Synthesis, characterization and in vivo studies of Cu(II)-64-labeled cross-bridged tetraazamacrocycle-amide complexes as models of peptide conjugate imaging agents

Article abstract of DOI:10.1021/jm070204r

Copper-64, a positron emitter suitable for positron emission tomography (PET), demonstrates improved in vivo clearance when chelated by the cross-bridged tetraazamacrocycle CB-TE2A compared to TETA. Good in vivo clearance was also observed for ⁶⁴

Full text of DOI:10.1021/jm070204r

J. Med. Chem. 2007, 50, 2527-2535
2527
Synthesis, Characterization and In Vivo Studies of Cu(II)-64-Labeled Cross-Bridged
Tetraazamacrocycle-amide Complexes as Models of Peptide Conjugate Imaging Agents
Jennifer E. Sprague,† Yijie Peng,‡ Ashley L. Fiamengo,† Katrina S. Woodin,‡ Evan A. Southwick,‡ Gary R. Weisman,‡
Edward H. Wong,‡ James A. Golen,§ Arnold L. Rheingold,| and Carolyn J. Anderson*,†
Mallinckrodt Institute of Radiology, Washington UniVersity School of Medicine, St Louis, Missouri, Department of Chemistry, UniVersity of
New Hampshire, Durham, New Hampshire, Department of Chemistry, UniVersity of Massachusetts, Dartmouth, Massachusetts, Department of
Chemistry and Biochemistry, UniVersity of California, La Jolla, San Diego, California
ReceiVed February 21, 2007
Copper-64, a positron emitter suitable for positron emission tomography (PET), demonstrates improved in
vivo clearance when chelated by the cross-bridged tetraazamacrocycle CB-TE2A compared to TETA. Good
in vivo clearance was also observed for 64Cu-CB-TE2A conjugated to a peptide, which converts one
coordinating carboxylate pendant arm to an amide. To better understand the in vivo stability of peptide-
conjugated CB-TE2A, cross-bridged monoamides were synthesized. Crystal structures of natCu(II)-CB-
TEAMA and natCu(II)-CB-PhTEAMA revealed hexadentate, distorted octahedral coordination geometry. In
vivo biodistribution showed clearance of all 64Cu-radiolabeled cross-bridged monoamides from liver and
bone marrow such that uptake at 24 h was <10% of uptake at 30 min. In contrast, >60% of 30 min uptake
from 64Cu-TETA was retained in these tissues at 24 h. Clearance of 64Cu-cross-bridged monoamides from
nontarget organs suggests good in vivo stability, thus supporting the use of CB-TE2A as a bifunctional
chelator without modifications to the macrocycle backbone.
Introduction
been reported by Weisman and Wong.16-18 A series of cross-
bridged Cu(II) complexes have been prepared and characterized.
Cross-bridged chelates were shown to have all four nitrogen
lone pairs convergent on a cleft to coordinate Cu(II). Octahedral
coordination geometry with the Cu(II) fully enveloped by the
chelator was achieved by the addition of two carboxylate
pendant arms to give Cu(II)-CB-TE2A (4,11-bis(carboxym-
ethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane; Figure 1).18
Recently, we have established the exceptional kinetic inertness
of this complex to aqueous acid decomplexation.19 Busch and
colleagues have reported additional cross-bridged Cu(II) com-
plexes that also demonstrate high kinetic stability.20-22 Studies
in our laboratory have confirmed improved in vivo stability of
64Cu-CB-TE2A compared to that of 64Cu-TETA.14,18,23
Targeting radioactive metals to tumors or other tissues of
interest can be accomplished by conjugation of a bifunctional
chelator to a peptide or monoclonal antibody.24-26 Conjugation
of a macrocyclic chelator such as TETA converts one carboxy-
late arm of the chelator to an amide by reaction with either the
N-terminus of a peptide or a lysine side chain. However,
conjugation of CB-TE2A to a peptide results in a single free
carboxylate to coordinate Cu(II); it was initially hypothesized
that this would decrease its in vivo stability. To overcome this
potential problem, Lewis et al. synthesized a CB-TE2A deriva-
tive in which a biotin molecule is covalently attached to the
macrocycle backbone, leaving both carboxylate arms intact.27
However, no kinetic or in vivo stability studies of Cu(II)-CB-
TE2A-Bz-biotin were reported. We reported the in vivo bio-
distribution of 64Cu-CB-TE2A conjugated to the cyclic soma-
tostatin analogue Y3-TATE (Tyr3-octreotate, fCYwKTCT-
OH).28 Favorable clearance properties of 64Cu-CB-TE2A-Y3-
TATE in liver, blood, and bone marrow were all suggestive of
improved in vivo stability of the 64Cu-CB-TE2A-Y3-TATE
relative to 64Cu-TETA-Y3-TATE.28
Copper-64 is a promising radionuclide for use in positron
emission tomography (PETa) imaging as well as radiotherapy
due to its half-life (t1/2 ) 12.7 h) and decay characteristics (â+
17.4%, Eâ+max ) 656 keV; â- 39%, Eâ-max ) 573 keV); it can
be produced in high yield and high specific activity on a
biomedical cyclotron.1-9 Increasing use of 64Cu and other copper
radioisotopes in nuclear medicine has generated a need for
strongly complexing chelators. Kinetic stability of Cu(II)
complexes has been shown to be more predictive of in vivo
stability than thermodynamic stability.10,11 Macrocyclic chelators
of Cu(II) such as TETA (1,4,8,11-tetraazacyclotetradecane-
1,4,8,11-tetraacetic acid; Figure 1) demonstrate higher kinetic
and thus in vivo stability relative to acyclic chelators such as
EDTA (ethylene diamine tetraacetic acid).10-13 However, bio-
distribution and metabolism studies in rats using TETA have
demonstrated significant transchelation of 64Cu to superoxide
dismutase in liver and albumin in blood resulting in high
background radioactivity.14,15
Several “cross-bridged” tetraamine ligands, where an ethylene
(-CH2CH2-) bridge connects two nonadjacent nitrogens, have
* To whom correspondence should be addressed. Mallinckrodt Institute
of Radiology, Washington University School of Medicine, 510 S. King-
shighway Boulevard, Campus Box 8225, St. Louis, MO 63110. Phone: 314-
362-8427. Fax: 314-362-9440. E-mail: andersoncj@wustl.edu.
† Washington University School of Medicine.
‡ University of New Hampshire.
