Rhenium and technetium complexes of thioamide derivatives of pyridylhydrazine that bind to amyloid-β plaques

Article abstract of DOI:10.1007/s00775-018-1590-4

Abstract: Age-associated deposition of amyloid-β in cerebral blood vessels, a condition referred to as cerebral amyloid angiopathy, can contribute to stroke and dementia. This research aimed to design new radioactive technetium-99?m complexes that bind to amyloid-β plaques that have the potential to assist in diagnosis of cerebral amyloid angiopathy using single-photon-emitted computed tomography (SPECT) imaging. Six new pyridylthiosemicarbazide ligands containing either benzofuran or styrylpyridyl functional groups that are known to selectively bind to amyloid plaques were prepared. Non-radioactive isotopes of technetium are not available so rhenium was used as a surrogate for exploratory chemistry. The new ligands were used to prepare well-defined [Re-oxo]³⁺ complexes where two pyridylthiosemicarbazide ligands were coordinated to a single metal ion to give bivalent complexes with two amyloid-β targeting functional groups. The interaction of the [Re-oxo]³⁺ complexes with synthetic amyloid-β_1-42 and with amyloid plaques in human brain tissue was investigated. Two ligands were selected to develop methods to prepare their [^99mTc-oxo]³⁺ complexes at the tracer level. These technetium-99?m complexes are likely to be isostructural to their rhenium-oxo analogues.

Full text of DOI:10.1007/s00775-018-1590-4

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-018-1590-4
ORIGINAL PAPER
Rhenium and technetium complexes of thioamide derivatives
of pyridylhydrazine that bind to amyloid‑β plaques
Scott P. Fletcher1 · Asif Noor1 · James L. Hickey1 · Catriona A. McLean2,3 · Jonathan M. White1 · Paul S. Donnelly1
Received: 8 May 2018 / Accepted: 22 June 2018
© SBIC 2018
Abstract
Age-associated deposition of amyloid-β in cerebral blood vessels, a condition referred to as cerebral amyloid angiopathy, can
contribute to stroke and dementia. This research aimed to design new radioactive technetium-99 m complexes that bind to
amyloid-β plaques that have the potential to assist in diagnosis of cerebral amyloid angiopathy using single-photon-emitted
computed tomography (SPECT) imaging. Six new pyridylthiosemicarbazide ligands containing either benzofuran or sty-
rylpyridyl functional groups that are known to selectively bind to amyloid plaques were prepared. Non-radioactive isotopes
of technetium are not available so rhenium was used as a surrogate for exploratory chemistry. The new ligands were used to
prepare well-defned [Re-oxo]3+ complexes where two pyridylthiosemicarbazide ligands were coordinated to a single metal
ion to give bivalent complexes with two amyloid-β targeting functional groups. The interaction of the [Re-oxo]3+ complexes
with synthetic amyloid-β1-42 and with amyloid plaques in human brain tissue was investigated. Two ligands were selected to
develop methods to prepare their [99mTc-oxo]3+ complexes at the tracer level. These technetium-99 m complexes are likely
to be isostructural to their rhenium-oxo analogues.
Graphical abstract
Keywords Technetium · Rhenium · Amyloid-β · Imaging
Introduction
Deposition of β-amyloid in cerebral blood vessels, referred
to as cerebral amyloid angiopathy (CAA), may contribute to
intracerebral haemorrhage (12% of clinical presentations) [1,
2]. Microhaemorrhages and microinfarcts may also occur
due to CAA [3]. β-amyloid (Aβ) is a 39–43 amino acid
peptide derived from the amyloid precursor protein. CAA
* Paul S. Donnelly
pauld@unimelb.edu.au
Extended author information available on the last page of the article
Vol.:(0123456789)
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JBIC Journal of Biological Inorganic Chemistry
results in changes to the morphology of the cerebral blood
vessels, the deposition and hardening of Aβ aggregates are
thought to contribute to microbleeds, haemorrhagic stroke
and vascular cognitive impairment [4, 5]. A non-invasive
test to help diagnose CAA has the potential to be clinically
useful [6].
blood vessels of the brain but not cross the blood–brain bar-
rier [6].
Latest developments have identifed 2-pyridylbenzofuran
derivatives as Aβ plaque imaging agents with lower non-
specifc binding and consequently superior sensitivity when
compared to benzothiazole and styrylpyridyl ligands. An
exhaustive synthetic chemistry programme led by AstraZen-
eca has identifed AZD 4694 as a lead candidate with better
sensitivity for amyloid due to low white matter binding and
high cortical binding (Fig. 1) [16, 17].
The deposition of aggregates of Aβ in brain tissue to
form extracellular plaques is also a pathological feature of
Alzheimer’s disease (AD) and in some instances CAA and
AD appear to be related. Recent developments have enabled
non-invasive assessment of amyloid burden in AD subjects
using positron emission tomography (PET) and radiolabeled
tracers that bind to Aβ plaques. One of the frst tracers used
for Aβ imaging in humans was a benzothiazole derivative
radiolabeled with the positron-emitting carbon-11 isotope
known by the trivial name of [11C]PIB (Pittsburgh com-
pound B; Fig. 1) [7–9]. [11C]PIB has also been investigated
for the detection of amyloid in CAA but the ability of [11C]
PIB to cross the blood–brain barrier makes it difcult to
distinguish Aβ deposits within blood vessels from those in
the cerebral parenchyma [4, 10]. Imaging agents that can
diferentiate between Aβ deposits on the walls of cerebral
blood vessels from Aβ plaques in the brain parenchyma may
help in non-invasive diagnosis of CAA [6].
Despite recent concerns over production-related short-
ages of technetium-99m, single-photon-computed emis-
sion tomography (SPECT) imaging retains its importance
to nuclear medicine. Technetium-99m is used in over 80%
of nuclear imaging procedures worldwide. The technetium-
99m isotope emits γ-rays (140 keV) with a half-life of 6 h.
The clinical development of technetium-99m complexes that
bind to Aβ plaques would result in signifcant developments
in diagnostic imaging of AD and CAA [18–20]. A wide
variety of technetium-99m complexes and their rhenium
analogues have been prepared that are capable of binding
to Aβ plaques [21–31]. An excellent recent comprehensive
review of the area is available [20].
Complexes where a tridentate ligand bearing either a
benzothioazole or stilbene derivatives is coordinated to the
[Tc(CO)3]+ core [28, 32] and [TcO]3+ complexes coordi-
nated to two bidentate hydroxyamamide ligands [33, 34] that
bind to Aβ plaques but do not cross the blood–brain barrier
have demonstrated the potential for diagnostic imaging of
CAA using technetium-99m complexes. In this work, we
prepare pyridylthiosemicarbazide ligands with appended
stilbene and benzofuran derivatives. The new ligands are
then used to form well-defned complexes where two substi-
tuted pyridylthiosemicarbazide ligands coordinate to a single
[MO]3+ core (M=Re or 99mTc) and investigate the Aβ bind-
ing of the new complexes. There are no stable isotopes of
technetium available so the group VII congener, rhenium,
was used in exploratory synthesis and characterization. The
relatively high molecular weight of these bivalent (possess-
ing two targeting groups) complexes and their nominal posi-
tive charge should preclude penetration of the blood–brain
barrier whilst retaining afnity for vascular Aβ.
The interaction between benzothiazole derivatives such
as [11C]-PIB and Aβ plaques and fbrils is thought to be
due to a combination of hydrophobic and hydrogen-bonding
interactions. Stilbene and styrylpyridyl derivatives are struc-
turally related to benzothiazoles and also exhibit selective
binding to Aβ plaques. Eforts focusing on the preparation
of stilbene or styrylpyridyl derivatives radiolabeled with
positron-emitting fuorine-18 resulted in the FDA approval
of [18F]AV45 (Florbetapir; Fig. 1) to detect the presence of
amyloid [11–15]. Pioneering work directed toward develop-
ing PET probes to help diagnose CAA investigated a series
of 18F-labelled styrylpyridyl derivatives with high molecu-
lar weights designed to selectively target Aβ plaques in the
Results and discussion
Modification of the terminal hydrazinic nitrogen of
hydrazinopyridyl derivatives with an additional thiourea
functional group results in a substituted pyridylthiocarbazide
ligand system that is capable of forming well-defned, sta-
ble complexes with two ligands bound to a single [MO]3+
(M = Re or 99mTc) cores. These new pyridylthiocarbazide
ligands can be considered as sulphur-containing variations
Fig. 1 Amyloid imaging agents, [11C]PIB, forbetapir and AZD 4694
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JBIC Journal of Biological Inorganic Chemistry
to 6-hydrazinonicotinamide (HYNIC) derivatives leading to
them being given the trivial name of SHYNIC ligands [35].
