Rhenium and technetium complexes that bind to amyloid-β plaques

Article abstract of DOI:10.1039/c4dt02969k

Alzheimer's disease is associated with the presence of insoluble protein deposits in the brain called amyloid plaques. The major constituent of these deposits is aggregated amyloid-β peptide. Technetium-99m complexes that bind to amyloid-β plaques could p

Full text of DOI:10.1039/c4dt02969k

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Rhenium and technetium complexes that bind to
amyloid-β plaques†
Cite this: DOI: 10.1039/c4dt02969k
a,b
a,b
c,e
David J. Hayne, Andrea J. North, Michelle Fodero-Tavoletti,
a,b
b,c
d
c,f
Jonathan M. White, Lin W. Hung, Angela Rigopoulos, Catriona A. McLean,
c
e
e
c,e
Paul A. Adlard, Uwe Ackermann, Henri Tochon-Danguy, Victor L. Villemagne,
b,c,g
a,b
Kevin J. Barnham
and Paul S. Donnelly*
Alzheimer’s disease is associated with the presence of insoluble protein deposits in the brain called
amyloid plaques. The major constituent of these deposits is aggregated amyloid-β peptide. Technetium-
9
9m complexes that bind to amyloid-β plaques could provide important diagnostic information on
amyloid-β plaque burden using Single Photon Emission Computed Tomography (SPECT). Tridentate
I
+
ligands with a stilbene functional group were used to form complexes with the fac-[M (CO)
3
]
(M = Re or
9
9m
Tc) core. The rhenium carbonyl complexes with tridentate co-ligands that included a stilbene func-
tional group and a dimethylamino substituent bound to amyloid-β present in human frontal cortex brain
tissue from subjects with Alzheimer’s disease. This chemistry was extended to make the analogous
9
9m
I
+
Received 26th September 2014,
Accepted 2nd December 2014
[
3
Tc (CO) ] complexes and the complexes were sufficiently stable in human serum. Whilst the lipophi-
licity (log D7.4) of the technetium complexes appeared ideally suited for penetration of the blood-brain
barrier, preliminary biodistribution studies in an AD mouse model (APP/PS1) revealed relatively low brain
uptake (0.24% ID g− at 2 min post injection).
DOI: 10.1039/c4dt02969k
1
www.rsc.org/dalton
relies on tests to establish progressive impairment of memory
Introduction
4
,5
and in at least one other area of cognition.
Molecular
Alzheimer’s disease (AD) is the major cause of neurodegenera- imaging techniques offer the possibility of identifying disease
tive dementia. A pathological hallmark of the disease is the associated pathology and the desire for earlier and more accu-
presence of extracellular senile plaques in the brain. The rate diagnosis of AD has led to the development of radioactive
major constituent of these plaques is an aggregated peptide tracers designed to cross the blood-brain barrier and bind with
called amyloid-β (Aβ), a 39–43 amino acid peptide derived some degree of selectivity to Aβ plaques.
1
,2
from the amyloid precursor protein (APP). The exact role of
The fluorescent dye Thioflavin-T that binds to Aβ plaques
Aβ plaques in dementia remains controversial, but extensive in vitro provided the structural inspiration for the development
cortical Aβ deposition is a common feature identified by post- of several radiolabelled benzothiazole and stilbene compounds
3
mortem analysis of AD subjects. Clinical diagnosis of AD that have been used with considerable success to quantify
plaque burden in human patients. It is thought that these
molecules enter a hydrophobic pocket or channel and bind to
a
School of Chemistry, University of Melbourne, Melbourne, 3010, Australia.
the plaques by way of a combination of hydrophobic and π–π
E-mail: pauld@unimelb.edu.au
1
1
b
interactions. The benzothiazole, 2-(4-[ C]methylamino-
Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road,
phenyl)-6-hydroxybenzo-thiazole, known as Pittsburgh com-
The University of Melbourne, Victoria 3010, Australia
c
11
Florey Institute of Neuroscience and Mental Health, University of Melbourne,
pound-B or [ C]PiB (Fig. 1), makes use of the short-lived
1
1
Parkville, Melbourne, Victoria 3010, Australia
positron-emitting isotope C to enable diagnostic imaging
d
Ludwig Institute for Cancer Research, Heidelberg, Victoria, Australia
6–8
using positron emission tomography (PET). There are some
e
Department of Molecular Imaging and Therapy, Austin Health, Melbourne,
similarities in the chemical structure of benzothiazoles, such
as PiB, and other plaque targeting groups such as stilbenes, in
that they have conjugated aromatic ring systems and are rela-
tively planar molecules. A variety of stilbene and styrylpyridine
derivatives are excellent probes for Aβ plaques and a stilbene
derivative radiolabelled with carbon-11 (4-N-methylamino-4′-
hydroxystilbene), known as SB-13 (Fig. 1), binds selectively to
Heidelberg, Victoria 3084, Australia
f
Department of Anatomical Pathology, The Alfred Hospital, Victoria 3181, Australia
g
Department of Pharmacology, University of Melbourne, Parkville, Melbourne,
Heidelberg, Victoria 3084, Australia
†
Electronic supplementary information (ESI) available: Images of tissue sections
2
from the frontal cortex of an age-matched control brain treated with [Re(CO)
3
L ]
4
3
and [Re(CO) L ]. CCDC 1025768 and 1025769. For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/c4dt02969k
This journal is © The Royal Society of Chemistry 2014
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+
I
I
3
ligands designed to bind to the [M(CO) ] core (M = Tc /Re )
that feature pendent stilbene functional groups designed to
bind to amyloid plaques. The ability of the new rhenium com-
plexes to bind to Aβ plaques in human brain tissue is assessed
9
9m
and the preliminary brain uptake of the
Tc analogues is
determined in both wild-type mice and a mouse model of Aβ
plaque pathology (APP/PS1).
11
18
Fig. 1 Chemical structures of [ C]PiB, SB-13 and F-AV45.
Results and discussion
Synthesis and characterisation
3
,9,10
Aβ plaques.
Preliminary studies of SB-13 in humans were
+
I
I
The fac-[M(CO) (H O) ] (M = Tc /Re ) cation can be considered
3
2
3
promising and stimulated continued explorations of stilbene
and styrylpyridine derivatives, as probes for Aβ plaques.
The longer half-life of fluorine-18 when compared to carbon-
1 ( C = 20.4 min, F = 109.7 min) encouraged efforts to
develop stilbene
labelled with fluorine-18 culminating in the recent FDA
approval of F-AV45 (florbetapir) (Fig. 1) to detect the pres-
ence of amyloid.
For imaging applications using carbon-11 or fluorine-18
radioisotopes the isotopes are produced by cyclotrons and the
radionuclides are attached covalently to plaque binding mole-
cules. Covalent attachment of radioisotopes to tracers requires
complicated synthetic manipulations and specialist equip-
ment. Despite the rapid increase in the number of hospitals
equipped with the requisite infrastructure for PET, single
photon emission computed tomography (SPECT) is the most
commonly used nuclear imaging technique. The most com-
a “semi metal aqua cation” where the carbonyl ligands stabil-
ize the low oxidation state and the trans effect increases the
lability of coordinated water molecules. Ligands containing an
3
,11
1
1
18
1
aromatic amine and carboxylate donors coordinate to fac-
1
2
13,14
or styrylpyridine
derivatives radio-
+
[
M(CO)
3
]
with rapid complexation kinetics producing com-
plexes with good stability in aqueous solvents. Tridentate
ligands bearing two pyridyl groups such as dipicolylamine
serve as a versatile and useful starting point for bifunctional
ligands where the amine nitrogen can be used to tether target-
ing groups. In this manuscript we present the synthesis of two
1
8
3
,5,15–17
1
2
pyridylamine-carboxylate tridentate ligands (HL , HL ) and
3
4
two dipyridylamine ligands (L , L ) that each feature stilbene-
like functional groups connected by a short alkyl linker
(
Fig. 2).
