Synthesis and characterization of a new lanthanide based MRI contrast agent, potential and versatile tracer for multimodal imaging

Article abstract of DOI:10.1016/j.tet.2014.06.126

In the present work a modular pathway towards the synthesis of a new versatile MRI contrast agent is reported and its physico-chemical properties are described. Two different functional groups were attached on two arms of the gadolinium 1,4,7,10-tetraazac

Full text of DOI:10.1016/j.tet.2014.06.126

Tetrahedron 70 (2014) 5450e5454
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and characterization of a new lanthanide based MRI
contrast agent, potential and versatile tracer for multimodal imaging
Satya Narayana Murthy Chilla , Sophie Laurent ,*, Luce Vander Elst a,b,
Robert N. Muller
a
a
a
a,b
Department of General, Organic and Biomedical Chemistry NMR and Molecular Imaging Laboratory, University of Mons, Avenue Maistriau, 19,
Mendeleiev Building, 7000 Mons, Belgium
Centre for Microscopy and Molecular Imaging (CMMI), Rue Adrienne Bolland 8, 6041 Charleroi-Gosselies, Belgium
b
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present work a modular pathway towards the synthesis of a new versatile MRI contrast agent is
reported and its physico-chemical properties are described. Two different functional groups were at-
tached on two arms of the gadolinium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA) in
order to get a platform able to bind one probe designed to target specific biological marker and a fluo-
rescent molecule likely to be used for optical imaging. The nuclear magnetic relaxation dispersion
Received 27 February 2014
Received in revised form 24 June 2014
Accepted 30 June 2014
Available online 3 July 2014
(
NMRD) profile, the oxygen-17 relaxometric NMR study and stability assessment versus transmetalation
Keywords:
of the Gd-complex show that this new contrast agent has a relaxivity and transmetalation stability
similar to GdeDOTA.
Imaging agents
Macrocyclic ligands
Gadolinium
Relaxivity
Ó 2014 Elsevier Ltd. All rights reserved.
GdeDOTA
1. Introduction
Ligand design has poor influence on the electron spin relaxation,
whereas
M
s can be tuned over several orders of magnitude by im-
3þ
In past two decades, clinical Magnetic Resonance Imaging (MRI)
has been developing very rapidly. However unambiguous medical
diagnosis often requires the help of MRI contrast agents (CAs).
These contrast agents are mainly paramagnetic complexes of
Gd(III) or superparamagnetic nanoparticles. The chemistry of nu-
merous paramagnetic complexes has been described in several
posing steric hindrance around the water-binding site in Gd
complexes of both linear (DTPA-type) and macrocyclic (DOTA-type)
ligands. Introduction of an extra methylene group on the backbone
1
of the ligands DOTA and DTPA leads to chelators TRITA and EPTPA,
3
þ
respectively, whose Gd chelates display a water exchange around
two orders of magnitude faster than the parent GdeDOTA and
reviews and books1 and derivatives designed to optimize chemical
,2
GdeDTPA.
complexation has largely contributed to the advances of magnetic
6e8
The development of multifunctional ligands for Gd
3þ
3
and magnetic properties have been proposed. The efficacy of
2
,3
a contrast agent is measured by its relaxivity (r
i
with i¼1,2), the
resonance imaging (MRI) in biomedical research. These ligands,
which could be more versatile, allow for conjugation of the Gd-
chelate with specific biological vectors or for the optimization of
their efficacy. Ideally, a multifunctional chelator should integrate
optimal properties for metal complexation with easy and versatile
synthetic possibility for conjugation. Grafting vectors on GdeDOTA,
as mono or diamide chelates has been reported. However, it is well
known that complexes bearing amide-chelating units are less sta-
ble and exhibit slower water exchange rates that are detrimental to
paramagnetic relaxation rate of water proton normalized to a 1 mM
concentration of Gd3 . Several tactics were devised to increase the
relaxivity of the paramagnetic metal complexes typically by in-
creasing: (i) the molecular size of the paramagnetic system by co-
valent or non-covalent interaction with macromolecules to slow
þ
4
down the rotational motion in the medium, (ii) the number of
5
coordinated water molecules (q), (iii) the number of paramagnetic
metal centres, linking the single complexes in multimeric systems.
The significant parameters determining the relaxivity of the com-
9e11
relaxivity.
