The synthesis of a 99mTc-labeled tetravalent targeting probe upon isonitrile coordination to 99mTcI for enhanced target uptake in saturable systems

Article abstract of DOI:10.1039/c9ra04311j

The presence of excess unlabeled ligands in the injectate hinders the target uptake of ^99mTc-labeled targeting vectors. To address the issue, we previously developed a chemical design which provides a ^99mTc-labeled trivalent RGD probe upon CN-βAla-Gly-Gly-c(RGDfK) (L_β) coordination to [^99mTc][Tc(CO)₃]⁺ core at pH 6.0. In this study, we extended our coordination mediated synthesis of the trivalent RGD probe to that of a tetravalent one. Our initial attempts reacting L_β with [^99mTc][Tc(CO)₃]⁺ core at pH 8.0 failed to provide [^99mTc][Tc(CO)₂(L_β)₄]⁺ due to the formation of multiple side products. A γ-aminobutylic acid (GABA) based isonitrile ligand CN-GABA-Gly-Gly-c(RGDfK) (L_G), on the other hand, avoided the side reaction and selectively provided [^99mTc][Tc(CO)₂(L_G)₄]⁺ (^99mTc-[L_G]₄) at pH 8.0. ^99mTc-[L_G]₄ exhibited higher binding affinity to integrin α_vβ₃ than its unlabeled ligand, and visualized U87MG tumor without tedious post-labeling purification. These results indicate that the metal coordination-mediated syntheses of ^99mTc-labeled multivalent probes have been successfully applied to a tetravalent one, which would allow a wider range of choices for designing novel ^99mTc-labeled multivalent probes of high in vivo target uptake.

Full text of DOI:10.1039/c9ra04311j

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The synthesis of a 99mTc-labeled tetravalent
targeting probe upon isonitrile coordination to
99mTcI for enhanced target uptake in saturable
systems†
Cite this: RSC Adv., 2019, 9, 26126
ab
a
b
Chun-wei Jen,a Hiromichi Akizawa
*
Yuki Mizuno,
Tomoya Uehara,
a
and Yasushi Arano
The presence of excess unlabeled ligands in the injectate hinders the target uptake of 99mTc-labeled
targeting vectors. To address the issue, we previously developed a chemical design which provides
a
99mTc-labeled trivalent RGD probe upon CN-bAla-Gly-Gly-c(RGDfK) (Lb) coordination to [99mTc]
[Tc(CO)3]+ core at pH 6.0. In this study, we extended our coordination mediated synthesis of the
trivalent RGD probe to that of a tetravalent one. Our initial attempts reacting Lb with [99mTc][Tc(CO)3]+
core at pH 8.0 failed to provide [99mTc][Tc(CO)2(Lb)4]+ due to the formation of multiple side products. A
g-aminobutylic acid (GABA) based isonitrile ligand CN-GABA-Gly-Gly-c(RGDfK) (LG), on the other hand,
avoided the side reaction and selectively provided [99mTc][Tc(CO)2(LG)4]+ 99mTc-[LG]4) at pH 8.0. 99mTc-
(
[LG]4 exhibited higher binding affinity to integrin avb3 than its unlabeled ligand, and visualized U87MG
tumor without tedious post-labeling purification. These results indicate that the metal coordination-
mediated syntheses of 99mTc-labeled multivalent probes have been successfully applied to a tetravalent
one, which would allow a wider range of choices for designing novel 99mTc-labeled multivalent probes
of high in vivo target uptake.
Received 8th June 2019
Accepted 11th August 2019
DOI: 10.1039/c9ra04311j
rsc.li/rsc-advances
designed chelator-vector conjugates (hereaer referred as
“ligands”) should be employed for radiolabeling reactions
(Fig. 1a).7 Owing to the extremely low concentration of metallic
Introduction
Medical applications of metallic radionuclides have been
rapidly expanding due to their unique roles in clinical nuclear
medicine.1–5 Metallic radionuclides emitting g photon or b+
particles are used for clinical nuclear imaging based on single
photon emission computed tomography (SPECT) or positron
emission tomography (PET), respectively, whereas ones emit-
ting bꢀ or a particles are used for internal radiotherapy of
malignant tumors. To achieve specic delivery of metallic
radionuclides to target tissues such as cancer, peptides or
antibody fragments which have high affinity to target molecules
are oen used as a targeting vector.6 Since these molecules
cannot form stable complexes with metallic radionuclides, they
need to be modied with proper chelating agents, and carefully
radionuclides (for example, 99mTc < 1 mM (ref. 8)), however,
these complexation reactions need to be performed with excess
ligands (z100 mM) to ensure high radiochemical yields in
a short reaction time. As a result, the reaction solutions would
contain not just the objective radiolabeled compounds but also
excess unlabeled ligands (Fig. 1a). Upon the injection of these
solutions into subjects, the target uptakes of the radiolabeled
compounds would be competitively inhibited by the excess
unlabeled ligands due to their similar binding affinities to
target molecules (Fig. 1a).9
To circumvent the issue, our group has developed a novel
chemical design concept of metal coordination mediated mul-
tivalency using the combination of [99mTc][TcI(CO)3]+ core and
isonitrile ligands (Fig. 1b).10,11 To test the validity of this design
concept, we selected c(RGDfK) peptides as a model targeting
vector, which has high affinity and specicity to integrin avb3.12
aLaboratory of Molecular Imaging and Radiotherapy, Graduate School of
Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba
260-8675, Japan
bLaboratory of Physical Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-
Tamagawagakuen, Machida, Tokyo 194-8543, Japan. E-mail: aki@ac.shoyaku.ac.jp
† Electronic supplementary information (ESI) available: HPLC methods, synthesis
scheme of the compounds 8a, 8b, 16, stability of M-½Lb0ꢁ3 (M ¼ 185/187Re and 99mTc)
at pH 6.0, in vitro stability of 99mTc-[LG]4, competitive inhibition curves of the
tested compounds, MS, 1H-NMR, 13C-NMR, IR, and/or purity check of key
compounds. See DOI: 10.1039/c9ra04311j
[
99mTc][TcI(CO)3(OH2)3]+ was rstly prepared as a labeling
precursor, whose water molecules were then substituted by CN-
bAla-Gly-Gly-c(RGDfK) (Fig. 2, Lb), a monovalent RGD ligand, at
slightly acidic conditions (pH 6.0), providing [99mTc][TcI(CO)3(-
Lb)3]+ (Fig. 2, 99mTc-[Lb]3), a 99mTc-labeled trivalent RGD probe.
99mTc-[Lb]3 exhibited higher binding affinity to integrin avb3
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Fig. 1 Schematic illustration of a conventional and a novel chemical design. (a) The reaction solution, along with the 99mTc-labeled probe,
contains excess unlabeled ligands whose binding affinity to a target molecule is similar to that of the 99mTc-labeled probe. Upon injection into
subjects without post-labeling purification, the target uptake of the 99mTc-labeled probe is competitively inhibited by the presence of the
unlabeled ligands. (b) The 99mTc-labeled trivalent probe would exhibit enhanced target binding affinity compared with its unlabeled ligand due to
the multivalent effect. This superior target binding affinity of the 99mTc-labeled probe would enable high in vivo target uptake of the radiotracer
without post-labeling purification. Adapted with permission from Mizuno et al., Purification-Free Method for Preparing Technetium-99m-
Labeled Multivalent Probes for Enhanced in Vivo Imaging of Saturable Sytems, J. Med. Chem., 2016, 59, 3331–3339. Copyright 2016 American
Chemical Society.10
yields. The aim of this study was thereby to demonstrate
whether our design concept of metal coordination mediated
multivalency could be extended to 99mTc-labeled tetravalent
probes. For this purpose, here we examined the inuence of the
structure of isonitrile ligands on the formation of their corre-
sponding 99mTc-labeled tetravalent probes.
