Gadolinium(III) complexes of dota-derived N-sulfonylacetamides (H 4(dota-NHSO2R) = 10-{2-[(R)sulfonylamino]-2-oxoethyl}-1,4, 7,10-tetraazacyclododecane-1,4,7-triacetic acid): A new class of relaxation agents for magnetic resonance imaging applications

Article abstract of DOI:10.1002/hlca.200590041

Four new ligands for lanthanide ions based on the H₃do3a (= 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) structure and bearing one N-sulfonylacetamide arm were synthesized, i.e., H₄dota-NHSO ₂R = 10-{2-[(R)sulfonylamino]-2-oxoethyl}-1,4,7,10- tetraazacyclododecane-1,4,7-triacetic acids 1a-e. A ¹⁵N-NMR study of the ¹⁵N-labelled Eu³⁺ complex of one such ligands, 1d, showed that the coordination of the N-sulfonylacetamide arm involves the carbonyl O-atom rather than the N-atom. The relaxometric properties of the corresponding Gd³⁺ complexes were investigated as a function of pH and temperature. These complexes have relaxivities in the range 4.5-5.3 mM ^-1 s^-1, at 20 MHz and 25°, and are characterized by a single H₂O molecule in their inner coordination sphere. The mean residence lifetime of this molecule is relatively long (500-700 ns) compared to other anionic complexes. The slow rate of H₂O exchange can be justified by the extensive delocalization of the negative charge on the N-sulfonylacetamide arm. The long residence time of the coordinated H ₂O allowed the observation of the effect of the prototropic exchange on the relaxivity. The study of the interaction between the complex [Gd(1e)]- and HSA revealed a weak affinity constant highlighting the importance of a localized negative charge on the complex to promote a strong interaction with the protein.

Full text of DOI:10.1002/hlca.200590041

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Helvetica Chimica Acta ± Vol. 88 (2005)
Gadolinium(III) Complexes of dota-Derived N-Sulfonylacetamides
(H4(dota-NHSO2R) ˆ 10-{2-[(R)sulfonylamino]-2-oxoethyl}-1,4,7,10-
tetraazacyclododecane-1,4,7-triacetic Acid): A New Class of Relaxation
Agents for Magnetic Resonance Imaging Applications
by Silvio Aime*a), Mauro Bottab), Giancarlo Cravottoc), Luca Frullanoa)1), Giovanni B. Giovenzanad),
Simonetta Geninatti Cricha), Giovanni Palmisanoe), and Massimo Sistie)
a
Á
) Dipartimento di Chimica I.F.M., Universita di Torino, Via P. Giuria 7, I-10125 Torino
(fax: ‡ 39-011-6707855; e-mail: silvio.aime@unito.it)
b
Á
) Dipartimento di Scienze dellꢀAmbiente e della Vita, Universita degli Studi del Piemonte Orientale
ꢁA. Avogadroꢀ, Spalto Marengo 33, I-15100 Alessandria
c
Á
) Dipartimento di Scienza e Tecnologia del Farmaco, Universita degli Studi di Torino, Via Giuria 9,
I-10125 Torino
d
Á
) Dipartimento di Scienze Chimiche Alimentari, Farmaceutiche e Farmacologiche, Universita degli Studi del
Piemonte Orientale ꢁA. Avogadroꢀ,Via Bovio 6, I-28100 Novara
e
Á
) Dipartimento di Scienze Chimiche e Ambientali, Universita degli Studi dellꢀInsubria, Via Valleggio 11,
I-22100 Como
Â
Dedicated to Professor Andre E. Merbach on the occasion of his 65th birthday
Four new ligands for lanthanide ions based on the H3do3a (ˆ1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
acid) structure and bearing one N-sulfonylacetamide arm were synthesized, i.e., H4dota-NHSO2R ˆ 10-{2-
[(R)sulfonylamino]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acids 1a ± e. A 15N-NMR study of
the 15N-labelled Eu3‡ complex of one such ligands, 1d, showed that the coordination of the N-sulfonylacetamide
arm involves the carbonyl O-atom rather than the N-atom. The relaxometric properties of the corresponding
Gd3‡ complexes were investigated as a function of pH and temperature. These complexes have relaxivities in the
range 4.5 ± 5.3 mmÀ1 sÀ1, at 20 MHz and 258, and are characterized by a single H2O molecule in their inner
coordination sphere. The mean residence lifetime of this molecule is relatively long (500 ± 700 ns) compared to
other anionic complexes. The slow rate of H2O exchange can be justified by the extensive delocalization of the
negative charge on the N-sulfonylacetamide arm. The long residence time of the coordinated H2O allowed the
observation of the effect of the prototropic exchange on the relaxivity. The study of the interaction between the
complex [Gd(1e)]- and HSA revealed a weak affinity constant highlighting the importance of a localized
negative charge on the complex to promote a strong interaction with the protein.
Introduction. ± Gadolinium complexes with polyaminopolycarboxylate ligands
have been under intense scrutiny in the last decade because of their possible application
as magnetic-resonance-imaging (MRI) contrast agents [1 ± 3]. The large majority of the
complexes considered for this purpose so far are based on octadentate ligands to ensure
a high thermodynamic (and kinetic) stability. A Gd3‡-bound H2O exchanging with
tissue water determines, via the enhancement of the relaxation (T1 and T2) of the H2O
protons, the intensity of the water signal and thus the contrast in the images.
1
)
Present address: Chemistry Department, Northwestern University, 2145 Sheridan Road, Evanston, IL,
60208-3113, USA.
ꢂ 2005 Verlag Helvetica Chimica Acta AG, Zürich

