6
S.-H. Moon et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
The elution gradient started from 0% MeCN (100% DW) for 5 min
and increase to 30% MeCN (70% DW) until 30 min. And than the
MeCN concentration was raised to 100% for 10 min. The retention
time of the final product 7 was 21 min. The purified product 7 was
collected and finally obtained as a white solid by freeze drying.
5.4. Radioisotope labeling
6
8
68
5.4.1. Ga-NOTA-GUL and Ga-DOTA-GUL labeling
68GaCl
in 0.1 M HCl solution (200 L, 111 MBq) was added to 1
M sodium acetate buffer (pH = 5.6, 200 L). NOTA-GUL in acetoni-
trile solution (10 L, 1 mg/1 mL) or DOTA-GUL in water (10 L, 1
3
l
l
Yield: 47.8 mg, 63% (2 steps overall yield). 1H NMR (DMSO d63
,
l
l
6
8
2
00 MHz) d 12.4 (br, 6H), 9.45 (s, 1H), 7.69 (s, 1H), 7.38 (d, J =
.10 Hz, 2H), 7.18 (d, J = 8.16 Hz, 2H), 6.76–6.41 (br, 2H), 6.30 (m,
mg/1 mL) was added and vigorously mixed for 1 min, respectively.
The NOTA-GUL reaction mixture was incubated for 10 min at room
6
8
H), 4.12–2.61 (m, 22H), 2.19 (m, 2H), 1.86 (m, 1H), 1.74–1.51
temperature. The labeling efficiency of Ga-NOTA-GUL was con-
firmed by ITLC-SG with 0.1 M Na CO as the TLC eluent and
+
+
(
m, 4H), 1.29 (m, 2H). MS (ESI ), (M+H) calcd for C32
H
47
N
7
O13S,
2
3
7
70.32; found 770.
detected with a radio-TLC scanner. The DOTA-GUL reaction mix-
ture was incubated for 10 min at 95 °C and cooled to room temper-
ature. The final compound was purified by PD-10 column
chromatography with distilled water. The labeling efficiency of
0
00
5
.2.6. Gallium 2,2 ,2 -(2-(4-(3-((S)-5-carboxy-5-(3-((S)-1,3-dicar-
boxypropyl)ureido)pentyl)thioureido)benzyl)-1,4,7-triazonane-1,4,7-
triyl)triacetate (8)
6
8
Ga-DOTA-GUL was evaluated by ITLC-SG with 0.1 M Na
the eluent and detected with a radio-TLC scanner.
2 3
CO as
3
GaCl 0.5 M in pentane (1.242 mL, 0.621 mmol) and 7 (47.8 mg,
0
5
.0621 mmol) were suspended in 1.0 M NaOAc buffer (2.0 mL, pH
.6) and the mixture was stirred for 8 h at room temperature.
6
8
5.4.2. Ga-NOTA-GUL kit labeling
68GaCl
in 0.1 M HCl solution (1.0, 2.0 or 3.0 mL) or 0.05 M HCl
The mixture was filtered through a 0.2-
and the filtrate was purified by silica gel column chromatography
ethyl acetate:n-hexane = 1:1, v/v) to obtain compound 8 as a
l
m pore syringe filter
3
solution (1.0, 2.0 or 3.0 mL) was added to the NOTA-GUL kit vial
(10 or 20 nmol). The vial was vigorously mixed for 1 min and then
was incubated for 10 min at room temperature. The labeling effi-
(
1
white solid. Yield: 268 mg, 58%. H NMR (DMSO d63, 600 MHz) d
6
8
1
7
2
1
8
2.4 (br, 3H), 9.45 (s, 1H), 7.69 (s, 1H), 7.38 (d, J = 8.10 Hz, 2H),
.18 (d, J = 8.16 Hz, 2H), 6.76–6.41 (br, 2H), 6.30 (m, 2H), 4.12–
.61 (m, 22H), 2.19 (m, 2H), 1.89 (m, 1H), 1.74–1.51 (m, 4H),
ciency of Ga-NOTA-GUL was evaluated by ITLC-SG with 0.1 M
Na CO as the eluent and detected with a radio-TLC scanner.
2
3
+
+
.29 (m, 2H). MS (ESI ), (M+H) calcd for
37.20; found 837.
C
32
H44GaN
7
O13S,
5.5. Biology study
5
.5.1. Stability test in human serum
0
00 000
68
Ga-NOTA-GUL (3.7 MBq, 100 l
L) or 68Ga-DOTA-GUL (3.7
5
.2.7. 2,2 ,2 ,2 -(2-(4-(3-((S)-6-(tert-butoxy)-5-(3-((S)-1,5-di-tert-
butoxy-1,5-dioxopentan-2-yl)ureido)-6-oxohexyl)thioureido)benzyl)-
,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (9)
(30 mg, 0.0615 mmol), p-SCN-Bn-DOTA (40 mg, 0.0726 mmol)
MBq, 100 lL) was added to a vial containing human serum (1
mL). After vortexing for 1 min, each mixture was incubated at
36.5 °C with shaking in a water bath. After 2 h, each reaction mix-
1
5
and trimethylamine (0.043 mL, 0.308 mmol) were suspended in
chloroform (1.0 mL). This solution was stirred overnight at room
temperature. This reaction mixture was dried under reduced
pressure. The product 9 was confirmed with MS by LC/MS system.
ture was checked by ITLC-SG with 0.1 M Na
detected with a radio-TLC scanner.
