Effects of targeting moiety, linker, bifunctional chelator, and molecular charge on biological properties of 64Cu-labeled triphenylphosphonium cations

Article abstract of DOI:10.1021/jm7015045

In this report, we present the synthesis and evaluation of six new ⁶⁴Cu-labeled triphenylphosphonium (TPP) cations. Biodistribution studies were performed using the athymic nude mice bearing U87MG human glioma xenografts to explore the impact of TPP moieties, linkers, bifunctional chelators (BFCs), and molecular charge on biological properties of ⁶⁴Cu radiotracers. On the basis of the results from this study, it is concluded that (1) mTPP (tris(4-methoxyphenyl)phosphonium) is a better mitochondrion-targeting molecule than TPP and 3mTPP (tris(2,4,6- trimethoxyphenyl)phosphonium); (2) DO3A (1,4,7,10-tetraazacyclododecane-4,7,10- triacetic acid) and DO2A (1,4,7,10-tetraazacyclododecane-4,7-diacetic acid) are suitable BFCs for the ⁶⁴Cu-labeling of TPP cations; (3) NOTA-Bn (S-2-(4-thioureidobenzyl)-1,4,7-triazacydononane-1,4,7-triacetic acid) has a significant adverse effect on the radiotracer tumor uptake and tumor-to-background ratios; and (4) monoanionic BFCs should be avoided to ensure that ⁶⁴Cu chelate has a neutral or negative charge. Considering the tumor uptake and tumor/liver ratios, ⁶⁴Cu(DO2A-xy-TPP)⁺ is the best candidate for more extensive evaluations in different tumor-bearing animal models.

Full text of DOI:10.1021/jm7015045

J. Med. Chem. 2008, 51, 2971–2984
2971
Effects of Targeting Moiety, Linker, Bifunctional Chelator, and Molecular Charge on Biological
Properties of 64Cu-Labeled Triphenylphosphonium Cations
Young-Seung Kim,† Chang-Tong Yang,† Jianjun Wang,† Lijun Wang,† Zi-Bo Li,‡ Xiaoyuan Chen,‡ and Shuang Liu*,†
School of Health Sciences, Purdue UniVersity, West Lafayette, Indiana, and Molecular Imaging Program at Stanford, Department of Radiology
& Bio-X, Stanford UniVersity, Stanford, California
ReceiVed December 3, 2007
In this report, we present the synthesis and evaluation of six new 64Cu-labeled triphenylphosphonium (TPP)
cations. Biodistribution studies were performed using the athymic nude mice bearing U87MG human glioma
xenografts to explore the impact of TPP moieties, linkers, bifunctional chelators (BFCs), and molecular
charge on biological properties of 64Cu radiotracers. On the basis of the results from this study, it is concluded
that (1) mTPP (tris(4-methoxyphenyl)phosphonium) is a better mitochondrion-targeting molecule than TPP
and 3mTPP (tris(2,4,6-trimethoxyphenyl)phosphonium); (2) DO3A (1,4,7,10-tetraazacyclododecane-4,7,10-
triacetic acid) and DO2A (1,4,7,10-tetraazacyclododecane-4,7-diacetic acid) are suitable BFCs for the 64Cu-
labeling of TPP cations; (3) NOTA-Bn (S-2-(4-thioureidobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid)
has a significant adverse effect on the radiotracer tumor uptake and tumor-to-background ratios; and (4)
monoanionic BFCs should be avoided to ensure that 64Cu chelate has a neutral or negative charge. Considering
the tumor uptake and tumor/liver ratios, 64Cu(DO2A-xy-TPP)+ is the best candidate for more extensive
evaluations in different tumor-bearing animal models.
Introduction
proposed as radiotracers for both myocardial perfusion imaging
and tumor imaging by positron emission tomography (PET).32–38
It has been reported that 3H-tetraphenylphosphonium (3H-TPP)
had a better tumor uptake than 99mTc-sestamibi, but its tumor
selectivity is very poor with the tumor/heart ratio , 1.0.32,36,37
The high uptake of 18F-BzTPP in the heart and liver may impose
a significant challenge for early detection of small tumors in
the chest and abdominal regions. Thus, there is an urgent need
for new radiotracers that have very high tumor selectivity and
high sensitivity to the changes in mitochondrial potentials at
the early stage of tumor growth.
Previously, we reported the synthesis and biological evalu-
ation of 64Cu(DO3A-xy-TPP)a, 64Cu(DO3A-xy-TPA), and 64Cu-
(DO3A-xy-mTPP) as PET radiotracers for tumor imaging using
athymic nude mice bearing subcutaneous U87MG human glioma
xenografts.39 The biodistribution data clearly show that 64Cu-
(DO3A-xy-TPP), 64Cu(DO3A-xy-TPA), and 64Cu(DO3A-xy-
mTPP) have relatively high tumor uptake with a long retention.
The most striking difference between 64Cu(DO3A-xy-TPP),
64Cu(DO3A-xy-TPA), 64Cu(DO3A-xy-mTPP), and 99mTc-Ses-
tamibi is that all three 64Cu radiotracers have much lower heart
uptake (<0.6% ID/g) than 99mTc-sestamibi (∼18% ID/g) at >30
min postinjection (p.i.). Their tumor/heart ratios are >40 times
Alteration in the mitochondrial potential (∆Ψm) is an
important characteristic of cancer and is caused directly by
mitochondrial dysfunction, such as DNA mutation and oxidative
stress.1–4 It has been demonstrated that the mitochondrial
transmembrane potential in carcinoma cells is significantly
higher than that in normal epithelial cells.5–9 For example, the
difference in ∆Ψm between the CX-1 colon carcinoma cell line
and the control green monkey kidney epithelial cell line CV-1
was approximately 60 mV (163 mV in tumor cells versus 104
mV in normal cells). The observation that the enhanced
mitochondrial potential is prevalent in tumor cell phenotype
provides the conceptual basis for the development of mitochon-
drion-targeting pharmaceuticals and imaging probes.1–3,10–13
Since plasma and mitochondrial transmembrane potentials are
negative, cationic molecules with appropriate structural features
can be driven electrophoretically through these membranes and
accumulate inside the energized mitochondria of tumor cells.1–4,11
Lipophilic organic cations, such as rhodamine-123 and 3H-
tetraphenylphosphonium (3H-TPP), have been used to measure
mitochondrial potentials in tumor cells.1,14–16 Cationic radiotrac-
ers, such as 99mTc-sestamibi and 99mTc-tetrofosmin, are also
able to localize in tumor cells due to the increased negative
mitochondrial potential. 99mTc-Sestamibi and 99mTc-Tetrofosmin
have been clinically used for imaging tumors of different origin
and the transport function of multidrug resistance P-glycoprotein
(MDR Pgp) by single photon emission computed tomography
(SPECT).17–31 However, their cancer diagnostic values are often
limited due to their insufficient tumor localization and high
uptake in the heart, liver, and muscle, which makes it difficult
to detect small lesions in the chest and abdominal regions.
Radiolabeled triphenylphosphonium (TPP) cations, such as
4-(18F-benzyl)triphenylphosphonium (18F-BzTPP), have been
a Abbreviations: DO3A-xy-TPP, triphenyl(4-((4,7,10-tris(carboxymethyl)-
1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)phosphonium; DO3A-
xy-TPA, triphenyl(4-((4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclodode-
can-1-yl)methyl)benzyl)arsonium; DO3A-xy-mTPP, tris(4-methoxyphenyl)-
(4-((4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)-
benzyl)phosphonium; DO3A-bu-TPP, triphenyl(4-((4,7,10-tris(carboxym-
ethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)-4-butyl)phosphonium;DO3A-
PEG2-TPP, triphenyl(2-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza-
cyclododecan-1-yl)ethoxy)ethoxy)ethyl)phosphonium; DO3A-xy-3mTPP,
tris(2,4,6-trimethoxyphenyl)(4-((4,7,10-tris(carboxymethyl)-1,4,7,10-tetra-
azacyclododecan-1-yl)methyl)benzyl)phosphonium; DO2A-xy-TPP, (4-((4,-
10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)-
triphenylphosphonium; DO2A-(xy-TPP)2, (4,4′-(4,10-bis(carboxymethyl)-1,-
4,7,10-tetraazacyclododecane-1,7-diyl)bis(methylene)bis(4,1-phenylene))-
bis(methylene)bis(triphenylphosphonium); NOTA-Bn-xy-TPP, triphenyl-
(4-((3-(4-((1,4,7-tris(carboxymethyl)-1,4,7-triazonan-2-yl)methyl)phenyl)th-
ioureido)methyl)benzyl)phosphonium.
* To whom correspondence should be addressed. Phone: 765-494-0236.
Fax: 765-496-1377. E-mail: lius@pharmacy.purdue.edu.
†
Purdue University.
Stanford University.
‡
10.1021/jm7015045 CCC: $40.75
2008 American Chemical Society
Published on Web 04/18/2008

