Development of a bifunctional chelating agent containing isothiocyanate residue for one step F-18 labeling of peptides and application for RGD labeling

Article abstract of DOI:10.1016/j.bmc.2012.07.050

We report herein a novel isothiocyanate active ligand for fluorine-18 labeling prepared by four step synthesis. It can be conjugated to a target molecule containing an amino functional group under weak basic conditions by way of thiourea bond formation. We explored the application of synthesized ligand by conjugating to well known α_vβ₃ integrin targeting peptide, c(RGDyK). The conjugated peptide showed good radiochemical yield and efficiency with an excellent radiochemical purity (97.1 ± 1.2%) in a short reaction time (10 min). Labeled peptide showed excellent in vitro and in vivo stability (>95%). α_vβ₃ integrin specific tumor uptake was observed both in biodistribution and small animal microPET studies on α_vβ₃-positive U87MG (human glioma cells) xenograft bearing mice. In general, successful application of synthesized ligand for labeling of RGD peptide could facilitate the possibility of using this ligand for labeling peptides containing an amino functional group.

Full text of DOI:10.1016/j.bmc.2012.07.050

Bioorganic & Medicinal Chemistry 20 (2012) 5941–5947
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of a bifunctional chelating agent containing isothiocyanate
residue for one step F-18 labeling of peptides and application for RGD labeling
⇑
Dinesh Shetty, Jae Min Jeong , Young Ju Kim, Ji Youn Lee, Lathika Hoigebazar, Yun-Sang Lee,
Dong Soo Lee, June-Key Chung
Department of Nuclear Medicine, Department of Radiation Applied Life Science and Institute of Radiation Medicine, Seoul National University College of Medicine, Seoul, Republic
of Korea
Clinical Research Institute and Cancer Research Institute, Seoul National University Hospital, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein a novel isothiocyanate active ligand for fluorine-18 labeling prepared by four step syn-
thesis. It can be conjugated to a target molecule containing an amino functional group under weak basic
conditions by way of thiourea bond formation. We explored the application of synthesized ligand by con-
Received 28 May 2012
Revised 21 July 2012
Accepted 23 July 2012
Available online 7 August 2012
jugating to well known
radiochemical yield and efficiency with an excellent radiochemical purity (97.1 1.2%) in a short reaction
time (10 min). Labeled peptide showed excellent in vitro and in vivo stability (>95%). vb3 integrin spe-
cific tumor uptake was observed both in biodistribution and small animal microPET studies on vb3-posi-
avb3 integrin targeting peptide, c(RGDyK). The conjugated peptide showed good
a
Keywords:
a
NODA
tive U87MG (human glioma cells) xenograft bearing mice. In general, successful application of
synthesized ligand for labeling of RGD peptide could facilitate the possibility of using this ligand for label-
ing peptides containing an amino functional group.
Al18
F
NO2A
c(RGDyK)
Ó 2012 Elsevier Ltd. All rights reserved.
Angiogenesis imaging
Bifunctional chelating agent
Aluminum fluoride
1. Introduction
[
18F]AlF labeling efficiency was reported if 1,4,7-triazacyclo-
nonane-1,4-diiacetic acid (NODA) with a benzyl group attached
to one of the nitrogen atoms of the cylcononane ring.12–16 Another
successful report of [18F]AlF labeling application was NODA-nitro-
imidazole conjugates for hypoxia imaging.17 To apply these pro-
18F-labeling of peptides has usually been performed in a multi-
step process by first labeling a suitable synthon and conjugating it
to the peptide via formation of amide, oxime, hydrazone, or by
using thiol reactive maleimide synthons.1–5 However, all these
methods require additional steps that include purification of radi-
olabeled synthons before conjugation and final products should be
re-purified to achieve high specific activity and purity as well as to
remove organic solvent.
cesses into labeling of biomolecules, we designed
a novel
bifunctional chelating agent that has an active conjugation site
for an amino group. Herein, we report an isothiocyanatobenzyl
NODA (NODA-Bz-SCN, 5) derivative that can be conjugated to bio-
molecules under mild conditions.
