A bifunctional chelator featuring the DOTAM-Gly-l-Phe-OH structural subunit: en route toward homo- and heterobimetallic lanthanide(III) complexes as PARACEST MRI contrast agents

Article abstract of DOI:10.1016/j.tetlet.2009.12.086

A new synthesis of a bifunctional chelator possessing DOTAM-Gly-l-Phe-OH and DO3A chelating cages interconnected by an oligoamide chain has been achieved via HBTU-mediated coupling from easily accessible building blocks. Both homo- and heterobimetallic lanthanide(III) complexes derived from this bifunctional chelator have been prepared in moderate yields. The CEST spectrum acquired for homobimetallic Eu³⁺ complex showed this molecule to be a promising PARACEST MRI contrast agent whereas the Eu³⁺/Tm³⁺ heterobimetallic complex lacked a useful CEST signal.

Full text of DOI:10.1016/j.tetlet.2009.12.086

Tetrahedron Letters 51 (2010) 1087–1090
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A bifunctional chelator featuring the DOTAM-Gly-L-Phe-OH structural subunit:
en route toward homo- and heterobimetallic lanthanide(III) complexes as
PARACEST MRI contrast agents
Mojmír Suchy a,b, Alex X. Li b, Robert Bartha b, Robert H. E. Hudson a,
*
´
a Department of Chemistry, The University of Western Ontario, London, Canada ON N6A 5B7
b Centre for Functional and Metabolic Mapping, Robarts Research Institute, The University of Western Ontario, London, Canada ON N6A 5K8
a r t i c l e i n f o
a b s t r a c t
Article history:
A new synthesis of a bifunctional chelator possessing DOTAM-Gly-L-Phe-OH and DO3A chelating cages
Received 19 November 2009
Revised 15 December 2009
Accepted 16 December 2009
Available online 23 December 2009
interconnected by an oligoamide chain has been achieved via HBTU-mediated coupling from easily acces-
sible building blocks. Both homo- and heterobimetallic lanthanide(III) complexes derived from this
bifunctional chelator have been prepared in moderate yields. The CEST spectrum acquired for homobime-
tallic Eu3+ complex showed this molecule to be a promising PARACEST MRI contrast agent whereas the
Eu3+/Tm3+ heterobimetallic complex lacked a useful CEST signal.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
DOTAM-Gly-
DO3A
L-Phe-OH
HBTU
Lanthanides
PARACEST
Since its first synthesis in 1961,1 the chemistry of cyclen,2 its
derivatives, and their complexes with transition metals, rare earth
elements, actinides, and lanthanides has experienced vigorous
development. These complexes are suitable for a variety of applica-
tions in modern chemistry, biochemistry, and medicine. As such,
cyclen-based complexes are nowadays being studied and used as
antimicrobial agents, synthetic nucleases, metal sensors, fluores-
cent, and luminescent probes as well as contrast agents (CAs) in
positron emission tomography (PET) or magnetic resonance (MR)
imaging.
A major category of MRI contrast agents are paramagnetic com-
plexes of Gd3+ which are capable of altering the T1 of water pro-
tons, resulting in increased brightness in T1-weighted images.3
More recently, a new group of MRI CAs was introduced that mod-
ulate signal by chemical exchange saturation transfer (CEST).4 Sub-
only one type of chelator unit capable of binding one lantha-
nide(III) cation. Almost all of these complexes are derivatives of
DOTA (1a, Fig. 1) or DOTAM (1b, Fig. 1). Curiously, to the best of
our knowledge no heterometallic MRI CAs containing two or more
different lanthanide(III) cations have been studied so far.
Based on the limited number of literature reports, the design
and synthesis of cyclen-derived lanthanide(III)-based heterome-
tallic complexes appears to be a challenging task. The first exam-
ple was described in 2003 by Faulkner and Pope.8 The Tb3+
complex of DO3A was first formed and two equivalents of this
complex was treated with DTPA-like chelator, followed by the
introduction of Yb3+. In the more recent work that appeared in
2006, Tremblay and Sames employed selective sequential metala-
tion based on the differences in kinetic stability of DOTA and
DTPA-derived complexes.9 Faulkner’s group has also accomplished
the synthesis of heterotetrametallic complex containing two dif-
ferent lanthanide(III) cations. For this work, two homobimetallic
complexes served as precursors, which were joined by an azo
(–N@N–) linkage.10 The same group of authors also investigated
the possibilities of the orthogonal protection/deprotection strategy
applied to the bifunctional DOTA-derived ligand; followed by a
sequential metallation using two different lanthanide(III) salts.11
Although not strictly lanthanide(III) containing, it is worth men-
tioning that very recently the Ugi multicomponent reaction has
been used to prepare a heterobimetallic complex (DO3A-based)
containing Gd3+ and Y3+ cations.12
sequently
a large number of paramagnetic ion-containing
lanthanide(III) complexes have been prepared which were capable
of inducing large hyperfine shifts of coordinated water protons as
well as other exchangeable protons present in proximity to the lan-
thanide(III) center.5 The term PARACEST6 MRI CAs has been coined
to describe these molecules.
Although several examples of multimeric PARACEST MRI CAs
appeared recently,7 the vast majority of these molecules contain
* Corresponding author. Tel.: +1 519 661 2111x86349; fax: +1 519 661 3022.
E-mail address: rhhudson@uwo.ca (R.H.E. Hudson).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.12.086