§ University of Massachusetts.
| University of California.
a Abbreviations: CB-TE2A, 4,11-bis(carboxymethyl)-1,4,8,11-tetraaza-
bicyclo[6.6.2]hexadecane; CB-TEAMA, 4-acetamido-11-carboxymethyl-
1,4,8,11-tetraazabicyclo[6.6.2]hexadecane; CB-MeTEAMA, 11-methylcar-
bamoylmethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadec-4-yl)-acetic acid;
CB-PhTEAMA, 11-phenylcarbamoylmethyl-1,4,8,11-tetraazabicyclo[6.6.2]-
hexadec-4-yl)-acetic acid; CH3CN, acetonitrile; D-Phe1-octreotide, fCFwK-
TCT-OH; EDTA, ethylene diamine tetraacetic acid; ID, injected dose; logP,
partition coefficients; PBS, phosphate buffered saline; PET, positron
emission tomography; PI, post-injection; RT, retention time; TFA, trifluo-
roacetic acid; TETA, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic
acid; Y3-TATE, Tyr3-octreotate, fCYwKTCT-OH.
Small molecule metabolites of 64Cu- and 67Cu-radiolabeled
peptides or antibodies have not been well characterized due to
the poor in vivo stability of Cu(II) chelates. As stated above,
10.1021/jm070204r CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/26/2007

2528 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Sprague et al.
Figure 1. Structures of TETA, CB-TE2A, CB-TEAMA, CB-MeTEAMA, and CB-PhTEAMA.
Scheme 1. Synthesis of 2
the primary radiometabolites of 64Cu-TETA and 67Cu-TETA-
conjugated peptides and antibodies in liver and blood result from
the dissociation of 64/67Cu from a peptide/antibody conjugate
and subsequent binding to proteins.15,29 This results in retention
of the transchelated 64Cu in nontarget tissues such as liver.15
However, the final metabolite of 111In-DTPA-D-Phe1-octreotide
(fCFwKTCT-OH), where 111In-DTPA forms a relatively stable
chelate, has been identified as 111In-DTPA-D-Phe.30 Similarly,
the kidney metabolite of 64Cu-chelate antibody conjugates was
reported to be 64Cu-chelate-lysine.31 It is likely that the peptide
portion of 64Cu-CB-TE2A-Y3-TATE will be degraded to yield
64Cu-CB-TE2A-D-Phe. To better understand the high in vivo
stability of 64Cu-CB-TE2A-Y3-TATE, three simplified monoa-
mide, monocarboxylate analogues of the CB-TE2A chelator
were synthesized (Figure 1). In this study, the in vivo stabilities
of 64Cu-CB-TEAMA, 64Cu-CB-MeTEAMA, and 64Cu-CB-
PhTEAMA were compared to those of 64Cu-TETA and 64Cu-
CB-TE2A.
Scheme 2. Synthesis of H[CB-TEAMA]‚2TFA
Scheme 3. Synthesis of H[CB-MeTEAMA]‚2TFA and
H[CB-PhTEAMA]‚4TFA
Results
Synthesis of Ligands CB-TEAMA, CB-MeTEAMA, and
CB-PhTEAMA. The title ligands were synthesized via synthetic
intermediate 2, which was prepared by statistical alkylation of
cross-bridged cyclam 1, as shown in Scheme 1. Thus, 1 was
alkylated with 1 equiv of t-butyl bromoacetate in CH3CN at
room temperature to give a mixture consisting of 2 (45%-50%
by NMR), dialkylated byproduct 3 (the precursor to CB-TE2A),
and unreacted 1. The desired mono-t-butyl ester 2 was isolated
by flash chromatography in 39% yield. Treatment of 2 with an
excess of 2-bromoacetamide in CH3CN at room temperature
gave cross-bridged cyclam 4 in 43% yield after workup (Scheme
2). Compound 4 was subsequently quantitatively deprotected
in 1:1 (v/v) CF3CO2H(TFA)/CH2Cl2 to salt 5‚2TFA. Similarly,
intermediates 6 and 7 were prepared by alkylation of 2 with
2-chloro-N-methylacetamide and 2-chloro-N-phenylacetamide
in yields of 98 and 73%, respectively (Scheme 3). TFA
deprotection of 6 and 7 gave 8 ([CB-MeTEAMA]H) and 9 ([CB-
PhTEAMA]H) in quantitative yields as TFA salts.
Copper(II) Complexes of CB-TEAMA and CB-PhTEA-
MA. The copper(II) complexes of CB-TEAMA and CB-
PhTEAMA were prepared and fully characterized as described
in the Experimental Section. Their X-ray crystal structures are
shown in Figure 2. In both complexes, Cu(II) is fully enveloped
by four nitrogens from the ligand cleft and two oxygens, one
from each pendant arm, resulting in a distorted octahedral
geometry. Amide coordination is through the amide pendant
arm’s carbonyl oxygen. For Cu(II)-CB-TEAMA, the Jahn-
Teller distortion is along the N(4)-Cu-O(1) axis, with
elongated Cu-N and Cu-O bond lengths of 2.196(6) and
2.401(5) Å, respectively. Strong equatorial coordination is
provided by N(2), N(3), O(2), and N(5). The trans-N(2)-Cu-
N(5) and cis-N(3)-Cu-N(4) angles of 174.3(2) and 87.1(2)°
are consistent with a reasonable ligand cleft-Cu(II) fit.
For Cu(II)-CB-PhTEAMA, the Jahn-Teller elongation is
along the N(1)-Cu-O(2) axis, with long Cu-N and Cu-O
bonds of 2.205(3) and 2.367(3) Å, respectively. The equatorial
coordination plane consists of N(2), O(3), N(3), and N(4). The
strongly bonded carboxylate is indicated by the short Cu-O(3)
distance of 1.950(3) Å. The intra-cleft trans-N(2)-Cu-N(3)
angle of 176.5(1)° and cis-N(1)-Cu-N(4) angle of 88.5(1)°
also confirm the good fit of Cu(II) within this cross-bridged
cyclam ligand cavity.

64Cu-Cross-Bridged Tetraazamacrocycle-amides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2529
Figure 2. ORTEP drawing of the Cu-CB-TEAMA and Cu-CB-PhTEAMA complexes. Hydrogens were omitted for clarity except on the primary
amide.
Table 1. Partition Coefficient for Each 64Cu Complex
of the 30 min uptake value. This resulted in an excretion of the
complexes in the urine (% ID in urine, 0-24 h PI, 64Cu-TETA,
chelate
LogP
71.6 ( 5.3% ID; 64Cu-CB-TE2A, 74.5 ( 6.1% ID; 64Cu-CB-
TEAMA, 66.9 ( 8.3% ID; 64Cu-CB-MeTEAMA, 63.0 ( 12.2%
ID; 64Cu-CB-PhTEAMA, 44.6 ( 2.1% ID).
64Cu-TETA
-3.58 ( 0.18
-2.07 ( 0.24
-2.35 ( 0.06
-2.08 ( 0.08
-1.35 ( 0.05
64Cu-CB-TE2A
64Cu-CB-TEAMA
64Cu-CB-MeTEAMA
64Cu-CB-PhTEAMA
In the liver, 64Cu-CB-MeTEAMA, initially (30 min PI) had
9.3-fold higher uptake than 64Cu-TETA (p < 0.01; Figure 3).
