Development of a PET radiotracer for non-invasive imaging of the reactive oxygen species, superoxide, in vivo

Article abstract of DOI:10.1039/c3ob42379d

Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide range of human disease states and drug toxicities, but development of imaging tools to study ROS biology in vivo remains a challenge. Here we synthesized and validated a nove

Full text of DOI:10.1039/c3ob42379d

Organic &
Biomolecular Chemistry
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Development of a PET radiotracer for non-invasive
imaging of the reactive oxygen species,
superoxide, in vivo†
Cite this: Org. Biomol. Chem., 2014,
12, 4421
Wenhua Chu,a Andre Chepetan,b Dong Zhou,a Kooresh I. Shoghi,a Jinbin Xu,a
Laura L. Dugan,b Robert J. Gropler,a Mark A. Mintuna and Robert H. Mach*a
Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide range of human
disease states and drug toxicities, but development of imaging tools to study ROS biology in vivo remains
a challenge. Here we synthesized and validated a novel PET tracer (12) and its 18F radiolabeled version
[
18F]12 to allow PET (positron emission tomography) imaging of superoxide in vivo. Initial analysis of ROS
reaction kinetics found that compound 12 was rapidly and selectively oxidized by superoxide, but not
other ROS. Cell culture studies in EMT6 cells exposed to the cancer chemotherapeutic agent Doxorubicin
(DOX), which activates the superoxide-generating enzyme, NADPH oxidase, showed that compound 12
was a sensitive and specific probe for superoxide in cells. The microPET imaging of heart in mice
with DOX-induced cardiac inflammation observed 2-fold greater oxidation of [18F]12 in the DOX-treated
mice compared to controls (p = 0.02), the results were confirmed by distribution studies on organs
subsequently removed from the mice and HPLC analysis of [18F] radioactivity compounds. These data
indicate that compound 12 is a useful PET tracer to imaging ROS in vivo.
Received 27th November 2013,
Accepted 9th May 2014
DOI: 10.1039/c3ob42379d
www.rsc.org/obc
Parkinson’s disease, Amyotrophic Later Sclerosis (ALS) and
Alzheimer’s disease,6–9 as well as end-organ fibrosis and
Introduction
Chronic inflammatory response in multiple diseases results in failure in diabetes.10 Collectively, ROS comprise the chemically
the activation of innate immune enzymes such as NADPH reactive products of molecular oxygen (O2) including the one-
oxidase to produce reactive oxygen species (ROS), which in electron reduction product of molecular oxygen: superoxide
turn have been shown in animal models to contribute to tissue (O2−).11 Superoxide can be produced by a number of sources
injury, fibrosis, remodeling and organ dysfunction for a range in vivo, including the mitochondrial electron transport chain
of pathology and disease. ROS are produced by normal cellular and NADPH oxidase(s), which are activated under conditions
metabolism, and are fundamental to cell biology in a broad of inflammation, ischemia, and tissue injury. Superoxide has
sense. However, while ROS are involved in all aspects of been shown to regulate distinct signaling cascades and to have
normal cellular function, disruption of the physiological specific molecular targets. The ability to detect elevated ROS
balance in ROS homeostasis caused by disease, injury, or in the intact animal or patient would provide a better under-
environmental stress (e.g., ionizing radiation, UV and heat standing of the links between ROS, tissue injury, and disease.
exposure) can lead to inappropriate cell signaling, dysfunction, Furthermore, the ability to selectively identify the ROS being
and overt cell death. At the organ level, these pathological generated will be critical to understanding the molecular cas-
responses to ROS can produce inflammation, fibrosis, tissue cades affected.
remodeling, and trigger other deleterious cascades in diseases
A variety of methods for evaluating ROS in cell culture and
processes such as ischemia and reperfusion of heart, lung, tissue slices have been developed, and these approaches have
brain and kidney,1–5 neurodegenerative diseases including advanced our understanding of both the physiological role of
ROS as signaling molecules and necessary intermediates in
cell metabolism, and as toxic species which can damage cells
aDepartment of Radiology, Washington University School of Medicine, St. Louis,
and tissues. Techniques currently employed include chemi-
luminescence assays,12,13 electron spin resonance (ESR) using
MO 63110, USA. E-mail: rmach@mail.med.upenn.edu
bDivision of Geriatric Medicine, Departments of Medicine and Neuroscience,
nitrone or nitroxide spin traps,14 and fluorescence-based
University of California–San Diego, La Jolla, CA 92093, USA
techniques with oxidizable fluorescent probes such as Peroxy
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ob42379d
Green 1, Peroxy Crimson 1,15,16 2,7-dichlorodihydrofluorescein
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Fig. 1 Structures of dihydroethidium, ethidium and 2-OH-ethidium.
diacetate (DCFH-DA),17–20 dihydrorhodamine 123 (DHR),21 biphenyl-2-amine (1) and 4-methoxybenzoyl chloride were
hydrocyanines22,23 (see ESI, Fig. S1†), and dihydroethidium heated in chlorobenzene to give compound 2. The nitro
(DHE, Fig. 1) and its analogs.24–30 Although there is an unmet groups of 2 were reduced to amines with ammonium formate
need for imaging tools to study the production of metabolic using Pd(OH)2 as a catalyst to afford 3. The amine groups of 3
and inflammatory ROS and/or reactive nitrogen species (RNS) were protected with methyl carbamate, then cyclized with
in humans or animal models, some considerable difficulties POCl3 to give 4 and 5, respectively. Compound 5 was depro-
must be overcome to develop such tools. First, ROS (and RNS) tected with 48% HBr under reflux to afford the intermediate 6.
are small molecules which have a relatively short lifetime, so it The amine groups of 6 were protected with t-butyl carbamate,
cannot be detected by conventional “radioligand bound to and the OH group of the phenol was propynylated with 3-bromo-
target” strategies. Second, fluorescence, chemiluminescence, prop-1-yne using K2CO3 as a base to give 7 and 8, respect-
optical, and electron spin resonance imaging are limited in their ively. Compound 8 was methylated with CH3I in THF to afford
ability to measure in vivo intracellular ROS due to substantial 9, which was reduced with NaBH3CN to give the precursor
attenuation of the output signal by the tissue within a few compound 10. Compound 10 underwent copper(I)-assisted 1,3-
centimeters. Given the drawbacks in the use of these probes, dipolar cycloaddition with 2-fluoroethyl azide (i.e., the “Click
especially for imaging in vivo ROS production in an intact reaction”) to yield 11, then the Boc group was deprotected to
animal or human, an alternative, sensitive method for detecting provide the DHE analog 12. Compound 14, the oxidized form
superoxide produced in a variety of biological systems is needed. of 12, was similarly synthesized from the intermediate 9 by
Positron emission tomography (PET) is a non-invasive Click reaction to afford 13, followed by deprotection of the Boc
imaging technique capable of measuring specific chemical or groups to give 14.
enzymatic reactions in tissues in vivo. Therefore, a radiolabeled
Selectivity studies
DHE analog as a PET tracer to image ROS in vivo could over-
come the disadvantages of existing DHE-based fluorescence
probes. Like DHE, a DHE analog used as a PET radiotracer will
not bind to DNA because it is a neutral molecule and its phe-
nanthridine moiety has a non-planar geometry. However, the
oxidation products of the DHE analog by ROS, ethidium or
2-OH ethidium analogs, can bind to DNA since these products
possess a positive charge and the corresponding phenanthri-
dine ring system has a planar geometry. Once these positively-
charged oxidized analogs bind to DNA in cells, they become
trapped and are retained within the cell for a longer duration
while the parent DHE analog washes out of the cell rapidly.
