Synthesis and functional evaluation of chiral dendrimer-triamine- coordinated Gd complexes as highly sensitive MRI contrast agents

Article abstract of DOI:10.1016/j.tetlet.2012.06.072

Novel chiral dendrimer-triamine-coordinated Gd complexes were synthesized and shown to have longitudinal relaxivity (r1) 3 times higher than that of clinically used Gd-DTPA. The pharmacokinetic differences between optical isomers were estimated from the affinity of 2-(R) and 2-(S) with bovine serum albumin (BSA), respectively, by a quartz crystal microbalance (QCM) measurement. As a result, the association constant K_a of 2-(S) was about 4 times higher than that of 2-(R), which means that 2-(S) is retained in the vascular retention for a longer time after administration. This result was also supported by T1-weighted MR images of mice before and after the intravenous injection of 2-(R) and 2-(S), as well as the time-course of the signal intensities (SI) at the blood vessels and quantification of Gd³⁺ concentration in the blood and urine.

Full text of DOI:10.1016/j.tetlet.2012.06.072

Tetrahedron Letters 53 (2012) 4580–4583
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and functional evaluation of chiral dendrimer–triamine-coordinated
Gd complexes as highly sensitive MRI contrast agents
Yuka Miyake a, Yu Kimura b, Syungo Ishikawa a, Hiroshi Tsujita a, Hiroki Miura a, Michiko Narazaki c,
Tetsuya Matsuda c, Yasuhiko Tabata d, Tetsuya Yano e, Akio Toshimitsu a,f, Teruyuki Kondo a,b,
⇑
a Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan
b Advanced Biomedical Engineering Research Unit, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan
c Graduate School of Informatics, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan
d Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaracho, Shogoin, Sakyo-ku 606-8507, Japan
e Canon Inc., 3-30-2 Shimomaruko, Ohta-ku, Tokyo 146-8501, Japan
f Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel chiral dendrimer–triamine-coordinated Gd complexes were synthesized and shown to have longi-
tudinal relaxivity (r1) 3 times higher than that of clinically used Gd-DTPA. The pharmacokinetic differ-
ences between optical isomers were estimated from the affinity of 2-(R) and 2-(S) with bovine serum
albumin (BSA), respectively, by a quartz crystal microbalance (QCM) measurement. As a result, the asso-
ciation constant Ka of 2-(S) was about 4 times higher than that of 2-(R), which means that 2-(S) is retained
in the vascular retention for a longer time after administration. This result was also supported by T1-
weighted MR images of mice before and after the intravenous injection of 2-(R) and 2-(S), as well as
the time-course of the signal intensities (SI) at the blood vessels and quantification of Gd3+ concentration
in the blood and urine.
Received 24 April 2012
Revised 11 June 2012
Accepted 15 June 2012
Available online 20 June 2012
Keywords:
Chiral dendrimer
MRI contrast agent
Gadolinium
Ó 2012 Elsevier Ltd. All rights reserved.
Drug design
Pharmacokinetics
Introduction
Therefore, there is a strong need for the development of highly
sensitive MRI contrast agents, and recently there has been growing
MRI has become a prominent non-invasive imaging technique
for disease diagnosis.1 Low-molecular-weight contrast agents
based on Gd-DTPA (DTPA = diethylenetriaminepentaacetic acid)
worldwide interest in the development of MRI contrast agents
that consist of Gd-functionalized dendrimer macromolecules.4
Dendrimers5 are a unique category of macromolecules with well-
controlled sizes, nanoscopic dimensions, and numerous peripheral
chemical groups to which Gd chelates can be coupled. Gd-
functionalized poly(amidoamine) (PAMAM)6 and poly(propylenei-
mine) (PPI)7 dendrimers have been reported and evaluated in
animal models for high-resolution MRI, in which dendrimers were
used as a core and Gd chelates were positioned in the periphery.8
Unfortunately, among the Gd-functionalized dendrimers that have
been reported for use as contrast agents, dendrimers were only
and
Gd-DOTA
(DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,
10-tetraacetic acid) have been approved by the U.S. Food and Drug
Administration (FDA) and the European Medicines Agency (EMEA),
and are widely used in the clinical diagnosis of tumors (Fig. 1).2
However, the non-specificity, low contrast efficiency, and rapid
renal excretion of these low-molecular-weight contrast agents
necessitate a high dosage (ca. 0.5 M), which imposes a great phys-
ical strain on the patient, and in some cases they may produce side
effects, such as osmotic pressure shock.3 The main reason for their
low contrast efficiency is that, among the nine coordination sites of
Gd, up to eight are solidly occupied with ionic chelating ligands,
and thus only one remains for coordination with free water mole-
cules, which is observed by MRI (Fig. 1). In addition, the rotational
motion of Gd metal in the center of existing small ligands cannot
be suppressed, and as a result the image contrast is considerably
reduced.
O
O
O
O
N
N
N
N
O
N
Gd
Gd
O
N
O
N
O
O
O
O
OH
O
O
O
O
H
O
O
O
H
H
H
Gd-DTPA
Gd-DOTA
⇑
Corresponding author. Fax: +81 75 383 7055.
E-mail address: teruyuki@scl.kyoto-u.ac.jp (T. Kondo).
Figure 1. Structure of Gd-DTPA and Gd-DOTA.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.06.072

