New Isomalabaricane-Derived Metabolites from a Stelletta Sp. Marine Sponge

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Article New Isomalabaricane-Derived Metabolites from a Stelletta sp. Marine Sponge

Sophia A. Kolesnikova 1,* , Ekaterina G. Lyakhova 1 , Anastasia B. Kozhushnaya 2 , Anatoly I. Kalinovsky 1, Dmitrii V. Berdyshev 1, Roman S. Popov 1 and Valentin A. Stonik 1,2

1 G.B. Elyakov Paciﬁc Institute of Bioorganic Chemistry, Far Eastern Branch of Russian Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, Russia; [email protected] (E.G.L.); [email protected] (A.I.K.); [email protected] (D.V.B.); [email protected] (R.S.P.); [email protected] (V.A.S.) 2 School of Natural Sciences, Far Eastern Federal University, Sukhanova Str. 8, 690000 Vladivostok, Russia; [email protected] * Correspondence: [email protected]; Tel.: +7-423-231-1168

Abstract: In continuation of our studies on a Vietnamese collection of a Stelletta sp., sponge we have isolated two new isomalabaricane triterpenoids, stellettins Q and R (1 and 2), and four new isomalabaricane-derived nor-terpenoids, stellettins S-V 3–6, along with previously known globostelletin N. Among them, compound 3 contains an acetylenic fragment, unprecedented in the isomalabaricane family and extremely rare in other marine sponge terpenoids. The structures and absolute conﬁgurations of all new compounds were established by extensive NMR, MS, and ECD analyses together with quantum-chemical modeling. Additionally, according to obtained new data we report the correction in stereochemistry of two asymmetric centers in the structures of two known isomalabaricanes, 15R,23S for globostelletin M and 15S,23R for globostelletin N.

Citation: Kolesnikova, S.A.; Keywords: isomalabaricanes; Stelletta sp.; marine sponge; terpenoid; structure elucidation 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. 1. Introduction Molecules 2021, 26, 678. https:// Malabaricanes are rather a small group of tricarbocyclic triterpenoids found in dif- doi.org/10.3390/molecules26030678 ferent 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- Academic Editor: Akihito Yokosuka 30, are known as metabolites of four genera of marine sponges—Stelletta, Jaspis, Geodia Received: 29 December 2020 and Rhabdastrella—belonging to the class Demospongiae. Some of them are highly cyto- Accepted: 25 January 2021 toxic against tumor cells [4]. Since the first isolation of three yellow highly conjugated Published: 28 January 2021 isomalabaricane-type triterpenoids from the marine sponge Jaspis stellifera in 1981 [5] more than 130 isomalabaricanes and related natural products have been reported from Publisher’s Note: MDPI stays neutral the abovementioned sponge genera. It was noticed that Stelletta metabolites are quite with regard to jurisdictional claims in different depending on the collection. Indeed, isomalabaricane triterpenoids were mainly published maps and institutional affil- found as very complex mixtures in tropical sponge samples, while boreal and cold-water iations. 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, Copyright: © 2021 by the authors. was unsuccessful, as the characteristic yellow pigments were not detected by thin layer Licensee MDPI, Basel, Switzerland. chromatography in the extracts of these sponges. This article is an open access article Additionally, in result of the chemical investigation of the sponge Stelletta tenuis, Li distributed under the terms and et al. identified two naturally occurring α-pyrones, namely gibepyrones C and F, along conditions of the Creative Commons with three isomalabaricane-type triterpenoids [7]. These α-pyrones were supposed to be Attribution (CC BY) license (https:// the oxidation products of the co-occurring stellettins [6]. Gibepyrone F had previously creativecommons.org/licenses/by/ been isolated from the fungal plant pathogen Gibberella fujikuroi [8], as well as from the 4.0/).

Molecules 2021, 26, 678. https://doi.org/10.3390/molecules26030678 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 12

Molecules 2021, 26, 678 2 of 13

been isolated from the fungal plant pathogen Gibberella fujikuroi [8], as well as from the sponge Jaspis stellifera [9].[9]. These These findings ﬁndings allow allow to to presume presume that that symbiotic symbiotic microorganisms microorganisms in the corresponding sponges are involved in the generation ofof somesome metabolites.metabolites. Diverse isomalabaricane-typeisomalabaricane-typenor nor-terpenoids,-terpenoids, containing containing less less than than 30 30 carbon carbon atoms atoms in intheir their skeleton skeleton systems,systems, have have been been found found together together with with isomalabaricanes isomalabaricanes several several times [10–12].times [10 Their–12]. Theirpresence presence could couldbe explaine be explainedd either either by oxidative by oxidative degradation degradation of C of30 metabolites or by precursor role of nor-terpenoids in the biosynthesis of these compounds C30 metabolites or by precursor role of nor-terpenoids in the biosynthesis of these com- [12,13].pounds However, [12,13]. However, the biogenesis the biogenesis of isomalabaricane of isomalabaricane compounds compounds in sponges in remains sponges to re-be mysteriousmains to be so mysterious far. so far. Recently, we have reported the isolation of two isomalabaricane-type isomalabaricane-type nornor-terpenoids,-terpenoids, cyclobutastellettolides A A and and B, B, and and seri serieses of ofknown known isomalabaricanes isomalabaricanes from from a Stelletta a Stelletta sp. [14]sp. [14We] Wesuppose suppose that that new new data data on on structural structural variety variety of of isomalabaricane derivativesderivatives supported with strong evidence on stereochemistry could someday shed light on their origin. In the present work, an investigatio investigationn of the chemical components of of a a Stelletta sp.sp. from Vietnamese waters was continued.continued. Herein Herein,, we report the isolationisolation andand structuralstructural elucidation of six new compounds 1–6 and known globostelletin N [[15].15].

2. Results and Discussion The frozen sample of a marine sponge Stelletta sp. was finely ﬁnely chopped and extracted with EtOH, then the extract was concentrated under reduced pressure and subjected to Sephadex LH-20LH-20 and and silica silica gel columngel column chromatography chromatography followed followed by normal- by andnormal- reversed- and reversed-phasephase HPLC procedures HPLC procedures (Figure S73) (Figur to afforde S73) new to afford stellettins new Q-V stellettins1–6 together Q-V 1 with–6 together known withglobostelletin known globostellet N [15] (Figurein N1 ).[15] (Figure 1).

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

Stellettin Q Q (1 (1) )was was isolated isolated as as a yellow a yellow oil oilwith with molecular molecular formula formula C32H C4432OH6 deduced44O6 de- byduced HRESIMS by HRESIMS (Figure (FigureS3). The S3). NMR The data NMR of data1 (Table of 1 1;(Table Figures1; Figures S4 and S4 S5) and were S5) closely were relatedclosely relatedto the spectral to the spectral characteristics characteristics of isomalabaricane of isomalabaricane globostelletin globostelletin K (Figures K (Figure 1 and1, S55Figures and S55S56) and initially S56) initiallyfound in found the marine in the sponge marine Rabdastrella sponge Rabdastrella globostellata globostellata [15] and[ 15also] and co- isolatedalso co-isolated from the from studied the studiedStellettaStelletta sp. [14].sp. [14].