A novel method for the one step 18F-labeling of peptides using
Integrins play important roles in the regulation of cellular acti-
[
18F]AlF (aluminum fluoride) complex formation with 1,4,7-triaza-
vation, migration, proliferation, survival, and differentiation.18,19
cyclononane-1,4,7-triacetic acid (NOTA) derivatives has been re-
ported, which did not need a drying step neither before nor after
the labeling.6,7 For radiofluorination strategies involving NOTA-
AlF chelation chemistry, the labeling procedure can be accom-
plished in water. From a biological perspective, these one-step
and water-compatible reactions are ideal for incorporation of
In particular, angiogenesis factor
a
vb3 integrin is important for
vb3 integrin
growth of various cancers.20–22 Therefore, imaging
a
expression in tumors is important for staging cancer and for the pa-
tient’s selection of anti-integrin treatments. Integrins represent a
subclass of cell adhesion molecules connecting the cytoskeleton
with the extracellular matrix (ECM) or other cells with the exposed
[
18F]fluoride into biomolecules. Although, NOTA has been proved
RGD tripeptide sequence.23–25 Integrin
avb3 is highly expressed on
to be an efficient chelating agent for labeling with 68Ga,8–11 it
was not so for [18F]AlF method. Recently, significant increase of
activated endothelial cells but not quiescent endothelial cells of
established vessels,26 making it a suitable target for anti-angiogenic
cancer management. After introduction of
tides,23,24 a variety of RGD based probes have been developed for
PET imaging of
vb3 integrin.27–30 Thus, RGD probes have been used
in clinical trials such as [18F]galacto-RGD and [18F]AH111585,
avb3 selective RGD pep-
⇑
a
Corresponding author. Tel.: +82 2 2072 3805; fax: +82 2 745 7690.
E-mail address: jmjng@snu.ac.kr (J.M. Jeong).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.050

5942
D. Shetty et al. / Bioorg. Med. Chem. 20 (2012) 5941–5947
which require multistep procedures for 18F-labeling. The former re-
quires four chemical reactions and three radio high performance li-
quid chromatography (radio-HPLC) purification steps,31–33 and the
latter requires two chemical reactions and two HPLC purification
steps.34–36 Due to the short half-life of 18F, repeated purification
steps can be critical to obtain a higher yield and a more reliable
method is required. Thus we synthesized 5, conjugated it with a
arginine–glycine–aspartic (RGD) peptide and studied its biological
activities to confirm the applicability of this agent.
(45 nmol) with [18F]fluoride (370 MBq) in 50
lL saline solution at
room temperature for 10 min. The ligand conjugated peptide, 6
(50 nmol), was mixed with the prepared [18F]AlF2+ solution, and
reactions were monitored at 95–110 °C after 10 min. Labeling effi-
ciencies were determined using Instant Thin Layer Chromatogra-
phy–Silica Gel (ITLC–SG) and labeled peptide was purified by
radio-HPLC (Fig. 1). We succeeded in purifying the labeled peptide
using HPLC or a C-18 cartridge but failed using an Alumina-N Car-
tridge (Fig. 2). The final radiochemical purity obtained was
97.1 1.2% and the labeling efficiency was 57.9 8.8%. The time
for whole labeling and purification procedure was less than
25 min. [18F]7 demonstrated higher stability than 95% for 4 h in
the prepared medium at room temperature and in human serum
at 37°C by ITLC–SG analysis (see Supplementary Fig. 2).
2. Results and discussion
2.1. Chemistry
The synthetic pathway of 5 is depicted in Scheme 1. The synthe-
sis began with formation of 1 from cyclononane according to a pre-
viously reported method.16 Nucleophilic substitution of 1 with 4-
nitrobenzylbromide resulted in 2, which was purified by column
chromatography. Tert-butyl protection was removed by acid
hydrolysis to yield 3. Successive catalytic hydrogenation of 3 under
basic condition produced 4 which were purified by prep-HPLC. Ba-
sic condition was crucial to avoid cleavage of benzyl carbon and
cyclononane nitrogen bonds. The amino group was converted into
isothiocyanate using thioposhgene in the solvents mixture. The fi-
nal product 5 was purified by prep-HPLC and obtained as a fine so-
lid. Easy handling and high stability of this agent at room
temperature enhance the possibility of general application for bio-
molecule conjugation. It is a common knowledge that reactive iso-
thiocyanate groups can be easily conjugated to amino groups of
biomolecules under mild basic conditions in aqueous buffer. The
high water solubility of the synthesized ligand was adequate for
conjugating with peptide or protein molecules.