This ligand system can be functionalized in a similar fash-
ion to HYNIC, for example, by attaching tumour targeting
peptides such as cylic-RGD or Tyr3-octreotate [36]. In this
work, we incorporate styrylpyridyl or benzofuran functional
groups, that are known to bind to Aβ plaques, into the ligand
framework with a view to forming [MO]3+ (M = 99mTc or
Re) complexes featuring two plaque targeting groups. It has
been suggested that such bivalent targeting molecules have
increased afnity for molecular targets.
derivative with the required E stereochemistry, 8, was pre-
pared via a Horner–Wadsworth–Emmons reaction with 7,
triethyl phosphite, sodium hydride and 4-(dimethylamino)
benzaldehyde. Reacting the bromo-styrilpyridine, 8, with
hydrazine yielded the substituted hydrazine 9 that was then
reacted with either methylisothiocyante or N,N-dimeth-
ylaminoethylisothiocyanate to form the H2L5 and H2L6,
respectively.
Dark red rhenium-oxo complexes, [ReO(HL1−6)2]+,
can be prepared by reacting the ligands with trans-
[ReOCl3(PPh3)2] in methanol (Fig. 4). The IR spectra
of the [ReO(H2L1−5)2]+ display the expected signals at
ν=955–969 cm−1 due to the presence of the Re-oxo core.
The electrospray ionisation mass spectra of each of the
The benzofuran-containing ligands, H2L1–4 (Fig. 2),
were synthesised from 5-methoxybenzofuran. Lithiation
of 5-methoxybenzofuran with n-butyllithium followed by
treatment with triisopropyl borate allowed isolation of the
corresponding boronic acid 1. This boronic acid was used in
a Suzuki coupling reaction with 2-chloro-5-iodopyridine to
form compound 2. Treatment of compound 2 with hydrazine
hydrate aforded the substituted hydrazine 4. Reaction of 4
with either methylisothiocyante or N,N-dimethylaminoeth-
ylisothiocyanate produced H2L1 and H2L2, respectively.
Deprotection of 2 with boron tribromide to produce 3, fol-
lowed by treatment with hydrazine and either methylisothi-
ocyante or N,N-dimethylaminoethylisothiocyanate allowed
isolated of H2L3 and H2L4. The N,N-dimethylaminoethyl
functional groups in H2L2 and H2L4 were added to improve
the solubility of the ligands and metal complexes in aque-
ous mixtures.
The synthesis of the styrylpyridyl ligands H2L5 and H2L6
started with reducing 6-bromonicotinaldehyde with sodium
borohydride to the alcohol 6, followed by chlorination with
thionyl chloride to form 7 (Fig. 3). The bromo-styrylpyridine
Fig. 3 Synthetic route of styrilpyridyl derivatives H2L5–6
Fig. 2 Synthesis of H2L1−4
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JBIC Journal of Biological Inorganic Chemistry
the diference electron density so this disordered counterion
and the disordered solvent were removed using the Squeeze
procedure [37]. The benzofuran functional group was disor-
dered over two orientations related by a 180° rotation about
the C8–C10 bond with occupancies close to 50:50.
The rhenium ion is in a distorted square pyramidal envi-
ronment with the dianionic thiosemicarbazide fragment
forming a N,N/S,S trans confguration about the Re-oxo
consistent with two other rhenium-oxo pyridylthiocarbazide
complexes characterised in our earlier work [35, 36]. The
selective formation of the N,N/S,S trans geometric isomer is
presumably due to a “trans efect”, although steric require-
ments may also play some role [38–40]. The Re–O1 bond
distance [1.625(1) Å] is relatively short even for rhenium(V)-
oxo bonds [41–43]. The Re–N4 bond length, 2.058(7) Å, is
shorter than typical Re–N bonds (ca. 2.15–2.18 Å) suggest-
ing some degree of multiple bond character [40, 42, 43].
The Re–S bond distance 2.281(2) Å is similar to the Re–S
bond distances in rhenium(V) complexes with amino thi-
olate ligands and Re–S bond distances in rhenium complexes
with thiosemicarbazonato ligands [40, 44, 45].
Fig. 4 Chemical structures of [ReO(HL1–6)2]+
complexes revealed signals for [ReO(HL1−6)2]+ with the
expected isotope patterns. The mass spectra of the com-
plexes with a terminal dimethylaminoethane functional
group, [ReO(HL2)2]+, [ReO(HL4)2]+ and [ReO(HL6)2]+,
also had signals that could be attributed to di- and trica-
tions, presumably due to protonation of the tertiary amine.
Each of the six complexes gives clear well-resolved 1H
NMR spectra consistent with a diamagnetic ground state.
The two coordinated ligands are magnetically equivalent.
Coordination to the Re-oxo core results in a general trend
for a downfeld shift in the chemical shift of the resonances
attributed to the aromatic protons when compared to the
free ligands.
Crystals of the formally charge neutral complex,
[ReO(HL6)(L6)], were isolated from the filtrate of the
reaction mixture and were characterised by X-ray crystal-
lography (Fig. 5c). In this instance, the thiosemicarbazide
fragment of each bidentate ligand still serves as a formal
dianionic bidentate ligands. The pyridyl functional groups
are not protonated but one of the ligands has a protonated
dimethylamino group that is involved in an intramolecular
hydrogen bond to the pyridyl functional group present on
the same ligand, to give an overall charge neutral complex.
The N3–C16 [1.265(1) Å] and N9–C37 [1.312(1) Å] bonds
are shorter than the other C–N bonds suggesting signifcant
double-bond character. Once again, the rhenium ion is in a
distorted square pyramidal environment with the dianionic
thiosemicarbazide fragment binding in a N,N/S,S trans con-
fguration about the Re-oxo. The Re–O1 bond [1.695(9) Å]
is within the expected range [41]. The Re–N (average
2.016 Å) and Re–S bonds (average 2.290 Å) are similar to
those found in [ReO(L4)2] and two other rhenium-oxo pyri-
dylthiocarbazide complexes characterised in our earlier work
[35, 36].
Crystals of [ReO(L4)2]3+ marginally suitable for analysis
by X-ray crystallography were isolated by vapour difusion
of diethyl ether to a mixture of [ReO(HL4)2]+ dissolved in
dimethylformamide that had been acidifed by addition of
HBF4. Addition of the acid to aid crystallisation resulted in
protonation of both the pyridyl and dimethylamine functional
groups but the coordinated thiosemicarbazide fragment still
serves as a dianionic ligand resulting in an overall charge
neutral zwitterionic ligand (Fig. 5a). The C3–N3 bond dis-
tance, 1.302(1) Å, is relatively short suggesting signifcant
sp2 hybridisation and C=N character and the C3–S1 bond
distance [1.757(9) Å] is consistent with thiol-like rather than
thione character. The complex lies on a crystallographic two-
fold axis of symmetry through the Re–O1 bond. The pro-
tonated pyridyl nitrogen forms a N–H···F hydrogen bond to
the BF4− counterion (N5···F4 2.79(1) Å, N4–H4···F4 147°)
while the protonated dimethylamino nitrogen N1 forms a
N–H···O hydrogen to a molecule of DMF (N1···O4 2.77(1)
Å, N1–H1···O4 151°) (Fig. 5b). The structure contained two
Interaction of [ReO(L1−6)2]+ with amyloid‑β1–42
and amyloid plaques present in human brain tissue
The interaction of the rhenium complexes, [ReO(HL1−6)2]+,
with synthetic Aβ1–42 was investigated using the fuorescent
probe thiofavin-T (ThT, (2-[p-(dimethylamino)phenyl]-
3,6-dimethyl-benzothiaolium chloride). The fuorescence
of ThT undergoes a 115-nm red shift in its excitation pro-
fle on binding to Aβ fbrils leading to fuorescence emis-
sion at λem =485 nm following excitation at λex =440 nm.
