Effect of the prosthetic group on the pharmacologic properties of 18F-labeled rhodamine b, a potential myocardial perfusion agent for positron emission tomography (PET)

Article abstract of DOI:10.1021/jm301453p

We recently reported the development of the 2-[¹⁸F]fluoroethyl ester of rhodamine B as a potential positron emission tomography (PET) tracer for myocardial perfusion imaging. This compound, which was prepared using a [¹⁸F]fluoroethyl prosthetic group, has significant uptake in the myocardium in rats but also demonstrates relatively high liver uptake and is rapidly hydrolyzed in vivo in mice. We have now prepared ¹⁸F-labeled rhodamine B using three additional prosthetic groups (propyl, diethylene glycol, and triethylene glycol) and found that the prosthetic group has a significant effect on the in vitro and in vivo properties of these compounds. Of the esters prepared to date, the diethylene glycol ester is superior in terms of in vitro stability and pharmacokinetics. These observations suggest that the prosthetic group plays a significant role in determining the pharmacological properties of ¹⁸F-labeled compounds. They also support the value of continued investigation of ¹⁸F-labeled rhodamines as PET radiopharmaceuticals for myocardial perfusion imaging.

Full text of DOI:10.1021/jm301453p

Article
pubs.acs.org/jmc
Effect of the Prosthetic Group on the Pharmacologic Properties of
18F‑Labeled Rhodamine B, a Potential Myocardial Perfusion Agent for
Positron Emission Tomography (PET)
Mark D. Bartholoma,†,‡,§ Vijay Gottumukkala,†,‡ Shaohui Zhang,†,‡ Amanda Baker,† Patricia Dunning,†
̈
Frederic H. Fahey,†,‡ S. Ted Treves,†,‡ and Alan B. Packard*,†,‡
†Division of Nuclear Medicine and Molecular Imaging, Boston Children’s Hospital, Boston, Massachusetts 02115, United States
‡Harvard Medical School, Boston, Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: We recently reported the development of the 2-
[18F]fluoroethyl ester of rhodamine B as a potential positron
emission tomography (PET) tracer for myocardial perfusion
imaging. This compound, which was prepared using a
[18F]fluoroethyl prosthetic group, has significant uptake in
the myocardium in rats but also demonstrates relatively high
liver uptake and is rapidly hydrolyzed in vivo in mice. We have
now prepared 18F-labeled rhodamine B using three additional
prosthetic groups (propyl, diethylene glycol, and triethylene
glycol) and found that the prosthetic group has a significant effect on the in vitro and in vivo properties of these compounds. Of
the esters prepared to date, the diethylene glycol ester is superior in terms of in vitro stability and pharmacokinetics. These
observations suggest that the prosthetic group plays a significant role in determining the pharmacological properties of 18F-
labeled compounds. They also support the value of continued investigation of 18F-labeled rhodamines as PET
radiopharmaceuticals for myocardial perfusion imaging.
have already been established for [18F]2-fluorodeoxyglucose
INTRODUCTION
■
([18F]FDG), demonstrating that production of 18F-labeled
Cardiovascular disease is a major health problem worldwide,
and noninvasive imaging modalities such as single-photon
radiopharmaceuticals at central sites is a reasonable alternative
to on-site production. An additional advantage of the 110 min
emission computed tomography (SPECT) and positron
half-life is that while it is long enough to allow distribution from
emission tomography (PET) play a key role in the diagnosis
central production facilities, it is still short enough to allow
and treatment planning for this disease. The limitations of
repeated (rest/stress) MPI studies of a patient on the same day.
single-photon tracers, including the absence of a standardized
Repeated single-day MPI studies are routinely performed using
attenuation correction method, which can result in attenuation
artifacts in obese patients or women with large or dense breast
tissue, the inability to perform quantitative measurements,
which are useful for the detection of balanced ischemia, the
99mTc MPI radiopharmaceuticals (t1/2 = 6 h),5 and recently, the
applicability of 18F MPI radiopharmaceuticals for single-day
MPI studies was demonstrated for flurpiridaz F 18.6,7
Several 18F-labeled compounds have been proposed as
higher spatial resolution and sensitivity of PET, and recurring
shortages of 99mTc, all argue for an increased role for PET in
possible MPI radiopharmaceuticals including quaternary
ammonium salts,8 tetraphenylphosphonium compounds,9−12
rotenone derivatives,13,14 and pyridazinone analogues, such as
BMS-747158-02 (flurpiridaz F 18, Lantheus Medical Imag-
ing).15−21 Of these compounds, flurpiridaz has been the subject
of the most extensive evaluation to date.6,22,23
myocardial perfusion imaging (MPI).1,2
Despite its advantages, the tracers that are currently available
for PET MPI suffer from significant practical limitations. The
short half-lives of 15O (2 min) and 13N (10 min) limit their use
to clinical centers with an on-site cyclotron. Rubidium-82 (t1/2
= 76 s) is generator produced, allowing its use at clinics without
access to cyclotrons, but the high cost of the generator limits its
use to facilities with high patient throughput. Other limitations
of 82Rb include less than optimal myocardial extraction and
high positron energy, which decreases spatial resolution.1 These
limitations have spurred interest in the development of 18F-
labeled myocardial perfusion tracers.3,4
Like the single-photon MPI tracers 99mTc-methoxyisobutyli-
sonitrile (99mTc-MIBI) and 99mTc-tetrofosmin and the 18F-
labeled tetraphenylphosphonium derivatives, rhodamine B is a
lipophilic cation (Figure 1). Other properties that rhodamine B
shares with 99mTc-MIBI and 99mTc-tetrofosmin include local-
ization in mitochondria24−27 and being a substrate for P-
The physical properties of 18F (β+, 0.635 MeV [97%]; t1/2
110 min) are nearly ideal for PET, and distribution networks
=
Received: October 8, 2012
Published: December 4, 2012
© 2012 American Chemical Society
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and found that only 2% of the diethylene glycol ester remained
intact at 30 min after injection compared to 6% of the
triethylene glycol ester.39 In combination, these results suggest,
somewhat unexpectedly, that changing the ester group has
minimal impact on the in vivo stability of these compounds.
Given the popularity of 2-fluoroethyl as a prosthetic group,
particularly in the preparation of 18F-labeled esters, and the
propensity of carboxylic esters to undergo in vivo hydrolysis,
especially in mice,40 it is somewhat surprising that there has not
been a more extensive evaluation of alternative prosthetic
groups that might be more resistant to in vivo degradation.
Here, we report our recent efforts to address this issue while at
the same time improving the pharmacologic properties of 18F-
labeled rhodamine B by evaluating the effect of changing the
prosthetic group on the biodistribution and serum stability of
the corresponding rhodamine B esters.
Figure 1. Rhodamine B.
glycoprotein, which is involved in multidrug resistance.28,29
Furthermore, it was found that nonradiolabeled rhodamine 123
accumulates in mouse heart.30 These properties suggested that
18F-labeled rhodamines are promising candidates for evaluation
as potential PET MPI radiopharmaceuticals.
We recently described the synthesis and initial biological
evaluation of an 18F-labeled rhodamine B ester, 2-[18F]-
fluoroethyl rhodamine B ([18F]3, Figure 2), as a potential
PET radiopharmaceutical for the evaluation of myocardial
perfusion.31,32 In these studies, we found that although [18F]3
did not accumulate in mouse myocardium, presumably because
of rapid in vivo hydrolysis of the 2-fluoroethylester prosthetic
group, it did demonstrate significant uptake in the rat heart.