Molecules 2021, 26, 678 3 of 13

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

1 2 No. 1 δH mult (J in Hz) δC δH mult (J in Hz) δC 1α 1.57, td (13.0, 3.9) 1.57, m 33.1 32.9 1β 1.38, m 1.36, m 2α 1.82, m 1.81, m 25.1 25.1 2β 1.71, m 1.68, m 3α 4.54, dd (11.7, 5.2) 80.8 4.53, dd (11.6, 5.2) 80.7 4 38.2 38.2 5α 1.73, m 46.5 1.75, m 46.4 6α 1.70, m 1.68, m 18.2 18.2 6β 1.48, m 1.48, m 7α 1.99, m 2.06, m 37.7 38.5 7β 2.00, m 1.93, m 8 44.2 43.8 9β 1.75, m 50.3 1.77, m 50.3 10 35.4 35.4 11α 2.13, m 2.15, m 36.4 36.4 11β 2.13, m 2.15, m 12 206.6 206.9 13 146.8 146.0 14 147.9 147.5 15 4.75, m 45.0 2 3.22, dt (9.2, 6.0) 48.0 16α 2.27, m 2.52, m 37.9 38.4 16β 3.02, ddt (19.4, 9.3, 2.5) 2,89, ddt (19.4, 9.2, 2.7) 17 6.80, br s 144.6 6.78, dd (4.3, 2.5) 143.6 18 1.79, s 16.2 2 2.06, s 15.7 19 1.02, s 22.4 1.01, s 22.4 20 195.6 195.1 21 2.29, s 26.9 2.30, s 27.0 22 146.6 146.7 23 3.86, br t (8.3) 47.5 3.95, m 48.0 24 6.57, br d (10.2) 145.9 6.59, dd (10.6, 1.5) 144.6 25 126.3 127.3 26 171.0 170.7 27 1.88, br s 12.6 1.86, d (1.3) 12.5 28 0.91, s 29.0 0.90, s 29.0 29 0.89, s 16.9 0.88, s 17.0 30 1.29, s 24.1 1.23, s 26.4 OAc 2.06, s 171.021.2 2.05, s 170.921.2 1 Assignments were made with the aid of HSQC, HMBC and ROESY data. 2 The values were found from HSQC experiment.

The detailed analysis of 2D spectra (COSY, HSQC, HMBC etc.) of 1 supported the main structure (Figure2 and Figures S6–S9). The signals of methyl group at δH 2.06, s; δC 21.2 and acetate carbonyl at δC 171.0, together with the HMBC correlation of axial proton H-3 at δH 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 δC 80.8 instead of ketone signal at δC 219.2 in 13C NMR spectrum of globostelletin K also demonstrated the 3-acetoxy-tricyclic core in 1, while the 3β-orientation of acetoxy group was conﬁrmed by strong correlations of H-3/H-5 and CH3-28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement with the signal of CH3-18 at δH 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. Molecules 2021, 26, x FOR PEER REVIEW 3 of 12

The detailed analysis of 2D spectra (COSY, HSQC, HMBC etc.) of 1 supported the MoleculesMoleculesMolecules 2021 20212021,, , 26, 2626,, , ,x x xx FOR FOR FORFOR PEER PEER PEERPEER REVIEW REVIEW REVIEWREVIEW 333 of ofof 12 1212 main structure (Figures 2 and S6–S9). The signals of methyl group at δH 2.06, s; δC 21.2 and acetate carbonyl at δC 171.0, together with the HMBC correlation of axial proton H-3 at δH 4.54, dd (11.7, 5.2) to that carbonyl, revealed the O-acetyl substitution at C-3 in the ring A. TheTheThe detailed detaileddetailed analysis analysisanalysis of ofof 2D 2D2D spec specspectratratra (COSY, (COSY,(COSY, HSQC, HSQC,HSQC, HMBC HMBCHMBC etc.) etc.)etc.) of ofof 1 11 supported supportedsupported the thethe Moreover, the signal of C-3 at δC 80.8 instead of ketone signal at δC 219.2 in 13C NMR mainmain structure structure (Figures (Figures 2 2 and and S6–S9). S6–S9). The The signals signals of of methyl methyl group group at at δ δHHH 2.06, 2.06, s; s; δ δCCC 21.2 21.2 and and mainspectrum structure of globostelletin (Figures 2 and K S6–S9). also demon The signalsstrated of methyl the 3-acetoxy-tricyclic group at δ 2.06, s; core δ 21.2 in and1, while the acetateacetateacetate carbonyl carbonylcarbonyl at atat δ δδCCC 171.0, 171.0, 171.0, together togethertogether with withwith the thethe HMBC HMBCHMBC corr corrcorrelationelationelation of ofof axial axialaxial proton protonproton H-3 H-3H-3 at atat δ δδHHH 3β-orientation of acetoxy group was confirmed by strong correlations of H-3/H-5 and CH3- 4.54,4.54,4.54, dd dddd (11.7, (11.7,(11.7, 5.2) 5.2)5.2) to toto that thatthat carbonyl, carbonyl,carbonyl, revealed revealedrevealed the thethe O-acetyl O-acetylO-acetyl substitution substitutionsubstitution at atat C-3 C-3C-3 in inin the thethe ring ringring A. A.A. 28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement1313 with the Moreover,Moreover,Moreover, the thethe signal signalsignal of ofof C-3 C-3C-3 at atat δ δδCCC 80.8 80.880.8 instead insteadinstead of ofof ketone ketoneketone signal signalsignal at atat δ δδCCC 219.2 219.2219.2 in inin 13CCC NMR NMRNMR spectrumspectrumspectrumsignal of of ofof CH globostelletin globostelletinglobostelletin3-18 at δH K KK1.79, also alsoalso demon demondemons andstrated stratedstratedits ROESY the thethe 3-acetoxy-tricyclic 3-acetoxy-tricyclic3-acetoxy-tricyclic correlation with core corecore CH in inin 1 131,-30., , while whilewhile As the thethe well as E 333configurationβββ-orientation-orientation-orientation of ofof acetoxyof acetoxyacetoxy 24(25)-double group groupgroup was waswas confirmed bond confirmedconfirmed was by by byfound strong strongstrong fromcorrelations correlationscorrelations the W of of-pathof H-3/H-5 H-3/H-5H-3/H-5 COSY and andand correlationCH CHCH33-3-- of 282828protons observed observedobserved H-24/CH in inin the thethe ROESY ROESY3ROESY-27. spectrum. spectrum.spectrum. The TheThe 13 1313ZZZ geometry geometrygeometry in inin 1 11 was waswas in inin agreement agreementagreement with withwith the thethe 3 H 3 Molecules 2021, 26, 678 signalsignalsignal ofofof CHCHCH33-183-18-18 atatat δδδHHH 1.79,1.79,1.79, sss andandand itsitsits ROESYROESYROESY correlationcorrelationcorrelation withwithwith CHCHCH33-30.3-30.-30. AsAsAs wellwellwell 4 asasas of 13EEE configurationconfiguration ofof 24(25)-double24(25)-double bondbond waswas foundfound fromfrom thethe WW-path-path COSYCOSY correlationcorrelation ofof configuration of 24(25)-doubleO bond was found from the OW-path COSY correlation of protonsprotonsprotons O H-24/CH H-24/CHH-24/CH33-27.3-27.-27. H H H H OO COOH HOOO O H OOO H H HHH HHH HHH H HHH O O COOHCOOHCOOH O O HHH COOH H HHH H HHH HHH HHH O 1 COOH OOO OOO 2 OOO OOO COOHCOOHCOOH HHH HHH Figure 2. Selected111 COSY ( ), HMBC ( ) and ROESY ( 222) correlationsOO Oof 1 and 2.

FigureFigureFigure 2. 2.2. Selected SelectedSelectedSelectedThe COSY COSYCOSY 1H- ( (( and),),),), HMBC HMBC HMBC13HMBCC-NMR ( ( (( ) )) )signalsand andandand ROESY ROESYROESYROESY of the( ((( side))) ) correlationscorrelations correlationscorrelations chain of ofof ofof 11 11 and as andand well 2 2 2.2. .. as the form of ECD curve

