*3.4. Single Crystal X-Ray Data of Compound* **4**

Crystal data of **4** (from CH2Cl2): C30H46N2O, M = 450.69, space group *P*21 (No. 4), monoclinic, *Z* = 2, *a* = 5.895(14) Å, *b* = 9.983(2) Å, *c* = 22.033(5) Å, *α* = 90◦, *β* = 95.971(6)◦, *γ* = 90◦, *V* = 1289.4(5)Å3, T = 173 K, <sup>μ</sup> (Mo *<sup>K</sup>*α) = 0.71073 mm−1. A crystal of dimensions of 0.18 × 0.08 × 0.05 mm<sup>3</sup> was measured on a Bruker APEX-II CCD diffractometer with a graphite monochromator (*ϕ*-*ω* scans, 2θmax = 55.18◦), Mo *K*α radiation. 9787 reflections were measured, 5785 independent reflections were observed (Rint = 0.0530). The final R1 values were 0.0625 (I >= 2σ (I)). The final wR2 values were 0.1278 (I >= 2σ (I)). The final R1 values were 0.1008 (all data). The final wR2 values were 0.1449 (all data). The goodness of fit on *F*<sup>2</sup> was 0.971. CCDC 1875789 for compound **4** contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/.

#### *3.5. Cytotoxicity Assay*

To identify novel antitumor inhibitors, compounds **1**–**4** were tested on five human cancer cell lines SW480, SMMC-7721, PC3, MCF-7 and K562 by using a CCK-8 assay. All cells were obtained from Centre of Drug Safety Evaluation and Research of Hunan Province. Those cells were cultured in a DMEM medium (high glucose) (Hyclone, Logan, UT, USA), which was supplemented with 10% fetal bovine serum (Sciencell, San Diego, CA, USA) in a humidified 5% CO2 atmosphere at 37 ◦C. CCK-8 was purchased from American Bimake Company (Bimake, Houston, TE, USA).

The cytotoxicity assay was performed according to the Cell Counting Kit-8 assay methods as described by elsewhere [23]. Briefly, all cells were seeded into 96-well plates at 3 × <sup>10</sup><sup>3</sup> cells per well and allowed to culture for 12 h before the addition of the drug. Then, each tumor cell line was exposed to the tested compounds at different concentrations (100–0 μM) for 72 h. DDP (Tokyo Chemical Industry, Tokyo, Japan) and 5-FU (Amresco, Portland, ME, USA) was used as positive control. After treatment, 10 μL of CCK-8 was added to each well, and the plates were incubated for an additional 12 h. OD450 absorbance was determined using a Spectramax-i3x (Molecular Devices, Sunnyvale, CA, USA). The experiments were performed in triplicate to obtained IC50 values.

#### **4. Conclusions**

In this study, two new steroidal alkaloids, hookerianine A (**1**) and hookerianine B (**2**), together with two known ones, scorucinine G (**3**) and epipachysamine D (**4**), were isolated from the stems and roots of *S. hookeriana*. To the best of our knowledge, four compounds were isolated from this plant for the first time. Two new compounds were shown to possess a 3*α* substituent, which were rarely reported. In addition, compound **1** represents the first example of pregnane-type steroidal alkaloid possessed a novel phenylacetyl group at C-3. Based on the preliminary structure-activity relationships study, we found that the different substituents at C-3 and the presence of double bond and epoxy group between C-16 and C-17 have an important effect on the cytotoxicity of steroidal alkaloids. Inspired by this, it deserves further structural modification and in-depth mechanism research on steroid alkaloids with those characteristics. The results suggested that these types of steroidal alkaloids may have the potential to be anticancer agents.

All of the 1H-NMR, 13C-NMR, 2D-NMR and HR-ESI-MS spectra of compound **1** and **2** are available in Supplementary Material.

**Supplementary Materials:** The following 1H-NMR, 13C-NMR, 2D-NMR, and HR-ESI-MS spectra are available as supporting data. Supplementary materials are available online.

**Author Contributions:** J.D. and J.W. designed the experiments and revised the paper; S.H. and J.W. performed the experiments, analyzed the data, and wrote the paper; S.H. and X.H. contributed to bioassay reagents and materials and analyzed the data; L.P. and J.D. revised the paper. All authors read and approved the final manuscript.

**Acknowledgments:** This research was supported by the National Natural Science Foundation of China [No. 30960529] and the Science and Technology Project of Guizhou Province [No. 2016-1015]. We are grateful to the Centre of Drug Safety Evaluation and Research of Hunan Province for measuring cytotoxicity.

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

#### **References**


**Sample Availability:** Samples of the compounds **1**–**4** are available from the authors.

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