Discovery of sulfonamide-tethered isatin derivatives as novel anticancer agents and VEGFR-2 inhibitors

Synthesis and characterization: Multiple sulfonamide-tethered isatin derivatives were synthesized and isolated as E/Z mixtures with reported yields (e.g., 68–88%) and melting points. Structural characterization was performed using 1H/13C NMR and EI-MS, with detailed chemical shift assignments and elemental analysis provided for each compound in the supplementary figures and spectra (e.g., compounds 6a–6i, 11a–11c, 12a–12c). In vitro cytotoxicity (SRB assay on T47D cells): Exponentially growing T47D cells were trypsinized, counted, and seeded at 5000 cells/100 μL per well in 96-well plates. After 24 h incubation at 37 °C (humidified atmosphere), cells were treated with test compounds at 0.01, 0.1, 1, 10, or 100 μM (or 1% DMSO vehicle) for 72 h. Media were removed and cells fixed with 10% trichloroacetic acid (4 °C, 1 h), washed with water, stained with 0.4% SRB for 30 min, and washed with 1% acetic acid. Protein-bound dye was dissolved in 10 mM Tris base, and OD was measured at 510 nm (Spectra Max Plus). Cell viability was expressed relative to untreated controls. NCI-60 primary anticancer screen: Test compounds were evaluated at a single concentration (10 μM) across 60 human tumor cell lines (nine cancer types) per NCI protocol, with 48 h incubation. Growth inhibition was quantified via SRB, reported as percent growth relative to untreated controls. VEGFR-2 inhibition assay: Per previously reported methods (references 4,5), VEGFR-2 kinase inhibitory activity was assessed (procedural details referenced in the main text cited methods). Carbonic anhydrase inhibition (CA I, II, IX): CO2 hydration rates were measured using an Applied Photophysics stopped-flow instrument. Phenol red (0.2 mM) served as indicator at 557 nm in 20 mM HEPES buffer (pH 7.5) with 20 mM Na2SO4. CO2 concentrations ranged from 1.7 to 17 mM. At least six traces of the first 5–10% of the reaction were used to determine initial velocities. Uncatalyzed rates were subtracted. Inhibitors were prepared as 0.1 mM stock solutions in water and diluted in buffer down to 0.01 nM. Enzyme and inhibitor were preincubated for 15 min at room temperature to allow E–I complex formation. Inhibition constants were determined using non-linear least-squares fits and the Cheng–Prusoff equation (PRISM 3), averaging at least three measurements. Annexin V-FITC apoptosis assay: Cells treated with compounds 11b and 12b for 24 h were trypsinized, washed with cold PBS, then stained with Annexin V-FITC and propidium iodide (PI) in binding buffer for 15 min at room temperature in the dark, and analyzed by flow cytometry. Cell cycle analysis: T47D cells (1 × 10^5 cells/well) were seeded in 6-well plates and incubated 24 h. Cells were treated for 24 h with vehicle (0.1% DMSO) or 10 μM of compounds 11b or 12b. Cells were harvested and fixed in ice-cold 70% ethanol at 4 °C for 12 h, ethanol removed, washed with cold PBS, then incubated in PBS containing RNase (1 mg/mL) for 30 min at 37 °C in the dark prior to PI staining and flow cytometric analysis (per references 8,9). Molecular dynamics (MD) simulations: GROMACS 2021 was used to simulate top docking poses. Ligand topologies were generated via CGenFF; protein parameters from CHARMM (CHARMM36 for protein). Systems were solvated in a cubic TIP3P water box with 10 Å padding and neutralized with Na+ and Cl−. Non-bonded interactions used a 12 Å cutoff and the Verlet cutoff scheme; long-range electrostatics were treated with PME. Energy minimization used steepest descent (5000 steps). Equilibration employed NVT and NPT ensembles for 125 ps at 300.15 K with positional restraints of 400 kJ mol−1 nm−2 (backbone) and 40 kJ mol−1 nm−2 (side chains). Production runs were 100 ns in NPT at 300.15 K and 1 bar, using Nose–Hoover thermostat and Parrinello–Rahman barostat; LINCS constrained H-bonds. V-rescale thermostat at 300 K with 1 ps coupling was also used. Trajectories were saved every 2 ps. Analyses included RMSD (gmx_rms), RMSF (gmx_rmsf), radius of gyration (gmx_gyrate), hydrogen bonds (gmx_hbond), protein–ligand center-of-mass distance (gmx_distance), visualization/contact analysis with VMD, and MM/PBSA binding energy estimation using g_mmpbsa.