[Carboxyl-11C]Labelling of Four High-Affinity cPLA2α Inhibitors and Their Evaluation as Radioligands in Mice by Positron Emission Tomography

Article abstract of DOI:10.1002/cmdc.201700697

Cytosolic phospholipase A2α (cPLA2α) may play a critical role in neuropsychiatric and neurodegenerative disorders associated with oxidative stress and neuroinflammation. An effective PET radioligand for imaging cPLA2α in living brain might prove useful for biomedical research, especially on neuroinflammation. We selected four high-affinity (IC₅₀ 2.1–12 nm) indole-5-carboxylic acid-based inhibitors of cPLA2α, namely 3-isobutyryl-1-(2-oxo-3-(4-phenoxyphenoxy)propyl)-1H-indole-5-carboxylic acid (1); 3-acetyl-1-(2-oxo-3-(4-(4-(trifluoromethyl)phenoxy)phenoxy)propyl)-1H-indole-5-carboxylic acid (2); 3-(3-methyl-1,2,4-oxadiazol-5-yl)-1-(2-oxo-3-(4-phenoxyphenoxy)propyl)-1H-indole-5-carboxylic acid (3); and 3-(3-methyl-1,2,4-oxadiazol-5-yl)-1-(3-(4-octylphenoxy)-2-oxopropyl)-1H-indole-5-carboxylic acid (4), for labelling in carboxyl position with carbon-11 (t_1/2=20.4 min) to provide candidate PET radioligands for imaging brain cPLA2α. Compounds [¹¹C]1–4 were obtained for intravenous injection in adequate overall yields (1.1–5.5 %) from cyclotron-produced [¹¹C]carbon dioxide and with moderate molar activities (70–141 GBq μmol^?1) through the use of Pd⁰-mediated [¹¹C]carbon monoxide insertion on iodo precursors. Measured logD_7.4 values were within a narrow moderate range (1.9–2.4). After intravenous injection of [¹¹C]1–4 in mice, radioactivity uptakes in brain peaked at low values (≤0.8 SUV) and decreased by about 90 % over 15 min. Pretreatments of the mice with high doses of the corresponding non-radioactive ligands did not alter brain time–activity curves. Brain uptakes of radioactivity after administration of [¹¹C]1 to wild-type and P-gp/BCRP dual knock-out mice were similar (peak 0.4 vs. 0.5 SUV), indicating that [¹¹C]1 and others in this structural class, are not substrates for efflux transporters.

Full text of DOI:10.1002/cmdc.201700697

Accepted Article
Title: [Carboxyl-11C]-Labeling of four high-affinity cPLA2a inhibitors
and their evaluation as radioligands in mice with positron
emission tomography
Authors: Martin J Fisher, Lindsay McMurray, Shuiyu Lu, Cheryl L
Morse, Jeih-San Liow, Sami S Zoghbi, Aneta Kowalski,
Geroge L Tye, Robert B Innis, Franklin I Aigbirhio, and Victor
W Pike
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of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201700697
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700697
A Journal of
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10.1002/cmdc.201700697
ChemMedChem
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[Carboxyl11C]-Labeling of four high-affinity cPLA2 inhibitors and their
evaluation as radioligands in mice with positron emission tomography
Martin J. Fisher[a], Lindsay McMurray[a], Shuiyu Lu[b]*, Cheryl L. Morse[b], Jeih-San Liow[b], Sami S.
Zoghbi[b], Aneta Kowalski[b], George L. Tye[b], Robert B. Innis[b], Franklin I. Aigbirhio[a], and Victor W.
Pike[b]
[a]
Dr. M. J. Fisher, Dr. L. McMurray, Prof. F. I. Aigbirhio
Molecular Imaging Chemistry Laboratory
Wolfson Brain Imaging Centre, University of Cambridge
Cambridge Biomedical Campus, Cambridge CB2 0QQ, UK
[b]
Dr. S. Y. Lu, Ms. C. L. Morse, Dr. J.-S. Liow, Dr. S. S. Zoghbi, Miss A. Kowalski, Mr. G. L. Tye, Dr. R. B. Innis, Dr. V. W. Pike
Molecular Imaging Branch
National Institute of Mental Health, National Institutes of Health
10 Center Drive, Room B3C346, Bethesda, MD 20892, USA
E-mail: shuiyu.lu@mail.nih.gov
Abstract: Cytosolic phospholipase A2 (cPLA2) may play a critical
role in neuropsychiatric and neurodegenerative disorders associated
with oxidative stress and neuroinflammation. An effective PET
radioligand for imaging cPLA2 in living brain might prove useful for
biomedical research, especially on neuroinflammation. We selected
four high-affinity (IC50 2.1 to 12 nM) indole-5-carboxylic acid-based
through increased vascular permeability and the recruitment of
granulocytes such as neutrophils to the site of injury or infection.
Increased cPLA2 activity and excessive production of
proinflammatory mediators, eicosanoids, and platelet activating
factor, may lead to neurodegenerative and neuropsychiatric
disease states, such as Alzheimer’s disease, Parkinson’s disease,
and traumatic brain injury.[7] Thus, cPLA2 is potentially an
attractive biomarker for quantifying disease and for designing
drugs to treat neuroinflammation and oxidative stress associated
disorders. The development of PET radioligands that specifically
target cPLA2 could help to evaluate and understand the role of
this enzyme in the pathophysiology of human diseases and would
be complementary to efforts to develop PET radioligands for other
neuroinflammation targets,[8] such as translocator 18 kDa protein
(TSPO),[8-10] COXs,[8,11,12] P2X purinoceptor 7 (P2X7),[8,13] and
inhibitors
of
cPLA2,namely
3-isobutyryl-1-(2-oxo-3-(4-
phenoxyphenoxy)propyl)-1H-indole-5-carboxylic acid (1); 3-acetyl-1-
(2-oxo-3-(4-(4-(trifluoromethyl)-phenoxy)phenoxy)propyl)-1H-indole-
5-carboxylic acid (2); 3-(3-methyl-1,2,4-oxadiazol-5-yl)-1-(2-oxo-3-(4-
phenoxyphenoxy)propyl)-1H-indole-5-carboxylic acid (3); and 3-(3-
methyl-1,2,4-oxadiazol-5-yl)-1-(3-(4-octylphenoxy)-2-oxopropyl)-1H-
indole-5-carboxylic acid (4), for labelling in carboxyl position with
carbon-11 (t1/2 = 20.4 min) to provide candidate PET radioligands for
imaging brain cPLA2. [11C]14 were obtained for intravenous
injection in adequate overall yields (1.1−5.5%) from cyclotron-
produced [11C]carbon dioxide and with moderate molar activities
(70−141 GBq/mol) through the use of Pd(0)-mediated [11C]carbon
monoxide insertion on iodo precursors. Measured logD7.4 values were
within a narrow moderate range (1.9−2.4). After intravenous injection
of [11C]14 in mice, radioactivity uptakes in brain peaked at low values
( 0.8 SUV) and decreased by about 90% over 15 min. Pre-treatments
of the mice with high doses of the corresponding non-radioactive
ligands did not alter brain time-activity curves. Brain uptakes of
radioactivity after administration of [11C]1 to wild type and P-gp/BCRP
dual knock-out mice were similar (peak 0.4 vs. 0.5 SUV), indicating
that [11C]1 and others in this structural class, are not efflux
transporters substrates.
