Isolation of Scalarane-Type Sesterterpenoids from the Marine Sponge Dysidea sp. and Stereochemical Reassignment of 12-epi-Phyllactone D/E

The chemical investigation of the marine sponge Dysidea sp., which was collected from Bohol province in the Philippines, resulted in the identification of 15 new scalarane-type sesterterpenoids (1–14, 16), together with 15 known compounds. The chemical structures of the new compounds were elucidated based on NMR spectroscopy and HRMS. The structure of 12-epi-phyllactone D/E (15) isolated during this study was originally identified in 2007. However, careful inspection of our experimental 13C NMR spectrum revealed considerable discrepancies with the reported data at C-9, C-12, C-14, and C-23, leading to the correction of the reported compound to the C-12 epimer of 15, phyllactone D/E. The biological properties of compounds 1–16 were evaluated using the MDA-MB-231 cancer cell line. Compound 7, which bears a pentenone E-ring, exhibits significant cytotoxicity with a GI50 value of 4.21 μM.

This family of scalarane derivatives is featured with a trans-fused 6/6/6/6 ring system and can be further categorized into three structural subgroups, namely scalarane, homoscalarane, and bishomoscalarane, based on the presence of single carbon substituents at C-20 and/or C-24 ( Figure 1). Among them, bishomoscalarane exhibits an exceptionally broad range of diversity in the carbon framework, arising from two distinctive sites: C-20 and C-24/C-25 ( Figure 2). Therefore, cyclopropane or alcohol/esters are frequently found at C-20 adjacent to the A ring [12]. The oxidation of C-24 and C-25 results in the formation of an extra E ring in the form of a lactone or cyclopentenone [18]; 24-oxo-25-norbishomoscalarane has also been identified as another feature of the D ring [8]. In addition, oxidation of the backbone usually occurs at C-3 [6], C-12 [19], and C-16 [12] to produce  The marine sponge Dysidea sp. is known to be a rich source of scalaranes, which exhibits useful pharmacological properties, such as anticancer and antimicrobial activities [17,[20][21][22][23][24]. In the course of our studies on bioactive natural products from marine organisms, we inspected the chemical components of Dysidea sp. collected from the Bohol province in the Philippines. As a result, we identified 15 new scalarane derivatives, including one scalarane, four 20,24-bishomo-25-norscalaranes, and 10 bishomoscalaranes (Figure 3), along with 14 known compounds ( Figure S1, Supplementary Materials). In this report, we disclose the structural assignment of the new scalarane sesterterpenoids and their pharmacological properties as anti-cancer agents. In addition, the C-12 configuration of compound 15, which was assigned by Li in 2007 [11], was reinvestigated because of the significant differences observed between the reported and experimental 13 C chemical shifts at C-9, C-12, C-14, and C- 23. the singlet methyl groups and methines from H 0.77 to C 51.4/50.3, H 0.82 to C 54.3/51.4, and H 1.04 to C 54.3, which are known as characteristic correlations occurring from the ring junctions of scalarane-type 6/6/6/6 fused-cyclic systems ( Figure 4). Additional HMBC correlations from the doublet methyl group at H 1.37 to C 80.4/44.9 and from the methine at H 2.34 to C 174.8/44.9 suggested the existence of a -valerolactone moiety. Therefore, our preliminary findings led to the hypothesis that compound 1 possessed a honulactone A-like scaffold (B+D type shown in Figure 2) [12]. While the △ 17,18 -olefin in honulactones is considered one of the structural features that forms the unsaturated lactone E-ring, the initially identified -valerolactone and DOU suggest the possibility of a saturated terminal lactone in compound 1. This speculation was confirmed by the 1 H-1 H COSY cross peak observed for H2-15-H2-16-H-17-H-18, as well as HMBC correlations from CH3-23 (H 1.04) to C-18 (C 52.5) and from H-18 (H 2.34) to C-13 (C 38.7). In addition, the cyclopropane moiety inferred from the 1 H NMR data was positioned at C-4 based on the HMBC correlations from H2-19 (H 0.57, and -0.49) to C-3 (C 33.2)/C-5 (C 50.3) and from CH3-27 (H 1.07) to C-4 (C 22.7), and the spin system for CH3-27-H-20 (H 0.72)-H2-19 (H 0.57, -0.49) in the 1 H-1 H COSY spectrum. Interpretation of the remaining HMBC correlations from CH3-4′ (H 1.23) to C-2′ (C 43.3)/C-3′ (C 64.5), H2-2′ (H 2.49/2.42) to C-1′ (C 171.7)/C-3′, and H-12 (H 5.41) to C-1′ elucidated the -hydroxyl butanoate group at C-12.