Results and discussion
Fig. 2 Reaction scheme of Lb and [99mTc][TcI(CO)3(OH2)3]+. A 99mTc-
99mTc-labeling reaction of Lb and Lb
0
labeled trivalent RGD probe [99mTc][TcI(CO)3(Lb)3]+ 99mTc-[Lb]3) is
(
generated from a reaction between [99mTc][TcI(CO)3(OH2)3]+ and Lb in
the molar ratio of 1 to 3.
Following the previous report,18 we at rst attempted synthe-
sizing
99mTc][TcI(CO)2(Lb)4]+ by reacting 99mTc][TcI(CO)3(-
[
[
OH2)3]+ with Lb at pH 8.0. Contrary to our expectation, however,
multiple side products were generated at this slightly basic
reaction condition (Fig. 4a). Since the formation of multiple
side products was not observed in the previous report18 where
MIBI was used as a coordinating ligand, we speculated that the
presence of RGD peptide might have caused some undesirable
side reactions. To verify this speculation, we reacted [99mTc]
[TcI(CO)3(OH2)3]+ at pH 8.0 with CN-bAla-Gly-Gly-NH-Bn (Fig. 3,
compared with its unlabeled monovalent ligand (Lb), due to the
multivalent effect.13–15 This superior target binding affinity of
99mTc-[Lb]3 enabled its high in vivo tumor uptake even in the
presence of excess unlabeled ligands (Fig. 1b). We also
demonstrated the applicability of this design concept to 99mTc-
labeled bivalent probes16 and 67Ga-labeled trivalent ones17 using
D-penicillamine and Schiff base as a chelating molecule,
respectively.
0
Lb ), where c(RGDfK) in Lb was replaced with benzylamine.
However, as shown in Fig. 4b, the formation of multiple side
products was again observed, suggesting that the side reaction
observed here was not caused by the presence of RGD peptides
but by other moieties in Lb.
Our design concept would not be restricted to bivalent or
trivalent probes but could be applied to other valent probes
such as tetravalent or hexavalent ones. For example, it was re-
ported that the reaction of methoxy-isobutyl-isonitrile (Fig. 3,
MIBI) with [99mTc][TcI(CO)3(OH2)3]+ at basic conditions gener-
ated [99mTc][TcI(CO)2(MIBI)4]+, where one CO molecule in
0
Stabilities of 99mTc=Re-½Lb ꢁ3 in acidic and basic solutions
[
99mTc][TcI(CO)3(MIBI)3]+ was further substituted by another
To gain an insight into the mechanism of the side0reaction, we
MIBI molecule.18 This result indicates that the preparation of
99mTc-labeled tetravalent RGD probes would be possible by
employing appropriate isonitrile ligands and reaction condi-
tions. However, it is still unknown if any isonitrile ligands could
provide 99mTc-labeled tetravalent probes in high radiochemical
0
evaluated the stabilities of 99mTc-½Lb ꢁ3 and Re-½Lb ꢁ3 at pH 8.0
(Fig. 5). Since technetium and rhenium share similar coordi-
nation chemistry due to their group homology, we employed
non-radioactive rhenium analogues to characterize their tracer
99mTc counterparts. 99mTc-½Lb ꢁ3 was prepared from a labeling
0
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Fig. 3 The chemical structures of Lb, LG, Lb0, LG0, c(RGDfK), and MIBI.
unique to geminal (2JHH ¼ 2 Hz) and vicinal (3JHH ¼ 10 and 17
Hz) couplings (Fig. S8b†), which proved the presence of an
alkene structure. The IR spectrum of compound A (Fig. S8c†)
also veried the presence of alkene (C]C, n ¼ 1647.88 cmꢀ1
)
and excluded the presence of isonitrile, whose IR peak should
appear at around 2150 cmꢀ1. On the other hand, compounds B
and D showed MS spectra (Fig. S7a and S6a,† respectively)
unique to rhenium isotopic patterns (the ratio of 185Re/187Re ¼
37/63). These results along with their m/z values indicated that
ꢀ
0
compounds B and D had structures of ½ReIðCOÞ3ðLb ÞðCNÞ2ꢁ
and ½ReIðCOÞ3ðLb Þ2ðCNÞꢁ; respectively (Fig. 5c).
0
The mechanism of the side reaction
0
The decomposition reaction of 99mTc=Re-½Lb ꢁ3 has the following
three characteristics: (1) one of the coordinated isonitrile
ligands is eliminated from the Tc/Re-complex with leaving an
isonitrile ion on the Tc/Re-complex, (2) the eliminated ligand
loses the isonitrile group, and its terminal structure is con-
verted to an alkene structure, (3) this reaction proceeds only at
high hydroxide ion concentration. These features possess the
characteristics of the “bimolecular elimination reaction”.19 We
thereby hypothesized that the side reaction observed at basic
conditions (Fig. 4) would also proceed via the following three
Fig. 4 HPLC analyses of 99mTc-labeling reactions under the condi-
tions of (a) [Lb] ¼ 150 mM, 110 ꢂC for 20 min at pH 8.0 (system 11) and (b)
0
½Lb ꢁ ¼ 1 mM, 100 ꢂC for 30 min at pH 8.0 (system 6).
0
reaction at pH 6.0 and was puried by HPLC. 99mTc-½Lb ꢁ3 was
simultaneous events: (1) the abstraction of the 2-proton in
then dissolved in aqueous solution at pH 8.0 containing
0
99mTc-½Lb ꢁ3 by
a
hydroxide ion, (2) the departure of
0
ꢂ
Re-½Lb ꢁ3; and the solution was heated at 100 C for 30 min. As
0
0
99mTcꢁ½TcIðCOÞ3ðLb Þ2ðCNÞꢁ from 99mTc-½Lb ꢁ3; and (3) the
0
0
shown in Fig. 5b, 99mTc-½Lb ꢁ3 and Re-½Lb ꢁ3 exhibited a similar
decomposition pattern upon heating at pH 8.0, whereas they
remained intact at pH 6.0 (Fig. S1†). These results indicate that
½
formation of an alkene structure (Fig. 6).
0
the decomposition of 99mTc-½Lb ꢁ3 under basic conditions
Design and synthesis of LG and 99mTc-labeling of LG
0
0
primarily accounts for the generation of the multiple peaks
observed in Fig. 4b.
According to the hypothesis proposed above, the high acidity of
0
the 2-proton in Lb (Fig. 6) would be essential for the side
We isolated compounds A, B and D (Fig. 5b) and determined
reaction to proceed. In addition, this high acidity of the 2-
proton can be partly attributed to the electron withdrawing
effect from the neighboring carbonyl group. We thereby newly
1
their chemical structures by H NMR, MS, and/or IR (Fig. 5c).
