A Copper Complex of a Thiosemicarbazone-Pyridylhydrazone Ligand Containing a Vinylpyridine Functional Group as a Potential Imaging Agent for Amyloid-β Plaques

Article abstract of DOI:10.1071/CH19311

Complexes containing positron-emitting radionuclides of copper have the potential to be of use for diagnostic imaging with positron emission tomography. Alzheimer's disease is characterised by the presence of amyloid-β plaques in the brain. A new thiosemicarbazone-pyridyl hydrazone tetradentate ligand with a pyridyl-4-vinylpyridine functional group was prepared with the aim of making a copper complex that binds to amyloid-β plaques to assist in the diagnosis of Alzheimer's disease. The ligand forms a charge neutral complex with copper(ii) that was characterised by X-ray crystallography and the electrochemical behaviour of the complex was investigated by cyclic voltammetry. The new ligand can be radiolabelled with positron-emitting copper-64 at room temperature in excellent radiochemical yields. The new complex interacts with synthetic amyloid-β fibrils and binds amyloid-β plaques present in post-mortem Alzheimer's disease brain tissue.

Full text of DOI:10.1071/CH19311

RESEARCH FRONT
CSIRO PUBLISHING
Aust. J. Chem. 2019, 72, 827–834
Full Paper
https://doi.org/10.1071/CH19311
A Copper Complex of a Thiosemicarbazone-
Pyridylhydrazone Ligand Containing a Vinylpyridine
Functional Group as a Potential Imaging Agent for
Amyloid-b Plaques*
A
A
B
Lachlan E. McInnes, Asif Noor, Peter D. Roselt,
C
A
A D
,
Catriona A. McLean, Jonathan M. White, and Paul S. Donnelly
A
School of Chemistry and Bio21 Molecular Science and Biotechnology Institute,
University of Melbourne, Melbourne, Vic. 3010, Australia.
B
Cancer Imaging, Peter MacCallum Cancer Centre, Melbourne, Vic. 3000, Australia.
C
Department of Anatomical Pathology, The Alfred Hospital, Vic. 3181, Australia, and
The Florey Institute of Neuroscience and Mental Health, University of Melbourne,
Melbourne, Vic. 3010, Australia.
D
Corresponding author. Email: pauld@unimelb.edu.au
Complexes containing positron-emitting radionuclides of copper have the potential to be of use for diagnostic imaging
with positron emission tomography. Alzheimer’s disease is characterised by the presence of amyloid-b plaques in the
brain. A new thiosemicarbazone-pyridyl hydrazone tetradentate ligand with a pyridyl-4-vinylpyridine functional group
was prepared with the aim of making a copper complex that binds to amyloid-b plaques to assist in the diagnosis of
Alzheimer’s disease. The ligand forms a charge neutral complex with copper(II) that was characterised by X-ray
crystallography and the electrochemical behaviour of the complex was investigated by cyclic voltammetry. The new
ligand can be radiolabelled with positron-emitting copper-64 at room temperature in excellent radiochemical yields. The
new complex interacts with synthetic amyloid-b fibrils and binds amyloid-b plaques present in post-mortem Alzheimer’s
disease brain tissue.
Manuscript received: 8 July 2019.
Manuscript accepted: 9 August 2019.
Published online: 25 September 2019.
Introduction
has been investigated as a hypoxia imaging agent as well as for
probing oxidative stress in amyotrophic lateral sclerosis.[3–9]
In addition, the structurally related compound [Cu(gtsm)]
(gtsm ¼ glyoxal-bis(4-methyl-3-thiosemicarbazone)) has been
investigated as a probe for measuring altered copper trafficking in
the brain during Alzheimer’s disease.[10]
Ligands containing thiosemicarbazone functional groups can
coordinate metal ions as anionic, resonance stabilised N,S donors
where the sulfur can act as a non-reducing thiolate-like donor.
Early work on bis(thiosemicarbazone) ligands included examples
by Robson, McFadyen, and Schaap where they isolated a range
of binuclear copper(II) and nickel(II) complexes derived from
Neurodegeneration associated with the onset of Alzheimer’s
disease (AD) leads to a decline in cognition, memory, attention,
and motor function. A key pathological hallmark of the disease
is the presence of extracellular protein deposits called amyloid
plaques. These plaques are comprised mostly of aggregated
amyloid-b peptide (Ab), which contains between 39 and 43
amino acids and is derived from the amyloid precursor pro-
tein.[11–15] Visualising these deposits in the brain using PET
imaging can provide an estimation of the Ab burden in patients,
which can assist in differential diagnosis of AD from other
neurodegenerative diseases with overlapping symptoms. Imag-
ing of Ab plaque burden could also serve as a tool to help
monitor the effectiveness of emerging therapeutic agents.[16,17]
Recent advances have led to three Ab plaque PET imaging
agents radiolabelled with fluorine-18 being approved for
a
binucleating ligand, 2-hydroxy-5-methylisopthalaldehyde
Continued interest in
bis(thiosemicarbazone).[1,2]
bis(thiosemicarbazone) ligands has focussed on their potential to
coordinate positron-emitting isotopes of copper in the develop-
ment of tracers for positron emission tomography (PET). PET
relies on the administration of a tracer that has been radiolabelled
with a positron-emitting isotope and there are positron-emitting
isotopes of copper that are available with a diverse range of
half-lives. Two of these isotopes, copper-60 (t1/2 ¼ 20 min)
and copper-62 (t1/2 ¼ 9.7 min) have relatively short radioactive
half-lives. There are also two positron-emitting isotopes with
longer half-lives, copper-61 (t1/2 ¼ 3.4 h) and copper-64 (t1/2
¼
12.7 h). The charge neutral, lipophilic complex [Cu(atsm)]
(atsm ¼ diacetyl-bis-(4-methyl-3-thiosemicarbazone)) (Fig. 1)
*This manuscript is dedicated to Professor Richard Robson in recognition of his enormous contributions to inorganic chemistry.
