DEA-Me for Beta Amyloid Imaging

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Chalcone Based Homodimeric PET Agent, C-(Chal)DEAMe for Beta Amyloid Imaging: Synthesis and Bioevaluation Kanchan Chauhan, Anjani K Tiwari, Nidhi Chadha, Ankur Kaul, Ajai Kumar Singh, and Anupama Datta Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01070 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 10, 2018

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Molecular Pharmaceutics

Chalcone Based Homodimeric PET Agent,

11

C-(Chal)2DEA-Me for Beta

Amyloid Imaging: Synthesis and Bioevaluation Kanchan Chauhan,a,b Anjani K. Tiwari,a Nidhi Chadha,a Ankur Kaul,a Ajai Kumar Singh,c and Anupama Dattaa* a.

Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine & Allied Sciences, DRDO, Brig. SK Mazumdar Marg, Delhi-110054, India

b. c.

Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Ensenada, Baja California, Mexico Department of Chemistry, Indian Institute of Technology, Delhi-110016, India

Abstract Homodimeric chalcone based

11

C-PET radiotracer,

11

C-(Chal)2DEA-Me was synthesized and

binding affinity towards beta amyloid (A) was evaluated. The computational studies revealed multiple binding of the tracer at the recognition sites of A fibrils. The bivalent ligand,

11

C-

(Chal)2DEA-Me displayed higher binding affinity compared to the corresponding monomer, 11CChal-Me and classical A agents. The radiolabeling yield with carbon-11 was 40-55% (decay corrected) with specific activity of 65‒90 GBq/μmol. A significant (p < 0.0001) improvement in the binding affinity of 11

11

C-(Chal)2DEA-Me with synthetic Aβ42 aggregates over the monomer,

C-Chal-Me demonstrates the utility of bivalent approach. The PET imaging and biodistribution

data displayed suitable brain pharmacokinetics of both the ligands with higher brain uptake in case of bivalent ligand. Metabolite analysis of healthy ddY mice brain homogenates exhibited high stability of the radiotracers in the brain with >93% intact tracer after 30 min post injection. Both chalcone derivatives were fluorescent in nature and demonstrated significant changes in the emission properties after binding with Aβ42. The preliminary analysis indicate high potential of 11

C-(Chal)2DEA-Me as in vivo Aβ42 imaging tracer and highlights the significance of bivalent

approach to achieve higher biological response for detection of early stages of amyloidosis. 1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Keywords: Amyloid beta42, Bivalent ligand, Chalcone, PET, Carbon-11 Introduction A number of debilitative pathological conditions involving protein misfolding represent a class of disorders that display aggregation of insoluble β-sheet forming rich, filamentous protein deposits in tissue. These protein deposits are specifically called amyloid beta (Aβ) aggregates.1,2 The well documented Aβ pathology include amyloid plaques in Alzheimer’s disease (AD),3 deposits in the walls of cerebral arteries and capillaries causing cerebral amyloid angiopathy (CAA),4 prion in transmissible prion disease, and amylin in diabetes type II.5,6 Also, traumatic brain injury (TBI), a major health issue induces neuropathological changes forming amyloid plaques deposited throughout the brain in moderate to severe TBI case.7,8 Within hours of brain injury, extensive plaque formation occurs in all the age groups. This is a growing concern as the progressive amyloid plaque pathology is implicated in syndromes of cognitive impairment, and is the epidemiological link with AD.3,7,9,10 The most aggregation prone and toxic species of Aβ plaques in AD is Aβ42 which is also a predominant form in TBI associated oligomers and plaques.11,12 As these Aβ deposits occur prior to the disease onset, their in vivo quantification may facilitate early diagnosis of the corresponding Aβ pathology. Non invasive nuclear imaging with positron emission tomography (PET) based radiotracers targeting Aβ protein aggregates in the living brain is gradually becoming more useful in clinical research and the development of drug.13 The PET tracers including B,14,15

18

F-flutemetamol,16

18

F-florebetapir,17

18

11

C-Pittsburgh compound-

F-florbetaben18 have successfully demonstrated

their potential for detecting Aβ aggregates in brain. Aβ42 exists in varied isoforms with diverse lengths and concentrations.19 Thus, further improvement in the binding affinity and selectivity of Aβ agents towards Aβ deposits may allow the determination of concentration and location for early

2 ACS Paragon Plus Environment

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diagnosis. In this regard, multivalent ligands are widely utilized in various biological systems for enhanced binding affinity towards their biological targets.20–23 The repeated β-sheet structure of Aβ aggregates possesses multiple binding sites. It is possible that the simultaneous interaction to the multiple recognition sites by a multivalent ligand may lead to thermodynamically more favorable binding. This would result in improved binding affinity over the corresponding monomer. Consequently, the multivalent approach has recently drawn quite attention for Aβ imaging. However, for most of these multivalent ligands, the molecular mass exceeds the general threshold value (600-700 Da) for BBB penetration, depicting nominal brain uptake or have not displayed improvement in the binding efficiency.24–29 In the search for new pharmacophores other than the molecules based on classical dye structures, Ono et al. introduced the first novel chalcone structure as Aβ42 specific SPECT tracer,

125

I-

DMIC.30 The chalcones belong to the class of naturally occurring flavanoids and structural analogues to curcumin that is known to have high BBB permeability and significant binding to Aβ protein in vivo. The electron donating groups at the terminal of chalcone scaffold are crucial in Aβ binding wherein dimethylamino group displayed highest binding efficiency over other functionalities. A number of radioligands based on chalcone core structure have been studied as 125

I, 18F, 99mTc, 68Ga-labeled Aβ tracer.29,31

In the present work, we have utilized a bivalent design for the development of chalcone based homodimeric

ligand,

(2E,2'E)-1,1'-((((methylazanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(4,1-

phenylene))bis(3-(4-(dimethylamino)phenyl)prop-2-en-1-one)

[(Chal)2DEA-Me]

for

PET

imaging of Aβ plaques after labeling with carbon-11 (11C) radioisotope. While selecting the framework the parameters like molecular weight, BBB permeability and brain pharmacokinetics were considered. The two chalcone moieties possessing terminal dimethylamino group have been



tethered with the help of a linker. The binding potential of the developed ligand was first examined through molecular modelling studies and then tested in vitro with synthetic Aβ42 peptide. The distribution of the radiolabeled ligand in the brain and other organs was evaluated in vivo in the healthy mice. The efficiency of the compound was compared with the 11C-radiolabeled monomeric unit. Experimental Methods Chemicals and Instrumentation. All reagents and solvents were commercially obtained from Sigma-Aldrich, Fischer-Scientific, or Merck and utilized without further purification unless otherwise specified. The reaction progress was monitored by thin-layer chromatography (Silica gel 60, F254, Merck). Column chromatography was carried out using silica MN60 (60−200 μm). 1

H NMR spectra were recorded on Bruker Avance II spectrometer (400 MHz and 300 MHz) with

TMS as the internal standard. Mass spectral data were obtained by Agilent 6310 ion trap mass spectrometer. HPLC trace analyses were performed on the JASCO HPLC system (JASCO). A NaI (Tl) scintillation detector system was used to monitor effluent radioactivity, and radioactivity measurements during synthesis and animal studies were performed with a Curiemeter (Aloka).