The NMR spectrum of compound A showed coupling constants
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0
prepared as a labeling precursor, mixed with LG and heated at
100 ꢂC for 30 min at pH 8.0. The HPLC analysis of this reaction
solution is presented in Fig. 7c, which shows a completely
different chromatographic prole from that in Fig. 4b. As
shown in Fig. 7c, the 99mTc-labeling reaction of LG at pH 8.0
0
generated only two peaks, and the retention time of the major
radioactivity
þpeak
was
similar
to
that
of
½ReIðCOÞ2ðLG0Þ4ꢁ ðRe-½LG0ꢁ4Þ characterized by ESI-MS and IR
(Fig. 7b and c). These results supported the validity of our
working hypothesis that the abstraction of the 2-proton by
a base is the key step for the side reaction. We also identied the
minor
½
radiþoactivity
peak
as
99mTcꢁ½TcIðCOÞ3ðLG0Þ3ꢁ
ð
99mTc-½LG0ꢁ3Þ based on its similar
HPLC retention time to that of ½ReIðCOÞ3ðLG0Þ3ꢁþ ðRe-½LG0ꢁ3Þ
(Fig. 7a and c).
A GABA-based isonitrile ligand with c(RGDfK) peptides
Based on the above ndings, we subsequently designed and
synthesized
a
GABA-based isonitrile ligand containing
c(RGDfK) peptides (LG, Fig. 3). The synthesis of LG was accom-
plished by a condensation reaction of CN-GABA-TFP (7b) and
H2N-Gly-Gly-c(RGDfK) (15), followed by HPLC purication
(Scheme S1†). Its corresponding rhenium complex, [ReI(CO)2(-
LG)4]+ (Re-[LG]4), was also synthesized as a surrogate compound
for its 99mTc counterpart.
As a result of preliminary evaluations on its 99mTc-labelling
reactions, we established a reaction condition which repro-
ducibly provides [99mTc][TcI(CO)2(LG)4]+ 99mTc-[LG]4) in over
(
90% radiochemical yields (Fig. 8). The ligand concentration
required to provide 99mTc-[LG]4 (200 mM) was lower than that
needed for 99mTc-[Lb]3 (350 mM), which is advantageous to
mitigate the competitive inhibition by unlabeled ligand in
vivo.9,20,21 Since isonitrile is more stable in basic solutions than
in acidic ones,22 the result that 99mTc-[LG]4 was prepared under
lower ligand concentration could be attributed to the improved
stability of the isonitrile moiety in LG during the labeling
reactions.
Fig. 5 The stability of M-½Lb0ꢁ3 (M ¼ 99mTc and 185/187Re) in basic solu-
tion. HPLC analyses (system 6) of M-½Lb0ꢁ3 (a) before heating and (b) after
heating at 100 ꢂC for 30 min at pH 8.0. (c) The chemical structures of the
products generated upon heating and the intact complex ðM-½Lb0ꢁ3Þ:
The stability of 99mTc-[LG]4 in an excess histidine solution
and murine plasma was evaluated by TLC and HPLC. HPLC
analyses were performed aer removing plasma protein from
the samples. The recovery rates of the radioactivity from plasma
samples to supernatant solutions were 96.8 ꢃ 0.4% (n ¼ 3) upon
addition of ethanol and protein precipitation. No decomposi-
tion of 99mTc-[LG]4 was observed at all even aer 6 h incubation
in both solutions (Table S1 and Fig. S2†). These results indicate
that 99mTc-[LG]4 possesses stability sufficient for in vivo
applications.
Fig.
6 A proposed reaction mechanism of the side reaction
proceeding at basic conditions.
In vitro binding affinities of RGD derivatives
To examine the differences in integrin avb3 binding affinities
between monovalent and tetravalent RGD peptides, we calcu-
lated the IC50 values of LG and Re-[LG]4 in a competitive binding
designed a g-aminobutyric acid (GABA) based isonitrile ligand
0
(LG0; Fig. 3). As the 2-proton in LG is no longer present next to
0
experiment using
[
125I]I-c(RGDyV) and U87MG cell as
the carbonyl group (Fig. 3), the 2-proton in LG would possess
much lower acidity.
a competed radioligand and an integrin avb3 positive cell line,
respectively. We included c(RGDyV) into the study as an internal
standard. As shown in Table 1 and Fig. S3†, Re-[LG]4 showed
99mTc-labeling reactions of LG were performed in a proce-
0
dure similar to that of Lb : [99mTc][TcI(CO)3(OH2)3]+ was
0
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tested in this study: (1) HPLC-puried 99mTc-[LG]4 (without the
unlabeled ligand (LG)), (2) unpuried 99mTc-[LG]4 (containing 5
nmol of LG) and (3) HPLC-puried 99mTc-[LG]4 with 5 nmol of
Re-[LG]4. The second sample represent our chemical design
where a 99mTc-labeled multivalent RGD probe prepared from
a monovalent ligand is injected into subjects without post-
labeling purication (Fig. 1b), whereas the third sample
imitates a conventional design where a 99mTc-labeled tetrava-
lent RGD probe prepared from a ligand already possessing
tetravalent RGD is injected into subjects without postlabeling
purication (Fig. 1a). Though the tumor uptake of 99mTc-[LG]4
was slightly impaired by the presence of 5 nmol of LG in the
injectate (5.00 ꢃ 0.84% ID gꢀ1 vs. 3.08 ꢃ 0.43% ID gꢀ1), its
tumor uptake value was signicantly higher than that of the
conventional design (3.08 ꢃ 0.43% ID gꢀ1 vs. 1.81 ꢃ 0.14% ID
gꢀ1).
The tumor to blood ratio of un-puried 99mTc-[LG]4 was not
higher than that of puried 99mTc-[LG]4 + Re-[LG]4. This is
attributed to the higher blood radioactivity level of unpuried
99mTc-[LG]4 (0.58 ꢃ 0.39% ID gꢀ1) than that of puried 99mTc-
[LG]4 + Re-[LG]4 (0.21 ꢃ 0.04% ID gꢀ1). Though further study
would be needed to elucidate the cause of the higher blood
radioactivity level, we are considering the possibility that the
difference in the normal tissue uptakes (Table 2) affected the
blood clearance of 99mTc-[LG]4, especially in the elimination
phase. Because blood clearance in the elimination phase can be
affected by redistribution from normal tissues, the higher
normal tissue uptakes could cause higher blood radioactivity
level in the elimination phase.
Fig. 7 HPLC analyses (system 7) of (a) Re-½LG0ꢁ3 and (b) Re-½LG0ꢁ4. (c) An
HPLC analysis (system 7) of a 99mTc-labeling reaction of LG under the
0
0
condition of LG ¼ 1 mM, 100 ꢂC for 30 min at pH 8.0.
Lastly, we performed a SPECT/CT imaging study using
unpuried 99mTc-[LG]4 containing 5 nmol of LG. A representative
SPECT/CT image is shown in Fig. 9, which clearly visualized
U87MG tumor despite the presence of excess unlabeled ligand
in the injectate, owing to the high tumor uptake of 99mTc-[LG]4.
