*3.3. Extraction and Isolation*

The frozen sponge was chopped and extracted with EtOH (1.7 L × 3) (Figure S73). The EtOH soluble materials (52.5 g) were concentrated, dissolved in distilled H2O (100 mL) and partitioned in turn with EtOAc (100 mL × 3). The EtOAc extracts were concentrated to a dark brown gum (15.7 g) that was further separated on a Sephadex LH-20 column (2 × 95 cm, CHCl3/EtOH, 1:1) to yield three fractions. Fraction 2 (10.2 g) was separated into nine subfractions using step-wise gradient silica gel column chromatography (4 × 15 cm, CHCl3→EtOH). Subfraction 2.4 (3.4 g) eluted with CHCl3/EtOH (80:1–10:1) was subjected to a silica gel column (4 × 15 cm, CHCl3/EtOH, 100:1→10:1) to obtain four subfractions. The fourth subfraction 2.4.4 (255.5 mg) was subjected to reversed-phase HPLC (YMC-Pack ODS-A, 70% EtOH) to give four subsubfractions (2.4.4.1-4) that were subjected to rechromatography. The HPLC fractionation of 2.4.4.1 (YMC-Pack ODS-A, 60% EtOH) gave cyclobutastellettolide A (7.7 mg, 0.004%), mixtures of globostelletins E+F (~2:1; 4.0 mg, 0.002%), K (3.4 mg, 0.002%), and M (1.7 mg, 0.002%), that were purified by HPLC procedures (Discovery HS F5-5, 60% EtOH), as it was reported previously [14]. One more component of this subsubfraction was purified (Discovery HS F5-5, 60% EtOH) to yield stellettin V (**6**, 2.6 mg, 0.001%). The subsubfraction 2.4.4.2, subjected to HPLC (Discovery HS F5-5, 70% EtOH) gave cyclobutastellettolide B [14] (1.6 mg, 0.004%) and stellettin T (**4**, 1.2 mg, 0.0006%), purified by HPLC (Discovery HS F5-5, 70% EtOH). The subsubfraction 2.4.4.3 contained cyclobutastellettolide B (1.4 mg) and stellettin Q (**1**, 0.9 mg, 0.0008%) isolated using reversed-phase HPLC (Discovery HS F5-5, 70% EtOH). The third subfraction 2.4.3 (641.5 mg) was divided four times (~160 mg × 4) using reversed-phase column chromatography (1 × 5 cm, YMC-Pack ODS-A, 50% EtOH and 100% EtOH) to yeild two subsubfractions. The subsubfraction eluted with 50% EtOH was separated by HPLC (YMC-Pack ODS-A, 70% EtOH) to afford a number of compounds and mixes for further purification. Then, stellettin U (**5**, 10.3 mg, 0.005%) as well as globostelletins N (9.2 mg, 0.004%) and M (1.5 mg) were isolated by HPLC (Discovery HS F5-5) in 80% MeOH. The HPLC procedures (Discovery HS F5-5) in 80% EtOH were used to obtain individual stellettins R (**2**, 2.1 mg, 0.001%), S (**3**, 6.9 mg, 0.003%) and portion of stellettin Q (**1**, 0.9 mg) that was purified by HPLC (Discovery HS F5-5) rechromatography in 65% CH3CN. Finally, one more portion of cyclobutastellettolide B (5.0 mg) was obtained from the subfraction by HPLC (Discovery HS F5-5) in 85% MeOH.

#### *3.4. Compound Characteristics*

Stellettin Q (**1**): Yellow oil; [*α*]D25–65.0 (*<sup>c</sup>* 0.1, CHCl3); ECD (*<sup>c</sup>* 8.6 × <sup>10</sup>−<sup>4</sup> M, EtOH) *<sup>λ</sup>*max (Δ*ε*) 195 (4.15), 229 (−27.92), 262 (12.78), 355 (−1.75) nm; 1H- and 13C-NMR data (CDCl3), Table 1; HRESIMS *m/z* 523.3065 [M–H]− (calcd for C32H43O6 523.3065).

