Purification-Free Method for Preparing Technetium-99m-Labeled Multivalent Probes for Enhanced in Vivo Imaging of Saturable Systems

Article abstract of DOI:10.1021/acs.jmedchem.6b00024

Metallic radionuclides provide target-specific radiolabeled probes for molecular imaging in radiochemical yields sufficient for administration to subjects without purification. However, unlabeled ligands in the injectate can compete for targeted molecules with radiolabeled probes, which eventually necessitates postlabeling purification. Herein we describe a "1 to 3" design to circumvent the issue by taking advantage of inherent coordination properties of technetium-99m (^99mTc). A monovalent RGD ligand possessing an isonitrile as a coordinating moiety (CN-RGD) was reacted with [^99mTc(CO)₃(OH₂)₃]⁺ to prepare [^99mTc(CO)₃(CN-RGD)₃]⁺ in over 95% radiochemical yields. This complex exhibited higher integrin α_vβ₃ binding affinity than its unlabeled monovalent ligand, primarily due to its multivalency. This compound visualized a murine tumor without removing unlabeled ligands, while a ^99mTc-labeled monovalent probe derived from a monovalent ligand could not. The metal coordination-mediated synthesis of radiolabeled multivalent probes thereby can be a useful approach for preparing ready-to-use target-specific probes labeled with ^99mTc and other metallic radionuclides of interest.

Full text of DOI:10.1021/acs.jmedchem.6b00024

Article
pubs.acs.org/jmc
Purification-Free Method for Preparing Technetium-99m-Labeled
Multivalent Probes for Enhanced in Vivo Imaging of Saturable
Systems
Yuki Mizuno,†,‡ Tomoya Uehara,† Hirofumi Hanaoka,†,§ Yota Endo,† Chun-Wei Jen,†
and Yasushi Arano*,†
†Department of Molecular Imaging and Radiotherapy, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana,
Chuo-ku, Chiba 260-8675 Japan
§
‡Research Fellow of Japan Society for the Promotion of Science and Department of Bioimaging Information Analysis, Graduate
School of Medicine, Gunma University, 3-39-15 Showa-chou, Maebashi 371-8511 Japan
S
* Supporting Information
ABSTRACT: Metallic radionuclides provide target-specific
radiolabeled probes for molecular imaging in radiochemical
yields sufficient for administration to subjects without
purification. However, unlabeled ligands in the injectate can
compete for targeted molecules with radiolabeled probes,
which eventually necessitates postlabeling purification. Herein
we describe a “1 to 3” design to circumvent the issue by taking
advantage of inherent coordination properties of technetium-
99m (99mTc). A monovalent RGD ligand possessing an
isonitrile as a coordinating moiety (CN-RGD) was reacted
with [99mTc(CO)3(OH2)3]+ to prepare [99mTc(CO)3(CN-
RGD)3]+ in over 95% radiochemical yields. This complex
exhibited higher integrin αvβ3 binding affinity than its unlabeled monovalent ligand, primarily due to its multivalency. This
compound visualized a murine tumor without removing unlabeled ligands, while a 99mTc-labeled monovalent probe derived from
a monovalent ligand could not. The metal coordination-mediated synthesis of radiolabeled multivalent probes thereby can be a
useful approach for preparing ready-to-use target-specific probes labeled with 99mTc and other metallic radionuclides of interest.
conjugate (hereafter referred as “ligand”) and hydrolysis of
the radiometal at extremely low radiometal concentrations (for
example 99mTc ≤ 1 μM).10 Therefore, a large excess of ligand
over metallic radionuclide is used to ensure high radiochemical
yields in short reaction times. As a result, the reaction solution
contains an excess of unlabeled ligands along with the objective
radiometal-labeled probes (Figure 1).
Most metallic radiopharmaceuticals currently used in clinical
settings are designed to image large capacity nonsaturable
systems such as bone, glomerular filtration, cerebral, or
myocardial perfusion, and the presence of unlabeled ligand in
the injectate does not affect the target localization of these
probes.12 When imaging saturable systems in the brain such as
neuronal receptors or transporters, the presence of unlabeled
ligand does not compete since the radiolabeled probes are
designed to achieve much higher penetration through the
blood-brain-barrier than their unlabeled charged ligands.13
However, when designing radiolabeled probes for saturable
systems in the peripheral tissues, the presence of unlabeled
ligand can compete with the radiolabeled probe for the targeted
INTRODUCTION
■
Radiolabeled molecular imaging probes have now become a
powerful tool for noninvasive monitoring of biological
processes at the cellular and/or molecular level.1−5 In these
radiolabeled probes, targeting vectors possessing high affinities
to specific biological targets are conjugated with gamma (γ) or
positron (β+) emitting radionuclides suitable for single photon
emission computed tomography (SPECT) or positron
emission tomography (PET) imaging, respectively. Among
the radionuclides, metallic radionuclides such as technetium-
99m (99mTc), gallium-67/68, and indium-111 have an
advantage over nonmetallic radionuclides such as carbon-11
and fluorine-18, as these metallic radionuclides are available via
a generator system and/or can provide objective radiolabeled
probes in radiochemical yields sufficient for administration to
subjects without purification.6−11 Indeed, most radiometal-
labeled probes used in clinical practice today are prepared in
>95% radiochemical yields and administered to patients
without any purification steps, which allows widespread
applications of the radiometal-labeled probes for molecular
imaging.
The synthesis of radiometal-labeled probes is a competitive
reaction between complexation with a chelators-vector
Received: January 7, 2016
© XXXX American Chemical Society
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Figure 1. A conceptual illustration of the three different molecular design strategies. (a) In a monovalent “1 to 1” design, both an unlabeled ligand
and a final radiolabeled probe possess the same monovalent targeting vector and display similar binding affinities to a target molecule. Upon in vivo
injection, the presence of the unlabeled ligand reduces the in vivo target uptake of the radiolabeled probe due to the competitive inhibition by the
unlabeled ligand. (b) In a multivalent “1 to 1” design, both a radiolabeled probe and an unlabeled ligand display higher binding affinities to a target
molecule due to the multivalent effect. The improved in vivo targeting of the radiolabeled probe is diminished in vivo due to the presence of the high
affinity unlabeled ligand in the injectate, resulting in the lower accumulation of the radiolabeled probe at the target. (c) In the in situ multivalent “1 to
3” design, an unlabeled monovalent ligand upon metal complexation results in a multivalent radiolabeled probe that has acquired in situ trivalency of
a targeting vector and displays higher binding affinity than its unlabeled monovalent construct. This “acquired” multivalency of the radio-labeled
probe results in higher affinity for the target and reduces the competitive inhibition from the unlabeled ligand, thus displaying enhanced in vivo target
localization.