(Figure 1S10) were13 analogous to those of globostelletin K [15] (Figure S57) suggesting the TheThe 11H-1H- and and 131313C-NMRC-NMR signals signals of of the the side side chain chain of of 11 asas well well as as the the form form of of ECD ECD curve curve sameTheTheThe stereochemistry H-H-H- and andand C-NMRC-NMRC-NMR of signals signals signalsthe side of ofof the thethechain. side sideside chainThis chainchain assignment of ofof 1 11 as asas well wellwell as asaswas the thethe inform formform a good of ofof ECD ECDECD agreement curve curvecurve with (Figure(Figure(Figure S10) S10) S10) were werewere analogous analogousanalogous to to to those thosethose of ofof globostelletin globostelletinglobostelletin K KK [15] [[15][15]15] (Figure (Figure(Figure(Figure S57) S57)S57)S57) suggesting suggestingsuggestingsuggesting the thethe the computational ECD results performed using density functional theory (DFT) with the samesamesame stereochemistry stereochemistry stereochemistry of of of the the the side sideside chain. chain.chain. This This This assignment assignment assignment was was was in in in a aa good goodgood agreement agreementagreement with withwith nonlocal exchange-correlation functional B3LYP [16], the polarization continuum model thethethe computational computational computational ECD ECD ECD results results results performed performedperformed using usingusing density densitydensity functional functionalfunctional theory theorytheory (DFT) (DFT)(DFT) with withwith the thethe nonlocalnonlocalnonlocal(PCM) [17] exchange-correlation exchange-correlation exchange-correlation and split-valence functional functional functional basis sets B3 B3 B3LYPB3 6-31G(d),LYPLYPLYP [16], [[16],[16],16], implementedthe thethe polarization polarizationpolarization in continuum continuum continuumthe Gaussian model modelmodel 16 package (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 package (PCM)(PCM)(PCM)of programs [17] [17][17] and andand split-valence [18] split-valencesplit-valence (Figure basis basisbasisS66). sets setssets The 6-31G(d), 6-31G(d),6-31G(d), 15R,23 implemented implementedSimplemented absolute configuration, in inin the thethe Gaussian GaussianGaussian 16 providing 1616 package packagepackage 13Z,24E of programs [18] (Figure S66). The 15R,23S absolute configuration, providing 13Z,24E ofofofgeometry programsprogramsprograms and [18][18][18] trans (Figure(Figure(Figure−syn S66).S66).−S66).trans TheTheThe-fused 151515RRR ,23tricyclic,23,23SSS absoluteabsoluteabsolute moiety configuration,configuration,configuration, with 3β-oriented providingprovidingproviding acetoxy 131313ZZZ,24,24,24 groupEEE fully geometry and trans−syn−trans-fused tricyclic moiety with 3β-oriented acetoxy group fully geometrygeometrygeometrysatisfies andthe andand transsimilarity transtrans−−−synsynsyn−−−trans transtransof the-fused-fused-fused experi tricyclic tricyclictricyclicmental moiety moietymoiety and with withtheoreticalwith 3 33βββ-oriented-oriented-oriented ECD acetoxy acetoxyacetoxy spectra group groupgroup of 1 fully(Figure fullyfully 3). In satisfies the similarity of the experimental and theoretical ECD spectra of 1 (Figure3). satisfiessatisfiessatisfiesdetailes, the thethe statistically similarity similaritysimilarity of of ofavereged the thethe experi experiexperi curvementalmentalmental (Figure and andand theoretical theoreticaltheoretical 3) follows ECD ECDECD the spectra spectraspectra shape of ofof 1 11the (Figure (Figure(Figure experimental 3). 3).3). In InIn one, In detailes, statistically avereged curve (Figure3) follows the shape of the experimental detailes,detailes,detailes, statistically statisticallystatistically avereged averegedavereged curve curvecurve (Figure (Figure(Figure 3) 3)3) follows followsfollows the thethe sh shshapeapeape of ofof the thethe experimental experimentalexperimental one, one,one, one,although although even even more more close close coincidencecoincidence was was indicated indicated for theoreticallyfor theoretically less probable less probable althoughalthoughalthough eveneveneven moremoremore closecloseclose coincidencecoincidencecoincidence waswaswas indicatedindicatedindicated forforfor theoreticallytheoreticallytheoretically lesslessless probableprobableprobable conformerconformer (Figure (Figure S67). S67). In addition,In addition, we couldwe could conclude conclude that the that presence the presence of 3-acetoxy of 3-acetoxy or or conformerconformerconformer (Figure (Figure(Figure S67). S67).S67). In InIn addition, addition,addition, we wewe could couldcould conclude concludeconclude that thatthat the thethe presence presencepresence of ofof 3-acetoxy 3-acetoxy3-acetoxy or oror 3-oxo3-oxo functions functions in in the the structures structures of the of correspondingthe corresponding compounds compounds insignificantly insignificantly affects affects 3-oxo3-oxo3-oxo functions functionsfunctions in inin the thethe structures structuresstructures of ofof the thethe co cocorrespondingrrespondingrresponding compounds compoundscompounds insignificantly insignificantlyinsignificantly affects affectsaffects thethe shape shape of of their their ECD ECD curves. curves. According According to obtained to obtained new data new we data pose we the pose same the 15 Rsame,23S 15R,23S thethethe shape shapeshape of ofof their theirtheir ECD ECDECD curves. curves.curves. According AccordingAccording to toto obtained obtainedobtained new newnew data datadata we wewe pose posepose the thethe same samesame 15 1515RRR,23,23,23SSS stereochemistrystereochemistry for for globostelletin globostelletin K (Figure K (Figure S69). S69). stereochemistrystereochemistrystereochemistry for forfor globostelletin globostelletinglobostelletin K KK (Figure (Figure(Figure S69). S69).S69).

FigureFigureFigure 3. 3.3. Comparison ComparisonComparison of ofof experimental experimentalexperimental and andand theoretical theoreticaltheoretical ECD ECDECD spectra spectraspectra of ofof stellettin stellettin stellettin Q QQ ( (1(11).).). FigureFigure 3. 3.Comparison Comparison of experimentalof experimental and theoreticaland theoretical ECD spectraECD spectra of stellettin of stellettin Q (1). Q (1). 11 1313 TableTableTable 1. 1.1. H1HH and andand 13CCC NMR NMRNMR data datadata of ofof 1 11 (700 (700(700 and andand 176 176176 MHz) MHz)MHz) and andand 2 22 (500 (500(500 and andand 126 126126 MHz) MHz)MHz) in inin CDCl CDClCDCl33.3 .. Stellettin R (2) has a molecular formula of C H O as it was established on the basis Table 1. 1H and 13C NMR data of 1 (700 and 176 32MHz)44 and6 2 (500 and 126 MHz) in CDCl3. of HRESIMS (Figure S11). Spectral data (Table1, Figure2, Figures S12 and S13) were consistent with known globostelletin M [15] (Figure1, Figures S59 and S60) possessing an isomalabaricane core connected with 13E double bond (CH3-18: δH 2.06, s). However, like stellettin Q, it contains 3β–acetoxy group (δH 4.53, dd (11.6, 5.2); δC 80.7; δH 2.05, s; δC 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 (δH 3.22, dt (9.2, 6.0); δC 48.0) and CH-23 (δH 3.95, m; δC 48.0) showed the values similar to those of globostelletin M and differed from globostelletin N (Figure1, 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). Molecules 2021, 26, x FOR PEER REVIEW 5 of 12

Molecules 2021, 26, 678 5 of 13 However, structure modeling as well as calculation of ECD spectra for possible stereoisomers of 2 demonstrated a good agreement between experimental and theoretical spectraHowever, for 15R structure,23S absolute modeling configuration as well as calculation(Figure 4) quite of ECD differ spectra from for 15 possibleS,23S reported forstereoisomers globostelletin of 2 demonstrated M [15]. This a good inconsiste agreementnce between encouraged experimental us to and re-investigate theoretical the stereochemistryspectra for 15R,23 ofS absolute co-isolated conﬁguration globostelletins (Figure M4 )and quite N. differ We have from obtained 15 S,23S reported NMR and ECD spectrafor globostelletin of the both M [15 compounds]. This inconsistence (Figures encouraged S59–S65) usand to re-investigatethey were identical the stereo- to those providedchemistry ofas co-isolated supplementary globostelletins data by M Li and et N. al We. [15]. have At obtained the same NMR time, and ECDour computational spectra resultsof the both suggested compounds globostelletin (Figures S59–S65) M to possess and they the were same identical 15R,23 toS those absolute provided configuration as (Figuresupplementary S70) of data cyclopentene by Li et al. [unit15]. At as the2, while same time,globostelletin our computational N has 15 resultsS,23R suggested stereochemistry globostelletin M to possess the same 15R,23S absolute conﬁguration (Figure S70) of cy- (Figure S71). Based on the data we believe that previously published research comprises clopentene unit as 2, while globostelletin N has 15S,23R stereochemistry (Figure S71). Based someon the datainaccuracies we believe and that previouslythe stereochemis publishedtry research of these comprises centres some inaccuraciesin corresponding isomalabaricanesand the stereochemistry should of these be centresrevised. in correspondingIt was noted isomalabaricanes that the isomalabaricane-type should be re- terpenoidsvised. It was undergo noted that a the photoisomerization isomalabaricane-type of terpenoids the sideundergo chain 13-dou a photoisomerizationble bond during the isolationof the side and chain storage 13-double [19,20]. bond We during consider the isolationcompounds and storage1 and 2 [ 19to, 20be]. the We 13 considerZ/E pair of the samecompounds 15R,231Sand isomer.2 to be the 13Z/E pair of the same 15R,23S isomer.

