Synthesis of [18F]PS13 and evaluation as a PET radioligand for cyclooxygenase-1 in monkey

Article abstract of DOI:10.1021/acschemneuro.0c00737

Cyclooxygenase-1 (COX-1) and its isozyme COX-2 are key enzymes in the syntheses of prostanoids. Imaging of COX-1 and COX-2 selective radioligands with positron emission tomography (PET) may clarify how these enzymes are involved in inflammatory conditions

Full text of DOI:10.1021/acschemneuro.0c00737

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Research Article
Synthesis of [18F]PS13 and Evaluation as a PET Radioligand for
Cyclooxygenase‑1 in Monkey
Carlotta Taddei, Cheryl L. Morse, Min-Jeong Kim, Jeih-San Liow, Jose Montero Santamaria,
Andrea Zhang, Lester S. Manly, Paolo Zanotti-Fregonara, Robert L. Gladding, Sami S. Zoghbi,
Robert B. Innis, and Victor W. Pike*
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ABSTRACT: Cyclooxygenase-1 (COX-1) and its isozyme COX-2
are key enzymes in the syntheses of prostanoids. Imaging of COX-
1 and COX-2 selective radioligands with positron emission
tomography (PET) may clarify how these enzymes are involved
in inflammatory conditions and assist in the discovery of improved
anti-inflammatory drugs. We have previously labeled the selective
high-affinity COX-1 ligand, 1,5-bis(4-methoxyphenyl)-3-(2,2,2-
trifluoroethoxy)-1H-1,2,4-triazole (PS13), with carbon-11 (t1/2
=
20.4 min). This radioligand ([11C]PS13) has been successful for
PET imaging of COX-1 in monkey and human brain and in
periphery. [11C]PS13 is being used in clinical investigations.
Alternative labeling of PS13 with fluorine-18 (t1/2 = 109.8 min) is
desirable to provide a longer-lived radioligand in high activity that
might be readily distributed among imaging centers. However, labeling of PS13 in its 1,1,1-trifluoroethoxy group is a radiochemical
challenge. Here we assess two labeling approaches based on nucleophilic addition of cyclotron-produced [18F]fluoride ion to gem-
difluorovinyl precursors, either to label PS13 in one step or to produce [18F]2,2,2-trifluoroethyl p-toluenesulfonate for labeling a
hydroxyl precursor. From the latter two-step approach, we obtained [18F]PS13 ready for intravenous injection in a decay-corrected
radiochemical yield of 7.9% and with a molar activity of up to 7.9 GBq/μmol. PET imaging of monkey brain with [18F]PS13 shows
that this radioligand can specifically image and quantify COX-1 without radiodefluorination but with some radioactivity uptake in
skull, ascribed to red bone marrow. The development of a new procedure for labeling PS13 with fluorine-18 at a higher molar
activity is, however, desirable to suppress occupancy of COX-1 by carrier at baseline.
KEYWORDS: Fluorine-18, PS13, radioligand, nucleophilic addition, PET, COX-1, brain imaging
and for developing anti-inflammatory drugs that have
improved target selectivity and reduced side-effect liability.
Although COX-2 plays the larger role in peripheral
inflammation, COX-1 is a key contributor to neuroinflamma-
tion.3 In healthy brain, COX-1 is almost exclusively localized to
resting microglia. In response to inflammatory stimuli, these
microglia become activated, and COX-1 expression increases.
Consequently, proinflammatory prostanoids arising from
COX-produced progenitor prostaglandins generate oxidative
stress. Ultimately, oxidative stress may lead to cytotoxicity and
neuronal loss.
INTRODUCTION
■
Cyclooxygenase-1 (COX-1) is a prostaglandin-endoperoxide
synthase that is constitutively expressed throughout the body.
COX-1 and its closely related isozyme, COX-2, play important
roles in normal physiology and in disease processes, especially
inflammation.1 These enzymes synthesize prostaglandins from
arachidonic acid in response to diverse stimuli on the path to a
group of important lipidic physiological mediators known as
the prostanoids. COXs are targets for inflammatory drugs,
notably aspirin and ibuprofen, that are widely prescribed for
peripheral diseases, such as rheumatoid arthritis. Chronic
inflammation in brain (neuroinflammation) is now recognized
to be associated with some neuropsychiatric disorders (e.g.,
Alzheimer’s disease, multiple sclerosis, and clinical depres-
sion).2−4 Effective tools for using nuclear medicine to
investigate these diseases and to assist in developing better
drug treatments are constantly sought. Radioligands for
imaging of COX-1 and COX-2 in vivo could be useful for
elucidating their roles in inflammation and neuroinflammation
Received: November 16, 2020
Accepted: January 14, 2021
Published: January 25, 2021
This article not subject to U.S.
Copyright. Published 2021 by American
Chemical Society
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Use of positron emission tomography (PET) with a well-
designed radioligand can quantify the regional distribution of
the binding of the radioligand to a protein target in vivo.
Numerous proteins in brain have been imaged in this manner,
including neurotransmitter receptors, transporters, plaques,
and enzymes.5,6 PET radioligand design is however challeng-
ing.5−10 Several early candidate PET radioligands failed to
image COX-1 in brain.11−13 In many cases, this was due to one
or more factors, such as limited ability to cross the blood-brain
barrier, relatively low COX-1 affinity (IC50 ≥ 20 nM), and high
nonspecific binding. Some success has been achieved in rat
with a prodrug approach in which a potent but poorly brain-
penetrant COX-1 inhibitor [11C]ketoprofen was delivered to
the brain as its methyl ester.13,14 Rapid hydrolysis in situ
generated [11C]ketoprofen for binding to COX-1. Never-
theless, this radiotracer was unsuccessful for imaging COX-1 in
human subjects affected by Alzheimer’s disease or mild
cognitive impairment.15 Moreover, a prodrug approach is not
readily amenable to biomathematical analysis because of the
difficulty of distinguishing hydrolysis from enzyme inhibition.
We recently produced a direct-acting, selective, and high-
affinity COX-1 radioligand, namely [11C]PS13 (Figure 1A).16
Two main approaches have been used for introducing
fluorine-18 into a 1,1,1-trifluoroethoxy group.22−28 Fawaz et
al.26 reported a direct one-step method based on nucleophilic
addition of the [18F]fluoride ion to a functionalized gem-
difluorovinyl precursor in the presence of a proton source, e.g.,
isopropyl alcohol (IPA). This method was used for the
synthesis of [18F]N-methyl-lansoprazole, a high-affinity PET
radioligand for tau protein (Figure 2A). Kramer et al.28
recently used a very similar approach for the synthesis of [18F]
N-methyl-lansoprazole for first-in-human studies. Rafique et
al.27 developed a two-step procedure to produce candidate 18F-
labeled radiotracers for PET imaging of neurofibrillary tangles
(Figure 2B). Their procedure involved [18F]fluoride ion
addition to 2,2-diifluorovinyl tosylate in the presence of a
proton source (e.g., H2O) followed by reaction of the
generated [18F]2,2,2-trifluoroethyl tosylate with a hydroxy
precursor in the presence of cesium carbonate as base. Another
example of this radiochemical approach is that of Riss et al.24
for the synthesis of an 18F-labeled astemizole derivative.
Herein, we report the synthesis of [18F]PS13 through each
of the two nucleophilic addition approaches (Figure 2C).
Reaction variables, such as temperature, reaction time, and
proton source, were investigated with attention to their effects
on decay-corrected radiochemical yield (RCY) and molar
activity (Am). [18F]PS13 was produced and evaluated with
PET imaging in healthy Macaca mulatta monkeys. Baseline and
self-blocking studies in brain and whole-body were performed
showing that [18F]PS13 can be an effective radioligand without
any issue of radiodefluorination or of other radiometabolites
within brain. However, a new method is needed for producing
[18F]PS13 at a much higher molar activity.
Figure 1. Chemical structures showing the labeling site for A
RESULTS AND DISCUSSION
■
[11C]PS13 and B [18F]PS13.
One-Step Labeling Approach. Initially, we investigated a
one-step approach for the synthesis of [18F]PS13, resembling
that taken by Fawaz et al.26 for labeling N-methyl-lansoprazole.
