Click chemistry toward bis(DOTA-derived) heterometallic complexes: Potential bimodal MRI/PET(SPECT) molecular imaging probes

Article abstract of DOI:10.1039/c3ra23260c

A general synthetic methodology has been developed for the preparation of DOTA-derived heterometallic omplexes as potential bimodal MRI/PET(SPECT) molecular probes. Both alkyne- and azide substituted DOTA-derived chelators have been synthesized and metallated with Gd³⁺ (MRI reporter) or cold isotopes of Cu²⁺, Ga³⁺ and In ³⁺ (potential PET or SPECT reporters). The Cu⁺-catalyzed Huisgen [3 + 2] cycloaddition has been utilized as a key step in the synthesis of potential bimodal MRI/PET(SPECT) molecular probes. A preliminary optimization of the reaction conditions has been carried out to make the reaction conditions compatible with the radioactive isotopes of Cu²⁺, Ga³⁺ and In³⁺ possessing short life times. The MRI sensitivity of the potential bimodal MRI/PET(SPECT) molecular probes has been determined and compared to that associated with a clinically used MRI contrast agent.

Full text of DOI:10.1039/c3ra23260c

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‘‘Click’’ chemistry toward bis(DOTA-derived)
heterometallic complexes: potential bimodal MRI/
Cite this: RSC Advances, 2013, 3,
3249
PET(SPECT) molecular imaging probes3
Mojm´ır Such´y,ab Robert Barthab and Robert H. E. Hudson*a
A general synthetic methodology has been developed for the preparation of DOTA-derived heterometallic
omplexes as potential bimodal MRI/PET(SPECT) molecular probes. Both alkyne- and azide substituted
DOTA-derived chelators have been synthesized and metallated with Gd3+ (MRI reporter) or ‘‘cold’’ isotopes
of Cu2+, Ga3+ and In3+ (potential PET or SPECT reporters). The Cu+-catalyzed Huisgen [3 + 2] cycloaddition
has been utilized as a key step in the synthesis of potential bimodal MRI/PET(SPECT) molecular probes. A
preliminary optimization of the reaction conditions has been carried out to make the reaction conditions
compatible with the radioactive isotopes of Cu2+, Ga3+ and In3+ possessing short life times. The MRI
sensitivity of the potential bimodal MRI/PET(SPECT) molecular probes has been determined and compared
to that associated with a clinically used MRI contrast agent.
Received 10th December 2012,
Accepted 1st January 2013
DOI: 10.1039/c3ra23260c
www.rsc.org/advances
molecular probes, capable of exploiting the complementarities
between the two imaging modalities.2c
Introduction
The first example of a discrete5 bimodal MRI/PET mole-
cular probe appeared in 2010, in a landmark publication by
Caravan, Sherry, Catana and coworkers.6 A Gd3+ derived
complex was subjected to Cu+-catalyzed Huisgen [3 + 2] click
cycloaddition (CuAAC) with 2-fluoroethylazide labeled with a
radioactive fluorine isotope (18F, t1/2 110 min) to form the
bimodal MRI/PET molecular probe 1 (Fig. 1), capable of
quantitative pH imaging.6 In more recent work, a DOTA-
derived chelator was coupled with a porphyrin moiety via a
peptide bond, followed by the metallation of the DOTA
chelation cage with Gd3+ and porphyrin subunit with Cu2+
(structure 2, Fig. 1).7 Although the authors did not use a
radioactive isotope of copper (64Cu, t1/2 12.7 h), they suggested
that their synthetic methodology would be fully compatible
with prospective radiolabeling chemistry. In addition, the
fluorescence response resulting from the presence of the
porphyrin moiety can be utilized for optical imaging.7
Magnetic resonance imaging (MRI) and positron emission
tomography (PET) are widely used complementary medical
imaging techniques. While MRI provides high-resolution,
(submillimeter) anatomical information it is recognized to
be limited by low sensitivity. Conversely, PET imaging is an
exceptionally sensitive technique which is limited to some
extent by its poor spatial resolution. It is now widely believed,
that integrated MRI/PET molecular imaging possesses a
tremendous potential for future clinical application. It has
been estimated that full human body scanning will be possible
within the next few years.1
Although a wide variety of molecular probes have been
developed for both MRI2 and PET,3 the synthesis of dual
probes capable of enhancing the contrast in bimodal MRI/PET
imaging is still in its infancy.4 It has been recently proposed,
that a simultaneous MRI/PET acquisition will have great
advantages, should chemists be able to develop the bimodal
A slightly different approach to bimodal MRI/SPECT (single
photon emission computer tomography) molecular probe has
been developed by Aime and coworkers.8 A pH sensitive
sulfonamide-modified DO3A chelator has been metallated
with Gd3+ (MRI reporter) and with a radioactive isotope of Ho3+
aDepartment of Chemistry, The University of Western Ontario, London, Ontario,
Canada. E-mail: robert.hudson@uwo.ca
bRobarts Research Institute, The University of Western Ontario, London, Ontario,
Canada
(
166Ho, t1/2 26.6 h). Assuming that both lanthanide(III)
Electronic supplementary information (ESI) available: 1H NMR spectra of
3
complexes (structures 3, Fig. 1) will have identical pharmaco-
kinetic properties; for their further use the authors proposed a
simultaneuos injection of the Gd3+ complex 3a along with a
small amount of the radiotracer labeled complex 3b.8 In a very
recent work, a conjugation of two L-histidine (His) residues to
central DTPA substructure resulted in the formation of a
compounds 6a, unmetallated version of 6b, 7a, ester deprotected version of 7a,
12 and 15. 13C NMR spectra of compounds 12 and 15. HR-ESI-MS spectra of
compounds 5a, 5b, 6a, 6b, unmetallated version of 6b, 7a, ester deprotected
version of 7a, 7b–7d, 13, DO3A-OtBu-Ac 2-chloroethylamine. Semi-preparative
HPLC chromatograms of crude reaction mixtures containing compounds 5a–5c.