−
ordered BF4 counterions which are hydrogen bonded to
the complex but a third BF4− counterion and several DMF
solvent molecules were extensively disordered. A sensible
model for the disordered BF4− could not be recognised from
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JBIC Journal of Biological Inorganic Chemistry
Fig. 5 a ORTEP representa-
tion of the cation present in
[ReO(H2L4)2](BF4)3.2DMF.
Ellipsoids are at the 30% level.
Anions and solvent removed
for clarity. b Hydrogen-
(a)
bonding interactions present in
[ReO(L4)2](BF4)3.2DMF. The
protonated pyridine nitrogen
forms a N–H···F hydrogen bond
to a BF4 counterion (N5···F4
2.79(1) Å, N4-H4···F4 147°)
while the protonated dimeth-
C14
O2
C7
N5
C6
C15
Re1
O1
S1
C8
C9
N4
C13
C11
C16
ylamino nitrogen N1 forms a
N–H···O hydrogen to a mol-
ecule of DMF (N1···O4 2.77(1)
Å, N1-H1···O4 151°). c ORTEP
representation of [ReO(HL6)
(L6)]
C5
N3
C10
C12
C3
C17
O3
N1
C18
N2
C2
C19
C1
(b)
(c)
Incubating Aβ1–42 (10 μM) in phosphate bufered saline at
37 °C leads to the formation of Aβ fbrils with the emis-
sion from ThT reaching a maximum value after about 13 h
(Fig. 6). Addition of [ReO(HL1)2]+ (20 μM) to a mixture of
Aβ1-42 only resulted in a modest reduction in fuorescence
suggesting this complex has little interaction with Aβ1–42
.
In comparison, addition of each of the other fve rhenium
complexes resulted in signifcant reductions in fuorescence
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JBIC Journal of Biological Inorganic Chemistry
human brain tissue using epi-fuorescent microscopy. Serial
sections (7 µm) of post-mortem samples of frontal cortex of
human brain tissue from clinically diagnosed Alzheimer’s
disease subjects were pretreated with bovine serum albu-
min to prevent nonselective binding and then treated with
[ReO(HL6)2]+ (10 μM in 15% DMSO/phosphate bufered
solution (PBS), 10 min). The localization of [ReO(HL6)2]+
on the treated brain tissue was measured by epi-fuorescent
microscopy (λex =405 nm, λem =461 nm, Fig. 7b) and com-
parison with the contiguous section immuno-stained with
an Aβ antibody (1E8), Fig. 7a, to identify plaques reveals
good co-localisation and binding to Aβ plaques. Amyloid-β
plaques are typically 40–60 µm in diameter so sequential
7-µm serial tissue sections often, but not always, contain
portions of the same plaque. The complex was not retained
in a healthy age-matched control tissue (Fig. 7c, d). The
complexes with a benzofuran functional group are not suf-
fciently fuorescent to permit their detection on brain tissue.
Fig. 6 Time-dependent changes in ThT fuorescence (λex =440 nm,
λem =480 mn) over 52 h for solutions of ThT (40 μM) and freshly
prepared Aβ1–42 (10 μM) at 37 °C (red). Following addition of
[ReO(HL1)2]+ (20 μM) orange; [ReO(HL5)2]+ (green); [ReO(HL3)2]+
(purple); [ReO(HL6)2]+ (red); [ReO(HL2)2]+ (black); [ReO(HL4)2]+
(pink)
Synthesis of [99mTcO(HL5−6)2]+
suggesting that these complexes either bind competitively
with ThT to Aβ1–42 fbrils or inhibit fbril formation. The
most signifcant reductions in fuorescence were seen fol-
lowing addition of [ReO(HL2)2]+, [ReO(HL6)2]+ and [ReO
(HL4)2]+.
The fact that the styrylpyridyl complex [ReO(HL6)2]+
revealed strong interactions in the Aβ1–42 fbril assay and
bound to Aβ plaques in human brain tissue encouraged us
to investigate the potential to prepare the technetium-99m
analogues. [99mTcO(HL5)2]+ and [99mTcO(HL6)2]+ were
prepared by adding aqueous [99mTcO4]− (1 mL in 0.9%
saline, ~1000 MBq) to a mixture of the ligands in ethanol
(1 mg mL−1) in the presence of tin chloride (1 mg mL−1) to
The styrilpyridyl functional group present in
[ReO(HL5)2]+ and [ReO(HL6)2]+ contributes to the com-
plexes being fuorescent (λem =460 nm, λex =370 nm) and
this fuorescence can be used to detect the complexes in
Fig. 7 Image a is human brain
tissue from an AD patient, c
from aged-matched healthy
control, both stained with
1E8 antibody and visualised
with bright-feld microscopy.
Images b and d are the contigu-
ous sections of these tissues,
respectively, and are stained
with [ReO(H2L6)2]Cl and were
visualised by epifuorescence,
(λex =405 nm, λem =461 nm)
microscopy. Scale bar75 μM
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JBIC Journal of Biological Inorganic Chemistry
serve as a reducing agent. The mixtures were left at room
temperature for 30 min then analysed by reversed-phase
HPLC with radioactivity detection (Fig. 8). The HPLC anal-
ysis revealed the little ‘free’ [99mTcO4]−, as using this mobile
phase, the anion elutes as a broad peak with the solvent front
(ca. 2 min). For both reactions, a single major radioactive
peak was evident that had a similar retention time to the non-
radioactive rhenium-oxo complexes (detected λ=254 nm)
suggesting structurally analogous [99mTcO(HL5)2]+ and
styrylpyridyl containing complexes, [ReO(H2L6)2]Cl, was
selected and shown to bind to amyloid plaques present in
post-mortem human brain tissue. Preliminary radiolabelling
experiments suggested it was relatively simple to prepare
[
99mTcO(HL5)2]+ and [99mTcO(HL6)2]+ in good radiochemi-
cal yields. The complexes reported here may warrant further
studies to further investigate their potential to be of use in
assisting in the diagnosis of cerebral amyloid angiopathy.
[
99mTcO(HL6)2]+ complexes. The slight diferences between
the retention times of the radioactive peak (99mTc complex)
and UV peak (Re complex) in the HPLC chromatograms are
partly due to the relative detector confguration but could
also refect the diference in polarity of the rhenium-oxo and
technetium-oxo cores.
Experimental
Materials and reagents
All reagents and solvents were obtained from standard com-
mercial sources and used without further purifcation. The
isotope 99mTc was received as pertechnetate eluted from a
technetium-99m generator as [99mTcO4]− in a saline solution.
Compounds 1 [16], 6 and 7–10 [46] were prepared according
to reported procedures.
Concluding remarks
Six new substituted pyridylthiosemicarbazide ligands con-
taining either benzofuran or styrylpyridyl functional groups
that are known to selectively bind to amyloid plaques were
prepared. The new ligands were used to prepare well-defned
[Re-oxo]3+ complexes where two substituted pyridylthio-
semicarbazide ligands were coordinated to a single metal
ion to give bivalent complexes with two amyloid-β targeting
functional groups. Each of the complexes were analysed by
NMR spectroscopy and mass spectrometry. Two rhenium
complexes were characterised by X-ray crystallography con-
frming the rhenium(V) was in a square pyramidal geometry
with an apical oxygen ligand and each thiosemicarbazide
ligand acting as a dianionic bidentate ligand selectively
forming the N,N/S,S trans geometric isomer. The interac-
tion of the complexes with synthetic Aβ1–42 was investi-
gated using ThT as a fuorescent probe for amyloid fbrils.