2.3. In vivo studies
Biodistribution experiment was performed in U87MG tumor-
bearing nude mice at 1 h after tail vein injection of [18F]7. Blocking
study was performed after injection of c(RGDyK) (3 mg/kg). The re-
sults are shown in Fig. 3. The tumor uptake of [18F]7 was
4.41 0.98% ID/g. The uptake values in the other major organs ex-
cept kidney were significantly lower than tumor uptake. High up-
takes in the kidneys and liver indicate tracer excretion via both the
renal and hepatobiliary routes. The tumor to muscle and the tumor
to blood ratios were 8.17 0.50 and 4.95 0.36% ID/g, respectively.
Low bone uptakes demonstrated in vivo stability of the agent. In
the blocking study, a decrease of radioactivity was observed in
all the organs, but it was most significant with tumor uptake,
which was reduced markedly from 4.41 0.98 to 1.33 0.20% ID/
g at 1 h post-injection (p = 0.00083)
The in vivo tumor targeting property of [18F]7 was evaluated in
U87MG tumor-bearing nude mice using dynamic small animal
microPET scans at 1 and 2 h post injection (Fig. 4A). Tumor uptake
was observed clearly at 1 and 2 h after injection but it decreased by
time. High bladder uptake demonstrated that the tracer was rap-
idly excreted via the renal system, and low soft tissue uptakes were
observed. The decreased tumor uptakes after blocking c(RGDyK)
In the present study, we conjugated 5 with c(RGDyK) peptide to
test the labeling efficiency and feasibility for angiogenesis imaging
(Scheme 2). The extended amino group of lysine in the peptide was
conjugated with 5 in the buffer solution (pH ꢀ9) with quantitative
yield. Conjugated product 6 was purified by prep-HPLC and was
eluted at 9.6 min without any other peaks (see Supplementary
Fig. 1).
(3 mg/kg) confirmed the integrin avb3-specific targeting of the la-
beled agent. For quantitative analysis of imaging data, we calcu-
lated standardized uptake value (SUV), which is defined as a
ratio of tissue radioactivity concentration (MBq/mL) at time t and
injected dose (MBq) at the time of injection divided by body weight
(g) in PET image. [18F]7 demonstrated tumor SUV of 7.42 0.49 and
2.2. Radiochemistry
To conduct the labeling, [18F]fluoride was prepared as previ-
ously described,37 and [18F]AlF2+ was prepared by mixing Al3+
OH
N
O
OtBu
N
O
OtBu
N
O
N
N
NH
ii
i
NO2
NO2
N
N
N
OH
OtBu
OtBu
3
2
O
1
O
O
OH
N
O
OH
N
O
N
N
iv
iii
NCS
NH2
N
HPLC
N
HPLC
OH
OH
5
4
O
O
Scheme 1. Synthetic pathway for the formation of NODA-SCN Chelator (5). (i) 4-nitro benzyl bromide, K2CO3, ACN, rt; (ii) 4 M HCl in dioxane, rt; (iii) Pd–C, 2 mM KOH in
MeOH, H2, rt; (iv) CSCl2, CaCO3, H2O/CHCl3, rt.

D. Shetty et al. / Bioorg. Med. Chem. 20 (2012) 5941–5947
5943
OH
HN
NH2
OH
N
O
N
+
CONH
O
NCS
O
NH
NH2
O
HN
N
N
H
OH
HO
HN
N
5
O
H
O
O
O
i
HO
N
S
N
OH
HN
N
HN
O
HN
HO
CONH
O
O
NH
NH2
O
HN
N
H
HO
HN
N
H
O
6
O
O
O
N
ii HPLC
Al
S
N
18F
N
OH
HN
HN
O
O
HN
CONH
O
O
NH
NH2
O
HN
N
H
HO
HN
N
O
H
7
O
Scheme 2. Synthetic pathway for the formation of NODA-SCN-RGD (6) and Al18F-NODA-SCN-RGD (7). (i) Na2CO3 buffer, pH 9, rt; (ii) (Al18F)2+, NaOAc buffer, pH 4, 110 °C,
10 min.
