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Jul 26, 2016 - afford [ReOL1] as dark-green crystals (116 mg, 0.24 mmol, 76%). 1H ..... mixture became bright red in color, triethylamine was added (0...
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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).

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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 structural integrity with concomitant impaired axonal transport and compromised synaptic function.1 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 © XXXX American Chemical Society

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

Figure 1. Chemical structures of the amyloid imaging agents 11C-PIB and Florbetapir. Received: April 19, 2016

A

DOI: 10.1021/acs.inorgchem.6b00972 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

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 instrument. Analytical radio-labeled high-performance liquid chromatography (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 concentrations 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). 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), 7.36 (m, 1H), 3.48 (m, 2H, OCNCH2), 2.89 (t, 3JHH = 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 mmol, 50%). 1H NMR (500 MHz, CDCl3): δ 8.56−8.54 (m, 1H, 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, 2H, CH2NHCO), 2.95 (t, 3JHH = 6.0 Hz, 2H, CH2CH2). 13C{1H}

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) remains 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 99m Tc 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 identification 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 chargeneutral 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.

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EXPERIMENTAL SECTION

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 B

DOI: 10.1021/acs.inorgchem.6b00972 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry NMR (126 MHz, CDCl3): δ 165.1, 158.2, 149.7, 148.3, 137.6, 128.9, 128.6, 126.5, 122.4, 122.4, 119.2, 116.6, 52.5, 48.2, 39.1. HRMS (ESILTQ-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/darkgreen crystalline precipitate formed. The precipitate was collected by filtration and washed with a small amount of water and then ethanol to afford [ReOL1] as dark-green crystals (116 mg, 0.24 mmol, 76%). 1H 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, 4 JHH = 1.4 Hz, 1H, PyH), 7.17−7.14 (m, 1H, ArH), 7.09 (dd, 3JHH = 7.4 Hz, 4JHH = 1.5 Hz, 1H, ArH), 6.95 (dd, 3JHH = 8.0 Hz, 4JHH = 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. ESIMS (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 dicyclohexylcarbodiimide (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 Nhydroxysuccinimidyl 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 afford H3L2 as a colorless powder (0.34 g, 1.2 mmol, 30%). 1H 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, 4 JHH = 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 Hz, 1H, ArH), 6.87 (ddd, 3JHH = 8.1 and 7.1 Hz, 4JHH = 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 (ESILTQ-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

[ReOL2] as a red powder (116 mg, 0.24 mmol, 76%). 1H NMR (500 MHz, CDCl3): δ 9.31 (ddd, 3JHH = 5.4 Hz, 4JHH = 1.5 Hz, 5JHH = 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, 3JHH = 8.2 and 7.1 Hz, 4JHH = 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. Synthesis of (E)-4-[2-(6-bromopyridin-3-yl)vinyl]-N,N-dimethylaniline (2). To anhydrous ethanol (200 mL) was added 6bromonicotinaldehyde (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 2bromo-5-(chloromethyl)pyridine (2.98 g, 14 mmol, 73%). To 2bromo-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, 3 JHH = 16.3 Hz, 1H, CHCH), 6.70 (m, 2H, ArH), 2.99 (s, 6H, 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. 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, 74%). 1H 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 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

∼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). X-ray Crystallography. Crystals were mounted in low-temperature 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 experiments 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

1H, CHCH), 6.72 (m, AA′BB′, 2H, ArH), 3.34 (s, 6H, N(CH3)2). 13 C{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. Synthesis of N-(2-Aminoethyl)-2-hydroxybenzamide Hydrochloride (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 1,2ethanediamine 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 powder (3.88 g, 19 mmol, 90%). 1H 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. Synthesis of (E)-5-[4-(Dimethylamino)styryl]-N-[2-(2hydroxybenzamido)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, 3 JHH = 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, 3 JHH = 8.1 Hz, 1H, PyH), 7.50−7.48 (m, AA′BB′, 2H, ArH), 7.42− 7.36 (m, 2H, ArH, CHCH), 7.15 (d, 3JHH = 8.2 Hz, 1H, ArH), 7.06−7.03 (m, 1H, ArH), 6.96 (d, 3JHH = 16.2 Hz, 1H, CHCH), 6.74−6.72 (m, AA′BB′, 2H, ArH), 5.53 (ddd, 2JHH = 12.5 Hz, 3JHH = 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

DOI: 10.1021/acs.inorgchem.6b00972 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry Scheme 1. Synthesis of H3L1, H3L2, [ReOL1], and [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.

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]. Both complexes are diamagnetic and give well-resolved 1H 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 resonances in both the 1H 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 downfield shift (Δδ = 1.79 ppm) in the 1H 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

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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 squarepyramidal 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 airsensitive and prone to oxidation, and this makes their incorporation into premade radiopharmaceutical kits challenging. 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-hydroxybenzamido)-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. Two different ligands with a N3O donor set of atoms, one with an amine and amide backbone H3L1 and one with a E

DOI: 10.1021/acs.inorgchem.6b00972 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry different pKa values of the amine (H3L1) and amide (H3L2) functional groups that are deprotonated upon coordination to the rhenium. 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

Table 1. Crystal Data, Data Collection, and Refinement Parameters for the Complexes [ReOL1], [ReOL2], and [ReOL3] [ReOL1] formula −1

M (g mol ) cryst size (mm3) cryst syst space group T (K) a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) U (Å3) Z Dcalcd (Mg m−3) wavelength (Å) abs coeff (mm−1) F(000) reflns measd indep reflns

Figure 2. ORTEP-3 representations26 (with 30% probability ellipsoids) of [ReOL1] and [ReOL2] (the SP-5-13-C enantiomer is shown for both complexes).

R [I > 2σ(I)] wR(F2) (all data)

both complexes, the rhenium ion is in a distorted squarepyramidal environment with the tetradentate ligand providing the donor atoms of the square plane, resulting in one 6membered 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 multiplebond 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− Npyridine 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). 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)-

[ReOL2]

[ReOL3]

C15H14N3O3Re

C15H12N3O4Re

C25H23N4O4Re· 2CHCl3 868.41 0.18 × 0.17 × 0.072

470.49 0.48 × 0.28 × 0.16 monoclinic P21/c 130.0(1) 12.4901(4) 8.0225(4) 13.8963(4) 90 100.626(3) 90 1368.56(9) 4 2.283

484.48 0.45 × 0.23 × 0.032 monoclinic P21/n 130.0(1) 7.5323(2) 18.7137(4) 10.3710(3) 90 111.091(3) 90 1363.94(7) 4 2.359

monoclinic C2/c 130.0(1) 28.6169(6) 15.2350(4) 14.5380(3) 90 103.953(2) 90 6151.2(2) 8 1.875

0.7107 (Mo Kα)

0.7107 (Mo Kα)

1.5418 (Cu Kα)

8.896

8.936

12.874

896 12472 5347 [Rint = 0.0319] 0.0272 0.0344

920 14504 5332 [Rint = 0.0306] 0.0244 0.0297

3392 10557 6215 [Rint = 0.0241] 0.0431 0.052

Table 2. Selected Bond Lengths in [ReOL1], [ReOL2], and [ReOL3] Re1−O1 Re1−O2 Re1−N1 Re1−N2 Re1−N3

[ReVOL1]

[ReVOL2]

[ReVOL3]

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 [H4L3]+, resulting in a peak at m/z 431.2072. The 1H and 13C 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. 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 environment, 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

DOI: 10.1021/acs.inorgchem.6b00972 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry 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

DOI: 10.1021/acs.inorgchem.6b00972 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

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