The first synthesis of [11C]oseltamivir: A tool for elucidating the relationship between Tamiflu and its adverse effects on the central nervous system

Article abstract of DOI:10.1002/jlcr.1604

Oseltamivir phosphate (Tamiflu) is an anti-influenza drug approved in many countries. Recently, in Japan, adverse effects on the central nervous system have been reported in younger patients administrated with Tamiflu. As a tool for elucidating the relationship between Tamiflu and its adverse effects, ¹¹C-labeled oseltamivir was synthesized through a two-step reaction involving [¹¹C]acetylation with [1-¹¹C]acetyl chloride. Starting from approximately 37.0 GBq of [¹¹C]CO₂, 1.2-1.8 GBq (n=5) of [¹¹C]oseltamivir was obtained at the end of synthesis (EOS) 36-39 min after the end of bombardment. Radiochemical purity and specific activity were greater than 98% and 2.7-6.3 GBq/μmol at EOS, respectively. Copyright

Full text of DOI:10.1002/jlcr.1604

Research Article
Received 27 August 2008,
Revised 14 January 2009,
Accepted 28 March 2009
Published online 3 July 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1604
The first synthesis of [11C]oseltamivir: a tool
for elucidating the relationship between
Tamiflu and its adverse effects on the central
nervous system
Ã
Takuya Arai,a Fujiko Konno,a Masanao Ogawa,a,b Ming-Rong Zhang,a and
Kazutoshi Suzukia
Oseltamivir phosphate (Tamiflus) is an anti-influenza drug approved in many countries. Recently, in Japan, adverse effects
on the central nervous system have been reported in younger patients administrated with Tamiflu. As a tool for elucidating
the relationship between Tamiflu and its adverse effects, 11C-labeled oseltamivir was synthesized through a two-step
reaction involving [11C]acetylation with [1-11C]acetyl chloride. Starting from approximately 37.0 GBq of [11C]CO2,
1.2–1.8 GBq (n = 5) of [11C]oseltamivir was obtained at the end of synthesis (EOS) 36–39 min after the end of bombardment.
Radiochemical purity and specific activity were greater than 98% and 2.7–6.3 GBq/lmol at EOS, respectively.
Keywords: [11C]oseltamivir; [1-11C]acetyl chloride; PET
Positron Emission Tomography (PET) is an advanced technology
to study drug biodistribution and interaction with target proteins
directly in experimental animals and humans using radiotracers
labeled with short-lived positron emitters such as 11C, 13N, 15O and
prodrug of the potent and selective neuraminidase inhibitor
18F. PET imaging has emerged as a powerful scientific and clinical
Ro 64-0802, is an anti-influenza drug approved in many countries
Introduction
Oseltamivir phosphate (Tamiflus, 1ꢀH3PO4, Figure 1), an ethyl ester
for the treatment and prevention of influenza types A and B. The
number of Tamiflu prescriptions has reached approximately
10 million in Japan, which accounted for 70–80% of the world
total in 2006. Recently, in Japan, abnormal behavior such as
suicide attempts, delirium and self-injury has been reported,
especially in younger patients administrated with Tamiflu.
According to the report by the Ministry of Health, Labor and
Welfare, abnormal behavior associated with Tamiflu administra-
tion was observed in 211 patients (0.002% of all patients), which
becomes a serious problem; however, the relationship between
Tamiflu and abnormal behavior has not been clearly elucidated.
Adverse effects on the central nervous system (CNS) are
generally induced by the accumulation of a drug and/or its
metabolite(s) in the brain through the blood–brain barrier (BBB).
