Synthesis and in vivo evaluation of [O-methyl-11C] 2-(4-methoxyphenyl)-N-methylbenzyl-N-(1-methylpiperidin-4-yl)acetamide as an imaging probe for 5-HT2A receptors

Article abstract of DOI:10.1002/jlcr.1124

2-(4-Methoxyphenyl)-N-(4-methylbenzyl)-N-(1-methylpiperidin-4-yl)acetamide (AC90179, 4), a highly potent and selective competitive 5-HT_2A antagonist, was labeled by [¹¹C]-methylation of the corresponding desmethyl analogue 5 with [

Full text of DOI:10.1002/jlcr.1124

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 1069–1077.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1124
Research Article
Synthesis and in vivo evaluation of [O-methyl-11C]
2-(4-methoxyphenyl)-N-(4-methylbenzyl)-N-(1-methyl-
piperidin-4-yl)acetamide as an imaging probe for
5-HT2A receptors
Jaya Prabhakaran1, Ramin V. Parsey1, Vattoly J. Majo1, Ronald L. Van
Heertum2, J. John Mann1,2 and J. S. Dileep Kumar1,*
1 Department of Psychiatry and Division of Neuroscience, Columbia University
& New York State Psychiatric Institute, 1051 Riverside Drive, Box: 42, New York,
NY 10032, USA
2 Department of Radiology, Columbia University, New York, NY10032, USA
Summary
2-(4-Methoxyphenyl)-N-(4-methylbenzyl)-N-(1-methylpiperidin-4-yl)acetamide (AC90179,
4), a highly potent and selective competitive 5-HT2A antagonist, was labeled by [11C]-
methylation of the corresponding desmethyl analogue 5 with [11C]methyl triflate. The
precursor molecule 5 for radiolabeling was synthesized from p-tolylmethylamine in
three steps with 46% overall yield. [11C]AC90179 was synthesized in 30 min (30 Æ 5%
yield, EOS) with a specific activity of 4500 Æ 500 Ci/mmol and >99% chemical and
radiochemical purities. Positron emission tomography studies in anesthetized baboon
revealed that [11C]4 Penetrates the blood–brain barrier (BBB) with a rapid influx
and efflux of the tracer in all brain regions. Due to lack of tracer retention or
specific binding, [11C]4 cannot be used as PET ligand for imaging 5-HT2A
receptors. Copyright # 2006 John Wiley & Sons, Ltd.
Received 26 May 2006; Revised 24 July 2006; Accepted 1 August 2006
Key Words: radiotracer; methyl triflate; positron emission tomography; 5-HT2A
receptors
*Correspondence to: J. S. Dileep Kumar, Department of Psychiatry and Division of Neuroscience,
Columbia University & New York State Psychiatric Institute, 1051 Riverside Drive, Box: 42, New York,
NY 10032, USA. E-mail: dk2038@columbia.edu
Contract/grant sponsor: National Institute of Health; contract/grant number: P50MH62185
Copyright # 2006 John Wiley & Sons, Ltd.

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J. PRABHAKARAN ET AL.