Taking into account all these features of Gd-chelates
plexes are the rotational correlation time (
s
R
), the water residence
(stability and physico-chemical properties), we preferably design to
prepare DOTA derivatives due to their well-recognized kinetic in-
ertness and thermodynamic stability, even if the synthesis of
macrocyclic chelates is more difficult and more time consuming
than the preparation of non-cyclic analogues. We synthesized and
time ( ) and the electron spin relaxation times (sS1,2).
s
M
*
Corresponding author. Tel.: þ32 65373525; fax: þ32 65373533; e-mail address:
sophie.laurent@umons.ac.be (S. Laurent).
http://dx.doi.org/10.1016/j.tet.2014.06.126
0
040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

S.N.M. Chilla et al. / Tetrahedron 70 (2014) 5450e5454
5451
R
R
R
characterized a new trendy and versatile agent GdeDOTAe[Amino
OH
OH
OBn
(S)
(
S)
(S)
Pentyl-Succinic Acid] APSA (GdeDOTAeAPSA), which provides
pendant functional groups for conjugation to selected probes.
These conjugations are preferably carried out via selective chem-
istry among amine and acid functionalities, which provide stable
compounds. This new derivative is obtained by selective alkylation
of two of the nitrogens of the macrocycle by acetate arms bearing
different functional groups allowing conjugation through amide or
urea linkage.
H
2
N
Br
Br
O
O
O
1
1
a, R = CH
b, R = (CH
2
CO(OBn)
NH CO (OBn)
2a, R = CH
2b, R = (CH
2
CO(OBn)
NH CO (OBn)
3, R = CH
4, R = (CH
2
CO(OBn)
NH CO (OBn)
2
)
4
2
)
4
2
)
4
O
OtBu
OBn
O
O
OBn
NH HN
NH HN
NH
N
N
N
N
N
Cbz-Cl, CHCl
3
tert-Butylbromoacetate
CO ACN
72%
97%
K
2
3
,
O
O
N
HN
OBn
OBn
O
OtBu
5
6
7
O
O
2
. Results and discussion
O
O
O
OtBu
OtBu
OtBu
OBn
OBn
OBn
OBn
(
R)
(R)
N
N
N
N
N
N
N
HN
N
The trans-DO2A 8 was synthesized by selective protection of N-1
Pd/C, H (g)
3, K
2
CO
3
,
ACN
4, K
2
CO
3
,
ACN
2
O
O
MeOH
56%
NH
38%
O
and N-7 nitrogens of cyclen by benzylchloroformate and further
NH
N
9
1%
1
2,13
(R)
alkylation and deprotection as previously described
derivatives 2a and 2b were synthesized from the benzyl esters of
aspartic acid and benzylurea derivative of -lysine by a method
analogous to Holmberg followed by Steglich esterification, which
provided S-dibenzyl 2-bromosuccinate and benzyl (2S)-6-
[(benzyloxy) carbonyl]amino}-2-bromohexanoate 4. The syn-
thetic building blocks 3 and 4, proved to be efficient alternatives to
unstable) reagents, such as alkene or tosylate-derivatives. Building
blocks 3 and 4 were obtained in high yield. They react easily with
the trans-DO2A 8 via S 2-type alkylation but the second alkylation
. The bromo
BnO
O
4
O
O
OtBu
OtBu
OtBu
O
L-
HN
8
9
BnO
10
L
O
O
O
O
O
OtBu
3
OH
OH
O
O
O-
O
OH
1
4
(R)
OH
(R)
{
(R)
N
N
N
N
O
GdCl
3
N
N
N
Pd/C, H
2
(g)
20%TFA in DCM
N
N
N
N
O
Gd3+
O
O
MeOH
95%
O
OH
N
(
(R)
HO
O
O
(R)
HO
(R)
4
1
O
4
+
NH
O
OtBu
4
H
2
N
HO
3
O
O
2
H N
N
1
12
Gd-DOTA-APSA
needs a little longer reaction time due to steric hindrance of the
fourth amine. The mono alkylation of 8 has been optimized by
varying the equivalents of bromide 3 in the presence of different
Scheme 1. Synthesis of GdeDOTAeAPSA.
