Synthesis and Preliminary PET Imaging Studies of a FAAH Radiotracer ([11C]MPPO) Based on α-Ketoheterocyclic Scaffold

Article abstract of DOI:10.1021/acschemneuro.5b00248

Fatty acid amide hydrolase (FAAH) is one of the principle enzymes for metabolizing endogenous cannabinoid neurotransmitters such as anandamide, and thus regulates endocannabinoid (eCB) signaling. Selective pharmacological blockade of FAAH has emerged as a potential therapy to discern the endogenous functions of anandamide-mediated eCB pathways in anxiety, pain, and addiction. Quantification of FAAH in the living brain by positron emission tomography (PET) would help our understanding of the endocannabinoid system in these conditions. While most FAAH radiotracers operate by an irreversible ("suicide") binding mechanism, a FAAH tracer with reversibility would facilitate quantitative analysis. We have identified and radiolabeled a reversible FAAH inhibitor, 7-(2-[¹¹C]methoxyphenyl)-1-(5-(pyridin-2-yl)oxazol-2-yl)heptan-1-one ([¹¹C]MPPO) in 13% radiochemical yield (nondecay corrected) with >99% radiochemical purity and 2 Ci/μmol (74 GBq/μmol) specific activity. The tracer showed moderate brain uptake (0.8 SUV) with heterogeneous brain distribution. However, blocking studies with a potent FAAH inhibitor URB597 demonstrated a low to modest specificity to the target. Measurement of lipophilicity, metabolite, and efflux pathway analysis were also performed to study the pharmacokinetic profile of [¹¹C]MPPO. In all, we reported an efficient radiolabeling and preliminary evaluation of the first-in-class FAAH inhibitor [¹¹C]MPPO with α-ketoheterocyclic scaffold. (Chemical Equation Presented).

Full text of DOI:10.1021/acschemneuro.5b00248

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Article
Synthesis and preliminary PET imaging studies of a FAAH
11
radiotracer ([C]MPPO) based on #-ketoheterocyclic scaffold
Lu Wang, Joji Yui, Qifan Wang, Yiding Zhang, Wakana Mori, Yoko Shimoda, Masayuki
Fujinaga, Katsushi Kumata, Tomoteru i Yamasak, Akiko Hatori, Benjamin H Rotstein,
Thomas Lee Collier, Chongzhao Ran, Neil Vasdev, Ming-Rong Zhang, and Steven H Liang
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.5b00248 • Publication Date (Web): 27 Oct 2015
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Synthesis and preliminary PET imaging studies of a FAAH
radiotracer ([11C]MPPO) based on αꢀketoheterocyclic scaffold
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Lu Wang,†,§ Joji Yui,‡,§ Qifan Wang,† Yiding Zhang,‡ Wakana Mori,‡ Yoko Shimoda,‡ Masayuki
Fujinaga,‡ Katsushi Kumata,‡ Tomoteru Yamasaki,‡ Akiko Hatori,‡ Benjamin H. Rotstein,†
Thomas Lee Collier,†, ¶ Chongzhao Ran,# Neil Vasdev,† Ming-Rong Zhang*,‡ and Steven H.
Liang*,†
† Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital & Department of Radiolo-
gy, Harvard Medical School, Boston, MA, 02114, United States
‡ Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, 263-8555, Japan
¶ Advion BioSystems, 10 Brown Road, Suite 101, Ithaca, New York, USA.
# Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital & Department of Radiolo-
gy, Harvard Medical School, Charlestown, MA, 02129, United States
§ These authors contribute equally to this work.
Supporting Information
ABSTRACT: Fatty acid amide hydrolase (FAAH) is one of the principle
enzymes for metabolizing endogenous cannabinoid neurotransmitters
such as anandamide, and thus regulates endocannabinoid (eCB) sig-
naling. Selective pharmacological blockade of FAAH has emerged as a
potential therapy to discern the endogenous functions of anandamide-
mediated eCB pathways in anxiety, pain and addiction. Quantification
of FAAH in the living brain by positron emission tomography (PET)
would help our understanding of the endocannabinoid system in these conditions. While most FAAH radiotracers oper-
ate by an irreversible (‘suicide’) binding mechanism, a FAAH tracer with reversibility would facilitate quantitative analy-
sis. We have identified and radiolabeled a reversible FAAH inhibitor, 7-(2-[11C]methoxyphenyl)-1-(5-(pyridin-2-yl)oxazol-
2-yl)heptan-1-one ([11C]MPPO) in 13% radiochemical yield (non-decay corrected) with >99% radiochemical purity and 2
Ci/µmol (74 GBq/µmol) specific activity. The tracer showed moderate brain uptake (0.8 SUV) with heterogeneous brain
distribution. However, blocking studies with a potent FAAH inhibitor URB597 demonstrated a low to modest specificity
to the target. Measurement of lipophilicity, metabolite and efflux pathway analysis were also performed to study the
pharmacokinetic profile of [11C]MPPO. In all, we reported an efficient radiolabeling and preliminary evaluation of the
first-in-class FAAH inhibitor [11C]MPPO with α-ketoheterocyclic scaffold.
KEYWORDS: PET, fatty acid amide hydrolase, FAAH, [11C]MPPO, radiotracer
INTRODUCTION
biosynthesized and released ‘on request’ in vivo. The sig-
naling network is modulated mainly via two G-protein-
coupled receptors, namely, cannabinoid receptors CB1
and CB2. The corresponding endocannabinoid ligands of
CB1/2 in mammals have been identified as anandamide
(AEA)2 and 2-arachidonoylglycerol (2-AG).3,4 Early efforts
have been focused on the direct pharmacological inter-
vention of endocannabinoid system by its natural agonist
Endocannabinoid signaling systems is a neuromodulatory
network which regulates mammalian neurophysiology,
including cognition and memory, motor function, anxie-
ty, pain perception, addiction and reward behaviors.1 En-
docannabinoids are endogenous small molecules (chemi-
cal messengers), and owing to their hydrophobic charac-
ter which preclude storage into synaptic vesicles, they are
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ꢀ9-tetrahydrocannabinol (the psychoactive component of
Cannabis sativa) and other synthetic cannabinoids; how-
ever, the concomitant detrimental effects on cognition
and motor control limit their use as therapeutic agents. In
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Figure 1. Representative PET radiotracers for imaging FAAH
Scheme 1. Mechanism of action and this work.
