Syntheses and relaxation properties of mixed gadolinium hydroxypyridinonate MRI contrast agents

Article abstract of DOI:10.1021/ic000563b

The tripodal ligand tris[(3-hydroxy-1-methyl-2-oxo-l,2-didehydropyridine-4-carboxamido)ethyl]amin e (TREN-Me-3,2-HOPO) forms a stable Gd³⁺ complex that is a promising candidate as a magnetic resonance imaging (MRI) contrast agent. However, its low water solubility prevents detailed magnetic characterization and practical applicability. Presented here are a series of novel mixed ligand systems that are based on the TREN-Me-3,2-HOPO platform. These new ligands possess two hydroxypyridinone (HOPO) chelators and one other chelator, the latter of which can be easily functionalized. The ligands described use salicylamide, 2-hydroxyisophthalamide, 2,3-dihydroxyterephthalamide, and bis(acetate) as the derivatizable chelators. The solution thermodynamics and relaxivity properties of these new systems are presented. Three of the four complexes (salicylamide-, 2-hydroxyisophthalamide-, and 2,3-dihydroxyterephthalamide-based ligands) possess sufficient thermodynamic stability for in vivo applications. The relaxivities of the three corresponding Gd³⁺ complexes range from 7.2 to 8.8 mM^-1 s^-1 at 20 MHz, 25 °C, and pH 8.5, significantly higher than the values for the clinically employed polyaminocarboxylate complexes (3.5-4.8 mM^-1 s^-1). The high relaxivities of these complexes are consistent with their faster rates of water exchange (<100 ns), higher molecular weights (>700), and greater numbers of inner-sphere coordinated water molecules (q = 2) relative to those of polyaminocarboxylate complexes. A mechanism for the rapid rates of water exchange is proposed involving a low energy barrier between the 8- and 9-coordinate geometries for lanthanide complexes of HOPO-based ligands. The pathway is supported by the crystal structure of La[TREN-Me-3,2-HOPO] (triclinic P1: Z = 4, a = 15.6963(2) A, b = 16.9978(1) A, c = 17.1578(2) A, α = 61.981(1)°, β = 75.680(1)°, γ = 71.600(1)°), which shows both 8- and 9-coordinate metal centers in the asymmetric unit, demonstrating that these structures are very close in energy. These properties make heteropodate Gd³⁺ complexes promising candidates for use in macromolecular contrast media, particularly at higher magnetic field strengths.

Full text of DOI:10.1021/ic000563b

Inorg. Chem. 2000, 39, 5747-5756
5747
Syntheses and Relaxation Properties of Mixed Gadolinium Hydroxypyridinonate MRI
Contrast Agents
Seth M. Cohen, Jide Xu, Emil Radkov, and Kenneth N. Raymond*
Department of Chemistry, University of California, Berkeley, California 94720
Mauro Botta
Dipartimento di Scienze e Tecnologie Avanzate, Universita` del Piemonte Orientale
“Amedeo Avogadro”, Corso Borsalino 54, 15100 Alessandria, Italy
Alessandro Barge and Silvio Aime*
Dipartimento di Chimica IFM, Universita` di Torino, Via P. Giuria 7, 10125 Torino, Italy
ReceiVed May 24, 2000
The tripodal ligand tris[(3-hydroxy-1-methyl-2-oxo-1,2-didehydropyridine-4-carboxamido)ethyl]amine (TREN-
Me-3,2-HOPO) forms a stable Gd3+ complex that is a promising candidate as a magnetic resonance imaging
(MRI) contrast agent. However, its low water solubility prevents detailed magnetic characterization and practical
applicability. Presented here are a series of novel mixed ligand systems that are based on the TREN-Me-3,2-
HOPO platform. These new ligands possess two hydroxypyridinone (HOPO) chelators and one other chelator,
the latter of which can be easily functionalized. The ligands described use salicylamide, 2-hydroxyisophthalamide,
2,3-dihydroxyterephthalamide, and bis(acetate) as the derivatizable chelators. The solution thermodynamics and
relaxivity properties of these new systems are presented. Three of the four complexes (salicylamide-,
2-hydroxyisophthalamide-, and 2,3-dihydroxyterephthalamide-based ligands) possess sufficient thermodynamic
stability for in vivo applications. The relaxivities of the three corresponding Gd3+ complexes range from 7.2 to
8.8 mM-1 s-1 at 20 MHz, 25 °C, and pH 8.5, significantly higher than the values for the clinically employed
polyaminocarboxylate complexes (3.5-4.8 mM-1 s-1). The high relaxivities of these complexes are consistent
with their faster rates of water exchange (<100 ns), higher molecular weights (>700), and greater numbers of
inner-sphere coordinated water molecules (q ) 2) relative to those of polyaminocarboxylate complexes. A
mechanism for the rapid rates of water exchange is proposed involving a low energy barrier between the 8- and
9-coordinate geometries for lanthanide complexes of HOPO-based ligands. The pathway is supported by the
crystal structure of La[TREN-Me-3,2-HOPO] (triclinic P1h: Z ) 4, a ) 15.6963(2) Å, b ) 16.9978(1) Å, c )
17.1578(2) Å, R ) 61.981(1)°, â ) 75.680(1)°, γ ) 71.600(1)°), which shows both 8- and 9-coordinate metal
centers in the asymmetric unit, demonstrating that these structures are very close in energy. These properties
make heteropodate Gd3+ complexes promising candidates for use in macromolecular contrast media, particularly
at higher magnetic field strengths.
Introduction
of attempts have been made to functionalize the ligand in order
to improve the water solubility of the Gd3+ complex. Initial
Complexes of gadolinium are important for their use as
commercial magnetic resonance imaging (MRI) contrast agents.
The novel complex Gd[TREN-Me-3,2-HOPO] has been shown
to possess several advantageous features for use as an MRI
contrast agent,1-4 including low toxicity, high stability (pGd
20.3), and high relaxivity (10 mM-1 s-1).5 To further exploit
the excellent properties of Gd[TREN-Me-3,2-HOPO], a number
studies focused on modifying the hydroxypyridinone (HOPO)
ring system6 and synthesizing substituted tris(2-aminoethyl)-
amine (TREN) scaffolds.7,8 An alternative ligand design is to
prepare mixed ligand systems that are more directly amenable
to substitution. Described here are tripodal ligands formed from
two HOPO chelators and a third binding group that can be easily
functionalized. The third chelating moiety of these “heteropo-
dands” can tune various properties of the ligand and the resulting
metal complex, including denticity, charge, solubility, and
stability.
* To whom correspondence should be addressed.
(1) Aime, S.; Botta, M.; Fasano, M.; Terreno, E. Acc. Chem. Res. 1999,
32, 941.
(2) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. ReV.
1999, 99, 2293.
(3) Aime, S.; Botta, M.; Fasano, M.; Terreno, E. Chem. Soc. ReV. 1998,
27, 19.
(4) Tweedle, M. F. In Relaxation Agents in NMR Imaging; Bunzli, J.-C.
G., Choppin, G. R., Eds.; Elsevier: Amsterdam, 1989; p 127.
(5) Xu, J.; Franklin, S. J.; Whisenhunt, D. W., Jr.; Raymond, K. N. J.
Am. Chem. Soc. 1995, 117, 7245.
(6) Johnson, A.; O’Sullivan, B.; Raymond, K. N. Inorg. Chem. 2000, 39,
2652.
(7) Hajela, S.; Botta, M.; Giraudo, S.; Xu, J.; Raymond, K. N.; Aime, S.,
in press.
(8) Hajela, S.; Johnson, A.; Sunderland, C. J.; Xu, J.; Cohen, S. M.;
Caulder, D. L.; Raymond, K. N. Manuscript in preparation.
10.1021/ic000563b CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/17/2000

5748 Inorganic Chemistry, Vol. 39, No. 25, 2000
Cohen et al.
