Synthesis and evaluation of [11C]RU40555, a selective glucocorticoid receptor antagonist

Article abstract of DOI:10.1002/jlcr.958

We demonstrated the synthesis of carbon-11 labeled 17-α-hydroxy-11- β-/4-/[methyl]-[1-methylethyl]-aminophenyl/-17α-[prop-1-ynyl] esta-4-9-diene-3-one (RU40555), a selective glucocorticoid receptor (GR) antagonist, and examined the in vivo profile of [

Full text of DOI:10.1002/jlcr.958

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 657–668.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.958
Research Article
Synthesis and evaluation of [11C]RU40555, a selective
glucocorticoid receptor antagonist
Takahiro Matsuya1,2,*, Hiroyuki Takamatsu1,2, Yoshihiro Murakami1,2,
Akihiro Noda1,2, Kazuhiko Osoda1, Rikiya Ichise2, Yuji Awaga2 and Shintaro
Nishimura1,2
1 Biopharmaceutical and Pharmacokinetic Research Laboratories, Fujisawa
Pharmaceutical Co., Ltd., 1-6, Kashima 2-chome, Yodogawa-ku, Osaka 532-8514,
Japan
2 The Medical and Pharmacological Research Center Foundation, Wo32, Inoyama-
Town, Hakui-City, Ishikawa 925-0613, Japan
Summary
We demonstrated the synthesis of carbon-11 labeled 17-a-hydroxy-11-b-/4-/[methyl]-
[1-methylethyl]-aminophenyl/-17a-[prop-1-ynyl]esta-4-9-diene-3-one (RU40555), a se-
lective glucocorticoid receptor (GR) antagonist, and examined the in vivo profile of
[11C]RU40555. [11C]RU40555 was synthesized by direct N-methylation with
[11C]CH3OTf at 608C for 5 min and an injectable solution of [11C]RU40555 was
obtained in 31 min at the end of bombardment. The decay-corrected radiochemical
yield was 19%, the specific radioactivity was 57.5 ꢀ 14.0 GBq/mmol, and the
radiochemical purity was more than 99% as determined by HPLC. In rat experiments,
the effects of adrenalectomy (ADX) on brain accumulation of [11C]RU40555 were
examined. ADX significantly decreased plasma corticosterone levels, and significantly
increased brain accumulation of [11C]RU40555. We succeeded in developing a rapid
automated synthesis method for [11C]RU40555, a GR antagonist, and showed
[11C]RU40555 had a potential as a PET tracer for mapping GR. Copyright # 2005
John Wiley & Sons, Ltd.
Key Words: [11C]RU40555; glucocorticoid receptor antagonist; PET
Introduction
It is well known that stress exposure activates the hypothalamic–pituitary–
adrenal (HPA) axis, and adrenal glucocorticoids secretion plays a role in
*Correspondence to: Takahiro Matsuya, The Medical and Pharmacological Research Center Foundation,
Wo32, Inoyama-Town, Hakui-City, Ishikawa 925-0613, Japan. E-mail: matsuya@mprcf.or.jp
Contract/grant sponsor: Ministry of Education, Culture, Sports, Science and Technology
Copyright # 2005 John Wiley & Sons, Ltd.
Received 25 October 2004
Revised 22 March 2005
Accepted 9 April 2005

658
T. MATSUYA ET AL.
*CH3
N
H
N
OH
OH
Method 1 or 2
O
O
Method 1. 11C-CH3I, DMI, 150 oC, 10 min
Method 2. 11C-CH3OTf, acetone, 60 oC, 5 min
Figure 1. The synthetic route for [11C]RU40555. Asterisk denotes position of
11C labeling
negative feedback control.1,2 In this negative feedback regulation, the
hippocampus, which is rich in glucocorticoid and mineralcorticoid receptors,3
plays a central role. Abnormality in this negative feedback control system
contributes to several kinds of psychiatric conditions, such as depression and
post-traumatic stress disorder (PTSD).4,5 Imaging of the glucocorticoid
receptor (GR) in the central nervous system is useful in pathological and
physiological studies in these psychiatric conditions. In order to perform GR
imaging, several PET tracers have been developed.6–10 However, these tracers
have not satisfied all the requirements as a PET tracer such as permeability
and/or metabolic stability,7–10 and none of them could achieve in vivo
imaging of the glucocorticoid receptor. Recently a selective GR antagonist,
RU40555 (17-a-hydroxy-11-b-/4-/[methyl]-[1-methylethyl]-aminophenyl/-17a-
[prop-1-ynyl]esta-4-9-diene-3-one) has been described11,12 (Figure 1). It was
reported that RU40555 blocked the suppressive effect of dexamethasone on
acute stress-induced corticosterone secretion in the feedback system, and that it
did not decrease corticosterone secretion when rats undergo stress.13,14 There-
fore, it was suggested that RU40555 could penetrate the blood–brain barrier,
and it may be a suitable candidate for a PET tracer for GR in the brain. In the
present study, we developed a synthesis method of carbon-11 labeled RU40555
as a PET tracer for GR, and we examined its brain accumulation in rats.
