N-[18F]fluoroethylpiperidin-4-ylmethyl butyrate: A novel radiotracer for quantifying brain butyrylcholinesterase activity by positron emission tomography

Article abstract of DOI:10.1016/j.bmcl.2004.01.080

In Alzheimer's disease, cerebral cortical butyrylcholinesterase (BChE) activity is reported to be elevated. Our aim was to develop a novel ¹⁸F-labeled tracer for quantifying cerebral BChE activity by positron emission tomography. With in vitro screening of N-[¹⁴C] ethylpiperidin-3- and 4-ylmethyl esters, N-[¹⁴C]ethylpiperidin-4- ylmethyl butyrate was selected as a lead for ¹⁸F-labeling, affording N-[¹⁸F]fluoroethylpiperidin-4-ylmethyl butyrate. The ¹⁸F-labeled butyrate showed the required properties for in vivo BChE measurement, that is, the lipophilic nature of the authentic ester, high specificity to BChE, a moderate hydrolysis rate, and the hydrophilic nature of the metabolite.

Full text of DOI:10.1016/j.bmcl.2004.01.080

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1927–1930
N-[18F]fluoroethylpiperidin-4-ylmethyl butyrate: a novel
radiotracer for quantifying brain butyrylcholinesterase activity by
positron emission tomography
Tatsuya Kikuchi,a,b Ming-Rong Zhang,a,c Nobuo Ikota,a Kiyoshi Fukushi,a
Toshimitsu Okamura,a,b Kazutoshi Suzuki,a Yasushi Aranob and Toshiaki Iriea,*
aDepartment of Medical Imaging, National Institute of Radiological Sciences, Chiba 263-8555, Japan
bDepartment of Molecular Imaging and Radiotherapy, University of Chiba, Chiba 263-8522, Japan
cSHI Accelerator Service, Tokyo 141-8686, Japan
Received 8 December 2003; revised 26 January 2004; accepted 27 January 2004
Abstract—In Alzheimer’s disease, cerebral cortical butyrylcholinesterase (BChE) activity is reported to be elevated. Our aim was to
develop a novel 18F-labeled tracer for quantifying cerebral BChE activity by positron emission tomography. With in vitro screening
of N-[14C]ethylpiperidin-3- and 4-ylmethyl esters, N-[14C]ethylpiperidin-4-ylmethyl butyrate was selected as a lead for 18F-labeling,
affording N-[18F]fluoroethylpiperidin-4-ylmethyl butyrate. The 18F-labeled butyrate showed the required properties for in vivo
BChE measurement, that is, the lipophilic nature of the authentic ester, high specificity to BChE, a moderate hydrolysis rate, and
the hydrophilic nature of the metabolite.
# 2004 Elsevier Ltd. All rights reserved.
There are two major forms of cholinesterases, acetyl-
cholinesterase (AChE, EC 3.1.1.7) and butyrylcholin-
esterase (BChE, EC 3.1.1.8), in the mammalian brain.
BChE is found in neurons and glial cells as well as in
neuritic plaques and tangles in Alzheimers’s disease
(AD).1 In AD, while AChE activity decreases progres-
sively, BChE activity is higher compared to age-mat-
ched controls.2 Two lipophilic acetylcholine analogues,
N-[11C]methylpiperidin-4-yl acetate ([11C]MP4A: 1)
and propionate([ 11C]MP4P: 2), have been developed3 and
clinically used to quantify regional cerebral AChE
activity in AD patients4,5 using positron emission
tomography (PET). Based on the same principle as 1 and
2 (thelipophilic substrateanaloguemethod), 11C-labeled
blood–brain barrier. Second, the tracer must have high
specificity for the target enzyme. Third, the hydrolysis
rate of the tracer in the brain region of interest must be
moderate.8 Finally, themetabolitemust behydrophilic
enough to be retained in the brain (metabolic trapping).
Among theconditions, optimization of thetracre’s
metabolic rate is very important, because reliability of
enzyme activity estimation is highly dependent on the
rate.8 Based on the computer-simulation study,8 it is
suggested that the metabolic rate of MP4A and MP4P
are appropriate for the human cerebral cortex.
The aim of this study was to develop an 18F-labeled
tracer with high specificity to and a moderate hydrolysis
rate with human cerebral BChE. Because of the longer
half-life( 18F, 110 min; 11C, 20 min) and shorter positron
range( 18F, 650 keV; 11C, 960 keV) of 18F compared to
11C, 18F is an attractivelable. Introduction of the
[18F]fluoromethyl group to the N-atom of piperidine is
not possible, due to instability of the fluoromethyl
group attached to the secondary amine.9 Another
method is N-[18F]fluoroethylation of the piperidine.
