Synthesis, radiosynthesis and preliminary in vivo evaluation of [ 123I]-(4-fluorophenyl) {1-[2-(2-iodophenyl)ethyl]piperidin-4-yl} methanone, a potential 5-HT2A-antagonist for SPECT brain imaging

Article abstract of DOI:10.1002/jlcr.845

Many people suffer from psychiatric illnesses like depression and anorexia. Relevant to these diseases is amongst others a malfunctioning of brain 5-HT_2A-receptors. To allow in vivo quantification of these receptors with Single Photon Emission

Full text of DOI:10.1002/jlcr.845

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 591–598.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.845
Research Article
Synthesis, radiosynthesis and preliminary in vivo
evaluation of [123I]-(4-fluorophenyl) {1-[2-(2-
iodophenyl)ethyl]piperidin-4-yl}methanone, a potential
5-HT2A-antagonist for SPECT brain imaging
P. Blanckaert1,*, M. Vandecapelle1, L. Staelens1, I. Burvenich1,
R. A. Dierckx2 and G. Slegers1
1 Department of Radiopharmacy, Ghent University, Harelbekestraat 72, B-9000 Gent,
Belgium
2 Division of Nuclear Medicine, Ghent University Hospital, De Pintelaan 185, B-9000
Gent, Belgium
Summary
Many people suffer from psychiatric illnesses like depression and anorexia. Relevant
to these diseases is amongst others a malfunctioning of brain 5-HT2A-receptors. To
allow in vivo quantification of these receptors with Single Photon Emission
Computerized Tomography (SPECT), a radiolabelled ligand with high 5-HT2A
affinity is needed.
This work reports the radiosynthesis of [123I]-(4-fluorophenyl) {1-[2-(2-iodopheny-
l)ethyl]piperidin-4-yl}methanone, the synthesis of its precursor, (4-fluorophenyl)
{1-[2-(2-bromophenyl)ethyl]piperidin-4-yl}methanone, and the preliminary in vivo
evaluation of the tracer. The precursor was synthesized with a total yield of 40%.
Radiolabelling was performed using a halogen exchange reaction and the yield was
70%. Radiochemical purity was >95%, and specific activity was at least 2.4 Ci/mmol.
Log P was measured to be 2.52. The tracer showed uptake in mice brain (3.5% I.D./g
tissue at 3 min post injection) and therefore will be evaluated further by regional brain
biodistribution and displacement studies in rabbits. Copyright # 2004 John Wiley &
Sons, Ltd.
Key Words: serotonin; 5-HT2A-receptor; SPECT; radiotracer
*Correspondence to: P. Blanckaert, Laboratory for Radiopharmacy, Faculty of Pharmaceutical Sciences,
Harelbekestraat 72, B-9000 Gent, Belgium. E-mail: peter.blanckaert@ugent.be
Copyright # 2004 John Wiley & Sons, Ltd.
Received 2 April 2004
Revised 23 April 2004
Accepted 26 April 2004

592
P. BLANCKAERT ET AL.
Introduction
Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter in
the central nervous system. Cerebral 5-HT-receptors have been implicated
in the pathogenesis of major mood and psychotic disorders, such as
depression and schizophrenia.1 Relevant to these diseases are problems
associated with the 5-HT2A G-protein-coupled receptor, found amongst
others in high densities in cortical areas, striatum and hippocampus in all
species.2 To evaluate 5-HT2A-receptor malfunctioning, in vivo quantification
by positron emission tomography (PET) or SPECT is needed. These
techniques provide highly sensitive methods for measuring in vivo neuro-
chemical and pharmacological effects at specific target–receptor proteins.
Several radioligands have been used to assess the 5-HT2A-receptor4 including
[11C]-N-methylspiperone, [11C]-MDL1009075, [18F]-setoperone, [123I]-R91150.
The PET-tracers are expensive, requiring a cyclotron on-site. [123I]-R91150
shows high aspecific binding, thereby limiting its in vitro use. Development
of better SPECT radiotracers with high specific brain uptake, selectivity for
5-HT2A-receptors, and low aspecific binding would therefore be very useful.
A log P value between 2 and 3 is desirable for minimizing aspecific binding
issues.
This work reports the synthesis and preliminary in vivo evaluation in mice
of
[
123I]-(4-fluorophenyl){1-[2-(2-iodophenyl)ethyl]piperidin-4-yl}methanone
([123I]-5b), a potential SPECT-tracer for in vivo imaging of the 5-HT2A-
receptor. The compound shows nanomolar affinity for the 5-HT2A-receptor6
(Ki=1.95 nM), 20-fold selectivity over the 5-HT2C-receptor and antagonistic
properties.
Results and discussion
Synthesis of the precursor molecule and the cold product as reference
compound is shown in Scheme 1.
The appropriate halogenated phenylacetic acid derivatives (1a, 1b) were
reduced to the alcohols (2a, 2b) with lithium aluminum hydride, after which
the hydroxyl moiety was replaced by bromine using phosphorus tribromide,
thus creating a much better leaving group. These ethylbromides (3a, 3b) were
then coupled with 4-(4-fluorobenzoyl)piperidine (4) by nucleophilic substitu-
tion.6
The iodinated and brominated products were both obtained in overall yields
of 40%. The radiolabelling was conducted by a Cu+ catalyzed nucleophilic
exchange on the bromoprecursor (Scheme 2). A radiochemical yield of 70%
was obtained.
The radiochemical purity of the tracer was >95%. Specific activity was
calculated to be at least 2.4Ci/mmol.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 591–598