.
order to circumvent this problem, targeting enzyme deg-
radation that controls endocannabinoid (AEA and 2-AG)
levels has emerged as a potential strategy to identify drug
candidates for medicinal uses.5-9 Two enzymes, fatty acid
amide hydrolase (FAAH) and monoacylglycerol lipase
(MAGL) are primarily responsible for the degradation of
AEA and 2-AG, respectively. In particular, FAAH is a ~60
kDa integral membrane enzyme that is highly expressed
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135; 9) with an established reversible binding mechanism
in the mammalian brain along with liver and kidney,10,11
(Scheme 1B).41,42 Briefly, the hydroxyl group in Ser241 resi-
due of FAAH is added to the carbonyl moiety of OL-135 to
form an enzyme-bound intermediate (10), which is fur-
ther stabilized by hydrogen bonding between
FAAH(Ser217) and the oxazole ring. The tetrahedral in-
termediate 10 is covalently bound to FAAH(Ser241), but in
a reversible manner, because the reaction cannot proceed
further to release a leaving group. On the basis of these
findings, we speculated that a positron-emitting analog of
OL-135 would provide a potential reversible PET ligand
for FAAH based on the existing mechanism of action.
Therefore, we selected a recently reported close analog of
OL-135, 7-(2-methoxyphenyl)-1-(5-(pyridin-2-yl)oxazol-2-
yl)heptan-1-one (MPPO; 11, Scheme 1C), which is amena-
ble for 11C-labeling and exhibited an excellent binding
profile43 to FAAH in vitro with IC50 value of 10 nM and
greater than 500 fold selectivity over triacylglycerol hy-
drolase (TGH) as well as > 105 fold selectivity over hydro-
lytic enzyme KIAA1363. Herein we describe our synthesis,
radiolabeling and preliminary evaluation of [11C]MPPO,
the first-in-kind α-ketoheterocyclic PET ligand targeting
FAAH.
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and functions as a serine hydrolase equipped with an un-
usual serine-serine-lysine (Ser241-Ser217-Lys142) catalytic
triad.12 Inhibition of FAAH increases the levels of AEA and
has been found to reduce anxiety, pain, addiction and
inflammation, as well as show anti-depressant and anal-
gesic effects in preclinical models13-15 without adverse ef-
fects in motility and behavior change observed with direct
CB1 interventions.16,17 Several reversible and irreversible
FAAH inhibitors including URB59716 and PF-04457845,18-21
JNJ-4216527922 and V15886623 have advanced to clinical
trials for release of osteoarthritis pain, cannabis with-
drawal, anxiety and schizophrenia.
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A positron emission tomography (PET) tracer for FAAH
is desirable to allow quantitative enzyme mapping, target
engagement and dose selection in clinical studies with
high sensitivity under minimal perturbation of the biolog-
ical state. As shown in Figure 1, there are continued ef-
forts in the search for suitable FAAH PET tracers, includ-
ing anadamide analogs,24 [11C-methyl]URB597 analog,25
[11C-carbonyl]URB694 ([11C]CURB; 1),26,27 [18F]DOPP
(2),28,29 [11C]PF-04457845 (4),30,31 [18F]PF-9811 (5),32 MK-3168
(6),30 and most recently [11C]MFTC (3).33 To date, only
two PET ligands have been reported in human studies,
namely [11C]CURB (1)34,35 and [11C]MK-3168 (6).36 Although
considerable advances have been made in the develop-
ment of FAAH tracers, the majority of the reported radi-
oligands are limited to suicide37 inhibitors (Figure 1A-B),
which feature a covalent irreversible binding mechanism.
For instance, [11C]CURB (1; Scheme 1A) binds to FAAH in
vivo by acylation of the Ser241 sidechain and concomitant
exclusion of unlabeled O-phenoxy fragment (7) to yield
11C-labeled FAAH (8). As the first radiotracer for FAAH in
humans, [11C]CURB has been successfully utilized to map
FAAH in the living brain using an irreversible two tissue
compartment model.34 However, based on the binding
mechanism, [11C]CURB only provides the measurement of
FAAH activity, as opposed to FAAH availability,29 which
could be extracted from a reversible tracer as used in a
ligand-receptor scenario in PET studies.38 A FAAH tracer
with reversibility would allow us to access key infor-
mation from PET quantifications, including volumes of
distribution, binding potentials, and monitor neurological
response to therapeutics (occupancy and displacement
studies).39,40 To date, [11C]MK-3168 (Figure 1C) is the only
reversible PET ligand for this target and has advanced to
human PET studies,31 but only preliminary proceedings
have been reported since 2012.22,36 Therefore there is an
urgent need for reversible FAAH radiotracers to address
these questions and provide a full quantitative assessment
of FAAH distribution, concentration, and activities in
brain, to establish it as a viable biomarker for the diagno-
sis and prognosis of patients with associated disorders in
endocannabinoid signaling system.
RESULTS AND DISCUSSION
Chemistry. With MPPO 11 as the target, we designed a
synthetic strategy for the synthesis (for detailed retrosyn-
thetic analysis, see Scheme S1 in Supporting Information).
As summarized in Scheme 2A, 5-(pyridin-2-yl)oxazole (14)
was constructed in a quantitative yield according to the
literature procedure.43-45 Protection of alcohol 15 with 3,4-
dihydro-2H-pyran in the presence of a catalytic amount of
PPTS afforded bromide 16 in 85% yield. Condensation of
2-methoxybenzaldehyde with Wittig salt 17 afforded the
mixture of Z/E olefins 18 in a yield of 56% over two steps.
