Synthesis and characterization of the copper(ii) complexes of new N 2S2-donor macrocyclic ligands: Synthesis and in vivo evaluation of the 64Cu complexes

Article abstract of DOI:10.1039/b808831d

The aim of this work was to prepare a novel class of ⁶⁴Cu(ii) labeled complexes with the new macrocyclic ligands 1,10-dithia-4,7- diazacyclododecane-3,8-dicarboxylic acid (NEC-SE, 1), 1,10-dithia-4,7- diazacyclotridecane-3,8-dicarboxylic acid (NEC-SP, 2) and 1,10-dithia-4,7- diazacyclotetradecane-3,8-dicarboxylic acid, (NEC-SB, 3) to evaluate the usefulness of these macrocycles for potential utility as ⁶⁴Cu(ii) chelators. The corresponding non-radioactive complexes [Cu(NEC-SE)] ·3H₂O (4), [Cu(NEC-SP)]·3H₂O (5) and [Cu(NEC-SB)] (6) were prepared and their ⁶⁴Cu-analogs, [ ⁶⁴Cu(NEC-SE)] (7) and [⁶⁴Cu(NEC-SP)] (8) and [ ⁶⁴Cu(NEC-SB)] (9) were produced in >98% radiochemical purity. Rats were injected with complex 7, 8 or 9 and were euthanized at 1, 4 and 24 h. All three complexes are cleared from the blood over the first hour following injection but there is poor clearance of this activity over 24 h. A similar pattern of retention was noted in the liver where the levels of activity in this tissue at 1 h are not statistically different from those at 24 h. Molecular mechanics and DFT studies were performed on the complexes in order to gain insight into the lower stability. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b808831d

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Synthesis and characterization of the copper(II) complexes of new N S -donor
2
2
6
4
macrocyclic ligands: synthesis and in vivo evaluation of the Cu complexes†
Grazia Papini, Simone Alidori, Jason S. Lewis,*‡ David E. Reichert,b,c Maura Pellei,a
a
a
b,c
a
b
b,c
a
Giancarlo Gioia Lobbia, Gr a´ inne B. Biddlecombe, Carolyn J. Anderson and Carlo Santini*
Received 27th May 2008, Accepted 16th September 2008
First published as an Advance Article on the web 4th November 2008
DOI: 10.1039/b808831d
6
4
The aim of this work was to prepare a novel class of Cu(II) labeled complexes with the new
macrocyclic ligands 1,10-dithia-4,7-diazacyclododecane-3,8-dicarboxylic acid (NEC-SE, 1),
1
,10-dithia-4,7-diazacyclotridecane-3,8-dicarboxylic acid (NEC-SP, 2) and 1,10-dithia-4,7-
diazacyclotetradecane-3,8-dicarboxylic acid, (NEC-SB, 3) to evaluate the usefulness of these
6
4
macrocycles for potential utility as Cu(II) chelators. The corresponding non-radioactive complexes
[
6
Cu(NEC-SE)]·3H
2
O (4), [Cu(NEC-SP)]·3H
2
O (5) and [Cu(NEC-SB)] (6) were prepared and their
Cu-analogs, [ Cu(NEC-SE)] (7) and [ Cu(NEC-SP)] (8) and [ Cu(NEC-SB)] (9) were produced in
4
64
64
64
>98% radiochemical purity. Rats were injected with complex 7, 8 or 9 and were euthanized at 1, 4 and
2
4 h. All three complexes are cleared from the blood over the first hour following injection but there is
poor clearance of this activity over 24 h. A similar pattern of retention was noted in the liver where the
levels of activity in this tissue at 1 h are not statistically different from those at 24 h. Molecular
mechanics and DFT studies were performed on the complexes in order to gain insight into the lower
stability.
Introduction
for use as bifunctional chelators, a large number of comparative
studies on macrocycles with different donors and ring sizes have
Copper radionuclides offer an attractive foundation for the
development of disease-targeting radiopharmaceuticals; with a
wide range of half-lives and decay schemes they have utility in
14–16
been recently published.
The majority of bifunctional ligands
used for complexing copper have centered around the tetraaza-
16
based macrocycles. In general, studies have involved conjuga-
6
0
61
62
both Positron Emission Tomography (PET) ( Cu, Cu, Cu and
tion of 1,4,7,10-tetraazacyclododecane-N,N¢,N¢¢,N¢¢¢-tetraacetic
acid (DOTA) or 1,5,8,11-tetraazacyclotetradecane-N,N¢,N¢¢,N¢¢¢-
tetraacetic acid (TETA) to the terminal amine of tumor-targeting
6
4
64
67
1,2
Cu) and targeted radiotherapy ( Cu and Cu). Attachment of
these nuclides to biomolecules, such as peptides and antibodies,
require conjugation of the nuclide to the biological entity via a
stable bifunctional ligand that can withstand metabolism and
demetalation in vivo; the in vivo stability of the metal chelate is
perhaps one of the most important considerations in the design of a
disease-targeting copper radiopharmaceutical. The identification
of kinetically inert radiocopper complexes, rather than copper
complexes with high thermodynamic stability, is the first stage in
64
+
peptides for subsequent labeling with Cu (t1/2 = 12.7 h, 17.4% b ,
-
17–21
4
1% EC, 40% b , Eavg = 278 keV) for PET imaging applications.
However, image quality is often less than optimal due to the slow
64
release of uncoordinated Cu by decomposition of the complex in
the blood or transchelation in the liver, causing elevated uptake in
4,22
non-target tissues such as the liver. New cross-bridged cyclam
4,23
chelates (e.g., CB-TE2A)
have recently been developed for
3,4
identifying a suitable bifunctional chelator for copper nuclides.
64
Cu and have shown significant metal-chelate stability and as
a consequence a lowering in non-target tissue accumulation.
