Synthesis, characterization and biological activity of99mTc- labeled piperidine analogues targeting sigma receptors

Article abstract of DOI:10.1002/jlcr.1750

Sigma receptors are expressed in high density in various types of cancer cells including brain tumours and are also involved in various diseases of central nervous system. This makes ligands that bind to these receptors, attractive molecular vectors for t

Full text of DOI:10.1002/jlcr.1750

Research Article
Received 2 December 2009,
Revised 4 February 2010,
Accepted 6 February 2010
Published online 30 March 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1750
Synthesis, characterization and biological
activity of 99mTc-labeled piperidine analogues
targeting sigma receptors
Drishty Satpati,a Ketaki Bapat,a Haladhar Dev Sarma,b Meera Venkatesh,a
Ã
and Sharmila Banerjeea
Sigma receptors are expressed in high density in various types of cancer cells including brain tumours and are also involved
in various diseases of central nervous system. This makes ligands that bind to these receptors, attractive molecular vectors
for targeting radiation to the specific sites with the purpose of imaging and therapy of neurological disorders. We report
synthesis of three derivatives of 4-amino-N-benzylpiperidine namely, 4-dithiocarbamato-N-benzylpiperidine, 4-iminodia-
cetato-N-benzylpiperidine and 4-(N-benzylpiperidine)-pyridin-2-ylmethyl-amino)-acetic acid and their radiolabeling with
technetium-99m. The in vivo evaluation of these radiolabeled compounds has been carried out in mice, for assessment of
their binding affinity with sigma receptors. Of the three complexes, [99mTcN]-4-dithiocarbamato-N-benzylpiperidine,
[
99mTcN]Pip-DTC exhibited the most promising characteristics with brain uptake of 0.6% ID/g at 5 min.p.i. that reduced to
0.3% ID/g after 2 h.p.i. Competition experiment carried out with [99mTcN]Pip-DTC complex, using (1)-pentazocine showed
its specificity towards sigma receptors, as was found to be evident from reduction in the brain uptake of this complex.
Introduction of iminodiacetate and pyridine moieties and subsequent radiolabeling did not result in complexes with
significant potential of targeting and binding with sigma receptors.
Keywords: benzylpiperidine; sigma receptors; [99mTc(CO)3(H2O)3]1; [99mTcN]21
imaging and subsequent therapeutic intervention in various
psychiatric diseases and various types of cancers. Positron
Introduction
The study of the central nervous system (CNS) and its related
neuropsychiatric disorders using diagnostic techniques of nuclear
medicine is a rapidly expanding area of research.1,2 Towards this,
the specific receptors, their subtypes, transmitters and enzymes
expressed on the CNS can serve as targets for development of
radiolabeled receptor-selective molecules. Among the various
neuroreceptors identified, sigma receptors belong to a unique
class of proteins different from opioid, N-methyl-D-aspartate
(NMDA) phencyclidine, dopaminergic and other known receptors
acting as neurotransmitter agents.3 Sigma receptors having high
affinity for neuroleptic drugs are known to be involved in several
disorders of the CNS like schizophrenia, depression, dementia,
etc.4,5 Besides being present in brain, sigma receptors are also over-
expressed in high density in different tumours like those of breast,
prostate, melanoma, non-small cell lung carcinoma and other
tumours of neural origin making them favourable targets for
developing radiotracers to be used in nuclear medicine for imaging
as well as subsequent therapy.6 On the basis of their pharmaco-
logical action, sigma receptors have been classified into two
subtypes as sigma-1 and sigma-2. Of the two subtypes, sigma-1
receptors are well studied and characterized. While sigma-1
receptors exhibit high binding affinity for [3H](1)-pentazocine
and other (1)-benzomorphans, di-o-tolyl guanidine (DTG) shows
high affinity towards both sigma-1 and sigma-2 sites.7,8
emission tomography (PET) as well as single photon emission
computed tomography (SPECT) has been used for imaging of
sigma receptors. In this respect, various radiolabeled agents
