Synthesis of Oxorhenium(V) and Oxotechnetium(V) Complexes That Bind to Amyloid-β Plaques

Article abstract of DOI:10.1021/acs.inorgchem.6b00972

Alzheimer's disease is characterized by the presence of amyloid plaques in the brain. The primary constituents of the plaques are aggregated forms of the amyloid-β (Aβ) peptide. With the goal of preparing technetium-99^m complexes that bind to Aβ plaques with the potential to be diagnostic imaging agents for Alzheimer's disease, new tetradentate ligands capable of forming neutral and lipophilic complexes with oxotechentium(V) and oxorhenium(V) were prepared. Nonradioactive isotopes of technetium are not available so rhenium was used as a surrogate for exploratory chemistry. Two planar tetradentate N₃O ligands were prepared that form charge-neutral complexes with oxorhenium(v) as well as a ligand featuring a styrylpyridyl functional group designed to bind to Aβ plaques. All three ligands formed complexes with oxorhenium(V), and each complex was characterized by NMR spectroscopy, mass spectrometry, and X-ray crystallography. The oxorhenium(V) complex with a styrylpyridyl functional group binds to Aβ plaques present in post-mortem human brain tissue. The chemistry was extrapolated to technetium-99^m at the tracer level for two of the ligands. The resulting oxotechnetium(V) complexes were sufficiently lipophilic and charge-neutral to suggest that they have the potential to cross the blood-brain barrier but exhibited modest stability with respect to exchange with histidine. The chemistry presented here identifies a strategy to integrate styrylpyridyl functional groups into tetradentate ligands capable of forming complexes with [M=O]³⁺ cores (M = Re or Tc).

Full text of DOI:10.1021/acs.inorgchem.6b00972

Article
pubs.acs.org/IC
Synthesis of Oxorhenium(V) and Oxotechnetium(V) Complexes That
Bind to Amyloid‑β Plaques
David J. Hayne,†,‡ Jonathan M. White,†,‡ Catriona A. McLean,§ Victor L. Villemagne,⊥,∥
Kevin J. Barnham,‡,⊥ and Paul S. Donnelly*,†,‡
‡
⊥
†School of Chemistry, Bio21 Institute, and Florey Institute of Neuroscience and Mental Health, University of Melbourne,
Melbourne, Victoria 3010, Australia
∥Department of Molecular Imaging & Therapy, Centre for PET, Austin Health, 145 Studley Road, Heidelberg, Victoria 3084,
Australia
§The Alfred Hospital, Melbourne, Victoria 3181, Australia
S
* Supporting Information
ABSTRACT: Alzheimer’s disease is characterized by the
presence of amyloid plaques in the brain. The primary
constituents of the plaques are aggregated forms of the
amyloid-β (Aβ) peptide. With the goal of preparing
technetium-99m complexes that bind to Aβ plaques with the
potential to be diagnostic imaging agents for Alzheimer’s
disease, new tetradentate ligands capable of forming neutral
and lipophilic complexes with oxotechentium(V) and oxorhenium(V) were prepared. Nonradioactive isotopes of technetium are
not available so rhenium was used as a surrogate for exploratory chemistry. Two planar tetradentate N3O ligands were prepared
that form charge-neutral complexes with oxorhenium(v) as well as a ligand featuring a styrylpyridyl functional group designed to
bind to Aβ plaques. All three ligands formed complexes with oxorhenium(V), and each complex was characterized by NMR
spectroscopy, mass spectrometry, and X-ray crystallography. The oxorhenium(V) complex with a styrylpyridyl functional group
binds to Aβ plaques present in post-mortem human brain tissue. The chemistry was extrapolated to technetium-99m at the tracer
level for two of the ligands. The resulting oxotechnetium(V) complexes were sufficiently lipophilic and charge-neutral to suggest
that they have the potential to cross the blood−brain barrier but exhibited modest stability with respect to exchange with
histidine. The chemistry presented here identifies a strategy to integrate styrylpyridyl functional groups into tetradentate ligands
capable of forming complexes with [MO]3+ cores (M = Re or Tc).
the role of Aβ plaques to the disease while assisting in diagnosis
and monitoring of emerging therapies. One of the first 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 compound
B; Figure 1).2,3 The interaction between benzothiazole
derivatives such as 11C-PIB and Aβ plaques and fibrils is
thought to be due to a combination of hydrophobic and
INTRODUCTION
■
Alzheimer’s disease (AD) is the most common form of
neurodegenerative dementia. The disease is characterized by
the presence of extracellular amyloid plaques and intracellular
neurofibrillary tangles (NFTs) in the brain. The major
constituents of the extracellular amyloid plaques are aggregated
forms of a peptide called amyloid-β (Aβ), a 39−43 amino acid
peptide derived from the amyloid precursor protein. These
amyloid plaques do not consistently correlate with cognitive
impairment, and some argue that smaller soluble oligomeric
species are the toxic form responsible for neuronal death.
However, oligomers and plaques are thought to be in
equilibrium. The NFTs include aggregated forms of a
hyperphosphorylated form of a microtubule-associated protein
called tau. The hyperphosphorylation of tau results in its
detachment from microtubules that consequently lose struc-
tural integrity with concomitant impaired axonal transport and
compromised synaptic function.1
Figure 1. Chemical structures of the amyloid imaging agents 11C-PIB
and Florbetapir.
In the past decade, diagnostic imaging of Aβ plaque burden
in patients has become possible using positron emission
tomography (PET) and radiolabeled tracers that bind to Aβ
plaques. These new imaging agents are providing insight into
Received: April 19, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.6b00972
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Article
MHz and 13C{1H} NMR at 101 MHz) or Varian FT-NMR 500 (1H
NMR at 500 MHz and 13C{1H} NMR at 126 MHz) spectrometer at
298 K. All chemical shifts were referenced to the internal solvent
residue and quoted in ppm relative to tetramethylsilane. Chemical &
MicroAnalytical Services Pty. Ltd., Victoria, Australia, carried out
elemental analyses for C, H, and N. Electrospray ionization mass
spectrometry (ESI-MS) spectra were recorded on an Agilent 6220
ESI-time-of-flight (TOF) liquid chromatography (LC)/MS instru-
ment. Analytical radio-labeled high-performance liquid chromatog-
raphy (HPLC) was performed using a Shimadzu 10AVP UV/visible
detector (Shimadzu, Kyoto, Japan), two LC-10ATVP solvent delivery
systems [for solvents A (0.1% trifluoroacetic acid in Milli-Q water) and
B (0.1% trifluoroacetic acid in acetonitrile)], and a Nacalai Tesque
Cosmosil 5C18-AR Waters column (4.6 mm i.d. × 150 mm; Nacalai
Tesque, Kyoto, Japan). The mobile phase used was a gradient
consisting of 5% solvent B at t = 0 to 100% solvent B after 20 min. All
runs were conducted at a constant total flow rate of 1 mL min−1, and
the absorbance was monitored at λ = 254 nm. The rhenium analogues
of 99mTc complexes were used to confirm the synthesis of complexes
by a comparison of the retention times upon analysis by reverse-phase
HPLC (RP-HPLC). Absorbance spectra were obtained on a Shimadzu
UV-1650PC UV/visible spectrophotometer, and emission spectra
were obtained on a Varian CARY Eclipse fluorescence spectrometer,
with both performed using capped quartz cuvettes. IR spectra were
recorded using a PerkinElmer Spectrum One Fourier transform
infrared spectrometer, with a zinc selenide/diamond universal ATR 60
sampling accessory. For the acquisition of high-resolution mass
spectrometry (HRMS) data, compounds in methanol at concen-
trations of 10−50 μM were directly infused at a sample flow rate of 5
μL min−1 into a Finnigan ESI source of a LTQ FT hybrid linear ion
trap mass spectrometer (Thermo, Bremen, Germany).