CH2), 3.08 (q, J ) 6.0 Hz, 4H, CH2), 3.24 (q, J ) 5.8 Hz, 2H, CH2),
3.57 (s, 6H, NCH3), 5.04 (s, 2H, CH2), 5.27 (s, 4H, CH2), 6.66 (d, J )
7.2 Hz, 2H, Ar H), 6.96 (d, J ) 8.2 Hz, 1H, Ar H), 7.05 (d, J ) 7.2
Hz, 2H, Ar H), 7.30 (m, 17H, Ar H), 7.76 (t, J ) 5.2 Hz, 2H, NH),
7.85 (br t, 1H, NH), 8.11 (d, J ) 6.0 Hz, 1H, Ar H). 13C NMR (400
MHz, CDCl3, 25 °C): δ 34.0, 37.2, 37.6, 37.7, 52.0, 52.2, 71.2, 74.7,
104.7, 112.7, 121.4, 121.9, 127.8, 128.6, 128.7, 128.8, 129.0, 130.8,
132.1, 132.2, 132.4, 135.8, 136.3, 146.2, 156.6, 159.5, 163.3, 165.2.
Anal. Calcd (found) for C48H50N6O8‚2H2O: C, 65.89 (66.13); H, 6.22
(5.93); N, 9.60 (9.97). (+)-FABMS: m/z 839 [M+ + H].
TREN-Me-3,2-HOPOSAM (2). 1 (1.8 mmol) was dissolved in 40
mL of 1:1 concentrated HCl/glacial acetic acid. After 18 h of stirring,
the solution was evaporated to dryness. The residue was coevaporated
with 4 × 20 mL of MeOH and then oven-dried, giving a pale yellow
solid. Yield: 79%. IR (KBr pellet): ν 1540, 1594, 3255 cm-1. 1H NMR
(300 MHz, CD3OD, 25 °C): δ 3.35 (s, 6H, NCH3), 3.59 (br s, 6H,
CH2), 3.76 (br s, 6H, CH2), 6.18 (br s, 2H, Ar H), 6.62 (br s, 2H, Ar
H), 6.71 (d, J ) 7.2 Hz, 2H, Ar H), 7.17 (br t, 1H, Ar H), 7.47 (br d,
1H, Ar H). 13C NMR (400 MHz, DMSO-d6, 25 °C): δ 34.4, 37.1,
52.1, 75.3, 103.0, 115.5, 116.6, 117.5, 119.0, 127.9, 128.5, 134.1, 148.0,
158.3, 159.8, 166.9, 169.8. Anal. Calcd (found) for C27H33N6O8Cl‚
MeOH‚0.5HCl: C, 51.32 (51.24); H, 5.77 (5.58); N, 12.82 (12.55).
(+)-FABMS: m/z 569 [M+ + H].
Figure 1. Diagram of the ligands examined in this study. The ligands
are designated TREN-Me-3,2-HOPO- followed by the suffixes indicated
in the figure.
This work details the syntheses of heteropodands that possess
sites for facile functionalization (Figure 1). These ligands use
an (Me-3,2-HOPO)-substituted TREN compound as an universal
intermediate. The syntheses, solution thermodynamics, and
relaxivity properties of these systems are described. The results
indicate that three of the ligand systems described have
properties desirable for new MRI imaging agents, including high
relaxivity and thermodynamic stability. Among the most notable
attributes is an extremely rapid water exchange rate (30-100
ns) that makes these complexes amenable to incorporation into
macromolecular structures. The rapid water exchange rate is
due to a low-energy barrier between the 8- and 9-coordinate
metal complexes. This hypothesis is supported by the crystal
structure of La[TREN-Me-3,2-HOPO], which shows both
coordination numbers at the metal ion in the same unit cell.
The findings suggest that an HOPO-based heteropodand ligand
strategy has substantial promise for designing MRI contrast
agents.
TREN-Me-3,2-HOPOBAC (3). (3,2-HOPO)2TREN9 (3.2 mmol),
benzyl 2-bromoacetate (10 mmol), and anhydrous K2CO3 (10 mmol)
were combined in dry THF (50 mL). The stirred mixture was warmed
to 60 °C overnight under N2(g). After the system had cooled to room
temperature, the reaction mixture was filtered, the filtrate was rotary-
evaporated, and the residue was applied to a flash silica gel column.
Elution with 0.5-4.0% CH3OH in CH2Cl2 produced a pale yellow,
thick oil as the pure benzyl-protected precursor. The ligand was
immediately deprotected by dissolving the protected precursor in glacial
acetic acid (20 mL) containing 20% Pd(OH)2 on charcoal catalyst (200
mg). The mixture was stirred under an atmosphere of H2(g) (400 psi)
at room temperature overnight. The solution was filtered, and the filtrate
was evaporated to dryness, affording a pale brown residue. The residue
was recrystallized from methanol to give 3 as a white powder. Yield:
53%. 1H NMR (500 MHz, D2O, 25 °C): δ 3.29 (br s, 4H, CH2), 3.37
(s, 6H, CH2), 3.40-3.42 (br m, 2H, CH2), 3.54 (br s, 4H, CH2), 3.79
(s, 6H, NCH3), 6.351 (d, J ) 4.3 Hz, 2H, Ar H), 6.839 (d, J ) 4.3 Hz,
2H, Ar H). 13C NMR (500 MHz, DMSO-d6, 25 °C): ν 36.33, 36.85,
51.08, 51.68, 55.77, 102.55, 116.67, 127.43, 148.12, 158.05, 165.83,
172.26. (+)-FABMS: m/z 565 [M+ + H]. Anal. Calcd (found) for
C24H32N6O10‚1.2H2O: C, 49.17 (49.68); H, 5.91 (6.15); N, 14.33
(13.98).
Experimental Section
Syntheses. General Information. Unless otherwise noted, starting
materials were obtained from commercial suppliers and used without
further purification. Tris(2-aminoethyl)amine was distilled under
vacuum from CaH2. Flash silica gel chromatography was performed
using Merck 40-70 mesh silica gel. Microanalyses were performed at
the Microanalytical Services Laboratory, College of Chemistry, Uni-
versity of California, Berkeley. Mass spectra were recorded at the Mass
Spectrometry Laboratory, College of Chemistry, University of Cali-
fornia, Berkeley. All NMR spectra were recorded either on an AMX
300 or 400 Bruker superconducting Fourier transform spectrometer or
on a DRX 500 Bruker superconducting digital spectrometer. Infrared
spectra were obtained using a Nicolet Magna IR 550 Fourier transform
spectrometer. The syntheses of the terephthalamide heteropodand,
TREN-Me-3,2-HOPOTAM, and the isophthalamide heteropodand,
TREN-Me-3,2-HOPOIAM, are described elsewhere.9
Protected TREN-Me-3,2-HOPOSAM (1). (3,2-HOPO)2TREN9 (2.2
mmol) was dissolved in 70 mL of dry CH2Cl2. This solution was added
to a solution of 2-(benzyoxybenzoyl)succinimide (2.6 mmol) dissolved
in 30 mL of dry CH2Cl2. After 2 h, the resulting solution was evaporated
to dryness, affording an amber oil. The oil was purified by silica column
chromatography with 0-8% MeOH in CH2Cl2 as the eluant. Removal
of solvent gave the product as a white foam. Yield: 78%. IR (film
from CH2Cl2): ν 1538, 1645, 3384 cm-1. 1H NMR (300 MHz, CDCl3,
25 °C): δ 2.25 (t, J ) 6.7 Hz, 4H, CH2), 2.36 (t, J ) 6.5 Hz, 2H,
HGd[TREN-Me-3,2-HOPOTAM]. TREN-Me-3,2-HOPOTAM (0.10
mmol) was dissolved in 10 mL of MeOH. Gd(NO3)3‚6H2O (0.10 mmol)
was added to the methanol solution, followed by an excess of pyridine,
which caused the precipitation of an off-white solid. The mixture was
heated to reflux for 2 h and was then evaporated to dryness. The residue
i
was then suspended in PrOH. This was followed by sonication, and
filtration to give the acid complex final product. (-)-FABMS: m/z
795 [M-].
