Monopicolinate-dipicolyl derivative of triazacyclononane for stable complexation of Cu2+ and 64Cu2+

Article abstract of DOI:10.1021/ic400174r

The synthesis and characterization of Hno1pa2py, a new tacn-based ligand, is reported. The complexation process with Cu²⁺ was proved to be very fast even in acidic medium. Potentiometric titrations allowed us to establish that Hno1pa2py exhibits an overall low basicity as well as a high selectivity for Cu²⁺ over Zn²⁺ cations. The copper(II) complex was synthesized and characterized using UV-vis and EPR spectroscopies and density functional theory (DFT) calculations. The studies clearly showed that the [Cu(no1pa2py)]⁺ complex is present in solution as a mixture of two isomers in which the ligand is coordinated to the metal center using a N ₅O donor set with the metal center in a distorted octahedral geometry. The very high kinetic inertness of the [Cu(no1pa2py)]⁺ complex was demonstrated by using acid-assisted dissociation assays as well as cyclic voltammetry. Preliminary investigations of ⁶⁴Cu complexation were performed to validate the potential use of such chelating agent for further application in nuclear medicine. The X-ray crystal structures of copper(II) complexes of L1, the ester derivative of Hno1pa2py, have been determined.

Full text of DOI:10.1021/ic400174r

Article
pubs.acs.org/IC
Monopicolinate-dipicolyl Derivative of Triazacyclononane for Stable
64
Complexation of Cu2+ and Cu2+
Melissa Roger,† Luís M. P. Lima,‡ Mathieu Frindel,§,† Carlos Platas-Iglesias,*,∥ Jean-Francois Gestin,§
̧
Rita Delgado,*,‡ Veronique Patinec,† and Raphael Tripier*,†
́
̈
†Universite
29238 Brest Cedex 3, France
‡Instituto de Tecnologia Química e Biolog
§Institut de Recherche Ther
apeutique IRT, UN Unite
Nantes-Angers, 8 quai Moncousu, BP70721 44007 Nantes Cedex, France
∥Departamento de Química Fundamental, Universidade da Coruna, Campus da Zapateira, Rua da Fraga 10, 15008 A Coruna, Spain
́
de Bretagne Occidentale, UMR-CNRS 6521, UFR des Sciences et Techniques, 6 avenue Victor le Gorgeu, C.S. 93837,
́
ica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras, Portugal
́
́
́ ́
INSERM 892-CNRS 6299, Centre de Recherche en Cancerologie
́
̃
̃
S
* Supporting Information
ABSTRACT: The synthesis and characterization of Hno1p-
a2py, a new tacn-based ligand, is reported. The complexation
process with Cu2+ was proved to be very fast even in acidic
medium. Potentiometric titrations allowed us to establish that
Hno1pa2py exhibits an overall low basicity as well as a high
selectivity for Cu2+ over Zn2+ cations. The copper(II) complex
was synthesized and characterized using UV−vis and EPR
spectroscopies and density functional theory (DFT) calculations.
The studies clearly showed that the [Cu(no1pa2py)]+ complex
is present in solution as a mixture of two isomers in which the
ligand is coordinated to the metal center using a N5O donor set
with the metal center in a distorted octahedral geometry. The
very high kinetic inertness of the [Cu(no1pa2py)]+ complex was
demonstrated by using acid-assisted dissociation assays as well as cyclic voltammetry. Preliminary investigations of 64Cu
complexation were performed to validate the potential use of such chelating agent for further application in nuclear medicine.
The X-ray crystal structures of copper(II) complexes of L1, the ester derivative of Hno1pa2py, have been determined.
which leads to the accumulation of 64Cu in organs.8 In order to
INTRODUCTION
■
overcome the lack of kinetic inertness of the tetraazamacrocy-
clic complexes, cage-like polyaza chelators such as bicyclic
hexaamines,9 dicarboxylic acid cross-bridged cyclen (H2cb-
do2a),10 cyclam (H2cb-te2a),11 and other derivatives12 were
subsequently developed (Chart 1). However, in spite of the
inertness of these complexes to the dissociation they exhibit
slow formation rates, which is a serious drawback for the aimed
biological applications. Introduction of picolinate arms on the
chelate framework has also been explored as a strategy for
improving thermodynamic stability and kinetic inertness.
Recently, the copper(II) complex of the noncyclic dipicolinate
diamine H2dedpa (also called H2bcpe) showed too low kinetic
and thermodynamic stability for in vivo applications.13,14
Nevertheless, the cyclic polyamines cyclen and cyclam bearing
one picolinate arm (Hdompa and Htempa, Chart 1)15 were
found to provide very attractive properties to their correspond-
ing copper(II) complexes, namely high thermodynamic
In the past years a great deal of research has been conducted to
develop radiopharmaceuticals for application in imaging and
therapy of tumors. Among the radioisotopes that can be
produced nowadays in high yield by a cyclotron is 64Cu (t1/2
12.7 h), which presents an interesting biologically viable half-
life decay and a low β+ energy suitable for positron emission
tomography (PET).1 Such characteristics have prompted the
design of a variety of 64Cu-based bifunctional chelating agents
(BCA) for imaging applications.2 A suitable BCA should
provide a fast complexation of 64Cu as well as a high
thermodynamic stability and kinetic inertness to avoid possible
transchelation and transmetalation in biological media.3
Moreover, bioreduction of Cu2+ to Cu+ constitutes an
additional possible pathway for the demetalation of the chelate
that should be considered in the design of an ideal BCA.4
The family of cyclic polyamines provides a wide range of
platforms useful for the design of progressively improved
BCAs.5 The easily accessible and well-known H4dota and
H4teta chelators proved their ability to form complexes of Cu2+
with very high thermodynamic stability (log KCuL > 21)6 but
presented limited kinetic inertness to avoid demetalation,7
Received: January 23, 2013
© XXXX American Chemical Society
A
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Chart 1. Structure of the Ligands Discussed in This Work
stability, kinetic inertness, fast complexation rates, and
electrochemical stability upon copper(II) reduction.
tion of either a nitrogen atom of the tacn unit or the carboxylic
acid group of the picolinate fragment.
Herein we report the synthesis and characterization of the
tacn-based ligand Hno1pa2py (Chart 1). The acid−base
properties of this ligand and the stability constants of its
complexes with Cu2+ and Zn2+ were determined. The complex
of Cu2+ was synthesized and characterized using cyclic
voltammetry, UV−vis and electron paramagnetic resonance
(EPR) spectroscopies, and density functional theory (DFT)
calculations. The kinetic stability of this complex was also
studied in acidic conditions, while preliminary 64Cu complex-
ation experiments were performed to validate the potential use
of such chelator for further applications in nuclear medicine.
Finally, structures of copper(II) complexes of the methyl ester
derivative of Hno1pa2py have been determined by X-ray
diffraction on single crystals obtained from solutions of the
complex at different pH values.
Trisubstituted triazamacrocyclic derivatives based on the
triazacyclononane (tacn) platform have also been applied in
copper(II) complexation studies, in particular those with acetic
acid (H3nota)16 or methylenephosphinic (H3trap, H3nopo)17,18
arms. It has been reported that a 64Cu- nota peptide conjugate
showed better in vivo stability than the 64Cu-dota homologue,19
offering an interesting way to explore development of new
BCAs. Recent studies with H3trap and H3nopo showed
neighboring properties to those of H3nota for 64Cu labeling.20
Another way to stabilize the complex of Cu2+ is by addition of
nitrogen donors, such as picolyl groups, as coordinating units. A
N5O coordinating triazacyclononane derivative (Hno1a2py),
bearing two picolyl and one acetic acid arms, showed capacity
to form a stable 64Cu complex with no evidence of
transchelation or demetalation.21 The bombesin bioconjugate
of its 64Cu complex also exhibited low activity accumulation in
the liver tissue and an extensive renal clearance.22 The
replacement of the carboxylate pendant arm by a picolinate
group on the previous N5O triazamacrocycle should enhance
the stability of its copper(II) complex. Indeed, azamacrocycles
with picolinate pendant arms have demonstrated their ability to
form stable complexes with different transition metal or
lanthanide cations.13−15,23 The new chelate should offer a N6
or N5O coordination mode to the metal center, thereby
providing the opportunity to prepare BCAs via functionaliza-
RESULTS AND DISCUSSION
■
Ligand Synthesis. The synthesis of the tacn derivative 1,4-
bis(pyridinemethyl)triazacyclononane (5) was achieved in five
steps involving the stepwise introduction of two picolyl groups
in the tacn unit (Scheme 1). The synthesis of 5 was already
described by two different groups with only slight modifica-
tions.22,24 In the present work a very similar approach to that
reported by Spiccia et al.22 was used, consisting of the
formation of an orthoamide intermediate 1 by reaction of tacn
B
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Scheme 1. Synthesis of Hno1pa2py
with N-dimethoxymethyl-N,N-dimethylamine. Addition of 2-
(chloromethyl)pyridine led to the corresponding ammonium
salt 2, whose neutral hydrolysis resulted in an intermediate with
only one secondary amine function available for N-alkylation
(3, Scheme 1). This intermediate was allowed to react with a
second equivalent of 2-(chloromethyl)pyridine to give 4,
followed by hydrolysis with 6 M HCl in methanol to give
compound 5 in 60% overall yield as calculated from tacn. This
yield represents a significant improvement when compared with
that reported by Spiccia et al. (46%), presumably because the
different intermediates shown in Scheme 1 were not isolated
and were used in the next step without further purification.
