Design of Ga-DOTA-based bifunctional radiopharmaceuticals: Two functional moieties can be conjugated to radiogallium-DOTA without reducing the complex stability

Article abstract of DOI:10.1016/j.bmc.2009.05.041

From the X-ray crystal structures of Ga-DOTA chelates, we were able to deduce that two free carboxylate groups of the radiogallium-DOTA complex may be utilized for coupling to functional moieties that recognize molecular targets for in vivo imaging withou

Full text of DOI:10.1016/j.bmc.2009.05.041

Bioorganic & Medicinal Chemistry 17 (2009) 4285–4289
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design of Ga–DOTA-based bifunctional radiopharmaceuticals: Two
functional moieties can be conjugated to radiogallium–DOTA without
reducing the complex stability
*
Takahiro Mukai , Jun Suwada, Kohei Sano, Mayumi Okada, Fumihiko Yamamoto, Minoru Maeda
Department of Biomolecular Recognition Chemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
From the X-ray crystal structures of Ga–DOTA chelates, we were able to deduce that two free carboxylate
groups of the radiogallium–DOTA complex may be utilized for coupling to functional moieties that
recognize molecular targets for in vivo imaging without reducing the radiogallium-complex stability.
Thus, we designed 2,20-[4,10-bis(2-{[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]amino}-2-oxoethyl)-
Received 18 March 2009
Revised 13 May 2009
Accepted 14 May 2009
Available online 21 May 2009
1,4,7,10-tetraazacyclododecane-1,7-diyl]diacetic acid (DOTA-MN2) (7), employing
a metronidazole
moiety as the recognition site of hypoxic lesions, based on the drug design concept of bifunctional radio-
pharmaceuticals. Coupling of DOTA-bis(tert-butyl)ester 5 with 1-(2-aminoethyl)-2-methyl-5-nitroimi-
dazole dihydrochloride, followed by deprotection, afforded the required 7 (DOTA-MN2). 67Ga-labeling
was carried out by reaction of DOTA-MN2 with 67Ga-citrate. When 67Ga–DOTA-MN2 was incubated in
phosphate-buffered saline or mouse plasma, no measurable decomposition occurred over a 24-h period.
In biodistribution experiments in NFSa tumor-bearing mice, 67Ga–DOTA-MN2 displayed not only a signif-
icant tumor uptake, but also rapid blood clearance and low accumulations in nontarget tissues, resulting
in high target-to-nontarget ratios of radioactivity. These results indicate the potential benefits of the drug
design of 67Ga–DOTA-MN2. The present findings provide helpful information for the development of radi-
ogallium-labeled radiopharmaceuticals for SPECT and PET studies.
Keywords:
Radiogallium
DOTA
Bifunctional radiopharmaceuticals
Metronidazole
Hypoxia
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
shipping of 68Ge/68Ga generators to a clinical site from which the
daughter 68Ga can be eluted on-site at any time on demand. De-
Positron emission tomography (PET) is a noninvasive medical
imaging technology that can be used to image the distribution of
positron-emitter-labeled agents with high resolution and sensitiv-
ity. As positron-emitters for PET, 11C, 13N, 15O and 18F have been
widely used, but their physical half-lives are very short (11C:
20.39 min, 13N: 9.96 min, 15O: 122 s, 18F: 109.8 min). Thus, the
use of PET requires large-scale and expensive equipment such as
an on-site cyclotron for the production of the short-lived posi-
tron-emitters, radiochemistry systems for the synthesis of PET
tracers, and incidental equipment, limiting the availability of PET
imaging.1,2
spite these advantages, there are few recent publications on the
development of 68Ga-labeled compounds except for 68Ga-labeled
peptides and proteins while a large number of 99mTc-labeled com-
pounds have been developed and used clinically. The development
of 68Ga-labeled compounds may be hampered by a lack of knowl-
edge of radiogallium-labeling chemistry.
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)
is well used as a chelator to form stable complexes with radiometals.