Computational Methodology. All the computational studies were performed with the molecular modeling software, Schrödinger, LLC, New York, NY, 2013 maestro 9.7. The docking study was performed by using coordinates of solution NMR 3D structure of Aβ fibrils of five chains (5 Aβ (1-42)); PDB: 2BEG)32 downloaded from protein data bank (PDB) www.rcsb.org and used to visualize the interaction of ligands with the fibrils model of β-sheets. The analysis was performed after preparing the protein structures using Preparation Wizard. The preparation of the Aβ fibrils 2BEG includes, i) pre-processing stage involving assignment of bond orders, addition of hydrogen


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and deletion of water molecules beyond 5 Å from hetero (het) groups; ii) minimization and optimization of protein structure in the presence of force field OPLS2005. Grid calculations were performed for the protein active site by generating the default size of grid box x = 33.8893 Å, y = 22.068 Å, and z = 10.424 Å. The ligands were prepared using the Ligprep v2.9 module, which involves generation of low energy conformers for docking analysis. Further, the potential binding pockets were identified for grid-generation without any constraints. Scaling factors were not applied to the Van der Waals radii and no constraints were applied while docking. Default settings were used for all the remaining parameters for docking of ligands via IFD methodology of Schrödinger software.33

Ligand Synthesis. Synthesis of tert-butyl-(2-bromoethyl)carbamate(1), (E)-1-(4-hydroxyphenyl)3-(4-isopropylphenyl)prop-2-en-1-one(2),

(E)-tert-butyl-2-(4-(3-(4-

(dimethylamino)phenyl)acryloyl)-phenoxy)ethylcarbamate(4),

and

(E)-1-(4-(2-

aminoethoxy)phenyl)-3-(4-(dimethylamino)phenyl)-prop-2-en-1-one(5) were similar as reported in our previous study.29

Synthesis of (E)-3-(4-(dimethylamino)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (ChalMe, 3). Under inert moisture free atmosphere, compound 2 (50 mg, 0.187 mmol) was dissolved in dry DMF (2 mL) containing sodium hydride (NaH; 0.935 mmol) and stirred for 10 min at ambient temperature. The reaction mixture was filtered and transferred to a dry reaction vial under inert atmosphere. Methyl iodide (34.97 μL, 0.561 mmol) was added dropwise and the reaction mixture was again heated for 30 min at 70 ˚C. Water (1 mL) to the reaction mixture and the compound was extracted with dichloromethane. The organic layers were combined, dried over Na2SO4 and



evaporated under vacuum. The final residue was purified by column chromatography using dichloromethane to give 3 (30.8 mg, yield 58.4%). δH (400 MHz; CD3OD; Me4Si): 3.06 (6H, s, 2 × CH3), 3.91 (3H, OCH3), 6.78 (2H, d, J = 8.8 Hz, 2 × CH), 7.05 (2H, d, J = 8.8 Hz, 2 × CH), 7.51 (1H, d, J = 15.2 Hz, CH), 7.61 (2H, d, J = 8.8 Hz, 2 × CH), 7.74 (1H, d, J = 15.2 Hz, CH), 8.07 (2H, d, J = 9.0 Hz, 2 × CH). δC (100 MHz; CDCl3; Me4Si): 36.22 (CH3), 51.52 (OCH3), 107.91, 109.73, 112.76, 118.92, 126.34, 126.61, 127.94, 141.03, 147.98, 159.03, 185.01 (CO); ESI-MS(+): m/z [M + 2H+] calcd (found): 283.1 (283.3).

Synthesis of (E)-1-(4-(2-bromoethoxy)phenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-1-one (6). The synthesis was performed according to the earlier reported procedure with some modifications.34 To the mixture of compound 2 (0.50 g, 1.872 mmol) and K2CO3 (0.500 g) in acetonitrile (30 mL) was added 1, 2-dibromoethane (3.51 g, 18.726 mmol) which was taken in excess to avoid the formation of disubstituted product. The reaction mixture was refluxed for 6 h. The reaction mixture was brought to room temperature, filtered and evaporated under vacuum to obtain a yellow residue. The crude product was purified by silica gel column chromatography using dichloromethane as mobile phase to give compound 5 (0.56 g, yield 81.1%). δH (300 MHz; CDCl3; Me4Si): 3.05 (6H, s, 2 × CH3), 3.65 (2H, t, J = 6.3 Hz, CH2), 4.35 (2H, t, J = 6.3 Hz, CH2), 6.69 (2H, d, J = 9.0, 2 × CH), 6.97 (2H, d, J = 9.0 Hz, 2 × CH), 7.32 (1H, d, J = 15.3, CH), 7.54 (2H, d, J = 8.7, 2 × CH), 7.78 (1H, d, J = 15.3, CH), 8.02 (2H, d, J = 8.7, 2 × CH); δ C (75 MHz; CDCl3; Me4Si): 40.07 (CH3), 28.66 (CH2), 67.81 (CH2), 111.80, 114.26, 116.56, 122.76, 130.25, 130.54, 132.49, 145.12, 151.92, 161.32, 188.78 (CO). ESI-MS (+): m/z [M + 2H+] calcd (found): 375.0 (375.9).


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Synthesis of (2E,2'E)-1,1'-(((azanediylbis(ethane-2,1-diyl))bis(oxy))bis(4,1-phenylene))bis(3(4-(dimethylamino)phenyl)prop-2-en-1-one) [(Chal)2DEA, 7]. The mixture of compound 4 (0.40 g, 1.29 mmol), 5 (0.48 g, 1.29 mmol) and K2CO3 (0.500 g) in acetonitrile (30 mL) was refluxed and reaction progress was monitored by thin layer chromatography. After the completion of the reaction, the reaction mixture was filtered, concentrated under vacuum and purified by column chromatography using 5% methanol in dichloromethane as mobile phase to give compound 6 (0.60 g, yield 78.1%). δH (300 MHz; CDCl3; Me4Si): 3.02 (12H, s, 4 × CH3), 3.12 (4H, t, J = 5.1 Hz, 2 × CH2), 4.16 (4H, t, J = 5.1 Hz, 2 × CH2), 6.66 (4H, d, J = 8.7, 4 × CH), 6.96 (4H, d, J = 8.7 Hz, 4 × CH), 7.31 (2H, d, J = 15.3, 2 × CH), 7.52 (4H, d, J = 8.7, 4 × CH), 7.75 (2H, d, J = 15.3, 2 × CH), 7.99 (4H, d, J = 8.7, 4 × CH); δC (75 MHz; CDCl3; Me4Si): 40.07 (CH3), 48.55 (CH2), 67.65 (CH2), 111.82, 114.17, 116.69, 122.85, 130.21, 130.49, 132.06, 144.93, 151.89, 162.14, 188.83 (CO). ESI-MS (+): m/z [M + H+] calcd (found): 604.3 (604.0).

Synthesis

of

(2E,2'E)-1,1'-((((methylazanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(4,1-

phenylene))bis(3-(4-(dimethylamino)phenyl)prop-2-en-1-one) [(Chal)2DEA-Me, 8]. The procedure was similar to the procedure followed for compound 3. Compound 8 was obtained as yellow solid (32.6 mg, yield 63.8%). δH (300 MHz; CDCl3; Me4Si): 2.95 (3H, s, NCH3), 2.98 (4H, t, J = 5.7 Hz, 2 × CH2), 3.02 (12H, s, 4 × CH3), 4.17 (4H, t, J = 5.7 Hz, 2 × CH2), 6.67 (4H, d, J = 9.0, 4 × CH), 6.94 (4H, d, J = 8.7 Hz, 4 × CH), 7.31 (2H, d, J = 15.3, 2 × CH), 7.52 (4H, d, J = 8.7, 4 × CH), 7.75 (2H, d, J = 15.3, 2 × CH), 7.99 (4H, d, J = 8.7, 4 × CH); δC (75 MHz; CDCl3; Me4Si): 40.11 (CH3), 43.41 (CH3), 56.44 (CH2), 66.22 (CH2), 111.82, 114.21, 116.63, 122.83, 130.28, 130.53, 131.95, 145.07, 151.91, 162.08, 188.98 (CO). ESI-MS(+): m/z [M+] calcd (found): 618.3 (618.0).



