Revival of TE2A; A better chelate for Cu(II) ions than TETA?

Article abstract of DOI:10.1039/b925703a

A highly effective synthetic route for TE2A was developed and the ⁶⁴Cu-labeled TE2A complexes showed higher kinetic inertness and faster clearance than most commonly used TETA analogs.

Full text of DOI:10.1039/b925703a

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Revival of TE2A; a better chelate for Cu(II) ions than TETA?w
a
b
a
c
Darpan N. Pandya, Jung Young Kim, Jeong Chan Park, Hochun Lee,
b
d
a
b
Prasad B. Phapale, Wonjung Kwak, Tae Hyun Choi, Gi Jeong Cheon,
d
a
Young-Ran Yoon and Jeongsoo Yoo*
Received (in Cambridge, UK) 8th December 2009, Accepted 25th February 2010
First published as an Advance Article on the web 23rd March 2010
DOI: 10.1039/b925703a
A highly effective synthetic route for TE2A was developed and
6
have been carried out so far. When considering that the
64
Cu–DO2A complex shows faster clearance than its DOTA
4
the
Cu-labeled TE2A complexes showed higher kinetic
1
counterpart and that the TE2A derivative, CB-TE2A, forms
1
inertness and faster clearance than most commonly used TETA
analogs.
7
extremely stable Cu(II) complexes, this oversight becomes all
the more mysterious.
Interest in the synthesis, structures and properties of metal
complexes based on various types of N-functionalized
polyazamacrocycles has increased tremendously over the last
few decades, due to their potential application in the field of
medical imaging and therapy (e.g. MRI contrast agents,
To the best of our knowledge, there is only one report on the
synthesis of TE2A in the literature, which was published by
1
4,16
Parker et al.
This synthetic procedure suffers from several
serious disadvantages, such as the co-formation of various
regioisomers (cis and two different trans) with other substituted
by-products of the cyclam, tedious column purification, harsh
reaction conditions and, therefore, a very low overall yield
from the cyclam (16%). Even though twenty years have passed
since the first report of TE2A, the regioselective synthesis of
1–3
diagnostic radiopharmaceuticals and radioimmunotherapy).
In particular, cyclen and cyclam backbones containing
4
N-acetic acid pendant arms e.g. DOTA, DO2A, TETA,
5
6
7
cross-bridged-DO2A (CB-DO2A),
cross-bridged-TE2A
6,8
0
(CB-TE2A) and their derivatives have been intensively studied
1,8-N,N -difunctionalized cyclams (e.g. TE2A) over the
as potential bifunctional chelates (BFCs) for imaging and
therapy (Fig. 1). The metal complexes of these BFC agents
are found to show different levels of kinetic inertness and
thermodynamic stability when used in vivo, depending on
unwanted formation of other regioisomers and other substituted
products is still a great challenge for the synthetic chemist.
Herein we report a new facile synthetic protocol for
TE2A and demonstrates its usefulness as an effective chelate
9
–12
64
their ligand structure.
The overall charge of the metal
for Cu.
complexes, which is determined mostly by the number of
coordinated acetate pendent arms and the oxidation state of
the metal ions, also has a huge effect on the clearance pattern
First, we protected the cyclam using formaldehyde to give
the tricyclic bis-aminal 2 containing two six-membered rings.
The alkylation of compound 2 with t-butyl bromoacetate
afforded the selective dialkylation at the 1,8-position without
the formation of any cis-disubstituted product. The product 3
having two non-adjacent quaternary nitrogen atoms is
1
3
of the complexes and activity uptake in non-target organs.
The relationship between their structure and in vivo physical/
biological properties is fairly well documented in the case of
cyclen-based BFCs. However, very few studies have been
carried out on some of their cyclam-based counterparts, in
spite of the structural similarity between cyclen and cyclam.
To our surprise, we found that although TE2A was first
insoluble in CH
(see Scheme 1).
CN and was isolated by simple filtration
3
The cleavage of the two aminal bridges and ester hydrolysis
of 3 could be achieved in one step by treating it with
5 equivalent of KOH in an ethanol–water mixture (1 : 1) at
80 1C for 8 h, and the crude product was then run through
Dowex ion-exchange resin (1X8, OH form) to afford the
hydrochloride salt of TE2A in excellent yield. This new
synthetic method for TE2A is very straightforward and very
easy to handle. No tedious column purification is needed and
the overall yield attained from the cyclam is 86%, which is
much higher than the value of 16% achieved in the previously
1
4
synthesized in 1988, only a few metal complexes of TE2A
1
were further reported by the same group in the early 1990s.