However, the strategy and yield obtained in this work are very
similar to those reported by Hasserodt et al. (59%),24 the only
difference being that 5 was obtained after basic, rather than
acid, hydrolysis of the formylated precursor. Reaction of
compound 5 with 6-chloromethylpyridine-2-carboxylic acid
methyl ester25 gave compound L1 with excellent yield (93%).
Finally, the hydrolysis of L1 with 6 M HCl led to Hno1pa2py
as a hydrochloride salt in 94% yield.
Figure 1. View of the crystal structure of [CuL1](ClO4)2·H2O (6).
Perchlorate anions, water molecules and hydrogen atoms are omitted
for clarity. The ORTEP plot is are at the 30% probability level.
Complexes of L1 with Cu2+ and Their X-ray Crystal
Structures. Attempts to obtain single crystals of the complex
of Cu2+ with Hno1pa2py suitable for X-ray diffraction analysis
were unsuccessful. However, single crystals of two complexes of
L1, which contains a methyl ester group instead of a
carboxylate function, were obtained. The complexes of L1
could actually be considered as a realistic model of any putative
conjugates where the carboxylate functional group is used for
conjugation with peptides/proteins. These crystals were
isolated after reaction of Hno1pa2py with Cu(ClO4)2·6H2O
in aqueous solutions at pH values 1.5 and 4.5, respectively,
followed by addition of methanol. A view of the structure of the
complex [CuL1](ClO4)2·H2O (6) obtained at pH 4.5 is shown
in Figure 1, while bond distances and angles of the metal
coordination environment are collected in Table 1. The
structure of complex [Cu(HL1)Cl](ClO4)2·H2O (7) obtained
at pH 1.5, together with bond distances, angles, and
description, is given in the Supporting Information (Figure
S1 and Tables S1, S2).
Crystals of compound [CuL1](ClO4)2·H2O (6) are
composed of the cationic entity [CuL1]2+, two perchlorate
anions, and a water molecule involved in hydrogen-bonding
interactions with the perchlorate anions. The metallic atom is
hexacoordinated, and the complex structure exhibits a distorted
octahedral geometry with the copper center coordinated to
three nitrogen atoms of the triazamacrocycle, two nitrogen
atoms from the picolyl substituents and the nitrogen atom of
C
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a
Table 1. Selected Bond Lengths (Å) and Angles (deg) of the Metal Coordination Environments in [CuL1](ClO4)2·H2O (6)
Cu(1)−N(1)
Cu(1)−N(2)
Cu(1)−N(3)
Cu(1)−N(4)
Cu(1)−N(5)
Cu(1)−N(6)
N(6)−Cu(1)−N(5)
N(6)−Cu(1)−N(3)
2.084(3)
2.235(3)
2.040(3)
2.504
N(5)−Cu(1)−N(3)
N(6)−Cu(1)−N(1)
N(5)−Cu(1)−N(1)
N(3)−Cu(1)−N(1)
N(6)−Cu(1)−N(2)
N(5)−Cu(1)−N(2)
N(3)−Cu(1)−N(2)
N(1)−Cu(1)−N(2)
164.87(12)
166.69(12)
97.03(12)
85.02(12)
101.56(12)
80.67(12)
84.78(12)
82.29(12)
2.009(3)
2.008(3)
96.17(12)
82.67(12)
a
See Figure 1 for labeling.
the picolinate ester group. The oxygen atom of the picolinate
ester group remains uncoordinated. The coordination poly-
hedron around the metal ion can be considered to be
composed of two nearly parallel planes: the nitrogen atoms
of the tacn unit (N1, N2, and N3) define the lower plane, while
the nitrogen atoms of the pyridyl units (N4, N5, and N6)
delineate the upper plane. The mean twist angle of the upper
and lower tripods amounts to 43.1°, which is evidence of an
important degree of distortion of the coordination polyhedron
from an octahedron (ideal value 60°) to a trigonal prism (ideal
value 0°). As a result of this distortion, several bond angles
(N1−Cu1−N2, 82.29°; N3−Cu1−N4, 97.91°; N2−Cu1−N4,
157.46°) differ significantly from those expected for an ideal
octahedral coordination. A smaller twist angle (26.6°) has been
reported for the [Cu(nota)]− complex,26 which is probably
related to the more important steric requirements of the
pendant arms of L1 compared to those of H3nota. The Cu−N
bond lengths are in the range 2.003−2.084 Å for N1, N3, N5,
and N6, and considerably longer for N2 (2.235 Å) and N4
(2.504 Å). A similar coordination environment has been
reported for [Cu(tacn3py)]2+.27
Table 2. Overall (βiH) and Stepwise (KiH) Protonation
Constants (in log Units) of Hno1pa2py, tacn, and H3nota at
298.2 0.1 K in 0.10 M KNO3
a
b
c,d
equilibrium reaction
L = no1pa2py−
log βiH
10.61(2)
L = tacn
L = nota3‑
c
d
d
L + H+ ⇄ HL
L + 2H+ ⇄ H2L
L + 3H+ ⇄ H3L
L + 4H+ ⇄ H4L
10.42
17.30
11.41; 11.73
c
15.86(5)
19.55(5)
21.16(9)
17.15; 17.47
c
d
d
<18.3
20.31; 20.63
c
22.02; 22.59
log KiH
c
d
L + H+ ⇄ HL
HL + H+ ⇄ H2L
H2L + H+ ⇄ H3L
H3L + H+ ⇄ H4L
10.61
10.42
6.88
11.41; 11.73
c
c
,
d
d
5.25
5.74
3.16
,
3.69
1.61
<1
c
d
1.71; 1.96
a
This work. Values in parentheses are standard deviations in the last
significant figures. I = 0.1 M KNO3.33 I = 0.1 M NaNO3.34 I = 0.1
b
c
d
M NaNO3.16a
The conformation adopted by the ligand in the [CuL1]2+
complex implies the occurrence of two helicities (one
belonging to the tacn moiety and the other associated with
the layout of the three pendant arms).28,29 Inspection of the
crystal structure data reveals that two Δ(λλλ) and Λ(δδδ)
enantiomers cocrystallize in equal amounts (racemate).
Identical conformations were observed in the X-ray structure
30
of [Cu(nota)]− and related complexes of Cu2+.31
Acid−Base Behavior of Hno1pa2py. The acid−base
reactions of the compound Hno1pa2py were studied in
aqueous solution and the protonation constants determined
by potentiometric titrations. The ligand has seven basic centers
(three tertiary amines of the tacn unit, three nitrogen atoms of
the pyridyl groups, and an oxygen atom of the carboxylate),
from which only four accurate constants could be determined
by potentiometric measurements (Table 2). The compound
exhibits low overall basicity because it has only one high
protonation constant, which corresponds to the protonation of
one amine in the macrocycle. The second one corresponds to
the protonation of another amine of the cycle, presenting a low
value for an amine protonation (log K = 5.25) due to the strong
electrostatic repulsions of charged amines placed at short
distance because of the small cavity size of the macrocycle. The
other two constants determined correspond to the protonation
of nitrogen atoms of the arms. Values of the same order were
found for other 9-membered macrocycles such as tacn32 and
H3nota3‑.16a,33 In the speciation diagram, shown in Figure 2, it
can be observed that the only species present in solution at
physiological pH is the monoprotonated one.
Figure 2. Speciation diagram of Hno1pa2py in aqueous solution for
CHno1pa2py = 10−3 M.