Journal compilation Ó CSIRO 2019
www.publish.csiro.au/journals/ajc

828
L. E. McInnes et al.
18F
N
S
N
S
N
S
N
S
O
O
N
N
N
N
3
Cu
Cu
NH
NH
N
H
N
H
N
H
N
H
[
18F]Florbetaben
18F
[Cu(atsm)]
[Cu(gtsm)]
N
3
N
N
N
S
N
[
18F]Florbetapir
Cu
N
N
H
S
N
H
18F
[Cu(atsm/a-stilbene)]
HO
HO
S
N
NH
[
18F]Flutemetamol
N
S
N
N
N
N
S
N
N
N
N
Cu
Cu
18F
N
H
N
H
N
N
O
NH
[
18F]NAV4694
O
Fig. 2. The clinically relevant Ab imaging agents, [18F]florbetaben,
18F]florbetapir, [18F]flutemetamol and [18F]NAV4694.
[
OH
N
[CuLSP
]
[CuLBF
]
expense of the electron donating dimethlylamino and hydroxy
groups present in [CuLSP] and [CuLBF].
Fig. 1. The chemical structures of the previously reported copper-based
amyloid targeting agents: the styryl-pyridyl based [CuLSP] and the pyridyl-
benzofuranyl based [CuLBF].
Results and Discussion
2-Bromo-5-(2-(pyridin-4-yl)vinyl)pyridine (5) was synthesised
using
a
Wadsworth–Emmons–Horner reaction between
clinical use; [18F]florbetapir, [18F]flutemetamol, and [18F]flor-
betaben and a fourth agent, [18F]NAV4694, is in Phase 3 clinical
trials for AD imaging (Fig. 2).[18–22] These conjugated,
aromatic, hydrophobic molecules bind to Ab fibrils and plaques
through a combination of non-covalent, hydrophobic, and p–p
interactions with the aromatic residues located on the surface of
aggregated Ab fibrils and plaques.[23–25]
2-bromopyridinyl-4-methylenediethylphosphonate, which was
generated in situ from 2-bromo-5-(chloromethyl)pyridine (3)
and 4-pyridinecarboxaldehyde (4). The resulting 2-bromo-
5-(2-(pyridin-4-yl)vinyl)pyridine (5) was then converted into the
substituted hydrazine 6 by treatment with hydrazine hydrate.[30]
The ligand H2L1 was afforded by the condensation of the
hydrazine with the asymmetric diacetyl-mono-4-methyl-3-thio-
semicarbazone (7) (Scheme 1). The ligand was characterised by
NMR spectroscopy, electrospray ionisation-orbital time of flight
mass spectrometry (ESI/O-TOF MS), and elemental analysis.
The ligand [H2L1] reacts with Cu(OAc)2ꢀH2O rapidly in
methanol to yield a dark purple complex (Scheme 1). The
resulting [CuL1] gave a single peak when analysed by reversed
phase HPLC (RP-HPLC) and analysis by ESI-MS gave a signal
with the expected copper isotope pattern at a m/z corresponding
to [CuIIL1 þ H]þ. The conditional stability constant of the
complex at pH 7.4 was determined to be (6.69 ꢁ 3) ꢂ 10ꢃ18 M
which is the same order of magnitude as the previously reported
values for this class of ligand.[28,29] Analysis of the UV-visible
spectroscopy of [CuL1] revealed an absorption at lmax 350 nm
and l 600 nm. The ligand H2L1 showed an emission maxima at
lem 471 nm (lex 382 nm), but this emission was significantly
quenched by the presence of copper(II) in [CuL1] (lem 411 nm,
The various positron-emitting isotopes of copper offer alter-
natives to fluorine-18 labelled tracers and a copper-based radio-
pharmaceutical to image Ab plaques is an attractive prospect.
A successful candidate radiopharmaceutical must cross the
blood–brain barrier (BBB), bind strongly to Ab plaques, remain
sufficiently stable to accumulate a reasonable signal at the target,
and any radioactive metabolites must not be BBB permeable.
Previously, we have used the bis(thiosemicarbazone) framework
to append a targeting group for Ab plaques. The compound,
[Cu(atsm/a-stilbene)] (Fig. 1), features a stilbene functionalgroup
appended to the framework through a hydrazone linkage and
showed good BBB penetration and selective uptake in an animal
model of AD (APP/PS1 transgenic mice) compared with a wild-
type control.[26] An alternative approach is to incorporate the
targeting group within the chelating domain, thereby negating the
need for a linker and reducing the molecular weight of the tracer.