NMRD profiles associated with compounds 5a–5c, 6b and Dotarem (16) acquired
at 25 uC and 37 uC. See DOI: 10.1039/c3ra23260c
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Herein, we have investigated the possibility of using ‘‘click’’
CuAAC-based chemistry to assemble the dual MRI/PET(SPECT)
molecular probes. Previously, we reported the development of
a library of alkyne modified DOTAM-derived complexes for use
in PARACEST (PARAmagnetic Chemical Exchange Saturation
Transfer) MRI10 and 1H MRS (magnetic resonance spectro-
scopy) thermometry.11 The present work describes the synth-
esis of alkyne- and azide-modified complexes based on the
DOTA (1,4,7,10-tetraazadodecane-1,4,7,10-tetraacetic acid) fra-
mework.12 The alkyne-derived building block was metallated
with Gd3+ (MRI reporter), while the azide-derived subunit was
used for the chelation of ‘‘cold’’ non-radiocative Cu2+, Ga3+
and In3+ cations. The ‘‘click’’ CuAAC reaction was utilized13 to
couple the two building blocks and a preliminary optimization
was carried out in order to obtain the reaction conditions
compatible with the limited life time of the radioactive
isotopes (64Cu, t1/2 12.7 h; 68Ga, t1/2 68 min; 111In, t1/2 67.9
h).3b The MRI sensitivity of the potential bimodal MRI/
PET(SPECT) molecular probes was evaluated and compared
to that associated with the commercially available MRI
contrast agent Dotarem.
Results and discussion
Retrosynthetic analysis of potential bimodal MRI/PET(SPECT)
molecular probes
The structural design and retrosynthetic analysis of potential
bimodal MRI/PET(SPECT) molecular probes 5 is depicted in
Scheme 1. Based on our previous experience,10 we decided to
use ‘‘click’’ CuAAC-based chemistry for the preparation of
probes 5. For this purpose we were required to prepare the
corresponding alkyne- and azide-functionalized intermediates.
The alkyne-modified building block 6b (Scheme 1) consists
of a DOTA framework metallated with Gd3+ and decorated with
a short dipeptide sequence containing O-propargyl-L-tyrosine
(Tyr). We have previously developed the synthesis of the
corresponding dipeptide linker and utilized it to form a library
of PARACEST MRI contrast agents.10a Another advantage
associated with the linker is the presence of the Tyr moiety,
which provides a UV chromophore that is useful during semi-
preparative HPLC purification of the molecular probes 5.
Taking into account the long term goal of these efforts to
prepare bimodal MRI/PET(SPECT) molecular probes contain-
ing ‘‘hot’’ radioactive isotopes, we decided to keep the
structure of the azide-modified subunit as simple as possible
to allow for a facile and rapid complexation. An azide-modified
building block (structures 7, Scheme 1) is represented by a
DOTA-derived complexes (Cu2+, Ga3+ and In3+) decorated with
a short alkyl azide appendage.
Fig. 1 Structures of bimodal MRI/PET(SPECT) molecular probes 1–4.
trifunctional ligand.9 The DTPA substructure was labelled with
Gd3+, while the His residues have been labelled with ‘‘cold’’
[Re(CO)3(H2O)]+ (structure 4a, Fig. 1) or with ‘‘hot’’
[
99mTc(CO)3(H2O)]+ 99mTc, t1/2 6.02 h, structure 4b, Fig. 1).
(
To account for the differences in sensitivity between MRI and
SPECT, a ‘‘cocktail mixture’’ containing a large amount of the
‘‘cold’’ molecular probe 4a and a tiny amount of the ‘‘hot’’
molecular probe 4b has been injected and subsequently
detected in mouse liver and kidneys.9
The evaluation of the different approaches to bimodal MRI/
PET(SPECT) molecular probes described above indicates, that
no general methodology for their preparation is currently
available and existing syntheses can be labour intense and
require the preparation of the variety of diverse synthetic
intermediates. It is easy to envision that a more general
approach should allow for preparation of bimodal MRI/
PET(SPECT) molecular probe libraries (with the possibility of
fine tuning their properties) from relatively few building
blocks.
Both, the alkyne- and the azide-modified subunits can be
obtained by a simple alkylation of a common starting material,
commercially available DO3A-OtBu (8, Scheme 1). Moreover,
the DOTA framework is known to form sufficiently stable
complexes with the metal ions used in this study.2b,3b
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Scheme 1 Retrosynthetic analysis of potential bimodal MRI/PET(SPECT) molecular probes 5a–5c.
The alkyne-modified building block 6b (83% yield,
Scheme 2) was purified by size exclusion chromatography
(SEC) as described in the Experimental, its identity was
confirmed by high resolution mass spectrometry (HR-MS)
and infra red (IR) spectrometry. The proper charged state
envelope was observed in the mass spectrum of 6b along with
Synthesis of the alkyne-modified building block 6c
The preparation of the N-chloroacetyl-Gly-L-Tyr(O-propargyl)OMe (9)
has been described previously.10a Alkylation of DO3A-OtBu (8,
Scheme 1) with electrophile 9 afforded the ligand 6a in excellent
yield (95%, Scheme 2).14 Refluxing the ligand 6a in t-BuOH/H2O
mixture in the presence of GdCl3?6H2O resulted in the removal of
protecting ester functionalities along with the metallation of the
DOTA framework to furnish the alkyne building block 6b.
Interestingly, if the same reaction is carried out in the absence of
GdCl3?6H2O using a catalytic amount of HCl, an unmetallated
version of the alkyne building block 6b can be obtained as described
in the Experimental.
the typical isotope pattern due to the presence of Gd3+ [see ESI
3
for more details]. In the IR spectrum, the shift of the
absorbtion band due to the presence of the peripheral
carbonyl groups (1617 cm21) confirms their involvement in
the Gd3+ chelation (1727 cm21 for non-metallated ligand 6a).