have been prepared using 11C, 18F, 99mTc and 123I as radio-
isotopes.9–13 Though PET radioligands studied out-number
those of SPET ligands, wide accessibility and attractive nuclear-
physical properties of technetium-99m provide strong impetus
to efforts in improving the availability and research for new
suitable SPET imaging agents. Technetium-99m is the ideal
diagnostic radionuclide in nuclear medicine due to its favour-
able decay characteristics (t1/2 = 6 h, Eg = 140 keV), easy avail-
ability and cost effectiveness.14 In the present studies, novel
[
99mTcN]21 and [99mTc(CO)3(H2O)3]1 cores have been chosen as
ideally suited precursors for designing of receptor-specific
radiotracers. [99mTcN]21 core forms stable complexes with N
and S donors in a tetradentate array. In this respect,
dithiocarbamates are ideally suited for complexation, leading
aRadiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai,
India
bRadiation Biology & Health Sciences Division, Bhabha Atomic Research Centre,
Mumbai, India
*Correspondence to: Sharmila Banerjee, Radiopharmaceuticals Division, Bhabha
Atomic Research Centre, Mumbai 400 085, India.
E-mail: sharmila@barc.gov.in
Radiopharmaceuticals designed to target sites expressing
sigma receptors may therefore serve as potential tools for
J. Label Compd. Radiopharm 2010, 53 198–204
Copyright r 2010 John Wiley & Sons, Ltd.

D. Satpati et al.
to formation of neutral complexes as a result of coordination of additional carbon atom corresponding to the carbon atom of
two molecules of dithiocarbamate moiety.15 99mTc(CO)3(H2O)3]1 the dithiocarbamate group. L2 was prepared in two steps as
[
core containing d6 Tc(I) center forms stable low spin complexes depicted in Scheme 1. Refluxing 1 with pyridine 2-carboxalde-
with N-containing ligands such as histidine, histamine, hyde resulted in the formation of imine, which was subse-
imidazole, iminodiacetic acid, diethylene triamine, Schiff’s quently reduced to the corresponding secondary amine using
bases.16 The use of
[ [
99mTcN]21 and 99mTc(CO)3(H2O)3]1 sodium borohydride as the reducing agent. The formation of the
precursors give high specific activity complexes emerging from reduced product was confirmed by disappearance of the imine
the requirement of low ligand concentration, thereby offering a proton at d8.0 observed in the 1H-NMR spectra of the imine and
promising advantage towards designing agents for targeting subsequent appearance of a two-proton singlet corresponding
receptors.
to pyridyl methyl protons at d2.9 and pyridine protons between
Several derivatives of piperidine, piperazine,17,18 benza- d8.54 and 7.18 in the aromatic region. The amine formed was
mides19 and alkylamine20 radiolabeled with suitable isotopes subsequently reacted with an equimolar amount of bromoacetic
have been reported in literature as potential radiopharmaceu- acid to yield the final product, L2. Formation of final product
ticals for imaging of sigma receptors. In the present studies, could be confirmed by the appearance of a peak at d3.55 in
4-amino-N-benzylpiperidine has been chosen as the parent 1H-NMR spectra corresponding to amino acetic acid group. Mass
molecule with the aim of preparing an agent for specific and 13C-NMR spectral characterization were also carried out for
targeting of sigma receptors. The N-benzyl piperidine template the ligand L2. Ligand L3 was prepared by dialkylation of the
is an integral unit of a host of ligands such as 2-[125I]BP, 2-[125I] amino group and was carried out by reaction of 1 with two
N-(N-benzylpiperidin-4-yl)-2-iodobenzamide, for use as CNS recep- equivalents of bromoacetic acid under alkaline conditions. The
tor-specific agents.13,19 As –NH2 group of piperidine in the 4- iminodiacetate formation was confirmed by 1H-NMR where a
amino-N-benzylpiperidine template is a pendant moiety, its facile two-proton singlet at d3.34 corresponding to ꢀNCH2COOH
yet versatile modes of derivatization to incorporate suitable group was observed. The compound was further characterized
functional groups provide possible routes for syntheses of different by mass and 13C-NMR spectra.