2972 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Kim et al.
Figure 1. TPP conjugates for preparation of the 64Cu-labeled TPP cations useful as tumor-selective PET radiotracers. The TPP moiety is used as
the “mitochondrion-targeting biomolecule” to carry 64Cu into tumor cells with higher mitochondrial potential than normal cells. DO3A, DO2A, and
NOTA are used as BFCs for 64Cu chelation. Different linkers are used to modify pharmacokinetics and improve tumor-to-background ratios of
64Cu radiotracers.
better than that of 99mTc-sestamibi at 120 min p.i. The muscle
uptake of 64Cu(DO3A-xy-TPP), 64Cu(DO3A-xy-TPA), and
64Cu(DO3A-xy-mTPP) is almost undetectable at >30 min p.i.
In contrast, 99mTc-sestamibi has a very high muscle uptake
(∼5% ID/g) over a 2 h study period. Results from in vitro assays
show that 64Cu(DO3A-xy-TPP) is able to localize in the
mitochondria of glioma cells.39 The tumor could be visualized
as early as 30 min p.i. by PET imaging of the tumor-bearing
mice administered with 64Cu(DO3A-xy-TPP). While the tumor
uptake of the 64Cu-labeled TPP cations is most likely due to
the enhanced negative mitochondrial potentials, their high tumor
selectivity has been attributed to their high hydrophilicity (log
P < -2.5). It was concluded that the 64Cu-labeled TPP cations
are very promising PET radiotracers that are sensitive to
mitochondrial potential differences between tumor cells and cells
in the normal organ. However, their high liver uptake remains
a significant challenge for them to be clinically useful as PET
radiotracers for tumor imaging.
of tumor cells. The main objective of this study is to determine
the ideal structural parameters for an optimal 64Cu radiotracer
that is sensitive to the early mitochondrial potential changes in
tumors of different origin.
Experimental Section
Materials and Instruments. Chemicals were purchased from
Aldrich (St. Louis, MO). DO3A(OBu-t)3 (1,4,7,10-tetraazacy-
clododecane-4,7,10-tris(tert-butyl acetate)), DO2A(OBu-t)2 (1,4,7,
10-tetraazacyclododecane-4,7-bis(tert-butyl acetate)), DOTA(OBu-t)3-
NHS(1,4,7,10-tetraazacyclododecane-1-(N-hydroxysuccinimideacetate)-
4,7,10-tris(tert-butyl acetate)), and p-SCN-Bn-NOTA (S-2-(4-
isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid)
were purchased from Macrocyclics Inc. (Dallas, TX). NMR (1H,
13C, and 31P) data were obtained using a Bruker DRX 300 MHz
FT NMR spectrometer. Chemical shifts are reported as δ in ppm
relative to TMS. Mass spectral data were collected using positive
mode on a Finnigan LCQ classic mass spectrometer, School of
Pharmacy, Purdue University. Elemental analysis was performed
by Dr. H. Daniel Lee using a Perkin-Elmer Series III analyzer,
Department of Chemistry, Purdue University. Cardiolite vials were
obtained as a gift from Bristol Myers Squibb Medical Imaging
(North Billerica, MA) and were reconstituted according to the
manufacturer’s package insert. 64Cu was produced using a CS-15
biomedical cyclotron at Washington University School of Medicine
by the 64Ni(p,n)64Cu nuclear reaction.
To improve the tumor/liver ratios of the 64Cu-labeled TPP
cations, we prepared 64Cu complexes with several new TPP
conjugates (Figure 1). Biodistribution studies were performed
using athymic nude mice bearing subcutaneous U87MG glioma
xenografts to explore the impact of TPP moieties, linkers,
bifunctional chelators (BFCs), and molecular charge on biologi-
cal properties of the 64Cu-labeled TPP cations. We are particu-
larly interested in their excretion kinetics from noncancerous
organs, such as heart, liver and muscle. The U87MG human
glioma cell line was chosen because it has no MDR1 Pgp
expression.40–43 This tumor-bearing animal model would allow
us to evaluate the tumor uptake of the 64Cu-labeled TPP cations
without complications from the MDR Pgp transport function
HPLC Methods. Method 1 used a LabAlliance HPLC system
equipped with a UV/vis detector (λ ) 254 nm) and Zorbax C18
semipreparative column (9.4 mm × 250 mm, 100 Å pore size).
The flow rate was 2.5 mL/min. The mobile phase was isocratic
with 90% solvent A (0.1% acetic acid in water) and 10% solvent
B (0.1% acetic acid in acetonitrile) at 0–5 min, followed by a
gradient mobile phase going from 90% solvent A and 10% solvent