Macrocyclic ligands form metal complexes which in general are
thermodynamically more stable than complexes with analogous
open-chain ligands and this has been termed the “macrocyclic
22,24
As mentioned, the dominant moieties for copper chelates have
been the tetraaza-based (N
studies done with N -based macrocycles. Thiaazacycloalkanes
exhibit interesting properties intermediate between those of cyclic
4
) macrocyclic ligands, with little or no
3,5–12
effect”.
In addition macrocyclic metal complexes are often
2
S
2
1
3
inert towards acid dissociation. Due to the interest in macrocycles
25
4-
polyamines and polythioethers. The discovery of (N
2
S ) donor
2
a
Dipartimento di Scienze Chimiche, Universit a` di Camerino, via S. Agostino
sites in biology based on cysteine-X-cysteine tripeptide motifs has
prompted the examination of small molecule models that build on
1
, 62032, Camerino, MC, Italy. E-mail: carlo.santini@unicam.it
b
Division of Radiological Sciences, Washington University School of
Medicine, St. Louis, MO 63110, USA. E-mail: lewisj2@mskcc.org
2-
a well known literature that is based on (N
evolved over several decades.
2
S
2
)
ligands, which
26,27
c
Although N,N-ethylene-di-L-
Alvin J. Siteman Cancer Center, Washington University School of Medicine,
28
St. Louis, MO, 63110, USA
cysteine (EC) has been known for a long time, it is surprising
that macrocyclic ligands based on the EC skeletons have not been
†
Electronic supplementary information (ESI) available: Complete biodis-
tribution data ( %ID/g and %ID/organ) for 19 harvested tissues at 1, 4
29
30
31
extensively investigated. EC copper(II), nickel(II), cobalt(III),
64
64
64
and 24 h for [ Cu(NEC-SE)] (7), [ Cu(NEC-SP)] (8) and [ Cu(NEC-SB)]
9) in male, Lewis rats. See DOI: 10.1039/b808831d
Current address: Department of Radiology, Memorial Sloan-Kettering
3
+
32,33
3+ 34–36
37
[
Tc(V)O] ,
[Re(V)O] ,
molybdenum(VI), indium(III) and
(
‡
38
gallium(III) complexes have been synthesized and characterized.
9
9m
Cancer Center, New York, NY, USA.
EC can be labeled with
Tc easily and efficiently with high
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 177–184 | 177

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3
9–43
radiochemical purity and stability.
Compared to metals with an
Results
oxidation state of +3 or higher, in the metal(II) complexes
copper(II) and nickel(II)) the EC acts as tetradentate N
to form complexes with nearly planar cis-NiN
Synthesis and characterization of the macrocyclic ligands
(
2
S
2
ligand
30
S
2
and cis-
The reaction scheme of the syntheses of 1,10-dithia-4,7-
diazacyclododecane-3,8-dicarboxylic acid (NEC-SE, 1), 1,10-
dithia-4,7-diazacyclotridecane-3,8-dicarboxylic acid (NEC-SP, 2)
and 1,10-dithia-4,7-diazacyclotetradecane-3,8-dicarboxylic acid,
(NEC-SB, 3) is shown in Fig. 1. The starting material ethylenedi-
cysteine was prepared by the sodium reduction of the precursor
L-thiazolidine-4-carboxylic acid in liquid ammonia according to
2
29
CuN
torially oriented on the l conformation NS chelate rings. The
non-macrocyclic N moiety has been used as a means to attach
radiocopper to biomolecules.
the rate of formation and dissociation of a series of N
2
S
2
units. The bulky carboxylic or ester groups are equa-
44
2
S
2
45,46
Siegfried and Kaden measured
macrocy-
2
S
2
2
+
2+ 15
cles with Cu and Ni . The ring size (from 12- to 16-membered
cycles) and the geometry (cis or trans) of the arrangement of the
donor groups was systematically varied in order to investigate how
these two parameters affect the mechanism of complex formation
and dissociation. In particular this study showed that the 14-
membered macrocycle (cis-arrangment) forms complexes in which
3
2
methods described by Blondeau et al. The ligands (1, 2 or 3) were
purified by washing with water and ethanol and were obtained in
1
13
high yield. Their authenticity was confirmed by H-NMR, C-
NMR, elemental analysis, IR and ESI-MS spectroscopy. The
novel ligands 1 and 2 are white, the ligand 3 is yellow. They
are microcrystalline solids, soluble in water solutions at pH > 7
and insoluble in common organic solvents; the ligand 3 is soluble
in methanol solution. The infrared spectra of 1–3 showed broad
2
+
2+
both Cu and Ni are ideally coordinated so that the dissociation
47–49
becomes extremely slow.
The marked dependence of the nature
of the backbone on the specificity of copper complexes of EC
led us to investigate further EC-proligands with extended 12-,
3- and 14-member backbones as ligands for the development of
new copper radiopharmaceuticals. It was hoped that these ligands
would better accommodate both Cu(I) and Cu(II). A series of 12-,
-macrocycles and the related Cu(I) and
Cu(II) derivatives were synthesized and their stabilities, redox and
spectroscopic properties were explored.
shown that complexes with the smaller rings react as flexible open-
-
1
absorptions in the range 3390–3412 cm due to the NH stretching
vibrations. The presence of the COO moiety is detected by intense,
1
-
1
broad absorptions in the range 1586–1633 cm , due to the ionized
-
COO stretching bands, and by medium or weak absorptions at
-
1
1
4- and 16-membered N
2
S
2
1726–1732 cm , attributable to the COOH un-ionized stretching
frequencies. This behavior is in accordance with the coexistence
of the predominant zwitterion together with a minority of the
un-ionized carboxylic groups. The formation of the macrocycle
is confirmed by the presence of the S(CH
15,50–52
In general it was
54
+
1
chain ligands directly with H , with the larger rings reacting in
2
) S protons in the H-
n
15
a similar manner as rigid ligands. It was shown that the 14-
membered macrocycle forms complexes in which the metal ions are
ideally coordinated so that their dissociation becomes extremely
slow. In view of a radiochemical application, N
with ring sizes varying between 12 and 16, as well as two 12-
membered N -rings with a pendant carboxylic and amino group
linked to the N atoms, respectively, were also synthesized, and
related Ag(I) complexes were tested in blood serum for their
stability.
