Gadolinium complexes of highly rigid, open-chain ligands containing a cyclobutane ring in the backbone: Decreasing ligand denticity might enhance kinetic inertness

Article abstract of DOI:10.1021/acs.inorgchem.9b02044

In an effort to explore novel ligand scaffolds for stable and inert lanthanide complexation in magnetic resonance imaging contrast agent research, three chiral ligands containing a highly rigid (1S,2S)-1,2-cyclobutanediamine spacer and different number of

Full text of DOI:10.1021/acs.inorgchem.9b02044

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Gadolinium Complexes of Highly Rigid, Open-Chain Ligands
Containing a Cyclobutane Ring in the Backbone: Decreasing Ligand
Denticity Might Enhance Kinetic Inertness
†
†
‡
§
†
́
̀
́
Oriol Porcar-Tost, Jose A. Olivares, Agnes Pallier, David Esteban-Gomez, Ona Illa,
,§
,‡
Carlos Platas-Iglesias,* Eva Toth,* and Rosa M. Ortun
̃
o*,†
́
́
†
‡
§
̀
̀
Departament de Química, Universitat Autonoma de Barcelona, 08193 Cerdanyola del Valles, Barcelona, Spain
́
́
́
́
Centre de Biophysique Moleculaire, UPR 4301, CNRS, Universite d’Orleans, rue Charles Sadron, 45071 Orleans Cedex 2, France
́
́
̃
a, Campus da Zapateira-Rua
Centro de Investigacions Científicas Avanzadas and Departamento de Química, Universidade da Corun
da Fraga 10, 15008 A Coruna, Spain
̃
S
* Supporting Information
ABSTRACT: In an effort to explore novel ligand scaffolds for stable and
inert lanthanide complexation in magnetic resonance imaging contrast agent
research, three chiral ligands containing a highly rigid (1S,2S)-1,2-
cyclobutanediamine spacer and different number of acetate and picolinate
groups were efficiently synthesized. Potentiometric studies show comparable
thermodynamic stability for the Gd3+ complexes formed with either the
octadentate (L3)4− bearing two acetate or two picolinate groups or the
heptadentate (L2)4− analogue bearing one picolinate and three acetate
groups (log KGdL = 17.41 and 18.00 for [Gd(L2)]− and [Gd(L3)]−,
respectively). In contrast, their dissociation kinetics is revealed to be very different: the monohydrated [Gd(L3)]− is
considerably more labile, as a result of the significant kinetic activity of the protonated picolinate function, as compared to the
bishydrated [Gd(L2)]−. This constitutes an uncommon example in which lowering ligand denticity results in a remarkable
increase in kinetic inertness. Another interesting observation is that the rigid ligand backbone induces an unusually strong
contribution of the spontaneous dissociation to the overall decomplexation process. Thanks to the presence of two inner-sphere
water molecules, [Gd(L2)]− is endowed with high relaxivity (r1 = 7.9 mM−1 s−1 at 20 MHz, 25 °C), which is retained in the
presence of large excess of endogenous anions, excluding ternary complex formation. The water exchange rate is similar for
[Gd(L3)]− and [Gd(L2)]−, while it is 1 order of magnitude higher for the trishydrated tetraacetate analogue [Gd(L1)]− (kex
298
= 8.1, 10, and 127 × 106 s−1, respectively). A structural analysis via density functional theory calculations suggests that the large
bite angle imposed by the rigid (1S,2S)-1,2-cyclobutanediamine spacer could allow the design of ligands based on this scaffold
with suitable properties for the coordination of larger metal ions with biomedical applications.
and Cu2+. Typically, complexes formed with macrocyclic
INTRODUCTION
■
ligands are kinetically more inert than the linear analogues,3
and their dissociation mechanisms are also different. Whereas
the acid-catalyzed pathway is the major contributor to the
dissociation of macrocyclic chelates (Chart 1),4 linear
complexes tend to dissociate via metal-assisted pathways.2a,5
For example, transmetalation reactions between [Gd(DTPA-
BMA)] and Cu2+ in the presence of citrate, phosphate, and
bicarbonate anions occur through dissociation of the
gadolinium complex assisted by endogenous ligands.5 Previous
work has shown that the incorporation of rigid moieties in the
structure of linear ligands results in significantly improved
kinetic inertness of their lanthanide complexes. A remarkable
example is the highly rigid [Gd(cddadpa)]− complex (Chart
1). It presents not only good thermodynamic stability but also
a kinetic inertness that is unprecedented for a linear ligand,
Contrast agents (CA) are paramagnetic or super-paramagnetic
substances that improve the sensitivity and the specificity of
magnetic resonance imaging (MRI) examinations. In the last
decades, there has been very active research to design more
efficient, selective, and safer CAs, which are valuable tools in
preclinical imaging, clinical diagnosis, and, more recently, in
theranostic approaches.1 Given its seven unpaired electrons
and slow electron spin relaxation, paramagnetic Gd3+ has the
greatest effect on nuclear relaxation times of surrounding
nuclei and is the most-used metal ion in MRI contrast agents.
To prevent in vivo toxicity of the complexes, which would be
related to the release of the free, noncomplexed metal, Gd3+
needs to be chelated in complexes of high thermodynamic
stability and kinetic inertness that ensure stable complexation
at physiological pH.2 Several mechanisms account for the
possible dissociation of Gd3+ complexes. These involve
spontaneous and acid-catalyzed processes, as well as
dissociation assisted by endogenous metal ions, such as Zn2+
Received: July 9, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.9b02044
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Inorganic Chemistry
Article
Chart 1. Previously Reported Ligands Related to Those Described and Studied in This Work
a
Scheme 1. Synthesis of Ligands H4cbdta, H4cbddapa, and H4cbddadpa (H4(L1), H4(L2), and (H4L3))
a
Reagents and conditions: (i) H2, Pd(OH)2, CH3OH, rt, 84%; (ii) (a) 2 M HCl in diethyl ether, CH2Cl2, rt, 4 h; (b) K2CO3, CH2Cl2, rt, 2 h, 80%;
(iii) tert-butyl bromoacetate, KI, DIPEA, DMF, rt; 18 h, 74%; (iv) 4 M HCl in dioxane, rt, 18 h, 77%; (v) (a) MeOH, rt, 2.5 h; (b) NaBH4,
CH3OH, 0 °C, 2 h, 87%; (vi) LiOH, 1:1 THF−H2O, rt, 4 h, quantitative; (vii) ClCO2Bn, NaHCO3, Na2CO3, 7:1 H2O−acetone, 0 °C, 18 h, 60%;
(viii) TBAI, NaH, THF, rt, 18 h, 62%; (ix) KI, DIPEA, DMF, rt, 30 h, 57%.
being comparable to those of clinically approved macrocyclic
complexes.6
netic relaxation,7 the most important being the number of
hydration water molecules (q), the water exchange rate, the
rotational dynamics of the complex, and its electron spin
relaxation.
Relaxivity is linearly proportional to the number of inner-
sphere water molecules; however, complexes with high
hydration numbers are often thermodynamically less stable,
increasing the risk of metal release. Moreover, for bishydrated
In the design of novel MRI agents, in addition to safety
issues, one must also consider the structural parameters that
affect the proton relaxivity r1 and, thus, the efficiency of a CA.
Indeed, relaxivity is related to a number of microscopic
parameters of the paramagnetic chelate as described by the
Solomon-Bloembergen-Morgan (SBM) theory of paramag-
B
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Table 1. Protonation Constants of (L1)4−, (L2)4−, (L3)4−, and Related Ligands, and Stability Constants of Their Metal
Complexes (25 °C, 0.15 M KCl)
11
2h
6
(L1)4−
(L2)4−
(L3)4−
edta4−
cdta4−
octapa4−
cddadpa4−
H
H
H
H
H
log K1
log K2
log K3
log K4
log K5
9.66 0.01
5.84 0.01
3.06 0.02
2.08 0.03
1.71 0.08
22.35
14.73 0.01
2.38 0.03
12.26 0.01
4.10 0.01
9.58 0.02
6.00 0.04
3.78 0.04
2.32 0.05
2.07 0.05
23.75
17.41 0.01
2.36 0.02
15.22 0.01
3.78 0.01
8.89 0.03
6.61 0.03
4.26 0.06
2.97 0.06
2.79 0.06
25.52
18.00 0.02
3.28 0.03
16.28 0.05
4.00 0.04
3.41 0.04
9.18
6.00
2.58
2.29
9.3612
5.9512
3.6212
2.5712
1.4912
22.9912
18.9713
1.6613
16.7514
2.5714
8.52
5.40
3.65
2.97
1.66
22.20
20.23
9.35
5.66
4.20
3.72
2.62
25.55
20.68
2.38
15.85
3.81
∑log KiH
log KGdL
log KGdHL
log KZnL
log KZnHL
log KZnH2L
20.05
16.28
b
b
14.61
19.32
a
pGd
13.4
16.2
17.4
15.4
18.0
20.0
19.7
a
b
pGd = −log[Gd3+free] at cL = 1 × 10−5 M; cGd = 1 × 10−6 M; pH 7.4. With La: log KLaL = 18.74 0.01 and log KLaHL = 2.8 0.1; with Lu: log
KLuL = 17.02 0.01 and log KLuHL = 3.85 0.1.
