A novel cholic acid-based contrast enhancement agent for targeted MRI

Article abstract of DOI:10.1016/j.bmcl.2008.01.044

The novel Gd(III) complexes of heptadentate ligands NE3TA and NE3TA-Bn were prepared, and their relaxivities were measured and favorably compared to the commercially available MRI contrast enhancement agent Gd(DOTA). NE3TA was conjugated with cholic acid (CA) to produce CA-NE3TA. TEM images of Gd(CA-NE3TA) indicate that the complex self-assembles forming nano-sized micelles and displays an over threefold increased relaxivity compared to Gd(DOTA). The new cholic acid-conjugated nanoparticle MR contrast enhancement agent, Gd(CA-NE3TA) possesses great promise for use in targeted MRI.

Full text of DOI:10.1016/j.bmcl.2008.01.044

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2505–2508
A novel cholic acid-based contrast enhancement
agent for targeted MRI
Hyun-Soon Chong,a,* Hyun A. Song,a Sooyoun Lim,a Keith Macrenaris,b Xiang Ma,a
Haisung Lee,a Phuong Buia and Thomas Meadeb
aChemistry Division, Biological, Chemical, and Physical Sciences Department, Illinois Institute of Technology, 3101 S. Dearborn St.,
LS 182, Chicago, IL 60616, USA
bDepartment of Chemistry, Biochemistry and Molecular and Cell Biology, Neurobiology and Physiology,
and Radiology Northwestern University, Evanston, IL, USA
Received 12 November 2007; revised 9 January 2008; accepted 14 January 2008
Available online 18 January 2008
Abstract—The novel Gd(III) complexes of heptadentate ligands NE3TA and NE3TA-Bn were prepared, and their relaxivities were
measured and favorably compared to the commercially available MRI contrast enhancement agent Gd(DOTA). NE3TA was con-
jugated with cholic acid (CA) to produce CA-NE3TA. TEM images of Gd(CA-NE3TA) indicate that the complex self-assembles
forming nano-sized micelles and displays an over threefold increased relaxivity compared to Gd(DOTA). The new cholic acid-con-
jugated nanoparticle MR contrast enhancement agent, Gd(CA-NE3TA) possesses great promise for use in targeted MRI.
Ó 2008 Published by Elsevier Ltd.
Magnetic resonance imaging (MRI) is a non-invasive and
high resolution imaging technique that has become a
powerful diagnostic tool in the clinic. The images due to
the MR signal of water protons provide a sharp contrast
between tissues with different proton relaxation times (T1
or T2). Signal intensity, i.e., relaxivity (1/T1 or 1/T2) re-
sults from proton exchange between a slowly exchanging
gadolinium-bound water molecule and bulk water.1 In or-
der to enhance contrast between tissues, paramagnetic
metal complexes have been introduced in vivo. The lan-
thanide Gd(III) is known to be an optimal paramagnetic
metal for MRI due to its high electronic spin (7/2) and
slow electronic relaxation rate.2 A number of Gd(III)
complexes such as Gd(DOTA) and Gd(DTPA) are clini-
cally approved for use in MRI.1 However, most contrast
agents have non-specific extracellular distribution and the
disadvantages of low relaxivity, low tissue specificity, and
rapid clearance.3 Considerable research efforts have been
directed towards developing safe Gd(III)-based MR con-
trast agents with high tissue specificity and sensitivity.
MRI is proven to be more sensitive and specific than other
medical tests for detecting liver malignancies and for
distinguishing them from benign lesions.4 The gadolinium
complexes of two DTPA analogues, benzyloxypropio-
nictetraacetate (BOPTA) and ethoxybenzyl-diethylenetri-
aminepentaacetic acid (EOB-DTPA) are the clinically
approved hepatobiliary agents.5 The Gd(III) complexes
provide low detection and characterization of metastatic
lesions, although the agents are useful in detection and
characterization of hepatocelluar lesions.6
In our continued effort to develop liver-specific MRI
contrast agents,7,8 we planned to use bile acid as a liver
or intestine targeting moiety. The property of amphifa-
cial bile acid to undergo enterohepatic circulation and
form helical aggregates makes it a useful shuttle system
to deliver various drugs to the liver and intestine with
favorable intestine absorption and pharmacokinetic
profile.9,10 Bile acids are efficiently taken up into the cells
by two types of carriers: apical sodium-dependent bile
salt transporters (ASBT) and Na+-independent carri-
ers.11 Experimental studies demonstrate that bile acids
enter liver and colon cancer cells which over express bile
acid transporters and carriers.11,12
Keywords: Chelating agents; Macrocyclic ligands; MRI contrast
agents; Gd(III) complexes; Cholic acid; CA-NE3TA; NE3TA;
NE3TA-Bn.
We have recently reported a new series of bimodal che-
lators, NETA, NPTA, NE3TA, NE3TA-Bn containing
both macrocyclic and acyclic components that have var-
ious biomedical applications.8,13,14 The Gd(III) com-
*
Corresponding author. Tel.: +1 312 567 3235; fax: +1 312 567
3494; e-mail: chong@iit.edu
0960-894X/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.01.044