3.77 0.57 at 1 and 2 h post injection, respectively, which de-
creased to 0.72 0.14 and 0.42 0.15, respectively, after blocking
(Fig. 4B).
present works, as researchers always tend to compare pharmacoki-
netics properties of labeled RGD monomers with dimers. Both in
biodistribution and PET imaging studies, [18F]7 showed similar
trend in tumor uptake and tumor to muscle ratio compared to
RGD dimer. Notably, however, the reported works could achieve
less than 25% radiochemical yield in the case of the RGD dimer,
which is lower than the values we obtained with our ligand
(57.9%). This difference of labeling yield is anticipated according
to recent literature.16 They used NOTA as a bifunctional chelating
agent, which has an interfering carbonyl oxygen atom for 18F bind-
ing with aluminum, but our ligand NODA-Bz-SCN does not have
such an interfering atom. Isothiocyanatobenzyl residue has been
applied for easy conjugation of ligands with peptides or proteins
containing amino groups. It is relatively stable compared to other
amine conjugating agents, such as activated esters, and showed
high conjugation yield. The formed thiourea bond is resistant to
most hydrolyzing enzymes in the biological system.
2.4. Discussion
The application of 18F-labeled RGD peptides for PET imaging of
tumor angiogenesis increased rapidly. However, most established
18F-labeling procedures require multiple drying steps to obtain ac-
tive [18F]fluoride and are time consuming and make automation of
the procedure difficult. Moreover, the reactions are usually per-
formed in organic solvents which should be removed for clinical
application. Therefore a straightforward single-step labeling strat-
egy to obtain radiofluorinated RGD peptides is a clinical require-
ment. With the emergence of the [18F]AlF labeling method, in
which water is used as a reaction solvent, drying steps could be
avoided. We tried to expand these findings for general 18F-labeling.
16,17 Interestingly, parallel works were reported recently on 18F la-
beled RGD dimers conjugated with NOTA that showed a similar
trend in biodistribution and PET imaging studies.38,39 Though our
findings are unique due to general ligand synthesis for biomolecule
conjugation, it might be necessary to compare those findings with
3. Conclusion
NODA-Bz-SCN was synthesized as a bifunctional chelating
agent for conjugation with peptide and labeling with 18F by a

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D. Shetty et al. / Bioorg. Med. Chem. 20 (2012) 5941–5947
Figure 1. HPLC profile of radiotracer [18F]7 before purification (A) and after purification (B). Samples were run on X-terraÒ 3.5
TFA in water for 30 min with a flow rate of 1.0 mL/min; tR = 11.2 min.
lm RP18 (4.6 Â 100 mm) column; EtOH/0.05%
Figure 2. Purification of [18F]7 using a C-18 cartridge. The reaction mixture was passed through the cartridge, washed with 10 mL distilled water and eluted with 1 mL EtOH.
chelating method, and it was successfully conjugated to RGD pep-
tide. The conjugated peptide was labeled with the [18F]AlF method
with high radiochemical efficiency. Biodistribution and microPET
imaging studies using tumor xenografted mice showed significant
according to a standard procedure. 1,4-bis(tert-butoxycarbonylm-
ethyl)-1,4,7-triazanonane (1) was synthesized according to previ-
ously reported method.16 All other chemicals were purchased
from Sigma/Aldrich (St. Louis, MO, U.S.A.). 1H and 13C NMR spectra
were recorded on 300-MHz, AL-300 FTNMR spectrometer JEOL (To-
kyo, Japan). Chemical shifts (d) were reported in ppm downfield
from tetramethylsilane and Coupling constants are reported in
hertz (Hz). The multiplicity is defined by s (singlet), d (doublet), t
(triplet), dd (doublet of doublets), br (broad), and m (multiplet).