Gd[TREN-Me-3,2-HOPOIAM]. TREN-Me-3,2-HOPOIAM (0.10
mmol) was dissolved in 10 mL of MeOH. Gd(NO3)3‚6H2O (0.10 mmol)
was added to the methanol solution, followed by an excess of pyridine.
The mixture was heated to reflux for 2 h and was then evaporated to
dryness. The residue was suspended in iPrOH, followed by sonication
and filtration. (+)-FABMS: m/z 781 [M+ + H].
Gd[TREN-Me-3,2-HOPOSAM]. 2 (0.20 mmol) was dissolved in
10 mL of MeOH. Gd(NO3)3‚6H2O (0.20 mmol) was added to the
methanol solution, followed by an excess of pyridine, which caused
the precipitation of an off-white solid. The mixture was refluxed for 2
h and was then evaporated to dryness. The residue was suspended in
iPrOH, followed by sonication and filtration. (+)-FABMS: m/z 724
[M+ + H].
KGd[TREN-Me-3,2-HOPOBAC]. To a solution of 3 (0.20 mmol)
in dry methanol (20 mL) was added 4 equiv of KOH solution in
methanol (0.1027 N) to neutralize the ligand. A solution of GdCl3‚
6H2O (0.18 mmol) in methanol (10 mL) was added slowly to the ligand
solution with stirring. The mixture was heated to reflux for 6 h under
(9) Cohen, S. M.; O’Sullivan, B.; Raymond, K. N. Inorg. Chem., in press.

Mixed Gd3+ Hydroxypyridinonate MRI Contrast Agents
Inorganic Chemistry, Vol. 39, No. 25, 2000 5749
Scheme 1
nitrogen. The solvent was then removed, and the residue was purified
twice on a Sephedax column eluted with methanol. The complex was
precipitated from a 3:1 mixture of THF/MeOH, washed with THF, and
vacuum-dried. (-)-FABMS: m/z 717 [M-].
The path length of the quartz cell (Hellma, Suprasil) was either 1 or
10 cm. Once again, all solutions were maintained at constant ionic
strength (0.1 M KCl), under a nitrogen atmosphere and at constant
temperature (25.0 ( 0.2 °C), by using a jacketed titration vessel fitted
to a Neslab RTE-111 water bath. To 40-50 mL of 0.1 M KCl were
added ligand and metal from prepared solutions (2.0 mM and 0.1 M,
respectively). After the p[H] was decreased to about 2.0 with 1.0 M
HCl, the ligands were titrated with standardized base to about p[H]
7.0-8.0. The thermodynamic reversibility was checked by cycling
titrations from low to high p[H] and back to low p[H]. The data
(absorbances at 151 wavelengths ranging between 250 and 400 nm,
p[H] values, and respective volumes of 20-60 spectra) were analyzed
to determine the formation constants using the factor analysis program
FINDCOMP and the nonlinear least-squares refinement program
REFSPEC.15 All values and errors (one standard deviation) reported
represent the average of at least four independent experiments.
Relaxivity Studies. 1H NMR. The longitudinal water proton
relaxation rate at 20 MHz was measured by using a Spinmaster
spectrometer (Stelar, Mede, Italy) operating at 0.5 T; the standard
inversion-recovery technique was employed (16 experiments, 4 scans).
A typical 90°-pulse width was 3.5 ms, and the reproducibility of the
T1 data was (0.5%. The temperature was controlled with a Stelar VTC-
91 air-flow heater equipped with a copper-constantan thermocouple
(uncertainty of (0.1 °C). The proton 1/T1 NMRD profiles were
measured on a Koenig-Brown field-cycling relaxometer over a con-
tinuum of magnetic field strengths from 0.000 24 to 1.2 T (correspond-
ing to 0.01-50 MHz proton Larmor frequencies). The relaxometer
operates under computer control with an absolute uncertainty in 1/T1
of (1%. Details of the instrument and of the data acquisition procedure
are given elsewhere.16
Structure Determination and Refinement of La[TREN-Me-3,2-
HOPO]. The X-ray data collection and structure solution and refinement
procedures were as previously described.10-12 Crystals were grown as
colorless rhomboids from a solution of the complex in DMSO diffused
with MeOH (Table 3). The complex crystallized in the space group
P1h (No. 2), with a ) 15.6963(2) Å, b ) 16.9978(1) Å, c ) 17.1578-
(2) Å, R ) 61.981(1)°, â ) 75.680(1)°, γ ) 71.600(1)°, and Z ) 4.
Data were corrected for Lorentz and polarization effects, and an
empirical absorption was applied using XPREP (ellipsoidal model; Tmax
) 0.931 and Tmin ) 0.702). Two different metal centers were found in
the asymmetric unit (see Results and Discussion and Figure 9). All of
the solvent molecules found, one DMSO (0.2 occupancy) and five
MeOH (varying occupancy of 0.25 to full) molecules, were severely
disordered. All non-hydrogen atoms except those of the disordered
groups and solvent were refined anisotropically. Hydrogen atoms were
assigned to idealized positions. Final refinement for 8738 observations,
917 parameters, and 52 restraints gave R1 ) 0.0710, wR2 ) 0.1659,
and GOF ) 1.038.
Potentiometric Titrations. All titrant solutions were prepared using
distilled water that was further purified by passing through a Millipore
Milli-Q reverse-osmosis cartridge system (resistivity 18 MΩ cm). The
titrants were degassed by boiling for 1 h while being purged with argon.
Solutions were stored under an atmosphere of purified argon (Ridox
oxygen scavenger, Fisher) and Ascarite II (A. H. Thomas) scrubbers
in order to prevent absorption of oxygen and carbon dioxide. Carbonate-
free 0.1 M KOH was prepared from Baker Dilut-It concentrate and
was standardized by titrating against potassium hydrogen phthalate using
phenolphthalein as an indicator. Solutions of 0.1 M HCl were similarly
prepared and were standardized by titrating against the KOH solution
to the phenolphthalein end point. The combined pH glass electrode
(Corning) filled with 3 M KCl (Corning filling solution) was calibrated
in hydrogen ion concentration units (p[H] ) -log [H+]) by titrating
2.000 mL of standardized HCl diluted in 50 mL of 0.100 M KCl
(Mallinckrodt, ACS grade) with 4.200 mL of standardized KOH.
Titrations were performed using a Dosimat 665 (Metrohm) piston buret
and an Accumet 925 (Fisher) pH-meter or a Titrino 702 SM buret with
a built in pH-meter. All solutions were maintained at constant ionic
strength (0.1 M KCl), under a nitrogen atmosphere and at constant
temperature (25.0 ( 0.2 °C), by using a jacketed titration vessel fitted
to a Neslab RTE-111 water bath. PC computers controlled both
instruments.13 All values and errors (one standard deviation) reported
represent the average of at least four independent experiments.
Spectrophotometric Titrations. The apparatus and methods for
spectrophotometric titrations have been described in detail elsewhere.14
17O NMR. Variable-temperature 17O NMR measurements were
recorded on JEOL EX-90 (2.1 T) and EX-400 (9.4 T) spectrometers
equipped with a 5 mm probe. A D2O external lock and solutions
containing 2.6% of the 17O isotope (Yeda) were used. The observed
transverse relaxation rates were calculated from the signal width at half-
height. Other details of the instrumentation, experimental methods, and
data analysis have been previously reported.3,17
Theory. The theory behind the water 1H and 17O relaxation properties
is presented in the Supporting Information and can be found in several
references.3,18-20
Results and Discussion
Synthesis. TREN-Me-3,2-HOPOSAM was synthesized as
shown in Scheme 1. Reaction of 2-(benzyoxybenzoyl)succin-
(15) Turowski, P. N.; Rodgers, S. J.; Scarrow, R. C.; Raymond, K. N. Inorg.
