Bispidine dioxotetraaza macrocycles: A new class of bispidines for 64Cu PET imaging

Article abstract of DOI:10.1021/ic500476u

The three new dioxo-tetraazamacrocyclic ligands with a fused, very rigid bispidine (3,7-diazabicyclo[3.3.1]nonane) group connecting the two tertiary amine donors, and ethyl, propyl, or benzene groups connecting the two amide donors are highly preorganized and lead to very stable, uncharged Cu ^II complexes. Solution spectroscopy and solid state structures indicate that these are square pyramidal with a solvent molecule occupying the apical position. Cyclic voltammetry defines a reversible Cu^III/II couple and a strongly negative irreversible Cu^II/I couple (ca. -2 V vs Fc/Fc⁺), indicating that the Cu^II complexes are very stable in solution. This is supported by superoxide dismutase (SOD) and human serum challenge experiments as well as the biodistribution, which all show that the benzene-based ligand has the highest in vitro and in vivo stability and that this was expected on the basis of the macrocycle ring size and shape and the highest degree of preorganization. This ligand is easy to functionalize for a possible coupling to biological vector molecules and/or fluorescence markers for PET (positron emission tomography) and multimodal imaging (i.e., PET and optical imaging).

Full text of DOI:10.1021/ic500476u

Article
pubs.acs.org/IC
Bispidine Dioxotetraaza Macrocycles: A New Class of Bispidines for
64Cu PET Imaging
Peter Comba,*,† Manja Kubeil,‡ Jens Pietzsch,‡,§ Henning Rudolf,† Holger Stephan,*,‡
and Kristof Zarschler‡
†Anorganisch-Chemisches Institut, Universitat Heidelberg, INF 270, D-69120 Heidelberg, Germany
̈
‡Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, D-01314 Dresden, Germany
§Fachrichtung Chemie und Lebensmittelchemie, Technische Universitat Dresden, D-01062 Dresden, Germany
̈
S
* Supporting Information
ABSTRACT: The three new dioxo-tetraazamacrocyclic ligands with a fused,
very rigid bispidine (3,7-diazabicyclo[3.3.1]nonane) group connecting the two
tertiary amine donors, and ethyl, propyl, or benzene groups connecting the two
amide donors are highly preorganized and lead to very stable, uncharged CuII
complexes. Solution spectroscopy and solid state structures indicate that these
are square pyramidal with a solvent molecule occupying the apical position.
Cyclic voltammetry defines a reversible CuIII/II couple and a strongly negative
irreversible CuII/I couple (ca. −2 V vs Fc/Fc+), indicating that the CuII
complexes are very stable in solution. This is supported by superoxide dismutase
(SOD) and human serum challenge experiments as well as the biodistribution,
which all show that the benzene-based ligand has the highest in vitro and in vivo
stability and that this was expected on the basis of the macrocycle ring size and
shape and the highest degree of preorganization. This ligand is easy to
functionalize for a possible coupling to biological vector molecules and/or fluorescence markers for PET (positron emission
tomography) and multimodal imaging (i.e., PET and optical imaging).
CuII complexes have been shown to have too low in vivo
INTRODUCTION
■
stabilities, for example, some are decomposed in vivo by
There is increasing interest in the development of applications
superoxide dismutase and ceruloplasmin.19−21 Also, the syn-
of radiotracers for cancer diagnosis. An important method is
thesis of polyaza macrocycles and the corresponding bio-
positron emission tomography (PET), where a radioactive
conjugates may be tedious (multiple step syntheses),22−25 and
positron emitting isotope is linked to a biological vector such as
complex formation may be slower than that required for in vivo
a protein, peptide, or aptamer that specifically accumulates in
PET imaging.4
cancer cells. With positron emitters, for example 64Cu ions, an
important focus is on the design of bifunctional chelators
Dioxotetraaza macrocyclic ligands have attracted interest for
a number of reasons: due to the macrocycle effect and the
(BFCs) that coordinate the β+ emitting metal ion with high
stabilization by two deprotonated amide nitrogen donors, they
stability and offer functional groups for conjugation to
are known to form highly stable and uncharged CuII complexes;
biological vector molecules. Because of its suitable half-lifetime
of 12.7 h and its decay scheme (β+, 19%; β−, 40%; and electron
and due to the deprotonated amide donors, these ligands are
also known to stabilize CuIII.26,27 In the context of PET
capture 40%), 64Cu is an ideal positron emitter for PET
imaging, it is specifically the high stability of the uncharged CuII
complexes that has attracted our attention.28−30 Another ligand
backbone known for its suitability as a BFC for 64Cu-based PET
imaging is the highly preorganized bispidine platform with its
rigid adamantane-derived 3,7-diazabicyclo[3.3.1]nonane back-
bone.31−35 This type of ligand, first discovered by Mannich and
Mohs,36 offers a widely variable donor set which allows the fine-
tuning of the electronic properties of the resulting com-
plexes.32,34 There is a wide range of applications of bispidine
transition metal chemistry,32,37,38 but there are only few reports
imaging, and the “matched-pair” 64Cu/67Cu offers possible
applications in diagnosis and therapy.1−5 There is a range of
well-known ligand systems for stable and well-defined CuII
complexes, and derivatives of these ligands have been used as
BFCs for PET imaging. Examples are ATSM (diacetyl-bis(N4-
methylthiosemicarbazone),6,7 derivatives of aza-macrocycle
ligand systems, including TETA and DOTA,8−13 derived
from the 14- and 12-membered cyclam- and cyclen-type aza-
macrocycles, and hexamine cage ligands.14−16 An advantage of
these systems is the high thermodynamic stability (macrocyclic
effect17,18) and facile methods for the attachment of
functionalities such as carboxylates, where a biofunctionaliza-
tion can be performed. However, some of the corresponding
Received: February 28, 2014
Published: June 6, 2014
© 2014 American Chemical Society
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Chart 1. Ligands Discussed
Scheme 1. Synthesis of the Macrocyclic Ligands H2LB, H2LE, and H2LP
on macrocyclic bispidines39,40 and uncharged CuII complexes of
bispidine-based ligands.41
Here, we combine the two motifs of the dioxotetraaza
macrocycles with the rigidity of the bispidine backbone and
report the synthesis of three derivatives of a new type of ligand
with varying ring size and preorganization, LB, LE, and LP (see
Chart 1), together with their CuII complexes.
mides)43 has found broad application in the synthesis of
dioxotetraaza macrocycles.28 1,2-Diaminoethane, 1,3-diamino-
propane, and o-phenylendiamine were condensed with
chloroacetyl chloride to yield the bis(α-chloroacetamide)
building blocks; [1 + 1] condensation with 5,7-dimethyl-1,3-
diazabicyclononane, the bispidine building block, resulted in
acceptable yields of the macrocycles H2LB (65%), H2LE (34%),
and H2LP (74%). The CuII complexes of the three macrocycles
were prepared by reaction of the ligand with an equimolar
amount of copper(II) acetate hydrate in methanol overnight;
suitable crystals for X-ray diffraction were obtained by ether
diffusion or evaporation of the methanolic complex solution at
ambient temperature.
RESULTS AND DISCUSSION
■
Synthesis. The three macrocycles H2LB, H2LE, and H2LP
were synthesized by cyclization of 5,7-dimethyl-1,3-diazabicy-
clononane42 with the bis(α-chloroacetamide) of the corre-
sponding diamine (Scheme 1). The use of bis(α-chloroaceta-
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Crystal Structures. The molecular structures of the ligands
are shown in Figure 1. In each of the structures, one of the
Figure 2. ORTEP (POV-ray) plots of the complexes [Cu(LB)(O)],
[Cu(LE)(OH2)], and [Cu(LP)(OH2)]; hydrogen atoms have been
omitted for clarity, and ellipsoids are shown at 50% probability.
