 8 ml dry DMF and stirred at room temperature for 24 h. The
reaction was quenched by adding 50 ml 0.05 N NaOH. After extraction with
ethyl acetate (3 ꢄ 25 ml) and drying over Na2SO4 the organic layer was
evaporated and the residue was purified by silica gel chromatography using
CH2Cl2/ethyl acetate (90:10) to give 3a (126 mg, 0.37 mmol, 72%) as a yellow
1
oil. TLC (ethyl acetate/hexane 3:7): Rf ¼ 0:60. H-NMR (CDCl3): d 1.49
(m, 9H, t-butyl), 3.02 (t, 4H), 3.58 (t, 4H), 4.21–4.30 (dt, 2H, FCH2), 4.71–4.83
(dt, 2H, FCH2), 6.82–7.02 (m, 4H).
4-(4-(2-Fluoroethoxy)phenyl)piperazine-1-carboxylic acid t-butyl ester
(3b). 3b was synthesized as described for 3a. Starting from 225 mg
(0.77 mmol) 2b, chromatographic isolation on silica gel yielded 86% of 3b as
a dark yellow oil after evaporation of the organic solvent (225 mg, 0.66 mmol).
TLC (ethyl acetate/hexane 3:7): Rf ¼ 0:75. 1H-NMR (CDCl3): d 1.49 (m, 9H,
t-butyl) 3.02 (t, 4H), 3.61 (t, 4H), 4.21–4.31 (dt, 2H, FCH2), 4.69–4.85 (dt, 2H,
FCH2), 6.81–7.01 (m, 4H).
1-(2-(2-Fluoroethoxy)phenyl)piperazine (4a). A solution of 3a (595 mg,
1.75 mmol) in 10 ml of concentrated hydrochloric acid was stirred at room
temperature for 15 min. Cleavage of the t-butyl ester was analysed by TLC.
The solution was carefully diluted with water and slowly adjusted to pH 8–10
with sodium hydroxide pellets. Extraction with ethyl acetate, drying over
Na2SO4 and removal of the solvent under reduced pressure yielded 60% of
4a (237 mg, 1.06 mmol) as a yellow oil. TLC (MeOH/CH2Cl2 3:1, 0.5%
triethylamine): Rf ¼ 0:20. 1H-NMR (CDCl3): d 3.09 (m, 8H), 4.18–4.31
(dt, 2H, FCH2), 4.68–4.86 (dt, 2H, FCH2), 6.82–7.02 (m, 4H).
1-(4-(2-Fluoroethoxy)phenyl)piperazine (4b). Ester cleavage of 3b to obtain
4b was performed as described above for the synthesis of 4a. Chromatography
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R. TIETZE ET AL.
on silica gel yielded 4b as a yellow oil (121 mg, 0.53 mmol, 50%). TLC
(MeOH/CH2Cl2 3:1, 0.5% triethylamine): Rf ¼ 0:20. 1H-NMR (CDCl3):
d 3.08 (m, 8H), 4.19–4.32 (dt, 2H, FCH2), 4.68–4.88 (dt, 2H, FCH2), 6.80–6.89
(m, 2H) 6.90–7.02 (m, 2H).
2-[4-(2-(2-Fluoroethoxy)phenyl)piperazin-1-ylmethyl]indole-5-carbonitrile (5a).
A mixture of 4a (237 mg, 1.05 mmol) and 1 (270 mg, 1.59 mmol) was dissolved
in 20 ml dry CH2Cl2, Na(OAc)3BH (335 mg, 1.59 mmol) was added to the
suspension in one portion. After stirring at room temperature for 24 h the
reaction was stopped by adding 30 ml saturated NaHCO3. The product was
extracted with ethyl acetate and the organic layer was dried over Na2SO4. The
solvent was evaporated and the residue was purified by silica gel chromato-
graphy in a two step procedure (a: CH2Cl2/ethyl acetate 90:10; b: CH2Cl2/
MeOH 95:5) yielding 5 as a pale yellow solid (98 mg, 0.26 mmol, 24%). M.p.:
1
1868C. TLC (CH2Cl2/MeOH 95:5): Rf ¼ 0:50. H-NMR (CDCl3): d 2.71
(t, 4H), 3.17 (t, 4H), 3.83 (s, 2H, CH2), 4.21–4.28 (dt, 2H, OCH2), 4.71–4.82
(dt, 2H, FCH2), 6.47–7.90 (m, 8H), 8.91 (s, 1H, NH). HPLC (Lichrosorb
RP18, 250 ꢄ 4:6, 1 ml/min, 30–80% CH3CN in water (0.1% TFA) in 25 min):
Rt ¼ 10:60 min. LC-MS (APCI): m/z 379.1 ½M þ Hꢅþ, 225.1 (fluoroethox-
yphenyl piperazinyl fragment [C12H17FN2O+H]+).
2-[4-(4-(2-Fluoroethoxy)phenyl)piperazin-1-ylmethyl]indole-5-carbonitrile (5b).