It was worthy of note that the synthesis of Wittig salt 17 in
organic solvents, such as THF, toluene or CH3CN, led to
low to modest yields of 18 (0-21%). The optimized reac-
tion parameters were identified as solvent-free conditions
in neat PPh3 at 120 °C for 4 h to generate 17, which was
utilized in the subsequent Wittig olefination immediately.
Compound 19 was obtained in 99% yield on exposure to a
Pd/C catalyzed hydrogenation of 18 and the subsequent
deprotection with TsOH yielded 20. Swern oxidation af-
forded aldehyde 21 in 83% yield. The synthesis of key in-
termediate 22 was not straightforward since base-
mediated condensation using KHMDS, LHMDS, nBuLi,
nBuLi/Bu3SnCl, nBuLi/ZnCl2/CuI or MeMgBr failed to
produce the desired product. A notable improvement was
achieved when iPrMgCl in THF was used, leading to the
secondary alcohol 22 in almost quantitative conversion.
Compound 22 was used without further purification in
the following Dess-Martin oxidation to generate MPPO
(11) in 51% yield. Demethylation of 11 with BBr3 in CH2Cl2
gave the radiolabeling precursor phenol (7-(2-
hydroxyphenyl)-1-(5-(pyridin-2-yl)oxazol-2-yl)heptan-1-
one; 23) in 58% yield after recrystallization.
With the goal to develop a novel and reversible FAAH
radiotracer, we aimed to design a 11C-labeled molecule
based on a potent and selective FAAH inhibitor, 7-
phenyl-1-(5-(pyridin-2-yl)oxazol-2-yl)heptan-1-one (OL-
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Scheme 2. Syntheses of standard and precursor, and radiolabeling of [11C]MPPO
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aReagents and conditions: (i) K2CO3, MeOH, 96%. (ii) 3,4-dihydro-2H-pyran, PPTS, CH2Cl2, 85%. (iii) PPh3, 120 °C. (iv) 2-methoxybenzaldehyde,
LiHMDS, THF, 56% over two steps. (v) Pd/C, H2, MeOH, 99%. (vi) p-Toluenesulfonic acid monohydrate, MeOH, 89%. (vii) (COCl)2, DMSO,
Et3N, CH2Cl2, 83%. (viii) 14, iPrMgCl, THF. (ix) Dess-Martin periodinane, CH2Cl2, 51% over two steps. (x) BBr3, CH2Cl2, 58%.
Radiolabeling. As shown in Scheme 2B, the methyl
ether was identified as the most convenient labeling site
for MPPO with carbon-11. The automated radiosynthesis
of [11C]MPPO was performed by the reaction of a phenolic
precursor 23 with [11C]CH3I in the presence of NaOH.
[11C]CH3I was bubbled through the reaction vial and after
5 min of reaction time at 80 °C, we observed highly selec-
tive conversion to [11C]MPPO. The reaction mixture was
purified by reverse phase HPLC, and reformulated in sa-
line for intravenous injection. [11C]MPPO was synthesized
in 13 ± 3% radiochemical yields based on [11C]CO2 (non-
decay corrected) for the subsequent preclinical PET imag-
ing studies. Specifically, starting from 350 - 530 mCi (13.0 -
19.6 GBq) of [11C]CO2, [11C]MPPO was obtained in 58 ± 17
mCi (2.15 ± 0.65 GBq) at end-of-synthesis (~29 min syn-
thesis time from end-of-bombardment) with > 99%
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mide 26 was obtained in a yield of 72% over two steps.
radiochemical purity (n = 10; for semi-preparative and
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analytical HPLC results, see Figure S1 in Supporting In-
formation). The specific activity was greater than 2
Ci/µmol (74 GBq/µmol) and no radiolysis was observed
within 90 min after formulation.
The resulting bromide 26 was subjected to nucleophilic
displacement with KF and 18-crown-6 in CH3CN to give
the corresponding fluoro analog 27 in 30% yield after re-
crystallization. The radiofluorination proceeded to gen-
erate [18F]27 in 3% isolated radiochemical yield with
greater than 1 Ci/µmol specific activity; however, further
evaluation was not pursued due to a rapid defluorination
in vivo (Scheme S2 in Supporting Information).
In a parallel approach, we also prepared an aliphatic
analog of OL-135 for radiolabeling. Swern oxidation of
alcohol 24 gave the corresponding aldehyde 25 in 70%
yield. After a sequential iPrMgCl-mediated condensation
and Dess-Martin oxidation, radiolabeling precursor bro-
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Table 1. Distribution of radioactivity in mice after injection of [11C]MPPO. Data are %ID/g (mean ± SD, n = 3)
Lipophilicity. Lipophilicity of radiolabeled tracer has
predictive utility for assessing blood-brain barrier perme-
ability, with an optimum logP range of 2.0–3.5.46-49
Using liquid-liquid partition between n-octanol and
PBS buffer (pH 7.4),47 logD7.4 of [11C]MPPO was deter-
mined to be 3.43 (n = 3), which is comparable with several
brain penetrant FAAH tracers, including [11C]CURB (1;
logD7.4 2.8),26 [11C]PF-04457845 (4; logD7.4 3.48).30
Whole body biodistribution studies in mice. The
kinetics and tissue distribution of [11C]MPPO was studied
in mice at several experimental time points (1, 5, 15, 30
and 60 min) post-tracer injection. The results are ex-
pressed as the percentage of injected dose per wet tissue
(%ID/g) in Table 1 (for biodistribution expressed as SUV
unit, see Table S1 in Supporting Information). At 1 min
post injection, a high uptake (> 3 %ID/g) was observed in
the heart, lungs, liver, kidneys and small intestine. After
the initial phase the radioactivity levels in most tissues
decreased rapidly, while the signals in the liver and small
intestine continually increased until 15 min and then de-
creased slowly. The radiotracer was efficiently cleared
from blood (1 min/60 min ratio of 6.1) and high uptake of
[11C]MPPO in the liver, kidney, and small intestine sug-
gests that hepatobiliary and urinary excretion, as well as
Figure 2. Representative coronal PET images (summed 0-30
min) and time-activity curves of [11C]MPPO in different rat brain
regions (n = 3, mean ± SEM).
