Synthesis, relaxometric and photophysical properties of a new pH-responsive MRI contrast agent: The effect of other ligating groups on dissociation of a p-nitrophenolic pendant arm

Article abstract of DOI:10.1021/ja048299z

Two gadolinium(III) chelates, GdNP-DO3A (1-methlyene-(p-NitroPhenol)-1,4,7, 10-tetraazacycloDOdecane-4,7,10-triAcetate) and GdNP-DO3AM (1-methlyene(p- NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triacetAMide), containing a single nitrophenolic pendant arm plus either three acetate or three amide pendant arms were synthesized and characterized. The properties of the gadolinium, terbium, and dysprosium complexes of these ligands were examined as a function of pH. The extent and mechanism of the changes in water relaxivity with pH of each gadolinium complex was found to differ substantially for the two complexes. The water relaxivity of Gd(NP-DO3A) increases from 4.1 mM ^-1 s^-1 at pH 9 to 7.0 mM^-1 s^-1 at pH 5 as a result of acid-catalyzed dissociation of the nitrophenol from the lanthanide. The nitrophenol group in Gd(NP-DO3AM) does not dissociate from the metal center even at pH 5; therefore, the very modest increase in relaxivity in this complex must be ascribed to an increase in prototropic exchange rate of the bound water and/or phenolic protons.

Full text of DOI:10.1021/ja048299z

Published on Web 07/10/2004
Synthesis, Relaxometric and Photophysical Properties of a
New pH-Responsive MRI Contrast Agent: The Effect of Other
Ligating Groups on Dissociation of a p-Nitrophenolic Pendant Arm
Mark Woods,†,‡ Garry E. Kiefer,*,§ Simon Bott,| Aminta Castillo-Muzquiz,‡
Carrie Eshelbrenner,‡ Lydie Michaudet,‡ Kenneth McMillan,§ Siva D. K. Mudigunda,†
Doug Ogrin, Gyula Tircso´,‡ Shanrong Zhang,# Piyu Zhao,‡ and A. Dean Sherry*,‡,#
Contribution from the Macrocyclics, 2110 Research Row, Suite 425, Dallas, Texas 75235,
Department of Chemistry, UniVersity of Texas at Dallas, P.O. Box 803066,
Richardson, Texas 75083, Dow Chemical Company, 2301 North Brazosport BouleVard,
Freeport, Texas 77541, Department of Chemistry, UniVersity of Houston, 136 Fleming Building,
Houston, Texas 77204-5003, Department of Chemistry, Rice UniVersity, P.O. Box 1892,
Houston, 77251-1892, and Rogers Magnetic Resonance Center, Department of Radiology,
UniVersity of Texas Southwestern Medical Center, 5323 Harry Hines BlVd.,
Dallas, Texas 75390-9085
Received March 24, 2004; E-mail: sherry@utdallas.edu; gkiefer@dow.com
Abstract: Two gadolinium(III) chelates, GdNP-DO3A (1-methlyene-(p-NitroPhenol)-1,4,7,10-tetraaza-
cycloDOdecane-4,7,10-triAcetate) and GdNP-DO3AM (1-methlyene(p-NitroPhenol)-1,4,7,10-tetraaza-
cycloDOdecane-4,7,10-triacetAMide), containing a single nitrophenolic pendant arm plus either three acetate
or three amide pendant arms were synthesized and characterized. The properties of the gadolinium, terbium,
and dysprosium complexes of these ligands were examined as a function of pH. The extent and mechanism
of the changes in water relaxivity with pH of each gadolinium complex was found to differ substantially for
the two complexes. The water relaxivity of Gd(NP-DO3A) increases from 4.1 mM-1 s-1 at pH 9 to 7.0
mM-1 s-1 at pH 5 as a result of acid-catalyzed dissociation of the nitrophenol from the lanthanide. The
nitrophenol group in Gd(NP-DO3AM) does not dissociate from the metal center even at pH 5; therefore,
the very modest increase in relaxivity in this complex must be ascribed to an increase in prototropic exchange
rate of the bound water and/or phenolic protons.
Introduction
nonspecific perfusion agents. Nonetheless, this family of agents
has proven to be quite valuable even with a modest relaxivity.1,2
The use of paramagnetic metal complexes as contrast media
in magnetic resonance imaging (MRI) is now widely accepted
in diagnostic radiology.1-3 Agents currently approved for clinical
use are based upon low-molecular weight chelates of gadolinium
that partition throughout all extra cellular space and catalyze
the relaxation of nearby water protons. Areas of regionally high
contrast agent concentration are delineated by enhanced positive
contrast in T1-weighted MR images. The relaxivity, the increase
in the rate of water proton relaxation rate per unit contrast agent,
of all current clinical agents is ∼4 mM-1 s-1. Much new
developmental effort has focused on methods of improving
relaxivity as a means of enhancing overall sensitivity of these
New paradigms for MR contrast agents will undoubtedly extend
their utility to measures of pH changes or thermal variations.
An elegant example of a potential next-generation functional
imaging agent was described by Meade et al.4 In that work, a
Gd(DO3A) derivative possessing a covalently linked â-galactose
moiety was prepared in such a way as to block external
coordination of water to the central gadolinium ion. Subsequent
exposure to â-galactosidase cleaves the saccharide from the
complex, opening up a vacant coordination site to allow water
direct access to the gadolinium center. Exchange of coordinated
water molecules with the bulk solvent is a primary mechanism
by which these chelates catalyze water proton relaxation so that
modulating the number of water molecules coordinated in the
inner sphere will modulate the relaxivity, in this case in response
to the presence of â-galactosidase.
† Macrocyclics.
‡ University of Texas at Dallas.
§ Dow Chemical Company.
| University of Houston.
Rice University.
# University of Texas Southwestern Medical Center.
(1) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Chem. ReV. 1999,
99, 2293-2352.
Modulation of the hydration state, q, has been used to create
agents that change relaxivity in response to pH and calcium
(2) Merbach, A. E.; Toth, E. The Chemistry of Contrast Agents in Medical
Magnetic Resonance Imaging; Wiley: New York, 2001.
(3) Sigel, A.; Sigel, H. The Lanthanides and Their Interrelations with
Biosystems; Dekker: New York, 2003; Vol. 40.
(4) Moats, R. A.; Fraser, S. E.; Meade, T. J. Angew. Chem., Int. Ed. 1997, 36,
726-728.
9
9248
J. AM. CHEM. SOC. 2004, 126, 9248-9256
10.1021/ja048299z CCC: $27.50 © 2004 American Chemical Society

Effect of Ligating Groups on Responsive MRI Agents
A R T I C L E S
Chart 1. Structures of 1-Methlyene-(p-NitroPhenol)-1,4,7,10-tetra-
azacycloDOdecane-4,7,10-triAcetate (NP-DO3A) and 1-Methlyene-
(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triacet-
AMide (NP-DO3AM)
Scheme 1. Synthesis of NP-DO3Aa
a Reagents and conditions: i. 2-hydroxyl-5-nitrobenzyl bromide/dioxane
(55%) ii. BrCH2CO2H/H2O/pH 8.5 (49%).
