All reagents were purchased from Sigma-Aldrich unless otherwise stated. 2-fluoroethyl-4-methylbenzene sulfonate was prepared using literature procedure . 1H NMR, proton-decoupled 13C NMR, and 19F NMR spectra were recorded on a Varian 400-MHz spectrometer; chemical shifts are reported in δ (ppm) with reference to either TMS or trichlorofluoromethane (CFCl3). Mass spectra were obtained from the University of Missouri Mass Spectrometry facility using nitrobenzyl alcohol (NBA) as matrix and analyzed via HRFab. Purity of the 7A and 7B were assessed using an HPLC (Waters system 600 equipped with dual λ-detector 2487 set to 254 and 280 nm) with a C-18 reversed-phase column (Phenomenex Luna® C18; 100 Å; 5 μm; 250 × 10 mm) using an eluent mixture of acetonitrile and water as a gradient system (75% acetonitrile in water over 20 min) at a flow of 3 mL/min.
Chemistry and radiochemistry
To the mixture of 6-methoxy-2-methyl benzothiazole (1.0 mmol) and 6-dimethylamino pyridine carbaldehyde (1.0 mmol) in DMSO was added 50% KOH and stirred at room temperature for 12 h. After the completion of the reaction, the reaction mixture was filtered, and the yellow solid obtained was used for the next reaction without purification. Yield 92% (0.28 g); yellow solid; Rf 0.62 (3:2 hexane-EtOAc); 1H NMR (400 MHz, CDCl3 ): 3.15 (s, 6H), 3.88 (s, 3H), 6.55 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 9.2 Hz, 1H), 7.14 (d, J = 16.0 Hz, 1H), 7.29 (t, J = 14.0 Hz, 2H), 7.71 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 8.29 (s, 1H); 13C NMR (100 MHz, CDCl3): 38.14, 55.79, 104.13, 106.05, 115.31, 118.0, 119.41, 123.02, 134.08, 134.22,148.93, 159.15, 165.33; HRMS (FAB) m/z calc. for C17H18N3OS: [M]+ + 1 312.1171; found: 312.1167.
The condensed product (0.28 g, 1.0 mmol) was taken in a 50-mL RB, dissolved in dry DCM under argon and subjected to −78°C using dry ice/acetone bath and stirred for 5 min. BBr3 (1 M in DCM, 5 mL, 5.0 mmol) was added drop wise maintaining the same temperature. The resulting mixture was slowly brought to room temperature and stirred overnight. The completion of the reaction was monitored by TLC. Once the reaction is completed, the flask is cooled to 0°C before addition of cold satd. sodium bicarbonate solution (5 mL). The reaction mixture is then extracted with ethyl acetate (4 × 25 mL), washed with water (2 × 50 mL), dried over anhydrous sodium sulfate, and the solvent evaporated under reduced pressure to give the red solid which was further purified by flash chromatography using hexane:EtOAc:MeOH (10:9:1) as eluent. Yield 86% (0.27 g); light red solid; Rf 0.24 (1:1 hexane-EtOAc);1H NMR (400 MHz, CDCl3): 3.03 (s, 6H), 6.65 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H), 7.24 to 7.32 (m, 2H),7.66 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 7.6 Hz, 1H), 8.29 (s, 1H), 9.82 (s, 1H); 13C NMR (100 MHz, CDCl3): 38.04, 106.51, 107.08, 116.11, 118.03, 119.58, 123.17, 134.13, 134.96, 135.60, 147.47, 149.36, 155.90, 159.25, 163.97; HRMS (FAB) m/z calc. for C16H16N3OS: [M]+ + 1 298.1014; found: 298.1015.