1H and 13C NMR spectra were acquired in CDCl3 and reference to
residual CHCl3 at 7.26 and 77.00 ppm, respectively, or in D2O refer-
enced to residual DOH at 4.65 ppm. Electrospray ionization mass
spectra (ESI–MS) were acquired on a Waters ESI ion trap spectrom-
eter (Milford, U.S.A.) for both positive and negative ion detection.
The samples were diluted 1 to 100 with methanol and injected di-
rectly into the source. Data are reported in the form of (m/z) versus
uptakes of the labeled peptide in
avb3 integrin positive tumors.
Blocking experiments demonstrated
avb3 integrin specific uptake
of the peptide. Synthesis of active ligand for general use and its
application in labeling of peptides could facilitate further steps to-
wards clinical applications of various labeled tracers.
4. Experimental section
4.1. General
18F was produced by the medical cyclotron, CYCLONEÒ 18/9
(IBA, Louvain-la-Neuve, Belgium) by 18O (p, n) 18F nuclear reaction

D. Shetty et al. / Bioorg. Med. Chem. 20 (2012) 5941–5947
5945
Figure 3. Biodistribution study (% D/g) of [18F]7 directly injected and with co-injection of cold c(RGDyK) (3 mg/kg) in male BALB/c nude mice (n = 8).
Figure 4. (A) Decay-corrected whole-body coronal microPET images of BALB/c nude mice bearing U87MG tumor and (B) SUVs of tumors and normal tissues (left shoulder) at
1 and 2 h post injection of [18F]7 and [18F]7 with c(RGDyK) as a blocking agent (3 mg/kg body weight). Arrows indicate tumors.
intensity. High resolution mass spectra were obtained on a Jeol,
JMS-AX505WA, HP 5890 series II spectrometer using fast atomic
bombardment (FAB+) ionization detection.
The solvent systems used were; solvent A (0.05% TFA solution in
H2O) and solvent B (EtOH). The flow rates were 1 mL/min for ana-
lytical HPLC and 7 mL/min for preparative HPLC, at the indicated
linear gradients. Waters Sep-Pak Light Accell Plus QMA cartridge
(Milford, U.S.A.) was used to obtain purified [18FÀ] solution. The
gamma scintillation counter was a Packard Cobra II (GMI, MN,
Preparative HPLC purification of the compounds was performed
on X-terraÒ 10
l
m RP18 (19 Â 250 mm) column. Analytical HPLC
was performed on X-terraÒ 3.5
l
m RP18 (4.6 Â 100 mm) column.

5946
D. Shetty et al. / Bioorg. Med. Chem. 20 (2012) 5941–5947
U.S.A.). Radio-thin layer chromatography (TLC) was counted using
a Bio-Scan AR-2000 System imaging scanner (Bioscan, DC, U.S.A.).
Instant TLC-silica gel (ITLC–SG) plates were purchased from Varian
Inc. (Agilent Technologies, Wilmington, U.S.A.). The biodistribution
experiments were performed in Seoul National University Hospital,
Seoul, Korea, which is fully accredited by AAALAC International
(2007, Association for Assessment and Accreditation of Laboratory
Animal Care International).
J = 9 Hz), 7.14–7.11 (d, 2H, J = 9 Hz), 4.22 (s, 2H), 3.41 (s, 4H),
3.34–3.13 (m, 4H), 3.12–2.60 (m, 8H). 13C NMR (D2O, 75 MHz,
25 °C): d 174.2 (CO), 133.1, 132.7, 127.0, 60.1, 56.9, 50.9, 50.6,
49.2. ESI-MS: m/z = 393.1 for [M+H]+. HRMS (FAB+) m/z calcd for
C
18H24N4O4S [M+H]+ 393.1518, found 393.1524.