Synthesis of the azide-modified building blocks 7b–7d
The synthesis of ligand 7a was found to be less straightfor-
ward. It was first decided to alkylate DO3A-OtBu (8) with an
electrophile possessing a protected terminal OH group, which
was expected to be converted to the azide functionality at the
end of the synthetic sequence. The primary amino group of
2-ethanolamine (10) was protected with a Boc group,15 while
the OH was benzylated.16 Literature procedures were used,15,16
the protected 2-ethanolamine derivative 11 was obtained in
95% overall yield (Scheme 3). The Boc group was removed,16
followed by the acylation with chloroacetyl chloride17 to give
electrophile 12 (47% yield based on 11, Scheme 3) after
purification by flash column chromatography (FCC). For
spectral characterization of electrophile 12 see ESI.
3
Alkylation of DO3A-OtBu (8) with electrophile 12 proceeded
smoothly,14 affording the OBn protected ligand 13 of sufficient
purity for the subsequent reaction (Scheme 3). The hydro-
genolytic removal (10% Pd/C) of the benzyl group proved to be
troublesome. Atmospheric pressure hydrogenation was found
to be sluggish (ca. 50% conversion after 7 days) while higher
pressure hydrogenation (ca. 40 psi) resulted in notable
decomposition. The rate of the hydrogenation at ambient
pressure was increased by the addition of AcOH; unfortunately
this lead to the formation of some inseparable impurities
(structures were not determined) in the reaction mixture along
with the desired alcohol. The isolated, crude residue was
subjected to treatment with SOCl2 (it was found to give better
results compared to MsCl), to effect hydroxyl-to-chloro func-
tional group exchange. The crude mixture containing the
desired chloro derivative was isolated and subjected to
Scheme 2 Synthesis of the alkyne building block 6b.
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Scheme 3 Synthesis of the azide building blocks 7b–7d.
reaction with NaN3 in DMF. The ligand 7a was obtained after
the purification by FCC in an overall yield 15%, based on 13.
We felt that this was not a satisfactory yield and the reaction
sequence was too laborious. In order to improve the chemical
route, we used a chlorine-based electrophile containing an
azido group. Thusly, 2-azidoethylamine (14) was prepared
from the HI salt of 2-iodoethylamine using the literature
procedure.18 Acylation of 14 with chloroacetyl chloride17
afforded N-chloroacetyl 2-azidoethylamine (15, Scheme 3) as
a slightly yellow oil in 55% yield after the FCC purification. It is
worth noting that synthesis of electrophile 15 has been
reported very recently using slightly different synthetic
procedure by Borbas and coworkers.19 Full spectroscopic
characterization of electrophile 15 is provided in the the
carbon longer side chain is also known, although no synthetic
details are available.20
Having synthesized the desired azido-modified ligand 7a, we
investigated the possibilities of its metallation with transition
metal cations relevant to PET(SPECT) (Cu2+, Ga3+, In3+). The
OtBu ester functionalities were removed by treatment with
trifluoroacetic acid (33% TFA) in CH2Cl2. It was found, that 1 h
reaction time was sufficient to achieve the complete deprotec-
tion (confirmed by 1H NMR and HR-MS). The volatiles were
removed by evaporation and the residues were subjected to
treatment with CuCl2?6H2O, Ga(NO3)3?H2O and InCl3?4H2O in
water (Scheme 3). The pH of the reaction mixtures was
adjusted to achieve the fast complex formation3b (see the
details in the Experimental). Optimization of the reaction
conditions was performed to achieve the full complexation
within 30 min (confirmed by HR-MS, see the Experimental for
the details). Complexes 7b (Cu2+), 7c (Ga3+) and 7d (In3+) were
purified by dialysis (see General experimental procedures), the
presence of an intact azido group was confirmed by IR
spectrometry, strong absorbtion band at ca. 2100 cm21 was
observed for each complex.
Experimental and ESI, the presence of an intact azido group
3
was confirmed by infra red (IR) spectrometry (strong absorb-
tion band at 2096 cm21). With 15 in hand we performed the
alkylation of the DO3A-OtBu (8, Scheme 3).14 This step was
found to proceed smoothly, providing the crude ligand 7a of
sufficient purity for the next steps in 98% yield. Analytical
samples of 7a were obtained upon purification by FCC, the
1
ligand 7a was characterized by H NMR spectroscopy and IR
Importantly, the reaction times for complex formation (30
min) are compatible with the short life times of radioactive
isotopes used in this study. The complexes 7b–7d were
prepared in larger amounts (ca. 30–40 mg) to allow for the
detailed investigation of the subsequent ‘‘click’’ reaction with
spectrometry (strong absorbtion band at 2098 cm21 confirmed
the presence of an intact azido group) and by HR-MS. The
azide 7a has been prepared only very recently by a slightly
different synthetic route,19 a related azide possessing one
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course this is still an undesirably long reaction time and
optimization is required, vide infra.
The Gd3+/Cu2+ heterometallic complex 5a (Scheme 4) was
isolated by semi-preparative HPLC (taking advantage of the
presence of the UV active Tyr scaffold), followed by the removal
of salts by dialysis (22% yield, see Experimental for details).
The reactions between the alkyne 6b and azides 7c and 7d
(Scheme 4) were carried out in similar fashion, heterometallic
complexes 5b (Gd3+/Ga3+, 19% yield) and 5c (Gd3+/In3+, 36%
yield) were purified in an analogous manner. Complexes were
produced in amounts (ca. 20 mg) sufficient for the character-
ization of their sensitivities in MRI.