Y. Miyake et al. / Tetrahedron Letters 53 (2012) 4580–4583
4581
used to slow the molecular tumbling and rotation of Gd. The intra-
vascular retention time is prolonged, but the principle of reducing
the 1H relaxation time of a water molecule is the same as that with
low-molecular-weight contrast agents.
In this study, we radically revised the concept of the established
methods that use dendrimers, so that they could be used as a li-
gand at the periphery of a Gd metal. As a result, we succeeded in
designing and synthesizing novel chiral dendrimer–triamine-
coordinated Gd complexes which are expected to be highly sensi-
tive MRI contrast agents.
2-(R)
2-(S)
Gd-DTPA
GdCl3·6H2O
Water
Figure 2. T1-weighted MR images of Gd-DTPA, GdCl3Á6H2O, 2-(R), 2-(S) (0.50 mM)
and water.
Results and discussion
Table 1
Longitudinal relaxivities (r1) of Gd-DTPA, 2-(R), 2-(S),
and GdCl3Á6H2O
As shown in Scheme 1, (R)- and (S)-chiral diols, which serve as
building blocks for chiral dendrimer–triamine ligands, were first
prepared by the asymmetric dihydroxylation reaction developed
by Sharpless and co-workers.9 Repeated binding of these chiral
diols gave the corresponding chiral dendrons in high yield and
with high enantioselectivity.10 Connection of the chiral dendrons
to 1,4,7-triazacyclononane gave 2nd-generation chiral dendri-
mer–triamine ligands, in which all 9 of the stereogenic centers
have an (R)- or (S)-configuration, respectively. Hydrolysis of the
acetal protecting groups and complexation of GdCl3Á6H2O gave no-
vel chiral dendrimer–triamine-coordinated Gd complexes, 2-(R)
and 2-(S),11 which are expected to be a highly sensitive MRI con-
trast agents.
Next, the longitudinal relaxivities (r1) of chiral dendrimer–
triamine-coordinated Gd complexes (2-(R) and 2-(S)), GdCl3Á6H2O,
and Gd-DTPA were calculated in vitro (Fig. 2 and Table 1). The r1
values of 2-(R) and 2-(S) were 11.4 and 11.1 mMÀ1 sÀ1, respec-
tively, which are approximately 3 times higher than that of Gd-
DTPA (r1 = 4.6 mMÀ1 sÀ1).
Entry
r1/mMÀ1 sÀ1
Gd-DTPA
2-(R)
2-(S)
GdCl3Á6H2O
4.6
11.4
11.1
14.6
Gd-DOTA have ionic chelating ligands that strongly suppress 8
coordination sites of Gd. On the other hand, the novel Gd com-
plexes have a triamine ligand and three chloride ligands, which
stably occupy 6 coordination sites of Gd. Accordingly, 3 coordina-
tion sites remain for water molecules, and thus the present Gd
complexes show longitudinal relaxivity that is 3 times higher than
that of Gd-DTPA. In addition, the central Gd is considered to be
covered through weak coordination by hydroxyl groups at the den-
drimer end, and they may dissociate only when a small, but highly
polar, water molecule approaches.12 The binding (coordination)
ability of 1,4,7-triazacyclononanes to Gd in 2-(R) and 2-(S) is con-
sidered to be high,13 and no ligand-free Gd3+ was formed, which
are strongly suggested by the following cytotoxicity examination.
As
mentioned
previously,
the
established
low-
molecular-weight Gd contrast agents such as Gd-DTPA and
K2OsO2(OH)4
(DHQD)2PHAL or
(DHQ)2PHAL
OH
MeO
p-MeC6H4SO3H
OMe
O
O
MeO
OH
OH
O
O
*
Cl
*
*
K3Fe(CN)6
K2CO3
acetone , r.t. , 4 h
Cl
nBu4NI
K2CO3
2-butanone
reflux
Cl
PMPO
tBuOH / H2O = 1/1
0 oC , 4 h
25
α
25
(
: 97%, [
R)
(S) : 97%, [
]
D = - 0.4780
: 83%, >99% ee (HPLC)
25
α
α
(R)
(R) : 94%, [
(S) : 90%, [
]
]
D = - 0.4994
D = + 0.5055
25
25
α
]
= + 0.4771
D
α
[
]
D = - 0.4911
: 82%, >99% ee (HPLC)
25
(S)
α
[
] D = + 0.4927
O
O
O
O
O
O
*
*
*
O
OH
OH
O
O
O
O
O
O
PPh3
NBS
O
3N HCl aq.
Cl
CAN
H2O : CH3CN = 1 : 2
*
*
*
PMPO
CH2Cl2
CH3CN
r.t.
KOH
toluene, reflux
oC
-10 o
C
O
O
O
0
O
O
O
25
α
(R) : 91%, [
(S) : 92%, [
]
]
= - 0.4215
D = + 0.4210
*
*
*
D
PMPO
HO
Br
25
α
25
25
25
α
α
α
) : 75%, [ ]
(
) : 61%, [
]
D = - 0.7150
D = + 0.7089
(
) : 71%, [
]
D = - 0.6945
(
R
D = - 0.6944
R
R
25
25
25
α
α
α
(S) : 65%, [
]
(S) : 70%, [
]
D = + 0.6969
(S) : 65%, [
] D = + 0.6909
O
OH
OH
O
O
OH
OH
OH
OH
O
H
H
HO
*
HO
N
N
*
*
*
*
*
N
H
O
O
O
O
O
OH
OH
1 M LiOEt / EtOH
O
80% CH3CO2H aq
48 h
O
GdCl3 6H2O
MeOH
O
*
HO
HO
*
O
*
O
O
O
*
0
oC
*
N
N
OH
OH
r.t.
N
N
*
N
N
*
O
*
*
O
OH
r.t.
OH
O
N
O
N
Cl3Gd
*
N
*
*
O
O
(
= C6H4)
(* = chiral center)
O
O
O
*
O
O
*
*
O
*
O
HO
HO
*
*
OH
*
*
*
O
OH
HO
25
HO
OH
OH
25
25
α
α
α
α
α
α
]
(R) : 66%, [
(S) : 60%, [
]
]
D = - 0.6379
D = + 0.6402
1-(R) : 80%, [
1-(S) : 80%, [
]
]
D = - 0.6310
D = + 0.6190
2-(R) : 93%, [
2-(S) : 95%, [
]
25
D = - 0.6645
D = + 0.6610
25
25
Scheme 1. Synthesis of novel chiral dendrimer–triamine-coordinated Gd complexes, 2-(R) and 2-(S).

