Synthesis of [18F]-labeled (6-fluorohexyl)triphenylphosphonium cation as a potential agent for myocardial imaging using positron emission tomography

Article abstract of DOI:10.1021/bc2004439

Lipophilic cations such as phosphonium salts penetrate the hydrophobic barriers of the plasma and mitochondrial membranes and accumulate in mitochondria in response to the negative inner-transmembrane potentials. Thus, as newly developed noninvasive imagi

Full text of DOI:10.1021/bc2004439

Article
pubs.acs.org/bc
Synthesis of [18F]-Labeled (6-Fluorohexyl)triphenylphosphonium
Cation as a Potential Agent for Myocardial Imaging using Positron
Emission Tomography
Dong-Yeon Kim,†,‡ Hee-Jung Kim,† Kook-Hyun Yu,*,† and Jung-Joon Min*,‡,§
†Department of Chemistry, Dongguk University-seoul, Seoul, Korea
‡Department of Nuclear Medicine, Chonnam National University Hwasun Hospital, Hwasun, Korea
§Laboratory of In Vivo Molecular Imaging, Department of Nuclear Medicine, Chonnam National University Medical School,
Gwangju, Korea
S
* Supporting Information
ABSTRACT: Lipophilic cations such as phosphonium salts
penetrate the hydrophobic barriers of the plasma and
mitochondrial membranes and accumulate in mitochondria
in response to the negative inner-transmembrane potentials.
Thus, as newly developed noninvasive imaging agents, [18F]-
labeled phosphonium salts may serve as molecular “voltage
sensor” probes to investigate the role of mitochondria,
particularly in myocardial disease. The present study reports
the radiosynthesis of (6-fluorohexyl)triphenylphosphonium
salt (3) as a potential agent for myocardial imaging by using
positron emission tomography (PET). The reference compound of (6-[18F]fluorohexyl)triphenylphosphonium salt ([18F]3) was
synthesized with 74% yield via three-step nucleophilic substitution reactions. The reference compound was radiolabeled via two-
step nucleophilic substitution reactions of no-carrier-added [18F]fluoride with the precursor hexane-1,6-diyl bis(4-
methylbenzenesulfonate) in the presence of Kryptofix 2.2.2 and K2CO3. The radiolabeled compound was synthesized with
15−20% yield. The radiochemical purity was >98% by analytical HPLC, and the specific activity was >6.10−6.47 TBq/μmol. The
cellular uptake assay showed 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 suggest that this
compound would be a promising candidate for myocardial imaging.
half-life, [18F]-labeled tracers would avoid this limitation and
INTRODUCTION
■
facilitate clinical protocols.
Nuclear medicine imaging by single-photon emission tomog-
raphy (SPECT) using [99mTc]-sestamibi, [99mTc]-tetrofosmin,
or 201thallium has been validated extensively. SPECT is a widely
used tool for the assessment of myocardial infarction (MI)
size1,2 for the risk stratification of patients. It also is an attractive
surrogate end point instead of mortality for determining the
efficacy of therapeutic strategies in clinical trials.1 However, the
technical limitations of SPECT imaging, such as low spatial
resolution and the assessment of only relative tracer
inhomogeneities resulting from soft tissue attenuation or
scatter, may compromise the delineation of small infarcts and
the diagnostic accuracy of SPECT.2
Positron emission tomography (PET) has several technical
advantages over SPECT, such as higher spatial resolution.
Owing to accurate attenuation correction, PET can provide
quantitative measures of myocardial tracer uptake.3 However,
the short half-life of currently used PET tracers for myocardial
imaging, 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
To address this need, we developed a [18F]-labeled
phosphonium 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 phenomenon is attributed to the highest
density and higher electrochemical 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 apoptosis underlies cell loss in heart
failure.9 Previous publications have reported that [18F]-labeled
phosphonium cations such as [18F]-fluorobenzyl triphenyl-
phosphonium ([18F]-FBnTP) are metabolically stable and
demonstrate excellent characteristics as cardiac imaging agents
in healthy and coronary artery disease models.5,7,10
Received: August 15, 2011
Revised: January 12, 2012
Published: January 22, 2012
© 2012 American Chemical Society
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Herein, we report the synthesis and characterization of a
novel [18F]-labeled phosphonium cation, (6-[18F]fluorohexyl)-
triphenylphosphonium salt ([18F]3) (Figure 1). Biological
The organic phase was washed twice with water and brine,
dried over sodium sulfate, and filtered. After evaporation of the
solvent, the solution was purified by column chromatography
(methylene chloride:n-hexane:acetone = 48:50:2) and recrystal-
lized from methylene chloride:n-hexane to yield 5.12 g (80%)
of compound 1. mp 77−79 °C; 1H NMR (500 MHz, CDCl3) δ
1.25−1.27 (m, 4H), 1.57−1.60 (m, 4H), 2.45 (s, 6H), 3.97 (t, J
= 6.3 Hz, 4H), 7.34 (d, J = 8.4 Hz, 4H), 7.77 (d, J = 8.4 Hz,
4H); 13C NMR (125 MHz, CDCl3) δ 21.60, 21.75, 28.26,
70.08, 127.96, 130.00, 133.01, 144.96; HRMS (ESI) m/z
calculated for C20H26O6S2Na [M+Na]+ 449.1064; found
449.1068.
