Gd complexes of DO3A-(biphenyl-2,2′-bisamides) conjugates as MRI blood-pool contrast agents

Article abstract of DOI:10.1021/ml300223b

We report the synthesis of DO3A derivatives of 2,2′-diaminobiphenyl (1a,b) and their Gd complexes of the type [Gd(1)(H₂O)]·xH ₂O (2a,b) for use as new MRI blood-pool contrast agents (BPCAs) that provide strong and prolonged vascular

Full text of DOI:10.1021/ml300223b

Letter
pubs.acs.org/acsmedchemlett
Gd Complexes of DO3A-(Biphenyl-2,2′-bisamides) Conjugates as MRI
Blood-Pool Contrast Agents
Ki-Hye Jung,†,# Hee-Kyung Kim,‡,# Ji-Ae Park,∥ Ki Soo Nam,† Gang Ho Lee,§ Yongmin Chang,*,‡,⊥
and Tae-Jeong Kim*,†
†Department of Applied Chemistry, ‡Department of Medical & Biological Engineering, and §Department of Chemistry, Kyungpook
National University, University Road 80, Daegu, 702-701, Korea
∥Molecular Imaging Research Center, Korea Institute of Radiological Medical Science, Nowon-gil 75, Seoul, 139-706, Korea
⊥Department of Radiology & Molecular Medicine, Kyungpook National University, Dongin-dong 2-ga, Daegu, 700-422, Korea
S
* Supporting Information
ABSTRACT: We report the synthesis of DO3A derivatives of 2,2′-
diaminobiphenyl (1a,b) and their Gd complexes of the type
[Gd(1)(H2O)]·xH2O (2a,b) for use as new MRI blood-pool
contrast agents (BPCAs) that provide strong and prolonged vascular
enhancement. Pharmacokinetic inertness of 2 compares well with
that of structurally related Dotarem, a DOTA-based MRI CA
currently in use. The R1 relaxivity in water reaches 7.3 mM−1 s−1,
which is approximately twice as high as that of Dotarem (R1 = 3.9
mM−1 s−1). They show interaction with HSA to give association
constants (Ka) in the order of two (∼102), revealing the existence of
the blood-pool effect. The in vivo MR images of mice obtained with
2 are coherent, showing strong signal enhancement in both heart, abdominal aorta, and small vessels. Furthermore, the brain
tumor is vividly enhanced for an extended period of time.
KEYWORDS: Gd chelates, DO3A, biphenyl, MRI BPCA, brain tumor
window for imaging is considerably reduced, thus limiting
acquisition of high-resolution images.3−5
agnetic resonance imaging (MRI) is a powerful
Mtechnique for noninvasive diagnosis of the human
To overcome such limitations inherent to ECF CAs, the
necessity for the development of blood-pool contrast agents
(BPCAs) has risen in the expectation that they reside in the
blood vessel for an extended period of time and thus are
eliminated much more slowly from the circulation than their
ECF counterparts. The BPCAs provide strong and prolonged
vascular enhancement. The BPCAs thus far developed may be
divided into three classes according to their mechanism of
action: (i) the noncovalent binding of low-molecular GdL to
HSA to prevent immediate leakage into the interstitial space,6−8
(ii) systems incorporating polymers or liposomes based on an
increase in the size of CAs, which slows down leakage through
endothelial pores,9−12 and (iii) systems based on nanoparticles,
involving a change in the route of elimination.13−15 The most
successful approach so far is the class (i) represented by MS-
325, a Gd−DTPA derivative containing a cholic acid moiety.
These complexes bind strongly and reversibly to HSA, leading
to not only high R1 relaxivity at clinical field strengths but also
much longer residence times in the blood as compared to ECF
agents. The lipophilic component represented by aromatic
Chart 1
anatomy, physiology, and pathophysiology on the basis of
superior spatial resolution and contrast useful in providing
anatomical and functional images of the human body.1 At the
clinical level, MRI techniques are mostly performed employing
Gd(III) chelates (GdL) to enhance the image contrast by
increasing the water proton relaxation rate in the body.2
Despite their wide and successful applications in clinics,
however, conventional Gd(III)-based low-molecular weight
CAs are mostly extracellular fluid (ECF) agents exhibiting rapid
extravasation from the vascular space. As a result, the time
Received: August 2, 2012
Accepted: October 23, 2012
Published: October 23, 2012
© 2012 American Chemical Society
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Chart 2
Table 1. Relaxivity Data of 2a, 2b, Dotarem, and MS-325 (64
a
MHz, 293 K)
R1 (mM−1 s−1
)
R2 (mM−1 s−1
)
b
c
b
c
water
PBS
HSA
water
PBS
HSA
2a
7.3
7.0
3.9
4.3
3.4
3.6
5.2
8.1
6.8
5.3
7.6
7.6
4.1
5.3
4.4
15.3
11.3
7.2
2b
Dotarem
MS-32522
19.0
5.9
37.0
a
b
c
Concentrations are given in [Gd]. PBS: pH 7.4. [HSA] = 0.67 mM
in PBS.
