Cytotoxic Furanoditerpenes from the Sponge Spongia tubulifera Collected in the Mexican Caribbean

Two new spongian furanoditerpenes, 3β-hydroxyspongia-13(16),14-dien-2-one (1) and 19-dehydroxy-spongian diterpene 17 (2), along with five known terpenes, the spongian furanoditerpenes 9-nor-3-hydroxyspongia-3,13(16),14-trien-2-one (3), 3β,19 dihydroxyspongia-13(16),14-dien-2-one (epispongiadiol) (4) and spongian diterpene 17 (5), the furanoditerpene ambliol C (6), and the sesterterpene scalarin (7), were isolated from the methanolic extract of the sponge Spongia tubulifera, collected in the Mexican Caribbean. The planar structures of the new compounds were elucidated by 1D/2D NMR and IR spectroscopic analysis, high resolution electrospray mass spectrometry (HRESIMS), and comparison of their spectral data with those reported in the literature. Absolute configurations were determined by comparison of the experimental electronic circular dichroism (ECD) spectrum with those calculated by time-dependent density functional theory (TDDFT). Compounds 1, 4, and 6 displayed weak cytotoxic activity against different human tumour cell lines.

In our continuing investigations of diterpenes from marine organisms [8,9], and in particular from marine sponges [10], we have focused our attention on the sponge Spongia tubulifera, collected in the Mexican Caribbean, because of the cytotoxic activity found in its methanolic extract. To the best of our knowledge, the only previous reports of S. tubulifera were a comparative study of the fatty acids composition of specimens of this sponge collected at Ahogado Reef near La Parguera, Puerto Rico [11] and the assays of the antimicrobial activity against Staphylococcus aureus and Candida albicans of the organic extracts of specimens collected at Urabá Gulf reefs in the Colombian Caribbean [12].

Results and Discussion
Specimens of the sponge S. tubulifera, collected by hand and scuba diving off the coast of the Mexican Caribbean, were extracted several times with CH 3 OH/CH 2 Cl 2 to give an extract which showed cytotoxic activity. The organic extract was subsequently partitioned between H 2 O/CH 2 Cl 2 , and the CH 2 Cl 2 portion was further fractionated into hexane, CH 2 Cl 2 , and aqueous methanolic fractions. The hexane fraction was submitted to silica gel flash chromatography using a gradient mixture of hexane and EtOAc to yield enriched terpene fractions that were then submitted repeatedly to reversed-phase HPLC separation (H 2 O/CH 3 OH mixtures) to yield 1-3 and 6. The CH 2 Cl 2 fraction was fractionated by solid phase extraction (SPE) with a RP-18 column using a stepped gradient from H 2 O, CH 3 OH, and CH 2 Cl 2 to yield enriched terpene fractions that were separated by RP-HPLC using H 2 O/CH 3 OH mixtures to afford 4, 5, and 7 ( Figure 1).
Compound 1 was obtained as a colorless white powder. The molecular formula of 1 was determined on the basis of the M +. peak at m/z 316.2014, observed in its HREIM spectrum (calculated for C 20 H 28 O 3 , 316.2038, 7 degrees of unsaturation) and from its 13 C NMR spectrum. Its IR spectrum displayed signals at 3500 and 1745 cm −1 , suggesting the presence of a hydroxyl group and a ketone carbonyl functionality, respectively.
Mar. Drugs 2019, 17, x FOR PEER REVIEW 2 of 10 five known terpenes, 3-7, and we evaluate their cytotoxic activity against a panel of five human tumour cell lines.

Results and Discussion
Specimens of the sponge S. tubulifera, collected by hand and scuba diving off the coast of the Mexican Caribbean, were extracted several times with CH3OH/CH2Cl2 to give an extract which showed cytotoxic activity. The organic extract was subsequently partitioned between H2O/CH2Cl2, and the CH2Cl2 portion was further fractionated into hexane, CH2Cl2, and aqueous methanolic fractions. The hexane fraction was submitted to silica gel flash chromatography using a gradient mixture of hexane and EtOAc to yield enriched terpene fractions that were then submitted repeatedly to reversed-phase HPLC separation (H2O/CH3OH mixtures) to yield 1-3 and 6. The CH2Cl2 fraction was fractionated by solid phase extraction (SPE) with a RP-18 column using a stepped gradient from H2O, CH3OH, and CH2Cl2 to yield enriched terpene fractions that were separated by RP-HPLC using H2O/CH3OH mixtures to afford 4, 5, and 7 ( Figure 1).
