Propylene cross-bridged macrocyclic bifunctional chelator: A new design for facile bioconjugation and robust 64cu complex stability

Article abstract of DOI:10.1021/jm500348z

The first macrocyclic bifunctional chelator incorporating propylene cross-bridge was efficiently synthesized from cyclam in seven steps. After the introduction of an extra functional group for facile conjugation onto the propylene cross-bridge, the two carboxylic acid pendants could contribute to strong coordination of Cu(II) ions, leading to a robust Cu complex. The cyclic RGD peptide conjugate of PCB-TE2A-NCS was prepared and successfully radiolabeled with ⁶⁴Cu ion. The radiolabeled peptide conjugate was evaluated in vivo through a biodistribution study and animal PET imaging to demonstrate high tumor uptake with low background.

Full text of DOI:10.1021/jm500348z

Article
pubs.acs.org/jmc
Propylene Cross-Bridged Macrocyclic Bifunctional Chelator: A New
64
Design for Facile Bioconjugation and Robust Cu Complex Stability
Darpan N. Pandya,†,∥ Nikunj Bhatt,†,∥ Gwang Il An,‡,∥ Yeong Su Ha,† Nisarg Soni,† Hochun Lee,§
Yong Jin Lee,‡ Jung Young Kim,‡ Woonghee Lee,† Heesu Ahn,† and Jeongsoo Yoo*,†
†Department of Molecular Medicine, BK21 Plus KNU Biomedical Convergence Program, Kyungpook National University, Daegu
700-422, South Korea
‡Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, South Korea
§Department of Energy Systems Engineering, Daegu Gyeongbuk Institute of Science & Technology, Daegu 711-873, South Korea
S
* Supporting Information
ABSTRACT: The first macrocyclic bifunctional chelator
incorporating propylene cross-bridge was efficiently synthe-
sized from cyclam in seven steps. After the introduction of an
extra functional group for facile conjugation onto the
propylene cross-bridge, the two carboxylic acid pendants
could contribute to strong coordination of Cu(II) ions, leading
to a robust Cu complex. The cyclic RGD peptide conjugate of
PCB-TE2A-NCS was prepared and successfully radiolabeled with 64Cu ion. The radiolabeled peptide conjugate was evaluated in
vivo through a biodistribution study and animal PET imaging to demonstrate high tumor uptake with low background.
attempted by several research groups. These modifications
include the variation of the length of carboxylate pendant arm
from the N atoms13 and the addition of extra rigidity by
introducing side-bridges14,15 or cross-bridge16 to the cyclic
framework.
INTRODUCTION
■
In recent years, bifunctional chelators (BFCs) have found
multiple applications in the field of medical imaging and
therapy.1−4 In particular, BFC plays a crucial role in the
development of target-specific radiopharmaceuticals in which a
targeting biomolecule (e.g., disease-specific peptide or anti-
body) is linked with a metallic radionuclide by BFC.5−7 The
ideal BFC requires the ability to form facile and strong
conjugations with biomolecules and to complex with radiometal
ions firmly in physiological conditions.8 Among many radio-
metal ions, 64Cu has becomes increasingly popular due to its
attractive decay mode (β+ 17%, β− 39%) and midlong half-life
(12.7 h), which allows both positron emission tomography
(PET) imaging and radiotherapy.9
Among these chemical modifications, replacing the trans-
positioned two acetic acid pendant arms to connect both
secondary amines with an ethylene linker and producing a cage-
like structure (i.e., ECB-TE2A, Figure 1) was found to be the
most effective method to increase the Cu-BFC stability, which
ultimately results in a reduction in the trans-metalation of Cu in
vivo.17,18 This ECB-TE2A could be used as a BFC, but because
the compound does not contain any other functional groups
that could be used for conjugation, modification with
biomolecules requires the sacrifice of one of its two acetic
acid functionalities. The utilization of the acetic acid
functionality for conjugation leads to reduced Cu-BFC complex
stability, as the group is not available for strong coordination
with Cu(II) ions.19 Furthermore, because only one carboxylate
group can coordinate to a Cu ion, the overall charge of the
complex changes from neutral to +1. Positively charged Cu
complexes demonstrate higher uptake in clearance organs such
as the liver and kidney, and the body clearance is slow in
comparison to that of neutral complexes.14,20 To solve this
conjugation problem, ECB-TE2A derivatives in which the
tetraaza-macrocyclic backbone or N-substituted pendant was
modified by an extra functional group were also introduced.
However, the total synthetic steps were drastically increased,
In the literature, various BFCs have been used for 64Cu
labeling. Most of these compounds are based on DOTA
(1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid),
TETA (1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N‴-tetraace-
tic acid), NOTA (1,4,7-triazacyclododecane-N,N′,N″-triacetic
acid), or their derivatives. The wide acceptance of poly-
azamacrocyclic chelators such as BFCs is due to their greater in
vivo stability in comparison to acyclic chelators such as EDTA
and DTPA.10 However, in recent studies, the in vivo stability of
Cu complexes of BFCs have been reported to be unsatisfactory,
and the chelating agents lose Cu(II) ions in vivo, which
increase the unnecessary radiation exposure to nontargeted
organs, as well as background noise.11,12 Therefore, the search
for a better BFC still continues.
To minimize the decomplexation of Cu-BFCs in vivo and to
improve the stability of Cu-BFC complexes, various mod-
ifications on the tetraaza-macrocyclic backbone have been
Received: March 5, 2014
Published: August 19, 2014
© 2014 American Chemical Society
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Figure 1. Structure of the bifunctional chelators.
Scheme 1. Seven-Step Synthesis of PCB-TE2A-NCS from Cyclam
and overall yields were significantly decreased, rendering the
use of these derivatives impractical.21,22
introduced as a form of glyoxal, while the propylene is
introduced as a 1,3-propanediol di-p-tosylate form in cross-
bridge reactions.
Recently, we reported that propylene cross-bridged TE2A
(PCB-TE2A) could form a more stable Cu complex than ECB-
TE2A and could be radiolabeled with 64Cu ions in milder
conditions.23 In addition, the synthetic process of PCB-TE2A is
advantageous, as it can be synthesized in higher overall yield
from commercially available cyclam in shorter times, compared
with ECB-TE2A. Furthermore, there is a better chance of
introducing extra functional groups onto a propylene bridge
than an ethylene bridge because the ethylene bridge is
In this study, we report a highly effective synthetic route for a
novel bifunctional chelator incorporating PCB-TE2A, in which
an extra functional group (NCS) for facile conjugation is
introduced onto the propylene cross-bridge, and the two
carboxylate pendants are saved for strong coordination to
Cu(II) ions. The chelator, PCB-TE2A-NCS, is conjugated with
a cyclic RGD peptide through the NCS functionality of the
BFC and radiolabeled with 64Cu ions. The in vivo targeting
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Scheme 2. Three-Step Synthesis of Intermediate for Propylene Cross-Bridge
affinity of 64Cu-labeled PCB-TE2A-NCS-c(RGDyK) was
examined in U87MG tumor models.