moieties in the chelate is responsible for the generation of
noncovalent interactions between the corresponding GdL and
HSA.16,17 The anionic phosphodiester moiety in MS-325 is also
known to contribute in a synergistic manner to the noncovalent
binding interaction with HSA.
We have also been involved for some time in the design and
synthesis of a new series of Gd-based MRI BPCAs.18−21 More
recent work includes the synthesis of a series of macrocyclic
DTPA-(biphenyl-2,2′-bisamides) (3) (Chart 1).
Figure 2. In vivo MR T1 weighted spin echo (SE) coronal images of
mice obtained by tail vein injection with 2a (0.1 mmol/kg): H, heart;
L, liver; K, kidney; B, bladder; and A, abdominal aorta (64 MHz).
as compared with their DO3A analogues, although those
stabilities are slightly higher than those of their DTPA
counterparts.21 Motivated by these findings and in an effort
to increase thermodynamic and kinetic stabilities, we have
developed further two new DO3A-(biphenyl-2,2′-bisamide)
chelates (1) and their Gd complexes (2) (Chart 2).
They form neutral Gd(III) complexes belonging to class (i)
of BPCAs and exhibit very high blood-pool enhancement due
to the presence of highly lipophilic biphenyl in the macrocyclic
chelate backbone.21 One disadvantage of these imaging probes,
however, is much lower thermodynamic and kinetic stabilities
Figure 1. Proton longitudinal paramagnetic relaxation rates of 2a and 2b as a function of [Gd] in PBS (pH 7.4) solutions of HSA (0.67 mM) at 64
MHz and 293 K.
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ACS Medicinal Chemistry Letters
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with protein. The extent of relaxation enhancement, an ε*
titration, is as expected in that the relaxation rate increase is
much larger in the presence of HSA, and the same rate does not
increase linearly with concentration, indicating the presence of
protein binding (cf. Figure S1 in the Supporting Informa-
tion).24 The association constants (Ka) characterizing the
interaction between HSA and 2a and 2b are 2.11 × 102 and
1.07 × 102 M−1, respectively. These values can be derived from
the so-called M-titration, proton longitudinal relaxation rate as
a function of the concentration of the paramagnetic substrate at
the fixed concentration of HSA (Figure 1). Fitting of the data
was carried out according to the literature method.25 Here, it is
worth noting that the MS-325, a well-known BPCA, shows a Ka
value of 6.1 × 103 M−1, greater by almost 1 order of magnitude
than those of the 2a and 2b.25 As is the case with MS-325, the
role of two aromatic units via a lipophilic interaction may be
invoked to rationalize the increase in the relaxivity in a PBS
solution, although such increases are not so dramatic.
Observations such as these in connection with the biphenyl
moiety have already been noted in our previous work with the
DTPA-biphenyl-2,2′-bis(amide) analogues (3).21 In short, 2a
may serve as a novel BPCA in that it carries macrocyclic DOTA
units, which would provide further thermodynamic and kinetic
stabilities than acyclic DTPA analogues. To the best of our
knowledge, 2a is the first example of BPCA possessing DOTA
as a chelate.
Figure 2 shows in vivo coronal images of mice obtained by
tail vein injection with 2a. The figure reveals clearly blood-pool
enhancement in heart and abdominal aorta, and the image lasts
as long as 1 h to be eventually excreted through kidney.
Supportive with these observations is the biodistribution data of
2a and the CNR profiles provided in Figures S3 and S4 in the
Supporting Information, respectively. Such slow renal clearance
as well as high stabilities (both thermodynamic and kinetic)
may compensate for the potential risk of nephrogenic systemic
fibrosis (NSF), which has often been reported with acyclic
DTPA series such as Omniscan.