In this current study we aimed to prepare three novel N
NMR at d 2.63–2.70 for 1, at d 1.72 and 2.49–2.77 for 2 and
1
3
1
at d 1.50 and 2.40–2.60 for 3. In the C{ H}NMR spectrum of
1 the chemical shifts of the S–CH carbons are observed at d
34.74 and 37.21; for 2 the corresponding C chemical shifts of
2
1
3
2
2
S -macrocycles
1
3
the S–CH
chemical shifts of the S–CH
In addition, in 2, the chemical shift of the C–CH
2
carbons are at d 33.56 and 37.07, while for 3 the
carbons are at d 30.52 and 33.44.
–C carbon is at
d 31.48, while for 3 the corresponding C chemical shift of the
C–CH CH –C carbons are at d 27.11. The ESI-MS negative-ion
C
2
S
2
2
2
1
3
53
2
2
S
2
spectrum of 1 in H
species [M - H] and [2M - H] at m/z 293 (100%) and 588
(80%), respectively. The ESI-MS negative-ion spectrum of 2 in
2
O/NH
4
OH is dominated by the deprotonated
2
-
-
macrocyclic ligands based on the L,L-ethylenedicysteine skeletons.
We present the synthesis and characterization of these novel
ligands, the non-radioactive copper complexes and the corre-
-
H
2
O/NH
4
OH is dominated by the deprotonated ligands [M - H]
6
4
2-
sponding Cu-analogs. We further report the in vivo character-
and [M - 2H] at m/z 307 (100%) and 153 (30%), respectively.
The ESI-MS negative-ion spectrum of 3 in H
dominated by the deprotonated species [M - 2H] at m/z
160 (100%).
6
4
istics of these Cu complexes in an animal model using acute
biodistribution and we discuss the usefulness of these agents as
bifunctional chelates for copper nuclides.
2
O/NH OH is
4
2
-
Fig. 1 The reaction schemes of the syntheses of 1,10-dithia-4,7-diazacyclododecane-3,8-dicarboxylic acid (NEC-SE, 1), 1,10-dithia-4,7-diazacyclotride-
cane-3,8-dicarboxylic acid (NEC-SP, 2) and 1,10-dithia-4,7-diazacyclotetradecane-3,8-dicarboxylic acid, (NEC-SB, 3).
1
78 | Dalton Trans., 2009, 177–184
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Fig. 2 The reaction schemes of the syntheses of Cu(NEC-SE) (4), Cu(NEC-SP) (5) and Cu(NEC-SB) (6).
6
4
Synthesis and characterization of the non-radioactive Cu(II)
complexes
reacting CuCl
2
and either 1, 2 or 3 in 0.1 M ammonium acetate
◦
buffer (pH 8.0) at 90 C for 1 h. It was necessary to dissolve 3
in 20 mL methanol prior to diluting with the 0.1 M ammonium
Ligands 1–3 were used in the preparation of the copper(II)
6
4
acetate buffer. The presence in solution of the Cu-complexes
was confirmed by radio-TLC by comparison with the TLC profile
of an authentic sample of non-radioactive complex (4, 5 or 6).
The complexes were produced in a radiochemical purity of >98%.
Compound 9 was additionally analyzed by radio-HPLC and was
complexes [Cu(NEC-SE)]·3H
2
O (4), [Cu(NEC-SP)]·3H
2
O (5), and
[
Cu(NEC-SB)] (6); the chemical scheme of the syntheses of the
compounds is given in Fig. 2. The stoichiometry of the compounds
was not affected by either reducing or increasing the amount
of the corresponding ligand. The blue copper complexes are
insoluble in common organic solvents and not very soluble in
water solutions at pH >7. The authenticity of 4–6 was confirmed
by elemental analysis and IR spectroscopy. The infrared spectra of
the derivatives 4–6 showed broad absorptions in the range 3397–
shown to have a radioactive peak at t
corresponded to the UV peak for the analogous cold complex.
R
= 12.61 min, which
In vivo data
-
1
3
412 cm due to the NH stretching vibrations. The presence of the
The biodistribution data (percent injected dose per gram, %ID/g)
6
4
64
64
COO moiety is detected by intense broad absorptions in the ranges
of [ Cu(NEC-SE)] (7) and [ Cu(NEC-SP)] (8) and [ Cu(NEC-
SB)] (9) in male Lewis rats is summarized in Table 1. Fig. 3 presents
a comparison between the biodistribution data (%ID/organ) of
1
-1
1
601–1617 cm- and 1358–1384 cm , due to the asymmetric and
symmetric stretching modes, respectively; the shift to blue of the
coordinated COO asymmetric stretchings with respect to the free
ligands being observed upon complex formation. The observed
range is in accordance with the values reported for coordinated
6
4
7, 8 and 9 and selected Cu–tetraazamacrocycles at 24 h post
56
injection. The data obtained from complexes 7, 8 and 9 are
indistinguishable. Although blood clearance of these complexes
is observed over the first hour post injection (7, 0.850 ± 0.051
%ID/g; 8, 0.820 ± 0.046 %ID/g; 9, 0.874 ± 0.140 %ID/g) the
clearance from the blood was poor over 24 h (7, 0.768 ± 0.040
%ID/g; 8, 0.774 ± 0.031 %ID/g; 9, 0.692 ± 0.151 %ID/g). The
kidney did demonstrate partial clearance of the radioactivity over
24 h but still remained at relatively high levels. The liver and kidney
uptakes of the complexes were high at 1 h post injection (liver: 7,
3.919 ± 0.363 %ID/g; 8, 3.151 ± 0.286 %ID/g; 9, 4.105 ± 0.465
54
COO stretching bands in amino-carboxylate metal complexes.
The magnitude of nasymCOO - nsymCOO (Dn) separation can be
used to explain the type of carboxylate structure present in the
-
1
solid state. Dn values for 4–6 are of about 250 cm , characteristic
of monodentate coordination compounds. In the low-frequency
region the copper–nitrogen stretching frequencies fall in the range
-
1
4
40–470 cm , while the copper–oxygen vibrations are detected in
-
1 55
the range 300–380 cm .
