N-[3-[4-(2-methoxyphenyl) piperaziny-1-yl]propyl]cyclam: Synthesized as a potential 5-HT1A receptor ligand and labelled with 99mTc-nitrido core

Article abstract of DOI:10.1002/jlcr.1532

This paper reports the synthesis of new potential 5-HT_1A receptor ligand N-[3-[4-(2-methoxyphenyl)piperaziny-1-yl]propyl]cyclam (MPPC) and radiolabelling of it with ^99mTc-nitrido core. The novel neutral complex ^99mTcN-MPPC

Full text of DOI:10.1002/jlcr.1532

Research Article
Received 30 January 2008,
Revised 16 June 2008,
Accepted 17 June 2008
Published online 3 September 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1532
N-[3-[4-(2-methoxyphenyl) piperaziny-1-
yl]propyl]cyclam: synthesized as a potential
5-HT1A receptor ligand and labelled with
99mTc-nitrido core
Ã
Fenglong Wang, Xuebin Wang , Shuye Yang, Jian Liu and Xianzhong Zhang
This paper reports the synthesis of new potential 5-HT1A receptor ligand N-[3-[4-(2-methoxyphenyl)piperaziny-1-yl]propyl]cy-
clam (MPPC) and radiolabelling of it with 99mTc-nitrido core. The novel neutral complex 99mTcN-MPPC combines 1,4,8,11-
tetraazacyclotetradecane (cyclam) ligand as chelate moiety for 99mTc-nitrido with a 1-(2-methoxyphenyl)piperazine moiety
derived from WAY 100635 via a 3-carbon alkyl chain. This provided a reliable and reproducible method for attaching the
technetium to the pharmacophore moiety of WAY 100635. 99mTcN-MPPC was prepared by a two-step procedure and the
radiochemical purity was found to be greater than 95%. It was hydrophilic and stable for at least
4 h at room temperature. In vivo stability study in normal rats showed that no degradation of 99mTcN-MPPC was found
in deproteinated blood samples at 2 h post-injection. This effective 99mTc-labelling strategy for obtaining neutral
99mTc nitrido complexes would be a useful tool to prepare new SPECT agents to image 5-HT1A receptor with cyclam conjugated
ligands.
Keywords: 5-HT1A receptor; technetium-99m; MPPC; 99mTcN-MPPC
structure of the cyclam and obtained a novel neutral complex
labelled with 99mTc(V)-nitrido core. This provided a reliable and
Introduction
The 5-HT1A receptor has been studied extensively due to its role
in a number of neuropsychiatric disorders such as schizophrenia,
Alzheimer’s disease, depression, hallucinogenic behavior, mo-
tion sickness, eating disorders and anxiety. It is therefore of
great interest, especially for diagnosis, to develop radioligands
of high selectivity and affinity for the receptor to allow brain
imaging. Despite the 11C- and 18F-labelled ligands for positron
emission tomography (PET) imaging and 123I-labelled ligands for
single photon emission computed tomography (SPECT) imaging
has played a vital role in studying the location and density of 5-
HT1A receptor, technetium-99m-labelled ligands would be more
desirable for the excellent properties of technetium-99m and its
ready availability.1–5
Based on the lead structure of N-[2-[4-(2-methoxy-
phenyl)-1-piperazinyl]-ethyl]-N-(2-pyridinyl)- cyclohexanecarbox-
amide (WAY100635), which is used in SPECT imaging and in
vitro autoradiographic studies of 5-HT1A receptors, considerable
progress has been made in the last few years for the
development of technetium-99m complexes.6–12 There are
several different approaches used for attaching the technetium
to the pharmacophore moiety of WAY 100635, such as the
mixed ligand complex approach (21111, 311, 411), tetra-
dentate N2S2 chelate approach and the tricarbonyl approach.
It is well known that the cyclic polyamines such as 1,4,8,11-
tetraazacyclotetradecane (cyclam) can be used as bifunctional
chelating agents of rhenium and technetium for labelling
bioactive ligands.13–18 In this study, we introduced the 1-(2-
methoxyphenyl) piperazine moiety of WAY 100635 into the
reproducible method for attaching the technetium to the
pharmacophore moiety of WAY 100635. This effective 99mTc-
labelling strategy would be a favorable method for preparation
of neutral 99mTc-nitrido complexes with cyclam conjugated
ligands to image 5-HT1A receptors.
Results and discussion
Chemistry
The synthesis of the cyclam chelating unit linked via a 3-carbon
alkyl chain to 1-(2-methoxyphenyl) piperazine was accomplished
as described in Scheme 1.