hydrogen-bonding interactions. Stilbene and styrylpyridine
derivatives are structurally related to benzothiazoles and also
exhibit selective binding to Aβ plaques. A group of stilbene and
styrylpyridine derivatives have selectivity for amyloid plaques
over other β-sheet aggregates such as NFTs and Lewy bodies
and therefore have the potential to be of use in the differential
diagnosis of AD from other conditions.4 Efforts focusing on the
preparation of stilbene or styrylpyridine derivatives radiolabeled
with positron-emitting fluorine-18 have culminated with the
recent FDA approval of 18F-AV45 (Florbetapir; Figure 1) to
detect the presence of amyloid.5−8
Despite the increase in the use of PET in clinical diagnosis,
single-photon-emission computed tomography (SPECT) re-
mains the most commonly used imaging modality and is still
considerably cheaper than PET. The most commonly used
radionuclide for SPECT imaging is techentium-99m 99mTc),
(
and it is estimated that this isotope is used in >85% of
diagnostic nuclear medicine scans.9 The nuclear properties of
99mTc are ideally suited for diagnostic procedures. The half-life,
6 h, is sufficiently long to allow for the preparation of pure
technetium complexes and for the accumulation in the target
tissue. The 99mTc isotope emits γ-rays (140 keV) that are close
to optimal for detection with commercial γ cameras. More
hospitals possess the requisite equipment for SPECT imaging,
the 99mTc isotope is readily available from generators, and
SPECT imaging is more economical than PET imaging.
A
99mTc-based Aβ plaque imaging agent could make the use
of diagnostic imaging for differential diagnosis and identi-
fication of individuals suitable for emerging therapies more
feasible.10−12 A major challenge in developing brain imaging
agents is making tracers that are capable of crossing the blood−
brain barrier. Detailed reviews discussing recent developments
in the pursuit of suitable 99mTc-based amyloid imaging probes
are available.13−15 Previous work in this area focused on making
tridentate ligands with a pendent stilbene functional group
capable of forming complexes with [Tc(CO)3]+. The [Re-
(CO)3]+ analogues formed with these ligands bound to Aβ
plaques in post-mortem human tissue, but the brain uptake of
the [Tc(CO)3]+ complexes was relatively low.16 Others have
also reported low brain uptake for several complexes designed
to bind to amyloid plaques featuring the organometallic
[Tc(CO)3]+ core.17−19 In this paper, we describe the synthesis
of tetradentate trianionic ligands designed to form charge-
neutral complexes with [TcO]3+ cores and prepare a
derivative featuring a styrylpyridyl functional group designed
to bind to Aβ plaques. A similar integrated approach of
incorporating the Aβ plaque binding functional group into
tetradentate ligands designed to bind to positron-emitting
isotopes of copper led to complexes that displayed good
interactions with Aβ plaques and crossed the blood−brain
barrier.20 A similar integrated approach with [MO]3+ (M = Tc
or Re) complexes incorporating 2-arylbenzothiazole plaque
targeting functional groups into tetradentate aminothiol ligands
resulted in complexes with reasonable affinities for Aβ1−40
fibrils.21 There are no stable isotopes of technetium available
so the group VII congener rhenium was used in exploratory
synthesis and characterization.
Synthesis of N-(2-Aminoethyl)picolinamide (1). This synthesis was
modified from a literature procedure.23 To 1,2-diaminoethane (33
mL) was added 2-ethyl picolinate (11.0 g, 73 mmol), and the reaction
mixture was heated at reflux for 3 h. The mixture was allowed to cool
to ambient temperature, and excess 1,2-diaminoethane was removed in
vacuo. To the residue was added water (75 mL), and the mixture was
adjusted to pH 5 (acetic acid). The mixture was extracted with
chloroform (3 × 100 mL), and then the aqueous phase was adjusted to
pH 13 by the addition of solid potassium carbonate and extracted with
chloroform (3 × 100 mL). The organic phase from the liquid
extraction at pH ∼13 was dried (K2CO3) and the solvent removed by
evaporation under reduced pressure to afford 1 as a light-yellow oil
(7.27 g, 44 mmol, 60%). 1H NMR (400 MHz, CDCl3): δ 8.49 (m, 1H,
PyH), 8.30 (s, 1H, OCNH), 8.13 (m, 1H, PyH), 7.78 (m, 1H, PyH),
3
7.36 (m, 1H), 3.48 (m, 2H, OCNCH2), 2.89 (t, JHH = 6.0 Hz, 2H,
CH2NH2), 1.19 (s, 2H). 13C{1H} NMR (101 MHz, DMSO-d6): δ
163.9, 150.0, 148.3, 137.7, 126.4, 121.8, 41.1, 40.6. ESI-MS (positive
ion). Calcd for C8H12N3O+ ([M + H]+): m/z 166.20. Found: m/z
166.10.
Synthesis of N-[2-[(2-Hydroxybenzyl)amino]ethyl]picolinamide
(H3L1). To ethanol (anhydrous, 130 mL) was added 1 (2.83 g, 17
mmol) and salicylaldehyde (2.10 g, 17 mmol). The mixture was stirred
for 30 min and then concentrated by evaporation under reduced
pressure, resulting in the formation of a yellow crystalline solid, which
was collected by filtration. This yellow crystalline solid (1.12 g, 4.2
mmol) was added to anhydrous ethanol (50 mL), the mixture was
sparged with N2, and then NaBH4 (1.57 g, 42 mmol) was added
portionwise. After 16 h, sodium hydroxide (1 M) was added to adjust
the mixture to pH ∼10 and then to pH ∼7−8 using hydrochloric acid
(HCl; 1 M). The mixture was extracted with ethyl acetate (100 mL)
and washed with water (3 × 50 mL), the organic phase was dried
(MgSO4), and the solvent was removed by evaporation under reduced
pressure to afford H3L1 as a colorless crystalline solid (1.12 g, 4.12
1
mmol, 50%). H NMR (500 MHz, CDCl3): δ 8.56−8.54 (m, 1H,
EXPERIMENTAL SECTION
■
PyH), 8.24 (s, 1H, NHCO), 8.18 (m, 1H, PyH), 7.85 (m, 1H,
PyH), 7.43 (ddd, 3JHH = 7.6 and 4.8 Hz, 4JHH = 1.2 Hz, 1H, PyH), 7.16
(m, 1H, ArH), 6.99 (m, 1H, ArH), 6.81 (dd, 3JHH = 8.1 Hz, 4JHH = 1.1
Hz, 1H, ArH), 6.77 (m, 1H, ArH), 4.04 (s, 2H, −NHCH2Ar), 3.66 (m,
General Procedures. All reagents and solvents were obtained
from commercial sources and used as received unless otherwise stated.