Figure 1. ORTEP (POV-ray) plots of the ligands H2LB, H2LE, and
H2LP; hydrogen atoms have been omitted for clarity, and ellipsoids are
shown at 50% probability.
Table 1. Selected Bond Distances and Angles of
[Cu(LB)(O)], [Cu(LE)(OH2)], and [Cu(LP)(OH2)]
amide protons is pointing toward the opposite tertiary amine,
stabilized by hydrogen bonding with N−H···N distances of
2.767(13) Å (H2LB), 2.183(13) Å (H2LE), and 2.908(8) Å
(H2LP) [hydrogen atoms are located at calculated positions,
and the corresponding N···N distances are 3.6360(19) Å
(H2LB), 3.067(2)Å (H2LE), and 3.7626(12) Å (H2LP)]. The
other amide proton is located proximal to the mean plane
defined by the four nitrogen donors of the macrocycle (N4
plane). The hole size of the macrocyclic ligands44 increases in
the order 1.875 Å (H2LB) < 1.962 Å (H2LE) < 2.042 Å (H2LP),
i.e., in terms of cavity size, H2LP appears to be the most suitable
ligand for CuII. Affected by the rigidity of both the bispidine
backbone and the benzyl ring as well as the planarity of the
amide groups, H2LB shows the highest degree of rigidity and
preorganization.45−47 Both H2LP and H2LB are largely planar
macrocyclic ligands, and H2LE, due to the puckered ethylene
bridge, shows a slightly more distorted N4 arrangement.
distance (esd) [Å]
[Cu(LB)(O)] [Cu(LE)(OH2)] [Cu(LP)(OH2)]
Cu−N(1)
Cu−N(2)
Cu−N(3)
Cu−N(4)
1.972(3)
1.898(2)
1.9935(8)
1.9006(8)
2.0193(17)
1.9356(16)
1.9332(14)
2.0352(17)
2.3942(16)
2.819(2)
1.898(3)
1.8890(9)
1.984(3)
1.9841(8)
Cu−Oaxial
2.300(2)
2.3239(8)
N(1)···N(4)
Cu···N4 (out-of-plane)
angle (esd) [deg]
2.862(4)
2.8783(11)
0.38(13)
[Cu(LB)(O)]
0.37(4)
[Cu(LE)(OH2)]
0.27(10)
[Cu(LP)(OH2)]
N(1)−Cu−N(2)
N(2)−Cu−N(3)
N(3)−Cu−N(4)
N(4)−Cu−N(1)
87.40(12)
85.19(12)
87.41(12)
92.71(12)
87.05(3)
85.82(4)
86.23(4)
92.71(3)
84.84(7)
96.94(8)
85.45(8)
88.08(7)
since a 13-membered macrocycle is known to be slightly too
small for CuII.48 The analysis of the unit cell reveals that the
coordination of the copper center is completed by an axial
amide oxygen of a neighboring molecule at 2.300 Å (see
Supporting Information). A square pyramidal coordination
geometry is also in agreement with the (solution) spectroscopic
data (see below; for this reason, the LB-derived complex is
described as [Cu(LB)(O)] in the crystal structure and generally
as [Cu(LB)(solv)] since we assume that, in solution, a solvent
Plots of the experimental molecular structures of the CuII
complexes of H2LB, H2LE, and H2LP appear in Figure 2, and
selected geometric parameters are listed in Table 1. The two
amides are deprotonated in all three complexes, leading to
neutral molecules. [Cu(LB)(O)] has a slightly distorted square
pyramidal coordination geometry with the copper center
displaced by 0.38 Å from the N4 plane; this is not unexpected
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molecule (e.g., OH2) coordinates in an apical position, leading
to a square pyramidal structure as in the crystal and with the
other ligands).
parameters discussed below. Interestingly, the electronic
properties of the dioxotetraaza macrocyclic ligand CuII
complexes not supported by bispidine platforms (electronic
spectra also given in Table 2) indicate that the optimum ring
size in that case is 14, and this is similar to what is observed for
tetraaza macrocycles.48 The increased rigidity of the bispidine-
type systems discussed here leads to a modification of the
structural and thermodynamic properties, and this also emerges
from the structural analysis above.
The experimental EPR spectra were recorded in frozen
solution in MeOH/EtOH (9:1) at X-band frequency; see
Figure 3 and Table 3 for the experimental and simulated
spectra and the spin Hamiltonian parameters, obtained by
simulation of the experimental spectra with the X-Sophe
software package.49 The relatively large values of Az >200 ×
10−4 cm−1, in combination with the relative small values of gz ≤
2.18, indicate a strong in-plane ligand field with relatively small
interactions to axial ligands, specifically for the H2LB- and H2LE-
based systems, and this is in agreement with the structural data
and solution electronic spectroscopic parameters. The small
variation of Ax/gx vs Ay/gy values indicate a small but significant
deviation from tetragonal symmetry.
Redox Properties. The electrochemical properties of the
CuII complexes H2LB, H2LE, and H2LP were studied in H2O,
MeOH, MeCN, and DMF to investigate the stabilization of the
CuII complexes and also a potential stabilization of CuIII
species. The experiments were performed in a three electrode
glass cell under argon atmosphere, using a glassy carbon
working electrode, a platinum wire as counter electrode, and an
Ag/AgCl or an Ag/AgNO3 reference electrode under aqueous
or nonaqueous conditions, respectively. In water as solvent, 3
M NaCl was used as supporting electrolyte, and the potentials
were normalized against K3[Fe(CN)6] (E = 298 mV); in
MeOH and DMF, 0.1 M Bu4NBF4, and in MeCN, 0.1 M
Bu4NPF6 were used as supporting electrolytes, and the
potentials were normalized against ferrocene, with −94 mV
(DMF), 120 mV (MeOH), and −87 mV (MeCN). The results
of the electrochemical measurements are given in Table 4.
The reversible potentials in MeCN, MeOH, H2O, and DMF
can be assigned to CuIII/II couples, and the corresponding redox
potentials (see Table 4) are in the expected range.26 While the
cavity size of the three macrocycles studied here increases in the
order LB < LE < LP, the energy needed to oxidize the CuII
complexes increases in the order LE < LB < LP, i.e., LE best
stabilizes high-valent copper ions. The CuII/I potentials could
only be measured in DMF, which offers the largest electro-
chemical window and showed a potential at < −2 V, indicating
a very high stabilization of the CuII complexes. The LP and LB
systems have very similar CuII/I potentials (slightly higher for
the LP-based couple), and the ethylene-bridged macrocycle has
the most positive potential.
Radiochemistry. The three macrocycles were evaluated as
potential ligands for radiolabeling with 64CuII. The radiolabeling
was monitored by radio-TLC, using neutral alumina plates and
2 M NH4OAc/MeOH (1:1) as mobile phase. [64Cu]CuCl2
remains at the starting position, and the complexes move with
Rf values of 0.75−0.79. Standard labeling experiments, using
MES/NaOH (pH 5.5) at ambient temperature, gave, as
expected, no evidence for 64CuII-labeled macrocyclic bispidine
ligands. Increasing the temperature to 50 °C and using a
slightly higher pH significantly increased the labeling efficiency.
The radiolabeling yields for H2LB, H2LE, and H2LP (100 μg
ligand/0.1 mL aqueous buffer) as a function of time are
The molecular structure of the other complex with a 13-
membered macrocycle, [Cu(LE)(OH2)], is similar to that of
[Cu(LB)(O)]. The distances of the donor atoms to the copper
center are on average 0.004 Å longer, and this is attributed to
the more flexible and approximately 0.118 Å longer ethyl bridge
(observed N···N distance in the experimental structures). The
emerging slightly larger cavity also leads to a decrease of the
displacement of the copper center from the N4 plane by 0.013
Å. In this structure, a water molecule is coordinated as an axial
ligand, completing the square pyramidal coordination sphere.
