2-Amino-3-(1-(4-(4-(2-methoxyphenyl) piperazine-1-yl)butyl)-1H-1,2,3- triazol-4-yl) propanoic acid: Synthesized, 99mTc-tricarbonyl labeled, and bioevaluated as a potential 5HT1A receptor ligand

Article abstract of DOI:10.1002/jlcr.2953

To develop a novel 5HT_1A receptor imaging agent, a new methoxyphenyl piperazine derivative was synthesized and radiolabeled with ^99mTc-tricarbonyl precursor. We used the Cu (I)-catalyzed cycloaddition of azide and terminal alkynes to

Full text of DOI:10.1002/jlcr.2953

Research Article
Received 15 March 2012,
Revised 1 July 2012,
Accepted 10 July 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2953
2-Amino-3-(1-(4-(4-(2-methoxyphenyl)
piperazine-1-yl)butyl)-1H-1,2,3-triazol-4-yl)
propanoic acid: synthesized, 99mTc-tricarbonyl
labeled, and bioevaluated as a potential 5HT1A
receptor ligand
Leila Hassanzadeh,a Mostafa Erfani,b and Seyed Esmaeil Sadat Ebrahimia
To develop a novel 5HT1A receptor imaging agent, a new methoxyphenyl piperazine derivative was synthesized and
radiolabeled with 99mTc-tricarbonyl precursor. We used the Cu (I)-catalyzed cycloaddition of azide and terminal alkynes to
synthesize 1, 2, 3 triazole as the metal chelating system. This synthesis provided reliable and reproducible method to attach
technetium to the methoxyphenyl piperazine moiety. 99mTc-tricabonyl labeling of ligand was performed at high radiochemical
purity (greater than 95%). The radiolabeled compound was stable at least 24h in room temperature. In vitro stability study in
human serum albumin showed more than 90% stability in 37ꢀC incubation for 6 h. Biodistribution studies in rat have shown
brain hippocampus uptake of 0.31Æ 0.02%ID/g at 5-min post-injection. The favorable in vitro/in vivo stability, lipophilicity,
and biodistribution profiles suggest that this radioconjugate is a good candidate for further exploration of its potential
clinical application.
Keywords: methoxyphenyl piperazine; 99mTc-tricarbonyl; click chemistry; 5HT1A receptor
the classic way with COgas and borohydride reduction or with
Introduction
an IsoLink kit.33,34 In the resulting precursor, the three water
The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) is
molecules can easily be replaced by ligands that contain suitable
involved in numerous central nervous system functions and psy-
donor atoms to form stable complexes.
chiatric disorders.1,2 The receptors that are activated by 5-HT
The click chemistry that is a Cu (I)-catalyzed cycloaddition of
have been divided into different classes which of these recep-
azides and terminal alkynes has only recently found applications
in the design of ligands for transition metals.22–30 This catalyzed
tors, the 5HT1A receptors have been implicated in the pathogen-
esis of depression, anxiety, schizophrenia, epilepsy, and eating
cycloaddition give high chemical yield under mild reaction
disorders.3 WAY100635 is a well-known high-affinity 5HT1A
conditions even in aqueous media. It is efficient, selective,
receptor antagonist.4,5 Many 11C and 18F radiolabeled derivatives
regiospecific, and devoid of side reactions. Their organometallic
99mTc (CO)3 complexes proved to be stable in in vivo studies.