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The theory describing the paramagnetic relaxation was developed by Bloembergen,
Solomon, and Morgan [4 ± 6] in the 1950s. With the support of this theory, it was early
suggested that the efficiency of a MRI contrast agent is strongly dependent on a
number of structural and dynamic parameters related to the coordination environment
around the metal center and to the tumbling rate of the complex. As the value of these
parameters for the commercial contrast agents is far from optimal, much research has
been undertaken with the aim of attaining the high efficiencies predicted by the theory.
The relaxation theory predicts that higher relaxation rates are obtained upon increasing
the rotational correlation time of the Gd3‡ complexes, which may be achieved by
noncovalent or covalent binding of low-molecular-mass Gd3‡ chelates to macro-
molecules or polymers [2]. In fact, many examples of lanthanide ligands functionalized
with moieties apt to interact with serum proteins, particularly HSA (human serum
albumin), are already known in the literature.
Here, we report the synthesis and physicochemical characterization of four new
ligands bearing an N-sulfonylacetamide pendant arm (see 1a ± e) and their lanthanide
complexes. The N-sulfonylacetamide moiety, possessing a negative charge at physio-
logic pH, represents an isosteric alternative to the carboxylic group. This offers the
possibility to modulate several properties of the resulting complexes (charge, size,
hydrophilicity, etc.) through a proper choice of the substituent at the S-atom by means
of a synthetically convenient way. In this work, we considered three complexes bearing
hydrophobic groups at the S-atom and investigated their binding affinity to HSA. HSA
is known to tightly bind anionic lipids and, in general, it is well-established that most of
the compounds interacting with HSA are hydrophobic organic anions [7][8]. It is thus
interesting to investigate both the structure and the interaction with HSA of these new
chelates.
We studied the protonation pattern of one of the ligands and the coordination
structure of its Eu3‡ complex. The relaxometric parameters and the interaction with
HSA of the gadolinium complexes were also assessed.

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Results and Discussion. ± Five N-sulfonylacetamide derivatives of H4DOTA, 1a ± e,
were prepared following the synthetic pathway outlined in the Scheme. First, the five 2-
bromo-N-sulfonylacetamides 3a ± e were synthesized by reacting bromoacetyl bromide
with the appropriate sulfonamide 2. Then, these building blocks were used to alkylate
do3a tri(tert-butyl) ester 4 (H3do3a ˆ 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
acid) to give 5a ± e, and finally the desired ligands 1a ± e were obtained after
deprotection with CF3COOH.
To gain insight into the coordination mode of this type of ligands, the 15N-labelled
ligand 1d was prepared starting from the commercially available p-toluenesulfon-
(15N)amide (ˆ4-methylbenzenesulfon(15N)amide).
Scheme
The Gd3‡ complexes were synthesized by stepwise addition of GdCl3 ´ 6 H2O to an
aqueous solution of the ligand up to the stoichiometric ratio, while maintaining the pH
of the solution at 6.0 with 1m NaOH. The formation of the complex was monitored by
measuring the solvent proton relaxation rate (1/T1) at 20 MHz and 258.

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Microscopic Protonation Sequence. The information obtainable from high-reso-
lution NMR spectroscopy can be used to assess the value of the protonation constants
and the sequence of protonation in polyaminocarboxylate ligands. The protonation
constants Kn can be determined by fitting the variation with pH of the chemical shift for
the various proton resonances, di, to Eqn. 1, where cn is the molar fraction of the n-
protonated species and dn are their intrinsic chemical shifts [9]. Combining Eqn. 1 with
Eqn. 2 thus allows the determination of Kn.
X
di ˆ
cndn
ꢀ1†
ꢀ2†
n
‡
cnÀ1‰H Š
Kn ˆ
cn
It is well known that the protonation state of a basic site influences the NMR
shielding constant of adjacent nuclei. In particular, the protonation of a basic site
induces a downfield shift of the 1H-NMR resonances of adjacent protons. The
magnitude of this shift depends upon the nature, the proximity, and the fraction of
protonation of the basic site. Sudmeier and Reilley determined a series of shielding
constants, or protonation shifts, for various model compounds and found for methylene
groups in the moieties CH2COOÀ, CH2NR2, and CH2CH2NR2 values of 0.2, 0.75, and
0.35 ppm, respectively [9][10].
1
The plot of the H-NMR resonances of ligand 1b (Fig. 1) as a function of pH are
reported in Fig. 2. Although, from this NMR titration experiment it is not possible to
precisely determine the number of protons involved in a given protonation step, by
analogy with similar data on related ligands, we may assume that, in the pH range 8 ± 10,
two protonations occur with similar constants (pK1 ꢁ pK2 ˆ 8.77 Æ 0.01). Likely, these
two protons are used to protonate two amino groups in ꢁtransꢀ position to each other to
minimize the electrostatic repulsion. This is consistent with the behavior reported for
other cyclen derivatives [11]. The fact that the protons 1, 4, 5, and 7 (see Fig. 1)
Fig. 1. H-NMR Spectrum of ligand 1b in D2O, at 400 MHz, 258, and pH 7.0. Arbitrary numbering.

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Fig. 2. pH Dependence of the 1H-NMR resonances of ligand 1b recorded at 400 MHz and 258. The curves
passing through the experimental values are the result of a best-fit procedure to Eqns. 1 and 2. Arbitrary
numbering of protons according to Fig. 1.
experience the largest shift indicates that the highest proton density is present on the N-
atoms a and c, although a significant amount of proton density is also located on the N-
atom b. The first protonation constant for this ligand is markedly lower than that
reported for H4dota (ˆ1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)
(pK1 ˆ 11.08 Æ 0.07) [11]. The next protonation event occurs at a pH close to 4
(pK3 ˆ 3.58 Æ 0.06). The trend in the chemical shift of the various resonances seems to
indicate that this proton is localized on the carboxyl group d. The upfield shift of the
protons 2, 3, and 6, instead of the expected downfield shift, indicates that the
protonation of the carboxylate group induces a shift of the proton density from the N-
atom b to a and c, whose protonated forms can be stabilized by H-bonding interaction
with the negatively charged acetate or N-sulfonylacetamide groups. The shift due to the
deprotonation of the amino group dominates the effect originating from the
protonation of the carboxylate group. This is confirmed by the simultaneous downfield
shift of the resonances 1, 4, 5, and 7 indicating an increased proton density on the N-
atoms a and c.
After the second protonation of the carboxylate groups d (pK4 ˆ 1.88 Æ 0.22), the
addition of the last two protons to the carboxylate e and to the N-sulfonylacetamide
group (pK5 ˆ 0.96 Æ 0.09, pK6 ˆ 0.45 Æ 0.40) restores the initial proton distribution on
the ring N-atoms. This is supported by the upfield shift of the resonances 1, 4, 5, and 7
due to the decreased proton density on the N-atoms a and c and by the downfield shift
of the resonances 2, 3, and 6 originating from the increased proton density on the N-
atom b.