Results and discussion
Chemistry
The methyl carbon bound to the aniline nitrogen of RU40555 was selected
as the position to be labeled because the labeling reagent, [11C]methyl
iodide ([11C]CH3I), is easily prepared and the sterically hindered methyl
group was expected to have sufficient metabolic stability. The precursor (4)
and RU40555 were synthesized using examples from the European patent15
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESIS AND EVALUATION OF [11C]RU40555
659
Figure 2. The synthetic route for precursor and RU40555
(Figure 2). Compound (1) was reacted with a compound, which was
prepared from compound (2), Mg and CuCl, to give compound (3) in a yield
of 75%. Compound (3) was unprotected by the use of 6 N H2SO4, resulting in
the precursor (4) in a yield of 90%. At first, the reaction of the precursor (4)
with [11C]CH3I in dimethylimidazolidinone was performed and purified
by HPLC. Total synthesis time was 40 min from the end of bombardment
(EOB). The decay-corrected radiochemical yield was 7.8 ꢀ 1.1%, calculated
from EOB, and the specific radioactivity was 13.1 ꢀ 5.5 Bq/mmol. The
radiochemical yield was not enough for animal studies. The reason for
this low yield was considered to result from the low reactivity of the amino
group of the precursor (4). The bulky isopropyl group may have further
decreased the reactivity of the aniline nitrogen and prevented the nucleophilic
reaction with the methylating agent. This information led us to use [11C]methyl
triflate ([11C]CH3OTf). Reaction of the precursor (4) with [11C]CH3OTf
in acetone gave [11C]RU40555 with better radiochemical yields. Total
synthesis time was 31 min after EOB. The decay-corrected radiochemical
yield was 19%, the radioactivity was 2.5 ꢀ 0.3 GBq, radiochemical purity
was more than 99%, and the specific radioactivity was 57.5 ꢀ 14.0 GBq/mmol
(n ¼ 4). The radiochemical purity and specific radioactivity were enough to
perform several kinds of animal experiments. Recently, Wilson et al. reported
that the aromatic amine function reacted much slower than the aliphatic
function, and that [11C]CH3OTf was superior to [11C]CH3I for alkylation of
aromatic amine.16 These results corresponded with our results. It was found
that [11C]CH3OTf could be applied to the methylation of a much hindered
nucleophile, such as N-isopropylaniline derivatives.
Whole body distribution and brain accumulation in rat
Typical time course images of whole body distribution following
[11C]RU40555 intravenous injection into rat is shown in Figure 4. Accumula-
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T. MATSUYA ET AL.
tion of radioactivity in the liver and then the small intestine was clearly
demonstrated. There was little accumulation in the bladder. Therefore, it was
considered that RU40555 and/or its metabolites were eliminated in the bile
acid. We hypothesized that [11C]RU40555 could penetrate the blood–brain
barrier and accumulate in the brain. The maximum brain accumulation of
[11C]RU40555 was observed 20 min after injection. Therefore, further ex vivo
experiments using rats were performed 20 min after injection.
Figure 5 showed the accumulation of [11C]RU40555 in the cortex, striatum,
hippocampus, cerebellum, and plasma 20 min after injection. The radioactivity
of plasma was the same as that of brain regions. It was widely distributed in
the brain, and the percentage of injected dose (%ID/kg/g) was about 0.03% in
the cortex, striatum, hippocampus, and cerebellum in normal rat. Percentages
of unchanged [11C]RU40555 in the plasma were 50.8% at 20 min. We
examined the effect of adrenalectomy (ADX) to avoid the effect of endogenous
corticosteroid in the present study. The plasma corticosterone level was
significantly decreased from 423ꢀ23 to 23ꢀ3 ng/mg by adrenalectomy
(n ¼ 5). These accumulations in the brain were significantly increased by
about 20% by ADX. It was reported that endogenous corticosterone occupied
28% of the GR of the hippocampus in normal conditions.17 Therefore, the
increase by ADX might result from avoidance of endogenous corticosterone
occupation.