Stellettin R (**2**): Yellow oil; [*α*]D25–24.0 (*<sup>c</sup>* 0.2, CHCl3); ECD (*<sup>c</sup>* 1.3 × <sup>10</sup>−<sup>3</sup> M, EtOH) *<sup>λ</sup>*max (Δ*ε*) 195 (4.06), 229 (−5.33), 252 (1.46), 273 (−0.48), 295 (0.47), 353 (−1.20) nm; 1Hand 13C- NMR data (CDCl3), Table 1; HRESIMS *m/z* 523.3068 [M–H]<sup>−</sup> (calcd for C32H43O6 523.3065).

Stellettin S (**3**): Slightly yellow oil; [*α*]D<sup>25</sup> + 31.5 (*<sup>c</sup>* 0.2, CHCl3); ECD (*<sup>c</sup>* 5.3 × <sup>10</sup>−<sup>3</sup> M, EtOH) *<sup>λ</sup>*max (Δ*ε*) 289 (0.34), 321 (−0.14) nm; 1H- and 13C-NMR data (CDCl3), Table 2; HRESIMS *m/z* 303.1966 [M–H]− (calcd for C19H27O3 303.1966).

Stellettin T (**4**): Slightly yellow oil; [*α*]D25–22.0 (*<sup>c</sup>* 0.1, CHCl3); ECD (*<sup>c</sup>* 3.4 × <sup>10</sup>−<sup>3</sup> M, EtOH) *<sup>λ</sup>*max (Δ*ε*) 208 (−0.98), 249 (0.58), 280 (−0.27), 321 (−0.29) nm; 1H- and 13C-NMR data (CDCl3), Table 2; HRESIMS *m/z* 351.2176 [M–H]− (calcd for C20H31O5 351.2177).

Stellettin U (**5**): Slightly yellow oil; [*α*]D<sup>25</sup> 0.0 (*<sup>c</sup>* 0.2, CHCl3); ECD (*<sup>c</sup>* 5.7 × <sup>10</sup>−<sup>3</sup> M, EtOH) *<sup>λ</sup>*max (Δ*ε*) 197 (−0.73), 232 (0.27), 261 (0.45), 295 (0.01), 321 (−0.34) nm; 1H- and 13C-NMR data (CDCl3), Table 2; HRESIMS *m/z* 337.2022 [M–H]<sup>−</sup> (calcd for C19H29O5 337.2020).

Stellettin V (**6**): Slightly yellow oil; [*α*]D<sup>25</sup> + 32.9 (*<sup>c</sup>* 0.17, CHCl3); ECD (*<sup>c</sup>* 7.7 × <sup>10</sup>−<sup>3</sup> M, EtOH) *λ*max (Δ*ε*) 210 (−0.04), 220 (0.08), 238 (−0.23), 269 (0.38), 291 (0.26), 330 (0.13), 357 (−0.09) nm; 1H- and 13C-NMR data (CDCl3), Table 2; HRESIMS *m/z* 337.2025 [M–H]<sup>−</sup> (calcd for C19H29O5 337.2020).

Globostelletin N (Figure 1): Slightly yellow oil; [*α*]D<sup>25</sup> + 17.8 (*c* 0.23, MeOH); ECD (*c* 1.2 <sup>×</sup> <sup>10</sup>−<sup>3</sup> M, EtOH) *<sup>λ</sup>*max (Δ*ε*) 196 (−4.18), 228 (9.44), 256 (−1.72), 289 (0.97), 340 (−1.97) nm; 1H- and 13C-NMR spectra (CDCl3) corresponded to previously reported data [15] (Figures S62–S64); HRESIMS *m/z* 479.2808 [M–H]− (calcd for C30H39O5 479.2803).

