Synthesis and in vivo evaluation of [O-methyl-11C] N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl) benzenesulfonamide as an imaging probe for 5-HT6 receptors

Article abstract of DOI:10.1016/j.bmc.2011.06.090

The serotonin receptor 6 (5-HT₆) is implicated in the pathophysiology of cognitive diseases, schizophrenia, anxiety and obesity and in vivo studies of this receptor would be of value for studying the pathophysiology of these disorders. Therefor

Full text of DOI:10.1016/j.bmc.2011.06.090

Bioorganic & Medicinal Chemistry 19 (2011) 5255–5259
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vivo evaluation of [O-methyl-11C] N-[3,5-dichloro-2-
(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide
as an imaging probe for 5-HT6 receptors
Fei Liu a, Vattoly J. Majo b, Jaya Prabhakaran b, Matthew S. Milak b,c, J. John Mann b,c,d, Ramin V. Parsey b,c
,
J.S. Dileep Kumar b,c,
⇑
a Division of Substance Abuse, Department of Psychiatry, Columbia University Medical Center, New York, USA
b Division of Molecular Imaging and Neuropathology, Department of Psychiatry, Columbia University Medical Center, New York, USA
c New York State Psychiatric Institute, 1051 Riverside Drive, Box #42, New York, NY 10032, USA
d Department of Radiology, Columbia University, Medical Center, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The serotonin receptor 6 (5-HT6) is implicated in the pathophysiology of cognitive diseases, schizophre-
nia, anxiety and obesity and in vivo studies of this receptor would be of value for studying the
pathophysiology of these disorders. Therefore, N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-
piperazinyl)benzenesulfonamide (SB399885), a selective and high affinity (pKi = 9.11) 5-HT6 antagonist,
has been radiolabeled with carbon-11 by O-methylation of the corresponding desmethyl analogue with
Received 14 April 2011
Revised 10 June 2011
Accepted 16 June 2011
Available online 19 July 2011
[
11C]MeOTf in order to determine the suitability of [11C]SB399885 to quantify 5-HT6R in living brain using
Keywords:
PET
Brain
Carbon-11
Radiotracer
Serotonin
PET. Desmethyl-SB399885 was prepared, starting from 1-(2-methoxyphenyl) piperazine hydrochloride,
in excellent yield. The yield obtained for radiolabeling of [11C]SB399885 was 30 5% (EOS) and the total
synthesis time was 30 min at EOB. PET studies with [11C]SB399885 in baboon showed fast uptake fol-
lowed by rapid clearance in the brain. Highest uptake of radioactivity of [11C]SB399885 in baboon brain
were found in temporal cortex, parahippocampal gyrus, pareital cortex, amygdala, and hippocampus.
Poor brain entry and inconsistent brain uptake of [11C]SB399885 compared to known 5-HT6R distribution
limits its usefulness for the in vivo quantification of 5-HT6R with PET.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
utility of the currently developed radiotracers for in vivo imaging
of 5-HT6R are limited in scope. The [18F]-labeled 5-HT6 ligand
The 5-hydroxytryptamine 6 receptor (5-HT6R) is the most re-
cently identified mammalian 5-hydroxytryptamine (5-HT) recep-
tor.1–3 In the central nervous system (CNS), an important role of
the 5-HT6R has been confirmed in cognitive functions, seizures,
feeding behavior, anxiety, epilepsy, dementia psychosis, addiction
and affective disorder.4–11 In vitro studies using 5-HT6R antago-
nists revealed that 5-HT6R are localized almost exclusively in the
CNS.12 Several human postmortem studies demonstrated that the
distribution of 5-HT6R appear to be similar to that in rats, with
the highest 5-HT6R densities found in the striatum, nucleus accum-
bens and olfactory tubercle, moderate densities in the amygdala,
hypothalamus, thalamus, hippocampus and cerebral cortex, but
none have been detected in the periphery.13–15
[
18F]12ST05 did not reveal any specific binding to the 5-HT6R in
the rats and cat, although the radioligand showed excellent brain
penetration.16 11C]GSK224558, an undisclosed Glaxo Smithkline’s
[
compound rapidly enters the porcine brain, but undergoes rapid
metabolism with peak regional tissue concentrations reached at
approximately 20 min post-injection. The research group at Glaxo
Smithkline also reported the synthesis and evaluation of a new
O
O
S
N
O
F
N
In vivo imaging of 5-HT6R with positron emission tomography
(PET) may facilitate the understanding of the pathophysiology of
diseases associated with the changes in this receptor and allow
occupancy studies to assist new drug development. However, the
N
N
S
O
N
O
18F
11CH3
11C]GSK215083
[
18F]12ST05
[
⇑
Corresponding author. Tel.: +1 (212) 543 1163; fax: +1 (212) 543 1054.