However, it is believed that Tamiflu and its active metabolite Ro
64-0802 do not easily pass through the BBB. Recent studies
demonstrated that the penetration of Tamiflu into the brain was
limited by the drug efflux transporter P-glycoprotein (P-gp) at
the BBB.1 On the other hand, P-gp expression level was
significantly lower in fetal or immature experimental animals
than in adults2,3 and its functional activity can be affected by
several factors, such as genetic polymorphisms of P-gp,4
drug–drug or drug–food interaction5–7 and CNS inflammation.8,9
These reports suggested that these factors can increase the
brain permeability of Tamiflu and its metabolites, leading to
adverse effects on the CNS.
tool to investigate the behavioral, therapeutic and toxic properties
of drugs. PET provides a new perspective on drug research to
directly assess both the pharmacokinetic and pharmacodynamic
properties in animals and humans. Recent studies on the adverse
effects of Tamiflu on the human CNS led us to synthesize 1
labeled with a positron emitter as a tool for studying the
distribution of the drug. In this paper, we report the first synthesis
of (3R,4R,5S)-ethyl 4-[carbonyl-11C]acetamido-5-amino-3-(pentan-
3-yloxy)cyclohex-1-ene-1-carboxylate ([11C]1) using an automated
production system. [1-11C]acetyl chloride ([11C]AcCl) was synthe-
sized by reacting [11C]CO2 with methyl magnesium bromide
(MeMgBr) in a polyethylene loop, followed by chlorination with
oxalyl chloride ((COCl)2) and distilled efficiently into the second
reaction vessel containing a solution of precursor 2. After the
[11C]acetylation of 2 (Figure 2), the Boc group in [11C]4 was easily
removed with 4 M HCl at 1201C in 3min (Scheme 1). We believe
aDepartment 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., 1-17-6 Osaki, Shinagawa-ku, Tokyo 141-8686,
Japan
*Correspondence to: Takuya Arai, Radiochemistry Section, Department of
Molecular Probes, Molecular Imaging Center, National Institute of Radiological
Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.
E-mail: arai_t@nirs.go.jp
J. Label Compd. Radiopharm 2009, 52 350–354
Copyright r 2009 John Wiley & Sons, Ltd.

I. Arai et al.
that our study will help to elucidate the apparent relationship and requires a remote-controlled system for radiation protec-
between Tamiflu and its adverse effects on the CNS.
tion. In addition, Tamiflu is rapidly hydrolyzed to its active form
Ro 64-0802 by carboxylesterase in vivo. Therefore, 11C-labeling
of the acetamide group in 1 with [11C]AcCl was considered to be
the most appropriate method.
Results and discussion
We initially considered to synthesize [11C]1 through a one-step
reaction between the diamine 3 and [11C]AcCl. In a preliminary
study using nonradioactive reagents, however, the acetylation of 3
with an equimolar amount of AcCl occurred preferentially in the
sterically less hindered 5-amino position, resulting in very poor yield
of 1.10 Therefore, precursor 2 protecting the 5-amino group with
the Boc group was synthesized from 1 by a new route (Scheme 2).
The two-step synthesis of 2 was accomplished with an overall yield
of 28%. The structural and stereochemical assignment of 2 was
confirmed by a comparison of 1H NMR, 13C NMR and optical
rotation data for 1 synthesized from 2 with those of an authentic
sample11. Physical and spectroscopic data for 4 were also identical
to those reported previously.12 Additionally, precursor 2 showed no
contamination by the target compound 1 in the HPLC analysis.
The synthesis of [11C]1 was achieved through a two-step
reaction consisting of the [11C]acetylation of 2 and the removal of
the Boc group in the resultant [11C]4 (Scheme 1). Figure 2 shows a
There are several restrictions on the production of radiotracers
labeled with a short-lived positron-emitting radionuclide. The
production of radiotracers must be achieved within a short time
O
O
NHAc
NHAc
NH
EtO C
NH · H PO
HO C
Tamiflu
Ro 64-0802
Figure 1. Chemical structures of Tamiflu and Ro 64-0802.
N2 (0.01% O2)
Target
Regulator
F. M.
P
P
To w a s t e
Vacuum pump
F. M.
N2 (0.01% O2)
(C) (B) (A)
N2 (0.01%
O2)
(E) (D)
To H P L C
To w a s t e
2nd reaction vessel 1st reaction vessel
Polyethylene loop
Stainless-steel coil
(Cooling air)
(Hot air) (Liq. N2)
(Hot air)
(Liq. N2)
(Hot air)
(Liq. N2)
(Hot air)
Figure 2. Schematic diagram of an automated production system for [11C]1. (a) (COCl)2/THF; (b) 1 M MeMgBr/THF; (C) DMF; (D) 4 M HCl; (E) 10% NH3 solution; F.M.: flow
meter; P: pressure gauge; : two-way solenoid valve; : three-way solenoid valve; : manual three-way valve; : cooler; : heater; : radioactivity sensor.