This ligand has eight coordinating donor atoms (four nitrogens and
four oxygens) and forms octa-coordinated complexes with metals
in +3 oxidation state.10 However, recent crystallographic studies
demonstrated a hepta-coordinated structure of Ga–DOTA com-
plexes; the four nitrogens of the cyclen ring and two oxygens of
the opposite carboxylate arms are coordinated to the metal.11,12
From these observations, we were able to deduce that two free car-
boxylate groups of the radiogallium–DOTA complex should be uti-
lized for coupling to functional moieties that recognize molecular
targets for in vivo imaging without reducing the radiogallium-com-
plex stability.
Similar to a c
-emitter produced by 99Mo/99mTc generator, 99mTc,
which is still the mainstay of diagnostic nuclear medicine, genera-
tor-produced positron-emitters are expected to contribute to the
progress of available PET studies.3,4 Among them, a metallic radio-
nuclide, 68Ga is of great interest because of its radiophysical prop-
erties (t1/2 = 68 min; b+ = 89% and EC = 11%; Eb+max = 1899 MeV).5–9
The long half-life of the parent 68Ge (t1/2 = 270.8 days) permits
To test this idea, we planned the development of a two func-
tional moieties-conjugated Ga–DOTA chelate, based on the concept
* Corresponding author. Tel.: +81 92 642 6651; fax: +81 92 642 6654.
E-mail address: mukai@phar.kyushu-u.ac.jp (T. Mukai).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.041

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T. Mukai et al. / Bioorg. Med. Chem. 17 (2009) 4285–4289
tyl-(oxycarbonyloxy)succinimide.18 Using this approach, as
COOH
N
outlined in Figure 2, DOTA-bis(tert-butyl)ester 5 was obtained in
good yields (82%). Acid-amine coupling of 5 with 1-(2-amino-
ethyl)-2-methyl-5-nitroimidazole dihydrochloride in the presence
of 1-hydroxybenzotriazole (HOBT), 1-ethyl-3-[3-(dimethylamino)
propyl] carbodiimide hydrochloride (EDC), and triethylamine in a
mixed solvent of CH2Cl2 and DMF gave 6 in 34% yield. Hydrolysis
of 6 with hydrochloric acid afforded the required 7 (DOTA-MN2)
in 95% yield. The 1H NMR and MS spectra were consistent with
the assigned structures.
A nonradioactive gallium-complex, Ga–DOTA-MN2 was pre-
pared by reacting DOTA-MN2 with Ga(NO3)3ꢀnH2O in 0.2 M ammo-
nium acetate buffer (pH 4.6). Heating was needed to complete the
reaction. In HPLC analyses, Ga–DOTA-MN2 and DOTA-MN2
showed well-separated peaks as shown in Figure 3. The structure
of Ga–DOTA-MN2 was assigned by ESI MS although the crystal
structure could not be determined. 67Ga-labeling was carried out
by reaction of DOTA-MN2 with 67Ga-citrate in an ammonium ace-
tate buffer (pH 5.8) at 95 °C. After purification by HPLC, 67Ga–
DOTA-MN2 was obtained with high radiochemical purity (over
96%) as determined by HPLC, cellulose acetate electrophoresis
and cellulose F TLC. Radiochemical yields of the final formulated
product were 35–59%.
CH3
CH3
O
O
N
N
N
N
N
N
N
H
N
H
N
NO2
O2N
COOH
Hypoxia recognition
Hypoxia recognition
Ga-chelation
Figure 1. Chemical structure of a bifunctional radiopharmaceutical, DOTA-MN2 (7).
of bifunctional radiopharmaceuticals. Bifunctional radiopharma-
ceuticals have the recognition site of the molecular target and che-
lation site for the radiometal independently in one molecule.13–16
In the present study, we designed and synthesized 2,20-[4,10-
bis(2-{[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]amino}-2-
oxoethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl]diacetic acid
(Fig. 1, DOTA-MN2), employing a metronidazole moiety as the rec-
ognition site of hypoxic lesions. To validate the utility of the drug
design, DOTA-MN2 was labeled with the easy-to-handle radioiso-
tope, 67Ga, and its in vitro stability and biodistribution in mice
were assessed.