cannabinoid receptor subtype
2
(CB2).[8,14] So far, only
[11C]arachidonic acid has been used with PET for pre-clinical and
clinical imaging[15,16] to explore brain cPLA2 activity.[17] However,
because of the involvement of arachidonic acid in neural functions
other than signal transduction, such as membrane synthesis and
remodelling, the incorporation rate (K*) has not been deemed
entirely specific to the cPLA2 signalling system.[17]
R2
O
O
N
OH
R1
O
R1
R2
O
Me
Me
1
2
3
4
PhO
(COiPr)
Introduction
O
4-CF3PhO
PhO
(COMe)
Me
Cytosolic phospholipase A2 (cPLA2) is one of the six members
of group IV phospholipase A2 (GIVA PLA2) enzyme family.[1-5] In
the arachidonic acid cascade, cPLA2 is activated by increases
O
N
(3-Me-1,2,4-oxadiazole)
Me
Me
N
O
N
in intracellular Ca2+ and kinase activation as
a result of
n-C8H17
extracellular stimulation, and hydrolyses phospholipids to
arachidonic acid and lysophospholipids in brain.[2,6] Arachidonic
acid is metabolized through the cyclooxygenase (COX) and
lipoxygenase (LOX) pathways to yield prostaglandins and
leukotrienes. These eicosanoids promote neuroinflammation
N
Figure 1. Structures of 14.
1
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Many selective inhibitors of cPLA2 are known.[3,4,18] We choose
a small group of indole-5-carboxylic acids[19,20] (14, Figure 1) for
PET radioligand development based on properties that are
suitable for this purpose, such as adequately low molecular
weight (< 500), moderate polar surface area, wide logP range
(2.6–6.3) and potent enzyme inhibitory ability (in the low nM
range).[21]
The importance of the carboxyl group as a component of the
pharmacophore needed for potent cPLA2 inhibitory activity is
highlighted by the lower potency seen when this group is replaced
with bioisosteres, such as hydroxamic acid, tetrazole, or acyl-
cyanamide.[22] cPLA2 strictly recognizes the “carboxyl group”
through interaction with the serine-228 residue of the active site.
Thus, ‘first generation’ radioligands for cPLA2 are likely to
contain a carboxyl group. Because of extensive ionization at
physiological pH, [11C]carboxylic acids (pKa ~ 4) do not generally
cross the blood-brain barrier (BBB), but some, such as
[11C]arachidonic acid (clogD, 4.89) do enter brain, even if only to
a limited extent.[17,23,24] In this study, the carboxyl group also
provided a generic site for labelling with carbon-11 (t1/2 = 20.4 min).
Here, we describe the labelling of 1−4 in their carboxyl groups, for
preliminary evaluation as PET radioligands for cPLA2 in mice.
subsequent reaction with neat epichlorohydrin in the presence of
KOH and tetrabutylammonium bromide afforded the epoxide 11
(Panel A, Figure 2). Iodo precursors for labeling (1619) were
prepared by treating an appropriate epoxide (911) intermediate
with a corresponding substituted phenol in the presence of DMAP
to obtain the intermediate alcohols 1215, followed by oxidation
with Dess-Martin periodinane reagent (Panel B, Figure 2). Only
the yields for compounds having the n-octyl chain (15 or 19) were
low. No further optimization was attempted because the quantities
of the final iodo-compounds were sufficient for several labeling
experiments.
Panel A
O
R
O
R
I
I
a
d
I
N
N
H
N
H
O
5
9
,
10
, 85%
87% R = Me
, 95%
b
iPr
6, 42%
N
N
O
O
N
O
OMe
N
I
d
c
I
I
N
Results and Discussion
N
N
H
H
O
Generally, [carbonyl11C]arylcarboxylic acids have been
synthesized from aryl halides, aryliodonium salts, arylboronic
acids, or arylboronic esters with either [11C]carbon monoxide or
[11C]carbon dioxide.[25-28] However, some methods suffer
drawbacks, such as reagent air/moisture sensitivity,[26]
requirement to use high amounts of precursor,[25] or low molar
activity.[29] [11C]Carbon monoxide has been gaining importance as
a useful primary labelling agent because: i) it can be synthesised
by passage of cyclotron-produced [11C]carbon dioxide over
heated molybdenum (875 °C) in high-yield and high molar
activity;[30] (ii) new apparatus[31-36] and techniques[37-41] have been
developed for increasing [11C]carbon monoxide availability in
small volumes of organic solvents; (iii) new methods for the
transition-metal mediated insertions of [11C]carbon monoxide into
radiotracers;[42-47] have expanded choice of reagents and
functional group tolerance. We chose to explore the Pd(0)-
mediated [11C]carbon monoxide insertion in iodo precursors for
the labeling of 1−4 because this method requires low amounts of
iodo precursors that should be stable and air-insensitive, and
because the method is expected to show high functional group
tolerance. In addition, we had access to an autoclave for
performing such reactions and experience with its use.[48,49]
7
8
11
, 75%
, 63%
, 46%
Panel B
I
R2
R2
N
OH
R1
OH
e
+
O
N
I
R1
O
9 11

12
%
, 51
, 77
, 73
, 27
13
14
15
%
%
%
R2
O
O
N
f
I
R1
R1 =
PhO
R2 =
COiPr
COMe
16
17, 52%
18
, 77%
19, 35%
, 96%
4-CF3PhO
PhO
3-Me-1,2,4-oxadiazole
3-Me-1,2,4-oxadiazole
n-C8H17
Figure 2. Syntheses of iodo precursors for radiolabelling 16–19. Conditions: a)
acetyl chloride or isobutyryl chloride, AlCl3, DCM, RT, 8 h. b) (1) pyridine,
trichloroacetyl chloride, dioxane, 80 C, 2.5 h; (2) MeOH, NaOH, 80 C, 30 min.
c) N-hydroxyacetamidine, NaH, THF, RT, 1 h. d) epichlorohydrin, KOH, TBAB,
RT, 1 h. e) ArOH, DMAP, DCM, 120 C, 1 h. f) Dess-Martin periodinane, DCM,
RT, 4 h.
