Synthesis and radiolabelling of [123I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl) butyl)-benzamide, a potential dopamine D3 antagonist for SPECT studies

Article abstract of DOI:10.1002/jlcr.671

Schizophrenia is a devastating mental disorder characterized by relapsing psychotic episodes accompanied with emotional, professional and social decline. The classical dopamine hypothesis of schizophrenia postulates that hyperactivity of dopaminergic neur

Full text of DOI:10.1002/jlcr.671

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 297–305.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.671
Research Article
Synthesis and radiolabelling of [123I]-4-iodo-
N-(4-(4-(2-methoxyphenyl)-piperazin-1-
yl)butyl)-benzamide, a potential dopamine
D3 antagonist for SPECT studies
L. Staelens1,*, F. Dumont1, F. De Vos2 and R. Oltenfreiter1,
M. Vandecapelle1, R.A. Dierckx2, G. Slegers1
1 Department of Radiopharmacy, Ghent University, Harelbekestraat 72,
9000 Gent, Belgium
2 Division of Nuclear Medicine, Ghent University Hospital, De Pintelaan 185,
9000 Gent, Belgium
Summary
Schizophrenia is a devastating mental disorder characterized by relapsing
psychotic episodes accompanied with emotional, professional and social
decline. The classical dopamine hypothesis of schizophrenia postulates
that hyperactivity of dopaminergic neurotransmission is responsible for the
positive symptoms of the disorder. More exactly hyperactivity of the
dopamine D3 receptor system is thought to be involved in the pathology of
schizophrenia. Therefore a new 123I-labelled compound was developed
which may allow in vivo visualization of the D3 receptor by SPECT.
[
123I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-benzamide was
synthesized and labelled by electrophilic aromatic substitution of the
tributylstannyl derrivative. The radiochemical yield was 82–85% and
the specific activity was >2.96 Ci/mmol. Copyright # 2003 John Wiley &
Sons, Ltd.
Key Words: Dopamine; D3 receptor; SPECT; radiotracer
*Correspondence to: L. Staelens, Laboratory of Radiopharmacy, Faculty of Pharmaceutical
Sciences, Harelbekestraat 72, 9000 Gent, Belgium E-mail: ludovicus.staelens@rug.ac.be
Copyright # 2003 John Wiley & Sons, Ltd.
Received 6 April 2002
Revised 30 September 2002
Accepted 13 October 2002

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L. STAELENS ET AL.
Introduction
The classical dopamine hypothesis of schizophrenia is based on the
correlation between clinical doses of antipsychotic drugs, with proto-
type haloperidol, and their potency to block the dopamine D2
receptor.1,2 The dopamine receptors have been divided into 5 subtypes
based on molecular biology data, the D1-like receptors D1 and D5 and
the D2-like receptors D2, D3 and D4. The latter class has recently
received substantial attention, particularly since this receptor class is
thought to be the target for atypical antipsychotic drugs, with prototype
clozapine. Atypical neuroleptics are known to bind to a much lesser
degree to the dopamine D2 receptors in the striatum than typical
neuroleptics although their antipsychotic efficacy is equal to that of
typical neuroleptics.3 Experimental studies have already shown a
predominant action of atypical neuroleptics in the limbic regions. D3,
D4 and D5 subtypes of dopamine receptors seem to be predominantly
located in the limbic system and cortical areas. Atypical neuroleptics
have a more selective action on these receptor subtypes than do typical
neuroleptics.4 In the past several radiotracers for the D2-like receptors
like [123I]-epidepride, [123I]-IBZM, [11C]-raclopride, [11C]-nemonapride
and [11C]-methylspiperone have been developed, however these showed
no selectivity between the different receptors of this class.5,6 Very few
radiotracers have been developed which show selectivity for the
dopamine D3 receptor like 7-OH-PIPAT.7
The aim of this study was to synthesize a tracer for SPECT studies of
the dopamine D3 receptor. In order to develop a tracer for these
receptors a number of demands have to be met. First of all the tracer
needs to have high affinity for the dopamine D3 receptor due to the low
density of these receptors, furthermore the selectivity over the other
dopamine receptor subtypes and other receptor types in general needs to
be good and finally the lipophilicity should not be too high in order to
avoid non-specific binding. Selection of [123I]-4-iodo-N-(4-(4-(2-meth-
oxyphenyl)-1-piperazinyl) butyl)-benzamide as a potential tracer for the
D3 receptor was based upon data collected by Murray et al.8 These data
indicate that the corresponding bromated derivative has high affinity for
the D3 receptor (pKi=9.3) and shows a 100 fold selectivity over the D2
(pKi=7.4) and D4 (pKi=7.3) receptors. Apart from dopamine receptors
there is some interaction with the 5-HT1A receptor (pKi=7.9) and the a1
receptor (pKi=7.9).8 From these data we can assume that the iodinated
derivative will also show selectivity for the D3 receptor moreover since
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A POTENTIAL DOPAMINE D3 ANTAGONIST
299
antagonists with larger groups in the same position also show D3
selectivity (e.g. aminobenzyl: pKi D3 = 9.7, pKi D2=7.7).8
Experimental
General
All reagents were purchased from commercial sources and were used
without further purification. Reactions were monitored by thin layer
chromatography (TLC, Polygram Sil G/UV254, Machery-Nagel, Ger-
many). Chromatographic purification of unlabelled compounds was
performed on silica gel using the solvent systems indicated in the text.
