Pycup - A bifunctional, cage-like ligand for 64Cu radiolabeling

Article abstract of DOI:10.1021/mp400686z

In developing targeted probes for positron emission tomography (PET) based on ⁶⁴Cu, stable complexation of the radiometal is key, and a flexible handle for bioconjugation is highly advantageous. Here, we present the synthesis and characterization of the chelator pycup and four derivatives. Pycup is a cross-bridged cyclam derivative with a pyridyl donor atom integrated into the cross-bridge resulting in a pentadentate ligand. The pycup platform provides kinetic inertness toward ⁶⁴Cu dechelation and offers versatile bioconjugation chemistry. We varied the number and type of additional donor atoms by alkylation of the remaining two secondary amines, providing three model ligands, pycup2A, pycup1A1Bn, and pycup2Bn, in 3-4 synthetic steps from cyclam. All model copper complexes displayed very slow decomplexation in 5 M HCl and 90 C (t_1/2: 1.5 h for pycup1A1Bn, 2.7 h for pycup2A, 20.3 h for pycup2Bn). The single crystal crystal X-ray structure of the [Cu(pycup2Bn)] ²⁺ complex showed that the copper was coordinated in a trigonal, bipyramidal manner. The corresponding radiochemical complexes were at least 94% stable in rat plasma after 24 h. Biodistribution studies conducted in Balb/c mice at 2 h postinjection of ⁶⁴Cu labeled pycup2A revealed low residual activity in kidney, liver, and blood pool with predominantly renal clearance observed. Pycup2A was readily conjugated to a fibrin-targeted peptide and labeled with ⁶⁴Cu for successful PET imaging of arterial thrombosis in a rat model, demonstrating the utility of our new chelator in vivo.

Full text of DOI:10.1021/mp400686z

Article
pubs.acs.org/molecularpharmaceutics
64
PycupA Bifunctional, Cage-like Ligand for Cu Radiolabeling
Eszter Boros,*,† Elena Rybak-Akimova,‡ Jason P. Holland,§,∥ Tyson Rietz,† Nicholas Rotile,†
Francesco Blasi,† Helen Day,† Reza Latifi,‡ and Peter Caravan*,†
†The Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard
Medical School, 149 Thirteenth Street, Suite 2301, Charlestown, Massachusetts 02129, United States
‡Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, Massachusetts 02155, United States
§Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital, Department of Radiology, Harvard Medical
School, 55 Fruit Street, White 427, Boston, Massachusetts 02114, United States
∥Life Sciences, Australian Nuclear Science and Technology Organisation, Kirrawee, New South Wales, 2232, Australia
S
* Supporting Information
ABSTRACT: In developing targeted probes for positron emission tomography (PET) based on 64Cu, stable complexation of
the radiometal is key, and a flexible handle for bioconjugation is highly advantageous. Here, we present the synthesis and
characterization of the chelator pycup and four derivatives. Pycup is a cross-bridged cyclam derivative with a pyridyl donor atom
integrated into the cross-bridge resulting in a pentadentate ligand. The pycup platform provides kinetic inertness toward 64Cu
dechelation and offers versatile bioconjugation chemistry. We varied the number and type of additional donor atoms by
alkylation of the remaining two secondary amines, providing three model ligands, pycup2A, pycup1A1Bn, and pycup2Bn, in 3−4
synthetic steps from cyclam. All model copper complexes displayed very slow decomplexation in 5 M HCl and 90 °C (t1/2: 1.5 h
for pycup1A1Bn, 2.7 h for pycup2A, 20.3 h for pycup2Bn). The single crystal crystal X-ray structure of the [Cu(pycup2Bn)]2+
complex showed that the copper was coordinated in a trigonal, bipyramidal manner. The corresponding radiochemical complexes
were at least 94% stable in rat plasma after 24 h. Biodistribution studies conducted in Balb/c mice at 2 h postinjection of 64Cu
labeled pycup2A revealed low residual activity in kidney, liver, and blood pool with predominantly renal clearance observed.
Pycup2A was readily conjugated to a fibrin-targeted peptide and labeled with 64Cu for successful PET imaging of arterial
thrombosis in a rat model, demonstrating the utility of our new chelator in vivo.
KEYWORDS: chelator, copper, positron emission tomography, fibrin
before achieving optimal target uptake. The longer half-life also
allows for remote production and shipment of the radionuclide
over large distances and makes this radionuclide attractive for
clinical application in PET. Several radiotracers based on 64Cu
are currently being evaluated in multicenter clinical trials.4
To utilize 64Cu for targeted imaging, stable complexation of
the metal to the targeting vector is key. Macrocyclic and cage-
like hexadentate ligand systems are among the current front
runners for stable coordination with minimal metal ion loss
even after multiple days of incubation under physiologically
INTRODUCTION
■
With the increased availability of cyclotrons capable of
producing radiometals, the application of copper radionuclides
for medical imaging has gained great interest.1 In recent years,
the long-lived isotope 64Cu (β+ 17.4%, Emax = 0.656 MeV, β−
39%, Emax = 0.573 MeV) has received the greatest attention for
this purpose.2 Copper-64 has near ideal emission properties for
positron emission tomography (PET); the β+ is low energy
which facilitates acquisition of high-resolution PET images.3
The longer half-life (t1/2 = 12.7 h), compared to commonly
used, but short-lived radionuclides 18F, 11C, or even 68Ga is
more applicable to developing targeted PET agents conjugated
to larger biomolecules, such as peptides, antibodies, and
antibody fragments that may require long circulation times
Received: November 13, 2013
Accepted: December 2, 2013
Published: December 2, 2013
© 2013 American Chemical Society
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Figure 1. Structures of various chelators used for labeling with 64Cu.
Figure 2. Structures of chelates investigated in this study (from left to right): pycup2A, pycup1A1Bn, pycup2Bn, and the peptide conjugate pep(12).
relevant conditions.3 Acyclic chelates often fall victim to rapid
decomplexation kinetics, resulting in the loss of the radiometal
ion, characterized in vivo by persistent uptake of the Cu
radionuclide in the liver and bound to plasma proteins.5−7 It is
important to note that 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid (DOTA; Figure 1), often considered the
workhorse ligand for radiometal-based nuclear medicine, has
been well-documented to lack sufficient kinetic inertness for
use as a ligand for Cu(II).8,9 The ligands CB-TE2A, CB-
TE1A1P, SarAr, and PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-
pentadeca-1(15),11,13-triene-3,6,9-triacetic acid; Figure 1)
have proven to be more suitable for the formation of kinetically
inert copper complexes.10−16 NOTA (1,4,7-triazacyclononane-
1,4,7-triacetic acid) can also provide an alternative in some
cases.17 While the kinetically inert complexation of 64Cu is one
requirement for a 64Cu radiopharmaceutical, the charge,
lipophilicity, and conjugation linker will each affect the behavior
of the radiolabeled bioconjugates in vivo, often in an
unpredictable manner.11,16,18,19 Therefore, it is crucial to have
an armamentarium of bifunctional chelators available. It was
our goal to develop a new bifunctional chelator which would
not only provide another alternative in the chelate tool-box, but
also overcome various shortcomings of the currently available
systems. Key requirements include: (1) facile chemical
synthesis; (2) ease of ligand modification to alter overall
charge; (3) slow decomplexation of the copper complex under
forcing conditions; (4) rapid radiolabeling; (5) high kinetic
inertness of the 64Cu complex with respect to transchelation;
and (6) bioconjugation and applicability to a target-specific
peptide.
modification of CB-TE2A-type structures, either by extension
of the cross-bridge20 or integration of a donor atom into the
cross bridge.21 We also took the potent copper chelator cyclam
as a base and used the previously established synthesis of 1,8-
(2,6-pyridinedimethylene)-1,4,8,11-tetraazacyclotetradecane
(here abbreviated as pycup) by Brandes et al.22 to furnish a
ligand that incorporates a pyridyl nitrogen donor atom into the
cross-bridge. Incorporation of this donor group for the purpose
of building a more sturdy ligand is based on the well-
documented preference of Cu(II) for softer nitrogen
donors.23−25
To our knowledge, while similar ligands have been evaluated
and found to be very inert chelators for Cu(II), the pycup
structure or synthesis of potential pycup derivatives have not
been further explored for this purpose. Closely related bis-
amido-derivatives have been explored for the purpose of
formation of multinuclear complexes in organic solvents.26−28
For our purpose, we hoped that incorporation of a donor ligand
into the cross-bridge would reduce the need for additional
pendant arm donor atoms for stable coordination. The pendant
arms can then be used as handles for bioconjugation, providing
a versatile, easily synthesized chelator. To understand the
impact of the number of donor atoms on coordination, we used
the two residual secondary amines on the pycup ligand to
introduce 0, 1, or 2 acetate arms as additional donors and
charge equilibrators (pycup2A, pycup1A1Bn, and pycup2Bn,
Figure 2). The corresponding copper complexes were used to
study acid stability, labeling properties, and the inertness of the
corresponding radiochemical complexes.