ligands without affecting the receptor binding properties. The
strategy in designing of the ligands were envisaged keeping in
mind that the net charges on the resultant radiolabeled complexes
Radiochemistry
[
99mTcN]21 known to form stable complexes with dithiocarba-
would be determining factors governing their biological specificity
as receptor-targeting agents. This paper reports the efforts to
prepare three different derivatives of 4-amino-N-benzylpiperidine 1
namely, 4-dithiocarbamato-N-benzylpiperidine (Pip-DTC) L1, 4-(N-
benzylpiperidine)-pyridin-2-ylmethyl-amino)-acetic acid (PPAA) L2
and 4-iminodiacetato-N-benzylpiperidine (Pip-IDA) L3. Ligand L1
has been radiolabeled with [99mTcN]21 core, whereas ligands L2 and
L3 have been radiolabeled with[99mTc(CO)3(H2O)3]1 core. In vivo
evaluation of 99mTc-complexes has been carried out to assess the
uptake in brain, which, in turn, reflects the ability to cross the
blood–brain barrier and bind to the receptors. Competition studies
with sigma receptor-specific ligand, (1)-pentazocine, have also been
carried out to study the receptor specificity of these complexes.
mates was used to radiolabel L1. Ligands L2 and L3 behaving as
tridentate ligands with N2O and NO2 as donor atoms, respec-
tively, were radiolabeled with the [99mTc(CO)3(H2O)3]1 core.
[
99mTcN]21 core was prepared using the kit following a
procedure reported earlier.22 The complex was then prepared
by incubation of L1 with the [99mTcN]21 core. The core as well as
the complex [99mTcN]Pip-DTC I could be obtained in 498%
radiochemical yield as determined by HPLC. In HPLC gradient
system, the nitrido core exhibited the retention time of 4 min
(Figure 1(a)), whereas the complex was eluted as a single species
with retention time of 15min (Figure 2(a)). Additional evidence
showing formation of the 99mTc-nitrido complex in good yields
(498%) were obtained from TLC and paper electrophoresis. TLC
was carried out in ethanol:chloroform:toluene:0.5M ammonium
acetate mixture (6:3:3:0.5 v/v) as well as in saline. In the former
solvent system, 99mTc-nitrido precursor as well as reduced
technetium remained at the origin (Rf = 0–0.1) corresponding to
498% of activity whereas 99mTcO4ꢀ moved to Rf = 0.4–0.6.
However, in saline, both 99mTcOꢀ4 and 99mTc-nitrido intermediate
corresponding to 498% of activity moved with the solvent front
(Rf = 0.8–1.0) with reduced technetium remaining at the origin.
Radiochemical purity of the complex (498%) could be deter-
mined by TLC in saline wherein the complex remained at the
origin (Rf = 0). In paper electrophoresis studies, while the 99mTc-
nitrido intermediate showed a movement of 5 cm towards the
anode, the complex did not show any movement indicating
possible neutrality of the complex. Lipophilicity of the radiola-
beled complex was determined by distribution in octanol and
water and the partition coefficient (log P) was found to be
1.770.2.