Figure 4.4.Comparison Comparison of of experimental experimental and and theoretical theoretical ECD spectraECD spectra of stellettin of stellettin R (2). R (2).

The molecular formula C19H28O3 of stellettin S (3) calculated from HRESIMS data 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, (Figureintriguing S19) due showed to the lack 3 to of be a a significant rather smaller part in molecule the molecule, then when classical compared C30-isomalabaricanes, with the intriguingmajority of due known to the isomalabaricanes lack of a significant and their part derivatives. in the molecule,when The 13C- and DEPTcompared NMR with the majorityspectra (Table of known2; Figures isomalabaric S21 and S22)anes exhibited and their 19 resonances, derivatives. including The 13C- those and of DEPT car- NMR spectrabonyl carbon (Table at 2;δ FiguresC 216.5 (C-3)S21 and and S22) carboxyl exhibited carbon 19 atresonances,δC 178.8 (C-12) including as well those as twoof carbonyl 1 13 carbondown-shifted at δC 216.5 quaternary (C-3) and carbons carboxyl at δC 88.1 carbon (C-13), at δ 77.8C 178.8 (C-14). (C-12)H- andas wellC-NMR as two spectra down-shifted (Figures S20 and S21) revealed five methyls, two methylene, two methine groups and seven quaternary carbons at δC 88.1 (C-13), 77.8 (C-14). 1H- and 13C-NMR spectra (Figures S20 quaternary carbons, suggesting an isoprenoid nature. In the HSQC spectrum (Figure S23) and S21) revealed five methyls, two methylene, two methine groups and seven quaternary four methyl singlets (δ 1.06, 1.08, 1.25, and 1.62) correlated with carbon signals at δ 21.6 carbons, suggesting anH isoprenoid nature. In the HSQC spectrum (Figure S23)C four methyl (CH3-29), 25.9 (CH3-28), 30.8 (CH3-30) and 23.3 (CH3-19), respectively, while singlet of one singlets (δH 1.06, 1.08, 1.25, and 1.62) correlated with carbon signals at δC 21.6 (CH3-29), more methyl group at δH 1.80 gave a cross-peak with high field signal at δC 3.7 (CH3-18). 25.9The further(CH3-28), inspection 30.8 (CH of 2D3-30) spectra and (Figure23.3 (CH5 and3-19), Figures respectively, S23–S26) revealedwhile singlet the bicyclic of one more methylframework group resembling at δH 1.80 the gave core ofa cross-peak globostelletin with A (Figure high field5), isolated signal from at δC the 3.7 sponge (CH3-18). The furtherRhabdastrella inspection globostellata of 2D[13 spectra]. (Figures 5 and S23–S26) revealed the bicyclic framework resemblingThis was the confirmed core of globostelletin by the key long-range A (Figure HMBC 5), isolated correlations from from the gem-dimethylsponge Rhabdastrella globostellatagroup (CH3-28 [13]. 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 CH -19 to C-1, C-9 and C-10; from methyl CH -30 to C-7, C-8 and C-9 as well This was3 confirmed by the key long-range HMBC3 correlations from gem-dimethyl as from the methylene of carboxymethyl group (CH2-11) to C-8, C-9, C-10 and carboxyl groupcarbon (CH C-123 (Figure-28 and5 and29) to Figure C-3, S24).C-4 and The empiricalC-5; from formula, H-5 to besidesC-1, C-4, bicyclic C-6, C-9 system and and C-10; from methyltwo carbonyls, CH3-19 required to C-1, two C-9 additional and C-10; degrees from methyl of unsaturation CH3-30 which to C-7, were C-8 accounted and C-9 foras well as froman acetylenic the methylene bond ina of short carboxymethyl side chain. The group NMR signals(CH2-11) of twoto C-8, quaternary C-9, C-10 carbons and at carboxyl carbonδC 88.1 (C-13),C-12 (Figures 77.8 (C-14) 5 and and S24). methyl The (CH empirica3-18) at lδ Hformula,1.80, δC besides3.7 were bicyclic finally attributed system and two carbonyls,to the methylacetylenic required two substituent additional at degrees C-8, that of was unsaturation confirmed bywhich HMBC were correlations accounted for an acetylenic bond in a short side chain. The NMR signals of two quaternary carbons at δC 88.1 (C-13), 77.8 (C-14) and methyl (CH3-18) at δH 1.80, δC 3.7 were finally attributed to the methylacetylenic substituent at C-8, that was confirmed by HMBC correlations from CH3-

MoleculesMolecules 2021 ,2021 26, x, 26FOR, x FORPEER PEER REVIEW REVIEW 3 of 312 of 12

The Thedetailed detailed analysis analysis of 2D of spec2D spectra (COSY,tra (COSY, HSQC, HSQC, HMBC HMBC etc.) etc.) of 1 of supported 1 supported the the mainmain structure structure (Figures (Figures 2 and 2 andS6–S9). S6–S9). The Thesignals signals of methyl of methyl group group at δ Hat 2.06, δH 2.06, s; δC s; 21.2 δC 21.2 and and acetateacetate carbonyl carbonyl at δ Cat 171.0, δC 171.0, together together with with the HMBCthe HMBC corr correlationelation of axial of axial proton proton H-3 H-3at δ Hat δH 4.54,4.54, dd (11.7, dd (11.7, 5.2) 5.2)to that to that carbonyl, carbonyl, revealed revealed the O-acetylthe O-acetyl substitution substitution at C-3 at C-3in the in ringthe ring A. A. 13 Moreover,Moreover, the thesignal signal of C-3 of C-3at δ atC 80.8 δC 80.8 instead instead of ketone of ketone signal signal at δ atC 219.2 δC 219.2 in 13 inC NMRC NMR MoleculesMolecules 20212021,, 2626,, 678x FOR PEER REVIEW 66 of of 1312 spectrumspectrum of globostelletin of globostelletin K also K also demon demonstratedstrated the 3-acetoxy-tricyclicthe 3-acetoxy-tricyclic core core in 1 ,in while 1, while the the 3β-orientation3β-orientation of acetoxy of acetoxy group group was was confirmed confirmed by strong by strong correlations correlations of H-3/H-5 of H-3/H-5 and andCH 3CH- 3- 28 observed28 observed in the in theROESY ROESY spectrum. spectrum. The The13Z 13geometryZ geometry in 1 inwas 1 was in agreement in agreement with with the the 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. from30 to C-8 CH 3andsignal-30 signal toC-13, C-8of alongCH andof 3CH-18 C-13, with 3-18at alongδthatatH 1.79,δ fromH with1.79, s CHand thats 3-18 andits from to ROESYits C-7, CH ROESY C-8,3-18 correlation C-9, to correlation C-7, C-13, C-8, with C-14 C-9, with CHand C-13, 3CH-30. CH3 C-14-30.3As-30. wellAs well as Eas E Analogous methylacetylenic substituent was characterized previously with similar andAnalogous CH3-30.configuration methylacetylenic Analogousconfiguration of methylacetylenic 24(25)-double of substituent24(25)-double bond substituentwas bond wascharacterized was found was found characterized from frompreviously the theW-path previouslyW -pathwith COSY COSYsimilar withcorrelation correlation of of chemical shifts in a series of synthetic alkynes [21]. similarchemical chemical shiftsprotonsprotons in shifts aH-24/CH series H-24/CH in aof series3 -27.synthetic3 -27. of synthetic alkynes alkynes [21]. [21].