64Cu-Labeled TPP Cations for Tumor Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2973
B at 5 min to 60% solvent A and 40% solvent B at 20 min. Method
2 used the LabAlliance HPLC system equipped with a UV/vis
detector (λ ) 254 nm), a ꢀ-ram IN-US detector, and Vydac C18
column (4.6 mm × 250 mm, 300 Å pore size). The flow rate was
1 mL/min with the mobile phase being isocratic with 90% solvent
A (10 mM ammonium acetate) and 90% solvent B (acetonitrile) at
0–5 min, followed by a gradient mobile phase going from 10% B
at 5 min to 60% B at 20 min.
Triphenyl(4-((4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacy-
clododecan-1-yl)methyl)-4-butyl)phosphonium Acetate (DO3A-
bu-TPP). To a solution containing DO3A(OBu-t)3 (52.0 mg, 0.1
mmol) and (4-bromobutyl)triphenylphosphonium bromide (47.8 mg,
0.1 mmol) and in anhydrous DMF (3 mL) was added triethylamine
(0.04 mL, 0.3 mmol). The reaction mixture was stirred at room
temperature for 24 h. After removal of volatiles, the residue was
dissolved in 12 N HCl (3 mL). After stirring at room temperature
for 10–20 min, volatiles were removed under reduced pressure. The
residue was dissolved in 4 mL of a 50% DMF/water mixture, and
the product was subjected to HPLC purification (method 1).
Fractions at ∼16.5 min were collected. The collected fractions were
combined and lyophilized to give a white powder. The yield was
35 mg (53%). The retention time was ∼16.5 min with the HPLC
purity >95%. 1H NMR (in D2O): 1.66 (m, 4H), 2.78–3.51 (m, 24H),
7.55–7.67 (m, 15H, aromatic). ES-MS: m/z ) 663.76 for [M +
H]+ (calcd 664 for [C36H48N4O6P]+). Anal. Calcd for
C36H48N4O6P·CH3CO2 ·H2O: C, 61.61; H, 7.21; N, 7.56. Found:
C, 61.15; H, 7.43; N, 7.35.
[C38H52N4O8P]+). Anal. Calcd for C35H46N4O6P·CH3CO2 ·H2O: C,
61.14; H, 7.07; N, 7.71. Found: C, 61.15; H, 7.63; N, 7.45.
4-Bromomethylbenzyl(tris-2,4,6-methoxyphenyl)phosphoni-
um Bromide (3mTPP-xy-Br). Tris(2,4,6-trimethoxyphenyl)phos-
phine (1.10 g, 2.0 mmol) in toluene (6 mL) was added dropwise to
a hot toluene solution (5 mL) containing R,R′-dibromo-p-xylene
(0.53 g, 2.0 mmol). The resulting reaction mixture was refluxed at
100 °C overnight. The white solid was filtered, washed with toluene
(5 mL) and ether (20 mL), and then dried under vacuum to give
the product: 3mTPP-xy-Br. The yield was 1.16 g (73%). 1H NMR
(in CDCl3): 3.59 (s, 18H, 2,6-OCH3); 3.83 (s, 9H, 4-OCH3); 4.55
(d, 2H, PCH2, JPH ) 15 Hz); 4.92 (s, 2H, CH2Br), 6.03 (s, 6H,
C6H2); 6.91–7.05 (m, 4H, C6H4). ESI-MS: m/z ) 715.2 for [M+
+
H] (calcd 715 for [C35H41O9PBr]+).
Tris(2,4,6-trimethoxyphenyl)(4-((4,7,10-tris(carboxymethyl)-
1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)phosphoni-
um Acetate (DO3A-xy-3mTPP). To a solution containing 3mTPP-
xy-Br (80.0 mg, 0.1 mmol) and DO3A(OBu-t)3 (52.0 mg, 0.1 mmol)
in 3 mL of anhydrous DMF was added triethylamine (0.07 mL,
0.5 mmol). The reaction mixture was stirred at room temperature
overnight. After removal of volatiles under vacuum, the residue
was dissolved in 2.2 mL of HCl solution (37%). After stirring at
room temperature for 10–20 min, volatiles were removed under
the reduced pressure. The residue was dissolved in 50% DMF/
H2O (3 mL), and the product was purified by HPLC (method 1).
Fractions at ∼23.8 min were collected. The collected fractions were
combined and lyophilized to give a white powder. The yield was
40.1 mg (41%). The HPLC retention time was ∼23.8 min with a
2,2′-(Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)bis(4-meth-
ylbenzenesulfonate) (TsO-PEG2-OTs). To a solution of 4-meth-
ylbenzene-1-sulfonyl chloride (8.4 g, 44.0 mmol) and HO-
(CH2CH2O)3H (3.0 g, 20.0 mmol, 2.67 mL) in dichloromethane
(20 mL) was slowly added triethylamine (5.1 g, 50.0 mmol). The
resulting mixture was stirred at room temperature for 6 h and then
heated and refluxed for another 2 h. The suspension was cooled to
room temperature. After removal of the solid by filtration, the filtrate
was washed by water (3 × 20 mL). Volatiles were removed, and
the light yellow solid was dissolved in dichloromethane (5 mL).
Upon addition of diethyl ether (25 mL), a white solid was formed.
The solid was collected by filtration and was dried under vacuum
overnight to give the expected product: TsO-PEG2-OTs. The yield
1
purity of >95%. H NMR (in D2O): 2.66–3.47 (m, 22H and 2H,
NCH2); 3.51 (s, 18H, 2,6-OCH3); 3.73 (s, 9H, 4-OCH3); 4.63 (d,
2H, PCH2, JPH ) 13 Hz); 6.09 (s, 6H, C6H2); 7.08(d, 2H, C6H4);
7.19 (d, 2H, C6H4). ESI-MS: m/z ) 981.20 for [M + H]+ (calcd
981 for [C49H66N4O15P]+). Anal. Calcd for C49H66N4O15P ·
CH3CO2 ·H2O: C, 57.84; H, 7.76; N, 7.29. Found: C, 58.15; H,
7.63; N, 7.45.
(4-((4,10-Bis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-
1-yl)methyl)benzyl)triphenylphosphonium Acetate (DO2A-xy-
TPP). TPP-xy-Br was prepared using the procedure described in a
previous report.39 To a solution of TPP-xy-Br (52.6 mg, 0.1 mmol)
and DO2A(OBu-t)2 (40 mg, 0.1 mmol) in anhydrous DMF (3 mL)
was added triethylamine (0.07 mL, 0.5 mmol). The reaction mixture
was stirred at room temperature overnight. Volatiles were removed
under vacuum, and the residue was dissolved in 2.2 mL of HCl
(37%). After stirring at room temperature for ∼15 min, volatiles
were removed completely. The residue was dissolved in 4 mL of
50% DMF/H2O (3 mL). The product was subjected to HPLC
purification (method 1). Fractions at ∼13.9 min were collected,
and the collected fractions were combined and lyophilized to give
a white powder. The yield was 33.0 mg (50.5%). The retention
time was ∼13.9 min with an HPLC purity of >95%. 1H NMR (in
D2O): 2.73–3.17 (m, 21H); 4.15 (s, 2H, NCH2); 4.68 (d, 2H, PCH2,
JPH ) 13 Hz); 6.92 (d, 2H, C6H4); 7.18 (d, 2H, C6H4); 7.43–7.76
(m, 15H, C6H5). ESI-MS: m/z ) 653.77 for [M + H]+ (654 calcd
for C38H46N4O4P). Anal. Calcd for C38H46N4O4P·CH3CO2 ·3.52O:
C, 61.92; H, 7.28; N, 7.22. Found: C, 62.01; H, 7.18; N, 7.35.
1
was 3.7 g (40%). H NMR (in CDCl3): 2.44 (s, 6H, OCH3), 3.52
(s, 4H), 3.65 (t, 4H, J ) 4.8 Hz), 4.14 (t, 4H, J ) 4.8 Hz), 7.34 (d,
4H, J ) 8.1 Hz), 7.79 (d, 4H, J ) 8.1 Hz). 13C NMR (in CDCl3):
21.63, 68.69, 69.22, 70.65, 127.93, 129.86, 132.87, 144.88.
Triphenyl(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethyl)phos-
phonium Tosylate (TPP-PEG2-OTs). To a hot toluene solution
(5 mL) containing TsO-PEG2-OTs (459.0 mg, 1.0 mmol) was added
dropwise triphenylphosphine (262.0 mg, 1.0 mmol) in toluene (10
mL). The resulting mixture was then refluxed for 18 h to get a
light yellow solution. Volatiles were removed under vacuum. The
residue was dissolved in 50% DMF/H2O and was subject to HPLC
purification (method 1). The fractions at ∼15 min were collected,
combined, and lyophilized to give a while solid. The yield was
103.0 mg (15%). 1H NMR (in CDCl3): 2.25 (s, 3H, CH3), 2.39 (s,
3H, CH3), 3.02–3.95 (m, 12H), 7.00 (d, 2H, J ) 7.8 Hz), 7.29 (d,
2H, J ) 8.1 Hz), 7.53–7.78 (m, 19H). 31P NMR: 26.15 (s). ESI-
MS: m/z ) 549.12 for [M + H]+ (calcd 549 for [C31H34O5PS]+).
Triphenyl(2-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetra-
azacyclododecan-1-yl)ethoxy)ethoxy)ethyl)phosphonium Acetate
(DO3A-PEG2-TPP). To a solution containing TPP-PEG2-OTs
(103.0 mg, 0.14 mmol) and DO3A(OBu-t)3 (73.5 mg, 0.14 mmol)
in anhydrous DMF (5 mL) was added triethylamine (145.0 mg,
1.43 mmol). The solution was kept at ∼60 °C for 3 h. After removal
of volatiles, the residue was dissolved in 12 N HCl (3 mL). The
resulting solution was stirred at room temperature for 2 h. Volatiles
were removed under reduced pressure. The residue was dissolved
in 50% DMF/H2O (3 mL) and purified by HPLC. Fractions at ∼14.3
min were collected. The collected fractions were combined and
lyophilized to give a pale yellow solid (31 mg, 28%). 1H NMR (in
D2O): 2.72–3.79 (m, 34H), 7.47–7.72 (m, 15H). 31P NMR: 26.65
(s). ESI-MS: m/z ) 723.20 for [M + H]+ (calcd 723 for
(4,4′-(4,10-Bis(carboxymethyl)-1,4,7,10-tetraazacyclododecane-
1,7-diyl)bis(methylene)bis(4,1-phenylene))bis(methylene)bis(triph-
enylphosphonium) Bisacetate (DO2A-(xy-TPP)2). DO2A-(xy-
TPP)2 was isolated from the same reaction mixture as for DO2A-
xy-TPP by collecting fractions at ∼17.0 min. The collected fractions
were combined and lyophilized to give a white powder. The yield
2+
for DO2A-(xy-TPP)2 was 38.3 mg (37.6%). The retention time
was ∼17.0 min with an HPLC purity of >95%. 1H NMR (in D2O):
2.75–3.23 (m, 20H); 4.20 (s, 4H, NCH2); 4.67 (d, 4H, PCH2, JPH
) 13 Hz); 6.91 (d, 4H, C6H4); 7.18 (d, 4H, C6H4); 7.37–7.76 (m,
30H, C6H5). ESI-MS: m/z ) 1018.79 for [M + H]+ (1019 calcd
for C64H68N4O6P2); m/z ) 1039.92 for [M + Na]+ (1041 calcd for
C64H67N4O6P2Na). Anal. Calcd for C64H68N4O4P2 ·2CH3CO2 ·6H2O:
C, 65.58; H, 6.96; N, 4.50. Found: C, 65.03; H, 6.53; N, 4.66.