%
ID/g; kidney: 7, 8.662 ± 0.551 %ID/g; 8, 9.438 ± 1.019 %ID/g;
6
4
Synthesis and characterization of the Cu(II) complexes
9, 8.895 ± 0.612 %ID/g), with little or no clearance observed for
6
4
64
The corresponding Cu-analogs, [ Cu(NEC-SE)] (7) and
Cu(NEC-SP)] (8) and [ Cu(NEC-SB)] (9) were produced from
either complex from the liver over 24 h (liver: 7, 3.314 ± 0.476
%ID/g; 8, 3.182 ± 0.188 %ID/g; 9, 3.296 ± 0.728 %ID/g; kidney:
6
4
64
[
64
64
64
Table 1 Biodistribution (percent dose per gram, %ID/g) of [ Cu(NEC-SE)] (7), [ Cu(NEC-SP)] (8) and [ Cu(NEC-SB)] (9) in male Lewis rats. Rats
were injected with ~40 mCi of complex and were euthanized at 3 time points (n = 4 per group at 1, 4 and 24 h). Following euthanasia, 19 tissues and
organs of interest were removed and weighed and the radioactivity present was measured in a g-counter. Given are the %ID/g values for selected organs.
The complete biodistribution data for all 19 tissues, including percent dose per organ (%ID/organ) values, can be found in the ESI†
64
64
64
[
Cu(NEC-SE)] (7), %ID/g
[ Cu(NEC-SP)] (8), %ID/g
[ Cu(NEC-SB)] (9), %ID/g
Tissue
1 h 4 h
24 h
1 h 4 h
24 h
1 h 4 h
24 h
Blood
Lung
Liver
0.850 ± 0.051 0.897 ± 0.080 0.768 ± 0.040 0.820 ± 0.046 0.835 ± 0.021 0.774 ± 0.031 0.874 ± 0.140 0.879 ± 0.112 0.692 ± 0.151
0.838 ± 0.065 0.768 ± 0.045 0.723 ± 0.036 0.776 ± 0.089 0.737 ± 0.009 0.771 ± 0.061 1.673 ± 0.225 0.949 ± 0.109 0.746 ± 0.143
3.919 ± 0.363 3.905 ± 0.540 3.314 ± 0.476 3.151 ± 0.286 3.418 ± 0.371 3.182 ± 0.188 4.105 ± 0.465 3.866 ± 0.635 3.296 ± 0.728
0.560 ± 0.025 0.731 ± 0.174 1.046 ± 0.106 0.483 ± 0.054 0.704 ± 0.014 1.078 ± 0.099 0.698 ± 0.168 0.815 ± 0.137 0.924 ± 0.207
Spleen
Kidney 8.662 ± 0.551 7.849 ± 1.099 4.906 ± 0.211 9.438 ± 1.019 7.818 ± 0.663 5.070 ± 0.518 8.895 ± 0.612 7.096 ± 1.059 4.738 ± 0.617
Muscle 0.380 ± 0.032 0.344 ± 0.012 0.309 ± 0.026 0.393 ± 0.055 0.352 ± 0.017 0.319 ± 0.031 0.411 ± 0.056 0.330 ± 0.050 0.302 ± 0.033
Fat
0.334 ± 0.054 0.266 ± 0.068 0.163 ± 0.026 0.318 ± 0.074 0.214 ± 0.024 0.153 ± 0.019 0.371 ± 0.073 0.218 ± 0.037 0.136 ± 0.035
0.672 ± 0.086 0.774 ± 0.096 1.041 ± 0.070 0.657 ± 0.025 0.736 ± 0.048 1.013 ± 0.078 0.839 ± 0.228 0.752 ± 0.059 0.923 ± 0.189
1.131 ± 0.072 1.274 ± 0.082 0.984 ± 0.076 1.099 ± 0.098 1.255 ± 0.040 0.999 ± 0.065 1.143 ± 0.195 1.155 ± 0.161 0.855 ± 0.188
Heart
Bone
This journal is © The Royal Society of Chemistry 2009
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6
4
Cu has been shown to dissociate in vivo from DOTA and DOTA-
16
conjugates and then undergoes metabolism and transchelation to
superoxide dismutase and other proteins causing increased blood
and liver accumulation.
22,24
The poor clearance of 7, 8 and 9
6
4
suggests that the Cu complexes are rapidly degraded in blood
6
4
and tissues and the Cu is sequestered by proteins and thereby
remaining trapped in these tissues hindering clearance. The cross-
bridged cyclam chelator, CBTE2A, has demonstrated improved in
vivo stability and consequently a reduction in transchelation when
4,16,22,24
compared to the non-bridged analog, TETA.
Conjugation
of this chelate to tumor-targeting peptides has caused an im-
provement in non-target organ clearance and higher tumor ratios
6
4
64
16,24
Fig. 3 Comparison between the biodistribution data (%ID/organ) of
compared with either the Cu-TETA and Cu-DOTA analogs.
In a recent report by Siegfried and Kaden, the kinetics of the
64
64
64
[
Cu(NEC-SE)] (7), [ Cu(NEC-SP)] (8) and [ Cu(NEC-SB)] (9) and
64
selected Cu-tetraazamacrocycles at 24 h post injection. Data for 7, 8 and
9
and Cu-CB-TE2A obtained in female Sprague-Dawley rats.
2+
formation and dissociation of Cu from a series N
2
S macrocycles
2
64
64
64
obtained in male Lewis Rats. Cu-Cyclam, Cu-TETA, Cu-DOTA
15
was studied. The ring size and the geometry of the arrangement
of the donor groups were varied and the kinetic characteristics of
the complexes were compared. Since all attempts to obtain crystal
structures of the copper complexes were unsuccessful, molecular
modeling was performed to gain insight into the geometries of
the complexes that may be contributing to in vivo instability.