2.2. Biological studies
The stability of 67Ga–DOTA-MN2 was investigated by incuba-
tion with phosphate-buffered saline (pH 7.4) or mouse plasma at
37 °C (Table 1). In both cases, the radiochemical purities of 67Ga–
DOTA-MN2 were unchanged for 24 h. These results indicate that
67Ga–DOTA-MN2 is highly stable in vitro although two carboxyl-
ates in the DOTA skeleton are conjugated with metronidazole
derivatives.
2. Results and discussion
2.1. Chemistry
The key step for the preparation of DOTA-bis-ester, a precursor
of DOTA-MN2, is regioselective N-deprotection of cyclen. At first,
N1,N7-di-protection of cyclen was performed by reaction with
benzyl chloroformate in aqueous media under strict pH control be-
tween 2 and 3, according to the method reported by Kovacs and
Sherry.17 While our study was in progress, León-Rodríguez et al. re-
ported the synthesis of N1,N7-di-protected cyclen 1 using tert-bu-
To assess the radioactivity pharmacokinetics, 67Ga–DOTA-MN2
was administered to normal mice (Table 2). At 30 min post-injec-
tion, the blood radioactivity was already at markedly low levels,
showing a fast blood clearance of 67Ga–DOTA-MN2. Only low
radioactivity, amounting to less than 0.9% of the injected dose/g,
COOtBu
COOtBu
N
N
N
H
N
H
(a)
(b)
(c)
(d)
NH
HN
BnOOC
N
N
COOBn
BnOOC
N
N
COOBn
NH
HN
H
N
H
N
N
N
COOtBu
COOtBu
1
2
3
COOtBu
COOtBu
COOtBu
CH3
CH3
O
N
N
N
N
N
N
N
O
(e)
(f)
N
N
N
N
BnOOC
N
N
COOBn
HOOC
N
N
COOH
N
N
H
N
H
NO2
O2N
4
5
6
COOtBu
COOtBu
COOtBu
COOH
N
CH3
CH3
N
(h)
(i)
O
O
Ga-DOTA-MN2
67Ga-DOTA-MN2
(g)
N
N
N
N
N
N
H
N
H
N
NO2
O2N
7
COOH
Figure 2. Synthesis of DOTA-MN2. Reagents and conditions: (a) tert-butyl-(oxycarbonyloxy)succinimide, CHCl3 (quant.); (b) benzyl bromoacetate, K2CO3, acetonitrile
(91.7%); (c) TFA, CH2Cl2 (97.0%); (d) tert-butyl bromoacetate, K2CO3, acetonitrile (92.7%); (e) Pd/C, H2, ethanol (quant.); (f) 1-(2-aminoethyl)-2-methyl-5-nitroimidazole
dihydrochloride, HOBt, EDC, triethylamine, CH2Cl2, DMF (33.9%); (g) concd HCl (95.4%); (h) Ga(NO3)3ꢀnH2O, 0.2 M NH4OAc buffer (pH 4.6); (i) 67Ga-citrate, 0.2 M NH4OAc
buffer (pH 5.8).

T. Mukai et al. / Bioorg. Med. Chem. 17 (2009) 4285–4289
4287
was detected in other tissues besides the kidney at any time. The
radioactivity level in the kidney was about 4% of the injected
dose/g at 30 min post-injection, but it was cleared rapidly from this
organ with time. At 24 h post-injection, 79% and 4% of the injected
radioactivity of 67Ga–DOTA-MN2 were recovered in the urine and
feces, respectively. In cellulose acetate electrophoresis and TLC
analyses, more than 85% of the radioactivity excreted in the urine
exhibited similar chromatographic behaviors to those of the
administered compound, 67Ga–DOTA-MN2 (Fig. 4). The distribu-
tion properties of 67Ga–DOTA-MN2 in mice were quite different
from those of 67Ga-citrate (Fig. 5). These findings indicate the high
in vivo stability and low nonspecific accumulation of 67Ga–DOTA-
MN2.