Scheme 2. Synthesis of NP-DO3AMa
and zinc ion concentrations.5-8 Lowe et al. employed a
toluenesulfonamide as a pH labile ligating group in a Gd(DO3A)
chelate;5 as the pH of a solution is reduced, the sulfonamide
becomes protonated and dissociates from the metal center. This
increases the hydration state from q ) 1 to q ) 2, and a
corresponding increase in relaxivity is observed. These authors
also reported that careful consideration must be given to the
ligating ability and pKa of the pH labile ligand to observe this
effect.5,9 We have recently observed a similar pH-sensitive
dissociation in complexes with one p-nitrophenolic ligating
group. The results of studies performed on the lanthanide(III)
complexes of two ligands, the triacetate (NP-DO3A) and
triamide (NP-DO3AM) (Chart 1) are reported herein. These
studies highlight the extent to which the choice of other ligating
groups in the complex can affect the success of this type of
responsive agent.
a
Reagents and conditions: i. (Boc)2O/CHCl3 (90%), ii. BnOCOCl/Et3N/
CHCl3 (98%), iii. HCl/MeOH (94%), iv. BrCH2CONH2/MeCN/K2CO3
(90%), v. H2/10% Pd on C/EtOH (99%), vi. 2-hydroxyl-5-nitrobenzyl
bromide/K2CO3/EtOH (65%).
trituration of the mono-alkylated from chloroform. The acetates
were then introduced into the mono-alkylated cyclen 1 using
bromoacetic acid in water at pH 8.5. After precipitation from
acidified water, NP-DO3A was obtained in an overall yield of
27%. The lanthanide complexes of NP-DO3A were synthesized
from the corresponding lanthanide chloride in water at pH 4.5.
Considering the low overall yield obtained in the synthesis
of NP-DO3A, a different approach was taken in the synthesis
of the triamide derivative, NP-DO3AM (Scheme 2). Here, three
cyclen amines were first protected as tert-butoxycarbamates
using di-tert-butyl dicarbonate.10 The remaining amine was then
orthogonally protected as a benzyloxycarbamate using benzyl
chloroformate. The mono-Cbz-protected cyclen 4 was obtained
by removal of the Boc groups using methanolic hydrochloric
acid. After liberation of the free base by base extraction, the
three amines of 4 were alkylated with bromoacetamide, using
standard conditions (K2CO3/MeCN). DO3AM was obtained
from the Cbz-protected DO3AM 5 by hydrogenolysis of the
benzyloxycarbamate with H2 and 10% palladium on carbon.
2-Hydroxy-5-nitrobenzyl bromide was then used to alkylate the
fourth nitrogen of DO3AM in ethanol, using K2CO3 as a base.
Results and Discussion
Synthesis. The ligand NP-DO3A was synthesized according
to the route shown in Scheme 1. 2-Hydroxy-5-nitrobenzyl
bromide was added to 2 equiv of cyclen in dioxane. Mono-
alkylated cyclen was separated from di-substituted cyclens by
column chromatography and residual cyclen removed by
(5) Lowe, M. P.; Parker, D.; Reany, O.; Aime, S.; Botta, M.; Castellano, G.;
Gianolio, E.; Pagliarin, R. J. Am. Chem. Soc. 2001, 123, 7601-7609.
(6) Li, W.-h.; Fraser, S. E.; Meade, T. J. J. Am. Chem. Soc. 1999, 121, 1413-
1414.
(7) Li, W.-h.; Parigi, G.; Fragai, M.; Luchinat, C.; Meade, T. J. Inorg. Chem.
2002, 41, 4018-4024.
(8) Hanaoka, K.; Kikuchi, K.; Urano, Y.; Nagano, T. J. Chem. Soc., Perkin
Trans. 2 2001, 1840-1843.
(9) Aime, S.; Batsanov, A. S.; Botta, M.; Howard, J. A. K.; Lowe, M. P.;
Parker, D. New J. Chem. 1999, 23, 669-670.
(10) Brandes, S.; Gros, C.; Denat, F.; Pullumbi, P.; Guilard, R. Bull. Soc. Chim.
Fr. 1996, 133, 65-73.
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A R T I C L E S
Woods et al.
Table 1. Crystal Structure Determination of
Table 2. Selected Angles (deg) and Bond Distances (Å) from the
-
-
Eu(NP-DO3AM)(H2O)(CF3SO2
)
2
Crystal Structure of Eu(NP-DO3AM)(H2O)(CF3SO2
)
2
formula
C23H35EuF6N8O13S2
Angles
N1-C2-C3-N4
formula mass
temperature
crystal system
space group
unit cell
a (Å)
b (Å)
c (Å)
961.67
293 K
triclcinic
P1h
56.7
60.4
61.0
64.4
13.7
3.3
26.7
74.4 (2)
65.4 (2)
63.9 (2)
64.8 (2)
68.2 (2)
75.0 (2)
68.0 (2)
70.9 (2)
N4-C5-C6-N7
N7-C8-C9-N10
N10-C11-C12-N1
N4-C41-C42-O41
N7-C71-C72-O71
N10-C101-C102-O101
N(1)-Eu(1)-O(15)
N(4)-Eu(1)-O(41)
N(7)-Eu(1)-O(71)
N(10)-Eu(1)-O(101)
O(15)-Eu(1)-O(1W)
O(41)-Eu(1)-O(1W)
O(71)-Eu(1)-O(1W)
O(101)-Eu(1)-O(1W)
11.042(2)
11.956(2)
13.723(3)
92.36
105.63
101.76(3)
1699.2(6)
2
R (deg)
â (deg)
γ (deg)
volume (Å3)
Z
final R (I > 2σ(I))
R (all data)
GOF
R1 ) 0.0422; wR2 ) 0.1741
R1 ) 0.0828; wR2 ) 0.2168
1.01
Bond Lengths
Eu(1)-O(1W)
Eu(1)-O(15)
Eu(1)-O(41)
Eu(1)-O(71)
Eu(1)-O(101)
Eu(1)-N(1)
Eu(1)-N(4)
Eu(1)-N(7)
Eu(1)-N(10)
2.486 (5)
2.242 (5)
2.382 (5)
2.403 (5)
2.387 (5)
2.643 (6)
2.685 (6)
2.745 (6)
2.676 (6)
a The full data set has been deposited with the Cambridge Crystal-
lographic Database.
donor atoms around the europium cation resembles that observed
in crystal structures of other complexes of this type. The
macrocycle adopts a [3333] conformation with each ethylene
bridge adopting a gauche conformation (average N-C-C-N
torsion angle, 60.6°). The four nitrogen atoms of the macrocycle
adopt a coplanar arrangement on one face of the europium ion.
The four oxygen atoms of the pendant arms adopt a similar
coplanar arrangement, sandwiching the europium ion. A water
molecule caps the complex sitting above the plane of the oxygen
atoms. The torsion angle between the N4 and O4 planes of 37°
defines the coordination geometry as a distorted monocapped
square antiprism. Examination of the data (Table 2) reveals that
a degree of distortion is present in the structure presumably the
result of incorporation of the six-membered chelate of the
phenol. The europium-nitrogen, -amide oxygen and -water
oxygen bond lengths are comparable with those reported for
other tetraamide derivatives of DOTA.11,12 The Eu-Ophen bond
distance is substantially shorter (0.15 Å) than the three
Eu-Oamide bonds, presumably reflecting the ionic nature of the
phenoxide oxygen atom. The two triflate anions lie above the
coordinated water molecule (Figure 1) with each triflate
positioned such that a hydrogen bond from one triflate oxygen
to one of the protons of coordinated water molecule is possible.
The distances between the triflate oxygen and the water oxygen
are 2.988 and 2.910 Å, respectively, consistent with hydrogen-
bonding interactions. These conclusions are supported by the
observations of Parker and co-workers who observed similar
hydrogen bonding interactions with triflate anions in related
tetraamide complexes.12
Figure 1. Crystal structure of Eu(NP-DO3AM)(H2O)(CF3SO2-)2 showing
a side view (upper) and top view (lower). Hydrogen atoms bonded to carbon
have been omitted for clarity. The structure shown is the ∆(λλλλ) conformer,
the Λ(δδδδ) conformer, cocrystallized in the P1h unit cell, is not shown.
