Radiosynthesis of [13N]dantrolene, a positron emission tomography probe for breast cancer resistant protein, using no-carrier-added [13N]ammonia

Article abstract of DOI:10.1016/j.bmc.2011.10.077

Dantrolene (1) is a substrate for breast cancer resistant protein, which is widely distributed in the blood-brain-barrier, intestine, gall bladder, and liver. PET study with 1 labeled with a positron emitter can be used to visualize BCRP and to elucidate

Full text of DOI:10.1016/j.bmc.2011.10.077

Bioorganic & Medicinal Chemistry 20 (2012) 305–310
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Bioorganic & Medicinal Chemistry
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Radiosynthesis of [13N]dantrolene, a positron emission tomography probe
for breast cancer resistant protein, using no-carrier-added [13N]ammonia
Katsushi Kumata a, Masanao Ogawa a,b, Makoto Takei a,c, Masayuki Fujinaga a, Yuichiro Yoshida a,b
,
Nobuki Nengaki a,b, Toshimitsu Fukumura a, Kazutoshi Suzuki a, Ming-Rong Zhang a,
⇑
a Department of Molecular Probes, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
b SHI Accelerator Service Co. Ltd, 1-17-6 Osaki, Shinagawa-ku, Tokyo 141-8686, Japan
c Tokyo Nuclear Service Co. Ltd, 7-2-7 Ueno, Taito-ku, Tokyo 110-0005, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Dantrolene (1) is a substrate for breast cancer resistant protein, which is widely distributed in the
blood–brain-barrier, intestine, gall bladder, and liver. PET study with 1 labeled with a positron emitter
can be used to visualize BCRP and to elucidate the effect of BCRP on the pharmacokinetics of drugs.
The objective of this study was to label 1 using nitrogen-13 (13N, a positron emitter; half-life:
Received 29 September 2011
Revised 27 October 2011
Accepted 29 October 2011
Available online 4 November 2011
9.9 min). Using no-carrier-added
[
13N]NH3 as the labeling agent, we synthesized [13N]dantrolene
([13N]1) for the first time. The reaction of carbomyl chloride 2b with [13N]NH3 gave an unsymmetrical
urea [13N]3, followed by cyclization of [13N]3 to afford [13N]1. Due to its instability, 2b was prepared
in situ by treating amine 5 with triphosgene in a ratio of 4 to 1 and used for subsequent [13N]ammonol-
ysis without purification.
Keywords:
[
13N]Dantrolene
PET
Breast cancer resistant protein
13N]Ammonia
Ó 2011 Elsevier Ltd. All rights reserved.
[
1. Introduction
the pharmacokinetic and pharmacodynamic profiles of drugs in the
living body and to elucidate the therapeutic efficacy, side and toxic
Dantrolene (1, 1-{[5-(4-nitrophenyl)-2-furyl]methylideneamino}
imidazolidine-2,4-dione; Scheme 1) is a skeletal muscle relaxant
that is currently used for specific and effective treatment of malig-
nant hyperthermia.1,2 It is also used for the management of neuro-
leptic malignant syndrome, muscle spasticity (e.g., after strokes, in
paraplegia, cerebral palsy, or patients with multiple sclerosis),
ecstasy intoxication, serotonin syndrome, and 2,4-dinitrophenol
poisoning.3–6 Recently, it has been shown that 1 is a substrate for
breast cancer resistance protein (BCRP).7 BCRP, one of the ATP-
binding cassette (ABC) transporters, was initially isolated from
atypical multidrug-resistant MCF-7 human breast cancer cells,
and is an efflux transporter with wide substrate specificity recog-
nizing molecules of either negative or positive charge, organic an-
ions, and sulfate conjugates.8,9 It has been reported that BCRP plays
an important role in the absorption (small intestine), distribution
(placenta and blood–brain-barrier), and elimination (liver and
small intestine) of drugs.10 BCRP, like other ABC transporters, is as-
sumed to limit the uptake of drugs and other xenobiotic com-
pounds from the blood into the brain.7,10,11
effects, and potential action mechanisms of drugs.12–15 The signifi-
cant effectiveness of PET motivated us to label 1 with a positron
emitter and to use this PET probe for imaging BCRP in the blood–
brain-barrier for the first time and examining the relationship be-
tween BCRP and brain uptake of 1 in rodents and primates.
