Development of a Positron Emission Tomography Radiotracer for Imaging Elevated Levels of Superoxide in Neuroinflammation

Article abstract of DOI:10.1021/acschemneuro.7b00385

Reactive oxygen species (ROS) are believed to play a major role in the proinflammatory, M1-polarized form of neuroinflammation. However, it has been difficult to assess the role of ROS and their role in neuroinflammation in animal models of disease becaus

Full text of DOI:10.1021/acschemneuro.7b00385

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Article
Development of a PET Radiotracer for Imaging
Elevated Levels of Superoxide in Neuroinflammation
Catherine Hou, Chia-Ju Hsieh, Shihong Li, Hsiaoju Lee, Thomas J. A. Graham, Kuiying Xu, Chi-Chang Weng,
Robert K. Doot, Wenhua Chu, Subhasish K. Chakraborty, Laura L. Dugan, Mark A. Mintun, and Robert Mach
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00385 • Publication Date (Web): 03 Nov 2017
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Development of a PET Radiotracer for Imaging Elevated Levels of
Superoxide in Neuroinflammation
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Catherine Hou,1 ChiaꢀJu Hsieh,1 Shihong Li,1 Hsiaoju Lee,1 Thomas J. Graham,1 Kuiying Xu,1
ChiꢀChang Weng,1 Robert K. Doot,1 Wenhua Chu,2 Subhasish K. Chakraborty,3 Laura L.
Dugan,3 Mark A. Mintun,2 Robert H. Mach1
1Department of Radiology, Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA
2Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO
3Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN
Keywords: reactive oxygen species, superoxide, positron emission tomography,
neuroinflammation
Corresponding author:
Robert H. Mach, Ph.D.
Britton Chance Professor of Radiology
University of Pennsylvania
Chemistry Building, Room 283
231 S. 34th St
Philadelphia, PA 19104
Email: rmach@mail.med.upenn.edu
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ABSTRACT
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Reactive oxygen species (ROS) are believed to play a major role in the proinflammatory, M1ꢀ
polarized form of neuroinflammation. However, it has been difficult to assess the role of ROS
and their role in neuroinflammation in animal models of disease because of the absence of
probes capable of measuring their presence with the functional imaging technique positron
emission tomography (PET). This study describes the synthesis and in vivo evaluation of
[18F]ROStrace, a radiotracer for imaging superoxide in vivo with PET, in an LPS model of
neuroinflammation. [18F]ROStrace was found to rapidly cross the bloodꢀbrain barrier (BBB) and
was trapped in the brain of LPSꢀtreated animals but not the control group. [18F]oxꢀROStrace,
the oxidized form of [18F]ROStrace, did not cross the BBB. These data suggest that
[18F]ROStrace is a suitable radiotracer for imaging superoxide levels in the CNS with PET.
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INTRODUCTION
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The term reactive oxygen species (ROS) has been used to describe a class of highly reactive
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ꢀꢀ
oxygenꢀcontaining molecules including superoxide radical anion (O2 ), hydrogen peroxide
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ꢀꢀ
(H2O2), hydroxyl radical (OH ) and hypochlorous acid (HOCl).1-3 While ROS have a role in
normal cellular processes, high levels of ROS can lead to oxidative stress and protein, lipid, and
DNA damage. Consequently, ROS levels are tightly regulated in a cell through the activity of the
enzymes, superoxide dismutase (SOD1, SOD2, SOD3), catalase, and NADH peroxidase.1, 3 An
imbalance between the rate of formation of ROS and the regulatory mechanisms resulting in
elevated ROS leads to a condition called oxidative stress.4 Oxidative stress has been
implicated in the pathogenesis of cardiovascular, neurodegenerative diseases and cancer.1
One major source of ROS is by oxidative phosphorylation in the mitochondria, and
mitochondrial dysfunction involving reduced Complex I activity is considered to be one of the
main sources of elevated ROS in Parkinson’s Disease (PD).2, 3 Another main source of ROS
are the Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidases (NOX1ꢀ5, DUOX1ꢀ2),
which are a family of enzymes that transfer electrons from the reduced form of NADPH to
molecular oxygen.3, 5 The NADPH oxidases have a widespread distribution throughout the body
and are thought to play a key role in host defense, cellular signaling, stress response and tissue
homeostasis. Within the CNS, NOX1 and NOX4 are expressed in low levels in neurons,
astrocytes and microglial cells, whereas NOX2 is expressed in high levels in microglial cells.
NOX2 is thought to be latent under normal conditions; upon microglial activation, NOX2
becomes activated through the formation of a multimeric protein complex leading to NOX2ꢀ
derived superoxide formation.5 This activation process is thought to play a key role in mediating
proinflammatory, M1ꢀpolarized microglia in neuroinflammation, and an increase in levels of
NOX2 and other markers of oxidative stress have been reported in postmortem samples of
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Alzheimer’s Disease (AD) brain and in transgenic mouse models of AD.4, 6 Similar results have
been reported in the substantia nigra of PD brain samples and in the 1ꢀmethylꢀ4ꢀphenylꢀ1,2,3,6ꢀ
tetrahydropyridine (MPTP) model of PD. In addition, the spinal cord of patients with amyotropic
lateral sclerosis (ALS) have been reported to have increased NOX2 expression and other
measures of oxidative stress such as the elevation of COXꢀ2 and other cytokines.7 These
results indicate that the proinflammatory form of neuroinflammation leading to the elevation of
superoxide and ROS represents a common pathway in neurodegenerative disorders.