To that end we have investigated hybrid thiosemicarbazone-
pyridylhydrazone ligands. This class of ligand can be radiola-
belled with copper-64 under mild conditions to give a single
radioactive species. Incorporation of functional groups such as
l
ex 331 nm) (Fig. 3).
Crystals of sufficient quality for X-ray analysis were grown
by diffusion vapours between a solution of [CuL1] in chloroform
and pentane (Fig. 4 and Table 1). The complex crystallises with
the CuII in a distorted square planar N3S environment tending
towards five-coordinate square pyramidal geometry due to a
styryl-pyridine, [CuLSP] (Fig. 1) or pyridyl-benzofuran, [CuLBF
]
(Fig. 1) intothe ligand framework can give complexes that bindto
Ab plaques.[27–29] Inthiswork, weincorporateda 4-vinylpyridine
functional group to investigate whether the complex binds to Ab
plaques with an additional pyridyl hydrogen bond acceptor at the
weakaxial interaction between the CuII and a sulfur ofanadjacent
II
ligand (Cu–S 2.932A), and the Cu being marginally out of the
0
˚
˚
N3S plane (,0.126 A). Of the N3S plane, the Cu–S bond is the

Imaging Amyloid-b Plaques with a Copper Complex
829
N
H
O
N
N
NaBH4
EtOH
SOCl2
Br
Br
Br
OH
Cl
1
2
3
O
H
N
4
N
N
Br
Br
O
P(OEt)2
NaH
N
P
O
O
5
H
N
H
N
O
NH
N
NH
N
S
S
7
N
H2N
HN
N
N
HN
NH2NH2
EtOH
AcOH
EtOH
N
N
H2L1
6
N
N
N
N
N
NH
Cu
NH
N
Et3N
N
H
S
·
Cu(OAc)2 2H2O
S
N
HN
N
MeOH
N
N
[CuL1]
Scheme 1. The synthesis of H2L1 and [CuL1].
(a)
(b)
(c)
1.0
0.8
0.6
0.4
0.2
0
600
600
400
200
0
400
200
0
400
200
0
H2L1
[CuL1]
400
200
0
300
400
500
600
700
300
400
500
600
300
400
500
600
Wavelength [nm]
Wavelength [nm]
Wavelength [nm]
Fig. 3. (a) UV-Visible spectra of H2L1 and [CuL1] (10 mM, DMF), (b) the excitation (red) and emission (blue) spectra of H2L1 (1 mM, DMF) and (c) the
excitation (red) and emission (blue) spectra of [CuL1] (10 mM, DMF).
˚
longest (2.2633(16) A), which is similar to the Cu–S bond found
in Cu(atsm) and previously reported thiosemicarbazone-
hydrazinopyridyl copper complexes.[27–29,31,32] Of the nitrogen
copper in vivo, resulting in poorer quality images due to
increased background signal. The electrochemical properties
of [CuL1] were investigated by cyclic voltammetry in anhy-
drous N,N-dimethylformamide (1 mM analyte) with tetrabuty-
lammonium hexafluorophosphate (0.1 M) as a supporting
electrolyte. Under these conditions, quasi-reversibþle reduction
˚
donors, Cu–Nthiosemicarbazanato (Cu–N3, 1.950(5) A) and
˚
Cu–Nhydrazone (Cu–N4, 1.951(5) A) were found to be shorter
˚
than Cu–Npyridyl (Cu–N6, 1.958(5) A). The C–S bond distance
˚
of the thiosemicarbazanato-arm (S–C1, 1.753(6) A) suggests a
more thiolate-like rather than thione-like character.
The biological activity and stability of copper bis(thiosemi-
carbazone) complexes can, in part, be related to the CuII/I
reduction potential.[33–35] The reducing environment inside
cells can lead to reduction of the copper(II) to copper(I) leading
to transfer of the copper to high affinity copper binding pro-
teins.[35] This instability can lead to dissociation of radioactive
1
0
0
of [CuL ] was found at E ¼ –1.18 V (versus Fc /Fc) which
corresponds to the CuII/I redox process (Fig. 5a), which is similar
0
0
to previously reported values for [Cu(atsm)] (E ¼ –1.17 V
versus Fcþ/Fc) under similar conditions.[3þ6] An irreversible
0
0
oxidation is seen at E ¼ 0.01 V (versus Fc /Fc) (Fig. 5b) that
could be due to either a CuIII/II process or a ligand-based process.
The copper-64 complex, [64Cu][CuL1], could be prepared at
room temperature (sodium acetate 0.1 M, pH 5, 30 min). The

830
L. E. McInnes et al.
The fluorescence of H2L1 was used to investigate its poten-
tial to bind to Ab plaques present in post-mortem brain tissue
from clinically diagnosed AD subjects. Brain tissue (7 mM thick)
from the frontal cortex of the AD subject was blocked for non-
specific binding with bovine serum albumin and then treated
with H2L1. The tissue was washed and then analysed by epi-
fluorescent microscopy. Ab plaques are typically 60 mm in
diameter, so 7 mm serial sections often contain components of
the same plaque. Comparison of the fluorescence from the tissue
that had been treated with H2L1 with the adjacent tissue section
that had been stained immunohistochemically with an Ab
specific antibody (1E8) revealed good co-localisation suggest-
ing that the ligand has some selectivity for Ab (Fig. 8).