´
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M. Suchy et al. / Tetrahedron Letters 51 (2010) 1087–1090
R
O
R
O
HO-Phe-Gly
Gly-Phe-OH
NHCbz
Cl
+
Cl H3N
Cl
N
N
N
N
O
N
N
N
O
O
Eu3+
9
8
O
R
O
R
O
N
O
K2CO3, DMF
71%
HO-Phe-Gly
Gly-Phe-OH
1a
1b
, R = OH (DOTA)
2
H
, R = NH2 (DOTAM)
N
Cl
NHCbz
O
EtO-Phe-Gly
Gly-Phe-OEt
O
7
O
O
O
N
N
N
N
i, K2CO3, MeCN
ii, H2, 10% Pd/C, EtOAc
90%, based on 6
+
O
N
N
N
N
O
O
O
O
O
O
O
O
O
O
N
N
4
+ 3b
EtO-Phe-Gly
Gly-Phe-OR
4
HN
O
O
N HN
3a
, R = OtBu
H2N
6
3b, R = H
DMF, HBTU
93%
HO-Phe-Gly
Gly-Phe-OH
O
Gly-Phe-OEt
O
EtO-Phe-Gly
O
N
N
N
Eu3+
O
N
O
N
N
N
N
O
N
O
OH
O
H
O
O
O
O
N
HO-Phe-Gly
N
H
N
O
O
H
N
H
N
H
N
N
N
H
EtO-Phe-Gly
N
H
O
N
N
H
O
Ph
3+ N
O
N
Ln
O
N
5a
, Ln = Eu
O
OH
Ph
N
5b, Ln = Tm
O
O
O
O
OH
10
i, TFA, CH2Cl2
ii, TmCl3.6H2O
MeCN/H2O
i, TFA, CH2Cl2
ii, NaOH, THF/H2O
iii, EuCl3.6H2O
MeCN/H2O
Figure 1. Structures of 1–5. The DOTAM-Gly-
red and DO3A-derived chelator is shown in blue.
L-Phe-OH chelator cage is indicated in
10
41%, based on
iii, EuCl3 . 6H2O
21% based on 10
MeCN/H2O, 18%
5a
We have recently developed a new temperature-responsive
5b
PARACEST MRI CA, Eu3+ DOTAM-Gly- -Phe-OH13 (2, Fig. 1) exhibit-
L
Scheme 1. Synthesis of bifunctional ligand 10 and complexes 5a, b.
ing a unique sensitivity in the physiological temperature range
(36–40 °C). As a part of our broad research program devoted to
the development of new PARACEST MRI CAs, we have also pre-
pared several asymmetrically substituted cyclen derivatives based
on the parent structure of 2.14 Among them, the carboxylic acid
derivative 3b, obtained after orthogonal deprotection of an ester
3a (Fig. 1), is well suited for amide bond formation via coupling
with primary amines. The accessibility of an amine counterpart 4
(Fig. 1), prepared by modifying a recently described synthetic pro-
tocol,15 prompted us to investigate the synthesis of bifunctional
chelator and its homo- (5a, Fig. 1) and heterobimetallic (5b,
Fig. 1) complexes. The results of these synthetic studies along with
the magnetic (PARACEST) properties of both complexes (Fig. 1) are
described herein.
The synthesis of the building block 3b has been accomplished
by a synthetic methodology developed in our laboratory.14 A mod-
ified synthesis of amine 4 was developed as indicated in Scheme 1
and described in Supplementary data. Acylation of mono-N-Cbz
ethylenediamine hydrochloride (9) with chloroacetyl chloride (8)
resulted in the formation of electrophile 7 in 71% yield. Alkylation
of commercially available DO3A tert-butyl ester (6) with electro-
phile 7, followed by a reductive removal of the Cbz group pro-
ceeded with the formation of amine 4 in high yield (94%, based
on 6, Scheme 1) and purity sufficient for the subsequent reaction.