of WAY100635 have been synthesized and evaluated for use in
positron emission tomography,6–9 Whereas its iodinated analogs
This reaction allows simultaneous formation of the chelating sys-
have been reported for use in single-photon emission computed
tem and conjugation to a biomolecule in a single high-yielding
tomography imaging of 5HT1A receptors.10,11
step. Unexpectedly, few examples of 1,2,3-triazole chelators are
Because 99mTc is the radionuclide of choice in diagnostic
nuclear medicine, many efforts have been focused on develop-
ing 99mTc-based radioligands for 5HT1A receptors. The successful
development of 99mTc-TRODAT as a radioligand for the dopa-
mine transporter has shown the feasibility of imaging specific
transporters in the brain with radiotracers based on 99mTc.12,13
Accordingly, many various radioligands have been made by
several groups to develop 99mTc complex for 5HT1A receptor
imaging,14–20 but there is no ideal 99mTc agent for imaging
central nervous system receptors.21 Over the last years, the radio-
chemistry of 99mTc-tricarbonyl complexes has been extended to
find useful applications in radiochemistry and nuclear medicine.
The basis for this kind of radiochemistry is the convenient prepara-
reported that were obtained via click chemistry,26,31 even though
triazoles are known to be potent ligand systems for various
transition metals.32
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of
Medical Sciences, PO Box 6446-14155, Tehran, Iran
bNuclear Science Research School, Nuclear Science & Technology Research Institute
(NSTRI), Atomic Energy Organization of Iran (AEOI), Karegar Avenue, PO Box 11365-
3486, Tehran, Iran
*Correspondence to: Mostafa Erfani, Nuclear Science Research School, Nuclear
Science & Technology Research Institute (NSTRI), Atomic Energy Organization
of Iran (AEOI), Karegar Avenue, PO Box 11365-3486, Tehran, Iran.
tion of the Tc-tricarbonyl precursor [99mTc(CO)3(H2O)3]+ either in E-mail: mgandomkar@aeoi.org.ir
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.

L. Hassanzadeh et al.
The present study demonstrates the application of click chem- The 99mTc (CO)3-triazole complex (4) has been characterized by
istry in the preparation of an organometallic 99mTc complex of a comparison with the corresponding rhenium complex (5), which
methoxyphenyl piperazine derivative, the pharmacophore moi- has been synthesized using [NEt4] [Re (CO) 3Br3] precursor using
ety of WAY100635. Methoxyphenyl piperazine has been deriva- conditions similar to 99mTc labeling. The ultraviolet-HPLC chro-
tized to 1-(3-azidopropyl)-4-(2-methoxyphenyl) piperazine that matogram retention time of rhenium complex (Figure 2c) was
undergoes [3 + 2] cycloaddition reaction with propargyl glycine. observed to be 18.62 min, which matches well with 18.82 min
The 1, 2, 3-triazole derivative thus formed has been radiolabeled g-HPLC chromatogram retention time of 99mTc (CO)3 complex
+
with [99mTc (CO) (H2O) ] core. And the properties of radioli- (4) (Figure 2b). The complex showed a retention time of
3
3
gand have been investigated by carrying out suitable in vitro 18.82 min that was separated from the [99mTc(CO)3(H2O)3]+ elut-
and in vivo studies.
ing out at 17.54 min. Because of closeness of their retention time
for both compounds, further investigation was performed. Corre-
sponding to this, after a 24-h period, the reaction mixture was
re-injected to HPLC, but no further [99mTc (CO)3(H2O)3]+ peak
was observed. This is a further indication that this observed peak
belongs to desired labeled ligand. According to the literature,
the labeled compound should possess a relatively small size with
lipophilic characteristic to cross the blood–brain barrier.10 The
calculated partition coefficient (log Poct/wat) for 99mTc (CO)3 com-
plex was 0.34 Æ 0.02 that seems not to be lipophilic enough to
cross blood–brain barrier; however, biodistribution experiments
showed a moderate brain uptake (Table 2).
