Radiosyntheses of two positron emission tomography probes: [11C]Oseltamivir and its active metabolite [11C]Ro 64-0802

Article abstract of DOI:10.1016/j.bmcl.2008.01.037

Oseltamivir phosphate (Tamiflu, 1·H₃PO₄ is an orally active anti-influenza drug, which is hydrolyzed by esterase to its carboxylate metabolite Ro 64-0802 (2) with potent activity inhibiting neuraminidase. In this study, fo

Full text of DOI:10.1016/j.bmcl.2008.01.037

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1260–1263
Radiosyntheses of two positron emission tomography probes:
[11C]Oseltamivir and its active metabolite [11C]Ro 64-0802
Fujiko Konno,a Takuya Arai,a Ming-Rong Zhang,a,* Akiko Hatori,a
Kazuhiko Yanamoto,a Masanao Ogawa,a,b Gukuto Ito,a,b Chika Odawara,a
Tomoteru Yamasaki,a Koichi Katoa and Kazutoshi Suzukia
aRadiochemistry Section, Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences,
4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
bSHI Accelerator Service Co., Ltd, 5-9-11 Kitashinagawa, Shinagawa-ku, Tokyo 141-8686, Japan
Received 10 December 2007; revised 8 January 2008; accepted 10 January 2008
Available online 13 January 2008
Abstract—Oseltamivir phosphate (Tamifluꢀ, 1ÆH3PO4 is an orally active anti-influenza drug, which is hydrolyzed by esterase to its
carboxylate metabolite Ro 64-0802 (2) with potent activity inhibiting neuraminidase. In this study, for the first time, we synthesized
carbon-11-labeled oseltamivir ([11C]1) and Ro 64-0802 ([11C]2) as two novel positron emission tomography probes and demon-
strated that [11C]1 had twofold higher radioactivity concentration in the mouse brains than [11C]2.
ꢁ 2008 Elsevier Ltd. All rights reserved.
Oseltamivir phosphate (Tamifluꢀ, 1ÆH3PO4, Scheme 1)
is an orally active anti-influenza drug for the treatment
of influenza types A and B.1 It is a prodrug which is
hydrolyzed by esterase to its carboxylate metabolite
Ro 64-0802 (2, Scheme 1), a potent inhibitor for neur-
aminidase.2 This drug is regularly prescribed as a treat-
ment for seasonal influenza in Japan and Japanese
consumption accounts for up to 75% of all Tamiflu
use worldwide. However, the safety of 1 is questioned
because suicidal or abnormal behaviors especially in
younger patients have been reported after Tamiflu
ingestion.3
barrier, they may enter the brain if the BBB is immature
or damaged. However, the relationship between the
presence of 1 and 2 in the CNS and the side effects of
Tamiflu has not been elucidated clearly.
Positron emission tomography (PET) is a useful way of
medical imaging using radioactive agents labeled with
positron emission nuclides such as carbon-11 (11C), fluo-
rine-18, nitrogen-13 and oxygen-15.5 PET is a non-inva-
sive imaging modality that provides functional
information on physiological, biochemical and pharma-
cological processes in vivo. PET can be used to assess
the drug action in humans from the drug development
and evaluation process to clinical therapy. It has the
ability to give pharmacokinetic and pharmacodynamic
information about a drug to determine the drug efficacy,
and proof of potential biochemical mechanisms of drug
action, including the side and toxic effects. Thus, posi-
tron emitter-labeled 1 and 2 may be useful to determine
their presence in the brain and to elucidate the cause of
their CNS side effects in humans.
The mechanisms for the abnormal behaviors remain
uncertain. A high dose of 1 damaged the brain in exper-
imental animals, and was likely caused by uptake of 1
and/or 2 into the central nervous system (CNS). It has
been reported that 2 affects the CNS because neuramin-
idase, a key enzyme inhibited by 2, plays a role in the
CNS development and impulse conduction.3 In vitro
study using brain slices also revealed that 2 had clear ef-
fects on neuronal excitability.4 Therefore, although it is
believed that 1 and 2 do not easily pass the blood–brain
The aim of this study was to synthesize ethyl (3R,4R,5S)-
4-[carbonyl-11C]acetamido-5-amino-3-(1-ethylpropoxy)-
cyclohex-1-ene-1-carboxylate ([11C]1, Scheme 1) and
(3R,4R,5S)-4-[carbonyl-11C]acetamido-5-amino-3-(1- ethyl-
propoxy)cyclohex-1-ene-1-carboxylic acid ([11C]2) using
[1-11C]acetyl chloride6 ([11C]AcCl) and to determine their
Keywords:
[ [
11C]Oseltamivir; 11C]Ro 64-0802; Positron emission
tomography; [11C]Acetyl chloride.
*
Corresponding author. Tel.: +81 43 206 4041; fax: +81 43 206
3261; e-mail: zhang@nirs.go.jp
0960-894X/$ - see front matter ꢁ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.037

