Bissubvilides A and B, CembraneâCapnosane Heterodimers from the

Article pubs.acs.org/jnp

Bissubvilides A and B, Cembrane−Capnosane Heterodimers from the Soft Coral Sarcophyton subviride Peng Sun,†,‡ Qing Yu,†,‡ Jiao Li,† Raffaele Riccio,§ Gianluigi Lauro,§ Giuseppe Bifulco,§ Tibor Kurtán,⊥ Attila Mándi,⊥ Hua Tang,† Tie-Jun Li,† Chun-Lin Zhuang,† William H. Gerwick,∥ and Wen Zhang*,† †

Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University, 325 Guo-He Road, Shanghai 200433, People’s Republic of China § Dipartimento di Farmacia, Università di Salerno, Via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy ⊥ Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary ∥ Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States S Supporting Information *

ABSTRACT: Two new biscembranoid-like compounds, bissubvilides A (1) and B (2), were isolated together with sarsolilide B (3), the proposed biogenetic precursor to 1, from the soft coral Sarcophyton subviride. The structures and absolute configurations were solved by spectroscopic analysis and TDDFT/ECD and DFT/NMR calculations. The bissubvilides represent a novel biscembranoid-like skeleton presumed to derive from a cembrane-type diene and a capnosane-type dienophile via a Diels− Alder reaction. These two molecules exerted no cytotoxicity against MG63 or A549 tumor cells or HuH7 tumor stem cells.

B

subviride resulted in the discovery of two novel cembrane− capnosane heterodimers, named bissubvilides A (1) and B (2), together with the known compound sarsolilide B (3).14 We report herein the isolation, structure elucidation, and bioactivities of these two new representatives of biscembranoid-like heterodimers.

iscembranoids are a structurally intriguing group of fused polycyclic marine natural products produced by soft corals of the genera Sarcophyton, Sinularia, and Lobophytum (family Alcyoniidae),1−5 as well as the sponge Petrosia nigricans.6 Since the first discovery of methyl sartortuoate in 1986,7 more than 80 biscembranoids have been reported. The structure of the reported biscembranoids is biosynthetically rationalized to be the Diels−Alder cycloaddition product of two cembrane monomers, serving as a 1,3-diene and a dienophile, respectively. The cembrane possessing a trisubstituted conjugated Δ21(34)/ Δ35-butadiene moiety serves as the 1,3-diene. The proposed dienophile is a cembranoid with a Δ1 or Δ1(14) double bond, which is usually conjugated with a carbonyl group or a C-20 carboxymethyl ester.8 Dienophiles derived from a cembranoid with a Δ1 double bond embedded in an α,β-unsaturated γlactone are quite rare; examples are provided by bislatumlides A−F from Sarcophyton latum.2,9 Most of the biscembranoids are favored in an endo-type Diels−Alder reaction. However, bislatumlides C and D are exceptions, being of the exo type.2 The complex and unique structures of these biscembranoids have broadly attracted the attention of synthetic organic chemists for their total synthesis.10 In the course of our ongoing search for bioactive secondary metabolites from South China Sea invertebrates,2,11,12 we collected the soft coral Sarcophyton subviride from the coast of Xisha, Hainan Province. Previous study of this species resulted in the isolation of nine polyhydroxysterols.13 Using a common workup for extraction and isolation,2 our investigation on S. © 2016 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Bissubvilide A (1) was obtained as an optically active, colorless powder. The molecular formula of 1 was established as Received: May 18, 2016 Published: October 5, 2016 2552

DOI: 10.1021/acs.jnatprod.6b00453 J. Nat. Prod. 2016, 79, 2552−2558

Journal of Natural Products

Article

Table 1. NMR Spectroscopic Data (1H 500 MHz, 13C 125 MHz, CDCl3) for Bissubvilides A (1) and B (2) bissubvilide A (1) no.