H2LP is the only one of the three ligands with a 14-
membered macrocyclic ring, known to be complementary for
CuII. Because of the larger cavity, the copper center is less
displaced from the center of the macrocyclic ring by 0.1 Å
compared to [Cu(LB)(O)] and [Cu(LE)(OH2)]. The mean
Cu−N distances are 0.044 Å (0.038 Å) longer than these in
[Cu(LB)(O)] ([Cu(LE)(OH2)]), but the distance of the two
bispidine nitrogens N(1) and N(4) decreases by 0.053 Å (0.059
Å). This emerges from the larger propylene bridge in
[Cu(LP)(OH2)], leading to a slightly expanded N(2)−Cu−
N(3) angle. Similar to [Cu(LE)(OH2)], [Cu(LP)(OH2)] has a
coordinated water molecule in the apical position, resulting in a
square pyramidal coordination geometry.
The structural reorganization parameter Σn6= 1|Δdn| offers the
opportunity to quantify the degree of preorganization of a
ligand. The structural reorganization is the sum of deviation of
donor atom···donor atom distances upon coordination of the
ligand to a metal ion.45 The comparatively small values of
Σn6= 1|Δdn| indicate a high degree of preorganization,45 and the
calculated structural reorganization parameters for the ligands
H2LB, H2LE, and H2LP validate the assumption that the degree
of preorganization decreases in the row H2LB (0.44) > H2LP
(0.91) > H2LE (1.21).
Electronic Properties. The UV−vis spectra were recorded
in methanolic solution, and the absorption maxima and
extinction coefficients are presented in Table 2 (see Supporting
Table 2. Maxima in Absorption and Extinction Coefficients
of the CuII Complexes of H2LB, H2LE, and H2LP (Measured
in MeOH) and Literature-Known Dioxo-Tetraaza
Macrocyclic Ligands
λ [nm]
ν [cm−1
]
ε [M−1·cm−1
]
[Cu(LB)(solv)]
483
476
524
620
520
505
520
20,700
21,010
19,080
16,130
19,230
19,800
19,230
149
[Cu(LE)(OH2)]
117
[Cu(LP)(OH2)]
189
[Cu(dioxo(12)aneN4)]
[Cu(dioxo(13)aneN4)]
[Cu(dioxo(14)aneN4)]
[Cu(dioxo(15)aneN4)]
15026
10026
10026
10026
Information for the spectra). Compared to the 13-membered
ring systems of [Cu(LB)(solv)] and [Cu(LE)(OH2)], the dd
transitions (asymmetric absorption band involving the t2g
→
dx2‑y2 transitions) of [Cu(LP)(OH2)] with a 14-membered
macrocyclic ligand are shifted by close to 2,000 cm−1 to lower
energy. This is due to the expected decrease of the ligand field
due to slightly longer bonds. This indicates that the solution
structures are similar to those observed in the crystals (see
above), and this also agrees with the frozen solution EPR
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ligand/0.1 mL aqueous buffer) after 90 min at 50 °C. The
specific activity obtained is 26 GBq/μmol for [64Cu(LB)(solv)];
for the other two complexes, [64Cu(LE)(OH2)] and [64Cu-
(LP)(OH2)], about 22 GBq/μmol was determined.
Stability Assays. The stability of the 64CuII-labeled
macrocyclic bispidine-amide ligands was studied in the presence
of a 3-fold excess of human superoxide dismutase (SOD) and
human serum, as described before.50 Figure 4 summarizes the
quantitative results of dissociated 64CuII in the presence of SOD
and human serum protein. The complex with the benzyl-ring-
based macrocycle [64Cu(LB)(solv)] shows the highest stability
toward SOD (transchelation of (1.5 0.5) %) and serum (2.3
0.9) % after 1 h of incubation at 37 °C. The radiocopper(II)
dissociation from [64Cu(LE)(OH2)] and [64Cu(LP)(OH2)] is
somewhat higher in the presence of excess SOD and human
serum (see Supporting Information for more details). That is,
the variation of the macrocycle ring size and preorganization of
the macrocyclic bispidine-amide ligands strongly influence the
stability of the copper(II) complexes. Obviously, the donor set
and overall charge of the ligand as well as the efficiency of the
shielding of the CuII center are also of importance, and this is
shown with the other 64CuII complexes subjected to the same
assays (see Figure 4 and Chart 1 for ligand structures; see
Supporting Information for more detail): increased shielding of
the CuII center and increased electron density at the pyridine
nitrogen donors of open-chain bispidine ligands lead to an
increase of the complex stability,34,37,51 and the same trend was
observed in an assay using an 80-fold excess of cyclam as
competing ligand.35,51 The three complexes 64Cu-L2, 64Cu-L3,
and 64Cu-LB have similar and high in vitro stabilities with less
than 5% of 64CuII dissociation in the SOD and human serum
assays. Importantly, these assays indicate that the stability of
[64Cu(LB)(solv)] toward transchelation to proteins and
enzymes is very similar to that of the open-chained bispidine
complexes (see above) and well-established 64CuII complexes
with other ligand systems; for example, the 64Cu-diamsar, 64Cu-
cyclam, and 64Cu-NOTA complexes have SOD and human
serum values of 0.6/1.7, 1.0/2.2, and 1.4/2.1.50
Lipophilicity. The log P values can be derived from the
distribution of neutral compounds in a two-phase octanol−
water system. For ionic compounds such as transition metal
complexes, the log D value at physiologically relevant pH is
more appropriate to predict the type of tissue in which the
substance will accumulate and which clearance path it will
take.52 As expected, the CuII complexes of dioxo-tetraaza-
macrocyclic bispidine ligands with aliphatic bridges, H2LP and
H2LE, are more hydrophilic (log D7.4 = −2.22 and −2.33; see
Table 6) than the CuII complex of H2LB, which, due to the
aromatic ring, is slightly lipophilic (log D7.4 = 0.21). As a
consequence of the slightly hydrophobic character, it is
expected that [64Cu(LB)(solv)] will be partially excreted via
the hepatobiliary route. The in vitro stability of [64Cu(LB)-
(solv)] in the presence of human serum and SOD is high and
comparable to that of the 64CuII complexes of the hexadentate
33
bis-amine-tetrakis-pyridine bispidine L2 and the pentadentate
Figure 3. X-band EPR spectra (experimental, black, bottom;
simulated, red, top) of the complexes (a) [Cu(LB)(solv)] at 106 K
and 9.441338 GHz, (b) [Cu(LE)(OH2)] at 107 K and 9.438382 GHz,
and (c) [Cu(LP)(OH2)] at 109 K and 9.448705 GHz.
ligand with p-methoxy substitution of the pyridinyl groups L3
(vide supra).35 The log D7.4 values of the latter radiotracers are
significantly lower (64Cu-L2, −2.77; 64Cu-L3, −2.44), and
therefore, there are now highly stable 64CuII bispidine
complexes with very different hydrophilicities available. This
will allow one to influence the biodistribution of the
biofunctionalized radiotracers by choosing the most appropriate
bifunctional bispidine chelator.
summarized in Table 5 and show that more than 95% of the
bispidine-based macrocycles are labeled with 64CuII after 1 h.