The three amine functions of cyclam were protected by an
excess of ethyl trifluoroacetate in methanol at room tempera-
ture. The preparation of 3-[4-(2-methoxyphenyl)piperaziny-1-
yl]propan-1-ol (2) was performed by alkylation of 1-(2-methox-
yphenyl)piperazine using 3-bromo-1-propanol as alkylating
agent. The reaction was carried out in anhydrous CH3CN in
the presence of K2CO3. 3-[4-(2-Methoxyphenyl)piperaziny-1-
yl]propyl methanesulfonate (3) was obtained by mesylation of
Key Laboratory of Radiopharmaceuticals of Ministry of Education, College of
Chemistry, Beijing Normal University, Beijing 100875, People’s Republic of China
*Correspondence to: X. Wang, Key Laboratory of Radiopharmaceuticals of
Ministry of Education, College of Chemistry, Beijing Normal University, Beijing
100875, People’s Republic of China.
E-mail: xbwang@bnu.edu.cn
J. Label Compd. Radiopharm 2008, 51 347–351
Copyright r 2008 John Wiley & Sons, Ltd.

F. Wang et al.
O
O
CF3
CF3
F3C
HN
HN
NH
NH
N
N
N
a
NH
O
1
O
CH3
N
O
CH3
N
CH3
O
b
c
(CH)3OMes
(CH)3OH
NH
N
3
2
O
O
CH3
N
O
CH3
N
O
N
N
N
N
CF3
CF3
F3C
N
N
N
N
d
e
N
N
(CH2)3
(CH2)3
O
MPPC
4
Scheme 1. Synthesis of N-3-[1-(2-methoxyphenyl)piperidin-4-yl]propylcyclam (MPPC). Reagents: (a) EtOTFA,Et3N,MeOH; (b) 1-(2-Methoxyphenyl)piperazine,
K2CO3, CH3CN; (c) CH3SO2Cl, CH2Cl2, Et3N; (d) 1, K2CO3, CH3CN; and (e) NaOH, MeOH.
2 with mesyl chloride and triethylamine in CH2Cl2. The synthesis
In vitro stability of the complex was evaluated by measuring
of N-[3-[4-(2-methoxyphenyl)piperaziny-1-yl]propyl]Tri-TFA-cy- the radiochemical purity (RCP) at different time points after
clam (4) was performed by alkylation of Tri-TFA cyclam (1) preparation at room temperature (25 1C). The RCP was still over
using 3 as alkylating agent.
95% after 4 h, which suggested that 99mTcN-MPPC complex
Three protected amine functions of 4 were deprotected in possessed a great stability in vitro.
MeOH/NaOH to give N-[3-[4-(2-methoxyphenyl)piperaziny-1-yl]
propyl]cyclam (MPPC).
In vivo stability was also tested in normal rats. No degradation
of 99mTcN-MPPC was found by HPLC analysis of deproteinated
blood samples at different time points post-injection.
Radiochemistry
Experimental
In order to label MPPC with 99mTc-nitrido core, the [99mTcN]21
intermediate was first prepared. The final 99mTcN-MPPC complex
was obtained in high yield (495%) by addition of [99mTcN]21
intermediate to MPPC ligand and heating under N2 at 1001C for
30 min. Quality control of the [99mTcN]21 intermediate and
99mTcN-MPPC was performed by TLC and HPLC. TLC was
performed on a polyamide film with saline and acetonitrile as
the mobile phase. Rf values for [99mTcN]21 intermediate, 99mTcN-
MPPC and other possible components are reported in Table 1.
The specific radioactivity of 99mTcN-MPPC was 240 TBq/mmol at
end of synthesis.
Materials
The SDH kit was provided by Beijing Shihong Pharmaceutical
Center, People’s Republic of China. A 99Mo/99mTc generator was
obtained from the China Institute of Atomic Energy (CIAE). All
other chemicals were of reagent grade and were used without
further purification. The TLC was performed on a polyamide
strip and eluted with saline and acetonitrile. Reversed-phase
high-performance liquid chromatography experiments were
performed on a SHIMADZU SCL-10AVP system with a liquid
scintillation analyzer (Packard Bioscience Co., USA). The ODS-C18
column (5 mm, 250 ꢁ 4.6 mm, Alltech Associates, Inc., USA) was
eluted at a flow rate of 1 mL/min according to the procedure
described in the experimental part. NMR spectra were recorded
on a Bruker Avance-500 (500 MHz) spectrometers.