O
O
O
[1-11C]AcCl, Et N, THF
4M HCl
H
N
H
N
Me
Me
NH
30°C, 3 min
120°C, 3 min
C
O
C
O
EtOOC
NHBoc
EtOOC
NHBoc
EtOOC
NH
2
[11C]4
[11C]1
Scheme 1. Synthesis of [11C]1.
J. Label Compd. Radiopharm 2009, 52 350–354
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

I. Arai et al.
O
Conc. HCl, EtOH
O
O
Boc2O, CH2Cl2
room temperature, 1 h
78%
90°C, 5 h
35%
NHAc
NH
NH
NH
NH
EtOOC
EtOOC
EtOOC
NHBoc
1
3
2
AcCl, Et N, CH Cl
O
Conc. HCl, EtOH
O
50°C, 1 h
90%
room temperature, 2 h
77%
NHAc
NHAc
EtOOC
NHBoc
EtOOC
NH
4
1
Scheme 2. Synthesis of precursor 2.
schematic diagram of an automated production system for [11C]1.
Synthesis involves the following steps: (1) trapping [11C]CO2 in a
stainless-steel coil; (2) transferring [11C]CO2 into the polyethylene
loop coated with MeMgBr; (3) passing (COCl)2/THF through the
loop and transferring the radioactive mixture into the first reaction
vessel; (4) distilling [11C]AcCl into the second reaction vessel
containing a solution of precursor 2; (5) [11C]acetylation of 2; (6)
adding 4 M HCl into the second reaction vessel to remove the Boc
group in the resultant [11C]4; (7) purification of [11C]1 by HPLC.
Starting from approximately 37.0 GBq of [11C]CO2, 1.2–1.8 GBq
(n=5) of [11C]1 was obtained at the end of synthesis (EOS).
The total synthesis time was 36–39 min from the end of
bombardment. Radiochemical purity and specific activity were
greater than 98% and 2.7–6.3 GBq/mmol at EOS, respectively. No
peaks corresponding to precursor 2 and other chemical impurities
were detected by HPLC. Its identity was confirmed by HPLC co-
injection with an authentic sample and LC-MS analysis. The retention
time of [11C]1 matched that of an authentic sample. Furthermore,
the LC-MS spectra showed an [M1H1] ion at m/z 313.2
corresponding to oseltamivir. Although the small radioactive peak
that had a retention time identical to that of possible by-product 5-
acetylamino derivative 5 was observed in HPLC analysis of the final
reaction mixture, [11C]1 and this impurity were completely separated
by semipreparative HPLC (Figure 3). Previously, we found that the
nonradioactive CO2 present in the MeMgBr solution acted as a
source of contamination of the [11C]CO2 and reduced the specific
activity of the product.13 Although only a small amount of MeMgBr
was used for the synthesis of [11C]AcCl, the specific activity of [11C]1
was unexpectedly low. The cause of this is currently under
investigation.
Figure 3. HPLC profiles of (a) the final reaction mixture and (b) [11C]1 isolated from the
reaction mixture by HPLC, co-injected with authentic samples of 1 and 5-acetylamino
derivative. HPLC conditions: column, CAPCELL PAK C18 (Shiseido, 4.6 mm
i.d. ꢁ 250 mm); eluent, MeCN/H2O/Et3N (30/70/0.1); flow rate, 1.5 mL/min; UV 254 nm.
