• 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2020-03
  • 2020-07
  • 2020-08
  • 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 AB-7-FUBAICA 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.