For mixed solvent systems, ratios are given with respect to volumes.
Proton NMR spectra were recorded on a Varian 300 MHz FT-NMR
spectrometer (Department of Medicinal Chemistry, Ghent University).
Chemical shifts were recorded in ppm (d) from an internal tetra-
methylsilane standard in either chloroform-d3, acetone-d6 or methyl-
sulfoxide-d6. High resolution mass spectroscopy performed using a
Micromass Q-Tof-2 mass spectrometer (Rega Institute for Medicinal
Research, Leuven University). [123I] Sodium iodide (in 0.05 M NaOH)
was purchased from DuPont Pharma (Brussels, Belgium). HPLC
analysis of the radioligand was performed using a Waters 515 HPLC
pump, a Waters 2487 UV detector (254 nm), and a Ludlum model 2200
sealer ratemeter. The column used was a reverse-phase base-deactivated
column (Nucleosil C18 BDS 4.6 Â 250 mm, 5 mm particle size; Alltech)
and the mobile phases and flow rates are indicated in the text below.
Synthesis of 2-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)-butyl)-isoin-
dole-1,3-dione (2)
1-(2-methoxyphenyl)-piperazine (2.3 g, 12 mmol) was dissolved in
dimethylformamide (10 ml). Anhydrous sodium carbonate (1.27 g,
12 mmol) was pulverized and added, the mixture was heated to 808C
during 15 min. N-4-bromobutylphtalimide (3.38 g, 12 mmol) was added
in small portions. The temperature was maintained at 808C during 3 h
and the reaction was monitored by TLC (dichloromethane/methanol/
ammonia, 97/3/5). The formation of a new product with Rf=0.64 was
observed. The reaction mixture was then purified by extracting with
ethyl acetate (100 ml) and the organic layer was washed with water
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(2 Â 100 ml). The organic layer was dried over anhydrous sodium
sulphate and evaporated under reduced pressure. 3.58 g of a yellow solid
was obtained. This solid was used without further purification.
Synthesis of 4-(4-(2-methoxyphenyl)-piperazin-1-yl)-butylamine (3)
2-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)-butyl)-isoindole-1,3-dione (2)
(3.54 g, 9 mmol) was dissolved in absolute ethanol (200 ml). After
addition of hydrazine hydrate (1 ml, 20 mmol) the mixture was refluxed
for 3 h. Subsequently the mixture was cooled to room temperature. The
reaction mixture was diluted with dichloromethane (100 ml) and washed
with water. The organic layer was isolated, dried with sodium sulphate
and evaporated under reduced pressure. The residue was analysed with
TLC (dichloromethane/methanol/ammonia: 87/13/5) and three pro-
ducts were observed, the starting product (Rf=0.9) and two newly
formed products (Rf: 0.5 and 0.2). The mixture was purified by column
chromatography (silica, 200 Â 30 mm) with dichloromethane/methanol/
ammonia (95/2/5) as eluent. The eluted fractions were collected and
1
analysed with mass spectrometry and H-NMR. The product with
Rf=0.2 was determined to be the desired product. The collected
fractions were evaporated to yield 1.8 g (yield=80%) of a white solid.
1H-NMR (d6-DMSO): 6.8–7.0 (m, 4 H, ArH), 3.9 (s, 3 H, OCH3), 3.2
(m, 4 H, piperazineH), 2.8 (t, 2 H, -CH2-NH2-), 2.6 (m, 4 H,
piperazineH), 2.4 (t, 2 H, N-CH2-CH2), 1.8–2.0 (2q, 4 H, -CH2-CH2-).
ESI-MS m/z: 264 ([M+H]+).