Results and discussion
Chemistry
Several molecular modelling studies have been reported for the
determination of the biologically active molecular unit respon-
sible for binding with sigma receptors. It is proposed that the
primary binding requires presence of a hydrophobic site offered
by a phenyl group and a hydrophilic site represented by the
nitrogen atom and its lone pair. The secondary binding site
requires the presence of an electronegative atom like oxygen or
sulphur.21 With the aim of preparing a 99mTc-labeled agent
specific for sigma receptors, three different derivatives of
4-amino-N-benzylpiperidine have been prepared by carrying
out functionalization of the amino group. (Scheme 1) Ligand L1,
a dithiocarbamate, was prepared by reaction of the parent
molecule, 4-amino-N-benzylpiperidine, with an equimolar
amount of carbon disulphide in the presence of sodium
hydroxide at room temperature. Formation of L1 could be
confirmed by 1H-NMR, 13C-NMR and mass spectra. A peak at
d213.6 in 13C-NMR spectra indicates the incorporation of an
The 99mTc-tricarbonyl synthon used for complexation was
prepared in-situ according to the reported procedure.23 It could
be prepared in 498% yield as determined by C-18 reverse
phase HPLC system. 99mTc(CO)3-PPAA II and [99mTc(CO)3-Pip-
IDA]ꢀ III complexes were prepared by incubation of ligands with
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D. Satpati et al.
Scheme 1. Syntheses of 4-amino-N-benzylpiperidine ligands.
the 99mTc-carbonyl core. Retention time of the precursor on retention times were found thereby indicating the stability of
HPLC was 13.7 min (Figure 1(b)), whereas that of complexes II the three complexes.
and III were 15.8 min and 11.6 min, respectively (Figure 2(b)
Challenge studies performed by incubation of complexes with
and (c)). The radiochemical yield of the complexes as excess amount of histidine and cysteine also did not lead to
determined by HPLC analyses was 498%. Optimization of the alterations in the retention time and pattern of the peak
pH for complexation was required for radiolabeling of the two obtained in HPLC system, indicating their kinetic inertness and
ligands with [99mTc(CO)3(H2O)3]1. Under optimized conditions it stability towards transchelation.
was found that pH of highly alkaline 99mTc-carbonyl precursor
had to be adjusted to 7 and 4 using PO34ꢀ buffer (pH 7.5,
0.5 M):HCl (1.0 M) (1:3, v/v) before addition of ligands L2 and L3,
Biodistribution studies
Pharmacokinetic pattern of the three complexes were studied
by carrying out biodistribution studies in normal Swiss mice.
(Table 1) The uptake of complex I in brain was higher than those
of the other complexes, being 0.6% ID/g at 5 min.p.i. with
retention till 30 min. The uptake reduced to 0.3% ID/g at 2 h.p.i.
The apparently low brain uptake of 0.6% ID/g, however,
compares well with many of the other reported agents for
brain receptor imaging.25 Results of competition studies at
30 min.p.i. for complexes I and II are given in Figure 3. In vivo
blocking studies wherein (1)-Pentazocine, a drug known to bind
specifically to sigma receptors, was administered 1 h prior to the
injection of the radiolabeled complex were carried out in Swiss
mice models. No radioactivity was observed in the mice brain
for preparation of complexes II and III, respectively. Logarithm
of partition coefficient (log P) is a parameter used to provide an
indication of the lipophilicity of the complexes. Log P values as
determined by extractions in octanol/saline were found to be
1.5670.18 and 0.9570.1 for the complexes II and III,
respectively. The log P value between 0.9 and 2.5 has been
suggested optimal for BBB crossing.24 As values of log P for
complexes under study lie within this range, they are expected
to bind to CNS receptors.
In vitro stability of the complexes was determined by
incubation of the complexes in water bath at 371C for 6 h. The
radiochemical purity of the complexes were checked after 6 h
using HPLC analyses where no observable change in the
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J. Label Compd. Radiopharm 2010, 53 198–204

D. Satpati et al.
Figure 1. HPLC pattern of precursors (a) [99mTcN]21 and (b) [99mTc(CO)3(H2O)3]1.
pre-administered with (1)-pentazocine in case of studies with
complex
I indicating complete blockage and hence the
probable specificity of complex I for binding with sigma
receptors. Uptake in liver and lungs was also reduced as these
organs are also known to express sigma receptors. These
observations are suggestive of receptor-mediated uptake of the
complex.
