Copper Bis(thiosemicarbazonato)-stilbenyl Complexes That Bind to Amyloid-β Plaques

Article abstract of DOI:10.1021/acs.inorgchem.0c01520

Alzheimer's disease is characterized by the presence of extracellular amyloid-β plaques. Positron emission tomography (PET) imaging with tracers radiolabeled with positron-emitting radionuclides that bind to amyloid-β plaques can assist in the diagnosis o

Full text of DOI:10.1021/acs.inorgchem.0c01520

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Article
Copper Bis(thiosemicarbazonato)-stilbenyl Complexes That Bind to
Amyloid‑β Plaques
Asif Noor, David J. Hayne, SinChun Lim, Jessica K. Van Zuylekom, Carleen Cullinane, Peter D. Roselt,
Catriona A. McLean, Jonathan M. White, and Paul S. Donnelly*
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ABSTRACT: Alzheimer’s disease is characterized by the presence of extracellular
amyloid-β plaques. Positron emission tomography (PET) imaging with tracers
radiolabeled with positron-emitting radionuclides that bind to amyloid-β plaques
can assist in the diagnosis of Alzheimer’s disease. With the goal of designing new
imaging agents radiolabeled with positron-emitting copper-64 radionuclides that
bind to amyloid-β plaques, a family of bis(thiosemicarbazone) ligands with
appended substituted stilbenyl functional groups has been prepared. The ligands
form charge-neutral and stable complexes with copper(II). The new ligands can be
radiolabeled with copper-64 at room temperature. Two lead complexes were
demonstrated to bind to amyloid-β plaques present in post-mortem brain tissue
from subjects with clinically diagnosed Alzheimer’s disease and crossed the blood-
brain barrier in mice. The work presented here provides strategies to prepare
compounds with radionuclides of copper that can be used for targeted brain PET
imaging.
INTRODUCTION
■
Alzheimer’s disease is characterized by the presence of
extracellular amyloid plaques. The major constituent of these
plaques are aggregated forms of the amyloid-β peptide (Aβ), a
39−43 amino acid peptide derived from the amyloid precursor
protein.1−5 The exact role amyloid plaques play in dementia
remains controversial,6 but noninvasive imaging of plaque
burden using positron emission tomography (PET) is a useful
tool to complement clinical and cognitive evaluation of
patients.7−10
Appropriately substituted stilbene derivatives are conjugated,
relatively planar, molecules that bind selectively to hydro-
phobic binding sites on amyloid-β aggregates through a
combination of noncovalent interactions.11,12 A stilbene
derivative radiolabeled with fluorine-18, [18F]florbetaben
(Figure 1), has received clinical approval to detect the
presence of amyloid and assist in diagnosis of Alzheimer’s
disease.13 In this work we append a stilbene functional group
to a copper chelator with the goal of developing amyloid
imaging agents that can take advantage of positron-emitting
copper radionuclides. Positron-emitting copper-64 has a
radioactive half-life of 12.7 h, potentially facilitating delivery
of tracers to facilities remote from the production site. Three
other positron-emitting radionuclides of copper are also
available. The relatively short-lived copper-62 radionuclide
(t1/2 = 9.7 min) can be provided by a 62Zn/62Cu generator
system and has been used in brain imaging studies in
humans.14−17 Copper-60 (t1/2 = 20 min) and copper-61 (t1/2
Figure 1. (a) [18F]Florbetaben; (b) thiosemicarbazone-styrylpyr-
idylhydrazone copper(II) complexes (R = CH3 or −(CH2)2N-
(CH3)2);19 (c) [Cu(atsm)]; and (d) [Cu(atsm/a-stilbene).23
= 3.4 h) can both be produced on biomedical cyclotrons in
high purity.18 A potential advantage of radiolabeling
Received: May 24, 2020
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Scheme 1. Synthesis of [CuL1‑6
]
specifically designed ligands with copper radionuclides is the
potential for the method to be a relatively simple alternative to
the conventional organic synthesis required to incorporate
fluorine-18 into tracers.
append stilbene functional groups to bis(thiosemicarbazone)
ligands. The complexes described in this work involve ligands
where the stilbene functional group is directly attached to one
thiosemicarbazone limb of the ligand through a stable N4-
substituted thiosemicarbazone functional group. The ability of
the copper complexes to interact with synthetic amyloid-β1−40
and to bind to amyloid-β plaques in human brain tissue was
investigated. Methods to make the radioactive copper-64
complexes were developed, and two candidates were selected
for evaluation of their ability to cross the blood−brain barrier
in mice.
A challenge in developing copper-based brain amyloid
imaging agents is designing copper complexes that are
sufficiently stable and capable of crossing the blood−brain
barrier. In an earlier work we developed hybrid thiosemicarba-
zone-pyridylhydrazone copper(II) complexes that incorporate
a styrylpyridyl (Figure 1b) or pyridyl-benzofuran functional
group and bind to amyloid-β plaques.19,20 The thiosemicarba-
zone-styrylpyridylhydrazone copper complexes with a methyl
functional group (Figure 1b; R = CH3) bound to amyloid-β
plaques in human brain tissue but did not cross the blood−
brain barrier in mice. Modification of the terminal functional
group to a −CH2CH2N(CH3)2 group (Figure 1b; R =
−CH2CH2N(CH3)2) resulted in improved, but still modest,
RESULTS AND DISCUSSION
■
Small modifications to the ligand framework of bis-
(thiosemicarbazone) ligands affect the biological activity of
their copper complexes.21,24,25 For example, copper(II)
complexes with electron-donating alkyl groups on the “di-
imine-like” backbone of the ligand have lower CuII/I reduction
potential and are therefore less susceptible to reduction when
compared to complexes with hydrogen groups on the
backbone.26−30 The nature of the substituents on the
backbone of bis(thiosemicarbazonato) copper(II) complexes
can also alter lipophilicity, membrane permeability, and
retention as well as altering noncovalent interactions with
serum proteins.31−35
brain uptake in mice of 1.1
tissue (% IA/g).
0.2% injected activity/gram
In this manuscript, we extend our work on bis-
(thiosemicarbazone) ligands that are conjugated to stilbene
functional groups with a goal of producing complexes which
bind to amyloid-β plaques but have superior brain uptake.
Tetradentate bis(thiosemicarbazone) ligands form charge-
neutral and lipophilic complexes with copper radionuclides.
One example, [Cu(atsm)] (Figure 1c), has been investigated
as a hypoxia imaging agent and crosses the blood−brain barrier
in humans.14−17,21,22 In an earlier Communication, we
reported a complex where a stilbene functional group was
appended to a bis(thiosemicarbazone) ligand using a
thiocarbohydrazone linkage, [Cu(atsm/a-stilbene)] (Figure
1d). The stilbene functional group in [Cu(atsm/a-stilbene)]
is attached to the bis(thiosemicarbazone) ligand through a
hydrazone linkage that was susceptible to hydrolysis, and this
ligand proved relatively difficult to radiolabel with copper-64
with effective labeling, requiring transmetalation of the zinc(II)
complex.23
A family of bis(thiosemicarbazone) ligands with appended
substituted stilbene functional groups were prepared with the
goal of providing stable, charge-neutral copper complexes that
could selectively bind to amyloid-β plaques. The binding of
stilbene derivatives to amyloid-β plaques is favored by the
addition of electron-donating substituents such as −NHCH3
or −N(CH3)2. Complexes with modifications to the bis-
(thiosemicarbazones) ligand framework were prepared to
investigate if these subtle changes can affect the ability of the
complexes to cross the blood−brain barrier. For each ligand,
alkyl (methyl or ethyl) functional groups remained on the di-
imine-like backbone of the ligand to retain the relative redox
stability of [Cu(atsm)].
In this work, we prepare a range of bis(thiosemicarbazone)
ligands where a different synthetic methodology is used to
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Figure 2. An ORTEP-3 representation of the crystal structure of [CuL1]. DMF (ellipsoids are shown at 40% probability). Bond lengths (Å), Cu1−
S1 2.2473(6), Cu1−N3 1.961(2), C2−N1 1.337(3), C2−N2 1.318(3), C5−N5 1.321(3), C5−N6 1.354(3), C6−N6 1.409(2), C17−N7 1.383(3),
C12−C13 1.335(3).
