Facile synthesis and evaluation of C-functionalized benzyl-1-oxa-4,7,10- triazacyclododecane-N,N′,N″-triacetic acid as chelating agent for 111In-labeled polypeptides

Article abstract of DOI:10.1016/j.bmc.2011.11.046

A 12-membered polyazamacrocycle, 1-oxa-4,7,10-triazacyclododecane-N, N′,N″-triacetic acid (ODTA), has been reported to provide an indium chelate of net neutral charge with thermodynamic stability higher than 1,4,7,10-tetraazacyclododecane-N,N′,N″,N?-tetra

Full text of DOI:10.1016/j.bmc.2011.11.046

Bioorganic & Medicinal Chemistry 20 (2012) 978–984
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Facile synthesis and evaluation of C-functionalized
0
00
benzyl-1-oxa-4,7,10-triazacyclododecane-N,N ,N -triacetic acid
1
11
as chelating agent for
In-labeled polypeptides
Hiroyuki Suzuki, Ayaka Kanai, Tomoya Uehara, Francisco L. Guerra Gomez, Hirofumi Hanaoka,
⇑
Yasushi Arano
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
a r t i c l e i n f o
a b s t r a c t
0
00
Article history:
A 12-membered polyazamacrocycle, 1-oxa-4,7,10-triazacyclododecane-N,N ,N -triacetic acid (ODTA), has
Received 11 October 2011
Revised 19 November 2011
Accepted 21 November 2011
Available online 1 December 2011
been reported to provide an indium chelate of net neutral charge with thermodynamic stability higher than
0
00
000
1,4,7,10-tetraazacyclododecane-N,N ,N ,N -tetraacetic acid (DOTA). However, neither synthetic procedure
for a C-functionalized ODTA (C-ODTA) nor its chelating ability with a trace amount of radioactive indium-
1
11
1
11 ( In) has been elucidated. We herein present a facile synthetic procedure for C-ODTA, and estimated
1
11
its ability as a chelating agent for radiolabeling peptides and proteins with
In. The synthetic procedure
Keywords:
C-ODTA
C-DOTA
involves the synthesis of a linear precursor using a para-substituted phenylalanine derivative as a starting
material. The following intramolecular cyclization reaction was best performed (>73% yield) when Boc-
protected linear compound and the condensation reagent, HATU, were simultaneously added to the reac-
Cyclization
Polyazamacrocycle
3
tion vessel at the same flow rate. The cyclic compound was then reduced with BH and alkylated with tert-
111
In
butyl bromoacetate. The synthetic procedure was straightforward and some optimization would be
required. However, most of the intermediate compounds were obtained easily in good yields, suggesting
that the present synthetic procedure would be useful to synthesize C-ODTA derivatives. The intramolecular
cyclization reaction might also be applicable to synthesize polyazamacrocycles of different ring sizes and
cyclic peptides. In 1 In radiolabeling reactions, C-ODTA provided
11
111
In chelates in higher radiochemical
1
11
yields at low ligand concentrations when compared with C-DOTA. The
In-labeled C-ODTA remained
unchanged in the presence of apo-transferrin. The biodistribution studies also showed that the 1 In-
labeled compound was mainly excreted into urine as intact. These findings indicate that C-ODTA would
be useful to prepare 1 In-labeled peptides of high specific activities in high radiochemical yields.
Ó 2011 Elsevier Ltd. All rights reserved.
11
11
1
. Introduction
HO
OH
HO
O
O
O
Monoclonal antibodies and peptides have been employed as a
targeting molecule for the delivery of radionuclides to tumor cells
in molecular imaging and targeted radionuclide therapy. Since
antibodies and peptides do not form stable chelates with metallic
radionuclides, a variety of bifunctional chelating agents have been
developed to apply metallic radionuclides of appropriate nuclear
properties to the purposes. Among them, a 12-membered polyaza-
N
N
N
O
N
N
N
N
O O
O O
HO
OH
HO
OH
0
00
000
macrocycle, 1,4,7,10-tetraazacyclododecane-N,N ,N ,N -tetraacetic
Figure 1. Structures of DOTA (left) and ODTA (right).
acid (DOTA; Fig. 1) has been used as a chelating agent for labeling
polypeptides with indium-111 (1 In) for SPECT imaging, yttrium-
11
1
in DOTA is converted to an active ester.6–8 The other involves
C-functionalized DOTA (C-DOTA) where a polypeptide binding site
is introduced at a benzyl group extended from the backbone
However, both DOTA derivatives require
high concentration of DOTA-conjugated polypeptides, elevated
2,3
9
0 or lutetium-177 for targeted radionuclide therapy, and euro-
4,5
pium for fluorescent imaging. Two major DOTA derivatives have
been developed to introduce DOTA molecules to polypeptides. One
is a N-functionalized DOTA where one of the four carboxylic acids
9
–11
carbon of DOTA.
1
11
temperature or long reaction time to provide
In-labeled poly-
⇑
Corresponding author. Tel.: +81 43 226 2896; fax: +81 43 226 2897.
E-mail address: arano@p.chiba-u.ac.jp (Y. Arano).
peptides in high radiochemical yields, due to the extremely slow
complex formation rates of the chelating agent. Efforts have been
0
968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.046

H. Suzuki et al. / Bioorg. Med. Chem. 20 (2012) 978–984
979
made to develop a chelating agent that provides kinetically inert
vided 3 in high yields. The intermediate compound 5 was obtained
by deprotection of the Boc group of 3 and subsequent alkylation of
the resulting 4 with tert-butyl bromoacetate, since our attempts to
prepare 5 with tert-butyl bromomalonate resulted in lower yields.