The pharmacologic properties of [18F]3 are, however, less than
optimal, particularly with respect to uptake and clearance from
the liver.
Prosthetic groups are a convenient way to radiolabel organic
species containing secondary amine or carboxylate moieties
with 18F,33−36 and by far the most commonly used prosthetic
group, particularly with respect to carboxylates, is 2-fluoroethyl,
which was used to prepare [18F]3. Henrikson and co-workers
measured the in vivo stability of the 18F-labeled methyl and
ethyl esters of carfentanil in mice and found that only 2% of the
methyl ester remained intact in the serum at 40 min after
injection while 5−6% of the ethyl ester remained intact at 40
min.37 Erlandsson and co-workers measured the stability of the
18F-labeled ethyl esters of the 4-chloro- and 4-bromometomi-
date in rats38 and found that that only 6% of the chloro
compound remained intact 30 min after injection while 39% of
the bromo compound remained intact at the same time point.
These investigators also measured the metabolic stability of the
diethylene and triethylene glycol esters of metomidate in rats
RESULTS AND DISCUSSION
■
In previous studies, we demonstrated the feasibility of using
[18F]3 as a myocardial perfusion agent in rats.32 The liver
uptake of this compound was, however, less than optimal and
interfered with the visualization of the myocardium. In the
present study, we evaluated three alternative prosthetic groups
to determine if, by changing the prosthetic group, we could
decrease the concentration of the compound in the liver and/or
increase the rate of clearance from the liver while
simultaneously maintaining or increasing tracer accumulation
in the heart.
Synthesis of 18F-Labeled Rhodamine B Esters. The
three tracers ([18F]4, propyl ester; [18F]5, diethylene glycol
ester; [18F]6, triethyleneglycol ester) were prepared in good
yield and high radiochemical purity using the one-pot reaction
(Figure 2) previously used to prepare [18F]3 followed by
purification by semipreparative high performance liquid
chromatography (HPLC).31,32 The identity of the tracers was
confirmed by analytical HPLC using the nonradioactive (19F)
compounds as reference materials.
The total synthesis time was 120 min, and the desired
products were obtained in >97% radiochemical purity in decay-
corrected yields of 18 1%, 19 1%, and 22 3% for [18F]4,
[18F]5, and [18F]6, respectively. The specific activities of
[18F]4, [18F]5, and [18F]6 are 50, 310, and 320 MBq/μmol
(1.35, 8.38, and 8.65 mCi/μmol), respectively (Table 1). The
low specific activities are a consequence of the formation of
byproducts in the reaction of the ditosyl precursor with
Figure 2. Radiosyntheses of the previously described ethyl ester [18F]3 and the new compounds [18F]4, [18F]5, and [18F]6: (a) K2CO3/K2.2.2,
ACN, 90 °C, 10 min; (b) DIPEA, ACN, 165 °C, 30 min.
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precursor significantly increases the overall synthesis time and
thus significantly lowers the overall radiochemical yield. Since
the low specific activities did not interfere with the character-
ization of the tracers, the additional HPLC purification step was
omitted in favor of increased radiochemical yield at the
developmental stage. Nonetheless, efforts to further increase
the specific activity of 18F-labeled rhodamine are currently
underway.
Table 1. In Vitro Stability, Partition Coefficients and Specific
Activities for the Three Rhodamine B Derivatives
a
compd serum stability (%)
log P
specific activity
b
[18F]4
[18F]5
[18F]6
99.6 0.7
2.15 0.01 50 MBq/μmol
(1.4 mCi/μmol)
c
96.1 0.8
99.0 0.4
97.1 1.6
d
b
1.90 0.01 310 MBq/μmol
(8.4 mCi/μmol)
The partition coefficients of [18F]4, [18F]5, and [18F]6 are
also summarized in Table 1.
c
96.1 2.0
97.1 1.1
92.1 1.2
Synthesis of Nonradioactive (19F) Compounds. The
nonradioactive 19F derivatives 4, 5, and 6 were prepared as
reference materials for the HPLC characterization of the 18F
compounds and for determination of the specific activity. The
corresponding NMR spectra and mass spectrometry data are
provided in Supporting Information (Figures S1−S11).
Compound 4 was obtained in 73% yield by reacting rhodamine
B chloride salt with commercially available 3-fluoropropan-1-ol
using the coupling reagents N-hydroxysuccinimide (NHS) and
N,N′-dicyclohexylcarbodiimide (DCC). Purification of the
crude reaction mixture by conventional silica gel column
chromatography was not successful; therefore, the product was
purified by semipreparative HPLC. In contrast, compounds 5
and 6 were prepared similarly to the 18F compounds, by
reacting rhodamine B lactone with the corresponding fluoro-
ethylene glycol esters 1 and 2, respectively. Precursors 1 and 2
were prepared in good yield (74−83%) from the corresponding
ditosylates and 1 M tetrabutylammonium fluoride (TBAF) in
d
be
,
1.99 0.01 320 MBq/μmol
(8.6 mCi/μmol)
ce
,
87.2 1.4
97.2 1.1
de
,
a
b
c
At 120 min unless otherwise stated. Mouse serum. Rat serum.
Human serum. At 15 min.
d
e
rhodamine B (i.e., 2-(2-tosylethoxy)ethyl rhodamine B and 2-
(2-hydroxyethoxy)ethyl rhodamine B, formed by in situ
hydrolysis of 2-(2-tosylethoxy)ethyl rhodamine B for [18F]5).
Because of the chemical similarity to the 18F-labeled product,
the only difference being a terminal -F vs -OH or -Tos group,
these byproducts are not separated in the final semipreparative
HPLC purification. We have recently shown for [18F]3 that
HPLC purification of the 18F-labeled tosyl precursor prior to
the reaction with rhodamine B lactone results in higher specific
activities.32 However, the additional HPLC purification of the
Figure 3. Coronal (top), sagittal (middle), and transverse (bottom) microPET images of [18F]4 (A), [18F]5 (B), and [18F]6 (C) in the rat.
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Figure 4. Time−activity curves (TACs) of the heart, liver, and lung constructed from microPET images of [18F]4 (A), [18F]5 (B), and [18F]6 (C) in
the rat.
tetrahydrofuran (THF) with minor modifications to a
procedure described by Mavel et al.41 Reaction of the
precursors with rhodamine B lactone in the presence of
diisopropylethylamine (DIPEA) gave 5 and 6 in 26% and 29%
yield after HPLC purification. All reference compounds were
characterized by proton and fluorine NMR spectroscopy and
mass spectrometry.
substituent, such as a fluorine atom or a methoxy group,
decreases the basicity of the corresponding alcohols (e.g., F-
CH2-CH2-OH ∼ CH3-O-CH2-CH2-OH < CH3-CH2-OH).43
As the propyl, diethylene glycol, and triethylene glycol
rhodamine esters are more stable than the ethyl ester, a similar
trend may be true for 18F-labeled rhodamines.
Small-Animal PET Imaging. Representative 60 min
(summed) small-animal PET images obtained with [18F]4,
[18F]5, and [18F]6 in rats are shown in Figure 3.
These PET images clearly show high and persistent retention
in the myocardium and high contrast between the myocardium
and blood, liver, bone, and lungs for all three compounds. In all
cases, including [18F]6 (Figure 3C), which showed greater
decomposition in rat serum than [18F]4 and [18F]5, the heart is
much more clearly defined than with [18F]3. For all three
compounds, the liver uptake is significantly lower than that of
[18F]3, with [18F]4 and [18F]6 being similar to each other and
[18F]5 being somewhat lower than [18F]4 and [18F]6. In all
three cases there is excretion of tracer through the gut and the
kidneys. A representative maximum intensity projection video
showing the distribution of [18F]5 is available as Supporting
Information.
