*3.5. Cell Culture*

Five cancer cell lines, including human prostate cancer cells (C4-2B), enzalutamideresistant C4-2B cells (C4-2B/ENZR), human breast cancer cells (MDA-MB-231) and human colon cancer cells (HCT-15 and RKO) used in this study were purchased from the Laboratory Animal Service Centre at Sun Yat-sen University (Guangzhou, China). Cell lines were

cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics (100 units/mL penicillin and 100 g/mL streptomycin). These cells were incubated at 37 ◦C in an atmosphere of 5% CO2.

#### *3.6. Cytotoxicity Assay*

The cells in logarithmic growth phase were seeded a in 96-well plates at a density of <sup>5</sup> × <sup>10</sup><sup>3</sup> cells/well for 24 h. Then, cells were treated with different concentrations of the compounds for an additional 48 h. Subsequently, 10 μL MTT (5 mg/mL) (Sigma, Saint Louis, MO, USA) were added to each well. After incubation in the incubator for 4 h, the suspension was discarded and the dark blue crystals were solubilized in dimethyl sulfoxide (DMSO). The absorbance of the solution was detected by a multifunction micro-plate reader (Molecular Devices, Flex Station 3, Molecular Devices, San Francisco, USA) at 450 nm. IC50 value was used to express the cytotoxic effect on the tested compounds.

#### **4. Conclusions**

In summary, two new highly oxygenated *ent*-abietane diterpenoids euphonoids H and I (**1**–**2**), together with two known analogues raserranes A (**3**) and B (**4**) were separated and identified from the EtOAc-soluble partition of the roots of *E. fischeriana*. Their structures were elucidated by comprehensive spectroscopic analysis, quantum chemical calculation and ECD calculations. All the compounds were isolated from *E. fischerian* for the first time. The two new compounds exhibited strong antiproliferative potency against the human prostate cancer cells C4-2B and C4-2B/ENZR, with IC50 values less than 10 μM. This study not only enriches the chemical diversity of *ent*-abietane diterpenoids in the *Euphorbia* species but also forms a basis for the discovery of bioactive natural products from Euphorbiaceae herbs.

The current results, together with others' previous discoveries, suggested that *ent*abietane diterpenoids with certain structural motifs might possess very strong anticancer activity against prostate cancer cell lines and this type of diterpenoids provided a promising skeleton for the development of anti-cancer agents for the treatment of prostate cancers.

**Supplementary Materials:** The following supporting information can be downloaded online: https: //www.mdpi.com/article/10.3390/molecules27217258/s1, Figures S1–S6: The 1D and 2D NMR (400 MHz) spectra of compound **1** in CDCl3, Figure S7: The HRESIMS spectrum of compound **1**, Figure S8: The IR spectrum of compound **1**, Figures S9–S14: The 1D and 2D NMR (400 MHz) spectra of compound **2** in CDCl3, Figure S15: The HRESIMS spectrum of compound **2**, Figure S16: The IR spectrum of compound **2**, Figures S17 and S18 and Tables S1–S9: NMR and ECD calculation method of compound **1**, Figures S19–S21 and Tables S10–S12: NMR and ECD calculation method of compound **2**, Figures S22–S25: 1H NMR and HRESIMS spectra of compound **1**–**2**, Table S13: NMR Data for compounds **1**–**2** in CDCl3.

**Author Contributions:** Original draft preparation, Q.-F.Z. and G.-B.X.; performing the experiments, Q.-F.Z. and X.-L.Y.; data analysis, G.-B.X.; review and editing, S.-G.L.; supervision, S.-G.L. and X.-L.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the New-shoot Talents Project of Guizhou Medical University (19NSP077), the Scientific Research Foundation for Innovative Talent of Guizhou Province ([2020]6011), National Natural Science Foundation of China (22167009) and the Project of Science and Technology Department of Guizhou Province ([2020]5006, ZK [2021]554).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data are available in the Supporting Information of the article.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are not available from the authors as the material has been used up for bioassays.