Figure 1. Schematic structure of (6-[18F]fluorohexyl)-
triphenylphosphonium salt ([18F]3).
Synthesis of 6-Fluorohexyl 4-Methylbenzenesulfo-
nate (2). Anhydrous acetonitrile (3.0 mL) was added to
tetrabutylammonium fluoride trihydrate (TBAF, 1.43 g, 4.54
mmol). The mixture was evaporated under reduced pressure to
remove the water. This procedure was repeated twice.
Compound 1 (1.94 g, 4.54 mmol) in 10.0 mL of anhydrous
acetonitrile was added to the reaction flask. The mixture was
stirred for 4 h at 85 °C in a closed tube. The solvent was
evaporated under reduced pressure. Column chromatography
(methylene chloride:n-hexane:acetone = 49:50:1) provided
studies, such as a cellular uptake assay, biodistribution studies,
and micro-PET imaging, were performed to test and optimize
the kinetics for radiolabeled phosphonium cation.
EXPERIMENTAL PROCEDURES
■
General. All commercial reagents and solvents were
purchased from Sigma-Aldrich or Merck, were of analytical
grade, and were used without further purification. [3H]-
tetraphenylphosphonium ([3H]-TPP) was purchased from
1
0.60 g (48% yield) of compound 2 as a yellow oil. H NMR
(500 MHz, CDCl3) δ 1.34−1.35 (m, 4H), 1.58−1.65 (m, 4H),
2.44 (s, 3H), 4.02 (t, J = 6.3 Hz, 2H), 4.39 (dt, J = 47.3, 6.0 Hz,
2H), 7.34 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 7.5 Hz, 2H); 13C
NMR (125 MHz, CDCl3) δ 21.73, 24.74, 28.82, 30.16, 70.49,
83.95, 127.98, 129.92, 133.21, 144.81; MS (FAB) m/z 275 [M
+H]+, 173 (100); HRMS (FAB) m/z calculated for
C13H20FO3S [M+H]+ 275.1117; found 275.1120.
1
Moravek Biochemicals, Inc. The H and 13C NMR spectra
were recorded on a JEOL ECA-500 FT-NMR spectrometer
(Advanced Radiation Technology Institute, Korea Atomic
Energy Research Institute). All chemical shifts were reported
on the ppm scale with tetramethylsilane as an internal standard.
Mass spectra were recorded on a JEOL JMS-AX505WA
spectrometer. Compounds were measured by electrospray
ionization (ESI) and fast atom bombardment (FAB) methods
at the National Center for Interuniversity Research Facilities
(NCIRF). Gravity column chromatography was performed on
Merck silica gel 60 (70−230 mesh ASTM). Thin layer
chromatography (TLC) was performed on Merck silica gel
60 F254 glass plates and was visualized by UV light. Purification
was achieved by HPLC, with a SP930D pump, UV730D UV
detector (Young-Lin Inc., Korea), and FC-3200 high energy
gamma detector (Bioscan, USA) to measure the radioactive
flow. The UV detection wavelength was 254 nm for all
experiments. Both semipreparative (Phenomenex Luna, C18,
10 mm × 250 mm) and analytical (Waters Atlantis C18, 4.6
mm × 250 mm) reverse-phase HPLC columns were used. A
CRC-712MH radioisotope calibrator (Capintec Instruments,
USA) was used for radioactivity measurements. [18F] analysis
was performed with a 1480 WIZARD 3 gamma counter
(Perkin-Elmer, USA), and [3H] was measured with an LS 6500
liquid scintillation counter (Beckman, USA). Normal rats were
imaged and analyzed by micro-PET (Inveon, Siemens). No-
carrier-added (n.c.a) [18F]fluoride was produced on a PETtrace
cyclotron (16.4 MeV, General Electric Company, USA) by
irradiation of a [18O]H2O water target.