N4
N3
N5
N2
S1
N1
Cu1
N6
Conclusion
A new thiosemicarbazone-pyridyl hydrazone tetradentate ligand
with a pyridyl-4-vinylpyridine functional group was prepared.
The ligand forms a charge neutral complex with copper(II),
[CuL1], that was characterised by X-ray crystallography. The
copper(II) complex is stable with a conditional stability constant
of (6.69 ꢁ 3) ꢂ 10ꢃ18 M and an investigation of the electro-
N7
chemical behaviour of the complex by cyclic voltammetry
Fig. 4. ORTEP representation (50 % ellipsoid probability) of [CuL1].
0
revealed a quasi-reversible CuII/I couple at E ¼ ꢃ1.18 V (versus
0
Solvent and hydrogen atoms bonded to carbon have been omitted for clarity.
Fcþ/Fc). The new ligand can be radiolabelled with positron-
emitting copper-64 at room temperature to give [64Cu][CuL1]
with high radiochemical purity. The [CuL1] complex interacts
with synthetic Ab fibrils and binds to amyloid-b plaques present
in post-mortem Alzheimer’s disease brain tissue.
1
Table 1. Summary of the crystallographic data for [CuL ] 2CHCl2
.
Parameter
[CuL1]ꢀ2CHCl3
Experimental
Empirical formula
Formula weight [g molꢃ1
Crystal system
C20H20Cl6CuN7S
666.73
]
General Procedures
Monoclinic
P21/c
All reagents and solvents were acquired from commercial
sources and used without further purification. Elemental anal-
ysis for C, H, and N was carried out by The Campbell Micro-
analytical Laboratory, The University of Otago, Dunedin, New
Zealand. NMR spectra were recorded on a Varian FT-500 NMR
(Varian, California, USA) (1H at 500 MHz and 13C{1H} at
125.7 MHz) at 297 K and referenced to internal solvent residue.
High resolution mass spectra were recorded on a Thermo Sci-
entific Exactive Plus OrbiTrap LC/MS (Thermo Fisher Scien-
tific, Massachusetts, USA) and calibrated to internal references.
Analytical RP-HPLC traces were acquired using an Agilent
1200 HPLC system equipped with an Alltech Hypersil BDS C18
analytical HPLC column (4.6 ꢂ 150 mm, 5 mm) with a flow rate
of 1 mL minꢃ1 and UV absorbance detection at 280 and 400 nm.
Retention times (Rt, min) were recorded using a gradient elution
of 5–100 % B in A (A ¼ 0.1 % trifluoroacetic acid (TFA),
B ¼ acetonitrile with 0.1 % TFA).
Space group
˚
a [A]
˚
b [A]
8.7212(9)
18.8894(14)
16.8703(18)
90
˚
c [A]
a [deg.]
b [deg.]
g [deg.]
93.526(10)
90
3
˚
V [A ]
2773.9(5)
4
Z
Temperature [K]
˚
130.00(10)
1.54184
15425
l [A]
Reflections collected
Independent reflections
R-factor [%]
5517
6.6
radiolabelled complex was characterised by RP-HPLC compar-
ing the UV-trace of [CuL1] and the scintillation detection of
[64Cu][CuL1] (Fig. 6). The straight-forward radiolabelling with
copper-64 suggests that this ligand could be easily adapted into a
kit-based formulation that has been so central to the success of
99mTc-labelled radiopharmaceuticals.
The affinity of [CuL1] for synthetic Ab1–40 was investigated
through fluorescence assays using the dye thioflavin T (ThT).[37]
ThT binds to amyloid fibrils resulting in a characteristic increase
in fluorescence at l 480 nm.[38] This assay is useful for screening
compounds but does not reliably report on molecules that bind to
different sites on Ab fibrils as does ThT.[23] Furthermore, fibril
morphology is variable and dependent on the method used for the
formation of fibrils.[39,40] Using this assay, the Ki of [CuL1] was
UV-Vis spectra were recorded on a Shimadzu UV1650-PC
spectrometer (Shimadzu, Kyoto, Japan) from 800 to 250nm.
Emission and excitationspectra wererecorded on a Varian CARY
eclipse fluorescence spectrophotometer in quartz cuvettes.
Cyclic voltammograms were recorded using a Metrohm
(Switzerland) AUTOLAB PGSTAT100 and analysed using
Autolab GPES V4.9 software. Measurements were carried out
using 1 mM of analyte in anhydrous DMF with tetrabutylam-
monium hexafluorophosphate (0.1 M) as supporting electrolyte.
A glassy carbon working electrode was used along with a Pt wire
counter electrode and a pseudo leakless Ag/Agþ working
electrode (EDAQ, New South Wales, Aþustralia). Measurements
0
0
were referenced versus ferrocene (Fc /Fc, E ¼ 0 V), where
found to be 4.3 ꢁ 1.6 mM (Fig. 7), which suggests only a
E0 refers to the midpoint of the reduction (Epc) and oxidation
[41,42]
relatively moderate affinity of the complex towards Ab1–40.