The microPET images also reveal high accumulation of each
tracer in the neck, as was observed for [18F]3, possibly
representing the parathyroid or salivary gland accumulation,
and in the muscles of the forelimbs. The bone uptake in all
cases is minimal, with the skeleton being barely visible,
suggesting minimal in vivo defluorination.
Serum Stability Studies. The stability of [18F]4, [18F]5,
and [18F]6 was measured in vitro at 37 °C in mouse, rat, and
human serum and in phosphate buffered saline (PBS) at 15, 30,
60, and 120 min. As expected, no degradation was observed for
any of the compounds in PBS for up to 2 h of incubation time.
The 3-[18F]fluoropropyl ester, [18F]4, remained >95% intact
for 2 h in all tested sera (Supporting Information Figure S14),
and a similar result was obtained for [18F]5 with 97% of the
compound present after 2 h (Supporting Information Figure
S15). These results are substantially different from those
obtained for [18F]3, which was rapidly degraded in mouse
serum with only 25% of the compound being intact after 2 h of
incubation time.
For [18F]6, a rather unusual degradation pattern was noted.
In all tested sera, up to 10% decomposition was observed after
the initial 15 min, but no further degradation occurred at later
time points (Supporting Information Figure S16). Further
degradation was, however, observed upon the addition of fresh
serum to the mixture. This pattern is different from that
observed for [18F]3, which decomposed continuously over the
2 h time period of the study. One possible explanation for this
effect would be that the compound responsible for the
degradation of the triethylene glycol ester is present in limited
amounts in all tested sera. If this is the case, this would probably
result in a more extensive degradation in vivo as the blood-to-
tracer ratio would be much higher and the protein responsible
for the degradation would not be depleted as quickly. This
phenomenon does not, however, negatively impact the ability
of this compound to accumulate in the heart, as [18F]6 shows
high and persistent uptake in the myocardium (vide infra).
The increased stability of [18F]4, [18F]5, and [18F]6
compared to the previously reported compound ([18F]3) is
presumably a consequence of the increasing basicity of the
corresponding fluoroalkyl prosthetic groups. For example, it
was found for benzoate esters that the rate of ester hydrolysis
decreases with increasing basicity (decreasing acidity) of the
alkyl leaving group.42 Furthermore, the acidity of alkyl alcohols
(e.g., methanol, ethanol, and n-propanol) decreases with
increasing alkyl chain length as exemplified by the correspond-
ing pKa values.43,44 Additionally, an electron withdrawing
Time−activity curves of [18F]4, [18F]5, and [18F]6, shown in
Figure 4, reveal rapid liver clearance and no significant washout
from the myocardium over the 60 min imaging period. Of the
three new compounds, the initial liver concentration of [18F]5
is lower and the liver clearance is faster compared to [18F]4 and
[18F]6. Thus, for [18F]5, the heart concentration (69 5 kBq/
cm3) at 5 min post injection (pi) is somewhat higher than that
in the liver (59 2 kBq/cm3) while for [18F]4 (66 6 kBq/
cm3 vs 65 6 kBq/cm3) and [18F]6 (16 2 kBq/cm3 vs 15
2 kBq/cm3) the concentrations are essentially equal. At 35 min
pi, the heart-to-liver ratio for [18F]5 increases to 4.2 while the
heart-to-liver ratios for [18F]4 and [18F]6 increase to 2.0 and
2.3, respectively. These differences can also be seen in a
comparison of the clearance rates of the tracers from the liver.
At 55 min pi, the liver concentration of [18F]5 is 25% of the 5
min value while the corresponding values for [18F]4 and [18F]6
are in the range 30−35%.
These observations are in agreement with the microPET
images (Figure 3), which show that the heart is much more
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Figure 5. Time-resolved microPET image of compound [18F]5 in the rat showing persistent retention in the heart and clearance of [18F]5 from the
liver.
a
Table 2. Rat Biodistribution Data for the Four Rhodamine B Esters at 60 min pi
compd
b
tissue
blood
[18F]3
[18F]4
[18F]5
[18F]6
0.08 0.03
2.06 0.61
1.78 0.65
0.89 0.07
5.63 0.78
8.31 0.81
2.40 0.53
0.20 0.02
0.16 0.04
0.45 0.08
25.8
0.05 0.01
1.32 0.13
1.58 0.19
0.55 0.11
3.82 0.32
8.26 1.62
2.53 0.67
0.20 0.07
0.30 0.01
0.65 0.08
26.4
0.25 0.02
2.51 0.16
2.06 0.16
0.49 0.04
3.53 0.57
9.28 0.66
1.69 0.42
0.36 0.04
0.54 0.07
0.77 0.04
10.0
0.19 0.01
1.03 0.13
0.56 0.05
0.25 0.03
0.83 0.10
3.24 0.43
0.44 0.09
0.24 0.03
0.32 0.05
0.38 0.07
5.4
heart
lung
liver
spleen
kidney
gut
skin
muscle
bone
c
heart/blood
c
heart/lung
1.6
0.8
1.2
1.8
c
heart/liver
2.3
2.4
5.1
4.1
c
heart/bone
4.6
2.0
3.3
2.7
a
b
c
Results are expressed as the mean %ID/g ESD for n = 5. Reference 32. Tissue ratios.
clearly defined with the diethylene glycol ester, [18F]5,
compared to the other esters.
Of the three compounds in the present study, the highest
heart concentration is observed for [18F]5 (2.51
0.16%
injected dose/gram) (%ID/g) followed by [18F]6 (1.32 0.13
%ID/g) and [18F]4 (1.03
0.13 %ID/g). The heart
Figure 5 shows the six 10-min frames for compound [18F]5
from which the time−activity curves (TACs) were derived.
This series of images shows that [18F]5 is essentially cleared
from the liver after 30 min, while the tracer is retained in the
myocardium through the 60 min scan.
concentration of [18F]5 is somewhat higher than that of the
previously described ethyl ester, [18F]3 (2.06 0.61 %ID/g),
but the difference is not statistically significant. The heart
concentration of [18F]5 is, however, significantly greater than
those of [18F]4 and [18F]6 (P < 0.0001). The liver
concentration of [18F]5 at 60 min pi (0.49 0.04 %ID/g) is
significantly lower than that of [18F]3 (0.89 0.07 %ID/g, P <
0.0001), higher than that of [18F]6 (0.25 0.03 %ID/g, P <
0.0001), and essentially equal to that of [18F]4 (0.55 0.11 %
ID/g). These increases in tracer uptake by the heart and
decreases in uptake by the liver for [18F]5 and [18F]6 lead to
significant improvements in the heart-to-liver ratios for these
compounds compared to the prototype compound. The heart-
to-liver ratio for [18F]3 was 2.3 while the ratios for [18F]5 and
[18F]6 are 5.1 and 4.1, respectively, suggesting that [18F]5 is the
preferred compound.
Small-animal PET images were also obtained for [18F]5 and
[18F]6 in mice to determine if the observed increase in in vitro
stability translated into improved myocardial uptake (Support-
ing Information, Figures S12 and S13). While both compounds
showed appreciable uptake in the myocardium, there was also
significant uptake in the kidneys, spleen, gall bladder, and
subsequently the intestine. This pattern of uptake, particularly
the high uptake in the gall bladder, suggests that while these
compounds are more stable in mice than [18F]3, they are not
stable enough to justify using mice for subsequent studies.