4.6. NODA-c(RGDyK) Conjugate (6)
A mixture of 5 (0.01 g, 0.026 mmol) and cRGDyK (0.016 g,
0.026 mmol) in 0.1 M sodium carbonate buffer (pH 9.5) was al-
lowed to react for 15 h at room temperature. After completion of
reaction, the reaction mixture was purified by RP-HPLC (100% A
for 5 min, 0 to 100% B for another 25 min). Product peak was eluted
at tR ꢀ9.6 min. Collected product fraction was lyophilized after
removing the EtOH to obtain product 6 as light yellow solid. 1H
NMR (D2O, 300 MHz, 25 °C): d 7.87–7.84 (d, 2H, J = 9 Hz), 7.66–
7.63 (d, 2H, J = 9 Hz), 7.34–7.31 (d, 2H, J = 6 Hz), 7.01–6.98 (br s,
2H), 5.10–4.98 (m, 2H), 4.73 (s, 4H), 4.44–4.40 (d, 2H, J = 12 Hz),
4.12–4.05 (br s, 2H), 3.89–2.95 (m, 24H), 2.45 (s, 2H), 2.32–1.12
(m, 16H). ESI-MS: m/z = 1012.5 for [M+H]+. HRMS (FAB+) m/z calcd
for C45H65N13O12S [M+H]+ 1012.4596, found 1012.1524.
4.2. 4,7-Bis(tert-butoxycarbonylmethyl)-1-(4-nitro-benzyl)-
1,4,7-triazanonane (2)
To a solution of 1 (0.2 g, 0.556 mmol) and K2CO3 (0.1 g,
0.556 mmol) in anhydrous acetonitrile (10 mL), 4-nitro benzyl bro-
mide (0.12 g, 0.556 mmol) in acetonitrile (2 mL) was added slowly.
Above reaction mixture was kept at room temperature with stir-
ring for 20 h and monitored through thin layer chromatography
(TLC, CH2Cl2/MeOH, 9/1, v/v). After completion, reaction mixture
was filtered and solvent was evaporated. Crude product was puri-
fied by flash column chromatography (CH2Cl2/MeOH). Product, 2
was eluted in 7% of methanol. 1H NMR (CDCl3, 300 MHz, 25 °C): d
8.11–8.08 (d, 2H, J = 9 Hz), 7.52–7.49 (d, 2H, J = 9 Hz), 3.25 (s,
4H), 3.74 (s, 2H), 2.90–2.70 (m, 12H), 1.40 (s, 18H). 13C NMR
(CDCl3, 75 MHz, 25 °C): d 171.3 (CO), 129.5, 123.5, 80.8, 61.7,
59.7, 55.5, 28.2. ESI-MS: m/z = 493.3 for [M+H]+.
4.7. Radiolabeling experiment
Stock solution of 2 mM AlCl3 was prepared by dissolving
AlCl3Á6H2O in 0.1 M sodium acetate buffer (pH 4). Sep-Pak Light
Accell Plus QMA cartridge was pre-conditioned by eluting with
0.4 M KHCO3 (5 mL), followed by water (10 mL). 18FÀ was loaded
to the cartridge by eluting aqueous 18F- solution produced by a
cyclotron and followed by washing with water (5 mL). The 18FÀ
loaded cartridge was eluted with saline (0.4 mL) to obtain 18FÀ
solution in saline. [18F]AlF was prepared by mixing 45 nmol stock
4.3. 4,7-Bis(carboxymethyl)-1-(4-nitro-benzyl)-1,4,7-
triazanonane (3)