molecule and significantly decreases the accumulation of the
radiolabeled probes at the target site. This is primarily due to
the presence of much higher amount of the unlabeled ligand
and similar binding affinity between the radiometal-labeled
probe and the unlabeled ligand14,15 (Figure 1a,b). This
competitive inhibition can be eliminated by a postlabeling
removal of the unlabeled ligands by HPLC or other purification
procedures. However, these additional steps significantly impair
the practical utility and advantages of the metallic radio-
nuclides.14
To address the issue, we envisioned a novel molecular design
using the unique behavior of metals to serve as a template for
coordinating multiple monovalent ligands (Figure 1c). Since
multivalent probes possessing multivalent targeting moieties on
a molecule exhibit higher binding affinities to the target than
their monovalent counterparts,16,17 the resulting multivalent
probes would decrease the level of competitive inhibition at the
target in the presence of unlabeled monovalent ligand. To
examine the validity of this in situ multivalent approach, we
applied the concept to the radiometal technetium-99m (99mTc),
the most widely available and frequently used radiometal in
diagnostic nuclear medicine. We then chose an isonitrile (CN-
Figure 2. Chemical structures of L1 (monovalent “1 to 1” design), L2
R)−[99mTc(CO)3]+ core combination as a model, because an
(in situ multivalent “1 to 3” design), and their metal complexes. Both
isonitrile group strongly coordinates the [99mTc(CO)3]+ core in
L1 and L2 act as monovalent ligands. Upon metal complexation, L1
generates a monovalent radiolabeled probe, M-[L1], while L2 results
in a trivalent radiolabeled probe, M-[L2]3.
a monodentate fashion with a metal to chelator ratio of
1:3.18−20 The cyclic RGDfK (-Arg-Gly-Asp-D-Phe-Lys-;
21
cRGDfK) peptide, a specific antagonist for integrin αvβ3
[
99mTc(CO)3(L2)3]+ (Figure 2, 99mTc-[L2]3). We introduced
was also selected as a model of a specific targeting vector. The
c(RGDfK) peptide was conjugated to the carboxyl residue of
terminal isonitrile β-Ala-Gly-Gly peptide to synthesize the
monovalent targeting ligand CN-β-Ala-Gly-Gly-c(RGDfK)
(Figure 2, L2) and its 99mTc-labeled trivalent complex,
the Gly-Gly spacer at each RGD motif to provide the
recommended distance between the targeting vector posi-
tion22,23 and to enhance the hydrophilicity of the molecule. To
comparatively assess the in situ multivalent “1 to 3” design
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a
Scheme 1. Synthesis of L2
a
Reagents and conditions: (a) formic acid, acetic anhydrate, 60%; (b) 2,3,5,6-tetrafluorophenol, DCC, DMF, 53%; (c) Burgess reagent, CH2Cl2,
83%; (d) di-tert-butyl dicarbonate, water, dioxane, 1 N NaOHaq, 42%; (e) c[R(pbf)GD(tBu)fK], DIC, HOBt, DMF; (f) TFA, TES, water, 59% over
steps e−f; (g) DIPEA, DMF, 49%.
(Figure 1c) against the conventional monovalent “1 to 1”
design methods (Figure 1a), the c(RGDfK) peptide was
separately conjugated with a cysteine derivative, which is known
to form a stable complex with [99mTc(CO)3]+ core in a 1:1
molar ratio24 (Figure 2, L1).
50 μM of L1 (20 nmol/400 μL) and heating at 110 °C for 30
min in a heating block. The HPLC retention times of the two
99mTc complexes were identical to the corresponding macro-
scopic and nonradioactive Re complexes characterized and
verified by ESI-MS separately (Figures 3 and S1).
The log D7.4 values of 99mTc-[L1] and 99mTc-[L2]3 were
−2.16 0.03 and −1.18 0.01, respectively.
RESULTS
■
Stability Assessment. The high stability of 99mTc-[L1] in
plasma solution has been reported elsewhere,24 whereas the
stability of 99mTc-[L2]3 was assessed in a 10 mM histidine
solution or in murine plasma at 37 °C. Under both conditions,
TLC analyses revealed more than 95% of 99mTc-[L2]3 remained
intact after 6 h incubation (Table 1). Additional HPLC analyses
Synthesis. The synthesis of the isocyanide-conjugated
c(RGDfK) (L2) is illustrated in Scheme 1. First, β-alanine
(β-Ala) was N-formylated, and its carboxylic acid was converted
to 2,3,5,6-tetrafluorophenol (TFP) ester. The formyl-β-Ala-
TFP was then treated with burgess reagent25 to provide
isocyano-β-Ala-TFP. This isocyanide-containing active ester
was conjugated with H2N-Gly-Gly-c(RGDfK) peptide where
the Gly-Gly spacer is introduced at the ε-amine residue of Lys
(K) in the peptide. The peptide construct CN-βAla-GlyGly-
c(RGDfK) was subsequently purified via preparative HPLC.
99mTc-Labeled Probes. The precursor complex [99mTc-
(OH2)3(CO)3]+ was prepared in >95% yields as described
previously26 with slight modification (see Experimental
Section). The 99mTc-[L1] and 99mTc-[L2]3 complexes were
prepared by mixing a L1 or L2 solution with freshly prepared
Table 1. In Vitro Stability of 99mTc-[L2]3 in 10 mM Histidine
a
Solution and Murine Plasma
percent of intact
time (h)
10 mM histidine
murine plasma
1
6
96.3 0.9
96.1 0.9
96.8 0.5
96.6 0.2
a
Results are expressed as mean SD of three experiments. Percent of
[
99mTc(OH2)3(CO)3]+, followed by heating the reaction
intact was determined by TLC analysis.
mixture. The optimized radiolabeling conditions to prepare
99mTc-[L2]3 were determined to be 150 μM of L2
concentration (40 nmol/265 μL), pH 6.0 at 110 °C for 20
min, followed by 100 °C for 20 min in a heating block. These
conditions consistently provided over 95% radiochemical yields
of the 99mTc-[L2]3 complex (Figure 3). Similarly, 99mTc-[L1]
was synthesized over 95% radiochemical yields at pH 6.0, using
performed at 6 h postincubation for both conditions indicated
no degradation of 99mTc-[L2]3 (>99%) (Figure 4a,b). The
recovery rate of radioactivity from plasma sample to super-
natant solution was 63.5
1.4% (n = 3) upon addition of
EtOH and protein precipitation.