Precursor synthesis
All iodo precursors for labeling were synthesised from
commercially available 5-iodoindole (Figure 2). Direct acylation
of 5-iodoindole with acetyl chloride or isobutyryl chloride gave the
methyl substituted (5) or isopropyl substituted (6) indole ketone,
respectively, in high and moderate yield, respectively. 5 and 6
were subsequently converted into the epoxides 9 and 10,
respectively, by treatment with neat epichlorohydrin in the
presence of KOH and tetrabutylammonium bromide. Reaction of
5-iodoindole with trichloroacetyl chloride in the presence of
pyridine, followed by methanolysis produced the methyl ester 7.
Condensation of 7 with N-hydroxyacetamidine in THF gave 8, and
Radiochemistry
Reaction of 16 with [11C]carbon monoxide in THF in the presence
of Pd(PPh3)4 afforded [11C]1 in useful yields (Figure 3). The bright
yellow color of Pd(PPh3)4 turned to brown over a period of several
weeks due to air and light sensitivity. [11C]Carbon monoxide
insertion efficiency decreased over this period when conducted
2
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with the same batch of Pd(PPh3)4, despite storage in a nitrogen-
protected glove-box. Attempts to use more air/moisture stable Pd
reagents, such as Pd(OAc)2 or PdCl2 with Xantphos, for labeling
reactions gave low yields. Efficiency of incorporation of
[11C]carbon monoxide could be restored by using fresh Pd(PPh3)4
or a slightly increased amount (510% mol) of slightly discolored
batch. Subsequently Pd(PPh3)4 in THF was used for other
labeling reactions. [11C]1−4 were obtained as formulated doses
for intravenous injection in useful yields (1.1−5.5% from
[11C]carbon dioxide) and with moderate molar activities (70−141
GBq/mol) (Table 1). Radiochemical purities exceeded 95%. The
identity of 11C-labeled radioligands was verified by co-injection of
reference compounds on analytical HPLC and by LC-MS analysis
of the carrier. Total synthesis time was around 45 min. Since the
quantities were adequate for small animal microPET imaging
studies, radiochemistry was not further optimized.
Table 1. Radiosynthesis parameters.
Radioligand
Prep. HPLC
method[a]
tR
(min)
Yield[b]
Molar activity
(GBq/mol)
(%)
1.7
3.1
1.1
5.5
[
[
[
[
11C]1
11C]2
11C]3
11C]4
I
9.5
114 (n = 5)
141 (n = 2)
109 (n = 3)
70 (n = 2)
I
10.3
9.0
I
II
12.5
[a] Preparative HPLC methods I and II are described in the Experimental
section. [b] Yields are from cyclotron-produced [11C]carbon dioxide.
In vitro measurements
In vitro experiments confirmed that [11C]14 possessed properties
considered favourable for brain PET radioligands (Table 2).
Measured logD7.4 values were within the range usually regarded
as desirable for brain radioligands,[21] and were consistently lower
than the calculated logP values because of the ionization of the
carboxyl groups.[19,20]
R2
O
O
N
I
R1
The values of plasma free fraction (fP) in pooled human plasma
were generally low, and especially for [11C]4 (Table 2). Although
higher plasma free fractions would be desirable, such low free
fractions are known among effective PET radioligands (e.g.,
[11C]MePPEP).[50] Radioligand stabilities in sodium phosphate
buffer (0.15 M) at pH 7.4 were excellent, except for [11C]4.
Stabilities in rat brain homogenates were also excellent. In vitro
analysis showed that [11C]1 was stable in rat and monkey whole
blood.
16 19

1) 11CO, Pd(PPh3)4, THF
130180 oC, 5 min
2) H2O
R2
O
N
O
OH
11C
R1
O
11
1 4
C] 
[
Figure 3. Radiosynthesis of [11C]14 through Pd(0)-mediated 11C-carbonylation
of corresponding iodo precursors.
Table 2. Radioligand in vitro properties.
[a]
Radioligand
IC50
logD
fP
(%)
Stability in buffer
(%)
Stability in rat brain homogenate
(%)
(nM)
[
[
[
[
11C]1
11C]2
11C]3
11C]4
12
2.10 ± 0.01
2.43 ± 0.01
2.07 ± 0.02
1.94 ± 0.15
1.46 ± 0.01
1.24 ± 0.01
n.d.[b]
98.9 ± 0.2
98.4 ± 0.2
99.7 ± 0.2
80.3 ± 3.4
96.9
97.5
n.d.
104
10
6.1
2.1
0.072 ± 0.001
[a] Data from references [19] and [20]. [b] n.d. = not determined.
Distribution of [11C]1 into cellular blood elements was affected by
plasma protein. More than 94% of [11C]1 was bound to blood cells
in the absence of autologous plasma, but the bound percentage
was considerably less in the presence of plasma (Figure 4). The
cellular blood uptake of [11C]1 may represent its possible
interaction with cPLA2 in peripheral blood cells.[51]
3
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with plasma
with PBS
0.6
0.4
0.2
0.0
100
KO mice, baseline (n = 2)
WT mice, baseline (n = 3)
KO mice, self-block (n = 3)
WT mice, self-block (n = 3)
50
0
0
10
20
30
40
Monkey
Rat
Time (min)
Figure 4. Distribution of [11C]1 in monkey and rat blood cells with or without
plasma. Values are mean ± SD for n = 3.
Figure 6. Brain time-activity curves for [11C]1 in wild type (WT) or efflux
transporter knock-out (KO) mice under baseline and self-block conditions.
Another possibility is that [11C]1−4 were excluded from brain by
efflux transporters, such as P-glycoprotein (P-gp) or breast
cancer resistance protein (BCRP).[52] Brain uptakes of [11C]1 in
wild type and dual P-gp/BCRP knock-out mice were similar (peak
SUV 0.4 vs. 0.5, Figure 6) under baseline or self-block conditions.
Lack of brain penetration was therefore not due to action of these
efflux transporters.
Peripheral organs are known to express higher concentrations of
cPLA2 than brain.[51,53-55] Figure 7 shows the time-activity curves
for [11C]2 in these organs under baseline conditions. Much higher
peak radioactivity was observed in peripheral organs, such as
heart (5.6 SUV), lungs (2.9 SUV) or kidneys (1.7 SUV) for all four
radioligands. No apparent blocking effects were observed when 2
was pre-administered at 2 mg/kg. These patterns of lacking
preblocking effects were also seen with the other three
radioligands injected into mice under baseline and self-blocking
conditions.