δC, type

1 2 3α 3β 4α 4β 5 6 7α 7β 8 9 10 11 12 13α 13β 14α 14β 15 16

57.3, C 38.4, CH 31.0, CH2

17 18 19 20 21 22 23 24α 24β 25α 25β 26 27 28α 28β 29 30 31 32α 32β 33α 33β 34 35 36α 36β 37 38 39 40 a

35.0, CH2 88.9, C 88.3, C 33.8, CH2 37.2, 82.9, 51.0, 117.1, 147.6, 26.6,

CH2 C CH CH C CH2

38.1, CH2 32.6, CH 24.1, CH3 20.7, 26.9, 25.1, 179.5, 54.1, 126.5, 134.6, 36.2,

CH3 CH3 CH3 C CH CH C CH2

29.1, CH2 67.7, CH 82.6, C 34.7, CH2 26.6, 135.2, 132.1, 30.0,

CH2 CH C CH2

29.8, CH2 130.5, C 127.2, C 38.7, CH2 19.3, 15.4, 21.9, 168.7,

CH3 CH3 CH3 C

bissubvilide B (2) δH (J in Hz)

1.78, 1.49, 1.88, 2.60, 1.72,

ova dd (13.0, 7.3) ov ov dd (15.2, 7.3)

ov ov ov ov ov d (6.8)

140.6, C 113.2, CH2 21.8, 27.4, 25.2, 180.0, 54.3, 126.6, 134.7, 36.3,

3.32, d (11.4) 4.98, d (11.4)

2.21, 2.03, 2.46, 5.88,

m ov brs brs

2.39, 2.62, 2.11, 2.75,

ov m m ov

2.28, 2.05, 1.78, 1.57, 1.30,

CH2 C CH CH C CH2

38.2, CH2

1.15, d (6.8) 1.12, s 1.29, s

ov ov ov ov d (9.2)

35.5, CH2

37.6, 83.2, 52.2, 123.7, 142.9, 24.7,

2.68, d (11.2) 5.48, d (11.2)

2.11, 1.99, 1.83, 1.32, 4.19,

57.7, C 38.1, CH 30.9, CH2

89.2, C 88.9, C 34.2, CH2

1.83, ov 2.33, ov 1.95, ov

1.93, 2.62, 1.63, 1.63, 2.27, 1.04,

δC, type

CH3 CH3 CH3 C CH CH C CH2

29.2, CH2 67.8, CH 82.8, C 34.9, CH2 26.7, 135.3, 132.2, 30.1,

CH2 CH C CH2

29.9, CH2 130.4, C 127.6, C 38.8, CH2

ov ov s s s

19.4, 15.5, 22.0, 168.9,

CH3 CH3 CH3 C

δH (J in Hz) 1.68, 1.44, 1.85, 2.50, 1.75,

m dd (13.5, 7.6) ov m ov

1.85, ov 2.38, ov 1.99, ov 2.82, d (11.4) 5.87, d (11.4) 2.34, 2.69, 1.65, 1.78,

ov m ov ov

5.09, 5.04, 1.96, 1.16, 1.29,

s s s s s

3.27, d (10.8) 4.98, d (10.8) 2.09, 1.99, 1.83, 1.31, 4.18,

ov ov ov ov d (8.9)

2.19, 2.05, 2.44, 5.88,

m ov brs ov

2.36, 2.62, 2.10, 2.72,

ov m ov ov

2.21, 1.99, 1.74, 1.56, 1.30,

ov ov s s s

Overlapped signals.

accounting for six double-bond equivalents. The remaining degrees of unsaturation were due to the presence of six rings in the molecule. The 1H NMR spectrum showed seven methyl groups that were attributed to two methyls of an isopropyl group (δH 1.04, H3-16; 1.15, H3-17; 3H each, d, J = 6.8 Hz),

C40H58O7, obtained by HRESIMS and NMR data, requiring 12 degrees of unsaturation. The 13C and DEPT spectra exhibited a total of 40 carbon resonances (Table 1) that were classified into 10 sp2 carbon atoms (2 OCO, 3 CHC, 1 CC) and 30 sp3 carbon atoms (4 OC, 1 OCH, 1 C, 4 CH, 13 CH2, 7 CH3), 2553