This difference in retention provides a method to quantify
ROS levels in vivo by PET imaging. To date, there have been no
reported non-invasive or minimally invasive methods to evalu-
ate ROS in the intact organism using PET. Here, we report the
novel development of a PET-based DHE analog which could be
used as a radiotracer for imaging superoxide levels in vivo.
After compound 12 was synthesized, its selective reactivity with
different reactive oxygen species was investigated and com-
pared to results obtained with DHE in a fluorescence plate
reader assay. Compound 12 was oxidized by superoxide gener-
ated through either xanthine oxidase metabolism of hypox-
anthine or by thermal decomposition of SIN-1 in the presence
of CPTIO. In the both test systems, oxidation of compound 12
was completely prevented by the addition of superoxide dismu-
tase (SOD) (Fig. 2). Compound 12 was also exposed to H2O2
alone or in the presence of horseradish peroxidase (HRP) to
generate hydroxyl free radical. The fluorescence intensity
results indicate that compound 12 has little reactivity with
hydroxyl radical (Fig. 2), or H2O2. These data suggest that 12
has good selectivity for superoxide.
Cell studies
As with DHE, compound 12 has no fluorescence at 595 nm,
but its oxidized form 14 has very strong fluorescence at
595 nm (data not shown). Therefore, fluorescence at 595 nm
can be used as an indicator of the oxidation of 12. In order to
help guide PET imaging studies, we explored the ability of 12
Results and discussion
Synthesis of 10 and 12
The synthesis of standard compound 12 and the precursor to detect ROS induced by the anticancer drug Doxorubicin
compound 10 are shown in Scheme 1 and 2. The 4,4′-dinitro- (DOX) using cancer cells growing under cell culture conditions
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Scheme 1 Synthesis of 12 and 14 (Part 1). Reagents: (a) 4-Methoxybenzoyl chloride, chlorobenzene, reflux; (b) HCOONH4, Pd(OH)2/C;
(c) ClCOOCH3; (d) POCl3; (e) 48% HBr.
as a model system. Mouse mammary adenocarcinoma (EMT6) Cell uptake studies of [18F]12
cells were first treated with 400 nM DOX, after 5 h, EMT6 cells
In order to be used as a PET radiotracer for imaging intracellu-
were then incubated with 2 µM DHE or 2 µM 12 for 60 min at
lar superoxide, the uncharged DHE analog 12 should cross
37 °C in the dark. Upon removal of the culture media, fluo-
membranes get in and out cell easily, but the oxidation pro-
ducts of 12 by ROS, 14, should not cross the cell membrane
and should have a low cell uptake. Furthermore, once inside
the cell, the compound 12 does not bind to DNA in the
rescence intensities of the cells were measured using absorp-
tion/emission wavelengths of 480/595 nm, respectively. These
results (Fig. 3) showed that the fluorescence intensity signifi-
cantly increased in EMT6 cells treated with DOX for both DHE
nucleus because it is both uncharged and non-planar, whereas
and 12 in comparison with non-treated EMT6 cells. The
oxidation of 12 to 14 leads to a charged, planar compound
which can intercalate into DNA. Once 14 binds to DNA in cells,
it becomes trapped intracellularly and is retained for a longer
time while the parent 12 washes out of the cell rapidly. This
difference in retention provides a method to quantify ROS
levels in vivo by PET imaging. Although it is not possible to
nuclear localization of the fluorescence in the DOX-treated
cells confirms that oxidation of 12 leads to trapping of the
probe by intercalation into DNA. These data indicate that 12
can measure DOX-induced superoxide production in intact
cancer cells.
measure the rate of washout of 12 and 14 from cells, it is
possible to measure the ability of 12 and 14 cross the mem-
Radiolabeling of 12
The radiotracer [18F]12 was synthesized using copper(I) cata- brane and enter the cell by using [18F]12 and [18F]14. There-
lyzed Click reaction with [18F]16 (Scheme 3). [18F]16 was fore, studies of the uptake kinetics of [18F]12 and its oxidized
efficiently synthesized by an improved vacuum distillation form, [18F]14, were carried out using EMT6 cells. Wild-type
method.31 The Boc groups of precursor 10 were first removed EMT6 cells have minimal expression of p-glycoprotein;32
with TFA to afford 17, then [18F]16 was distilled into the solu- therefore, active transport of [18F]12 and [18F]14 from tumor
tion of 17 in DMF, followed by the addition of a mixture of cells is not expected to occur. In this experiment, the non-
copper sulfate and sodium ascorbate as the Cu(I) source. [18F]12 charged DHE analog, 12, should be cell-permeable and enter
was obtained with a yield of 42.8% (non-decay corrected) the cell easily, while its oxidized state, 14, which has a posi-
after reversed phase HPLC purification and solid phase extrac- tive charge, should be not cross the cell membrane. There-
tion. Ascorbic acid was added to the final dose to stabilize fore, radiolabeling of [18F]12 and [18F]14 allowed us to easily
[18F]12 from autoxidation. (For analytical HPLC conditions see measure the kinetics of cellular uptake in both the
ESI, Fig. S3 and S4†). The total synthesis time was 120 min. uncharged (12) and charged (14) forms of the molecule. A 5.6
The final dose in 10% ethanol–saline had a specific activity of × 106 Bq mL−1 aliquot of either [18F]12 or [18F]14 was incu-
1.3–2.4 × 1010 Bq µmol−1 with a radiochemical purity of bated with EMT6 cells for 30 min at 37 °C, then the radio-
90–100%. [18F]14, the oxidized form of [18F]12, was generated activity within the cells was counted. The results are shown
by oxidation of [18F]12 with CuSO4 in DMSO.