Results and discussion
The 1-(2-methoxyphenyl) piperazine unit of WAY100635 is a
moiety that has favorable affinity toward 5HT1A receptors and
was chosen as the parent molecule for derivatization to an azide
in preparation of a clicked 1, 2, 3-triazole. Because of good results
in previous studies, propyl spacers were used for linkage of 1-(2-
methoxyphenyl) piperazine.9 The 1-(2-methoxyphenyl) pipera-
zine was reacted with 3-bromo-1-propanol as the alkylation
agent to prepare 3-(4-(2-methoxyphenyl) piperazine-1yl)propa-
nol 1. The reaction was carried out in anhydrous CH3CN in the
presence of K2CO3 by refluxing overnight (Figure 1). By treating
compound 1 with methanesulfonyl chloride in the anhydrous
CH2Cl2 in the presence of Et3N, the methyl 3-(4-(2-methoxyphe-
nyl) piperazine-1-yl) propane-1-sulfonate 2 was obtained. Com-
pound 2 was converted to an azide in the presence of NaN3
and it was ready to click reaction via azide complex.
The radioligand was found to be stable even after a 24-h
period. In serum stability studies, nearly 4.36 Æ 0.09% of the
activity was associated with the precipitate obtained after ethanol
500 mV
190
450
400
350
300
250
200
150
100
50
All the intermediates and final product have been character-
ized by infrared (IR), NMR, and ESI-mass spectroscopy (MS).
1
In H NMR, a downfield shift has been observed for C-1 proton
from d 3.85–3.83 ppm (corresponding to compound 1) to d
4.38–4.35 ppm, indicating the desired mesylation of compound
1. The SN2 azidation reaction was performed completely. Corre-
sponding to this in IR spectra, the stretching band at 2095 cmÀ1
(characteristic of N3 functionality) indicated the desired derivati-
zation. In addition, the C-1 proton shift from d 4.38–4.35 ppm in
mesylated compound to d 3.39–3.37 ppm in azide compound
was a further confirmation of azidation reaction.
0
(a)
0.00
30.0 mV *1000
25.0
5.00
10.00
15.00
20.00
25.00
min
190
On the basis of click reaction, a one-pot procedure was chosen
for labeling the compound 3 with [99mTc (CO) 3(H2O) 3] + precur-
sor. The (3 + 2) cycloaddition and labeling was performed using
compound 3, propargyl glycine, Cu(OAc)2, sodium ascorbate,
and [99mTc(CO)3(H2O)3]+ that were heated in 100 ꢀC (Figure 1).
20.0
15.0
10.0
5.0
O
CH3
OMe
N
OMe
N
OMe
N
(b)
S
0.0
0.00
(a)
(c)
O
N
N
(b)
OH
5.00
10.00
15.00
20.00
25.00
min
NH
O
2
1
OMe
OMe
(d)
N+
O
N
N
N
N-
N
N
N
N
N
NH
O
3
Tc
4
OC
CO
CO
(c)
Figure 1. Synthesis of radioligand. Reagents: (a) 3-bromo-1-propanol and K2CO3,
CH3CN; (b) CH3SO2Cl, Et3N, and CH2Cl2; (c) NaN3 and dimethylformamide; and (d)
propargyl glycine, [99mTc(CO)3(H2O)3]+, copper acetate, sodium ascorbate, and
tBuOH/H2O.
Figure 2. HPLC profile of (a) [99mTc(CO)3(H2O)3]+ , (b) 99mTc (CO)3-triazole complex,
and (c) Re(CO)3-triazole complex.
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012

L. Hassanzadeh et al.
addition, indicating the low binding of complex with serum
proteins. The ethanol fraction was characterized by HPLC where
a single peak was observed at the same time (18.82min, >90%)
as that of the complex, indicating no decomposition of the
complex and therefore its stability under 37ꢀC incubation.
Biological evaluation of radiolabeled ligand was performed in
rat. The results are shown in Table 1. In biodistribution studies,
clearance from the blood circulation was fast. The remaining
activity in the blood decreased from 2.22 Æ 0.35 %ID/g at 2 min
to 0.42 Æ 0.20 %ID/g at 30 min. Whole-body clearance showed
significant accumulation of activity in kidneys and liver 2 min
after injection (7.56 Æ 1.09 and 6.15 Æ 1.04 %ID/g, respectively).