chelates, the coordinated water molecules can be replaced by
endogenous anions, thereby decreasing the efficiency of the
CA.8 Usually, complexes with more than one hydration water
also present lower kinetic inertness, although it has been
observed that the incorporation of cyclohexane or pyridine
moieties, for instance, in [Gd(CyPic3A)]− or [Gd(HYD)]−,
respectively, leads to a remarkable kinetic inertness for these
bishydrated complexes, which is comparable to that of the
monohydrated [Gd(DTPA)]2− (Chart 1).9 Furthermore,
lowering the ligand basicity, for example, with hydrazine
functions in [Gd(HYD)]−, was also identified as an important
factor to improve kinetic inertness.9b
In the objective of inducing ligand rigidity, we incorporated
a cyclobutane ring in the scaffold of linear chelators. Here we
present the synthesis of three new ligands, H4cbdta,
H4cbddapa, and H4cbddadpa, that will be referred to as
H4(L1), H4(L2), and H4(L3), respectively (Scheme 1; see
Experimental Section for full names), and the investigation of
their Gd3+ complexes with respect to thermodynamic stability,
kinetic inertness, and relaxometric properties. In addition to
the assessment of the role of a highly rigid ligand backbone, the
comparison of complexes [Gd(L1)]−, [Gd(L2)]−, and [Gd-
(L3)]− provides information about the influence of the
picolinate moiety and of increasing ligand denticity on these
properties. Indeed, (L1)4−, (L2)4−, and (L3)4− are potentially
hexa-, hepta-, and octadentate, respectively; therefore,
coordination of 3, 2, and 1 inner-sphere water molecules is
expected in their Gd3+ complexes. This experimental assess-
ment was completed with density functional theory (DFT)
calculations on [Gd(L2)]− and [Gd(L3)]− to gain further
insight into the relationship between structure and coordina-
tion chemistry of these novel and highly rigid chelators.
diisopropylethyl amine (DIPEA) and potassium iodide led to
3, which, by acidolysis of tert-butyl esters, provided H4(L1) in
54% yield for the two steps.
Alternatively, reductive amination of methyl 6-formylpicoli-
nate, 5, with amine 4 gave compound 6 in 87% yield.
Subsequent removal of tert-butyl carbamate followed by
alkylation led to 7 in 74% yield. Finally, saponification of the
methyl ester with LiOH followed by acidolysis of the three tert-
butyl esters afforded H4(L2) in 77% yield.
The synthetic route to H4(L3) was slightly different, since
attempts to introduce the second picolinate unit by reductive
amination, both in one-pot reaction and in a sequential
manner, failed probably due to the severe steric constriction
imposed by the cyclobutane ring. Neither the attempt to do it
by alkylation of the second amine with methyl chloromethyl-
picolinate, 10, was satisfactory. The order of introduction of
the substituents by alkylation reactions was then reversed.
Symmetric dicarbamate 8 was prepared in 60% yield from 1 by
removal of the tert-butyl carbamate and reaction of the free
amine with benzyl chloroformate. Compound 8 reacted with
tert-butyl bromoacetate using NaH as a base and in the
presence of tetrabutylammonium iodide (TBAI) in anhydrous
tetrahydrofuran (THF) for 18 h. Compound 9 was then
obtained in 62% yield. Hydrogenolysis of the benzyl
carbamates and subsequent reaction with 10 led to 11 (57%
yield), which, after full deprotection, provided H4(L3) in 70%
yield.
[Gd(L1)]−, [Gd(L2)]−, and [Gd(L3)]− complexes were
synthesized in aqueous solution by reaction of equimolar
amounts of the corresponding ligand and GdCl3·6H2O
followed by adjustment of the pH to ∼7. The high-resolution
mass spectrometry (HRMS) spectra of the complexes showed
the expected peaks in each case confirming their formation
(see the Supporting Information).
RESULTS AND DISCUSSION
■
Synthesis of the Ligands and of the Gd3+ Complexes.
Ligands H4(L1), H4(L2), and H4(L3) were synthesized
according to Scheme 1, starting from chiral and orthogonally
protected (1S,2S)-1,2-cyclobutanediamine, 1, which was
previously described.10
Ligand Protonation Constants and Stability Con-
stants of the Metal Complexes. The protonation constants
of (L1)4−, (L2)4−, and (L3)4−, as well as the stability constants
of their complexes with Gd3+ and Zn2+, were determined by
pH-potentiometric titrations. For (L3)4−, the stability con-
stants with La3+ and Lu3+ were also assessed. The global
Sequential deprotection of diamine 1 by catalytic hydro-
genolysis of the benzyl carbamate and acid hydrolysis of the
tert-butyl carbamate afforded free diamine 2 at room
temperature (rt) in 64% yield for the two steps. Alkylation
of 2 with tert-butyl bromoacetate (4.4 equiv) in the presence of
basicity of the three ligands increases in the order of (L1)4−
<
(L2)4− < (L3)4− (Table 1), in accordance with the subsequent
incorporation of one and two picolinate units, respectively, in
L2 and L3.
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Figure 1. Plot of kobs values for the dissociation of [Gd(L2)]− (0.1138 mM) as a function of the Cu2+ ion concentration at pH 3.5, 3.8, 4.1, and 4.9
(a) and as a function of proton concentration (b). The red line on (b) represents the fit to eq 1.
Table 2. Dissociation Rate Constants of [Gd(L2)]− and of Some Relevant Gd3+Complexes Used as MRI Contrast Agents
15
9b
2h
6
2a
ligand
(L2)4−
PY4−
HYD4−
octapa4−
cddadpa4−
DTPA4−
k0 [s−1
]
3.0 × 10−4
0.49
k1 [M−1 s−1
]
]
0.17
520
0.85
9.8
11.8
0.016
0.58
k2 [M−1 s−1
1.58 × 103
2.5 × 104
22.5
9.7 ×·104
0.93
k3 [M−1 s−1
]
2.4 × 10−3
6.8 × 10−4
1.49 × 105
Cu
a
t1/2 [h]
0.64
2.8 × 104
5.3 × 103
0.15
202
a
t1/2 = ln 2/kobs, where kobs was calculated by using pH 7.4 and cCu2+ = 1 μM.
The first protonation constant (log K1), corresponding to
more stable for each of the three ligands than those of the
endogenous Zn2+ cation. This aspect can be important to limit
potential Zn2+ transmetalation of the Gd3+ complexes leading
to Gd3+ release.
the protonation of a backbone nitrogen, is similar for (L1)4−
(9.66) and (L2)4− (9.58), while it is smaller for (L3)4− (8.89).
A similar tendency was observed for the tetraacetate edta4−
(edta = ethylenediaminetetraacetic acid) with respect to the
bispicolinate derivative octapa4− (octapa = 6,6′-[(ethane-1,2-
diyl-bis((carboxymethyl)azanediyl))bis(methylene)]-
dipicolinic acid), the latter having a considerably lower log K1
value (8.52 vs 9.18).2h The rigidity of the ligand also affects the
first protonation constant; with identical pending groups, log
K1 is typically higher for the rigidified ligands containing a
cyclobutyl or a cyclohexyl moiety instead of the flexible
ethylene bridge between the two amines (Table 1).
Dissociation Kinetic Studies. Kinetic inertness of metal
complexes is a key parameter for their safe in vivo application.
It is usually described by assessing the rate constants of the
different pathways that can contribute to the overall
dissociation. These involve spontaneous, acid- or metal-
catalyzed processes (as depicted in Figure S4 in the Supporting
Information), which are characterized by rate constants k0, k1,
and k2, or k3, respectively. While macrocyclic chelates are
typically endowed with higher kinetic inertness, with rigidified
open-chain ligands such as cddadpa6 or HYD9b (Chart 1),
kinetic inertness comparable to that of macrocyclic Gd3+
complexes could be achieved.
Ligands (L1)4−, (L2)4−, and (L3)4− form both non-
protonated and monoprotonated mononuclear complexes
with Gd3+ and Zn2+ ions. In addition, (L3)4− forms a
diprotonated Zn2+ complex as well. These protonated
complexes are observed at acidic pH (see species distribution
diagrams in Supporting Information) and can be attributed to
the protonation of the carboxylate groups. At pH 7, only the
nonprotonated complexes exist for any of the three systems.