These results again demonstrated the utility of our design
concept for achieving high in vivo target uptake of 99mTc-labeled
probes without tedious post-labeling purication.
Fig. 8 HPLC analyses (system 9) of Re-[LG]4 and a 99mTc-labeling
reaction performed under the condition of [LG] ¼ 200 mM, 85 ꢂC for
1 h, and 37.5 mM P.B. (pH 8.0).
Table 1 The IC50 values of each compound
Conclusion
Compound
IC50, nM
95% C.I.a
In this study, we have found a way to prepare a 99mTc-labeled
tetravalent RGD probe upon isonitrile ligands coordination to
99mTcI in the molar ratio of 4 to 1. Our initial attempts reacting
a bAla-based isonitrile ligand (Lb) with [99mTc][Tc(CO)3]+ core at
pH 8.0 failed to provide [99mTc][Tc(CO)2(Lb)4]+ due to the
bimolecular elimination reaction. GABA-based isonitrile
ligands (LG and LG0), on the other hand, avoided the side reac-
tion and provided 99mTc-labeled tetravalent probes from reac-
tions at basic conditions. In the SPECT/CT study, 99mTc-[LG]4
clearly visualized integrin avb3 positive tumor without tedious
post-labeling purication to remove its excess unlabeled ligand.
These results clearly indicate that the metal coordination-
mediated synthesis of 99mTc-labeled bivalent and trivalent
probes has been successfully extended to that of tetravalent
LG
Re-[LG]4
c(RGDyV)
107
2.59
34.8
90.2ꢀ126
1.90ꢀ3.54
27.5ꢀ44.0
a
C.I. ¼ condential interval.
much lower IC50 value than those of LG and c(RGDyV), indi-
cating that the metal complex has gained enhanced binding
affinity due to the multivalent effect. This superior binding
affinity is essential for 99mTc-[LG]4 to achieve high in vivo target
uptake when injected into subjects without post-labeling
purication.
In vivo study of 99mTc-[LG]4
We evaluated the biodistribution of 99mTc-[LG]4 in U87MG- ones, which would lead to a wider range of choices for designing
bearing nude mice at 1 h post injection, and these results are novel 99mTc-labeled multivalent probes of high in vivo target
summarized in Table 2. There were three different samples uptake. In addition, because 99mTc-hexakis-isonitriles such as
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Table 2 Biodistribution at 1 h postinjection in U87MG-bearing micea
Un-puried 99mTc-[LG]4 (LG ¼ 5
Puried 99mTc-[LG]4 + Re-[LG]4 (Re-
[LG]4 ¼ 5 nmol)
Tissue
Puried 99mTc-[LG]4 (LG ¼ 0 nmol)
nmol)
Blood
Liver
Spleen
Kidney
Pancreas
Heart
0.26 ꢃ 0.04
1.85 ꢃ 0.23
2.16 ꢃ 0.31
13.05 ꢃ 0.87
0.93 ꢃ 0.07
1.10 ꢃ 0.13
2.25 ꢃ 0.47
4.57 ꢃ 2.43
4.01 ꢃ 0.89
0.74 ꢃ 0.17
5.00 ꢃ 0.84*
19.0 ꢃ 2.31
2.72 ꢃ 0.49
7.10 ꢃ 2.04
0.58 ꢃ 0.39
1.29 ꢃ 0.14
1.29 ꢃ 0.12
13.12 ꢃ 1.87
0.46 ꢃ 0.06
0.52 ꢃ 0.05
1.60 ꢃ 0.11
4.21 ꢃ 2.01
3.34 ꢃ 1.06
0.44 ꢃ 0.12
3.08 ꢃ 0.43
6.15 ꢃ 3.03
2.35 ꢃ 0.40
7.19 ꢃ 2.12
0.21 ꢃ 0.04
0.95 ꢃ 0.14
0.95 ꢃ 0.09
12.28 ꢃ 1.14
0.27 ꢃ 0.05
0.36 ꢃ 0.07
0.84 ꢃ 0.25
2.36 ꢃ 0.83
1.99 ꢃ 0.65
0.26 ꢃ 0.23
1.81 ꢃ 0.14*
8.81 ꢃ 1.48
1.93 ꢃ 0.22
9.60 ꢃ 4.11
Lung
Stomach
Intestine
Muscle
Tumorb
T/Blood
T/Liver
T/Muscle
a
b
The results were expressed as percent injected dose per gram (% ID gꢀ1) ꢃ SD (n ¼ 5 or 6). Statistical analysis was performed using one-way
analysis of variance followed by Tukey's multiple-comparison test (* different from “un-puried 99mTc-[LG]4 (LG ¼ 5 nmol)”, p < 0.01).
reagents were purchased from Wako Pure Chemical Industries,
Ltd. (Tokyo, Japan) and were used without further purication.
Mass spectrometry was carried out using a JMS-T100LP (JEOL
Ltd., Tokyo, Japan) or an Agilent 6130 Series Quadrupole LC/MS
1
spectrometer (Agilent technologies, Tokyo). H NMR/13C NMR
spectra was recorded on a JEOL ECS-400 (400 MHz/100 MHz,
respectively) spectrometer (JEOL Ltd, Tokyo). Infrared (IR)
spectra were recorded on a FT/IR-4700 (Jasco Co., Tokyo, Japan).
Boc-Gly-Gly-NH-Bn (2)
Boc-Gly-Gly-OH (1) (180 mg, 0.775 mmol) and benzylamine (77
mL, 0.705 mmol) were dissolved in dichloromethane (4 mL). To
this
stirred
mixture
was
added
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl,
270 mg, 1.41 mmol), and the mixture was stirred at room
temperature for 1 h. The solvent was removed in vacuo and the
residue was dissolved in chloroform (15 mL) and washed with
5% citric acid (10 mL) and 5% NaHCO3 (10 mL). The organic
layer was then dried over magnesium sulfate. Aer removing
magnesium sulfate by lter, the organic solvent was removed in
vacuo to afford the compound 2 as a white solid (189 mg, 76%).
1H NMR (400 MHz, DMSO-d6): d 1.37 (s, 9H, tBu), 3.57 (d, 2H,
HaGly), 3.74 (d, 2H, HaGly), 4.29 (d, 2H, CH2-phenyl), 7.05 (t, 1H,
NH), 7.21–7.33 (m, 5H, Harom), 8.11 (t, 1H, NH), 8.28 (t, 1H, NH).
13C NMR (100 MHz, DMSO-d6): d 28.16 (C(CH3)3), 41.96 (CaGly),
42.07 (CH2-phenyl), 43.42 (CaGly), 78.16 (C(CH3)3), 126.73
(Carom), 127.10 (Carom), 128.22 (Carom), 139.24 (Carom), 155.91
(CO), 168.79 (CO), 169.74 (CO). ESI-MS, m/z: 322.18 [M + H]+,
found 322.24.
Fig. 9 A SPECT/CT image of a U87MG tumor-bearing male nude
mouse over 30–94 min post i.v. injection of 3.7 MBq of 99mTc-[LG]4
containing 5 nmol of LG. Red circle indicates the U87MG tumor.