64Cu-complexes have been used for the imaging of a number
of protein targeting vectors, such as somatostatin ana-
logues,29−31 bombesin derivatives,10,16,32 and RGD pepti-
des.11,17,33 We demonstrated that pycup2A could be used as
a bifunctional ligand by conjugating it to a known fibrin-
Inspired by the variety of macrocyclic ligand scaffolds, we
designed a new ligand to have the capability to fulfill all criteria
listed above. Recently published work on new Cu(II) chelators
has shown that highly inert Cu-complexes can be obtained by
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targeting peptide.34 This conjugate was radiolabeled and
subjected to the same kinetic inertness challenges as the
model compounds, followed by its evaluation as a PET probe
for thrombus imaging in a rat model.
g, 0.33 mmol) was dissolved in MeCN (10 mL). K2CO3 (10
equiv, 0.45 g, 3.3 mmol). tert-Butyl-bromoacetate (1.5 equiv,
0.098 g, 0.074 μL, 0.51 mmol) was added to the mixture, and
the reaction was stirred at room temperature overnight. The
mixture was then filtered, and the filtrate was reduced and
purified using preparative HPLC (method A). The desired
product eluted at 17.7 min. All fractions containing the pure
product were pooled, and the organic solvent was removed by
rotary evaporation. The aqueous residue was lyophilized to
afford the product (0.04 g, 0.075 mmol, yield: 22%) as a
colorless oil. Using this method, no monoalkylated product was
isolated. 1H NMR (CDCl3, 500 MHz, ppm): 8.02 (m, 1H), 7.5
(m, 2H), 4.23−2.97 (m, 24H), 2.05−1.92 (m, 4H), 1.43 (s,
18H). 13C NMR (CDCl3, 125 MHz, ppm): 165.7, 160.8,
123.23, 119.4, 84.1, 56.6, 53.9, 52.3, 27.8, 20.4, 19.8. LC-ESI-
MS: calcd for C29H50N5O4: 532.4. Found: 532.3 [M + H]+, Rt =
7.1 min (method C).
EXPERIMENTAL SECTION
■
General Methods and Materials. 1H and 13C NMR
spectra were recorded on a Varian 500 NMR system.
Chemicals were supplied by Aldrich Chemical Co., Inc., and
were used without further purification. Solvents (HPLC grade)
were purchased from various commercial suppliers and used as
received. Cyclam was received from Alfa Aesar (Ward Hill, MA,
USA). 2,6-Bis(bromomethyl)pyridine was purchased from
Sigma Aldrich, USA. Electronic spectra were recorded between
360 and 900 nm in 10 nm intervals on a Spectra max M2
(Molecular devices), using the SoftMax Pro software.
Purification via high-performance liquid chromatography
(HPLC) of intermediates was performed using HPLC methods
A and B after injection of the filtered, crude mixture onto
preparative HPLC (Rainin, Dynamax, column: 250 mm
Phenomenex C18). Method A: mobile phase A = 50 mM
ammonium acetate in water; mobile phase B = 10% 50 mM
ammonium acetate in water, 90% MeCN; flow-rate 15 mL/
min, 0−1 min: 5% B, 1−3 min: 5−40% B, 3−15 min: 40−80%
B, 15−16 min: 80−95% B, 16−20 min: 95% B, 20−21 min:
95−5% B, 21−23 min: 5% B. Purification of the ligand−peptide
conjugate FBP12 was done using method B: Mobile phase A =
0.1% trifluoroacetic acid in H2O; mobile phase B = 0.1%
trifluoroacetic acid in MeCN, flow rate 15−18 mL/min, 0−4
min: 5% B, 4−33 min: 5−40% B, 33−37 min: 40−95% B, 37−
42 min: 95% B, 42−43 min: 95−5% B, 45−48 min: 5% B.
HPLC purity analysis (both UV and MS detection) was carried
out on an Agilent 1100 system (column: Phenomenex Luna,
C18(2) 100/2 mm) with UV detection at 220, 254, and 280
nm by using method C: A gradient of A (10 mM ammonium
acetate in water) to 95% B (50 mM ammonium acetate in
water, 90% MeCN), flow rate 0.8 mL/min. 0−1 min: 5% B, 1−
10 min: 5−95% B, 10−12 min: 95% B, 12−12.5 min: 95−5%
B, 12.5−15 min: 5% B. Determination of radiochemical yield
and purity was measured using method D using an Agilent
1100 Series HPLC unit with a Carroll/Ramsey radiation
detector with a silicon PIN photodiode (column for reaction
control and DOTA challenge: Phenomenex Luna 5u C18(2),
150 mm × 4.6 mm × 5 μm; column for plasma stability and
blood draw analysis: Polaris C18 A 150 mm × 4.6 mm × 5
μm). Mobile phase A was 0.1% trifluoroacetic acid in H2O and
mobile phase B 0.1% trifluoroacetic acid in MeCN, flow rate 0.8
mL/min. 0−1 min: 5% B, 1−10 min: 5−95% B. 10−12 min:
95% B, 12−12.5 min 95−5% B, 15 min: 5% B.
1,8-(2,6-Pyridinedimethylene)-1,4,8,11-tetraazacyclotetra-
decane-2,4-diacetic Acid (pycup2A). 1,8-(2,6-Pyridinedi-
methylene)-2,4- (tert-butyldiacetato)-1,4,8,11-tetraazacyclote-
tradecane (0.04 g, 0.075 mmol) was dissolved in a 1:1 mixture
of dichloromethane and trifluoroacetic acid (3 mL). The
reaction mixture was stirred overnight at room temperature.
The solvent was removed in vacuo, and the oily residue was
brought up a minimal amount of H2O and lyophilized to afford
the product as a white solid (0.045 g, 0.075 mmol, quantitative
1
yield, with product as di-TFA salt). H NMR (CD3OD, 500
MHz, ppm): 8.15 (s, br, 1H), 7.62 (s, br, 2H), 4.90−3.97 (m,
4H), 3.87−1.85 (m, 20H). 13C NMR (CD3OD, 125 MHz,
ppm): 171.9, 159.8, 140.7, 122.9, 55.2, 53.8, 53.2, 53.0, 52.8,
52.5, 51.6, 51.2, 50.2, 49.1, 48.2, 46.4, 44.6, 19.2. LC-ESI-MS:
calcd for C21H34N5O4: 420.3. Found: 420.3 [M + H]+, Rt = 1.1
min (method C).
1,8-(2,6-Pyridinedimethylene)-4-benzyl-1,4,8,11-tetraaza-
cyclotetradecane (pycup1Bn). 1,8-(2,6-Pyridinedimethylene)-
1,4,8,11-tetraazacyclotetradecane (0.187g, 0.61 mmol) was
dissolved in MeCN (20 mL). K2CO3 (0.18 g, 1.3 mmol) and
benzylbromide (0.068 g, 0.047 mL, 0.4 mmol) were added to
the solution, and the reaction mixture was stirred at room
temperature overnight. Subsequently, the solid components
were filtered off, and the solvent was partially removed in vacuo.
The residue was then purified using preparative HPLC
(method A). The desired product eluted at 8.6 min. All
fractions containing the pure product were pooled, and the
organic solvent was removed by rotary evaporation. The
aqueous residue was lyophilized to afford the product (0.131 g,
0.35 mmol, yield: 55%) as a yellow semisolid. 1H NMR
(CD3OD, 400 MHz, ppm): 7.74 (m, 1H), 7.36 (m, 5H), 7.20
(d, 2H), 4.03 (d, 1H), 4.02 (s, 2H), 3.98 (d, 1H), 3.45 (dd,
2H), 3.21−2.37 (m, 18H), 1.72 (m, 2H). 13C NMR (CD3OD,
100 MHz, ppm): 159.6, 157.8, 137.9, 139.3, 128.3, 127.9, 121.0,
120.2, 138.9, 121.0, 58.8, 57.9, 53.7, 52.8, 52.1, 52.0, 48.8, 24.8,
24.2. LC-ESI-MS: calcd for C24H36N5: 394.3. Found: 394.3 [M
+ H]+, Rt = 5.6 min (method C).
1,8-(2,6-Pyridinedimethylene)-2,4-dibenzyl-1,4,8,11-tet-
raazacyclotetradecane (pycup2Bn). 1,8-(2,6-Pyridinedi-
methylene)-2,4-dibenzyl-1,4,8,11-tetraazacyclotetradecane was
isolated from the reaction for the formation of 1,8-(2,6-
pyridinedimethylene)-4-benzyl-1,4,8,11-tetraazacyclotetrade-
cane as a side product but can be selectively synthesized by
alkylation with 2 equivalents of benzyl bromide. The product
was purified using preparative HPLC (method A). The desired
product eluted at 18 min. All fractions containing the pure
1,8-(2,6-Pyridinedimethylene)-1,4,8,11-tetraazacyclotetra-
decane (pycup). The high dilution intramolecular dialkylation
of cyclam with 2,6-bis(bromomethyl)pyridine was conducted as
previously reported in literature by Brandes et al.22 The product
(0.22 g, 0.73 mmol) was afforded in a 14% yield starting from
1
cyclam (1 g, 5 mmol). H NMR (CD3OD, 400 MHz, ppm):
7.83 (m, 1H), 7.28 (d, 2H), 4.15 (d, 2H), 3.82 (d, 2H), 3.31−
2.84 (m, 16H), 2.62 (m, 2H), 2.07 (m, 2H). 13C NMR
(CD3OD, 100 MHz, ppm): 156.8, 138.9, 121.0, 55.8, 54.3,
52.2, 48.7, 23.1. LC-ESI-MS: calcd for C17H30N5: 304.4. Found:
304.4 [M + H]+, Rt = 0.5 min (method C).
1,8-(2,6-Pyridinedimethylene)-2,4- (tert-butyldiacetato)-
1,4,8,11-tetraazacyclotetradecane (pycup2AtBu). 1,8-(2,6-
Pyridinedimethylene)-1,4,8,11-tetraazacyclotetradecane (0.102
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product were pooled, and the organic solvent was removed by
all starting materials had been consumed and only trace
amounts of ligand−peptide conjugates containing only one
chelate were observed. The product was isolated using
preparative HPLC (method B), eluting at 24.1 min. The
corresponding fractions containing pure product were
collected, and the solvent was removed in vacuo to afford the
product as a white powder (0.002 g, 0.8 μmol, 10% purified
yield). LC-ESI-MS: calcd for C113H160ClN27O21S2: 2331.1.