The aim of this study was to label 1 with 13N (positron emitter,
half-life: 9.965 min; 100% b+, decay) and to synthesize 1-{[5-
(4-nitrophenyl)-2-furyl]methylideneamino}-3-[13N]imidazolidine-
2,4-dione ([13N]dantrolene, [13N]1, Scheme 1). Although 1 has been
previously labeled with [11C]COCl2 in our laboratory,16 a useful
13N-labeled PET ligand has several favorable features. For example,
PET imaging using an 13N-ligand can be repeated on the same indi-
vidual within a shorter period of time and often results in much
lower radiation burden on the subject than using an 11C-ligand.
So far, no 13N-labeled PET ligands except [13N]NH3
have
17–19
been developed for clinical use. A main reason is that the labeling
technique using 13N and [13N]NH3 has not been well established.
O
O
Positron emission tomography (PET) is a useful molecular imag-
ing tool using radioactive probes labeled with positron-emitting
isotopes, such as 11C, 18F, 13N, and 15O. PET can be used to investigate
O
CH
N
N
O
CH
N
N
NH
13NH
O2N
O2N
O
O
dantrolene
[
13N]dantrolene
(1)
([13N]1)
⇑
Corresponding author. Tel.: +81 43 382 3708; fax: +81 43 206 3261.
E-mail address: zhang@nirs.go.jp (M.-R. Zhang).
Scheme 1. Chemical structures of dantrolene (1) and [13N]dantrolene ([13N]1).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.077

306
K. Kumata et al. / Bioorg. Med. Chem. 20 (2012) 305–310
Radiosynthesis with 13N must be accomplished within a short time
compatible with its short half-life, using only a limited step se-
quence. Another characteristic of radiosynthesis involves the effi-
cient production and application of anhydrous [13N]NH3 with
high specific activity as a labeling agent.20–22 Because of its short
half-life and the ease of contamination by nitrogen carrier in air,
it was difficult to produce 13N-labeled ligands with high specific
activity (>37 GBq/mmol) and enough radioactivity to carry out
conventional PET studies.
containing H2O and EtOH (10 mM). The irradiated solution was
quickly passed through a pre-conditioned cation exchange column
to concentrate [13N]NH3 onto this column. The [13N]NH3 was re-
leased with aqueous KOH from the column, dried by flowing
through another column filled with CaO, and then trapped into a
cooled vial containing various solvents and solutions. The specific
activity of anhydrous [13N]NH3 in the present study was estimated
to 37–100 GBq/l
mol (n > 20) at the end of [13N]NH3 production.
Ten years ago, Suzuki et al. developed an automated synthesis
system for producing anhydrous [13N]NH3 (>37 GBq/mmol).23,24
Using this synthesis system, we synthesized [13N]thalidomide25
2.3. [13N]Ammonolysis
2.3.1. [13N]Ammonolysis of 4-nitrophenylcarbamate 2a with
and
[
13N]carbamazepine,26 two therapeutic drugs. We used
[
13N]NH3
small-animal PET to determine the pharmacokinetics in vivo of
two PET probes. To widen the usefulness of the labeling technique
with high specific activity [13N]NH3, we selected 1 as a target com-
pound in the present study, with the expectation that [13N]1 could
become a promising PET probe for imaging BCRP. Here, we synthe-
sized [13N]1 using no-carrier-added [13N]NH3 equipped with an
entirely-automated synthesis system for the first time.
