Synthesis of novel WAY 100635 derivatives containing a norbornene group and radiofluorination of [18F]AH1.MZ as a serotonin 5-HT1A receptor antagonist for molecular imaging

Article abstract of DOI:10.1002/jlcr.1589

5-HT_1A receptors are involved in a variety of psychiatric disorders and in vivo molecular imaging of the 5-HT_1A status represents an important approach to analyze and treat these disorders. We report herein the synthesis of three new

Full text of DOI:10.1002/jlcr.1589

Research Article
Received 10 December 2008,
Accepted 20 January 2009
Published online 3 March 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1589
Synthesis of novel WAY 100635 derivatives
containing a norbornene group and
radiofluorination of [18F]AH1.MZ as a
serotonin 5-HT1A receptor antagonist for
molecular imaging
Ã
¨
Matthias M. Herth, Vasko Kramer, and Frank Rosch
5-HT1A receptors are involved in a variety of psychiatric disorders and in vivo molecular imaging of the 5-HT1A status
represents an important approach to analyze and treat these disorders. We report herein the synthesis of three new
fluoroethylated 5-HT1A ligands (AH1.MZ, AH2.MZ and AH3.MZ) as arylpiperazine derivatives containing a norbornene
group. AH1.MZ (Ki = 4.2 nM) and AH2.MZ (Ki = 30 nM) showed reasonable in vitro affinities to the 5-HT1A receptor, whereas
AH3.MZ appeared to be non-affine toward the 5-HT1A receptor. The receptor profile of AH1.MZ and AH2.MZ showed
selectivity within the 5-HT system. 18F-labelling via [18F]FETos to [18F]AH1.MZ was carried out in radiochemical yields of
470%. The final formulation of injectable solutions including [18F]FETos synthon synthesis, radiosynthesis and semi-
preparative high-performance liquid chromatography (HPLC) separation took no longer than 130 min and provided
[
18F]AH1.MZ with a purity of 498% as indicated by analytical HPLC analyses.
Keywords: radiofluorination; WAY 100635; [18F]AH1.MZ; 5-HT1A antagonist; PET
in vivo studies have been performed with several 5-HT1A
Introduction
selective antagonists such as [11C]WAY 10063513 and [18F]p-
MPPF.14 The chemical structures of WAY 100635 and p-MPPF are
presented in Scheme 1.
Serotonin (5-hydroxytryptamine, 5-HT) is an important neuro-
transmitter that is involved in physiological and pathophysio-
logical processes in both the peripheral and the central nervous
systems. Therefore, the 5-HT system is one of the most
important targets for medicinal chemistry.1–4 Alterations in
serotonin neurotransmission have been implicated in a number
of human disorders such as migraine, schizophrenia, depression
and anxiety as well as in normal human functions such as
learning, sleep, sexual activity and appetite. Especially, it is the 5-
HT1A receptor that may be involved in these various physiolo-
gical processes.5–10 A major task in the clinical routine is
depression, because it affects an estimated 121 million people
worldwide11 while the molecular basis for depression is not fully
being understood. However, deficits in the activity of serotonin-
mediated neurons in the brain are clearly central to the
disease.12 In vivo studies to quantitatively determine 5-HT1A
receptor availability would provide a significant advance in the
understanding of the mentioned disorders and conditions.
Positron emission tomography (PET) is an appropriate tool to
measure in vivo directly, non-invasively and repetitively the
binding potential, the receptor availability, uptake kinetics of
radio tracers for neuroreceptors as well as to evaluate the
efficacy of drugs directed to these molecular targets.
Fiorino et al. synthesized new arylpiperazines containing a
norbornene group as 5-HT1A receptor antagonists and reported
their outstanding in vitro selectivity for the 5-HT1A receptor.15
4-[3-[4-(o-Methoxyphenyl)piperazin-1-yl]propoxy]-4-aza-tricyclo-
[5.2.1.02,6]dec-8-ene-3,5-dione (1) showed even affinity in the
subnanomolar range for 5-HT1A receptors and moderate to no
affinity for other relevant receptors (Table 1).
Therefore, we decided to slightly modify promising ligands by
replacing a methoxy- by a fluoroethoxy group for labelling
purposes, to alter the position of the fluoroethoxy group within
the phenylic ring (Scheme 2) and to determine the affinities of
the new compounds toward several 5-HT receptors. In addition,
we report the optimized labelling and purification procedure of
the promising candidate [18F]AH1.MZ.
Institute of Nuclear Chemistry, University of Mainz, Fritz-Strassmann-Weg 2, D-
55128 Mainz, Germany
A number of neurotransmitter analogs labelled with b1
emitter containing radioligands were synthesized as radio-
pharmaceuticals for imaging the 5-HT1A receptor. To date,
*Correspondence to: Matthias M. Herth, Institute of Nuclear Chemistry,
University of Mainz, Fritz-Strassmann-Weg 2, D-55128 Mainz, Germany.