To the solution of alcohol (0.15 g, 0.4 mmol) and 2-(Bromoethoxy)-tert-butyldimethylsilane (0.096 g, 0.4 mmol) in DMF (5 mL) was added Cs2CO3 (0.20 g, 0.6 mmol). The resulting mixture was stirred at 140°C for 6 h. Following the completion of the reaction (monitored by TLC), it was quenched with the addition of ice cold water and extracted with ethyl acetate (3 × 25 mL). The organic layer was washed with water (2 × 50 mL) and dried over anhydrous sodium sulfate and the solvent evaporated under reduced pressure to give crude products which were purified by PTLC using hexane:EtOAc (60:40) as eluent; yield 54% (0.12 g); yellow solid; Rf 0.44 (2:3 hexane-EtOAc); 1H NMR (400 MHz, CDCl3): 0.12 (s, 6H), 0.92 (s, 9H), 3.15 (s, 6H), 4.02 (bs, 1H), 4.11 (bs,1H), 6.56 (d, J = 8.8Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.14(d, J = 16.4 Hz, 1H), 7.24 to 7.32 (m, 2H), 7.72 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 8.8 Hz, 1H), 8.29 (s, 1H); 13C NMR (100 MHz, CDCl3): 31.04, 42.24, 68.44, 76.22, 110.76, 112.22, 121.21, 124.34, 129.86, 140.02, 142.74, 149.96, 152.44, 162.68, 172.86; HRMS (FAB) m/z calc. for C24H34N3OSSi: [M]+ + 1 456.2140; found: 456.2145.
To the solution of TBDMS-protected compound (0.05 g, 0.1 mmol) in THF was added TBAF (1 M in THF, 0.5 mL, 0.5 mmol) and stirred at RT for 6 h. Following completion of the reaction (monitored by TLC), the solvent was evaporated under reduced pressure to obtain a crude product which was purified by PTLC using hexane:EtOAc (75:25) as eluent. 1H NMR (400 MHz, CD3COCD3): 3.17 (s, 6H), 3.96 (bs, 2H), 4.20 (bs, 2H), 6.65 (d, J = 8.9, Hz, 1H), 6.71 (d, J = 8.8 Hz, 1H), 7.10 (t, J = 8.2 Hz, 1H), 7.23 (d, J = 16.4 Hz, 1H), 7.43 (d, J = 16.4 Hz, 1H), 7.57 to 7.93 (m, 2H), 8.36 to 8.42 (m, 1H) 13C NMR (100 MHz, CD3COCD3 ): 37.08, 60.41, 70.33, 104.64, 105.07, 105.82, 115.79, 116.09, 117.76, 119.44, 122.88, 123.21, 133.65, 134.23, 149.04, 150.26, 157.26.
Pyridine (0.08 g, 1 mmol) and DMAP (0.0012 g, 0.01 mmol) were added to a solution of alcohol (0.08 g, 0.2 mmol) in DCM (10 mL) at 0°C. Thereafter, p-toluene-sulfonylchloride (0.076 g, 0.4 mmol) dissolved in DCM (2 mL) was added, and the resulting solution was stirred at room temperature for 7 h and quenched by the addition of water (15 mL). The resulting mixture was extracted with DCM (3 × 5 mL), and organic extracts were combined, dried over Na2SO4, filtered, and concentrated. Finally, the residue was purified by Prep TLC using the eluent mixture (Hex/EtOAc = 60:40) to obtain the compound as a viscous yellow liquid. Yield 57% (0.06 g); yellow viscous liquid; Rf 0.41 (1:1 hexane-EtOAc): 1H NMR (400 MHz, CDCl3): 2.43 (s, 3H), 3.15 (s, 6H), 4.22 (m, 2H), 4.41 (bs, 2H), 6.52 (d, J = 8.4 Hz, 1H), 6.54 (d, J = 8.4 Hz, 1H), 6.68 (d, J = 12.6 Hz, 1H), 6.81 (d, J = 12.6 Hz, 1H), 6.91(d, J = 8.8 Hz, 1H), 7.08 to 7.17 (m, 1H), 7.30 to 7.34 (m, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 9.6 Hz, 1H), 7.83 (d, J = 9.6 Hz, 1H), 7.95 (d, J = 8.8 Hz, 1H), 8.31 (d, J = 10.0 Hz, 1H) 13C NMR (100 MHz, CDCl3 ): 21.63, 38.08, 66.08, 67.99, 104.97, 105.05, 106.05, 115.53, 115.68, 117.82, 120.96, 123.03, 123.46, 127.97, 129.84, 134.10, 134.27, 134.45, 137.89, 144.99, 148.98, 149.76, 156.02. HRMS (FAB) m/z calc. for C25H25N3O4S2Na: [M]+ + Na 518.1184; found: 518.1176.
