New positron emission tomography (PET) radioligand for imaging σ-1 receptors in living subjects

Article abstract of DOI:10.1021/jm300371c

σ-1 receptor (S1R) radioligands have the potential to detect and monitor various neurological diseases. Herein we report the synthesis, radiofluorination, and evaluation of a new S1R ligand 6-(3-fluoropropyl)-3-(2- (azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one ([¹⁸F]FTC-146, [ ¹⁸F]13). [¹⁸F]13 was synthesized by nucleophilic fluorination, affording a product with >99% radiochemical purity (RCP) and specific activity (SA) of 2.6 ± 1.2 Ci/μmol (n = 13) at end of synthesis (EOS). Positron emission tomography (PET) and ex vivo autoradiography studies of [¹⁸F]13 in mice showed high uptake of the radioligand in S1R rich regions of the brain. Pretreatment with 1 mg/kg haloperidol (2), nonradioactive 13, or BD1047 (18) reduced the binding of [¹⁸F]13 in the brain at 60 min by 80%, 82%, and 81%, respectively, suggesting that [ ¹⁸F]13 accumulation in mouse brain represents specific binding to S1Rs. These results indicate that [¹⁸F]13 is a promising candidate radiotracer for further evaluation as a tool for studying S1Rs in living subjects.

Full text of DOI:10.1021/jm300371c

Article
pubs.acs.org/jmc
New Positron Emission Tomography (PET) Radioligand for Imaging
σ‑1 Receptors in Living Subjects
Michelle L. James,†,# Bin Shen,†,# Cristina L. Zavaleta,† Carsten H. Nielsen,†,‡ Christophe Mesangeau,§
Pradeep K. Vuppala,∥ Carmel Chan,† Bonnie A. Avery,∥ James A. Fishback,⊥ Rae R. Matsumoto,⊥
Sanjiv S. Gambhir,† Christopher R. McCurdy,§ and Frederick T. Chin*,†
†Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Stanford University, Palo Alto, California 94305-5484,
United States
‡Cluster for Molecular Imaging and Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet, University of
Copenhagen, Copenhagen, Denmark
∥
§Department of Medicinal Chemistry and Department of Pharmaceutics, The University of Mississippi, University, Mississippi
38677-1848, United States
⊥Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, West Virginia
26506-9500, United States
S
* Supporting Information
ABSTRACT: σ-1 receptor (S1R) radioligands have the
potential to detect and monitor various neurological diseases.
Herein we report the synthesis, radiofluorination, and
evaluation of a new S1R ligand 6-(3-fluoropropyl)-3-(2-
(azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one ([18F]FTC-
146, [18F]13). [18F]13 was synthesized by nucleophilic
fluorination, affording a product with >99% radiochemical
purity (RCP) and specific activity (SA) of 2.6 1.2 Ci/μmol
(n = 13) at end of synthesis (EOS). Positron emission
tomography (PET) and ex vivo autoradiography studies of
[18F]13 in mice showed high uptake of the radioligand in S1R
rich regions of the brain. Pretreatment with 1 mg/kg
haloperidol (2), nonradioactive 13, or BD1047 (18) reduced
the binding of [18F]13 in the brain at 60 min by 80%, 82%, and 81%, respectively, suggesting that [18F]13 accumulation in mouse
brain represents specific binding to S1Rs. These results indicate that [18F]13 is a promising candidate radiotracer for further
evaluation as a tool for studying S1Rs in living subjects.
intracellular and extracellular Ca2+ levels and thus have a broad
INTRODUCTION
■
range of neuromodulatory effects.15,16 Certain S1R agonists
Initially the σ receptor (SR) was thought to belong to the
have been shown to regulate endothelial cell proliferation,17
opioid class of receptors;1 however, further pharmacological
improve cognition,18,19 provide neuroprotection,20 and act as
evaluation classified it as a distinct molecular entity, resulting in
antidepressant agents,4,21 while antagonists inhibit cocaine-
its recognition as a separate family of receptors.2 There are two
induced seizures22 and attenuate neuropathic pain,23 high-
known SR subtypes, namely, σ-1 and σ-2 (S1R and S2R,
respectively).3 At present, S1Rs are the most well characterized
lighting the potential of S1Rs as both a diagnostic and
of the two subtypes.4
therapeutic target.
There are numerous different compounds that target SRs,
Despite initial controversy and conflicting ideas, recent key
many of which fall into the following three classes of
discoveries concerning the SR have helped elucidate various
compounds: (1) benzomorphans, such as (+)-pentazocine (1,
Figure 1) and (+)-N-allylnormetazocine (NANM) that
biological aspects about this molecular chaperone and its
putative functional roles.5−7 Mainly located at the endoplasmic
preferentially bind S1Rs (compared to their (−)-enantiomers),
reticulum of cells, S1Rs have been implicated in a host of
(2) endogenous neurosteroids like progesterone (an antagonist
biochemical processes and pathological conditions including
of S1Rs), and (3) butyrophenones, such as the antipsychotic
neurodegenerative diseases, psychiatric disorders, pain sensiti-
agent haloperidol (2, Figure 1) that displays high affinity for
zation, drug addiction, digestive function, regulation of smooth
muscle contraction, and ischemia.4,5,8−13 S1Rs are also highly
expressed in most known human cancers (e.g., breast, lung,
Received: March 17, 2012
colon, ovarian, prostate, brain).9,14 Agonists for S1Rs influence
© XXXX American Chemical Society
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Figure 1. Selected σ-1 receptor (S1R) ligands and radioligands.
a
Scheme 1
a
Reagents and conditions: (a) 3-chloropropionyl chloride, AlCl3, DMF, 85 °C; (b) Et3SiH, CF3COOH, rt; (c) KF, TBAF, reflux; (d) 2-
(hexamethyleneimino)ethyl chloride, K2CO3, DMF, 55 °C.
both SR subtypes.24,25 Over the past two decades, several
groups have reported the development of high affinity S1R
ligands,25−33 and of these, some have been labeled with
radioisotopes (Figure 1) for use in positron emission
tomography (PET) studies.
vesicular acetylcholine transporter (VAChT, Ki = 50 nM),41
and is therefore not entirely specific for S1Rs.11,42,43 In order to
accurately investigate and characterize the in vivo localization
and role of S1Rs in both normal and disease states, a highly
selective and specific imaging agent/technique is essential.
With the aim of synthesizing a new, selective fluorinated PET
radioligand for studying S1Rs in living subjects, we identified a
lead compound from the benzothiazolone class of compounds
originally reported by Yous and colleagues in 2005.33 SN56 (9,
Figure 1, Supporting Information Table 1) from this class was
reported to have high affinity (Ki = 0.56 nM) and apparent high
selectivity for the S1R (S2R/S1R > 1000). More recently, a
tritiated version of this compound, [3H]9, was assessed in
vitro44 and results suggested that it may be a favorable
alternative to the S1R radioligand [3H]1. We devised a strategy
for modifying 9 in a way that would allow incorporation of a
fluorine-18 radiolabel without greatly altering the structure of
the molecule in the hope of maintaining its high affinity and
selectivity for the S1R. Our target molecule, 6-(3-fluoropropyl)-
3-(2-(azepan-1-yl)ethyl)benzo[d]thiazol-2(3H)-one 13 (FTC-
146)45 (Scheme 1) contains a fluoropropyl in place of the
propyl group on 9.