Within 30 min, activity decreased in kidney and liver with a mod-
erate clearance (3.51 Æ 0.47 and 4.11 Æ 0.59 %ID/g, respectively).
This favorable clearance is probably due to the balance in lipo-
philicity for this radioligand and indicates both renal excretion
and hepatobiliary metabolism for tracer. The distribution of the
activity in different parts of rat brain regions showed mild overall
accumulation of radiotracer in the brain (Table 2). The radioactiv-
ity concentration of hippocampus and cerebellum at 5-min post-
injection was 0.31 Æ 0.02 and 0.10 Æ 0.01 %ID/g, respectively
(Figure 3). The uptake in cortex was 0.25 Æ 0.01 %ID/g at 5 min.
At 30-min post-injection, the retention of activity in hippocam-
pus was observed (0.26 Æ 0.05 %ID/g), whereas the uptake in
cerebellum decreased gradually (0.05 Æ 0.01 %ID/g). The cortex
activity remained almost constant (0.19 Æ 0.01 %ID/g). The results
showed that distribution of activity in different parts of the brain
Figure 3. Uptake of radioactivity in regions of the brain in rat 5-min post-injection
of 99mTc (CO)3-triazole complex.
was correlated with the location of 5HT1A receptors in the brain.
The retention of activity in hippocampus could be caused by
specific binding of the radioligand to the 5HT1A receptors and
tolerable lipophilicity of this complex (becauseof a 3-carbon length
spacer and carbonyl itself as a precursor).
In blocking investigation, the uptake in different parts of the
brain was decreased, which for hippocampus and cortex was
statistically significant (P < 0.05) (0.31–0.15 and 0.25–0.16%,
Table 1. Biodistribution of 99mTc-methoxyphenyl piperazine-triazole (with 3-carbon spacer) in normal rat (%ID/g Æ standard
deviation, n = 3)
Post-injection time (min)
Organs
2
5
5 block
15
30
Blood
Kidney
Spleen
Intestine
Liver
2.22 Æ 0.35
7.56 Æ 1.09
0.99 Æ 0.13
2.82 Æ 0.52
6.15 Æ 1.04
1.83 Æ 0.39
1.47 Æ 0.17
2.02 Æ 0.4
1.95 Æ 0.42
9.98 Æ 1.45
1.23 Æ 0.18
1.31 Æ 0.08
5.59 Æ 0.91
1.43 Æ 0.25
0.85 Æ 0.37
0.97 Æ 0.25
2.52 Æ 0.21
2.04 Æ 0.25
3.36 Æ 0.12
4.47 Æ 0.53
0.87 Æ 0.07
0.63 Æ 0.24
0.42 Æ 0.2
9.34 Æ 1.21
1.15 Æ 0.28
1.76 Æ 0.54
6.51 Æ 0.99
1.17 Æ 0.23
0.92 Æ 0.15
3.51 Æ 0.47
1.62 Æ 0.12
6.73 Æ 1.27
4.11 Æ 0.59
0.78 Æ 0.12
0.58 Æ 0.10
Lung
Heart
Table 2. Biodistribution of 99mTc-methoxyphenyl piperazine-triazole (with 3-carbon spacer) in different regions of the brain in
normal rat (%ID/g Æstandard deviation), n = 3
Region of brain
2 min
5 min
5 mina
15 min
30 min
Cortex
0.22 Æ 0.04
0.26 Æ 0.02
0.13 Æ 0.02
0.12 Æ 0.01
0.15 Æ 0.01
2
0.25 Æ 0.01
0.31 Æ 0.02
0.10 Æ 0.01
0.13 Æ 0.06
0.11 Æ 0.05
3.1
0.16 Æ0.02b
0.15 Æ 0.04b
0.11 Æ 0.02
0.09 Æ 0.01
0.09 Æ 0.03
1.36
0.22 Æ 0.06
0.20 Æ 0.02
0.09 Æ 0.03
0.10 Æ 0.02
0.09 Æ 0.01
2.2
0.19 Æ 0.01
0.26 Æ 0.05
0.05 Æ 0.01
0.08 Æ 0.06
0.08 Æ 0.06
5.2
Hippocampus
Cerebellum
ROB
Brain
Hippocampus/cerebellum
Brain/blood
0.07
0.06
0.05
0.09
0.19
ROB, rest part of the brain except cortex, hippocampus, and cerebellum.