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NOE correlations of H-2/H-4α and H-4α/H-11 indicated an α orientation of these protons, and correlations of H-4β/H3-19, H3-19/H-7β, H-7β/H-10, and H-10/H3-18 suggested that these protons have the same β orientation. An α orientation for the hydroxy group at C-6 was suggested by comparison of the C-5 to C-10 NMR data with those of sarsolilides B and C,14 which was supported by pyridine-induced solvent deshielding shifts of H-4α and H-8α (Table S1).15 In the lower portion of 1, an E configuration of the Δ22 double bond was determined by the 13C NMR chemical shift of C-38 (δC 15.4 Hz)16 and by a NOE correlation of H-22 with H-24β. The Z configuration of the Δ30 double bond was assigned by a NOE correlation between H-30 and H-32β. The NOE correlations of H-21/H33α, H-21/H 3-38, H3 -38/H-25α, and of H-25α/H3 -39 indicated an α orientation of these protons, while the NOE correlations of H-22/H-24β and H-24β/H-26 indicated a β orientation of these latter protons. The distinct NOE correlations between H-22 and H-3β and between H-21 and H2-14 led to the assignment of the relative configuration of the central cyclohexene ring. Thus, the relative configuration was suggested to be (1R*,2R*,5R*,6R*,9S*,10S*,21S*,26S*,27R*)1. Compound 1 is proposed to result from a Diels−Alder cycloaddition between the co-isolated sarsolilide B (3),14 a capnosane-type monomer, and a proposed cembranoid-type intermediate (4) (Scheme 1). There should be eight possible

two vinyl methyls (δH 1.78, H3-37; 1.57, H3-38; 3H each, s), and three tertiary methyls (δH 1.12, H3-18; 1.29, H3-19; 1.30, H3-39; 3H each, s). The HMQC spectrum allowed assignment of all protons and the corresponding carbons in the molecule (Table 1). These NMR data displayed similar signals to biscembranoid data reported in the literature.2,9 Analysis of the COSY spectrum readily delineated the isolated proton spin systems, i.e., H2-4 to H2-36, H2-7 to H2-8, H-10 to H-11, H2-13 to H2-14, H-15 to H3-16/17, H-21 to H22, H2-24 to H-26, H2-28 to H-30, and H2-32 to H2-33 (Figure 1). The HMBC correlations from H3-18 to C-8, C-9, and C-10,

Scheme 1. Plausible Diels−Alder Reaction Leading to 1

Figure 1. Key COSY (bold), HMBC (blue), and NOE (red) correlations of 1.

from the allylic proton H-10 (δH 2.68, d, J = 11.2 Hz) to C-6, C-9, and C-12, and from H2-7 to C-6 and C-8 revealed the presence of a cyclopentane ring. The isopropyl group was located at the trisubstituted Δ11 double bond, as indicated by the HMBC correlations from H3-16/17 to C-12. The longrange HMBC correlation from H3-19 and H-21 (δH 3.23, d, J = 11.4 Hz) to C-20 (δC 179.4) revealed the formation of an εlactone functionality. Further analyses of 2D NMR data revealed a capnosane-based skeleton for the upper-portion structure of 1, which is similar to sarsolilides B (3) and C previously isolated from Sarcophyton trocheliophorum.14 The HMBC cross-peaks from H-21 to C-1, C-2, C-14, C-20, and C33 and from H3-37 to C-34, C-35, and C-36 constructed a cyclohexene ring in the core of 1. The lower portion of 1 was elucidated by connection of the isolated fragments by HMBC correlations from H3-38 to C-22, C-23, and C-24, from H3-39 to C-26, C-27, and C-28, from H-30 to C-32, and from H-33 to C-34. The diagnostic long-range 4J HMBC cross-peak between H3-39 and C-40 via an O-atom constructed a lactone ring, suggesting the presence of an α,β-unsaturated ε-lactone. The proposed planar structure of 1 as a cembrane−capnosane heterodimer was thus fully assigned by detailed analysis of the 2D NMR data as shown in Figure 1. The relative configuration of 1 was deduced on the basis of coupling constants and NOE correlations. In the upper portion of 1, an E configuration of the Δ11 double bond was deduced by the NOE correlation between H-11/H-17 (Figure 1). The

