## Materials and Methods
### Evaluation of the Antioxidant Potential of the Bicyclic Thiazolpyrimidine Derivatives
#### 2.3.2 Redox Status Methods  
Analyses were performed in the human serum pool harvested from healthy volunteers. Investigated compounds dissolved in DMSO in volume of 50 \(\upmu\)L were added in serum aliquots (450 \(\upmu\)L) in duplicates. Three different concentrations were prepared (100%, 50%, 25%) for every substance. An additional set of samples was prepared with the addition of the prooxidant terc-bythil hydroperoxide (TBH) in order to get information about the antioxidative potency of the compounds. The samples were incubated at 37 \({}^{\circ}\)C, 2 h in a water bath. The control sample was native serum pool. Advanced Oxidation Protein Products (AOPP)  
AOPP was assayed in 20 mM phosphate buffer with pH 7.4 in reaction with glacial acetic acid and 1.16 M potassium-iodide according to [PERSON] et al., [22]. The complex formed has an absorbance maximum at 340 nm. The calibration of this reaction is performed with Chloramine T as a standard, with a concentration range 10-100 \(\upmu\)mol/L. This compound also has a specific absorption maximum at 340 nm. From the obtained parameters, the Oxy Score is calculated that represents the resulting action and is then used for further analysis. Total Oxidative Status (TOS)  
TOS was measured by using a spectrophotometric method optimized by [PERSON], 2005 [23], and [PERSON] et al., 2015 [24]. All oxidants such as hydrogen-peroxide and lipid-hydroperoxide from the sample have the capability to oxidize the ferrous ion-o-dianisidine complex to a ferric ion. The reagent is composed of 5 mM ferrous ammonium sulfate and 10 mM _o_-dianisidinedihydrochloride. The oxidation is performed in high concentration of glycerol molecules (1.35 mol/L). The second reaction is based on the ferric ion reaction with xylenol orange (150 \(\upmu\)mol/L) in an acidic medium, which makes a colored complex. The intensity of color is proportional to the total amount of oxidant molecules present in the sample. Absorbance measurement is at 560 nm. The assay is calibrated with hydrogen peroxide (water solution, 10-200 \(\upmu\)mol/L) and the results are expressed in micromolar hydrogen peroxide equivalent per liter (\(\upmu\)mol H\({}_{2}\)O\({}_{2}\) equiv/L). Paraoxonase-1 Enzyme Activity (PON1)  
Serum PON1 activity was measured kinetically using paraoxon (POase activity) and diazinon-O-analog (DZOase activity) as substrates, by the method of [PERSON], 1999 [25], and the PON1 phenotype was determined both graphically and by using the anti-mode method with DZOase/POase activity ratio calculation. POase activity determination is based on serum PON1 enzyme activity on 1.2 mM paraoxon as a substrate, and its conversion to a \(p\)-nitropol compound; the rate of this conversion could be followed at 405 nm, because in alkaline conditions (10 mmol/L Tris-HCl, pH 8.5, 2 mmol/L CaCl\({}_{2}\)) \(p\)-nitropol has an intense yellow color. DZOase activity was measured upon PON1 enzyme reaction with a diazinone-O-analog (1 mM); after that it decomposed on diethyl-phosphate and 2-isopropyl-4-methyl-6-pyrimidinol (IMHP); the rate of this conversion could be followed at 270 nm, where IMHP has a characteristic absorption maximum. Total SH-groups Content  
The levels of SH-groups were measured by [PERSON]'s method [26] modified by [PERSON] et al., 2015 [24], using 10 mM dinitrodithiobenzoic acid (DTNB) as a reagent. DTNB reacts with aliphatic thiol compounds in a basic environment (pH 9.0) and this reaction generates 1 mole \(p\)-nitropol anion per mole of thiol. The absorbance was measured at 412 nm. The calibration of the method was achieved with the reduced glutathione in the concentration range from 0.1-1.0 mM. Total Antioxidant Status (TAS)  
TAS was measured by a spectrophotometric method using 10 mmol/L 2,2'-azino-bis(3-ethylbenzothazoline-6-sulphonic acid (ABTS) as a chromogen [27]. The reduced ABTS molecule is oxidized to ABTS\({}^{+}\) radical cation using hydrogen peroxide in acidic medium (an acetate buffer 30 mmol/L, pH 3.6).