Production of 11C-iodomethane. 11C-CO2 was produced by the nuclear reaction 14N(p,α)11C in nitrogen gas atmosphere encompassing 0.01% oxygen with 18-MeV protons using cyclotron CYPRIS HM-18 (Sumitomo Heavy Industry, Tokyo, Japan).35 Subsequently, it was transferred into the reaction vial consisting of 0.4 M LiAlH4 in dry THF (300 μL) at 0˚C under inert atmosphere. THF was evaporated and 57% HI (300 μL) was added to the reaction mixture to give 11

C-CH3I which was distilled and transferred via stream of N2 gas (flow rate 30 mL/min) into the

vessel containing DMF (500 μL) at ambient temperature.

Radiolabeling of Chal (2) with

11

C. Radiolabeling was performed according to the reported

literature.35 Prior to the addition of 11C-CH3I, the precursor 2 (1 mg, 3.74 μmol ) was dissolved in dry DMF, NaH (18.7 μmol) was added and the reaction mixture was stirred at room temperature for 5 min. The solution of cyclotron produced 11C-CH3I in DMF was added to the reaction mixture and it was heated for 3 min at 80 ˚C. The reaction mixture was then cooled on an ice bath and quenched with the addition of water (500 μL). HPLC separation of the final mixture was performed on Shiseido Capcell Pak UG80 C18 column (10 mm i.d. × 250 mm) using CH3CN/CH3CO2NH4 buffer solution (pH 4.3, 50mM) (30/70, v/v) at 5.0 ml/min and 370 nm. To a flask containing ethanol (300 μL) and Tween 80 (75 μL), the fraction containing 11C-Chal-Me (retention time, tR = 8.35 min) was collected. The resulting solution was evaporated to dryness, redissolved in the sterilized saline solution and filtered with 0.22 μm Millipore filter. The identity of the radiolabeled product was established by co-injection of cold Chal-Me on a reversed phase analytical HPLC with Capcell Pak UG80 C18 column (4.6 mm i.d. × 250 mm) using mobile phase as CH3CN/CH3CO2NH4 buffer solution (pH 4.3, 50mM) (30/70, v/v) at 1.0 ml/min (tR = 8.15 min).


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By comparing the examined radioactivity with that of the mass of the compound measured at UV, the specific activity of the ligand was calculated.

Radiolabeling of (Chal)2DEA (7) with

11

C. Radiolabeling of compound 7 was performed

according to the procedure used for radiolabeling of Chal (2).

11

C-(Chal)2DEA-Me was obtained

as product. Retention time (tR) of the final radiolabeled ligand on radio HPLC with similar gradient system was found to be 10.47 min and its identity was again examined by the coinjection of the cold ligand (Chal)2DEA-Me (tR = 9.79 min).

In Vitro Binding Studies with Aβ Aggregates. Solid form of Aβ42 was purchased from SigmaAldrich and aggregation was carried out by gently dissolving the peptide (0.25 mg mL−1) in a pH 7.4 buffer solution containing 1 mM EDTA and 10 mM sodium phosphate. The solution was incubated at 37 °C for 42 h with gentle and constant shaking. The specificity of the developed radioligands to bind with Aβ plaques was examined by previously described procedure with some modifications.30,36 The binding assay on Aβ42 aggregates was performed by mixing 100 μL of Aβ42 aggregates (0‒1050 nM) with 100 μL of an appropriate concentration of radioligands and 900 μL of PBS in 12 mm × 75 mm borosilicate glass tubes. Non-specific binding was estimated in the presence of 1μM (Chal)2DEA-Me/ Chal-Me. For the blocking assay, the study was performed in the presence of 100-fold excess of unlabeled DMIC and (Chal)2DEA-Me. Concentration of 50 nM was maintained for Aβ42 in the final solution. The reaction mixture was incubated at 37 °C for 20 min, and then filtered through GF/B Whatman filters by vacuum filtration to separate bound and free radioactivity. The bound activity present in the filter was measured in gamma scintillation counter. The difference between total binding (in the absence of non-



radiolabeled ligand) and non specific binding was calculated to obtain specific binding.

Small Animal PET Imaging. The PET imaging was performed on a small-animal PET scanner (Inveon; Siemens Medical Solutions). The anesthesization of healthy Sprague-Dawley (SD) (male, 240-330 g) rats was done with isoflurane (1‒2 %) and a 40˚C water circulation system (T/Pump TP401, Gaymar Industrie) was used for maintaining their body temperature during the experiments. The 11C-(Chal)2DEA-Me or 11C-Chal-Me (35-37 MBq) were intravenously injected through the tail vein and immediately the emission scan was acquired for 60 min. A group of three animals was used for each experiment and a single PET scan was acquired with one animal.

Image Analysis. The data modelling of PET was done in 3-D sinograms which by Fourier sinograms were converted to 2-D sinograms (frames × min: 4 × 1, 8 × 2, 8 × 5). For dynamic image rebuilding, back-projection filtered with Hanning’s filter and a Nyquist cut-off frequency (0.5 cycle/pixel) was used. Analyses of the PET images was done using using ASIPro VM™ (Analysis Tools and System Setup/Diagnostics Tool; Siemens Medical Solutions) with the MR imaging template as the reference. Time-activity curves (TACs) were generated for contralateral and ipsilateral striatum. Decay-correction was done for radioactivity uptake in the brain where injection time was considered as a reference and it was expressed as standardized uptake value. It was then normalized for the body weight and injected radioactivity. The standardized uptake value (SUV) was calculated as: SUV = (radioactivity per cubic centimetre tissue/injected radioactivity) × grams body weight.


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General Method for Metabolite Analysis. The normal ddY mice were injected with 29‒42 MBq of the ligand of interest through the tail vein. At the time point of 10 and 30 min, the mice were sacrificed by cervical dislocation and the whole brain sample was obtained. Immediately after the dissection, the brain homogenates were prepared by mechanically homogenizing the nitrogenfrozen brain samples and adding PBS (1 mL). The mixture was vortexed vigorously and MeCN was added to it. The mixture was centrifuged for 5 min, the supernatant was separated and analyzed through radio-HPLC under following conditions:37 Capcell Pack UG80C18 column, 4.6 mm i.d. × 250 mm; MeCN/H2O/Et3N, 7/3/0.01 (v/v/v); flow rate, 0.8 mL/min. The percentage intact tracer was calculated. The activity of the waste fractions of HPLC was measured with auto-gamma counter.

Biodistribution. Normal ddY mice (n = 3) (male, 34-36 g) were injected with saline solution of 11

C-(Chal)2DEA-Me (5.6 MBq in 100 μL per mouse) directly through the tail vein.37 The mice

were sacrificed by cervical dislocation at the time framework of 1‒60 min. The organs of interest were quickly removed, rinsed with chilled saline and weighed. A gamma counter (1480 Wizard 3, Perkin-Elmer, Waltham, MA, USA) was used for the measurement of the radioactivity in each organ and the data was expressed as percentage injected dose per gram of the organ (% ID/g). All radioactivity calculations were decay corrected.