The identity of the heterometallic complexes 5a–5c was
confirmed by HR-MS spectrometry. Proper charge state
envelopes along with isotope patterns due to the presence of
heavy metals have been observed in the mass spectra
associated with 5a–5c (see Fig. 2 and ESI for details). Also
3
worthy of note, no absorption band which can be attributed to
the presence of the azide group (ca. 2100 cm21) was present in
the IR spectra associated with complexes 5a–5c.
Optimization of the reaction conditions for the ‘‘click’’ CuAAC
reaction between an alkyne 6b and azides 7b–7d
Scheme 4 ‘‘Click’’ CuAAC reaction between the alkyne building block 6b and
the azide building blocks 7b–7d.
Bearing in mind that the long term goal of this work is the
preparation of bimodal MRI/PET(SPECT) molecular probes
containing ‘‘hot’’ radioactive isotopes, we decided to perform
an optimization of the reaction conditions used to perform the
‘‘click’’ CuAAC chemistry. Thus a solution (in 1 ml of water)
containing 1025 mol of the alkyne building block 6b; 1025 mol
of the azide precursors 7b, 7c or 7d; 2.5 6 1026 mol
CuSO4?5H2O; 1.25 6 1025 mol of sodium ascorbate and
1025 mol of DMEDA was incubated for 1 h at 80 uC. Samples
were withdrawn prior to the placement of the reaction vial to a
heating block, then after 15 min and 1 h, followed by an HPLC
analysis. In the case of Gd3+/Cu2+ heterometallic complex 5a, a
complete conversion was observed in the sample taken prior to
the placement of the vial in the heating block. Compared to
other results discussed below, this observation indicates, that
the Cu2+ cation present in the structure of azide 7b might be
involved in catalyzing the ‘‘click’’ CuAAC reaction between the
alkyne 6b and the azide 7b.
Surprisingly, quite different results were obtained when
alkyne 6b was incubated with azide 7c which contains Ga3+.
The formation of Gd3+/Ga3+ heterometallic complex 5b was
found to proceed rather sluggishly with only ca. 10%
conversion to 5b was observed after 1 h at 80 uC (Fig. 3).
Taking into consideration the short life time of the widely
available radioactive isotope of gallium (68Ga, t1/2 68 min), it
appears clear that further optimization of the reaction
conditions (e. g. microwave irradiation22) is required to make
this methodology feasible for the preparation of the radio-
alabelled complex 5b. It may also be possible to accelerate the
reaction by use of internal alkynes in the so-called strain-
promoted Cu-free cycloaddition reaction.23
an alkyne precursor 6b. Yet more optimization will be required
when using complexes 7b–7d in the ‘‘real life’’ radiolabeling,
wherein it is expected, that no purification of 7b–7d will be
carried out. Subsequent ‘‘click’’ reaction of 7b–7d with an
alkyne precursor 6b is expected to be carried out in ‘‘one-pot’’
fashion, followed by the high pressure liquid chromatography
(HPLC) purification of the desired bimodal MRI/PET(SPECT)
molecular probes.
‘‘Click’’ reaction between an alkyne precursor 6b and azide
precursors 7b–7d
The methodology for the ‘‘click’’ CuAAC chemistry of DOTA-
derived alkynes has been recently developed in our labora-
tory.10 The reactions were carried out in a mixture of i-PrOH/
H2O, using excess of the azide (1.5–8 equivalents) in the
presence of CuSO4?5H2O (0.25 equivalents) and sodium
ascorbate (1.2 equivalents). We decided to use the identical
conditions to carry out the first reaction between an alkyne
building block 6b and a Cu2+ derived azide building block 7b
(1.2 equivalents have been used, Scheme 4). Very slow progress
in the reaction was observed (HR-MS spectrometry) even after
extended period of heating to 70 uC (48 h). Neither addition of
CuSO4?5H2O and sodium ascorbate, nor increasing the
temperature to 80 uC appeared to speed up the reaction.
It is well documented that addition of agents capable of the
formation of transient Cu chelates can be used to increase the
rate of the ‘‘click’’ CuAAC reactions.13 We turned our attention
to N,N9-dimethyl ethylenediamine (DMEDA) an inexpensive
and widely available reagent often used for this puropse.21
Addition of 1.2 equivalents (based on the alkyne 6b) of DMEDA
was found to increase the rate of the reaction dramatically,
resulting in the completion of the reaction in 18 h at 60 uC as
indicated by HR-MS (complete consumption of alkyne 6b). Of
On the other hand, incubation of the alkyne 6b with an In3+
containing azide 7d revealed ca. 70% conversion to the Gd3+/
In3+ heterometallic complex 5c after incubation for 15 min at
80 uC (Fig. 3), full conversion was observed in the sample
withdrawn after 1 h.
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Fig. 2 HR-ESI-MS spectrum of Gd3+/In3+ heterometallic complex 5c showing a proper charge state envelope (701.7, M2+ charge state; 468.1, M3+ charge state) and
isotope pattern. Insets show the agreement between the observed (bottom) and simulated (top) charge states. HR-ESI-MS spectra of associated with complexes 5a
and 5b can be found in the ESI.3
To summarize the results from this study, our methodology is
suitable for the preparation of radiolabeled heterometallic
complexes related to Gd3+/Cu2+ complex 5a and Gd3+/In3+
complex 5c, while more work is warranted to make the presented
methodology compatible with the preparation of radiolabeled
heterometallic complexes related to Gd3+/Ga3+ complex 5b.
Fig. 3 HPLC chromatograms of reaction mixtures before the placement in a heating block (panels A, C) and after being incubated for 15 min at 80 uC (panels B, D).
The slow formation of Gd3+/Ga3+ heterometallic complex 5a (tR 7.6 min) from the alkyne building block 6b (tR 5.3 min, panels A, B) is shown at the top, while the
rapid formation (ca. 70% conversion after 15 min at 80 uC) of Gd3+/In3+ heterometallic complex 5a (tR 9.6 min) from the alkyne building block 6b (tR 5.6 min, panels C,
D) is shown at the bottom.