Dry tetrabutylammonium fluoride (0.065 g, 0.25 mmol) was dissolved in dry acetonitrile (5 mL) under argon and treated with the acetonitrile solution of tosylated precursor 6 (0.050 g, 0.1 mmol) and refluxed at 110°C. The progress of the reaction was monitored via the TLC. Following completion of the reaction, the solvent was evaporated, and the residue was extracted with EtOAc (2 × 10 mL). Combined organic extracts were dried with Na2SO4, filtered, and evaporated, and the residue was purified using thin-layer chromatography employing a mobile eluent mixture (Hex:EtOAC = 80:20) to obtain 7A (0.021 g; 60%; bright yellow solid; Rf = 0.42; 3:2, EtOAc-hexane).1H NMR (400 MHz, CDCl3): δ 3.14 (s, 6H), 4.20 to 4.33 (m, 2H), 4.79 (dd, J = 47.6, 8.0 Hz, 2H), 6.51 to 6.57 (m, 1H), 6.67 to 6.82 (m, 1H), 7.08 (dd, J = 9.2, 2.6 Hz, 1H), 7.23 to 7.34 (m, 1H), 7.30 to 7.34 (m, 1H), 7.70 to 7.97 (m, 2H), 8.31 (dd, J = 8.8, 2.0 Hz 1H); 13C NMR (100 MHz, CDCl3 ): δ 38.10, 38.13, 67.63, 67.84, 81.01, 82.70, 105.32, 106.05, 115.67, 117.86, 119.34, 123.12, 134.23, 134.33, 148.98, 156.36, 159.17, 165.74; 19F NMR (282 MHz, CFCl3 ): −224 ppm; HRMS (FAB) m/z calc. for C18 H19 FN3 OS: [M + 1]+ 344.1232; found: 343.1230.
Radiochemical synthesis 18F-7B
For radiolabeling, tosylated precursor (5, 3 mg) dissolved in dry acetonitrile (400 μL) was transferred into an amber-colored vial containing anhydrous kryptofix 2, 2, 2 (Sigma Chemicals, Perth, WA, AU)/K2CO3/18F-fluoride, obtained using standard procedures. The reaction mixture was heated to 100°C for 20 min in an oil bath, cooled in ice-cold water, and diluted to 5% acetonitrile in water. The crude mixture was loaded on a C-18 sep-Pak cartridge (Waters, Milford, MA, USA), primed with ethanol (5 mL) and water (10 mL). The C-18 sep-Pak was washed with water (5 mL × 6) and 25% acetonitrile (5 mL × 4) and finally eluted with 100% acetonitrile (1 mL).
The crude mixture was purified using high-performance liquid chromatography (dual λ detection set at 254 and 280 nm) equipped with a radiodetector (Bioscans) using a C-18 column (Phenomenex (Torrance, CA, USA); 5-μm 250 × 10 mm) using an eluent gradient acetonitrile 75% to 95% over 20 min (flow rate: 3 mL/min). The fraction of 18F-7B eluting at 9.5 min (radiochemical purity >95%; radiochemical yield: 30%; specific activity: 1,200 to 1,400 Ci/mmol) was collected, concentrated, resuspended in ethanol/saline, and employed for bioassays.
Human serum (Sigma-Aldrich, St. Louis, MO, USA) was thawed. Serum (100 μL) was taken, diluted with saline to 10%, and incubated with HPLC-purified fraction of 18F-7B (100 μCi each) at 37°C for 30 and 60 min. Samples were periodically withdrawn and analyzed on radio-HPLC using eluent mixture described above and radio-TLC using an eluent mixture of ethanol/saline in a ratio of 90:10 (to allow mobility of parental compound and other hydrophobic metabolites off the surface to analyze protein binding) and 10:90 (to analyze hydrophilic metabolites).
