Synthesis and characterization of nitroimidazole derivatives for 68Ga-labeling and testing in tumor xenografted mice

Article abstract of DOI:10.1021/jm100545a

Radiolabeled nitroimidazole (NI) derivatives have been used for imaging hypoxic tissues. We synthesized NI derivatives conjugated with bifunctional chelating agents such as 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and isothiocyanatobenzyl-NOTA (SCN-NOTA) via ethyleneamine bridge by formation of amide and thiourea bond, respectively. We proved that amide oxygen of Ga-NOTA-NI contributes to the formation of metal complex by X-ray crystallography. We labeled them with ⁶⁸Ga and found that both ⁶⁸Ga-NOTA-NI and ⁶⁸Ga- SCN-NOTA-NI were labeled in high efficiency (>96%) and were stable at room temperature in the prepared medium and at 37 A C in human serum. In vitro cell uptake experiments using CHO and CT-26 cell lines showed significantly increased uptakes of both of the agents in hypoxic condition. Biodistribution study in CT-26 xenografted mice showed increasing tumor to muscle ratios. ⁶⁸Ga-NOTA-NI showed lower intestine uptake than ⁶⁸Ga-NOTA-SCN-NI due to hydrophilicity. Also, ⁶⁸Ga-NOTA-NI showed higher tumor uptake than ⁶⁸Ga-NOTA-SCN-NI in an animal PET study. In conclusion, we successfully developed ⁶⁸Ga labeled NI derivatives for hypoxic tissue imaging.

Full text of DOI:10.1021/jm100545a

6378 J. Med. Chem. 2010, 53, 6378–6385
DOI: 10.1021/jm100545a
Synthesis and Characterization of Nitroimidazole Derivatives for 68Ga-Labeling and
Testing in Tumor Xenografted Mice
Lathika Hoigebazar,†,‡ Jae Min Jeong,*,†,‡ Soo Young Choi,§ Jae Yeon Choi,†,‡ Dinesh Shetty,†,‡
Yun-Sang Lee,†,‡ Dong Soo Lee,†,‡ June-Key Chung,†,‡ Myung Chul Lee,†,‡ and Young Keun Chung§
†Department of Nuclear Medicine, Radiation Applied Life Sciences, Institute of Radiation Medicine,
Seoul National University College of Medicine, 101 Daehangno, Jongno-gu, Seoul 110-744, Korea,
‡Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-744, Korea, and
§Intelligent Textile System Research Centre and Department of Chemistry, Seoul National University, Seoul, Korea
Received May 5, 2010
Radiolabeled nitroimidazole (NI) derivatives have been used for imaging hypoxic tissues. We syn-
thesized NI derivatives conjugated with bifunctional chelating agents such as 1,4,7-triazacyclononane-
1,4,7-triacetic acid (NOTA) and isothiocyanatobenzyl-NOTA (SCN-NOTA) via ethyleneamine bridge
by formation of amide and thiourea bond, respectively. We proved that amide oxygen of Ga-NOTA-NI
contributes to the formation of metal complex by X-ray crystallography. We labeled them with 68Ga and
found that both 68Ga-NOTA-NI and 68Ga- SCN-NOTA-NI were labeled in high efficiency (>96%)
and were stable at room temperature in the prepared medium and at 37 °C in human serum. In vitro cell
uptake experiments using CHO and CT-26 cell lines showed significantly increased uptakes of both of
the agents in hypoxic condition. Biodistribution study in CT-26 xenografted mice showed increasing
tumor to muscle ratios. 68Ga-NOTA-NI showed lower intestine uptake than 68Ga-NOTA-SCN-NI due
to hydrophilicity. Also, 68Ga-NOTA-NI showed higher tumor uptake than 68Ga-NOTA-SCN-NI in an
animal PET study. In conclusion, we successfully developed 68Ga labeled NI derivatives for hypoxic
tissue imaging.
Introduction
The detection of hypoxia is important for treatment plan-
ning, such as in cases of cancer or myocardial ischemia.1-3
Since NIa drugs have wide clinical applications, especially for
the targeting of hypoxic tumors,4-16 the inclusion of the NI
moiety has become an important consideration during drug
development. In particular, 2-NI (1) can be reduced to form a
reactive chemical species, which can bind to cell components
in the absence of sufficient oxygen,5,17-19 and thus, the deve-
lopment of radiolabeled NI derivatives for the imaging of
hypoxia remains an active field of research to improve cancer
therapy result.
