Structure-relaxivity relationships of serum albumin targeted MRI probes based on a single amino acid Gd complex

Article abstract of DOI:10.1021/jm4000177

The Gd(III) complex of DO3A-N-α-aminopropionate, Gd(DOTAla), was used to generate a small library of putative MRI probes targeted to human serum albumin (HSA). Ten compounds were synthesized via multistep organic synthesis, and the corresponding Gd complexes were investigated for their affinity to HSA, lipophilicity, and relaxivity in the absence and presence of HSA. Negative charge and moderate lipophilicity correlate with increased HSA affinity and relaxivity.

Full text of DOI:10.1021/jm4000177

Brief Article
pubs.acs.org/jmc
Structure−Relaxivity Relationships of Serum Albumin Targeted MRI
Probes Based on a Single Amino Acid Gd Complex
Eszter Boros and Peter Caravan*
The Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard
Medical School, 149 Thirteenth Street, Suite 2301, Charlestown, Massachusetts 02129, United States
S
* Supporting Information
ABSTRACT: The Gd(III) complex of DO3A-N-α-aminopropionate, Gd(DOTAla), was used to generate a small library of
putative MRI probes targeted to human serum albumin (HSA). Ten compounds were synthesized via multistep organic
synthesis, and the corresponding Gd complexes were investigated for their affinity to HSA, lipophilicity, and relaxivity in the
absence and presence of HSA. Negative charge and moderate lipophilicity correlate with increased HSA affinity and relaxivity.
the probe binds to HSA. This latter effect is due to the decrease
of the rotational diffusion rate (molecular tumbling rate) of the
Gd complex when it binds to the large HSA protein.10 This
slower tumbling rate of the paramagnetic Gd ion better
approximates the proton Larmor frequency and results in
increased relaxation. Tissue contrast will be greatest when both
effects are maximized, i.e., when the probe is highly bound to
HSA and when the relaxivity of the bound probe is optimized.
Additionally, a comparably low relaxivity of the unbound probe
is desirable, since this provides clearer delineation of target
bound and unbound probe in vivo.
We have recently explored the application of a single amino
acid type ligand system for Gd, termed DOTAla.19 We have
shown that DOTAla (DO3A-N-α-aminopropionate) deriva-
tives form kinetically inert Gd(III) complexes with respect to
Gd dechelation, We hypothesized that this ligand scaffold
would provide a convenient platform for the development of
novel HSA targeting scaffolds. The overall charge under
physiological conditions, the chirality of the α-carbon on
DOTAla, and the type and combination of HSA-binding
groups used are freely variable. This allows for the establish-
ment of a structure−relaxivity relationship for high relaxivity
contrast agents based on the DOTAla scaffold and for HSA
targeting in general.
INTRODUCTION
■
Magnetic resonance imaging (MRI) has established itself as one
of the main noninvasive imaging techniques in the clinic. This
technique relies on the interaction of the proton nuclei of water
with the applied magnetic field.1 Contrast agents (or MR
probes) can alter this interaction by modulating the
longitudinal (T1) or transverse (T2) relaxation time of the
surrounding proton nuclei.2,3 Contrast agents containing the
strongly paramagnetic Gd(III) ion are suitable for shortening
the T1 of the proton nuclei of H2O in their immediate vicinity;
the Gd-induced change in T1 normalized to contrast agent
concentration is termed relaxivity, r1. Commercial, small-
molecule, nontargeted Gd-based probes have relaxivities of
3.5−6.9 mM−1 s−1 when measured in blood plasma on clinical
scanners.4,5 Subsequently, the low sensitivity of MRI
commands high concentrations of these compounds in order
to achieve visible contrast. Developments in recent years have