Brain uptake of complex II was found to be 0.5% ID/g
5 min.p.i, which reduced to 0.2% ID/g 2 h.p.i. There was no
decrease in the radioactivity in the brain on carrying out
blocking studies with (1)-pentazocine which could be attrib-
Figure 2. HPLC pattern of 99mTc-complexes (a) [99mTcN]Pip-DTC (I), (b) 99mTc(CO)3-
PPAA (II), (c) [99mTc(CO)3-PipIDA]ꢀ (III).
The proposed structure of the complex I has been shown in
uted possibly to its less specific binding to sigma receptors as scheme 1 where two dithiocarbamate molecules each behaving
compared with that of complex I (Figure 3). The presence of the as a bidentate ligand coordinates with technetium resulting in
additional aromatic moiety (pyridine ring) besides the benzyl the formation of a neutral complex. Such a speculation can be
group leading to steric hindrance may be the probable cause for substantiated by earlier reports where the involvement of two
decrease in the binding affinity.
DTC moieties have been proposed.26 Ligand L2 acts as a
Complex III showed negligible brain uptake of 0.18% ID/g at tridentate ligand for complexation with the 99mTc(CO)3-pre-
5 min.p.i. Blood uptake was less compared with other lipophilic cursor with pyridine nitrogen, carboxyl oxygen and iminodiace-
derivatives. Within 30 min of injection of the complex, the tate nitrogen acting as coordinating groups. Ligand L3 has a
radioactivity cleared from all the major organs whereby studies tridentate array of donors in the iminodiacetate moiety as the
at 2 h.p.i were not carried out with this complex. Brain uptake chelating groups which replace the three labile aqua molecules
studies are consistent with the observed log P values.
of [99mTc(CO)3(H2O)3]1 by a nitrogen and two carboxyl oxygen
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D. Satpati et al.
Table 1. Biodistribution studies of complexes I, II and III in normal Swiss mice
Complex Time (p.i.)
Blood
Liver
Intestine
Kidney
Heart
Brain
Lungs
Spleen
I
5 min
30 min
2 h
5 min
30 min
2 h
870.6
3.370.6
270.06
8.370.8
3.670.5
1.870.2
2.270.25
1.470.6
3172.0
25.474.0
1370.5
20.376.0
1673.3
16.471.0 10.871.7 12.272.5
3471.5
972.0
2.670.4
5.671.0
771.0
5.370.6 24.674.9
8.870.4 19.673.2
2273.0
1170.8
7.871.0
1071.0
7.471.0
5.771.5
9.670.2
6.870.5
5.470.9
1.470.17
0.470.2
0.670.05
0.4370.03
0.370.05
0.570.02
0.4670.2
0.2170.12
0.1870.03
0.0670.02
2372.0
15.672.8
7.270.8
14.771.4
9.273.7
5.370.5
2.370.54
0.770.06
1070.7
970.6
870.5
5.671.6
5.47.1.7
3.970.8
0.970.2
0.470.05
II
III
5 min
30 min
2.870.3
2074.0
670.4
270.7
%ID/g of organ (mean7standard deviation), n = 3.
could be seen implying that introduction of the iminodiacetic
acid moiety strongly affects the receptor affinity.
Experimental
General
All chemical reagents were of commercial grade. Carbon
monoxide in 0.5 L refillable canisters was obtained from M/s
Alchemie Gases & Chemicals, Mumbai, India. 99mTcO4ꢀ was
eluted from an in-house 99Mo/99mTc column generator using
normal saline. 4-amino-N-benzylpiperidine was purchased from
Fluka Chemie GmbH. Commercial kit for preparation of 99mTc-
nitrido species was obtained from CIS Bio International.
Electrophoresis experiments were carried out using 0.025 M
phosphate buffer (pH 7.5) at 300 V/cm for 1 h. HPLC analyses
were performed on a Jasco PU 1580 system with a Jasco 1575
tunable absorption detector and a radiometric detector system.
