N-[18F]fluoroethylpiperidin-4ylmethyl acetate, a novel lipophilic acetylcholine analogue for PET measurement of brain acetylcholinesterase activity

Article abstract of DOI:10.1021/jm049100w

The reduction of acetylcholinesterase (AChE) activity in the brain has been measured in dementia disorders such as Alzheimer's disease and dementia with Lewy bodies using ¹¹C-labeled acetylcholine analogues, N-[ ¹¹C]methylpiperidin-4-yl acetate and propionate, and positron emission tomography (PET). Our aim was to develop an ¹⁸F-labeled acetylcholine analogue useful for brain AChE mapping with PET, since ¹⁸F, with a longer half-life, has advantages over ¹¹C. In a preliminary study, a series of N-[¹⁴C]ethylpiperidin-3-yl or -4-ylmethanol esters (acetyl and propionyl esters) were newly designed and evaluated in vitro regarding the reactivity with and specificity to AChE using purified human enzymes, leading to a novel ¹⁸F-labeled acetylcholine analogue, N-[¹⁸F]fluoroethylpiperidin-4-ylmethyl acetate. In rat experiments, the ¹⁸F-labeled candidate showed desirable properties for PET AChE measurement: high brain uptake of the authentic ester, high AChE specificity, a moderate hydrolysis rate, and low membrane permeability (metabolic trapping) of the metabolite.

Full text of DOI:10.1021/jm049100w

J. Med. Chem. 2005, 48, 2577-2583
2577
N-[18F]Fluoroethylpiperidin-4ylmethyl Acetate, a Novel Lipophilic Acetylcholine
Analogue for PET Measurement of Brain Acetylcholinesterase Activity
Tatsuya Kikuchi,†,§ Ming-Rong Zhang,†,‡ Nobuo Ikota,† Kiyoshi Fukushi,† Toshimitsu Okamura,†,§
Kazutoshi Suzuki,† Yasushi Arano,§ and Toshiaki Irie*,†
Department of Medical Imaging, National Institute of Radiological Sciences, Chiba 263-8555, Japan, SHI Accelerator Service,
Tokyo 141-8686, Japan, and Department of Molecular Imaging and Radiotherapy, University of Chiba 263-8522, Japan
Received November 9, 2004
The reduction of acetylcholinesterase (AChE) activity in the brain has been measured in
dementia disorders such as Alzheimer’s disease and dementia with Lewy bodies using 11C-
labeled acetylcholine analogues, N-[11C]methylpiperidin-4-yl acetate and propionate, and
positron emission tomography (PET). Our aim was to develop an 18F-labeled acetylcholine
analogue useful for brain AChE mapping with PET, since 18F, with a longer half-life, has
advantages over 11C. In a preliminary study, a series of N-[14C]ethylpiperidin-3-yl or -4-
ylmethanol esters (acetyl and propionyl esters) were newly designed and evaluated in vitro
regarding the reactivity with and specificity to AChE using purified human enzymes, leading
to a novel 18F-labeled acetylcholine analogue, N-[18F]fluoroethylpiperidin-4-ylmethyl acetate.
In rat experiments, the 18F-labeled candidate showed desirable properties for PET AChE
measurement: high brain uptake of the authentic ester, high AChE specificity, a moderate
hydrolysis rate, and low membrane permeability (metabolic trapping) of the metabolite.
Introduction
showed a much lower hydrolysis rate and lower specific-
ity to AChE than [11C]MP4A and [11C]MP4P.10,11
It has been reported that primary alcohol esters are
more rapidly hydrolyzed by cholinesterases than sec-
ondary alcohol esters.12 Modification of the ester group
on N-[18F]fluoroethylpiperidines from the secondary to
the primary ester form is expected to produce an 18F-
labeled acetylcholine analogue with appropriate AChE
reactivity. In this study, we synthesized a series of
N-[14C]ethylpiperidin-3-yl and -4-ylmethanol esters (pri-
mary alcohol esters) and evaluated their reactivity and
specificity for AChE in vitro using purified human
enzymes to determine the compound most suitable for
18F-labeling. After the screening, the selected pip-
eridinemethanol ester was labeled with [18F]fluoroethyl
bromide and its reactivity and AChE specificity were
evaluated in vitro using rat tissue and an AChE specific
inhibitor. Furthermore, an in vivo study was also
performed in rat for evaluating the retention of the
metabolite in the brain.
Rationale. Figure 1 shows a compartmental model
for the lipophilic acetylcholine analogue illustrating the
principle of PET measurement of AChE activity in the
brain based on the metabolic trapping of the acetylcho-
line analogue. In this model, the k3 represents the first-
order hydrolysis rate of the acetylcholine analogue with
AChE. In PET, the k3 value is not measured directly
but is estimated through mathematical analysis from
the time-activity curve of tracer in the brain measured
by PET. Therefore, the accuracy of AChE measurement
with PET depends on the tracer used for k3 estimation.