This synthetic route of introducing the additional functional
group onto the carbon atom of the cross-bridge for facile
conjugation is highly advantageous in terms of overall yield and
total reaction time in comparison with the previously reported
synthetic routes (Figure 1). PCB-TE2A-NCS was synthesized
in 60% overall yield starting from the readily available cyclam
with total 6 days of reaction time, which is far superior than 7%
overall yield with 33 days reaction time for ECB-TE2A-NHS
and 9% overall yield with 41 days reaction time for ECB-TE2A-
NCS (Table 1).21,22 In previously reported syntheses, the extra
RESULTS AND DISCUSSION
■
PCB-TE2A-NCS was successfully synthesized in seven steps
from a commercially available cyclam, as shown in Scheme 1.
The trans-dialkylated 1,8-N,N′-bis(carbo-tert-butoxymethyl)-
1,4,8,11-tetraazacyclotetradecane (4) was synthesized in
quantitative yield from cyclam by a regioselective protection/
alkylation/deprotection strategy.23
For the propylene cross-bridge precursor, a suitably tailored
nitro-derivative of a ditosyl ester of 1,3-propane diol (11) was
synthesized from diethyl malonate in three steps by
modifications of reported procedures (Scheme 2).24,25 In
brief, a condensation reaction of diethyl malonate with 4-nitro
benzyl bromide was carried out in the presence of K2CO3 to
afford diethyl 2-(4-nitrobenzyl)malonate (9). In this reaction, a
mild base and excess of diethyl malonate were used to prevent
the formation of dialkylated byproduct. The intermediate 9 was
reduced using sodium borohydride to furnish the diol, which
was recrystallized from EtOAc/hexane to generate pure 2-(4-
nitrobenzyl)propane 1,3-diol (10) as an off-white powder. The
alcohol groups of 10 were tosylated by treatment with p-
toluenesulfonyl chloride in the presence of pyridine to afford
pale yellow oil, which was crystallized from ethanol to yield a
white solid (11).
Table 1. Comparison of the Syntheses of the Bifunctional
Chelators Containing an Extra Functional Group for Facile
Conjugation
bifunctional
chelator
total steps for
synthesis
overall
yield
total time for
synthesis
PCB-TE2A-NCS
7
8
60%
7%
6 days
ECB-TE2A
33 days
-NHS21
ECB-TE2A
-NCS22
13
9%
41 days
functional group was introduced either at the CH2 carbon of
the cyclam backbone or by the modifying carboxylic acid
pendent arm. The introduction of an extra functional group on
the ethylene cross-bridge is difficult because this bridge was
introduced by using glyoxal.16
The introduction of a propylene cross-bridge with additional
functionality to the two newly generated secondary amines was
carried out by treating compound 4 with 11 in dried toluene in
the presence of anhydrous Na2CO3. The removal of tosylate
salts of the obtained brown colored oil was achieved by
treatment with 20% NaOH, and the crude product was purified
by column chromatography to obtain salt-free compound 5.
Deprotection of tert-butyl group was achieved by treating
compound 5 with a 1:1 (v/v) mixture of trifluoroacetic acid
(TFA) and methylene dichloride at room temperature to afford
the TFA salt of PCB-TE2A-NO2 (6) as an oil, which was
further triturated with Et2O to provide an off-white solid. For
the reduction of the nitro group, 6 was treated with 10% Pd/C
in water under H2 (g) at room temperature for 12 h. The
resulting reaction mixture was then filtered off and washed with
water, and the combined filtrates were evaporated under
vacuum to yield an oily residue, which was triturated with Et2O
to provide an off-white solid (7). The NH2 group of 7 was
substituted by NCS by reaction with thiophosgene in a CHCl3
and 0.5 M HCl solution. The reaction mixture was stirred at
room temperature, followed by layer separation and washing of
the CHCl3 layer with water. The combined aqueous layers were
washed with CHCl3 to remove unreacted thiophosgene. Finally,
the aqueous layer was lyophilized to afford white solid (8) (for
more information, see the Supporting Information).
Although we have already proven that PCB-TE2A formed
ultrastable Cu complexes as ECB-TE2A, the in vitro stability of
the Cu complex of PCB-TE2A-NCS was evaluated to verify the
influence of the structural modification on its Cu complex
stability. As a model Cu complex, the compound 7 was used
instead of PCB-TE2A-NCS (8) because the NCS group of 8
was observed to be converted to NH2 during complexation
reaction, which was confirmed by mass analysis of reaction
mixture, and the expected Cu-PCB-TE2A-NCS complex could
not be isolated. To a solution of PCB-TE2A-NH2 and
Cu(ClO4)2·6H2O in methanol, 1 M solution of NaOH was
added, and the reaction mixture was refluxed for 2 h, cooled,
and filtered through celite bed. The filtrate was subjected to
diethyl ether diffusion. The deposited blue crystals were
collected and dried to obtain Cu-PCB-TE2A-NH2 in 88% yield.
To evaluate the kinetic stability of Cu-PCB-TE2A-NH2,
acidic decomplexation and cyclic voltammetry experiments
were performed, and the results were compared with those of
Cu-PCB-TE2A.13,26 An acidic decomplexation study was
carried out in extremely harsh conditions (12 M HCl at 90
°C), and the degradation pattern was monitored by repeated
sample injection into an HPLC (Figure 2A,B). As the
decomplexation reaction proceeds, the peak area for intact
Cu-PCB-TE2A-NH2 at 6.6 min should decrease, and the peak
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Figure 2. Time-dependent UV-HPLC chromatograms of Cu-PCB-TE2A-NH2 (A) and Cu-PCB-TE2A (B) during acidic decomplexation in 12 M
HCl at 90 °C. Cyclic voltammograms (scan rate 100 mV/s) of Cu-PCB-TE2A-NH2 (C) and Cu-PCB-TE2A (D).
Scheme 3. Conjugation of PCB-TE2A-NCS to c(RGDyK) Peptide
area for free copper (at 2.0 min) should increase. However, the
Cu-PCB-TE2A-NH2 did not demonstrate any decomposition
in 12 M HCl even after 7 days, and it remained intact, as
observed in the case of Cu-PCB-TE2A. This study indicates
that the further alkylation of the propylene cross-bridge did not
affect the stability of the Cu complex of the PCB-TE2A core
and also strongly implies that this high stability of Cu-PCB-
TE2A-NH2 complex will be maintained even after conjugation
with biomolecules through NCS group, as the conjugated
group on the propylene cross-bridge is located far from the Cu
coordination core, which contains two carboxylate groups that
can coordinate strongly to Cu(II) ions.
The electrochemical behavior of Cu-PCB-TE2A-NH2 was
also examined by a cyclic voltammetry study to identify any
possibility of decomplexation by reducing Cu(II) to Cu(I) in
physiological conditions, as the two oxidation states of Cu ions
favor different coordination geometries. As shown in Cu-PCB-
TE2A (Figure 2D), Cu-PCB-TE2A-NH2 generated quasi-
reversible reduction/oxidation peaks (E1/2 = −0.84 V) without
any oxidation peak for free Cu(I) ions to Cu(II), which
indicates that PCB-TE2A-NH2 can even complex with Cu(I)
ions firmly (Figure 2C).
validated αvβ3 integrin ligand, and its radiolabeling efficiency
with 64Cu was also verified.