PET imaging
The measured moderate lipophilicities (logD values) of the four
radioligands might suggest that they should be able to cross the
blood-brain barrier. However, in wild type mice after intravenous
administration of [11C]1−4, peak radioactivities in brain were low
( 0.8 SUV) and declined by > 90% within 15 min (Figure 5).
Ligand pretreatment with 1 at 2 mg/kg in wild type mice did not
alter the shapes of brain time-activity curve for [11C]1 (Figure 6),
and therefore provided no evidence for specific binding of the
radioligand to cPLA2. The other radioligands, [11C]23, were
also tested in this manner. The shapes of the resulting time-
activity curves were similar to those under baseline conditions and
again were not suggestive of the presence of specific binding.
0.8
[
[
[
[
11C]1 (n = 3)
11C]2 (n = 4)
11C]3 (n = 3)
11C]4 (n = 3)
0.6
0.4
0.2
0.0
6
Heart (n = 4)
Lung (n = 4)
4
Kidney (n = 3)
Brain (n = 4)
2
0
10
20
30
40
50
Time (min)
0
Figure 5. Brain time-activity curves for [11C]14 in wild type mice.
0
10
20
30
40
Time (min)
A primary factor underlying the lack of brain uptake of [11C]1−4 is
likely the extensive ionization of the carboxyl groups to negatively
charged carboxylate groups at physiological pH. A possibility is
that ligand lipophilicity must be increased even further to allow
brain entry. For example [11C]arachidonic acid, which does get
into brain to a low extent, has a high clogD value of 4.9.
Figure 7. Time-activity curves for [11C]2 in different organs of wild type mice
under baseline conditions.
It is also difficult at this stage to ascertain whether the low uptake
and fast wash-out may also be attributed to the lack of cPLA2
enzyme density (Bmax) in mouse brain, the insufficient radioligand
affinity or other confounding factors.[21,52]
4
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water and extracted with ethyl acetate. The combined organics were dried
(Na2SO4) and concentrated in vacuo. The product was purified by
recrystallization from ethyl acetate to afford 5 (3.80 g, 13.3 mmol, 85%).
1H NMR (DMSO-d6, 300 MHz) δH 12.14−11.98 (bs, 1H), 8.52 (dd, J 0.5,
1.6, 1H), 8.32 (s, 1H), 7.49 (dd, J 8.52, 1.6, 1H), 7.33 (dd, J 8.5, 0.5, 1H),
2.45 (s, 3H). 13C NMR (DMSO-d6, 75 MHz) δC 193.1, 136.2, 135.5, 131.2,
130.1, 128.2, 116.3, 115.0, 86.6, 27.6. m/z (ES+) [M+H]+ = 284.9 (100%).
Conclusions
The Pd(0)-mediated 11C-carbonylation of aryl iodides proved
effevtive for producing structurally complex [11C]arylcarboxylic
acids. All tested radioligands [11C]14 showed low brain
penetration and lack of retention in mouse brain in vivo. [11C]1 is
not a brain P-gp or BRCP efflux transporter substrate. The results
show that [11C]14 are ineffective brain PET radioligands for
cPLA2. They also provide no cPLA2-specific binding in
periphery. Successful PET imaging of brain cPLA2 remains
desirable, but will likely require radioligands with no carboxyl
group, higher brain penetration and higher enzyme affinity – a
considerable medicinal chemistry challenge. Better animal
models of cPLA2 activated inflammation with deep
understanding of the underlying biology are also needed.
1-(5-Iodo-1H-indol-3-yl)-2-methylpropan-1-one (6): To a solution of 5-
iodoindole (2.83 g, 11.0 mmol) and AlCl3 (2.62 g, 19.8 mmol) in DCM (60
mL) was added isobutyryl chloride (2.08 mL, 19.8 mmol) and the solution
stirred at room temperature for 8 h. The reaction mixture was quenched by
addition of water and extracted with ethyl acetate. The combined organics
were dried (Na2SO4) and concentrated in vacuo. The product was purified
by recrystallization from ethyl acetate to afford 6 (1.46 g, 13.3 mmol, 42%).
1H NMR (DMSO-d6, 300 MHz) δH 12.14−12.00 (bs, 1H), 8.56 (d, J 1.7, 1H),
8.32 (d, J 3.14, 1H), 7.48 (dd, J 8.5, 1.7, 1H), 7.32 (dd, J 8.5, 0.5, 1H)
3.50−3.36 (m, 1H), 1.12 (d, J 6.7, 6H). 13C NMR (DMSO-d6, 75 MHz) δC
199.9, 136.3, 134.7, 131.2, 130.4, 128.7, 114.9, 114.6, 86.5, 36.3, 20.2.
m/z (ES+) [M+H]+ = 312.9 (100%).
Experimental Section
Methyl 5-iodo-1H-indole-3-carboxylate (7): To a solution of 5-iodoindole
(4.20 g, 17.0 mmol) in dioxane (70 mL) was added pyridine (13.7 mL) and
trichloroacetyl chloride (9.5 mL, 85.0 mmol) and the solution stirred at 80
C for 2.5 h. The cooled reaction mixture was then poured into water and
extracted with ethyl acetate. The combined organics were washed with
brine, dried (Na2SO4) and concentrated in vacuo. The residue was
dissolved in anhydrous methanol (100 mL) and NaOH (235 mg, 5.88
mmol) was added and the reaction mixture heated at 80 C for 30 min. The
cooled reaction mixture was concentrated in vacuo and the partitioned
between water and ethyl acetate and extracted with ethyl acetate. The
combined organics were washed with brine, dried (Na2SO4) and
concentrated in vacuo. The product was purified by recrystallization from
ethyl acetate to afford 7 (3.85 g, 12.7 mmol, 75%). 1H NMR (DMSO-d6,
300 MHz) δH 8.32 (d, 1H), 8.09 (s, 1H), 7.49 (dd, 1H), 7.35 (d, 1H), 3.81
(s, 3H).
Materials and Methods
All radiochemistry was performed in a lead-shielded hot-cell for radiation
safety to personnel. Radioactivity was measured with a calibrated dose
calibrator (Atomlab 300, Biodex Medical Systems, Shirley, NY) or a -
counter (Wallac Wizard 3", 1480 automatic -counter; PerkinElmer,
Waltham, MA). Radioactivity measurements were corrected for physical
decay. Radiochemical yields are calculated for formulated radioligand from
[
11C]carbon dioxide. Reagents and solvents are used as purchased unless
otherwise specified.
