3.10.1. Cytotoxicity Assay of Compound **1** with NCI-H460 Cell Line

NCI-H460 hypotriploid human cells (ATCC HTB-177) were grown in monolayers to near confluence in flasks and then seeded into wells at 3.33 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/mL of Roswell Park Memorial Institute (RPMI) medium with standard fetal bovine serum (FBS), 180 μL/well, and incubated for 24 h at 37 ◦C in 96-well plates. Cells were exposed to compound **1** at ten half log concentrations, the highest being 21.9 μM with 1% dimethyl sulfoxide (DMSO) present, while the lowest was 0.7 nM. Plates were incubated for an additional 48 h and then stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), for 25 min, after which the optical densities were recorded at 630 and 570 nm for each well on a SpectraMax M2 microplate reader with SoftMax® Pro Microplate Data Acquisition and Analysis Software (Molecular Devices, LLC, Version No. M2, Sunnyvale, CA, USA). The test samples were compared with a negative control of 1% DMSO and a positive control of doxorubicin (0.1 μg/mL and 1 μg/mL), both in RPMI medium. Due to the limited availability of compound **1**, fully toxic concentrations were not reached; hence, the resultant dose–response curve was incomplete. Nevertheless, the IC50 value for compound **1** is greater than 21.9 μM.