The trans-fused cyclic scaffold in 1 was determined from the NOESY cross peaks observed between H-11 (H 1.71) and CH3-21 (
The trans-fused cyclic scaffold in 1 was determined from the NOESY cross peaks observed between H-11β (δ H 1.71) and CH 3    Compound 2 was isolated as a colorless oil, and its molecular formula was determined to be C31H46O6 by HRESIMS (m/z [M + Na] + 537.3167, calcd 537.3187), corresponding to nine degrees of unsaturation. Analysis of the 1D and 2D NMR spectra obtained for 2 indicated a similar carbon framework to 1, but the higher oxidation state of the lactone in E ring appeared as a major difference. HMBC correlations from CH3-23 (δH 1.22) to C-18 (δC 133.7) and from CH3-26 (δH 1.56) to C-17 (δC 162.9) revealed an α,β-unsaturated lactone in the E ring, which was responsible for the one degree higher DOU than that of 1. In addition, the 13 C chemical shift of C-24 (δC 104.4) was characteristic of a ketal carbon atom, of which the position was confirmed by HMBC correlations from CH3-26 to C-24. The β-configuration of OH-24 was determined by the NOESY correlation observed between H-16α (δH 2.28) and CH3-26 ( Figure S3, Supplementary Materials).
Compound 3 was isolated as a mixture of two inseparable epimers. The molecular formula of 3 was deduced to be C31H44O6 by HRESIMS (m/z [M + Na] + 535.3011, calcd 535.3030), corresponding to 10 degrees of unsaturation. An initial inspection of the 13 C NMR spectrum revealed that most of the peaks were split into a doublet-like shape, indicating a 1:1 mixture of diastereomers. The 1D and 2D NMR spectra obtained for compound 3 exhibited most of the structural features of 2, except for one more disubstituted olefin observed at δH (6.38/6.37)/δC (138.84/138.80) and δH (6.29/6.25)/δC (118.6/118.4). The location of the double bond was determined to be △ 15,16 using the consecutive 1 H-1 H COSY correlations observed for H-14 (δH 2.69/2.62)-H-15 (δH 6.38/6.37)-H-16 (δH 6.29/6.25). The splittings observed in the 13 C NMR spectrum were most prominent at CH3-26 (ΔδC 1.13 ppm), informing a mixture of C-24 epimers. This phenomenon has often been observed in the case of 24-homoscalaranes, which possess both an △ 15,16 -olefin and 24-hydroxy pentenolide E-ring [25,26]. Since the △ 15,16 -olefin increases the planarity of the D-ring and renders the C-24 stereocenter more isolated, the 24R* and 24S* diastereomers exhibit almost identical spectroscopic and chromatographic behaviors to give an inseparable mixture. Compound 2 was isolated as a colorless oil, and its molecular formula was determined to be C 31 H 46 O 6 by HRESIMS (m/z [M + Na] + 537.3167, calcd 537.3187), corresponding to nine degrees of unsaturation. Analysis of the 1D and 2D NMR spectra obtained for 2 indicated a similar carbon framework to 1, but the higher oxidation state of the lactone in E ring appeared as a major difference. HMBC correlations from CH 3 -23 (δ H 1.22) to C-18 (δ C 133.7) and from CH 3 -26 (δ H 1.56) to C-17 (δ C 162.9) revealed an α,β-unsaturated lactone in the E ring, which was responsible for the one degree higher DOU than that of 1. In addition, the 13 C chemical shift of C-24 (δ C 104.4) was characteristic of a ketal carbon atom, of which the position was confirmed by HMBC correlations from CH 3 -26 to C-24. The β-configuration of OH-24 was determined by the NOESY correlation observed between H-16α (δ H 2.28) and CH 3 -26 ( Figure S3, Supplementary Materials).