focused on the increase of relaxivity of the contrast agent to
decrease the administered dose, as well as the specific targeting
of these compounds to suitable in vivo targets.6−8
Human serum albumin (HSA) is the major protein
component of blood plasma and serves as a transporter for
endogenous, hydrophobic compounds such as fatty acids and
bilirubin as well as a vehicle for small lipophilic drug
molecules.9 HSA provides an excellent target for Gd-based T1
agents for the purpose of blood pool imaging due to its high
concentration in the bloodstream and variety of binding
sites.10,11 HSA-binding Gd complexes have been widely
explored, and one such compound, gadofosveset (aka MS-
325 and sold as Vasovist or Ablavar), is approved for clinical
use.12 While gadofosveset provides greatly enhanced relaxivity
in the presence of HSA compared to other commercial agents,
its relaxivity is below the theoretical maximum at clinical field
strengths such as 1.5 T.13 Furthermore, the ligand used to
coordinate Gd is the acyclic polyaminocarboxylate DTPA, a
ligand with limited kinetic inertness.14 This has led to intense,
further investigation of HSA-targeted MR agents over the past
two decades.15−18 T1 enhanced contrast arising from HSA
targeting relies on two distinct effects: local probe accumulation
due to HSA binding, and a multifold increase in relaxivity when
RESULTS AND DISCUSSION
■
Chemistry. DOTAla or Fmoc-DOTAla served as conven-
ient intermediates for probe synthesis, placing the metal
complex at the C- or the N-terminus, if only one R group was
conjugated (Gd(1), Gd(2), Gd(4), Gd(9)), or at the center of
the corresponding artificial tripeptide structure. We chose R
groups like iodinated phenols or ibuprofen derivatives with
known affinity for HSA20 or to modify charge. For attachment
to the C-terminus, glycine methyl ester, (S)-methylbenzyl-
amine, and 3,5-diiodotyrosine were used, while for attachment
to the N-terminus (S)-methylbenzylcarboxylate, 3,5-diiodotyr-
osine, ibuprofen, or acetamide capping was utilized. The
Received: January 4, 2013
Published: February 7, 2013
© 2013 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
synthesis of each ligand was performed using typical solution
phase peptide synthesis (Supporting Information).
Table 1. Numbering Scheme of Compounds Synthesized and
Investigated for This Study
The HATU-mediated amide bond formation with N-
methylmorpholine as the corresponding base was followed by
HPLC purification and Fmoc deprotection using solid-support
piperazine. For coupling of more than one R group, this
sequence was repeated with an additional purification step after
the second Fmoc deprotection. Peptide coupling on the
DOTAla scaffold results in formation of diastereomers, which
were separated at a precursor stage in the case of ligand H3(6),
affording diastereomers H3(6a) and H3(6b). All other ligand
systems were isolated as isomer mixtures. The final Fmoc
deprotection step was generally followed by TFA-catalyzed
deprotection of tert-butyl esters to afford the ready to chelate
ligand; in the case of ligand H3(3), the tert-butyl protected
precursor for H3(2) was acetylated using acetic anhydride. The
corresponding crude intermediate was purified using prepara-
tive HPLC, and subsequently the tert-butyl esters were
removed using the same acid catalyzed deprotection conditions
as used for all other ligand systems. Complex formation was
achieved by dissolution of the ligand in H2O (Scheme 1),
compd
X
Y
Gd(1)
Gd(2)
Gd(3)
Gd(4)
Gd(5)
Gd(6a)
Gd(6b)
Gd(7)
Gd(8)
Gd(9)
H
Gly-OMe
H
(S)-NHC(CH3)Bn
(S)-NHC(CH3)Bn
OH
COCH3
Tyr(I)2-H
Tyr(I)2-H
Tyr(I)2-H
Tyr(I)2-H
(S)-COC(CH3)Bn
CO-Ibu
Gly-OMe
(S)-NHC(CH3)Bn
(S)-NHC(CH3)Bn
Tyr(I)2-OH
Tyr(I)2-OH
H
CO-Ibu
similar pathway (predominantly renal), while compounds with
longer tR might rely more on hepatic clearance.