A family of six ligands, H2L1−6, were prepared by selective
transamination reactions of substituted dissymmetric bis-
(thiosemicarbazone) precursors where one limb of the
precursor bears one -N4-methyl-N4-phenyl-3-thiosemicarba-
zone functional group (Scheme 1).36−39 Reaction of these
precursors with primary amines, such as amino-stilbenes,
involves nucleophilic attack of the primary amine on the
thiocarbonyl carbon atom with the elimination of N-
methylaniline to give the new ligands in excellent yields. In
each case, the E-conformation of the double bond in the
stilbene functional group was confirmed by 1H NMR
spectroscopy and a characteristic doublet (3JHH = 17 Hz).
The reaction of the ligands with copper(II) acetate
monohydrate at room temperature allows isolation of red-
brown charge-neutral copper(II) complexes. The electrospray
ionization mass spectra all reveal signals that can be attributed
to the protonated mono cations with the expected isotope
patterns.
indicative of double bond character, and the C2−S1 distance
(1.766(3) Å), indicative of more thiol-like rather than thione-
like character.
Copper(II) bis(thiosemicarbazonato) complexes are stable
(Ka = 1018), but reduction to a copper(I) complex results in
less stable complexes where copper(I) can dissociate from the
ligand. The electrochemical properties of each of the
complexes were investigated by cyclic voltammetry in
anhydrous dimethylformamide. Each complex exhibited a
quasi-reversible redox process at a glassy carbon electrode
that could be attributed to a CuII/I process ranging from Em =
−1.09 to Em = −1.16 V vs ferricinium/ferrocene (Fc+/Fc)
(Figure 3, Table 1), similar to what is found in [Cu(atsm)]
(Em ≈ −1.16 V). The similar reduction potentials of [CuL1−6
]
to [Cu(atsm)] suggest they should display a similar resistance
to reduction in vivo as [Cu(atsm)].26,29 Each complex also
displayed two essentially irreversible and partially overlapping
processes at more positive potentials, i.e., E ∼ 0−0.2 V vs Fc+/
Fc. One of these processes may involve oxidation of the
stilbene functional group as the free ligands show a similar
oxidation (Figure S22). The second overlapping process could
be due to either a CuIII/II process or oxidation of a ligand-based
orbital with π-character on bis(thiosemicarbazonato) ligand
(Figure 3). There is an oxidative process in [Cu(atsm)] at Em
= 0.15 V.39,41,42
Absorption spectra of H2L1−6 and [CuL1−6] (10 μM) were
measured in phosphate buffered saline (pH 7.4) with 20%
DMSO to assist in solubility. Each ligand and copper(II)
complex has an absorbance at λ = 360 nm due to the
functionalized stilbene. Complexation with copper(II) leads to
an absorption at λ = 495 nm due to the characteristic metal−
ligand-charge transfer transitions of bis(thiosemicarbazonato)-
copper(II) complexes (Figure S21). The absorption spectra for
CuL1−6 remain unchanged in the presence of a large excess of
glutathione (1 mM, Figure S21) showing that the complexes
are resistant to reduction and decomplexation in the presence
of this abundant biological reductant and copper binding
ligand.
The ligands H2L1−6 could be radiolabeled with copper-64 at
room temperature in 30 min to give [64Cu][CuL1−6] in high
radiochemical yields (>95%) without the need for further
purification. The radiolabeled complexes were characterized by
comparing their HPLC traces with their nonradioactive
[
63/65Cu] analogues (Figure 4). The HPLC traces (C18
It was possible to grow crystals of [CuL1] of suitable quality
for analysis by X-ray crystallography. The X-ray crystal
structure of [CuL1] (Figure 2) reveals that the copper(II)
ion is in the expected distorted square planar environment with
a dianionic tetradentate N2S2 ligand with three 5-membered
chelate rings. The Cu−N bond lengths (Cu−N3 1.961(2) Å)
and Cu−S (Cu1−S1 2.2473(6) Å) are similar to those found
in [Cu(atsm)].28,39,40 The double deprotonation of the ligand
is evident with relatively short C2−N2 distance (1.318(3) Å),
reversed phase column) of the compounds show a general
trend of increased retention time with increasing molecular
weight and expected lipophilicity, for example, [CuL2] (R1 =
R2 = CH3) has a shorter retention time than [CuL6] (R1 = R2
= CH2CH3) (Figure 4).
Estimations of lipophilicity made by measuring octanol−
water partition coefficients (log P) or octanol-buffer
distribution coefficients (log D7.4) are often useful in
identifying compounds that are likely to cross the blood−
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Figure 4. RP-HPLC chromatograms of [64Cu][CuL1−6] (red)
(radiation detection) and [natCu][CuL1−6] (blue, λabs = 280 nm,
offset for clarity).
Figure 3. Cyclic voltammograms of [CuL1−6] in DMF at a glassy
carbon working electrode, with a scan rate of 100 mV/s, 1 mM
+
−
Table 2. Log D7.4 for [64Cu][CuL1−6
]
analyte, and electrolyte = 0.1 M NBu4 PF6 . Potentials quoted versus
Fc+/Fc.
complex
log D7.4
Table 1. Summary of the CuII/I Reduction Potentials of
[64Cu][CuL1]
[64Cu][CuL2]
[64Cu][CuL3]
[64Cu][CuL4]
[64Cu][CuL5]
[64Cu][CuL6]
1.6 0.2
1.61 0.01
1.5 0.1
1.59 0.05
1.44 0.04
1.56 0.03
Complexes [CuL1−6
]
complex
Em (CuII/I) vs (Fc+/Fc)
Ic/Ia
peak separation (mV)
[CuL1]
[CuL2]
[CuL3]
[CuL4]
[CuL5]
[CuL6]
−1.16
−1.16
−1.15
−1.09
−1.14
−1.15
1.14
1.20
1.15
1.04
1.41
1.13
95
72
76
65
81
73
either by binding directly to the fibrils in competition with
ThT or by inhibiting fibril formation.
All six complexes displayed similar stability, CuII/I reduction
potentials, radiolabeling efficiency with copper-64, and log D7.4
values, and all were shown to interact with Aβ1−40, making the
selection of two lead candidates for more detailed inves-
tigations somewhat arbitrary. A low molecular weight (<500
Da) is an important consideration for penetration of the
blood−brain barrier,45 which suggests that the complexes
featuring the bis(thiosemicarbazone) framework with the
lowest molecular weight, either CuL1 or CuL2 (Figure 2),
would be worth further investigation. The complex where all
the functional groups were methyl, [CuL2] (MW = 529 Da),
was selected over [CuL1] based on the relative ease of
synthesis of the dimethylamine-substituted stilbene precursor.
The largest bis(thiosemicarbazone) framework, with all ethyl
substituents on the bis(thiosemicarbazone) fragment, CuL6
(MW = 571 Da), was also selected for further evaluation. The
relatively simple switch of methyl to ethyl substituents on
bis(thiosemicarbazone) copper(II) complexes can alter the
extent to which the complexes bind to serum proteins which
can also affect biodistribution, but this was not investigated in
this work.34,35,51
brain barrier through passive diffusion. Most compounds that
cross the blood-brain barrier have a log(D) in the range of 1−
3.43−45 The log D7.4 values for [64Cu][CuL1−6] (Table 2) are
all similar, ranging from 1.4−1.6. In this instance, this method
is less able to discriminate complexes of similar lipophilicities
than analysis by HPLC (Figure 4). The lipophilicity of each
complex is within the range required for penetration of the
blood−brain barrier and similar to that of [Cu(atsm)] (log D7.4
= 1.49
0.08)46 which crosses the blood−brain barrier in
humans,14,15,47,48 although it is acknowledged that many other
factors are also important such as efflux by P-glycoprotein.49,50
The dye Thioflavin-T (ThT, (2-[p-(dimethylamino)-
phenyl]-3,6-dimethyl-benzothiaolium chloride) can be used
to indicate the formation of Aβ1−40 fibrils. The emission from
ThT undergoes a λ = 115 nm red shift to λem = 485 nm upon
interaction with amyloid fibrils. The incubation of a mixture of
Aβ1−40 (4 μM) and ThT (2 μM) in PBS at 37 °C leads to the
formation of Aβ fibrils that can be monitored by measuring the
increase in fluorescence from ThT (Figure 5a). Addition of
[CuL1−6] (1 μM) results in a significant reduction in ThT
fluorescence (Figure 5a). The reduced ThT fluorescence
suggests that each of the complexes interact by Aβ1−40 fibrils
The interactions of [CuL2] and [CuL6] with preformed
mature Aβ fibrils were also investigated. A mixture of Aβ1−40
(100 μM) was gently agitated in phosphate buffered saline
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Figure 5. (a) Time-dependent changes in ThT fluorescence (λex = 440 nm, λem = 480 nm) for solutions of Aβ1−40 (4 μM) and ThT (2 μM)
incubated with and without Cu(II) complexes [CuL1−6] (1 μM) over 20 h at 37 °C. (b) Aβ1−40 aggregation assay using mature fibrils (5 μM),
where the data indicate the change in ThT fluorescence (5 μM) incubated for 1 h in the presence and absence of compounds. TEM micrographs of
Aβ1−40 fibrils (10 μM) (c), Aβ1−40 fibrils (10 μM) in the presence of [CuL2] (10 nM) (d), and Aβ1−40 fibrils (10 μM) in the presence of [CuL6]
(10 nM) (e).