E-mail: herthm@uni-mainz.de
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M. M. Herth et al.
Scheme 1. Chemical structures of WAY 100635 and p-MPPF.
Table 1. Affinities of (1) for 5-HT1A, 5-HT2A, 5-HT2C, D1, D2, a1 and a2 receptors
5-HT1A
0.021
5-HT2A
5-HT2C
D1
D2
a1
a2
(1)
4104
4104
4104
4104
75.3
3650
Ki values in nM are based on the means of four experiments.
Scheme 2. Structures of novel compounds (2)–(4) compared with the original (1).
starting heterocycle endo-N-hydroxy-5-norbornene-2,3-dicar-
boximide with 1-bromo-3-chloropropane in the presence of
NaOH in absolute ethanol as reported by Fiorino et al.15
Results and discussion
Chemistry
Organic synthesis of WAY 100635 derivatives containing a
norbornene group has been described by Fiorino et al.15. Owing
to the necessary structural replacement of a methoxy- by a
fluoroethoxy group for labelling purposes, a similar synthesis
route was applied, but hydroxyphenylpiperazines were used as
starting materials for both precursors and reference compounds.
Receptor characterization
The potential of new fluoroethylated derivatives related to their
affinity and selectivity was examined by determining affinities
(Ki’s) to 5-HT receptors by radioligand binding assays through
NIMH Psychoactive Drug Screening Program (PDSP). The results
are summarized in Table 2.
The synthesis strategy, shown in Scheme 3, employs
a
protection/deprotection approach of the secondary amine and
the phenolic hydroxy group followed by alkylation of the key
intermediates (5) or (6) performed in MeCN in the presence of
K2CO3 and NaI under reflux. By means of Finkelstein exchange,
alkylation yields were improved by the increased leaving
tendency of iodine. However, (10) was synthesized by the
AH1.MZ (2) (Ki = 4.2 nM) and AH2.MZ (3) (Ki = 30 nM) reveal a
low to moderate nanomolar affinity to the 5-HT1A receptor,
whereas AH3.MZ (4) shows no affinity toward the 5-HT1A
receptors. Medium receptor affinity toward 5-HT2B (Ki = 144 nM)
could be detected for the o-fluoroethylated compound (2),
whereas the m-substituted ligand (3) lost this affinity.
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M. M. Herth et al.
Scheme 3. General synthesis strategy.
Table 2. Affinities of novel compounds (2)–(4) to 5-HT neuroreceptors
Original structure (1)a
AH1.MZ (2)
AH2.MZ (3)
AH3.MZ (4)
5-HT1A
5-HT1B
5-HT1D
5-HT1E
5-HT2A
5-HT2B
5-HT2C
5-HT3
0.021
—
—
4.270
3076
410.000
n.d.
410.000
410.000
n.d.
410.000
22297490
35707333
410.000
n.d.
343757
410.000
382759
599760
14479
410.000
410.000
17677279
410.000
n.d.
—
17387238
44067794
11507140
410.000
n.d.
410.000
410.000
552753
4104
—
4104
—
5-HT5A
5-HT6
5-HT7
—
—
—
410.000
410.000
410.000
Ki values in nM7SEM of human receptors are based on the means of four experiments. n.d., not determined.
aKi values reported by Fiorino et al.
However, AH1.MZ and AH2.MZ showed a reasonable in vitro production was performed in an automated module according
affinity profile, with high to moderate affinity to the 5-HT1A to Bauman et al.17 and used for [18F]fluoroalkylation (Scheme 4).
receptor and selectivity to other 5-HT receptors. The
The [18F]fluoroalkylation of the precursor (7) was optimized
outstanding affinity and selectivity of the reference compound only due to temperature variation resulting in radiochemical
4-[3-[4-(o-methoxyphenyl)piperazin-1-yl]propoxy]-4-aza-tricyclo- yields of 470% (Figure 1). Final reaction conditions were 1201C,
[5.2.1.02,6]dec-8-ene-3,5-dione (1) is lost by introducing a 7 mmol of precursor (7) and 7 mmol of 5 N NaOH dissolved in
fluoroethyl group (Table 2).
1 mL of dry DMSO with a reaction time of 20 min.
The final formulation of injectable solutions including
[18F]FETos synthon synthesis, radiosynthesis and a semi-pre-
parative high-performance liquid chromatography (HPLC) se-
paration (mBondapak C18 7.8 ꢀ 300 mm column, flow rate 8 mL/
min, eluent: MeCN/0.25 M NH4Ac buffer, pH 4.3 adjusted with
Radiochemistry
The 18F-labelling of the precursor (7) was carried out similar to
that reported in Herth et al.16 The necessary [18F]FETos synthon
J. Label Compd. Radiopharm 2009, 52 201–207
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M. M. Herth et al.
Scheme 4. Radiosynthesis of [18F]AH1.MZ.