However, the N-fluoroethyl derivatives of 1 and 2 are
reported to have low reactivity to BChE.10 Furthermore,
the concentration of BChE activity in the human cere-
bral cortex is much lower than that of AChE. There-
butyrylcholineanaloguse such as
N-[11C]methylpiper-
idin-4-yl butyrate([ 11C]MP4B: 3) have been developed
as potential tracers for the measurement of cerebral
BChE activity by PET.6,7
In the lipophilic substrate analogue method, the ideal
tracer must satisfy the following conditions: First, the
unchanged tracer must be lipophilic enough to cross the
Keywords: Positron emission tomography.
* Corresponding author. Tel.: +81-43-206-3191; fax: +81-43-251-
7147; e-mail: t_irie@nirs.go.jp
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.080

1928
T. Kikuchi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1927–1930
fore, to develop an 18F-labeled tracer with moderate
BChE-reactivity, the ester structure needs to be mod-
ified so as to have increased reactivity with BChE.
Primary alcohol esters are known to be hydrolyzed by
cholinesterases more rapidly than secondary alcohol
esters.11 We have therefore designed a series of primary
esters (butyrate and valerate) of N-[14C]ethylpiper-
idinemethanols (Table1 ), which are used to identify a
structuremost suitablefor 18F-labeling. The 14C-labeled
esters are modified from 3 in three respects: first, con-
version from the secondary ester to the primary ester to
increase the hydrolysis rate by BChE; second, change of
theacyl group from thebutyryl ( 4, 5R, 5S) to thevaleryl
group (6, 7R, 7S); and third, changeof theposition of
an acyl group on thepiperidinering including optical
isomers.
Scheme 1. Reagents and conditions: (a) lipase PS, vinyl acetate,
CHCl3; (b) NaOHaq, MeOH; (c) butyryl chloride or valeryl chloride,
pyridine, CH2Cl2; (d) 4M-HCl/AcOEt; (e) 30% CH3CHO, sodium
triacetoxy-borohydride, CH2Cl2 for 4–7S, and 1-fluoro-2-tosilethane,
K2CO3, dimethylformamide, 80 ꢂC for 8; (f) [14C]C2H5I, K2CO3,
acetone, 70 ꢂC or 1-bromo-2-[18F]fluoroethane, DMF, 130 ꢂC. R=
butyryl or valeryl, R0=ethyl of fluoroethyl.
The esters12 were prepared from corresponding N-Boc-
piperidinol or piperidinemethanols as starting materials
human enzymes, AChE (human erythrocytes) and
BChE (human serum).6 In a pH 7.4 phosphatebuffer
containing AChE or BChE, thefirst-ordre hydrolysis
rateconstant of 14C or 18F-labeled ester was measured
and normalized for unit enzyme concentration, afford-
ing kAChE and kBChE, thenormalizde rateconstant
(unit; minꢀ1Unitꢀ1mLꢀ1) of the ester for AChE and
BChE. Concentrations of AChE and BChE activity in
the human cerebral cortex were measured by Ellman’s
method ([AChE]=0.6 Unit/g tissue, [BChE]=0.1 Unit/
g tissue). The AChE-rate and BChE-rate of each ester
were calculated as: AChE-rate=kAChEꢁ[AChE] and
BChE-rate=kBChEꢁ[BChE]. Total-rate was defined as:
Total-rate=AChE-rate+BChE-rate. The BChE speci-
ficity (%) of each ester was defined by the ratio of
BChE-rateto total-ratemultiplied by 100 (%).
as shown in Scheme 1. Theoptical separation of the
R
and S isomers of N-Boc-3-piperidinemethanol was per-
formed by the literature method with lipase PS.13 By the
calculation from literature optical rotations of N-Boc-3-
piperidinemethanols, the optical purity was 90% ee for
R isomer and more than 99% ee for S isomer. N-
[14C]ethyl and N-[18F]fluoroethyl labeling was performed
with [14C]ethyl iodide (55 Ci/mol) and [18F]fluoroethyl
bromide(20 Ci/ mmol), respectively. Since the radio-
labeled compounds display no special absorption on
UV spectrum (200–400 nm), the specific activity of them
were estimated same specific activity as [14C]ethyl iodide
and [18F]fluoroethyl bromide used for radiolabeling.