Although not statistically significant, liver uptake was 7.4- and
5.8-fold higher for 64Cu-CB-TEAMA and 64Cu-CB-PhTEAMA,
respectively, compared to 64Cu-TETA. It was expected that liver
uptake would reflect lipophilicity as determined by partition
coefficients. Consistent with this hypothesis, all cross-bridged
monoamides showed significantly higher 30 min liver uptake
than the more hydrophilic 64Cu-TETA. However, 30 min liver
uptake of 64Cu-CB-TE2A was even lower than for 64Cu-TETA,
despite a similar partition coefficient to 64Cu-CB-MeTEAMA.
This may be explained by the complex in vivo bioavailability,
which reflects ongoing excretion as 64Cu-CB-TE2A clears
rapidly via the kidneys.
By 24 h, the liver uptake fell to 23.4 ( 5.0% for 64Cu-CB-
TE2A, 6.7 ( 0.9% for 64Cu-CB-TEAMA, 7.8 ( 5.5% for
64Cu-CB-MeTEAMA, and 1.3 ( 0.3% for 64Cu-CB-PhTEAMA
compared to 30 min uptake (Figure 3). In contrast, 64Cu-TETA
was retained in the liver to a much greater extent than cross-
bridged complexes, resulting in a decrease in liver uptake to
66.2 ( 11.6% of 30 min uptake at 24 h (p < 0.001). Similarly,
bone marrow uptake fell significantly between 30 min and 24
h for 64Cu-CB-TE2A, 64Cu-CB-TEAMA, and 64Cu-CB-Ph-
TEAMA. Although marrow uptake for 64Cu-CB-MeTEAMA
does appear to decrease between 30 min and 24 h, this decrease
is not statistically significant (p ) 0.06) due to high variability
in 30 min uptake. In contrast, no significant change in marrow
uptake was observed for 64Cu-TETA over this period.
Solid-state IR as well as solution electronic spectral data are
fully consistent with these structures. The asymmetric carboxy-
late stretch for Cu-CB-PhTEAMA is found at 1628 cm-1, while
the amide carbonyl stretch appears at 1677 cm-1. Corresponding
bands are observed at 1627 and 1677 cm-1 for Cu-CB-
TEAMA. Both complexes exhibit a single broad d-d band at
648 nm (ꢀ ) 55 M-1cm-1) and 625 nm (ꢀ ) 35 M-1cm-1),
respectively, in their aqueous solution electronic spectra.
Radiochemistry. Consistent with previously reported results
for radiolabeling CB-TE2A with 64Cu(II),14 radiolabeling CB-
TEAMA, CB-MeTEAMA, and CB-PhTEAMA in aqueous
solution (0.1 M NH4OAc, pH 8.0) resulted in the formation of
several kinetically favored impurities, as evidenced by the
presence of multiple peaks on radio-HPLC. However, using
basic conditions in ethanol with Cs2CO3, as described by
Boswell et al. for radiolabeling CB-TE2A,14 single peaks were
observed on radio-HPLC for 64Cu-CB-TEAMA (RT ) 17.1
min, 2.2-3.2 mCi/µmol), 64Cu-CB-MeTEAMA (RT ) 18.4
min, 53-73 mCi/µmol), and 64Cu-CB-PhTEAMA (RT ) 26.9
min; 51-74 mCi/µmol).
Partition Coefficients. The partition coefficients (octanol/
PBS) were determined for each complex to evaluate relative
lipophilicity (Table 1). The order of lipophilicity was as
follows: 64Cu-CB-PhTEAMA > 64Cu-CB-MeTEAMA ∼
64Cu-CB-TE2A > 64Cu-CB-TEAMA > 64Cu-TETA.
Biodistribution Studies. To examine the in vivo properties
of 64Cu-CB-TEAMA, 64Cu-CB-MeTEAMA, and 64Cu-CB-
PhTEAMA, biodistribution studies were carried out in male
Lewis rats. For comparison, biodistribution was also performed
for 64Cu-TETA and 64Cu-CB-TE2A. Figure 3 shows the
radioactivity uptake to 24 h postinjection (PI) in several organs.
All five complexes cleared rapidly from blood between 30 min
and 2 h PI (<15% of 30 min activity remaining at 2 h).
However, between 2 and 24 h, blood-associated radioactivity
decreased significantly more for each monoamide cross-bridged
complex than for 64Cu-TETA or 64Cu-CB-TE2A (% 2 h uptake
remaining at 24 h ) 100 × [% ID/g24h/% ID/g2h]; 64Cu-TETA,
80.5 ( 12.5%; 64Cu-CB-TE2A, 61.9 ( 7.5%; 64Cu-CB-
TEAMA, 23.4 ( 5.4%; 64Cu-CB-MeTEAMA, 17.2 ( 6.3%;
64Cu-CB-PhTEAMA, 19.3 ( 3.5%; p < 0.01). Kidney uptake
decreased slowly between 30 min and 24 h for all five
complexes such that 24 h kidney uptake was reduced to <10%
To further evaluate uptake and excretion of these 64Cu
complexes by the liver, radioactivity uptake in the gut was
examined (Figure 4). Gastrointestinal tract biodistribution was
performed without the removal of fecal contents. The GI tract
was divided into four sections: stomach, proximal intestine,
middle intestine, and distal intestine. The distal intestine included
the cecum to the rectum; the remaining intestine (stomach to
cecum) was divided in half to give the proximal and middle
regions. For all cross-bridged complexes, minimal stomach
uptake, high 30 min proximal intestine uptake that decreased
with time, and increasing uptake in more distal parts of the
intestine with time were suggestive of excretion by the liver
into bile, with subsequent clearance as feces. At all time-points,
intestinal uptake was highest for 64Cu-CB-PhTEAMA, followed
by 64Cu-MeTEAMA and 64Cu-CB-TEAMA. This pattern of
uptake was not observed for 64Cu-TETA. Radioactivity in the
feces was <0.1% ID at 30 min, 1 h, and 2 h PI for all complexes.

2530 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Sprague et al.
Figure 3. Selected organ biodistribution of 64Cu-TETA (1), 64Cu-CB-TE2A ([),64Cu-CB-TEAMA (b), 64Cu-CB-MeTEAMA (9), 64Cu-CB-
PhTEAMA (2) in 33-40 day old, male Lewis rats (30 min to 24 h, n ) 4; 64Cu-TETA 1 h marrow, n ) 3, excluded by Q-test; 64Cu-CB-TE2A
2 h, n ) 3, animal died prior to sacrifice). Note differences in scale.