FD mass spectrometry (MS) was performed on a Finnigan
MAT90-Spectrometer.
Radio-TLC plates were scanned and detected using
Canberra Packard Instant Imager.
a
Chemistry
4-(3-Chloropropoxy)-4-aza-tricyclo-[5.2.1.02,6]dec-8-ene-3,5-dione
(10)
4-(3-Chloropropoxy)-4-aza-tricyclo-[5.2.1.02,6]dec-8-ene-3,5-dione
(10) was synthesized as described by Fiorino et al.15
Figure 1. [18F]fluoroalkylation of 7 mmol of precursor (7) to [18F]AH1.MZ at
different reaction temperatures using DMSO and 7 mmol of 5 N NaOH.
General procedure for boc-protection
To a solution of 15.7 mmol of N-hydroxyphenylpiperazine and
27 mmol of NaHCO3 dissolved in 50 mL of THF/H2O/dioxane
(1:1:1), 18.8 mmol of Boc2O was added and stirred overnight.
Later, the solution was diluted with H2O, extracted with CH2Cl2,
dried over Na2SO4 and concentrated. Purification by column
chromatography (PE:EtOAc 7:3) gave the final pure product.
Tert-butyl 4-(2-hydroxyphenyl)piperazine-1-carboxylate (11a): A
total of 2.8 g of N-2-hydroxyphenylpiperazine (15.7 mmol), 2.3 g
of NaHCO3 (27 mmol) and 4.1 g of Boc2O (18.8 mmol) yielded
acetic acid (25/75)) took no longer than 130 min and provided
[18F]AH1.MZ with a radiochemical purity of 498% as indicated
by analytical HPLC analyses (ET 250/8/4 Nucleosil 5 C18, flow rate
1 mL/min, eluent: MeCN/0.05 Na2HPO4 buffer, pH 7.4 adjusted
with H3PO4 (40:60)). Thereby, amounts of ꢁ3 GBq of [18F]fluor-
ine were used as starting radioactivity. A typical specific activity
of 5 GBq/mmol could be observed for these low-scale activities.
In conclusion, a new 18F-labelled compound could be
obtained as an injectable solution in overall radiochemical
yields of about 25% within 130 min.
1
2.43 g (8.9 mmol, 57%) of the pure product. H-NMR (300 MHz,
CDCl3), d [ppm] = 7.135–7.054 (m, 2H), 6.959–6.928 (m, 1H),
6.875–6.19 (m, 1H), 3.597–3.566 (t, 4H), 2.837–2.805 (t, 4H), 1.470
(s, 9H). MS (FD) m/z (% rel. int.): 278.2 (100.0 [M]1), 279.2 (17.27
[M11]1). Rf = 0.6 (PE/EtOAc 7:3).
Experimental
General
Tert-butyl 4-(3-hydroxyphenyl)piperazine-1-carboxylate (11b): A
total of 5 g of N-3-hydroxyphenylpiperazine (28.04 mmol), 4.11 g
of NaHCO3 (48 mmol) and 7.32 g of Boc2O (33.6 mmol) yielded
Chemicals were purchased from ABX, Acros, Aldrich, Fluka,
Merck or Sigma and were used without further purification.
Moisture sensitive reactions were carried out under an argon or
a nitrogen atmosphere using dry solvents over molecular sieve.
Flash chromatographies were conducted on silica gel 60
(0.040–0.063 mm, Acros) columns. TLCs were run on pre-coated
plates of silica gel 60F254 (Merck). HPLC was performed on the
following systems:
Analytical HPLC: System equipped with a Sykam S 1100
Solvent Delivery System, S 8110 Low Pressure Gradient Mixer,
Rheodyne 9725i Inject Valve, Linear UVIS-205 Absorbance
Detector, Axxiom Chromatography 900-200 Pyramid, Pyramid
2.07, loop: 20 mL).
1
6.98 (25.05 mmol, 89%) of the pure product. H-NMR (300 MHz,
CDCl3), d [ppm] = 7.129–7.074 (t, 1H), 6.524–6.401 (m, 3H),
3.606–3.572 (t, 4H), 3.123–3.088 (t, 4H), 1.465 (s, 9H). MS (FD) m/z
(% rel. int.): 278.1 (100.0 [M]1), 279.1 (10.78 [M11]1). Rf = 0.52
(PE/EtOAc 7:3).
Tert-butyl 4-(4-hydroxyphenyl)piperazine-1-carboxylate (11c): A
total of 2.8 g of N-4-hydroxyphenylpiperazine (15.7 mmol), 2.3 g
of NaHCO3 (27 mmol) and 4.1 g of Boc2O (18.8 mmol) yielded
1
4.01 g (14.4 mmol, 92%) of the pure product. H-NMR (300 MHz,
CDCl3), d [ppm] = 7.101–6.784 (m, 4H), 3.702–3.669 (t, 4H),
3.111–3.054 (t, 4H), 1.459 (s, 9H). MS (FD) m/z (% rel. int.): 278.1
(100.0 [M]1), 279.1 (15.56 [M11]1). Rf = 0.35 (PE/EtOAc 7:3).