[ReOCl3(PPh3)2] was prepared according to literature procedures.22
NMR spectra were acquired on an Agilent 400-MR (1H NMR at 400
3
2H, CH2NHCO), 2.95 (t, JHH = 6.0 Hz, 2H, CH2CH2). 13C{1H}
B
DOI: 10.1021/acs.inorgchem.6b00972
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Article
1
NMR (126 MHz, CDCl3): δ 165.1, 158.2, 149.7, 148.3, 137.6, 128.9,
[ReOL2] as a red powder (116 mg, 0.24 mmol, 76%). H NMR (500
3
4
5
128.6, 126.5, 122.4, 122.4, 119.2, 116.6, 52.5, 48.2, 39.1. HRMS (ESI-
MHz, CDCl3): δ 9.31 (ddd, JHH = 5.4 Hz, JHH = 1.5 Hz, JHH = 0.7
Hz, 1H, PyH), 8.45 (m, 1H, PyH), 8.39 (dd, 3JHH = 7.9 Hz, 4JHH = 1.8
Hz, 1H, ArH), 8.31 (ddd, 3JHH = 7.7 Hz, 4JHH = 1.4 Hz, 5JHH = 0.7 Hz,
1H, PyH), 8.10 (ddd, 3JHH = 7.7 and 5.4 Hz, 4JHH = 1.4 Hz, 1H, PyH),
7.40 (ddd, JHH = 8.2 and 7.1 Hz, JHH = 1.8 Hz, 1H, ArH), 7.06 (m,
2H, ArH), 5.57 (m, 1H, CHH), 4.14−4.10 (m, 2H, CH2), 3.98 (m,
1H, CHH). 13C{1H} NMR (126 MHz, CDCl3): δ 177.5, 170.2, 169.4,
155.0, 150.1, 145.8, 132.8, 131.8, 128.7, 124.9, 122.7, 121.7, 119.9,
61.2, 55.3. ESI-MS (positive ion). Calcd for [C15H13N3O4Re]+ ([M +
H]+): m/z 486.05. Found: m/z 486.050. IR (ν in cm−1): 983 (Re
O). Crystals suitable for X-ray diffraction were grown from a mixture
of the complex dissolved in DMF layered with diethyl ether.
+
LTQ-FT). Calcd for C15H18N3O2 ([M + H]+): m/z 272.1394.
Found: m/z 272.1391.
Synthesis of [ReOL1]. To a flask charged with [ReOCl3(PPh3)2]
(250 mg, 0.30 mmol), H3L1 (84 mg, 0.31 mmol), and sodium acetate
(302 mg, 3.7 mmol) was added ethanol (anhydrous, deoxygenated, 8
mL), and the mixture was heated at reflux for 3 h. The now dark-green
mixture was allowed to cool to ambient temperature, and a black/dark-
green crystalline precipitate formed. The precipitate was collected by
filtration and washed with a small amount of water and then ethanol to
3
4
1
afford [ReOL1] as dark-green crystals (116 mg, 0.24 mmol, 76%). H
NMR (500 MHz, DMSO-d6): δ 9.29−9.28 (m, 1H, PyH), 8.41 (m,
1H, PyH), 8.14−8.12 (m, 1H, PyH), 8.05 (ddd, 3JHH = 7.7 and 5.2 Hz,
Synthesis of (E)-4-[2-(6-bromopyridin-3-yl)vinyl]-N,N-dimethyla-
niline (2). To anhydrous ethanol (200 mL) was added 6-
bromonicotinaldehyde (5.13 g, 28 mmol), and the mixture was
sparged with N2 for 30 min before NaBH4 (1.23 g, 32 mmol) was
added in small portions and then stirred at ambient temperature.
When the reaction was complete by thin-layer chromatography
analysis (SiO2, 20% ethyl acetate in PET spirits), the mixture was
adjusted to pH 2 (1 M HCl) and then to pH 10 (saturated NaHCO3).
The mixture was concentrated by evaporation under reduced pressure
and extracted with ethyl acetate (200 mL). The organic phase was
washed with brine (100 mL) and dried (MgSO4), and the solvent was
removed under reduced pressure to afford a brown oil, which was used
without further purification To the oil was added thionyl chloride (30
mL, 414 mmol) dropwise at −60 °C. After 1 h, the reaction mixture
was allowed to warm to ambient temperature, and excess thionyl
chloride was removed in vacuo. To the residue was added a saturated
aqueous solution of NaHCO3 (50 mL), and the mixture was extracted
with ethyl acetate (3 × 50 mL). The organic phase was dried
(Na2SO4) and the solvent removed by evaporation under reduced
pressure. The residue was purified by flash chromatography (SiO2,
33% ethyl acetate in PET spirits) and the solvent removed by
evaporation under reduced pressure to afford colorless crystals of 2-
bromo-5-(chloromethyl)pyridine (2.98 g, 14 mmol, 73%). To 2-
bromo-5-(chloromethyl)pyridine (1.71 g, 8.3 mmol) was added
triethyl phosphite (15 mL, 87 mmol). The reaction mixture was
heated at reflux for 4 h, then the mixture was allowed to cool to
ambient temperature, and excess triethyl phosphite was removed in
vacuo. To the resultant oil was added anhydrous tetrahydrofuran (50
mL), 4-(dimethylamino)benzaldehyde (1.13 g, 7.57 mmol), and
sodium hydride (60% w/w in mineral oil, 0.95 g, 24 mmol). After 12 h,
a saturated aqueous solution of NaHCO3 (50 mL) was added and the
mixture was extracted with ethyl acetate (100 mL). The organic phase
was washed with a saturated aqueous solution of NaHCO3 (3 × 50
mL) and dried (Na2SO4), and the solvent was removed to afford 2 as a
yellow powder (1.68 g, 5.5 mmol, 49%). 1H NMR (400 MHz,
CDCl3): δ 8.40 (m, 1H, PyH), 7.64 (m, 1H, PyH), 7.40 (m, 3H, PyH
and ArH), 7.07 (m, AB, 3JHH = 16.3 Hz, 1H, CHCH), 6.78 (m, AB,
3JHH = 16.3 Hz, 1H, CHCH), 6.70 (m, 2H, ArH), 2.99 (s, 6H,
3
4JHH = 1.4 Hz, 1H, PyH), 7.17−7.14 (m, 1H, ArH), 7.09 (dd, JHH
=
4
3
4
7.4 Hz, JHH = 1.5 Hz, 1H, ArH), 6.95 (dd, JHH = 8.0 Hz, JHH = 1.1
Hz, 1H, ArH), 6.65 (m, 1H, ArH), 4.89 (m, AB, 1H, CHH), 4.51−
4.47 (m, 1H, CH2CHH), 4.37 (m, 1H, CHHCH2), 4.08 (m, AB, 1H,
CHH), 3.78 (m, 1H, CH2CHH), 3.44 (m, 1H, CHHCH2). 13C{1H}
NMR (126 MHz, DMSO-d6): δ 175.5, 175.1, 171.9, 157.8, 156.2,
141.7, 137.9, 136.9, 128.8, 125.1, 121.7, 120.8, 115.1, 70.1, 54.2. ESI-
MS (positive ion). Calcd for [C15H15N3O3Re]+ ([M + H]+): m/z
472.07. Found: m/z 472.065. IR (ν in cm−1): 950 (ReO). Crystals
suitable for X-ray diffraction were grown from a mixture of the
complex dissolved in dichloromethane and layered with n-pentane.
Synthesis of N-Hydroxysuccinimidyl Ester of Salicyclic Acid. This
synthesis was modified from a literature procedure.24 To a mixture of
salicylic acid (1.06 g, 7.6 mmol) and N-hydroxysuccinimide (1.77 g, 15
mmol) in tetrahydrofuran (50 mL) was added dicyclohexylcarbodii-
mide (3.07, 15 mmol). The reaction mixture was stirred at ambient
temperature for 12 h, and a colorless precipitate was observed and
removed by filtration. The solvent was removed from the filtrate by
evaporation under reduced pressure, the residue was dissolved in ethyl
acetate (100 mL) and washed with aqueous citric acid (5% w/w, 2 ×
50 mL), and the organic phase was filtered to remove a colorless
precipitate. The organic phase was washed with a saturated aqueous
solution of NaHCO3 (2 × 50 mL), citric acid (5% w/w in water, 2 ×
50 mL), and brine (50 mL) and dried (MgSO4), and the solvent
removed by evaporation under reduced pressure to afford N-
hydroxysuccinimidyl ester of salicyclic acid as a colorless powder
(1.62 g, 6.9 mmol, 91%). 1H NMR (400 MHz, CDCl3): δ 9.50 (s, 1H,
OH), 7.99 (m, 1H, ArH), 7.57 (m, 1H, ArH), 7.03 (m, 1H, ArH), 6.97
(m, 1H, ArH), 2.91 (s, 4H, CH2CH2).