HPLC studies demonstrated that the reaction resulted
in a single complex. The retention time of the 99mTcN-MPPC
complex was found to be about 2.20 min, while that of
[
99mTcN]21 intermediate was 10.50 min. The HPLC chromato-
grams of [99mTcN]21 intermediate and 99mTcN-MPPC complex
are shown in Figure 1. The paper electrophoresis pattern of
99mTcN-MPPC remained at the point of spotting, suggesting that
it is a neutral complex.
Synthesis of Tri-TFA cyclam (1)
Ethyl trifluoroacetate (4.80 mL, 40 mmol) was added dropwise to
The partition coefficient (log P) of 99mTcN-MPPC was a mixture of cyclam (2.00 g, 10 mmol) and Et3N (1.38 mL,
ꢀ1.6470.03 at pH 7.4, which indicated that it is a hydrophilic 10 mmol) in methanol (20 mL) at room temperature. The
complex.
addition continued over a period of 5 min. The homogeneous
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J. Label Compd. Radiopharm 2008, 51 347–351

F. Wang et al.
Table 1. Rf values for some selected complexes
99mTcO4]ꢀ
[
99mTcO2 ꢂ nH2O
½
99mTcNꢃ2inþt
99mTcN-MPPC
Saline
Acetonitrile
0.1
0.3–0.5
0.1
0.1
0.8–1.0
0.1
0.6–0.7
0.1
Figure 1. The HPLC chromatograms of [99mTcN]21 intermediate (A) and 99mTcN-MPPC (B).
reaction mixture was cooled with an ice-water bath to control in CH2CI2 (20 mL), and the reaction mixture was then stirred at
the mild exotherm. Stirring was continued under N2 for 5 h. room temperature overnight. The solution was washed with
Volatiles were removed in vacuo. The residue was passed saturated aqueous ammonium chloride (30 mL) and brine (30 mL)
through a small silica gel plug (25 g), eluted with 100% EtOAc. and then dried (Na2SO4). The solvent was then evaporated in
The eluent was concentrated to give the product as a white vacuo and the residue was purified by column chromatography
1
1
foam. H NMR (500 MHz, CDCl3) d: 3.79–3.51 (m, 12H), 2.95 (m, on silica gel (MeOH/ CH2CI2, 1:15) to give a white solid. H NMR
2H), 2.72 (m, 2H), 2.18 (m, 2H), 1.86 (m, 2H)
(500MHz, CDCl3) d: 6.98–6.89 (m, 4H), 3.73 (s, 3H), 3.45–3.32 (m,
6H), 3.20–3.13(m, 7H), 2.39(m, 2H), 1.42 (m, 2H).
Synthesis of 3-[4-(2-methoxyphenyl)piperaziny-1-yl]propyl
methanesulfonate (3)
Synthesis of N-[3-[4-(2-methoxyphenyl)piperaziny-1-yl]pro-
pyl]Tri-TFA-cyclam (4)
1-(2-methoxyphenyl)piperazine (0.96 g, 5 mmol) and 3-bromo-1-
propanol (1.04 g, 7.5 mmol) were dissolved in anhydrous CH3CN To a stirred solution of 1 (0.97 g, 2 mmol) and anhydrous K2CO3
(20 mL). K2CO3 (1.04 g, 7.5 mmol) was added and the mixture (0.42 g, 3 mmol) in anhydrous CH3CN (20 mL) was added 3
was heated under reflux for about 7 h. The solid phase was (0.65 g, 2 mmol) and the mixture was heated under reflux for
removed by filtration. The solvent was then evaporated in vacuo about 9 h. The solid phase was removed by filtration and the
to obtain 2, which was used for the next step reaction without solvent was then evaporated in vacuo. The residue was purified
further purification.
by column chromatography on silica gel (MeOH/ CH2CI2, 1:16)
1
To the solution of 2 (1.25 g, 5 mmol) and Et3N (1 mL, 7.5 mmol) to give a white solid. H NMR (500 MHz, CDCl3) d: 7.07-6.89 (m,
in CH2CI2 (50 mL), cooled to 0–5 1C, was added dropwise with 4H), 3.90 (s, 3H), 3.16–3.04 (m, 12H), 2.95–2.79(m, 16H),
stirring a solution of methanesulfonyl chloride (0.87 g, 7.5 mmol) 1.82–1.84(m, 6H)
J. Label Compd. Radiopharm 2008, 51 347–351
Copyright r 2008 John Wiley & Sons, Ltd.
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F. Wang et al.
Synthesis of N-[3-[4-(2-methoxyphenyl)piperaziny-1-yl]pro- In vitro stability study
pyl]cyclam (MPPC)
The stability of 99mTcN-MPPC in the reaction mixture was
assayed by measuring the RCP by TLC and HPLC at 1, 2 and 4 h
after preparation at room temperature (25 1C).