Semi-preparative HPLC: HPLC system equipped with a Dionex
P 680A pump, software Dionex Chromeleon vers., a Raytest 6.6
NaI scintillation counter (Gabi) and a Linear UVD170U (254 nm)
absorbance detector. Reversed-phase HPLC was carried out for
analytical separations using an ET 250/8/4 Nucleosil 5 C18
column and for semi-preparative applications a mBondapak C18
7.8 ꢀ 300 mm column was used. Three hundred and 400 MHz
NMR spectra were recorded on a Bruker 300 MHz-FT-NMR-
Spectrometer AC 300 or a Bruker-Biospin DRX 400 MHz spectro-
meter. Chemical shifts were reported in parts per million (ppm).
Alkylating procedure for 1,2-bromofluoroethane
NaH (7.19 mmol) was gradually added to the corresponding
phenolic derivatives (7.19 mmol) dissolved in 50 mL of dry, cold
DMF (01C) and stirred for 30 min. To the resulting mixture 1,2-
bromofluoroethane (7.19 mol) was added slowly and later stirred
for 20 h at 601C. After evaporation of the solvent, the residue
was taken up in EtOAc, washed with H2O and finally extracted
3 ꢀ with EtOAc. The combined organic extracts were dried
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M. M. Herth et al.
(Na2SO4), filtered and evaporated. Chromatography of the at room temperature, the solutions were diluted with 30 mL of
residue gave the pure product.
ether and carefully neutralized with NH4OH and ice bath
Tert-butyl 4-(2-(2-fluoroethoxy)phenyl)piperazine-1-carboxylate cooling. The layers were separated and the aqueous layer was
(12a): A total of 2 g of tert-butyl 4-(2-hydroxyphenyl)piperazine- extracted 3 ꢀ with ether. The combined organic extracts were
1-carboxylate (7.19 mmol), 189.4 mg of NaH (7.19 mmol) and washed with water, dried (Na2SO4), filtered and evaporated to
916 mg of 1,2-bromofluoroethane (0.52 mL; 7.19mmol) yielded afford a viscous oil. Silica gel column chromatography of the
2.04g (5.8mmol, 86%) of the pure product. 1H-NMR (300MHz, residues gave the pure products.
CDCl3), d [ppm]= 6.968–6.842 (m, 4H), 4.852–4.825 (m, 1H),
1-(2-(2-Fluoroethoxy)phenyl)piperazine (5a): A total of 1 g of
4.694–4.667 (m, 1H), 4.304–4.276 (m, 1H), 4.209–4.182 (m, 1H), tert-butyl 4-(2-(2-fluoroethoxy)phenyl)piperazine-1-carboxylate
3.591 (bs, 4H), 3.029 (bs, 4H), 1.459 (s, 9H). MS (FD) m/z (% rel. int.): (2.4 mmol) and 10 mL of TFA yielded 435 mg (1.55 mmol, 64%)
324.2 (100.0 [M]1), 325.2 (11.16 [M11]1). Rf = 0.54 (PE/EtOAc 7:3). of the pure product. 1H-NMR (300 MHz, CDCl3), d [ppm] =
Tert-butyl 4-(2-(3-fluoroethoxy)phenyl)piperazine-1-carboxylate 7.073–6.856 (m, 4H), 4.861–4.819 (m, 1H), 4.702–4.661 (m, 1H),
(12b): A total of 2 g of tert-butyl 4-(3-hydroxyphenyl)pipera- 4.313–4.246 (m, 1H), 4.235–4.142 (m, 1H), 3.374 (bs, 8H). MS (FD)
zine-1-carboxylate (7.19 mmol), 189.4 mg of NaH (7.19 mmol) m/z (% rel. int.): 223.5 (100.0 [M]1), 224.5 (13.32 [M11]1).
and 916 mg of 1,2-bromofluoroethane (0.52 mL; 7.19 mmol) Rf = 0.92 (CH3Cl/MeOH 8:1).
yielded 1.92 g (5.5 mmol, 81%) of the pure product. 1H-NMR
1-(2-(3-Fluoroethoxy)phenyl)piperazine (5b): A total of 1 g of
(300 MHz, CDCl3), d [ppm] = 7.189–7.135 (t, 1H), 6.566–6.403 (m, tert-butyl 4-(2-(2-fluoroethoxy)phenyl)piperazine-1-carboxylate
3H), 4.815–4.788 (m, 1H), 4.657–4.630 (m, 1H), 4.241–4.212 (m, (2.4 mmol) and 10 mL of TFA yielded 653 mg (2.31 mmol, 95%)
1H), 4.148–4.119 (m, 1H), 3.562–3.528 (t, 4H), 3.128–3.094 (t, 4H), of the pure product. 1H-NMR (300MHz, CDCl3),
d
1.461 (s, 9H). MS (FD) m/z (% rel. int.): 323.9 (100.0 [M]1), 324.9 [ppm] = 7.183–7.128 (t, 1H), 6.656–6.354 (m, 3H), 4.812–4.4.784
(17.82 [M11]1). Rf = 0.7 (PE/EtOAc 7:3).
