Isomeric trimethylene and ethylene pendant-armed cross-bridged tetraazamacrocycles and in vitro/in vivo comparisions of their copper(II) complexes

Article abstract of DOI:10.1021/ic200014w

Ethylene cross-bridged tetraamine macrocycles are useful chelators in coordination, catalytic, medicinal, and radiopharmaceutical chemistry. Springborg and co-workers developed trimethylene cross-bridged analogues, although their pendant-armed derivatives

Full text of DOI:10.1021/ic200014w

ARTICLE
pubs.acs.org/IC
Isomeric Trimethylene and Ethylene Pendant-armed Cross-bridged
Tetraazamacrocycles and in Vitro/in Vivo Comparisions of their
Copper(II) Complexes
Antoinette Y. Odendaal,† Ashley L. Fiamengo,‡ Riccardo Ferdani,‡ Thaddeus J. Wadas,‡ Daniel C. Hill,†
Yijie Peng,† Katie J. Heroux,§ James A. Golen,|| Arnold L. Rheingold,§ Carolyn J. Anderson,*,‡ Gary R.
Weisman,*,† and Edward H. Wong*,†
†Department of Chemistry, University of New Hampshire, 23 Academic Way, Durham, New Hampshire 03824, United States
‡Mallinckrodt Institute of Radiology, Department of Chemistry, Department of Biochemistry and Molecular Biophysics, Washington
University School of Medicine, 510 Kingshighway Boulevard, Campus Box 8225,
St. Louis, Missouri 63110, United States
§Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, 92093, United States
Department of Chemistry and Biochemistry, University of Massachusetts, Dartmouth, North Dartmouth, Massachusetts, 02747,
United States
S Supporting Information
b
ABSTRACT: Ethylene cross-bridged tetraamine macrocycles are useful
chelators in coordination, catalytic, medicinal, and radiopharmaceutical
chemistry. Springborg and co-workers developed trimethylene cross-
bridged analogues, although their pendant-armed derivatives received little
attention. We report here the synthesis of a bis-carboxymethyl pendant-
armed cyclen with a trimethylene cross-bridge (C3B-DO2A) and its
isomeric ethylene-cross-bridged homocyclen ligand (CB-TR2A) as well
as their copper(II) complexes. The in vitro and in vivo properties of these
complexes are compared with respect to their potential application as 64Cu-radiopharmaceuticals in positron emission tomography
(PET imaging). The inertness of Cu-C3B-DO2A to decomplexation is remarkable, exceeding that of Cu-CB-TE2A. Electrochemical
reduction of Cu-CB-TR2A is quasi-reversible, whereas that of Cu-C3B-DO2A is irreversible. The reaction conditions for preparing
64Cu-C3B-DO2A (microwaving at high temperature) are relatively harsh compared to 64Cu-CB-TR2A (basic ethanol). The in vivo
behavior of the 64Cu complexes was evaluated in normal rats. Rapid and continual clearance of 64Cu-CB-TR2A through the blood, liver,
and kidneys suggests relatively good in vivo stability, albeit inferior to 64Cu-CB-TE2A. Although 64Cu-C3B-DO2A clears continually,
the initial uptake is high and only about half is excreted within 22 h, suggesting poor stability and transchelation of 64Cu to proteins in the
blood and/or liver. These data suggest that in vitro inertness of a chelator complex may not always be a good indicator of in vivo stability.
’ INTRODUCTION
A limited number of pendant-armed derivatives of these ligands
have been investigated, though none with ionizable functional
groups.6,7 We were intrigued whether such a trimethylene cross-
bridged tetraamine derivative would have distinct in vitro or in vivo
behavior compared to its most closely related ethylene cross-bridged
analogues. We report here the synthesis and characterization of a dica-
rboxymethyl pendant-armed cyclen with such a cross-bridge, C3B-
DO2A (1), as well as its isomeric ethylene cross-bridged homocyclen
ligand, CB-TR2A (2) (Figure 2). Their copper(II) complexes have
been prepared and fully characterized. Finally, 64Cu radiolabeling and
animal biodistribution studies have been carried out for comparison
with each other, and with the widely used chelator CB-TE2A, with
the overall goal of developing the optimal 64Cu-cross-bridged com-
plex for conjugation to biomolecules as potential PET imaging agents.
The family of ethylene cross-bridged tetraamine macrocyclic
ligands (Figure 1a) has become valuable as metal chelators in
coordination, catalytic, as well as medicinal chemistry.1,2 Their
pendant-armed derivatives have been especially useful in endowing
corresponding metal complexes with remarkable kinetic inertness.
One lead bifunctional chelator for copper radiometals has been the
bis-carboxymethyl pendant-armed cross-bridged cyclam, CB-TE2A
(Figure 1b). Its 64Cu-labeled bioconjugates have significantly
improved in vivo behavior and superior inertness toward radio-
metal loss and are of interest for positron emission tomography
(PET) imaging applications.2,3 We have been developing a
second generation of cross-bridged ligands aimed at improving
the kinetics of complexation and decreasing nontarget organ
accumulation.4 Springborg and co-workers previously reported
an interesting series of trimethylene cross-bridged tetraazama-
crocycles and their coordination chemistry (Figure 1c, 1d).1,5
Received: January 4, 2011
Published: March 07, 2011
r
2011 American Chemical Society
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The respective copper(II) complexes of 1 and 2 were prepared
by refluxing Cu(ClO4)2 overnight with the ligand in a 95%
ethanol solution that was adjusted to pH 8. Isolated crude
complexes were recrystallized from methanol/diethyl ether to
give crystalline products.
1
Ligand and Complex Spectroscopic Studies. The H and
13C{1H} NMR spectral data for C3B-DO2A (1) and CB-TR2A
(2) are detailed in the Experimental Section. The NMR spectra
of H2-1 exhibit the expected time-averaged 2-fold symmetry in
solution. The 13C{1H} NMR spectrum exhibits 6 resonances and
1
the H NMR spectrum shows the expected singlet for the
enantiotopic hydrogens of the acetate methylenes (N-CH2-
CO2H). On the other hand, H2-2 (TFA salt) is completely
asymmetric as shown by NMR spectroscopy. All of the carbon
resonances are unique and every methylene hydrogen pair is
diastereotopic. The two distinct N-CH2-CO2H resonances ap-
pear as AB and AX multiplets.