Figure 6. (a) Images of human brain tissue from an AD subject and (b) images of human brain tissue from aged-matched non-AD control. Images
stained with 1E8 antibody were visualized with bright-field microscopy, and those of the contiguous section were stained with [CuL2] or [CuL6]
and visualized using epi-fluorescence microscopy (λex = 405 nm, λem = 461 nm).
(pH 7.4) at 37 °C for 48 h to promote the formation of Aβ1−40
fibrils and then ThT (100 μM) was added, and the mixture
was incubated at 37 °C for a further 60 min. Aliquots of this
mixture (5 μM fibrils) were then used for the following assays.
An increase fluorescence (λem = 480 nm) from ThT confirmed
the presence of Aβ1−40 fibrils, and the fluorescence intensity
was similar to that in the presence of DMSO (5%) that is
required to assist in the solubility of [CuL1−6] (Figure 5 b).
Addition of [Cu(atsm)] at either 0.1 μM or 1 μM did not
significantly alter the emission intensity of ThT, suggesting no
interaction with Aβ1−40 fibrils and that the presence of
paramagnetic CuII (d9) did not interfere with the assay. In
contrast, addition of either [CuL2] or [CuL6] (0.1 μM) to
Aβ1−40 fibrils (20 μM of fibrils, 20 μM ThT) resulted in a
reduction in ThT fluorescence by 40% and 27%, respectively
(Figure 5b), suggesting that the complexes are capable of
binding to Aβ1−40 fibrils and displacing ThT. Addition of
[CuL2] and [CuL6] at 1 μM of [CuL2] and [CuL6] resulted in
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a further reduction in fluorescent intensity from ThT by 67%
and 58%, respectively (Figure 5b). The free ligands H2L2 and
H2L6 were also capable of displacing ThT from Aβ1−40 fibrils,
leading to similar reductions in fluorescence intensity as their
respective metal complexes which provided further reassurance
that the presence of paramagnetic copper(II) in the complexes
does not interfere with the assay (Figure 5b).
The interaction of [CuL2] and [CuL6] with Aβ1−40 fibrils
was also investigated using transmission electron microscopy
(TEM). A mixture of Aβ1−40 (100 μM) was gently agitated for
48 h in phosphate buffered saline (pH 7.4) at 37 °C to
promote the formation of Aβ fibrils, and then aliquots (10 μM)
of fibrils were treated with either [CuL2] or [CuL6] for 2 h at
37 °C. The TEM images show the expected morphology of the
fibrils (10 μM of fibrils) formed for the untreated sample
(Figure 5c), while the images for the solutions treated with
either [CuL2] or [CuL6] (10 μM of fibrils, 10 nM of complex)
reveal a dramatic change in morphology to what appears to be
amorphous irregular aggregates (Figure 5d, e).
Figure 7. Brain uptake measured by an ex-vivo biodistribution
analysis. Mice were euthanized at 2 and 60 min after injection. Tissues
were harvested, weighed, and radioactivity quantified on a gamma
counter. Brain uptake expressed as % injected dose/gram tissue. Data
represents mean SEM of n = 3 mice/group.
Table 3. Biodistribution Data Expressed as Injected Activity
a
Normalized to the Mass of the Organ (% IA/g)
The ligands H2L2 and H2L6 are fluorescent due to the
presence of the stilbene functional group. Coordination of
copper(II) to the ligands significantly reduces this fluores-
cence, but the copper(II) complexes remain sufficiently
fluorescent (λem = 440 nm, λex = 365 nm, Figure S20) to
detect their binding to amyloid-β plaques in brain tissue using
epi-fluorescence microscopy. Post-mortem samples of frontal
cortex of human brain tissue (7 μm serial sections) from
clinically diagnosed Alzheimer’s disease subjects were pre-
treated with 2% bovine serum albumin to reduce nonspecific
binding and then treated with either [CuL2] or [CuL6] (10
μM). The selective retention of the complexes on the treated
brain tissue was assessed using epi-fluorescence microscopy
(λex = 405 nm, λem = 461 nm, Figure 6). Amyloid-β plaques are
typically 40−60 μm in diameter, so contiguous serial sections
of tissue (7 μm) often contain portions of the same plaque.
The complexes bound selectively to amyloid-β plaques that
could be identified by comparison with the contiguous section
of tissue where the amyloid-β plaques had been stained with an
amyloid-β specific antibody (1E8). Importantly, neither
[CuL2] or [CuL6] showed any evidence of significant retention
on brain tissue from age-matched control subjects (non-AD
post-mortem tissue, Figure 6).
[64Cu][CuL2]
[64Cu][CuL6]
2 min
60 min
2 min
60 min
blood
lungs
heart
22.0(2.4)
95.5(20.4)
13.8(1.7)
35.5(2.3)
7.0(0.3)
1.0(0.1)
16.1(0.9)
2.2(0.6)
1.8(0.1)
14.3(3.5)
3.8(0.3)
50.8(1.2)
8.2(0.5)
1.2(0.1)
33.0(2.0)
0.34(0.04)
16.8(2.7)
67.4(7.7)
13.1(2.9)
50.3(4.2)
7.8(0.5)
0.7(0.1)
59.7(22.5)
1.1 (0.2)
1.5(0.1)
29.7(5.1)
2.3(0.1)
67.9(2.9)
4.3(0.2)
0.49(0.01)
125.9(12.8)
0.22(0.02)
liver
kidneys
muscle
spleen
brain
a
Standard error given in brackets (n = 3).
compounds could be related to the large blood volume in the
lungs and could relate to the activity present in blood.55
CONCLUSION
■
Amyloid imaging agents that use the range of positron-emitting
radionuclides of copper that are available could increase the
number of facilities that could perform diagnostic PET
scans.19,20,23,56−62 Incorporating positron-emitting radionu-
clides of copper(II) into specifically designed ligands can be
relatively simple, fast, and efficient when compared to the
incorporation of fluorine-18 into organic molecules through
the formation of covalent C−F bonds. However, a major
challenge in using metal complexes for brain imaging
applications is the difficulty in preparing metal complexes
that cross the blood−brain barrier yet retain selectivity for
particular targets. In this work a family of bis-
(thiosemicarbazone) ligands with appended substituted
stilbene ligands have been prepared that form charge-neutral
and stable complexes with copper(II). The new ligands could
be radiolabeled with [64Cu]CuII at room temperature, and the
complexes have a lipophilicity, as estimated by measurements
of log D7.4, within a range that is consistent with compounds
that cross the blood−brain barrier. Two leads complexes, that
were selected for further evaluation, bound to amyloid plaques
present in post-mortem brain tissue for AD subjects. These
two compounds cross the blood−brain barrier in mice to a
similar degree to [64Cu][Cu(atsm)],52 a complex that has been
used for brain imaging in humans.14−17,48
The potential of [64Cu][CuL2] and [64Cu][CuL6] to cross
the blood−brain barrier was evaluated in wild-type mice
(Balb/c). The radiolabeled tracers were administered via tail
vein injection, and then the mice were euthanized at either 2 or
60 min postinjection. The activity accumulated in each organ
was counted and expressed as a percentage of the injected
activity normalized to the mass of the organ (% IA/g). Both
the tracers show good brain uptake after 2 min postinjection,
[64CuL2] = 2.2
0.6% IA/g; [64CuL6] = 1.1
0.2% IA/g
(Figure 7 and Table 3). For both compounds the degree of
brain uptake is similar to that of [64Cu]Cu(atsm) in wild-type
mice (∼0.9
0.4% ID/g)52 which correlates to sufficient
uptake for imaging the brain in humans. Wild-type mice do not
possess Aβ plaques, so an ideal tracer should clear from their
brains rather than be retained.12 Following the initial uptake in
the brain, both tracers display good clearance at 60 min
postinjection ([64CuL2] = 0.34 0.04% IA/g; [64CuL6] = 0.22
0.02% IA/g). The high level of uptake in the liver for both
compounds is expected for lipophilic compounds that clear
primarily through hepatobiliary metabolism.53,54 The high
degree of uptake in the lungs at 2 min postinjection for both
The brain uptake of [64Cu][CuL2] is better than the
copper(II) hybrid thiosemicarbazone-styrylpyridylhydrazone
copper complexes with a −CH2CH2N(CH3)2 functional
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112.5, 30.6. HRMS (ESI) (+ve ion) m/z calcd for [C15H17N2O2]+:
255.11 [M + H]+; found, 255.113.