5
No systematic studies on the use of TE2A as a potential BFC
a
Department of Molecular Medicine and Nuclear Medicine,
Kyungpook National University, Daegu 700-422, Republic of Korea.
E-mail: yooj@knu.ac.kr; Fax: +82-53-426-4944;
Tel: +82-53-420-4947
Molecular Imaging Research Center, Korea Institute of Radiological
and Medical Sciences, Seoul 139-706, Republic of Korea
Department of Applied Chemistry, Kumoh National Institute of
b
16
reported syntheses. TE2A (4) and the intermediate (3) were
c
fully characterized by NMR spectroscopy, elemental analysis
and HR-MS.
Technology, Yangho-dong 1, Gumi, Kyeongbuk 730-701,
Republic of Korea
Department of Molecular Medicine, Kyungpook National University
d
School of Medicine & Clinical Trial Center, Kyungpook National
University Hospital, Daegu 700-422, Republic of Korea
w Electronic supplementary information (ESI) available: Experimental
procedures for the synthesis, acidic decomplexation, cyclic voltammetry,
radiolabeling and biodistribution studies of TE2A. Half-lives for
acid decomplexation and reduction potentials of Cu–DOTA and
Cu–CB-TE2A. See DOI: 10.1039/b925703a
Fig. 1 Some commonly used bifunctional chelators.
Chem. Commun., 2010, 46, 3517–3519 | 3517
This journal is ꢀc The Royal Society of Chemistry 2010

View Article Online
Scheme 1 Three-step synthesis of TE2A from cyclam.
The prepared TE2A was complexed with Cu(ClO
4
)
2
ꢁ6H
2
O
to afford its Cu(II) complex, Cu–TE2A, in 89% yield. The
Cu–TETA complex was also prepared and characterized
according to the literature method for comparison studies.
Then, in order to evaluate the kinetic stabilities of the
copper(II) complexes, which are known to be more important
than their thermodynamic stabilities in determining the in vivo
Fig. 2 Time-dependant HPLC chromatograms of Cu–TE2A (a) and
Cu–TETA (b) during acidic decomplexation in 5 M HCl at 90 1C.
Cyclic voltammograms (scan rate 100 mV s , 0.1 M NaOAc, pH 7) of
ꢂ1
Cu–TE2A (c) and Cu–TETA (d).
structure of the Cu–TE2A complex revealed that the Cu(II)
ion is strongly coordinated by the four nitrogen atoms and the
1
7
stabilities of Cu–tetraazamacrocycle complexes,
acidic
decomplexation and cyclic voltammetry experiments were
1
performed.
two axial positions are occupied by two carboxylate oxygen
14,15
8
atoms in the octahedral coordination geometry.
In this
The half-lives for the acid-assisted decomplexation of
Cu–TE2A and Cu–TETA under pseudo first-order conditions
at high HCl concentrations are summarized in Table 1. In spite
of the structural similarity of the two ligands of Cu–TE2A
and Cu–TETA, they showed dramatically different kinetic
inertnesses to acidic decomplexation. The kinetic inertness of
Cu–TE2A in 5 M HCl was at least one-order of magnitude
higher than that of Cu–TETA. The half-life of Cu–TE2A in
case, a strong N4 in-plane ligand field is attained, which
endows the complex with high kinetic inertness and thermo-
dynamic stability. In contrast, the core coordination sphere of
the Cu–TETA complex involves N O coordination in the
2
2
equatorial plane and two ring N atoms in the axial positions,
2
1
5,20
which may be the factor that increases its kinetic lability.
Encouraged by the higher in vitro kinetic stability of
Cu–TE2A, we decided to test the in vivo stability of the metal
6
4
1
2 M HCl (90 1C) was only 2.6 min, but this is still more than
complexes labelled with radioactive copper ions. The Cu
radionuclide (t1/2 = 12.7 h) is one of most widely studied
radiometal ions in PET (positron emission tomography)
imaging, and also shows good potential in the targeted
radiotherapy of cancer, thanks to its attractive decay mode
twice the value of 1.1 min for Cu–TETA. The progress
of acidic decomplexation in 5 M HCl at 90 1C was also
monitored by repeated injection of a reaction fraction into
an HPLC system (Fig. 2a–b). As the integral of the intact
Cu(II) complex peak decreased, the peak for free Cu(II) ions
gradually increased. Whereas 3% of the Cu–TE2A complex
still remained intact after 6 h, all of the Cu–TETA complexes
were decomposed within 40 min.
