Synthesis and evaluation of novel carbon-11 labeled oxopurine analogues for positron emission tomography imaging of translocator protein (18 kDa) in peripheral organs

Article abstract of DOI:10.1021/jm200516a

To develop a PET ligand for imaging TSPO in peripheral organs, we designed three novel oxopurine analogues [¹¹C]3a-c (LogD: 1.81-2.17) by introducing a pyridine ring in place of a benzene ring in the lead compound [¹¹C]2 (LogD: 3.48). The desmethyl precursors 10 for radiosynthesis were synthesized by reacting glycine 7 with picolylamines 4, followed by hydrolysis and by Curtius rearrangement with diphenylphosphoryl azide. Methylation of 10a-c with methyl iodide produced unlabeled compounds 3a-c. The radiosynthesis of [¹¹C]3a-c was performed by reacting 10a-c with [¹¹C]methyl iodide. Compounds 3a-c displayed high or moderate in vitro binding affinities (K_i: 5-40 nM) for TSPO. PET with [ ¹¹C]3a-c in rats showed high uptake in the lung, heart, and kidney, which are organs with high TSPO expression. Metabolite analysis with [ ¹¹C]3a showed that radioactivity in these organs mainly corresponded with unchanged [¹¹C]3a. PET with [¹¹C]3a using a rat model of lung inflammation showed a significant signal in the lipopolysaccharide- treated lung.

Full text of DOI:10.1021/jm200516a

ARTICLE
pubs.acs.org/jmc
Synthesis and Evaluation of Novel Carbon-11 Labeled Oxopurine
Analogues for Positron Emission Tomography Imaging of
Translocator Protein (18 kDa) in Peripheral Organs
Katsushi Kumata,† Joji Yui,† Akiko Hatori,† Masayuki Fujinaga,† Kazuhiko Yanamoto,† Tomoteru Yamasaki,†
Kazunori Kawamura,† Hidekatsu Wakizaka,† Nobuki Nengaki,†,‡ Yuichiro Yoshida,†,‡ Masanao Ogawa,†,‡
Toshimitsu Fukumura,† and Ming-Rong Zhang*,†
†Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku,
Chiba 263-8555, Japan
‡SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan
S Supporting Information
b
ABSTRACT: To develop a PET ligand for imaging TSPO in
peripheral organs, we designed three novel oxopurine analogues
[11C]3aꢀc (LogD: 1.81ꢀ2.17) by introducing a pyridine ring
in place of a benzene ring in the lead compound [11C]2 (LogD:
3.48). The desmethyl precursors 10 for radiosynthesis were
synthesized by reacting glycine 7 with picolylamines 4, followed
by hydrolysis and by Curtius rearrangement with diphenylpho-
sphoryl azide. Methylation of 10aꢀc with methyl iodide
produced unlabeled compounds 3aꢀc. The radiosynthesis of [11C]3aꢀc was performed by reacting 10aꢀc with [11C]methyl
iodide. Compounds 3aꢀc displayed high or moderate in vitro binding affinities (Ki: 5ꢀ40 nM) for TSPO. PET with [11C]3aꢀc in
rats showed high uptake in the lung, heart, and kidney, which are organs with high TSPO expression. Metabolite analysis with
[11C]3a showed that radioactivity in these organs mainly corresponded with unchanged [11C]3a. PET with [11C]3a using a rat
model of lung inflammation showed a significant signal in the lipopolysaccharide-treated lung.
’ INTRODUCTION
PET ligand used for imaging TSPO in neuroinflammation.10
However, [11C]1 had several limitations, such as relatively low
brain uptake, high nonspecific binding, and high lipophilicity.11
To investigate TSPO precisely using a PET ligand with improve-
ment over [11C]1, several dozen 11C and 18F-labeled radio-
ligands have been reported.12ꢀ16 We have developed a new PET
ligand, N-benzyl-N-ethyl-2-(7,8-dihydro-7-[11C]methyl-8-oxo-
2-phenyl-9H-purin-9-yl)acetamide ([11C]AC-5216, [11C]2;
Scheme 1), for imaging TSPO in the human brain.17,18 However,
[11C]2, like [11C]1, had rather slow kinetics in the brain and did
not show a decrease of radioactivity in the brain within the PET
scan, which limited its usefulness for quantitative analysis of
TSPO.18,19 High lipophilicity (LogD: 3.48) and potent binding
affinity (Ki: 0.6 nM for TSPO)20 of [11C]2 were considered the
partial causes of the shortcomings.
Translocator protein (18 kDa) (TSPO, formerly known as the
peripheral-type benzodiazepine receptor, PBR) is located on the
mitochondrial outer membrane in several organs, including the
lung, heart, kidney, and endocrine organs such as the adrenal,
testis, and pituitary gland.1 A low level of TSPO was also found in
microglia cells of the brain.1ꢀ3 It has been demonstrated that
TSPO has wide functions related to the regulation of cholesterol
transport and synthesis of steroid hormones, porphyrin transport
and heme synthesis, apoptosis, cell proliferation, anion transport,
regulation of mitochondrial functions, immunomodulation, and
inflammation.1,3
Visualization of the immune response with molecular imaging
probes specific to immune cells could enhance understanding of
the cellular mechanism of various inflammatory diseases. Ima-
ging probes using various modalities have been developed to
visualize and detect autoimmune disease, neuroinflammation,
inflammatory arthritides, cardiovascular system, and cancer.4ꢀ7
Among these imaging probes, positron emission tomography
(PET) ligands for TSPO are becoming potential and specific
tools which have been used to visualize inflammation and
elucidate the relationalship between TSPO and various inflam-
matory diseases.5ꢀ9
In this study, using [11C]2 as a lead compound, we designed
three novel oxopurine analogues [11C]3aꢀc (Scheme 1) and
evaluated their potential as PET ligands for imaging TSPO. As
shown in the chemical structures, a pyridine ring was introduced
into [11C]3aꢀc instead of a benzene ring in [11C]2. This
structural change may decrease lipophilicity of [11C]3aꢀc, so
these radioligands may be expected to show improved kinetics
(R)-(1-(2-Chlorophenyl)-N-[11C]methyl,N-(1-methylpropyl)-
isoquinoline ([11C](R)-PK 11195, [11C]1, Scheme 1) was the first
Received: April 28, 2011
Published: July 26, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
and have lower nonspecific binding in the targeted tissues than
[11C]2.
using PET with [11C]3a in a rat model which was induced by
lipopolysaccharide (LPS).
On the other hand, with the recognition that TSPO plays an
important role in regulating immune response,1,3,4 we used
[11C]3aꢀc to elucidate in vivo specific binding for TSPO in
the peripheral organs and to visualize lung inflammation in the
present study. To our knowledge, there were few reports of PET
studies with TSPO radioligands for imaging lung inflammation.21,22
Because TSPO is present in the peripheral organs at high density
(Bmax: 141 pmol/mL in lung, 393 pmol/mL in heart, and 158
pmol/mL in kidney),23 moderate binding affinity of a PET ligand
may be enough to determine in vivo specific binding for TSPO in
these organs.