Five of the new complexes either bind competitively with
ThT to Aβ1–42 fbrils or inhibit fbril formation. One of the
Instrumentation
1H, 13C, COSY, HSQC, and HMBC spectra were all
recorded using a Varian FT-NMR 400 or Varian FT-NMR
1
500 spectrometer (Varian, California USA). All H NMR
spectra were acquired at 400 MHz or 500 MHz and 13C spec-
tra were acquired at 101 MHz and 126 MHz. The reported
peaks were all referenced to solvent peaks in the order of
parts per million at 25 °C. ESI-MS—all ESI-MS data were
recorded on an Agilent 6510 ESI-TOF LC/MS Mass Spec-
trometer (Agilent, California USA). Absorbance spectra
were recorded using a Shimadzu UV-1650 PC spectropho-
tometer (Shimadzu, Sydney NSW). Emission spectra were
recorded on a Varian CAREY Eclipse fuorescence spec-
trophotometer (Varian, California USA). Infrared spectra
Fig. 8 a Reversed-phase HPLC chromatograms of [ReO(HL5)2]+
grams of [ReO(HL6)2]+ (blue, UV detection at λ=254 nm) and
(blue, UV detection at λ=254 nm) and
[
99mTcO(HL5)2]+
[
99mTcO(HL6)2]+ (red, radiation detection)
(red, radiation detection).
b Reversed-phase HPLC chromato-
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JBIC Journal of Biological Inorganic Chemistry
were obtained from a Perkin Elmer Spectrum One FTIR
spectrometer (Waltham, Massachusetts USA).
stirred for a further 4 h. An of-white-brown precipitate was
observed before H2O (20 mL) was added slowly, followed
by the slow addition of NaHCO3 (20 mL, sat. aq.). The solid
was collected by fltration, washed with H2O (10 mL), cold
ethanol (1–2 mL) and pentane (2×10 mL). The brown solid
was dissolved in ethyl acetate (30 mL) and was taken to dry-
ness in vacuo to aford a brown powder. (228 mg, 80%). 1H
NMR [CD3OD, 400 MHz]: δ (ppm) 8.84 (d, J=2.5 Hz, 1H,
11), 8.22 (dd, J=8.4, 2.5 Hz, 1H, 12), 7.52 (d, J=8.4 Hz,
1H, 13), 7.35 (d, J=8.8 Hz, 1H, 4), 7.23 (s, 1H, 8), 6.98 (d,
J=2.5 Hz, 1H, 7), 6.82 (dd, J=8.9, 2.5 Hz, 1H, 3). 13C{1H}
NMR [CD3OD, 101 MHz]: δ (ppm) 155.0, 153.4, 151.4,
151.1, 146.6, 136.3, 130.9, 127.6, 125.8, 115.4, 112.4,
106.8, 104.9. ESI-MS (+ve ion): m/z 246.0343 (experimen-
tal), 246.0322 (calculated for [C13H9ClNO2]+).
Non-radioactive analytical HPLC was performed on an
agilent 1100 Series. Samples were analysed using Column
1: SGE ProteCol™ c18 HPH 125 column (4.6× 150 mm,
5 µm, 120 Å). Gradient elution followed System A: gradient
elution of Bufer A (0.1% TFA in H2O) and Bufer B (0.1%
TFA in CH3CN) from 0 to 100% B to A over 20 min and UV
detection at λ=254 nm. Analytical HPLC traces of radiola-
belled compounds were acquired using a Shimadzu 10 AVP
UV–Vis spectrophotometer (Shimadzu, Kyoto Japan) and a
sodium iodide scintillation detector with two LC-10ATVP
solvent delivery systems for solvent A and B. Peak separa-
tion was achieved using Column 2: Nacalai Tesque Cosomo-
sil 5C18-AR Waters column (4.6×150 mm, 5 µm) (Kyoto,
Japan) at a fow rate of 1 mL min−1. System A: gradient
elution of Bufer A (0.1% TFA in H2O) and Bufer B (0.1%
TFA in CH3CN) from 0 to 100% B to A over 20 min and UV
detection at λ=254 nm.
2‑Hydrazinyl‑5‑(5‑methoxybenzofuran‑2‑yl)pyridine (4)
To a fask charged with 2-(6-chloro-3-pyridinyl)-5-methoxy-
benzofuran (300 mg, 1.16 mmol), hydrazine hydrate (5 mL,
30%, 48.3 mmol) was added dropwise at 0 °C. The mixture
was heated at refux overnight and cooled to 0 °C leading
to the formation of colourless precipitate. The precipitate
was collected by fltration and washed with H2O (10 mL),
cold ethanol (1–2 mL), diethyl ether (2×10 mL) and pen-
tane (2 × 10 mL). The solid was dried at 50 °C in vacuo
Syntheses of benzofuran and styrylpyridyl
derivative ligands
2‑Chloro‑5‑(5‑methoxybenzofuran‑2‑yl)pyridine (2)
A mixture of 5-methoxy-2-benzofuranyl-boronic acid
(474 mg, 2.47 mmol), 2-chloro-5-iodopyridine (562 mg,
2.35 mmol), palladium(II)-bis(triphenylphosphine)dichlo-
ride (52.0 mg, 0.074 mmol) and potassium carbonate
(682 mg, 4.94 mmol) was heated at refux in ethanol (17 mL)
for 4 h under an atmosphere of dinitrogen. The solvent was
then removed in vacuo and the product was extracted from
a brine solution with ethyl acetate. The organic layer was
dried with MgSO4 and the ethyl acetate was removed in
vacuo. The crude product was purifed by fash chromatogra-
phy (DCM). DCM was removed under vacuum producing a
beige-yellow solid which was collected and dried at 50 °C in
vacuo for 4 h (554 mg, 91%). 1H NMR [CDCl3, 400 MHz]:
δ (ppm) 8.84 (d, J=2.4 Hz, 1H), 8.05 (dd, J=8.3, 2.5 Hz,
1H), 7.42 (d, J = 9.4 Hz, 1H), 7.39 (s, 1H), 7.051 (s, 1H),
7.046 (s, 1H), 6.94 (dd, J =8.9, 2.6 Hz, 1H), 3.86 (s, 3H).
13C{1H} NMR [CDCl3, 101 MHz]: δ (ppm) 156.5, 152.6,
150.9, 150.3, 146.2, 134.6, 129.3, 125.8, 124.5, 114.3,
112.0, 103.6, 56.1. ESI-MS (+ve ion): m/z 259.9567 (experi-
mental), 260.0478 (calculated for [C14H11ClNO2]+).
1
for 4 h to aford a colourless powder (275 mg, 93%). H
NMR [DMSO-d6, 400 MHz]: δ (ppm) 8.52 (d, J=2.3 Hz,
1H), 7.91 (dd, J = 8.8, 2.4 Hz, 1H), 7.89 (s, 1H), 7.45 (d,
J = 8.9 Hz, 1H), 7.08 (d, J = 2.6 Hz, 1H), 7.07 (s, 1H),
6.83–6.80 (m, 2H), 4.27 (s, 2H), 3.78 (s, 3H). 13C{1H} NMR
[DMSO-d6, 101 MHz]: δ (ppm) 161.7, 155.7, 155.3, 148.7,
144.4, 133.4, 129.8, 114.9, 111.8, 111.2, 106.0, 103.1, 99.0,
55.5. ESI-MS (+ve ion): m/z 256.1060 (experimental),
256.1086 (calculated for [C14H14N3O2]+).
2‑(6‑Hydrazinylpyridin‑3‑yl)benzofuran‑5‑ol (5)
To a fask charged with 2-(6-chloro-3-pyridinyl)-5-benzo-
furanol (200 mg, 0.814 mmol), hydrazine hydrate (6 mL,
30%, 57 mmol) was added dropwise at 0 °C. The mixture
was heated at refux overnight and then cooled to 0 °C. The
of-white precipitate was collected by fltration and washed
with H2O (10 mL), cold ethanol (1–2 mL), diethyl ether
(2×10 mL) and pentane (2×10 mL). The solid was dried
at 50 °C in vacuo for 4 h to aford a colourless powder
1
2‑(6‑Chloropyridin‑3‑yl)benzofuran‑5‑ol (3)
(185 mg, 95%). H NMR [DMSO-d6, 400 MHz]: δ (ppm)
9.13 (s, 1H), 8.49 (d, J = 2.2 Hz, 1H), 7.88 (dd, J = 8.9,
2.4 Hz, 1H), 7.86 (s, 1H), 7.33 (d, J = 8.7 Hz, 1H), 6.99
(s, 1H), 6.87 (d, J=2.4 Hz, 1H), 6.80 (d, J=8.8 Hz, 1H),
6.67 (dd, J=8.7, 2.5 Hz, 1H), 4.24 (s, 2H). 13C{1H} NMR
[DMSO-d6, 101 MHz]: δ (ppm) 161.6, 154.9, 153.4, 148.0,
144.3, 133.3, 129.9, 115.1, 112.1, 110.9, 106.0, 104.9, 98.8.
