## 3 Results
### Molecular Docking  
The knowledge of the target and respective binding site of the molecule under study is essential in drug development [49]. Three steroidal targets have been studied by molecular docking: ER\(\alpha\), ST, and 17\(\beta\)-HSD type 1. ER\(\alpha\) is involved in the control of many physiological processes, such as cell proliferation [50]. ST and 17\(\beta\)-HSD type 1 are also relevant for the regulation of cell replication by adjusting steroid hormone levels [51]. In fact, ST makes the conversion of 3-sulfated steroids into their hydroxylated analogs, including the transformation of E1 sulfate into E1. In addition, 17\(\beta\)-HSD type 1 reduces the 17-carbonyl group of androstane and estrane molecules to more potent 17\(\beta\)-hydroxysteroids [52]. Therefore, their deregulation is involved in cancer development, particularly in hormone-dependent breast cancers.  
Molecular docking studies were made against ER\(\alpha\), ST, and 17\(\beta\)-HSD1 by means of AutoDock vina executable. The results obtained for compounds **2**, **4-6** are summarized in Table 3. For all the tested compounds, binding energy values were similar for ER\(\alpha\) and higher than those determined for E2, which suggests a weak affinity to this target. In addition, by analyzing the interactions between these compounds and the macromolecule, marked differences were observed when compared with the interactions performed by E2. However, regarding the results for compound **5**, which presented the best affinity value, it is possible to verify some common interactions between this compound and the reference compound (E2). In fact, although compound **5** does not establish the essential  
Figure 6: HepaRG cycle distribution after treatment with compound **4** (at 50 \(\upmu\)M) for 24 h. 5-Fluorouracil ((5-FU), 50 \(\upmu\)M) was used as positive control and untreated cells as a negative control. Each bar represents the mean \(\pm\) SD (originating from two independent experiments). \(***\)\(p\) < 0.001 vs. control (two-way ANOVA post-hoc Bonferroni test).  
Figure 7: HepaRG relative fluorescence intensity after treatment with compound **4** (50 \(\upmu\)M) for 48 and 72 h, obtained by the carboxyfluorescein succinimidyl ester assay. Each bar represents the median with a range of two samples. * \(p\) < 0.05; ** \(p\) < 0.01 vs. control (two-way ANOVA post-hoc Bonferroni test).  
conventional hydrogen bonds with His524 and Glu353 residues, there are some interactions in common, such as Van der Waals interactions with Leu387, Met388, and Leu391 residues and alkyl and pi-alkyl interactions with Phe404, as it is shown in Figure 8. In addition, despite the absence of interaction with Glu353, this compound establishes a Van der Waals interaction with Hist524, a weaker interaction when compared with the conventional hydrogen bond. Concerning the 17/B-HSD1 enzyme, the lowest energy was obtained with compounds **2** and **6** (Figure 9). Although the energy values were similar to those previously obtained with the ligand DHT, these compounds do not perform the essential interaction for inhibitory activity, which is the hydrogen bond with His221 [53]. However, both compounds seem to interact with Leu149, Pro187, and Val143 residues through alkyl and pi-alkyl interactions, similarly to DHT. In addition, compounds **2** and **6** establish a conventional hydrogen bond with Ser222, although the biological significance of this interaction is unclear. Furthermore, their binding mode is very similar, and both structures are practically overlapping within the macromolecule. Concerning ST enzyme, in addition to the weak affinity energies obtained, no relevant interactions with the amino acid involved in the binding site (Leu74, Arg98, Thr99, Val101, Leu103, Leu167, Val177, Phe178, Thr180, Gly181, Thr484, and Phe488) [54] were observed.