#### **4. Conclusions**

To summarize, the present report describes the isolation and structural elucidation of six metabolites **1**–**6** from a tropical marine sponge belonging the genus *Stelletta*. A combination of NMR methods, supported with computational quantum-chemical modeling allowed us to establish the structures and absolute stereochemistry of two isomalabaricanes **1** and **2**, while the structures and configurations of four isomalabaricane-derived terpenoids **3**–**6** were suggested on the basis of spectral data and biogenetic considerations. Stellettin S (**3**) represents the first acetylene-containing isomalabaricane-related compound. Additionally, according to new data the absolute stereochemistry of the C-15 and C-23 asymmetric centers of known globostelletins M and N were corrected.

**Supplementary Materials:** The following are available online. Figure S1: article title, authors affiliations and contact information, Figure S2: contents of Supplementary Materials, Figures S3–S10: HRESIMS, 1H- and 13C-NMR, HSQC, HMBC, COSY, ROESY spectra (CDCl3, 700 MHz) and ECD spectrum (EtOH) of stellettin Q (**1**), respectively, Figures S11–S18: HRESIMS, 1H- and 13C-NMR, HSQC, HMBC, COSY, ROESY spectra (CDCl3, 500 MHz) and ECD spectrum (EtOH) of stellettin R (**2**), respectively, Figures S19–S27: HRESIMS, 1H- and 13C-NMR, DEPT, HSQC, HMBC, COSY, ROESY spectra (CDCl3, 700 MHz) and ECD spectrum (EtOH) of stellettin S (**3**), respectively, Figures S28–S35: HRESIMS, 1H- and 13C-NMR, HSQC, HMBC, COSY, ROESY spectra (CDCl3, 700 MHz) and ECD spectrum (EtOH) of stellettin T (**4**), respectively, Figures S36–S44: HRESIMS, 1H- and 13C-NMR, DEPT, HSQC, HMBC, COSY, ROESY spectra (CDCl3, 700 MHz) and ECD spectrum (EtOH) of stellettin U (**5**), respectively, Figures S45–S53: HRESIMS, 1H- and 13C-NMR, DEPT, HSQC, HMBC, COSY, ROESY spectra (CDCl3, 700 MHz) and ECD spectrum (EtOH) of stellettin V (**6**), respectively, Figures S54–S57: HRESIMS, 1H- and 13C-NMR spectra (CDCl3) and ECD spectrum (EtOH) of globostelletin K, respectively, Figures S58–S61: HRESIMS, 1H- and 13C-NMR spectra (CDCl3) and ECD spectrum (EtOH) of globostelletin M, Figures S62–S65: HRESIMS, 1H and 13C NMR spectra (CDCl3) and ECD spectrum (EtOH) of globostelletin N, respectively, Figure S66: theoretical modeling details, Figures S67 and S68: optimized geometries and statistical weights of main and minor conformations of stellettin Q (**1**) and stellettin R (**2**), respectively, Figures S69–S71: the computational ECD results for globostelletins K, M and N, respectively. Figure S72: the computational ECD results calculated for all possible 15,23-stereoisomers of studied globostelletins K, M, N, and new stellettins Q (**1**) and R (**2**), Figure S73: the isolation scheme.

**Author Contributions:** Conceptualization, S.A.K., E.G.L.; methodology, S.A.K. and E.G.L.; formal analysis, S.A.K., E.G.L. and D.V.B.; investigation, S.A.K., E.G.L. and A.B.K.; data curation S.A.K. and E.G.L.; writing—original draft preparation, S.A.K. and E.G.L.; writing—review and editing, S.A.K., E.G.L. and V.A.S.; visualization, S.A.K. and E.G.L.; NMR data providing, A.I.K.; HRESI MS spectra

acquisition and interpretation, R.S.P.; supervision, V.A.S.; project administration, V.A.S.; funding acquisition, V.A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the RSF (Russian Science Foundation), grant number 20-14- 00040.

**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". Authors thank the Vietnamese Academy of Science and Technology for collaboration and participation in prеvious joint publication [14].

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

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

#### **References**


*Article*