Here, we report: (1) the chemical synthesis of novel unlabeled
compounds 3aꢀc, (2) the radiosynthesis of [11C]3aꢀc, (3) the
in vitro binding affinity (Ki) with TSPO and lipophilicity, (4) the
kinetics and in vivo specific binding for TSPO in the peripheral
organs of rats using small-animal PET with [11C]3aꢀc, (5) the
in vivo metabolite analysis with [11C]3a in the plasma, liver, lung,
heart, and kidney, and (6) the visualization of lung inflammation
’ RESULTS AND DISCUSSION
Chemistry. Novel oxopurine analogues 3aꢀc were synthe-
sized as shown in Scheme 2. Picolylamines 4aꢀc were synthesized
by reducing Schiff’s base6, which was prepared by condensation of
pyridinecarboxaldehyde 5 with methyl- or ethylamine. The glycine
compound 7, which was prepared according to procedures
described previously,24 reacted with 4aꢀc in the presence of
(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexa-
fluorophosphate (BOP) to afford amide 8. Hydrolysis of 8 with
NaOH gave carboxylic acid 9, followed by Curtius rearrange-
ment with diphenylphosphoryl azide to produce 10 in chemical
yields of 46ꢀ73%. The unlabeled compounds 3aꢀc were
prepared by reacting 10 with methyl iodide (MeI) in N,N-
dimethylforamide (DMF) using K2CO3 as a base.
Radiosynthesis of [11C]3aꢀc was performed using a home-
made automated synthesis system.25 The labeling agent
[11C]methyl iodide ([11C]MeI) was prepared by reducing
cyclotron-produced [11C]carbon dioxide ([11C]CO2) with
lithium aluminum hydride (LiAlH4), followed by iodination with
57% hydroiodic acid.25,26 The produced [11C]MeI was purified
by distillation and trapped in a DMF solution of desmethyl
precursor 10aꢀc and NaOH (Scheme 3). [11C]Methylation of
10aꢀc with [11C]MeI proceeded efficiently for 5 min at 60 °C.
HPLC purification of the reaction mixtures gave [11C]3a with
31 ( 9% (n = 7), [11C]3b with 38 ( 11% (n = 7), and [11C]3c
with 25 ( 6% (n = 6) radiochemical yields (decay-corrected)
based on [11C]CO2, respectively (Supporting Information: Figure 1).
Starting from 11.2ꢀ16.3 GBq of [11C]CO2, 1.2ꢀ2.3 GBq of
[11C]3aꢀc was produced with 22 ( 3 min (n = 20) of synthesis
times from the end of bombardment. Identities of these radio-
active products were confirmed by coinjection with unlabeled
3aꢀc on the analytic HPLC chromatograms. In the final
formulated solutions, the radiochemical purity of [11C]3aꢀc
was higher than 99% and specific activity was 70ꢀ165 GBq/
μmol, as determined by comparison of the assayed radioactivity
Scheme 1. Chemical Structures of PET Ligands for TSPO
Scheme 2. Syntheses of 3aꢀca
a Reagents and conditions: (a) methyl- or ethylamine, ethanol, room temperature, 10 h; (b) NaBH4, ethanol, room temperature, 12 h; (c) BOP, DMF,
room temperature, 3 h; (d) NaOH, ethanol, room temperature, 12 h; (e) diphenylphosphoryl azide, Et3N, DMF, 100 °C, 6h;(f) MeI, NaH,DMF, 50°C, 5 h.
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Scheme 3. Radiosyntheses of [11C]3aꢀca
values of [11C]3aꢀc lie in the range normally considered as a
favorable PET ligand.28 The decreased lipophilicity may improve
kinetics and reduce nonspecific binding in the targeted
regions while performing PET imaging with [11C]3aꢀc.
Small-Animal PET Studies. The in vivo uptake and kinetics in
the peripheral organs of rats were examined using small-animal
PET with [11C]3aꢀc.
Figure 1 shows the PET summation images between 0 and 30
min after intravenous injection of [11C]3a (A), [11C]3b (B), or
[11C]3c (C) in the lung, heart, kidney, liver, and cardiac blood
pool of the rat whole body. Higher uptake was found in the lung,
heart, and kidney than in the liver. This distribution pattern
of radioactivity was consistent with the distribution of TSPO
expression in the peripheral organs of rodent animals.23 As seen
in these images, radioactivity also accumulated in the small
intestine, suggesting a putative excreting pathway after the
radioligands were injected to the rats.
a Reagents and conditions: [11C]MeI, DMF, 60 °C, 5 min.
Table 1. In Vitro Binding Affinity (Ki) for TSPO and
Lipophilicity
Figure 2 shows the timeꢀactivity curves (TACs) in all
examined regions of the rat whole body after injection of
[11C]3a (A), [11C]3b (B), or [11C]3c (C). The uptake of
radioactivity in the heart, lung, and kidney peaked at 1ꢀ3 min
after injection and then decreased with time in all regions. The
radioactivity level in the lung, heart, and kidney decreased from
3.4ꢀ5.0 SUV, 4.6ꢀ5.5 SUV, and 3.3ꢀ4.0 SUV at 2 min to
0.5ꢀ0.8 SUV, 0.9ꢀ2.0 SUV, and 1.0ꢀ2.0 SUV at 27.5 min after
injection. On the other hand, uptakes in the cardiac blood pool
and liver decreased from 3.0ꢀ3.4 SUV and 1.6ꢀ2.0 SUV at
2 min after injection to 0.4ꢀ0.9 SUV and 0.5ꢀ1.2 SUV at the
end of PET scans.
For comparison, the TACs in the corresponding regions were
determined after injection of [11C]2 (Figure 2D). The radio-
activity in the lung peaked at 1 min (7.6 SUV) after injection and
thereafter decreased to 2.3 SUV at 27.5 min. In the heart and
kidney, radioactivity peaked at 10 min (heart, 4.2 SUV; kidney,
2.8 SUV), respectively. After the peaked uptake, radioactivity in
both organs as well as in the blood and liver remained almost
the same level until the end of the PET scans. On the other
hand, PET with [11C]1 showed a similar kinetics to [11C]2
(Supporting Information: Figure 3).
ligand
R1
R2
Ki (nM)a
cLogDc
LogDb
3a
3b
3c
2
CH3CH2
CH3CH2
CH3
4-pyridyl
3-pyridyl
3-pyridyl
5.1 ( 0.3
13.2 ( 0.8
40.0 ( 6.3
0.6 ( 0.1
0.7 ( 0.0
2.38
2.38
1.85
3.32
5.10
2.03
2.17
1.81
3.48
3.74
1
a Values of in vitro binding affinity are the mean ( SD measured in
duplicate brain homogenates. b cLogD values were calculated with Pallas
3.4 software. c LogD values were determined in the octanol/phosphate
buffer (pH = 7.40) by the shaking flash method (n = 3; maximum
range, ( 5%).
with the mass measured from the carrier UV peak on HPLC
(Supporting Information: Figure 2). No significant peaks corre-
sponding to 10aꢀc were observed in the HPLC chromatograms
of final products. [11C]3aꢀc did not show radiolysis at room
temerature for 90 min after formulation, indicating radiochemi-
cal stability within the time of at least one PET scan.