E-mail address: dk2038@columbia.edu (J.S. Dileep Kumar).
Figure 1. 5-HT6 PET tracers available in literature.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.090

5256
F. Liu et al. / Bioorg. Med. Chem. 19 (2011) 5255–5259
generation of 5-HT6R antagonists based on the 3-benzenesulfonyl-
8-piperazine-1-yl-quinoline scaffold namely [11C]GSK215083.17,18
[
by reverse phase high performance liquid chromatography (RP-
HPLC) giving an average radiochemical yield of 30 5% (EOS, n
= 4). The chemical identity of [11C]SB399885 was confirmed by
co-injection with a sample of standard SB399885 on analytical
RP-HPLC. Chemical and radiochemical purities of [11C]SB399885
were found to be >95% with a specific activity 2500 500 Ci/mmol
(EOS, n = 4). Average time required for the [11C]-labeling was
30 min.
11C]GSK215083 (Fig. 1), is currently evaluated in human and
demonstrated uptake and retention in the human brain, and the
highest BP was observed in caudate and putamen followed by
the frontal cortex. However, treatment with selective 5-HT2A
antagonist ketanserin decreased 90% of specific binding of
[
11C]GSK215083 in cortical regions thereby demonstrating non-
specific binding.19,20
SB399885, (N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-
3-(1-piperazinyl)benzenesulfonamide), is a brain-penetrating, po-
tent and selective 5-HT6R antagonist (pKi = 9.11). SB399885 also
possesses excellent selectivity (>200-fold) over 5-HT1A, 5-HT1B, 5-
2.2. Determination of unchanged radioligand in plasma
Figure 2 shows the time course of the unmetabolized fraction
of [11C]SB399885 in baboon plasma. Only polar metabolites were
observed and the fraction of unmetabolized [11C]SB399885 was
99% at 2 min, 94% at 4 min, 71% at 12 min, 45% at 30 min, 32% at
60 min and 27% at 90 min. The [11C]SB399885 exhibits 1% and 5%
free fraction (fp or unbound radiotracer) in human and baboon
plasma respectively.
HT1D, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT4, 5-HT7, a1B
,
D2, D3 and D4 receptors as well as ion channels and enzymes.21–23
In addition the lipophilicity of SB399885 was adequate for BBB
penetration. The attractive affinity, selectivity and moderate lipo-
philicity (ClogP value 3.5 0.68 measured with ACD log PDB
program) and the presence of C-11 labeling site prompted us to
develop [11C]SB399885 as a PET tracer for 5-HT6R. Herein, we de-
scribe preparation of the desmethyl precursor, radiosynthesis of
2.3. PET imaging studies with [11C]SB399885 in baboon
[
11C]SB399885 and its in vivo evaluation in non-human primate
PET images in baboon show that [11C ]SB399885 enter the BBB
and the radioactivity is not retained in brain. The time activity
curves (TACs) demonstrate the higher uptake of radioactivity is
found in temporal cortex, parahippocampal gyrus, pareital cortex,
amygdala, and hippocampus, whereas, prefrontal cortex, anterior
cingulate, striatum and cerebellum shows lower uptake of radioac-
tivity (Fig .3).
with PET.
2. Results
2.1. Chemistry and radiochemistry
The precursor phenolic alcohol 5 was synthesized from com-
mercially available 1-(2-methoxyphenyl)piperazine hydrochloride
in four steps (Scheme 1).