Introduction
Alteration of serotonergic 5-HT2A receptors (5-HT2AR) has been reported in
schizophrenia, stress, aggression, suicide and major depression.1–3 These
receptors are also targets for atypical antipsychotic drugs (APDs).4–6 In vivo
imaging of 5-HT2AR with positron emission tomography (PET) may further
our understanding of the pathophysiology of diseases associated with the
changes in this receptor and allow occupancy studies to assist new drug
development. The utility of currently available radiotracers for in vivo imaging
of 5-HT2AR are limited by high non-specific binding, lipophilic metabolites or
slow kinetics (Figure 1).7–12 [18F]altanserin has lipophilic radiometabolites that
cross the blood-brain barrier (BBB) and slow kinetics.10,11 Determining a
suitable reference region for [18F]setoperone is difficult.13 [11C]M100907 is a
selective 5-HT2A PET tracer, but has the disadvantage of slow kinetics making
quantification less reliable.12 Given the limitations of the existing tracers, we
sought to develop 5-HT2A receptor imaging agents that have faster washout to
facilitate modeling within the window of time allowed by the half-life of
carbon-11, without radiolabeled nonpolar metabolites, and with high
selectivity for the 5-HT2A receptor. We describe the radiosynthesis and
in vivo evaluation of [11C] 2-(4-methoxyphenyl)-N-(4-methylbenzyl)-N-
(1-methylpiperidin-4-yl)acetamide ([11C]AC-90179 or [11C]4 ) in baboon using
PET. AC-90179, is a selective, competitive 5-HT2AR antagonist (Ki = 2.5 nM)
and a 5-HT2A receptor inverse agonist (Ki = 2.1 nM) and with antipsychotic
properties indicated by behavior pharmacology.14,15 Functional high-through-
put screening of many 5-HT2A antagonists indicates they are also inverse
agonist activity at 5-HT2AR.14 Thus the favorable in vitro profile, lipophilicity
of 2.8 (clogP, calculated with ACD/ log P DB program) indicating the ability
to penetrate BBB, and the presence of a potential site for [11C]-labeling
prompted us to develop [11C] AC-90179 as a candidate ligand for in vivo
quantification of 5-HT2AR binding.
Results and discussion
The synthesis of [11C]AC-90179 ([11C]4) was achieved from the precursor
molecule, 2-(4-hydroxyphenyl)-N-(4-methylbenzyl)-N-(1-methylpiperidin-4-yl)
acetamide (5). The phenolic alcohol 5 was synthesized from p-tolylmethylamine
H
N
N
O
S
S
O
OH
18F
O
H311C
N
18F
N
N
N
O
O
O
F
[
18F]Altanserin
[
18F]Setoperone
[
11C]M100907
Figure 1. 5-HT2A receptor PET tracers in human use
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1069–1077
DOI: 10.1002/jlcr

SYNTHESIS AND EVALUATION OF AC90179
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N
N
N
a
b
NH2
O
N
N
+
O
H
O
1
2
3
4
N
N
N
c
d
N
O
O
O11CH3
OH
11C]4
5
[
Scheme 1. Synthesis and radiosynthesis of [11C]4: (a) NaBH4, methanol, 74%;
(b) 4-methoxyphenylacetyl chloride, triethylamine, THF, 85%; (c) BBr3,
CH2Cl2, 80%; (d) i. NaOH, [11C]CH3OTf, 5 min, acetone, ii. HPLC purification
(30% EOS)
(1) in three steps (Scheme 1). Reductive amination of
1
with
1-methylpiperidone (2) afforded 1-methyl-N-(4-methylbenzyl)piperidin-4-
amine (3) in 74% yield. Condensation of compound 3 with 4-methoxyphe-
nylacetyl chloride in presence of triethylamine provided the standard
compound 4 in 85% yield. The radiolabeling precursor 5 was synthesized by
a facile demethylation of the methoxy group in compound 4 with boron
tribromide (BBr3) in 80% yield.
Precursor 5 was subjected to radiomethylation using [11C]methyl triflate
(MeOTf) in presence of aqueous NaOH in acetone to provide [11C]4. The
radiolabeled product was separated from the reaction mixture by reverse phase
high performance liquid chromatography (RP-HPLC) with an average yield of
30 Æ 5% (EOS, n = 6). The chemical identity of [11C]4 was confirmed by
co-injection with an authentic sample of standard 4 on analytical RP-HPLC.
Chemical and radiochemical purities of [11C]4 were found to be >99% with a
specific activity 4500 Æ 500 Ci/mmol (EOB, n = 4). Average time required
for the [11C]-labeling was 30 min (EOB). Partition coefficient (log Po/w) of
[11C]4 was determined by the standard shake flask method16 and found to
be 2.5.