During the present PET scans, it was found that [11C]3aꢀc
displayed improved kinetics in the lung, heart, and kidney in
comparison with [11C]2 and [11C]1. This result was partly due to
the lower lipophilicity of [11C]3aꢀc (LogD: 1.81ꢀ2.17) than
[11C]2 (LogD: 3.48) and [11C]1 (LogD: 3.74). Introduction of a
pyridine ring to [11C]3aꢀc contributed to the decrease in
lipophilicity and washout of radioactivity from the lung, heart,
and kidney. The rank order of the decreasing rate of radioactivity
in these organs after the radioligand injection was [11C]3a <
[11C]3b < [11C]3c. Among the three radioligands, [11C]3a
showed the highest uptake and moderate kinetics in the lung,
heart and kidney.
To determine in vivo specific binding for TSPO in the
peripheral organs, we performed inhibitory experiments using
TSPO-selective ligand 2 or 1. Figure 3 shows the TACs after
injection of [11C]3aꢀc by pretreatment with 2 (AꢀC) or 1
(DꢀF) in all regions examined. In the 2-treated groups as
compared to the control, the uptake of radioactivity in the heart,
lung, and kidney fell to low levels immediately after injection.
Significant inhibition by 1, a standard TSPO ligand, was also
examined in the lung, heart, and kidneys within the PET scans.
On the other hand, the liver uptake by treatment with 1 or 2
increased about 1.5-fold, compared to the control at 27.5 min after
In Vitro Binding Assays. In vitro binding affinities (Ki) of
3aꢀc for TSPO were measured from competition for the binding
of TSPO-selective [11C]1 using rat brain homogenates. As
shown in Table 1, 3aꢀc showed high or moderate binding
affinity (Ki: 5ꢀ40 nM) for TSPO, although the affinity was
weaker than those of 1 and 2. Lower electron density and
lipophilicity of the pyridine ring in 3aꢀc than those of the
benzene ring in 2 may be the main causes of the decrease in the
binding affinity for TSPO. The rank order of Ki was 3a > 3b > 3c,
suggesting that N-ethyl-N-(4-pyridylmethyl)amino unity is pre-
ferred for binding to TSPO. Because TSPO is present in the lung,
heart, and kidney with a high density (Bmax > 140 pmol/mL),24
the Ki values of 3aꢀc may be enough to elucidate the in vivo
specific binding for TSPO in the peripheral organs using PET
with [11C]3aꢀc.
Computation and Measurement of Ligand Lipophilicities.
The computed values of lipophilicities at pH = 7.4 (cLogD) for
[11C]3aꢀc were 1.85ꢀ2.38 (Table 1). These values were similar
to the corresponding measured values (LogD) of 1.81ꢀ2.17,
which were measured by the Shake Flask method.27 The lipophilicity
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Figure 1. Representative coronal PET images of rat whole body at the levels of the lung, heart, kidney, liver, and cardiac blood pool acquired between
0 and 30 min after intravenous injection of [11C]3a ((A) 16 MBq, 0.15 nmol), [11C]3b ((B) 17 MBq, 0.18 nmol), and [11C]3c ((C) 16 MBq, 0.13 nmol)
in the isofurane-anesthetized rat, which was placed in the prone position. L and R indicate left and right side of the rats.
Figure 2. Timeꢀactivity curves in the isoflurane anesthetized rat lung (b), heart (2), kidney (9), liver (]), and cardiac blood pool (4) after
intravenous injection of [11C]3a (A), [11C]3b (B), [11C]3c (C), and [11C]2 (D). PET scans were performed for 30 min after the injection of each
radioligand. Data are the means ( SD (n = 4).
injection. The increased liver uptake may be due to displacement of
specially bound radioligand from heart, lung, kidney, etc., by 1 and 2.
The maximum inhibitory percentages accounting for total
uptakes in the organs by treatment with 2 or 1 during PET scans
with [11C]3aꢀc were calculated (Supporting Information: Table 1).
High inhibitory percentages were determined in the lung
(66ꢀ85%), heart (80ꢀ92%), and kidney (55ꢀ74%). These
results indicated the presence of high in vivo specific binding
for TSPO in the peripheral organs. The rank order of specific
binding for TSPO in these organs was [11C]3a > [11C]3b >
[11C]3c. The in vivo specific binding level of [11C]3aꢀc for
TSPO was proportional to the sequence of their in vitro binding
affinity (Table 1). Among the three radioligands, [11C]3a,
showing the highest binding affinity for TSPO, exhibited the
highest in vivo specific binding for TSPO in the peripheral
organs. Because of its in vitro binding affinity, kinetics, and
in vivo specific binding, [11C]3a was selected for further
investigation.
Metabolite Analysis. In the imaging study, the presence of
significant quantities of radiolabeled metabolites in the target
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Figure 3. Timeꢀactivity curves in the isoflurane anesthetized rat lung (b), heart (2), kidney (9), liver (]), and cardiac blood pool (4) after
intravenous injection of [11C]3a (A,D), [11C]3b (B,E), and [11C]3c (C,F) in pretreatment experiments, in which unlabeled 2 (1 mg/kg, AꢀC) or 1 (3
mg/kg, DꢀF) was given at 1 min before radioligand. PET scans were performed for 30 min after the injection of each radioligand. Data are the means (
SD (n = 4).
Table 2. Percentages of Unchanged [11C]3a in the Plasma
tissues may produce an insurmountable barrier to the proper
quantification of images and interpretation of the results.
and Peripheral Tissues of Rats at 30 min after Injectiona
Here, we performed metabolite analysis in the plasma, heart,
% of unchanged
lung, liver, and kidney of rats at 30 min after injection of
tissue
[
11C]3a
[11C]3a.