O O O O O O H H H H H H H H COOHCOOH H H H H H H H H H H O OO O O OO O COOHCOOH H H H H 1 1 2 2 O O

FigureFigureFigure 5. 2. Selected SelectedSelected 2. Selected COSY COSY COSY ( ( ) )(),and andHMBC ),HMBC HMBC ( ( () ( and) correlations)) and correlationsROESY ROESY ( of () of 3correlations and3)and correlations 4 and4 and structure of structure1 andof 1 and2of. known of2. known globostelletin A. The The1H- 1andH- and13C-NMR 13C-NMR signals signals of the of sidethe side chain chain of 1 ofas 1well as well as the as formthe form of ECD of ECD curve curve Interestingly, the NMR signal of CH3-19 (δH 1.62, s) was notably downfield shifted in Interestingly,(Figure(Figure S10) thethe S10) NMR NMRwere were signalanalogoussignal analogous ofof CHCH to33 -19-19those to ((δthoseδHH of 1.62, globostelletin of s)globostelletin was notably K [15] K downfielddownfield [15](Figure (Figure S57) shifted S57) suggesting inin suggesting the the comparison with that in a number of isomalabaricanes and their derivatives spectra. We comparisonsame withsame stereochemistry that stereochemistry in a numbernumber of oftheof of isomalabaricanesisomalabaricanes theside side chain. chain. This This andassignment assignment theirtheir derivativesderivatives was was in a in good spectra.spectra. a good agreement We agreement with with explained it by the joint influence of the methylacetylene and carboxymethyl groups. The explainedthe it by computationalthe the computational joint influenceinfluence ECD ECD ofresults the results methylacetylene performed performed using using andand density carboxymethylcarboxymethyl density functional functional groups.groups.theory theory (DFT) TheThe (DFT) with with the the quantum chemical calculations (Figure S66) of the chemical shifts for structure 3 quantum chemicalnonlocalchemicalnonlocal calculationsexchange-correlation calculations exchange-correlation (Figure (Figure S66) functional S66) of functional the of chemical B3theLYP B3chemicalLYP shifts[16], [16], the for shifts structurepolarizationthe polarization for 3structureconfirmed continuum continuum 3 model model theconfirmed down-shifted the down-shifted position of position the proton of the signal proton of CH signal-19 andof CH afforded3-19 and its afforded theoretical its confirmed(PCM) the(PCM) down-shifted [17] [17]and andsplit-valence split-valenceposition basisof thebasis sets proton sets6-31G(d), 6-31G(d), signal3 implemented of implemented CH3-19 inand the in afforded Gaussianthe Gaussian its 16 package 16 package chemicaltheoretical shift chemical value ofshiftδ value1.69 ppm. of δH 1.69 ppm. theoreticalof chemical programsof programs shift [18]H value [18](Figure of(Figure δH S66). 1.69 S66). ppm.The The15 R ,2315RS,23 absoluteS absolute configuration, configuration, providing providing 13Z ,2413ZE,24 E The relative stereochemistry of 3 was determined by by ROESY ROESY experiment experiment (Figures (Figure6 6 The relativegeometrygeometry stereochemistry and andtrans trans−syn− −synoftrans 3− transwas-fused -fuseddetermined tricyclic tricyclic moiety by moietyROESY with with experiment3β-oriented 3β-oriented (Figuresacetoxy acetoxy group6 group fully fully and FigureS26). A S26). trans A-fusiontrans-fusion of the ofbicyclic the bicyclic system system was shown was shown by key by NOE key NOEinteractions. interactions. The and S26). satisfiesA transsatisfies-fusion the similaritythe of similarity the bicyclic of the of experi thesystem experimental wasmental shown and andtheoretical by theoretical key NOE ECD ECDinteractions. spectra spectra of 1 Theof(Figure 1 (Figure 3). In 3). In Thecorrelations correlations between between CH CH3-19/CH-19/CH3-29, -29,CH3-28/H-5, CH -28/H-5, H-5/H H-5/Ha-11, CHa-11,3-19/H-9, CH -19/H-9, and CH and3- correlationsdetailes, betweendetailes, statistically statisticallyCH3-19/CH3 avereged avereged3-29, 3 CHcurve3 -28/H-5,curve (Figure3 (Figure H-5/H 3) follows 3)a follows-11, theCH shthe3-19/H-9,ape sh 3apeof the ofand experimentalthe CH experimental3- one, one, CH30/H3-30/Hb-11 showed-11 showed the β-orientations the β-orientations of CH3 of-19 CH and3-19 H-9, and whereas H-9, whereas H-5, CH H-5,2-11, CH and2-11, CH and3-30 30/Hb-11 showedalthoughb although the even β-orientations even more more close ofclose CHcoincidence 3coincidence-19 and H-9,was whereaswas indicated indicated H-5, for CH fortheoretically2-11, theoretically and CH less3-30 less probable probable CHwere-30 α-oriented. were α-oriented. The chair The conformation chair conformation of the ring of theB with ring equatorial B with equatorial positions positions of H-9 and of were3 α-oriented.conformerconformer The (Figurechair (Figure conformation S67). S67). In addition, In ofaddition, the wering couldwe B with could conclude equatorial conclude that positions that the presencethe presenceof H-9 of and 3-acetoxy of 3-acetoxy or or H-9CH3-30 and corresponded CH3-30 corresponded to the long-range to the long-range COSY correlation COSY correlation between betweenH-9 and H-9Hβ-7 and (Figures Hβ-7 CH3-30 corresponded3-oxo3-oxo functions functions to the in long-rangethe in structuresthe structures COSY of the correlationof cotherresponding corresponding between compounds H-9 compounds and H insignificantlyβ-7 (Figuresinsignificantly affects affects (Figure5 and S25)5 and together Figure S25) with together ROESY with correlations ROESY correlations H-5/Ha-11 H-5/Hand Hαa-7/H-11 andb-11. HTakingα-7/H binto-11. 5 and S25)the together shapethe shape of with their of their ROESYECD ECD curves. correlations curves. According According H-5/H to obtained ato-11 obtained and new Hα new-7/H data bdata-11. we poseweTaking pose the intosamethe same 15R ,2315RS,23 S Takingconsideration into consideration the relative the stereochemistry relative stereochemistry of theof compound the compound 3 along3 along with with above Molecules 2021, 26, x FOR PEER REVIEW considerationstereochemistry stereochemistrythe relative for stereochemistry globostelletinfor globostelletin K (Figureof K the(Figure S69).compound S69). 3 of3 12 along with above mentioned absolute stereochemistry stereochemistry of of the the C C3030 congenerscongeners 1 and1 and 2 as2 aswell well as the as the fact fact of co- of mentioned absolute stereochemistry of the C30 congeners 1 and 2 as well as the fact of co- co-isolation of cyclobutastellettolides A and B [14] with the same absolute configurations isolation of cyclobutastellettolides A and B [14] with the same absolute configurations we we suggested the 5S, 8R, 9R, 10R absolute stereochemistry of stellettin S (3). suggested the 5S,8R,9R,10R absolute stereochemistry of stellettin S (3). The detailed analysis of 2D spectra (COSY, HSQC, HMBC etc.) of 1 supported the main structure (Figures 2 and S6–S9). The signals of methyl group at δH 2.06, s; δC 21.2 and acetate carbonyl at δC 171.0, together with the HMBC correlation of axial proton H-3 at δH 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 δC 80.8 instead of ketone signal at δC 219.2 in 13C NMR spectrum of globostelletin K also demonstrated the 3-acetoxy-tricyclic core in 1, while the 3β-orientation of acetoxy group was confirmed by strong correlations of H-3/H-5 and CH3- 28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement with the signal of CH3-18 at δH 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. FigureFigure 3. Comparison 3. Comparison of experimental of experimental and andtheoretical theoretical ECD ECD spectra spectra of stellettin of stellettin Q (1 ).Q (1). O O 1 13 O TableTable 1. 1H 1. and H and13C NMR C NMR data data of 1 (700of 1 (700and and176 MHz)176 MHz) and and2 (500 2 (500and and126 MHz)126 MHz) in CDCl in CDCl3. 3. H H H H COOH H H H H H O O O O COOH H H 1 2 O

Figure 2. Selected COSY ( ), FigureHMBC 6. ( Selected) and ROESY ROESY ( ( ))) correlationscorrelationscorrelations ofofof 313. .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 15R,23S absolute configuration, providing 13Z,24E geometry and trans−syn−trans-fused tricyclic moiety with 3β-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 15R,23S stereochemistry for globostelletin K (Figure S69).