A
To estimate the tumor accumulation of radioactivity, 67Ga–
DOTA-MN2 was administered to NFSa tumor-bearing C3H/He mice
(Table 3). This model is widely used in investigations of hypoxic tu-
mors.19–22 The normal tissue distributions of 67Ga–DOTA-MN2 in
tumor-bearing mice were very similar to those in normal mice. Sig-
nificant accumulations of the radioactivity in the tumor were ob-
served, and the tumor-to-blood and tumor-to-muscle ratios
increased up to 6 h post-injection. Previously, Yang et al. developed
B
a
99mTc-labeled metronidazole derivative that 99mTc-ethylenedi-
cysteine chelate was conjugated with two metronidazole moie-
ties.23 This tracer recognized and identified hypoxic lesions such
as tumor hypoxia in rats and infarction areas in patients with acute
ischemic stroke.23,24 Likewise, the tumor accumulation of 67Ga–
DOTA-MN2 would be due to the recognition of the hypoxic lesions
by the metronidazole molecules. This is supported by the observa-
tion that the tumor accumulation of 67Ga–DOTA-MN2 was signifi-
cantly higher than that of 67Ga–DOTA (0.23 0.04% injected dose
per gram at 1 h postinjection). These results indicate that the drug
0
10
20
30
Retention Time (min)
Figure 3. Typical HPLC profiles of Ga–DOTA-MN2 (solid line) and DOTA-MN2
(broken line) (A) and 67Ga–DOTA-MN2 (B). The analyses were performed with a
Cosmosil 5C18-PAQ column (4.6 ꢁ 250 mm) at a flow rate of 0.6 mL/min with a
mixture of water and acetonitrile (92:8) containing 0.1% TFA.
A
Table 1
In vitro stability of 67Ga–DOTA-MN2 in phosphate-buffered saline (pH 7.4) and mouse
plasma at 37 °C
Incubation media
Radiochemical purity (%)
6 h 12 h
1 h
3 h
24 h
Phosphate-buffered
saline
Mouse plasma
97.6 2.5 96.5 1.3 96.0 1.0 95.8 0.9 96.4 1.7
97.7 0.2 98.0 0.8 96.5 1.2 96.8 0.8 96.2 1.0
Each value represents the mean SD of three samples.
-4
-2
0
2
4
6
Distance from the origin (cm)
B
Table 2
Biodistribution of radioactivity after intravenous administration of 67Ga–DOTA-MN2
in normal mice
Tissue
Uptake (% injected dose per gram tissue)
30 min
1 h
3 h
6 h
24 h
Blood
1.08 0.18
0.35 0.08
0.31 0.05
0.36 0.07
0.29 0.05
4.09 1.02
0.45 0.09
0.48 0.07
0.84 0.13
0.31 0.03
0.42 0.35
0.14 0.02
0.11 0.03
0.14 0.03
0.15 0.04
2.31 0.84
0.22 0.01
0.09 0.01
0.25 0.03
0.28 0.18
0.07 0.06
0.11 0.04
0.09 0.04
0.09 0.07
0.09 0.03
1.51 0.42
0.19 0.03
0.05 0.02
0.09 0.03
0.16 0.18
0.03 0.00
0.11 0.04
0.06 0.07
0.07 0.05
0.07 0.03
0.99 0.18
0.20 0.03
0.02 0.01
0.05 0.01
0.17 0.16
0.01 0.00
0.02 0.02
0.01 0.02
0.03 0.01
0.02 0.00
0.18 0.03
0.07 0.01
0.02 0.02
0.02 0.02
0.01 0.03
Spleen
Pancreas
Stomach
Intestine
Kidney
Liver
Heart
Lung
Muscle
0
0.2
0.4
0.6
0.8
1
Rf value
Figure 4. Chromatographic analyses of urine samples for 24 h after injection of
67Ga–DOTA-MN2. These samples were analyzed by cellulose acetate electrophore-
sis (A) and cellulose F TLC (B). Cellulose acetate electrophoresis was run in an
electrostatic field of 0.8 mA/cm for 30 min in veronal buffer (I = 0.06, pH 8.6).
Cellulose F TLC was developed with a mixture of water, acetonitrile, and 0.1 M
citrate (40:60:1). The peak of 67Ga–DOTA-MN2 was observed at 1 cm cathode on
cellulose acetate electrophoresis or Rf value of ca. 0.7 on TLC.