2974 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Kim et al.
4-Aminomethylbenzyltris(phenyl)phosphonium Bromide (TPP-
xy-NH2). Phthalimide potassium (204.0 mg, 1.1 mmol) and TPP-
xy-Br (526.0 mg, 1.0 mmol) were mixed in acetonitrile (20 mL).
The suspension was heated to reflux for 22 h. After filtration,
volatiles in the filtrate were removed under vacuum. The yellow
residue was dissolved in dichloromethane (100 mL) and washed
by water (30 mL). The organic phase was separated and dried over
anhydrous MgSO4. After removal of volatiles, the residue was
recrystallized in the MeOH/ether mixture to give a light yellow
solid (260.0 mg, 44%). 1H NMR (in CDCl3): 4.75 (s, 2H, CH2N),
5.38 (d, 2H, PCH2, JPH ) 14.4 Hz), 7.05 (dd, 2H, J ) 8.1, 2.1
Hz), 7.14 (d, 2H, J ) 8.1 Hz), 7.58–7.88 (m, 19H). The intermediate
product (260.0 mg, 0.44 mmol) and 0.53 mL of aqueous hydrazine
(55%, 8.8 mmol) were added into ethanol (20 mL). The resulting
solution was refluxed for 20 h. Volatiles were removed under
vacuum to get a light yellow solid, which was then dissolved in
dichloromethane. Water was used to extract the product, and the
water phase was dried under vacuum to give the expected product,
TPP-xy-NH2, as a yellow oil (120 mg, 59.0%). 1H NMR (in CDCl3):
3.79 (s, 2H, CH2N), 4.63 (br s, 2H, NH2), 5.20 (d, 2H, PCH2, JPH
) 14.4 Hz), 6.94 (dd, 2H, J ) 7.8, 1.8 Hz), 7.06 (d, 2H, J ) 7.8
Hz), 7.56–7.68 (m, 15H).
Triphenyl(4-((3-(4-((1,4,7-tris(carboxymethyl)-1,4,7-triazonan-
2-yl)methyl)phenyl)thioureido)methyl)benzyl)phosphonium Ac-
etate (NOTA-Bn-xy-TPP). TPP-xy-NH2 (5.0 mg, 0.01 mmol) and
p-Bn-SCN-NOTA (4.9 mg, 0.01 mmol) were dissolved in 2 mL of
the 50% DMF/H2O mixture. The pH was adjusted to 8.5 with 1 N
NaOH. The mixture was stirred at room temperature overnight.
After purification (method 2), the collected fractions at ∼15.1 min
were combined and lyophilized to give a white powder (4.9 mg,
50.9%). 1H NMR (in D2O): 2.42–3.82 (m, 19H), 4.42 (s, 2H,
CH2NH), 4.51 (d, 2H, PCH2, JPH ) 13.8 Hz), 6.71 (m, 2H),
6.82–7.16 (m, 6 H), 7.37–7.52 (m, 12H), 7.61(m, 3H). 31P NMR:
25.80 (s). ESI-MS: m/z ) 832.29 for [M]+ (calcd 832 for
[C46H51N5O6PS]+). Anal. Calcd for C46H51N5O6PS·CH3CO2 ·H2O:
C, 63.35; H, 6.20; N, 7.70. Found: C, 63.28; H, 6.04; N, 7.86.
Cu(DO2A-xy-TPP)OAc. DO2A-xy-TPP (45.7 mg, 0.07 mmol)
and Cu(OAc)2 ·2H2O (20.0 mg, 0.10 mmol) were dissolved in 5
mL of H2O and 0.5 mL of 0.5 M NH4OAc buffer (pH ) 6.0). The
solution was heated at 100 °C for 45 min. After cooling to room
temperature, diethyl ether (25 mL) was added to the filtrate above
slowly. The precipitate was separated and dried under vacuum
overnight before being submitted for elemental analysis. The yield
was 33.6 mg (67%). IR (cm-1, KBr pellet): 1613 (s, νCdO), 1715
(s, νCdO), and 3412 (bs, νO-H). ESI-MS (positive mode): m/z )
715.30 for [M + H]+ (716 calcd for [C38H44CuN4O4P]+). Anal.
Calcd for C38H43CuN4O4 ·CH3CO2 ·2H2O: C, 59.69; H, 6.12; N,
7.24. Found: C, 59.97; H, 6.37; N, 7.00.
Partition Coefficient. All 64Cu radiotracers were purified by
HPLC. HPLC purification is needed to eliminate potential interfer-
ence from other radioimpurities. After complete removal of volatiles
in the HPLC mobile phase, the residue was dissolved in a mixture
of 3 mL of saline and 3 mL of n-octanol in a round-bottom flask.
The mixture was vigorously stirred for 20 min at room temperature
and was then transferred to a 15 mL Falcon conical tube. The tube
was centrifuged at 12 500 rpm for 5 min. Samples in triplets from
n-octanol and aqueous layers were obtained and were counted on
a γ-counter (Perkin-Elmer Wizard 1480). The log P value was
reported as an average of the data obtained in three independent
measurements.
Animal Model and Biodistribution Protocol. Biodistribution
studies were performed using athymic nude mice bearing U87MG
human glioma xenografts in compliance with the NIH animal
experiment guidelines (Principles of Laboratory Animal Care, NIH
Publication No. 86-23, revised 1985). The animal protocol has been
approved by Purdue University Animal Care and Use Committee
(PACUC). Female athymic nu/nu mice were purchased from Harlan
(Charles River, MA) at 4–5 weeks of age. The mice were
orthotopically implanted with 5 × 106 U87MG human glioma cells
into the mammary fat pad. Four weeks after inoculation, the tumor
size was in the range 0.3–0.8 g, and animals were used for
biodistribution and imaging studies. Sixteen tumor-bearing mice
(20–25 g) were randomly divided into four groups, each of which
had four animals. The HPLC-purified 64Cu radiotracer (∼2.5 µCi
dissolved in 0.1 mL of saline) was administered to each animal
via tail vein. Four animals were euthanized by sodium pentobarbital
overdose (100–200 mg/kg), exsanguinations, and opening of the
thoracic cavity at 5, 30, 60, and 120 min postinjection (p.i.). Blood
samples were withdrawn from the heart through a syringe. Organs
were excised, washed with saline, dried with absorbent tissue,
weighed, and counted on a γ-counter (Perkin-Elmer Wizard 1480).
Organs of interest included tumor, brain, heart, spleen, lungs, liver,
kidneys, muscle, and intestine. The organ uptake was calculated
as a percentage of the injected dose per gram of organ tissue (%
ID/g).
Calibration of microPET. Scanner activity calibration was
performed to map between microPET image units and units of
activity concentration. A preweighed 50 mL centrifuge tube was
filled with a solution containing 64CuCl2 (∼9.3 MBq as determined
by the dose calibrator), which was used to simulate the whole body
of the mouse. This tube was weighed, centered in the scanner
aperture, and imaged for a 30 min static image. From the sample
weight and assuming a density of 1 g/mL, the activity concentration
in the bottle was calculated in units of µCi/mL. Eight planes were
acquired in the coronal section. A rectangular region of interest
(ROI) (counts/pixel/s) was drawn on the middle of 8 coronal planes.
Using these data, a calibration factor was obtained by dividing the
known radioactivity in the cylinder (µCi/mL) by the image ROI.
This calibration factor was determined periodically and did not vary
significantly with time.
MicroPET Imaging. MicroPET imaging of the tumor-bearing
mice was performed using a microPET R4 rodent model scanner
(Concorde Microsystems, Knoxville, TN). The U87MG tumor-
bearing mice (n ) 3) were imaged in the prone position in the
microPET scanner. The tumor-bearing mice were injected with
∼250 µCi of 64Cu(DO3A-bu-TPP) via the tail vein, then anesthe-
tized with 2% isoflurane and placed near the center of the FOV,
where the highest resolution and sensitivity are obtained. Multiple
static scans were obtained at 0.5, 1.0, 2, and 20 h p.i. The images
were reconstructed by a two-dimensional ordered subsets expecta-
tion maximum (OSEM) algorithm. No correction was necessary
for attenuation or scatter. At each microPET scan, the ROIs were
drawn over the tumor and major organs on decay-corrected whole-
body coronal images. The average radioactivity concentration within
the tumor or an organ was obtained from mean pixel values within
the multiple ROI volume, which were converted to counts/mL/
min by using the calibration constant C. Assuming a tissue density
of 1 g/mL, the ROIs were converted to counts/g/min and were then
64Cu-Labeling and Dose Preparation. To a 5 mL vial were
added 50 µg of the TPP conjugate dissolved in 0.5 mL of 0.1 M
NaOAc buffer (pH ) 6.9) and 0.12 mL of 64CuCl2 solution (1.0–2.0
mCi) in 0.05 N HCl. The final pH in the reaction mixture was
5.0–5.5. The reaction mixture was then heated at 100 °C for 30
min. After cooling to room temperature, a sample of resulting
solution was analyzed by radio-HPLC (method 2). The radiochemi-
cal purity (RCP) was >90% for all six 64Cu radiotracers with the
specific activity of ∼50 Ci/mmol. All 64Cu radiotracers were
purified by HPLC before being used for biodistribution studies.
The dose solution was prepared by dissolving the HPLC-purified
radiotracer in saline to 10–25 µCi/mL. For the imaging study,
64Cu(DO3A-bu-TPP) was first prepared and the resulting reaction
mixture was used without purification. The mixture was diluted
with saline to a concentration of ∼5 mCi/mL.
Solution Stability. The solution stability of 64Cu(DO3A-bu-
TPP), 64Cu(DO2A-xy-TPP)+, and 64Cu(NOTA-Bn-xy-TPP) were
studied using the EDTA challenge experiment. The 64Cu radiotrac-
ers were first prepared and then purified by HPLC. After removal
of volatiles in the HPLC mobile phase, they were dissolved in 25
mM phosphate buffer (pH ) 7.4) containing EDTA (1 mg/mL) to
1 mCi/mL. Samples of the resulting solution were analyzed by
radio-HPLC at 0, 1, 2, 4, and 12 h post-purification.