Figure 4. In vitro stability of 99mTc-[L2]3 in histidine solution and
murine plasma. (a) HPLC analysis 6 h postincubation (a) in 10 mM
histidine solution and (b) in murine plasma. Plasma proteins were
precipitated with EtOH prior to HPLC analysis.
Figure 3. HPLC analysis of 99mTc-[L2]3 coinjected with Re-[L2]3. The
retention time of 99mTc-[L2]3 was identical to that of Re-[L2]3, which
was verified by ESI-MS.
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Binding Affinity to Integrin αvβ3. The integrin αvβ3
binding affinities of L1, Re-[L1], L2, Re-[L2]3, and the
c(RGDyV) ligand were determined by displacement of 125I-
c(RGDyV) bound to integrin αvβ3 positive U87MG human
glioma cells at 37 °C (Figure 5). The IC50 values calculated
(without L1); and (5) unpurified 99mTc-[L1] containing 5
nmol of L1. The results are summarized in Table 3. The tumor
accumulation displayed the following order: purified 99mTc-
[L2]3 > unpurified 99mTc-[L2]3 > purified 99mTc-[L1] >
purified 99mTc-[L2]3 + Re-[L2]3 > unpurified 99mTc-[L1].
The tumor uptake of 99mTc-[L2]3 was significantly reduced in
the presence of nonradioactive Re-[L2]3, an illustration of the
integrin αvβ3 receptor blocking and specificity of 99mTc-[L2]3 in
vivo. Unpurified 99mTc-[L2]3 displayed significantly higher
tumor uptake and tumor to background ratios comparatively
not just to unpurified 99mTc-[L1] but also to purified 99mTc-
[L1], demonstrating the advantage of “1 to 3” design strategy
over the conventional “1 to 1” design.
SPECT Imaging. Figure 6 illustrates the SPECT/CT
imaging of tumor-bearing mice administered with unpurified
Figure 5. In vitro inhibition curves of 125I-c(RGDyV) bound to
U87MG glioma cells by L1, Re-[L1], L2, Re-[L2]3, and c(RGDyV) at
37 °C.
from the displacement curves of 125I-c(RGDyV) are illustrated
in Table 2. Both Re-[L1] and L1 showed similar integrin αvβ3
receptor affinities, while Re-[L2]3 displayed 8-fold higher
binding affinity for integrin αvβ3 than its monovalent ligand L2.
Table 2. IC50 Values of Each Compound
a
Figure 6. SPECT/CT images of U87MG tumor-bearing male nude
mice over 45−75 min post i.v. injection of (a) 7.4 MBq of 99mTc-[L2]3
coinjected with 5 nmol of L2 and (b) 9.6 MBq of 99mTc-[L1]
coinjected with 5 nmol of L1. Images are shown at the same signal
intensity scale. Red circle indicates the U87MG tumor.
design
compound
IC50, nM
95% C.I.
L1
43.3
38.4
25.9
3.16
41.2
34.4−54.5
28.4−51.9
19.9−33.7
2.14−4.68
34.9−48.7
“1 to 1”
Re-[L1]
L2
“1 to 3”
Re-[L2]3
c(RGDyV)
reference
a
C.I. = confidential interval.
99mTc-[L2]3 (7.4 MBq) containing 5 nmol of L2 (Figure 6a)
and a comparative unpurified 99mTc-[L1] (9.6 MBq) containing
5 nmol of L1 (Figure 6b) over 45−75 min postinjection. Clear
visualization of the tumor was demonstrated in the “1 to 3”
design (Figure 6a), whereas tumor was barely visualized in the
“1 to 1” design (Figure 6b). These images correlated well with
the biodistribution results at the similar time point.
Biodistribution. The biodistribution of radioactivity at 1 h
postadministration in nude mice bearing U87MG xenografts
was determined in the following 5 samples: (1) HPLC-purified
99mTc-[L2]3 (without L2); (2) HPLC-purified 99mTc-[L2]3 in
the presence of 5 nmol Re-[L2]3; (3) unpurified 99mTc-[L2]3
containing 5 nmol of L2; (4) HPLC-purified 99mTc-[L1]
a
Table 3. Biodistribution at 1 h Postinjection in Tumor-Bearing Mice
99mTc-[L2]3 (“1 to 3” design)
99mTc-[L1] (“1 to 1” design)
purified
unpurified
purified + Re-[L2]3
purified
unpurified
L2 = 0 nmol
L2 = 5 nmol
Re-[L2]3 = 5 nmol
L1 = 0 nmol
L1 = 5 nmol
blood
liver
0.30 0.03
2.13 0.50
10.91 0.57
0.77 0.07
6.28 0.34
0.73 0.21
0.33 0.08
1.38 0.18
10.66 1.29
0.79 0.47
4.27 0.79
0.30 0.04
3.80 0.57
12.17 4.01
2.77 0.35
12.98 2.80
0.67 0.04
1.39 0.14
7.37 0.60
0.64 0.26
4.52 1.38
0.24 0.04
0.50 0.07
7.63 1.05
3.84 0.22
0.89 0.39
25.62 3.88
0.30 0.03
0.97 0.10
6.83 0.95
3.02 0.48
1.54 1.01
28.52 4.14
0.18 0.05
kidney
stomach
b
b
intestine
muscle
c
d
d
e
d
tumor
7.18 0.60
24.06 3.89
3.58 1.10
10.37 2.48
1.80 0.14
2.77 0.51
5.55 0.48
0.36 0.06
9.12 1.43
1.14 0.11
1.18 0.08
0.17 0.01
6.90 1.60
T/blood
T/liver
2.70 0.20
1.31 0.20
7.67 1.68
T/muscle
a
b
c
The results were expressed as percent injected dose per gram (%ID/g) SD (n = 5). Expressed as %ID. Statistical analysis was performed using
d
e
one-way analysis of variance followed by Tukey’s multiple-comparison test (different from 99mTc-[L2]3 (L2 = 5 nmol), p < 0.001. Different from
99mTc-[L2]3 (L2 = 5 nmol), p < 0.05).
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solution and in murine plasma. More than 95% of 99mTc-[L2]3
remained intact for 6 h in 10 mM histidine solutions as well as
in murine plasma (Table 1, Figure 4a,b), suggesting high in vivo
stability of 99mTc-[L2]3.35 The high stability of 99mTc-[L2]3
against a ligand exchange reaction is consistent with the fact
that the coordination of an isonitrile to [99mTc(CO)3]+ core
provides a kinetically inert complex against the substitution due
to the strong bonding of an isonitrile toward a low-valent metal
center.36 These chemical features were indeed one of the bases
for choosing an isonitrile as the coordinating moiety in our
present “1 to 3” design strategy.