Examining S1Rs in living subjects with PET is an important
step toward understanding the receptor’s functional role and
involvement in disease. PET radioligands specific for S1Rs
could potentially provide a noninvasive means of (1)
investigating the distribution and function of these sites in
living subjects, in both normal and disease states, (2) assessing
receptor occupancy to determine optimal doses of therapeutic
drugs, (3) early detection and staging of S1R-related disease(s),
and (4) monitoring therapeutic response. Some existing S1R
radioligands include [11C]SA4503 (3),34 [18F]FM-SA4503
(4),35 [18F]FPS (5),36 [18F]SFE (6),37,38 [11C]13 (7),30 and
[18F]fluspidine (8)39 (Figure 1). Table 1 within the Supporting
Information highlights the affinity and selectivity profiles of
these compounds and compares them to 1 and 2. At present,
the only radioligand being routinely used in S1R clinical
research is 3; however, this compound also displays affinity for
the emopamil binding protein (EBP, Ki = 1.7 nM)40 and the
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a
Scheme 2. Radiosynthesis of [18F]13
a
Reagents and conditions: (a) benzoic acid, K2CO3, DMF, 110 °C; (b) (2-(hexamethyleneimino)ethyl chloride, K2CO3, DMF, 65 °C; (c) NaOH,
H2O, MeOH, reflux; (d) p-toluenesulfonyl chloride, Et3N, DCM, rt; (e) Kryptofix-222/K+/[18F]F−, DMSO, 150 °C.
To the best of our knowledge, no compounds from the
benzothiazolone class have been evaluated as PET radioligands
for S1Rs. Since 13 has an entirely different scaffold from other
known S1R radiotracers and was born out of a class of highly
selective S1R ligands, we believe studies using this compound
may generate valuable and novel information about the S1R.
In this paper, we report the synthesis of 13, the
radiosynthesis of [18F]13, and the preliminary evaluation of
this new S1R radioligand through the use of cellular uptake
assays, PET imaging of mice, ex vivo autoradiography, and
radiometabolite studies.
respectively. All initial binding assays were performed using
brain homogenates because the CNS is a primary target for the
radiotracer being developed. However, since many reports in
the literature utilize liver for SR binding assays, the compounds
were also tested under these conditions. Results from rat liver
homogenate binding assays were as follows: 13 Ki = 0.96
0.21 nM (S1R) and 467
60 nM (S2R) (Supporting
Information Table 2), 2 Ki = 3.3 0.6 nM (S1R) and 57.2
2.4 nM (S2R), 9 Ki = 1.61 0.10 nM (S1R) and 247 14
nM (S2R). In a NovaScreen and in-house profile of 59 targets,
13 displayed >10 000-fold selectivity for S1R compared to all
other tested targets (Supporting Information Table 2). At 10
000 nM screening concentration, 13 exhibited <50% displace-
ment of the radioligand, and at 100 nM screening
concentration, it exhibited <50% displacement for 10 targets,
including α2-adrenoceptors, histamine H2 receptors, muscarinic
M2 receptors, peripheral muscarinic receptors, neuronal (α-
bungarotoxin insensitive) nicotinic receptors, opioid receptors,
norepinephrine transporters, calcium L type channels, sodium
channels site 2, and acetylcholine esterase. Therefore, even for
targets where there was modest displacement at the screening
concentrations of 13, this still represented a >10 000-fold
preference for S1Rs.
RESULTS
■
Synthesis, log D, and in Vitro Binding of 13.
Compound 13 was prepared according to Scheme 1.
Compounds 10 and 11 were synthesized using known
procedures.44 The fluorinated intermediate 12 was successfully
prepared from 11 via halogen exchange using tetra-n-
butylammonium fluoride (TBAF) and potassium fluoride.
Following this, 12 was alkylated with 2-(hexamethyleneimino)-
ethyl chloride in the presence of potassium carbonate in DMF
to afford 13.
The experimental pKa (10.4) of 13 was determined46 to be
slightly higher than the calculated pKa (9.36), and the
experimental log DPBS,pH7.4 SD was measured to be 1.45
0.04.
In Vitro Half-Life Studies in Mouse and Rat Liver
Microsomes. The metabolic stability of 13 was evaluated in
mouse and rat liver microsomes. First, 13 was incubated in the
presence of an NADPH-generating system at 37 °C for 60 min.
The reaction was initiated by adding cofactors and quenched at
designated time points (0, 5, 10, 15, 30, 45, 60 min) by addition
of an equal volume of ice-cold acetonitrile. 13 was found to
have a half-life of 4.2 min with a clearance of 0.55 mL min−1 g−1
in mouse, and a half-life of 12.6 min with a clearance of 0.18
mL min−1 g−1 in rat liver microsomes.
By use of previously described methods,44 13 was subjected
to radioligand binding assays and found to demonstrate high
affinity (Ki = 0.0025 nM) and superior selectivity for S1Rs
(>145 000-fold selectivity for S1R compared to S2R) in rat
brain homogenates (Supporting Information Table 2). The
affinity of 2 was determined in the same assay to serve as a
reference standard, and it was found to have a Ki of 3.35 0.79
nM for S1R and a Ki of 80.63 14.08 nM for S2R in rat brain
homogenates. In the same assay, 9 displayed a Ki of 1.7 0.1
and 627 115 nM in rat brain homogenates for S1R and S2R,
Radiochemistry. The strategy for generating [18F]13
involved the preparation of a tosylate precursor 17 and its
subsequent radiolabeling with fluorine-18 (Scheme 2).
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Figure 2. Cell uptake results for (A) [18F]13 and (B) [3H]1. Uptake of [18F]13 and [3H]1 was determined in control CHO cells and CHO cells
transfected with σ-1 receptor (S1R) cDNA following incubation for either 30 or 120 min. Results are expressed as counts per minute (CPM)
recorded in a sample from a particular well per CPM recorded in medium per amount of protein (μg) present in a sample from that well.
Compound 11 was reacted with benzoic acid to yield 14, which
was then alkylated with 2-(hexamethyleneimino)ethyl chloride.
Hydrolysis of the intermediate 15 afforded the corresponding
alcohol 16. The tosylate precursor was then prepared by
reacting the alcohol with p-toluenesulfonyl chloride in the
presence of triethylamine. [18F]13 was successfully synthesized
via nucleophilic substitution using an automated GE TRACER-
lab FXFN radiosynthesis module. Fluorine-18 (half-life of 109.8
min) radiolabeling was accomplished by reaction of tosylate
precursor (17) with cyclotron-produced 18F-fluoride as a
Kryptofix-222/K+/[18F]F− complex in DMSO at 150 °C for
Figure 3. Western blot analysis of σ-1 receptor (S1R) expression in
control CHO cells, CHO cells transfected with S1R cDNA, and a
positive control cell line (JAR cells) known to contain S1R protein.
Cell lysates (50 μg of protein) were subjected to gel electrophoresis
followed by immunoblot analysis with S1R specific antibody S-18
(400:1): lane 1, control CHO cells (transfected with empty S1R
vector); lane 2, CHO cells overexpressing S1R (transfected with vector
containing S1R cDNA); lane 3, positive control cell lysate for S1R as
supplied by Santa Cruz Biotechnology (JAR cells). Blot was also
stained for α-tubulin as a protein loading control.
15 min. Semi-preparative reverse-phase high performance liquid
chromatography (HPLC) of the crude reaction mixture
afforded [18F]13 in 3.7
1.9% yield (n = 13) at end of
bombardment (EOB) (and 2.5 1.2% yield at EOS), >99%
radiochemical purity (RCP) with a specific activity (SA) of 3.9
1.9 Ci/μmol (EOB) (and SA of 2.6 1.2 Ci/μmol at EOS)
in a total synthesis time of 75 min. The formulated version of
[18F]13 in saline/ethanol (9:1, total 10 mL) was shown to be
stable for at least 5.5 h via analytical reverse-phase HPLC.