PCB-TE2A-NCS was simply conjugated to c(RGDyK) by
treating c(RGDyK) with PCB-TE2A-NCS in 0.1 M Na2CO3
(pH 9.5) and stirring overnight in the dark, followed by
purification by semipreparative HPLC (Scheme 3). The
collected fraction was combined and lyophilized to afford the
final product as white powder in 72% yield. The purified PCB-
TE2A-NCS-c(RGDyK) was confirmed by analytical HPLC and
ESI mass spectrometry [m/z calculated for C52H79N14O12S,
1122.56; found m/z, 1122.49 for [M]−].
The purified PCB-TE2A-NCS-c(RGDyK) conjugate was
radiolabeled with 64Cu by incubation with 64CuCl2 in 0.1 M
NaOAc buffer (pH 8.0) at 80 °C for 1 h, and the radiochemical
yield and purity were confirmed by radio-TLC and HPLC,
respectively. The PCB-TE2A-NCS-c(RGDyK) was radio-
labeled with 64Cu at >95% radiochemical yield, and the
radiochemical purity was over 98% after HPLC purification
(Figure 3). The specific activity of 64Cu-PCB-TE2A-NCS-
c(RGDyK) was calculated to be in the range of 900−1050 μCi/
μg. The logP values calculated for 64Cu-PCB-TE2A-NCS-
c(RGDyK) and 64Cu-ECB-TE2A-c(RGDyK) were −2.81 and
−2.98, respectively, indicating that 64Cu-PCB-TE2A-NCS-
c(RGDyK) is more lipophilic than 64Cu-ECB-TE2A-c-
(RGDyK). The purified conjugate was also evaluated for
After confirming the high in vitro stability of the Cu complex
of PCB-TE2A-NH2, we tested the feasibility of conjugating
PCB-TE2A-NCS with cyclic RGD peptide, which is a well-
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of Cu(II) ions from chelator than PCB-TE2A-NCS analogue
(24.0% vs 5.5%). In case of liver and kidney samples, though
the difference observed is marginal, still 64Cu-PCB-TE2A-NCS-
c(RGDyK) shows slightly less demetalation of 64Cu ions
compared to the 64Cu-ECB-TE2A-c(RGDyK) (10.0% vs 10.6%
in liver and 8.7% vs 12.3% in kidney for 64Cu-PCB-TE2A-NCS-
c(RGDyK) and 64Cu-ECB-TE2A-c(RGDyK), respectively). In
addition to free 64Cu ion peaks, only intact 64Cu-PCB-TE2A-
NCS-c(RGDyK) and 64Cu-ECB-TE2A-c(RGDyK) peaks were
seen in HPLC chromatograms (Figure 5). Higher in vivo
stability of Cu complex of PCB-TE2A-NCS conjugate could be
partially attributed to the availability of both carboxylate
pendant arms for the strong coordination to 64Cu.
The in vivo behavior and tumor targeting efficacy of 64Cu-
labeled PCB-TE2A-NCS-c(RGDyK) conjugate was evaluated
by a biodistribution study in U87MG tumor-bearing nude mice.
The biodistribution data for 64Cu-PCB-TE2A-NCS-c(RGDyK)
is displayed in Figure 6. First, the PCB-TE2A-NCS-conjugated
RGD peptide was rapidly cleared from the body by both the
kidney and liver. The blood activity at 1 h after injection was
only 0.25 0.03 %ID/g, while the kidney and liver activities
were 4.95 0.15 and 3.21 0.28 %ID/g, respectively, at the
same time. The tumor also exhibited the second highest uptake
after the kidney (3.65 0.08 %ID/g) at 1 h postinjection. The
αvβ3 receptor-mediated specific uptake of radiolabeled RGD
peptide was confirmed by a blocking study. At the coinjection
of cold c(RGDyK) peptide (5 mg/kg), tumor uptake was
reduced to one-fourth of that of the nonblocked sample (3.65
0.08 vs 0.90 0.11 %ID/g), while other organs exhibited
comparable uptakes. The activities in the clearance organs
gradually decreased over time, and about one-third of the 1 h
activities were observed in the liver and kidneys at 24 h (1.06
0.21 and 1.86 0.37 %ID/g, respectively). However, the tumor
exhibited much more prolonged retention of radiolabeled RGD
peptide, mostly because of specific binding of peptide to αvβ3
receptor.29−31 The tumor uptake was even slightly increased at
4 h, and 75% of the activity was remaining at 24 h, compared to
1 h (3.72 0.36 %ID/g at 4 h and 2.73 0.79 %ID/g at 24 h).
At 24 h postinjection, the highest activity was found in the
tumor (red color in Figure 6). The tumor-to-organ ratios were
46.2, 20.7, 7.2, 4.1, 1.5, and 2.6 for blood, muscle, bone,
intestine, kidney, and liver, respectively, at 24 h. Blood and
muscle uptake at 24 h was only 0.06 0.01 and 0.14 0.08 %
ID/g, respectively. Our 64Cu-PCB-TE2A-NCS-c(RGDyK)
demonstrated higher tumor uptakes than previously reported
for 64Cu-AmBaSar-c(RGDyK) and 64Cu-DOTA-c(RGDyK)27
but showed comparable tumor uptakes with 64Cu-NODAGA-
c(RGDfK) and 64Cu-CB-TE2A-c(RGDfK).29 Even though
Fani et al. used c(RGDfK) instead of c(RGDyK), it would be
worthy to compare their biodistribution data with the current
one because they also used the same U87MG tumor model,
and ethylene cross-bridged TE2A (CB-TE2A) closely resem-
bles our propylene cross-bridged TE2A (PCB-TE2A). Tumor
uptakes of both 64Cu-PCB-TE2A-NCS-c(RGDyK) and 64Cu-
CB-TE2A-c(RGDfK) are very comparable (3.65 0.08 vs 3.66
Figure 3. UV-HPLC chromatogram (218 nm) of PCB-TE2A-NCS-
c(RGDyK) (top) compared with radio-HPLC chromatogram of 64Cu-
PCB-TE2A-NCS-c(RGDyK) (bottom).
serum stability. In fetal bovine serum (FBS), the conjugate did
not present any sign of decomposition up to 24 h at 37 °C.
To check the effect of conjugation of c(RGDyK) with PCB-
TE2A-NCS on integrin binding affinity, in vitro affinity assay
was carried out by using PCB-TE2A-NCS-c(RGDyK) (Figure
4). The 50% inhibitory concentration (IC50) calculated for
Figure 4. In vitro inhibition of 125I-echistain binding to integrin on
U87MG cells by c(RGDyK) and PCB-TE2A-NCS-c(RGDyK).
