**4. Conclusions**

Four new polyhydroxylated steroids were isolated along with two previously known related steroids from the Vietnamese starfish *A. laevigatus* and their chemical structures were elucidated. Two new compounds have the common 5α-cholestane skeleton, while the other two new compounds have the 5β-cholestane skeleton, which are rare among starfish steroids. Previously, only two steroid compounds with the *cis*-A/B ring junction were known from two species of the starfish *L. clathrata* and *T. novaecaledoniae*. Three of the substances that were isolated from *A. laevigatus* did not show cytotoxic effects against normal JB6 Cl41 and human colorectal cancer HT-29 and breast cancer MDA-MB-231 cells, however they suppressed cell proliferation and colony formation of cancer HT-29 and MDA-MB-231 cells.

**Supplementary Materials:** The following are available online. Copies HRESIMS (Figures S1, S9, S16, and S23), 1H-NMR (Figures S2, S10, S17, and S24), 13C-NMR (Figures S3, S11, S18, and S25), COSY (Figures S4, S12, S19, and S26), HSQC (Figures S5, S13, 20, and S27), HMBC (Figures S6, S14, S21, and S28), and ROESY (Figures S7, S15, S22, and S29) spectra of compounds **1**, **2**, **3**, and **4**, respectively. Copies 1H-NMR (Figure S8) spectra of (*R*) and (*S*)-MTPA esters of **1**, 1D TOCSY (Figure S30) spectrum of **4**.

**Author Contributions:** A.A.K. isolated the metabolites, elucidated their structures, and prepared manuscript; D.T.H. and T.T.T.T. isolated the metabolites and elucidated their structures; N.V.I. and T.V.M. analyzed the compounds and edited the manuscript; O.S.M. carried out biological evaluation of compounds and analysis of the results; A.I.K. performed the acquisition and interpretation of NMR spectra; R.S.P. did the acquisition and interpretation of mass spectra; N.T.T.H. and P.Q.L. helped with collection of animals and manuscript editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partly funded by the RFBR (Russian Foundation for Basic Research), grant number 20-03-00014 and the VAST (Vietnam Academy of Science and Technology), grant numbers QTRU02.03/18-19 and QTRU04.05/18-19.

**Acknowledgments:** The study was carried out on the equipment of the Collective Facilities Center "The Far Eastern Center for Structural Molecular Research (NMR/MS) of PIBOC FEB RAS". The authors are grateful to Do Cong Thung (Institute of Environment and Marine Resources of the VAST, Hai Phong, Vietnam) for species identification of the starfish.

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