Cell Uptake Studies. Uptake of [18F]13 in Chinese
hamster ovary (CHO) cells was compared to the uptake of
the known S1R ligand [3H]1. Control CHO cells (transfected
with a vector not containing the S1R gene (to serve as a
negative control)) and CHO cells transfected with a vector
containing S1R cDNA (to serve as a positive control for S1R
expression in cells) were used for the uptake assays. Cells were
exposed to [18F]13 or [3H]1 for 30 and 120 min (triplicate for
each time point). The incubated cells were subsequently
washed, lysed, and counted for radioactivity. All collected data
were normalized for amount of protein present in each well.
Data for both uptake assays (Figure 2) showed that there was a
small increase in uptake for both radioligands between 30 and
120 min in control CHO cells. This increase was more
pronounced in CHO cells transfected with S1R cDNA and
numerically higher at both 30 and 120 min compared with
negative control CHO cells. The uptake of [18F]13 in cells
transfected with S1R cDNA was 4-fold higher than uptake in
control CHO cells at 120 min (Figure 2).
administration of [18F]13 and terminated 60 min later. Graphs
depicting uptake of [18F]13 in whole mouse brain as a function
of time for baseline and blocking studies are displayed in Figure
4. The baseline time−activity curve (TAC) (Figure 4)
Western Blot. Western blot analysis was performed using
Image J (image processing and analysis software in Java) and
showed that the level of S1R expression in the CHO cells
transfected with the S1R cDNA was approximately 4.3 times
greater than that found in the control CHO cells that had been
transfected with an empty vector (Figure 3).
Figure 4. Time−activity curves (TACs) from mouse positron
emission tomography (PET) studies. TACs represent accumulation
of [18F]13 in whole mouse brain as a function of time for baseline (n =
3), preblock with 2 (n = 3), preblock with 13 (n = 3), and preblock
with 18 (n = 3). Baseline studies involved iv administration of [18F]13
(95−125 μCi), whereas blocking studies involved pretreatment of
mice with 2 (1 mg/kg), 13 (1 mg/kg), or 18 (1 mg/kg) 10 min prior
to iv administration of [18F]13 (95−125 μCi).
PET Imaging in Mice. The in vivo kinetics of [18F]13 in
normal mice were assessed using small animal PET. Dynamic
brain PET scanning was commenced 1 min prior to
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demonstrated that [18F]13 entered the brain rapidly, peaked
within the first few minutes, and then gradually decreased over
the remaining time of the scan. Pretreatment with 2 (1 mg/kg),
13 (1 mg/kg), or N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-
(dimethylamino)ethylamine (BD1047, 18, 1 mg/kg) 10 min
prior to radioligand administration reduced the binding of [18F]
13 in the brain at 60 min by 80%, 82%, and 81%, respectively
(Figure 4). PET images provided visual evidence that [18F]13
rapidly crossed the blood−brain barrier (BBB) and appeared to
accumulate in known S1R rich regions (Figure 5, sagittal brain
Figure 6. Graph depicting the percentage of intact radiotracer ([18F]
13) in mouse blood, liver, and brain at 30 and 60 min after injection.
than that observed in plasma (34% at 30 min and 27% at 60
min, compared to 60% and 50% for plasma) while the
percentage of intact tracer in the brain remained 100%
throughout the duration of our studies.
DISCUSSION
■
Figure 5. Sagittal mouse PET images and ex vivo autoradiography of
sagittal brain sections (12 μm) obtained 60 min after administration of
[18F]13. Sections used for autoradiography were stained with Nissl for
anatomical correlation (right). Baseline study involved injection of
radiotracer only ([18F]13, 200 μCi), whereas the blocked study
involved pretreatment with known σ-1 receptor ligand 18 (1 mg/kg)
10 min prior to radiotracer administration. PET images (5 min static
scans) were acquired just prior to perfusing mice and harvesting brain
tissue for autoradiography. White dotted lines indicate location of
mouse brain in sagittal PET images: Cb = cerebellum, Ctx = cortex,
FN = facial nucleus, H = hippocampus, Mb = midbrain, Ob = olfactory
bulb.
Since S1Rs are believed to be intimately associated with
numerous psychiatric conditions and neurodegenerative
diseases, radioligands specific for S1Rs have the potential to
serve as novel diagnostic tools and may be useful in assessing
treatment effectiveness. The present study describes the
synthesis and radiolabeling of a new S1R PET radioligand
from the benzothiazolone class of compounds, together with its
preliminary in vitro and in vivo characterization using cell
uptake studies, metabolic stability tests, PET imaging of mice,
and ex vivo autoradiography.
Compound 13 was successfully synthesized (Scheme 1) and
found to have an experimental log D of 1.45 0.04. We also
investigated the in vitro binding properties of 13, and it was
found to display high affinity (Ki = 0.0025 nM, Supporting
Information Table 2) and superior selectivity for S1Rs (>145
000-fold selectivity for S1Rs compared to S2Rs) in rat brain
homogenate when compared to its parent 9. In vitro binding of
13 was also evaluated in liver homogenates and found to have
Ki of 0.96 0.21 nM (S1R) and 467 60 nM (S2R). The
affinity of 2 for S1R and S2R was determined in the same
binding assays to serve as a standard reference. The Ki of 2 in
rat brain for S1R and S2R was found to be 3.35 0.79 and
PET image). There also seemed to be some accumulation in
the snout area and spine over the course of the study. Since the
spatial resolution of PET and partial volume effects did not
allow for accurate determination of radioligand localization in
specific brain regions, ex vivo autoradiography was performed
(see Figure 5 and below description).
Ex Vivo Autoradiography. The distribution of [18F]13 in
the mouse brain was further evaluated by ex vivo auto-
radiography at 60 min after injection of tracer. In brief, mice
were perfused with saline and brains were snap frozen,
sectioned (12 μm), and incubated with phosphor imaging
film overnight. Figure 5 shows the autoradiography results for
selected 12 μm sagittal brain sections from both the baseline
and blocking study (the latter of which was pretreated with 18
(1 mg/kg) 10 min prior to injection of [18F]13). From these
images it can be seen that [18F]13 mainly accumulated in the
midbrain, facial nucleus, cortex, and hippocampus. There was
also some accumulation in the cerebellum, albeit lower than
that observed in the midbrain and cortex. The corpus callosum
appeared to be devoid of [18F]13 binding. Autoradiography
results from the blocking study depicted a dramatic reduction
in [18F]13 uptake in the mouse brain. A similar pattern of
radioligand distribution can be seen when comparing the
sagittal brain PET images with the autoradiography results for
both the baseline and blocking study (Figure 5).