0

Imaging Amyloid-b Plaques with a Copper Complex
831
(a)
(b)
1 µA
2 µA
–1.6
–1.4
–1.2
–1.0
–0.8
–1.6
–1.2
–0.8
–0.4
0
0.4
Potential ([V] vs Fc+/Fc)
Potential ([V] vs Fc+/Fc)
Fig. 5. Cyclic voltammogram of [CuL1] in dimethylformamide at a glassy carbon working electrode from (a) ꢃ0.8 to ꢃ1.5 V
(versus Fcþ/Fc) at a scan rate of 200 mV sꢃ1 and (b) from 0.5 to ꢃ1.5 V (versus Fcþ/Fc) at a scan rate of 200 mV sꢃ1. Arrows indicate
scanning direction.
within the WINGX suite of programs.[45] [CuL1]ꢀ2CHCl3 CCDC
1936998.
[64Cu]CuCl2 was obtained from either the Sir Charles
Gardiner Hospital Radiopharmaceutical Production and Devel-
opment Centre (Perth, WA) or from Austin Health Department
of Molecular Imaging and Therapy (Heidelburg, Victoria) as a
formulation in 0.1 M HCl.
Radiolabelling of [64Cu][CuL1] was achieved by the addition
of a DMSO stock solution of H2L1 (2 mL, 1 mg mLꢃ1) to a
solution of [64Cu]CuCl2 (10 MBq, 0.1 M HCl) that had been
buffered with sodium acetate (0.1 M, pH 5). The reaction was
incubated at room temperature for 30 min and an aliquot
removed for characterisation by radio-RP-HPLC. Radio-RP-
HPLC was performed using a Shimadzu SPD-10ATvP HPLC
2.5 × 105
2.0 × 105
1.5 × 105
1.0 × 105
5 × 103
4 × 103
3 × 103
2 × 103
1 × 103
0
5.0 × 104
0
0
5
10
15
20
Time [min]
˚
system equipped with a Phenomenex Luna C18 100 A column
Fig. 6. Comparative RP-HPLC traces of [64Cu][CuL1] (red, scintillation)
and [CuL1] (black, UV 350 nm).
(4.6 ꢂ 150 mm, 5 mm) with a 1 mL minꢃ1 flow rate. Chromato-
grams were acquired with a UV-vis detector (254 and 350 nm)
and scintillation detector in series. Retention times are recorded
in minutes using a gradient elution method of 5–100 % B in A
over 15 min followed by holding at 100 % B for a further 3 min.
Buffer A was 0.05 % TFA, buffer B was 0.05 % TFA in
acetonitrile.
80000
60000
40000
20000
0
Synthetic Ab1–40 was prepared using a Liberty microwave
peptide synthesiserfromCEMCorporation(USA). Forcoupling,
5 equivalents of Fmoc amino acid and O-(1H-6-chlorobenzo-
triazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HCTU), plus 10 equivalents of N,N-diisopropylethylamine
(DIEA) were used. Residues 28–42 were coupled once at 758C
for 5 min and residues 1–27 were double coupled under the same
conditions except for Fmoc-His(Trt)-OH which was double
coupled at 508C. Fmoc cleavage was performed using a solution
of 0.1 M oxyma pure in 20 % piperidine/DMF at 908C for 1 min,
except for residues 23–27 which were deprotected for 10 min.
Cleavage of the Mmt protecting group at Lys-28 was affected
via four multiple treatments with 1 % TFA in DCM. After
synthesis, the resin-bound peptide was deprotected and cleaved
with a cocktail of 2 % water/2 % thioanisole/1 % triisopropylsi-
lane in TFA for 3 h. The resin was filtered and the volume of
TFA reduced to ,2 mL via nitrogen aspiration, followed by a
precipitation step and two further washes with diethyl ether.
Crude material was purified using RP-HPLC on an Agilent
1100 instrument, which was equipped with a degasser, auto-
sampler, fraction collector, and multichannel UV-vis detector.
Mobile phases were A ¼ 10 mM NH4OAc in water, pH 9.2 and
–2
–1
0
1
2
3
log10[CuL1] [µM]
Fig. 7. Competitive inhibition of thioflavin T binding to Ab1–40 fibrils
with [CuL1].
(Epa) peaks of a quasi- or fully reversible process where
0
0
E ¼ (Epc þ Epa)/2.
All X-ray crystal structure data was collected on an Oxford
Diffraction SuperNova CCD diffractometer using Cu-Ka radia-
tion, and the temperature during collection was maintained at
130.00(10) K using an Oxford Cryosystems cooling device. The
structure was solved by direct methods using SHELXT and
refined using least-squares methods using SHELXL.[43,44] Ther-
mal ellipsoid plots were generated using ORTEP-3 integrated

832
L. E. McInnes et al.
(a)
(b)
0
µm 100
0
µm 100
Fig. 8. (a) Epi-fluorescence microscopy (lex 340–380 nm, lem 425 nm) of human brain tissue treated with H2L1 (1 mM) at 20 ꢂ magnification. (b) Brightfield
microscopy of the adjacent tissue immunohistochemically stained with 1E8 antibody. Scale bars indicate 100 mm.
B ¼ 10 mM NH4OAc in 80 % acetonitrile/20 % water, pH 9.2.