The dotted lines indicate the hydrogen-bonding interactions of the coordi-
nated water molecule and the triflate anions. An ORTEP rendering of the
structure is included in the ESI.
After crystallization from methanol and acetonitrile the ligand
NP-DO3AM was obtained in an overall yield of 48%. The
lanthanide complexes of NP-DO3AM were prepared from
equimolar quantities of the ligand NP-DO3AM and either the
lanthanide chloride or triflate at 60 °C in water.
Crystal Structure. An X-ray-quality crystal of the
europium(III) salt of NP-DO3AM (Eu(NP-DO3AM)) was
grown from aqueous solution (pH 5) by slow evaporation at
room temperature (see structure determination data in Table 1).
The structure obtained by X-ray diffraction is shown in Figure
1. The complex crystallizes in the P1h space group with two
complexes, replete with coordinated water molecules, and four
triflate counterions per unit cell. Since there are only two
counterions per complex present in the unit cell, the overall
formal charge of the complex is 2+. This indicates that the
phenol is deprotonated in this structure. The arrangement of
(11) Aime, S.; Barge, A.; Bruce, J. I.; Botta, M.; Howard, J. A. K.; Moloney,
J. M.; Parker, D.; de Sousa, A. S.; Woods, M. J. Am. Chem. Soc. 1999,
121, 5762-5771.
(12) Dickins, R. S.; Howard, J. A. K.; Maupin, C. L.; Moloney, J. M.; Parker,
D.; Riehl, J. P.; Siligardi, G.; Williams, J. A. G. Chem. Eur. J. 1999, 5,
1095-1105.
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Effect of Ligating Groups on Responsive MRI Agents
A R T I C L E S
Gd(NP-DO3AM) when the relaxivity data are fitted to eq 1.
[GdLH]
Ka )
(1)
[GdL-][H+]
That the pKa values of Ln(NP-DO3AM) complexes are lower
than those of the corresponding acetate analogues may easily
be rationalized in terms of the electron-donating ability of the
other pendant arms in the complex. Acetates are ionic electron
donors and therefore much more effective electron donors than
the nonionic amides. Given the increased electron donation to
the central lanthanide in Ln(NP-DO3A) complexes, it is
expected that the lanthanide will be less electron deficient in
these complexes than in the corresponding Ln(NP-DO3AM)
complexes. Ln(NP-DO3A) complexes will therefore have less
demand for electron density from the phenolic group, raising
the pKa of the phenolic proton This, in part, accounts for the
higher pH range over which the relaxivity is enhanced for
Gd(NP-DO3A). However, it is apparent that a difference in the
mechanism by which these two complexes enhance relaxivity
exists. The relaxivity enhancement of Gd(NP-DO3A) occurs
as the phenolic group is being protonated, whereas the enhance-
ment observed for Gd(NP-DO3AM) occurs at below the pH at
which the phenol is protonated. We postulate that the origin of
this difference in mechanism is also the result of differences in
the electron-donating ability of the other ligating groups present
in these two complexes.
Luminescence and NMR Measurements. Lanthanide-based
luminescence is an invaluable tool for probing the structure and
behavior of lanthanide complexes, giving information on the
extent of hydration, interactions with cations and anions, as well
as structural and electronic aspects of a complex. Luminescence
is especially useful for measuring the hydration state, q, of
lanthanide complexes using Horrocks’ method15,16 or a modified
method reported by Parker and co-workers.17 The intensity of
lanthanide-based luminescent emission may often be enhanced
by excitation of the lanthanide ion via a sensitizing chromo-
phore. However, the p-nitrophenol substituent proves to be a
hindrance rather than a help in this regard. Europium and
terbium are the most commonly studied lanthanide ions due to
their long-lived excited states. But, due to the comparative ease
with which the Eu3+ ion may be reduced (Eox ≈ 0.8 V) the
study of europium by luminescence in this case was not feasible.
Cyclic voltammograms of Gd(NP-DO3AM) over the pH range
4.1-7.6 show that the first oxidation of the p-nitrophenol moiety
occurs at 128 mV (referenced to Ag/AgCl) and is insensitive
to pH. Thus ligand-metal charge transfer (LMCT) from the
readily oxidized p-nitrophenol group rapidly quenches the
excited state of the Eu3+ ion. Europium-based emission in these
ligands is so weak that a study of these complexes was
precluded. This limited study to the Tb3+ complexes.
Figure 2. Relaxivity pH profiles of Gd(NP-DO3A) (filled diamonds) and
Gd(NP-DO3AM) (open squares) recorded at 25 °C and 20 MHz.
Relaxivity Measurements. The effect of pH upon the
relaxivity of the two corresponding gadolinium complexes was
examined at 25 °C and 20 MHz (Figure 2). The pH profile of
Gd(NP-DO3A) shows a fairly substantial relaxivity enhancement
(71%) on passing from high to low pH. This relaxivity
enhancement was found to be reversible, and measurements
repeated after incubation for 2 days confirmed that the enhance-
ment was not the result of dissociation of the gadolinium ion.
The relaxivity pH profile is noticeably different from that
observed for Gd(DOTA)- which remains flat across this same
pH range.13 The form of the change is also radically different
from that of Gd(DO3A) wherein the pH profile remains flat
until below pH 4 then, rises dramatically at lower pH values.13
Rather, the relaxivity enhancement observed for Gd(NP-DO3A)
is consistent with a q ) 1, anionic complex at high pH that
becomes a q ) 2, neutral complex at low pH as the phenol
becomes protonated and dissociates. The relaxivity observed
at high pH (r1 ) 4.1 mM-1 s-1) is comparable with that of
Gd(DOTA)- (r1 ) 4.2 mM-1 s-1 14
)
and at low pH (r1
)
)
7.0 mM-1 s-1) with that measured for Gd(DO3A) (r1
6.9 mM-1 s-1).
An enhancement in the relaxivity of Gd(NP-DO3AM) was
also observed at lower pH values, but in this case the
enhancement was more modest, ∼25%. The much smaller
relaxivity enhancement observed for Gd(NP-DO3AM) is more
difficult to understand in terms of phenol dissociation. The
relaxivity value at high pH (r1 ) 2.8 mM-1 s-1) falls within
the range expected for a dicationic, q ) 1 complex.11 The
relaxivity at low pH (r1 ) 3.4 mM-1 s-1), however, falls short
of that observed for the q ) 2 Gd(DO3AM)3+ (r1 ) 4.7 mM-1
s-1, pH 4 and 9).
The relaxivity pH profiles in Figure 2 were fitted to a simple
protonation equilibrium (eq 1). The pKa value of Gd(NP-DO3A)
was determined by potentiometric titration at 7.36 ((0.04), and
a good fit for the data was obtained using this value. In contrast
the pKa value determined by potentiometric titration for
Yb(NP-DO3AM) was significantly lower 7.22 ((0.05). How-
ever, this value does not readily allow the relaxivity data to be
fitted, in fact an apparent pKa value of 6.48 is obtained for
Nonradiative deactivation of the excited state of terbium by
proximate OH oscillators causes some quenching of luminescent
emission. The more OH oscillators present the greater the extent
of this quenching effect, and so lanthanides with more coordi-
nated water molecules will have shorter luminescent lifetimes.
