## 5 Conclusions  
C-ring modifications in the E1 acetate scaffold originated molecules with higher cytotoxic activities than E1 acetate. Of these, the 9\(\alpha\)-hydroxy,11\(\beta\)-nitrooxy derivative **5** showed to be the most cytotoxic molecule against hormone-dependent cancer cells. In addition, the introduction of 11\(\beta\)-nitrooxy group originated a drastic reduction in HepaRG cell viability and increased the proliferation of T47-D cells in _E-screening assay_. Therefore, similarly to previous studies, it was difficult to dissociate the estrogenic effect from the cytotoxic activity. Importantly, 9\(\alpha\)-hydroxyestrone acetate **4** promoted a selective cytotoxic effect on HepaRG cells, induced an arrest at G\({}_{0}\)/G\({}_{1}\) phase, and, interestingly, did not promote T47-D proliferation. This last finding is of major importance since it has been described that usually, estrogen derivatives could stimulate cell proliferation through the interaction with their receptors and consequently stimulate tumor growth. Overall, this preliminary study demonstrated that this structural modification can be of interest to develop new anticancer esterase derivatives without estrogenic activity.  
**Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12073579/s1](https://www.mdpi.com/article/10.3390/app12073579/s1)]([https://www.mdpi.com/article/10.3390/app12073579/s1](https://www.mdpi.com/article/10.3390/app12073579/s1)). Figure S1. IR spectrum of compound **2**; Figure S2. 11A-NMR spectrum of compound **2** in CDCl3; Figure S3. 11A-NMR spectrum of compound **2** in CDCl3; Figure S4. IR spectrum of compound **3**; Figure S5. 11A-NMR spectrum of compound **3** in DMSO-\(d_{6}\); Figure S6. 11A-NMR spectrum of compound **3** in DMSO-\(d_{6}\); Figure S7. IR spectrum of compound **4**; Figure S8. 11A-NMR spectrum of compound **4** in CDCl3; Figure S9. 11A-NMR spectrum of compound **4** in CDCl3; Figure S10. IR spectrum of compound **5**; Figure S11. 11A-NMR spectrum of compound **5** in CDCl3; Figure S12. 11C-NMR spectrum of compound **5** in CDCl3; Figure S13. IR spectrum of compound **6**; Figure S14. 11A-NMR spectrum of compound **6** in CDCl3; Figure S15. 11C-NMR spectrum of compound **6** in CDCl3.  
**Author Contributions:** Data curation, A.O.S., S.S. and G.A.; Formal analysis, C.C., M.M., V.B., A.O.S. and S.S.; Funding acquisition, A.F., S.S. and G.A.; Investigation, C.C., M.M., P.P., V.B., A.O.S. and S.S.; Methodology, C.C., M.M., A.O.S. and S.S.; Project administration, A.F. and S.S.; Resources, A.O.S., A.F. and G.A.; Software, V.B. and S.S.; Supervision, A.F., S.S. and G.A.; Validation, A.O.S., S.S. and G.A.; Writing--Original draft, C.C., V.B. and S.S.; Writing--Review and editing, C.C., M.M., A.O.S., S.S. and G.A. All authors have read and agreed to the published version of the manuscript.  
**Funding:** The authors acknowledge the support provided by FEDER funds through the POCI-COMPETE 2020--Operational Programme Competitiveness and Internationalisation in Axis I--Strengthening research, technological development, and innovation (project POCI-01-0145-FEDER-007491) and National Funds by FCT--Foundation for Science and Technology (Project UID/Multi/00709/2013).  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** Not applicable.  
**Acknowledgments:** The authors acknowledge the support provided by FEDER funds through the POCI-COMPETE 2020--Operational Program Competitiveness and Internationalization in Axis I (project POCI-01-0145-FEDER-007491) and National Funds by FCT--Foundation for Science and Technology (project UID/Multi/00709/2019). The authors also would like to thank the support provided by FEDER funds through the \"Programa Operacional do Centro\" (CENTRO-01-0145-FEDER-000013).  
**Conflicts of Interest:** The authors declare no conflict of interest.