DISCUSSION
■
High and specific in vivo target uptake is a fundamental
requirement for molecular-targeted radiolabeled probes to be
practical as a molecular imaging tool. Thus, the concept of the
multivalent effect has been applied to develop radiolabeled
probes of high and specific in vivo targeting abilities, and the
effectiveness of this design has been well demonstrated in many
targeting vectors such as RGD peptides,15,22,23,27−29 bomb-
esin,30 somatostatine,31 and Lys-Glu urea motif,32 whose
corresponding target molecules are known to overexpress in
various pathological conditions.
Figure 5 and Table 2 illustrate the in vitro integrin αvβ3
binding affinities in cell-binding assays of the metal complexes
(Re-[L1] and Re-[L2]3) and the uncomplexed ligands (L1, L2).
Re-[L2]3, a trivalent RGD metal complex, displayed an order of
magnitude (ca. 8) higher binding affinity than its unlabeled
ligand L2 (3.16 vs 25.9 nM), while in the “1 to 1” design, both
the metal complex (Re-[L1]) and its unlabeled ligand (L1)
showed similar IC50 values (38.4 vs 43.3 nM). The enhanced
binding affinity of Re-[L2]3 is attributable to the presence of
trivalent RGD motif in Re-[L2]3 which is similar in magnitude
to trivalent RGD peptides reported previously.28 This clearly
demonstrates that increased binding affinity toward a targeted
molecule can be achieved by metal complexation of multiple
monovalent ligands such that the metal complex is transformed
and acts as a multivalent species.
When a ligand containing multivalent targeting vectors is
chelated with a metallic radionuclide of interest (multivalent “1
to 1”; Figure 1b), the radiolabeled probe possesses enhanced
binding affinity to the target molecule. However, the unlabeled
ligand present in the solution can result in a greater level of in
vivo competition at the target site, since both the labeled probe
and the unlabeled ligand possess similar binding affinities,
which would paradoxically impair the improved binding affinity
of the radiolabeled multivalent probe and again compel
postlabeling purification steps. Radiolabeling of a monovalent
targeting vector (monovalent “1 to 1”; Figure 1a), on the other
hand, would display a lower in vivo targeting ability, although
the degree of competitive inhibition by the unlabeled ligand
observed here would not be as significant as observed in the
multivalent “1 to 1” design.
The biodistribution studies in tumor bearing nude mice were
conducted to compare the in vivo targeting potential and
pharmacokinetics of the 99mTc-labeled probes derived from the
“1 to 1” design and the in situ multivalent “1 to 3” design
(Table 3). Although the postlabeling purification is not
compatible with the original purpose in this study, we included
the HPLC-purified 99mTc-labeled probes into the biodistribu-
tion studies to elucidate the in vivo behavior of the 99mTc-
labeled probes free from the competitive inhibitions caused by
the presence of the unlabeled ligands. The higher tumor uptake
of purified 99mTc-[L2]3 than that of purified 99mTc-[L1] (7.18
The in situ multivalent “1 to 3” design presented herein
(Figure 1c) differs from the two approaches (monovalent and
multivalent “1 to 1”) as the multivalency itself is induced only
upon complexation to the radiometal and not from the
uncomplexed ligand, thus preserving the significantly greater
binding affinity of the resulting radiolabeled multivalent probe
and diminishing the inhibition by the presence of the unlabeled
ligand.
Since the quantitative formation of the mixed ligand complex
is a prerequisite for the in situ multivalent “1 to 3” design, we
initially verified that the model L2 ligand coordinates the
[
99mTc(CO)3]+ metal core with a metal to ligand ratio of 1:3 to
0.60 vs 2.77
0.51%ID/g) is consistent with the results
form [99mTc(CO)3(L2)3]+ (99mTc-[L2]3) in high radiochemical
yield. To establish the identity of the tracer 99mTc-[L2]3
complex on macroscopic scale, we also synthesized and
characterized the nonradioactive rhenium analogue, since
technetium and rhenium share similar coordination chemistry
due to their group homology, such that rhenium complexes are
routinely used as nonradioactive surrogates of the respective
99mTc-complexes.33,34 The HPLC profile in the bottom of
Figure 3 shows that heating the reaction mixture of L2 and
obtained in in vitro binding study where Re-[L2]3 showed
higher integrin αvβ3 binding affinity than did Re-[L1].
Additionally, the tumor uptake of 99mTc-[L2]3 was significantly
blocked with the co-injection of 5 nmol Re-[L2]3 to 1.80
0.14%ID/g, demonstrating that the accumulation of 99mTc-
[L2]3 in the tumor was indeed integrinαvβ3 specific. The
biodistribution results of the unpurified 99mTc-labeled probes,
where 5 nmol of the unlabeled ligands were co-injected,
represent a practical clinical situation where 99mTc-labeled
probes are administered to subjects without purification.
Though the tumor uptake of 99mTc-[L2]3 was partially blocked
by the presence of the unlabeled monovalent ligand, the tumor
uptake of this unpurified 99mTc-[L2]3 was still ca. 3-fold higher
than that of unpurified 99mTc-[L1] and was also higher than
that of purified 99mTc-[L1] (3.80 0.57, 1.14 0.11, and 2.77
0.51%ID/g, respectively), thus demonstrating the enhanced
in vivo targeting and localization ability of the “1 to 3” designed
99mTc-labeled probe compared to the conventional monovalent
“1 to 1” designed approach. In vivo SPECT/CT imaging
studies similar to the clinical scenario were also conducted in a
murine tumor model by injecting the unpurified 99mTc-labeled
probes. Figure 6 depicts the comparative SPECT/CT images
obtained in the two approaches and also support the superiority
of the “1 to 3” design strategy.
[
99mTc(CO)3(OH2)3]+ complex produced a single radioactive
peak with >95% radiochemical yields. This peak was identified
as the objective mixed ligand complex 99mTc-[L2]3 by
comparative HPLC retention time profile with that of rhenium
counterpart (Figure 3, Re-[L2]3), indicating that the isonitrile−
[
99mTc(CO)3]+ core combination is an appropriate model
platform that induces in situ multivalency of a targeting vector
upon complexation reaction. The ligand L1, which is a model
ligand for the monovalent “1 to 1” conventional approach, has
been previously described24 and coordinated the [99mTc-
(CO)3]+ core with a metal to ligand ratio of “1:1” to form
the [99mTc(CO)3(L1)] complex (Figure 2 99mTc-[L1], Figure
S1).