The radiochemical purities of the labeled compounds
were more than 98%. Though primary esters at the
second position of the piperidine or pyrrolidine ring are
6,14
With BChE (Fig. 1A), thehydrolysis rateof each of the
esters synthesized ranged from 0.31 (4) to 0.85 minꢀ1
gꢀ1 mLꢀ1 (5S). For thepositional effect, the3yl-esters
(butyrate; 5R and 5S, valerate; 7R and 7S) were hydro-
lyzed more rapidly than corresponding 4yl-esters (buty-
rate; 4, valerate; 6). With AChE (Fig. 1B), each ester
showed a much lower hydrolysis rate compared to with
BChE, ranging from 0.0024 (6) to 0.017 minꢀ1gꢀ1mLꢀ1
(5R). The same positional effect was observed as BChE,
i.e., the hydrolysis rate of 3-yl ester was larger than that
of 4-yl ester. Each of the butyryl esters (4, 5R, 5S)
showed a 2–4 times higher hydrolysis rate than that of
the corresponding valeryl esters (6, 7R, 7S). Regarding
the chiral effect, R isomers of butyryl and valeryl ester
(5R, 7R) showed higher hydrolysis compared to the
corresponding S isomers (5S, 7S). Ignoring other effects
such as hydrolysis by other enzymes,6 each of the esters
examined showed more than 97% of BChE-specificity
(Fig. 1C). The total-rate of each ester in the human
cerebral cortex was almost same as BChE-rate (Fig.
known to migrateto theN atom,
all theprimary
esters synthesized in this study were quite stable
(decomposition rate; <0.0001 minꢀ1) in a phosphate
buffer (pH=7.4, 0.1M).
To estimate the BChE specificity and hydrolysis rate of
the designed esters in the human brain, we used purified
Table 1. Structureof compounds
Compd
R1
R2
R3
Reference compounds
1 (MP4A)
2 (MP4P)
3 (MP4B)
MeH
MeH
MeH
OCOCH
OCOC
OCOC
3
2H5
3H7
1A), sinceAChE-ratewas ngeligibl.e Compound
showed an estimated hydrolysis rate of 0.32 minꢀ1
ꢀ41
Evaluated compounds
H
g
4
Et
Et
Et
Et
Et
CH2OCOC3H7
H
H
CH2OCOC4H9
mLꢀ1, being the closest to the rate of 1 (0.16 minꢀ1
5R
5S
6
7R
7S
8
(R)CH2OCOC3H7
(S)CH2OCOC3H7
H
g
ꢀ1 mLꢀ1, Table2 ), one of established esters, suggesting
that 4 may bemost suitablefor 18F-labeling.
(R)CH2OCOC4H9
(S)CH2OCOC4H9
H
H
H
Et
CH2CH2F
Based on these results from in vitro screening of N-
[14C]ethyl labeled esters, N-[18F]fluoroethylpiperidin-4-
CH2OCOC3H7

T. Kikuchi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1927–1930
1929
Figure 1. Thehydrolysis rates (min ꢀ1) of primary esters, butyryl esters (4, 5R and 5S) and valeryl esters (6, 7R and 7S) of N-[14C]ethyl piper-
idinemethanols with pure human BChE (panel A; BChE-rate) and AChE (panel B; AChE-rate) at the unit tissue concentration (g/mL) and BChE
specificity (%) (panel C) of each ester estimated for the human cerebral cortex.
ylmethyl butyrate (8) was finally synthesized and its
biochemical and physicochemical properties were com-
pared with those of a [11C]MP4A, [11C]MP4P and
[11C]MP4B (Table2 ). Thetotal-rate(0.20 min ꢀ1) of 8
estimated for the human cerebral cortex was almost the
sameas that of MP4A (0.16 min ꢀ1), suggesting that 8
would havean appropriatehydrolysis ratein thehuman
brain. TheBChE-specificity of 8 was estimated as 98%,
indicating that 8 satisfies the required conditions for in
vivo BChE measurement by PET, so far as biochemical
properties are concerned. The partition coefficients (log
P) of authentic ester and metabolite of 8 were 0.62 and
ꢀ1.3, respectively, suggesting that the ester form is suf-
ficiently lipophilic and its metabolite is moderately
hydrophilic. Actually in our preliminary study, high
initial uptake was observed in rat cerebral cortex after
intravenous injection of 8 (1.2% dose/g tissue at 1 min),
whileits mteabolitehardly crossde theblood–brain
barrier (<0.1% dose/g tissue at 1–30 min) after intra-
venous injection.