Synthesis of (6-Fluorohexyl)triphenylphosphonium
Salt (3). Compound 3 was prepared by the modification of a
previously reported method.12 Triphenylphosphine (1.0 g, 3.81
mmol) dissolved in 10.0 mL anhydrous acetonitrile was added
to compound 2 (1.05 g, 3.81 mmol). The solution was refluxed
for 19 h. The solvent was evaporated under reduced pressure.
Column chromatography (methylene chloride:methanol:ethyl
acetate = 8:1:1) provided 1.03 g (74%) of compound 3 as a
powder. Retention factor (Rf): 0.45−0.50 (methylene chlor-
1
ide:methanol = 9:2); mp 220−223 °C; H NMR (500 MHz,
CDCl3) δ 1.31−1.36 (m, 2H), 1.50−1.61 (m, 6H), 2.28 (s,
3H), 3.56−3.61 (m, 2H), 4.33 (dt, J = 47.2, 6.1 Hz, 2H), 7.04
(d, J = 7.8 Hz, 2H), 7.63−7.79 (m, 17H); 13C NMR (125
MHz, CDCl3) δ 21.60, 22.01, 22.56, 24.75, 29.90, 84.03,
118.08, 118.75, 126.22, 128.32, 130.55, 133.61, 135.03. 138.50,
144.67; MS (FAB) m/z 365 [M]+, 365 (100); HRMS (FAB)
m/z calculated for C24H27FP [M]+ 365.1834; found 365.1834.
R a d i o l a b e l i n g o f ( 6 - F l u o r o h e x y l ) -
triphenylphosphonium Salt ([18F]3). [18F]Fluoride was
produced by an 18O(p, n)18F reaction on a PETtrace cyclotron.
The activity was extracted from H218O by an anion exchanger
and was eluted by aqueous potassium carbonate (25.0 mmol)
into the reaction vessel. The radioactive solution was dried
together with 4.0 mg of Kryptofix 2.2.2 in 1.0 mL of acetonitrile
under nitrogen at 100 °C. The solution was evaporated at 100
°C by bubbling nitrogen gas. The residue was dried by
azeotropic distillation with acetonitrile (1 mL, 3 times). Then,
4.0 mg of compound 1 dissolved in 1.0 mL of anhydrous
acetonitrile were added. The mixture was heated for 5 min at 90
°C in the closed state. Radio-TLC showed a >80% yield of
compound [18F]2. The solution was passed through a small
silica Sep-Pak cartridge. Six milligrams of triphenylphosphine
dissolved in 1.0 mL of toluene were added to the reaction
Synthesis of Hexane-1,6-diyl Bis(4-methylbenzenesul-
fonate) (1). Compound 1 was prepared by the modification of
a previously reported method.11 Hexane-1,6-diol (1.77 g, 15.0
mmol) in 30.0 mL of anhydrous pyridine was added to 4-
methylbenzene-1-sulfonyl chloride (8.58 g, 45.0 mmol) at 0 °C.
The mixture was stirred at room temperature for 3 h, quenched
with 3.0 mL of water, and stirred for a further 30 min.
Methylene chloride and 1.0 M HCl were added to the reaction
mixture, and the pyridine was extracted from the organic phase.
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Figure 2. (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 = 50:50 at a flow rate of 3.0 mL/min. UV impurities were not observed. (B) Analytical HPLC chromatogram of [18F]3
coinjected with the nonradioactive compound 3.
vessel, and the solution was heated to 220 °C for 3 min with no
separation step (Scheme 2).