Above pH 10, a slight precipitation was observed in the
[Gd(L1)]− and [Gd(L2)]− samples, possibly due to the
formation of hydroxo complexes. The stability constants of
those complexes could not be calculated, and therefore only
experimental data below that point were used to fit the curves
and calculate the stability constants.
The kinetic inertness of [Gd(L2)]− was investigated by
monitoring the exchange reaction with Cu2+, which is a
physiologically relevant metal ion with high efficiency to
promote transmetalation of Gd3+ complexes in general. The
transmetalation was followed by UV−vis spectrophotometry in
the pH range of 3.5−4.9 at three different Cu2+ concentrations,
corresponding to 20-, 30-, and 40-fold excess of the exchanging
metal ion (Figure S5 in the Supporting Information).
Unfortunately, for [Gd(L3)]− even at pH 6.1, the dissociation
was too fast to be followed by conventional UV−vis
spectroscopy or relaxometry, thus preventing a quantitative
study. On the one hand, indeed, at pH 6.1 full dissociation was
observed at 1 min following the mixing of the [Gd(L3)]−
complex and 20 equiv of Cu2+. On the other hand, [Gd(L1)]−
was not studied in view of its lower stability. For [Gd(L2)]−,
the pseudo-first-order rate constants, kobs, as a function of the
pH and the Cu2+ ion concentration, are shown in Figure 1.
The dissociation is independent of Cu2+ concentration,
while it is strongly accelerated with decreasing pH, indicating
that spontaneous and acid-catalyzed processes are responsible
for the dissociation. This is in contrast to the dissociation of
[Gd(DTPA)]2− (Chart 1) and some related open-chain
The stability constants (log K) of the complexes with Gd3+
and Zn2+ ions follow the same order as their basicity, that is,
(L3)4− > (L2)4− > (L1)4−. This result was expectable owing to
the extra coordinating sites provided by the picolinate units
with respect to acetates. However, the stability of [Gd(L3)]−
remains lower than that of the octapa4− and cddadpa4−
(cddadpa = 6,6′-[(cyclohexane-1,2-diylbis((carboxymethyl)-
azanediyl)]bis(methylene)dipicolinic acid) analogues, suggest-
ing that the cyclobutane ring imposes severe constraint and
that this prevents the ligand from properly adapting to
lanthanide coordination. Nevertheless, the Gd3+ complexes are
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complexes, where metal-assisted pathways also represent a
significant contribution.2a
contribution of the individual pathways and estimate the half-
life for physiological conditions. One can simply conclude that,
in this case, the addition of a second picolinate function is
detrimental for the kinetic inertness (as experimentally
assessed at pH 6 and 4). We note that a decrease in kinetic
inertness upon increasing the ligand denticity has been already
reported for linear Mn2+ complexes.16 In general, such a
situation can occur when the additional donor atoms of the
ligand (i) contribute to more efficient proton-assisted
dissociated pathways, since they provide more protonation
sites and/or (ii) allow for the formation of dinuclear species,
which would not be possible without those donor atoms.
Luminescence Studies to Assess Hydration Numbers
and Anion Binding. Inner-sphere proton relaxivity is linearly
proportional to the number q of inner-sphere water molecules
in the Gd3+ complexes.2 Hydration numbers have been
determined on the corresponding [Eu(L1)]−, [Eu(L2)]−,
and [Eu(L3)]− analogues, by measuring luminescence life-
times in H2O and D2O solutions (Table 3, see Figure S7 for
The proton concentration dependence of the kobs values
could be fitted to eq 1, resulting in rate constants k0, k1, and k2,
corresponding to the spontaneous dissociation (k0) and to the
proton-catalyzed dissociation of the nonprotonated (k1) and
the monoprotonated complex (k2). These rate constants are
shown and compared to those of some related complexes in
Table 2.
kobs = k0 + k1[H+]+k2[H+]2
(1)
The k1 = 0.49 M−1 s−1 value for [Gd(L2)]− is similar to k1 for
2a
the clinically approved MRI agent [Gd(DTPA)]2− and the
pyridine derivatives GdPY15 and GdHYD,9b while it is 1 order
of magnitude higher than that for the cyclohexane-derivative
[Gd(cddadpa)]−.6 The constant k2 is much lower than for
[Gd(DTPA)]2−, whereas for [Gd(cddadpa)]−, this dissocia-
tion pathway was not important at all. When comparing to the
pyridine derivatives GdPY and GdHYD, [Gd(L2)]− has similar
k1 but much higher k2. Overall, the major difference between
[Gd(L2)]− and all the other Gd complexes listed in Table 2 is
in the rate constant characterizing the spontaneous dissocia-
tion, k0. For [Gd(L2)]−, k0 = 3 × 10−4 s−1 is higher than the
close-to-zero values reported for the other complexes, where k0
could be most often neglected in the analysis of the kobs rate
constants.
Table 3. Luminescence Decay Lifetimes (τ) and Calculated
Hydration Numbers (q)
a
b
c
complex
τH2O (ms)
τD2O (ms)
q
q
[Eu(L1)]−
[Eu(L2)]−
[Eu(L3)]−
0.241
0.405
0.544
0.760
2.16
1.984
3.1
2.1
1.3
2.8
1.9
1.1
The dissociation half-life t1/2 was calculated using the
available rate constants for physiological conditions (pH 7.4
and 1 μM Cu2+ concentration). Among the complexes with a
rigidified ligand skeleton containing a pyridine (GdPY,
GdHYD), a cyclohexane (Gdcddadpa), or a cyclobutane
([Gd(L2)]−) in the ligand backbone, [Gd(L2)]− has the
shortest t1/2 and, thus, the lowest kinetic inertness. This is a
direct consequence of the importance of the spontaneous
dissociation pathway (k0) probably induced by the presence of
the cyclobutane ring, while not present at all for the three other
complexes (GdPy, GdHYD, and Gdcddadpa) with a rigidified
backbone. At pH 7.4, this spontaneous pathway represents
100% of the overall dissociation, and even at pH 4, the
spontaneous pathway is responsible for 80% of the overall rate.
The importance of spontaneous dissociation is very unusual in
general for lanthanide poly(aminocarboxylate) complexes. This
represents an unexpected effect of further increasing the steric
constraint in the ligand structure with respect to cyclohexane
or pyridine derivatives. In addition, the picolinate function also
likely contributes to reduce kinetic inertness, though at
physiological pH this effect has no consequence for [Gd-
(L2)]−. Such an influence of the picolinate was previously
reported for [Gd(octapa)]− as compared to [Gd(edta)]− (t1/2
= 0.15 vs 55 h, respectively; pH 7.4 and 1 μM Cu2+). To
explain this difference between [Gd(octapa)]− and [Gd-
(edta)]−, two factors have been evoked: (a) picolinates
increase the rate of the metal-ion-catalyzed dissociation
pathway,2h as a result of their higher denticity, which favors
the formation of the key dinuclear intermediate, GdLCu, in
Cu-assisted dissociation; (b) the protonated picolinate
complex has a significant kinetic activity in the decomplex-
ation. For [Gd(L2)]−, we could not detect the metal-assisted
pathway, but the high kinetic activity of the protonated
complex at lower pH is an important factor, as it is evidenced
by the high value of k2 (again, this has no effect at pH 7.4). For
the bis(picolinate) derivative [Gd(L3)]− complex, it was
impossible to derive dissociation rate constants and analyze the
a
Concentrations of complexes were 0.2 mM, 0.1 M Hepes buffer, pH,
b
pD = 7, 25 °C. The q values were obtained from eq 2 with A = 1.2
and B = 0.25. eq 2 with A = 1.11 and B = 0.31.
c
the absorption spectra of L2 and [Eu(L2)]−, as well as the
emission and excitation spectra of [Eu(L2)]− as an
example).17,18 All luminescence decay curves were mono-
exponential (Figure S8). The following empiric equation (eq
2) is used to calculate q from the differences of luminescence
decay lifetime in H2O and D2O, τH2O and τD2O, (A = 1.2 and B
= 0.2519 or A = 1.11 and B = 0.3120).
i
j
y
z
1
1
j
j
z
z
q = A
−
− B
j
j
j
z
z
z
τ
τ
H2O
D2O
(2)
k
{
As expected on the basis of ligand denticity, the hydration
numbers (q) are 3, 2, and 1 for [Eu(L1)]−, [Eu(L2)]−, and
[Eu(L3)]−, respectively. Figure 2 shows the structures for the
three hydrated Gd3+ complexes.