Compound 3 was isolated as a mixture of two inseparable epimers. The molecular formula of 3 was deduced to be C 31 H 44 O 6 by HRESIMS (m/z [M + Na] + 535.3011, calcd 535.3030), corresponding to 10 degrees of unsaturation. An initial inspection of the 13 C NMR spectrum revealed that most of the peaks were split into a doublet-like shape, indicating a 1:1 mixture of diastereomers. The 1D and 2D NMR spectra obtained for compound 3 exhibited most of the structural features of 2, except for one more disubstituted olefin observed at δ H (6.38/6.37)/δ C (138.84/138.80) and δ H (6.29/6.25)/δ C (118.6/118.4). The location of the double bond was determined to be 15,16 using the consecutive 1 H-1 H COSY correlations observed for H-14 (δ H 2.69/2.62)-H-15 (δ H 6.38/6.37)-H-16 (δ H 6.29/6.25). The splittings observed in the 13 C NMR spectrum were most prominent at CH 3 -26 (∆δ C 1.13 ppm), informing a mixture of C-24 epimers. This phenomenon has often been observed in the case of 24-homoscalaranes, which possess both an 15 olefin and 24-hydroxy pentenolide E-ring [25,26]. Since the 15,16 -olefin increases the planarity of the D-ring and renders the C-24 stereocenter more isolated, the 24R* and 24S* diastereomers exhibit almost identical spectroscopic and chromatographic behaviors to give an inseparable mixture.
Compound 4 was isolated as an inseparable mixture and its molecular formula was determined to be C 32 H 46 O 6 by HRESIMS (m/z [M + Na] + 549.3163, calcd 549.3187), indicating 10 degrees of unsaturation. The NMR spectra of 4 were only discriminated from those of 3 by the extra methylene group observed at δ H 1.50 and δ C 29.5/29.4, which was also supported by the mass difference of +14. The extra methylene group was observed in the ester side chain located at C-12, which formed a 3-hydroxypentanoate moiety, as supported by the spin system for H 2 .6), eight methylenes, four methines, and seven methyl groups. In addition, the HMBC correlation from the singlet methyl at δ H 2.22 to δ C 197.7 suggested the presence of a methyl ketone moiety, instead of the lactone E-ring observed in compounds 1-4, leading to the conclusion that 5 had a B+F type skeleton, as shown in Figure 2.
Detailed interpretation of the combined spectral data of 5 revealed that the features related to the A-B-C ring system were identical to those of 1-4. As anticipated, the methyl ketone was positioned at C-17 to form an unsaturated ketone in the D ring on the basis of HMBC correlations between CH 3 -26 (δ H 2.22) and C-17 (δ C 135.1), and H-18 (δ H 6.72) and C-17/C-24 (δ C 197.7) (Figure 4)  Compound 8 was isolated as a yellow oil, and its molecular formula was determined to be C 34 H 52 O 8 by HRESIMS (m/z [M + Na] + 611.3541, calcd 611.3554), corresponding to nine degrees of unsaturation. The 1 H NMR spectrum obtained for compound 8 showed similar patterns to that of 5. However, the upfield peaks observed for the cyclopropane moiety in 5 were substituted by an oxymethine at δ H 5.35, a methyl singlet at δ H 1.09, and an acetate at δ H 2.03, suggesting the C+F type scaffold shown in Figure 2. Therefore, the connectivity of C-27-C-20-C-4-C-19 was determined using the HMBC correlations observed from CH 3 -19 (δ H 0.99) to C-20 (δ C 73.2) and from CH 3 -27 (δ H 1.09) to C-4 (δ C 39.4)/C-20 ( Figure 6). In addition, the acetate at δ H 2.03 exhibited a HMBC correlation with C-20 to be located at C-20. The relative configuration at C-20 was assigned as 20R* from the NOESY correlations observed between H-20 (δ H 5.35) and H-2β (δ H 1.47)/CH 3    Compound 9 was isolated as a colorless oil, and its molecular formula was determined to be C 30 H 48 O 6 by HRESIMS (m/z [M + NH 4 ] + 522.3810, calcd 522.3789) corresponding to seven degrees of unsaturation. Analysis of the 1D and 2D NMR data provided almost identical features to those of 8 to determine the carbon skeleton of compound 9. In this case, only one ester carbon atom (δ C 172.2) was observed in the 13 C NMR spectrum, and the acetate groups shown in the 1 H NMR spectrum of 8 disappeared. This information indicated that compound 9 was the deacetylation product of 8. Accordingly, the upfield shifts of H-20 (δ H 4.32) and H-16 (δ H 4.55) were the major differences, compared to compound 8.