(m, 1H), 4.654–4.626 (m, 1H), 4.237–4.179 (m, 1H), 4.144–4.117
Tert-butyl 4-(2-(4-fluoroethoxy)phenyl)piperazine-1-carboxylate (m, 1H), 3.342 (bs, 1H), 3.181–3.148 (t, 4H), 3.060–3.027 (t, 4H). MS
(12c): A total of 2g of tert-butyl 4-(4-hydroxyphenyl)piperazine-1- (FD) m/z (% rel. int.): 223.6 (100.0 [M]1), 224.6 (10.02 [M11]1).
carboxylate (7.19 mmol), 189.4 mg of NaH (7.19 mmol) and 916 mg 1-(2-(4-Fluoroethoxy)phenyl)piperazine (5c): A total of 1 g of
of 1,2-bromofluoroethane (0.52 mL; 7.19 mmol) yielded 1.55 g tert-butyl 4-(2-(2-fluoroethoxy)phenyl)piperazine-1-carboxylate
(4.79 mmol, 67%) of the pure product. 1H-NMR (300 MHz, CDCl3), d (2.4 mmol) and 10 mL of TFA yielded 676 mg (2.39 mmol, 99%)
[ppm] = 6.942–6.810 (m, 4H), 4.801–4.772 (m, 1H), 4.643–4.614 (m, of the pure product. 1H-NMR (300 MHz, CDCl3), d [ppm] =
1H), 4.210–4.182 (m, 1H), 4.117–4.089 (m, 1H), 3.573–3.540 (t, 4H), 6.898–6.830 (m, 4H), 4.851–4.739 (m, 1H), 4.640–4.612 (m, 1H),
3.012–2.979 (t, 4H), 1.460 (s, 9H). MS (FD) m/z (% rel. int.): 323.9 4.229–4.162 (m, 1H), 4.115–4.086 (m, 1H), 3.039 (s, 8H), 2.306
(100.0 [M]1), 324.9 (17.23 [M11]1). Rf = 0.76 (PE/EtOAc 7:3).
(bs, 1H). MS (FD) m/z (% rel. int.): 224.1 (100.0 [M]1), 225.1
(1.12 [M11]1).
1-[2-(Tert-butyl-diphenyl-silanyloxy)-phenyl]-piperazine (6a): A
total of 1.19 g of 4-[2-(tert-butyl-diphenyl-silanyloxy)-phenyl]-
piperazine-1-carboxylic acid tert-butylester (2.31 mmol) and
8.7 mL of TFA yielded 0.91 g (2.2 mmol, 94%) of the pure
product. 1H-NMR (300 MHz, CDCl3), d [ppm] = 7.660–7.629 (d,
4H), 7.466–7.389 (m, 6H), 6.727–6.691 (m, 2H), 6.619–6.593 (m,
2H), 3.213 (bs, 8H), 1.049 (s, 9H). MS (FD) m/z (% rel. int.): 416.2
(100.0 [M]1), 417.2 (32.98 [M11]1).
General procedure for TBDPS-protection
Tert-butyldiphenylsilyl chloride was added to a solution of the
corresponding phenolic derivative, imidazole, and 30 mL of THF
at 01C. This solution was stirred at 01C for 30 min, then stirred at
401C for 1 h, cooled, diluted with brine and extracted with
CH2Cl2. Standard workup gave crude silyl ether, which was
purified by silica gel column chromatography.
4-[2-(Tert-butyl-diphenyl-silanyloxy)-phenyl]-piperazine-1-carboxylic
acid tert-butylester (9a): A total of 2.27 g of tert-butyldiphenylsilyl
chloride (8.27 mmol), 2.30 g of 4-(2-hydroxyphenyl)piperazine-1-
carboxylate (8.27 mmol) and 1.11 g of imidazole (16.2 mmol)
General procedure for N-alkylation
A
4-(3-chloropropoxy)-4-aza-tricyclo-[5.2.1.02,6]dec-8-ene-3,5-
1
dione (10) (0.006 mol) and NaI (0.009 mol) were stirred in
50 mL of dry MeCN under reflux for 30 min. Then, the
appropriate arylpiperazine (0.03 mol) and anhydrous K2CO3
(0.009 mol) were added. The reaction mixture was stirred under
reflux for 24 h. After cooling, the mixture was filtered,
concentrated to dryness and the residue was dissolved in water
(50 mL). The solution was extracted several times with CH2Cl2.
