## 4 Discussion
However, in the _E-screening_ assay, compound **5** also revealed estrogenic activity in T47-D cells, which seems to be in concordance with the literature [22; 58]. In this context and as an example, [PERSON] et al. [18] synthesized 11\(\beta\)-estradiol carboxylates, esters, and ethers and showed that when 11\(\beta\)-chain had four or five non-hydrogen atoms, the estrogenic effect diminished, and a SERM (selective estrogen receptor modulator) was obtained. In addition, other structure-activity relationship studies showed that large hydrophobic substituents at 11\(\beta\) side chains originated antiestrogenic compounds and that different chemical groups in this chain promoted different affinities to ER [16; 17]. Thus, the potential estrogenic activity associated with compound **5** can be explained by the small size of the 11\(\beta\)-nitrooxy group. On the other hand, in our study, compound **4**, which only has a 9\(\alpha\)-hydroxyl group, did not show a T47-D proliferation. Interestingly, [PERSON] and co-workers [21] demonstrated that 3-methoxyesta-1,3,5(10),9(11)-tetra-17-one was an estrogenic compound with affinity to ER\(\alpha\) and ER\(\beta\). Otherwise, the 9-hydroxymethyl-11-keto analog showed low cytotoxicity against MCF-7 cells and also was an estrogenic compound. Thus, the size of the group at C9 seems to be important for estrogenic and cytotoxic activities. Concerning the cell cycle effects of these compounds, some studies showed that several steroids [59; 60] as well as non-steroids [61; 62; 63] lead to cell cycle arrest. In this context and as an example, 11\(\alpha\)-substituted 2-methoxyestradiol derivatives led to a cell cycle arrest at G\({}_{2}\)/M and demonstrated an important antiestrogenic effect [5]. In addition, as an example of non-steroidal compounds interfering in the cell cycle, 2-benzyl1-(3,4,5-trimethoxyphenyl)-1\(H\)-naphtho [1,2-\(e\)][1,3]oxazin-3(2\(H\))-one also induced G\({}_{2}\)/M phase arrest on A2780 cancer cell line and in silico studies showed a relevant molecular interaction with tubulin [63]. Remarkably, in our study, compound **4** induced a G\({}_{0}\)/G\({}_{1}\) phase arrest, probably because it interferes with many proteins of the cell cycle such as cyclin-dependent kinases and others [64; 65], which are important for the cellular regulation of DNA replication. However, additional studies are required to clarify the biological mechanisms. The estrogenicity absence of compound **4** associated with an arrest in G\({}_{0}\)/G\({}_{1}\) showed that this steroid may be a suitable starting point to develop potentially improved anticancer drugs. In addition, the CFSE assay showed that compound **4** promoted a low number of cell divisions of HepaRG cancer cells, being less proliferative. This result is in agreement with the cell cycle analysis results. Finally, A\({}^{9,11}\)-E1 acetate (compound **6**)showed weaker antiproliferative activity in all studied cell lines when compared with analog **3**, which has a 3-hydroxyl group [24]. Molecular docking studied the interactions between these C-ring oxidized E1 acetate analogs and proteins that interact with them [66; 67]. Few studies were reported, including docking with C-ring modified steroids [21; 68]. Concerning ER\(\alpha\) results, weaker binding energies for compound **6** were observed, compared with E2 and with compound **3**, according to our previous report [24]. In addition, compounds **4** and **5** also had weaker binding energies to ER\(\alpha\) than E1 and E2. These results were expected because nonpolar groups are preferable in C9 and C11 positions for the interaction with ER\(\alpha\)[11; 69]. Thus, the cytotoxicity originated by compound **5** can be associated with another mechanism of action than the interaction with ER\(\alpha\). In this context, another possible reason is the generation of nitric oxide, which has a cytotoxic effect [70].