Synthesis and biological evaluation of a radioiodinated spiropiperidine ligand as a potential σ1 receptor imaging agent

Article abstract of DOI:10.1002/jlcr.1777

We report the synthesis and evaluation of 1′-(4-[¹²⁵I] iodobenzyl)-3H-spiro[isobenzofuran-1,4′-piperidine] ([¹²⁵I] Spiro-I) as a potential SPECT tracer for imaging of σ₁ receptors. [¹²⁵I]Spiro-I was prepared in 55-65% isolated radiochemical yield, with radiochemical purity of >99%, via iododestannylation of the corresponding tributyltin precursor. In receptor binding studies, Spiro-I displayed low nanomolar affinity for σ₁ receptors (σ₁: K_i = 2.75±0.12 nM; σ₂: K_i = 340 nM) and high subtype selectivity (σ₂/σ₁ = 124). Biodistribution in mice demonstrated relatively high concentration of radioactivity in organs known to contain σ₁ receptors, including the lung, kidney, heart, spleen, and brain. Administration of haloperidol 5 min prior to injection of [¹²⁵I]Spiro-I significantly reduced the concentration of radioactivity in the above-mentioned organs. These findings suggest that the binding of [¹²⁵I]Spiro-I to σ₁ receptors in vivo is specific. Copyright

Full text of DOI:10.1002/jlcr.1777

Research Article
Received 25 May 2009,
Revised 18 March 2010,
Accepted 23 March 2010
Published online 17 June 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1777
Synthesis and biological evaluation of a
radioiodinated spiropiperidine ligand as a
potential r1 receptor imaging agent
Ã
Rui-Qin Chen,a Yan Li,a Qiu-Yan Zhang,a Hong-Mei Jia,a
b
c
Winnie Deuther-Conrad, Dirk Schepmann, Jorg Steinbach,
b
¨
Peter Brust, Bernhard Wunsch, and Bo-Li Liu
b
c
a
¨
We report the synthesis and evaluation of 10-(4-[125I]iodobenzyl)-3H-spiro[isobenzofuran-1,40-piperidine] ([125I]Spiro-I) as a
potential SPECT tracer for imaging of r1 receptors. [125I]Spiro-I was prepared in 55–65% isolated radiochemical yield, with
radiochemical purity of 499%, via iododestannylation of the corresponding tributyltin precursor. In receptor binding
studies, Spiro-I displayed low nanomolar affinity for r1 receptors (r1: Ki = 2.7570.12 nM; r2: Ki = 340 nM) and high subtype
selectivity (r2/r1 = 124). Biodistribution in mice demonstrated relatively high concentration of radioactivity in organs known
to contain r1 receptors, including the lung, kidney, heart, spleen, and brain. Administration of haloperidol 5 min prior to
injection of [125I]Spiro-I significantly reduced the concentration of radioactivity in the above-mentioned organs. These
findings suggest that the binding of [125I]Spiro-I to r1 receptors in vivo is specific.
Keywords: s1 receptor; spiropiperidine; Iodine-125
density of cerebral and cerebellar s1 receptors was found to be
reduced in patients with early stage Alzheimer’s disease as
compared with healthy subjects.10 However, of the s1 tracers
reported to date none has ideal properties for imaging.
[11C]SA4503 was found to have high nonspecific binding,13
while [18F]FPS and [123I]TPCNE were found to have irreversible
Introduction
Sigma receptors represent a distinct class of proteins, of which
two subtypes (s1 and s2) are known.1 They are expressed in the
central nervous system (CNS), endocrine, immune, and certain
peripheral tissues, and are believed to play an important role in
regulating and integrating nervous, endocrine, and immune
responses.2–3 The s1 receptor is believed to regulate glutamate
NMDA receptor function and the release of neurotransmitters
such as dopamine in the CNS. In the healthy brain, s1 receptors
have been implicated in learning and memory. Sigma-1
receptors are also linked to a number of brain disorders,
including schizophrenia, depression, Alzheimer’s disease, as well
as ischemia.4–7 It is noteworthy that the human s1 receptor gene
is located on chromosome 9, band p13, a region known to be
associated with different psychiatric disorders.8 Development of
radiotracers for in vivo imaging of s1 receptors will allow studies
of their involvement in pathological processes in the living
brain, and may provide much needed diagnostic tools for the
above-mentioned diseases.
