**1. Introduction**

Malabaricanes are rather a small group of tricarbocyclic triterpenoids found in different tropical flowering terrestrial plants [1–3]. Isomalabaricanes, which differ from malabaricanes in the configuration of C-8 asymmetric center and have an α-oriented CH3- 30, are known as metabolites of four genera of marine sponges—*Stelletta*, *Jaspis*, *Geodia* and *Rhabdastrella—*belonging to the class Demospongiae. Some of them are highly cytotoxic against tumor cells [4]. Since the first isolation of three yellow highly conjugated isomalabaricane-type triterpenoids from the marine sponge *Jaspis stellifera* in 1981 [5] more than 130 isomalabaricanes and related natural products have been reported from the abovementioned sponge genera. It was noticed that *Stelletta* metabolites are quite different depending on the collection. Indeed, isomalabaricane triterpenoids were mainly found as very complex mixtures in tropical sponge samples, while boreal and cold-water sponges contain mostly alkaloids and lipids. From a chemo-ecological point of view, this indicates that studied sponges are able to produce different types of secondary metabolites in order to adapt to the various living conditions [6]. In confirmation, our attempt to find isomalabaricanes in a cold-water *Stelletta* spp., collected in 2019 in the Sea of Okhotsk, was unsuccessful, as the characteristic yellow pigments were not detected by thin layer chromatography in the extracts of these sponges.

Additionally, in result of the chemical investigation of the sponge *Stelletta tenuis*, Li et al. identified two naturally occurring α-pyrones, namely gibepyrones C and F, along with three isomalabaricane-type triterpenoids [7]. These α-pyrones were supposed to be the oxidation products of the co-occurring stellettins [6]. Gibepyrone F had previously been isolated from the fungal plant pathogen *Gibberella fujikuroi* [8], as well as from the

**Citation:** Kolesnikova, S.A.; Lyakhova, E.G.; Kozhushnaya, A.B.; Kalinovsky, A.I.; Berdyshev, D.V.; Popov, R.S.; Stonik, V.A. New Isomalabaricane-Derived Metabolites from a *Stelletta* sp. Marine Sponge. *Molecules* **2021**, *26*, 678. https:// doi.org/10.3390/molecules26030678

Academic Editor: Akihito Yokosuka Received: 29 December 2020 Accepted: 25 January 2021 Published: 28 January 2021

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sponge *Jaspis stellifera* [9]. These findings allow to presume that symbiotic microorganisms in the corresponding sponges are involved in the generation of some metabolites.

Diverse isomalabaricane-type *nor*-terpenoids, containing less than 30 carbon atoms in their skeleton systems, have been found together with isomalabaricanes several times [10–12]. Their presence could be explained either by oxidative degradation of C30 metabolites or by precursor role of *nor*-terpenoids in the biosynthesis of these compounds [12,13]. However, the biogenesis of isomalabaricane compounds in sponges remains to be mysterious so far.

Recently, we have reported the isolation of two isomalabaricane-type *nor*-terpenoids, cyclobutastellettolides A and B, and series of known isomalabaricanes from a *Stelletta* sp. [14] We suppose that new data on structural variety of isomalabaricane derivatives supported with strong evidence on stereochemistry could someday shed light on their origin.

In the present work, an investigation of the chemical components of a *Stelletta* sp. from Vietnamese waters was continued. Herein, we report the isolation and structural elucidation of six new compounds **1**–**6** and known globostelletin N [15].

#### **2. Results and Discussion**

The frozen sample of a marine sponge *Stelletta* sp. was finely chopped and extracted with EtOH, then the extract was concentrated under reduced pressure and subjected to Sephadex LH-20 and silica gel column chromatography followed by normal- and reversedphase HPLC procedures (Figure S73) to afford new stellettins Q-V **1**–**6** together with known globostelletin N [15] (Figure 1).

**Figure 1.** Structures of compounds **1**–**6** and globostelletins K, M, and N.

Stellettin Q (**1**) was isolated as a yellow oil with molecular formula C32H44O6 deduced by HRESIMS (Figure S3). The NMR data of **1** (Table 1; Figures S4 and S5) were closely related to the spectral characteristics of isomalabaricane globostelletin K (Figure 1, Figures S55 and S56) initially found in the marine sponge *Rabdastrella globostellata* [15] and also co-isolated from the studied *Stelletta* sp. [14].


**Table 1.** 1H and 13C NMR data of **1** (700 and 176 MHz) and **2** (500 and 126 MHz) in CDCl3.

<sup>1</sup> Assignments were made with the aid of HSQC, HMBC and ROESY data. <sup>2</sup> The values were found from HSQC experiment.