The molecular modeling studies provide support for the role of
the carboxylic acid groups in coordinating the copper atoms.
The preferred structure for 7 is a square pyramidal complex
with the base formed from one of the N atoms, two O atoms
each donated by one of the carboxylate groups, and one of
the S atoms. The apical position is filled by the remaining ring
64
4,56
7, 4.906 ± 0.211 %ID/g; 8, 5.070 ± 0.518 %ID/g; 9, 4.738 ± 0.617
%
ID/g). The poor clearance of these complexes suggests that the
6
4
complexes are dissociating in tissues and Cu remains trapped in
these tissues hindering clearance.
57
Discussion
In this current study we prepared three novel N
2
2
S macro-
cyclic ligands 1,10-dithia-4,7-diazacyclododecane-3,8-dicarboxy-
lic acid (NEC-SE, 1), 1,10-dithia-4,7-diazacyclotridecane-3,8-
dicarboxylic acid (NEC-SP, 2), and 1,10-dithia-4,7-diazacyclo-
tetradecane-3,8-dicarboxylic acid (NEC-SB, 3) based on the L,L-
ethylenedicysteine skeleton. It is anticipated that the use of macro-
˚
nitrogen, and the remaining ring S is 2.957 A from the copper.
The complex retains an essentially planar arrangement of the N
and S atoms, planes defined by N–Cu–N and S–Cu–S intersect
◦
◦
40
with a 27 angle comparable to the 21 found by Schugar.
cyclic N
2
S
2
ligands would result in an increase in in vivo metal–
Complex 8 prefers an octahedral coordination arrangement with
the equatorial positions filled by the ring nitrogens and sulfurs,
and the axial positions filled by carboxylate oxygens. The N and
S atoms are even more planar than in 7 with the N–Cu–N and
S–Cu–S planes separated by only 13.2 . Complex 9 was found to
favor a distorted tetrahedral arrangement with the copper being
chelate stability and therefore lead to an improvement in target to
non-target tissue ratios. This has been demonstrated before and
3,5–12
has been termed to “macrocycle-effect”.
characterized all the non-radioactive (4–6) and the Cu complexes
7–9) of these three ligands (Fig. 1 and 2). We further studied the
We synthesized and
6
4
◦
(
in vivo biodistribution of complexes 7, 8 and 9 in healthy rats to
coordinated by the carboxylates and both ring sulfurs. As with 8,
the N–Cu–N and S–Cu–S planes are separated by only 13.4 . The
◦
compare the biokinetics of these three compounds with previously
6
4
reported data obtained with selected Cu-tetrazamacrocycles.
The biodistribution of complexes 7, 8 and 9 were very similar
to each other and very few statistical differences between the data
exist. All three complexes were cleared from the blood over the
first hour following injection but there was poor clearance of
this activity over 24 h (Table 1). A similar pattern of retention
is noted in the liver where the levels of activity in this tissue at
structures of these models are shown in Fig. 4, and a structural
summary in Table 2.
The NBO analysis provides a method for analyzing the strength
of the bonding interactions present in the complexes; an examina-
tion of the relative strengths for each donor–copper interaction
Table 2 Structural features of B3LYP optimized complexes
1
h are not statistically different from those at 24 h. The kidney
did demonstrate partial clearance of the complexes over 24 h but
still remained at relatively high levels. The retention of activity in
tissues was similar to that observed with Cu-cyclam (Fig. 3)
Complex 7
Complex 8
Complex 9
6
4
29
Cu–L ( A˚ )
N9
N12
S3
S6
Cu–L ( A˚ )
N10
N13
S3
S7
O18
O20
Cu–L ( A˚ )
N11
N14
S3
S8
O19
O21
6
4
4
2.193
1.997
2.957
2.341
1.948
2.053
2.154
2.150
2.553
2.550
1.967
1.968
2.213
2.188
2.565
2.540
1.953
1.955
and Cu-monooxo-tetrazamacrocyclic complexes. However, on
6
4
64
comparison with Cu-TETA and Cu-DOTA, the uptake and
retention of 7, 8 and 9 are orders-of-magnitude higher (Fig. 3).
This could in part be attributed to the kinetics of these hydrophilic
complexes, but is more likely due to very rapid dissociation of the
O17
O19
6
4
◦
◦
◦
Cu from the chelates in vivo. This results in a biodistribution
Angle ( )
Angle ( )
Angle ( )
6
4
N–Cu–N 91.94
S–Cu–S 98.77
N–Cu–N 88.21
S–Cu–S 103.55
N–Cu–N 84.66
S–Cu–S 112.21
being more similar to “free” Cu than the biokinetics of intact
complexes. This same dissociation phenomenon is observed
although slower) with N4-macrocyclic chelates such as DOTA.
N
2
S
2
O–Cu–N 86.02, 79.03 O–Cu–N 81.73, 81.49 O–Cu–N 81.62, 81.05
(
1
80 | Dalton Trans., 2009, 177–184
This journal is © The Royal Society of Chemistry 2009

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copper vary greatly. All three systems contain three types of donor
groups: nitrogen, sulfur, and oxygen; no one type of metal donor
interaction seems strong enough to override the others. In complex
7
it appears that macrocyclic constraints prevent optimal overlap
of the donor atoms with the copper center, most likely lowering
the in vivo stability of the complex. Complexes 8 and 9 appear to be
equally well coordinated with the available donor atoms, although
it does appear that there may be less than maximal coordination
with the sulfur donors when compared with the strength of the Cu–
S6 bond found in complex 7. This would suggest that the lower
stability may arise from weaker Cu–S interactions than would be
found in a non-macrocyclic ligand.
Conclusions
We prepared three novel N
2
2
S macrocyclic ligands NEC-SE (1),
NEC-SP (2) and NEC-SB (3) based on the L,L-ethylenedicysteine
skeleton. From ligands 1, 2 and 3 we have synthesized and
characterized the non-radioactive copper complexes (4–6) and the
6
4
analogous Cu complexes (7–9). To the best of our knowledge,
6
4
this is the first report of Cu labeled to this form of (N
2
2
S )
macrocyclic ligand. It is however evident that the biological
behavior of these complexes is less than ideal and additional study
is required to improve the in vivo stability of these agents. In fact,
the new ligands described in this manuscript are susceptible to
further functionalization, for example by modification of the ring
dimensions, and/or, by change of the donor atoms set.