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Based on the above discussion, an imaging probe capable of measuring levels of superoxide in
brain would be a valuable tool for measuring the proꢀinflammatory, M1ꢀpolarized form of
neuroinflammation. Furthermore, since neuroinflammation is thought to represent a hallmark in
the pathophysiology of a number of neurodegenerative diseases 7 a PET radiotracer for
measuring superoxide levels in the CNS could provide insight into the relationship between the
formation of ROS and other pathological features associated with neurodegeneration (e.g., Aβ
and tau formation in AD).8
The fluorescent probe dihydroethidium (DHE) has been used to measure superoxide levels in
cells and tissues using microscopy and optical imaging techniques.9-11 Our group has previously
reported the synthesis of an 18Fꢀlabeled analogue of DHE, [18F]FDMT, and its evaluation in an
animal model of adriamycinꢀinduced model of cardiotoxicity (Figure 1).12 The results of this
study were significant since it revealed for the first time the ability to image ROS in a living
organism using positron emission tomography (PET). While this tracer was able to image ROS
levels in peripheral organs in vivo, it did not cross the bloodꢀbrainꢀbarrier to allow imaging of
elevated superoxide levels associated with proinflammatory microglia activation. In the current
study, we report the in vivo evaluation of an 18Fꢀlabeled analogue of DHE, [18F]ROStrace
(Scheme 1), which is capable of crossing the bloodꢀbrainꢀbarrier. We also describe the series
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of in vivo validation studies confirming the ability of [18F]ROStrace to image superoxide levels in
a murine model of neuroinflammation.
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RESULTS
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Chemistry and reactivity with reactive oxygen species. The synthesis of ROStrace and its
precursor for radiolabeling is shown in Scheme 1. The hydroxyethyl was introduced to the
known hydroxy compound diꢀtertꢀbutyl (6ꢀ(4ꢀhydroxyphenyl)phenanthridineꢀ3,8ꢀdiyl)dicarbamate
12 to form compound 2, which was methylated with iodomethane, reduced by sodium
borohydride and then carefully tosylated to give the precursor compound 3. The standard
compound, ROStrace (6), was synthesized (Scheme 1) under a similar procedure. Once the
compounds were reduced, they are very labile especially in solution. All the following
procedures were performed in a rapid manner under low temperature.
The reactivity of DHE and ROStrace for superoxide, hydrogen peroxide and hydroxyl radical
was measured using an in vitro assay as previously reported.12 Two different methods were
used to generate superoxide, xanthine oxidase metabolism of hypoxanthine or by thermal
decomposition of 3ꢀmorpholinosyndnomine (SINꢀ1) in the presence of 2ꢀ(4ꢀcarboxyphenyl)ꢀ
4,4,5,5,tetramethylꢀimidazolineꢀ1ꢀoxylꢀ3ꢀoxide (CPTIO). In the both systems, oxidation of
ROStrace was completely prevented by the addition of superoxide dismutase (SOD) (Figure 2).
ROStrace was also exposed to hydrogen peroxide alone or in the presence of horseradish
peroxidase (HRP) to generate hydroxyl free radical. The fluorescence intensity results indicate
that ROStrace has little reactivity with hydrogen peroxide or hydroxyl radical (Figure 2). These
data suggest that ROStrace has good selectivity for superoxide. Since its chemical reactivity is
similar to DHE, which is selectively oxidized to ethidium by superoxide radical (Figure 2), we
believe ROStrace will behave like DHE in vivo and measure brain levels of superoxide radical.13
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Radiochemistry. [18F]ROStrace was synthesized (Scheme 2) in an 8ꢀ10% radiochemical yield
starting from [18F]fluoride ion, with molar activity >74 GBq/ꢁmol. In order to confirm the
mechanism of uptake of radioactivity in the LPSꢀtreated mice, [18F]oxꢀROStrace was also
synthesized. This was accomplished by the quantitative conversion of [18F]ROStrace to [18F]oxꢀ
ROStrace by treatment with cupric sulfate (Scheme 2).
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In vivo evaluation. The in vivo evaluation of [18F]ROStrace was performed using an LPSꢀ
treated mouse model of neuroinflammation. MicroPET imaging studies were conducted in both
control (N = 15) and LPSꢀtreated animals (N = 16). The sagittal images revealed a much
higher brain uptake of [18F]ROStrace in the LPSꢀtreated animals relative to the control group
(Figure 3a). Timeꢀactivity curves from the control mice demonstrated that [18F]ROStrace has a
high initial brain uptake, peaking within the first minute, followed by a rapid washout. In LPSꢀ
treated animals, activity continued to increase in the brain, peaking around three minutes postꢀ
injection (Figure 3b). Levels of brain activity in both control and LPSꢀtreated mice stabilized after
about 20 minutes. However, the retention of radioactivity in brain was higher in the LPSꢀtreated
mice.