PCB-TE2A-NCS-c(RGDyK) was comparable with that of
reference standard c(RGDyK) (61.5 7.14 and 29.97 5.41
nM, respectively), which indicates that the conjugation of PCB-
TE2A-NCS with c(RGDyK) has minimal effect on its affinity
toward αvβ3 integrin.
The in vivo stability study of 64Cu-PCB-TE2A-NCS-
c(RGDyK) was carried out with little modification in the
published procedures.27,28 For the comparison, 64Cu-labeled
tracer ECB-TE2A-c(RGDyK) was prepared as per a reported
process.29 A comparative HPLC blood, liver, and kidney
analysis data of two tracers are displayed in Figure 5. The
results clearly showed that 64Cu-PCB-TE2A-NCS-c(RGDyK)
is more robust in rat blood and that the demetalation of free
64Cu ions from PCB-TE2A-NCS chelator is lesser compared to
ECB-TE2A analogue. While 64Cu-ECB-TE2A-c(RGDyK)
showed 24.0% of demetalated free 64Cu ion peak in rat blood
sample at 1 h postinjection, 64Cu-PCB-TE2A-NCS-c(RGDyK)
showed only 5.5% of free copper ion peak. 64Cu-labeled ECB-
TE2A complex showed more than 4 times higher demetalation
0.58 at 1 h; 2.73
0.79 (24 h) vs 2.99
0.79 (18h),
respectively). Both tracers cleared fast and showed high tumor-
to-background ratios (tumor-to-blood, 14.6 vs 7.48; tumor-to-
muscle, 19.2 vs 13.6 at 1 h, for PCB-TE2A and CB-TE2A
analogues, respectively). 64Cu-PCB-TE2A-NCS-c(RGDyK)
showed higher liver and kidney uptakes than 64Cu-CB-TE2A-
c(RGDfK) at 1 h (liver, 3.21 0.27 vs 1.57 0.54; kidney,
4.95 0.14 vs 2.14 0.53, respectively), while 64Cu-CB-TE2A-
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Figure 5. In vivo stability of radiotracers in rat at 1 h postinjection. HPLC profiles of (A) blood, (B) liver, and (C) kidney for 64Cu-PCB-TE2A-
NCS-c(RGDyK) and (D) blood, (E) liver, and (F) kidney for 64Cu-ECB-TE2A-c(RGDyK). Free 64Cu2+ ions ( ), intact 64Cu-PCB-TE2A-NCS-
c(RGDyK) ( ), and intact Cu-ECB-TE2A-c(RGDyK) ( ).
●
64
■
▲
Figure 6. Biodistribution data of 64Cu-PCB-TE2A-NCS-c(RGDyK) at 1 h, 1 h blocking, and 4 and 24 h postinjection in U87MG tumor bearing
nude mice (n = 3).
c(RGDfK) showed much higher bone uptake than PCB-TE2A
counterpart (2.57 0.23 vs 0.33 0.02). Higher liver uptake
could be partially attributed to more lipophilicity of benzyl-
NCS linker of PCB-TE2A-NCS chelator32 (logP values: −2.81
and −2.98 for 64Cu-PCB-TE2A-NCS-c(RGDyK) and 64Cu-
ECB-TE2A-c(RGDyK), respectively), and higher bone uptake
of 64Cu-CB-TE2A-c(RGDfK) could be partially attributed to
intrinsic bone uptake property of demetalated free 64Cu ions.
All other organ uptakes are comparable. Rapid body clearance
and high tumor uptake indicate that PCB-TE2A-NCS chelator
complexes well with 64Cu ions and that its conjugation with the
RGD peptide through a thiourea bond is stable in physiological
conditions.
The animal PET imaging data further confirmed the
biodistribution data of 64Cu-PCB-TE2A-NCS-c(RGDyK).
Coronal PET images collected at 1, 4, and 24 h are shown in
Figure 7. The U87MG xenograft tumor on the right flank of
nude mouse was clearly observed even at 1 h after injection
Figure 7. PET image of 64Cu-PCB-TE2A-NCS-c(RGDyK) at 1, 4, and
24 h postinjection in U87MG tumor-bearing nude mice. White arrow,
tumor; yellow arrowhead, liver; green arrowhead, intestine.
(3.80 0.25 %ID/g). However, high activities were found in
the abdominal region, especially in the liver and intestines (3.74
0.35 and 3.62 0.36 %ID/g, respectively). At 4 h, liver
activity was decreased, but more activity was found in the
intestines (4.67 0.44 %ID/g), which is in good agreement
with the biodistribution data. Most of the abdominal activities
in the liver, intestine, and kidneys were cleared from body at 24
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(t, 1H, J = 7.7 Hz), 4.13−4.21 (m, 4H), 7.38−7.40 (dd, 2H, J = 10
Hz), 8.13−8.15 (dd, 2H, J = 10 Hz). 13C NMR (125.8 MHz, CDCl3):
δ 15.54, 35.85, 54.64, 63.34, 125.27, 131.38, 147.19, 148.50, 169.78.
HRMS (FAB) calculated for C14H18NO6, 296.1134 [(M + H)+];
found, 296.1138 [(M + H)+].
h (liver 1.71 0.08, kidney 1.52 0.16, and muscle 0.12
0.01 %ID/g), but the tumor activity was still similar to that
observed at 1 h (3.49
0.10 %ID/g at 24h), resulting in
unambiguously clear tumor imaging with low background. The
advantage of Cu-64 as a radioiostope is its midlong half-life
(12.7 h), which allows late imaging at time points when most
activities clear from nonspecific organs, leaving only specific
binding in the target organ(s) or tissue(s). As observed with
our targeted chelator, the tumor can be much more easily
identified at later time points than at earlier ones.
Synthesis of 2-[(4-Nitrophenyl)methyl]propan-1,3-diol (10).
To a suspension of NaBH4 (14.35 g, 379.3 mmol) in EtOH (200 mL)
was added dropwise a solution of 9 (11.2 g, 37.93 mmol) in EtOH
(100 mL), and the mixture was refluxed for 16 h. After the addition of
aqueous NH4Cl solution in small portions (5%, 150 mL), the crude
solution was distilled under vacuum to remove EtOH. CH2Cl2 (200
mL) was added, and the mixture was filtered. When the two layers
were separated, the aqueous phase was extracted with CH2Cl2 (3 × 50
mL), and then the combined organic phases were washed with
aqueous NaHCO3 (5%, 100 mL), dried over MgSO4, and
concentrated under vacuum. The resulting yellow oil was recrystallized
from EtOAc/hexane to yield the off-white powder of 10 (6.81 g, 85%
CONCLUSIONS
■
PCB-TE2A-NCS containing an extra functional group for easy
and selective conjugation with biomolecules was synthesized in
an efficient way by introducing the NCS group at the propylene
cross-bridge. The synthesized PCB-TE2A-NH2 was demon-
strated to form robust Cu complexes by keeping both
carboxylic acid pendants available for strong complexation
with Cu(II) ions. PCB-TE2A-NCS was easily conjugated with
peptide, and the prepared conjugate was radiolabeled with 64Cu
in high yield. Biodistribution, in vivo stability, and animal-PET
imaging studies confirmed the high in vivo stability and
excellent tumor targeting efficacy of the RGD peptide-
conjugated PCB-TE2A-NCS chelator. Various propylene
cross-bridged BFCs containing other functional groups for
specific conjugation could be developed by employing our
synthetic strategy. In addition, the introduction of new linker
instead of benzyl group on PCB-TE2A could lead to more
favorable physiological properties of 64Cu-labeled tracer.