HSA Binding. To determine the binding of the complexes
to HSA under physiologically relevant conditions, a well-
established ultrafiltration assay was employed.21 HSA, a 67 kDa
protein, constitutes about 4.5% of plasma (0.67 mM); hence, a
4.5% w/v solution of HSA in HEPES (50 mM) was used for all
related studies. Protein-complex binding was determined by
ultrafiltration across a membrane with a MW 5000 cutoff and
compared with the filtrate of a solution containing no HSA.
The amounts of unbound and total Gd complex were measured
directly by ICP-MS. A constant complex concentration of 0.1
mM was used. Binding, expressed as the fraction bound to HSA
as a percentage, is found in Table 2. HSA has a great number of
positively charged arginine and lysine residues within the drug
binding pockets, which in turn will increase affinity for smaller,
negatively charged compounds.22 This is clearly showcased
when comparing Gd(6a/b) and Gd(7). The diastereoisomers
Gd(6a) and Gd(6b) possess a terminal, protonated amine and
show similar and low affinity to HSA: only 37% binding on
average. On the other hand, Gd(7), which differs from Gd(6a/
b) only by replacement of the terminal amine with a terminal
carboxylate, shows greatly enhanced binding by almost 2-fold
(69%), compared to Gd(6a/b). Both diastereomers Gd(6a)
and Gd(6b) show only small difference in percent binding,
indicating that orientation of the metal complex alone has only
little influence. Complexes with a net negative charge (Gd(7),
Gd(8), Gd(9)) were found to have greatly increased affinity as
opposed to the neutral (Gd(3)) or cationic complexes (Gd(1),
Gd(2), Gd(4), (Gd(5), Gd(6a), Gd(6b)). Acetylation of the
primary amine (Gd(3)) also leads to only a slight, non-
significant increase in affinity to HSA compared to the
nonacetylated probe Gd(2). Combination of anionic charge
and two strongly HSA binding R groups provides the highest %
HSA binding (Gd(8)). The high % HSA binding of Gd(8)
could be explained by the occurrence of two separate binding
events with either R group or by binding of both R groups
simultaneously. In general, there was a positive correlation
between lipophilicity (tR) and affinity (% bound), although
there was considerable scatter, r2 = 0.72 (Figure S1).
Scheme 1. General Structure and Synthetic Scheme for
Derivatives H3(1)−H4(9) and the Corresponding Gd
a
Complexes Gd(1) through Gd(9)
a
(i) GdCl3·6H2O, H2O/MeOH (1:1), pH 6.5.
which results in an acidic solution (pH < 3). 0.9 equiv of
GdCl3·6H2O was added, and the pH was slowly adjusted to 6.5
using an aqueous NaOH solution (0.1 M). Complex formation
was monitored by LC−MS. Subsequent filtration and
lyophilization afforded the complex as an off-white solid. For
further studies, such as binding to HSA, relaxivity, and
lipophilicity estimates using a HPLC method, the solid was
redissolved in HEPES buffer (50 mM) and filtered. The
complex concentration in solution was determined using ICP-
MS. The final set of compounds includes derivatives bearing R
groups with presumed weak binding to HSA (Gd(1), Gd(2),
Gd(3)), one strongly HSA binding R group (Gd(4), Gd(5),
Gd(9)), or two strongly HSA binding R groups (Gd(6a/6b),
Gd(7), Gd(8)) (Table 1).
Lipophilicity. We utilized a common HPLC method with
an aqueous mobile phase at pH 6.8 to estimate lipophilicity of
the metal complexes at close to physiological pH. We assumed
that the measured retention time (tR) would provide us with a
good approximation for the corresponding log D of the metal
complexes. Table 2 summarizes the retention times measured,
including the clinically used compound MS-325. Comparison
of retention times can also provide clues on how these
compounds might distribute and clear in vivo. As the biological
behavior of MS-325 is well-known, one can hypothesize that
compounds with shorter tR than MS-325 might clear using a
Lipophilicity must also be considered in the context of
overall charge, which is also a dominant factor for predicting
affinity. For instance cationic Gd(6b) and anionic Gd(7) have
similar lipophilicity but the anionic Gd(7) has ∼2-fold higher
affinity for HSA.