Syntheses of non-radioactive standards 14
All reference compounds were synthesized in-house based on previously
reported methods.[19,20] Compounds were identified with HRMS, 1H NMR
and 13C NMR spectroscopy. Compound purities were determined with
analytical HPLC and exceeded 98%.
5-(5-Iodo-1H-indol-3-yl)-3-methyl-1,2,4-oxadiazole (8): To a solution of N-
hydroxyacetamidine (956 mg, 12.9 mmol) in THF (150 mL) was added
NaH (60% dispersion in mineral oil, 511 mg) and the mixture stirred at
room temperature for 1 h. After addition of 7 (3.85 g, 12.7 mmol), the
mixture was heated under reflux for 24 h. Water and ethyl acetate were
added and THF was removed in vacuo. The concentrated mixture was
extracted with ethyl acetate and the combined organics were dried
(Na2SO4) and concentrated in vacuo. The product was purified by
recrystallization from ethyl acetate to afford 8 (2.67 g, 8.17 mmol, 63%).
1H NMR (DMSO-d6, 300 MHz) δH 8.44 (dd, J 1.7, 0.5, 1H), 8.35 (s, 1H),
7.56 (dd, J 8.5, 1.7, 1H), 7.42 (dd, J 8.5, 0.5, 1H), 2.41 (s, 3H). 13C NMR
(DMSO-d6, 75 MHz) δC 172.4, 167.3, 136.1, 131.7, 131.5, 129.2, 128.8,
127.2, 115.5, 99.7, 86.3. m/z (ES+) [M+H]+ = 324.9 (100%).
Syntheses of iodo precursors 1619
General Procedure A: alkylation of indoles. A mixture of the appropriate
indole (1.0 equiv.), powdered KOH (2.2 equiv.), and tetrabutylammonium
bromide (10 mol%), was stirred in neat epichlorohydrin (18 equiv.) at room
temperature for 1 h before being directly subjected to flash column
chromatography.
General Procedure B: ring-opening of epoxides. A mixture of the
appropriate epoxide (1.0 equiv.), the appropriate substituted phenol (1.0
equiv.), and 4-dimethylaminopyridine (20 mol%) was dissolved in a small
volume of DCM before being concentrated in vacuo. The resulting mixture
was heated at 120 C for 1 h before being directly subjected to flash
column chromatography.
1-(5-Iodo-1-(oxiran-2-ylmethyl)-1H-indol-3-yl)ethan-1-one (9): Prepared
according to General Procedure A using 5 (3.56 g, 12.5 mmol), KOH (1.54
mg, 27.5 mmol), tetrabutyl ammonium bromide (402 mg, 1.25 mmol) and
epichlorohydrin (18 mL, 225 mmol). Purification by flash column
chromatography eluting with EtOAc/pet ether (10:90 to 50:50) afforded 9
(3.7 g, 10.9 mmol, 87%). 1H NMR (CDCl3, 300 MHz) δH 8.65 (dd, J 0.5,
1.7, 1H), 7.61 (s, 1H), 7.46 (dd, J 1.7, 7.6, 1H), 7.05 (d, J 8.7, 1H), 4.47
(dd, J 5.9, 15.2, 1H), 3.26-3.20 (m, 1H), 2.78 (t, J 4.2, 1H), 2.40 (s, 3H).
13C NMR (DMSO-d6, 75 MHz) δC 192.8, 135.4, 132.0, 131.4, 128.3, 116.7,
111.7, 86.9, 50.6, 48.4, 45.0, 27.5. m/z (ES+) [M+H]+ = 342.3 (100%).
General Procedure C: oxidation of secondary alcohols. To a mixture of
the appropriate alcohol (1.0 equiv.) in dry DCM was added Dess-Martin
periodinane (1.5 equiv.). The reaction mixture was stirred at room
temperature for 4 h, followed by the addition of sodium thiosulfate in
saturated NaHCO3 solution. After stirring for 10 min, the reaction mixture
was extracted with DCM. The combined organics were dried (MgSO4) and
concentrated in vacuo. The product was purified with flash column
chromatography.
1-(5-Iodo-1-(oxiran-2-ylmethyl)-1H-indol-3-yl)-2-methylpropan-1-one (10):
Prepared according to General Procedure A using 6 (1.44 g, 4.60 mmol),
KOH (567 mg, 10.12 mmol), TBAB (148 mg, 0.460 mmol) and
epichlorohydrin (4.2 mL, 82.8 mmol). Then purified by flash column
chromatography eluting with EtOAc/pet ether (10:90 to 50:50) afforded 10
(1.61 g, 4.37 mmol, 95%). 1H NMR (CDCl3, 300 MHz) δH 8.72 (d, J 1.6,
1-(5-Iodo-1H-indol-3-yl)ethan-1-one (5): To a solution of 5-iodoindole (4.00
g, 15.6 mmol) and AlCl3 (3.73 g, 28.1 mmol) in DCM (60 mL) was added
acetyl chloride (2.31 mL, 28.1 mmol) and the solution stirred at room
temperature for 8 h. The reaction mixture was quenched by addition of
5
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10.1002/cmdc.201700697
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1H), 7.66 (s, 1H), 7.46 (dd, J 1.7, 8.6, 1H), 7.06 (d, J 8.6, 1H), 4.45 (dd, J
2.4, 15.2, 1H), 3.99 (dd, J 5.9, 15.2, 1H), 3.26−3.19 (m, 1H), 2.76 (t, J 4.2,
1H), 2.37 (dd, J 2.5, 4.6, 1H), 1.15 (d, J 6.7, 6H). 13C NMR (DMSO-d6, 75
MHz) δC 199.0, 135.3, 133.6, 130.9, 130.7, 127.8, 114.1, 110.6, 85.9, 49.3,
47.4, 43.9, 36.2, 18.7. m/z (ES+) [M+H]+ = 370.3 (100%).
1H), 4.24−4.13 (m, 1H), 3.94−3.84 (m 2H), 2.41 (s, 3H), 1.58-1.45 (m, 2H),
1.32−1.18 (m, 12H), 0.89−0.81 (m, 3H). 13C NMR (DMSO-d6, 75 MHz) δC
172.2, 167.3, 156.9, 136.6, 135.2, 135.0, 131.4, 129.6, 128.9, 127.4, 114.8,
114.5, 98.8, 86.6, 70.0, 68.3, 50.1, 34.7, 31.7, 31.6, 29.3, 29.1, 29.0,22.5,
14.4, 11.7. m/z (ES+) [M+H]+ = 588.5 (100%).