Deficient DMEM (Dulbecco’s modified Eagle medium) High
Glucose (containing 5.3 mmol/L KCl and 110.34 mmol/L
NaCl; Life Technologies) plus 5% fetal bovine serum (FBS),
1% penicillin-streptomycin, and 1% L-glutamine. Cells were
incubated at 37 °C for 0.5, 1, or 2 h with radioactive culture
medium and [18F]3 (0.74 MBq/200 μL). The radioactive
medium was removed, and the radioactivity was measured. The
wells were washed three times with cold phosphate-buffered
saline. The cells were harvested, and the cell-associated
radioactivity was determined. The mitochondrial membrane
potential-dependent cellular uptake of [18F]3 was further
assessed with H9c2 cells treated with carbonyl cyanide m-
chlorophenylhydrazone (CCCP), which is a protonphore that
selectively abolishes the mitochondrial membrane potential.14
The compound was dissolved in dimethyl sulfoxide and diluted
to the desired concentration with low K+ HEPES buffer. The
final concentration of dimethyl sulfoxide was <0.1%. CCCP
solution (0.50 μM) was added to 5.0 × 104 cells at 30 min
before the start of the experiment. After incubation with [18F]3,
radioactivity was determined by a gamma counter.15 The
mitochondrial membrane potentials of both cell lines were
assessed by a cell uptake study with [3H]-TPP (37 kBq/200
μL). The [3H]-TPP uptake assay is a standard method for
measuring mitochondrial membrane potential in vitro.7 After
incubation with [3H]-TPP, radioactivity was determined with a
liquid scintillation counter. Triplicate samples were obtained for
all uptake studies. Data are expressed as the accumulation ratio
(%) SD calculated by dividing the radioactivity in the pellet
by the total radioactivity (supernatant + pellet).
The solution was cooled and injected into a semipreparative
HPLC column system for purification (mobile phase,
acetonitrile:phosphate-buffered saline = 50:50; flow rate, 3
mL/min; 254 nm; tR: 19.5 min) (Figure 2A). For identification
of the radioproduct, the collected HPLC fraction was
coinjected with cold compound 3. [18F]3 was dried, made
isotonic with sodium chloride, and passed through a 0.20 μm
membrane filter into a sterile multidose vial for in vitro and in
vivo experiments. The total reaction time of [18F]3 was within
60 min, and the overall decay-corrected radiochemical yield was
approximately 15−20%. Radiochemical purity was >98% by the
analytical HPLC system (with the same isocratic as used for the
semipreparative HPLC system). The specific activity was
>6.10−6.47 TBq/μmol (Figure 2B).
In Vitro Stability Study. For the labeling stability test,
[18F]3 (0.37 MBq/100 μL) was incubated with human serum
(1.0 mL) in a 37 °C water bath for 4 h and then was analyzed
by chromatography on ITLC-sg strips developed with
methylene chloride:methanol = 9:2. After they were developed,
the chromatographic strips were scanned on an automatic TLC
scanner, and TLC was performed at various time points. All
experiments were performed in triplicate.
Partition Coefficient. After the complete removal of
volatiles of [18F]3, the residue was dissolved in a mixture of
3 mL of saline and 3 mL of n-octanol in a round-bottom flask.
The mixture was vigorously stirred for 20 min at room
temperature and then was transferred to a 15 mL Falcon
conical tube. The tube was centrifuged at 3000 rpm for 5 min.
Samples in triplets from n-octanol and aqueous layers were
obtained and were counted on a gamma counter. The log P
value was reported as an average of the data obtained in three
independent measurements.13
Murine Biodistribution Studies. To confirm the activity
of [18F]3 in vivo, biodistribution studies were performed with a
murine model. Biodistribution in different organs was assessed
in BALB/c mice (18−20 g, Orient, Kyunggido, Korea) at 10,
30, 60, and 120 min after i.v. injection of 7.4 MBq radiotracer.
Blood, heart, lung, liver, spleen, stomach, intestine, kidney,
pancreas, bone, and muscle were sampled from mice, and the
radioactivity of each organ was measured with a gamma
counter. Radioactivity determinations were normalized by the
Cell Lines, Culture Conditions, and Cell Uptake
Studies. [18F]3 accumulation was measured in cell culture
with rat embryonic cardiomyoblast (H9c2) and mouse normal
fibroblast (NIH/3T3) cell lines. All cells were grown in
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a
Scheme 1. Synthesis of (6-Fluorohexyl)triphenylphosphonium Salt
a
Reagents and conditions: (a) 4-methylbenzene-1-sulfonyl chloride, pyridine, room temp, 3 h; (b) tetra butyl ammonium fluoride, acetonitrile, 85
°C, 4 h; (c) triphenylphosphine, acetonitrile, reflux, 19 h.
a
Scheme 2. Radiosynthesis of (6-Fluorohexyl)triphenylphosphonium Salt
a
Reagents and conditions: (d) [18F]KF, Kryptofix 2.2.2, acetonitrile, 90 °C, 5 min; (e) triphenylphosphine, toluene, 220 °C, 3 min.
weight of the tissue and the amount of radioactivity injected to
obtain the % ID/g.