UV-Vis Spectroscopic Measurements using Aβ Aggregates. For the fluorescence studies, stock solution of pre-aggregated Aβ42 (0.2 μM) was prepared and added to 150 μL of the probe dissolved in PBS buffer (pH 7.4) to attain a final concentration of 10 nM. The solution was transferred to a 96 well microplate and the fluorescence emission spectrum



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of 3 and 8 bound to background or Aβ42 were collected from the 500–700 nm range. To calculate the ratio of the probe to background signal, peak intensity for probe bound to Aβ was divided by an average of background measurements. A minimum of 10 measurements was taken for 3 and 8 bound to background and to Aβ. The experiment was also performed in the presence of 2,2'-((azanediylbis(ethane-2,1diyl))bis(azanediyl))bis(1-(4-(2-methoxyphenyl)piperazin-1-yl)ethanone) (Bis-mpp) and bovine serum

albumin.

The

concentration

range

of

2,2'-((azanediylbis(ethane-2,1-

diyl))bis(azanediyl))bis(1-(4-(2-methoxyphenyl)piperazin-1-yl)ethanone) was 5-20 nM for the experiment. For carrying out the studies in the presence of BSA, BSA was first dissolved in buffer, filtered through a filter (0.2 μm) into sterile tubes and incubated in a water bath at 60-75°C without agitation for 4 days.38 It was taken in 5-20 nM concentration range for the studies. The final concentration of (Chal)2DEA-Me and Aβ42 was 10 nM each in the competitive binding studies.

Results and Discussion Amyloidosis disorders caused due to the aberration in protein conformation and metabolism converts non-pathogenic peptides and proteins into insoluble fibrils which can eventually lead to various neurodegenerative disorders due to β- amyloid peptide misfolding. In Aβ pathology, a significant plaque formation takes place before the onset of the clinical symptoms and a major impediment in the management of the disease is inability to diagnose the disease at the possible earliest stage. Thus, there is a demand of ligands targeted towards Aβ plaques for improved diagnostic accuracy to increase certainty, and identify patients at risk of cognitive impairment in the early stages. So far, PET radiopharmaceuticals labeled with short lived radionuclides 11C and 18

F have been widely utilized in Aβ quantification.39,40 The


11

C-tracers are particularly widely

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utilized in clinical trials due to the low radiation doses offered by 11C to the healthy control group as well as the patient. The half life of 11C is adequate enough for multistep synthetic procedures and short enough for repetitive studies in the same subject on same day. As brain is the most delicate organ, therefore low radiation burden is favored and hence, there has been a brief history of utilizing 11C-labeled radiopharmaceuticals in imaging neurological disorders.41-43 In the present work, the hypothesis for enhanced binding affinity and brain uptake with the use of bivalent ligand approach in Aβ imaging has been evaluated. Based on our earlier work, chalcone with the terminal dimethylamino group has been used as Aβ binding pharmacophore.29 The designed ligand, 11C-(Chal)2DEA-Me holds highly conjugated rigid aromatic ring system through a flexible linker which may allow the simultaneous binding at two recognition sites resulting in high binding affinity towards Aβ aggregates.

Molecular Docking Studies. The computational study is aimed to gain insight on the interaction between Aβ proteins and proposed ligands. For the purpose three dimensional structure of Aβ fibrils [5 Aβ (1-42); PDB: 2BEG] was used.27 It is a five chain structure with sequence from human and has two alpha helical regions ranging from residues 8‒25 and 28‒38 joined by beta turn. There are small stretches known in Aβ peptide which are known to be responsible for amyloid formation. In PDB 2BEG, amino acid stretches from 16‒21 (KLVFFA) and 32‒36 (IGLMV) were found to be amyloidgenic and used for docking analysis after grid generation. Monomeric unit (Chal-Me) was taken into consideration and was evaluated to compare with bivalent ligand, (Chal)2DEA-Me. The molecular docking pose analysis of the monomeric (Chal-Me) and the bivalent chalcone ((Chal)2DEA-Me) analogues was performed with Aβ42 fibrils to understand the mode of binding that might also be effective in inhibition of Aβ aggregates. The binding interaction of bivalent ligand was compared with the corresponding monomer and the classical ligands. The induced fit 13 ACS Paragon Plus Environment


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docking (IFD) of the test ligands were performed at the site of amino acid stretches and the docking results are shown in the Table 1 and Figure 1.

Table 1. The thermodynamic parameters of IFD docking Glide

Glide

Glide

Glide

Gscore Evdw

Ecoul

Energy

Einternal Score

-12.79

-73.36

-4.23

-77.59

15.91

-243.96

-8.11

-50.89

-2.37

-53.26

2.47

-233.02

DMIC

-9.36

-54.75

-4.67

-59.43

4.03

-239.07

AV-45

-9.11

-53.00

-3.17

-56.17

0.10

-234.35

PIB

-8.08

-46.75

-5.94

-52.69

0.16

-234.10

Ligands (Chal)2DEA-Me (Dimer) Chal-Me (Monomer)

XP

IFD

In beta amyloid structure, parallel orientation of β-sheets generate hydrophobic pockets along the length of the filament where aromatic molecules intercalate and interact via π-π interaction, Hbonding or hydrophobic interactions. In the study, all the molecules entered the hydrophobic channel of residues along its long axis parallel to the axis of Aβ fibrils whereas in case of dimer, simultaneous interactions with two recognition sites were found. Through the moderate flexible structure, (Chal)2DEA-Me accommodates and tightly binds to the hydrophobic cavity in 3D Aβ fibrils displaying the highest binding score (Gscore) of ‒12.790 as shown in Figure 1B and Table 1.


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Figure 1. The binding mode depicted by 3D docking pose and 2D view of ligand interaction diagram of (A) Chal-Me and (B) (Chal)2DEA-Me associated with amyloid beta fibril (2BEG) respectively

When compared with (Chal)2DEA-Me (Figure 1B), the C=O group of monomer (Chal-Me)


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displays interactions of with the Met35 of chain D (Figure 1A) and occupies binding pocket with good pose score of ‒8.113. The known Aβ ligands such as iodinated derivative of chalcone (DMIC), Florbetapir (AV-45) and Pittsburgh compound-B (PIB) bind with the protein with Gscore of ‒9.11, ‒9.36, ‒8.08 and ‒6.401 respectively (Table 1 and Supporting Information, Figure S1). The vicinity of residues, Asp23, Chain D and E; Glu22, Chain E and D; Leu34, Chain E; Ala21, Chain D; Met35, Chain E and Ala21, Chain D can be seen enclosing the chalcone moieties in all chalcone based ligands (DMIC, (Chal)2DEA-Me and Chal-Me). Also, the comparison of interaction energies, electrostatic and van der Waals energies in terms of Emodel, of (Chal)2DEAMe with the monomer and known ligands displayed the better ligand binding free energy towards Aβ fibril’s amyloidgenic stretch. Hence, in comparison to known ligands, (Chal)2DEA-Me showed the highest binding score by induced fit of two monomeric units stretched onto the hydrophobic units and high binding affinity. The computational analysis could provide a meaning insight on mode of binding at the hydrophobic pocket of Aβ fibrils which was further authenticated through various in vitro and in vivo studies.

Ligand Synthesis and Radiolabeling. The pivotal role of chalcones in targeting Aβ42 amyloids prompted us to utilize its activity in bivalent ligand approach and bioevaluate the fate of the bivalent ligand in PET imaging after labeling with 11C. The bivalent hypothesis was explored and the ligand was developed from an optimized synthetic strategy wherein the two units of chalcones were linked via a linker for designing the bivalent ligand. Monomeric conjugate of chalcone was also synthesized for the comparative studies. The synthetic route employed is shown in Scheme 1.