the intestinal reuptake pathway, may dominate the whole
body distribution of radioactivity. The present result indi-
cates that the distribution of [11C]MPPO was in agreement
with the distribution of FAAH in mice as reported previ-
ously,50-52 with high expression in the liver, brain, testes,
kidneys and spleen. In addition, rapid clearance of radio-
activity from lungs, heart and muscle was observed,
which is consistent with low FAAH expression in these
organs in mice.50
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Figure 4. Reversible binding of [11C]MPPO in the brain. (n = 3;
mean ± SD). a. SUV data from PET imaging of [11C]MFTC in rat
whole brain; b. SUV data from PET imaging of [11C]MPPO in rat
whole brain.
including cerebral cortex, cerebellar cortex, cerebellar
nuclei and pons, indicating certain specificity of
[11C]MPPO, albeit modest, to FAAH in the rat brains, alt-
hough there was only a marginal difference of regional
brain uptake between baseline and blocking.
[11C]MPPO, as hypothesized in Scheme 1B, may show a
reversible binding profile to FAAH (Figure 4). The radio-
activity reached the maximum uptake of 0.65 SUV, follow
by a steady washout in rat brains (5 min/60 min ratio of
5.1). Compared with an irreversible FAAH radiotracer
[11C]MFTC,33 within 60 min post injection, no significant
decrease of [11C]MFTC in rat brain uptake was observed (5
min/60 min ratio of 0.9).
Figure 3. Representative PET images (0-30 min) and time-
activity curves (baseline and blocking) of [11C]MPPO in rat
brains. (n = 3; mean ± SEM).
Metabolite analysis. To evaluate the in vivo stability
of [11C]MPPO, the fraction of radiometabolites in the
plasma and brain homogenate of Sprague-Dawley rats
was evaluated post-tracer injection. The percentages of
unchanged [11C]MPPO and the corresponding radiome-
tabolites, as determined by radio-HPLC, are shown in
Figure 5. Analysis of rat brain homogenates and plasma 15
min post-tracer injection showed that 75% and 90% me-
tabolism occurred with the radioactivity associated with
hydrophilic metabolites. These results indicates a rapid in
vivo metabolism of [11C]MPPO, which may contribute to
the low specific binding and/or rapid washout in PET
imaging studies.
The total level of initial brain uptake of [11C]MPPO was
moderate to high with 1.87 %ID/g and 2.27 %ID/g at 1 min
and 5 min post-tracer injection, respectively. The radioac-
tivity washout from the brain was rapid with 0.88 %ID/g
at 60 min time point (5 min/60 min ratio of 2.6). These
results indicate moderate brain uptake (0.8 SUV) of
[11C]MPPO. Therefore we further evaluated [11C]MPPO as
a suitable reversible PET tracer for FAAH neuroimaging
in PET imaging studies in Sprague-Dawley rats.
PET imaging studies in rats. Representative PET im-
ages of rat brain after injection of [11C]MPPO are shown in
Figure 2A. PET images in normal rats showed moderate
brain penetration and accumulation of radioactivity in the
brain. The highest radioactivity was seen in the cerebellar
nuclei (0.87 SUV), followed by cerebral cortex, hippo-
campus, thalamus, striatum while the lowest uptake was
observed in the pons. As shown in the time-activity
curves of different brain regions, radioactivity in brain
tissues increased rapidly after the injection of [11C]MPPO,
peaked at 1.5 min (0.87 SUV in cerebellar nuclei), and
gradually washed out over 60 min. The distribution pat-
tern of [11C]MPPO was similar to that for [11C]CURB26 or
[11C]MFTC33 and the distribution of FAAH in the rat
brain.51-54
As shown in Figure 3, pretreatment with a potent FAAH
inhibitor, URB597 (3 mg/kg, 30 min iv before injection),
successfully abolished the difference of radioactivity up-
takes in different regions of interest. The radioactivity
distribution became fairly uniform in all brain regions,
Figure 5. Radiometabolites of [11C]MPPO in rats. (n = 3; mean ±
SD)
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radiochemical yield, high radiochemical purity and high
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specific activity. The lipophilicity, whole body distribu-
tion, brain penetration, efflux pump and metabolism
studies were evaluated to determine the suitability of
[11C]MPPO as a FAAH radiotracer. Preliminary PET imag-
ing studies showed low to moderate level of specific bind-
ing to FAAH in murine brains. Although [11C]MPPO is less
likely pursued for in vivo mapping of FAAH in the brain,
the radiosynthesis and preliminary evaluation by PET
imaging studies offers a roadmap for the investigation of
other FAAH radiotracer candidates based on the α-
ketoheterocyclic drug scaffolds with reversible binding
profiles. In the future, radioligands with higher affinity
and stability with minimal off-target binding will need to
be developed to allow in vivo imaging of FAAH.
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METHODS
Materials and Methods
Chemistry. General Conditions. All the chemicals em-
ployed in the syntheses were purchased from commercial
vendors and used without further purification. Thin-layer
chromatography (TLC) was conducted with 0.25 mm sili-
ca gel plates (60F-254) and visualized by exposure to UV
light (254 nm) or stained with potassium permanganate.