3. Discussion
Protection of piperazine moiety by trichloroacetyl chloride in
the presence of diisopropylethylamine provided 1-(2-methoxy-
phenyl)-4-trichloroacetylpiperazin 2 in 87% yields. The crude
product was treated with excess of chlorosulfonic acid to give
the key intermediate 3-(4-trichloroacetylpiperazin-1-yl)-4-meth-
oxybenzenesulfonyl chloride (3) in 77% overall yield. Coupling of
compound 3 with 2-amino-4,6-dichlorophenol in the presence
of pyridine afforded the corresponding trichloroacetylsulfonamide
4. Removal of the protecting group under basic condition gave the
final compound, desmethyl SB399885 precursor 5, N-(3,5-di-
chloro-2-hydroxyphenyl)-4-methoxy-3-piperazin-1-ylbenzene-
sulfonamide. The synthesis of [11C]SB399885 was achieved by
O-alkylation of the normethyl moiety of the precursor using
The affinity (pKi = 9.11) and selectivity of SB399885 for 5HT6R
makes [11C]SB399885 promising candidate as a PET tracer. The
presence of an aromatic methoxy function gives easy access to
[
[
[
11C]-methylation of the corresponding desmethyl compound with
11C]CH3I or [11C]CH3OTf. The optimum reaction condition for
11C]-labeling was found to be [11C]MeOTf in acetone containing
precursor and aqueous NaOH, providing [11C]SB399885 in high
yield and specific activity suitable for imaging studies. The lipo-
philicity value of 3.5 0.5, measured in terms of log Po/w, is favour-
able for BBB penetration of
[
11C]SB399885. Radiolabeled
metabolites found in baboon during the time of PET scanning
elutes before the parent compound on a reverse phase HPLC col-
umn, indicating higher polarity than [11C]SB399885. These metab-
olites are unlikely to cross the BBB because of their high polarity
and hence the image obtained could be attributed to the parent
[
11C]MeOTf in acetone at room temperature for 5 min (Scheme 2).
The radiolabeled product was separated from the reaction mixture
Scheme 1. Synthesis of 5. Reagents and conditions: (a) CCl3COCl, CH2Cl2, rt, 4 h: 87%; (b) ClSO2OH, CH2Cl2, rt, 1 h: 77.5%; (c) 2-amino-4,6-dichlorophenol, CH2Cl2, pyridine,
0 °C, overnight: 96%; (d) KOH, THF, reflux, 20 h, 74%.

F. Liu et al. / Bioorg. Med. Chem. 19 (2011) 5255–5259
5257
Scheme 2. Radiosynthesis of [11C]SB399885. Reagents and conditions: (a) [11C]CH3OTf, NaOH, acetone, rt, 5 min.
120
100
80
60
40
20
0
4. Materials and methods
4.1. General
The commercial chemicals and solvents used in the synthesis
were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO),
Fisher Scientific Inc. (Springfield, NJ), or Lancaster (Windham,
NH) and were used without further purification. Analytical grade
reagents were purchased from standard commercial sources. 1H
NMR spectra were recorded on a Bruker PPX 300 and 400 MHz
spectrometer. Spectra were recorded in CDCl3 or CD3OD and chem-
ical shifts and reported in ppm relative to TMS as internal standard.
The mass spectra were recorded on JKS-HX 11UHF/HX110 HF
Tandem Mass Spectrometer in the FAB+ mode. Thin layer
chromatography (TLC) was performed using Silica gel 60 F254
plates from E Merck. HPLC analyses were performed using a
Waters 1525 binary HPLC system (analytical: Phenomenex, Prod-
0
20
40
60
80
100
Time (min)
Figure 2. Unmetabolized parent fraction of [11C]SB399885 in baboon plasma.
