## 2 Materials and Methods
### Cytotoxicity Assays  
The cytotoxic effects of the compounds S1-S4 were assessed using the XTT assay, detecting the reduction of tetrazolium salt by mitochondrial succinate dehydrogenase.  
The XTT--Tetrazolium Hydroxide Kit (Xenometrix AG, Allschwil, Switzerland)--was employed according to the manufacturer's protocol.  
The cells were introduced into 96-well culture plates at a concentration of \(1\times 10^{5}\) mL\({}^{-1}\) and allowed to adhere. Then, the cells were exposed to varying concentrations of S1-S4 (from 0 to 600 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro m}\) mL\({}^{-1}\)) for either 24 or 72 h. Negative controls consisted of the culture medium alone, while positive controls contained 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). After exposure, the medium from each well was collected for subsequent cytokine analysis, and the cell monolayers were washed thoroughly (at least three times) with PBS to eliminate any residual substances. Fresh sDMEM containing a mixture of XTT reagents (XTTII and XTTI; 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazol-5-carboxyanilide inner salt with buffer) was then added at the ratio of 1:100. After 3 h of incubation, the content was mixed. A FLUOstar Omega multi-mode plate reader (BMG Labtech, Ortenberg, Germany) was used to measure the absorbance. Measurement was performed at 480 nm (690 nm as the reference wavelength).  
All reagents used in the XTT assay were part of the kit. Two independent repetitions of the entire procedure were performed. Each determination was performed in triplicate. The 50% inhibitory concentration (IC\({}_{50}\)) was calculated from the concentration-response curves with the use of the standard Hill equation, as described in [15]. The following parameters were estimated: the dissociation constant (K), the Hill exponent (E), benchmark dose (BMD), and IC\({}_{50}\).  
The CellTiter-Glo(r) Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was conducted in accordance with the manufacturer's instructions to assess the quantity of metabolically active cells. Cells were plated at a density of \(1\times 10^{7}\) mL\({}^{-1}\) in 96-well plates and treated with varying concentrations of the compounds under investigation for either 24 or 72 h. Following treatment, a lyophilized reagent containing luciferin and luciferase was introduced, and the plates were gently shaken on an orbital shaker for 2 min to facilitate cell lysis. The samples were then incubated for 10 min at room temperature to ensure the luminescence signal stabilized before measurement with the multi-mode plate reader.  
The CytoTox-ONE Homogeneous Membrane Integrity Assay Kit (Promega, USA), utilizing a fluorometric method to measure the release of LDH from cells with damaged membranes, was also employed. The assay was performed following the manufacturer's guidelines. Cells were seeded at a density of \(1\times 10^{5}\) mL\({}^{-1}\) and subjected to different concentrations of the test compounds for either 24 or 72 h. After the treatment period, 50 \(\mathrm{\SIUnitSymbolMicro m}\) of the culture medium from the treated cells was mixed with 50 \(\mathrm{\SIUnitSymbolMicro m}\) of the CytoTox-ONE reagent. This mixture was incubated in the dark for 10 min at room temperature, and the reaction was halted by adding 50 \(\mathrm{\SIUnitSymbolMicro m}\) of the stop solution. Fluorescence readings were taken using a multi-mode plate reader at an excitation wavelength of 560 nm and an emission wavelength of 590 nm.