The ideal tracer must satisfy the following conditions:
First, the unchanged tracer must be sufficiently lipo-
philic to cross the blood-brain barrier. Second, the
tracer must have a high specificity for the target
enzyme. Third, the hydrolysis rate of the tracer must
be moderate. It is known that precise estimation of k3
Biochemical analyses of postmortem brain tissues
have revealed that the activity of acetylcholinesterase
(AChE) was lower in Alzheimer’s disease compared with
age-matched controls.1 For quantitative measurement
of AChE activity in the brain using positron emission
tomography (PET), 11C-labeled lipophilic acetylcholine
analogues, N-[11C]methylpiperidin-4-yl acetate ([11C]-
MP4A) and propionate ([11C]MP4P), were developed2
and applied to PET studies of dementia disorders such
as Alzheimer’s disease,3,4 Parkinson’s disease with
dementia,5 and dementia with Lewy bodies.6,7
Since 18F has a longer half-life (110 min vs 20 min)
than 11C, 18F-labeled derivatives of [11C]MP4A and [11C]-
MP4P have advantages over [11C]MP4A and [11C]MP4P
such as better image quality and delivery of tracer to
other PET centers. Though displacement of N-[11C]-
methyl group of [11C]MP4A and [11C]MP4P with [18F]-
fluoromethyl group would provide only a minimal
biochemical change, due to the molecular similarity and
biosteric property of the N-CH2F and N-CH3 groups, it
is not applicable to the piperidine ring due to the
instability of the fluoromethyl group attached to the
secondary amine.8 Recently, using [18F]fluoroethyl bro-
mide as an 18F-labeling reagent, 18F-labeled derivatives
of [11C]MP4A and [11C]MP4P such as N-[18F]fluoroeth-
ylpiperidin-4-yl acetate ([18F]FEtP4A),9 N-[18F]fluoro-
ethylpiperidin-3-yl acetate, and N-[18F]fluoroethylpip-
eridin-4-yl propionate10 were synthesized. Though the
N-[18F]fluoroethyl moiety in the derivatives was resis-
tant to in vivo defluorination, all of the compounds
* Corresponding author: Phone: +81-43-206-3191, Fax: +81-43-
251-7147, E-mail: t_irie@nirs.go.jp.
† National Institute of Radiological Sciences.
‡ SHI Accelerator Service.
§ University of Chiba.
10.1021/jm049100w CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/01/2005

2578 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Kikuchi et al.
Table 1. Structure of Compounds
compound
R1
R2
R3
Secondary Esters
MP4A
MP4P
Me
Me
H
H
H
H
OCOCH3
OCOCH2CH3
OCOCH3
FEtP4A CH2CH2F
1
Et
OCOCH3
Primary Esters
2
Et
H
CH2OCOCH3
3R
3S
4
Et
(R)CH2OCOCH3
(S)CH2OCOCH3
H
(R)CH2OCOCH2CH3
(S)CH2OCOCH2CH3
H
H
Et
H
Figure 1. Schematic diagram representing the rationale of
tracer design for measuring AChE activity in the brain using
11C/18F-labeled lipophilic acetylcholine analogue and PET. In
this model, K1 and k2 represent the rate constants of transfer
of the lipophilic analogue across the blood-brain barrier
(BBB), k3 represents the hydrolysis rate of the ester by AChE
(an index of AChE activity) and k4 and k5 represent the rate
constants of transfer of the hydrophilic metabolite across the
BBB. Both k4 and k5 must be small.
Et
CH2OCOCH2CH3
5R
5S
6
Et
H
H
Et
CH2CH2F
CH2OCOCH3
cerebellum, and striatum) were measured at 1, 5, 15,
30, and 60 min after intravenous administration of [18F]-
6 and the metabolite of [18F]6 in rats. The k5, the
elimination rate constant of radioactivity from the brain,
was estimated from the curve during 15 and 60 min
after intravenous injection of [18F]6 in rats. The brain
uptake was expressed as % dose per g tissue after
correction for cerebral blood pool (4%) radioactivity.
is possible under the condition that the ratio of k3 to k2
is about 0.5.13 Finally, the radiolabeled metabolite must
be sufficiently hydrophilic not to cross the blood-brain
barrier; thus both k4 and k5 must be as small as possible.
Synthesis. The esters evaluated in this study were
prepared from corresponding N-Boc piperidinol or
piperidinemethanols as starting materials. The optical
separation of the R and S isomers of N-Boc-3-
piperidinemethanol was performed by the literature
method with lipase PS.14 The optical purity was 90%
for R isomer and more than 99% for S isomer. N-[14C]-
ethyl and N-[18F]fluoroethyl labeling was performed
with [14C]ethyl iodide (2.0 MBq/µmol) and [18F]fluoro-
ethyl bromide (740 GBq/µmol), respectively. The radio-
chemical purities of the labeled compounds were more
than 98%. Though some primary esters at the second
position of the piperidine or pyrrolidine ring are known
to migrate to the N atom,15,16 all the primary esters
synthesized in this study were stable (decomposition
rate; <0.0001 min-1) in a pH 7.4 phosphate buffer.
In Vitro Study. The hydrolysis rate with and speci-
ficity to AChE of the 14C and 18F-labeled esters were
evaluated in vitro using two assay systems, either a
pure enzyme system or a crude brain tissue homogenate
system. In the former system, since it is known that
BChE hydrolyzes acetylcholine at about one-third the
rate of AChE, BChE (human serum) and AChE (human
erythrocytes) were used to examine the quantitative
relationships between the structure and hydrolysis rate
of the esters. In the latter system, to evaluate the AChE
specificity of esters more rigorously, the hydrolysis rate
of the esters was measured in rat (Wistar, male, 8
weeks) brain tissue homogenates with or without a
specific inhibitor of AChE (BW284c51, 30 nM). The
octanol-water partition coefficients of the esters and
the metabolites were measured using 14C- and 18F-
labeled compounds.
Results and Discussion
In preliminary studies, to determine the primary ester
structure suitable for 18F-labeling, a series of N-[14C]-
ethylpiperidin-3-yl or -4-ylmethanol esters were syn-
thesized and evaluated in vitro with respect to reactivity
with and specificity to AChE. The 14C-labeled esters
were derived from the previously reported 18F-labeled
acetylcholine analogue, [18F]FEtP4A, by modifying the
structure in three different ways (Table 1): (1) conver-
sion from the secondary acetyl ester to the primary ester
to increase the hydrolysis rate by AChE; (2) conversion
of the acyl group from acetyl (2, 3R, 3S) to propionyl
(4, 5R, 5S) to slightly decrease the rate; (3) positional
change of an acyl group on the piperidine ring (3-yl, 4-yl)
including optical isomers (R, S) to further modify the
hydrolysis rate as well as the specificity to AChE.