Found: 1165.1 [M + 2H]2+. Rt = 4.5 min (method D).
rotary evaporation. 1H NMR (CD3OD, 400 MHz, ppm): 7.38−
7.28 (m, 13H), 4.11−3.52 (m, 12H), 2.99−2.72 (m, 16H). 13
C
NMR (CD3OD, 100 MHz, ppm): 159.3, 138.2, 130.8, 128.7,
128.5, 121.1, 58.2, 53.4, 53.3, 22.3. LC-ESI-MS: calcd for
C31H42N5: 484.3. Found: 484.4 [M + H]+, Rt = 8.7 min
(method C).
1,8-(2,6-Pyridinedimethylene)-4-benzyl-8-(tert-butylaceta-
to)-1,4,8,11-tetraazacyclotetradecane (pycup1AtBuBn). 1,8-
(2,6-Pyridinedimethylene)-4-benzyl-1,4,8,11-tetraaza-cyclote-
tradecane (0.032 g, 0.081 mmol) was dissolved in a 1:3 mixture
of DMF and MeCN (4 mL) and heated to 50 °C. K2CO3 (0.1
g, 0.72 mmol) and KI (0.1 g, 0.6 mmol) were added to the
solution, followed by a portion-wise addition of tert-butyl-
bromoacetate (2.5 equiv, 0.03 mL, 0.039 g, 0.2 mmol) every 24
h (0.01 mL portions). After 72 h, no starting material was
detected by LCMS. The solution was diluted with H2O (1 mL),
and the reaction mixture was filtered and purified with
preparative HPLC (modified method A, with hold of 95% B
solvent for 7 min instead of 4). The desired product elutes with
a retention time of 17 min. The corresponding fractions were
pooled, and the MeCN portion of the solvent was removed by
rotary evaporation. The residual solvent was removed by
lyopohilization to afford the product as a light yellow semisolid
Nonradioactive Copper Complexes. The general
procedure was as follows: Ligand (0.04 mmol) was dissolved
in MeOH (1 mL). Cu(ClO4)2·6H2O (0.04 mmol) was
dissolved in MeOH (1 mL) and added dropwise to the ligand
solution. The ligand was subsequently deprotonated using 2
equiv of NaOH (1 M aqueous solution). Upon addition of the
base, a stark change of color from light to dark blue/green was
observed. Solid components of the complexation reaction were
removed using a 0.2 μm filter. The filtrate was collected and
purified using solid phase extraction (C18 sep pak). The
fractions containing the product were collected, and the solvent
was removed in vacuo to afford the desired copper complex as a
blue ([Cu(pycup2Bn)][ClO4]2), turquoise ([Cu-
(pycup1A1Bn)]), or green powder ([Cu(pycup2Bn)][ClO4]).
All complexes were analyzed for purity using LC-MS, method
C. Na[Cu(pycup2A)] LC-ESI-MS: calcd for C21H34CuN5O4:
1
(0.013 g, 0.025 mmol, yield: 31%). H NMR (CD3OD, 500
MHz, ppm): 7.83 (m, 1H), 7.45−7.22 (m, 7H), 4.32−3.95 (m,
6H), 3.46−2.33 (m, 20H), 1.51 (m, 1H), 1.47 (s, 9H), 1.28 (m,
1H). 13C NMR (CD3OD, 125 MHz, ppm): 170.5, 138.6, 131.7,
129.4, 129.3, 128.7, 127.4, 121.9, 120.3, 80.9, 59.9, 57.3, 55.6,
53.9, 53.2, 53.1, 52.9, 51.9, 49.8, 49.4, 47.1, 26.3, 21.5. LC-ESI-
MS: calcd for C30H46N5O2: 508.4. Found: 508.4 [M + H]+, Rt =
6.1 min (method C).
482.18. Found: 482.2 [M + 2H]+, Rt = 1.5 min. UV−vis: λmax
=
710 nm, ε = 116 M−1 cm−1. [Cu(pycup1A1Bn)] LC-ESI-MS:
calcd for C26H36CuN5O2: 513.22. Found: 513.2 [M]+, Rt = 7.1
min. UV−vis: λmax = 680 nm, ε = 133 M−1 cm−1.
[Cu(pycup2Bn)][ClO4 ]2 LC-ESI-MS: calcd for
C31H41ClCuN5: 581.23. Found: 581.2 [M + Cl−]+, Rt = 7.0
min. UV−vis: λmax = 670 nm, ε = 268 M−1 cm−1.
[Cu(FBP12)][ClO4]2 LC-ESI-MS: Found: 581.2 [M +
2H]2+, Rt = 4.5 min.
1,8-(2,6-Pyridinedimethylene)-4-benzyl-1,4,8,11-tetraaza-
cyclotetradecane-8-acetic acid (pycup1A1Bn). 1,8-(2,6-Pyr-
idinedimethylene)-4-benzyl-8-(tert-butyl-acetato)-1,4,8,11-tet-
raazacyclotetradecane (0.003 g, 6 μmol) was dissolved in a 1:1
mixture of dichloromethane and trifluoroacetic acid (3 mL).
The reaction mixture was stirred overnight at room temper-
ature. The solvent was removed in vacuo, and the oily residue
was brought up a minimal amount of H2O and lyophilized to
afford the product as a white solid (0.002 g, 5.9 μmol,
X-ray Crystallography and Structural Refinement
Details. A blue plate crystal of the macrocyclic copper(II)
complex was mounted on the Mitogen mount using paraton oil.
X-ray intensity data were measured at 100 K with a Bruker D8
Quest diffractometer (equipped with a Photon CMOS detector
and a Triumph monochromator) using Mo Kα radiation (λ =
0.71073 Å). The raw data frames were integrated with the
SAINT+ program; corrections for Lorenz and polarization
effects were applied. An empirical absorption correction based
on multiple measurements of equivalent reflections was applied
using the program SADABS. The structure was solved by the
intrinsic phasing method (SHELX 2013)35 and refined by full-
matrix least-squares on F2 with SHELXL.36 All non-hydrogen
atoms (except for disordered solvent) were refined anisotropi-
cally; hydrogens were placed in calculated positions. Benzyl
groups of the macrocycle and chloride counterions were refined
as disordered. Sheets of macrocyclic cations are separated by
layers filled with disordered solvent molecules (which were not
individually modeled, but rather collectively taken into account
using the SQUEEZE module of Platon) and chloride
counterions; the overall stoichiometry (3Cl:Cu) suggests a
singly protonated species must be present in the solvent.
Crystallographic information is available in the cif. Note: Atom
numbering in cif differs from given numbering in manuscript.
Computational Details. All calculations were conducted
using density functional theory (DFT) as implemented in the
Gaussian03W suite of ab initio quantum chemistry programs.37
Geometry optimizations and vibrational frequency calculations
were performed by using the unrestricted B3LYP exchange and
1
quantitative yield, with product as the nTFA salt). H NMR
(CD3OD, 400 MHz, ppm): 7.91 (m, 1H), 7.65−7.21 (m, 7H),
4.81−1.72 (m, 28H). 13C NMR (CD3OD, 125 MHz, ppm):
178.1, 160.6, 160.2, 131.7, 131.1, 130.1, 129.1, 128.8, 121.8,
58.1, 54.3, 52.9, 52.5, 50.9, 21.0. LC-ESI-MS: calcd for
C26H38N5O2: 452.3. Found: 452.3 [M + H]+, Rt = 3.2 min
(method C).
pep(12). pycup2A·2TFA (0.015 g, 0.03 mmol) and PyBOP
(benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluoro-
phosphate, 0.014 g, 0.035 mmol) were dissolved in DMF
(dimethylformamide, 1 mL). DIPEA (N,N-diisopropylethyl-
amine) was added in 2 μL aliquots in order to adjust the pH of
the solution to 7. After 5 min, Pep2 (0.012 g, 0.008 mmol,
synthesized using standard solid phase methods; sequence H-
[Phe-His-Cys-Hyp-Tyr(3-Cl)-Asp-Leu-Cys-His-Ile-Gln cyclic
(2 → 8) disulfide]-1-(4-(aminomethyl)benzyl) acetamide)
was dissolved in DMF and added dropwise to the preactivated
ligand solution. Subsequently, the pH of the reaction mixture
was readjusted to 7 using additional 2 μL aliquots of DIPEA.
The reaction was then allowed to stir overnight at room
temperature. After 24 h, the crude reaction mixture was
analyzed using LCMS and was found to contain product, while
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correlation functionals and the double-ζ LANL2DZ basis set
for all atoms.38,39 Normal self-consistent field (SCF) and
geometry convergence criteria were employed, and structures
were optimized in the gas phase without the use of symmetry
constraints. Harmonic frequency analysis based on analytical
second derivatives was used to characterize the optimized
geometries as local minima.
Acid Stability of Nonradioactive Metal Complexes. A
sample of 1 mg of the copper complex was dissolved in 1 mL
(affording a ∼2 × 10−3 M solution). A portion of 1 mL of 10 M
HCl was added, and the absorption spectrum (400−900 nm) of
the corresponding solution was measured immediately (t0).
The solution was then incubated in an oil bath at 90 °C. The
absorption spectrum was remeasured at different time points,
and data were fitted with a first-order dissociation kinetics
model ([A] = [A]0e−kt) to estimate the half-life using the Igor
Pro software package.
Radioactive Copper Complexes. The general procedure
was as follows: 64CuCl2 (0.1−0.4 mCi, in 30 μL, purchased
from University of WisconsinMadison) was diluted with 270
μL of pH 8 sodium citrate (10 mM). A sample of 10 μL of a 0.1
mM ligand solution (in HEPES pH 7.4) was added (affording a
final ligand concentration of <0.01 mM), and the reaction
mixture was heated at 70 °C for 15 min. Subsequent reaction
monitoring was done by analytical HPLC, method D. Under
these conditions, all pycup derivatives form the desired
radiochemical complex with quantitative yields. These crude
radiochemical reaction mixtures were stable over at least 48 h
(evaluated by HPLC); no decomplexation or loss of copper is
observed. Observed retention times: 64Cu(DOTA): 4.8 min;
[Cu(pycup2A)]: 6.6 min; [Cu(pycup1A1Bn)]+: 8.8 min;
[Cu(pycup2Bn)]2+: 8.9 min; free 64Cu in serum: 4.2 min.