Lanthanide chelates containing more than one inner-sphere
water molecule are often prone to ternary complex formation
with endogenous anions, such as carbonate, phosphate, or
citrate.21−23 These anions replace the hydration water
molecules and lead to a drastic relaxivity decrease of the
Gd3+ complexes.24 It has been previously shown that, for
bis(hydrated) complexes, the inner-sphere structure and the
respective position of the two inner-sphere water molecules are
primordial to induce or to prevent ternary complex
formation.25 While GdDO3A easily undergoes ternary complex
formation, several bis(hydrated), linear complexes, such as
GdHYD, GdPY, etc., proved to be resistant.26
The formation of ternary complexes between [Gd(L1)]−,
[Gd(L2)]−, or[Gd(L3)]− and carbonate and phosphate, two
abundant physiological anions (22−29 and 1.12−1.45 mM
concentrations, respectively, in the blood), was studied by
measuring the luminescence lifetime of their related Eu3+
E
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Figure 2. Structures and q values for [Gd(L1)(H2O)3]−, [Gd(L2)(H2O)2]−, and [Gd(L3)(H2O)]− complexes. Charges are omitted for clarity.
complexes in H2O and D2O. These two anions can interact
differently with lanthanide complexes; phosphate has been
demonstrated to bind in a monodentate way, while carbonate
typically binds in a bidentate manner. The luminescence
emission decays of the Eu3+ complexes were recorded in H2O
and D2O, in the presence of 10 and 50 equiv of carbonate and
phosphate (50 equiv are above the physiological concen-
trations of this anion in human plasma), and the hydration
numbers were calculated according to eq 2 (Table 4).
2H2O system. The calculated structures of [Gd(L2)(H2O)2]−·
4H2O and [Gd(L3)(H2O)]−·2H2O (Figure 3) show hepta-
Table 4. q Values for [Eu(L1)]−, [Eu(L2)]−, and [Eu(L3)]−
in the Absence or in the Presence of 10 and 50 Equivalents
a
of Phosphate and Carbonate, Respectively
a
q
phosphate
carbonate
complex
[Eu(L1)]−
[Eu(L2)]−
[Eu(L3)]−
anion free [EuL]−
10 eq
50 eq
2.4
2.1
10 eq
50 eq
2.1
2.0
3.1
2.1
1.3
2.4
2.2
1.3
2.0
2.1
1.3
1.2
1.3
a
The q values were obtained from eq 2 with A = 1.2 and B = 0.25; 0.2
mM EuL, 0.1 M Hepes buffer, pH, pD = 7.4, 25 °C.
The q values of [Eu(L2)]− and [Eu(L3)]− did not decrease
with the addition of 10 and 50 equiv of phosphate or
carbonate, excluding the formation of ternary Eu3+ complexes
with these anions. In contrast, the number of water molecules
in [Eu(L1)]− complex decreased by 22% when phosphate was
added and up to 35% in the presence of carbonate (Figure S6
in the Supporting Information). These results suggest that
Figure 3. Calculated structure (a) and (c), and electrostatic potential
(b) and (d) of [Gd(L2)(H2O)2]− and [Gd(L3)(H2O)]−, respectively
Electrostatic potentials (hartree) are mapped on the molecular
surfaces defined by the 0.001 electrons·bohr−3 contour of the
electronic densities.
and octadentate coordination of the ligands to the metal ion, as
expected. Our calculations predict rather long Gd−N distances
involving the amine nitrogen atoms of the ligands, an effect
that is more striking in the case of the complex of (L3)4−
(Figure 3, Table 5). These Gd−N distances are considerably
longer than those calculated for the [Gd(cddadpa)(H2O)]−
complex (2.81 and 2.75 Å), reflecting a better accommodation
of the Gd3+ ion in the cavity of the latter. We also notice that
the stability of the [Lu(L3)(H2O)]− complex is 1 order of
magnitude lower than that of the Gd3+ analogue (Table 1).
This can be attributed to the larger bite angle imposed by the
cyclobutanediamine unit, as estimated from the N−Gd−N
angles of 69.1 and 65.7° calculated for the structurally related
[Gd(L3)(H2O)]− and [Gd(cddadpa)(H2O)]− complexes.
The analysis of the electrostatic potential calculated with
DFT on isodensity surfaces defined by a 0.001 a.u. contour of
the electron density28 provides additional valuable information
to rationalize the different dissociation kinetic profiles of
[Gd(L2)(H2O)2]− and [Gd(L3)(H2O)]−. As for other Ln3+
stability follows the trend [Eu(L3)]− ≈ [Eu(L2)]−
≫
[Eu(L1)]−. Moreover, this tendency is confirmed by the
time dependence of the luminescence intensity shown in
Figure S8.
DFT Calculations. DFT calculations were performed to
gain insight into the relation between molecular structure and
the thermodynamic and kinetic properties of [Gd(L2)]− and
[Gd(L3)]− complexes. On the grounds of previous studies, our
calculations included two explicit second-sphere water
molecules hydrogen-bonded to each of the coordinated
water molecules, while bulk solvent effects were introduced
by using a polarized continuum model (PCM). The use of
mixed cluster-PCM models is important to achieve a better
description of the solution structures of Gd3+ complexes with
polyamino polycarboxylate ligands, in particular, regarding the
Gd−Owater distances and the spin density at the O nuclei of the
water molecule.27 For the sake of comparison, we also
performed DFT calculations on the [Gd(cddadpa)(H2O)]−·
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Table 5. Bond Distances of the Gd3+ Coordination
Environments (Å) Obtained with DFT Calculations
This orientation of the inner-sphere water molecules,
together with the negative charge of the complex, is likely
responsible for the lack of binding of carbonate and phosphate
described above.
GdL2
GdL3
Gdcddadpa
NMRD and 17O NMR Studies. According to the Solomon-
Bloembergen-Morgan theory of paramagnetic relaxation, the
relaxivity is related to a number of microscopic parameters of
the paramagnetic chelate, which involve the number of
hydration water molecules, the water exchange rate, the
rotational dynamics of the complex, and its electron spin
relaxation. To describe these parameters, nuclear magnetic
relaxation dispersion (NMRD) profiles were recorded for
[Gd(L1)]−, [Gd(L2)]−, and [Gd(L3)]− complexes in the field
range of 0.01−80 MHz at 25, 37, and 50 °C. The NMRD
curves reflect the magnetic field dependency of the proton
relaxivity and are helpful to distinguish between different
relaxation mechanisms. The profiles of all three complexes
(Figure 4) have the shape typical of low molecular weight
chelates with a single dispersion between 1 and 10 MHz. The
relaxivity values decrease with increasing temperature, which is
consistent with fast rotation of the complex that limits the
relaxivity. The relaxivity values measured at 20 MHz and 25 °C
(Table 6) are in coherence with the size and the hydration
number of the chelates.
Gd1−N1
Gd1−N2
Gd1−N3
Gd1−N4
Gd1−O1
Gd1−O2
Gd1−O3
Gd1−O4
Gd1−O1w
Gd1−O2w
2.744
2.917
2.789
2.785
3.034
2.875
2.701
2.419
2.390
2.392
2.361
2.519
2.599
2.748
2.805
2.646
2.446
2.394
2.450
2.381
2.570
2.423
2.389
2.422
2.417
2.482
2.474
poly(aminocarboxylate) complexes,29 the surfaces of the
complexes present a hydrophilic region containing the
carboxylate groups and coordinated water molecules, and a
hydrophobic side containing the pyridyl rings and cyclobutane
rings. In the [Gd(L3)(H2O)]−·2H2O complex, three coordi-
nated carboxylate oxygen atoms are placed in the same region
of the complex surface, rendering a more negative electrostatic
potential than for the [Gd(L2)(H2O)]− complex (O1, O3, and
O4, Figure 3). This result is in agreement with the higher
protonation constant determined with potentiometric meas-
urements for [Gd(L3)(H2O)]− (KGdHL = 3.28) compared with
[Gd(L2)(H2O)2]− (log KGdHL = 2.36). Thus, the much faster
complex dissociation of [Gd(L3)(H2O)]− is likely related to
the tendency of this complex to protonate, which together with
the long Gd−N distances provides a low-energy path for
complex dissociation. Similarly, the lack of contribution of a
metal-assisted pathway to the dissociation of [Gd(L2)-
(H2O)2]− must be related to its rather open structure, which
yields a molecular surface with lower negative electrostatic
potential and thus a lower tendency to form the key dinuclear
intermediate.
Compared with other complexes described in the literature,
we can observe that the relaxivity of bis(hydrated) [Gd(L2)]−
is very similar to that of [Gd(CyPic3A)]− under the same
conditions; an identical trend is observed comparing
monohydrated complexes [Gd(L3)]− and [Gd(cddadpa)]−.
Both [Gd(L2)]− and [Gd(L3)]− present higher values for
relaxivity, almost twice that of [Gd(DTPA)]−, for instance.
The relaxivities were also measured in the presence of
physiological concentration (0.6 mM) of human serum
albumin (HSA) at 20 and 60 MHz, 37 °C. These values are
30−40% higher than those recorded in the absence of HSA
(see Supporting Information), indicating a weak binding to the
protein.
