Potential Diagnostic Imaging of Alzheimer's Disease with Copper-64

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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

Potential Diagnostic Imaging of Alzheimer’s Disease with Copper-64 Complexes That Bind to Amyloid‑β Plaques Lachlan E. McInnes,† Asif Noor,† Kai Kysenius,‡,∥ Carleen Cullinane,§,⊥ Peter Roselt,§ Catriona A. McLean,○,∥ Francis C. K. Chiu,◆ Andrew K. Powell,◆ Peter J. Crouch,‡ Jonathan M. White,† and Paul S. Donnelly*,†

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School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, ‡Department of Pharmacology and Therapeutics, and ∥The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Victoria, Australia, 3010 § Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia, 3000 ⊥ The Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia, 3000 ○ Department of Anatomical Pathology, The Alfred Hospital, Melbourne, Victoria, Australia, 3181 ◆ Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia, 3052 ABSTRACT: Amyloid-β plaques, consisting of aggregated amyloid-β peptides, are one of the pathological hallmarks of Alzheimer’s disease. Copper complexes formed using positron-emitting copper radionuclides that cross the blood−brain barrier and bind to specific molecular targets offer the possibility of noninvasive diagnostic imaging using positron emission tomography. New thiosemicarbazone-pyridylhydrazone based ligands that incorporate pyridyl-benzofuran functional groups designed to bind amyloid-β plaques have been synthesized. The ligands form stable complexes with copper(II) (Kd = 10−18 M) and can be radiolabeled with copper-64 at room temperature. Subtle changes to the periphery of the ligand backbone alter the metabolic stability of the complexes in mouse and human liver microsomes, and influenced the ability of the complexes to cross the blood−brain barrier in mice. A lead complex was selected based on possessing the best metabolic stability and brain uptake in mice. Synthesis of this lead complex with isotopically enriched copper-65 allowed us to show that the complex bound to amyloid-β plaques present in post-mortem human brain tissue using laser ablation-inductively coupled plasma-mass spectrometry. This work provides insight into strategies to target metal complexes to amyloid-β plaques, and how small modifications to ligands can dramatically alter the metabolic stability of metal complexes as well as their ability to cross the blood−brain barrier.

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parallel orientation of β sheet structures, and certain aromatic molecules can bind to these hydrophobic pockets through π−π interactions.14,15 Derivatives of 2-pyridylbenzofuran also bind to Aβ plaques but display less nonspecific binding when compared to styrylpyridine and benzothiazole based ligands. Detailed structure−activity studies identified [18F]NAV4694 (formerly [18F]AZD4694, Figure 1) as a lead candidate with better sensitivity for amyloid due to low white matter binding and high cortical binding.16−20 A diverse range of positron-emitting isotopes of copper are available that offer the potential to be of use in varied brainimaging applications. There are two comparatively short-lived isotopes, copper-60 (t1/2 = 20 min) and copper-62 (t1/2 = 9.7 min), as well as two isotopes copper-61 (t1/2 = 3.4 h) and copper-64 (t1/2 = 12.7 h) with longer radioactive half-lives. The

INTRODUCTION Alzheimer’s disease (AD) is a common form of neurodegenerative disease resulting in cognitive decline in episodic memory, attention, and language. A pathological hallmark of the disease is the presence of extracellular plaques comprised mostly of aggregated amyloid-β peptide (Aβ), a 39−43 amino acid peptide derived from the amyloid precursor protein.1−8 Molecular imaging using positron emission tomography (PET) and radiolabeled tracers that bind to amyloid plaques allows assessment of the Aβ burden in patients to assist in early differential diagnosis of the disease as well as monitoring the progress of emerging therapies.9,10 Recent advances have led to at least three PET imaging agents, labeled with positronemitting fluorine-18 gaining clinical approval, [18F]florbetapir, [18F]flutemetamol, and [18F]florbetaben (Figure 1).11−13 These aromatic hydrophobic molecules interact with Aβ fibrils and Aβ plaques through a combination of noncovalent interactions. Small channels and hydrophobic pockets, that extend along the length of the filament, are generated by the © XXXX American Chemical Society

Received: December 12, 2018

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DOI: 10.1021/acs.inorgchem.8b03466 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

Figure 1. Structures of clinically relevant Aβ imaging agents.

development of copper-based brain imaging agents requires developing ligands capable of forming copper complexes that are sufficiently stable in the body and capable of crossing the blood−brain barrier. A single ligand framework would be suitable for the full range of copper isotopes and would serve as a valuable platform to develop versatile imaging agents. In principle, the rapid and simple incorporation of a radioactive metal ion into a specific targeting ligand is an attractive alternative to the conventional organic synthesis that is required to incorporate fluorine-18 or carbon-11 into tracers. The selectivity of the complex can be achieved by grafting the complex to a targeting molecule that binds to specific molecular targets in vivo. It is preferable that the metal complexes can be prepared from “kit formulations”, and this has been important to the success of technetium-99m radiopharmaceuticals as used in single photon emission computed tomogaphy.21 Preparing stable copper complexes that are capable of crossing the blood−brain barrier is challenging. Pioneering work has highlighted the potential of copper(II) complexes of bis(thiosemicarbazone) ligands such as [Cu(atsm)] for brain imaging applications as either perfusion or hypoxia selective agents (Figure 2).22−26 [Cu(atsm)] is a relatively low molecular weight complex that is charge neutral, lipophilic, and capable of crossing the blood−brain barrier in humans.27 A related copper complex, [Cu(gtsm)] (Figure 2), that is also capable of crossing the blood−brain barrier in mice, has been used to probe changes in copper metabolism in animal models of amyloid pathology.28,29 There is considerable interest in developing copper-based Aβ plaque imaging agents. 30−36 To develop a bis(thiosemicarbazone) copper complex that could be used for Aβ plaque imaging, we prepared a bis(thiosemicarbazonato)copper(II) complex with an appended stilbene functional group, [Cu(atsm/a-stilbene)] (Figure 2).30 The [64Cu][Cu(atsm/a-stilbene)] complex crossed the blood−brain barrier and displayed increased uptake and retention in the brain of transgenic mice (APP/PS1), which are used as a model of the amyloid pathology associated with AD, when compared to wild-type controls. Inspired by the potential of charge neutral bis(thiosemicarbazone)copper(II) complexes for brain imaging applications, we prepared hybrid thiosemicarbazono-pyridylhydrazone ligands that incorporated styrylpyridine Aβ plaque

Figure 2. Structures of [Cu(atsm)], [Cu(gtsm)], [Cu(atsm/astilbene)], [CuLSP1], and [CuLSP2].

targeting groups, [CuLSP1] and [CuLSP2].31 The integrated ligand design, where the pyridyl functional group is used to coordinate to the metal ion as well as being a vital component of the functional groups required to bind Aβ plaques, was used to minimize the molecular weight of the complexes and increase the likelihood of crossing the blood−brain barrier. [CuLSP1] bound to amyloid-β plaques in human brain tissue collected from AD subjects, but micro-PET studies, in wildtype mice following administration [64Cu][CuLSP1], were disappointing with no radioactivity evident in the brain 5 min postinjection. Modification of the ligand to incorporate a dimethylaminoethane functional group to one limb of the ligand, [64Cu][CuLSP2] (Figure 2), improved the brain uptake with 1.1 (0.2)% of the injected dose per gram accumulating in the brain at 5 min postinjection. In this manuscript we report the synthesis of hybrid thiosemicarbazonato-benzofuran ligands and their copper complexes. The shift from styrylpyridyl to benzofuran derivatives for targeting Aβ targeting was inspired by the low nonspecific white matter binding [18F]NAV4694 leading to improved diagnostic imaging. Copper complexes with simple modifications of the ligand platform were prepared with a view to systematically improve metabolic stability and blood−brain barrier penetration. A goal was to design and identify a single ligand platform that could, in principle, be readily adapted with different binding groups to act as a starting point for copperbased brain imaging.

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RESULTS Synthesis and Characterization. Four new thiosemicarbazone-hydrazinopyridylbenzofuran ligands with either methyl or ethyl substituents on the backbone or thiosemicarbazone limb of the ligand were prepared. The benzofuran derivative 2(6-chloropyridin-3-yl)-5-methoxybenzofuran (3) was prepared by a palladium catalyzed Suzuki coupling reaction between the boronic acid of 5-methoxybenzofuran (2) and 2-chloro-5iodopyridine (Figure 3). The methoxy functional group in 3 B


Article

Inorganic Chemistry

Figure 3. Synthesis of H2L1−4 and [CuL1−4].