Studies of the Copper(II) and Zinc(II) Complexes with
Hno1pa2py. Determination of Stability Constants. The
stability constants of the complexes formed by Hno1pa2py
with Cu2+ and Zn2+ metal ions were determined using
potentiometric titrations at T = 298.2 K and in 0.10 M
KNO3 aqueous solutions (Table 3). The stability of the
complex formed with Zn2+ was investigated taking into account
that this cation exists in biological media in significant
concentrations and generally forms thermodynamically stable
complexes with ligands containing nitrogen and oxygen donor
atoms. The formation of complexes with both Cu2+ and Zn2+
was found to be fast, with almost no free metal ion present in
D
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Table 3. Overall (βM H L ) and stepwise (KM H L ) stability constants (in log units) of complexes of Hno1pa2py, tacn and H3nota
m
h
l
m
h l
with Cu2+ and Zn2+ cations, at 298.2 0.1 K in I = 0.10 0.01 M KNO3
a
b
d
,e
e,f
equilibrium reaction
L = no1pa2py‑
L = tacn
L = nota3‑
log βM
mHhLl
f
Cu2+ + L + H+ ⇄ CuHL
Cu2+ + L ⇄ CuL
Cu2+ + L ⇄ CuLOH + H+
Cu2+ + 2L ⇄ CuL2
23.67(3)
24.37
c
d
e
f
20.96(5)
15.52; 15.4
21.63
d
9.65(4)
9.97
e
27.4
Zn2+ + L + H+ ⇄ ZnHL
19.0(1)
16.49(6)
5.30(7)
d
e
e
Zn2+ + L ⇄ ZnL
11.62; 11.3
18.3
Zn2+ + L ⇄ ZnLOH + H+
Zn2+ + 2L ⇄ ZnL2
e
20.5
log KM
CuL + H+ ⇄ CuHL
Cu2+ + L ⇄ CuL
CuL + L ⇄ CuL2
CuLOH + H+ ⇄ CuL
ZnL + H+ ⇄ ZnHL
Zn2+ + H+ ⇄ ZnHL
ZnLOH + H+ ⇄ ZnL
Zn2+ + 2L ⇄ ZnL2
mHhLl
f
2.71
2.74
c
d
e
f
20.96
15.52; 15.4
21.63
e
12.01
d
11.31
2.51
5.55
d
e
e
16.49
11.19
11.62; 11.3
18.3
9.2
a
b
The charges of L and complexes are omitted to comprise the three different ligands considered. Values in parentheses are standard deviations in
c
the last significant figures. The value of KCuno1pa2py was determined by competition with H4edta by titration in Hno1pa2py/Cu2+/H4edta 1/0.9/1.2.
I = 0.1 M NaNO3.33 I = 0.1 M.35 I = 0.1 M NaNO3.16a
d
e
f
Figure 3. Speciation diagrams of Cu2+ (left) and Zn2+ (right) in presence of Hno1pa2py. [Cu2+]tot = [Hno1pa2py]tot = 10−3 M and [Zn2+]tot
[Hno1pa2py]tot = 10−3 M.
=
solution above pH 2 for Cu2+ and above pH 4 for Zn2+ (see the
speciation diagrams shown in Figure 3). For the complexes of
Cu2+ it was not possible to determine the stability constants by
direct potentiometry due to the almost complete formation of
the complex at low pH, and therefore, they were obtained by
competition titrations using H4edta (ethylenediaminetetracetic
acid) for which reliable constant values are available for our
experimental conditions.34
The stability constants of the complexes of Hno1pa2py with
the studied metal ions are very high, being >105 larger than
those of the parent amine tacn,33,35 although slightly lower than
those of H3nota.6,16a On the other hand, Hno1pa2py exhibits a
higher selectivity for Cu2+ over Zn2+ than H3nota. However,
comparison of the complexation ability of ligands with different
basicity is not correct if the competition of the ligands for the
proton is not taken into account. Thus, the pM values
calculated for the complexes of Cu2+ and Zn2+ with
Hno1pa2py and related ligands at physiological pH are
shown in Table 4. The pCu value for the complex of
Hno1pa2py is slightly larger than that for the H3nota analogue,
Besides the [M(no1pa2py)]+ species, the potentiometric
data also evidence formation of monoprotonated complexes at
very low pH, while at high pH (>10) deprotonation of one
water molecule leads to the additional formation of hydroxo
complexes.
E
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and furthermore, the former ligand is more selective for Cu2+ in
presence of Zn2+.
unit (N2 and N1) and the donor atoms of the picolinate arm
N4 and O1 (mean deviation form planarity 0.12 and 0.10 Å for
the Zn and Cu complexes, respectively). The trans angle N5−
M−N3 (157.8 and 165.2° for the Zn and Cu complexes,
respectively; Table 5) deviates considerably from the expected
value for a regular octahedron (180°). Three of the cis angles of
this equatorial plane are considerably smaller than the ideal
value of 90°, while the fourth cis angle of the equatorial plane is
considerably larger [N2−M−O1 = 128.4° (Zn) and 123.8°
(Cu)]. All these data point to important distortions of the
metal coordination environments for both complexes (Table
5).
a
Table 4. Calculated pM Values for the Complexes of
Hno1pa2py, tacn, and H3nota
metal ion
Hno1pa2py
tacn
H3nota
Cu2+
Zn2+
17.75
13.28
14.24
8.17
17.61
14.28
a
Values calculated at pH = 7.4 for 100% excess of ligand with [M2+]tot
= 1 × 10−3 M, based on the constants of Tables 2 and 3.
DFT Calculations. Attempts to obtain single crystals of the
complexes of Hno1pa2py with Cu2+ and Zn2+ suitable for X-
ray diffraction studies were unsuccessful. To obtain information
on the structure in solution of these complexes, the
[Zn(no1pa2py)]+ and [Cu(no1pa2py)]+ systems were char-
acterized by means of density functional theory (DFT)
calculations with the TPSSh model. In these calculations
solvent effects (water) were taken into account by using a
polarizable continuum model (PCM). Test calculations
performed on the [CuL1]2+ system provide a minimum energy
conformation that resembles the corresponding X-ray crystal
structure. The calculated bond distances between the metal ion
and the coordinating donor atoms of the ligand are in very
good agreement with the ones found in the crystal structure
(see Supporting Information, Table S3) with an average
unsigned deviation of only 2.4%. The largest deviation
corresponds to the Cu−N1 distance, which amounts to 2.167
and 2.084 Å in the calculated and experimental structures,
respectively. Thus, it was concluded that our DFT calculations
provide a good structural agreement with the reference X-ray
data.
The octahedral coordination environment is even more
distorted in the case of the Δ(δδδ) form, particularly in the case
of the Zn2+ complex. One of the equatorial planes of the
octahedron is delineated by N1, N3, N4, and O1 [mean
deviation from planarity 0.26 (Zn) and 0.24 Å (Cu)]. The trans
angles N2−M−N6 [150.2 and 158.4° for the Zn and Cu
complexes, respectively], as well as the cis angles N3−M−O1
[133.5° (Zn) and 137.3° (Cu)] point to heavily distorted
octahedral environments.
In the case of [Cu(no1pa2py)]+, EPR measurements point
to the presence of two complex species in solution (see below),
which presumably correspond to the Λ(δδδ) and Δ(δδδ)
isomers. For the Zn2+ analogue the presence of two species in
solution is also compatible with the experimentally observed
1
13C and H NMR spectra recorded in D2O in the temperature
range 5−45 °C, which show broad signals that point to the
presence of relatively fast intramolecular conformational
exchange processes (Supporting Information, Figures S3 and
S4). Most likely this exchange process corresponds to the
Λ(δδδ) ↔ Δ(δδδ) interconversion, which involves the rotation
of the three pendant arms of the ligand. Additional 13C and 1H
NMR spectra were recorded in d7-DMF solution at different
temperatures ranging from 45 °C to −40 °C (Figure S4,
Supporting Information). However, the spectra recorded at
−40 °C showed broad signals, indicating that the slow-
exchange region for the dynamic process responsible for line-
Our DFT calculations provided two minimum energy
conformations for the [Zn(no1pa2py)]+ and [Cu-
(no1pa2py)]+ systems with relative energies below 1.0 kcal
mol−1. These minimum energy structures differ by the overall
folding of the ligand around the metal ion, which results in
Λ(δδδ) or Δ(δδδ) conformations (Figure 4). In both
1
broadening is not achieved even at −40 °C. The H NMR
spectrum recorded at this temperature showed two set of
signals in the range 7.5−9.0 ppm with clearly different
intensities, which is in line with the presence of two complex
species in solution.
Electronic, Infrared, and X-band EPR Spectra of the
Copper(II) Complex of Hno1pa2py. The electronic spectra of
the complex of Cu2+ with Hno1pa2py recorded at pH 6.85 and
9.40 exhibit a broad band in the visible region centered at 652
nm (ε = 117 M−1 cm−1) due to the copper(II) d−d transitions.
Upon lowering the pH to 1.95, the maximum has a slight blue
shift to 641 nm with the ε value unchanged.
The infrared spectrum of the prepared [Cu(no1pa2py)]-
(ClO4) complex exhibits the characteristic carbonyl frequencies
for a coordinated carboxylate group of the picolinate moiety.