relative stereoisomers in theory arising from a Diels−Alder reaction (1a−d for endo-type and 1e−h for exo-type, 1a corresponding to isomer 1, Scheme S1, Supporting Information). In order to corroborate the proposed relative configuration of 1, a combined QM/NMR approach was employed comparing the experimental and calculated chemical shift NMR parameters. An extensive conformational search at the empirical level (molecular mechanics, MM) for all of the possible stereoisomers was carried out. The selected conformers were submitted to a geometry and energy optimization step at the density functional level (DFT), and they were subsequently used for the computation of the 13C and 1H NMR chemical shifts. These calculations accounted for the contributions of each conformer on the basis of the related energies on the final Boltzmann distribution. Then, for each atom the experimental and calculated 13C and 1H NMR chemical shifts were compared as Δδ values, and subsequently, the mean absolute errors (MAEs) for all possible stereoisomers were computed (Tables S2−S5, Supporting Information).17,18 Among them, stereoisomer 1a showed the 13C MAE as the lowest single value among the various isomers; this isomer had the third lowest 1H MAE (Figure 2). To unambiguously assign the relative configuration of 1, we relied on the recently introduced DP4+ approach,19 for which the combination of 2554



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secondary hydroxy group at C-26.22 The (R)- and (S)-MPA esters of 1 were respectively prepared, and the significant ΔR−S values for those protons close to C-26 were observed (Figure 4). The NMR comparison of the (R)- and (S)-MPA diesters revealed a 26S configuration based on Mosher’s rule, which is consistent with the assigned absolute configuration of 1.

Figure 2. 13C (opaque and transparent blue bars) and 1H (opaque and transparent red bars) mean absolute errors (MAE) histograms related to compounds 1a−h. For each compound, MAE bars are reported for all the possible relative stereoisomers. The lowest MAEs are highlighted with opaque colored bars. Figure 4. ΔδR−S values (in ppm) for the MPA esters of 1.

both 13C/1H chemical shift data are compared with the experimental data and afford a clear enhancement in the resulting final performance. The application of this methodology confirmed isomer 1a as the most probable isomer (DP4+ probability, all data = 100%), thereby corroborating the proposed relative configuration. The absolute configuration of 1 was elucidated by timedependent density functional theory (TDDFT)/electronic circular dichroism (ECD) calculation. 20,21 In order to reproduce the experimental conditions, the conformations of 1a from MM calculations were further submitted to a new optimization of geometries at the DFT level using the integral equation formalism polarizable continuum model (IEFPCM) in MeOH to reproduce the experimental environment. ECD spectra were predicted at the TDDFT (NStates = 40) MPW1PW91/6-31G(d,p) level and MeOH IEFPCM. Final ECD spectra for the identified diastereoisomers were built considering the influence of each conformer on the total Boltzmann distribution taking into account the relative energies. Conformational analysis of (1R,2R,5R,6R,9S,10S,21S,26S,27R)-1 was performed combining conformational search and molecular dynamics at different temperatures so as to generate the most representative conformers. The Boltzmann-weighted ECD spectrum of 1 was calculated and showed a good superposition with the experimental curve (Figure 3), in particular, the negative band at ca. 230 nm. The absolute configuration was thus assigned as (1R,2R,5R,6R,9S,10S,21S,26S,27R)-1. The established absolute configuration of 1 was further confirmed by using the modified Mosher’s method22 on the