Experimental
Materials and methods
Tokyo, Japan). [11]Carbon dioxide was produced by the
bombardment of dry N2 gas (1.5 MPa; Nippon Sanso,
Tokyo, Japan) containing 0.01% O2 (Nippon Sanso) with a beam
Carbon-11 was produced by the 14N(p, a)11C nuclear reaction
using the CYPRIS HM-18 cyclotron (Sumitomo Heavy Industry,
www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 350–354

I. Arai et al.
(15–20 mA) of 18 MeV protons (14.2 MeV on target). Radioactivity Synthesis of (3R,4R,5S)-ethyl 4-amino-5-(tert-butoxycarbonylami-
no)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxylate (2)
was measured by
a dose calibrator (IGC-3R Curiemeter;
Aloka, Tokyo, Japan). HPLC was performed using a JASCO HPLC
system (JASCO, Tokyo, Japan): the eluate was monitored for
radioactivity (NaI (Tl) scintillation detector system) and UV
absorption at 254 nm. For analytical HPLC, CAPCELL PAK C18
column (4.6 mm i.d. ꢁ 250 mm, Shiseido, Tokyo, Japan) was
used and eluted with MeCN/H2O/Et3N (30/70/0.1) at a flow
rate of 1.5 mL/min (1; retention time = 6.6 min). For semipre-
parative HPLC, XBridge C18 column (10 mm i.d. ꢁ 250 mm,
Waters Co., Milford, MA, USA) was used and eluted with
MeCN/H2O/Et3N (30/70/1) at a flow rate of 5.0 mL/min (1;
retention time = 7.2 min). LC-MS was performed in positive
ion mode on an Agilent 1100 Series HPLC system (Agilent
Technologies, Walldbron, Germany) coupled with an
Applied Biosystem 4000 QTrap mass spectrometer (Applied
Biosystems, Darmstadt, Germany). The LC system was
equipped with an XBridge C18 column (3 mm i.d.
ꢁ 100 mm, Waters Co.), eluted with MeCN/5 mM HCOOH
To a solution of 3 (46.5 mg, 0.17 mmol) in CH2Cl2 (3 mL) was
added di-tert-butyldicarbonate (41.4 mg, 0.19 mmol) in CH2Cl2
(1.5 mL). The reaction mixture was stirred at room temperature
for 1 h. After evaporation of the solvent, the residue was purified
by column chromatography on silica gel with CHCl3 to give 2
(49.5 mg, 78%) as colorless prisms: m.p. 51–521C; IR (ATR) nmax
1699, 1715 cmꢂ1 1H NMR (300 MHz, CDCl3) d (ppm) 0.93 (t,
;
J = 7.3 Hz, 6H), 1.29 (t, J = 7.2 Hz, 3H), 1.45 (s, 9H), 1.48–1.63 (m,
6H), 2.16–2.27 (m, 1H), 2.76–2.89 (m, 2H), (m, 1H), 3.40 (quintet,
J = 5.7 Hz, 1H), 3.72 (br, 1H), 3.82–3.85 (m, 1H), 4.20 (q, J = 7.2 Hz,
2H), 5.01 (br, 1H), 6.81 (s, 1H); 13C NMR (75 MHz, CDCl3) d (ppm)
9.5, 9.6, 14.2, 25.7, 26.3, 28.3, 55.1, 60.8, 78.5, 81.2, 129.3, 136.6,
155.7, 166.3; HRMS (FAB): m/z calculated for C19H35N2O5 [M1H1]:
371.2546, found 371.2581; ½aꢃ2D7 ꢂ27.3 (c 0.36, CHCl3).
Synthesis of (3R,4R,5S)-ethyl 4-acetamido-5-(tert-butoxycarbonyla-
mino)-3-(pentan-3-yloxy)cyclohex-1-ene1-carboxylate (4)
(25/75) at
a flow rate of 0.3 mL/min (retention time =
5.2 min).
To a solution of 2 (49.5 mg, 0.13 mmol) and Et3N (23.6 mL,
Melting points (m.p.) were uncorrected. 1H NMR and 13C NMR 0.17 mmol) in CH2Cl2 (3 mL), acetyl chloride (10.2 mL, 0.14 mmol)
spectra were recorded on a JNM-GX-270 spectrometer. High- in CH2Cl2 (2 mL) was added dropwise at 01C. The reaction
resolution FAB mass spectra (HRMS (FAB)) were obtained on a mixture was stirred at room temperature for 2 h. After
JEOL NMS-SX 102-SX spectrometer. Optical rotations were evaporation of the solvent, the residue was purified by column
recorded on a JASCO P-2200 digital polarimeter. Column chromatography on silica gel with 40% AcOEt/hexane to gave 4
chromatography was performed on Merck Kieselgel gel 60 (42.4 mg, 77%) as colorless needles: m.p. 140–1411C; IR (ATR)
1
F254 (70–230 mesh). TLC was carried out on Merck Kieselgel 60 nmax 1670, 1690, 1705 cmꢂ1; H NMR (300 MHz, CDCl3) d (ppm)
F254 plates.