Experimental
Methods and materials
Chemicals and analysis. All syntheses and handling were
carried out under an atmosphere of dry oxygen-free dinitrogen,
using standard Schlenk techniques or a glove box. All solvents
were dried, degassed and distilled prior to use. Elemental analyses
Fig. 4 The DFT (B3LYP/LACVP*) optimized structures of Cu(NEC-
SE) (7), Cu(NEC-SP) (8) and Cu(NEC-SB) (9).
(
C, H, N, S) were performed in house with a Fisons Instruments
provides a means to determine the predominate interactions
1108 CHNS-O Elemental Analyser. Melting points were taken on
an SMP3 Stuart Scientific Instrument. IR spectra were recorded
for each complex. The copper center in complex 7 is strongly
-
1
-1
coordinated by one of the nitrogens (N12 242.62 kcal mol ) and
as neat from 4000 to 600 cm with a Perkin-Elmer Spectrum
-
1
-1
-1
sulfur (S6 679.65 kcal mol ) and oxygen (O17 246.44 kcal mol ),
One System and as Nujol mulls from 600 to 100 cm with a
-
1
while the other nitrogen (N9 161.36 kcal mol ) and oxygen (O19
73.08 kcal mol ) display weaker interactions with the copper.
Perkin-Elmer System 2000 FT-IR instrument. IR annotations
-
1
1
used: m = medium, mbr = medium broad, s = strong, sbr = strong
1
13
Complex 8 is more balanced in terms of its bonding interactions,
broad, sh = shoulder, w = weak. H- and C-NMR spectra were
-
1
with both nitrogens (N10 and N14, 138.7 and 154.61 kcal mol )
recorded on a Oxford-400 Varian spectrometer (400.4 MHz for
-
1
1
13
and sulfurs (S3 and S7, 370.61 and 375.46 kcal mol ) having
relatively equal interactions. In a similar trend both oxygen donors
H, 100.1 MHz for C). Chemical shifts (d) are quoted relative
to internal SiMe
4
. NMR annotations used: br = broad, m =
(
O18 and O20) have respective bond strengths of 183.27 and 183.04
multiplet, qn = quintet, t = triplet. Electrospray mass spectra (ESI-
MS) were obtained in positive- or negative-ion mode on a Series
1100 MSD detector HP spectrometer, using an acetone mobile
phase. The compounds were added to reagent grade methanol
to give solutions of approximate concentration 0.1 mM. These
solutions were injected (1 mL) into the spectrometer via a HPLC
HP 1090 Series II fitted with an autosampler. The pump delivered
the solutions to the mass spectrometer source at a flow rate of
-
1
kcal mol . Complex 9 also shows relatively equivalent interaction
strengths between pairs of donor atoms and the copper center, the
two nitrogens (N11 and N14) have energies of 113.58 and 130.29
kcal mol , while the two sulfurs (S3 and S8) have energies of 351.91
and 376.49 kcal mol . The two oxygen donors also interact in an
-
1
-
1
-
1
equivalent fashion (O19 and O21) 175.58 and 174.6 kcal mol .
The stability of these complexes therefore appear to be a balance
of favorable donor–copper interactions and steric strain caused
by their macrocyclic nature. In all cases the macrocycle is able to
adopt a conformation in which the copper can sit in the plane of
the ring, however the interactions between the donor atoms and
-
1
300 mL min , and nitrogen was employed both as a drying and
nebulizing gas. Capillary voltages were typically 4000 V and 3500 V
for the positive- and negative-ion mode, respectively. Confirmation
of all major species in this ESI-MS study was aided by comparison
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 177–184 | 181

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of the observed and predicted isotope distribution patterns, the
latter calculated using the IsoPro 3.0 computer program.
ethanol (3 ¥ 25 mL) and dried under reduced pressure to yield
◦
1
derivative 2 in 72% yield. Melting point, 212–215 C. H NMR
O/NaOD, 293 K): d 1.72 (qn, 2H, CH ). 2.54 (br, 4H, CH N),
.61–2.77 (m, 8H, CH
O/NaOD, 293 K): d 31.48 (CH
CH S), 48.57 (CH N), 65.55 (CH), 181.68 (CO
(
2
(
(
3
D
2
2
2
Radiochemistry. All chemicals unless otherwise stated were
13
1
2
S), 3.22 (t, 2H, CH), C{ H}NMR
2 2
purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO).
D
2
), 33.56 (CH
S), 37.07
H). IR (cm ):
-
2
Water was distilled and then deionized (18 MX cm ) by passing
through a Milli-Q water filtration system (Millipore Corp., Mil-
ford, MA). Radioactivity was counted with a Beckman Gamma
-1
2
2
2
-
412br (nNH), 1732m (nasymCOOH), 1588s (nasymCOO ), 633w,
5
H
73w, 536sbr, 439m, 373s, 279w. ESI-MS (major negative-ions,
8
000 counter containing a NaI crystal (Beckman Instruments,
2-
2
-
O/NH
4
OH), m/z (%): 153 (30) [M - 2 H] , 307 (100) [M -
Inc., Irvine, CA). Care was taken to ensure all buffers and
vials were as free of metal contaminants as possible. The non-
radioactive copper complexes (4, 5 and 6) were used as fully
characterised non-radioactive standards to confirm the identity of
H] . Anal. Calcd. for C11
H
20
N
2
O
4
2
S : C, 42.84; H, 6.54; N, 9.08; S,
2
0.79%. Found: C, 42.75; H, 6.61; N, 8.97; S, 20.33%.