4582
Y. Miyake et al. / Tetrahedron Letters 53 (2012) 4580–4583
In contrast to amine-terminated dendrimer Gd-MRI contrast
60
50
40
30
20
10
agents, such as Gd-functionalized PAMAM and PPI dendrimers,
no cytotoxic effect was observed for either 2-(R) or 2-(S) with
L929 cells (Fig. 3).14
Thus, contrast enhancement by 2-(R) and 2-(S) was evaluated
in vivo. Figure 4 shows T1-weighted MR images of mice before
and after intravenous injection of 2-(R), 2-(S), and Gd-DTPA
(0.10 mmol Gd/kg). Since most of the injected Gd-DTPA was ex-
creted through the kidney, and accumulated in the bladder within
30 min, little contrast enhancement was observed except for the
kidney. In contrast, no accumulation of 2-(R) or 2-(S) in specific or-
gans, such as the liver and kidney, was observed with high and pro-
longed contrast enhancement throughout the entire bodies of
mice. They also showed improved vascular retention and a moder-
ate renal excretion rate (completely excreted after 24 h).
0
1
2
3
Concentration / 10-10
M
Figure 5. Frequency change of the electrodes coated with BSA after addition of 2-
(R) (circular symbol) and 2-(S) (triangular symbol).
To accurately discuss the pharmacokinetic differences between
optical isomers, the affinities of 2-(R) and 2-(S) with bovine serum
albumin (BSA), which is a model of plasma protein, were estimated
by a quartz crystal microbalance (QCM) measurement (Fig. 5).15 As
Figure 6. Time-course of the signal intensities (SI) at the blood vessels in MR
images after injection of 2-(R) (circular symbol) and 2-(S) (triangular symbol).
Figure 3. Viabilities of L929 cells exposed Gd-DTPA, GdCl3Á6H2O, 2-(R), and 2-(S) at
a result, association constant Ka of 2-(S) (4.02 Â 1010 MÀ1) was
about 4 times higher than that of 2-(R) (9.61 Â 109 MÀ1), which
means that 2-(S) is retained in vasculature for longer after admin-
istration in a mouse body. This result was also supported by T1-
weighted MR images of mice before and after intravenous injection
of 2-(R) and 2-(S) (Fig. 4).
0.25 mM.
More directly, a measurement of the time-course of the signal
intensities (SI) at the blood vessels in MR images indicated that
the rate of clearance of 2-(R) was faster than that of 2-(S)
(Fig. 6). In addition, the concentrations of Gd3+ in the blood and ur-
ine, 60 min after the injection of 2-(R) and 2-(S), were quantified
by an atomic absorption spectroscopy, which showed that 30.2%
of 2-(R) and 20.6% of 2-(S) were transferred to urine, while 22.9%
of 2-(R) and 27.8% of 2-(S) were retained in the blood, respectively.
All results obtained strongly support that 2-(S) is retained in vascu-
lature for longer than 2-(R) after administration in a mouse body.
Generally, drugs administered in the blood interact with plasma
proteins with equilibrium between associated and dissociated
states. Upon glomerular filtration, which is an important metabolic
pathway, only a chiral contrast agent in the dissociated state can
be excreted. Therefore, the difference in the affinities of 2-(R)
and 2-(S) for plasma protein might affect their metabolism and re-
sult in the difference in their distributions throughout the body.
Conclusions
In conclusion, we have synthesized the first chiral dendrimer–
triamine-coordinated Gd contrast agents, and the pharmacokinetic
differences between the optical isomers, 2-(R) and 2-(S), were
Figure 4. T1-weighted MR images before and after intravenous injection of Gd-
DTPA, 2-(R), and 2-(S).