Table 2 shows the percentages of unchanged [11C]3a
plasma
liver
23.4 ( 0.8
43.6 ( 1.2
92.1 ( 2.2
93.8 ( 1.4
94.7 ( 3.5
(retention time (tR): 7.8 min) in these tissues, as determined
by analytical radio-HPLC. In addition to the unchanged [11C]3a,
a polar radioactive peak (tR: 2.6 min) corresponding to a
radiolabeled metabolite appeared on the front line of HPLC
chromatograms of the plasma and liver. On the other hand, more
than 90% of the total radioactive component was identified as the
unchanged [11C]3a in the lung, heart, and kidney at 30 min after
injection, respectively. This result indicated that despite the
presence of radiolabeled metabolite in the plasma and liver, the
specific binding for TSPO examined in the lung, heart, and
kidney was mainly due to [11C]3a itself and was not influenced
by the radiolabeled metabolite. This finding further supported
that [11C]3a is a potent PET ligand for imaging TSPO in the
lung, heart, and kidney.
lung
heart
kidney
a Data are the means of ( SD (n = 3) in each tissue.
organs, we performed PET study with [11C]3a using a rat model
of lung inflammation. By injecting LPS (5 mg/kg) intratrache-
ally to rats, inflammation was induced in the lungs.29,30 It has
been reported that TSPO expression was augmented in the
lungs of the LPS-treated rats, although the cellular sources
enriching TSPO expression in the lung have not been deter-
mined clearly.30,31
Visualization of Lung Inflammation. To elucidate the
Figure 4 shows representative PET summation images of LPS-
treated (A) and control (vehicle) (B) rat lungs between 0 and
usefulness of [11C]3a for imaging inflammation in the peripheral
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’ SUMMARY
To visualize TSPO in the peripheral organs and lung inflam-
mation, three novel oxopurine analogues 3aꢀc were developed
and labeled with 11C. [11C]3aꢀc were synthesized by [11C]-
methylation of the desmethyl precursor 10aꢀc with [11C]MeI in
high radiochemical yields. Compounds 3aꢀc exhibited high or
moderate in vitro binding affinity (Ki: 4ꢀ50 nM) with TSPO and
suitable lipophilicity (LogD: 1.81ꢀ2.17) as a favorable PET
ligand. Compared to lead compound [11C]2, PET with [11C]-
3aꢀc demonstrated that the kinetics in the lung, heart, and
kidney of rats had improved. Among the three radioligands,
[11C]3a had the highest in vitro binding affinity for TSPO,
uptake, and in vivo specific binding and displayed moderate
kinetics in the lung, heart, and kidney. Only unchanged [11C]3a
was found in these organs despite the presence of radiolabeled
metabolite in the plasma and liver at 30 min after the radioligand
injection.
Figure 4. Representative coronal PET images of LPS-treated (A) and
control (B) lungs acquired between 0 and 30 min after intravenous
injection of [11C]3a (17 MBq, 0.15 nmol). Timeꢀactivity curves (C)
were obtained in LPS-treated (O) and control (b) lung of inflammatory
rats after injection of [11C]3a. Data are the means ( SD (n = 4).
PET with [11C]3a provided an observatory signal in the lung
of LPS-treated rats. Thus, [11C]3a may be a useful PET ligand for
the visualization of TSPO-related disease such as inflammation.
Because of the suitable kinetics, the utilization of PET with
[11C]3a would contribute to the study of TSPO function and
quantitative evaluation of in vivo binding parameters and recep-
tor occupancy of TSPO therapeutic compounds.
Table 3. Biodistribution (% ID/g Tissue: mean ( SD, n = 4)
in Rats after Injection of [11C]3a
tissue
control
LPS-treated
blood
0.06 ( 0.00
0.02 ( 0.00
1.65 ( 0.12
2.10 ( 0.22
0.53 ( 0.04
1.78 ( 0.12
1.88 ( 0.16
0.38 ( 0.04
7.49 ( 2.55
1.57 ( 0.46
0.11 ( 0.03
0.08 ( 0.01
0.02 ( 0.00
3.54 ( 0.73
2.17 ( 0.17
1.03 ( 0.28
1.97 ( 0.15
1.88 ( 0.37
0.37 ( 0.07
5.27 ( 1.32
2.68 ( 1.36
0.10 ( 0.01
brain
lung
’ EXPERIMENTAL SECTION
heart
liver
Materials and Methods. All chemical reagents and solvents were
purchased from commercial sources (Sigma-Aldrich, MO; Wako Pure
Chemical Industries, Osaka, Japan; Tokyo Chemical Industries, Tokyo,
Japan) and used as supplied. Compound 1 was purchased from Tocris
Bioscience (Bristol, UK) and 2 was prepared in our laboratory.17 1H
NMR (300 MHz) spectra were recorded on a JEOL-AL-300 spectro-
meter (JEOL, Tokyo) with tetramethylsilane as an internal standard. All
chemical shifts (δ) were reported in parts per million (ppm) downfield
relative to the chemical shift of tetramethylsilane. Signals are quoted as s
(singlet), d (doublet), dd (double doublet), dt (double triplet), dq
(double quartet), t (triplet), q (quartet), or m (multiplet). Fast atom
bombardment mass spectra (FAB-MS) and high-resolution mass
spectra (HRMS) were obtained on a JEOL-NMS-SX102 spectrometer
(JEOL) and recorded on the spectrometer. Melting points were
measured using a micro melting point apparatus (MP-500P, Yanaco,
Tokyo) and are uncorrected. Column chromatography was performed
using Wako gel C-200 (70ꢀ230 mesh). High performance liquid
chromatography (HPLC) was performed using a JASCO HPLC system
(JASCO, Tokyo). Chemical purity (g95%) of compounds was assayed
by analytical HPLC (10: Capcell Pack C18, 4.6 mm ID ꢁ 250 mm,
UV at 254 nm, MeCN/H2O 4/6. 3: Capcell Pack C18, 4.6 mm ID ꢁ
250 mm, UV at 254 nm, MeCN/H2O 4.5/5.5.). Carbon-11 (11C) was
produced by the 14N (p,R) 11C nuclear reaction using a CYPRIS HM-18
cyclotron (Sumitomo Heavy Industry, Tokyo). If not otherwise stated,
radioactivity was measured with an IGC-3R Curiemeter (Aloka, Tokyo).
Radioligands [11C]1 and [11C]2 were synthesized according to the
procedures described previously.10,17 In radio-HPLC purifiaction and
analysis, effluent radioactivity was monitored using a NaI (Tl) scintilla-
tion detector system.
kidney
spleen
pancreas
adrenal
small intestine
muscle
30 min after injection of [11C]3a. The accumulation of radioactivity
was observed in the lungs of LPS-treated rats, whereas no such
accumulation was found in the lungs of control rats. Differences
in the TACs were significant between LPS-treated and control
lungs (Figure 4C). As calculated from the TACs, the values
of area under TACs between 0 and 30 min (AUC0ꢀ30 min
=
SUV ꢁ min) in the lungs were 43.5 ( 3.5 for the control and
118.6 ( 11.5 for the LPS-treated rats, respectively. On the
other hand, the corresponding values of AUC0ꢀ30 min in the
lungs for [11C]1 were 62.8 ( 4.0 for the control and 86.0 ( 2.4
for the LPS-treated rats, respectively (Supporting Informa-
tion: Figure 4).
To support the present PET results, the radioactivity concen-
trations were measured in the control and LPS-treated rats by the
dissection method. Table 3 shows the radioactivity in various
tissues of two groups of rats at 30 min after injection of [11C]3a.