With building blocks 3b and 4 in hand, we investigated the possi-
bilities of the amide bond formation. HBTU in warm (60 °C) DMF in
the presence of a base (DIPEA) afforded superior results, and
orthogonally protected bifunctional ligand 10 was prepared in
93% yield as indicated in Scheme 1 and described in Supplemen-
tary data.
of chelator 10 have been cleaved after subjecting the ligand 10 to
TFA-mediated removal of tert-butyl ester functionalities. Despite
this unexpected problem, we were able to prepare homo- and het-
erobimetallic complexes 5 in moderate yield.
To prepare a homobimetallic complex 5a, we first deprotected
the tert-butyl ester functionalities and then subjected the partially
deprotected molecule to saponification conditions in order to re-
move ethyl ester protecting groups. The mixture of the desired
deprotected ligand along with the products of the hydrolysis on
the oligoamide chain has been subjected to exhaustive metallation
with EuCl3Á6H2O. The homobimetallic complex 5a was purified by
HPLC and was obtained in 21% yield, based on the ligand 10
(Scheme 1). The absence of free Eu3+ in the product was confirmed
by Xylenol Orange test.16 Complex 5a was characterized by HR-
ESI-MS as described in Supplementary data.
To overcome the problem associated with the oligoamide linker
hydrolysis, we decided to retain the ethyl ester protecting groups
while trying to prepare heterobimetallic complex 5b. Thus, tert-bu-
tyl ester-deprotected ligand was subjected to metalation with one
equivalent of TmCl3Á6H2O (Scheme 1). This reaction was carried
out at low temperature (0 °C) and monometallic complex was ob-
tained in 41% yield after HPLC purification. Notably, no trace of the
bimetallic complex was found (UPLC-ESI-HRMS). Treatment of the
monometallic complex with 2.5 equiv of EuCl3Á6H2O, followed by
HPLC purification afforded heterobimetallic complex 5b in 18%
yield; overall yield based on the ligand 10 was 7% (Scheme 1).
The product was characterized by HR-ESI-MS and it did not contain
any free lanthanide(III) cations as indicated by Xylenol Orange
test.16
An oligoamide linker joining the two chelating cages present in
10 was found to be sensitive toward both acid and base-catalyzed
hydrolysis. The unwanted cleavage occurred on the amidic bond
connecting the phenylalanine unit with ethylene diamine side
chain. A similar behavior was previously observed with cysta-
To confirm that the sequential metalation took place as indi-
cated above, that is that DO3A unit was metalated first, we initially
turned to 1H NMR spectrometry. As depicted in Supplementary
data, the spectra of both molecules, the unmetalated ligand and
the corresponding monometalated Tm3+ complex, exhibit
mine-modified DOTAM-Gly-L-Phe-OH. In fact, significant portions