Compound 10 was isolated as a yellow oil, and its molecular formula was determined to be C 33 Figure 2) [12]. A detailed analysis of the 1 H NMR spectrum identified an oxymethine group at δ H 4.44 as a major difference from honulactone C. The location of the oxymethine was determined to be C-16, as indicated by the HMBC correlations from H-16 (δ H 4.44) to C-17 (δ C 162.1)/C-18 (δ C 135.6) and 1 H-1 H COSY cross peak for H 2 -15 (δ H 1.91, 1.84)-H-16 ( Figure 6). The configuration of the OH-16 group was assigned as α-orientation based on the small coupling constant observed for H-16 (dd, J H-15-H-16 = 4.7, 1.4 Hz), and compound 10 was named as 16α-hydroxyhonulactone C [12].
Compound 11 was isolated as a yellow oil. Its molecular formula was determined as C 33 H 50 O 8 by HRESIMS (m/z [M + Na] + 597.3396, calcd 597.3398), corresponding to nine degrees of unsaturation. The 1 H and 13 C NMR data of 11 were almost identical to those of 10, but a ketal moiety (δ C 104.4) was observed instead of one doublet methyl group and two oxymethines in compound 10. As shown in compounds 2-4, the hemiketal functionality in the scalarane-type scaffold usually occurs at C-24 in the E-ring, which was also applicable in this case, as indicated by the HMBC correlations from CH 3 -26 (δ H 1.56) to C-17 (δ C 162.9)/C-24 (δ C 104.4). The α-orientation of the hydroxyl group at C-24 was determined by the NOESY correlation between H-16β (δ H 2.33) and CH 3 -26. Thus, compound 11 was named 24α-hydroxyhonulactone C [12].
Compound 12 was isolated as an inseparable mixture. Its molecular formula was determined as C 33 H 48 O 8 by HRESIMS (m/z [M + Na] + 595.3241, calcd 595.3241), corresponding to 10 degrees of unsaturation. Compared to 11, two more sp 2 methines at δ C 138.9/138.7 and δ H 6.38, and δ C 118.44/118.35 and δ H 6.28/6.26 were observed in the 13  As discussed in the cases of 3 and 4, the presence of the olefin at 15,16 and the hemiketal at C-24 rendered compound 12 an inseparable mixture of C-24 epimers.
Compound 13 was isolated as an amorphous solid. Its molecular formula was determined as C 31 H 48 O 6 by HRESIMS (m/z [M + Na] + 539.3325, calcd 539.3343), corresponding to eight degrees of unsaturation. Inspection of the 1 H NMR spectrum of 13 revealed most of the structural features of the bishomoscalarane-type skeletons. Precise analysis of the 13 C NMR and HSQC data revealed the presence of a triplet methyl group (δ H 0.67/δ C 8.80) and ketal carbon (δ C 104.4), suggesting the A+D type skeleton shown in Figure 2. While most of the spectral data of 13 were identical to phyllofolactone H, the ketal carbon indicated the oxidation of C-24 to give a 24-hydroxy pentenolide E ring. This insight can be confirmed by the HMBC correlation from CH 3 -26 (δ H 1.48) to C-17 (δ C 163.0)/C-24 (δ C 104.4). The configuration of OH-24 was determined to be α-orientation by the NOESY correlation between H-16β (δ H 2.33) and CH 3 -26. Thus, compound 13 was named 24α-hydroxyphyllofolactone H [19].