Using purified human AChE and BChE, the relative
hydrolysis rates of N-[14C]ethylpiperidinemethanol es-
ters were measured (Figure 2). With AChE (Panel A),
all the primary esters synthesized showed a rather
narrow range of hydrolysis rates (AChE-rates), ranging
from 0.30 to 0.85 min-1 in unit enzyme concentration
per mL. Among the six esters, the AChE rates were
different from each other (P < 0.01, Games-Howel test)
in descending order of 3R > 2 > 5R > 5S > 3S > 4.
Under the same condition, the hydrolysis rate of [11C]-
MP4A was 0.59 min-1 (data not shown). The acetyl
esters (2, 3R, 3S) showed AChE-rate of about 2-fold that
of the corresponding propionyl esters (4, 5R, 5S). As for
the positional effect between 4yl- and 3yl-esters, no
consistent tendency was observed. Regarding the enan-
tiomer effect, R isomers of acetyl and propionyl ester
(3R, 5R) showed 1.8-fold and 1.4-fold higher AChE-rates
than the corresponding S isomers (3S, 5S). It is also
known that in the case of the secondary esters, N-[14C]-
methylpiperidin-3-yl acetate ([14C]MP3A) and pro-
pionate ([14C]MP3P), R isomers are more rapidly hy-
In Vivo Study. To evaluate bidirectional transport
rates of the hydrophilic metabolite of [18F]6 across the
blood-brain barrier (k4 and k5 in Figure 1), rat in vivo
studies were performed. Time courses of radioactivity
concentration in three brain regions (cerebral cortex,

Novel Lipophilic Acetylcholine Analogue
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2579
Table 2. Comparison of [18F]6 with Previously Synthesized
[
18F]- or [11C]-Labeled N-Alkylpiperidinol Esters
total
ratea
AChE
log P
log P
compd
specificity,b %
esterb
metabolitec
[
[
[
[
18F]6
1.67
1.73
0.41
0.10
86
97
82
65
0.04
-0.30
-0.22
-0.11
-1.3
-2.2
-2.2
-1.9
11C]MP4A
11C]MP4P
18F]FEtP4A
a The first-order hydrolysis rate constant (min-1) in rat cerebral
cortical homogenate at a unit tissue concentration (g/mL) without
inhibitor. b The AChE-mediated hydrolysis rate was measured
using AChE-specific inhibitor, BW284c51. c Partition coefficients
of authentic esters and their metabolites measured in a mixture
of 1-octanol/phosphate buffer (0.1 M, pH 7.4). The values for
[
18F]FEtP4A are literature values.
Figure 2. Relative hydrolysis rates of N-[14C]ethyl-
piperidinemethanol esters, acetates (2, 3R and 3S) and pro-
pionates (4, 5R and 5S), with pure human AChE (Panel A;
AChE-rate) and BChE (Panel B; BChE-rate), and the ratio of
the AChE-rate to BChE-rate (Panel C) (mean(SD, triplicate).
In each panel, the highest value is taken as 100. The AChE-
rates and BChE-rates of the six esters were significantly
different from each other (P < 0.01, Games-Howel test).
drolyzed by AChE than S isomers.17 The reported R/S
ratios of [14C]MP3A and [14C]MP3P are 3 and 8,
respectively, which are much larger than the observed
R/S ratios (1.4-1.8 times) of primary esters examined
in this study. This difference may be associated with
the more flexible nature of the primary ester bond
compared with the secondary ester bond.
Figure 3. Comparison of enzymatic hydrolysis rates of
secondary (gray bar) and primary (dotted bar) alcohol acetyl
esters with different N-alkyl groups (MP4A; N-CH3, 1 and 2;
N-C2H5, FEtP4A and 6; N-C2H4F) measured in rat cerebral
cortical homogenates. The values (mean(SD, n)3) are pre-
sented by taking the rate of [11C]MP4A as 1. The rates were
significantly different from each other (P < 0.01, Games-Howel
test).
Regarding the BChE-rate (Figure 2; Panel B), com-
pletely inverse structure-activity relationships were
observed: (1) a wide range (about 220-fold) of BChE-
rates among the esters, (2) higher rates of the propionyl
esters than those of acetyl esters, and (3) inverse
stereospecificity of BChE, that is, S isomer > R isomer
with BChE versus R isomer > S isomer with AChE.
Because of such differences in the AChE- and BChE-
rates among the N-[14C]ethyl esters, the dynamic range
of the ratio of AChE-rate/BChE-rate reached almost
400-fold among the six derivatives (Figure 2; Panel C).
Of the six 14C-labeled esters examined, 2 showed the
highest value of the AChE-rate/BChE-rate ratio. In
addition, the hydrolysis rate of 2 was almost the same
as that of [11C]MP4A (one of the established tracers in
human PET studies), which is known to have the
hydrolysis rate appropriate for AChE in the human
cerebral cortices. Based on these results, the structure
of 2 was considered to be most suitable for 18F-labeling,
and thus N-[18F]fluoroethylpiperidin-4-ylmethyl acetate,
[18F]6, was synthesized.
value as that of [11C]MP4A. In the present method, since
the accuracy of AChE measurement is highly dependent
on the ratio of the hydrolysis rate (k3) to the washout
rate (k2) of tracer in the target brain region, we must
select a tracer with an appropriate k3/k2 ratio, which is
proportional to k3 when k2 is constant. In human
cerebral cortices which is the major target region in the
diagnosis of Alzheimer’s disease, [11C]MP4A is known
to have a suitable k3/k2 ratio. The result indicates that
the total hydrolysis rate of [18F]6 may be suitable for
the human cerebral cortex. With AChE specificity,
though the value (86%) of [18F]6 was lower than that
(97%, data not shown) of corresponding N-C2H5 com-
pound 2, the value was intermediate between [11C]-
MP4A (97%) and [11C]MP4P (82%), and much higher
than that (65%) of [18F]FEtP4A. These results suggest
that [18F]6 may be a promising candidate as a tracer
for quantification of human brain AChE activity in vivo
using PET, so far as biochemical properties are con-
cerned.