[
11C]SB399885. The distribution of [11C]SB399885 binding in the
baboon did not correspond to distribution of 5-HT6R reported for
rats and human in which striatum, shows higher binding whereas
cortical regions, amygdala, thalamus and hippocampus shows
moderate binding.28,29 Highest uptake of radioactivity of
igy ODS 4.6 Â 250 mm, 5
l
m column; semipreparative: Phenome-
nex, Prodigy ODS(3) 10 Â 250 mm, 10
lm column). Flash column
chromatography was performed on Silica gel (Fisher 200–400
mesh) using the solvent system indicated. [11C]carbon dioxide
was produced from an RDS112 cyclotron (Siemens, Knoxville, TN)
and [11C]Methyl iodide was synthesized using the reaction of
[
11C]SB399885 in baboon were found in temporal cortex, parahip-
pocampal gyrus, pareital cortex, amygdala, and hippocampus. The
reason for the discrepancies of [11C]SB399885 binding regions in
baboon is not clear and may be attributed to a species difference
or the ligand can be a P-glycoprotein substrate which prevent
the brain uptake of the radioligand. The poor brain accumulation
of [11C]SB399885 and the lack of selectivity to ROI in brain limits
the potential of [11C]SB399885 as an in vivo imaging agent to
quantify 5-HT6R. However, a structure–activity relationship study
of SB399885 may provide a better PET ligand with in vivo kinetics
that permit valid and reliable measurement of 5-HT6R binding.
[
11C]CO2 with lithium aluminum hydride, followed by hydroiodic
acid and was then converted to [11C]methyl triflate at 200 °C by
passing through a glass column impregnated with silver triflate.24
For detection of radiolabeled [11C]SB399885, gamma ray detector
(Bioscan Flow-Count fitted with a NaI detector) was used in series
with the UV detector (Waters Model 996 set at 254 nm). Data
acquisition for both the analytical and preparative systems was
accomplished using a Waters Empower Chromatography System.
CER
ACN
AMY
CAU
PAR
PFC
PIP
PUT
TEM
HIP
0.05
0.045
0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
THA
0
10
20
30
40
50
60
70
80
90
Time (min)
Figure 3. Time activity curves of [11C]SB399885 in baboon. AMY = Amygdala; ACN = Anterior cingulate, CAU = Caudate; CER = Cerebellum; HIP = Hippocampus; PAR =
Parietal cortex; PFC = Prefrontal cortex, PIP = Parahippocampal gyrus; PUT = Puteman; TEM = Temporal cortex; THA = Thalamus.

5258
F. Liu et al. / Bioorg. Med. Chem. 19 (2011) 5255–5259
PET studies were performed in baboon with an ECAT EXACT HR+
scanner (Siemens, Knoxville, TN). All animal experiments were
carried out with the approval of the Institutional Animal Care
and Use Committee of Columbia University Medical Center and
New York State Psychiatric Institute. Metabolite analyses were per-
formed using Phenomenex Prodigy column (ODS3, 4.6 Â 250 mm,
1H NMR (CDCl3, 400 MHz): 7.82–7.78 (1H, m), 7.59–7.55 (1H, d,
J = 5.6), 7.43 (2H, m), 7.06 (1H, m), 6.94–6.90 (1H, d, J=), 5.32 (1H,
s), 4.04–4.02 (4H, m), 3.94 (s, 3H), 3.11 (4H, m). HRMS calculated
(FAB+): C19H18Cl5N3O5S, 574.9410, Observed, 574.9400
4.2.4. N-(3,5-Dichloro-2-hydroxyphenyl)-4-methoxy-3-
piperazin-1-yl-benzenesulfonamide (5)
10
lm. The free fractions and metabolites were measured using
Packard Instruments Gamma Counter (Model E5005, Downers
Grove, IL).
A 1 M solution of potassium hydroxide (0.6 ml) was added over
5 min to a rapidly stirred solution of N-(3,5-Dichloro-2-hydroxy-
phenyl)-4-methoxy-3-[4-(2,2,2-trichloro-ethanoyl)-piperain-1-
yl]-benzenesulfonmide (4) (100 mg) in tetrahydrofuran (15 mL) at
room temperature. After stirring for 20 h, the stirred, ice-cooled
mixture was adjusted to pH 7.0 by the addition of concentrated
hydrochloric acid to afford the title compound (5) as a light brown
solid which was extracted with dichloromethane (2 X 10 ml) and
washed with water (10 ml), brine (10 ml) and dried (62 mg, 74%).