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H.-S. Chong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2505–2508
HO2C
CO2H
N
CO2H
HO2C
CO2H
N
N
N
CO2H
N
N
N
N
N
NH
O
OH
N
HO2C
CO2H
N
N
CO2H
H
CO2H
R
DOTA
CA-NE3TA
R = H, NE3TA
R = Bn, NE3TA-Bn
HO
OH
Figure 1. MRI contrast enhancement agents in preclinical and clinical use.
15
NE3TA–Bn is a heptadentate ligand with a benzyl
group which can be further modified for conjugation
to a targeting moiety. Both NE3TA and NE3TA-Bn
can produce neutral Gd(III) complexes that have an
advantage of less protein interaction and a potentially
more favorable in vivo tissue distribution, and the corre-
sponding Gd(III) complexes may provide enhanced
relaxivity due to increase in hydration number (q) when
compared to that of DOTA (Fig. 1) and DTPA.
10
5
0
Herein, we report the synthesis and characterization of
the new Gd(III) complexes, Gd(NE3TA), Gd(NE3TA-
Bn), and Gd(CA-NE3TA). Transmission electron
microscopy (TEM) images of cholic acid analogues,
CA-NE3TA and Gd(CA-NE3TA) were obtained.
Gd(CA-NE3TA) Gd(NE3TA)
Gd(NE3TA-Bn) Gd(DOTA)
Figure 2. Relaxivity of the new Gd(III) complexes.
plexes of octadentate NETA and NPTA are stable in
both serum and mice and possess enhanced relaxivity
compared to those of DOTA.8 In the present study,
the Gd(III) complexes of heptadentate NE3TA and
NE3TA-Bn were prepared as potential MRI contrast
agents. Heptadentate NE3TA contains four amines
and three carboxylates as potential donor groups.
Gd(NE3TA) and Gd(NE3TA-Bn) were synthesized by
reacting GdCl3 with NE3TA and NE3TA-Bn, respec-
tively. NE3TA or NE3TA-Bn were mixed with GdCl3
in a molar ratio of 1–0.9, and the resulting mixture
was adjusted to pH 7 and heated to 90 °C and stirred
until no free Gd(III) ions were detected using Arsen-
t
-BuO2C
t-BuO2C
CO2t-Bu
N
CO2t-Bu
N
N
N
N
N
CO2t-Bu
NH
O
S
OH
O
CO2t-Bu
NH
OH
N
H
H2N
S
N
(2)
3
CH2Cl2, 48 h Reflux
(67%)
HO
OH
1
4M HCl
HO
OH
1,4-dioxane
24 h
SH
CH2Cl2
DCC
DMAP
(85%)
(97%)
S
N
HO2C
CO2H
N
N
O
N
OH
OH
NH
O
OH
CO2H
N
H
HO
OH
HO
OH
CA-NE3TA (4)
Scheme 1. Synthesis of CA-NE3TA.