Compound 1 was obtained as a colorless white powder. The molecular formula of 1 was determined on the basis of the M +. peak at m/z 316.2014, observed in its HREIM spectrum (calculated for C20H28O3, 316.2038, 7 degrees of unsaturation) and from its 13 C NMR spectrum. Its IR spectrum displayed signals at 3500 and 1745 cm −1 , suggesting the presence of a hydroxyl group and a ketone carbonyl functionality, respectively.     Figure 2, indicated that 1 has a similar structure to 3α-hydroxyspongia-13(16),14-dien-2-one isolated from an unidentified Spongia collected in Australia [13]. The differences of the proton and carbon chemical shifts at C-3, e.g., δ H /δ C 3.90 (d, J = 1.5 Hz)/ 83.1 in 1 instead of δ H /δ C 4.36 (d, J = 1.5 Hz)/ 80.1 in 3α-hydroxyspongia-13(16),14-dien-2-one, suggested that they differed only in the stereochemistry at C-3, and thus, 1 must be its 3β isomer. The NOESY correlations from H-3 at δ H 3.90 to H-5 at δ H 1.62 and H-18 at δ H 1.21 indicated that these protons were in the same face of the molecule, confirming the β-orientation of the hydroxyl group at C-3. The relative configuration of the remaining stereogenic centers in 1 was also confirmed by its NOESY correlations ( Figure 2). These data indicated that 1 is a new spongian furanoditerpene derivative with a 3β-hydroxyspongia-13(16),14-dien-2-one structure.  Figure 2, indicated that 1 has a similar structure to 3α-hydroxyspongia-13(16),14-dien-2-one isolated from an unidentified Spongia collected in Australia [13]. The differences of the proton and carbon chemical shifts at C-3, e.g., δH/δC 3.90 (d, J = 1.5 Hz)/ 83.1 in 1 instead of δH/δC 4.36 (d, J = 1.5 Hz)/ 80.1 in 3α-hydroxyspongia-13(16),14-dien-2one, suggested that they differed only in the stereochemistry at C-3, and thus, 1 must be its 3β isomer. The NOESY correlations from H-3 at δH 3.90 to H-5 at δH 1.62 and H-18 at δH 1.21 indicated that these protons were in the same face of the molecule, confirming the β-orientation of the hydroxyl group at C-3. The relative configuration of the remaining stereogenic centers in 1 was also confirmed by its NOESY correlations ( Figure 2). These data indicated that 1 is a new spongian furanoditerpene derivative with a 3β-hydroxyspongia-13(16),14-dien-2-one structure. The absolute configurations of the stereogenic carbons of 1 were determined by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by timedependent density functional theory (TDDFT) calculations. Overall, the two possible enantiomers for 1, (3R,5R,8R,9R,10R)-1 and (3S,5S,8S,9S,10S)-1, were initially submitted to a conformational search with the Maestro Suite (Schrödinger). Four conformers were found within a 10.0 kcal/mol energy threshold from global minimum. All these conformers were geometrically optimized by a density functional theory (DFT) method at the HSEH1PBE/cc-pVDZ functions (see computational details in the experimental section). The resulting ECD spectra were combined by Boltzmann weighting to give a composite spectrum for each enantiomer. Comparison of the experimental and calculated ECD spectra for 1 showed excellent agreement with the (3R,5R,8R,9R,10R)-1 enantiomer ( Figure 3). Thus, the absolute configurations of C-3, C-5, C-8, C-9, and C-10 were determined as 3R, 5R, 8R, 9R, and 10R, respectively. The absolute configurations of the stereogenic carbons of 1 were determined by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by time-dependent density functional theory (TDDFT) calculations. Overall, the two possible enantiomers for 1, (3R,5R,8R,9R,10R)-1 and (3S,5S,8S,9S,10S)-1, were initially submitted to a conformational search with the Maestro Suite (Schrödinger). Four conformers were found within a 10.0 kcal/mol energy threshold from global minimum. All these conformers were geometrically optimized by a density functional theory (DFT) method at the HSEH1PBE/cc-pVDZ functions (see computational details in the experimental section). The resulting ECD spectra were combined by Boltzmann weighting to give a composite spectrum for each enantiomer. Comparison of the experimental and calculated ECD spectra for 1 showed excellent agreement with the (3R,5R,8R,9R,10R)-1 enantiomer ( Figure 3). Thus, the absolute configurations of C-3, C-5, C-8, C-9, and C-10 were determined as 3R, 5R, 8R, 9R, and 10R, respectively.   Table S2).   Table S2).