in Fig. 4. As expected, uptake in EMT6 cells of the oxidized
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Scheme 2 Synthesis of 12 and 14 (Part 2). Reagents: (f) (Boc)2O; (g) 3-bromoprop-l-yne, acetone, K2CO3; (h) CH3I, THF; (i) NaBH3CN, MeOH;
( j) N3CH2CH2F, CuSO4, sodium ascorbate, DMF; (k) EtOAc–HCl (3 : 1).
form, [18F]14, was much lower than uptake of the uncharged MicroPET, biodistribution and stability studies of [18F]12
form, [18F]12, indicating that the neutral species rapidly
A mouse model of DOX-induced cardiotoxicity was used for
enters the cell whereas the oxidized form [18F]14 does not
cross the cell membrane easily because of its permanent
positive charge.
the in vivo evaluation of [18F]12. It is currently believed that
chemotherapy-induced cardiotoxicity of DOX is caused via the
generation of superoxide and other ROS in cardiac tissue.33–35
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Fig. 3 Fluorescence imaging of ROS induced by anticancer drug doxo-
rubicin treatment in EMT6 cells. Control cells (A and C) and doxo-
rubicin-treated EMT6 cells (B and D) were imaged using either 2 µM DHE
(A and B) or 2 µM 12 (C and D). Doxorubicin-treated cells displayed
increased fluorescence signal for both compounds. Scale bar represents
100 μm.
Fig. 2 Compounds 12 and DHE for selective reactivity with superoxide
radical.
Scheme 3 Synthesis of [18F]12 and [18F]14. Reagents: (l) [18F]KF, K222, K2CO3, CH3CN, 85 °C, 5 min; (m) TFA; (n) CuSO4, sodium ascorbate, DMF,
DIPEA; (o) Cu++
.
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contained very low levels of [18F]14. However, little or no [18F]12
was present in the heart and only the oxidized form of [18F]12,
[18F]14, was detected. These results show that [18F]12 was
stable in the blood of DOX-treated mice and was oxidized to
[18F]14 by of DOX-induced generation of ROS in the hearts of
treated mice (see ESI, Fig. S6†). HPLC analysis results con-
firmed that the 60 min uptake in the heart of DOX-treated
mice measured by microPET and biodistribution comes from
[18F]14, the oxidized form of [18F]12. To assess the contri-
bution of the oxidized compound, [18F]14 was injected into
saline-treated and DOX-treated mice. As observed in Fig. 5,
there were no differences in the kinetics of [18F]14 in saline
and DOX treated mice. In both saline and DOX treated mice,
[18F]14 clears cardiac tissue within 2 s and reaches steady-state
above background levels, but well below SUV levels of [18F]12
(Fig. 5). The background uptake may be attributed to non-
specific uptake and binding of [18F]14 to DNA, proteins and
other moieties due to its electrostatic charge.
Fig. 4 EMT6 cell uptake assay of [18F]12 and the oxidized compound
[
p ≪ 0.001, student t test.
18F]14. Uptake of [18F]12 was significantly higher than that of [18F]14
Discussion
It is currently believed that elevated levels of ROS such as
superoxide play a contributing role in a wide variety of patho-
logical conditions. However, much of the evidence supporting
this hypothesis has been generated in cellular or animal
models of disease using fluorescent, chemiluminescent, and
optical imaging techniques to measure ROS levels. Although
valuable information has been gained in these preclinical
studies, there is a need to develop a strategy for measuring
ROS levels which could be used in translational imaging
studies in humans. The goal of the current study was to
develop a radiotracer which could be used to study disease-
associated increases in ROS with the molecular imaging tech-
nique, PET. For this study, we synthesized a 18F-labeled analog
of DHE (i.e., [18F]12) a well-characterized fluorescent probe for
measuring superoxide levels in vitro and in vivo, and con-
Fig. 5 MicroPET imaging of mice heart in untreated and DOX-treated
mice with [18F]12 and [18F]14.
DOX-treated mice and saline-treated controls were injected ducted a series of studies aimed at characterizing its ability to
with [18F]12 for dynamic microPET imaging studies. Initial measure ROS in vivo with PET. The results of our in vitro
uptake of the radioactive counts in heart was similar between studies suggest that compound 12 behaves in a manner very
treated and untreated mice. However, 1 h later, the radioactive similar to DHE. That is, the parent, neutral compound crosses
uptake was on average ∼2 fold higher in the DOX-treated mice the cell membrane and is oxidized by ROS (i.e., superoxide) to
in heart than that in the control hearts (n = 4) (Fig. 5). Post- form a planar, permanently charged species which is retained
microPET biodistribution data were obtained to further in the cell by intercalation into DNA.
confirm the microPET results (see ESI, Fig. S5†), and these
DOX is a highly effective anticancer drug that is frequently
data were in good agreement with the microPET data in that employed to treat hematological and solid tumors including
the radioactive uptake in the heart of DOX-treated mice was leukemia, breast cancer, and soft tissue sarcomas. However,
higher than that of control mice.
the therapeutic effectiveness of DOX is compromised by its
To further verify the result of the kinetics and stablity of dose dependent and cumulative cardiotoxic side effects which
[18F]12 in vivo and in vitro, blood and heart samples from include cardiomyopathy. The mechanisms responsible for
control mice were incubated in vitro for 5 and 60 min. Results DOX-induced cardiomyopathy remain unclear, but it is widely
of HPLC anaysis showed that [18F]12 is stable in saline-treated accepted that the DOX-induced cardiotoxicity is mainly attribu-
control mice, where only a minimal amount of the oxidized ted to the initial formation of superoxide and other ROS.33–35
form of [18F]12, [18F]14 was observed at 60 min (see ESI, It has previously been reported that DOX induces NADPH
Fig. S6†). After 60 min injection of [18F]12 in DOX treated oxidase-2 (Nox2, the respiratory burst oxidase) in heart muscle.
mice, HPLC analysis demonstrated that the blood samples Therefore, DOX-induced ROS production in the mouse heart
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provided a valuable model for the in vivo evaluation of [18F]12. active species in the hearts of DOX-treated mice at 60 min is
In the microPET imaging study, [18F]12, like DHE, does not [18F]14, the in vitro stability of [18F]12 was evaluated by HPLC
bind to DNA because it is a neutral molecule and its phenan- in blood and heart at 5 and 60 min. HPLC analysis showed
thridine moiety has a non-planar geometry, thus it easily gets that [18F]12 is stable in blood and hearts of untreated mice for
in and out of cells in uptake studies. In contrast, [18F]14, the 60 min. In contrast, the HPLC analysis in vivo at 60 min
oxidized form of [18F]12, should be trapped inside cells showed that the parent compound, [18F]12, was the main com-
because it can bind to DNA due to its permanent positive ponent in blood and it had been completely oxidized to [18F]14
charge and its planar geometry. Once positively-charged [18F]14 in heart in DOX-treated mice. This is consistent with the
is bound to DNA, it is trapped intracellularly and retained hypothesis that the initial cardiac uptake in the microPET
for a longer time whereas the parent compound, [18F]12, imaging studies in both the DOX-treated and untreated mice
washes out of the cell quickly. Since [18F]14 is formed by the is from [18F]12, and the prolonged retention and higher SUVs
ROS-based oxidation of [18F]12, the difference in retention of in the DOX-treated animals is caused by the higher levels of
radioactivity between two tissues or two conditions (i.e., ROS (and subsequent formation of [18F]14) in the drug-treated
control versus patient group) should provide an effective group. The biodistribution data and the HPLC analysis results
method to quantify differences in ROS levels in vivo with PET confirmed that the presence of the oxidized form of [18F]12,
imaging.
