Synthesis of [18F]-labeled (2-(2-fluoroethoxy)ethyl) triphenylphosphonium cation as a potential agent for myocardial imaging using positron emission tomography

Article abstract of DOI:10.1016/j.bmcl.2011.11.005

[¹⁸F]-labeled molecular probe for the detection of myocardial perfusion deficit is driving particular interest due to its high clinical applicability. Thus, we synthesized (2-(2-[¹⁸F]fluoroethoxy)ethyl) triphenylphosphonium salt ([

Full text of DOI:10.1016/j.bmcl.2011.11.005

Bioorganic & Medicinal Chemistry Letters 22 (2012) 319–322
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of [18F]-labeled (2-(2-fluoroethoxy)ethyl)triphenylphosphonium
cation as a potential agent for myocardial imaging using positron
emission tomography
Dong-Yeon Kim a,b, Hee-Jung Kim a, Kook-Hyun Yu a, , Jung-Joon Min b,c,
⇑
⇑
a Department of Chemistry, Dongguk University-Seoul, Seoul, Republic of Korea
b Department of Nuclear Medicine, Chonnam National University Hwasun Hospital, Hwasun, Republic of Korea
c Laboratory of In Vivo Molecular Imaging, Department of Nuclear Medicine, Chonnam National University Medical School, Gwangju, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
18F]-labeled molecular probe for the detection of myocardial perfusion deficit is driving particular inter-
Article history:
[
est due to its high clinical applicability. Thus, we synthesized (2-(2-[18F]fluoroethoxy)ethyl)triphenyl-
phosphonium salt ([18F]3) that specifically accumulates in myocardium according to mitochondrial
membrane potential. Here, we evaluated the performance of [18F]3 as a mitochondrial voltage sensor
in vitro and in vivo. The [18F]3 was synthesized with 20ꢀ30% radiochemical yield and radiochemical pur-
Received 11 September 2011
Revised 25 October 2011
Accepted 2 November 2011
Available online 10 November 2011
ity was >98% by analytical HPLC. Specific activity was >6.7 TBq/lmol. The cellular uptake assay showed
Keywords:
preferential uptake of [18F]3 in cardiomyocytes. The results of biodistribution and micro-PET imaging
studies of [18F]3 in mice and rats showed preferential accumulation in the myocardium. The results sug-
gest that this compound would be a promising candidate for myocardial imaging.
Ó 2011 Elsevier Ltd. All rights reserved.
Mitochondrial voltage sensor
Myocardial perfusion agent
[
18F]-labeled
Phosphonium salt
Positron emission tomography (PET)
Myocardial perfusion imaging by single-photon emission
tomography (SPECT) using [99mTc]-sestamibi, [99mTc]-tetrofosmin,
or 201thallium has been validated extensively.1 However, the tech-
nical limitations of SPECT, such as low spatial resolution and rela-
tive tracer inhomogeneities resulting from soft tissue attenuation
or scatter, may compromise the detection of small lesions and
the diagnostic accuracy of SPECT.2 Positron emission tomography
(PET) has several technical advantages over SPECT, such as higher
spatial resolution and quantitative measurement of myocardial
tracer uptake.3 However, the short half-life of currently used PET
cardiac tracers, including [13N]-ammonia, 82rubidium, and [15O]-
water, limit the widespread clinical use of PET due to the need
for a nearby cyclotron or generator. Because of their longer half-
life, [18F]-labeled tracers would avoid this limitation and facilitate
clinical protocols.
nomenon is attributed to the highest density and higher electro-
chemical membrane potential of the mitochondria in
cardiomyocytes.5–7 Loss of the mitochondrial membrane potential
is an early event in cell death caused by myocardial ischemia.8
Cumulative evidence suggests that mitochondria-controlled apop-
tosis underlies cell loss in heart failure.9 Previous publications
reported that [18F]-labeled phosphonium cations such as [18F]-flu-
orobenzyl triphenylphosphonium ([18F]-FBnTP) are metabolically
stable and demonstrate excellent characteristics as cardiac imag-
ing agents in healthy and coronary artery disease models.5,7,10
In this Letter, we report the synthesis and characterization of a
novel
[
18F]-labeled phosphonium cation (2-(2-[18F]fluoroeth-
oxy)ethyl)triphenylphosphonium cation ([18F]3), which is a deriva-
tive of a previously reported radiolabeled tetraphenylphosphonium
salt.11 Biological studies, such as cellular uptake assay, biodistribu-
tion studies, and micro-PET imaging, were performed to test and
optimize the kinetics for radiolabeled phosphonium cation.
The total synthesis of compounds 3 and [18F]3 is shown in
Schemes 1 and 2. The reference compound was synthesized via
three-step procedures and was analyzed by 1H, 13C NMR, electro-
spray ionization (ESI) high resolution mass spectroscopy to con-
firm the identity (for experimental details see Supplementary
data).
To address this need, we developed a [18F]-labeled phospho-
nium cation.4 Similar to SPECT tracers such as [99mTc]-sestamibi
and
[
99mTc]-tetrofosmin, phosphonium cations accumulate in
cardiomyocytes to a higher degree than in normal cells.5 This phe-
⇑
Corresponding authors. Address: Department of Nuclear Medicine, Chonnam
National University Medical School, 160 Ilsimri, Hwasun, Jeonnam 519-763,
Republic of Korea. Tel.: +82 61 379 8476; fax: +82 61 379 8455 (J.J.). Address:
Department of Chemistry, Dongguk University-Seoul, 30 Pildong-ro 1-Gil, Jung-gu,
Seoul 100-715, Republic of Korea. Tel.: +82 2 2260 3709; fax: +82 2 2268 8204
(K.H.).
For the synthesis of reference compound, compound 1 was pre-
pared by four kinds of the modification of a previously reported
method.12–15 Compound 1 was synthesized with 75–85% yield,
E-mail addresses: yukook@dongguk.edu (K.H. Yu), jjmin@jnu.ac.kr (J.J. Min).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.005

