2.4. Compound Toxicity and Stability Testing

Toxicity of compounds 5 and 7 was evaluated in vitro on the U-87 cell line. Briefly, U-87 cells were seeded in 96-well plates (cat. No. 92096, TPP Techno Plastic Products, Trasadingen, Switzerland) at 2000 cells/well in DMEM FluoroBrite containing 10% FBS and incubated for 24 h. Cells were washed and further grown in DMEM FluoroBrite with 5% KnockOut™ Serum Replacement (Gibco, Waltham, MA, USA). Compounds 5 and 7 were added to the wells in a 1:2 dilution series starting at 20 µM and ending at 0.16 µM. To evaluate the effect of the compound on cell growth, confluence was determined every hour for 17h using the Incucyte^® S3 Live-Cell Analysis System (Sartorius, Göttingen, Germany). Next, cells were stained with the LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells (Invitrogen, Carlsbad, CA, USA; cat. # L3224) and cell viability was evaluated. Data were normalized to the mean confluence of wells containing no inhibitors. To measure the stability of the compounds, fresh blood was collected from heathy mice by cardiac puncture with a heparin-coated needle and syringe and 30UI/mL heparin was added. Next, compound 5, compound 7 or vehicle (DMSO) was added to the blood at a final concentration of 500 µM and incubated at 37 °C. At indicated time periods, samples were taken, diluted ½ in phosphate-buffered saline (PBS) and centrifugated at 500× g for 5 min to collect plasma. The cell pellet was washed twice with PBS and cells were lysed in radioimmunoprecipitation assay buffer (RIPA) buffer (25 mM Tris, 150 mM NaCl, 1% NP-40, 1% Sodium Deoxycholate, 0.1% SDS, pH 7.6). To determine the remaining inhibitory activity of the compounds, the plasma and cell lysates were incubated with active recombinant MMP-9 (0.5 nM) and the remaining MMP-9 activity was measured using the OmniMMP substrate peptide. Background inhibition was subtracted and results were expressed as percentage inhibition.