kinetics due to their high affinity for s1 receptors ([18F]FPS11,14
:
KD = 0.570.2 nM; TPCNE12,15: Ki = 0.67 nM). In order to overcome
the drawback of slow binding kinetics, a lower affinity tracer (in
the nanomolar range) needs to be developed. In the context of
routine clinical applications, imaging with SPECT have some
advantages over PET such as more widespread availability, no
need for an on-site cyclotron and lower cost. Considering
[
123I]TPCNE as the only SPECT radiotracer used for human
imaging so far and the drawback related to its use, our interest is
focused on the development of s1 receptor probes for SPECT
with improved pharmacokinetic profiles.
Many compound classes with diverse structures, such as aKey Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry
of Education, College of Chemistry, Beijing Normal University, Beijing, 100875,
China
arylacetamides, phenylethylamines, benzamides, piperazine,
and piperidines, possess affinity for s1 receptors. In the past
few years, several potential tracers for imaging s1 receptor
bForschungszentrum Dresden-Rossendorf, Institute of Radiopharmacy/
Neuroradiopharmaceuticals Leipzig, 04318 Leipzig, Germany
expression with PET and SPECT have been reported. Among
them, [11C]SA4503,9–10 [18F]FPS,11 and [123I]TPCNE12 have been
evaluated in human studies. For instance, [11C]SA4503 was used
to investigate Parkinson’s disease (PD) patients with PET and this
tracer could be used as an indicator of presynaptic dopaminer-
gic damage in PD.9 In a recent study using [11C]SA4503, the
cInstitute of Pharmaceutical and Medicinal Chemistry of Westfa¨lische Wilhelms-
¨
Universitat Munster, 48149 Munster, Germany
¨
¨
*Correspondence to: Hong-Mei Jia, College of Chemistry, Beijing Normal
University, Beijing, 100875, China.
E-mail: hmjia@bnu.edu.cn
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Copyright r 2010 John Wiley & Sons, Ltd.

R.-Q. Chen et al.
Recently, a new class of spirocyclic piperidines was reported bromide provided compound 2 (Spiro-I). Compound 2 reacted
to have low nanomolar affinity for s1 receptors and high with bis(tributyltin) for 6 h in anhydrous triethylamine at reflux
selectivity over the s2 receptor subtype.16–20 Furthermore, when under nitrogen in the presence of palladium catalyst, followed by
tested against 15 different receptors and transporters the purification to afford yellow-light oil 3 in 87% yield.
reported compounds were found to lack activity at submicro-
[
125I]Spiro-I was prepared via an iododestannylation reaction
molar concentrations. Spirocyclic piperidines are therefore using chloramine-T as the oxidizing agent. The reaction was
attractive lead compounds for SPECT tracer development. Based quenched with sodium metabisulfite, and the resulting mixture
on a QSAR model of spirocyclic piperidines recently developed was purified using a C18 Sep-Pak cartridge. After removing
in our group,21 the affinity of 10-(4-iodobenzyl)-3H-spiro[isoben- inorganic salts including [125I]NaI by washing with water,
zofuran-1,40-piperidine] (Spiro-I) was predicted to be in the low
[
125I]Spiro-I was eluted with ethanol. The product was further
nanomolar range. We have previously reported the crystal purified by radio-HPLC using a reversed-phase column and
structure of this compound.22 In the present study, we have mobile phase consisting of acetonitrile and water (90:10 v/v)
synthesized [125I]Spiro-I, determined its log D value, and with a flow rate of 2 mL/min.
evaluated its potential as a putative SPECT tracer for imaging
In order to identify the radiotracer, the stable Spiro-I was co-
of s1 receptors by in vitro radioligand binding assays and injected and co-eluted with the radioactive product. Their HPLC
biodistribution studies in mice.
profiles using acetonitrile and water (85:15 v/v) as mobile phase
at a flow rate of 1 mL/min are presented in Figure 2. From Figure
2, the retention times of stable Spiro-I and [125I]Spiro-I were
observed to be 4.62 and 5.00 min, respectively. The difference in
retention time was in good agreement with the time lag which
is due to the distance between the UV and radioactivity detector
Results and discussion
Chemistry and radiochemistry
The synthetic route of [125I]Spiro-I is shown in Figure 1. of our HPLC system. After purification by HPLC, no significant
Compound 1 was synthesized according to the method reported chemical impurities were detected by analytical RP-HPLC (UV,
in the literature.23 Alkylation of compound 1 with 4-iodobenzyl 231 nm). The isolated radiochemical yield of [125I]Spiro-I was
Figure 1. Synthetic route to [125I]Spiro-I.