The 20 carbon signals observed in the 13 C NMR spectrum of 2 along with the presence of two α-furan proton signals (δ H 7.09 and 7.06) and four tertiary methyl groups (δ H 1.28, 1.23, 1.22, and 1. 16) in its 1 H NMR spectrum were indicative of a spongian furanoditerpene structure. The planar structure of 2 was established by a combination of 1D and 2D NMR spectroscopy. Comparison of the NMR data of 2 with those of 1 (see Table 1) revealed that they shared the same framework at the B, C, and D rings but differed in the A-ring. Signals in the 13 C NMR spectrum of 2 for the conjugated ketone carbonyl group at δ C 201.2 (C-3) and two sp 2 carbons, the non-protonated carbon at δ C 144.3 (C-2) and the methine carbon at δ C 128.3 (C-1), were consistent with the presence of a conjugated α,β-unsaturated ketone moiety.
The key 1 H-13 C long range correlations between the olefinic proton at δ H 6.54 (H-1) and the olefinic carbon at δ C 144.3 (C-2), the ketone carbonyl carbon at δ C 201.2 (C-3) and the carbon at δ C 54.5 (C-5), along with the HMBC correlation from the methyl singlet at δ H 1.22 (H-20) to the olefinic carbon at δ C 128.3 (C-1) placed the α,β-unsaturated ketone in the A-ring (Figure 2). The exchangeable proton signal at δ H 5.93 was indicative of an enolized α-diketone moiety in the A-ring. The NMR data for this part of the molecule (see Table 1) are in agreement with those observed for other diterpenes containing the same A-ring in the tetracyclic framework such as spongian diterpene 17 (5), previously reported from the nudibranch Doriprismatica (= Glossodoris) atromarginata [14]. The diagnostic HMBC correlations displayed by the α-furan proton signals and the methyl groups Me-17 and Me-18 displayed in Figure 2 confirm 2 as a new spongian furanoditerpene that was named 19-dehydroxy-spongian diterpene 17.
As in 1, the absolute configurations of the stereogenic carbons of 2 were determined by comparison of the experimental to those generated by TDDFT on the two possible enantiomers. The two possible enantiomers for 2, (5R,8R,9R,10R)-2 and (5S,8S,9S,10S)-2, were initially submitted to a conformation search with the Maestro Suite (Schrödinger). Thus, 4 conformers were found within a 10.0 Kcal/mol energy threshold from a global minimum. All these conformers were geometrically optimized by density functional theory method at the HSEH1PBE/cc-pVDZ function (see computational details in experimental section). As shown in Figure 4, the calculated ECD spectra for the (5R,8R,9R,10R)-2 and its experimental data were almost identical. Thus, the absolute configurations of C-5, C-8, C-9, and C-10 of 2 were determined as 5R, 8R, 9R, and 10R, respectively. The IR spectrum of 2 shows absorptions from the hydroxyl (3505 cm −1 ) and a conjugated ketone carbonyl (1650 cm −1 ) groups. The 20 carbon signals observed in the 13 C NMR spectrum of 2 along with the presence of two αfuran proton signals (δH 7.09 and 7.06) and four tertiary methyl groups (δH 1.28, 1.23, 1.22, and 1.16) in its 1 H NMR spectrum were indicative of a spongian furanoditerpene structure. The planar structure of 2 was established by a combination of 1D and 2D NMR spectroscopy. Comparison of the NMR data of 2 with those of 1 (see Table 1) revealed that they shared the same framework at the B, C, and D rings but differed in the A-ring. Signals in the 13 C NMR spectrum of 2 for the conjugated ketone carbonyl group at δC 201.2 (C-3) and two sp 2 carbons, the non-protonated carbon at δC 144.3 (C-2) and the methine carbon at δC 128.3 (C-1), were consistent with the presence of a conjugated α,βunsaturated ketone moiety.