320
D.Y. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 319–322
Scheme 1. Synthesis of (2-(2-fluoroethoxy)ethyl)triphenylphosphonium salt (3). Reagents and conditions: (a) 4-methylbenzene-1-sulfonyl chloride, pyridine, room temp,
3 h; (b) tetrabutylammonium fluoride, acetonitrile, 85 °C, 6 h; (c) triphenylphosphine, acetonitrile, reflux, 19 h.
Scheme 2. Radiosynthesis of (2-(2-[18F]fluoroethoxy)ethyl)triphenylphosphonium salt ([18F]3) Reagents and conditions: (d) [18F]KF, Kryptofix 2.2.2, cetonitrile, 90 °C, 5 min;
(e) triphenylphosphine, toluene, 220 °C, 3 min.
and was treated thereafter with tetrabutylammonium fluoride
(TBAF) in acetonitrile, to generate the fluoro compound 2 with a
56% yield. Finally, we performed a nucleophilic substitution reac-
tion with triphenylphosphine,16 in which the p-tosyloxy residue
was chosen as the leaving group. Synthesis yield of 3 was 75%
the UV peak of reference compound 3 (upper side of Fig. 2B) and
the radio peak of [18F]3 were detected with the same retention
time (lower side of Fig. 2B), indicating high purity of [18F]3.
The total radiolabeling reaction time of [18F]3 was under 50 min
and radiochemical yield ranged 20–30% in the labeled compound
(EOS; corrected for decay). Radiochemical purity was >98% by ana-
and the methylene groups in the
x-position of the alkyl side chain
were split into a doublet of triplets by fluorine in NMR (2JHF
:
lytical HPLC. The specific activity was >6.7 TBq/l
mol. [18F]3 had
47.0 Hz). We also confirmed 4-methylbenzenesulfonate as a coun-
ter ion by NMR.
the necessary characteristics for use in the clinic because the total
reaction time for the radiosynthesis of [18F]3 was under 50 min.
For synthesis of [18F]-labeled compound, 1 was reacted with
For in vitro stability test, [18F]3 (0.37 MBq/100
lL) was incu-
n.c.a.
[
18F]fluoride in the presence of potassium carbonate
bated with human serum (1.0 mL) in a 37 °C water bath for 4 h
and then analyzed by chromatography on ITLC-sg strips developed
with methylene chloride: methanol (9:2 v/v). After development,
the chromatographic strips were scanned on an automatic TLC
scanner. The percentage of the remaining [18F]3 (Rf = 0.40–0.45)
was >95%, indicating the relatively high in vitro stability of the
radiotracer (Fig. 2).
(K2CO3) and the phase-transfer agent Kryptofix 2.2.2. Radio-TLC
showed a >90% of compound [18F]2. Then, [18F]2 was reacted with
triphenylphosphine to yield target compound with no separation
step. The solution was cooled and injected into a semipreparative
HPLC column system equipped with a UV detector and a high en-
ergy gamma detector for purification of [18F]3 (mobile phase, ace-
tonitrile:phosphate-buffered saline (45/55 v/v); flow rate, 3 mL/
min; 254 nm; tR; 14.9 min). A chromatogram of crude [18F]3 was
obtained by measuring UV absorption at 254 nm (Fig. 1A, upper
side) and by using the high energy gamma detector (Fig. 1A, lower
side). Generally, the [18F]-labeled compound was not detected by
the UV detector because it was present at very low levels. The ab-
sence of an overlapping region in Figure 1A indicated high specific
activity of [18F]3. For the identification of the radioproduct, the
collected HPLC fraction (Fig. 1A, lower side) was coinjected with
reference compound 3. No peaks corresponding to those of impu-
rities were observed in the analytical UV chromatogram in which
To determine the lipophilicity of [18F]3, the compound was dis-
solved in a mixture of 3 mL of saline (0.9% NaCl aqueous solution)
and 3 mL of n-octanol in a round-bottom flask. The mixture was
vigorously stirred for 20 min at room temperature and was then
transferred to a 15 mL Falcon conical tube. The tube was centri-
fuged at 3000 rpm for 5 min. Samples in triplets from n-octanol
and aqueous layers were obtained and were counted on a gamma
counter. LogP value of [18F]3 was 0.89 0.02. Although there is lit-
tle information available with regard to the optimal lipophilicity
needed for the high myocardial selectivity of a radiotracer, a pre-
dictive model has been reported for selective accumulation of