Figure 2. HPLC co-elution profiles of Spiro-I and [125I]Spiro-I, with retention time of 4.62 and 5.00 min, respectively.
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 569–574

R.-Q. Chen et al.
55–65% (n = 5). The radiochemical purity was higher than 99%. interest at 15, 30, 60, 120, and 240 min after intravenous
The specific activity of the n.c.a. Na[125I] was 42200 Ci/mmol at administration of 1 mCi [125I]Spiro-I is summarized in Table 1.
the time of delivery. No attempt was made to measure the
[
125I]Spiro-I showed a high initial brain uptake, with slow
specific activity of [125I]Spiro-I. To prepare a suitable solution of clearance and high brain-to-blood ratios. The radioactivity
[
125I]Spiro-I for in vivo use, the eluted radioactive peak concentration in the brain peaked at 15 min post-injection
corresponding to [125I]Spiro-I was collected. After the solvent (p.i.) with 5.2870.41 %ID/g, and slowly cleared thereafter with
was removed in vacuo, the product was re-dissolved in less than 4.6670.38 %ID/g at 30 min, 4.1070.33 %ID/g at 60 min,
0.1 mL ethanol and diluted with sterile saline to provide 2.7070.23 %ID/g at 120 min and 1.5270.16 %ID/g at 240 min
approximately 1 mCi of radioactivity per 0.1 mL of solution.
p.i.. In contrast to the high uptake and retention in the brain, the
The in vitro stability of [125I]Spiro-I was evaluated by measuring blood radioactivity levels were low with 1.2670.25 %ID/g at
the radiochemical purity (RCP) at different time points. The results 15 min and 0.5870.10 %ID/g at 240 min p.i., resulting in high
showed that [125I]Spiro-I was chemically stable in 0.9% NaCl brain-to-blood ratios (4.19, 5.18, 5.06, and 3.70 at 15, 30, 60, and
solution at room temperature. After 6 h of incubation with mouse 120 min p.i., respectively). Compared with its 18F-labelled analog,
plasma at 371C, the RCP of [125I]Spiro-I was still higher than 99%,
which suggests that [125I]Spiro-I possesses high stability in vitro.
The apparent distribution coefficient of [125I]Spiro-I was
[
125I]Spiro-I showed higher uptake in the brain and a similar
clearance rate.20 In terms of clearance rate in the brain,
125I]Spiro-I may be superior to [123I]TPCNE, the most promising
[
determined by measuring the distribution of the radiotracer SPECT imaging agent of s1 receptor until now. [125I]Spiro-I
between 1-octanol and 0.05 mol/L sodium phosphate buffer at showed slow washout from the brain, while nearly no brain
pH = 7.4. Considering the significant influence of small quantities radioactivity of [123I]TPCNE was decreased at 4 h p.i. (1.0670.08
of radioactive impurity on the apparent log p value of the and 1.0770.20 %ID/g at 0.33 and 4 h p.i., respectively).15
radiotracer using the counting method,24 pre-washing of the Second, high uptakes (3.41–12.13 %ID/g at 15 min p.i.) were
1-octanol layer containing [125I]Spiro-I with aqueous buffer was observed in organs known to contain s receptors, including the
performed. It was found that the log D value was o2.0 without lungs, kidneys, heart, spleen, and liver, followed by slow
pre-washing, while it increased to nearly 3.0 after pre-washing clearance over time. Third, the accumulation of radioactivity in
for one time. With two successive washing procedures, the log D the thyroid at 240 min p.i. was quite low suggesting that
value of [125I]Spiro-I was finally determined to be 3.0670.11
(n = 4) at pH 7.4, from which one can expect a good blood-brain
barrier penetration of this radiotracer.