80.63
14.08 nM, respectively, which was comparable to
affinities reported for this compound in the literature,48−51 thus
validating our assay conditions and technique. Our findings
concerning the in vitro binding data for 13 demonstrated that
the small structural modification made to 9 in order to form 13
led to an improvement in affinity and selectivity for S1Rs. In
fact, both the affinity and selectivity (S1R vs S2R) of 13 are
higher than values reported for most other known S1R ligands
(Supporting Information Table 1). The large increase in S1R
affinity found in rat brain assay when comparing 13 with its
parent 9 is unlikely to be explained through classical structure−
activity relationships. One possible explanation for the large
increase in affinity could be that in addition to 13 binding S1Rs,
it may interact with one or more proteins when engaged, which
could affect the affinity at the S1R site. This is because S1Rs are
ligand-gated chaperones that participate in protein−protein
interactions to modulate activity at various cellular targets, such
as ion channels, receptors, and signaling pathways. The reason
for the difference in affinity of 13 in the rat brain and rat liver
assay is unknown but thought to be related to differences in the
Ex Vivo Determination of Radiometabolites: Mouse
Plasma, Liver, and Brain. The percentage of intact [18F]13 in
mouse plasma, liver, and brain (at 30 and 60 min after
administration of tracer) was assessed via HPLC. Figure 6
shows that the percentage of intact [18F]13 in liver was lower
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assay environment, most likely related to lipid composition
and/or variations in protein partners that may be present in
liver versus brain.
uptake in mouse brain within the first few minutes) appear
similar to that reported for [18F]4 and [18F]8 in normal mice.39
On the other hand, the binding profile of [18F]13 at later time
points is quite different from the reported uptake levels for
other known S1R radioligands at corresponding times. For
example, [18F]13 reached its maximum uptake in mouse brain
within the first few minutes of imaging and gradually washed
out to 65% of its maximum at 60 min after injection, whereas
[18F]4 reached its maximum uptake in the brain at 30 min after
injection and did not experience significant washout over the
remainder of the study (120 min after injection). Biodis-
tribution studies with [18F]8 demonstrated that it reached
maximum uptake in the mouse brain at 30 min after injection
and then washed out to a level 81% of its maximum at 60 min
after injection. Future studies involving the use of available S1R
tracers in the same animal (and/or animal model of disease) are
needed to properly understand the relative advantages/
disadvantages of each tracer.
Since there was some observed bone uptake in our mouse
PET studies, we had reason to suspect possible defluorination
of [18F]13. Further investigation of the metabolic profile of 13
revealed that the major metabolites were hydroxylation
(oxidation) products and that a minor metabolite (<10%,
data not shown) of a defluorinated product was apparent at all
time points. The molecular formula suggests a radical
defluorination mechanism, although this needs to be confirmed
through more detailed studies. Importantly, these results are
from in vitro measurements and do not indicate the actual in
vivo metabolic profile of this compound and cannot predict the
metabolism of [18F]13 in different species.
Bone uptake has also been reported in studies using other
S1R tracers35,39 and has been postulated to be due to high
levels of S1Rs in highly proliferative tissues (e.g., bone
marrow). Furthermore, Mavlyutov and colleagues showed
that S1Rs are expressed in the mouse spinal cord.53 We are
currently evaluating [18F]13 in rats and non-human primates,
and preliminary results in rats show less bone uptake. Both rat
and monkey data will be reported in a later publication. Future
studies will involve further characterization of [18F]13,
especially concerning its selectivity and specificity for S1Rs.
Binding to EBP and VAChT will be determined, along with
PET imaging and ex vivo autoradiography in S1R knockout
mice. Other studies will involve comparative analysis of [18F]13
with other known S1R radioligands in the same animal models
of disease (e.g., Alzheimer’s disease, neuropathic pain) to
determine which is most suitable for clinical applications.
In conclusion, we have successfully prepared a new
benzothiazolone 18F-labeled S1R ligand, [18F]13, that demon-
strates promise as a specific PET radiotracer for visualizing
S1Rs in living subjects, and therefore, it warrants further
investigation.
Radiosynthesis of [18F]13 was achieved by nucleophilic
aliphatic [18F]fluorination of compound 17 (Scheme 2). Since
heating the reaction (precursor concentration 2 mg/mL) at 150
°C for 15 min afforded high purity product in sufficient yields
(2−5%, 1−10 mCi/mL) for preliminary in vitro and in vivo
investigations, no further optimizations were pursued at this
stage.
It is typical to differentiate between S1R and S2Rs using
benzomorphan-type opiates such as the well-known selective
S1R radioligand [3H]1. For this reason we selected [3H]1 as
the “gold standard” S1R ligand to compare with [18F]13 in cell
uptake studies. Results obtained from cell uptake studies
demonstrated a 4-fold higher uptake of [18F]13 in CHO cells
transfected with S1R cDNA compared to control CHO cells at
120 min (Figure 2). This was comparable to the 3.6-fold
greater uptake of [3H]1 in transfected CHO cells versus uptake
in control cells at 120 min, indicating that [18F]13 behaves
similarly to [3H]1 and that it might be a more sensitive marker
of S1R levels. Western blot results (Figure 3) verified that the
level of [18F]13 uptake in cell assays (at 120 min) correlated to
the level of S1R protein levels and therefore highlight its
potential as a radioligand for accurately identifying and
visualizing S1Rs.
Following these encouraging in vitro cell uptake results, the
in vivo kinetics and binding of this radiofluorinated ligand were
evaluated in living, normal mice using small animal PET.
Baseline TACs (Figure 4) showed that [18F]13 rapidly crossed
the BBB, reached a maximum uptake of ∼17% ID/g within the
first few minutes, and then slowly washed out of the brain
throughout the remainder of the scan to a level of 6% ID/g at
60 min. Pretreating mice with 2 (1 mg/kg) or 13 (1 mg/kg) 10
min prior to radioligand administration led to a marked
reduction of [18F]13 binding in the brain (80% or 82%
reduction, respectively, at 60 min) (Figure 4). Comparable
results have been reported for other S1R-related rodent
blocking studies with 2.35,52 Since 2 also displays affinity for
dopamine receptor type 2 (D2), S2R, and VAChT, we also
performed blocking studies with 18 (a known S1R selective
antagonist structurally unrelated to 13). Results from these
blocking studies demonstrated 81% reduction in [18F]13
uptake in the brain at 60 min, indicating that [18F]13
accumulation in mouse brain likely represents specific S1R
binding. Ex vivo autoradiography (Figure 5) showed that [18F]
13 mainly accumulated in midbrain (especially facial nucleus),
cortical regions, and hippocampus and to a lesser extent in the
cerebellum and thalamus, which are all known to contain
S1Rs.34 Our autoradiography results were similar to that seen in
other S1R ex vivo autoradiography studies.28,39 PET images and
autoradiography results from the blocking studies with 18 (1
mg/kg) demonstrate notable reduction of [18F]13 accumu-
lation in mouse brain (Figure 5). The PET signal present
outside the brain after blocking with 18 could be due to
peripheral defluorination of [18F]13 leading to uptake of
[18F]fluoride ion in jaw bone and spine. Importantly, ex vivo
radiometabolite analysis revealed that [18F]13 in brain
remained intact throughout the duration of our studies (Figure
6), meaning that the PET signal observed in the brain was
entirely due to intact [18F]13.
EXPERIMENTAL SECTION
■
Chemistry. Unless otherwise stated, reagents and starting materials
were obtained from commercial suppliers and were used without
purification. Precoated silica gel GF Uniplates (Analtech) were used
for thin-layer chromatography (TLC). Column chromatography was
performed on silica gel 60 (Sorbent Technologies). H and 13C NMR
1
spectra were obtained on a Bruker APX400 at 400 and 100 MHz,
respectively. The high-resolution mass spectra were recorded on a
Waters Micromass Q-Tof Micro mass spectrometer with a lock spray
source. The mass spectra were recorded on a WATERS ACQUITY
Ultra Performance LC with ZQ detector in ESI mode.
Although [18F]13 is yet to be evaluated alongside other
fluorinated S1R radioligands, its initial kinetics (i.e., rapid
F
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The purity of final compounds (>97%) was verified by HPLC on a
Waters 2695 system equipped with a Waters X-Bridge C18 column
and a Waters 996 UV detector set at 254 nm. The following conditions
were used: a flow rate of 1 mL/min, a gradient run from 50% eluent A
(H2O), 40% eluent B (acetonitrile), 10% eluent C (H2O/triethyl-
amine, 98.2:0.2) to 0% eluent A, 90% eluent B, 10% eluent C for 12
min. Chemical names were generated using ChemDraw Ultra
(CambridgeSoft, version 10.0).
