*2.1. Structure Elucidation of Compounds* **1**−**6**

The concentrated methanol extract of *A. laevigatus* was subjected to sequential separation by column chromatography using Amberlite XAD-2 silica gel followed by reversed-phase HPLC on Discovery C18 and YMC-Pack Pro C18 columns to yield four new polyhydroxylated steroids (**1**−**4**), along with two known compounds **5** and **6** (Figure 1). The known polyhydroxylated steroids were identified by analysis of their 1H-, 13C-NMR, and ESIMS spectra and comparison with those reported earlier for (24*S*)-5α-cholestane-3β,5,6β,15α,24-pentaol (**5**) and related sulfated compounds previously isolated from the starfish *Luidia clathrata* and *Henricia leviuscula* [16,17], and (25*S*)-5α-cholestane-3β,5,6β,15α,16β,26-hexaol (**6**) from the starfish *L. clathrata* [18].

**Figure 1.** Structures of compounds **1**−**6** isolated from *A. laevigatus.*

The molecular formula of compound **1** was established to be of C27H48O7 from the [M + Na]<sup>+</sup> sodium adduct ion peak at *m*/*z* 507.3292 in the (+)HRESIMS spectrum and the [M – H]– deprotonated ion peak at *m*/*z* 483.3321 in the (–)HRESIMS spectrum (Figure S1). The 1H- and 13C-NMR spectroscopic data of **1** displayed the proton and carbon chemical shifts of two angular methyl groups CH3-18 (δ<sup>H</sup> 1.00 s and δ<sup>C</sup> 16.5) and CH3-19 (δ<sup>H</sup> 1.17 s and δ<sup>C</sup> 17.3), one oxygenated methylene CH2-26 (δ<sup>H</sup>

3.42 dd (*J* = 10.5, 5.7), 3.30 m and δ<sup>C</sup> 68.4), four oxygenated methines CH-3 (δ<sup>H</sup> 4.00 m and δ<sup>C</sup> 68.3), CH-6 (δ<sup>H</sup> 3.49 t (*J* = 3.0) and δ<sup>C</sup> 76.5), CH-15 (δ<sup>H</sup> 3.86 d (*J* = 3.3) and δ<sup>C</sup> 85.6), and CH-16 (δ<sup>H</sup> 3.96 dd (*J* = 8.1, 3.3) and δ<sup>C</sup> 82.1), and two oxygenated tertiary carbons C-5 (δ<sup>C</sup> 76.6) and C-14 (δ<sup>C</sup> 82.7) (Tables 1 and 2, Figures S2 and S3). These signals were similar to the corresponding resonances in the NMR spectra of co-occuring compound **6,** except for the proton and carbon resonances of C-14 and CH-15. Therefore, the doublet of doublets of H-15 at δ<sup>H</sup> 3.73 (*J* = 10.4, 2.4) in the 1H-NMR spectrum of **6** became doublet at δ<sup>H</sup> 3.86 d (*J* = 3.3) in the 1H-NMR spectrum of **1** suggesting the presence of an additional hydroxyl group at C-14 in the steroid nucleus of **1** as compared with **6**. In addition, the value of the *J*16,17 = 8.1 Hz and the existence of the ROESY cross-peak H-16/H-17 indicated the α-orientation of the proton H-16 and, accordingly, 3β,5,6β,14,15α,16β,26-heptahydroxy substitution in **1**. An analysis of the COSY, HSQC, HMBC, and ROESY spectra ascertained all the proton and carbon signals in **1** (Tables 1 and 2, Figures S4). The COSY and HSQC experiments led to the assignment of the proton atom sequences at C-1 to C-4, C-6 to C-12 through C-11, C-15 to C-17, C-17 to C-21 through C-20, C-20 to the end of the side chain. The key HMBC correlations H-4/C-2; H-6/C-8, C-10; H-8/C-7, C-9, C-14; H-16/C-17; H3-18/C-12, C-13, C-14, C-17; H3-19/C-1, C-5, C-9, C-10; H3-21/C-17, C-20, C-22; H2-24/C-23, C-25, C-26; and H3-27/C-24, C-25, C-26 confirmed the total structure of the molecule of **1** (Figure 2). The signal shape and coupling constants of protons H-3, H-6, H-15, and H-16 and the presence of the key ROESY cross-peaks Hα-4/H-6; H-8/H-15; H-16/H-17; H-17/H3-21; H3-18/H-8, H-15; and H3-19/Hβ-2, Hβ-4, H-8 confirmed the 3β,6β,15α,16β relative configurations of the oxygenated carbons and common 5α-cholestane skeleton in **1** (Figure 2). The 20*R-*configuration was assigned based on the ROESY correlations of H3-18/H-20 and H-16/H-22 and the downfield chemical shift of H3-21 at δ<sup>H</sup> 0.90 [19]. The absolute configuration of the asymmetric center C-25 was defined by examination of 1H-NMR spectra of (*R*)- and (*S*)-MTPA derivatives obtained by reaction of **1** with *S*-(+)- and *R*-(−)-MTPA chlorides, respectively. The 1H-NMR spectrum of 3,15,26-tri**-**(*R*)-MTPA ester of **1** showed H2-26 signals as two close double doublets at δ<sup>H</sup> 4.15 and 4.18, while that of 3,6,15,26-tetra-(*S*)-MTPA ester of **1** displayed two well-separated double doublets at δ<sup>H</sup> 4.08 and 4.24 (Figure S8). These values were comparable with those of (*R*)- and (*S*)-MTPA derivatives obtained from other (25*S*)-26-hydroxy steroids [20]. Thus, the structure of compound **1** was determined to be the (25*S*)-5α-cholestane-3β,5,6β,14,15α,16β,26-heptaol.