The positron emission tomography (PET) tracer [18F]fluoro-
misonidazole (FMISO)20-23 (Figure 1) was the first NI agent
Figure 1. NI agents previously developed for the imaging of
hypoxia.
used for imaging hypoxia. However, it requires at least a 2 h wait
before image acquisition due to its slow washout from normoxic
tissues, which is a serious fault because 18F labeled agents have
relatively short half-lives (110 min).18 Agents more hydrophilic
than [18F]FMISO have been developed, such as [18F]fluoro-
erythronitroimidazole (FETNIM),24,25 [18F]1-R-D-(2-deoxy-
2-fluoroarabinofuranosyl)-2-nitroimidazole (FAZA),6,8,18,26-28
[18F]2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoro-
propyl)acetamide (EF-5),16,29-32 and [124I]1-R-D-(2-deoxy-
2-fluoroarabinofuranosyl)-2-nitroimidazole (IAZA),7 to
improve target to nontarget ratio by increasing excretion
rates (Figure 1).
*To whom correspondences should be addressed. Phone: 82-2-2072-
3805. Fax: 82- 2-745-7690. E-mail: jmjng@snu.ac.kr.
a Abbreviations: NI, nitroimidazole; SCN-NOTA, isothiocyanatobenzyl-
1,4,7-triazacyclononane-1,4,7-triacetic acid; PET, positron emission tomo-
graphy; FMISO, fluoromisonidazole; FETNIM, fluoroerythronitroimida-
zole; FAZA, 1-R-D-(2-deoxy-2-fluoroarabinofuranosyl)-2-nitroimidazole;
EF-5, 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acet-
amide; IAZA, 1-R-D-(2-deoxy-2-fluoroarabinofuranosyl)-2-nitroimidazole;
ATSM, diacetyl-bis(N4-methylthiosemicarbazone); DMF, dimethylforma-
mide; TLC, thin layer chromatography; DCC, N,N0-dicyclohexylcarbodi-
imide; ITLC-SG, instant thin layer chromatography silica gel; TEA,
triethylamine; RP-HPLC, reverse phase-high performance liquid chromato-
graphy.
Furthermore, although a 64Cu-diacetyl-bis(N4-methylthio-
semicarbazone) (ATSM) has been developed that is cleared
rapidly from normoxic tissues, the production of 64Cu requires
r
pubs.acs.org/jmc
Published on Web 08/06/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6379
Scheme 1. Synthesis of NOTA-2-NI-N-ethylamine (3) and SCN-NOTA-2-NI-N-ethylamine (4)a
a (i) tert-Butyl 2-bromoethylcarbamate, K2CO3, DMF, room temp; (ii) HCl/MeOH, room temp; (iii) NOTA, H2O/DMF, DCC, pyridine, room temp,
overnight; (iv) SCN-NOTA, CHCl3, TEA, room temp, overnight.
Table 1. Crystallographic Experimental Data of Ga-3
a special solid target and expensive target material, and hence,
its use is likely to be limited.33-35
parameter
In fact, the productions of 18F, 64Cu, and 124I require
empirical formula
formula weight
temperature
C18H24GaN7O10
568.16
293(2) K
cyclotron and chemical processing, which are expensive and
difficult to operate and maintain. In the present study, we
undertook to develop 68Ga-labeled NI derivatives for hypoxia
imaging by PET because 68Ga can be produced using a
relatively easily available generator system,36-39 and a simple
kit can be produced to help routine labeling procedure.40
Unlike the radioactive halogens, the labeling of 68Ga re-
quires the use of bifunctional chelating agents that can con-
jugate to biomolecules and form thermodynamically stable
complexes with Ga. The triazamacrocyclic ligand, NOTA,
has been reported to form highly stable complexes with small
˚
0.710 73 A
wavelength
crystal system
space group
unit cell dimensions
orthorhombic
Pbca
˚
a = 14.1280(5) A, R = 90°
˚
b = 14.8639(5) A, β = 90°
˚
c = 21.2809(7) A, γ = 90°
4468.9(3) A
3
˚
volume
Z
8
calculated density
absorption coefficient
F(000)
1.689 Mg/m3
1.304 mm-1
2336
cations like Ga ,
3þ 41,42 and thus, various peptides conjugated
with NOTA derivatives have been developed.43,44 In previous
studies, 67Ga-labeled NI derivatives of NOTA were prepared
with a functional group atthe ring ethylene backbone.45 In the
present study, we synthesized a new NOTA-NI (3) conjugate
for labeling with 68Ga. In addition, we also synthesized SCN-
NOTA-NI (4) conjugate as a control and compared the in
vivo and in vitro properties of these two derivatives.