Table 2 shows the biochemical properties of [18F]6 as
compared with two established tracers, [11C]MP4A and
[11C]MP4P, and the previously developed [18F]FEtP4A
performed using rat brain tissue homogenates. For the
total hydrolysis rate, [18F]6 showed almost the same
Figure 3 shows a summary of the relationships
between the ester structure and the hydrolysis rate of
ester in rat brain homogenates. In the case of secondary
esters, the conversion of N-alkyl group from N-CH3 in

2580 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Kikuchi et al.
0.035 min-1 (half time; 20 min), which was about 3-fold
larger than those of [11C]MP4A and [11C]MP4P in rats
(half time; 60 min).18 As for [11C]MP4A and [11C]MP4P,
large species difference in the k5 value is known between
rats and humans; k5 in humans is almost 10-fold smaller
than in rats.19 Based on such a species difference, in
humans, half time of metabolite clearance might be
longer than the 20 min observed in rats. Considering
the duration time of PET scan (about 60 min), the
metabolite of 6 formed within the brain might have a
sufficiently longer residence time for k3 estimation in a
human PET study. Furthermore, the rank order of 18
F
uptake from 6 during the stationary phase, striatum >
cortex > cerebellum, was consistent with the known
distribution of the AChE activity in the brain and was
different from that of the blood flow, cortex > striatum
> cerebellum.
Figure 4. Time-activity curves of 18F uptake in the three
brain regions following intravenous administration of [18F]6
and the 18F-labeled metabolite of 6 in rats. Square; cerebral
cortex, diamond; striatum, circle; cerebellum.
Conclusion
[14C]MP4A to N-C2H5 in 1 and N-C2H4F in FEtP4A
resulted in 8-fold and 16-fold reductions in the hydroly-
sis rate, respectively, indicating that the hydrolysis rate
of FEtP4A is too low. Apparently, introduction of the
fluorine atom to the N-ethyl group reduced the hydroly-
sis rate by about 2-fold. On the other hand, conversion
of the ester structure from the secondary ester to the
primary ester resulted in a 16-fold increase in the
hydrolysis rate, in both cases of N-ethyl and N-fluoro-
ethyl compounds. The same fluorine effect, i.e., 50%
reduction in the rate, was also observed with the
primary esters examined. Because of such compensating
effects of dual modifications, i.e., ester conversion from
the secondary to primary form (16-fold increase) and
conversion of N-alkyl group from N-CH3 to N-C2H4F (16-
fold decrease), 6 had consequently almost the same
hydrolysis rate as [11C]MP4A.
We have developed a novel 18F-labeled radiotracer,
[18F]fluoroethylpiperidin-4-ylmethyl acetate (6), as a
tracer for the quantification of AChE activity in the
brain using PET, through a simple screening test of the
N-[14C]ethyl esters synthesized using purified human
enzymes in vitro. In experiments using rats, it was
confirmed that 6 possessed desirable properties for
accurate and quantitative measurement of AChE activ-
ity using PET based on the metabolic trapping principle.
Experimental Section
Synthesis. [14C]Ethyl iodide (2.04 MBq/µmol) was obtained
from Amersham International Ltd. All starting materials for
chemical synthesis were purchased from Aldrich Chemical Co.
Other chemicals were of the highest grade available com-
mercially. Authentic samples were analyzed by 1H and 13C
nuclear magnetic resonance (1H and 13C NMR) spectroscopy
using a JNM AL-300 (300 MHz) spectrometer and also by
elemental analysis. Optical rotations were measured on a
JASCO DIP-360 polarimeter.
Preparation of (R)- and (S)-N-tert-butoxycarbonyl 3-
piperidinemethanol. (R)- and (S)-N-tert-Butoxycarbonyl 3-
piperidinemethanol were prepared with the reported method.1
The racemic N-tert-butoxycarbonyl 3-piperidinemethanol (5.0
g, 23 mmol) was stirred in 100 mL CHCl3 for 2 h at room
temperature with 15 mL vinyl acetate and 2 g lipase PS
(pseudomonas sp.; Amano Pharmaceutical Co., Ltd., Japan).
The residue, that was obtained by filtering to remove the
insoluble solid and evaporated under diminished pressure to
remove the solvent, was then purified by column chromatog-
raphy (silica gel C-200, hexane/ethyl acetate ) 1/3) to yield
(S)-N-tert-butoxycarbonylpiperidin-3-ylmethyl acetate (2.5 g,
42%, [R]2D0 +17.8° (c ) 1, CHCl3)).
Additionally, about 2.8 g of recovered hydroxyl compounds
enriched with nonreacted R isomer was treated twice with
lipase PS in the same manner as described above and purified
by column chromatography to obtain optically pure (R)-N-tert-
butoxycarbonyl 3-piperidinemethanol (2.0 g, 40%, [R]2D0 -11.2°
(c ) 1, CHCl3)).
(S)-N-tert-Butoxycarbonylpiperidin-3-ylmethyl acetate (2.0
g, 7.8 mmol) was dissolved in 5 mL EtOH, and 2.5 mL 2 M
NaOH was added and reacted for 2 h at room temperature.
Then 50 mL ethyl acetate was added and washed with water.
The organic layer was dried over anhydrous MgSO4, and the
residue that was obtained by evaporating the solvent was
purified by column chromatography (silica gel C-200, hexane/
ethyl acetate ) 1/1), and yielding (S)-N-tert-butoxycarbonyl
3-piperidinemethanol (1.6 g, 97%, [R]2D0 +12.5° (c ) 1, CHCl3)).
