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.683

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.683

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 411–419.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.683
Research Article
1
23
Synthesis and radiolabelling of [ I]-4-iodo-
N-(4-(4-(2-methoxyphenyl)-piperazin-1-
yl)butyl)-benzamide, a potential dopamine
D antagonist for SPECT studies
3
1
,
1
2
1
L. Staelens *, F. Dumont , F. De Vos , R. Oltenfreiter ,
1
M. Vandecapelle , R. A. Dierckx and G. Slegers
2
1
1
Department of Radiopharmacy, Ghent University, Harelbekestraat 72,
000 Gent, Belgium
9
2
Division of Nuclear Medicine, Ghent University Hospital, De Pintelaan 185,
000 Gent, Belgium
9
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.
1
23
Therefore, a new
I-labelled compound was developed which may allow in
1
23
vivo visualization of the D3 receptor by SPECT. [ I]-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; D receptor; SPECT; radiotracer
3
*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 3 October 2002

4
12
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 D
1
2
,2
receptor.
The dopamine receptors have been divided into five
subtypes based on molecular biology data, the D -like receptors D
1
1
and D and the D -like receptors D , D and D . The latter class has
5
2
2
3
4
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 D receptors in the striatum than typical
2
neuroleptics although their antipsychotic efficacy is equal to that of
3
typical neuroleptics.
Experimental studies have already shown a predominant action of
atypical neuroleptics in the limbic regions. D , D and D subtypes of
3
4
5
dopamine receptors seem to be predominantly located in the limbic
system and cortical areas. Atypical neuroleptics have a more selective
4
action on these receptor subtypes than do typical neuroleptics.
1
In the past several radiotracers for the D -like receptors like [ I]-
23
2
1
23
11
11
11
epidepride, [ I]-IBZM, [ C]-raclopride, [ C]-nemonapride and [ C]-
methylspiperone have been developed; however these showed no
5
,6
selectivity between the different receptors of this class.
Very few
radiotracers have been developed which show selectivity for the
7
dopamine D receptor like 7-OH-PIPAT.
3
The aim of this study was to synthesize a tracer for SPECT studies of
the dopamine D receptor. In order to develop a tracer for these
3
receptors a number of demands have to be met. First of all, the tracer
needs to have high affinity for the dopamine D receptor due to the low
3
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
1
23
avoid non-specific binding. Selection of [ I]-4-iodo-N-(4-(4-(2-meth-
oxyphenyl)-1-piperazinyl) butyl)-benzamide as a potential tracer for the
8
D receptor was based upon data collected by Murray et al. These data
3
indicate that the corresponding bromated derivative has high affinity for
the D receptor (pK ¼ 9:3) and shows a 100-fold selectivity over the D
3
i
2
(
pK ¼ 7:4) and D4 (pK ¼ 7:3) receptors. Apart from dopamine
i
i
receptors there is some interaction with the 5-HT
8
receptor
1
A
(
pK ¼ 7:9) and the a receptor (pK ¼ 7:9). From these data, we can
i
1
i
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 411–419

A POTENTIAL DOPAMINE D
3
ANTAGONIST FOR SPECT STUDIES
413
assume that the iodinated derivative will also show selectivity for the
D3 receptor moreover since antagonists with larger groups in
the same position also show D3 selectivity (e.g. aminobenzyl:
8
pK D ¼ 9:7; pK D ¼ 7:7).
i
3
i
2
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-d , acetone-d or methyl-
3
6
sulfoxide-d . High resolution mass spectroscopy was performed using a
6
Micromass Q-Tof-2 mass spectrometer (Rega Institute for Medicinal
123
Research, Leuven University). [ I] 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
scaler ratemeter. The column used was a reverse-phase base-deactivated
column (Nucleosil C18 BDS 4.6 ꢀ 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)-iso-
indole-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,
2 mmol) was pulverized and added, the mixture was heated to 808C
1
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 R 0.64 was
f
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 411–419

4
14
L. STAELENS ET AL.
observed. The reaction mixture was then purified by extracting with
ethyl acetate (100 ml) and the organic layer was washed with water
(2 ꢀ 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 (R : 0.9) and two newly
f
formed products (R : 0.5 and 0.2). The mixture was purified by column
f
chromatography (silica, 200 ꢀ 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 R
f
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.
1
H-NMR (d -DMSO): 6.8-7.0 (m, 4 H, ArH), 3.9 (s, 3 H, OCH ), 3.2
6
3
(m, 4 H, piperazineH), 2.8 (t, 2 H, -CH -NH -), 2.6 (m, 4 H,
2 2
piperazineH), 2.4 (t, 2 H, N-CH -CH ), 1.8-2.0 (2q, 4 H, -CH -CH -).
2
2
2
2
+
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,
0.68 mmol) was added and the temperature of the mixture was cooled
1
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,
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 411–419