In the Ga-NOTA complex, all three carboxyl groups
of NOTA bind with gallium. However, in 3, one of these
carboxyl groups formed an amide group, and thus, only two
carboxyl groups remain for gallium chelation. Accordingly,
we wondered whether 3 forms a stable complex with gallium,
and if so, which atom of the amide bond would bind with
gallium. To confirm this, we performed X-ray crystallography
using Ga-3 crystals produced at pH 3 and pH 5 in aqueous
solution.
crystal size
range for data collection
limiting indices
0.51 mm ꢀ 0.38 mm ꢀ 0.11 mm
2.40-27.51°
-18 e h e 18, -19 e k e 19,
-27 e l e 27
8963/5048 [R(int) = 0.0345]
reflections collected/unique
completeness to θ = 27.51
absorption correction
max and min transmission
refinement method
98.2%
empirical
0.8698 and 0.5560
full-matrix least-squares on F2
5048/0/326
data/restraints/parameters
goodness-of-fit on F2
1.059
final R indices [I > 2σ(I)]
R indices (all data)
extinction coefficient
R1 = 0.0398, wR2 = 0.0966
R1 = 0.0686, wR2 = 0.1076
0.0038(4)
The subsequent deprotection of the amine was achieved
using 1.25 M HCl in methanol (MeOH), which gave 2 in high
yield (84%). Reaction completion was confirmed by thin
layer chromatography (TLC), and the crude product was
purified by recrystallization from MeOH.
Results and Discussion
Chemistry. To synthesize the 68Ga-labeled nitroimidazole
derivatives 3 and 4, we prepared 2-(2-nitroimidazolyl)ethyl-
amine (2) in the form of an HCl salt (Scheme 1). tert-Butyl
2-(2-nitroimidazolyl)ethylcarbamate was synthesized by the
N-alkylation of 1 with tert-butyl 2-bromoethylcarbamate in
MeCN (35%). K2CO3 was used as the base, and the reaction
was performed at room temperature overnight. We observed
that conjugation yields increased up to 80% when this
reaction was carried out in dimethylformamide (DMF),
which we attribute to be the higher solubility of 1 in DMF.
The crude product was purified by recrystallization from
EtOAc.
To synthesize 3, acid/amine coupling was carried out in a
water and DMF mixture (1:1 vol/vol) using N,N0-dicyclo-
hexylcarbodiimide (DCC) as a coupling agent in the presence
of pyridine. To synthesize 4, SCN-NOTA and amine were
conjugated in chloroform (CHCl3) using triethylamine
(TEA) as a base. The formation of products during both
reactions was monitored by mass spectrometry in electro-
spray ionization positive mode (MS/ESIþ). Products were
purified by reverse phase high performance liquid chroma-
tography (RP-HPLC) to obtain products 3 and 4 at yields
of 52% and 73%, respectively. According to mass analysis,
we did not find even a trace amount of disubstituted or

6380 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Hoigebazar et al.
Figure 2. ORTEP drawing of Ga-3. Hydrogen atoms and counterions have been omitted for clarity. Gallium binds with oxygen atom of amide
bond and not with nitrogen.
Figure 3. In vitro cell uptake studies of (A) 68Ga-3 and (B) 68Ga-4 under normoxic and hypoxic conditions by CHO and CT26 cells. p values
represent comparisons between uptakes under normoxic and hypoxic conditions (t-test): (//) p < 0.01, (/) p < 0.05, n = 4 at each time point.
trisubstitued product of NOTA-NI, as was expected by the
reaction of acid amine coupling (Supporting Information
p S9).
with either amide nitrogen (N4) or oxygen (O5). Both crystals at
pH 3 and 5 produced identical diffraction patterns, and gallium
was found to be coordinated with the amide oxygen (O5) of
NOTA (Figure 2). Direct binding of NI residue and gallium
atom was not observed. This is important because the intact NI
group is essential for the in vivo binding of the agent in hypoxic
tissue. In addition, we found one counterion (CO32-) per unit
cell derived from Na2CO3 buffer used during complexation. Ga-
3 complex showed Pbca symmetry with eight molecules per unit
cell (Z). Three nitrogen atoms (from the backbone ring) and
three oxygen atoms (two from the carboxylic acid and one from
the amide oxygen of the modified carboxylic acid) were found to
coordinate with the gallium atom to produce a distorted octa-
hedral geometry, which was evidenced by a compression of N-
Ga-N angles (average 84.7°) and expansion of O-Ga-O
angles (average 93.3°). The average bond angle of trans N-
Ga-O was 166.5°, which is similar to that found in Ga-NOTA
(167°).41,46,47 The average Ga-N bond length in Ga-3 was
Crystallography. Crystallography was performed only on
Ga-3 and not on Ga-4 because 4 possesses three carboxyl
groups and thus is deemed to be almost certain to form an
octahedral crystal structure with gallium. However, as men-
tioned above, 3 has only two carboxyl groups (one formed an
amide bonded to ethylnitroimidazole), and thus, it was not
clear whether it would form a stable complex with gallium.