In the cyclization reaction, we observed two key parameters that
significantly affected the synthetic yields. When the cyclization
reaction was conducted by an addition of a DMF solution of HATU
to a mixed solution of 8a, DIEA and HOAt in DMF, many side-prod-
ucts impaired the yields. However, when a DMF solution of 8a and
a DMF solution of HATU were simultaneously added to a mixed
solution of DIEA and HOAt in DMF, the reduction of side-products
gave improved yields in the cyclization reaction (>73%). This would
be attributable to rapid reaction rates of HATU-activated 8a and ra-
pid degradation rates of HATU in the presence of excess base.2
The protecting group of the secondary amine 8 also affected the
reaction yields. The best yield was achieved when the amine was
protected with Boc group. The linear compound without a protect-
ing group 8b or with trifluoroacetyl protecting group 8c resulted in
lower cyclization yields (data not shown). These results suggest
that the presence of a protecting group with an appropriate molec-
ular size would render the two reaction groups closer each other.
Following deprotection and subsequent reduction of the two
11–14
radiometal chelates in high radiochemical yields.
Although
DTPA derivatives provide 1 In chelate of high in vivo stability in
higher radiochemical yields than DOTA derivatives, 1 In-labeled
polypeptides exhibit long residence time of radioactivity in the li-
ver (for intact antibodies) or kidney (for polypeptides smaller than
11
11
1
5,16
albumin) when they are used as bifunctional chelating agents.
Contrary to DOTA and DTPA, a 12-membered polyazamacrocycle,
0
00
1
-oxa-4,7,10-triazacyclododecane-N,N ,N -triacetic acid (ODTA;
Fig. 1), provides an indium chelate of net neutral charge similar to
6
7
0
00
a
Ga chelate of C-functionalized 1,4,7-triazacyclononane-N,N ,N -
triacetic acid (NOTA) with thermodynamic stability higher than that
1
7
of In-DOTA. Such characteristics of ODTA were considered useful
to reduce the non-target radioactivity levels when recalling short
1,22
6
7
residence time of Ga-NOTA-conjugated methionine from the kid-
9
ney. However, neither the synthetic procedure for C-functionalized
ODTA (C-ODTA) nor its complexation ability with a trace amount of
1
11
In has been elucidated, which stimulated us to investigate the
synthetic procedure of C-ODTA and its complexation ability
1
11
with
In.
2
2
. Results and discussion
3
amide bonds in 9a with BH -THF, 11 was obtained in 26% yield.
When the reduction products were treated with aqueous HCl, the
ester bond was readily cleaved, which was contrary to the C-DOTA
derivatives so far reported. The use of methanol–HCl solution pro-
vided 11 at an expense of low synthetic yields. The C-ODTA deriv-
ative 12 was then obtained in 43% yield by the reaction of 11 with
tert-butyl bromoacetate. The present synthetic procedure was
.1. Synthesis of C-ODTA
Among a variety of cyclization methods so far reported,10,18–20
we employed an intramolecular cyclization procedure, as shown
in Schemes 1 and 2. The condensation reaction between N-Boc-
0
2
,2 -oxybis(ethylamine) 1 and N-Tfa-p-nitrophenylalanine 2 pro-
H
H N
NH2
H N
N
2
2
O
O
1
Boc
a
b
H
N
O N
2
O
O
c
Tfa
Tfa
NH
N
H
Boc
O N
O
OH
O N
O
OH
2
2
3
Tfa
NH2
N
H
d
2
H
N
H
N
O N
O
2
O N
2
O
O
O
O
Tfa
Tfa
NH
O
NH3Cl
e
N
H
N
H
4
5
f
H
N
H
N
O N
2
O
O N
O
O
2
O
O
O
O
NH Cl
Tfa
N
2
N
H
g
N
H
Boc
OH
OH
6
7
h
H
N
O N
2
O
O
N
NH2
Boc
OH
8
Scheme 1. Synthetic procedure for the linear precursor of C-ODTA. Reagents and conditions: (a) (Boc)
2%; (c) IBCF, NMM, THF, ꢀ15 °C for 30 min, rt for 1 h, 81%; (d) 4 M HCl/ethyl acetate, rt, 1 h, 98%; (e) tert-butyl bromoacetate, TEA, DMF, 0 °C to rt, 24 h, 53%; (f) 4 M HCl/ethyl
acetate, rt, 3 h, 93%; (g) (Boc) O, TEA, DMF, 0 °C to rt, 3 h, 79%; (h) 25% NH aq, rt, 6 h.
2 2
O, methanol, THF, 0 °C for 1 h, rt for 24 h, 89%; (b) (Tfa) O, TFA, rt, 15 h,
8
2
3

9
80
H. Suzuki et al. / Bioorg. Med. Chem. 20 (2012) 978–984
H
H
O N
2
O
N
O N
2
O
N
N
O
N
O
O
Tfa
N
a
N
H
R
H
R
OH
O
8
8
8
a: R=Boc
b: R=H
9a: R=Boc
9b: R=H
b
c: R=Tfa
9c: R=Tfa
H
H
H
H
O
N
N
O
N
N
O
N
O N
2
O N
2
c
N
H2Cl
H
O
1
11
0
d
O
HO
HO
O
O
N
O
N
O
N
O N
2
O N
2
e
N
N
N
O O
O O
13
O
O
OH
12
Scheme 2. Synthetic procedure for C-ODTA. Reagents and conditions: (a) HATU, HOAt, DIEA, DMF, rt, 20 h, 9a, 74%, 9b, N.D., 9c, 48%; (b) 4 M HCl/ethyl acetate, rt, 3 h, 85%; (c) (1)
M BH -THF, 0 °C for 1 h, reflux for 24 h, (2) HCl/methanol, reflux, 6 h, 26%; (d) tert-butyl bromoacetate, Na CO , acetonitrile, 0 °C to rt, 24 h, 43%; (e) 10% anisole/TFA, 6 h, rt, 42%.
1
3
2
3
straightforward and some optimization would be required. How-
ever, most of the intermediates were obtained in good yields, sug-
gesting that this procedure would be useful to prepare C-ODTA
derivatives. The cyclization reaction might be applicable to a syn-
thesis of C-functionalized polyazamacrocycles of different ring
sizes or cyclic peptides.