5-(5-Iodo-1-(oxiran-2-ylmethyl)-1H-indol-3-yl)-3-methyl-1, 2, 4-oxadiazole
(11): Prepared according to General Procedure A using 8 (2.60 g, 7.95
mmol), KOH (978 mg, 17.4 mmol), tetrabutyl ammonium bromide (260 mg,
0.807 mmol) and epichlorohydrin (11 mL, 140 mmol). Then purified by
flash column chromatography eluting with EtOAc/pet ether (10:90 to
40:60) followed by recrystallization from ethyl acetate afforded 11 (1.40 g,
3.67 mmol, 46%). 1H NMR (DMSO-d6, 300 MHz) δH 8.45−8.43 (m, 1H),
8.38 (s, 1H), 7.64−7.59 (m, 2H), 4.70 (dd, J 3.4, 15.2, 1H), 4.35 (dd, J 6.5,
15.2, 1H), 3.42-3.35 (m, 1H) 2.80 (dd, J 4.1, 4.8, 1H), 2.58 (dd, J 2.6, 4.8
1H), 2.41 (s, 3H). 13C NMR (DMSO-d6, 75 MHz) δC 172.0, 167.4, 135.6,
135.4, 134.6, 131.7, 128.9, 127.4, 114.6, 99.2, 86.9, 50.7, 48.7, 44.0 11.7.
m/z (ES+) [M+H]+ = 382.3 (100%).
1-(5-Iodo-1-(2-oxo-3-(4-phenoxyphenoxy)propyl)-1H-indol-3-yl)-2-
methylpropan-1-one (16): Prepared according to General Procedure C
using 12 (1.46 g, 2.63 mmol), Dess-Martin periodinane reagent (1.66 g,
3.92 mmol) in DCM (30 mL). Purification by flash column chromatography
eluting with EtOAc/pet ether (10:90 to 30:70) afforded 16 (1.39 g, 2.5 mmol,
96%). 1H NMR (CDCl3, 300 MHz) δH 8.84 (d, 1H), 7.67 (1H, s), 7.55 (dd,
1H), 7.31−7.38 (m, 2H), 6.87−7.14 (m, 8H), 5.27 (s, 2H), 4.69 (s, 2H), 3.26
(sept, 1H), 1.27 (d, 6H). 13C NMR (DMSO-d6, 75 MHz) δC 200.9, 199.6,
158.3, 154.4, 150.6, 138.5, 137.2, 131.4, 130.4, 128.8, 123.2, 121.0, 117.9,
116.5, 114.0, 113.9, 87.1, 71.8, 53.3, 20.1. HRMS (ESI-TOF) calcd for
C
27H25NIO4 [M+H]+ 554.0815, found 554.0823.
1-(3-acetyl-5-iodo-1H-indol-1-yl)-3-(4-(4-
1-(1-(2-Hydroxy-3-(4-phenoxyphenoxy)propyl)-5-iodo-1H-indol-3-yl)-2-
methylpropan-1-one (12): Prepared according to General Procedure B
using 10 (1.90 g, 5.15 mmol), 4-phenoxyphenol (958 mg, 5.15 mmol), and
4-dimethylaminopyridine (121 mg, 0.992 mmol). Purification by flash
column chromatography eluting with EtOAc/pet ether (20:80 to 30:70)
afforded 12 (1.46 g, 2.63 mmol, 51%). 1H NMR (DMSO-d6, 300 MHz) δH
8.45 (s, 1H), 8.32 (s, 1H), 7.51−7.48 (m, 2H), 7.39−7.28 (m, 2H), 7.39−7.28
(m, 3H), 7.10−7.05 (m, 1H), 6.95−6.86 (m, 4H), 6.81−6.76 (m, 1H), 5.50
(d, J 5.2, 1H), 4.48 (dd, 3.7, J 14.1, 1H), 4.36-4.20 (m, 2H), 3.96−3.89 (m,
2H), 3.40−3.32 (m, 1H), 6.37 (dd, J 1.7, 6.8, 6H). 13C NMR (DMSO-d6, 75
MHz) δC 199.5, 158.4, 155.1, 150.1, 138.4, 136.9, 131.1, 130.4, 130.3,
130.2, 129.1, 129.0, 123.1, 121.4, 121.1, 117.8, 117.3, 116.3, 114.0, 113.5,
86.9, 70.3, 68.1, 50.0, 36.6, 20.2, 20.1. m/z (ES+) [M+H]+ = 556.5 (100%).
(trifluoromethyl)phenoxy)phenoxy)propan-2-one (17): Prepared according
to General Procedure C using 13 (4.64 g, 7.80 mmol), Dess-Martin
periodinane reagent (4.92 g, 11.6 mmol) in DCM (100 mL). Purification by
flash column chromatography eluting with EtOAc/pet ether (40:60 to
60:40) afforded 17 (2.4 g, 4.05 mmol, 52%). 1H NMR (DMSO-d6, 300 MHz)
δ
H 8.78 (s, 1H), 7.64 (s, 1H), 7.53−7.59 (m, 3H), 6.93−7.10 (m, 6H), 6.89
(d, 1H), 5.27 (s, 2H), 4.71 (s, 2H), 2.51 (s, 3H). 13C NMR (DMSO-d6, 75
MHz,) δC 200.8, 192.8, 161.7, 155.3, 149.0, 139.2, 137.1, 131.4, 130.2,
128.3, 127.9, 127.8, 126.6, 123.5, 123.0, 122.1, 117.5, 116.7, 115.9, 113.9,
87.2, 53.3, 27.7. HRMS (ESI-TOF) calcd for C26H19F3INO4 [M+H]+
594.0380, found 594.0384.
=
1-(5-Iodo-3-(3-methyl-1,2,4-oxadiazol-5-yl)-1H-indol-1-yl)-3-(4-
phenoxyphenoxy)propan-2-one (18): Prepared according to General
Procedure C using 14 (590 mg, 1.04 mmol), Dess-Martin periodinane
reagent (656 mg, 1.54 mmol) in DCM (3 mL). Recrystallization from ethyl
acetate afforded 18 (450 mg, 0.796 mmol, 77%). 1H NMR (CDCl3, 300
MHz) δH 8.68 (d, 1H), 7.83 (s, 1H), 7.60 (dd, 1H), 7.31−7.38 (m, 2H), 7.10
(tt, 1H), 6.91−7.06 (m, 7H), 5.31 (s, 2H,), 4.71 (s, 2H), 2.49 (s, 3H). 13C
NMR (75 MHz, DMSO-d6) δC 200.8, 172.0, 167.4, 158.3, 154.4, 150.6,
137.0, 135.3, 131.7, 130.4, 128.9, 127.9, 123.2, 121.0, 117.9, 116.5, 114.4,
99.5, 86.9, 71.7, 53.5, 11.7. HRMS (ESI-TOF) calcd for C26H20IN3O4
[M+H]+ 566.0565, found 566.0571.