As a substrate for [13N]ammonolysis, 4-nitrophenylcarbamate
2a was synthesized according to Scheme 3. Condensation of 5-(4-
nitrophenyl)-2-furaldehyde (4) with ethyl hydrazinoacetate
hydrochloride gave a Schiff’s base 5 in 69% yield. Reaction of 5 with
4-nitrophenylcarbamoyl chloride at room temperature for 12 h
afforded 2a in a chemical yield of 64%.
Conditions for the [13N]ammonolysis of 2a with no-carrier-
added [13N]NH3 were examined. Reactions of 2a with [13N]NH3
were performed in anhydrous THF, dichloroethane (DCE), CH3CN
and DMF in the presence of various bases (K2CO3, pyridine, Et3N,
DMAP, i-Pr2NEt and lutidine), respectively. HPLC was used to ana-
lyze the incorporation efficiency of [13N]3 in the reaction mixture.
Among all conditions investigated, when i-Pr2NEt and DCE were
used, 5a reacted with [13N]NH3 at 75 °C for 3 min to afford
2. Results and discussion
2.1. Synthetic route
Regarding the chemical structure of 1 with a hydantoin ring, we
planned to label this compound using [13N]NH3 as the labeling
agent. This approach involves (1) the production of anhydrous
[
13N]3 with the highest radioactivity-incorporating yield (decay-
13N]NH3,22,23 (2) [13N]ammonolysis of 4-nitrophenylcarbamate
corrected) of 60% in the reaction mixture. Thus, DCE and i-Pr2NEt
were used as a solvent and base for the following [13N]ammonoly-
sis in the present study.
[
2a or carbamoyl chloride 2b with [13N]NH3, and (3) cyclization of
unsymmetrical urea [13N]3 to construct the [13N]hydantoin ring,
as shown in the retro-synthetic route (Scheme 2).
2.3.2. One-pot synthesis of [13N]3 via carbamoyl chloride 2b
To achieve higher efficiency of [13N]ammonolysis in a short per-
iod of time, the more reactive carbomyl chloride 2b was used as
another substrate in place of 2a (Table 1). Amine 5 was firstly trea-
ted with triphosgene to give 2b in situ. After excess COCl2 was re-
moved, 2b was not separated from the reaction mixture due to its
2.2. Production of anhydrous [13N]NH3
Anhydrous [13N]NH3 gas used for radiosynthesis was produced
by the nuclear 16O (p,
a
)
13N reaction22,23 by irradiating a target
O
CH2COOEt
CH2COOEt
Ammonolysis
13NH3
Cyclization
N N
O
CH N N
O
O
CH
N
N
O
CH
O
13NH
C
13NH2
C
R
O2N
O2N
O2N
O
[
13N]1
[
13N]3
2a: R =
2b: R = Cl
O
NO2
Scheme 2. Retrosynthesis of [13N]1.
OCOCl
HCl
.
CH2COOEt
H2NNHCH2COOEt
NO2
b
N
N
O
N
NHCH2COOEt
O
CHO
O
CH
O
CH
C
O
a
O2N
O2N
O2N
4
5
2a
NO2
CH2COOEt
13NH3
N
N
O
O
CH
C
13NH2
c
O2N
[
13N]3
Scheme 3. Chemical synthesis and [13N]ammonolysis of 2a. Reagents and conditions: (a) DMF, H2O, room temperature, 24 h, 69%; (b) Et3N, CH2Cl2, room temperature, 12 h,
64%; (c) base (K2CO3, pyridine, Et3N, DMAP, i-Pr2NEt or lutidine), solvent (THF, DCE, CH3CN or DMF), 75 °C, 3 min, 2–60%.