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Coordination Mode. The magnitude of the lanthanide-induced shift (LIS) can be
used to help identify of the donor atoms of the ligand in a complex. The LIS, the
difference between the chemical-shift values of a nucleus in the paramagnetic complex
and in a corresponding diamagnetic derivative, is given by the sum of a contact (Dc), a
pseudocontact (Dp), and a diamagnetic (Dd) contribution (Eqn. 3) [12][13].
For the atoms directly bound to the paramagnetic Ln3‡ ions, the contact
contribution is generally very large and normally dominates the LIS. Therefore, for
paramagnetic Ln3‡ complexes with ligands containing multifunctional groups, the
donor sites can be easily recognized by the magnitude of their LIS. However, a large
LIS is invariantly accompanied by extensive line-broadening, which may prevent the
detection of the resonance.
D ˆ Dc ‡ Dp ‡ Dd
(3)
To investigate the coordination geometry of the N-sulfonylacetamide pendant arm,
ligand 1d was synthesized and studied by 15N-NMR. The d (15N) of the 15N-labelled 1d
and of the corresponding La3‡ and Eu3‡ complexes shows diamagnetic and para-
magnetic contributions of ‡ 11 and À 23 ppm, respectively (Fig. 3). From literature
data on N-atoms directly coordinated to Eu3‡, a 15N LIS of the order of À 1500 ppm
was expected [14]. Furthermore, the linewidth at half height of the 15N resonance for
[Eu(1d)]À results to be only 20.3 Hz. Both the small LIS and paramagnetically induced
line broadening clearly indicate that the N-sulfonylacetamide group coordinates the
Ln3‡ cations through the carboxyl group. Thus, we may safely suggest a coordination
geometry of the type represented in Fig. 4.
Fig. 3. 15N-NMR Spectra of [La(1d)]À, [Eu(1d)]À, and 1d at 40.53 MHz and 258

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Fig. 4. Schematic representation of the lanthanide coordination by the N-sulfonylacetamide arm
Relaxometric Characterization of the Gd3‡ Complexes. The ability of a paramagnetic
complex to accelerate the NMR relaxation rates of the nuclei of the solvent water
molecules is commonly expressed in terms of relaxivity, rip (i ˆ 1,2) [1 ± 3]. This
parameter represents the enhancement of the proton relaxation rate of an aqueous
1 mm solution of the paramagnetic complex with respect to pure water.
The relaxivities of the four complexes, as measured at 20 MHz and 258, are 4.5, 5.0,
5.1, and 5.3 mmÀ1 sÀ1 for [Gd(1a)]À, [Gd(1b)]À, [Gd(1c)]À, and [Gd(1e)]À, respec-
tively. These values are similar to those typical for complexes of similar size and with
one inner-sphere H2O molecule and suggest the presence of a single water molecule
(q ˆ 1) coordinated to the metal center also for [Gd(1a ± e)]À. The complexes
[Gd(1a)]À, [Gd(1b)]À, and [Gd(1c)]À showed a similar pH dependence of the
relaxivity at 20 MHz and 258. The fairly constant relaxivity in the pH range 2 ± 12
implies that no changes in the structure and in the hydration number occur in this
interval. On the other hand, at very low and very high pH values, there is a clear
increase in relaxivity.
This enhancement of rip is too small to be ascribed to a change in the hydration
number of the complex. Most likely, it arises from acid- and base-catalyzed activation of
the prototropic exchange, leading to a more-efficient paramagnetic relaxation.
However, for the change in relaxivity observed at pH < 2, a contribution due to partial
decomplexation of the metal chelate cannot be completely ruled out.
The activation of the proton exchange on the coordinated H2O molecule
(prototropic exchange process) has the effect to shorten the effective residence
lifetime, tM, of the water protons. This phenomenon has already been observed in Gd3‡
complexes characterized by an exceedingly long residence lifetime of the H2O
molecule, where the activation of the prototropic exchange is able to remove the
limiting effect on the relaxivity of a long tM value [15 ± 18]. The occurrence of a tM value
long enough so as to limit the relaxivity was confirmed by the temperature dependence
of the relaxivity for the complexes [Gd(1a)]À, and [Gd(1b)]À (Fig. 5).
The profile can be split in two regions; at high temperatures (T> 308), the
exponential decrease in relaxivity reflects the exponential decrease of the reorien-
tational correlation time, tR, with temperature. At lower temperatures (T< 308),
the relaxivity profile flattens out as a result of the increase in tM that becomes
comparable to or shorter than T1M and thus quenches the relaxivity (T1M ꢂ tM; slow
exchange regime). The comparison with the analogous profile of [Gd(dota)]À indicates
that the N-sulfonylacetamide complexes are characterized by a slower rate of H2O
exchange.

Helvetica Chimica Acta ± Vol. 88 (2005)
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À
À
Fig. 5. Temperature dependence of r1p for [Gd(1a)]À
(
(
), and [Gd(dota)]
(
).
~
!
*
), [Gd(1b)]
To emphasize the effects associated with the activation of the prototropic exchange,
the pH dependence of the relaxivity for the complexes [Gd(1a)]À, and [Gd(1b)]À was
also measured at low temperature (0.18), where the H2O-exchange rate is lower
(Fig. 6). The exchange lifetime of H2O protons is strongly pH-dependent, being very
long at neutral pH (on the order of ms) and significantly shorter (of the order of ns)
either in acidic or basic conditions. The prototropic exchange process may be
adequately treated as a pseudo-first-order mechanism, characterized by a rate constant
kip, the values of which for pure water at 301 K were reported to be in the range 4.1 À
7.1 ´ 109 sÀ1 mÀ1 and 2.2 À 4.3 ´ 109 sÀ1 mÀ1 for the acidic (kex,a) and basic (kex,b) catalysis,
respectively [19]. When the exchange of the entire H2O molecule is fast, the
contribution arising from the prototropic exchange can be neglected. However, when
the exchange of the H2O molecule is slow, the prototropic exchange can contribute
significantly to the observed relaxivity under basic or acidic conditions. The pH
dependence of the relaxivity can, therefore, be fitted by using Eqn. 4, where ro1s and r1os
are the inner- and outer-sphere components of the relaxivity, Ct is the molar
concentration of the metal complex, q the hydration number, and T1M the longitudinal
relaxation time of the protons of the bound H2O molecule [1 ± 3].
It is assumed that T1M does not vary with pH. The pH dependence of the relaxivity
of the complexes [Gd(1a)]À and [Gd(1b)]À was measured at 0.18 and 20 MHz, and the
values of T1M, tM, kex,a, and kex,b obtained from the best fit of the data are reported in
Table 1 and compared with those obtained for the complex [Gd(dota)]À under identical
experimental conditions. In the pH region around neutrality, the relaxivity is mainly
dominated by the exchange rate of the whole H2O molecule and thus is slightly higher