Excess Ligand Challenge Experiment. A 100 μL aliquot
of the radiolabeled ligand or peptide conjugate (ligand
concentration <0.01 mM) was added to 100 μL of DOTA (1
mM, HEPES buffer, pH 7.4), affording a solution with over
100-fold excess DOTA. The solution was then incubated for 24
h at 37 °C or for 48 h at 70 °C. The 10 μL aliquots were
removed at specific time points and analyzed for transchelation
using HPLC, method D.
PET-CT and Biodistribution of FBP12. Four male Wistar
rats (350−400 g) were anesthetized with isoflurane (1−2% in
70% N2O and 30% O2), and the body temperature was kept at
37.5 °C. The right femoral vein was cannulated for probe
injection. The right femoral artery was cannulated for blood
sampling. An incision was made in the neck, and the right
common carotid artery was isolated. Intramural thrombus was
produced by crushing the right common carotid artery with
hemostat clamps for 5 min. Probes were injected within 20 min
of thrombus formation. The rat was then placed in a cradle and
positioned in a Gamma Medica Triumph Micro PET/CT/
SPECT. Each rat was injected with approximately 0.3−0.4 mL
and 300−500 μCi of the dose solution, and the animal
underwent a 90 min dynamic PET scan with the field of view
centered on the neck. The total activity injected was calculated
by subtracting the activity in the syringe before injection from
the activity remaining in the syringe after injection as measured
on a dose calibrator (Capintec CRC-25PET). After the PET
imaging, a CT image of the same anatomical region was
obtained. The CT scan was performed with a continuous
infusion of CT contrast (Isovue, Bracco Diagnostics Inc.,
Princeton NJ, USA infused at 0.3 mL/min) to delineate the
major blood vessels.
During the imaging study serial blood draws were collected
from the femoral artery at 0, 2, 5, 10, 15, 30, 60, and 120 min
postinjection into EDTA blood tubes. Blood was weighed, and
radioactivity in the blood was measured on a gamma counter
(Packard, CobraII Auto gamma) to assess clearance of total
64Cu. Animals were euthanized two hours post injection, and
the organ distribution of 64Cu was quantified ex vivo. The
thrombus (located in the right carotid artery), the contralateral
carotid artery, blood, urine, intra-abdominal organs, brain, left
rectus femoris muscle, and left femur bone were collected from
all animals. The tissues were weighed, and radioactivity in each
tissue was measured on a gamma counter, along with a weighed
aliquot of the injected dose solution. The radioactivity in each
tissue was reported as percent-injected dose per gram (%ID/g).
The right and left carotid arteries were further analyzed by
autoradiography using a multipurpose film and a Perkin-Elmer
Cyclone Plus Storage Phosphor system. Data were analyzed by
using the Student’s t-test where P-values of less than 0.05 were
considered statistically significant. Uncertainties are expressed
as the standard error of the mean.
Rat Plasma Stability. A 100 μL aliquot of the radiolabeled
ligand or peptide conjugate (ligand concentration <0.01 mM)
was added to 400 μL of rat plasma (Lampire Biological
Laboratories). The solution was then incubated for 24 h at 37
°C. The 100 μL aliquots were removed at specific time points,
diluted with 200 μL HEPES buffer, and filtered using a 0.2 μm
filter. A 100 μL aliquot of this filtrate was analyzed for
transchelation using HPLC, method D.
Animal Models. All experiments were performed as
previously published, in accordance with the NIH Guide for
the Care and Use of Laboratory Animals, and were approved by
the Massachusetts General Hospital Subcommittee on
Research Animal Care.
PET Image Analysis. The PET data were dynamically
reconstructed with varying time windows. The volume of
interest (VOI) analysis of the reconstructed and coregistered
PET and CT data was performed with Amide.40 The CT image
was used as guidance to identify the VOI regions. VOI of
carotid arteries were determined guided by contrast agent
present in the blood vessel and the region of high activity from
the PET image (from the thrombus). The contralateral artery
VOI was chosen to be the same size as the VOI derived for the
thrombus in the ipsilateral artery.
Biodistribution of [64Cu(pycup2A)]. Three BALB/c mice
were injected with ∼5 μCi of radiochemically pure [64Cu-
(pycup2A)] through a tail vein catheter. The animals then were
sacrificed 2 h post injection, and organs were harvested and
weighed; their corresponding activity was counted using a
Packard, CobraII Auto gamma scintillation well counter. The
radioactivity in each tissue was reported as percent-injected
dose per gram (%ID/g), which was calculated by dividing the
counts of 64Cu per gram of tissue by the total counts of the
injected dose.
RESULTS AND DISCUSSION
■
Preparation of Ligands and Complexation with Cu(II).
All intermediates and final products were characterized by using
1H, 13C NMR spectroscopy; the connectivity was confirmed
using proton−proton and proton−carbon correlation 2D NMR
(HSQC and HMBC). The synthesis of pycup derivatives differs
considerably from the synthesis of other cross-bridged
systems.41 Unlike most alternative caged or cross-bridged
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a
Scheme 1. Synthesis Scheme of Model Ligand Systems Synthesized
a
(a) CHCl3, Na2CO3, rt, 24 h. (b) Benzyl bromide (2.5 equiv), MeCN, rt, 18 h. (c) benzyl bromide (0.65 equiv), MeCN, rt, 18 h. (d) (i) tert-
butylbromoacetate (1.5 equiv), 50 C, 24 h. (ii) DCM/TFA (1:1), rt, 16 h.
systems, synthesis of pycup is facile and does not require metal-
templating or multistep protecting group chemistries. Instead,
the pycup ligand can be furnished in one step using
intramolecular cross-bridging using 2,6-bis(bromomethyl)-
pyridine.22
afford the complexes as turquoise or blue microcrystalline
powders.
Solid State Crystal Structure of [Cu(pycup2Bn)]2+.
Crystals suitable of X-ray diffraction were obtained by
dissolution of the [Cu(pycup2Bn)]2+ complex in 5 M HCl
and heating at 60 °C. The single-crystal X-ray structure of the
macrocyclic complex revealed well-defined sheets of macro-
cyclic cations separated by layers filled with disordered solvent
molecules (which were not individually modeled, but rather
collectively taken into account using the SQUEEZE module of
Platon) and chloride counterions; the overall stoichiometry
(3Cl:Cu) suggests a singly protonated species must be present
in the solvent. Relevant bond lengths and angles are provided
in Table 1; the molecular structure is shown in Figure 3.
A low yield of only 14% for intramolecular cyclization was
obtained which is likely due to competing intermolecular
reactions that were observed even under high dilution
conditions. Importantly, the corresponding cyclen analogue
cannot be formed under the same reaction conditions. Single or
double alkylated products such as pycup2AtBu and pycup2Bn
can be synthesized under standard conditions using 0.5−2.5
equiv of alkyl bromide (tert-butyl-bromoacetate or benzyl
bromide) as an electrophile in the presence of inorganic base
(K2CO3). The low solubility of the pycup scaffold in
acetonitrile was alleviated by the addition of DMF. Single
alkylated products were separated from the double-alkylated
products by preparative HPLC using a neutral mobile phase
and gradient method. In the case of pycup1AtBu1Bn, pycup1Bn
was alkylated under the same conditions as above, but with the
addition of KI and heating in order to increase the efficiency of
the second, less favored alkylation. The pycup1AtBu1Bn
product was isolated using preparative HPLC as described
above. In the case of pycup2AtBu and pycup1AtBu1Bn, the tert-
butyl protection group was removed using a 1:1 mixture of
DCM and TFA overnight. All derivatives were synthesized
within a maximum of four easily reproducible steps (Scheme
1).
Subsequent complexation of the ligands with Cu(ClO4)2·
6H2O at pH 5−6 (adjusted using 0.1 M NaOH or HCl)
provided the corresponding copper complexes as confirmed by
LC-MS. Immediate complex formation occurs upon adjustment
of the pH as was evident from the color change of the solution
from light blue ([Cu(H2O)6]2+) to a turquoise or dark blue
depending on the Cu complex environment. Purification of Cu
complexes was achieved using reverse-phase chromatography
with C18 sep-pak cartridges where the separation was
monitored by observation of the color change of the eluate
from light blue (uncomplexed copper) to dark blue or
turquoise (copper complex). The complexes were loaded
onto the prerinsed cartridge in H2O and eluted using a 1:1
mixture of H2O/EtOH. Fractions containing the complexes
were analyzed using LC-MS. The solvent was evaporated to
Figure 3. Molecular structure of the cation [Cu(pycup2Bn)]2+
(thermal ellipsoids are drawn at 50% probability level). Hydrogen
atoms, counterions, and solvent molecules are omitted for clarity.
The coordination geometry of copper is typical of copper(II)
complexes with a N-donor polydentate ligand: the metal is five-
coordinate, all five nitrogen donors of the macrocycle are
bound to the central copper cation, and no additional solvent
or anion is present within the bonding distance of the metal.
Copper−nitrogen bond lengths range from 1.954(3) Å (Cu−
N1) to 2.154(2) Å (Cu−N5); these distances are common for
coordination bonds formed by copper(II) and neutral nitrogen
donors.6 The shortest Cu−N contact is between the copper
center and the pyridine nitrogen (N1), followed by the contact
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Figure 4. DFT optimized structures of (A) trans-[Cu(pycup1A1H)]+, (B) cis-[Cu(pycup1A1H)]+, and (C) [Cu(pycup2Bn)]2+ . Selected atoms
have been numbered, and hydrogen atoms bonded to carbon have been omitted for clarity. Gray = carbon; blue = nitrogen, cyan = copper; red =
oxygen; white = hydrogen.