[18F]14, in heart tissue of DOX-treated mice. All of the data
Our microPET imaging results show that the initial uptake suggest that [18F]12 is capable of imaging ROS levels in intact
of radioactivity in heart was similar between DOX-treated and cells and tissues using the in vivo imaging technique, PET.
untreated mice, suggesting that the DOX treatment does not
In conclusion, abnormal production or inadequate elimin-
alter tissue penetration of the uncharged [18F]12. However, at ation of ROS has been linked to a broad array of disease con-
60 min, the radioactive uptake is ∼2 fold higher in the DOX- ditions, including Alzheimer’s disease, diabetes, heart failure,
treated mice heart than that in the control hearts (n = 4). cancer, neurodegeneration and the aging process itself. The
These imaging results can be explained by the ROS induced by pharmaceutical industry has actively pursued therapeutic
DOX in the hearts of DOX-treated mice. The initial cardiac attempts to diminish oxidative stress produced by ROS in
uptake represents [18F]12 which is delivered to the hearts of patients and in animal models of disease. There is a growing
both treated and untreated mice. After 60 min, most of the consensus that there is a need to develop tools which will
[18F]12 has washed out in the hearts of untreated mice where allow researchers to better understand the temporal, spatial,
there is minimal ROS production, but the [18F] signal remains and biochemical contributions of ROS to normal biology as
elevated in the hearts of DOX-treated mice, consistent with well as disease pathology. To date, techniques to carry out
intracellular oxidation of [18F]12 to [18F]14 by ROS, which due such studies in humans using non-invasive imaging tech-
to its positive charge, then binds to DNA and is retained in the niques do not exist. Thus, there is a pressing need to develop
heart for an extended duration. Indeed, when the oxidized new imaging approaches which can allow clinical measure-
form of [18F]12, [18F]14, was injected to saline and DOX treated ment of ROS. The current study demonstrates the development
mice, there were no differences in the kinetics of [18F]14 of a novel PET tracer to image and dynamically monitor bio-
between the groups, suggesting that retention of [18F]12 is logical ROS. Our data suggest that compound 12 is a useful
specific to the reaction with superoxide to produce [18F]14, in and sensitive probe to detect superoxide in vitro and in vivo.
particular in DOX-treated mice where one would expect higher Furthermore, the radiolabeled molecule [18F]12 is a promising
ROS levels. Therefore, the activity retained in the hearts at PET tracer for the non-invasive imaging of ROS in vivo with
60 min in the DOX-treated mice represents [18F]14. Similarly, PET.
consistent with the microPET imaging results, the time-activity
curve (TAC) showed that there was a significantly higher SUV
in the myocardium of DOX-treated mice compared to hearts
from untreated mice. The difference between treated and
untreated mice at 60 min reflects the increased level of the
Experimental section
General methods and materials
ROS in the heart of DOX-treated mice. Finally, the low fluo- All chemicals were obtained from standard commercial
rescence of the untreated EMT cells and high accumulation of sources and used without further purification. All reactions
fluorescent signal in the nucleus of DOX-treated cells is con- were carried out using standard air-free and moisture-free
sistent with our proposed mechanism of trapping of [18F]12 in techniques under an inert nitrogen atmosphere with dry sol-
the hearts of DOX-treated mice. This study is also a good vents unless otherwise stated. Flash column chromatography
example of the advantage of having a duo-function, optical/ was conducted using Scientific Adsorbents, Inc. silica gel, 60A,
PET probe in characterizing the mechanism of accumulation “40 Micron Flash” (32–63 µm). Melting points were deter-
of a PET radiotracer in tissue.
mined by using MEL-TEMP 3.0 apparatus and are uncorrected.
The 60 min biodistribution data indicate the DOX-treated Routine 1H and 13C NMR spectra were recorded at 300 MHz on
mice have a two-fold higher retention in heart tissue compared a Varian Mercury-VX and 400 MHz on Agilent Technologies
to untreated mice, which is consistent with the results of the spectrometers. All chemical shifts are reported as a part per
microPET study. In order to further confirm that the radio- million (ppm) downfield from tetramethylsilane (TMS). All
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coupling constants (J) are given in Hertz (Hz). Splitting pat- 2H), 7.59 (d, J = 1.8 Hz, 1H), 7.48–7.41 (m, 3H), 7.32 (d, J = 8.7
terns are typically described as follows: s, singlet; d, doublet; t, Hz, 2H), 7.30 (d, J = 8.7 Hz, 1H), 6.99 (d, J = 9.0 Hz, 2H), 3.81
triplet; m, multiple. ESI/MS was performed on a Waters ZQ (s, 3H), 3.68 (s, 3H), 3.65 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
4000 single quadrupole mass spectrometer equipped with an δ 165.0, 161.8, 154.0, 138.4, 137.9, 135.2, 133.0, 131.9, 130.2,
electrospray ionization (ESI) LC-MS interface. Elemental analy- 129.3, 128.9, 126.6, 117.9, 116.4, 113.6, 55.4, 51.6, 51.5.
sis (C, H, N) was determined by Atlantic Microlab, Inc., Nor- Analysis (calcd, found for C24H23N3O6·0.5CH3CH2OCH2CH3):
cross, GA. High performance liquid chromatography (HPLC) C (64.19, 63.96), H (5.80, 5.86), N (8.64, 8.47).
was performed with an ultraviolet detector operating at
Dimethyl 6-(4-methoxyphenyl)phenanthridine-3,8-diyldicar-
251 nm and a well-scintillation NaI (Tl) detector and associ- bamate (5). A mixture of 4 (2.70 g, 6.0 mmol) and POCl3
ated electronics for radioactivity detection. An Alltech Econosil (25 mL) was heated to reflux for 6 h. The mixture was cooled to
C18 250 × 10 mm semi-preparative column and Altech Altima 23 °C, then poured into ice (200 g), neutralized with Na2CO3 to
C18 250 × 4.6 mm analytical column were used for preparation pH = 8, and extracted with ethyl acetate (75 mL × 2); the com-
and analysis respectively.