Flash column chromatography was performed using silica
gel (particle size 0.040-0.063 mm). H-Nuclear magnetic
resonance (NMR) spectra were obtained at 400 or 500
MHz on Bruker spectrometers in CDCl3 solutions at room
temperature with tetramethylsilane (TMS, δ = 0) as an
Figure 6. PET images (0-60 min) and time-activity curves in
wild-type and PgP/BCRP KO mice (n = 3; mean ± SEM).
PET imaging studies in PgP/Bcrp knockout mice.
Another possible reason for relatively a low-to-moderate
brain uptake and/or specific binding of [11C]MPPO in the
brain could be insufficient brain penetration due to ATP-
binding cassette (ABC) efflux transporters, including P-
glycoprotein (PgP) and breast cancer resistance protein
(Bcrp) at the blood-brain barrier.40,55
13
internal standard. C NMR spectra were obtained at 100
or 125 MHz and 19F-NMR spectra were obtained at 376
MHz. Chemical shifts (δ) are reported in ppm and cou-
pling constants are reported in Hertz. The multiplicities
are abbreviated as follows: s = singlet, d = doublet, t =
triplet, m = multiplet, br = broad signal, dd = doublet of
doublets, etc. For all the HRMS measurements, the ioni-
zation method is ESI and the mass analyzer type is TOF.
In order to evaluate the interaction between [11C]MPPO
and ABC transporters, we carried out PET imaging studies
and compared radiotracer behavior, including brain up-
take and washout on wild-type and PgP/Bcrp knockout
mice (ABCB1a/1b-/-ABCG2-/-), in cerebral cortex and cere-
bellum regions. As shown in Figure 6, peak brain uptake
in cerebral cortex and cerebellum was 0.95 SUV and 1.04
SUV in PgP/Bcrp KO mice, respectively, representing a
marginal difference in comparison to the uptakes of cere-
bral cortex (0.74 SUV) and cerebellum (0.78 SUV) in wild
type controls. In addition, brain uptakes in cerebral cor-
tex and cerebellum of [11C]MPPO was not noticeably in-
creased in PgP/Bcrp KO mice compared with that of wild
type mice (ratio of KO/WT in AUC[cerebral cortex]= 1.2
and ratio of KO/WT in AUC[cerebellum]= 1.2); therefore
these results indicates [11C]MPPO lacks intensive interac-
tions with PgP/Bcrp efflux pump on the murine blood-
brain barrier and is not likely a substrate for Pgp/Bcrp in
mice.
2-((7-(2-methoxyphenyl)hept-6-en-1-yl)oxy)tetrahydro-2H-
pyran (18). Compound 16 (3.9 g, 14.7 mmol) and PPh3 (5.8
g, 22.3 mmol) were placed in an oven-dried reaction ves-
sel charged with a stir bar, which was filled with argon.
The mixture was heated at 120 °C for 4 h, then cooled
down to ambient temperature, followed by addition of
THF (40 mL). The resulting mixture was stirred at room
temperature for 10 min, then LiHMDS (1.0 M in THF, 18.4
mL, 18.4 mmol) was added slowly. The reaction was
stirred for 1 h and cooled down to 0 °C. A solution of 2-
methoxybenzaldehyde (1.8 g, 13.5 mmol) in THF (20 mL)
was added into the reaction. The mixture was stirred
overnight at room temperature, then diluted with EtOAc
(70 mL), and washed with H2O (30 mL x 1) and brine (30
mL x 1), dried over anhydrous Na2SO4, and concentrated.
The residue was purified by flash chromatography on sili-
ca gel (hexanes to ethyl acetate gradient column) to yield
the compound 18 as a yellow oil (2.3 g, 56% over two
1
steps). Rf = 0.5 (Hexanes/EtOAc = 50/1); H NMR showed
CONCLUSION
the compound exists as a 1:0.6 mixture of Z- and E- prod-
ucts, which are used for the next step without further
We have efficiently synthesized
a
new α-
ketoheterocyclic FAAH radioligand, [11C]MPPO, in good
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ACS Chemical Neuroscience
Page 8 of 21
separation; HRMS calcd for C19H29O3 (M + H)+ 305.2117,
found 305.2121.
39.0, 29.7, 29.4, 28.9, 28.8, 23.8; HRMS calcd for
C21H23N2O3 (M + H)+ 351.1709; found 351.1702.
1
2
3
4
5
6
7
8
9
7-(2-methoxyphenyl)-1-(5-(pyridin-2-yl)oxazol-2-yl)heptan-
1-one (11). To a solution of the compound 14 (219 mg, 1.5
mmol) in anhydrous THF (7 mL) at 0 °C was added
iPrMgCl (2 M in THF, 0.9 mL, 1.8 mmol), the mixture was
stirred at 0 °C for 1 h. A solution of compound 21 (330 mg,
1.5 mmol) in THF (4 mL) was added into the reaction, and
the mixture was allowed to room temperature and stirred
for 3 h and quenched with saturated aqueous NH4Cl (10
mL) and extracted with EtOAc (3 x 10 mL). The organic
layers were combined, washed with water (1 x 10 mL) and
brine (1 x 10 mL), dried over Na2SO4, filtered and evapo-
rated under reduced pressure. The residue was dissolved
immediately into CH2Cl2 (10 mL). Dess-Martin perio-
dinane (1.1 g, 2.6 mmol) was added to a solution and the
resultant suspension was stirred at rt for 2 h. Then the
reaction was quenched with saturated aqueous Na2SO3
solution (5 mL) and NaHCO3 solution (5 mL), and the
mixture was stirred for another 30 min. Then the aqueous
layer was extracted with CH2Cl2 (3 x 7 mL). The combined
organic portion was washed with brine (5 mL), dried over
Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel (hex-
anes to ethyl acetate gradient column) to yield the com-
pound 11 as a white solid (278 mg, 51% over two steps). Rf
= 0.7 (Hexanes/EtOAc = 2/1); 1H NMR (400 MHz, CDCl3) δ
8.68 (d, J = 4.8 Hz, 1H), 7.90-7.87 (m, 2H), 7.82 (td, J = 8.0,
1.6 Hz, 1H), 7.35-7.31 (m, 1H), 7.20-7.13 (m, 2H), 6.91-6.84
(m, 2H), 3.83 (s, 3H), 3.13 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 7.6
Hz, 2H), 1.85-1.77 (m, 2H), 1.66-1.58 (m, 2H), 1.50-1.40 (m,
12-bromo-1-(5-(pyridin-2-yl)oxazol-2-yl)dodecan-1-one (26).