By 24 h PI, amounts of 64Cu excreted as feces were as follows:
64Cu-TETA ) 3.1 ( 0.7% ID, 64Cu-CB-TE2A ) 3.5 ( 0.9%
ID, 64Cu-CB-TEAMA ) 6.8 ( 1.8% ID, 64Cu-CB-MeTEAMA
) 11.2 ( 1.2% ID, and 64Cu-CB-PhTEAMA ) 39.3 ( 3.7%
ID.
served for Cu(II)-CB-TE2A.18 For both monoamides, the amide
pendant arm was coordinated via the carbonyl oxygen. Although
coordination geometries may change in solution, this increases
the likelihood that a peptide-conjugated Cu(II)-CB-TE2A could
also retain this distorted octahedral coordination geometry, thus
accounting for the favorable in vivo clearance properties seen
with 64Cu-CB-TE2A-Y3-TATE.28
Discussion
Somewhat surprisingly, the logP values of 64Cu-CB-Me-
TEAMA (-2.08 ( 0.08) and 64Cu-CB-TE2A (-2.07 ( 0.24)
were shown to be very similar. It was expected that the
additional methyl group on 64Cu-CB-MeTEAMA would result
in a more lipophilic complex than 64Cu-CB-TE2A. However,
the difference in charge (64Cu-CB-MeTEAMA ) +1, 64Cu-
CB-TE2A ) 0) likely balances out the additional methyl group,
resulting in similar partition coefficients. This is in contrast to
a previously reported logP value for 64Cu-CB-TE2A (-2.42 (
0.04).23 However, the radiolabeling method for 64Cu-CB-TE2A
has since been optimized, and the ligand used in the original
study may have contained trace radio-impurities such as 64Cu-
TE3A (predicted charge ) -1),33 which would be less lipophilic
than 64Cu-CB-TE2A. The order of lipophilicity for the remaining
complexes was consistent with predictions.
Copper-64-CB-TEAMA, 64Cu-CB-MeTEAMA, and 64Cu-
CB-PhTEAMA cleared over time from blood, liver, and marrow
despite initial high uptake in some organs. Previous studies have
associated retention of radioactivity in nontarget tissues with
poor in vivo stability and transchelation of 64Cu to proteins.3,15
High liver uptake at 30 min, with subsequent excretion into
feces, was not unexpected for 64Cu-CB-PhTEAMA given the
structure and lipophilicity. Liver uptake and fecal clearance were
similar for 64Cu-CB-MeTEAMA and 64Cu-CB-TEAMA, with
fecal clearance being significantly less for both compared to
64Cu-CB-PhTEAMA (p < 0.001). To account for the relatively
high initial liver uptake with resultant low fecal excretion, we
The group at Washington University has previously shown
that the 64Cu-labeled cross-bridged chelator CB-TE2A has
favorable biodistribution properties and good in vivo stability
relative to 64Cu-TETA.14,23 Subsequently, CB-TE2A was con-
jugated to the peptide Y3-TATE to evaluate its use as a
bifunctional chelator.28 Despite conversion of one carboxylate
arm of CB-TE2A to a secondary amide upon peptide conjuga-
tion, it was found that 64Cu-CB-TE2A-Y3-TATE had superior
in vivo clearance properties, suggestive of improved in vivo
stability relative to 64Cu-TETA-Y3-TATE.28 To investigate the
in vivo biodistribution characteristics of the conjugated cross-
bridged chelate independent of a peptide, three monoamide
derivatives of CB-TE2A have been synthesized as models of a
CB-TE2A-peptide conjugate.
High kinetic stability of a Cu(II) chelate has been shown to
be predictive of in vivo stability.10,11 It has been shown that the
half-life of Cu(II)-CB-TE2A in 5 M HCl (90 °C) is ap-
proximately 2000-fold longer than for Cu(II)-TETA, indicating
much higher kinetic stability.32 Preliminary kinetic stability
studies of Cu(II)-CB-TEAMA revealed that the harsh conditions
caused hydrolysis of the amide bond, resulting in conversion
back to Cu(II)-CB-TE2A. It was, therefore, not possible to
determine directly the kinetic stability of these Cu(II) cross-
bridged monoamide complexes.
Spectral data as well as X-ray crystal structures of both Cu-
(II)-CB-TEAMA and Cu(II)-CB-PhTEAMA confirmed their
distorted octahedral coordination geometry as previously ob-

64Cu-Cross-Bridged Tetraazamacrocycle-amides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2531
Figure 4. Gut biodistribution of 64Cu-TETA, 64Cu-CB-TE2A, 64Cu-CB-TEAMA, 64Cu-CB-MeTEAMA, and 64Cu-CB-PhTEAMA in 33 to 40 day
old, male Lewis rats (30 min to 24 h, n ) 4; 64Cu-CB-TE2A 2 h, n ) 3, animal died prior to sacrifice). Intestines were removed and divided into
three parts for counting without removal of fecal contents. Note differences in scale.
propose that 64Cu-CB-TEAMA and 64Cu-CB-MeTEAMA may
and in organic solvents are potentially explosive and should be
prepared and handled only in small quantities and with great
care. All work involving the use of radioactive materials at
Washington University is conducted under Radiation Safety Com-
mittee approved authorizations in accordance with the University’s
Nuclear Regulatory Commission license and Missouri State reg-
istrations.
Copper-64 was produced on a CS-15 biomedical cyclotron at
Washington University School of Medicine according to published
procedures.9 Copper chloride (CuCl2) was purchased from Johnson
Matthey (West Deptford, NJ). All other chemicals were purchased
from Sigma-Aldrich Chemical Co. (St. Louis, MO). All solutions
were prepared using ultrapure water (18 MΩ/cm2 resistivity).
IR spectra were recorded on a Nicolet MX-1 FT spectrophoto-
meter. UV-vis data were collected using a Cary 50 Bio UV-vis
spectrophotometer. Elemental analyses were performed at Atlantic
Microlab, Inc. (Norcross, GA). Analytical reversed-phase HPLC
was performed on a Waters 600E (Milford, MA) chromatography
system with a Waters 991 photodiode array detector and an Ortec
Model 661 radioactivity detector (EG&G Instruments, Oak Ridge,
TN). Radioactive samples were counted using a Beckman 8000
have undergone enzymatic modification in the liver, such as
hydrolysis of the amide to form 64Cu-CB-TE2A. This might
allow secretion of the radio-metabolite(s) into blood, with
subsequent clearance via the kidneys.
The observation that all cross-bridged complexes demonstrate
continuous clearance from blood, liver, and bone marrow is
highly suggestive of improved in vivo stability compared to
64Cu-TETA, as was shown previously for 64Cu-CB-TE2A.14,23
These data further support the observation that CB-TE2A can
be used as a bifunctional chelator for 64Cu-radiolabeled peptides
without additional modifications to the macrocycle backbone.
However, no studies have been conducted to examine the in
vivo stability properties of 64Cu-CB-TE2A conjugates to larger
macromolecules, which would require imaging at later time
points.
Experimental Section
Materials and Methods. Caution! Although we did not experi-
ence any difficulties, metal perchlorate salts with organic ligands

2532 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Sprague et al.
automated well-type gamma counter (Fullerton, CA). ES-MS was
accomplished using a Waters Micromass ZQ (Milford, MA). Male
Lewis rats (21 d old, 40-50 g) were purchased from Charles River
Laboratories (Boston, MA).