2 3
bases. Although using K CO resulted in the worst selectivity of
1
2
mono versus bis alkylation, it resulted in the best overall yields for
the monoprotection reaction to yield 9. The final alkylation of 9 was
performed alike but by using excess of the bromo analogue under
refluxing condition and yielded 10. Compounds 9 and 10 were
obtained with an excellent optical purity of 100% (determined by
chiral HPLC). The debenzylation of 10 was performed under
hydrogenolysis yielding 11 quantitatively. The final deprotection
procedure with TFA afforded the DOTA-(APSA) chelator 12. Purifi-
cation of 12 performed by RP-chromatography yielded the tri-
fluoroacetate salt (analytical purity) with a reasonable yield. It is to
be noted that the ethyl esters of the bromo analogues of 3 and 4
were synthesized and tested but during the final basic hydrolysis,
a semi-hydrolyzed product precipitation occurred, leading to
incompletion of reaction.
1
0
8
6
4
2
0
Gd-DOTA-APSA
Gd-DOTA
0
.01
0.1
1
10
100
1000
Proton Larmor Frequency (MHz)
3 2
Finally complexation was performed with GdCl $6H O by
Fig. 1. 1H NMRD profiles of GdeDOTAeAPSA (triangles) & GdeDOTA (white circles).
The lines represent simulations using the best-fit parameters (see Table 1).
maintaining pH between 6.2 and 6.7. The excess of gadolinium ions
was removed by using chelex. The absence of excess ions was
confirmed by xylenol orange test. The complex was purified by
reverse phase chromatography.
The functional groups (e.g., eNH , COOH) of our complex allow
2
selective conjugation to a wide variety of organic moieties or (bio)
macromolecules, via amine as well as carboxylic acid functions
The water residence time was determined from variable-
17
temperature O NMR studies. The temperature dependence of
17
the reduced O transverse relaxation rates (1/T2r) (Fig. 2) is typical
of a water residence time of the order of 100 ns at 310 K. The
(
Scheme 1), for purposes of targeting and/or optimization of
R
s . It is
16
M
analysis of the data using the usual equations gives a s value of
to be noted that a metal chelator with one amine group, DO3A-N-
aminopropionate (a-amino-DOTA), has already been reported.
a
-
1
5
73 ns, a value lower than that of GdeDOTA (Table 1) but larger than
that reported for GdeDOTMA, a more crowded macrocyclic Gd
The straightforward synthesis and the versatility of further conju-
gation of our new chelator make this system an excellent multi-
functional ligand for the development of imaging agents. The full
synthesis and characterization of the ligand are described in the
Experimental section. We should note, however, that the non-
complexed ligand is not compatible with an extended use in con-
jugation reactions like peptide couplings.
17
complex. The NMRD profile was fitted using the inner sphere
SolomoneBloembergeneMorgan) and outer sphere (Freed)
theories. As expected considering the molecular weight of
GdeDOTAeAPSA, its value of is increased as compared to
GdeDOTA (Table 2). Finally the value of SO is decreased and similar
to that of GdeHPDO A.
(
s
R
s
3
Proton relaxivity measurements in water revealed that the
GdeDOTAeAPSA chelate is stable in the pH range extending from 3
to 8. The magnetic field dependence of the proton longitudinal
2.1. Transmetalation
water proton relaxivities (r
1
NMRD profile) measured at 310 K
The Gd3 chelates can be sensitive to transmetalation by en-
þ
dogenous ions, such as Cu , Ca and Zn . Among the three
metals mentioned, Zn has a high affinity for the Gd complexes.
2þ
2þ
2þ
shows that the relaxivity of our new complex is slightly higher than
that of the parent compound GdeDOTA (Fig. 1).
2þ

5452
S.N.M. Chilla et al. / Tetrahedron 70 (2014) 5450e5454
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1
e+6
Gd-DOTA-APSA
Gd-DOTA
Gd-DTPA
1e+5
0.0026
0.0028
0.0030
0.0032
0.0034
0.0036
0.0038
-
1
1
/T(K
)
2000
4000
6000
8000
Time (min)
Fig. 2. Temperature dependence of the reduced 17O transverse relaxation rate of Gd
DOTAeAPSA (26.156 mM).
Fig. 3. Relaxation rate RP
(t)/RP 2þ
1 0
(t ) versus time of 2.5 mM Zn aqueous solution for
1
GdeDOTAeAPSA (2.5 mM), GdeDOTA (2.5 mM) and Magnevist (2.5 mM).