The synthesis of the tridentate ligands HL and L utilised
2
4
aldehyde 2, (E)-4-(4-N,N-dimethylaminostyryl)benzaldehyde,
that was prepared by formylation of E-4-(4-bromostyryl)-N,N-
dimethylaniline (2a). Precursor 2a was prepared by a Horner–
Wadsworth–Emmons reaction between diethyl-4-bromobenzyl-
9
9m
monly used radioisotope for SPECT is
from convenient generators. The 6 hour half-life of
Tc, which is available
9
9m
Tc is
sufficiently long to allow for the preparation of pure techne-
tium complexes and for accumulation in target tissue, but
short enough to not deliver excessive radiation doses to the
phosphonate
Scheme 1). The methyl ester of L was prepared by reductive
amination of 2-picolylamine with E-4-stilbenecarboxaldehyde
1) to give pyridyl amine 3 followed by an aza-Michael addition
and
4-N,N-dimethylaminobenzaldehyde
1
(
9
9m
patient. A
Tc based radiotracer for determining plaque
(
burden suitable for SPECT imaging would be of considerable
clinical utility by increasing the number of centres that are
3
with methyl acrylate (Scheme 2). The synthesis of L involved
aza-Michael addition between pyridyl amine 3 and 2-vinylpyri-
dine. Reductive amination of aldehyde 2 with 2-picolylamine
followed by aza-Michael additions with either methyl acrylate
1
8–20
able to perform diagnostic scans.
plexes based on tetradentate N
benzothiazole plaque binding groups designed to form neutral
Several technetium com-
2 2
S ligands fused to phenyl-
2
4
or 2-vinylpyridine furnished either the methyl ester of L or L
V
3+
complexes with the [Tc O] have been synthesized. Some of
V
3+
these [Tc O] complexes show considerable promise and
2
0–22
warrant further investigation.
‘
An alternative technetium
V
3+
I
+
core’ to the [Tc O] is the low valent [Tc (CO) ] and inno-
3
vations in synthetic methodology have led to the possibility of
+
I
I
preparing fac-[M(CO) (H O) ] (where M = Tc or Re ) using
3
2
3
2
3–25
conditions amenable to radiopharmaceutical applications.
The “carbonyl core” approach exploits the stability of the
metal tricarbonyl core by substituting the water ligands with
ligands designed to modify biodistribution. In the absence of
non-radioactive isotopes of technetium the Group VII conge-
ner rhenium, which by virtue of the lanthanide contraction
has similar ionic radii to technetium, is often used as a surro-
gate to guide synthetic developments. This manuscript
describes the synthesis and characterisation of tridentate Fig. 2 Chemical structures of ligands H
1
–2
3–4
2
L
and L
.
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Scheme 1 Synthesis of precursor 2a by Wadsworth-Emmons-Horner
reaction followed by formylation of 2a to give aldehyde 2.
1
1
I
1
Fig. 3 H NMR spectra of MeL (red) and fac-[Re (CO)
3
L ] (black).
Methylene protons and signals are highlighted with green marks and
ethylene with blue.
1
H NMR spectroscopy confirmed the E-stereochemistry of the
3
stilbene pendant groups was retained ( JHH > 16 Hz). Upon
coordination to the metal the methylene and ethylene protons
1
of each ligand become diasterotopic, for example, the H NMR
1
spectrum of the methyl ester of L has singlets for each of the
two methylene groups and two multiplets for the proton pairs
within the ethylene that split into in four AB doublets for each
methylene proton and four multiplets for the ethylene protons
upon coordination (Fig. 3).
I
1
The crystal structures of fac-[Re (CO)
Re (CO)
3
3
L ] and fac-
L ] show distorted octahedral geometry about the
metal centre with carbonyl groups arranged facially (Table 1).
I
3 +
[
I
1
Compound fac-[Re (CO)
3
L ] crystallises in the triclinic space
group P ˉ1 (Fig. 4). The asymmetric unit contains two neutral com-
I
1
plexes and acetonitrile solvent molecules. In fac-[Re (CO) L ],
3
the remaining coordination sphere of the metal is completed
by an NNO donor atom set provided by the pyridyl nitrogen, ter-
tiary amine and carboxylate groups of the ligand. The rhenium
ion is in the equatorial plane defined by two carbonyl groups
and the nitrogen donor atoms of the ligand with apical sites
Scheme 2 (a) Reductive amination of (E)-stilbene-carboxyaldehyde
derivatives (aldehydes 1 or 2) to give pyridylamines 3 or 4 respectively. occupied by the remaining carbonyl group and carboxylato
1
(
b) Synthesis of proligand MeL by aza-Michael addition of 3 with methyl oxygen. Coordination of the ligand sees the formation of a five
acrylate.
membered chelate ring containing two nitrogen donor atoms
N1, N2) and a methylene carbon (C6) and a six membered
(
chelate ring that includes the two ethylene carbons (C7, C8), ter-
Scheme 2). The methyl esters of L and L , were hydrolysed to tiary amine nitrogen (N2), and carboxylato oxygen (O1). The
1
2
(
1
2
give the corresponding carboxylic acids, HL and HL .
bond distances between rhenium and the two N donor atoms
and fac- differ, with bond Re–N1 being 2.186(5) Å and Re–N2 2.262(5) Å
I
1–2
The rhenium complexes fac-[Re (CO)
3
L
]
I
3–4 +
[
Re (CO) L ] were prepared by microwave irradiation of an in length. The Re–N1 bond length is within the range of similar
3
I
I
5 3 3
3
aqueous mixture of [Re (CO) (CF SO )] with the respective fac-[Re (CO) NNO] complexes but bonds Re–O1 and Re–N2 are
ligands in methanol. The use of microwave irradiation offers slightly longer, at 2.142(4) Å and 2.262(5) Å, compared to
the advantage of high product yields with relatively short reac- 2.107–2.132 Å and 2.232–2.255 Å respectively for similar
26–31
tion times of about 30 minutes. For each complex, analysis by complexes.
This is possibly due to the inclusion of a six
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I
1
I
3 +
Table 1 Crystallographic data for fac-[Re (CO)
3
3
L ] or fac-[Re (CO) L ]
I
1
I
3
fac-[Re (CO)
.5MeCN
3
L ]·
fac-[Re (CO)
3
L ]-
0
3 3
CF SO
Formula
M
C
28
H
24.50
N
2.50
5
O Re
32 27 3 3 6
C H F N O ReS
824.82
662.20
Colour and Habit
Crystal size (mm )
System
Space group
T (°K)
a (Å)
b (Å)
c (Å)
α (°)
β (°)
Pink rod
0.13 × 0.06 × 0.04
Triclinic
Pˉ1
Colourless block
0.36 × 0.27 × 0.21
Monoclinic
3
1
P2 /c
130.0 K
130.0 K
13.3527(3)
14.7033(3)
17.0611(3)
90
91.244(2)
90
3348.79(12)
4
1.636
10.6380(7)
12.5358(7)
19.0696(13)
95.970(5)
96.832(5)
99.253(5)
2472.4(3)
4
I
3 +
Fig. 5 An ORTEP representation of fac-[Re (CO) L ] with 30% prob-
3
−
ability thermal ellipsoids, disordered counterions ( OTf) were removed
γ (°)
by a squeeze procedure and hydrogen atoms are omitted for clarity.
Selected bond lengths (Å): Re–N1 2.213(4), Re–N2 2.251(4), Re–N3
2.162(5), Re–C29 1.919(5), Re–C30 1.905(5), Re–C31 1.922(6).
3
U/Å
Z
D
calcd (Mg m−3
)
1.779
Wavelength (Å)
1.5418 (Cu-Kα)
9.980
0.7107 (Mo-Kα)
3.754
Absorption coefficient
−
1
(mm
)
F(000)
Reflections measured
1300
17 732
1624
22 309
have similar absorbance spectra each displaying two maxima
at λmax = 300 nm and λmax = 312 nm. Both ligands fluoresce at
Independent reflections
9825 [Rint
.0456]
0.04
=
7471 [Rint
0.0272]
0.0378
=
0
λem = 355 nm following excitation at λ = 312 nm. The absor-
R [I > 2σ(I)]
ex
2
I
1
I
3 +
wR(F ) (all data)
0.0525
0.0509
3 3
bance spectra for fac-[Re (CO) L ] and fac-[Re (CO) L ] have
maxima at λmax = 300 and λmax = 312 nm, and an emission
with a maximum at λem = 360 nm (Fig. 6a). There is an
increase in molar absorptivity upon coordination, from
3
−1
−1
3
−1
−1
2
7.0(±0.2) × 10 M cm and 30.6(±0.3) × 10 M cm for the
1
3
3
−1
methyl ester of L and L respectively to 43.5(±0.9) × 10 M
cm for fac-[Re (CO)
Re (CO) L ] . The introduction of the electron donating di-
methylamino functional group (HL and L ) leads to a batho-
−
1
I
1
3
−1
−1
3
L ] and 42.9(±0.3) × 10 M cm for fac-
I
3 +
[
3
2
4
chromic shift of approximately 40 nm in the absorbance
2
4
maxima (λ 355 nm for HL , λ 345 nm for HL ) and larger
I
2
Stokes shifts of 90–100 nm. Complexes fac-[Re (CO) L ] and
fac-[Re (CO) L ] are also fluorescent (λem = 472 nm for fac-
3
Re (CO) L ] and λ 464 nm for fac-[Re (CO) L ] (Fig. 6b)). The
3 3
3
I
4 +
I
2
I
4 +
[
electronic spectra of stilbene and its derivatives are concen-
tration dependent leading to excimer formation and stilbenes
are photochromic but such detail was not investigated for
these derivatives.