2
.2. Radiolabeling of C-ODTA and C-DOTA with 111In
A
C-ODTA provided two radioactive 111In peaks at retention times
of 11.1 (complex A) and 11.4 min (complex B), as shown in Figure
. Both complexes remained unchanged when the radioactive
2
peaks were isolated and re-analyzed by HPLC (data not shown).
Similar results were observed by the reaction of C-ODTA with a
stoichiometrically equivalent amount of non-radioactive indium.
Both complexes exhibited similar mass spectra, suggesting that
3
+
2+
both are stereoisomers as also observed with Y and Cu chelates
of C-DOTA.
2
3,24
0
2
4
6
8
10 12 14 16 18 20
Table 1 shows the radiochemical yields of 111In-C-ODTA and
In-C-DOTA when the reaction was performed in 1 or 10 lM
1
11
B
111
solution of C-DOTA or C-ODTA with
radiochemical yields of
InCl
In-C-ODTA were determined by combin-
ing the two complexes. As expected from higher stability constant
3
at 37 or 100 °C. The
1
11
1
7
111
of ODTA than DOTA for indium, C-ODTA provided
In com-
plexes in much higher radiochemical yield (>95%) than did C-DOTA
Table 1
Radiochemical yields of C-ODTA and C-DOTA
Radiochemical yields (%)
0
2
4
6
8
10 12 14 16 18 20
37 °C
100 °C
Retention time (min)
C-ODTA
C-DOTA
C-ODTA
C-DOTA
Figure 2. HPLC profiles of 11In-C-ODTA (A) and urine samples collected for 6 h
postinjection of 1 In-C-ODTA (B). 111In-C-ODTA exhibited two radioactivity peaks
at retention times of 11.1 min (complex A) and 11.4 min (complex B). When each
complex was separated by HPLC and injected to mice, both complex A and complex
B were excreted in the urine intact.
1
1
10
l
l
M
M
N.D.
57.5 ± 0.73
N.D.
75.9 ± 2.48
96.4 ± 1.60
N.D.
22.8 ± 7.99
96.1 ± 0.46
11
N.D.: not determined.
Results are expressed as means (SD) of five experiments each point.

H. Suzuki et al. / Bioorg. Med. Chem. 20 (2012) 978–984
981
Table 2
complex A (retention time of 11.1 min) and B (retention time of
1.4 min) exhibited rapid elimination rates from the blood with
Stability of 111In-C-ODTA and 111In-C-DOTA against apo-transferrin (aTf)
1
Time (h)
Percent of intact radiolabeled complex
aTf (0.1 mM) aTf (1.0 mM)
low radioactivity levels in any organs (Table 3). The majority of
the radioactivity was excreted in the urine (Table 3), indicating
their rapid urinary excretion. However, a small difference was ob-
C-ODTA
C-DOTA
C-ODTA
C-DOTA
1
11
served in the liver uptake;
In-C-ODTA complex B registered a
1
6
98.3 ± 0.70
98.5 ± 0.49
98.4 ± 0.19
98.0 ± 0.25
97.4 ± 1.05
98.4 ± 0.59
98.2 ± 0.32
98.1 ± 0.14
98.0 ± 0.21
97.8 ± 0.20
98.3 ± 0.24
98.7 ± 0.29
98.1 ± 0.42
98.8 ± 0.36
97.8 ± 0.43
98.5 ± 0.22
98.0 ± 0.19
98.0 ± 0.25
97.3 ± 0.19
95.7 ± 0.81
slightly higher initial radioactivity levels in the liver than those
111
of
In-C-ODTA complex A. This would be attributable to the dif-
2
7
4
2
1
11
ferent stereostructures of the two
prior studies
The analysis of urine samples collected for 6 h postinjection of
In-C-ODTA, as suggested from
and from their retention times on HPLC.
2
3,24
1
20
Results are expressed as means (SD) of five experiments each point.
1
11
each
In-C-ODTA exhibited a single HPLC profile at a retention
1
11
time identical to each
high in vivo stability of
environment.
In-C-ODTA (Fig. 2B). This reinforced
1
11
In-C-ODTA under physiological
(
<25%) when the reaction was conducted in 1 lM at 100 °C. How-
ever, both C-DOTA and C-ODTA showed poor radiochemical yields
under milder (37 °C) reaction conditions, suggesting that while
111
C-ODTA would possess complexation rate with
In superior to
3. Conclusion
C-DOTA, C-ODTA still requires harsh conditions not applicable to
heat-sensitive proteins.
A facile synthetic procedure for C-ODTA has been established.
Since the majority of synthetic intermediates were obtained in
high yields and purities, the present procedure may be useful to
a large-scale synthesis of C-ODTA. The cyclization reaction may
2
.3. Stability of 111In-C-ODTA and 111In-C-DOTA in apo-
transferrin solution
also be applicable to prepare polyazamacrocycles of other ring
The stability of 111In-C-ODTA and 111In-C-DOTA were compared
in the presence of apo-transferrin (aTf) after removing free C-ODTA
111
sizes and cyclic peptides. C-ODTA provided two net neutral
In
complexes in higher radiochemical yields than C-DOTA at an ele-
1
11
111
or C-DOTA from the
In complexes. As shown in Table 2, both
vated temperature. The stability of
to that of
In-C-ODTA was comparable
In-C-DOTA. The findings in this study indicate that
C-ODTA possesses an advantage over both DO3A and C-DOTA in
1
11
In-C-ODTA and 111In-C-DOTA remained intact (>95%) during
111
1
11
the experimental intervals, indicating that C-ODTA provides
In
1
11
chelate stable enough for in vivo applications.
preparing
In-labeled peptides of high specific activities without
post-labeling purification for molecular imaging. The net neutral
1
11
2
.4. Biodistribution and metabolic studies
charge of
In-C-ODTA may also be useful to decrease the radioac-
tivity levels in non-target tissues.