ablocked,
bTwo-tailed t-test showed statistically significant difference in 5-min post-injection between cortexes and hippocampus blocked
and unblocked in rats brain.
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Copyright © 2012 John Wiley & Sons, Ltd.
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L. Hassanzadeh et al.
respectively). The clearance of nonspecific uptake from brain and Synthesis of 1-(3-azidopropyl)-4-(2-methoxyphenyl)
specific affinity to 5HT1A receptors in brain could be the main piperazine (3)
reason for the blocking results. There were no significant
To a stirred solution of compound 2 (189.1 mg, 0.69 mmol) in 10-ml dry
changes in activity of other major organs with blocking agent.
To determine the affinity of the 99mTc (CO)3-triazole complex
toward binding with 5HT1A receptor, the in vitro studies were
carried out in rat homogenized hippocampus membrane at
37 ꢀC temperature. The percentage of binding of the complex
dimethylformamide, sodium azide (138 mg, 2.07 mmol) was added, and
the stirring was continued at 100 ꢀC overnight. Water was then added
to the reaction mixture and the aqueous phase extracted with diethyl
ether (3 Â 10 ml). The organic extract was washed with water, brine,
and dried over Na2SO4. The solvent was removed under vacuum, and
in membrane receptors was found to be 39 Æ 2.64% when the product was purified by silica-gel column using 1% MeOH/CH3Cl as
the eluting solvent. Yield: 87% colorless crystals 1H NMR (500 MHz,
100 ml (6.6 MBq) of HPLC purified tracer was added. The binding
in competition studies in the presence of 0.2 nM of 5HT1A full
CDCL3) d: 7.03–6.86 (m, 4H), 3.87 (s, 3H), 3.39–3.37 (t, 2H), 3.11 (broad s,
4H), 2.66 (broad s, 4H), 2.50–2.52 (t, 2H), 1.86–1.80 (m, 2H). IR (KBr, n cmÀ1):
agonist 8-OH-DPAT was 13 Æ 0.89%. The results show the non-
2095. ESI-MS m/z: 275 [M] (calcd for C14H21N5O: 275.17).
specific binding in the presence of blocking agent is less than
35% of total binding. On the other hand, the binding for 99mTc
Preparation of [99mTc (CO)3(H2O)3] + precursor
(CO)3-triazole complex is receptor mediated and specific.
+
The precursor [99mTc (CO)3(H2O)3]
was prepared according to the
reported procedure.33 In a closed vial, 4.5 mg Na2CO3, 5.5 mg NaBH4,
and 20-mg sodium potassium tartrate were added, and the vial was
flushed with COgas, and after that, the 30-mCi activity that was eluted
from a commercial 99Mo/99mTc generator was added and heated to
90 ꢀC for 30 min. After cooling the vial to room temperature, pH was
neutralized to 7 through adding HCl (1 N). Radiochemical purity of the
precursor was checked by RP-HPLC.
Experimental
All reagents were obtained from known commercial sources such as
Sigma-Aldrich (Munich, Germany) and Fluka (Munich, Germany) and
used without further purification. The 99mTcO4À was eluted from an in-
house 99Mo/99mTc column generator using 0.9% saline. IR spectra were
recorded as KBr pellets on Perkin Elmer (Shelton, USA) spectrometer.