Ligand Synthesis. Synthetic reactions were run under a nitrogen
atmosphere. Solvents were removed by rotary evaporation under
reduced pressure (water aspirator) and vacuum line (mechanical
pump). 1,4,8,11-Tetraazabicyclo[6.6.2]hexadecane (cross-bridged
cyclam 1) was prepared as previously reported.18
53.23, 54.98, 55.11, 58.11, 58.13, 58.70, 58.93, 116.4 (q, J ) 291.4
Hz, CF3COOH), 163.1 (q, J ) 35.2 Hz, CF3COOH), 170.05,
171.77; IR (CH3CN) 3681, 3540, 3164, 3003, 2944, 1694, 1444,
1422, 1376, 1202, 1039, 918 cm-1; HRFABMS, m/z (M + H)+
exact mass for C16H35N5O3, 342.2505; found, 342.2500 (error -0.5
mmu/-1.4 ppm).
4-Carbo-t-butoxymethyl-11-(N-methylacetamido)-1,4,8,11-
tetraazabicyclo[6.6.2]hexadecane (6). Monoarmed ligand 2 (18
mg, 0.052 mmol) was dissolved in dry CH3CN (4 mL), Na2CO3
(13 mg, 0.1226 mmol), KI (23 mg, 0.1385 mmol), and 2-chloro-
N-methyl acetamide (13 mg, 0.1209 mmol) were added sequentially,
and the solution was stirred for 14 h at room temperature. Solvent
was removed to yield crude product as a solid, which was dissolved
in water (2 mL) and adjusted to pH 3 (3 M HCl with cooling). The
aqueous phase was extracted with benzene (2 × 25 mL), and the
retained aqueous phase was adjusted to pH 14 with solid KOH
(with cooling). The basic aqueous phase was extracted with benzene
(2 × 25 mL), the combined extracts were dried (Na2SO4), and the
benzene was removed to yield 6, an oil (21 mg, 0.051 mmol, 98%,
>94% purity by 13C NMR): 1H NMR (C6D6, 400 MHz) δ 1.05-
1.40 (m, 4H), 1.40 (s, 9H, C(CH3)3), 2.02-2.16 (m, 2H), 2.17-
2.36 (m, 2H), 2.36-2.66 (m, 8H), 2.64 (d, 3H, J ) 5.1 Hz), 2.68-
2.94 (m, 5H), 2.86 and 3.07 (AB, 2H, J ) 16.2 Hz), 3.02 and 3.15
(AB, 2H, J ) 16.4 Hz), 3.24 (td, 1H, J ) 12.1, 4.3 Hz), 3.40 (td,
1H, J ) 11.9, 4.3 Hz), 4.11 (ddd, 1H, J ) 16.6, 10.0, 4.3 Hz),
6.59 (br s, 1H, NH); 13C{1H} NMR (C6D6, 100.5 MHz) δ 25.77,
28.32, 28.53, 28.58. 51.96, 52.70, 53.83, 54.02, 56.29, 57.18, 57.76,
57.95, 58.81, 60.23, 61.58, 80.34, 171.53, 171.69; IR (CCl4) 3407,
4-Carbo-t-butoxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]-
hexadecane (2). 1,4,8,11-Tetraazabicyclo[6.6.2]hexadecane (1; 100
mg, 0.441 mmol) was dissolved in dry CH3CN (4 mL), sodium
carbonate (47.0 mg, 0.441 mmol) was added in one portion, and
t-butyl bromoacetate (0.065 mL, 0.44 mmol) was added in one
portion by syringe. The solution was stirred for 14 h at room
temperature, followed by solvent removal. NMR indicated this crude
product to be a mixture of monoester 2, diester 3, and residual
tetraamine 1. Purification by flash chromatography (SiO2, MeOH/
CH2Cl2 ) 1.5:10) yielded an oil that was dissolved in water (4
mL), adjusted to pH 14 (solid KOH), and extracted with benzene
(3 × 50 mL). The combined extracts were dried (Na2SO4) and the
solvent was removed to yield 2 as a light yellow oil (60.0 mg,
0.172 mmol, 39%): 1H NMR (C6D6, 500 MHz) δ 1.20-1.32 (m,
2H), 1.38 (s, 9H, C(CH3)3), 1.41-1.52 (m, 2H), 1.94-2.01 (dm,
1H, J ) 15.6 Hz), 2.08-2.14 (dm, 1H, J ) 14.7 Hz), 2.19 (ddd,
1H, J ) 13.4, 10.0, 3.4 Hz), 2.24-2.29 (dm, 1H, J ) 12.9 Hz),
2.30-2.40 (m, 3H), 2.45 (td, 1H, J ) 13.2, 3.7 Hz), 2.53 (td, 1H,
J ) 11.5, 5.4 Hz), 2.58-2.65 (ddm, 1H, J ) 13.4, 5.9 Hz), 2.65-
2.80 (m, 5H), 2.85-2.94 (m, 1H), 2.97-3.04 (m, 1H), 3.00 and
3.11 (AB, 2H, J ) 16.4 Hz), 3.15-3.26 (m, 2H), 3.41-3.48 (m,
1H), 4.69 (m, 1H, NH); 13C{1H} NMR (C6D6, 100.5 MHz) δ 25.73,
28.21 (C(CH3)3), 28.35, 48.79, 49.03, 49.07, 50.20, 53.67, 55.63,
55.71, 57.36, 57.59, 59.37, 60.75, 79.92 (OC(CH3)3), 171.24
(CdO); IR (neat) 1158, 1214, 1255, 1296, 1366, 1391, 1458, 1492,
1736(CdO), 2801, 2914, 3236 (NH) cm-1; HRFABMS (M + H)+
exact mass calcd for C18H33N4O2, 341.2917; found, 341.2915 (error
-0.1 mmu/-0.3 ppm).
4-Acetamido-11-carbo-t-butoxymethyl-1,4,8,11-tetraazabicyclo-
[6.6.2]hexadecane (4). 4-Carbo-t-butoxymethyl-1,4,8,11-tetraaza-
bicyclo[6.6.2]hexadecane (2; 30 mg, 0.086 mmol) was dissolved
in dry CH3CN (4 mL). Na2CO3 (9.2 mg, 0.086 mmol) was then
added, followed by 2-bromoacetamide (12 mg, 0.086 mmol). The
mixture was stirred for 14 h at room temperature and then solvent
was removed. The residue was dissolved in water (2 mL), adjusted
to pH 14 (solid KOH) with cooling, and extracted with benzene
(2 × 20 mL). The combined extracts were dried (Na2SO4) and
solvent was removed to give 4 as an oil (15.0 mg, 0.0372 mmol,
43%): 1H NMR (C6D6, 400 MHz) δ 1.01-1.40 (m, 4H), 1.39
(s, 9H, C(CH3)3), 2.00 (dt, 1H, J ) 12.3, 4.1 Hz), 2.05-2.68 (m,
13H), 2.68-2.86 (m, 3H), 2.79 and 3.04 (AX, 2H, J ) 16.4 Hz),
3.02 and 3.14 (AB, 2H, J ) 16.4 Hz), 3.20 (td, 1H, J ) 8.0, 4.1
Hz), 3.36 (td, 1H, J ) 12.1, 4.5 Hz), 4.08 (ddd, 1H, J ) 13.3, 8.2,
3.9 Hz), 6.37 (br s, 1H), 6.42 (br s, 1H); 13C{1H} NMR (C6D6,
100.5 MHz) δ 28.04, 28.22, 28.40. 51.67, 52.27, 53.46, 53.86,
55.89, 56.76, 57.24, 57.52, 57.55, 58.65, 59.94, 61.06, 79.96,
171.18, 174.44; IR (CCl4) 3448, 2978, 2922, 2809, 1739, 1689,
1368, 1155, 1125 cm-1; HRFABMS (M + H)+ exact mass calcd
for C20H40O3N5, 398.3131; found, 398.3106 (error -2.5 mmu/
-6.3 ppm).