group (Figure 1b; R = −CH2CH2N(CH3)2) that we reported
previously.19 The ligands presented in this work are easier to
radiolabel than the H2atsm/a-stilbene derivative (Figure 1d)
we reported in a preliminary Communication.23 A direct
comparison of the brain uptake for [64Cu][CuL2] and
[64Cu]Cuatsm/a-stilbene (Figure 1d) is not possible because
a different method of quantification (PET imaging) was used
in the earlier work.23
Future work on the complexes described here could include
investigating their uptake and retention in murine models of
amyloid pathology and quantifying their affinity for amyloid-
β1−40. An advantage of ligands designed to bind copper(II) is
that it is possible to use them for any of the four positron-
emitting radionuclides of copper that are available. This work
focused on copper-64, but extrapolation of this chemistry to
copper-60, copper-61, or copper-62 should be relatively
straightforward. The ligands described here could be readily
adapted to kit-based formulations, similar to those for
diagnostic agents based on technetium-99.
Synthesis of (E)-4-Amino-4′-methylaminostilbene. To (E)-4-
nitro-4′-methylaminostilbene (0.99 g, 3.9 mmol) was added ethanol
(20 mL), and the mixture was sparged with N2 (1 h). Stannous
chloride was then added (4.40 g, 23 mmol) to the mixture, and the
mixture was heated at reflux. After 1.5 h, the reaction was cooled to
ambient temperature and adjusted to pH 9 (1 M NaOH). The
mixture was extracted into ethyl acetate (3 × 80 mL), and the
combined organic phase was washed with brine (3× 100 mL), dried
(MgSO4), and filtered; the solvent was then removed under reduced
1
pressure to afford a yellow solid (0.75 g, 3.3 mmol, 87%). HNMR
(400 MHz; CDCl3): δ 7.32 (m, 4H, ArH), 6.86 (m, 2H, CH2
CH2), 6.67 (m, 2H, ArH), 6.60 (m, 2H, ArH), 3.68 (s, 3H, NH2,
NHMe), 2.86 (s, 3H). 13C NMR (101 MHz; CDCl3): δ 148.6, 145.5,
129.0, 127.48, 127.36, 127.28, 125.6, 124.7, 115.4, 112.6, 30.9. HRMS
(ESI) (+ve ion) m/z calcd for [C15H17N2O2]+: 225.14 [M + H]+;
found, 225.137.
Synthesis of (E)-4-Nitro-4′-dimethylaminostilbene. To 4-
nitrobenzyl phosphanate was added dimethylformamide (dry, 10
mL), and the mixture was stirred under an atmosphere of N2. To the
dark yellow solution was added sodium hydride (60% w/w in mineral
oil, 0.41 g, 10 mmol), and the mixture became dark red in color. After
15 min, 4-dimethylbenzaldehyde was added to the reaction mixture
and the mixture was stirred for 1 h. Ethanol was added (2 mL) to this
mixture and then dH2O (50 mL) was added to afford a red
precipitate.The precipitate was isolated by filtration and washed with
dH2O. The red solid was dissolved in dichloromethane and washed
with dH2O (3 × 200 mL) and then brine (1 × 100 mL). The organic
phase was dried (MgSO4) and filtered, and the solvent was removed
under reduced pressure to afford a red solid (1.75 g, 6.5 mmol, 90%).
1H NMR (400 MHz; CDCl3): δ 8.18 (m, 2H, ArH), 7.56 (m, 2H,
ArH), 7.45 (m, 2H, ArH), 7.21 (d, 3JHH = 16.2, 1H, CHCH), 6.93
EXPERIMENTAL SECTION
■
General Procedures. Unless otherwise stated, all reagents were
purchased from commercial sources and used without further
purification. Flash column chromatography was carried out using
silica gel (40−63 μm) as the stationary phase. Analytical TLC was
performed on precoated silica gel plates (0.25 mm thick, 60F254,
Merck, Germany) and visualized under UV light. H and 13C NMR
1
spectra were recorded either on a 400 MHz Varian/Agilent 400-MR
or 500 MHz AR spectrometer at 298 K. Chemical shifts are reported
in parts per million (ppm) and referenced to the residual solvent peak.
Coupling constants (J) are reported in Hertz (Hz). Standard
abbreviations indicating multiplicity were used as follows: m =
multiplet, quint. = quintet, q = quartet, t = triplet, d = doublet, s =
singlet, br = broad. ESI-MS spectra were recorded on an Agilent 6510
ESI-TOF LC/MS mass spectrometer or a Thermo Fisher OrbiTRAP
infusion mass spectrometer. 4-Methyl-4-phenyl-3-thiosemicarba-
3
(d, JHH = 16.2, 1H, CHCH), 6.72 (m, 2H, ArH), 3.02 (s, 6H,
N(CH3)2). 13C NMR (101 MHz; CDCl3): δ 151.0, 145.2, 133.8,
128.5, 126.2, 124.43, 124.30, 121.7, 112.3, 40.4. HRMS (ESI) (+ve
ion) m/z calcd for [C16H17N2O2]+: 269.32 [M + H]+; found, 269.129.
Synthesis of (E)-4-Amino-4′-dimethylaminostilbene. To (E)-
4-nitro-4′-dimethylaminostilbene (1.7 g, 6.5 mmol) was added
ethanol (40 mL), and the mixture was sparged with N2 (1 h).
Stannous chloride was added (7.6 g, 40 mmol) to this mixture, and
the mixture was heated at reflux. After 1.5 h, the reaction was cooled
to ambient temperature and adjusted to pH 9 (1 M NaOH). The
mixture was extracted into dichloromethane (300 mL), and the
organic phase was washed with brine (150 mL), dried (MgSO4), and
filtered; the solvent was then removed under reduced pressure to
36
zide,63 4-methylaminobenzaldehyde,64 and H2‑ATSM/M2 were
prepared according to reported procedures. UV−vis spectroscopy
was performed on a Shimadzu UV1650-PC spectrometer (Shimadzu,
Kyoto, Japan), and a Varian Cary Eclipse fluorescence spectrometer
was used for fluorescence spectroscopy. Cyclic voltammograms were
recorded using a Metrohm (Switzerland) AUTOLAB PGSTAT100
instrument and analyzed using Autolab GPES V4.9 software.
Measurements were carried out using 1 mM analyte in anhydrous
DMF with tetrabutylammonium hexafluorophosphate (0.1 M) as a
supporting electrolyte. A glassy carbon working electrode was used
along with a Pt wire counter electrode and a pseudo-leakless Ag/Ag+
working electrode (EDAQ, New South Wales, Australia). Measure-
ments were referenced to internal ferrocene (Fc/Fc+, Em = 0.00 V
where Em = (Epc + Epa)/2.
1
afford a yellow solid (1.1 g, 4.6 mmol, 71%). H NMR (400 MHz;
CDCl3): δ 7.38 (m, 2H, ArH), 7.30 (m, 2H, ArH), 6.86 (m, 2H,
CHCH), 6.72 (m, 2H, ArH), 6.67 (m, 2H, ArH), 3.67 (br, 2H,
NH2), 2.97 (s, 6H, N(CH3)2). 13C NMR (101 MHz; CDCl3): δ
149.9, 145.5, 129.1, 127.30, 127.22, 126.7, 125.5, 124.8, 115.4, 112.8,
40.7. HRMS (ESI) (+ve ion) m/z calcd for [C16H19N2]+: 239.15 [M
+ H]+; found, 239.15.
