10.1002/cmdc.201800105
ChemMedChem
FULL PAPER
analytical column (5 μM, 120 Å, 150 mm × 4.60 mm i.d) was used, with an
isocratic elution method (total flow = 1 mL/min, solvent system = 60%
acetonitrile and 40 % water).
transplanted on the surface of the chorioallantoic membrane (CAM) as a
25 μL hanging drop. Vascularized tumours appeared approximately 3 days
after inoculation on the surface of the CAM. 2 or cDDP were administered
intravenously on treatment days 1 and 2, after which tumour size and
toxicity was monitored daily over a period of 8 days. Tumours were
Reduction study of 2: A stock solution of 2 (0.205 mM) in phosphate
buffer (200 mM, pH 7.4) was made and its concentration was determined
accurately by ICP-OES. Separately, a solution of sodium L-ascorbate (0.3
mM) in phosphate buffer (200 mM, pH 7.4) was made. The stock solution
of 2 (100 μL) and phosphate buffer (1800 μL, 200 mM, pH 7.4) were mixed
in a HPLC vial. The reduction was started by adding the sodium L-
ascorbate solution (100 μL) into the vial, capping it, and shaking vigorously
for 5 s. The vial was then placed in the HPLC autosampler, and the batch
run was initiated. Injections (50 μL) were performed at 10 min intervals,
and detection was by UV (214 and 254 nm). The area of the starting
material peak was monitored over time. A Shimpack VP-ODS C18
analytical column (5 μM, 120 Å, 150 mm × 4.60 mm i.d) was used, with an
isocratic elution method (total flow = 1 mL/min, solvent system = 40%
acetonitrile and 60 % water).
2
measured daily, volume = (the largest diameter)
diameter) X 0.5.
X (perpendicular
Statistical Analysis: Statistical analysis was performed based on a two-
way ANOVA with post-hoc Dunnett's multiple comparisons test performed
in GraphPad Prism.
Acknowledgements
This study was supported by the European Union (ERC-2015-
StG-LS7-680209) to PNS, Ministry of Education (Singapore)
research grant (R143-000-680-114) to WHA, and National
University of Singapore Research Scholarship to KL.
MTT Assay: The cytotoxicity of the compounds was determined by a
colorimetric
microculture
(3-(4,5-dimethythiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. The cells were harvested from
culture flasks by trypsinisation and seeded into Cellstar 96-well
microculture plates (seeding density = 6000 cells/well). After the cells had
resumed exponential growth for 24 h, they were exposed to the
compounds under investigation at different concentrations in media for 72
h. The compounds (1, 2, cDDP, and EA) were diluted separately in
complete medium to the desired concentrations, and this solution (100 μL)
was added to each well and serially diluted to other wells. After exposure
for 72 h, compound solutions were replaced with MTT in media (100 μL, 5
mg/mL) and incubated for an additional 45 min. Subsequently, the medium
was aspirated and the purple formazan crystals formed in viable cells were
dissolved in DMSO (100 μL/well). Optical densities (570 nm) were
measured with a microplate reader. The quantity of viable cells was
expressed in terms of treated/control (T/C) values by comparison to
untreated control cells, and 50% inhibitory concentrations (IC50) were
calculated from concentration-effect curves by interpolation. Evaluation
was based on means from at least three independent experiments, each
comprising six replicates per concentration level.
Keywords: Medicinal chemistry • Cancer • Cytotoxicity •
Platinum • Redox chemistry
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