MRI probes based on C6-peracetate β-cyclodextrins: Synthesis, gadolinium complexation and in vivo relaxivity studies

Article abstract of DOI:10.1016/j.poly.2018.03.013

The initial synthesis of two β-cyclodextrin derivatives bearing seven carboxylate ligands was optimized in order to improve the production of contrast agents. A speciation study using potentiometric analysis was performed on a gadolinium(III) complex. The

Full text of DOI:10.1016/j.poly.2018.03.013

Accepted Manuscript
MRI probes based on C6-peracetate β-cyclodextrins: synthesis, gadolinium
complexation and in vivo relaxivity studies
Anais Biscotti, Cécile Barbot, Lionel Nicol, Paul Mulder, Célia Sappei, Marie-
Hubert Roux, Isabelle Déchamps-Olivier, François Estour, Géraldine Gouhier
PII:
S0277-5387(18)30138-4
https://doi.org/10.1016/j.poly.2018.03.013
POLY 13079
DOI:
Reference:
To appear in:
Polyhedron
Received Date:
Accepted Date:
31 January 2018
5 March 2018
Please cite this article as: A. Biscotti, C. Barbot, L. Nicol, P. Mulder, C. Sappei, M-H. Roux, I. Déchamps-Olivier,
F. Estour, G. Gouhier, MRI probes based on C6-peracetate β-cyclodextrins: synthesis, gadolinium complexation
and in vivo relaxivity studies, Polyhedron (2018), doi: https://doi.org/10.1016/j.poly.2018.03.013
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MRI probes based on C6-peracetate -cyclodextrins: synthesis, gadolinium
complexation and in vivo relaxivity studies
a
a
b
b
b
b
Anais Biscotti , Cécile Barbot , Lionel Nicol , Paul Mulder , Célia Sappei , Marie-Hubert Roux ,
c
a
a
Isabelle Déchamps-Olivier , François Estour , Géraldine Gouhier *
a
Normandie Univ, COBRA, UMR 6014, FR 3038, INSA Rouen, CNRS, IRIB, IRCOF 1 rue Tesnière 76821
Mont-Saint-Aignan, France; Fax: 33-235522959; Phone: 33-235522909; E-mail:
geraldine.gouhier@univrouen.fr
b
Normandie Univ, INSERM U1096, IRIB, UFR de Médecine et de Pharmacie, 76821 Université de Rouen,
France
c
Institut de Chimie Moléculaire de Reims, UMR CNRS 7312, UFR des Sciences Exactes et Naturelles BP
1039, 51687 Reims Cedex 2, France
Abstract
The initial synthesis of two -cyclodextrin derivatives bearing seven carboxylate ligands was
optimized in order to improve the production of contrast agents. A speciation study using
potentiometric analysis was performed on a gadolinium(III) complex. The in vivo myocardic activity
was evaluated on mice. This study highlights the best efficiency for the cyclodextrin derivative having
free hydroxyl groups and validated the biomedical potential of the flexible cyclodextrin scaffold as a
cardiac MRI probe.
Keywords
Contrast Agent – Cyclodextrin –– MRI in vivo Study – Association Constant – Organic Synthesis
1
. Introduction
Medical imaging constitutes an essential field of current medicine. Its applications are numerous;
they allow a precise diagnosis of pathology to be established, help surgeons during an intervention
or follow the efficiency of a treatment [1]. The available techniques (scanner, scintigraphy,
echography, magnetic resonance imaging (MRI), fluorescence) are evolving with the aim of
decreasing the examination time, improving the quality of the images and ensuring the safety of the
patient. Among these techniques, MRI is often used when a doubt persists after radiographies,
ultrasounds or scans. This technique offers detailed images of organs and tissues with high spatial
resolution. The acquisition of these high-defined images is based on the nuclear magnetic resonance
of the protons of water molecules contained in the body, in the presence of an outside magnetic
field and electromagnetic excitement. Nevertheless, in spite of its numerous advantages, MRI suffers
from a weak sensibility to distinguish healthy and altered tissues. Therefore, the administration of a
contrast agent is required to significantly improve the contrast of images in the diagnosis of cerebral
or cardiac dysfunctions. Indeed, the use of paramagnetic or super-paramagnetic agents as MRI
probes modifies the rate of relaxation of the protons in the neighbourhood of the water molecules
[
2,3]. Ten million clinical scans are performed each year with the administration of contrast agents
to improve the efficiency of the medical diagnostic. For this purpose, chelates of gadolinium, as
1

Gd(III) ions, are the most used derivatives because the metallic ion, having seven unpaired electrons,
strongly enhances the paramagnetic properties. However, the Gd(III) ion is toxic in its free form, and
thechelates have to be stable under physiological conditions in order to prevent the release of the
metallic ion in the organism [4,5]. Thus, the cyclic 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid (DOTA) and the linear diethylenetriamine pentaacetic acid (DTPA) contrast agents
are the most common chelating ligands clinically used. Their efficiencies are characterized by a
-
1 -1
relaxivity parameter noted r , with a value of 3.83 and 4.02 mM s for DOTA and DTPA (37°C, 20
1
MHz), respectively. In order to improve the in vivo relaxivity value and consequently the quality of
the image and to reduce the injection concentration, extensive efforts have been made to modify
the structure of these contrast agents. Further investigations use new scaffolds to extend the scope
of MRI applications. Among the interesting compounds, cyclodextrins are promising structures [6].
Indeed, these natural macrocycles composed of (1-4)-linked-D-glucose units can be easily modified
by introduction of contrast agent on the primary alcohol functions. Therefore, the lower face of
theses macrocyclic oligosaccharides was functionalized with DTPA [7,8], DOTA [913] and others
chelates [14] by click chemistry or using a peptide linker. Thus, the increase of mass of the MRI
probe decreases the reorientational correlation times ( ) and undergoes an enhancement of the
r
relaxivity. Moreover, cyclodextrins are soluble in water and present a hydrophobic cavity in which a
lipophilic guest can be included to form a so-called inclusion complex. Such intermolecular inclusion
complexes between lipophilic DOTA and DTPA in presence of -cyclodextrins were studied in MRI
and an enhancement of the relaxivity value was observed [15]. This structural modularity and the
remarkable properties of cyclodextrins were also exploited by our group and two contrast agents, 1
and 2, based on native and permethylated -cyclodextrins bearing seven acetate ligands on the
primary face have been synthesized, as shown in Fig. 1 [16,17].
Fig. 1. Structures of the contrast agents 1 and 2.
An in vitro study confirmed the positive impact of the cyclodextrin scaffold on the relaxivity values
-
1
-1
®
-1 -1
(
[
6.53 and 4.56 mM s for 1 and 2, respectively) compared to commercial DOTAREM (3.5 mM s )
16]. Despite the intensive effort focused on the use of cyclodextrins for MRI imaging, only a few in
vivo MRI studies using contrast agents based on Gd-cyclodextrin entities have been described.
Modified cyclodextrins [18-20], multimodal probes [21,22] and nanoparticles [23] were studied to
visualize cancer metastasis in small animals. Profiles of the retention of polyrotaxanes [24-26] and
the -cyclodextrin core dendrimer [27] in organs were also reported.
2