2918, 2809, 1739, 1684, 1559, 1457, 1368, 1155, 1125 cm-1
;
HRFABMS (M + H)+ exact mass calcd for C21H42N5O3, 412.3288;
found, 412.3274 (error -1.4 mmu/-3.3 ppm).
(11-Methylcarbamoylmethyl-1,4,8,11-tetraazabicyclo[6.6.2]-
hexadec-4-yl)-acetic acid‚2TFA (8‚2TFA). 4-(Carbo-t-butoxy-
methyl)-11-(N-methylacetamido)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane
6 (14 mg, 0.034 mmol) was dissolved in a mixture of TFA and
CH2Cl2 (TFA/CH2Cl2 ) 1:1, 8 mL), the solution was stirred for
16 h, and the solvent was removed to yield 8 as a TFA salt (20
mg): 1H NMR (400 MHz, D2O, HOD peak set at 4.80) δ 1.70-
1.80 (dm, 2H, J ) 17.0 Hz), 2.30-2.46 (m, 2H), 2.70-2.85 (m,
2H), 2.74 (s, 3H, NCH3), 3.00-3.38 (m, 14H), 3.44-3.76 (m, 4H),
3.57 and 4.11 (AX, 2H, J ) 17.4 Hz), 3.58 and 4.06 (AX, 2H, J
) 16.1 Hz); 13C{1H} NMR (100.5 MHz, D2O) δ 19.41 (2C), 25.95,
47.51, 47.87, 48.26, 48.32, 52.82, 53.29, 55.13 (2C), 58.07 (2C),
58.61, 58.93, 116.43 (q, JCF ) 292.2 Hz, CF3COOH), 163.05 (q,
JCF ) 35.2 Hz, CF3COOH), 167.65, 171.74; IR (CH3CN) 3638-
3538 (br), 3164, 3002, 2944, 1682, 1444, 1418, 1376, 1201, 1132,
1039 cm-1; HRFABMS m/z (M + H)+ exact mass calcd for
C17H34N5O3, 356.2662; found, 356.2664 (error +0.2 mmu/+0.5
ppm). The product is a di-TFA salt on the basis of the mass (no
evidence of impurities by NMR).
4-(Carbo-t-butoxymethyl)-11-(N-phenylacetamido)-1,4,8,11-
tetraazabicyclo[6.6.2]hexadecane (7). Monoarmed ligand 2 (20
mg, 0.058 mmol) was dissolved in dry CH3CN (4 mL), and Na2-
CO3 (74 mg, 0.698 mmol), KI (13.4 mg, 0.0807 mmol), and
2-chloro-N-phenylacetamide (37 mg, 0.218 mmol) were added in
single portions. The solution was stirred for 14 h at room
temperature and solvent was removed by rotary evaporation to yield
a solid, which was dissolved in water (2 mL). The pH was adjusted
to 3 (3 M HCl with cooling), and the acidic solution was extracted
with benzene (2 × 25 mL). The retained aqueous phase was
adjusted to pH 14 (solid KOH with cooling) and extracted with
benzene (2 × 25 mL), the combined benzene extracts were dried
(Na2SO4), and the solvent was removed to yield 7 as an oil (20
mg, 0.042 mmol, 73%): 1H NMR (C6D6, 500 MHz) δ 1.03-1.40
(m, 4H), 1.40 (s, 9H, C(CH3)3), 2.00-2.03 (m, 2H), 2.08 (dt, 1H,
J ) 13.0, 3.9 Hz), 2.12-2.23 (m, 2H), 2.24-2.35 (m 2H), 2.48-
2.55 (m, 2H), 2.55-2.66 (m, 4H), 2.71 (td, 1H, J ) 8.8, 3.6 Hz),
2.76-2.86 (m, 2H), 2.85 and 3.09 (AX, 2H, J ) 16.4 Hz), 2.97
(td, 1H, J ) 11.7, 4.2 Hz), 3.01 and 3.13 (AB, 2H, J ) 16.4 Hz),
3.25 (td, 1H, J ) 13.2, 4.6 Hz), 3.33 (td, 1H, J ) 12.0, 4.4 Hz),
4.17-4.24 (m, 1H), 6.92 (tt, 1H, J ) 8.6, 1.0 Hz), 7.14-7.22 (m,
4-Acetamido-11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]-
hexadecane‚2TFA (5‚2TFA). 4-Carbo-t-butoxymethyl-11-aceta-
mido-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (4; 15 mg, 0.0372
mmol) was dissolved in a mixture of CF3CO2H (TFA) and CH2Cl2
(TFA/CH2Cl2 ) 1:1, 6 mL), and the solution was stirred for 14 h
at room temperature. Solvent was then removed to give 5 as a TFA
salt (2 equiv TFA calculated on the basis of mass; 20 mg): 1H
NMR (D2O, 400 MHz, HOD peak set to 4.80) δ 1.72-1.80 (dm,
2H, J ) 17.4 Hz), 2.30-2.45 (m, 2H), 2.70-2.84 (m, 2H), 3.00-
3.36(m, 16H), 3.42-3.72 (m, 4H), 3.56 and 4.08 (AX, 2H, J )
17.4 Hz), 3.63 and 4.08 (AX, 2H, J ) 16.1 Hz); 13C{1H} NMR
(D2O, 100.5 MHz) δ 19.46, 19.47, 47.56, 47.82, 48.20, 48.30, 52.91,

64Cu-Cross-Bridged Tetraazamacrocycle-amides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2533
Table 2. Crystal Data for [Cu-CB-TEAMA]ClO4‚NaClO4 and [Cu-CB-PhTEAMA]ClO4‚MeOH
[Cu-CB-TEAMA]ClO4‚NaClO4
[Cu-CB-PhTEAMA]ClO4‚MeOH
empirical formula
formula weight
temperature (K)
crystal system
C16H28Cl2CuN5NaO11
623.86
213(2)
orthorhombic
P212121
C23H36ClCuN5O8
609.56
100(2)
orthorhombic
Pbca
space group
unit cell dimensions (Å)
a ) 10.011(5)
b ) 12.346(6)
c ) 20.652(11)
2553(2)
a ) 21.134(5)
b ) 10.7258(16)
c ) 23.111(4)
5238.6(15)
4
volume (Å3)
Z
4
density (calcd; Mg/m3)
absorption coefficient (mm-1
crystal size (mm3)
crystal color, habit
reflections collected
independent reflections
1.623
1.145
0.20 × 0.20 × 0.05
blue, plate
12 979
4363 [R(int) ) 0.0415]
99.1%
4363/0/325
1.029
1.546
0.993
0.40 × 0.30 × 0.08
blue, plate
22 661
4613 [R(int) ) 0.0432]
99.0%
4613/0/343
1.049
)
completeness to theta ) 25.00°
data/restraints/parameters
goodness-of-fit on F2
final R indices [I > 2sigma(I)] (%)
R1 ) 6.57, wR2 ) 16.83
R1 ) 5.42, wR2 ) 13.03
absolute structure parameter
0.51(3)
NA
largest diff. peak, hole (e.Å-3
)
0.893, -0.476
1.038, -0.461
2H), 7.91 (dd, 2H, J ) 8.6, 1.0 Hz), 9.12 (br s, 1H, NH); 13C{1H}
NMR (C6D6, 100.5 MHz) δ 28.30, 28.57(C(CH3)3), 28.69, 51.87,
52.49, 53.84, 54.10, 56.13, 57.22, 57.46, 58.03, 58.36, 59.43, 60.40,
62.44, 80.39, 119.31, 124.24, 129.84, 139.48, 169.63, 171.49; IR
(neat) 703, 739,1126, 1156, 1265, 1368, 1443, 1521, 1601, 1684,
1732, 2818, 2925, 2979, 3053, 3308 cm-1; HSFABMS (M + H)
exact mass calcd for C26H44N5O3, 474.3444; found, 474.3440 (error
-0.4 mmu/-0.8 ppm).