The UPLC/MS/MS mass spectrophotometer consisted of a Waters
Micromass Quattro Micro triple−quadrupole system (Manchester,
U.K.). The system was controlled by MassLynx software, version 4.0.
Ionization was performed in the positive electrospray mode. The MS/
MS parameters for the analysis were as follows: capillary voltage 4.95
kV, cone voltage 31 V, extractor voltage 5 V, RF lens voltage 0.5 V.
The source and desolvation temperatures were 110 and 400 °C,
respectively, and the desolvation and cone gas flows were 252 and 76
L/h, respectively. The selected mass-to-charge (m/z) ratio transition
of the 13 ion [M + H]+ used in the single ion recording (SIR) was m/z
337.03 The dwell time was set at 500 ms. Calculated pKa and log P
were determined using PALLAS 3.1.2.4 software from CompuDrug
Chemistry, Ltd. (Sedona, AZ, U.S.).
For the reported radiochemistry, semipreparative HPLC separations
were performed on Dionex 680 pump with KANUR UV detector K-
2001 (for purification of [18F]13). Analytical HPLC was performed on
Lab Alliance with model 500 UV detector. Radioactivity in HPLC
eluates was detected with a model 105S single-channel radiation
detector (Carroll & Ramsey Associates). The UPLC system consisted
of Water’s Acquity UPLC instrument (Milford, MA, U.S.) equipped
with a binary solvent manager, vacuum degasser, thermostated column
compartment, and an autosampler. Chromatographic separations were
performed on a Waters Acquity UPLC BEH C18 column (1.7 μm, 2.1
mm × 50 mm). For the metabolite studies, an isocratic method was
developed using a mobile phase containing 0.1% formic acid in water/
0.1% formic acid in methanol (50:50, v/v). For the metabolite
separation, a linear gradient method was developed with a mobile
phase containing 0.1% formic acid in water (A) and 0.1% formic acid
in acetonitrile (B). The linear gradient elution program was as follows:
0−80% B over 6 min, followed by an isocratic hold at 80% B for
another 4 min. At 10 min, B was returned to 0% in 2 min and the
column was equilibrated for 3 min before the next injection. The total
run time for each injection was 15 min. The flow rate was 0.2 mL/min.
The column temperature was maintained at 25 °C, and the injection
volume was 10 μL. For metabolite studies an Agilent 1200 HPLC
system with autosampler and Gabi radioactivity detector (Raytest) was
used.
removed in vacuo, and the residue was purified by chromatography on
a silica gel column using methylene chloride/methanol (9.5:0.5) as the
eluent. 3-(2-(Azepan-1-yl)ethyl)-6-(3-fluoropropyl)benzo[d]thiazol-
2(3H)-one was isolated as a hydrochloride salt (white solid, 0.120 g,
80%) by addition of HCl/dioxane. 1H NMR (D2O): δ 7.34 (br s, 1H),
7.26−7.24 (m, 1H), 7.16−7.14 (m, 1H), 4.46 (dt, J = 47.2, 4.5 Hz,
1H), 4.28 (t, J = 4.8 Hz, 2H), 3.49−3.37 (m, 6H), 2.70−2.66 (m, 2H),
1.97−1.66 (m, 11H). 13C NMR (D2O): δ 173.02 (CO), 137.92
(Cq), 133.68 (Cq), 127.31 (CHar), 122.67 (CHar), 122.09 (Cq),
110.90 (CHar), 84.33 (d, J = 157.6 Hz, CH2), 55.23 (CH2), 53.46
(CH2), 37.47 (CH2), 31.34 (d, J = 18.8 Hz, CH2), 30.30 (d, J = 5.5
Hz, CH2), 25.61 (CH2), 23.37 (CH2). HRMS (ESI+) calculated for
C18H26N2OFS [M + H]+ 337.1750, found 337.1764.
3-(2-Oxo-2,3-dihydrobenzo[d]thiazol-6-yl)propyl Benzoate
(14). K2CO3 (5.31 g, 38.4 mmol) and benzoic acid (9.38 g, 76.8
mmol) were added, under mechanical stirring, to a solution of 11
(3.51 g, 15.4 mmol) in anhydrous DMF (250 mL). The reaction
mixture was heated at 110 °C for 6 h. After cooling, the mixture was
poured into 100 mL of a 2.5 N HCl solution in water, extracted with
ethyl acetate (3 × 70 mL), and the organic phase was washed with
brine. The solvent was dried over Na2SO4 and evaporated in vacuo,
and the residue was purified via chromatography on a silica gel column
using a gradient of petroleum ether/ethyl ether (4:6 to 6:4) as the
eluent. The product was then recrystallized in toluene to give 2.97 g
(62%) of 3-(2-oxo-2,3-dihydrobenzo[d]thiazol-6-yl)propyl benzoate as
1
a white solid. H NMR (DMSO-d6): δ 11.70 (br s, 1H), 7.91 (d, J =
7.6 Hz, 2H), 7.63 (t, J = 7.5 Hz, 1H), 7.49 (t, J = 7.6 Hz, 2H), 7.41 (s,
1H), 7.12 (d, J = 8.1 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 4.25 (t, J = 6.3
Hz, 2H), 2.71 (t, J = 7.4 Hz, 2H), 2.03−1.97 (m, 2H). 13C NMR
(DMSO-d6): δ 170.00, 165.70, 135.77, 134.42, 133.22, 129.76, 129.10,
128.64, 126.58, 123.44, 122.18, 111.34, 64.02, 31.34, 29.93. MS (ESI−)
m/z 312 (M+ − 1, 100), 190 (52) .
3-(3-(2-(Azepan-1-yl)ethyl)-2-oxo-2,3-dihydrobenzo[d]-
thiazol-6-yl)propyl Benzoate (15). K2CO3 (0.75 g, 5.47 mmol) and
2-(hexamethyleneimino)ethyl chloride hydrochloride (0.47 g, 2.37
mmol) were added, under mechanical stirring, to a solution of 14
(0.57 g, 1.82 mmol) in anhydrous DMF (10 mL). The reaction
mixture was heated at 65 °C for 2 h. After cooling, the mixture was
poured into 80 mL of water, extracted with ethyl acetate (3 × 60 mL),
and the combined organic layers were washed with brine and dried.
The solvent was removed in vacuo, and the residue was chromato-
graphed on a silica gel column using diethyl ether as the eluent to give
0.72 g (90%) of 3-(3-(2-(azepan-1-yl)ethyl)-2-oxo-2,3-dihydrobenzo-
[d]thiazol-6-yl)propyl benzoate as a colorless oil. A sample was
1
prepared as a hydrochloride salt for analysis. H NMR (DMSO-d6): δ
11.29 (br s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.66−7.57 (m, 3H), 7.50 (t,
J = 7.6 Hz, 2H), 7.28 (d, J = 8.0 Hz, 1H), 4.43−4.40 (m, 2H), 4.27 (t,
J = 6.0 Hz, 2H), 3.44−3.18 (m, 6H), 2.77 (t, J = 7.2 Hz, 2H), 2.06−
1.56 (m, 10H). 13C NMR (DMSO-d6): δ 168.74 (CO), 165.52 (CO),
136.68 (Cq), 134.23 (Cq), 133.07 (CHar), 129.59 (Cq), 128.92
(CHar), 128.50 (CHar), 126.78 (CHar), 122.45 (CHar), 121.44 (Cq),
111.36 (CHar), 63.83 (CH2), 53.62 (CH2), 52.05 (CH2), 37.02
(CH2), 31.10 (CH2), 29.72 (CH2), 25.58 (CH2), 22.88 (CH2). HRMS
(ESI+) calculated for C25H31N2O3S [M + H]+ 439.2055, found
439.2056.