Complexation of Ga(III) with 3 was performed in aqueous
solution by mixing stoichiometric amounts of 3 and Ga(NO3)3
3
xH2O at pH 3 or 5. Complex formation was monitored by MS/
ESIþ, and the Ga-3 produced was purified by RP-HPLC.
Crystals of Ga-3 were obtained by allowing the water solution
to evaporate slowly at room temperature and analyzed using
graphite-monochromated Mo KR radiation and an Enraf-
Nonius CCD single-crystal X-ray diffractometer at room tem-
perature (data are summarized in Table 1). We investigated the
crystal structure to ensure the possibility of metal coordination
˚
2.09 A, which is the same as that of Ga-NOTA, and the average
47
˚
˚
Ga-O bond length was 1.94 A (1.93 A for Ga-NOTA).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6381
Figure 4. Histograms showing biodistribution at 10, 30, 60, and 120 min postinjection of (A) 68Ga-3 and (B) 68Ga-4 in mice bearing CT-26
xenograft which are reported as mean percentage injected dose per gram tissue ( standard deviation (% ID/g ( SD); n = 4 at each time point.
Radiochemistry. 68Ga was eluted from a 68Ge/68Ga-
generator using 0.1 N HCl. Radiolabeling was conducted
at pH 3 in a boiling water bath for 10 min. Free 68Ga3þ was
removed using an alumina cartridge. Labeling efficiencies
were checked by instant thin layer chromatography silica
gel (ITLC-SG) (Supporting Information Figure 1) and
found to be 96.0 ( 0.7% and 96.3 ( 5.5% for 68Ga-3 and
68Ga-4, respectively. Low partition coefficients (log P)
were observed for 68Ga-3 (-2.71) and 68Ga-4 (-2.27),
indicating that both of them are hydrophilic. However,
68Ga-3 was found to be a little more hydrophilic than
68Ga-4, which was expected because of the absence of the
aromatic ring. Both agents showed low protein binding
(0.06 ( 0.02% at 10 min and 0.12 ( 0.04% at 60 min
for 68Ga-3, 1.66 ( 0.04% at 10 min and 2.45 ( 0.06% at
60 min for 68Ga-4), which is desirable for imaging agents,
and this finding was also consistent with the literature
which reported that 68Ga-labeled NOTA derivatives
showed low protein binding.48 Furthermore, both labeled
agents were found to be stable up to 240 min at room
temperature (Supporting Information Figure 2).
significantly higher uptakes of 68Ga-3and 68Ga-4under hypoxic
than under normoxic conditions after 1 h (Figure 3). The diffe-
rence between the uptakes in hypoxic and normoxic conditions
was significant in CHO and CT26 cell lines at 60 min (1.8 (
0.1 times in CHO, 1.5 ( 0.6 times in CT26 for 68Ga-3; 5.6 (
1.5 times in CHO, 2.6 ( 0.3 times in CT26 for 68Ga-4). Cell
uptake results obtained for 68Ga-3 and 68Ga-4 were comparable
to those of [18F]FAZA and [18F]FMISO. According to the
literature, uptake of [18F]FAZA and [18F]FMISO under hypo-
xic conditions was increased significantly in comparison to nor-
moxic conditions (1.4 ( 0.3 times for [18F]FAZA and 1.5 ( 0.4
times for [18F]FMISO) at 20 min and (2.7 ( 0.4 times for
[18F]FAZA and 3.0 ( 0.6 times for [18F]FMISO) at 100 min.18
Animal Studies in Mice Bearing CT-26 Xenografts. For
68Ga-3 and 68Ga-4, biodistribution studies were performed
at different time points (10, 30, 60, and 120 min) after intra-
venous injection of these labeled derivatives (37 KBq) into
mice bearing CT-26 xenografts (Figure 4). As the tumors
grow, hypoxia essentially builds up.12,13 So the tumor uptake
can be used as an indicator of the agents taken up to hypoxia.
The highest uptakes were demonstrated in kidneys (16.43 (
2.41% ID/g for 68Ga-3 and 28.53 ( 16.59% ID/g for 68Ga-4
at 10 min) for both derivatives, indicating that they are
probably excreted via kidneys. The higher liver and intestine
Cell Uptake Study. CHO (a Chinese hamster ovarian
cancer cell line) and CT-26 (a mouse colon cancer cell line)
were used for the cell uptake study. Both cell lines showed

6382 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Hoigebazar et al.
