## 2 Results and Discussion
### Lipophilicity and Protein Affinity
The results depended on the algorithm and base data used in the log \(P\) calculation [41]. In this group of compounds, the differences were particularly pronounced. The log \(P\) values obtained by the numerical methods C log \(P\) and M log \(P\) were smaller than log k\({}_{\rm w}\)--obtained by RP C18 HPLC or IAM chromatography--but they were colinear, which was observed for some groups of compounds [42]. The relationship between log k\({}_{\rm w}\) and C log \(P\) is expressed by the following equation:  
\[\text{Log k}_{\rm w}=0.50818(\pm 0.0539)\text{Clog }P+2.3107\ (0.0504)\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \  
1-hydroxybutyl substituent. The lowest lipophilicity was found for the compounds with amine and two hydroxyl groups (**8g**, **8h** and **8k**). Their properties were the result of large polar surface areas (tPSAs) (Table 6). The compounds being the pairs of enantiomers had the identical lipophilic-hydrophilic characterization. The tPSAs of the compounds were on the borderline or above the range indicated as beneficial for potential drugs [43]. The plasma protein affinity of the compounds was analyzed using immobilized human serum albumin (HSA) and \(\alpha_{1}\)-glycoprotein (AGP), which are the main blood proteins. Measurements were performed under the isocatric conditions using the propan-2-ol/acetate ammonium buffer at pH = 7.4 (15:85, _v/v_) as the mobile phase. The log k values of all compounds are presented in Table 7. Based on the calibration curve log K = f(log k) (Equations (4) and (5)) of a set of drugs of the known percentage of protein binding, the log K values of the compounds were calculated. Next, they were converted to a percentage of protein plasma binding (% PPB) [44]. The results are presented in Table 7. They show that the compounds bound poorly to the plasma proteins, particularly to AGP. The calculated pKa values of the compounds and the analysis of various microspecies distributions indicated the molecular forms of the compounds at pH = 7.4 and, therefore, they bound poorly to the proteins [45; 46]. In the case of only compound **8m**, 4% of the cationic form was found at pH = 7.4. This was associated with the piperazine ring protonation. This was revealed by the greater extent of compound-to-AGP binding. Thus, this confirms a general trend that glycoproteins bind bases better than the other microspecies. The quantitative structure-binding relationship analysis confirmed the positive contribution of lipophilicity in the HSA binding. The compounds of the highest lipophilicity exhibited the strongest affinity for HSA (compounds **8i**, **8j** and **8m**) [46]. The HSA and AGP binding was also largely correlated with the molar refractivity (MR) of the compounds. Summing up, the above-described chromatographic studies (IAM and RP-18) and the calculated in silico log \(P\) values gave very different values of log k\({}_{\text{w}}\) and log \(P\), but they were collinear and described well the lipophilicity changes in the test series of compounds. The studied compounds were characterized by a small affinity for plasma proteins, which could be advantageous in potential in vivo studies.