The two coordinated water molecules in [Gd(L2)(H2O)2]−·
4H2O present relatively similar calculated Gd−Owater distances
(2.482 and 2.474 Å, Figure 3). The ligand wraps around the
Gd3+ ion resulting in a set of five donor atoms of the ligand
arranged in a relatively planar fashion (O1, O3, N1, N2, and
N3), with one of the carboxylate ligands containing O2 and
O4 coordinating above and below this plane, respectively. The
two coordinated water molecules approach the metal ion from
different sides of the mean plane. As a result, the oxygen atoms
of these water molecules define a rather open O−Gd−O angle
of 88.4°.
The NMRD studies have been complemented by variable-
temperature 17O transverse relaxation rate and chemical shift
measurements, which allow, respectively, direct assessment of
the water exchange rate and estimation of the hydration
number. Figure 5 shows the variable-temperature, reduced
transverse 17O relaxation times and chemical shifts for the
three gadolinium complexes recorded at 54.2 MHz (9.4 T).
The luminescence lifetime measurements indicated 3, 2, and 1
inner-sphere water molecules for [Eu(L1)]−, [Eu(L2)]−, and
[Eu(L3)]−, respectively, and the 17O chemical shifts measured
on the Gd3+ analogues are in accordance with this. Indeed, the
Figure 4. NMRD profiles of 1.88 mM [Gd(L1)]−, 1.91 mM [Gd(L2)]−, and 2.13 mM [Gd(L3)]− in water at pH = 7 and temperatures of 25, 37,
and 50 °C. Curves represent the simultaneous fit as described in the text.
G
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Table 6. Relaxivity Values, r1 (mM−1 s−1), for [Gd(L1)]−, [Gd(L2)]−, and [Gd(L3)]−, and Related Gd3+ Complexes from the
Literature, at 25° C
9a
6
30
[Gd(L1)]−
[Gd(L2)]−
[Gd(L3)]−
[Gd(CyPic3A)]−
[Gd(cddadpa)]−
[Gd(DTPA)]−
a
b
r1
r1
q
8.8
8.1
3
7.9
7.4
2
5.8
5.5
1.2
8.3
7.9
2
5.6
4.3
1
1
a
b
20 MHz (0.47 T). 60 MHz (1.41 T).
Figure 5. Reduced transverse 17O NMR relaxation rates (top) and 17O NMR chemical shifts (bottom) of the following aqueous solutions: 15.3
mmol kg−1 [Gd(L1)]−, 14.5 mmol kg−1 [Gd(L2)]−, and 6.46 mmol kg−1 [Gd(L3)]− at pH = 6.5. Curves represent the simultaneous fit.
Table 7. Relaxivities (20 MHz, 25°C) and Parameters Obtained from the Simultaneous Fitting of 17O NMR and NMRD Data
for [Gd(L1)]−, [Gd(L2)]−, and [Gd(L3)]− and Literature Values for Related Complexes [Gd(HYD)]−,9b [Gd(CyPic3A)]−,9a
30
and [Gd(DTPA)]2−
9b
9a
30
[Gd(L1)]−
[Gd(L2)]−
[Gd(L3)]−
[Gd(HYD)]−
[Gd(CyPic3A)]−
[Gd(DTPA)]2−
kex (106 s−1
)
127 15
21.7 3.6
10.0 2.3
36.1 1.6
8.1 0.5
27.6 1.7
7.8
44
29.1
3.3
298
ΔH‡ (kJ mol−1
)
43.5
+33
92.6
2.1
21.0
−4.0
0.55
2
51.6
+53
58
ΔS‡ (J mol−1 K−1
)
−17
66
15
7
+10
92
4
−1
5
298
τRH (ps)
2
2
3
119
2
298
τV (ps)
2.6 0.5
20.1 1.3
−3.0 0.4
0.46 0.13
2
3.8 0.2
20.5 0.7
−3.1 0.3
0.40 0.02
1
ER (kJ mol−1
)
23.8 0.6
−3.1 0.2
0.44 0.07
3
17.3
−3.8
A/ℏ (106 rad s−1
)
Δ2 (1020 s−2
)
q
2
1
scalar coupling constants fitted for the three chelates are
approximately A/ℏ ≈ −(3.0−3.1) × 106 rad s−1, at the lower
limit of typical values reported for Gd3+ complexes. DFT
calculations performed on the [Gd(L2)(H2O)2]−·4H2O and
[Gd(L3)(H2O)]−·2H2O systems (see computational details
below) provide A/ℏ values of −3.8 × 106 and −2.7 × 106 rad
s−1, in reasonable agreement with the experimental data. This
also confirms the hydration number of the complexes
determined by luminescence lifetime measurements. The A/
ℏ determined for [Gd(L3)(H2O)]−·2H2O is somewhat higher
than those determined for [Gd(octapa)(H2O)]− using 17O
NMR measurements (A/ℏ = −2.3 × 106 rad s−1)2i and DFT
calculations (A/ℏ = −2.5 × 106 rad s−1).31 The relatively low
values of A/ℏ determined for these series of complexes might
be related to a rather efficient delocalization of the spin density
through the aromatic picolinate units.
have different water exchange rates. While the tris(hydrated)
[Gd(L1)]− is in the fast exchange regime in the entire
temperature range (1/T2r increases with decreasing temper-
ature), [Gd(L2)]− and [Gd(L3)]− are in the fast exchange
regime at high temperatures and in an intermediate range at
lower temperatures. The temperature dependency of the
chemical shifts follows the same trend. The transverse
relaxation rate of the coordinated water oxygen, 1/T2m,
determines 1/T2r in the fast exchange region. In turn, 1/T2m
is influenced by the following parameters: the water exchange
rate kex, the longitudinal electronic relaxation rate 1/T1e, and
the scalar coupling constant A/ℏ.
The reduced transverse 17O relaxation rates and chemical
shifts were fitted together with the NMRD profiles according
to the SBM theory. The following parameters were calculated:
the water exchange rate kex298, the activation enthalpy ΔH‡ and
entropy ΔS‡, the scalar coupling constant A/ℏ, the rotational
correlation time τRH, its activation energy ER, and the
The difference in the temperature dependence of the
transverse 17O relaxation rates shows that the three complexes
H
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parameters referring to electron spin relaxation, namely, Δ2
and τv298. The distances between the Gd3+ ion and the protons
in the inner and outer coordination sphere were fixed to typical
values, rGdH = 3.1 Å and aGdH = 3.6 Å, respectively, and the
diffusion coefficient and its activation energy were fixed to
DGdH298 = 26 × 10−10 m2 s−1 and EDGdH = 20 kJ mol−1. Reliable
information on dynamic processes, like water exchange and
rotational correlation times for small complexes, can be
obtained by application of the SBM approach to the analysis
of the NMRD data at medium and high magnetic fields,
provided that detailed information about electron spin
relaxation is not required.32 Consequently, only relaxivity
values above 6 MHz were included in the fitting. The number
of water molecules q directly coordinated to Gd3+ was fixed to
3, 2, and 1 for [Gd(L1)]−, [Gd(L2)]−, and [Gd(L3)]−,
respectively.
forms a stable, monohydrated complex with Gd3+, which is
very labile. In contrast, the complex with the heptadentate
(L2)4− ligand presents a much higher kinetic inertness together
with a rather high relaxivity associated with the presence of two
coordinated water molecules. While the inertness of the
complex is not good enough to conceive any in vivo
application as an MRI contrast agent, this complex represents
a rare case in which lowering ligand denticity causes a
noticeable increase in kinetic inertness.34 A structural analysis
suggests that the large bite angle of the (1S,2S)-1,2-
cyclobutanediamine spacer can make ligands based on this
scaffold more suitable for the coordination of bulkier metal
ions. Future work will expand the family of ligands presented
here and will explore their coordination properties toward
other metal ions with relevant biomedical applications.
The equations used are given in the Supporting Information,
and the parameters obtained are shown in Table 7.
EXPERIMENTAL SECTION
■
Tetra(tert-butyl) 2,2′,2″,2′′′-[{(1S,2S)-cyclobutane-1,2-diyl}-
bis(azanediyl)]tetraacetate (3). To a solution of 2 (65.6 mg, 0.75
mmol), prepared according to ref 10, potassium iodide (480 mg, 2.89
mmol, 3.85 equiv), and diisopropylethylamine (1.08 mL, 6.2 mmol,
8.3 equiv) in dimethylformamide (DMF) (2 mL) was added tert-butyl
bromoacetate (0.49 mL, 3.3 mmol, 4.4 equiv), and the reaction
mixture was stirred at room temperature for 18 h under N2
atmosphere. Then, the solution was diluted with CH2Cl2 (20 mL)
and washed with saturated K2CO3 (2 × 5 mL) and brine (1 × 5 mL).