Synthetic procedures for 6-(3-chloropropanoyl)benzo[d]thiazol-
2(3H)-one (10) and 6-(3-chloropropyl)benzo[d]thiazol-2(3H)-one
(11) are described in the Supporting Information.
6-(3-Fluoropropyl)benzo[d]thiazol-2(3H)-one (12). A mixture
of 11 (0.30 g, 1.32 mmol), KF (0.23 g, 3.95 mmol), and TBAF (1 M in
THF, 3.95 mL, 3.95 mmol) in THF (10 mL) was heated at reflux for 4
h. After completion of the reaction, the reaction mixture was
partitioned between ethyl acetate and water, and the organic layer
was washed with brine and dried. The solvent was removed in vacuo,
and the residue was purified by chromatography on a silica gel column
using petroleum ether/ether (8:2) as the eluent to give 0.010 g (36%)
3-(2-(Azepan-1-yl)ethyl)-6-(3-hydroxypropyl)benzo[d]-
thiazol-2(3H)-one (16). To a solution of 16 (0.67 g, 1.53 mmol) in
methanol (10 mL) was added a solution of sodium hydroxide (0.15 g,
3.75 mmol) in water (10 mL). The mixture was heated at 90 °C for 1
h, concentrated in vacuo, poured into 1 N HCl (20 mL), and extracted
with ethyl acetate (10 mL). The pH of the aqueous layer was adjusted
to 10 with potassium carbonate, and the mixture was extracted with
ethyl acetate (3 × 20 mL). The combined organic layers were washed
with brine, dried, and evaporated. The residue was chromatographed
on a silica gel column using methylene chloride/methanol (9.7:0.3) as
the eluent to give 0.47 g (92%) of 3-(2-(azepan-1-yl)ethyl)-6-(3-
hydroxypropyl)benzo[d]thiazol-2(3H)-one as a white solid. A sample
1
of 6-(3-fluoropropyl)benzo[d]thiazol-2(3H)-one as a white solid. H
NMR (CDCl3): δ 10.33 (br s, 1H), 7.23 (s, 1H), 7.10 (s, 2H), 4.45
(dt, J = 47.2, 5.8 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H), 2.00 (dquint, J =
25.2, 6.8 Hz, 2H). 13C NMR (CDCl3): δ 173.26, 136.39, 133.69,
126.83, 124.09, 122.13, 111.79, 82.77 (d, J = 164.2 Hz), 32.13 (d, J =
19.7 Hz), 31.01 (d, J = 5.2 Hz). MS (ESI−) m/z 210 (M+ − 1).
3-(2-(Azepan-1-yl)ethyl)-6-(3-fluoropropyl)benzo[d]thiazol-
2(3H)-one Hydrochloride (13). K2CO3 (0.180 g, 1.30 mmol) and 2-
(hexamethyleneimino)ethyl chloride hydrochloride (0.080 g, 0.40
mmol) were added, under mechanical stirring, to a solution of 12
(0.090 g, 0.43 mmol) in anhydrous DMF (2 mL). The reaction
mixture was heated at 55 °C for 2 h. After cooling, the mixture was
poured into 10 mL of water, extracted with ethyl acetate (3 × 20 mL),
washed with saturated aqueous NaCl, and dried. The solvent was
1
was prepared as a hydrochloride salt for analysis. H NMR (DMSO-
d6): δ 11.35 (br s, 1H), 7.46 (d, J = 1.2 Hz, 1H), 7.36 (d, J = 8.4 Hz,
1H), 7.50 (dd, J = 8.0, 1.2 Hz, 1H), 4.31 (t, J = 6.8 Hz, 2H), 3.80 (br s,
G
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2H), 3.53 (s, 1H), 3.39−3.29 (m, 6H), 2.60 (t, J = 7.6 Hz, 2H), 1.79
(br s, 4H), 1.68 (quint, J = 8.0 Hz, 2H), 1.58 (br s, 4H). 13C NMR
(DMSO-d6): δ 170.04 (CO), 138.34 (Cq), 134.45 (Cq), 127.41
(CHar), 122.92 (CHar), 121.91 (Cq), 111.58 (CHar), 60.26 (CH2),
54.60 (CH2), 53.07 (CH2), 37.70 (CH2), 34.50 (CH2), 31.47 (CH2),
25.99 (CH2), 23.58 (CH2). HRMS (ESI+) calculated for C28H27N2O2S
[M + H]+ 335.1793, found 335.1786.
monoamine transporters as well as opioid, NMDA, dopamine D2, and
serotonin 5-HT2 receptors) were use for in-house assays, and full
competition assays were conducted if there was >30% displacement of
the radioligand at the 10 000 nM screening concentration. For the in-
house assays, at least 10 concentrations of 13 were tested to generate
IC50 values, which were converted to Ki values using the Cheng−
Prufsoff equation.
In Vitro Half-Life Studies in Mouse and Rat Liver Micro-
somes. Nonradioactive 13 was incubated in the presence of an
NADPH-generating system at 37 °C for 60 min in test tubes. The
basic incubation mixture consisted of 5 mM substrate, 1 mg/mL
microsomal protein, 3 mM MgCl2, 1 mM NADP, 5 mM glucose 6-
phosphate, 1 IU/mL glucose 6-phosphate dehydrogenase, and 100
mM Tris HCl buffer (pH 7.4) in a final volume of 1 mL. The reaction
was initiated by adding cofactors and quenched at designated time
points (0, 5, 10, 15, 30, 45, 60 min) by addition of an equal volume of
ice-cold acetontrile. The mixture was centrifuged at 3000 rpm for 10
min, and the supernatant was analyzed by UPLC/MS/MS.
3-(3-(2-(Azepan-1-yl)ethyl)-2-oxo-2,3-dihydrobenzo[d]-
thiazol-6-yl)propyl 4-Methylbenzenesulfonate (17). A solution
of p-toluenesulfonyl chloride (0.22 g, 1.26 mmol) in methylene
chloride (10 mL) was slowly added to a solution of 16 (0.38 g, 1.15
mmol) and triethylamine (0.34 mL, 2.42 mmol) in methylene chloride
(20 mL). The mixture was stirred for 3 days at room temperature, and
the solvent was evaporated. The residue was purified by chromatog-
raphy on a silica gel column using a gradient of methylene chloride/
methanol (10:0 to 9.7:0.3) as the eluent to give 0.50 g (89%) of 3-(3-
(2-(azepan-1-yl)ethyl)-2-oxo-2,3-dihydrobenzo[d]thiazol-6-yl)propyl
1
4-methylbenzenesulfonate as a pale yellow oil. H NMR (DMSO-d6):
δ 7.78 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.30 (s, 1H), 7.20
(d, J = 8.4 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 4.00−3.93 (m, 4H), 2.71
(t, J = 6.8 Hz, 2H), 2.59−2.55 (m, 6H), 2.41 (s, 3H), 1.88−1.85 (m,
2H), 1.45 (br s, 8H). 13C NMR (DMSO-d6): δ 168.36, 144.68, 135.24,
135.11, 132.32, 129.98, 127.41, 126.45, 122.10, 121.25, 111.11, 69.77,
54.84, 54.21, 40.64, 30.13, 29.80, 27.93, 26.27, 20.95. MS (ESI+) m/z
489 (M+ + 1, 100), 335 (4).
Determination of pKa for 13. The pKa of 13 was determined
using the potentiometric titration method.46 A solution of 0.01 M
sodium hydroxide was prepared and the pH measured as 11.9.