K. Kumata et al. / Bioorg. Med. Chem. 20 (2012) 305–310
307
made using no-carrier-added [13N]NH3 to achieve [13N]ammonoly-
sis by the one-pot synthesis of [13N]3 from 5 (Table 2). Our ap-
proach was to control the amount of triphosgene relative to
amine 5 to guarantee the complete consumption of COCl2. Table 2
shows the one-pot synthesis results of [13N]3 according to the
Table 1
One-pot synthesis of [13N]3 by reacting 5 with triphosgene, followed by [13N]ammo-
nolysis-1
1)
2) removing
triphosgene
3) 13NH3
(6 μmol)
triphosgene
5
[ 2b ]
[
13N]3
i-Pr2NEt (6 μmol),
°
75 C, 3 min
reaction conditions listed. When 1
mol COCl2) was treated with 3
mol 5, [13N]3 was formed but
its yield was not reproducible. When 0.75 mol triphosgene
(2.5 mol COCl2) was used, high and reproducible efficiency of
13N]ammonolysis was achieved. This result suggested that no
lmol triphosgene (equal to
(3 μmol)
75 C, 30 min
3
l
l
l
[
13N]NH3 (nmol)
Addition of NH3 (nmol)
Yield* (%)
l
[
ꢀ1
ꢀ1
ꢀ1
None
100
10,000
No product
34
75
COCl2 was left in the reaction mixture after acylation of 5. Further
decreasing the amount of triphosgene decreased the yield of
13N]3. Thus, the 1–4 ratio of triphosgene to 5 was suitable for
*
Radioactivity-incorporating yield (decay-corrected) for [13N]3 was measured by
[
analytical HPLC. All results are the mean (n = 3) with a maximum range of 5%.
achieving high incorporation efficiency of [13N]3 via 2b in situ.
2.5. Cyclization of [13N]3
instability. The reaction mixture containing 2b was directly used to
react with [13N]NH3 by the one-pot synthetic procedure.
The cyclization of [13N]3 was attempted without any further
purification by the addition of conventional coupling agents, such
as 1,10-carbonyldiimidazole (CDI),26,27 1,10-carbonyl-di-(1,2,4-tri-
zole) diimidazole (CDT),27 trifluoromethanesulfonic acid anhydride
((CF3SO2)2O),28 or bis(trimethylsilyl)trifluoroacetamide (BSTFA),29
in a one-pot reaction, followed by heating the reaction mixtures
for at least 3 min at 100–120 °C. However, these treatments did
not give [13N]1 presumably due to perturbation of the reaction
mixture by excess 5 and i-Pr2NEt.
Table 1 shows [13N]ammonolysis efficiency according to the
conditions listed. When no-carrier added [13N]NH3 was used, no
[
13N]3 was produced in the reaction mixture. By the addition of
100 nmol NH3 to the mixture, [13N]3 was produced in 34% yield.
Addition of 104 nmol NH3 increased the [13N]ammonolysis yield
to 75%. The difference in results between the addition of NH3 and
non-addition of NH3 was explained by the reaction scale of PET
chemistry. Since the NH3 carrier contained in the no-carrier-added
(cyclotron-produced) [13N]NH3 for the present radiosynthesis was
only about 1 nmol, [13N]NH3 was easily exhausted by trace COCl2
and HCl, resulting from the acylating process. Although as much
unreacted COCl2 as possible was removed before [13N]ammonoly-
sis, trace COCl2 left in the mixture was enough to react with
Next, [13N]3 was separated from the reaction mixture and then
cyclized (Scheme 4B). Semi-preparative HPLC purification (eluent:
CH3CN/H2O, 4/6) for the reaction mixture gave a radiochemically
pure fraction corresponding to [13N]3 (Fig. 1A). When the fraction
was heated at 95 °C to remove all solvents, we unexpectedly found
that [13N]3 was gradually converted to [13N]1 during the heating
process. As shown in Figure 1, heating [13N]3 in CH3CN and H2O
for 1 min and 2 min gave [13N]1 in 17% (B) and 74% (C) accounting
for the total radioactivity in the HPLC fraction, respectively. After
heating the radioactive fraction for 3 min, [13N]3 was efficiently
converted to [13N]1 (Fig. 1D). The efficient cyclization of [13N]3 in
a short period of time pave the way for the rapid synthesis of
[
13N]NH3, which stopped the subsequent [13N]ammonolysis. Utili-
zation of the carrier improved the reaction efficiency, but the
yielded [13N]3 with too low specific activity was not useful for
PET study, especially for brain imaging.