Relaxivity. Relaxivity of all compounds synthesized was
determined from the slope of 1/T1 versus Gd concentration in
mM measured at 0.47 and 1.41 T in the absence and presence
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Table 2. Summary of Lipophilicity (as Retention Times), Relaxivity (r1) in 50 mM HEPES Buffer (pH 7.4, 37 °C) in the
Absence (r1free) or Presence (r1obs) of 4.5% HSA at 0.47 and 1.41 T and Percent of 0.1 mM Probe Concentration Bound to 4.5%
a
w/v HSA
0.47 T
1.41 T
lipophilicity, tR (min) r1 (mM−1 s−1
)
r1 (mM−1 s−1), 4.5% HSA r1 (mM−1 s−1
)
r1 (mM−1 −1), 4.5% HSA % bound to 4.5% HSA
s
obs
free
obs
free
Gd(1)
Gd(2)
Gd(3)
Gd(4)
Gd(5)
Gd(6a)
Gd(6b)
Gd(7)
Gd(8)
Gd(9)
MS-325
7.0
4.5
4.8
5.2
6.1
6.9
7.1
6.8
6.5
7.1
5.6
6.6
8.4
4.1
3.9
4.5
5.8
6.3
6.6
6.1
6.0
6.7
4.8
5.5
7.6
7
6.0
7.1
5.9
7
12.4
8.9
8.6
6.3
21
20
25
34
39
69
90
70
88
23.7
20.9
18.5
18.4
30.7
31.8
37.2
17.4
16.7
10.9
11.7
21.8
21.1
22.4
23.8
11.0
12.7
14.1
12.3
17
14.7
11.2
b
b
b
b
b
42
a
b
The error in the binding and relaxivity data is estimated to be 10% of the measured value. From ref 23.
of 4.5% w/v HSA in HEPES buffer, at compound
concentrations of 0.05−0.4 mM (without HSA) and 0.025−
0.15 mM (in presence of HSA). In the absence of HSA,
relaxivity shows a strong positive correlation (r2 = 0.94) with
molecular weight, as expected (Figure S2), since the heavier
complexes will tumble more slowly. The observed relaxivity
r1obs in the presence of HSA is the population-weighted average
bound
of the relaxivity due to the free (r1free) and bound (r1
)
probe, eq 1,
r obs = f r free + f r bound
1
1
1
b
(1)
f
where fb and ff are the mole fractions of bound and free, HSA
unbound probe, respectively.
If the relaxivity of the HSA-bound probe is the same for all
probes, then one would expect a linear relationship between
percent of bound probe and observed relaxivity for all
complexes. This would be true if HSA binding conferred
similar rotational dynamics on all probes and did not alter water
Figure 1. Correlation plots of observed relaxivity in HSA versus
percent binding to HSA. Compounds were split into two categories:
C-terminal aryl substituent (filled symbols) (Gd(2), Gd(3), Gd(6a),
Gd(6b), Gd(7), Gd(9)) and C-terminal aliphatic substituent (open
symbols) (Gd(1), Gd(4), Gd(5), Gd(8)).