Synthesis of N-[2-(2-Hydroxybenzamido)ethyl]picolinamide
(H3L2). To N,N-dimethylformamide (DMF; anhydrous, 15 mL) was
added the N-hydroxysuccinimidyl ester of salicylic acid (0.95 g, 4.0
mmol), 1 (0.72 g, 4.4 mmol), and triethylamine (3 mL). After 48 h,
the solvent was removed in vacuo. The residue was dissolved in
dichloromethane and washed with water, and the organic phase was
dried (MgSO4) and concentrated by evaporation under reduced
pressure to afford a colorless crystalline solid, which was collected by
filtration and washed with cold methanol and then diethyl ether to
N(CH3)2). 13C{1H} NMR (101 MHz, CDCl3): δ 150.7, 148.3, 139.3,
134.6, 133.5, 131.9, 128.07, 127.93, 124.6, 118.8, 112.4, 40.5. ESI-MS
(positive ion). Calcd for [C15H16BrN2]+ ([M + H]+): m/z 305.05.
Found: m/z 305.047.
1
afford H3L2 as a colorless powder (0.34 g, 1.2 mmol, 30%). H NMR
(500 MHz, CDCl3): δ 12.49 (s, 1H, OH), 8.57−8.53 (m, 2H, NH and
PyH), 8.22 (m, 1H, PyH), 8.06 (s, 1H, NH), 7.87 (m, 1H, PyH), 7.55
(dd, 3JHH = 8.0 Hz, 4JHH = 1.5 Hz, 1H, ArH), 7.46 (ddd, 3JHH = 7.6 Hz,
4JHH = 4.8 Hz, 5JHH = 1.2 Hz, 1H, PyH), 7.36 (ddd, 3JHH = 8.4 Hz, 4JHH
= 7.1 Hz, 5JHH = 1.4 Hz, 1H, ArH), 6.94 (dd, 3JHH = 8.3 Hz, 4JHH = 1.1
Synthesis of Lithium (E)-5-[4-(Dimethylamino)styryl]picolinate
(3). To a flask charged with 2-bromo-5-[4-(dimethylamino)styryl]-
pyridine (1.17 g, 3.8 mmol) was added dry, deoxygenated diethyl ether
(40 mL). The mixture was cooled to −40 °C, and n-butyllithium (2.2
M in n-hexanes, 1.9 mL, 4.2 mmol) was added dropwise. After 1.5 h,
an excess of solid carbon dioxide was added and the mixture was
allowed to warm to ambient temperature. After 45 min, water (40 mL)
was added and the mixture was partitioned with ethyl acetate (100
mL). A brown precipitate remained in the aqueous layer upon
separation of the two phases. After centrifugation of the aqueous layer,
the supernatant was decanted from the brown precipitate, which was
then lyophilized to afford 3 as an orange powder (0.78 g, 2.9 mmol,
3
4
Hz, 1H, ArH), 6.87 (ddd, JHH = 8.1 and 7.1 Hz, JHH = 1.1 Hz, 1H,
ArH), 3.78 (m, 2H, CH2CH2), 3.69 (m, 2H, CH2CH2). 13C{1H}
NMR (126 MHz, CDCl3): δ 170.7, 167.0, 161.8, 149.2, 148.4, 137.7,
134.1, 126.8, 126.2, 122.5, 118.9, 118.4, 114.3, 42.5, 39.2. HRMS (ESI-
+
LTQ-FT). Calcd for C15H16N3O3 ([M + H]+): m/z 286.1186.
Found: m/z 286.1184.
Synthesis of [ReOL2]. To a flask charged with [ReOCl3(PPh3)2]
(261 mg, 0.31 mmol), H3L2 (89 mg, 0.31 mmol), and sodium acetate
(291 mg, 3.6 mmol) was added anhydrous, deoxygenated ethanol (8
mL), and the mixture was heated at reflux for 3 h. The mixture was
allowed to cool to ambient temperature, and a dark-red solid was
collected by filtration and washed with water and methanol to afford
1
74%). H NMR (500 MHz, DMSO-d6): δ 8.54 (m, 1H, PyH), 8.05
(m, 1H, PyH), 7.93 (m, 1H, PyH), 7.46 (m, AA′BB′, 2H, ArH), 7.30
(m, AB, 3JHH = 16.5 Hz, 1H, CHCH), 7.04 (m, AB, 3JHH = 16.5 Hz,
C
DOI: 10.1021/acs.inorgchem.6b00972
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1H, CHCH), 6.72 (m, AA′BB′, 2H, ArH), 3.34 (s, 6H, N(CH3)2).
13C{1H} NMR (101 MHz, DMSO-d6): δ 167.5, 154.4, 150.3, 145.8,
134.2, 132.8, 131.3, 1279, 124.4, 123.4, 119.3, 112.1, 39.9. ESI-MS
(positive ion). Calcd for [C16H17N2O2]+ ([M + H]+): m/z 269.13.
Found: m/z 269.128.
∼1000 MBq). A solution of stannous chloride in ethanol (1 mg mL−1,
20 μL) was added, and the mixture was left at ambient temperature for
45 min. The mixture was analyzed by RP-HPLC to confirm the
synthesis of the desired complex.
Estimation of log D. To a vial containing 1-octanol (5 mL) and
phosphate-buffered saline (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 separate. The 1-octanol layer was used in
the subsequent steps and each measurement was carried out in
triplicate. An aliquot of the 1-octanol layer (900 μL) was added to PBS
(900 μL, 20 mM, pH 7.4). The two phases were mixed by mechanical
shaking (5 min) and then separated by a centrifuge (5 min at 13200
rpm, Eppendorf 5415 D centrifuge). A 500 μL aliquot of the organic
phase and 500 μL from the aqueous phase were analyzed for
radioactivity in counts per minute (PerkinElmer, Wizard 1470
automatic gamma counter), enabling calculation of the partion
coefficient (D).
Synthesis of N-(2-Aminoethyl)-2-hydroxybenzamide Hydrochlor-
ide (4). To 1,2-diaminoethane (16 mL) was added methyl salicylate
(3.18 g, 21 mmol), and the mixture was heated at reflux for 3 h. The
mixture was allowed to cool to ambient temperature, and excess 1,2-
ethanediamine was removed in vacuo. To the residue was added 0.01
M HCl (50 mL), and the mixture was adsorbed to a column of Dowex
50Wx2 resin (H+ form). The column was washed with water (200
mL) and eluted with 1.5 M HCl (300 mL). The acidic eluent was
evaporated to dryness under reduced pressure to afford 4 as a colorless
1
powder (3.88 g, 19 mmol, 90%). H NMR (400 MHz, D2O): δ 7.69
(m, 1H, ArH), 7.44 (m, 1H, ArH), 6.97 (m, 2H, ArH), 3.67 (t, 3JHH
=
5.8 Hz, 2H, CH2), 3.21 (t, 3JHH = 5.8 Hz, 2H, CH2). ESI-MS (positive
ion). Calcd for [C9H13N2O2]+ ([M + H]+): m/z 181.10. Found: m/z
181.123.