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Fig. 4 Chemical structure of Dotarem (Gd3+ DOTA, 16).
MRI sensitivity associated with the potential bimodal MRI/
PET(SPECT) molecular probes 5a–5c
Since the detection sensitivity associated with MRI is several
orders of magnitude lower than imaging modalities involving
radioisotopes (PET, SPECT), the ability to detect the bimodal
MRI/PET(SPECT) molecular probes by MRI therefore repre-
sents a limiting factor. To obtain more information on the
MRI sensitivity associated with the molecular probes 5a–5c we
measured their relaxivities and compared them to those
Fig. 5 NMRD profiles in water at a concentration of 2.0 mM and at 37 uC. Top:
Dotarem, 16. Bottom: Gd3+/In3+ heterometallic complex 5c.
associated with
a clinically used MRI contrast agent
Dotarem2b (Gd3+ DOTA, 16, Fig. 4).
NMRD profiles associated with heterometallic complexes
5a–5c were acquired using fast field cycling NMR in water at 25
uC and 37 uC (see Experimental for details). The dependence of
the relaxivity associated with 5a–5c on the strength of the
by UV light, phosphomolybdic acid staining (compound 7a) or
anisaldehyde staining (compound 15). Size exclusion chroma-
tography (SEC) was peformed using BIO-GEL P2, 45–90 mm
mesh resin (15 g, per 0.23 mmol of compound). Ten fractions
(10 ml each) were collected and identified by UV and I2
vapours, fractions containing complex 6b were combined and
lyophilized. The absence of free Gd3+ in 6b was verified by a
xylenol orange test.26 Dialysis was performed for 5 days against
water using cellulose ester membrane with molecular weight
cut off 500 Da. HPLC analysis and purification was performed
using a Resolve CN column (particle size 10 mm; 8 6 100 mm
Radial-Pak cartridge). Mobile phase: Method A: 90% H2O/10%
MeCN – 31% H2O/69% MeCN over 13 min, linear gradient and
flow rate 3 ml min21. Ultra performance liquid chromato-
graphy (UPLC) was performed using a BEH C18 column
(particle size 1.7 mm; 1.0 id 6 100 mm) and HR-ESI-MS
detector. Mobile phase: Method B: 100% H2O – 100% MeCN
over 6 min, linear gradient, flow rate 0.25 ml min21. NMR
magnetic field (Fig. 5, ESI ) correlates well with the same
3
measurement carried out for Dotarem (16, Fig. 5). The shape
of the curves confirms the presence of one molecule of water
exchanging with Gd3+ centre for all of the complexes 5a–5c
(Fig. 5, ESI ).24
3
The relaxivity of the complexes 5a–5c at 20 MHz (ca. 5.0–5.5
mmol21 21) is slightly higher, compared to the relaxivity of
s
Dotarem (16, ca. 4.5 mmol21 21).2b This is likely attributable
s
to the increase in molecular size of the probes 5a–5c, resulting
in a somewhat slower rate of molecular tumbling in solution.25
The remaining NMRD profiles associated with complexes 5a–
5c acquired at 25 uC and 37 uC can be found in the ESI.
3
Overall, the MRI sensitivity of the potential bimodal MRI/
PET(SPECT) molecular probes 5a–5c compares well with the
MRI sensitivity of clinically used MRI contrast agent Dotarem
(16). The presence the second metal ion does not affect the
MRI sensitivity of complexes 5a–5c.
1
spectra were recorded on 400 MHz spectrometer; for H (400
MHz), d values were referenced as follows: CDCl3 (7.27 ppm),
D2O (4.75 ppm), for 13C (100 MHz) CDCl3 (77.0 ppm). Mass
spectra (MS) were obtained using an electron spray ionization
Experimental
(ESI ) Time-of-Flight (TOF) instrument, or chemical ionization
3
(CI) (compound 15). FTIR spectra (in KBr) were acquired using
an IR spectrometer. Longitudinal relaxation rates in the range
from 0.01–42 MHz were acquired using a Stelar fast field-
cycling nuclear magnetic resonance relaxometer at 25 uC and
37 uC. The samples [complexes 5a–5c, Dotarem (16)] were
dissolved in water to achieve the final concentration 2.0 mM.
General experimental procedures
All reagents were commercially available unless otherwise
stated. All solvents were HPLC grade and used as recieved.
Water was deionized (18.2 MV cm21 Millipore water) and
CH2Cl2 and THF were dried over Al2O3 in a solvent purification
system. Organic extracts were dried with Na2SO4 and solvents
were removed under reduced pressure using a rotary evapora-
tor. Aqueous solutions were lyophilized. Flash column
chromatography (FCC) was carried out using silica gel, mesh
size 230–400 Å. Thin layer chromatography (TLC) was carried
out on Al backed silica gel plates, compounds were visualized
Alkylation of DO3A-OtBu (8) with N-chloroacetyl-Gly-L-
Tyr(O-propargyl)-OMe (9)
DO3A-OtBu (8, 257 mg, 0.5 mmol) was dissolved in MeCN (10
ml), followed by the addition of K2CO3 (180 mg, 1.3 mmol) and
N-chloroacetyl-Gly-L-Tyr(O-propargyl)-OMe
(9,
prepared
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according to the literature,10a 183 mg, 0.5 mmol). The mixture
was stirred for 18 h at 60 uC. The solvent was evaporated, the
residue was partitioned between water (30 ml) and EtOAc (2 6
30 + 20 ml). The combined organic extract was dried and was
concentrated. The residue was triturated with hexanes,
affording DO3A-OtBu-Gly-L-Tyr(O-propargyl)-OMe (6a, 400
mg, 95%), colorless solid. 1H NMR (CDCl3) d 9.45 (m, D2O
exch., 2H), 7.18–6.77 (br m, 4H), 4.63 (m, 3H), 3.84–2.17 (br m,
68.2, 42.6, 39.7. HRMS (ESI ) m/z; found 228.0793 [M + H]+
3
(calcd 228.0791 for C11H15ClNO2). FTIR: n 3299, 3066, 2950,
1720, 1660, 1536, 1452, 1272, 1106, 1028, 713 cm21
.