Table 1. Comparison of the Various Geometric Parameters between the Experimental X-ray Single Crystal Structure and DFT
Optimized Structure of the [Cu(pycup2Bn)]2+ Complex and Selected Optimized Structural Parameters for the Two Geometric
Isomers of [Cu(pycup1A1H)]+
parameters [r: Å; a: deg] X-ray structure DFT structure [Cu(pycup2Bn)]2+ DFT structure trans-[Cu(pycup1A1H)]+ DFT structure cis-[Cu(pycup1A1H)]+
r(Cu−N1)
r(Cu−N2)
r(Cu−N3)
r(Cu−N4)
r(Cu−N5)
r(Cu−O1)
1.953
2.053
2.154
2.053
2.154
2.003
2.115
2.208
2.115
2.208
2.113
2.362
2.060
2.314
2.102
1.970
76.3
1.921
2.258
2.420
2.312
2.011
2.037
83.0
a(N1−Cu−N2)
a(N1−Cu−N3)
a(N2−Cu−N3)
a(N2−Cu−N4)
a(N3−Cu−N5)
a(N1−Cu−N5)
a(N1−Cu−O1)
a(N2−Cu−O1)
a(N3−Cu−O1)
a(N4−Cu−O1)
a(N5−Cu−O1)
81.0
80.9
124.8
87.4
124.0
86.8
92.0
85.1
82.5
74.1
161.9
110.3
124.8
161.8
112.1
124.0
154.7
169.4
98.3
155.5
108.4
165.4
83.5
176.9
106.1
86.4
100.8
168.0
97.9
99.1
83.4
83.3
a
RMSD/Å
0.1164
a
NB: The mass weighted RMSD value was calculated based on the atomic positions of all heavy atoms. The two benzyl groups showed significant
distortion in the experimental structure. For these atoms, one ring position was selected and used as an input geometry and subsequently for
comparison with the DFT optimized structure.
with the adjacent tertiary amines of the pyridine bridge (N2,
N4); the longest contacts are with the benzylated cyclam
nitrogens (N3, N5); this is consistent with the relative ligand
field strength of these nitrogen donors and the overall
conformation of the bridged cyclam. The coordination
geometry of copper in the pyridine-bridged cyclam is nearly
trigonal, with the trigonal plane formed by the pyridine
nitrogen (N1) and two tertiary amines from the benzylated
fragment of cyclam (N3, N4) and the tertiary amine bridgehead
nitrogens (N2, N4) occupying apical positions (with the
corresponding bond angle, N2−Cu−N4, 161.90(15)°. The
trigonal distortion parameter τ, which ranges from 1 for ideal
trigonal bipyramidal geometry to 0 for ideal tetragonal
pyramid,42 is 0.86 for the macrocyclic cation described herein.
The cyclam macrocyclic ring is folded, although both six-
membered chelate rings adopt chair conformations. This
represents a stark contrast to the structures reported of the
strapped bis-amide complexes by Hegedus and coworkers.28 In
that case, the six-membered metallacycles cannot fold into chair
conformation due to the sp2-hybridization of the two amide-
carbons, which subsequently leads to copper adopting
octahedral coordination with an ethyl acetate coordinated as
an additional, monodentate ligand.
Computational Study of [Cu(pycup2Bn)]+2 and [Cu-
(pycup1A1H)]+. As we were only able to obtain single crystals
suitable for X-ray diffraction for the [Cu(pycup2Bn)]2+
complex, we employed density functional theory (DFT;
B3LYP/LANL2DZ) calculations to investigate the optimized
Cu(II) coordination environment when chelated by pycup
derivatives with acetate groups available for coordination. The
DFT optimized structure of [Cu(pycup2Bn)]2+ was compared
to that of the experimental single crystal X-ray structure (Figure
4 and Table 1).
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The comparison of the experimental and DFT calculated
structure of [Cu(pycup2Bn)]2+ revealed a weighted root-mean-
squared-displacement (RMSD) between all heavy atoms (C, N,
and Cu) of only 0.1164 Å. All other bond lengths and angles lie
within standard errors for DFT calculations with the largest
deviation in bond length of only 0.062 Å for the r(Cu−N2) and
r(Cu−N4) bonds.
The small RMSD value between the calculated and the
experimental structures confirms that the calculations faithfully
reproduce the geometric structure of this class of complexes
and provides confidence in our subsequent analysis of the
computational data.
Table 2. Half-Lives for the Dissociation of Cu(II)
Complexes in 5 M HCl and 90 °C
a
ligand
t1/2
pycup2A
pycup1A1Bn
pycup2Bn
DOTA
2.7 h
1.5 h
20.3 h
<3 min
<3 min3
156 h3
∼40 h3
b
NOTA
CB-TE2A
SarAr
C3B-TE2A
[Cu(L1)]
4.3 h
The DFT optimized geometric isomers of [Cu-
(pycup1A1H)]+ revealed that, in the gas phase, the trans
isomer is more stable than the cis isomer with a standard free
energy difference of ΔG(trans−cis) = −83 kJ mol−1. This
energy difference is enthalpic in nature with ΔH(trans−cis) =
−85 kJ mol−1. Examination of the structures reveals that both
geometric isomers of Cu(pycup1A1H) are highly distorted with
asymmetry observed in the Cu−N bond lengths (Table 1). For
example, in trans-[Cu(pycup1A1H)]+ the N2 and N4 atoms are
both cis to the pyridine N1 atom yet the Cu−N2 and Cu−N4
bond lengths are 2.362 Å and 2.311 Å, respectively. A similar
trend is observed in the geometry of the cis-[Cu-
(pycup1A1H)]+ isomer. The calculations predict that, in the
trans-[Cu(pycup1A1H)]+ isomer, a strong Jahn−Teller dis-
tortion is observed across the macrocyclic ring involving the
two bridge-head nitrogen atoms N2 and N4.
Acid Stability. For suitable cross comparison with
previously investigated systems from literature, we subjected
the three model complexes to acid-mediated decomplexation
under strongly forcing conditions. Dissolution of the copper
complex in 5 M HCl and followed by measurement of the UV−
vis spectrum (Figure S4) between 400 and 900 nm provided
the t = 0 data points, as we had previously found that all three
complexes exhibited very slow decomplexation at room
temperature. The acidic solution was then heated at 90 °C,
and samples were removed at specific time points and
monitored by UV−vis spectroscopy.
a
DOTA was measured as a control, values found for previously
published ligands NOTA, CB-TE2A, SarAr, C3B-TE2A,20 and
[Cu(L1)].21 The authors report no decomplexation for up to 7 days.
b
nation or exchange of the metal ion with the proton trapped by
the ligand at lower pH ranges becomes much more difficult. We
preformed the 64Cu(citrate) complex to avoid formation of
colloidal Cu(OH)2 at elevated pH and added the 64Cu(citrate)
complex to pycup ligand solutions in pH 7.4 HEPES buffer.
Ligand concentrations as low as 10 μM were able to provide
quantitative yields within 15 min at 70 °C, affording specific
activities of 100 mCi/μmol. We observed same radiolabeling
conditions for all derivatives synthesized. Lower temperatures
such as 60 °C provide quantitative yields within 30 min. We
hypothesize that the rigid preorganized structure of pycup is the
reason for the need to label at increased temperatures. The
cross-bridged structure can likely not invert as readily as more
flexible ligand systems such as triethylenetetramine (TETA) or
DOTA. Figure 5 shows crude radiolabeling traces obtained
Under these conditions, we observed a half-life of 2.7 h for
dissociation of copper from the complex with pycup2A and 1.5
h for dissociation of copper from pycup1A1Bn. The copper
complex of pycup2Bn proved to be much more kinetically inert
with a half-life of 20.3 h.
All Cu(pycup) derivatives were found to be more stable to
decomplexation than the DOTA (reproduced as a control) and
NOTA (from ref 3) complexes. The Cu(pycup) derivatives
were inferior to, but more comparable with, the highly inert
complexes [Cu(CB-TE2A)] and [Cu(SarAr)]2+ (Table 2). Our
results are comparable to what Denat and co-workers obtained
for DO2A type derivatives with an ether crossbridge.21 We
hypothesize that the protonation of the acetate arm(s) results
in initial aquation of the copper complexes of pycup2A and
pycup1A1Bn, and this results in eventual dissociation of the
metal ion. Collectively, these data indicated that integration of
the pyridyl ligand moiety into the cross-bridge was advanta-
geous for increased kinetic inertness.
Figure 5. Radio-HPLC traces of the crude reaction mixture after 15
min at 70 °C are shown for the radiochemical complexes
[64Cu(pycup2A)] (blue), [64Cu(pycup1A1Bn)]+ (red), and [64Cu-
(pycup2Bn)]2+ (green). One-minute offsets of the red and green trace
are used for clearer delineation of the product peaks.
Radiolabeling. Radiolabeling conditions for 64Cu can range
from a pH of 5 to above 7, depending on the ligand used. We
observed radiolabeling yielding only up to 20% product under
conditions below pH 7 for all pycup derivatives, which
prompted us to use more basic conditions above pH 7. Due
to the proton-sponge nature of the pycup ligand,22 deproto-
using the conditions described above. Nevertheless, the
radiolabeling conditions require shorter reaction time at a
lower temperature as CB-TE2A conjugates.
DOTA Ligand Challenge and Rat Plasma Challenge of
Model Complexes. Table 3 summarizes the percentage
transchelation under different conditions at select time points.
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Table 3. Summary of Stability Data of Radiochemical 64Cu Complexes of the Investigated Ligand Systems Using Excess DOTA
a
Challenge and Plasma Challenge
ligand [time point, % complex stability]
challenge experiment
pycup2A
pycup1A1Bn
pycup2Bn
2 h
95
12 h
96
24 h
95
2 h
99
12 h
96
24 h
96
2 h
99
12 h
95
24 h
93
>100 equiv DOTA (37 °C)
>100 equiv DOTA (70 °C)
rat plasma (37 °C)
2 h
97
24 h
95
48 h
95
2 h
98
24 h
95
48 h
95
2 h
97
24 h
91
48 h
91
0.5 h
98
12 h
98
24 h
98
0.5 h
99
12 h
99
24 h
98
0.5 h
97
12 h
98
24 h
97
a
Data are provided as % intact radiochemical complex at the corresponding time point.