taken up in 10 mL of water and its pH adjusted to approximately
8. Copper perchlorate hexahydrate (0.080 g, 0.22 mmol) was
dissolved in 5 mL of water and added to give a light-blue solution.
This was heated to 80 °C overnight to give a dark-blue solution.
After cooling to room temperature, this was evaporated to dryness
under reduced pressure. The residue was treated with absolute
ethanol and centrifuged to remove a small amount of insoluble
material. Ether diffusion into the supernatant yielded 32 mg (31%)
of the desired complex. X-ray quality crystals were grown from
an acetonitrile solution of this product by ether diffusion. IR: 3475-
(11-Phenylcarbamoylmethyl-1,4,8,11-tetraazabicyclo[6.6.2]-
hexadec-4-yl)-acetic acid‚4TFA (9‚4TFA). 4-(Carbo-t-butoxy-
methyl)-11-(N-phenylacetamido)-1,4,8,11-tetraazabicyclo[6.6.2]-
hexadecane (7; 140 mg, 0.296 mmol) was dissolved in a mixture
of TFA and CH2Cl2 (TFA/CH2Cl2 ) 1:1, 20 mL), and the solution
was stirred for 16 h. Solvent was then removed to yield 8 as a
tetra-TFA adduct on the basis of mass (255 mg, quant; no impurities
by NMR): 1H NMR (CD3CN, 500 MHz, CHD2CN central peak
set to 1.94) δ 1.63-1.72 (m, 2H), 2.20-2.34 (m, 2H), 2.68-2.76
(dm, 2H, J ) 12.5 Hz), 2.94-3.02 (dm, 2H, J ) 14.7 Hz), 3.03-
3.22 (m, 12H), 3.27 (td, 1H, J ) 12.9, 3.2 Hz), 3.40-3.61 (m,
4H), 3.62-3.72 (m, 2H), 4.05 (A of AX, 1H, J ) 16.6 Hz), 4.23
(A of AX, 1H, J ) 16.6 Hz), 7.12-7.17 (m, 1H), 7.31-7.36 (m,
2H), 7.57-7.61 (m, 2H), 10.02 (s, 1H, NH), 10.90 (br s, 2H), 11.06
(br s, 3H); 13C{1H} NMR (CD3CN, 125.7 MHz, CD3CN central
peak set to 1.39) δ 20.73, 20.81, 48.77, 48.98, 49.39, 49.43, 53.73,
53.91, 56.66, 57.70, 59.42, 60.40, 117.25 (q, JCF ) 290.5 Hz, CF3-
CO2), 121.24, 125.88, 129.94, 138.79, 160.95 (q, JCF ) 36.4 Hz,
CF3CO2), 166.73, 171.91; IR (CH3CN) 3648, 3607, 3550, 3002,
3380 (w, br), 1677 (s, CO), 1628 (s, COO), 1098 (vs, ClO4) cm-1
.
Visible electronic spectrum (H2O): λmax 648 nm (ꢀ ) 55 M-1cm-1).
Anal. Calcd for CuC16H30N5O3(ClO4)‚0.3NaClO4: C, 35.58; H,
5.60; N, 12.96; Cl, 8.53%. Found: C, 35.52; H, 5.73; N, 12.89;
Cl, 8.54%.
X-ray Crystallography. Crystallographic data are collected in
Table 2. Both space groups were uniquely determined from
systematic absences. Data collections for both structures were
performed on a Bruker D8 platform diffractometer equipped with
an APEX CCD detector using MoK(R) radiation. Data were
corrected for absorption using empirical methods (SADABS). All
nonhydrogen atoms were refined with anisotropic thermal param-
eters and hydrogen atoms were placed in idealized locations, except
for those associated with the hydroxyl proton in the solvent MeOH
for the PhTEAMA complex. Those hydrogens were ignored, but
were included in computations of intensive properties. For the CB-
TEAMA complex structure, refinement of the Flack parameter
yielded a value of 0.51, which is interpreted as an indication that
crystallization occurred as a racemic twin (further refinement with
TWIN and BASF ) 0.51 yielded identical results). All software
was obtained from the SMART, SAINT, and SHELXTL Bruker
libraries (Bruker-AXS, Madison, WI).
Radiolabeling with 64Cu. Cu(II) complexes of CB-TEAMA,
CB-MeTEAMA, and CB-PhTEAMA were prepared according to
the method described previously for radiolabeling CB-TE2A-Y3-
TATE with 64Cu.28 Briefly, CuCl2 + 64Cu (30 µCi) was added to
each monoamide (1:1) in NH4OAc, pH 8.0. The reaction mixture
was heated for 30 min at 95 °C. Complex formation was followed
by radio-TLC. After sufficient time for radio-decay, formation of
the correct complex was demonstrated by ES-MS (Cu(II)-CB-
PhTEAMA (C22H34CuN5O3Cu) calcd m/z (M+), 479.20; found,
479.08; Cu(II)-CB-MeTEAMA (C17H32CuN5O3Cu) calcd m/z (M+),
417.18; found, 417.04). HPLC analysis of the monoamide com-
plexes was accomplished using an Agilent Technologies C8 column
(3.0 × 150 mm; Palo Alto, CA)33 with the following gradient:
2944, 1680, 1632, 1446, 1428, 1375, 1201, 1039, 918, 749 cm-1
;
HRFABMS (M + H)+ exact mass calcd for C22H36N5O3, 418.2818;
found, 418.2811 (error -0.7 mmu/-1.7 ppm).