A C-18 reversed phase HiQ Sil (5 m, 250 ꢁ 4 mm) column has
been used. About 25 mL of the test solution was injected into the
column and the elution was monitored by observing the
radioactivity profile. The flow rate was maintained at 1 mL/min.
The gradient system consisting of eluting solvents H2O
(solvent A) and acetonitrile (solvent B) with 0.1% trifluoroacetic
acid was used (0–28 min, 90% A-10% A; 28–30 min, 10% A;
30–32 min, 10% A-90% A). NMR spectra were obtained using
Varian VXR 300S spectrophotometer operating at 300 MHz for
1H and at 75 MHz for 13C. Mass spectra were recorded on QTOF
Micromass Instrument using electron spray ionization (ESI) in
positive mode. All the animal experiments were carried out in
compliance with the relevant National laws as approved by the
local committee on the conduct and ethics of animal
experimentation.
Chemical synthesis
4-dithiocarbamato-N-benzylpiperidine (L1)
Figure 3. Effect of (1)-pentazocine on distribution of radioactivity (30 min.p.i.) of
(a) [99mTcN]Pip-DTC (I) and (b) 99mTc(CO)3-PPAA (II).
To a solution of 4-amino-N-benzylpiperidine (1) (50 mg,
0.26 mmol) in diethyl ether (10 mL), carbon disulphide
(15 mL, 0.25 mmol) was added along with sodium hydroxide
to form the complex. Complexes I and II are neutral with similar (4.8mg, 0.12 mmol). A white precipitate was obtained immediately
lipophilicity thus showing similar brain uptake. However, no on addition of carbon disulphide. The reaction mixture was stirred
significant blocking was observed in competition studies for at room temperature overnight. After 24 h, solvent was removed
complex II indicating lower receptor affinity of the complex. The under reduced pressure and the product was purified using
insignificant brain uptake of complex III was expected due to its ammonia:methanol (5:95) as eluting solvent on a silica gel column.
residual negative charge and lower lipophilicity. Besides this
feature no uptake in other organs expressing sigma receptors benzyl), 2.21 (m, 1H, piperidine), 2.09 (m, 4H, piperidine), 1.48
1H-NMR (d ppm, CDCl3): 7.40 (s, 5H, aromatic), 4.18 (s, 2H,
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D. Satpati et al.
(m, 4H, piperidine); 13C-NMR (d ppm, CD3OD) 31.78 (2C, Radiochemistry
piperidine), 35.58 (2C, piperidine), 53.43 (C, piperidine), 64.03
(C, benzyl), 128.47 (C, phenyl), 129.36 (2C, phenyl), 130.82 (2C,
[
99mTcðCOÞ3ðH2O3Þ]1 precursor
phenyl), 138.58 (C, phenyl), 213.6 (C, NHCS2); MS (ESI): mass
calculated for C13H17N2S2Na; 288, found 289 [M1H]1.
The synthon was prepared by using modified procedure
reported by Alberto et al.23 An aqueous solution of NaBH4
(5.5 mg), Na2CO3 (4 mg) and Na/K tartrate (15 mg), in 0.5 mL
double distilled water was prepared. Carbon monoxide gas was
purged through this solution for 5 min and after adding
99mTcOꢀ4 (1 mL, 37 MBq) to the solution, it was heated at 801C
for 15 min. The reaction mixture was then cooled on an ice bath
for 10 min. and pH of the reaction mixture was adjusted to 8
with 300 mL of 0.5 M phosphate buffer (pH 7.5): 1 M HCl (1:3 v/v).
The synthon was characterized by HPLC.
{4-(N-benzylpiperidine)}-pyridin-2-ylmethyl-amine (2)
To a solution of 1 (190 mg, 1 mmol) in dry methanol (10 mL), 2-
pyridine carboxaldehyde (100 mg, 1 mmol) was added and the
reaction mixture was refluxed for 24 h. Formation of the product
was confirmed by TLC in 15% MeOH/CHCl3 Rf (product) = 0.3.