I
1
Fig. 4 An ORTEP representation of fac-[Re (CO)
3
L ] with 30% prob-
ability thermal ellipsoids, hydrogen atoms and solvent atoms omitted for
clarity. Selected bond lengths (Å): Re–N1 2.186(5), Re–N2 2.262(5), Re–
O1 2.142(4), Re–C25 1.927(7), Re–C26 1.913(7), Re–C27 1.928(7).
I
1–2
I
3–4 +
Binding of fac-[Re (CO)
plaques in human brain tissue. Frontal cortex brain tissue col-
membered chelate ring instead of two five membered chelate lected from subjects with clinically diagnosed AD and age
rings seen in other complexes with the same donor atom set.
matched control subjects were used to determine the plaque-
L ] crystallises in the monoclinic binding capacity of the new rhenium complexes. Amyloid-β
3
L
] & fac-[Re (CO)
3
L
]
to Aβ
I
3 +
Compound fac-[Re (CO)
3
space group P2 /c (Fig. 5). The asymmetric unit contains one plaques are typically 30–50 μm in diameter so 5 μm serial sec-
1
cationic complex with the asymmetric ligand forming both a tions of tissue often contain comparable Aβ plaque distri-
five and six membered chelate ring upon coordination. Bond bution. Aβ plaques were stained with an Aβ antibody (1E8) and
I
+
Re–N3 (2.162(5) Å) is within the range of other fac-[Re (CO) ]
contiguous sections of brain tissue were treated with a solution
3
I
1
complexes containing two pyridyl groups and a tertiary amine of a chosen Re complex. The complexes fac-[Re (CO) L ] or fac-
Re–Npy = 2.155–2.187 Å) but Re–N1 is longer (2.213(4) Å). This [Re (CO) L ] showed little to no plaque binding within brain
3
I
3 +
(
3
is again likely due to the six membered chelate ring with the tissue from AD positive subjects. The complexes that con-
bond Re–N2 (2.251(4) Å) also being longer relative to similar tained the electron-donating dimethylamino functional group
2
6,27,32–36
complexes (Re–Namine 2.216–2.242 Å).
were more promising. Epifluorescent microscopy of tissue sec-
1
–2
3–4
I
2
I
4 +
Ligands HL and L are fluorescent due to the presence tions treated with fac-[Re (CO) L ] and fac-[Re (CO) L ] reveal
of the stilbene functional group. The methyl ester of L and L
3
3
1
3
good correlation of the complexes to Aβ plaques stained with
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I
1
I
3 +
Fig. 6 Absorbance (solid line) and emission (dotted line) spectra of a) fac-[Re (CO)
3
3
L ] (5 μm) and fac-[Re (CO)
3
L ] (2 μm) in acetonitrile, λex =
I
2
3 3
I
4 +
12 nm, and (b) fac-[Re (CO) L ] (5 μM) and fac-[Re (CO) L ] (5 μM) in acetonitrile, λex = 355 nm.
1
Fig. 8 Representative RP-HPLC trace of (a) fac-[M(CO)
Re , black; M =
3
L ] (where M =
I
99m
I
Tc , red). Absorbance measured at 254 nm.
Fig. 7 Tissue sections from the frontal cortex of an AD affected brain
×10 magnification). Images on the left are immunohistologically stained
(
using 1E8 antibody and viewed by bright-field microscopy. The match-
ing adjacent slides treated with the rhenium complex and viewed by
99m
I
1
99m
I
2
3 3
affording fac-[ Tc (CO) L ] and fac-[ Tc (CO) L ] in >98%
radiochemical purity without purification (Fig. 8). A mixture of
products was formed when reacting fac-[ Tc (CO)
with the ligands containing two pyridyl functional groups, L
epifluorescence microscopy are on the right (λex = 359 nm, λem
=
9
9m
I
+
461 nm.
3 2
(H O)]
3
4
and L . Radiochemical purity of the desired complexes after
E8 antibody on the adjacent slide (Fig. 7) especially with the incubation at 70 °C for 20 min was ∼50% and ∼40%, for fac-
1
9
9m
3 +
99m
4 +
plaque cores. Encouragingly there was no evidence of non-
[
3 3
Tc(CO) L ] and fac-[ Tc(CO) L ] respectively. The com-
specific binding in the tissue from aged matched control sub- plexes were found to be stable over several hours allowing
I
2
I
4 +
jects after treatment with fac-[Re (CO) L ] or fac-[Re (CO) L ]
sufficient time for purification by ‘Sep-Pak’ reverse phase C18
3
3
(
see ESI†).
Synthesis of fac-[ Tc (CO)
columns. The use of aqueous ethanol improved the radio-
9
9m
I
1–2
99m
I
3–4 +
99m
4 +
3
L
] & fac-[ Tc (CO)
3
L
] . chemical purity of fac-[ Tc(CO)
3
L ] to 80%.
The lipophilicity of the complexes was estimated by
9
9m
I
1–2
Synthesis of technetium complexes, fac-[ Tc (CO) L ] and
3
9
9m
I
3–4 +
99m
I
1–2
fac-[ Tc (CO) L ] , involved addition of the tridentate measuring the partitioning of fac-[ Tc (CO)
3
L
] and fac-
] between 1-octanol and PBS (pH 7.4) to give
mixture to pH 6 and incubation of the mixture at 70 °C for a log D7.4 value. It is difficult to predict if small molecules will
0 min. The RP-HPLC profile of the technetium complexes be capable of crossing the blood-brain barrier but lipophilicity
3
9
9m
I
+
99m
I
3–4 +
ligands to fac-[ Tc (CO)
3
(H
2
O)] , adjustment of the reaction
[
Tc (CO) L
3
2
was compared to the respective analogous rhenium complex. is an important criterion to be considered. Compounds with a
The slight difference in the retention times between the log P of 1–3.5 are considered to have the best potential to cross
3
7
UV-Vis detection of the Re complexes and the radioactive peak the blood-brain barrier. The log D7.4 values ranged from
9
9m
I
4 +
99m
I
1
of the technetium complexes is at least partially attributed to 1.0 for fac-[ Tc (CO)
L ] to 2.1 for fac-[ Tc (CO) L ]. The
3
3
the configuration of the detectors. Both carboxylate containing addition of the dimethylamino functional group results in
1
2
ligands, HL and HL , labelled with high radiochemical yield, complexes that are less lipophilic (Table 2).
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9
9m
I
1–2
99m
I
3–4 +
Table 2 Log D7.4 values of fac-[
Tc (CO)
3
L
] and fac-[
Tc (CO)
3
L
]
9
9m
I
1
99m
I
2
99m
I
3 +
99m
I
4 +
fac-[ Tc (CO)
3
L ]
fac-[ Tc (CO)
3
L ]
fac-[ Tc (CO)
3
L ]
3
fac-[ Tc (CO) L ]
Log D7.4
2.1
1.7
1.8
1.0
9
9m
2
Table 3 Biodistribution of fac-[
Tc(CO)
3
L ] in APP/PS1 transgenic
mice and wild-type-mice(n = 3); Average %dose/g (standard deviation)
Wild-type
min
APP/PS1
2
30 min
Ave (std)
2 min
Avg (std)
30 min
Avg (std)
Ave (std)
Blood
Brain
Liver
Spleen
Heart
Stomach
Kidney
Intestine
12.87 (3.82)
0.25 (0.05)
26.95 (7.84)
7.88 (3.80)
5.43 (1.84)
1.69 (0.81)
25.32 (9.24)
3.15 (0.91)
9.75 (0.43)
0.21 (0.02)
26.13 (4.69)
8.97 (4.77)
4.25 (0.62)
3.77 (0.70)
19.60 (1.09)
4.39 (1.71)
11.71 (0.62)
0.24 (0.02)
24.64 (5.70)
6.67 (1.07)
5.25 (0.48)
2.06 (0.93)
24.76 (4.29)
3.15 (1.02)
8.47 (1.04)
0.19 (0.01)
20.32 (0.65)
5.96 (0.97)
3.59 (0.20)
2.19 (1.68)
18.45 (0.90)
4.15 (0.59)
Fig. 9 Coronal and sagittal co-register SPECT/CT images of an APP/
9
9m
PS1 transgenic mouse after intravenous tail injection of fac-[
Tc
4
+
9
9m
I
2
99m
I
4 +
(CO) L ] (∼3.5 MBq in 200 μL of 10% ethanol in saline (0.9%)). Amount
3
Stability of fac-[ Tc (CO)
3
L ] & fac-[ Tc (CO)
3
L ] in
of radioactivity is shown by the scale bar on the right.
blood serum and in vivo biodistribution. The complexes fac-
I
2
I
4 +
3 3
[Tc (CO) L ] and fac-[Tc (CO) L ] were added to human sera
and incubated at 37 °C with aliquots of the mixture taken
for analysis by RP-HPLC. Proteins were removed by addition of
acetonitrile to incipient precipitation followed by centrifu-
gation and the supernatant was analysed by HPLC. The neutral
9
9m
I
1
complex, fac-[ Tc (CO) L ] remains stable in serum for over
3
three hours but there is some degradation of the cationic
9
9m
I
4 +
3
complex, fac-[ Tc (CO) L ] , evident after incubation in
serum but the complex still retained ∼60% radiochemical
purity.