Figure 3. A typical preparative HPLC chromatogram of the purification of
[11C]RU40555 obtained using a UV-detector (k=254 nm) and a radiodetector
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SYNTHESIS AND EVALUATION OF [11C]RU40555
661
Figure 4. A typical dynamic image of whole body distribution of [11C]RU40555
following intravenous injection (n=3). Each image represents an accumulative
image for 5 min. The upper left image represents 0–5 min, and the bottom right
image represents 55–60 min
Normal
ADX
0.0600%
0.0500%
0.0400%
0.0300%
0.0200%
0.0100%
0.0000%
*
*
*
*
Cortex
Hippocampus
Striatum
Cerebellum
Plasma
Figure 5. The accumulation of [11C]RU40555 in the cortex, striatum,
hippocampus, cerebellum, and plasma 20 min after injection. ADX=adrenalect-
omy. Each column represents the mean of five animals and the bar represents SD.
*P50.05 versus normal rats
Reul et al. reported the distribution of GR in rats.3 According to their
in vitro autoradiography study, high levels of GR were in the cortical,
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T. MATSUYA ET AL.
amygdaloid, lateral, medial thalamic and hypothalamic regions, and low
levels of GR were in the hindbrain and cerebellum. These observations
were different from the present results in that the distribution of [11C]RU40555
was widespread in all regions in this study. This discrepancy may be due
to the differences between in vitro and in vivo results, because it is known
that GR shows internalization when agonist and antagonist bind to the
receptor.18
Though further experiments are necessary to evaluate the ability of
[11C]RU40555 to act as a GR imaging agent, [11C]RU40555 showed some
potential as a PET tracer.
Experimental
Materials and reagents
Acetone, tetrahydrofuran (THF), hexane, and ethyl acetate were purchased
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Silver triflate was
purchased from Aldrich Japan (Tokyo, Japan). Graphpac GC material was
purchased from Alltech Japan (Tokyo, Japan). Sterile filters were purchased
from Millipore Japan (Tokyo, Japan). Hydroxypropyl-b-cyclodextrine was
purchased from TOKYO KASEI KOGYO Co., Ltd. (Tokyo, Japan). 5a, 10a-
epoxy-17a-ethynyl-17b-hydroxy-estr-9(11)-ene-3-one 3-(cyclic 1,2-ethanediyl
acetal) was synthesized according to the literature.15
Animals
All animals were kept under a natural light/dark cycle and had unlimited
access to water and food. In the case of ADX rats, 0.9% saline was substituted
for water. All experiments using rats were performed in accordance with the
institutional guidelines of The Medical and Pharmacological Research Center
Foundation.
Male SD rats (8 weeks old) were used in the present study. Normal and
ADX rats were purchased from Japan SLC (Shizuoka, Japan). ADX was
performed at 5 weeks of age.
Analysis
HPLC analysis was performed with the Agilent 1100 HPLC system (Agilent
Technologies Japan, Tokyo) and an Aloka positron detector RLC-700 (Aloka
Co., Ltd., Tokyo, Japan).
HPLC analysis of RU40555
Column: YMC-Pack C18 Pro (4.6 ꢁ 150 mm, 5 mm) (YMC Co., Ltd., Kyoto,
Japan).
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SYNTHESIS AND EVALUATION OF [11C]RU40555
663
Mobile phase: (acetonitrile:THF ¼ 6 : 1): 0.05 M NH4OAc-0.1% AcOH ¼
65:35 (v/v).
Flow rate: 1 ml/min.
Wavelength: 254 nm.
Preparation of 4-(N-isopropyl-N-trimethylsilylamino)phenyl bromide (2). A
solution of N-isopropyl-4-bromoaniline (2.47 g, 11.5 mmol) in THF (7 ml) was
cooled in a dry ice bath. A solution of n-BuLi (1.58 M hexane solution,
7.23 ml, 11.4 mmol) was added dropwise with stirring at ꢂ788C for 15 min.
TMSCl (1.38 g, 12.7 mmol) was added at the same temperature. The reaction
mixture was stirred at ꢂ788C for 30 min and the solution was allowed to warm
to room temperature. The mixture was concentrated in vacuo. The residue was
subjected to vacuum distillation (bp 81–838C at 0.55 mmHg) to yield (2)
1
(1.55 g, 47%). H-NMR (300 MHz, CDCl3) d0.00 (9H, s), 0.96 (6H, d), 2.45–
2.48 (1H, m), 6.88 (2H, d, J¼ 8:8 Hz), 7.38 (2H, d, J¼ 8:8 Hz).