was cleaved with boron tribromide to give the chlorosubstituted benzofuran derivative (4), which was reacted with hydrazine hydrate to give 2-(6-hydrazinopyridin-3-yl)-5methoxybenzofuran (5) that could then be reacted with one of four different substituted mono-keto thiosemicarbazones to give H2L1−4 (Figure 3). The ligands were characterized by a combination of mass spectrometry, NMR spectroscopy, reversed phase high performance liquid chromatography (RP-HPLC), and elemental analysis. The ligands H2L1−4 react with copper acetate in methanol to give charge neutral, purple CuII complexes (Figure 3). An immediate color change to dark purple following the addition of the copper acetate suggests complexation of CuII is rapid. Analysis by electrospray mass spectrometry revealed that each complex gave a peak at a m/z value corresponding to [CuIILx + H+]+ with the expected isotope pattern, and each complex gave a single peak when analyzed by reversed-phase HPLC. The electronic spectra of [CuL1−4] in dimethylformamide display ligand centered absorptions at about λabs = 330 nm and broad absorptions at about λabs = 600 nm presumably due to metal− ligand charge transfer transitions. The copper(II) complexes are stable with respect to dissociation of the metal ion from the ligand, and competition experiments versus Na2H2EDTA (EDTA = ethylendiaminetetracetic acid) gave apparent conditional dissociation constants for [CuL1−4] at pH 7.4 of 10−18 M (Table 1). The copper complexes were also stable in the presence of human serum albumin, a protein with picomolar affinity for copper(II).37 Crystals of suitable quality for analysis by X-ray crystallography were isolated for both [CuL3] and [CuL4] (Figure 4, Table 1. Conditional Metal Ion Stabilities of [CuL1−4] at pH 7.4 against EDTA and in Vitro Metabolic Degradation HalfLife (in Vitro t1/2) in Liver Microsomes complex [CuL1] [CuL2] [CuL3] [CuL4]

Kd(7.4) (×10−18 M)

in vitro t1/2 mouse liver microsomes (min)

in vitro t1/2 human liver microsomes (min)

± ± ± ±

3 11 15 21

4 12 11 29

3.9 2.7 1.2 1.3

0.6 0.6 0.4 0.4

Figure 4. ORTEP representations (ellipsoids at the 40% level) of (a) [CuL3]·acetone with solvent and hydrogen atoms bound to carbon omitted for clarity. (b) Dimer found in [CuL3]·acetone. Cu1−S1 2.275(1) Å, Cu1−S1′ 2.792(2) Å. (c) [CuL4] hydrogen atoms bound to carbon not shown.

C


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Inorganic Chemistry Table 2). Both complexes crystallized as charge neutral complexes with formally dianionic tetradentate ligands. In Table 2. X-ray Crystallographic Data for [CuL3] and [CuL4] empirical formula formula weight/g mol−1 crystal system space group a/Å b/Å c/Å α/(deg) β/(deg) γ/(deg) V/Å3 Z temperature/K λ/Å reflections collected independent reflections R-factor/%

[CuL3]·acetone

[CuL4]

C24H28CuN6O3S 544.12 monoclinic P21/c 13.0608(13) 18.165(7) 10.8078(14) 90 108.934(13) 90 2425.4(11) 4 130.00(10) 0.71073 20977 7914 8.58

C22H23CuN6O2S 499.06 orthorhombic Fdd2 44.021(4) 40.539(2) 5.0328(4) 90 90 90 8981.4(12) 16 130.00(10) 1.54184 5164 3128 4.82

Figure 5. Cyclic voltammograms of [CuL1−4] in dimethylformamide at a glassy carbon working electrode, scan rate 200 mV s−1, 1 mmol L−1 analyte, electrolyte = 0.1 mol L−1 tetrabutylammonium hexafluorophosphate. Potentials quoted versus Fc+/Fc. Arrows indicate the scanning direction.

Table 3. Summary of the CuII/I Reduction Potentials of [CuL1‑4] compound

both complexes, copper(II) is a distorted square planar N3S environment, but in the case of [CuL3] an additional weaker axial interaction between CuII and a sulfur of an adjacent ligand (Cu−S1′ 2.792(2) Å) results in a dimer where the CuII tends toward five-coordinate square pyramidal (Figure 4b). The Cu−S1 bond distances (2.275(1) Å in [CuL3] and 2.229(2) Å in [CuL4]) are similar to those found in [Cu(atsm)]38,39 and in our earlier reports of copper(II) complexes of thiosemicarbazone-hydrazinopyridyl ligands.31,40 In both complexes the Cu−Nhydrazone bonds (Cu−N4, 1.937(4) Å in [CuL3], 1.948(5) Å in [CuL4]) and Cu−Nthiosemicarbazanato bonds (Cu−N3, 1.9394 Å in [CuL3], 1.9595 Å in [CuL4]) are shorter than the Cu−Npyridyl interactions (Cu−N6, 1.975(4) Å in [CuL3], 1.960(6) Å in [CuL4]). The C−S bond distances (1.782(5) Å and 1.767(7) Å) suggest more thiol-like rather than thione-like bonding, consistent with the deprotonation of the thiosemicarbazone limb of the ligand.38 The bond angles within the deprotonated hydrazone limb of the ligand, N4− N5−C5 109.6(3)° in [CuL3] and N4−N5−C6 109.7(5) in [CuL4], are smaller in than in three previously reported complexes where the hydrazone limb remains protonated with the analogous bond angle averaging 114.4°.31,40 Electrochemistry. The biological activity of copper(II) complexes of bis(thiosemicarbazones) can be partially related to their redox chemistry. Cyclic voltammetry in dimethylformamide at a glassy carbon electrode revealed that [CuL1−4] undergo a quasi-reversible process, tentatively attributed to a CuII/I process at −1.16 V vs ferrocenium/ferrocene (Fc+/Fc) (Figure 5, Table 3). In addition, a second quasi-reversible process was observed at ∼0.05 V vs Fc+/Fc, which could be due to either a formal CuII/III process or a ligand-based oxidation, and it is acknowledged that thiosemicarbazonato ligands are redox noninnocent.41−45 In Vitro Metabolic Stability of [CuL1−4] Using Liver Microsomes. The metabolic stability of [CuL1−4] was investigated in human and mouse liver microsomes, and the formation of likely metabolites was monitored. The copper complexes (1 μM) were incubated with either mouse or human liver microsomes at 37 °C (microsomal protein

1

[CuL ] [CuL2] [CuL3] [CuL4]

E°′ (CuII/I) (vs Fc+/Fc) −1.16 −1.16 −1.17 −1.16

V V V V

Ic/Ia

peak separation (mV)

1.53 1.82 1.39 1.33

78 72 71 66

concentration 0.4 mg mL−1) in the presence and absence of a nicotinamide adenine dinucleotide hydrogen phosphate (NADPH)-regenerating system, were NADPH is the cofactor for CYP450-mediated metabolism. The reactions were quenched at various time points over a 60 min incubation period by the addition of acetonitrile resulting in precipitation of proteins. The samples were centrifuged, and the supernatant was analyzed for the original copper complexes using HPLC/ MS. The concentration versus time data was used to calculate a first-order degradation rate constant (k) for depletion of [CuL1−4] and an in vitro-metabolic degradation half-life (in vitro t1/2 = ln(2)/k) (Table 1). Each complex was stable in liver microsomes in the presence of NADPH, but addition of a NADPH-regenerating system led to extensive metabolism. The nature of the alkyl substituents on both the backbone of the ligand (R2 in Figure 3) and the terminal substituents on the thiosemicarbazonato limb of the ligand (R1 in Figure 3) had a significant effect on the rate of degradation. The complex with all methyl substituents, [CuL1], is the least stable with the shortest in vitro t1/2, whereas the complex where each substituent is the longer ethyl functional group, [CuL4], is the most stable of the four complexes (Table 1). For a qualitative indication of the metabolic products, the copper complexes were incubated with liver microsomes at a higher concentration of copper complex (50 μM) and protein (2 mg mL−1) in the presence of the NADPH-regenerating system and uridine-5′-diphosphoglucuronic acid trisodium salt (UDPGA) required for phase II glucuronidation. For each of the four copper complexes, a metabolite with a mass increase of 16 amu, presumably due to oxygenation of the ligand, could be identified as well as a metabolite with a mass reduction of 63.5 amu, presumably due to loss of the CuII ion from the ligand. In the case of [CuL3] and [CuL4], two additional metabolites D


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Inorganic Chemistry

Figure 6. HPLC chromatograms of [64Cu][CuL1−4] (red) radiation detection and comparative nonradioactive analogue, [CuL1−4] (black), UV detection (λabs = 254 nm).

the brain more effectively (0.77 ± 0.30% IA/g at 30 m.p.i.) than [64Cu][CuL1] (1.06 ± 0.09% IA/g at 30 m.p.i.). The liver uptake and retention of [64Cu][CuL4] are also less than the other three complexes consistent with the increased metabolic stability of [CuL4] in mouse liver microsomes. Interaction with Synthetic Amyloid-Beta and Amyloid-Beta Plaques in Human Brain Tissue. Two complexes, one with a dimethyl backbone, [CuL2], and one with a diethyl backbone, [CuL4], were selected to investigate their interaction with synthetic Aβ1−42 by transition electron microscopy (TEM). Images of Aβ1−42 fibrils formed in the absence of the compounds show the expected morphology (Figure 7A).30,46−48 Addition of either [CuL2] or [CuL4] (1 equiv) to Aβ1−42 results in dramatic changes in the structural morphology as identified by the TEM images (Figure 7B,C), resulting in structures reminiscent of clusters of curvilinear irregular protofibrils.49 To assess the Aβ plaque binding capacity of [CuL4], consecutive sections of formalin-fixed human brain tissue (7 μm-thick) from both an AD subject and an age-matched nonAD control (AMC) were either stained for amyloid plaques using immunohistochemistry (Figure 8A,B) or analyzed for elemental composition using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) (Figure 8C−G). The 1E8 amyloid-β antibody identified amyloid-β plaques present predominantly in the gray matter region of the frontal cortex in the brain tissue from the AD subject (an approximately 3 mm thick layer on the surface of the cortex containing neuronal cell bodies, Figure 8A,B). In the consecutive section, the boundaries between gray and white matter were evident by quantitative elemental imaging of phosphorus-31, which is more concentrated in white matter than gray matter as myelin has a high phospholipid content (Figure 8C).50,51 To determine the selective binding of [CuL4] to plaques, the brain tissue was treated with nonradioactive isotopically enriched [65Cu][CuL4] to facilitate differentiation of the compound from endogenous background copper present in the brain. Naturally occurring copper-65 is less abundant than copper-63, with a stable 65Cu/63Cu ratio of 0.46:1. An increase in this ratio is indicative of an increase in copper-65 concentration, presumably due to binding of [65Cu][CuL4]. Laser ablation-ICP-MS analysis of the brain tissue treated with