Biodistribution Studies. The results of the 60 min
biodistribution studies in rats carried out for [18F]4, [18F]5,
and [18F]6 are summarized in Table 2.
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The blood activity for all three tracers is very low at 60 min
after injection (<0.25 %ID/g). The heart-to-blood ratios for
[18F]4, [18F]5, and[18F] 6 are 26.4, 10.0, and 5.4, respectively.
The bone concentration for all three tracers is less than 1 %ID/
g, consistent with minimal in vivo defluorination. There is
significant accumulation of each tracer in the kidney and gut,
suggesting a combination of renal and hepatobiliary excretion.
There is also significant accumulation of all three tracers in the
lungs and the spleen. This may be due to aggregation, as the
apparent specific activity of these compounds is relatively low
(Table 1) and rhodamines are known to aggregate even at
submicromolar concentrations.45 Efforts are currently underway
to increase the specific activity of the compound, which, if this
hypothesis is correct, will reduce tracer uptake in these
nontarget organs. It is, however, important to note that this
does not interfere with visualization of the heart.
These results are somewhat disparate from those obtained by
Erlandsson et al. in their studies of ethyl esters of chloro- and
bromometomidate and of the of the triethylene and diethylene
glycol esters of metomidate.38,39 These investigators found that
the ethyl ester was slightly more stable in vivo than the
diethylene glycol ester and that the difference between stability
of the triethylene and diethylene glycol esters was minimal.
CONCLUSIONS
■
On the basis of the promising initial results with [18F]3, we
expanded our efforts to develop a myocardial perfusion tracer
based on 18F-labeled rhodamines. The focus of the current
effort was to improve the stability and pharmacokinetics
compared to the original compound. Therefore, we evaluated
different prosthetic groups: propyl, diethylene glycol, and
triethylene glycol. The elongation of the prosthetic group by
only one methylene group (propyl vs ethyl) resulted in a tracer,
[18F]4, that was significantly more stable in mouse, rat, and
human serum than the prototype compound. This compound
also showed improved cardiac imaging characteristics compared
to [18F]3. Similarly, the myocardial uptake of [18F]5, the
diethylene glycol ester, was significantly increased while the
clearance of nontarget tissues (e.g., liver, blood, lung) was also
improved. Unfortunately, however, the triethylene glycol ester
showed no additional improvement, and current efforts are
therefore being directed toward further studies on [18F]5.
In combination, these results confirm our original premise:
18F-labeled rhodamines are promising candidates for continued
evaluation as PET myocardial perfusion tracers. They also
suggest that further examination of the role of prosthetic groups
in the biodistribution and in vivo stability of 18F-labeled
compounds is warranted.
The results of the biodistribution studies of [18F]4, [18F]5,
and [18F]6 may also be compared to other 18F-labeled
myocardial perfusion tracers currently under development.
There are several reports of biodistribution studies in rats of
flurpiridaz, an 18F-labeled MPI agent that is currently in late-
stage clinical trials. In Wistar rats, this compound exhibited
heart uptake of 2.06 %ID/g at 60 min pi, a result that is
somewhat lower than the uptake of [18F]5 (2.51 %ID/g).16 In
Sprague−Dawley rats, however, flurpiridaz F 18 showed heart
uptake with 3.3 %ID/g at 60 min pi.17,46 In the Wistar rat study,
flurpiridaz F 18 cleared quickly from the liver (0.99 %ID/g at
10 min pi vs 0.38 %ID/g at 60 min pi), the 60 min value being
somewhat lower than that observed for [18F]5 (0.49 %ID/g).16
As noted above, the heart-to-liver ratio is of particular
importance for a myocardial perfusion tracer. The heart-to-liver
ratio for [18F]5 at 60 min pi (5.1) is similar to that of flurpiridaz
F 18, 5.4 in Wistar rats and 3.7 in Sprague−Dawley rats,16,46
and the heart-to-blood ratio of flurpiridaz F 18 at 60 min pi
(13.7) is slightly higher than that of [18F]5 (10.0).13
EXPERIMENTAL SECTION
■
General. Rhodamine B lactone (>97%) was purchased from MP
Biomedical (Solon, OH). Rhodamine B chloride salt was obtained
from Sigma-Aldrich (St. Louis, MO). Propane-1,3-diol ditosylate was
purchased from Acros Organics (Fair Lawn, NJ), and diethylene glycol
ditosylate and triethylene glycol ditosylate were purchased from TCI
(Waltham, MA). For the radiosynthesis, extra dry reagent grade
acetonitrile (Thermo Scientific) and Kryptofix (K2.2.2) (98%)
(Sigma-Aldrich) were used. Potassium carbonate (99.97%) was
purchased from Alfa Aesar (Ward Hill, MA). Other solvents and
reagents were of the highest grade commercially available and used
without further purification. Tetrabutylammonium fluoride (1 M in
THF) was purchased from Sigma-Aldrich. 3-Fluoropropanol was
obtained from Oakwood Products, Inc. (West Columbia, SC). The
purity of the nonradioactive (19F) reference compounds was ≥95% as
determined by analytical HPLC and NMR. Thin-layer chromatog-
raphy (TLC) was performed using silica gel IB-F coated plastic sheets
from J. T. Baker (Phillipsburg, NJ). Nuclear magnetic resonance
spectra were obtained using a Varian 600 MHz VNMRS system or a
400 MHz Varian 400-MR system (Palo Alto, CA). Chemical shifts are
given as parts per million (ppm) and are reported relative to
tetramethylsilane. Coupling constants are reported in hertz (Hz). The
multiplicity of the NMR signals is described as follows: s = singlet, d =
duplet, t = triplet, q = quartet, m = multiplet. High-resolution mass
spectra (ESI-MS mode) were obtained at the University of Illinois
Mass Spectrometry Facility using a Micromass 70-VSE spectrometer.
Fluorine-18 (as F− in water) was purchased from Cardinal Healthcare
(Woburn, MA) and Brigham and Women’s Hospital (Boston, MA).
Purification and Quality Control. Analytical HPLC was carried
out using a Hitachi 7000 system including an L-7455 diode array
detector, an L-7100 pump, and a D-7000 interface. The radiometric
HPLC detector comprised Canberra nuclear instrumentation modules
and was optimized for 511 keV photons. An LaChrom PuroSphere
Another 18F-labeled myocardial perfusion tracer currently in
clinical trials is 4-[18F]fluorophenyl)triphenylphosphonium
(18F-FTPP), an 18F-labeled analogue of the tetraphenylphos-
phonium cation.12 This compound exhibits heart uptake of 1.64
%ID/g at 5 min, 1.51 %ID/g at 30 min, and 1.57 %ID/g at 60
min pi in Sprague−Dawley rats.12 At 60 min pi, the liver
concentration of 18F-FTPP is 0.17 %ID/g, significantly lower
than that of [18F]5 (0.48 %ID/g) and flurpiridaz F 18 (0.38 %
ID/g). Thus, at 60 min pi, the heart-to-liver ratio of 18F-FTPP
is 9.2 compared to 5.1 for [18F]5 and 5.4 for flurpiridaz F 18.
Fluorine-18-FTPP is rapidly cleared from blood with the heart-
to-blood ratio increasing from 11 at 5 min to 75 at 60 min pi.