bined ethyl acetate was washed with water (50 mL), saturated
N-(4,4′-Dinitrobiphenyl-2-yl)-4-methoxybenzamide (2). A NaCl (50 mL), and dried over Na2SO4. After evaporation of the
solution of 4,4′-dinitrophenyl-2-amine 1 (10.37 g, 40 mmol) ethyl acetate under reduced pressure, the crude product was
and 4-methoxybenzyl chloride (8.53 g, 50 mmol) in toluene purified by flash chromatography with hexane–EtOAc to afford
(200 mL) was heated to reflux for 24 h, then cooled to 23 °C. 1.97 g (76%) of 5 as yellow solid, m.p. 207.9–208.7 °C. 1H NMR
The solid was filtered out, washed with ether, and recrystal- (300 MHz, DMSO-d6) δ 9.96 (s, 1H), 9.93 (s, 1H), 8.62 (d. J = 9.0
lized from methanol to afford 12.7 g (81%) of 2 as a white Hz, 1H), 8.52 (d, J = 9.3 Hz, 1H), 8.28 (s, 1H), 8.12 (s, 1H), 7.87
solid, m.p. 210.2–211.6 °C. 1H NMR (300 MHz, DMSO-d6) δ (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 8.1 Hz,
10.21 (s, 1H), 8.45 (d, J = 2.4 Hz, 1H), 8.27 (d, J = 8.4 Hz, 2H), 2H), 7.05 (d, J = 8.4 Hz, 2H), 3.79 (s, 3H), 3.65 (s, 3H), 3.59 (s,
8.20 (dd, J = 8.4 Hz, 2.1 Hz, 1H), 7.80–7.71 (m, 5H), 7.01 (d, J = 3H). 13C NMR (75 MHz, DMSO-d6) δ 160.6, 160.3, 154.7, 144.0,
9.0 Hz, 2H), 3.81 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.1, 139.8, 138.4, 132.5, 131.7, 128.9, 125.1, 123.8, 123.3, 119.3,
162.2, 147.4, 147.0, 144.6, 141.4, 136.6, 131.6, 129.8, 129.6, 119.0, 117.0, 115.8, 114.2, 55.9, 52.5, 52.4. Analysis (calcd,
125.8, 123.6, 121.9, 120.8, 113.7, 55.4. Analysis (calcd, found found for C24H21N3O5): C (66.81, 66.78), H (4.91, 4.80), N (9.74,
for C20H15N3O6): C, (61.07, 61.11), H (3.84, 3.76), N (10.68, 9.63).
10.53).
4-(3,8-Diaminophenanthridin-6-yl)phenol (6). A solution of
N-(4,4′-Diaminobiphenyl-2-yl)-4-methoxybenzamide (3). A 5 (1.73 g, 4 mmol) in 48% HBr (100 mL) was heated under
mixture of 2 (3.93 g, 10.0 mmol), ammonium formate (6.31 g, reflux for 3 days, then neutralized with Na2CO3 to pH = 8, and
100 mmol), and 20% Pd(OH)2/C (200 mg) in methanol (50 mL) extracted with CH2Cl2 (50 mL × 2); the combined CH2Cl2 was
and ethyl acetate (50 mL) was heated to reflux for 3 h. The cata- washed with water (50 mL), NaCl (50 mL) and dried over
lyst was filtered out through celite, and the filtrate was evapor- Na2SO4. After evaporation of the CH2Cl2 under reduced
ated under reduced pressure. The crude product was dissolved pressure, the crude product was purified by flash chromato-
in ethyl acetate (100 mL), washed with saturated NaHCO3 graphy with ethyl acetate to afford 1.10 g (91%) of 6 as brown
(50 mL), water (50 mL), NaCl (50 mL), and dried over Na2SO4. solid, m.p. 201.4–202.3 °C. 1H NMR (300 MHz, DMSO-d6)
After evaporation of the ethyl acetate under reduced pressure, δ 9.69 (s, 1H), 8.30 (d, J = 8.7 Hz, 1H), 8.21 (d, J = 9.0 Hz, 1H),
the crude product was purified by flash chromatography with 7.46 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 8.7 Hz, 1H), 7.08 (s, 1H),
hexane–EtOAc (1 : 3) to afford 3.03 g (91%) of 3 as white solid, 7.03 (s, 1H), 6.95 (m, 3H), 5.37 (s, 2H), 5.33 (s, 2H). 13C NMR
m.p. 161.4–162.6 °C. 1H NMR (300 MHz, CDCl3) δ 8.05 (s, 1H), (75 MHz, DMSO-d6) δ 159.6, 158.0, 148.2, 146.8, 144.2, 131.9,
7.99 (d, J = 2.1 Hz, 1H), 7.60 (d, J = 9.0 Hz, 2H), 7.17 (d, J = 8.7 131.3, 125.4, 125.3, 122.9, 122.4, 121.2, 117.4, 115.4, 110.7,
Hz, 2H), 7.02 (d, J = 8.4 Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 6.77 109.0. Analysis (calcd, found for C19H15N3O·0.5H2O): C (73.53,
(d, J = 8.7 Hz, 2H), 6.48 (dd, J = 8.0 Hz, 2.1 Hz, 1H), 3.81 (s, 73.23), H (5.20, 5.51), N (13.54, 12.34).
3H), 3.79 (s, 4H). 13C NMR (75 MHz, CDCl3) δ 164.4, 162.2,
tert-Butyl 6-(4-hydroxyphenyl)phenanthridine-3,8-diyldicar-
146.2, 145.8, 135.9, 130.7, 130.5, 128.6, 128.0, 127.2, 122.5, bamate (7). A mixture of 6 (1.05 g, 3.5 mmol) and di-tert-butyl
115.5, 113.9, 110.6, 106.9, 55.3. Analysis (calcd, found for dicarbonate (1.82 g, 8.4 mmol) in tert-butanol (50 mL) was
C20H19N3O2): C, (72.05, 71.94), H (5.74, 5.68), N (12.60, 12.53).