Figure 5. Small animal PET images of mice bearing a CT-26 xenograft on the right shoulder. (A) 68Ga-3 (13.3 MBq/0.1 mL) and (B) 68Ga-4
(12.6 MBq/0.1 mL) are injected through tail vein at 30 and 60 min postadministration. (C) and (D) show SUVs for 68Ga-3 and 68Ga-4,
respectively. “Normal” indicates muscle.
uptake of 68Ga-4 (12.41 ( 1.09 and 9.21 ( 0.72% ID/g at
1 h, respectively) than 68Ga-3 (3.59 ( 0.21 and 0.66 ( 0.12%
ID/g at 1 h, respectively) was attributed to the higher
lipophilicity of 4. The initial tumor uptakes were 2.47 (
0.47 and 2.37 ( 0.29% ID/g at 10 min postinjection for 68Ga-3
and 68Ga-4, respectively. These activities declined over time
for both derivatives. It was 1.25 ( 0.11% ID/g for 68Ga-3
and 1.34 ( 0.17% ID/g for 68Ga-4 at 30 min, 0.73 ( 0.18%
ID/g for 68Ga-3 and 0.61 ( 0.06% ID/g for 68Ga-4 at 60 min,
and 0.51 ( 0.10% ID/g for 68Ga-3 and 0.42 ( 0.07% ID/g
for 68Ga-4 at 120 min. In addition, the tumor to blood uptake
ratios of 68Ga-3 (0.62 ( 0.01) and 68Ga-4 (0.73 ( 0.12) were less
compared to those of [18F]FAZA (3.27)6 and [18F]FMISO
(1.19)49 at 60 min, whereas the tumor to muscle uptake ratios
of 68Ga-3 (2.13 ( 0.58) and 68Ga-4 (1.64 ( 0.39) were compar-
able to those of F-18 labaled NI derivatives (1.69 for [18F]FAZA
and 1.65 for [18F]FMISO)6,49 at the same time point (Figure 4).
These results could open the possibility of 68Ga-labeled NI
derivatives, especially 68Ga-3, for PET hypoxia tumor imaging.
A small animal PET study was performed, and images
were obtained at 30 and 60 min after injecting 68Ga-3 (13.3
MBq/0.1 mL) or 68Ga-4 (12.6 MBq/0.1 mL) into a mouse
bearing CT-26 xenografts via tail vein. Both agents showed
high liver, kidney, and bladder uptakes as predicted from the
biodistribution study. 68Ga-3 demonstrated better tumor
uptake than 68Ga-4 (Figure 5). To perform a quantitative
analysis, we calculated standardized uptake value (SUV),
which is defined as a ratio of tissue radioactivity concentra-
tion (MBq/mL) at time T and injected dose (MBq) at the time
of injection divided by body weight (g) in PET image. 68Ga-3
(0.30 ( 0.2) demonstrated a higher SUV value than 68Ga-4
(0.19 ( 0.1) at 60 min postinjection. From literature SUV
values of [18F]FAZA is 0.68 ( 0.2 and [18F]FMISO is 1.07 (
0.3.18 The tumor to muscle SUV ratios of 68Ga-3 and 68Ga-4
were 5.7 ( 2.5 and 3.95 ( 1.3 at 60 min postinjection, res-
pectively, which were higher than the values of [18F]FAZA
(2.4 ( 0.6) and [18F]FMISO (2.7 ( 0.6).18
The high uptake of these 68Ga-labeled derivatives in liver
and kidney is the drawback of these derivatives, since many
tumors are located in abdominal area. To solve these pro-
blems, development of a more hydrophilic agent is required
because hydrophilic agents generally demonstrate reduced
liver uptake and increased kidney excretion rate. If we add
carboxyl or hydroxyl groups at the linker between nitroimi-
dazole and NOTA, more hydrophilic agents might be pro-
duced. Especially, introduction of carboxyl group would
make the product more negatively charged and hence may
facilitate the rapid clearance through the kidneys. Or chan-
ging the bifunctional chelating agent would be another
possibility for improvement.
Conclusion
We successfully synthesized NI conjugates of NOTA via
formation of amide (3) or thiourea bond (4). Furthermore,
amide carbonyl was found to participate in the formation of
Ga-3. Both 3 and 4 were labeled by 68Ga at high yields in a
straightforward manner, and both labeled agents were found
to be stable and to have low protein binding. 68Ga-3 was
found to have a lower log P value than Ga-4, which is
attributed to its higher hydrophilicity. 68Ga-3 and 68Ga-4
showed elevated uptakes in hypoxic cells in vitro. In addition,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6383
biodistribution and PET studies showed that both have high
tumor to muscle ratios. Thus, both agents, but especially
68Ga-3, were found to be potential agents for the PET imaging
of hypoxic tissue.