Scheme 1. Synthetic scheme for (Chal)2DEA-Me and Chal-Me


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The facile synthetic methodology utilized classical organic chemistry including condensation and nucleophilic substitution reactions. Commercially available N,N-dimethylbenzaldehyde and 4hydroxyacetophenone undergo Claisen-Schmidt condensation reaction in basic medium to give Eisomer of the chalcone (2). The O-methylation of compound 2 with methyliodide in the presence of sodium hydride at the hydroxyl position gave the monomer, Chal-Me (3) in 58.4% yield. The compound 2 was again functionalized to corresponding amino and bromo intermediates (5 & 6) to give bivalent ligand with C2 symmetry. The intermediate 5 was obtained by the conjugation of bromoethylamine-boc (4) with compound 2 followed by the cleavage of boc group.

The

intermediate 6 was obtained by the reaction of dibromoethane with compound 2. The conjugation of compounds 5 and 6 gave the dimeric compound (Chal)2DEA (7) in 78.1% yield. The Nmethylation of compound 7 was achieved under the conditions employed for the methylation of compound 3 and it gave final product, (Chal)2DEA-Me (8) in 63.8% yield. The proposed structure and stoichiometry of the chalcone derivatives were validated by NMR and mass spectrometry. The purity of the final products was found to be >98%. The introduction of C-11 radioisotope is commonly done by the reaction with

11

C-CH3I via

nucleophilic substitution reaction. The protocols for 11C-CH3I preparation are well established. In the present study, wet chemistry method has been utilized and radiolabeling of ligand was performed in the presence of sodium hydride.35 The radiolabeling of precursors 2 and 7 by 11CCH3I was done via O-11C-methylation and N-11C-methylation respectively to produce 11C-ChalMe and

11

C-(Chal)2DEA-Me. The tracers were isolated by reversed phase HPLC in high

radiochemical yields (40‒55%, decay corrected) and purity (>98%) (Supporting Information, Figure S14 and S15). The identity of 11C-Chal-Me and 11C-(Chal)2DEA-Me was established by co-injection of non-radiolabled Chal-Me and (Chal)2DEA-Me respectively. The overall



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preparation time for both the tracers was 18‒20 min from end of bombardment (EOB) and the specific activity was calculated as 65‒90 GBq/μmol at the end of synthesis (EOS).

In Vitro Binding Study with Aβ42 Aggregates. Aggregation of cytotoxic amyloid-beta peptide (Aβ) into pathological depositions in the brain is the main reason for progressive cognitive decline. The 11C labeled compounds 2 and 7 were evaluated for in vitro binding affinity with Aβ aggregates and in vivo brain pharmacokinetics. The evaluation of binding affinity of chalcone derivatives towards Aβ aggregates was performed in solution. Aβ42 was chosen for the study as it is the prime amyloid present in Aβ plaques.44 The quantitative comparison of 11C-(Chal)2DEAMe and 11C-Chal-Me was performed with different concentration of synthetic Aβ42 aggregates. Figure 2A shows the binding affinity as the percentage of total aggregate bound activity. In terms of aggregate bound activity, both ligands exhibited high binding while the order of binding of 11C(Chal)2DEA-Me (12.43 ± 1.7 %) was ~1.6 fold greater than the monomer 11C-Chal-Me (7.78 ± 1.03 %) indicating enhanced binding affinity with bivalent ligand approach. The percentage binding of the bivalent ligand was further enhanced to ~2-fold at higher concentration of Aβ42 aggregates. The binding efficiency of Chal-Me was observed to be similar to the chalcone based 99m

Tc derivative34 while (Chal)2DEA-Me had better binding efficiency than that of the

99m

Tc

complex. The non-specific activity was measured in the presence of excess of non-radiolabeled Chal-Me/(Chal)2DEA-Me 1.5-2.8 %. Furthermore, the blocking assay in the presence of large concentration of DMIC, displayed reduction of binding of 11C-Chal-Me and 11C-(Chal)2DEA-Me by ~72% and ~80% respectively (Figure 2B). The results thereby indicate the high and specific binding of both the ligands towards Aβ aggregates.


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Figure 2(A). Comparative binding assay and; (B). Blocking assay of the radiolabeled ligands with Aβ42 aggregates. Results are expressed as mean ± SEM (n = 3). ****p < 0.0001 using 2-way ANOVA

Bonferroni’s test.

UV-Vis Spectroscopic Measurements and Binding assay. Chalcone with push-pull conjugation system undergoes intra charge transfer resulting in delocalization of electrons within the molecule. The electronic structure with the donor and acceptor groups separated by the polarizable group possesses an appropriate range of the absorption and emission bands. An Aβ optical imaging probe should possess essential properties like (i) molecular mass in the range of 600‒700 Da, (ii) high specificity and binding affinity towards Aβ plaques, (iii) emission wavelength above 450 nm, (iv) straightforward synthesis and (v) a significant alteration in the emission properties upon binding with Aβ aggregates.45 Thus, the perspective was evaluated for the developed ligands, Chal-Me and (Chal)2DEA-Me by understanding the photophysical properties pre and post binding, with synthetic Aβ42 aggregates. In the UV-vis spectroscopic measurement, Chal-Me exhibited absorbance at 410 nm and emission at 530 nm while (Chal)2DEA-Me gave absorbance at 430 nm and emission at 580 nm (Figure 3). 21 ACS Paragon Plus Environment


Figure 3. Absorbance and fluorescence spectra of Chal-Me and (Chal)2DEA-Me

Both the ligands met the essential criteria to serve as useful candidates for Aβ optical imaging. Therefore, the UV-vis properties of the ligands were measured in the presence of synthetic Aβ42 aggregates. The emission intensity of the free probe was compared with the emission intensity of probe after mixing with Aβ42 aggregates. Chal-Me showed 6.2 while (Chal)2DEA-Me displayed 8.5 fold increase in emission intensity upon binding with Aβ42 aggregates with a hypsochromic shift of 10-20 nm (Figure 4). A remarkable enhancement in the emission intensity and the hypsochromic shift in the emission spectra suggest the high binding potential. These changes in the emission properties are beneficial for obtaining high contrast in optical imaging. The results depict that the developed ligands possess potential for Aβ optical imaging as well and can be further explored in this direction.


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Figure 4. Emission spectra of Chal-Me, (Chal)2DEA-Me before and after mixing with Aβ42 aggregates

To rule out the possibilities of false positive results, the fluorescence property of the ligands was explored to study the variation in binding potential of (Chal)2DEA-Me in the presence of ligand with similar physicochemical properties, bivalent methoxy phenyl piperazine derivative (Bismpp). Treatment with Bis-mpp did not affect the emission intensity of the ligand mixed with Aβ42 aggregates. Also, the fluorescence data suggested that the presence of BSA led to a negligible variation in the fluorescence emission intensity (± 3%) thus highlighting insignificant binding of the bivalent ligand to β rich protein, BSA (Figure S16).