NaBH4 was added to the above mixture and refluxed for 5 h.
After completion of the reaction, methanol was evaporated in a
rotary evaporator. The product was extracted in CHCl3
(3 ꢁ 10 mL), washed with water thrice and dried over anhydrous
Na2SO4. The solution was filtered and CHCl3 was evaporated to
yield a yellow solid. The product was characterized by TLC in 1%
NH3/methanol. Rf (product) = 0.7.
[
99mTcN]21 core
The kit vial, containing succinic dihydrazide (5.0 mg), stannous
chloride dihydrate (100mg), 1,2-diaminopropane–N,N,N0,N0-tetra-
acetic acid (5 mg), sodium dihydrogen phosphate (0.5 mg) and
disodium hydrogen phosphate (5.8 mg) in freeze dried form, was
used for preparing the precursor. The kit vial stored at 41C was
allowed to attain ambient temperature. The freshly eluted
99mTcOꢀ4 (1 mL, 37 MBq) was added to the vial, vortexed and
allowed to stand at room temperature for 20 min. The 99mTc-
nitrido intermediate thus prepared was characterized by TLC.
1H-NMR (d ppm, CDCl3): 8.54 (d, 1H, pyridine), 7.64 (t, 1H,
pyridine), 7.33 (s, 5H, phenyl), 7.33 (d, 1H, pyridine), 7.18 (t, 1H,
pyridine), 3.94 (s, 2H, benzyl), 2.98 (s, 2H, pyridylmethyl), 2.58 (m,
1H, piperidine), 2.20 (m, 2H, piperidine), 1.54 (t, 2H, piperidine).
{4-(N-benzylpiperidine)}-pyridin-2-ylmethyl-amino)-acetic acid (L2)
To a solution of 2 (195 mg, 0.7 mmol) in methanol (10 mL),
bromoacetic acid (97 mg, 0.7 mmol) was added along with
potassium carbonate (0.1 mg, 0.07 mmol) and stirred at room
temperature for 5 days. The solvent was removed under
vacuum. The residue was extracted in chloroform (3 ꢁ 10 mL)
and the combined extracts were washed with water and dried
over anhydrous sodium sulphate. TLC (silica): 20% MeOH/CHCl3
Rf (product) = 0.5.
[
99mTcN]Pip-DTC (I)
To 0.1 mL solution of L1 (50 mg, 0.2 mM) in methanol, 1 mL of
[
99mTcN]21 precursor prepared using the kit vial was added. The
reaction mixture was incubated at room temperature for 30 min.
The complex was characterized by TLC and HPLC.
1H-NMR (d ppm, CDCl3): 8.54 (d, 1H, pyridine), 7.64 (t, 1H,
pyridine), 7.33 (s, 5H, phenyl), 7.32 (d, 1H, pyridine), 7.18 (t, 1H,
pyridine), 3.94 (s, 2H, benzyl), 3.55 (s, 2H, –CH2COOH), 2.90 (s,
2H, pyridylmethyl), 2.58 (m, 1H, piperidine), 2.09 (m, 4H,
piperidine), 1.54 (t, 4H, piperidine); 13C-NMR (1H-decoupled
d ppm, CD3OD) 28.3 (2C, piperidine), 32.90 (2C, piperidine), 53.8
(C, piperidine), 59.5 (C, pyridylmethyl), 58.8 (C, –CH2COOH) 62.9
(C, benzyl), 123.6 (C, pyridine), 125 (C, pyridine), 128.6 (C,
phenyl), 129.3 (2C, phenyl), 130.9 (2C, phenyl), 138.5 (C, phenyl),
99mTcðCOÞ3-PPAA (II)
To 0.1 mL solution of L2 (400 mg, 0.6 mM) in methanol, 0.4 mL of
[
99mTc(CO)3(H2O)3]1 precursor was added. pH was adjusted to 7
and the reaction mixture was heated at 1001C for 20 min. The
complex was characterized by HPLC.