1HNMR (CDCl3, 400 MHz): 7.48–7.38 (2H, m), 7.25–7.23 (m,
1H), 7.18 (m,1H), 6.97–6.95 (m, 1H), 6.85–6.77 (1H, d, J = ), 5.27
(m,1H), 3.97–3.86 (m, 4H), 3.83 (3H, s), 3.12–3.02 (m,4H), 1.80
4.2. Synthesis
Synthesis of desmethyl-SB399885 (5) was based on a published
procedure with few modifications.25
4.2.1. 1-(2-Methoxyphenyl)-4-trichloroacetylpiperazin (2)
A solution of trichloroacetyl chloride (0.5 ml, 4.46 mmol) in
anhydrous dichloromethane (5 ml) was added over 10 min to a
stirred solution of 1-(2-methoxyphenyl) piperazine hydrochloride
(1) (1.00 g, 4.37 mmol) in anhydrous dichloromethane (5 ml) in
the presence of diisopropylethylamine (1.6 ml, 8.92 mmol) at room
temperature under nitrogen atmosphere. The reaction mixture was
stirred for 4 h at room temperature under N2. Then the reaction
was quenched with water (5 ml) and extracted with CH2Cl2. The
combined organic layers were washed with water (3 Â 15 ml)
and brine (10 ml), and dried over anhydrous magnesium sulfate.
The solvent was evaporated under reduced pressure to give the
title compound (2) as light brown oil (1.28 g, 87%).
(1H,s). HRMS calculated (FAB+):
Observed, 432.0556.
C17H20O4N3Cl2S, 432.0552,
4.3. Radiosynthesis of [O-methyl-11C]-N-[3,5-dichloro-2-
(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)
benzenesulfonamide([11C]SB399885)
The precursor desmethyl-SB399885 (5) (0.5–1.0 mg) was
dissolved in 400
ll of acetone in a capped 1-ml V-vial. NaOH
1H NMR (CDCl3, 400 MHz): 7.09–7.06 (1H, m), 6.96–6.90 (m,
3H) 4.13 (3H, m), 3.89 (s, 3H), 3.19–3.18 (4H, m), 1.32–1.26 (1H
m). HRMS calculated (FAB+): C13H15Cl3N2O2, 336.0199: Observed,
336.0188.
(8 ll, 5 N) was added and the resultant solution was allowed to
stand for 1 min. [11C]CH3OTf was transported by a stream of argon
(20–30 ml/min) into the vial over approximately 5 min at room
temperature. At the end of the trapping, the product mixture was
diluted with 0.5 ml of deionized water and directly injected onto
4.2.2. 3-(4-Trichloroacetylpiperazin-1-yl)-4-
a
semipreparative RP-HPLC (Phenomenex, prodigy C18,
methoxybenzenesulfonyl chloride (3)
10 Â 250 mm, 10 m), and eluted under isocratic conditions with
l
A solution of 1-(2-methoxyphenyl)-4-trichloroacetylpiperazine
(2) (1.00 g, 2.96 mmol) in anhydrous dichloromethane (7 ml) was
added over 30 min to ice-cooled chlorosufonic acid (6 ml, mmol).
After stirring for 0.5 h at 0 °C, the reaction mixture was stirred for
1 h at room temperature under nitrogen atmosphere. Then the reac-
tion mixture was poured into a mixture of ice-water (20 ml) and
dichloromethane (20 ml) with rapid stirring. The layers were sepa-
rated and the organic phase was washed with water (4 Â 15 ml) and
brine (10 ml), and dried over anhydrous magnesium sulfate. The
solvent was evaporated under reduced pressure to give the title
compound (3) as viscous oil (1.03 g, 77.5%).
a mobile phase composed of acetonitrile/0.1 M AMF ammonium
formate buffer 30:70 (v/v) at a flow rate of 10 mL/min. The product
fraction with a retention time of 6–7 min based on a gamma
detector was collected, diluted with 100 ml of deionized water,
and passed through a classic C-18 Sep-Pak Ò cartridge, and washed
with 10 ml water. The yield of formation of [11C]SB399885 is
typically in the range of 100–200 mCi (30% yield, based on
[
11C]CH3OTf at end of synthesis (EOS)). The product fraction was
reconstituted with 1 ml of absolute ethanol and 9 ml of sterile
saline. The saline solution of [11C]SB399885 was passed through a
sterile 0.22 lm membrane filter into a vented sterile, pyrogen-free
bottle. Aliquots of the formulated solution was analyzed by
analytical HPLC column using UV and gamma detectors
1H NMR (CDCl3, 400 MHz): 7.80–7.76 (1H, m), 7.50–7.49 (m,
1H), 7.06–7.02 (m, 1H), 4.04 (4H, m), 4.02 (s, 3H), 3.25–3.22 (4H,
m). HRMS calculated (FAB+): C13H14Cl4N2O4S, 433.9428, Observed,
433.9431.