9
9m
2
3
The biodistribution of fac-[ Tc(CO) L ] was investigated
in both wild-type (WT) and 10 month old APP/PS1 transgenic
mice. The APP/PS1 transgenic (Tg) amyloid model results in
high Aβ plaque levels in the brain. The accumulation of radio-
activity was predominately in the liver and kidneys. Brain
−
1
uptake was detected at 2 min post injection (0.25% ID g for
−
1
Fig. 10 Time activity plot of selected organs from transgenic mice
WT and 0.24% ID g for Tg) but with no statistically signifi-
cant difference between wild-type and transgenic mice at
either 2 minutes or 30 minutes post injection (mpi) (2 mpi, p =
9
9m
4 +
administered fac-[
5 min and 62 min.
3
Tc(CO) L ] . Activity was recorded at 13 min,
3
0
.429; 30 mpi, p = 0.136) (Table 3). An effective radiotracer
for Aβ plaques would record higher radioactivity for brain
tissue from the transgenic mice, particularly at 30 min post Retention of activity within the thyroid may indicate instability
9
9m
4
injection.
3
of the complex, fac-[ Tc(CO) L ], with respect to oxidation,
9
9m VII
− 38
Micro-SPECT images of transgenic mice (3 × APP/PS1) were resulting in formation of pertechnetate ([ Tc
O
] ). No
L ] by intrave- uptake is apparent in the cranial region indicating no or very
3
4
9
9m
I
4 +
acquired after administering fac-[ Tc (CO)
nous tail injection with anatomical information afforded by low brain uptake of fac-[ Tc (CO)
X-ray computed tomography (CT). Post-mortem immunohisto- time confirms the lack of fac-[ Tc (CO) L ] uptake across
3
9
9m
I
4 +
3
L ] . A plot of activity over
9
9m
I
4 +
chemical staining of brain tissue samples confirmed the pres- the blood-brain barrier in vivo (Fig. 10). The majority of activity
ence of Aβ plaques. Visual inspection of the co-registered was shown to be within the bladder. A large amount of activity
SPECT/CT images reveal that activity due to accumulation of was also recorded within the gall bladder and liver indicating
9
9m
Tc is concentrated within the thoracic region with activity excretion of the compound by both the urinary and hepatobili-
also detected in the abdomen and the thyroid (Fig. 9). ary system.
Dalton Trans.
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Paper
wave reactions were carried out using a Biotage Initiator micro-
wave reactor. Nuclear magnetic spectra were acquired on an
Conclusion
1
13
1
Four tridentate ligands designed to coordinate to fac- Agilent 400-MR ( H NMR at 400 MHz and C{ H} NMR at
I
+
99m
I
+
1
[
3 3
Re (CO) ] and fac-[ Tc (CO) ] and bind to Aβ plaques via a 101 MHz) or Varian FT-NMR 500 spectrometer ( H NMR at
1
3
1
stilbene functional group were synthesised. The rhenium com- 500 MHz and C{ H} NMR at 126 MHz) at 298 K. All chemical
plexes were characterised by a combination of NMR, electronic shifts were referenced to the internal solvent residue and
spectroscopy and mass spectrometry, and two rhenium com- quoted in ppm relative to TMS. Chemical & Micro Analytical
plexes were characterised by X-ray crystallography. The com- Services Pty. Ltd., Victoria, Australia, carried out elemental
plexes with an electron-donating dimethylamino substituent analyses for C, H and N. Analytical radio-HPLC was performed
I
2
on the stilbene functional group, fac-[Re (CO)
3
L ] and fac- using a Shimadzu 10AVP UV–Vis detector (Shimadzu, Kyoto,
Re (CO) L ] , bound to Aβ plaques present in post-mortem Japan), two LC-10ATVP solvent delivery systems (for solvent A
I
4 +
[
3
brain tissue collected from human AD subjects. Analogous (0.1% trifluoroacetic acid in MilliQ H
2
O) & B (0.1% trifluoro-
9
9m VII
−
technetium complexes were prepared from [ Tc
4
O ] using acetic acid in acetonitrile)), a Nacalai Tesque Cosmosil 5C18-
an ‘Isolink’ kit that uses potassium boranocarbonate as both a AR Waters column (4.6 mm I.D. × 150 mm) (Nacalai Tesque,
reducing agent and a source of carbon monoxide. The neutral Kyoto, Japan). The mobile phase used was a gradient consist-
9
9m
I
1
99m
I
2
complexes [ Tc (CO)
3 3
L ] and [ Tc (CO) L ] were more ing of 5% solvent B at t = 0 to 100% solvent B after 20 min. All
stable in human serum than the cationic complexes, runs were conducted at a constant total flow rate of 1 mL
9
9m
I
3
99m
I
4
−1
[
Tc (CO)
3 3
L ] and [ Tc (CO) L ] and all four technetium min and the absorbance was monitored at λ 254 nm. The Re
9
9m
complexes have log D7.4 values that suggested appropriate lipo- analogues of
Tc complexes were used to confirm synthesis
philicity for penetration of the blood-brain barrier via non- of complexes by comparison of retention times when analysed
facilitated processes. Preliminary biodistribution and micro- by RP-HPLC. Absorbance spectra were obtained on a Shimadzu
SPECT imaging experiments revealed that although neutral UV-1650PC UV/visible spectrophotometer and emission
9
9m
I
2
[
[
Tc (CO) L ] displayed higher brain uptake than cationic spectra were obtained on a Varian CARY Eclipse fluorescence
3
9
9m
I
4
3
Tc (CO) L ], the degree of brain uptake in a transgenic spectrometer, with both performed using capped quartz cuv-
murine model of amyloid pathology was relatively low (0.24% ettes. Infrared spectra were recorded using a Perkin-Elmer
−
1
ID g ). Effective penetration of the blood-brain barrier is a Spectrum One FTIR spectrometer, with a zinc selenide/
major challenge in the development of metal-based imaging diamond universal ATR 60 sampling accessory.
agents designed to offer insight into the molecular aspects of
Crystals of compounds were mounted in low temperature
neurodegeneration. Compounds that bind to Aβ plaques are oil then flash cooled. Intensity data were collected at 130 K on
often lipophilic and consequently display high levels of depo- an X-ray diffractometer with CCD detector using either Cu-Kα
sition in the lungs and liver and also susceptible to metab- (λ = 1.54184 Å) or Mo-Kα (λ = 0.71073 Å) radiation. Data were
4
2
olism by P450 enzymes. Nonspecific binding to albumin and reduced and corrected for absorption. The structures were
other serum proteins can also decrease the dose of a particular solved by direct methods and difference Fourier synthesis
4
3
tracer that enters the brain. It would be of interest to investi- using the SHELX suite of programs as implemented within
4
4
gate if these technetium complexes are substrates for either the WINGX software. Thermal ellipsoid plots were generated
1
p-glycoprotein or breast cancer resistant protein, as both func- using ORTEP-3 software. In both structures fac-[Re(CO)
3
L ]
the trans-stilbene moiety was dis-
The relative small size and low molecular weight of ordered with two orientations of the alkene occupying the
3
3 3 3
tion as efflux pumps that act to remove substrates from the and fac-[Re(CO) L ]CF SO
3
9–41
brain.