In vitro stability studies of the mixed ligand complex 99mTc-
[L2]3 were conducted in a physiological ligand (histidine)
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In comparing tumor uptake of unpurified 99mTc-[L2]3 and
purified 99mTc-[L2]3 + Re-[L2]3, it is also worth noting that the
purified 99mTc-[L2]3 + Re-[L2]3 condition acts as a mimic of a
scenario of a multivalent “1 to 1” design where the unlabeled
ligand is a trivalent RGD and can compete with the 99mTc-
labeled trivalent RGD (Figure 1b). The higher tumor uptake of
unpurified 99mTc-[L2]3 than that of purified 99mTc-[L2]3 + Re-
[L2]3 thereby indirectly demonstrates that the in situ
multivalent “1 to 3” design strategy is superior in in vivo
targeting potential not only to the monovalent “1 to 1” design
strategy (Figure 1a) but also to the multivalent “1 to 1” design
strategy (Figure 1b).
While these studies illustrate the potential for this novel “1 to
3” multivalent design to provide higher-affinity imaging probes
upon metal complexation, it is with the caveat that the results
obtained here are all based on the specific experimental
conditions (cell line, animal model, amount of unlabeled ligand,
etc.), and the expression level of target proteins37 and/or the
amount of unlabeled ligands38 can intricately affect the degree
of competitive inhibition on the target uptake of such
radiolabeled probes. Thus, careful consideration should be
taken when this design is expanded or applied to other systems,
experimental models, or conditions. Nevertheless, this study for
the first time has demonstrated the usefulness and potential of
the in situ multivalent strategy for the development of target-
specific metal-based radiolabeled probes that exhibit high target
uptake despite the presence of unlabeled ligand. Thus, this
strategy would enable the preparation of radiolabeled probes of
high target uptake without postlabeling purification steps,
which may accelerate the practice of molecular imaging by
radiolabeled probes in preclinical and/or clinical settings.
systems (1−9) were used for various preparative and analytic studies
and are detailed in the Supporting Information.
Boc-Gly-Gly-OH (2). H-GlyGly-OH (50.0 mg, 0.38 mmol) was
dissolved in dioxane/water/1N NaOHaq (760 μL/380 μL/380 μL),
and di-tert-butyl dicarbonate (92.0 mg, 0.42 mmol) was added to this
mixture in an ice bath. The reaction mixture was stirred at room
temperature for 1 h, and the solvent was evaporated in vacuo. The
residue was dissolved in water and washed with hexane. The pH was
adjusted to 2 using 5% aqueous citric acid and extracted with ethyl
acetate, and the organic layer was dried over magnesium sulfate. After
removing the solvent in vacuo, the residue was purified with silica gel
column chromatography (chloroform/methanol/acetic acid =10/1/
1
0.1) to afford compound 2 as a white powder (39.6 mg, 42%). H
NMR (400 MHz, CD3OD): δ 1.46 (s, 9H, tBu), 3.76 (s, 2H, CH2),
3.92 (s, 2H, CH2COOH). ESI-MS, m/z: 255.10 [M + H]+, found
255.10.
H-Gly-Gly-c(RGDfK) (3). The compound 1 (78.5 mg, 0.086 mmol),
2 (20.0 mg, 0.086 mmol), and HOBt (33.0 mg, 0.216 mmol) was
dissolved in DMF (1 mL) and stirred in an ice bath for 10 min. To this
stirred mixture, DIC (33 μL, 0.216 mmol) was added dropwise and
stirred in an ice bath for 10 min, followed by stirring at room
temperature for 2 h. After removing the solvent in vacuo, the residue
was redissolved in TFA/TES/water (9 mL/0.5 mL/0.5 mL) and
stirred at room temperature for 3 h. The solvent was evaporated in
vacuo, and diethyl ether was added to form the precipitate. The
precipitate was washed with diethyl ether 3 times, purified by
preparative HPLC using system 5, and lyophilized to obtain
compound 3 as a white powder (2 steps, 36.2 mg, 58.5%). ESI-MS,
m/z: 718.36 [M + H]+, found 718.33.
Formylamino-β-alanine-OH (4). β-alanine (5.00 g, 56.2 mmol) was
dissolved in formic acid/acetic anhydrate (25 mL/13 mL) and stirred
at 95 °C under N2 atmosphere for 3 h. After the solvent was
evaporated in vacuo, the residue was purified with silica gel column
chromatography (chloroform/methanol = 5/1) to afford compound 4
as a white powder (3.97 g, 60.1%). 1H NMR (400 MHz, D2O): δ 2.43
(t, 2H, CH2CO), 3.30 (t, 2H, NHCH2), 7.83 (s, 1H, formylamino).
ESI-MS, m/z: 118.05 [M + H]+, found 118.09.
CONCLUSION
■
We have developed an in situ multivalent “1 to 3” design to
prepare 99mTc-labeled trivalent probes from isonitrile-derivat-
ized monovalent ligands without postlabeling purification steps.
This design concept would provide a kit formulation for on-site
synthesis of target-specific 99mTc-labeled trivalent probes, which
allows the full appreciation of the advantage of the metallic
radionuclide. In addition, this design may not only be limited to
the isonitrile−[99mTc(CO)3]+ core combination but may also
be applicable to other ligand−metal combinations that form
mixed ligand complexes of high in vivo stability. Similarly, as
the multivalent effect is a universal phenomenon among many
kinds of small molecule−protein interactions, this design could
be expanded to a variety of biomolecules for molecular imaging
and/or targeted radionuclide therapy.
2,3,5,6-Tetrafluorophenyl Formylamino-β-alanine (5). 4 (1.00 g,
7.6 mmol) and TFP (1.39 g, 8.4 mmol) were dissolved in DMF (10
mL) and stirred in an ice bath for 10 min. To this stirred mixture,
DCC (1.72 g, 8.4 mmol) was added and stirred in an ice bath for 10
min, followed by stirring at room temperature overnight. After
removing dicyclohexylurea by filtration, the solvent was removed in
vacuo and purified with silica gel column chromatography (ethyl
1
acetate) to afford compound 5 as a white powder (1.21 g, 53%). H
NMR (400 MHz, CDCl3): δ 2.95 (t, 2H, CH2CO), 3.68 (q, 2H,
NHCH2), 6.25 (br, 1H, NH), 7.00 (m, 1H, Harom), 8.17 (s, 1H,
formylamino). ESI-MS, m/z: 266.04 [M + H]+, found 266.04.