Bissubvilide B (2) was obtained as an optically active, colorless solid. The molecular formula was determined to be C40H56O7 by HRESIMS, showing two mass units less than that of 1. Analysis of the NMR data of 2 revealed a close similarity to compound 1. The only difference was that the isopropyl group in 1 was replaced by an isopropylene (δH = 5.09, 1H, s, H-16a and 5.04, 1H, s, H-16b, and 1.96, 3H, s, H-17) in 2. This was further supported by HMBC correlations from H2-16 and H3-17 to C-15 (δC = 140.6) and C-12 (δC = 142.9). The relative configuration of 2 was suggested to be the same as that of 1 by the highly similar NMR data and NOE correlations. The proposed relative configuration of 2 was strongly supported by calculation of NMR parameters (Scheme S1, Figure S1, and Tables S6−S9, Supporting Information). Conformational analysis of (1R,2R,5R,6R,9S,10S,21S,26S,27R)-2 was performed at various levels applying both the conformational search and high-temperature molecular dynamics for generation of conformers and various DFT levels of theory for optimization and ECD calculations (Tables S10 and S11, Supporting Information). While the 203 and 240 nm ECD transitions could be reproduced by all ECD levels computed for the B97D/TZVP PCM/MeOH reoptimized MMFF conformers, all ECD calculations showed a positive Cotton effect (CE) at ca. 280 nm that was missing from the experimental spectrum (Figures 5 and S2). In order to confirm the ECD results, specific rotation values were calculated for the low-energy conformers of (1R,2R,5R,6R,9S,10S,21S,26S,27R)-2 with three DFT functionals. All of the low-energy conformers gave positive specific rotation values with all applied functionals. Further the Boltzmann-averaged values were found to be +113.9, +89.5, and +117.9 at the B3LYP/TZVP, BH&HLYP, and PBE0/TZVP levels with the PCM solvent model for MeOH, respectively. This was in good agreement with the experimental data of +120, and therefore the absolute configuration of 2 could be unambiguously determined as (1R,2R,5R,6R,9S,10S,21S,26S,27R)-2. Capnosanes are rarely occurring diterpenoids in soft corals and are sometimes regarded as cembranoid derivatives with an irregular carbobicyclic skeleton. Since the first report in 1986,23 capnosanes have so far been found in six species of soft corals including Cespitularia sp.,23 Alcyonium coralloides,24 Sarcophyton trocheliophorum,14,25,26 Sarcophyton elegans,27 Sarcophyton solidum,28 Sarcophyton tortuosum,29 and Sinularia pavida.14,30 Among them, sarsolilides A−C represent the only three

Figure 3. Comparison between the experimental ECD curve and the TDDFT/ECD spectrum of (1R,2R,5R,6R,9S,10S,21S,26S,27R)-1 at the MPW1PW91/6-31G(d,p) level with PCM for MeOH. 2555