The radioactivity level in LPS-treated lungs was 2.1-fold higher
than that in control lungs, which was similar to the PET result. In
addition to the lungs, increased radioactivity was seen in the
liver and small intestine. No significant difference in uptake in
the other regions was determined between the control and LPS-
treated rats. PET study with [11C]3a for LPS-treated rats
showed that radioactivity increased in the lung in response to
lung inflammation. Radioactive signals in LPS-treated lungs
may be due to the increase in TSPO expression compared to the
control lungs.
Chemistry. N-Ethyl-4-pyridylmethylamine (4a). A mixture of
4-pyridinecarboxaldehyde (5a; 1.52 g, 10 mmol) and ethylamine
(3 mL) in ethanol (5 mL) was stirred for 10 h at room temperature.
After the reaction, NaBH4 (0.38 g, 10 mmol) was added to this mixture.
The reaction mixture was continuously stirred at room temperature for
12 h and then evaporated in vacuo to remove ethanol. The residue was
extracted with dichloromethane (CH2Cl2) and water, and the organic
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Journal of Medicinal Chemistry
ARTICLE
layer was dried over Na2SO4 and evaporated. The crude product was
purified by silica gel column chromatography (CH2Cl2/methanol: 10/1)
to give 4a (2.30 g, 70.6%) as a colorless liquid. 1H NMR (CDCl3, δ):
8.54 (2H, d, J = 4.4 Hz), 7.26 (2H, d, J = 5.1 Hz), 3.81 (2H, s), 2.68 (2H,
q, J = 7.0 Hz), 1.14 (3H, t, J = 7.0 Hz). FABꢀMS: m/z 137 (M + H).
N-Ethyl-3-pyridylmethylamine (4b). The procedure described for
the synthesis of 4a was applied to 3-pyridinecarboxaldehyde (5b) and
ethylamine to give 4b (73.4%) as a colorless liquid. 1H NMR (CDCl3,
δ): 8.56 (1H, s), 8.50 (1H, d, J = 3.7 Hz), 7.68 (1H, d, J = 7.7 Hz),
7.23ꢀ7.30 (1H, m), 3.81 (2H, s), 2.69 (2H, q, J = 7.0 Hz), 1.14 (3H, t,
J = 7.0 Hz). FABꢀMS: m/z 137 (M + H).
4-(2-(N-Methyl,N-3-pyridylmethylamino)-2-oxoethylamino-2-phenyl-
pyrimidine-5-carboxylic Acid (9c). The procedure described for the
synthesis of 9a was applied to 8c to give 9c (68.5%) as a colorless solid;
mp 154ꢀ155 °C. 1H NMR (DMSO-d6, δ): 8.96ꢀ9.04 (2H, m),
8.53ꢀ8.61 (2H,m), 8.31ꢀ8.46 (2H, m), 7.45ꢀ7.68 (4H, m), 7.19ꢀ7.32
(1H, m), 4.67 (2H, d, J = 7.3 Hz), 4.48ꢀ4.57 (2H, m), 4.40 (2H, q, J = 7.1
Hz), 3.09 (3H, s), 1.42 (3H, t, J = 7.1 Hz). FABꢀMS: m/z 378 (M + H).
N-Ethyl-N-(4-pyridylmethyl)-2-(8-oxo-2-phenyl-7,8-dihydro-9H-purin-
9-yl)acetamide (10a). Diphenylphosphoryl azide (0.22 mL, 1 mmol) was
added to a solution of 9a (0.39 g, 1 mmol) and triethylamine (Et3N;
0.14 mL, 1 mmol) in DMF (5 mL). The reaction mixture was heated at
100 °C for 6 h and then evaporated to remove DMF. The residue was
extracted with ethyl acetate, and the organic layer was washed with brine and
dried over Na2SO4. After the solvent was removed, the crude product was
purified by column chromatography (CH2Cl2/methanol: 95/0.5) and
recrystallized from methanol to give 10a (0.18 g, 46.9%) as colorless solid;
mp 212ꢀ213 °C. 1H NMR (DMSO-d6, δ): 11.50 (1H, s), 8.65 (1H, d, J =
5.9 Hz), 8.27ꢀ8.38 (4H, m), 7.41ꢀ7.52 (4H, m), 7.22 (1H, d, J = 5.5 Hz),
4.56ꢀ4.96 (4H, m), 3.47 (2H, dq, J = 7.0, 71.7 Hz), 1.16 (3H, dt, J = 7.0,
68.9 Hz). HRMS (FAB) calcd for C21H20N6O2, 389.1726; found, 389.1757.
N-Ethyl-N-(3-pyridylmethyl)-2-(8-oxo-2-phenyl-7,8-dihydro-9H-purin-
9-yl)acetamide (10b). The procedure described for the synthesis of 10a
was applied to 9b to give 10b (73.1%) as a colorless solid; mp 203ꢀ204 °C.
1HNMR(DMSO-d6, δ):9.87(1H, s), 8.49ꢀ8.66 (2H, m), 8.31 (2H, d, J=
3.7 Hz), 8.14 (1H, d, J = 8.5 Hz), 7.39ꢀ7.64 (4H, m), 7.11 (1H, m),
4.63ꢀ4.87 (4H, m), 3.47 (2H, d, J = 6.2 Hz), 1.11ꢀ1.38 (3H, m). HRMS
(FAB) calcd for C21H20N6O2, 389.1726; found, 389.1757.
N-Methyl-N-(3-pyridylmethyl)-2-(8-oxo-2-phenyl-7,8-dihydro-9H-
purin-9-yl)acetamide (10c). The procedure described for the synthesis
of 10a was applied to 9c to give 10c (73.4%) as a colorless solid; mp
206ꢀ207 °C. 1H NMR (DMSO-d6, δ): 11.53 (1H, s), 8.57ꢀ8.62 (1H, m),
8.47ꢀ8.49 (1H, m), 8.29ꢀ8.38 (2H, m), 7.62ꢀ7.87 (1H, m), 7.48ꢀ7.52
(2H, m), 7.13ꢀ7.32 (3H, m), 4.57ꢀ4.92 (4H, m), 3.25 (3H, d, J = 46.6
Hz). HRMS (FAB) calcd for C20H18N6O2, 375.1569; found, 375.1599.