2-(6-Chloro-3-pyridinyl)-5-methoxybenzofuran (300 mg,
1.16 mmol) was dissolved in DCM (5 mL). To this mixture,
boron tribromide in DCM (1 M, 2.0 mL, 2.00 mmol) was
added dropwise at 0 °C under an atmosphere of dinitrogen.
The reaction was allowed to reach room temperature and
1 3

JBIC Journal of Biological Inorganic Chemistry
ESI-MS (+ve ion): m/z 242.1100 (experimental), 242.0930
A brown precipitate formed that was collected by fltration,
washed with diethyl ether (2×5 mL) followed by pentane
(2×5 mL). The solid was dried at 50 °C in vacuo for 4 h to
aford a brown powder (119 mg, 83%). 1H NMR [DMSO-d6,
400 MHz]: δ (ppm) 9.40 (s, 1H), 9.17 (s, 1H), 8.73 (s, 1H),
8.62 (d, J=2.0 Hz, 1H), 8.16 (d, J=3.4 Hz, 1H), 8.04 (dd,
J=8.7, 2.2 Hz, 1H), 7.37 (d, J=8.8 Hz, 1H), 7.12 (s, 1H),
6.90 (d, J=2.3 Hz, 1H), 6.70 (dd, J=8.8, 2.4 Hz, 1H), 6.62
(d, J=8.7 Hz, 1H), 2.88 (d, J=4.4 Hz, 3H). 13C{1H} NMR
[DMSO-d6, 126 MHz]: δ (ppm) 182.57, 159.3, 154.1, 153.5,
148.2, 144.4, 134.1, 129.7, 118.1, 112.7, 111.1, 106.6, 105.1,
100.2, 39.5, 30.8. m/z 315.0913 (experimental), 315.0916
(calculated for [C15H15N4O2S]+). λmax(DMSO) nm (ε/dm3
mol−1 cm−1) 332 (35323). λex =333 nm (λem =401 nm). RP-
HPLC (Column 2, System A): RT (min) 8.2.
(calculated for [C13H12N3O2]+).
H2L1
A mixture of 2-(6-hydrazinyl-3-pyridinyl)-5-methoxyben-
zofuran (80 mg, 0.313 mmol) and methyl isothiocyanate
(46 mg, 0.63 mmol) was heated at refux in ethanol (4 mL)
for 4 h. A colourless precipitate was observed and collected
by fltration. The product was washed with diethyl ether
(2×5 mL) followed by pentane (2×5 mL). The solid was
dried at 50 °C in vacuo for 4 h to aford a colourless powder
1
(84 mg, 82%). H NMR [DMSO-d6, 400 MHz]: δ (ppm)
9.41 (s, 1H), 8.75 (s, 1H), 8.65 (d, J = 1.9 Hz, 1H), 8.16
(s, 1H), 8.07 (dd, J=8.6, 2.1 Hz, 1H), 7.49 (d, J=8.9 Hz,
1H), 7.20 (s, 1H), 7.12 (d, J=2.5 Hz, 1H), 6.86 (dd, J=8.9,
2.5 Hz, 1H), 6.63 (d, J = 8.7 Hz, 1H), 3.79 (s, 3H), 2.88
(d, J=4.4 Hz, 3H); 13C{1H} NMR [DMSO-d6, 101 MHz]:
δ (ppm) 182.6, 159.4, 155.8, 154.5, 148.9, 144.5, 134.1,
129.6, 117.9, 112.4, 111.4, 106.5, 103.3, 100.4, 55.6, 30.8;
ESI-MS (+ve ion): m/z 329.1073 (experimental), 329.1072
(calculated for [C16H17N4O2S]+). λmax(DMSO) nm (ε/
dm3 mol−1 cm−1) 329 (42268), 423 (29920). λex =332 nm
(λem =400 nm). RP-HPLC (Column 2, System A): RT (min)
10.3.
H2L4
A mixture of 2-(6-hydrazinyl-3-pyridinyl)-5-benzofuranol
(110 mg, 0.456 mmol) and N,N-dimethylaminoethyliso-
thiocyanate (119 mg, 0.912 mmol) was heated at refux
in ethanol (8 mL) for 4 h. The of-white precipitate that
formed was collected by fltration and washed with cold
ethanol (1–2 mL), diethyl ether (2 × 5 mL) followed by
pentane (2×5 mL). The solid was dried at 50 °C in vacuo
1
for 4 h to aford a colourless powder (127 mg, 75%). H
H2L2
NMR [DMSO-d6, 400 MHz]: δ (ppm) 9.46 (s, 1H), 9.18 (s,
1H), 8.74 (s, 1H), 8.63 (d, J=2.0 Hz, 1H), 8.05 (dd, J=8.7,
2.0 Hz, 1H), 7.97 (s, 1H), 7.37 (d, J=8.8 Hz, 1H), 7.13 (s,
1H), 6.90 (d, J=2.4 Hz, 1H), 6.71 (dd, J=8.8, 2.4 Hz, 1H),
6.62 (d, J = 8.7 Hz, 1H), 3.53 (q, J = 6.1 Hz, 2H), 2.36 (t,
J = 6.7 Hz, 2H), 2.11 (s, 6H). 13C{1H} NMR [DMSO-d6,
126 MHz]: δ (ppm) 181.8, 159.3, 154.0, 153.5, 148.2, 144.4,
134.1, 129.7, 118.3, 112.7, 111.1, 106.6, 105.1, 100.3, 57.5,
45.1, 41.3. ESI-MS (+ve ion): m/z 372.1493 (experimental),
372.1494 (calculated for [C18H22N5O2S]+). λmax(DMSO)
nm (ε/dm3 mol−1 cm−1) 332 (33994). λex = 332 nm
(λem =396 nm). RP-HPLC (Column 2, System A): RT (min)
8.2.
A mixture of 2-(6-hydrazinyl-3-pyridinyl)-5-methoxyben-
zofuran (50 mg, 0.196 mmol) and N,N-dimethylaminoeth-
ylisothiocyanate (90 mg, 0.691 mmol) was heated at refux
in ethanol (4 mL) for 4 h. A colourless precipitate formed
that was collected by fltration and washed with diethyl ether
(2×5 mL) followed by pentane (2×5 mL). The solid was
dried at 50 °C in vacuo for 4 h to aford a colourless powder
1
(64 mg, 85%). H NMR [DMSO-d6, 400 MHz]: δ (ppm)
9.46 (s, 1H), 8.77 (s, 1H), 8.65 (d, J = 1.7 Hz, 1H), 8.08
(dd, J=8.7, 1.7 Hz, 1H), 7.97 (s, 1H), 7.49 (d, J=8.9 Hz,
1H), 7.21 (s, 1H), 7.12 (d, J=2.5 Hz, 1H), 6.86 (dd, J=8.9,
2.6 Hz, 1H), 6.62 (d, J = 8.7 Hz, 1H), 3.79 (s, 3H), 3.53
(q, J = 6.1 Hz, 2H), 2.35 (t, J = 6.7 Hz, 2H), 2.11 (s, 6H);
13C{1H} NMR [DMSO-d6, 101 MHz]: δ (ppm) 186.1, 159.5,
155.8, 154.4, 148.9, 144.5, 134.2, 129.6, 118.2, 112.5, 111.4,
106.6, 103.3, 100.5, 57.6, 55.6, 45.1, 41.3; ESI-MS (+ve
ion): m/z 386.1650 (experimental), 386.1651 (calculated for
[C19H24N5O2S]+). λmax(DMSO) nm (ε/dm3 mol−1 cm−1) 325
(34397), 421 (26264). λex =330 nm (λem =398 nm).