could be identified, with either a mass loss of 14 or 28 amu consistent with N-demethylation and N-deethylation, as well as a mass increase of 176 amu consistent with glucuronidation. Radiochemistry and Biodistribution in Mice. Radioactive copper-64 complexes, [64Cu][CuL1−4], could be readily prepared in minutes at room temperature by adding H2L1−4 to a solution of 64Cu2+ buffered in acetate buffer at pH 5. The identity of the radiolabeled product was confirmed by comparison of the HPLC profiles of both the radiolabeled and nonradiolabeled analogues (Figure 6). The radiochemical purity of each radiolabeled complex was determined to be >95% by radio-TLC. The distribution coefficients (log D7.4) were measured by extracting the lipophilic component with octanol from an aliquot of the reaction mixture diluted in PBS, before repartitioning the octanol layer with PBS and determining the ratio of activity in each layer to limit the influence on hydrophilic contaminants and are summarized below (Table 4). Each complex gave Log D7.4 values similar to the BBB permeable copper complex [64Cu][Cu(atsm)]. Table 4. Log D7.4 Values for [64Cu][Cu(atsm)] and [64Cu][CuL1‑4] complex

log D7.4

[64Cu][Cu(atsm)] [64Cu][CuL1] [64Cu][CuL2] [64Cu][CuL3] [64Cu][CuL4]

1.73 1.84 1.81 1.88 1.48

To assess the ability of each complex to cross the blood− brain barrier each, radioactive complex was administered to wild-type mice (C57Bl/6) via tail-vein injection. The mice were euthanized at 2 and 30 min post injection (m.p.i.), and the activity accumulated in each organ was counted and expressed as a percentage of the injected activity normalized to the mass of the organ (% IA/g) (Table 5). The two compounds with the highest brain uptake were [64Cu][CuL4] (1.54 ± 0.60% IA/g at 2 m.p.i.) and [64Cu][CuL1] (1.39 ± 0.06% IA/g at 2 m.p.i.). As these mice do not contain Aβ plaques, it is ideal if the initial high brain uptake is followed by effective clearance. Both [64Cu][CuL1] and [64Cu][CuL4] show similar initial brain uptake, but [64Cu][CuL4] clears from E


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Inorganic Chemistry Table 5. Biodistribution Data Expressed As Injected Activity Normalized to the Mass of the Organ (% IA/g)a [CuL1] blood lungs heart liver kidneys muscle spleen brain

[CuL2]

[CuL3]

[CuL4]

2 min

30 min

2 min*

30 min

2 min

30 min

2 min*

30 min*

5.34 (0.74) 22.14 (3.36) 9.71 (0.53) 24.56 (3.13) 13.36 (2.91) 2.18 (0.57) 35.47 (15.24) 1.39 (0.06)

2.53 (0.46) 14.96 (6.28) 7.63 (0.24) 28.3 (3.32) 9.72 (1.26) 1.24 (0.18) 28.01 (15.64) 1.06 (0.09)

7.80 (0.99) 18.89 (4.29) 10.78 (4.29) 36.96 (11.19) 11.24 (3.82) 1.87 (0.70) 51.94 (24.92) 1.06 (0.43)

1.60 (0.21) 6.89 (0.71) 4.01 (0.82) 26.70 (6.11) 7.47 (0.69) 0.74 (0.12) 26.31 (7.97) 0.49 (0.09)

6.51 (1.47) 16.56 (2.69) 13.64 (1.51) 32.78 (5.81) 10.16 (2.03) 2.48 (0.48) 23.16 (9.86) 0.77 (0.19)

2.02 (0.06) 9.59 (2.33) 6.96 (0.22) 33.60 (4.21) 12.15 (0.92) 1.93 (0.12) 21.84 (5.56) 0.73 (0.04)

3.86 (1.19) 16.76 (9.20) 6.91 (1.56) 16.57 (4.15) 5.63 (1.33) 1.59 (0.51) 14.66 (6.61) 1.54 (0.60)

1.54 (0.12) 6.37 (0.49) 7.12 (1.75) 19.27 (1.05) 10.40 (2.02) 1.79 (0.21) 9.32 (3.77) 0.77 (0.30)

Standard error given in brackets (n = 3). *Indicates experiments where n = 4.

a

Figure 7. [CuL2] and [CuL4] change the aggregation profile of Aβ1−42. Transition electron microscopy of (A) Aβ1−42 (10 μM), (B) Aβ1−42 (10 μM) in the presence of [CuL2] (10 μM), and (C) Aβ1−42 (10 μM) in the presence of [CuL4] (10 μM).

Figure 8. (A) Immunohistochemistry staining for Aβ plaques (1E8) from AD brain sections. (B) Magnified image of the gray and white matter border in the 1E8 stained section. (C) LA-ICP-MS image for phosphorus (31P) content of an adjacent section, with dashed line to indicate the border of gray and white matter. (D) An example of tissue measured by LA-ICP-MS showing the natural 65Cu/63Cu ratio (0.46). (E) [65Cu][CuL4] stained aged-match control tissue, (F) [65Cu][CuL4] stained AD tissue. (G) [65Cu][CuLSP1] stained AD tissue. Dashed line indicates the border between gray and white matter.

the vehicle confirmed a consistent ratio of 65Cu/63Cu of 0.45, reflecting the natural abundance of each isotope (Figure 8D; Table 6). Treatment of an aged-match control and AD subject tissues with [65Cu][CuL4] (10 μM) resulted in a significant increase in the 65Cu/63Cu ratio in both the age-matched control (Figure 8E) and the AD subject (Figure 8F). In the tissue from the AD subject, the phosphorus-31 images (Figure 8B,C) can be used to delineate between white and gray matter, and the preferential binding of [65Cu][CuL4] to the gray matter, that is high in amyloid-β plaques, is demonstrated by the increase in the 65Cu/63Cu ratio (Figure 8F). In contrast, the frontal cortex brain tissue from an age-matched control

subject shows relatively uniform distribution with a smaller increase in copper-65 content (Figure 8E; Table 6). The benzofuran based tracer [18F]NAV4694 displays a relatively low degree of nonspecific binding to white matter when compared to the styrylpyridine-based tracer [18F]Florobetapir, and stilbene-based [18F]Florobetaben (Figure 1). Treatment of the brain tissue with [65Cu][CuL4] resulted in an average 65Cu/63Cu ratio of 2.71 for gray matter and 1.89 for white matter. In comparison, the styrylpyridyl based compound, [65Cu][CuLSP1], had less differentiation between gray and white matter with 65Cu/63Cu ratios of 1.73 and 1.42, respectively (Figure 8F,G; Table 6). Thus, our data indicate F


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Inorganic Chemistry

[CuL4], were characterized by X-ray crystallography, confirming the copper(II) is in the expected four-coordinate distorted square planar environment, but in the solid state weak axial interactions with the sulfur from an adjacent molecule result in [CuL3 ] forming a dimer. In both complexes double deprotonation of the ligand results in an overall charge neutral complex. The biological behavior of the bis(thiosemicarbazonato) copper(II) complexes can, in part, be related to their redox chemistry. The copper(II) complexes are generally stable with respect to loss of the metal ion in vivo, but reduction to copper(I) can lead to release of the metal from the ligand, particularly in the presence of high affinity copper(I) binding proteins.24,58−61 It is important that copper(II) complexes designed to bind to Aβ plaques are sufficiently resistant to reduction to copper(I). Complexes [CuL1−4] undergo a quasireversible CuII/I reduction at E0′ = −1.16 V (vs Fc+/Fc) so they are approximately 30 mV harder to reduce than [Cu(atsm)] (E0′ = −1.13 V under the same conditions). The second quasi-reversible process at ∼0.05 V (vs Fc+/Fc) could be due to either a CuII/III process or a ligand-based oxidation as thiosemicarbazones are redox noninnocent ligands.43 Although each of the complexes exhibited similar chemical stability in vitro with respect to dissociation of copper(II), their stability in liver microsomes was dramatically different. Liver microsomes prepared from liver homogenates contain cytochrome P450 enzymes, the major enzymes involved in drug metabolism.62 Each of the complexes are stable in the microsomal control samples in the absence of NADPH, but are metabolized in liver microsomes in the presence of NADPH. Substituting the methyl substituents on the ligand for longer ethyl substituents improved the metabolic stability of the complexes in liver microsomes. The complex with four ethyl substituents, [CuL4], is the most stable of the four complexes with an in vitro metabolic half-life of 29 and 21 min in human and mouse liver microsomes, respectively. Metabolites identified included species due to oxidation, loss of copper from the ligand, N-dealkylation, and glucuronidation. Each of the new ligands can be radiolabeled with copper-64 at room temperature to give complexes with high radiochemical yields. The ability of [64Cu][CuL1−4] to cross the blood−brain barrier was assessed in wild-type mice (C57Bl/6). The two complexes with the highest uptake at 2 m.p.i. were [64Cu][CuL1] and [64Cu][CuL4]. Of the four new complexes, [64Cu][CuL4] is the most promising as the relatively high initial uptake in the brain is accompanied by fast clearance. As wild-type mice do not possess Aβ plaques, an ideal tracer should clear from the brain rather than be retained. The reduced uptake and retention in the liver of [64Cu][CuL4] and higher initial uptake in the brain when compared to the other three tracers are consistent with the prediction of increased metabolic stability in liver microsomes. The combination of the in vitro metabolic stability studies and biodistribution studies identified [CuL4] as a lead candidate for further Aβ plaque binding studies in human brain tissue. Previously, we have used epi-fluorescent microscopy to probe the binding of copper complexes to Aβ plaques in human brain tissue.30,31 In this instance, the lack of sufficient fluorescent emission from [CuL4] required us to employ an alternative methodology based on elemental mapping using laser ablation-inductively coupled plasmamass spectrometry. A sample of nonradioactive isotopically