In conclusion, we have developed a novel [18F]-labeled
compound, N-[18F]fluoroethylpiperidin-4-ylmethyl buty-
rate, which satisfies the required conditions for BChE
measurement by PET.
Acknowledgements
Wethank thecrwe of theCyclotron Scetion and
Radiopharmaceuticals Section of the National Institute
of Radiological Sciences for their support in the oper-
ation of thecyclotron and theproduction of radio-
isotopes.
Table 2. Comparison of N-[18F]fluoroethylpiperidine-4-ylmethyl
butyrate( 8) with established PET tracers, [11C]MP4A and [11C]MP4P,
and a previously-developed BChE-Tracer, [11C]MP4B
Compd
Total-ratea
Specificity to
BChEb (%)
Log Pc
(ester)
Log Pc
(metabolite)
References and notes
8
0.200
0.033
0.035
0.160
98
98
—
—
0.62
0.21
ꢀ1.3
ꢀ2.2
ꢀ2.2
ꢀ2.2
1. Guillozet, A. L.; Smiley, J. F.; Mash, D. C.; Mesulam,
M. M. Ann. Neurol. 1997, 42, 909.
2. Perry, E. K.; Perry, R. H.; Blessed, G.; Tomlinson, E.
Neuropath. Appl. Neurobiol. 1978, 4, 273.
MP4B
MP4P
MP4A
ꢀ0.22
ꢀ0.30
3. Irie, T.; Fukushi, K.; Akimoto, Y.; Tamagami, H.;
Nozaki, T. Nucl. Med. Biol. 1994, 21, 801.
4. Iyo, M.; Namba, H.; Fukushi, K.; Shinotoh, H.;
Nagatsuka, S.; Suhara, T.; Sudo, Y.; Suzuki, K.; Irie, T.
Lancet 1997, 349, 1805.
a The estimated hydrolysis rate in the human cerebral cortex (unit;
minꢀ1gꢀ1mLꢀ1) calculated in the same manner as in Figure1 .
b Calculated by the ratio of BChE-rate to total-rate.
c Partition coefficients measured in a mixture of 1-octanol/phosphate
buffer (0.1M, pH 7.4).

1930
T. Kikuchi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1927–1930
5. Kuhl, D. E.; Koeppe, R. A.; Minoshima, S.; Snyder, S. E.;
Ficaro, E. P.; Foster, N. L.; Frey, K. A.; Kilbourn, M. R.
Neurology 1999, 52, 691.
6. Kikuchi, T.; Fukushi, K.; Ikota, N.; Ueda, T.; Nagatsuka,
S.; Arano, Y.; Irie, T. J. Labelled. Cpd. Radiopharm. 2001,
44, 31.
7. Snyder, S. E.; Gunupudi, N.; Sherman, P. S.; Butch, E. R.;
Skaddan, M. B.; Kilbourn, M. R.; Koeppe, R. A.; Kuhl,
D. E. J. Cereb. Blood. Flow. Metab. 2001, 21, 132.
8. Fukushi, K.; Nagatsuka, S.; Irie, T. J. Labelled. Cpd.
Radiopharm. 2003, 46 (Suppl 1), S347.
Compound 6: N-Ethylpiperidin-4-ylmethyl valerate; 1H
NMR (d, CDCl3): 0.85–0.94 (6H, m, 2ꢁCH3), 1.25–1.36
(6H, m, 3ꢁCH2), 1.54–1.69 (3H, m, 2ꢁCH, CH), 1.81–
1.89 (2H, m, 2ꢁCH), 2.27 (2H, t, CH2), 2.36 (2H, q, CH2),
2.90–2.94 (2H, m, 2ꢁCH), 3.89 (2H, t, CH2). 13C NMR
(CDCl3): 12.10, 13.66, 22.24, 27.40, 28.90, 34.02, 35.40,
52.56, 52.90, 68.61, 173.90. Anal. calcd for (C13H25NO2):