Preparation of 4-(N-isopropyl-N-methylamino)phenyl bromide (5). The reac-
tion mixture of N-isopropyl-4-bromoaniline (20 g, 93.4 mmol) and HCHO
(28.1 g, 934 mmol) in formic acid (35.2 ml, 934 mmol) was stirred at 1208C for
2 h. The mixture was concentrated in vacuo. The mixture was diluted with ethyl
acetate and washed with saturated aqueous sodium bicarbonate solution and
brine. The extracts were dried over magnesium sulfate, filtered and
concentrated in vacuo. The desired product was obtained by distillation
1
(108–1158C, 0.2 mmHg) (19 g, 89%). H-NMR (300 MHz, CDCl3) d1.51 (6H,
d, J¼ 6:6 Hz), 2.69 (3H, s), 4.03 (1H, q, J¼ 6:6 Hz), 6.64 (2H, d, J¼ 8:8 Hz),
7.28 (2H, d, J¼ 8:8 Hz); MS (ES+) m/e 228.2.
Preparation of 5a-17b-dihydroxy-11b-[4[(N-isopropyl-N-trimethylsilylamino)-
phenyl]-17a-ethynyl-esta-9-ene-3-one 3-(cyclic 1,2-ethanediyl acetal) (3). A
mixture of magnesium (129 mg, 5.31 mmol) in THF (3 ml) was stirred at room
temperature. A solution of 4-(N-isopropyl-N-trimethylsilylamino)phenyl
bromide (1.552 g, 5.42 mmol) in THF (10 ml) was added dropwise with
stirring at room temperature. The reaction mixture was stirred and heated to
reflux for 25 min. The Grignard solution was added to a solution of CuCl
(35.2 mg, 0.355 mmol) in THF (5 ml) at 6–78C. After stirring for 30 min, a
mixture of 5a, 10a-epoxy-17a-ethynyl-17b-hydroxy-estr-9(11)-ene-3-one 3-
(cyclic 1,2-ethanediyl acetal) (658 mg, 1.78 mmol) in THF (15 ml) was added
dropwise at 6–78C. After stirring at 6–78C for 30 min, the mixture was
concentrated in vacuo. The residue was diluted with ethyl acetate and washed
with saturated aqueous sodium bicarbonate solution and brine. The extracts
were dried over magnesium sulfate, filtered and concentrated in vacuo. The
residue was purified by silica gel column chromatography eluted with a
mixture of hexane and ethyl acetate (7:3) to give the desired product as an
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T. MATSUYA ET AL.
1
amorphous solid (671 mg, 75%). H-NMR (300 MHz, CDCl3) d0.48 (3H, s),
1.16–1.37 (12H, m), 1.41–1.81 (4H, m), 1.82–1.93 (7H, m), 1.89 (3H, s), 1.92–
2.40 (2H, m), 2.44–2.54 (1H, m), 3.58 (1H, q, J¼ 5:8 Hz), 3.87–4.06 (4H, m),
4.20–4.27 (1H, m), 4.43 (1H, s), 6.54 (2H, brs), 7.00 (2H, d, J¼ 8:4 Hz); MS
(ES+) m/e 506.5.
Preparation of 5a-17b-dihydroxy-11b-[4[(N-isopropyl-N-methylamino)phe-
nyl]-17a-ethynyl-esta-9-ene-3-one 3-(cyclic 1,2-ethanediyl acetal) (6). A solu-
tion of 4-(N-isopropyl-N-methylamino)phenyl bromide (10.5 g, 46 mmol) in
THF (10 ml) was added to a suspension of Mg (1.09 g, 44.6 mmol) in THF
(3 ml) dropwise at room temperature. The reaction mixture was stirred at
reflux for 25 min. The Grignard solution was added to a solution of CuCl
(294 mg, 2.97 mmol) in THF (50 ml), at 6–78C. After stirring for 30 min, a
solution of 5a, 10a-epoxy-17a-ethynyl-17b-hydroxy-estr-9(11)-ene-3-one 3-
(cyclic 1,2-ethanediyl acetal) (5.51 g, 14.9 mmol) in THF (50 ml) was added
dropwise at 6–78C. After stirring at 6–78C for 30 min, the mixture was
concentrated in vacuo, the residue was diluted with ethyl acetate and washed
with aqueous 1 N hydrogen chloride solution, saturated aqueous sodium
bicarbonate solution and brine. The extracts were dried over magnesium
sulfate, filtered and concentrated in vacuo. The residue was filtered and washed
1
with IPE to give the desired product as a white powder (11.6 g, 100%). H-
NMR (300 MHz, CDCl3) d0.48–2.07 (3H, s), 1.09–1.17 (6H, m), 1.25–1.40
(2H, m), 1.40–1.60 (2H, m), 1.60–1.64 (1H, m), 1.64–1.78 (3H, m), 1.80 (1H,
brs), 1.82–1.92 (2H, m), 1.88 (3H, s), 1.93–2.07 (4H, m), 2.07–2.39 (5H, m),
2.44–2.55 (1H, m), 2.69 (3H, s), 3.88–4.10 (3H, m), 4.21–4.28 (1H, m), 4.43
(1H, s), 6.69 (2H, d, J¼ 7:7 Hz), 7.04 (2H, d, J¼ 7:7 Hz); MS (ES+) m/e 520.5.