1-(1-(2-Hydroxy-3-(4-(4-(trifluoromethyl)phenoxy)phenoxy)propyl)-5-iodo-
1H-indol-3-yl)ethan-1-one (13): Prepared according to General Procedure
B using 9 (3.53 g, 10.3 mmol), 4-(4-trifluoromethylphenoxy)phenol (2.62 g,
10.3 mmol), and 4-dimethylaminopyridine (253 mg, 2.07 mmol).
Purification by flash column chromatography eluting with EtOAc/pet ether
(20:80 to 50:50) afforded 13 (4.71 g, 7.92 mmol, 77%). 1H NMR (DMSO-
d6, 300 MHz) δH 8.56−8.53 (m, 1H), 8.32 (s, 1H), 7.74−7.78 (m, 2H),
7.14−7.01 (m, 6H), 5.34 (d, J 5.2, 1H), 4.48 (dd, J 3.5, 13.9, 1H), 4.37-4.17
(m, 2H), 4.00-3.92 (m, 2H), 2.41 (s, 3H). 13C NMR (DMSO-d6, 75 MHz) δC
192.7, 161.8, 156.0, 148.6, 139.2, 136.8, 131.1, 130.2, 128.6, 127.8, (q, J
3.6), 122.1, 117.4, 116.5, 115.3, 114.0, 89.9, 70.5, 68.2, 50.0, 27.6. m/z
(ES+) [M+H]+ = 596.4 (100%).
1-(5-Iodo-3-(3-methyl-1,2,4-oxadiazol-5-yl)-1H-indol-1-yl)-3-(4-
octylphenoxy)propan-2-one (19): Prepared according to General
Procedure C using 15 (274 mg, 0.467 mmol), Dess-Martin periodinane
reagent (294 mg, 0.695 mmol) in DCM (10 mL). Recrystallization from
ethyl acetate afforded 19 (110 mg, 0.162 mmol, 35%). 1H NMR (DMSO-d6,
300 MHz) δH 8.66 (d, 1H), 7.57 (dd, 1H), 7.18 (d, 2H), 6.85−6.90 (m, 3H),
5.29 (s, 2H), 4.71 (s, 2H), 2.59 (t, 2H), 2.48 (s, 3H), 1.57−1.66 (m, 2H),
1.27−1.36 (m, 10H), 0.89 (t, 3H). 13C NMR (DMSO-d6, 75 MHz) δC 200.9,
172.0, 167.4, 156.1, 136.9, 135.6, 135.3, 131.7, 129.7, 128.9, 127.3, 114.9,
114.4, 99.4, 86.9, 53.5, 34.7, 31.7, 31.6, 29.3, 29.1, 29.0, 22.5, 14.4, 11.7.
HRMS (ESI-TOF) calcd for C28H32IN3O3 [M+H]+ 586.1555, found 586.1561.
1-(5-Iodo-3-(3-methyl-1,2,4-oxadiazol-5-yl)-1H-indol-1-yl)-3-(4-
phenoxyphenoxy)propan-2-ol (14): Prepared according to General
Procedure B using 11 (600 mg, 1.57 mmol), 4-phenoxyphenol (293 mg,
1.58 mmol), and 4-dimethylaminopyridine (37 mg, 0.303 mmol).
Purification by flash column chromatography eluting with EtOAc/pet ether
(30:70) afforded 14 (650 mg, 1.15 mmol, 73%). 1H NMR (DMSO-d6, 300
MHz) δH 8.48−8.42 (m, 1H), 8.34 (s, 1H), 7.58 (d, J 1.2, 2H), 7.36−7.32 (m,
2H), 7.08 (dt, J 1.2, 7.46, 1H), 6.95−6.89 (m, 2H), 5.53 (d, J 5.3, 1H), 4.54
(dd, J 3.6, 14.2, 1H), 4.38 (dd, J 7.5, 14.2, 1H), 4.27−4.16 (m, 1H), 3.93
(dd, J 2.2, 5.3, 1H), 2.41 (s, 3H). 13C NMR (DMSO-d6, 75 MHz) δC 172.1,
167.3, 158.4, 155.1,150.1, 136.6, 135.2, 131.4, 130.4, 128.9, 127.5, 123.1,
121.1, 117.8, 116.3, 114.6, 98.9, 86.7, 70.4, 68.3, 50.1, 11.7. m/z (ES+)
[M+H]+ = 568.3 (100%).
Radiochemistry
No-carrier-added [11C]carbon dioxide (~89 GBq) was prepared with the
14N(p,)11C nuclear reaction by bombarding a nitrogen-1% oxygen gas
target (initial pressure 300 psi) with a proton beam (16 MeV, 45 A) from
a cyclotron (PETrace, GE) for 40 min. Radiochemistry was performed on
a modified Synthia platform controlled by in-house developed software
1-(5-Iodo-3-(3-methyl-1,2,4-oxadiazol-5-yl)-1H-indol-1-yl)-3-(4-
octylphenoxy)propan-2-ol (15): Prepared according to General Procedure
B using 11 (690 mg, 1.81 mmol), 4-octylphenol (373 mg, 1.81 mmol), and
4-dimethylaminopyridine (94 mg, 0.770 mmol). Purification by flash
column chromatography eluting with EtOAc/pet ether (20:80 to 40:60)
afforded 15 (285 mg, 0.486 mmol, 27%). 1H NMR (DMSO-d6, 300 MHz) δH
8.43 (t, J 1.2, 1H), 8.33 (s, 1H), 7.56 (s, 2H), 7.09 (d J 8.6, 2H), 6.85 (d, J
8.6, 2H), 5.49 (d, J 5.6, 1H), 4.54 (dd J 3.6, 14.3, 1H), 4.36 (dd, J 7.6, 14.3,
based on Labview.[48,49,56] 11C]Carbon dioxide was first collected in a
[
stainless steel trap filled with molecular sieves (13X; Grace, Chicago, IL)
at room temperature. The trap was purged with helium at 80 mL/min for 60
s to remove oxygen. [11C]Carbon dioxide was released in a helium stream
(16 mL/min) at ~ 270 °C and concentrated in a liquid nitrogen cryogenic
trap filled with silica gel. [11C]Carbon dioxide was then released from the
6
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10.1002/cmdc.201700697
ChemMedChem
FULL PAPER
first cryo-trap in helium (10 mL/min) by warming the trap with a halogen
lamp and then passed over molybdenum wire (99.97%, 0.05 mm diameter,
Strem Chemicals, Newburyport, MA) in a quartz tube (22 cm length, 0.7
cm i.d.) heated at 875 °C to produce [11C]carbon monoxide. [11C]Carbon
monoxide was first concentrated cryogenically on silica gel in a second
stainless steel trap cooled with liquid nitrogen and then released in helium
into the autoclave by warming the trap with a halogen lamp.
room temperature for 30 min. The stability of [11C]1 and it distribution into
cellular blood elements was determined in freshly obtained anticoagulated
(heparin) blood. The stability of [11C]14 was obtained by dividing the
tissue radiochromatographic composition by the radiochemical purity of
the radioligand.