Y. Miyake et al. / Tetrahedron Letters 53 (2012) 4580–4583
4583
Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem. Rev. 1994, 94, 2483–
2547.
clarified. The influence of the dendrimer generations on contrast
ability in MRI is now under investigation.
10. Chang, H.-T.; Chen, C.-T.; Kondo, T.; Siuzdak, G.; Sharpless, K. B. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 182–186.
11. 1-(R): White powder. 1H NMR (CDCl3, 400 MHz): d (ppm): 1.98–2.08 (br s, 12H,
OH), 2.83 (br s, 12H, –N(CH2)2N–), 3.49–3.56 (m, 15H), 3.70–4.34 (s, 9H,
–CH(OH)–), 4.32–4.44 (m, 12H, –C6H4CH2–), 4.59–4.64 (m, 9H), 7.16–7.47 (m,
36H, –C6H4–). ESI TOF MS m/z for C87H105N3O18 [M+H]+: 1480.7858.
1-(S): White powder. ESI TOF MS m/z for C87H105N3O18 [M+H]+: 1480.7228.
Preparation of 2-(R) from the reaction of 1-(R) with GdCl3Á6H2O. A solution of
Acknowledgments
This work was partly supported by the Creation of Innovation
Centers for Advanced Interdisciplinary Research Areas Program
‘Innovative Techno-Hub for Integrated Medical Bio-imaging’, and
a Grant-in-Aid for the Global COE Program ‘Integrated Material Sci-
ence’ from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan. YK acknowledges financial support
from Adaptable and Seamless Technology Transfer Program
through Target-driven R&D (A-STEP), Japan Science and Technol-
ogy Agency (JST). TK acknowledges financial support from the Dai-
wa Securities Health Foundation.
GdCl3Á6H2O (46 mg, 125
solution of 1-(R) (185 mg, 125
stirred at room temperature for 24 h. Then, the solvent, MeOH, was removed
under vacuum to give 2-(R) (202 mg, 116 mol, 93%) as a white powder. 2-(R):
l
mol) in MeOH (1.5 mL) was added dropwise to a
l
mol) in MeOH (1.0 mL), and the mixture was
l
MALDI TOF MS (matrix: DCTB) m/z for C87H105ClGdN3O18 [M-2Cl]+: 1669.6334
(7%), 1670.6258 (46%), 1671.6252 (75%), 1672.6242 (100%), 1673.6264 (80%),
1674.6282 (84%), 1675.6292 (54%), 1676.6334 (25%), 1677.6376 (3%), 1678.
6410 (2%). The same procedure gave 2-(S) in 95% yield. 2-(S): White powder.
ESI-TOF MS m/z for C87H105Cl3GdN3O18 [M]+: 1741.5671 (13%), 1742.5467
(40%), 1743.5668 (100%), 1744.5619 (60%), 1745.5624 (19%), 1746.2629 (14%),
1747.5433 (18%).
12. The increase in the coordination number of free water to the present novel MRI
contrast agents, 2-(R) and 2-(S), was shown by preliminary XAFS
measurements using the SPring 8 synchrotron radiation facility.
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