All of the reagents and materials were purchased from Sigma Chemical Co. (St. Louis, MO, USA), Bide Pharmatech Co. (Shanghai, China), and Energy Chemical Co. (Shanghai, China). Reactions took place opening to the atmosphere unless otherwise specified. The reactions were monitored by thin-layer chromatography (TLC) using 200 μM silica gel (China National Pharmaceutical Group Co., China) or Dionex UltiMate 3000 HPLC. 1H and 13C NMR spectra were recorded on a Bruker Avance III 400 spectrometer. High-resolution mass spectra (HRMS) were performed on an Agilent G6520 Q-TOF mass spectrometer. 18F-Fluoride was produced using a Sumitomo 10MeV cyclotron. 18F-Fluoride was produced from 18O-water and the 18O(p,n)18F reaction. The synthesis and characterization of PVBO and the precursor are presented in supplemental materials.
In vitro relative binding affinity of the reference compound PVBO towards ERα and ERβ
Relative binding affinities were determined by a competitive radiometric binding assay using 3H-estradiol ([2,4,6,7-3H]-17β-estradiol, 70-115 Ci/mmol, Perkin-Elmer, Waltham, MA). Data are presented as the relative binding affinity (RBA) with the RBA of estradiol towards ERα and ERβ set to 100%. The experiment and data analysis were performed according to a previously described binding assay protocol.
Molecular docking for PVBO was performed against ERα using the highest resolution X-ray crystal structure (PDB entry 6VIG, 1.45 Å) by Schrodinger suit. As for ERβ, molecular docking was performed with the crystal structure of ERβ complexed with ERB-041 (2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3-benzoxazol-5-ol) (PDB entry 1X7B), because PVBO was derived from ERB-041. All the water molecules as well as the ligand were removed by PyMOL. Before docking, the Schrödinger protein preparation wizard module was used to prepare the 3D structures of protein, optimize 3D structures with pH = 7.0, and restrain minimization converge heavy atoms to RMSD of 0.30 Å, using force field OPLS_2005. The docking grid of 20 Å was generated over the co-crystallized ligand with the Receptor grid generation module. PVBO and ERB-041 were pre-processed by the Ligprep, with Epik to generate the proper protonation states at pH 7.0, with at most 32 stereoisomers generated. The compounds were then docked to the protein using the “extra precision” glide docking.
Starting from unlabeled precursor 12, 18F-PVBO was obtained using a two-step radiosynthetic method. The 18F-fluoride solution was passed through a QMA SepPak Light anion exchange cartridge (Waters) and eluted from the cartridge into a vial with MeCN (aq) (1.1 ml) containing Krypotofix 222 (13 mg) and K2CO3 (3 mg). The solvents were evaporated at 120 °C and then dried 3 more times by adding 1 ml of MeCN. Then, precursor 12 was dissolved in 1 ml of MeCN and added to the vial. The reaction system was sealed and heated at 120 °C for 20 min. After the solvents were evaporated, 1 ml of DCM and 0.25 ml of TFA were added, and the mixture was heated at 60 °C for 5 min to remove the methoxymethyl ether group. The product was purified by HPLC (Column: XBridge BEH C18 OBD Prep Column, 5 μm, 10 mm × 250 mm; eluent: acetonitrile in 0.3% phosphoric acid solution; flow: 3 ml/min.18F-FES was produced as previously described.
Distribution coefficients (Log D7.4)
HPLC-purified 18F-PVBO (100 μCi/tube) was added to a mixture of n-octanol/PBS pH 7.4 (0.5 mL/0.5 mL). After vortexing at room temperature for 10 min, the tubes were shaken in a water bath at 37°C for 30 min. After standing for stratification, 0.2 mL of solution was drawn from both phases. The radioactivity was counted using an automated gamma counter (Wizard 2, model 2480, PerkinElmer). The experiments were performed in triplicate.
In vitro stability
The stability of 18F-PVBO was measured in PBS or fetal bovine serum (FBS). Briefly, 18F-PVBO (100 μL, 37 MBq/mL) was added to 500 μL of PBS or FBS and incubated at 37 °C for 1 h or 2 h. Stability was evaluated by HPLC.
Cell culture and animal models
The human prostate cancer cell line DU-145 and the mouse embryonic fibroblast cell line NIH/3T3 were purchased from the American Type Culture Collection (Manassas, USA). Cells were cultured in the Minimum Essential Medium (MEM, Gibco, Carlsbad, USA) or Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, USA) supplemented with 10% FBS (Gibco, Carlsbad, USA) in a 37°C incubator with 5% CO2.
Athymic female nude mice (6-8 weeks, n = 10, Slaccas, Shanghai, China) and female BALB/c mice (10-12 weeks, n = 15, Slaccas, Shanghai, China) were used in this study. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. After one week of acclimatization, DU-145 cells (1 ×106 cells/mouse) were injected into the right thigh subcutaneously.