[
99mTc][Tc(MIBI)6]+ also possess very high in vivo stability, we
are currently working to apply our metal coordination mediated
multivalency concept to hexavalent ones, and are going to
report the results in the near future elsewhere.
Experimental section
General
H2N-Gly-Gly-NH-Bn$CF3CO2H (3)
[
185/187Re(CO)3(OH2)3]Br (19),23 Boc-Gly-Gly-OH (1),10 CN-bAla-
TFP (7a),10 CN-bAla-Gly-Gly-c(RGDfK) (Lb)10 and H2N-Gly-Gly- The compound 2 (104 mg, 0.324 mmol) was dissolved in tri-
c(RGDfK)$CF3CO2H (15)10 were synthesized according to the uoroacetic acid (TFA, 2.5 mL) and stirred at room temperature
previous reports. Technetium-99m as Na99mTcO4 was eluted in for 1 h. Aer removing TFA in vacuo, the compound 3 was
saline solution on daily basis from a 99Mo/99mTc generator successively used in the following condensation reactions
(FUJIFILM RI Pharma Co., Ltd., Tokyo, Japan). All other without further purication.
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4-Formylaminobutanoic acid (5b)
CN-GABA-Gly-Gly-NH-Bn ðLG0Þ (8b)
4-Aminobutanoic acid (2.00 g, 19.4 mmol) was dissolved in To a solution of CN-GABA-TFP (7b) (106.8 mg, 0.409 mmol) and
formic acid/acetic anhydrate (20 mL/15 mL) and stirred at 95 ꢂC H2N-Gly-Gly-NH-Bn$CF3CO2H (3) (100.2 mg, 0.299 mmol) in
under N2 atmosphere for 30 min. Aer the solvent was evapo- DMF (2 mL) was added NaHCO3 (75.2 mg, 0.895 mmol). The
rated in vacuo, the residue was washed with diethyl ether to mixture was stirred at room temperature for 6 h. Aer removing
afford the compound 5b as a white solid (1.00 g, 39%). 1H NMR NaHCO3 by ltration, the solvent was removed in vacuo and the
(400 MHz, DMSO-d6): d 1.62 (m, 2H, CH2CH2CH2), 2.22 (t, 2H, crude product was puried with silica gel column chromatog-
CH2CO), 3.08 (q, 2H, NHCH2), 7.99 (s, 1H, formylamino), 8.02 (s, raphy (chloroform/methanol ¼ 20/1) to afford the compound 8b
1
1H, NH). ESI-MS, m/z: 130.05 [M ꢀ H]ꢀ, found 130.09.
as a white solid (60 mg, 63%). H NMR (400 MHz, DMSO-d6):
d 1.79 (m, 2H, CH2CH2CH2), 2.27 (t, 2H, CH2CH2CH2), 3.50 (m.
2H, CNCH2), 3.72 (d, 2H, HaGly), 3.74 (d, 2H, HaGly), 4.28 (d, 2H,
CH2-phenyl), 7.21–7.33 (m, 5H, Harom), 8.23 (t, 1H, NH), 8.28–
8.32 (m, 2H, NH). ESI-MS, m/z: 339.14 [M + Na]+, found 339.16.
IR (ATR, n/cmꢀ1): 2147.35 (s, C^N).
2,3,5,6-Tetrauorophenyl 4-formylaminobutanoate (6b)
The compound 5b (525 mg, 4.00 mmol) and 2,3,5,6-tetrauoro
phenol (TFP, 797 mg, 4.80 mmol) were dissolved in chloroform
(8 mL). To this stirred mixture was added EDC HCl (1.53 g, 8.00
mmol), and the mixture was stirred at room temperature for 2
hours. The solvent was removed in vacuo and puried with silica
gel column chromatography (hexane/ethyl acetate ¼ 1/1) to
0
0
½ReIðCOÞ3ðLb Þ3ꢁCF3CO2 ðRe-½Lb ꢁ3Þ (9)
[ReI(CO)3(OH2)3]Br (19) (9.7 mg, 0.024 mmol) and Lb (8a)
(65 mg, 0.215 mmol) were dissolved in 0.1 M acetate buffer (pH
6.0, 21.5 mL). Aer heating at 100 ꢂC for 3 h, the crude was
puried by preparative HPLC using system 1 and lyophilized to
afford the compound 9 as a white powder (30 mg, 97%). 1H
NMR (400 MHz, DMSO-d6): d 2.70 (t, 2H, CH2CH2CO), 3.76 (d,
2H, HaGly), 3.81 (d, 2H, HaGly), 4.09 (t, 2H, CH2CH2CO), 4.28 (d,
2H, CH2-phenyl), 7.21–7.33 (m, 5H, Harom), 8.27 (t, 1H, NH), 8.36
(t, 1H, NH), 8.43 (t, 1H, NH). 13C NMR (100 MHz, DMSO-d6):
0
1
afford the compound 6b as a white powder (929 mg, 83%). H
NMR (400 MHz, DMSO-d6): d 1.81 (m, 2H, CH2CH2CH2), 2.81 (t,
2H, CH2CO), 3.18 (q, 2H, NHCH2), 7.95 (m, 1H, Harom), 8.03 (s,
1H, formylamino), 8.11 (s, 1H, NH). ESI-MS, m/z: 302.04 [M +
Na]+, found 301.97.
2,3,5,6-Tetrauorophenyl 4-isocyanobutanoate (CN-GABA-
TFP) (7b)
d 33.89 (CH2CH2CO), 41.06 (CH2CH2CO), 42.02, 42.08, (CaGly
,
The compound 6b (717 mg, 2.57 mmol) and Burgess reagent24
(918 mg, 3.85 mmol) were dissolved in dichloromethane (24
mL) and stirred at 50 ꢂC for 30 minutes. Aer removing the
solvent in vacuo, the residue was puried with silica gel chro-
CH2-phenyl), 126.79 (Carom), 127.18 (Carom), 128.26 (Carom),
139.31 (Carom), 158.24 (q, CN), 168.80 (CO), 168.96 (CO), 169.10
(CO), 182.71 (ReCO). ESI-MS, m/z: 1177.35 [M]+, found 1177.56.
IR (ATR, n/cmꢀ1): 2244.74 (w, C^N), 2212.92 (m, C^N),
2057.67, 1990.18 (s, C^O).
matography (hexane/ethyl acetate
¼
10/1) to afford the
compound 7b as a dark yellow oil (404 mg, 60%). 1H NMR (400
MHz, CDCl3): d 2.16 (br, 2H, CH2CH2CH2), 2.91 (t, 2H, CH2CO), ½ReIðCOÞ3ðLG0Þ3ꢁCF3CO2 ðRe-½LG0ꢁ3Þ (10)
3.59 (m, 2H, NCH2), 7.02 (m, 1H, Harom). IR (ATR, n/cmꢀ1):
2149.28 (s, C^N).