The required gem-difluoroalkene precursor (5) was synthesized
by modifying known methods16,23,26,29,30 (Figure 3). Thus, 2-
(4-methoxyphenyl)hydrazine-1-carboxamide (1) was treated
with 4-methoxybenzoyl chloride to yield 2-(4-methoxybenzo-
yl)-2-(4-methoxyphenyl)hydrazine-1-carboxamide (2). Cycli-
zation of 2 under basic conditions gave 1,5-bis(4-methox-
yphenyl)-1H-1,2,4-triazol-3-ol (3). Alkylation of 3 with 1,1,1-
trifluoro-2-iodoethane yielded 1,5-bis(4-methoxyphenyl)-3-
(2,2,2-trifluoroethoxy)-1H-1,2,4-triazole (4). Defluorination
of 4 by treatment with n-butyllithium gave 3-(2,2-difluor-
ovinyloxy)-1,5-bis(4-methoxyphenyl)-1H-1,2,4-triazole (5).
All steps proceeded in moderate to high chemical yields.
A major issue in the labeling of a 1,1,1-trifluoroethoxy group
with [18F]fluoride ion is the labeling of the gem-difluoroalkene
precursor through overall fluorine isotope exchange, with the
extent of exchange dependent on the type of an added proton
source and its concentration and other reaction condi-
tions.22,23,27 Nucleophilic addition of no-carrier-added
(NCA) [18F]fluoride ion to a gem-difluorovinyl substrate,
1,1-difluoroethene, in acetonitrile in the absence of an added
proton source was reported earlier to produce [1-18F]1,1,1,2-
tetrafluoroethane.31 The [18F]fluoride ion was used as its K+
complex of the cryptand 4,7,13,16,21,24-hexaoxa-1,10-
diazabicyclo[8.8.8]hexacosane (K+-K 2.2.2 complex).
Throughout the current study, we also used the [18F]fluoride
ion as its K+-K 2.2.2 complex. In our attempt to label PS13 by
treating 5 with [18F]fluoride ion in the absence of an added
PET in monkey showed that [11C]PS13 is able to enter the
brain from plasma and to bind avidly to COX-1.17 A large
proportion of [11C]PS13 brain uptake could be preblocked by
intravenous administration of PS13 itself or S-ketoprofen
methyl ester as a source of COX-1 selective ketoprofen but
could not be preblocked by a selective COX-2 ligand, MC-
1.17,18 The COX-1 specific signal in monkey brain is readily
quantifiable without interference from radiometabolites.
[11C]PS13 is similarly effective for imaging and quantifying
constitutive COX-1 in human brain.19 Furthermore,
[11C]PS13 shows some promise for imaging COX-1 in
periphery, including tumors.20 These findings encourage
further application of [11C]PS13 for the study of COX-1
expression in human inflammatory diseases and their drug
treatments.
An 18F-labeled version of PS13 (Figure 1B) could be useful
because the longer half-life of fluorine-18 (109.8 min) versus
that of carbon-11 (20.4 min) would allow distribution of
multiple doses to remote off-site PET imaging facilities that
lack radioisotope production or radiochemistry capability. No-
carrier-added fluorine-18 can be produced up to a very high
activity (>1 TBq) as [18F]fluoride ion by the 18O(p,n)18F on
18O-enriched water.21 Typically, a PET radiotracer dose for
PET imaging in a human participant is only about 700 MBq.
Thus, efficient fluorine-18 chemistry might provide several
doses of radiotracer from a single radiosynthesis. PS13 has a
1,1,1-trifluoroethoxy group (−OCH2CF3) in its structure,
which is a potential site for labeling with fluorine-18.
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Figure 2. Strategies for 18F-labeling of a 1,1,1-trifluoroethoxy group: A, a one-step approach (Fawaz et al.);26 B, a two-step approach (Rafique et
al.);27 and C, radiosynthesis approaches investigated in this work for the synthesis of [18F]PS13.
Figure 3. One-step approach for the synthesis of [18F]PS13 including precursor 5 synthesis. Reagents and conditions: (i) 4-methoxybenzoyl
chloride, toluene, pyrimidine, 110 °C, 2.5 h; (ii) KOH (10%, aq.), EtOH, 60 °C, 1.5 h; (iii) 1. K2CO3, DMF, RT, 10 min and 2. 1,1,1-trifluoro-2-
iodoethane, 100 °C, 3 h; (iv) n-BuLi (1.6 M in hexanes), THF, −78 °C, 45 min; (v) [18F]F−, K 2.2.2, K2CO3, DMSO, proton source, thermal
heating, 10−20 min.
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Figure 4. Representation of the 19F/18F addition−elimination process for 5 in the absence of an added proton source.
Table 1. Conditions Screening for the Synthesis of [18F]PS13 via the One-Step Approach
c
d
time
(min)
proton source
yield ratio of [18F]PS13 to
yield of [18F]PS13
(%)
Am of [18F]PS13
(GBq/μmol)
a
b
c
entry
1
2
temp (°C)
proton source
(μL)
[18F]5
90
90
20
20
20
20
20
2
20
20
20
20
2
IPA
IPA
tert-BuOH
IPA
IPA
(NH4)2CO3(aq.)
NH4Cl(aq.)
NH4Cl(aq.)
NH4Cl(aq.)
NH4Cl(aq.)
NH4Cl(aq.)
IPA
10.8
21.6
21.6
21.6
21.6
1.6
1.6
3.2
6.4
16
1:5
1:2.5
1:5
1:2
1:2.5
1:5
1:0.9
1:0.4
1:0.4
0:0
e
3
4
5
6
90
130
130 (MW)
130
130
130
130
130
130 (MW)
130
f
7
8
0.8
3.3
0.8
0
14.2
16.8
8.6
e
9
10
11
12
13
1.6
21.6
21.6
1:10
g
20
10
5.9
4.5
0.6
1.0
g
130
IPA
a
b
c
Reactions (n = 1) were performed with 5 (1.5 mg) in DMSO (300 μL), unless otherwise noted. Volume per reaction. Yield ratios and yields
d
e
f
were determined from radiochromatograms acquired during HPLC analysis of the crude reaction mixture. Decay-corrected to ERP. n = 2. n = 4.
g
Reaction used 3 mg of precursor 5.
proton source (Figure 4), unwanted [18F]5 was formed in
strong preference over [18F]PS13. Fawaz et al.,26 in their study
of nucleophilic addition of [18F]fluoride ion to a gem-
difluorovinyl substrate, improved the ratio of the 18F-
trifluoromethylated product to the 18F-labeled vinyl product
and the overall yield by, for example, adding IPA as a proton
source. With the aim of improving the production of
[18F]PS13 over [18F]5, we explored different proton sources
and reaction conditions.
Initially, reaction temperature and the use of IPA or tert-
butanol as a proton source were studied (Table 1, entries 1−
5). [18F]PS13 and [18F]5 were obtained in a 1:5 ratio of
nonisolated yields when precursor 5 (1.5 mg) was treated with
[18F]fluoride ion in the presence of 10.8 μL of IPA in DMSO
(300 μL) at 90 °C for 20 min (entry 1). By doubling the
amount of IPA to 21.6 μL, the ratio of [18F]PS13 to [18F]5
doubled to 1:2.5 (entry 2). tert-Butanol gave no advantage over
IPA as a proton source (entry 3). The ratio of [18F]PS13 to
[18F]5 increased to 1:2 when the temperature was increased to
130 °C in the presence of IPA (21.6 μL) (entry 4). Use of
microwave (MW) heating instead of thermal heating gave no
improvement in the [18F]PS13 yield (entry 5). Therefore, the
best conditions for producing [18F]PS13 using IPA as the
proton source were those in entry 4.
μL) at 130 °C for 20 min (entry 6). When NH4Cl(aq.) was
used, the ratio of [18F]PS13 to [18F]5 increased to 1:0.9 (entry
7). [18F]PS13 was obtained as the major 18F-labeled product
by doubling the amount of NH4Cl(aq.) to 3.2 μL per reaction
(entry 8). By increasing NH4Cl(aq.) to 6.4 μL, the ratio of
[18F]PS13 to [18F]5 increased further to 1:0.4 (entry 9).
Nonetheless, another increase of NH4Cl(aq.) to 16 μL gave no
18F-labeled products (entry 10). This was likely due to severe
quenching of the nucleophilicity of the [18F]fluoride ion
through a high degree of aqueous solvation.32,33 Microwave
heating gave [18F]5 as the major labeled product (entry 11)
and was not investigated further. Therefore, treatment of 5
(1.5 mg) in the presence of 3.2 or 6.4 μL of NH4Cl(aq.) in
DMSO (300 μL) at 130 °C for 20 min (entries 8 and 9) was
best for selective formation of [18F]PS13.
