## 2 Materials and Methods
### Chemistry
#### 2.2.2 In Vitro Cytotoxicity Screening  
The potential of **6a-g** to reduce the in vitro viability of the human Caucasian hepatocyte carcinoma (HepG2) cell line population to 50% of its maximum viability was described with inhibitory concentrations (IC\({}_{50}\) values, in uM units). The methods for in vitro estimation of IC\({}_{50}\) parameters are published in [43; 44] and, in addition, are also provided in the Supplementary Materials. The IC\({}_{50}\) value for a particular molecule (Table 3) was acquired from a nonlinear regression analysis based on the dependence between a semi-logarithmic plot of incubation concentration and percentage of absorbance (_A_) value relative to untreated controls [43; 44]. These IC\({}_{50}\) descriptors were calculated using Microsoft Excel software, version 2016 (Microsoft Corp., Redmont, WA, USA). \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Entry**} & \multirow{2}{*}{**\(\mathbf{log}\)\(k_{\text{w}}\)**} & \multirow{2}{*}{\(S\)} & \multicolumn{2}{c}{**1 \(\chi^{2}\)red**} & \multicolumn{2}{c}{**2 \(RSS\)**} & \multicolumn{2}{c}{**3 \(R\)**} & \multicolumn{2}{c}{**4 \(Adj.\)\(R^{2}\)**} & \multicolumn{2}{c}{**5 \(RMSE\)**} & \multicolumn{2}{c}{**6 \(F\)**} & \multicolumn{2}{c}{**7 _Prob \textgreater{}F_**} \\ \hline
6a & 4.1170 & 4.7782 & 0.0036 & 0.0179 & 0.9945 & 0.9867 & 0.0598 & 447.63 & 4.37 \(\times\) 10\({}^{-6}\) \\
6b & 4.8419 & 5.5848 & 0.0017 & 0.0066 & 0.9976 & 0.9939 & 0.0408 & 821.64 & 8.82 \(\times\) 10\({}^{-6}\) \\
6c & 4.2672 & 4.9826 & 0.0040 & 0.0201 & 0.9943 & 0.9863 & 0.0635 & 431.66 & 4.78 \(\times\) 10\({}^{-6}\) \\
6d & 4.8631 & 5.5062 & 0.0020 & 0.0097 & 0.9977 & 0.9945 & 0.0441 & 1089.80 & 4.79 \(\times\) 10\({}^{-7}\) \\
6e & 5.0882 & 5.5840 & 0.0004 & 0.0016 & 0.9997 & 0.9992 & 0.0176 & 7051.83 & 4.54 \(\times\) 10\({}^{-9}\) \\
6f & 4.9260 & 5.3504 & 0.0023 & 0.0114 & 0.9972 & 0.9932 & 0.0478 & 876.96 & 8.23 \(\times\) 10\({}^{-7}\) \\
6g & 5.2184 & 5.9042 & 0.0011 & 0.0056 & 0.9989 & 0.9973 & 0.0335 & 2180.52 & 8.51 \(\times\) 10\({}^{-8}\) \\ \hline \hline \end{tabular}  
* \(\chi^{2}\)red, reduced chi-square; \({}^{2}\)_RSS_, residual sum of squares: \({}^{3}\)\(R\), correlation coefficient; \({}^{4}\)_Adj._\(R^{2}\), adjusted coefficient of determination; \({}^{5}\)_RMSE_, root mean square error (standard deviation); \({}^{6}\)_F_, [PERSON]’s significance ratio ([PERSON]’s _F_-test); \({}^{7}\)_Prob \textgreater{} \(F\)_, probability of obtaining the _F_-ratio (significance of a whole model).  
\end{table} 
Table 2: Extrapolated log \(k_{\text{w}}\) parameters and \(S\) (slope) data (RP-HPLC) of the **6a–g** set together with the calculated values of statistical descriptors (\(\chi^{2}\)red, \(RSS\), \(R\), \(Adj.\)\(R^{2}\), \(RMSE\), \(F\), and _Prob \textgreater{}F_), which defined the linearity between log \(k\) and \(\varphi_{\text{M}}\) for a particular molecule. The indication of a significance level of the _F_-ratio (_Prob \textgreater{}F_ parameter): extremely significant in all cases. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Entry**} & \multicolumn{4}{c}{**MIC (μM)**} & \multicolumn{2}{c}{**IC\({}_{50}\) (μM)**} \\ \cline{2-5}  & _Mtb_ H\({}_{37}\)Ra & _MK DSM_ & _M_S_ & _MM_ & _HepG2_ \\ \hline
6a & 69.58 & 69.58 & 139.16 & 69.58 & 83.08 \\
6b & 33.76 & 33.76 & 67.52 & 33.76 & 33.10 \\
6c & 33.48 & 66.96 & 133.92 & 66.96 & 49.36 \\
6d & 32.36 & 8.09 & 64.73 & 15.12 & 43.35 \\
6e & 3.78 & 15.13 & 30.25 & 15.12 & 29.39 \\
6f & 15.15 & 15.15 & 15.15 & 30.36 & 54.71 \\
6g & 3.63 & 14.54 & 14.54 & 8.09 & 22.18 \\ INH & 58.33 & 29.16 & 43.75 & 29.16 & nd \\ \hline \hline \end{tabular}  
* nd = not determined.  
\end{table} 
Table 3: The in vitro biological evaluation of the **6a–g** set and isoniazid (INH) reference drug including their activity (MIC values, in μM units) against _M. tuberculosis_ H\({}_{37}\)Ra ATCC 25177 (_Mtb_ H\({}_{37}\)Ra), _M. kansasii_ DSM 44162 (_MK_ DSM), _M. smgematis_ ATCC 700084 (_MS_), and _M.