1
yield). H NMR (500 MHz, CD3OD): δ 1.92−1.98 (m, 1H), 278−
2.79 (d, 2H, J = 6.5 Hz), 3.53−3.54 (d, 4H, J = 5.5 Hz), 7.44−7.46
(dd, 2H, J = 8.5 Hz), 8.12−8.14 (dd, 2H, J = 9 Hz). 13C NMR (125.8
MHz, CD3OD): δ 33.42, 44.93, 61.22, 122.89, 129.80, 146.30, 148.88.
HR MS (FAB) calculated for C10H14NO4, 212.0923 [(M + H)+];
found, 212.0923 [(M + H)+].
Synthesis of 2-[(4-Nitrophenyl)methyl]propan-1,3-diol bis-
(4-methylbenzenesulfonate) (11). A solution of p-toluenesulfonyl
chloride (14.51 g, 76.11 mmol) in pyridine (30 mL) was added
dropwise to a stirred solution of 10 (6.43 g, 30.44 mmol) in pyridine
(20 mL), while the temperature was maintained below 0 °C. After the
completion of the addition, the reaction mixture was stirred at room
temperature for 10 h, and then it was poured into ice-cold aqueous 5
M HCl solution (100 mL). The aqueous phase was extracted with
CH2Cl2 (3 × 100 mL), and then the combined organic phase was
washed with brine (2 × 100 mL), dried over MgSO4, and evaporated
under reduced pressure to afford pale yellow oil, which was further
crystallized from ethanol (100 mL) to obtain the white solid of
EXPERIMENTAL SECTION
■
1
compound 11 (14.08 g, 89% yield). H NMR (500 MHz, CDCl3): δ
Materials and Methods. Cyclam was purchased from CheMatech
(Dijon, France). All other reagents and solvents were purchased from
Sigma-Aldrich (St. Louis, MO) and were used as received. Copper-64
was produced at KIRAMS (Seoul, Korea) by the 64Ni(p,n)64Cu
nuclear reaction using an MC50 Cyclotron (Scanditronix, Sweden).
Instrumentation. All 1H NMR and 13C NMR spectra were
measured on a Varian Unity Inova 500 MHz instrument. High-
resolution mass spectra (HRMS) were recorded on a JEOL JMS700 or
Quattro Premier XE mass spectrometer. Elemental analyses were
carried out at Kyungpook National University, Korea. UV−vis spectra
were acquired on a Shimadzu UV−vis spectrophotometer (UV-
1650PC). Analytical HPLC traces were acquired using a Waters 600
series HPLC system and Waters Grace smart RP C18 column (4.6 ×
250 mm, 5 μm), with the mobile phase consisting of 0.1% TFA/H2O
(solvent A) and 0.1% TFA/acetonitrile (solvent B) and a gradient
consisting of 1% B to 70% B in 20 min with a 1 mL/min flow rate. The
radio-TLC measurements were performed using a Bioscan 2000
imaging scanner (Bioscan, Washington, DC, USA).
2.32−2.34 (m, 1H), 2.46 (s, 6H, 2 × Ar−CH3), 2.72−2.74 (d, 2H, J =
7.5 Hz), 3.85−3.99 (m, 4H), 7.13−7.15 (dd, 2H, J = 8 Hz), 7.33−7.35
(dd, 4H, J = 8.5 Hz), 7.70−7.72 (dd, 4H, J = 8 Hz), 8.00−8.02 (dd,
2H, J = 9 Hz). 13C NMR (125.8 MHz, CDCl3): δ: 21.58, 32.93, 39.66,
67.73, 123.70, 127.82, 129.74 129.97, 132.08, 145.09, 145.34, 146.70.
HR MS (FAB) calculated for C24H26NO8S2, 520.1100 [(M + H)+];
found, 520.1097 [(M + H)+].
Synthesis of 4,11-N,N′-Bis(carbo-tert-butoxymethyl)-16-(4-
nitrobenzyl)-1,4,8,11-tetraazabicyclo[6.6.3]heptadecane (5). A
solution of 1,8-N,N′-bis(carbo-tert-butoxymethyl)-1,4,8,11-tetraazacy-
clotetradecane 4 (5.23 g, 12.20 mmol), 2-[(4-nitrophenyl)methyl]-
propan-1,3-diol bis(4-methylbenzenesulfonate) 11 (6.34 g, 12.20
mmol) and anhydrous Na2CO3 (2.97 g, 28.06 mmol) in anhydrous
toluene (200 mL) was refluxed for 2 days. The solvent was evaporated
from the reaction mixture under reduced pressure, and CH2Cl2 (200
mL) was added. The resulting brown slurry was filtered off to remove
Na2CO3 through a celite pad and was washed with CH2Cl2 (2 × 25
mL). The solvent was evaporated from the combined filtrate and wash
materials under reduced pressure. The residue was dissolved in 20% aq
NaOH (100 mL). After stirring for 4 h, the resultant solution was
extracted with CHCl3 (3 × 100 mL). The combined extracts were
washed by brine and dried over MgSO4, and the solvent was
evaporated under reduced pressure. The resulting residue was purified
via column chromatography on alumina (basic) and eluted with
CH2Cl2/methanol (20:1) to afford the off-white powder of compound
5 (5.45 g, 74% yield). 1H NMR (500 MHz, CDCl3): δ 8.14−8.12 (dd,
2H, J = 9 Hz), 7.60−7.59 (dd, 2H, J = 8.5 Hz), 4.09 (t, 1H, J = 11.5
Hz), 3.64−3.14 (m, 9H), 3.11−2.54 (m, 16H) 2.42−2.20 (m, 3H),
1.74−1.52 (m, 2H), 1.48−1.45 (d, 18H, J = 15 Hz). 13C NMR (125.8
MHz, CDCl3): δ 171.11, 169.84, 147.15, 146.89, 130.44, 123.90,
81.86, 81.68, 61.12, 60.92, 57.59, 56.75, 56.49, 55.25, 52.66, 50.96,
49.54, 47.99, 36.46, 31.44, 28.41, 28.22, 24.13, 22.23. HRMS (FAB)
calculated for C32H54N5O6, 604.4074 [(M + H)+]; found, 604.4077
[(M + H)+].
Synthesis of Diethyl-2-[(4-nitrophenyl)methyl]-
propanedioate (9). 4-Nitrobenzyl bromide, (10.57 g, 48.93 mmol)
was added to a stirred solution of diethyl malonate, (52 mL, 54.86 g,
342.51 mmol) and K2CO3 (14.21 g, 102.8 mmol) in acetone (40 mL).