The strong bands around 1635−1598 cm−1 and at 1381 cm−1
were, respectively, assigned to antisymmetric and symmetric
stretching vibrations of the carboxylate group, thus pointing to
the involvement of the carboxylate in the coordination of the
metal.
The X-band EPR spectra of frozen aqueous samples of the
complex at 90 K showed a pH dependence, indicating the
presence of two paramagnetic species in equilibrium through-
out the entire pH range. At pH 6.85 (Figure 5), the EPR
spectra of both paramagnetic species exhibit three well resolved
Figure 4. Geometries of the [Cu(no1pa2py)]+ system optimized in
aqueous solution at the TPSSh/SVP level.
structures the metal ion is six-coordinated in a heavily distorted
octahedral environment. In the Λ(δδδ) conformation the
nitrogen atom of one of the picolyl pendant arms (N6) remains
uncoordinated, while for the Δ(δδδ) form N5 does not
coordinate to Cu2+ or Zn2+. According to our calculations the
Δ(δδδ) form is more stable than the Λ(δδδ) one by 0.22 kcal
mol−1 (Cu) and 0.98 kcal mol−1 (Zn).
In the Λ(δδδ) conformation one of the equatorial planes of
the octahedron is defined by two nitrogen atoms of the tacn
F
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Table 5. Bond Distances (Å) and Angles (deg) Obtained for [M(no1pa2py)]+ Complexes (M = Zn, Cu) with TPSSh/SVP
Calculations in Aqueous Solution
Λ(δδδ), Cu
Λ(δδδ), Zn
Δ(δδδ), Cu
Δ (δδδ), Zn
M−N1
M−N2
M−N3
M−N4
M−N5
M−O1
2.377
2.069
2.174
2.056
2.077
2.009
83.56
79.74
76.31
100.16
152.15
84.08
158.99
81.24
123.84
98.00
165.22
96.87
96.31
76.84
89.96
2.339
2.217
2.263
2.097
2.183
2.086
97.82
80.18
74.72
97.82
151.14
80.47
153.47
77.43
128.36
102.93
157.82
98.14
97.75
77.66
94.02
M−N1
M−N2
M−N3
M−N4
M−N6
M−O1
2.453
2.150
2.133
2.146
2.066
2.143
78.79
78.93
71.15
83.56
142.81
83.55
106.79
158.44
94.93
145.32
79.87
137.30
94.66
75.92
88.07
2.328
2.232
2.246
2.091
2.209
2.093
80.08
78.83
73.87
110.96
146.75
80.26
113.68
150.20
96.28
146.13
75.24
133.52
96.10
77.63
88.61
N1−M−N2
N1−M−N3
N1−M−N4
N1−M−N5
N1−M−O1
N2−M−N3
N2−M−N4
N2−M−N5
N2−M−O1
N3−M−N4
N3−M−N5
N3−M−O1
N4−M−N5
N4−M−O1
N5−M−O1
N1−M−N2
N1−M−N3
N1−M−N4
N1−M−N6
N1−M−O1
N2−M−N3
N2−M−N4
N2−M−N6
N2−M−O1
N3−M−N4
N3−M−N6
N3−M−O1
N4−M−N6
N4−M−O1
N6−M−O1
obtained by simulation of the spectra are compiled in Table 6.36
These values indicate three different principal values for the g
and A parameters of each species, with gz > (gx + gy)/2 and the
lowest g value ≥2.035, which is characteristic of mononuclear
2
2
copper(II) complexes in dx −y ground state and elongation of
the axial bonds. Elongated rhombic-octahedral, tetragonal, or
distorted square pyramidal symmetries would be consistent
with these data.37 At the indicated pH, the percentages of the
two species are 69% (A) and 31% (B). By increasing the pH to
9.40 no changes of the EPR parameters were observed, but the
percentages of each species changed to 36% (A) and 64% (B).
The two species exhibit quite different EPR parameters, with
the A species presenting a higher gz and a lower Az than the B
form.
Theoretical calculations of the g-tensors were performed with
DFT on the Λ(δδδ) and Δ(δδδ) conformations of the
[Cu(no1pa2py)]+ complex described above. These calculations
provided gx = 2.083, gy = 2.074, and gz = 2.209 for the Λ(δδδ)
form, and gx = 2.077, gy = 2.122, and gz = 2.195 for the Δ(δδδ)
isomer. On the basis of these data we assign the A and B species
observed in the EPR spectrum to the Λ(δδδ) and Δ(δδδ)
isomers, respectively. Our calculations appear to overestimate
to a certain extent gx and gy, while gz is slightly underestimated
in the case of the Λ(δδδ) isomer. However, an overall good
agreement is observed between the experimental and calculated
g-values, which confirms that our DFT calculations provide
molecular geometries in good agreement with the actual
structure of the complex in solution.
Kinetic Inertness of the Copper(II) Complex of Hno1pa2py.
A common method to ascertain the kinetic stability of metal
complexes is the study of their acid-assisted dissociation. We
have studied the dissociation of the copper(II) complex of
Hno1pa2py in aqueous solution under pseudo-first-order
conditions by monitoring the decrease of the d−d transition
band in the visible spectroscopic range. Due to the high
inertness shown by the complex in preliminary assays, the
conditions finally used to determine its half-life were in 3 M
Figure 5. X-band EPR spectra of the complex of Cu2+ with
Hno1pa2py in aqueous solution at pH 6.85 and 9.40. Dashed lines
represent the simulated spectra in each case.
lines of the four expected at low field from the coupling of the
3
unpaired electron spin with the copper nucleus (I = /2). The
fourth line of both species is overlapped by the strong band at
the high field part of the spectra. No superhyperfine splitting
due to coupling with the nitrogen atoms of the ligand was
observed. Spectroscopic visible data (λmax), the hyperfine
coupling constants Ai (i = x, y, and z), and the g values
G
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a
Table 6. EPR Parameters for the Copper(II) Complexes of Hno1pa2py at 90 K in 1.0 M NaClO4 Frozen Aqueous Solution
EPR parameters
b
b
b
[Cu(no1pa2py)]+
pH
gx
gy
gz
Ax
Ay
Az
A expt
6.85
2.042
2.083
2.036
2.077
2.069
2.074
2.104
2.122
2.236
2.209
2.198
2.195
<5
<5
9
160.4
191.2
Λ(δδδ) theor
B expt
6.85
11
Δ(δδδ) theor
a
b
Complex concentration at ca. 2 × 10−3 M. Ai × 104 (cm−1).
aqueous HCl at 90 °C. The half-life value (t1/2) of 204 min (3.4
h) indicates a very high degree of kinetic inertness for the
studied complex. Dissociation kinetics studies in acidic media
are uncommon for complexes of Cu2+ with 1,4,7-triazacyclo-
nonane derivatives except for H3nota, while being usual for
complexes of cross-bridged derivatives of cyclen and cyclam
such as H2cb-do2a and H2cb-te2a.3 Half-lives have also been
reported for complexes of monopicolinate derivatives of cyclen
and cyclam, Hdompa and Htempa.15 In Table 7 is shown a
Table 7. Acid-Assisted Dissociation Inertness of Selected
Copper(II) Complexes
ligand
conditions
half-life (t1/2), min
ref
Hno1pa2py
H3nota
3 M HCl, 90 °C
5 M HCl, 30 °C
1 M HCl, 30 °C
5 M HCl, 90 °C
2 M HCl, 25 °C
1 M HCl, 25 °C
204
<3
this work
3
3
H2cb-do2a
H2cb-te2a
Hdompa
Htempa
240
9240
1.4
Figure 6. Cyclic voltammogram of [Cu(no1pa2py)]+ in aqueous
solution at neutral pH recorded at 100 mV s−1.
3
15
15
behavior of Hno1pa2py with 64Cu2+ following literature
investigations.4,38 Radiolabeling of Hno1pa2py by copper-64
was investigated with solutions prepared as described in the
Experimental Section. The “carrier added” solution was not
obtained by addition of cold copper, but rather results from the
presence of cold competitors [Cu (27.76%), Fe (14.30%), Ni
(5.24%)] in the radioactive copper solution (specific activity:
2.32 GBq/μmol). The chelation was achieved at 6 < pH < 7, by
reaction of solutions of 64CuCl2 with Hno1pa2py at 10, 1, and
0.1 mM concentrations in 0.1 M ammonium acetate. Reaction
mixtures were stirred at room temperature during 5, 15, and 30
min, and radiolabeled solutions were analyzed on TLC plates
and by means of HPLC experiments. No Hno1pa2py
concentration lower than 0.1 mM was investigated since at
this concentration the specific radiolabeling activity of 364
MBq/μmol was already quite low for an application in
immunodiagnostic. This result has to be associated with the
specific activity of the copper solution as described above. A
great improvement will be reached when higher copper-64
specific activity solutions are used.