The key 1 H-13 C long range correlations between the olefinic proton at δH 6.54 (H-1) and the olefinic carbon at δC 144.3 (C-2), the ketone carbonyl carbon at δC 201.2 (C-3) and the carbon at δC 54.5 (C-5), along with the HMBC correlation from the methyl singlet at δH 1.22 (H-20) to the olefinic carbon at δC 128.3 (C-1) placed the α,β-unsaturated ketone in the A-ring (Figure 2). The exchangeable proton signal at δH 5.93 was indicative of an enolized α-diketone moiety in the A-ring. The NMR data for this part of the molecule (see Table 1) are in agreement with those observed for other diterpenes containing the same A-ring in the tetracyclic framework such as spongian diterpene 17 (5), previously reported from the nudibranch Doriprismatica (= Glossodoris) atromarginata [14]. The diagnostic HMBC correlations displayed by the α-furan proton signals and the methyl groups Me-17 and Me-18 displayed in Figure 2 confirm 2 as a new spongian furanoditerpene that was named 19-dehydroxyspongian diterpene 17.

General Experimental Procedures
Optical rotations were measured on a JASCO DIP-1000 polarimeter, with a Na (589 nm) lamp and filter. IR spectra were measured on a FTIR Bruker Vector 22 spectrometer. 1 H, 13 C, and 2D NMR spectra were recorded on a Bruker Avance 500 spectrometer at 500 and 125 MHz, respectively, using CDCl 3 . Low resolution electrospray mass spectrometry (LRESIMS) and high resolution electrospray mass spectrometry (HRESIMS) experiments were performed on the Applied Biosystems QSTAR Elite system. LREIMS and HREIMS were performed on the Mass Spectrometer Thermo MAT95XP. HPLC separations were performed on the Agilent 1100 liquid chromatography system equipped with a solvent degasser, quaternary pump, and diode array detector (Agilent Technologies, Waldbronn, Germany) using a semipreparative reversed phase column Luna C18, 5 µ, 100 Å, 250 × 10 mm. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) were used for TLC analysis, and the spots were visualized under a UV light (254 nm) or by heating the plate pretreated with H 2 SO 4 /H 2 O/AcOH (1:4:20).

Animal Material
The sponge Spongia tubulifera was collected by hand and traditional scuba diving off the coast of the Mexican Caribbean (18 •

Extraction and Isolation
Sliced bodies of S. tubulifera (wet weight, 157.7 g; dry weight, 40.2 g) were exhaustively extracted with CH 3 OH-CH 2 Cl 2 (1:1, 3 × 1.5 L) at 25 • C for 24 h each extraction. The combined extracts were concentrated under reduced pressure to give 12.0 g of a crude residue that was first partitioned between CH 2 Cl 2 /H 2 O (1:1 v/v). The resulting aqueous portion was extracted with n-butanol (200 mL) to yield the n-butanol fraction (1.44 g). The organic phase was concentrated under reduced pressure and partitioned between 10% aqueous CH 3 OH (400 mL) and hexane (2 × 400 mL) to give, after removing the solvent under reduced pressure, 526 mg of the hexane fraction. The H 2 O content (% v/v) of the methanolic fraction was adjusted to 50% aqueous CH 3 OH, and the mixture was extracted with CH 2 Cl 2 (100 mL) to afford, after removing the solvent under reduced pressure, 1.51 g of the CH 2 Cl 2 fraction and 2.38 g of the remaining aqueous methanolic fraction. The hexane fraction (526 