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

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

Molecules 2021, 26, 678 7 of 13

1 13 Table 2. H and C NMR data (700 and 176 MHz) of 3–6 in CDCl3.

3 4 5 6 No. 1 δH mult (J in Hz) δC δH mult (J in Hz) δC δH mult (J in Hz) δC δH mult (J in Hz) δC 1α 1.76, m 1.33, m 1.78, m 1.79, m 35.9 34.3 35.5 35.6 1β 1.57, ddd (13.3, 6.3, 3.7) 1.28, m 1.57, ddd (13.5, 6.0, 4.0) 1.60, m 2α 2.33, m 1.72, m 2.31, m 2.32, dt (15.4, 4.5) 34.8 23.3 34,8 34.8 2β 2.70, ddd (15.6, 12.3, 6.4) 1.68, m 2.65, ddd (15.4, 12.6, 6.1) 2.66, ddd (15.4, 12.6, 6.1) 3 216.5 α: 4.44, dd (11.2, 5.1) 80.3 216.1 216.0 4 47.7 37.6 47.5 47.5 5α 1.33, dd (12.4, 2.7) 47.5 1.01, dd (11.9; 2.7) 46.2 1.40, dd (12.6; 3.0) 46.7 1.39, dd (12.7; 3.0) 46.8 6α 1.47, dq (13.6, 3.2) 1.65, m 1.46, m 1.47, m 21.1 17.9 20.4 20.5 6β 1.83, qd (13.2, 3.4) 1.47, m 1.79, m 1.78, m 7α 1.25, td (13.2, 3.7) 1.51, m 1.11, m 1.08, m 36.5 30.0 31.2 31.5 7β 1.74, m 1.59, m 2.20, br d (15.1) 2.23, br d (14.2) 8 34.1 52.5 44.5 44.5 9β 2.22, t (5.1) 51.2 2.31 br d (8.0) 49.4 2.78 br t (5.0) 48.3 2.74 br t (4.6) 48.3 10 38.5 38.6 38.5 38.4 11a 2.40, dd (18.1, 5.8) 2.58, dd (18.9, 7.9) 2.41, dd (17.7, 5.3) 2.48, dd (18.1, 5.4) 31.1 34.1 31.1 30.5 11b 2.33, dd (17.7, 4.8) 1.70, br d (18.9) 2.30, m 2.38, dd (18.1, 4.8) 12 178.8 175.2 2 173.7 177.6 3 13 88.1 213.0 183.8 178.0 3 14 77.8 2.12, s 24.7 15–17 18 1.80, s 3.7 19 1.62, s 23.3 1.27, s 24.6 1.24, s 20.8 1.14, s 20.7 20–27 28 1.08, s 25.9 0.89, s 27.9 1.07, s 25.7 1.07, s 25.7 29 1.06, s 21.6 0.87, s 16.2 1.00, s 21.5 0.99, s 21.5 30 1.25, s 30.8 1.31,s 24.6 1.17, s 27.8 1.12, s 27.7 170.9 OAc 2.05, s 21.2 4.23, dq (10.9, 7.1), H 4.15, q (7.1), 2H, 60.8 60.6 OEt 4.13, dq (10.9, 7.1), H 1.26, t (7.1), 3H 14.1 14.0 1.31, t (7.1), 3H 1 Assignments were made with the aid of HSQC, HMBC and ROESY data. 2 The values were found from HMBC experiment. 3 These signals could be interchanged. Molecules 2021, 26, 678 8 of 13

Stellettin T (4) with the molecular formula C20H32O5 seemed to be another isomalabaricane-type derivative. The ispection of NMR data (Table2; Figure5 and Figures S29–S33) revealed the same type of 9-carboxymethyl substituted bicyclic core as was deduced for compound 3. It contains 3β-acetoxy group, confirmed with the signals of CH-3 (δH 4.44, dd (11.2, 5.1); δC 80.3), methyl of acetate group (δH 2.05, s; δC 21.2) and 13 acetate carbon (δC 170.9). According to the C NMR spectrum and molecular formula, compound 4 has one carbonyl less side chain then known globostelletin A [13]. Based Molecules 2021, 26, x FOR PEER REVIEW 8 of 12 on this data and HMBC correlations from CH3-14 (δH 2.12, s) and CH3-30 (δH 1.31, s) to C-13 (δC 213.0), the acetyl was connected with C-8 (δC 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 configurationsspectral analysis at (Figure C-5, C-8, S43). C-9 Correlation and C-10 identical between to H-9 those (δ ofH 2.78, co-isolated br t (5.0)) isomalabaricanes. and CH3-19 (δH 1.24, Thes) indicated structures their of stellettinsβ-orientation. U ( 5Meanwhile,) and V (6) a corresponded ROESY correlation to the between same C19 H-5H30 O(δ5H molecular1.40, dd (12.6, formula 3.0))/H deduceda-11 (δH from2.41, dd HRESIMS (17.7, 5.3)) (Figures and H S36b-11 and(δH 2.30, S45). m)/CH In comparison3-30 (δH 1.17, with s) MoleculesMolecules 2021 2021, 26,, x26 FOR, x FOR PEER PEER REVIEW REVIEW 3 of3 12of 12 co-isolatedconfirmed metabolites,the α-orientation the spectral of H-5, data -CH of compounds2-COOEt and5 andCH63-30.revealed The above-mentioned bicyclic core with ketoresults group were at in C-3, agreement gem-dimethyl with the group spatial at structure C-4 and two of isomalabaricane angular methyls derivatives. at C-8 and C-10 (TableCompound2). Additionally, 6 was an1H- isomer and 13 ofC-NMR compound spectra 5, ofdiffered compound by the5 NMR(Figures signals S37 and(Figures S38) demonstratedS46 and S47) ofThe signals carboxylicThe detailed detailed of two carbonsanalysis carbonyls analysis ( δofC 177.62D (ofδC 2Dspec173.7 and spectra 178.0), and tra(COSY, 183.8)(COSY, methyl HSQC, and HSQC, C-19 one HMBC ethoxy(δ HMBCH 1.14, etc.) group s), etc.) ofmethylene ( 1δof Hsupported 14.15, supported the the qCH (7.1);2-11δ (CmainδH60.8 2.48,main structure and dd structure δ(18.1,H 1.26, (Figures 5.4) (Figures t (7.1); and 2 and2.38,δ 2C and14.1). S6–S9). dd S6–S9). (18.1, HMBC The 4.8))The signals experiment signalsand ofethoxy methyl of methyl (Figure group group 7group (andδH at 4.23, δ FigureatH 2.06,δ dqH 2.06, (10.9, S41)s; δ s;C 21.2 δC 21.2 and and allowed7.1); 4.13, toacetate dq placeacetate (10.9, carbonyl the carbonyl7.1) carboxy and at δ1.31,at groupC 171.0, δC 171.0,t (7.1)). at together C-8 together The and with ethylkey with HMBCthe ester the HMBC at HMBCcorrelations C-11 corr on correlation theelation (Figure basis of axialofof 7)congruousaxial satisfiedproton proton H-3 H-3 at δ atH δH correlationsthe proposed4.54,4.54, from ddstructure dd(11.7, methylenes (11.7, 5.2) of 5.2)6 to. However, that to –CH that carbonyl,2-CH carbonyl, since3 (δ revealed Hthe 4.15,revealed values q the (7.1), of the O-acetyl carboxyl 2H)O-acetyl and substitution carbons CHsubstitution2-11 shifts ( δatH C-3 2.41,atfor C-3 in6 ddare the in the ring ring A. A. 13 (17.7,close, 5.3)distinguishingMoreover, andMoreover, 2.30, m)the their tothesignal carboxyl signal correlations of C-3of C-12 C-3 at ( δatandC .80.8δ 173.7)C direct80.8 instead andinstead ester also of positioningketoneof from ketone methyl signal signal without CH at 3δat-30C 219.2δ (Cdataδ 219.2H 1.17, infor 13in C NMRC NMR s)isomer to carboxyl 5spectrum broughtspectrum C-13 someof (δ C globostelletinof183.8). globostelletinuncertainty. The relative K Toalso K avoidalso demon stereochemistry demon futurestratedstrated difficulties the of the 3-acetoxy-tricyclic5 was 3-acetoxy-tricyclic with determined structurally bycore ROESYcorerelated in 1 in, while 1 , while the the spectralesters we analysis3 βcalculated-orientation3β-orientation (Figure carbon of S43). acetoxy of chemacetoxy Correlation groupical group shift was between valueswas confirmed confirmed for H-9 two (byδH isomersstrongby2.78, strong br correlations t5 (5.0))correlationsand 6 and (Figure of CH H-3/H-5 of3 -19 S66).H-3/H-5 (δ HIt and and CH CH3- 3- 1.24,was shown, s) indicated28 observed28 that observed theoretical their inβ the-orientation.in the ROESY δC ROESYC-13 spectrum.(5 Meanwhile, )spectrum. = 192.7 The and The a13 δ ROESYCZ 13C-12 geometryZ geometry ( correlation5) = 182.4 in 1in wasgave 1 between was in the agreementin Δ agreement H-5δC(13-12) (δH = with with the the 1.40,10.3 ddppm (12.6,signal signalclose 3.0))/H of toCHof experimentalaCH3-11-183 -18 (δatH δat2.41,H δ1.79,H dd value1.79, (17.7,s and s Δand 5.3))δ C(13-12)its its andROESY =ROESY H10.1b-11 correlation (ppm,δcorrelationH 2.30, while m)/CHwith withtheoretical CH3 -30CH3-30. (δ3-30.H As 1.17,and Aswell well as asE E s)experimental confirmedconfigurationconfiguration theΔδC(13-12)α-orientation for of compound24(25)-doubleof 24(25)-double of H-5, 6 were -CH bond 2 bondof-COOEt 0.4was ppmwas found and found(clcd CH from δ 3Cfrom-30. C-13 the The the(W6)-path above-mentioned=W 183.2,-path COSY δ COSYC C-12 correlation (correlation6) of of results= 182.8). were protonsprotons in agreement H-24/CH H-24/CH3 with-27.3-27. the spatial structure of isomalabaricane derivatives.