The present study is focused on in vivo experiments on mice in order to evaluate the efficiency of
the cyclodextrins 1 and 2, represented in Fig. 1, as myocardic contrast agents. Indeed, cardiovascular
disease is the main cause of death in the world [28]. Thus, patients with a cardiovascular disease or a
high risk need an early detection and a personalized diagnostic. Cardiac MRI is an imaging technique
that has undergone significant development in the past decade. The indications of an MRI
examination in cardiology are more and more numerous. To reach a selective biodistribution of the
contrast agent in the myocardic area, new probes bearing a recognition moiety have to be designed.
Beforehand, the MRI properties of the Gd complexes 1 and 2 have to be evaluated in vivo, as well as
their stability under physiological conditions and the first kinetic parameters of their elimination.
These studies require large amounts of the contrast agents, thus the initial synthesis of the two
precursors of 1 and 2 was first optimized for scaling up.
2
. Experimental Section
2
.1. Materials and methods
All solvents and reagents were purchased from commercial sources and used without further
purification. Reactions were monitored by thin-layer chromatography (TLC) on a plate of silica gel 60
F
254 (E. Merck, Darmstadt, Germany) and detection by charring with sulfuric acid. Column
1
13
chromatographies were performed on silica gel 60 (0.063-0.200 mm, E. Merck). H (300 MHz) and C
(
75.5 MHz) NMR spectra were recorded on a Bruker AVANCE 300. Chemical shifts in the NMR
spectra are reported in parts per million from TMS. IR spectra were recorded on a Perkin-Elmer IRFT
650 spectrometer. ESI-MS data were acquired using a HCT Ultra Ion Trap mass spectrometer
Bruker Daltonics, Bremen, Germany) or using a LCT Premier XE (Waters, Manchester, UK) for the
1
(
complexes. Experiments were achieved in the positive or negative ion mode using a protonated or
deprotonated molecule of bombesin as an internal reference (m/z 1619.8229 and 1617.8073
respectively). All MRI results were performed on a Biospec 47/40 4,7 T Avance III (Bruker Ettlingen)
with a 72 mm volume resonator and a 4 channel mouse heart receiver coil.
2
2
.2. Syntheses
.2.1. Heptakis-(6-O-tert-butyldimethylsilyl)-cyclomaltoheptaose 4
Under a nitrogen atmosphere, -cyclodextrin 3 (10 g, 9.43 mmol) was dissolved in 200 mL of
anhydrous pyridine and tert-butyldimethylsilyl chloride (11.7 g, 83 mmol) was added. The solution
was stirred at room temperature for 17 hours. The crude product was poured into 1.5 L of water.
The white precipitate obtained was then filtered and washed twice with 200 mL of water. The solid
was recrystallized in methanol. The pure white product 4 was obtained in 85% yield (15.5 g, 8.02
1
mmol). TLC (DCM/MeOH, 85:15, v:v) Rf = 0.43. White powder, m.p. > 260 °C. H NMR (300 MHz,
CDCl
3
), ppm: 5.05 (s, 7H, 7 x H-1), 4.45 (m, 7H, 7 x H-3), 4.10
(
m, 7H, 7 x H-6), 4.02-3.79 (m, 28H, 7 x H-4, 7 x H-2, 7 x H-5, 7 x H-6), 1.01 (s, 63H,
1
3
SiC(CH
3
)
3
), 0.17 (s, 42H, Si(CH
3
)
2
). C NMR (75 MHz, CDCl ), ppm:102.7 (7C, 7 xC-1), 82.4, 74.2,
3
7
4.0 (21C, 7 x C-4, 7 x C-2, 7 x C-3), 72.1 (7C, 7 x C-5), 62.2 (7C, 7 x C-6), 26.3 (21C, 7 x SiC(CH ) ), 18.5
3
3
3

+
-
(
7C, 7 x SiC(CH
3
)
3
), -4.8 (14C, 7 x Si(CH
3
)
2
). LRMS ESI-MS+ (m/z): 1955.1 [M+Na] . IR (ATR-D) max (cm
1
): 2928.1 (C-Halkyl).
2
.2.2. Heptakis-(6-O-tert-butyldimethylsilyl-2,3-di-O-benzyl)-cyclomaltoheptaose 5
NaH (60% in mineral oil, 0.83 g, 20 mmol) was dissolved in 5 mL of anhydrous DMF and the mixture
was stirred under an argon atmosphere at 0-5 °C for 15 minutes. A solution of 4 (1 g, 0.52 mmol) in
DMF (50 mL) was added dropwise to the NaH solution. The mixture was stirred for 30 minutes.
Benzyl bromide (2.85 mL, 24 mmol) was then added dropwise, maintaining a temperature of 0 to 5
°
C. After warming to room temperature, the mixture was stirred for 18 hours. The reaction was
quenched by the addition of MeOH at a temperature of 0 to 5 °C. The reaction medium was then
dissolved in 50 mL of pentane, washed with water (3 x 30 mL) and brine (50 mL). The organic layer
was dried over anhydrous Na
2
SO . After filtration, the solvent was removed under reduced pressure
4
and the residue was purified by chromatography on silica gel (pentane/DCM, 10:1 to 6:4, v/v) to give
compound 5 (1.36 g, 0.43 mmol) in 82% yield. TLC (Pentane/DCM, 9:1, v:v) Rf = 0.63. White powder,
1
m.p. > 260 °C. H NMR (300 MHz, CDCl
3
) , ppm: 7.19-6.95 (m, 70H, Har), 5.15-4.66 (m, 14H, 7 x CH
-Ar), 3.88-
), 0.04 (s, 42H,
), ppm: 139.3, 138.5, 138.3, (14C, 14 x C=CH), 128.0, 127.9, 127.7,
2
-
Ar), 4.62-4.42 (m, 14H, H-6), 4.40-4.20 (m, 7H, 7 x H-5), 4.15-3.92 (m, 14H, 7 x H-3, 7 x CH
.58 (m, 14H, 7 x H-4, 7 x CH
2
3
2
-Ar), 3.50-3.31 (m, 7H, H-2), 0.89 (s, 63H, SiC(CH
)
3 3
1
3
Si(CH
3
)
2
). C NMR (75 MHz, CDCl
3
1
7
6
27.5, 127.3, 126.8, (70C, 14 x C-Ar), 97.9, 97.2 (7C, 7 x C-1), 80.9, 79.3, 78.0, 75.5, 74.1, 73.0, 72.7,
2.6, 72.5, 72.45, 72.40, 72.2 (42C, 7 x C-2, 7 x C-3, 7 x C-4, 7 x C-5, 14 x CH -CAr), 62.6, 62.54, 62.50,
), -4.8, -5.2 (14C, 7 x Si(CH ).
2
2.4, 62.2 (7C, 7 x C-6), 26.0 (21C, 7 x SiC(CH
3
)
3
), 18.3 (7C, 7 x SiC(CH
3
)
3
)
3 2
2
+
-1
LRMS ESI-MS+ (m/z): 1618.9 [M+2Na] . IR (ATR-D) max (cm ): 2928.1 (C-Halkyl), 2856.3 (C-Halkyl),
1497.1 (C=C), 1454.7 (C=C), 1027.0 (C-O), 879.0 (C-O). Elemental analysis: Anal. Calcd for
C
182
H
252
O35Si
7
: C, 68.39; H, 7.95; Found: C, 70.81; H, 7.94%.
2
.2.3. Heptakis-(2,3-di-O-benzyl)-cyclomaltoheptaose 6
To a solution of compound 5 (0.9 g, 0.28 mmol) in 4 ml of THF, tetra-butylammonium fluoride in THF
1M, 4 mL, 4 mmol) was added at room temperature. The mixture was refluxed for 2 hours. The
solvent was removed under reduced pressure and the residue was dissolved in 5 mL of methylene
chloride. The organic layer was washed with 3 x 5 mL of brine and dried over MgSO . After filtration,
(
4
the solvent was evaporated under reduced pressure to give compound 6 (0.65 g, 0.27 mmol) in 95%
1
yield. TLC (DCM/AcOEt/MeOH, 5:5:1, v:v:v) Rf = 0.43. White powder, m.p. > 260 °C. H NMR (300
MHz, CDCl
3
), ppm: 7.07 (s, 70H, 70 x H-Ar), 4.95 (m, 7H, H1), 4.82-4.34 (m, 28H, CH
2
-Ar), 4.03-3.86
(
(
m, 21H, 7 x H-3, 7 x H-5, 7 x H-6), 3.77-3.70 (m, 7H, 7 x H-6), 3.58-3.48 (m, 7H, 7 x H-4), 3.41-3.39
1
3
m, 7H, 7 x H-2). C NMR (75 MHz, CDCl
3
), ppm: 139.1, 138.3 (CHAr -C), 128.1, 127.9, 127.7, 127.2,
1
7
2
26.8 (70C, 70 x CHAr-C), 98.1 (7C, 7 x C-1), 80.6, (7C, 7 x C-5), 79.3 (7C, 7 x C-4), 78.6 (7C, 7 x C-2),
5.0 (7C, 7 x CH
2
Ar), 73.0 (7C, 7 x C-3), 72.6 (7C, 7 x CH
2
-Ar), 61.9 (7C, 7 x C-6). LRMS ESI-MS+ (m/z):
+
-1
418.3 [M+Na] . IR (ATR-D) max (cm ): 3339.0 (O-H), 3029.2 (C-HAr), 2926.3 (C-Halkyl), 2867.4 (C-
H
alkyl), 1496.7 (C=C), 1454.0 (C=C), 1025.0 (C-O).
2.2.4. Heptakis-(6-O-allyl-2,3-dibenzyl)-cyclomaltoheptaose 7
To a solution of compound 6 (130 mg, 0.054 mmol) in 30 mL of THF, NaH (60% in mineral oil, 43 mg,
.08 mmol) was added at room temperature under an argon atmosphere. The mixture was stirred
1
for 30 minutes. Allyl iodide (319 mg, 1.9 mmol) was slowly added to the mixture and the reaction
was stirred for 18 hours at 50 °C. The reaction was quenched by the addition of 15 mL MeOH. The
4