Considering the difference between radiosynthesis in PET
chemistry and conventional chemical synthesis, more efforts were
Table 2
[
13N]1 using an automated system.
One-pot synthesis of [13N]3 by reacting 5 with triphosgene, followed by [13N]ammo-
nolysis-2
2.6. Automated synthesis of [13N]1
2) 13NH3
1)
triphosgene
According to the optimized reaction conditions examined
above, we synthesized [13N]1 using an entirely-automated synthe-
sis system30 (Scheme 5).
5
[ 2b ]
[13N]3
i-Pr2NEt (6 μmol),
75°C, 3 min
(3 μmol)
75 C, 30 min
Amine 5 (3 lmol) was reacted with triphosgene (0.75 lmol) in
DCE in the presence of i-Pr2NEt to give a mixture containing 2b.
This mixture was immediately set to a reaction vial for the subse-
quent [13N]ammonolysis. After irradiation, [13N]NH3 was recov-
ered from the cyclotron target, dried and trapped in the above
mixture. The reaction vial was heated at 75 °C for 3 min. Semi-pre-
parative HPLC purification for the [13N]ammonolysis mixture gave
a radioactive fraction of [13N]3. This fraction was collected and
Triphosgene (mmol)
Yield* (%)
1
0.75
0.3
0–34
86
53
Radioactivity-incorporating yield (decay-corrected) for [13N]3 was measured by
analytical HPLC. All results are the mean (n = 3) with a maximum range of 5%.
*
A: One-pot Reaction
CH2COOEt
O
a
b
c
N
N
O
O
CH
N
N
O
CH
13NH
5
2b
C
13NH2
O2N
O2N
O
[
13N]3
[
13N]1
B: After separation of [13N]3
d
[
13N]3
[
13N]1
Scheme 4. Cyclization of [13N]3. Reagents and conditions: (a) i-Pr2NEt, DCE, 75 °C, 30 min (b) [13N]NH3, DCE, 75 °C, 3 min; (c) condensation agent (CDI, CDT, (CF3SO2)2O or
BSTFA), DCE, 100–120 °C, 1–3 min; (d) CH3CN, H2O, 95 °C, 1–3 min, 17–100%.

308
K. Kumata et al. / Bioorg. Med. Chem. 20 (2012) 305–310
A
D
B
[13N]3
[13N]3
[13N]1
1 min
95°C
95°C
1 min
C
[13N]1
[13N]1
1 min
[13N]3
95°C
Figure 1. HPLC analytical chromatograms reflecting the cyclization process of [13N]3 (retention time: 5.5 min) to form [13N]1 (retention time: 3.1 min) in CH3CN and H2O at
95 °C: (a) before heating the HPLC fraction containing [13N]3; (b) heating [13N]3 for 1 min; (c) heating [13N]3 for 2 min; (d) heating [13N]3 for 3 min. The analytical conditions
were as follows: Shiseido Capcell Pak C18 (4.6 mm / ꢁ 250 mm), H2O/CH3CN (6/4), 1.0 mL/min, 254 nm. The identity of [13N]3 or [13N]1 was confirmed by co-injection with
the non-radioactive 3 or 1.
16O (p, α)13
N
1) production and concentration of [13N]NH3 on resin AG 50W-X8
2) release of [13N]NH3 with 2 N KOH
3) drying of [ [13N]NH3 with CaO
CH2COOEt
13NH3
triphosgene
N
N
O
O
CH
5
2b
C
13NH2
O2N
[
13N]3
4) semi-preparative HPLC purification
5) cyclization by hearting HPLC fraction containing [13N]3
6) removal of HPLC solvents
7) formulation
O
O
CH
N N
13NH
O2N
O
[
13N]1
Scheme 5. Automated synthesis of [13N]1 using no-carrier-added [13N]NH3.
heated for 3 min at 95 °C in a sealed flask. After all solvents were
removed, [13N]1 was obtained as a formulated product.
was detected by HPLC. Moreover, the radiochemical purity of
13N]1 remained >95% after maintaining the product at room tem-
perature for at least 40 min, and this product was stable for per-
forming evaluation. These analytical results were in compliance
with our in-house quality control/assurance specifications.