stirred 2 days at 23 °C. After evaporation of the solvent under
Dimethyl 2-(4-methoxybenzamido)biphenyl-4,4′-diyldicarba- reduced pressure, the crude product was purified by flash
mate (4). A mixture of 3 (2.67 g, 8 mmol), methyl chlorofor- chromatography with CH2Cl2–EtOAc (3 : 1) to afford 1.47 (84%)
1
mate (1.66 g, 17.6 mmol) and triethylamine (2.02 g, 20 mmol) of 7 as white solid, m.p. 212.7–213.3 °C. H NMR (300 MHz,
in CH2Cl2 (75 mL) was stirred overnight at 23 °C. The mixture DMSO-d6) δ 9.80 (s, 1H), 9.73 (s, 1H), 9.71 (s, 1H), 8.68 (d, J =
was washed with water (50 mL), NaCl (50 mL) and dried over 9.3 Hz, 1H), 8.57 (d, J = 9.3 Hz, 1H), 8.32 (d, J = 1.8 Hz, 1H),
Na2SO4. After evaporation of the CH2Cl2 under reduced 8.18 (d, J = 1.8 Hz, 1H), 8.01 (d, J = 7.5 Hz, 1H), 7.79 (dd, J = 9.0
pressure, the crude product was purified by flash chromato- Hz, 1.8 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.4 Hz,
graphy with hexane–EtOAc (1 : 2) to afford 3.31 g (92%) of 4 as 2H), 1.54 (s, 9H), 1.48 (s, 9H). 13C NMR (75 MHz, DMSO-d6)
1
white solid, m.p. 130.7–132.0 °C. H NMR (300 MHz, DMSO- δ 160.7, 158.6, 153.4, 144.0, 140.1, 138.7, 131.8, 131.0, 128.8,
d6) δ 9.77 (s, 1H), 9.64 (s, 1H), 9.63 (s, 1H), 7.79 (d, J = 8.7 Hz, 125.0, 123.5, 123.1, 119.2, 118.7, 116.9, 115.9, 115.6, 80.0,
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28.8, 28.7. Analysis (calcd, found for C29H31N3O5·0.75H2O): 67.2, 55.7, 37.0.28.3, 28.2. Analysis (calcd, found for
C (67.62, 67.92), H (6.36, 6.61), N (8.16, 7.74).
C33H37N3O5): C (71.33, 71.47), H (6.71, 6.79), N (7.56, 7.50).
tert-Butyl 6-(4-(prop-2-ynyloxy)phenyl)phenanthridine-3,8-
tert-Butyl 6-(4-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)-
diyldicarbamate (8). A mixture of 7 (1.25 g, 2.5 mmol), 80% methoxy)phenyl)-5-methyl-5,6-dihydrophenanthridine-3,8-diyl-
propargyl bromide in toluene (744 mg, 5.0 mmol) and K2CO3 dicarbamate (11). A mixture of 10 (278 mg, 0.5 mmol), 1-azido-
(1.04 g, 7.5 mmol) in acetone (50 mL) was heated under reflux 2-fluoroethane (1.05 mmol), sodium ascorbate (495 mg,
for 8 h. After evaporation of the solvent, ethyl acetate (75 mL) 2.5 mmol), and CuSO4·5H2O (62.5 mg, 0.25 mmol) in DMF
was added, washed with water (50 mL × 2), NaCl (50 mL) and (10 mL) was stirred overnight at 23 °C. The reaction mixture
dried over Na2SO4. After evaporation of the solvent under was diluted with ethyl acetate (75 mL), and washed with water
reduced pressure, the crude product was purified by flash (50 mL × 2), saturated NaCl (50 mL), and dried over Na2SO4.
chromatography with CH2Cl2–EtOAc (10 : 1) to afford 1.28 g After evaporation of the solvent under reduced pressure, the
(95%) of 8 as white solid, m.p. 129.5–130.3 °C. 1H NMR crude product was purified by flash chromatography with
(300 MHz, CDCl3) δ 8.46 (d, J = 9.0 Hz, 1H), 8.37 (d, J = 9.0 Hz, CH2Cl2–EtOAc (100 : 5) to afford 229 mg (71%) of 11 as white
1
1H), 8.05 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.90 (s, solid, m.p. 124.7–125.5 °C. H NMR (300 MHz, CDCl3) δ 7.64
1H), 7.79 (d, J = 2.1 Hz, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.08 (d, J = (m, 2H), 7.57 (d, J = 8.1 Hz, 1H), 7.18 (s, 2H), 7.01 (d, J = 8.4
8.7 Hz, 2H), 6.95 (s, 1H), 6.89 (s, 1H), 4.74 (d, J = 2.4 Hz, 2H), Hz, 2H), 6.73 (m, 4H), 6.56 (s, 2H), 5.21 (s, 1H), 5.07 (s, 2H),
2.55 (t, J = 2.4 Hz, 1H), 1.55 (s, 9H), 1.50 (s, 9H). 13C NMR 4.74 (dt, J = 47.7 Hz, 4.2 Hz, 2H), 4.60 (dt, J = 27.6 Hz, 4.2 Hz,
(75 MHz, CDCl3) δ 160.5, 158.0, 152.7, 152.6, 143.8, 138.5, 2H), 2.82 (s, 3H), 1.50 (s, 9H), 1.48 (s, 9H). 13C NMR (75 MHz,
136.9, 133.0, 131.0, 129.2, 125.2, 122.9, 122.4, 119.3, 119.0, CDCl3) δ 157.6, 152.5, 144.9, 144.5, 139.1, 137.0, 135.7, 134.2,
117.6, 116.5, 114.8, 80.9, 80.8, 78.5, 75.7, 55.9, 28.3, 28.2. Ana- 128.0, 125.5, 123.6, 123.0, 122.6, 117.9, 117.0, 116.4, 114.6,
lysis (calcd, found for C32H33N3O5·0.5H2O): C (70.06, 70.10), 107.5, 102.4, 81.3 (d, J = 171.9 Hz), 80.4, 80.2, 67.1, 61.8, 50.4
H (6.25, 6.10), N (7.66, 7.64).
3,8-Bis(tert-butoxycarbonylamino)-5-methyl-6-(4-(prop-2- C35H41FN6O5): C (65.20, 65.28), H (6.41, 6.56), N (13.03, 12.58).