The detailed analysis of 2D spectra (COSY, HSQC, HMBC etc.) of **1** supported the main structure (Figure 2 and Figures S6–S9). The signals of methyl group at *δ*<sup>H</sup> 2.06, s; *δ*<sup>C</sup> 21.2 and acetate carbonyl at *δ*<sup>C</sup> 171.0, together with the HMBC correlation of axial proton H-3 at *δ*<sup>H</sup> 4.54, dd (11.7, 5.2) to that carbonyl, revealed the O-acetyl substitution at C-3 in the ring A. Moreover, the signal of C-3 at *<sup>δ</sup>*<sup>C</sup> 80.8 instead of ketone signal at *<sup>δ</sup>*<sup>C</sup> 219.2 in 13C NMR spectrum of globostelletin K also demonstrated the 3-acetoxy-tricyclic core in **<sup>1</sup>**, while the 3*β*-orientation of acetoxy group was confirmed by strong correlations of H-3/H-5 and CH3-28 observed in the ROESY spectrum. The 13*Z* geometry in **1** was in agreement with the signal of CH3-18 at *δ*<sup>H</sup> 1.79, s and its ROESY correlation with CH3-30. As well as *E* configuration of 24(25)-double bond was found from the *W*-path COSY correlation of protons H-24/CH3-27.

**Figure 2.** Selected COSY ( ), HMBC ( ) and ROESY ( ) correlations of **1** and **2**.

The 1H- and 13C-NMR signals of the side chain of **1** as well as the form of ECD curve (Figure S10) were analogous to those of globostelletin K [15] (Figure S57) suggesting the same stereochemistry of the side chain. This assignment was in a good agreement with the computational ECD results performed using density functional theory (DFT) with the nonlocal exchange-correlation functional B3LYP [16], the polarization continuum model (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 package of programs [18] (Figure S66). The 15*R*,23*S* absolute configuration, providing 13*Z*,24*E* geometry and *trans*−*syn*−*trans*-fused tricyclic moiety with 3*β*-oriented acetoxy group fully satisfies the similarity of the experimental and theoretical ECD spectra of **1** (Figure 3). In detailes, statistically avereged curve (Figure 3) follows the shape of the experimental one, although even more close coincidence was indicated for theoretically less probable conformer (Figure S67). In addition, we could conclude that the presence of 3-acetoxy or 3-oxo functions in the structures of the corresponding compounds insignificantly affects the shape of their ECD curves. According to obtained new data we pose the same 15*R*,23*S* stereochemistry for globostelletin K (Figure S69).

**Figure 3.** Comparison of experimental and theoretical ECD spectra of stellettin Q (**1**).

Stellettin R (**2**) has a molecular formula of C32H44O6 as it was established on the basis of HRESIMS (Figure S11). Spectral data (Table 1, Figure 2, Figures S12 and S13) were consistent with known globostelletin M [15] (Figure 1, Figures S59 and S60) possessing an isomalabaricane core connected with 13*E* double bond (CH3-18: *δ*<sup>H</sup> 2.06, s). However, like stellettin Q, it contains 3*β*–acetoxy group (*δ*<sup>H</sup> 4.53, dd (11.6, 5.2); *δ*<sup>C</sup> 80.7; *δ*<sup>H</sup> 2.05, s; *δ*<sup>C</sup> 170.9; 21.2). Concerning to the relative configuration of the cyclopentene unit in the side chain of **2**, the ROESY cross-peaks between H-15/H-24 and H-23/CH3-18 ascertained a *trans*-relationship of the vicinal protons H-15 and H-23. Careful examination of the chemical shifts for CH-15 (*δ*<sup>H</sup> 3.22, dt (9.2, 6.0); *δ*<sup>C</sup> 48.0) and CH-23 (*δ*<sup>H</sup> 3.95, m; *δ*<sup>C</sup> 48.0) showed the values similar to those of globostelletin M and differed from globostelletin N (Figure 1, Figures S63 and S64) isolated by Li et al. [15] and co-isolated by us. Moreover, the ECD spectrum of **2** (Figure S18) displayed the same curve and peaks as those published for globostelletin M (Figure S61).