A POTENTIAL DOPAMINE D
3
ANTAGONIST FOR SPECT STUDIES
415
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 ꢀ 30 mm)
starting with pure dichloromethane and gradually (steps of 0.5%,
00 ml of each eluent) changing to dichloromethane/methanol (98/2).
3
The eluted fractions were evaporated, a white solid was obtained (2.7 g
yield = 90%).
1
H-NMR (d -DMSO): 7.8-7.9(dd, 4 H, ArH), 6.7-7.0 (m, 4 H, ArH),
6
3
.8 (s, 3 H, OCH ), 3.7 (t, 2 H, -CH -NH-CO), 3.3 (m, 4 H,
3 2
piperazineH), 2.9 (m, 4 H, piperazineH), 2.8 (t, 2 H, N-CH -CH ),
2
2
+
.6-1.8 (2q, 4 H, -CH -CH -). ESI-MS m/z: 446-448 ([M+H] ).
2 2
1
Synthesis of N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-4-tri-
butylstannyl-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). Hexabu-
tylditin (10.44 g) and a catalytic amount ( ꢁ 3 mg) of tetrakistriphenyl-
phosfinepalladium 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 (R 0.65). The mixture was purified on a silica column
f
(silica, 200 ꢀ 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%).
1
H-NMR (d -CHCl ): 7.5-7.7 (dd, 4 H, ArH), 7 (m, 4 H, ArH), 3.9 (s,
1
3
3
H, OCH ), 3.1 (t, 2 H, -CH -NH-CO), 2.7 (s, 4 H, piperazineH), 2.9
3 2
(m, 4 H, piperazineH), 2.8 (t, 2 H, N-CH -CH ), 1.3-1.5 (2q, 4 H, -CH -
2 2 2
CH -), 0.8-0.9 (t, 27 H, tributylstannyl). EXACT MASS m/z: 658.3392
2
+
[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)-butylamine (3) (1.00 g, 3.80 mmol)
was dissolved in dichloromethane (10 ml). Triethylamine (0.61 g,
.03 mmol) was added and the temperature of the mixture was cooled
6
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
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 411–419

4
16
L. STAELENS ET AL.
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/
ammonia: 87/13/5) of the mixture showed a newly formed product (Rf
0
.8). Purification was conducted on a silica column (silica, 200 ꢀ 30 mm)
starting with pure dichloromethane and gradually (steps of 0.5%,
00 ml of each eluent) changing to dichloromethane/methanol (98/2).
3
The eluted fractions were evaporated, a white solid was obtained (1.5 g
yield = 88%).
1
H-NMR (d -DMSO): 7.9-8.0 (dd, 4 H, ArH), 6.7-7.1 (m, 4 H, ArH),
6
3
.9 (s, 3 H, OCH ), 3.7 (t, 2 H, -CH -NH-CO), 3.3 (m, 4 H,
3 2
piperazineH), 2.9 (m, 4 H, piperazineH), 2.8 (t, 2 H, N-CH -CH ),
2
2
+
1
.6-1.8 (2q, 4 H, -CH -CH -). EXACT MASS m/z: 494.1309 ([M+H] )
2 2
(calculated: 494.1306).
123
Synthesis of [ I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)-
butyl)-benzamide ( I-1)
123
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-
123
solved in ethanol (50 ml). n.c.a. [ I]NaI in sodium hydroxide solution
(
15 ml 0.01 M), chloramine T (282 mg, 1mmol) 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. The mixture was purified by HPLC with ethanol/acetate
9
buffer (0.05 M, pH 5) (35/65) as mobile phase at a flow rate of 1 ml/min.
The radiolabelled product was collected (R : 16 min) (Figure 1) and
t
analysed with the same HPLC system.
123
Figure 1. Radiochromatogram of a [ I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-pi-
123
perazin-1-yl)butyl)-benzamide ( I-1) synthesis
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 411–419

A POTENTIAL DOPAMINE D
3
ANTAGONIST FOR SPECT STUDIES
417
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.
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
O
N
OCH3
NH
O
OCH3
N
N
Br
N
O
+
N
I
O
2
II
O
Br
NH
NH2
OCH3
N
OCH3
N
N
N
III
4
3
V
IV
O
O
Sn(Bu)3
NH
I
NH
OCH3
N
OCH3
N
N
N
5
1
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) hexabutylditin, tetrakistriphenylphosfinepalladium and toluene,
(V) 4-iodobenzoylchloride, triethylamine and dichloromethane
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 411–419

4
18
L. STAELENS ET AL.
O
O
123I
Sn(Bu)3
NH
NH
OCH3
N
OCH3
N
N
N
VI
123I-1
5
123
Scheme 2. [ I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-benz-
1
23
amide. (VI) [ I]-NaI, chloramine T, acetic acid, sodiummetabisulphite and
ethanol
by deprotection of the formed phtalimide moiety using hydrazine
hydrate. The primary amine was then coupled to the appropriate
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 re .ı njecting 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.
Conclusion
123
We reported the synthesis and radiolabelling of [ I]-4-iodo-N-(4-(4-(2-
methoxyphenyl)-piperazin-1-yl)butyl)-benzamide as a potential radio-
tracer for in vivo visualization of the dopamine D receptor antagonist.
3
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.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 411–419

A POTENTIAL DOPAMINE D
3
ANTAGONIST FOR SPECT STUDIES
419
References
1
2
3
. Creese I, Burt D, Snyder S. Science 1976; 192(4238): 481–483.
. Seemen P, Lee T. Science 1975; 188(4194): 1217–1219.
. Br u. cke T, Podreka I, Wenger S. J Cerebral Blood Flow Metab 1991; 11 (S2):
S877.
4
. Sokoloff P, Giros B, Martres M, Bouthenet M, Schwartz J. Nature 1990;
3
47: 146–151.
. Leslie WD, Abrams CR, Greenberg CR, Hobson D. J Nucl Med 1996; 37:
589–1591.
5
1
6
. Ishiwata K, Hayakawa N, Ogi N, et al. Ann Nucl Med 1999; 13, 161–167.
. Chumpradit S, Kung MP, Vessotskie JM, Kung HF. J. Label Compd
Radiopharm 1995; 36: 1051–1062.
7
8
. Murray PJ, Harrison LA, Johnson MR et al. Bioorg. Med Chem Lett 1995;
5
: 219–222.
. Pan D, Gatley SJ, Chen R, Ding Y-S. J Label Comp Radiopharm 1996; 6:
23–532.
9
5
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 411–419