The solid-state IR spectra of Cu-C3B-DO2A (11) and Cu-CB-
TR2A (12) showed carboxylate stretching bands at 1616 and
1609 cm-1, respectively, both consistent with coordinated
pendant arms. Their d-d electronic spectral maxima in aqueous
solutions are at 614 nm (85 M-1 cm-1) and 608 nm (32 M-1
cm-1) respectively, also typical of six-coordinate Cu(II) with
N4O2 coordination spheres.
Figure 1. (a) Cross-bridged tetraazamacrocycles; (b) CB-TE2A; (c-
d) Trimethylene cross-bridged tetraazamacrocycles.
Electrochemical and Acid Inertness Studies. Cyclic voltam-
metry of Cu-C3B-DO2A (11) in 0.1 N sodium acetate revealed
an irreversible reduction wave with a peak potential at -1.05 V
(Ag/AgCl, scan rate 200 mV/s) with a large copper stripping peak
in the return scan. Under the same conditions, Cu-CB-TR2A
(12) exhibited a quasi-reversible reduction wave at -0.95 V with a
peak-to-peak separation of 138 mV. Our previous results indi-
cated that Cu(II)-complexes of ethylene cross-bridged cyclam
and its derivatives typically have quasi-reversible reductions while
cyclen analogues display irreversible reductions.11,12 Thus, elec-
trochemically Cu-C3B-DO2A (11) behaves as expected for a
cross-bridged cyclen complex while the isomeric homocyclen-
based complex Cu-CB-TR2A (12) is more similar to cross-
bridged cyclam complexes. The possible relevance of Cu(II)
reduction potentials to in vivo radio-copper loss from tetraaza-
macrocyclic chelators and their bioconjugates has been
postulated.12 It was hypothesized thatCu(II) reduction potentials
lower than about -0.6 V (Ag/AgCl) may be necessary to avoid
reduction by common bioreductants. If indeed so, neither of these
complexes should be susceptible to in vivo Cu(II) reduction.
Acid inertness data of Cu(II)-tetraamine complexes provide a
convenient measure of their resistance toward demetalation in
aqueous solution. We have found acid inertness half-lives ob-
tained under pseudo first-order conditions to be useful first
predictors for in vivo viability of 64Cu-labeled chelator
complexes.2 In particular, Cu-CB-TE2A was shown to have a
half-life of more than 6 d even in 5 M HCl at 90 °C.11 Studies in 5
M HCl solutions at 30 °C revealed that Cu-C3B-DO2A (11) is
indefinitely inert while Cu-CB-TR2A (12) dissociated with a
half-life of 10.8(4) h. Remarkably, the Cu-C3B-DO2A (11)
complex only demetalated in 12 M HCl at 90 °C with a half-life of
1.1(1) d. To our knowledge, this represents the most acid-inert
copper amine complex studied. Since Springborg reported the
half-life of the parent trimethylene cross-bridged copper complex
in 5 M HCl at 25 °C to be almost 6 days while we have found the
isomeric Cu-CB-homocyclen complex to be significantly less
inert (half-life only 4.8 h in 1 M HCl, 30 °C),5,13 the trimethylene
bridge does appear to impart greater acid inertness. Here,
Figure 2. Isomeric dicarboxymethyl pendant-armed cross-bridged li-
gands 1 and 2.
’ RESULTS AND DISCUSSION
Synthesis. The ligand C3B-DO2A (1) was synthesized by
N-alkylation of the parent trimethylene cross-bridged Spring-
borg ligand (3, C3B-cyclen)6 with t-butyl bromoacetate
(Scheme 1) to give diester 4 as a hydrobromide salt. Deprotec-
tion of this product with excess trifluoroacetic acid (TFA)
provided H2-C3B-DO2A, also as a hydrobromide (H2-1 HBr).
3
The synthetic route used for preparation of CB-TR2A (2) is
shown in Scheme 2. The approach from homocyclen to cross-
bridged homocyclen has been communicated previously,8 but we
include synthetic details and characterization data herein.
Condensation of homocyclen with aqueous glyoxal in MeCN
leads to a mixture of two constitutionally isomeric cis-fused
tetracyclic bisaminals, 5 (major) and 6 (minor), which were
separated chromatographically.9 The major isomer (5), the
desired compound, was shown to be cis-fused by virtue of
dynamic NMR spectra indicative of enantiomerization
(selected variable temperature 13C{1H} NMR spectral data are
included in the experimental details for compound 5).9,10 Highly
regio- and stereoselective dibenzylation of 5 gives rac salt 7
through benzylation on the most sterically available nitrogen
lone pairs. Double reductive ring expansion with NaBH4 gives
dibenzyl cross-bridged homocyclen 8, which is debenzylated to
cross-bridged homocyclen 9 by hydrogenolysis. In analogy to our
synthesis of CB-TE2A3 and C3B-DO2A (1) (vide supra), 9 was
di-N-alkylated with t-butyl bromoacetate to give bis-ester-armed
derivative 10, which was deprotected with TFA to give the
desired ligand as H2-CB-TR2A (H2-2) as a TFA salt.
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Scheme 1. Synthesis of H2-C3B-DO2A (H2-1)
Scheme 2. Synthesis of H2-CB-TR2A (H2-2)
attachment of the two enveloping carboxymethyl pendant arms
further enhanced the acid inertness of Cu-C3B-DO2A (11) even
beyond that of Cu-CB-TE2A.
Table 1. Crystal Data for Cu-CB-TR2A (12) and Cu-C3B-
DO2A (11)a
Cu-CB-TR2A
Cu-C3B-DO2A
X-ray Structural Data. A summary of crystal data for the two
X-ray diffraction studies can be found in Table 1. While the
quality of the Cu-CB-TR2A (12) crystal used was relatively poor,
nonetheless three distinct six-coordinated distorted octahedral
complexes with N4O2 coordination spheres were found. Each of
these provides a pendant arm oxygen to bridge two of three
sodium cations which together form a central core for this
assembly. Two of these, complexes 12A and 12B are well-defined
(Figure 3). Complex 12A has a [333]/[2233] ligand conforma-
tion while complex 12B adopts instead a [333]/[2323] con-
formation, the distinction mainly being the sign of the N-C-
C-N torsion angle of the short bridge. Complex 12C is
conformationally disordered but possesses the same basic Cu
coordination sphere as 12A and 12B.