Alkylation of DO3A-OtBu (8) with N-chloroacetyl-O-benzyl
2-ethanolamine (12), formation of azide modified ligand 7a
DO3A-OtBu (8, 515 mg, 1 mmol) was dissolved in MeCN (15
ml), followed by the addition of K2CO3 (359 mg, 2.6 mmol) and
N-chloroacetyl-O-benzyl 2-ethanolamine (12, 228 mg, 1 mmol).
The mixture was stirred for 18 h at 60 uC. The solvent was
evaporated, the residue was partitioned between water (40 ml)
and EtOAc (40 + 30 ml). The combined organic extract was
dried and was concentrated. The residue was triturated with
hexanes, providing DO3A-OtBu-Ac-O-benzyl 2-ethanolamine
(13, 670 mg, 95%) of sufficient purity for the subsequent
32H), 1.42 (m, 27H). HRMS (ESI ) m/z; found 845.5052 [M + H]+
3
(calcd 845.5024 for C43H69N6O11). FTIR: n 3244, 2974, 2821,
1727, 1674, 1510, 1368, 1306, 1227, 1162, 1107, 584 cm21
.
Metallation of ligand 6a with GdCl3?6H2O
DO3A-OtBu-Gly-L-Tyr(O-propargyl)-OMe (6a, 192 mg, 0.23
mmol) was dissolved in a mixture of t-BuOH (6 ml) and H2O
(3.5 ml). GdCl3?6H2O (85 mg, 0.23 mmol) was added and the
mixture was refluxed with stirring for 48 h. t-BuOH was
evaporated and the aqueous solution was subjected to size
exclusion chromatography (SEC) as described in General
experimental procedures. The fractions containing the product
were combined and lyophilized to afford Gd3+ DO3A-OH-Gly-L-
Tyr(O-propargyl)-OH (6b, 155 mg, 83%), colorless solid.
reaction. HRMS (ESI ) m/z; found 706.4772 [M + H]+ (calcd
3
706.4755 for C37H64N5O8).
DO3A-OtBu-Ac-O-benzyl 2-ethanolamine (13, 653 mg, 0.93
mmol) was dissolved in t-BuOH (10 ml) and AcOH (1 ml) and
10% Pd/C (1 g) was added. The mixture was stirred vigorously
for 48 h at rt under an atmosphere of H2. The catalyst was
filtered off using a small plug of CELITE, the filter was washed
with MeOH and the filtrate was concentrated to leave 625 mg
of an oily residue containing inseparable mixture of desired
DO3A-OtBu-Ac 2-ethanolamine and some other unknown
impurity. The crude mixture was dissolved in CH2Cl2 (10
ml), followed by cooling the reaction mixture to 0 uC. SOCl2
(220 ml, 3.04 mmol) was added dropwise (over ca. 1 min
period), the cooling bath was removed and the mixture was
stirred for 18 h at rt. The mixture was diluted with CH2Cl2 (10
ml) and was washed with saturated NaHCO3 solution (20 ml).
The aqueous phase was extracted with CH2Cl2 (2 6 10 ml),
combined organic extract was dried and was concentrated to
leave a brown solid residue (610 mg), containing the mixture
of the desired DO3A-OtBu-Ac 2-chloroethylamine along with
other unknown impurity. The residue was used for the next
HRMS (ESI ) m/z; found 816.1987 [M + H]+ (calcd 816.1976
3
for C30H40N6O11Gd). FTIR: n 3392, 3256, 3066, 2870, 1614,
1511, 1398, 1323, 1242, 1085, 1022, 980, 904, 721, 591 cm21
.
An unmetallated version of the complex 6b can be obtained,
if the reaction is carried out under the same experimental
conditions replacing GdCl3?6H2O with diluted HCl (1 M
solution, 250 ml), followed by the removal of t-BuOH by
evaporation and lyophilizing the aqueus phase. DO3A-OH-Gly-
L-Tyr(O-propargyl)-OH (150 mg, 98%) is obtained as pale
brown solid. Analytical samples can be obtained upon
purification by SEC as described above. 1H NMR (D2O) d
7.12 (m, 2H), 6.92 (m, 2H), 4.69 (m, 2H), 4.54 (m, 1H), 3.77–
2.87 (br m, 29H). HRMS (ESI ) m/z; found 663.2985 [M+ H]+
3
(calcd 663.2990 for C30H43N6O11).
step without further purification, HRMS (ESI ) m/z; found
3
N-Chloroacetyl-O-benzyl 2-ethanolamine (12)
634.3972 [M + H]+ (calcd 634.3947 for C30H57ClN5O7).