Chem. 1988, 27, 474.
(16) Koenig, S. H.; Brown, R. D., III. NMR Spectroscopy of Cells and
Organism; CRC Press: Boca Raton, FL, 1987.
(17) Aime, S.; Botta, M.; Fasano, M.; Paoletti, S.; Terreno, E. Chem.s
Eur. J. 1997, 3, 1499.
(18) Banci, L.; Bertini, I.; Luchinat, C. Nuclear and Electron Relaxation;
VCH: Weinheim, Germany, 1991.
(19) Freed, J. H. J. Chem. Phys. 1978, 68, 4034.
(20) Powell, D. H.; Dhubhghaill, O. M. N.; Pubanz, D.; Helm, L.; Lebedev,
Y. S.; Schlaepfer, W.; Merbach, A. E. J. Am. Chem. Soc. 1996, 118,
9333.
(10) SAINT: SAX Area-Detector Integration Program, Version 4.024;
Siemens Industrial Automation, Inc.: Madison, WI, 1994.
(11) SHELXTL: Crystal Structure Analysis Determination Package; Si-
emens Industrial Automation, Inc.: Madison, WI, 1994.
(12) SMART: Area-Detector Software Package; Siemens Industrial Au-
tomation, Inc.: Madison, WI, 1994.
(13) Rodgers, S. J.; Lee, C. W.; Ng, C. Y.; Raymond, K. N. Inorg. Chem.
1987, 26, 1622.
(14) Kappel, M.; Raymond, K. N. Inorg. Chem. 1982, 21, 3437.

5750 Inorganic Chemistry, Vol. 39, No. 25, 2000
Cohen et al.
Scheme 2
Table 1. Protonation and Formation Constants for Heteropodand Ligands and Gd3+ Complexes as Measured by Potentiometric or
Spectrophotometric Titration (I ) 0.1 (KCl); T ) 25 °C)a
TREN-Me-3,2-HOPOb,c TREN-Me-3,2-HOPOBACd TREN-Me-3,2-HOPOSAMb TREN-Me-3,2-HOPOIAMb TREN-Me-3,2-HOPOTAMb
log K1
log K2
log K3
log K4
log K5
8.20(1)
6.95(3)
5.80(3)
4.96(5)
n/a
9.53(4)
7.30(2)
6.53(1)
3.23(3)
n/a
9.01(2)
7.86(1)
6.02(1)
5.24(1)
n/a
8.35(2)
7.20(2)
5.99(1)
5.05(5)
n/a
11.42(7)
8.14(3)
7.05(1)
5.75(1)
4.98(6)
log â110
log â111
log â112
20.3(2)
23.8(1)
n/a
14.8(2)
22.2(3)
25.3(3)
17.3(2)
23.6(1)
27.9(1)
16.5(1)
21.2(2)
26.6(1)
24.1(1)
29.7(2)
34.3(2)
pGd
20.3
13.7
16.1
16.2
20.1
a Numbers in parentheses correspond to the estimated standard deviations calculated from the variance/covariance matrix of at least four independent
measurements. b Protonation constants measured by potentiometric titration; formation constants measured by spectrophotometric titration. c From
ref 5. d Protonation and formation constants measured by potentiometric titration.
imide21 with the (Me-3,2-HOPO)2TREN intermediate9 gave the
benzyl-protected ligand (1). Deprotection with 1:1 glacial acetic
acid/concentrated hydrochloric acid afforded the free ligand (2)
as the hydrochloride salt.
HOPOIAM has protonation constants very similar to those of
the parent compound, consistent with the similar protonation
constants of the isophthalamide and HOPO chelators.24 Finally,
TREN-Me-3,2-HOPOBAC shows an intermediate behavior,
with the first three pKa’s being higher than those of the parent
compound but with the last pKa being much lower due to the
strongly acidic acetate groups.
The synthesis of TREN-Me-3,2-HOPOBAC is shown Scheme
2. The (Me-3,2-HOPO)2TREN intermediate was mixed with an
excess of benzyl 2-bromoacetate and anhydrous K2CO3 in dry
THF. After the mixture was heated overnight at 60 °C, the
protected ligand was isolated by flash silica column chroma-
tography. Immediate deprotection by reductive hydrogenation
(glacial acetic acid, 400 psi of H2(g), 20% Pd(OH)2/C) afforded
the free ligand as a white powder. The preparations of the other
two ligands in this study were similar and are described in detail
elsewhere.9 This methodology allows for the preparation of gram
quantities of the desired ligands. In addition, the thiazolide-
activated intermediates in the synthesis of TREN-Me-3,2-
HOPOIAM and TREN-Me-3,2-HOPOTAM react with a variety
of amines for attachment of solubilizing, bioactive, or macro-
molecular components.9 Simple modifications of the salicyl-
amide and bis(acetate) syntheses would also allow for facile
derivatization.
Ligand Protonation Constants. The protonation constants
for all of the heteropodands were determined by potentiometric
titration.9,13 These values were required for further determination
the stabilities of the heteropodand ligands with Gd3+. The pKa’s
and standard deviations for ligands are listed Table 1. The
titration results clearly demonstrate the influence of the mixed
chelator design on the protonation behavior of the ligands
relative to the parent compound. TREN-Me-3,2-HOPOSAM and
TREN-Me-3,2-HOPOTAM are more basic than TREN-Me-3,2-
HOPO.5 This is consistent with the basicity of the simple
chelators, whose methylamide derivatives have pKa’s of 8.03,
11.1/6.1, and 6.12 for the salicylamide, terephthalamide, and
hydroxypyridinone chelators, respectively.22,23 TREN-Me-3,2-
Stability Constants of the Ligands with Gd3+. Of the
heteropodate lanthanide complexes, only TREN-Me-3,2-HOPO-
BAC was sufficiently soluble to permit determination of the
formation constants by potentiometry (Table 1). The overall
formation constant (log â110) of Gd[TREN-Me-3,2-HOPOBAC]-
is 14.8(2), substantially lower than that of the parent complex
(20.3).5,6 The conditional stability constant (pGd at 1 mM Gd3+
,
10 mM ligand, pH 7.4) of a metal-ligand complex is typically
considered a better gauge of physiologically relevant complex
stability than the overall stability constant.25 The pGd value of
13.7 for Gd[TREN-Me-3,2-HOPOBAC]- is significantly lower
than that of the parent complex (20.3)5,6 and slightly lower than
that of Gd[DTPA-BMA], which has the lowest conditional
stability constant of all the commercially used MRI agents.
The stabilities of the heteropodand ligands TREN-Me-3,2-
HOPOIAM, TREN-Me-3,2-HOPOTAM, and TREN-Me-3,2-
HOPOSAM with Gd3+ were measured by spectrophotometric
titration.15 The strong UV absorptions (π f π*) of the aromatic
components of the ligands were monitored to follow the
complexation reactions. Typical experimental conditions com-
prised 20-60 spectra, monitored between 200 and 400 nm, for
a titration of each heteropodand in the presence of an equimolar
(22) Cohen, S. M.; Meyer, M.; Raymond, K. N. J. Am. Chem. Soc. 1998,
120, 6277.
(23) Garrett, T. M.; Miller, P. W.; Raymond, K. N. J. Am. Chem. Soc.
1989, 28, 128.
(24) Cohen, S. M. Ph.D. Dissertation, University of California, Berkeley,
CA, 1998.