Urinea
Fecesa
78.50 4.99
4.33 2.58
Each value represents the mean SD of four animals.
a
Expressed as % injected dose.

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T. Mukai et al. / Bioorg. Med. Chem. 17 (2009) 4285–4289
complex for multifunctional radiotracers. To explore these ideas,
we are currently developing Ga–DOTA-based multivalent ligands
and multifunctional tracers.
20
15
10
5
A
B
3. Conclusions
We successfully designed and synthesized a metronidazole-
derivatized radiogallium–DOTA chelate, 67Ga–DOTA-MN2, based
on the previous findings obtained by X-ray crystallography of
Ga–DOTA chelates and the drug design concept of bifunctional
radiopharmaceuticals. As expected, 67Ga–DOTA-MN2 exhibited
high stability although two carboxylates in the DOTA skeleton
were conjugated with metronidazole derivatives. Furthermore,
67Ga–DOTA-MN2 showed not only a significant tumor uptake but
also rapid blood clearance and low accumulations in nontarget tis-
sues of the radioactivity after its administration in mice, resulting
in the high and increasing target-to-nontarget ratios of the radio-
activity. Although further studies are needed, radiogallium–
DOTA-MN2 should be a potential radiopharmaceutical for in vivo
hypoxia imaging. Also, these results suggest that two functional
moieties such as a metronidazole in this study can be conjugated
to radiogallium–DOTA chelate without reducing the complex sta-
bility. The present findings provide useful information about the
chemical design of radiogallium-labeled radiopharmaceuticals for
SPECT and PET studies.
0
20
15
10
5
0
Figure 5. Biodistribution of radioactivity at 30 min (A) and 6 h (B) post-injection of
67Ga–DOTA-MN2 (open columns) or 67Ga-citrate (closed columns) in normal mice.
Each value represents the mean SD of four animals.
4. Experimental
4.1. General procedure
Chemical reagents and solvents were of commercial quality and
were used without further purification unless otherwise noted. All
melting points were determined on a Yanaco melting point appa-
ratus and are uncorrected. 1H NMR spectra were obtained on a Var-
ian Unity 400 (400 MHz), and the chemical shifts are reported in
parts per million downfield from tetramethylsilane. Infrared (IR)
spectra were recorded with a Shimadzu FTIR-8400 spectrometer.
High-resolution mass spectra were obtained with an Applied Bio-
systems Mariner System 5299 spectrometer (ESI MS). Column
Table 3
Biodistribution of radioactivity after intravenous administration of 67Ga–DOTA-MN2
in NFSa tumor-bearing C3H/He mice
Tissue
Uptake (% injected dose per gram tissue)
1 h
3 h
6 h
Blood
0.59 0.30
0.15 0.03
0.09 0.06
0.21 0.10
0.37 0.29
2.77 0.89
0.25 0.03
0.17 0.13
0.55 0.15
0.25 0.13
0.49 0.12
0.06 0.01
0.10 0.04
0.08 0.05
0.09 0.02
0.09 0.03
1.20 0.18
0.15 0.04
0.05 0.04
0.13 0.02
0.05 0.01
0.22 0.04
0.05 0.01
0.11 0.03
0.07 0.01
0.07 0.02
0.06 0.01
1.04 0.18
0.20 0.08
0.04 0.01
0.10 0.02
0.05 0.03
0.20 0.04
Spleen
Pancreas
Stomach
Intestine
Kidney
Liver
Heart
Lung
Muscle
Tumor
chromatography was performed on Silica Gel 60N (63–210 lm,
Kanto Chemical), and the progress of the reaction was monitored
by TLC on Silica Gel 60F 254 plates (Merck). 67Ga was supplied
by Fujifilm RI Pharma Co., Ltd. as 67Ga-citrate. Cellulose acetate
electrophoresis (Separax-SP) was run in an electrostatic field of
2.5 mA/cm for 20 min or 0.8 mA/cm for 30 min in veronal buffer
(I = 0.06, pH 8.6). Cellulose F TLC was developed with a mixture
of water, acetonitrile, and 0.1 M citrate (40:60:1).
