224
N. Li et al. / European Journal of Medicinal Chemistry 69 (2013) 223e231
9
9m
ꢃ
4
2
. Materials and methods
-Bromopropionitrile,
TcO
was added. The mixture was shaken vigorously and heated
ꢂ
to 75 C for 15 min. Radio-TLC or Radio-HPLC showed radiolabeling
yield and radiochemical purity >95% and >98%.
3
4-nitroimidazole,
triethylamine,
phosphate-buffered saline (PBS), sodium tartrate, hydroxylamine,
and bovine serum albumin (BSA) were purchased from Sigmae
.2. Evaluation of 99mTc-N4IPA
2
2
9
9m
Aldrich (St. Louis, MO, USA). Na[
TcO
4
] was eluted from a
9
9
99m
Mo/
Tc generator (China Isotope and Radiation Co. Ltd, Beijing,
.2.1. Partition coefficient of 99mTc-N4IPA
China) using saline. Other commercial materials were purchased
from VWR International (San Diego, CA, USA). All reagents were
analyzed before use. All synthesized chemicals were characterized
The partition coefficient of 99mTc-N4IPA complex was deter-
mined by measuring the distribution of radioactivity in 1-octanol
and 0.01 M PBS. Concisely, a 10
was added to a vial which contained 1 mL of 1-octanol and 0.99 mL
of PBS. After the mixture had been vortexes for 8 min, the vial was
centrifuged for 5 min to ensure complete separation of layers. Then,
99m
mL sample of
Tc-N4IPA in PBS
1
by H NMR. Melting points were determined on WRS-IA apparatus
and were uncorrected.
Radiochemical purity was checked both with radioactivity thin-
layer scanner (Bioscan, IAR-2000, Washington DC, USA), and HPLC
10
mL of each layer was pipetted into the test tubes, to calculate
(
Agilent, USA), with Zorbax SB-C18 (4.6 mm ꢁ 250 mm, 5
using gradient program as follows: 0e10 min: 0%e20%B; 10e
5 min: 20%e90%B; 15e20 min: 90%e50%B; 20e21 min: 50%e10%
B and the flow rate 1.0 mL/min. Solvent A was PBS (pH 6.0, 10 mM)
and solvent B was CH CN. The radioactivity was measured using an
mm)
log P ¼ log (counts in octanol/counts in water) [19].
1
.2.2. The in vitro stability of 99mTc-N4IPA
The radiochemical purity measured by radio-HPLC at different
2
3
times after preparation represent the in vitro stability of the radi-
automated gamma scintillation counter (Perkin Elmer, 1470-002,
USA). Autoradiogram was captured using a high-sensitivity imaging
film (BAS-SR2025, Fuji Photo Film, Japan), and scanned using
computerized imaging analysis system (PLA5100, Fuji medical-
system, USA, Stanford, CT). In vivo SPECT imaging was performed
on a SPECT camera (Siemens, E cam, Germany).
99m
oligand.
Tc-N4IPA (0.2 mL, 14.8 MBq) was added to a test tube
with 1 mL 0.1 M PBS. The mixture was incubated by shaking the test
ꢂ
tube at 25 and 37 C in a water bath. The radiochemical purity was
measured at 10 min, 1, 2, 4, 8 and 12 h by radio-HPLC. The same
experiment was repeated while 5% BSA was added in the incuba-
tion mixture.
Chinese hamster ovary (CHO) cell line was provided by the
Department of Genetic Research, Beijing Cancer Hospital. Human
glioma U87 and human lung cancer A549 cells were provided by
the College of Life Sciences, Beijing University. Dulbecco’s Modified
Eagle’s Medium (DMEM) was purchased from Gibco BRL Life
Technologies (Grand Island, NY, USA) and fetal bovine serum, from
Hyclone (Logan, UT, USA). The mice were kept under specific
pathogen free conditions and were handled and maintained ac-
cording to Institutional Animal Care and Use Committee guidelines.
.2.3. Blood retention of 99mTc-N4IPA in mice
2
99m
Tc-N4IPA (0.2 mL, 14.8 MBq) was injected intravenously into
the tail vein of healthy female BABL/c mice (18e22 g, n ¼ 7), 10
blood was collected at a different location of the tail vein at 2 min,
5 min, 30 min, 1, 2, 4, 6, 8, 11, 21, 24 h post injection. The radio-
activity of the samples was measured with a counter. The blood
Tc-N4IPA was expressed as percentage of injected
dose per gram of tissue (%ID/g).
