Cytotoxicity Evaluation of Compounds 1 and 2

Compounds 1 and 2 were assayed (at concentrations of 16 μg/mL, 32 μg/mL, 64 µg/mL, and 128 μg/mL) against a human epithelial colorectal (Caco-2) cell line (American Type Culture Collection, ATCC HTB-37) using the MTS assay [26 (link)]. Cells were cultured in DMEM supplemented with penicillin-streptomycin, nonessential amino acids (1%), and FBS (10%), at 37 °C with CO₂ (5%). The cells were incubated with compounds (in triplicate) or DMSO (negative control) in a 96-well tissue culture-treated plate at 37 °C with CO₂ (5%) for two hours. The MTS assay reagent (Promega, Madison, WI, USA) was subsequently added to each well and plates were incubated for four hours at 37 °C with CO₂ (5%). The quantity of viable cells (at OD₄₉₀) after treatment was expressed as a percentage of the viability of DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity data was analyzed via a two-way ANOVA, with post hoc Dunnet’s multiple comparisons test (n = 3, P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).