**Figure 2.** (**A**) COSY and key HMBC correlations for compound **1**; (**B**) Key ROESY correlations for compound **1**. Colors reveal the atoms of hydrogen (blue), oxygen (red) and carbon (grey) and their bonds.


**Table 1.** 1H-NMR data of compounds **<sup>1</sup>**−**<sup>4</sup>** (CD3OD, <sup>δ</sup> in ppm, *<sup>J</sup>* in Hz) *<sup>a</sup>*.

*<sup>a</sup>* Assignments from 700 MHz COSY, HSQC, HMBC (8 Hz), and ROESY (250 ms) data.

The molecular formula of compound **2** was established to be of C27H48O6 from the [M + Na]<sup>+</sup> sodium adduct ion peak at *m*/*z* 491.3337 in the (+)HRESIMS spectrum and the [M –H]– deprotonated ion peak at *m*/*z* 467.3379 in the (–)HRESIMS spectrum (Figure S9). Along with mass-spectra, the 1H-, 13C-, and DEPT NMR spectra revealed the presence of a hexahydroxy substitution in **2**. Similarity of the corresponding proton and carbon signals, as well as coupling constants in the NMR spectra of **2** and **1,** indicated that compound **2** has the same 14,15α,16β-trihydroxy substitution in the steroid C/D rings and 26-hydroxy cholestane side chain (Tables 1 and 2, Figures S10 and S11).

**Table 2.** 13C-NMR data of compounds **<sup>1</sup>**−**<sup>4</sup>** (CD3OD).



**Table 2.** *Cont.*

However, most of the proton and carbon chemical shifts of steroid A/B rings in the NMR spectra of **2** were quite different from those of **1**. The characteristic proton and carbon resonances of angular methyl group CH3-19 (δ<sup>H</sup> 1.04 s and δ<sup>C</sup> 16.1), an oxygenated methine CH-3 (δ<sup>H</sup> 3.53 m and δ<sup>C</sup> 72.4), and an oxygenated methine CH-6 (δ<sup>H</sup> 3.77 q (*J* = 2.6) and δ<sup>C</sup> 72.8) observed in the NMR spectra of **2** testified to the 3β,6β-dihydroxy substitution in 5α-cholestane nucleus in **2** [21]. The COSY and HSQC spectroscopic data ascertained the proton sequences at C-1 to C-8, C-8 to C-12 through C-11, C-15 to C-17, C-17 to C-21 through C-20, and C-20 to the end of the side chain (Figures S12 and S13). The key HMBC cross-peaks H-6/C-8, C-10; H-16/C-13, C-15; H3-18/C-12, C-13, C-14, C-17; H3-19/C-1, C-5, C-9, C-10 and the key ROESY cross-peaks Hα-4/H-6; H-5/H-3, Hα-7, H-9; H-16/H-17, H-22; H-17/H3-21; H3-18/H-8, H-11β, H-15, H-20; and H3-19/Hβ-1, Hβ-2, Hβ-4, H-8 confirmed the 3β,6β,15α,16β-tetrahydroxy pattern in 5α/9α/10β/13β steroid nucleus in **2** (Figures S14 and S15). The configuration at C-25 was determined as (*S*) by analogy with co-occurring compound **1** and similarity of the proton and carbon chemical shifts of the both side chains in the NMR spectra. Therefore, the structure of steroid **2** was established as the (25*S*)-5α-cholestane-3β,6β,14,15α,16β,26-hexaol.