Brain tissue sections (8 mm) were deparaffined (xylene,
3 ꢂ 3 min) followed by rehydration (2 min soakings in 100,
90, 70, and 0 % v/v ethanol/water). The hydrated sections were
then washed in PBS (5 min). Autofluoresence of the tissue was
quenched by treatment with potassium permanganate (0.25 %
w/v in PBS, 20 min) followed by washing with PBS (2 ꢂ 2 min).
The brown coloured tissue was then washed with potassium
metabisulfite and oxalic acid (both 1 % w/v in PBS, 6 min). The
tissue sections were then washed with PBS (3 ꢂ 2 min) followed
by blocking with bovine serum albumin (BSA, 2 % w/v in PBS,
10 min), rinsed briefly with PBS and then covered with [CuL1]
(10 mM in 30 % DMSO in PBS, 10 min). The sections were then
washed with BSA solution again to remove non-specifically
bound complex. The sections were then washed with PBS
(3 ꢂ 2 min), dipped in distilled water, and mounted with non-
fluorescent mounting medium (DAKO).
˚
A Phenomonex Jupiter 10m C4, 300A, 4.6 ꢂ 150 mm column
was used for purification using a gradient elution of 20–60 %
buffer B in A over 40 min with a 5 mL minꢃ1 flow rate with
detection at l 230 nm. Isolated fractions were lyophilised on
a Christ freeze dryer and the identity of the peptide confirmed
by mass spectrometry. The purity was found to be . 95 %
by HPLC.
The ThT binding assay was performed as follows. Lyophi-
lised Ab1–40 was dissolved in 1,1,1,3,3,3-hexafluoropropan-
2-ol to a theoretical concentration of 1 mg mLꢃ1 and sonicated
in an ice bath for 1 h. The mixture was then allowed to incubate
at room temperature for 1 h. The solvent was then allowed to
evaporate overnight. Drying was completed by centrifugal
vacuum for 10 min to obtain a clear peptide film which was
either used fresh or stored at ꢃ808C for future experiments.
Synthetic amyloid fibrils were obtained by first dissolving the
obtained peptide film (100 mg) in NaOH (60 mM, 20 mL), and
then vortexing the solution before sonicating in an ice bath for
10 min. The mixture was then diluted with MilliQ water (70 mL)
followed by phosphate buffered saline (PBS) (ꢂ 10, 10 mL). The
mixture was then centrifuged (14000 g) for 5 min at 48C and the
supernatant transferred to a fresh centrifuge tube. The concen-
tration of Ab1–40 was determined by making a 1 : 50 dilution and
measuring the absorbance at 214 nm by UV-vis spectroscopy
and determined using e214 ¼ 92 ¼ 1462 Mꢃ1 cmꢃ1. The solution
was then allowed to incubate for 3 days at 378C with constant
agitation to induce fibril formation. The assay was performed
by incubating Ab1–40 (1 mM) with a constant concentration of
ThT (2 mM) in a medium of 20 % DMSO in PBS. Increasing
concentrations of [CuL1] in a DMSO stock solution were added
to give increasing final concentrations of the compound in
the assay (50 nM – 100 mM). Mixtures were thoroughly mixed
before being added to a 96 well plate and analysed using a
FLUOstar Omega (BMG Labtech) using a excitation filter of
440 nm and an emission filter of 480 nm and performed in
triplicate and blanked to a well containing the assay components
excluding Ab1–40. The results were analysed using GraphPad
Prism 6 (Graph Pad) using a one site–fit Ki model using a Kd
value of 6.18 mM for ThT that had been determined under the
same assay conditions (20 % DMSO in PBS).
(6-Bromopyridin-3-yl)methanol (2)
This compound was synthesised as previously reported and gave
spectra that corresponded well with literature values.[30]
A solution of 6-bromonicotinaldehyde (1) (5.10 g, 27.4mmol)
was dissolved in anhydrous ethanol (150mL) under an atmo-
sphere of nitrogen gas. To this mixture was added sodium
borohydride (1.25 g, 33.0mmol). The reaction mixture was
stirred at room temperature for 24h. The pH of the reaction
mixture was then adjusted to pH 2 by the addition of HCl (1M)
and then to pH 10 by the addition of a saturated solution of
NaHCO3. The solvent was removed under reduced pressure and
the residue extracted with ethyl acetate (100 mL þ 50 mL). The
combined organic extracts were washed with brine (2ꢂ 75mL),
dried over MgSO4, and evaporated to dryness under reduced
pressure. The resultant solid was purified by column chromatog-
raphy (SiO2, 30% ethyl acetate/petroleum spirits) to yield an
off-white solid (3.11 g, 16.5mmol, 60% yield). m/z (ESI/O-TOF)
[C6H6BrNOþH]þ 187.97093, calcd 18797055. dH (400 MHz,
CDCl3) 8.32 (d, 4JHH 2.3, 1H, ArH), 7.59 (dd, 3JHH 8.2, 4JHH 2.4,
3
1H, ArH), 7.47 (d, JHH 8.2, 1H, ArH), 4.70 (s, 2H, CH2),
2.37 (s, 1H, OH).
2-Bromo-5-(chloromethyl)pyridine (3)
Paraffin preserved brain tissue blocks were provided by the
Victorian Brain Bank Network.