The two 111In-C-ODTA complexes were separated by HPLC, and
each complex was injected to normal mice. Both 1 In-C-ODTA
11
4
. Experimental
Table 3
Biodistribution of radioactivity after intravenous injection of 111In-C-ODTAa
4.1. General
Time after injection
1
Proton nuclear magnetic resonance ( H-NMR) spectra were re-
corded on a JEOL JNM-ALPHA 400 spectrometer (JEOL Ltd, Tokyo,
Japan). Fast atom bombardment mass spectra (FAB-MS) were ta-
ken on a JEOL JMS-AX500 mass spectrometer (JEOL Ltd, Tokyo).
Electrospray ionization mass spectrometry (ESI-MS) was carried
out using an Agilent 6130 Series Quadrupole LC/MS electrospray
system equipped with diode array detector (Agilent technologies,
Tokyo). Elemental analyses were performed by PE-2400 (Perkin-El-
mer Japan, Tokyo). The melting points were measured using a Tho-
mas Hoover capillary melting point apparatus and were reported
uncorrected. HPLC was performed with a Unison US-C18 column
(4.6 ꢁ 150 mm, Imtakt, Kyoto, Japan) at flow rate of 1 mL/min with
a gradient mobile phase starting from 100% A (0.1% aqueous triflu-
oroacetic acid (TFA)) and 0% B (methanol with 0.1% TFA) to 70% A
and 30% B at 20 min. The radiochemical yields were also deter-
1
h
6 h
24 h
Complex A
0.00 ± 0.00
0.09 ± 0.02
0.02 ± 0.01
0.80 ± 0.09
0.01 ± 0.01
0.01 ± 0.01
0.02 ± 0.01
0.03 ± 0.02
0.13 ± 0.12
0.90 ± 0.57
Blood
Liver
Spleen
Kidney
Pancreas
Heart
0.12 ± 0.03
0.22 ± 0.03
0.06 ± 0.01
3.01 ± 1.36
0.05 ± 0.02
0.07 ± 0.02
0.17 ± 0.02
0.12 ± 0.09
0.03 ± 0.01
1.07 ± 0.09
0.00 ± 0.00
0.04 ± 0.01
0.02 ± 0.02
0.30 ± 0.04
0.01 ± 0.01
0.01 ± 0.01
0.01 ± 0.01
0.02 ± 0.02
0.29 ± 0.24
2.09 ± 1.43
86.4 ± 2.29
1.71 ± 1.00
Lung
Bone
Stomachb
b
Intestine
b
Urine
Feces
b
Complex B
Blood
Liver
0.15 ± 0.10
1.42 ± 0.23
0.08 ± 0.03
4.11 ± 1.60
0.05 ± 0.03
0.07 ± 0.03
0.21 ± 0.08
0.15 ± 0.07
0.09 ± 0.05
2.40 ± 0.54
0.00 ± 0.00
0.60 ± 0.12
0.07 ± 0.02
1.33 ± 0.22
0.02 ± 0.01
0.01 ± 0.01
0.03 ± 0.01
0.02 ± 0.01
0.29 ± 0.35
2.76 ± 1.53
0.00 ± 0.00
0.28 ± 0.08
0.02 ± 0.02
0.32 ± 0.15
0.01 ± 0.01
0.01 ± 0.01
0.02 ± 0.01
0.01 ± 0.01
0.42 ± 0.26
1.59 ± 0.64
83.0 ± 6.25
3.76 ± 1.93
mined by TLC (Merck Silica gel 60) developed with a mixture of
Spleen
Kidney
Pancreas
Heart
Lung
111
methanol and water (3:7).
3
InCl (74 MBq/mL) was purchased
by Nihon Medi-Physics Co. Ltd (Tokyo). Chemicals purchased from
commercial sources were of reagent grade or higher and used
without further purification.
Bone
Stomach
b
0
4
.2. N-(tert-Butyloxycarbonyl)-2,2 -oxybis(ethylamine) (1)
b
Intestine
b
Urine
Fecesb
This compound was synthesized according to the procedure de-
scribed previously with slight modifications. To a cooled (0 °C)
solution of 2,2 -oxybis(ethylamine) (10.0 mL, 94.1 mmol) in meth-
anol (1000 mL) was added dropwise a solution of di-tert-butyl
2
5
Results are expressed as means (SD) of five animals each point.
Tissue radioactivity is expressed as percent of injected dose per gram of wet
tissue.
0
a
b
Expressed as percent of injected dose per tissue.
2
dicarbonate ((Boc) O; 10.3 g, 47.0 mmol) in THF (300 mL), and

9
82
H. Suzuki et al. / Bioorg. Med. Chem. 20 (2012) 978–984
the mixture was stirred for 1 h at the same temperature and addi-
tional 24 h at room temperature. After removing the solvent in
vacuo, the residue was dissolved in 1 N NaOH (200 mL), and ex-
tracted with chloroform (300 mL ꢁ 3). The organic phases were
4.6. tert-Butyl 12-Trifluoroacetyl-11-(4-nitrobenzyl)-10-oxo-
3,9,12-triaza-6-oxadodecanate (5)
To a chilled (0 °C) suspension of 4 (5.63 g, 13.1 mmol) and tri-
ethylamine (TEA; 2.75 mL, 19.7 mmol) in DMF (30 mL) was added
dropwise tert-butyl bromoacetate (1.93 mL, 13.1 mmol). After the
addition, the mixture was stirred for 24 h at room temperature.
The solvent was removed in vacuo, and the residue was dissolved
in ethyl acetate (50 mL). The organic phase was washed with 5%
4
combined, dried over anhydrous MgSO . The solvent was removed
1
in vacuo to provide 1 as a colorless oil (6.88 g, 71.5%). H NMR
CDCl ): d 1.39 (9H, s, Boc), 2.79–2.82 (2H, t, CH ), 3.24–3.28 (2H,
dd, CH ), 3.41–3.43 (2H, t, CH ), 3.44–3.47 (2H, t, CH ), 5.00 (1H,
d, NH). ESI-MS (M+H) : m/z 205, found: 205.