Preparation of N-Ethylpiperidin-4-yl Acetate (1). Gen-
eral Method. N-tert-Butoxycarbonyl-4-hydroxypiperidine (1.0
To evaluate the lipophilicity of [18F]6 and its metabo-
lite in vitro, partition coefficients (Log P) of [18F]6 and
its metabolite were measured in a mixture of 1-octanol
and a phosphate buffer (0.1 M, pH 7.4) and compared
with those of the reference compounds (Table 2). With
the ester form, the Log P values were in the decreasing
order of [18F]6 > [18F]FEtP4A > [11C]MP4P > [11C]-
MP4A and with the metabolite, [18F]6 > [18F]FEtP4A
> [11C]MP4P ) [11C]MP4A (the metabolites of [11C]-
MP4A and [11C]MP4P are identical), which correlated
well with the molecular volume. Since [11C]MP4A and
[11C]MP4P are known to have relatively high extraction
fractions by the brain tissues, it is expected that [18F]6
is sufficiently lipophilic to cross the blood-brain barrier.
To examine whether the metabolite from [18F]6 is
hydrophilic enough not to cross the blood-brain barrier,
we have performed rat in vivo studies to measure
transport rates (k4 and k5 in Figure 1) of the 18F-labeled
metabolite across the blood-brain barrier. When the
18F-labeled metabolite was intravenously injected in rats
(Figure 4; lower curves), the uptake of 18F was almost
the same among the three brain regions and remained
low until 60 min after injection, indicating that k4
(transport rate of the metabolite from blood to brain;
Figure 1) is slow. When [18F]6 was injected in rats
(Figure 4; upper curves), the uptake of 18F was much
higher than that of the metabolite and it was different
among the three regions. The estimated k5 (elimination
rate of the metabolite from brain to blood; Figure 1) was

Novel Lipophilic Acetylcholine Analogue
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2581
g, 5.0 mmol) was dissolved in 10 mL methylene chloride, and
pyridine (790 mg, 10 mmol) was added. To this solution, acetyl
chloride (630 mg, 8.0 mmol) was added and the reaction
mixture was kept at room temperature for 3 h. After adding
100 mL ethyl acetate, the organic layer of the mixture was
washed with water, 5% HCl, water, saturated NaHCO3, and
finally water. The organic solution was then dried over
anhydrous MgSO4. The residue that was obtained by evapo-
rating the solvent was purified by column chromatography
(silica gel C-200, hexane/ethyl acetate ) 4/1), yielding N-tert-
butoxycarbonylpiperidin-4-yl acetate (1.01 g, 83%). The com-
pounds were assigned according to the previously reported
NMR spectra.1
N-tert-Butoxycarbonylpiperidin-4-yl acetate (500 mg, 2.06
mmol) was dissolved in 5 mL ethyl acetate, and 5 mL 4 M
HCl/ethyl acetate was added under ice-cold condition. After
the solution was allowed to stand for 1 h at room temperature,
ethyl acetate was evaporated under vacuum at less than room
temperature, yielding piperidin-4-yl acetate hydrochloride (380
mg). This compound was dissolved in 5 mL chloroform, and 5
mL saturated NH3-chloroform solution was added under ice-
cold conditions. After the mixture was reacted for 5 min at
room temperature, the solution was filtered to remove the
insoluble solid and the solvent was then evaporated under
diminished pressure, yielding piperidin-4-yl acetate. The
compounds were assigned according to the reported NMR
spectra.1
Piperidin-4-yl acetate (200 mg, 1.4 mmol) was dissolved in
40 mL methylene chloride and 10 mL 30% acetaldehyde and
triacetoxy borohydride (850 mg, 4.0 mmol) were added. The
solution was allowed to stand for 2 h at room temperature.
After adding 100 mL ethyl acetate, the organic layer of the
mixture was washed with water. The organic solution was then
dried over anhydrous MgSO4. The residue that was obtained
by evaporating the solvent was purified by column chroma-
tography (Chromatorex NH silica gel, hexane/ethyl acetate )
4/1) to yield N-ethylpiperidin-4-yl acetate (1) (165 mg, 69%).
N-Ethylpiperidin-4-yl acetate (1): 1H NMR (δ, CDCl3): 1.03
(3H, t, CH3), 1.59-1.70 (2H, m, 2×CH), 1.82-1.89 (2H, m,
2×CH,), 1.99 (3H, s, CH3), 2.11-2.19 (2H, m, 2×CH), 2.35 (2H,
q, CH2), 2.63-2.69 (2H, m, 2×CH), 4.68-4.74 (1H, m, CH).
13C NMR (CDCl3): 12.19, 21.29, 30.81, 50.47, 52.15, 70.43,
170.52. Anal. (C9H17NO2): C, H, N. IR (neat) cm-1 1705.
Other N-ethyl compounds (2-5S) were synthesized by the
same manner as described above from the corresponding
N-tert-butoxycarbonylpiperidinemethanols as a starting mate-
rial. The reference compounds (MP4A, MP4P, and FEtP4MA)
were prepared using the method in the literature including
radiolabeling.2,3
28.40, 35.34, 44.05, 46.93, 66.15, 79.42, 154.79, 174.38. Anal.
(C14H25NO4): C, H, N. IR (neat) cm-1 1692, 1742.
Piperidin-4-ylmethyl acetate: 1H NMR (δ, CDCl3): 1.13-
1.22 (2H, m, 2×CH), 1.63-1.74 (3H, m, 2×CH, CH), 2.00 (3H,
s, CH3), 2.05 (1H, s, NH), 2.51-2.61 (2H, m, 2×CH), 3.03-
3.09 (2H, m, 2×CH), 3.85-3.88 (2H, m, CH2). 13C NMR
(CDCl3): 20.83, 29.83, 46.03, 53.64, 69.02, 171.1. Anal. (C8H15-
NO2): C, H, N. IR (neat) cm-1 3374, 1736.
Piperidin-4-ylmethyl propionate: 1H NMR (δ, CDCl3): 1.09
(3H, t, CH3), 1.07-1.12 (1H, m, CH), 1.39-1.48 (1H, m, CH),
1.59-1.65 (1H, m, CH), 1.66 (1H, s, NH), 1.71-1.79 (2H, m,
2×CH), 2.24-2.35 (3H, m, CH, CH2), 2.46-2.55 (1H, m, CH),
2.92-2.97 (1H, m, CH), 3.02-3.06 (1H, m, CH), 3.80-3.93 (2H,
m, CH2). 13C NMR (CDCl3): 9.10, 25.90, 27.49, 27.76, 36.63,
46.84, 49.89, 67.05, 174.42. Anal. (C9H17NO2): C, H, N. IR
(neat) cm-1 1734, 3372.