Synthesis of (E)-N-Methyl-2-(4-oxohexan-3-ylidene)-
hydrazine-1-carbothioamide. To a flask charged with 3,4-
hexadione (7.827 g, 68.6 mmol) was added dH2O (200 mL) and a
catalytic amount of acetic acid (5 drops). The mixture was cooled to 5
°C and 4-methyl-3-thiosemicarbazide (6.086 g, 57.9 mmol) was
added in portions over 2 h. A white precipitate was observed, and the
mixture was stirred for 1 h further after the final addition. The white
precipitate was isolated by filtration, washed with dH2O and then
Synthesis of (E)-4-Nitro-4′-methylaminostilbene. 4-Nitro-
benzyl phosphanate (2.1 g, 7.7 mmol) was dossolved in
dimethylformamide (dry, 10 mL), and then sodium hydride (60%
w/w in mineral oil, 0.39 g, 9.8 mmol) was added. The suspension was
stirred for 15 min followed by the addition of 4-methylaminobenzal-
dehyde (0.94 g, 7.0 mmol). The mixture was stirred for 2 h, and then
ethanol (2 mL) was added, followed by dH2O (50 mL), to afford a
red precipitate. The precipitate was isolated by filtration and washed
with dH2O. The red solid was dissolved in dichloromethane and
washed with dH2O (3 × 200 mL) and then brine (1 × 100 mL). The
organic phase was dried (MgSO4) and filtered, and the solvent was
removed under reduced pressure to afford a red solid (1.4 g, 5.5
1
pentane, and dried at the pump (quantitative yield). H NMR (400
MHz; DMSO-d6): δ 10.81 (s, 1H, NH), 8.56 (m, 1H, NH), 3.05 (d,
3JHH = 4.5 Hz, 3H, CH3), 2.95 (q, 3JHH = 7.3 Hz, 2H, CH2), 2.59 (q,
3
3JHH = 7.5 Hz, 2H, CH2), 0.97 (t, JHH = 7.3 Hz, 3H, CH3), 0.86 (t,
1
3JHH = 7.5 Hz, 3H, CH3). 13C NMR (101 MHz; DMSO-d6): δ 200.0,
mmol, 79%). H NMR (400 MHz; CDCl3): δ 8.18 (m, 2H, ArH),
7.56 (m, 2H, ArH), 7.41 (m, 2H, ArH), 7.19 (d, 3JHH = 16.2 Hz, 1H,
179.0, 149.0, 31.4, 29.3, 16.4, 10.2, 8.1; HRMS (ESI) (+ve ion) m/z
calcd for [C8H16N3OS]+: 202.10 [M + H]+; found, 202.101.
Synthesis of (E)-N-Ethyl-2-(4-oxohexan-3-ylidene)-
hydrazine-1-carbothioamide. To a flask charged with 3,4-
3
CHCH), 6.92 (d, JHH = 16.2 Hz, 1H, CHCH), 6.60 (m, 2H,
ArH), 3.98 (br, J = 0.5 Hz, 1H, NH), 2.89 (s, 3H, CH3). 13C NMR
(101 MHz; CDCl3): δ 150.1, 133.9, 128.7, 126.2, 125.5, 124.3, 121.8,
G
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Article
hexadione (7.107 g, 62.3 mmol) was added dH2O (200 mL) and a
catalytic amount of acetic acid (5 drops). The mixture was cooled to 5
°C and 4-ethyl-3-thiosemicarbazide (6.104 g, 51.2 mmol) was added
in portions over 2 h. A white precipitate was observed, and the
mixture was stirred for 1 h further after the final addition. The white
precipitate was isolated by filtration, washed with dH2O and then
Synthesis of H2L2. To a flask charged with H2ATSM-M2 (0.23,
0.82 mmol) and (E)-4-amino-4′-dimethylaminostilbene (0.20 g, 0.84)
was added acetonitrile (17 mL). The reaction was heated at reflux for
18 h, and an orange precipitate was observed. The precipitate was
isolated from the hot reaction mixture by filtration, washed with
acetonitrile and then diethyl ether, and dried in vacuo (0.26 g, 0.56
mmol, 67%). 1H NMR (400 MHz; DMSO-d6): δ 10.58 (s, 1H, NH),
10.30 (s, 1H, NH), 9.94 (s, 1H, NH), 8.41 (d, J = 4.5 Hz, 1H), 7.54
(q, J = 11.7 Hz, 4H), 7.43 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 16.3 Hz,
1H), 6.97 (d, J = 16.4 Hz, 1H), 6.73 (d, J = 8.9 Hz, 2H), 3.04 (d, J =
4.5 Hz, 3H), 2.93 (s, 6H), 2.29 (s, 3H), 2.25 (s, 3H). 13C NMR (126
MHz, DMSO-d6): δ 178.94, 176.86, 150.38, 149.71, 148.19, 137.84,
135.49, 128.85, 127.91, 125.91, 125.83, 125.44, 123.49, 112.70, 31.69,
12.50, 12.23. HRMS (ESI) (+ve ion) m/z calcd for [C23H30N7S2]+:
468.2004 [M + H]+; found, 468.1993.
1
pentane, and dried at the pump (quantitative yield). H NMR (400
MHz; DMSO-d6): δ 10.74 (s, 1H, NH), 8.59 (m, 1H, NH), 3.62 (m,
3H, CH2), 2.95 (q, 3JHH = 7.3 Hz, 2H, CH2), 2.59 (q, 3JHH = 7.5 Hz,
3
3
2H, CH2), 1.16 (t, JHH = 7.1 Hz, 3H, CH3), 0.98 (t, JHH = 7.3 Hz,
3
3H, CH3), 0.86 (t, JHH = 7.5 Hz, 3H, CH3). 13C NMR (101 MHz;
DMSO-d6): δ 8.08, 10.18, 14.11, 16.45, 29.32, 38.75, 148.99, 178.03,
199.90. HRMS (ESI) (+ve ion) m/z calcd for [C9H18N3OS]+: 216.12
[M + H]+; found, 216.117.
Synthesis of DTSM-N-methyl-N-phenyl. To a flask charged
with 4-methyl-4-phenyl-3-thiosemicarbazide (1.506 g, 8.3 mmol) and
(E)-N-methyl-2-(4-oxohexan-3-ylidene)hydrazine-1-carbothioamide
(1.664, 8.3 mmol) was added EtOH (50 mL) and a catalytic amount
of acetic acid (8 drops). The mixture was heated at reflux for 1.5 h. A
white precipitate was observed and was collected by filtration after the
reaction cooled to ambient temperature. The precipitate was washed
with a minimum of ethanol and then pentane and dried at the pump.
Additional product was collected after the filtrate was stirred at
Synthesis of H2L3. To a flask charged with DTSM-N-methyl-N-
phenyl (334 mg, 0.92 mmol) and (E)-4-amino-4′-methylaminostil-
bene (206 mg, 0.92 mmol) was added acetonitrile (10 mL), and the
mixture was heated at reflux for 6 h. A yellow precipitate was isolated
by hot filtration, washed with acetonitrile and then pentane, and dried
1
in vacuo (133 mg, 0.30 mmol, 33%). H NMR (400 MHz; DMSO-
d6): δ 10.73 (s, 1H, NH), 10.44 (s, 1H, NH), 9.88 (s, 1H, NH), 8.33
(m, 1H, NH), 7.57 (m, 2H, ArH), 7.50 (m, 2H, ArH), 7.35 (m, 2H,
3
3
1
ArH), 7.07 (d, JHH = 16.4 Hz, 1H, CHCH), 6.91 (d, JHH = 16.4
Hz, 1H, CHCH), 6.54 (m, 2H, ArH), 5.88 (m, 1H, NH), 3.03 (m,
3H, CH3), 2.95 (m, 4H, 2CH2), 2.70 (m, 3H, CH3), 0.94 (m, 6H,
2CH3). 13C NMR (126 MHz; DMSO-d6): δ 178.5, 176.3, 152.1,
150.6, 149.7, 137.2, 135.2, 128.8, 127.6, 125.4, 125.0, 124.6, 122.2,
111.7, 31.3, 29.6, 17.19, 17.14, 11.00, 10.82. HRMS (ESI) (+ve ion)
m/z calcd for [C24H32N7S2]+: 482.2161 [M + H]+; found, 482.2153.