mixture was then stirred at room temperature overnight and
filtered. The aqueous solution was evaporated in vacuo and
purified by RP-HPLC (100% of A for 5 min and 0-60% of C
for 30 min) to obtain pure compound 3 as a white solid (25 mg,
52%). After purification, the product obtained was injected into
an analytical HPLC column with the same conditions used
for the purification and found to be single peak, and also purity
was confirmed by HRMS. 1H NMR (300 MHz, D2O): δ 7.31 (s,
1H), 7.02 (s, 1H), 4.46 (br, 2H), 3.78 (br, 4H), 3.56 (br, 4H), 3.25
(br, 4H), 3.14 (br, 4H), 2.96 (br, 4H). 13C NMR (75 MHz, D2O):
δ 172.2, 172.0, 145.3, 129.3, 128.1, 58.3, 57.0, 51.3, 50.7, 50.4,
50.0, 39.2. MS (ESIþ), (M þ H)þ: 442.1 observed, 442.21 cal-
culated. HRMS: 442.2055 observed, 442.2050 calculated for
C17H28N7O7.
Experimental Section
Chemistry. NOTA was purchased from ChemaTech (Dijon,
France) and SCN-NOTA from Futurechem (Seoul, Korea).
HPLC grade MeCN and EtOH were purchased from Fischer
Scientific Korea Ltd. (Seoul, Korea). All other reagents were
purchased from Sigma-Aldrich (St. Louis, MO) and were used
without further purification. 68Ge/68Ga generator was pur-
1
chased from Eckert and Ziegler (Berlin, Germany). H NMR
and 13C NMR spectra were recorded on a AL 300 FT NMR
spectrometer (300 MHz for 1H and 75 MHz for 13C; Jeol Ltd.,
Tokyo, Japan) and were referenced internally using residual
solvent signals. 1H NMR peaks are described as s for singlet, d
for doublet, t for triplet, q for quartet, m for multiplet, or br for
broad, and coupling constants are presented in Hz. 1H chemical
shift values are expressed as δ values (parts per million) relative
to tetramethylsilane (the internal standard). HPLC was perfor-
med using a XTerra RP18 (10 mm ꢀ 250 mm) column (Waters
Corporation, Milford, MA). The solvent systems used were A
(10 mM HCl in H2O), B (H2O), C (MeCN), and D (EtOH), and
the flow rates for analytical and preparative HPLC were 1 and
5 mL/min with suggested linear gradients, respectively. The
purities of synthesized compounds were confirmed to be higher
than 95% by analytical HPLC (Supporting Information). ESI
data were recorded on a Waters ESI ion trap spectrometer
(Milford, MA) in positive ion detection mode and high resolu-
tion mass spectra (HRMS) on a Jeol, JMS-AX505WA, HP5890
series II spectrometer by fast atomic bombardment (FABþ)
ionization detection simultaneously. Samples were diluted 1-50
times with water or MeCN and injected directly into the source.
Crystallographic analysis was conducted at the organometallic
laboratory at Seoul National University.
tert-Butyl 2-(2-Nitroimidazolyl)ethylcarbamate. K2CO3 (1.83 g,
13.26 mmol) was added to a stirring solution of 1(1 g, 8.84 mmol) in
DMF (3 mL). tert-Butyl 2-bromoethylcarbamate (1.98 g, 8.84
mmol) was then added dropwise, and the reaction mixture was
stirred at room temperature overnight. The reaction mixture was
than filtered, the solid obtained was washed with MeOH, and
residual solvent was evaporated. The solid obtained was dissolved
in water, extracted with EtOAc, and the organic layer was evapo-
rated in vacuo to obtain the solid. Finally the crude compound
was recrystallized from EtOAc to afford pure compound as a
dark yellow solid (1.9 g, 84%), and purity was confirmed by
TLC. 1H NMR (CD3OD): δ 7.35 (s, 1H), 7.10 (s, 1H), 4.51-4.54
(t, J = 9 Hz, 2H), 3.47-3.50 (t, J = 9 Hz, 2H), 1.28-1.39 (s, 9H).
13C NMR (CD3OD): δ 158.4, 131.9, 129, 128.3, 80.4, 51.2, 40.8,
28.7. MS (ESIþ), (M þ Na)þ: 279.1 observed, 279.11 calculated for
C10H16N4NaO4.