SYNTHESIS, RADIOSYNTHESIS AND
593
OH
Br
OH
LiAlH4, dry THF
RT, 12 h
PBr3
O
80˚C, 3 h
R
R
R
(2a,b)
(1a): R = Br
(1b): R = I
(3a,b)
O
O
DMF, K2CO3
90˚C, 22 h
F
(3a,b)
+
N
F
R
N
H
(4)
(5a,b)
Scheme 1. Synthesis of precursor and reference compound
O
O
n.c.a. Na123I/Cu+
N
135˚C, 60 min
F
N
F
Br
123I
Scheme 2. Radiosynthesis of [123I]-5b
Identification of the collected tracer was performed by comparing retention
times on HPLC between the radioactive labelled product and the unlabelled
iodinated molecule 5b.
Tracer stability at room temperature was tested by reinjecting the tracer into
the HPLC system; the radiochemical purity was >95% at 24 h after synthesis.
The log P value was measured by the shake-flask method to be 2.52, a
suitable value for blood–brain–barrier penetration.11
Biodistribution studies demonstrated brain uptake of the compound
in mice (3.5% I.D./g tissue at 3 min p.i.) (Figure 1). Other organs are not
shown.
Experimental
General
All reagents were purchased from commercial sources (Sigma Aldrich Fluka,
Acros Organics, Belgium) and were used without further purification, unless
stated otherwise. 4-(4-Fluorobenzoyl)piperidine was commercially available
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 591–598

594
P. BLANCKAERT ET AL.
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
blood
brain
0
50
100
150
200
Time (min)
Figure 1. Biodistribution of [123I]-5b in NMRI mice
as the p-toluenesulfonate. It was extracted as the base and purified by
recrystallization from acetonitrile.
Reactions were monitored by thin layer chromatography under UV
(254 nm) where possible (TLC, Polygram Sil G/UV254, Machery-Nagel,
Germany).
Purification of unlabelled compounds was achieved by column chromato-
graphy with silica gel (Sigma Aldrich, 200-400 mesh), using the solvent systems
indicated in the text. For mixed solvent systems, ratios are given with respect
to volumes.
1H-NMR spectra were recorded on a Varian 300 MHz FT-NMR spectro-
meter (Department of Medicinal Chemistry, Ghent University). Chemical
shifts were recorded in ppm (d) from an internal tetramethylsilane standard in
either chloroform-d3, acetone-d6 or dimethylsulfoxide-d6.
Mass spectrometry was performed on a Waters Micromass ZMD mass-
spectrometer with electrospray-ionization probe. Samples were dissolved in
methanol.
n.c.a. (no carrier added) [123I] NaI (in 0.05 M NaOH) was purchased from
Bristol-Myers-Squibb (Brussels, Belgium). HPLC purification and analysis of
the radioligand were performed using a Waters 515 HPLC pump, a Waters
2487 UV detector (254 nm), and a Ludlum 2200 scaler ratemeter. Purification
and analysis of radioligand were performed on a reversed-phase analytical
HPLC column (Alltech Alltima C18 4.6 Â 250 mm, 5 mm). Mobile phase was
50/50 acetonitrile/phosphate buffer (0.02 M, pH 6.5). A flow rate of 4 ml/min
was applied.
For biodistribution studies, male NMRI (Naval Medical Research Institute)
mice were used. Our research protocol was approved by the ethical committee
(ECP 03/22).
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 591–598