(2JHF: 47.2 Hz). We also confirmed 4-methylbenzenesulfonate
as counterions by NMR.
For synthesis of the [18F]-labeled compound, 1 was reacted
with nca [18F]fluoride in the presence of potassium carbonate
(K2CO3) and the phase-transfer agent Kryptofix 2.2.2. Then,
[18F]2 was reacted with triphenylphosphine to yield the target
compound with no separation step. After purification of the
[18F]-labeled target compound by semiprep HPLC, [18F]3 was
compared with the reference compound by analytical HPLC to
determine the identity (Figure 2A and B). Figure 2B revealed a
single peak, suggesting the formation of one product ([18F]3)
identical to the reference compound. Radiochemical yield
ranged 15−20% in the labeled compound (EOS; corrected for
decay). Radiochemical purity was >98% by analytical HPLC.
The specific activity was >6.10−6.47 TBq/μmol.
Micro-PET Imaging Studies. Normal rats were imaged
with micro-PET at 0.5 and 1.0 h after i.v. injection of 37 MBq
of [18F]3. Eight-week-old male Sprague−Dawley rats (250−
260 g, Orient, Kyunggido, Korea) were used for this study.
Animal care, all experiments, and euthanasia were performed in
accordance with protocols approved by the Chonnam National
University Animal Research Committee and the Guide for the
Care and Use of Laboratory Animals published by the National
Institutes of Health (NIH publication 85−23, revised 1985).
Rats were anesthetized by ketamine hydrochloride (100 mg/
kg) and xylazine hydrochloride (2.5 mg/kg).
RESULTS AND DISCUSSION
■
When the radiotracer (0.37 MBq [18F]3) was incubated in
human serum at 37 °C for 4 h, the percentage of the remaining
[18F]3 was >95%, indicating the relatively high in vitro stability
of the radiotracer (Supporting Information).
Synthesis and Radiochemistry. 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
1
and was analyzed by H, 13C NMR, and FAB high-resolution
A previous publication has reported the synthesis of the (3-
[18F]fluoropropyl)triphenylphosphonium cation, with 12%
radiochemical yield.12 In this previous study, the whole
radiosynthesis was completed in 56 min, with an average
specific radioactivity of 15.5 GBq/μmol. The radiochemical
characteristics of [18F]3 and (3-[18F]fluoropropyl)-
triphenylphosphonium cation were quite similar, except that
[18F]3 had a significantly higher specific activity. In the current
study, we further used the fluoroalkylphosphonium cation
derivative ([18F]3) in biological studies, including a cellular
uptake assay, biodistribution studies, and micro-PET imaging.
mass spectroscopy to confirm the identity.
For synthesis of the reference compound, compound 1 was
first prepared by commercially available hexane-1,6-diol reacted
with an excess of 4-methylbenzene-1-sulfonyl chloride in
pyridine. Compound 1 was synthesized with an 80% yield
and was treated thereafter with TBAF in acetonitrile, to
generate the fluoro compound 2. Finally, a nucleophilic
substitution reaction was performed with triphenylphosphine,
in which the p-tosyloxy residue was chosen as the leaving
group. Methylene groups in the ω-position of the alkyl side
chain were split into a doublet of triplets by fluorine in NMR
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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. The heart was visible with excellent heart-to-
liver and heart-to-lung contrast at each time point after i.v. injection.
(log P = 1.1), their membrane diffusion kinetics are so fast they
tend to localize in mitochondria-rich organs such as the heart.16
Furthermore, because the alkyl group increases the hydro-
phobicity, the hydrophobic interaction between the triphenyl-
phosphonium cation and the lipid core is attractive due to the
hydrophobicity of the lipophilic phosphonium cation and
increased entropy.17−19
In Vitro Cell-Uptake Assays. The mitochondrial mem-
brane potential of H9c2 cells, which was assessed by a cellular
[3H]-TPP uptake assay, was significantly higher than that of
NIH/3T3 cells (Figure 3A). 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 3B. The uptake increased from 2.37
0.13% at 0.5 h to 3.28 0.08% at 2 h. The uptake was about 6-
fold higher than that of NIH/3T3 cells. When incubated with
0.5 μM of CCCP, cellular uptake of [18F]3 was significantly
inhibited (P < 0.05) at all incubation time points (Figure 3B).