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for [Gd(dota)]À, this complex being characterized by a faster rate of exchange. At high
(usually > 9) or low pH (usually < 3) values, the H2O-exchange rate is no longer a
factor limiting the relaxivity due to the activation of the prototropic exchange. Under
these conditions, r1p is primarily controlled by tR, and then the higher relaxivity
observed for [Gd(1a)]À and [Gd(1b)]À can be explained in terms of the higher relative
molecular mass of these two complexes.
Ct Á q
os
r1p ˆ ro1s ‡ ri1s ˆ
‡ r1
ꢀ4†
ꢀ
ꢁ
1
‡
55:56 Á T1M
‡
kex ‡ kex a Á ‰H Š ‡ kex b Á ‰OHÀŠ
Fig. 6. pH Dependence of the longitudinal water proton relaxivity (r1p) for a 1 mm solution (20 MHz, 0.18) of
[Gd(1a)]À
(
ꢀ), [Gd(1b)]À, (.), and [Gd(dota)]À
(
). The lines through the data points are the result of a best
8
fitting procedure to Eqn. 4.
Table 1. Best-Fit Parameters Obtained from the Analysis of the pH Dependence of the Relaxivity for [Gd(1a)]À,
[Gd(1b)]À and [Gd(dota)]À at 0.18 and 20 MHz
[Gd(1a)]À
[Gd(1b)]À
[Gd(dota)]À
T1M [10À6 s]
kex [105 sÀ1
kexb [108 sÀ1
kexa [107 sÀ1
ro2s [sÀ1MÀ1
2.83 Æ 0.05
2.65 Æ 0.06
2.38 Æ 0.31
1.63 Æ 0.21
3.80 Æ 0.05
2.34 Æ 0.05
2.45 Æ 0.07
4.10 Æ 0.61
1.41 Æ 0.21
3.96 Æ 0.07
3.4 Æ 0.13
]
6.02 Æ 0.07
0.79 Æ 0.16
0.43 Æ 0.07
4.12 Æ 0.09
]
]
]
Determination of the Water-Exchange Lifetime tM. An accurate estimate of the
exchange lifetime of the H2O molecules directly coordinated to the metal center, in a
Gd3‡ chelate, can be obtained from the temperature dependence of the transverse
relaxation rate of the H2O 17O nuclei [1][20][21].

Helvetica Chimica Acta ± Vol. 88 (2005)
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Fig. 7 shows the temperature dependence of the paramagnetic contribution to the
transverse relaxation rate, R2, for [Gd(1b)]À, measured at pH 7 and at 2.12 T and
normalized to 50 mm concentration. For comparison, the corresponding profile of the
complex [Gd(dota)]À is also shown [22]. The curves exhibit a maximum that is
characteristic of the changeover between the fast-exchange region, at high temper-
ature, where R2 is mainly dominated by electronic relaxation, and the slow-exchange
region, at low temperature, where the transverse relaxation rate is dominated by tM.
The relaxation rates measured for the two complexes are similar but the maximum for
[Gd(1b)]À is shifted towards higher temperatures, to indicate a slower rate of H2O
exchange. A best-fit procedure of the experimental data obtained for the three
complexes investigated ([Gd(1a)]À, [Gd(1b)]À, and [Gd(1c)]À) according to the
standard theory provides values for the exchange lifetimes on the order of a few
hundred ns (Table 2) at 258 [19]. The calculated exchange rates are slower than that
reported for the negatively charged [Gd(dota)]À, but rather similar to those observed
for several neutral Gd3‡ complexes of monoamide derivatives of dota [2][23]. A
possible explanation for this behavior can be the high delocalization of the negative
charge on the N-sulfonylacetamide moiety, which results in a higher enthalpy of
activation for the dissociation of the GdÀOw bond with respect to the corresponding
carboxylate derivative.
Fig. 7. Temperature dependence of the transverse water 17O relaxation rate at 9.4 T and pH 7 for 50 mm solutions
of [Gd(1b)]À (.) and [Gd(dota)]À
(
*
). The solid curve through the data points was calculated with the
parameters reported in Table 2.
For Gd-based MRI contrast agents, a long exchange lifetime represents a drawback
since it often limits the attainable relaxivities. However, this disadvantage turns into an
advantage for contrast agents based on the chemical exchange saturation transfer