The initial analysis of the group data involved a comparison of the average %ID/cm3 from 40 –
60 min postꢀinjection of the radiotracer for the control versus LPSꢀtreated animals. This time
interval was chosen because it represents a plateau phase in the timeꢀactivity curves. A graph
of the average %ID/cm3 for the two groups (Figure 3c) revealed a statistically significant
difference between the control and LPSꢀtreated animals (P < 0.0001) even though there was
some overlap between individual subjects in the LPSꢀtreated group and the control group. When
the LPSꢀtreated mice were grouped as a whole and compared to the control group, a 156%
increase in brain uptake was observed.
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It was also observed that not all animals responded to the LPSꢀtreatment in the same manner.
That is, some animals displayed more severe symptoms than others, while others appeared to
be unaffected by the dose of LPS. In order to characterize the animals to the degree of
“sickness” following the LPSꢀtreatment, a scoring mechanism based on criteria outlined by
Carstens and Moberg14 for recognizing pain and distress in laboratory animals was used. Using
this method, animals were assigned a condition score based on visual observation of signs of
distress including hunched posture, decreased motion, piloerection, lack of grooming, and
discharge from the eyes. The animals were then grouped into three levels of sickness: level 1
animals displayed mild symptoms and appeared fairly normal; level 2 animals displayed a
moderate response including some of the stated symptoms; level 3 animals displayed
practically all the symptoms and with more severity. The results of this analysis are shown in
Figure 4. Animals with a condition score of 2 or 3 had a significantly higher uptake of
[18F]ROStrace relative to the control group and level 1 animals. These data indicate that the
difference in response to LPS as measured via the observational studies is predictive of the
relative amount of neuroinflammation in the CNS.
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To further confirm the mechanism of retention of [18F]ROStrace in the CNS, radiometabolite
analysis of homogenates from brains obtained after completion of the microPET imaging studies
was conducted. The results of the HPLC studies indicated that the activity in brain was primarily
the oxidized species, [18F]oxꢀROStrace, while a much smaller amount of the parent compound
was detected (68% vs 10%) 2 hr after an iv injection of [18F]ROStrace. Blood levels of the
oxidized species are much lower (27%) at the same time point, suggesting that [18F]oxꢀ
ROStrace is not entering the brain from the circulation (Table 1).
We believe the neutral species, [18F]ROStrace, enters the brain where it is trapped upon
oxidation to [18F]oxꢀROStrace by superoxide. To demonstrate that the higher retention of
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[18F]ROStrace in LPSꢀtreated mice is due to oxidation by LPSꢀinduced ROS in the brain, and
that the peripherally oxidized species, [18F]oxꢀROStrace, is not able to enter the brain, mice
were imaged after an injection of [18F]oxꢀROStrace. [18F]oxꢀROStrace showed no brain uptake
in the untreated mice (Figure 5). These data demonstrate that the neutral species,
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[18F]ROStrace, crosses the bloodꢀbrain barrier and is oxidized to [18F]oxꢀROStrace after entering
the brain. LPS treatment is known to cause some disruption of the blood brain barrier, and our
LPSꢀtreated mice displayed a small amount of brain uptake when injected with [18F]oxꢀ
ROStrace at 24 hours post LPS treatment. The same imaging procedure performed one week
after LPS treatment resulted in no uptake. These data confirm the trapping mechanism of
[18F]ROStrace in the brain is due to oxidation by superoxide to [18F]oxꢀROStrace (Figure 5).
Ex vivo autoradiography studies were conducted on brain sections harvested after completion of
the PET imaging studies. In these studies, animals were injected with DHE (15 mg/kg, i.v.) 30
min prior to sacrifice so that a comparison can be made between the distribution of the
radiotracer, which is primarily [18F]oxꢀROStrace and the fluorescent signal from oxidized DHE
(i.e., ethidium / 2ꢀOHꢀethidium). The results of this study indicate a similar pattern of distribution
between the radiotracer and oxidized DHE fluorescence (Figure 6). We also obtained
fluorescence images in mice 30 minutes after injection of cold ROStrace (14 mg/kg, i.v.). As
expected, this also provided a similar pattern of distribution in the brain. These results further
support the mechanism of uptake and trapping of [18F]ROStrace in the CNS under conditions of
elevated ROS.
DISCUSSION
The goal of this study was to develop a PET radiotracer for imaging superoxide levels in the
CNS that are produced during the proinflammatory, M1ꢀpolarized form of neuroinflammation
that is thought to be a hallmark feature of neurodegeneration.7, 8, 15 Our group previously
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reported an 18Fꢀlabeled analogue of the fluorescent probe DHE and demonstrated that it was
capable of imaging superoxide that is produced in myocardial tissues in an animal model of
Adriamycinꢀinduced cardiotoxicity.12 Although this probe is capable of imaging superoxide in
peripheral organs, its inability to cross the bloodꢀbrain barrier prevented its use as a means of
imaging superoxide produced under conditions of neuroinflammation and mitochondrial
dysfunction which are thought to occur in most neurodegenerative disorders.