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and H2O. The Et2O solution was concentrated under reduced pressure to give a dark brown residue (10.0 g). The residue was subjected to CC on silica gel with a gradient elution of CH2Cl2−MeOH (from 100:1 to 1:1) to give 16 fractions (Fr. 1−16). Fraction 8 (76.0 mg) was fractionated by Sephadex LH-20 (CH2Cl2−MeOH, 2:1) and then purified on preparative HPLC to give 3 (1.5 mg, 61% MeOH−H2O, 2.0 mL/min, tR = 43.0 min). Fr. 9 (116.0 mg) was fractionated by Sephadex LH-20 (CH2Cl2−MeOH, 2:1) to give seven subfractions (subfr. 9a−g). Subfr. 9a was further purified on preparative HPLC to give 1 (3.5 mg, 78% MeOH−H2O, 1.5 mL/min, tR = 45.1 min) and 2 (1.8 mg, 78% MeOH−H2O, 1.5 mL/min, tR = 61.2 min). Bissubvilide A (1): white, amorphous powder; [α]20.0D +129 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (4.35), 230 (3.93) nm; ECD (MeOH, c 3.0 × 10−4) λmax (Δε) 202 (+10.48), 233 (−2.91) nm; IR (film) νmax 3417, 2957, 2931, 1682, 1651, 1621, 1446, 1347 cm−1; 1H and 13C NMR spectroscopic data, Table 1; HRESIMS [M + H]+ m/z 651.4263 (calcd for C40H59O7, 651.4255). BissubvilideB (2): white, amorphous powder; [α]20.0D +120 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (4.59), 238 (4.35) nm; ECD (MeOH, c 2.0 × 10−4) λmax (Δε) 203 (+9.70), 240 (−1.29) nm; IR (film) νmax 3427, 2929, 2857, 1713, 1674, 1451, 1374, 1238 cm−1; 1H and 13C NMR spectroscopic data, Table 1; HRESIMS [M + H]+ m/z 649.4109 (calcd for C40H57O7, 649.4099). Preparation of (R)-MPA Ester of 1. To a solution of 1 (0.8 mg, 1.2 μmol) with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 2.4 mg, 12 μmol) and 4-(dimethylamino) pyridine (DMAP, 1 crystal) in dry CH2Cl2 (1.0 mL) was added (R)-αmethoxyphenylacetic acid (MPA, 0.4 mg, 2.4 μmol) for 24 h at room temperature. The reaction solvent was removed under reduced pressure, and the reaction mixture was subjected to column chromatography on silica (acetone−petroleum ether, 3:1) to afford the (R)-MPA ester of 1 (0.5 mg, yield 55%): 1H NMR (500 MHz, CDCl3) δH 5.88 (br s, 1H, H-30), 5.56 (d, J = 7.8 Hz, 1H, H-26), 5.47 (d, J = 11.6 Hz, 1H, H-11), 5.11 (d, J = 10.7 Hz, 1H, H-22), 3.27 (d, J = 10.7 Hz, 1H, H-21), 2.76 (m, 1H, H-33β), 2.68 (d, J = 11.6 Hz, 1H, H-10), 2.61 (m, 1H, H-4α), 2.61 (m, 1H, H-13β), 2.58 (m, 2H, H-29), 2.56 (m, 1H, H-32β), 2.36 (m, 1H, H-32α), 2.35 (m, 1H, H-28α), 2.32 (m, 1H, H-7β), 2.28 (m, 1H, H-36α), 2.26 (m, 1H, H-15), 2.06 (m, 1H, H-36β), 2.04 (m, 1H, H-33α), 2.01 (m, 1H, H-24β), 1.94 (m, 2H, H-8), 1.93 (m, 1H, H-13α), 1.92 (m, 1H, H-28β), 1.87 (m, 1H, H-24α), 1.84 (m, 1H, H-3β), 1.79 (m, 3H, H-37), 1.78 (m, 1H, H7α), 1.73 (m, 1H, H-2), 1.69 (m, 1H, H-4β), 1.67 (m, 1H, H-14α), 1.64 (m, 1H, H-25α), 1.59 (m, 1H, H-14β),1.54 (m, 1H, H-3α), 1.49 (s, 3H, H-38),1.36 (m, 1H, H-25β), 1.32 (s, 3H, H-39), 1.23 (s, 3H, H-19), 1.17 (d, 3H, J = 6.8 Hz, H-17), 1.11 (s, 3H, H-18), 1.05 (d, 3H, J = 6.8 Hz, H-16); ESIMS [M + H]+ m/z 799.28. Preparation of (S)-MPA Ester of 1. The same reaction of 1 (0.8 mg, 1.2 μmol) with EDC (2.4 mg, 12 μmol), DMAP (1 crystal) in dry CH2Cl2 (1.0 mL), and (S)-MPA (0.4 mg, 2.4 μmol) afforded the (S)MPA ester of 1 (0.4 mg, yield 40%): 1H NMR (500 MHz, CDCl3) δH 5.76 (br s, 1H, H-30), 5.49 (d, J = 10.7 Hz, 1H, H-11), 5.47 (d, J = 7.8 Hz, 1H, H-26), 5.35 (d, J = 12.5 Hz, 1H, H-22), 3.31 (d, J = 12.5 Hz, 1H, H-21), 2.76 (m, 1H, H-33β), 2.68 (d, J = 10.7 Hz, 1H, H-10), 2.62 (m, 1H, H-13β), 2.60 (m, 1H, H-4α), 2.54 (m, 1H, H-32β), 2.43 (m, 2H, H-29), 2.34 (m, 1H, H-32α), 2.33 (m, 1H, H-7β), 2.27 (m, 1H, H-36α), 2.26 (m, 1H, H-15), 2.26 (m, 1H, H-28α), 2.09 (m, 1H, H24β), 2.07 (m, 1H, H-36β), 2.03 (m, 1H, H-33α), 1.94 (m, 1H, H24α), 1.93 (m, 2H, H-8), 1.92 (m, 1H, H-13α), 1.85 (m, 1H, H-3β), 1.80 (m, 1H, H-28β), 1.79 (m, 1H, H-7α), 1.79 (m, 3H, H-37), 1.78 (m, 1H, H-25α),1.74 (m, 1H, H-2), 1.70 (m, 1H, H-4β), 1.68 (m, 1H, H-14α), 1.58 (m, 1H, H-14β), 1.56 (m, 1H, H-25β), 1.53 (m, 1H, H3α), 1.53 (s, 3H, H-38), 1.25 (s, 3H, H-19), 1.17 (d, 3H, J = 6.8 Hz, H-17), 1.12 (s, 3H, H-39), 1.11 (s, 3H, H-18), 1.04 (d, 3H, J = 6.8 Hz, H-16); ESIMS [M + H]+ m/z 799.43. Computational Section for NMR Prediction. Maestro 10.231 was used to build the chemical structures of the possible relative diastereoisomers of 1 and 2, and MacroModel 10.832 was used to optimize the structures using the OPLS force field and the PolakRibier conjugate gradient algorithm (PRCG, maximum derivative less than 0.001 kcal/mol). Starting from the obtained 3D structures, we