Synthesis of Cu-CB-PhTEAMA. An amount of 9‚4TFA (0.082
g, 0.99 mmol) was dissolved in 3 mL methanol. Copper perchlorate
hexahydrate (0.056 g, 0.15 mmol) dissolved in 2 mL of methanol
was added to give a light-blue solution. NaOH (6 equiv, 0.40 mL,
1.0 M) was added and the light-blue suspension was refluxed for
5 h to give a clear solution. Cooling to room temperature gave a
light-blue precipitate that was filtered off and dried under reduced
pressure to give 0.052 g (59% yield) of the complex. X-ray quality
crystals were grown from a methanol solution of this product by
ether diffusion. IR: 1677 (s, CO), 1628 (s, COO), 1116 (vs, ClO4)
cm-1. Visible electronic spectrum (H2O): λmax 648 nm (ꢀ ) 55
M-1cm-1). Anal. Calcd for CuC22H34N4O3(ClO4)(H2O): C, 44.22;
H, 6.07; N, 11.72; Cl, 5.93. Found: C, 44.47; H, 5.80; N, 11.68;
Cl, 5.98.
Synthesis of Cu-CB-TEAMA. An amount of 5‚2TFA (0.076
g, 0.19 mmol) was stirred in 10 mL of TFA overnight. After
removal of volatiles under reduced pressure, the oily residue was

2534 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Sprague et al.
(4) Lewis, J. S.; Lewis, M. R.; Cutler, P. D.; Srinivasan, A.; Schmidt,
M. A.; Schwartz, S. W.; Morris, M. M.; Miller, J. P.; Anderson, C.
J. Radiotherapy and dosimetry of 64Cu-TETA-Tyr3-octreotate in a
somatostatin receptor-positive, tumor-bearing rat model. Clin. Cancer
Res. 1999, 5, 3608-3616.
(5) Lewis, J. S.; Srinivasan, A.; Schmidt, M. A.; Anderson, C. J. In vitro
and in vivo evaluation of 64-Cu-TETA-Tyr3-octreotate. A new
somatostatin analog with improved target tissue uptake. Nucl. Med.
Biol. 1999, 26, 267-273.
(6) Zimmermann, K.; Grunberg, J.; Honer, M.; Ametamey, S.; Schubiger,
P. A.; Novak-Hofer, I. Targeting of renal carcinoma with Cu-64/67-
labeled anti-L1-CAM antibody chCE7: Selection of copper ligands
and PET imaging. Nucl. Med. Biol. 2003, 30, 417-427.
(7) Wu, A. M.; Yazaki, P. J.; Tsai, S.; Nguyen, K.; Anderson, A.-L.;
McCarthy, D. W.; Welch, M. J.; Shively, J. E.; Williams, L. E.;
Raubitschek, A. A.; Wong, J. Y. C.; Toyokuni, T.; Phelps, M. E.;
Gambhir, S. S. High-resolution microPET imaging of carcinoem-
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(8) Rogers, B. E.; Bigott, H. M.; McCarthy, D. W.; Manna, D. D.; Kim,
J.; Sharp, T. L.; Welch, M. J. MicorPET imaging of a gastrin-
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solvent A, H2O (0.1% formic acid); solvent B, CH3CN (0.1% formic
acid; solvent B); 0% B to 50% B in 40 min (0.5 mL/min flow
rate).
All monoamides, as well as CB-TE2A, were radiolabeled (no
carrier added) with 64Cu according to the method described by
Boswell et al. for formation of 64Cu-CB-TE2A.14 TETA was
radiolabeled with 64Cu in 0.5 M NH4OAc, pH 6.5 (30 min at RT).
Radio-HPLC for 64Cu-CB-TEAMA, 64Cu-CB-MeTEAMA, and 64
-
Cu-CB-PhTEAMA was performed as described above. For 64Cu-
CB-TE2A and 64Cu-TETA, radio-HPLC was performed using an
isocratic method (0.1% formic acid in H2O).
Partition Coefficients. The partition coefficients (logP) of
64Cu-TETA, 64Cu-CB-TE2A, 64Cu-CB-TEAMA, 64Cu-CB-Me-
TEAMA, and 64Cu-PhTEAMA were determined using previously
described methods.19 Briefly, 4 µL of 64Cu-labeled complex was
added to 500 µL octanol + 500 µL PBS (obtained from a saturated
octanol-PBS solution, n ) 4). Solutions were shaken for 1 h at
room temperature. For each sample, 50 µL was removed from each
phase and counted separately in a gamma counter. The partition
coefficient was calculated as a ratio of counts in the octanol fraction
to counts in the PBS fraction.
Biodistribution. All animal experiments were performed in
compliance with the Guidelines for the Care and Use of Research
Animals established by Washington University’s Animal Studies
Committee. Male Lewis rats (33-40 d old) were injected intrave-
nously with 64Cu-radiolabeled complexes (43 µCi, 1 nmol ligand)
via the tail vein. Tissue biodistribution data were obtained at 0.5,
1, 2, and 24 h PI according to previously described methods.5 Rats
in the 24 h group were maintained in metabolism cages; food and
water were administered ad libitum. Urine and feces were collected.
Statistical Methods. All data are presented as mean ( standard
deviation. For statistical classification, one-way ANOVA was used
to determine statistically significant differences between the five
complexes. Subsequently, Tukey’s multiple comparison test (post-
test) was used to compare individual data sets. Student’s t-test (two-
tailed, unpaired) was used to compare individual time points for a
single complex. All statistical analysis was performed using
GraphPad PRISM (San Diego, CA). All p values less than 0.05
were considered significant.
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Acknowledgment. The authors acknowledge Todd A. Per-
kins and Deborah Sultan for production of 64Cu and Laura
Meyer, Chris Sherman, Nicole Fettig, and Lori Strong for their
excellent technical assistance. This research was supported by
NCI Grant R01 CA93375. The production of 64Cu at Wash-
ington University School of Medicine is supported by the NCI
Grant R24 CA86307. J.E.S. was supported by the Department
of Defense Breast Cancer Research Program Grant W81XWH-
04-1-0396.
Supporting Information Available: X-ray crystallographic
information for Cu-CB-TEAMA and Cu-CB-PhTEAMA, including
experimental crystal data, refinement details, final atomic position
parameters, atomic thermal parameters, and complete bond distances
and angles. This material is available free of charge via the Internet
at http://pubs.acs.org.
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