The combined organic layers were dried over anhydrous
Na2SO4, and the solvent was removed under vacuum. The
crude mixtures were purified by silica gel column chromato-
graphy.
4-(3-f4-[2-(Tert-butyl-diphenyl-silanyloxy)-phenyl]-piperazin-1-ylg
-propoxy)-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dion (8): A total of
2.16 g of 4-(3-chloropropoxy)-4-aza-tricyclo-[5.2.1.02,6]dec-8-ene-
3,5-dione (8.47mmol), 1.125 g of NaI (7.5 mmol), 0.91 g of 1-[2-
(tert-butyl-diphenyl-silanyloxy)-phenyl]-piperazine (2.31 mmol)
and 5.4g of K2CO3 (7.5 mmol) yielded 1.33 g (2.1 mmol, 90%)
of the pure product. 1H-NMR (300 MHz, CDCl3), d [ppm] =
7.725–7.693 (m, 4H), 7.423–7.304 (m, 6H), 6.941–6.909 (dd, 1H),
yielded 1.33 g (2.57 mmol, 31.2%) of the pure product. H-NMR
(300 MHz, CDCl3), d [ppm] = 7.716–7.694 (d, 4H), 7.432–7.320 (m,
6H), 6.994–6.412 (m, 4H), 3.573 (bs, 4H), 3.043 (bs, 4H), 1.472 (s,
9H), 1.088 (s, 9H). MS (FD) m/z (% rel. int.): 516.2 (100.0 [M]1),
517.2 (42.08 [M11]1). Rf = 0.65 (PE/EtOAc 4:1).
4-[4-(Tert-butyl-diphenyl-silanyloxy)-phenyl]-piperazine-1-car-
boxylic acid tert-butylester (9b): A toal of 2.27 g of tert-
butyldiphenylsilyl chloride (8.27 mmol), 2.30 g of 4-(4-hydroxy-
phenyl)piperazine-1-carboxylate (8.27 mmol) and 1.11 g of imi-
dazole (16.2 mmol) yielded 1.1 g (2.12 mmol, 25%) of the pure
product. 1H-NMR (300 MHz, CDCl3), d [ppm] = 7.657–7.631 (d,
4H), 7.466–7.384 (m, 6H), 6.723–6.593 (dd, 4H), 2.955–2.889 (m,
8H), 1.471 (s, 9H), 1.008 (s, 9H). MS (FD) m/z (% rel. int.): 415.9
(100.0 [M]1), 416.9 (31.68 [M11]1). Rf = 0.62 (CHCL3/MeOH 5:1).
General procedure for boc-deprotection
The starting material (2.4 mmol) was carefully and gradually
dissolved in trifuoroacetic acid (TFA) (10 mL). After 2 h of stirring
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M. M. Herth et al.
6.826–6.771 (m, 1H), 6.598–6.545 (t, 1H), 6.454–6.427 (d, 1H),
6.158–6.152 (m, 2H), 4.073–4.005 (t, 2H), 3.413 (bs, 3H),
3.251–3.105 (m, 6H), 2.693 (bs, 4H), 2.003–1.885 (m, 2H),
1.768–1.732 (d, 1H), 1.508–1.477 (d, 2H), 1.077 (s, 9H). MS (FD)
m/z (% rel. int.): 635.9 (100.0 [M]1), 635.9 (62.64 [M11]1).
Rf = 0.45 (CHCl3/MeOH 8:2).
4-(3-f4-[2-(2-Fluoroethoxy)-phenyl]-piperazin-1-ylg-propoxy)-4-
aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dion (2): A total of 1.53 g of 4-
(3-chloropropoxy)-4-aza-tricyclo-[5.2.1.02,6]dec-8-ene-3,5-dione
(6.01 mmol), 0.79 g of NaI (5.32 mmol), 0.356 g of 1-(2-(2-
fluoroethoxy)phenyl)piperazine (1.64 mmol) and 3.83 g of
K2CO3 (5.32 mmol) yielded 260 mg (0.58 mmol, 38%) of the pure
product. 1H-NMR (300 MHz, CDCl3), d [ppm] = 6.999–6.815 (m,
4H), 6.162–6.123 (t, 2H), 4.838–4.810 (t, 1H), 4.679–4.651 (t, 1H),
4.277–4.249 (t, 1H), 4.182–4.155 (t, 1H), 4.063–4.025 (t, 2H), 3.395
(s, 2H), 3.297 (bs, 4H), 3.192 (s, 2H), 2.951–2.873 (m, 6H), 2.082
(bs, 2H), 1.754–1.723 (d, 1H), 1.495–1.460 (d, 1H). MS (FD) m/z (%
rel. int.): 443.3 (100.0 [M]1), 444.3 (26.04 [M11]1). Rf = 0.4 (CHCl3/
MeOH 12:2).