PET imaging studies in baboon show that [11C]4 penetrates BBB and
accumulates in brain (Figure 2). The time activity curves of several brain
regions were examined and demonstrated an uptake of [11C]4 that peaked in
most regions within the first 10 min and then declined rapidly. Only slight
retention of [11C]4 was seen in anterior cingulate, thalamus, putamen and
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

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J. PRABHAKARAN ET AL.
Figure 2. MRI and later time phase PET images of [11C]4 in baboon brain (Top
row: MRI; bottom row: PET scan: sum of 14–40 min frames, left column:
sagittal, middle column: coronal, right column: axial views)
hippocampus in comparison to cerebellum with binding ratios of 1.24, 1.17,
1.13, 1.12 at 115 min, respectively (Figure 3).
It appears there is no quantifiable specific binding detected by this tracer
despite the fact that it has high affinity and selectivity in vitro and penetrates
the BBB. It is possible that [11C]4 is a substrate for p-glycoproteins and is
actively and rapidly extruded from the brain which may contribute to the low
retention of radiotracer in brain. The low injected mass of the carrier
(0.68 Æ 0.02 mg) along with high [11C]4 specific activity (3400 Æ 500 Ci/mmol,
at the time of injection) in the range of 0.034 Æ 0.001 mg/kg baboon weight,
given a Bmax of 520 fmol/mg/protein in frontal cortex of postmortem human
brain,17 the possibility of self blocking or occupancy of the 5-HT2A receptor
sites by 4 is unlikely. A structure affinity relationship study of compound 4
may provide a better PET ligand with in vivo kinetics that permit valid and
reliable measurement of 5-HT2AR binding.
Experimental section
All commercial reagents and solvents were used without further purification
unless otherwise specified.1H NMR spectra were recorded on a Bruker PPX
300 MHz spectrometer. Spectra were recorded in CDCl3 and chemical shifts
are reported in ppm relative to TMS, which is used as the internal standard.
Mass spectra were recorded on JKS-HX 11UHF/HX110 HF Tandem Mass
Spectrometer in the EI+ mode. HPLC analyses were performed using Waters
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1069–1077
DOI: 10.1002/jlcr

SYNTHESIS AND EVALUATION OF AC90179
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0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
CER
ACN
HIP
OCC
PUT
THA
0
20
40
60
80
100
120
Time (min.)
Figure 3. Time activity curves of the radioactivity in baboon after the injection of
[11C]4. (CER = cerebellum, ACN = anterior cingulate, HIP = hippocampus,
OCC = occipital cortex, PUT = putamen, THA = thalamus)
1525 HPLC system (column: Phenomenex, Prodigy ODS(3) 4.6 Â 250 mm,
5 mm for analytical and Phenomenex C18, 10 Â 250 mm, 10 mm for semi-
preparative RP-HPLC). Flash column chromatography was performed on
silica gel (Fisher 200–400 mesh) using the solvent system indicated.
[11C]MeOTf was synthesized from [11C]CO2 according to a reported
procedure.17
1-methyl-N-(4-methylbenzyl)piperidin-4-amine (3)
Sodium borohydride (1.6 g, 43 mmol) was added in small portions to a
solution of benzylamine 1 (3 g, 25 mmol) and 1-methylpiperidone 2 (3.2 g,
28.5 mmol) in methanol (95 ml) and the mixture was stirred at room
temperature for 16 h. Water (50 ml) was added to the reaction mixture and
the products were extracted into dichloromethane (4 Â 30 ml). The dichlor-
omethane layer was separated, washed with brine and dried over anhydrous
magnesium sulfate. Solvent was evaporated under reduced pressure to obtain
the piperidine amine (3) in 83% yield (4.5 g).