Table 6. Average 65Cu/63Cu Ratios Determined for Both Gray and White Matter ROIs in Diagnosed Alzheimer’s Disease (AD) Cases and Aged Match Control (AMC) Cases sample AD vehicle AMC vehicle AD [65Cu][CuL4] AMC [65Cu] [CuL4] AD [65Cu] [CuLSP1] AMC [65Cu] [CuLSP1]

gray matter avg ratio (65Cu/63Cu)

white matter avg ratio (65Cu/63Cu)

0.45 0.45 2.71 1.11

0.45 0.45 1.89 1.10

1.73

1.42

1.16

1.15

improved differentiation between white matter and the amyloid plaque-rich gray matter for our newly synthesized compound [65Cu][CuL4] when compared to [65Cu][CuLSP1].

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DISCUSSION The suite of positron-emitting isotopes of copper that are available offers considerable potential for the development of new PET imaging agents. The relatively short-lived isotope, copper-60 (t1/2 = 20 min), is relatively easy to produce with a small medical cyclotron, and copper-62 (t1/2 = 9.7 min) is available from a convenient generator.52 The availability of copper-62 from a portable generator is particularly attractive for brain or perfusion imaging that involves acquiring images shortly after injection of the tracer.27,53,54 Copper-61 (t1/2 = 3.4 h) and copper-64 (t1/2 = 12.7 h) can be produced in hospitalbased cyclotrons.52,55,56 Copper-64 has a relatively low energy of positron-emission and lacks interfering gamma emissions allowing for acquisition of images of comparable quality to those obtained with fluorine-18.57 The relatively long half-life of copper-61 and copper-64 allows PET imaging to be carried out at a site remote from the cyclotron facility used to generate the radionuclide and facilitate centralized production and distribution of Good Manufacturing Processes (GMP) grade tracers. A major challenge in developing copper-based brain imaging agents is designing copper complexes that can cross the blood−brain barrier. Inspired by the success of bis(thiosemicarbazonato) complexes, such as Cu(atsm) (Figure 2), in brain imaging applications, we designed hybrid thiosemicarbazone-pyridylhydrazone ligands that form stable, charge neutral, and lipophilic complexes with copper(II) that can be readily modified to selectively bind to Aβ plaques. A pyridyl-benzofuran derived Aβ plaque targeting group was selected as [18F]NAV4696 has emerged as a leading candidate for diagnostic imaging of Aβ plaques as it displays high cortical binding in AD subjects but low nonspecific binding to white matter.20 It is important to consider the metabolic stability of compounds designed as PET tracers, and for applications involving imaging in the brain it is preferable that the major radioactive metabolites do not cross the blood−brain barrier. Complexes with either methyl- or ethyl-substituents on the backbone of the ligand (R1 and R2, Figure 3) were prepared to probe whether such simple modifications could be used to dramatically alter metabolic stability, serum protein interactions, and the degree of penetration through the blood− brain barrier. All four ligands readily form charge neutral copper(II) complexes. Two of the complexes, [CuL3] and G


Inorganic Chemistry

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enriched [65Cu][CuL4] was used for the analysis to improve the sensitivity of the technique. The complex preferentially binds to areas of the brain identified as being rich in Aβ plaques as measured by immunohistochemistry in the contiguous section of brain tissue (Figure 8). The benzofuran containing complex, [65Cu][CuL4], appears to bind with improved differentiation between white and gray matter when compared to the styryl-pyridine containing complex [65Cu][CuLSP1], with the potential to offer better sensitivity for amyloid due to low white matter binding and high cortical binding similar to [18F]NAV4694.20 On the basis of these results, [CuL4] radiolabeled with any of the available positronemitting isotopes could be of use in assessing amyloid pathology in AD patients using positron emission tomography.

Article

EXPERIMENTAL SECTION

General Procedures. All standard reagents and solvents were acquired from commercial sources and used without further purification. Isotopically enriched copper-65 (99.7%) was purchased from Trace Sciences International (Ontario, Canada). Elemental analysis for C, H, and N was carried out by The Campbell Microanyalitical Laboratory, The University of Otago, Dunedin, New Zealand. NMR spectra were recorded on a Varian FT-500 NMR (California, USA) (1H at 500 MHz and 13C{1H} at 125.7 MHz) at 297 K or an Agilent MR400 NMR (California, USA) (1H at 400 MHz and 13C{1H} at 101 MHz) at 297 K and referenced to internal solvent residue. High resolution mass spectra were recorded on a Thermo Scientific Exactive Plus OrbiTrap LC/MS (Thermo Fisher Scientific, Massachusetts, USA) and calibrated to internal references. Analytical RP-HPLC traces were acquired using an Agilent 1200 HPLC system equipped with an Alltech Hypersil BDS C18 analytical HPLC column (4.6 × 150 mm, 5 μm) with a flow rate of 1 mL/min and UV absorbance detection at 280 and 400 nm. Retention times (Rt/min) were recorded using a gradient elution of 5−100% B in A (A = 0.1% trifluoroacetic acid (TFA), B = acetonitrile with 0.1% TFA) over 30 min. UV−vis spectra were recorded on a Shimadzu UV1650-PC spectrometer (Shimadzu, Kyoto, Japan) from 800 to 250 nm. Fluorescence emission spectra were recorded on a Varian Cary Eclipse fluorescence spectrometer. Emission and excitation spectra were recorded on a Varian CARY eclipse fluorescence spectrophotometer in quartz cuvettes. Cyclic voltammograms were recorded using an Metrohm (Switzerland) AUTOLAB PGSTAT100 and analyzed using Autolab GPES V4.9 software. Measurements were carried out using 1 mM of analyte in anhydrous DMF with tetrabutylammonium hexafluorophosphate (0.1 M) as a supporting electrolyte. A glassy carbon working electrode was used along with a Pt wire counter electrode and a pseudo leakless Ag/Ag+ working electrode (EDAQ, New South Wales, Australia). Measurements were referenced to internal ferrocene (Fc/Fc+, E°′ = 0.00 V where E′ refers to the midpoint of the reduction (Epc) and oxidation (Epa) peaks of a quasi- or fully reversible process where E′ = (Epc + Epa)/2. All X-ray crystal structure data were obtained on an Oxford Diffraction SuperNova CCD diffractometer using Cu-Κα radiation, and the temperature during collection was maintained at 130.00(10) K using an Oxford Cryosystems cooling device. The structure was solved by direct methods using SHELXT and refined using leastsquares methods using SHELXL.63,64 Thermal ellipsoid plots were generated using ORTEP-3 integrated within the WINGX suite of programs.65 [CuL3]·acetone CCDC 1881051; [CuL4] CCDC 1881050. Synthetic Procedures. 5-Methoxybenzofuran (1) was purchased from Matrix Scientific (United States), and 5-methoxybenzofuran boronic acid (2),16,17 diacetyl-mono-4-methyl-3-thiosemicarbazone (6), diacetyl-mono-4-ethyl-3-thiosemicarbazone (7), 66,67 and [H2LSP1]31 were prepared according to literature procedures. 2-(6-Chloropyridin-3-yl)-5-methoxybenzofuran (3). 5-Methoxybenzofuran boronic acid (2) (1.16 g, 6.04 mmol), 5-iodo-2chloropyridine (1.30 g, 5.43 mmol), bis(triphenylphosphine)palladium dichloride (123 mg, 0.04 mmol), and potassium carbonate (1.50 mg, 10.85 mmol) were suspended in ethanol (100 mL), and the mixture was heated at reflux for 3 h. The reaction mixture was then filtered while still hot, and the filtrate was evaporated to dryness under reduced pressure. The remaining residue was extracted into ethyl acetate (2 × 40 mL) and washed with brine (3 × 30 mL). The organic phase was evaporated to dryness under reduced pressure to yield a beige powder which was purified by flash chromatography (SiO2, CH2Cl2) to give -(6-chloropyridin-3-yl)-5-methoxybenzofuran (3) (888 mg, 3.42 mmol, 63%). 1H NMR (500 MHz; CDCl3): δH/ ppm 8.85 (dd, 4JHH = 2.5 Hz, 5JHH = 0.7 Hz, 1H, PyrH), 8.06 (dd, 3 JHH = 8.3 Hz, 4JHH = 2.5 Hz, 1H, PyrH), 7.43−7.39 (m, 2H, ArH + PyrH), 7.05 (m, 2H, ArH), 6.94 (dd, 3JHH = 8.9 Hz, 4JHH = 2.6 Hz, 1H, ArH), 3.86 (s, 3H, CH3O). 13C{1H} NMR (126 MHz; CDCl3):