I
+
the [Tc (CO)
3
] core makes it attractive for the synthesis of lipo- same site in the crystal. This was refined as a two-site disorder
1
philic and neutral molecules capable of crossing the blood- model with occupancies of ca. 63 : 37 for fac-[Re(CO)
3
L ] and
3
brain barrier and providing diagnostic information on Aβ 73 : 27 for fac-[Re(CO) L ]CF SO , equivalent carbon–carbon
3
3
3
plaque burden central to the progression of Alzheimer’s bonds were restrained to have the same bond distances, and
disease. In this work tridentate ligands with a stilbene func- equivalent carbon atoms restrained to have similar thermal
1
tional group were used to form complexes featuring the parameters. The triflate counter-ion of fac-[Re(CO) L ]CF SO
3
3
3
I
+
[
Re (CO)
3
] core that bound to Aβ plaques in human subjects. was disordered and could not be modelled satisfactorily. The
This chemistry was extended to make complexes based on contributions from this counter-ion were removed using the
9
9m
I
+
45
[
Tc (CO) ] and further investigations aimed at structural squeeze procedure, which calculated 232 electrons unac-
3
modifications for improving the brain uptake of these com- counted for in the void volume of the unit cell. This is reason-
plexes is warranted.
ably consistent with four triflate anions in the unit cell. fac-
I
1
I
3
[
Re (CO) L ]·0.5MeCN: CCDC 1025768. fac-[Re (CO) L ]CF SO :
3 3 3 3
CCDC 1025769.
E)-4-(4-Bromostyryl)-N,N-dimethylaniline
from a literature procedure. To a dry round bottom flask was
added 4-bromobenzyl bromide (3.57 g, 14.2 mmol) and triethyl
(
(2a). Modified
Experimental
General synthetic chemistry procedures
4
6
All reagents and solvents were obtained from commercial phosphite (7.5 mL, 43.1 mmol). The reaction mixture was
sources and used as received unless otherwise stated. Micro- heated at reflux for 2.5 h then unreacted triethyl phosphite
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Paper
Dalton Transactions
was removed by evaporation under reduced pressure and the 139.83, 137.5, 136.5, 136.2, 128.8, 128.7, 128.6, 128.4, 127.6,
residue was used without further purification. DMF (8 mL), 126.7, 126.6, 122.5, 122.0, 54.6, 53.3. ESI-MS (+ve ion) m/z
+
sodium hydride (80% w/w, 1.01 g, 33.7 mmol) and 4-dimethyl- [M + H] 301.170 (experimental), 301.17 (calculated for
+
aminobenzldehyde (2.10 g, 14.1 mmol) were added to the
residue and the mixture was stirred at ambient temperature
C
21
H
21
N
2
).
(E)-N,N-Dimethyl-4-(4-((pyridin-2-ylmethylamino)methyl)-
for 12 h. The reaction was quenched by the addition of styryl)aniline (4). To anhydrous ethanol (50 mL) was added
ethanol then water to afford a green precipitate. The precipi- 2-picolylamine (0.44 g, 9.7 mmol) and 2 (0.36 g, 1.4 mmol).
tate was collected by filtration, dissolved in dichloromethane The reaction mixture was set stirring and heated at reflux.
(40 mL), washed with water (3 × 40 mL), and the organic phase After 1 h, sodium borohydride (0.70 g, 18.5 mmol) was added
was then dried (MgSO ). The MgSO was removed by filtration in small portions and the reaction was heated at reflux again.
4
4
and petroleum spirits (boiling range 40–60 °C) were added to After 6 hours, the reaction was cooled to ambient temperature
the filtrate to give (E)-4-(4-bromostyryl)-N,N-dimethylaniline as and the mixture was adjusted to pH 12 (1 M NaOH) and then
1
yellow plate-like crystals (3.75 g, 12.4 mmol, 88%). H NMR pH 7 (1 M HCl). After extracting the reaction mixture with
(
400 MHz; CDCl ): δ 7.43 (m, 4H, ArH), 7.34 (m, AA′BB′, 2H, ethyl acetate the organic layer was washed with brine (2 ×
3
3
3
ArH), 7.03 (d, JHH = 16.3, 1H, CHvCH), 6.84 (d, JHH = 16.3, 100 mL), and then with water (3 × 100 mL), dried over MgSO
H, CHvCH), 6.72 (m, AA′BB′ 2H, ArH), 2.99 (s, 6H, N(CH ). filtered and the solvent was evaporated under reduced
ESI-MS (+ve ion) [M + H] m/z 302.071 (experimental), 302.05 pressure. The resultant waxy yellow residue was triturated with
4
,
1
3 2
)
+
+
(
calculated for C16
H
17BrN ).
petroleum benzene (boiling range 40–60%) and isolated by fil-
4
-(4-Dimethylamino)styryl)benzaldehyde (2). Modified from tration followed by purification by silica chromatography
4
6
a literature procedure. To anhydrous and deoxygenated THF (chloroform) to afford compound 4 as a yellow powder (0.33 g,
(
(
5 mL) was added (E)-4-(4-bromostyryl)-N,N-dimethylaniline 0.97 mmol, 68%). Calc. for C23
0.48 mg, 1.6 mmol) and the mixture was cooled to −78 °C. To N, 11.92. Found: C, 78.7; H, 7.30; N, 12.10. H NMR (500 MHz;
25 3 2
H N ·0.5H O: C, 78.37; H, 7.44;
1
the reaction mixture was added n-butyl lithium (1.2 M, 2 mL, CDCl ): δ 8.57 (m, 1H, pyH), 7.64 (m, 1H, PyH), 7.45–7.40 (m,
3
2
.4 mmol) dropwise and then DMF (2.6 mL). The mixture was 5H), 7.32 (m, 3H, PyH, ArH), 7.17–7.15 (m, 1H, PyH), 7.03 (d,
3
3
stirred for 3.5 h at −78 °C and then the reaction was diluted
JHH = 16.2, 1H, CHvCH), 6.91 (d, JHH = 16.3, 1H, CHvCH),
with diethyl ether (1 mL) followed by water to afford a bright 6.72 (m, 2H, ArH), 3.94 (s, 2H, CH ), 3.84 (s, 2H, CH ), 2.98 (s,
2
2
1
13
yellow precipitate. The reaction mixture was extracted with 6H, N(CH
3
)
2
). { H} C NMR (126 MHz; CDCl
3
): δ 160.0, 150.2,
dichloromethane (150 mL), washed with brine (100 mL), then 149.5, 138.8, 137.2, 136.5, 128.7, 128.6, 127.7, 126.2, 126.0,
water (3 × 100 mL), the organic phase was then dried (MgSO4), 124.4, 122.5, 122.0, 112.6, 54.7, 53.4, 40.6. ESI-MS (+ve ion)
+
filtered and the solvent removed by evaporation under reduced [M + Na] m/z 344.212, (experimental), 344.21 (calculated for
+
pressure to afford 4-(4-dimethylamino)styryl)benzaldehyde as
C
23
H
26
N
3
).
1
an orange crystalline powder (0.32 mg, 1.3 mmol, 82%).
H
(E)-Methyl 3-((pyridin-2-ylmethyl)(4-styrylbenzyl)amino)pro-
NMR (400 MHz; CDCl ): δ 7.43 (m, 4H), 7.33 (d, J = 8.4, 2H), panoate (methyl ester of L ). To ethanol (30 mL) was added 3
.03 (d, JHH = 16.3, 1H), 6.84 (d, JHH = 16.3, 1H), 6.72 (m, (0.40 g, 1.3 mmol), methyl acrylate (0.25 mL, 2.8 mmol) and
H), 2.99 (s, 6H). ESI-MS (+ve ion) [M + H] m/z 252.155 (experi- acetic acid (0.20 mL, 3.5 mmol). The mixture was heated at
1
3
3
3
7
2
+
mental), 252.14 (calculated).
E)-1-(Pyridin-2-yl)-N-(4-styrylbenzyl)methanamine
reflux for 26 h then cooled to ambient temperature and the
(3). To solvent was removed by evaporation under reduced pressure.