In summary, the use of IPA produces predominantly [18F]5
over [18F]PS13 (entries 1, 2, 4, and 5), whereas NH4Cl(aq.)
favors the formation of [18F]PS13 over [18F]5 (entries 7−9).
The absence of water when using IPA favors fluorine isotope
exchange by addition−elimination and causes [18F]5 to prevail
over [18F]PS13. Although the water in NH4Cl(aq.) is expected
to suppress the nucleophilicity of the free fluoride ion
(radioactive and nonradioactive) leading to a slower addition
reaction, the ready availability of protons favors formation of
[18F]PS13 over elimination of the fluoride ion (Figure 3).
Despite dominant formation of [18F]PS13 over [18F]5 when
using NH4Cl(aq.) as a proton source, [18F]PS13 was obtained in
only low nonisolated yields in the 0.8−3.3% range and with
molar activities in the 8.6−16.8 GBq/μmol range (Table 1,
entries 7−9). Low yields in fluorine-18 chemistry can still be
Alternative proton sources, such as a saturated aqueous
solution of ammonium carbonate [(NH4)2CO3(aq.)] or of
ammonium chloride [NH4Cl(aq.)], were also investigated
(entries 6−11). [18F]PS13 was formed in a low 1:5 ratio to
[18F]5 when 5 (1.5 mg) was treated with the [18F]fluoride ion
in the presence of 1.6 μL of (NH4)2CO3(aq.) in DMSO (300
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Figure 5. Two-step approach for the synthesis of [18F]PS13.
useful because of the very high activities of [18F]fluoride that
can be produced. However, radioligands that are intended to
bind to low density binding sites with high affinity usually need
to be produced with high molar activity to avoid excessive
target occupancy by carrier in vivo and to avoid consequent
violation of the radiotracer principle. In our laboratory, the
molar activities of 18F-labeled radiotracers typically exceed 75
GBq/μmol at the end of the synthesis, as recently reported for
the synthesis of [18F]LSN3316612, a radioligand for imaging
O-linked-β-N-acetyl-glucosamine hydrolase in human brain.34
The highest molar activity for [18F]PS13 (16.8 GBq/μmol,
corrected to the end of the radionuclide production (ERP))
was obtained when using 3.2 μL of NH4Cl(aq.) per reaction
(entry 8). In this case, [18F]PS13 was obtained in only 3.3%
yield. Again, this suggests that the water present in the
NH4Cl(aq.) suppresses the nucleophilicity of the free [19F]/
[18F]fluoride ion.
In a reaction performed with 5 (3 mg) and IPA (21.6 μL) in
DMSO (300 μL) at 130 °C for 20 min, [18F]PS13 was
obtained in a nonisolated yield of 5.9% with a molar activity of
0.6 GBq/μmol (decay-corrected to ERP) (entry 12). A shorter
reaction time of 10 min gave [18F]PS13 in a lower nonisolated
yield of 4.5% but with a higher molar activity of 1 GBq/μmol
(entry 13). This indicates that fluorine isotope exchange
increases over time, thereby reducing the molar activity. In
summary, with this one-step approach, the molar activity of
[18F]PS13 was much lower, and the yield was only slightly
higher with IPA than with NH4Cl(aq.) as a proton source.
Two-Step Approach. With the aim of increasing the yield
of [18F]PS13 and its molar activity, we investigated a two-step
strategy (Figure 5) resembling that reported by Rafique et al.27
(Figure 2B).
Table 2. Time Study for Step 1 (Two-Step Approach) with
IPA as a Proton Source
a
b
c
entry
time (min)
[18F]6 yield (%)
Am of [18F]6 (GBq/μmol)
1
2
3
4
1
2.5
5
54
56
61
63
1.3
0.8
0.4
0.3
10
a
Reactions (n = 1) were performed with 2,2-difluorovinyl 4-
methylbenzenesulfonate (2.5 mg) and IPA (38 μL) in DMSO (500
b
μL) at 85 °C. Yields were determined from radiochromatograms
c
acquired during HPLC analysis of the crude reaction mixture. Decay-
corrected to ERP.
aimed at optimizing temperature, type of proton source, and
molar activity.
Temperature was studied in single reactions performed in
DMSO (100 μL) with 0.5 mg of 2,2-difluorovinyl 4-
methylbenzenesulfonate and IPA (7.6 μL) for 1 min. At 45,
85, and 130 °C, the yields of [18F]6 were 9.4, 22, and 6.6%,
respectively. Therefore, 85 °C was used for subsequent
reaction optimization. Saturated NH4Cl(aq.) (1.1 μL) was
compared with IPA (7.6 μL) as a proton source in triplicate
reactions using 0.5 mg of the precursor at 85 °C for 1 min in
DMSO (100 μL). [18F]6 was obtained in moderate yields (59
4%) but with a lower molar activity (1.7 0.5 GBq/μmol)
when using IPA as a proton source. With NH4Cl(aq.) (1.1 μL)
as a proton source, [18F]6 was obtained in much lower yield
(14 5%) but with higher molar activity (22 1 GBq/μmol).
Therefore, NH4Cl(aq.) was selected as the preferred proton
source for obtaining [18F]6 in a moderately useful yield but
with optimal molar activity.
Step 2 Optimization. We proceeded to explore the second
step required for the synthesis of [18F]PS13. First, we studied
the reaction of [18F]6 with the alcohol precursor 3 (3 mg, 1
equiv) in DMF (300 μL) with potassium carbonate (11.6 mg,
8.4 equiv) as a base at 130 °C for 20 min (Figure 5).
Conditions like those in the synthesis of reference PS13
(Figure 3, step (iii)) were applied. [18F]PS13 was obtained in a
nonisolated yield of 30 2.4% from [18F]fluoride ion and with
a molar activity of 2.8 0.5 GBq/μmol (n = 3) when IPA (7.6
μL) was used as the proton source in Step 1. A lower
[18F]PS13 yield of 12 1.4% and only a comparable molar
Step 1 Optimization. Initially we focused on optimizing the
addition of the [18F]fluoride ion to 2,2-difluorovinyl 4-
methylbenzenesulfonate to give [18F]2,2,2-trifluoroethyl tosy-
late ([18F]6) as a labeling synthon (Figure 5).
The reaction time with IPA as a proton source was
investigated (Table 2). For reactions between 1 and 10 min,
yields of [18F]6 increased with time over a narrow moderate
range (54−63%; entries 1−4). However, the molar activity
decreased rapidly with time from 1.3 GBq/μmol after 1 min
(entry 1) to 0.3 GBq/μmol after 10 min (entry 4). Therefore,
a 1-min reaction time was selected for subsequent experiments
activity of 3.2
2 GBq/μmol were achieved by using
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Figure 6. PET studies of brain in monkey A with [18F]PS13 (carrier PS13:6.6 nmol/kg at baseline). Panel A: regional VT images from baseline scan
(top row) and blocked scans (with PS13, 0.3 mg/kg, i.v.) (middle row), and corresponding MRI images (bottom row). Left column: coronal
images; middle column: sagittal images; right column: horizontal images. Highest radioactivity uptake occurred in the prefrontal and parietal
cortices. Panel B: whole brain time-activity curves at baseline and after preblock obtained with [18F]PS13. Corresponding curves obtained in a
different monkey with [11C]PS13 from the study in ref 17 with a much lower PS13 carrier dose (0.19 nmol/kg at baseline) are shown for
comparison.
NH4Cl(aq.) (1.1 μL) as the proton source in Step 1. It should
be noted that these results were obtained using a low starting
radioactivity amount (≤1.2 GBq) and that syntheses were
interrupted between Step 1 and Step 2 to withdraw an aliquot
for HPLC analysis of the Step 1 outcome. This may be a
reason for the observed molar activity to fall from Step 1 to
Step 2.
target COX-1, and therefore of a diminished COX-1 specific
PET signal. Nonetheless, we performed experiments in
monkey to characterize [18F]PS13 as a PET radioligand. One
reason was to assess whether further radiochemical research
would be warranted to find a method of labeling that could
deliver adequate yield with a reliably higher molar activity. The
spectrum of radiometabolites that is generated from a PET
radioligand depends on the molecular position of the
radiolabel.35 One position of the radiolabel may give
troublesome radiometabolites, whereas another position may
not. We especially wished to know whether [18F]PS13 is
extensively defluorinated in monkey in vivo, because avid bone
uptake of the [18F]fluoride ion in skull can be problematic for
quantification of radioligand binding in nearby brain regions.