1H NMR (300 MHz, CDCl3) d: 7.16 (m, 4 H), 3.64 (s, 2 H), 2.69 (m, 2 H),
2.43 (m, 1 H), 2.38 (s, 3 H), 2.17 (s, 3 H), 2.02-1.99 (m, 4 H), 1.61 (m, 2 H);
HRMS (EI+) calculated for: C14H23N2: 219.1861; Found: 219.1852
Copyright # 2006 John Wiley & Sons, Ltd.
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2-(4-methoxyphenyl)-N-(4-methylbenzyl)-N-(1-methylpiperidin-4-yl)acetamide
(4)
To a solution of piperidine amine 3 (350 mg, 1.60 mmol) and triethylamine
(0.45 ml, 3.2 mmol) in THF (20 ml), 4-(methoxyphenyl)acetyl chloride (325 mg,
3.2 mmol) was added and the solution was stirred at room temperature for 2 h.
The reaction was diluted with water and the products were extracted into ethyl
acetate. The ethyl acetate layer was washed with water, saturated brine and
then dried over anhydrous magnesium sulfate. The solvent was evaporated
under reduced pressure and product was purified by silica gel column
chromatography using ethyl acetate and hexane (1:1) as the eluent to obtain
methoxyacetamide (4) in 70% yield (411 mg) as a viscous solid.
1H NMR (300 MHz, CDCl3) d: 7.24-7.04 (m, 6 H), 6.82 (m, 2 H), 4.65 (m,
1 H), 4.43 (m, 2 H), 3.78 (brs, 4 H), 3.55 (s, 1 H), 2.81 (m, 2 H), 2.31-2.22 (m,
6 H), 2.16 (m, 2 H), 1.81-1.43 (m, 4 H); HRMS (EI+) calculated for:
C23H31O2N2: 367.2386; Found: 367.2375
2-(4-hydroxyphenyl)-N-(4-methylbenzyl)-N-(1-methylpiperidin-4-yl)acetamide
(5)
Methoxyacetamide 4 (127 mg, 0.35 mmol) was dissolved in anhydrous
dichloromethane (2 ml) and a 1 M solution of BBr3 in dichloromethane
(1.4 ml) was added to it. The solution was stirred for 1 h at room temperature
after which it was quenched by adding methanol drop wise at 08C. The
products were extracted into ethyl acetate, washed with water and brine and
dried over anhydrous magnesium sulfate. Solvent was evaporated under
reduced pressure and the product was purified using silica gel column
chromatography. Upon elution with a mixture of ethyl acetate and hexane
(60:40), the phenolicacetamide (5) was isolated in 80% yield as a viscous solid
(100 mg).
1H NMR (300 MHz, CDCl3) d: 7.24-6.7 (m, 8 H), 4.67 (m, 1 H), 4.44
(m, 2 H), 3.52 (s, 2 H), 3.15 (brs, 2 H), 2.85 (m, 1 H), 2.40-2.24 (m, 7 H), 1.78-
1.49 (m, 4 H); HRMS (EI+) calculated for: C22H29O2N2: 353.2229; Found:
353.2204
Radiosynthesis of (O-methyl[11C]2-(4-methoxyphenyl)-N-(4-methylbenzyl)-
N-(1-methyl- piperidin-4-yl)acetamide ([11C]4)
[11C]MeOTf18 was trapped into an acetone (400 ml) solution containing 0.5 mg
of precursor 5 and 10 ml of 5 N NaOH at room temperature for 5 min. At the
end of the trapping, the reaction mixture was heated on a water bath at 608C
for 4 min and then directly injected onto a semi-preparative RP-HPLC
(Phenomenex C18, 10 Â 250 mm, 10 mm) and eluted with acetonitrile: 0.1 M
ammonium formate: acetic acid solution (30:69.5:0.5 v/v/v) at a flow rate of
12.5 ml/min. The product fraction with a retention time of 9–10 min based on
Copyright # 2006 John Wiley & Sons, Ltd.