+
ꢂ
21
(b 19%, b 40%). TE2A was quantitatively radiolabeled
with Cu by the addition of CuCl to a solution of TE2A in
6
4
64
2
0.1 M NH OAc (pH 6.8), followed by 20 min incubation at
4
30 1C. Within 5 min, the labeling yield of TE2A reached
more than 90%. The radiolabeling yield was measured by
radio-TLC and doubly confirmed by a radio-HPLC system.
Another important mechanism leading to the loss of the
copper(II) ion from chelates in biological systems is the
reduction of Cu(II) to Cu(I) and subsequent demetallation.
Therefore, the reduction potential of Cu(II) complexes might
6
4
The radiolabeled Cu–TE2A did not show any noticeable
degradation at 37 1C for 24 h in serum stability studies. We
further carried out side by side comparative biodistribution
provide
a good guideline for predicting their in vivo
1
7,18
64
stabilities.
Both the Cu–TE2A and Cu–TETA complexes
studies using the Cu–labeled TE2A and TETA in order to
6
evaluate the in vivo stability and clearance of Cu–TE2A
4
yielded irreversible reduction voltammograms in 0.1 M
aqueous sodium acetate solution adjusted to pH 7 (Fig. 2c–d).
However, in our experiment, the reduction potential of
Cu–TE2A was ꢂ1.10 V (vs. Ag/AgCl), while that of Cu–TETA
was only ꢂ0.88 V, which means that Cu–TE2A is more
difficult to reduce to the Cu(I) complex than Cu–TETA, by
a factor of about 200 mV, and that Cu–TE2A might be more
kinetically inert to reduction and subsequent demetallation of
(Fig. 3). Currently, TETA is one of the most commonly used
6
ligands for radiolabeling studies of Cu, because of the easy
4
radiolabeling and excellent in vivo stability of the labeled
6
4
complex. Although
Cu-labeled CB-TE2A was recently
reported to show superior in vivo stability and higher resistance
to transmetallation as compared to TETA, the utilization of
CB-TE2A as a BFC for the radiolabeling of heat-sensitive
biomolecules such as antibodies is limited by the harsh labeling
1
9
the Cu(I) ions under physiological conditions.
6
This dramatic difference in the kinetic inertness to acidic
decomplexation of the two Cu(II) complexes might be due to
their different coordination geometries. The X-ray crystal
conditions at elevated temperature (95 1C).
The biodistribution data clearly showed that the
6
4
Cu–TE2A complex was excreted from the body faster than
3
518 | Chem. Commun., 2010, 46, 3517–3519
This journal is ꢀc The Royal Society of Chemistry 2010

View Article Online
a
Table 1 Half-lives for acid decomplexation and reduction potentials of copper(II) complexes
Complex
5 M HCl, 50 1C
5 M HCl, 90 1C
12 M HCl, 90 1C
Ered/V vs. Ag/AgCl
Cu–TETA
Cu–TE2A
4.1(3) h
92.6(2) h
4.7(4) min
46.2(8) min
1.1(3) min
2.6(5) min
ꢂ0.88 (irrev)
ꢂ1.10 (irrev)
a
Half-lives are mean values of 2–3 experiments.
project of MOST and KOSEF. The Korea Basic Science
Institute (Daegu) is acknowledged for the NMR and MS
measurements.
Notes and references
1
2
3
V. Alexander, Chem. Rev., 1995, 95, 273–342.
S. Liu, Adv. Drug Delivery Rev., 2008, 60, 1347–1370.
T. K. Nayak and M. W. Brechbiel, Bioconjugate Chem., 2009, 20,
8
25–841.
L. M. De Leon-Rodriguez and Z. Kovacs, Bioconjugate Chem.,
008, 19, 391–402.
J. Huskens, D. A. Torres, Z. Kovacs, J. P. Andre, C. F. G.
C. Geraldes and A. D. Sherry, Inorg. Chem., 1997, 36, 1495–1503.
T. J. Wadas and C. J. Anderson, Nat. Protoc., 2006, 1, 3062–3068.
C. A. Boswell, X. Sun, W. Niu, G. R. Weisman, E. H. Wong,
A. L. Rheingold and C. J. Anderson, J. Med. Chem., 2004, 47,
1465–1474.
4
5
2
64
Fig. 3 Biodistribution data of Cu-labelled TETA and TE2A at 24 h
6
7
post-injection (five Sprague-Dawley rats per time point).
6
its TETA counterpart; the activity uptake of Cu–TE2A
4
8
9
E. H. Wong, G. R. Weisman, D. C. Hill, D. P. Reed, M. E. Rogers,
J. S. Condon, M. A. Fagan, J. C. Calabrese, K.-C. Lam, I. A. Guzei
and A. L. Rheingold, J. Am. Chem. Soc., 2000, 122, 10561–10572.