Preparation of human brain homogenates
Well-established literature procedures were used for preparation of AD homogenates [11, 26]. Grey matter was isolated from frozen postmortem frontal cortex tissue by dissection with a scalpel. To prepare insoluble fractions, dissected tissue was sequentially homogenized in four buffers (3 mL/g wet weight of tissue) with glass dounce tissue grinders (Kimble, Vineland, NJ, USA): 1) high salt (HS) buffer: 50 mM Tris-HCl pH 7.5, 750 mM NaCl, 5 mM EDTA; 2) HSbuffer with 1%Triton X-100; 3) HSbuffer with 1%Triton X-100 and 1 M sucrose; and 4) phosphate-buffered saline (PBS). Homogenates were centrifuged at 100,000×g; after each homogenization step, the pellet was resuspended, and homogenized in the next buffer in the sequence. For comparison in initial binding studies, unfractionated tissue homogenates were also prepared by homogenization of tissue in only PBS.
Preparation of Aβ1-42 fibrils
Aβ1-42 fibrils were obtained using literature procedures described earlier . Briefly, synthetic Aβ1-42 peptide (1 mg) (Bachem, Torrance, CA) was initially dissolved in DMSO (50 μL) and diluted with the addition of mQ-H2O (925 μL). Finally, 1 M Tris-HCl (25 μL; pH 7.6) was added to the peptide solution to obtain a final peptide concentration (222 μM; 1 mg/mL) . Thereafter, the peptide solution was incubated for 30 h at 37°C with shaking at 1,000 rpm in an Eppendorf Thermomixer. The fibril formation was confirmed by ThioT fluorescence. For determining the concentration of fibrils, the fibril reaction mix was centrifuged at 15,000×g for 15 min to separate the fibrils from the monomer. The concentration of Aβ monomer in the supernatant was determined in a BCA protein assay.
Binding assays were performed using previously described methods . Briefly, a fixed concentration (1 μM/well) of Aβ1-42 fibrils was incubated for 1 h at 37°C with increasing concentrations of 18F-7B (1.5 to 100 nM) in 30 mM Tris-HCl pH 7.4, 0.1% BSA in a reaction volume of 150 μL. A fixed ratio of hot:cold (18F-7B and 7A) was used for all radioligand concentrations. The exact hot:cold (18F-7B:7A) ratio was measured in each experiment by counting an aliquot of a sample (2 μL) of the radioligand preparation in a scintillation counter. Binding of 18F-7B to human brain homogenates was assessed by incubating samples of insoluble fraction (5-μg insoluble protein/well) from AD subjects, with increasing concentrations of 18F-7B (1.5 to 100 nM). Nonspecific binding was determined in parallel experiments utilizing 7A (2.5 μM) for Aβ1-42 fibrils and 7A (5 μM) for AD tissue as a competitor. Bound and free radioligand were separated by vacuum filtration through glass fiber 96-well filter plates (Millipore Multiscreen FB filter plate), followed by washes using ice-cold assay buffer (3 × 150 μL). Glass fiber filters containing the bound ligand were mixed with Optiphase Supermix scintillation cocktail (150 μL; PerkinElmer, Waltham, MA, USA) and counted immediately. All data were obtained in triplicate and analyzed by curve fitting to a one-site binding model using nonlinear regression employing Graphpad Prism software (version 4.0) to determine K
d and B
max values. B
max values were calculated in pmol/gram wet weight of brain tissue . To calculate binding potential (BP) where BP = B
max was converted from pmol/gram brain tissue to units of nanomolar by assuming 1 g of brain tissue = 1 mL .