4-(3-f4-[2-(3-Fluoroethoxy)-phenyl]-piperazin-1-ylg-propoxy)-4-
aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dion (3): A total of 1.53 g of 4-
(3-chloropropoxy)-4-aza-tricyclo-[5.2.1.02,6]dec-8-ene-3,5-dione
(6.01 mmol), 0.79 g of NaI (5.32 mmol), 0.356 g of 1-(2-(3-
fluoroethoxy)phenyl)piperazine (1.64 mmol) and 3.83 g of
K2CO3 (5.32 mmol) yielded 460 mg (1.02 mmol, 52%) of the pure
product. 1H-NMR (300 MHz, CDCl3), d [ppm] = 7.190–7.035 (m,
1H), 6.585–6.339 (m, 3H), 6.227–6.058 (s, 2H), 4.791–4.776 (t, 1H),
4.647–4.618 (t, 1H), 4.228–4.171 (t, 1H), 4.135–4.107 (t, 1H),
4.029–3.986 (m, 2H), 3.399 (bs, 2H), 3.157 (bs, 6H), 2.566 (bs, 6H),
1.897–1.806 (m, 2H), 1.752–1.722 (d, 1H), 1.490–1.461 (d, 1H). MS
(FD) m/z (% rel. int.): 443.0 (100.0 [M]1), 444.0 (33.19 [M11]1).
Rf = 0.44 (CHCl3/MeOH 8:1).
Radiochemistry
[18F]FETos synthesis
To a dried Kryptofixs2.2.2./[18F]fluoride complex, 4 mg of ethyle-
neglycol-1,2-ditosylate in 1 mL of acetonitrile was added and
heated under stirring in a sealed vial for 3 min. Purification of the
crude product was accomplished using HPLC (Lichrosphere RP18-
EC5, 250 ꢀ 10 mm, acetonitrile/water 50:50, flow rate 5 mL/min, Rf:
8 min). After diluting the HPLC fraction containing the [18F]FETos
with water (HPLC fraction/water 1:4), the product was loaded on a
C18-Sepac cartridge, dried with a nitrogen stream and eluted with
1.2 mL of DMSO. The whole preparation time was about 40 min
and the overall radiochemical yield was between 60 and 80%.17
Radiolabelling of [18F]AH1.MZ
[18F]FETos diluted in 0.8 mL of dry DMSO was added to a solution
of 3 mg of 4-f3-[4-(2-hydroxy-phenyl)-piperazin-1-yl]-propoxyg-4-
aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dion (7) (7mmol) and 1.5mL of
5 N NaOH (7 mmol) dissolved in 0.2 mL of dry DMSO. The solution
remained at 1201C for 20 min and was quenched with 1 mL of H2O.
Reactants and by-products were separated from [18F]AH1.MZ by
semi-preparative HPLC (mBondapak C18 7.8 ꢀ 300 mm column, flow
rate 8 mL/min, eluent: MeCN/NH4Ac buffer, pH 4.3 adjusted with
acetic acid (25/75)). The retention times of [18F]AH1.MZ, [18F]FETos
and 4-f3-[4-(2-hydroxy-phenyl)-piperazin-1-yl]-propoxyg-4-aza-tri-
cyclo[5.2.1.02,6]dec-8-ene-3,5-dion (7) were 5.02, 13.4 and
9.72 min, respectively. The collected product was diluted with
water (4:1), passed through a conditioned strataX-cartridge (1 mL
of EtOH, 1 mL of H2O), washed with 10 mL of H2O and eluted with
at least 1 mL of EtOH. Finally, EtOH was removed in vacuo and
[18F]AH1.MZ was dissolved in 1 mL of saline.
4-(3-f4-[2-(4-Fluoroethoxy)-phenyl]-piperazin-1-ylg-propoxy)-4-
aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dion (4): A total of 1.53 g of 4-
(3-chloropropoxy)-4-aza-tricyclo-[5.2.1.02,6]dec-8-ene-3,5-dione
(6.01 mmol), 0.79 g of NaI (5.32 mmol), 0.356 g of 1-(2-(4-
fluoroethoxy)phenyl)piperazine (1.64 mmol) and 3.83 g of
K2CO3 (5.32 mmol) yielded 200 mg (0.44 mmol, 27%) of the pure
product. 1H-NMR (300 MHz, CDCl3), d [ppm] = 6.883–6.815 (m,
4H), 6.141 (s, 2H), 4.792–4.764 (t, 1H), 4.633–4.606 (t, 1H),
4.199–4.171 (t, 1H), 4.106–4.078 (t, 1H), 4.033–3.991 (t, 2H), 3.403
(bs, 2H), 3.159 (bs, 3H), 3.087–3.055 (t, 4H), 2.614–2.531 (m, 6H),
1.909–1.818 (m, 2H), 1.755–1.726 (d, 1H), 1.496–1.466 (d, 1H). MS
(FD) m/z (% rel. int.): 442.9 (100.0 [M]1), 443.9 (43.73 [M11]1).
Rf = 0.52 (CHCl3/MeOH 8:1), 0.6 (EtOH).