(R)- and (S)-Piperidin-3-ylmethyl acetate; R isomer: [R]D20
+13.9° (c ) 1, CHCl3), S isomer: [R]20 -15.4° (c ) 1, CHCl3).
1H NMR (δ, CDCl3): 1.06-1.11 (1H,Dm, CH), 1.30-1.48 (1H,
m, CH), 1.59-1.65 (1H, m, CH), 1.72-1.76 (2H, m, 2×CH),
1.99 (3H, s, CH3), 2.00 (1H, s, NH), 2.27-2.34 (1H, m, CH),
2.46-2.54 (1H, m, CH), 2.93-3.06 (2H, m, 2×CH), 3.78-3.91
(2H, m, 2×CH). 13C NMR (CDCl3): 20.78, 25.74, 27.67, 36.44,
46.72, 49.73, 67.16, 171.01. Anal. (C8H15NO2): C, H, N. IR
(neat) cm-1 1735.
(R)- and (S)-Piperidin-3-ylmethyl propionate; R isomer:
[R]2D0 +10.5° (c ) 1, CHCl3), S isomer: [R]20 -11.9° (c ) 1,
CHCl3). 1H NMR (δ, CDCl3): 0.86 (3H, t, CH3D), 1.06-1.12 (1H,
m, CH), 1.36-1.49 (1H, m, CH), 1.61-1.71 (1H, m, CH), 1.76-
1.80 (2H, m, 2×CH), 1.86 (3H, s, CH3), 2.05 (1H, s, NH), 2.31-
2.36 (1H, m, CH), 2.41-2.53 (1H, m, CH), 2.99-3.12 (2H, m,
2×CH), 3.81-3.96 (2H, m, 2×CH). 13C NMR (CDCl3): 16.52,
21.28, 24.90, 28.10, 37.22, 48.23, 49.30, 66.79, 172.25. Anal.
(C9H17NO2): C, H, N. IR (neat) cm-1 1735.
N-Ethylpiperidin-4-ylmethyl acetate (2): 1H NMR (δ,
CDCl3): 1.02 (3H, t, CH3), 1.20-1.34 (2H, m, CH2), 1.59-1.68
(3H, m, 2×CH, CH), 1.79-1.87 (2H, m, 2×CH), 1.99 (3H, s,
CH3), 2.33 (2H, q, CH2), 2.88-2.92 (2H, m, 2×CH), 3.87 (2H,
d, CH2). 13C NMR (CDCl3): 12.06, 20.82, 28.83, 35.28, 52.51,
52.84, 68.78, 171.06. Anal. (C10H19NO2): C, H, N. IR (neat)
cm-1 1736.
N-Ethylpiperidin-4-ylmethyl propionate (4): 1H NMR (δ,
CDCl3): 1.04 (3H, t, CH3), 1.10 (3H, t, CH3),1.28-1.32 (2H,
m, CH2), 1.59-1.67 (3H, m, 2×CH, CH), 1.80-1.88 (2H, m,
2×CH), 2.28 (2H, q, CH2), 2.35 (2H, q, CH2), 2.89-2.93 (2H,
m, 2×CH), 3.89 (2H, d, CH2). 13C NMR (CDCl3): 9.11, 12.10,
27.50, 28.88, 35.38, 52.55, 52.89, 68.66, 174.49. Anal. (C11H21-
NO2): C, H, N. IR (neat) cm-1 1723.
(R)- and (S)-N-Ethylpiperidin-4-ylmethyl acetate (3R, 3S);
N-tert-Butoxycarbonylpiperidin-4-ylmethyl acetate: 1H NMR
(δ, CDCl3): 1.08-1.22 (2H, m, 2×CH), 1.43 (9H, s, t-Bu), 1.64-
1.68 (2H, m, 2×CH), 1.72-1.81 (1H, m, CH), 2.03 (3H, s, CH3),
2.63-2.71 (2H, m, 2×CH), 3.90 (2H, d, CH2), 4.07-4.11 (2H,
m, 2×CH). 13C NMR (CDCl3): 20.82, 28.37, 28.61, 35.47, 43.28,
68.40, 79.35, 154.72, 171.02. Anal. (C13H23NO4): C, H, N. IR
(neat) cm-1 1689, 1738.
R isomer: [R]2D0 +7.8° (c ) 0.4, CHCl3), S isomer: [R]20 -8.7°
D
1
(c ) 1, CHCl3). H NMR (δ, CDCl3): 1.01 (3H, t, CH3), 1.24-
1.32 (3H, m, CH, CH2), 1.50-1.69 (2H, m, 2×CH), 1.75-1.88
(2H, m, 2×CH) 1.98 (3H, s, CH3), 2.33 (2H, q, CH2), 2.78-
2.87 (2H, m, 2×CH), 3.78-3.84 (1H, m, CH), 3.89-3.94 (1H,
m, CH). 13C NMR (CDCl3): 11.97, 20.88, 24.81, 27.40, 38.83,
52.75, 53.62, 56.77, 67.37, 171.11. Anal. (C10H19NO2): C, H,
N. IR (neat) cm-1 1718.
N-tert-Butoxycarbonylpiperidin-4-ylmethyl propionate: 1H
NMR (δ, CDCl3): 1.09-1.23 (5H, m, CH3, 2×CH), 1.42 (9H, s,
t-Bu), 1.63-1.67 (2H, m, 2×CH), 1.72-1.81 (1H, m, CH), 2.30
(2H, q, CH2), 2.62-2.71 (2H, m, 2×CH), 3.90 (2H, d, CH2),
4.07-4.10 (2H, m, 2×CH). 13C NMR (CDCl3): 9.11, 27.49,
28.40, 28.65, 35.56, 43.34, 68.26, 79.37, 154.76, 174.42. Anal.