N-Ethyl-N-(4-pyridylmethyl)-2-(7-methyl-8-oxo-2-phenyl-7,8-dihy-
dro-9H-purin-9-yl)acetamide (3a). A mixture of 10a (72 mg, 0.18
mmol), MeI (13 μL, 0.21 mmol) and sodium hydride (NaH; 60% in
mineral oil, 9 mg, 0.24 mmol) in DMF (1 mL) was stirred at 50 °C for
5 h. After DMF was removed on reduced pressure, the residue was
quenched with ethyl acetate and washed with water and a saturated NaCl
solution. After the organic layer was dried over Na2SO4, the solvent was
removed to give a crude product, which was recrystallized from methanol
to give 3a (0.99 g, 33.3%) as a colorless solid; mp 184ꢀ185 °C. 1H NMR
(DMSO-d6, δ): 8.54ꢀ8.66 (2H, m), 8.28ꢀ8.37 (3H, m), 7.40ꢀ7.50
(4H, m), 7.22 (1H, d, J = 5.1 Hz), 4.50ꢀ5.01 (4H, m), 3.15 (3H, d, J =
5.1 Hz), 3.45 (2H, dq, J = 7.0, 73.1 Hz), 3.15 (3H, d, J = 5.1Hz), 1.12 (3H,
dt, J = 7.0, 70.4 Hz). HRMS (FAB) calcd for C22H22N6O2, 403.1862;
found, 403.1836.
N-Methyl-3-pyridylmethylamine (4c). The procedure described for
the synthesis of 4a was applied to 5b and methylamine to give 4c
(66.3%) as a colorless liquid. 1H NMR (CDCl3, δ): 8.55ꢀ8.56 (1H,
m), 8.51 (1H, dd, J = 1.5, 4.0 Hz) 7.67ꢀ7.70 (1H, m), 7.25ꢀ7.29
(1H, m), 3.77 (2H, s), 2.46 (3H, s), 1.99 (1H, s). FABꢀMS: m/z 123
(M + H).
Ethyl 4-(2-(N-Ethyl,N-4-pyridylmethylamino)-2-oxoethylamino-2-
phenylpyrimidine-5-carboxylate (8a). The BOP reagent (2.00 g, 4.4
mmol) was added to a mixture of 2-(5-ethoxycarbonyl)-2-phenylpyr-
imidine-4-ylamino)acetic acid24 (7; (1.20 g, 4.0 mmol), Et3N (0.61 mL,
4.4 mmol) and 4a (0.62 g, 4.5 mmol) in DMF (25 mL). The reaction
mixture was stirred at room temperature for 3 h and then evaporated in
vacuo to remove DMF. The residue was extracted with ethyl acetate and
water, and the organic layer was dried over Na2SO4 and evaporated. The
crude product was purified by open column chromatography (n-hexane/
ethyl acetate: 2/1) to give 8a (0.73 g, 38.5%) as a colorless solid; mp
124ꢀ126 °C. 1H NMR (CDCl3, δ): 9.00 (1H, s), 8.39ꢀ8.46 (4H, m),
7.79 (1H, dd, J = 8.1, 59.0 Hz), 7.24ꢀ7.49 (5H, m), 4.68 (2H, d, J =
12.8 Hz), 4.57 (2H, d, J = 4.8 Hz), 4.41 (2H, q, J = 7.1 Hz), 3.27 (2H, q,
J = 7.3 Hz), 1.34ꢀ1.44 (6H, m). FABꢀMS: m/z 420 (M + H).
Ethyl 4-(2-(N-Ethyl,N-3-pyridylmethylamino)-2-oxoethylamino-2-
phenylpyrimidine-5-carboxylate (8b). The procedure described for
the synthesis of 8a was applied to 7 and 4b to give 8b (19.0%) as a
1
colorless solid; mp 121ꢀ122 °C. H NMR (CDCl3, δ): 8.95ꢀ9.01
(2H, m), 8.50ꢀ8.59 (2H, m), 8.34 (2H, dd, J = 6.4, 25.5 Hz), 7.58ꢀ7.68
(1H, m), 7.45ꢀ7.50 (3H, m), 7.15ꢀ7.19 (1H, m), 4.67 (2H, d, J = 6.6
Hz), 4.48 (2H, dd, J = 4.4, 13.6 Hz), 4.41 (2H, q, J = 7.2 Hz), 3.39ꢀ3.53
(2H, m), 1.15ꢀ1.44 (6H, m). FABꢀMS: m/z 420 (M + H).
Ethyl 4-(2-(N-Methyl,N-3-pyridylmethylamino)-2-oxoethylamino-
2-phenylpyrimidine-5-carboxylate (8c). The procedure described for
the synthesis of 8a was applied to 7 and 4c to give 8c (46.9%) as a
colorless solid; mp 136ꢀ137 °C. 1H NMR (CDCl3, δ): 8.96ꢀ9.04 (2H,
m), 8.53ꢀ8.61 (2H,m), 8.31ꢀ8.46 (2H, m), 7.45ꢀ7.68 (4H, m),
7.19ꢀ7.32 (1H, m), 4.67 (2H, d, J = 7.3 Hz), 4.48ꢀ4.57 (2H, m),
4.40 (2H, q, J = 7.1 Hz), 3.09 (3H, s), 1.42 (3H, t, J = 7.1 Hz). FABꢀMS:
m/z 406 (M + H).
4-(2-(N-Ethyl,N-4-pyridylmethylamino)-2-oxoethylamino-2-phenyl-
pyrimidine-5-carboxylic Acid (9a). A mixture of 8a (0.71 g, 1.7 mmol)
and NaOH (1 mL, 5 M) in ethanol (50 mL) was stirred at room
temperature for 12 h. After ethanol was removed, HCl (1 M) was added
slowly to neutralize the reaction mixture. The obtained precipitate was
collected and washed with water to give 10a (80.1%) as a colorless solid;
N-Ethyl-N-(3-pyridylmethyl)-2-(7-methyl-8-oxo-2-phenyl-7,8-dihy-
dro-9H-purin-9-yl)acetamide (3b). The procedure described for the
synthesis of 3a was applied to 10b to give 3b (49.2%) as a colorless solid;
mp 185ꢀ187 °C. 1H NMR (DMSO-d6, δ): 8.64 (1H, s), 8.52 (1H, s),
8.24ꢀ8.36 (3H, m), 7.72 (1H, dd, J = 7.9, 52.2 Hz), 7.38ꢀ7.46 (3H, m),
7.12ꢀ7.17 (1H, m), 4.88 (2H, d, J = 18.0 Hz), 4.66 (2H, d, J = 22.0 Hz),
3.45ꢀ3.52 (5H, m), 1.27 (3H, dt, J = 7.2, 69.3 Hz. HRMS (FAB) calcd
for C22H20N6O2, 403.1862; found, 403.1836.
N-Methyl-N-(3-pyridylmethyl)-2-(7-methyl-8-oxo-2-phenyl-7,8-di-
hydro-9H-purin-9-yl)acetamide (3c). The procedure described for the
synthesis of 3a was applied to 10c to give 3c (54.6%) as a colorless solid;
mp 171ꢀ172 °C. 1H NMR (DMSO-d6, δ): 8.53ꢀ8.67 (2H, m), 8.25ꢀ
8.36 (3H, m), 7.62ꢀ7.78 (1H, m), 7.38ꢀ7.46 (3H, m), 7.18ꢀ
7.23 (1H, m), 4.90 (2H, d, J = 8.8 Hz), 4.66 (2H, d, J = 23.8 Hz), 3.50
(3H, s), 2.99 (3H, s). HRMS (FAB) calcd for C21H22N6O2, 389.1726;
found, 389.1757.