Compound 5b was synthesized as described for 5a starting from 4b (125 mg,
0.56 mmol) and 1 (140 mg, 0.83 mmol). The pale yellow solid 5b was obtained in
a yield of 27% (56 mg, 0.15 mmol). M.p.: 2058C. TLC (CH2Cl2/MeOH 95:5):
1
Rf ¼ 0:55. H-NMR (CDCl3): d 2.68 (t, 4H), 3.22 (t, 4H), 3.74 (s, 2H, CH2)
4.09–4.20 (dt, 2H, OCH2), 4.34–4.77 (dt, 2H, FCH2), 6.46 (s, 1H), 6.88 (m, 4H),
7.42 (s, 2H), 7.91 (s, 1H), 8.90 (s, 1H, NH). HPLC (Lichrosorb RP18, 250 ꢄ 4:6,
1 ml/min, 30–80% CH3CN in water (0.1% TFA) in 25 min): Rt ¼ 10:13 min.
LC-MS (APCI): m/z 379.1 ½M þ Hꢅþ, 225.0 (fluoroethoxyphenyl piperazinyl
fragment [C12H17FN2O+H]+).
2-[4-(2-Hydroxyphenyl)piperazin-1-ylmethyl]indole-5-carbonitrile (6a). 1-(2-
Hydroxyphenyl)piperazine (397 mg, 2.23 mmol) and 1 (253 mg, 1.49 mmol)
were dissolved in 10 ml dry CH2Cl2 and Na(OAc)3BH (1263 mg, 5.96 mmol)
was added to the suspension. After stirring at room temperature for 1 h, the
reaction was terminated by adding 30 ml saturated NaHCO3. The crude
product was purified by silica gel chromatography using CH2Cl2/MeO H
(95:5) as eluant yielding 32% (159 mg, 0.48 mmol) of 6a as a pale yellow solid.
M.p.: 1488C. TLC (CH2Cl2/MeOH 95:5): Rf ¼ 0:30. 1H-NMR (CDCl3):
d 2.44 (t, 4H), 3.33 (t, 4H), 3.74 (s, 2H, CH2), 6.43–8.05 (m, 8H), 8.87 (s, 1H,
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SYNTHESIS OF 18F-LABELLED D4 RADIOLIGANDS
67
NH), 11.69 (s, 1H, OH). LC-MS (APCI): m/z 333.1 ½M þ Hꢅþ, 179.0
(hydroxyphenyl piperazinyl fragment [C10H14N2O+H]+).
2-[4-(4-Hydroxyphenyl)piperazin-1-ylmethyl]indole-5-carbonitrile (6b). 6b was
synthesized as described above for compound 6a. The reaction yielded 6b
as a pale yellow solid (183 mg, 0.55 mmol, 36%). M.p.: 1498C. TLC (CH2Cl2/
1
MeOH 95:5): Rf ¼ 0:50. H-NMR (CDCl3): d 2.53 (t, 4H), 3.31 (t, 4H), 3.73
(s, 2H, CH2), 6.51 (s, 1H), 6.59 (d, 2H), 6.82 (d, 2H), 7.39–7.98 (m, 3H), 8.79
(s, 1H, NH), 11.70 (s, 1H, OH). LC-MS (APCI): m/z 333.1 ½M þ Hꢅþ, 179.0
(hydroxyphenyl piperazinyl fragment [C10H14N2O+H]+).
Radiochemistry
1-[18F]Fluoro-2-tosyloxyethane39. No-carrier-added
[18F]fluoride
(200–
650 MBq) was delivered on a QMA-cartridge and eluted with a solution of
15 mg Kryptofix1 2.2.2/15 ml 1 M K2CO3 in 1 ml acetonitrile/water (8:2). The
solution was evaporated using a stream of nitrogen at 858C and co-evaporated
to dryness with CH3CN (2 ꢄ 200 ml). Following the procedure described by
Block et al.,39 4.5 mg (12 mmol) bistosyloxyethane in 500 ml anhydrous
acetonitrile were added to the reaction vessel and the mixture was stirred for
3 min at 908C. The reaction was quenched by dilution with 500 ml water. The
prosthetic group 1-[18F]fluoro-2-tosyloxyethane was isolated by gradient
reversed-phase HPLC (Lichrosorb RP18, 125 ꢄ 8 mm, 4 ml/min, CH3CN/
H2O (40/60) (0.1% TFA)). The product fraction was diluted 1:10 with water
and fixed on a C18-cartridge (Waters Sep-Pak1Plus), dried in a nitrogen
stream and eluted with 1 ml DMF in a reaction vessel. 1-[18F]Fluoro-2-
tosyloxyethane was obtained in a radiochemical yield (RCY) of 84% as
determined by radio-HPLC from a sample withdrawn from the reaction
mixture. Radio-TLC (ethyl acetate/hexane 7:3): Rf ¼ 0:87. Radio-HPLC
(Lichrosorb RP18, 250 ꢄ 4:6 mm, 1 ml/min, 30–80% CH3CN in water (0.1%
TFA) in 25 min): Rt ¼ 14:34 min.