Preparation
of
17-b-hydroxy-11-b-/4-/[1-methylethyl]-aminophenyl/-17a-
[prop-1-ynyl]est a-4-9-diene-3-one (4). A solution of 6 N H2SO4 (4.42 ml,
26.5 mmol) was added to a solution of ketal (3) (670 mg, 1.32 mmol) in acetone
(26 ml) dropwise with cooling on an ice bath. The reaction mixture was stirred
at 08C for 1 h. The mixture was concentrated in vacuo. The residue was diluted
with ethyl acetate and washed with saturated aqueous sodium bicarbonate
solution and brine. The extracts were dried over magnesium sulfate, filtered
and concentrated in vacuo. The residue was purified by silica gel column
chromatography eluted with a mixture of hexane and ethyl acetate (1:1) to give
a gum. The desired product was obtained by freeze-drying from benzene
1
(530 mg, 90%). H-NMR (300 MHz, CDCl3) d0.56 (3H, s), 1.10–2.09 (18H,
m), 2.15–2.52 (6H, m), 2.53–2.63 (2H, m), 2.72–2.84 (1H, m), 3.58 (1H, q,
J¼ 6:2 Hz), 4.33 (1H, d, J¼ 7:0 Hz), 5.75 (1H, s), 6.52 (2H, d, J¼ 8:4 Hz), 6.94
(2H, d, J¼ 8:4 Hz); MS (ES+) m/e 444.5.
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SYNTHESIS AND EVALUATION OF [11C]RU40555
665
Preparation of RU40555 (7). A solution of 6N H2SO4 (50 ml, 300 mmol) was
added to a solution of ketal (6) (7.8 g, 15 mmol) in acetone (300 ml) dropwise
while cooling on an ice bath. The reaction mixture was stirred at 08C for 1 h.
The mixture was concentrated in vacuo. The residue was diluted with ethyl
acetate and washed with saturated aqueous sodium bicarbonate solution and
brine. The extracts were dried over magnesium sulfate, filtered and
concentrated in vacuo. The residue was purified by silica gel column
chromatography eluted with a mixture of hexane and ethyl acetate (7:3) to
give a gum. The desired products were recrystallized from ethanol and water
to give a light yellow powder (6.0 g, 87%). 1H-NMR (300 MHz, CDCl3) d0.56
(3H, s), 1.14 (6H, m), 1.19–1.55 (2H, m), 1.62–1.82 (3H, m), 1.89 (3H, s), 1.91–
2.06 (2H, m), 2.18–2.53 (7H, m), 2.54–2.61 (2H, m), 2.70 (3H, s), 2.72–2.84
(1H, m), 3.98–4.10 (1H, m), 4.35 (1H, d, J¼ 6:6 Hz), 5.76 (1H, s), 6.70 (2H, d),
6.99 (2H, d, J¼ 8:8 Hz); MS (ES+) m/e 458.5.
Preparation of [11C]CH3I and [11C]CH3OTf
Production of [11C]CO2 was accomplished via the 14N(p, a)11C nuclear
reaction by proton bombardment (12 MeV, 35 mA) of a nitrogen target using a
cyclotron-target system (OSCAR, JFE Plant & Service Corporation,
Yokohama, Japan). [11C]CH3I was made from reduction with LiAlH4 and
reaction with HI. Then [11C]CH3I was converted to [11C]CH3OTf by passing
through a glass column (6 cm length, 5 mm internal diameter) heated to 2008C
and containing silver triflate-impregnated graphitized carbon (300 mg).