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CONCLUSION The versatility of the hybrid thiosemicarbazone-pyridylhydrazone ligand platform allows for incorporation of different functional groups as demonstrated by the incorporation of either benzofuranyl or styrylpyridine containing functional groups that bind to amyloid-β plaques. The radioactive copper(II) complexes could be rapidly prepared in high radiochemical yield at room temperature, and the resulting complexes are stable with respect to dissociation of metal ion. Simple modifications to the ligand framework can improve metabolic stability and the degree of brain uptake in wild-type mice. The complex with three ethyl substituents, [CuL4], displays the best stability in liver microsomes, crosses the blood−brain barrier with fast clearance, and binds to amyloidβ plaques in post-mortem human brain tissue. Analysis of postmortem brain tissue treated with isotopically enriched [65Cu][CuL4] by laser ablation-inductively coupled plasmamass spectrometry demonstrated that the compound bound to amyloid-β plaques present in the frontal cortex of an AD subject with good differentiation between white and gray matter indicative of low nonspecific binding. This approach, of using isotopically enriched metal isotopes and imaging human tissue using laser ablation-inductively coupled plasma-mass spectrometry, could be useful for investigating the interactions of other metal complexes in tissue samples. The work presented here adds to the recent progress in the development of the chemistry required to realize the potential of copper-based brain imaging agents.30−33 When compared to the preparation of fluorine-18 imaging agents, radiolabeling specifically designed ligands with copper(II) is relatively simple, fast, and efficient. The ligands discussed here could easily be incorporated into kit-based formulations where solutions of radioactive copper(II) are simply added to preprepared vials containing ligand. A similar kit-based approach was central to the widespread adoption and success of technetium-99m containing single photon computed emission tomographic imaging agents. Although this work focused on using copper-64, it is likely that a single ligand platform could be used with any of the diverse range of positron-emitting copper isotopes, further increasing the versatility of this approach to brain imaging. The work described in this manuscript provides insight into strategies to target metal complexes to amyloid-β plaques but also highlights that small modifications to ligands can dramatically alter metabolic stability and the ability of metal complexes to cross the blood−brain barrier. H


Article

Inorganic Chemistry δC/ppm 156.5 (ArC), 152.6 (PyrC), 150.9 (ArC), 150.3 (ArC), 146.2 (PyrC), 134.6 (PyrC), 129.3 (ArC), 125.9 (PyrC), 124.5 (ArC), 122.4 (PyrC), 114.3 (ArC), 112.0 (ArC), 103.6 (ArC), 56.1 (CH3O). MS(ESI/O-TOF) (m/z): [M + H]+ calcd for [C14H10ClNO2+H]+, 260.0473; found m/z 260.0474 RP-HPLC: Rt 15.87 min. 2-(6-Chloropyridin-3-yl)-benzofuran-5-ol (4). A degassed solution of 2-(6-chloropyridin-3-yl)-5-methoxybenzofuran) (3) (119 mg, 0.46 mmol) in dichloromethane (6 mL) under and atmosphere of N2 gas was cooled to 0 °C. To this mixture, boron tribromide (1.0 M in CH2Cl2, 2 mL) was added dropwise. After the addition was complete, the reaction mixture was allowed to warm to ambient temperature and was stirred for 2.5 h. The reaction was then quenched with water and aqueous saturated NaHCO3, resulting in the precipitation of a beige solid. This solid was collected by filtration and washed with water. The product was recrystallized from ethyl acetate/pentane, and the solid was washed with pentane to give 2-(6-chloropyridin-3-yl)benzofuran-5-ol (4) as a beige powder. (65 mg, 0.26 mmol, 57%). 1H NMR (500 MHz; CD3OD): δH/ppm 8.85 (d, 4JHH = 2.5 Hz, 1H, PyrH), 8.24 (dd, 3JHH = 8.4 Hz, 4JHH = 2.4 Hz, 1H, PyrH), 7.54 (d, 3 JHH = 8.4 Hz, 1H, PyrH), 7.37 (d, 3JHH = 8.8 Hz, 1H, ArH), 7.25 (s, 1H, ArH), 6.98 (d, 4JHH = 2.5 Hz, 1H, ArH), 6.83 (dd, 3JHH = 8.8, 4 JHH = 2.5 Hz, 1H, ArH). 13C{1H} NMR (126 MHz; CD3OD): δC/ ppm 155.0 (ArC), 153.4 (PyrC), 151.4 (PyrC), 151.2 (ArC), 146.6 (PyrC), 136.3 (PyrC), 130.9 (ArC), 127.6 (ArC), 125.8 (ArC), 115.4 (ArC), 112.4 (ArC), 106.8 (ArC), 105.0 (ArC). MS(ESI/O-TOF) (m/z): Cald for [C13H8ClNO2 + H]+, 246.0317; found, 246.0924. 2-(6-Hydrazinopyridin-3-yl)-benzofuran-5-ol (5). To a flask charged with 2-(6-chloropyridin-3-yl)-benzofuran-5-ol (4) (120 mg, 0.49 mmol) was added hydrazine hydrate (65%, 5 mL). The resultant suspension was heated at reflux under an atmosphere of N2 for 4 h, and then the mixture was then allowed to cool to room temperature. A precipitate formed that was collected by filtration and washed with water followed by pentane to give 2-(6-hydrazinopyridin-3-yl)benzofuran-5-ol (5) as an off-white solid (76 mg, 0.32 mmol, 65%), 1 H NMR (500 MHz; DMSO-d6): δH/ppm 9.12 (s, 1H, OH), 8.49 (dd, 4JHH = 2.4, 5JHH = 0.7 Hz, 1H, PyrH), 7.88 (dd, 3JHH = 8.8, 4JHH = 2.4 Hz, 1H, PyrH), 7.86 (s, 1H, NH), 7.33 (ddd, 3JHH = 8.8, 4JHH = 0.8, 5JHH = 0.5 Hz, 1H, ArH), 6.99 (d, 4JHH = 0.9 Hz, 1H, ArH), 6.87 (dd, 4JHH = 2.5, 5JHH = 0.4 Hz, 1H, ArH), 6.80 (d, 3JHH = 8.8 Hz, 1H, PyrH), 6.66 (dd, 3JHH = 8.7, 4JHH 2.5 Hz, 1H, ArH), 4.25 (s, 2H, NH2). 13C{1H} NMR (126 MHz; DMSO-d6): δC/ppm 161.6 (PyrC), 154.9 (ArC), 153.4 (ArC), 148.0 (ArC), 144.3 (PyrC), 133.3 (PyrC), 129.9 (ArC), 115.1 (PyrC), 112.1 (ArC), 110.9 (ArC), 106.0 (PyrC), 104.9 (ArC), 98.8 (ArC). MS(ESI/O-TOF) (m/z): Calcd for [C13H11N3O2+H]+, 242.0924; found 242.0924 Dipropionyl-mono-4-methyl-3-thiosemicarbazone (8). To a mixture of water (100 mL) acidified with HCl (conc., 10 drops) was added 3,4-hexanedione (18.0 mL, 148 mmol), and the mixture was cooled in an ice bath. To this mixture was added 4-methyl-3thiosemicarbazide (3.06 g, 29.1 mmol) portion-wise over 10 min. The mixture was stirred for 4 h and then extracted with chloroform (3 × 50 mL). The combined organic extracts were dried over MgSO4, filtered, and concentrated under reduced pressure. Addition of pentane to the point of turbidity and cooling to −20 °C resulted in the precipitation of a crystalline solid. This solid was collected by filtration and washed with pentane to give dipropionyl-mono-4methyl-3-thiosemicarbazone (8) as colorless crystals (4.80 g, 23.85 mmol, 82%). 1H NMR (400 MHz, CDCl3): δH/ppm 8.85 (s, 1H, NNH(C = S)N), 7.53 (s, 1H), 3.27 (d, 3JHH = 4.9 Hz, 3H, NHCH3), 2.82 (q, 3JHH = 7.3 Hz, 2H, CH3CH2), 2.51 (q, 3JHH = 7.7 Hz, 2H, CH3CH2), 1.10 (t, 3JHH = 7.3 Hz, 3H, CH3CH2), 1.03 (t, 3JHH = 7.7 Hz, 3H, CH3CH2). 13C{1H} NMR (101 MHz, CDCl3): δC/ppm 199.0 (CO), 179.3 (CS), 149.2 (CN), 31.7 (CH2), 30.0 (CH3CH2), 16.6 (CH3NH), 9.9 (CH3CH2), 8.2 (CH3CH2). MS(ESI/ O-TOF) (m/z): Calcd for [C8H15N3OS + H]+, 202.1009; found, 202.1010 (experimental), Dipropionyl-mono-4-ethyl-3-thiosemicarbazone (9). To a mixture of 3,4-hexanedione (3.0 mL, 28.0 mmol) acidified with HCl (conc., 5 drops) and cooled to 5 °C was added 4-ethyl-3thiosemicarbazide (3.15 g, 26.4 mmol) portion-wise over 1 h. After