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M. Suchy et al. / Tetrahedron Letters 51 (2010) 1087–1090
extensive signal broadening and are therefore uninformative. In
fact, in both spectra only three groups of broad unresolved signals
at ca. 1.00–1.50 ppm, 2.50–4.50 ppm, and 7.00–8.50 ppm can be
observed, precluding any structural assignment. It is also impor-
tant to point out that none of the characteristic resonances17 due
to the presence of paramagnetic Tm3+ were observed.
The problem was solved by performing a time course study
(Fig. 2). DOTA (1) as a model compound for the DO3A chelator
along with ligand 3b was treated with one equivalent of
TmCl3Á6H2O at 0 °C in aq MeCN. Samples were withdrawn after
24, 48, 72, and 120 h and were analyzed by HR-ESI-MS. It was
found that the metalation of DOTA (1) was completed after 24 h,
whereas significant portions (46%) of ligand 3b remained unmeta-
lated even after 120 h (Fig. 2). Based on the results of this kinetic
study it may be reasonably assumed that the first site of the meta-
lation in ligand 10 is the DO3A cage. This observation is consistent
with our experience with the metalation of a disulfide dimer
Figure 4. CEST spectrum of heterobimetallic complex 5b (20 mM, water, rt,
saturation power B1 14 lT, presaturation time TS 10 s).
derived from DOTAM-Gly-
L-Phe-OH was found to be quite
sluggish.7f
Eu3+ center. This may occur by acceleration of the rate of relaxation
of water at the Eu3+ center by the proximal Tm3+, but determina-
tion of the mechanism for the observed effect will require further
study. This result highlights that the choice of lanthanides and
their chelating ligand in the heterobimetallic complexes can pro-
foundly affect the resulting CEST properties.
After we obtained the desired bimetallic complexes 5, we eval-
uated the PARACEST effect. A CEST spectrum of the homobimetallic
complex 5a was acquired following a previously established exper-
imental protocol.13b The CEST effect (ca. 30%) due to the bound
water18 was observed at ca. 45 ppm (Fig. 3), which is consistent
with the data for the parent CA 2.13b It is important to point out
that the presence of the second Eu3+ slightly decreased the overall
CEST effect associated with 5a. Since Eu3+-DOTA does not have a
detectable off-resonance CEST effect,19 it was therefore expected
that 5a would not have a greater CEST effect than 2.
In summary we have developed a concise synthetic strategy to-
ward a bifunctional chelator featuring DOTAM-Gly-L-Phe-OH sub-
unit. We were able to prepare homo- and heterobimetallic
lanthanide(III) complexes derived from this chelator in moderate
chemical yield.
One of the drawbacks of our current design is the unwanted
hydrolysis of the oligoamide chain connecting the two chelating
units. Other possibilities (e.g., Cu+-catalyzed Huisgen ‘click’ dipolar
cycloaddition20) are being explored in order to interconnect the
two chelating subunits with a structural element that is not prone
to facile hydrolysis.
The CEST spectrum of the heterobimetallic complex 5b shows a
marked reduction in the signal due to bound water at the Eu3+ cen-
ter (ca. 45 ppm), Figure 4. It is evident that the chelated Tm3+ has a
large effect on the signal generated by water coordinated at the
The PARACEST spectrum of the Eu3+-containing homobimetallic
complex 5a was determined. The results were consistent with
those for the parent CA 2, although the signal due to bound water
was slightly diminished. In contrast, the signal due to bound water
at the Eu3+ center was obliterated by the presence of a proximal
coordinated Tm3+. The synthesis of other heterobimetallic com-
plexes and evaluation of their PARACEST properties are currently
in progress and will be reported in due course.
Acknowledgment
Figure 2. Amount of unmetalated material present (%) in the reaction mixture after
metalation of DOTA (1, blue curve,
TmCl3Á6H2O.
)
and ligand 3b (red curve,
) with
We thank the Ontario Institute for Cancer Research (OICR) for
the financial support of this work.
Supplementary data
Supplementary data (full experimental details and spectro-
scopic characterization of new compounds 5a, 5b, 7, and 10) asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2009.12.086.
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l

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M. Suchy et al. / Tetrahedron Letters 51 (2010) 1087–1090
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