Autoradiography and immunohistochemistry
Frozen AD frontal cortex sections (12 μm) were obtained using a TBS Minotome PLUS Cryostat. The tissue sections were thaw-mounted onto Superfrost Plus (Fisherbrand 12-550-15 (Fisherbrand, Leicestershire, UK)) microscope slides, allowed to air dry for 10 to 15 min, and stored at −80°C. For autoradiography, brain sections were brought to room temperature for 5 min and then pre-incubated in assay buffer (30 mM Tris-HCl, pH 7.4 + 0.1% BSA) for 10 min at RT. Sections were incubated with 18F-7B (300 μL/slide; 10 nM) in the assay buffer for 60 min at room temperature. For determining nonspecific binding, adjacent sections were incubated in the additional presence of 2.5 μM cold ligand 7A. Sections were then washed at RT for 1 min in 30 mM Tris-HCl pH 7.4, 2 min in 70% ethanol/30 mM Tris-HCl, 1 min in 30% ethanol/30 mM Tris-HCl, and 1 min in 30 mM Tris-HCl (washing protocol using literature precedents . Sections were then dried and exposed to phosphor-imaging screen (BAS-MS 2025) for 30 min. Autoradiography images were obtained on a Fuji Bio-Imaging System FLA-7000 (Tokyo, Japan) and analyzed using MultiGauge software (Fuji, Tokyo, Japan). Following exposure to a phosphor-imaging screen, the sections were then blocked for 60 min at room temperature with 3% milk-0.25% Tween 20-PBS buffer. Immunostaining for Aβ plaques was carried out using monoclonal antibody HJ3.4 (directed against the N-terminus of human Aβ) conjugated to Alexa 568 (generously provided by John Cirrito). The sections were incubated overnight in antibody diluted 1:250 in 0.5% milk-PBS-0.25% Tween 20 at 4°C, washed for 5 min × 3 with PBS-0.25% Tween 20, and air dried [30, 31]. Finally, sections were cover-slipped and scanned with the NanoZoomer 2.0-HT System (Olympus) at 20× resolution using TRITC fluorescence filter, and images were acquired using NDP scan 2.5 software (Olympus).
All animal procedures were approved by the Washington University Animal Studies Committee. Pharmacokinetics of 18F-7B in brain and other critical tissues of normal male 12-week-old FVB (wild type (WT); 28 to 36 g) mice were determined as previously described . Briefly, 18F-7B (740 kBq) was dissolved in 100-μL saline containing 10% ethanol. All animals were anesthetized by isofluorane inhalation and injected with radiotracer 18F-7B (740 kBq, 100 μL) via bolus injection through a tail vein. Animals were sacrificed by cervical dislocation under anesthesia at 5, 30, 60, and 120 min after injection (n = 3 each). Blood samples were obtained by cardiac puncture, organs then harvested rapidly, and all tissue samples analyzed for γ-activity. Data are expressed as the percentage injected dose (%ID) per gram of tissue (tissue kBq (injected kBq)−1 (g tissue)−1 × 100).
For imaging, female double-transgenic mouse (BL6/FVB APP/PS1, 22.6 months old, n = 3) and a female wild-type mouse (BL6/FVB; 22.6 months old, n = 3) were anesthetized with isoflurane (1.5% to 2.5%) in oxygen at flow rate of 1 to 2 L/min via an induction chamber and maintained with a nose cone. Following anesthesia, the mice were secured with their heads in the center of the field of view, fixed in the scanner in a prone head-first position (HFP), and placed in an acrylic-imaging tray. MicroPET imaging was performed using Inveon PET/CT scanner (Siemens Medical Solutions, Malvern, PA, USA) following intravenous tail-vein injection of HPLC-purified [18F]7B (12.95 MBq; 28 μL; 35% ethanol in saline) employing the catheter system in a slow bolus, followed by flushing with isotonic saline solution. PET dynamic data acquisition was performed over 75 min starting immediately following injection of the tracer. The emission data were normalized and corrected for attenuation, scatter, and decay. Attenuation correction was obtained using the co-registered CT data. The image volume consisted of 256 × 256 × 159 voxels, with a size of 0.39 × 0.39 × 0.8 mm3 per voxel for the Inveon scanner. For anatomical visualization, PET images were co-registered with CT images from an Inveon PET/CT scanner. For analysis, brain uptake (Bq/L) was normalized to injected dose and weight of animals. For analysis, PET images were initially reconstructed in 52 dynamic frames and the second time as four frames of 30 min each, with 3D-MAP reconstruction algorithm for incorporating resolution recovery (20 iterations, β value of 0.0043). For evaluation of uptake ratios of cortex and cerebellum, the regions of interest (ROI) were drawn in the frontal cortex (target) and cerebellum (reference) regions in both transgenic and WT mice. Additionally, SUV values in time-activity curves were normalized to the average activity measured in those regions between 1 to 2 min post-injection. All image data were processed and analyzed using Inveon Research Workspace 4.1 software (Siemens, Malvern, PA, USA). All PET and CT image datasets were scaled to calibrated kBq/cc.