(15) Horrocks, W. D., Jr.; Sudnick, D. R. J. Am. Chem. Soc. 1979, 101, 334-
340.
(13) Zhang, X.; Chang, C. A.; Brittain, H. G.; Garrison, J. M.; Telser, J.;
Tweedle, M. F. Inorg. Chem. 1992, 31, 5597-5600.
(14) Toth, E.; Pubanz, D.; Vauthey, S.; Helm, L.; Merbach, A. E. Chem. Eur.
J. 1996, 2, 1607-1615.
(16) Horrocks, W. D., Jr.; Sudnick, D. R. Acc. Chem. Res. 1981, 14, 384-392.
(17) Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker, D.; Royle,
L.; de Sousa, A. S.; Williams, J. A. G.; Woods, M. J. Chem. Soc., Perkin
Trans. 2 1999, 493-504.
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A R T I C L E S
Woods et al.
of Tb3+
.
Measurement of the triplet energy level of
Gd(NP-DO3AM) at 77 K indicates a smaller energy gap, 1700
cm-1 at pH 4.1. However, this energy gap closes as the pH
rises until at pH 7.0 the gap is just 500 cm-1 (Figure 4). Such
a small gap increases the extent of BET thereby, quenching
nearly all of the terbium-based luminescent intensity. Since the
luminescent lifetime of Tb(NP-DO3AM) at high pH is so short,
accurate measurement is not feasible. Thus, measuring the
hydration state of the complexes by Horrocks’ method is not
viable.
In light of the BET inherent in the luminescent behavior of
the terbium complexes a second method of determining the
hydration state was employed. The contact shift arising from
exchange of water molecules coordinated to Dy3+ gives rise to
a shift in the bulk water resonance in the 17O NMR spectrum.
The hydration state may be determined by monitoring the
dysprosium-induced shift of the bulk water resonance as a
function of the concentration of the dysprosium complex.19 The
hydration states of Dy(NP-DO3A) and Dy(NP-DO3AM) were
determined under both basic and acidic conditions (Support-
ing information). As anticipated, the hydration state of
Dy(NP-DO3A) at pD 4 was determined to be 1.8, whereas at
pD 9 the value of q was 0.92. In contrast, the hydration state of
Dy(NP-DO3AM) does not vary significantly between the two
pHs, with values of q ) 1.1 and 1.2 being determined at pD 4
and 9, respectively. These results help us understand the origin
of the relaxivity enhancement in each complex.
In the case of Gd(NP-DO3A) an increase in proton concen-
tration results in protonation of the phenoxide oxygen with
subsequent dissociation of the phenolic pendant arm from the
metal center. This vacates a coordination site, thereby allowing
coordination of a second water molecule and an increase in
relaxivity. Thus, the relaxivity enhancement closely correlates
with the pKa of the phenol. The origin of the relaxivity
enhancement observed for Gd(NP-DO3AM) is more difficult
to pin down, but the dissociation of the nitrophenol pendant
arm from the metal center can be ruled out. Of course, exchange
of coordinated water molecules with the bulk solvent is not the
only means by which inner sphere relaxivity may be effected.
It is sufficient that only protons in the inner sphere exchange
with the bulk; thus, a change in the number of exchangeable
protons on the complex, or in the rate that they exchange, would
be sufficient to effect a change in relaxivity. More slowly
exchanging systems may be probed by the use of saturation
transfer NMR experiments. In an attempt to identify any site
that may be in slow exchange, saturation transfer experiments
were performed in H2O, at 0 °C presaturating at increments
across the frequency range from +60 to -60 ppm. The effect
of presaturation upon the intensity of the bulk water signal was
monitored, any decrease in bulk water intensity being indicative
of a two-pool exchange process. At both low and high pH (4
and 9) no saturation transfer effects were observed, suggesting
that a change from a slow-exchange regime was not involved
in the relaxivity enhancement mechanism.
Figure 3. Effect of pH on the terbium emission lifetime of Tb(NP-DO3A)
at (λex ) 270 nm, λem ) 545 nm) (open circles), shown with the relaxivity
profile of Gd(NP-DO3A) (filled diamonds).
The lifetime of terbium-based emission measured for
Tb(NP-DO3A) (λex ) 270 nm, λem ) 545 nm) at high pH was
around twice as long as that measured at low pH (Figure 3).
This observation is consistent with a change in the number of
proximate OH oscillators with changing pH. However, an
increase in the number of proximate OH oscillators is not the
only mechanism by which a change in emission intensity may
be observed as a function of changing pH. If the energy gap
between the triplet energy level of an adjacent aromatic
chromophore and the terbium excited state (5D4) is very small,
then energy may not only pass from the chromophore to terbium
ion but also back again. This back energy transfer (BET) will
result in an increase in ligand-based emission at the expense of
terbium-based emission. The lifetimes recorded for Tb(NP-
DO3A) at both low and high pH (τTb ) 0.25-0.58 ms) are
considerably shorter than those observed for other terbium
complexes (typical lifetimes are τTb ≈ 1.0 ms for q ) 3
complexes and ∼3.0 ms for q ) 0 complexes).17 Such short
luminescent lifetimes suggest that another nonradiative decay
mechanism takes place in this system besides the vibronic
quenching of OH oscillators. To investigate this further, the
triplet energy level of Gd(NP-DO3A) was measured at 77 K.
At pH 4.0 the T1 energy level lies some 2000 cm-1 above the
5D4 state of Tb and 1800 cm-1 higher at pH 8.3 (Figure 4). An
energy gap of this size is small enough to allow a considerable
amount of BET, hence, the shorter than expected luminescent
lifetimes.18 However, since no substantial change in the triplet
energy level is observed with pH and the change in lifetime
mirrors the relaxivity profile of Gd(NP-DO3A), it seems likely
that a change in the number of OH oscillators is related to the
relaxation switching effect. This is further supported by the
observation that the pH range over which terbium emission
decreases is identical to that over which the relaxivity is
enhanced.
In contrast to the observation for Tb(NP-DO3A), when the
terbium-based luminescence of Tb(NP-DO3AM) was measured
as a function of pH, the intensity of emission was found to drop
rapidly with rising pH until at pH 7 almost no emission intensity
could be detected. This trend is the reverse of that observed for
Tb(NP-DO3A); it is also a much more dramatic change and
one which cannot be explained by changes in the hydration state
The fact that the relaxivity enhancement of Gd(NP-DO3AM)
does not directly correspond to a pKa in the complex may,
however, hold a clue to the origin of the relaxivity change. By
plotting the relaxivity profile of Gd(NP-DO3AM) alongside the
(19) Alpoim, M. C.; Urbano, A. M.; Geraldes, C. F. G. C.; Peters, J. A. J. Chem.
Soc., Dalton Trans. 1992, 463-467.
(18) Parker, D. Coord. Chem. ReV. 2000, 205, 109-130.
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9252 J. AM. CHEM. SOC. VOL. 126, NO. 30, 2004

Effect of Ligating Groups on Responsive MRI Agents
A R T I C L E S
Figure 4. Jablonski diagrams of the terbium complexes of NP-DO3A (left) and NP-DO3AM (right) showing that the proximity of the triplet energy level
of the nitrophenol group causes substantial back energy transfer in these complexes.
enhancement. The similarity of this pKa value (7.22) with that
of the phenol in Gd(NP-DO3A) (7.36) leads us to conclude that
the observed pKa is that of the phenol. Deprotonation of the
phenol improves the electron-donating capacity of the phenol
in this complex which in turn appears to substantially raise the
pKa of the coordinated water molecule. The pKa of the
coordinated water molecule in Yb(NP-DO3AM) was determined
to be 10.20 ((0.04), substantially higher than the value of 7.90
determined for tricationic tetraamide complexes by Parker and
co-workers.11 In this respect it is expected that prototopic
exchange of the water protons are not significantly involved in
the mechanism of relaxivity enhancement for this complex. The
differences in the magnitude and pH of the relaxivity enhance-
ments observed for these two complexes can readily be
accounted for by the radical change in mechanism observed
Figure 5. Relaxivity pH profile of Gd(NP-DO3AM) plotted alongside the
data from the potentiometric titration of Yb(NP-DO3AM). The pKa of the
complex at 7.22 corresponds to the onset of the relaxivity enhancement as
when the nature of other ligating groups in the chelate are
altered.
the pH is lowered.