SYNTHESIS, RADIOSYNTHESIS AND
595
Synthesis of precursor and reference compound
2(2-Bromophenyl)ethanol (2a). Lithiumaluminumhydride (2.85 g, 75 mmol)
was slowly added to dry tetrahydrofuran (100 ml) under nitrogen. The
suspension was stirred and cooled to 08C. A solution of 2-bromophenylacetic
acid (1a) (10.7 g, 50 mmol) in dry tetrahydrofuran (50 ml) was added dropwise.
The addition funnel was rinsed with dry tetrahydrofuran, and the ice-bath was
removed. The mixture was stirred at ambient temperature for 12 h. The
reaction mixture was cooled to 08C, and quenched by adding ethylacetate
(50 ml) dropwise, followed by methanol (20 ml). 15% NaOH in water (30 ml)
was added to the mixture, which was then filtered over celite. The precipitate
was washed with tetrahydrofuran (3 Â 50 ml). The filtrate was evaporated
under reduced pressure. Dichloromethane (250 ml) was added to the residue
and the organic layer was separated, washed with brine (100 ml), dried over
anhydrous Na2SO4 and the solvent was removed under reduced pressure. A
yellowish oil was obtained (6.81 g, 69%).
1H-NMR (d6-DMSO, d): 7.56 (d, 1 H, Br–ArH), 7.26 (d, 1 H, Br–ArH),
7.14-7.06 (q, 2 H, Br–ArH), 3.88 (t, 2 H, R–CH2–OH), 2.87 (t, 2 H, Ar–CH2–
R), 1.54 (s, 1 H, – OH).
ESI-MS m/z: 201-203 ([M+H]+).
2(2-Iodophenyl)ethanol (2b). 2b was prepared in exactly the same manner as
2a, starting from 2-iodophenylacetic acid. Yield was 60%.
1H-NMR (d6-DMSO, d): 7.75 (d, 1H, I–ArH), 6.90 (d, 1H, I–ArH), 6..94-
7.37 (q, 2H, I–ArH), 3.86 (t, 2H, R–CH2–OH), 2.87 (t, 2H, Ar–CH2–R), 1.54
(s, 1H, – OH).
ESI-MS m/z: 249 ([M+H]+).
1-Bromo-2(2-bromoethyl)benzene (3a). 2-(2-Bromophenyl)ethanol (2a) (5 g,
25 mmol) was added to a 25 ml vial, and the vial was cooled to 08C under
nitrogen. Phosphorus tribromide (8.12 g, 2.8 ml, 30 mmol) was added
dropwise. The reaction mixture was heated at 808C for 2 h. The mixture was
poured onto crushed ice, saturated NaHCO3-solution (100 ml) was added, and
the mixture was stirred for 30 min. The mixture was extracted with chloroform
(3 Â 100 ml) and the combined extracts washed once with saturated NaHCO3-
solution (100 ml) and once with brine (100 ml). The solution was dried over
anhydrous Na2SO4, filtered and evaporated under reduced pressure. A
yellowish oil was obtained (6.31 g, 96%).
1H-NMR (d6-DMSO, d): 7.79 (d, 1H, Br–ArH), 7.25 (d, 1H, Br–ArH), 7.18-
7.09 (q, 2H, Br–ArH), 3.48 (t, 2H, R–CH2–Br), 2.96 (t, 2H, Ar–CH2–R).
1-Iodo-2(2-bromoethyl)benzene (3b). 3b was prepared in exactly the same
manner as 3a, starting from 2b. Yield was 90%.
1H-NMR (d6-DMSO, d): 8.10 (d, 1H, I–ArH), 7.65 (d, 1H, I–ArH), 7.03-
6.92 (q, 2H, I–ArH), 3.52 (t, 2H, R–CH2–Br), 3.05 (t, 2H, Ar–CH2–R).
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 591–598