The organic layer was dried over MgSO4, filtered, and evaporated
under reduced pressure. Purification by column chromatography (1:3
ethyl acetate (EtOAc)/hexane) afforded 3 (288 mg, 0.53 mmol, 70%
While there is a slight increase in the water exchange rate
from [Gd(L3)]− to [Gd(L2)]− (kex298 = 8.1 × 106 and 10.0 ×
106 s−1, respectively), [Gd(L1)]− has 1 order of magnitude
faster water exchange (kex298 = 127 × 106 s−1), which is only 6
times lower than that of the aqua ion [Gd(H2O)8]3+ ion (800
× 106 s−1). The reason for this very fast exchange is likely the
high flexibility of the inner coordination sphere around the
binding site of the three hydration water molecules, despite the
rigid ligand structure. In addition, the negative value of the
activation entropy points to an associatively activated
mechanism for [Gd(L1)]−, which means that the incoming
water molecule has also a role in the rate-determining step. On
the one hand, while only the determination of activation
volumes via variable-pressure 17O NMR measurements would
allow for a precise assessment of the water exchange
mechanism, activation entropies also provide a hint.33 On
the other hand, [Gd(L2)]− and [Gd(L3)]− are characterized
by an interchange or slightly dissociatively activated water
exchange mechanism (the activation entropy is close to zero or
has a small positive value), in contrast to the high positive ΔS‡
value for [Gd(DTPA)]2−, clearly indicating a dissociative
mechanism.
The calculated values of the rotational correlation time,
τRH298, were 66, 92, and 119 ps, for [Gd(L1)]−, [Gd(L2)]−,
and [Gd(L3)]−, respectively, in accordance with their
increasing size. The small differences in the relaxivity of the
three complexes can be related to the opposing effects of the
decreasing hydration number and the increasing size, hence,
rotational correlation time in the order of [GdL1]−, [Gd-
(L2)]−, and [Gd(L3)]−. The water exchange rate has no
influence on relaxivity for such small complexes; their relaxivity
is only limited by fast rotation.
yield) as a yellow oil. [α]2D0: + 8 (c = 1.0, CH3OH); H NMR (360
1
t
MHz, CDCl3): δ 1.44 (s, 28H, Bu, H3, H4), 1.79 (m, 2H, H3′, H4′),
3.32 (m, 2H, H1, H2), 3.41 (m, 1H, H5), 3.46 (m, 3H, H5), 3.49 (m,
3H, H5), 3.54 (m, 1H, H5); 13C NMR (90 MHz, CDCl3): δ 20.3 (C3,
C4), 28.1 (tBu), 52.9 (C5), 62.9 (C1, C2), 80.6 (C-tBu), 171.1 (CO).
IR (ATR): ν 2978, 2931, 1729 cm−1; HRMS (electrospray ionization
(ESI)) m/z calcd for C28H51N2O8 [M + H]+: 543.3640. Found:
543.3647.
2,2′,2″,2′′′-[{(1S,2S)-Cyclobutane-1,2-diyl}bis(azanediyl)]-
tetraacetic acid (H4cbdta, H4(L1)). A solution of compound 3 (180
mg, 0.33 mmol) in 4 M HCl in dioxane (12 mL) was stirred at room
temperature for 18 h. Then, the solvent was evaporated under
reduced pressure, and a small amount of water (2 mL) was added, and
the mixture was evaporated to dryness. This process was repeated
once with the addition of water and twice with an addition of diethyl
ether (2 mL) to afford the desired ligand (110 mg, 0.25 mmol, 77%
1
yield) as a yellow solid. H NMR (600 MHz, D2O): δ 1.67 (m, 2H,
H3, H4), 1.97 (m, 2H, H3, H4), 3.88 (m, 8H, H5), 3.94 (m, 2H, H1,
H2); 13C NMR (150 MHz, D2O): δ 18.1 (C3, C4), 52.5 (C5), 61.7
(C1, C2), 171.2 (CO). HRMS(ESI) m/z calcd for C12H18N2O8Na [M
+ Na]+: 341.0961. Found: 341.0962.
Methyl 6-[{(1S,2S)-cyclobutane-1,2-diylbis[(2-tert-
butoxycarbonyl)azanediyl]} (methylene)]picolinate (6). Alde-
hyde 5 (0.124 g, 0.75 mmol, 1 equiv), prepared according to ref 2h,
was added to a solution of 410 (140 mg, 0.75 mmol) in CH3OH (5
mL), and the reaction mixture was stirred at room temperature for 2.5
h. Small aliquots of this reaction were removed and concentrated to
dryness for NMR analysis to confirm full Schiff base formation. Then,
the reaction was diluted with CH3OH (5 mL) and cooled to 0 °C,
and then NaBH4 (31 mg, 0.81 mmol) was added. After it was stirred
for 2 h at 0 °C, the reaction was quenched with saturated (satd)
NaHCO3 and extracted with CH2Cl2. The organic layer was dried
over MgSO4, filtered, and evaporated under reduced pressure to
afford 6 (218 mg, 0.65 mmol, 87% yield) as a yellow oil. [α]2D0: +5 (c
CONCLUSIONS
■
In this work, we have described efficient syntheses of three
different ligands containing a rigid (1S,2S)-1,2-cyclobutanedi-
amine spacer and a different number of acetate and picolinate
groups. We have shown that this versatile spacer can be easily
functionalized with different coordinating groups, providing a
new structural entry for ligand design to coordination chemists.
We expected that the rigid nature of the spacer could provide
Gd3+ complexes with high kinetic inertness. Although detailed
dissociation kinetic data could not be obtained for [Gd(L3)]−,
thermodynamic and luminescence studies as well as computa-
tional calculations suggest that the octadentate ligand (L3)4−
1
t
= 1.0, CH3OH); H NMR (400 MHz, CDCl3): δ 1.42 (s, 11H, Bu,
H3, H4), 1.95 (m, 1H), 2.12 (m, 1H), 2.87 (br s, 2H, NH), 3.15 (m,
1H), 3.79 (m, 1H), 3.98 (s, 3H, Me), 4.01 (m, 2H, H5), 7.55 (m,
1H), 7.78 (m, 1H), 7.97 (m, 1H); 13C NMR (90 MHz, CDCl3): δ
23.3, 23.5, 28.3, 52.2, 52.8, 61.5, 64.5, 77.2, 123.7, 125.7, 137.6, 146.9,
I
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154.9, 160.3, 165.5; IR (attenuated total reflectance (ATR)): ν 3117,
2975, 1687 cm−1; HRMS(ESI) m/z calcd for C17H25N3O4Na [M +
Na]+: 358.1737. Found: 358.1724.
HRMS(ESI): m/z calcd for C24H24N2O6Na [M + Na]+: 377.1472.
Found: 377.1466.
Di-tert-butyl 2,2′-[(1S,2S)-cyclobutane-1,2-diyl)bis-
(benzyloxycarbonylazanediyl)] diacetate (9). To a solution of
anhydrous THF (8 mL) containing previously washed 60% NaH in
mineral oil (280 mg, 7 mmol, 10 equiv), TBAI (1.55 g, 4.20 mmol, 6
equiv) was added under nitrogen atmosphere. At the same time, a
solution of anhydrous THF (10 mL) containing diprotected amine 8
(250 mg, 0.70 mmol) under nitrogen atmosphere was prepared. After
that, the second solution was added using a cannula connected to the
first one. Finally, tert-butyl bromoacetate (0.620 mL, 4.20 mmol, 6
equiv) was added, and the mixture was stirred at room temperature
for 24 h (reaction was monitored by TLC). Then, the reaction was
quenched by adding 10 mL of water, and THF was removed under
vacuum. Next, more water was added (10 mL), and the crude was
extracted with dichloromethane (3 × 30 mL). The organic layer was
dried over magnesium sulfate, and the solvent was removed under
vacuum. The residue was purified by column chromatography (3:1
hexane−EtOAc) to afford dialkylated diamine 9 (250 mg, 62% yield)
as a brown oil along with the corresponding monoalkylated product
(64 mg, 21% yield) that was submitted to further alkylation under
Methyl 6-[{(1S,2S)-cyclobutane-1,2-diyltris[(2-tert-butoxy-2-
oxoethyl)azanediyl]} (methylene)] picolinate (7). Diamine 6
(218 mg, 0.65 mmol) was dissolved in CH2Cl2 (5 mL). Then, a
solution of 1 M HCl in EtOAc (11.25 mL, 11.25 mmol, 15 equiv) was
added, and the reaction was stirred at rt for 4 h. The solvent was
evaporated under reduced pressure. Then, the crude product was
dissolved in CH2Cl2 (20 mL) and stirred over an excess of K2CO3
(0.83 g, 6 mmol). After 2 h, the solution was filtered and evaporated.