Compound 2 (0.1 g, 0.204 mmol) was dissolved in 4 M HCl in
1,4-dioxane solution (8 mL) and kept for 24 h stirring at room tem-
perature. Completion of the reaction was monitored by ESI+ mass
analysis. After the completion solvent was evaporated to obtain 3
as hydrochloride salt. 1H NMR (D2O, 300 MHz, 25 °C): d 8.06–
8.03 (d, 2H, J = 9 Hz), 7.58–7.55 (d, 2H, J = 9 Hz), 4.37 (s, 2H), 3.58
(s, 4H), 3.50–3.30 (m, 6H), 3.15–2.90 (m, 6H). 13C NMR (D2O,
75 MHz, 25 °C): d 173.1 (CO), 149.0, 132.8, 124.8, 60.7, 56.7, 50.6,
49.8. ESI-MS: m/z = 381.3 for [M+H]+.
AlCl3 (22.5
370 MBq). After adjusting pH to 4 by adding glacial acetic acid
(4 L), the mixture was incubated at room temperature for
10 min. The prepared [18F]AlF solution (72.5
L) was added to solu-
l lL,
L) and the eluted 18F- saline solutions (50
l
l
tion of 6 (50 nmol) in 0.1 M sodium acetate buffer (1 mL). Reaction
mixture was heated to 110 °C on heating block for 10 min. The
labeled compound was either purified by HPLC or passing through
C-18 light cartridge to remove the unchelated [18F]AlF resulted to
obtain labeled peptide [18F]AlF-NODA-RGD (7). Labeling efficiency
was measured by ITLC–SG, eluted with 60% MeCN solution. Chro-
matography strips were monitored using a TLC scanner. Unlabeled
4.4. 4,7-Bis(carboxymethyl)-1-(4-amino-benzyl)-1,4,7-
triazanonane (4)
Compound 3 (0.06 g, 0.156 mmol) was dissolved in 2 mM KOH
solution in methanol (10 mL). Palladium on charcoal (Pd–C, 10%)
was introduced to above solution under N2. Above mixture was
stirred under H2 atmosphere for 8 h. Reaction mixture was filtered
through celite and solvent was evaporated. Crude product was re-
dissolved in water and purified by RP-HPLC (100% A for 5 min, 0 to
80% B for another 25 min). Eluted product peak (tR ꢀ15 min) was
evaporated completely to obtain product 4 as white solid. 1H
NMR (D2O, 300 MHz, 25 °C): d 7.26–7.24 (d, 2H, J = 6 Hz), 6.88–
6.85 (d, 2H, J = 9 Hz), 3.73 (s, 2H), 3.23 (s, 4H), 2.80–2.55 (m,
12H), 2.10 (s, 2H, –NH). 13C NMR (D2O, 75 MHz, 25 °C): d 180.4
(CO), 146.1, 131.8, 128.1, 122.7, 61.5, 60.5, 52.2, 51.2. ESI-MS: m/
z = 351.3 for [M+H]+.
[
18F]AlF remained at the origin and 7 moved to the solvent front.
Stabilities in prepared medium at room temperature and in human
serum at 37 °C were checked for 240 min. Extent of decomposition
was checked by ITLC eluted with the same system above.
4.8. Biodistribution in U87MG xenografted BALB/c nude mice
Each BALB/c nude mouse was injected subcutaneously with
2 Â 105/0.1 mL of U87MG (human glioma cells) cells in the right
shoulder. After 11 days, labeled peptide
[
18F]7 (0.148 MBq/
0.1 mL) was injected intravenously into each mouse through tail
vein. For blocking studies, cold c(RGDyK) was injected (3 mg/kg
body weight) along with labeled peptide. Mice were sacrificed
60 min post injection. Blood, muscle, bone and other organs were
separated immediately and weighed. Counts were obtained with
4.5. 4,7-Bis(carboxymethyl)-1-(4-isothiocyanato-benzyl)-1,4,7-
triazanonane (5)
c-scintillation counter. Percentages of injected dose per tissue
Thiophosgene (0.01 mL) was added dropwise into the mixture
of 4 (0.04 g, 0.112 mmol), CaCO3 (0.012 g, 0.172 mmol) and H2O
(3 mL) in CHCl3 (3 mL). The mixture was stirred overnight. After fil-
tration, solvent was removed under vacuum and purified by RP-
HPLC (100% A for 5 min, 0 to 100% B for another 25 min). Product
peak was eluted at tR ꢀ24 min. Collected fraction was evaporated
to yield 5. 1H NMR (D2O, 300 MHz, 25 °C): d 7.32–7.29 (d, 2H,
weight (% ID/g) were calculated. Statistical analyses were per-
formed by Student’s t-test.
4.9. PET imaging in U87MG xenografted BALB/c nude mice
BALB/c mice bearing glioma xenografts were injected with
[
18F]7 (11.1 MBq/0.1 mL) through a tail vein. For inhibition exper-

D. Shetty et al. / Bioorg. Med. Chem. 20 (2012) 5941–5947
5947
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