64Cu-Labeled TPP Cations for Tumor Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2975
Chart 1. Synthesis of DO3A-Conjugated TPP and TPA Cations
divided by the total administered activity to obtain an imaging ROI-
derived percentage administered activity per gram of tissue (%
ID/g).
DO3A-PEG2-TPP were prepared according to Chart 1. They
were designed to examine the linker effects on organ uptake
and excretion kinetics of their 64Cu complexes. DO2A-xy-TPP
and DO2A-(xy-TPP)2 were isolated from the same reaction
(Chart 2) between DO2A(t-Bu)2 and TPP-xy-Br in 1:1 molar
ratio in the presence of triethylamine. DO2A-xy-TPP was
designed to explore the impact of molecular charge of 64Cu
chelates (64Cu-DO3A versus 64Cu-DO2A) on biological proper-
ties of 64Cu radiotracers. DO2A-(xy-TPP)2 was designed to test
if the extra TPP moiety will result in any added benefits with
respect to the tumor uptake and T/B ratios. NOTA-Bn-xy-TPP
was prepared according to Chart 3 by reacting p-SCN-Bn-NOTA
with TPP-xy-NH2 in a mixture of DMF/water (50:50 ) v/v)
under slightly basic conditions (pH ) 8.5). It was designed to
examine the impact of BFCs (DO3A versus NOTA-Bn) on
biodistribution characteristics of the 64Cu radiotracer. All six
newly synthesized TPP conjugates were purified by HPLC
(method 1) and had an HPLC purity of >95% before being
used for 64Cu-labeling. After lyophilization, they were all
obtained as their acetate salts since the HPLC mobile phases
contain 1% acetic acid. They all have been characterized by
NMR (1H, 13C, and 31P), ESI-MS, and elemental analysis (C,
H, and N).
Metabolism. The metabolic stability of 64Cu(DO2A-xy-TPP)+,
64Cu(DO3A-PEG2-TPP), and 64Cu(NOTA-Bn-xy-TPP) was evalu-
ated in normal athymic nude mice. Two animals were used for
each radiotracer. Each mouse was administered with the 64Cu
radiotracer at a dose of 100 µCi in 0.2 mL of saline via tail vein.
The urine samples were collected at 30 and 120 min p.i. by manual
void and were mixed with equal volume of acetonitrile. The mixture
was centrifuged at 8000 rpm. The supernatant was collected and
filtered through a 0.20 µm Millex-LG syringe-driven filter unit to
remove the precipitate and large proteins. The filtrate was analyzed
by radio-HPLC (method 2). The feces samples were collected at
120 min p.i. and were suspended in the 50% acetonitrile aqueous
solution. The mixture was vortexed for 5–10 min. After centrifuging
at 8000 rpm for 5 min, the supernatant was collected and passed
through a 0.20 µm Millex-LG syringe-driven filter unit to remove
the precipitate or particles. The filtrate was then analyzed by radio-
HPLC (method 2).
Stability of 64Cu(DO3A-PEG2-TPP) in Liver. The liver tissue
was harvested at 120 min p.i. from the mouse administered with
64Cu(DO3A-PEG2-TPP) (∼100 µCi), counted in a γ-counter for
the total liver radioactivity, cut into small pieces, and then
homogenized. The homogenate was mixed with 2 mL of saline.
After centrifuging at 8000 rpm for 5 min, the supernatant was
collected and counted with a γ-counter to determine the radioactivity
recovery. After filtration through a 0.20 µm Millex-LG syringe-
driven filter unit to remove the precipitate or particles, the filtrate
was then analyzed by radio-HPLC (method 2).
Data and Statistical Analysis. The biodistribution data and target-
to-background (T/B) ratios are reported as an average plus the standard
variation based on results from four tumor-bearing mice at each time
point. Comparison between two different radiotracers was made using
the two-way ANOVA test (GraphPad Prim 5.0, San Diego, CA). The
level of significance was set at p < 0.05.
Synthesis and Characterization of Cu(DO2A-xy-TP-
P)(OAc). Cu(DO2A-xy-TPP)+ is used as a model compound
for structural characterization of 64Cu(DO2A-xy-TPP)+. It was
prepared by reacting DO2A-xy-TPP with 1 equiv of Cu(II)
acetate in 0.5 M ammonium acetate buffer (pH ) 6), was
isolated as its acetate salt, and has been characterized by IR,
ESI-MS, and elemental analysis. The IR spectrum of Cu(DO2A-
xy-TPP)(OAc) shows a broad band at 3432 cm-1 due to the
crystallization water, a strong band at 1721 cm-1 due to the
acetate counterion, and a strong band at 1637 cm-1 from two
coordinated carboxylate groups. The ESI-MS spectrum of
Cu(DO2A-xy-TPP)+ displays a molecular ion at m/z ) 715.30
for [M + H]+. Attempts to grow crystals for structural
Results
Synthesis of TPP Conjugates. Synthesis of TPP conjugates
was straightforward. DO3A-bu-TPP, DO3A-xy-3mTPP, and

2976 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Kim et al.
Chart 2
Chart 3
Table 1. HPLC Retention Time and log P Values for the 64Cu-Labeled
TPP Cations
determination of Cu(DO2A-xy-TPP)(OAc) by X-ray crystal-
lography were unsuccessful. Unlike DO3A, which forms anionic
Cu-DO3A chelate,44 DO2A-xy-TPP has only two acetate
chelating arms. Coordination of these two carboxylate-O atoms
will result in formation of a neutral Cu-DO2A chelate. Thus,
Cu(DO2A-xy-TPP)+ is cationic with a positive charge on TPP.
On the basis of elemental analysis and IR spectroscopic data,
we believe that Cu(DO2A-xy-TPP)+ exists as its acetate salt
form in the solid state.
retention
compound
RCP (%)
time (min)
log P value
64Cu(DO3A-xy-TPP)
>95
>98
>95
>96
>97
>97
>95
>98
15.6
20.9
15.5
18.2
17.5
18.2
22.0
17.4
-2.67 ( 0.21
-2.02 ( 0.01
-2.98 ( 0.36
-2.81 ( 0.07
-1.89 ( 0.02
-2.51 ( 0.18
-1.23 ( 0.01
-1.90 ( 0.15
64Cu(DO3A-xy-mTPP)
64Cu(DO3A-bu-TPP)
64Cu(DO3A-PEG2-TPP)
64Cu(DO3A-xy-3mTPP)
64Cu(DO2A-xy-TPP)+
64Cu(DO2A-(xy-TPP)2)2+
64Cu(NOTA-Bn-xy-TPP)
Radiochemistry. All 64Cu radiotracers were prepared by
reacting 64CuCl2 with the TPP conjugate in 0.1 M NaOAc buffer
(pH ) 6.9) at 100 °C for 30 min and were analyzed using the
same reversed-phase HPLC method (method 2). The RCP was
>90%, with the specific activity being >50 Ci/mmol for all
new 64Cu radiotracers. Their partition coefficients were deter-
mined in a 50%:50% (v/v) mixture of n-octanol and 25 mM
phosphate buffer (pH ) 7.4). Their calculated log P values and
HPLC retention times are listed in Table 1. As the number of
methoxy groups increases, the lipophilicity of 64Cu radiotracer
was significantly increased from 64Cu(DO3A-xy-TPP) (log P
) –2.67 ( 0.21)39 to 64Cu(DO3A-xy-mTPP) (log P ) –2.02
( 0.05),39 and 64Cu(DO3A-xy-3mTPP) (log P ) –1.89 ( 0.02).
Replacing xylene (xy) with a butylene (bu) linker resulted in a
reduction of lipophilicity for the 64Cu radiotracer: 64Cu(DO3A-
bu-TPP) (log P ) –2.98 ( 0.36). Substitution of xylene with
the PEG2 linker did not change the lipophilicity of 64Cu(DO3A-
PEG2-TPP) (log P ) –2.81 ( 0.07) vs 64Cu(DO3A-xy-TPP)
(log P ) –2.67 ( 0.21). 64Cu(NOTA-Bn-xy-TPP) (log P )
–1.90 ( 0.15) is more lipophilic than 64Cu(DO3A-xy-TPP), due
to the presence of an extra aromatic benzoyl group. Since
NOTA-Bn-xy-TPP has the same number of acetate chelating
arms as DO3A-xy-TPP, it is expected to form the 64Cu chelate,
in which 64Cu is coordinated either by N3O3 donor atoms in
the distorted octahedral coordination geometry or by N3O2 donor
atoms in the square-pyrimidal coordination sphere with one
carboxylate-O atom being uncoordinated and deprotonated. It
is reasonable to believe that 64Cu(NOTA-Bn-xy-TPP) exists in
solution as its Zwitterion form.