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In the time interval between our initial publication and this current study, a number of ROSꢀ
based radiotracers have been reported in the literature. These include: 1) [18F]PCꢀFLT, which
measures extracellular and intracellular levels of H2O2;16 2) [11C]VitaminꢀC, which measures
extracellular levels of H2O2;17 3) the hydrocyanine dye derivative, [18F]ROS1, which measures
superoxide and hydroxyl radicals;18 4) [18F]5ꢀFluoroaminosuberic acid, which provides an
indirect measure of intracellular levels of ROS by imaging the increase in expression of the
cystine transporter (system xcꢀ);19 and 5) [11C]DHQ1, a dihydroisoquinoline analogue capable of
measuring intracellular levels of superoxide.20 [11C]DHQ1 is capable of crossing the bloodꢀbrain
barrier and has potential for imaging increased ROS levels in the CNS as a consequence of
neuroinflammation.20 A number of radiolabeled analogues of dihydroꢀmethidium (a.k.a,
hydromethidine) have been synthesized and evaluated in vitro and in vivo, including
[3H]hydromethidine and [11C]hydromethidine.21, 22 [3H]Hydromethidine is oxidized by superoxide
and hydroxyl radicals and has been evaluated in animal models of stroke and cisplatinꢀinduced
nephrotoxicity.23, 24 A recent paper on the radiosynthesis and preliminary in vivo evaluation of
[11C]methidium has also been reported.22 It should also be noted that our group reported the in
vitro properties of methidium towards various forms of ROS in the patent literature (US
9,035,057), but did not pursue radiolabeling this compound because of our interest in
developing an 18Fꢀlabeled radiotracer for imaging ROS.
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In the current study, we report the synthesis, in vitro characterization, and in vivo evaluation of
[18F]ROStrace in an LPSꢀtreatment model of neuroinflammation. In vitro studies indicate that it
is oxidized to the charged species, [18F]oxꢀROStrace, by superoxide since it has low reactivity
for hydrogen peroxide and hydroxyl radical (Figure 2). Our in vivo studies included not only the
uptake of the parent radiotracer, [18F]ROStrace, in LPSꢀtreated versus control mice, but a
detailed metabolite analysis study confirming the mechanism of uptake and trapping of the
radiotracer in the CNS. This trapping mechanism was also confirmed by the observation that
the oxidized species, [18F]oxꢀROStrace, is not capable of crossing the bloodꢀbrain barrier. The
distribution of radioactivity in the LPSꢀtreated animals also matched the distribution of the
fluorescence signal from treatment of the animals postꢀPET with DHE, which further supports
the mechanism of trapping of the radiotracer in the CNS. Our results revealed that the relative
uptake and retention of [18F]ROStrace correlated with the level of sickness induced by LPS,
which suggest that this radiotracer is capable of measuring different levels of neuroinflammation
in rodent models of neurodegeneration. Taken collectively, our data demonstrates that
[18F]ROStrace is the first 18Fꢀlabeled radiotracer capable of measuring elevated levels of
superoxide which occur during the “neurotoxic” or proinflammatory form of neuroinflammation.
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CONCLUSION
[18F]ROStrace is a promising PET radiotracer for imaging elevated superoxide levels in the
CNS under conditions of neuroinflammation. We are currently using this radiotracer in a panel
of transgenic mouse models of AD, PD and other neurodegenerative disorders.
METHODS
General. All chemicals were purchased from commercial sources and were used without
further purification. NMR spectra were taken on a Bruker DMX 500 MHz. Mass spectroscopy
data and compound purity analysis were acquired using ESI technique on 2695 Alliance LCMS.
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Purification of target compounds were carried out on a Biotage Isolera One with a dualꢀ
wavelength UVꢀVIS detector. Chemical shifts (δ) in the 1HꢀNMR spectra were referenced by
assigning the residual solvent peaks. Elemental analyses were performed by Atlantic
Microlabs, Inc. (Atlanta, GA) and were within ± 0.4% unless otherwise noted. Measurement of
reactivity for ROS was conducted using the published method.12
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Di-tert-butyl (6-(4-(2-hydroxyethoxy)phenyl)phenanthridine-3,8-diyl)dicarbamate (1). Diꢀtertꢀ
butyl (6ꢀ(4ꢀhydroxyphenyl)phenanthridineꢀ3,8ꢀdiyl)dicarbamate 12 (501 mg, 1 mmol), 2ꢀ
bromoethanol (400 mg, 3.22 mmol), potassium carbonate (690 mg, 5 mmol) and DMF (6 mL)
were put in a sealed vial and heated to 95°C overnight. The mixture was diluted with water (50
mL) and extracted with ethyl acetate. The organic layer was washed with H2O/NaHCO3
aqueous/brine (2 x each) and dried with Na2SO4. After filtration, the filtrate was condensed and
the residue was applied to FC (dichloromethane/ethyl acetate 0ꢀ40%) yielding compound 1 (340
mg, 62%) and the recovered starting material. 1H NMR (500 MHz, DMSOꢀd6) δ ppm 1.45 (s, 9
H), 1.51 (s, 3H), 3.79 (dt, J = 5.1, 4.9 Hz, 2H), 4.11 (t, J = 4.9 Hz, 2H), 4.92 (t, J = 5.5 Hz, 1H),
7.13 (d, J = 8.6 Hz, 2H), 7.65 (d, J = 8.6 Hz, 2H), 7.77 (dd, J = 8.9, 2.0 Hz, 1H), 8.00 (d, J = 8.6
Hz, 1H), 8.17 (s, 1H), 8.25 (s, 1H), 8.56 (d, J = 9.1 Hz, 1H), 8.67 (d, J = 9.2 Hz, 1H), 9.67 (s,
1H), 9.69 (s, 1H). 13C NMR (500 MHz, DMSOꢀd6) δ ppm 28.06, 28.12, 59.59, 69.69, 79.34,
114.11, 115.13, 116.29, 118.14, 118.69, 122.46, 122.67, 122.93, 124.38, 128.10, 131.04,
+
131.86, 138.08, 139.45, 143.31, 152.76, 152.78, 159.06, 159.83. HRMS: Calcd. for C31H36N3O6
[M+H]+ 546.2599. Found 546.2626.