The reaction mixture heated at 45 °C for 1 h. The mixture was allowed
to cool to room temperature, and the resulting yellow slurry was
filtered off to remove K2CO3 and washed with acetone (2 × 20 mL).
The solvent was evaporated from the combined filtrate and wash liquid
under reduced pressure. The excess diethylmalonate was removed by
horizontal distillation (2 h, 80−130 °C and 0.005 mbar). The yellow
oily residue was dissolved in ethanol (200 mL). The precipitated white
solid (dialkylated product) was filtered and washed with ethanol (2 ×
20 mL). The solvent was evaporated from the combined filtrate and
wash material under reduced pressure to provide a solid, which was
recrystallized from hexane/Et2O (1:1) to afford white crystalline
1
needles of 9 (12.72 g, 88% yield). H NMR (500 MHz, CDCl3): δ
1.20−1.23 (t, 6H, J = 7 Hz), 3.31−3.32 (d, 2H, J = 7.5 Hz), 3.65−3.68
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Article
Synthesis of 4,11-N,N′-Bis(carboxymethyl)-16-(4-nitroben-
zyl)-1,4,8,11-tetraazabicyclo[6.6.3]heptadecane·2TFA (11·
2TFA) (6). Compound 5 (2.56 g, 4.24 mmol) was dissolved in a 1:1
(vol/vol) mixture of CF3CO2H (TFA) and CH2Cl2 (70 mL). The
mixture was stirred at room temperature for 24 h. The solvent was
removed under reduced pressure to yield an oily residue, which was
triturated with Et2O to provide the off-white solid of 6 as a TFA salt
(2.93 g, 96% yield). 1H NMR (500 MHz, D2O): δ 8.12−8.10 (dd, 2H,
J = 8.5 Hz), 7.47−7.45 (dd, 2H, J = 8.5 Hz), 3.92−3.22 (m, 12H),
3.20−2.52 (m, 12H), 2.48−2.05 (m, 5H), 1.79 (br s, 1H), 1.53 (br s,
1H). 13C NMR (125.8 MHz, D2O): δ 174.07, 169.66, 162.36 (q, JCF
= 35.7 Hz, CF3COOH), 146.64, 146.51, 130.15, 123.98, 113.01 (q,
JCF = 292.3 Hz, CF3COOH), 60.46, 59.44, 56.79, 55.68, 53.43, 51.50,
48.99, 47.68, 36.14, 31.64, 22.78, 16.70. HRMS (FAB) calculated for
C24H38N5O6, 492.2822 [(M + H)+]; found, 492.2819 [(M + H)+].
Synthesis of 4,11-N,N′-Bis(carboxymethyl)-16-(4-aminoben-
zyl)-1,4,8,11-tetraazabicyclo[6.6.3]heptadecane·2TFA (7·2TFA)
(7). To a solution of compound 6 (4.26 g, 5.92 mmol) in water (50
mL) was added 10% Pd/C (1.28 g). The resulting mixture was stirred
under H2 (g) at room temperature for 12 h. The reaction mixture was
filtered through celite pad, which was washed with water (2 × 20 mL).
The combined filtrate was evaporated under vacuum to generate an
oily residue, which was triturated with Et2O to provide the off-white
solid of compound 7 (4.01 g, 98% yield). 1H NMR (500 MHz, D2O):
δ 7.36−7.35 (dd, 2H, J = 8.5 Hz), 7.27−7.26 (dd, 2H, J = 8 Hz),
4.10−2.51 (m, 27 H), 2.50−1.51 (m, 6H). 13C NMR (125.8 MHz,
D2O): δ 173.69, 169.14, 162.2 (q, JCF = 34.9 Hz, CF3COOH),
139.09, 130.34, 128.19, 122.99, 112.74 (q, JCF = 289.1 Hz,
CF3COOH), 60.09, 59.11, 56.27, 55.17, 52.58, 51.24, 48.63, 47.27,
35.31, 30.95, 22.45, 16.18. HRMS (FAB) calculated for C24H40N5O4,
462.3080 [(M + H)+]; found, 462.3085 [(M + H)+].
AgCl (sat. KCl) reference electrode and Pt wire counter electrode.
The samples (1 mM) were run in 0.2 M phosphate buffer adjusted to
pH 7.0 with glacial acetic acid at a scan rate of 100 mV/s. The
solutions were deoxygenated for 15 min with argon prior to use and
kept under argon atmosphere during measurement.
Conjugation of PCB-TE2A-NCS to RGD Peptide. A solution of
PCB-TE2A-NCS (8.61 μmol, 4.33 mg) was mixed with c(RGDyK)
(2.87 μmol, 1.78 mg) in 0.1 M Na2CO3 buffer (pH 9.5). After stirring
at room temperature overnight in the dark, the PCB-TE2A-NCS
conjugated to c(RGDyK) peptide was isolated by semipreparative
HPLC [Waters μbondapak C18; 10 μm, 7.8 × 300 mm; flow rate 3
mL/min, with the mobile phase starting from 95% solvent A (0.1%
TFA in water) and 5% solvent B (0.1% TFA in acetonitrile) (0−2
min) to 35% solvent A and 65% solvent B at 32 min]. The peak
containing the PCB-TE2A-NCS-c(RGDyK) conjugate was collected at
a retention time of 17.2 min. The collected fraction was combined and
lyophilized to afford the final product as a white powder. PCB-TE2A-
NCS-c(RGDyK) was obtained in 72% yield with 15.7 min retention
time on analytical HPLC [Grace smart RP C18; 5 μm, 4.6 × 250 mm;
flow rate 1 mL/min, with the mobile phase consisting of 0.1% TFA/
H2O (solvent A) and 0.1% TFA/acetonitrile (solvent B), and a
gradient consisting of 1% B to 70% B in 20 min]. The purified PCB-
TE2A-NCS-c(RGDyK) compound was identified by electrospray mass
spectrometry (m/z calculated for C52H79N14O12S, 1122.56; found m/z,
1122.49 for [M]−).
64Cu Radiolabeling of PCB-TE2A-NCS-c(RGDyK). 64Cu (0.5−2
mCi) in 100 μL of 0.1 M NaOAc buffer (pH 8.0) was added to 5 μg of
PCB-TE2A-NCS-c(RGDyK) in 100 μL of 0.1 M NaOAc buffer (pH
8.0). The reaction mixture was incubated at 80 °C for 1 h. The
reaction was monitored by radio-TLC using Whatman MKC18F TLC
plates developed with 30:70 10% NH4OAc/methanol (64Cu-PCB-
TE2ANCS-c(RGDyK), Rf = 0.9). The 64Cu-labeled peptide was
further purified by reverse-phase HPLC (RP-HPLC) using a Grace
smart RP C18 column (5 μm, 4.6 × 250 mm) eluted with a mobile
phase consisting of 0.1% TFA/H2O (solvent A) and 0.1% TFA/
acetonitrile (solvent B), and a gradient consisting of 1% B to 70% B in
20 min at a flow rate of 1 mL/min. 64Cu-PCB-TE2A-NCS-c(RGDyK)
(retention time [tR] 17.1 min) was collected in 1−2 mL of HPLC
solvent. The radiochemical purity of the radiolabeled peptide was
measured using the same HPLC conditions as above.