Compound 15 was isolated as an inseparable mixture. Its molecular formula was determined to be C 32 H 48 O 6 by HRESIMS (m/z [M + Na] + 551.3366, calcd 551.3343), corresponding to nine degrees of unsaturation. The MS data indicated an additional methylene relative to 14, which was further supported by the change observed in the coupling pattern of the terminal methyl group of the side chain at C-12 from a doublet to triplet. The 13 C NMR and HSQC data identified the methylene group at δ H 1.51/1.25 and δ C 29.5/29.4, which were involved in the spin system for H 2 -2 -H-3 -H 2 -4 -CH 3 -5 in the 1 H-1 H COSY spectrum to confirm the presence of the 3-hydroxypentanoate side chain. The orientation of the ester at C-12 was assigned as α by the NOESY signal between H-12 (δ H 5.55/5.49) and CH 3 -23 (δ H 1.06/1.05), as well as the small coupling constant observed for H-12 (dd, J = 2.3, 1.8 Hz), to identify 12-epi-phyllactone D/E. Interestingly, the identified structure was previously isolated as a mixture of C-24 epimers by Li et al. in 2007 [11], but our experimental 13 C NMR data showed some discrepancies with the previously reported data at C-9 (∆ 4.28 ppm), C-11 (∆ 2.7 ppm), C-12 (∆ 2.08 ppm), C-14 (∆ 4.46 ppm), and C-23 (∆ 4.35 ppm) (Figure 8a). In addition, another identification of 12-epi-phyllactone D/E was reported by Andersen et al. in 2009 [13]. Although they acquired almost identical experimental NMR data with ours rather than those reported by Li, the isolated compound was estimated to be same as Li's without consideration of the differences in NMR data (Tables S17 and S18, Supplementary  Materials). Therefore, we investigated the variations in 13 C chemical shifts depending on the orientation of the substituents at C-12.  (Figure 8a). In addition, another identification of 12-epi-phyllactone D/E was reported by Andersen et al. in 2009 [13]. Although they acquired almost identical experimental NMR data with ours rather than those reported by Li, the isolated compound was estimated to be same as Li's without consideration of the differences in NMR data (Tables S17 and S18, Supplementary Materials). Therefore, we investigated the variations in 13 C chemical shifts depending on the orientation of the substituents at C-12.  Figure 8b). However, the reported chemical shifts for 15 were better aligned with those of phyllactone D/E. Furthermore, the differences in the 13 C NMR chemical shifts observed between isolated 15 and compounds Phyllactone D (17) and E (18), the reported 12β-epimers of 15, were selected for comparison [25]. While C-12 in phyllactones D and E was observed at δ C 75.1 and 75.8, Mar. Drugs 2021, 19, 627 9 of 15 respectively, the corresponding chemical shifts of the reported and isolated 15 were observed at δ C 75.3 and 73.2/73.1, respectively. The deviations observed for isolated 15 from phyllactone D/E became more obvious at C-9, C-14, and C-23 ( Figure 8b). However, the reported chemical shifts for 15 were better aligned with those of phyllactone D/E. Furthermore, the differences in the 13 C NMR chemical shifts observed between isolated 15 and compounds 3, 4, 12, and 14, which share an identical substructure for the B-E ring system, showed negligible values (< 0.5 ppm) around the C-ring (Table S19, Supplementary Materials). Accordingly, isolated 15 is more likely to be the 12α-epimer. Even though Li determined the 12a-configuration observing the NOESY signal between H-12 and CH 3 -23 and J H-12-H-13 calculation (3.0, 2.5 Hz), the NMR database suggests that the compound previously reported by Li is presumed to be a mixture of phyllactone D (17) 13 C and HSQC NMR spectra showed characteristic peaks for the aldehyde carbon atom at δ C 196.4, two carbonyl carbons at δ C 169.6 and 169.6, one trisubstituted olefin at δ C 145.8 and 124.2, and one oxymethine at δ C 76.9. The HMBC correlation between the two methyl groups at δ C 33.3 and 21.4 was identified as a characteristic feature of the 4-dimethyl-sesterterpenoid scaffold ( Figure 9). The aldehyde at δ H 9.41 exhibited a HMBC correlation with C-18 (δ C 58.7) to be located at C-25. Additional HMBC correlations from H-18 (δ H 3.07) to C-16 (δ C 145.8)/C-17 (δ C 124.2)/C-24 (δ C 169.6), along with the 1 H-1 H COSY cross peak for H-14-H 2 -15-H-16, indicated the presence of the acid at C-24 and trisubstituted olefin at C-16. The acetate group (δ C 21.3/δ H 1.95) was positioned at C-12, as indicated by the HMBC correlation from H-12 (δ H 4.80) to C-1 (δ C 169.6) and 1 H-1 H COSY cross peak for H 2 -11-H-12. Thus, the planar structure of 16 was found to be the deacetalization product of scalarin (19) [3]. The NOESY correlations between CH 3 -23 (δ H 0.86) and H-12/H-18 determined the configuration of the C-12 acetate and C-18 formyl groups as α. Whereas scalarin (19) exists only in its hemiacetal form, the formation of 18-epi-19 or 19 via the acetalization of 16 was not observed. To rationalize the observed difference in reactivity, 18-epi-16 was proposed as a plausible precursor of scalarin, and geometrical optimization of 16 and 18-epi-16 was performed at the B3LYP/6-31G** level of theory. The atomic distance between O-24 to C-25 was calculated to be 3.37 Å for 16 and 2.68 Å for 18-epi-16 ( Figure 10). This result suggests that 18-epi-16 can undergo acetalization to form scalarin because the β-orientation of C-25 increases its proximity to the acid at C-24. However, the acetalization of the 25α-formyl group in 16 will be restricted due to its remoteness to OH-24 to exist as its aldehyde form.