obs
exchange kinetics. We did observe a good correlation of r1
with % bound to a first approximation, but we found that much
better correlations could be obtained if we grouped the
compounds in terms of whether or not they had a C-terminal
aryl substituent. Compounds with a C-terminal aryl substituent
(Gd(2), Gd(3), Gd(6a), Gd(6b), Gd(7), Gd(9)) showed only
moderate enhancement in relaxivity in presence of HSA. On
the other hand, derivatives with no C-terminal aryl substituent
(Gd(1), Gd(4), Gd(5), Gd(8)) provided considerably more
enhancement of relaxivity in the presence of HSA (higher
r1bound). This is shown graphically in Figure 1 where
extrapolation to 100% bound gives the estimated r1bound. It
was found that for the group containing an aryl substituent on
the C-terminus, the theoretical maximum relaxivity is
considerably lower (37.8 mM−1 s−1) than for compounds
with an aliphatic C-terminus (50.6 mM−1 s−1). There are
several possible reasons for this difference: (1) the two groups
of compounds could bind to different sites on HSA, and each
site would have different rotational dynamics; (2) there may be
more internal motion associated with compounds’ C-terminal
aryl group, and this in turn results in lower relaxivity; (3) there
could be other changes in compound hydration or water
exchange kinetics upon binding. While the physical studies
required to elucidate these differences are beyond the scope of
this report, this work illustrates the need to optimize affinity
and relaxivity when developing targeted MR probes.
For the C-terminal aryl group, it was found that (S)-
methylbenzylamine constitutes a rather poor binding group,
which does not lead to great increase of binding or relaxivity.
Predominant factors with influence on relaxivity are charge and
choice of the HSA binding R group. The binding pockets on
HSA are often described as deep, “sock-shaped” cavities with a
high density of cationic charge facilitating binding of smaller,
anionic molecules. This is readily showcased with compounds
from the aliphatic C terminus group. Binding and observed
relaxivity are highest for one of the smallest compounds of the
entire compound series, Gd(9). The monoanionic charge of the
complex likely increases the possibility for tight binding of the
complex itself through hydrogen bonding to the binding
pocket, while the strongly HSA binding R group is already in
place and is able to bind simultaneously and further rigidify the
structure of the bound probe.
MR Imaging. To illustrate how observed relaxivity in
presence of HSA compares to relaxivity in the unbound state, a
T1 weighted image was acquired for a series of 0.1 mM probe
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1
details and characterization of intermediates using H and 13C NMR
and LC−ESI-MS are in the Supporting Information, together with
ESI-MS data and synthetic procedure for Gd complexes. The ligands
H3(1)−H4(9) were obtained using general procedure 3 (Supporting
Information). The solvent was removed in vacuo, and the product was
isolated as the trifluoroacetate salt. For the formation of Gd complexes
Gd(1)−Gd(9), general procedure 4 was used (Supporting Informa-
tion).
concentration in the absence of presence of 4.5% HSA (Figure
2). The clinically used contrast agent MS-325 is shown as a
2,2′,2″-(10-(2-Amino-3-((2-methoxy-2-oxoethyl)amino)-3-oxo-
propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic Acid,
1
H3(1). H NMR (D2O, 500 MHz, ppm): 3.64−3.63 (m, 5H), 3.51
(s, 3H), 3.51−2.58 (m, 22H). 13C NMR (D2O, 125 MHz, ppm):
175.8, 171.6, 170.4, 170.3, 58.1, 56.7, 53.9, 52.9, 52.4, 50.4, 48.9, 46.0,
41.22. LC−ESI-MS calcd for C20H37N6O9: 505.25. Found: 505.3 [M +
H]+.
Figure 2. T1 weighted image of compounds MS-325, Gd(8), and
Gd(9) with measured signal intensity listed beneath each sample.
Samples contain 0.1 mM compound, with or without 4.5% w/v HSA.
TR/TE/flip angle = 25 ms/4.7 ms/70°.
2,2′,2″-(10-(2-Amino-3-oxo-3-(((S)-1-phenylethyl)amino)propyl)-
1
1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic Acid, H3(2). H
NMR (D2O, 500 MHz, ppm): 7.45−7.37 (m, 5H), 4.98−4.96 (m,
1H), 3.51 (m, 1H), 3.19−2.26 (m, 23H), 1.51 (t, 3H). 13C NMR
(D2O, 125 MHz, ppm): 179.0, 176.6, 128.8, 128.7, 126.0, 125.8, 59.3,
49.4, 21.1. LC−ESI-MS calcd for C25H41N6O7: 537.3. Found: 537.3
[M + H]+.
reference (far left), showing increased signal in presence of
HSA when compared with the MS-325 solution without HSA.