Conjugation and Radiolabeling of ECB-TE2A-c(RGDyK).
ECB-TE2A was conjugated with c(RGDyK) peptide by using standard
NHS activation method. First, cyclic(RGDyK) peptide was synthe-
sized following typical solid phase synthesis (Fmoc protection). Then,
ECB-TE2A (3.4 mg, 9.9 × 10−6 mole) and c(RGDyK) (3.4 mg, 5.5 ×
10−6 mole) were dissolved in anhydrous DMSO. To this solution, 2 M
N,N′-diisopropylcarbodiimide (7.5 μL) and of 2 M N,N′-diisopropyl
ethylamine (20 μL) was added. Resulted mixture was stirred at room
temperature for 15 h. The crude reaction mixture was purified by
preparative HPLC to afford pure ECB-TE2A-c(RGDyK), which was
characterized by electrospray mass spectrometry (m/z calculated for
C43H70N13O11, 944.53; found m/z, 944.8 for [M + H]+).
Synthesis of 4,11-N,N′-Bis(carboxymethyl)-16-(4-isothiocya-
natobenzyl)-1,4,8,11-tetraazabicyclo[6.6.3]heptadecane (8).
To a solution of compound 7 (1.21 g, 1.75 mmol) in 0.5 M HCl
(50 mL) was carefully added thiophosgene (CSCl2, CAUTION!)
(4.03 mL, 6.04 g, 52.5 mmol) in CHCl3 (50 mL). The reaction
mixture was stirred for 3 h at room temperature, and the layers were
allowed to separate. The aqueous layer was removed, and the organic
CHCl3 layer was then washed with water (2 × 50 mL). The combined
aqueous layers were washed with CHCl3 (3 × 50 mL) to remove the
unreacted thiophosgene. Finally, the aqueous layer was lyophilized to
1
afford the white solid of 8 (0.86 g, 97% yield). H NMR (500 MHz,
D2O): δ 7.30 (s, 2H), 7.07(s, 2H), 4.01−2.81 (m, 18 H), 2.80−1.89
(m, 12 H), 1.29 (br s, 1H). 13C NMR (125 MHz, DMSO-d6): δ
173.81, 168.70, 138.26, 134.23, 130.54, 128.80, 125.97, 60.26, 59.05,
55.56, 55.03, 52.58, 51.32, 48.45, 47.52, 35.56, 31.06, 22.88, 16.25.
HRMS (FAB) calculated for C25H38N5O4S, 504.2645 [(M + H)+];
found, 504.2647 [(M + H)+].
Synthesis of Cu-PCB-TE2A-NH2. To a solution of PCB-TE2A-
NH2 7 (156 mg, 0.34 mmol) and Cu(ClO4)2·6H2O (126 mg, 0.34
mmol) in 20 mL of methanol was added 1 M aqueous solution of
NaOH (2.41 mL, 2.41 mmol). The resulting clear blue solution was
refluxed for 2 h, cooled, and filtered through a celite bed. The filtrate
was subjected to diethyl ether diffusion. The deposited blue crystals
were collected and dried (155 mg, 88% yield). HRMS (FAB):
calculated for C24H38CuN5O4, 523.2220 [(M + H)+]; found, 523.2219
[(M + H)+]. Elemental analysis: calculated for C24H38CuN5O4·3H2O,
C 47.94, H 7.50, N 12.71; found, C 47.37, H 6.83, N 11.26. Visible
electronic spectrum: λmax(H2O)/635 nm; λmax(5 M HCl)/651 nm.
Acid Decomplexation Studies by HPLC. The sample
concentration of the copper complex (Cu-PCB-TE2A-NH2) studied
was 3 mmol in 12 M HCl. The UV HPLC spectrum in 12 M HCl at
90 °C was recorded at specific time points by injecting an aliquot (20
μL) onto a reverse-phase Xbridge C18 column (4.6 × 150 mm, 5 μm)
using an isocratic method (0.1% TFA/H2O−MeOH 91−9, and 1 mL/
min flow rate). The decreasing absorbance in the UV region (280 nm)
was used to monitor the progress of the decomplexation reaction.
Electrochemical Studies. Cyclic voltammetry was conducted
with a Biologic model SP-150 with a three-electrode configuration.
The working electrodes were a glassy carbon (diameter = 3 mm), Ag/
64Cu-labeled ECB-TE2A-c(RGDyK) was prepared by following a
published procedure.29 Briefly, 10 μg of ECB-TE2A-c(RGDyK) was
incubated with 64CuCl2 (1−2 mCi) in 0.1 M NH4OAc buffer (pH 8.0)
at 95 °C for 30 min. The radiotracer was purified by HPLC by
following similar conditions as those described for 64Cu-PCB-TE2A-
NCS-c(RGDyK). Radiochemical purity was >90% before HPLC
purification and was over 99% after HPLC purification.
Specific Activity Measurement. For specific activity measure-
ment, a nonradioactive Cu-PCB-TE2A-NCS-c(RGDyK) was prepared
by incubating PCB-TE2A-NCS-c(RGDyK) with excess CuCl2 (10 mol
equivalent) at 90 °C for 1 h. The pH of the reaction mixture was
adjusted to 8.0 by 0.1 M NaOH. Various amount of Cu-PCB-TE2A-
NCS-c(RGDyK) was injected into HPLC, and a calibration plot was
prepared for area under the peak for Cu-PCB-TE2A-NCS-c(RGDyK)
at 220 nm wavelength versus known amount of Cu-PCB-TE2A-NCS-
RGDyK injected. Peak area obtained in UV chromatogram at 220 nm
for the purified 64Cu-PCB-TE2A-NCS-c(RGDyK) was used to
calculate specific activity (μCi/μg).
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Journal of Medicinal Chemistry
Article
Determination of Partition Coefficient. The logP values of
64Cu-PCB-TE2A-NCS-c(RGDyK) and 64Cu-ECB-TE2A-c(RGDyK)
were determined by adding 5 μL of the labeled tracer (∼50 μCi) to a
mixture of 500 μL of 1-octanol and 500 μL of water. The resulting
solutions were vigorously vortexed for 5 min at room temperature,
then centrifuged for 5 min to ensure complete separation of layers.
From each of the six sets, a 100 μL aliquot was removed from each
phase into screw tubes and counted separately in a gamma counter.
The partition coefficient was calculated as a ratio of counts in the 1-
octanol fraction to counts in the water fraction. The logP values were
reported in an average of six measurements. This experiment was
carried out in duplicate.