(C14H25NO4): C, H, N. IR (neat) cm-1 1697, 1722.
(R)- and (S)- N-tert-Butoxycarbonylpiperidin-3-ylmethyl
acetate; R isomer: [R]D20 -16.2° (c ) 1, CHCl3), S isomer
[R]2D0 +17.8° (c ) 1, CHCl3). The compounds were assigned
according to the reported NMR spectra1.
(R)- and (S)-N-Ethylpiperidin-4-ylmethyl propionate (5R,
5S); R isomer: [R]D20 +8.0° (c ) 1.1, CHCl3), S isomer: [R]20
D
-8.9° (c ) 1.1, CHCl3).1H NMR (δ, CDCl3): 0.98-1.10 (6H,
m, 2×CH3) 1.19-1.33 (3H, m, CH, CH2), 1.54-1.69 (3H, m,
3×CH), 1.74-1.97 (2H, m, 2×CH), 2.22-2.36 (3H, m, CH,
CH2), 2.77-2.87 (2H, m, 2×CH), 3.78-3.95 (2H, m, 2×CH).
13C NMR (CDCl3): 9.11, 11.95, 24.81, 27.40, 27.51, 35.87,
52.71, 53.60, 56.77, 67.14, 174.41. Anal. (C11H21NO2): C, H,
N. IR (neat) cm-1 1731.
Preparation of N-Fluoroethylpiperidin-4-ylmethyl
Acetate (6). Piperidin-4-ylmethyl acetate (500 mg, 3.2 mmol),
1-fluoro-2-tosylethane (1.38 g, 6.35 mmol), and 150 mg K2CO3
were dissolved in 4 mL dimethylformamide for 2.5 h at 80 °C.
After adding 100 mL ethyl acetate, the organic layer of the
mixture was washed with water, 27% NH3, and finally
saturated NaCl. The organic solution was then dried over
anhydrous MgSO4. The residue that was obtained by evapo-
(R)- and (S)- N-tert-Butoxycarbonylpiperidin-3-ylmethyl pro-
pionate; R isomer: [R]D20 -14.7° (c ) 1, CHCl3), S isomer:
[R]2D0 +16.3° (c ) 1, CHCl3). H NMR (δ, CDCl3): 1.12 (3H, t,
1
CH3), 1.19-1.25 (1H, m, CH), 1.43 (10H, s, t-Bu, CH), 1.60-
1.67 (1H, m, CH), 1.73-1.79 (2H, m, 2×CH), 2.29 (2H, q, CH2),
2.59 (1H, broad s, CH), 2.74-2.81 (1H, m, CH), 3.83-3.99 (4H,
m, CH2, 2×CH). 13C NMR (CDCl3): 9.13, 24.43, 27.23, 27.50,

2582 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Kikuchi et al.
rating the solvent was purified by column chromatography
(silica gel C-200, chloroform/ethyl acetate ) 1/1) to yield
N-fluoroethylpiperidin-4-ylmethyl acetate (472 mg, 73%).
interval, 200 µL EtOH was added to each tube to stop the
reaction. 5 µL of the solution was then applied to a silica gel
TLC plate and developed with a mixture of ethyl acetate:2-
propanol:ammonia (100:10:1; vols). The air-dried TLC plate
was covered with a 5 µm thick foil and placed in a cassette in
contact with the imaging phosphor plate for 1 h. The radio-
activities corresponding to the Rf value of the unchanged esters
and metabolites were quantitated using the BAS 1800 system.
The enzymatic hydrolysis rate by AChE (AChE rate) of each
compound was calculated as: AChE rate ) (-ln(A2/A1))/(T2 -
T1)/C, where A1 and A2 represent the 14C radioactivity of the
unchanged ester remaining at times T1 and T2, respectively;
and C represents the enzyme concentration (Unit/mL) in the
reaction solution. The hydrolysis rates by BChE (BChE rate)
were measured in the same manner. Nonenzymatic hydrolysis
rates of each ester were negligibly slow (<0.0001 min-1). The
assay was performed in triplicate, and the statistical compari-
son, Games-Howel test (p < 0.01) was made.
N-Fluoroethylpiperidin-4-ylmethyl acetate (6): 1H NMR (δ,
CDCl3): 1.21-1.37 (2H, m, 2×CH), 1.62-1.70 (3H, m, CH,
CH2), 2.02 (3H, s, CH3), 2.01-2.08 (2H, m, 2×CH), 2.61 (1H,
m, CH), 2.71 (1H, m, CH), 2.93 (2H, d, CH2), 3.88-3.91 (2H,
m, 2×CH), 4.46 (1H, m, CH), 4.62 (1H, m, CH). 13C NMR
(CDCl3): 20.90, 28.73, 34.93, 53.56, 58.30, 58.52, 68.70, 80.78,
83.04, 171.10. Anal. (C10H18FNO2): C, H, N. IR (neat) cm-1
1735.
N-[14C]Ethylation (14C-labeled 1-5S). A mixture of precursor
ester (demethyl piperidinemethanol ester, 100 µmol), 5 mg K2-
CO3, and [14C]ethyl iodide (10 MBq) was heated in 2 mL
acetone at 50 °C with a water bath for 2 h. An ethyl acetate
solution containing anhydrous HCl (in excess of ester) was
added to the reaction solution, and the mixture was dried
under nitrogen gas. The residue was dissolved in 200 µL
MeOH and was subjected to thin-layer chromatography (TLC)
with a silica gel plate and a mixture of ethyl acetate:2-
propanol:ammonia (100:10:1; vols) as a developing solvent. The
radioactive zone with Rf value corresponding to the authentic
compound was collected and extracted with a 1:2 mixture of
MeOH and chloroform. Radiochemical yields based on [14C]-
methyl iodide were more than 70% and purities were more
than 99%.