We performed baseline and self-blocking PET experiments
on brain in two monkeys. In many respects, results in the two
monkeys were quite comparable and are exemplified here for
monkey A (see the Supporting Information for imaging results
from monkey B). In the baseline experiment, [18F]PS13
binding was highest in the prefrontal and parietal cortices
(Figure 6A). The whole brain time-activity curve at baseline
peaked early at 3.79 SUV and then declined smoothly to the
end of the scan (Figure 6B). In the preblock experiment, peak
radioactivity uptake was similar, but subsequent radioactivity
decline was faster, indicating the presence of some COX-1
specific binding in the baseline experiment. These time-activity
curves are compared with those of [11C]PS13 of higher molar
activity (∼105 GBq/μmol) for another monkey, as published
previously.17 The baseline whole brain radioactivity curve for
[18F]PS13 is very similar to that for [11C]PS13. An intravenous
PS13 dose of 0.3 mg/kg was used in the preblock experiment
with [18F]PS13. In the experiment with [11C]PS13, the
preblocking PS13 dose was 1.0 mg/kg, i.v., and gave a
somewhat faster decline in radioactivity after peak but down to
a similar level to that for [18F]PS13 at 90 min.
With the aim to obtain [18F]PS13 in useful activity yield
with the highest achievable molar activity, use of NH4Cl(aq.) for
Step 1 was chosen for producing [18F]PS13 for PET
experiments in monkey.
The use of potassium carbonate as a base in Step 2 posed
problems in scale-up of the process to a higher starting activity.
After Step 1, the reagents for Step 2 were added together to the
reaction vial through a needle controlled with an automated
robotic arm (see Methods section). The reagents had to be in
an almost homogeneous solution to allow smooth addition
without compromising the apparatus function through the
robot needle blockade. Ammonium carbonate [(NH4)2CO3]
has higher solubility in organic solvents (e.g., DMF) than
potassium carbonate. Therefore, ammonium carbonate was
tested as a base for Step 2. [18F]PS13 was obtained in a higher
nonisolated yield (17%) when using (NH4)2CO3 rather than
K2CO3 (12 1.4%, n = 3). We therefore selected (NH4)2CO3
as a base for Step 2 in scale-up of the synthesis of [18F]PS13
for PET imaging experiments in monkey. Separation of
[18F]PS13 was achieved by reversed phase HPLC (Supporting
Information, Figure S1). Radiochemically pure [18F]PS13 was
reproducibly obtained (Supporting Information, Figure S2)
and formulated ready for intravenous injection in an isolated
decay-corrected yield of 7.9
2.7% from the starting
[18F]fluoride ion with a molar activity of 7.9
μmol (n = 6).
2.2 GBq/
Stability of Formulated [18F]PS13. The radiochemical
purity of formulated [18F]PS13 kept at RT for 1 and 4 h was
analyzed with radio-HPLC. [18F]PS13 maintained a radio-
chemical purity of greater than 98% with no evidence of
radiodefluorination.
In our study of monkey A, the dose of [18F]PS13 was 23.6
MBq/kg, and the carrier dose was 6.6 nmol/kg. 18F-Labeled
ligands are typically administered at lower doses of radio-
activity per kg in humans than in nonhuman primates for
imaging low density targets in brain. For example, in the study
of [18F]LSN3316612, the radioactive dose was, on average,
about 190 MBq in subjects that on average weighed 75 kg,
equivalent to an average dose of 2.53 MBq/kg.34 Thus, by
PET Experiments in Monkey. In this study, [18F]PS13
was only obtainable for intravenous injection with a moderate
molar activity. This implies that a dose administered to
monkey in a PET experiment would have a moderately high
amount of carrier, with a risk of appreciable occupancy of the
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Figure 7. Plasma clearance and metabolism of [18F]PS13 in monkey A. Panel A: reversed phase HPLC analysis of radiometabolites in plasma at
180 min after intravenous radioligand injection at baseline. Panel B: time-course of the percentage of radioactivity in plasma represented by
unchanged [18F]PS13 under baseline and preblocked conditions. Panel C: time-course for unchanged [18F]PS13 in plasma under baseline and
preblocked conditions.
Figure 8. Analysis of PET data from monkey A under baseline and preblock conditions. Panel A: regional VT values. Panel B: regional VT values
adjusted for normalized plasma free fraction (Supporting Information, Table S1). Panel C: VT values determined from PET data for different
durations of scan data from time of radioligand injection, normalized to the value at the end of scanning (180 min). Panel D: Lassen plot showing
VND as the X-axis intercept and occupancy (slope of plot) of the available COX-1 (that not already occupied by carrier at baseline) by the blocking
agent PS13 (0.3 mg/kg, i.v.).
using a similar radioactive dose of [18F]PS13 in humans, the
dose of carrier (in nmol/kg) might be reduced almost 10-fold,
to about 0.66 nmol/kg, and the possible issue of enzyme
occupancy at baseline might be reduced. By comparison, the
typical dose of carrier in our human experiments with
[11C]PS13 has been at 0.11 0.06 nmol/kg.19
the radioactivity in plasma (∼20 min) was similar to that for
[11C]PS13 (28 min).17 The time-courses for unchanged
radioactivity and [18F]PS13 in plasma in the baseline and
preblock experiments were similar (Figure 7B and Figure 7C).
Plasma free fractions (f P) for [18F]PS13 in these PET
experiments were low (1.14−2.79%) but accurately measurable
(Supporting Information, Table S1).
In these experiments with [18F]PS13, radioactivity cleared
rapidly from plasma. Several radiometabolites emerged in
plasma that were less hydrophobic than [18F]PS13, as judged
by their faster elution in reversed phase HPLC analysis (Figure
7A). Negligible radioactivity eluted at the solvent front,
indicating the absence of [18F]fluoride ion as a radio-
metabolite. The time for radiometabolites to reach 50% of
Brain time-activity curves from [18F]PS13 were well fitted
with the two-tissue compartment model and gave regional total
distribution volumes (VT’s) (Figure 8A) and their adjustments
for plasma free fractions (VT/f P’s) (Figure 8B). These were
consistent with those obtained in our previous study with
[11C]PS13.17 VT ranged from 4.47 in the limbic region to 8.03
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in the frontal cortex at baseline. In the preblock experiment,
they decreased between 25 and 55% across brain regions, as
did VT/fP values. Moreover, at baseline whole brain and
regional VT values rapidly reached stable values with respect to
the duration of PET data used in their determination (Figure
8C), indicating that radioactivity within brain was not
contaminated by radiometabolites. The occupancy of COX-1
achieved with PS13 as a preblocking agent may be estimated
graphically with a Lassen plot where the X-axis is VT at baseline
and the Y-axis is the decrease in VT from baseline for several
brain regions under the preblock conditions. Major assump-
tions of the Lassen plot are that the radioligand and blocking
agent bind selectively to the target, in this case COX-1, and
that target occupancy and nonspecific binding are uniform
across brain. The Lassen plot for monkey A showed the
occupancy of COX-1 (slope of curve) by the 0.3 mg/kg
intravenous dose of PS13 to be 83% and the nondisplaceable
volume of distribution, VND (X-axis intercept), to be 2.70 mL/
cm3. By comparison, in the study17 of [11C]PS13 using a
higher intravenous blocking dose of 1.0 mg/kg, occupancy was
87 4%, based on the slopes of Lassen plots, and VND was 2.5
mL/cm3. The strong correlation coefficient (Figure 8D)
indicates that the major assumptions of the Lassen plots
were well satisfied for [18F]PS13, as they were for [11C]PS13.17
The study in monkey B gave results comparable to those in
monkey A with respect to radioligand brain uptake and
distribution (Supporting Information, Figure S3) and the
emergence of radiometabolites in plasma at baseline
(Supporting Information, Figure S4A) and under preblock
conditions (Supporting Information, Figures S4B and 4C).