596
P. BLANCKAERT ET AL.
(4-Fluorophenyl){1-[2-(2-bromophenyl)ethyl]piperidin-4-yl}methanone (5a).
To a solution of 4-(4-fluorobenzoyl)piperidine (4) (2.07 g, 10 mmol) in dry
DMF (50 ml) under a nitrogen atmosphere was added 1-bromo-2-(2-
bromoethyl)benzene (3a) (3.93 g, 15 mmol), followed by K2CO3 (5.5 g,
40 mmol). The mixture was heated at 908C for 22 h. After cooling to ambient
temperature, the mixture was filtered, and the precipitate was washed with
DMF (20 ml). The solvent was evaporated under reduced pressure, and the
residue was purified by column chromatography with 20:80:10 EtOAc/hexane/
Et3N to give 5a as a yellow solid (2.32 g, 60%).
1H-NMR (d6-DMSO, d): 7.90 (q, 2H, F–ArH), 7.60 (d, 1H, Br–ArH), 7.22
(d, 1H, Br–ArH), 7.13 (m, 2H, F–ArH).7.09-7.00 (q, 2H, Br–ArH), 3.30-3.20
(m, 4H), 3.10 (t, 1H), 2.80 (t, 2H, Ar–CH2–CH2), 2.70 (t, 2H, Ar–CH2), 2.10-
1.92 (m, 4H).
ESI-MS m/z: 389/391 [M+H]+.
(4-Fluorophenyl){1-[2-(2-iodophenyl)ethyl]piperidin-4-yl}methanone (5b). 5b
was prepared in exactly the same manner as 5a, starting from 3b. Yield was
52%.
1H-NMR (d6-DMSO, d): 7.90 (q, 2H, F–ArH), 7.76 (d, 1H, I–ArH), 7.28 (t,
1H, I–ArH), 7.03-6.92 (m, 2H, F–ArH), 6.93-6.77 (m, 2H, I–ArH), 3.30-3.20
(m, 4H), 3.10 (t, 1H), 2.90 (t, 2H, Ar–CH2–CH2), 2.60 (t, 2H, Ar–CH2 ), 2.10-
1.92 (m, 4H).
ESI-MS m/z: 438 [M+H]+.
Radiosynthesis
The nucleophilic exchange labelling on the bromoprecursor was performed
using a Cu+-catalysed method7–9. Two stock solutions were prepared:
Solution A containing 32.5 mg CuSO4 /10 ml water; Solution B, containing
9 mg SnSO4, 25 mg gentisinic acid and 50 mg citric acid /10 ml water. Both
solutions were flushed with nitrogen to remove oxygen. The bromoprecursor
5a (1 mg, 2.6 mmol) was dissolved in 100 ml ethanol under nitrogen; 500 ml of
solution B and 65 ml of solution A were added to the vial. The vial was flushed
with nitrogen, sonified for 15 min, and [123I] NaI (0.1–5mCi) was added.
The vial was heated at 1358C for 1 h in an aluminum heating mantle, and
cooled to ambient temperature. After cooling, the reaction mixture was
injected onto an HPLC column for purification (Alltech Alltima RPC18,
4.6 Â 250 mm), with 50/50 acetonitrile/phosphate buffer (0.02 M, pH6.5) as
eluent at 4 ml/min. The desired radiolabelled product (Rt=19.3 min) was
collected, and diluted with water to bring the acetonitrile concentration below
10%. The mixture was passed through a Sep-Pak cartridge (Waters Sep-Pak
Light tC18), and rinsed with 5 ml saline. The cartridge was previously
activated with 1 ml methanol and rinsed with 1 ml saline. The tracer was eluted
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 591–598