Effect of Endogenous Anions and Complex Stability.
The decidedly small relaxivity enhancement observed for
Gd(NP-DO3AM) means that this complex has less viability as
a contrast agent for MR imaging. The more substantial increase
observed for Gd(NP-DO3A) on the other hand could render
this a useful basis for the design of a practical responsive MR
imaging agent. However, one feature of lanthanide complexes
in which two coordination sites of the metal ion are available
for water molecules is that they can bind endogenous anions,
displacing the inner sphere water molecules.18,20-23 Clearly such
binding is undesirable since it results in a lowering of relaxivity
data from the potentiometric titration (Figure 5) it can be seen
that the pKa of 7.22 ((0.05) determined from the potentiometric
titration lies close to the onset of the relaxivity enhancement.
We can imagine that around its pKa value the oxygen of the
phenol, which remains coordinated to the metal center, is
transiently protonated; that is to say that, although the phenol
is protonated, the protons are in extremely rapid exchange with
the bulk solvent and do not reside on the complex long enough
to be efficiently relaxed. As the pH is lowered further, the rate
of this exchange slows down to such an extent that the protons
reside on the complex long enough for relaxation by gadolinium
to occur before they are replaced. In this way the protonated
phenol pendant arm begins to contribute to the overall relaxivity
of the complex through prototopic exchange but only at pH
values below the pKa of the phenol. Once the rate of prototopic
exchange in the complex plateaus, so does the relaxivity
(20) Botta, M.; Aime, S.; Barge, A.; Bobba, G.; Dickins, R. S.; Parker, D.;
Terreno, E. Chem. Eur. J. 2003, 9, 2102-2109.
(21) Bruce, J. I.; Dickins, R. S.; Govenlock, L. J.; Gunnlaugsson, T.; Lopinski,
S.; Lowe, M. P.; Parker, D.; Peacock, R. D.; Perry, J. J. B.; Aime, S.;
Botta, M. J. Am. Chem. Soc. 2000, 122, 9674-9684.
(22) Dickins, R. S.; Aime, S.; Batsanov, A. S.; Beeby, A.; Botta, M.; Bruce, J.
I.; Howard, J. A. K.; Love, C. S.; Parker, D.; Peacock, R. D.; Puschmann,
H. J. Am. Chem. Soc. 2002, 124, 12697-12705.
(23) Lowe, M. P.; Parker, D. Chem. Commun. 2000, 707-708.
9
J. AM. CHEM. SOC. VOL. 126, NO. 30, 2004 9253

A R T I C L E S
Woods et al.
and perturbation of the inflection point. To assess the effect of
endogenous anions upon the relaxivity, 1 mM solutions of
Gd(NP-DO3A) were titrated against citrate, acetate, lactate, and
phosphate at both pH 4 and 9. In the presence of up to 40 equiv
of each anion, no effect upon relaxation rate was observed,
suggesting that none of these endogenous anions bind to the
complex in either the q ) 1 or q ) 2 hydration states
(Supporting Information). Although not measured here, the
effect of carbonate binding has also been reported to have
deleterious effects upon relaxivity.5,18 However, the effects of
carbonate are unlikely to represent a serious drawback to the
use of Gd(NP-DO3A) in vivo since the carbonates are only
observed to bind in a bidentate manner. Thus, carbonate is only
likely to affect the complex at lower pH when the phenolic
pendant arm has dissociated; however, the amount of bicarbon-
ate in solution diminishes as the pH is lowered. Recently, Parker
and co-workers described an elegant solution to bicarbonate
binding that could easily be incorporated into this chelate.24 The
onset of carbonate binding was found to be prevented by
incorporating anionic substituents into the chelate to leave it
formally negatively charged.
Figure 6. Complex Lu(NP-DO3A) is stable over the pH range 4-11 for
up to 24 h, with greater than 98% of the complex remaining intact in this
pH range. Dissociation of the complex is only observed at pH 3 or below.
using a 3-fold excess of the ligand. The pH was adjusted to 7.4
and ZnCl2 added such that the solution was 3 mM in zinc. The
solution was incubated for 24 h, and aliquots were removed
after 5 min, 2 h, 16 h, and 24 h. The extent of complex dissoci-
ation measured using the method described above. Over the
course of the experiment the extent of demetalation by Zn2+
was found to be less than 2%. The stability over such a wide
pH range and in response to Zn2+ ions suggests that the lanth-
anide complexes of NP-DO3A should exhibit favorable in vivo
stabilities.
One further consideration as to whether a complex may be
used in vivo is the stability of the complex. In this regard good
kinetic stability is a more important feature of a complex than
thermodynamic stability. This is because ultimately it is only
important that the rate of complex dissociation is slow enough
that the complex is excreted from the body before dissociation
occurs. Octadentate ligands such as DOTA and HP-DO3A form
extremely kinetically and thermodynamically stable complexes
with gadolinium, and it is reasonable to assume that in
octadentate form, NP-DO3A will also form highly stable
complexes. However, under acidic conditions NP-DO3A be-
comes a heptadentate ligand, and such ligands are generally
known to be less kinetically inert than their octadentate
counterparts. Since the demetalation of these complexes is
believed to be a cation-mediated process, usually via protons
or Zn2+ ions, the kinetic stability of the complex was assessed
by examining the extent of dissociation induced by the pres-
ence of these two cations. Accordingly, a solution of
177Lu(NP-DO3A) was synthesized using a 5-fold excess of
ligand. After verification that better than 98% of the lanthanide
ion had been complexed, aliquots of the solution were taken
and adjusted to different pH values over the range 1-11. After
incubation, the amount of 177Lu liberated from the complex after
5 min, 2 h, 6 h, and 24 h was determined at each pH point by
eluting a sample through a Sephadex G-50 column. The
radioactivities of the eluent and the column were then compared.
The intact complex elutes, but free lutetium is retained by the
column, so that by comparing the radioactivity of the two the
percentage of free lutetium present may be determined. The
results (Figure 6) show unequivocally that lanthanide complexes
of NP-DO3A are kinetically inert over a pH range from 4 to
11. At lower pH values (pH 1-3) dissociation of the lutetium
ion is observed. The extent of dissociation does not alter
significantly as a function of time, suggesting that the equilib-
rium position is achieved rapidly. A similar technique was used
to assess the effect of Zn2+ upon the kinetic stability of the
complex. A 1 mM solution of 177LuNP-DO3A was prepared
Conclusions
We have successfully demonstrated that a methylene nitro-
phenol pendant arm may be employed as a pH-responsive
pendant arm in DO3A-type complexes. The pH range over
which this pendant arm dissociates from the central lanthanide
ion renders it ideal as the basis of new pH-responsive con-
trast media for MRI. In addition, the relaxivity profile of
Gd(NP-DO3A) appears to be unaffected by endogenous anions,
and the complex is found to be inert to demetalation over the
pH range 2-11 and in response to Zn2+ ions. Substitution of
amide pendant arms for acetates has several deleterious effects
on the complex. The reduced electron-donating capacity of the
amide-ligating groups renders the central lanthanide ion too
electron deficient to release the nitrophenol. In consequence,
the relaxivity enhancement of [Gd(NP-DO3AM)] is rather
modest and appears to arise from prototopic exchange of the
phenolic proton as it becomes protonated at lower pH.