[
Starting from cyclotron-produced [13N]NH3 with total radioac-
tivity of 6.6–7.5 GBq at a beam current of 15 lA after 25 min pro-
ton bombardment, [13N]1 was obtained with 270–480 MBq (n = 7)
as an injectable solution at the end of synthesis. This amount of
radioactivity is enough for in vitro and in vivo evaluation by animal
experiments. The automated reaction sequence took 17 2 min
from the end of bombardment.
3. Conclusions
[
13N]1, a PET probe for BCRP, was synthesized using no-carrier-
Identity, radiochemical purity and specific activity of [13N]1
were measured using analytical HPLC. Identity was confirmed by
co-injecting with non-radioactive 1. The radiochemical purity of
added [13N]NH3 equipped with an automated system for the first
time. The entire automated synthesis was reproducible and reli-
able, giving [13N]1 in sufficient radiochemical yield and purity for
animal experiments. PET studies with [13N]1 will be used to visu-
alize BCRP in the blood–brain-carrier for the first time and to elu-
cidate the influence of BRCP on rodent brain uptakes of drugs.
[
13N]1 was higher than 98% and specific activity was about
30 GBq/lmol at the end of synthesis, as determined from the mass
measured from the HPLC UV analysis. In the final product solution,
no significant peak relative to 5 and its decomposition products

K. Kumata et al. / Bioorg. Med. Chem. 20 (2012) 305–310
309
4.2. Production system of automated synthesis
4. Experimental section
Radiosynthesis was performed using an automated unit for the
quick production of 13N-labeled compounds using anhydrous
[
volume of the production line and required amount of reagent,
which decreased the radioactivity loss in the line to a low level.
The system enabled one to carry out all the procedures from pro-
duction of [13N]NH3 to formulation of [13N]1.
Melting points were measured using a micro melting point
apparatus (MP-500P, Yanaco, Tokyo) and are uncorrected. Nuclear
magnetic resonance (1H NMR) spectra were recorded on a JNM-
GX-270 spectrometer (JEOL, Tokyo) with tetramethylsilane as an
internal standard. All chemical shifts (d) were reported in parts
per million (ppm) downfield from the standard. High resolution
(HR) MS (FAB) was obtained on a JEOL NMS-SX102 spectrometer
(JEOL). Column chromatography was performed on Merck Kiesel-
gel gel 60 F254 (70–230 mesh). Nitrogen-13 (13N) was produced
13N]NH3.22,23,30 This system was designed to minimize the inner
4.3. Production of anhydrous [13N]NH3 gas
by the 15O (p, 13N nuclear reaction using a CYPRIS HM-18 cyclo-
a)
Before irradiation, an aqueous ethanol solution (10 mM) was
saturated with pure oxygen gas by bubbling for about 30 min. This
solution was loaded into the target chamber. The target was irradi-
tron (Sumitomo Heavy Industry, Tokyo). Radioactivity was mea-
sured with a dose calibrator (IGC-3R Curiemeter, Aloka, Tokyo).