[
125I]Spiro-I is relatively stable to in vivo deiodination.
In order to verify the specific binding of [125I]Spiro-I to s
receptors in vivo, the effects of preinjection of haloperidol
In vitro radioligand competition studies
The s1 and s2 receptor affinities of Spiro-I were determined in
competition experiments with the radioligands (1)-[3H]pentazo-
cine and [3H]ditolylguanidine using guinea pig brain and rat liver
preparations, respectively. In the s1 assay Spiro-I showed high
affinity (Ki = 2.7570.12nM) and the corresponding binding curve
is displayed in Figure 3. The low s2 affinity of Spiro-I (Ki = 340 nM)
indicates a high subtype selectivity (s2/s1 = 124), which suggests
that it is a promising s1 receptor ligand for neuroimaging.
Biodistribution and blocking studies in mice
In vivo biodistribution studies of [125I]Spiro-I were performed in
female ICR mice. The uptake of radioactivity in the organ of
Figure 3. Binding curve of Spiro-I in competitive binding assay.
Table 1. Biodistribution of [125I]Spiro-I in female ICR micea
Organ
15 min
30 min
60 min
120 min
240 min
Blood
Brain
Heart
Liver
Spleen
Lung
Kidney
Thyroid
Stomachb
Muscle
Small intestine
1.2670.25
5.2870.41
3.4170.40
12.1371.46
6.1770.23
8.2070.88
11.2471.68
3.2270.82
2.8070.49
1.9470.27
16.9371.62
0.9070.06
4.6670.38
2.5270.25
11.7071.89
6.6870.70
6.8170.32
9.1670.47
4.0671.28
4.1470.84
1.4270.18
26.5174.85
0.8170.13
4.1070.33
2.0370.27
8.9271.11
6.6670.47
5.6370.78
7.6470.94
3.8070.91
4.3170.99
1.4370.22
16.2973.31
0.7370.27
2.7070.23
1.3770.15
7.4871.10
4.4070.42
3.8670.49
5.5671.31
2.1870.83
5.0171.22
1.3570.25
12.9470.43
0.5870.10
1.5270.16
0.7670.07
4.2270.49
2.7370.56
2.6070.27
3.4970.80
1.3970.52
1.7970.25
0.8970.54
9.3873.75
aData are means of %ID/g of tissue7SD, n = 5.
b%ID/organ.
J. Label Compd. Radiopharm 2010, 53 569–574
Copyright r 2010 John Wiley & Sons, Ltd.
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R.-Q. Chen et al.
(0.1 mL, 1.0 mg/kg) on the biodistribution of radioactivity in Melting point was recorded on an X-6 micro melting point
various organs of female ICR mice were examined. Either saline apparatus (Beijing Taike Co., LTD, China) and was uncorrected.
or haloperidol was injected 5 min prior to the radiotracer
HPLC analyses were performed on a Shimadzu SCL-10AVP
injection. The results of blocking studies at 60 min p.i. are given system (SHIMADZU Corporation, Japan) which consisted of a
in Table 2. Of particular interest was a significant reduction binary pump with on-line degasser, a model SPD-10AVP UV-VIS
(84%, po0.001) in the brain accumulation of radioactivity at detector operating at a wavelength of 231 nm, and a Packard
60 min p.i.. Moreover, the concentration of radioactivity in 500TR series flow scintillation analyzer (Packard BioScience Co.,
organs known to possess s receptors was significantly reduced USA). The samples were analyzed on a Agilent TC-C18(2) column
with heart by 55%, spleen by 60%, and lung by 60%. These data (150mmꢁ 4.6 mm, 5 mm) using acetonitrile and water (85:15 v/v)
suggest that the binding of [125I]Spiro-I to s receptors was as mobile phase at a flow rate of 1 mL/min. HPLC separation was
specific in vivo.
carried out on an Alltech HPLC system with Alltech HPLC pump
Currently, there are no SPECT tracers for imaging of s1 model 626, a linear UVIS-201 detector operating at a wavelength
receptors in clinic use, and only one putative tracer, [123I]TPCNE, of 231 nm, and a Bioscan flow count. The sample was separated
has been evaluated in human studies. Unfortunately, [123I]TPCNE on an Alltech Alltima RPC-18 column (250mmꢁ 4.6 mm, 5 mm)
suffers from irreversible binding in the human brain suggesting using acetonitrile and water (90:10 v/v) as mobile phase at a flow
that a tracer with less avid binding to s1 receptors will be better rate of 2 mL/min.