Elemental analysis calcd (%) for C24H31N7S2·1.2H2O·MeCN: C
57.37, H 6.74, N 20.58; found C 57.25, H 6.40, N 20.59.
ambient temperature over 48 h (1.211 g, 3.3 mmol, 40%). H NMR
(500 MHz, DMSO-d6): δ 11.08 (s, 3H, NH), 10.40 (s, 1H), 9.35 (d, J
= 5.1 Hz, 1H, NH), 8.30 (q, J = 4.3 Hz, 1H, NH), 7.60−7.48 (m, 2H,
ArH), 7.40 (dd, J = 12.4, 4.7 Hz, 3H, ArH), 3.56 (s, 3H, CH3), 3.03
(d, 3H, CH3), 2.96−2.76 (m, 4H, CH2CH3), 1.00−0.79 (m, 6H,
CH2CH3). 13C NMR (126 MHz, DMSO-d6): δ 178.99, 175.08,
169.15, 150.51, 143.37, 130.47, 128.44, 127.13, 43.28, 31.75, 17.67,
17.56, 11.55, 11.48. HRMS (ESI) (+ve ion) m/z calcd for
[C16H25N6S2]+: 356.1582 [M + H]+; found, 365.1575
Synthesis of H2L4. To a flask charged with DTSM-N-methyl-N-
phenyl (94 mg, 0.26 mmol) and (E)-4-amino-4′-dimethylaminos-
tilbene (62 mg, 0.26 mmol) was added acetonitrile (5 mL), and the
mixture was heated at reflux for 6 h. A yellow precipitate was isolated
by hot filtration, washed with acetonitrile and then pentane, and dried
in vacuo (30 mg, 0.06 mmol, 24%). 1H NMR (400 MHz; DMSO-d6):
δ 10.73 (s, 1H, NH), 10.44 (s, 1H, NH), 9.89 (s, 1H, NH), 8.32 (m,
1H, NH), 7.59 (m, 2H, ArH), 7.52 (m, 2H, ArH), 7.43 (m, 2H, ArH),
7.11 (d, 3JHH= 16.3 Hz, 1H, CHCH), 6.97 (d, 3JHH = 16.3 Hz, 1H,
Synthesis of DTSE-N-methyl-N-phenyl. To a flask charged with
4-methyl-4-phenyl-3-thiosemicarbazide (1.521 g, 8.4 mmol) and (E)-
N-ethyl-2-(4-oxohexan-3-ylidene)hydrazine-1-carbothioamide (1.807,
8.4 mmol) was added EtOH (50 mL) and a catalytic amount of acetic
acid (6 drops). The mixture was heated until the reagents had
dissolved and then was left to stir at ambient temperature for 72 h. A
white precipitate was observed and was collected by filtration, washed
with a minimum of ethanol and then pentane, and dried at the pump.
Additional product was collected after the filtrate was stirred at
ambient temperature over 48 h (1.370 g, 3.62 mmol, 43%). 1H NMR
(400 MHz; DMSO-d6): δ 11.09 (s, 1H, NH), 10.40 (s, 1H), 9.34 (d, J
= 4.3 Hz, 1H, NH), 8.29 (t, J = 5.8 Hz, 1H, NH), 7.51 (m, 2H, ArH),
7.39 (m, 3H, ArH), 3.58 (m, 5H, CH3, CH2CH3), 2.84 (m, 4H,
3
CHCH), 6.73 (m, 2H, ArH), 3.04 (d, JHH = 4.4 Hz, 3H, CH3),
2.98 (m, 10H, 2CH2, N(CH3)2), 0.95 (m, 6H, 2CH3). 13C NMR (101
MHz; DMSO-d6): δ 178.5, 176.3, 152.1, 150.7, 149.9, 137.3, 135.0,
128.4, 127.5, 125.5, 125.02, 124.99, 123.0, 112.2, 40.0, 31.3, 17.21,
17.15, 11.01, 10.83. HRMS (ESI) (+ve ion) m/z calcd for
[C25H34N7S2]+: 496.2317 [M + H]+; found, 496.2311. Elemental
analysis calcd (%) for C25H33N7S2·0.7H2O: C 59.07, H 6.82, N 19.29;
found C 58.75, H 6.40, N, 19.48.
3
CH2CH3), 1.12 (t, J HH= 7.1 Hz, 3H), 0.90 (m, 6H, CH2CH3). 13C
NMR (126 MHz; DMSO-d6): δ 178.8, 177.5, 174.6, 152.5, 150.0,
142.8, 130.0, 128.0, 126.7, 42.8, 38.6, 17.2, 14.3, 11.07, 11.03. HRMS
(ESI) (+ve ion) m/z calcd for [C17H27N6S2]+: 379.1739 [M + H]+;
found, 379.1733
Synthesis of H2L5. To a flask charged with DTSE-N-methyl-N-
phenyl (80 mg, 0.21 mmol) and (E)-4-amino-4′-methylaminostilbene
(50 mg, 0.22 mmol) was added acetonitrile (5 mL), and the mixture
was heated at reflux for 6 h. A yellow precipitate was isolated by hot
filtration, washed with acetonitrile and then pentane, and dried in
vacuo (33 mg, 0.07 mmol, 32%). 1H NMR (400 MHz; DMSO-d6): δ
10.75 (s, 1H, NH), 10.39 (s, 1H, NH), 9.88 (s, 1H, NH), 8.33 (m,
1H, NHz), 7.57 (m, 2H, ArH), 7.50 (m, 2H, ArH), 7.35 (m, 2H,
Synthesis of H2L1. To a flask charged with H2ATSM-M2 (0.24 g,
0.88 mmol) and (E)-4-amino-4′-methylaminostilbene (0.20 g, 0.88)
was added acetonitrile (17 mL). The reaction was heated at reflux for
18 h, and an orange precipitate was observed. The precipitate was
isolated from the hot reaction mixture by filtration, washed with
acetonitrile and then diethyl ether, and dried in vacuo to afford an
orange powder (0.28 g, 0.63 mmol, 72%). 1H NMR (400 MHz;
DMSO-d6): δ 10.57 (s, 1H, NH), 10.30 (s, 1H, NH), 9.94 (s, 1H,
NH), 8.41 (m, 1H, NH), 7.56 (m, 2H, ArH), 7.50 (m, 2H, ArH), 7.35
(m, 2H, ArH), 7.07 (d, 3JHH = 16.3 Hz, 1H, CHCH), 6.91 (d, 3JHH
3
3
ArH), 7.07 (d, JHH = 16.3 Hz, 1H, CHCH), 6.91 (d, JHH = 16.3
Hz, 1H, CHCH), 6.54 (m, 2H, ArH), 5.88 (m, 1H, NH), 3.60 (m,
2H, CH2), 2.97−2.87 (m, 4H, 2CH2), 2.70 (m, 3H, CH3), 1.15 (m,
3H, CH3), 0.93 (m, 6H, 2CH3). 13C NMR (101 MHz; DMSO-d6): δ
177.9, 176.7, 152.4, 151.0, 150.2, 137.6, 135.6, 129.2, 128.0, 125.8,
125.5, 125.0, 122.7, 112.1, 39.0, 30.0, 17.77, 17.60, 14.8, 11.43, 11.28.
HRMS (ESI) (+ve ion) m/z calcd for [C25H34N7S2]+: 496.2317 [M +
H]+; found, 496.2310. Elemental analysis calcd (%) for C25H33N7S2·
H2O·MeCN: C 58.46, H, 6.53, N, 20.20; found C 58.17, H 6.53, N
20.26.
3
= 16.3 Hz, 1H, CHCH), 6.54 (d, JHH = 8.6 Hz, 2H, ArH), 5.88
3
3
(m, 1H, NH), 3.04 (d, JHH = 4.5 Hz, 3H, CH3), 2.70 (d, JHH = 5.0
Hz, 3H, CH3), 2.29 (s, 3H, CH3), 2.25 (s, 3H, CH3). 13C NMR (101
MHz; DMSO): δ 11.78, 12.05, 29.60, 31.24, 111.65, 122.24, 124.57,
125.34, 125.39, 127.59, 128.77, 135.18, 137.25, 147.74, 149.24,
149.71, 176.42, 178.49. HRMS (ESI) (+ve ion) m/z calcd for
[C22H28N7S2]+: 454.1848 [M + H]+; found, 454.1849. Elemental
analysis calcd (%) for C22H27N7S2.0.5H2O: C 57.12, H 6.10, N 21.19;
found C 57.07, H 6.05, N 21.15.