Experimental Section
General Remarks. All solvents and reagents were purchased from
1
commercial sources and used as received. H and 13C NMR spectra
were recorded on a JEOL Eclipse 270 spectrometer at 270.17 and 67.93
MHz, respectively, or on a Bruker Avance spectrometer at 250.13 and
62.89 MHz, respectively. 17O NMR spectra were recorded on a Varian
Inova 500 spectrometer operating 67.72 MHz. Relaxation measurements
were made using an MRS-6 NMR Analyzer from the Institut “Jozˇef
Stefan”, Ljubljana, Slovenjia, operating at 20 MHz. Luminescence
spectra were recorded on a Perkin-Elmer LS55 or an Edinburgh
Instruments nF 900 fluorimeter. The pH of luminescence samples were
measured using a Fisher Accumet 925 pH meter equipped with an Orion
8103 Ross combination pH electrode. The pH of luminescence samples
was altered by addition of solid lithium hydroxide monohydrate or
p-toluenesulfonic acid. Triplet energy levels were determined from the
measured emission spectra of frozen samples of the corresponding
(24) Messeri, D.; Lowe, M. P.; Parker, D.; Botta, M. Chem. Commun. 2001,
2742-2743.
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Effect of Ligating Groups on Responsive MRI Agents
A R T I C L E S
79.1 (C(CH3)3), 79.3 (C(CH3)3), 155.4 (CdO), 155.6 (CdO). νmax/cm-1
3312 (NH), 2976, 2932, 2815, 1690 (CdO), 1463, 1417, 1365, 1248,
1172, 1088, 774. m/z (ESI+); 473 (100% [M + H]+), 495 (5% [M +
Na]+). Anal. Found C ) 58.3%, H ) 9.7%, N ) 11.7%. C23H44N4O6
requires C ) 58.5%, H ) 9.3%, N ) 11.9%.
gadolinium(III) complex at 77 K. Singlet energy levels were obtained
by comparing the excitation spectra and the absorption spectra of the
complexes recorded on a Hewlett-Packard 8453 UV-visible spectrom-
eter. Cyclic voltammograms were recorded on a BAS CV-50W
voltammetric analyzer using a glassy carbon working electrode,
platinum wire auxiliary electrode, and Ag/AgCl reference electrode.
Samples were prepared at 0.6 mM concentrations using a 50 mM
solution of tetrabutylammonium perchlorate in acetonitrile and a buffer
solution of either phosphate-citrate or tris buffer.
:
1-Benzyloxycarbonyl-4,7,10-tris-tert-butoxycarbonyl-1,4,7,10-
tetraazacyclododecane (3). To a cooled solution (0 °C) of the tri-
Boc-protected cyclen 2 (6.6 g, 14.0 mmol) and triethylamine (2.4 mL,
16.7 mmol) in chloroform (150 mL) was added benzyl chloroformate
(2.9 g, 16.7 mmol). The resulting solution was allowed to warm to
room temperature and stirred for 8 h. The precipitates were removed
by filtration, and the solvents were removed in vacuo. The residue was
purified by column chromatography over silica gel eluting with 30%
ethyl acetate in hexanes to afford the title compound as a colorless
solid (8.3 g, 98%). Rf ) 0.5 (SiO2, 30% EtOAc hexanes). Mp ) 76.5-
1-Methlyene-(p-nitrophenol)-1,4,7,10-tetraazacyclododecane (1).
To a stirred dioxane solution (20 mL) of 1,4,7,10-tetraazacyclododecane
(2.3 g, 14.0 mmol) was added a solution of 2-hydroxy-5-nitrobenzyl
bromide (1.2 g, 7.0 mmol) in dioxane (15 mL). After 5 min, K2CO3
(1 g, 7.2 mmol) was added and the reaction stirred for 1 h at ambient
temperature. The reaction mixture was then filtered and the filtrate
concentrated in vacuo to give a yellow semisolid which was purified
by column chromatography over silica gel eluting with CHCl3/MeOH/
NH4OH (3:2:1). The title compound was isolated from the slower-
moving yellow fraction which also contained cyclen starting material.
The resulting yellow oil was triturated with CHCl3 (100 mL) to remove
cyclen and filtered to afford the title compound as a crystalline yellow
solid (1.2 g, 55%): mp 142-145 °C. 1H NMR (250 MHz, D2O): δ )
7.84 (2H, m, 4-Ph,6-Ph), 6.30 (1H m, 3-Ph), 3.39 (2H, s, NCH2Ar),
2.70-2.87 (16H, m br, ring CH2). 13C NMR (62.9 MHz, D2O): δ )
45.3 (ring CH2), 45.5 (ring CH2), 47.2 (ring CH2), 51.7 (ring CH2),
56.3 (NCH2Ar), 122.0 (4-Ph), 128.3 (5-Ph), 130.9 (6-Ph), 131.9 (1-
Ph), 135.6 (3-Ph), 180.1 (2-Ph). νmax/cm-1: 3356 br (NH/OH), 2994,
2957, 2849, 1588, 1472, 1460, 1434, 1273, 1241, 1168, 1132, 1086.
HRMS (MALDI): C15H26N5O3 ([MH]+) requires 324.2037, found
324.2033.
1
77.5 °C. H NMR (270 MHz, CDCl3): δ ) 7.28 (5H, m, Ph), 5.04
(2H, s, OCH2Ph), 3.44 (6H, s br, NCH2CO), 3.32 (16H, m br, ring
CH2), 1.38 (9H, s, C(CH3)3), 1.35 (18H, s, C(CH3)3). 13C NMR (67.5
MHz, CDCl3): δ ) 28.4 (C(CH3)3), 28.5 (C(CH3)3), 50.3 (br, ring CH2),
67.2 (OCH2Ph), 79.9 (C(CH3)3), 128.1 (4-Ph), 128.3 (3-Ph) 128.5 (2-
Ph), 136.5 (1-Ph), 156.6 (br, CdO). νmax/cm-1: 3064, 3029, 3004, 2976,
2933, 1694 (CdO), 1468, 1411, 1366, 1248, 1165, 1102, 1028, 776.
A suitable analysis of this compound could not be obtained.
1-Benzyloxycarbonyl-1,4,7,10-tetraazacyclododecane Trihydro-
chloride (4). To a cooled solution (0 °C) of the protected cyclen 3
(8.0 g, 13.2 mmol) in methanol (10 mL) was added concentrated HCl
(10 mL) dropwise. The resulting solution was allowed to warm to room
temperature and stirred for 8 h. The reaction was concentrated at room
temperature under reduced pressure to a volume of 15 mL, and the
precipitates were removed by filtration. The residue was then crystal-
lized from a mixture of water and tetrahydrofuran to afford the title
compound as a colorless crystalline solid (5.1 g, 94%). Mp ) 191.5-
192.5 °C. 1H NMR (270 MHz, CD3OD): δ ) 7.40 (5H, m, Ph), 5.15
(2H, s, OCH2Ph), 3.14 (16H, m, ring CH2). 13C NMR (67.5 MHz, CD3-
OD): δ ) 43.0 (ring CH2), 44.7 (ring CH2), 45.4 (ring CH2), 46.7
(ring CH2), 68.8 (OCH2Ph), 128.5 (3-Ph), 128.9 (4-Ph), 129.0 (2-Ph),
135.8 (1-Ph), 158.5 (CdO). νmax/cm-1: 3430 (NH), 2997, 2806, 1709
(CdO), 1622, 1447, 1261, 1162, 767, 741. m/z (ESI+); 307 (100%
[M + H]+), 329 (3% [M + Na]+). Anal. Found C ) 45.9%, H )
7.5%, N ) 12.8%. C16H29N4O2‚3HCl‚0.4H2O requires C ) 45.4%, H
) 7.1%, N ) 13.2%.