Synthesis of 4-bromo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-
benzamide (4)
4-(4-(2-methoxyphenyl)-piperazin-1-yl)-butylamine (3) (1.77 g, 6.73 mmol)
was dissolved in dichloromethane (18 ml). Triethylamine (1.08 g,
10.68 mmol) was added and the temperature of the mixture was cooled
to 08C. 4-Bromobenzoylchloride (1.66 g, 7.57 mmol) was dissolved in
dichloromethane (1.4 ml) and added dropwise at such a rate as to
maintain the temperature below 108C. Then the mixture was stirred at
room temperature for 2 h. The reaction mixture was diluted with
dichloromethane (100 ml) and washed with a saturated sodium
bicarbonate solution. The organic layer was then washed with water,
dried with anhydrous sodium sulphate and the solvent was removed
under reduced pressure. TLC analysis (dichloromethane/methanol/
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A POTENTIAL DOPAMINE D3 ANTAGONIST
301
ammonia: 87/13/5) of the mixture showed a newly formed product
(Rf=0.8). Purification was conducted on a silica column (silica,
200 Â 30 mm) starting with pure dichloromethane and gradually (steps
of 0.5%, 300 ml of each eluent) changing to dichloromethane/methanol
(98/2). The eluted fractions were evaporated, a white solid was obtained
1
(2.7 g yield = 90%). H-NMR (d6-DMSO): 7.8–7.9(dd, 4 H, ArH),
6.7–7.0 (m, 4 H, ArH), 3.8 (s, 3 H, OCH3), 3.7 (t, 2 H, -CH2-NH-CO),
3.3 (m, 4 H, piperazineH), 2.9 (m, 4 H, piperazineH), 2.8 (t, 2 H, N-CH2-
CH2), 1.6–1.8 (2q, 4 H, -CH2-CH2-). ESI-MS m/z: 446-448 ([M+H]+).
Synthesis of N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-4-
tributylstannyl-benzamide (5)
4-bromo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-benzamide
(4) (2.7 g, 6.04 mmol) was dissolved in dry toluene (100 ml). Hexa-
butylditin (10.44 g) and a catalytic amount ( Æ 3 mg) of tetrakistri-
phenylphosfinepalladium were added and the mixture was refluxed
under nitrogen for 15 h. The reaction mixture was analysed by TLC
(dichloromethane/methanol/ammonia: 87/13/5) and showed a newly
formed product (Rf=0.65). The mixture was purified on a silica column
(silica, 200 Â 30 mm) using dichloromethane/methanol (99/1) as eluent.
The eluted fractions were collected, a yellowish, oily substance was
obtained (2.8 g yield=71%).
1H-NMR (d1-CHCl3): 7.5-7.7 (dd, 4 H, ArH), 7 (m, 4 H, ArH), 3.9 (s,
3 H, OCH3), 3.1 (t, 2 H, -CH2-NH-CO), 2.7 (s, 4 H, piperazineH), 2.9
(m, 4 H, piperazineH), 2.8 (t, 2 H, N-CH2-CH2), 1.3–1.5 (2q, 4 H, -CH2-
CH2-), 0.8–0.9 (t, 27 H, tributylstannyl). EXACT MASS m/z: 658.3392
([M+H]+) (calculated: 658.3394).
Synthesis of 4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-
benzamide (1)
4-(4-(2-methoxyphenyl)-piperazin-1-yl)-butylamin (3) (1.00 g, 3.80 mmol)
was dissolved in dichloromethane (10 ml). Triethylamine (0.61 g,
6.03 mmol) was added and the temperature of the mixture was cooled
to 08C. 4-Iodobenzoylchloride (1.14 g, 4.28 mmol) was dissolved in
dichloromethane (0.8 ml) and added dropwise at such a rate as to
maintain the temperature below 108C. Then the mixture was stirred at
room temperature for 2 h. The reaction mixture was diluted with
dichloromethane (100 ml) and washed with a saturated sodium
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L. STAELENS ET AL.
bicarbonate solution. The organic layer was then washed with water,
dried with anhydrous sodium sulphate and the solvent was removed
under reduced pressure. TLC analysis (dichloromethane/methanol/
ammonia: 87/13/5) of the mixture showed a newly formed product
(Rf=0.8). Purification was conducted on a silica column (silica,
200 Â 30 mm) starting with pure dichloromethane and gradually (steps
of 0.5%, 300 ml of each eluent) changing to dichloromethane/methanol
(98/2). The eluted fractions were evaporated, a white solid was obtained
(1.5 g yield=88%). 1H-NMR (d6-DMSO): 7.9–8.0 (dd, 4 H, ArH), 6.7–
7.1 (m, 4 H, ArH), 3.9 (s, 3 H, OCH3), 3.7 (t, 2 H,-CH2-NH-CO), 3.3 (m,
4 H, piperazineH), 2.9 (m, 4 H, piperazineH), 2.8 (t, 2 H, N-CH2-CH2),
1.6–1.8 (2q, 4 H, -CH2-CH2-). EXACT MASS m/z: 494.1309 ([M+H]+)
(calculated: 494.1306).