(
anhydrous ethanol (80 mL) was added 2-picolylamine (1 mL, The crude product was eluted through a plug of silica using
9
4
.7 mmol) and trans-4-stilbene-carboxyaldehyde (0.98 g, ethyl acetate, the solvent removed and the residue dried
.7 mmol). The reaction mixture was heated at reflux under an in vacuo to afford the methyl ester of L as a yellow oil (0.32 g,
1
atmosphere of N . After 30 min sodium borohydride (0.71 g, 0.83 mmol, 62%). Calc. for C H N O : C, 77.69; H, 6.78; N,
2
25 26 2 2
1
1
3
9 mmol) was added in small portions and the reaction was 7.25. Found: C, 77.5; H, 6.7; N, 7.2. H NMR (500 MHz; CDCl ):
heated at reflux again. After 6 hours at reflux the reaction was δ 8.51 (ddd, JHH = 4.9, JHH = 1.8, JHH 0.9 Hz, 1H, PyH), 7.66
3
4
5
cooled to ambient temperature, the mixture was adjusted to (m, 1H, PyH), 7.52–7.49 (m, 3H, ArH), 7.47–7.45 (m, 2H, ArH),
3
pH 10 (1 M NaOH) then to pH 7 (1 M HCl). The reaction 7.37–7.32 (m, 4H, ArH), 7.25 (m, 1H, PyH), 7.15 (ddd, JHH
=
3
3
mixture was extracted with dichloromethane and the organic 7.4, JHH = 4.9, JHH = 1.2 Hz, 1H, PyH), 7.09 (s, 2H, CHvCH),
phase was washed (3 × 100 mL, H O). The organic phase was 3.77 (s, 2H, CH ), 3.65 (s, 2H, CH ), 3.63 (s, 3H, CH ), 2.88 (t,
2
2
2
3
3
3
dried (MgSO
4
), filtered, the solvent removed by evaporation
J
HH = 7.1 Hz, 2H, CH
2
–CH
2
), 2.54 (t, JHH = 7.1 Hz, 2H, CH
): δ 173.0, 159.9, 149.0, 138.6,
vacuum to yield a colourless wax (1.3 g, 4.4 mmol, 93%). Calc. 137.5, 136.5, 136.4, 129.3, 128.8, 128.6, 128.5, 127.7, 126.6,
2
–
1
13
2 3
under reduced pressure, and the residue was dried under CH ). { H} C (126 MHz; CDCl
for C21
H
20
N
2
·0.75H
2
O: C, 80.35; H, 6.90; N, 8.98. Found: C, 126.6, 123.0, 122.1, 60.0, 58.3, 51.6, 49.7, 32.8. ESI-MS (+ve ion)
1
+
8
0.4; H, 6.7; N, 8.9. H NMR (500 MHz; CDCl
3
): δ 8.62 (d, J = m/z [M + H] 387.211 (experimental), 387.21 (calculated for
+
4
.7, 1H), 7.68 (m, 1H, PyH), 7.57–7.52 (m, 4H, ArH), 7.41–7.35 C H N O ).
2
5
26 2 2
(
m, 5H, ArH, PyH), 7.30 (m, 1H, ArH), 7.21 (m, 1H, PyH), 7.15
(E)-Methyl 3-((4-(4-(dimethylamino)styryl)benzyl)(pyridine-
2
(s, 2H, CHvCH), 3.99 (s, 2H, CH
2
), 3.90 (s, 2H, CH
2
), 2.27 (s, 2-ylmethyl)amino)propanoate (methyl ester of L ). To ethanol
1
13
1
H, NH). { H} C NMR (126 MHz; CDCl ): δ 159.8, 149.4, (10 mL) was added 4 (0.40 g, 1.3 mmol), methyl acrylate
3
Dalton Trans.
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Paper
(
0.25 mL, 2.8 mmol) and acetic acid (0.20 mL, 3.5 mmol). The
(E)-N,N-Dimethyl-4-(4-(((2-(pyridin-2-yl)ethyl)(pyridin-2-
4
reaction mixture was shielded from light and stirred at ylmethyl)amino)methyl)styryl)aniline (L ). To ethanol (10 mL)
ambient temperature under nitrogen atmosphere. After 48 h, was added 4 (0.31 g, 0.90 mmol), 2-vinylpyridine (0.25 mL,
the solvent and unreacted methyl acrylate were removed 2.3 mmol) and acetic acid (0.25 mL, 4.4 mmol). The reaction
in vacuo to afford the methyl ester of L as a yellow oil. (0.32 g, mixture was shielded from light and stirred at ambient temp-
0
2
1
.74 mmol, 98%). H NMR (400 MHz; CDCl
3
): δ 8.50 (m, 1H, erature under nitrogen atmosphere for 11 days, the solvent
3
PyH), 7.65 (m, 1H, PyH), 7.49 (d,
.43–7.39 (m, 4H, ArH), 7.29 (m, AA′BB′, 2H, ArH), 7.14 (ddd, pressure to afford L as a yellow oil, (0.38 g, 0.74 mmol, 97%),
JHH = 7.9, 1H, PyH), and excess 2-vinylpyridine were removed under reduced
4
7
3
3
4
3
J
HH = 7.4, JHH = 4.9, JHH = 1.0, 1H, PyH), 7.02 (d, JHH = 16.3, which was purified by silica chromatography (CHCl
3
3
–CH OH).
3
1
H, CHvCH), 6.89 (d, JHH = 16.3, 1H, CHvCH), 6.71 (m, AA′ Calc. for C30
H N ·0.5H O: C, 78.74; H, 7.37; N, 12.24. Found:
32 4 2
1
BB′, 2H, ArH), 3.76 (s, 2H, CH ), 3.62 (s, 2H), 2.98 (s, 6H, C, 79.0; H, 7.3; N, 12.2. H NMR (500 MHz; CDCl ): δ 8.48 (m,
2
3
3
3
N(CH
CH ). ESI-MS (+ve ion) [M + H] m/z 430.259 (experimental), 2H, PyH), 7.25 (m, AA′BB′, 2H, ArH), 7.09 (m, 2H, PyH), 7.01 (d,
30.25 (calculated for C H N O ).
3 2 2
) ), 2.87 (t, JHH = 7.1, 2H, CH ), 2.53 (t, JHH = 7.1, 2H, 2H, PyH), 7.56 (m, 2H, PyH), 7.42–7.38 (m, 4H, ArH), 7.36 (m,
+
2
+
3
3
4
JHH = 16.3, 1H), 6.89 (d, J = 16.3, 1H), 6.70 (m, AA′BB′, 2H),
HH
2
7
32
3
2
(
E)-3-((4-(4-(Dimethylamino)styryl)benzyl)
(pyridine-2-yl- 3.82 (s, 2H, CH
2
), 3.70 (s, 2H, CH
2
), 2.98 (m, 10H, CH
). { H} C NMR (126 MHz; CDCl ): δ 160.8, 160.4,
nol and distilled water (1 : 1, 14 mL) was added the methyl 150.2, 149.3, 148.9, 138.0, 137.0, 136.4), 136.2, 129.2, 128.5,
2 2
–CH ,
2
1
13
methyl)amino)propanoic acid (HL ). To a mixture of metha- N(CH
3
)
2
3
2
ester of L (0.25 g, 0.58 mmol), CsCO
the reaction mixture was set stirring and heated at reflux for 60.2, 58.4, 54.2, 40.6, 36.2. ESI-MS (+ve ion) [M + H] m/z
3
(.40 g, 1.1 mmol) and 127.6, 126.0, 126.0, 124.4, 123.5, 122.9, 121.9, 121.2, 112.6,
+
1
h. The reaction mixture was then extracted into dichloro- 449.283 (experimental), 449.27 (Calculated).
I
1
methane by gradual addition of HCl to the aqueous layer. The
organic phase was dried (MgSO
evaporation under reduced pressure to afford HL as a light mixture was heated at reflux for 30 min then the mixture was
fac-[Re (CO)
3
L ]. To aqueous NaOH (1 M, 3 mL) was added
1
4
) and the solvent removed by the methyl ester of L (40 mg, 0.10 mmol). The reaction
2
yellow powder (0.16 g, 0.39 mmol, 67%). Calc. for allowed to cool to ambient and transferred to a microwave
I
C
6
26
H
29
N
3
O
2
·H
2
O: C, 72.03; H, 7.21; N, 9.69. Found: C, 72.0; H, reaction flask. Methanol (7 mL) and [Re (CO)
5
OTf] (51 mg,
1
.8; N, 9.7. H NMR (500 MHz; CDCl ): δ 8.59 (m, 1H, PyH), 0.11 mmol) were added and the pH adjusted to ∼pH 7 by
3
7
.67 (m, 1H, PyH), 7.43 (m, 4H, ArH), 7.26 (m, AA′BB′, 2H, addition of HCl (1 M). The flask was capped and irradiated at
3
ArH), 7.23 (m, 2H, PyH), 7.04 (d, JHH = 16.3, 1H, CH
2
vCH
2
), 150 °C (3 × 10 min) shaking between each heating cycle. The
3
6
.88 (d, J = 16.3, 1H, CH vCH ), 6.71 (m, AA′BB′, 2H, ArH), solvent volume was reduced by evaporation under a stream of
HH 2 2
3
.85 (s, 2H, CH
2
), 3.78 (s, 2H, CH
2
), 2.97 (m, 8H, CH
2
,
nitrogen until the point of turbidity. The flask was sealed and
). C NMR (126 MHz; stored at 4 °C for 2 days and colourless crystals were observed.