2-(4-(3,8-Bis((tert-butoxycarbonyl)amino)-5-methyl-5,6-dihydrophenanthridin-6-yl)phenoxy)ethyl
4-methylbenzenesulfonate (3). Compound 2 (120 mg, 0.22 mmol) was suspended in a mixture
of toluene (9 mL) and iodomethane (1 mL) and kept stirring at 70°C overnight. The mixture was
condensed under vacuum and the residue was dissolved in 5 mL of methanol. The mixture was
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cooled with ice bath and excess of NaBH4 (~30 mg) was added. The mixture was kept stirring at
0°C for 10 min and condensed under vacuum below 10°C. The residue was applied to a preꢀ
equilibrated column of silica and eluted with dichloromethane/ethyl acetate (0ꢀ40%). The
combined fractions were condensed below 10°C yielding the methylated compound (67 mg,
54%) as slightly brown solid. This compound (67 mg, 0.12 mmol) was then dissolved in
dichloromethane (3 mL) under 0°C, pꢀtoluenesulfonyl chloride (84 mg, 0.44 mmol) and
trimethylamine (120 mg, 1.2 mmol) were added. The reaction was stirred at 0°C for 2 hr and the
reaction mixture was applied to a preꢀequilibrated column of silica gel and eluted with
dichloromethane /ethyl acetate (0ꢀ15%). The proper fractions were combined and condensed
below 10°C yielding the title compound as a slightly red solid (65 mg, 76%). 1H NMR (500 MHz,
CDCl3) δ ppm 1.48 (s, 9 H), 1.50 (s, 3H), 2.40 (s, 3H), 2.83 (s, 3H), 4.01 (t, J = 4.8 Hz, 2H), 4.28
(t, J = 4.8 Hz, 2H), 4.92 (t, J = 5.5 Hz, 1H), 5.22 (s, 1 H), 6.47 (s, 2H), 6.54 (d, J = 8.7 Hz, 2H),
6.72 (s, 2H), 6.98 (d, J = 8.7 Hz, 2H), 7.15 (s, 1H), 7.17 (s, 1H), 7.28 (d, J = 8.1 Hz, 2H), 7.57
(d, J = 8.8 Hz, 1H), 7.64 (d, J = 9.0 Hz, 1H), 7.76 (d, J = 8.3 Hz, 2H). 13C NMR (500 MHz,
CDCl3) δ ppm 21.56, 28.29, 28.33, 37.06, 65.29, 67.18, 68.04, 80.30, 80.45, 102.35, 107.54,
114.45, 116.34, 117.01, 117.86, 122.66, 123.03, 125.50, 127.92, 127.97, 128.10, 128.95,
129.77, 132.84, 134.35, 135.72, 137.01, 139.14, 144.86, 152.52, 157.38. HRMS: Calcd. for
C39H46N3O8S+ [M+H]+ 716.3000. Found 716.3016.
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Di-tert-butyl (6-(4-(2-fluoroethoxy)phenyl)phenanthridine-3,8-diyl)dicarbamate (4). Diꢀtertꢀbutyl
(6ꢀ(4ꢀhydroxyphenyl)phenanthridineꢀ3,8ꢀdiyl)dicarbamate1 (1.04 g, 2.07 mol) was dissolved in
MeCN, 2ꢀfluoroethyltosylate (902 mg, 4.14 mmol) and K2CO3 (855 mg, 6.22 mmol) were added.
The reaction was heated to 80°C and allowed to stir overnight. The reaction mixture was filtered
and concentrated to give a residue that was purified by FC (DCM/Ethyl acetate 0ꢀ50%) to afford
the desired compound as offꢀwhite foam (800mg, 70%). 1H NMR (500 MHz, DMSOꢀd6) δ ppm
1.45 (s, 9H), 1.51 (s, 9H), 4.36 (dt, J = 30.1, 3.8 Hz, 2H), 4.81 (dt, J = 47.9, 3.8Hz, 2H), 7.16 (d,
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J = 8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 7.77 (dd, J = 9.0, 2.2 Hz, 1H), 8.00 (d, J = 8.7 Hz, 1H),
8.56 (d, J = 9.1 Hz, 1H), 8.67 (d, J = 9.1 Hz, 1H), 9.67 (s, 1H), 9.69 (s, 1H). 13C NMR (500 MHz,
DMSOꢀd6) δ ppm 28.08, 28.14, 67.15, 67.31, 79.38, 81.52, 82.85, 114.18, 115.06, 116.27,
118.18, 118.73, 122.54, 122.69, 123.01, 124.39, 128.12, 131.14, 132.32, 138.13, 139.49,
143.32, 152.78, 152.81, 158.52, 159.79. HRMS: Calcd. for C31H35FN3O5 [M+H]+ 548.2555;
Found 548.2545.