[
99mTcðCOÞ3-PipIDA]ꢀ (III)
138.6 (C, pyridine), 149.6 (C, pyridine), 161 (C, pyridine), 177.7 (C, To 0.1 mL solution of L3 (500 mg, 0.83 mM) in methanol, 0.4 mL
–CH2COOH); MS (ESI) mass calculated for C20H25N3O2; 339,
of [99mTc(CO)3(H2O)3]1 precursor was added. pH was adjusted to
4 and the reaction mixture was heated at 1001C for 20 min. The
complex was characterized by HPLC.
found 340 [M1H]1.
4-iminodiacetato-N-benzylpiperidine (L3)
Biodistribution and blocking studies
To a solution of 1 (38 mg, 0.18 mmol) in methanol (3 mL),
Biodistribution studies were carried out in normal Swiss mice
(20–25 g, 4–5 weeks old). The radiolabeled complex (0.1 mL,
3–7 MBq) was injected via the tail vein. After 5 min, 30 min and
2 h post-injection, animals (n = 3) were sacrificed. All major
organs were excised, weighed and counted for radioactivity in a
NaI(Tl) flat geometry detector. Radioactivity has been expressed
as percentage of injected dose per gram of tissue. To determine
the receptor specificity, blocking studies were carried out where
animals were administered intraperitoneal injection of 25 mg of
(1)-pentazocine 1 h prior to the administration of radiolabeled
complex. After 5 min post-injection of the complex, animals
were sacrificed and percent radioactivity associated with each
organ was estimated.
bromoacetic acid (50 mg, 0.36 mmol) was added along with
potassium carbonate (0.07 mg, 0.5 mmol). The reaction mixture
was stirred at room temperature for 5 days. Subsequently,
solvent was removed under rotary evaporation. TLC (silica): 20%
MeOH/CHCl3 Rf (product) = 0.7.
1H-NMR (d ppm, D2O): 7.39 (s, 5H, phenyl), 3.86 (s, 2H, benzyl),
3.34 (s, 4H, –CH2COOH), 2.28 (m, 1H, piperidine), 1.98 (m, 4H,
piperidine), 1.62 (t, 4H, piperidine); 13C-NMR (d ppm, CD3OD)
27.92 (2C, piperidine), 30.0 (2C, piperidine), 52.62 (C, piperidine),
58.8 (2C, –CH2COOH), 62.83 (C, benzyl), 128.60 (C, phenyl),
129.45 (2C, phenyl), 130.73 (2C, phenyl), 137.94 (C, phenyl),
177.87 (2C, –CH2COOH); MS (ESI): mass calculated for
C16H22N2O4; 306, found 307 [M1H]1.
J. Label Compd. Radiopharm 2010, 53 198–204
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

D. Satpati et al.
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Conclusion
Three different ligands derived from the parent molecule
4-amino-N-benzylpiperidine have been radiolabeled with differ-
ent Tc-99m cores and studied for their capability to target
receptors in vivo. The three 99mTc-complexes could be prepared
in 498% radiochemical yields. However, the significant uptake
in the brain was observed only for one of the complex,
[
99mTcN]Pip-DTC which could be completely blocked on
injection of sigma-1 receptor specific drug, (1)-pentazocine.
99mTcN]Pip-DTC has also shown promising results towards in
[
vivo binding with sigma receptor albeit with uptake in liver.
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F. Refosco, R. Pasqualini, A. Duatti, Inorg. Chem. 1999,
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Acknowledgements
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The authors are grateful to Dr V. Venugopal, Director, Radio-
chemistry and Isotope Group for his constant support and
encouragement. Authors are thankful to Dr K. Kothari for her
help and CIS Bio International for providing 99mTc-nitrido kits as
free samples as a part of IAEA’s coordinated research project.
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J. Label Compd. Radiopharm 2010, 53 198–204