Tumor-to-blood ratio
Tumor-to-muscle ratio
0.95 0.30
2.36 0.96
3.63 1.10
4.05 0.64
4.55 0.44
4.42 1.45
Each value represents the mean SD of four or five animals.
4.2. Chemistry
design of 67Ga–DOTA-MN2 based on bifunctional radiopharmaceu-
ticals makes both high in vivo stability of Ga–DOTA chelate and
significant recognition of the hypoxic lesions by metronidazole
molecules possible.
4.2.1. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid,
1,7-bis(tert-butyl) ester (5)
Compound 5 was prepared in five steps from cyclen according
to the literature.18
A specific accumulation of radiotracer in the target tissues is
crucial for successful in vivo imaging. For receptor-targeted imag-
ing, ligands with high receptor affinity are prerequisite. Recently,
multivalent (bivalent and tetravalent) ligands showed markedly
increased binding affinity with their respective receptors com-
pared to monovalent ligands.25 The multivalent concept has been
used for the development of receptor-targeted radiotracers.26 The
present study suggest that two carboxylate groups of Ga–DOTA
complex should be used for coupling to two or more ligand moie-
ties without reducing the complex stability. Furthermore, two dif-
ferent functional moieties would be attached to the Ga–DOTA
4.2.2. Di-tert-butyl 2,20-[4,10-bis(2-{[2-(2-methyl-5-nitro-1H-
imidazol-1-yl)ethyl]amino}-2-oxoethyl)-1,4,7,10-
tetraazacyclododecane-1,7-diyl]diacetate (6)
1-(2-Aminoethyl)-2-methyl-5-nitroimidazole dihydrochloride
was prepared from metronidazole according to the literature.27
Under argon, to a solution of 5 (60.0 mg, 0.12 mmol) in dry CH2Cl2/
DMF = 5:1 (6 mL) was added triethylamine (0.132 mL, 0.93 mmol),
1-(2-aminoethyl)-2-methyl-5-nitroimidazole
dihydrochloride
(113 mg, 0.47 mmol), HOBt (71.4 mg, 0.47 mmol), and EDC
(89.4 mg, 0.47 mmol) in an ice bath. After stirring for 48 h at room

T. Mukai et al. / Bioorg. Med. Chem. 17 (2009) 4285–4289
4289
temperature, chloroform (30 mL) was added. The mixture was
washed with distilled water, and then dried and evaporated in
vacuo. The residue was recrystallized from methanol to obtain 6
(32.3 mg, 33.9%). 6: White solid; mp 186–189 °C; 1H NMR
(400 MHz, CDCl3) d: 1.43 (s, 18H), 2.41 (m, 8H), 2.50 (s, 6H), 2.69
(m, 8H), 3.04 (s, 4H), 3.09 (s, 4H), 3.60 (m, 4H), 4.48 (t, J = 6.41 Hz,
4H), 7.93 (s, 2H), 8.58 (m, 2H); IR (KBr) cmꢂ1: 2981, 1743, 1665,
1365, 1261, 1055; ESI MS (m/z): 821.4657 (M+H)+.
interest were removed and weighed, and radioactivity counts were
determined with an auto well gamma counter (ARC-370M; Aloka).
4.3.3. Biodistribution study in tumor-bearing mice
Syngeneic NFSa fibrosarcoma cells were inoculated into the
right hind leg muscle of female C3H/He mice (5 weeks old). When
tumors were approximately 0.5 cm in diameter, the animals were
intravenously injected with 67Ga–DOTA-MN2 (22 kBq). The biodis-
tribution of radioactivity was monitored at 1, 3 and 6 h post-
injection.
4.2.3. 2,20-[4,10-Bis(2-{[2-(2-methyl-5-nitro-1H-imidazol-1-
yl)ethyl]amino}-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-
1,7-diyl]diacetic acid (7) (DOTA-MN2)
Acknowledgments
A mixture of 6 (17.0 mg, 20.7 lmol) and concd HCl (3.0 mL) was
stirred at room temperature for 2 h, and then concentrated in va-
cuo. After the residue was dissolved in a small amount of methanol,
dry ether was added to precipitate 7 (14.0 mg 95.4%). Compound 7:
White solid; mp 152–155 °C; 1H NMR (400 MHz, D2O) d: 2.63 (s,
6H), 3.00–3.20 (m, 16H), 3.60–3.70 (m, 12H), 4.58 (t, J = 5.86 Hz,
4H), 8.32 (s, 2H); IR (KBr) cmꢂ1: 3218, 1731, 1681, 1545, 1372,
1195, 1087; ESI MS (m/z): 709.3381 (M+H)+.