O O O O O O H H H H H H H H COOHCOOH H H H H H H H H H H O O O O O O O O COOHCOOH H H H H 1 1 2 2 O O

FigureFigureFigure 2. 7. SelectedSelected 2. Selected COSY COSY COSY ( ( ())), and HMBC), HMBC HMBC ( ( () and)) ) correlationsand correlationsROESY ROESY ( of( of) 5correlations 5and) and correlations6 .6. of 1 of and 1 and 2. 2.

CompoundROESY correlationsTheThe6 1wasH- 1 H-and an and isomerof13C-NMR 6 13 supportedC-NMR of compoundsignals signals the of relative theof5, differedthe side side stereoch chain bychain theofemistry 1of NMR as 1 wellas signals similarlywell as theas (Figures the form to form that of S46 ECDof ECD curve curve and S47) of carboxylic carbons (δ 177.6 and 178.0), methyl C-19 (δ 1.14, s), methylene compound(Figure (Figure5. In S10)fact, S10) werewe weredetected analogous analogous Cexpe tocted thoseto NOEthose of interactionsglobostelletinof globostelletin H-9 K [15] (KδH [15] 2.74, (Figure (Figure br t S57)(4.6))/CH S57) suggesting suggesting3- the the CH -11 (δ 2.48, dd (18.1, 5.4) and 2.38, dd (18.1, 4.8)) and ethoxy group (δ 4.23, dq (10.9, 19 (2δH 1.14,sameH sames); stereochemistryH-5 stereochemistry (δH 1.39, dd (12.7;of theof 3.0))/Hthe side side chain.a-11 chain. ( δThisH 2.48, This assignment dd assignment (18.1, 5.4)) was was andin Hain H good ba-11 good (agreementδH agreement2.38, with with 7.1); 4.13, dq (10.9, 7.1) and 1.31, t (7.1)). The key HMBC correlations (Figure7) satisﬁed dd (18.1,the 4.8))/CHthe computational computational3-30 (δH ECD1.12, ECD results s). results Therefore, performed performed derivatives using using density density5 and functional functional6 possess theory theorythe (DFT) same (DFT) with with the the the proposed structure of 6. However, since the values of carboxyl carbons shifts for 6 are stereochemistrynonlocalnonlocal as exchange-correlation other exchange-correlation co-isolated isomalabaricanes. functional functional B3 LYPB3 AlthoughLYP [16], [16], the compoundsthe polarization polarization 5 continuumand continuum 6 are model model close, distinguishing their correlations and direct ester positioning without data for isomer rather artificial(PCM)(PCM) [17] products [17] and and split-valence derivedsplit-valence during basis basis setsEt OHsets 6-31G(d), 6-31G(d),extraction, implemented implemented the isolated in the in pair the Gaussian Gaussianof esters 16 package16 package 5 brought some uncertainty. To avoid future difﬁculties with structurally related esters we allowed ofto programsreliablyof programs establish [18] [18] (Figure the(Figure position S66). S66). The of The the15 Rether15,23RS,23 groupabsoluteS absolute based configuration, configuration, on the chemical providing providing shifts 13 Z13,24ZE,24 E calculated carbon chemical shift values for two isomers 5 and 6 (Figure S66). It was shown, of C-12 andgeometrygeometry C-13. and and trans trans−syn−syn−trans−trans-fused-fused tricyclic tricyclic moiety moiety with with 3β-oriented 3β-oriented acetoxy acetoxy group group fully fully that theoretical δ C-13 (5) = 192.7 and δ C-12 (5) = 182.4 gave the ∆δ = 10.3 ppm Bothsatisfies compoundssatisfies Cthe the similarity were similarity supposed of theof the experi to experiC bemental themental half-ester and and theoretical derivativestheoretical ECD ECDofC(13-12) spectrathe spectra hypothetical of 1of (Figure 1 (Figure 3). 3).In In close to experimental value ∆δ = 10.1 ppm, while theoretical and experimental dicarboxylicdetailes,detailes, acid. statistically The statistically anhydrous avereged C(13-12)avereged form curve of curve the (Figure acid(Figure was3) follows 3) reported follows the theshbyape shRaviape of theetof al.the experimental [5] experimental as a one, one, ∆δ for compound 6 were of 0.4 ppm (clcd δ C-13 (6) = 183.2, δ C-12 (6) = 182.8). productC(13-12) althoughof ozonolysisalthough even even of moreisomal more closeabaricane close coincidence coincidence precursor Cwas [22,23]. was indicated indicated Moreover, for C for theoreticallyRavi theoretically et al. obtained less less probable probable ROESY correlations of 6 supported the relative stereochemistry similarly to that of dimethylconformer andconformer monomethyl (Figure (Figure S67).esters S67). In of addition,In the addition, acid weand wecould did could not conclude pointconclude the that placethat the the presenceof presenceesterification of 3-acetoxyof 3-acetoxy or or compound 5. In fact, we detected expected NOE interactions H-9 (δ 2.74, br t (4.6))/CH - in the case3-oxo of3-oxo the functions latter.functions in thein the structures structures of theof the corresponding corresponding compounds Hcompounds insignificantly insignificantly3 affects affects 19 (δH 1.14, s); H-5 (δH 1.39, dd (12.7; 3.0))/Ha-11 (δH 2.48, dd (18.1, 5.4)) and H -11 (δH Amongthethe shape isolated shape of their ofnew their ECD compounds ECD curves. curves. According 1– According6, we find to obtained tostellettin obtained new S new(3 data) the data we most wepose poseintriguing, theb the same same 15R 15,23RS,23 S 2.38, dd (18.1, 4.8))/CH3-30 (δH 1.12, s). Therefore, derivatives 5 and 6 possess the same stereochemistrystereochemistry for forglobostelletin globostelletin K (Figure K (Figure S69). S69). stereochemistrysince occurrences as of other acetylene-containing co-isolated isomalabaricanes. isoprenoids are Although rare and compounds not so far reported5 and 6 are 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

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

1 13 TableTable 1. 1 H1. andH and 13C NMRC NMR data data of 1 of (700 1 (700 and and 176 176MHz) MHz) and and 2 (500 2 (500 and and 126 126MHz) MHz) in CDCl in CDCl3. 3.