64Cu-Labeled TPP Cations for Tumor Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2977
Table 2. Solution Stability Data for the HPLC-Purified 64Cu-Labeled
TPP Cations in the Presence of Excess EDTA (1 mg/mL in 25 mM
phosphate buffer, pH ) 7.5)
Comparison with 99mTc-Sestamibi. Figures 2 and 3 compare
the organ uptake and T/B ratios between the 64Cu radiotracers
and 99mTc-sestamibi in the tumor, heart, liver, and muscle. The
biodistribution data and T/B ratios for 99mTc-sestamibi were
obtained from our previous report.39 99mTc-Sestamibi has been
clinically used for tumor imaging and for monitoring the MDR
transport function in tumors of different origin.17–29 This
comparison allows us to demonstrate that the 64Cu-labeled TPP
cations described in this study have significant advantages over
99mTc-sestamibi with respect to their organ uptake and T/B
ratios.
The most striking differences between 64Cu-labeled TPP
cations and 99mTc-sestamibi are their uptake in the heart and
muscle (Figure 2) and their tumor/heart, tumor/lung, and tumor/
muscle ratios (Figure 3). For example, the heart uptake was
<1% ID/g for all six 64Cu radiotracers at >30 min p.i., while
the heart uptake of 99mTc-sestamibi was 19.22 ( 7.62 at 5 min
p.i. and 19.19 ( 5.32% ID/g at 120 min p.i.39 The tumor/heart
ratios of 64Cu(DO3A-bu-TPP), 64Cu(DO3A-PEG2-TPP), and
64Cu(DO2A-xy-TPP)+ were steadily increased from ∼1 at 5
min p.i. to ∼4 at 120 min p.i. 64Cu(DO2A-xy-TPP)+ and 99mTc-
sestamibi had very similar tumor uptake over the 2 h period
(Figure 2), but the tumor/heart ratio of 64Cu(DO2A-xy-TPP)+
at 120 min p.i. was about 40-fold better than that of 99mTc-
sestamibi. The tumor/heart ratios of 64Cu(NOA-Bn-xy-TPP) and
64Cu(DO3A-(xy-TPP)2)2+ were very low due to their low tumor
uptake. 64Cu(DO3A-xy-3mTPP) had the best tumor/heart ratio
at >60 min p.i. The muscle uptake of the 64Cu-labeled TPP
cations was almost undetectable at >30 min p.i. In contrast,
99mTc-sestamibi has a high muscle uptake (4.84 ( 1.22 and
5.45 ( 1.24% ID/g at 5 and 120 min p.i., respectively). The
lung uptake of the 64Cu radiotracers was also significantly lower
(p < 0.01) than that of 99mTc-sestamibi at 5–60 min p.i. (Tables
SI-VI), and their tumor/lung ratios were better than those of
time post-purification
0.5 h
1 h
2 h
4 h
12 h
64Cu(DO3A-xy-TPP)
64Cu(DO2A-xy-TPP)+
64Cu(NOTA-Bn-xy-TPP)
99
99
98
99
98
98
98
98
99
98
97
98
97
95
97
Solution Stability. The EDTA challenge experiment was used
to study solution stability of 64Cu(DO2A-xy-TPP)+, 64Cu(DO3A-
PEG2-TPP), and 64Cu(NOTA-Bn-xy-TPP). These three 64Cu
radiotracers were selected because they represent three different
types of BFCs for 64Cu chelation. The purpose of these studies
was to demonstrate that the 64Cu radiotracer remains intact
before being injected into the tumor-bearing mice. Table 2
summarizes the solution stability data for the HPLC-purified
64Cu(DO2A-xy-TPP)+, 64Cu(DO3A-PEG2-TPP), and 64Cu(NO-
TA-Bn-xy-TPP) in the presence of excess EDTA (3 mM, pH
) 7.4). It is quite clear that all three 64Cu radiotracers remain
stable for >12 h in the presence of excess EDTA, which is
completely consistent with their metabolic stability during
excretion from normal mice.
Biodistribution Data. Biodistribution characteristics of all
six new 64Cu radiotracers were evaluated in the athymic nude
mice bearing subcutaneous U87MG human glioma xenografts.
Detailed biodistribution data and T/B ratios for these six 64Cu
radiotracers are listed in Tables SI-SVI. The organ uptake is
expressed as an average % ID/g plus the standard variation based
on results from four tumor-bearing mice (n ) 4) at each time
point. The main objective of these studies was to explore the
impact of TPP moieties, linkers, bifunctional chelators, and
overall molecular charge on biodistribution characteristics and
excretion kinetics of the 64Cu-labeled TPP cations.
Figure 2. Direct comparison of organ (tumor, heart, liver, and muscle) uptake between the 64Cu-labeled TPP cations and 99mTc-sestamibi in
athymic nude mice (n ) 4) bearing U87MG human glioma xenografts. The most striking difference between the 64Cu-labeled TPP cations and
99mTc-sestamibi is their uptake in the heart and muscle.