We would like to thank Fujifilm RI Pharma Co., Ltd, Tokyo, Japan
for donating the 67Ga-citrate. We also thank Dr. Koichi Ando and
Dr. Sachiko Koike of the National Institute of Radiological Sciences,
Chiba, Japan for their kind gift of NFsa. This work was supported in
part by the Grant-in-Aid for Young Scientists (B) (18790894) from
the Ministry of Education, Culture, Sports, Science and Technology
of Japan and a Research Grant from the Research Foundation for
Pharmaceutical Sciences.
4.2.4. Ga–DOTA-MN2
DOTA-MN2 (1.0 mg, 0.141 mmol) and Ga(NO3)3ꢀnH2O (3.0 mg)
were dissolved in 0.2 M NH4OAc buffer (pH 4.6, 0.5 mL). The mix-
ture was stirred and heated at 95 °C for 1 h. After cooling to room
temperature, Ga–DOTA-MN2 was purified by HPLC performed
with a Cosmosil 5C18-PAQ column (10 ꢁ 250 mm) at a flow rate
of 3 mL/min with a mixture of water and acetonitrile (90:10) con-
taining 0.1% TFA. ESI MS (m/z): 775.2523 (M+H)+.
References and notes
1. Frick, M. P.; Gupta, N. C.; Sunderland, J. J.; Best, M. A.; Rysavy, J. A.; Shiue, C. Y.
Semin. Nucl. Med. 1992, 22, 182.
2. Keppler, J. S.; Conti, P. S. Am. J. Roentgenol. 2001, 177, 31.
3. Knapp, F. F., Jr.; Mirzadeh, S. Eur. J. Nucl. Med. 1994, 21, 1151.
4. Pagani, M.; Stone-Elander, S.; Larsson, S. A. Eur. J. Nucl. Med. 1997, 24, 1301.
5. Mirzadeh, S.; Lambrecht, R. M. J. Radioanal. Nucl. Chem. 1996, 202, 7.
6. Fani, M.; André, J. P.; Maecke, H. R. Contrast Media Mol. Imaging 2008, 3, 67.
7. Antunes, P.; Ginj, M.; Zhang, H.; Waser, B.; Baum, R. P.; Reubi, J. C.; Maecke, H.
Eur. J. Nucl. Med. Mol. Imaging 2007, 34, 982.
8. Zhernosekov, K. P.; Filosofov, D. V.; Baum, R. P.; Aschoff, P.; Bihl, H.; Razbash, A.
A.; Jahn, M.; Jennewein, M.; Rösch, F. J. Nucl. Med. 2007, 48, 1245.
9. Win, Z.; Al-Nahhas, A.; Rubello, D.; Gross, M. D. Q. J. Nucl. Med. Mol. Imaging
2007, 51, 244.
10. Benetollo, F.; Bombieri, G.; Calabi, L.; Aime, S.; Botta, M. Inorg. Chem. 2003, 42,
148.
11. Heppeler, A.; Froidevauz, S.; Mäcke, H. R.; Jermann, E.; Béhé, M.; Powell, P.;
Hennig, M. Chem. Eur. J. 1999, 5, 1974.
12. Viola, N. A.; Rarig, R. S., Jr.; Ouellettea, W.; Doyle, R. P. Polyhedron 2006, 25,
3457.
13. Ogawa, K.; Mukai, T.; Arano, Y.; Hanaoka, H.; Hashimoto, K.; Nishimura, H.;
Saji, H. J. Labelled Compd. Radiopharm. 2004, 47, 753.