High-resolution fast bombardment MS was performed using an Agilent
1100 (Alto, USA)/Bruker Daltonic (Ion trap) VL (Bremen, Germany) instru-
ment. 1H NMR spectra were obtained on Bruker 500 MHz instrument
using CDCl3 as the solvent.
Preparation of 99mTc (CO)3-1-(3-azidopropyl)-4-
(2-methoxyphenyl) piperazine (4)
Monitoring of all reactions was performed with analytical reversed-
phase HPLC (RP-HPLC) on a JASCO 880-PU intelligent pump HPLC system
(Tokyo, Japan) equipped with a multiwavelength detector and a flow-
through Raytest GABI g-detector. CC 250/4.6 Nucleosil 120–5 C-18 col-
umn from Teknokroma (Barcelona, Spain) was used for HPLC. The HPLC
solvents consisted of 0.1% trifluoroacetic acid/water (solvent A) and
acetonitrile (solvent B). The followed gradient program was used in HPLC
system: 0 min 95% A, 5 min 95% A, 25 min 0% A, 27 min 0% A, 30 min
95%A, flow = 1 ml/min, g = 280 nm.
Compound 3 (5 mg, 1.6 Â 10À2 mmol) was dissolved in tBuOH/H2O, then
aqueous solutions of propargyl glycine (100 ml, 0.1 M), copper acetate
(20 ml, 0.01 M) and sodium ascorbate (40 ml, 0.01 M) were added. The
[
99mTc (CO)3(H2O)3]+ precursor (100 ml, 10 MCi) was added in the reaction
mixture. The reaction mixture was heated at 100 ꢀC for 1h. The complex
prepared was characterized by HPLC.
Preparation of Re (CO)3-1-(3-azidopropyl)-4-(2-methoxy phenyl)
piperazine (5)
Quantitative gamma counting was performed on an ORTEC model
4001 M g-system (London, England) well counter.
Compound 3 (5 mg, 18 mmol), [NEt4]2[ReBr3 (CO)3] (13.5 mg, 18 mmol),
propargyl glycine (2.03 mg, 18 mmol), copper (II) acetate (101mg, 0.56mmol),
and sodium ascorbate (112 mg, 0.56 mmol) were dissolved in t-butanol/H2O
(1 : 1, 2 ml). The reaction mixture was incubated in a sealed vial at 100 ꢀC
for 1 h. The [NEt4]2[ReBr3 (CO)3] precursor was prepared from rhenium
pentacarbonyl bromide [Re(CO)5Br], using a published procedure.35
The compound 5 was characterized by ESI-MS m/z: 657 [M] (calcd for
C22H26N6O6Re: 657.15).
Synthesis of 3-(4-(2-methoxyphenyl) piperazine-1yl)
propanol (1)
A 1-(2-Methoxyphenyl) piperazine (0.96 g, 5 mmol) and 3-bromo-1-
propanol (1.04 g, 7.50mmol) were dissolved in anhydrous CH3CN (20 ml).
K2CO3 (1.04 g, mmol) was added, and the mixture was heated under reflux
overnight. The solid phase was removed by filtration. The product was
purified on a silica-gel column using MeOH/CH2Cl2 1: 15 as the eluting
solvent. Yield: 95.2%; white solid; 1H NMR (CDCl3) d: 7.03–6.86 (m, 4H), 3.87
(s, 3H), 3.85–3.83 (T, 2H), 3.11 (broad s, 4H), 2.76 (broad s, 4H), 2.73–2.71
(T, 2H), 1.80–1.75 (m, 2H).
Partition coefficient and in vitro stability study
The partition coefficient of the complex was determined by measuring
the activity that partitioned between the 1-octanol and water. A 1-ml
1-octanol and 1-ml water and labeled complex (100 ml, 89 mCi) were
mixed in a centrifuge tube. The mixture was vortexed at room tempera-
ture for 5 min and then centrifuged at 2000 r/min for 5 min. The counts in
0.1-ml samples of both organic and inorganic layers were determined by
Synthesis of methyl 3-(4-(2-methoxyphenyl) piperazine-1-yl)
propane-1-sulfonate (2)
To the solution of 1 (625 mg, 2.51 mmol) and Et3N (419.3 ml, 3.01 mmol) in a well-type gamma counter. The measurement was repeated three times.