The DU-145 cell line and NIH/3T3 cell line were cultured in MEM or DMEM supplemented with 10% FBS in a 37°C incubator with 5% CO2. MTT was used to assess the cytotoxicity of different concentrations (0, 10, 25, 50, 75, and 100 μM) of the unlabeled PVBO on these two cell lines. Briefly, 100 μL of cells were seeded in a 96-well plate at a cell density of 5000 cells/well and incubated for 24 h. The cells were treated with PVBO at different concentrations (0, 10, 25, 50, 75, and 100 μM) and incubated for 24 h. Ten microliters of MTT solution were added to each well and incubated for another 4 h. Then, the MTT solution was discarded, 150 μL of DMSO was added to each well, and the absorbance at 490 nm was measured with a microplate reader (Synergy H1 Hybrid, Biotek, Winooski, USA).
Cell uptake was performed using DU145 cells in the presence and absence of the ERβ selective drug ERB-041. DU145 cells were seeded in 12-well plates at a cell density of 1×105 cells/well and incubated for 24 h. Fresh serum-free medium (1 ml) containing 18F-PVBO (37 MBq) was added to each well. To inhibit the uptake of 18F-PVBO, DU145 cells were pretreated with the ERβ-selective ERB-041 for 1 h and then incubated with 18F-PVBO (37 MBq) at 37°C for 15, 30, 60, and 120 min. Each well was washed with PBS three times before cell lysis with NaOH. The results were measured using an automatic gamma counter (Wizard 2, model 2480, PerkinElmer). Cell uptake is presented as the percentage of the total added radioactivity dose.
Immunohistochemical (IHC) staining
DU145 tumors were fixed with formalin. IHC staining was performed on 4 μm sections taken from paraffin-embedded tumor tissues. After rehydration, the sections were incubated with a 1:200 dilution of the primary antibody, rabbit anti-estrogen receptor alpha antibody (ab3575, Abcam, Cambridge, UK) and rabbit anti-estrogen receptor β antibody (ab3576, Abcam, Cambridge, UK) at 4°C overnight. Then, the sections were incubated with a biotinylated secondary antibody (ab205718, Abcam, Cambridge, UK) and treated with Avidin-biotin-peroxidase. The 3-amino-9-ethylcarbazole substrate chromogen was used, followed by tissue counterstaining with hematoxylin. Sections were examined and photographed using a confocal microscope (Olympus, Tokyo, Japan)
The membranes were incubated with 1:1000 dilutions of primary rabbit anti-estrogen receptor alpha (ab3575, Abcam, Cambridge, UK), rabbit anti-estrogen receptor β (ab3576, Abcam, Cambridge, UK) and anti-GAPDH (1:50000, 60004-1-lg, Proteintech, Rosemont, USA) antibodies at 4 °C overnight. The next day, after washing with PBS three times for 10 min each, the membranes were incubated with the secondary antibodies for 1 h at room temperature. Finally, the target proteins were visualized using a LAS4000 enhanced chemiluminescence system (GE Healthcare, Wisconsin, USA).
Micro-PET/CT (Inveon, Siemens Company, Germany) studies were carried out in DU145-bearing athymic female nude mice using 18F-PVBO or 18F-FES. The in vivo targeting specificity of 18F-PVBO was evaluated in a blocking study with the administration of unlabeled PVBO (5 μmol/kg) or ERB-041 (5 μmol/kg) 30 min before 18F-PVBO injection. The dose of 18F-PVBO was 7.02 ± 0.93 MBq per mouse. The dose of 18F-FES was 6.51 ± 0.64 MBq per mouse. A 120 min dynamic PET scan was immediately performed for 2 h after injecting the radiotracer through the tail vein. A 10 min CT scan was acquired for anatomic localization. The relative position of the organ was defined based on the anatomy of CT. The uptake values were expressed as the percentage of the injected dose per gram of tissue (%ID/g).
BALB/c mice were anesthetized via inhalation of 2% isoflurane and injected with 3-5 MBq radiotracer through the tail vein for the biodistribution study. The mice were sacrificed at 30 min, 60 min and 120 min after injection. Blood was withdrawn by cardiac puncture, and the organs/tissues were collected, weighed and evaluated using an automatic gamma counter (Wizard 2, model 2480, PerkinElmer). Uptake values were expressed as percentage of injected dose per gram (%ID/g).
Histological analysis of major organs
The mice were sacrificed at 7 days after 18F-PVBO PET imaging. Tissue samples from the heart, liver, spleen, lung, kidney, and brain were collected for pathological examination. H&E staining of the major organs of nude mice was performed based on a standard procedure.
GraphPad Prism 5 (GraphPad Software, CA, USA) and SPSS 24.0 software (SPSS, Inc., Chicago, IL) were used for statistical analyses. The data are expressed as the mean ± standard deviation (SD). Differences between groups were analyzed using a two-sided unpaired Student’s t-test. Differences were considered statistically significant at p < 0.05 (*p < 0.05; ** p < 0.01; *** p < 0.001).