1
mp 244ꢀ245 °C. H NMR (DMSO-d6, δ): 13.4 (1H, br), 8.83ꢀ8.87
(2H, m), 8.23ꢀ8.66 (3H, m), 7.48ꢀ7.57 (5H, m), 4.68 (2H, s), 4.55 (2H,
dd, J = 5.3, 77.2 Hz), 3.48 (2H, dq, J =7.0, 33.4 Hz), 1.16 (3H, dt, J = 7.0,
47.4 Hz). FABꢀMS: m/z 392 (M + H).
4-(2-(N-Ethyl,N-3-pyridylmethylamino)-2-oxoethylamino-2-phenyl-
pyrimidine-5-carboxylic Acid (9b). The procedure described for the
synthesis of 9a was applied to 8b to give 9b (98.4%) as a colorless solid;
1
mp 163ꢀ164 °C. H NMR (DMSO-d6, δ): 8.86 (2H, t, J = 7.9 Hz),
8.52ꢀ8.67 (2H, m), 8.35 (2H, dd, J = 7.2, 26.6 Hz), 7.85ꢀ7.92 (1H, m),
7.37ꢀ7.56 (4H, m), 4.50ꢀ4.78 (4H, m), 3.38ꢀ3.50 (2H, m), 1.13 (3H,
dt, J = 7.0, 55.7 Hz). FABꢀMS: m/z 392 (M + H).
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Radiochemistry. N-Ethyl-N-(4-pyridinylmethyl)-2-(7-[11C]methyl-
8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide ([11C]3a). [11C]MeI
for labeling was synthesized from cyclotron-produced [11C]CO2 as described
previously.26 Briefly, [11C]CO2 was bubbled into 0.4 M LiAlH4 in anhydrous
tetrahydrofuran (THF, 300 μL). After evaporation of THF, the remaining
complex was treated with 57% hydroiodic acid (300 μL). The produced
[11C]MeI was transferred under helium gas flow with heating into a reaction
vessel containing desmethyl precursor 10a (0.6ꢀ0.8 mg), NaOH (3ꢀ5 μL,
0.1 M), and anhydrous DMF (300 μL) cooled to ꢀ15 to ꢀ20 °C. After
radioactivity reached a plateau, the reaction vessel was warmed to 60 °C and
maintained for 5 min. MeCN/H2O (4/6, 500 μL) was added to the reaction
mixture to stop the reaction, and the mixture was applied to the semipre-
parative HPLC system. HPLC purification was completed on a Capcell Pack
then centrifuged at 9000g for 10 min at 4 °C. The pellet was suspended
in Tris-HCl buffer (10 mM, pH 7.4, containing 320 μM sucrose) and
centrifuged at 9000g for 10 min at 4 °C. The resulting pellet was then
washed with 50 mM Tris-HCl buffer (pH 7.4) by centrifugation at
12000g for 10 min at 4 °C. Finally, the crude mitochondrial pellet was
resuspended in 50 mM Tris-HCl buffer (pH 7.4) at a concentration of
100 μg original wet tissue/mL and used for binding assays.
Each crude mitochondrial preparation (100 μL) was incubated with
[11C]1 (final concentration 3.4 ( 0.6 nM) and various concentrations of
tested ligands (1, 2, 3aꢀc) in a total volume of 1 mL at room
temperature for 30 min, and the reaction was terminated by rapid
filtration through 0.3% polyethylenimine-pretreated Whatman GF/C
glass fiber filters, using a cell harvester (M-24, Brandel, Gaithersburg,
MD). The filters were immediately washed three times with 5 mL of ice-
cold Tris-HCl buffer (50 mM), and the filter-bound radioactivity was
quantified using a γ counter (Wallac 1470 WIZARD 300, PerkinElmer,
Waltham, MA). Nonspecific binding was determined in the presence of
1 (10 μM). All assays were performed in duplicate, except for total
binding and nonspecific binding, which were in quadruplicate. Specific
binding at each compound concentration was calculated as a percentage
in relation to the control specific binding, and which were converted to
probit values to determine the IC50 of each compound. The IC50 value
was further converted to Ki according to the ChengꢀPrusoff equation.33
PET Studies on Normal Rats. PET scans were performed using a
small-animal Inveon PET scanner (Siemens Medical Solutions USA,
Knoxville, TN), which provides 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. Before scans, the rats were anesthetized with 5% (v/v)
isoflurane and maintained thereafter by 1ꢀ2% (v/v) isoflurane. Emis-
sion scans were acquired for 30 min in three-dimensional list mode with
an energy window of 350ꢀ750 keV, immediately after intravenous
injection of [11C]3aꢀc (16ꢀ18 MBq/200 μL, 0.13ꢀ0.24 nmol). To
determine in vivo specific binding, unlabeled 1 (3.0 mg/kg) and 2 (1.0
mg/kg dissolved in 300 μL saline containing 10% ethanol and 5%
polysorbate 80) were injected at 1 min before injection of [11C]3aꢀc.
Four rats were used for each experiment. All list-mode acquisition data
were sorted into three-dimensional sinograms, which were then Fourier
rebinned into two-dimensional sinograms (frames ꢁ min: 2 ꢁ 2, 1 ꢁ 4,
4 ꢁ 5). Dynamic images were reconstructed with filtered back-projec-
tion using a Hanning’s filter, with a Nyquist cutoff of 0.5 cycle/pixel.
Regions of interest were placed on the lung, heart, kidney, liver, small
intestine, and cardiac blood pool using ASIPro VM (Analysis Tools and
System Setup/Diagnostics Tool, Siemens Medical Solutions USA).
Regional uptake of radioactivity was decay-corrected to the injection
time and was expressed as the standardized uptake value (SUV), which
was normalized to the injected radioactivity and body weight. SUV =
(radioactivity per mL tissue/injected radioactivity) ꢁ g body weight. For
comparison, PET with [11C]1 and [11C]2 (15ꢀ20 MBq/200 μL,
0.15ꢀ0.18 nmol) was performed according to the same procedures
described above.
C
18 column (10 mm ID ꢁ 250 mm; Shiseido, Tokyo) using a mobile phase
of MeCN/H2O (4/6) at a flow rate of 5.0 mL/min. The tR for [11C]3a was
8.0 min, whereas that for unreacted 10a was 4.9 min. The radioactive fraction
corresponding to the desire product was collected in a sterile flask, evaporated
to dryness in vacuo, redissolved in 3 mL of sterile normal saline, and passed
through a 0.22 μm Millipore filter for analysis and animal experiments. The
synthesis time was 23 min from the end of bombardment.