Figure 5. Comparison between the experimental ECD curve and the TDDFT/ECD spectra of (1R,2R,5R,6R,9S,10S,21S,26S,27R)-2 at various levels with PCM for MeOH computed for the B97D/TZVP PCM/MeOH conformers.

capnosanes containing an α,β-unsaturated ε-lactone moiety.14,28 Bissubvilides A and B represent a novel heterodimeric skeleton formed by the coupling of a capnosane and a cembrane. These two new natural products were evaluated in vitro for their cytotoxic activity toward several tumor cell lines. Compounds 1 and 2 exhibited no cytotoxicity against human osteosarcoma MG-63 (IC50 > 30 μM) or A549 lung cancer (IC50 > 25 μM) cells or Huh7 human hepatology cancer stem cells (IC50 > 50 μM).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in MeOH with an Autopol IV polarimeter at the sodium D line (590 nm). UV absorption spectra were recorded with a Varian Cary 100 UV/vis spectrophotometer; wavelengths are reported in nm. ECD spectra were recorded with a Jasco-715 spectropolarimeter. Infrared spectra were recorded in thin polymer films on a Nexus 470 FT-IR spectrophotometer (Nicolet); peaks are reported in cm−1. NMR spectra were recorded at 300 K on a Bruker Avance DRX-500 NMR spectrometer. Chemical shifts are reported with the use of the residual CDCl3 signals (δH 7.26 ppm; δC 77.0 ppm) and pyridine-d6 signals (δH 8.74, 7.58, 7.22; δC 150.3, 135.9, 123.9) as internal standards for 1H and 13C NMR spectra. The 1H and 13C NMR assignments were supported by COSY, HMQC, HMBC, and NOESY experiments. The mass spectra and high-resolution mass spectra were performed on a Q-TOF Micro mass spectrometer, resolution 5000. An isopropyl alcohol solution of sodium iodide (2 mg/mL) was used as a reference compound. Semipreparative HPLC was performed on an Agilent 1100 system equipped with a refractive index detector using a YMC Pack ODS-A column (5 μm, 250 × 10 mm). Commercial silica gel (Yantai, 200−300 and 400−500 mesh) was used for column chromatography (CC). Precoated silica gel plates (Yantai, HSGF-254) were used for analytical thin-layer chromatography (TLC). Spots were detected on TLC under UV or by heating after spraying with an anisaldehyde sulfuric acid reagent. Animal Material. Specimens of the soft coral Sarcophyton subviride were collected in June 2012 from the coast of Xisha Island in the South China Sea, at a depth of 15 m, and frozen immediately after collection. The specimen was identified by Dr. Xiubao Li (South China Sea Institute of Oceanology, Chinese Academy of Sciences, China). A voucher specimen (GE-15) was deposited in the Research Center for Marine Drugs, Second Military Medical University, China. Extraction and Isolation. Fresh specimens of S. subviride (2.1 kg) were immediately frozen, cut into pieces, and exhaustively extracted with acetone at room temperature (6 × 1.5 L). The organic extracts were evaporated to give a residue that was partitioned between Et2O 2556