Gd(8), which has the highest affinity for HSA among the
compounds investigated, provides high signal in the presence of
HSA (right). However, Gd(8) also had the highest relaxivity in
the absence of HSA, and this results in the highest signal in the
absence of HSA compared to MS-325 or Gd(9). This high
relaxivity of Gd(8) in the unbound state results in lower
contrast between the samples with and without HSA (two
tubes on right). Gd(9) provides the greatest contrast between
the free and HSA-bound states (middle two tubes) due to a
considerably shorter rotational correlation time of the unbound
molecule but also a comparably high observed relaxivity in the
presence of HSA. This large change in relaxivity upon binding
and resultant increase in contrast for Gd(9) is highly
advantageous for in vivo applications, making it an interesting
candidate for future animal studies.
2,2′,2″-(10-(2-Acetamido-3-oxo-3-(((S)-1-phenylethyl)amino)-
propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic Acid,
1
H3(3). H NMR (CD3OD, 500 MHz, ppm): 8.4 (s, 1H) 7.29−7.15
(m, 5H), 4.95 (s, 1H), 4.01−2.85 (m, 24H), 1.91 (s, 3H), 1.39 (t, 3H).
1H NMR (CD3OD, 125 MHz, ppm): 178.3, 154.4, 147.3, 143.2, 128.2,
126.8, 125.9, 56.0, 55.7, 53.3, 50.6, 49.1, 48.4, 29.3, 15.6. LC−ESI-MS
calcd for C27H43N6O8: 579.3. Found: 579.3 [M + H]+.
2,2′,2″-(10-(2-(2-Amino-3-(4-hydroxy-3,5-diiodophenyl)-
propanamido)-2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-
1,4,7-triyl)triacetic Acid, H4(4). 1H NMR (D2O, 500 MHz, ppm):
7.8−7.66 (d, 2H), 4.33 (d, 1H), 4.34 (d, 1H), 3.89−2.54 (m, 24H).
13C NMR (D2O, 125 MHz, ppm): 174.9, 169.5, 154.3, 140.9, 131.1,
124.7, 84.8, 54.4, 51.9, 47.9, 35.2, 28.3. LC−ESI-MS calcd for
C26H39I2N6O10: 849.1. Found: 849.1 [M + H]+.
2,2′,2″-(10-(2-(2-Amino-3-(4-hydroxy-3,5-diiodophenyl)-
propanamido)-3-((2-methoxy-2-oxoethyl)amino)-3-oxopropyl)-
1
1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic Acid, H3(5). H
CONCLUSION
■
NMR (D2O, 500 MHz, ppm): 7.69 (s, 2H), 4.62 (d, 1H), 4.18 (s,
1H), 3.94 −2.80 (m, 30H). 13C NMR (D2O, 125 MHz, ppm): 171.0,
170.6, 170.1, 160.8, 160.5, 154.9, 140.8, 140.3, 129.4, 117.5, 115.1,
84.4, 84.0, 61.9, 56.1, 53.8, 53.1, 51.5, 51.3, 40.9. LC−ESI-MS calcd for
C29H44I2N7O11: 920.1. Found: 920.1 [M + H]+.
Ten derivatives of Gd(DOTAla) (DO3A-N-α-aminopropio-
nate) were synthesized with different relaxivities and affinities
for human serum albumin. We found that changes in affinity
are dominated by complex charge and choice of binding groups.
A combination of anionic charge and two strong binding
groups provides greatest affinity (Gd(8)). However, while the
combination of strong HSA binding substituents might increase
the fraction bound to HSA, it does not necessarily result in the
highest observed relaxivity. We found that the small,
monoanionic complex Gd(9) showed the highest relaxivity in
the presence of HSA, possibly due to a more efficient decrease
of rotational freedom. Gd(9) also provides the greatest increase
in relaxivity in the presence of HSA when compared to the
unbound probe. It compares well to values found for MS-325.