Substrate 4 (0.36 g, 0.5 mmol) was dissolved in methanol (10 mL)
and NaOH (0.40 g, 10 mmol) was added at 01C. The mixture was
stirred for 30 min at room temperature. The solid phase was
removed by filtration and the solvent was evaporated in vacuo
to obtain a white solid. Water (20 mL) and chloroform (10 mL)
were added. The organic phase was separated and washed with
water (3 ꢁ 10 mL). The aqueous phases were reextracted with
chloroform (15 mL) and the combined organic solutions were
dried over Na2SO4. Solvent was evaporated in vacuo to yield a
white solid. 1H NMR (500 MHz, CDCl3) d: 7.04–6.83 (m, 4H),
3.99(s, 3H), 3.37–2.72 (m, 28H), 1.76–1.81 (m, 6H).
In vivo stability study
The experiments in rats were carried out in compliance with the
national laws relating to the care and experiments on laboratory
animals. In vivo stability study of the 99mTcN-MPPC in normal
male Wistar rats (body mass 250–260 g) was performed by
injecting the tracer (0.2 mL, 2.0–3.7 MBq) into the tail vein. Rats
were sacrificed at 5, 60 and 120 min post-injection. Blood
samples taken were centrifuged for 15 min at 5000 r/min to
collect serum. The serum was transferred (1 mL) into a tube and
mixed with acetonitrile (1 mL). The resulting mixture was
vortexed for 10 s, and centrifuged for 15 min at 5000 r/min.
The supernatant liquid was analyzed by HPLC following the
same procedure as described above for preparation of the
99mTcN-MPPC.
Preparation of the 99mTcN-MPPC
The 99mTcN-MPPC was prepared by a two-step procedure.
First, 1 mL saline-containing Na99mTcO4 (about 740 MBq)
was added into a SDH kit and kept for 15 min at room
temperature to obtain the [99mTcN]21 intermediate. Second,
0.1 mL [99mTcN]21 (about 74 MBq) intermediate was added to
the 0.1 mL ethanol containing 1.5 mg MPPC ligand. The pH of
the solution was adjusted to 11–12 by addition of 0.9 mL
of buffer (composition: 50 mL of 0.15 M Na2HPO4175 mL of
0.1 M NaOH) and the reaction mixture was heated under N2 at
100 1C for 30 min.
Conclusions
We used bifunctional chelating agent 1,4,8,11-tetraazacyclote-
tradecane (cyclam) to attach the technetium-99m to 1-(2-
methoxyphenyl)piperazine, an important pharmacophore for
reported 5-HT1A PET and SPECT ligands and obtained MPPC
ligand for the first time. This provided a new method for
attaching the technetium to the pharmacophore moiety of WAY
100635 and preparing neutral technetium-99m complexes for 5-
HT1A receptor imaging. 99mTcN-MPPC complex was prepared by
using two-step procedure with high radiochemical purity
(495%). It was a neutral and hydrophilic complex with high in
vitro and in vivo stability.
The [99mTcN]21 intermediate and final 99mTcN-MPPC complex
were characterized by TLC and HPLC. TLC was performed on a
polyamide film with saline and acetonitrile as the mobile phase.
HPLC was performed as following: about 5 mL of the 99mTcN-
MPPC complex solution was injected into the column, C18
reverse phase column was eluted at flow rate of 1 mL/min and
water (solvent A) and acetonitrile (solvent B) were used as the
mobile phase in the following gradient (0–20 min, 100–20% B,
20–35 min, 20–100% B).
In further study, we will characterize and determine the
structure of 99mTcN-MPPC using Re model compound. At the
same time, the feasibility of this 99mTc-nitrido complex for 5-
HT1A receptor imaging will be evaluated in the future.
Determination of the partition coefficient for the complex
The partition coefficient of the complex was determined by
measuring the activity that partitioned between the 1-octanol
and aqueous phosphate buffer (0.025 mol/L, pH 7.4) under strict
equilibrium conditions. 2 mL 1-octanol and 2 mL 99mTcN-MPPC
phosphate buffer were mixed in a centrifuge tube. The mixture
was vortexed at room temperature for 5 min and then
centrifuged at 5000 r/min for 5 min. The counts in 0.1 mL
samples of both organic and inorganic layers were determined
by a well type gamma counter. The measurement was repeated
three times. The partition coefficient (P) was calculated using the
following equation
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (20401004). The authors wish to acknowl-
edge the support of Beijing Shihong Pharmaceutical Center for
the donation of SDH kits.
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P = (cpm in octanol-cpm in background)/(cpm in buffer-cpm
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The final partition coefficient value was expressed as log P.
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