2-[4-(2-(2-[18F]Fluoroethoxy)phenyl)piperazin-1-ylmethyl]indole-5-carbonitrile
(½18Fꢅ5a). 3.6mg (10.8mmol) 2-[4-(2-hydroxyphenyl)piperazin-1-ylmethyl]indole-
5-carbonitrile (6a) were dissolved in 250 ml dry DMSO and 50ml sodium
methoxide in dry methanol (8 mg/ml) were added to the reaction vial. The
reaction mixture was stirred for 3 min at 1208C under a nitrogen atmosphere.
Subsequently, 1-[18F]fluoro-2-tosyloxyethane in 50 ml dry DMF (5–20 MBq) were
added to the reaction mixture. The progression of the reaction was analysed by
radio-HPLC. The radiochemical yield of [18F]5a was 81 ꢀ 5% after 5–10 min at
1208C. Radio-HPLC (Lichrosorb RP18, 250 ꢄ 4:6 mm, 1 ml/min, 30–80%
CH3CN in water (0.1% TFA) in 25 min): Rt ¼ 10:66 min.
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R. TIETZE ET AL.
2-[4-(4-(2-[18F]Fluoroethoxy)phenyl)piperazin-1-ylmethyl]indole-5-carbonitrile
(½18Fꢅ5b). 3.6mg (10.8mmol) 2-[4-(4-hydroxyphenyl)piperazin-1-ylmethyl]indole-
5-carbonitrile (6b) were dissolved in 250 ml dry DMF. 113ml 1.4N tetrabuty-
lammonium hydroxide in dry MeOH were added to the reaction vial. The
reaction mixture was stirred for 3 min at 1208C under a nitrogen atmosphere.
Subsequently, 50 ml of the 1-[18F]fluoro-2-tosyloxyethane solution in dry DMF
(5–20 MBq) were added to the reaction mixture at 1408C. The progression of the
reaction was analysed by radio-HPLC. The radiochemical yield of [18F]5b was
47 ꢀ 4% after 5 min at a reaction temperature of 1408C. Radio-HPLC
(Lichrosorb RP18, 250 ꢄ 4:6, 1 ml/min, 30–80% CH3CN in water (0.1% TFA)
in 25 min): Rt ¼ 10:24 min.
Optimization of the 18F-fluoroethylation procedure. The 18F-fluoroethylation of
6a and 6b was optimized by repeating the reaction with varying parameters as
indicated in Table 1.
Receptor binding experiments and data analysis
Receptor binding studies were performed as described previously.38 In brief,
the dopamine D1 receptor assay was done with porcine striatal membranes at
a final protein concentration of 40 mg/assay tube and the radioligand
[3H]SCH23390 at 0.3 nM (Kd ¼ 0:5 nM). Competition experiments with the
human D2long, D2short, D3 and D4,4 receptors were run with preparations of
membranes from CHO cells expressing the corresponding receptor and
[3H]spiperone at a final concentration of 0.5 nM. The assays were carried out
at a protein concentration of 6–30 mg/assay tube and Kd values of 0.10 nM for
D
2long, D2short and D3 and 0.10–0.13 nM for D4,4. Serotonin 5-HT1A, 5-HT2
and adrenergic a1 binding were measured utilizing porcine cortical membranes
and the selective radioligands [3H]8-OH-DPAT, [3H]ketanserin and [3H]pra-
zosin, respectively, each at a final concentration of 0.5 nM. The resulting
competition curves were analysed by nonlinear regression using the algorithms
in PRISM (GraphPad Software, San Diego, USA). 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.43
Conclusion
In conclusion, two isomeric fluoroethoxy substituted derivatives of 5-cyano-
indole were synthesized and characterized as high-affinity dopamine D4
receptor ligands in vitro. A para-fluoroethoxy substituent at the phenylpiper-
azine moiety was tolerated by D4 receptors and more advantageous than
an ortho substituent with respect to dopamine D4 receptor selectivity. For
both 18F-labelled derivatives of 5-cyano-indole the reaction conditions for
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SYNTHESIS OF 18F-LABELLED D4 RADIOLIGANDS
69
18F-fluoroethylation using [18F]fluoroethyl tosylate were successfully opti-
mized providing potential PET imaging probes for the dopamine D4 receptor.
The methodology of 18F-fluoroethylation could also be applied to alternative
lead compounds of the FAUC series, such as derivatives of pyrazolo[1,5-
a]pyridine, in order to provide D4 radioligand candidates for PET with
improved D4 receptor affinity and selectivity.
Acknowledgements
We gratefully acknowledge Wilhelm Hamkens and the cyclotron staff for
producing and delivering [18F]fluoride (PET Net GmbH, Erlangen). This work
was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG, PR
677/2).
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