(Phenomenex C18, Prodigy ODS3, 4.6 Â 250 mm, 5
lm; mobile
phase: acetonitrile/0.1 M AMF, 35/65 (v/v), flow rate: 2 ml/min,
retention time: 8.1 min) to determine the specific activity and
chemical and radiochemical purity.
4.2.3. N-(3,5-Dichloro-2-hydroxyphenyl)-4-methoxy-3-[4-
(2,2,2-trichloro-ethanoyl)-piperain-1-yl]-benzenesulfonmide
(4)
4.4. Partition coefficient measurement
A
solution of 3-(4-trichloroacetylpiperazin-1-yl)-4-meth-
oxybenzenesulfonyl chloride (3) (512 mg, 1.17 mmol) in dry
dichloromethane (15 ml) was added dropwise to a mixture of 2-
amino-4,6-dichlorophenol (276 mg, 1.55 mmol) in dry dichloro-
methane (5 ml) and dry pyridine (0.1 ml) at 0 °C under nitrogen
atmosphere. After stirring overnight at room temperature, the
reaction mixture was washed with 1 M hydrochloric acid (2 ml),
water (2 Â 15 ml) and brine 10 (ml), and dried over anhydrous
magnesium sulfate. The solvent was evaporated under reduced
pressure, and the product was purified by silica gel column chro-
matography using 1% methanol in methylene chloride as eluent
to afford the compound (4) as a brown solid (655 mg, 96%).
Partition coefficient (log Po/w) of [11C]SB399885 was measured
by mixing 0.1 mL of the radioligand formulation with 5 gram each
of 1-octanol and freshly prepared PBS buffer (pH 7.4) in a culture
tube.26 The culture tube was shaken mechanically for 5 min fol-
lowed by centrifugation for 5 min. Radioactivity per 0.5 g each of
1-octanol and aqueous layer was measured using a well counter.
The partition coefficient was determined by calculating the ratio
of counts/g of 1-octanol to that of buffer. 1-Octanol fractions were
repeatedly portioned with fresh buffer to get consistent values for
partition coefficient. All the experimental measurements were
performed in triplicate.

F. Liu et al. / Bioorg. Med. Chem. 19 (2011) 5255–5259
5259
4.5. PET studies in baboons
[
11C]SB399885 is 30 min from EOB using [11C]MeOTf in acetone,
with a 30% yield at EOS with excellent chemical and radiochemical
purities and high specific activity. PET studies of [11C]SB399885 in
anesthetized baboon showed that the tracer penetrates the BBB
and the influx into brain was high for the first few minutes, but
radioactivity then declined rapidly. The distribution of
PET studies were performed according to a protocol approved
by the Institutional Animal Care and Use Committee of Columbia
University Medical Center and New York State Psychiatric
Institute.
[
11C]SB399885 was also found to be not identical to 5-HT6R
4.5.1. General imaging procedure
distribution reported for rat and human. This suggests that
11C]SB399885 is not a useful PET tracer for imaging 5-HT6R in
baboon. However, structure activity relationship study of
11C]SB399885 may provide ligands for identifying the optimal
PET scans were performed in two male baboons with an ECAT
EXACT HR+ scanner (CPS/Knoxville, TN). The fasted animal was
immobilized with ketamine (10 mg/kg, im) and anesthetized
with 1.5–2.0% isoflurane via an endotracheal tube. Core temper-
ature was kept constant at 37 °C with a heated water blanket. An
intravenous infusion line with 0.9% NaCl was maintained during
the experiment and used for hydration and radiotracer injection.