Preparation of [11C]RU40555 from [11C]CH3I
Desmethyl-RU40555 was prepared as previously described. [11C]RU40555 was
synthesized by bubbling [11C]CH3I into a reaction vessel containing 3.0 mg
desmethyl-RU40555 in dimethylimidazolidinone (0.22 ml) at 108C (Figure 1).
The solution was heated at 1508C for 10 min.
Preparation of [11C]RU40555 from [11C]CH3OTf
The synthesis route of [11C]RU40555 is shown in Figure 1. Desmethyl-
RU40555 was prepared as previously described. [11C]RU40555 was synthe-
sized by bubbling [11C]CH3OTf into a reaction vessel containing 1.0 mg
desmethyl-RU40555 in acetone (0.22 ml) at ꢂ208C (Figure 1). The solution
was heated at 608C for 5 min.
Separation and preparation of [11C]RU40555
The reaction mixture was purified by reversed-phase HPLC. The fraction
containing [11C]RU40555 was collected (Figure 3) and concentrated under
reduced pressure. The residue was dissolved in 3% hydroxypropyl-b-
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T. MATSUYA ET AL.
cyclodextrine saline solution and passed through a sterile membrane filter
(MILLEX-GV, 0.22 mm) into a sterile vial. The retention time of the product
(7 min) was identical to that of the authentic sample.
Preparative HPLC was performed with a Lab-Quatec IP-7500 pump system
(Lab-Quatec. Co., Ltd., Tokyo, Japan).
Column: YMC-Pack C18Pro (10 ꢁ 250 mm, 5 mm)
Mobile phase: Acetonitrile: 0.1% acetic acid-0.05 M NH4OAc ¼ 70 : 30m >
(v/v)
Flow late: 6 ml/min
Wavelength: 254 nm
Retention time: RU40555 (11 min)
Whole body distribution of [11C]RU40555 in rat
Whole body distribution was examined with a new planar positron imaging
system (PPIS) with a spatial resolution less than 2.1 mm FWHM. This system
has been developed for studies in small animals and plants (Hamamatsu
Photonics, Shizuoka, Japan).19 Rats (n ¼ 3) were anesthetized with intra-
peritoneal injection of pentobarbital (25 mg/kg), and fixed on an acrylic plate,
which was the center of the detector units. A 60-min emission scan consisting
of 12 frames was obtained following [11C]RU40555 (15 MBq) intravenous
injection (Figure 4).
Accumulation of [11C]RU40555 in the rat brain
Normal (n ¼ 5) and ADX (n ¼ 5) rats were anesthetized with intraperitoneal
injection of pentobarbital (25 mg/kg), and [11C]RU40555 (50 MBq) was
intravenously injected. Twenty minutes after injection, arterial blood was
corrected for plasma corticosterone level measurement from the aorta
abdominalis, and then the rats were quickly decapitated. The cerebral cortex,
striatum, hippocampus, and cerebellum were removed, and their radioactivity
was counted using an auto well gamma counter (1480 WIZARD; Wallac Oy,
Turku, Finland). Data are represented as the percentage of injected dose
(%ID/kg/g) (Figure 5).
Analysis of plasma contents of unchanged [11C]RU40555 was performed
20 min after injection. Acetonitrile (0.1 ml) was added to the plasma sample
(0.1 ml), mixed at room temperature, and centrifuged for 3 min at 12 000g. The
supernatant was spotted onto TLC plates, and developed (MeOH:
H2O ¼ 80 : 20). After drying, the TLC plate was exposed to an imaging plate
(Fuji Film, Tokyo, Japan) for 45 min, and was analyzed for the contents of
nonmetabolites and metabolites of [11C]RU40555 using an imaging plate
reader (BAS1800, Fuji Film, Tokyo, Japan).
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SYNTHESIS AND EVALUATION OF [11C]RU40555
667
Conclusion
A selective GR antagonist, [11C]RU40555, was successfully synthesized by
using [11C]MeOTf. It was found that [11C]RU40555 could cross the blood
brain barrier. Though further experiments are necessary, we showed that
[11C]RU40555 has the potential to be used as a PET tracer for GR.
Acknowledgements
This study was performed in part through Special Coordination Funds for
Promoting Science and Technology from the Ministry of Education, Culture,
Sports, Science and Technology, the Japanese Government. The authors
thank Prof. Kiyofumi Yamada (Kanazawa University Graduate School of
Nature Science & Technology) for fruitful discussions. The authors also thank
Shigeo Hayashi for operation of the cyclotron.
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