Noteworthy are the distinct internal “axial” N-Cu-N angles
of 191.3(2)° and 183.2(2)° respectively, with each cation sunken
into the ligand cavity but with the [333]/[2233] ligand con-
formation engulfing the cation more. Corresponding “equatorial”
N-Cu-N angles are similar at 81.3(2)° and 80.2(2)° respec-
tively. Jahn-Teller elongations in both structures are along
select N-Cu-O axes; N(8)-Cu(2)-O(6) in 12A and
N(3)-Cu(1)-O(2) in 12B. Copper-nitrogen bond distances
in both structures range from 1.96 to 2.24 Å while copper-
oxygen distances are between 1.96 and 2.51 Å (Table 2).
chemical formula
molecular weight
space group
color
C
45H78Cl3Cu3N12Na3O27
C21H30CuF12N4O6
726.03
1585.13
P2(1)/c
blue
P2(1)/n
blue
a (Å)
20.2068(19)
24.613(2)
13.0914(12)
90.00
12.2973(7)
13.3899(8)
17.5444(10)
90.00
b (Å)
c (Å)
R (deg)
β (deg)
105.8090(10)
90.00
106.581(3)
90.00
γ (deg)
V (Å3)
6264.6(10)
4
2768.7(3)
4
Z
Dcacld (g/cm3)
1.681
1.742
T (K)
173(2)
100(2)
μ (mm-1
)
1.252 (Mo KR)
13599
2.293 (Cu KR)
5164
unique data, Rint
parameters/restraints
R1 (I > 2σ(I))
R1 (all data)
804/40
399/0
0.0784
0.0497
0.1396
0.0558
wR2 (all data)
a For full details, see the Supporting Information.
0.2026
0.1299
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The X-ray structure of Cu-C3B-DO2A (11) is shown in
Figure 4. Its good metal-ligand fit is indicated by observed
internal “axial” and “equatorial” N-Cu-N angles of 173.6(1)°
and 97.5(1)° respectively. Similar numbers were found in a
Cu(II) complex of the parent trimethylene cross-bridged cyclen
(170.5° and 99.0°).5 The first angle indicates that the cation is
protruding slightly from the ligand cavity while the latter value is
substantially larger than those found in reported ethylene cross-
bridged ligand Cu(II) complexes (typically around 80°) as a
result of its trimethylene cross-bridging.14,15 Its Jahn-Teller
elongation is along the N(1)-Cu(1)-O(3) axis which deviates
substantially from linearity at 159°. The copper-nitrogen bond
distances range from 2.0 to 2.2 Å, similar to those found in Cu-
CB-TR2A (12), while the Cu-O distances are 2.00 and 2.45 Å.
In light of these observations, no significant structural insight can
be gleaned to account for the very disparate acid inertness
between these two complexes.
Radiochemistry. Consistent with the extremely high acid
inertness of its Cu-complex, radiolabeling of C3B-DO2A was
found to be very challenging and ultimately required rather harsh
conditions for successful labeling. Radiolabeling was attempted
under a variety of aqueous conditions, varying concentration
(0.5-1 μg/μL), buffer (0.1-1.25 M NH4OAc, 0.1-0.5 M NH4-
citrate, and 0.1-0.5 Na2HPO4), pH (4.5-8), and temperature
(25-95 °C). Reactions were monitored over time to assess the
rate of complexation. All reactions had very disappointing yields
that rarely exceeded 50% within 2 h, even at high temperature. At
longer time points (3-18 h) the yields were generally better;
however, they rarely were >70%. Furthermore, instability of the
64Cu-C3B-DO2A complex was observed in the form of second-
ary peak formation. Although in our hands, radiolysis has not
been commonly observed for small molecules, reactions were
carried out in the presence of two different radiolytic scavengers
Figure 3. X-ray structure of 12: views of complexes 12A and 12B (50%
Figure 4. X-ray structure of complex 11 (50% thermal ellipsoids;
thermal ellipsoids; hydrogens omitted for clarity).
hydrogens omitted for clarity).
Table 2. Selected Bond Data for Cu-CB-TR2A (12) and Cu-C3B-DO2A (11)a
Cu-CB-TR2A
A
B
Cu-C3B-DO2A
Cu-N Bond Lengths (Å)
Cu(2)-N(5)
Cu(2)-N(6)
Cu(2)-N(7)
Cu(2)-N(8)
1.975(5)
1.988(5)
2.051(5)
2.228(6)
Cu(1)-N(1)
1.968(5)
1.956(6)
2.244(6)
2.041(5)
Cu(1)-N(1)
Cu(1)-N(2)
Cu(1)-N(3)
Cu(1)-N(4)
2.201(3)
2.003(3)
2.013(3)
2.002(3)
Cu(1)-N(2)
Cu(1)-N(3)
Cu(1)-N(4)
Cu-O Bond Lengths (Å)
Cu(2)-O(6)
Cu(2)-O(7)
2.513(6)
1.960(4)
Cu(1)-O(2)
Cu(1)-O(3)
2.501(5)
1.981(4)
Cu(1)-O(1)
Cu(1)-O(3)
2.003(2)
2.449(2)
N-Cu-N Angles Inside Ligand Cleft (deg)
N(5)-Cu(2)-N(6)
N(7)-Cu(2)-N(8)
191.3(2)
81.3(2)
N(1)-Cu(1)-N(2)
N(3)-Cu(1)-N(4)
183.2(2)
N(2)-Cu(1)-N(3)
N(1)-Cu(1)-N(4)
173.6(1)
97.5(1)
80.2(2)
a Estimated standard deviations in parentheses. For full details, see the Supporting Information.
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Lewis rats to examine their in vivo properties. The blood,
liver, and kidney clearance of these agents are plotted in
Figure 5 along with previously published data of 64Cu-CB-
DO2A and 64Cu-CB-TE2A for comparison.18 Although 64Cu-
CB-TR2A demonstrated rapid clearance from the blood suggest-
ing that it is stable for the limited time it remains in the
bloodstream, significantly more activity was observed in the
blood at 24 h for 64Cu-CB-TR2A compared to 64Cu-CB-TE2A
(64Cu-CB-TE2A vs 64Cu-CB-TR2A (%ID/g; p value): 0.003 (
0.0006 vs 0.01 ( 0.002; p = 0.003). Levels of 64Cu-CB-TR2A
remaining in the blood at 24 h were comparable to 64Cu-CB-
DO2A (0.01 ( 0.002 vs 0.011 ( 0.004).