[ReI(CO)3(OH2)3]Br (19) (2.6 mg, 6.4 mmol) and LG (8b) (20 mg,
63.2 mmol) were dissolved in 0.1 M phosphate buffer (P.B., pH
7.0, 6.32 mL). Aer heating at 100 ꢂC for 3 h, the crude was
puried by preparative HPLC using system 3 and lyophilized to
afford the compound 10 as a white powder (8.9 mg, quant.). 1H
NMR (400 MHz, DMSO-d6): d 1.93 (m, 2H, CH2CH2CH2), 2.29 (t,
2H, CH2CH2CH2), 3.75 (d, 4H, HaGly), 3.98 (t, 2H, CNCH2), 4.28
(d, 2H, CH2-phenyl), 7.10–7.33 (m, 5H, Harom), 8.23 (t, 1H, NH),
8.29 (t, 1H, NH), 8.34 (t, 1H, NH). ESI-MS, m/z: 1219.40 [M]+,
found 1219.47. IR (ATR, n/cmꢀ1): 2238.95 (w, C^N), 2208.09 (m,
C^N), 2053.82, 1981.50 (s, C^O).
0
0
CN-bAla-Gly-Gly-NH-Bn ðLb Þ (8a)
CN-bAla-TFP (7a) was synthesized as previously reported.10 To
a solution of the compound 7a (120 mg, 0.486 mmol) and H2N-
Gly-Gly-NH-Bn$CF3CO2H (3) (108.6 mg, 0.324 mmol) in N,N-
dimethylformamide (DMF, 2 mL) was added NaHCO3 (40.8 mg,
0.486 mmol). The mixture was stirred at room temperature for
1 h. Aer removing NaHCO3 by ltration, the solvent was
removed in vacuo and the crude compound was puried with
silica gel column chromatography (chloroform/methanol ¼ 20/
1) to afford the compound 8a as a white solid (70 mg, 72%). 1H
½ReðCOÞ2ðLG0Þ4ꢁCF3CO2 ðRe-½LG0ꢁ4Þ (11)
NMR (400 MHz, DMSO-d6): d 2.56 (br, 2H, CH2CH2CO), 3.64 (br, Re-½LG0ꢁ3 (10) (3.6 mg, 0.0027 mmol) and LG0 (8b) (4.7 mg, 0.0149
2H, CH2CH2CO), 3.74 (d, 2H, HaGly), 3.77 (d, 2H, HaGly), 4.29 (d, mmol) were dissolved in 0.1 M P.B. (pH 8.0, 1.47 mL). The
2H, CH2-phenyl), 7.21–7.33 (m, 5H, phenyl), 8.23 (t, 1H, NH), mixture was heated at 100 ꢂC for 6 h, aer which time additional
8.29 (t, 1H, NH), 8.37 (t, 1H, NH). 13C NMR (100 MHz, DMSO-d6): LG (8b) (4.7 mg, 0.0149 mmol) was added. Aer heating at
0
d 34.47 (CH2CH2CO), 37.47 (t, CH2CH2CO), 41.97 (CaGly), 42.07 100 ꢂC for another 6 h, the crude product was puried by
(CaGly), 42.23 (CH2-phenyl), 126.76 (Carom), 127.17 (Carom), preparative HPLC using system 3 and lyophilized to afford the
128.25 (Carom), 139.32 (Carom), 155.73 (t, CN), 168.78 (CO), compound 11 as a white powder (0.4 mg, 10%). ESI-MS, m/z:
169.15 (2C, CO). ESI-MS, m/z: 325.13 [M + Na]+, found 325.19. IR 1507.56 [M]+, found 1507.65. IR (ATR, n/cmꢀ1): 2228.34 (w,
(ATR, n/cmꢀ1): 2150.24 (s, C^N).
C^N), 2160.85 (s, C^N), 1996.93, 1953.54 (s, C^O).
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99mTc][TcI(CO)3(OH2)3]+
30%) as an orange solid. The spectrum data of the compound
[
12, 13 and 14 are shown down below.
[
99mTc][TcI(CO)3(OH2)3]+ was prepared according to a reported
method25 with slight modications. Briey, a kit containing
0.285 mg sodium tetraborate decahydrate (Na2B4O7$10H2O),
0.715 mg sodium carbonate (Na2CO3), 1 mg sodium(+)-tartrate
dihydrate (C4H4Na2O6$2H2O), and 0.45 mg sodium boranocar-
bonate [Na2(H3BCO2)] was ꢀpurged with N2 atmosphere before
adding 300 mL [99mTc]TcO4 (ꢄ333 MBq) to a sealed vial. Aer
the mixture was heated for 14 min at 100 ꢂC, the vial was
allowed to stand at room temperature for 3 min. The pH of the
solution was then adjusted to 6, 7, or 8 with 1 M AcOH. Ten min
aer the neutralization, AgNO3 (7.1 mg, 0.042 mmol) was added
to the solution to precipitate out Clꢀ as AgCl. Aer ltering the
solution through 0.45 mm Cosmonice Filter W (NACALAI TES-
QUE, Inc., Kyoto), the solution was used0for labeling reactions
with Lb and LG. For the reactions with Lb and LG0; 1N HCl was
used for neutralization and [99mTc][TcI(CO)3(OH2)3]+ solution
was used without the removal of Clꢀ.
The eliminated ligand (compound A in Fig. 5b) (12)
1H NMR (400 MHz, DMSO-d6): d 3.75 (d, 2H, HaGly), 3.83 (d, 2H,
HaGly), 4.28 (d, 2H, CH2-phenyl), 5.61 (dd, 1H, J ¼ 2, 10 Hz,
H
alkene), 6.08 (dd, 1H, J ¼ 2, 17 Hz, Halkene), 6.31 (dd, 1H, J ¼ 10,
17 Hz, Halkene), 7.21–7.33 (m, 5H, Harom), 8.27 (t, 1H, NH), 8.34
(t, 1H, NH), 8.44 (t, 1H, NH). ESI-MS, m/z: 298.12 [M + Na]+,
found 298.10. IR (ATR, n/cmꢀ1): 1647.88 (s, C]C).
0
½ReðCOÞ3ðLb Þ2ðCNÞꢁ (compound D in Fig. 5b) (13)
1H NMR (400 MHz, DMSO-d6): d 2.69 (t, 2H, CH2CH2CO), 3.75
(d, 2H, HaGly), 3.80 (d, 2H, HaGly), 4.07 (t, 2H, CH2CH2CO), 4.28
(d, 2H, CH2-phenyl), 7.21ꢀ7.33 (m, 5H, Harom), 8.26 (t, 1H, NH),
8.30 (t, 1H, NH), 8.44 (t, 1H, NH). ESI-MS, m/z: 924.21 [M + Na]+,
found 924.38.
99mTc-labeling reaction of Lb
ꢀ
0
½ReðCOÞ3ðLb ÞðCNÞ2ꢁ (compound B in Fig. 5b) (14)
ESI-MS, m/z: 627.10 [M + 2H]+, found 627.22.
A 5 mL of freshly prepared [99mTc][TcI(CO)3(OH2)3]+ (ꢄ5.55 MBq)
was added to a vial containing 95 mL of Lb solution (0.1 M P.B.,
pH 8.0) to reach the nal ligand concentration of 150 mM. The
ꢂ
99mTc-labeling reaction of LG
0
mixture was heated at 110 C for 20 min under N2 atmosphere
in a heating block. The reaction solution was analyzed by HPLC
using system 11.