Small Animal PET Imaging and Biodistribution. Significant plaque accumulation takes place prior to the earliest clinical symptoms and understanding of the pharmacokinetics of any Aβ specific probe in normal brain is a crucial parameter in deciding its fate in imaging Aβ pathology. An ideal Aβ probe should possess high initial uptake with a rapid washout from the normal brain so as to achieve high signal to noise ratio in brain. The potential of the dimeric ligand to penetrate the blood brain barrier and its pharmacokinetics was understood by dynamic PET brain imaging of normal SD rat after intravenous administration through the tail vein. The results were compared with the corresponding monomeric unit. The μPET images and the time activity curves displaying the brain uptake kinetics of the two ligands are shown in the Figure 5. The brain regions depicted the higher initial uptake of

11

C-(Chal)2DEA-Me (SUV ~ 3.6) when compared to

11

C-Chal-Me

(SUV ~ 2.1). In the absence of the Aβ plaques in normal brain, the ligands did not exhibit prolonged retention or any specific binding. Both the ligands displayed a rapid washout however, on comparing the time activity curve, the washout was faster in case of the monomer.



Figure 5. PET images and time activity curves for 11C-(Chal)2DEA-Me and 11C-Chal-Me in healthy rats

The biodistribution study of the radiolabeled tracer in normal mice is a method to measure the potential of the probe to penetrate the BBB and washout kinetics from the brain. The biodistribution results in healthy ddY mice were found to be concordant with μPET imaging. A significant high brain uptake of the dimer with a comparatively slower washout in comparison to monomer was seen (Figure 6-8). The brain2min/brain30min ratio for 11C-Chal-Me was 5.08 while for 11

C-(Chal)2DEA-Me was 2.83. Although the washout of the dimer was slower, it was comparable

to that of PiB.46 This rapid washout is crucial for obtaining high contrast images in AD brain. The activity depletion was also found to be fast in blood. The pattern for the route of excretion was similar in both the ligands. Both the renal and the hepatobiliary route of excretion were observed as evident by high uptake in liver and kidney with slow washout. The uptake in the intestine increased with time. The high liver uptake can be attributed to the lipophilic nature of the probe and was analogous to the other reported chalcone derivatives.29,31 Notably, the other non-targeted organs did not show any significant uptake.


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20

2 min 10 min 30 min

15

% ID/g

60 min 10

5

ra in B

St om ac h

SI

id ne y K

Sp le en

Li ve r

Lu ng

t ea r H

B

lo od

0

Organs Figure 6. Biodistribution profile of 11C-Chal-Me in normal ddY mice at various time points after intravenous injection in tail vein.

15

2 min 10 min 30 min

10

60 min

% ID/g 5

ra in B

ac h St om

SI

id ne y K

Sp le en

Li ve r

Lu ng

t ea r H

lo od

0

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Organs

Figure 7. Biodistribution profile of 11C-(Chal)2DEA-Me in normal ddY mice at various time points after intravenous injection in tail vein.



Figure 8. Comparison of the brain uptake of Chal-Me and (Chal)2DEA-Me in normal ddY mice. Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 using a 2-way ANOVA with

Bonferroni’s test. Metabolite Study. After the intravenous administration, a radiotracer immediately comes into the contact of a wide range of metabolizing enzymes present in blood and tissues. Without any exception, almost all the radiotracers are metabolized by the enzymes to form radiometabolites. These troublesome radiometabolites are the insurmountable barrier in the accurate quantification of Aβ proteins in the brain.47 Therefore, the fate of the developed radioligands was analyzed by testing their stability in plasma and brain homogenates. Table 2 shows the percentage of intact tracer after 10 and 30 min. Two radiometabolites corresponding to demethylation were observed and eluted at the earlier time points. Both the ligands displayed high stability in the brain with >93% intact tracer after 30 min. The study therefore, justifies that the brain uptake was mainly due to

11

C-Chal-Me and

11

C-(Chal)2DEA-Me. The radioligands showed a comparatively lower

stability in plasma. However, the biodistribution data revealed a fast depletion of the activity from blood.


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Table 2. Percentage of intact tracer in plasma and brain homogenates of ddY mice Time after the injection (min)

11

C-Chal-Me

11

C-(Chal)2DEA-Me

Plasma

Brain

Plasma

Brain

10

40.3

98.6

37.6

98.7

30

25.8

94.8

23.9

93.3

CONCLUSIONS A major impediment to the management of A pathology is the ability to diagnose the disease at the right time as significant plaque accumulation take place prior to the earliest clinical symptoms. 11

C-labeled homodimeric ligand with two units of chalcone has been developed for improved

binding towards Aβ42 plaques. The bivalent ligand with its unique structure follows the empirical rules for Aβ imaging. The two chalcone moieties tethered with a flexible linker are able to bind to the two recognition sites simultaneously. The computational studies show the higher binding affinity, and thermodynamic stability in comparison to the monomeric and classical ligands. In vitro binding study with synthetic Aβ displays the higher binding of the bivalent ligand. It also demonstrated higher brain uptake with high SUV values and revealed optimum brain pharmacokinetics. Taken together, these preliminary studies suggest that the developed ligand, 11

C-(Chal)2DEA-Me has potential to be utilized as in vivo Aβ imaging tracer and the bivalent

approach may be a useful way to achieve higher biological response towards Aβ pathologies like TBI and AD. Also, the high sensitivity of the fluorescent properties of the bivalent ligand can further be exploited towards the optical imaging of Aβ aggregates.

Associated content



Supporting Information. 3D docking posse depicting the binding and 2D view of ligand interaction diagram of: DMIC; AV-45; and PIB with amyloid beta fibril (2BEG). 1H, 13C NMR, and mass spectra of compounds 3-8. HPLC chromatograms of 11C-Chal-Me and 11C-(Chal)2DEA-Me. Data from fluorescence experiments used for control studies with BSA and Bis-Mpp. This material is available free of charge via the Internet at http://pubs.acs.org. Author Information Corresponding Author *Tel.: +91-11-23905123. E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest.

Acknowledgements We thank Dr. Anil K Mishra, Scientist G, Additional Director, Head DCRS and Dr. A.K. Singh, Scientist H, Director, Institute of Nuclear Medicine and Allied Sciences, Delhi for providing necessary facilities. This work was supported by Defence Research and Development Organization, Ministry of Defence, under project TD-16-17/INM-321. The authors also acknowledge Dr. M. R. Zhang, NIRS, Japan and his team for the technical support provided in optimization of radiobiochemical studies.

References (1)

(2) (3)

(4)

Sipe, J. D.; Benson, M. D.; Buxbaum, J. N.; Ikeda, S.; Merlini, G.; Saraiva, M. J. M.; Westermark, P. Nomenclature 2014: Amyloid Fibril Proteins and Clinical Classification of the Amyloidosis. Amyloid 2014, 21 (4), 221–224. Eisenberg, D.; Jucker, M. The Amyloid State of Proteins in Human Diseases. Cell 2012, 148 (6), 1188–1203. Edwards, G.; Moreno-Gonzalez, I.; Soto, C. Amyloid-Beta and Tau Pathology Following Repetitive Mild Traumatic Brain Injury. Biochem. Biophys. Res. Commun. 2017, 483 (4), 1137–1142. Thal, D. R.; Walter, J.; Saido, T. C.; Fändrich, M. Neuropathology and Biochemistry of Aβ and Its Aggregates in Alzheimer’s Disease. Acta Neuropathol. (Berl.) 2015, 129 (2), 167– 28 ACS Paragon Plus Environment

Page 28 of 33

Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(5) (6) (7)

(8)

(9)

(10) (11)

(12)

(13)

(14)

(15)

(16)

(17) (18)