However, in this monkey, much less reduction of total binding
(VT) was achieved in the preblocking experiment with the
same preblocking regimen used for monkey A. Reductions in
VT were absent in cerebellum, limbic region, thalamus, and
striatum. Only modest reductions in VT (≤35%) were seen for
whole brain and frontal, parietal, temporal, and occipital
cortices (Supporting Information, Figure S5A). These regional
reductions in VT were much lower than in a control experiment
in the same monkey with [11C]PS13 of much higher molar
activity (221 GBq/μmol) and much lower carrier dose (0.1
nmol/kg) when administered with the same blocking dose (0.3
mg/kg) at 15 to 5 min before the scan (Supporting
Information, Table S2). Reduction in VT for whole brain was
2-fold higher at 44% for [11C]PS13 and 22% for [18F]PS13
(Supporting Information, Table S2). These data suggest that
occupancy of COX-1 at baseline was already substantial in the
experiment with [18F]PS13 (although the carrier dose was
lower than in the baseline experiment for monkey A).
Nonetheless, as for monkey A, VT from [18F]PS13 in monkey
B showed stability with respect to the duration of PET data
needed for its calculation (Supporting Information, Figure
S5B).
Figure 9. Summed (0−180 min) whole-body MIP PET image
obtained with [18F]PS13 in monkey.
followed the areas of red marrow. The molecular identity of
this radioactivity uptake however remains unknown. The
distribution of [18F]PS13 uptake in major organs was quite
consistent with that previously reported for [11C]PS1318, as
well as with constitutive COX-1 expression measured in post-
mortem human studies.36,37
In view of the unusually low COX-1 specific signal found
with PET in the brain of monkey B, an improved procedure or
method for synthesizing [18F]PS13 at an appreciably higher
molar activity is needed to avoid concerns over COX-1
occupancy at baseline. Certain strategies might be applied with
the current radiochemistry to improve molar activity. One
strategy would be to perform the radiochemistry with a very
high amount of the cyclotron-produced [18F]fluoride ion. In
our setting, we usually start with a relatively low radioactivity
(∼10 GBq) having a moderate molar activity (75−200 GBq/
μmol), but other facilities are capable of producing and using a
much higher activity of the [18F]fluoride ion in a safe manner.
Generally, the amount of the carrier fluoride ion accompanying
a cyclotron production of [18F]fluoride ion does not increase
greatly with the activity produced. Hence, exceptionally high
molar activity can be achieved under high-level radioactivity
production conditions. For example, there are reports of 18F-
labeled products having molar activities at the end of synthesis
of >740 GBq/μmol from 185 GBq of [18F]fluoride ion38,39 and
an unusually high molar activity of 4.7−5.9 TBq/μmol from
220 GBq of [18F]fluoride ion.40 The dominant source of carrier
in our method for producing [18F]PS13 is from the release of
the fluoride ion from the gem-difluorovinyl precursor during
the labeling reaction, and this quantity of carrier will be a quite
constant quantity. Therefore, the amount of carrier in a
labeling reaction will be relatively fixed, and the molar activity
should increase with starting radioactivity. This approach could
be successful for producing [18F]PS13 as an efficacious PET
radioligand in high activity, as has been achieved for other
radiotracers. Technical improvement, such as performing the
radiolabeling in a low microvolume with a smaller amount of
the precursor, might also foster higher molar activity.41
In both baseline experiments and in one self-blocking study
(monkey B), some radioactivity uptake in skull was observed
(Supporting Information, Figure S6). At first, this was
suspected to be due to radiodefluorination of [18F]PS13.
However, a summed whole body maximum-intensity projec-
tion (MIP) PET image of monkey at baseline showed
radioactivity uptake in the areas of red marrow, such as
vertebrae, pelvic bones, skull, and the proximal parts of the
humeri, but not in rapidly metabolizing peripheral bone
(Figure 9). This indicated that no radiodefluorination of
[18F]PS13 had occurred and that the distribution of uptake
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similar literature procedure.16 2-(4-Methoxyphenyl)hydrazine-1-car-
boxamide (1) (2 g, 11.04 mmol, 1 equiv) was placed in an oven-dried
argon-flushed round-bottomed flask (100 mL) with anhydrous
toluene (22 mL). Pyrimidine (1.12 mL, 13.81 mmol, 1.25 equiv)
and a solution of 4-methoxybenzoyl chloride (2.36 g, 13.81 mmol,
1.25 equiv) in anhydrous toluene (11 mL) were added slowly to the
flask under an argon atmosphere (balloon) and then refluxed (∼110
°C) for 1.5 h with magnetic stirring. The solution was cooled, poured
into a conical flask containing a mixture of EtOAc−THF (9:1 v/v;
450:50 mL) and water (100 mL), and left under vigorous magnetic
stirring for 2 h. The resultant precipitate was filtered off to give 2-(4-
methoxybenzoyl)-2-(4-methoxyphenyl)hydrazine-1-carboxamide (2)
(2.19 g, 6.96 mmol) in 63% yield. 1H NMR and LC-MS (ESI)
analyses of 2 agree with literature values;16,30 1H NMR (d6-DMSO): δ
8.87 (br, 1H), 7.49 (br, 2H), 7.28 (d, 2H), 6.89 (m, 4H), 3.77 (s,
3H), 3.73 (s, 3H); LC-MS(ESI): m/z = 316.2 [M]+. Compound 2
was used without further purification for the synthesis of 3, as follows.
Precursor 2 (2 g, 6.34 mmol, 1 equiv) was added to a solution of
KOH(aq.) (10% w/v; 17 mL) and ethanol (8.5 mL) in a round-
bottomed flask (100 mL). This mixture was heated to 60 °C and left
for 1.5 h under an argon atmosphere (balloon) with magnetic stirring.
The solvent was then removed by rotary evaporation. Cold water (5
mL) was added, and the pH of the mixture was adjusted to 2 with 1
M HCl (∼25 mL) under magnetic stirring. The whitish precipitate
was filtered off, washed with cold water (3 × 30 mL), and desiccated
to give 3 (1.62 g, 5.44 mmol) in 86% yield. Compound 3 was stored
under desiccation until future use. 1H NMR and LC-MS(ESI)
analyses of 3 agree with literature values:16,30 1H NMR (d6-DMSO): δ
11.25 (br, 1H), 7.33 (d, 2H), 7.29 (d, 2H), 7.02 (d, 2H), 6.94 (d,
2H), 3.77 (s, 3H), 3.73 (s, 3H); LC-MS(ESI): m/z = 298.2 [M]+.
(2,2-Difluorovinyloxy)-1,5-bis(4-methoxyphenyl)-1H-1,2,4-tria-
zole (5). This compound was prepared according to similar literature
procedures.16,22,26 The alcohol precursor 3 (400 mg, 1.34 mmol, 1
equiv) was placed in an oven-dried argon-flushed round-bottomed
flask (25 mL) with anhydrous DMF (4 mL) and K2CO3 (929 mg,
6.72 mmol, 5 equiv). This mixture was stirred for 10 min at RT. Then
1,1,1-trifluoro-2-iodoethane (663 μL, 6.72 mmol, 5 equiv) was added
slowly under an argon atmosphere (balloon). The mixture was heated
to 100 °C, left for 3 h under magnetic stirring, and then cooled.
EtOAc (140 mL) and water (30 mL) were then poured into the
reaction flask. The organic phase was separated off and washed with
water (1 × 60 mL) and brine (1 × 60 mL). The organic layers were
collected and dried (MgSO4). The solvent was removed by rotary
evaporation. Silica gel flash chromatography (hexanes/EtOAc) of the
crude product gave 1,5-bis(4-methoxyphenyl)-3-(2,2,2-trifluoroe-
CONCLUSIONS
■
Of the two approaches explored for labeling PS13 with
fluorine-18, the two-step approach gave the best compromise
between overall radiochemical yield and molar activity. Despite
the low molar activity obtained in producing [18F]PS13 for
evaluation with PET in monkey, this radioligand gave a sizable
COX-1 specific signal in the brain of one monkey with time-
stable VT values, indicating absence of radiometabolites. Some
low radioactivity uptake was seen in skull in some instances but
not due to radiodefluorination. Brain regional VT values and
VND were comparable with those previously measured in
monkey with [11C]PS13. Nonetheless, an improved procedure
or method for labeling PS13 with the NCA [18F]fluoride ion
remains desirable to ensure that the molar activity can be
reliably higher to avoid any risk of unacceptable occupancy of
COX-1 at baseline. Radioactivity uptake in red marrow of skull
may remain an issue for quantitative PET imaging in human
subjects, especially for quantification of radioligand uptake in
regions near skull.
METHODS
■
General Materials and Methods. Water from a purification
apparatus (Milli-Q; Waters Corp; Columbia, MD) was used in
syntheses and radiosyntheses, unless otherwise stated. Other solvents
and chemicals were purchased from Aldrich Chemical Co.