Synthesis of [123I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)-
butyl)-benzamide (123I-1)
The iodination was conducted by electrophilic aromatic substitution of
the tributylstannyl derivative (5). N-(4-(4-(2-methoxyphenyl)-piperazin-
1-yl)butyl)-4-tributylstannyl-benzamide (5) (650 mg, 1 mmol) was dis-
solved in ethanol (50 ml). n.c.a. [123I]NaI in sodium hydroxide solution
(15 mL 0.01 M), chloramine T (282 mg, 1 mmol) and glacial acetic acid
(5 ml) were added. The mixture was stirred and left to react for 10 min at
room temperature. Afterwards an aqueous solution of sodium
metabisulphite (15 ml 285 mg, 1.5 mmol) was added to quench the
reaction.9 The mixture was purified by HPLC with ethanol/acetate
buffer (0.05 M, pH 5) (35/65) as mobile phase at a flow rate of 1 ml/min.
The radiolabelled product was collected (Rt=16 min) and analysed with
the same HPLC system (see Figure 1).
Determination of the lipophilicity
Lipophilicity (log P) was determined by adding 100 mCi of the labelled
compound to a mixture of 100 ml water and 100 ml octanol. After this
mixture was shaken thoroughly, 50 ml of the octanol was taken and
another 50 ml water is added. The mixture was shaken again and 1 ml of
both the water and the octanol were counted for radioactivity. Log P
was calculated by dividing the counts in the octanol by the counts in the
water.
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A POTENTIAL DOPAMINE D3 ANTAGONIST
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Figure 1. Radiochromatogram of a [123I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-pi-
perazin-1-yl)butyl)-benzamide (123I-1) synthesis
Results and discussion
Synthesis of the precursor molecule and the cold product is shown in
Scheme 1. These molecules were synthesized by coupling 1-(2-
methoxyphenyl)-piperazine and N-4-bromobutylphtalimide followed
by deprotection of the formed phtalimide moiety using hydrazine
hydrate. The primary amine was then coupled to the appropriate
Scheme 1. Synthesis of N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-4-tri-
butylstannyl-benzamide and 4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)-
butyl)-benzamide (I) sodium carbonate and dimethylformamide, (II) hydrazine
hydrate and ethanol, (III) 4-bromobenzoylchloride, triethylamine and dichlor-
omethane, (IV) hexabutyiditin, tetrakistriphenylphosfinepalladium and toluene,
(V) 4-iodobenzoylchloride, triethylamine and dichloromethane
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L. STAELENS ET AL.
halogen benzoylchloride. The iodinated product was synthesized in
good overall yield of about 53%. Further reaction of the bromine
compound with hexabutylditin in the presence of tetrakistriphenyl-
phosfinepalladium gave the tributylstannyl precursor. The overall yield
for the precursor synthesis was about 38%.
The radiolabelling of the precursor molecule was conducted by
electrophilic iododestannylation of the tributylstannyl precursor as
shown in Scheme 2 and gave a radiochemical yield of 82–85%. The
radiochemical purity of the collected fraction was >95%. Since no UV
signal was obtained from the synthesized product specific activity was
obtained by determination of the detection limit of the UV detector, this
showed the specific activity to be >2.96 Ci/mmol. Stability was tested by
.
leaving the product in solution at room temperature and reınjecting it
into the HPLC system. Up to 48 h after synthesis the radiochemical
purity remained >95%. The total amount of radioactivity obtained was
up to 10 mCi, sufficient to perform SPECT studies in human volunteers.
The log P value was measured to be 2.2.
Scheme 2. [123I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-benza-
mide. (VI) [123l]-NaI, chloramine T, acetic acid, sodiummetabisulphite and
ethanol
Conclusion
We reported the synthesis and radiolabelling of [123I] 4-iodo-N-(4-(4-(2-
methoxyphenyl)-piperazin-1-yl)butyl)-benzamide as a potential radio-
tracer for in vivo visualization of the dopamine D3 receptor antagonist.
The compound was labelled in a good yield (82–85%) and high specific
activity (>2.96 Ci/mmol) and will be evaluated by means of in vitro and
in vivo experiments.
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