SCN-NOTA-2-NI-N-ethylamine (4). A mixture of SCN-
NOTA (0.05 g, 0.11 mmol) and 2 (0.026 g, 0.13 mmol) in CHCl3
(1 mL) containing TEA (0.034 g, 0.33 mmol; used as a base) was
stirred overnight at room temperature. The resulting product
was purified by RP-HPLC (30-60% of C with A for 30 min) to
afford 4 as light yellow solid (49 mg, 73%). The product purity
was confirmed by analytical HPLC (with the same conditions)
1
as well as by HRMS. H NMR (D2O): δ 7.13-7.06 (d, 3H),
6.92-6.87 (d, 3H), 4.39 (br, 3H), 3.80-3-69 (br, 6H), 3.41-
3.34 (q, 1H), 3.21-3.13 (br, 7H), 2.97-2.90 (br, 3H), 2.61-2.45
(br, 1H), 1.03-0.98 (t, 1H), 0.93-0.88 (t, 1H). 13C NMR (D2O):
δ 181.1, 176.4, 172.3, 171.0, 145.2, 130.9, 129.4, 128.4, 128.3, 126.8,
58.0, 56.4, 54.3, 52.5, 50.1, 47.2, 43.9, 43.2, 43.0, 34.0, 30.7. MS
(ESIþ), (M þ H)þ: 607.1 observed, 607.23 calculated. HRMS:
607.2307 observed, 607.2302 calculated for C25H35N8O8S.
Ga-NOTA-NI Complex (Ga-3). Ga(NO3)3 xH2O and 3 were
3
dissolved in water at a stoichiometric ratio of 1:1, and the pH
was adjusted to 5 by adding 0.5 M Na2CO3 solution using
microfine pH test paper. This mixture was then heated in a
boiling water bath for 30 min. In addition, the same procedure
was repeated at pH 3. MS/ESIþ was used to confirm reaction
completion and was purified by RP-HPLC (15-30% of D with
B for 30 min) and lyophilized to afford Ga-3 as a white solid. The
1
purity confirmation was done by analytical HPLC. H NMR
(D2O): δ 7.35 (s, 1H), 7.11 (s, 1H), 4.57 (br, 2H), 4.10 (br, 2H),
3.90 (br, 2H), 3.60-3.74 (q, 4H), 3.34 (br, 6H), 3.03 (br, 4H),
2.80 (br, 2H). MS (ESIþ), (M þ H)þ: 508.1 observed, 508.11
calculated C17H25GaN7O7.
X-ray Diffraction Analysis. Data Reduction. Single crystal
diffraction data were obtained using an Enraf-Nonius CCD
single-crystal X-ray diffractometer at room temperature using
˚
graphite-monochromated Mo KR radiation (λ = 0.710 73 A).
Preliminary orientation matrices and unit cell parameters were
obtained from the peaks of the first 10 frames and then refined
using whole data sets. Frames were integrated and corrected for
Lorentz and polarization effects using DENZO-SMN.50
Structure Solution and Refinement. The structures of the
crystals were solved by direct methods using SHELXS-97 and
refined by full-matrix least-squares with SHELXL-97.51 Draw-
ings were prepared using ORTEP52 software. All non-hydrogen
atoms were refined anisotropically. All hydrogen atoms are
treated as idealized contributions.
2-(2-Nitroimidazolyl)ethylamine (2). A sample of 1.25 M HCl
in MeOH (3 mL) was added to a solution of tert-butyl 2-
(2-nitroimidazolyl)ethylcarbamate (1.9 g) in MeOH (2 mL) at
room temperature under continuous stirring. After 5 h the
resulting product 2 was filtered and washed with MeOH and
evaporated in vacuo. The solid obtained was recrystallized from
MeOH to afford pure compound 2 as a pale yellow solid (1.2 g,
84%). TLC was used to confirm the purity, where the product
appeared as a single spot in TLC. 1H NMR (CD3OD): δ 7.56 (s,
1H), 7.21 (s, 1H), 4.75-4.79 (t, J = 12 Hz, 2H), 3.47-3.51 (t,
J = 12 Hz, 2H). 13C NMR (CD3OD): δ 145.2, 128.8, 128.5, 47.2,
39.7. MS (ESIþ), (M þ H)þ: 157.1 observed, 157.07 calculated
for C5H9N4O2.
Radionuclide Labeling. NaOAc buffer (1 M, pH 5, 0.1 mL)
was added to 3 (22 μg) in 0.1 mL of 68GaCl3 (39.6-245.3 MBq),
which was eluted from a 68Ge/68Ga-generator using 0.1 M HCl
to adjust the pH to ∼3, and then the mixture was heated at 100
°C for 10 min. The reaction mixture was analyzed by ITLC-SG
(German Science, Ann Arbor, MI) using 0.1 M Na2CO3 solu-
tion as eluant to check labeling efficiency (Supporting Informa-
tion Figure 1). 68Ga-4 (50 nmol) was labeled using 68GaCl3
(150.2-355.2 MBq) and the same procedure.