mg) was subjected to a flash chromatography column on silica gel using a stepped gradient from hexane to EtOAc to give 14 fractions (FHC1-C14). Separation of the fraction FHC2, eluted with hexane/EtOAc (9:1, 93.9 mg), by RP-HPLC with a mobile phase consisting of an isocratic at 100% CH 3 OH at a flow rate of 2.0 mL/min afforded 6 (13.5 mg; t R = 8.4 min). Separation of the fraction FHC3, eluted with hexane/EtOAc (9:1, 20.0 mg), by RP-HPLC (isocratic 100% CH 3 OH, flow rate 2.0 mL/min) gave 2 (2.6 mg; t R = 9.7 min) and 6 (2.6 mg; t R = 8.9 min). Separation of the fraction FHC4, eluted with hexane/EtOAc (8:2, 9.0 mg), by RP-HPLC with a mobile phase consisting of 5 min gradient from 90% to 95% of CH 3 OH in H 2 O, followed by a 10 min isocratic at 95% of CH 3 OH in H 2 O and, finally, a 5 min gradient from 95% to 100% of CH 3 OH in H 2 O at a flow rate of 2.0 mL/min yielded 2 (2.0 mg; t R = 12.6 min). Separation of the fraction FHC5, eluted with hexane/EtOAc (8:2, 21.0 mg), by RP-HPLC with a mobile phase consisting of 5 min gradient from 90% to 95% of CH 3 OH in H 2 O, followed by a 15 min isocratic at 95% of CH 3 OH in H 2 O and, finally, a 10 min gradient from 95% to 100% of CH 3 OH in H 2 O at a flow rate of 2.0 mL/min afforded 2 (1.7 mg; t R = 13.0 min) and 3 (1.5 mg; t R = 12.0 min). Separation of the fraction FHC7, eluted with hexane/EtOAc (8:2, 27.2 mg), by RP-HPLC (isocratic 100% CH 3 OH, flow rate 2.0 mL/min) yielded 1 (3.0 mg; t R = 16.8 min). Separation of the fraction FHC8, eluted with hexane/EtOAc (8:2, 20.9 mg), by RP-HPLC with a mobile phase consisting of 5 min gradient from 90% to 95% of CH 3 OH in H 2 O followed by a 15 min isocratic at 95% of CH 3 OH in H 2 O and, finally, a 1 min gradient from 95% to 100% of CH 3 OH in H 2 O at a flow rate of 2.0 mL/min afforded 1 (1.8 mg; t R = 11.0 min). The dicloromethane fraction (1.51 g) was subjected to solid phase extraction (SPE) with RP-18 column (Merck KGaA) using a stepped gradient from H 2 O to CH 3 OH and then CH 2 Cl 2 , to give 6 fractions: H 2 O (100%), H 2 O/CH 3 OH (2:1, 1:1, and 1:2), CH 3 OH (100%), and CH 2 Cl 2 100%. The fraction eluted with H 2 O/CH 3 OH (1:2) was submitted to RP-HPLC separation using a mobile phase consisting of 20 min gradient from 50% to 100% of CH 3 OH in H 2 O followed by a 10 min isocratic at 100% of CH 3 OH at a flow rate of 2.0 mL/min to afford 4 (9.2 mg; t R = 10.0 min). Separation of the fraction eluted with CH 3 OH (100%) by RP-HPLC using a mobile phase consisting of 30 min gradient from 80% to 100% of CH 3 OH in H 2 O at a flow rate of 2.0 mL/min afforded 7 (5.0 mg; t R = 26.7 min) and 5 (3.1 mg; t R = 29.7 min).

Conclusions
In summary, two new spongian furanoditerpenes, 1 and 2, together with five known terpenes, four furanoditerpenes 3-6 and one sesterterpene 7, were isolated from sponge Spongia tubulifera collected in the Mexican Caribbean as its metabolic components. The absolute configurations of the new compounds 1 and 2 were determined by comparison of experimental and calculated ECD spectra. Compounds 1, 4, and 6 displayed weak cytotoxic activity against a panel of five human tumor cell lines. This work represents the first chemical study of the secondary metabolites from S. tubulifera.