(Milwaukee, WI), Acros Organics BVBA (Geel, Belgium), and
Enamine Ltd. (Kiev, Ukraine) and used as received. [11C]PS13 for a
control experiment in monkey B (see the Supporting Information)
was produced as described previously.17
1H- (400 MHz), 13C- (100 MHz), and 19F-NMR (376.49 MHz)
spectra were recorded at RT on an Avance-400 spectrometer (Bruker;
1
Billerica, MA). H and 13C chemical shifts are reported in δ units
(ppm) downfield relative to the chemical shift for tetramethylsilane
and 19F chemical shifts relative to that for CFCl3. Abbreviations br, s,
d, t, and m denote broad, singlet, doublet, triplet, and multiplet,
respectively. LC-MS for compound characterization was performed
on an LCQ Deca instrument (Thermo Fisher Scientific Corp.;
Waltham, MA) equipped with a Synergi Fusion-RP column (4 μm,
150 × 2 mm; Phenomenex; Torrance, CA). Flash chromatography
was performed on a semiautomated apparatus (CombiFlash Rf + UV;
Teledyne ISCO Inc.; Lincoln, NE).
γ-Radioactivity from 18F was measured with a calibrated dose
calibrator (Atomlab 300; Biodex Medical Systems, USA) or for low
levels (<40 kBq) with a well-type γ-counter (model 1080 Wizard;
PerkinElmer; Boston, MA) having an electronic window set between
360 and 1,800 keV. 18F Radioactivity measurements were corrected
for background and physical decay. All radiochemistry with fluorine-
18 was performed in lead-shielded hot-cells for personnel radiation
protection.
1
thoxy)-1H-1,2,4-triazole (4) (366 mg, 0.96 mmol) in 72% yield. H
NMR, 13C NMR, 19F NMR, and LC-MS(ESI) analyses of 4 agree
with literature values:16 1H NMR (CDCl3): δ 7.36−7.34 (d, 2H),
7.19−7.18 (d, 2H), 6.87−6.85 (d, 2H), 6.77−6.74 (d, 2H), 4.70−
4.64 (q, 2H), 3.78−3.73 (d, 6H); 13C NMR (CDCl3): δ 166.22,
161.03, 159.87, 153.46, 131.09, 130.30, 127.07, 124.36, 121.61,
119.66, 114.64, 113.98, 66.27−65.16, 55.59−55.34; 19F NMR
(CDCl3), δ 74.2; LC-MS(ESI): m/z = 380.1 [M]+.
A semiautomated apparatus (Synthia)42 was used for all fluorine-18
radiochemistry. Dedicated recipes were created in Autorad software
and followed step by step for the syntheses of [18F]PS13. The HPLC
apparatus for [18F]PS13 separation comprised a pump (P4.1S;
Knauer; Berlin, Germany), a UV absorbance detector (UVD2.1S;
Knauer), and a radioactivity detector (flow-count; Eckert & Ziegler;
Berlin, Germany). Clarity Chromatography Station software (Data-
Apex; Prague, Czech Republic) was used to record the chromato-
grams. Radio-HPLC equipment for analyses comprised a pump
(DGU-20A3R; Shimadzu; Columbia, MD), a UV absorbance
detector (CBM-20A; Shimadzu), and a radioactivity detector (flow-
count; Eckert & Ziegler).
All animals used in this study were handled in accordance with the
Guide for the Care and Use of Laboratory Animals43 and the National
Institute of Mental Health Animal Care and Use Committee.
Results of statistical analyses are presented as mean SD.
Syntheses. 1,5-Bis(4-methoxyphenyl)-1H-1,2,4-triazol-3-ol (3).
The alcohol precursor 3 was prepared in two steps according to a
Compound 4 (200 mg, 0.53 mmol, 1 equiv) was placed in an oven-
dried argon-flushed round-bottomed flask (25 mL) with anhydrous
THF (2 mL). The flask was placed in a dry-ice/acetone cooling bath
(∼−78 °C). Then n-BuLi (450 μL, 1.1 mmol, 2.1 equiv) was added
dropwise under an argon atmosphere (balloon) with magnetic
stirring. The reaction was left at −78 °C under magnetic stirring
for 45 min. Then the reaction was quenched with water−THF (1:1 v/
v; 5 mL) and left to warm to RT. The organic phase was extracted
from the aqueous phase with EtOAc (2 × 20 mL). The combined
organic layers were washed with brine (1 × 30 mL), dried (MgSO4),
and concentrated under vacuum. The crude product was purified with
silica gel flash column chromatography (hexanes/EtOAc) to give 5
1
(106 mg, 0.30 mmol) in 56% yield. H NMR (CDCl3): δ 7.46−7.44
(d, 2H), 7.31−7.28 (d, 2H), 6.97−6.94 (d, 2H), 6.86−6.84 (d, 2H),
6.80−6.75 (q, 1H), 3.87−3.83 (d, 3H); 13C NMR (CDCl3): δ 130.30,
127.47, 114.56, 113.90, 105.47−105.02, 55.37, 55.13; 19F NMR
(CDCl3): δ 96.04, 116.94; LC-MS(ESI): m/z = 360.1 [M]+.
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Production of [18F]Fluoride Ion. The NCA [18F]fluoride ion was
produced with the 18O(p,n)18F reaction by irradiation of [18O]water
(95 atom %; 1.8 mL) with protons (14.1 MeV, 20−25 μA) generated
with a PETtrace cyclotron (GE; Milwaukee, WI). Labeling experi-
ments for radiochemistry optimization started with about 0.2−1.2
GBq of [18F]fluoride ion, whereas radiotracer productions for monkey
studies started with about 10−15 GBq.
The remaining stock solution for Step 1 was stored at −20 °C up to
one month.
For Step 2, the alcohol precursor 3 (2.5−3 mg, 8.41−10.09 μmol, 1
equiv), anhydrous DMF (300 μL), and (NH4)2CO3 (12.5−13 mg,
130−135 mmol, 15 equiv) were placed in an oven-dried tapered
crimp-capped V-vial (0.9 mL; Thermo Scientific, Waltham, MA). This
vial was sealed with a PTFE-silicone septum and placed in the Synthia
apparatus.
An empty oven-dried 5 mL V-vial (part # 95050; Alltech) sealed
with a screw-cap (Alltech Associates Inc.) and PTFE-silicone septum
was placed in the oven of the Synthia apparatus that would be used
for the azeotropic drying of the [18F]fluoride ion and subsequent
reaction.
One-Step Synthesis of [18F]PS13. Reagent Preparation and
Apparatus Setup. A stock solution of K 2.2.2 (37.64 mg, 0.1 mmol, 2
equiv) and K2CO3 (6.91 mg, 0.05 mmol, 1 equiv) in water (50 μL)
plus anhydrous acetonitrile (450 μL) was loaded into a glass screw-
neck 1 mL V-vial (12 × 32 mm, part # 186002802; Waters Corp.)
and sealed with a bonded PTFE-silicone septum (12 × 32 mm, part #
186000274; Waters Corp.). An aliquot of this stock solution (100 μL)
was placed in an oven-dried 3 mL V-vial (part # 95030; Alltech
Associates Inc., Deerfield, IL) and sealed with a PTFE-silicone septum
(Alltech Associates Inc.). This vial was then placed in a lead-shielded
pot and prepared for receiving the cyclotron-produced [18F]fluoride
ion in 18O-enriched water. The remaining K 2.2.2/K2CO3 stock
solution was stored at −20 °C for not more than one month.
The gem-difluoroalkene 5 (1.5 mg, 4.2 μmol, 1 equiv), proton
source (IPA or NH4Cl(aq.)), and anhydrous DMSO (300 μL) were
placed in a glass screw-cap V-vial (1 mL, 12 × 32 mm, part #
186002802 Waters Corp.). The vial was sealed with a bonded PTFE-
silicone septum (part # 186000274; Waters Corp.) and placed in the
Synthia apparatus.
Synthesis [18F]PS13. The V-vial containing the cyclotron-produced
[18F]fluoride ion was measured for radioactivity. The vial was then
placed in position in the Synthia apparatus. The recipe for the two-
step synthesis of [18F]PS13 was started by selecting the prompt
command in the Autorad software. After a programmed acetonitrile
wash of the Synthia apparatus, the desired activity volume (50−500
μL) was withdrawn from the [18F]fluoride ion vial by the robotic arm,
placed in the reaction vial present in the apparatus oven, and then
subjected to three azeotropic dryings with acetonitrile at 110 °C
under with nitrogen flow at reduced pressure. Then the activity in the
reaction vial was measured, and the solution containing the reagents
for the synthesis of ([18F]6) was added to the reaction vial and heated
at 85 °C for 1 min. Reagents for Step 2 were added immediately to
the reaction vial, which was then heated to 130 °C for 20 min. Then
the radioactivity in the reaction vial was measured.