Almost quantitative labeling of the macrocyclic bispidine
ligands was also achieved at lower concentrations (5 μg
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Table 3. Simulated g- and A-values (10−4 cm−1) of the Complexes [Cu(LB)(solv)], [Cu(LE)(OH2)], and [Cu(LP)(OH2)]
(Simulations by X-Sophe49) and Literature-Known CuII Complexes of Dioxo-tetraaza Macrocyclic Ligands
[Cu(LB)(O)]
[Cu(LE)(OH2)]
[Cu(LP)(OH2)]
[Cu(o-13N4)]68
[Cu(o-14N4)]68
gz
2.154
2.046
2.018
209.8
25.1
2.157
2.047
2.019
208.8
22.6
2.179
2.044
2.037
203.9
26.0
2.171
2.046
2.046
202.8
28.5
2.176
2.049
2.049
207.4
37.1
gx
gy
Az
Ax
Ay
27.4
22.6
24.3
28.5
37.1
AN,z
AN,x
AN,y
10.9
9.9
11.0
11.7
12.3
13.2
12.9
13.0
13.1
Table 4. CuIII/II (in MeCN, H2O, MeOH, and DMF) and
CuII/I Potentials (in DMF, Irreversible) of the CuII
Complexes of H2LB, H2LE, and H2LP
MeCN
a
H2O
MeOH
a
b
b
[mV]
[mV]
[mV]
DMF [mV]
[Cu(LB)(solv)]
[Cu(LE)(OH2)]
310
121
451
219
−17
−88
266
−29; −2264
(irrev.)
71
118; −2116
(irrev.)
[Cu(LP)(OH2)]
418
−258; −2253
(irrev.)
a
b
vs Fc/Fc+. vs K3[Fe(CN)6]).
Table 5. Radiochemical Yield (RCY) as a Function of Time
a
for the 64Cu-Labeling of H2LB, H2LE, and H2LP at 50 °C
Figure 4. 64Cu transchelation [% of control] in SOD (black) and
human serum (gray) for 64Cu-labeled H2LE, H2LP, H2LB, L1, L2, L3,
and EDTA, determined after incubation of the complexes for 1 h at 37
°C; each point represents the mean and standard deviation of three
samples.
duration [min]
RCY [%]
[64Cu(LB)(solv)]
[64Cu(LE)(OH2)]
[64Cu(LP)(OH2)]
5
78 (2)
93 (2)
>95
30
60
5
83 (2)
96 (2)
>98
30
60
5
pathway). At 24 h p.i., nearly the entire activity is found in feces
and urine. The overall fast clearance from the liver and other
organs and tissues indicates a high stability of the 64CuII
complex of H2LB, and this is in full agreement with the
stability assays reported above.
80 (4)
94 (2)
>98
30
60
a
100 μg ligand/100 μL MES-NaOH buffer at pH 6.5. Shown are the
averages of two independent experiments with standard deviation in
parentheses.
CONCLUSIONS
■
The ligands H2LB, H2LE, and H2LP are a new class of
macrocyclic bispidine ligands with variable macrocycle hole
shape and size and different degrees of preorganization.
Because of deprotonation of the amide functions upon
coordination, the CuII complexes are uncharged. Cyclic
voltammetry reveals a high stability of the CuII species and
also shows that CuIII complexes are stabilized. The (frozen)
solution EPR and electronic spectra of the CuII compounds
indicate that the complexes are square-pyramidal in solution
with strong in-plane ligand fields and weak interactions to axial
ligands, and this is in agreement with the solid state structural
data. Radiotracer experiments with 64CuII show a relatively
rapid labeling within 1 h at 50 °C with >95% yield and specific
activities of 26 GBq/μmol. Challenge experiments with SOD
and human serum indicate a high stability of the slightly
lipophilic complex [64Cu(LB)(solv)]. Biodistribution studies
showing rapid blood and tissue clearance support the high
complex stability in vivo. In terms of labeling, specific activity,
and stability, [64Cu(LB)(solv)] is well suited for PET
application, and preliminary experiments indicate that efficient
biofunctionalization of H2LB with targeting peptides or
Biodistribution. The biodistribution data of [64Cu(LB)-
(solv)] in rats demonstrate at 5 min post injection (p.i.)
predominant activity accumulation in kidneys, liver, and
intestine. In this early distribution phase, more than half of
the injected dose (61 7) % ID is already eliminated via the
hepatobiliary system into the intestine (intestine plus feces). At
this time point, the white adipose tissue (WAT), muscle, and
skin also show a significant accumulation of activity. This
biodistribution pattern in the early distribution phase is
expected from the modest hydrophobicity of the complex.
Subsequently, [64Cu(LB)(solv)] is efficiently eliminated from
blood and tissues (% ID/g for most organs and tissues <0.13 at
60 min p.i. and <0.07 at 24 h p.i., respectively; see Supporting
Information for details) with only substantial but minor residual
activity in both the kidneys (0.39 0.02) % ID/g and the liver
(0.42 0.04) % ID/g at 60 min p.i. and only in the kidneys
(0.16 0.02) % ID/g after 24 h. The hepatobiliary pathway
nearly exclusively contributes to the fast clearance of radio-
activity observed with (80 6) % ID found in the intestine at
60 min p.i. (also accounting for activity in the excretion
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Table 6. Experimental Octanol−Water Partition Coefficients (log Do/w) of the 64Cu Bispidine-dioxo-tetraaza Macrocyclic
a
Ligand Complexes at Different pH Values
pH 7.2
pH 7.4
pH 7.6
[64Cu(LB)(solv)]
[64Cu(LE)(O2H)]
[64Cu(LP)(O2H)]
0.21 (0.01)
0.21 (0.01)
0.22 (0.02)
−2.35 (0.03)
−2.22 (0.01)
−2.33 (0.03)
−2.22 (0.01)
−2.27 (0.02)
−2.15 (0.01)
a
Shown are the averages of three independent experiments with the standard deviation in parentheses.
EPR measurements were performed on a Bruker ELEXSYS-E-500
instrument with liquid nitrogen, in frozen methanol/ethanol (9:1)
solution. The spin Hamiltonian parameters were obtained by
simulation of the experimental data with the XSophe software
package.53,54 ESI-MS spectra were recorded on a Bruker ApexQe
hybrid 9.4 T FT-ICR.
Elemental analyses were obtained from a CHN-O-vario EL by the
“Mikroanalytische Labor”, Department of Organic Chemistry,
University of Heidelberg.
Crystal structural data and deteremination details are given as
Supporting Information. Data collection was performed with a Bruker
AXS Smart 1000 CCD diffractometer (Mo−Kα radiation, sealed tube,
graphite monochromator) or with an Agilent Technologies Super-
nova-E CCD diffractometer (Mo− or Cu−Kα radiation, microfocus
tube multilayer mirror optics). Correction for air and detector
absorption were performed, as well as Lorentz and polarization
effects.55,56 Semiempirical multiscan methods56−59 were used to
correct the absorption by the crystal or an analytical56,60 or numerical
treatment.56,60,61 Using Olex2,62 structures were solved by charge
flipping63,64 or conventional direct methods.65,66 Structures were
refined by full-matrix least-squares methods based on F2 against unique
reflections.65,66 All non-hydrogen atoms were given anisotropic
displacement parameters and were put at calculated positions and
refined with a riding model. CCDC 988221-988226 contains the
supplementary crystallographic data for this Article. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
Figure 5. 64Cu biodistribution of [64Cu(LB)(solv)] in male Kyoto-
Wistar rats, four animals per time point (mean
SD). Insert: I*,
intestine plus feces; I, intestine; U, urine; and U*, urine plus feces.
antibodies is possible by introducing a carboxylic acid linker at
the aromatic bridge.