According to the presence of the [M + Na]<sup>+</sup> sodium adduct ion peak at *m*/*z* 491.3341 in the (+)HRESIMS spectrum and the [M – H]– deprotonated ion peak at *m*/*z* 467.3380 in the (–)HRESIMS spectrum, the molecular formula C27H48O6 of compound **3** has been found to be identical to that of **2** (Figure S16). The detailed comparison of the 1H- and 13C-NMR spectra of compounds **3** and **2** has revealed that the proton and carbon resonances belonging to the steroid C/D rings and side chain of **3** are close to those of **2** indicating the 14,15α,16β,26-tetrahydroxy substitution in **3**, while the proton and carbon signals of the steroid A/B rings of **3** substantially differed from those of **2** (Tables 1 and 2, Figures S17 and S18). The proton and carbon signals in the NMR spectroscopic data attributable to the A/B rings of **3** showed the presence of angular methyl group CH3-19 (δ<sup>H</sup> 1.13 s and δ<sup>C</sup> 26.3) and two oxygenated methines CH-3 (δ<sup>H</sup> 3.99 br. q (*J* = 2.7) and δ<sup>C</sup> 67.1) and CH-6 (δ<sup>H</sup> 3.66 q (*J* = 2.7) and δ<sup>C</sup> 74.3). The signal of CH3-19 in the 13C-NMR spectrum of **3** was shifted from δ<sup>C</sup> 16.1 to 26.3 in comparison with that of **2**. This fact strongly testified to *cis*-A/B ring fusion in **3** [16,20]. The coupling constant *J* = 2.7 of the broad quartet of H-3 corresponded well to the 3β-hydroxyl group in 5β-cholestane nucleus, and the coupling constant *J* = 2.7 of the quartet of H-6 indicated the 6β-hydroxyl group in **3** [16]. All the proton

and carbon signals associated with the steroid nucleus and side chain were assigned by 2D experiments (Tables 1 and 2, Figure 3, Figures S19). Proton and carbon chemical shifts of the steroid A/B rings of **3** were similar to the corresponding data of (25*S*)-5β-cholestane-3β,6β,15α,16β,26-pentaol isolated from the starfish *L. clathrata* [16]. The key ROESY cross-peaks H3-19/Hβ-1, H-5, H-8; H3-18/H-8, H-15, H-20; Hα-4/H-6, Hα-7; Hβ-4/H-6; H-8/H-15; H-16/H-17; and H-17/H3-21 and proton coupling constants confirmed the 3β,6β,15α,16β relative configurations of the oxygenated carbons and the 5β-cholestane skeleton of **3** (Figure 3). As a result, steroid **3** was proved to be the *cis*-A/B ring fusion isomer of steroid **2** and its structure was established as (25*S*)-5β-cholestane-3β,6β,14,15α,16β,26-hexaol.

**Figure 3.** (**A**) COSY and key HMBC correlations for compound **3**; (**B**) Key ROESY correlations for compound **3**. Colors reveal the atoms of hydrogen (blue), oxygen (red) and carbon (grey) and their bonds.

The molecular formula of compound **4** was established to be of C27H48O7 from the [M + Na]<sup>+</sup> sodium adduct ion peak at *m*/*z* 507.3290 in the (+)HRESIMS spectrum and the [M – H]– deprotonated ion peak at *m*/*z* 483.3327 in the (–)HRESIMS spectrum (Figure S23). Data of the mass-spectra and the 1H- and 13C-NMR spectra showed the presence of seven hydroxyl groups in **4**. The examination of the 1H-, 13C-, and 2D NMR spectra of steroids **4** and **3** revealed that both compounds have the identical 14,15α,16β-trihydroxy substitution and 26-hydroxy cholestane side chain, but the proton and carbon resonances of the steroid A/B rings of **4** differed from those of **3** (Tables 1 and 2, Figures S24). The deshielded shift of the signal of CH3-19 at δ<sup>C</sup> 25.6 in the 13C-NMR spectrum and the existence of the ROESY cross-peak H3-19/H-5 immediately showed a 5β-cholestane skeleton in **4**. The proton connectivities from C-1 to C-9 in A/B rings were ascertained using the COSY and HSQC experiments. The 1H- and 13C-NMR spectroscopic data, referred to the steroid A/B rings of **4**, revealed the proton and carbon chemical shifts of three oxygenated methines, including CH-3 (δ<sup>H</sup> 3.70 br. q (*J* = 3.7) and δ<sup>C</sup> 72.2), CH-4 (δ<sup>H</sup> 3.66 t (*J* = 3.7) and δ<sup>C</sup> 75.3), and CH-6 (δ<sup>H</sup> 4.00 q (*J* =3.3) and δ<sup>C</sup> 73.3). The irradiation of the proton H-5 in the 1D TOCSY experiment gave an enhancing signal of the neighboring proton H-4, that confirmed the presence of an additional hydroxyl group at C-4 in **4** as compared with **3** (Figure S30). Small values of the coupling constants of the protons H-3, H-4, and H-6 showed the absence of their axially axial interaction with neighboring protons. As a result, the 3β,4α,6β-trihydroxy

pattern and the *cis*-A/B ring fusion were determined. Accordingly, the structure of **4** was established as (25*S*)-5β-cholestane-3β,4α,6β,14,15α,16β,26-heptaol.

Compounds **3** and **4** have the 5β-cholestane skeleton, which are rare among starfish steroids. Previously, only two steroid compounds with the *cis*-A/B ring junction, (25*S*)-5β-cholestane-3β,6β,15α,16β,26-pentaol from the starfish *L. clathrata* [17] and (25*S*)-5β-cholestane-3α,6β,15α,16β,26- pentaol from the starfish *Tremaster novaecaledoniae* [20], were found.
