Discovery of selective, antimetastatic and anti-cancer stem cell metallohelices via post-assembly modification

A remarkable array of mechanistic and pharmacological behaviours is discovered via click derivatisation of asymmetric, optically pure helicate-like compounds.


S4
The compounds 5-(chloromethyl)-2,2'-bipyridine, 1, 2 5-hydroxypicolinaldehyde, 3 and phenylglycinol 4 were synthesised by reported methods. S-phenylglycinol (1.00 g, 7.3 mmol) was dissolved in dry THF (20 ml) and was added dropwise to a stirred suspension of sodium hydride (0.36 g, 15.0 mmol) in dry THF (10 ml) under inert argon atmosphere. The solution was stirred for 1 h at room temperature. A solution of 5-(chloromethyl)-2,2'-bipyridine (1.82 g, 7.3 mmol) in dry THF (20 ml) was then added dropwise and the solution was stirred for 1 h at room temperature before being heated to reflux (65 ˚C) S5 for a further 2 h. The reaction mixture was then cooled to ambient temperature and brine (40 ml) was added. The product was extracted with diethyl ether (4 × 60 ml), dried over sodium sulphate and the solvent was removed under reduced pressure to leave a dark yellow oil which was further purified by silica gel flash chromatography (petroleum ether/EtOAc/triethylamine, 8:8:1) to give the pure product as a white solid (1.77 g, 98% yield). Sodium azide (1.64 g, 25.2 mmol) was added to a solution of benzyl bromide (2.0 ml, 16.8 mmol) in DMSO (25 mL). The mixture was stirred overnight at ambient temperature. Brine (75 mL) was added into the solution before extracting the product into diethyl ether (3 × 150 ml
After cooling to room temperature, brine (8 mL) was then added into the solution and the product was extracted with diethyl ether (3×10 mL). The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure to give the 4-(bromomethy)benzonitrile as colourless oil (0.71 g, 87.5% yield). 1

HOOC N 3
A solution of 4-chloromethyl benzoic acid (1 g, 5.86 mmol) and sodium azide (1.14 g, 17.54 mmol) in DMSO (100 mL) was heated at 80 °C for 2 days. After cooling to room temperature, brine (100 mL) was added into the solution, and the mixture was diluted with diethyl ether (300 mL). The organic phase was dried over anhydrous sodium sulphate. The solvent was removed under reduced pressure to give the pure product as a white solid (0.87 g, 84.3% yield). 1                          Intens.

Chemosensitivity (MTT assay)
The cells were seeded in 96-well tissue culture plates at a density of 1×10 4 A2780/cisR cells/ml, Supplementary Figure 24

Apoptosis assay (Annexin V assay)
Cells were prepared and treated as described for cell cycle assay.

Rubidium based assay for detection of Na + /K + ATPase activity
The cells were seeded on 6-well plates (5×10 5 cells/dish) and incubated overnight.

Resistance to detachment assay
Following procedure was used to measure the ability of cells to resist detachment by trypsin.
HCT116 or A2780 cells were seeded at a density of 5 × 10 3 cells/well in 200 µL complete medium in a 96-well plate. After 2 days at 37 °C in 5% CO 2 complete medium was replaced with serum-starved medium containing 0.1% w/v BSA (bovine serum albumin). After another 24 h, the medium was removed, and the cells were treated for 3 h with 10 µM and 20 µM metallohelices in the serum-starved medium. Following the incubation the medium was removed again, the cells were washed with PBS, and 30 µL of (0.005% w/v) trypsin solution was added to each well, and the plates were incubated at 37 °C for 12 min. At the end of the incubation, the trypsin solution was removed, and wells were washed with PBS. Cells that were S49 still adherent to the plates were fixed with 200 µL of 10% (w/v) cold TCA (trichloracetic acid) at 4 °C for 1 h. The adherent cells were detected by the sulforhodamine B (SRB) assay.
Absorbance values were plotted against compound concentration.

Re-adhesion assay
The ability of HeLa and HCT116 cells treated with metallohelices to re-adhere after detachment was studied using the re-adhesion assay. The cells were seeded at a density of 2×10 5 cells/well (6-well plate) in 3 mL of the complete medium. After 48 h the medium was replaced with a serum-starved medium (0.1% w/v BSA), and the cells were grown for another 24 h. Then the medium was removed, and the cells were treated with metallohelices (10 µM) in the same medium for 3 h. The medium was removed, and the cells were washed twice with PBS. The cells were trypsinized (0.05% trypsin, 5 min), collected by centrifugation, resuspended in serum-starved medium supplemented with 0.1% w/v BSA and kept for 30 min at room temperature for surface receptor reconstitution. The cells were seeded in octuplicates on 96-well plate at a density of 2×10 4 cells in 0.1 mL/well. Cells were incubated (37 °C, 5% CO 2 ) to adhere for 30 min. Following the incubation, the medium was removed, and the wells were twice gently washed twice with PBS. The adhered cells were determined by the sulforhodamine B (SRB) test.

Cell invasion test
For the invasion test, HCT116 p53 +/+ were seeded at a density of 3×10 5 cells in T25 flasks.
Cells were incubated for 72 h at 37 °C in a 5% CO 2 humidified atmosphere in the complete culture medium (10% FBS), followed by 24 h incubation in a serum-free medium supplemented with 0.1% w/v. These viability tests confirm that non-toxic doses are used (cell viability = ca 90% in all cases), and thus inhibition of gap closure indicates migration inhibition.