Additionally, more tissue associated radioactivity was observed
in the kidneys of rats receiving 64Cu-CB-TR2A at 24 h p.i.
compared to the other two CB chelators (64Cu-CB-TR2A vs
64Cu-CB-TE2A vs 64Cu-CB-DO2A: 0.18 ( 0.05, 0.012 ( 0.003
and 0.064 ( 0.007%ID/g, respectively; p < 0.0001). Similarly,
there was significantly greater accumulation in the liver of mice
receiving 64Cu-CB-TR2A compared to 64Cu-CB-DO2A or 64Cu-
CB-TE2A (0.075 ( 0.014, 0.024 ( 0.002, and 0.056 ( 0.012%
ID/g, respectively; p = 0.0003). While the increased 64Cu in the
liver from 64Cu-CB-TR2A may be attributed to hepatobilary
clearance, transchelation of copper to liver proteins is also a likely
possibility.17 Boswell et al. reported the transchelation of radio-
copper to liver proteins such as superoxide dismutase and
metallotheinein after injection of 64Cu labeled CB-chelators.18
At this time it is unclear why CB-TR2A was less stable in vivo
than CB-DO2A despite its comparatively low reduction potential
and its relative kinetic acid inertness. 64Cu-C3B-DO2A exhibited
very slow clearance from the liver compared to CB-TR2A, CB-
DO2A, and CB-TE2A (percent reduction at 22 or 24 h PI: 22.2,
66.4, 69.5 and 90.2% clearance, respectively). Kidney clearance
showed a similar pattern out to 22 or 24 h PI (64Cu-C3B-DO2A,
50.4%; 64Cu-CB-TR2A, 89.2%; 64Cu-CB-DO2A, 95.6%; and
64Cu-CB-TE2A, 99.1%). The poor bioclearance of 64Cu-C3B-
DO2A compared to the other radiolabeled cross-bridged chela-
tors is surprising. Previously, in vitro assays of Cu(II)-complex
acid inertness and resistance to Cu(II)/Cu(I) reduction have
provided useful indicators of their in vivo stability and bioclear-
ance behavior.1,12 The very slow hepatic clearance of 64Cu-C3B-
DO2A observed here suggests significant radiometal loss in the
liver from what was predicted to be a very inert complex. Close
examination of the X-ray structure of Cu-C3B-DO2A revealed
that the trimethylene cross-bridge led to a larger equatorial N-
Cu-N angle of 98° instead of the 80-88° typical for its
ethylene-bridged analogues but no other noteworthy differences
in Cu-chelator bonding parameters. The possibility that an
incompletely formed, perhaps out-of-cavity, 64Cu complex being
the actual radiolabeled product can account for the observed
radiocopper loss in vivo. However, the very harsh radiolabeling
conditions (2 h, microwave 100 °C, pH 3) mitigate against such a
kinetic product. Other contributing factors affecting in vivo
clearance including lipophilicity, susceptibility to metabolic
degradation, or formation of easily reduced ternary complexes
as a consequence of this trimethylene cross-bridge may be
implicated but will require additional SAR studies for
validation.19
Figure 5. Biodistribution of 64Cu-11 and 64Cu-12 compared to pub-
lished data for 64Cu-CB-DO2A, 64Cu-CB-TE2A.
with no significant benefit. Carrier-added reactions did not
improve radiolabeling yields.
After many failed attempts at radiolabeling 1 with high yields
under aqueous conditions, the standard conditions for radiola-
beling CB-TE2A were attempted,16 by dissolving the compound
(0.5-3 mg) in ethanol (50-100 mL), preincubating it in the
presence of an excess of Cs2CO3 and then heating the reaction at
95 °C with 100-130 μCi of 64Cu. Unfortunately, the yields were
still relatively low (30-70% after 1 h) and did not exceed 85%
even after several hours. Replacing Cs2CO3 with Li2CO3 resulted
in extremely low yields (3-6%) at all time points (1-18 h).
Again, carrier-added reactions did not generate any improvement
in radiolabeling yield.
In an attempt to speed up the reaction kinetics, a microwave
reactor was employed. In all reactions 1.0 μg C3B-DO2A was
incubated with ∼100 μCi 64Cu in a total volume of 300 μL. Many
different reactions were performed where solvent, pH, tempera-
ture, and reaction time were varied, and it was found that the
shortest reaction that consistently provided a greater than 97%
radiochemical purity consisted of microwave heating a solution
of 1 in Milli-Q water at pH = 3 to 100 °C for 2 h.
In contrast, CB-TR2A was successfully labeled with 64Cu by the
method employed for CB-TE2A16 (radiochemical purity g98%)
at room temperature in basic ethanolic solution. A single peak
corresponding to 64Cu-CB-TR2A was confirmed by radio-TLC.
Biodistribution Studies. The biodistribution of 64Cu-C3B-
DO2A and 64Cu-CB-TR2A were determined in normal, juvenile
’ SUMMARY
The trimethylene cross-bridged chelator C3B-DO2A (1) and
its ethylene cross-bridged isomer CB-TR2A (2) have been
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synthesized and characterized as were their copper(II) com-
plexes. Both complexes have distorted octahedral N4O2 coordi-
nation spheres and should be resistant to biological reduction.
While Cu-CB-TR2A (12) is reasonably resistant to acid decom-
plexation, Cu-C3B-DO2A (11) is remarkably inert. At the same
time, successful radio-copper labeling of CB3B-DO2A (11)
required relatively harsh conditions. Contrary to the highly inert
in vitro behavior of Cu-C3B-DO2A (11), biodistribution studies
of 64Cu-C3B-DO2A revealed very poor in vivo clearance data,
significantly inferior to those of 64Cu-CB-TE2A and even 64Cu-
CB-DO2A.
55.8, 81.0, 171.4. MS (ESIþ): m/z 441.1 (MHþ), 385.1 (MHþ
1tBu), 329.1 (MHþ - 2tBu).