An empty oven-dried 5 mL V-vial (part # 95050; Alltech Associates
Inc.) was sealed with a PTFE-silicone septum and screw cap (Alltech)
and placed in the oven of the Synthia apparatus that would be used
for drying the [18F]fluoride ion and subsequent reaction.
HPLC Separation of [18F]PS13. The HPLC apparatus for
purification of [18F]PS13 was started with a prompt command of
the Autorad software. The crude reaction mixture was quenched with
water (∼1 mL), mixed in the robotic arm syringe, and injected onto a
Luna PFP(2) column (5 μm, 100 Å, 250 × 10 mm; Phenomenex),
eluted with acetonitrile−water (65:35 v/v) at 2 mL/min with eluate
monitored for radioactivity and absorbance at 254 nm (Supporting
Information, Figure S1). The retention time of [18F]PS13 was about
23 min. After each use, the HPLC column was washed for at least 30
min with acetonitrile−water (65:35 v/v).
Synthesis of [18F]PS13. The V-vial containing the cyclotron-
produced [18F]fluoride ion was measured for radioactivity upon
receipt. This vial was then placed in position in the Synthia apparatus.
The recipe for the one-step synthesis of [18F]PS13 was started with a
prompt command in the Autorad software. After programmed
washing of the apparatus with acetonitrile, the desired volume (50−
500 μL) was withdrawn from the [18F]fluoride ion vial by the robotic
arm and placed in the reaction vial present in the apparatus oven. This
solution was taken to dryness by three additions of acetonitrile and
evaporation at 110 °C under nitrogen flow and reduced pressure.
Then the radioactivity in the reaction vial was measured. The solution
containing the reagents for the synthesis of [18F]PS13 was then added
to the vial containing the dried [18F]fluoride ion and heated at 130 °C
for 20 min. Then the reaction was quenched with acetonitrile−water
(50:50 v/v; 0.5 mL). The activity in the reaction vial was measured.
An aliquot of the crude reaction mixture was analyzed with radio-
HPLC to evaluate the radiochemical yield, radiochemical purity, and
molar activity of [18F]PS13.
Formulation of [18F]PS13 for Intravenous Injection. The pure
[18F]PS13 was collected in a sterile round-bottomed flask within a
TRACERlab FX2 N apparatus (GE Healthcare; Chicago, IL) and
diluted with sterile water (∼40 mL; USP grade; Hospira; Lake Forest,
IL) before being passed through into a C18 Sep-Pak cartridge that
had been previously eluted with ethanol (USP grade; 10 mL; Warner
Graham; Cockeysville, MD) followed by water (HPLC grade; 10 mL;
EMD Millipore Corp.; Burlington, MA). The Sep-Pak cartridge was
then washed with sterile water (USP grade; 10 mL), before eluting off
the adsorbed [18F]PS13 with ethanol (USP grade; 1 mL) followed
with saline for injection (0.9% w/v, USP grade, 10 mL; Fresenius
Kabi; Lake Zurich, IL) into a round-bottomed flask. This solution was
then passed through a Millex LG sterile filter (0.2 μm; EMD
Millipore) into a sealed sterile vial (Hospira). A small aliquot of this
formulated product (∼500 μL) was withdrawn into a sterile syringe
(1 mL; Air-Tite Products Co. Inc.; Virginia Beach, VA), placed in a
separate vial, and measured for radioactivity. This aliquot was used for
HPLC analysis.
Analysis of [18F]PS13. [18F]PS13 was analyzed on a Luna PFP(2)
column (5 μm 100 Å, 250 × 4.6 mm; Phenomenex, Torrance, CA)
eluted with acetonitrile−water (45:55 v/v) at 1.8 mL/min with eluate
monitored for radioactivity and absorbance at 254 nm. After each use,
the HPLC column was washed for at least 30 min with acetonitrile−
water (65:35 v/v).
Two-Step Synthesis of [18F]PS13. Reagent Preparation and
Apparatus Setup. An oven-dried 3 mL V-vial was loaded with stock
K 2.2.2/K2CO3 solution (100 μL), as described above for the one-
step method, and the Synthia apparatus was prepared in readiness for
receipt of the cyclotron-produced [18F]fluoride ion in 18O-enriched
water.
Analysis of [18F]PS13. [18F]PS13 was analyzed on a Luna PFP(2)
column (5 μm, 100 Å, 250 × 4.6 mm; Phenomenex), eluted with
acetonitrile−water (1:1 v/v) at 2 mL/min with eluate monitored for
absorbance at 254 nm (Supporting Information, Figure S2). After
each use, the HPLC column was washed for at least 30 min with
acetonitrile−water (65:35 v/v).
A stock solution of the precursor for Step 1 was prepared from 2-
difluorovinyl 4-methylbenzenesulfonate (4 μL, 22.2 μmol, 1 equiv),
saturated NH4Cl(aq.) (10.6 μL), and anhydrous DMSO (1 mL). This
was then placed in an oven-dried glass screw-cap 1 mL V-vial (12 ×
32 mm; part # 186002802; Waters Corp.) and sealed with a bonded
PTFE-silicone septum (part # 186000274; Waters Corp.). An aliquot
of this solution (100 μL) was placed in an oven-dried glass screw-cap
1 mL V-vial (12 × 32 mm; part # 186002802; Waters Corp.) and
sealed with a bonded PTFE-silicone septum (part # 186000274;
Waters Corp.). This vial was then loaded into the Synthia apparatus.
Measurement of Molar Activity (Am) of [18F]PS13. Aliquots
(20 μL) of reference PS13 solution at five different known
concentrations (9.8 × 10−3−9.8 × 10−2 mM) were each analyzed
twice with radio-HPLC to obtain a calibration curve of average peak
area (mAU × s) versus concentration.
At the end of synthesis, an aliquot (20 μL) of the formulated
[18F]PS13 was analyzed with radio-HPLC. The peak area of PS13
eluting with [18F]PS13 was used to determine the concentration of
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Research Article
carrier in the formulated [18F]PS13 dose from the calibration curve.
The value (μmol/mL) obtained was multiplied by the total volume of
the formulated [18F]PS13 dose to determine the total amount of
carrier (μmol). The activity of the [18F]PS13 dose at the end of
synthesis divided by the total amount of carrier gave the molar activity
of [18F]PS13. This value was then decay-corrected to ERP.
Stability of [18F]PS13 in Buffer and in Monkey Whole Blood
and Plasma in Vitro. The stability of [18F]PS13 to incubation in
sodium phosphate buffer (0.15 M, pH 7.4) for 1 and 4 h at RT was
assessed by reversed phase HPLC on an Xterra column (7.8 × 300
mm, 10 μm; Waters Corp.), eluted at 4.0 mL/min with methanol−
water−triethylamine (85:20:0.1, by vol.).
was measured with reversed phase HPLC on an Xterra column (7.8 ×
300 mm, 10 μm; Waters Corp.), eluted at 4.0 mL/min (for monkey
A) or 3.5 mL/min (for monkey B) with methanol−water−
triethylamine (85:20:0.1, by vol.), after separating plasma from
whole blood, as previously described.45
PET Brain Data Analysis. Total distribution volume (VT), a sum of
a specific binding component known as the specific volume of
distribution (VS) and a nondisplaceable volume of distribution (VND),
may serve as an index of binding site density that equals the ratio at
equilibrium of the concentration of the radioligand in tissue to that in
plasma.46 VT values, uncorrected for radioactivity in blood (5% of
brain volume), were estimated for different brain regions with Logan
graphical analysis47,48 by using the PET brain time−activity curves
and the measured radiometabolite-corrected arterial input functions.
Kinetic analyses were performed at the voxel level, so that the
resulting VT values for [18F]PS13 could be shown as parametric PET
images. The temporal stabilities of VT in whole brain in the baseline
experiments were assessed by estimating VT from progressively time-
truncated data sets. Standard Error (i.e.,% SE or identifiability) was
expressed as a percentage. A smaller percentage indicates better
identifiability.48 Additionally, regional brain VT data were used to
generate a Lassen plot49,50 to estimate COX-1 occupancy in the
preblock conditions and to estimate VND. The PET image analysis,
including kinetic modeling, was performed with PMOD 3.9 (PMOD
Technologies).