As previously noted, it is well-known that ethyl esters are
susceptible to rapid hydrolysis in mouse serum,47 and it appears
likely that the three esters in the present study (propyl,
diethylene glycol, and triethylene glycol) undergo a similar
decomposition process, albeit to a different extent depending
on the ester moiety. Irrespective of the mechanism, the rate of
decomposition of the three new compounds is apparently slow
enough to allow accumulation of the tracers in the heart, in
contrast to the results observed for the ethyl ester. The net
result is that using diethylene glycol as the prosthetic group
results in higher in vitro stability and significantly improves the
pharmacokinetics of 18F-labeled rhodamines. In this context, it
is worth noting that this is one of the few examples where
diethylene glycol (or triethylene glycol) has been used as a
prosthetic group for labeling a compound with 18F.
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Star C18e column (4 mm × 30 mm, 3 μm) was used for analytical
measurements. The solvent system consisted of 0.1% trifluoroacetic
acid (TFA) in water (solvent A) and 0.1% TFA in acetonitrile (solvent
B) at a flow rate of 1 mL/min at room temperature. The solvent
gradient was 0−15 min (30−70% B), 15−25 min (70% B). The serum
stability graph was created from the raw HPLC data using GraphPad
Prism (GraphPad Software, La Jolla, CA). For semipreparative HPLC,
an ISCO system comprising an ISCO V4 variable wavelength UV−
visible detector (operated at λ = 550 nm), ISCO 2300 HPLC pumps, a
radiometric γ detector similar to that described above, and a Grace
Apollo C18 column (10 mm × 250 mm, 5 μm) was used.
Semipreparative HPLC method A (isocratic) consisted of the
following: 40% solvent A, 60% solvent B; flow rate, 5 mL/min;
room temperature. Semipreparative HPLC method B (gradient)
consisted of the following: 0−10 min (40% B); 10−30 min (40−50%
B); 30−35 min (50−100% B); 35−40 min (100% B); flow rate, 5 mL/
min; room temperature. Radiofluorination yields were determined by
thin-layer chromatography using silica gel plates and chloroform/
methanol (8:1 v/v) as the solvent. After development, the TLC strips
were cut into 1 cm pieces and counted with a Packard Cobra γ
counter.
Synthesis of Reference Compounds. 2-(2-Fluoroethoxy)-
ethyl Tosylate (1). Diethylene glycol ditosylate (500 mg, 1.21 mmol)
was dissolved in 5 mL of anhydrous THF under argon. The solution
was heated to 80 °C followed by the addition of 2.42 mL of 1 M TBAF
in THF. Product formation was monitored by TLC using a mixture of
ethyl acetate/hexane 1:1 (v/v). After 15 min of heating, the starting
material was fully converted as confirmed by TLC, and the reaction
was stopped by cooling in an ice bath. After rotary evaporation to
remove solvent and the difluorodiethylene glycol formed during the
reaction, the remaining pale-yellow oil was purified by silica gel
column chromatography using ethyl acetate/hexane 1:1 (v/v) as the
eluent. Fractions containing the product were combined and the
solvent was removed by rotary evaporation to provide 1 as a colorless
oil. Yield: 262 mg (83%). 1H NMR (CDCl3, 400 MHz): δ 7.80 (2H, d,
J = 8.26), 7.35 (2H, d, J = 8.20), 4.48 (dt, 2H, J1 = 48, J2 = 4.20), 4.18
(t, 2H, J = 4.80), 3.71 (m, 3H), 3.63 (m, 1H), 2.45 (s, 3H).
incomplete conversion, and thus, the crude product was prepurified by
silica gel flash chromatography using the same conditions as described
for 4. At this point, the purity of the product was >95% (by NMR). In
order to obtain a high purity reference material, a small fraction of the
crude product was further purified by semipreparative HPLC using the
same conditions as for the radioactive compound (HPLC method B).
1
Yield (crude): 132 mg (26%). H NMR (CDCl3, 400 MHz): δ 8.33
(dd, 1H, J = 1.14, 7.75), 7.82−7.72 (m, 2H), 7.31 (dd, 1H, J = 0.95,
7.42), 7.07 (d, 2H, J = 9.14), 6.84−6.80 (m, 4H), 4.45 (dt, 2H, J1 = 47,
J2 = 3.8), 4.39 (m, 1H), 4.17 (m, 2H), 3.64−3.51 (m, 12H), 1.32 (t,
12H, J = 7.11). 19F NMR (CDCl3, 600 MHz): −75.6 ppm. HRMS m/
+
z (%): calcd for C32H38FN2O4 [M+] 533.2816, found 533.2808
(100%).
Rhodamine B 2-(2-(2-Fluoroethoxy)ethoxy)ethyl Ester (6).
Compound 6 was prepared and purified by a procedure similar to that
described for compound 5 except that 2 was used instead of
compound 1. HPLC analysis revealed incomplete conversion, and
thus, the crude product was prepurified by silica gel flash
chromatography using the same conditions as described for 4. The
purity of the product was >95% (by NMR). In order to obtain a high
purity reference material, a small fraction of the crude product was
further purified by semipreparative HPLC using the same conditions
as for the radioactive compound (HPLC method B). Yield (crude):
1
29%. H NMR (CDCl3, 400 MHz): δ 8.34 (dd, 1H, J = 0.92, 7.75),
7.81−7.72 (m, 2H), 7.30 (dd, 1H, J = 0.77, 7.37), 7.06 (d, 2H, J =
9.79), 6.80 (m, 4H), 4.53 (dt, 2H, J1 = 48, J2 = 4.1), 4.17 (m, 2H), 3.74
(m, 1H), 3.66 (m, 1H), 3.62−3.53 (m, 14H), 1.32 (t, 12H, J = 7.11).
19F NMR (CDCl3, 600 MHz): −75.6 ppm. HRMS m/z (%): calcd for
+
C34H42FN2O5 [M+] 577.3078, found 577.3073 (100%).
Radiosyntheses of 18F-Labeled Rhodamines. The rhodamine
B esters [18F]4, [18F]5, and [18F]6 were prepared by a one-pot two-
step procedure as previously described for [18F]3.31,32 Briefly, after
addition of Kryptofix and K2CO3 to an aqueous [18F]fluoride solution,
the mixture was azeotropically dried in a Pierce vial using acetonitrile.
Propane-1,3-diol ditosylate, diethylene glycol ditosylate, or triethylene
glycol ditosylate in 0.5 mL of anhydrous acetonitrile was added to the
dried residue, and the mixture was heated at 90 °C for 10 min in a
sealed Pierce vial. After rapid cooling of the mixture to room
temperature, a solution of rhodamine B lactone in 0.8 mL of
anhydrous acetonitrile and DIPEA (3−5 drops) were added into the
vial. Further heating at 160 °C for 30 min using a 30G ventilation
needle gave the crude 18F-labeled rhodamine, which was purified by
semipreparative HPLC. Method A was used to purify [18F]4 (tR = 22.5
min), and method B was used to purify [18F]5 (tR = 20.5 min) and
[18F]6 (tR = 21.5 min). HPLC fractions containing the radioactive
product were combined and dried under a stream of nitrogen at 65 °C.
The radioactive product was then redissolved in 10% EtOH/PBS for
the serum stability studies, 10% EtOH/water for the partition
coefficient measurements, or 10% EtOH/saline for animal experi-
ments. The integrity and purity of the radioactive product were
confirmed by analytical HPLC. The dry down process did not affect
the purity of the compounds.
2-(2-(2-Fluoroethoxy)ethoxy)ethyl Tosylate (2). Compound 2
was prepared and purified by a procedure similar to that described for
1 except that triethylene glycol ditosylate was used as the starting
material instead of diethylene glycol ditosylate. Yield: 249 mg (74%).
1H NMR (CDCl3, 400 MHz): δ 7.80 (2H, d, J = 8.31), 7.35 (2H, d, J
= 8.42), 4.54 (dt, 2H, J1 = 48, J2 = 4.20), 4.17 (m, 2H), 3.75 (m, 1H),
3.71−3.65 (m, 3H), 3.61 (m, 4H), 2.45 (s, 3H) ppm.