H2L5
A mixture of (E)-4-(4-hydrazinylstyryl)-N,N-dimethylani-
line (60 mg, 0.237 mmol) and methyl isothiocyanate (35 mg,
0.474 mmol) was heated at refux in ethanol (4 mL) for 4 h.
The light green-brown precipitate that formed was collected
by fltration and washed with diethyl ether (2×5 mL) fol-
lowed by pentane (2×5 mL). The solid was dried at 50 °C in
vacuo for 4 h to aford a light green-brown powder (66 mg,
H2L3
1
85%). H NMR [DMSO-d6, 400 MHz]: δ (ppm) 9.32 (s,
A mixture of 2-(6-hydrazinyl-3-pyridinyl)-5-benzofuranol
(110 mg, 0.456 mmol) and methyl isothiocyanate (67 mg,
0.912 mmol) was heated at refux in ethanol (4 mL) for 4 h.
1H), 8.41 (s, 1H), 8.21 (d, J = 1.0 Hz, 1H), 8.11 (s, 1H),
7.85 (dd, J=8.7, 1.5 Hz, 1H), 7.38 (d, J=8.6 Hz, 2H), 6.98
(d, J = 16.4 Hz, 1H), 6.88 (d, J = 16.5 Hz, 1H), 6.71 (d,
1 3

JBIC Journal of Biological Inorganic Chemistry
J=8.7 Hz, 2H), 6.52 (d, J=8.7 Hz, 1H), 2.92 (s, 6H), 2.88
(d, J=4.3 Hz, 3H). 13C{1H} NMR [DMSO-d6, 126 MHz]:
δ (ppm) 182.6, 158.1, 149.7, 146.1, 133.9, 127.1, 126.6,
125.8, 125.2, 120.3, 112.3, 106.8, 40.0, 30.7. ESI-MS (+ve
ion): m/z 328.1594 (experimental), 328.1596 (calculated for
[C17H22N5S]+). λmax(DMSO) nm (ε/dm3 mol−1 cm−1) 368
(36009). λex =370 nm (λem =458 nm). RP-HPLC (Column
2, System A): RT (min) 11.0.
m/z 857.1252 (experimental), 857.1333 (calculated for
[C32H30N8O5ReS2]+). λmax(DMSO) nm (ε/dm3 mol−1 cm−1)
329 (42268), 429 (30317). λex = 332 nm (λem = 391 nm).
RP-HPLC (Column 2, System A): RT (min) 18.3. IR: ῡmax
(cm−1) 961 (ʋRe=O), 3102 (ʋN–H).
[ReO(HL2)2]Cl
[ReO(HL2)2]Cl was synthesised by general procedure with
H2L2 (50 mg, 0.129 mmol), oxotrichlorobis(triphenylph
osphine)rhenium(V) (50 mg, 0.060 mmol) and methanol
H2L6
1
A mixture of (E)-4-(4-hydrazinylstyryl)-N,N-dimethylani-
line (75 mg, 0.294 mmol) and N,N-dimethylaminoethyli-
sothiocyanate (115 mg, 0.881 mmol) was heated at refux
in ethanol (4 mL) for 4 h. A light green-brown precipitate
was observed, collected by fltration and washed with die-
thyl ether (2×5 mL) followed by pentane (2×5 mL). The
solid was dried at 50 °C in vacuo for 4 h to aford a light
(4 mL), to obtain a red-brown powder (41 mg, 70%). H
NMR (DMSO-d6, 400 MHz]: δ (ppm) 10.65 (s, 2H), 8.77
(s, 2H), 8.55 (s, 2H), 7.92 (s, 2H), 7.76 (s, 2H), 7.60 (d,
J=8.9 Hz, 2H), 7.48 (s, 2H), 7.22 (d, J=2.2 Hz, 2H), 6.96
(dd, J=8.9, 2.2 Hz, 2H), 3.92 (d, J=52.1 Hz, 4H), 3.82 (s,
6H), 2.84 (s, 12H). ESI-MS (+ve ion): m/z 971.2484 (exper-
imental), 971.2490 (calculated for [C38H44N10O5ReS2]+).
λmax(DMSO) nm (ε/dm3 mol−1 cm−1) 323 (34397), 424
(26264). λex =320 nm (λem =385 nm). IR: ῡmax (cm−1) 966
(ʋRe=O), 2620 (ʋN–H).
1
green-brown powder (68.4 mg, 61%). H NMR [DMSO-
d6, 400 MHz]: δ (ppm) 9.38 (s, 1H), 8.43 (s, 1H), 8.21 (d,
J = 1.9 Hz, 1H), 7.91 (s, 1H), 7.86 (dd, J = 8.7, 2.0 Hz,
1H), 7.38 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 16.4 Hz, 1H),
6.88 (d, J=16.4 Hz, 1H), 6.71 (d, J=8.8 Hz, 2H), 6.52 (d,
J=8.7 Hz, 1H), 3.52 (q, J=6.1 Hz, 2H), 2.92 (s, 6H), 2.35
(t, J = 6.7 Hz, 2H), 2.10 (s, 6H). 13C{1H} NMR [DMSO-
d6, 126 MHz]: δ (ppm) 181.8, 158.1, 149.8, 146.2, 134.0,
127.1, 126.8, 126.0, 125.2, 120.3, 112.3, 106.8, 57.6, 45.1,
41.2, 40.0. ESI-MS (+ve ion): m/z 385.2155 (experimental),
385.2174 (calculated for [C20H29N6S]+). λmax(DMSO) nm (ε/
dm3 mol−1 cm−1) 369 (35496). λex =370 nm (λem =461 nm).
RP-HPLC (Column 2, System A): RT (min) 10.3.
[ReO(HL3)2]Cl
[ReO(HL3)2]Cl was synthesised by general procedure with
H2L3 (100 mg, 0.318 mmol), oxotrichlorobis(triphenylph
osphine)rhenium(V) (126 mg, 0.151 mmol) and methanol
(10 mL), to obtain a red-brown powder (110 mg, 84%). 1H
NMR (DMSO-d6, 400 MHz]: δ (ppm) 8.53 (s, 4H), 7.94 (d,
J=9.6 Hz, 2H), 7.47 (d, J=8.9 Hz, 2H), 7.43 (s, 2H), 7.00
(d, J=2.4 Hz, 2H), 6.81 (dd, J=8.8, 2.4 Hz, 2H), 3.07 (s,
6H). 13C{1H} NMR [DMSO-d6, 126 MHz]: δ (ppm) 167.4,
153.9, 150.8, 148.6, 138.0, 133.1, 129.2, 128.7, 123.4,
118.4, 114.1, 111.5, 105.6, 103.7, 33.3. ESI-MS (+ve ion):
[M]+ m/z 829.1015 (experimental), 829.1020 (calculated for
[C30H26N8O5ReS2]+). λmax(DMSO) nm (ε/dm3 mol−1 cm−1)
329 (41299), 429 (29950). λex = 330 nm (λem = 390 nm).
RP-HPLC (Column 2, System A): RT (min) 14.1. IR: ῡmax
(cm−1) 954 (ʋRe=O), 3356 (ʋN–H), 3148 (ʋO–H).
General procedure for the syntheses of Re(V)
complexes
A mixture of ligands, H2L1−6 (2.1 eq.) and oxotrichlorobis-
(triphenyl-phosphine)rhenium(V), [ReOCl3(PPh3)2], [47]
(1 eq.) was heated at refux in methanol (20 mL) for 4 h. A
dark red-brown precipitate was collected by fltration and
washed with cold methanol (2–5 mL), diethyl ether (2 ×
5–20 mL) followed by pentane (2 × 5–20 mL). The solid was
dried at 50 °C in vacuo for 4 h to aford a red-brown powder.