To a solution of the compound 14 (500 mg, 3.42 mmol) in
anhydrous THF (15 mL) at 0 °C was added iPrMgCl (2 M
in THF, 2.1 mL, 4.2 mmol), the mixture was stirred at 0 °C
for 1 h. A solution of aldehyde 25 (1.1 g, 4.2 mmol) in THF
(10 mL) was added into the reaction, and the mixture was
allowed to room temperature and stirred for 3 h and
quenched with saturated aqueous NH4Cl (7 mL) and ex-
tracted with EtOAc (3 x 10 mL). The organic layers were
combined, washed with water (1 x 10 mL) and brine (1 x 10
mL), dried over Na2SO4, filtered and evaporated under
reduced pressure. The residue was dissolved immediately
into CH2Cl2 (30 mL). Dess-Martin periodinane (1.77 g, 4.18
mmol) was added to a solution and the resultant suspen-
sion was stirred at rt for 2 h. Then the reaction was
quenched with saturated aqueous Na2SO3 solution (7 mL)
and NaHCO3 solution (7 mL), and the mixture was stirred
for another 30 min. Then the aqueous layer was extracted
with CH2Cl2 (3 x 15 mL). The combined organic portion
was washed with brine (15 mL), dried over Na2SO4, fil-
tered, and concentrated in vacuo. The residue was puri-
fied by flash chromatography on silica gel (hexanes to
ethyl acetate gradient column) to yield the compound 26
as a white solid (997 mg, 72% over two steps). Rf = 0.25
10
11
12
13
14
15
16
17
18
19
20
21
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23
24
25
26
27
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35
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1
(Hexanes/EtOAc = 10/1); H NMR (400 MHz, CDCl3) δ
8.67-8.65 (m, 1H), 7.87-7.85 (m, 2H), 7.80 (td, J = 7.6, 1.6
Hz, 1H), 7.32-7.29 (m, 1H), 3.39 (t, J = 6.8 Hz, 2H), 3.10 (t, J
= 7.6 Hz, 2H), 1.88-1.80 (m, 2H), 1.79-1.73 (m, 2H), 1.42-1.27
13
(m, 14H); C NMR (100 MHz, CDCl3) δ 188.6, 157.4, 153.3,
13
4H); C NMR (100 MHz, CDCl3) δ 188.5, 157.4, 153.2, 150.1,
150.1, 146.3, 137.1, 126.8, 124.1, 120.4, 39.1, 34.1, 32.8, 29.4,
29.3, 29.2, 29.1, 28.7, 28.1, 24.0; HRMS calcd for
C20H28BrN2O2 (M + H)+ 407.1334; found 407.1331.
146.3, 137.1, 131.0, 129.7, 126.8, 124.1, 120.3, 120.2, 110.1, 55.2,
39.1, 30.0, 29.6, 29.2, 29.0, 23.9; HRMS calcd for
C22H25N2O3 (M + H)+ 365.1865; found 365.1859.
12-fluoro-1-(5-(pyridin-2-yl)oxazol-2-yl)dodecan-1-one (27).
A solution of 18-crown-6 (486 mg, 1.84 mmol) in anhy-
drous CH3CN (5.5 mL) was heated at 85 °C for 10 min,
then KF (110 mg, 1.88 mmol) was added into the hot solu-
tion. And the mixture was stirred at 85 °C for 20 min. Af-
ter that, compound 26 (150 mg, 0.37 mmol) was added,
and the vessel was sealed tightly. The mixture was stirred
at 95 °C for 42 h, then quenched with saturated aqueous
NH4Cl (10 mL) and extracted with Et2O (3 x 10 mL). The
combined organic portion was washed with brine (2 × 10
mL), dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was purified by flash chromatography
on silica gel (hexanes to ethyl acetate gradient column) to
yield the crude compound, which was recrystallized using
10% CH2Cl2/pentane to yield pure compound 27 as a
white solid (38 mg, 30%). Rf = 0.25 (Hexanes/EtOAc =
7-(2-hydroxyphenyl)-1-(5-(pyridin-2-yl)oxazol-2-yl)heptan-
1-one (23). To a solution of the compound 11 (428 mg, 1.17
mmol) in anhydrous CH2Cl2 (15 mL) at -78 °C was added
BBr3 (0.35 mL, 3.53 mmol), the mixture was allowed to
room temperature and stirred for 1 h, and quenched with
saturated aqueous NaHCO3 (7 mL) and 10% NaOH (7
mL). The aqueous layer was extracted with CH2Cl2 (3 x 7
mL). The combined organic portion was washed with
brine (10 mL), dried over Na2SO4, filtered, and concen-
trated in vacuo. The residue was purified by flash chroma-
tography on silica gel (hexanes to ethyl acetate gradient
column) to yield the crude product, which was re-
crystallized using 10% CH2Cl2/Hexanes to yield pure com-
pound 23 as a white solid (238 mg, 58%). Rf = 0.4 (Hex-
anes/EtOAc = 2/1); 1H NMR (500 MHz, CDCl3) δ 8.68 (d, J
= 4.8 Hz, 1H), 7.89-7.86 (m, 2H), 7.82 (td, J = 8.0, 1.6 Hz,
1H), 7.34-7.31 (m, 1H), 7.12-7.10 (m, 1H), 7.10-7.04 (m, 1H),
6.85 (t, J = 7.5 Hz, 1H), 6.76 (dd, J = 8.0, 0.5 Hz, 1H), 5.35-
5.30 (m, 1H), 3.11 (t, J = 7.0 Hz, 2H), 2.62 (t, J = 7.5 Hz, 2H),
1.81-1.77 (m, 2H), 1.68-1.63 (m, 2H), 1.45-1.42 (m, 4H); 13C
NMR (125 MHz, CDCl3) δ 188.5, 157.3, 153.5, 153.1, 150.0,
146.2, 137.1, 130.2, 128.5, 126.9, 126.8, 124.1, 120.6, 120.4, 115.1,
1
10/1); H NMR (400 MHz, CDCl3) δ 8.67-8.65 (m, 1H),
7.88-7.85 (m, 2H), 7.80 (td, J = 7.6, 2.0 Hz, 1H), 7.33-7.29
(m, 1H), 4.43 (dt, J = 47.2, 6.4 Hz, 2H), 3.11 (t, J = 7.2 Hz,
2H), 1.79-1.70 (m, 2H), 1.69-1.61 (m, 2H), 1.40-1.24 (m,
14H); 13C NMR (100 MHz, CDCl3) δ 188.6, 157.4, 153.3, 150.1,
146.3, 137.1, 126.8, 124.1, 120.4, 84.2 (d, JC-F = 163.1 Hz), 39.1,
30.5, 30.3, 29.4, 29.3, 29.2, 29.1, 25.1, 25.0, 24.0; 19F NMR
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ACS Chemical Neuroscience
light cycle and were allowed free access to food pellets
(376 MHz, CDCl3) δ -217.9; HRMS calcd for C20H28FN2O2
(M + H)+ 347.2135; found 347.2129.