Molecules 2021, 26, 678 9 of 13

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 15R,23S configuration for globostelletin M instead of previously reported 15S,23S [15] as well as 15S,23R stereochemistry for globostelletin N instead of 15R,23R [15].

3. Materials and Methods 3.1. General Experimental Procedures Optical rotations were measured on Perkin-Elmer 343 digital polarimeter (Perkin Elmer, Waltham, MA, USA). UV-spectra were registered on a Shimadzu UV-1601PC spec- trophotometer (Shimadzu Corporation, Kyoto, Japan). ECD spectra were obtained on a Chirascan plus instrument (Applied Photophysics Ltd., Leatherhead, UK). 1H-NMR (500.13 MHz, 700.13 MHz) and 13C-NMR (125.75 MHz, 176.04 MHz) spectra were recorded in CDCl3 on Bruker Avance III HD 500 and Bruker Avance III 700 spectrometers (Bruker BioSpin, Bremen, Germany). The 1H- and 13C-NMR chemical shifts were referenced to the solvent peaks at δH 7.26 and δC 77.0 for CDCl3. HRESIMS analyses were performed using a Bruker Impact II Q-TOF mass spectrometer (Bruker). The operating parameters for ESI were as follows: a capillary voltage of 3.5 kV, nebulization witH-Nitrogen at 0.8 bar, dry gas ﬂow of 7 L/min at a temperature of 200 ◦C. The mass spectra were recorded within m/z mass range of 100–1500. The instrument was operated using the otofControl (ver. 4.1, Bruker Daltonics) and data were analyzed using the DataAnalysis Software (ver. 4.4, Bruker Daltonics). Column chromatography was performed on Sephadex LH-20 (25–100 µm, Pharmacia Fine Chemicals AB, Uppsala, Sweden), silica gel (KSK, 50–160 mesh, Sorbﬁl, Krasnodar, Russia) and YMC ODS-A (12 nm, S-75 um, YMC Co., Ishikawa, Japan). HPLC were carried out using an Agilent 1100 Series chromatograph equipped with a differential refractometer (Agilent Technologies, Santa Clara, CA, USA). The reversed-phase columns YMC-Pack ODS-A (YMC Co., Ishikawa, Japan, 10 mm × 250 mm, 5 µm and 4.6 mm × 250 mm, 5 µm) and Discovery HS F5-5 (SUPELCO Analytical, Bellfonte, PA, USA, 10 mm Molecules 2021, 26, 678 10 of 13

× 250 mm, 5 µm) were used for HPLC. Yields are based on dry weight (212.1 g) of the sponge sample.

3.2. Animal Material The Stelletta sp. sponge sample (wet weight 1.3 kg) was collected by SCUBA diving at the depth of 7–12 m near Cham Island (15◦54.30 N, 108◦31.90 E) in the Vietnamese waters of the South China Sea during the 038-th cruise of R/V “Academik Oparin” in May 2010. The species was identiﬁed and described [14] by Dr. Boris B. Grebnev from G. B. Elyakov Paciﬁc Institute of Bioorganic Chemistry, FEB RAS (PIBOC, Vladivostok, Russia). A voucher specimen (PIBOC O38-301) has been deposited at the collection of marine invertebrates in PIBOC.

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 25 −4 Stellettin Q (1): Yellow oil; [α]D –65.0 (c 0.1, CHCl3); ECD (c 8.6 × 10 M, EtOH) 1 13 λmax (∆ε) 195 (4.15), 229 (−27.92), 262 (12.78), 355 (−1.75) nm; H- and C-NMR data − (CDCl3), Table1; HRESIMS m/z 523.3065 [M–H] (calcd for C32H43O6 523.3065). 25 −3 Stellettin R (2): Yellow oil; [α]D –24.0 (c 0.2, CHCl3); ECD (c 1.3 × 10 M, EtOH) 1 λmax (∆ε) 195 (4.06), 229 (−5.33), 252 (1.46), 273 (−0.48), 295 (0.47), 353 (−1.20) nm; H- 13 − and C- NMR data (CDCl3), Table1; HRESIMS m/z 523.3068 [M–H] (calcd for C32H43O6 523.3065). Molecules 2021, 26, 678 11 of 13

25 −3 Stellettin S (3): Slightly yellow oil; [α]D + 31.5 (c 0.2, CHCl3); ECD (c 5.3 × 10 1 13 M, EtOH) λmax (∆ε) 289 (0.34), 321 (−0.14) nm; H- and C-NMR data (CDCl3), Table2; − HRESIMS m/z 303.1966 [M–H] (calcd for C19H27O3 303.1966). 25 −3 Stellettin T (4): Slightly yellow oil; [α]D –22.0 (c 0.1, CHCl3); ECD (c 3.4 × 10 M, 1 13 EtOH) λmax (∆ε) 208 (−0.98), 249 (0.58), 280 (−0.27), 321 (−0.29) nm; H- and C-NMR − data (CDCl3), Table2; HRESIMS m/z 351.2176 [M–H] (calcd for C20H31O5 351.2177). 25 −3 Stellettin U (5): Slightly yellow oil; [α]D 0.0 (c 0.2, CHCl3); ECD (c 5.7 × 10 M, 1 EtOH) λmax (∆ε) 197 (−0.73), 232 (0.27), 261 (0.45), 295 (0.01), 321 (−0.34) nm; H- and 13 − C-NMR data (CDCl3), Table2; HRESIMS m/z 337.2022 [M–H] (calcd for C19H29O5 337.2020). 25 −3 Stellettin V (6): Slightly yellow oil; [α]D + 32.9 (c 0.17, CHCl3); ECD (c 7.7 × 10 M, EtOH) λmax (∆ε) 210 (−0.04), 220 (0.08), 238 (−0.23), 269 (0.38), 291 (0.26), 330 (0.13), 1 13 − 357 (−0.09) nm; H- and C-NMR data (CDCl3), Table2; HRESIMS m/z 337.2025 [M–H] (calcd for C19H29O5 337.2020). 25 Globostelletin N (Figure1): Slightly yellow oil; [ α]D + 17.8 (c 0.23, MeOH); ECD (c 1.2 −3 × 10 M, EtOH) λmax (∆ε) 196 (−4.18), 228 (9.44), 256 (−1.72), 289 (0.97), 340 (−1.97) nm; 1 13 H- and C-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 com- bination 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: 1 13 HRESIMS, H- and C-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: 1 13 HRESIMS, H- and C-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 1 13 S54–S57: HRESIMS, H- and C-NMR spectra (CDCl3) and ECD spectrum (EtOH) of globostelletin 1 13 K, respectively, Figures S58–S61: HRESIMS, H- and C-NMR spectra (CDCl3) and ECD spectrum 1 13 (EtOH) of globostelletin M, Figures S62–S65: HRESIMS, H and C 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 Molecules 2021, 26, 678 12 of 13

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.

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