Radiochemistry. The production of 64Cu was performed at a PET
cyclotron as described in detail elsewhere.67 The yields of the nuclear
reaction 64Ni(p,n)64Cu were between 3.6−5.2 GBq (EOB) with
specific activities of 150−250 GBq/μmol Cu. The bispidine ligands
were radiolabeled by adding 50−200 MBq of [64Cu]CuCl2 to 0.1 to
100 μg of H2LB, H2LE, and H2LP dissolved in 100 μL of 0.05 M 2-[N-
morpholino]ethansulfonic acid (MES)-NaOH buffer (pH 5.5, 6.0, and
6.5). The radiolabeling yield was determined by radio-TLC (stationary
phase, neutral alumina plates (Merck, F254); mobile phase, methanol/
2 M ammonium acetate (1/1, v/v); 64CuCl2, Rf = 0; and 64Cu−
bispidine complexes, Rf = 0.75−0.79). Radio-TLC chromatograms
were scanned using a radioisotope thin layer analyzer (Rita Star,
raytest). The labeling efficiency was investigated as a function of pH,
time, temperature, and ligand concentration. For biodistribution
experiments, H2LB (10 μg dissolved in 250 μL of 1 M MES/NaOH
buffer (pH 6.5)) was radiolabeled with [64Cu]CuCl2 (120 MBq) for
90 min at 50 °C. Radio-TLC confirmed full complexation. The
radiochemical purity of [64Cu(LB)(solv)] was also determined by
radio-HPLC on a HPLC Hewlett-Packard System (Series 1100);
activity detector, Raytest Ramona Star; column, Zorbax (Agilent)
300SB-C18, 300 Å, 5 μm, 250 mm × 9.4 mm; gradient, eluent A
(water + 0.1% TFA) and eluent B (acetonitrile + 0.1% TFA), 5 min
95% A, 10 min 95% A to 95% B, 10 min 95% B, and 1 mL/min
([64Cu(LB)(solv)]; tR = 13.6 min).
EXPERIMENTAL SECTION
■
General Procedures and Spectroscopic Measurements. All
reactions were carried out under air. Chemicals and solvents were
purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen,
Germany), ABCR GmbH & Co. KG (Karlsruhe, Germany), or
Merck KGaA (Darmstadt, Germany) and were used without further
purification. The bispidine building block (1,5-dimethyl-3,7-
diazabicyclo[3.3.1]nonane) was synthesized as published.42
Electrochemistry was performed on a CH Instruments CHI660D
electrochemical workstation, equipped with a CH Instruments
Picoamp Booster. All electrochemical measurements were performed
in a glass cell, covered with a Teflon cap, situated in a Faraday cage. All
complex solution were prepared in degassed solvents saturated with
Ar. The supporting electrolyte for nonaqueous solutions [dimethyl-
formamide (DMF) and methanol (MeOH)] was 0.1 M tetra-n-
butylammonium tetrafluoroborate, and for acetonitrile (MeCN), 0.1
M tetra-n-butylammonium hexafluorophosphate was used. All spectra
in nonaqueous media were recorded with an Ag/AgNO3 reference
electrode, a glassy carbon working electrode, and a platinum wire as
counter electrode. Aqueous solutions contained 3 M sodium chloride
(NaCl) as supporting electrolyte and were performed with an Ag/
AgCl reference electrode, a glassy carbon working electrode and a
platinum wire as counter electrode. Electrochemical measurements in
nonaqueous media were normalized versus ferrocene, where ferrocene
had the following potentials: DMF (−94 mV), MeOH (120 mV), and
MeCN (−87 mV); in aqueous media, potentials were normalized
against potassium hexacyano ferrate (K3[Fe(CN)6]) with 298 mV.
Potentials marked with a * are assigned as irreversible.
Determination of the Distribution Ratio log Do/w at 25
1
°C. Information about the lipophilicity of the 64CuII-labeled bispidine
dioxotetraaza macrocycles H2LB, H2LE, and H2LP was obtained by
distribution experiments in buffer/1-octanol mixtures. Aqueous
solutions of the bispidine ligands (1 mM in MES/NaOH, pH 6.5)
were pre-equilibrated in the presence of 100 μM Cu(NO3)2, spiked
with [64Cu]CuCl2 (3 MBq) for 1 h at 50 °C. Full complexation was
checked by radio-TLC to give no evidence of free 64CuII. Then, 50 μL
of the 64CuII-labeled bispidine complexes, dissolved in MES/NaOH
UV−vis-NIR spectra were recorded with a Jasco V-570 UV−vis-
near-IR spectrometer as methanolic solutions. NMR spectra were
1
recorded on a Bruker Avance I (200 MHz), and chemical shifts of H
and 13C are referenced to solvent resonances (CDCl3).
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buffer, was taken and added to 450 μL of 0.05 M 4-(2-hydroxyethyl)-
1-piperazine ethanesulfonic acid (HEPES)-NaOH solutions (pH 7.2,
7.4, and 7.6). The aqueous buffered solutions (HEPES/NaOH buffer)
consist of 100 μM bispidine ligands, 10 μM Cu(NO3)2, and 300 kBq
[64Cu]CuCl2. The distribution experiments in 1-octanol/buffer
2,10-Dioxo-6,19-dimethyl-1,4,8,11-tetraazatetracyclo-
[12.3.3.16,19.012,17]-heneicosa-12,14,16-triene (H2LB). 1,5-Di-
methyl-diazabicyclo[3.3.1]nonane (0.77 g; 5.00 mmol; 1 equiv) and
N,N′-bis(chloroacetyl)-1,2-phenylendiamine (1.30 g; 5.00 mmol; 1
equiv) were refluxed with Na2CO3 (5.30 g; 50.00 mmol; 10 equiv) in
acetonitrile (250.00 mL) for 24 h. After cooling to room temperature,
the solid was removed by filtration, and the solvent was evaporated.
The residue was redissolved in a small amount of ethanol, and after the
addition of water, the solution was left to crystallize uncovered at room
temperature. The product (1.11 g; 3.24 mmol; 65% yield) forms
systems were performed at 25
1 °C in microcentrifuge tubes (2
cm3) by means of mechanical shaking for 30 min. The phase ratio
V(1‑octanol)/V(aq) was 1:1 (0.5 cm3 each). All samples were centrifuged
and the phases separated, and the copper(II) concentration was
determined in both phases radiometrically using γ-radiation [64Cu,
NaI(Tl) scintillation counter automatic gamma counter 1480, Wizard
3″, PerkinElmer]. The results are the average values of three
independent experiments.
1
colorless block shaped crystals that were dried in vaccuo. H NMR
(CDCl3, 200 MHz): δ [ppm] = 0.80 (s, CH3, 6H), 1.16 (s, CH2, 2H),
2.14−2.19 (d, CH2,ax,eq, J = 10.7 Hz, 4H), 2.76−2.82 (d, CH2,ax,eq, J =
11.0 Hz, 4H), 2.97 (s, NCH2CO, 4H), 7.06−7.11 (m, Harom, 2H),
7.93−7.98 (m, Harom, 2H), 9.49 (bs, NH, 2H). 13C NMR (CDCl3, 100
MHz): δ [ppm] = 25.77, 33.10, 46.18, 62.56, 63.90, 121.30, 124.80,
127.81, 169.20. Elemental analysis: calcd C(66.65), H(7.65),
N(16.36); exp C(66.73), H(7.65), N(16.36). Mass spectrometry:
HR-ESI MS (MeOH) m/z = 343.21299 (calcd 343.21340)
[H2LB+H]+, 365.19490 (calcd 365.19535) [H2LB+Na]+, 381.16887
(calcd 381.16928) [H2LB +K]+, 707.40063 (calcd 707.40092) [2 H2LB
+Na]+, 1049.60673 (1049.60650) [3 H2LB +Na]+.
In Vitro Stability Studies in the Presence of Human Serum
and SOD. The detailed procedure of the in vitro assays in SOD and
human serum was published previously.50 The radiolabeling
conditions of the ligands H2LE, H2LP, and H2LB are described above.