N-Boc-O-benzyl 2-ethanolamine (11) was prepared from
2-ethanolamine (10) using a literature protocol.15,16 Removal
of the Boc protecting group from 11 was carried out as
described in the literature.16 A crude O-benzyl 2-ethanolamine
(3.59 g, 23.7 mmol) was dissolved in dry THF (70 ml), the
solution was cooled to 0 uC, followed by the addition of K2CO3
(6.56 g, 47.5 mmol). A solution of chloroacetyl chloride (2.83
ml, 35.6 mmol) in dry THF (10 ml) was added dropwise (over
ca. 10 min period) to a stirred reaction mixture, while the
temperature was maintained at 0 uC. The stirring continued
for 1 h at 0 uC and for 3 h at room temperature (rt). The
reaction was quenched by slow addition of water (5 ml), the
solvent was evaporated and the residue was partitioned
between saturated NaHCO3 solution (50 ml) and EtOAc (50 +
30 ml). The combined organic extract was dried and was
concentrated, the residue was subjected to FCC on 50 g SiO2,
hexanes-acetone (2 : 1). Evaporation of the eluate afforded
N-chloroacetyl-O-benzyl 2-ethanolamine (12, 2.54 g, 47%) as
The residue obtained in the previous step was dissolved in
DMF (3 ml), followed by the addition of NaN3 (195 mg, 3
mmol). The mixture was stirred for 4 h at 80 uC, was cooled to
rt and was diluted with saturated NaHCO3 solution (40 ml),
followed by the extraction with EtOAc (2 6 30 ml). Combined
organic extract was washed with brine (2 6 60 ml), was dried
and was concentrated. The residue was subjected to FCC on 60
g SiO2, CH2Cl2–MeOH (9 : 1), the fractions containing the
product were identified by phosphomolybdic acid staining.
Evaporation of the eluate afforded the azide modified ligand
7a (96 mg, 15% based on 13) as colorless solid. 1H NMR
(CDCl3) d 9.24 (br s, D2O exch., 1H), 3.42–2.23 (br m, 28H), 1.45
(m, 27H). HRMS (ESI ) m/z; found 641.4367 [M + H]+ (calcd
3
641.4350 for C30H57N8O7). FTIR: n 3176, 2976, 2820, 2098,
1729, 1668, 1558, 1454, 1368, 1308, 1266, 1163, 1108, 976, 852,
591 cm21
.
N-Chloroacetyl 2-azidoethylamine (15)
1
slightly yellow oil. H NMR (CDCl3) d 7.33 (m, 5H), 6.95 (br s,
2-Azidoethylamine (14, prepared according to the literature,18
976 mg, 11.34 mmol) was placed in the round bottom flask
which was flushed with N2. Dry THF (25 ml) was added and the
D2O exch., 1H), 4.53 (s, 2H), 4.03 (s, 2H), 3.58 (m, 2H), 3.52 (m,
2H); 13C NMR (CDCl3) d 165.9, 137.7, 128.5, 127.9, 127.8, 73.2,
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resulting solution was cooled to 278 uC, followed by the
addition of Et3N (4.1 ml, 29.47 mmol). Chloroacetyl chloride
(1.17 ml, 14.74 mmol) was added dropwise (via syringe, over
ca. 3 min period), the mixture was stirred in the atmosphere of
N2 while the cooling bath was allowed to warm up gradually
and was completely removed after 2 h. After being stirred for
further 2 h (N2 atmosphere) was the mixture diluted with brine
(80 ml) and was extracted with EtOAc (3 6 30 ml). Combined
organic extract was dried and was concentrated, the residue
was subjected to FCC on 60 g SiO2, hexanes-acetone (2 : 1), the
fractions containing the product were identified by anisalde-
hyde staining. Evaporation of the eluate afforded
N-chloroacetyl 2-azidoethylamine (15, 1.01 g, 55%) as slightly
yellow oil. 1H NMR (CDCl3) d 6.90 (br s, D2O exch., 1H), 4.07 (s,
2H), 3.50 (m, 4H); 13C NMR (CDCl3) d 166.2, 50.5, 42.5, 39.1.
HRMS (CI) m/z; found 163.0380 [M + H]+ (calcd 163.0386 for
C4H8ClN4O); LRMS (CI) m/z (rel. abundance): 163 [M+] (83),
120 (18), 106 (100), 72 (29). FTIR: n 3294, 3077, 2937, 2096,
534.1612 for C18H31N8O7Cu). FTIR: n 3211, 3052, 2937, 2101,
1671, 1554, 1249, 590 cm21
.
Ga3+ modified azide building block 7c (30 mg, 53%), pale
yellow solid. HRMS (ESI ) m/z; found 539.1471 [M 2 2H]+
3
(calcd 539.1493 for C18H30N8O7Ga). FTIR: n 3375, 2963, 2102,
1669, 1605, 1404, 1312, 1088, 925, 593 cm21
.
In3+ modified azide building block 7d (31 mg, 53%),
colorless solid. HRMS (ESI ) m/z; found 585.1262 [M 2 2H]+
3
(calcd 585.1276 for C18H30N8O7In). FTIR: n 3400, 2963, 2108,
1629, 1388, 1328, 1087, 931, 585 cm21
.
‘‘Click’’ CuAAC reaction between the alkyne building block 6b
and the azide building blocks 7b–7d
Separate mixtures containing the alkyne building block (6b, 68
mg, 8.3 6 1025 mol) and the azide building blocks (7b and 7c,
54 mg; 7d, 59 mg; 0.1 mmol each) in i-PrOH (0.5 ml) and H2O
(1.5 ml) were treated with CuSO4?5H2O (0.1 M solution, 170 ml,
1.67 6 1025 mol), sodium ascorbate (0.1 M solution, 670 ml, 6.7
6 1025 mol) and DMEDA (11 ml, 0.1 mmol). The mixtures were
stirred for 18 h at 60 uC, were cooled to rt and were transferred
into centrifuge tubes. Insoluble portions were removed by
centrifugation (10,000 rpm for 2 min), the supernatants were
transferred into HPLC vials and were subjected to the HPLC
purification (Method A) as described in General experimental
procedures. The fractions containing the desired heterometallic
complexes 5a–5c were concentrated, followed by dialysis (see
General experimental procedures) and lyophilization.
1655, 1534, 1434, 1262, 1102, 762 cm21
.