the addition was complete, the mixture was allowed to warm to room temperature. The precipitate that formed was collected by filtration and washed with water to give dipropionyl-mono-4-ethyl-3thiosemicarbazone (9) as a colorless powder (5.31 g, 21.1 mmol, 80%). 1H NMR (400 MHz; CDCl3): δH/ppm 8.75 (s, 1H, NNH(C = S)N), 7.47 (s, 1H, CH3CH2NH), 3.80−3.73 (m, 2H, CH3CH2NH), 2.82 (q, 3JHH = 7.3 Hz, 2H, CH3CH2), 2.51 (q, 3JHH = 7.7 Hz, 2H, CH3CH2), 1.31 (t, 3JHH = 7.3 Hz, 3H, CH3CH2NH), 1.11 (t, 3JHH = 7.3 Hz, 3H, CH3CH2), 1.03 (t, 3JHH = 7.7 Hz, 3H, CH3CH2NH). 13 C{1H} NMR (101 MHz; CDCl3): δC/ppm 199.0 (CO), 178.1 (CS), 149.1 (CN), 39.9 (CH3CH2), 30.0 (CH3CH2), 16.6 (CH3CH2), 14.3 (CH3CH2), 9.9 (CH3CH2), 8.2 (CH3CH2). MS(ESI/O-TOF): Cald for [C9H17N3OS + H]+ 216.1165; found, 216.1161. Diacetyl-2-(2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol)-(4methyl-3-thiosemicarbazone), H2L1. A suspension of 2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol (5) (50 mg, 0.21 mmol) and diacetyl-mono-4-methyl-3-thiosemicarbazone (6) (37 mg, 0.21 mmol) in ethanol (10 mL) was acidified with acetic acid (four drops). The mixture was heated at reflux under an atmosphere of N2 for 4 h and then allowed to cool to room temperature. The precipitate that formed was isolated by filtration and washed with diethyl ether to give H2L1 as a yellow powder (55 mg, 0.14 mmol, 67%). 1H NMR (500 MHz; DMSO-d6): δH/ppm 10.25 (s, 1H, Pyr-NH-N), 10.16 (s, 1H, NNH(C = S)N), 9.19 (s, 1H, OH), 8.69 (dd, 4JHH = 2.3 Hz, 5JHH = 0.6 Hz, 1H, PyrH), 8.33 (q, 3JHH = 4.5 Hz, 1H, CH3−NH-C = S), 8.11 (dd, 3JHH = 8.7 Hz, 4JHH = 2.3 Hz, 1H, PyrH), 7.37 (m, 2H, PyrH +ArH), 7.18 (d, 5JHH = 0.8 Hz, 1H, ArH), 6.91 (d, 4JHH = 2.4 Hz, 1H, ArH), 6.72 (dd, 3JHH = 8.7 Hz, 4JHH 2.5 Hz, 1H, ArH), 3.04 (d, 3JHH = 4.6 Hz, 3H, CH3-NH), 2.24 (m, 6H, N = C−CH3), 13C{1H} NMR (126 MHz; DMSO-d6): δC/ppm 178.4 (NH-(CS)-NH, 156.8 (PyrC), 154.0 (ArC), 153.6 (ArC), 148.5 (N = C-CH3), 148.3 (ArC), 145.4 (N = C-CH3), 144.3 (PyrC), 134.3 (PyrC), 129.7 (ArC), 118.5 (PyrC), 112.8 (ArC), 111.1 (ArC), 107.2 (PyrC), 105.2 (ArC), 100.5 (ArC), 11.5 (CH3−C = N), 11.1(CH3−C = N). Anal. Calcd for C19H20N6O2S: C, 57.56; H, 5.08; N, 21.20. Found: C, 57.24; H, 5.19; N, 21.50. MS(ESI/O-TOF) (m/z): Calcd for [C19H20N6O2S+H]+, 397.1441; found 397.1444. RP-HPLC: Rt = 10.95 min. Diacetyl-2-(2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol)-(4ethyl-3-thiosemicarbazone), H2L2. A suspension of 2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol (5) (143 mg, 0.59 mmol) and diacetylmono-4-ethyl-3-thiosemicarbazone (7) (126 mg, 0.67 mmol) in ethanol (10 mL) was acidified with acetic acid (three drops) and heated at reflux for 1 h. The mixture was then stirred at ambient temperature for 16 h. A precipitate was collected by filtration and washed with diethyl ether to give H2L2 as a yellow powder (207 mg, 0.50 mmol, 85%). 1H NMR (500 MHz, d6-DMSO): δH/ppm 10.26 (s, 1H, Pyr-NH-N), 10.10 (s, 1H, NNH(C = S)N), 9.18 (s, 1H, OH), 8.69 (d, 4JHH = 2.3 Hz, 1H, PyrH), 8.35 (t, 3JHH = 5.9 Hz, 1H, CH3CH2NH), 8.11 (dd, 3JHH = 8.8 Hz, 4JHH = 2.3 Hz, 1H, PyrH), 7.37 (m, 2H, PyrH + ArH), 7.19 (s, 1H, ArH), 6.91 (d, 4JHH = 2.4 Hz, 1H, ArH), 6.72 (dd, 3JHH = 8.7 Hz, 4JHH = 2.5 Hz, 1H, ArH), 3.61 (quintet, 3JHH = 6.7 Hz, 2H, CH3CH2NH), 2.25 (m, 6H, CH3), 1.15 (t, 3JHH = 7.1 Hz, 3H, CH3CH2NH). 13C{1H} NMR (126 MHz; DMSO-d6): δC/ppm 177.3 (CS), 156.8 (PyrC), 154.0 (ArC), 153.5 (ArC), 148.5 (PyrC), 148.3 (CN), 145.3 (CN), 144.3 (PyrC), 134.3 (PyrC), 129.7 (ArC), 118.5 (ArC), 112.8 (ArC), 111.1 (PyrC), 107.1 (ArC), 105.1 (ArC), 100.4 (ArC), 38.5 (CH3CH2), 14.4 (CH3CH2), 11.5 (CH3), 11.1 (CH3). Anal. Calcd for C20H22N6O2S: C, 58.52; H, 5.40; N, 20.47. Found: C, 58.16; H, 5.49; N, 20.71. MS(ESI/O-TOF) (m/z): Calcd for [C20H23N6O2S]+ 411.1598, found 411.1600 (experimental). RP-HPLC: Rt = 11.21 min. Dipropionyl-2-(2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol)-(4methyl-3-thiosemicarbazone), H2L3. A suspension of 2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol (5) (222 mg, 0.92 mmol) and dipropionyl-mono-4-methyl-3-thiosemicarbazone (8) (203 mg, 1.01 mmol) in ethanol (20 mL) was acidified with acetic acid (5 drops) and heated at reflux for 2 h and then allowed to cool to room temperature. The precipitate that formed was collected by filtration and washed with pentane to give H2L3 as a yellow powder (255 mg, I