A 20 mL solution of freshly prepared [99mTc][TcI(CO)3(OH2)3]+0
(ꢄ22.2 MBq) was added to a vial containing 380 mL of LG
solution (0.1 M P.B., pH 8.0) to reach the nal ligand concen-
tration of 1 mM. The mixtures were heated at 100 ꢂC for 30 min
under N2 atmosphere in a heating block. The reaction solutions
were analyzed by HPLC using system 7.
99mTc-labeling reaction of Lb
0
A 20 mL solution of freshly prepared [99mTc][TcI(CO)3(OH2)3]+
0
(ꢄ22.2 MBq) was added to a vial containing 380 mL of Lb
solution (0.1 M P.B., pH 6.0 or 8.0) to reach the nal ligand
concentration of 1 mM. The mixtures were heated at 100 ꢂC for
30 min under N2 atmosphere in a heating block. The reaction
solutions were analyzed by HPLC using system 6.
CN-GABA-Gly-Gly-c(RGDfK) (LG) (16)
To a solution of the compound 15 (13.8 mg, 16.6 mmol) and N,N-
diisopropylethylamine (4.9 mL, 28.8 mmol) in DMF (180 mL) was
added the compound 7b (4.0 mg, 16 mmol). The reaction
mixture was stirred at room temperature for 1 h, aer which
time diethyl ether was added to the solution to form precipitate.
The precipitate was collected and puried with preparative
HPLC using system 4, and lyophilized to afford the compound
16 as a white powder (7.0 mg, 52%). ESI-MS, m/z: 813.40 [M +
H]+, found 813.49.
0
Stability of 99mTc=Re-½Lb ꢁ3 in acidic and basic solutions
0
99mTc-½Lb ꢁ3 was prepared by the reaction at pH 6.0 which was
described above and puried by HPLC (system 6). The eluent of
the objective fraction was evaporated in vacuo and the residue
was dissolved in 0.1 M P.B. (pH 6.0 or 8.0) containing 40 nmol of
0
0
0
Re-½Lb ꢁ3 to prepare 111 kBq 99mTc-½Lb ꢁ3/40 nmol Re-½Lb ꢁ3/100
mL sample solutions. The sample of pH 6.0 was analyzed by
HPLC (system 6) before heating. Both solutions were then
heated at 100 ꢂC for 30 min in a heating block and analyzed by
HPLC (system 6) aer heating.
[ReI(CO)3(LG)3]CF3CO2 (Re-[LG]3) (17) and [ReI(CO)2(LG)4]
CF3CO2 (Re-[LG]4) (18)
L
G (16) (6.0 mg, 7.4 mmol) and [ReI(CO)3(OH2)3]Br (19) (0.3 mg,
Characterization of three peaks generated upon heating
0.7 mmol) were dissolved in 50 mM P.B. (pH 8.0, 720 mL). The
mixture was heated at 110 ꢂC for 2 h, aer which time additional
0
Re-½Lb ꢁ3
0
Re-½Lb ꢁ3 (9) was prepared by the method described above. LG (1.5 mg, 1.9 mmol) was added to the solution. Aer heating at
0
Twenty nine mg of Re-½Lb ꢁ3 (0.225 mmol) was dissolved in 5 mL 110 ꢂC for another 2 h, the crude was puried with preparative
of 0.1 M P.B. (pH 8.0) and the mixture was heated at 100 ꢂC for HPLC using system 5 and lyophilized to afford the compounds
5 h. The crude product was puried by preparative HPLC using 17 and 18 as white powders (0.5 mg and 25% for the compound
system 2 to afford the compound 12 (1.4 mg, 23% calculated 17, and 0.2 mg and 8% for the compound 18). ESI-MS (17), m/z:
from the Re-complex) as a white solid, the compound 14 1355.06 [M + H]2+, found 1355.26. ESI-MS (18), m/z: 874.13 [M +
(1.8 mg, 13%) as a purple oil and the compound 13 (6.1 mg, 3H]4+, found 874.4.
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99mTc-labeling reaction of LG
Diego, CA). All the binding experiments were carried out with
quadruplet samples.
LG (16) (20 nmol) was dissolved in 75 mL of 50 mM P.B. at pH 8.0.
A 25 mL solution of freshly prepared [99mTc][TcI(CO)3(OH2)3]+
(ꢄ27.8 MBq) was added to this LG solution, followed by heating
Animal model
ꢂ
All animal procedures were performed in accordance with the
institutional guidelines approved by the Chiba University
Animal Care Committee and experiments were approved by the
Chiba University Animal Care Committee. BALBc nu/nu male
mice (Japan SLC, Inc., Shizuoka, Japan) of 6 week-old were
xenograed by subcutaneous injection of U87MG human glio-
blastoma cells (5 ꢅ 106 cells per 50 mL of culture medium) into
their right hind legs. The mice were subjected to biodistribution
studies as well as SPECT/CT imaging studies when the tumor
weight reached 0.1–0.5 g.
at 85 C for 1 h under N2 atmosphere. The resulting solution
was analyzed by HPLC using system 9.
Stability assessment
The unlabeled ligand in 99mTc-[LG]4 was removed by HPLC
using system 9. The radioactive peak was collected and the
solvent was removed in vacuo. The residue was reconstituted in
0.001% v/v TritonX-100 in D-PBS(ꢀ), and 10 mL of the 99mTc-
[LG]4 solution was mixed with 190 mL of histidine solution to
reach the nal histidine concentration of 10 mM. Similarly 10
mL of the 99mTc-[LG]4 solution was mixed with 190 mL of freshly
prepared murine plasma. Aer incubation at 37 ꢂC for 1 and 6 h,
1 mL of aliquots were drawn and analyzed with radio-TLC (n ¼ 3)
Biodistribution
Male nude mice bearing tumor xenogras of U87MG were
injected via the tail vein with 100 mL (11.1 kBq) of 99mTc-[LG]4 (LG
¼ 0 nmol), 99mTc-[LG]4 (LG ¼ 5 nmol) or 99mTc-[LG]4 (Re-[LG]4 ¼ 5
nmol). 99mTc-[LG]4 (LG ¼ 0 nmol) was prepared by diluting an
HPLC-puried 99mTc-[LG]4 solution with D-PBS (ꢀ). 99mTc-[LG]4
(Re-[LG]4 ¼ 5 nmol) was prepared by adding Re-[LG]4 to an
HPLC-puried 99mTc-[LG]4 solution. Un-puried 99mTc-[LG]4
solution was used for 99mTc-[LG]4 (LG ¼ 5 nmol). The animals
were sacriced and dissected at 1 h aer administration. The
tissues of interest were removed and weighed, and the radio-
activity was determined with an autowell gamma counter. The
results are presented as the percentage injected dose per gram
(% ID gꢀ1) or percentage injected dose per tissue (% ID). Values
were expressed as mean ꢃ SD for a group of 5 or 6 animals.
