General
All reagents and solvents used were obtained from commercially available sources and used with no further purification. 4-Fluorophthalic anhydride was purchased from Alfa Aesar (Ward Hill, MA, USA) while 4-nitrophthalic anhydride was from Frinton Laboratories Inc. (Hainesport, NJ, USA). Nuclear magnetic resonance (NMR) spectra were performed on a Bruker Avance DPX 400 (Bruker Corporation, Billerica, MA, USA) operating at 400 MHz for 1H NMR spectra and 100 MHz for 13C NMR spectra. 18F-HF was produced on a GE PET trace via the 18O(p, n)18F nuclear reaction (RPA Hospital, Camperdown, NSW, Australia). Haloperidol was obtained from Tocris Bioscience (Bristol, UK), (+)-pentazocine from Research Biochemicals Incorporated (Natick, MA, USA) and σ ligands synthesised in house at LifeSciences, ANSTO (Lucas Heights, NSW, Australia). For cell uptake studies, drugs were dissolved in PBS buffer or in saline for injection for the animal studies, with the help of a few drops of 0.5% acetic acid. Semi-preparative high-performance liquid chromatography (HPLC) purification was performed with a Waters 600 HPLC controller (Waters Company, Milford, MA, USA) and pump, an in-line UV detector (Waters 486, 254 nm) and a single sodium diode crystal flow radioactivity detector (Carrol & Ramsey Associates, Berkeley, CA, USA) using a Pheonomenex Bondclone (Lane Cove, New South Wales, Australia) (C18, 10 μm, 300 × 7.8 mm) eluting at 3 mL/min with 30% MeCN/70% water containing 0.1% TFA.
Purity analysis and specific activity
Purity analysis and specific activity of 18F-SIG343 and 18F-SIG353 were performed on a Varian 9002 pump (Varian Medical Systems, Palo Alto, CA, USA), a linear UV-VIS detector (λ = 221 nm) in series with an Ortec ACE Mate Scint 925 γ-detector (Ortec, South Illinois Ave., Oak Ridge, TN, USA) on a Phenomenex Synergi Max-RP (C12, 4 μm, 250 × 4.6 mm) eluting at 1 mL/min with 40% MeCN/60% ammonium acetate (0.1 M) as the mobile phase. The identity of the labelled compounds was confirmed by co-injection with the authentic compounds on HPLC. For specific activity calculations, the radioactivity of the injected product for the radiochemical analysis was measured with a Capintec R15C dose calibrator (Capintec, Inc., Ramsey, NJ, USA), while the mass of SIG343 and SIG353 was determined by comparing the area of the UV absorbance peak corresponding to the carrier product with a calibrated standard curve relating its mass to UV absorbance.
Lipophilicity
The lipophilicity of SIG343 and SIG353 were assessed using RP-HPLC by determining the logP
7.5 value using literature procedures [46]. Samples, dissolved in methanol, were analysed using a C18 column (RP C18, Xterra, 5 μm, 4.6 × 150 mm) and a mobile phase consisting of MeOH and phosphate buffer (0.1 M, pH 7.5) compounds were estimated by a comparison of its retention time to that of standards of known log P values.
Chemistry
Synthesis of SIG343 and SIG353
2-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-5-fluoroisoindoline-1,3-dione, SIG343
A mixture of 4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl) butan-1-amine (132 mg, 0.5 mmol) [47], 4-fluorophthalic anhydride (83 mg, 0.5 mmol) and p-xylene (3 mL) was stirred and gently boiled under a stream of nitrogen. As the xylene evaporated, more was added to maintain the volume. Within a few minutes, a viscous pale yellow gum had formed, but this redissolved slowly, disappearing completely after 1.25 h to form a pale yellow solution. Heating was continued for a total of 2 h (approximately 3 mL of additional xylene required), then the hot solution was treated with charcoal, filtered through celite and evaporated. The crystalline residue was recrystallised from 95% ethanol to give 177 mg (85.9%) of large colourless plates. 1H NMR (CDCl3) δ 1.62 (m, 2H), 1.76 (m, 2H), 2.52 (t, J = 7.5 Hz, 2H), 2.68 (t, J = 5.8 Hz, 2H), 2.79 (t, J = 5.7 Hz, 2H), 3.52 (s, 2H), 3.72 (t, J = 7.0 Hz, 2H), 3.82 (s, 3H), 3.83 (s, 3H), 6.50 (s, 1H), 6.57 (s, 1H), 7.36 (ddd, J = 8.8, 8.2, 2.4 Hz, 1H), 7.50 (dd, J = 7.0, 2.1 Hz, 1H), 7.83 (dd, J = 8.2, 4.5 Hz, 1H). 13C NMR (CDCl3) δ 24.5, 26.5, 28.6, 38.1, 51.0, 55.7, 55.8, 55.9, 57.6, 109.5, 111.2 (d, J = 24.5 Hz), 111.5, 121.0 (d, J = 23.0 Hz), 125.6 (d, J = 9.6 Hz), 126.1, 126.6, 127.8 (d, J = 2.3 Hz), 135.1 (d, J = 9.2 Hz), 147.2, 147.5, 166.4 (d, J = 230.8 Hz), 167.1 (d, J = 3.0 Hz), 167.8. Anal. C H N (C23H25FN2O4); theoretical C, 66.98; H, 6.11; N, 6.79; found C, 67.07; H, 6.16; N, 6.73.