Article

Inorganic Chemistry 0.58 mmol, 63%). 1H NMR (500 MHz, DMSO-d6): δH/ppm 10.41 (s, 1H, Pyr-NH), 10.30 (s, 1H, NNH(C = S)N), 9.18 (s, 1H, OH), 8.69 (dd, 4JHH = 2.4 Hz, 5JHH = 0.7 Hz, 1H, PyrH), 8.23 (q, 3JHH = 4.3 Hz, 1H, CH3NH), 8.12 (dd, 3JHH = 8.8 Hz, 4JHH = 2.4 Hz, 1H, PyrH), 7.38 (d, 3JHH = 8.7 Hz, 1H, ArH), 7.33 (d, 3JHH = 8.8 Hz, 1H, PyrH), 7.17 (d, 5JHH = 0.8 Hz, 1H, ArH), 6.91 (d, 4JHH = 2.5 Hz, 1H, ArH), 6.72 (dd, 3JHH = 8.7 Hz, 4JHH = 2.5 Hz, 1H, ArH), 3.04 (d, 3JHH = 4.6 Hz, 3H, CH3NH), 2.89 (m, 4H, CH3CH2), 0.98 (m, 6H, CH3CH2). 13 C{1H} NMR (126 MHz; DMSO-d6): δC/ppm 178.4 (CS), 157.0 (PyrC), 154.0 (ArC), 153.5 (PyrC), 151.4 (CN), 148.6 (CN), 148.2 (ArC), 144.2 (PyrC), 134.3 (PyrC), 129.7 (ArC), 118.4 (ArC), 112.8 (ArC), 111.1 (ArC), 106.9 (PyrC), 105.1 (ArC), 100.4 (ArC), 31.2 (NH-CH3), 17.32 (CH2CH3), 16.40 (CH2CH3), 11.04 (CH2CH3), 10.70 (CH2CH3). Anal. Calcd for C21H24N6O2S·2H2O: C, 54.77; H, 6.13; N 18.25. Found: C, 54.62; H, 5.96; N, 18.53. MS(ESI/O-TOF) (m/z): Calcd for [C21H24N6O2S + H]+ 425.1754, found, 425.1757. RP-HPLC: Rt = 12.07 min. Dipropionyl-2-(2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol)-(4ethyl-3-thiosemicarbazone), H2L4. A suspension of 2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol (5) (117 mg, 0.54 mmol) and dipropionyl-mono-4-ethyl-3-thiosemicarbazone (9) (119 mg, 0.49 mmol) in ethanol (5 mL) was acidified with acetic acid (two drops) and heated at reflux for 4 h. The mixture was then evaporated to dryness under reduced pressure. The resultant yellow residue was triturated in pentane and a yellow solid collected by filtration to give H2L4 as a yellow powder (159 mg, 0.36 mmol, 73%). 1H NMR (500 MHz; DMSO-d6): δH/ppm 10.44 (s, 1H, PyrNH), 10.27 (s, 1H, NNH(C = S)N), 9.18 (s, 1H, OH), 8.69 (d, 4JHH = 1.7 Hz, 1H, PyrH), 8.23 (t, 3JHH = 5.7 Hz, 1H, CH3CH2NH), 8.12 (dd, 3JHH = 8.6 Hz, 4JHH = 1.6 Hz, 1H, PyrH), 7.38 (d, 3JHH = 8.7 Hz, 1H, ArH), 7.33 (d, 3JHH = 8.8 Hz, 1H, PyrH), 7.18 (s, 1H, ArH), 6.92 (d, 4JHH = 2.1 Hz, 1H, ArH), 6.72 (dd, 3JHH = 8.7 Hz, 4JHH = 2.3 Hz, 1H, ArH), 3.61 (quintet, 3JHH = 6.6 Hz, 2H, CH3CH2NH), 2.91−2.84 (m, 4H, CH3CH2), 1.16 (t, 3JHH = 7.1 Hz, 3H, CH3CH2NH), 0.99 (m, 6H, CH3CH2). 13C{1H} NMR (126 MHz; DMSO-d6): δC/ppm 177.4 (CS), 156.9 (PyrC), 154.0 (ArC), 153.5 (ArC), 151.3 (CN), 148.6 (CN), 148.3 (ArC), 144.2 (PyrC), 134.4 (PyrC), 129.7 (ArC), 118.5 (PyrC), 112.8 (ArC), 111.1 (ArC), 107.0 (PyrC), 105.1 (ArC), 100.4 (ArC), 38.5 (CH3CH2NH), 17.4 (CH3CH2), 16.6 (CH3CH2), 14.4 (CH3CH2NH), 11.06 (CH3CH2), 10.68 (CH3CH2). Anal. Calcd for C22H26N6O2S·H2O: C, 57.88; H, 6.18; N, 18.41. Found: C, 57.86; H, 5.88; N, 18.23. MS(ESI/O-TOF) (m/z): Calcd for [C22H26N6O2S + H]+ 439.1911, found 439.1914. RP-HPLC: Rt = 12.31 min. [Cu(diacetyl-2-(2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol)-(4methyl-3-thiosemicarbazone))], [CuL1]. A suspension of H2L1 (39 mg, 0.09 mmol) in chloroform (20 mL) was heated before copper acetate monohydrate (25 mg, 1.3 mmol) was added. The mixture was then heated at reflux for an additional 4 h, and then allowed to cool to room temperature. The mixture was filtered, isolating a purple solid that was washed with water, followed by ethanol, and then air-dried to yield a purple powder (36 mg, 0.07 mmol, 78% yield). Anal. Cald for C19H18CuN6O2S·CHCl3·CH3CH2OH: C, 47.02; H, 4.79; N, 14.15. Found C, 46.65; H, 4.26; N, 14.24. MS(ESI/O-TOF) (m/z): Calcd for [C19H18CuN6O2S + H]+ 458.0578 (experimental), 458.0581 (calculated). RP-HPLC: Rt = 10.24 min. [Cu(diacetyl-2-(2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol)-(4ethyl-3-thiosemicarbazone))], [CuL2]. To a suspension of H2L2 (46 mg, 0.11 mmol) in methanol (5 mL) was added copper acetate monohydrate (35 mg, 0.18 mmol), and the purple mixture was stirred at room temperature for 1 h. The precipitate that formed was collected by filtration and washed with diethyl ether to give [CuL2] as a dark purple solid (55 mg, 0.11 mmol, 99% yield). Anal. Calcd for C20H20CuN6O2S·1.5H2O: C, 48.14; H, 4.65; N, 16.84. Found: C, 48.46; H, 4.45; N, 17.05. MS(ESI/O-TOF) (m/z): Calcd for [C20H20CuN6O2S + H]+ 472.0737; found, 472.0735. RP-HPLC: Rt = 12.42 min. [Cu(dipropionyl-2-(2-(6-hydrazinopyridin-3-yl)-benzofuran-5ol)-(4-methyl-3-thiosemicarbazone))], [CuL3]. To a suspension of H2L3 (98 mg, 0.23 mmol) in methanol (5 mL) and triethylamine (3

drops) was added copper acetate monohydrate (56 mg, 0.28 mmol). The purple mixture was stirred at room temperature for 1 h, and the mixture was evaporated to dryness under reduced pressure. The residue was then dissolved in the minimum volume of acetone, and the mixture was added dropwise to a rapidly stirring equal volume of pentane. The solid that formed was collected by filtration and washed with pentane to give [CuL3] as a dark purple solid (49 mg, 0.10 mmol, 43%). Anal. Calcd for C21H22CuN6O2S·2CH3OH: C, 50.22; H, 5.50; N, 15.28. Found: C, 50.59; H, 4.94; N, 15.18. MS(ESI/OTOF) (m/z): Calcd for [C21H22CuN6O2S + H]+ 486.0894; found, 486.0892. RP-HPLC: Rt = 13.06 min. Crystals of [CuL3]· (CH3COCH3) sufficient quality for analysis by X-ray crystallography were grown by exchange of vapors between an acetone solution of the complex and pentane. [Cu(dipropionyl-2-(2-(6-hydrazinopyridin-3-yl)-benzofuran-5ol)-(4-ethyl-3-thiosemicarbazone))], [CuL4]. To a suspension of H2L4 (58 mg, 0.13 mmol) in methanol (5 mL) and triethylamine (2 drops) was added copper acetate monohydrate (40 mg, 0.20 mmol). The purple reaction mixture was stirred at room temperature for 1 h. The precipitate that formed was collected by filtration washed with diethyl ether to give [CuL4] as purple powder (57 mg, 0.11 mmol, 85%). Anal. Calcd for C22H24CuN6O2S·CH3OH: C, 51.92; H, 5.30; N, 15.79. Found: C, 51.75; H, 4.81; N, 15.97. MS(ESI/O-TOF) (m/ z): Calcd for [C22H24CuN6O2S + H]+ 500.1050; found, 500.1048. RP-HPLC: Rt = 13.21 min. Crystals [CuL4] of sufficient quality for analysis by X-ray crystallography analysis grown from a diffusion of vapors between an acetone solution of the complex and pentane. [65Cu][Cu(diacetyl-2-((E)-4-(2-(6-hydrazinopyridin-3-yl)vinyl)N,N-dimethylaniline)-(4-methyl-3-thiosemicarbazone))], [65Cu][CuLSP1]. H2LSP1 was prepared as reported.31 To a suspension of H2LSP1 (11 mg, 0.03 mmol) in a mixture of methanol (5 mL), dimethyl sulfoxide (1 mL) and triethylamine (4 drops) were added [65Cu]CuCl2 (8 mg, 0.06 mmol). The mixture was stirred at room temperature overnight, and a purple solid was precipitated by the addition of water. The precipitate was collected by filtration and washed with water to give [65Cu][CuLSP1] as a purple powder (15 mg, 0.03 mmol, 99%). HR-MS(ESI/O-TOF) (m/z): Calcd for [C21H2565CuN7S + 2H]2+ 237.0658; found, m/z 237.0659; calcd for [C21H2565CuN7S + H]+ 473.1243; found, 473.1240. RP-HPLC: Rt = 10.49 min. [65Cu][Cu(dipropionyl-2-(2-(6-hydrazinopyridin-3-yl)-benzofuran-5-ol)-(4-ethyl-3-thiosemicarbazone))], [65Cu][CuL4]. To a suspension of H2L4 (22 mg, 0.05 mmol) in methanol (4 mL), triethylamine (three drops) was added [65Cu]CuCl2 (12 mg, 0.09 mmol). The mixture was stirred at room temperature for 16 h, and then a purpled solid was precipitated by addition of water. The precipitate was collected by filtration and washed with water, ethanol and diethyl ether to give [65Cu][CuL4] as a dark purple powder (18 mg, 0.04 mmol, 80%). HR-MS(ESI/O-TOF) (m/z): Calcd for [C22H2465CuN6O2S+H]+ 502.1032; found, 502.1051. RP-HPLC: Rt = 13.42 min. Determination of Conditional Copper Binding Affinities. Conditional metal−ligand binding affinities were determined using a modified procedure to that previously published (Xaio, Wedd, 2010). Briefly, the Kd was determined for each complex by incubating a standard solution of copper (AAS standard, Sigma-Aldrich, 10 μM) with ligand (12 μM) and increasing concentrations of Na2H2EDTA (0−150 μM) in DMSO/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (60% DMSO, 20 mM HEPES, pH 7.4) for 24 h at room temperature. The concentration of the corresponding copper complex was determined by measuring the absorbance at l = 537 nm. The Kd was then determined using eq 1 and K′a[CuEDTA] = 1.10 × 10−16 M.