The ease of functionalization of the DOTAla scaffold does not
limit applications of this moiety to HSA but ultimately provides
an optimal platform for exploration of targeted, high relaxivity
Gd-based T1 agents.
2,2′,2″-(10-(2-(2-Amino-3-(4-hydroxy-3,5-diiodophenyl)-
propanamido)-3-oxo-3-(((S)-1-phenylethyl)amino)propyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic Acid, H3(6a/b). 1H NMR
(CD3OD, 500 MHz, ppm): 8.38 (d, 1H) 7.65 (d, 2H), 7.33−7.24 (m,
5H), 4.98 (d, 1H), 4.76−4.03 (m, 5H), 3.64−2.50 (m, 18H), 1.51−
1.39 (t, 3H). 13C NMR (CD3OD, 125 MHz, ppm): 169.7, 169.2,
160.2, 159.9, 154.9, 142.9, 140.6, 129.7, 128.1, 126.8, 125.9, 117.4,
114.8, 89.4, 81.2, 81.1, 53.8, 53.3, 51.3, 50.9, 49.0, 48.2, 34.3. LC−ESI-
MS calcd for C34H48I2N7O9: 952.2. Found: 952.2 [M + H]+.
2,2′,2″-(10-(3-(((S)-1-Carboxy-2-(4-hydroxy-3,5-diiodophenyl)-
ethyl)amino)-3-oxo-2-((S)-2-phenylpropanamido)propyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic acid, H3(7). 1H NMR
(CD3OD, 500 MHz, ppm): 7.97 (d, 1H), 7.61−7.28 (m, 7H), 4.66
(m, 1H), 4.02−2.61 (m, 26H), 1.59 (m, 3H). 13C NMR (CD3OD, 125
MHz, ppm): 171.0, 168.2, 161.2, 156.8, 142.4, 141.1, 139.9, 127.1,
126.8, 122.2, 121.3, 117.4, 83.9, 65.1, 49.2, 31.2. LC−ESI-MS calcd for
C35H47I2N6O11: 981.1. Found: 981.1 [M + H]+.
EXPERIMENTAL SECTION
2,2′,2″-(10-(3-((1-Carboxy-2-(4-hydroxy-3,5-diiodophenyl)ethyl)-
■
1
Chemistry. H and 13C NMR spectra were recorded on a Varian
amino)-2-(2-(4-isobutylphenyl)propanamido)-3-oxopropyl)-
1
1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic Acid, H3(8). H
11.7 T NMR system. The purity of tested compounds as determined
by analytical HPLC with absorbance at 220 nm was >95%. Estimation
of lipophilicity of complexes Gd(1)−Gd(9) and MS-325 was also
carried out using method C: mobile phases A (ammonium formate, 20
mM, pH 6.8) and B (9:1 MeCN/20 mM ammonium formate); flow
rate 0.8 mL/min; gradient, 0−40 min, 0−95% B; 40−43 min, 95% B;
43−43.5 min, 95−0% B; 43.5−45 min, 0% B. Further experimental
NMR (D2O, 500 MHz, ppm): 7.52 (d, 2H), 7.16−7.00 (m, 4H), 4.48
(m, 1H), 3.86−2.85 (m, 22H), 1.78 (m, 2H), 1.78 (m, 1H), 1.21 (m,
3H) 0.81 (m, 6H). 13C NMR (D2O, 125 MHz, ppm): 171.0, 170.6,
161.2, 153.2, 142.1, 140.6, 139.9, 129.3, 128.8, 128.1, 126.8, 83.9, 65.1,
53.4, 34.6, 30.0, 29.4, 27.4, 24.7, 22.3, 21.5, 21.3. LC−ESI-MS calcd for
C39H55I2N6O11: 1037.2. Found: 1037.1 [M + H]+.