2-(5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-5-fluoroisoindoline-1,3-dione, SIG353
4-Fluorophthalic anhydride and 5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentan-1-amine were treated under the same reaction conditions for the synthesis of SIG343 to give the title compound as colourless crystals. 1H NMR (CDCl3) δ 1.41 (m, 2H) 1.71 (m, 4H), 2.60 (m, 2H), 2.84 (m, 4H), 3.66 (s, 2H), 3.68 (t, J = 7.1 Hz, 2H), 3.82 (s, 3H), 3.83 (s, 3H), 6.51 (s, 1H), 6.57 (s, 1H), 7.36 (ddd, J = 8.8, 8.2, 2.4 Hz, 1H), 7.50 (dd, J = 7.0, 2.4 Hz, 1H), 7.83 (dd, J = 8.2, 4.7 Hz, 1H).
Synthesis of the radiolabelling precursors
2-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-5-nitroisoindoline-1,3-dione (1)
4-Nitrophthalic anhydride and 4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butan-1-amine were treated under the same reaction conditions for the synthesis of SIG343 to give the title compound as bright orange crystals. 1H NMR (CDCl3) δ 1.64 (m, 2H), 1.81 (m, 2H), 2.53 (t, J = 7.5 Hz, 2H), 2.68 (t, J = 5.8 Hz, 2H), 2.79 (t, J = 5.8 Hz, 2H), 3.51 (s, 2H), 3.79 (t, J = 7.2 Hz, 2H), 3.82 (s, 3H), 3.83 (s, 3H), 6.48 (s, 1H), 6.56 (s, 1H), 8.01 (d, J = 7.9 Hz, 1H), 8.58 (dd, J = 7.9, 2.4 Hz, 1H), 8.64 (d, J = 2.4 Hz, 1H). Anal. C H N (C23H25N3O6); theoretical C, 62.86; H, 5.73; N, 9.56; found C, 63.03; H, 5.64; N, 9.44.
2-(5-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-5-nitroisoindoline-1,3-dione (2)
4-Nitrophthalic anhydride and 5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl) pentan-1-amine were treated under the same reaction conditions for the synthesis of SIG343 to give the title compound as bright orange crystals. 1H NMR (d6-DMSO) δ 1.95 (m, 2H), 2.14 (m, 2H), 2.27 (m, 2H), 2.98 (t, J = 7.0 Hz, 2H), 3.13 (t, J = 5.7 Hz, 2H), 3.22 (t, J = 5.7 Hz, 2H), 3.55 (s, 2H), 3.69 (t, J = 7.0 Hz, 2H), 4.29 (s, 6H), 7.11 (s, 1H), 7.14 (s, 1H), 8.53 (d, J = 7.9 Hz, 1H), 9.04 (d, J = 1.8 Hz, 1H), 9.09 (dd, J = 7.9, 1.8 Hz, 1H).