H.-S. Chong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2505–2508
2507
azoIII (AAIII) assay. Relaxivity of the aqueous Gd(III)
complexes (pH 7) was measured on a Bruker MQ60
NMR analyzer, and the concentration of the Gd(III)
complexes was measured by ICP-MS. The relaxivity
data (Fig. 2) indicate that the Gd(III) complexes of
NE3TA (3.94 mMÀ1 sÀ1) and NE3TA-Bn (3.74 mMÀ1s À1
)
provided higher relaxivity as compared to Gd(DOTA)
(2.97 mMÀ1s À1), probably due to increase in q. NE3TA
was successfully conjugated with the most common bile
acid, cholic acid (Scheme 1). A number of reports have
shown that conjugation of cholic acid to various drugs
via the amide bond little affects interaction of the conju-
gates with the human bile acid transporters.9,12 Amphi-
facial cholic acid is a good targeting moiety, and its
conjugation to MR contrast agent is expected to form
helical globular aggregates15 with a favorable pharma-
cokinetic and relaxivity profile.1 Cholic acid was preac-
tivated with 2-mercaptothiazoline to afford 1, which
was further reacted with a functionalized NE3TA (2)16
containing an amino group to provide 3. The t-butyl
group of 3 was removed by treatment with 4 M HCl
in 1,4-dioxane to provide CA-NE3TA (4). TEM image
of CA-NE3TA (10 lM aqueous solution) indicates that
the bile acid conjugated NE3TA forms discrete spherical
micells in nanometer size (ꢀ10–50 nm, Fig. 3) due to the
presence of hydrophobic cholic acid moiety surrounded
by hydrophilic NE3TA ligand. The Gd(III) complex of
CA-NE3TA was prepared as described above. Gd
(CA-NE3TA) self-assembles into nano-sized micells as
evidenced by TEM measurement (ꢀ10 nm, Fig. 4).
Interestingly, when the complex Gd(CA-NE3TA) was
taken in low concentration (10 lM aqueous solution),
it was shown to rapidly agglomerate into stacked spher-
ical micells (Fig. 5). Aggregation of Gd(CA-NE3TA) as
evidenced by TEM images is proposed to result from
heating during the complexation reaction and/or gado-
linium induced cross linking.17 T1 relaxivity of
Gd(CA-NE3TA) in aqueous solution (0.8 mM) was
measured to 10.85 mMÀ1 sÀ1. The relaxivity data
Figure 4. TEM image unstained of Gd(CA-NE3TA) (100 lM aqueous
solution); (inset, scale bar in 10 nm).
Figure 5. TEM image unstained of Gd(CA-NE3TA) (10 lM aqueous
solution).
indicate that incorporation of cholic acid with NE3TA
produced more than threefold increase in T1 relaxivity.
This increase in relaxivity may be a result of slower
molecular rotation due to aggregation of the complex
as evidenced by TEM image.
In summary, we have prepared a series of novel Gd(III)
complexes, Gd(NE3TA), Gd(NE3TA-Bn), and Gd(CA-
NE3TA). Relaxivity of Gd(NE3TA) and Gd(NE3TA-
Bn) is high when compared to that of the commercially
available MR contrast agent Gd(DOTA). Both the cho-
lic acid-based NE3TA, CA-NE3TA and the correspond-
ing Gd(III) complex, Gd(CA-NE3TA) self assemble
forming nano-sized micells as shown by TEM images.
Figure 3. TEM image unstained of CA-NE3TA (10 lM aqueous
solution).

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H.-S. Chong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2505–2508
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Gd(CA-NE3TA) displayed much increased relaxivity as
compared to Gd(DOTA). The new cholic acid-conju-
gated Gd(III) complex, Gd(CA-NE3TA) possesses
promise for use in targeted MRI. Studies of serum sta-
bility and cellular uptake and in vivo biodistribution,
MRI of Gd(CA-NE3TA) in mice are underway.
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M. W. J. Org. Chem. 2001, 66, 7745.
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Acknowledgment
This research was supported by the National Institutes
of Health (K22CA102637 to C.H.S. and 1R01-EB00-
5866 to T.M.).
Supplementary data
Full Experimental details. Supplementary data associ-
ated with this article can be found, in the online version,
at doi:10.1016/j.bmcl.2008.01.044.
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