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Table 2. Best-Fit Parameters Obtained from the Analysis of the Temperature Dependence of the 17O-NMR
Transverse Relaxation Rate, R2, of the Gd3‡ Complexes in Aqueous Solution at pH 7
25
q
D2
[sÀ2/1019
tv
tM
DHm
[kJ molÀ1
DHv
[kJ molÀ1
]
]
[ps]
[ns]
]
[Gd(1a)]À
[Gd(1b)]À
[Gd(1c)]À
[Gd(dota)]À
1
1
1
1
1.7 Æ 0.5
2.1 Æ 0.3
3.7 Æ 0.4
1.4 Æ 0.2
9 Æ 3
507 Æ 20
746 Æ 35
613 Æ 40
244 Æ 16
52 Æ 0.5
44 Æ 0.7
59 Æ 0.8
50 Æ 0.3
2.7 Æ 2
9.0 Æ 3
1.8 Æ 1
1.0 Æ 0.6
15 Æ 2
14 Æ 3
7 Æ 2
(CEST) mechanism. This new class of contrast agents is based on paramagnetic Ln3‡
cations other than Gd3‡ that are able to shift the resonance of the coordinated H2O
protons substantially away from the resonance of the bulk H2O, without inducing an
extreme line broadening [24][25]. All the complexes so far investigated are based on
tetramide derivatives of dota. The H2O-exchange lifetimes typical of the mono-N-
sulfonylacetamide derivatives of dota investigated in this study are still too short to find
a successful application in CEST MRI procedures. Nevertheless, the observed values
suggest that the replacement of acetate with N-sulfonylacetamide arms could slow the
H2O exchange enough to make the appropriate complexes potentially useful for CEST
applications.
Finally, the measurement of the exchange lifetime by 17O-NMR at pHs 3.5 and 7.0
for [Gd(1a)]À afforded very similar values, thus confirming once more that the
relaxivity change as a function of pH at 20 MHz arises exclusively from the activation of
the prototropic exchange.
Binding to HSA. MRI Contrast agents that are currently used in clinical practice are
Gd3‡ complexes characterized by a relative molecular mass between 500 and 600.
Complexes of this size are characterized by a relaxivity that is mainly controlled by the
fast molecular mobility in solution. The most widely adopted strategy to reduce the
molecular rotational tumbling, and thus increase the relaxivity, exploits the non-
covalent binding of the small paramagnetic complexes to macromolecular substrates
[26 ± 28]. HSA has been widely employed to this purpose because it represents the
most-abundant serum protein, and it can be used as a target by contrast agents designed
for angiographic applications [2][27]. Strong interaction between HSA and the
contrast agent is pursued not only for increasing the relaxivity but also for attaining
longer residence lifetimes in the bloodstream.
The interaction between paramagnetic complexes and HSA can be conveniently
investigated at a fixed frequency by means of the proton-relaxation-enhancement
(PRE) method [26 ± 28]. The enhancement of the NMR H2O proton relaxation in the
presence of the macromolecular substrate is used to obtain reliable values for the
binding constant (KA) and the relaxivity of the macromolecular adduct (r1b). It is
known that the presence of hydrophobic substituents on the surface of the complex and
an overall negative charge play an important role in promoting a strong interaction with
HSA. This prompted us to study the noncovalent binding of [Gd(1e)]À with HSA.
This interaction was studied by measuring, at the fixed frequency of 20 MHz and at
258, the longitudinal H2O proton relaxation rate of a 1.98 ´ 10À4 m aqueous solution
(50 mm HEPES) of the complex in the presence of increasing concentrations of the

Helvetica Chimica Acta ± Vol. 88 (2005)
599
protein (Fig. 8). The observed relaxation rate can be expressed in terms of Eqn. 5,
free
bound
lp
where r and r
are the relaxivities of the free complex and of the complex ± HSA
lp
adduct at 258, respectively, and R1d represents the diamagnetic contribution (charges
are omitted for the sake of clarity). The interaction between the complex and the
protein can be represented by the equilibrium of Eqn. 6, characterized by the formation
constant KA (Eqn. 7), where n represents the number of equivalent and independent
interaction sites. By combining Eqn. 5 with Eqn. 7, it is possible to evaluate the product
bound
KA ´ n and r
. The affinity constant of [Gd(1e)]À for HSA is rather weak (KA ´ n ˆ 4 ´
lp
102), whereas the relaxivity of the macromolecular adduct, [Gd(1e)] ± HSA, is
24.4 mmÀ1 sÀ1. The KA value found for this complex is significantly smaller than those
found for other anionic complexes. On the basis of the available information, it is not
possible to ascribe the limited binding to structural constraints or to the charge
delocalization in the N-sulfonylacetamide moiety.
Ro1bs ˆ rfree‰Gd À ꢀ1e†Š ‡ rbound‰Gd À ꢀ1e† À HSAŠ ‡ R1d
ꢀ5†
ꢀ6†
1p
1p
‰Gdꢀ1e†Š ‡ HSA $ ‰Gdꢀ1e†Š À HSA
‰‰Gdꢀ1e†Š À HSAŠ
KA
ˆ
ꢀ7†
‰‰Gdꢀ1e†ŠŠ Á n Á ‰HSAŠ
Fig. 8. Plot of the water proton relaxation rate of a 0.1 mm [Gd(1e)]À solution as function of the HSA
concentration (20 MHz, 258, in 50 mm phosphate buffer)
The relaxivity of the [Gd(1e)] ± HSA supramolecular adduct is very close to the
value that is predicted by the Solomon ± Bloembergen ± Morgan theory by using the
parameters determined for the other Gd3‡ complexes studied and by using a tr value of