suited for clinical applications.12 With a Ki value of 2.75 nM,
Spiro-I may therefore possess more suitable binding affinity to
s1 receptors than TPCNE (Ki = 0.67 nM).12,15 Moreover, Spiro-I has
lower s2 receptor affinity (Ki = 340 nM vs Ki = 38.8 nM, respec-
tively), and better subtype selectivity (s2/s1 = 124 vs s2/s1 = 58).
In biodistribution studies, [125I]Spiro-I was found to have high
brain uptake, high specific binding to s1 receptors, as well as
limited in vivo deiodination. It is noteworthy that [125I]Spiro-I
showed clearance from the brain after the peak uptake at
15 min. The results warrants further evaluation of [125I]Spiro-I as
a putative tracer for imaging s1 receptors with SPECT.
Chemistry
10-(4-iodobenzyl)-3H-spiro[isobenzofuran-1,40-piperidine](2)
Compound 1 (178mg, 0.94mmol) was dissolved in anhydrous
acetonitrile (50 mL). K2CO3 (1.3g, 94mmol), KI (120 mg,
0.72 mmol), and 4-iodobenzyl bromide (280mg, 0.94 mmol) were
added. The mixture was refluxed for 8 h. After cooling the
inorganic salts were filtered off and the solvent was removed
under reduced pressure. The residue was purified by chromoto-
graphy (ethyl acetate: petroleum ether =1:4 v/v) to afford white
solid: yield 30%. The solid was crystallized from ether/petroleum
ether as colorless crystal. Melting point: 118.7–119.61. IR (KBr,
cmꢀ1): 2940, 1049, 758. 1H-NMR(400 MHz, CDCl3)d(ppm): 7.64–7.66
(d, J = 8.2Hz, 2H), 7.12–7.28 (m, 6H), 5.06 (s, 2H), 3.55 (s, 2H), 2.83
(d, J = 10.4 Hz, 2H), 2.45 (t, J= 11.6 Hz, 2H), 1.97–2.04 (m, 2H), 1.76
(d, J= 12.2Hz, 2H). 13C-NMR (400 MHz, CDCl3)d: 145.61, 138.95,
137.39, 131.29, 127.81, 127.61, 127.38, 121.08, 120.83, 92.47, 84.58,
70.76, 62.67, 50.09, 36.53. EI-MS (m/z): 405(M1).
Experimental
General
[
125I]NaI (specific activity as I: 17.4 Ci/mg, solvent: 1.0Eꢀ05 mol/L
NaOH, pH = 8–11) was bought from PerkinElmer Life and
Analytical Sciences. All other reagents and chemicals were
purchased from commercial sources and used without further
1
purification. H NMR and 13C NMR spectra were recorded on a
Bruker Avance-500 (500 MHz) or a Bruker Avance III (400 MHz) NMR
spectrometer. Chemical shift (d) are reported in ppm downfield
from tetramethylsilane and coupling constants (J) are reported in
Hertz (Hz). MS spectra were obtained by GC2000/TRACETM EI/MS
(Finigan Co., USA) or Quattro micro API ESI/MS (Waters, USA).
10-(4-(tributylstannyl)benzyl)-3H-spiro[isobenzofuran-1,40-piperi-
dine] (3)
Compound 2 (98 mg, 0.24 mmol) was dissolved in anhydrous
triethylamine (10 mL) under nitrogen. Bis(tributyltin) (0.36 mL,
Table 2. Effects of preinjection of haloperidol (0.1 mL, 1.0 mg/kg) 5 min prior to the injection of [125I]Spiro-I on the
biodistribution of radioactivity in female ICR micea
Organ
60 min(control)
60 min(blocking)
% blocking
pb
Blood
Brain
Heart
Liver
Spleen
Lung
Kidney
Thyroid
Stomachc
Muscle
Small intestine
1.3570.23
4.0370.51
2.7170.22
7.9470.84
5.3570.57
6.6270.13
5.8270.27
2.4670.66
5.9370.79
1.4970.21
10.3472.45
1.5470.09
0.6370.11
1.2370.21
5.4270.43
2.1470.27
2.6370.34
4.4870.52
1.4570.17
5.5670.69
0.8770.14
8.3271.69
114
ꢀ84
ꢀ55
ꢀ32
ꢀ60
ꢀ60
ꢀ23
ꢀ41
ꢀ6
0.176
o0.001
o0.001
o0.001
o0.001
o0.001
0.002
0.052
0.494
0.004
0.261
ꢀ42
ꢀ20
aData are means of %ID/g of tissue7SD, n = 4–5;
bp values for the control vs blocking group at 60 min p.i. calculated by Student’s t test (independent, two-tailed).