4,10-Bis-(carboxymethyl)-1,4,7,10-tetrazazabicyclo-
-
[5.5.3]pentadecane Hydrobromide (H2-1 HBr). The com-
3
pound 4 HBr (0.47 g, 0.90 mmol) was dissolved in a 1:1 mixture of
3
dichloromethane (DCM, 15 mL) and trifluoroacetic acid (TFA, 15 mL),
and the reaction mixture was stirred at room temperature under argon
for 3 days. DCM and TFA were then evaporated off, the remaining oil
was dissolved in ethanol, and the desired product slowly precipitated
with diethyl ether. After removal of trace solvents under high vacuum,
H2-1 HBr (0.34 g) was obtained as a slightly off-white solid in a 92%
3
yield. 1H NMR (D2O, 300 MHz): δ 1.82 (m, 2H, NCH2CH2CH2N),
2.95-3.25 (m, 20H, NCH2CH2N and NCH2CH2CH2N), 3.61 (s, 4H,
NCH2C(O)). 13C{1H} NMR (D2O, 75 MHz): δ 18.2, 51.7, 53.8, 53.9,
60.2, 173.1. MS (ESIþ): m/z 329.1 (MHþ). Elem. analysis: calcd for
’ EXPERIMENTAL SECTION
1
General Procedures. H and 13C{1H}-NMR spectra were ac-
C15H28N4O4 HBr: C 44.02, H 7.14, N 13.69; found: C 43.94, H 6.92,
quired on a Varian Mercury 400 MHz spectrometer operating at 399.75
and 100.51 MHz respectively, on a Varian INOVA 500 MHz spectro-
meter operating at 500 and 125.67 MHz respectively, or on a Bruker
spectrometer operating at 360 and 90.56 MHz respectively or on a
Varian Gemini operating at 300 and 75 MHz respectively. Variable
temperature dynamic 13C{1H} NMR spectra of 5 were acquired on a
JEOL FX-90Q spectrometer operating at 22.5 MHz. IR spectra were
recorded using KBr pellets on a Nicolet MX-1 FT-IR Spectrophot-
ometer. ESI-MS was performed on a Thermofinnigan LCQ Mass
Spectrometer coupled to a Picoview electrospray source or on a Waters
Micromass ZQ 4000. FAB-MS data were acquired on the JEOL JMS-
AX505HA Mass Spectrometer at the University of Notre Dame.
Electronic spectra were measured using a Cary 219 spectrophotometer.
Elemental analyses were performed at Atlantic Microlab Inc.
Norcross, GA.
All solvents were reagent grade and from commercial sources and
were used without further purification. All metal complexation reactions
were performed in standard Schlenk glassware under a nitrogen atmo-
sphere. Recrystallizations were conducted in closed containers without
precautions to exclude either air or moisture. Bulk solvent removal was
by rotary evaporation under reduced pressure, and trace solvent removal
from solids was by vacuum pump evacuation.
Caution! Perchlorate salts of metal complexes containing organic ligands
as solids or in organic solvents are potentially explosive. Although no
problems were encountered by us, cautious handling of only small amounts
of these compounds should be the rule.
Ligand Synthesis. C3B-cyclen (3) was synthesized according to a
procedure previously published by Springborg et al. in 1995.20 Briefly,
cyclen (1,4,7,10-tetraazacyclododecane) was treated with 2 equiv of p-
toluenesulfonyl chloride (TsCl) in pyridine: the only product obtained
was cyclen-1,7-bis-p-toluenesulfonamide. The 3-carbon cross bridge was
then introduced by alkylation of the 2 secondary nitrogens with 1 equiv
of 1,3-propanediol di-p-toluenesulfonate. The N-protecting tosyl groups
were removed from the crude product in refluxing HBr/CH3COOH (3
3
N 13.30.
cis-Decahydro-5H-2a,4a,7a,9a-tetraazacyclopenta-
[cd]phenalene (5). Aqueous glyoxal (3.7 mL 40% (w/w); 25.1
mmol) was added dropwise over 1 h under nitrogen to a stirred solution
of 1,4,7,10-tetraazacyclotridecane (homocyclen) (3.42 g, 18.36 mmol)
in MeCN (100 mL). The reaction mixture was then heated to 60 °C for
2.5 h. Solvent was removed under reduced pressure to give a yellow oil,
which was dissolved in CHCl3 and dried (Na2SO4). After concentration
of the solution, the crude product was purified by flash chromatography
(basic alumina, 10% EtOH/Et2O) to give the product as a clear oil (Rf
0.29) which crystallized upon storage at -10 °C. (1.91 g, 50%): mp 28-
30 °C; 1H NMR (360 MHz, CDCl3) δ 1.20-1.32 (dm, 1H, J = 8.1 Hz)
1.90-3.70 (br m, 19H) 2.88 (d, J = 2.8 Hz, CH) 3.37 (d, J = 2.8 Hz, CH)
(Note: the 1H NMR spectrum exhibited severe dynamic exchange
broadening.); 13C{1H} NMR (22.5 MHz, CDCl3, -39 °C) δ 19.79
(CH2CH2CH2), (Note: three pairs of peaks (labeled A, B, and C) each
broadened upon warming and coalesced to a singlet by 55 °C; see
below) 45.51 and 53.18 (A), 49.35 and 51.82 (B), 51.82 and 54.76 (C),
49.93, 75.39 (CH), 77.74 (CH); 13C{1H} NMR (22.5 MHz, CDCl3,
55 °C) δ 20.35, (Note: the following peaks (labeled A, B, and C)
correspond to three pairs of coalesced resonances.) 49.82 (br) (A),
50.97 (B), 53.83 (C), 50.41, 76.13 (CH), 78.17 (CH); MS, m/z 208
(Mþ); IR (CCl4) 2930, 2883, 2842, 2795, 2790, 1320, 1278, 1161, 1130,
1112, 1095 cm-1. Anal. Calcd for C11H20N4: C, 63.43; H, 9.68; N,
26.90. Found: C, 63.17; H, 9.98; N, 27.02. A minor component (0.20 g,
1
5%), was also isolated during the flash chromatography (Rf 0.07): H
NMR (360 MHz, CDCl3) δ 1.40 (dtt, 1H, J = 15.0, 5.6, 2.0 Hz) 2.02
(dtt, 1H, J = 15.0, 10.7, 2.9 Hz) 2.45-2.85 (m, 8H) 2.92-3.08 (m, 3H)
3.13 (ddd, 2H, J = 13.7, 5.7, 2.6 Hz) 3.27-3.40 (m, 2H) 3.59 (s, 2H,
CHCH); 13C NMR (90.56 MHz, CDCl3) δ 25.11, 49.33, 51.31, 51.63,
52.31, 80.94. These spectra are consistent with the constitutionally
isomeric cis-fused tetracyclic bisaminal 6.
(9br,9cr)-Decahydro-2a,7a-bis(phenylmethyl)-5H-
4a,9a-diaza-2a,7a-diazoniacyclopenta[cd]phenalene
Di-
days), and the crude product was recrystallized to give pure 3 3HBr.
4,10-Bis-(carbo-tert-butoxymethyl)-1,4,7,10-
3
bromide (7). To a stirred solution of purified homocyclen-glyoxal
adduct 5 (1.49 g, 7.17 mmol) in MeCN (35 mL) at room temperature
was added benzyl bromide (12.40 g, 72.50 mmol), all in one portion.
The reaction mixture was stirrred at room temperature for 3 weeks.