X-ray Crystallography. Crystals were mounted in low-temper-
ature oil and then flash-cooled. The intensity data were collected at
130 K on an X-ray diffractometer with a CCD detector using either Cu
Kα (λ = 1.54184 Å) or Mo Kα (λ = 0.71073 Å) radiation. Data were
reduced and corrected for absorption. The structures were solved by
direct methods and difference Fourier synthesis using the SHELX25
suite of programs, as implemented within the WINGX software.26
Thermal ellipsoid plots were generated using ORTEP-3 software.26
Binding of [ReOL3] to Aβ1−42 Fibrils. Aβ1−42 was prepared
according to literature procedures.27 A 1 mg mL−1 stock solution of
Aβ1−42 peptide was made by adding aqueous sodium hydroxide (24
μL, 60 mM) to Aβ1−42 (120 μg), and the mixture was sonicated in an
ice water bath for 5 min. The sample was mixed after the addition of
water (Milli-Q, 84 μL) and 10× PBS (12 μL) and placed in a
centrifuge for 5 min (14000 g at 4 °C), and the supernatant was
retained and kept on ice. The Aβ concentration A214 was determined
for a 1:100 dilution of the supernatant (Milli-Q water; ε214 nm = 94586
cm−1 M−1). The supernatant was incubated for 3 days at 37 °C with
stirring to allow fibril formation. The binding affinity (Ki) of [ReOL3]
for Aβ1−42 fibrils was estimated using fluorescence competition
assay.28,29 The samples for the competition assay were made up in a
solvent mixture of 15% DMSO in PBS with the concentrations of
Aβ1−42 and Thioflavin T fixed at 2 and 1 μM, respectively. The
concentration of the complex [ReOL3] in each sample was varied over
a logarithmic scale, from 0.0050 to 50 μM, and repeated in triplicate.
The samples were left at ambient temperature for 2 h before the
fluorescence of each sample was recorded. The data were analyzed
using GraphPad Prism software with the IC50 value calculated using a
one-site competitive binding linear regression. The Ki value was then
calculated using the Cheng−Prousoff equation Ki = IC50/(1 + [L]/
Kd), where [L] and Kd refer to the concentration and dissociation
constant of Thioflavin T (1000 nM and 750 nm, respectively).28,29
Analysis of the Ability of Complexes To Bind to Aβ Plaques
in Human Brain Tissue. The Health Sciences Human Ethics
Subcommittee, The University of Melbourne, approved all experi-
ments using human brain tissue. Brain tissue was collected at autopsy.
Brain tissue from the frontal cortex was sourced by the Victoria Brain
Bank Network and preserved by paraffin embedding. Prof. Catriona
McLean provided diagnosis of AD pathology according to standard
National Institute of Aging and Reagan Institute Working Group on
Diagnostic Criteria for the Neuropathological Assessment of
Alzheimer’s Disease (1997) criteria. Brain tissue (5 μm serial sections)
was “deparaffinized” (xylene, 3 × 2 min) prior to rehydration [soaked
for 2 min in 100%, 90%, 70%, and 0% (v/v) ethanol/water]. The slides
were then washed in PBS (5 min), and the autofluorescence was
quenched with potassium permanganate (0.25% in PBS, 20 min)
before washing again with PBS (2 × 20 min). Samples were then
treated with a solution of potassium metabisulfite and oxalic acid (1%
each in PBS) until the tissue changed from brown to colorless and
then washed with PBS (3 × 2 min). The sections were treated with
bovine serum albumin (2% BSA in PBS, pH 7.4, 10 min) and then
treated with a mixture of [ReOL3] (10 μM, 15% DMSO/PBS, 10
min). The sections were treated with BSA to remove any
Synthesis of (E)-5-[4-(Dimethylamino)styryl]-N-[2-(2-
hydroxybenzamido)ethyl]picolinamide (H3L3). To 3 (0.43 g, 1.6
mmol) in DMF (15 mL) cooled to 0 °C was added 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxide hexafluorophosphate (HATU; 0.57 g, 1.5 mmol). The reaction
mixture became bright red in color, triethylamine was added (0.8 mL,
5.8 mmol), and the mixture was heated to 40 °C. After 6 h, 4 (0.32 g,
1.5 mmol) was added, and the mixture was stirred at ambient
temperature for 12 h. The solvent was removed under reduced
pressure, the residue was dissolved in ethyl acetate and filtered, and the
filtrate was partitioned with brine. The organic phase was washed (3 ×
150 mL, brine), dried (NaSO4), and filtered, and the solvent was
removed by evaporation under reduced pressure to afford H3L3 as an
orange powder (0.37 g, 0.9 mmol, 58%). 1H NMR (500 MHz,
CDCl3): δ 9.16 (m, 1H, PyH), 8.39 (dd, 3JHH = 7.9 Hz, 4JHH = 1.7 Hz,
1H, ArH), 8.30 (dd, 3JHH = 8.2 Hz, 4JHH = 1.9 Hz, 1H, PyH), 8.10 (d,
3JHH = 8.1 Hz, 1H, PyH), 7.49 (m, AA′BB′ 2H, ArH), 7.39 (m, 2H,
ArH and CHCH), 7.15 (m, 1H, PyH), 7.05 (m, 1H, ArH), 6.96 (m,
AB, 3JHH = 16.2 Hz, 1H, CHCH), 6.73 (m, AA′BB′, 2H, ArH), 5.53
(m, 1H, CHHCH2), 4.04 (m, 2H, CH2CH2), 3.92 (m, 1H,
CHHCH2), 3.07 (s, 6H, N(CH3)2). 13C{1H} NMR (126 MHz,
CDCl3): δ 177.9, 170.2, 169.6, 151.7, 150.7, 147.6, 140.5, 139.3, 137.2,
132.8, 131.8, 129.3, 124.5, 123.0, 122.8, 121.5, 120.1, 115.8, 112.2,
+
61.2, 55.3, 40.3. HRMS (ESI-LTQ-FT). Calcd for C25H27N4O3 ([M
+ H]+): m/z 431.2078. Found: m/z 431.2072.
Synthesis of [ReOL3]. To a flask charged with H3L3 (100 mg, 0.23
mmol), [ReOCl3(PPh3)2] (196 mg, 0.23 mmol), and sodium acetate
(179 mg, 2.2 mmol) was added anhydrous, deoxygenated ethanol (6
mL). The reaction mixture was heated at reflux for 1.5 h and changed
from green to dark red in color. Upon cooling to ambient temperature,
a brown precipitate formed. The precipitate was collected by filtration
and washed with ethanol and diethyl ether. The precipitate was
recrystallized from dichloromethane and n-pentane to afford [ReOL3]
as red crystals (81 mg, 0.13 mmol, 57%). 1H NMR (500 MHz,
CDCl3): δ 9.16 (m, 1H, PyH), 8.39 (dd, 3JHH = 7.9 Hz, 4JHH = 1.7 Hz,
1H, ArH), 8.30 (dd, 3JHH = 8.2 Hz, 4JHH = 1.9 Hz, 1H, PyH), 8.10 (d,
3JHH = 8.1 Hz, 1H, PyH), 7.50−7.48 (m, AA′BB′, 2H, ArH), 7.42−
3
7.36 (m, 2H, ArH, CHCH), 7.15 (d, JHH = 8.2 Hz, 1H, ArH),
3
7.06−7.03 (m, 1H, ArH), 6.96 (d, JHH = 16.2 Hz, 1H, CHCH),
2
3
6.74−6.72 (m, AA′BB′, 2H, ArH), 5.53 (ddd, JHH = 12.5 Hz, JHH
=
7.2 Hz, 3JHH = 2.7 Hz, 1H, CHH), 4.07−4.00 (m, 2H, CH2), 3.92 (m,
1H, CHH), 3.07 (s, 6H, N(CH3)2). 13C{1H} NMR (126 MHz,
CDCl3): δ 177.9, 170.2, 169.6, 151.7, 150.7, 147.6, 140.5, 139.3, 137.2,
132.8, 131.8, 129.3, 124.5, 123.0, 122.8, 121.6, 120.1, 115.8, 112.2,
61.2, 55.4, 40.3. ESI-MS (positive ion). Calcd for [C25H24N4O4Re]+
([M + H]+): m/z 631.14. Found: m/z 631.137. IR (ν in cm−1): 970
(ReO). Crystals suitable for X-ray diffraction were grown from a
mixture of [ReOL3] dissolved in chloroform layered with n-pentane.