c%ID/organ.
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J. Label Compd. Radiopharm 2010, 53 569–574

R.-Q. Chen et al.
0.71 mmol), tetrakis(triphenylphosphine) Palladium(0) (11 mg, tube. After adding 0.50 mL buffer to the octanol fraction and
0.01 mmol) were added. The mixture was refluxed for 6 h. After 0.05 mL of 1-octanol to the aqueous fraction, activity in both
cooling the inorganic salts were filtered off and the solvent was tubes was measured in an automatic g-counter (Wallac 1470
removed under reduced pressure. The residue was purified by Wizard, USA). Accurate volumes of each counted phase were
chromotography (ethyl acetate:petroleum ether = 1:4 v/v) to determined by weight and known densities. The distribution
afford yellow-light oil, yield 87%. IR(KBr, cmꢀ1): 2922, 1464, 1049. coefficient was determined by calculating the ratio of cpm/mL
1H-NMR(500 MHz, CDCl3)d(ppm): 7.47ꢀ7.17(m, 8H), 5.10 (s, 2H), of 1-octanol layer to that of buffer layer and expressed as log D.
3.61(s, 2H), 2.90(d, J = 10.2 Hz, 2H), 2.47(t, J = 11.5 Hz, 2H), 2.05 Samples from the 1-octanol layer were re-distributed until
(t, J = 12.2 Hz, 2H), 1.80 (d, J = 13.2 Hz, 2H), 1.60ꢀ1.54 (m, 6H), consistent distribution coefficient values were obtained. The
1.39ꢀ1.34 (m, 6H), 1.10ꢀ1.06 (m, 6H), 0.93ꢀ0.90 (m, 9H). measurement was carried out in triplicate and repeated four
13C-NMR (400 MHz, CDCl3)d: 145.78, 140.36, 138.95, 136.40, 129.00, times.
127.52, 127.32, 126.61, 121.02, 120.85, 84.76, 70.72, 63.49, 50.20,
36.60, 29.10, 27.39, 13.68, 9.56. ESI (m/z): 570.5 ([M1H]1).
In vitro radioligand competition studies
s1 and s2 receptor competition binding assays were performed
as previously reported.16 The s1 receptor asssay was performed
using a guinea pig brain membrane preparation as receptor
material and (1)-[3H]pentazocine as radioligand. Nonspecific
binding was determined with 10 mM unlabeled (1)-pentazocine.
The s2 receptor affinity was determined using rat liver
membrane preparation with the radioligand [3H]ditolylguani-
dine in the presence of 2 mM (1)-pentazocine to mask
s1-binding sites. Nonspecific binding was determined with
10 mM ditolylguanidine. Ki values were calculated according to
Cheng and Prusoff and represent data from at least three
independent experiments, each performed in triplicate. The
results are given as mean7standard error of the mean
Radiochemistry
To a solution of the tributyltin precursor (3) in ethanol (1 mg/
mL), 100 mL solution of the precursor, 2 mL Na125I (795 mCi, carrier
free, specific activity 42200 Ci/mmol), 100 mL chloramine-T
(2.0 mg/mL), and 100 mL HCl (1 mol/L) were added to a sealed
vial. The reaction mixture was kept at room temperature for
10 min. The reaction was quenched with 100 mL Na2S2O5
solution (50 mg/mL). The pH value of the mixture was adjusted
to 7.0–7.2 with NaOH solution (1 mol/L). The mixture was then
passed across a C18 Sep-Pak cartridge. After elution with water,
inorganic salts including [125I]NaI was separated from the
product. The crude product was recovered into a sample vial
by slowly flushing the cartridge with 10 mL of absolute ethanol.