182. Greenwald, J.; Riek, R. Biology of Amyloid: Structure, Function, and Regulation. Structure 2010, 18 (10), 1244–1260. Westermark, P.; Andersson, A.; Westermark, G. T. Islet Amyloid Polypeptide, Islet Amyloid, and Diabetes Mellitus. Physiol. Rev. 2011, 91 (3), 795–826. Cruz-Haces, M.; Tang, J.; Acosta, G.; Fernandez, J.; Shi, R. Pathological Correlations between Traumatic Brain Injury and Chronic Neurodegenerative Diseases. Transl. Neurodegener. 2017, 6, 20. Johnson, V. E.; Stewart, W.; Smith, D. H. Widespread τ and Amyloid-β Pathology Many Years after a Single Traumatic Brain Injury in Humans. Brain Pathol. Zurich Switz. 2012, 22 (2), 142–149. Bird, S. M.; Sohrabi, H. R.; Sutton, T. A.; Weinborn, M.; Rainey-Smith, S. R.; Brown, B.; Patterson, L.; Taddei, K.; Gupta, V.; Carruthers, M.; Lenzo, N.; Knuckey, N.; Bucks, R. S.; Verdile, G.; Martins, R. N. Cerebral Amyloid-β Accumulation and Deposition Following Traumatic Brain injury—A Narrative Review and Meta-Analysis of Animal Studies. Neurosci. Biobehav. Rev. 2016, 64, 215–228. Gupta, R.; Sen, N. Traumatic Brain Injury: A Risk Factor for Neurodegenerative Diseases. Rev. Neurosci. 2015, 27 (1), 93–100. Mondello, S.; Buki, A.; Barzo, P.; Randall, J.; Provuncher, G.; Hanlon, D.; Wilson, D.; Kobeissy, F.; Jeromin, A. CSF and Plasma Amyloid-β Temporal Profiles and Relationships with Neurological Status and Mortality after Severe Traumatic Brain Injury. Sci. Rep. 2014, 4. DeKosky, S. T.; Abrahamson, E. E.; Ciallella, J. R.; Paljug, W. R.; Wisniewski, S. R.; Clark, R. S. B.; Ikonomovic, M. D. Association of Increased Cortical Soluble abeta42 Levels with Diffuse Plaques after Severe Brain Injury in Humans. Arch. Neurol. 2007, 64 (4), 541–544. Pontecorvo, M. J.; Mintun, M. A. PET Amyloid Imaging as a Tool for Early Diagnosis and Identifying Patients at Risk for Progression to Alzheimer’s Disease. Alzheimers Res. Ther. 2011, 3 (2), 11. Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G.-F.; Debnath, M. L.; Klunk, W. E. Synthesis and Evaluation of 11C-Labeled 6-Substituted 2-Arylbenzothiazoles as Amyloid Imaging Agents. J. Med. Chem. 2003, 46 (13), 2740–2754. Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.; Blomqvist, G.; Holt, D. P.; Bergström, M.; Savitcheva, I.; Huang, G.; Estrada, S.; Ausén, B.; Debnath, M. L.; Barletta, J.; Price, J. C.; Sandell, J.; Lopresti, B. J.; Wall, A.; Koivisto, P.; Antoni, G.; Mathis, C. A.; Långström, B. Imaging Brain Amyloid in Alzheimer’s Disease with Pittsburgh Compound-B. Ann. Neurol. 2004, 55 (3), 306–319. Koole, M.; Lewis, D. M.; Buckley, C.; Nelissen, N.; Vandenbulcke, M.; Brooks, D. J.; Vandenberghe, R.; Van Laere, K. Whole-Body Biodistribution and Radiation Dosimetry of 18F-GE067: A Radioligand for in Vivo Brain Amyloid Imaging. J. Nucl. Med. Off. Publ. Soc. Nucl. Med. 2009, 50 (5), 818–822. Yang, L.; Rieves, D.; Ganley, C. Brain Amyloid Imaging — FDA Approval of Florbetapir F18 Injection. N. Engl. J. Med. 2012, 367 (10), 885–887. Becker, G. A.; Ichise, M.; Barthel, H.; Luthardt, J.; Patt, M.; Seese, A.; Schultze-Mosgau, M.; Rohde, B.; Gertz, H.-J.; Reininger, C.; Sabri, O. PET Quantification of 18F-Florbetaben Binding to β-Amyloid Deposits in Human Brains. J. Nucl. Med. Off. Publ. Soc. Nucl. Med. 2013, 54 (5), 723–731. 29 ACS Paragon Plus Environment


(19) Fissers, J.; Waldron, A.-M.; De Vijlder, T.; Van Broeck, B.; Pemberton, D. J.; Mercken, M.; Van Der Veken, P.; Joossens, J.; Augustyns, K.; Dedeurwaerdere, S.; Stroobants, S.; Staelens, S.; Wyffels, L. Synthesis and Evaluation of a Zr-89-Labeled Monoclonal Antibody for Immuno-PET Imaging of Amyloid-β Deposition in the Brain. Mol. Imaging Biol. MIB Off. Publ. Acad. Mol. Imaging 2016, 18 (4), 598–605. (20) Chauhan, K.; Arun, A.; Singh, S.; Manohar, M.; Chuttani, K.; Konwar, R.; Dwivedi, A.; Soni, R.; Singh, A. K.; Mishra, A. K.; Datta, A. Bivalent Approach for Homodimeric Estradiol Based Ligand: Synthesis and Evaluation for Targeted Theranosis of ER(+) Breast Carcinomas. Bioconjug. Chem. 2016, 27 (4), 961–972. (21) Krall, N.; Pretto, F.; Neri, D. A Bivalent Small Molecule-Drug Conjugate Directed against Carbonic Anhydrase IX Can Elicit Complete Tumour Regression in Mice. Chem. Sci. 2014, 5 (9), 3640–3644. (22) Rook, Y.; Schmidtke, K.-U.; Gaube, F.; Schepmann, D.; Wünsch, B.; Heilmann, J.; Lehmann, J.; Winckler, T. Bivalent Beta-Carbolines as Potential Multitarget AntiAlzheimer Agents. J. Med. Chem. 2010, 53 (9), 3611–3617. (23) Haviv, H.; Wong, D. M.; Silman, I.; Sussman, J. L. Bivalent Ligands Derived from Huperzine A as Acetylcholinesterase Inhibitors. Curr. Top. Med. Chem. 2007, 7 (4), 375– 387. (24) Liu, K.; Guo, T. L.; Chojnacki, J.; Lee, H.-G.; Wang, X.; Siedlak, S. L.; Rao, W.; Zhu, X.; Zhang, S. Bivalent Ligand Containing Curcumin and Cholesterol as Fluorescence Probe for Aβ Plaques in Alzheimer’s Disease. ACS Chem. Neurosci. 2012, 3 (2), 141–146. (25) Iikuni, S.; Ono, M.; Watanabe, H.; Matsumura, K.; Yoshimura, M.; Harada, N.; Kimura, H.; Nakayama, M.; Saji, H. Enhancement of Binding Affinity for Amyloid Aggregates by Multivalent Interactions of 99mTc-Hydroxamamide Complexes. Mol. Pharm. 2014, 11 (4), 1132–1139. (26) Iikuni, S.; Ono, M.; Watanabe, H.; Matsumura, K.; Yoshimura, M.; Kimura, H.; IshibashiUeda, H.; Okamoto, Y.; Ihara, M.; Saji, H. Imaging of Cerebral Amyloid Angiopathy with Bivalent 99mTc-Hydroxamamide Complexes. Sci. Rep. 2016, 6. (27) Zha, Z.; Choi, S.; Zhu, L.; Song, J.; Wu, Z.; Ploessl, K.; Kung, H. Multidentate 68GaPolypegylated Styrylpyridines for Imaging of β-Amyloid Deposition in Cerebral Angiopathy (CAA). J. Nucl. Med. 2016, 57 (supplement 2), 1808–1808. (28) Zha, Z.; Choi, S. R.; Ploessl, K.; Lieberman, B. P.; Qu, W.; Hefti, F.; Mintun, M.; Skovronsky, D.; Kung, H. F. Multidentate 18F-Polypegylated Styrylpyridines As Imaging Agents for Aβ Plaques in Cerebral Amyloid Angiopathy (CAA). J. Med. Chem. 2011, 54 (23), 8085–8098. (29) Chauhan, K.; Datta, A.; Adhikari, A.; Chuttani, K.; Singh, A. K.; Mishra, A. K. 68Ga Based Probe for Alzheimer’s Disease: Synthesis and Preclinical Evaluation of Homodimeric Chalcone in β-Amyloid Imaging. Org. Biomol. Chem. 2014, 12 (37), 7328–7337. (30) Ono, M.; Haratake, M.; Mori, H.; Nakayama, M. Novel Chalcones as Probes for in Vivo Imaging of Beta-Amyloid Plaques in Alzheimer’s Brains. Bioorg. Med. Chem. 2007, 15 (21), 6802–6809. (31) Ono, M.; Saji, H. SPECT Imaging Agents for Detecting Cerebral β-Amyloid Plaques. Int. J. Mol. Imaging 2011, 2011, 543267. (32) Lührs, T.; Ritter, C.; Adrian, M.; Riek-Loher, D.; Bohrmann, B.; Döbeli, H.; Schubert, D.; Riek, R. 3D Structure of Alzheimer’s Amyloid-beta(1-42) Fibrils. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (48), 17342–17347. 30 ACS Paragon Plus Environment