Precipitate was collected by suction filtration, washed with MeCN (2 ꢀ
30 mL), then CH2Cl2 (3 ꢀ 15 mL). Residual solvent was removed under
vacuum. This gave product as white solid (3.60 g, 90%): mp 222-
225 °C (dec); 1H NMR (360 MHz, D2O, MeCN secondary ref set at
2.06 ppm) δ 1.86-1.97 (dm, 1H, J = 15.0 Hz, H6-eq) 2.15-2.33 (m, 1H,
H6-ax) 2.78 (td, 1H, J = 12.2, 2.9 Hz) 3.09-3.92 (m, 13H) 4.21-4.39
(m, 2H), 4.78 and 5.09 (AB, 2H, J = 13.3 Hz, CH2Ph) 4.87 and 5.18 (AB,
2H, J = 13.0 Hz, CH2Ph), 4.95 and 5.12 (AB, 2H, J = 1.1 Hz, CHCH),
7.30-7.90 (m, 10H, Ph); 13C{1H} NMR (90.56 MHz, D2O, MeCN
secondary ref set at 1.70 ppm) δ 19.40 (CH2CH2CH2), 43.37, 46.34,
tetraazabicyclo[5.5.3]pentadecane Hydrobromide (4 HBr).
The compound C3B-cyclen 3HBr (3 3HBr, 0.45 g, 0.99 mmol) was
3
3
3
dissolved in MeCN (25 mL). Na2CO3 (0.45 g, 4.2 mmol) and tert-butyl
bromoacetate (0.34 mL, 2.3 mmol) were then added, and the reaction
was heated to 60 °C for 3 days. After cooling, solids were removed by
filtration, and the filtrate was evaporated under reduced pressure.
Residual solvent was removed under high vacuum, and the product
was triturated with diethyl ether to give 4 HBr (0.42 g) in 81% yield as
3
the residual solid. This material was taken on without further purifica-
tion. 1H NMR (DMSO-d6, 300 MHz): δ 1.32 (s, 18H, C(CH3)3), 1.68
(m, 2H, NCH2CH2CH2N), 2.65-3.05 (m, 20H, NCH2CH2N and
NCH2CH2CH2N), 3.38 (s, 4H, NCH2C(O)), 12.80 (br s, inside N-H).
13C{1H} NMR (DMSO-d6, 75 MHz): δ 21.4, 28.5, 50.2, 53.3, 54.1,
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47.68, 48.94, 51.25, 52.84, 61.05, 61.16, 61.37, 63.34, 76.81 (CH), 78.82
(CH), 125.56 (1C), 126.62 (1C), 130.28 (2C), 130.52 (2C), 132.18
(2C), 133.18 (2C), 134.10 (2C); IR (KBr) 3062, 3050, 3026, 3018,
2996, 2957, 2915, 2886, 2864, 2844, 2821, 2807, 1480, 1456, 1437, 1283,
1212, 1146, 1110, 1055, 769, 711, 706 cm-1. Anal. Calcd for
C6D6) δ 27.14 (CH2CH2CH2), 47.68, 48.56, 48.89, 49.02, 52.85, 53.27,
53.59, 54.20, 55.97, 59.41; MS, m/z 212 (Mþ); IR (KBr) 3474, 2948,
2929, 2907, 2894, 2845, 2699, 1577, 1464, 1399, 1356, 1342, 812, 612,
523 cm-1; HRMS exact mass calcd for C11H25N4 213.2079, found
213.2078.
C25H34N4Br2 0.5H2O: C, 53.68; H, 6.31; N, 10.02. Found: C, 53.94;
H, 6.10; N, 9.97.
4,11-Bis(phenylmethyl)-1,4,8,11-tetraazabicyclo-
4,11-Bis-(carbo-tert-butoxymethyl)-1,4,8,11-
3
tetraazabicyclo[6.5.2]pentadecane (10). To a stirred solution of
9 (35.0 mg, 0.165 mmol) in MeCN (8 mL), sodium carbonate (50.0 mg,
0.472 mmol) was added in one portion, followed by tert-butyl bromoa-
cetate (0.050 mL, 0.34 mmol) via syringe. The mixture was stirred for 3
days at room temperature. Solvent was removed under reduced pressure
to yield crude product, which was subjected to flash chromatography
(SiO2, CH2Cl2:MeOH = 10:1) to yield product as light yellow oil (40.0
mg, 0.0909 mmol, 55%): 1H NMR (500 MHz, C6D6) δ 1.30-1.46 (m,
2H), 1.40 (s, 18H), 2.25 (dt, 1H, J = 13.9, 2.9 Hz), 2.50-2.62 (m, 5H),
2.72-2.88 (m, 10H), 2.88-2.95 (m, 1H), 2.96-3.03 (m, 1H), 3.09 and
3.17 (AB, 2H, J = 16.4 Hz), 3.20 (br s, 2H), 3.24-3.31 (m, 1H), 3.59
(ddd, 1H, J = 12.5, 9.8, 4.9 Hz); 13C{1H} NMR (125.7 MHz, C6D6) δ
28.36, 28.60, 28.61, 51.24, 53.34, 55.08, 55.82, 56.86, 57.39, 57.52 (2C),
57.66, 57.74, 59.84, 60.22, 80.17, 80.22, 171.52, 172.08; IR(neat) 2976,
2924, 2852, 2800, 1734, 1719, 1457, 1367, 1152, 1124; HRFABMS, m/z
(M þ H)þ exact mass for C23H45N4O4: 441.3441; Found: 441.3456
(error þ1.5 mmu/þ0.3 ppm)
[6.5.2]pentadecane (8). NaBH4 (13.10 g, 0.340 mol) was added in
small portions over 20 min to a stirred solution of dibenzyl tetracyclic
bisaminal dibromide salt 7 (3.05 g, 5.55 mmol) in 95% EtOH (75 mL).
The reaction mixture was stirred at room temperature for 10 days. Excess
NaBH4 was then decomposed by slow addition of 150 mL 3 M HCl.