and water. The animal experiments were approved by the
Animal Ethics Committee of Massachusetts General Hos-
pital and/or the National Institute of Radiological Scienc-
es.
1
Radiolabeling of [11C]MPPO. Carbon-11 CO2 was pro-
duced by 14N(p, α)11C nuclear reaction using a CYPRIS
HM18 cyclotron (Sumitomo Heavy Industry, Tokyo, Ja-
pan). [11C]Methyl iodide ([11C]CH3I) was synthesized from
cyclotron-produced [11C]CO2. Briefly, [11C]CO2 was bub-
bled into a solution of LiAlH4 (0.4 M in THF, 300 μL).
After evaporation, the remaining reaction mixture was
treated with hydroiodic acid (57% aqueous solution, 300
μL). The resulting [11C]CH3I was transferred under helium
gas with heating into a pre-cooled (-15 to -20 °C) reaction
vessel containing precursor 23 (1 mg), NaOH (6 μL, 0.5 M)
and anhydrous DMF (300 μL). After the radioactivity
reached a plateau during transfer, the reaction vessel was
warmed to 80 °C and maintained for 5 min. CH3CN /
0.05M NH4OAc (v/v, 2/8, 1 mL) was added to the reaction
mixture, which was then injected to a semi-preparative
HPLC system. HPLC purification was completed on a
Capcell Pak C18 column (10 mm ID × 250 mm; Shiseido,
Tokyo) using a mobile phase of CH3CN / 0.05M NH4OAc
(v/v, 2/8) at a flowrate of 5.0 mL/min. The retention time
for [11C]MPPO was 9.5 min, whereas that for unreacted 23
was 5.1 min. The radioactive fraction corresponding to the
desired product was collected in a sterile flask, evaporated
to dryness in vacuo, re-dissolved in sterile normal saline
(3 mL), and passed through a 0.22 μm Millipore filter. The
synthesis time was ca. 27 min from end-of-bombardment.
Radiochemical and chemical purity were measured by
analytical HPLC (Capcell Pak C18, 4.6 mm ID × 250 mm,
UV at 254 nm; CH3CN / 0.05M NH4OAc (v/v, 2/8) ; reten-
tion time 8.5 min, 1.0 mL/min). The identity of [11C]MPPO
was confirmed by the co-injection with unlabeled MPPO.
The specific activity of [11C]MPPO was calculated based on
a mass calibration curve at 254 nm. Radiochemical yield
was 41% decay-corrected based on [11C]CO2 with >99%
radiochemical purity and 2.2 Ci/μmol specific activity.
Measurement of Lipophilicity. The LogD value was
measured by mixing [11C]MPPO (radiochemical purity:
100%; approximately 150,000 cpm) with n-octanol (3.0 g)
and a sodium phosphate-buffered saline (PBS, 3.0 g; 0.1
M, pH 7.40) in a test tube. The tube was vortexed for 3
min at room temperature, followed by centrifugation at
3500 rpm for 5 min. An aliquot of 0.65 mL PBS and 0.65
mL n-octanol was removed, weighted, and its radioactivi-
ty was counted using a 1480 Wizard autogamma counter
(PerkinElmer, Waltham, MA), respectively. Each sample
from the remaining organic layer were removed and re-
partitioned until consistent LogD value was obtained. The
LogD value was calculated by comparing the ratio of
cpm/g of n-octanol to that of PBS and expressed as LogD
= Log [cpm/g (n-octanol)/cpm/g (PBS)]. All assays were
performed in triplicate.
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Biodistribution in mice.
A saline solution of
[11C]MPPO (2 MBq, 0.03 nmol/200 µL) was injected into
ddy mice via tail vein. Three mice were sacrificed at 1, 5,
15, 30, and 60 min after injection. Whole brain, heart,
lung, liver, spleen, kidneys, small intestine (including
contents), muscle, testes, and blood samples were quickly
removed and weighed. The radioactivity present in these
tissues was measured using a gamma counter, and all ra-
dioactivity measurements were decay corrected. The up-
take of each organ is expressed as a percentage of the in-
jected dose per gram of wet tissue (% ID/g).