Synthesis of [99mTcOL2] and [99mTcOL3]. To a vial was added ligand
(H3Lx) (100 μL of a ∼1.5 mg sample in 1 mL of ethanol), 100 μL of
aqueous NaHCO3 (1 M), and generator eluent ([99mTcO4]−, 100 μL,
D
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Article
Scheme 1. Synthesis of H3L1, H3L2, [ReOL1], and [ReOL2]
diamide backbone H3L2, were prepared. Ligand H3L1 was
synthesized by the reductive amination of 1 with salicaldehyde
(Scheme 1). The diamide variant, H3L2, was prepared by the
reaction of 1 with the N-hydroxysuccinimidyl-activated ester of
salicylic acid (Scheme 1). The reaction of either H3L1 or H3L2
with [ReOCl3(PPh3)2] leads to the formation of dark-green
[ReOL1] or red [ReOL2], respectively (Scheme 1), and the
complexes can be identified by ESI-MS as their protonated
monocations, [ReOHL1]+ (m/z 472.06) and [ReOHL2]+ (m/z
486.05). The visible spectrum of dark-green [ReOL1] (1.0
mmol L−1, CH3CN) is dominated by a broad absorbance with
λmax = 594 nm (ε = 331 M−1 cm−1) that tails to λ ≈ 750 nm,
whereas the absorption spectrum of red [ReOL2] (1.0 mmol
L−1, CH3CN) features a broad absorption with λmax = 556 nm
(ε = 163 M−1 cm−1) that tails to λ ≈ 680 nm. The IR spectra of
both complexes show the characteristic ReO stretch at 950
cm−1 for [ReOL1] and 983 cm−1 for [ReOL2].
nonspecifically bound complex, washed in PBS (3 × 2 min) and then
distilled water, and mounted with nonfluorescent mounting media
(Dako). Images were obtained on a Leica (Bannockburn, IL) DM1RB
microscope fitted with a Carl Zeiss AxioCam MR color camera.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Rhenium Oxo
Complexes. Ligands containing a mixture of amide, amine,
and mercapto donor atoms are commonly used as tetradentate
N2S2 bis(amino)bis(thiol) ligands to form stable square-
pyramidal complexes with [TcVO]3+ cores. A simple way to
incorporate Aβ plaque targeting functional groups into
bis(amino)bis(thiol) ligands is via N-alkylation of one of the
amine functional groups. A potential disadvantage of this
approach is that the formation of [TcO]3+ complexes with this
type of ligand can lead to a mixture of syn and anti
diastereomers with respect to the arrangement of the
substituent on the nitrogen and the metal oxo core. A further
complication is that thiol-containing ligands are often air-
sensitive and prone to oxidation, and this makes their
incorporation into premade radiopharmaceutical kits challeng-
ing. The use of semirigid tetradentate ligands, lacking tertiary
amines, for complexation with [TcO]3+ has the advantage of
precluding the formation of syn and anti diastereomers.30,31
With the goal of preparing non-thiol-based planar ligands
capable of forming lipophilic neutral complexes with the [Re/
TcO]3+ core that bind to Aβ plaques, we focused on trianionic
N3O donor ligands capable of coordinating through amide,
phenolate, and pyridyl functional groups. A tetradentate
diamide−phenol−pyridine-containing ligand, 1-(2-hydroxyben-
zamido)-2- pyridinecarboxamido)benzene, based on amides
derived from 1,2-diaminobenzene forms a charge-neutral
lipophilic complex with [99mTcO]3+ that displays significant
uptake in the brain of mice (1.95% injected dose at 1 min
postinjection). This complex reacts rapidly with glutathione to
form a hydrophilic metal species, and this reactivity could result
in metabolic trapping of the radioactive metal in the brain due
to the relatively high cerebral glutathione concentration.32,33 In
this work, we prepare tetradentate amide-containing ligands
with a flexible aliphatic backbone derived 1,2-diaminoethane
with the hope that they would be less susceptible to reactions
with glutathione.
1
Both complexes are diamagnetic and give well-resolved H
NMR spectra consistent with rhenium(V) in an essentially
square-pyramidal geometry with a spin-paired d2 electron
configuration. Coordination of the oxorhenium(V) ion to the
ligands results in a general downfield shift for most of the
1
resonances in both the H and 13C NMR spectra compared to
the spectra of the metal-free ligands. Upon coordination of the
metal ion, both the methylene and ethylene protons in
[ReOL1] become diastereotopic, resulting in three distinct
AB spin systems. The resonance attributed to one of the
geminal protons on the backbone ethylene group closest to the
phenolate limb of the ligands undergoes a particularly large
1
downfield shift (Δδ = 1.79 ppm) in the H NMR spectrum:
[ReOL1] (δ = 5.57 ppm) compared to the metal-free ligand
H3L2 (δ = 3.78 ppm). The significant change in the magnetic
environment of this proton is possibly due to its close proximity
(syn) with the oxo ligand bound to the rhenium ion.
Interestingly, [ReOL1] was unstable in the solvent used for
NMR analysis (CDCl3) when left to stand for 24 h, whereas
[ReOL2] remained stable for at least several weeks. The
difference in stability between [ReOL1] and [ReOL2] was also
evident by HPLC analysis of the complexes. A single peak
could be observed in the chromatogram for [ReOL2], but
samples of [ReOL1] decomposed on the column when an
acidic mobile phase was used (0.1% trifluoroacetic acid/water/
acetonitrile), and this difference in stability may reflect the
Two different ligands with a N3O donor set of atoms, one
with an amine and amide backbone H3L1 and one with a
E
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different pKa values of the amine (H3L1) and amide (H3L2)
functional groups that are deprotonated upon coordination to
the rhenium.