Rhodamine B 3-Fluoropropyl Ester (4). Rhodamine B (chloride
salt, 200 mg, 0.42 mmol), NHS (53 mg, 0.46 mmol), DCC (95 mg,
0.46 mmol), and DIPEA (80 μL, 0.46 mmol) were mixed in 10 mL of
anhydrous acetonitrile under an inert atmosphere (argon). After 3 h,
32 mg (0.42 mmol) of 3-fluoropropanol was added and stirring was
continued overnight. The solvent was removed by rotary evaporation
under reduced pressure, and the dark purple residue was prepurified by
silica gel flash chromatography using ethyl acetate/acetone 1:2 (v/v).
The crude product was eluted with methanol. The purity of the crude
product was >95% according to NMR characterization. In order to
obtain a high purity reference material, a small fraction of the crude
product was further purified by semipreparative HPLC using the same
conditions as for the radioactive compound (method A). Yield (crude
product): 165 mg (73%). 1H NMR (CDCl3, 600 MHz): δ 8.29 (d, 1H,
J = 7.71), 7.81 (t, 1H, J = 7.50), 7.75 (t, 1H, J = 7.58), 7.33 (d, 1H, J =
7.25), 7.07 (m, 2H), 6.81 (m, 4H), 4.36 (dt, 2H, J1 = 47, J2 = 5.73),
4.17 (t, 2H, J = 6.18), 3.60 (q, 8H, J = 6.95, 14.03), 1.88 (m, 2H), 1.32
(t, 12H, J = 6.89) ppm. 19F NMR (CDCl3, 600 MHz): −75.8 ppm.
Serum Stability Studies. For each experiment, 3.7−5.6 MBq
(100−150 μCi) [18F]4, [18F]5, or [18F]6 in 20 μL of 10% EtOH/PBS
was added to 200 μL of serum in a 1.5 mL Eppendorf tube, which was
equilibrated at 37 °C in a water bath prior to the addition. The tubes
were shaken and incubated at 37 °C. At selected time points (15, 30,
60, and 120 min), 50 μL aliquots were taken from the serum solution
and 200 μL of cold (4 °C) absolute EtOH was added. The samples
were stored on ice for 5 min before filtering using Eppendorf
centrifuge filters (molecular weight cutoff of 30 kDa) at 13 200 rpm for
5 min at 4 °C. Cold water (200 μL) was added to the filtrate and an
aliquot analyzed by HPLC. For the control, an aliquot from the 10%
EtOH/PBS stock solution was taken, diluted with water and its purity
confirmed by HPLC. The percentage of intact 18F-labeled rhodamine
ester was calculated from the chromatograms. All experiments were
performed on three separate batches of compound, and three samples
were obtained at each time point for each batch of compound.
Partition Coefficient Measurements. For each determination,
3.7−5.6 MBq (100−150 μCi) of [18F]4, [18F]5, or [18F]6 in 20 μL
+
HRMS m/z (%): calcd for C31H36FN2O3 [M+] 503.2710, found
503.2704 (100%).
Rhodamine B 2-(2-Fluoroethoxy)ethyl Ester (5). Rhodamine B
lactone (354 mg, 0.80 mmol) was dissolved in 10 mL of acetonitrile,
and the solution was heated to 80 °C before adding 262 mg (1.00
mmol)of 1 dissolved in 2 mL of acetonitrile and 0.5 mL (2.94 mmol)
of DIPEA. The mixture was refluxed for 16 h, allowed to cool to room
temperature, and evaporated to dryness. HPLC analysis revealed
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10% EtOH/water was added to a mixture of 480 μL of water and 500
μL of octanol in 1.5 mL Eppendorf tubes. Samples were vortexed for 1
min and then centrifuged at 13 200 rpm for 5 min. From each sample,
three 100 μL samples of both the water and the organic layer were
transferred into plastic tubes, and the samples were counted with a
Packard Cobra γ counter. Experiments were performed in triplicate.
Small-Animal PET Imaging Studies. All in vivo experiments
(PET imaging and biodistribution) with [18F]4, [18F]5, and [18F]6
were performed in rats because of the higher in vivo stability of the
previously reported compound [18F]3 in rats compared to mice.
Animal studies were carried out under a protocol approved by the
Children’s Hospital Boston Institutional Animal Care and Use
Committee. For animal studies, the HPLC-purified 18F-labeled
rhodamine esters [18F]4, [18F]5, and [18F]6 were redissolved in
10% EtOH/saline. Before injection, the solution was filtered by
centrifugation through a sterilized Eppendorf filter tube (0.2 μm). For
PET imaging studies, a group of at least five animals were scanned.
Animals were injected with 100 μL of [18F]4, [18F]5, or [18F]6 (3.7−
5.6 MBq, 100−150 μCi) and anesthetized with isoflurane (2−4% in
air). Data acquisition was initiated as quickly as possible after injection
of the tracer. Imaging was performed using a Siemens Focus 120
microPET scanner. Data were acquired for 60 min in list mode and
reconstructed into either a single 60 min image or six 10 min frames.
For reconstruction, the 3D PET data were rebinned into 2D sinograms
and reconstructed using the 2D ordered subset expectation max-
imization (2D OSEM) iterative algorithm to generate an image with a
volume of 128 × 128 × 95 voxels (0.866 × 0.866 × 0.796 mm3).
Image analysis was performed using the ASIPro software package
(Siemens Medical Solutions). Time−activity curves (TACs) were
constructed by manually drawing regions of interest (ROI) within the
left ventricular myocardium and the chest and medially in the liver. All
ROIs were then copied on each of the frames, and time−activity
curves of mean pixel values within the ROI were generated.
ACKNOWLEDGMENTS
■
These studies were supported by the Children’s Hospital
Boston Radiology Foundation and NIH Grant 1 R01
HL108107-01 (A.B.P.). The 70-VSE mass spectrometer was
purchased in part with a grant from the Division of Research
Resources, National Institutes of Health (Grant RR 04648).
ABBREVIATIONS USED
■
PET, positron emission tomography; SPECT, single photon
emission computed tomography; MPI, myocardial perfusion
imaging; HPLC, high performance liquid chromatography;
TLC, thin layer chromatography; ID, injected dose; ESI,
electrospray ionization; TBAF, tetrabutylammonium fluoride;
DIPEA, diisopropylethylamine; PBS, phosphate buffered saline;
FDG, fluorodeoxyglucose
REFERENCES
■
(1) Bengel, F. M.; Higuchi, T.; Javadi, M. S.; Lautamaki, R. Cardiac
positron emission tomography. J. Am. Coll. Cardiol. 2009, 54, 1−15.
(2) Machac, J. Cardiac positron emission tomography imaging.
Semin. Nucl. Med. 2005, 35, 17−36.
(3) Higuchi, T.; Bengel, F. M. Cardiovascular nuclear imaging: from
perfusion to molecular function: non-invasive imaging. Heart 2008, 94,
809−816.
(4) Beller, G. A.; Watson, D. D. A welcomed new myocardial
perfusion imaging agent for positron emission tomography. Circulation
2009, 119, 2299−2301.
(5) Hansen, C. L.; Goldstein, R. A.; Akinboboye, O. O.; Berman, D.