Synthesis of 1,10-dithia-4,7-diazacyclotetradecane-3,8-dicar-
boxylic acid, (NEC-SB, 3). Ethylendicysteine (5.50 g,
0.50 mmol) was added to a methanol/water (4 : 1) solution
100 mL) of NaOH (3.28 g, 82.01 mmol) and the solution was
stirred at room temperature for 2 h. 1,4-dibromobutane (4.43 g,
0.50 mmol) were added and the mixture was stirred for 48 h
6
4
64
the Cu analogs (7, 8 and 9). Identification of the Cu complexes
was performed using radio thin-layer chromatography (radio-
TLC). Radio-TLC detection was accomplished using a BIOSCAN
AR2000 Imaging Scanner (Washington, DC). TLC was performed
2
(
2
using glass-backed silica gel and a 10% aqueous (CH
3
COO)NH
4
to obtain a clear yellow solution. The solution was acidified to
pH 2. The precipitate which separated out was filtered, washed
with water (3 ¥ 50 mL) and ethanol (2 ¥ 25 mL) and dried under
reduced pressure to yield derivative 3 in 70% yield. Melting point,
solution : methanol (1 : 1) as mobile phase. Reaction progress
and purity were also monitored by analytical reversed-phase
HPLC, which was performed on a Waters 600E (Milford, MA)
chromatography system with a Waters 991 photodiode array
detector and an Ortec Model 661 radioactivity detector (EG &
G Instruments, Oak Ridge, TN). A Vydak C-18 column was
employed with a gradient that changes from 0.1% TFA in water to
◦
1
2
(
02 C (decomposition). H NMR (D
br, 4H, CH ), 2.54 (br, 4H, CH N), 2.64 (m, 8H, CH
O/NaOD, 293 K): d 27.11 (CH
S), 45.73 (CH N), 61.90 (CH), 179.46
2
O/NaOD, 293 K): d 1.50
S), 3.02 (t,
),
2
2
2
1
3
1
2
3
H, CH). C{ H}NMR (D
0.52 (CH S), 33.44 (CH
2
2
5
2
: 95 0.1%TFA in Water : 0.1 %TFA in CH
5 min. Unreacted Cu was eluted in the void volume, t < 1.0 min.
3
CN over the course of
2
2
2
-
1
6
4
(CO H). IR (cm ): 3397br (nNH), 1726m (nasymCOOH), 1633s
2
R
-
(
nasym COO ), 634w, 616w, 569m, 552m, 536m, 457w, 376w, 280m.
ESI-MS (major negative-ions, H
2
O/NH
4
OH), m/z (%): 160 (100)
: C, 44.70; H, 6.88; N,
Ligand synthesis and characterization
2
-
[
M - 2 H] . Anal. Calcd. for C12
H
22
N
2
O
4
S
2
Synthesis of 1,10-dithia-4,7-diazacyclododecane-3,8-dicar-
8.69; S, 19.89%. Found: C, 44.55; H, 6.72; N, 8.38; S, 19.44%.
boxylic acid (NEC-SE, 1). Ethylenedicysteine was synthesised
2
6
according to literature methods. Ethylenedicysteine (5.50 g,
0.50 mmol) and 1,2-dibromoethane (3.85 g, 20.50 mmol) were
Complex synthesis and characterization
2
Synthesis of [Cu(NEC-SE)]·3H
pared by stirring 1 (0.293 g, 1.0 mmol) and (CH
0.200 g, 1.0 mmol) in 30 mL of H O for 6 h at room temperature.
The resulting blue solid was filtered off, washed with water (3 ¥
2
O (4). Complex 4 was pre-
added to 100 mL of a NaHCO
3
solution (6.89 g, 82.01 mmol). The
◦
3
COO) Cu
2
mixture was gradually heated up to 100 C and was stirred for 24 h
to obtain a clear pale yellow solution. The solution was cooled to
room temperature and acidified to pH 2. The resulting precipitate
which separated out was filtered and washed with water (3 ¥
(
2
5
0 mL) and ethanol (3 ¥ 25 mL) and dried under reduced
◦
pressure to yield 4 in 85% yield. Melting point, 179–182
C
5
0 mL) and ethanol (3 ¥ 15 mL) and dried under reduced pressure
-
1
◦
1
(decomposition). IR (cm ): 3409br (nNH), 3210w (nOH), 1610 s
asymCOO), 1362 s (nsymCOO), 644mbr, 556br, 528br, 467br, 430w,
400w, 377m, 337m, 310br. Anal. Calcd. for C10 : C,
9.30; H, 5.41; N, 6.83; S, 15.64%. Found: C, 29.41; H, 5.48; N,
to yield derivative 1 in 65% yield. Melting point, 225–228 C. H
NMR (D O/NaOD, 293 K): d 2.49 (br, 4H, CH N), 2.63–2.70
S), 3.10 (t, 2H, CH). C{ H}NMR (D O/NaOD,
93 K): d 34.74, 37.21 (CH S), 49.26 (CH N), 65.75 (CH),
H). IR (cm ): 3390br (nNH), 1730sh (nasymCOOH),
586s (nasymCOO ), 610br, 532s, 459m, 399w, 376sbr, 267w, 255w.
(
n
2
2
1
3
1
H
22CuN
2
O
7
S
2
(
m, 8H, CH
2
2
2
2
1
1
2
2
-
1
6.60; S, 15.46%.