Table 1
Parameters obtained by the theoretical fitting of the 17O experimental NMR data (q
was fixed to 1)
3. Conclusion
GdeDOTAa
A new contrast agent was synthesized and characterized. This
contrast agent shows promising relaxivity, which could further be
optimized by reducing s and provides versatile functional groups
M
for getting multimodal contrast agent by grafting targets and
chromophores. The modular synthetic pathway described can be
further implemented in the synthesis of wide variety of specific
MRI contrast agents.
Parameters
GdeDOTAeAPSA
3
10
s
m
[ns]
[kJ mol
73ꢃ11
52.7ꢃ0.23
61.4ꢃ0.46
ꢁ3.64ꢃ0.13
1.79ꢃ0.12
2.7ꢃ0.2
122ꢃ10
50.1ꢃ0.2
48.7ꢃ0.2
ꢁ3.42ꢃ0.03
1.94ꢃ0.09
11.4ꢃ0.5
4.0ꢃ4.4
s
ꢁ1
]
D
H
s
ꢁ1
D
S
[kJ mol
]
6
ꢁ1
)
A/Z (10 rad/s
2
0 ꢁ2
B [10
s ]
2
98
s
v
[ps]
[kJ molꢁ1]
E
V
20.0ꢃ3.1
a
Ref. 18.
4. Experimental section
Table 2
Parameters obtained by 1H NMRD relaxivity profile of Gd-complex in water at 37 Ca
4.1. General
ꢀ
GdeDOTAeAPSAa
GdeDOTAa,b
Cyclen was purchased from Chematech (France) while all
Parameters
chemicals were purchased from SigmaeAldrich (Belgium), Across
s
s
s
v
[ps]
[ps]
(so) [ps]
8.4ꢃ1.3
73.5ꢃ3.5
193ꢃ18
7ꢃ1
53ꢃ1.3
404ꢃ24
(
Belgium). All chemicals were used without further purification. All
R
1
13
H and C NMR spectra were recorded on Bruker Avance 500 MHz
ꢀ
a
spectrometer at 25 C using 5 mm sample tubes. The chemical
The
following
parameters
were
fixed:
(D
is
the
diffusion
coefficient¼3ꢂ10 m2
ꢁ
9
s
ꢁ1
, d is the distance of closest approach¼0.36 nm, r is the
shifts are given in (d) parts per million. The purification of the
distance between the proton of the inner sphere water molecule and Gd
compounds was performed on KP-silica cartridges from Biotage
over Biotage flash chromatography instrument, Uppsala, Sweden.
For the ligand purification KP-C18 Biotage cartridges were used. The
pH values of the solution were adjusted using aqueous solution 1 M
NaOH and HCl. Mass spectra were obtained on Q-TOF Ultima mass-
spectrometer (Micromass, Manchester UK). Samples were dis-
1
7
ion¼0.31 nm, q is fixed to 1 and
m
s was fixed to the value determined by O NMR).
b
Ref. 18.
Therefore, this metal ion is able to replace a significant amount of
Gd , which may result in release of the toxic Gd aqua ion in the
body. To investigate the kinetic stability of GdeDOTAeAPSA, the
proton longitudinal relaxation rate of a mixture of Gd complex
and an equal amount of ZnCl in phosphate buffer was as usually
3
þ
3þ
ꢁ
1
solved in H
2
O/MeOH and injected at flow rate 5
m
l min . The cone
ꢀ
3
þ
voltage was 40 V (T¼90 C). The compounds 2a, 2b, 6, 7 and 8 were
synthesized as reported in Ref. 13. Chiral HPLC was performed on
2
ꢀ
Ò
11
Alliance Waters 2695, ChiralPak
AD-RH column
5
mm
monitored at 20 MHz and 37 C. Upon transmetalation by a dia-
magnetic Zn2 ion in phosphate buffer, the released Gd pre-
þ
3þ
(4.6ꢂ150 mm), mobile phase: acetonitrile/water (40/60), flow rate:
0.5 ml/min, detection by Waters PDA 486.
cipitates as GdPO , which does not contribute to the relaxivity.