An arterial line was placed for obtaining arterial samples for the
input function. The head was positioned at centre of the field of
view, and a 10 min transmission scan was performed before
the tracer injection. For each scan, 5 1 mCi (S.A. of 2300 250
Ci/mmol) of [11C]SB399885 was injected as an iv bolus and emis-
sion data were collected for 90 min in 3-D mode. Plasma samples
were taken every 10 s for the first 2 min, using an automatic
system, and thereafter manually for a total of 30 samples over
90 min.
[
[
PET tracer for imaging 5HT6R.
References and notes
1. Woolley, M. L.; Marsden, C. A.; Fone Kevin, C. F. Curr. Drug Targets: CNS Neurol.
Disord. 2004, 3, 69.
2. Monsma, F. J.; Shen, Y.; Ward, R. P.; Hamblin, M. W.; Sibley, D. R. Mol.
Pharmacol. 1993, 43, 320.
3. Plassat, J. L.; Amlaiky, N.; Hen, R. Mol. Pharmacol. 1993, 44, 229.
4. Mitchell, E. S.; Neumaier, J. F. Pharmacol. Ther. 2005, 108, 320.
5. Reavill, C.; Rogers, D. C. Curr. Opin. Investig. Drugs. 2001, 2, 104.
6. Russell, M. G. N.; Dias, R. Curr. Top. Med. Chem. 2002, 2, 643.
7. Yoshioka, M.; Matsumoto, M.; Togashi, H.; Mori, K.; Saito, H. Life Sci. 1998, 62,
1473.
8. Svenningsson, P.; Tzavara, E. T.; Qi, H. S.; Carruthers, R.; Witkin, J. M.; Nomikos,
G. G.; Greengard, P. J. Neurosci. 2007, 27, 4201.
9. Otano, A.; Frechilla, D.; Cobreros, A.; Cruz-Orive, L. M.; Insausti, A.; Insausti, R.;
Hamon, M.; Del Rio, J. J. Neurosci. 1999, 92, 1001.
10. Routledge, C.; Bromidge, S. M.; Moss, S. F.; Price, G. W.; Hirst, W. D.; Newman,
H.; Riley, G.; Gager, T.; Stean, T.; Upton, N.; Clarke, S. E.; Brown, A. M.;
Middlemiss, D. N. Br. J. Pharmacol. 2000, 130, 1606.
11. Woolley, M. L.; Bentley, J. C.; Sleight, A. J.; Marsden, C. A.; Fone, K. C. F.
Neuropharmacology 2001, 41, 210.
12. Hirst, W. D.; Minton, J. A. L.; Bromidge, S. M.; Moss, S. F.; Latter, A. J.; Riley, G.;
Goutledge, C.; Middlemiss, D. N.; Price, G. W. Br. J. Pharmacol. 2000, 130, 1597.
13. Boess, F. G.; Riemer, C.; Bös, M.; Bentley, J.; Bourson, A.; Sleight, A. J. Mol.
Pharmacol. 1998, 54, 577.
4.5.2. Protein binding and metabolite analyses
The protein binding of [11C]SB399885 in baboon blood samples
were determined as described elsewhere.27 The percentage of
radioactivity in plasma as unchanged [11C]SB399885 was deter-
mined by HPLC. The HPLC system consisted of a pump (Renin),
and an injector equipped with a sensitive
c-detector. The injection
volume loaded on the HPLC column was 10–1000
ll. Blood sam-
ples were taken at 2, 6, 12, 30, 60, and 90 min after radioactivity
injection for metabolite analysis. The supernatant liquid obtained
after centrifugation of the blood sample at 2,000 rpm for 1 min
was transferred (0.5 ml) into a tube and mixed with acetonitrile
(0.7 ml). The resulting mixture was vortexed for 10 s, and centri-
fuged at 14,000 rpm for 4 min. The supernatant liquid (1 ml) was
removed and the radioactivity was measured in a well-counter
and the majority (0.8 ml) was subsequently injected onto the HPLC
column [column: Phenomenex, Prodigy ODS (3) 4.6 Â 250 mm,
14. Gerard, C.; Martres, M. P.; Lefevre, K.; Miquel, M. C.; Verge, D.; Lanfumey, L.;
Doucet, E.; Hamon, M.; el Mestikawy, S. Brain Res. 1997, 746, 207.