These results clearly demonstrate that [18F]3 accumulates in
the cells through the mitochondrial membrane potential.
Biodistribution Studies and Micro-PET Imaging. 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 >20% ID/g at 10 min
after radiotracer injection. The heart-to-blood ratio of [18F]3
Figure 3. (A) Cell uptake of [3H]-TPP in H9c2 and NIH/3T3 cells at
0.5, 1.0, or 2.0 h in a 37 °C incubator. Uptake by H9c2 cells was higher
than uptake by NIH/3T3 cells for the entire time. This finding means
that the mitochondria membrane potential of H9c2 cells was higher
than that of NIH/3T3 cells. (B) Cell uptake of [18F]3 in H9c2 cells,
H9c2 cells treated with 0.5 μM protonphore CCCP, and NIH/3T3
cells at 0.5, 1.0, or 2.0 h in a 37 °C incubator. Uptake by H9c2 cells
was about 6-fold higher than uptake by NIH/3T3 cells. When
incubated with 0.5 μM CCCP, the cellular uptake of [18F]3 was
significantly inhibited (P < 0.05). All results are expressed as a
percentage of applied radioactivity and are the means SD of three
measurements.
Lipophilicity of Radiolabeled Compound. To assess the
lipophilicity of [18F]3, we measured the log P value of [18F]3,
which was 1.78
0.05. Although there is little information
available with regard to the optimal lipophilicity needed for the
high myocardial selectivity of a radiotracer, a predictive model
has been reported for the selective accumulation of
phosphonium cations in myocardial cells.16 For lipophilic
cationic radiotracers (log P = 0.5−1.5) such as 99mTc-sestamibi
a
Table 1. Normal Biodistribution Studies in BALB/c Mice at 10, 30, 60, and 120 min after i.v. Injection of [18F]3
10 min
30 min
60 min
120 min
Blood
0.72 0.14
21.84 2.26
6.57 1.25
5.13 1.73
6.11 2.22
6.48 0.48
15.24 2.90
56.63 12.20
12.32 2.84
4.24 1.15
5.22 0.86
30.95 4.80
4.58 1.61
3.36 0.30
5.43 1.76
0.29 0.02
20.47 4.35
4.52 0.57
1.74 0.11
3.29 0.08
5.58 1.11
11.06 1.66
20.72 1.20
9.73 0.32
4.16 0.49
5.55 1.15
71.87 21.63
11.90 3.37
4.50 0.44
4.91 0.67
0.19 0.03
23.20 2.70
3.70 0.12
1.30 0.12
2.94 0.19
5.58 1.69
11.29 2.10
12.68 3.13
10.09 1.13
4.53 0.54
7.30 1.36
126.76 22.51
17.83 1.18
6.27 0.65
5.13 0.35
0.14 0.01
20.09 3.12
2.84 0.17
0.81 0.20
2.37 0.57
5.07 2.13
3.47 0.88
11.47 1.84
9.91 1.68
4.14 0.28
7.45 1.86
138.61 8.10
25.53 5.88
7.07 0.98
4.83 0.44
Heart
Lung
Liver
Spleen
Stomach
Intestine
Kidney
Pancreas
Normal Muscle
Bone
Heart/blood
Heart/liver
Heart/lung
Heart/muscle
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 >20% ID/g for the whole time after radiotracer injection. The heart-to-blood ratio of [18F]3 was >138, whereas the heart-to-lung, heart-to-liver,
and heart-to-normal muscle ratios were >7, >25, and >4, respectively, at 120 min.
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(8-[18F]fluorooctyl)triphenylphosphonium cation (a longer-
chain analogue) showed higher liver uptake and delayed
clearance (data not shown).
CONCLUSION
■
In the current study, we suggested the feasibility of a [18F]-
labeled phosphonium cation as a PET myocardial agent.
Alkyltriphenylphosphonium cations are better candidates for
the general delivery of compounds to mitochondria because
they accumulate to a greater extent within the mitochondria in
cells.20 The [18F]3 compound was synthesized very easily from
the reaction of triphenylphosphine with appropriate precursors,
which have the proper cationic activity and lipophilicity to
penetrate the mitochondrial 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 functional studies with diverse in vitro
and small animal models.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Figure 5. (A) Time−activity curves generated from dynamic PET
images. [18F]3 accumulated specifically in the heart. The [18F]3 in
liver was washed out rapidly but was retained in the myocardium for
the whole time. (B) Contrast ratio of myocardium-to-liver and
myocardium-to-lung for 1.0 h after i.v. injection of [18F]3.