Figure 3. In vivo MR T1 weighted SE coronal images of brain tumor
(C6-glyoma cell) obtained with 2a (0.1 mmol/kg) (64 MHz).
The synthesis of 1 and 2 is straightforward as described in
Scheme S1 in the Supporting Information. The synthesis of 2
proceeds in two steps: That is, initial formation of the DO3A-
diaminobiphenyl conjugate (1) followed by complexation with
GdCl3. For the preparation of the mono-Gd analogue (2b), it is
initially required to protect one amino group in 2,2′-
bis(amino)biphenyl with di-tert-butyl dicarbonate. The for-
mation of 1 and 2 was confirmed by microanalytical,
spectroscopic (NMR and MS), and HPLC methods (cf. ESI).
Table 1 shows the proton relaxivities, R1 and R2, of 2a and 2b
along with Gd-DOTA (Dotarem) and MS-325 for comparative
purposes. Measurements were made in three different media:
water, PBS (pH 7.4), and a PBS solution of HSA. R1 relaxivities
of 2a and 2b in water are almost twice as high as that of
structurally analogous Dotarem. In PBS, however, the same
relaxivities drop to almost half the original values. The
observations such as these may be expected when invoking
possible displacement of the inner-sphere water molecule in 2
by the phosphate anion of PBS. Related observations such as a
R1 drop in the PBS solution of HSA have already been noted
and rationalized in terms of the inner-sphere water molecule
displaced by a donor atom from a carboxylate on the protein or
the phosphate anion of PBS.23 R1 relaxivities of 2a and 2b are
almost doubled in the PBS solution of HSA, demonstrating the
existence of interaction with HSA, although the interactions are
not as strong as the one observed with MS-325.
The proton relaxation enhancement (PRE) was measured to
obtain more detailed information about such an interaction
p
p
Figure 4. Evolution of longitudinal relaxation rates R1 (t)/R1 (0) as a function of time for various MRI CAs {[Gd]0 and [ZnCl2]0 = 2.5 mM in PBS
(pH 7.4) at 128 MHz and 293 K}.
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Figure 5. IMR-90 cell viability for 2 and Dotarem at various concentrations.
An additional feature of 2a is that it is capable of enhancing
the brain tumor for as long as an hour, which is rarely observed
with an ordinary ECF agent such as Dotarem (cf. Figure 3).
An additional advantage of 2a, when compared with existing
MS-325, lies in the fact that the former incorporates DOTA as
a chelate backbone rather than DTPA. Thus, greater enhance-
ment in kinetic stabilities would be expected with 2 than their
DTPA-based counterparts without a significant loss of the
blood-pool effect. Indeed, such an expectation is demonstrated
biodistribution, and CNR profiles. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +82-53-420-5471. E-mail: ychang@knu.ac.kr (Y.C.). Tel:
+82-53-950-5587. Fax: +82-53-950-6594. E-mail: tjkim@knu.
ac.kr (T.-J.K.).
Author Contributions
p
p
#These authors contributed equally.
in Figure 4, which shows the plots of R1 (t)/R1 (0) as a
P
function of time for various MRI CAs. The relative value of R1
Funding
P
P
at any time t, R1 (t)/R1 (0), is therefore a good estimator of the
extent of transmetalation by zinc. Among endogenous ions,
only Zn2+ can displace a significant amount of Gd because the
concentration of the former in blood is relatively high.26 Its
evolution over time gives relevant information about the
kinetics of the reaction. Quantitative transmetalation kinetic
data are provided (Figure S2 and Table S1 in the Supporting
Information). A dramatic increase in kinetic stability is
observed with 2 as compared with the plots exhibited by
DTPA-based CAs including 3, a new type of BPCA recently
developed by us.21 In addition, the kinetic stability of 2
compares well with that of Dotarem. Little change in R1
observed in the pH range 1−7 demonstrates that 2 is stable
enough under highly acidic conditions (Figure S7 in the
Supporting Information).
This work was partially supported by NRF (Grant Nos. 2010-
0024143 and 2011-0015353) and The Advanced Medical
Technology Cluster for Diagnosis & Prediction, KNU from
MKE (Grant No. RTI04-01-01). The KNU Research Fund
(2012) is also gratefully acknowledged.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank M.-K. Kang for her assistance in the biodistribution
experiment.
■
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* Supporting Information
■
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