2,3,5,6-Tetrafluorophenyl isocyano-β-alanine (6). The compound
5 (0.30 g, 1.1 mmol) and Burgess reagent (0.26 g, 1.1 mmol) were
dissolved in dichloromethane (10 mL) and stirred at 50 °C for 3 h.
After removing the solvent in vacuo, the residue was purified with silica
gel chromatography (chloroform/acetonitrile = 3/1) to afford
compound 6 as a white powder (0.23 g, 83.1%). 1H NMR (400
MHz, CDCl3): δ 3.11 (t, 2H, CH2CO), 3.81 (t, 2H, NCH2), 7.02 (m,
1H, Harom). 13C NMR (100 MHz, CDCl3): δ 33.37 (s, CH2CO), 36.71
(t, NCH2), 103.74 (t, CaromH), 128−147 (multiple peaks, CaromF),
158.61 (s, CN), 165.58 (s, CO).
CN-βAla-GG-RGD (L2) (7). The compound 3 (9.6 mg, 13.4 μmol)
and DIPEA (3.4 μL, 20 μmol) were dissolved in DMF (200 μL) and
stirred in an ice bath for 10 min. The compound 6 (4.0 mg, 16 μmol)
was added to this mixture and stirred at room temperature for 1 h. The
precipitate formed upon addition of diethyl ether was collected,
purified with preparative HPLC using system 3, and lyophilized to
obtain compound 7 as a white powder (5.2 mg, 49%). The >95%
purities were determined by HPLC using system 9 (Figure S2). ESI-
MS, m/z: 799.39 [M + H]+, found 799.44.
EXPERIMENTAL SECTION
■
General. All commercially available chemicals were of analytical
grade and were used without further purification. Fmoc-protected
amino acids and H-Gly-Trt (2-Cl) resin were purchased from
Watanabe Chemical Industries, Ltd. (Hiroshima, Japan). c[R(pdf)-
GD(tBu)fK] (1) and [Re(CO)3(OH2)3]Br (10) were synthesized
according to the previous report.21,39 All other reagents were
purchased from Wako Pure Chemical Industries, Ltd. (Tokyo).
Technetium-99m as Na99mTcO4 was eluted in saline solution on a
99
daily basis from a
Mo/99mTc generator (Ultra-Techne Kow,
FUJIFILM RI Pharma Co., LTD., Tokyo). Mass spectrometry was
carried out using an AccuTOF LC-plus (JMS-T100LP, JEOL Ltd.,
1
Tokyo). H NMR spectra was recorded on a JEOL ECS-400 (400
MHz) spectrometer (JEOL Ltd., Tokyo). All compounds tested in
inhibition assay, biodistribution study, and SPECT study were
prepared in >95% purities as determined by HPLC. Different HPLC
L-Cysteine, S-(2-carboxyethyl)-N-(tert-butoxycarbonyl), 1-methyl
ester (8). N-(tert-butoxycarbonyl)-L-cysteine methyl ester (117.7 mg,
0.5 mmol) and DIPEA (350 μL, 2 mmol) were dissolved in DMF (150
F
DOI: 10.1021/acs.jmedchem.6b00024
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
μL) and stirred in an ice bath for 10 min. To this stirred mixture, 3-
iodo-propionic acid (100 mg, 0.5 mmol) dissolved in DMF (150 μL)
was added and stirred in an ice bath for 10 min, followed by stirring at
room temperature for 3 h. After removing the solvent in vacuo, the
residue was purified with silica gel column chromatography (chloro-
form/methanol/acetic acid = 30/1/0.1) to afford compound 8 as a
of n-octanol and 25 mM phosphate buffer (pH 7.4) pre-incubated for
24 h to reach equilibrium. The mixtures were vortexed for 1 min,
followed by being left to stand for 1 min. This procedure was repeated
3 times. The mixtures were then centrifuged at 3000 rpm for 5 min.
The samples from both n-octanol and aqueous layers were obtained
and counted in an autowell γ counter. The log D7.4 values were
reported as mean SD of 3 experiments.
1
pale yellow powder (93.8 mg, 61%). H NMR (400 MHz, CDCl3): δ
Stability Assessment. The unlabeled ligand in 99mTc-[L2]3 was
removed by the HPLC using system 6. 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 μL of
the 99mTc-[L2]3 solution was mixed with 190 μL of histidine solution
to reach the final histidine concentration of 10 mM. Similarly 10 μL of
this 99mTc-[L2]3 solution was mixed with 190 μL of freshly prepared
murine plasma. After incubation at 37 °C for 1 and 6 h, 1 μL of
aliquots were drawn and analyzed with radio-TLC (n = 3) (Silica gel
60 F254, Merck Ltd., Tokyo) developed with MeOH:10% w/w
AcONH4 in water = 1:1. For HPLC analysis of the histidine sample,
the incubation solution was directly analyzed by radio-HPLC using
system 6. For HPLC analysis of the plasma sample, 200 μL of EtOH
was added to 100 μL of the plasma incubate to precipitate the
proteins. The sample was centrifuged at 15,000 g for 5 min at 4 °C.
The supernatant was collected, and the pellet was washed with 300 μL
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 6. The recovery rate of
radioactivity from the plasma sample was determined by following
equation, (the radioactivity of the supernatant)/(the original radio-
activity of the plasma sample) × 100 (n = 3). The radioactivity was
determined using an autowell γ counter (WIZARD 1480, PerkinElmer
Japan Co., Ltd., Yokohama, Japan).
1.45 (s, 9H, tBu), 2.64 (t, 2H, CH2CO), 2.80 (t, 2H, SCH2), 3.00 (br,
2H, CH2S), 3.77 (s, 3H, OCH3), 4.54 (br, 1H, α-proton). ESI-MS, m/
z: 330.10 [M + Na]+, found 330.09.
3-L-Cysteine Propionic Acid-c(RGDfK), 1-Methyl Ester (L1) (9). The
compound 1 (21 mg, 0.023 mmol), 8 (7.8 mg, 0.025 mmol), and
HOBt (8.44 mg, 0.063 mmol) were dissolved in DMF (200 μL) and
stirred in an ice bath for 10 min. To this stirred mixture, DIC (8.8 μL,
0.063 mmol) was added and stirred in an ice bath for 10 min, followed
by stirring at room temperature for 1 h. After removing the solvent in
vacuo, the residue was redissolved in TFA/TES/water (9 mL/0.5 mL/
0.5 mL) and stirred at room temperature for 3 h. The solvent was
evaporated in vacuo, and diethyl ether was added to form the
precipitate. The precipitate was washed with diethyl ether 3 times,
purified by preparative HPLC using system 4, and lyophilized to
obtain compound 9 as a white powder (2 steps, 3.6 mg, 18%). The
>95% purities were determined by HPLC using system 6 (Figure S4).
