*2.4. In Vitro Analysis*

## 2.4.1. Hydrogen Peroxide Scavenging Activity (*HPSA*)

The Manolov et al. method was used to assess the hydrogen peroxide scavenging capability [17]. A 43 mM solution of H2O2 was prepared in a potassium phosphate buffer solution (0.2 M, pH 7.4). The analysis of the samples was carried out as follows: in test tubes, 0.6 mL H2O2 (43 mM), 1 mL sample/standard with different concentrations (20–1000 μg/mL), and 2.4 mL potassium phosphate buffer solution were mixed. The mixture was stirred and incubated in the dark for 10 min at 37 ◦C. Absorbance was measured at 230 nm with a spectrophotometer (Camspec M508, Leeds, UK) against a blank solution containing phosphate buffer and H2O2 without the sample. Ascorbic acid and quercetin were used as standards. The percentage *HPSA* of the samples was evaluated by comparing it with a blank sample and calculated using the following formula:

$$I\_\prime\%(HPSA) = \left[\frac{A\_{blank} - (A\_{TS} - A\_{CS})}{A\_{blank}}\right] \times 100\%$$

where *Ablank* is the absorbance of the blank sample, *ACS* is the absorbance of the control sample, and *ATS* is the absorbance of the test sample.

#### 2.4.2. Inhibition of Albumin Denaturation (*IAD*)

*In vitro*, analysis of anti-inflammatory activity was assessed as inhibition of albumin denaturation (*IAD*). The analysis was performed according to Manolov et al. method [18] with minor modifications. The experiment was performed with human albumin. The solution of albumin (1%) was prepared in distilled water (pH 7.4). The tested compounds/standard were dissolved firstly in PBS, so the final concentration of the stock solution was 1000 μg/mL. Then a series of working solutions with different concentrations (20–500 μg/mL) in PBS were prepared. The reaction mixture was containing 2 mL test sample/standard of different concentrations and 1 mL albumin (1%). The mixture was incubated at 37 ◦C for 15 min and then heated at 70 ◦C for 15 min in a water bath. After cooling, the turbidity was measured at 660 nm with a spectrophotometer (Camspec M508, Leeds, UK). Ibuprofen and ketoprofen were used as standards. The experiment was performed three times. Percentage inhibition of albumin denaturation (*IAD*) was calculated against the control. The control sample is albumin with the same concentration dissolved in distilled water.

$$\%IAD = \left[\frac{A\_{blank} - A\_{sample}}{A\_{blank}}\right] \times 100$$

2.4.3. Determination of Lipophilicity as cLlogP

The lipophilicity of the compounds was calculated using the software: ACD/ChemSketch/ LogP Predictor v.14.08.

## 2.4.4. Molecular Docking

The molecular docking of the ligands was performed using AutoDock Vina 1.1.2 (ADV) and AutoDock 4.2 (AD) [19,20] against the human serum albumin (HSA), deposited with the entry code 7JWN in the Protein Data Bank [21]. Two software were chosen for molecular docking because although their names are similar, the principle on which they work is different. Because it is known that molecular docking studies may generate false positive results, the use of two software that works differently can be used for cross-validation of the results [22]. Supplementary, AD possesses an intrinsic tool that performs a rapid clustering analysis to confirm that the best binding pose is one of the most found poses from the total of poses generated.

For each compound, two ligand files were created, one for each R and S isomer using Avogadro 1.2.0 and following the previously reported protocol [23,24].

The preparation of the macromolecule as the target was performed according to the standard procedure previously reported by our group—removal of the co-crystallized molecules, addition of the polar hydrogen atoms and addition of charges [25].

The final preparation of the files of ligands and macromolecules was performed using AutoDockTools 1.5.6 [20].

Four main potential binding sites were targeted in the molecular docking study— Sudlow site I (subdomain IIA), Sudlow II (subdomain IIIA), site III and cleft. The reason for this choice is that, to the best of our knowledge, the other binding sites reported in the literature have a limited role in drug binding, and the sites we have chosen are the most important in drug binding to albumin [26–31].

The search space for each potential binding site was set as a cube, with sides equal to 20 for ADV and 54 for AD (spacing = 0.375). The cartesian coordinates of the center of the search space for each site were set as follows: for site Sudlow1x= 30.62, y = 25.50, z = 12.43, for site Sudlow 2 x = 5.95, y = 18.22, z = 21.06, for site 3 x = 30.15, y = 26.98, z = 37.99 and for cleft site x = 20.89, y = 21.74, z = 22.43.

In order to obtain results with higher reproducibility, ADV was requested to generate 20 poses for each ligand in each binding site, while AD was requested to generate 200 poses in order to perform the cluster analysis in the limit of 2 Å root mean square deviation of the coordinates of atoms.

The visualization of the results of the molecular docking study was performed using Chimera 1.10.2 [32].