(
3
2
2
2
2
+
NaHCO
3
(20 mL) and saturated NaCl solution (5 mL) successively,
SO . After removing the solvent in
4
.3. N-Trifluoroacetyl-4-nitrophenylalanine (2)
and dried over anhydrous Na
2
4
vacuo, the residue was subjected to column chromatography on
silica gel using a mixture of chloroform/methanol (60:1) as an elu-
ent to provide 5 as a pale yellow solid (3.53 g, 53.1%) melting at
Toa solutionof 4-nitro-
50 mL) was added dropwise a mixture of trifluoroacetic anhydride
(Tfa) O; 8.9 mL, 63.0 mmol) and TFA (20 mL) for 2 h with stirring at
0 °C. The mixture was stirred for additional 15 h at room tempera-
ture. The solvent was removed in vacuo, and the residue was azeotr-
oped with toluene in vacuo. The residue was dissolved in 5% NaHCO
L-phenylalanine (5.26 g,25.0 mmol)inTFA
(
(
6
1
t
2
55–58 °C. H NMR (CDCl
CH ), 3.13–3.18, 3.30–3.36 (2H, dq, CH
lapped, CH ), 4.85–4.87 (1H, dd, CH), 7.38–7.40 (2H, d, aromatic),
7.55 (1H, s, NH), 8.12–8.14 (2H, d, aromatic). ESI-MS (M+H) : m/z
507, found: 507. Anal. Calcd for C21 O: C, 48.09; H,
ꢂH
5.96; N, 10.68. Found: C, 48.06; H, 5.74; N, 10.44.
3
): d 1.42 (9H, s, Bu), 2.85–2.88 (2H, m,
2
2
), 3.41–3.58 (8H, over-
2
+
3
(
100 mL), and washed with chloroform (50 mL ꢁ 3). The aqueous
H
29
N
4
O
7
F
3
2
phase was then acidified with 10% citric acid (100 mL), and extracted
with ethyl acetate (300 mL ꢁ 3). The organic phases were combined,
dried overanhydrous MgSO
4
. After removing the solvent invacuo, the
4.7. 12-Trifluoroacetyl-11-(4-nitrobenzyl)-10-oxo-3,9,12-triaza-
6-oxadodecanoic acid hydrochloride (6)
resulting pale yellow solid was collected, washed with hexane and
dried in vacuo to provide 2 (6.27 g, 81.7%) melting at 139–140 °C.
1
H NMR (CDCl
3
): d 3.27–3.31, 3.42–3.48 (2H, dq, CH
2
), 4.93–4.96
Compound 5 (3.43 g, 6.77 mmol) was dissolved in 4 M HCl/
ethyl acetate (30 mL), and the mixture was stirred for 3 h. After
removing the solvent in vacuo, the resulting white solid was col-
lected, and washed with diethyl ether, and then dried in vacuo to
(
1H, dd, CH), 6.77–6.78 (1H, d, NH), 7.30–7.33 (2H, d, aromatic),
ꢀ
8
.17–8.19 (2H, d, aromatic). ESI-MS (MꢀH) : m/z 305, found: 305.
Anal. Calcd for C11
C, 42.57; H, 2.89; N, 8.80.
9 2 5 3 2
H N O F ꢂ0.2H O: C, 42.65; H, 3.06; N, 9.04. Found:
provide 6 as a white solid (3.07 g, 93.0%) melting at 188–189 °C.
1
2 2
H NMR (D O): d 3.03–3.36 (10H, overlapped, CH ), 3.51–3.55
4
.4. 1-(tert-Butyloxycarbonyl)-10-trifluoroacetyl-9-(4-nitro-
(2H, m, CH
2
), 4.51–4.55 (1H, t, CH), 7.31–7.33 (2H, d, aromatic),
ꢀ
benzyl)-8-oxo-1,7,10-triaza-4-oxadecane (3)
8.03–8.06 (2H, d, aromatic). ESI-MS (MꢀH) : m/z 449, found:
4
49. Anal. Calcd for C17
21
H N
4
O
7
F
3
ꢂHCl: C, 41.94; H, 4.55; N,
To a chilled solution (ꢀ15 °C) of 2 (1.41 g, 4.61 mmol) in THF
11.51. Found: C, 41.88; H, 4.58; N, 11.28.
(20 mL) was added N-methylmorpholine(NMM; 0.51 mL, 4.61 mmol)
and subsequently isobutylchloroformate (IBCF; 0.61 mL, 4.61 mmol)
under N atmosphere. After 5 min, a solution of 1 (0.857 g, 4.19 mmol)
4.8. 3-(tert-Butyloxycarbonyl)-12-trifluoroacetyl-11-(4-nitro-
benzyl)-10-oxo-3,9,12-triaza-6-oxadodecanoic acid (7)
2
in THF (10 mL) was added dropwise at the same temperature, and the
mixture was stirred for 30 min at the same temperature and then at
room temperature for 1 h. After removing the solvent invacuo, the res-
idue was dissolved in ethyl acetate (50 mL). The organic phase was
To a chilled (0 °C) solution of 6 (3.07 g, 6.31 mmol) and TEA
(1.32 mL, 9.46 mmol) in DMF (20 mL) was added dropwise a solu-
tion of (Boc) O (1.65 g, 7.56 mmol) in DMF (10 mL). After the addi-
2
washed with 5% NaHCO
rated NaCl solution (30 mL ꢁ 1), successively and dried over anhy-
drous MgSO . After removal of the solvent in vacuo, the residue was
3
(30 mL ꢁ 2), 5% citric acid (30 mL ꢁ 3), satu-
tion, the mixture was stirred for 3 h at room temperature. The
solventwasremovedinvacuo, andtheresiduewasdissolvedinethyl
acetate (40 mL). The organic phase was washed with 5% citric acid
4
subjected to flash chromatography on silica gel using a mixture of
(20 mL ꢁ 3), and dried over anhydrous MgSO
4
. After removing the
solvent in vacuo, the residue was subjected to column chromatogra-
phyonsilicagelusinga mixtureof chloroform/methanol(20:1)asan
chloroform/methanol (100:1) as an eluent to provide 3as apale yellow
1
solid (1.67 g, 80.6%) melting at 128–129 °C. H NMR (CDCl
3
): d 1.44
(
9H, s, Boc), 3.17–3.51 (10H, overlapped, CH
2
), 4.76–4.78 (1H, dd,
eluent to provide 7 as a pale yellow solid (2.73 g, 78.6%) melting at
1
CH), 6.89 (1H, d, NH), 7.35–7.37 (2H, d, aromatic), 8.14–8.16 (2H, d, aro-
matic). ESI-MS (M+Na) : m/z 515, found: 515. Anal. Calcd for
3
70–71 °C. H NMR (CDCl ): d 1.45 (9H, s, Boc), 3.11–3.73 (10H, over-
+
lapped, CH
2
), 3.81–3.87, 4.09–4.16 (2H, dq, CH ), 5.06–5.18 (1H, m,
2
C
20
H
27
N
4 7 3
O F : C, 48.78; H, 5.53; N, 11.38. Found: C, 48.46; H, 5.30;
CH), 7.39–7.42 (2H, d, aromatic), 7.69–7.71, 7.91–7.93 (1H, dd, NH),
8.13–8.15 (2H, d, aromatic). ESI-MS (M+Na) : m/z 573, found: 573.
+
N, 11.21.
Anal. Calcd for C22
Found: C, 47.79; H, 5.43; N, 9.74.
H
29
N
4
O
9
F
3
ꢂ0.2H
2
O: C, 47.69; H, 5.35; N, 10.11.
4
.5. 10-Trifluoroacetyl-9-(4-nitrobenzyl)-8-oxo-1,7,10-triaza-4-
oxadecane hydrochloride (4)
4
.9. 10-(tert-Butyloxycarbonyl)-5,8-dioxo-6-(4-nitrobenzyl)-1-
Compound 3 (1.52 g, 3.09 mmol) was dissolved in 4 M HCl/ethyl
acetate (15 mL), and the mixture was stirred for 1 h at room temper-
ature. After removing the solvent in vacuo, the resulting white solid
wascollectedandwashedwithdiethylether, andthendriedinvacuo
oxa-4,7,10-triaza-cyclododecane (9a)
Compound 7 (2.12 g, 3.85 mmol) was dissolved in 25% NH
3
aqueous solution (20 mL). After the mixture was stirred for 6 h at
room temperature, the solvent was removed in vacuo. The residue
(8a) was dissolved in DMF (35 mL) to prepare solution ‘A’. O-(7-
1
toprovide4asa whitesolid(1.30 g, 98.4%)meltingat211–212 °C. H
NMR (D
CH ), 3.47–3.49 (2H, t, CH
d, aromatic), 8.04–8.06 (2H, d, aromatic). ESI-MS (M+H) : m/z 393,
found: 393. Anal. Calcd for C15
ꢂ1.4HCl: C, 40.63; H, 4.64;
N, 12.64. Found: C, 40.80; H, 4.52; N, 12.62.
2 2
O): d 2.96–2.99 (2H, t, CH ), 3.07–3.36 (6H, overlapped,
0
0
2
2
), 4.51–4.55 (1H, t, CH), 7.31–7.34 (2H,
Azabenzotriazol-1-yl)-N,N,N ,N -tetramethyluronium hexafluoro-
phosphate (HATU; 2.20 g, 5.78 mmol) was dissolved in DMF
(35 mL) to prepare solution ‘B’. Both the solution ‘A’ and ‘B’ were
loaded into gas-tight syringes, and the two syringes were locked
+
20 4 5 3
H N O F

H. Suzuki et al. / Bioorg. Med. Chem. 20 (2012) 978–984
983
onto syringe pump. To a mixed solution of N,N-diisopropylethyl-
amine (DIEA; 2.68 mL, 15.4 mmol) and HOAt (786 mg, 5.78 mmol)
in DMF (1.5 L) was added each solution ‘A’ and ‘B’ simultaneously
at a flow rate of 1.2 mL/h with stirring. After the addition, the mix-
ture was stirred for another 20 h at room temperature. After
removing the solution in vacuo, the residue was dissolved in ethyl
acetate (100 mL) and washed three times with a mixture of 5%
After the addition, the mixture was stirred for 24 h at room temper-
ature under the same conditions. The reaction mixture was filtered,
and the filtrate was evaporated in vacuo. The residue was dissolved
in a mixture of saturated NaHCO
NaCl solution (4 mL), and extracted with chloroform (20 mL ꢁ 3).
The combined organic phases were dried over anhydrous MgSO
3
solution (10 mL) and saturated
4
,
and the solvent was removed in vacuo. The residue was subjected
to flash chromatography on silica gel using a mixture of chloro-
NaHCO
over anhydrous MgSO
3
(40 mL) and saturated NaCl solution (10 mL), and dried
. The solvent was removed in vacuo, and
4
form/methanol/25% NH
3
aqueous solution (120:10:1) as an eluent
1
the residue was subjected to column chromatography on silica
gel using a mixture of chloroform/methanol (20:1) as an eluent
to provide 12 as a red-brown oil (165 mg, 42.5%). H NMR (CDCl
3
):
d 1.36 (9H, s, Bu), 1.40 (9H, s, Bu), 1.44 (9H, s, Bu), 2.27–3.59
(23H, overlapped, CH, CH ), 7.42–7.44 (2H, d, aromatic), 8.08–8.10
(2H, d, aromatic). ESI-MS (M+H) : m/z 651, found: 651. Anal. Calcd
for C33 : C, 60.90; H, 8.36; N, 8.61. Found: C, 60.54; H, 8.50;
N, 8.22.