(25) Cacheris, W. P.; Quay, S. C.; Rocklage, S. M. Magn. Reson. Imaging
1990, 8, 467.
(21) Cohen, S. M.; Raymond, K. N. Inorg. Chem. 2000, 39, 3624.

Mixed Gd3+ Hydroxypyridinonate MRI Contrast Agents
Inorganic Chemistry, Vol. 39, No. 25, 2000 5751
equilibrium constants which were fixed to the literature values.29
The calculated stability constants for the metal-ligand com-
plexes are provided in Table 1. As judged from the pGd values,
all of these ligands show satisfactory stability for consideration
as MRI contrast agents. The two salicylate-derived compounds,
TREN-Me-3,2-HOPOIAM and TREN-Me-3,2-HOPOSAM, have
pGd values of 16.2 and 16.1, respectively. This is lower than
the value of the parent compound5,6 but is competitive with those
of commercially used MRI contrast agents. TREN-Me-3,2-
HOPOTAM has a pGd value of 20.1, very similar to that of
the parent compound.5 The stability of Gd[TREN-Me-3,2-
HOPOTAM]- is enhanced, in part, by the additional intramo-
lecular hydrogen bond formed by the 2,3-dihydroxyterephthal-
amide unit.30
Relaxometry. At 20 MHz, 25 °C, and pH 8.5 the relaxivities
of the Gd3+ heteropodate complexes are 8.8, 7.2, 7.7, and 5.6
mM-1 s-1 for the terephthalamide, isophthalamide, salicylate,
and bis(acetate) complexes, respectively. Although smaller than
the parent compound,6,7 these complexes have relaxivities much
higher than those of all currently available commercial contrast
agents.2,3 The latter are monoaquo, enneacoordinated polyami-
H
nocarboxylate complexes with r1p values in the range 3.5-
4.8 mM-1s-1, under identical experimental conditions. At 20
MHz, the relaxivity of the aqueous solutions of small Gd3+
H
H
chelates under the rapid-exchange condition (T1M . τM , see
eq 3, Supporting Information) receives comparable contributions
H
H
os
is
from R1p
(∼60%) and R1p
(∼40%). The outersphere
Figure 2. Representative data from spectrophotometric titration
experiments: experimental (top) and calculated (bottom) spectra for a
titration of TREN-Me-3,2-HOPOIAM with Gd3+. Factor analysis
component has a slight dependence on the molecular size of a
complex, through the parameters a and D, but its values are
expected to be very similar for all of these systems (2.0-2.5
mM-1 s-1 at 25 °C).2-4 The inner-sphere term at 20 MHz is
dominated by the τR/rH6 ratio (see eq 4, Supporting Information)
and then increases with the molecular weight of the metal
chelates. It follows that the large relaxivity enhancement
observed for the heteropodand complexes reported here could
be attributed to (a) a different hydration structure (parameter q
in eq 3, Supporting Information), (b) a longer reorientational
correlation time (τR) due to the increased molecular weight, or
(c) a shorter distance (rH) for the inner-sphere water molecule-
2+
elucidated the presence of four species: LH4 (dotted line), MLH2
(dotted-dashed line), MLH+ (dashed line), and ML (solid line). I )
0.1 M (KCl); T ) 25 °C.
amount of Gd3+. All three systems displayed substantial spectral
changes upon metal complexation (see Figure 2 and Supporting
Information). The absorbance maxima of the spectra generally
shifted to lower energy as the pH was raised (pH ∼4-5) with
complicated multiple shoulder features at the longer wavelength
ends of the transitions. Further increases in pH caused all of
the spectral maxima to undergo substantial shifts to longer
wavelengths. This large movement in the absorption wavelength
maxima was coupled with a significant increase in the intensities
of the bands. The final spectra corresponded to the fully formed
Gd3+ complexes. At high pH (pH > 8), there was no change in
the UV absorption spectra.
The speciations of TREN-Me-3,2-HOPOTAM, TREN-Me-
3,2-HOPOIAM, and TREN-Me-3,2-HOPOSAM with Gd3+ are
similar to that reported for the parent compound,5 with the
observation of an extra species, the diprotonated complex
GdLH2.6 In fact, this species is also formed in the parent system,
as has been revealed by recent investigations.6,26 The latter
studies used a combination of spectral factor analysis,27,28
comparison of calculated and observed spectral band shapes,
and superior potentiometric calibration procedures6 to more
accurately examine the solution speciations. A close derivative
of the parent compound with improved water solubility has been
prepared, and thermodynamic analysis of its Gd3+ coordination
chemistry has established the presence of an equivalent GdLH2
species.6 The nonlinear least-squares refinement of the overall
formation constant â110 included the ligand protonation constants
obtained by potentiometric titration and the metal hydrolysis
H
(s). More insight can be gained by plotting r1p versus the
molecular weight (Figure 3) for the heteropodate complexes,
for the commercial MRI contrast agents Gd[DTPA]2- (DTPA
) 1,1,4,7,7-pentakis(carboxymethyl)-1,4,7-triazaheptane), Gd-
[DTPA-BMA] (DTPA-BMA ) 1,7-bis[(N-methylcarbamoyl)-
methyl]-1,4,7-tris(carboxymethyl)-1,4,7-triazaheptane), Gd-
[DOTA]- (DOTA ) 1,4,7,10-tetrakis(carboxymethyl)-1,4,7,-
10-tetraazacyclododecane), and Gd[HP-DO3A] (HP-DO3A )
10-(2-hydroxypropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tet-
raazacyclododecane), and for Gd[DO3A] (DO3A ) 1,4,7-tris-
(carboxymethyl)-1,4,7,10-tetraazacyclododecane), a macrocyclic
complex with two inner-sphere water molecules (q ) 2). The
H
linear relationship observed confirms the dependence of r1p
on the tumbling rates of the complexes and the close similarity
of the outer-sphere contributions and excludes significant
variations in the distance values (rH). Figure 3 also shows that
the heteropodands can be divided into two groups with one (q
) 1) and two (q ) 2) inner-sphere water molecules. The
relaxivity of Gd[DTPA-BMA] does not follow the expected
behavior because of a limiting (long) τM value.31,32
(29) Martell, A. E.; Motekaitis, R. M. Determination and Use of Stability
Constants; VCH: New York, 1988.
(30) Hou, Z. Ph.D. Dissertation, University of California, Berkeley, CA,
1995.
(26) O’Sullivan, B.; Raymond, K. N. Unpublished results.
(27) Tauler, R.; Smilde, A.; Kowalski, B. J. Chemom. 1995, 9, 31.
(28) Geladi, P.; Smilde, A. J. Chemom. 1995, 9, 1.

5752 Inorganic Chemistry, Vol. 39, No. 25, 2000
Cohen et al.
On the basis of the constants determined by potentiometric
titration (Table 1), the complex undergoes a transition between
pH 6 and 8, with the dominant species at pH 8 being the
unprotonated (ML-) complex, but by pH 6, the monoprotonated
complex (MLH) is the sole (>90%) component (see the
Supporting Information). The protonation likely occurs at the
acetate bridgehead nitrogen, breaking coordination to the metal
ion. This reduces the denticity of the ligand from hepta- to
hexadentate and provides an additional site for water coordina-
tion, subsequently increasing relaxivity. The pH-dependent
relaxivity is the result of a structural reorganization of the ligand
around the metal center caused by a change in the protonation
state of the complex.
The ligands TREN-Me-3,2-HOPOSAM, TREN-Me-3,2-HO-
POTAM, and TREN-Me-3,2-HOPOIAM were expected to bind
in a hexadentate fashion to the lanthanide ion, allowing for
coordination of at least two inner-sphere water molecules. The
relaxivity data (vide infra) support this hypothesis, suggesting
that the lanthanide complexes are 8-coordinate (like the parent
complex)5 with two inner-sphere water molecules (q ) 2).