HPLC was performed using a JASCO HPLC system (JASCO, Tokyo):
effluent radioactivity was monitored using a NaI (Tl) scintillation
detector system. Dantrolen (1) was purchased from Aldrich–Sigma
(Milwaukee, WI). If not otherwise stated, chemicals were pur-
chased from Aldrich–Sigma and Wako Pure Industries (Osaka) with
the highest grade commercially available.
ated at 15 lA for 15–25 min with 18 MeV protons (15.7 MeV on
target) from the cyclotron. The irradiated solution was quickly
passed through the preconditioned column (1 mm / ꢁ 40 mm)
filled with cation exchange resin AG 50W-X8 and [13N]NH3 in irra-
diated water was concentrated on this column. Then, [13N]NH3 was
eluted with aqueous KOH (30 lL, 2 N) under He gas flow, desic-
cated through the small column (3 mm / ꢁ 30 mm, kept at
150 °C) filled with CaO (250 mg), and flowed into a vial (Pyrex,
5 mL) containing various anhydrous organic solvents (1 mL) cooled
at ꢂ15°C. The production time of [13N]NH3 was about 4 min from
the end of bombardment.
4.1. Chemical synthesis
4.1.1. Ethyl 2-{2-[5-(4-nitrophenyl)furfurylidene]hydrazino}
acetate (5)
Ethyl hydrazinoacetate hydrochloride (8.8 g, 57 mmol) in water
(20 mL) was added to a solution of 5-(4-nitrophenyl)-2-furalde-
hyde (4; 12.4 g, 57 mmol) in DMF (100 mL). This mixture was stir-
red at room temperature for 24 h. After DMF and H2O were
removed under reduced pressure, the obtained residue was
washed with water and extracted with CH2Cl2. The organic layer
was washed with saturated NaCl, dried over Na2SO4 and removed.
The crude product was purified by column chromatography on sil-
ica gel under n-hexane/AcOEt (3:1) as eluent to give 5 (12.5 g, 69%)
as a yellow powder; mp: 104–105 °C. 1H NMR (300 MHz, CDCl3) d:
8.23 (2H, d, J = 8.8 Hz), 7.82 (2H, d, J = 8.8 Hz), 7.57 (1H, s), 6.90
(1H, d, J = 3.7 Hz), 6.62 (1H, d, J = 3.7 Hz), 4.24 (2H, q, J = 7.2 Hz),
4.07 (2H, s), 1.31 (3H, t, J = 7.0 Hz). HRMS (FAB) calcd for
4.4. [13N]Ammonolysis of 2a using no-carrier-added [13N]NH3
In a hot cell, a solution of [13N]NH3 (5–18 MBq) in DCE (100
was added to a reaction vial (Pyrex, 5 mL) containing 2a (1.45 mg,
mol), anhydrous THF, CH3CN, DMF or DCE (200 L), and various
bases (K2CO3, pyridine, Et3N, DMAP, i-Pr2NEt or lutidine: 6 mol).
lL)
3
l
l
l
Each reaction mixture was sealed and heated at 75 °C for 3 min
in a hot oil bath. The reaction vial was rapidly cooled and the effi-
ciency of [13N]ammonolysis was evaluated for each reaction.
The incorporation yield (decay-corrected) of radioactivity for
[
13N]3 was measured by analytical HPLC using the following condi-
C
15H16N3O5, 318.1090; found, 318.1053.
tion: Shiseido Capcell Pak C18 column: 4.6 mm / ꢁ 250 mm;
CH3CN/H2O: 4/6; flow rate: 1.0 mL/min; 254 nm; retention time:
5.6 min. The identity of [13N]3 was confirmed by co-injection with
the non-radioactive 3.
4.1.2. 4-Nitrophenyl 1-(2-ethoxy-2-oxoethyl)-2-[(5-(4-
nitrophenyl)furan-2-yl)methylene]hydrazinecarboxylate (2a)
4-Nitrophenyl chloroformate (240 mg, 1.2 mmol) was added
slowly to a mixture of 5 (317 mg, 1.0 mmol) and Et3N (0.21 mL,
1.5 mmol) in dry CH2Cl2 (3 mL) at 0 °C. The reaction mixture was
stirred at room temperature for 12 h. After removal of CH2Cl2
and Et3N under reduced temperature, the crude product was puri-
fied by column chromatography on silica gel under hexane/AcOEt
(from 4/1 to 3/1) to give 2a (309 mg, 64%) as an orange powder;
mp: 142–144 °C. 1H NMR (300 MHz, CDCl3) d: 8.26–8.33 (4H, m),
7.88 (2H, d, J = 8.8 Hz), 7.60 (1H, s), 7.45 (1H, s), 6.89–6.97 (2H,
m), 4.80 (2H, s), 4.32 (2H, q, J = 7.1 Hz), 1.35 (3H, t, J = 7.1 Hz).