D.Y. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 319–322
321
Figure 1. (A) Purification of [18F]3 on semipreparative HPLC. The reaction mixture was injected on a C18 column and was eluted with acetonitrile: phosphate-buffered
saline = 45:55 at a flow rate of 3.0 mL/min. A chromatogram of crude [18F]3 was obtained by performing UV absorption at 254 nm (Fig. 1A, upper side) and by using a high
energy gamma detector (Fig. 1A, lower side). (B) Analytical HPLC chromatogram of [18F]3 coinjected with the nonradioactive compound 3.
phosphonium cations in myocardial cells.17 For lipophilic cationic
radiotracers (logP = 0.5–1.5) such as 99mTc-sestamibi (logP = 1.1),
their membrane diffusion kinetics are so fast that they tend to
localize in mitochondria-rich organs such as the heart.17 Further-
more, because the alkyl group increases the hydrophobicity, the
hydrophobic interaction between the triphenylphosphonium cat-
ion and the lipid core is attractive, due to the hydrophobicity of
the lipophilic phosphonium cation and increased entropy.18–20
Cell uptake values of [18F]3 in H9c2 and NIH/3T3 cells over
incubation periods of 0.5, 1, and 2 h are shown in Figure 3. The up-
take increased from 0.47% 0.03% at 0.5 h to 0.68% 0.02% at 2 h.
The uptake was about twofold higher than that of NIH/3T3 cells.
The mitochondrial membrane potential-dependent cellular uptake
of [18F]3 was further assessed using the H9c2 cells treated with
Figure 2. In vitro human serum stability of [18F]3.
Table 1
Normal biodistribution studies in BALB/c mice at 10, 30, 60, 120 min after i.v.
injection of [18F]3a
10 min
30 min
60 min
120 min
Blood
Heart
Lung
Liver
0.64 0.09
35.23 6.80
7.69 1.18
3.57 1.10
3.35 1.39
3.75 1.16
9.14 4.58
0.22 0.03
19.33 5.11 19.96 2.08 24.27 5.47
0.23 0.02
0.24 0.02
5.18 0.85
2.42 0.24
2.81 1.07
3.98 0.90
8.52 2.62
4.01 0.74
2.03 0.17
1.95 1.52
4.72 0.92
4.67 2.10
5.55 1.13
1.33 0.26
1.23 0.05
2.49 0.83
2.66 0.61
9.28 5.05
5.44 0.87
3.20 0.94
2.28 0.13
Spleen
Stomach
Intestine
Kidney
Pancreas
Normal muscle
Bone
36.05 14.41 23.67 9.53 16.86 2.73
8.80 3.74
6.46 1.85
337 1.14
11.08 6.01
4.58 0.49
5.96 2.23
5J27 0.64
235 0.64
8.05 2.39
3.71 0.59
7.35 2.12
4.42 1.15
2.44 0.48
9.87 1.10 19.16 7.52
5.05 0.61 4.36 0.31
Heart/liver
Heart/lung
a
Data are expressed as the percentage administered activity (injected dose) per
gram of tissue (% ID/g, n = 12). The myocardial uptake of [18F]3 was >35% ID/g at
10 min after radiotracer injection. The heart-to-blood ratio of [18F]3 was >55,
whereas the heart-to-lung and heart-to-liver ratios were >4 and >11, respectively,
at 10 min.
Figure 3. Cell uptake of [18F]3 in H9c2 cells, H9c2 cells treated with 0.5
protonphore CCCP, and NIH/3T3 cells at 0.5, 1.0, or 2.0 h in a 37 °C incubator. All
results are expressed as percentage of applied radioactivity, and are the
means SD of three measurements (P <0.05).
lM
a