Synthesis of H2L6. To a flask charged with DTSE-N-methyl-N-
phenyl (83 mg, 0.22 mmol) and (E)-4-amino-4′-dimethylaminos-
tilbene (55 mg, 0.23 mmol) was added acetonitrile (5 mL), and the
H
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mixture was heated at reflux for 6 h. A yellow precipitate was isolated
by hot filtration, washed with acetonitrile and then pentane, and dried
at the pump (38 mg, 0.07 mmol, 32%). 1H NMR (400 MHz; DMSO-
d6): δ 10.76 (s, 1H, NH), 10.39 (s, 1H, NH), 9.89 (s, 1H, NH), 8.33
(m, 1H, NH), 7.59 (m, 2H, ArH), 7.52 (m, 2H, ArH), 7.43 (m, 2H,
Then, an aliquot was analyzed by radio-HPLC to determine the
radiochecmical yield and purity (0−100% of buffer B to A at 1 mL/
min over 15 min, A = 0.05% TFA in Milli-Q and B = in 0.05% TFA in
acetonitrile, Luna C18 column 4.6 × 150 mm, 5 μm). The log(D7.4)
for each tracer was measured according to reported procedure. For
log D7.4 measurements, an aliquot (2 μL) of the radioactive reaction
mixture was added to a partition of 1-octanol (500 μL) that had been
saturated with PBS and to PBS (498 μL) that had been saturated with
1-octanol. The mixture was vortexed briefly and then allowed to
separate over the course of 30 min to 1 h. An aliquot (400 μL) of the
1-octanol layer was taken and repartitioned against PBS (400 μL).
The mixture was then vortexed briefly and allowed to separate over 30
min, and an aliquot (2 μL) of each layer was collected; the amount of
radioactivity in each layer was quantified using a Capintec (Captus
4000e) gamma counter. Log D7.4 was then calculated by calculating
the log10 of the ratio of the counts between the 1-octanol layer and the
PBS layer. Each partition was repeated in triplicate and reported as
the average.
3
3
ArH), 7.11 (d, JHH = 16.3 Hz, 1H, CHCH), 6.97 (d, JHH = 16.4
Hz, 1H, CHCH), 6.72 (m, 2H, ArH), 3.60 (m, 2H, CH2) 2.97 (m,
3
10H, CH2, CH2, N(CH3)2), 1.15 (t, JHH = 7.1 Hz, 3H, CH3), 0.95
(m, 6H, CH3,CH3). 13C NMR (101 MHz; DMSO-d6): δ 177.5, 176.3,
152.0, 150.6, 149.9, 137.3, 135.0, 128.4, 127.5, 125.5, 125.02, 124.98,
123.0, 112.2, 40.0, 38.6, 17.34, 17.16, 14.3, 10.99, 10.85. HRMS (ESI)
(+ve ion) m/z calcd for [C26H36N7S2]+: 510.2474 [M + H]+; found,
510.2473. Elemental analysis calcd (%) for C26H35N7S2·0.2H2O: C
60.83, H, 6.95, N, 19.10; found C 60.57, H 6.56, N 19.45.
General Procedure for the synthesis of Cu(II) Complexes
H2L1−6. To a flask charged with ligands H2L1−6 (1 equiv) and
Cu(OAc)2·H2O (1.1 equiv) was added dimethylformamide (2 mL).
The reaction mixture was stirred under an atmosphere of N2. After 2 h
H2O was added to the red mixture and a very fine precipitate was
observed which was collected by centrifugation and analyzed by
HRMS and elemental analyses.
In Vivo Biodistribution of [64Cu]CuL2 and [64Cu]CuL6 in Balb/
c Mice. All animal experiments were performed with the approval of
the Peter MacCallum Cancer Centre Animal Experimentation Ethics
Committee and in accordance with the Australian code for the care
and use of animals for scientific purposes, eighth edition, 2013. For in
vivo biodistribution studies [64Cu][CuL2] and [64Cu][CuL6] were
radiolabeled using the above procedure, and the final doses were
diluted in 0.1 M ammoniumn acetate buffer containing 10% DMSO
and 8% ethanol (pH 7.0, 3−4 MBq, 150 μL each). Mice (Balb/c) (n
= 3 for each time point) were injected via an intravenous tail vein
injection. Mice were euthanized at each time point. Organs of interest
were removed and weighed, and the radioactivity present in each
organ was quantified using a Capintec (Captus 4000e) gamma
counter. The results are presented as the percentage injected activity
per gram of tissue (% IA/g).
UV−vis Spectroscopy and Glutathione (GSH) Challenge.
UV−vis and fluorescence spectra were recorded in DMSO using (5
μM) solutions of the complexes [CuL1−6]. A broad absorption
centered at λmax = 480 nm due to characteristic MLCT transitions that
occur in typical bis(thiosemicarbazonato)copper(II) complexes was
observed (Figure S19). The emission spectra (λex = 365 nm) shows
strong emission bands centered at 420−440 nm for all samples
attributed to typical fluorescence from the stilbene functional group
(Figure S20). The stability of the complexes was tested in the
presence of glutathione as the reductant. Stock solutions (1 mM) of
complexes [CuL1−6] were prepared in DMSO and then diluted in
PBS to a final concentration of 10 μM in PBS with 20% DMSO.
Aliquots of the samples were treated with GSH (1 mM) for 1 h at RT,
and UV−vis spectra were recorded for both treated and untreated
samples where no change was observed (Figure S21).
Synthesis of [CuL1]. Following the general procedure, the
following parameters were obtained: H2L1 (100 mg, 0.22 mmol)
and Cu(OAc)2.H2O (46 mg, 0.25 mmol); the product was isolated as
a brown powder (93 mg, 0.18 mmol, 82%). HRMS (ESI) (+ve ion)
m/z calcd for [C22H26CuN7S2]+: 515.0987 [M + H]+; found,
515.0974. Elemental analysis calcd (%) for C22H25CuN7S2·0.5H2O:
C 50.41, H 5.00, N 18.71; found C 50.19, H 5.14, N 18.60.
Synthesis of [CuL2]. Following the general procedure, the
following parameters were obtained: H2L2 (97 mg, 0.21 mmol) and
Cu(OAc)2·H2O (48 mg, 0.26 mmol); the product was isolated as a
brown powder (101 mg, 0.19 mmol, 91%). HRMS (ESI) (+ve ion)
m/z calcd for [C23H28CuN7S2]+: 529.1144 [M + H]+; found,
529.1130. Elemental analysis calcd (%) for for C23H27CuN7S2: C
52.20, H 5.14, N 18.53; found C 52.01, H, 5.20, N 18.60.
Synthesis of [CuL3]. Following the general procedure, the
following parameters were obtained: H2L3 (47 mg, 0.096 mmol)
and Cu(OAc)2.H2O (22 mg, 0.11 mmol); the product was isolated as
a brown powder (39 mg, 0.073 mmol, 76%). HRMS (ESI) (+ve ion)
m/z calcd for [C24H30CuN7S2]+: 543.1300 [M + H]+; found,
543.1291. Elemental analysis calcd (%) for C24H29CuN7S2·H2O: C
51.36, H 5.57, N 17.47; found C 51.34; H 5.20; N 17.86.
Synthesis of [CuL4]. Following the general procedure, the
following parameters were obtained: H2L4 (23 mg, 0.046 mmol)
and Cu(OAc)2·H2O (10 mg, 0.052 mmol); the product was isolated
as a brown powder (24 mg, 0.043 mmol, 93%). HRMS (ESI) (+ve
ion) m/z calcd for [C25H32CuN7S2]+: 557.1457 [M + H]+; found,
557.1444. Elemental analysis calcd (%) for C25H31CuN7S2: C 53.89,
H 5.61, N 17.60; found C 53.54, H 5.90, N 17.62.
In Vitro Aβ Binding Assays. Synthetic Aβ1−40 (500 μg, Bachem,
4014442) was dissolved in HFIP (500 μL), and the mixture was
incubated on ice for 1 h and then left in a fumehood to evaporate
overnight followed by drying by high speed vacuum centrifugation to
remove residual HFIP and moisture. The peptide was dissolved in 60
mM NaOH (100 μL) and incubated for 5 min at room temperature.
Then, Milli-Q water (350 μL) was added, the mixture was
ultrasonicated for 5 min on ice. The solution was buffered with 10
× PBS (pH 7.4, 50 μL) and centrifuged for 5 min, and an aliquot of
supernatant was analyzed by UV spectrometry to determine the
concentration (λabs = 214, ε = 94526 M−1 cm−1); the concentration
was found to be 148 μM. Stock solutions of ThT (1 mM in PBS) and
complexes [CuL1−6] (1 mM in DMSO) were prepared freshly; final
samples (100 μL, in triplicates) were prepared on ice by mixing
Aβ1−40 (final concentration, 4 μM), ThT (final concentration, 4 μM),
and [CuL1−6] (final concentration, 1 μM), and the control consisted
of Aβ1−40 (final concentration, 4 μM) and ThT (final concentration, 4
μM). The samples were prepared on a 96-well plate and incubated at
37 °C in a FLUOstar Omega filter-based multimode microplate
reader. The ThT fluorescence intensity (440/480 nm excitation/
emission filters) of each sample was recorded every 10 min with
orbital shaking before each cycle.