0.88 (t, J = 7.3 Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H), 1.29 (t, J = 7.2 Hz,
Chemicals and solvents were obtained commercially and used 3H), 1.43 (s, 9H), 1.48–1.54 (m, 4H), 1.99 (s, 3H), 2.25–2.34 (m, 1H),
without further purification. Oseltamivir phosphate was pur- 2.74 (dd, J = 17.8, 5.3 Hz, 1H), 3.36 (quintet, J = 5.6 Hz, 1H),
chased from Sequoia Research Products Ltd. (Oxford, UK), 3.74–3.85 (m, 1H), 3.96–3.98 (m, 1H), 4.03–4.16 (m, 1H), 4.20–4.26
MeMgBr in THF (about 1.0 M) from Kanto Chemical Co., Inc. (m, 2H), 5.14 (d, J = 9.0 Hz, 1H), 5.88 (d, J = 9.2 Hz, 1H), 6.79 (s, 1H);
(Tokyo, Japan) and (COCl)2 and 2,6-di-tert-butylpyridine from 13C NMR (75 MHz, CDCl3) d (ppm) 9.2, 9.5, 14.1, 23.3, 25.7, 26.1,
Sigma-Aldrich Chemical Co., Ltd. (Milwaukee, WI, USA) at the 28.3, 30.9, 49.1, 54.4, 60.9, 75.8, 79.6, 82.2, 129.3, 137.6, 156.3,
highest grade available.
165.9, 170.8; MS (FAB): m/z 413 [M1H1]; ½aꢃD27 ꢂ94.4 (c 0.20,
CHCl3).
Chemistry
Synthesis of (3R,4R,5S)-ethyl 4-acetamido-5-amino-3-(pentan-3-
yloxy)cyclohex-1-ene-1-carboxylate (1)
Synthesis of (3R,4R,5S)-ethyl 4,5-diamino-3-(pentan-3-yloxy)cyclo-
hex-1-ene-1-carboxylate (3)
To a solution of 4 (40 mg, 0.10 mmol) in EtOH (2 mL) was added
concentrated HCl (1 mL). The reaction mixture was stirred at
501C for 1 h. After evaporation of the solvent, the residue was
dissolved with H2O (2 mL) and washed with AcOEt (1 mL). The
layers were separated and the aqueous phase was basified with
saturated NaHCO3 and extracted with CHCl3 (3 mL ꢁ 3). The
combined organic extracts were dried over Na2SO4, filtered and
evaporated. The residue was purified by column chromatogra-
phy on silica gel with 5% MeOH/CHCl3 to give 1 (27.2 mg, 90%)
as colorless prisms: m.p. 107–1081C; 1H NMR (300 MHz, CDCl3) d
(ppm) 0.90 (t, J = 7.3 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H), 1.29 (t,
J = 7.0 Hz, 3H), 1.46–1.57 (m, 4H), 2.05 (s, 3H), 2.10–2.21 (m, 1H),
2.76 (dd, J = 17.6, 5.1 Hz, 1H), 3.23 (quintet, J = 5.5 Hz, 1H),
3.31–3.38 (m, 1H), 3.48–3.57 (m, 1H), 4.21 (dd, J = 14.3, 7.0 Hz,
2H), 4.21–4.26 (m, 1H), 5.68 (d, J = 8.1 Hz, 1H), 6.79 (s, 1H); 13C
NMR (75 MHz, CDCl3) d (ppm) 9.3, 9.5, 14.2, 23.7, 25.7, 26.2, 33.6,
49.2, 59.0, 60.8, 74.8, 81.6, 129.6, 137.5, 166.3, 170.9; MS (FAB):
m/z 313 [M1H1]; ½aꢃD26 ꢂ60.9 (c 0.36, CHCl3). Comparison of 1
To a solution of 1 (114 mg, 0.37 mmol) in EtOH (2 mL) was
added concentrated HCl (1 mL). The reaction mixture was
stirred at 901C for 5 h. After evaporation of the solvent,
the residue was dissolved with H2O (3 mL) and washed with
AcOEt (2 mL). The layers were separated and the aqueous
phase was basified with saturated NaHCO3 and extracted with