The slurry containing product and K2CO3 could be carried directly
through to the next step (assuming 100% deprotected amine). Then,
the mixture was dissolved in DMF (2 mL) under N2 atmosphere. KI
(0.312 g, 1.88 mmol, 2.89 equiv), DIPEA (0.71 mL, 4.10 mmol, 6.3
equiv), and tert-butyl bromoacetate (0.315 mL, 2.14 mmol, 3.3 equiv)
were added, and the reaction mixture was stirred at rt for 18 h. Then,
the solution was diluted with CH2Cl2 (20 mL) and washed with
saturated K2CO3 (2 × 5 mL) and brine (1 × 5 mL). The organic layer
was dried over MgSO4, filtered, and evaporated under reduced
pressure. Purification by column chromatography (1:3 to 1:1 mixtures
of EtOAc/hexane) affords 7 (263 mg, 0.455 mmol, 74% yield) as a
1
similar conditions. [α]D = +2.0 (c = 1.0 in MeOH); H NMR (400
t
yellow oil. [α]2D0: + 17 (c = 1.0, CH3OH); H NMR (400 MHz,
1
MHz, CDCl3) δ 1.27−1.52 (m, 18H, Bu), 1.52−1.73 (m, 2H, H3,
t
H4), 1.94−2.13 (m, 2H, H3′, H4′), 3.68−4.18 (m, 4H, H5), 4.37−4.60
(m, 2H, H1, H2), 5.12 (m, 4H, CH2−Ph), 7.33 (m, 10H, Ar); IR
CDCl3): δ 1.43 (m, 29H, Bu, H3, H4), 1.82 (m, 2H, H3, H4), 3.32
(m, 3H), 3.48 (m, 5H), 3.99 (s, 3H, Me), 4.02 (m, 1H, H6), 4.09 (m,
1H, H6′), 7.80 (t, 1H, J = 7.6 Hz, H8), 7.91 (d, 1H, J = 7.6 Hz), 7.98
(d, 1H, J = 7.5 Hz); 13C NMR (100 MHz, CDCl3): δ 18.6, 20.5, 28.0,
28.1, 52.7, 54.1, 56.8, 61.8, 63.3, 80.6, 123.4, 126.1, 137.2, 147.0,
161.2, 165.9, 170.9; IR (ATR): ν 2978, 1722 cm−1; HRMS(ESI) m/z
calcd for C30H47N3O8Na [M + Na]+: 600.3255. Found: 600.3267.
6-[{(1S,2S)-Cyclobutane-1,2-diyltris[(carboxymethyl)-
azanediyl]}(methylene)] picolinic acid (H4 cbddapa, H4(L2)). A
solution of compound 7 (180 mg, 0.31 mmol) was dissolved in THF/
H2O (1:1, 5 mL). Then LiOH (0.052 g, 1.25 mmol, 4 equiv) was
added, and the reaction mixture was subsequently stirred at room
temperature for 4 h and concentrated to dryness under reduced
pressure. The resultant residue was dissolved in 4 M HCl in dioxane
(8 mL) and stirred at room temperature for 18 h. The solvent was
evaporated under reduced pressure. A small amount of water (2 mL)
was added, and the mixture was evaporated to dryness. This process
was repeated once with the addition of water and twice with an
addition of diethyl ether (2 mL) to afford the desired ligand (110 mg,
0.25 mmol, 77% yield) as a yellow solid. 1H NMR (600 MHz, D2O):
δ 1.75 (m, 2H, H3, H4), 2.06 (m, 2H, H3′, H4′), 3.76 (m, 1H), 3.75
(m, 1H), 3.83 (m, 1H), 4.00 (m, 4H), 4.16 (m, 1H), 4.32 (m, 2H,
H6), 8.01 (d, 1H, J = 7.7 Hz), 8.28 (d, 1H, J = 6.2 Hz), 8.46 (t, 1H, J
= 7.6 Hz, H8). 13C NMR (150 MHz, D2O): δ 17.3, 18.2, 52.0, 53.1,
53.4, 60.6, 62.3, 126.2, 129.0, 146.6, 154.0, 163.0, 168.7, 174.25.
HRMS (ESI) m/z calcd for C17H21N3O8Na [M + Na]+: 418.1226.
Found: 418.1221.
Dibenzyl [(1S,2S)-cyclobutane-1,2-diyl]dicarbamate (8). To
an ice-cooled solution of 110 (0.160 g, 0.73 mmol) in water (30 mL)
and acetone (4 mL), NaHCO3 (0.120 g, 1.45 mmol, 2 equiv) and
Na2CO3 (0.230 g, 2.20 mmol, 3 equiv) were added. The mixture was
stirred until the complete dissolution of the carbonates. Then, benzyl
chloroformate (0.2 mL, 1.20 mmol, 1.6 equiv) was added, and the
mixture was stirred at 0 °C (reaction was monitored by thin-layer
chromatography (TLC)). After 18 h, the reaction was extracted with
EtOAc (4 × 50 mL), and the organic layer was dried over magnesium
sulfate. The solvent was removed under vacuum, and the excess of
benzyl chloroformate was lyophilized. The residue was purified by
column chromatography (2:1 hexane−EtOAc) to afford diprotected
amine 8 (0.155 g, 0.44 mmol, 60% yield) as a white solid. mp 70−73
°C (EtOAc); [α]D = −10.0 (c = 1.0 in MeOH); 1H NMR (400 MHz,
CDCl3): δ 1.54 (m, 2H, H3, H4), 2.17 (m, 2H, H3′, H4′), 3.93 (m, 2H,
H1, H2), 5.11 (m, CH2−Ph), 5.19 (br. 2H, NH), 7.37 (s, 10H, Ar);
13C NMR (100 MHz, CDCl3): δ 23.5 (C3, C4), 53.4 (C1, C2), 66.7
(ATR): ν 2978 (CHst), 1743 (CO), 1709 (CO) cm−1
;
HRMS(ESI):m/z calcd for C24H24N2O6Na [M + Na]+: 605.2833.
Found: 605.2830.
Dimethyl 6,6′-[{(1S,2S)-cyclobutane-1,2-diylbis[(2-tert-bu-
toxy-2-oxoethyl)azane-diyl]}bis(methylene)] dipicolinate
(11). Dialkylated diamine 9 (230 mg, 0.73 mmol), KI (365 mg,
2.20 mmol, 1.5 equiv), and methyl 6-(chloromethyl)picolinate, 10,35
(300 mg, 1.60 mmol, 1.1 equiv) were dissolved in anhydrous DMF
(10 mL) under nitrogen atmosphere. After that, DIPEA (0.820 mL,
4.70 mmol, 3.2 equiv) was added, and the reaction was stirred at
room temperature for 30 h. Then, EtOAc (30 mL) was added, and
washes with saturated NaHCO3 (3 × 20 mL), brine (3 × 20 mL), and
water (1 × 20 mL) were performed. The final organic layer was dried
over magnesium sulfate, and the solvent was removed under vacuum.
The residue was purified by column chromatography over silica gel
with a gradient of solvents (3:1 to 1:1 mixtures of hexane−EtOAc) to
afford 11 (270 mg, 0.42 mmol, 57% yield) as a brown oil. [α]D
=
1
+18.0 (c = 1.0 in MeOH); H NMR (400 MHz, CDCl3): δ 1.39 (s,
t
18H, Bu), 1.41−1.50 (m, 2H, H4R, H3S), 1.73−1.89 (m, 2H, H4S,
H3R), 3.25 (s, 4H, H5), 3.41 (m, 2H, H1, H2), 3.98 (s, 6H, Me), 4.05
(s, 4H, H6), 7.73 (m, 2H, Ar), 7.86 (m, 2H, Ar), 7.96 (m, 2H, Ar);
13C NMR (100 MHz, CDCl3): δ 19.3 (C3, C4), 28.1 (CH3-tBu), 52.8
(CH3), 53.8 (C5), 57.0 (C6), 62.6 (C1, C2), 80.8 (C-tBu), 123.4 (Ar),
126.2 (Ar), 137.2 (Ar), 147.1 (Ar), 161.3 (Ar), 165.9 (CO), 170.8
(CO); IR (ATR): ν 2977 (CHst), 2951 (CHst), 1721 (CO), 1589
(CO) cm−1; HRMS (ESI): m/z calcd for C24H24N2O6Na [M +
Na]+: 613.3232. Found: 613.3231.
6,6′-[{(1S,2S)-Cyclobutane-1,2-diylbis[(carboxymethyl)-
azanediyl]}bis(methylene)] dipicolinic Acid (H4cbddadpa,
H4(L3)). Compound 11 (150 mg, 0.245 mmol) was dissolved in
THF/H2O (1:1, 5 mL), LiOH (31.0 mg, 0.740 mmol, 3 equiv) was
added, and the reaction mixture was stirred at room temperature for 4
h. Then, the mixture was concentrated to dryness under reduced
pressure, and the resultant residue was dissolved in 4 M HCl in
dioxane (3 mL) and stirred at room temperature for 18 h. Then, the
solvent was evaporated under reduced pressure. A small amount of
water (3 mL) was added, and the mixture was evaporated to dryness.