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D.Y. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 319–322
Figure 4. Micro-PET imaging (corona) of rats after i.v. injection of [18F]3. Shown are images at 0.5 h (A) and 1.0 h (B) after injection of [18F]3. H, heart; L, liver.
carbonyl cyanide m-chlorophenylhydrazone (CCCP) which is pro-
tonphore that selectively abolishes the mitochondrial membrane
at GIST and Nuclear R&D Program of the Korean Ministry of
Education, Science and Technology.
potential. When incubated with 0.5 lM of CCCP, cellular uptake
of [18F]3 was significantly inhibited (P <0.05) at all incubation time
points (Fig. 3).21,22 These results clearly demonstrate that [18F]3
accumulates in the cells through the mitochondrial membrane
potential.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.005.
The in vivo biodistribution of [18F]3 was examined in BALB/c
mice (Table 1). High levels of radioactivity accumulated in the
heart. The myocardial uptake of [18F]3 was >35% ID/g at 10 min
after radiotracer injection. The heart-to-blood ratio increased from
>55 at 10 min to >102 at 120 min, indicating rapid clearance of the
compound from the blood. The heart-to-lung ratio and the heart-
to-liver ratio were >4 and >11, respectively, indicating that [18F]3
is optimal as a cardiac imaging agent.
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Coronal micro-PET imaging was performed in rats at 0.5 h
(Fig. 4A) and 1 h (Fig. 4B) after i.v. injection of [18F]3. The results
demonstrated good visualization of the heart, with excellent
heart-to-background contrast at each time point. Micro-PET imag-
ing revealed rapid washout from the liver and lung after i.v. injec-
tion of [18F]3. However, [18F]3 was retained at a constant level in
the myocardium for up to 1.0 h after injection.
In the current study, we suggested the feasibility of a [18F]-la-
beled phosphonium cation as a PET myocardial agent. Alkyltriphe-
nylphosphonium cations are better candidates for the general
delivery of compounds to mitochondria, because they accumulate
to a greater extent within the mitochondria in cells.23 The [18F]3
compound was synthesized very easily from the reaction of tri-
phenylphosphine with appropriate precursor, which have the
proper cationic activity and lipophilicity to penetrate the mito-
chondrial membrane and accumulate inside. [18F]3 revealed good
myocardial uptake, high myocardial selectivity, and fast clearance
from other organs. Future studies will be directed toward func-
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This research was supported by the National Research Founda-
tion of Korea (NRF) (No. 2010-0028750), by the Bio R&D program
(2010-0020658), and in part by the Bioimaging Research Center