However, structure modeling as well as calculation of ECD spectra for possible stereoisomers of **2** demonstrated a good agreement between experimental and theoretical spectra for 15*R*,23*S* absolute configuration (Figure 4) quite differ from 15*S*,23*S* reported for globostelletin M [15]. This inconsistence encouraged us to re-investigate the stereochemistry of co-isolated globostelletins M and N. We have obtained NMR and ECD spectra of the both compounds (Figures S59–S65) and they were identical to those provided as supplementary data by Li et al. [15]. At the same time, our computational results suggested globostelletin M to possess the same 15*R*,23*S* absolute configuration (Figure S70) of cyclopentene unit as **2**, while globostelletin N has 15*S*,23*R* stereochemistry (Figure S71). Based on the data we believe that previously published research comprises some inaccuracies and the stereochemistry of these centres in corresponding isomalabaricanes should be revised. It was noted that the isomalabaricane-type terpenoids undergo a photoisomerization of the side chain 13-double bond during the isolation and storage [19,20]. We consider compounds **1** and **2** to be the 13*Z*/*E* pair of the same 15*R*,23*S* isomer.

**Figure 4.** Comparison of experimental and theoretical ECD spectra of stellettin R (**2**).

The molecular formula C19H28O3 of stellettin S (**3**) calculated from HRESIMS data (Figure S19) showed **3** to be a rather smaller molecule then classical C30-isomalabaricanes, intriguing due to the lack of a significant part in the molecule, when compared with the majority of known isomalabaricanes and their derivatives. The 13C- and DEPT NMR spectra (Table 2; Figures S21 and S22) exhibited 19 resonances, including those of carbonyl carbon at *δ*<sup>C</sup> 216.5 (C-3) and carboxyl carbon at *δ*<sup>C</sup> 178.8 (C-12) as well as two down-shifted quaternary carbons at *δ*<sup>C</sup> 88.1 (C-13), 77.8 (C-14). 1H- and 13C-NMR spectra (Figures S20 and S21) revealed five methyls, two methylene, two methine groups and seven quaternary carbons, suggesting an isoprenoid nature. In the HSQC spectrum (Figure S23) four methyl singlets (*δ*<sup>H</sup> 1.06, 1.08, 1.25, and 1.62) correlated with carbon signals at *δ*<sup>C</sup> 21.6 (CH3-29), 25.9 (CH3-28), 30.8 (CH3-30) and 23.3 (CH3-19), respectively, while singlet of one more methyl group at *δ*<sup>H</sup> 1.80 gave a cross-peak with high field signal at *δ*<sup>C</sup> 3.7 (CH3-18). The further inspection of 2D spectra (Figure 5 and Figures S23–S26) revealed the bicyclic framework resembling the core of globostelletin A (Figure 5), isolated from the sponge *Rhabdastrella globostellata* [13].

This was confirmed by the key long-range HMBC correlations from gem-dimethyl group (CH3-28 and 29) to C-3, C-4 and C-5; from H-5 to C-1, C-4, C-6, C-9 and C-10; from methyl CH3-19 to C-1, C-9 and C-10; from methyl CH3-30 to C-7, C-8 and C-9 as well as from the methylene of carboxymethyl group (CH2-11) to C-8, C-9, C-10 and carboxyl carbon C-12 (Figure 5 and Figure S24). The empirical formula, besides bicyclic system and two carbonyls, required two additional degrees of unsaturation which were accounted for an acetylenic bond in a short side chain. The NMR signals of two quaternary carbons at *δ*<sup>C</sup> 88.1 (C-13), 77.8 (C-14) and methyl (CH3-18) at *δ*<sup>H</sup> 1.80, *δ*<sup>C</sup> 3.7 were finally attributed to the methylacetylenic substituent at C-8, that was confirmed by HMBC correlations from CH3-30 to C-8 and C-13, along with that from CH3-18 to C-7, C-8, C-9, C-13, C-14 and CH3-30. Analogous methylacetylenic substituent was characterized previously with similar chemical shifts in a series of synthetic alkynes [21].

**Figure 5.** Selected COSY ( ) and HMBC ( ) correlations of **3** and **4** and structure of known globostelletin A.

Interestingly, the NMR signal of CH3-19 (*δ*<sup>H</sup> 1.62, s) was notably downfield shifted in comparison with that in a number of isomalabaricanes and their derivatives spectra. We explained it by the joint influence of the methylacetylene and carboxymethyl groups. The quantum chemical calculations (Figure S66) of the chemical shifts for structure **3** confirmed the down-shifted position of the proton signal of CH3-19 and afforded its theoretical chemical shift value of *δ*<sup>H</sup> 1.69 ppm.