Stability Study. The above prepared 68Ga-3 and 68Ga-4 were
stored at room temperature for 0, 30, 60, 120, and 240 min and
then analyzed by ITLC-SG: 0.1 M Na2CO3 (68Ga3þ remained
at the origin, and labeled products moved with the solvent
front) and 0.1 M HCl (68Ga3þ moved with the solvent front,
NOTA-2-NI-N-ethylamine (3). A solution of 2 (0.021 g, 0.1
mmol) in DMF (3 mL) and DCC (0.034 g, 0.16 mmol) solution
in pyridine (0.5 mL) were sequentially added to NOTA (0.050 g,
0.16 mmol) solution in water (3 mL) with stirring. The reaction

6384 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Hoigebazar et al.
and labeled products remained at the origin) were used as
eluants.
(GE Healthcare, Princeton, NJ). Emission data were acquired
for 20 min. The acquired three-dimensional emission raw
data were reconstructed to temporally framed sonograms by
Fourier rebinning using ordered subsets expectation maximiza-
tion (OSEM) reconstruction algorithm without attenuation
correction. For the PET images, data were analyzed in 20 frames
(10 times with 1 min frames).
Data Analysis. AsiPRO VM 5.0 software (Concorde Micro-
systems, Knoxville, TN) was used to perform image and region
of interest (ROI) analyses with the PET data sets. For the ROI
(value/pixel) analyses, 1.5 mm radius of sphere per tumor and
muscle were collected, and ROI and standard deviation were
determined. Once ROI determination was done, SUV was
calculated using the formula
Partition Coefficients. Na2PO4 buffer (0.1 M, pH 7.4, 3 g) was
added to octanol (3 g), and then 68Ga-3 (1.1 MBq/10 μL) or
68Ga-4 (1.2 MBq/10 μL) was added, mixed vigorously, and
centrifuged (3000 rpm for 5 min). Radioactivities of octanol
fraction (0.5 g) and 0.1 M Na2PO4 buffer fraction (0.5 g) were
measured using a γ counter, and log P values were calculated.
Serum Protein Binding. Human serum protein binding frac-
tions were determined using a previously reported method with
minor modifications.48 PD-10 columns were preconditioned by
loading 1.0 mL of 1% bovine serum albumin in 0.1 M DTPA
and successive washing with 100 mL of phosphate buffered
saline (PBS). 68Ga-3 (1.3 MBq/10 μL) or 68Ga-4 (1.1 MBq/
10 μL) was mixed with human serum (1 mL) and incubated for
10 or 60 min at 37 °C. And then each mixture was loaded onto a
preconditioned PD-10 column and eluted with PBS; 30 fractions
(fraction size 0.5 mL) were collected per sample in 5 mL test
tubes. Radioactivity of each fraction was measured using a
γ counter and expressed as cpm (count per minute). To check
for the presence of protein in each fraction, aliquots (2 μL) from
each test tube were spotted on a filter paper and stained with
Coomassie blue. Protein bound fractions appeared at the void
volume and free fractions at the bed volume.
SUV ¼ CCF=ðinjected dose=weight of mouseÞ
where CCF (decay corrected activity concentration) was calcu-
lated by the formula
CCF ðMBq=mLÞ ¼ radioactivity ðmCi=mLÞ
ꢀ branching ratio ꢀ RIO ðvalue=pixelÞ
where the branching ratio for 68Ga is 0.891.
In Vitro Cell Uptake Study. Cell uptake studies were carried
out using the CHO and CT-26 cell lines. Both cell lines were
maintained in DMEM culture medium enriched with 10% fetal
bovine serum (both from Welgene Inc., Korea) and containing
a 1% antibiotics mixture (penicillin, streptomycin, and ampho-
tericin B: (10 000 IU/10 mg)/25 μg/mL, Mediatech Inc.) in 5%
CO2 in an incubator at 37 °C. The cells were subcultured over-
night in 24-well plates (2 ꢀ 105 cells/well). And then preincuba-
tion was performed under normoxic (5% CO2 in air) or hypoxic
(5% CO2 in 95% N2) conditions for 4 h. 68Ga-3 (0.37 MBq/
100 μL) or 68Ga-4 (0.44 MBq/100 μL) was added to the wells and
incubated for 5 or 60 min. Wells were then washed with DMEM
(Dulbecco’s modified Eagle medium), and the cells were dis-
solved in 0.5% of SDS (sodium dodecyl sulfate) in PBS
(phosphate buffered saline) (0.5 mL). The tracer uptakes were
measured using a γ-counter (Packard, Canberra Co.), and total
protein concentrations in samples were determined using the
bicinchoninic acid method (Pierce).
Acknowledgment. This work was supported by NRF of
Korea (Grants R0A-2008-000-20116-0 and 2009-0082087),
funded by Ministry of Education, Science and Technology.
Note Added after ASAP Publication. This paper was pub-
lished on the Web on August 6, 2010 with several minor textual
errors. The revised version was published on August 11, 2010.
Supporting Information Available: Labeling experiments and
stability data, crystallographic information, and analytical and
spectral characterization data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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