4
Therefore, the overall relaxivity of the solution decreases over time
with a rate depending on the rate of transmetalation. This decrease
in relaxivity is a good estimation of the kinetic lability of the Gd
4.2. Experimental procedures
3
þ
complexes (Fig. 3). For example, Magnevist shows significant
decomplexation during a time period of 5 days. On the contrary,
GdeDOTAeAPSA is virtually inert towards transmetalation with
4.2.1. Synthesis of (S)-dibenzyl 2-bromosuccinate (3). A solution of
2a (9.3 g, 32.1 mol), benzyl alcohol (5 mL, 48.2 mol), N,N-dicyclo-
hexylcarbodiimide (DCC) (10.0 g, 48.5 mol) and 4-dimethylami-
nopyridine (DMAP) (catalytic) in CH Cl (25 mL) was stirred at
2
þ
the Zn ion at pH 7.0 for extended periods of time, appearing
2
2
3
þ
therefore similar to Gd
GdeHPDO A
3
complexes like GdeDOTA and
room temperature for 3 h. The precipitated dicyclohexylurea was
filtered off and the filtrate was washed with water, dried over an-
hydrous Na SO and evaporated under reduced pressure. The crude
8,11
(Fig. 3). These data show that the replacement of
two acetate by aminobutylacetate and succinate arms does not
2
4
lower significantly neither the thermodynamic nor the kinetic
product was purified by flash chromatography to give 3 (7.29 g) as
19
1
stabilities as compared to GdeDOTA.
3
colourless oil yield 60%. H NMR (500 MHz, CDCl ) d: 7.35e7.28 (m,

S.N.M. Chilla et al. / Tetrahedron 70 (2014) 5450e5454
5453
1
1
0H), 5.23e4.99 (m, 4H), 4.69e4.43 (m,1H), 3.30 (dd, J¼17.2, 8.9 Hz,
pressure and yielded 12 quantitatively. H NMR (500 MHz, D
2
O)
d:
13
1
H), 3.00 (dd, J¼17.2, 6.1 Hz, 1H); C NMR (500 MHz, CDCl
3
) d:
4.32e4.21 (m, 1H), 4.1e3.42 (m, 8H), 3.09e2.9 (m, 15H), 2.89e2.61
(m, 5H), 1.65e1.52 (m, 4H), 1.38 (s br, 2H). C (500 MHz, D
176.07, 170.43, 169.23, 60.43, 57.49, 57.04, 56.56, 56.25, 52.41, 51.15,
13
169.38, 168.81, 135.32, 135.07, 128.69, 128.57, 128.59, 128.40, 128.26,
2
O)
d:
þ
6
7.90, 67.04, 39.81, 38.18; ESI-MS (C18
H17BrO
4
); m/z: 378 [MþH] .
5
0.56, 49.84, 46.61, 45.46, 45.37, 44.12, 43.21, 38.68, 38.42, 28.19,
4
.2.2. Syntheis of benzyl (2S)-6-{[(benzyloxy) carbonyl]amino}-2-
25.94, HRMS (ESI): calcd for C22
534.2799.
H
40
N
5
O
10 [MþH]þ 534.2775; found
bromohexanoate (4). A solution of compound 2b (1.03 g, 2 mmol),
benzyl alcohol (0.33 mL, 3 mmol), N,N -dicyclohexyl carbodiimide
0
4.2.6. Preparation of Gd3 complex of DOTA-[APSA]. The complex
was obtained by adding portion wise GdCl $6H O to a solution of
(0.1 g, 0.18 mmol) in (5 mL) H O, maintaining the pH between 6.2
þ
(
DCC) (0.678 g, 3.2 mmol) and 4-dimethylaminopyridine (0.03 g,
0
3
.3 mmol) in CH
2
Cl
2
(10 mL) was stirred at room temperature for
3
2
h. The precipitated dicyclohexylurea was filtered off and the fil-
2
trate was washed with 5% aq AcOH (10 mL) and water (3ꢂ10 mL),
dried over anhydrous Na SO and evaporated under vacuum. The
crude product was purified by column chromatography to give 4
and 6.7 by addition of 1 M NaOH. The final pH of the solution after
stirring 52 h was 6.3. The excess of the free lanthanide was removed
2
4
3
as Gd(OH) precipitate, which appeared at pH 9 after addition of
1
(
0.77 g, 60%) as pale yellow oil. H NMR (500 MHz) d: 7.51e7.27 (m,
1 M NaOH. The resulting solution was treated with chelex-100 to
3
þ
3þ
1
1
0H), 5.26e5.03 (m, 4H), 4.71 (d, J¼11.9 Hz, 1H), 4.24 (t, J¼7.2 Hz,
remove free Gd ions. The absence of free Gd ions was con-
firmed by a xylenol orange test. The pH of the supernatant was
decreased to 7 and the solution was freeze-dried. The complex was
dissolved in water, purified by reverse phase flash chromatography
and freeze-dried, yielding 80 mg of GdeDOTAeAPSA as a white
H), 3.17 (dd, J¼12.4, 6.1 Hz, 2H), 2e2.1 (m br, 2H), 1.7 (s br, 2H), 1.5
þ
(
s br, 2H) ESI-MS (C21
H
24BrNO
4
) m/z¼434 [MþH] .