Analogous to Gd[TREN-Me-3,2-HOPOBAC]-, these complexes
also show pH-dependent relaxivity profiles. The relaxivity
increases with a lowering of pH from 5 to 3 for Gd[TREN-
Me-3,2-HOPOTAM]-, from 6 to 3 for Gd[TREN-Me-3,2-
HOPOIAM], and from 7 to 4 for Gd[TREN-Me-3,2-HO-
POSAM]. This behavior reflects the increase of the hydration
number from q ) 2 to q ) 3 upon protonation of the ligand
and subsequent rearrangement of the coordination polyhedra
(see the Supporting Information). Ligand protonation likely
occurs at the 2-hydroxyisophthalamide, 2,3-dihydroxytereph-
thalamide, and salicylamide chelators for Gd[TREN-Me-3,2-
HOPOIAM], Gd[TREN-Me-3,2-HOPOTAM]-, and Gd[TREN-
Me-3,2-HOPOSAM], respectively (unpublished data).24 Proto-
tropic exchange may also contribute to the increase in relaxivity
at lower pH values.
Figure 3. Plots of relaxivity (20 MHz, 25 °C) versus molecular weight
for several Gd3+ MRI contrast agents. For these compounds, relaxivity
increases linearly with molecular weight. The linear relationship gives
a strong indication of the number of inner-sphere water molecules for
these complexes.
NMRD Profiles. Nuclear magnetic resonance dispersion
(NMRD) profiles can be used to determine the values of the
parameters that contribute to the relaxivity of a Gd3+ complex.2,3
The experiment involves measuring the magnetic field strength
(Larmor frequency) dependence of the solvent proton longitu-
dinal relaxation rate in the presence of a Gd3+ complex. This
field dependence is measured at several temperatures to improve
the fitting of several parameters. Figure 5 shows the NMRD
profiles and calculated fittings for the complexes at 15, 25, and
39 °C.
Figure 4. Plots of proton relaxivity (mM-1 s-1; filled circles) at 25
°C and 20 MHz and of the 17O transverse relaxation rate (s-1; open
circles) at 25 °C and 12 MHz versus pH for Gd[TREN-Me-3,2-
HOPOBAC]-.
The ligand TREN-Me-3,2-HOPOBAC is expected to bind
in a heptadentate fashion, using four HOPO oxygens, two acetate
oxygens, and the acetate tertiary nitrogen. The presence of a
single metal-bound water molecule in Gd[TREN-Me-3,2-
HOPOBAC]- suggests that this heptadentate ligand forms an
overall 8-coordinate lanthanide complex like the parent com-
pound.5 The relaxivity of Gd[TREN-Me-3,2-HOPOBAC]- is
strongly dependent upon solution pH (Figure 4). The relaxivity
is constant between pH 8 to 11, markedly increases upon
lowering the pH, and below pH 6 reaches a plateau of about
10.4 mM-1 s-1. The increase in relaxivity corresponds to an
increase of one water molecule (q ) 2) in the Gd3+ inner
coordination sphere. This is clearly confirmed by the fact that
the profile of the 17O transverse relaxation rate vs pH, which
only reports the effects associated with the metal-bound water
Numerous papers are available on the details of the fitting
of NMR dispersion profile data.2,3 For the purposes of this work,
the experimental data and parameters obtained from these data
will be presented and discussed in the context of (1) how HOPO-
based complexes compare with commercial MRI contrast agents,
(2) how the heteropodate structures compare with the parent
complex (and a more soluble analogue of the parent complex,
Gd[SerTREN-Me-3,2-HOPOTREN]),7,8 and (3) the overall
efficacy of these complexes as MRI contrast media.
As shown in Figure 5, the NMRD profiles of the HOPO-
based ligands first drop (in the range from 1 to 10 MHz) and
then rise with increasing magnetic field strength (increasing
Larmor frequency). These profiles are distinct from the NMRD
profiles of commercial polyaminocarboxylate complexes, which
begin to drop off at ∼1 MHz and continue to drop with
increasing field strength. This difference is important because
clinical MRI experiments are performed at frequencies between
20 and 60 MHz,2-4 a region where polyaminocarboxylate
complexes reach a minimum in relaxivity and HOPO-based
1
molecules, reproduces quite closely the H relaxivity behavior
for Gd[TREN-Me-3,2-HOPOBAC]-, also shown in Figure 4.
The observed relaxivity behavior can be explained in terms of
the speciation of the lanthanide complex as a function of pH.
(31) Gonzalez, G.; Pagliarin, R.; Sisti, M.; Terreno, E. J. Phys. Chem. 1994,
98, 53.
(32) Aime, S.; Botta, M.; Fasano, M.; Paoletti, S.; Anelli, P. L.; Uggeri,
F.; Virtuani, M. Inorg. Chem. 1994, 33, 4707.

Mixed Gd3+ Hydroxypyridinonate MRI Contrast Agents
Inorganic Chemistry, Vol. 39, No. 25, 2000 5753
Table 2 lists the parameters obtained from fitting the NMRD
profiles for all four Gd3+ complexes. The parameters are (1)
the zero-field value of the electronic relaxation time (τSO, which
is the value of τ1s at zero magnetic field strength), (2) the
correlation time of the fluctuation of the transient zero-field
splitting (τV), (3) the rotational correlation time (τR), and (4)
the distance between the water protons and the metal-centered
unpaired electron spin (rH).3
The metal-proton distances of the coordinated water mol-
ecules (rH) for the HOPO-based complexes are slightly smaller
than those found in commercial contrast agents.2-4 The shorter
distances between the paramagnetic centers and the water
protons do make small contributions to the higher relaxivities
of the HOPO-based compounds. Furthermore, rH is strongly
correlated with the rotational correlation time (τR) and thus its
variation may be insignificant. More importantly, all of the
HOPO-based systems have very long rotational correlation times
(τR) relative to those of the polyaminocarboxylate complexes.
This contributes significantly to their high relaxivities, as shown
previously in Figure 3, where the increasing molecular weights
of the complexes correspond to a linear increase with relaxivity.
Finally, the NMRD fittings indicate that Gd[TREN-Me-3,2-
HOPOIAM], Gd[TREN-Me-3,2-HOPOTAM]-, and Gd[TREN-
Me-3,2-HOPOSAM] have q values of 2, whereas Gd[TREN-
Me-3,2-HOPOBAC]- has a q values of 1. In the cases
of
Gd[TREN-Me-3,2-HOPOIAM],
Gd[TREN-Me-3,2-
HOPOTAM]-, and Gd[TREN-Me-3,2-HOPOSAM], the dou-
bling of the q values nearly doubles the values of the relaxivities
of the former compounds over those of the polyaminocarboxy-
late complexes.
17O NMR VT Relaxivity Behavior. The residence water
lifetime τM is a measure of how long a water molecule remains
bound to the lanthanide center before dissociating into the bulk
solvent. This value can be determined by measuring the
transverse 17O NMR relaxation time at various temperatures.
Typically, the residence lifetime is not a limiting parameter for
the relaxivity of a complex at physiological temperature.31,32
However, recent studies have shown that, for macromolecular
systems with extremely long rotational correlation times, the
water exchange rate can become the primary limiting factor for
attaining high relaxivity.1,34 In these macromolecular systems,
long rotational correlation times were obtained with noncovalent
HSA-DOTA adducts and dendrimer-DOTA conjugates. How-
ever, the water molecules bound to the lanthanide complexes
did not dissociate quickly enough (large τM), diminishing the
effect of the long τR in relaxing the bulk solution.
A primary goal of the heteropodand design was attachment
to macromolecular structures and therefore prompted the
determination the water exchange rates for these compounds.