CDCl ): δ 173.6, 156.5, 150.3, 149.3, 138.2, 137.2, 133.9, 130.0, The solvent was decanted and the crystals washed with H O
3
13
3 2 2
N(CH ) ), 2.58 (t, JHH = 6.2, 2H, CH
3
2
I
1
1
4
29.4, 127.8, 126.4, 125.7, 123.8, 123.7, 122.9, 58.1, 57.8, 49.3, and dried in vacuo to afford fac-[Re (CO)
0.6, 31.1. ESI-MS (+ve ion) [M + H] m/z 416.234 (experi- crystalline powder (40 mg, 0.062 mmol, 57%). Calc. for
3
L ] as a colourless
+
+
mental), 416.23 (calculated for C H N O ).
C H N O Re: C, 50.54; H, 3.61; N, 4.37. Found: C, 50.52;
27 23 2 5
2
6
30
3
2
−
1
−1
(
E)-2-(Pyridin-2-yl)-N-(pyridin-2-ylmethyl)-N-(4-styrylbenzyl)- H, 3.67; N, 4.30. IR (thin film) ν(CO) 2017 cm , 1903 cm ,
3
−1 1
ethanamine (L ). To ethanol (40 mL) was added 2-vinylpyri- 1873 cm . H NMR (500 MHz; CDCl
dine (0.35 mL, 3.2 mmol), 3 (0.48 g, 1.6 mmol) and acetic acid 7.93 (m, 1H, PyH), 7.62 (m, AA′BB′, 2H, ArH), 7.54 (m, AA′BB′,
0.40 mL, 7.0 mmol). The reaction mixture was heated at reflux 2H, ArH), 7.47 (m, 1H, PyH), 7.39 (m, 3H, PyH, ArH), 7.32 (m,
3
): δ 8.92 (m, 1H, PyH),
(
3
3
for 36 h. The crude product was loaded onto a plug of silica 3H, ArH), 7.20 (d, JHH = 16.4 Hz, 1H, CHvCH), 7.13 (d, JHH
=
and washed with 10% ethyl acetate in dichloromethane, 10% 16.3 Hz, 1H, CHvCH), 4.88 (m, AB, 1H, CH ), 4.65 (m, AB, 1H,
2
MeOH in ethyl acetate then eluted using 15% MeOH in ethyl CH
2
), 4.56 (m, AB, 1H, CH
–CH ), 2.82 (m, 1H, CH
reduced pressure and the residue dried to afford L as a col- 2.23 (m, 1H, CH –CH ). { H} C NMR (126 MHz; CDCl ): δ
2
), 4.02 (m, AB, 1H, CH
2
), 2.92 (m,
acetate. The solvent was removed by evaporation under 1H,CH
2
2
2
–CH ), 2.41 (m, 1H, CH
2
2
–CH ),
2
3
1
13
2
2
3
ourless wax (within crude: 0.39 g, 0.96 mmol, 99%). Calc. for 197.2, 195.9, 194.1, 177.2, 158.7, 153.8, 139.8, 139.2, 136.8,
·0.5H O: C, 77.90; H, 5.88; N, 13.63. Found: C, 77.5; 132.5, 130.9, 129.5, 129.0, 128.4, 127.3, 127.1, 126.9, 126.2,
C
20
H
17
N
3
2
1
+
H, 5.8; N, 13.9. H NMR (500 MHz; CDCl ): δ 8.49 (m, 2H, PyH), 123.2, 66.9, 66.0, 49.8, 33.5. ESI-MS (+ve ion) [M + H] m/z
3
+
7
7
5
2
24 2 5
.57 (m, 2H, PyH), 7.50 (m, 2H, ArH), 7.42 (m, AA′BB′, 2H), 643.108 (experimental), 643.12 (calculated for C27H N O Re ).
.36 (m, 3H), 7.28 (m, AA′BB′, 2H), 7.27 (m, 1H, PyH), 7.10 (m, Crystals suitable for X-ray diffraction were grown by evapor-
H,PyH, CHvCH), 3.82 (s, 2H, CH ), 3.71 (s, 2H,CH ), 3.04 (m, ation of a solution of the complex dissolved in acetonitrile.
2
2
1
13
I
2
H, CH
2
–CH
2
), 2.95 (m, 2H, CH
2
–CH
2
). { H} C NMR
fac-[Re (CO)
3
L ]. To a microwave reaction flask equipped
I
(126 MHz; CDCl
3
): δ 160.5, 159.9, 149.1, 148.8, 138.7, 137.5, with
a
stirrer bar was added [Re (CO)
5
OTf] (41 mg,
1
1
36.6, 136.4, 129.3, 128.8, 128.8, 128.6, 128.5, 127.7, 126.6, 0.10 mmol), and methanol (2.5 mL). The reaction flask was
26.5, 123.6, 123.0, 122.1, 121.3, 59.9, 58.4, 54.1, 359. ESI-MS sealed and the mixture was heated to 150 °C using MW
+
2
(+ve ion) m/z [M + H] 406.228 (experimental), 406.23 (calcu- irradiation (2 × 15 min). L (42 mg, 0.10 mmol) was added
+
lated for C H N ).
to the reaction mixture and the MW vessel was sealed and
2
8
28 3
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Dalton Transactions
irradiated to 150 °C (15 min) then at 130 °C (10 min). A pre- Found: C, 47.4; H, 3.7; N, 6.4. IR ν(CO) 2020 cm 1, 1903 cm−1
−
,
cipitate was formed upon reducing the solvent volume under a 1882 cm− . H NMR (500 MHz; CDCl ): δ 9.04 (m, 1H, PyH),
1 1
3
stream of nitrogen. The precipitate was isolated by filtration to 8.91 (m, 1H, PyH), 7.96 (m, 1H, PyH), 7.87 (m, 1H, PyH), 7.65
I
2
3
afford fac-[Re (CO) L ] as a yellow powder (30 mg, 0.44 mmol, (m, 1H, PyH), 7.59 (m, AA′BB′, 2H, ArH), 7.52 (m, 2H, PyH),
−
1
−1
−1
3
4
C
4%). IR ν(CO) 2021 cm , 1906 cm , 1886 cm . Calc. for 7.43 (m, 5H, ArH, PyH), 7.12 (d, J = 16.2 Hz, 1H, CHvCH),
HH
3
29
H
28
N
3
O
5
Re·2.5H
2
O: C, 47.73; H, 4.56; N, 5.76. Found: 6.92 (d,
C, 47.8; H, 4.5; N, 5.7. H NMR (500 MHz; CDCl
J
HH = 16.3 Hz, 1H, CHvCH), 6.75 (m, AA′BB′, 2H,
1
3
): δ 8.93 (m, ArH), 4.84 (m, AB, 1H, CHH), 4.69 (m, AB, 1H, CHH), 4.55 (m,
1
H, PyH), 7.92 (m, 1H, PyH), 7.56 (m, AA′BB′, 2H, ArH), 7.43 AB, 1H, CHH), 4.38 (m, AB, 1H, CHH), 3.29 (m, 3H, CH –
2
13
3
1
(
m, 4H, ArH, PyH), 7.29 (m, AA′BB′, 2H, ArH), 7.13 (d, JHH
=
CHH), 3.01 (s, 6H, N(CH
HH = 16.3, 1H, CHvCH), NMR (126 MHz; CDCl ): δ 195.8, 193.7, 193.5, 162.4, 159.4,
.74–6.72 (m, AA′BB′,2H, PyH), 4.87 (m, AB, 1H, CH ), 4.63 (m, 154.9, 151.8, 150.4, 141.3, 140.7, 140.4, 132.6, 131.0, 128.1,
3 2 2
) ), 2.19 (m, 1H, CH –CHH). { H} C
3
1
6
6.3, 1H, CHvCH), 6.92 (d,
J
3
2
AB, 1H, CH
2
), 4.54 (m, AB, 1H, CHH), 4.01 (m, AB, 1H, CHH), 127.9, 127.5, 127.0, 126.8, 125.5, 125.2, 123.0, 112.7, 67.7, 66.9,
+
3
.01 (s, 6H, N(CH ) ), 2.93 (m, 1H, CHH–CH ), 2.82 (m, 1H, 51.3, 40.7, 34.8. ESI-MS (+ve ion) [M] m/z 719.199 (experi-
3 2 2
+
CH –CHH), 2.37 (m, 1H, CH –CHH), 2.22 (m, 1H, CHH–CH ). mental), 719.20 (calculated for C H N O Re ).