The other bispidine derivatives (L1, L2, and L3) were radiolabeled as
follows: to each ligand (10 nmol, stock solution of 1 mg/mL diluted in
H2O), was added [64Cu]CuCl2 (10−14 MBq in 100 μL of 0.1 M
MES/NaOH-buffer, pH 5.5) followed by 20 min of incubation at 25
°C. Formation of the complexes was verified by radio-TLC, using
methanol/2 M ammonium acetate (pH 6) (1/1, v/v) on a neutral
alumina plate (64CuCl2, Rf = 0; 64Cu-bispidine complexes, Rf = 0.75−
0.79). The radiochemical yield was higher than 99%; otherwise, they
were rejected. For SOD experiments, 64Cu-labeled ligands (0.1 nmol,
1.5 μL) or [64Cu]CuCl2 as reference were added to SOD (0.3 nmol,
10 μg). The mixtures were incubated for 1 h at 37 °C followed by
adding 1 volume of native sample buffer. The samples were separated
using nonreducing and nondenaturing polyacrylamide gel electro-
phoresis (PAGE), and after electrophoresis, the gel was exposed for 10
min to an imaging plate (Fujifilm). Following electronic auto-
radiography using a radioluminography laser scanner, the gel was
stained with PageBlue protein staining solution (Thermo Fisher
Scientific) according to the manufacturer’s instructions. Quantitative
analysis of average band intensities was performed with the Advanced
Image Data Analysis (AIDA) program (Raytest). For serum
experiments, aliquots of human serum “off the clot” (Biochrom AG)
were filtered using syringe filters with a pore size of 0.45 μm. After
mixing filtered serum (220 μL) with 1 M HEPES/NaOH buffer at pH
7.4 (45 μL), [64Cu]Cu-labeled ligands were added. Following
incubation for 1 h at 37 °C, 2× Laemmli sample buffer (400 μL,
Bio-Rad Laboratories) was added. Importantly, the Laemmli buffer
was not supplemented with any reducing agent, and the samples were
not heated. The mixtures were separated using nonreducing SDS−
polyacrylamide gel electrophoresis (SDS−PAGE). After electro-
phoresis and exposure of the gel to an imaging plate (Fujifilm) for
10 min, electronic autoradiography was accomplished using a
radioluminography laser scanner. Finally, the gel was stained with
PageBlue protein staining solution (Thermo Fisher Scientific)
according to the manufacturer’s instructions. As for the SOD
challenge, quantitative analyses of average band intensities were
performed with the AIDA software.
2,10-Dioxo-6,15-dimethyl-1,4,8,11-tetraazatricyclo-
[8.3.3.16,15]heptadecane (H2LE). 1,5-Dimethyldiaza-bicyclo[3.3.1]-
nonane (0.77 g; 5.00 mmol; 1 equiv) and N,N′-bis(chloroacetyl)-1,2-
ethylendiamine (1.06 g; 5.00 mmol; 1 equiv) were refluxed with
Na2CO3 (5.30 g; 50.00 mmol; 10 equiv) in acetonitrile (250.00 mL)
for 24 h. After cooling to room temperature, the solid was removed by
filtration, and the solvent was evaporated. The product (0.50 g; 1.70
mmol; 34% yield) was crystallized as small white crystals from hot
1
water. H NMR (CDCl3, 200 MHz): δ [ppm] = 0.77 (s, CH3, 6H),
1.09 (s, CH2, 2H),1.87−1.94 (d, CH2,ax,eq, J = 11.3 Hz, 2H), 1.98 (s,
NCH2CO, 2H), 2.29−2.36 (d, CH2ax,eq, J = 10.8 Hz, 2H), 2.49−
2.55 (d, CH2,ax,eq, J = 10.8 Hz, 2H), 2.79−2.85 (d, CH2ax,eq, J = 11.3
Hz, 2H), 2.89 (s, NCH2CO, 2H), 3.20−3.22 (m, NHCH2,
2H),3.39−3.45 (m, CH2NH, 2H). 13C NMR (CDCl3, 100 MHz): δ
[ppm] = 25.46, 32.88, 37.53, 41.39, 42.79, 46.10, 58.76, 60.59, 62.54,
65.05, 171.33, 174.11. Elemental analysis: calcd (H2LE +1 H2O) calcd
C(57.65), H(9.03), N(17.93); obsd C(57.54), H(9.17), N(17.75).
Mass spectrometry: HR-ESI MS (MeOH) m/z = 295.21298 (calcd
295.21340) [H2LE+H]+, 611.40053 (calcd 611.40092) [2 H2LE+Na]+,
905.60640 (calcd 905.60650) [3 H2LE+Na]+.
2,10-Dioxo-6,16-dimethyl-1,4,-8,11-tetraazatricyclo-
[9.3.3.16,16]oxtadecane (H2LP). 1,5-Dimethyldiaza-bicyclo[3.3.1]-
nonane (0.77 g; 5.00 mmol; 1 equiv) and N,N′-bis(chloroacetyl)-1,3-
propylendiamine (1.13 g; 5.00 mmol; 1 equiv) were refluxed with
Na2CO3 (5.30 g; 50.00 mmol; 10 equiv) in acetonitrile (250.00 mL)
for 24 h. After cooling to room temperature, the solid was removed by
filtration, and the solvent was evaporated. The product (1.14 g; 3.71
mmol; 74% yield) was crystallized as small colorless needles from
aqueous ethanol by evaporation at room temperature. 1H NMR
(CDCl3, 200 MHz): δ [ppm] = 0.78 (s, CH3, 6H), 1.09 (s, CH2, 2H),
1.70−1.78 (quint, J = 5.56, CH2CH2CH2, 2H), 1.96−2.02 (d, J = 10.7
Hz, CH2ax,eq, 4H), 2.67−2.73 (d, J = 10.9 Hz, CH2ax,eq, 4H), 2.82 (s,
NCH2CO, 4H), 3.37−3.45 (q, J = 5.18 Hz, CH2, 4H). 13C NMR
(CDCl3, 100 MHz): δ [ppm] = 25.08, 28.57, 32.89, 39.72, 46.53,
62.36, 64.58, 170.96. Elemental analysis: (H2LP+1 H2O) calcd
C(58.87), H(9.26), N(17.16); exp C(58.89), H(9.26), N(17.10).
Mass spectrometry (HR-ESI MS (MeOH)): m/z = 309.22859 (calcd
309.22905) [H2LP+H]+, 331.21054 (calcd 311.21100) [H2LP+Na]+,
347.18449 (calcd 347.18493) [H2LP+K]+, 617.44981 (calcd
617.45028) [2 H2LP+H]+.
Biodistribution Experiments. All animal experiments were
carried out in male Wistar rats (Wistar-Kyoto strain; aged 7−8
weeks, 140−195 g; Harlan Winkelmann GmbH, Borchen, Germany)
according to the guidelines of the German Regulations for Animal
Welfare. The protocol was approved by the local Ethical Committee
for Animal Experiments. The biodistribution experiments were
performed as published in detail elsewhere.35,51 The injection volume
of [64Cu(LB)(solv)] (0.4−2.6 MBq; radiochemical purity, >99% after
HPLC separation; specific activity, 12 MBq/μg [64Cu(LB)(solv)];
dissolved in electrolyte solution E-153 (Serumwerk Bernburg,
Germany) at pH 7.2) was 0.5 mL. The 64Cu activity concentration
in organs and tissues was calculated as either the percentage of the
injected dose per gram tissue (% ID/g tissue) or, for both intestine
and urine, the percentage of the injected dose (% ID).
[CuII(LB)(O)]. The ligand H2LB (50 mg; 146.01 μmol; 1 equiv) was
dissolved in MeOH (1 mL) and a solution of Cu(OAc)2 × 2 H2O
(29.2 mg; 146.01 μmol) in MeOH (1 mL) was added while stirring.