SYNTHESIS, RADIOSYNTHESIS AND
597
with 1 ml ethanol. An aliquot was reinjected onto the same HPLC system for
quality control and stability testing. For biodistribution studies, the tracer was
formulated in an ethanol/saline solution (containing less then 10% ethanol),
and filtered through a 0.2 mm filter (Schleicher&Schuell, FP 013/AS).
Since no UV-signal was obtained from the synthesized product, specific
activity was calculated by determination of the detection limit of the UV-
detector, using a calibration curve with cold 5b.
Determination of partition coefficient
Determination of the partition coefficient was performed according to
published literature.10,11 Briefly, about 10 ml (0.1 mCi) of the radioligand was
added to a separatory funnel containing n-octanol (100 ml) and phosphate
buffer (0.02 M, pH 7.4, 100 ml). The mixture was shaken manually for 3 min
and the layers were separated. The aqueous layer was discarded, to remove
any hydrophilic impurities present. The n-octanol layer (100 ml) was
transferred to a second separatory funnel containing phosphate buffer
(100 ml). The mixture was shaken for 3 min, and the layers were separated.
A 5 ml aliquot of both layers was counted for radioactivity. The aqueous layer
was discarded. Once again, the n-octanol layer (95 ml) was transferred to a new
separatory funnel containing phosphate buffer (95 ml), the funnel shaken for
3 min, the layers separated, and a 5 ml aliquot of both layers was taken and
counted for radioactivity. This process was repeated once more. The
radioactivity counts were decay-corrected, and the partition coefficient was
calculated: P=counts in n-octanol/counts in buffer. Reported log P value
represents the mean of three determinations.
Biodistribution study in NMRI mice
A biodistribution study of the radiotracer was performed in mice. Adult white
male NMRI mice weighing 20–25 g were each injected with 1-2mCi of the
radiotracer [123I]-5b in the tail vein. The mice were sacrificed at selected time
points after injection (n=3 per time point). Blood and tissues of different
organs (brain, heart, lung, liver, kidney, e.a.) were rapidly removed and
weighed. Radioactivity of the samples was measured in an automated g-
counter (Cobra, Packard Canberra). Tissue radioactivity concentrations were
expressed as percent of injected dose per gram of tissue (% I.D./g tissue).
Conclusion
This work reported the synthesis and radiolabelling of (4-fluorophenyl) {1-[2-
(2-iodophenyl)ethyl]piperidin-4-yl} methanone, a potential radiotracer for
in vivo visualization of the 5-HT2A-receptor with SPECT. The precursor was
synthesized in an overall yield of 40%. The tracer was labelled in good yield
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 591–598

598
P. BLANCKAERT ET AL.
(70%). The specific activity was at least 2.4 Ci/mmol. Log P was 2.52. The
tracer showed good brain uptake in mice (3.5% I.D./g tissue at 3 min p.i.).
Regional biodistribution and displacement studies in rabbit brain are further
required to demonstrate specific binding in brain regions expressing 5-HT2A-
receptors.
References
1. Lundkvist C, Halldin C, Ginovart N, Nyberg S, Swahn CG, Carr AA, Brunner F,
Farde L. Life Sci 1996; 58: L187–L192.
2. Saxena PR. Pharmacol Therapeut 1995; 66: 339–368.
3. Runyon SP, Peddi S, Savage JE, Roth BL, Glennon RA, Westkaemper RB.
J Med Chem 2002; 45: 1656–1664.
4. Oh SJ, Ha HJ, Chi DY, Lee HK. Current Med Chem 2001; 8: 999–1034.
5. Ullrich T, Rice KC. Abstracts Papers Am Chem Soc 2000; 220: U556–U556.
6. Fu X, Tan PZ, Kula NS, Baldessarini R, Tamagnan G, Innis RB, Baldwin RM.
J Med Chem 2002; 45: 2319–2324.
7. Mertens J, Gysemans M, Hermanne A, Terriere D. Eur J Nucl Med 1992; 19:
626–626.
8. Galinier E, Garreau L, Dognon AM, Ombettagoka JE, Frangin Y, Chalon S,
Besnard JC, Guilloteau D. Eur J Med Chem 1993; 28: 927–933.
9. Farah K, Farouk N. J Label Comp & Radiopharm 1997; 39: 915–926.
10. Huang YY, Hwang DR, Zhu ZH, Bae SA, Guo NN, Sudo YS, Kegeles LS,
Laruelle M. Nucl Med Biol 2002; 29: 741–751.
11. Wilson AA, Jin L, Garcia A, DaSilva JN, Houle S. Appl Rad Isotopes 2001; 54:
203–208.
12. Azizian H, Eaborn C, Pidcock A. J Organometall Chem 1981; 215: 49–58.
13. Staelens L, Dumont F, De Vos F, Oltenfreiter R, Vandecapelle M, Dierckx RA,
Slegers G. J Label Comp Radiopharm 2003; 46: 297–305.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 591–598