Thin layer radiochromatograms, which present the measured
radioactive emission on a storage phosphor screen after 30 min
incubation time, are presented in Figure 7 (see Supporting
Information, Figure S5, for 5 and 15 min). In each case, free
64Cu2+ was not observed, and an outstanding 100% radio-
labeling of Hno1pa2py with 64Cu2+ was observed. Three
radiolabeled species with different abundances (∼60% + 25% +
15%) were observed for all incubation times.
The HPLC radiochromatogram obtained from a 1 mM
ligand solution after a 30 min of incubation time is compared to
that of 64Cu-acetate as a reference in Figure 8 (chromatograms
B and C). The diagrams confirm the results initially obtained
on TLC plates, showing that three radiolabeled species are
formed in comparable ratios, while free 64Cu was not detected.
32
comparison of the reported half-lives for selected copper(II)
complexes. Overall, and in spite of the various experimental
conditions used, it is possible to conclude that the inertness of
the complex of Cu2+ with Hno1pa2py is clearly higher than
those with Hdompa and Htempa and somewhat lower than
that of [Cu(cb-te2a)], which is one of the most inert
copper(II) complexes of polyaza ligands known.
Electrochemistry of the Copper(II) Complex of Hno1p-
a2py. A relevant pathway for the dissociation of copper(II)
complexes of macrocyclic ligands has been suggested to be the
reduction to Cu+ followed by demetalation of the complex.1b
Thus, the electrochemical behavior of the copper(II) complex
of Hno1pa2py at neutral pH was studied by using cyclic
voltammetry in aqueous solution (Figure 6). A quasireversible
red
reduction system was observed at E1/2 = −675 mV versus
Ag/AgCl, with a ΔEp = 78 mV (Epc = −714 mV, ipc = 1.465 ×
10−5 A, Epa = −636 mV, ipa = 1.368 × 10−5 A). This result
shows that the electrogenerated copper(I) complex is stable on
the CV time scale, which demonstrates the capacity of
Hno1pa2py to adapt at least transiently to the copper(I)
coordination requirements thus avoiding demetalation upon
reduction of the metal ion. This is in contrast to the irreversible
reduction found for complexes of most macrocyclic compounds
including H3nota, cyclen, cyclam, and H2cb-do2a,1b while
complexes of monopicolinate counterparts Hdompa and
Htempa as well as of H2cb-te2a present quasireversible
reductions. The reduction process observed for the copper(II)
complex of Hno1pa2py (Epc = −518 mV versus NHE, upon
conversion) places it slightly outside the estimated −400 mV
(NHE) threshold for typical bioreductants.1b
Copper-64 Radiolabeling. The high stability of the
copper(II) complexes encouraged us to further examine the
H
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Figure 7. Thin layer radiochromatogram of [64Cu(no1pa2py)]+ and %
of 64Cu emission: (A) 10 mM ligand and 30 min incubation time; (B)
1 mM ligand and 30 min incubation time; (C) 0.1 mM ligand and 30
min incubation time; (D) copper-64 acetate as reference.
The same experiment was then performed with the “cold”
complex [Cu(no1pa2py)]+, and the equivalent UV-HPLC
chromatogram obtained is also presented in Figure 8
(chromatogram A). It showed two major species with relative
abundances of 72% (lower Rf) and 29% (higher Rf), indicating
that the third species observed upon 64Cu complexation is
related to side effects of the radiolabeling technique, for
instance radiolysis phenomena associated to the radiation
emitted by copper-64. Indeed, ionizing radiation generates
radical species, including OH•, which can react with the copper
complex and explain the formation of hydroxide species even at
neutral pH values.39 Even so, the HPLC chromatograms shown
in Figure 8 indicate the presence of two complex species when
using cold Cu2+. The presence of several 64Cu radiolabeled
species has been previously observed for other polyazamacro-
cycles, and attributed to the formation of dimers or the
presence of impurities.40 The formation of three different
radiolabeled species may reflect the coordination versatility of
the ligand: first of all, the macrocycle can act as a N5O or N6
donor; additionally, Jahn−Teller effects may result in 4 or 5-
coordinate copper centers (as previously seen in the crystal
structure of the methyl ester derivative). The crystal structure
of [Cu(HL1)Cl]+ also revealed that coordinating anions can
also participate in the primary coordination sphere. Therefore,
a large excess of acetate in the experimental workup could also
result in the formation of ternary complexes with this anion and
such effect has to be considered.
Figure 8. (A) HPLC UV-chromatogram of [Cu(no1pa2py)]+. (B)
HPLC radiochromatogram of 1 mM Hno1pa2py labeled with 64Cu2+
(30 min incubation time). (C) HPLC radiochromatogram of copper-
64 acetate.
CONCLUSIONS
cyclic backbone was obtained following an easy-to-run
procedure involving orthoamide chemistry. The final chelator
was obtained in a six-step procedure with good overall yields.
The ligand exhibits a low overall basicity due to the small
macrocyclic cavity and the introduction of aromatic groups.
The copper(II) complexation process was found to be very fast,
including in acid medium. Besides, the thermodynamic stability
of the complexes of Cu2+ was found to be relatively high
compared to other tacn-based ligands, and the pCu value
showed high selectivity of Hno1pa2py for Cu2+ over Zn2+. A
detailed investigation of the structure of the complexes by using
EPR and UV−vis spectroscopies and 1H and 13C NMR
experiments at variable temperatures, supported by additional
DFT calculations, indicates that at neutral pH the complex is
■
Macrocyclic chelators and their metal complexes have wide-
spread applications in the biomedical sciences, including
radiopharmaceutical chemistry. The use of copper radio-
nuclides in radiopharmaceuticals is increasing, and the
challenge still persists to develop new chelators able to meet
the very strict metal binding specifications for applications such
as PET imaging or radiotherapy. Picolinate groups appended
on macrocycles proved to yield efficient chelators for
copper(II). In this work we have presented the synthesis of a
new ligand, Hno1pa2py, based on the tacn framework
functionalized with two picolyl and one picolinate pendant
arms, and designed for stable complexation of Cu2+ ions in
aqueous solution. Selective N-functionalization of the macro-
I
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3
tacn); 3.74 (s, 4 H, CH2-py); 6.99 (dd, 2 H, J = 6.6 Hz, 3J = 4.9 Hz,
Harom); 7.36 (d, 2 H, 3J = 7.6 Hz, Harom); 7.47 (dd, 2 H, 3J = 7.6 Hz, 3J
= 6.6 Hz, Harom); 8.37 (d, 2 H, 3J = 4.9 Hz, Harom). 13C NMR (CDCl3,
100 MHz): δ 46.3, 51.5, 52.4 (tacn); 63.0 (CH2-py); 121.6, 122.8,
136.0, 148.6 (Carom); 159.9 (Cq).
present in solution as a mixture of two isomers: Λ(δδδ) and
Δ(δδδ). The two isomers present very distorted octahedral
coordination environments in which the ligand uses a N5O
donor set, with one of the picolyl pendant arms remaining
uncoordinated.
The investigation of the kinetic stability of the complexes in
acidic solutions underlines a high kinetic inertness of the
[Cu(no1pa2py)]+ complex compared to other copper(II)
complexes of macrocyclic ligands reported in the literature. In
addition, CV experiments performed in aqueous solution
confirmed its inertness by emphasizing a good stability of the
complexes upon reduction of the metal ion to Cu+ at neutral
pH.
The Hno1pa2py ligand consequently appears as a very
attractive candidate for the design of Cu2+-based radiopharma-
ceuticals for application in PET imaging and encouraged us to
investigate, as a preliminary study, the ability of this ligand to
chelate the 64Cu radionuclide. Radiolabeling with 64Cu was
demonstrated to be very efficient with a quantitative complex-
ation of the hot metal. Thus, this ligand scaffold is promising
for the preparation of chelates for PET applications provided
that the minor species observed in solution can be separated,
for instance by HPLC workup.
1-Methylpicolinate-4,7-bis(pyridin-2-ylmethyl)-1,4,7-triaza-
cyclononane (L1). Sodium carbonate (88 mg, 0.8 mmol) and a
solution of 6-chloromethylpyridine-2-carboxylic acid methylester (62
mg, 0.3 mmol) in acetonitrile (10 mL) were added to a solution of 5
(100 mg, 0.3 mmol), and the mixture was stirred at 50 °C for 3 days.