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Di-tert-butyl (6-(4-(2-fluoroethoxy)phenyl)-5-methyl-5,6-dihydrophenanthridine-3,8-
diyl)dicarbamate (5). A screwꢀcap vial containing a solution of Diꢀtertꢀbutyl (6ꢀ(4ꢀ(2ꢀ
fluoroethoxy)phenyl)phenꢀanthridineꢀ3,8ꢀdiyl)dicarbamate (4, 45 mg, 0.082 mmol) in toluene
(1.5 mL) was treated with 0.50 mL of iodomethane. The vial was briefly purged with nitrogen,
sealed and heated to 95°C for 6 h. The reaction mixture was then moved to a 75°C bath and
allowed to stir overnight. The reaction mixture was condensed and taken up in EtOH (2 mL).
The solution was treated with excess NaBH4 (7 mg) and concentrated to give a residue that was
purified by FC (DCM/Ethyl acetate 0ꢀ30%) to give the desired product as a colorless solid (35
mg, 75%). 1H NMR (500 MHz, CDCl3) δ ppm 1.48 (s, 9H), 1.50 s, 9H), 2.85 (s, 3H), 4.08 (dt, J =
30.1, 4.2 Hz, 2H), 4.67 (dt, J = 47.4, 4.2 Hz, 2H), 5.24 (s, 1H), 6.45 9s, 1H), 6.46 (s, 1H), 6.69ꢀ
6.73 (m, 4H), 7.03 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 8.7 Hz, 1H). 13C
NMR (500 MHz, CDCl3) δ ppm 28.30, 28.34, 37.08, 66.86, 67.03, 67.20, 80.42, 80.73, 81.16,
82.52, 102.36, 107.48, 114.48, 117.04, 117.84, 122.69, 123.05, 125.52, 128.06, 134.15, 135.78,
136.99, 139.11, 144.87, 152.56, 157.80. HRMS: Calcd. for C32H39FN3O5 [M+H]+ 564.2868;
Found 564.2880.
6-(4-(2-Fluoroethoxy)phenyl)-5-methyl-5,6-dihydrophenanthridine-3,8-diamine (6). Diꢀtertꢀbutyl
(6ꢀ(4ꢀ(2ꢀfluoroethoxy)phenyl)ꢀ5ꢀmethylꢀ5,6ꢀdihydrophenanthridineꢀ3,8ꢀdiyl)dicarbamate (5, 20
mg, 0.035 mmol) was dissolved in EtOAc (1.5 mL) and concentrated HCl (37%, 0.5 mL) was
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added. The reaction mixture was purged with nitrogen and left stirred at r.t. for 30 min. The
reaction mixture was neutralized with 7N NH3 in methanol and concentrated under high vacuum
to give a roseꢀcolored solid. The solid was dissolved in DCM and applied to FC (ethyl acetate)
to afford the title compound as colorless solid (12 mg, 93%). 1H NMR (500 MHz, CDCl3) δ ppm
2.80 (s, 3H), 4.10 (dt, J = 30.2, 4.2 Hz, 2H), 4.68 (dt, J = 47.4, 4.2 Hz, 2H), 5.13 (s, 1H), 5.92 (d,
J = 2.2 Hz, 1H), 6.18 (dd, J = 8.1, 2.1 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 6.61 (dd, J = 8.4, 2.4
Hz, 1H), 6.72 (d, J = 8.7 Hz, 2H), 7.05 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 8.2 Hz, 1H), 7.52 (d, J =
8.4 Hz, 1H). 13C NMR (500 MHz, CDCl3) δ ppm 37.03, 66.87, 67.03, 67.43, 81.18, 82.54, 99.52,
104.81, 112.98, 114.27, 114.41, 115.09, 122.17, 122.69, 122.95, 128.09, 134.61, 135.60,
144.47, 144.85, 146.47, 157.70. HRMS: Calcd. for C22H23N3OF [M+H]+ 364.1820; Found
364.1831.
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Radiochemistry. The radiosynthesis of [18F]ROStrace was accomplished on an AllꢀinꢀOne
module (Trasis, Belgium) with full automation. Briefly, [18F] fluoride (800 mCi) was produced by
proton irradiation of enriched 18O water via the reaction of 18O (p, n) 18F by the Cyclone
cyclotron (IBA, Belgium). The [18F] fluoride in 18O water solution is delivered to the hotcell and
trapped on a preꢀactivated SepꢀPak® Light QMA Carb cartridge (Waters) and eluted to the
reaction vial by 1 mL eluent containing 2 mg potassium carbonate and 7 mg Kryptofix in a
mixture of 0.85 mL acetonitrile and 0.15 mL water. The residual water was evaporated
azotropically with 1 mL acetonitrile at 100°C under a stream of nitrogen gas and vacuum. A
solution of 4 mg tosylate precursor in 0.6 mL methyl sulfide (DMSO) was added to the reaction
vial for a 10 min reaction, followed by a 10 min deꢀprotection with 1.0 mL 1 N HCl. The reaction
mixture was cooled to 50 °C and neutralized with 0.8 mL 1 N NaOH. The crude product was
diluted with 5 mL mobile phase and passed through an Alumina N Light cartridge
(Waters) and a 0.45 ꢁm nylon filter to the HPLC loop for high performance chromatography
(HPLC). A Phenomenex Luna C18 (2) semiꢀpreparative column (10 X 250 mm) with mobile
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phase acetonitrile/water/TFA (15:85:0.1 in volume) was used for HPLC purification. At a flow
rate of 5 mL/min, the product was eluted at 18 min and diluted with water to 21 mL. The
intermediate (oxidized byproduct) was then eluted with 65% acetonitrile with water. The diluted
product solution was passed through a SepꢀPak® Plus C18 cartridge (Waters). The trapped
product was rinsed with water to waste and then eluted with ethanol (0.6 mL, with 0.1% ascorbic
acid) followed by 8 mL normal saline to the final production vial through a 0.2 ꢁM nylon filter.