Evaporation of solvent under reduced pressure gave a white solid which
was dissolved in H2O (100 mL), adjusted to pH 14 with solid KOH
(with cooling), and extracted with benzene (6 ꢀ 50 mL). The combined
extracts were dried (Na2SO4), and solvent was removed under reduced
1
pressure to yield 1.44 g (quant) of product as a viscous oil; H NMR
(360 MHz, C6D6) δ 1.37-1.60 (m, 2H, CH2CH2CH2), 2.26-2.49 (m,
5H), 2.53-2.73 (m, 7H), 2.78 (ddd, 1H, J = 13.7, 4.3, 2.6 Hz), 2.82-
3.04 (m, 5H), 3.09-3.21 (m, 1H), 3.26 and 3.64 (AB, 2H, J = 13.4 Hz,
CH2Ph), 3.60 and 3.62 (AB, 2H, J = 14.4 Hz, CH2Ph), 3.88 (ddd, 1H, J =
12.7, 8.2, 4.9 Hz), 7.07-7.41 (m, 10H, CH2Ph); 13C{1H} NMR (90.56
MHz, C6D6) δ 28.59 (CH2CH2CH2), 52.39, 53.78, 55.43, 55.84, 56.36,
56.43, 57.04, 57.93, 59.04, 59.48, 60.82, 60.99, 126.93, 126.98, 128.41,
128.42, 129.08, 129.39, 140.92, 141.03; MS, m/z 392 (Mþ); IR (neat)
3077, 3053, 3020, 2919, 2863, 2800, 2784, 2745, 2721, 2702, 1448, 1364,
1110, 1046, 1025, 729, 696 cm-1; HRMS exact mass calcd for C25H37N4
393.3018, found 393.3012. The 13C and 1H NMR spectra showed the
material to be >98% purity.
4,11-Bis-(carboxymethyl)-1,4,8,11-tetraazabicyclo-
[6.5.2]pentadecane (H2-2 3.7TFA). A solution of 10 (96.0 mg,
3
0.218 mmol) in trifluoroacetic acid (3.4 mL, 0.046 mol) and CH2Cl2
(3.4 mL) was stirred under N2 for 2 days. The reaction mixture was then
concentrated under aspirator pressure and residual solvent was removed
under vacuum to give 0.1635 g (0.2179 mmol, quant) of H2-CB-TR2A
(H2-2) as a brown oil with 3.7 equiv of TFA by mass. 1H NMR (MeCN-d3,
500 MHz) δ 1.69-1.76 (dm, 1H, J = 16.8 Hz), 2.14-2.28 (m, 1H),
2.82-3.52 (m, 22-23H), 3.53 and 3.89 (AX, 2H, J = 17.6 Hz,
NCH2CO2), 3.86 and 4.05 (AB, 2H, J = 17.4 Hz, NCH2CO2), 11.64
(br s, 6H, includes all COOH peaks); 13C{1H} NMR (MeCN-d4,
125.68 MHz) δ 20.94, 48.58, 50.04, 50.40, 52.25, 53.36 (br), 53.85,
55.16 (br), 55.69, 56.37, 57.59, 58.21, 58.40, 117.08 (q, 1JCF = 283.2 Hz,
F3CCOOH), 160.67 (q, 2JCF = 37.0 Hz, F3CCOOH), 170.88, 172.99.
(1H NMR integration from 2.82 to 3.52 ppm was slightly high, likely
1,4,8,11-Tetraazabicyclo[6.5.2]pentadecane (9). Hydroge-
nolysis of 8 was carried out in a glass apparatus designed for the
exclusion of O2 and for measurement of H2 uptake with maintenance
of constant pressure. 10% Pd/C (0.20 g) and glacial HOAc (60 mL)
were added to a 125 mL hydrogenation flask which was connected to the
apparatus. The system was evacuated (water aspirator) and flushed with
nitrogen four times. The system was then evacuated, filled with hydro-
gen, and catalyst was equilibrated under H2 (767 mmHg) for 1.5 h. To
this was added a solution of 8 (1.11 g, 2.83 mmol) in glacial HOAc
(5 mL). The mixture was stirred for 19 h under H2 (767 mmHg). (The
theoretical H2 uptake for this reaction was 138 mL. The observed uptake
was 144 mL.) The apparatus was then evacuated and flushed with
nitrogen four times, the reaction flask was removed from the apparatus,
the contents were filtered through Celite, and the catalyst and Celite
were washed with glacial HOAc (2 ꢀ 5 mL). The combined filtrate and
washings were concentrated under reduced pressure to give a light
yellow oil which was dissolved in H2O (50 mL), adjusted to pH 14 with
KOH, and extracted with benzene (5 ꢀ 50 mL). The combined extracts
were dried (Na2SO4), and solvent was removed under reduced pressure
to give an oil. After kugelrohr distillation (90-120 °C air bath
temperature, 0.03 mmHg), the product oil solidified upon standing to
give 0.494 g (82% crude yield) of solid product. This material contained
minor impurities and was therefore further purified. The solid was
dissolved in absolute EtOH (5 mL) containing conc HCl (1 mL). A
hydrochloride salt crystallized, was filtered, washed with cold (0-5 °C)
absolute EtOH (2 ꢀ 1 mL), and air-dried. This solid was dissolved in
H2O (10 mL), adjusted to pH 14 with solid KOH (with cooling), and
extracted with benzene (6 ꢀ 15 mL). The combined extracts were dried
(Na2SO4), and solvent was removed under reduced pressure to give an
oil. This oil was kugelrohr distilled (100 °C air bath temperature, 0.03
mmHg) and subsequently solidified upon standing to give 0.283 g
(47%) of pure product as a white solid: mp 28-30 °C; 1H NMR (360
MHz, C6D6) δ 1.26-1.51 (m, 2H CH2CH2CH2), 2.10-2.90 (m, 21H),
3.30 (ddd, 1H, J = 12.1, 7.5, 2.4 Hz); 13C{1H} NMR (90.56 MHz,
because of H2O). This material was complexed with Cu(ClO4)2 6H2O
without further purification (vide infra).
Complex Synthesis. Cu-C3B-DO2A (11). An amount of 100 mg
(0.203 mmol) of the C3B-DO2A (1) as a trifluoroacetate salt was
3
combined with 75 mg (0.203 mmol) of Cu(ClO4)2 6H2O in 8 mL of
3
95% ethanol. The pH was adjusted to 8.5 with 0.1 N NaOH. This
suspension was refluxed under nitrogen for 24 h to give a dark blue
solution. After thorough removal of volatiles under reduced pressure, a
dark-blue sticky solid was obtained. This was extracted with 4 mL of
methanol and centrifuged. The supernatant was then placed in vials
inside a glass container for diethylether diffusion. A total crop of 75 mg of
dendritic dark-blue crystals formed (64% yield). Elem. analysis: calcd for
C15CuH26N4O4 1.33NaClO4 1.33H2O: C 31.24, H 5.01, N 9.71, Cl
3
3
8.18%. Found: C 31.05, H 5.21, N 9.45, Cl 8.06%. Electronic spectrum
(aq.) λmax 614 nm (ε = 84.6 M-1 cm-1). FT-IR (KBr) νCOO 1687,
1616 cm-1. X-ray crystals were grown from a hexafluoro-i-propanol
solution upon diethylether diffusion.