t
t
t
to provide 9a as a white solid (1.24 g, 73.5%) melting at 102–
2
1
+
1
03 °C. H NMR (CDCl
3
): d 1.41 (9H, s, Boc), 2.98–3.65 (10H, over-
), 3.89–3.94 (1H, m, CH ), 4.39–4.43 (1H, d, CH ), 4.57–
.63 (1H, dd, CH), 6.61 (1H, m, NH), 7.41–7.43 (2H, d, aromatic),
.73–7.75 (1H, d, NH), 8.14–8.16 (2H, d, aromatic). ESI-MS
lapped, CH
2
2
2
54 4 9
H N O
4
7
(
+
M+Na) : m/z 459, found: 459. Anal. Calcd for C20
H
28
N
4
O: C,
4.13. 6-(4-Nitrobenzyl)-1-oxa-4,7,10-triazacyclododecane-
0
00
5
5.04; H, 6.47; N, 12.84. Found: C, 54.71; H, 6.56; N, 12.50.
N,N ,N -triacetic acid (13) (C-ODTA)
4
.10. 5,8-Dioxo-6-(4-nitrobenzyl)-1-oxa-4,7,10-triazacyclodode-
Compound 12 (54.7 mg, 0.253 mmol) was dissolved in a mix-
ture of TFA (1.8 mL) and anisole (0.2 mL), and the solution was
stirred for 6 h at room temperature. The solvent was concen-
trated in vacuo, and diethyl ether (3 mL) was added. The precip-
cane (10)
Compound 9a (1.23 g, 2.83 mmol) was dissolved in 4 M HCl/ethyl
acetate (10 mL), and the solution was stirred for 3 h at room temper-
ature before the solvent was removed in vacuo. The resulting white
solid was dissolved in water (10 mL), and 1 N NaOH was added to
bring the pH of the solution to 11. The aqueous solution was ex-
tracted with ethyl acetate (80 mL ꢁ 3), and the combined organic
itate was collected and dried in vacuo to provide C-ODTA as a
1
pale yellow solid (28.9 mg, 41.7%). H NMR (D
2
O): 2.64–4.07
(23H, overlapped, CH, CH
8.09 (2H, d, aromatic). ESI-MS (M+H) : m/z 483, found: 483. Anal.
Calcd for C21 COOH: C, 40.71; H, 4.78; N,
2
), 7.36–7.38 (2H, d, aromatic), 8.07–
+
H
30
N
4
O
9
ꢂ1.5H
2
Oꢂ2CF
3
phases were dried over anhydrous MgSO
4
. After removing the sol-
7.60. Found: C, 40.81; H, 4.58; N, 7.66.
vent in vacuo, the resulting white solid was collected and washed
with diethyl ether, and then dried in vacuo to provide 10 (899 mg,
4.14. 111In radiolabeling of C-ODTA and C-DOTA
1
8
2
5.2%) melting at 231–232 °C. H NMR (DMSO): d 2.58 (2H, s, CH
2
),
), 3.07–3.31 (6H, over-
), 4.35–4.40 (1H, m, CH), 7.50–
.69–2.73 (1H, d, CH
2
), 2.95–3.01 (1H, dd, CH
), 3.33–3.53 (2H, m, CH
2
To a 1 M acetate buffer (pH 4.5, 15
(10 L). After the mixture was allowed to stand for 5 min at room
temperature, a solution of C-ODTA or C-DOTA in 0.1 M acetate buf-
fer (pH 4.5, 25 L) was added. The final chelate concentrations
were 10 M. The mixture was incubated for 1 h at 37 °C.
In radiolabeling of C-ODTA and C-DOTA was also conducted
l
L) was added 111InCl
3
lapped, CH
2
2
l
7
.52 (2H, d, aromatic), 7.65–7.68 (1H, dd, NH), 8.13–8.15 (2H, d, aro-
+
matic), 8.32–8.34 (1H, d, NH). ESI-MS (M+H) : m/z 337, found: 337.
Anal. Calcd for C15 O: C, 52.86; H, 6.06; N, 16.44.
ꢂ0.25H
Found: C, 53.25; H, 6.08; N, 16.05.
l
H
20
N
4
O
5
F
3
2
l
1
11
at 100 °C for 10 min under similar reaction conditions as stated
4
.11. 6-(4-Nitrobenzyl)-1-oxa-4,7,10-triazacyclododecane (11)
above except for using 0.1 or 1 M acetate buffer (pH 3.0) with
the final chelate concentration of 1 or 10 lM.
To a chilled (0 °C) suspension of 10 (870 mg, 2.33 mmol) in THF
(
3
10 mL) was added BH -THF (1 M, 29.5 mL, 29.5 mmol) under N
2
4.15. Stability assessments of 111In-C-ODTA and 111In-C-DOTA
atmosphere. The mixture was stirred for 1 h at 0 °C, and then
heated to reflux for 24 h. The solution was cooled to 0 °C, quenched
with methanol (15 mL) for 1 h, and then evaporated to dryness.
The residue was dissolved in methanol (30 mL) again, and the sol-
vent was evaporated. The residue was dissolved in methanol satu-
rated with HCl gas (5 mL). The solution was refluxed for 6 h. After
the solution was cooled to 0 °C, 1 N NaOH (30 mL) was added, and
extracted with chloroform (30 mL ꢁ 3). The combined organic
111In-C-ODTA and 111In-C-DOTA were prepared as described
above with the final chelate concentration of 10
l
M. After incuba-
1
11
tion for 10 min at 100 °C, the
In-labeled compounds were sub-
jected to HPLC purification to remove free chelates. The
radioactive fractions were collected and evaporated to dryness.