This compound was synthesised as previously reported and
analysis matched with literature values.[30]

Imaging Amyloid-b Plaques with a Copper Complex
833
(6-Bromopyridin-3-yl)methanol (2) (2.93 g, 11.3mmol) was
dissolved in dichloromethane (40 mL) and treated with an excess
of thionyl chloride (7mL). The reaction was monitored by TLC
(33 % ethyl acetate in petroleum spirits; Rf 0.81) and once
complete, volatiles were removed by evaporation. The residue
was then suspended in saturated NaHCO3 (50 mL) and extracted
with ethyl acetate (2ꢂ 50mL). The organic extracts were com-
bined, dried over MgSO4, and evaporated to dryness to yield a
brown oil, which yielded a crystalline white solid upon standing.
The solid was suspended in pentane and isolated by filtration,
washedwithpentane, and air-dried togive a crystalline colourless
solid (2.13 g, 10.3mmol, 91% yield). dH (400 MHz, CDCl3) 8.37
(d, 4JHH 1.9, 1H, ArH), 7.60 (dd, 3JHH 8.2, 4JHH 2.4, 1H, ArH),
7.50 (d, 3JHH 8.2, 1H, ArH), 4.54 (s, 2H, CH2).
(2.65 g, 25.2 mmol) was added portion-wise over the course of
10 min, resulting in the precipitation of a white solid. The
reaction mixture was allowed to warm to room temperature
and the mixture was extracted with chloroform (2 ꢂ 40 mL) and
the combined organic extracts dried over MgSO4, filtered, and
concentrated. The solution was then diluted with pentane,
precipitating a colourless solid that was collected by filtration,
washed with pentane, and air-dried to yield a white solid (3.68 g,
21.2 mmol, 84 % yield). m/z (ESI/O-TOF) [C6H11N3OSþH]þ
174.06963; calcd 174.06956. dH (400 MHz, DMSO-d6) 10.63
3
(s, 1H, NNH(C=S)N), 8.62 (d, JHH 3.9, 1H, CH3NH), 3.05
(d, 3JHH 4.6, 3H, CH3NH), 2.42 (s, 3H, CH3), 1.96 (s, 3H, CH3).
Diacetyl-2-((E)-5-(2-(6-hydrazinopyridin-3-
yl)vinyl)pyridine)-(4-ethyl-3-thiosemicarbazone) (H2L1)
(E)-2-Bromo-5-(2-(pyridin-4-yl)vinyl)pyridine (5)
A suspension of diacetyl-mono-4-methyl-3-thiosemicarbazone
(7) (48 mg, 0.28 mmol) and (E)-2-hydrazino-5-(2-(pyridine-4-
yl)vinyl)pyridine (6) (46 mg, 0.22 mmol) in ethanol (4 mL) was
acidified with acetic acid (4 drops) and heated at reflux for 2 h,
resulting in the precipitation of an orange solid. The reaction was
allowed to cool to room temperature and the solid isolated by
filtration, which was then washed with diethyl ether and air-
dried to yield an orange powder (52 mg, 0.14 mmol, 64 % yield).
Anal. Calc. for C18H21N7S: C 58.83, H 5.76, N 26.68 %. Found:
C 57.23, H 5.76, N 26.01. m/z (ESI/O-TOF) [C18H21N7SþH]þ
368.18530; calcd 368.16519. Rt (RP-HPLC) 8.79 min. dH
(500 MHz, DMSO-d6) 10.20 (s, 1H, PyrNH), 10.16 (s, 1H,
A solution of 2-bromo-5-(chloromethyl)pyridine (3) (1.05 g,
5.09 mmol) in triethylphosphite (9mL) was heated to reflux for
4 h. After that time the solvent was removed by evaporation and
the resultant residue dissolved in anhydrous tetrahydrofuran
(30 mL). To this, 4-nicotinaldehyde (4) (521 mg, 4.86 mmol) was
then added, followed by sodium hydride (60 % w/w in mineral
oil, 617 mg, 15.4mmol). The reaction was then stirred for 16h at
room temperature under an atmosphere of nitrogen gas. The
reaction was then quenched by the addition of saturated NaHCO3
(25 mL) and then extracted with ethyl acetate (2ꢂ 25mL). The
combined organic extracts were dried over MgSO4, filtered, and
the filtrateevaporatedtodrynessunder reducedpressuretoyield a
yellow solid that was suspended in pentane, isolated by filtration,
and air-dried to yield a yellow powder (436 mg, 1.67 mmol, 34%
yield). m/z (ESI/O-TOF) [C12H9BrN2þH]þ 261.00249; calcd
261.00219. dH (500 MHz, CDCl3) 8.61 (m, 2H, PyrH), 8.49
(d, 4JHH 2.5, 1H, PyrH), 7.72 (dd, 3JHH 8.3, 4JHH 2.6, 1H, PyrH),
7.51 (d, 3JHH 8.3, 1H, PyrH), 7.36 (m, 2H, PyrH), 7.21 (d, 3JHH
3
4
NNH(C=S)N), 8.53 (dd, JHH 4.6, JHH 1.5, 2H, PyrH), 8.42
4
(d, JHH 2.0, 1H, PyrH), 8.32 (q, JHH 4.3, 1H, CH3NH), 8.04
3
3
(dd, JHH 8.8, JHH 2.2, 1H, PyrH), 7.53–7.49 (m, 3H,
4
3
3
PyrH þ CH=CH), 7.31 (d, JHH 8.8, 1H, PyrH), 7.16 (d, JHH
16.5, 1H, CH=CH), 3.04 (d, 3JHH 4.6, 3H, CH3NH), 2.24 (s, 6H,
CH3C=N). dC (126 MHz, DMSO-d6) 178.4 (C=S), 156.9
(PyrC), 149.9 (PyrC), 148.5 (C=N), 147.8 (PyrC), 145.3 (C=N),
144.7 (PyrC), 135.4 (PyrC), 130.1 (CH=CH), 124.4 (PyrC),
123.5 (CH=CH), 120.5 (PyrC), 107.2 (PyrC), 31.2 (CH3NH),
11.5 (CH3C=N), 11.0 (CH3C=N).