4.2.3. Synthesis of dibenzyl (2R)-2-[4,10-bis(2-tert-butoxy-2-ox-
oethyl)-1, 4, 7,10-tetraazacyclododecan-1-yl]succinate
9). Compound 3 (0.532 g, 1.4 mmol) and (0.3 g, 2.26 mmol) K CO
3
powder. HRMS (ESI): calcd for
707.1569; found 707.1545.
C
22
H36GdN
5
O
10Na [MþNa]þ
(
2
are added to a solution of (562 mg, 1.4 mmol) 8 in 100 mL ACN, after
which the mixture is heated at 50 C for 8 h. The solids are filtered
ꢀ
3þ
4.2.7. Transmetalation. The stability of the Gd
complexes was
1
off over a pad of Celite and the solvent is removed in vacuo.
Chromatographic purification (silica gel, 98/1.5/0.5 DCM/MeOH/
TEA) yielded 555 mg of 9 as slight yellow oil. Yield 56% with 100%
determined by a transmetalation method monitoring the H lon-
ꢀ
11
gitudinal relaxation rates of water during 5 days at 37 C. The
measurements were performed on a Bruker Minispec mq-20 spin
analyzer at 20 MHz (Bruker, Karlsruhe, Germany) using 7 mm
sample tubes containing 2.5 mM of Gd complexes and 2.5 mM of
ZnCl in 300 l of phosphate buffer solution (26 mM KH PO
41 mM Na HPO
, pH¼7).
optical purity. H NMR (500 MHz, CDCl
3
)
d
: 7.48e7.12 (m, 8H), 4.04
3
þ
(
(
1
dd, J¼10.0, 4.0 Hz, 1H), 3.41e3.11 (m, 4H), 2.92e2.4 (m, 14H), 2.49
13
dd, J¼23.6, 9.9 Hz, 4H), 1.46 (s br, 18H); C (500 MHz, CDCl
71.89, 171.41, 170.91, 135.78, 135.73, 128.52, 128.48, 128.33, 128.28,
28.26,128.17,127.12,126.75, 80.88, 66.47, 66.42, 57.13, 56.08, 53.41,
3
),
d
:
2
m
2
4
,
2
4
1
5
C
2.03, 51.69, 50.00, 49.86, 46.81, 28.20. HRMS (ESI): calcd for
4.2.8. 17O relaxometric measurements. 17O NMR measurements
were performed at 11.75 T on 350 L samples contained in 5 mm
o.d. tubes on a Bruker Avance 500 spectrometer (Karlsruhe, Ger-
þ
38 57
H N
4
O
8
[MþH] 697.4176; found 697.4150.