Cu-CB-TR2A (12). To a solution of 2 3.7 TFA (0.1635 g, 0.2179
3
mmol) in 95% EtOH (15 mL) was added Cu(ClO4)2 6H2O (136 mg,
3
0.366 mmol), and the pH of the reaction mixture was adjusted to 8 with
1 M NaOH. The reaction was then refluxed under N2 for approximately
23 h. The reaction mixture was centrifuged, and the supernatant
collected and concentrated under aspirator pressure. Residual solvent
was removed under vacuum to give 264 mg of a blue solid. This solid was
dissolved in 95% EtOH (15 mL), and the blue solution was placed in a
diethyl ether diffusion chamber. A powdery blue solid was collected to
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Inorganic Chemistry
ARTICLE
give 55.3 mg (44% yield) of purified product. Elem. analysis: calcd for
Cu(C15H26N4O4) 2H2O 1.2NaClO4: C, 31.45; H, 5.28; N, 9.78; Cl,
7.43. Found C, 31.49; H, 5.14, N, 9.68, Cl, 7.10; IR (KBr) 3536, 3490,
3430, 3394, 3364, 1610, 1485, 1437, 1397, 1320, 1098, 631 cm-1. UV-
vis (H2O) λmax 608 (ε = 32.1 M-1 cm-1); Cyclic voltammetry
conducted in 0.1 sodium acetate: indicated a quasi-reversible reduction
counter. The percent injected dose per gram (%ID/g) and percent
injected dose per organ (%ID/organ) were calculated by comparison to
a weighed and counted standard. Rats were allowed food and water ad
libitum. Animals in the 24 h group for 64Cu-C3B-DO2A and 64Cu-
NOTA were maintained in metabolism cages; urine and feces were
collected.
3
3
with EF = -0.950 V (Ag/AgCl). A 0.13 M solution of Cu-CB-TR2A 2
All biodistribution data are presented as the mean ( standard
deviation. Group comparisons were made using standard ANOVA
methods. Post hoc testing of individual group differences was accom-
plished with the Bonferroni test. Groups with p < 0.05 were considered
significantly different. GraphPad Prism software (version 5.02; San
Diego, CA) was used for all statistical analyses.
3
(H2O) 1.2(NaClO4) in 95% EtOH was placed in a diethylether
3
diffusion chamber. Plate-like blue crystals were collected for X-ray
structural analysis, which yielded an empirical formula of Cu-CB-
TR2A H2O NaClO4.
3
3
X-ray Crystallography. Cu-CB-TR2A (11). A blue plate crystal
was mounted in a Nylon loop, and X-ray diffraction data collected on a
Bruker D8 diffractometer equipped with an APEX CCD detector at
173 K. The monoclinic space group P21/c was uniquely assigned from
systematic absences. The structure was solved by direct methods. Except
as noted, non-hydrogen atoms were refined with anisotropic thermal
parameters, and hydrogen atoms were included as idealized contribu-
tions. The structure was found to be extensively disordered with regard
to both the macrocycle and the counterions. Satisfactory disorder
models could be created for both, but extensive restraint was necessary
because of limited resolution in the diffraction data. The perchlorate ions
were refined as rigid tetrahedra, and several carbon atoms in the
heterocycle were refined with isotropic thermal parameters. Addition-
ally, the thermal parameters for C37 and its disorder partner, C37a were
refined with the EADP command.
’ ASSOCIATED CONTENT
1
Supporting Information. Listings of H and 13C{1H}
S
b
NMR spectra of synthetic intermediates and ligands. This
material is available free of charge via the Internet at http://
pubs.acs.org. X-ray crystallographic files in CIF format. These
X-ray files have also been deposited with the Cambridge Crystal-
lographic Data Centre (CCDC reference numbers 806127 and
806128) and are available from the Director, Cambridge Crystal-
lographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
U.K.
’ AUTHOR INFORMATION
Cu-C3B-DO2A (12). A blue rod 0.15 ꢀ 0.10 ꢀ 0.10 mm in size was
mounted on a Cryoloop with Paratone oil. Data were collected in a
nitrogen gas stream at 100(2) K using j and ω scans. Crystal-to-
detector distance was 60 mm and exposure time was 10 s per frame using
a scan width of 0.5°. Data collection was 99.9% complete to 67.00° in q.
A total of 26635 reflections were collected covering the indices, -14 e
h e 14, -16 e k e 16, -21 e l e 16. A total of 5166 reflections were
found to be symmetry independent, with an Rint of 0.0299. Indexing and
unit cell refinement indicated a primitive, monoclinic lattice. The space
group was found to be P2(1)/n (No. 14). The data were integrated using
the Bruker SAINT software program and scaled using the SADABS
software program. Solution by direct methods (SIR-97) produced a
complete heavy-atom phasing model consistent with the proposed
structure. All non-hydrogen atoms were refined anisotropically by full-
matrix least-squares (SHELXL-97). All hydrogen atoms were placed
using a riding model. Their positions were constrained relative to their
parent atom using the appropriate HFIX command in SHELXL-97.
Radiochemistry. The highest radiochemical purity with greatest
consistency of yield was obtained using a microwave assisted reaction. A
solution of 64CuCl2 (750 μCi in 50 μL Milli-Q water, pH 3) was added
to 4.61 μg (14.0 nmol) C3B-DO2A dissolved in 250 μL of Milli-Q water
(pH 3). The sealed vial containing the reaction mixture was placed in the
microwave reactor, stirred for 2 min, and then heated at 100 °C for 2 h.
Complex formation was confirmed by radio-TLC (Rf ∼ 0.8, C18 silica
plates, eluent: methanol/10% ammonium acetate 7:3, Rf (64Cu-acetate = 0).
Radiochemical purity was determined by HPLC and found to be g98%
before use in animal studies (Complex retention time 10.55 min; Agilent
C8 column; isocratic, 0.1% TFA in water; 0.5 mL/min).
Corresponding Author
*E-mail: andersoncj@wustl.edu (C.J.A.), gary.weisman@unh.
edu (G.R.W.), ehw@cisunix.unh.edu (E.H.W.).
’ ACKNOWLEDGMENT
We acknowledge financial support from NIH NCI-
CA093395.
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