This process was repeated twice with water and twice with the
addition of diethyl ether (3 mL) to afford the desired ligand (100 mg,
0.184 mmol, 75% yield) as a brown solid. [α]D = +36.0 (c = 1.0 in
1
H2O); H NMR (400 MHz, D2O): δ 1.72−1.82 (m, 2H, H4R, H3S),
2.04−2.17 (m, 2H, H4S, H3R), 3.71−3.91 (m, 4H, H5), 4.16 (m, 2H,
H1, H2), 4.46 (s, 4H, H6), 7.76 (m, 2H, Ar), 8.04 (m, 2H, Ar), 8.17
(m, 2H, Ar); 13C NMR (100 MHz, D2O): δ 17.7 (C3, C4), 52.1 (C5),
55.1 (C6), 51.5 (C1, C2), 125.9 (Ar), 128.7 (Ar), 143.8 (Ar), 144.2
(CH2−Ph), 128.1 (Ar), 128.5 (Ar), 136.3 (Ar), 155.6 (CO); IR
(ATR): ν 3306 (NHst), 2975 (CHst), 1682 (CO) cm−1
;
J
DOI: 10.1021/acs.inorgchem.9b02044
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(Ar), 152.0 (Ar), 164.1 (CO), 171.0 (CO); IR (ATR): ν 3377
(OHst), 2945 (CHst), 1720 (CO), 1616 (CO) cm−1; HRMS
(ESI): m/z calcd for C24H24N2O6Na [M + Na]+: 495.1486. Found:
495.1478.
Sample Preparation of the Metal Complexes. The ligand
concentrations were determined based on pH-potentiometric titration
curves. Gd3+, Zn2+, and Eu3+ (for luminescence lifetimes) concen-
trations were determined by titrating the metal solutions with
standardized Na2H2edta in urotropine buffer (pH 5.6−5.8) in the
presence of xylenol orange as an indicator. The GdL complexes were
prepared by mixing the ligand and the metal and adjusting the pH to
7.
Meiboom-Gill spin−echo technique. To eliminate susceptibility
corrections to the chemical shifts, the sample was placed in a glass
sphere fixed in a 10 mm NMR tube. To improve sensitivity, H217O
(10% H217O, CortecNet) was added to achieve ∼1% 17O content in
the sample. The pH of the samples was 6.5, and the GdL complex
concentrations were the following: 15.3 mmol/kg ([Gd(L1)]−), 13.7
mmol/kg ([Gd(L1)]−), and 6.46 mmol/kg ([Gd(L1)]−). To avoid
any free Gd3+, some ligand excess was used (6% L1, 5% L2, and 5%
L3).
DFT Calculations. All calculations presented in this work were
performed employing DFT within the hybrid meta-generalized
gradient approximation (hybrid meta-GGA), with the TPSSh
exchange-correlation functional.38 Geometry optimizations were
conducted with the Gaussian0939 program package using the large-
core approximation with the quasirelativistic effective core potential
proposed by Dolg et al.40 and the associated [5s4p3d]-GTO valence
basis set for Gd. The Standard 6-311G(d,p) basis set was used for all
other atoms. Bulk water solvent effects were included by using the
integral equation formalism variant of the polarizable continuum
model (IEFPCM),41 using the universal force field (UFF)42 radii
scaled by a factor of 1.1 to construct the solute cavity. Analytical
second derivatives were calculated to confirm that the optimized
geometries correspond to local energy minima on the potential energy
surface.
Hyperfine coupling constants A/ℏ were computed using the all-
electron calculations with the second-order Douglas-Kroll-Hess
(DKH2) method43 as implemented in ORCA (release 4.0.1.2).44
The basis sets used for these calculations included the SARC2-DKH-
QZVP basis set for Gd45 and the DKH-def2-TZVP basis set for all
other atoms.46 The RIJCOSX approximation47 was employed to
accelerate the calculations using the SARC2-DKH-QZVP/JK auxiliary
basis sets for Gd and auxiliary basis sets for all other atoms generated
automatically by ORCA using AutoAux procedure.48 Solvent effects
were introduced with the universal solvation model based on solute
electron density and on a continuum model (SMD).49
Protonation constants of ligands, stability constants of complexes,
and protonation constants of complexes are described and defined in
eqs 3−5.
[HiL]
Ki =
[Hi−1L][H+]
(3)
[ML]
[M][L]
KML
=
(4)
[M(HiL)]
[M(Hi−1L)][H+]
KMH L
=
i
(5)
where [M], [L], and [ML] are the equilibrium concentrations of free
metal ion, deprotonated ligand, and deprotonated complex,
respectively. Experimental data were refined using the computer
software Hyperquad 2008.36 Species distribution plots were calculated
taking the experimental constants using the computer software
HySS.37 The ionic product of water used at 25 °C was pKw = 13.77,
while the ionic strength was kept at 0.1 M. Fixed values were used for
pKw and total concentrations of metal, ligand, and acid.
Kinetic Measurements. The rates of the metal exchange
reactions of [Gd(L2)]− were studied by following the formation of
[Cu(L2)]− using conventional UV−vis spectrophotometry. The
exchange reactions were followed at 245 nm in the pH range of
3.35−4.90. The concentration of the complex was 0.11 mM, while
Cu2+ ion was added at high excess (10−40 equiv) to ensure pseudo-
first-order conditions. The temperature of the samples was kept at 25
°C, and the ionic strength of the solutions was kept constant by using
0.15 M NaCl. To keep the pH constant, 50 mM methylpiperazine
buffer was used. The pseudo-first-order rate constants (kobs) were
calculated by fitting the absorbance versus time data to the
monoexponential function (eq 6).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b02044.
1H and 13C NMR spectra of the new products, HRMS
spectra of the gadolinium complexes, potentiometry
studies, details on kinetic inertness studies, anion
binding studies, luminescence studies, equations used
for the analysis of the 17O and NMRD data, references,
optimized Cartesian coordinates of the complexes
obtained with DFT calculations (PDF)
obst
At = (A0 − Ae)e−k + Ae
(6)
where A0, At, and Ae are the absorbance at time = 0 s, at time t, and at
equilibrium, respectively. The fittings were performed with Origin 9.1
software by using standard least-squares procedure.
1
Relaxometric Measurements. H NMRD profiles of aqueous
1.88 mM [Gd(L1)]−, 1.91 mM [Gd(L2)]−, and 2.13 mM [Gd(L3)]−
solutions (pH = 7) were measured at 25, 37, and 50 °C on a Stelar
SMARTracer Fast Field Cycling NMR relaxometer (0.00024−0.24 T,
0.01−10 MHz 1H Larmor frequency) and a Bruker WP80 NMR
electromagnet adapted to variable-field measurements (0.47−1.88 T,
20−80 MHz), controlled by the SMARTracer PC-NMR console. The
temperature was controlled by a VTC91 temperature control unit and
maintained by a gas flow. The temperature was determined according
to previous calibration with a Pt resistance temperature probe.
To avoid any free Gd3+, some ligand excess was used (6% L1, 5%
L2, and 5% L3).
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: carlos.platas.iglesias@udc.es. (C.P.-I.)
́
*E-mail: eva.jakabtoth@cnrs.fr. (E.T.)
*E-mail: rosa.ortuno@uab.es. (R.M.O.)
ORCID
Rosa M. Ortuno: 0000-0001-7635-7354
̃
Notes
The authors declare no competing financial interest.
17O NMR Studies. Variable-temperature 17O NMR measurements
of aqueous solutions of GdL complexes were performed on a Bruker
Advanced 400 MHz spectrometer using a 10 mm broad band fluorine
observation (BBFO) probe (9.4 T, 54.2 MHz) in the temperature
range of 1−75 °C. The temperature was calculated according to
published calibration routines with ethylene glycol and MeOH.
Acidified water (HClO4, pH 3.3) was used as diamagnetic reference.
Transverse 17O relaxation times were obtained by the Carl-Purcell-
ACKNOWLEDGMENTS
O.P.-T. and J.A.O. thank Univ. Autonoma de Barcelona for
predoctoral fellowships and O.P.-T. also for a travel grant.
Financial support from Spanish Ministerio de Economıa y
Competitividad (Grant No. CTQ2016-77978-R, AEI/FEDER,
UE) is gratefully acknowledged. Authors thank A. Martınez-
■
̀
́
́
K
DOI: 10.1021/acs.inorgchem.9b02044
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
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́
́
́
́
́
T.; Kalman, F. K.; Esteban-Gomez, D.; Toth, E.; Platas-Iglesias, C.
Approaching the Kinetic Inertness of Macrocyclic Gadolinium(III)-
Based MRI Contrast Agents with Highly Rigid Open-Chain
Derivatives. Chem. - Eur. J. 2016, 22, 896−901.
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Supercomputacion de Galicia for providing the supercomput-
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