■
*Jung-Joon Min, M.D., Ph.D., Department of Nuclear
Medicine, Chonnam National University Medical School, 160
Ilsimri, Hwasun, Jeonnam 519-763, Republic of Korea; Phone
82-61-379-8476; Fax 82-61-379-8455; E-mail: jjmin@jnu.ac.kr.
Kook-Hyun Yu, Ph.D., Department of Chemistry, Dongguk
University-Seoul, 30 Pildong-ro 1-Gil, Jung-gu, Seoul, 100-715,
Republic of Korea; Phone 82-2-2260-3709; Fax 82-2-2268-
8204; E-mail: yukook@dongguk.edu.
was >30 at 10 min, indicating rapid clearance of the compound
from the blood. The heart-to-lung, heart-to-liver, and the heart-
to-normal muscle ratios were >3, >4, and >5, respectively, at 10
min after radiotracer injection. Notably, the heart-to-blood and
heart-to-liver ratios increased to >138 and >25 at 120 min after
radiotracer injection, indicating that [18F]3 is optimal as a
cardiac imaging agent.
Notes
The authors declare no competing financial interest.
Coronal micro-PET imaging was performed in rats at 0.5 h
(Figure 4A) and 1 h (Figure 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.
Time−activity curves for the myocardium, liver, and lung
revealed rapid washout from the liver and lung after i.v.
injection of [18F]3. However, [18F]3 was retained at a constant
level in the myocardium for up to 1.0 h after injection (Figure
5A). The myocardium-to-liver and myocardium-to-lung uptake
ratios quickly reached 5.0 after [18F]3 injection (Figure 5B).
The rationale for preparing the 6-carbon spacer group was to
decrease lipophilicity. Previous publications have reported that
[18F]-FBnTP is metabolically stable and demonstrates excellent
characteristics as a cardiac imaging agent in healthy
mice.7However, the myocardium-to-liver uptake ratio in the
time-activity curve reached 1.0 at approximately 25 min after i.v.
injection of [18F]-FBnTP, indicating delayed washout from the
liver. Clearance of the radiotracer from the liver would be
dependent on the lipophilicity of the compound. Thus, in the
current study, several kinds of radiolabeled phosphonium salts
were synthesized to assess the appropriate lipophilicity by
different carbon chain lengths; however, highly lipophilic
structures (such as benzene rings) were not adopted for the
radiolabeling of phosphonium salts. Compared to [18F]3, the
(3-[18F]fluoropropyl)triphenylphosphonium cation12 (a short-
er-chain analog) showed lower myocardial uptake, whereas the
ACKNOWLEDGMENTS
■
This research was supported by the National Research
Foundation of Korea (NRF) (No. 2011-0029941), by the
Brain Korea 21 Project funded by the Ministry of Education,
Science, and Technology, and in part by the Nuclear R&D
Program of the Korean Ministry of Education, Science and
Technology.
REFERENCES
■
(1) Schwaiger, M., and Melin, J. (1999) Cardiological applications of
nuclear medicine. Lancet 354, 661−6.
(2) Gibbons, R. J., Valeti, U. S., Araoz, P. A., and Jaffe, A. S. (2004)
The quantification of infarct size. J. Am. Coll. Cardiol. 44, 1533−42.
(3) Knuuti, J., and Bengel, F. M. (2008) Positron emission
tomography and molecular imaging. Heart 94, 360−7.
(4) Kim, D., Yu, K., Bom, H., and Min, J. (2007) Synthesis of (4-
[18F]Fluorophenyl)triphenylphosphonium as a Mitochondrial Voltage
Sensor for PET. Nucl. Med. Mol. Imaging 41, 561−565.
(5) Madar, I., Ravert, H., Dipaula, A., Du, Y., Dannals, R. F., and
Becker, L. (2007) Assessment of severity of coronary artery stenosis in
a canine model using the PET agent 18F-fluorobenzyl triphenyl
phosphonium: comparison with 99mTc-tetrofosmin. J. Nucl. Med. 48,
1021−30.