ynyloxy)phenyl)phenanthridinium iodide (9). A mixture of 8 6-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-
(d, J = 20.5 Hz), 37.0, 28.3, 28.2. Analysis (calcd, found for
(1.08 g, 2.0 mmol) and CH3I (5 mL) in THF (15 mL) was heated 5-methyl-5,6-dihydrophenanthridine-3,8-diamine (12). A mixture
under reflux for 5 days. After removing of the solvent, ethyl of 11 (161 mg, 0.25 mmol) in ethyl acetate (6 mL) and 37%
ether (30 mL) was added, the precipitate solid was filtered out HCl (2 mL) was stirred for 3 h. After evaporation of the solvent
and washed with ethyl ether to afford 1.12 g (82%) of 9 as under reduced pressure, the product was dried in vacuum for
1
yellow solid, m.p. 209.4–210.0 °C. H NMR (300 MHz, DMSO- 8 h to afford 138 mg (100%) of 12 (HCl salt) as pale yellow
1
d6) δ 10.27 (s, 1H), 9.98 (s, 1H), 9.01 (d, J = 8.4 Hz, 1H), 8.93 (d, solid, m.p. 106.0–106.5 °C. H NMR (400 MHz, DMSO-d6, HCl
J = 8.7 Hz, 1H), 8.71 (s, 1H), 8.40 (d, J = 8.7 Hz, 1H), 8.02 (d, J = salt) δ 8.19 (s, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.88 (d, J = 8.4 Hz,
9.0 Hz, 1H), 7.77 (s, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.1 1H), 7.27 (dd, J = 8.2 Hz, 2.0 Hz, 1H), 7.08 (d, J = 1.6 Hz, 1H),
Hz, 2H), 5.01 (s, 2H), 4.16 (s, 3H), 3.71 (s, 1H), 1.55 (s, 9H), 7.01 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 6.73 (dd, J = 8.4
1.44 (s, 9H). 13C NMR (75 MHz, DMSO-d6) δ 163.6, 159.9, Hz, 1.6 Hz, 1H), 6.56 (d, J = 1.6 Hz, 1H), 5.61 (s, 1H), 5.02 (s,
153.3, 153.2, 142.9, 140.8, 136.0, 131.4, 130.2, 129.1, 126.0, 2H), 4.77 (dt, J = 46.8 Hz, 4.4 Hz, 2H), 4.67 (dt, J = 27.6 Hz, 4.0
125.5, 124.7, 124.1, 122.0, 120.9, 118.4, 116.1, 81.2, 80.7, 79.4, Hz, 2H), 2.80 (s, 3H). 13C NMR (100 MHz, DMSO-d6, HCl salt)
56.5, 43.9, 28.7, 28.6. Analysis (calcd, found for δ 172.4, 158.2, 145.7, 143.2, 137.0, 133.8, 131.6, 130.0, 128.1,
C33H36IN3O5·0.5H2O): C (57.40, 57.47), H (5.40; 5.34), N (6.08, 125.4, 124.8, 124.4, 115.1, 112.1, 107.1, 82.3 (d, J = 167.3 Hz),
5.91).
65.3, 64.9, 50.4 (d, J = 20.2 Hz), 36.8. Analysis (calcd, found for
tert-Butyl 5-methyl-6-(4-(prop-2-ynyloxy)phenyl)-5,6-dihydro- C25H25FN6O·2.5HCl·0.5H2O): C (55.13, 55.49), H (5.27, 5.34),
phenanthridine-3,8-diyldicarbamate (10). NaBH3CN (126 mg, N (15.43, 15.11).
2.0 mmol) was added to a solution of 9 (682 mg, 1.0 mmol) in
3,8-Bis(tert-butoxycarbonylamino)-6-(4-((1-(2-fluoroethyl)-
methanol (15 mL) and the mixture was stirred 3 h at 23 °C. 1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-methylphenanthridinium
After evaporation of the methanol under reduced pressure, iodide (13). This was prepared according to the same proce-
ethyl acetate (75 mL) was added, washed with water (30 mL), dure for compound 11, except using compound 9 (170 mg,
NaCl (30 mL), and dried over Na2SO4. After removal of the 0.25 mmol), 1-azido-2-fluoroethane (0.5 mmol), sodium ascor-
solvent under reduced pressure, the crude product was puri- bate (248 mg, 1.25 mmol), and CuSO4·5H2O (32 mg,
fied by flash chromatography with CH2Cl2–ether (100 : 5) to 0.13 mmol) in DMF (10 mL). The reaction mixture was diluted
afford 468 mg (84%) of 10 as white solid, m.p. 215.4–216.0 °C. with water (75 mL), then extracted with CH2Cl2 (50 mL × 2),
1H NMR (300 MHz, CDCl3) δ 7.64 (d, J = 8.7 Hz, 1H), 7.57 (d, the extracted CH2Cl2 was combined, washed with water
J = 8.1 Hz, 1H), 7.18 (s, 1H), 7.17 (d, J = 8.1 Hz, 1H), 7.02 (d, J = (50 mL), NaCl (50 mL) and dried over Na2SO4. After evapor-
9.0 Hz, 2H), 6.77–6.70 (m, 4H), 6.52 (s, 2H), 5.23 (s, 1H), 4.55 ation of the CH2Cl2, the crude product was recrystallized from
(d, J = 2.1 Hz, 2H), 2.83 (s, 3H), 2.46 (t, J = 2.1 Hz, 1H), 1.50 (s, ethyl acetate to afford 13 as yellow solid (105 mg, 55%),
9H), 1.48 (s, 9H). 13C NMR (75 MHz, CDCl3) δ 157.0, 152.5, m.p. 156.0–157.9 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.23, 9.93,
144.9, 139.1, 137.0, 135.7, 134.5, 127.9, 125.5, 123.0, 122.7, 8.97 (d, J = 9.6 Hz, 1H), 8.95 (d, J = 10.0 Hz, 1H), 8.68 (s, 1H),
117.9, 117.0, 116.4, 114.7, 107.5, 102.4, 80.4, 80.3, 78.6, 75.4, 8.38 (d, J = 7.2 Hz, 1H), 8.37 (s, 1H), 7.99 (d, J = 8.8 Hz, 1H),
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7.75 (s, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), (1 mg per 10 mL) in water (10 mL). The Sep-Pak was rinsed
5.32 (s, 2H), 4.84 (dt, J = 47.2 Hz, 4.8 Hz, 2H), 4.75 (dt, J = 27.6 with the solution of ascorbic acid (10 mL), and then [18F]12
Hz, 4.0 Hz, 2H), 4.13 (s, 3H), 1.51 (s, 9H), 1.41 (s, 9H). 13C was eluted with ethanol (containing ascorbic acid, 1 mg
NMR (100 MHz, DMSO-d6) δ 163.5, 160.5, 153.1, 153.0, 145.5, mL−1). The dose was formulated in 10% ethanol–saline (con-
142.7, 140.6, 137.9, 135.8, 131.3, 130.0, 128.8, 128.4, 125.9, taining 0.1 mg ascorbic acid mL−1). Total synthesis time was
125.3, 124.1, 123.9, 121.8, 120.6, 118.2, 115.8, 82.4 (d, J = 167.3 90 min starting from [18F]fluoride, yield 42.8% (non-decay cor-
Hz), 81.0, 80.5, 61.9, 50.6 (d, J = 19.3 Hz), 43.8, 28.5, 28.4.
rected) starting from [18F]16, radiochemical purity up to 99%,
3,8-Diamino-6-(4-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)- and the specific activity at the end of synthesis is 1.3–2.4 ×
methoxy)phenyl)-5-methylphenanthridinium chloride (14). This 1010 Bq µmol−1
.