Table 1. Crystal Data, Data Collection, and Refinement
Parameters for the Complexes [ReOL1], [ReOL2], and
[ReOL3]
Dark-green blocklike crystals of [ReOL1] suitable for analysis
by X-ray crystallography were grown from a solution of the
complex in dichloromethane, and red crystals of [ReOL2] were
grown by diffusion of diethyl ether into a mixture of the
complex dissolved in DMF (Figure 2 and Tables 1 and 2). In
[ReOL1]
[ReOL2]
[ReOL3]
formula
C15H14N3O3Re
C15H12N3O4Re
C25H23N4O4Re·
2CHCl3
M (g mol−1
)
470.49
484.48
868.41
cryst size
(mm3)
0.48 × 0.28 ×
0.16
0.45 × 0.23 ×
0.032
0.18 × 0.17 × 0.072
cryst syst
monoclinic
P21/c
monoclinic
P21/n
monoclinic
C2/c
space group
T (K)
130.0(1)
12.4901(4)
8.0225(4)
13.8963(4)
90
130.0(1)
7.5323(2)
18.7137(4)
10.3710(3)
90
130.0(1)
28.6169(6)
15.2350(4)
14.5380(3)
90
a (Å)
b (Å)
c (Å)
α (deg)
β (deg)
γ (deg)
U (Å3)
Z
100.626(3)
90
111.091(3)
90
103.953(2)
90
1368.56(9)
4
1363.94(7)
4
6151.2(2)
8
Dcalcd (Mg
2.283
2.359
1.875
m−3
)
wavelength
(Å)
0.7107 (Mo Kα) 0.7107 (Mo Kα) 1.5418 (Cu Kα)
abs coeff
8.896
8.936
12.874
(mm−1
)
F(000)
896
920
3392
reflns measd
indep reflns
12472
14504
10557
Figure 2. ORTEP-3 representations26 (with 30% probability ellipsoids)
of [ReOL1] and [ReOL2] (the SP-5-13-C enantiomer is shown for
both complexes).
5347 [Rint
0.0319]
=
5332 [Rint
0.0306]
=
6215 [Rint =
0.0241]
R [I > 2σ(I)]
0.0272
0.0344
0.0244
0.0297
0.0431
0.052
wR(F2) (all
data)
both complexes, the rhenium ion is in a distorted square-
pyramidal environment with the tetradentate ligand providing
the donor atoms of the square plane, resulting in one 6-
membered and two 5-membered chelate rings and an apical oxo
ligand. Both complexes crystallize in centrosymmetric space
groups, with both SP-5-13-C and SP-5-13-A enantiomers
present in the unit cell.34 In [ReOL1], the Re−N1 bond length
[1.908(2) Å] is shorter than the Re−N2 bond [1.978(3) Å],
but both bonds are shorter than typical Re−N single bonds
(approximately 2.13 Å),35 suggesting some degree of multiple-
bond character consistent with deprotonation of the amine and
amide, respectively, and consistent with other complexes that
feature a Re−Namide bond.30,33 The Re−Npyridine bond in
[ReOL2] [2.086(2) Å] is marginally shorter than the Re−
Table 2. Selected Bond Lengths in [ReOL1], [ReOL2], and
[ReOL3]
[ReVOL1]
[ReVOL2]
[ReVOL3]
Re1−O1
Re1−O2
Re1−N1
Re1−N2
Re1−N3
1.697(2)
1.968(2)
1.908(2)
1.978(3)
2.130(2)
1.681(2)
1.969(2)
1.973(2)
1.955(2)
2.086(2)
1.690(4)
1.961(4)
1.965(5)
1.945(5)
2.080(5)
methyl]phosphonate and 4-(dimethylamino)benzaldehyde in
the presence of sodium hydride (Scheme 2). Ligand H3L3 was
characterized by ESI-MS with the protonated ligand cation
N
pyridine bond in [ReOL1] [2.130(2) Å], but both are similar to
the Re−Npyridine bond length found in [ReO2(pyridine)4]+.36
The Re−Ophenolate bond distance is 1.968(2) Å in [ReOL1] and
1.969(2) Å in [ReOL2]. The Re−Ooxo bond in [ReOL2] is
shorter, 1.681(2) Å, than the analogous bond in [ReOL1],
1.697(2) Å, as suggested by IR spectroscopy (ReO stretches
ν = 983 and 950 cm−1, respectively).
[H4L3]+, resulting in a peak at m/z 431.2072. The H and 13C
1
NMR spectra of H3L3 were as expected. The rhenium complex
[ReOL3] was prepared by reacting H3L3 with [ReOCl3(PPh3)2]
in the presence of sodium acetate (Scheme 2). In ESI-MS, the
complex gave the expected peak for [ReOHL3]+ with the
expected isotope pattern at m/z 631.137.
The superior stability of [ReOL2] with a diamide backbone
compared to [ReOL1], which has an amine−amide backbone,
led us to focus on the synthesis of a ligand featuring a diamide
backbone where the pyridyl donor forms part of an integrated
Aβ plaque targeting styrylpyridyl functional group, H3L3. The
ligand was prepared by a HATU-mediated coupling of 3, which
was prepared by lithiation of 2 and a reaction with solid carbon
dioxide, with 4 (Scheme 2). The brominated styrylpyrdine
starting material, 2, with the required E configuration about the
double bond, was prepared by a Horner−Wadsworth−
Emmons reaction between diethyl [(6-bromopyridin-3-yl)-
Red crystals of [ReOL3] suitable for analysis by X-ray
crystallography were grown from a mixture of the complex
dissolved in chloroform (Figure 3 and Table 1). As expected,
the rhenium ion is in a distorted square-pyramidal environ-
ment, with the square plane comprised of three Re−N bonds
and one Re−Ophenolate bond, and an apical oxo ligand, with both
SP-5-13-A and SP-5-13-C enantiomers present in the unit
cell.34 The Re−Ooxo bond is again shorter [1.690(4) Å] than
the Re−Ooxo bond in [ReOL1], and this is reflected in the Re
O IR stretch (ν = 970 cm−1). The addition of the extra
styrylpyridine functional group results in very little change to
F
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Scheme 2. Synthesis of H3L3 and [ReOL3]
plaque. Human brain tissue (5 μm serial sections) collected
from subjects with clinically diagnosed AD was pretreated with
BSA to prevent nonselective binding and then treated with
solutions of [ReOL3]. Localization of the compound on the
treated brain tissue was measured by epifluorescent microscopy
(λex = 359 nm; λem = 461 nm) and compared to the contiguous
section immuno-stained with an Aβ antibody (1E8) that
selectively binds to Aβ plaques (Figure 4a,b) revealing that this
sample of brain tissue has well-defined neuritic dense-cored
Figure 3. ORTEP-3 representation26 (with 30% ellipsoids) of
[ReOL3]·2CHCl3 (solvent and minor component of disorder on
styrylpyridine; double bond omitted for clarity).
the coordination environment of the rhenium ion, leading to
bond lengths and angles similar to those found in [ReOL2].
Binding of [ReOL3] to Aβ1−42 Fibrils and Aβ Plaques in
Human Brain Tissue. The affinity of [ReOL3] for Aβ1−42
fibrils was estimated to be Ki = 855 nM using a fluoresencence
competition assay against Thioflavin T. Competitive dye
binding assays for quantifying the interaction of molecules to
Aβ fibrils are prone to false positives and rely on an assumption
that the compound being tested, [ReOL3] in this case, binds to
the same binding site(s) on the fibrils as the dye (Thioflavin T
in this case).37 Despite these limitations, they still provide
useful information with respect to potential interactions with
bonafide Aβ plaques in human subjects.
Amyloid plaques in human AD subjects are complicated
heterogeneous aggregates that contain a mixture of proteins
and chemical species, so it is valuable to assess the amyloid
plaque binding capacity in human subjects with clinically
diagnosed AD. The complex [ReOL3] is fluorescent because of
the styrylpyridine functional group, and this fluorescence
permits detection of the compound on human tissue using
epifluorescent microscopy. Aβ plaques are typically 40−60 μm
in diameter, so 5 μm serial sections often comprise the same Aβ
Figure 4. Frontal cortex serial tissue sections (5 μm) from human
subjects viewed at 10× magnification: (a) tissue from a subject with
clinically diagnosed AD with the Aβ plaques stained with a 1E8
antibody; (b) serial section to part a treated with [ReOL3] and then
visualized with epifluorescent microscopy (λex = 359 nm; λem = 461
nm), showing that [ReOL3] binds to Aβ plaques; (c) tissue from an
age-matched healthy brain control subject treated with 1E8 antibody;
(d) serial section to part c treated with [ReOL3] visualized with
epifluorescent microscopy (λex = 359 nm; λem = 461 nm), showing low
nonspecific binding in healthy brain tissue.