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dx.doi.org/10.1021/jm4000177 | J. Med. Chem. 2013, 56, 1782−1786

Journal of Medicinal Chemistry
Brief Article
2,2′,2″-(10-(2-Carboxy-2-(2-(4-isobutylphenyl)propanamido)-
ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic Acid,
H4(9). 1H NMR (D2O, 500 MHz, ppm): 7.26 (d, 2H), 7.20 (d,
2H), 3.73 −2.83 (m, 26H), 2.43 (m, 2H), 1.78 (t, 1H), 1.41 (d, 3H),
0.81 (m, 6H). 13C NMR (D2O, 125 MHz, ppm): 177.5, 176.9, 141.9,
138.5, 130.1, 127.3, 70.2, 54.4, 46.5, 44.8, 30.7, 27.8, 22.8. LC−ESI-MS
calcd for C30H48N5O9: 622.3. Found: 622.4 [M + H]+.
Supkowski, R. M.; Horrocks, W. D.; McMurry, T. J.; Lauffer, R. B. The
Interaction of MS-325 with Human Serum Albumin and Its Effect on
Proton Relaxation Rates. J. Am. Chem. Soc. 2002, 124, 3152−3162.
(13) Caravan, P.; Farrar, C. T.; Frullano, L.; Uppal, R. Influence of
Molecular Parameters and Increasing Magnetic Field Strength on
Relaxivity of Gadolinium- and Manganese-Based T1 Contrast Agents.
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(16) Henoumont, C.; Elst, L. V.; Laurent, S.; Muller, R. N. Synthesis
and Physicochemical Characterization of Gd-C4-Thyroxin-DTPA, a
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Serum Albumin by Proton Relaxometry, NMR Diffusometry, and
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(17) Chang, Y.-H.; Chen, C.-Y.; Singh, G.; Chen, H.-Y.; Liu, G.-C.;
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Characterization of Carbon Backbone Modified [Gd(TTDA)-
(H2O)]2− Derivatives. Inorg. Chem. 2011, 50, 1275−1287.
(18) Nivorozhkin, A. L.; Kolodziej, A. F.; Caravan, P.; Greenfield, M.
T.; Lauffer, R. B.; McMurry, T. J. Enzyme-Activated Gd3+ Magnetic
Resonance Imaging Contrast Agents with a Prominent Receptor-
Induced Magnetization Enhancement. Angew. Chem., Int. Ed. 2001, 40,
2903−2906.
(19) Boros, E.; Polasek, M.; Zhang, Z.; Caravan, P. Gd(DOTAla)A
Single Amino Acid Gd-Complex as a Modular Tool for High Relaxivity
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(20) Peters, T. All about Albumin; Academic Press: San Diego, CA,
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(21) McMurry, T. J.; Parmelee, D. J.; Sajiki, H. S., D. M.; Ouellet, H.
S.; Walovitch, R. C.; Tyeklar, Z.; Dumas, S.; Bernard, P.; Nadler, S.;
Midelfort, K.; Greenfield, M.; Troughton, J.; Lauffer, R. B. The Effect
of a Phosphodiester Linking Group on Albumin Binding, Blood Half-
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(22) Aime, S.; Gianolio, E.; Longo, D.; Pagliarin, R.; Lovazzano, C.;
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ChemBioChem 2005, 6, 818−820.
ASSOCIATED CONTENT
* Supporting Information
Detailed experimental and characterization of intermediates, Gd
complexes, and list of nonstandard abbreviations. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: 617-643-0193. E-mail: caravan@nmr.mgh.harvard.edu.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
E.B. acknowledges the Swiss National Science Foundation for a
fellowship for prospective researchers. Nathaniel Kenton is
acknowledged for synthesis of DOTAla and Fmoc-DOTAla
according to ref 19. This work was supported in part by Award
R01EB009062 from the National Institute of Biomedical
Imaging and Bioengineering (NIBIB) and Award
P41RR14075 from the National Center for Research Resources
(NCRR).
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