solvent was removed under reduced pressure to give a yellow oil. The reaction mixture was then
dissolved in 30 mL of methylene chloride, washed with water (3 x 300 mL) and brine (100 mL). The
organic layer was dried over anhydrous Na
2
SO . After filtration, the solvent was removed under
4
reduced pressure and the residue was purified by chromatography on silica gel (cyclohexane/ethyl
acetate, 10:0 to 8:2, v:v) to give compound 7 (131 mg, 0.049 mmol) in 90% yield. TLC
1
(
cyclohexane/AcOEt, 8:2, v:v) Rf = 0.47. Colourless solid, m.p. > 260 °C. H NMR (300 MHz, CDCl
3
),
ppm: 7.28-7.13 (m, 83H from CDCl
CH=CH , 7 x H-5, 7 x H-1), 4.80-4.76 (m, 7H, 7 x CH
.92 (m, 42H, 7 x CH -CH=CH , 7 x H-4, 7 x H-6, 7 x CH
.50-3.48 (m, 7H, 7 x H2). C NMR (75 MHz, CDCl ), ppm:139.3 (7C, 7 x C-Ar), 138.3, (7C, 7 x C-Ar),
34.8 (7C, 7 x CH=CH ), 128.1, 128.0, 127.9, 127.5, 127.2, 126.9 (70C, 14 x CH-Ar), 116.9 (7C, 7 x
), 98.5 (7C, 7 x C-1), 80.9 (7C, 7 x C-3), 78.7 (14C, 7 x C-2, 7 x C-2), 75.4 (7C, 7 x CH -CH=CH
2.8 (7C, 7 x CH -Bn), 72.2 (7C, 7 x CH
3
, 70 x Har), 5.91-5.78 (m, 7H, CH=CH2, 5.26-5.07 (m, 28H, 7 x
-CH=CH ), 4.54-4.43 (m, 14H, 7 x CH -Ar), 3.95-
-Ar, 7 x H-3), 3.62-3.59 (m, 7H, 7 x H-6),
2
a
H
b
2
2
3
3
1
b
H
a
2
2
1
3
3
2
CH=CH
7
2
2
2
),
-
2
2
-Bn), 71.2 (7C, 7 x C-5), 69.0 (7C, 7 x C-6). IR (ATR-D) max (cm
1
): 2920.4 (C-Halkyl), 2854.0 (C-Halkyl), 1497.0 (C=C), 1454.0 (C=C), 1454.0 (C=C), 1027.4 (C-O). LRMS
+
ESI-MS+ (m/z): 2699.5 [M+Na] . Elemental analysis: Anal. Calcd for C161
H O35: C, 72.26; H, 6.85.
182
Found: C, 73.07; H, 6.66%.
2
.2.5. Heptakis-(6-O-dihydroxypropyl-2,3-di-benzyl)-cyclomaltoheptaose 8
Compound 7 (130 mg, 0.049 mmol) was dissolved in 3 mL of acetone, and 4-methylmorpholine-N-
oxyde monohydrate was added (171 mg, 1.47 mmol). The reaction medium was diluted with 2.5 mL
of water, followed by addition of osmium tetroxide (1.24 L of a 4 wt % solution in water, 4.9 mol).
The cloudy mixture was stirred at room temperature. After 1 hour, 2.5 mL of water were added. The
reaction was stirred for 48 hours at room temperature under an argon atmosphere. The reaction
was then quenched with a thiosulfate solution (5 mL) and the acetone was evaporated. The resulting
mixture was extracted with CH
2
Cl
2
(3 x 5 mL). The combined organic layers were dried over MgSO .
4
After filtration, the solvent was removed under reduced pressure. Compound 8 (142 mg, 0.049
1
mmol) was obtained as a grey solid in 99% yield. Grey powder, m.p. > 260°C. H NMR (300 MHz,
CDCl
x CH
MHz, CDCl
C-1), 80.2 (7C, 7 x C-3), 78.4, 75.1, 72.7, 71.3, 70.6, (42C, 7 x CH
3
), ppm: 7.15 (m, 70 x HAr), 5.01-4.43 (m, 42H, 7 x H-1, 7 x H-5, 14 x CH
-CH-OH, 7 x CH-CH -OH, 7 x H-3, 7 x H-2, 14 x H-6), 2.57-2.36 (s, 7H, 7 x CH
), ppm: 139.1, 138.2 (7C, 14 x C-Ar), 128.2, 127.2, 127.0, (70C, 14 x CH-Ar), 99.3 (7C, 7 x
-Ar, 7 x C-5, 7 x C-2, 7 x CH -CH-OH, 7
2
-Ar), 3.94-3.49 (m, 70H,
1
3
7
2
2
2
-OH). C NMR (75
3
2
2
-
1
x CH-CH
2
-OH), 63.5 (7C, 7 x C-6). IR (ATR-D) max (cm ): 3391.9 (O-H), 2921.4 (C-Halkyl), 2852.1 (C-
+
H
alkyl), 1456.0 (C=C), 1025.5 (C-O). LRMS ESI-MS+ (m/z): 2936.6 [M+Na] . Elemental analysis: Anal.
Calcd for C161
196
H O49: C, 66.33; H, 6.78; O, 26.89. Found: C, 66.98; H, 6.88%.
2
.2.6. Heptakis-(6-O-hydroxyethyl-2,3-di-O-benzyl)-cyclomaltoheptaose 9
Compound 8 (500 mg, 0.17 mmol) was dissolved in water (50 mL) and CH Cl (5 mL). The cloudy
mixture was strongly stirred at a temperature of 0 to 5 °C. NaIO (1.3 g, 6 mmol) was added. The
mixture was stirred for 2 hours at room temperature. The reaction medium was then cooled to 0 °C
and NaBH (519 mg, 13 mmol) was added portionwise. The mixture was strongly stirred for 1 hour at
°C and then for a further 30 minutes at room temperature. The excess NaBH was quenched with
HCl 2N. The resulting mixture was extracted with CH Cl (3 x 5 mL). The combined organic layers
were dried over MgSO . After filtration, the solvent was removed under reduced pressure. The
2
2
4
4
0
4
2
2
4
intermediate product 9 (459 mg, 0.17 mmol) was obtained quantitatively. Grey powder, m.p. > 260
1
°
C. H NMR (300 MHz, CDCl
3
), ppm: 7.36-6.91 (m, 70H, HAr), 5.14-3.34 (m, 105 H, 7 x H-1, 7 x H-2, 7
5