S.; Botvinick, E. H.; Churchwell, K. B.; Cooke, C. D.; Corbett, J. R.;
Cullom, S. J.; Dahlberg, S. T.; Druz, R. S.; Ficaro, E. P.; Galt, J. R.;
Garg, R. K.; Germano, G.; Heller, G. V.; Henzlova, M. J.; Hyun, M. C.;
Johnson, L. L.; Mann, A.; McCallister, B. D., Jr.; Quaife, R. A.; Ruddy,
T. D.; Sundaram, S. N.; Taillefer, R.; Ward, R. P.; Mahmarian, J. J.
Myocardial perfusion and function: single photon emission computed
tomography. J. Nucl. Cardiol. 2007, 14, e39−60.
(6) Woo, J.; Tamarappoo, B.; Dey, D.; Nakazato, R.; Le Meunier, L.;
Ramesh, A.; Lazewatsky, J.; Germano, G.; Berman, D. S.; Slomka, P. J.
Automatic 3D registration of dynamic stress and rest (82)Rb and
flurpiridaz F 18 myocardial perfusion PET data for patient motion
detection and correction. Med. Phys. 2011, 38, 6313−6326.
(7) Lazewatsky, J.; Maddahi, J.; Berman, D.; Bhat, G.; Sinha, S.;
Devine, M.; Case, J.; Ehlgen, A. Development of a method for the
determination of dose ratio and minimum inter-injection interval for a
one-day rest-stress protocol with BMS747158 PET myocardial
perfusion agent. J Nucl. Med. Meet. Abstr. 2010, 51, 600.
(8) Studenov, A. R.; Berridge, M. S. Synthesis and properties of 18F-
labeled potential myocardial blood flow tracers. Nucl. Med. Biol. 2001,
28, 683−693.
(9) Ravert, H. T.; Madar, I.; Dannals, R. F. Radiosynthesis of 3-
[18F]fluoropropyl and 4-[18F]fluorobenzyl triarylphosphonium ions. J.
Labelled Compd. Radiopharm. 2004, 47, 469−476.
(10) Madar, I.; Ravert, H. T.; Du, Y.; Hilton, J.; Volokh, L.; Dannals,
R. F.; Frost, J. J.; Hare, J. M. Characterization of uptake of the new
PET imaging compound 18F-fluorobenzyl triphenyl phosphonium in
dog myocardium. J. Nucl. Med. 2006, 47, 1359−1366.
(11) Madar, I.; Ravert, H.; Dipaula, A.; Du, Y.; Dannals, R. F.; Becker,
L. 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. 2007, 48, 1021−
1030.
(12) Shoup, T. M.; Elmaleh, D. R.; Brownell, A. L.; Zhu, A.;
Guerrero, J. L.; Fischman, A. J. Evaluation of (4-[(18)F]fluorophenyl)-
triphenylphosphonium ion. A potential myocardial blood flow agent
for PET. Mol. Imaging Biol. 2011, 13, 511−517.
(13) Martarello, L.; Greenamyre, J. T.; Goodman, M. M. Synthesis
and evaluation of a new fluorine-18 labeled rotenoid as a potential
Biodistribution Studies. For each compound, a total of five
animals were injected with 1.1−2.2 MBq (30−60 μCi) of the 18F-
labeled rhodamine in 100 μL of 10% EtOH/saline via the tail vein. At
60 min pi, animals were sacrificed by CO2 asphyxia and weighed.
Selected tissues were then excised, weighed, and assayed for 18F. The
percent injected dose per gram (%ID/g) for each tissue was calculated
by comparison of the tissue counts to a standard sample prepared from
the injectate.
Data Analysis. Statistical analysis was performed by unpaired two-
tailed t test using Prism software (GraphPad Software Inc.).
ASSOCIATED CONTENT
■
S
* Supporting Information
MS (ESI+), 1H NMR, and 19F NMR spectra of the
nonradioactive reference compounds; HPLC profiles of the
nonradioactive and 18F-labeled compounds; PET images of
[18F]5 and [18F]6 in the mouse; data of the serum stability
studies; maximum intensity projection (MIP) of [18F]5. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (617) 355-7539. Fax: (617) 730-0619. E-mail: alan.
packard@childrens.harvard.edu.
Present Address
§Department of Nuclear Medicine, University Hospital
Freiburg, 79106 Freiburg, Germany.
Notes
The authors declare no competing financial interest.
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PET probe of mitochondrial complex I activity. J. Labelled Compd.
Radiopharm. 1999, 42, 1039−1051.
sestamibi imaging in paediatric neuroblastoma and ganglioneuroma
and its relation to P-glycoprotein. Eur. J. Nucl. Med. 1999, 26, 396−
403.
(30) Vora, M. M.; Dhalla, M. In vivo studies of unlabeled and
radioiodinated rhodamine-123. Nucl. Med. Biol. 1992, 19, 405−410.
(31) Heinrich, T. K.; Gottumukkala, V.; Snay, E.; Dunning, P.; Fahey,
F. H.; Treves, S. T.; Packard, A. B. Synthesis of fluorine-18 labeled
rhodamine B: a potential PET myocardial perfusion imaging agent.
Appl. Radiat. Isot. 2010, 68, 96−100.
(32) Gottumukkala, V.; Heinrich, T. K.; Baker, A.; Dunning, P.;
Fahey, F. H.; Treves, S. T.; Packard, A. B. Biodistribution and stability
studies of [18F]fluoroethylrhodamine B, a potential PET myocardial
perfusion agent. Nucl. Med. Biol. 2010, 37, 365−370.
(33) Coenen, H. H. Fluorine-18 labeling methods: features and
possibilities of basic reactions. Ernst Schering Res. Found. Workshop
2007, 62, 15−50.
(14) Marshall, R. C.; Powers-Risius, P.; Reutter, B. W.; O’Neil, J. P.;
La Belle, M.; Huesman, R. H.; VanBrocklin, H. F. Kinetic analysis of
18F-fluorodihydrorotenone as a deposited myocardial flow tracer:
comparison to 201Tl. J. Nucl. Med. 2004, 45, 1950−1959.
(15) Yalamanchili, P.; Wexler, E.; Hayes, M.; Yu, M.; Bozek, J.;
Kagan, M.; Radeke, H. S.; Azure, M.; Purohit, A.; Casebier, D. S.;
Robinson, S. P. Mechanism of uptake and retention of F-18 BMS-
747158-02 in cardiomyocytes: a novel PET myocardial imaging agent.
J. Nucl. Cardiol. 2007, 14, 782−788.
(16) Huisman, M. C.; Higuchi, T.; Reder, S.; Nekolla, S. G.; Poethko,
T.; Wester, H. J.; Ziegler, S. I.; Casebier, D. S.; Robinson, S. P.;
Schwaiger, M. Initial characterization of an 18F-labeled myocardial
perfusion tracer. J. Nucl. Med. 2008, 49, 630−636.
(17) Purohit, A.; Radeke, H.; Azure, M.; Hanson, K.; Benetti, R.; Su,
F.; Yalamanchili, P.; Yu, M.; Hayes, M.; Guaraldi, M.; Kagan, M.;
Robinson, S.; Casebier, D. Synthesis and biological evaluation of
pyridazinone analogues as potential cardiac positron emission
tomography tracers. J. Med. Chem. 2008, 51, 2954−2970.
(18) Yu, M.; Guaraldi, M.; Kagan, M.; Mistry, M.; McDonald, J.;
Bozek, J.; Yalamanchili, P.; Hayes, M.; Azure, M.; Purohit, A.; Radeke,
H.; Casebier, D. S.; Robinson, S. P. Assessment of 18F-labeled
mitochondrial complex I inhibitors as PET myocardial perfusion
imaging agents in rats, rabbits, and primates. Eur. J. Nucl. Med. Mol.
