br General procedure for urea synthesis from
5.1.2. General procedure for urea synthesis from isocyanates
The appropriately substituted aryl isocyanate (1.40 mmol) was added to a suspension of amine 13a (1.33 mmol) in anhydrous tetra-hydrofuran (15 mL) under a nitrogen atmosphere. The mixture was stirred at room temperature for 4 h and then concentrated under re-duced pressure. The residue was purified on silica gel by stepwise gradient elution with dichloromethane/ethyl acetate (100:0 to 70:30).
5.1.3. General procedure for urea synthesis from anilines
A substituted aniline (3.0 mmol) and CDI (3.0 mmol) were dissolved in anhydrous dichloromethane (15 mL) and the reaction was stirred at room temperature for 1 h under a nitrogen atmosphere. The solvent was then removed under reduced pressure, yielding the crude carbamoyli-midazoles as solids. The carbamoylimidazoles were then dissolved in dichloromethane (5 mL), and added to a solution of amine 13a or 13b (2.0 mmol) in dichloromethane (15 mL). The reaction was then left to stir for 1 h at room temperature. Dichloromethane (50 mL) was then added, and the organic layer was washed with water (3 × 100 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The product was purified on silica gel by step-wise gradient elution with dichloromethane/ethyl acetate (100:0 to 90:10).
5.1.4. General procedure for ester hydrolysis
1.5 M NaOH (10 mL). The solution was stirred at 40 °C for 4 h. The ethanol was removed under reduced pressure, and the residue was acidified with 1 M HCl. The resulting suspension was filtered and the solid washed with water (10 mL) and ethanol (5 mL), yielding the carboxylic acids as solids.
5.2. Biochemical methods
Biochemicals were obtained as follows: propidium iodide and horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgGs were from Sigma Chemical (St. Louis, MO, USA); Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), L-glutamine, trypsin/EDTA, penicillin and streptomycin were from Thermo Fisher Scientific (Waltham, MA, USA); phosphate-buffered saline (PBS) was from Amresco (Solon, OH, USA); reagents for electrophoresis were from Bio-Rad (Richmond, CA, USA); anti-cyclin B1 (Cat no 4138, Dilution 1:1000), anti-cyclin E1 (Cat no 4129, Dilution 1:1000), anti-cyclin D1 (Cat no 2978, Dilution 1:1000), anti-CDK6 (Cat no 3136, Dilution 1:2000), anti-p38 MAP kinase (Cat no 9212, Dilution 1:1000), anti-
5.2.2. Cell culture and viability assays
Human MDA-MB-231 breast cancer cells were obtained from ATCC (Manassas, VA, USA) and grown at 37 °C in a humidified AB-7-FUBAICA of 5% CO2 in air in DMEM that was supplemented with 10% FBS and 1% penicillin/streptomycin. The control MCF10A cell line was a gift from Prof Christine Clarke (Westmead Institute for Medical Research, Westmead, NSW, Australia). Confluent cells (80–90%) were harvested using Trypsin/EDTA, washed in PBS and then seeded in 24-well plates (7.5 × 104 cells/well). To study early signalling events serum was re-moved after 24 h and cells were treated with various concentrations of CTU analogues in DMSO (final concentration 0.1%) for 24 h; control cells were treated with DMSO alone. ATP formation and caspase-3/7 activity were assessed using the CellTiter-Glo® and Caspase-Glo® assays, respectively (Promega; Annandale, NSW, Australia) as described pre-viously (Cui et al., 2011). BrdU incorporation (5 × 103 cells/well) was estimated in similar fashion using the Roche cell proliferation ELISA kit (Sigma-Aldrich).
MDA-MB 231 cells were seeded in 6-well plates (15 × 104 cells/ well) (Rawling et al., 2017). Twenty-four hours after serum removal the cells were treated with CTU analogues (10 μM, 24 h). Treated cells were fixed overnight at −20 °C in 80% ethanol, followed by addition of 0.1% NP40 and 0.1 mg/mL RNAse A in 0.1 M PBS. After staining with pro-pidium iodide (50 μg/mL) cells were incubated on ice for 1 h and were then subjected to flow cytometry in an FC500 instrument (Beckman Coulter Australia, Lane Cove, NSW); cell cycle distribution data were obtained using MXP software.
5.2.4. Western immunoblot analysis
MDA-MB 231 cells were seeded in 6-well plates (1.5 × 105 cells/ well). After 24 h of serum starvation, cells were treated with CTU analogues (10 μM, 24 h); control cells were treated with solvent alone. Following treatments, cells were washed with PBS and lyzed in Laemmli buffer (31.25 mM Tris-HCl, pH 6.8; 1% sodium dodecylsulfate; 12.5% glycerol; 0.005% bromophenol blue; 2.5% β-mercaptoethanol). Protein extracts (Marcus et al., 1985) were electrophoresed on 12% sodium dodecylsulfate-polyacrylamide gels (Murray, 1991). After transfer to nitrocellulose (Whatman, Dassel, Germany) the membranes were incubated with 5% nonfat dry milk in Tris-buffered saline con-taining Tween (10 mM Tris; 100 mM NaCl; 0.1% Tween 20), washed in the same buffer and incubated overnight at 4 °C with primary anti-bodies as described previously (Cantrill et al., 1989). Detection was performed using IRDye conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibody (1:10,000 dilution, 1 h, room temperature; Li-Cor Biosciences, Lincoln, NE) and analyzed using an Odyssey Infrared Imaging System (Li-Cor Biosciences). Preliminary experiments estab-lished that the signal responses on densitometric analysis were linearly related to protein loading under these conditions.