+
x H-3, 7 x H-4, 7 x H-5, 14 x H-6, 14 x CH
IR (ATR-D)  max (cm ): 2920.7 (C-Halkyl), 2851.9 (C-Halkyl), 1455.5 (C=C), 1025.8 (C-O).
2
-Ar, 7 x CH
2
-CH
2
-OH). LRMS ESI-MS+ (m/z): 2727.1 [M+Na] .
-
1
2
.2.7. Heptakis-(6-O-ethoxycarboxymethyl-2,3-di-O-benzyl)-cyclomaltoheptaose 10
The product 9 (459 mg, 0.17 mmol) was solubilized in a mixture of acetone/water (1:1, v/v). KBr
0.33 mL, 1 mmol), TEMPO (156 mg, 1 mmol) and a saturated NaHCO solution (0.16 mL) were
(
3
successively added. The solution was then cooled to 0 °C and NaClO (0.2 mL) was added. The pH was
adjusted by addition of NaOH (0.5M) and the mixture was stirred for 48 hours at room temperature.
The reaction was quenched with MeOH. The resulting mixture was extracted with diethyl ether (3 x
5
mL). The solvents were evaporated under reduced pressure and the residue was diluted in EtOH
(15 mL). DCC (90 mg, 0.44 mmol) and HOBt (67 mg, 0.44 mmol) were added and the mixture was
stirred for 18 hours under an argon atmosphere. After filtration, the solvent was removed under
reduced pressure. The crude product was purified by flash chromatography on silica gel
(
CH
2
Cl
2
/MeOH, 10:0 to 9:1, v/v). Compound 10 (80 mg, 27 mol) was obtained in 57% yield as a grey
1
solid. TLC (DCM/MeOH, 25:1, v:v) Rf = 0.54, Grey powder, m.p. > 260 °C. H NMR (300 MHz, CDCl
3
),
ppm: 7.47-6.89 (m, 70H, H-Ar), 5.30-3.47 (m, 105H, 7 x H1, 7 x H-2, 7 x H-3, 7 x H-4, 7 x H-5, 14 x H-6,
-
1
1
2
1
6
4 x CH
2
-Ar, 7 x CH
2
-CO
2
CH
3
, 7 x O-CH
2
CH
3
), 1.26-1.24 (m, 21H, 7 x O-CH
2
-CH ). IR (ATR-D) max (cm ):
3
920.70 (C-Halkyl), 2851.94 (C-Halkyl), 1645.90 (C=O), 1455.00 (C=C), 1454.52 (C=C), 1377.20 (C=C),
025.78 (C-O). Elemental analysis: Anal. Calcd for C168
6.52; H, 6.34%.
H O49: C, 67.28; H, 6.59; O, 26.14. Found: C,
196
2
.2.8. Heptakis-(6-O-ethoxycarboxyethyl)-cyclomaltoheptaose 11
Palladium on charcoal, 10% (100 mg) was added to a solution of compound 10 (500 mg, 0.17 mmol)
in methanol (15 mL). The suspension was purged three times with nitrogen and then subjected to a
flow of hydrogen. The reaction mixture was stirred for 18 hours at room temperature. The
suspension was filtered with a syringe nylon filter and washed with methanol. The filtrate was
concentrated to dryness to afford compound 11 (256 mg, 0.15 mmol) in 87% yield. TLC (DCM/MeOH,
1
8
5:15, v:v) Rf = 0.35. White powder, m.p. > 260 °C. H NMR (300 MHz, D
2
O), ppm: 4.95 (m, 7H, 7 x
CO), 1.25 (m, 21H,
O), ppm: 173.6 (7 x C=O), 96.8 (7C, 7 x C-1), 80.7 (7C, 7 x C-2), 79.9,
H-1), 4.16-3.48 (m, 56H, 7 x H-5, 7 x H-4, 7 x H-3, 7 x H-2, 7 x H-6, 7 x -OCH
2
1
3
OCH
2
CH
3
). C NMR (75 MHz, D
2
7
6.8 (7C, 7 x C-4, 7 x C-3), 70.2 (7C, 7 x C-5), 69.9 (7C, 7 x OCH
2
CO), 59.4 (14C, 14 x CH
3
) 68.7 (7C, 7 x
+
C-6). LRMS ESI-MS+ (m/z): 1759.6 [M+Na] . Elemental analysis: Anal. Calcd for C70
112
H O49: C, 48.39;
H, 6.50. Found: C, 47.85; H, 6.02%.
2
.2.9. Heptakis-(6-O-ethoxycarboxymethyl-2,3-O-methyl)-cyclomaltoheptaose 12
Under a nitrogen atmosphere, compound 11 (100 mg, 5.76 mol) was dissolved in 10 mL of
anhydrous THF. NaH (60% in mineral oil, 5.36 mg 0.14 mmol) was slowly added at 0 °C. Iodomethane
(0.02 mL, 0.29 mmol) was added dropwise over 1 hour and the reaction mixture was further stirred
for 18 hours at room temperature. The excess sodium hydride was decomposed by addition of
methanol (1 mL). The solvents were removed under reduced pressure. The crude product was
triturated in cyclohexane (5 mL) and the remaining solids were filtered off. The solvent was
evaporated under reduced pressure and the crude product was purified by flash chromatography on
silica gel.
The gradient elution was as follows:
6

-
-
initial conditions, 100% CH
2
Cl
/MeOH, 95:5, v/v), 20 min
Cl /MeOH, 95:5, v/v), 20 min.
2
, 0-20 min
linear increase to (CH
2
Cl
2
2
-
isocratic conditions, (CH
2
2
.2.10. Heptakis-(6-O-carboxymethyl)-cyclomaltoheptaose 13
Under a nitrogen atmosphere, compound 11 (150 mg, 0.86 mol) was solubilized in MeOH (3 mL).
An aqueous solution of sodium hydroxide 0.5 M (1 mL) was added. The reaction mixture was stirred
for 18 hours at room temperature. The solvent was removed under reduced pressure and the
solution was filtered on Dowex 50WX8-100 and freeze-dried to give compound 13 (83 mg, 0.54
1

