## 2 Results and Discussion
### Lipophilicity and Protein Affinity  
Considering the lack of cytotoxicity against the selected tumor cell lines and against several in vitro tests we undertook for the studies presented above, which were expected to explain the reason for these results, we undertook studies on the detailed analysis of the physicochemical properties of our compound as lipophilicity and protein affinity. At the beginning, the UV-Vis spectra of compound **8g** were made to find out the influence of a pH solution on the electronic structure of the compound and its retention in the chromatographic system. The lack of influence of the pH solution on the electron structure of the compounds was demonstrated. As a pH = 7.4 was recommended for the IAM chromatographic studies, C-18 chromatography evaluation was made at this physiological pH. There were regular changes in the retention of compounds in a function of the organic modifier (MeOH, ACN) content in the mobile phase for both chromatographic systems. That relationship is described by the Soczewinski-Wachtmeister equation [32]:  
\[\text{log k}=\text{log k}_{\text{w}}+\text{S}\ \varphi \tag{1}\]  
where \(\varphi\) is the volume fraction of the organic modifier in the mobile phase, \(\text{log k}_{\text{w}}\) is the intercept and \(S\) is the slope of the regression curve. \(\text{Log k}_{\text{w}}\) refers to the retention parameter of a compound with pure water as the mobile phase. The S and \(\text{log k}_{\text{w}}\) quantities, estimated by the extrapolation procedure, are commonly applied as lipophilicity descriptors [33; 34; 35]. The obtained data are presented in Table 5. The estimated \(\text{log k}_{\text{w}}\) values for both phases were significantly different, but they were highly correlated. The relationship between the \(\text{log k}_{\text{w}}\) values is expressed by the following equation:  
\[\text{log k}_{\text{w(IAM)}}=0.7821(\pm 0.0545)\ \text{log k}_{\text{w(RP-18)}  
The differences between the log k\({}_{\rm w(RP-18)}\) and log k\({}_{\rm w(IAM)}\) values were the result of various interactions of considered pyrazolo[4,3-\(e\)][1,2,4]triazine sulfonamides on the octadecyl and IAM phases. [PERSON] and [PERSON] assumed that partitioning was the principal retention mechanism in IAM retention, including both hydrophobic and polar interactions with the solvated layer(s) of the stationary phases and ionizable groups of immobilized phospholipids [36]. The studies showed that the compounds were characterized by weaker affinity to the immobilized phospholipids than to the octadecyl phase. Significant electrostatic interactions with the IAM phase resulted in different retention of compounds and different values of log k\({}_{\rm w}\) in the IAM phase compared with those obtained using the C-18 one. A similar trend was observed for other pyrazolo[4,3-\(e\)][1,2,4]triazines [37]. A significant though relatively stable difference between the log k\({}_{\rm w}\) values for both phases (\(\Delta\) log k\({}_{\rm w}\) in the range of 1-1.3) may suggest that these were the result of mainly heterocycling-sulphonamide moiety interactions (unmodified element of the considered compounds) (Table 5). The molar refractivity (MR) and the polar surface area (tPSA) were estimated according to the fragmentation method introduced by [PERSON] [38]. ACD log \(P\) was calculated using ACD/Labs methodology [39]. C log \(P\) and M log \(P\) were estimated for comparison (Table 6). Using the selected methods, the calculated log \(P\) values proved to be significantly different but colinear (correlation coefficient r was in the range of 0.93-0.98). Particularly, the Moriguchi estimation of the log \(P\) values gave low values. The obtained log \(P\) values expressed the lipophilicity changes of the studied set of compounds, but they did not describe the actual octanol-water coefficient distribution value. This phenomenon was observed in some groups of new synthesized and studied compounds as well as applied drugs [40].