2978 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Kim et al.
Impact of 64Cu Chelate. Because of its d9 electron config-
uration, Cu(II) complexes are often kinetically labile with respect
to dissociation. The BFCs for copper radionuclides have been
focused on macrocyclic chelators that form Cu(II) complexes
with very high thermodynamic stability and kinetic inertness.45–50
Since the 64Cu chelate is a major part of the 64Cu radiotracer,
we performed biodistribution studies on 64Cu(DO2A-xy-TPP)+,
64Cu(DO2A-(xy-TPP)2)2+, and 64Cu(NOTA-Bn-xy-TPP) in
athymic nude mice bearing U87MG human glioma xenografts.
Figure 5 shows the impact of the 64Cu chelate on both tumor
uptake and T/B ratios of 64Cu radiotracers. Biodistribution data
and T/B ratios of 64Cu(DO3A-xy-TPP) were obtained from our
previous report.39 64Cu(DO3A-xy-TPP) and 64Cu(DO2A-xy-
TPP)+ have very similar lipophilicity (log P ) –2.67 ( 0.21
and –2.51 ( 0.18, respectively) in spite of their difference in
molecular charge. Their tumor uptake and tumor/heart and
tumor/lung ratios compared well over the 2 h period. However,
the liver uptake of 64Cu(DO2A-xy-TPP)+ was significantly
lower (p < 0.01) and its tumor/liver ratios were much better
than that 64Cu(DO3A-xy-TPP) at all four time points. Because
of the extra xy-TPP moiety, 64Cu(DO2A-(xy-TPP)2)2+ is most
likely dicationic, and its lipophilicity (log P ) –1.23 ( 0.01)
is higher than that of 64Cu(DO2A-xy-TPP)+ (log P ) –2.51 (
0.18). As a result, 64Cu(DO3A-(xy-TPP)2)2+ has a much higher
liver uptake and lower tumor uptake than 64Cu(DO2A-xy-TPP)+
and 64Cu(DO3A-xy-TPP). 64Cu(NOTA-Bn-xy-TPP) has the
same overall molecular charge as 64Cu(DO3A-xy-TPP), but its
tumor uptake and tumor/heart ratios were significantly lower
(p < 0.01) than those of 64Cu(DO3A-xy-TPP) at all four time
points. These data clearly demonstrated that the 64Cu chelates
have a significant impact on the radiotracer tumor uptake and
T/B ratios.
Linker Effects. Figure 6 illustrates the linker effects on tumor
uptake and tumor/heart ratios of 64Cu radiotracers. The tumor
uptake of 64Cu(DO3A-bu-TPP) (1.29 ( 0.58% ID/g) was
significantly lower (p < 0.01) than that of 64Cu(DO3A-xy-TPP)
(2.51 ( 0.38% ID/g) at 120 min p.i., but their tumor/heart ratios
were almost identical (4.80 ( 1.04 and 4.25 ( 0.23, respec-
tively). Substitution of xylene with the PEG2 linker also led to
a significant reduction of the radiotracer in the liver and higher
tumor/liver ratios, even though 64Cu(DO3A-PEG2-TPP) and
64Cu(DO3A-xy-TPP) have similar lipophilicity (Table 1).
Figure 3. Direct comparison of tumor-to-background ratios between
the 64Cu-labeled TPP cations and 99mTc-sestamibi in athymic nude mice
bearing U87MG human glioma xenografts. The most significant
difference between the 64Cu-labeled TPP cations and 99mTc-sestamibi
is their tumor/heart, tumor/lung, and tumor/muscle ratios.
PET Imaging. We performed a microPET imaging study on
64Cu(DO3A-bu-TPP) using athymic nude mice (n ) 3) bearing
U87MG human glioma xenografts. Figure 7 illustrates the
coronal microPET images (A) and the activity accumulation
quantification (B) in several organs of the tumor-bearing mice
administered with ∼250 mCi of 64Cu(DO3A-bu-TPP). The
tumor was clearly visualized as early as 30 min p.i. with very
high tumor-to-background contrast. No significant radioactivity
accumulation was detected in the brain. The radioactivity
accumulation in the heart and muscle was also very low. After
normalization, the tumor uptake of 64Cu(DO3A-bu-TPP) is 1.29
( 0.57%, 1.22 ( 0.73%, 1.31 ( 0.55%, and 1.14 ( 0.56%
ID/g at 3 min, 1.4 h, 3.9 h, and 20.5 h p.i., respectively. These
data are completely consistent with those obtained in the ex
vivo biodistribution study (Table SI).
99mTc-sestamibi (Figure 3) over the 2 h study period. Among
the six new 64Cu radiotracers, 64Cu(DO2A-xy-TPP)+ is probably
the best considering its uptake in the tumor and liver.
Impact of TPP Cations. Figure 4 shows the impact of TPP
cations on the radiotracer tumor uptake and T/B ratios. Bio-
distribution data and T/B ratios for 64Cu(DO3A-xy-TPP) and
64Cu(DO3A-xy-mTPP) were obtained from our previous report.39
64Cu(DO3A-xy-mTPP) has the liver uptake significantly lower
(p < 0.01) than that of 64Cu(DO3A-xy-TPP). Even though the
tumor uptake of 64Cu(DO3A-xy-3mTPP) is the lowest, its
tumor/heart ratio is the best at >60 min p.i. The addition of
extra methoxy groups on the TPP improves the radiotracer
clearance from the liver and lungs, but it also resulted in a
significant washout from the tumor. As a result, the tumor/lung
ratios of 64Cu(DO3A-xy-TPP), 64Cu(DO3A-xy-mTPP), and
64Cu(DO3A-xy-mTPP) were well within the experimental error
(Figure 4). It seems that mTPP is better than TPP and 3mTPP
as the mitochondrion-targeting molecules considering both the
tumor uptake and tumor/liver ratios of their corresponding 64Cu
radiotracers.
Metabolic Properties. Metabolism studies were performed
using normal athymic nude mice on 64Cu(DO2A-xy-TPP)+,
64Cu(DO3A-PEG2-TPP), and 64Cu(NOTA-Bn-xy-TPP). These
three 64Cu radiotracers were selected because of BFCs for 64Cu
chelation. Each mouse was administered with ∼100 µCi of the
64Cu radiotracer. Since they were excreted from renal and
hepatobiliary routes, we collected both urine and feces samples

64Cu-Labeled TPP Cations for Tumor Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2979
Figure 4. Impact of TPP moieties on tumor uptake and tumor/heart ratios of the 64Cu-labeled TPP cations in nude mice bearing U87MG human
glioma xenografts (n ) 4).
and analyzed them by a reversed-phase HPLC method to
determine if they are able to retain their chemical integrity.
Figure 8 illustrates radio-HPLC chromatograms of 64Cu(DO3A-
PEG2-TPP) (left) and 64Cu(DO3A-Bn-xy-TPP) (right) in saline
before injection (A), in the urine at 30 min p.i. (B), in the urine
at 120 min p.i. (C), and in the feces at 120 min p.i. (D). There is
no significant metabolite detectable in both urine and feces samples
from the mice administered with either 64Cu(DO3A-PEG2-TPP)
or 64Cu(NOTA-Bn-xy-TPP). Similar metabolic stability was also
observed for 64Cu(DO2A-xy-TPP)+ (Figure SI).
for their biodistribution characteristics and excretion kinetics
and found that 64Cu(DO3A-bu-TPP), 64Cu(DO2A-xy-TPP)+,
and 64Cu(DO3A-PEG2-TPP) have a tumor uptake comparable
to that of 99mTc-sestamibi (Figure 2), but their T/B ratios are
much better than those of 99mTc-sestamibi at all four time points
(Figure 3). For example, the tumor/heart ratio of 64Cu(DO2A-
xy-TPP)+ is 1.13 ( 0.21 at 5 min p.i. and increases to 3.39 (
0.23 at 120 min p.i., while the tumor/heart ratio of 99mTc-
sestamibi is <0.2 during the 2 h period. The tumor/heart, tumor/
lung, and tumor/muscle ratios of 64Cu(DO2A-xy-TPP)+ are 25,
20, and 160 times, respectively, better than those 99mTc-
sestamibi at 120 min p.i. The tumor/heart and tumor/liver ratios
of 64Cu(DO3A-bu-TPP) are more than 37- and 235-fold higher
than those 99mTc-sestamibi.
Transchelation of 64Cu(DO3A-PEG2-TPP) in the Liver.
We also examined the stability of 64Cu(DO3A-PEG2-TPP) in
the liver. About 50% of total liver radioactivity was recovered
in the homogenate. The supernatant was analyzed using the
reversed-phase HPLC method (method 2). Figure SII shows the
radio-HPLC chromatogram of the liver homogenate. There are
two radiometric peaks at 5 and 10 min with no intact
64Cu(DO3A-PEG2-TPP), suggesting that it underwent extensive
tranchelation in the liver during the 2 h study period.
While the enhanced mitochondrial potential provides the
electrochemical driving force for cationic radiotracers to localize
in the energized mitochondria of tumor cells, its lipophilicity
is one of the main factors that control the diffusion kinetics for
radiotracers to cross both plasma and mitochondrial membranes.
For more lipophilic radiotracers, such as 99mTc-sestamibi (log
P ) 1.09 ( 0.15), their membrane diffusion kinetics are fast
and they can localize quickly in the tumor and mitochondrion-
rich organs, such as the heart, liver, and kidneys, and their tumor
Discussion
In this study, we evaluated six new 64Cu-labeled TPP cations
using athymic nude mice bearing U87MG glioma xenografts