F. Konno et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1260–1263
1261
O
O
H
H
prodrug
in vivo
N
R
N R
EtO
HO
NH2
NH2
O
O
Oseltamivir (1): R = -COCH3
11C]1: R = -11CO CH3
Scheme 1. Chemical structures of oseltamivir (1), Ro 64-0802 (2) and their labeled compounds [11C]1, [11C]2.
Ro 64-0802 (2): R = -COCH3
[
[
11C]2: R = -11CO CH3
radioactivity concentrations in mouse brains. The label-
ing strategy did not change their chemical structures
and maintained their pharmacological profiles. So far,
chemical syntheses of non-radioactive 1 have been suc-
cessfully performed7 and a method for the synthesis of
[11C]1 has also been reported;8 however, radiosyntheses
of [11C]1 and [11C]2 have not been achieved. Radiosynthe-
sis for PET probes involving short-lived positron emitters
such as 11C (20.4 min), which is different from those used
in conventional organic synthesis, requires practical
methods and techniques under radiation protection.
The radiosynthesis must be accomplished within a short
time compatible to the half-life, using only a limited step
sequences. Another important characteristic of radiosyn-
thesis is the efficient production and application of the
radioactive labeling agents such as [11C]AcCl,9
[11C]CH3I10 and [11C]CH3NO2,11 which is not commer-
cially available like the non-radioactive AcCl, CH3I and
CH3NO2.
the analytic data previously reported.7c On the other
hand, the non-radioactive 2 was prepared by hydrolyz-
ing 1 with 2 N NaOH in almost quantitative yield.
Radiosyntheses of [11C]1 and [11C]2 were carried out as
shown in Scheme 3. The radioactive agent [11C]AcCl
was synthesized by reacting methylmagnesium bromide
(CH3MgBr) with [11C]CO2 on the inner surface of a nar-
row polyethylene tube, followed by chlorination with
oxalyl chloride.9 The radiochemically pure [[11C]AcCl
was purified by distillation and trapped in a solution
of THF containing 3 and Et3N at À15 ꢂC. After this re-
agent trapping ended, the acetylation required a further
5 min at 80 ꢂC to give [11C]5. Without purifying this
mixture, 6 N HCl (300 lL) was added to the reaction
to eliminate the Boc group of [11C]5. After the treat-
ment, the radioactive mixture was purified by HPLC
(Waters XBridge Prep C18 5 lm column, 10 mm ID
·50 mm, CH3CN/H2O/Et3N (30/70/1), 5 mL/min,
254 nm) to afford [11C]1. These sequences were accom-
plished using an automated synthesis system. Moreover,
[11C]1 was hydrolyzed with 1% NaOH for 5 min at
100 ꢂC to give [11C]2.
Here, we report: 1) chemical synthesis of a precursor (3)
for radiosynthesis using a new route, 2) radiosyntheses
of [11C]1 and [11C]2, 3) radioactivity concentrations of
[11C]1 and [11C]2 in the brain and blood of mice.
The identity, radiochemical purity and specific activity
of [11C]1 and [11C]2 were determined using HPLC and
radio-TLC. In the HPLC analysis, the retention time
was 6.6 min for [11C]1 (CAPCELL PAK C18 column:
4.6 mm ID ·250 mm, CH3CN/H2O/Et3N (30/70/0.1),
1.5 mL/min, 254 nm) and 5.2 min for [11C]2 (CAP-
CELL PAK C18 column: 4.6 mm ID ·250 mm,
CH3OH/pH 6.8 phosphate buffer (4/6), 1 mL/min,
210 nm), respectively. The identity was confirmed by
co-injecting with the corresponding non-radioactive
sample. The radiochemical purity of [11C]1 or [11C]2
was higher than 98% and the specific activity was
about 4 GBq/lmol as determined from the mass mea-
Precursor 3 for radiosynthesis was prepared using a new
route from 1, as shown in Scheme 2. The diamine 4 was
prepared by heating 1 with concentrated HCl in EtOH.
Excess treatment with HCl led to hydrolysis of the ester
group. Direct acetylation of 4 using an equimolar
amount of AcCl was simulated for radiosynthesis but
the desired 1 was not afforded. This result was supported
by that the preferable acylation took place in the steri-
cally less hindered 5-amine position.7d Therefore, the
5-amine group was firstly protected with Boc group to
give precursor 3. The structure of 3 was assigned by
NMR study and was further derived to 5, identical to
O
O
O
NH2
NH2
NH2
NHCOCH3
NHBo c
HCl
a
Boc2O
b
AcCl
c
1
EtO
EtO
EtO
NHBoc
O
O
O
4
3
5
NaOH
d
2
Scheme 2. Chemical synthesis. Reagents and conditions: (a) EtOH, 90 ꢂC, 5 h, 37%; (b) CH2Cl2, 25 ꢂC, 1 h, 78%; (c) cat. DMAP, pyridine, 90 ꢂC,
1 h, 95%; (d) EtOH, 90 ꢂC, 2 h, 99%.