After shaking well, the final product is ready for quality control (QC) and animal studies. The
yield ranged from 8% to 20% (decay corrected to the start of synthesis) in an average time of 65
min from receipt of 18Fꢀfluoride in 18O water solution from the cyclotron.
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For the oxidized analog, [18F]oxꢀROStrace, [18F]ROStrace was oxidized by adding 2 mL 25 mM
cupric sulfate aqueous solution. HPLC purification utilized a Sunfire C18 column (Waters, 10 x
250mm) with a mobile phase of 35% acetonitrile in 20 mM ascorbic acid solution. The retention
time is approximately 10 min with a 5 mL/min flow rate. The enrichment and final formulation is
the same as for [18F]ROStrace.
Animal models. All animal studies were performed under protocols approved by the University
of Pennsylvania Institutional Animal Care and Use Committee. Balb/c mice (Charles River) were
injected ip with 5 mg/kg LPS from E coli 0111:B4 (Sigma Aldrich) 24 hours prior to imaging
studies. Mice were maintained on anesthesia of 2% isoflurane and placed on a warm pad for
scanning. Animals were euthanized at the completion of the scanning procedure by cervical
dislocation and brain and blood extracted for HPLC analysis of the identity of the radioactive
species present or autoradiography.
Condition Assessment. The effect of LPS treatment on the animals was evaluated by visual
observation of indicators including hunched posture, decreased motion, piloerection, lack of
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grooming, and discharge from the eyes. This method was developed by combining different
animal condition scoring systems and adapting them for common symptoms of LPS treatment.
Condition scores ranged from 0 (normal) to 4 (moribund) and were obtained by scoring each
animal for the presence of each indicator and whether it was mild or severe (0, 1 or 2). After all
indicators were added together, the possible totals (0 to 10) were subdivided into the final
condition scores, 0 ꢀ 4. No treated animals were scored 0 or 4 in this study. Animals
consistently lost approximately 10% body weight at 24 hrs postꢀLPS treatment, even level 1
animals, so weight was measured but not included as a factor.
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PET imaging. Mouse imaging was performed on the Philips MOSAIC HP small animal PET
scanner, developed in collaboration with Philips Medical Systems. Dynamic images were
acquired up to 60 minutes, after an i.v. injection of 200ꢀ300 ꢁCi [18F]ROStrace or [18F]oxꢀ
ROStrace. Slice thickness was 1/2 mm and FOV 12.8 cm. Data reconstruction was performed
using 3DꢀRAMLA protocol with decay correction turned on and normalization set for efficiency.
Timeꢀactivity curves were constructed by plotting the normalized percent injected dose per cm3
(normalized %ID/cm3) versus time for a regionꢀofꢀinterest encompassing the entire brain. When
making comparisons between control and LPSꢀtreated animals, the average %ID/cm3 tissue for
the 40 – 60 min period postꢀinjection of [18F]ROStrace was used since this represents a plateau
phase in the timeꢀactivity curves (i.e., ∆ %ID/cm3 = 0). Three different regionsꢀofꢀinterests
(whole brain, cerebrum and cerebellum) were used for the data analysis shown in Figures 3 and
4.
Ex vivo autoradiography. Immediately after imaging, mice were injected i.v. with 15 mg/kg
DHE (Sigma Aldrich) and brains harvested 30 minutes later and sectioned on a cryostat
microtome. Sections were exposed to phosphor plates to obtain autoradiographic [18F]ROStrace
and fluorescent DHE images (GE Typhoon).
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Metabolite analysis. Blood samples and brains were collected from mice immediately postꢀ
scan and acetonitrile/PBS (1:1) was added at twice the volume of each sample. The blood
solution was vortexed and brains were homogenized with a pellet pestle, followed by
centrifugation at 3000 g for 10 min. After separating the supernatant from the pellet, each
portion was counted with a gamma counter (Perkin Elmer Wizard 2480) to determine the
extraction efficiency. Part of the supernatant (> 200 ꢁL) was diluted with the same volume of
water and then filtered with a 0.45 ꢀm nylon filter, and 200 ꢁL of the filtered supernatant was
injected into an analytical metabolite HPLC (1200 series, Agilent Technologies) equipped with
an Agilent SBꢀC18 column (5 ꢁm, 25 cm x 4.6 mm) and a fraction collector (Waters). The
gradient mobile phase consisted of acetonitrile/0.1% TFA solution and the flow rate was 1
mL/min. A fraction was collected every 30 seconds and counted with the gamma counter or
directly with the LabLogic Laura PosiꢀRAM radiodetector.