ESI-MS, m/z: 793.37 [M + H]+, found 793.39.
[Re(CO)3(L2)3]+ (Re-[L2]3) (11). The rhenium compound 10 (0.683
mg, 1.69 μmol) and 7 (L2, 13.5 mg, 16.9 μmol) were dissolved in 0.1
M A.B. (pH 6.0, 1 mL). The mixture was heated at 90 °C for 3 h and
purified by preparative HPLC using system 1. The fractions containing
the desired product were collected and lyophilized to obtain
compound 11 as a white powder (4.1 mg, 90%). The >95% purities
were determined by HPLC using system 6 (Figure S3). ESI-MS, m/z:
1334.04 [M + H]2+, found 1334.41
Binding Affinity to Integrin αvβ3. U87MG human glioma cells
were grown in Dulbecco’s modified 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/mL, Sigma-Aldrich Japan K.K.) at
37 °C in humidified atmosphere containing 5% CO2. Multiscreen DV
filter plates (Merck Millipore, MA) were seeded with 2 × 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
incubated at 37 °C with 125I-c(RGDyV) in the presence of increasing
concentrations of c(RGDyV), L1, Re-[L1], L2, or Re-[L2]3. The total
incubation volume was adjusted to 200 μL. After incubatation for 1 h
at 37 °C, the plates were filtered and washed twice with 200 μL of the
ice-cold binding buffer. The polyvinylidene fluoride filters were
collected, and the radioactivity was determined using an autowell γ
counter. The IC50 values were calculated by fitting the data by
nonlinear regression using GraphPad Prism (GraphPad Software, Inc.,
San Diego, CA). All the binding experiments were carried out with
quadruplet samples.
Animal Model. Animal studies were conducted in accordance with
the institutional guidelines approved by the Chiba University Animal
Care Committee. BALBc nu/nu male mice (Japan SLC, Inc., Shizuoka,
Japan) 6 weeks old were xenografted by subcutaneous injection of
U87MG human glioblastoma cells (5 × 106 cells/50 μL of culture
medium) into their right hind legs. The mice were subjected to
biodistibution studies as well as SPECT/CT imaging studies when the
tumor weight reached 0.1−0.5 g.
Biodistribution. Male nude mice bearing tumor xenografts of
U87MG were injected via the tail vein with 100 μL (11.1 kBq) of
99mTc-[L1] (L1 = 5 nmol) as the monovalent “1 to 1” design, 99mTc-
[L2]3 (Re-[L2]3 = 5 nmol) as the multivalent “1 to 1” design, or
99mTc-[L2]3 (L2 = 5 nmol) as the “1 to 3” design. In case of
multivalent “1 to 1” design, Re-[L2]3 was added to the HPLC-purified
99mTc-[L2]3 solution. In addition, HPLC-purified 99mTc-[L1] and
99mTc-[L2]3 were also evaluated using similar methods. The animals
were sacrificed and dissected at 1 h after administration. The tissues of
interest were removed and weighed, and the radioactivity was
determined with an autowell gamma counter. The results are
[Re(CO)3(L1)] (Re-[L1]) (12). The rhenium compound 10 (3.77 mg,
9.33 μmol) and L1 (7.4 mg, 9.33 μmol) were dissolved in 0.1 M A.B.
(pH 6.0, 1 mL). The mixture was heated at 70 °C for 3 h and purified
by preparative HPLC using system 2. The fractions containing the
desired product were collected and lyophilized to obtain compound 12
as a white powder (2.3 mg, 23.5%). The >95% purities were
determined by HPLC using system 7 (Figure S5). ESI-MS, m/z:
1049.28 [M + H]+, found 1049.49.
[
99mTc(CO)3(OH2)3]+. [99mTc(CO)3(OH2)3]+ was prepared accord-
ing to a reported method with slight modifications.26 Briefly, 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
boranocarbonate [Na2(H3BCO2)] was purged with N2 atmosphere
−
before adding 300 μL 99mTcO4 (111 MBq) to a sealed vial. The
mixture was heated for 7 min at 100 °C, and the pH was adjusted to 6
with 1 N HClaq. The purities were determined by radio-HPLC using
system 6.
Preparation of 99mTc-[L2]3. A 100 μL (37 MBq) solution of
freshly prepared [99mTc(CO)3(OH2)3]+ was added to a vial containing
165 μL (0.1 M A.B., pH 6.0) of L2 solution to reach the final ligand
concentration of 150 μM (40 nmol/265 μL). The mixture was then
heated at 110 °C for 20 min, followed by 100 °C for 20 min under N2
atmosphere in a heating block. The radiochemical purities were
determined by radio-HPLC using system 6.
Preparation of 99mTc-[L1]. A 150 μL (55.5 MBq) solution of
freshly prepared [99mTc(CO)3(OH2)3]+ was added to a vial containing
250 μL (0.1 M A.B., pH 6.0) of L1 solution to reach the final ligand
concentration of 50 μM (20 nmol/400 μL). The mixture was heated at
110 °C for 30 min under N2 atmosphere in a heating block. The
radiochemical purities were determined by radio-HPLC using system
7.
Determination of Log D7.4 Values. 99mTc-[L1] and 99mTc-[L2]3
were purified by HPLC (systems 7 and 6, respectively), and the
eluents of the two compounds were evaporated in vacuo. Then, the
residues were dissolved in 25 mM phosphate buffer (pH 7.4) to
prepare 3.7 MBq/mL sample solutions. Ten μL (37 kBq) of each
sample solution was added to an equal volume mixture (3 mL:3 mL)
G
DOI: 10.1021/acs.jmedchem.6b00024
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Journal of Medicinal Chemistry
Article
presented as the percentage injected dose per gram (%ID/g) or
percentage injected dose per tissue (%ID). Values were expressed as
mean SD for a group of 5 animals.
(6) Liu, S.; Edwards, D. S. 99mTc-labeled small peptides as diagnostic
radiopharmaceuticals. Chem. Rev. 1999, 99, 2235−2268.
(7) Liu, S. Bifunctional coupling agents for radiolabeling of
biomolecules and target-specific delivery of metallic radionuclides.
Adv. Drug Delivery Rev. 2008, 60, 1347−1370.