600
Helvetica Chimica Acta ± Vol. 88 (2005)
30 ns. This represents the maximum attainable value in the presence of the relatively
low H2O-exchange rate shown by [Gd(1e)]À.
Conclusions. ± The N-sulfonylacetamide group represents an isosteric equivalent of
the acetic acid group and thus acts as a good donor group for lanthanide(III) cations. A
new series of Ln complexes (Ln ˆ Eu, Gd) containing an N-sulfonylacetamide moiety
at the do3a scaffold were prepared and characterized in terms of solution coordination
mode and relaxometric behavior. The replacement of an acetic acid by an N-
sulfonylacetamide moiety on the dota basic structure results in a substantial decrease of
the rate of H2O exchange. Even though these complexes have a net negative charge at
neutral pH, the exchange lifetimes are similar to those of related neutral complexes.
The high delocalization of the charge on the N-sulfonylacetamide moiety may be at the
origin of this effect. The long values of the mean residence lifetimes of the coordinated
H2O molecule (tM) allowed the observation of the effects of the prototropic exchange
at high and low pH.
The slow H2O exchange, typical of these complexes, represents a serious drawback
for the development of Gd-based MRI contrast agents but at the same time, it provides
new insights for the design of novel CEST agents.
The authors thank Dr. Joop Peters, TU Delft, The Netherlands, for useful discussions. Financial support
from MUIR (FIRB project) and from Bracco Imaging SpA, Milano, Italy, is gratefully acknowledged.
Experimental Part
General. HSA (crystallized and lyophilized) was purchased from Sigma, St. Louis, USA, and was used
without any further purification. The molecular mass was assumed to be 69 kDa [17]. p-Toluenesulfon(15N)a-
mide (> 99% 15N) was purchased from Isotec Inc., Miamisburg, USA, and used as received. All other chemicals
were of anal. grade and were purchased from Sigma-Aldrich, St. Louis, USA. M.p.: Büchi 520 apparatus;
uncorrected. NMR Spectra: Bruker AC-200 (200 (1H) and 50.2 MHz (13C) resp.) and Jeol EX-400 (400 MHz (1H
and 15N)) spectrometers; d in ppm, J in Hz; NMR pH titration in D2O (99.8%), the pD being adjusted with DCl
or CO2-free NaOD (Sigma).
The longitudinal H2O proton relaxation rates were measured on the Stelar Spinmaster spectrometer (Stelar,
Mede (PV), Italy) operating at 20 MHz, by means of the standard inversion-recovery technique (16
experiments, 2 scans). A typical 908 pulse width was 3.5 ms, and the reproducibility of the T1 data was 0.5%. The
temp. was controlled with a Stelar VTC-91 air-flow heater equipped with a copper-constantan thermocouple
(uncertainty of 0.18).
Variable-temp. 17O-NMR measurements were recorded on a Jeol EX-400 (9.4 T) spectrometer, equipped
with a 5-mm probe, by using D2O as external lock. Experimental settings were: spectral width 10880 Hz, pulse
width 7 ms, acquisition time 10 ms, 256 scans, no sample spinning. Solns. containing 2.6% of 17O isotope (Yeda,
Israel) were used. The transverse relaxation rates (Ro2bs ) were calculated from the signal width at half-height.
The paramagnetic contribution, R2, was obtained by subtracting from the data the signal width at half-height of a
soln. of H2O at pH 7 with identical 17O enrichment (R2 ˆ Ro2bse À R2w).
Mass Spectra: VG 7070-EQ spectrometer; CI (isobutene) for ligands 1a ± e, FAB (m-nitrobenzyl alcohol or
glycerol) for the other compounds; in m/z. Elemental analyses were performed with a Perkin-Elmer 240
apparatus.
Naphthalene-2-sulfonamide (2e). Naphthalene-2-sulfonyl chloride (2.49 g, 11.0 mmol) and (NH4)2CO3
were mixed and finely ground. The mixture was heated for 15 min at 1008, resulting in extensive foaming. The
cooled residue was washed thoroughly with H2O to remove ammonium salts. The crude product was purified by
recrystallization from H2O: 2.28 g (88%) of 2e. M.p. 217 ± 2198. 1H-NMR (CDCl3, 200 MHz): 8.20 (s, 1 H), 8.02
(br. s, 1 H); 7.91 (s, 1 H); 7.47 ± 7.37 (m, 4 H); 7.14 ± 7.02 (m, 2 H). 13C-NMR (CDCl3, 50.2 MHz): 140.6; 133.7;
131.5; 128.5; 127.8; 127.7; 127.3; 126.9; 125.7; 121.8.