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Statistical Analysis Methods. Nonparametric MannꢀWhitney test was used to compare the
normalized %ID/cm3 difference between control and LPSꢀtreated groups. Multiple group
comparison among control and each condition level group was calculated by Nonparametric
KruskalꢀWallis test with Dunn’s post hoc test. Spearman’s rank correlation was used to calculate
the correlation between normalized %ID/mm3 and condition scale in each regionꢀofꢀinterest. P
value < 0.05 was considered as a statistically significant difference.
Author Contributions
R.H.M., L.L.D. and M.A.M. conceived of the project; C.H. and R.H.M. managed the study.
T.J.G., K.X. and W.C. conducted organic synthesis; S.L. and T.J.G. conducted radiochemistry;
S.L. and H.L. performed analytical QC. S.K.C. and L.L.D. conducted the in vitro ROS assay.
C.H. prepared the animals and performed the imaging studies; CꢀC.W. performed the
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autoradiography. CꢀJ.H. and R.K.D. processed images and performed the PET data analysis.
C.H., CꢀJ.H., S.L., K.X. and R.H.M. wrote the manuscript.
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Funding Sources
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This research was funded by a grant from the Michael J. Fox Foundation (RHM), and NIH grant
AG033679ꢀ05 (LLD).
Conflict of Interest
The radiotracer described in this manuscript is covered under US Patent Number 9,035,057 B2
(RHM, WC, LLD, MAM, RHM, inventors). There are no other conflicts of interest.
ACKNOWLEDGEMENT
We thank Eric Blankemeyer and the University of Pennsylvania Small Animal Imaging Facility
for assistance with microPET imaging.
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J., Capretta, A., and Valliant, J. F. (2017) Preparation of an 18 F-Labeled Hydrocyanine
Dye as a Multimodal Probe for Reactive Oxygen Species, Chemistry (Weinheim an der
Bergstrasse, Germany) 23, 254-258.
[19] Yang, H., Jenni, S., Colovic, M., Merkens, H., Poleschuk, C., Rodrigo, I., Miao, Q., Johnson, B.
F., Rishel, M. J., Sossi, V., Webster, J. M., Benard, F., and Schaffer, P. (2017) 18F-5-
Fluoroaminosuberic Acid as a Potential Tracer to Gauge Oxidative Stress in Breast
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[20] Okamura, T., Okada, M., Kikuchi, T., Wakizaka, H., and Zhang, M. R. (2015) A (1)(1)C-
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[21] Abe, K., Takai, N., Fukumoto, K., Imamoto, N., Tonomura, M., Ito, M., Kanegawa, N., Sakai,
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Table 1
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Polar
[18F]ROStrace
[18F]ox-ROStrace
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Blood
7.0 ± 1.9
10.0 ± 2.5
31.8 ± 7.3
68.3 ± 2.2
27.0 ± 2.3
23.8 ± 8.2
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Figure 1. Structures of Dihydroethidium, [18F]FDMT, and [18F]ROStrace.
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Figure 2. DHE and ROStrace are selectively oxidized by superoxide and hydroxyl radicals, but
not by H2O2. ROStrace was exposed to superoxide (generated by two methods: xanthine
oxidase metabolism of hypoxanthine or by thermal decomposition of SINꢀ1 in the presence of
CPTIO); hydrogen peroxide alone; or in the presence of horseradish peroxidase (HRP) to
generate hydroxyl free radical. Oxidation of ROStrace was prevented by the addition of
superoxide dismutase (SOD).
Figure 3. [18F]ROStrace microPET imaging in control and LPSꢀtreated mice. (A) Representative
control and LPSꢀtreated mouse images 40ꢀ60 min post [18F]ROStrace injection. (B) Time activity
curves demonstrate high initial brain uptake followed by plateau after 40 min. %ID/cm3 were
obtained from 40ꢀ60 min summed images (C) to show differential uptake in control vs. LPSꢀ
treated mice.
Figure 4. Stratification of LPSꢀtreated mice based on condition score suggests that higher brain
uptake of [18F]ROStrace correlates with greater severity of symptoms from LPS treatment.
Figure 5. Demonstration that [18F]oxꢀROStrace does not cross the blood brain barrier. (A)
Mechanism of brain uptake and retention of [18F]ROStrace. (B) microPET images and time
activity curve comparing brain uptake of [18F]ROStrace vs [18F]oxꢀROStrace.
Figure 6. The distribution pattern of [18F]ROStrace is comparable with the fluorescent signal
from DHE or ROStrace. (A) Direct comparison of ex vivo [18F]ROStrace autoradiography with
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DHE fluorescence in the same brain section. Distribution is also comparable with ROStrace
fluorescence performed in separate animals. ARG: autoradiography, DHE: dihydroethidium, CX:
cortex, HP: hippocampus, CB: cerebellum. (B) LPS treated mice showed significantly higher
[18F]ROStrace uptake (* P < 0.0001) compared with nonꢀtreated animals by ex vivo
autoradiography.
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Figure 1
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ACS Chemical Neuroscience
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Scheme 1
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Figure 2
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Superoxide
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ACS Chemical Neuroscience
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Scheme 2
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LPS Treated
Control
Figure 3
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Whole Brain
Cerebrum
Cerebellum
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Figure 4
ACS Chemical NeuLroPsSciternecaeted (2)
LPS treated (1)
LPS treated (3)
Control (0)
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