was prepared according to the same procedure for compound
Conversion of [18F]12 to [18F]14
12, except using compound 13 (77 mg, 0.1 mmol) in ethyl
acetate (3 mL) and 37% HCl (1 mL) and stirred for 3 h. After
evaporation of the solvent under reduced pressure, the
product was dried in vacuum for 8 h to afford 55 mg (100%) of
14 (HCl salt) as red solid, m.p. 133.9–135.3 °C. 1H NMR
(400 MHz, DMSO-d6, HCl salt) δ 8.80 (d, J = 6.8 Hz, 1H), 8.74
(d, J = 7.2 Hz, 1H), 8.41 (s, 1H), 7.76–7.41 (m, 8H), 5.28 (s, 2H),
4.87–4.71 (m, 4H), 4.00 (s, 3H). 13C NMR (100 MHz, DMSO-d6,
HCl salt) δ 161.2, 160.3, 131.3, 128.6, 125.9, 124.3, 115.8, 82.4
(d, J = 167.3 Hz), 61.9, 50.6 (d, J = 17.8 Hz), 43.3.
To a solution of [18F]12 in DMSO (300 µL) was added a solu-
tion of CuSO4·5H2O (1 mg) in water (10 µL). The mixture was
shaken and checked by HPLC. [18F]12 was consumed quickly,
and [18F]14 was formed as the major product (90%). The reac-
tion mixture was diluted with water (10 mL) for solid phase
extraction (Waters, C18 Classic). The [18F]14 was eluted with
DMSO for cell study (4.8 × 106 Bq per 100 µL DMSO).
In vivo imaging of doxorubicin (DOX)-induced cardiotoxicity
Animal model. All animal studies were carried out in
accordance with the National Institute of Health Guide for the
Care and Use of Laboratory Animals under protocols approved
by the Washington University in St. Louis Animal Study Com-
mittee (IACUC). DOX has been shown to induce the formation
of ROS, chief among them is the generation of the superoxide
radical.32 To that end, male C57BL/6 mice were divided into
2 groups (4 mice per group). The untreated group received 200
µL of saline intraperitoneal (i.p.) while the DOX-treated mice
received a single i.p. injection of 20 mg kg−1 DOX-HCl salt.
Five days post-treatment, 60 min dynamic PET imaging was
performed as described below.
Pre-clinical PET imaging. Mice were anesthetized by inhala-
tion of 2–2.5% isoflurane administered via an induction
chamber. Dynamic PET acquisition was started after a bolus
injection of [18F]12 (9.3–11.1 × 106 Bq per 100 µL) via a tail
vein catheter. PET imaging was performed on either the micro-
PET Inveon or Focus-220. Scanners were cross-calibrated to
ensure reliability of the data. Body temperature was main-
tained by a heat lamp. At the conclusion of imaging, the bio-
distribution of [18F]12 was determined in multiple organs. In
addition, HPLC analysis was performed to determine the pres-
ence of parent and metabolites species in heart and blood. To
evaluate the kinetics and cardiac uptake of the oxidized form,
[18F]14 was similarly injected to untreated and DOX treated
mice for comparison against the kinetics of [18F]F12.
Radiosynthesis of [18F]12
Deprotection of 10. Into a 10 mL Pyrex tube with screw cap
was loaded 10 (2 mg, 3.51 µmol). The tube was flushed with
argon before and after the addition of TFA (500 µL) and
ascorbic acid (1 mg, 5.68 µmol) in water (100 µL), and then the
tube was capped and wrapped with aluminum foil. The tube
was shaken to dissolve 10, and the reaction was allowed to
proceed at ambient temperature for 15 min. TFA and water
were then removed under vacuum (∼1 torr) at ambient temp-
erature to afford 17 as a pale yellow residue. This was done 1 h
before labeling.
Radiosynthesis of [18F]12. DMF (300 µL) and N,N-diisopro-
pylethylamine (10 µL) were added to the previously prepared
tube to dissolve 17, and then the tube was cooled in a dry ice
trap. [18F]fluoroethylazide 16 (∼1.3 × 109 Bq) in acetonitrile
(200 µL), starting from [18F]fluoride (1.9 × 109 Bq), was distilled
directly to the tube according to the literature. The tube was
removed from the trap, and warmed up to ambient tempera-
ture in a water bath. Sodium ascorbate (15 mg, 76 µmol) in
water (50 µL) and CuSO4·5H2O (5 mg, 20 µmol) in water (50
µL) was mixed to form a yellow slurry, which was added to the
tube containing [18F]16 and 17. The reaction mixture was
shaken occasionally at ambient temperature over 10 min, and
then a solution of 10% acetonitrile–water–0.1% TFA (4 mL)
was added to the mixture. The solution was injected through a
nylon filter (0.45 µm) for HPLC purification. Semi-preparative
HPLC purification was carried out using an Agilent SB-C18 250
× 9.4 mm 5 µ column with 17% acetonitrile–water–0.1% TFA
as the mobile phase at a flow rate of 4 mL min−1 and UV at
Author contributions
280 nm. [18F]12 was eluted at 14 min, and the desired HPLC W.C. conducted the organic synthesis and wrote the paper,
fraction (∼5.5 × 108 Bq) was collected in a 50 mL tube contain- A.C. conducted the in vitro assays measuring the reactivity of the
ing ascorbic acid (5 mg) in water (∼10 mL). The collected probes, D.Z. conducted the radiolabeling reactions and in vivo
eluent was further diluted with water to 50 mL, and passed metabolism studies, K.I.S. designed the in vivo imaging,
through a C18 Sep-Pak (Waters, Classic), which had been pre- metabolism studies and analyzed the microPET image data,
treated with ethanol (10 mL) and a solution of ascorbic acid J.X. conducted the cell culture experiments, L.L.D. supervised
4430 | Org. Biomol. Chem., 2014, 12, 4421–4431
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Paper
the in vitro evaluation of the imaging probes and edited the 14 S. Dikalov, J. Jiang and R. P. Mason, Free Radical Res., 2005,
paper, R.J.G. M.A.M. designed the in vivo imaging study, and
39, 825–836.
R.H.M. designed the imaging probes and edited the paper.
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Competing financial interests
The authors declare no competing financial interests.
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Acknowledgements
The authors would like to thank Fanjie Zhang for her technical
assistance. This research was funded by grants AG36045 and
5R01EB12284 awarded by the National Institutes of Health,
the Charles and Joanne Knight Alzheimer’s Research Initiative
of the Washington University Alzheimer’s Disease Research
Center (RHM), and 5R01AG033679 and the Larry L. Hillblom
Foundation (LLD).
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