2
2
2
33 33 4 3
1
13
{
H} C NMR (126 MHz; CDCl
3
): δ 197.2, 195.9, 194.2, 177.3,
Staining of human brain tissue
1
1
3
6
58.8, 153.7, 150.7, 140.1, 139.7, 132.4, 131.0, 128.3, 128.0,
26.5, 126.1, 125.1, 123.2, 122.7, 112.5, 66.9, 66.0, 49.7, 40.5, The Health Sciences Human Ethics Sub-committee, The Uni-
3.5. ESI-MS(+ve ion) [M + H] m/z 686.164 (experimental), versity of Melbourne, approved all experiments using human
+
86.17 (calculated for C29
H
29
N
3
O
5
Re).
brain tissue (Ethics Approval No. 1341145). Brain tissue was
I
3
fac-[Re (CO) L ]CF SO . A microwave reaction flask was collected at autopsy. Brain tissue from the frontal cortex was
3
3
3
3
I
charged with L (50 mg, 0.11 mmol), [Re (CO)
5
OTf] (43 mg, preserved by formalin fixation and paraffin embedding. AD
1
.1 mmol) and methanol (3 mL). The flask was capped and pathology was confirmed according to standard National Insti-
the reaction mixture was irradiated at 150 °C (3 × 10 min), tute of Aging and Reagan Institute Working Group on Diagnos-
shaking between each heating cycle. To the mixture was added tic Criteria for the Neuropathological Assessment of
diethyl ether and colourless crystals were observed. The reac- Alzheimer’s Disease (1997) criteria. The brain tissue samples
tion mixture was stored at −18 °C overnight and the resultant of age-matched Human control (HC) were subject to the above
I
3
crystals were isolated by filtration to afford fac-[Re (CO)
CF SO as colourless crystals (63 mg, 0.074 mmol, 70%). Calc. carried out (xylene, 3 × 2 min) prior to rehydration (soaked for
for C H F N O ReSN ·H O: C, 45.60; H, 3.47; N, 4.99. Found: 2 min in 100%, 90%, 70%, then 0% ethanol–water v/v). The
3
L ]- criteria. Deparaffinization of AD and HC sections (5 μm) was
3
3
3
2
27
3
3
6
3
2
−
1
−1
C, 45.7; H, 3.3; N, 4.6. IR ν(CO) 2027 cm , 1901 cm
,
slides were then washed in PBS (5 min) and the autofluores-
): δ 9.03 (m, 1H, pyH), cence was quenched with potassium permanganate (0.25% in
.92 (m, 1H, PyH), 7.93 (m, 1H, PyH), 7.86 (m, 1H, PyH), 7.67 PBS, 20 min) before washing again with PBS (2 × 20 min).
1
1
1
8
869 cm− . H-NMR (500 MHz; CDCl
3
(
7
m, 3H, PyH, ArH), 7.52 (m, 4H, ArH, PyH), 7.45 (m, 2H, ArH), Samples were then treated with a solution of potassium meta-
3
.39 (m, 3H, ArH, PyH), 7.30 (m, 1H, PyH), 7.20 (d, JHH = 16.4 bisulfite and oxalic acid (1% each in PBS) until the tissue
3
Hz, 1H, CHvCH), 7.13 (d, J
= 16.3 Hz, 1H, CHvCH), 4.86 changed from brown in colour to colourless then washed in
HH
(
m, AB, 1H, CHH), 4.71 (m, AB, 1H, CHH), 4.57 (m, AB, 1H, PBS (3 × 2 min). The sections were blocked with bovine serum
CHH), 4.43 (m, AB, 1H, CHH), 3.31 (m, 3H, CH –CHH), 2.19 albumin (2% BSA in PBS, pH 7.4, 10 min) then treated covered
m, 1H, CH –CHH). { H} C NMR (126 MHz; CD OD): δ 196.8, with filtered compound (50 μM, 15% DMSO–PBS, 60 min). The
2
1
13
(
2
3
1
1
1
94.9, 194.5, 164.4, 161.5, 156.6, 153.4, 142.0, 141.8, 140.5, sections were treated with BSA to remove any non-specifically
38.4, 134.0, 131.5, 131.2, 129.8, 129.1, 128.5, 128.0, 127.9, bound complex and washed in PBS (3 × 2 min), then distilled
27.7, 127.6, 126.2, 125.7, 121.8 (q), 68.1, 67.9, 51.8, 35.6. water and finally mounted with non-fluorescent mounting
+
ESI-MS (+ve ion) m/z [M] 676.159 (experimental), 676.16 (cal- media (Dako). Images were obtained on a Leica (Bannockburn,
culated). Crystals suitable for X-ray diffraction were grown by IL) DM1RB microscope fitted with a Carl Zeiss AxioCam MR
exchange of vapours between n-pentane and a solution of the colour camera.
complex in dichloromethane.
I
4
Radiochemistry
General synthesis of [ Tc (CO) L ] complexes. The fac-
3 3 3
fac-[Re (CO) L ]CF SO . To a microwave reaction flask
I
99m
I
1–4
equipped with a stirrer bar was added [Re (CO) OTf] (40 mg,
5
3
4
99m
I
+
0
.080 mmol), L (40 mg, 0.090 mmol), and methanol (5 mL).
[
3 2
Tc (CO) (H O)] starting material was prepared as directed
The reaction vessel was sealed and the mixture was heated to by the manufacturer instructions supplied with the Isolink™
VII
−
1
30 °C using microwave irradiation (15 min) followed by kit (Covidien). In short, [Tc O ] (1000 MBq, in 0.9% saline
4
further irradiation to 140 °C (15 min). The solvent volume was solution (1 mL)) eluted from a Gentech® generator was added
reduced under a stream of nitrogen and diethyl ether (5 mL) to an Isolink™ kit (Mallinckrodt Medical B.V., The Nether-
was added, and the mixture was stored at −18 °C for 12 h. The lands) and was heated in a bath of boiling water. After 20 min,
precipitate that formed was collected by filtration to afford fac- the kit was removed from the bath, let cool to ambient temp-
I
4
[
2
Re (CO)
3
L ]CF
3 3
SO as a yellow powder (15 mg,0.017 mmol, erature and aliquots from the kit, containing fac-
9
9m
I
+
99m
1%). Calc. for C H N F O ReS: C, 47.05; H, 3.72; N, 6.46.
[
Tc (CO) (H O) ] , was used for subsequent
Tc labelling
3
4
32
4
3
6
3
2
3
Dalton Trans.
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Paper
of ligands. The radiolabeling of ligands was carried out by scanner (Mediso nanoScan SPECT/CT) was accomplished by
9
9m
I
+
addition of fac-[ Tc (CO) (H O) ] (100 μL) to a solution of 3 × 10 min static SPECT scans at approximately 2, 25 and
3
2
3
the chosen ligand in dimethyl sulfoxide or ethanol (100 μL, 50 min post injection with one CT scan (25 kVp, 0.45 s,
mg mL− ). The reaction mixture was then adjusted to pH 6 0.99 mA, helical scan with 720 projections). To confirm the
1
1
by addition of 1 M HCl and incubated at 70 °C for 20 min. presence of Aβ plaques, brain tissue samples were immunohis-
Measurement of Log D7.4 values. To a vial containing tochemically stained ex vivo, using 1E8 antibody.
1
-octanol (5 mL) and PBS (5 mL, 20 mM, pH 7.4) was added
the radiotracer (50 μL). The mixture was shaken by hand for
3
min and the fractions were allowed to seperate. The
1
-octanol layer used in the subsequent steps and each
Acknowledgements
measurement was carried out in triplicate. An aliquot of the
-octanol layer (900 μL) was added to PBS (900 μL, 20 mM, The Australian Research Council is acknowledged for financial
pH 7.4). The two phases were mixed by mechanical shaking support. Covidien Ltd. provided the ‘Isolink’ kits used in this
5 min) then separated by centrifuge (5 min at 13 200 rpm, research. Dr Nouria Salehi (Royal Melbourne Hospital,
1
(
Eppendorf 5415 D centrifuge). A 500 μL aliquot of the organic Melbourne, Australia) is acknowledged for assistance in analys-
phase and 500 μL from the aqueous phase was taken. The ing biodistribution data. The Victorian Brain Bank is acknowl-
radioactive decay of each aliquot was measured in counts per edged for the provision of the brain tissue used in this study.
minute (cpm) (Perkin-Elmer, Wizard 1470 automatic gamma
counter) enabling calculation of the partition coefficient (D)
and log D7.4
In vivo biodistribution of fac-[ Tc (CO)
.
9
9m
I
2
3
L ] in mice. All
Notes and references
methods conformed to the Australian National Health and
Medical Research Council published code of practice for
animal research and The Howard Florey Animal Ethics Com-
mittee approved experimentation. All mice were housed
according to standard animal care protocols and fed standard
laboratory chow and tap water ad libitum. Mice were kept on a
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2 : 12 hour light dark cycle and all testing was performed
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9
9m
I
2
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3
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9
9m
I
4 +
tion of fac-[ Tc (CO)
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9m
I
+
1
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4
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−
1
9
9m
I
+
[
Tc (CO)
3
(H
2
O)
3
]
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9m
I
4 +
7
1
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3
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