After stirring at room temperature overnight, the purple precipitate
was separated by filtration, redissolved in MeOH, and subjected to
ether diffusion. The analytically pure complex crystallized as long red-
purple needles (33 mg, 81.68 μmol, 56% yield). Elemental analysis:
([CuII(LB)]+1 H2O) calcd C(55.09), H(6.47), N(12.85); expd
Syntheses. N,N′-Bis(chloroacetyl)-1,2-phenylenediamine, N,N′-
bis(chloroacetyl)-1,2-ethylenediamine, and N,N′-bis(chloroacetyl)-
1,3-propylenediamine were prepared as described in the literature.43,28
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Article
C(54.96), H(6.29), N(13.02). UV−vis spectroscopy: λ = 483 nm
(20704 cm−1), ε = 149 M−1 cm−1; cyclic voltammetry 310 mV
(MeCN), −17 mV (MeOH), −29 mV, −2264 mV* (DMF), 219 mV
(H2O).
DEDICATION
■
Dedicated to Professor Bernd Johannsen on the occasion of his
75th birthday.
[CuII(LE)(OH2)]. The ligand H2LE (50 mg; 169.84 μmol; 1 equiv)
was dissolved in MeOH (1 mL) and a solution of Cu(OAc)2 × 2H2O
(34.0 mg; 169.84 μmol) in MeOH (1 mL) was added while stirring.
After stirring at room temperature overnight, the purple precipitate
was separated by filtration, redissolved in MeOH, and subjected to
ether diffusion. The analytically pure complex crystallized as long red
purple needles (33 mg, 81.68 μmol, 56% yield). Elemental analysis:
([CuII(LE)]+1.5 MeOH) calcd C(49.06), H(7.48), N(13.87); exp
C(49.10), H(7.56), N(14.05). UV−vis spectroscopy: λ = 476 nm
(21008 cm−1), ε = 189 M−1 cm−1; cyclic voltammetry 121 mV
(MeCN), −88 mV (MeOH), 118 mV, −2116 mV* (DMF), 71 mV
(H2O).
REFERENCES
■
(1) Shokeen, M.; Anderson, C. J. Acc. Chem. Res. 2009, 42, 832−841.
(2) Blower, P. J.; Lewis, J. S.; Zweit, J. Nucl. Med. Biol. 1996, 23,
957−980.
(3) Schmidt-Ott, W.-D. Z. Phys. 1959, 154, 286−293.
(4) Cooper, M. S.; Ma, M. T.; Sunassee, K.; Shaw, K. P.; Williams, J.;
Paul, R. L.; Donnelly, P. S.; Blower, P. J. Bioconjugate Chem. 2012, 23,
1029.
(5) Cutler, C. S.; Hennkens, H. M.; Sisay, N.; Huclier-Markai, S.;
Jurisson, S. S. Chem. Rev. 2013, 113, 858−883.
(6) Holland, J. P.; Barnard, P. J.; Collision, D.; Dilworth, J. R.; Edge,
R.; Green, J. C.; Heslop, J. M.; McInnes, E. J. L.; Salzmann, C. G.;
Thompson, A. L. Eur. J. Inorg. Chem. 2008, 22, 3549.
(7) Bu, H. X.; Zhand, Z. H.; An, D. L.; Chen, Y. T.; Shinoya, M.;
Kimura, E. Inorg. Chem. 1996, 241 (1), 125.
[CuII(LP)(OH2)]. The ligand H2LP (50 mg; 162.12 μmol; 1 equiv)
was dissolved in MeOH (1 mL), and a solution of Cu(OAc)2 × 2 H2O
(32.0 mg; 162.12 μmol) in MeOH (1 mL) was added while stirring.
After stirring at room temperature overnight, the purple solution was
evaporated to dryness and the residue redissolved in MeOH and
subjected to ether diffusion. The analytically pure complex crystallized
as long red purple needles (33 mg, 81.68 μmol, 56% yield). Suitable
crystals were obtained by slow evaporation of a methanolic solution.
Elemental analysis: ([CuII(LP)]+2 H2O) calcd C(47.34), H(7.45),
N(13.80); exp C(47.32), H(7.16), N(13.80). UV−vis spectroscopy: λ
= 524 nm (19084 cm−1), ε = 117 M−1 cm−1; cyclic voltammetry 451
mV (MeCN), 266 mV (MeOH), −254 mV, −2253 mV* (DMF), 418
mV (H2O).
(8) Martell, A. E.; Motekaitis, R. J.; Clarke, E. T.; Delgado, R.; Sun,
Y.; Ma, R. Supramol. Chem. 1996, 20 (3−4), 353.
(9) Chaves, S.; Delgado, R.; Frausto da Silva, J. J. R. Talanta 1992, 39
(3), 249.
(10) Delgado, R.; Frausto da Silva, J. J. R. Talanta 1982, 29 (10),
815.
(11) Woodin, K. S.; Heroux, K. J.; Boswell, C. A.; Wong, E. H.;
Weisman, G. R.; Niu, W.; Tomellini, S. A.; Anerson, C. J.; Zakharov, L.
N.; Reingold, A. L. Inorg. Chem. 2005, 4829.
(12) Wong, E. H.; Weisman, G. R.; Hill, D. C.; Reed, D. P.; Rogers,
M. E.; Condon, J. S.; Fagan, M. A.; Calabrese, J. C.; Lam, K.-C.; Guzei,
I. A.; Rheingold, A. L. J. Am. Chem. Soc. 2000, 122, 10561−10572.
(13) Boswell, C. A.; Sun, X.; Niu, W. J.; Weisman, G. R.; Wong, E.
H.; Rheingold, A. L.; Anderson, C. J. J. Med. Chem. 2004, 47, 1465−
1474.
ASSOCIATED CONTENT
■
S
* Supporting Information
Details of the crystallographic experiments, a plot of the crystal
structure of [CuII(LB)(O)], spectra of the metal-free ligands,
UV−vis spectra and CV’s of the complexes reported, and
details of the SOD and human serum assays and the
biodistribution experiments. This material is available free of
charge via the Internet at http://pubs.acs.org. CCDC 988221-
988226 contains the supplementary crystallographic data which
can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
(14) Geue, R. J.; Hambley, T. W.; Harrowfield, J. M.; Sargeson, A.
M.; Snow, M. R. J. Am. Chem. Soc. 1984, 106, 5478−5488.
(15) Sargeson, A. M. Coord. Chem. Rev. 1996, 151, 89−114.
(16) Di Bartolo, N. M.; Sargeson, A. M.; Donlevy, T. M.; Smith, S. V.
J. Chem. Soc., Dalton Trans. 2001, 2303−2309.
(17) Cabbiness, D. K.; Margerum, D. W. J. Am. Chem. Soc. 1969, 61,
6540−6541.
(18) Cabbiness, D.; Margerum, D. W. J. Am. Chem. Soc. 1970, 92,
2152−2153.
(19) Jones-Wilson, T. M.; Deal, K. A.; Anderson, C. J.; McCarthy, D.
W.; Kovacs, Z.; Motekaitis, R. J.; Sherry, A. D.; Martell, A. E.; Welch,
M. J. Nucl. Med. Biol. 1998, 25, 523−530.
AUTHOR INFORMATION
■
Corresponding Authors
*(P.C.) Fax: +49-6221-546617. E-mail: peter.comba@aci.uni-
heidelberg.de.
(20) Bass, L. A.; Wang, M.; Welch, M. J.; Anderson, C. J. Bioconjugate
Chem. 2000, 11, 527−532.
(21) Odendaal, A. Y.; Fiamengo, A. L.; Ferdani, R.; Wadas, T. J.; Hill,
D. C.; Peng, Y.; Heroux, K. J.; Golen, J. A.; Rheingold, A. L.;
Anderson, C. J.; Weisman, G. R.; Wong, E. H. Inorg. Chem. 2011, 50,
3078−3086.
*(H.S.) Fax: +49-351-2603232. E-mail: h.stephan@hzdr.de.
Notes
The authors declare no competing financial interest.
(22) Lattuada, L.; Barge, A.; Cravotto, G.; Giovenzana, G. B.; Tei, L.
Chem. Soc. Rev. 2011, 40, 3019−3049.
