## 2 Results and Discussion
### Susceptibility to Metabolic Transformation
#### 2.5.1 Phase I Metabolism of **8m**, **8i** and **8j** with Rat Liver Microsomes  
Phase I metabolism was considered in the first step of the studies on transformations of **8m**, **8i** and **8j** compounds. Each compound was incubated with rat liver microsomes (RLMs) and NADPH as a cofactor of phase I metabolism, and the reaction mixtures were monitored by HPLC analysis with UV-Vis detection. The chromatograms recorded after 60 min of incubation are shown for each compound in Figure 4. The chromatographic peaks were analyzed by their ESI-MS spectra, and the m/z values related to the HPLC bands are presented in Table 8. The UV-Vis spectra of **8m**, **8i** and **8j** and their metabolites obtained with RLM in the presence of NADPH are presented in Figure 5. \begin{table}
\begin{tabular}{c c c c c c c} \hline  & \multicolumn{3}{c}{**HSA**} & \multicolumn{3}{c}{**AGP**} \\ \cline{2-7}  & **log k\({}_{\text{HSA}}\)** & **log K\({}_{\text{HSA}}\)** & **\% HSA** & **log k\({}_{\text{AGP}}\)** & **log K\({}_{\text{AGP}}\)** & **\% AGP** \\ \hline
8a & \(-0.78.4\) & \(-0.420\) & \(27.8\) & \(-1.841\) & \(-1.647\) & \(2.3\) \\
8b & \(-0.822\) & \(-0.459\) & \(26.1\) & \(-0.6199\) & \(-0.396\) & \(28.9\) \\
8c & \(-0.442\) & \(-0.075\) & \(46.1\) & \(-0.519\) & \(-0.293\) & \(34.1\) \\
8d & \(-0.782\) & \(-0.418\) & \(27.9\) & \(-0.483\) & \(-0.257\) & \(36.0\) \\
8e & \(-2.052\) & \(-1.702\) & \(2.0\) & \(-0.966\) & \(-0.751\) & \(15.2\) \\
8f & \(-1.771\) & \(-1.418\) & \(3.7\) & \(-0.986\) & \(-0.771\) & \(14.6\) \\
8g & \(-0.926\) & \(-0.564\) & \(21.6\) & \(-0.762\) & \(-0.543\) & \(22.5\) \\
8h & \(-1.248\) & \(-0.890\) & \(11.5\) & \(-1.675\) & \(-1.477\) & \(3.3\) \\
8i & \(-0.205\) & \(0.165\) & \(60.0\) & \(-0.297\) & \(-0.066\) & \(46.6\) \\
8j & \(-0.186\) & \(0.1831\) & \(61.0\) & \(-0.384\) & \(-0.155\) & \(41.6\) \\
8k & \(-0.917\) & \(-0.555\) & \(22.0\) & \(-1.646\) & \(-1.447\) & \(3.5\) \\
8l & \(-0.694\) & \(-0.329\) & \(32.2\) & \(-0.494\) & \(-0.268\) & \(35.4\) \\
8m & \(-0.197\) & \(0.173\) & \(60.4\) & \(-0.222\) & \(0.010\) & \(51.0\) \\ \hline \end{tabular}
\end{table} 
Table 7: The log k values of the compounds obtained using the human serum albumin (HSA) and \(\alpha_{1}\)-glycoprotein (AGP) columns, with the log K values obtained from a calibration curve (log binding equilibrium constant) and calculated % plasma protein binding (% PPB) (% HSA, % AGP). \begin{table}
\begin{tabular}{c c c} \hline
**Compd.** & & **ESI-MS m/z** \\ \hline
**8m** & 432.1 & [8m + H\({}^{+}\)] \\ P1 & 448.1 & [8m + 16 + H\({}^{+}\)] \\ P2 & 404.1 & [8m-28 + H\({}^{+}\)] \\ \hline
**8i** & 449.1 & [8i + H\({}^{+}\)] \\ P3 & 465.1 & [8i + 16 + H\({}^{+}\)] \\ P4 & 435.1 & [8i-14 + H\({}^{+}\)] \\ P5 & 421.1 & [8i-28 + H\({}^{+}\)] \\ \hline
**8j** & 449.1 & [8] + H\({}^{+}\)] \\ P6 & 465.1 & [8] + 16 + H\({}^{+}\)] \\ P7 & 435.1 & [8j-14 + H\({}^{+}\)] \\ P8 & 421.1 & [8j-28 + H\({}^{+}\)] \\ \hline \end{tabular}
\end{table} 
Table 8: ESI-MS m/z obtained after **8m**, **8i** and **8j** metabolism with rat liver microsomes (RLMs) and NADPH as cofactors. Figure 4: Metabolism of **8m** (**a**), **8i** (**b**) and **8j** (**c**) with rat liver microsomes (RLMs) and NADPH as cofactors. Representative HPLC chromatograms of the incubations of 0.05 mM **8m**, **8i** and **8j** at 37 \({}^{\circ}\)C with 2 mg/mL of RLM in the presence of 2 mM NADPH. Figure 5: UV-Vis spectra of the metabolites of **8m** (**a**), **8i** (**b**) and **8j** (**c**) formed with RLM and NADPH. Spectra were recorded directly during HPLC analysis by means of a multidiode array detector in an ammonium formate pH 3.4/methanol mobile phase. After 60 min of incubation with **8m**, we observed in Figure 4a two metabolite peaks of very low intensities: P1 at Rt near 17 min and P2 near 19 min, whereas Rt of the substrate was much lower. Analyses of the metabolite spectra also indicated the strong changes in comparison with that of the substrate **8m**. Bathochromic shifts were observed in the spectra of both metabolites P1 and P2.