Similarly, 0.01 M hydrochloric acid solution was prepared and the pH
measured as 2.07. To 50 mL of a 1 mM 13 solution, 0.1 mL volume of
sodium hydroxide was added and the pH recorded (Mettler Toledo
SevenEasy pH meter S20) until the pH of the solution became
constant. To the same sample, 0.1 mL portions of hydrochloric acid
were added and pH recorded until it became constant. A titration
curve was then plotted as pH versus volume of base/acid added. The
intersection point of these two curves was noted as the pKa value of
10.4.
Determination of Partition Coefficient (log D) for 13. By use
of the stir-flask method,47 n-octanol and water/PBS, pH 7.4 (equal
quantity), were added to a glass vial (25 mL). The contents were
sealed and stirred continuously for 24 h at 25 °C to achieve mutual
saturation of the phases. Water/PBS, pH 7.4, phase was brought into a
vessel together with a Teflon-coated magnetic stirring bar. The n-
octanol phase containing the known quantity of test substance was
poured very carefully on top of the aqueous phase in order to avoid
emulsion formation as far as possible. The vessel was not shaken;
instead the system was stirred for an extended period of time (at least
36 h) allowing equilibrium to be reached. The contents were allowed
to separate upon standing and then centrifuged. An aliquot of aqueous
layer was taken and diluted (1000 times) for quantitative analysis by
UPLC/MS/MS.
In Vitro Radioligand Binding Assays. Competition binding
assays were performed as previously described.54−56 Briefly, radio-
ligands were used to tag the targeted sites under standard conditions,
with S2R binding performed in the presence of 300 nM 1 to mask
radioligand binding to S1Rs. In addition, SR binding was performed
for nonradioactive 13, 2, and 9 in rat liver homogenates using a 96-well
platform.57 Nonradioactive 13 was initially evaluated at non-SR sites
using a screening concentration of 10 000 nM. If <50% displacement
of the radioligand was observed, then the results were reported as Ki
>10 000 nM. If >50% displacement was observed, for assays run by
NovaScreen/Caliper Life Sciences (Hanover, MD), a second screening
concentration of 100 nM was examined, and Ki values were estimated
based on radioligand displacement of the two screening concentrations
of 13. If the estimated Ki yielded a selectivity ratio relative to S1R of
less than 100-fold, then full competition curves were run; all targets
with moderate affinity for non-SR sites from the NovaScreen assays
greatly exceeded the 100-fold selectivity criterion, and it was not
necessary to run full competition assays. A panel of seven non-SR sites
that have been historically problematic for earlier σ ligands (three
Radiosynthesis of [18F]13. No carrier added-aqueous [18F]-
fluoride ion was produced on a PETtrace cyclotron (GE Healthcare,
Sweden) by irradiation of a 1.6 mL water target using a 16 MeV
proton beam on 95% enriched [18O]H2O by the [18O(p,n)18F] nuclear
reaction. [18F]Fluoride in [18O]H2O was transferred to a GE
TRACERlab FXFN synthesizer and passed through an anion exchange
resin (QMA cartridge in carbonate form, prepared by washing with 1
mL of EtOH and 1 mL of water) under vacuum. Trapped
[18F]Fluoride ions were then eluted from the QMA cartridge and
transferred to the reactor using an eluent solution containing 3.5 mg of
K2CO3 and 15 mg of Kryptofix 222 (K222: 4,7,13,16,21,24-hexaoxa-
1,10-diazabicyclo[8.8.8]hexacosan) in acetronitrile (0.9 mL) and water
(0.1 mL) mixture. The solution was then evaporated at 65 °C under
helium flow and vacuum, followed by heating at 88 °C under vacuum.
Tosylate precursor 17, 3-(2-oxo-3-(2-(azepan-1-yl)ethyl)-2,3-
dihydrobenzo[d]thiazol-6-yl)propyl 4-methylbenzenesulfonate (2
mg) was dissolved in DMSO (1 mL) and added to the dry
Kryptofix-222/K+[18F]F− complex. The mixture was allowed to react
at 150 °C for 15 min. Upon completion, the reaction mixture was
diluted with sterile water (8 mL) and passed through a C18 Sep-Pak
cartridge. The C18-trapped, radiolabeled product was then eluted from
the C18 Sep-Pak with acetonitrile (1.5 mL) and sterile water (1.5 mL).
The resulting crude mixture was then injected onto two serial HPLC
Phenomenex Gemini C-18, 5 μm (10 mm × 250 mm) semipreparative
reversed-phase column. A mobile phase of H2O (0.1% TEA)/
acetonitrile (0.1% TEA), (pH 8): (20/80, v:v) was used with a flow
rate of 5.0 mL/min, and the retention time (tR) of [18F]13 was 13 min.
Tosylate precursor and decomposed hydroxyl derivative were eluted at
17.5 and 10 min, respectively. The radioactive fraction corresponding
to [18F]13 was collected in a round-bottom flask containing sterile
water (15 mL) and then passed through a C18 Sep-Pak. A further 10
mL of sterile water was passed through the C18 Sep-Pak. The trapped,
purified radiolabeled product was eluted from the C18 Sep-Pak using
ethanol (1 mL) and saline (9 mL). The formulated solution was then
filtered through a sterile 13 mm Millipore GV 0.22 μm filter into a
sterile pyrogen free evacuated 30 mL vial. Solutions in saline
containing no more than 10% ethanol by volume were used for the
studies described in this article.
Quality Control of [18F]13. For determination of specific activity
and radiochemical and chemical purity, an aliquot of the final solution
of known volume and radioactivity was injected onto an analytical
reversed-phase HPLC column (Phenomenex Gemini C18 5 μm, 4.6
mm × 250 mm). A mobile phase of H2O (0.1% TEA)/acetonitrile
(0.1% TEA) (20:80, v/v) at a flow rate of 1.0 mL/min was used to
elute [18F]13 with a tR of 8.33 min. The area of the UV absorbance
peak measured at 254 nm corresponding to the carrier product was
measured (integrated) on the HPLC chromatogram and compared to
a standard curve relating mass to UV absorbance.
Cell Uptake Studies Using Transfected Cells. CHO cells were
grown in Ham’s F-12 medium. For uptake studies CHO cells were
transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.)
and either pcDNA (empty vector, negative control) or S1R gene
H
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Journal of Medicinal Chemistry
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(OPRS1, accession number NM_005866.2, OriGene, Rockville, MD,
U.S.) following directions from the manufacturer. The cells were
harvested, and 2 × 105 cells were seeded per well in 24-well plates.
Twenty-four hours later, CHO cells were transfected with either 0.8 μg
of pcDNA (empty vector, control) or 0.8 μg of σ-1 DNA. The
medium was refreshed 12 h later. Twenty-four hours after initial
transfection, Ham’s F-12 medium was prepared containing enough
[18F]13 for 2 μCi per well. After 30 and 120 min uptake, medium from
each of the triplicate wells was aspirated and cells were washed twice
with cold PBS (500 μL). Following this, cells were lyzed with 1 N
NaOH (500 μL). A portion of each lysate (250 μL) was transferred to
a glass tube, and activity was measured with a Cobra II γ counter
(Packard-Perkin-Elmer, Waltham, MA, U.S.). Protein content from
each well was measured by Bradford assay. The same protocol was
followed for [3H]1 (NEN Life Science Products, Boston, MA) except
the activity was measured with a liquid scintillation counter (Beckman
Coulter LS 6500, Brea, CA, U.S.) following addition of scintillation
fluid.
sagittal brain sections were cut using a cryostat microtome HM500
(Microm, Germany). The sections were mounted on microscope
slides (Fisherbrand Superfrost Plus microscope slides), air-dried for 10
min, and then exposed to 18F-sensitive storage phosphor screens
(Perkin-Elmer, U.S.) for 12 h at 4 °C. The image plates were analyzed
using a Typhoon 9410 variable mode imager (Amersham Biosciences,
U.S.), and image data were visualized and processed by Image J (image
processing and analysis software in Java). Anatomy of brain sections
was confirmed by Nissl staining (cresyl violet acetate, Sigma Aldrich).