(6) Min, J. J., Biswal, S., Deroose, C., and Gambhir, S. S. (2004)
Tetraphenylphosphonium as a novel molecular probe for imaging
tumors. J. Nucl. Med. 45, 636−43.
436
dx.doi.org/10.1021/bc2004439 | Bioconjugate Chem. 2012, 23, 431−437

Bioconjugate Chemistry
Article
(7) Madar, I., Ravert, H., Nelkin, B., Abro, M., Pomper, M., Dannals,
R., and Frost, J. J. (2007) Characterization of membrane potential-
dependent uptake of the novel PET tracer 18F-fluorobenzyl
triphenylphosphonium cation. Eur. J. Nucl. Med. Mol. Imaging 34,
2057−65.
(8) Kroemer, G. (2003) Mitochondrial control of apoptosis: an
introduction. Biochem. Biophys. Res. Commun. 304, 433−5.
(9) Cesselli, D., Jakoniuk, I., Barlucchi, L., Beltrami, A. P., Hintze, T.
H., Nadal-Ginard, B., Kajstura, J., Leri, A., and Anversa, P. (2001)
Oxidative stress-mediated cardiac cell death is a major determinant of
ventricular dysfunction and failure in dog dilated cardiomyopathy. Circ.
Res. 89, 279−86.
(10) Madar, I., Ravert, H. T., Du, Y., Hilton, J., Volokh, L., Dannals,
R. F., Frost, J. J., and Hare, J. M. (2006) Characterization of uptake of
the new PET imaging compound 18F-fluorobenzyl triphenyl
phosphonium in dog myocardium. J. Nucl. Med. 47, 1359−66.
(11) Yu, K. H., Kim, Y. S., Kim, S. W., Park, J. H., Yang, S. D.,
Herdering, W., and Knoechel, A. (2003) Synthesis of [18F]-
Fluoroclofilium as a potential cardiac imaging agent for PET studies.
J. Label. Compd. Radiopharm. 46, 1151−1160.
(12) Ravert, H. T., Madar, I., and Dannals, R. F. (2004)
Radiosynthesis of 3-[18F]fluoropropyl and 4-[18F]fluorobenzyl triar-
ylphosphonium ions. J. Label. Compd. Radiopharm. 47, 469−476.
(13) Kim, Y. S., Yang, C. T., Wang, J., Wang, L., Li, Z. B., Chen, X.,
and Liu, S. (2008) Effects of targeting moiety, linker, bifunctional
chelator, and molecular charge on biological properties of 64Cu-labeled
triphenylphosphonium cations. J. Med. Chem. 51, 2971−84.
(14) Steen, H., Maring, J. G., and Meijer, D. K. (1993) Differential
effects of metabolic inhibitors on cellular and mitochondrial uptake of
organic cations in rat liver. Biochem. Pharmacol. 45, 809−18.
(15) Cheng, Z., Winant, R. C., and Gambhir, S. S. (2005) A new
strategy to screen molecular imaging probe uptake in cell culture
without radiolabeling using matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry. J. Nucl. Med. 46, 878−86.
(16) Zhou, Y., and Liu, S. (2011) 64Cu-Labeled Phosphonium
Cations as PET Radiotracers for Tumor Imaging. Bioconjugate Chem.
(17) Demura, M., Kamo, N., and Kobatake, Y. (1987) Binding of
lipophilic cations to the liposomal membrane: thermodynamic
analysis. Biochim. Biophys. Acta 820, 303−308.
(18) Ono, A., Miyauchi, S., Demura, M., Asakura, T., and Kamo, N.
(1994) Activation energy for permeation of phosphonium cations
through phospholipid bilayer membrane. Biochemistry 33, 4312−8.
(19) Smith, R. A., Kelso, G. F., James, A. M., and Murphy, M. P.
(2004) Targeting coenzyme Q derivatives to mitochondria. Methods
Enzymol. 382, 45−67.
(20) Ross, M. F., Kelso, G. F., Blaikie, F. H., James, A. M., Cocheme,
H. M., Filipovska, A., Da Ros, T., Hurd, T. R., Smith, R. A., and
Murphy, M. P. (2005) Lipophilic triphenylphosphonium cations as
tools in mitochondrial bioenergetics and free radical biology.
Biochemistry (Mosc.) 70, 222−30.
437
dx.doi.org/10.1021/bc2004439 | Bioconjugate Chem. 2012, 23, 431−437