The relative stereochemistry of **3** was determined by ROESY experiment (Figure 6 and Figure S26). A *trans*-fusion of the bicyclic system was shown by key NOE interactions. The correlations between CH3-19/CH3-29, CH3-28/H-5, H-5/Ha-11, CH3-19/H-9, and CH3-30/Hb-11 showed the *β*-orientations of CH3-19 and H-9, whereas H-5, CH2-11, and CH3-30 were *α*-oriented. The chair conformation of the ring B with equatorial positions of H-9 and CH3-30 corresponded to the long-range COSY correlation between H-9 and H*β*-7 (Figure 5 and Figure S25) together with ROESY correlations H-5/Ha-11 and Hα-7/Hb-11. Taking into consideration the relative stereochemistry of the compound **3** along with above mentioned absolute stereochemistry of the C30 congeners **1** and **2** as well as the fact of co-isolation of cyclobutastellettolides A and B [14] with the same absolute configurations we suggested the 5*S,* 8*R,* 9*R,* 10*R* absolute stereochemistry of stellettin S (**3**).

**Figure 6.** Selected ROESY ( ) correlations of **3**.


**Table 2.** 1H and 13C NMR data (700 and 176 MHz) of **3**–**6**

 in CDCl3.

43

Stellettin T (**4**) with the molecular formula C20H32O5 seemed to be another isomalabaricane-type derivative. The ispection of NMR data (Table 2; Figure 5 and Figures S29–S33) revealed the same type of 9-carboxymethyl substituted bicyclic core as was deduced for compound **3**. It contains 3*β*-acetoxy group, confirmed with the signals of CH-3 (*δ*<sup>H</sup> 4.44, dd (11.2, 5.1); *δ*<sup>C</sup> 80.3), methyl of acetate group (*δ*<sup>H</sup> 2.05, s; *δ*<sup>C</sup> 21.2) and acetate carbon (*δ*<sup>C</sup> 170.9). According to the 13C NMR spectrum and molecular formula, compound **4** has one carbonyl less side chain then known globostelletin A [13]. Based on this data and HMBC correlations from CH3-14 (*δ*<sup>H</sup> 2.12, s) and CH3-30 (*δ*<sup>H</sup> 1.31, s) to C-13 (*δ*<sup>C</sup> 213.0), the acetyl was connected with C-8 (*δ*<sup>C</sup> 52.5). The key ROESY correlations H-3/H-5, H-5/Ha-11, H-14/CH3-19, H-9/CH3-19 and H-9/CH3-29 (Figure S34) suggested configurations at C-5, C-8, C-9 and C-10 identical to those of co-isolated isomalabaricanes.

The structures of stellettins U (**5**) and V (**6**) corresponded to the same C19H30O5 molecular formula deduced from HRESIMS (Figures S36 and S45). In comparison with co-isolated metabolites, the spectral data of compounds **5** and **6** revealed bicyclic core with keto group at C-3, gem-dimethyl group at C-4 and two angular methyls at C-8 and C-10 (Table 2). Additionally, 1H- and 13C-NMR spectra of compound **5** (Figures S37 and S38) demonstrated signals of two carbonyls (*δ*<sup>C</sup> 173.7 and 183.8) and one ethoxy group (*δ*<sup>H</sup> 4.15, q (7.1); *δ*<sup>C</sup> 60.8 and *δ*<sup>H</sup> 1.26, t (7.1); *δ*<sup>C</sup> 14.1). HMBC experiment (Figure 7 and Figure S41) allowed to place the carboxy group at C-8 and ethyl ester at C-11 on the basis of congruous correlations from methylenes –CH2-CH3 (*δ*<sup>H</sup> 4.15, q (7.1), 2H) and CH2-11 (*δ*<sup>H</sup> 2.41, dd (17.7, 5.3) and 2.30, m) to carboxyl C-12 (*δ*C. 173.7) and also from methyl CH3-30 (*δ*<sup>H</sup> 1.17, s) to carboxyl C-13 (*δ*<sup>C</sup> 183.8). The relative stereochemistry of **5** was determined by ROESY spectral analysis (Figure S43). Correlation between H-9 (*δ*<sup>H</sup> 2.78, br t (5.0)) and CH3-19 (*δ*<sup>H</sup> 1.24, s) indicated their *β*-orientation. Meanwhile, a ROESY correlation between H-5 (*δ*<sup>H</sup> 1.40, dd (12.6, 3.0))/Ha-11 (*δ*<sup>H</sup> 2.41, dd (17.7, 5.3)) and Hb-11 (*δ*<sup>H</sup> 2.30, m)/CH3-30 (*δ*<sup>H</sup> 1.17, s) confirmed the *α*-orientation of H-5, -CH2-COOEt and CH3-30. The above-mentioned results were in agreement with the spatial structure of isomalabaricane derivatives.