[ReO(HL4)2]Cl
[ReO(HL4)2]Cl was synthesised by general procedure with
H2L4 (100 mg, 0.269 mmol), oxotrichlorobis(triphenylph
osphine)rhenium(V) (107 mg, 0.128 mmol) and methanol
[ReO(HL1)2]Cl
[ReO(HL1)2]Cl was synthesised by general procedure
with H2L1 (150 mg, 0.457 mmol), oxotrichlorobis(triph
enylphosphine)rhenium(V) (181 mg, 0.218 mmol) and
methanol (15 mL), to obtain a red-brown powder (130 mg,
(10 mL), to obtain a red-brown powder (97 mg, 80%). H
1
NMR [DMSO-d6, 400 MHz]: δ (ppm) 10.69 (s, 2H), 9.36
(s, 2H), 8.74 (s, 2H), 8.53 (s, 2H), 7.92 (s, 2H), 7.81 (s, 2H),
7.48 (d, J=8.7 Hz, 2H), 7.42 (s, 2H), 7.00 (d, J=1.9 Hz,
2H), 6.81 (dd, J=8.8, 1.8 Hz, 2H), 3.95 (d, J=53.4 Hz, 4H),
3.23–3.19 (m, 4H), 2.84 (s, 12H). 13C{1H} NMR [DMSO-
d6, 126 MHz]: δ (ppm) 166.7, 153.9, 151.0, 148.6, 133.4,
132.2, 129.3, 123.3, 118.5, 114.1, 111.5, 105.6, 103.7, 54.7,
1
67%). H NMR [DMSO-d6, 400 MHz]: δ (ppm) 8.56 (s,
4H), 7.94 (d, J = 9.7 Hz, 2H), 7.59 (d, J = 9.0 Hz, 2H),
7.51 (s, 2H), 7.22 (d, J = 2.5 Hz, 2H), 6.97 (dd, J = 8.9,
2.5 Hz, 2H), 3.82 (s, 6H), 3.08 (s, 6H). ESI-MS (+ve ion):
1 3

JBIC Journal of Biological Inorganic Chemistry
42.3, 41.0. ESI-MS (+ve ion): [M]+ m/z 943.2225 (experi-
mental), 943.2177 (calculated for [C36H40N10O5ReS2]+).
λmax(DMSO) nm (ε/dm3 mol−1 cm−1) 323 (38583), 423
(29700). λex =324 nm (λem =390 nm). RP-HPLC (Column
2, System A) RT (min) 9.9. IR: ῡmax (cm−1) 969 (ʋRe=O),
2560 (ʋN–H), 3176 (ʋO–H).
temperature. To this solution, seven parts of deionised water
was added and vortex briefy followed by ultrasonication
for 5 min on ice. One part of 10× PBS was added, vortexed
briefy and centrifuged for 5 min. Supernatant was trans-
ferred to a fresh tube and kept on ice. The concentration of
the stock solution was determined by measuring the absorb-
ance at 214 nm (ε=75,887 M−1 cm−1).
[ReO(HL5)2]Cl
Stock solutions of ThT (1 mM in PBS), and Re (V) com-
plexes (1 mM in DMSO) were freshly prepared. The fnal
sample was composed of Aβ1–42 (1 eq., 10 µM), ThT (4 eq.,
40 µM) and the Re complexes (2 eq., 20 µM) to make a
fnal volume of 500 µL in PBS with 2.5% DMSO. Samples
were transferred on a 96-well plate kept over ice having 150
µL in each well to analyse in triplicates. The ThT fuores-
cence intensity of each sample was recorded every 12.5 min
at 37 °C using an OPTIMA fuorescent plate reader with
440/480 10 nm excitation/emission fltres over a period
of 52 h.
[ReO(HL5)2]Cl was synthesised by general procedure with
H2L5 (150 mg, 0.458 mmol), oxotrichlorobis(triphenylph
osphine)rhenium(V) (182 mg, 0.218 mmol) and methanol
1
(15 mL), to obtain a red-brown powder (97 mg, 50%). H
NMR [DMSO-d6, 400 MHz]: δ (ppm) 8.55 (dd, J = 9.6,
1.9 Hz, 2H), 8.21 (s, 2H), 7.84 (d, J = 9.4 Hz, 2H), 7.54
(d, J = 8.2 Hz, 6H), 7.28 (d, J = 16.4 Hz, 2H), 7.20 (d,
J=16.5 Hz, 2H), 6.94 (s, 4H), 3.07 (s, 6H), 3.00 (s, 12H).
ESI-MS (+ve ion): m/z 855.2306 (experimental), 855.2380
(calculated for [C34H40N10OReS2]+). λmax(DMSO) nm (ε/
dm3 mol−1 cm−1) 353 (41915), 467 (33322). λex =370 nm
(λem =453 nm). RP-HPLC (Column 2, System A): RT (min)
13.6. IR: ῡmax (cm−1) 955 (ʋRe=O), 3017 (ʋN–H).
Human brain tissue staining
Parafn-preserved brain tissue blocks were provided by the
Victorian Brain Bank Network. Brain tissue was collected
at autopsy. The National Neural Tissue Resource Centre
performed sourcing and preparation of human brain tissue.
AD pathologic diagnosis was made according to standard
National Institute on Ageing-Reagan Institute criteria. Deter-
mination of age-matched human control (HC) cases was
subject to the above criteria. The AD and HC brain tissues
sections (7 µm) were frst deparafned (xylene, 3 × 2 min)
followed by rehydration (soaking in a series of 100, 90, 70,
and 0% v/v ethanol/water baths). The hydrated tissue sec-
tions were washed in phosphate bufer saline (PBS, 5 min).
Autofuorescence of tissues was quenched using potassium
permanganate (0.25% in PBS, 20 min) and washed with PBS
(2 × 2 min) to remove the excess. The now brown-coloured
sections were washed with potassium metabisulfte and
oxalic acid (1% in PBS) until brown colour 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 [ReO(HL6)2]Cl (10 µm
in 15% v/v DMSO/PBS, 10 min). The sections were treated
with BSA again to remove any unbound compound. Finally,
the sections were washed with PBS (3 × 2 min), deionised
water, and mounted with non-fuorescent mounting media
(Dako). Florescence images were visualised using a Leica
DM IL LED Fluorescence microscope ftted with a Leica
DFC310FX colour camera.
[ReO(HL6)2]Cl
[ReO(HL6)2]Cl was synthesised by modifed general proce-
dure with H2L6 (90 mg, 0.234 mmol), oxotrichlorobis(triphe
nylphosphine)rhenium(V) (93 mg, 0.111 mmol) and metha-
nol (9 mL). The solvent was removed under reduced pres-
sure and the remaining solid was heated in methanol:acetone
(10:90 40 mL). A dark brown solid was collect by hot fl-
1
tration (30 mg, 27%). H NMR [DMSO-d6, 400 MHz]: δ
(ppm) 10.57 (s, 1H), 8.45 (d, J=75.3 Hz, 4H), 7.85 (s, 2H),
7.48 (d, J=7.9 Hz, 4H), 7.20 (t, J=17.6 Hz, 4H), 6.76 (d,
J = 8.3 Hz, 4H), 3.94 (d, J = 85.8 Hz, 4H), 2.96 (s, 12H),
2.82 (s, 12H). ESI-MS (+ve ion): m/z 485.1782 (experi-
mental), 485.1805 (calculated for [C40H55N12OReS2]2+).
λmax(DMSO) nm (ε/dm3 mol−1 cm−1) 355 (36231), 425
(32650). λex =370 nm (λem =462 nm). RP-HPLC (Column
2, System A): RT (min) 11.7.