Integrin Receptor Binding Assay. The cell-binding assay of the
PCB-TE2A-NCS-c(RGDyK) was compared with c(RGDyK) (refer-
ence molecule) using 125I-echistatin on U87MG cells as described
previously.33 U87MG cells (2 × 106/100 μL) were resuspended in
binding buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1
mM MnCl2, and 0.1% bovine serum albumin). For the assay, equal
volumes of nonradioactive ligand PCB-TE2A-NCS-c(RGDyK) and
radioactive ligand (0.1 μCi 125I-echistatin, PerkinElmer, Branford, CT)
were added. Increased concentrations (10−5 to 10−14 M) of the ligands
were added to each tube. The tubes were incubated for 60 min at
room temperature, and then the reaction medium was removed and
the cells were washed three times with PBS. The cells were harvested,
and the bound 125I-echistatin was counted in a gamma counter, 1480
WIZARD (Wallac). Data was analyzed with GraphPad Prism 5
(GraphPad softwere, Inc., San Diego, CA) to determine the IC50 value.
Experiments were carried out with triplicate samples.
analyzed using a gamma counter. The percent of injected dose per
gram (%ID/g) was calculated by comparison to a weighted, counted
standard. The receptor-mediated localization of the radiotracer
(blocking study) was investigated by injection of nonradioactive
c(RGDyK) peptide at 5 mg/kg along with 64Cu-PCB-TE2A-NCS-
c(RGDyK) in the same tumor model, and biodistribution was carried
out at 1 h postinjection.
MicroPET Imaging in U87MG Tumor Bearing Nude Mice.
Small animal PET scans and image analysis were performed using an
Inveon PET/CT scanner (Siemens). The imaging studies were carried
out on female nude mice bearing xenograft U87MG tumors on their
right flank. The mice were injected via the tail vein with 64Cu-PCB-
TE2A-NCS-cRGDyK (∼500 μCi). At 1, 4, and 24 h after injection, the
mice were anesthetized with 1% to 2% isoflurane, positioned in the
prone position, and imaged. The images were reconstructed by a 2-
dimensional ordered-subsets expectation maximum (OSEM) algo-
rithm, and no corrections were necessary for attenuation or scatter. An
ROI was placed on the liver, kidney, muscle, and tumor in the coronal
microPET images for quantification purpose.
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR (1H and 13C) and HR-MS spectra of all synthesized
compounds, HR-MS of Cu-PCB-TE2A-NH2, ESI mass spectra
of PCB-TE2A-NCS-c(RGDyK) and ECB-TE2A-c(RGDyK),
and biodistribution data of 64Cu-PCB-TE2A-NCS-c(RGDyK).
This material is available free of charge via the Internet at
http://pubs.acs.org.
Animal Models. All animal experiments were conducted in
compliance with the Animal Care and Use Committee requirements of
Kyungpook National University. Xenograft tumor models of U87MG
cell lines were prepared using 6 week-old BALB/c nu/nu female nude
mice. A total of 5 × 106 U87MG cells were inoculated subcutaneously
into the right flank of the mice. Tumors of appropriate size usually
grew within 15 d after the tumor cell implantation.
AUTHOR INFORMATION
■
Corresponding Author
*(J.Y.) E-mail: yooj@knu.ac.kr.
In Vivo Stability. The in vivo stability of 64Cu-PCB-TE2A-NCS-
c(RGDyK) was evaluated by following reported processes.27,28 For
direct comparison purposes, in vivo stability of 64Cu-ECB-TE2A-
c(RGDyK) was also evaluated in the same method. Approximately 1
mCi of radiolabeled conjugates (64Cu-PCB-TE2A-NCS-c(RGDyK)
and 64Cu-ECB-TE2A-c(RGDyK)) were injected intravenously in
Sprague−Dawley rats. The blood was collected from the heart at 1
h postinjection and immediately centrifuged for 10 min at 13,000 rpm.
To the supernatant (200 μL), a 100 μL mixture of acetonitrile−water−
trifluoroacetic acid (50:45:5) was added, followed by mixing, keeping
at 4 °C for 10 min and centrifugation for 10 min at 13,000 rpm. The
supernatant was then filtered through a 0.22 μm filter, and the filtrate
was injected to reverse-phase HPLC [Grace smart RP C18 column (5
μm, 4.6 × 250 mm) eluted with a mobile phase consisting of 0.1%
TFA/H2O (solvent A) and 0.1% TFA/acetonitrile (solvent B), and a
gradient consisting of 1% B to 70% B in 20 min at a flow rate of 1 mL/
min]. The eluent was collected with a fraction collector (1.0 mL/
fraction), and the radioactivity of each fraction was measured with a
gamma counter.
Author Contributions
∥These authors (D.N.P., N.B., and G.I.A.) contributed equally
to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by an R&D program through the
National Research Foundation of Korea funded by the Ministry
o f S c i e n c e , I C T
&
F u t u r e P l a n n i n g ( N o .
2013R1A2A2A01012250, 2013M2A2A6042317, and
2014032805), and the Basic Research Laboratory (BRL)
Program (2013R1A4A1069507). The Korea Basic Science
Institute (Daegu) is acknowledged for providing assistance with
the NMR and MS measurements.
For the liver and kidney, samples were first homogenized well with
homogenizer, and then 5 mL of PBS was added and vortexed for 5 min
followed by centrifugation for 10 min at 13,000 rpm. To the
supernatant (200 μL), a 100 μL mixture of acetonitrile−water−
trifluoroacetic acid (50:45:5) was added, followed by mixing, keeping
at 4 °C for 10 min and centrifugation for 10 min at 13,000 rpm. The
supernatant was then filtered through a 0.22 μm filter, and the filtrate
was injected to reverse-phase HPLC; collected fractions were
measured with a gamma counter as per previously described for
blood sample.
ABBREVIATIONS USED
■
BFCs, bifunctional chelators; DOTA, 1,4,7,10-tetraazacyclodo-
decane-N,N′,N″,N‴-tetraacetic acid; TETA, 1,4,8,11-tetraaza-
cyclotetradecane-N,N′,N″,N‴-tetraacetic acid; NOTA, 1,4,7-
triazacyclododecane-N,N′,N″-triacetic acid; DTPA, diethylene
triamine pentaacetic acid; ECB-TE2A, 4,11-bis-
(carboxymethyl)-1,4,8,11-tetraazabicyclo[6,6,2]hexadecane;
PCB-TE2A, 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo-
[6,6,3]hexadecane; NCS, isothiocyanate; EtOAc, ethyl acetate;
Et2O, diethyl ether; c(RGDyK), cyclic Arg-Gly-Asp-DTyr-Lys
peptide; FBS, fetal bovine serum; U87MG, Human glioblasto-
ma-astrocytoma epithelial-like cell line; KIRAMS, Korea
Institute of Radiological and Medical Sciences; tR, retention
time in chromatography
Biodistribution of 64Cu-PCB-TE2A-NCS-c(RGDyK). Tumor-
bearing BALB/c nude mice (n = 3) were injected via tail-vein with
64Cu-PCB-TE2A-NCS-c(RGDyK) (ca. 20 μCi in 150 μL saline per
mice). The animals were sacrificed at 1, 4, and 24 h postinjection. The
organs and tissues of interest (blood, heart, muscle, fat, bone, spleen,
kidney, intestine, liver, and tumor) were collected, weighted, and
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Journal of Medicinal Chemistry
Article
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