15. Ward, R. P.; Hamblin, M. W.; Lachowicz, J. E.; Hoffman, B. J.; Sibley, D. R.; Dorsa,
D. M. Neuroscience 1995, 64, 1105.
16. Tang, S.; Verdurand, M.; Joseph, B.; Lemoine, L.; Daoust, A.; Billard, T.; Fournet,
G.; Le Bars, D.; Zimmer, L. Nucl. Med. Biol. 2007, 34, 995.
17. Martarello, L.; Ahmed, M.; Chuang, A. T.; Cunningham, V. J.; Jakobsen, S.;
Johnson, C. N. J. Label. Compd. Radiopharm. 2005, 48, S7.
18. Gee, A. D.; Martarello, L.; Johnson, C. N.; Witty, D. R. WO2006053785A1.
19. Huiban, M.; Passchier, J.; Martarello, L.; Cunningham, V. J.; Jacobson, S.; Gee, A.
D. Neuroimage 2006, 31, T13.
20. Parker, C. A.; Cunningham, V. J.; Martarello, L.; Rabiner, E. A.; Searle, G. E.; Gee,
A. D.; Davy, M.; Johnson, C. N.; Ahmed, M.; Grunn, R. N.; Laurelle, M.
Neuroimage 2008, 41, T20.
21. Hirst, W. D.; Stean, T. O.; Rogers, D. C.; Sunter, S.; Pugh, P.; Moss, S. F.;
Bromidge, S. M.; Riley, G.; Smith, D. R.; Bartlett, S.; Heidbreder, C. A.; Atkins, A.
R.; Lacroix, L. P.; Dawson, L. A.; Foley, A. G.; Regan, C. M.; Upton, N. Eur. J.
Pharmacol. 2006, 553, 109.
22. Wesolowska, A.; Nikiforuk, A. Neuropharmacology 2007, 52, 1274.
23. Foley, A. G.; Hirst, W. D.; Gallagher, H. C.; Barry, C.; Hagan, J. J.; Upton, N.;
Walsh, F. S.; Hunter, A. J.; Regan, C. M. Neuropharmacology 2008, 54, 1166.
24. Jewett, D. M. Appl. Radiat. Isot. 1992, 43, 1383.
25. Bromidge, S. M.; Clarke, S. E.; Gager, T.; Griffith, K.; Jeffrey, P.; Jennings, A. J.;
Joiner, G. F.; King, F. D.; Lovell, P. J.; Moss, S. F.; Newman, H.; Riley, G.; Rogers,
D.; Routledge, C.; Serafinowska, H.; Smith, D. R. Bioorg. Med. Chem. Lett. 2001,
11, 55.
26. Wilson, A. A.; Jin, L.; Garcia, A.; DaSilva, J. N.; Houle, S. Appl. Radiat. Isot. 2001,
54, 203.
10
rate: 2 ml/min, retention time: 6 min] connected to a Waters guard
column (Resolve™ 10 m, 90 Å) equipped with a radioactivity
lm; mobile phase: acetonitrile/0.1 M AMF, 35:65 (v/v), flow
l
detector. The metabolite and free fractions were collected using a
Bioscan gamma detector. All the acquired data were then subjected
to correction for background radioactivity and physical decay to
calculate the percentage of the parent compound in the plasma
at different time points. In order to reaffirm that the retention time
of the parent had not shifted during the course of the metabolite
analysis, a quality control sample of [11C]SB399885 was injected
at the beginning and the end of the study. The percentage of
radioactive parent obtained was used for the measurement of
metabolite-corrected arterial input functions.
27. Gandelman, M. S.; Baldwin, R. M.; Zoghbi, S. S.; Zea-Ponce, Y.; Innis, R. B. J.
Pharm. Sci. 1994, 83, 1014.
28. Roberts, J. C.; Reavill, C.; East, S. Z.; Harrison, P. J.; Patel, S.; Routledge, C.; Leslie,
R. A. Brain Res. 2002, 934, 49.
5. Conclusion
29. East, S. Z.; Burnet, P. W. J.; Leslie, R. A.; Roberts, J. C.; Harrison, P. J. Synapse
2002, 45, 191.
The radiosynthesis of [11C]SB399885, a 5-HT6R antagonist has
been achieved. Total time required for the synthesis of