̀
(23) Denat, F.; Brandes, S.; Guilard, R. Synlett 2000, 5, 561−574.
ACKNOWLEDGMENTS
■
(24) Chong, H.-S.; Song, H. A.; Kang, C. S.; Le, T.; Sun, X.; Dadwal,
M.; Lee, H.; Lan, X.; Chen, Y.; Dai, A. Chem. Commun. 2011, 47,
5584−5586.
Financial support by the German Science Foundation (DFG),
the University of Heidelberg, and the Helmholtz Association
(funding through the Helmholtz Virtual Institute Nano-
Tracking, Agreement Number VH-VI-421, and the Helm-
holtz-Portfolio Topic “Technologie und Medizin − Multi-
(25) Lewis, E. A.; Allan, C. C.; Boyle, R. W.; Archibald, S. J.
Tetrahedron Lett. 2004, 45, 3059−3062.
(26) Kimura, E. J. Coord. Chem. 1986, 15, 1−28.
(27) Sharma, S. K.; Hundal, G.; Gupta, R. Eur. J. Inorg. Chem. 2010,
621−636.
modale Bildgebung zur Aufklarung des In-vivo-Verhaltens von
̈
polymeren Biomaterialien”) are gratefully acknowledged. We
are grateful to Professor H. Wadepohl for the collection of the
X-ray data and helpful discussions, and to Madlen Matterna,
Janine Partsch, and Regina Herrlich for technical assistance.
(28) Antunes, P.; Delgado, R.; Drew, M. G. B.; Felix, V.; Maecke, H.
Inorg. Chem. 2007, 46 (8), 3144−3153.
(29) Barnard, P. J.; Holland, J. P.; Bayly, S. R.; Wadas, T. J.;
Anderson, C. J. Inorg. Chem. 2009, 48, 7117−7126.
6706
dx.doi.org/10.1021/ic500476u | Inorg. Chem. 2014, 53, 6698−6707

Inorganic Chemistry
Article
(30) Vin Tan, K.; Pellegrini, P. A.; Skelton, B. W.; Hogan, C. F.;
Greguric, I.; Barnard, P. J. Inorg. Chem. 2014, 53, 468−477.
(31) Comba, P.; Nuber, B.; Ramlow, A. J. Chem. Soc., Dalton Trans.
1997, 347−352.
(66) Sheldrick, G. M. Acta Crystallogr., Sect. A 2008, 64, 112−122.
(67) Thieme, S.; Walther, M.; Pietzsch, H.-J.; Henniger, J.; Preusche,
S.; Maeding, P.; Steinbach, J. Appl. Radiat. Isot. 2012, 70, 602−608.
(68) Miyoshi, K.; Tanaka, H.; Kimura, E.; Tsuboyama, S.; Murata, S.;
Shimizu, H.; Ishizu, K. Inorg. Chim. Acta 1983, 78, 23.
(32) Comba, P.; Kerscher, M.; Schiek, W. Prog. Inorg. Chem. 2007,
55, 613−704.
(33) Juran, S.; Walther, M.; Stephan, H.; Bergmann, R.; Steinbach, J.;
Kraus, W.; Emmerling, F.; Comba, P. Bioconjugate Chem. 2009, 20,
347−359.
(34) Comba, P.; Morgen, M.; Wadepohl, H. Inorg. Chem. 2013, 52,
6481−6501.
(35) Comba, P.; Hunoldt, S.; Morgen, M.; Pietzsch, J.; Stephan, H.;
Wadepohl, H. Inorg. Chem. 2013, 52, 8131−8143.
(36) Mannich, C.; Mohs, P. Chem. Ber. 1930, B63, 608−612.
(37) Comba, P.; Kerscher, M. Coord. Chem. Rev. 2009, 253, 564−
574.
(38) Comba, P. Oxidation Catalysis with High-Valent Nonheme Iron
Complexes, in Molecular Catalysis; Gade, L. H., Hofmann, P., Eds.;
Wiley-VCH: Weinheim, Germany, 2014.
(39) Comba, P.; Pritzkow, H.; Schiek, W. Angew. Chem. 2001, 113,
2556.
(40) Islam, M. J. l.; Miller, E. J.; Gordner, J. S.; Patel, D.; Wang, Z.
Tetrahedron Lett. 2013, 2133−2136.
(41) Comba, P.; Daumann, L.; Lefebvre, J.; Linti, G.; Martin, B.;
Straub, J.; Zessin, T. Aust. J. Chem. 2009, 62, 1238−1245.
(42) Kuznetsov, A. I.; Basargin, E. B.; Ba, M. K.; Moskovin, A. S.;
Miroshnichenko, I. V.; Bosnikov, M. Y. Chem. Heterocycl. Compd.
1989, 647.
(43) Bradshaw, J. S.; Krakowiak, K. E.; An, A.; Izatt, R. M. J.
Heterocyclic Chem. 1990, 27, 2113−2116.
(44) Comba, P.; Okon, N.; Remenyi, R. J. Comput. Chem. 1999, 20,
781−785.
(45) Comba, P.; Fath, A.; Kuhner, A.; Nuber, B. J. Chem. Soc., Dalton
̈
Trans. 1997, 1889−1898.
(46) Comba, P. Coord. Chem. Rev. 1999, 182, 343−371.
(47) Comba, P.; Schiek, W. Coord. Chem. Rev. 2003, 238−239, 21−
29.
(48) Comba, P.; Hambley, T. W.; Hitchman, M. A.; Stratemeier, H.
Inorg. Chem. 1995, 34, 3903−3911.
(49) Hanson, G. R.; Gates, K. E.; Noble, C. J.; Griffin, M.; Mitchell,
A.; Benson, S. J. Inorg. Biochem. 2004, 98, 903−916.
(50) Zarschler, K.; Kubeil, M.; Stephan, H. RSC Adv. 2014, 4,
10157−10164.
(51) Comba, P.; Emmerling, F.; Jakob, M.; Kraus, W.; Kubeil, M.;
Morgen, M.; Pietzsch, J.; Stephan, H. Dalton Trans. 2013, 42, 6142−
6148.
(52) Comba, P.; Martin, B.; Sanyal, A.; Stephan, H. Dalton Trans.
2013, 42, 11066−11073.
(53) Wang, D.; Hanson, G. R. J. Magn. Reson. A 1995, 117, 1−8.
(54) Wang, D.; Hanson, G. R. Appl. Magn. Reson. 1996, 11, 401−415.
(55) Bruker SAINT Bruker AXS; Bruker: Madison, WI, 1997−2008.
(56) SCALE3 ABSPACK CrysAlisPro; Agilent Technologies UK Ltd.,
Oxford, U.K., 2011−2013.
(57) Blessing, R. H. Acta Crystallogr., Sect. A 1995, 51, 33−38.
(58) Sheldrick, G. M. SADABS-2004-2008, SADABS-2004/1; Bruker
AXS: Karlsruhe, Germany, 2004−2008.
(59) Sheldrick, G. M. TWINABS; Bruker AXS: Karlsruhe, Germany,
2004−2008.
(60) Clark, R. C.; Reid, J. S. Acta Crystallogr., Sect. A 1995, 51, 887−
897.
(61) Sheldrick, G. M. SADABS; Bruker AXS: Karlsruhe, Germany,
2004−2012.
(62) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.;
Puschmann, H. J. Appl. Crystallogr. 2009, 42, 339−341.
(63) Palatinus, L.; Chapuis, G. J. Appl. Crystallogr. 2007, 40, 786−
790.
(64) Palatinus, L. SUPERFLIP; EPF Lausanne: Switzerland, 2007.
(65) Sheldrick, G. M. SHELXS-97; University Gottingen: Gottingen,
̈
̈
Germany, 1997.
6707
dx.doi.org/10.1021/ic500476u | Inorg. Chem. 2014, 53, 6698−6707