The stability of [18F]PS13 in monkey whole blood and plasma was
also measured with HPLC, essentially as previously described for
[11C]PS13.17
Plasma Free Fraction Determinations. Plasma free fractions
(fp) of [18F]PS13 in monkeys studied with PET were determined by
an ultrafiltration method,44 as previously described17 for [11C]PS13.
PET Experiments in Monkey. For each PET scanning session,
the monkey was immobilized with ketamine and maintained under
anesthesia with 1.6% isoflurane in oxygen. An intravenous perfusion
line, filled with saline (0.9% w/v), was used for bolus injection of
[18F]PS13. PET serial dynamic images were obtained on a Focus 220
PET camera (Siemens Medical Solutions; Knoxville, TN) or a
Biograph mCT camera (Siemens Healthneers; Erlangen, Germany).
Brain Scans. Two male rhesus (Macaca mulatta) monkeys
(monkey A, 12.2 kg; monkey B, 13.8 kg) were scanned at baseline
for up to 180 min to measure uptake of radioactivity into brain and to
determine activity distribution after bolus intravenous injection of
[18F]PS13 (monkey A, 287 MBq, PS13 carrier 6.2 nmol/kg; monkey
B, 128 MBq, PS13 carrier 3.0 nmol/kg) using a Focus 220 PET
camera (Siemens Medical Solutions; Knoxville, TN). Preblock
experiments were also performed in the same monkeys in which
PS13 (0.3 mg/kg, i.v.) was administered intravenously between 15
and 5 min before [18F]PS13 (monkey A, 266 MBq; PS13 carrier 6.6
nmol/kg; monkey B, 281 MBq; PS13 carrier, 6.6 nmol/kg). All
monkeys had T1-weighted magnetic resonance imaging (TR/TE/x =
24 ms/3 ms/300), acquired on a 1.5-T Horizon instrument (GE
Medical Systems; Waukesha, WI).
The time-stability of VT values was evaluated by fitting regional
time-activity curves for PET data with truncated acquisition times
using the two-tissue compartment model, ranging from 180 to 60 min.
The ratio of the regional VT values from the truncated scan to that
from the 180 min measurement was computed for each region of
interest.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00737.
NMR spectra, radiochromatograms for radioligand
separations and analyses, monkey PET, and metabolism
data (PDF)
Whole-Body Scan. A third rhesus monkey (Macaca mulatta)
(monkey C; 10.3 kg, female) was injected with [18F]PS13 (166 MBq;
PS13 carrier 5.0 nmol/kg, i.v.) for whole-body PET imaging on a
Biograph mCT camera (Siemens Healthneers; Erlangen, Germany)
for 180 min.
AUTHOR INFORMATION
■
Image Analysis. PET brain images were reconstructed using
Fourier rebinning plus two-dimensional filtered back-projection. PET
images were initially coregistered to each monkey’s T1-weighted
magnetic resonance image (MRI) and then to a standardized monkey
MRI template using the Fuse It module of PMOD 3.9 (PMOD
Technologies; Zurich, Switzerland). A set of 33 predefined brain
regions of interest from the template was then applied to the
coregistered PET image to obtain regional decay-corrected time−
activity curves for frontal cortex, temporal cortex, parietal cortex,
occipital cortex, striatum, thalamus, limbic region, cerebellum, and
brain stem. Data reported for whole brain data are from gray matter.
All PET images were corrected for attenuation and scatter.
Radioactivity concentrations were expressed as standardized uptake
value (SUV), which normalizes for subject weight and injected
radioactivity, according to the equation
Corresponding Author
Victor W. Pike − Molecular Imaging Branch, National
Institute of Mental Health, National Institutes of Health,
Bethesda, Maryland 20892-1003, United States;
orcid.org/0000-0001-9032-2553; Phone: +1 301 594
5986; Email: pikev@mail.nih.gov
Authors
Carlotta Taddei − Molecular Imaging Branch, National
Institute of Mental Health, National Institutes of Health,
Bethesda, Maryland 20892-1003, United States
Cheryl L. Morse − Molecular Imaging Branch, National
Institute of Mental Health, National Institutes of Health,
Bethesda, Maryland 20892-1003, United States
Min-Jeong Kim − Molecular Imaging Branch, National
Institute of Mental Health, National Institutes of Health,
Bethesda, Maryland 20892-1003, United States
Jeih-San Liow − Molecular Imaging Branch, National Institute
of Mental Health, National Institutes of Health, Bethesda,
Maryland 20892-1003, United States
SUV = (% injected dose per g) × body weight in g
Whole-body PET images were reconstructed with ordered subset
expectation maximization (OSEM) with time-of-flight and resolution
recovery.
Analysis of [18F]PS13 Radiometabolites in Monkey Plasma. To
determine a radiometabolite-corrected arterial input function for brain
PET scans, blood samples (0.5−1 mL each) were drawn from the
monkey femoral artery into heparin-treated Vacutainer tubes at 15 s
intervals until 120 s, followed by 0.5−4 mL samples at 3, 5, 10, 30, 60,
90, 120, 150, and 180 min. The concentration of parent radioligand
Jose Montero Santamaria − Molecular Imaging Branch,
National Institute of Mental Health, National Institutes of
Health, Bethesda, Maryland 20892-1003, United States
527
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ACS Chem. Neurosci. 2021, 12, 517−530

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pubs.acs.org/chemneuro
Research Article
Andrea Zhang − Molecular Imaging Branch, National
Institute of Mental Health, National Institutes of Health,
Bethesda, Maryland 20892-1003, United States
Lester S. Manly − Molecular Imaging Branch, National
Institute of Mental Health, National Institutes of Health,
Bethesda, Maryland 20892-1003, United States
Paolo Zanotti-Fregonara − Molecular Imaging Branch,
National Institute of Mental Health, National Institutes of
Health, Bethesda, Maryland 20892-1003, United States
Robert L. Gladding − Molecular Imaging Branch, National
Institute of Mental Health, National Institutes of Health,
Bethesda, Maryland 20892-1003, United States
Sami S. Zoghbi − Molecular Imaging Branch, National
Institute of Mental Health, National Institutes of Health,
Bethesda, Maryland 20892-1003, United States
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Robert B. Innis − Molecular Imaging Branch, National
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00737
Author Contributions
C.T.: Radiosynthesis studies via the two methodologies to
produce [18F]PS13, synthesis of [18F]PS13 for PET brain
studies in monkey, and manuscript design; C.L.M.: assistance
in producing [18F]PS13 for PET brain studies in monkey and
synthesis of [18F]PS13 for PET whole-body studies in monkey;
M.-J.K., P. Z.-F., J.-S.L., and R.L.G.: PET imaging and data
analysis; S.S.Z., A.Z., L.S.M., and J.M.S.: metabolite analysis;
V.W.P. and R.B.I. project conception and supervision. All
authors contributed to the writing of this manuscript (overseen
by R.B.I. and V.W.P.).
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Funding
This study was supported by the Intramural Research Program
of NIH (National Institute of Mental Health; project number
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the NIH Clinical PET Center (Director: Dr. Peter
Herscovitch) for fluorine-18 production. The authors thank
Dr. Prachi Singh and Dr. Stal Shrestha for their previous
related work on [11C]PS13 and Dr. Shuiyu Lu for his technical
assistance in the radiosynthesis of [18F]PS13.
ABBREVIATIONS
■
Am, molar activity; COX-1, cyclooxygenase-1; COX-2, cyclo-
oxygenase-2; ERP, end of radioisotope production; fp, plasma
free fraction; IPA, isopropyl alcohol; K 2.2.2, 4,7,13,16,21,24-
hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane; MW, microwave;
MIP, maximum intensity projection; MRI, magnetic resonance
image; PET, positron emission tomography; PTFE, polytetra-
fluoroethylene; RCY, radiochemical yield; RT, room temper-
ature; VS, specific volume of distribution; SUV, standardized
uptake value; VND, nondisplaceable volume of distribution; VT,
total distribution volume
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S., Zoghbi, S. S., Fujita, M., Innis, R. B., and Pike, V. W. (2018) 3-
Substituted 1,5-diaryl-1H-1,2,4-triazoles as prospective PET radio-
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