mol) in 63% yield as a yellow solid. Yellow powder, m.p. > 260 °C. H NMR (300 MHz, D
2
O) , ppm:
5
3
1
.02 (m, 7H, 7 x H-1), 4.24-4.09 (m, 14H, 7 x CH
2
CO), 3.93-3.80 (m, 28H, 7 x H-5, 7 x H-4, 14 x H-6),
.64-3.57 (m, 14H, 7 x H-3, 7 x H-2). C-NMR (75 MHz, D O), ppm: 173.9 (7 x C=O), 101.8 (7C, 7 x C-
1
3
2
), 81.2 (7C, 7 x C-2), 72.9, (7C, 7 x C-4, 7 x C-5), 70.4 (7C, 7 x C-5), 69.6 (OCH
2
CO), 68.7 (7C, 7 x C-6).
-
LRMS ESI-MS- (m/z): 1539 [M-H] . Elemental analysis: Anal. Calcd for C56
84
H O49: C, 43.64; H, 5.49.
Found: C, 42.30; H, 5.87%.
2
.2.11. Heptakis-(6-O-carboxymethyl-2,3-O-methyl)-cyclomaltoheptaose 14
Compound 12, obtained in the previous step, was solubilized in MeOH (3 mL). An aqueous solution
of sodium hydroxide 0.5 M (1 mL) was added. The reaction mixture was stirred for 18 hours at room
temperature. The reaction medium was concentrated to remove MeOH under reduced pressure and
the resulting solution was filtered on Dowex 50WX8-100 and freeze-dried to give compound 14 (5
1
mg, 2.93 mol) in 51% yield as a white solid. White powder, m.p. > 260 °C. H NMR (300 MHz,
CDCl
3
), ppm: 5.28 (m, 7H, 7 x H-1), 4.15-4.03 (m, 14H, OCH
2
CO), 3.85 (m, 14H, 7 x 7 x H-4, 7 x H-6),
), 3.43 (s, 21H, 7 x CH ), 3.28
O), ppm: 173.6 (7C, 7 x CO), 96.8 (7C, 7 x C-1), 80.7 (7C, 7 x C-
), 79.9, 76.8 (7C, 7 x C-4, 7 x C-3), 70.2 (7C, 7 x C-5), 69.9 (7C, 7 x OCH CO), 68.7 (7C, 7 x C-6), 59.4
). LRMS ESI-MS- (m/z): 1735.8 [M-H] . Elemental analysis: Anal. Calcd for C70
3.74 (m, 14H, 7 x H-5, 7 x H-6), 3.71 (m, 7H, 7 x H-5), 3.52 (s, 21H, 7 x CH
3
3
1
3
(m, 7H, 7 x H-2). C NMR (75 MHz, D
2
2
2
-
(14C, 14 x CH
3
H O49: C,
112
48.39; H, 6.50. Found: C, 47.90; H, 6.36%.
2
.2.12. Gadolinium/lanthane complexes 1:1 1/2 from compounds 13/14
A solution of lanthanide chloride hexahydrate LnCl
3
.6H O (0.497 ml, 7.7 mM, Ln = Gd, La) was
2
prepared in MilliQ-water. The lanthanide solution was added to a water solution of 13/14 under
stoichiometric conditions. After each addition, the pH was adjusted to 7 with 1 M NaOH. The
complex was incubated at 25 °C, for 18 hours, and the pH increased to 8.5. Precipitation of the
residual free lanthanide occurred in the Ln(OH)
membrane filter and was lyophilized.
3
form. The sample was passed through a 0.45 µm
2
.2.13. Heptakis-(6-O-carboxymethyl)-cyclomaltoheptaose Gadolinium 1 was isolated as a white-
-
1
yellow solid. IR max (cm ): 3366, 2936, 1744, 1559, 1139, 1040. LRMS (ESI-MS-): m/z 563 [M+Gd-
3
-
4-
-
6H] , 422 [M+Gd-7H] . HRMS (ESI-MS-): m/z Calcd for C56
H
77
O
49Gd : 422.8198. Found: 422.8178.
2
.2.14. Heptakis(6-O-carboxymethyl-2,3-di-O-methyl)-cyclomaltoheptaose Gadolinium 2 was isolated
-
1
-
as a white solid. IR max (cm ): 3392, 1589, 1324, 1019. LRMS (ESI-MS-): m/z 1890 [M+Gd-4H] , 944
2
-
3-
4-
[
1
M+Gd-5H] , 629 [M+Gd-6H] , 471 [M+Gd-7H] . HRMS (ESI- MS-): m/z Calcd for C70
890.5200. Found: 1890.5232.
108
H O49Gd:
7

2
.2.15. Heptakis-(6-O-carboxymethyl)-cyclomaltoheptaose lanthane 13:La was isolated as a white-
yellow solid. In this case, the (-O-CH -CO) proton and carbon signals shifted respectively by 0.36 and
.2 ppm. The carbonyl functions shifted by 3.9 ppm. The C NMR spectrum showed only one signal
2
1
3
1
1
for (C=O) at δ 177.8 ppm. H NMR (300 MHz, D
CH CO, H-6), 3.67-3.49 (m, 28 H, H-5, H-4, H-3, H-2). C NMR (75 MHz, D
01.8 (C-1), 80.9 (C-2), 72.9 (C-4 or C-3), 71.8 (C-4 or C-3), 70.3 (C-5), 69.5 (O-CH
2
O) δ, ppm: 4.97 (s, 7 H, H-1), 3.84-3.79 (m, 28 H,
1
3
2
2
O) δ, ppm: 177.8 (C=O),
1

5
1
2
-CO), 68.8 (C-6). IR
-
1
-
2-
max (cm ): 3318, 1587, 1250, 1087, 1011. LRMS (ESIMS-): m/z 1675 [M+La-4H] , 837 [M+La-5H] ,
3
-
4-
58 [M+La-6H] , 418 [M+La-7H] . HRMS (ESI-MS-): m/z Calcd for C56
675.2860.
H O49La: 1675.2832 Found:
80
2
.2.16. Heptakis(6-O-carboxymethyl-2,3-di-O-methyl)-cyclomaltoheptaose lanthane 14:La was
1
isolated as a white solid. H NMR (300 MHz, D
OCH
2
O) δ, ppm: 5.28 (s, 7 H, H-1), 4.13-3.89 (m, 14 H,
), 3.46
2
CO), 3.75-3.62 (m, 35 H, H-6a, H-5, H-4, H-6b, H-3), 3.54 (s, 21 H, CH
3
1
3
(
2
s, 21 H, CH
3
), 3.30 (dd, J = 3 Hz, J = 9 Hz, 7 H, H-2). C NMR (75 MHz, D
), 79.8 (C-3), 77.5 (C-4), 70.5 (C-5), 71.2 (-OCH CO-), 69.6 (C-6), 59.4 (CH
). IR max (cm ): 3321, 1563, 1389,1031. LRMS (ESI-MS-): -): m/z 1871 [M+La4H] , 935
2
O) δ, ppm: 97.1 (C1), 80.7 (C-
2
3
),
-
1
-
5
[
7.9 (CH
3
2
-
3-
4-
M+La-5H] , 623 [M+La-6H] 467 [M+La-7H] . HRMS (ESI-MS-): m/z Calcd for C70
108
H O49La:
1871.5023. Found: 1871.5056.
2
.3. In vivo MRI Study
The investigations using animals conforms to the Guide for the Care and Use of Laboratory Animals
published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and has
been submitted for approval by the local ethical committee CENOMEXA n°54. To measure T1 (Table
1
), the sequence of measure of drip FAIR FISP was used and a series of images after adiabatic
inversion was acquired [29,30]. Two series of 32 images were generated with a time of repetition of
0 seconds between the impulses of inversion. The post-treatment was performed with ParaVision®
1
software 5.1 (Bruker Ettlingen). The correction of relaxation time was done according to the heart
rhythm of the animal to obtain the definitive mapping of T1. The experiments were performed on
three mice and 10 minutes after injection in the jugular vein.
Table 1
Relaxation time in various myocardiac areas of mice without any injection of contrast agent
(control), injection of commercial contrast agent Magnevist® and contrast agents 1 and 2.
Heart zones
Control
T1 (ms)
Magnevist®
T1 (ms)
Compound 1
Compound 2
(ms)
T1 (ms)
T
1
1
Left ventricle
Right ventricle
1738
1793
864
852
273
266
668
642
2
8

3
Septum
Left ventricular wall
Muscle
1141
1187
1346
926
886
575
695
859
856
4
5
1103
1127
1145
2
.4. Potentiometric equilibrium studies
All solutions were prepared with distilled water, filtered on 0.45 µm pore membranes (Pall Supor )
and degassed by argon saturation in order to remove all dissolved CO . Alkali metal ions have strong