Plasma free fraction
[
11C]14 were each prepared in similar manner. The following is a typical
The plasma free fraction (fp) of [11C]14 was measured by ultrafiltration
through membrane filters (Centrifree; Millipore), as previously
described.[58] Briefly, 740 kBq (~ 4.4 L) of [11C]14 was added to 700 L
of pooled human plasma. The mixture was incubated at room temperature
for 10 min. The ultrafiltration components that contained high radioactivity
were allowed time until radioactivity decayed to within the optimal range of
the -counter before they were counted. Quantification of the ultrafiltrates
was carried out gravimetrically.
procedure. Iodo precursor 1619 (1.52.0 mg, 2.53.6 µmol) and
Pd(PPh3)4 (0.81.2 µmol) in THF (80 µL) were loaded into a reagent loop.
The mixture was pumped into the autoclave and allowed to react with
[
11C]carbon monoxide at 180 °C (130 C for [11C]4) for 5 min. The
radioactive reaction mixture was collected in a vented glass V-vial (5 mL),
and quenched with H2O (3 mL). 11C-Labeled product was purified by semi-
preparative HPLC, equiped with a Luna C18 column (10 m, 10  250 mm,
Phenomenex, Torrance, CA). Absorbance was monitored at 254 nm
(System Gold 166, Beckman Coulter Inc., Pasadena, CA) while
PET imaging
radioactivity was monitored with
a pindiode detector (Bioscan Inc.,
Washington DC). Semi-prep HPLC method (I) was eluted at 6 mL/min with
a linear gradient of 5 mM aqueous NH4OH (A)–MeCN (B), starting with
10% B for 1 min, increasing to 55% B in 14 min, held for 3 min, then
increased to 85% B in 2 min and held at 85% B until the end of run. Semi-
prep HPLC method (II) was eluted at 6 mL/min with a linear gradient of 5
mM aqueous NH4OH (A)–MeCN (B), starting with 10% B for 1 min,
increasing to 60% B in 14 min, held for 3 min, then increased to 85% B in
2 min and held at 85% B until the end of run. The radioligand fraction was
collected and concentrated in the presence of ascorbic acid (0.15 mg)
under vacuum at 80 °C as soon as the fraction collection began, and for a
further 1 min after fraction collection ended. The radioactive residue was
reconstituted in ethanol/saline solution (1:9, 10 mL), ascorbic acid (0.15
mg) and TWEEN (12 mg) to obtain dose for injection (up to 1.5 GBq).
When required the formulation was sterilized by filtration through a 0.22
m sterile filter (Millipore-MP, Waters Corp., Milford, MA). An aliquot (50
or 100 L) of the formulated product was analysed with radio-HPLC on a
Gemini-NX column (5 m, 4.6 × 250 mm; Phenomenex), eluted at 3
mL/min with a linear gradient of NH4OH (A, 2.5 mM)MeCN(B) from 10 to
55% B over 12 min, to obtain radiochemical purity, chemical purity and
molar activity. Retention times (tR) for [11C]14 are 5.6, 5.7, 5.2 and 7.9
min, respectively. The identity of 11C-labeled radioligands was verified by
co-injection of reference standards on analytical HPLC and by LC-MS
analysis of the carrier.
PET imaging experiments were performed in accordance with the Guide
for Care and Use of Laboratory Animals[59] and were approved by the
National Institute of Mental Health Animal Care and Use Committee. All
mice were anesthetized with 1.5% isoflurane. Scans were acquired using
microPET Focus 120 (Siemens Medical Solutions, Knoxville, TN) for a
duration of 100 min. [11C]14 was individually injected (~ 7.4 MBq)
intravenously (i.v.) through a tail vein catheter. Blocking agent (2 mg/kg)
was administered i.v. at 30 min before radioligand injection also via tail
vein catheter. Images were reconstructed using Fourier rebinning followed
by 2D OSEM without attenuation correction. No scatter correction was
applied.
Acknowledgements
This work was supported by the Intramural Research Program of
the National Institutes of Health (NIMH; Project number: ZIA-
MH002793) and a UK Medical Research Council grant (PI FIA;
Project number: MR/K02308X/1) including salary support for MF
and LM. We thank the NIH Clinical Center PET Department
(Chief: Dr. Peter Herscovitch) for regular supply of cyclotron-
produced carbon-11. We are grateful to Dr. Stanley Rapoport for
stimulating discussions.
Lipophilicity (logD7.4) measurements and stability in aqueous buffer
The value for the distribution coefficient (logD7.4) of [11C]14 between 1-
octanol and sodium phosphate buffer (0.15 M, pH 7.4) was determined
with a technique described previously,[57] but with correction for radioligand
instability in the buffer phase. The radioligand in ethanol/saline was placed
in 0.15 M sodium phosphate buffer for the duration of the study followed
by the determination of its radiochemical purity to obtain information on its
stability in buffer. The radiochemical composition of the aqueous buffer
remaining after their extraction with 1-octanol was determined using high-
performance liquid chromatography (HPLC) on a X-Terra C18 column (10
m, 7.8  300 mm, Waters Corp.) and a mobile phase of MeOH:H2O:Et3N
(92.5:7.5:0.1, v/v) at 4.0 mL/min. The HPLC system consisted of Beckman
Gold (Beckman Coulter, Inc.) analytic pumps equipped with an in-line
photodiode-array detector and a flow-through NaI scintillation detector-rate
meter (Bioscan). Data from the radioanalysis were collected and stored
with Bio-Chrome Lite software (Bioscan) and analyzed after decay
correction of the radiochromatograms.
Keywords: cPLA2, neuroinflammation, radiopharmaceuticals,
Carbonylation, carbon-11
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