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Small-animal PET study in rats. PET scans were per-
formed using a small-animal Inveon PET scanner (Sie-
mens Medical Solutions USA, Knoxville, TN), which pro-
vides 159 transaxial slices 0.796 mm (center to center)
apart, a 10 cm transaxial field of view (FOV), and a 12.7 cm
axial FOV. Prior to imaging studies, Sprague-Dawley rats
were anesthetized with 5% (v/v) isoflurane and main-
tained by 1-2% (v/v) isoflurane. To inject [11C]MPPO, a 24-
gauge needle with catheter (Terumo Medical Products,
Tokyo) was placed into the rat tail vein. Emission scans
were acquired for 90 min in three-dimensional list mode
with an energy window of 350-750 keV, immediately after
intravenous injection of [11C]MPPO (40-51 MBq, 0.7-1
nmol/200 μL). For the pretreatment studies, unlabeled
URB597 (3.0 mg/kg) dissolved in 300 μL saline containing
10% ethanol and 5% Tween® 80) were injected at 30 min
before the injection of [11C]MPPO. Three rats were used
for each experiment.
PET dynamic images were reconstructed with filtered
back-projection using a Hanning’s filter, a Nyquist cutoff
of 0.5 cycle/pixel. The PET images were reconstructed
using ASIPro VM software (Analysis Tools and System
Setup/Diagnostics Tool, Siemens Medical Solutions). All
list-mode acquisition data were sorted into three-
dimensional sinograms, which were then Fourier re-
binned into two-dimensional sinograms (1 min × 4 scans,
2 min × 8 scans, 5 min × 8 scans). Regions of interest were
placed on the whole brain, cerebral cortex and cerebellum
using ASIPro VM. Regional brain uptake of radioactivity
was decay-corrected to the injection time and was ex-
pressed as the standardized uptake value (SUV), which
was normalized to the injected radioactivity and body
weight. SUV = (radioactivity per mL tissue/injected radio-
activity) × g body weight.
Small-animal PET study in mice. A wide-type or
Pgp/Bcrp knockout FAB mouse was secured in a custom-
designed chamber, placed in the Inveon PET scanner, and
prepared as described above. A 29-gauge needle with 12–
15 cm of polyethylene 10 tube prepared in house was
placed into the tail vein of mouse to inject [11C]MPPO (18
MBq, 0.12 nmol/100 µL). A dynamic emission scan in 3D
list mode was performed for 60 min (1 min × 4 scans, 2
min × 8 scans, 5 min × 8 scans). Three mice were used for
Animal experiments. DdY mice (male; 7 weeks old;
34–36 g), and Sprague-Dawley (male; 7 weeks old; 210–230
g) rats were purchased from Japan SLC (Shizuoka, Japan).
Wild-type (male; 17–18 weeks old; 30–32 g) and Pgp/Bcrp
knockout (Abcb1a/1b-/-Abcg2-/-; male; 17–18 weeks old; 31–
33 g) FAB mice were purchased from Taconic Farm (Hud-
son, NY). These animals were housed under a 12 h dark-
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PET, positron emission tomography; AEA, anandamide; 2-
each experiment, respectively. The PET data were treated
as above.
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3
4
5
6
7
8
AG, 2-arachidonoylglycerl; FAAH, fatty acid amide hydrolase;
MAGL, monoacylglycerol lipase; SUV, standardized uptake
value, TAC, time-activity curve; %ID/g, percentage of inject-
ed dose per gram of wet tissue; SUV, standard uptake value;
KO, knockout; PgP, P-glucoprotein; Bcrp, breast cancer re-
Metabolite assay for rat plasma and brain homog-
enate. Following the intravenous injection of [11C]MPPO
(82 MBq, 1.1 nmol/200 µL), Sprague-Dawley rats (n = 3)
were sacrificed by cervical dislocation at 15 min. Blood
and whole brain samples were quickly removed and the
blood samples were centrifuged at 15,000 g for 2 min at 4
°C to separate the plasma. The supernatant (0.5 mL) was
then collected in a test tube containing CH3CN (0.5 mL)
and the resulting mixture was vortexed for 15 s and centri-
fuged at 15,000 g for 2 min for deproteinization. The rat
brain was homogenized in an ice-cooled CH3CN/H2O (1/1,
1 mL) solution. The homogenate was centrifuged at
150,000 rpm for 2 min at 4 °C and the supernatant was
collected. The recovery of radioactivity into the superna-
tant was > 90% based on the total radioactivity in the
brain homogenate.
sistance
hexaoxacyclooctadecane;
protein;
18-crown-6,
PPTS,
1,4,7,10,13,16-
Pyridinium p-
toluenesulfonate; LiHMDS, Lithium bis(trimethylsilyl)amide;
THF, tetrahydrofuran; DMF, dimethylformamide; DMSO,
Dimethyl sulfoxide.
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An aliquot of the supernatant (100–500 µL) obtained
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time: 4.6 min) to total radioactivity (corrected for decay)
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ASSOCIATED CONTENT
Supporting Information
Characterization of all new compounds. This material is
available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Tel: +81 433 823 709. Fax: +81-43-206-3261. E-mail:
zhang@nirs.go.jp.
* Tel: +1 617 726 6106. Fax: +1 617 726 6165. E-mail:
liang.steven@mgh.harvard.edu.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We would like to thank the staff at the radiochemistry pro-
gram, Nuclear Medicine and Molecular Imaging, Massachu-
setts General Hospital, MA, USA and National Institute of
Radiological Sciences, Chiba, Japan for their support with
cyclotron operation, radioisotope production, radiosynthesis
and animal experiments. S.H.L is a recipient of an NIH career
development award (NIH/NIDA K01DA038000).
ABBREVIATIONS
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Figure 1.Representative PET radiotracers for imaging FAAH.
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Figure 4.Reversible binding of [11C]MPPO in the brain. (n = 3; mean ± SD).
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Scheme 1. Mechanism of action and this work.
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Scheme 3. Radiolabeling of FAAH radiotracers.
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