Biological Activity
The cytotoxicity of compounds 1-16 against MDA-MB-231 (a human breast cancer cell line) was evaluated to elucidate their potential as anticancer agents . Compounds 1-6, 8,  11, and 13-15 exhibited moderate cytotoxicity with GI 50 values ranging from 40 to 72 µM. Compounds 9, 10, 12, and 16 were inactive toward the cancer cell line (Table 1). Among the bishomoscalaranes, the highest anticancer activity was exhibited by compound 7, which has a relatively rare cyclopentenone E-ring (B+E type scaffold in Figure 2), with a GI 50 value of 4.2 µM. The highly diversified structures of the isolated scalaranes provided some information on their structure-activity relationship (SAR). The presence of the 15,16 -olefin generally had a detrimental effect that reduced the cytotoxicity in the range of 12-30 µM, as shown by the sets of 2 and 3 (B+D type), 13 and 14 (C+D type), and 13 and 14 (A+D type). Comparing 3 with 4 and 14 with 15, the homologation of one methylene group at C-4 was beneficial toward increasing the activity to~20 µM. A series of compounds 2, 11, and 13, which only differ at the C-4 substituent, indicated the disadvantageous effect of oxidation at C-20 on the anticancer activity. The negative effect of oxidation at C-20 was also observed in the inactive series of compounds 9, 10, and 12.

General Experimental Procedures
Specific optical rotations were collected on a Rudolph Research Analytical (Autopol III) polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). IR spectra were measured on a JASCO FT/IR-4100 spectrophotometer (JASCO Corporation, Tokyo, Japan). The 1D and 2D NMR spectra were taken in CDCl 3 using a Bruker 600 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) at 297.1 K. 1 H NMR spectra were collected after 64-128 scans, and 13 C NMR spectra were collected at a range of 10,000-15,000 scans depending on the sample concentrations. The mixing time for NOESY experiments was set as 0.3 s. Chemical shifts were reported in parts per million relative to CHCl 3 residue (δ H 7.26, δ C 77.1) in CDCl 3 . High resolution mass-spectra were obtained on a Sciex X500R Q-TOF spectrometer (Framingham, MA, USA) equipped with an ESI source. MPLC was performed using the TELEDYNE ISCO CombiFlash Companion with the TELEDYNE ISCO RediSep Normal-phase Silica Flash Column (Teledyne ISCO, Lincoln, NE, USA). HPLC was performed on a PrimeLine Binary pump (Analytical Scientific Instruments, Inc., El Sobrante, CA, USA) utilizing silica columns (YMC-Pack Silica, 250 × 10 mm I.D., or 250 × 4.6 mm I.D., 5 µm; YMC Co. Ltd., Kyoto, Japan), the Shodex RI-101 (Shoko Scientific Co. Ltd., Yokohama, Japan), or the UV-M201.

Biological Material
The marine sponge used in this study was collected in March 2016 from the Bohol province in the Philippines (N 9 • 43 31.39 E 124 • 32 19.86 ) at a depth of 15 m using scuba diving. The sponge was directly kept frozen at −20 • C until identified as Dysidea sp. and chemically analyzed. A voucher sample (163PIL-267) has been stored at the Marine Biotechnology Research Center, Korea Institute of Ocean Science & Technology (KIOST).