2
tendencies to form partially dissociated complexes in solutions with carboxylate ions. The affinity of
this association increases with the number of carboxylic acid groups. Therefore, potentiometric
studies were performed using tetramethylammonium chloride (NMe
Tetramethylammonium chloride ( 99%, Fluka), tetramethylammonium hydroxide pentahydrate (
7%, Sigma) and glacial acetic acid (RPE, Carlo Erba) were used without further purification.
4
Cl) as a background electrolyte.
9
Automatic titrations were carried out with a 905 Metrohm Titrando apparatus equipped with an 800
Dosino 2 mL-burette, connected to a computer piloted by the Tiamo 2.2 software to control the
titrant delivery, data acquisition and to check for e.m.f. stability. The burette can deliver base
additions as low as 0.2 L. Sample solutions were titrated in a double-walled, glass-reaction vessel,
fitted with a sealed lid containing ports for the glass combination electrode (Metrohm #6.0234.100),
a Teflon anti-diffusion burette tip and a Teflon gas line. All titrations were done at 25  0.1 °C in a
thermostated cell using a thermocirculator bath (Julabo HE cryostat), under an argon stream to
avoid the dissolution of carbon dioxide. All equilibrium measurements were carried out in 4 mL
sample volumes under magnetic stirring. The ionic strength was adjusted to 0.1 N with NMe
4
Cl. 0.01
M HCl was prepared from standard Titrisol (Merck) and adjusted to I = 0.1 M with NMe Cl. The
4
measured potential of this solution before each sample acquisition was used to calculate the
standard potential for the hyperquad program in order to transform the potential curves into pH
curves. Carbonate-free NMe
phthalate (RPE, Carlo Erba) by potentiometry, recording the e.m.f. (mV) after each base addition.
The ionic product of water was determined by titrating an acetic solution in 0.1 M NMe Cl at
= 13.78. This value was used in the
4
OH solution (ca. 0.05 M) was standardized against potassium hydrogen
4
different concentrations. Under these conditions, pK
calculations.
w
2
.4.1. Protonation constants determination
The ligand stock solution was prepared by the dissolution of a weighted amount of the solid
hydrochloride salt of the ligand in an appropriate amount of 0.1 M NMe Cl solution. Titrations with
NMe OH were carried out in the presence of extra HCl to ensure that all the carboxylate groups
4
4
9

were protonated. The initial volume of the measured solution was 4 mL and ligand concentrations
-
3
were 0.3-1.0 x 10 M. Before the first point acquisition, the reaction cell was sealed, the solution
was stirred and sparged with argon for 10 minutes. Finally, the sample solution was titrated to ca.
pH 12 with standardized NMe
account three tracks, the volume of each base addition being fixed respectively at 10, 2 and finally
0 µL for the first to the last track. The electrode was monitored for up to 6 minutes or until its drift
4
OH in a monotonic mode. The method was written taking into
1
was less than 0.4 mV/min, before recording a mV reading. The protonation constants of 13 were
determined from eight titrations. The concentration of the ligand was calculated from
potentiometric data (e.m.f.) during the determination of the protonation constants, processed with
the general computation program HYPERQUAD [31]. In HYPERQUAD, the stability constants, β, (not
log β) are the parameters that are refined and by default, a standard deviation is labelled as
excessive if it is more than 33% of the parameter value. Although this value was user-definable, the
default value of 33% was used for this work.
The concentration protonation constants  are defined by the equation:
h
h

h
 [H
h
L]/[H] [L]
(with L = ligand, H = proton; the charges are omitted)
As the dissociation constant, K is commonly defined by:
a
K
a
 [H][Hh-1L]/[H L]
h
It is evident that pK
a
log logh1.
h
2
.4.2. Complex stability constants
Stock solutions of the metal chloride were prepared from commercially available reagents
gadolinium (III) chloride hydrate, Strem Chemicals) of purity higher than 99.9% in distilled water and
diluted to the required concentrations. The ionic strength was adjusted to 0.1 M with NMe Cl. The
(
4
exact concentration of the metal chloride stock solution was determined by complexometric
titrations with the ethylenediaminetetraacetic acid disodium salt dihydrate (Titriplex® III, Merck) in
acetate buffer (50 mM, pH 5.8) using xylenol orange indicator (Merck, ACS reagent). [32] The
stability constants of 1 were determined from seventeen titrations, from solutions containing 0-2
-
3
equivalents of metal, with a ligand concentration equal to 0.6-1.010 M and an HCl concentration,
-
3
when added, of 2.5 to 7.510 M. The same method was used for the metal-ligand stability
constants. The volumes of the increments were fixed at 4 µL.
The stability constants of the complexes are expressed by the general formula:
1
0

m
l
h
β
h
= [M
m
L
l
H ]/[M] [L] [H]
h
and the dissociation constant Kmlh can be defined similarly to the case of free ligand as:
K
mlh  [H][M
m
L
l
H
h-1]/[M
m
L
l
h
H ]
Analogously, pKmlh logmlh logmlh1
.
(with M = metal ion, L = ligand, H = proton; the charges are omitted)
The computer program HYSS was used to obtain the speciation distributions curves [31,33].
Logarithmic values of the protonation constants of 13 and of the overall stability constants of the
Gd(III) complexes were determined by potentiometric titrations; I = 0.1 M (NMe
3.78. Formation constants of the soluble gadolinium hydroxylated species GdH-1, GdH-2 and GdH-3
were taken from Kragten et al [34]. Their respective values were -7.3, -14.6, and -21.9. The pGd has
4
Cl), T = 298 K, pKw =
1
-
6
been calculated as a function of pH for a standard set of conditions (initial concentration: [Gd] = 10
-
5
M, [L] = 10 M). The variation of pGd (-log [Gdfree]) with pH gave useful information about the
stability of the complex under different pH conditions. The pGd value was calculated as a function of
-
6
-5
pH with a standard set conditions (initial concentration: [Gd] = 10 M, [L] = 10 M). pGd represents
the coordinated properties of the ligand for the metal and so has to be as high as possible to ensure
negligible dissociation of the toxic free Gd(III) ions. The affinity of the ligand 13 toward Gd(III) ions as
a function of pH was obtained by comparing the amount of free metal relative to the total amount of
metal introduced.
3
. Results and discussion
3
.1. Scale up of contrast agents 1 and 2
Larger amounts of contrast agents 1 and 2 were required in order to perform in vivo studies.
Therefore, the previously synthetic pathways had to be modified to improve the reaction yields [16].
The limiting step was the introduction of carboxylate functions on the primary face of the native -
CD, because a direct substitution of C-6 alcohols was not possible. Thus, the synthesis of 1 involved
allylation of secondary hydroxyl groups to avoid this problem. As the deprotection step was tricky
and time consuming to purify the corresponding product, we decided to develop a convergent
synthesis of 1 and 2. The new strategy is now based on the introduction of the carboxylate groups
using the methodology developed by Kraus, i.e. oxidation of allylic groups on the primary face of the
β-cyclodextrin 3 [35]. Using this approach, compounds 1 and 2 are obtained from the same precursor
1
1

11 (Scheme 1). This latest compound is prepared in a multi-step synthesis involving selective
benzylation of secondary hydroxyls groups (compound 6, Scheme 1) followed by the introduction of
allylic groups on the primary face (compound 7, Scheme 1). As compound 7 is soluble in organic
solvents, the use of sodium periodate and sodium borohydride as reagents to quantitatively access
to 9 required a heterogeneous mixture of solvents and high speed stirring (Scheme 2). The
subsequent esterification of 9, followed by catalytic hydrogenation of 10 finally led to the desired
derivative 11 in 23% yield on a gram scale.
1
2