(Silica gel 60 F254
, Merck Ltd., Tokyo) developed with
MeOH : 10% w/w AcONH4 in water ¼ 1 : 1. For HPLC analysis of
the histidine sample, the incubation solution was directly
analyzed by HPLC using system 9. For HPLC analysis of the
plasma sample, 200 mL of EtOH was added to 100 mL of the
plasma sample to precipitate the proteins. The sample was
ꢂ
centrifuged at 15 000g for 5 min at 4 C. The supernatant was
collected and, the pellet was washed with 300 mL of 66% v/v
EtOH in D-PBS(ꢀ) and centrifuged. This washing step was
repeated twice. The supernatants of both centrifugation steps
were combined and analyzed by HPLC using system 9. The
recovery rate of radioactivity from the plasma sample was
determined by following equation, (the radioactivity of the
supernatant)/(the original radioactivity of the plasma sample) ꢅ
100 (n ¼ 3). The radioactivity was determined using an autowell
g counter (WIZARD 1480, PerkinElmer Japan Co., Ltd., Yoko-
hama, Japan).
Small animal SPECT/CT imaging study
SPECT images were acquired over 30–94 min aer intravenous
administration of 99mTc-[LG]4 (100 mL, 3.7 MBq, LG ¼ 5 nmol) to
male BALBc nu/nu mice bearing U87MG xenogras via the tail
vein. CT scans were performed before SPECT scans for
anatomic reference. The mice were anaesthetized with 1–2% (v/
v) isourane (DS Pharma Animal Health, Osaka, Japan) and
positioned on the animal bed where anesthesia was continu-
ously delivered via a nose cone system. SPECT imaging and X-
ray CT imaging were performed by use of small animal
SPECT/CT system (Triumph Lab SPECT4/CT, TriFoil Imaging
Inc., Chatsworth, CA) equipped with a ve pinhole (0.5 mm)
collimator. Data acquisition was performed for 64 min at 240 s
per projection with stepwise rotation of 16 projections over
360ꢂ.
Binding affinity to integrin avb3
U87MG human glioma cells were grown in Dulbecco's Modied
Eagle Medium (Sigma-Aldrich Japan K.K., Tokyo) supplemented
with 10% v/v fetal bovine serum (Nippon Biosupply Center,
Tokyo), and 1% v/v penicillin–streptomycin (10 000 unit–10 mg
mLꢀ1, Sigma-Aldrich Japan K.K.) at 37 C in humidied atmo-
ꢂ
sphere containing 5% CO2. Multiscreen DV lter plates (Merck
Millipore, MA) were seeded with 2 ꢅ 105 cells in the binding
buffer (20 mM Tris, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2,
1 mM MnCl2, 0.1% BSA, pH 7.4) and the mixtures were incu-
bated at 37 ꢂC with [125I]I-c(RGDyV) in the presence of
increasing concentrations of c(RGDyV), LG, Re-[LG]3, or Re-[LG]4.
The total incubation volume was adjusted to 200 mL. Aer
incubation for 1 h at 37 ꢂC, the plates were ltered, and washed
twice with 200 mL of the ice cold binding buffer. The poly-
vinylidene uoride (PVDF) lters were collected and the radio-
Conflicts of interest
There are no conicts to declare.
Acknowledgements
activity was determined using an autowell g counter. The IC50 Y. M. was supported by JSPS Research Fellowships for Young
values were calculated by tting the data by nonlinear regres- Scientists. This work was supported in part by JSPS Research
sion using GraphPad Prism (GraphPad Soware, Inc., San Fellows Program (No. 264896). We thank Prof. Atsushi Nishida
26134 | RSC Adv., 2019, 9, 26126–26135
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(Chiba University) for valuable discussion on the mechanism of 13 M. Mammen, S. K. Choi and G. M. Whitesides, Angew.
the side reaction.
Chem., Int. Ed., 1998, 37, 2754–2794.
14 L. L. Kiessling, J. E. Gestwicki and L. E. Strong, Curr. Opin.
Chem. Biol., 2000, 4, 696–703.
15 S. Bhatia, L. C. Camacho and R. Haag, J. Am. Chem. Soc.,
2016, 138, 8654–8666.
16 Y. Taira, T. Uehara, M. Tsuchiya, H. Takemori, Y. Mizuno,
S. Takahashi, H. Suzuki, H. Hanaoka, H. Akizawa and
Y. Arano, Bioconjugate Chem., 2018, 29, 459–466.
17 H. A. Holik, T. Uehara, S. Nemoto, T. Rokugawa,
Y. Tomizawa, A. Sakuma, Y. Mizuno, H. Suzuki and
Y. Arano, Bioconjugate Chem., 2018, 29, 2909–2919.
18 X. Chen, Y. Guo, Q. Zhang, G. Hao, H. Jia and B. Liu, J.
Organomet. Chem., 2008, 693, 1822–1828.
References
1 C. S. Cutler, H. M. Hennkens, N. Sisay, S. Huclier-Markai and
S. S. Jurisson, Chem. Rev., 2013, 113, 858–883.
2 B. M. Zeglis, J. L. Houghton, M. J. Evans, N. Viola-Villegas
and J. S. Lewis, Inorg. Chem., 2014, 53, 1880–1899.
3 D. Brasse and A. Nonat, Dalton Trans., 2015, 44, 4845–4858.
4 S. Bhattacharyya and M. Dixit, Dalton Trans., 2011, 40, 6112–
6128.
5 T. J. Wadas, E. H. Wong, G. R. Weisman and C. J. Anderson,
Chem. Rev., 2010, 110, 2858–2902.
6 M. Morais, A. Paulo, L. Gano, I. Santos and J. D. G. Correia, J.
Organomet. Chem., 2013, 744, 125–139.
7 S. Liu, Adv. Drug Delivery Rev., 2008, 60, 1347–1370.
8 S. Liu and S. Chakraborty, Dalton Trans., 2011, 40, 6077–
6086.
19 J. F. Bunnett, Angew. Chem., Int. Ed. Engl., 1962, 1, 225–235.
20 W. C. Eckelman, A. G. Jones, A. Duatti and R. C. Reba, Drug
Discov. Today, 2013, 18, 984–991.
21 W. C. Eckelman, Eur. J. Nucl. Med. Mol. Imaging, 1995, 22,
249–263.
9 J. R. Ballinger, Q. J. Nucl. Med. Mol. Imaging, 2002, 46, 224–
232.
22 K. Sung and C. C. Chen, Tetrahedron Lett., 2001, 42, 4845–
4848.
10 Y. Mizuno, T. Uehara, H. Hanaoka, Y. Endo, C.-W. Jen and
Y. Arano, J. Med. Chem., 2016, 59, 3331–3339.
11 Y. Mizuno, T. Uehara, H. Hanaoka, Y. Endo, C.-W. Jen and
Y. Arano, J. Med. Chem., 2017, 60, 6768–6769.
12 R. Haubner, H. J. Wester, U. Reuning, R. Senekowitsch-
23 N. Lazarova, S. James, J. Babich and J. Zubieta, Inorg. Chem.
Commun., 2004, 7, 1023–1026.
24 S. M. Creedon, H. K. Crowley and D. G. McCarthy, J. Chem.
Soc., Perkin Trans. 1, 1998, 1015–1017.
25 R. Alberto, K. Ortner, N. Wheatley, R. Schibli and
A. P. Schubiger, J. Am. Chem. Soc., 2001, 123, 3135–3136.
¨
Schmidtke, B. Diefenbach, H. Kessler, G. Stocklin and
M. Schwaiger, J. Nucl. Med., 1999, 40, 1061–1071.
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