CHCl3 (5 mL ꢁ 3). The combined organic extracts were dried
over Na2SO4, filtered and evaporated. The residue was
purified by column chromatography on silica gel with 2%
MeOH/CHCl3 to give 3 (34.9 mg, 35%) as a pale yellow oil:
1
IR (ATR) nmax 1714, 3292, 3373 cmꢂ1; H NMR (300 MHz, CDCl3)
d (ppm) 0.923 (t, J = 7.3 Hz, 3H), 0.93 (t, J = 7.3 Hz, 3H), 1.28 (t,
J = 7.2 Hz, 3H), 1.44–1.69 (m, 4H), 1.97–2.08 (m, 1H), 2.60–2.82
(m, 3H), 3.37 (quintet, J = 5.7 Hz, 1H), 3.77–3.81 (m, 1H),
4.18 (q, J = 7.2 Hz, 2H), 6.78 (s, 1H); 13C NMR (75 MHz, CDCl3) d
(ppm) 9.4, 9.7, 14.1, 25.7, 26.4, 34.0, 51.1, 58.5, 60.7, 79.0,
80.9, 129.4, 137.6, 166.5; HRMS (FAB): m/z calculated for
C14H27N2O3 [M1H1]: 271.2022, found 271.2056; ½aꢃ2D2 ꢂ40.3 (c
0.55, CHCl3).
1
with an authentic sample revealed that H NMR, 13C NMR and
optical rotation data were identical. For an authentic sample,
m.p. 107–1081C; 1H NMR (300 MHz, CDCl3) d (ppm) 0.90
J. Label Compd. Radiopharm 2009, 52 350–354
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

I. Arai et al.
(t, J = 7.3Hz, 3H), 0.91 (t, J = 7.3Hz, 3H), 1.29 (t, J = 7.0 Hz, 3H), 3 min to facilitate the [11C]acetylation of 2. Into the second
1.46–1.57 (m, 4H), 2.05 (s, 3H), 2.09–2.21 (m, 1H), 2.76 (dd, reaction vessel, 4 M HCl (300 mL) was added to remove the Boc
J = 17.2, 5.1Hz, 1H), 3.23 (quintet, J = 5.5 Hz, 1H), 3.31–3.38 (m, group in the resultant [11C]4. After reaction at 1201C for 3 min,
1H), 3.48–3.56 (m, 1H), 4.21 (dd, J = 14.3, 7.0Hz, 2H), 4.21–4.24 (m, the reaction mixture was treated with 10% NH3 solution (500 mL)
1H), 5.62 (d, J = 8.1Hz, 1H), 6.79 (s, 1H); 13C NMR (75 MHz, CDCl3) and applied onto a semipreparative HPLC column. A radioactive
d (ppm) 9.3, 9.5, 14.2, 23.7, 25.7, 26.2, 33.7, 49.2, 59.1, 60.8, 74.8, fraction with a retention time of approximately 7 min was
81.6, 129.6, 137.5, 166.3, 170.9; ½aꢃ2D7 ꢂ60.8 (c 0.36, CHCl3).
collected, evaporated and dissolved in sterile water for injection.
(3R,4R,5S)-ethyl 5-acetamido-4-amino-3-(pentan-3-yloxy)cyclohex- Analysis of [11C]1
1-ene-1-carboxylate (5)
The identity of [11C]1 was confirmed by HPLC co-injection with
Colorless needles; m.p. 160–1611C; 1H NMR (300MHz, CDCl3) d
(ppm) 0.92 (t, J= 7.3 Hz, 3H), 0.93 (t, J=7.3Hz, 3H), 1.29 (t, J=7.0Hz,
3H), 1.47–1.62 (m, 4H), 1.98 (s, 3H), 2.26–2.34 (m, 1H), 2.76–2.83 (m,
1H), 2.90–2.95 (m, 1H), 3.42 (quintet, J= 5.9 Hz, 1H), 3.81–3.82 (m,
1H), 4.02–4.11 (m, 1H), 4.20 (dd, J= 14.3, 7.0 Hz, 2H), 6.44 (d,
J= 7.7 Hz, 1H), 6.85 (s, 1H); 13C NMR (75MHz, CDCl3) d (ppm) 9.5,
14.1, 23.4, 25.8, 26.2, 29.5, 48.4, 54.0, 60.8, 77.9, 81.4, 129.4, 135.9,
166.3, 170.0; MS (FAB): m/z 313 [M1H1]; ½aꢃD27 ꢂ24.7 (c 0.35, CHCl3).