C 68.68, H 11.08, N 6.16. Found: C 68.70, H 11.07, N
6.15. IR (neat) cmꢀ1 1706.
Compound 7R and 7S: (R)- and (S)-N-Ethylpiperidin-4-
ylmethyl valerate; R isomer; [a]2D0 +7.0ꢂ (c=1.1, CHCl3),
S isomer; [a]D20 ꢀ7.7ꢂ (c=1.1, CHCl3). 1H NMR (d,
CDCl3): 0.86–0.94 (6H, m, 2ꢁCH3) 1.28–1.37 (3H, m,
CH, CH2), 1.55–1.67 (6H, m, 4ꢁCH, CH2), 1.82–1.90
(2H, m, 2ꢁCH), 2.25–2.37 (4H, m, 2ꢁCH2), 2.70–2.81
(2H, m, 2ꢁCH), 3.82–3.95 (2H, m, 2ꢁCH). 13C NMR
(CDCl3): 11.97, 13.67, 18.88, 22.23, 27.05, 27.40, 34.00,
35.90, 52.74, 53.65, 56.78, 67.09, 173.81. Anal. calcd for
(C13H25NO2): C 68.68, H 11.08, N 6.16. Found: C 68.69,
H 11.08, N 6.15. IR (neat) cmꢀ1 1712.
Compound 8: N-fluoroethylpiperidin-4-ylmethyl butyrate;
1H NMR (d, CDCl3): 0.87 (3H, t, CH3), 1.27–1.36 (2H, m,
2ꢁCH), 1.51–1.66 (5H, m, 3ꢁCH, CH2), 1.96–2.03 (2H,
m, 2ꢁCH), 2.18–2.23 (2H, m, 2ꢁCH), 2.56 (1H, m, CH),
2.66 (1H, m, CH), 2.88–2.92 (2H, m, 2ꢁCH), 3.84–3.87
(2H, d, CH2), 4.41 (1H, m, CH), 4.56 (1H, m, CH). 13C
NMR (CDCl3): 13.53, 18.33, 28.67, 34.92, 36.03, 53.50,
58.21, 58.47, 68.33, 80.68, 82.89, 173.54. Anal. calcd for
(C12H22FNO2): C 62.31, H 9.59, N 6.06. Found: C 62.31,
H 9.60, N 6.05. IR (neat) cmꢀ1 1735.
9. Bohme, H.; Hilp, M. Chem. Ber. 1970, 103, 104.
¨
10. Shao, X.; Butch, E. R.; Kilbourn, M. R.; Snyder, S. E.
Nucl. Med. Biol. 2003, 30, 491.
11. Whittaker, V. P. Physiol. Rev. 1951, 31, 312.
12. Compound 4: N-Ethylpiperidin-4-ylmethyl butyrate; 1H
NMR (d, CDCl3): 0.89–0.95 (6H, m, 2ꢁCH3), 1.10–1.37
(4H, m, 2ꢁCH2), 1.58–1.69 (3H, m, 2ꢁCH, CH), 1.81–1.89
(2H, m, 2ꢁCH), 2.25 (2H, t, CH2), 2.36 (2H, q, CH2), 2.90–
2.94 (2H, m, 2ꢁCH), 3.89 (2H, t, CH2). 13C NMR (CDCl3):
12.10, 13.67, 18.46, 28.92, 35.42, 36.19, 52.57, 52.91, 68.60,
173.72. Anal. calc for (C12H23NO2): C 67.57, H 10.87, N
6.57. Found: C 67.55, H 10.86, N 6.55. IR (neat) cmꢀ1 1718.
Compound 5R and 5S: (R)- and (S)-N-Ethylpiperidin-4-
ylmethyl butyrate; R isomer; [a]2D0 +7.3ꢂ (c=1.1, CHCl3),
S isomer; [a]D20 ꢀ7.9ꢂ (c=1.1, CHCl3). 1H NMR (d,
CDCl3): 0.89–1.10 (6H, m, 2ꢁCH3) 1.27–1.37 (3H, m,
CH, CH2), 1.56–1.71 (4H, m, 4ꢁCH), 1.78–1.92 (2H, m,
2ꢁCH), 2.25 (2H, t, CH2), 2.36 (2H, q, CH2), 2.76–2.89
(2H, m, 2ꢁCH), 3.82–3.99 (2H, m, 2ꢁCH). 13C NMR
(CDCl3): 11.99, 13.66, 18.45, 24.86, 27.42, 35.92, 36.18,
52.75, 53.67, 56.79, 67.09, 173.66. Anal. calcd for
(C12H23NO2): C 67.57, H 10.87, N 6.57. Found: C 67.56,
H 10.86, N 6.58. IR (neat) cmꢀ1 1716.
13. Wirz, B.; Walther, W. Tetrahedron: Asymmetry 1992, 3,
1049.
14. Shao, X.; Lisi, J. M.; Butch, E. R.; Kilbourn, M. R.;
Snyder, S. E. Nucl. Med. Biol. 2003, 30, 293.