Hydrolysis Rate and Specificity with Rat Cerebral
Cortical Homogenate. Experimental animals used in the
present study were treated and handled according to the
“Recommendations for Handling of Laboratory Animals for
Biomedical Research”, compiled by the Committee on Safety
and Ethical Handling Regulations for Laboratory Animal
Experiments, NIRS.
Tissue homogenates were prepared as follows. Rats (Wistar,
male, 8 weeks, 210-230 g, n ) 3) were anesthetized with
diethyl ether and killed by decapitation. The brain was
removed and dissected, and the cerebral cortex was obtained
and weighed. The tissues were homogenized in ice-cold phos-
phate buffer (0.1 M, pH 7.4). The homogenates were diluted
to the appropriate concentration, and 100 µL homogenate of
cerebral cortex was placed in the reaction tubes. Each com-
pound solution (15 kBq in 20 µL buffer) was added to each
tube to initiate the reaction. The following procedure was the
same as that described above. Diethyl ether, used as anesthe-
sia, did not affect the measurement of enzyme activity. The
specificity of compounds for AChE was measured by addition
of BW284c51 (30 nM). The AChE specificities of the compounds
in the cerebral cortex homogenate were measured from the
ratio of the noninhibited hydrolysis rate to inhibited hydrolysis
rate. Statistical comparison was made using the Games-
Howel test (p < 0.01).
Partition Coefficient. 18F-labeled 6, MP4A, MP4P, and
their metabolites were added to a 1:1 mixture of 1-octanol and
0.1 M phosphate buffer (pH 7.4), shaken vigorously, and
allowed to equilibrate for 1 h at room temperature. The
concentrations and purity of the radioactivity in the organic
and aqueous phases were measured with a gamma or liquid
scintillation counter and TLC, respectively. The partition
coefficient was calculated as the ratio of the concentration in
the organic phase to that in the aqueous phase.
N-[18F]Fluoroethylation (18F-labeled 6). N-[18F]Fluoroeth-
ylpiperidin-4-ylmethyl acetate was prepared with reported
method using piperidin-4-ylmethyl acetate and 1-bromo-2-[18F]-
fluoroethane. [18F]F- was produced by the 18O (p, n) 18F
reaction on 10-20 atom % H218O using 18 MeV protons (14.2
MeV on target) from the cyclotron and separated from [18O]-
H2O using Dowex 1-X8 anion-exchange resin in an irradiating
room. The [18F]F- was eluted from the resin with aqueous
potassium carbonate (3.3 mg/0.3 mL) into a glass vial contain-
ing CH3CN (1.5 mL)/Kryptofix 2.2.2. (25 mg) and transferred
into a reaction vessel in a hot cell. After the [18F]F- was dried
to remove H2O and CH3CN from the reaction vessel, 2-bro-
moethyl triflate (BrCH2CH2OTf) (8 µL) in o-dichlorobenzene
(500 µL) was added to the radioactive solution. Then, the
resulting 1-bromo-2-[18F]fluoroethane in this vessel was dis-
tilled under a helium flow (90-100 mL/min) at 130 °C for 2
min and bubbled into another vessel containing a solution of
anhydrous DMF (300 µL) and piperidin-4-ylmethyl acetate (1
mg) at -20 °C. After maximum radioactivity was reached into
the solution, the reaction mixture was warmed to 130 °C and
maintained for 30 min. The content was diluted with a high
performance liquid chromatography (HPLC) mobile phase (500
µL) and applied onto a semipreparative column (10 mm ID ×
250 mm, Megapack SIL C18) attached to the JASCO HPLC
system. Elution with 5 mM CH3COONH4 (pH ) 4.9)/CH3CN
(9/1) at flow rate of 4 mL/min gave a radioactive fraction
corresponding to pure 18F-labeled 6 (tR ) 13 min). The fraction
was collected in a rotary evaporator and evaporated to dryness
at 90 °C under reduced pressure. The residue was redissolved
in 10 mL of saline and passed through a Millipore filter (GS,
0.22 µm) to provide an injectable solution of 18F-labeled 6. All
of the above procedures were carried out automatically by the
use of a previously reported system. Total synthesis time was
92-96 min from the end of bombardment. At the end of
synthesis, 347-420 MBq of 18F-labeled 6 was obtained. The
radiochemical purity of 18F-labeled 6 was assayed by analytical
HPLC (column: Finepak Sil C18-10, JASCO, 4.6 mm ID ×
250 mm, Megapack SIL C18). The mobile phase was 50 mM
CH3COONH4 (pH ) 3.1)/CH3CN (3/7). The tR was 7.3 min for
18F-labeled 6. The radiochemical purity of the compound was
consistently more than 98%.
Brain Regional Kinetics of Compound 6 and Its
Metabolite. 18F-Labeled 6 or metabolite (370 kBq/200 µL)
were injected intravenously to rats (Wistar, male, 8 weeks,
210-230 g, n ) 3 per time point), and the rats were
anesthetized with diethyl ether and killed by decapitation at
1, 5, 15, 30, and 60 min after injection. The striatum, cerebral
cortex, and cerebellum were dissected out and weighed, and
the radioactivity was measured with a gamma counter. The
brain uptake of radioactivity was calculated as % dose per g
tissue.
Acknowledgment. We thank the crew of the Cy-
clotron Section and Radiopharmaceuticals Section of the
National Institute of Radiological Sciences for their
support in the operation of the cyclotron and the
production of radioisotopes.
Hydrolysis Rate with Pure Human AChE and BChE.
Purified enzymes were obtained from Sigma Chemical Co. The
purified human AChE was added to 0.5% Triton X-100
phosphate buffer (0.1 M, pH 7.4), and then the solution (100
µL) was placed in tubes and preincubated at 37 °C for 30 min.
Each 14C-labeled ester solution (15 kBq in 20 µL buffer) was
added to each tube to initiate the reaction. At a designated
Supporting Information Available: Elemental analysis.
This material is available free of charge via the Internet at
http://pubs.acs.org.

Novel Lipophilic Acetylcholine Analogue
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2583
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