3
16.4, 1H, CH=CH), 7.07 (d, JHH 16.4, 1H, CH=CH). dC
(126 MHz, CDCl3) 150.6 (PyrC), 149.1 (PyrC), 143.6 (PyrC),
141.9 (PyrC), 135.5 (PyrC), 131.4 (PyrC), 129.1 (PyrC), 128.4
(PyrC), 128.2 (CH=CH), 121.1 (CH=CH).
[Cu(diacetyl-2-((E)-5-(2-(6-hydrazinopyridin-3-
yl)vinyl)pyridine)-(4-ethyl-3-thiosemicarbazone))] ([CuL1])
(E)-2-Hydrazino-5-(2-(pyridin-4-yl)vinyl)pyridine (6)
A suspension of diacetyl-2-((E)-5-(2-(6-hydrazinopyridin-3-yl)-
vinyl)pyridine)-(4-ethyl-3-thiosemicarbazone) (H2L1) (23 mg,
0.06mmol) in methanol (4mL) was alkalised with triethylamine
(4 drops). To this, copper acetate monohydrate (19 mg,
0.10mmol) was added and the reaction heated to reflux for 2 h.
The reaction was then allowed to cool to room temperature
and diluted with water. The mixture was filtered, washed with
water, and air-dried to yield a dark coloured solid (24 mg,
0.06mmol, quant. yield). Anal. Calc. for C18H19CuN7S: C 47.41,
H 4.86, N 21.50 %. Found: C 47.19, H 4.36, N 20.91. m/z (ESI/O-
TOF) [C18H19CuN7Sþ2H]2þ 215.04301, [C18H19CuN7SþH]þ
429.07867; calcd 215.04321, 429.07914. Rt (RP-HPLC)
9.28min. Crystals of sufficient quality for X-ray analysis were
grown from exchange of vapours of pentane and a solution of the
complex dissolved in chloroform.
A suspension of (E)-2-bromo-5-(2-(pyridine-4-yl)vinyl)pyridine
(5) (372 mg, 1.42 mmol) in hydrazine monohydrate (4 mL) and
ethanol (8 mL) was heated at reflux for 16 h. The reaction mix-
ture was then allowed to cool to room temperature and the sol-
vent volume was reduced by approximately half by evaporation,
resulting in the precipitation of a yellow solid that was isolated
by filtration, washed with water, and air-dried to yield a yellow
powder (55 mg, 0.26 mmol, 18 % yield). m/z (ESI/O-TOF)
[C12H12N4 þ H]þ 213.11373; calcd 213.11347. dH (500 MHz,
4
DMSO-d6) 8.48 (m, 2H, PyrH), 8.18 (d, JHH 2.1, 1H, PyrH),
3
7.84 (m, 2H, NH þ PyrH), 7.46 (m, 2H, PyrH), 7.41 (d, JHH
3
16.4, 1H, CH=CH), 6.97 (d, JHH 16.4, 1H, CH=CH), 6.75
3
(d, JHH 8.8, 1H, PyrH), 4.24 (s, 2H, NH2). dC (126 MHz,
DMSO-d6) 161.7 (PyrC), 149.9 (PyrC), 148.3 (PyrC), 144.9
(PyrC), 133.9 (PyrC), 130.6 (CH=CH), 121.2 (CH=CH), 120.9
(PyrC), 120.3 (PyrC), 106.4 (PyrC).
Conflicts of Interest
Diacetyl-mono-4-methyl-3-thiosemicarbazone (7)
The authors declare no conflicts of interest.
This compound was synthesised as previously reported.[46]
A solution of 2,3-butanedione (2.40 mL, 27.6 mmol) in
water (50 mL) was acidified with HCl (conc., 5 drops) and
cooled in an ice bath. To this, 4-methyl-3-thiosemicarbazide
Acknowledgements
The authors recognise financial support from the Australian Research
Council. The authors thank the Victorian Brain Bank Network for the

834
L. E. McInnes et al.
provision of human brain tissue and Prof. Rodney J. Hicks (Peter MacCallum
Cancer Centre) for radiochemistry laboratory facilities.
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[24] G. Kuang, N. A. Murugan, Y. Tu, A. Nordberg, H. Agren, J. Phys.
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