82.81 (CO
2
-
Synthesis of [Cu(NEC-SP)]·3H
2
O (5). Complex 5 was pre-
COO) Cu
O at room temperature for
h. The resulting blue precipitate was filtered off, washed with
ESI-MS (major negative-ions, H
[
2
O/NH
4
OH), m/z (%): 293 (100)
: C,
0.80; H, 6.16; N, 9.52; S, 21.78%. Found: C, 41.05; H, 6.26; N,
pared by stirring 2 (0.308 g, 1.0 mmol) and (CH
3
2
-
-
M – H] , 588 (60) [2M - H] . Anal. Calcd. for C10
H
18
N
2
O
4
S
2
(
6
0.200 g, 1.0 mmol) in 30 mL of H
2
4
9
.37; S, 21.53%.
water (3 ¥ 50 mL) and ethanol (3 ¥ 25 mL) and dried under
◦
reduced pressure to yield 5 in 77% yield. Melting point, 173–176 C
Synthesis of 1,10-dithia-4,7-diazacyclotridecane-3,8-dicar-
boxylic acid (NEC-SP, 2). The ligand NEC-SP, 2, was prepared
analogously to 1. Ethylenedicysteine (5.50 g, 20.50 mmol) and
-
1
(
(
2
decomposition). IR (cm ): 3412br (nNH), 3162wbr (nOH), 1617s
asymCOO), 1384s (nsymCOO), 640br, 460 m, 445sh, 377w, 350br,
68w, 255w. Anal. Calcd. for C11 : C, 31.16; H, 5.71;
n
H
24CuN
2
O
7
S
2
1
,3-dibromopropane (4.14 g, 20.50 mmol) were added to a
N, 6.61; S, 15.12%. Found: C, 31.11; H, 5.58; N, 6.41; S, 14.97%.
NaHCO
3
(6.89 g, 82.01 mmol) solution (100 mL). The mixture
◦
was gradually heated up to 100 C and stirred for 24 h to
obtain a clear yellow solution. The solution was cooled to
room temperature and acidified to pH 2. The precipitate which
separated out was filtered, washed with water (3 ¥ 50 mL) and
Synthesis of [Cu(NEC-SB)] (6). Complex 6 was prepared by
stirring 3 (0.322 g, 1.0 mmol) and (CH
3
COO)
2
Cu (0.200 g,
1.0 mmol) in 30 mL of H O at room temperature for 6 h. The
2
resulting blue precipitate was filtered off, washed with water
1
82 | Dalton Trans., 2009, 177–184
This journal is © The Royal Society of Chemistry 2009

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(
3 ¥ 50 mL) and ethanol (3 ¥ 25 mL) and dried under reduced
NMR
ESI-MS Electrospray ionization mass spectroscopy
IR Infra-red
Nuclear magnetic resonance
◦
pressure to yield 6 in 67% yield. Melting point, 187 C (decomposi-
tion). IR (cm ): 3397br (nNH), 3209br (nOH), 1601s (nasymCOO),
-
1
1
3
358s (nsymCOO), 617m, 592w, 568w, 553br, 400br, 385br, 330br,
01w, 280m. Anal. Calcd. for C12 : C, 37.54; H, 5.25;
NEC-SE 1,10-Dithia-4,7-diazacyclododecane-3,8-dicarboxylic
H
20CuN
2
O
4
S
2
acid
N, 7.30; S, 16.70%. Found: C, 37.35; H, 5.07; N, 7.19; S, 16.53%.
NEC-SP 1,10-Dithia-4,7-diazacyclotridecane-3,8-dicarboxylic
acid
6
4
64
Synthesis of radiocomplexes [ Cu(NEC-SE)] (7), [ Cu(NEC-
NEC-SB 1,10-Dithia-4,7-diazacyclotetradecane-3,8-
dicarboxylic acid
6
4
SP)] (8) and [ Cu(NEC-SB)] (9). Copper-64 was produced and
5
8 64
purified as previously described.
CuCl (200 mCi) was diluted
2
PET
TETA
Positron emission tomography
1,5,8,11-Tetraazacyclotetradecane-N,N¢,N¢¢,N¢¢¢-
tetraacetic acid
in 100 mL of 0.1 M ammonium acetate buffer (pH 7.5). 0.25 mg
of 1, 2, or 3 was dissolved in 100 mL of the same buffer, and then
added to the Cu solution. It was necessary to dissolve 3 in 20 mL
methanol prior to diluting with the 0.1 M ammonium acetate
buffer. After vigorous stirring of the resulting mixture at 90 C for
6
4
TLC
Thin layer chromatography
◦
1
h, deprotonation and coordination of the enantiomerically pure
macrocycle to the metal center occurred. The presence in solution
of the labeled complexes was established by TLC (7, R
= 0.32; 8,
Acknowledgements
We are very grateful for the technical assistance of Christo-
pher Sherman. Thanks also to Tom Voller and the WUSTL
cyclotron facility staff for radionuclide production. This work
was partially supported by grants from the National Institutes
of Health/National Cancer Institute (NIH/NCI) (CA86307 and
CA93375), by University of Camerino (FAR 2007), and by
Ministero dell¢Istruzione dell¢Universit a` e della Ricerca (PRIN
f
R
f
= 0.35; 9, R = 0.55) by comparison with the TLC profile of an
f
authentic sample of the corresponding non-radioactive complex.
The complexes were produced in a radiochemical purity of >98%.
For the biodistribution studies the radioactive complexes were
diluted with 0.9% saline.
2
007).
Biodistribution studies
All animal experiments were conducted in compliance with the
Guidelines for the Care and Use of Research Animals estab-
lished by Washington University’s Animal Studies Committee.
Male Lewis rats (30-day old, Charles River Laboratories, Inc.,
Wilmington, MA) were injected with either complex 7, 8 or 9
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3
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(
4
1
~40 mCi in 150 mL). Rats were examined at 3 time points (n =
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9 tissues and organs of interest were removed and weighed,
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D. K. Cabbiness and D. W. Margerum, J. Am. Chem. Soc., 1969, 91,
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%ID/organ) were then calculated on comparison to known
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standards.
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9
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59
geometry optimized in the program PCModel , using the GMX
60
force field. This is a MM2-like valence force field and uses a
Urey-Bradley force field about metal centers. Monte Carlo based
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1
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62
AMPAC and optimized using the SAM/1D Hamiltonian. The
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calculations (B3LYP/LACVP*) were then performed on each
1
1
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63
complex followed by a NBO analysis.
Abbreviations
2
DOTA
EC
1,4,7,10-Tetraazacyclododecane-N,N¢,N¢¢,N¢¢¢-
tetraacetic acid
N,N-Ethylene-di-L-cysteine
2
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 177–184 | 183

View Article Online
2
2
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