Ex Vivo Determination of Radiometabolites: Mouse Plasma,
Liver, and Brain. At 30 and 60 min after iv injection of 630−690 μCi
[18F]13, blood, brain, and liver from Balb C mice (n = 2 per time
point) were obtained. Blood samples were collected via cardiac
puncture and were immediately centrifuged at 1800 g for 4 min at
room temperature to separate plasma. Each plasma sample (100 μL)
was transferred to a tube containing acetonitrile (300 μL) and mixed
thoroughly. Water (40 μL) was then added to each tube and after
mixing was centrifuged at 9400g for 4 min. The resulting supernatants
were collected, and an aliquot of each supernatant (100 μL) was
analyzed via the same HPLC method used for quality control of [18F]
13 (fitted with a highly sensitive positron detector for radioactivity).
The percent ratio of intact [18F]13 (tR = 8.33 min) to the total
radioactivity (decay corrected to the time of dose administration) on
the HPLC chromatogram was calculated as % = [(peak area for [18F]
13)/(total peak area)] × 100. Brain and liver samples were
homogenized in 500 μL of ice-cold acetonitrile, and protein
precipitates were sedimented by centrifugation (9400g, 4 min). The
resulting supernatants were analyzed by HPLC as described above.
Samples (100 μL) from each supernatant were measured in a γ
counter to assess the extraction efficiency into supernatant liquids.
Pelleted cells were washed twice with 1 mL of EtOH and then
counted. The activity in the supernatants was compared to that in the
corresponding pellets to afford the percentage of the tracer bound to
serum proteins. No attempt was made to enhance the efficiency of
[18F]fluoride ion extraction by adding carrier fluoride ion prior to
extractions.
Western Blot. Cell lysates from 2 × 106 cells were prepared by
scraping cells in ice-cold harvesting buffer (RIPA lysis buffer, Santa
Cruz Biotechnology). The lysates were centrifuged (14 000 rpm, 20
min at 4 °C), and supernatants were collected. The protein
concentration of the supernatant was determined by Bradford assay.
Equal amounts of protein (50 μg) were loaded onto 10% SDS−
polyacrylamide minigels. After gel electrophoresis, proteins were
transferred to a nitrocellulose membrane and blocked at room
temperature using 5% nonfat milk blocking buffer (15 mL of 1× TBS,
0.01% Tween 20, and 0.75 g of milk powder). Following this, the
membrane was incubated overnight at 4 °C with goat polyclonal anti-
S1R (S-18, sc-22948, Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
primary antibody. The primary antibody was diluted 1:400 in 5%
nonfat milk blocking buffer. After the sample was washed three times
with TBST (TBS with 0.01% Tween 20), bovine anti-goat-IgG
horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnol-
ogy, Inc., Santa Cruz, CA) diluted 1:5000 in TBST was added, and the
mixture was incubated for 1 h at room temperature. After the mixture
was washed three times with TBST, S1R protein was visualized using
ECL reagent (Pierce, Rockford, IL, U.S.) and images were obtained
using film. Blot was also stained for α-tubulin as a protein loading
control. Image J (image processing and analysis software in Java) was
used for Western blot analysis.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures for compounds 10 and 11, tables
outlining affinity and selectivity of some S1R ligands, and on/
off-target binding data for compound 13. This material is
available free of charge via the Internet at http://pubs.acs.org.
Animals. All experimental procedures involving animals were
performed under humane conditions following approval from the
Stanford University or University of Mississippi animal research
internal review board. Animals had access to food and H2O ad libitum
and were kept under a 12 h light/dark cycle.
AUTHOR INFORMATION
Corresponding Author
*Phone: (650) 725-4182. E-mail: chinf@stanford.edu.
■
Small-Animal PET Imaging in Mice. All PET imaging was
performed on a microPET R4 model scanner (Siemens) fitted with a
computer-controlled bed, 10.8 cm transaxial and 8 cm axial field of
view (FOV), no septa and operated exclusively in three-dimensional
list mode. PET images were reconstructed with two-dimensional
OSEM (ordered subsets expectation maximization) and analyzed using
AMIDE (a medical image data examiner) software.58 Normal Balb C
mice (20−30 g) were anesthetized using isoflurane gas (3% for
induction and 1−3% for maintenance). Acquisition of the PET data in
list mode was commenced just prior to iv administration of [18F]13
(95−125 μCi in 100 μL of 0.9% saline) via the tail vein and was
continued for a period of 60 min. Blocking studies involved
pretreatment of mice with 2 (1 mg/kg), 13 (1 mg/kg), or 18 (1
mg/kg) 10 min prior to tracer administration.
Ex Vivo Autoradiography. Sagittal brain sections were obtained
60 min following iv injection of 200−250 μCi [18F]13 for both
baseline and blocking studies. Baseline study involved injection of
radiotracer only, whereas blocking study involved pretreatment with σ-
1 specific ligand 18 (1 mg/kg) 10 min prior to radiotracer
administration. Each mouse underwent a 5 min static brain PET
scan just prior to perfusion. After each mouse was perfused with saline
(10 mL), the brain was removed, snap-frozen in isopentane (in liquid
nitrogen), and embedded in optimal cutting temperature (OCT)
compound (Tissue-Tek, Sakura, U.S.). Subsequently, 12 μm thick
Author Contributions
#M.L.J. and B.S. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was supported in part by the NCI ICMIC P50
Grant CA114747 (S.S.G.) and NIDA Grant DA023205
(C.R.M.). We appreciate the technical assistance of Jamaluddin
Shaikh (West Virginia University, Morgantown, WV) during
some of the binding assays.
ABBREVIATIONS USED
■
AMIDE, a medical image data examiner; BBB, blood−brain
barrier; Cb, cerebellum; cDNA, complementary deoxyribonu-
cleic acid; CHO, Chinese hamster ovary; CPM, counts per
minute; Ctx, cortex; DCM, dichloromethane; DMF, dimethyl-
formamide; DMSO, dimethyl sulfoxide; EBP, emopamil
binding protein; ECL, enhanced chemiluminescence; EOB,
I
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Article
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end of bombardment; EOS, end of synthesis; ESI, electrospray
ionization; FN, facial nucleus; FOV, field of view; H,
hippocampus; HPLC, high performance liquid chromatogra-
phy; HRMS, high-resolution mass spectrometry; ID/g, injected
dose per gram; iv, intravenous; Kryptofix 222, 4,7,13,16,21,24-
hexaoxa-1,10-diazabicyclo[8.8.8]hexacosan; LC, liquid chroma-
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magnetic resonance; Ob, olfactory bulb; OCT, optimal cutting
temperature; OSEM, ordered subsets expectation maximiza-
tion; PBS, phosphate buffered saline; PET, positron emission
tomography; QMA, quaternary methylammonium; RCP,
radiochemical purity; S1R, σ-1 receptor; SA, specific activity;
SR, σ receptor; S2R, σ-2 receptor; TAC, time−activity curve;
TBAF, tetra-n-butylammonium fluoride; TBS, Tris buffered
saline; TEA, triethylamine; THF, tetrahydrofuran; TLC, thin-
layer chromatography; tR, retention time; UPLC, ultra-
performance liquid chromatography; UV, ultraviolet; VAChT,
vesicular acetylacholine transporter
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