Kd([CuLx]K ′a([CuEDTA]) =

([Lx]Total /[CuLx]) − 1 ([EDTA]Total /[CuEDTA]) − 1

(1)

Interaction of Complexes with Human Serum Albumin. The interaction of each complex with human serum albumin was investigated by emission spectroscopy. Solutions were prepared consisting of [CuLx] (where x = 1−4, 10 mM) in DMSO/PBS J


Article

Inorganic Chemistry

counter. Log D7.4 was then calculated by calculating the log10 of the ratio of the counts between the 1-octanol layer and the PBS layer. Each partition was repeated in triplicate and reported as the average. For in vivo studies, the ligands H2L1−4 were dissolved in DMSO (1 mg/mL), and an aliquot (2 μL) was added to a solution of [64Cu]CuCl2 (0.1 M HCl) buffered in sodium acetate (0.1 M, pH 5) and incubated at room temperature for 30 min. An aliquot (2 μL) was removed for quality control by diluting in ethanol (50 μL) and analyzed by comparative radio-RP-HPLC. The remaining reaction mixture was diluted with ammonium acetate buffer (0.1 M, pH 8), ethanol (8%), and DMSO (10%) to give a final volume of 100 μL with between 3 and 4 MBq of activity for each dose. Biodistribution measurements were carried out as described previously.31 The mice were injected intravenously with [64Cu][CuLx] (maximum volume of 100 μL) prepared according to the above procedure. Three animals were subsequently sacrificed at 2 min postinjection and 30 min postinjection, respectively. The organs of interest were removed and weighed, and the radioactivity was measured with an automatic counter. The percentage injected activity per gram (% IA/g) was calculated by a comparison of the tissue counts to suitably diluted aliquots of the injected material. Preparation of Synthetic Aβ1−42 Fibrils. Synthetic Aβ1−42 was synthesized using microwave-assisted solid-phase peptide synthesis (Liberty Blue peptide synthesizer, Biotage) using standard F-moc coupling procedures as reported previously.70 Synthetic peptide was then dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol to a theoretical concentration of 1 mM. The mixture was sonicated in an ice bath to aid dissolution, and then incubated in an ice bath for 60 min. The solvent was then allowed to evaporate, and drying was completed by the aid of a speed vac to obtain a clear peptide film. Aliquots were stored at −80 °C until required. For the preparation of Aβ1−42 fibrils, the clear peptide films (400 μg) were dissolved in aqueous NaOH (80 μL, 60 mM) with the aid of vortex mixing and then sonicated in an ice water bath for 10 min. To this water (280 μL) and PBS (10× , 40 μL) was added. After mixing thoroughly on a vortex mixer, the mixture was centrifuged for 5 min at 14000g. the supernatant was transferred to a clean tube, and the concentration was determined by UV−visible spectroscopy using ε214 (Aβ1−42) = 95 426 M−1 cm−1. (TM Ryan, Metallomics, 2015) Aβ mixtures were then incubated at 37 °C for 3 days with constant swirling to induce fibrillization. Transition Electron Microscopy of Amyloid Fibrils. Fibrillar Aβ1−42 (10 mM) was incubated in the absence and presence of both [CuL2] (10 mM) and [CuL4] (10 mM) in 10% DMSO in PBS and incubated for 1 h at 37 °C. An aliquot of each solution was spotted onto a carbon-coated copper grid and allowed to incubate for 2 min before excess solution was blotted off the grid. Samples were analyzed using an FEI Tecnai F20 operating at a voltage of 200 kV. Staining of Human AD Brain Tissues for Laser Ablation ICPMS. Paraffin preserved brain tissue blocks were provided by the Victorian Brain Bank Network. Both AD and HC brain tissue sections (7 μm) were deparaffined (xylene, 3 × 3 min) followed by rehydration (2 min soakings in 100%, 90%, 70%, and 0% v/v ethanol/water). The hydrated sections were then washed in phosphate buffered saline (PBS, 5 min) and then blocked with BSA (2%, PBS, pH 7.4). Tissue sections were then washed briefly with PBS and then covered with [65Cu][CuL4] (10 mM, 30% DMSO in PBS, 10 min). The sections were then washed with BSA (2%, PBS) to remove nonspecifically bound complex. Sections were then washed with PBS (3 × 2 min), dipped in distilled water and sections were allowed to air-dry. LA-ICP-MS experiments were carried out as described previously.71 Briefly, sections were placed in a 10 × 10 cm ablation cell together with matrix-matched elemental standards for quantitative analysis. Brain sections were ablated with the NWR213 ablation system (Kennelec Scientific) using a series of rasters using a 60 μm square laser spot size and a scanning speed of 240 μm s−1. Argon gas flow (1.2 L min−1) was used to transfer the vaporized material into the 8800 QQQ-ICP-MS (Agilent) and analyzed for the elements 13C, 31 P, 63Cu, and 65Cu. Data were analyzed using the Iolite analysis

(10%) and in both the presence of human serum albumin (0.2% m/v) and absence. Solutions were then allowed to incubate at room temperature for 2 h before being analyzed by emission spectroscopy with λex = 335 nm. Metabolic Stability. Microsomal stability assays were performed by incubating the compound (1 μM) with human or mouse liver microsomes (Xenotech LLC, Kansas, USA) suspended in phosphate buffer (0.1 M, pH 7.4) at a final microsomal protein concentration of 0.4 mg/mL at 37 °C. Metabolic stability assays were conducted in the presence of a NADPH-regenerating system (1 mg/mL NADP, 1 mg/ mL glucose-6-phosphate, 1 U/mL glucose-6-phosphate dehydrogenase and 0.67 mg/mL MgCl2). For each complex, the reaction was initiated by adding an aliquot of a stock solution (50% acetonitrile/ water) to the microsomal reaction matrix (final organic solvent content: 1% acetonitrile). Samples were also incubated in the absence of the NADPH-regenerating system to monitor for non-cytochrome P450-mediated metabolism in the microsomal matrix. The reaction was quenched at various time points by the addition of acetonitrile, containing diazepam as an internal standard. NADPH was also added to control samples after quenching to ensure equivalent sample matrix in all cases. To facilitate metabolite detection, incubations were performed at a higher concentration of substrate (50 μM) and microsomal protein (2 mg/mL). The reaction was initiated by the addition of both NADPH and UDPGA as cofactors in this reaction. Quenched samples were centrifuged for 4 min at 4500 rpm, and the supernatant was removed and analyzed by LC/MS using a Waters (Massachusetts, USA) Xevo G2 QTOF coupled to a Waters Acquity UPLC with an Ascentis Express C8 column (50 × 2.1 mm, 2.7 μm, Supelco, Pennsylvania, USA) using a gradient of acetonitrile−water with 0.05% formic acid as buffer and a 0.4 mL/min flow rate. The concentration of each peak was then determined using a standard sample prepared at the incubation concentration using authentic samples under the same LC/MS conditions. Compound concentration versus time data for both human and mouse liver microsomes were fitted to a monoexponential decay function to determine the first-order rate constant (k) for substrate depletion. The degradation rate constant (k) was used to calculate an in vitro metabolic degradation half-life (in vitro t1/2 = ln2/k).68 In the case of [CuL1] and [CuL2], there was an apparent deviation from first-order kinetics at later time points, and therefore only the initial rate of loss was used in the determination of degradation half-life ([CuL1]: 2−5 min, [CuL2]: 2−15 min). Radiolabeling with 64Cu: Synthesis of [64Cu][CuL1−4]. [64Cu]CuCl2 was obtained from either the Sir Charles Gardiner Hospital Radiopharmaceutical Production and Development Centre (Perth, WA) or from Austin Health Department of Molecular Imaging and Therapy (Heidelberg, Victoria) dissolved in 0.1 M HCl. Radiolabeling of [64Cu][CuL1−4] was achieved by the addition of a DMSO stock solution of H2L1−4 (2 μL, 1 mg./mL) to a solution of [64Cu]CuCl2 (10 MBq, 0.1 M HCl) that had been buffered with sodium acetate (0.1 M, pH 5). The reaction was incubated at room temperature for 30 min, and an aliquot was removed for characterization by radio-RP-HPLC. Radio-RP-HPLC was acquired using a Shimadzu SPD-10ATvP HPLC system equipped with a Phenomenex Luna C18 100 Å column (4.6 × 150 mm, 5 μm) with a 1 mL/min flow rate. Chromatograms were acquired with a UV−vis detector (254 and 350 nm) and scintillation detector in series. Retention times are recorded in minutes using a gradient elution method of 5−100% B in A over 15 min followed by holding at 100% B for a further 3 min. Buffer A was 0.05% TFA, buffer B was 0.05% TFA in acetonitrile. Log D7.4 measurements were performed using an adjusted procedure to that previously reported.69 A 2 μL aliquot of the radioactive reaction mixture was added to a partition of 1-octanol (500 μL) that had been presaturated with PBS and PBS (498 μL) that had been presaturated with 1-octanol. The mixture was vortexed briefly and then allowed to separate over the course of 30 min to 1 h. A 400 μL aliquot of the 1-octanol layer was taken and repartitioned against PBS (400 μL). The mixture was then vortexed briefly and allowed to separate over 30 min, and a 50 μL aliquot of each layer was taken and counted using a Wizard2 (PerkinElmer, US) gamma K


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Inorganic Chemistry

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software package operating under the Igor Pro suite, with carbon (13C) and phosphorus (31P) channels used for signal normalization. Images used for quantitation were constructed for a ratio of the 65Cu and 63Cu, achieving a pixel-by-pixel value.

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ASSOCIATED CONTENT

Accession Codes

CCDC 1881050−1881051 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

ORCID

Jonathan M. White: 0000-0002-0707-6257 Paul S. Donnelly: 0000-0001-5373-0080 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Australian Research Council. The Victorian Brain Bank Network for the provision of human tissue. We thank Susan Jackson and Kerry Warren for their technical expertise in performing the in vivo biodistribution study. Kai Kysenius is a former recipient of the Sigrid Juselius Postdoctoral Fellowship (Helsinki, Finland). Prof. Rodney J. Hicks (Peter MacCallum Cancer Centre) for radiochemistry laboratory and preclinical imaging facilities.

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REFERENCES

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Potential Diagnostic Imaging of Alzheimer's Disease with Copper-64

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