Alkylation of DO3A-OtBu (8) with N-chloroacetyl
2-azidoethylamine (15)
DO3A-OtBu (8, 257 mg, 0.5 mmol) and N-chloroacetyl
2-azidoethylamine (15, 89 mg, 0.55 mmol) were dissolved in
MeCN (4 ml), followed by the addition of K2CO3 (180 mg, 1.3
mmol). The mixture was stirred for 18 h at 60 uC. The solvent
was evaporated, the residue was partitioned between water (20
ml) and EtOAc (2 6 20 ml). Combined organic extract was
dried and was concentrated, the residue was triturated with
hexanes to afford the azide modified ligand 7a (314 mg, 98%).
Analytical samples can be obtained by FCC as described above,
for the spectral characterization of 7a see above.
Gd3+/Cu2+ heterometallic complex 5a (25 mg, 22%), pale blue
solid. HPLC: t 8.4 min. HRMS (ESI ) m/z; found 1351.3684 [M 2
3
R
2H]+ (calcd 1351.3666 for C48H72N14O18CuGd). FTIR: n 3394,
2925, 1616, 1395, 1322, 1242, 1085, 589 cm21
.
Gd3+/Ga3+ heterometallic complex 5b (22 mg, 19%), color-
less solid. HPLC: t 7.9 min. HRMS (ESI ) m/z; found 1357.3512
3
R
[M 2 4H]+ (calcd 1357.3489 for C48H70N14O18GaGd). FTIR: n
Metallation of the azide modified ligand 7a with CuCl2?2H2O,
Ga(NO3)3?H2O and InCl3?4H2O
3390, 2926, 1673, 1615, 1397, 1318, 1085, 937, 587 cm21
.
Gd3+/In3+ heterometallic complex 5c (42 mg, 36%), colorless
A solution of azide modified ligand 7a (192 mg, 0.3 mmol) in
CH2Cl2 (3 ml) was treated with TFA (1.5 ml). The mixture was
stirred at 18 h at rt, the solvent was evaporated and the excess
TFA was removed by coevaporation with PhCH3 (2 6 20 ml) and
CH2Cl2 (2 6 20 ml), providing fully deprotected azide modified
ligand (141 mg, quantitative) as colorless solid. 1H NMR (D2O) d
solid. HPLC: t 8.7 min. HRMS (ESI ) m/z; found 1402.3400 [M
3
R
2 3H]+ (calcd 1402.3331 for C48H71N14O18GdIn). FTIR: n 3396,
2923, 1621, 1391, 1326, 1085, 936, 584 cm21
.
Optimization of the reaction conditions for the ‘‘click’’
reaction between the alkyne building block 6b and the azide
building blocks 7b–7d
3.73 (m, 8H), 3.37–3.24 (br m, 20H). HRMS (ESI ) m/z; found
3
473.2464 [M + H]+ (calcd 473.2472 for C18H33N8O7).
Separate vials containing solutions of the alkyne building
block 6b (1025 mol); the azide precursors 7b, 7c or 7d (1025
mol each); CuSO4?5H2O (2.5 6 1026 mol); sodium ascorbate
(1.25 6 1025 mol) and DMEDA (1025 mol) in 1 ml of H2O were
incubated for 1 h at 80 uC in a heating block. Samples (100 ml)
were withdrawn prior to the placement of the reaction vial to
the heating block, then after being incubated at 80 uC for 15
min and 1 h. The samples were diluted with H2O to reach the
total volume of 500 ml, followed by an HPLC analysis (Method
A, General experimental procedures).
Separate solutions of deprotected azide modified ligand (47
mg, 0.1 mmol) in water (1.5 ml) were treated with CuCl2?2H2O
(19 mg, 0.11 mmol), Ga(NO3)3?H2O (28 mg, 0.11 mmol) and
InCl3?4H2O (32 mg, 0.11 mmol) and the pH of each reaction
was adjusted (1 M NaOH) to reach the optimum complexation
kinetics (pH 5.5 for Cu2+, pH 3 for Ga3+ and pH 6.5 for In3+).3b
The mixtures were stirred for 30 min at 95 uC, were cooled to
room temperature and were neutralized (pH 7, 1 M NaOH).
The aqueous solutions were transferred into dialysis bags and
were dialyzed (see General experimental procedures). The
solutions were then transferred into centrifuge tubes, were
frozen and were lyophilized to provide azide building blocks
7b–7d as solid residues.
Conclusions
Cu2+ modified azide building block 7b (36 mg, 67%), blue
With the aim to establish a general methodology for the
synthesis of dual MRI/PET(SPECT) molecular probes, we have
solid. HRMS (ESI ) m/z; found 534.1628 [M 2 H]+ (calcd
3
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presented the preparation of an alkyne modified Gd3+ derived
complex along with a synthesis of a small series of azide
modified complexes metallated with metal ions relevant to
PET and SPECT (Cu2+, Ga3+, In3+). ‘‘Click’’ CuAAC chemistry
was utilized to connect these building blocks. Crucially, a
common starting material which is commercially available
(DO3A-OtBu) was used to assemble both the alkyne- and azide
functionalized counterparts.
Ontario Institute for Cancer Research) and NSERC (National
Science and Engineering Council of Canada).
References
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A preliminary optimization of the reaction conditions for
the metallation of the azide modified building block with
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preliminary optimization for the ‘‘click’’ CuAAC chemistry
utilized to connect the respective building blocks. This
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Gd3+/In3+ heterometallic complexes; however, it is not suitable
for the Gd3+/Ga3+ complex for which more study is required.
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radioactive isotopes. The results of these studies will be
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Acknowledgements
We thank Dr Mohamed Moustafa for the preparation of
2-iodoethylammonium iodide, a precursor to 14. We are
indebted to Drs. Francisco Martinez and Timothy J. Scholl
(Department of Medical Biophysics, The University of Western
Ontario, London, Ontario, Canada) for the acquisition of
NMRD profiles. We thank Mr. Pierangello Gobbo for the
assistance with the acquisition of FTIR spectra. Mr. Mark
Milne is thanked for fruitful discussions.
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