*3.6. Molecular Docking*

Encouraged by previous reports from our group where docking techniques were applied with good results [47,48], in the present work, docking was employed to search possible imidazole receptors.

Many solid tumors are characterized by aberrant signal transduction through di fferent receptors belonging to the ErbB family of receptor tyrosine kinases, where the EGFR and HER2 receptors belong; therefore, one therapeutic approach in oncotherapy is the inhibition of one or both of these receptors [49,50]. The ErbB receptors and their ligands are overexpressed in the majority of solid neoplasms; EGFR and ErbB-3 are found on average in 50% to 70% of lung, colon, and breast carcinomas [51]. HER2 is mainly related with breast cancer (is expressed in 30% of primary breast carcinomas [51]), but is also related with ovary, colon, lung, uterine cervix, and esophagus cancers, amongs<sup>t</sup> others [52]. As co-expression of di fferent ErbB receptors occurs commonly, 87% of EGFR positive tumors also express HER2 [51]. Due to all of the above, EGFR and HER2 receptors have been selected in the literature [53] to relate in vitro anti-cancer activity to in silico docking calculations. In this last reference, the results from the docking of imidazole derivatives against EGFR and HER2 showed a general good agreemen<sup>t</sup> with their cytotoxic results. They evaluated two imidazoles that are reported in the presented work, **11** and **12**, with generally closely related results; having the same docking algorithm and protocol, di fferences may arise due to di fferent ligand preparation as this step can influence the final result [54]. In the present work, the proposed docking protocol was employed for an initial screening for both EGFR and HER2 as potential cancer-mediated receptors for the synthesized imidazole derivatives.

The binding energies results from the docking analysis are shown in Table 3, which includes imidazole as a negative control and lapatinib, an EGFR and HER2 inhibitor [55], as the positive control. All synthesized compounds showed better results than the imidazole, suggesting the derivatization improved their affinity for these receptors. Although lapatinib showed the best result against both enzymes compared to our compounds, it was closely followed by some products. From the synthesized compounds, **11**, **12**, **5**, **9**, and **7** presented the best results interacting with both EGFR and HER2, as they were in the first five places with lower binding energies. After that, there were variations in the order in which the synthesized products interacted with the selected receptors. Comparing the results for the docking in each receptor, against the in vitro results for each of the evaluated cell lines, there was little agreemen<sup>t</sup> between them. This can be explained in several ways, one could be the use of a specific docking algorithm, while each one presents differences in the way results are achieved. The employment of different algorithms with the present work dataset of ligand structures and GI50 values could be further explored to find the most suitable algorithm for the synthesized ligands. On other hand, it could be possible that the biological receptors where the compounds interact are different to those in EGFR and HER2, explaining the little correlation shown. Additionally, it has been reported that docking results could be significantly improved with post-docking energy refining through semi-empirical methods such as PM7 [56].

The compound that represented good agreemen<sup>t</sup> between its in vitro and in silico results was **11**, bearing a *p*-nitro substitution (Figure 4). It showed −9.11 and −9.19 kcal/mol binding energy with EGFR and HER2, respectively, having the second-best affinity with both receptors. On the other hand, it was the first or second most active compound against the six evaluated cell lines. This suggests that **11** could be one potential lead compound for further derivatization in the search for new active antiproliferative agents.


**Table 3.** Docking scores of the synthesized triphenyl imidazoles with their controls.

**Figure 4.** Compound **11** (orange) depicted with both of the analyzed receptors. Highlighted are the residues at distances <5.0 Å from **11**, with some surfaces active to show the cavity in which the docking analysis put the best docked pose. (**a**) EGFR, with the near residues in purple. (**b**) HER2, with the near residues in cyan.

### *3.7. In Silico Drug-Likeness Prediction*

As can be seen from Table 4, the calculations from the SwissADME website allow for the analysis of which synthesized compounds have better pharmacokinetics and drug-like properties.

All of them had a TPSA between the limits suggested for good bioavailability (20–130 Å2). The vast majority are inhibitors to cytochrome enzymes, which could affect the metabolism and present drug–drug interactions [27], **13** being the least CYP inhibitor, followed by **11**, **12**, and **9**. Although their water solubility was moderate, all of them are predicted to have a high gastrointestinal (GI) absorption (although this can be partially limited for **1**–**10**, being P-gp substrates). The exception to this is compound **13**, which is poorly soluble and has low GI absorption. The grea<sup>t</sup> majority seem to be able to be permeate the blood–brain barrier (BBB), although this was not the case for compounds **9**, **11**, **12**, and **13**. However, as all the BBB permeant compounds are also P-gp substrates, they would be pumped out from the brain and we would expect no interactions with the central nervous system due to this. Due to these enlisted data, we could expect the synthesized compounds to be, in general, suitable for oral administration.

Lipinski's rule of five [57] can be applied as a first filter, which accounts for the physicochemical properties related to the drug-likeness of a molecule. The molecular weight, number of H-bond donors and acceptors, and lipophilicity are in general accordance to the Lipinski rule. Only compounds **9** and **13** presented a violation, in both cases related to their very high lipophilicity.

Considering the predicted pharmacokinetics and drug-likeness, compounds **11** and **12** with nitro substitution can be considered as promising lead compounds for further studies, which can be additionally supported by the fact they were amongs<sup>t</sup> the most active in vitro compounds, both as AChE inhibitors, **12** as an XO inhibitor, and **11** as part of the antiproliferative imidazoles in cancer cell lines.



MW = Molecular weight; TPSA = Topological polar surface area; Log P = Logarithm of the partition coefficient; ESOL Log S = ESOL model logarithm of molar solubility in water; ESOL class = Solubility class in Log S scale; MS = Moderately soluble; PS = Poorly soluble; GI = Gastrointestinal; BBB = Blood–brain barrier; P-gp = Permeability glycoprotein; CYP = Cytochrome.