B. E. Rogers, C. J. Anderson, J. M. Connett, L. W. Guo,
W. B. Edwards, E. L. C. Sherman, K. R. Zinn and M. J. Welch,
Bioconjugate Chem., 1996, 7, 511–522.
6
was lower than that of Cu–TETA in all organs at 24 h
4
6
post-injection (Fig. 3), which suggests that Cu–TE2A is more
4
6
stable under physiological conditions than Cu–TETA and
4
that the transchelation of the free copper ion from the
13,22
6
4
Cu–TE2A complex is minimized.
The higher stability
1
1
0 R. Delgado, V. Felix, L. M. P. Lima and D. W. Price, Dalton
Trans., 2007, 2734–2745.
1 T. M. Jones-Wilson, K. A. Deal, C. J. Anderson, D. W. McCarthy,
Z. Kovacs, R. J. Motekaitis, A. D. Sherry, A. E. Martell and
M. J. Welch, Nucl. Med. Biol., 1998, 25, 523–530.
64
and faster clearance pattern of Cu–TE2A might be attributed
to its core coordination sphere and overall charge. While the
6
4
64
overall charge of Cu–TE2A is neutral, that of Cu–TETA is
ꢂ2 under physiological conditions because of the presence of
two non-coordinated carboxylate groups.
12 A. Bianchi, L. Calabi, C. Giorgi, P. Losi, P. Mariani, D. Palano,
P. Paoli, P. Rossi and B. Valtancoli, J. Chem. Soc., Dalton Trans.,
2
001, 917–922.
3 J. Yoo, D. E. Reichert and M. J. Welch, J. Med. Chem., 2004, 47,
625–6637.
In conclusion, the facile synthesis of TE2A, superior kinetic
stability of the Cu–TE2A complex compared to that of
1
6
6
4
Cu–TETA and quantitative labelling with
Cu at low
14 I. M. Helps, D. Parker, J. Chapman and G. Ferguson, J. Chem.
Soc., Chem. Commun., 1988, 1094–1095.
temperature are expected to lead to the widespread use of
TE2A as a new chelator for various copper(II) radionuclides.
Furthermore, many new bifunctional chelators could be
derived based on the TE2A backbone and utilized to label
disease-specific peptides and antibodies. Easy access to TE2A
would also allow for the further structural modification of
various metal complexes and enable its application in
diverse research fields, such as optical imaging probes, MRI
reagents, and catalysis, in addition to medical imaging and
radiotherapy.
1
5 J. Chapman, G. Ferguson, J. F. Gallagher, M. C. Jennings and
D. Parker, J. Chem. Soc., Dalton Trans., 1992, 345–353.
6 I. M. Helps, D. Parker, J. R. Morphy and J. Chapman,
Tetrahedron, 1989, 45, 219–226.
1
1
7 M. Shokeen and C. J. Anderson, Acc. Chem. Res., 2009, 42, 832–841.
8 K. S. Woodin, K. J. Heroux, C. A. Boswell, E. H. Wong,
G. R. Weisman, W. Niu, S. A. Tomellini, C. J. Anderson,
L. N. Zakharov and A. L. Rheingold, Eur. J. Inorg. Chem.,
2005, 4829–4833.
1
1
9 K. J. Heroux, K. S. Woodin, D. J. Tranchemontagne, P. C.
B. Widger, E. Southwick, E. H. Wong, G. R. Weisman,
S. A. Tomellini, T. J. Wadas, C. J. Anderson, S. Kassel,
J. A. Golen and A. L. Rheingold, Dalton Trans., 2007, 2150–2162.
This work was supported by MEST/KOSEF (Nuclear R&D
Program, M2080600010808M060010810), a Korea Research
Foundation Grant funded by the Korean Government
20 J. D. Silversides, C. C. Allan and S. J. Archibald, Dalton Trans.,
007, 971–978.
2
2
1 T. J. Wadas, E. H. Wong, G. R. Weisman and C. J. Anderson,
Curr. Pharm. Des., 2007, 13, 3–16.
(
MOEHRD, Basic Research Promotion Fund) (KRF-
008-331-E00262), and the Brain Korea 21 Project in 2009.
2
2
2 L. A. Bass, M. Wang, M. J. Welch and C. J. Anderson,
Bioconjugate Chem., 2000, 11, 527–532.
6
The production of Cu was supported partially by the QURI
4
This journal is ꢀc The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 3517–3519 | 3519