4.2.5. 67Ga–DOTA-MN2
To a solution of DOTA-MN2 (50
lg) in 0.2 M NH4OAc buffer (pH
5.8, 50 L) was added 30
l
l
L of 67Ga-citrate (0.73 MBq). The mix-
ture was stirred and heated at 95 °C for 2 h. After cooling to room
temperature, 67Ga–DOTA-MN2 was purified by HPLC performed
with a Cosmosil 5C18-PAQ column (4.6 ꢁ 250 mm) at a flow rate
of 0.6 mL/min with a mixture of water and acetonitrile (92:8) con-
taining 0.1% TFA. The radiochemical purity was determined by
HPLC, cellulose acetate electrophoresis and cellulose F TLC.
14. Ogawa, K.; Mukai, T.; Arano, Y.; Ono, M.; Hanaoka, H.; Ishino, S.; Hashimoto, K.;
Nishimura, H.; Saji, H. Bioconjugate Chem. 2005, 16, 751.
15. Ogawa, K.; Mukai, T.; Arano, Y.; Otaka, A.; Ueda, M.; Uehara, T.; Magata, Y.;
Hashimoto, K.; Saji, H. Nucl. Med. Biol. 2006, 33, 513.
16. Ogawa, K.; Mukai, T.; Inoue, Y.; Ono, M.; Saji, H. J. Nucl. Med. 2006, 47, 2042.
17. Kovacs, Z.; Sherry, A. D. Synthesis 1997, 7, 759.
18. De Leon-Rodriguez, L. M.; Kovacs, Z.; Esqueda-Oliva, A. C.; Miranda-Olvera, A.
D. Tetrahedron Lett. 2006, 47, 6937.
19. Halpern, H. J.; Yu, C.; Peric, M.; Barth, E.; Grdina, D. J.; Teicher, B. A. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 13047.
20. Ando, K.; Koike, S.; Ohira, C.; Chen, Y. J.; Nojima, K.; Ando, S.; Ohbuchi, T.;
Kobayashi, N.; Shimizu, W.; Urano, M. Int. J. Radiat. Biol. 1999, 75, 505.
21. Yamamoto, F.; Aoki, M.; Furusawa, Y.; Ando, K.; Kuwabara, Y.; Masuda, K.;
Sasaki, S.; Maeda, M. Biol. Pharm. Bull. 2002, 25, 616.
22. Mahy, P.; De Bast, M.; Gillart, J.; Labar, D.; Grégoire, V. Eur. J. Nucl. Med. Mol.
Imaging 2006, 33, 553.
23. Yang, D. J.; Ilgan, S.; Higuchi, T.; Zareneyrizi, F.; Oh, C. S.; Liu, C. W.; Kim, E. E.;
Podoloff, D. A. Pharm. Res. 1999, 16, 743.
24. Song, H. C.; Bom, H. S.; Cho, K. H.; Kim, B. C.; Seo, J. J.; Kimm, C. G.; Yang, D. J.;
Kim, E. E. Stroke 2003, 34, 982.
4.3. Biological studies
4.3.1. In vitro stability
67Ga–DOTA-MN2 was diluted with 20 mM phosphate-buffered
saline (pH 7.4) or mouse plasma, and the solutions were incubated
at 37 °C. After 1, 3, 6, 12 and 24 h of incubation, the samples were
drawn and the radioactivity was analyzed by cellulose acetate
electrophoresis.
4.3.2. Biodistribution study in normal mice
Animal experiments were conducted in accordance with our
institutional guidelines and were approved by the Animal Care
and Use Committee, Kyushu University. Biodistribution experi-
ments were performed by intravenously administering 67Ga–
DOTA-MN2 or 67Ga-citrate into 6-week-old male ddY mice (28–
30 g). 67Ga–DOTA-MN2 and 67Ga-citrate were diluted with
20 mM phosphate-buffered saline (pH 7.4). Groups of four mice
25. Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Angew. Chem., Int. Ed. 2006, 45,
2348.
26. Liu, S. Mol. Pharm. 2006, 3, 472.
27. Hay, M. P.; Wilson, W. R.; Moselen, J. W.; Palmer, B. D.; Denny, W. A. J. Med.
Chem. 1994, 37, 381.
were administered 100
(22 kBq) prior to euthanasia at selected time points. Tissues of
lL
of each 67Ga-labeled compound