The radioactive residue dissolved in 0.1 M sodium bicarbonate buf-
fer (pH 7.4, 15
(30.0 mg/210 L or 3.0 mg/210
buffer (pH 7.4). The mixture was incubated at 37 °C. After incuba-
tion for 1, 6, 24, 72, 120 h, a 5–10 L aliquot of each sample was
lL) was added to a solution of apo-transferrin
phases were dried over anhydrous MgSO
4
, and the solvent was re-
l
lL) in 0.1 M sodium bicarbonate
moved in vacuo before the residue was subjected to flash chroma-
tography on silica gel using a mixture of chloroform/methanol/25%
l
NH
brown oil (184 mg, 25.5%). H NMR (CDCl
overlapped, CH, CH ), 3.46–3.52 (2H, m, CH ), 3.56–3.63 (2H, over-
), 7.31–7.33 (2H, d, aromatic), 8.11–8.13 (2H, d,
3
aqueous solution (12:4:1) as an eluent to provide 11 as a
drawn, and the % of intact complex were determined by TLC.
1
3
): d 2.39–3.02 (11H,
2
2
4.16. In vivo studies
lapped, CH
aromatic).
2
Animal studies were conducted in accordance with our institu-
tional guidelines and were approved by Chiba University Animal
1
11
4
.12. Tris(tert-butyl) 6-(4-nitrobenzyl)-1-oxa-4,7,10-triazacy-
Care Committee.
In-C-ODTA was prepared as described above,
0
00
clododecane-N,N ,N -triacetate (12)
and subjected to HPLC purification to separate the two peaks. Bio-
distribution studies were performed by intravenous administra-
tion of a 0.1 mL of phosphate-buffered saline (1 mM, pH 7.4)
To a chilled suspension (0 °C) of 11 (184 mg, 0.595 mmol) and
Na
2
CO
3
(189 mg, 1.79 mmol) in acetonitrile (5 mL) was added tert-
solution of each radioactive fraction (0.3
lCi/100 lL) to 6-week-
butyl bromoacetate (262
l
L, 1.79 mmol) under N atmosphere.
2
old male ddY mice (Japan SLC Inc., Shizuoka, Japan). Groups of five

9
84
H. Suzuki et al. / Bioorg. Med. Chem. 20 (2012) 978–984
mice were used for the experiments. Organs of interest were re-
moved, weighed, and the radioactivity counts were determined
6. Mier, W.; Hoffend, J.; Krämer, S.; Schuhmacher, J.; Hull, W. E.; Eisenhut, M.;
Haberkorn, U. Bioconjugate Chem. 2005, 16, 237.
7.
Mukai, T.; Namba, S.; Arano, Y.; Ono, M.; Fujioka, Y.; Uehara, T.; Ogawa, K.;
with an auto-well
c counter at 1, 6, and 24 h post-injection. To
Konishi, J.; Saji, H. J. Pharm. Pharmacol. 2002, 54, 1073.
8. Anelli, P. L.; Lattuada, L.; Gabellini, M.; Recanati, P. Bioconjugate Chem. 2001, 12,
determine the amounts and routes of excretion from the body,
mice were housed in metabolic cages after injection of 1 In-C-
11
1081.
9.
Wu, C.; Jagoda, E.; Brechbiel, M.; Webber, K. O.; Pastan, I.; Gansow, O.;
ODTA (0.3 lCi/100 lL). Urine and feces were collected for 24 h
Eckelman, W. C. Bioconjugate Chem. 1997, 8, 365.
postinjection, and the radioactivity counts were determined. Mice
10. Renn, O.; Meares, C. F. Bioconjugate Chem. 1992, 3, 563.
11. Chappell, L. L.; Ma, D.; Milenic, D. E.; Garmestani, K.; Venditto, V.; Beitzel, M. P.;
Brechbiel, M. W. Nucl. Med. Biol. 2003, 30, 581.
were also housed in metabolic cages after injection of 111In-C-
ODTA (5
jection, and were analyzed by HPLC after filtration through a
0 kDa cutoff ultrafiltration membrane (Microcon-10, Millipore).
lCi/100 lL). Urine samples were collected for 6 h postin-
1
1
1
1
1
2. Chong, H. S.; Garmestani, K.; Ma, D.; Milenic, D. E.; Overstreet, T.; Brechbiel, M.
W. J. Med. Chem. 2002, 45, 3458.
3. Chong, H. S.; Milenic, D. E.; Garmestani, K.; Brady, E. D.; Arora, H.; Pfiester, C.;
Brechbiel, M. W. Nucl. Med. Biol. 2006, 33, 459.
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Melichar, F. Appl. Radiat. Isot. 2009, 67, 21.
5. Tsai, S. W.; Li, L.; Williams, L. E.; Anderson, A. L.; Raubitschek, A. A.; Shively, J. E.
Bioconjugate Chem. 2001, 12, 264.
6. Camera, L.; Kinuya, S.; Garmestani, K.; Brechbiel, M. W.; Wu, C.; Pai, L. H.;
McMurry, T. J.; Gansow, O. A.; Pastan, I.; Paik, C. H., et al Eur. J. Nucl. Med. 1994,
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1
Acknowledgments
This work was supported in part by a Grant-in-Aid for Technol-
ogy Development Program for Advanced Measurement and Analy-
sis from Japan Science and Technology Agency and Special Funds
for Education and Research (Development of SPECT Probes for
Pharmaceutical Innovation) from the Ministry of Education, Cul-
ture, Sports, Science and Technology, Japan. This work was dedi-
cated to our distinguished colleague, the late Mr. Yuichiro Taira.
1
1
7. Delgado, R.; Quintino, S.; Teixeira, M.; Zhang, A. J. Chem. Soc., Dalton Trans.
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20. Mishra, A. K.; Gestin, J. F.; Benoist, E.; Faivre-Chauvet, A.; Chatal, J. F. New J.
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