The solvent was evaporated under reduced pressure and the
residue was purified by radio-HPLC. As analyzed by HPLC, the
final product was obtained with a radiochemical purity of
499%. The isolated radiochemical yield of [125I]Spiro-I was
55–65% (n = 5). In order to identify the radioactive product, the
stable Spiro-I was co-injected and co-eluted with the radioactive
product.
Biodistribution and blocking studies in mice
Experiments in female ICR mice (n = 5, 18–22 g) were carried out
in compliance with the national laws related to the care and
experiments on laboratory animals. The HPLC-purified
[
125I]Spiro-I was injected via tail vein (0.1 mL, 1 mCi). The mice
were sacrificed by cervical fracture at various time points.
Samples of blood and organs of interest were removed,
weighed and counted in an automatic g-counter (Wallac 1470
Wizard, USA). The results were expressed in terms of the
percentage of the injected dose per gram (%ID/g) of blood or
organs.
For blocking studies, mice were injected via tail vein with
either saline (0.1 mL) or haloperidol (0.1 mL, 1.0 mg/kg) 5 min
prior to radiotracer injection. The animals were sacrificed by
cervical fracture at 60 min p.i. Blood or organs were isolated and
analyzed as described above for the biodistribution study.
Significant differences between control and test groups were
determined by Student’s t test (independent, two-tailed). The
criterion for significance was pp0.05.
In vitro stability
The in vitro stability of [125I]Spiro-I was evaluated by monitoring
the RCP at different time points. [125I]Spiro-I in 0.9% NaCl was
kept at room temperature for up to 6 h. The RCP of [125I]Spiro-I
was determined by radio-HPLC chromatography at 1, 2, 3, 4, 5,
and 6 h.
To 1.0 mL of fresh mouse plasma, 0.1 mL of [125I]Spiro-I
solution (188 mCi) was added and incubated at 371C. At 1, 2, 3, 4,
5, and 6 h, 0.15 mL aliquots were withdrawn and treated with
0.15 mL methanol to precipitate the proteins. Samples were
then centrifuged at 3000 rpm for 10 min. The supernatants of
each sample were analyzed by radio-HPLC.
Conclusion
Determination of log D value
[
125I]Spiro-I has been synthesized and evaluated as a potential
The log D value of [125I]Spiro-I was determined by measuring
the distribution of the radiotracer between 1-octanol and
0.05 mol/L sodium phosphate buffer at pH = 7.4. The two phases
were pre-saturated with each other. 1-octanol (3 mL) and
phosphate buffer (3 mL) were pipetted into a 10 mL plastic
centrifuge tube. 1.5 mL of a solution of HPLC-purified [125I]Spiro-I
(1 mCi) in ethanol was then added. The tube was vortexed for
5 min, followed by centrifugation for 10 min (3000 rpm, Anke
TDL80-2B, China). About 50 mL of the 1-octanol layer was
weighed in a tared tube. The 1-octanol layer was removed and
about 500 mL of the buffer layer was weighed in a second tared
tracer for imaging of s1 receptors with SPECT. The radiotracer
has been prepared in good radiochemical yield and high
radiochemical purity. In vitro competition binding assays
showed that Spiro-I exhibit low nanomolar affinity for s1
receptors and high subtype selectivity. The log D value of
[
125I]Spiro-I was found to be 3.0670.11, which is within the
range expected to give high brain uptake. In biodistribution
studies [125I]Spiro-I was found to have high initial brain uptake
followed by slow clearance. The binding pattern of the tracers in
vivo was found to be in good agreement with the known
distribution of s1 receptors, and blocking studies confirmed
J. Label Compd. Radiopharm 2010, 53 569–574
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

R.-Q. Chen et al.
high specific binding. The results warrants further evaluation of
125I]Spiro-I as a putative tracer for imaging s1 receptors with
SPECT.
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[
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the corrections. The authors are grateful to Prof. Frank Wuest for
his helpful suggestions of the organic synthetic part. This work
was supported by the National Natural Science Foundation of
China (No. 20501004).
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