Compound 12 (Scheme 3) was itself obtained after methylation of the secondary face. Saponification
of 11 and 12 gave the corresponding compounds 13 and 14, respectively. Addition of the gadolinium
chloride hexahydrate salt under controlled pH provided the desired contrast agents 1 and 2 [16].
Probes 1 and 2 were characterized by mass spectrometry analysis [16]. The nonacoordination of the
Gd centre was proved by luminescence experiments from the corresponding europium complexes,
showing respective hydration degrees of 2.75 and 2.43
[
16].
1
3

Scheme 3. Synthesis of contrast agents 1 and 2 from precursor 11.
1
H NMR spectroscopy of the lanthane complex [14:La] revealed a broadened peak with a chemical
shift of 0.27 ppm for the signal that corresponds to the methylene groups in the -position of the
carboxylate functions. This shielding is consistent with the Dehaen’s study on a DTPA derivative [36].
1
3
Similar comparisons of the C NMR spectra showed a deshielding of 4 ppm for the peak
corresponding to the carbonyl groups, as represented in Fig. 2.
1
3
Fig. 2. C NMR spectra of compound 14 and its lanthane complex [14:La] in D O.
2
1
4

The same results were obtained with complexes 14 and [14:La], with a shielding from 4.14 to 3.78
ppm for the signal of the methylene groups and a deshielding from 173 to 177 ppm for the peak of
the carbonyl groups [16].
3
.2. Stability study of the complex 1
Potentiometric analysis was also performed on the contrast agent 1 (GdL). The acidity constants
(pK
a
) of the seven carboxylic acid functions (listed in Table 2) were firstly determined from the
©
titration curves of 13 alone, with fitting by the Hyperquad program. An example of a titration curve
of 13 is shown in Fig. 3.
Fig. 3. Potentiometric titrations in presence or absence of 13 with [L] = 0.001 M, in NMe
.1 M, in presence of excess HCl ([HCl] = 0.01 M).
4
Cl
0
The successive pKa values of the Brönsted pairs increased according to the successive dissociation
into a polyprotic acid. Nevertheless, the delta pKa values of the pairs LH5/LH4 and LH6/LH5 differ
from the h “statistical factor” pKn+1-pKn = 0.6, which suggest that the involved dissociable groups
interact with each other.
Secondly the stability constants were adjusted from titration curves obtained by mixing 13 and the
lanthanide in a known stoichiometry, as represented in Fig. 4.
1
5

pH
µmol OH added
-
Fig. 4. Potentiometric titrations in the presence or absence of Gd(III) ions with 13, [L] = 0.001 M in
NMe Cl 0.1 M with varying Gd/L ratios.
4
Based on the results, the best chemical model corresponds to the presence of GdL and GdL .
2
Computations were monitored taking into account protonated as well as soluble hydroxy species.
+
+
Formation constants of the soluble gadolinium hydroxylated species Gd(OH) , Gd(OH)
2
and Gd(OH)
3
were respectively fixed at -7.3, -14.6 and -21.9 [4]. The stability constants of the complex are listed in
Table 2.
Table 2. Protonation constants of the ligand 13 and stability constants of the complex Gd(III)ligand 1.
Ligand
Species
LH
pK
a
mlh
log mlh
5.79 (2)
10.59 (2)
011
012
LH/L
5.79
4.80
LH
LH
LH
LH
LH
LH
2
3
4
5
6
7
LH
LH
LH
LH
LH
LH
2
3
4
5
6
7
/LH
/LH
/LH
/LH
/LH
/LH
013
014
015
016
017
15.00 (3)
19.14 (2)
22.53 (3)
26.09 (2)
28.43 (2)
2
3
4
5
6
4.41
4.14
3.39
3.56
2.34
1
6

Hydroxylated species
Species
GdH -1
mlh
101
log

mlh
-7,3
GdH -2
GdH -3
-14,6
-21,9
1
0
0
2
3
1
Gd -ligand
Species
mlh
log mlh
pKmlh
GdLH
GdL
111
110
111
12.08 (2)
6.99 (5)
-0.9 (1)
5.09
7.89
8.60
GdLH-1
GdLH-2
GdLH-3
GdLH-4
112
113
114
121
120
-9.5 (1)
9.8
9.67
-
-19.3 (4)
-28.97 (9)
16.3 (4)
11.4 (1)
GdL
2
2
H
4.9
-
GdL
As an example: pK110 corresponds to GdL, pK11-1 to GdLH-1, pK121 to GdL H.
2
The difference between the successive stability constants of the Gd(III)-ligand showed that pK11 from
GdL, pK11-1 from GdLH-1, pK11-2 from GdLH-2 and pK11-3 from GdLH-3 were higher than the acidity
constants from the ligand. The complexation effectively decreased the acidity.
The evaluation of pKa values for such a complex system was thus difficult due to the overlapping of
the species in equilibrium inside the pH range. Successive deprotonation formed negatively charge
species and increased the pKa values, underlining the presence of intramolecular interactions
between the carboxylic acid functions. Finally, the distribution curves are represented in Fig. 5.
1
7

%
sp
eci
es
for
me
d
rel
ati
ve
to
1
00
Gd
GdLH
GdL
80
60
40
20
0
Gd
GdLH
-
2
GdLH
-1
GdH
-
3
GdL
2
GdLH
-3
GdL H
2
GdH
-2
2
3
4
5
6
7
8
9
pH
Fig. 5. Distribution curves of the contrast agent 1 with [Gd]total = 6.139 mM, [L]total = 6.149 mM
conditions corresponding to the complex preparation).
(
Complexation between ligand 13 and the Gd(III) ion was effective between pH 3 and 9, in the
presence of [Gd]total = 6.139 mM and [L]total = 6.149 mM. At pH 6.45, gadolinium was predominantly
in the GdL (83.7%) and GdL
more acidic pH, GdLH was predominant, at pH 4 (86.7%) in equilibrium with GdL (7%) and free Gd
6.2%), with the latter representing 100% of the species at pH 2.6. Hydroxylated GdlH-1 and GdLH-2
2
(4.7%) complexed forms, and in GdLH and GdLH-1 minoritary forms. At a
(
appeared at pH values over 6. Indeed, the gadolinium atom in the presence of carboxylic groups
completed its coordination sphere with hydroxyl groups. These species also entered in competition
with the hydroxylated forms GdH-1, GdH-2 and GdH-3. To prevent free gadolinium from remaining in
the solution, the pH of the samples containing the gadolinium complex was increased to pH 8.5. The
speciation curves then showed that the residual free gadolinium precipitates in the Gd(OH) form.
3
Filtration of the sample eliminated this precipitate. The logarithmic formation constant of the
monometal complex 1 was evaluated as 7.89 (software Hyperquad®).
The variation of pGd (-log [Gdfree]) with pH, presented in Fig. 6, gave useful information about the
stability of the complex under different pH conditions. At pH 7.4, under biological consideration, the
pGd value is 4.85. This value is much lower in comparison to the pGd values for the Gd-DTPA (19.1)
and Gd-DOTA (18.9) complexes, confirming the lowest stability of the gadolinium-carboxylate
complex 1.
1
8

9
8
7
6
5
4
3
2
pG
d
2
3
4
5
6
7
8
9
pH
-
6
Fig. 6. pGd = -log [Gd]free, with [Gd]total = 10 M, [ligand]total = 10 [Gd]total.
The affinity of ligand 13 toward the Gd(III) ion as a function of pH was obtained by comparing the
amount of free metal relative to the total metal introduced, as represented in Fig. 7.
lo
g1
0
0
(
d
G -1
fre
e / -
Gd
2
tot -3
al)
-
-
-
4
5
6
2
3
4
5
6
7
8
9
pH
Fig. 7. Log (Gd free/Gd total) = f (pH) with [Gd]total = 6.139 mM, [L]total = 6.149 mM (conditions of the
complex preparation).
The complexation of gadolinium with the ligand begins around pH 3, related to the decrease of the
log10(Gd free/Gd total) = f(pH) curve, the apparition of a plateau corresponding to preponderant
GdLH, GdL and GdL complexes. After pH 7, the GdL hydroxylated forms become predominant and
2
are in competition with hydroxylated gadolinium species.
1
9