Imaging 2009, 36, 63−72.
(19) Yu, M.; Bozek, J.; Guaraldi, M.; Kagan, M.; Azure, M.;
Robinson, S. P. Cardiac imaging and safety evaluation of BMS747158,
a novel PET myocardial perfusion imaging agent, in chronic
myocardial compromised rabbits. J. Nucl. Cardiol. 2010, 17, 631−636.
(20) Mou, T.; Jing, H.; Yang, W.; Fang, W.; Peng, C.; Guo, F.;
Zhang, X.; Pang, Y.; Ma, Y. Preparation and biodistribution of
[18F]FP2OP as myocardial perfusion imaging agent for positron
emission tomography. Bioorg. Med. Chem. 2010, 18, 1312−1320.
(21) Sherif, H. M.; Nekolla, S. G.; Saraste, A.; Reder, S.; Yu, M.;
Robinson, S.; Schwaiger, M. Simplified quantification of myocardial
flow reserve with flurpiridaz F 18: validation with microspheres in a pig
model. J. Nucl. Med. 2011, 52, 617−624.
(22) Yu, M.; Nekolla, S. G.; Schwaiger, M.; Robinson, S. P. The next
generation of cardiac positron emission tomography imaging agents:
discovery of flurpiridaz F-18 for detection of coronary disease. Semin.
Nucl. Med. 2011, 41, 305−313.
(23) Maddahi, J.; Czernin, J.; Lazewatsky, J.; Huang, S. C.; Dahlbom,
M.; Schelbert, H.; Sparks, R.; Ehlgen, A.; Crane, P.; Zhu, Q.; Devine,
M.; Phelps, M. Phase I, first-in-human study of BMS747158, a novel
18F-labeled tracer for myocardial perfusion PET: dosimetry, biodis-
tribution, safety, and imaging characteristics after a single injection at
rest. J. Nucl. Med. 2011, 52, 1490−1498.
(24) Lacerda, S. H.; Abraham, B.; Stringfellow, T. C.; Indig, G. L.
Photophysical, photochemical, and tumor-selectivity properties of
bromine derivatives of rhodamine-123. Photochem. Photobiol. 2005, 81,
1430−1438.
(25) Reungpatthanaphong, P.; Dechsupa, S.; Meesungnoen, J.;
Loetchutinat, C.; Mankhetkorn, S. Rhodamine B as a mitochondrial
probe for measurement and monitoring of mitochondrial membrane
potential in drug-sensitive and -resistant cells. J. Biochem. Biophys.
Methods 2003, 57, 1−16.
(34) Kilbourn, M. R.; Shao, X. In Fluorine-18 Radiopharmaceuticals;
John Wiley & Sons Ltd.: Hoboken, NJ, 2009; pp 361−388.
(35) Lasne, M.-C.; Perrio, C.; Rouden, J.; Barre, L.; Roeda, D.; Dolle,
F.; Crouzel, C. Chemistry of β+-emitting compounds based on
fluorine-18. Top. Curr. Chem. 2002, 222, 201−258.
(36) Cai, L.; Lu, S.; Pike, V. W. Chemistry with [18F]fluoride ion.
Eur. J. Org. Chem. 2008, 2853−2873.
(37) Henriksen, G.; Platzer, S.; Marton, J.; Hauser, A.; Berthele, A.;
Schwaiger, M.; Marinelli, L.; Lavecchia, A.; Novellino, E.; Wester, H. J.
Syntheses, biological evaluation, and molecular modeling of 18F-
labeled 4-anilidopiperidines as mu-opioid receptor imaging agents. J.
Med. Chem. 2005, 48, 7720−7732.
(38) Erlandsson, M.; Karimi, F.; Lindhe, O.; Langstrom, B. (18)F-
labelled metomidate analogues as adrenocortical imaging agents. Nucl.
Med. Biol. 2009, 36, 435−445.
(39) Erlandsson, M.; Hall, H.; Laangstroem, B. Synthesis and in vitro
evaluation of 18F-labelled di- and tri(ethylene glycol) metomidate
esters. J. Labelled Compd. Radiopharm. 2009, 52, 278−285.
(40) Kronauge, J. F.; Noska, M. A.; Davison, A.; Holman, B. L.;
Jones, A. G. Interspecies variation in biodistribution of technetium (2-
carbomethoxy-2-isocyanopropane)6+. J. Nucl. Med. 1992, 33, 1357−
1365.
(41) Zea-Ponce, Y.; Mavel, S.; Assaad, T.; Kruse, S. E.; Parsons, S.
M.; Emond, P.; Chalon, S.; Giboureau, N.; Kassiou, M.; Guilloteau, D.
Synthesis and in vitro evaluation of new benzovesamicol analogues as
potential imaging probes for the vesicular acetylcholine transporter.
Bioorg. Med. Chem. 2005, 13, 745−753.
(42) Nomura, Y.; Kubozono, T.; Hidaka, M.; Horibe, M.; Mizushima,
N.; Yamamoto, N.; Takahashi, T.; Komiyama, M. Predominant role of
basicity of leaving group in α-effect for nucleophilic ester cleavage.
Bioorg. Chem. 2004, 32, 26−37.
(43) Mock, W. L.; Zhang, J. Z. Concerning the relative acidities of
simple alcohols. Tetrahedron Lett. 1990, 31, 5687−5688.
(44) Silva, C. O.; Da, S. E. C.; Nascimento, M. A. C. Ab initio
calculations of absolute pKa values in aqueous solution. Part 2.
Aliphatic alcohols, thiols, and halogenated carboxylic acids. J. Phys.
Chem. A 2000, 104, 2402−2409.
(45) Lopez, A. I.; Ruiz, O. P. Dimeric states of rhodamine B. Chem.
Phys. Lett. 1982, 87, 556−560.
(46) Yu, M.; Guaraldi, M. T.; Mistry, M.; Kagan, M.; McDonald, J.
L.; Drew, K.; Radeke, H.; Azure, M.; Purohit, A.; Casebier, D. S.;
Robinson, S. P. BMS-747158-02: a novel PET myocardial perfusion
imaging agent. J. Nucl. Cardiol. 2007, 14, 789−798.
(26) Hu, Y.; Moraes, C. T.; Savaraj, N.; Priebe, W.; Lampidis, T. J.
Rho(0) tumor cells: a model for studying whether mitochondria are
targets for rhodamine 123, doxorubicin, and other drugs. Biochem.
Pharmacol. 2000, 60, 1897−1905.
(27) Lampidis, T. J.; Hasin, Y.; Weiss, M. J.; Chen, L. B. Selective
killing of carcinoma cells “in vitro” by lipophilic−cationic compounds:
a cellular basis. Biomed. Pharmacother. 1985, 39, 220−226.
(28) Rusiecka, I.; Skladanowski, A. C. Induction of the multixeno-
biotic/multidrug resistance system in various cell lines in response to
perfluorinated carboxylic acids. Acta Biochim. Pol. 2008, 55, 329−337.
(29) De Moerloose, B.; Van de Wiele, C.; Dhooge, C.; Philippe, J.;
Speleman, F.; Benoit, Y.; Laureys, G.; Dierckx, R. A. Technetium-99m
(47) Kronauge, J. F.; Chiu, M. L.; Cone, J. S.; Davison, A.; Holman,
B. L.; Jones, A. G.; Piwnica, W. D. Comparison of neutral and cationic
myocardial perfusion agents: characteristics of accumulation in
cultured cells. Int. J. Radiat. Appl. Instrum., Part B 1992, 19, 141−148.
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