To assess the in vitro stability of the formed radiolabeled model
complexes, we conducted two different in vitro challenge
experiments. We first used over 100 equiv of DOTA to assess
transchelation with a potent copper chelator (log K for
[Cu(DOTA)]2− = 22.2)3 at 37 °C in pH 7.4. We found that
within 24 h, all complexes were stable with <7% decom-
plexation observed (Table 3). To determine the thermody-
namic equilibrium state of this transchelation reaction, we also
conducted the same experiment at 70 °C, over a time frame of
48 h. We observed a maximum transchelation to DOTA of
≤9% which persisted after 15 h incubation demonstrating that
all 64Cu(pycup) complexes investigated are at least an order of
magnitude more thermodynamically stable than [Cu(DOTA]2−
at pH 7.4.
As a second challenge experiment, we incubated the
radiolabeled complexes in rat plasma at 37 °C and quantified
the percentage of intact radiochemical complex at 30 min, 1 h,
and 24 h. We found that the model complexes were all highly
stable under these conditions even after 24 h (≥97% intact).
Biodistribution of [64Cu(pycup2A)]. We next evaluated
the biodistribution of a nonderivatized pycup derivative. We
radiolabeled pycup2A, using conditions described above. Balb/c
mice (n = 3) were injected with equivalent radioactive doses of
[64Cu(pycup2A)], sacrificed, and organs harvested 2 h post
injection. Biodistribution studies revealed only background
levels of activity were retained in the animals at 2 h p.i.
Importantly, liver accumulation of radioactivity was very low
(1.14 0.21%ID/g) providing additional confirmation of the
stability of our Cu-complexes in vivo.6
We observed low levels of activity in the kidneys (2.05
0.19%ID/g) and very high levels of radioactivity in the urine,
consistent with rapid renal clearance. Almost all of the activity
cleared the animals by 2 h. The biodistribution of [64Cu-
(pycup2A)] shows a favorable clearance profile and low uptake
in nontarget organs, which provides a firm basis for the
development of targeted agents using the pycup technology
(Table 4).
Bioconjugation. To demonstrate that our new ligand
system can be easily conjugated to biomolecules, we linked
pycup2A to a fibrin-binding specific peptide via amide bond
formation with a carboxylate of pycup2A. The fibrin-binding
peptide originated from a phage display screen by Kolodzjej et
al.43 and has been used in other fibrin targeted imaging
probes.34,44 Fibrin is a principal component of blood clots45
and represents a suitable imaging target for the detection of
thrombus (blood clot). Thrombus is implicated in devastating
cardiovascular diseases such as stroke, myocardial infarction,
and deep-vein thrombosis.
Table 4. Biodistribution Data for [64Cu(pycup2A)] in Balb/c
Mice (n = 3) at 2 h Post-Injection Expressed as Percent
Injected Dose per Gram of Organ (%ID/g)
organ/tissue
average (% ID/g, std. dev.)
blood
liver
0.04 0.01
1.14 0.20
0.05 0.01
0.21 0.01
0.12 0.04
0.32 0.07
2.05 0.19
heart
lung
spleen
intestine
kidney
DMF and added to the PyBOP activated ligand pycup2A. The
pH was adjusted to 7, and the reaction mixture was allowed to
stir overnight. The excess pycup2A provided essentially
exclusive formation of the desired, diamidated product. We
observed that product only formed when the pH was
maintained at 7 or slightly below, likely due to partial
protonation of the terminal amines of the peptide under
more acidic conditions. Purification of the peptide using
preparative HPLC, and an acidic mobile phase provided the
desired product in moderate yields. Successful conjugate
(structure shown in Figure 6) was confirmed by detection of
the [M + 2H]2+ ion using LC-MS. Formation of the
corresponding copper complex was performed using the same
method as described in the Experimental Section for the model
complexes.
Thrombus Imaging Using FBP12. We have been
interested in fibrin targeting for some time34,46,47 and recently
characterized several fibrin-targeted peptides conjugated with
DOTA-monoamide and labeled with 64Cu for thrombus PET
imaging.34 In that study, we found that the 64Cu(DOTA-
monoamide)-labeled peptide (termed FBP2 and analogous to
the structure in Figure 6) showed good thrombus uptake in a
rat model of acute arterial thrombosis, and that the thrombi
were readily apparent in PET images. We also found however,
that FBP2 was not fully stable in vivo and there was some
transmetalation of 64Cu from the probe to plasma proteins.
This transmetalation resulted in higher blood background and
limited the thrombus:background levels that could be achieved.
Here, we tested the hypothesis that a more inert copper chelate
should result in greater thrombus:background levels. We
evaluated the 64Cu-labeled bioconjugate FBP12 using the
same crush injury model of carotid artery thrombosis as used
previously.34
PET images with the pycup derivative FBP12 clearly
delineate the thrombus in the right carotid artery. Figure 5
shows three orthogonal PET images taken through the region
of high activity denoted by the yellow arrow. CT angiography
The fibrin-binding peptide, with two terminal amine
functional groups, was dissolved in a minimal amount of
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Figure 6. Chemical structure of the pep(12) conjugate.
Figure 7. Fused PET/CT sagittal (left), coronal (middle), and transaxial (right) images (30−90 min p.i.) of a rat with a carotid artery thrombus
using FBP12. The yellow arrow denotes the location of the engineered clot with high activity; the white arrow indicates the surgical incision site
which also shows high activity due to microthrombi.
confirmed that this region of high activity was localized to the
right common carotid artery, the site at which the thrombus
was induced. This model also results in some microthrombosis
around the surgical site.34,48 Figure 7 shows that the surgical
site is strongly enhanced by FBP12 (white arrows). It is
apparent from the sagittal (left) and axial (right) images that
the enhancement of the surgical site is superficial (white
arrow), while the carotid thrombus is deeper in the neck
(yellow arrow). The coronal (middle) and axial (right) images
also demonstrate that there is very little activity in the
contralateral artery.
The images were quantitatively analyzed by taking a volume
of interest (VOI) that covered the focal activity in the right
carotid and comparing it to the same sized VOI in the left
carotid. Time activity curves (Figure S7, Supporting Informa-
tion) showed increased initial activity in both vessels after
injection, but the signal in the left carotid clears rapidly while
signal clears more slowly from the right carotid. The ratio of
right carotid−left carotid VOI was 2.96 0.42 at 90 min post
injection. We also analyzed the carotid artery segments ex vivo
using autoradiography (Figure S8, Supporting Information).
Autoradiography confirmed higher activity in the clot
containing vessel, and the ratio of activity (right carotid−left
carotid) in the autoradiography images was 1.5 0.19. Ex vivo
biodistribution data (Table 5) showed higher activity in the
thrombus than in the blood, brain, heart, muscle, bone, and
contralateral artery. At 2 h post injection, most of the activity
had cleared the animal.
Table 5. Biodistribution and Clot-to-Organ Ratios of Probe
FBP12 in a Rat Model of Arterial Thrombus at 2 h Post-
Injection (n = 4)
organ/tissue average (% ID/g, std. dev.) clot:organ ratio, std. dev.
clot
0.38 0.08
0.23 0.04
0.09 0.07
0.07 0.01
0.01 0.00
0.09 0.01
0.11 0.04
0.38 0.11
0.64 0.32
0.50 0.07
2.53 0.09
2.75 0.05
1.00 0.00
1.67 0.21
6.63 1.59
5.39 0.65
42.48 4.90
4.31 0.65
4.24 0.92
1.14 0.26
0.83 0.19
0.76 0.04
0.15 0.02
0.14 0.02
contra
muscle
bone
brain
blood
heart
intestine
lungs
spleen
liver
kidney
As expected, we observed about half the activity in the blood
at 2 h for FBP12 vs FBP2 (0.09 0.01 vs 0.164 0.016,
respectively). However, we also saw less uptake in the
thrombus for FBP12, and this limited the overall efficacy. An
analysis of blood sampled at different times post injection
indicated that the probe FBP12 underwent rapid metabolism,
and this also limited overall efficacy. A functional assay was
used to determine the fraction of active fibrin binding probe
present (Figure S5). We also performed HPLC analysis of the
plasma (Figure S6). Both analyses showed that only 50% of the
plasma activity at 15 min post injection was intact probe.
We then looked at the stability of radiolabeled FBP12
incubated with plasma (Figure S3). This in vitro assay clearly
showed that the complex underwent significant degradation in
plasma after 30−60 min. Because the model compounds were
stable in plasma, the plasma instability of FBP12 can be traced
to degradation of the peptide and not release of 64Cu.
Comparison of the HPLC traces of FBP12 in plasma with
plasma samples spiked with 64CuCl2 confirmed that the
Although clear delineation the thrombus in the right carotid
artery (Figure 7) as well as the microthrombi induced through
injury at the surgical site was possible, the quantitative analysis
showed that pycup derivatized FBP12 showed similar efficacy
to the DOTA conjugate FBP2 reported previously. It was
anticipated that the greater kinetic inertness conferred by the
pycup chelator compared to DOTA-monoamide would result
in less transchelation of 64Cu to plasma proteins and reduced
background.
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Notes
decomposition products were not equivalent to plasma protein-
bound copper.
The authors declare the following competing financial
interest(s): P.C. has equity in Factor 1A, LLC, the company
which holds the patent rights to the peptides used in these
probes.
Future work will address the root of this peptide instability
and focus on medicinal chemistry approaches to prepare more
metabolically robust peptides.