Radiochemistry
An aqueous 18F-fluoride solution (18F-HF, 6 to 8 GBq) was added to a 10-mL vial containing anhydrous acetonitrile (1 mL), Kryptofix 2.2.2 (2.0 mg; Sigma-Aldrich Corporation, St. Louis, MO, USA) and K2CO3 (0.7 mg). The solvent was evaporated under a stream of nitrogen at 100°C under vacuum. This azeotropic drying was repeated twice by further addition of anhydrous acetonitrile (2 × 1 mL). The nitro precursors (1) or (2) (2 mg) was dissolved in DMF (0.5 mL) and added to the dried K222.K2CO3.K18F complex. The reaction was stirred and heated at 150°C for 5 min before the reaction mixture was diluted with mobile phase (500 μL) and purified by semi-preparative reverse-phase chromatography. The peak with the retention time corresponding to SIG343 (16 min) or SIG353 (21 min) was collected and diluted with water (10 mL) and then trapped on a Waters C18 Light Sep Pak®. The trapped radiotracer was eluted off the cartridge with ethanol (1 mL). The ethanol was concentrated in vacuo and diluted with saline for in vivo studies while it was diluted with PBS (pH 7.2) for cell studies.
In vitro studies
Radioreceptor binding assays
The test compounds were solubilised in DMSO and diluted in 50 mmol/L Tris-HCl (pH 8.0). Membrane homogenates were prepared from male Sprague-Dawley rat brains as previously described [48, 49]. The binding of σ ligands to σ1 and σ2 receptors was determined according to literature methods with minor modification [50]. Briefly, the percentage of inhibition was determined by incubating, in triplicate, aliquots of diluted rat brain membrane (300 μg of protein) with 10-11 to 10-5 mol/L concentrations of the studied drugs in 50 mmol/L Tris-HCl (pH 8.0) with 3H-(+)-pentazocine (3 nmol/L) at 37°C for 2.5 h for σ1 receptors or with 3H-DTG (10 nmol/L) and (+)-pentazocine (1 μmol/L) at 25°C for 1.5 h for σ2 receptors. In both assays, non-specific binding was determined in the presence of haloperidol (10 μmol/L). After incubation, the reaction was terminated by rapid filtration using a Brandel 48-well cell harvester (Brandel, Gaithersburg, MD, USA) over Whatman GF/B glass fibre filters that were soaked in a solution of 0.5% polyethyleneimine at 4°C for at least 2 h before use. Filters were washed three times with 5 mL of ice-cold wash buffer (50 mmol/L Tris HCl, pH 7.4). The filters were collected and the amount of bound radioactivity was measured using a β-scintillation counter (Tri-Carb 2100TR, Packard Instrument Co., Downers Grove, IL, USA). The percentage inhibition of the studied drugs, at concentrations of 10-5 and 10-7 mol/L, was also determined for the cross activity at a number of other neuroreceptors (Caliper LifeSciences, MA, USA). The IC
50 values were then converted to apparent K
i values using the Cheng-Prusoff equation and radioligand K
d values [51].
Cell culture
A375 (human amelanotic melanoma) cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% foetal calf serum (Invitrogen, CA, USA) and 2 mM l-glutamine (Sigma). Cells were maintained in 175 cm2 flasks in at 37°C in 5% CO2:95% atmosphere humidified incubator and grown to sub-confluent monolayers before being detached using trypsin for use in animal models.
Cell uptake and inhibition studies
In 24-well culture plates, 2.5 × 105 cells were seeded in complete medium and left to attach overnight. The following day, the number of cells per well was counted in triplicate. Before incubation with the radiotracer, the growth media was removed and the cells were washed once with warm PBS to remove all traces of growth media. 18F-SIG343 or 18F-SIG353 was formulated in warm PBS containing 0.1% Tween-80. Freshly prepared radiotracer (0.37 MBq in 500 μL) was added and the cells were incubated at 37°C for 2, 15, 30, 60 and 120 min. Uptake was terminated by removing the tracer solution and washing cells with ice-cold PBS. Subsequently, the cells were lysed with 500 μL of 0.2 N NaOH. The radioactivity in the cell lysate was measured with a Wallac 1480 γ-counter (PerkinElmer, MA, USA). The results were expressed as percentage of applied dose per 1 × 105 cells. All activities were corrected for decay. Optimal uptake time will be selected for inhibition cell uptake studies.