Aβ fbril binding assays
AB1–42 was synthesised using solid-phase peptide synthe-
sizer (Liberty Blue peptide synthesizer, Biotage) using
F-moc chemistry on a 0.1 mM scale using reported proce-
dure [48]. Synthetic peptide was dissolved in HFIP (1 mg
per 221.7 μL HFIP) incubated for 1 h on ice then allowed to
evaporate overnight in fume hood. The residue was further
dried under high vacuum to remove any residual HFIP and
moisture and then stored at −80 °C. For a 1 mg mL−1 stock
solution in PBS, the HFIP-treated peptide was dissolved in
two parts of 60 mM NaOH and incubated for 3 min at room
Radiolabelling with 99mTc
Radiolabelling with Techentium-99m was carried out with
the addition of ligands, H2L2−10 in EtOH (1 mg mL−1) to
1 3

JBIC Journal of Biological Inorganic Chemistry
a solution of tin chloride in EtOH (1 mg mL−1). 100 µL
aliquot of eluted sodium pertechnetate ([99mTcO4]−, 1 ml in
0.9% saline, ~1000 MBq) was added and the mixture was
left for 30 min. 20 µL aliquot was used to assess the purity
of the radiochemical product using RP-HPLC.
3. Hecht M, Krämer LM, von Arnim CAF, Otto M, Thal DR (2018)
Acta Neuropathol 135:681–694
4. Johnson KA, Gregas M, Becker JA, Kinnecom C, Salat DH,
Moran EK, Smith EE, Rosand J, Rentz DM, Klunk WE, Mathis
CA, Price JC, DeKosky ST, Fischman AJ, Greenberg SM (2007)
Ann Neurol 62:229–234
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M, Skovronsky D, Kung HF (2011) J Med Chem 54:8085–8098
7. Klunk WE, Wang Y, Huang G-F, Debnath ML, Holt DP, Mathis
CA (2001) Life Sci 69:1471–1484
Crystallography
Intensity data were collected with an Oxford Difraction
SuperNova CCD difractometer using Cu–Kα radiation,
and the temperature during data collection was maintained
at 130.0(1) K using an Oxford Cryosystems cooling device.
The structures were solved by direct methods and diference
Fourier synthesis [49]. Thermal ellipsoid plots were gener-
ated using the programme ORTEP-3 integrated within the
WINGX suite of programmes [50].
8. Mathis CA, Bacskai BJ, Kajdasz ST, McLellan ME, Frosch MP,
Hyman BT, Holt DP, Wang Y, Huang G-F, Debnath ML, Klunk
WE (2002) Bioorg Med Chem Lett 12:295–298
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Cowie TF, Dickinson KL, Maruf P, Darby D, Smith C, Wood-
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Mathis CA, Price JC, Masters CL, Villemagne VL (2007) Neurol-
ogy 68:1718–1725
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RNK, Becker JA, Garcia-Alloza M, Prada C, Frosch MP, Rosand
J, Viswanathan A, Smith EE, Johnson KA (2008) Ann Neurol
64:587–591
Crystal data for [ReO(H2L4)2](BF4)3·2DMF:
C42H55B2F8N12O7ReS2 M = 1263.92 T = 130.0 K,
λ = 1.54184 Å, space group C 2/c, a = 16.937(3)
b =24.270(3), c =16.9438(19) Å, β = 111.774(17)°
V =6467.9(17) Å3, Z = 4, Dc = 1.298 Mg M−3 μ(Cu–Kα)
4.899 mm−1, F(000) = 2544, crystal size 0.41 × 0.03 ×
0.02 mm3, 18209 refections measured θmax = 67.5°, 5829
independent refections [R(int) = 0.0865], the fnal R was
0.0706[I > 2σ(I) 4123 data] and wR(F2) was 0.2144 (all
data), GOOF=0.957.
11. Kung HF, Lee C-W, Zhuang Z-P, Kung M-P, Hou C, Ploessl K
(2001) J Am Chem Soc 123:12740–12741
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Singh K, Kung HF, Kung M-P, Houle S (2004) Am J Geriatr
Psychiatry 12:584–595
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Z-P, Kung M-P, Kung HF (2003) Nucl Med Biol 30:565–571
14. Kung HF, Choi SR, Qu W, Zhang W, Skovronsky D (2010) J Med
Chem 53:933–941
15. Zhu L, Ploessl K, Kung HF (2014) Chem Soc Rev 43:6683–6691
16. Swahn B-M, Wensbo D, Sandell J, Sohn D, Slivo C, Pyring D,
Malmstroem J, Arzel E, Vallin M, Bergh M, Jeppsson F, Johnson
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Lett 20:1976–1980
Crystal data for [ReO(HL6)(L6)]: C40H53N12ORe S2
0.0.5(H2O) M=976.26, T=130.0 K, λ=1.54184 Å, space
group P-1, a = 10.2115(12) b =13.740(2)), c =18.614(3
Å, α = 103.960(13)° β = 94.436(10)° γ = 94.116(11)°
V =2515.9(6) Å3, Z = 2, Dc = 1.289 Mg M−3 μ(Cu–Kα)
5.287 mm−1, F(000) = 992, crystal size 0.15 × 0.07 ×
0.02 mm3, 16704 refections measured θmax = 67.5°, 9062
independent refections [R(int) = 0.1240], the fnal R was
0.0846 [I > 2σ(I) 4316 data] and wR(F2) was 0.2071 (all
data), GOOF=0.899.
17. Swahn B-M, Sandell J, Pyring D, Bergh M, Jeppsson F, Jureus A,
Neelissen J, Johnstroem P, Schou M, Svensson S (2012) Bioorg
Med Chem Lett 22:4332–4337
18. Hickey JL, Donnelly PS (2012) Coord Chem Rev 256:2367–2380
19. Hayne DJ, Lim SC, Donnelly PS (2014) Chem Soc Rev
43:6701–6715
20. Chen K, Cui M (2017) Med Chem Comm 8:1393–1407
21. Hayne DJ, White JM, McLean CA, Villemagne VL, Barnham KJ,
Donnelly PS (2016) Inorg Chem 55:7944–7953
CCDC deposition numbers: CCDC1840665 and CCDC
1841151.
22. Hayne DJ, North AJ, Fodero-Tavoletti M, White JM, Hung LW,
Rigopoulos A, McLean CA, Adlard PA, Ackermann U, Tochon-
Danguy H, Villemagne VL, Barnham KJ, Donnelly PS (2015)
Dalton Trans 44:4933–4944
Acknowledgements The Australian Research Council (Grant Nos.
DP160100288, FT130100204) for funding this research. The Victorian
Brain Bank for provision of human brain tissue. We acknowledge the
generous support of Prof. Andrew M. Scott, A/Prof. Henri Tochon-
Danguy, A/Prof. Uwe Ackermann, Dr. FT Lee, Nick Alexopoulos, and
David Thomas of the Austin Hospital, Heidelberg, Victoria, Australia,
for providing access to 99mTc and radiochemistry facilities. Associate
Professors Kevin Barnham and Victor Villemagne are acknowledged
for their ongoing advice and guidance in this area of research.
23. Chan CY, Noor A, McLean CA, Donnelly PS, Barnard PJ (2017)
Chem Commun 53:2311–2314
24. Zhang X, Hou Y, Peng C, Wang C, Wang X, Liang Z, Lu J, Chen
B, Dai J, Liu B, Cui M (2018) J Med Chem 61:1330–1339
25. Li Z, Cui M, Dai J, Wang X, Yu P, Yang Y, Jia J, Fu H, Ono M,
Jia H, Saji H, Liu B (2013) J Med Chem 56:471–482
26. Ono M, Fuchi Y, Fuchigami T, Kobashi N, Kimura H, Haratake
M, Saji H, Nakayama M (2010) ACS Med Chem Lett 1:443–447
27. Ono M, Ikeoka R, Watanabe H, Kimura H, Fuchigami T, Hara-
take M, Saji H, Nakayama M (2010) Bioorg Med Chem Lett
20:5743–5748
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Afliations
Scott P. Fletcher1 · Asif Noor1 · James L. Hickey1 · Catriona A. McLean2,3 · Jonathan M. White1 · Paul S. Donnelly1
1
2
3
School of Chemistry and Bio21 Molecular Science
and Biotechnology Institute, University of Melbourne,
Melbourne, VIC 3010, Australia
The Florey Institute of Neuroscience and Mental Health,
University of Melbourne, Parkville, Melbourne, VIC 3052,
Australia
Department of Anatomical Pathology, The Alfred Hospital,
Melbourne, VIC 3181, Australia
1 3