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SYNTHESIS AND EVALUATION OF AC90179
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g-detector was collected, diluted with 100 ml of deionized water, and passed
through a classic C-18 Sep-Pak1 cartridge. Reconstitution of the product in
1 ml of absolute ethanol afforded [11C]4 in 30% yield, based on [11C]CO2 at
EOS). A portion of the ethanol solution was analyzed by analytical HPLC
(Phenomenex C18; mobile phase: acetonitrile/0.1 M ammonium formate/
acetic acid solution, 30:69.5: 0.5 v/v/v, flow rate: 2 ml/min, retention time:
9 min) to determine the specific activity and radiochemical purity.
Measurement of partition coefficient (log Po/w
)
Partition coefficient of [11C]4 was measured by mixing 0.1 ml of the
radioligand formulation with 5 g each of 1-octanol and freshly prepared
PBS buffer (pH = 7.4) in a culture tube.16 The culture tube was shaken
mechanically for 5 min followed by centrifugation (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 layers. 1-Octanol fractions were repeatedly
portioned with fresh buffer to get consistent values for partition coefficient.
All the experimental measurements were performed in triplicate.
PET studies in baboons
PET studies were done in two baboons (22 and 20 kg) (Papio anubis) with an
ECAT EXACT HR+ scanner (Siemens, Knoxville, TN). For each scanning
session, the fasted animal was immobilized with ketamine (10 mg/kg, i.m.),
and anesthetized with 1.5–2.0% isoflurane via an endotracheal tube. Core
temperature was kept constant at 378C with a heated water blanket. An i.v.
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 determination of the input function for
quantification purposes. In each scanning session, the head was positioned
so that the brain was in the center of the field of view, and a 10 min
transmission scan was performed before the first tracer injection. For baboon
scans 5.0 Æ 0.5 mCi (mass = 0.68 Æ 0.02 mg, specific activity 3300 Æ 500 Ci/
mmol) of [11C]4 were injected as an i.v. bolus and emission data were collected
for 120 min in 3D mode over the following time frames: 2 Â 0.5, 3 Â 1, 5 Â 2,
4 Â 4, 9 Â 10 min. Plasma samples were taken at every 10 s for the first 2 min,
using an automatic system, and thereafter manually for a total of 34 samples
over 2 h.
Image processing and analysis
The PET data was reconstructed with attenuation correction using the
transmission data, and scatter correction was done using model-based scatter
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correction.19 The reconstruction filter and estimated image filter were Shepp
0.5, the axial (Z) filter was all pass 0.4, and the zoom factor was 4.0. The final
image resolution at the center of the field of view was 5.1 mm FWHM.20
A
T1-weighted magnetic resonance image (MRI) of the animal’s head was
acquired on a GE 1.5-T Signa Advantage system. Regions of interests were
drawn on the MRI using the MEDX software (Sensor Systems, Inc., Sterling,
VA). ROIs included cerebellum, hippocampus, occipital cortex, anterior
cinulate cortex, putamen, and thalamus. The PET data were co-registered to
the MRI using the software AIR,21 and time-activity curves (TACs) were
generated for each ROI.
Conclusion
The radiosynthesis of [11C]AC-90179, a 5-HT2AR competitive antagonist has
been achieved. Total time required for the synthesis of [11C]4 is 30 min from
EOB using [11C]MeOTf in acetone, with a 30% yield at EOS based on
[11C]MeOTf. The chemical and radiochemical purities are >99% and specific
activity is >4000 Ci/mmol. PET studies of [11C]4 in baboon showed that the
tracer penetrates the BBB, but tracer washout from the brain is rapid and was
not retained to a quantifiable degree in any brain region. Thus, [11C]4 is not a
useful PET tracer for quantifying 5-HT2AR binding in baboon.
Acknowledgements
National Institute of Health (P50 MH62185) provided funding. The authors
also thank Mr Norman Simpson for his assistance in the radiolabeling
experiments.
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