The speciation study experiments confirmed the presence in solution of monometallic GdL, with its
additional protonated or hydroxylated forms. The species with one gadolinium ion coordinated for
two ligands, GdL
2
, and its mono-protonated form, GdL H, were also detected and became
2
predominant in case of a Gd:L molar ratio below 0.5. Such complexes have been already observed by
Bonnet’s team, the formation of ML being controlled by adjusting the pH value [18]. In our case, the
diagram is more complex and no range of pH is favourable to the formation of the single metallic
species GdL. Consequently, at physiological pH (pH = 7.4), the required conditions for MRI imaging,
and in the presence of a 1:1 Gd:L ratio, the monometallic complex GdL 1 (62.3%) is at equilibrium
with GdLH-1 (20.2%) and GdL (8%). The logarithmic formation constant of the contrast agent 1 was
2
determined to be 7.89 by potentiometry studies. Contrast agent 2, bearing the same carboxylate
ligands, a similar stability behaviour was assumed. These values were in accordance with the stability
constant of an acetate peranhydrocyclodextrin (log  = 7.5) [18].
3
.3. In vivo preliminary study
The efficiency tests of contrast agents 1 and 2 were carried out on mice. The first MRI in vivo studies
focused on the heart area of mice at 4.7 Tesla and confirmed the in vitro relaxometry results
obtained at 0.5 Tesla. In order to avoid uncertainty in the results due to the viscosity of 1 and 2, the
probes were administrated via the jugular vein and the MRI signal was observed ten minutes after
injection. The contrast agents 1, 2 and commercial linear Magnevist® were injected at the usual
-
1
concentration (0.10 mmol kg ) and compared with images obtained without any contrast agent
control). Relaxivity values were calculated from the concentration injected. The images provided
(
from semi-quantitative maps obtained by a recovery T1-weighted pulse sequence were observed in
five cardiac zones (1-5, Fig. 8).
®
Witness
Magnevist
Contrast agent 1
Fig. 8. Myocardiac MRI of mice, 4.7 Tesla; 1: Left ventricle, 2: Right ventricle, 3: Septum, 4:
Left ventricle wall, 5: Muscle, 6: Liver.
Table 3. Relaxivity values for cardiac areas of mice without contrast agent (control) and in the
presence of commercial Magnevist®, and contrast agents 1 and 2 (Bruker, 4.7 Tesla).
2
0

®
Control
Magnevist
Compound 1
Compound 2
Left ventricle
1)
8
.70
.17
1
.61
.47
6.09
25.40
(
Right ventricle
2)
9
1
6.21
5.49
26.10
10.60
(
Septum (3)
3.93
3.67
6.14
6.17
Left
5.87
8.25
4.01
ventricular wall
(4)
Muscle (5)
2.90
4.16
3.90
A natural contrast is obtained in the different areas with the control (Tables 1 and 3) depending on
the local concentration of water. The injection of Magnevist® decreased the relaxation time twofold
in the ventricles and to a lesser extent in other areas. In any case, the resulting r values remained
1
-
1 -1
relatively homogeneous (Table 3, 4.16-6.21 mM s ), and the contrast agent was easily diffused
through the muscle and the cardiac walls. In the case of chelate 1, an intense imaging signal is
especially observed in the ventricles, leading to an important enhancement (about 320%) of r (Table
1
-
1 -1
3
, zone 2, 26.10 mM s ). The obtained values in the other areas were more weakly modified,
showing a compartmentation effect, likely due to the difficulty of the cyclodextrin derivative 1
crossing through the membranes. A similar effect was observed for 2, but the probe 2 improved the
-
1 -1
contrast less efficiently than 1 (Table 3, zone 2, 9.17 mM s ). Thus, these results proved different
behaviours of the synthesized probes due to the methylation or not of the hydroxyl groups of
cyclodextrin.
The kinetic of elimination of the contrast agents 1 and 2 was studied for 3 days after injection. The
study showed a regular and fast elimination of the contrast agents, mainly during the first hour.
After five hours a plateau is reached and the usual T1 value obtained in the absence of contrast
agent was observed, suggesting the total excretion from the body after 24 hours.
After injection of 2, no visible toxic effect due to the potential release of free gadolinium was
observed in mice over seven days following the administration of the contrast agent. However, we
noticed a dilation of the right ventricle, but with no embolism, consecutive to a high lung blood
pressure. A study is planned to evaluate more deeply the toxicity of the product. Despite this weak
stability constant, no effect related to an acute toxicity was detected in mice. The T1 recovery rate of
1
and 2 were similar compared to the DOTA ligand, showing a time residence in the body compatible
with the usual commercial agent. With the contrast agents 1 and 2 having the same stability, the
higher signal variation with 1 seems to be mainly due the presence of hydroxyl groups, considering
the variation of mass between the two adducts is negligible. The hydroxylated derivative 1 could
form easier hydrogen bonds with water molecules than 2 and that would cause a larger decrease of
the relaxation time due to the hydrogen bonding interactions between the secondary hydration
sphere of the macrocycle and the inner sphere of the gadolinium atom. Thus, 1 positively modifies
the MRI signal and induces a larger increase of the relaxivity r compared to the value obtained with
1
2
1

-
2
. This in vivo observation confirmed the in vitro results previously published (+43%, 6.5 and 4.7 mM
s for probes 1 and 2, respectively at 0.5 Tesla) [16]. The higher enhancement of relaxivity in vivo
1
-1
observed in the presence of 1 (for instance +185% in the right ventricle, Table 3, zone 2) is due to
interactions with biomolecules present in blood, while in vitro measurements were performed in Tris
buffer.
In opposition to the vascular permeability of DOTA, a confinement in the vascular space was
observed for contrast agents 1 and 2 due to the cyclodextrin scaffold, as previously observed by
Bonnet [18]. Such a property can find application for cardiac diagnosis after heart attack. Indeed in
this case the macrocycle MRI probe could diffuse into the coronary walls providing new and more
precise vital diagnosis by quantifying the blood volume of the damaged vessel.
4
. Conclusion
The syntheses of -cyclodextrins derivatives bearing seven carboxylate ligands on the primary face
were successfully optimized, which provided sufficient amounts of the contrast agents in order to
evaluate their biomedical potential. In vivo MRI properties investigated for the cardiac area in mice
showed a high enhancement of relaxivity for both cyclodextrin probes, with a stronger effect for the
hydroxylated derivative due interactions with the second coordination hydration sphere. Although
the monometallic complexes at physiological pH testified a relatively low stability constant, the
preliminary in vivo studies did not highlight any toxicity and underlined the correct elimination rate.
Work is currently in progress to develop more stable contrast agents, useful for the diagnosis of
heart failure.
Acknowledgements
Normandy University, INSA Rouen, CNRS are also acknowledged for their financial support. We are
grateful to Interreg AIChem for the PhD grant (A.B.) and Labex SynOrg for the PhD grant (C. S.). We
thank Eva Toth and Célia Bonnet from CNRS, CBM, Orléans, France for their interactive discussions.
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2
4

1
00
Gd
GdLH
GdL
8
6
4
2
0
0
0
0
0
GdLH-2
GdH-3
GdLH-1
GdL
2
GdLH-3
GdL2H
GdH-2
2
3
4
5
6
7
8
9
pH
2
5