Small Animal SPECT/CT Imaging Study. SPECT images were
acquired over 45−75 min after intravenous administration of 99mTc-
[L1] (100 μL, 9.6 MBq, L1 = 5 nmol) or 99mTc-[L2]3 (100 μL, 7.4
MBq, L2 = 5 nmol) to male BALBc nu/nu mice bearing U87MG
xenografts via the tail vein. CT scans were performed before SPECT
scans for anatomic reference. The mice were anaesthetized with 1−2%
(v/v) isoflurane (DS Pharma Animal Health, Osaka, Japan) and
positioned on the animal bed where anesthesia was continuously
delivered via a nose cone system. SPECT imaging and X-ray CT
imaging were performed by use of small animal SPECT/CT system
(Triumph LabSPECT4/CT, TriFoil Imaging Inc., Chatsworth, CA)
equipped with a five pinhole (0.5 mm) collimator. Data acquisition
was performed for 30 min at 30 s per projection with stepwise rotation
of 60 projections over 360°.
(8) Bartholomae, M. D.; Louie, A. S.; Valliant, J. F.; Zubieta, J.
Technetium and gallium derived radiopharmaceuticals: comparing and
contrasting the chemistry of two important radiometals for the
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development of diagnostic and therapeutic radiopharmaceuticals.
Dalton Trans. 2011, 40, 6112−6128.
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preparation of 99mTc radiotracers. Dalton Trans. 2011, 40, 6077−6086.
(11) Arano, Y. Recent advances in 99mTc radio pharmaceuticals. Ann.
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(12) Eckelman, W. C. Radiolabeling with technetium-99m to study
high-capacity and low-capacity biochemical systems. Eur. J. Nucl. Med.
1995, 22, 249−263.
ASSOCIATED CONTENT
■
(13) Kung, M. P.; Stevenson, D. A.; Plossl, K.; Meegalla, S. K.;
Beckwith, A.; Essman, W. D.; Mu, M.; Lucki, I.; Kung, H. F.
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00024.
[
99mTc]TRODAT-1: A novel technetium-99m complex as a dopamine
transporter imaging agent. Eur. J. Nucl. Med. 1997, 24, 372−380.
(14) Ballinger, J. R. The influence of carrier on 99mTc
radiopharmaceuticals. Q. J. Nucl. Med. 2002, 46, 224−232.
(15) Oxboel, J.; Brandt-Larsen, M.; Schjoeth-Eskesen, C.;
Myschetzky, R.; El-Ali, H. H.; Madsen, J.; Kjaer, A. Comparison of
two new angiogenesis PET tracers 68Ga-NODAGA-E[c(RGDyK)]2
and 64Cu-NODAGA-E[c(RGDyK)]2; in vivo imaging studies in
human xenograft tumors. Nucl. Med. Biol. 2014, 41, 259−267.
(16) Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Synthetic
multivalent ligands in the exploration of cell-surface interactions. Curr.
Opin. Chem. Biol. 2000, 4, 696−703.
HPLC methods, HPLC chromatograms of 99mTc-[L1]
and Re-[L1], purity checks and characterizations of the
compounds 7, 9, 11, 12, and 1H NMR and/or 13C NMR
of the compounds 4-6 (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +81-43-226-2896. E-mail: arano@chiba-u.jp. Fax: +81-
43-226-2897.
(17) Mammen, M.; Choi, S. K.; Whitesides, G. M. Polyvalent
interactions in biological systems: Implications for design and use of
multivalent ligands and inhibitors. Angew. Chem., Int. Ed. 1998, 37,
2754−2794.
Notes
The authors declare no competing financial interest.
(18) Chen, X.; Guo, Y.; Zhang, Q.; Hao, G.; Jia, H.; Liu, B.
Preparation and biological evaluation of 99mTc-CO-MIBI as myocardial
perfusion imaging agent. J. Organomet. Chem. 2008, 693, 1822−1828.
(19) Hao, G.; Zang, J.; Liu, B. Preparation and biodistribution of
novel 99mTc(CO)3-CNR complexes for myocardial imaging. J. Labelled
Compd. Radiopharm. 2007, 50, 13−18.
ACKNOWLEDGMENTS
■
This work was supported in part by the Grants-in-Aids for the
Development of Systems and Technology for Advanced
Measurement and Analysis Program (Development of
SPECT probe for molecular imaging) from Japan Science
and Technology Agency, JST, and JSPS Research Fellows
Program (no. 264896).
(20) Hao, G. Y.; Zang, J. Y.; Zhu, L.; Guo, Y. Z.; Liu, B. L. Synthesis,
separation and biodistribution of 99mTc-CO-MIBI complex. J. Labelled
Compd. Radiopharm. 2004, 47, 513−521.
(21) Haubner, R.; Wester, H. J.; Reuning, U.; Senekowitsch-
ABBREVIATIONS USED
Schmidtke, R.; Diefenbach, B.; Kessler, H.; Stocklin, G.; Schwaiger,
M. Radiolabeled ανβ3 integrin antagonists: A new class of tracers for
tumor targeting. J. Nucl. Med. 1999, 40, 1061−1071.
(22) Shi, J.; Wang, L.; Kim, Y. S.; Zhai, S.; Liu, Z.; Chen, X.; Liu, S.
Improving tumor uptake and excretion kinetics of 99mTc-labeled cyclic
arginine-glycine-aspartic (RGD) dimers with triglycine linkers. J. Med.
Chem. 2008, 51, 7980−7990.
(23) Wang, L.; Shi, J.; Kim, Y. S.; Zhai, S.; Jia, B.; Zhao, H.; Liu, Z.;
Wang, F.; Chen, X.; Liu, S. Improving tumor-targeting capability and
pharmacokinetics of 99mTc-Labeled cyclic RGD dimers with PEG4
linkers. Mol. Pharmaceutics 2009, 6, 231−245.
̈
■
A.B., acetate buffer; Boc, tert-butoxycarbonyl; tBu, tert-butyl;
DIC, N,N′-diisopropylcarbodiimide; DIPEA, N,N-diisopropy-
lethylamine; DMF, N,N-dimethylformamide; HOBt, 1-hydrox-
ybenzotriazole; TES, triethylsilane; TFA, trifluoroacetic acid;
TFP, 2,3,5,6-tetrafluoro phenol; pbf, 2,2,4,6,7-pentamethyldihy-
drobenzofuran-5-sulfonyl
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DOI: 10.1021/acs.jmedchem.6b00024
J. Med. Chem. XXXX, XXX, XXX−XXX