The pycup ligand and its bifunctional derivatives represent a
new class of chelators for 64Cu. While we have prepared
derivatives with pendant carboxylates and successfully con-
jugated one of these to a peptide, it is apparent that other
conjugation strategies can be employed. The stability data with
[Cu(pycup2Bn)]2+ suggest that the pentadentate pycup ligand
itself is sufficient for robust labeling and formation of an inert
complex with copper. This allows one or both of the secondary
amines on pycup to be modified with a functional group for
bioconjugation and/or chemical modification to alter overall
charge or lipophilicity. The ease of synthesis of pycup and its
derivatives, including differentiation of one of the secondary
amines, results in tremendous flexibility in using pycup
derivatives to not only chelate 64Cu but also to modify the
pharmacokinetic properties of the conjugate. Because deriva-
tization with a mixture of donor arms is easily attainable, the
pycup ligand could be linked easily to multiple targeting
moieties simultaneously, using bioorthogonal functional group
chemistry. The possibility for asymmetric derivatization of the
two secondary nitrogens also opens up additional possibilities
for the development of trifunctional derivatives. Such an
approach could establish pycup as a convenient chelator and
conjugation tool for multimodal systems. These research
endeavors as well as conjugation of pycup to other peptides
are ongoing.
ACKNOWLEDGMENTS
■
Dr. Peter Mueller (X-ray diffraction facility, MIT chemistry
department) is warmly acknowledged for assistance with
elucidation of the solid state crystal structure of [Cu(py-
cup2Bn)]2+. P.C. acknowledges the National Heart, Lung, and
Blood Institute for funding (NHLBI, award HL109448) and an
instrumentation grant from the National Center for Research
Resources (NCRR, award RR029495) for the micro-PET/
SPECT/CT. E.B. and J.P.H. thank the Swiss National Science
Foundation and the Australian National Science and Technol-
ogy Organization for support, respectively. E.R.A. acknowledges
the US Department of Energy Office of Basic Energy Science.
The X-ray diffractometer at Tufts was funded by the NSF
(MRI: CHE-1229426).
REFERENCES
■
(1) Holland, J.; Williamson, M.; Lewis, J. Unconventional nuclides
for radiopharmaceuticals. Mol. Imaging 2010, 9, 1−20.
(2) Wadas, T. J.; Wong, E. H.; Weisman, G. R.; Anderson, C. J.
Copper Chelation Chemistry and its Role in Copper Radiopharma-
ceuticals. Curr. Pharm. Des. 2007, 13, 3−16.
(3) Wadas, T. J.; Wong, E. H.; Weisman, G. R.; Anderson, C. J.
Coordinating Radiometals of Copper, Gallium, Indium, Yttrium, and
Zirconium for PET and SPECT Imaging of Disease. Chem. Rev. 2010,
110, 2858−2902.
(4) Copper 64-ATSM and PET/CT Scan in Predicting Disease
Progression in Patients With Newly-Diagnosed Stage IB, Stage II,
Stage III, or Stage IVA Cervical Cancer Who Are Undergoing
Radiation Therapy and Cisplatin Treatment. http://clinicaltrials.gov/
show/NCT00794339 (accessed July 5, 2013).
(5) Boros, E.; Cawthray, J. F.; Ferreira, C. L.; Patrick, B. O.; Adam,
M. J.; Orvig, C. Evaluation of the H2dedpa Scaffold and its cRGDyK
Conjugates for Labeling with 64Cu. Inorg. Chem. 2012, 51, 6279−6284.
(6) Boswell, C. A.; Sun, X.; Niu, W.; Weisman, G. R.; Wong, E. H.;
Rheingold, A. L.; Anderson, C. J. Comparative in Vivo Stability of
Copper-64-Labeled Cross-Bridged and Conventional Tetraazamacro-
cyclic Complexes. J. Med. Chem. 2004, 47, 1465−1474.
(7) Bailey, G. A.; Price, E. W.; Zeglis, B. M.; Ferreira, C. L.; Boros, E.;
Lacasse, M. J.; Patrick, B. O.; Lewis, J. S.; Adam, M. J.; Orvig, C.
H2azapa: a Versatile Acyclic Multifunctional Chelator for 67Ga, 64Cu,
111In, and 177Lu. Inorg. Chem. 2012, 51, 12575−12589.
CONCLUSION
■
In conclusion, we successfully developed the cross-bridged
bicyclic ligand system pycup for rapid chelation of 64Cu and
subsequent formation of a kinetically inert complex. Pycup
provides an N5 ligand donor set, which can be easily modulated
by alkylation of the two secondary amines present. The
addition of donor groups such as acetates does not impact the
radiolabeling properties of the ligand or the thermodynamic
and kinetic inertness of the corresponding copper complexes
significantly. Conjugation to a target-specific peptide and
subsequent PET imaging in vivo is easily attainable, as
exemplified by the conjugation to a fibrin-binding peptide for
the imaging of thrombus in rats. The flexible and facile
functionalization of the ligand pycup provides future oppor-
tunities for conjugation to more than one targeting vector.
Pycup is well-suited for conjugation to other commonly used
targeting vectors, providing radiochemists with a new mean for
visualization of biomolecules using 64Cu.
(8) Sibbons, K. F.; Shastri, K.; Watkinson, M. The application of
manganese complexes of ligands derived from 1,4,7-triazacyclononane
in oxidative catalysis. Dalton Trans. 2006, 5, 645−661.
(9) Maheshwari, V.; Dearling, J. L. J.; Treves, S. T.; Packard, A. B.
Measurement of the rate of copper(II) exchange for 64Cu complexes of
bifunctional chelators. Inorg. Chim. Acta 2012, 393, 318−323.
(10) Ma, M. T.; Cooper, M. S.; Paul, R. L.; Shaw, K. P.; Karas, J. A.;
Scanlon, D.; White, J. M.; Blower, P. J.; Donnelly, P. S. Macrobicyclic
Cage Amine Ligands for Copper Radiopharmaceuticals: A Single
Bivalent Cage Amine Containing Two Lys3-bombesin Targeting
Peptides. Inorg. Chem. 2011, 50, 6701−6710.
(11) Cai, H.; Li, Z.; Huang, C.-W.; Shahinian, A. H.; Wang, H.; Park,
R.; Conti, P. S. Evaluation of Copper-64 Labeled AmBaSar Conjugated
Cyclic RGD Peptide for Improved MicroPET Imaging of Integrin αvβ3
Expression. Bioconjugate Chem. 2010, 21, 1417−1424.
ASSOCIATED CONTENT
■
S
* Supporting Information
Radio-HPLC traces of competition studies, UV−vis absorption
spectra, experimental details and results of the functional fibrin
binding assay, plasma and blood stability of FBP12, VOI
analysis, and ex vivo autoradiography. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
(12) Huang, S.; Joso, R.; Fuchs, A.; Barner, L.; Smith, S. V. Probing
Solid−Liquid Interfaces Using Radiotracer Technology: Character-
ization of Functionalized Microspheres. Chem. Mater. 2008, 20, 5375−
5380.
Corresponding Authors
*E-mail: eboros@nmr.mgh.harvard.edu.
*E-mail: caravan@nmr.mgh.harvard.edu.
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Article
(13) Wong, E. H.; Weisman, G. R.; Hill, D. C.; Reed, D. P.; Rogers,
M. E.; Condon, J. P.; Fagan, M. A.; Calabrese, J. C.; Lam, K.-C.; Guzei,
I. A.; Rheingold, A. L. Cross-bridged cyclams and pendant arm
derivatives. J. Am. Chem. Soc. 2000, 112, 10561.
(14) Wei, L.; Ye, Y.; Wadas, T. J.; Lewis, J. S.; Welch, M. J.; Achilefu,
S.; Anderson, C. J. 64Cu-Labeled CB-TE2A and diamsar-conjugated
RGD peptide analogs for targeting angiogenesis: comparison of their
biological activity. Nucl. Med. Biol. 2009, 36, 277−285.
Applications of Somatostatin Receptor (hsst2) Expressing Tumors.
Bioconjugate Chem. 2002, 13, 530−541.
(30) Krenning, E. P.; Kwekkeboom, D. J.; Bakker, W. H.; Breeman,
W. A. P.; Kooij, P. P. M.; Oei, H. Y.; Vanhagen, M.; Postema, P. T. E.;
Dejong, M.; Reubi, J. C.; Visser, T. J.; Reijs, A. E. M.; Hofland, L. J.;
Koper, J. W.; Lamberts, S. W. J. Somatostatin Receptor Scintigraphy
with [In-111-Dtpa-D-Phe(1)]- and [I-123-Tyr(3)]-Octreotide - the
Rotterdam Experience with More Than 1000 Patients. Eur. J. Nucl.
Med. 1993, 20, 716−731.
(15) Tircso,
Sherry, A. D.; Kovac
́
G.; Benyo,
́
E. T.; Suh, E. H.; Jurek, P.; Kiefer, G. E.;
(31) Guo, Y.; Ferdani, R.; Anderson, C. J. Preparation and Biological
Evaluation of 64Cu Labeled Tyr3-Octreotate Using a Phosphonic
Acid-Based Cross-Bridged Macrocyclic Chelator. Bioconjugate Chem.
2012, 23, 1470−1477.
(32) Carlucci, G.; Ananias, H. J. K.; Yu, Z.; Hoving, H. D.; Helfrich,
W.; Dierckx, R. A. J. O.; Liu, S.; de Jong, I. J.; Elsinga, P. H. Preclinical
Evaluation of a Novel 111In-Labeled Bombesin Homodimer for
Improved Imaging of GRPR-Positive Prostate Cancer. Mol.
Pharmaceutics 2013, 10, 1716−1724.
(33) Jeong, J. M.; Hong, M. K.; Chang, Y. S.; Lee, Y.-S.; Kim, Y. J.;
Cheon, G. J.; Lee, D. S.; Chung, J.-K.; Lee, M. C. Preparation of a
Promising Angiogenesis PET Imaging Agent: 68Ga-Labeled c-
(RGDyK) Isothiocyanatobenzyl-1,4,7-Triazacyclononane-1,4,7-Tria-
cetic Acid and Feasibility Studies in Mice. J. Nucl. Med. 2008, 49,
830−836.