After cooling, the solution was filtered and the filtrate evaporated
under vacuum. The residue was purified by column chromatography
using neutral alumina (CHCl3/MeOH 98/2), yielding a brown oil
1
(137 mg, 93%). H NMR (CDCl3, 400 MHz): 2.86 (s, 12 H, tacn);
3.80 (s, 4 H, CH2-py); 3.92 (s, 2 H, CH2-pic); 3.95 (s, 3 H, CH3); 7.11
(dd, 2 H, 3J = 4.8 Hz, 3J = 7.4 Hz, Harom‑py); 7.46 (d, 2 H, 3J = 7.6 Hz,
3
Harom‑py); 7.61 (t, 2 H, J = 7.6 Hz, Harom‑py); 7.78−7.76 (m, 2 H,
Harom‑pic); 7.96 (dd, 1 H, 3J = 3.6 Hz, 3J = 5.2 Hz, Harom‑pic); 8.47 (d, 2
H, J = 4.8 Hz, Harom‑py). 13C NMR (CDCl3, 100 MHz): 52.8 (CH3);
3
55.8 (tacn); 64.6 (CH2); 121.8, 123.1 (Carom‑py); 123.4, 126.4
(Carom‑pic); 136.2 (Carom‑py); 137.1 (Carom‑pic); 147. 0 (Cq-CO2CH3);
148.8 (Carom‑py); 160.3, 161.4 (Cq-CH2); 165.9 (CO2CH3). MALDI-
TOF: m/z 461.2 [M + 1]+; 462.2 [M + 2]+; 463.2 [M + 3]+.
1-Picolinic Acid-4,7-bis(pyridin-2-ylmethyl)-1,4,7-triazacy-
clononane Hydrochloride (Hno1pa2py). A solution of L1 (570
mg, 1.24 mmol) in hydrochloric acid (6 M, 10 mL) was stirred at 55
°C for 2 days, and then evaporated under reduced pressure. Addition
of diethyl ether lead to precipitation of the desired compound as an
off-white powder (776 mg, 94%). 1H NMR (D2O, 400 MHz): 2.89 (s,
4 H, tacn); 2.91 (bs, 4 H, tacn); 3.42 (bs, 4 H, tacn); 4.16 (s, 4 H,
CH2-py); 4.61 (s, 2 H, CH2-pic); 7.83 (d, 1 H, 3J = 7.6 Hz, Harom‑pic);
7.89 (dd, 2 H, J = 6.6 Hz, Harom‑py); 8.06 (d, 2 H, J = 8.0 Hz,
Harom‑py); 8.18 (t, 1 H, 3J = 7.6 Hz, Harom‑pic); 8.31 (d, 1 H, 3J = 7.6 Hz,
Harom‑pic); 8.48 (dd, 2 H, 3J = 6.6 Hz, 3J = 8.0 Hz, Harom‑py); 8.56 (d, 2
H, 3J = 6.6 Hz, Harom‑py). 13C NMR (D2O, 100 MHz): 47.5, 52.8, 54.2
(tacn); 58.0 (CH2-py); 61.2 (CH2-pic); 129.2, 130.7 (Carom‑pic); 129.5,
132.1 (Carom‑py); 143.6, 144.3 (Carom‑py); 150.2, 150.8 (Carom‑pic); 153.4,
154.3 (Cq‑arom); 170.3 (Cq-CO2H). MALDI-TOF: m/z 447.2 [M +
1+]. Anal. Calcd for C25H30N6O2·6HCl·2H2O: C, 42.81; H, 5.75; N,
11.98. Found: C, 42.57; H, 5.72; N, 11.73.
EXPERIMENTAL SECTION
■
Materials and Methods. Reagents were purchased from ACROS
Organics and from Aldrich Chemical Co. 1,4,7-Triazacyclononane
(tacn) was purchased from CheMaTech (Dijon, France). 6-
Chloromethylpyridine-2-carboxylic acid methylester was synthesized
according to a published method.25 Acetonitrile, toluene, and water
solvents were distilled before use. Elemental analyses were performed
at the Service de Microanalyse, CNRS, 69360 Solaize, France. NMR
and MALDI mass spectra were recorded at the “Services communs” of
3
3
1
the University of Brest. H and 13C NMR spectra were recorded with
Bruker Avance 500 (500 MHz), Bruker Avance 400 (400 MHz), or
Bruker AMX-3 300 (300 MHz) spectrometers; NMR spectra of all the
previously described derivatives are given in the Supporting
Information (Figures S6−S10). MALDI mass spectra were recorded
with an Autoflex MALDI TOF III LRF200 CID spectrometer.
Caution! Although no problem was found during our experiments, salts
of perchlorate and their metal complexes are potentially explosive and
should be handled with great care and in small quantities.
1,4-Bis(pyridin-2-ylmethyl)-1,4,7-triazacyclononane (5). N-
Dimethoxymethyl-N,N-dimethylamine (910 μL, 6.8 mmol, 1.1
equiv) was added to a solution of tacn (880 mg, 6.81 mmol) in
chloroform (2 mL) and toluene (8 mL) The reaction mixture was
heated to reflux with stirring for 2 h, and then the solvent was
evaporated under reduced pressure yielding a clear oil. This crude
product was dissolved in acetonitrile (20 mL) and added dropwise to a
solution of 2-chloromethylpyridine (950 mg, 7.48 mmol, 1.1 equiv) in
acetonitrile (50 mL). The reaction mixture was stirred at room
temperature (rt) for one week, and then the solvent was evaporated
under reduced pressure to yield a brown powder, which was dissolved
in water and stirred at reflux during 3 h. The solvent was evaporated
under reduced pressure yielding a brown oil, which was dissolved in
acetonitrile (20 mL). A solution of 2-chloromethylpyridine (950 mg,
7.48 mmol, 1.1 equiv) in acetonitrile (20 mL) and potassium
carbonate (2.5 equiv) were added. The mixture was stirred at rt for 4
days and filtered, and the solvent was evaporated under reduced
pressure to yield a brown oil. The resulting product was dissolved in
methanol (30 mL), hydrochloric acid (6 M, 30 mL) was added, and
the mixture was refluxed with stirring during 3 h. Distilled water was
added (40 mL), and the solution was extracted with chloroform (40
mL). The pH of the aqueous layer was adjusted to ∼12 with NaOH
pellets, and then extracted with chloroform (3 × 50 mL). Organic
layers were recombined, dried under MgSO4, and filtered. The filtrate
was evaporated under reduced pressure yielding a brown oil. The
residue was purified by column chromatography using neutral alumina
Preparation of [CuL1](ClO4)2]. Cu(ClO4)2·6H2O (40 mg, 0.108
mmol) was added to a solution of Hno1pa2py (50 mg, 0.108 mmol)
in water (5 mL), and the pH of the solution was adjusted to ≈4.5 with
an aqueous HCl solution (1 M). The mixture was heated at 40 °C
during 14 h. The solution was evaporated; the resulting blue solid
dissolved in a minimum quantity of methanol, and diethylether was
added. The precipitate formed was filtered to yield a turquoise powder.
Single crystals suitable for X-ray diffraction analysis were obtained
upon slow evaporation of an aqueous solution of the turquoise powder
(73 mg, 83%). Anal. Calcd for C26H32Cl2CuN6O10·1.5H2O·KCl: C,
34.73; H, 3.92; N, 9.35. Found: C, 34.75; H, 3.88; N, 8.85.
Preparation of [Cu(no1pa2py)](ClO4). Cu(ClO4)2·6H2O (100
mg, 0.270 mmol) was added to a solution of Hno1pa2py·6HCl·2H2O
(185 mg, 0.265 mmol) in water (25 mL). The solution was slowly
neutralized by addition of diluted aqueous KOH, and then stirred at rt
overnight. The turquoise solution obtained was evaporated and dried
under vacuum, the residue was taken in 25 mL of dry MeCN, and the
colorless insoluble matter was separated by filtration. This isolation
procedure was repeated 3 times in progressively smaller volumes of
dry MeCN until no more inorganic matter separated. The clear
solution was then evaporated and dried under vacuum to yield a
turquoise solid (146 mg, 88%). Anal. Calcd for
C25H29ClCuN6O6·0.5CH3CN: C, 49.64; H, 4.89; N, 14.47. Found:
C, 49.90; H, 5.26; N, 14.54. Selected IR bands (KBr, cm−1):
νas(COO−) 1635 (s) and 1598 (s), νs(COO−) 1381 (m); ν(Cl−O)
1
(CHCl3/MeOH 95/5) to yield 5 as a brown oil (1.99 g, 60%). H
−
NMR (CDCl3, 400 MHz): 2.51 (m, 8 H, tacn); 2.62 (m, 4 H, m,
(ClO4 ) centered at 1100 (vs).