2980 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Kim et al.
Figure 5. Impact of BFCs and their 64Cu chelates on tumor uptake and tumor/heart ratios of the 64Cu-labeled TPP cations in nude mice (n ) 4)
bearing U87MG human glioma xenografts.
selectivity is low. In contrast, the 64Cu-labeled TPP cations, such
as 64Cu(DO2A-xy-TPP)+ (log P ) –2.51 ( 0.18), have very
low lipophilicity and slow membrane penetration kinetics, as
demonstrated in the in vitro assays using glioma cells and the
isolated mitochondria from glioma cells.39 As a result, most of
the 64Cu-labeled TPP cations tend to localize in the tumor, where
the mitochondrial potential is elevated as compared to normal
tissues such as the heart and muscle. It is not surprising that
the 64Cu radiotracers with log P values < –1.5 all have low
uptake in the heart and muscle with very high tumor/heart and
tumor/muscle selectivity.
In addition to lipophilicity, there are many other factors
contributing to the differences in biological properties of 64Cu
radiotracers. The 64Cu radiotracer described in this study
contains a TPP cation, a linker, and a 64Cu chelate (64Cu +
BFC), all of which affect its biodistribution and excretion
kinetics from noncancerous organs. For example, introduction
of methoxy groups on the TPP cation improves the radiotracer
clearance from liver and lungs (Figure 4), but it also results in
a significant washout from the tumor. Considering both tumor
uptake and T/B ratios of 64Cu radiotracers, mTPP is better than
TPP and 3mTPP as the mitochondrion-targeting molecule.
Replacing xylene with the butylene linker results in a significant
reduction of the tumor uptake, but the tumor/heart and tumor/
lung ratios of the 64Cu radiotracer remained relatively unchanged
(Figure 6). More importantly, the use of a butylene linker results
in a significant improvement of the tumor/liver ratio at 120 min
(Figure 6) even though 64Cu(DO3A-bu-TPP) has a lower tumor
uptake than 64Cu(DO3A-xy-TPP) at >30 min p.i. This is further
confirmed by the PET imaging study (Figure 7). 64Cu(DO3A-
xy-TPP) and 64Cu(DO2A-xy-TPP)+ have very similar lipophi-
licity (log P ) –2.67 ( 0.21 versus –2.51 ( 0.18) despite their
charge difference. Their tumor uptake and tumor/heart and
tumor/lung ratios compared well over the 2 h study period.
However, the positive charge of 64Cu(DO2A-xy-TPP)+ results
in a dramatic reduction in liver uptake, which leads to much
better tumor/liver ratios than those of 64Cu(DO3A-xy-TPP)
(Figure 5). It seems that an overall neutral molecular charge is
not required to achieve high tumor uptake and high T/B ratios.
Addition of an extra TPP moiety makes 64Cu(DO3A-(xy-
TPP)2)2+ more lipophilic than 64Cu(DO2A-xy-TPP)+. While the
dicationic character might explain the low tumor uptake of
64Cu(DO3A-(xy-TPP)2)2+ (Figure 5), its high liver uptake is
probably related to its high lipophilicity. From this point of view,
monoanionic BFCs should be avoided to ensure that the 64Cu
chelate has a neutral or negative charge in order to maintain
the high radiotracer tumor uptake.

64Cu-Labeled TPP Cations for Tumor Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2981
Figure 6. Impact of linkers on tumor uptake and tumor/heart ratios of 64Cu radiotracers in nude mice bearing U87MG human glioma xenografts
(n ) 4).
at >30 min p.i. Since 64Cu(NOTA-Bn-xy-TPP) (log P ) –1.81
( 0.02) has the same lipophilicity as 64Cu(DO3A-xy-3mTPP)
(log P ) –1.90 ( 0.15), it is reasonable to believe that its low
tumor uptake is probably caused by the 64Cu-NOTA-Bn chelate.
More detailed studies are needed to clarify the impact of the
coordination chemistry in the 64Cu chelate on tumor uptake and
T/B ratios of the 64Cu-labeled TPP cations.
Significant metabolic degradation has been detected in the
feces of athymic nude mice administered with 64Cu(DO3A-xy-
TPP), and the metabolic instability is probably caused by
cleavage of the TPP moiety.39 For 64Cu(DO3A-PEG2-TPP) and
64Cu(NOTA-Bn-xy-TPP), however, there was very little me-
tabolism (<5%) detected in urine and feces samples (Figure
8), suggesting that the linker and BFC have a significant impact
on the metabolic stability of 64Cu radiotracers and that the 64Cu
radiotracer is able to maintain its integrity during excretion.
Similar metabolic stability was observed for 64Cu(DO2A-xy-
Figure 7. Decay-corrected whole-body coronal microPET images (top)
and the radioactivity accumulation quantification (n ) 3, mean ( SD)
TPP)+ (Figure SI).
in selected organs (bottom) of athymic nude mice bearing U87MG
tumor administered with ∼250 µCi of 64Cu(DO3A-bu-TPP). Arrows
The radioactivity detected in the urine and feces samples
represents only the portion excreted from both renal and
hepatobiliary routes. The remaining radioactivity is still “trapped”
in organ tissues. We examined the stability of 64Cu(DO3A-
PEG2-TPP) in the liver and found that there was no intact
indicate the presence of glioma tumors.
Among all the 64Cu radiotracers evaluated in this animal
model, 64Cu(NOTA-Bn-xy-TPP) has the lowest tumor uptake

2982 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10
Kim et al.
Figure 8. Radio-HPLC chromatograms of 64Cu(DO3A-PEG2-TPP) (left) and 64Cu(DO3A-Bn-xy-TPP) (right) in saline before injection (A), in the
urine at 30 min p.i. (B), in the urine at 120 min p.i. (C), and in the feces at 120 min p.i. (D). Each mouse was administered with ∼100 µCi of the
64Cu radiotracer.
64Cu(DO3A-PEG2-TPP) in the liver homogenate (Figure SII).
While the identity of the two radiometric peaks at 5 and 10
min (Figure SII) is unclear at this moment, the HPLC
chromatographic pattern of 64Cu(DO3A-PEG2-TPP) in the liver
homogenate is very similar to that of 64Cu-labeled TETA-
octreotide and that of 64Cu-DOTA in the rat liver homogenate.51,52
On the basis of the literature data from the in vivo stability
studies of 64Cu complexes of tetraazamacrocycles,51–55 it is
reasonable to believe that the presence of two radiometric peaks
at 5 and 10 min is most likely caused by transchelation of 64Cu
from 64Cu(DO3A-PEG2-TPP) to superoxide dismutase (SOD),
which is a homodimeric enzyme abundant in the liver and
kidneys.56
The metabolism of the 64Cu-labeled biomolecules (anti-
bodies and small peptides) and 64Cu complexes of tet-
raazamacrocycles has been investigated extensively.51–64

64Cu-Labeled TPP Cations for Tumor Imaging
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 10 2983
Supporting Information Available: Detailed biodistribution
data and T/B ratios for six new 64Cu-labeled TPP cations are listed
in Tables SI-SVI. Figure SI shows the radio-HPLC chromatograms
of 64Cu(DO2A-xy-TPP)+ in saline before injection (A) and in the
urine at 30 min p.i. (B). Figure SII illustrates the radio-HPLC
chromatogram of 64Cu(DO3A-PEG2-TPP) in the liver homogenate
at 120 min p.i. These materials are available free of charge via the
Internet at http://pubs.acs.org.
These studies clearly demonstrate that the kinetic inertness
of the 64Cu chelate is of particular importance for the in vivo
stability of 64Cu radiopharmaceuticals,51 and their instability
is often caused by transchelation of 64Cu from the 64Cu-BFC
chelate to the proteins in high concentrations, namely, SOD
in the liver.51,56 For target-specific 64Cu radiotracers, the
tumor uptake is predominantly determined by receptor
binding affinity of the targeting biomolecule (antibody or
small peptide). Thus, the BFCs for 64Cu chelation should be
those that form the 64Cu-BFC chelates with high kinetic
inertness in order to minimize liver radioactivity accumula-
tion. For the 64Cu-labeled TPP cations as described in this
study, however, the BFC contributes greatly to not only the
radiotracer tumor uptake but also their excretion kinetics from
noncancerous organs, such as liver and lungs. The BFCs for
64Cu chelation should be those that result in the 64Cu
radiotracer with high tumor uptake and the best T/B,
particularly tumor/heart and tumor/liver, ratios. From this
point of view, both DO3A and DO2A are suitable as BFCs
for 64Cu-labeling of TPP cations. It must be noted that the
radioactivity “trapped” inside the liver due to transchelation
of 64Cu is only a small portion of the injected radioactivity.
Whenever possible, one has to consider the radioactivity
excreted from renal and hepatobiliary routes (urine and feces
samples), as well as the radioactivity “trapped” in various
organ tissues.
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Conclusion
In summary, we evaluated six new 64Cu-labeled TPP cations
for their biodistribution characteristics using athymic nude mice
bearing U87MG glioma xenografts. We found that most of the
new 64Cu radiotracers have significant advantages over 99mTc-
sestamibi with respect to their uptake in the heart and muscle.
We also found that the TPP moieties, linkers, BFCs, and
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groups on the TPP cation improves the radiotracer clearance
from the liver and lungs, but it also results in a significant
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the tumor/heart and tumor/lung ratios of the 64Cu radiotracer
remained relatively unchanged. Substitution of DO3A with
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Acknowledgment. The authors would like to thank Dr.
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