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(33) Schrodinger, LLC, New York, NY; Glide, Protein Prep Wizard, Lig Prep, 2014. (34) Ono, M.; Ikeoka, R.; Watanabe, H.; Kimura, H.; Fuchigami, T.; Haratake, M.; Saji, H.; Nakayama, M. Synthesis and Evaluation of Novel Chalcone Derivatives with (99m)Tc/Re Complexes as Potential Probes for Detection of β-Amyloid Plaques. ACS Chem. Neurosci. 2010, 1 (9), 598–607. (35) Kikuchi, T.; Minegishi, K.; Hashimoto, H.; Zhang, M.-R.; Kato, K. The Use of Tetrabutylammonium Fluoride to Promote N- and O-(11) C-Methylation Reactions with iodo[(11) C]methane in Dimethyl Sulfoxide. J. Label. Compd. Radiopharm. 2013, 56 (13), 672–678. (36) Li, Z.; Cui, M.; Dai, J.; Wang, X.; Yu, P.; Yang, Y.; Jia, J.; Fu, H.; Ono, M.; Jia, H.; Saji, H.; Liu, B. Novel Cyclopentadienyl Tricarbonyl Complexes of (99m)Tc Mimicking Chalcone as Potential Single-Photon Emission Computed Tomography Imaging Probes for β-Amyloid Plaques in Brain. J. Med. Chem. 2013, 56 (2), 471–482. (37) Tiwari, A. K.; Yui, J.; Fujinaga, M.; Kumata, K.; Shimoda, Y.; Yamasaki, T.; Xie, L.; Hatori, A.; Maeda, J.; Nengaki, N.; Zhang, M.-R. Characterization of a Novel Acetamidobenzoxazolone-Based PET Ligand for Translocator Protein (18 kDa) Imaging of Neuroinflammation in the Brain. J. Neurochem. 2014, 129 (4), 712–720. (38) Holm, N. K.; Jespersen, S. K.; Thomassen, L. V.; Wolff, T. Y.; Sehgal, P.; Thomsen, L. A.; Christiansen, G.; Andersen, C. B.; Knudsen, A. D.; Otzen, D. E. Aggregation and Fibrillation of Bovine Serum Albumin. Biochim. Biophys. Acta BBA - Proteins Proteomics 2007, 1774 (9), 1128–1138. (39) Klohs, J.; Rudin, M. Unveiling Molecular Events in the Brain by Noninvasive Imaging. Neurosci. Rev. J. Bringing Neurobiol. Neurol. Psychiatry 2011, 17 (5), 539–559. (40) Rowe, C. C.; Ackerman, U.; Browne, W.; Mulligan, R.; Pike, K. L.; O’Keefe, G.; TochonDanguy, H.; Chan, G.; Berlangieri, S. U.; Jones, G.; Dickinson-Rowe, K. L.; Kung, H. P.; Zhang, W.; Kung, M. P.; Skovronsky, D.; Dyrks, T.; Holl, G.; Krause, S.; Friebe, M.; Lehman, L.; Lindemann, S.; Dinkelborg, L. M.; Masters, C. L.; Villemagne, V. L. Imaging of Amyloid Beta in Alzheimer’s Disease with 18F-BAY94-9172, a Novel PET Tracer: Proof of Mechanism. Lancet Neurol. 2008, 7 (2), 129–135. (41) Tripathi, M.; Sharma, R.; Varshney, R.; Jaimini, A.; Jain, J.; Souza, M. M. D.; Bal, J.; Pandey, S.; Kumar, N.; Mishra, A. K.; Mondal, A. Comparison of F-18 FDG and C-11 Methionine PET/CT for the Evaluation of Recurrent Primary Brain Tumors. Clin. Nucl. Med. 2012, 37 (2), 158–163. (42) Mestre-Fusco, A.; Trampal, C.; Intriago, B.; Wessling, H.; Fuertes, J.; Suárez-Piñera, M.; Díaz-Fernández, M. L. Assessment of Rhabdoid Brain Tumor by F-18 FDG PET, C-11 Methionine PET and MRI. Clin. Nucl. Med. 2012, 37 (2), e33–e35. (43) Kimura, H.; Kawai, T.; Hamashima, Y.; Kawashima, H.; Miura, K.; Nakaya, Y.; Hirasawa, M.; Arimitsu, K.; Kajimoto, T.; Ohmomo, Y.; Ono, M.; Node, M.; Saji, H. Synthesis and Evaluation of (-)- and (+)-[11C]galanthamine as PET Tracers for Cerebral Acetylcholinesterase Imaging. Bioorg. Med. Chem. 2014, 22 (1), 285–291. (44) Glabe, C. Does Alzheimer Disease Tilt the Scales of Amyloid Degradation versus Accumulation? Nat. Med. 2000, 6 (2), 133–134. (45) Park, Y. D.; Park, J. H.; Hur, M. G.; Kim, S. W.; Min, J.-J.; Park, S.-H.; Yoo, Y. J.; Yoon, Y.-J.; Yang, S. D. Fluorescent 2-Styrylpyridazin-3(2H)-One Derivatives as Probes Targeting Amyloid-Beta Plaques in Alzheimer’s Disease. Bioorg. Med. Chem. Lett. 2012, 22 (12), 4106–4110 31 ACS Paragon Plus Environment


(46) Mathis, C. A.; Wang, Y.; Holt, D. P.; Huang, G.-F.; Debnath, M. L.; Klunk, W. E. Synthesis and Evaluation of 11C-Labeled 6-Substituted 2-Arylbenzothiazoles as Amyloid Imaging Agents. J. Med. Chem. 2003, 46 (13), 2740–2754. (47) Pike, V. W. PET Radiotracers: Crossing the Blood-Brain Barrier and Surviving Metabolism. Trends Pharmacol. Sci. 2009, 30 (8), 431–440.


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DEA-Me for Beta Amyloid Imaging

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