Synthesis of [CuL5]. Following the general procedure, the
following parameters were obtained: H2L5 (14 mg, 0.027 mmol)
and Cu(OAc)2.H2O (7.3 mg, 0.037 mmol); the product was isolated
as a brown powder (12 mg, 0.022 mmol, 81%). HRMS (ESI) (+ve
ion) m/z calcd for [C25H32CuN7S2]+: 557.1457 [M + H]+; found,
557.1444. Elemental analysis calcd (%) for C25H31CuN7S2·0.5H2O: C
52.24, H 6.03, N 17.25; found, C 53.24, H 5.65, N 17.13.
Synthesis of [CuL6]. Following the general procedure, the
following parameters were obtained: H2L6 (5 mg, 0.009 mmol) and
Cu(OAc)2.H2O (2 mg, 0.095 mmol); the product was isolated as a
brown powder (4.5 mg, 0.010 mmol, 78%). HRMS (ESI) (+ve ion)
m/z calcd for [C26H34CuN7S2]+: 571.1613 [M + H]+; found,
571.1600. Elemental analysis calcd (%) for C26H33CuN7S2: C 54.67,
H 5.82, N 17.16; found, C 54.43, H 5.94, N 17.01.
Synthesis of [64Cu]CuL1−6. [64Cu]CuCl2 in 0.1 M HCl was
obtained from was obtained from the Sir Charles Gardiner Hospital
Radiopharmaceutical Production and Development Centre (Perth,
WA, Australia). Aliquots of the [64Cu]CuCl2 solution (10 μL, 12.5
MBq) were buffered with sodium acetate (1 M, 10 μL, pH 4.5); each
of the ligands H2L1−6 (2 μg in 10 μL of DMSO) was added, and the
reaction mixture was left to stand at room temperature for 30 min.
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For the assay with preformed fibrils, a stock solution of Aβ1−40 (100
μM) was gently agitated for 48 h in PBS at 37 °C to promote the
formation of Aβ fibrils. Then, the solution was treated with an
equivalent amount of ThT (100 μM) for 1 h. The solution was
diluted, and an aliquot (5 μM fibrils) was treated with the copper
complexes, [CuL2] and [CuL6] as well as the free ligands H2L2 and
H2L6 (1 μM and 0.1 μM) for 1 h at 37 °C. Then, the ThT
fluorescence intensity of each sample was recorded using the plate
reader.
TEM Imaging of Aβ Fibrils. A stock solution of Aβ1−40 (100 μM)
was incubated in PBS at 37 °C and agitated at 300 rpm for 48 h to
promote the formation of fibrils, which was confirmed using ThT
fluorescence response. The fibrils (10 μM) were then incubated and
agitated with the Cu(II) complexes (10 nM) for 2 h at 37 °C. The
solutions were spotted onto 300-mesh carbon-coated copper grids
and allowed to incubate for 2 min; excess solution was blotted off.
Samples were then negatively stained with uranyl acetate (0.5% w/v, 2
min) and analyzed on a FEI Tecnai F20 TEM transmission electron
microscope at a voltage of 200 kV.
chemistry of free ligands; and biodistribution analyses
(PDF)
Accession Codes
CCDC 1982494 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Author
■
Paul S. Donnelly − School of Chemistry and Bio21 Molecular
Science and Biotechnology Institute, University of Melbourne,
Parkville, Victoria 3010, Australia; orcid.org/0000-0001-
5373-0080; Email: pauld@unimelb.edu.au
Treatment of Human AD Brain Tissues with [CuL2] and
[CuL6]. Experiments using post-mortem human brain tissue were
performed with the approval of the University of Melbourne, Faculty
of Science Human Ethics Advisory Group (Ethics ID 1341145).
Human brain tissues (provided by the Victorian Brain Bank,
Melbourne, Australia) were collected by autopsy from the frontal
cortex and preserved by formalin fixation and paraffin embedding.
The brain tissue sections (7 μm) were first deparaffined (xylene, 3 × 2
min) and then rehydrated (soaked for 2 min in a series of 100%, 90%,
70%, and 0% v/v ethanol/water). The hydrated tissue sections were
washed in phosphate buffer saline (PBS, 5 min). Autofluorescence of
the tissue was quenched using potassium permanganate (KMnO4)
(0.25% in PBS, 20 min), and the sections were washed with
potassium metabisulfite and oxalic acid (1% in PBS) until the brown
color was removed, followed by washing with PBS (3 × 2 min). The
sections were blocked with bovine serum albumin (2% BSA in PBS,
pH 7.0, 10 min) and covered with a solution of [CuL2] or [CuL6]
complexes (10 μM in 15% v/v DMSO/PBS, 10 min). The sections
were treated with BSA again (4 min) to remove any Cu(II) complex
nonspecifically bound to the tissue and then were washed with PBS (3
× 2 min) and deionized water. The coverslips were mounted using
Dako fluorescence mounting medium (Agilent/Dako, cat# S3023).
Fluorescence images were visualized using a Leica DM IL LED
microscope.
Authors
Asif Noor − School of Chemistry and Bio21 Molecular Science
and Biotechnology Institute, University of Melbourne, Parkville,
Victoria 3010, Australia
David J. Hayne − School of Chemistry and Bio21 Molecular
Science and Biotechnology Institute, University of Melbourne,
Parkville, Victoria 3010, Australia; orcid.org/0000-0003-
2756-9427
SinChun Lim − School of Chemistry and Bio21 Molecular
Science and Biotechnology Institute, University of Melbourne,
Parkville, Victoria 3010, Australia
Jessica K. Van Zuylekom − Research Division, Peter
MacCallum Cancer Centre, Melbourne, Victoria 3000,
Australia
Carleen Cullinane − Research Division, Peter MacCallum
Cancer Centre, Melbourne, Victoria 3000, Australia; The Sir
Peter MacCallum Department of Oncology, University of
Melbourne, Parkville, Victoria 3010, Australia
Peter D. Roselt − Centre for Cancer Imaging, Peter MacCallum
Cancer Centre, Melbourne, Victoria 3000, Australia
Catriona A. McLean − The Florey Institute of Neuroscience and
Mental Health, University of Melbourne, Parkville, Victoria
3010, Australia; Department of Anatomical Pathology, The
Alfred Hospital, Victoria 3181, Australia
Jonathan M. White − School of Chemistry and Bio21 Molecular
Science and Biotechnology Institute, University of Melbourne,
Parkville, Victoria 3010, Australia; orcid.org/0000-0002-
0707-6257
X-ray Crystallography. The molecular structure of [CuL1] was
confirmed by X-ray crystallography. Single crystals suitable for X-ray
diffraction were obtained by slow evaporation of compound dissolved
in DMF. Intensity data for CuL1 were collected with an Oxford
Diffraction SuperNova CCD diffractometer using Cu−Kα radiation,
and the temperature during all data collections was maintained at
130.0(1) K using an Oxford Cryosystems cooling device. The
structure was solved by direct methods using SHELXT and refined
using the least-squares method using SHELXL.65,66 Thermal ellipsoid
plots were generated using the program Mercury integrated with the
WINGX suite of programs.67 Crystal data for CuL1:
C22H25CuN7S2Cu·(DMF) M = 588.24, T = 130.0(2) K, λ =
1.54184 Å, monoclinic, space group P21/c, a = 9.1130(2) Å, b =
8.9380(2) Å, c = 33.5671(7)) Å, β = 96.493(2)°, V = 2716.57(10) Å3,
Z = 4, Dc = 1.438 Mg M−3 μ(Cu−K α) = 2.852 mm−1, F(000) = 504,
crystal size = 0.27 × 0.14 × 0.03 mm; θmax = 76.54°, 11453 reflections
measured, 5314 independent reflections (Rint = 0.0262), the final R =
0.0358 [I > 2σ(I), 4432 data] and wR(F2) = 0.1032 (all data), GOOF
= 1.055; CCDC accession code 1982494.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c01520
Funding
The Australian Research Council is acknowledged for financial
support (DP160100288, LP150100656).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The human brain tissue was provided by The Victorian Brain
Bank Network. We acknowledge the Mass Spectrometry and
Proteomics Facility (MSPF) at the Bio21 Institute, University
of Melbourne, for the use of their mass spectrometer and
HPLC systems. Professor Rodney J. Hicks (Peter MacCallum
Cancer Centre) is thanked for his generous support of this
research by provision of radiochemistry facilities. We thank
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01520.
■
sı
1H NMR spectra; HR-ESMS spectra; absorption/
emmision spectroscopy and GSH challenge; electro-
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