**Figure 7.** Selected COSY ( ) and HMBC ( ) correlations of **5** and **6**.

Compound **6** was an isomer of compound **5**, differed by the NMR signals (Figures S46 and S47) of carboxylic carbons (*δ*<sup>C</sup> 177.6 and 178.0), methyl C-19 (*δ*<sup>H</sup> 1.14, s), methylene CH2-11 (*δ*<sup>H</sup> 2.48, dd (18.1, 5.4) and 2.38, dd (18.1, 4.8)) and ethoxy group (*δ*<sup>H</sup> 4.23, dq (10.9, 7.1); 4.13, dq (10.9, 7.1) and 1.31, t (7.1)). The key HMBC correlations (Figure 7) satisfied the proposed structure of **6**. However, since the values of carboxyl carbons shifts for **6** are close, distinguishing their correlations and direct ester positioning without data for isomer **5** brought some uncertainty. To avoid future difficulties with structurally related esters we calculated carbon chemical shift values for two isomers **5** and **6** (Figure S66). It was shown, that theoretical *δ*<sup>C</sup> C-13 (**5**) = 192.7 and *δ*<sup>C</sup> C-12 (**5**) = 182.4 gave the Δ*δ*C(13-12) = 10.3 ppm close to experimental value Δ*δ*C(13-12) = 10.1 ppm, while theoretical and experimental Δ*δ*C(13-12) for compound **6** were of 0.4 ppm (clcd *δ*<sup>C</sup> C-13 (**6**) = 183.2, *δ*<sup>C</sup> C-12 (**6**) = 182.8).

ROESY correlations of **6** supported the relative stereochemistry similarly to that of compound **5**. In fact, we detected expected NOE interactions H-9 (δ<sup>H</sup> 2.74, br t (4.6))/CH3- 19 (δ<sup>H</sup> 1.14, s); H-5 (δ<sup>H</sup> 1.39, dd (12.7; 3.0))/Ha-11 (δ<sup>H</sup> 2.48, dd (18.1, 5.4)) and Hb-11 (δ<sup>H</sup> 2.38, dd (18.1, 4.8))/CH3-30 (δ<sup>H</sup> 1.12, s). Therefore, derivatives **5** and **6** possess the same stereochemistry as other co-isolated isomalabaricanes. Although compounds **5** and **6** are

rather artificial products derived during EtOH extraction, the isolated pair of esters allowed to reliably establish the position of the ether group based on the chemical shifts of C-12 and C-13.

Both compounds were supposed to be the half-ester derivatives of the hypothetical dicarboxylic acid. The anhydrous form of the acid was reported by Ravi et al. [5] as a product of ozonolysis of isomalabaricane precursor [22,23]. Moreover, Ravi et al. obtained dimethyl and monomethyl esters of the acid and did not point the place of esterification in the case of the latter.

Among isolated new compounds **1**–**6**, we find stellettin S (**3**) the most intriguing, since occurrences of acetylene-containing isoprenoids are rare and not so far reported in the isomalabaricane series. To date, several biosynthetic pathways leading to the alkyne formation in natural products has been supported with identified and characterized gene clusters. In the first case, acetylenases, a special family of desaturases, catalyze the dehydrogenation of olefinic bonds in unsaturated fatty acids to afford acetylenic functionalities [24,25]. Next, acetylenases are also used to form the terminal alkyne in polyketides [26]. One more biosynthetic route results in a terminal alkyne formation in acetylenic amino acids and involves consequent transformations by halogenase BesD, oxidase BesC and lyase BesB [27]. Finally, two recent papers describe the molecular basis for the formation of alkyne moiety in acetylenic prenyl chains occurring in a number of meroterpenoids [28,29]. The abovementioned reports highlight hot trends in a scientific search for enzymatic machineries leading to the biologically significant and synthetically applicable acetylene bond in natural compounds. We believe that isolation of the new terpenoidal alkyne **3** could inspire further investigations of the *Stelletta* spp. sponges and associated microorganisms through genome mining.

According to obtained new data we also report the correction in stereochemistry of two asymmetric centers in globostelletins M (Figure S70) and N (Figure S71). Really, their ECD and NMR spectra in comparison with those of globostelletin K and stellettins Q and R (Figures S59–S71) clearly show rather 15*R*,23*S* configuration for globostelletin M instead of previously reported 15*S*,23*S* [15] as well as 15*S*,23*R* stereochemistry for globostelletin N instead of 15*R*,23*R* [15].