J
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X-ray Diffraction Studies. X-ray diffraction data were collected
with a circular diffractometer X-Calibur-2 CCD 4 (OXFORD
DIFFRACTION), including a four circle goniometer (KM4) and a
two-dimensional CCD detector (SAPPHIRE 2). Three dimensional X-
ray diffraction data were collected on an X-CALIBUR-2 CCD 4-circle
diffractometer (Oxford Diffraction). Data reduction, including
interframe scaling, Lorentz, polarization, empirical absorption, and
detector sensitivity corrections, was carried out using attached
programs of Crysalis software41 (Oxford Diffraction). Complex
scattering factors were taken from the program SHELX9742 running
under the WinGX program system.43 The structure was solved by
direct methods with SIR-9744 and refined by full-matrix least-squares
on F2. All hydrogen atoms were included in calculated positions and
refined in riding mode. Crystal data and details on data collection and
refinement are summarized in Table 8. CCDC 919095 and 919096
0.75 for the mononuclear copper(II) complexes investigated here] and
the actual value of ⟨S2⟩.49 The results obtained indicate that spin
contamination is negligible for systems investigated in this work [⟨S2⟩
− S(S + 1) < 0.0020]. The stationary points found on the potential
energy surfaces as a result of geometry optimizations were tested to
represent energy minima rather than saddle points via frequency
analysis. The default values for the integration grid (75 radial shells
and 302 angular points) and the SCF energy convergence criteria
(10−8) were used in all calculations. The relative free energies of the
different conformations of [Cu(no1pa2py)]+ and [Zn(no1pa2py)]+
complexes were calculated in aqueous solution at the TPSSh/SVP
level, and they include non-potential-energy contributions (zero point
energies and thermal terms) obtained through frequency analysis.
Throughout this work solvent effects were included by using the
polarizable continuum model (PCM), in which the solute cavity is
built as an envelope of spheres centered on atoms or atomic groups
with appropriate radii. In particular, the integral equation formalism
(IEFPCM) variant as implemented in Gaussian 09 was used.50
Calculation of EPR parameters (g-tensor) was performed in
aqueous solution with the TPSSh functional. In these calculations
we used the aug-cc-pVTZ-J basis set of Sauer for Cu,51 and for the C,
H, N, and O atoms we used the EPR-III basis sets of Barone,52 which
is a triple-ζ basis set including diffuse functions, double d-polarizations,
and a single set of f-polarization functions, together with an improved
s-part to better describe the nuclear region. The g-tensor was
calculated as a correction, Δg, to the free electron g-value (ge = 2.002
3193) that represents contributions of three gauge including atomic
orbitals (GIAO) magnetic shielding terms:
Table 8. Crystal Data and Refinement Details for
[CuL1](ClO4)2·H2O
[CuL1](ClO4)2·H2O
formula
MW
C26H34Cl2CuN6O11
741.03
cryst syst
space group
T/K
monoclinic
P21/c
170(2)
a/Å
8.5089(2)
22.8400(18)
15.8525(3)
90
b/Å
c/Å
Δg = ge + gRMC + gDC + gOZ/SOC
α/deg
β/deg
γ/deg
90.901(2)
90
Here gRMC is the relativistic mass correction, gDC is the diamagnetic
correction, and gOZ/SOC represents the orbital Zeeman and spin−orbit
coupling contributions.53
V/Å3
3080.4(3)
1532
F(000)
Z
Potentiometric Studies. Equipment and Work Conditions. The
potentiometric setup consisted of a 50 mL glass-jacketed titration cell
sealed from the atmosphere and connected to a separate glass-jacketed
reference electrode cell by a Wilhelm type salt bridge filled with 0.1 M
KNO3 electrolyte. An Orion 720A+ measuring instruments fitted with
a Metrohm 6.0123.100 glass electrode and a Metrohm 6.0733.100 Ag/
AgCl reference electrode was used for the measurements. The ionic
strength of the experimental solutions was kept at 0.10 0.01 M with
KNO3; temperature was controlled at 298.2 0.1 K using a Huber
CC3-K6 compact cooling and heating bath thermostat and a
previously calibrated Orion 91-70-06 ATC-probe. Atmospheric CO2
was excluded from the titration cell during experiments by slightly
bubbling purified nitrogen on the experimental solution. Titrant
solutions were added through capillary tips at the surface of the
experimental solution by a Metrohm Dosimat 665 automatic buret.
Titration procedure is automatically controlled by software after
selection of suitable parameters, allowing for long unattended
experimental runs. The titrant was a KOH solution prepared at ca.
0.1 M from a commercial ampule of analytical grade, and its accurate
concentration was obtained by application of the Gran’s method54
upon titration of a standard HNO3 solution. Ligand solutions were
prepared at ca. 2.0 × 10−3 M, and the Cu2+ and Zn2+ solutions were
prepared at ca. 0.05 M from analytical grade nitrate salts and
standardized by complexometric titrations with H4edta (ethyl-
enediaminetetraacetic acid).55 Sample solutions for titration contained
approximately 0.04 mmol of ligand in a volume of 30.00 mL. In
complexation titrations metal cations were added at 0.9 equiv of the
ligand amount. In competition titrations H4edta was additionally
added at 1.2 equiv.
4
λ, Å (Mo Kα)
0.710 73
1.598
D
calc/g cm−3
μ/mm−1
0.952
θ range/deg
Rint
2.84−28.28
0.0204
reflns measd
unique reflns
reflns obsd
GOF on F2
26 979
7636
6270
1.027
a
R1
0.0522
b
wR2 (all data)
largest differences peak and hole/e Å−3
0.1444
1.054 and −0.645
a
b
R1 = ∑||Fo| − |Fc||/∑|Fo|. wR2 = {∑[w(||Fo|2 − |Fc|2)2]/∑[
4
w(Fo )]}1/2
.
contain the supplementary crystallographic data for this Article. These
data can be obtained free of charge from the Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Computational Details. All calculations presented in this work
were performed employing the Gaussian 09 package (Revision
B.01).45 Full geometry optimizations of the [CuL1]2+, [Cu-
(no1pa2py)]+, and [Zn(no1pa2py)]+ complexes were performed in
aqueous solution employing DFT within the hybrid meta-GGA
approximation with the TPSSh exchange-correlation functional.46
Input geometries were generated from the crystallographic data of
[CuL1](ClO4)2. For geometry optimization purposes we used the
standard Ahlrich’s valence double-ξ basis set with polarization
functions (SVP).47 No symmetry constraints have been imposed
during the optimizations. In the case of copper complexes calculations
were performed by using an unrestricted model, and therefore spin
contamination48 was assessed by a comparison of the expected
difference between S(S + 1) for the assigned spin state [S(S + 1) =
Measurements. The electromotive force of the sample solutions
was measured after calibration of the electrode by titration of a
standard HNO3 solution at 2.0 × 10−3 M in the work conditions. The
[H+] of the solutions was determined by measurement of the
electromotive force of the cell, E = E°′ + Q log [H+] + Ej. The term
pH is defined as −log[H+]. E°′ and Q were determined by acid region
of the calibration curves. The liquid-junction potential, Ej, was found
K
dx.doi.org/10.1021/ic400174r | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
to be negligible under the experimental conditions used. The value of
Kw = [H+][OH−] was found to be equal to 10−13.78 by titrating a
solution of known hydrogen-ion concentration at the same ionic
strength in the alkaline pH region, considering E°′ and Q valid for the
entire pH range. The protonation constants of H4edta and the
thermodynamic stability constants of its copper(II) complex used in
competition titration refinements were taken from the literature.34
Each titration consisted of 50−70 equilibrium points in the range pH
2.5−11.5, and at least two replicate titrations were performed for each
particular system. Backtitrations were always performed at the end of
each direct complexation titration in order to check if equilibrium was
attained throughout the full pH range.
the d−d transition band of the complex in the visible range at 660 nm,
at 90 °C and 3 M HCl without control of ionic strength. The
experimental data were fitted by exponential regression, after
correction for background absorbance due to the weakly absorbing
free Cu2+ in the presence of excess Cl− anions, in order to calculate the
half-life of the complex.
Copper-64 Radiolabeling. Copper-64 dichloride in 0.1 M
hydrochloric acid was obtained from the ARRONAX cyclotron
(Saint-Herblain, France). Radiochemical purity was determined by
gamma spectroscopy, and chemical purity was controlled by ICP-OES.
For radiolabeling solutions, ammonium acetate, hydrochloric acid
30%, and sodium hydroxide pellets were purchased as trace select
grade from Fluka Analytical. Ammonium chloride ACS reagent
(Sigma-Aldrich), methanol HPLC grade (Fisher Chemical), and
acetonitrile gradient grade (Fisher Scientific) were used for the
analytical controls. Water (18.2 MΩ cm) for aqueous solutions was
obtained from a Milli-Q gradient system (Millipore). Hno1pa2py
copper-64 chelation was achieved by addition of 5 μL carrier-added
64CuCl2 solution (1.8 MBq, 11.5 ppm of total copper for 44 ppm total
metal, specific activity of 2.32GBq/μmol), and 5 μL of 0.1 M sodium
hydroxide to 50 μL of Hno1pa2py solutions in 0.1 M ammonium
acetate at different concentration levels (10 mM; 1 mM; 0.1 mM).