m
4
5
.2.4. Synthesis of dibenzyl(2R)-2-[7-((1R)-1-[(benzyloxy)carbonyl]-
-{[(benzyloxy)carbonyl]amino}pentyl)-4,10-bis (2-tert-butoxy-2-
many). Temperature was regulated by air or nitrogen flow con-
trolled by a Bruker BVT 3200 unit. O transverse relaxation times of
17
oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]succinate
10). Compound 4 (0.465 g, 1.08 mmol) and K CO (0.2 g,
.4 mmol) are added to a solution of (500 mg, 0.72 mmol) 8 in
60 mL) ACN, after which the mixture is heated under reflux con-
distilled water (pH 6.5e7) were measured using a CPMG sequence
ꢀ
(
1
(
2
3
and a subsequent two-parameter fit of the data points. The 90 and
ꢀ
17
180 pulse lengths were 27.5 and 55 ms, respectively. The O T of
2
water in complex solution was obtained from line width mea-
ditions for 18 h. The solids are filtered off over a pad of Celite and
the solvent is removed in vacuo. Chromatographic purification
surements. All spectra were proton decoupled. The data are pre-
R
sented as the reduced transverse relaxation rate {1/T
2
¼55.55/([Gd
p
(
silica gel, 83/15/2 Acetone/MeOH/TEA) yielded 286 mg of 10 as
yellow oil. Yield 38% with 100% optical purity. H NMR (500 MHz,
CDCl
complex]ꢂqꢂT
2
), where: [Gd complex] is the molar concentration
of the complex, q is the number of coordinated water molecules
p
3
)
d
: 7.37e7.22 (m, 20H), 5.33e4.91 (m, 9H), 4.27 (dd, J¼7.5,
.3 Hz, 1H), 3.43e3.03 (m, 9H), 2.83e2.13 (m, 17H), 1.92e1.33 (m,
3H); 13C (500 MHz, CDCl
: 175.96, 174.17, 173.30, 173.24, 173.17,
and T
2
is the paramagnetic transverse relaxation rate}. The fitting of
16
4
2
the experimental data was performed as previously described.
3
)
d
The sample concentration was determined by ICP-AES on a Jobi-
þ
1
1
6
4
71.41, 156.51, 136.83, 135.38, 135.23, 134.88, 128.68, 128.65, 128.53,
28.41, 128.26, 128.03, 127.93, 82.16, 82.06, 67.47, 67.04, 66.82,
6.37, 61.03, 58.39, 56.04, 53.95, 52.84, 52.59, 48.93, 48.77, 47.67,
7.23, 46.05, 44.92, 44.81, 44.35, 41.90, 40.52, 29.88, 29.26, 28.98,
nYvon JY 70 instrument (Longjumeau, France) and was further
1
confirmed by H relaxometry of a decomplexed sample.
4.2.9. Proton NMRD. Proton nuclear magnetic relaxation disper-
sion (NMRD) profiles were measured on a Stelar Spinmaster FFC
(Mede, Italy) fast field cycling NMR relaxometer over a magnetic
field range from 0.24 mT to 1.0 T. Measurements were performed
on 0.6 mL samples contained in 10 mm o.d. Pyrex tubes. Additional
relaxation rates at 20 and 60 MHz were obtained on a Minispec
mq20 and a Minispec mq60, respectively.
2
8.18, 27.93, 26.94, 26.18, 24.95, 24.47, 22.52, 22.08. HRMS (ESI):
calcd for C59
H
80
N
5
O
12 [MþH]þ 1050.5803; found 1050.5787.
4
.2.5. Synthesis of (2R)-2-[7-[(1R)-5-amino-1-carboxypentyl]-4,10-
bis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl] succinic acid
12). 10% Pd/C (50 mg) is added to a solution of (280 mg,
.27 mmol) 10 in (20 mL) MeOH and the resulting suspension is
shaken for 6 h under H atmosphere. The suspension was filtered
over a Celite pad and the solvent was removed in vacuo. Crude 11
163 mg) was obtained as slight yellow oil. The crude was pro-
ceeded for TFA hydrolysis without further purification. The crude
was dissolved in (10 mL) 20% TFA in CH Cl stirred for 16 h at room
temperature. The reaction mixture was concentrated and washed
with Et
O (2ꢂ25 mL) and the residue was dried under reduced
(
0
2
Acknowledgements
(
This work was supported by the ARC (AUWB-2010-10/15-
UMONS-5) Programs of the French Community of Belgium and the
FNRS (Fond National de la Recherche Scientifique). The support and
sponsorship concerted by COST3 Actions (D38 and TD1004) and of
the EMIL program are acknowledged. The authors thank the Centre
2
2
2

5454
S.N.M. Chilla et al. / Tetrahedron 70 (2014) 5450e5454
for Microscopy and Molecular Imaging (CMMI, supported by the
European Regional Development Fund and the Walloon Region).
The authors thank Prof. Anne-Lise Hantson from Department of
Applied Chemistry and Biochemistry, University of Mons for pro-
viding chiral HPLC analyses.
8. Laurent, S.; Vander Elst, L.; Vroman, A.; Muller, R. N. Helv. Chim. Acta 2007, 90,
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