(34) Ciesienski, K. L.; Yang, Y.; Ay, I.; Chonde, D. B.; Loving, G. S.;
Rietz, T. A.; Catana, C.; Caravan, P. Fibrin-Targeted PET Probes for
the Detection of Thrombi. Mol. Pharmaceutics 2013, 10, 1100−1110.
(35) Sheldrick, G. M. Phase annealing in SHELX-90: direct methods
for larger structures. Acta Crystallogr. 1990, A46, 467.
̀
s, Z. (S)-5-(p-Nitrobenzyl)-PCTA, a Promising
Bifunctional Ligand with Advantageous Metal Ion Complexation
Kinetics. Bioconjugate Chem. 2009, 20, 565−575.
(16) Ait-Mohand, S.; Fournier, P.; Dumulon-Perreault, V.; Kiefer, G.
́ ́
E.; Jurek, P.; Ferreira, C. L.; Benard, F.; Guer
in, B. Evaluation of 64Cu-
Labeled Bifunctional Chelate-Bombesin Conjugates. Bioconjugate
Chem. 2011, 22, 1729−1735.
(17) Dumont, R. A.; Deininger, F.; Haubner, R.; Maecke, H. R.;
Weber, W. A.; Fani, M. Novel 64Cu- and 68Ga-Labeled RGD
Conjugates Show Improved PET Imaging of ανβ3 Integrin Expression
and Facile Radiosynthesis. J. Nucl. Med. 2011, 52, 1276−1284.
(18) Altai, M.; Strand, J.; Rosik, D.; Selvaraju, R. K.; Eriksson
Karlstrom, A.; Orlova, A.; Tolmachev, V. Influence of Nuclides and
̈
Chelators on Imaging Using Affibody Molecules: Comparative
Evaluation of Recombinant Affibody Molecules Site-Specifically
Labeled with 68Ga and 111In via Maleimido Derivatives of DOTA
and NODAGA. Bioconjugate Chem. 2013, 24, 1102−1109.
(19) Cooper, M. S.; Ma, M. T.; Sunassee, K.; Shaw, K. P.; Williams, J.
D.; Paul, R. L.; Donnelly, P. S.; Blower, P. J. Comparison of 64Cu-
Complexing Bifunctional Chelators for Radioimmunoconjugation:
Labeling Efficiency, Specific Activity, and in Vitro/in Vivo Stability.
Bioconjugate Chem. 2012, 23, 1029−1039.
(36) Sheldrick, G. M. SHELXL 97, Program for Crystal Structure
Analysis (Release 97-2); University Gottingen: Gottingen, 1997.
̈
̈
(37) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.;
Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.;
Barone, V.; et al. Gaussian 09; Gaussian, Inc.: Wallingford, CT, 2009.
(38) Becke, A. Density-functional thermochemistry. III. The role of
exact exchange. J. Chem. Phys. 1993, 98, 5648−5652.
(20) Pandya, D. N.; Dale, A. V.; Kim, J. Y.; Lee, H.; Ha, Y. S.; An, G.
I.; Yoo, J. New Macrobicyclic Chelator for the Development of
Ultrastable 64Cu-Radiolabeled Bioconjugate. Bioconjugate Chem. 2012,
23, 330−335.
́ ̀
(21) Esteves, C. V.; Lamosa, P.; Delgado, R.; Costa, J.; Desogere, P.;
Rousselin, Y.; Goze, C.; Denat, F. Remarkable Inertness of Copper(II)
Chelates of Cyclen-Based Macrobicycles with Two trans-N-Acetate
Arms. Inorg. Chem. 2013, 52, 5138−5153.
(39) Lee, C.; Yang, W.; Parr, R. Development of the Colle-Salvetti
correlation-energy formula into a functional of the electron density.
Phys. Rev. B 1988, 37, 785−789.
(22) Brandes, S.; Denat, F.; Lacour, S.; Rabiet, F.; Barbette, F.;
Pullumbi, P.; Guilard, R. Synthesis of Macropolycyclic Ligands Based
on Tetraazacycloalkanes. Eur. J. Org. Chem. 1998, 11, 2349−2360.
(23) Roger, M.; Lima, L. M. P.; Frindel, M.; Platas-Iglesias, C.;
Gestin, J.-F.; Delgado, R.; Patinec, V.; Tripier, R. Monopicolinate-
dipicolyl Derivative of Triazacyclononane for Stable Complexation of
Cu2+ and 64Cu2+. Inorg. Chem. 2013, 52, 5246−5259.
(40) Loening, A. M.; Gambhir, S. S. AMIDE: A Free Software Tool
for Multimodality Medical Image Analysis. Mol. Imaging 2003, 2, 131.
(41) Heroux, K. J.; Woodin, K. S.; Tranchemontagne, D. J.; Widger,
P. C. B.; Southwick, E.; Wong, E. H.; Weisman, G. R.; Tomellini, S. A.;
Wadas, T. J.; Anderson, C. J.; Kassel, S.; Golen, J. A.; Rheingold, A. L.
The long and short of it: the influence of N-carboxyethyl versus N-
carboxymethyl pendant arms on in vitro and in vivo behavior of
copper complexes of cross-bridged tetraamine macrocycles. Dalton
Trans. 2007, 21, 2150−2162.
́
(24) Lima, L. M. P.; Esteban-Gomez, D.; Delgado, R.; Platas-Iglesias,
C.; Tripier, R. Monopicolinate Cyclen and Cyclam Derivatives for
Stable Copper(II) Complexation. Inorg. Chem. 2012, 51, 6916−6927.
(25) Gasser, G.; Tjioe, L.; Graham, B.; Belousoff, M. J.; Juran, S.;
(42) Addison, A. W.; Rao, T. N.; Reedijk, J.; van Rijn, J.; Verschoor,
G. C. Synthesis, structure, and spectroscopic properties of copper(II)
compounds containing nitrogen-sulphur donor ligands; the crystal and
molecular structure of aqua[1,7-bis(N-methylbenzimidazol-2[prime or
minute]-yl)-2,6-dithiaheptane]copper(II) perchlorate. J. Chem. Soc.,
Dalton Trans. 1984, 7, 1349−1356.
(43) Kolodziej, A. F.; Nair, S. A.; Graham, P.; McMurry, T. J.;
Ladner, R. C.; Wescott, C.; Sexton, D. J.; Caravan, P. Fibrin Specific
Peptides Derived by Phage Display: Characterization of Peptides and
Conjugates for Imaging. Bioconjugate Chem. 2012, 23, 548−556.
(44) Botnar, R. M.; Buecker, A.; Wiethoff, A. J.; Parsons, E. C.;
Katoh, M.; Katsimaglis, G.; Weisskoff, R. M.; Lauffer, R. B.; Graham,
P. B.; Gunther, R. W.; Manning, W. J.; Spuentrup, E. In vivo magnetic
resonance imaging of coronary thrombosis using a fibrin-binding
molecular magnetic resonance contrast agent. Circulation 2004, 110,
1463−1466.
Walther, M.; Kunstler, J.-U.; Bergmann, R.; Stephan, H.; Spiccia, L.
̈
Synthesis, Copper(II) Complexation, 64Cu-Labeling, and Bioconjuga-
tion of a New Bis(2-pyridylmethyl) Derivative of 1,4,7-Triazacyclo-
nonane. Bioconjugate Chem. 2008, 19, 719−730.
(26) Wynn, T.; Hegedus, L. S. Synthesis and Structural Studies of
1,8-Pyridine-Capped 5,12-Dioxocyclams. J. Am. Chem. Soc. 2000, 122,
5034−5042.
(27) Achmatowicz, M.; Hegedus, L. S.; David, S. Synthesis and
Structural Studies of 5,12-Dioxocyclams Capped by 4-Substituted
Pyridines Across the Amine Nitrogens. J. Org. Chem. 2003, 68, 7661−
7666.
(28) Gil, J. M.; David, S.; Reiff, A. L.; Hegedus, L. S. Synthesis and
Complexation of “Back-to-Back” Capped Bis-dioxocyclams. Inorg.
Chem. 2005, 44, 5858−5865.
(29) Eisenwiener, K.-P.; Prata, M. I.; Buschmann, I.; Zhang, H.-W.;
(45) Monroe, D. M.; Hoffman, M. What Does It Take to Make the
Perfect Clot? Arterioscler. Thromb. Vasc. Biol. 2006, 26, 41−48.
(46) Overoye-Chan, K.; Koerner, S.; Looby, R.; Kolodziej, A.; Zech,
S.; Deng, Q.; Chasse, J.; McMurry, T.; Caravan, P. EP-2104R: a fibrin-
Santos, A. C.; Wenger, S.; Reubi, J. C.; Macke, H. R. NODAGATOC,
̈
a New Chelator-Coupled Somatostatin Analogue Labeled with
[
67/68Ga] and [111In] for SPECT, PET and Targeted Therapeutic
628
dx.doi.org/10.1021/mp400686z | Mol. Pharmaceutics 2014, 11, 617−629

Molecular Pharmaceutics
Article
specific gadolinium-Based MRI contrast agent for detection of
thrombus. J. Am. Chem. Soc. 2008, 130, 6025−6039.
(47) Uppal, R.; Ciesienski, K. L.; Chonde, D. B.; Loving, G. S.;
Caravan, P. Discrete Bimodal Probes for Thrombus Imaging. J. Am.
Chem. Soc. 2012, 134, 10799−10802.
(48) Uppal, R.; Ay, I.; Dai, G.; Kim, Y.; Sorensen, A.; Caravan, P.
Molecular MRI of intracranial thrombus in a rat ischemic stroke
model. Stroke 2010, 41, 1271−1277.
629
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