CH2Cl2 (25 ml), cooled to 0–5 ꢀC, was added dropwise with stirring a The partition coefficient (P) was calculated using the following equation.
solution of methane sulfonyl chloride (193.4 ml, 1.26 mmol) for 30 min. P = (count per minute in octanol–count per minute in background)/
The reaction mixture was then stirred at same temperature and was (count per minute in water–count per minute in background). The final
monitored with thin layer chromatography to complete the reaction. partition coefficient value was expressed as log P.
The reaction mixture was washed with cold water, cold 10% HCl solution,
The stability of 99mTc (CO)3 complex was assayed by monitoring the
NaHCO3 (sat.), and NaCl (sat.) and then dried with Na2SO4. The solvent HPLC elution profile and determined the radiochemical purity after
was then evaporated in vacuum, and the residue was purified by column incubation at room temperature for 24 h. To determine the in vitro serum
chromatography on silica-gel using 1 : 15 MeOH/CH2Cl2 as eluting stability, 100 ml of radiolabeled complex was incubated with 1-ml human
solvent. Yield: 95%; slightly yellow oil; 1H NMR (500 MHz, CDCl3) d: serum at 37 ꢀC for 24 h. A 1-ml ethanol was added to the aforementioned
7.06–6.85 (m, 4H), 4.38–4.35 (T, 2H), 3.87 (s, 3H), 3.80–3.55 (T, 2H), 3.51 solution. The precipitate was separated by centrifugation. The supernatant
(broad s, 4H), 3.15 (broad s, 4H), 3.05 (s, 3H), 2.32–2.26 (M, 2H).
was injected in HPLC to determine the stability of the complex.
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012

L. Hassanzadeh et al.
Biodistribution study
Acknowledgements
Animal experiments were performed in compliance with the regulations
of our institution and with generally accepted guidelines governing such
work. A group of three rats (220–280 g) received 20 MBq of high specific
activity radiotracer in 0.3 ml of buffered saline via a tail vein. The radiola-
beled complex was purified by RP-HPLC and collected in a vial. The
solvent was removed under vacuum. The radioligand was diluted by
saline for injection. The HPLC chromatogram revealed any decomposition
of complex. Groups of three rats per time point with results expressed
(mean Æ standard deviation) as the percentage injected dose per gram of
wet tissue (%ID/g). For blocking studies, rats received a tail vein injection
of a solution (50 ml) of 8-OH-DPAT the putative blocker (2 mg/kg) 1 min
before administration of the radiotracer and were dissected. The brain
was rapidly removed, chilled, and dissected. Samples from different brain
regions (cortex, hippocampus, and cerebellum) were collected, weighed,
and counted.
The authors wish to thank Ms. Zoghi, Mr. Mirfallah, and Mr. Talebi
of the Radioisotope Division (AEOI) for their assistance.
Conflict of Interest
The authors did not report any conflict of interest.
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Conclusion
In this study, a new methoxyphenyl piperazine derivative was
successfully synthesized and radiolabeled in high yield specific
activity (23.13 MBq/mmol) by [99mTc (CO)3(H2O)3]+ precursor
via click chemistry as a potential 5HT1A receptor imaging agent.
Preliminary biological evaluation showed moderate brain
uptake. Accumulation in the different parts of the brain in rat,
followed by excretion via the kidney and liver, were observed
for the prepared conjugate. Receptor binding studies indicated
specific binding in rat brain membrane. It would be best to
modify the complex to increase the lipophilicity and respec-
tively brain uptake.
J. Label Compd. Radiopharm 2012
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