G
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plaques. Colocalization of the immuno-stained plaques and the
rhenium complex is clearly evident, demonstrating that the new
compound binds to Aβ plaques in human brains. Coordination
of the Npyridyl atom to the metal ion does not compromise the
ability of the styrylpyridyl functional group to selectively
interact with Aβ plaques. Importantly, there is no nonspecific
binding of [ReOL3] to an age-matched healthy control brain
(Figure 4d).
reduction of [99mTcO4]−, by varying the pH and buffers used,
we were not able to improve the radiochemical yield. The
complex [99mTcOL2] was relatively stable to ligand exchange in
the presence of glutathione (1 mM) with <5% decomposition
after 1 h but was more susceptible to ligand exchange in the
presence of histidine (1 mM), with ∼16% of the complex being
converted to an unidentified more hydrophilic complex after 1
h.
The UV/visible spectrum of an aqueous mixture of [ReOL2]
[50% PBS (pH 7.4) and 50% dimethyl sulfoxide] did not
change in the presence of excess histidine and glutathione,
suggesting that the complexes were relatively stable. The UV/
visible spectrum of [ReOL3] did not change in the presence of
excess histidine, but a subtle and rapid change in the spectrum
upon the addition of excess glutathione was evident with a
small shift in the major absorption from λabs ≈ 440 to 450 nm.
The cause of this minor change in the electronic spectrum is
not known but is not due to the formation of “free” H3L3. It is
possible that either noncovalent interactions between the ligand
and tripeptide or interactions of glutathione with the nominal
sixth coordination site of the metal ion cause this small change
in the energy of this charge-transfer band. After this minor
initial change, the spectrum was stable for at least 2 h.
For these complexes to be useful brain and Aβ plaque
imaging agents, it is essential that they are able to cross the
blood−brain barrier. Lipophilicity is an important parameter to
consider with respect to the potential of a compound to
penetrate the blood−brain barrier. The lipophilicity can be
estimated by calculating log Poct/water (where P = partition
coefficient) by determining the partitioning of a compound
between octanol and water. For compounds with ionizable
functional groups, log Doct/water (where D = distribution
coefficient) is preferred, where partitioning of the complex
between octanol and a buffer of known pH is determined. In
general, compounds with log(P/D) values between 0.9 and 3.0
are required for penetration of the blood−brain barrier, but
complicating issues include nonspecific binding to proteins and
recognition by efflux proteins.38,39 As expected, [TcOL3] (log
Synthesis of [99mTcOL2] and [99mTcOL3]. The stability of
[ReOL2] and [ReOL3] and the ability of [ReOL3] to bind to
Aβ plaques in human brain tissue encouraged preliminary
investigations in the synthesis of their analogous 99mTc
complexes at the “tracer” level. Adding tin(II) chloride to
reduce [99mTcO4]− in the presence of an aqueous mixture of
H3L2 resulted in the formation of a complex with a crude
radiochemical yield of ∼90% that was tentatively assigned to
[TcOL2] by comparing the radioactive HPLC trace (Figure 5)
D
oct/water = 1.87 0.02) was more lipophilic than [TcOL2] (log
Doct/water = 1.44
0.02). The PET imaging agent 11C-PIB
(Figure 1), which is useful in characterizing Aβ plaque burden,
has log P = 1.3.2 There are two technetium complexes that are
routinely used as cerebral perfusion imaging agents, [99mTcO-
(HMPAO)] (HMPAO = hexamethylpropyleneamine oxime)
and [99mTcO(ECD)] (ECD = ethylcysteinate dimer), and their
log P values are 1.9 and 1.6, respectively.40 Although estimation
of the lipophilicity is often used in assessing the potential of
compounds to cross the blood−brain barrier, there are many
other factors that contribute to the effective penetration of the
blood−brain barrier, and an ex vivo biodistribution study is a
better method to assess brain uptake.33
CONCLUDING REMARKS
■
With the goal of preparing planar ligands capable of forming
lipophilic neutral complexes with a [Re/TcO]3+ core,
tetradentate N3O ligands with three acidic protons were
synthesized. A ligand based on a mixed amide−amine
backbone, H3L1, was compared to a ligand with a diamide
backbone, H3L2. Both ligands readily formed neutral complexes
with [ReO]3+, with the rhenium ion in a distorted square-
pyramidal environment with the tetradentate ligand providing
the donor atoms of the square plane, resulting in one 6-
membered and two 5-membered chelate rings and an apical oxo
ligand. The superior stability of [ReOL2], with a diamide
backbone, led us to focus on the synthesis of a ligand featuring
a diamide backbone, where the pyridyl donor forms part of an
integrated Aβ plaque targeting styrylpyridine functional group,
H3L3. As expected, the rhenium ion in [ReOL3] is in a distorted
square-pyramidal environment, with the square plane com-
prised of three Re−N bonds and one Re−Ophenolate bond, and
an apical oxo ligand. This complex binds to Aβ plaques in
human brain tissue despite coordination of the pyridyl nitrogen
atom to the metal ion.
Figure 5. Comparative RP-HPLC analysis of [99mTcOL2] (black) with
[ReOL2] (red).
to that of the rhenium congener [ReOL2] (detected by
absorbance in the UV region). The small difference in the
retention times between the traces for [99mTcOL2] and
[ReOL2] is, in part, due to the detector configurations but
could also reflect the difference in polarity between the
oxorhenium(V) and oxotechnetium(V) cores.30 The crude
radiochemical yield for the preparation of a complex tentatively
assigned to [99mTcOL3] (assigned based on a comparison of its
HPLC traces with those of [ReOL3]) was always consistently
lower (∼60%) compared to the radiochemical yields of
[TcOL2]. Despite repeating the synthesis in the presence of
varying amounts of either sodium tartrate or sodium citrate,
commonly used additives to improve the radiochemical yields
of complexes prepared by the tin(II) chloride mediated
Extrapolation of the chemistry to 99mTc at the tracer level
appeared simple for the parent ligand, H3L2, and [TcOL2]
could be prepared rapidly in high radiochemical yield, but the
radiochemical yield for the synthesis of [TcOL3] was lower.
Both [TcOL2] and [TcOL3] were sufficiently lipophilic and
H
DOI: 10.1021/acs.inorgchem.6b00972
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.6b00972.
Crystallographic data for [ReOL1] (CIF)
Crystallographic data for [ReOL2] (CIF)
Crystallographic data for [ReOL3]·2CHCl3 (CIF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: pauld@unimelb.edu.au.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
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Notes
The authors declare no competing financial interest.
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Que, L. JBIC, J. Biol. Inorg. Chem. 2004, 9, 39.
ACKNOWLEDGMENTS
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Financial support from the Australian Research Council (ARC)
is acknowledged. P.S.D. is an ARC Future Fellow. V.L.V. is an
NHMRC Senior Research Fellow. The Victorian Brain Bank
Network is acknowledged for the provision of human tissue.
We acknowledge the generous support of Prof. Andrew M.
Scott, A/Prof. Henri Tochon-Danguy, A/Prof. Uwe Acker-
mann, Dr. FT Lee, Nick Alexopoulos, and David Thomas of the
Austin Hospital, Heidelberg, Victoria, Australia, for providing
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