1-Methlyene-(p-nitrophenol)-1,4,7,10-tetrazacyclododecane-
4,7,10-triacetic Acid (NP-DO3A). The mono-alkylated cyclen (1) (0.2
g, 0.6 mmol) was dissolved in water (5 mL) and the pH adjusted to 5
using HCl (12 N). A solution of bromoacetic acid (309 mg, 2.2 mmol)
in water (10 mL) was added in one portion and the pH adjusted to and
maintained at 8.5 by addition of 45% KOH solution. Once the pH was
stabilized at 8.5 for 1 h, the pH was raised to 11 and maintained for 6
h. The solution was then cooled in an ice bath and acidified to pH 2
with concentrated HCl. A saturated solution of KCl was then added to
facilitate crystallization of the desired product. After stirring for 1 h
the colorless precipitate was filtered and dried in vacuo to afford the
title compound as a colorless solid (150 mg, 49%): mp 230-235 °C
1-Benzyloxycarbonyl-4,7,10-tris(carbamoylmethyl)-1,4,7,10-
tetraazacyclododecane (5). The trihydrochloride salt 4 (4.1 g, 9.8
mmol) was dissolved in aqueous sodium hydroxide solution (20 mL,
1 N) and extracted with chloroform (3 × 20 mL). The organic extracts
were dried (Na2SO4), and the solvents were removed in vacuo. The
resultant solid was dissolved in acetonitrile (60 mL), and anhydrous
potassium carbonate (5.2 g, 39.0 mmol) was added. The solution was
then heated to 60 °C for 20 min before bromoacetamide (5.4 g, 39.4
mmol) was added, and the reaction mixture stirred at 70 °C for a further
18 h. The solvents were removed in vacuo, and the residue was
dissolved in water (10 mL) and extracted with dichloromethane (3 ×
50 mL). The product was purified by flash column chromatography
over silica gel eluting with chloroform/methanol (5:2) to yield the title
compound as a colorless solid (4.21 g, 90%). Rf ) 0.45 (SiO2, CHCl3/
MeOH, 5:2). Mp ) 154-154.5 °C. 1H NMR (270 MHz, CDCl3): δ )
7.30 (5H, m, Ph), 7.01 (1H, s br, NH), 6.93 (2H, s br, NH), 6.81 (1H,
s br, NH), 6.70 (2H, s br, NH), 5.06 (2H, s, OCH2Ph), 3.44 (6H, s br,
NCH2CO), 3.25-2.55 (16H, m, ring CH2). 13C NMR (67.5 MHz,
CDCl3): δ ) 53.3 (ring CH2), 53.6 (ring CH2), 54.2 (ring CH2), 58.3
(br, NCH2CO), 67.5 (OCH2Ph), 128.3 (3-Ph and 4-Ph), 128.7 (2-Ph),
1
dec. H NMR (250 MHz, D2O): δ ) 7.87 (2H, m, Ph-4, Ph-6), 6.38
(1H, m, Ph-3), 3.39 (2H, s, NCH2Ar), 3.01-2.90 (16H, m br, ring CH2),
2.74 (2H, s, NCH2CO2), 2.67 (4H, s, NCH2CO2). 13C NMR (62.9 MHz,
D2O): δ ) 51.3 (ring CH2), 55.0 (ring CH2), 55.4 (ring CH2), 58.3
(NCH2CO2), 60.6 (NCH2CO2), 62.1 (NCH2Ar), 121.2 (4-Ph), 128.4
(5-Ph), 130.6 (6-Ph), 132.9 (1-Ph), 137.5 (3-Ph), 175.5 (2-Ph), 182.1
(CdO), 183.4 (CdO). νmax/cm-1: 3378 br (OH), 3075, 2961, 2861,
1717 (CdO), 1595, 1526, 1456, 1395, 1341, 1288, 1234, 1094. HRMS
(MALDI) C21H32N5O9 ([MH]+) requires 498.2202, found 498.2180.
1,4,7-Tris-tert-butoxycarbonyl-1,4,7,10-tetraazacyclododecane (2).10
A solution of di-tert-butyl dicarbonate (23.0 g, 106 mmol) in chloroform
(130 mL) was added dropwise over the course of 2 h to a solution of
cyclen (6.0 g, 35 mmol) and triethylamine (15 mL, 106 mmol) in
chloroform (170 mL) at 0 °C. The resulting solution allowed to warm
to room temperature and stirred for 18 h. The reaction mixture was
then washed with water (2 × 100 mL), the organic layer was dried
over Na2SO4, and the solvents were removed in vacuo. The residue
was purified by column chromatography over a silica gel eluting with
diethyl ether to afford the title compound as a colorless solid (14.8 g,
90%). Rf ) 0.3 (SiO2, Et2O). Mp ) 72-73.5 °C. 1H NMR (270 MHz,
CDCl3): δ ) 3.50 (4H, s br, ring CH2), 3.15 (8H, s br, ring CH2), 2.71
(4H, s br, ring CH2), 1.33 (9H, s, C(CH3)3), 1.31 (18H, s, C(CH3)3).
13C NMR (67.5 MHz, CDCl3): δ ) 28.5 (C(CH3)3), 28.7 (C(CH3)3),
45.9 (ring CH2), 48.9 (ring CH2), 49.5 (ring CH2), 51.0 (ring CH2),
136.5 (1-Ph), 156.9 (NCO2), 174.6 (CdO), 174.7 (CdO). νmax/cm-1
:
3414 (NH), 2937, 2822, 1670 (CdO), 1473, 1456, 1261, 1164, 1118,
995, 755, 604. m/z (ESI+); 478 (100% [M + H]+).
1,4,7,10-Tetraazacyclododecane-1,4,7-triacetamide (DO3AM). Ben-
zyloxycarbonyl-protected DO3AM 5 (2.2 g, 4.6 mmol) was dissolved
9
J. AM. CHEM. SOC. VOL. 126, NO. 30, 2004 9255

A R T I C L E S
Woods et al.
in ethanol (50 mL), and 10% palladium on carbon (0.5 g) was added.
The reaction mixture was then shaken on a Parr hydrogenator under a
hydrogen pressure of 45 psi at room temperature for 12 h. The reaction
was filtered, and the solvents were removed in vacuo to afford the
title compound as a colorless solid (1.63 g, 99%). Mp ) 232-234 °C.
1H NMR (270 MHz, CD3OD): δ 3.19 (4H, s, CH2CO), 3.13 (2H, s,
CH2CO), 2.73-2.57 (16H, m br, ring CH2), 13C NMR (67.5 MHz, CD3-
OD): δ 44.9 (ring CH2), 51.2 (ring CH2), 51.4 (ring CH2), 52.4 (ring
[
153EuL]+); Gd(NP-DO3AM), 650 (100% [GdL]+); an appropriate
isotope pattern was observed; Tb(NP-DO3AM), 651 (100% [TbL]+);
Dy(NP-DO3AM), 654 (100% [DyL]+); an appropriate isotope pattern
was observed.