The specific uptake of the radiotracers into the cancer cells was examined in the presence of σ ligands as competitors (final concentration 5 μM): (+)-pentazocine (σ1), haloperidol (non-selective σ1/σ2) and unlabelled SIG343 or SIG353 (σ2). Cells were prepared as described for uptake studies. Prior to the addition of the radiotracer, 400 μL of blocking drug (6.25 μmol/L in PBS) or PBS (for controls) was added to the wells. Freshly prepared radiotracer (0.37 MBq in 100 μL) was added, and the cells were incubated at 37°C for 15 min as the optimal time previously determined by the cell uptake studies. Uptake was terminated as described for uptake studies. The percentage of uptake in the treatment groups relative to the control group was determined.
Ethics
Animal experiments were performed according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the ANSTO Animal Care and Ethics Committee.
In vivo studies
A375 tumour-bearing mice model
Female Balb/C nude mice aged 5 to 6 weeks old were obtained from the Animal Resource Centre (Perth, Australia). The animals were kept at a constant temperature of 22°C ± 2°C on a 12/12 h light/dark cycle with lights on at 09:00 am. Food and water were freely available. After a week of acclimatisation, mice were injected subcutaneously in the left flank with 1 × 106 A375 human amelanotic melanoma cells, in 100 μL of Ca2+/Mg2+-free phosphate buffered saline. The procedure was performed without anaesthetic. For in vivo studies, A375 tumour-bearing mice were used 26 days after tumour inoculation.
Biodistribution studies
Tumour-bearing mice were used to examine the tissue distribution of radioactivity after intravenous injection into the tail vein of 1 MBq of 18F-SIG343 or 18F-SIG353 in 100 μL of saline. At 15, 30, 60, and 120 min post injection of the radiotracer, groups of mice (n = 5) were sacrificed by CO2 administration followed by cervical fracture and dissection. Selected organs were weighed, and the radioactivity was measured using a γ-counter. The percentage of injected dose (%ID) was calculated by comparison with suitable dilutions of the injected dose. Radioactivity concentrations were expressed as percentage of injected dose per gram of wet tissue (%ID/g), assuming a uniform density of 1 g/cm3. Data were corrected for the radioactivity decay and tail injected dose and normalised for the standard mouse body weight (20 g). The remaining activity in the carcass was also determined in order to obtain the total activity in the mouse (background activity) at each time point.
Blocking studies
Blocking studies were performed to investigate the specific uptake of the tracer via σ2 receptor mechanism. Haloperidol and the unlabelled compound SIG343 or SIG353 (1 mg/kg) were administered by intravenous injection 5 min prior to radiotracer administration. Control mice received saline only. Five minutes after injection of the blocking drug, 18F-SIG343 or 18F-SIG353 (1 MBq/100 μL of saline) was injected, and groups of mice (n = 5) were sacrificed 30 min post injection of the radiotracer. Organs were processed as described for the biodistribution study. Radioactivity concentrations in the organs in the treatment groups were compared to that measured in control mice.
Metabolite studies
The amount of intact 18F-SIG343 and 18F-SIG353 in the plasma, urine, tumour and brain cortex was quantified by thin layer chromatography (TLC) and radio-HPLC analysis. Mice were injected with 20 MBq of the radiotracer in 100 μL of saline. Mice were sacrificed 15, 60 and 120 min post injection of radiotracer. Whole organ samples of brain cortex and tumour samples (10 to 60 mg, minced) were added to unlabelled SIG343 or SIG353 (5 μL; 1 mg/mL), KF (5 μL; 1 mg/mL), MeCN (0.3 mL) and water (0.2 mL). Samples were exposed to an ultrasonic probe (Ultrasonic processor, Misonix Inc., Farmingdale, NY, USA) for 2 min before being centrifuged (5,000 rpm, 5 min). Plasma (50 μL) or urine (10 μL) samples were added to unlabelled SIG343 or SIG353, KF and 0.5 mL MeCN. Plasma samples were centrifuged (Heraeus Biofuge PrimoR, Thermo Fisher Scientific, Hudson, NH, USA; 5,000 rpm, 5 min), and the supernatant was removed and the radioactivity of the precipitated pellets was measured using a gamma counter to determine the extraction efficiency. If necessary, multiple extractions were performed to ensure maximum recovery of the radioactivity. An appropriate amount of the supernatant, based on the activity level (cpm), was collected (approximately 100 μL), diluted with water (up to 1.5 mL) for HPLC analysis or evaporated to dryness under vacuum for TLC analysis.