Radiochemical purity was assayed by analytical HPLC (Capcell Pack
C18, 4.6 mmIDꢁ 250 mm, UV at 254 nm; MeCN/H2O, 4/6; tR, 7.8 min).
The identity of [11C]3a was confirmed by coinjection with unlabeled 3a.
The specific activity of [11C]3a was calculated by comparison of the
assayed radioactivity to the mass associated with the carrier UV peak at
254 nm. Radiochemical yield (decay-corrected), 35% based on [11C]CO2;
radiochemical purity, >99%; specific activity, 100 GBq/μmol.
N-Ethyl-N-(3-pyridinylmethyl)-2-(7-[11C]methyl-8-oxo-2-phenyl-7,
8-dihydro-9H-purin-9-yl)acetamide ([11C]3b). The procedure de-
scribedfor the synthesis of[11C]3awas appliedto 10b to give[11C]3b; tR,
8.3 min for purification and 8.0 min for analysis; synthesis time, 24 min;
radiochemical yield (decay-corrected), 34% based on total [11C]CO2;
radiochemical purity, >99%; specific activity, 106 GBq/μmol.
N-Methyl-N-(3-pyridinylmethyl)-2-(7-[11C]methyl-8-oxo-2-phenyl-
7,8-dihydro-9H-purin-9-yl)acetamide ([11C]3c). The procedure de-
scribed for the synthesis of [11C]3a was applied to 10c to give
[11C]3c; tR, 8.1 min for purification and 6.2 min for analysis; synthesis
time, 22 min; radiochemical yield (decay-corrected), 23% based on total
[11C]CO2; radiochemical purity, > 99%; specific activity, 98 GBq/μmol.
Measurement and Computation of Lipophilicity. The LogD
values were measured by mixing [11C]3aꢀc (radiochemical purity,
100%; about 200000 cpm) with n-octanol (3.0 g) and sodium phosphate
buffer (PBS; 3.0 g, 0.1 M, pH 7.4) 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 1 mL PBS and 1 mL n-octanol was removed,
weighted, and counted, respectively. Samples from the remaining
organic layer were removed and repartitioned until consistent LogD
values were 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. Meanwhile, the values of cLogD of [11C]3aꢀc were computed
using Pallas 3.4 software (CompuDrug, Sedona, AZ).
Evaluation. Male SpragueꢀDawley rats (6ꢀ8 weeks old) were
purchased from Japan SLC (Shizuoka, Japan). The rats were housed
under a 12-h darkꢀlight cycle and were allowed free access to food
pellets and water. The animal experiments were approved by the Animal
Ethics Committee of the National Institute of Radiological Sciences
(Chiba, Japan).
In Vitro Binding Assays for TSPO. For this assay, the method
described previously was used.32 Rats (n = 5) were sacrificed by
decapitation under ether anesthesia. The whole brains were rapidly
dissected and homogenized in 10 volumes of ice-cold Tris-HCl buffer
(10 mM, pH 7.4, containing 320 μM sucrose). The homogenate was
centrifuged at 77g for 10 min at 4 °C. The supernatant was collected and
Metabolite Assay for Rat Tissue. After intravenous injection of
[11C]3a (37 MBq/100 μL, 0.32 nmol) into rats (n = 3), these rats were
sacrificed by cervical dislocation at 30 min. Blood (0.7ꢀ1.0 mL), lung,
heart, liver, and kidney samples (0.5ꢀ1.0 g) were removed quickly. The
blood sample was centrifuged at 15000 rpm for 1 min at 4 °C to separate
plasma, and 250 μL of plasma was collected in a test tube containing
MeCN (500 μL) and a solution of the authentic unlabeled 3a (0.5 mg/
5.0 mL MeCN, 10 μL). After the tube was vortexed for 15 s and
centrifuged at 15000 rpm for 2 min for deproteinization, the supernatant
was collected. The extraction efficiency of radioactivity into the MeCN
supernatant ranged from 81% to 95% of the total radioactivity in the
plasma. On the other hand, the lung, heart, kidney, and liver were dissected
and homogenized together in an ice-cooled MeCN/H2O (4/6, 1.0 mL)
solution. The homogenate was centrifuged at 15000 rpm for 1 min at 4 °C
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Journal of Medicinal Chemistry
ARTICLE
and supernatant was collected. The recovery of radioactivity into the
supernatant was 71ꢀ85% based on the total radioactivity in the tissue
homogenate.
acetonitrile; MeI, methyl iodide; [11C]MeI, [11C]methyl -
iodide; NaBH4, sodium borohydride; NaH, sodium hydride;
PBR, peripheral-type benzodiazepine receptor; PBS, phosphate
buffer solution; PET, positron emission tomography; SUV, stan-
dardized uptake value; TAC, timeꢀactivity curve; THF, tetra-
hydrofuran; tR, retention time; TSPO, translocator protein
(18 kDa)
An aliquot of the supernatant (100ꢀ500 μL) obtained from the
plasma or tissue homogenate was injected into the HPLC system for
radioactivity and analyzed under the same conditions described above.
The percent ratio of [11C]3a (tR, 7.8 min) to total radioactivity
(corrected for decay) on the HPLC chromatogram was calculated as
% = (peak area for [11C]3a/total peak areas) ꢁ 100.
Preparation of Rat Model of Lung Inflammation. Rats were intra-
tracheally injected with lipopolysaccharide (LPS, Escherichia coli O127,
B8, Sigma-Aldrich; about 125 μg in 50 μL saline, 5.0 mg/kg), respec-
tively. Control (vehicle) rats received treatment with 50 μL of saline.
Twenty-four hours after LPS treatment or control, these rats were used
for PET and biodistribution studies.
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’ ASSOCIATED CONTENT
S
Supporting Information. HPLC separation charts of
b
[11C]3aꢀc. HPLC analysis charts of [11C]3aꢀc. PET study
with [11C]1 in normal rat: timeꢀactivity curves after injection of
[11C]1. PET study with [11C]3aꢀc: maximum inhibitory per-
centages (%) of total uptake by pretreatment with 1 or 2 after
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injection of [11C]1. This material is available free of charge via
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: 81-43-382-3708. Fax: 81-43-206-3261. E-mail: zhang@
nirs.go.jp.
’ ACKNOWLEDGMENT
We are grateful to the staff of National Institute of Radiological
Sciences for support with the cyclotron operation, radioisotope
production, radiosynthesis, and animal experiments.
’ ABBREVIATIONS USED
AUC, area under timeꢀactivity curve; BOP, (benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate;
CH2Cl2, dichloromethane; [11C]CO2, [11C]carbon dioxide;
DMF, N,N-dimethyl formamide; DMSO, dimethyl sulfoxide;
Et3N, triethylamine; FAB-MS, fast atom bombardment mass
spectra; FOV, field of view; HPLC, high-performance liquid
chromatography; HRMS, high-resolution mass spectra; LiAlH4,
lithium aluminum hydride; LPS, lipopolysaccharide; MeCN,
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