1262
F. Konno et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1260–1263
O
CH3Mg Br
a
(COCl)2
NH11COCH3
NHBoc
3
CH311COOMgBr
NaOH
CH311COCl
11CO2
b
c
EtO
O
[
11C]5
HCl
d
[
11C]1
[
11 C]2
e
Scheme 3. Radiosynthesis. Reagents and conditions: (a) THF, À5 to 0 ꢂC; (b) THF, 75 ꢂC, min; (c) THF, 80 ꢂC, 5 min; (d) 80 ꢂC, 5 min, 10% from
11CO2; (e) 100 ꢂC, 5 min, 100%.
sured from the HPLC UV analysis. In the final prod-
uct solution, no significant peak relative to 3 and its
decomposition product was detected by HPLC. The
two products were further determined by radio-TLC
Table 1. Radioactivity (% of injection dose/g tissue: mean SD, n = 3)
in the brain and blood of mice at 1, 5,15, 30 and 60 min after injection
Tissue
Brain
Time (min)
[
11C]1
[
11C]2
1
5
0.20 0.01
0.15 0.01
0.14 0.01
0.10 0.01
0.07 0.02
0.35 0.03
0.21 0.04
with
a mixture as developing solvents: CHCl3/
CH3OH/AcOH (5/2/0.1), Rf 0.8 ([11C]1), 0.4 ([11C]2).
Co-elution with authentic standards on TLC sup-
ported their identity. Moreover, the radiochemical
purity of the products remained >95% after mainte-
nance of the preparations at 25 ꢂC for 3 h, and they
were stable for performing evaluation. From the end
of bombardment, the synthesis times were 30 min for
[11C]1 and 35 min for [11C]2, respectively. Starting
from the cyclotron-produced [11C]CO2 with total
radioactivity of 30–37 GBq, [11C]1 or [11C]2 was ob-
tained with about 740 MBq (n > 3) as an injectable
solution. This amount of radioactivity is enough for
animal experiments.
15
30
60
0.07
0.07 0.01
0.03
0
0
Blood
1
5
2.89 0.35
1.58 0.08
1.36 0.05
0.79 0.06
0.37 0.04
6.23 0.34
3.31 0.58
1.31 0.51
0.59 0.03
0.22 0.04
15
30
60
the BBB and be retained in the brain, although 1 has
been reported to
substrate.13
a substrate of P-glycoprotein
In conclusion, [11C]1 and its active metabolite [11C]2
were synthesized for the first time as two novel PET
probes. Preliminary evaluation showed that these probes
had low uptakes into the mouse brain, although it was
unclear whether the low concentrations in the brain
are enough to induce the side CNS effects of Tamiflu.
The next step of our study is to determine the detail dis-
tribution pattern and to acquire PET images of animal
and human brains of different ages. We believe that
our study is helpful to elucidate the cause of the side ef-
fects of Tamiflu using the two PET probes.
The preliminary study on the radioactivity concentra-
tions of [11C]1 and [11C]2 in the mouse brain and blood
was performed. A solution of [11C]1 and [11C]2 (average
of 20 MBq/400 lL) was injected into Wister mice (30 g,
7 weeks, male) through the tail vein. Three mice for each
time point were sacrificed by cervical dislocation at 1, 5,
15, 30 or 60 min after injection. The whole brain and
blood samples were quickly removed. The radioactivi-
ties present in these tissues were measured and expressed
as a percentage of the injected dose per gram of wet tis-
sue. All radioactivity measurements were corrected for
decay.
Acknowledgments
Table 1 summarizes the uptakes of [11C]1 and [11C]2 in
the brain and blood. As can be seen, [11C]1 and [11C]2
had relatively low uptakes into the mouse brain and
their radioactivity concentration in the brain was much
lower than some useful PET probes12 developed by us
for clinical brain imaging. Despite their low uptakes into
the brain, [11C]1 had higher levels in the brain than
[11C]2 from 15 to 60 min after injection. Within this per-
iod, the two probes displayed slow washout of radioac-
tivity from the brains, respectively, and the
concentration of [11C]1 was about twofold higher than
that of [11C]2. The ratios of brain/blood radioactivity,
which is an index determining the actual accumulation
of a radioligand in the brain, increased from 0.07 to
0.19 for [11C]1 and from 0.06 to 0.14 for [11C]2 at the
end of this experiment, respectively. Compared to
[11C]2, this result revealed that [11C]1 could pass through
The authors are grateful to the crew of the Cyclotron
Operation Section and Department of Molecular
Probes, Molecular Imaging Center of National Institute
of Radiological Sciences (NIRS) for their support in
the operation of the cyclotron and production of
radioisotopes. This study was supported in part by a
Grant-in-Aid for the Molecular Imaging Program from
the Ministry of Education, Culture, Sports, Science and
Technology, Japanese Government.
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