In vitro receptor and monoamine transporter binding
Binding assays were performed by the NIMH PDSP at the
Department of Biochemistry, Case Western Reserve University,
Cleveland, OH, USA (Bryan Roth, Director). Compounds AH1.MZ
(2), AH2.MZ (3) and AH3.MZ (4) were assayed for their affinities
for the 5-HT receptor family. Competitive binding experiments
were performed in vitro using cloned human receptors.
Reported values of the inhibition coefficient (Ki) are mean7SD
of four separate determinations.
Conclusion
4-f3-[4-(2-Hydroxy-phenyl)-piperazin-1-yl]-propoxyg-4-aza-tricy-
clo[5.2.1.02,6]dec-8-ene-3,5-dion (7): A solution of 0.68 g of
4-(3-f4-[2-(tert-butyl-diphenyl-silanyloxy)-phenyl]-piperazin-1-ylg-
propoxy)-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dion (1.14mmol)
and 0.21 g of NH4F (2.8 mmol) in anhydrous MeOH (30 mL) was
stirred for 2 h at 701C. After evaporation of the solvent, the
residue was taken up in NH4OH and extracted 3 ꢀ with CHCl3.
The combined organic extracts were washed with brine, dried
with Na2SO4 and evaporated. Chromatography (CHCl3/MeOH
10:1) of the residue yielded 380 mg (0.96 mmol, 84 %) of the pure
product. 1H-NMR (300 MHz, CDCl3), d [ppm] = 7.154–7.124 (dd,
1H), 7.073–7.018 (m, 1H), 6.937–6.900 (dd, 1H), 6.855–6.793 (m,
1H), 6.154–6.141 (t, 2H), 4.041–3.998 (t, 2H), 3.408 (bs, 2H),
3.175–3.162 (m, 2H), 2.893–2.863 (t, 4H), 2.617 (m, 6H),
1.912–1.820 (m, 2H), 1.762–1.732 (d, 1H), 1.500–1.471 (d, 1H).
MS (FD) m/z (% rel. int.): 396.8 (100.0 [M]1), 397.8 (40.03 [M11]1).
Rf = 0.67 (CHCl3/MeOH 5:1).
Fiorino et al.15 reported about an outstanding high affine
(Ki = 0.021 nM) and selective compound, 4-[3-[4-(o-methoxyphenyl)-
piperazin-1-yl]propoxy]-4-azatricyclo-[5.2.1.02,6] dec-8-ene-3,5-dione,
for the 5-HT1A receptor subtype. By replacing the methoxy- by a
fluoroethoxy group of the parent compound, three different
reference compounds (2)–(4) were obtained enabling a labelling
strategy with [18F]FETos. In vitro evaluation of these ligands showed
high to moderate affinities to the 5-HT1A receptor of AH1.MZ
(Ki = 4.2 nM) and of AH2.MZ (Ki = 30 nM), but not any affinity toward
the 5-HT1A receptor of the p-substituted fluoroethylated com-
pound (AH3.MZ) (Ki410.000 nM). The receptor profile of AH1.MZ
and AH2.MZ demonstrates selectivity within the 5-HT system.
However, the outstanding affinity and selectivity of the literature
reference compound 4-[3-[4-(o-methoxyphenyl)piperazin-1-yl]pro-
poxy]-4-aza tricyclo-[5.2.1.02,6]dec-8-ene-3,5-dione (1) is mainly lost
by introducing a fluoroethyl group. Nevertheless, compound
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 201–207

M. M. Herth et al.
AH1.MZ may provide potential for molecular imaging of the 5-HT1A
receptor system. Because of this potential, 18F-labelling via
[18F]FETos to [18F]AH1.MZ was carried out and optimized. Radio-
chemical yields were 470%. The final formulation of injectable
solutions including [18F]FETos synthon synthesis, radiosynthesis
and a semi-preparative HPLC separation took no longer than
130 min and provided [18F]AH1.MZ with a purity 498%.
In the near future, a broad receptor screening, in vitro
autoradiography and in vivo PET experiments are planned to
verify the potential for non-invasive molecular imaging of
[18F]AH1.MZ as an 18F-tracer for the 5-HT1A system.
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¨
The authors wish to thank Sabine Hohnemann for the syntheses of
[18F]FETos. Financial support by Friedrich-Naumann-Stiftung and by
the European Network of Excellence (EMIL) is gratefully acknowl-
edged. Ki determinations were generously provided by the National
Institute of Mental Health’s Psychoactive Drug Screening Program,
Contract ] NO1MH32004 (NIMH PDSP). The NIMH PDSP is directed
by Bryan L. Md, PhD, at the University of North Carolina at Chapel
Hill and Project Officer Jamie Driscol at NIMH, Bethesda, MD, USA.
[16] M. M. Herth, F. Debus, M. Piel, M. Palner, G. M. Knudsen,
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Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org