Crystal Structure Determination of Eu(NP-DO3AM)(H2O)-
(CF3SO3)2. The structure is presented in Figure 1. Crystals were grown
from aqueous solution at pH 5 by slow evaporation at room temper-
ature: C23H35EuF6N8O13S2, mass ) 961.67, triclinic, P1h, a ) 11.042
(2) Å, b ) 11.956 (2) Å, c ) 13.723 (3) Å, R ) 92.36 (3)°, â )
105.63 (3)°, γ ) 101.76 (3)°, U ) 1699.2 (6) Å3, Z ) 2, Dø ) 1.880
gcm-3, λ(Mo KR) ) 0.71073 Å, µ ) 2.076 mm-1, F(000) ) 964, T )
293 K. The sample was studied on a Bruker CCD 1000. The data
collection gave 4844 unique reflections. Refinement was performed
using a full-matrix least-squares method on F2 using SHELXTL, version
5. This procedure afforded a final R (I > 2σ(I)) of R1 ) 0.0422; wR2
) 0.1741. For all data R1 ) 0.0828; wR2 ) 0.2168.
CH2), 56.4 (CH2CO), 57.9 (CH2CO), 175.2 (CdO), 175.4 (CdO). νmax
/
cm-1: 3390 (NH), 3177 (NH), 2967, 2832, 1668 (CdO), 1611, 1454,
1330, 1286, 1107, 1001, 925, 776, 609. m/z (ESI+): 344 (100% [M
+ H]+), 366 (63% [M + Na]+). Anal. Found C ) 43.4%, H ) 8.8%,
N ) 25.0% C14H29N7O3‚2.5H2O requires C ) 43.3%, H ) 8.8%, N )
25.2%.
1-Methlyene-(p-nitrophenol)-1,4,7,10-tetraazacyclododecane-
4,7,10-triacetamide (NP-DO3AM). To a solution of DO3AM (0.13
g, 0.38 mmol) in ethanol (100 mL) was added 2-hydroxy-5-nitrobenzyl
bromide (0.09 g, 0.38 mmol) and the mixture heated to 60 °C. After 1
h potassium carbonate (0.05 g, 0.38 mmol) was added and the reaction
stirred at 60 °C for a further 12 h. After cooling to room temperature
the reaction mixture was filtered, and the solvents were removed in
vacuo. The residue was then crystallized for a mixture of methanol
and acetonitrile to afford the ligand as a pale-yellow solid (1.2 g, 65%).
Kinetic Stability Profiles of 177Lu(NP-DO3A). A stock 177Lu
solution was prepared by adding 2 µL of a 0.3 mM solution of
177LuCl3 in 0.1 N HCl to 2 mL of a 0.3 mM solution of LuCl3 also in
0.1 N HCl. A similar stock solution of the ligand was prepared in
deionized water. The complex was prepared at a 5:1 ligand/metal ratio
by combining appropriate aliquots of each standard solution at pH 2.
The pH was then adjusted to pH 7 using 0.1 N NaOH. The extent of
complex formation was determined by passing a sample of the complex
solution through a Sephadex G-50 column eluting with 4:1 saline
(0.85% NaCl/NH4OH). Two 3-mL fractions were collected, and the
amount of radioactivity in the combined elutions was compared to that
remaining on the resin. Only free metal was retained on the resin. Once
the complexation reaction had reached >98% completion, a pH stability
profile was then generated. Aliquots were taken from the complexation
reaction at pH 7, and the pH adjusted to high and low pH points. The
solutions were then allowed to equilibrate, and aliquots were removed
at intervals (5 min, 2 h , 6 h, and 24 h). The amount of free lutetium
versus the amount of complex present in each individual sample pH
point was then measured by using the radioactivity counting method
described above. To assess the effect of Zn2+ the 177Lu complex was
prepared with a 3:1 ligand/metal ratio, the pH was adjusted to 7.4, and
3 equiv of ZnCl2 were added. Aliquots were removed from the reaction
at (5 min, 2 h, 16 h,and 24 h), and the extent of transmetalation was
measured using the same radioactivity counting method.
1
Mp ) 103-105 °C. H NMR (270 MHz, D2O): δ ) 8.09 (1H, s,
3
3
3-Ph), 7.99 (1H, d, JH-H ) 9 Hz, 5-Ph), 6.47 (1H, d, JH-H ) 9 Hz,
6-Ph), 4.43 (2H, s, CH2Ar), 3.20 (4H, s, CH2CO), 3.12 (2H, s, CH2-
CO), 2.52-2.82 (16H, m br, ring CH2N). 13C NMR (67.5 MHz,
CDCl3): δ ) 44.7 (ring CH2N), 49.5 (ring CH2N), 51.2 (ring CH2N),
53.4 (ring CH2N), 56.1 (CH2CO), 66.4 (CH2CO), 67.7 (CH2Ar), 118.7
(2-Ph), 126.6 (5-Ph), 127.0 (3-Ph), 127.3 (6-Ph), 134.7 (4-Ph), 173.2
(1-Ph), 176.3 (CdO), 176.6 (CdO). m/z (ESI+): 495 (100% [M +
H]+). Anal. Found C ) 46.0%, H ) 7.4%, N ) 20.8% C21H34N8O6‚
3H2O requires C ) 46.0%, H ) 7.4%, N ) 20.4%.
Preparation of the Lanthanide(III) Complexes of NP-DO3A. An
aqueous lanthanide chloride solution (0.4 mmol in 1.5 mL) was added
in 200-µL aliquots to a solution of NP-DO3A (0.2 g, 0.4 mmol) in
water (5 mL, pH 2). The pH of the solution was maintained between
4 and 5 throughout the addition process via the addition of 1 N KOH.
Complexation progress was monitored by reverse phase HPLC (PRP-1
10 µm/250 mm × 4.1 mm, 30% MeOH, λmax ) 254 nm, flow rate of
1 mL/min; ligand tR ) 2.6 min, chelate tR ) 10.9 min). HRMS
(MALDI): C21H29N5O9Eu ([EuLH]+) requires 648.1278, found
648.1288; C21H29N5O9Gd ([GdLH]+) requires 653.1260, found
653.1227; C21H29N5O9Tb ([TbLH]+) requires 654.1221, found 654.1212;
C21H29N5O9Dy ([DyLH]+) requires 659.1257, found 659.1234.
Preparation of the Lanthanide(III) Complexes of NP-DO3AM.
To a solution of NP-DO3AM (50 mg, 0.1 mmol) in water (4 mL) was
added a solution of the appropriate lanthanide chloride or triflate (0.1
mmol) in aqueous solution (4 mL). The reaction was heated at 60 C
for 18 h. After cooling to room temperature the complex was dried by
lyophilization. The complexes were employed without further purifica-
tion. m/z (ESI+); Eu(NP-DO3AM), 643 (100% [151EuL]+), 645 (100%
Acknowledgment. We thank the National Institutes of Health
(RR-02584, CA-84697, CA96019, and EB-04285), the Robert
A Welch Foundation (AT-584), and the Texas Advanced
Technology Program for financial assistance.
Supporting Information Available: ORTEP rendering of the
crystal structure of Eu(NP-DO3AM), 1H NMR spectra of each
europium complex, hydration state determination by 17O NMR
and anion titration data. This material is available free of charge
via the Internet at http://pubs.acs.org.
JA048299Z
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9256 J. AM. CHEM. SOC. VOL. 126, NO. 30, 2004