mL
1
g
99m
retention of
2
.1. Synthesis and radiolabeling of 99mTc-N4IPA
2
.3. In vitro cell uptake of 99mTc-N4IPA
1
-(4-Nitroimidazole-1-yl)-propanhydroxyiminoamide (N4IPA)
The internalization of 99mTc-N4IPA in CHO tumor cells was
was synthesized according to a reported procedure with some
modification [10,15]. Briefly, 3-Bromopropionitrile (2.66 g,
0.0 mmol) was added to the suspension of 4-nitroimidazole
1.13 g, 10.0 mmol) in triethylamine (10 mL), and the mixture was
refluxed in an oil-bath for 4 h. 1-(2-Cyanoethyl)-4-nitroimidazole
1.33 g, 8 mmol, yield 80%) was obtained as a white solid by
performed as described [20,21]. CHO cells were grown in suspen-
ꢂ
sion at 37 C in DMEM plus 10% fetal calf serum, were spun down
2
(
from suspension and resuspended in fresh medium at the desired
6
cell density (1 ꢁ10 /mL). Cells (20 mL) were equilibrated for 45 min
ꢂ
in special glass vials with stirring at 37 C under a continuous flow
(
ꢂ
1
of a pre-humidified gas mixture of 95% N
exposure) or 95% air þ 5% CO (aerobic exposure). After equilibra-
Tc-N4IPA (0.2 mL, 14.8 MBq) was added to make the final
2
2
þ 5% CO (hypoxia
recrystallization from methanol. m.p. 109e110 C. H NMR (CDCl
00 MHz) ), 4.39 (t, 2H, J ¼ 3.6 Hz, CH
: 3.17 (t, J ¼ 4.0 Hz, 2H, CH
3
,
2
5
7
C
C
3
d
2
2
),
9
9m
tion,
.93 (s, 1H, imi-H), and 8.47 (s, 1H, imi-H). ESI-HRMS calcd for
þ
radioactivity of 0.25 MBq/mL in each vial. After 5 min, 1, 2, 3, 4 h,
samples (0.2 mL, 5 repeat each) were removed from the vials to
6
H
H
7
N
N
4
O
O
2
[M þ H] 167.0564, found 167.0532. Anal. Calcd for
6
6
4
2
: C, 43.38; H, 3.64; N, 33.72; found C 43.83, H 3.57, N
1
mL centrifugal tubes without disturbing the oxygenation status of
cells in the vials. Those vials were centrifugated at 1500 r/min for
min, to separate the cells from the supernatant. 180 L of the
supernatant was carefully aspirated, and its radioactivity was
counted as that in the supernatant (Cs), and the remaining 20 L of
3.40. 1-(2-Cyanoethyl)-4-nitroimidazole (0.83 g, 5 mmol), and
hydroxylamine (0.66 g, 20 mmol), were mixed in 10 mL methanol,
and the mixture was stirred at room temperature for 10 h. The
mixture was placed at 0 C overnight, and a white crystalline
powder precipitated. It was further purified by recrystallization
5
m
ꢂ
m
ꢂ
the samples was counted as that the residual (Cr). The percentage
of radioactivity uptake in cells was calculated as follows:
uptake ¼ (Cr ꢃ Cs/9)/(Cr þ Cs) ꢁ 100%. Cell viability was confirmed
by methylene blue staining before and after the uptake experiment,
to be greater than 90% as the requirement of the experiment.
from water, to a pale green crystals, yield 75%. m.p. 145.0e146.0 C;
H NMR (CD
1
3
OD, 500 MHz)
2 2
d: 2.4e2.6 (m, 4H, NH and CH ), 4.2 (t,
2
1
1
2
4
H, J ¼ 3.6 Hz, CH ), 7.63 (s, 1H, imi-H), 8.11 (s, 1H, imi-H). 8.38 (s,
2
H, NeOH). 1 C NMR (CD
3
3
OD, 500 MHz)
d
:31.98, 44.51, 121.67,
þ
37.50, 146.77, and 149.40. ESI-HRMS calcd for C
00.0778, found 200.0772. Anal. Calcd for C
6
9
H
N
10
N
5
O
3
[M þ H]
6
H
5
O
3
: C, 36.18; H,
.55; N, 35.16; found C, 36.25; H, 4.52; N, 35.41.
2.4. Biodistribution of 99mTc-N4IPA
9
9m
Tc-N4IPA was synthesized as follows: N4IPA solution
L, 1 mg/mL) and sodium tartrate solution (25 L, 1 mg/mL)
were added into 1 mL of 0.1 M PBS (pH ¼ 7.4). Then freshly pre-
pared stannous chloride solution (10 L, 1 mg/mL) and 370 MBq
(
200
m
m
A suspension of U87 cells (human glioma) or A549 cells (human
7
lung cancer) in 0.1 mL (1 ꢁ10 /mL) was injected subcutaneously at
m
the left axilla of six week-old BALB/c mice (female, 20e25 g). When