HRMS (FAB) calcd for C22H19N4O9; 483.1152; found, 483.1163.
4.5. [13N]Ammonolysis of 2b using no-carrier-added [13N]NH3
4.5.1. One-pot synthesis by removing excess COCl2
A solution of triphosgene (1.78 mg, 6
added to a reaction vial containing a mixture of 5 (1.08 mg, 3
and i-Pr2NEt (1 mL, 6 mol) in DCE (100 L). The mixture was
lmol) in DCE (100
lL) was
l
mol)
l
l
heated at 75 °C under N2 for 30 min. After excess COCl2 was re-
moved from the reaction mixture with N2 flow, an anhydrous
[
13N]NH3 solution (30–150 MBq) in DCE (200
lL) was added to
the mixture containing 2b. The incorporation yield (decay-cor-
rected) of radioactivity for [13N]3 was measured by analytical
HPLC. For comparison, NH3 solution with a designated concentra-
4.1.3. Ethyl 2-{1-carbamoyl-2-[(5-(4-nitrophenyl)furan-2-
yl)methylene]hydrazinyl}ethanoate (3)
tion (1 mM or 100 mM) in DCE (100 lL) was mixed with the
To a solution of anhydrous ammonia (about 5%) in DMF (10 mL)
2a (100 mg, 0.2 mmol) was added. The reaction mixture was stir-
red at room temperature for 12 h. After removal of DMF under re-
duced pressure, the crude product was purified by column
chromatography on silica gel under CH2Cl2/CH3OH (from 0% to
3%) to give 3 (47 mg, 65.2%) as a yellow powder. 1H NMR
(DMSO-d6) d: 8.28 (2H, d, J = 8.8 Hz), 7.84 (2H, d, J = 8.8 Hz), 7.30
(1H, s), 6.95 (1H, d, J = 3.7 Hz), 6.79 (1H, d, J = 3.7 Hz), 4.78 (2H,
s), 4.26 (2H, q, J = 7.2 Hz), 1.30 (3H, t, J = 7.1 Hz); HRMS (FAB) calcd
for C16H17N4O6, 361.1148; found, 361.1100.
no-carrier-added (cyclotron-produced) [13N]NH3 in advance for
performing [13N]ammonolysis.
4.5.2. One-pot synthesis by controlling the amount of
triphosgene
A solution of triphosgene (90–270 mg, 0.3–1
(100 L) was added to a reaction vial containing 5 (1.08 mg,
mol) and i-Pr2NEt (1 mL, 6 mol) in DCE (100 L). The mixture
was heated at 75 °C under N2 for 30 min. Then, an anhydrous solu-
tion of [13N]NH3 (30–180 MBq) in DCE (200
L) was added into the
lmol) in DCE
l
3
l
l
l
l

310
K. Kumata et al. / Bioorg. Med. Chem. 20 (2012) 305–310
reaction vial. The reaction mixture was continuously heated for
3 min at 75 °C. The radioactivity-incorporating yield (decay-cor-
rected) for [13N]3 was measured by analytical HPLC.
References and notes
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A
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3
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We thank the staff of the Cyclotron Operation Section and
Department of Molecular Probes, National Institute of Radiological
Sciences (NIRS) for their assistance in the operation of the cyclotron
and production of radioisotopes. This study was partially supported
by a consignment expense for Molecular Imaging Program on Re-
search Base for PET Diagnosis from the Ministry of Education, Cul-
ture, Sports, Science and Technology (MEXT), Japanese Government.