an authentic sample and LC-MS analysis. Radiochemical purity
analysis of [11C]1 was performed on an analytical HPLC column.
Specific activity was calculated by determining the area of the
UV absorbance peak of the carrier ligand in an aliquot of known
radioactivity compared with the area of a standard sample.
Conclusion
Through a two-step reaction consisting of the [11C]acetylation of
2 with [11C]AcCl, followed by the removal of the Boc group in
the resultant [11C]4, [11C]1 was synthesized repeatedly and
reliably in adequate radiochemical yield and purity suitable for
routine use in animal and human studies.
Radiochemistry
Automated production system for [11C]1
The automated production system used for the synthesis of
[11C]1 was developed at the National Institute of Radiological
Sciences (NIRS). The unit is versatile and equipped for the
synthesis of multiple PET radiopharmaceuticals (Figure 2).14
The coil is a stainless-steel trap (SUS316; length: 1.5 m; o.d.: 18 in;
i.d.: 1 mm) and the loop is a polyethylene tube (length: 40 cm; o.d.:
Acknowledgement
The authors thank the cyclotron crew of NIRS and Mr M. Takei,
Tokyo Nuclear Service, for their technical support in the operation
of the cyclotron and production of radioisotopes. We also thank Mr
K. Furutsuka and T. Ito, SHI Accelerator Service, for their excellent
assistance in the MS analysis. This study was partially supported by
a consignment grant for the Molecular Imaging Program on
Research Base for PET Diagnosis from the Japanese Ministry of
Education, Culture, Sports, Science and Technology (MEXT).
1
16 in; i.d.: 0.75mm). Major parts, such as the coil, loop, reaction
1
vessels and solenoid valves, are connected by 16 in Teflon tubes.
Quick cooling and heating (monitored by a temperature sensor)
during these processes is realized by using a cooling device
(Vortex, OH, USA) with liquid N2 and a heating device (Nakazawa
Seisakusho, Chiba, Japan) with hot air, respectively.
Synthesis of [11C]AcCl
References
During the production of [11C]CO2, MeMgBr (1 M in THF, 500 mL)
was passed through the polyethylene loop (cooled between ꢂ5
and 01C) previously flushed with N2. N2 (3 mL/min) was then
passed through the loop for 30 s to remove the excess MeMgBr
solution and to leave a thin film of MeMgBr. After irradiation,
[11C]CO2 was carried from the target with a stream of N2
(1000 mL/min) and trapped in the stainless-steel coil cooled
between ꢂ170 and ꢂ1651C. After trapping of [11C]CO2, this coil
was heated at 501C and concentrated [11C]CO2 was transferred
in an N2 stream (3.0 mL/min) into the loop (maintained between
ꢂ5 and 01C) coated with MeMgBr. By passing (COCl)2/THF (10/
400, 400 mL) through the loop, the radioactive mixture was
transferred into the first reaction vessel containing 2,6-di-t-
butylpyridine (30 mL).
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Synthesis of [11C]1
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After the addition of DMF (300 mL) into the first reaction vessel,
synthesized [11C]AcCl was distilled into the second reaction
vessel (maintained between ꢂ10 and ꢂ51C) containing a
solution of 2 (2 mg) and Et3N (30 mL) in THF (300 mL) by heating
the first reaction vessel at 1201C in an N2 stream (150 mL/min).
The entire distillation step lasted for approximately 5 min. After
distillation, the second reaction vessel was heated at 301C for
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J. Label Compd. Radiopharm 2009, 52 350–354