2 3
CO as the eluent and
5.5.2. In vitro cell binding assay using human prostate cancer cell
The PSMA positive human prostate cancer cell line 22Rv1 was
purchased from American Type Culture Collection (ATCC, Manas-
sas, VA, USA) and grown in ATCC-formulated RPMI 1640 (WEL-
GENE Inc., Daegu, Korea) mixed with 10% (v/v) heat-inactivated
+
+
78 8
MS (ESI ), (M+H) calcd for C48H N O15S, 1039.53; found 1040.
5
.2.8. (((1S)-1-Carboxy-5-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-
,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)pentyl)
-glutamic acid (10)
The crude mixture of 9 was dissolved in trifluoroacetic acid/
MeCl solution (v/v: 1/1, 2.0 mL) and stirred at room temperature
1
Ò
carbamoyl)-
L
fetal bovine serum (Gibco , Grand Island, NY, USA) and 1% (v/v)
Ò
antibiotic-antimycotic (100ꢁ) (Gibco , Grand Island, NY, USA) in
2
a humidified incubator at 37 °C with 5% carbon dioxide.
for 4 h. The reaction mixture was dried under reduced pressure
and the residue was purified with an HPLC system using MeCN
and DW eluents. The elution gradient started from 0% MeCN
For the competition binding assay, 22Rv1 cells were plated in
5
24-well plates at approximately 2 ꢁ 10 cells/well and incubated
for 24 h in
2
a humidified incubator at 37 °C with 5% CO .
(
3
1
100% DW) for 5 min and increase to 30% MeCN (70% DW) until
0 min. And than the MeCN concentration was raised to 100% for
0 min. The retention time of the final product 10 was 23 min.
Ga-NOTA-GUL was serially diluted in culture medium containing
0.5% bovine serum albumin. Serially diluted Ga-NOTA-GUL solu-
tions were added to the cells in the presence of 1.85 kBq/0.5 mL
1
25
The purified product 10 was collected and finally obtained as a
white solid by freeze drying. Yield: 23.0 mg, 43% (2 steps overall
yield). H NMR (DMSO d63, 600 MHz) d 12.9 (br, 1H), 12.5 (br,
of
pentyl)ureido)pentanedioic acid ( I-MIP-1072) and incubated
for 1 h in a humidified incubator at 37 °C with 5% CO . After 1 h,
I labeled (S)-2-(3-((S)-1-carboxy-5-((4-iodobenzyl)amino)
1
25
1
2
6
2
4
1
H), 9.44 (s, 1H), 7.71 (s, 1H), 7.63 (s, 1H), 7.37 (d, J = 13.56 Hz,
H), 7.18 (br, 2H), 6.31–6.35 (br, 3H), 6.30 (m, 2H), 5.75 (s, 1H),
.12–4.02 (m, 4H), 4.12–2.61 (m, 20H), 2.29–2.19 (m, 4H), 1.74–
the media were aspirated and the cells were washed twice with
fresh media. The cells were dissolved in 0.5% sodium dodecylsul-
fate and transferred into 5-mL plastic test tubes. Radioactivity
+
.66 (m, 2H), 1.56–1.51 (m, 2H), 1.27–1.23 (m, 2H). MS (ESI ), (M
was counted with a c-scintillation counter.
+
+H) calcd for C36
H
54
N
8
O
15S, 871.34; found 871.
5.5.3. Biodistribution study using a xenograft model
5
.3. Formulation of NOTA-GUL kit
Specific pathogen-free 4-week-old male BALB/c nude mice were
6
used for all animal studies. The 22Rv1 cells (5 ꢁ 10 cells in 0.1 mL
RPMI-1640 medium) were subcutaneously injected into the left
flank of each mouse. The xenografted mice were used for in vivo
study after 2–3 weeks. The long diameter of the tumors was
7
(1 mg) was dissolved in distilled water (25 mL) and filtered
through a 0.2- m sterile syringe filter. The solution aliquots (0.2
mL or 0.4 mL) were dispensed into sterilized 10-mL vials. Next,
.0 M sodium acetate buffer solution (0.5 mL, pH 5.5) was added
to each vial. The vials were freeze-dried for 2 days and stored at
47 °C until use.
l
1
between 1.5 and 2.0 cm.
For comparison with 68Ga-NOTA-GUL and
68
Ga-DOTA-GUL,
6
8
21
ꢀ
Ga-PSMA-11 was prepared by a published method. All three
Moon, Sung-Hyun