If the water exchange rates of the heteropodand derivatives were
not sufficiently rapid, then any macromolecular derivatives
would suffer from the same limitations as the previously studied
DOTA- and DTPA-based macromolecular systems.33-37 Vari-
able-temperature 17O NMR experiments require higher concen-
Figure 5. Nuclear magnetic relaxation dispersion (NMRD) profiles
for the Gd3+ heteropodate complexes at 15 (circles), 25 (squares), and
39 °C (triangles). From top to bottom: Gd[TREN-Me-3,2-HOPOIAM]
(pH 9.0), Gd[TREN-Me-3,2-HOPOTAM]- (pH 7.6), Gd[TREN-Me-
3,2-HOPOSAM] (pH 9.0), and Gd[TREN-Me-3,2-HOPOBAC]- (pH
9.2).
(33) Aime, S.; Botta, M.; Fasano, M.; Geninatti Crich, S.; Terreno, E.
Coord. Chem. ReV. 1999, 321, 185.
(34) Toth, E.; Pubanz, D.; Vauthey, S.; Helm, L.; Merbach, A. E. Chem.s
Eur. J. 1996, 12, 1607.
(35) Aime, S.; Botta, M.; Fasano, M.; Crich, S. G.; Terreno, E. J. Bioinorg.
Chem. 1996, 1, 312.
(36) Aime, S.; Botta, M.; Geninatti Crich, S.; Giovenzana, G. B.; Pagliarin,
R.; Piccinini, M.; Sisti, M.; Terreno, E. J. Bioinorg. Chem. 1997, 2,
470.
complexes increase in relaxivity. The shape of these profiles is
due to the higher molecular weight of the HOPO-based chelates
and the subsequent increase in the rotational correlation time
(τR) that allows the field dependence of the electronic relaxation
time to become visible (see eqs 5-7, Supporting Information).
On a much larger scale, similar NMRD profiles have been
shown for polyaminocarboxylate ligands that are noncovalently
bound to human serum albumin (HSA).3,33
(37) Wiener, E. C.; Brechbiel, M. W.; Brothers, H.; Magin, R. L.; Gansow,
O. A.; Tomalia, D. A.; Lauterbur, P. C. Magn. Reson. Med. 1994, 31,
1.

5754 Inorganic Chemistry, Vol. 39, No. 25, 2000
Cohen et al.
Table 2. Parameters Obtained from Nuclear Magnetic Resonance Dispersion (NMRD) Profile Fittings
Gd[TREN-Me-3,2-HOPOBAC]- Gd[TREN-Me-3,2-HOPOSAM] Gd[TREN-Me-3,2-HOPOTAM]- Gd[TREN-Me-3,2-HOPOIAM]
T (°C)
τ
15
25
39
15
110
1
39
39
11
77
1
15
72
21
103
2
25
67
20
94
2
39
65
16
66
2
15
42
20
160
2
25
41
18
125
2
39
38
13
96
2
15
47
14
123
2
25
52
16
99
2
39
49
11
74
2
SO (ps)a 43
τV (ps) 18
τR (ps) 131
q
1
rH (Å)
2.95
2.95
2.95
3.10
3.10
3.10
3.05
3.05
3.05
3.10
3.10
3.10
a τSO is related to ∆2 and τV by the relation τSO ) (12∆2τV)-1
.
Figure 6. Plots of the 17O transverse relaxation rate versus temperature
for Gd[TREN-Me-3,2-HOPOTAM]- (21.6 mM, pH 8.5, 9.4 T; filled
circles) and Gd[DO3A] (50 mM, pH 7.5, 2.1 T; open circles).
trations than NMRD measurements. Only the most soluble
complexes Gd[TREN-Me-3,2-HOPOTAM]- and Gd[TREN-
Me-3,2-HOPOBAC]- could be evaluated by 17O NMR methods
at 9.4 and 2.1 T, respectively (Figure 6). For comparison, the
data for Gd[DO3A] (at 2.1 T) are presented, which demonstrate
a typical profile for an intermediate-exchange system with a
τM of about 160 ns at 25 °C.38 The profile for Gd[TREN-Me-
3,2-HOPOTAM]- (and Gd[TREN-Me-3,2-HOPOBAC]-; data
not shown) is notably distinct from that of Gd[DO3A] and is
indicative of an extremely rapid water exchange process
characterized by a τM (at 25 °C) of about 10-15 ns (see the
Supporting Information). The values of the exchange rates
cannot be determined with high accuracy by the experiment
because the 17O relaxation is not limited by the short τM but
rather by the longitudinal electron spin relaxation time (τ1s).
The water exchange rates are only 1 order of magnitude slower
than that of the aqua ion and 1-2 orders of magnitude faster
than those found for commercial MRI contrast agents.2-5 These
rapid exchange rates are also nearly optimal for a slowly rotating
MRI contrast agent.34 The data suggest that macromolecular-
based derivatives of the heteropodand ligands will not be limited
by water exchange, as was found for DTPA- and DOTA-based
systems. This is further illustrated by the simulated NMRD
profiles of Gd[TREN-Me-3,2-HOPOTAM]- and Gd[DOTA]-
as functions of increasing τR (Figure 7).34,37
1
Figure 7. Simulated H relaxivity profiles (mM-1 s-1) at 25 °C for
Gd[TREN-Me-3,2-HOPOTAM]- (top) and Gd[DOTA]- (bottom) with
varying reorientational correlation times τR. The lower curve in each
case represents the fitted experimental NMRD profile.
where the relaxivity increases with decreasing temperature (τR
becomes longer and T1MH decreases); (2) slow exchange (τM
.
T1MH), where the relaxivity depends on 1/τM and therefore
decreases with decreasing temperature; and (3) intermediate
exchange (τM and T1M have the same order of magnitude),
H
where there is little dependence of the relaxivity on temperature.
The temperature-dependent relaxivity profiles for Gd[TREN-
Me-3,2-HOPOIAM] and Gd[TREN-Me-3,2-HOPOSAM] in-
crease exponentially with decreasing temperature (see the
Supporting Information), indicating that they lie in the rapid-
exchange regime (are not dependent on τM) and that the water
exchange lifetimes are well below 100 ns.
As first suggested when the Gd[TREN-Me-3,2-HOPO]
complex was reported,5 the reason for the rapid water exchange
rate seems likely to be a low-energy barrier between a
9-coordinate tris(aqua) complex intermediate and the observed
8-coordinate bis(aqua) complex, both of which have similar
stabilities. This is the same reason that water exchange for the
lanthanide aqua ions is so rapid. The early-lanthanide aqua
complexes are 9-coordinate, and the smaller late-lanthanide aqua
ions are 8-coordinate. In both cases, the intermediate (dissocia-
tive for coordination number 9 and associative for coordination
Although the lanthanide complexes of TREN-Me-3,2-
HOPOIAM and TREN-Me-3,2-HOPOSAM were too insoluble
to directly study by 17O NMR experiments, their exchange rates
could be estimated by an indirect method. The temperature
1
dependence of the H relaxivity of a complex can yield useful
qualitative information about the water exchange rate when the
solubilities of the samples preclude analysis of the 17O NMR
data.38 Different exchange regimes are possible (see eq 3,
Supporting Information): (1) rapid exchange (τM , T1MH),
(38) Aime, S.; Botta, M.; Crich, S. G.; Giovenzana, G.; Pagliarin, R.; Sisti,
M.; Terreno, E. Magn. Reson. Chem. 1998, 36, S200.