Reaction mixtures were stirred at rt for 5, 15, and 30 min, and
radiolabeled solutions were analyzed on TLC plates (silica gel on
TLC-PET foils, Fluka analytical) and HPLC. A mixture of ammonium
chloride (20% in water) and methanol (1:1) was used for TLC
analyses. TLC plates were revealed on a storage phosphor screen using
a Cyclone Plus phosphor imager (Perkin-Elmer). HPLC analyses were
performed using an Eckert & Ziegler HPLC Module, Knauer pumps
K120, Knauer HPLC Degasser, and a reverse phase HPLC column
(ACE C18.3 μm, 150 mm × 3 mm) with the following elution
gradient: isocratic mode ammonium acetate buffer (50 mM, pH 4.5)
during 2.5 min; then addition of acetonitrile from 0 to 10% in the
ammonium acetate buffer, for 15 min at a flow rate of 0.5 mL/min;
and finally 10% of acetonitrile during 2.5 min. Radioactive and UV
(227 nm) profiles were monitored with an Eckert & Ziegler detector
shielding module and a Knauer smartline UV 2520 using Modular-Lab
software.
Calculations. The potentiometric data were refined with the
HYPERQUAD software,56 and speciation diagrams were plotted using
the HySS software.57 The overall equilibrium constants βiH and βM H L
h l
m
are defined by βM H L = [MmHhLl]/[M]m[H]h[L]l (βiH = [HhLl]/
m
h l
[H]h[L]l and βMH L= βML(OH) × Kw). Differences, in log units, between
−1
the values of protonated (or hydrolyzed) and nonprotonated constants
provide the stepwise (log K) reaction constants (being KM H L =
m
h l
[MmHhLl]/[MmHh−1Ll][H]). The errors quoted are the standard
deviations calculated by the fitting program from all the experimental
data for each system.
Spectroscopic Studies. A stock solution of the complex of
Hno1pa2py with Cu2+ was prepared at 2.86 × 10−3 M. The spectra in
the visible region were measured on a Unicam UV4 spectrometer in
aqueous solutions at 298 K. Sample solutions of the complex were
measured at pH 1.95, 6.85, and 9.40, after adjustment by addition of
KOH or HNO3. EPR spectra were recorded on a Bruker EMX 300
spectrometer operating at the X-band and equipped with a
continuous-flow cryostat for liquid nitrogen. Sample solutions of the
complex at ca. 2.0 × 10−3 M and 1 M in NaClO4 were prepared at pH
values of 1.90, 2.41, 3.00, 6.85, and 9.40 by addition of aqueous
NaClO4 to the stock solution followed by pH adjustment with HNO3
or KOH (see Figure S6 of the Supporting Information). The EPR
spectra of the frozen solutions at 90 K were recorded at an attenuated
microwave power of 2.0 mW and a microwave frequency of 9.5 GHz.
Experimental EPR spectra were simulated using the SpinCount
software36 to determine the relevant parameters. The infrared
spectrum (400−4000 cm−1) of the [Cu(no1pa2py)](ClO4) complex
was acquired on a Bruker IFS 66/S spectrometer as a KBr pellet.
Electrochemical Studies. Cyclic voltammograms were performed
in aqueous solution at rt with a BAS CV-50W voltammetric analyzer
operated with BAS data acquisition software. Experiments were run in
a glass cell BAS MF-1082 placed inside a BAS C-2 cell stand (Faraday
cage). The three-electrode setup consisted of a reference Ag/AgCl
electrode (BAS MF-2052) filled with aqueous 3 M NaCl solution, a
platinum wire auxiliary electrode (BAS MW-1032), and a glassy
carbon working electrode (BAS MF-2012). A solution of the Cu2+
complex of HL2 at neutral pH was prepared at 1.43 × 10−3 M in 0.10
M NMe4NO3 electrolyte. Sample solutions were degassed by bubbling
N2 prior to all measurements, and were kept under a N2 stream during
the measurements. Between each scan the working electrode was
electrocleaned by multicycle scanning in the supporting electrolyte
solution, polished on alumina 1 and 0.05 μm, cleaned with water and
sonicated before use, according to standard procedures. Cyclic
voltammograms with sweep rate ranging from 10 to 200 mV s−1
were recorded in the region from 0 to −1000 mV, with the voltage
being ramped from 0 to −1000 mV and then back to 0 mV. At this
potential range the ligand was found to be electroinactive. The half-
wave potentials, E1/2’s, were obtained by averaging the anodic and
cathodic peak potentials. All potential values are reported relative to
the Ag/AgCl reference electrode in aqueous 3 M NaCl unless
otherwise noted.
ASSOCIATED CONTENT
* Supporting Information
■
S
Structure, bond distances, angles, and description of the
structure of complex [Cu(HL1)Cl](ClO4)2·H2O (7) obtained
at pH 1.5. X-ray crystal structures of [CuL1](ClO4)2·H2O (6)
and [Cu(HL1)Cl](ClO4)2·H2O (7) in CIF format. NMR
spectra (1H and 13C) of precursors and ligands L1 and
1
Hno1pa2py. H and 13C NMR spectra of [Zn(no1a2py)]+ at
different temperatures, X-band EPR spectra of the complex of
Cu2+ with Hno1pa2py, experimental (X-ray) and calculated
(UTPSSh/SVP) bond lengths (Å) and angles (deg) of the
metal coordination environment in [CuL1]2+, and TLC
experiments with 64Cu. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: carlos.platas.iglesias@udc.es (C.P.-I); delgado@itqb.
unl.pt (R.D.); raphael.tripier@univ-brest.fr (R.T.).
■
Notes
The authors declare no competing financial interest.
Kinetic Inertness of the Complex of Cu2+ in Acidic Media
(HCl). The measurement of the acid-assisted dissociation of the
complex of Cu2+ with Hno1pa2py was performed under pseudo-first-
order conditions by addition of concentrated aqueous solutions of HCl
to an aqueous solution of the preformed complex at ca. 2 × 10−3 M.
Dissociation was followed by the decrease with time in the intensity of
ACKNOWLEDGMENTS
R.T. acknowledges the Minister
et de la Recherche, the Centre National de la Recherche
■
̀
e de l′Enseignement Super
́
ieur
Scientifique, the ANR program αRIT/βPET, and especially the
financial support from the Region Bretagne, France. R.T. also
́
L
dx.doi.org/10.1021/ic400174r | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(19) Ait-Mohand, S.; Fournier, P.; Dumulon-Perreault, V.; Kiefer, G.
thanks the “Service Commun” of NMR facilities of the
University of Brest. C.P.-I. acknowledges Centro de Super-
E.; Jurek, P.; Ferreira, C. L.; Ben
́
ard, F.; Guer
́
in, B. Bioconjugate Chem.
2011, 22, 1729−1735.
computacion
́
de Galicia (CESGA) for providing the computer
o para a
̌
(20) Simece
̌
k, J.; Wester, H. J.; Notni, J. Dalton Trans. 2012, 41,
facilities. R.D. and L.M.P.L. acknowledge Fundaca
Ciencia e a Tecnologia (FCT), with coparticipation of the
̂
̧
̃
13803−13806.
(21) Fani, M.; Del Pozzo, L.; Abiraj, K.; Mansi, R.; Tamma, M. L.;
Cescato, R.; Waser, B.; Weber, W. A.; Reubi, J.-C.; Maecke, H. R. J.
Nucl. Med. 2011, 52, 1110−1118.
European Community funds FEDER, POCI, QREN, and
COMPETE for the financial support under Project PTDC/
QUI/67175/2006. L.M.P.L. thanks FCT also for a postdoctoral
fellowship (SFRH/BPD/73361/2010).
(22) Gasser, G.; Tjioe, L.; Graham, B.; Belousoff, M. J.; Juran, S.;
Walther, M.; Kunstler, J. U.; Bergmann, R.; Stephan, H.; Spiccia, L.
̈
Bioconjugate Chem. 2008, 19, 719−730.
(23) Palinkas, Z.; Roca-Sabio, A.; Mato-Iglesias, M.; Esteban-Gomez,
D.; Platas-Iglesias, C.; de Blas, A.; Rodriguez-Blas, T.; Toth, E. Inorg.
Chem. 2009, 48, 8878−8889.
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dx.doi.org/10.1021/ic400174r | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
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dx.doi.org/10.1021/ic400174r | Inorg. Chem. XXXX, XXX, XXX−XXX