The TLC sample was reconstituted in methanol (25 to 50 μL) and mixed before being applied to the concentrating zone of the silica TLC plate. In a separate lane, the corresponding 18F-labelled and 19F-standard was also spotted. The TLC solvent systems of EtOAc/MeOH (70:30) were utilised for 18F-SIG343 (rf 0.50) and 18F-SIG353 (rf 0.55). The UV of the standard was identified using a UV lamp, while the movement and integration of the radioactive spots were visualised and measured using a phosphorimager (BAS 2500 Phosphorimager, Fujifilm, Tokyo, Japan) with Fujifilm Multigage 3.0 software. The intact radiotracer was identified as the radioactive spot containing the identical rf value to the corresponding 19F-standard seen under the UV lamp. The integration of the active spot in relation to all the activity in the TLC lane gave the percentage of intact radiotracer.
Radio-HPLC analysis was performed following the method of Hilton et al. [52]. A pre-column (Waters Oasis HLB, 25 μm, 20 × 3.9 mm) and a reversed phase HPLC column (Phenomenex Bondclone C18, 10 μm, 250 × 4.6 mm or Phenomenex Synergi Max-RP 80A C18, 4 μm, 250 × 4.6 mm) in series, with a switching valve between columns was utilised. The pre-column was washed with 1% acetonitrile in water for 3 min at 1.5 mL/min and then the solvent direction was switched to include the HPLC column. Both columns in series were then eluted over 10 min. The radioactivity peak corresponding to the authentic radiotracer was compared to the total activity registered in the radiochromatogram to give the fraction of unchanged radiotracer in the sample.
PET/CT imaging studies
The mouse was anaesthetised via inhalant isoflurane (Forthane, Abbott Laboratories, IL, USA) (5% induction, 1% to 3% maintenance in 200 mL/min oxygen). Respiration and heart rates of the animal were monitored (BioVet; m2m Imaging Corp, Cleveland, OH, USA) during the entire scanning period. The core body temperature of the animals was maintained via a temperature-controlled heating pad. The mouse was injected intravenously with 4.6 to 16.7 MBq of 18F-SIG343 or 18F-SIG353, with a constant mass of the unlabelled compounds of 0.06 nmol in 100 μL of saline (n = 3). A 120-min PET scan was performed on an Inveon multimodality positron emission tomography-computed tomography (PET/CT) system (Siemens Medical Solutions, Knoxville, TN, USA) followed by a 10-min CT scan on each subject for anatomical information. Image acquisition commenced simultaneously with radiotracer injection. The data was histogrammed into 25 consecutive frames, and activity volumes were reconstructed with an iterative reconstruction (OSEM/MAP) including attenuation and scatter correction, achieving a reconstructed spatial resolution of 1.5 mm [53]. Briefly, each individual PET scan was co-registered to its respective CT (automatic and visual quality control) (Anatomist/Brainvisa, version 3.1.4). Regional activity data (Bq/cc) were extracted from nine selected volumes/regions of interest (VOIs/ROIs) where σ receptor distribution was reported and the tail and expressed as percentage of injected dose per cubic centimetres (%ID/cc).
Data and statistical analyses
Significant outliers were identified by Grubb tests and removed from the raw data set. Subsequently, data were analysed using the GraphPad Prism 5.04 (GraphPad Software Inc., San Diego, CA, USA) statistical package software. Significance was set at P ≤ 0.05 for all statistical analyses.
In the cell uptake blocking studies, separate one-way ANOVAs [drug treatment as a factor (control, (+)-pentazocine, haloperidol and unlabelled SIG343 or SIG353)], followed by Bonferroni's post hoc tests were performed to statistically determine the significant changes in the uptake percentage of the radiotracers into the cells compared to controls.
Selected tumour-to-organ (tumour-to-blood and tumour-to-muscle) uptake ratios (TORs), were calculated by dividing the tumour radioactivity concentration (%ID/g) by the radioactivity concentration in the respective organ at time t. Comparisons of organ radioactivity concentrations (%ID/g) and TORs in the biodistribution studies or %ID/cc in PET imaging studies between the two radiotracers were performed using separate unpaired, two-tailed Student's t tests, matched organs and time points.
Meanwhile, for the blocking studies, separate within-group two-way ANOVAs [drug treatment (control, haloperidol and unlabelled SIG343 or SIG353) × regions] followed by Bonferroni's post hoc tests were performed for each radiotracer to determine any statistically significant changes in the uptake (%ID/g) amongst selected ROIs compared to the control.