Mixed Gd3+ Hydroxypyridinonate MRI Contrast Agents
Inorganic Chemistry, Vol. 39, No. 25, 2000 5755
Figure 8. Water exchange pathways for Gd3+ complexes (aminocarboxylate and hydroxypyridinonate) that interconvert between 8- and 9-coordinate
(upper panel). Free energy diagrams for water exchange: (left) 9-coordinate ground state; (right) 8-coordinate ground state; (middle) CN 8 and 9
equal in energy (lower panel).
number 8) is readily accessible (Figure 8). The commercial
contrast agents have 9-coordinate ground state geometries with
no 8-coordinate geometries observed, even for the smaller late
lanthanides. Hence, the complexes exchange water through
dissociative processes that go to relatively unstable 8-coordinate
intermediates. In a series where the larger metal ions are
9-coordinate and the smaller ones are 8-coordinate, there will
be an ionic radius where 9- and 8-coordinate complexes are
equal in energy. At that point, the associative (8- to 9-coordinate)
and dissociative (9- to 8-coordinate) exchange rates are identical
and the rate of exchange is maximum.
Our expectation that the energy difference between the
8-coordinate and 9-coordinate structures for the HOPO com-
plexes is very low is here confirmed. As previously described,5
the structure of Gd[TREN-Me-3,2-HOPO] is an 8-coordinate
square antiprism with six HOPO oxygen donors and two water
molecules. However, the crystal structure of same ligand
complexed with an early-lanthanide ion, namely, La[TREN-
Me-3,2-HOPO], has both an 8- and a 9-coordinate lanthanide
metal center in the unit cell (Figure 9). The coordination
geometry of one metal center (La(2)) is a square antiprism
comprising the six HOPO oxygens, one disordered solvent
(mixed DMSO and methanol), and an amide oxygen from the
neighboring La(1)[TREN-Me-3,2-HOPO] molecule. The other
metal center (La(1)) is a monocapped square antiprism com-
posed of the six HOPO oxygens, one methanol molecule, and
two amide oxygens from the neighboring La(2)[TREN-Me-3,2-
HOPO] molecule (Table 3).10-12 The presence of both coordina-
tion numbers in the same crystal lattice indicates that the
coordination environments of these two metal ions (La(1) and
La(2)) must be very similar in energy. The structure demon-
strates that the ligand can easily accommodate both coordination
numbers for the lanthanide ions. The La3+ and Gd3+ structures
taken together show that the TREN-Me-3,2-HOPO ligand allows
free interchange between 8- and 9-coordinate complexes. The
stability of both coordination numbers is consistent with the
rapid rate of water exchange for the HOPO-based complexes:
the ligand provides a low barrier for interconversion between
the two states, resulting in rapid solvent exchange (vide infra).
Further confirmation of this proposed mechanism can be
obtained by the use of variable-pressure NMR experiments,
which are capable of distinguishing between an associative and
a dissociative exchange mechanism by measuring the volume
of activation.39
Conclusions
The syntheses, solution thermodynamics, and relaxivity results
for four novel HOPO-based heteropodand ligands have been
presented. Potentiometric and spectrophotometric titrations have
shown that at least three of the four complexes possess
satisfactory thermodynamic stabilities to be considered as MRI
contrast agents. All of the compounds show high relaxivities
relative to those of commercially available contrast agents,
although the heteropodate complexes have somewhat lower
relaxivities than those found for tris(hydroxypyridinonate)
complexes.5,7 The high relaxivities of these compounds can be
attributed, in part, to several common features, including long
rotational correlation times and short metal-water bond dis-
tances. The complexes also have rapid water exchange rates,
as determined by 17O NMR and variable-temperature relaxivity
measurements. The rapid exchange rates can be explained in
terms of a low-energy barrier between the 8- and 9-coordinate
complexes, as evidenced by the structure of La[TREN-Me-3,2-
HOPO]. These rapid exchange rates also make the heteropodate
complexes attractive candidates for preparing macromolecular
MRI contrast agents.
Despite these common features, the compounds can be
divided into two classes on the basis of the number of
coordinated inner-sphere water molecules. A q value of 2 was
inferred for Gd[TREN-Me-3,2-HOPOIAM], Gd[TREN-Me-3,2-
(39) Frey, U.; Merbach, A. E.; Powell, D. H. In SolVent Exchange on Metal
Ions: A Variable NMR Approach; Delpuech, J.-J., Ed.; John Wiley &
Sons: Chichester, U.K., 1995; p 263.

5756 Inorganic Chemistry, Vol. 39, No. 25, 2000
Cohen et al.
Figure 9. Structural diagrams (ORTEP) of La[TREN-Me-3,2-HOPO]. The asymmetric unit (top) contains two complexes of La[TREN-Me-3,2-
HOPO] connected by amide oxygen coordination. The La(1) complex (bottom right, 9-coordinate monocapped square antiprism) is viewed down
the C4 axis, and the La(2) complex (bottom left, 8-coordinate square antiprism) is viewed perpendicular to the C4 axis. The structure suggests that
the 8- and 9-coordinate geometries are very similar in energy, allowing for rapid solvent exchange.
Table 3. Crystal Data and Structure Refinement Details for
La[TREN-Me-3,2-HOPO]
changes that make the metal centers more accessible to water
molecules and increases relaxation of the bulk solvents.
fw
temp
844.24
146(2) K
triclinic
P1h
a ) 15.6963(2) Å
R ) 61.981(1)°
b ) 16.9978(1) Å
â ) 75.680(1)°
c ) 17.1578(2) Å
γ ) 71.600(1)°
3807.69(7) Å3, 4
1.473 g cm-3
The mixed ligand design strategy is a facile and straightfor-
ward means to access novel ligand structures with several
tunable properties. This method has produced several promising
leads for the development of new MRI contrast agents. Starting
with HOPO-based systems, we prepared complexes that possess
high thermodynamic stabilities, high relaxivities, and rapid water
exchange rates. Continued investigations into heteropodand
“mixed” ligand systems, with other novel binding groups
substituting for the third HOPO chelator, should contribute to
the further development of new MRI contrast agents.
crystal system
space group
unit cell dimensions
V, Z
density (calcd)
crystal size
no. of reflns collected
no. of indep reflns
no. of data/restraints/params
goodness-of-fit on F2a
final R indices [I > 2σ(I)]b
R indices (all data)b
largest diff peak and hole
0.20 × 0.10 × 0.06 mm
20 526
Acknowledgment. S.M.C. thanks Dr. Brendon O’Sullivan
for help with the solution thermodynamics analysis. This work
was supported in part by NIH Grant AI11744 and by the
Director, Office of Energy Research, Office of Basic Energy
Sciences, Chemical Sciences Division, U.S. Department of
Energy, under Contract No. DE-AC03-76F00098. S.A. and M.B.
acknowledge the MURST and CNR (Legge 95/95) for financial
support.
12 855 [R(int) ) 0.020]
8738/52/917
1.038
R1 ) 0.0710, wR2 ) 0.1659
R1 ) 0.1131, wR2 ) 0.1880
+2.034 and -1.873 e Å-3
a GOF ) [∑w(|Fo| - |Fc|)2/(No - Nv)]1/2, where w ) 1/(σ2|Fo|). b R1
) ∑||Fo| - |Fc||/∑|Fo|; wR2 ) [∑w(Fo - Fc2)2/∑wFo ]1/2
.
2
4
HOPOTAM]-, and Gd[TREN-Me-3,2-HOPOSAM], which
contributes to the particularly high relaxivities of these com-
plexes. However, Gd[TREN-Me-3,2-HOPOBAC]- has only one
inner-sphere water molecule. In addition, all of the complexes
display pH-dependent relaxivity profiles. The pH dependence
can be correlated to the solution thermodynamics, which indicate
that the metal complexes become protonated near or below
neutral pH. Protonation of the metal complexes causes structural
Supporting Information Available: Plots of potentiometric titration
data, spectrophotometric titration data, species distributions of metal
complexes, and relaxivity versus pH/temperature data, a listing of
parameters obtained from 17O relaxation data as a function of
temperature, and text plus equations presenting the theory behind
relaxation measurements. This material is available free of charge via
the Internet at http://pubs.acs.org.
IC000563B