Helvetica Chimica Acta ± Vol. 88 (2005)
601
2-Bromo-N-sulfonylacetamides 3a ± e: General Procedure. The sulfonamide 2a ± d (2 mmol) was suspended
in anh. toluene (5 ml) (chlorobenzene for 2e), bromoacetyl bromide (3 ± 4 mmol) was carefully added dropwise,
and the mixture was refluxed for 4 ± 5 h. The product crystallized from the mixture upon cooling. It was
separated by filtration and recrystallized from toluene in the case of 3a ± c. For the isolation of 3d, e, the mixture
was evaporated; the residue redissolved in sat. NaHCO3 soln., the soln. filtered, and then the desired product
precipitated by slow addition of conc. HCl soln.; further purification was achieved by recrystallization from
toluene.
2-Bromo-N-(methylsulfonyl)acetamide (3a): Yield 47.5%. Colorless crystals. M.p. 102 ± 1048. 1H-NMR
.
‡
(CDCl3, 200 MHz): 9.05 (s, 1 H); 3.96 (s, 3 H); 3.36 (s, 2 H). EI-MS: 217, 215 (M ). Anal. calc. for C3H6BrNO3S
(216.05): C 16.68, H 2.80, N 6.48; found C 16.50, H 3.01, N 6.45.
2-Bromo-N-[(4-nitrophenyl)sulfonyl]acetamide (3b). Yield 33.5%. Colorless crystals. M.p. 163 ± 1658.
1H-NMR (CDCl3, 200 MHz): 9.01 (s, 1 H); 8.22 (d, J ˆ 8.8, 2 H); 8.09 (d, J ˆ 8.8, 2 H); 3.65 (s, 2 H). 13C-NMR
.
‡
(CDCl3, 50.2 MHz): 165.2 (s); 150.4 (s); 144.0 (s); 129.5 (d); 123.8 (d); 27.7 (t). EI-MS: 324, 322 (M ). Anal.
calc. for C8H7BrN2O5S (323.12): C 29.74, H 2.18, N 8.67; found C 29.52, H 2.21, N 8.80.
2-Bromo-N-[(4-methylphenyl)sulfonyl]acetylamide (3c): Yield 91.5%. Colorless crystals. M.p. 114 ± 1168.
1H-NMR (CDCl3, 200 MHz): 9.36 (s, 1 H); 7.98 (d, J ˆ 8.2, 2 H); 7.36 (d, J ˆ 8.2, 2 H); 3.85 (s, 2 H); 2.45 (s, 3 H).
13C-NMR (CDCl3, 50.2 MHz): 164.2 (s); 145.6 (s); 134.5 (s); 129.7 (d); 128.5 (d); 27.8 (t); 21.6 (t). EI-MS: 293,
.
‡
291 (M ). Anal. calc. for C9H10BrNO3S (292.15): C 37.00, H 3.45, N 4.79; found C 36.91, H 3.61, N 4.67.
2-Bromo-N-[4-methylphenyl)sulfonyl]acetyl(15N)amide (3d). Yield 70.5%. Colorless crystals. M.p. 115 ±
1168. 1H-NMR (CDCl3, 200 MHz): 9.38 (s, 1 H); 7.98 (d, J ˆ 8.1, 2 H); 7.38 (d, J ˆ 8.1, 2 H); 3.84 (s, 2 H); 2.47 (s,
3 H). 13C-NMR (CDCl3, 50.2 MHz): 163.6 (s); 145.6 (s); 134.6 (s); 129.6 (d); 128.5 (d); 27.8 (t); 21.6 (t). EI-MS:
.
‡
294, 292 (M ). Anal. calc. for C3H6Br15NO3S (293.14): C 36.88, H 3.44, N 5.12; found C 36.75, H 3.61, N 5.15.
2-Bromo-N-(naphthalen-2-ylsulfonyl)acetamide (3e): Yield 87.7%. White crystals. M.p. 132 ± 1348.
1H-NMR (CDCl3, 200 MHz): 9.63 (br. s, 1 H); 8.72 (s, 1 H); 8.06 ± 7.91 (m, 4 H); 7.73 ± 7.63 (m, 2 H); 3.84 (s,
2 H). 13C-NMR (CDCl3, 50.2 MHz): 187.9 (s); 135.3 (s); 134.9 (s); 131.4 (s); 129.2 (d); 129.1 (d); 128.9 ± 127.3
.
‡
(4 d); 28.0 (t). EI-MS: 329, 327 (M ). Anal. calc. for C12H10BrNO3S (328.18): C 43.92, H 3.07, N 4.27; found
C 44.03, H 3.21, N 4.09.
Synthesis of 5a ± e: General Procedure. To a soln. of 4 (1 mmol) in DMF, finely powdered K2CO3 (3 mmol)
followed by 3a ± e (1.5 mmol) were added, and the heterogeneous mixture was heated at 70 ± 1008 for 4 ± 15 h
(TLC (SiO2, CH2Cl2/MeOH/conc. NH3 soln. 9 :1:0. 1) monitoring). The mixture was diluted with CH2Cl2
(10 ml), filtered, and evaporated. Chromatography (silica gel) of the solid residue afforded pure 5a ± e.
10-{2-[(Methylsulfonyl)amino]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic Acid Tri(tert-bu-
tyl) Ester (5a): Yield 69.0%. White amorphous solid. M.p. 76 ± 788. 1H-NMR (CDCl3, 200 MHz): 3.01 (s, 3 H);
3.40 ± 1.90 (m, 24 H); 1.41 (s, 27 H). 13C-NMR (CDCl3, 50.2 MHz): 177.2 (s); 172.1 (s); 172.0 (s); 81.6 (s); 81.5
‡
(s); 59.2 (t); 55.8 (t); 55.5 (t); 50.5 (br. s (4 t)); 39.3 (q); 27.8 (q); 27.6 (q). CI-MS: 650 ([M ‡ H] ). Anal. calc. for
C29H55N5O9S (649.84): C 53.60, H 8.53, N 10.78; found C 53.52, H 8.70, N 10.80.
10-{2-{[(4-Nitrophenyl)sulfonyl]amino}-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic Acid
Tri(tert-butyl) Ester (5b): Yield 85.2%. Pale yellow amorphous solid. M.p. 63 ± 658. 1H-NMR (CDCl3,
200 MHz): 8.27 (d, J ˆ 8.8, 2 H); 8.04 (d, J ˆ 8.8 Hz, 2 H); 3.20 ± 2.20 (m, 24 H); 1.45 (s, 18 H); 1.43 (s, 9 H).
13C-NMR (CDCl3, 50.2 MHz): 176.8 (s); 172.0 (s); 169.6 (s); 151.6 (s); 148.1 (s); 128.8 (d); 122.6 (d); 81.9 (s);
‡
81.5 (s); 59.3 (t); 57.0 (t); 55.2 (t); 50.1 (br. s (4 t)); 28.0 (q); 27.7 (q). CI-MS: 757 ([M ‡ H] ). Anal. calc. for
C34H56N6O11S (756.91): C 53.95, H 7.46, N 11.10; found C 53.84, H 7.37, N 11.19.
10-{2-{(4-Methylphenyl)sulfonyl]amino}-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic Acid
Tri(tert-butyl) Ester (5c): Yield 53.2%. White amorphous solid. M.p. 75 ± 778. 1H-NMR (CDCl3, 200 MHz):
7.97 (d, J ˆ 8.0, 2 H); 7.02 (d, J ˆ 8.0, 2 H); 3.05 ± 2.87 (m, 8 H); 2.16 ± 2.05 (m, 16 H); 1.45 (s, 18 H); 1.41 (s, 9 H).
13C-NMR (CDCl3, 50.2 MHz): 176.1 (s); 172.0 (s); 171.8 (s); 142.7 (s); 138.9 (s); 129.1 (d); 126.3 (d); 81.6 (s);
‡
81.4 (s); 59.7 (t); 56.1 (t); 55.3 (t); 50.2 (br. s (4 t)); 27.8 (q); 27.7 (q); 22.0 (q). CI-MS: 726 ([M ‡ H] ). Anal.
calc. for C35H59N5O9S (725.94): C 57.91, H 8.19, N 9.65; found C 57.78; H 8.26; N 9.70.
10-{2-{[(4-Methylphenyl)sulfonyl](15N)amino}-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
Acid Tri(tert-butyl) Ester (5d): Yield 64.6%. White amorphous solid. M.p. 73 ± 778. 1H-NMR (CDCl3,
200 MHz): 7.98 (d, J ˆ 8.0); 7.04 (d, J ˆ 8.0, 2 H); 3.20 ± 2.10 (m, 24 H); 1.48 (s, 18 H); 1.42 (s, 9 H). 13C-NMR
(CDCl3, 50.2 MHz): 176.2 (s); 171.9 (s); 171.8 (s); 142.5 (s); 138.8 (s); 129.1 (d); 126.5 (d); 81.5 (s); 81.4 (s); 59.4
‡
(t); 56.2 (t); 55.5 (t); 50.3 (br. s (4 t)); 27.8 (q); 27.7 (q); 22.0 (q). CI-MS: 727 ([M ‡ H] ). Anal. calc. for
C35H5915NN4O9S (726.93): C 57.83, H 8.18, N 9.77; found C 57.67, H 8.32, N 9.69.
10-{2-[(Naphthalen-2-ylsulfonyl)amino]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic Acid Tr-
(tert-butyl) Ester (5e): Yield 59.8%. White amorphous solid. M.p. 90 ± 938. 1H-NMR (CDCl3, 200 MHz): 8.60 (d,

602
Helvetica Chimica Acta ± Vol. 88 (2005)
J ˆ 1.4, 1 H); 8.26 (dd, J1 ˆ 8.6, J2 ˆ 1.7, 1 H); 7.85 ± 7.70 (m, 2 H); 7.64 (d, J ˆ 8.6, 1 H); 7.48 ± 7.34 (m, 2 H);
3.20 ± 2.10 (m, 24 H); 1.46 (s, 18 H); 1.45 (s, 9 H). 13C-NMR (CDCl3, 50.2 MHz): 176.2 (s); 172.0 (s); 171.8 (s);
143.0 (s); 134.0 (s); 132.4 (s); 129.0 (2 d); 127.3 ± 126.3 (4 d); 125.5 (d); 81.7 (s); 81.5 (s); 59.7 (t); 56.0 (t); 55.3 (t);
‡
50.0 (br. s (4 t)); 27.8 (q); 27.7 (q). CI-MS: 762 ([M ‡ H] ). Anal. calc. for C38H59N5O9S (761.98): C 59.90,
H 7.80, N 9.19; found C 59.92, H 8.00, N 9.12.
Hydrolysis of 5a ± e to 1a ± e: General Procedure. A soln. of 5e ± e (1 mmol) in CF3COOH (5 ml) was
stirred for 24 h at r.t. The mixture was then evaporated, the residue redissolved in MeOH (3 ml), and the
product precipitated by addition of an excess of Et2O. The solid was washed twice with Et2O and finally dried in
vacuo: 1a ± e.
10-{2-[(Methylsulfonyl)amino]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic Acid (1a): White
1
amorphous solid. M.p. 200 ± 2018 (dec.). H-NMR (D2O, 200 MHz): 3.72 (s, 4 H); 3.56 (s, 4 H); 3.45 (s, 4 H);
3.25 ± 2.92 (m, 16 H); 3.11 (s, 3 H). 13C-NMR (D2O, 50.2 MHz): 176.1 (s); 173.9 (s); 172.2 (s); 58.0 (t); 56.3 (t);
‡
‡
55.4 (t); 53.5 (br. s (2 t)); 50.6 (br. s (2 t)); 43.0 (q). FAB-MS (pos.): 504 ([M ‡ Na] ), 482 ([M ‡ H] ). Anal.
calc. for C17H31N5O9S (481.53): C 42.40, H 6.49, N 14.54; found C 42.15, H 6.57, N 14.43.
10-{2-{[(4-Nitrophenyl)sulfonyl]amino}-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic Acid
1
(1b): White amorphous solid. M.p. 95 ± 1008 (dec.). H-NMR (D2O, 200 MHz): 8.28 (d, J ˆ 8.1, 2 H); 8.04 (d,
‡
‡
J ˆ 8.1, 2 H); 3.80 ± 3.40 (m, 8 H); 3.30 ± 2.70 (m, 16 H). FAB-MS (pos.): 611 ([M ‡ Na] ), 589 ([M ‡ H] ).
Anal. calc. for C22H32N6O11S (588.59): C 44.89, H 5.48, N 14.28; found C 44.62, H 5.49, N 14.09.
10-{2-{[(4-Methylphenyl)sulfonyl]amino}-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic Acid
1
(1c): White amorphous solid. M.p. 175 ± 1798 (dec.). H-NMR (D2O, 200 MHz): 7.66 (d, J ˆ 8.2, 2 H); 7.25 (d,
J ˆ 8.2, 2 H); 3.56 ± 3.34 (m, 8 H); 3.16 ± 2.80 (m, 16 H); 2.23 (s, 3 H). 13C-NMR (D2O, 50.2 MHz): 175.8 (s);
172.8 (s); 172.7 (s); 148.5 (s); 137.2 (s); 132.3 (d); 130.0 (d); 59.7 (t); 56.7 (t); 55.8 (t); 53.1 (br. s (2 t)); 50.7 (br. s
‡
‡
(2 t)), 23.2 (q). FAB-MS (pos.): 580 ([M ‡ Na] ), 558 ([M ‡ H] ). Anal. calc. for C23H35N5O9S (557.62):
C 49.54, H 6.33, N 12.56; found C 49.45, H 6.48, N 12.53.
10-{2-{[(4-Methylphenyl)sulfonyl](15N)amino}-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
Acid (1d): White amorphous solid. M.p. 177 ± 1818 (dec.). 1H-NMR (D2O, 200 MHz): 7.70 (d, J ˆ 8.1, 2 H); 7.30
(d, J ˆ 8.1, 2 H); 3.60 ± 3.46 (m, 8 H); 3.19 ± 2.84 (m, 16 H); 2.27 (s, 3 H). 13C-NMR (D2O, 50.2 MHz): 175.7 (s);
172.7 (s); 172.6 (s); 148.6 (s); 137.2 (s); 132.3 (d); 130.0 (d); 57.9 (t); 56.7 (t); 55.7 (t); 53.0 (br. s (2 t)); 50.8 (br. s
(2 t)); 23.2 (q). FAB-MS (pos.): 581 ([M ‡ Na] ), 559 ([M ‡ H] ). Anal. calc. for C23H3515NN4O9S (558.61):
‡
‡
C 49.45, H 6.32, N 12.71; found C 49.44, H 6.51, N 12.63.
10-{2-[(Naphthalen-2-ylsulfonyl)amino]-2-oxoethyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic Acid
1
(1e): White amorphous solid. M.p. 203 ± 2068 (dec.). H-NMR (D2O, 200 MHz): 8.26 (m, 2 H); 7.73 ± 7.63 (m,
‡
3 H); 7.39 (m, 2 H); 3.01 ± 2.81 (m, 8 H); 3.53 ± 2.68 (m, 16 H). FAB-MS (pos.): 616 ([M ‡ Na] ), 594 ([M ‡
‡
H] ). Anal. calc. for C26H35N5O9S (593.66): C 52.60, H 5.94, N 11.80; found C 52.47, H 6.07, N 11.73.
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