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performed exhaustive conformational searches at the empirical molecular mechanics level, with the Monte Carlo Multiple Minimum (MCMM) method (50 000 steps) in order to allow a full exploration of the conformational space, and the low mode conformational search (LMCS) method (50 000 steps) was used to integrate the conformational sampling. Furthermore, we performed molecular dynamics simulations at 450, 600, 700, and 750 K, with a time step of 2.0 fs, an equilibration time of 0.1 ns, and a simulation time of 10 ns. For each diastereoisomer, we minimized (PRCG, maximum derivative less than 0.001 kcal/mol) and compared all of the conformers obtained from the previously mentioned conformational searches. We used the “Redundant Conformer Elimination” module of Macromodel 10.832 to select nonredundant conformers, excluding the conformers differing more than 21.0 kJ/mol (5.02 kcal/mol) from the most energetically favored conformation and setting a 0.5 Å RMSD (root-mean-square deviation) minimum cutoff for saving structures. Next, we optimized the obtained conformers at the quantum mechanical (QM) level by using the MPW1PW91 functional and the 6-31G(d) basis set. After this step at the QM level, the newly obtained geometries were visually inspected in order to remove further possible redundant conformers, and then those selected were accounted for in the subsequent computation of the 13C and 1H NMR chemical shifts, using the MPW1PW91 functional and the 6-31G(d,p) basis set. Final 13C and 1 H NMR spectra for each of the investigated diastereoisomers were built considering the influence of each conformer on the total Boltzmann distribution taking into account the relative energies. All QM calculations were performed using the Gaussian 09 software package.33 Computational Section for ECD Spectra and Specific Rotation Prediction. For compound 1, an exhaustive conformational search was performed, following the same protocol as described for the generation of conformers used in the prediction of NMR parameters. In this case, a constant dielectric term of MeOH, mimicking the presence of the solvent, was used in the calculations to reduce artifacts. The conformers selected were submitted to a further optimization of the geometries at the DFT using the MPW1PW91 functional, the 631G(d) basis set, and the integral equation formalism version of the polarizable continuum model (MeOH IEFPCM). ECD spectra were predicted at the TDDFT (NStates = 40) MPW1PW91/6-31G(d,p) level and MeOH IEFPCM. Final ECD spectra for the identified diastereoisomers were built considering the influence of each conformer on the total Boltzmann distribution taking into account the relative energies. A Gaussian band-shape function was applied with the exponential half-width of 0.25 eV to simulate the ECD curve, using SpecDisc software.34 For compound 2, mixed torsional/low-frequency mode conformational searches were carried out by means of the Macromodel 9.9.223 software using the Merck Molecular Force Field (MMFF) with an implicit solvent model for CHCl3.35 Geometry reoptimizations were carried out at the B97D/TZVP level36,37 with the PCM solvent model for MeOH. TDDFT/ECD and specific rotation calculations were run with various functionals (B3LYP, BH&HLYP, PBE0) and the TZVP basis set as implemented in the Gaussian 09 package.33 ECD spectra were generated as sums of Gaussians with 4200 cm−1 half-height widths (corresponding to ca. 24 at 240 nm), using dipole-velocitycomputed rotational strength values.38 Boltzmann distributions were estimated from the B97D energies. The MOLEKEL software package was used for visualization of the results.39 Cytotoxicity Assay. The cytotoxicities of the isolated compounds were evaluated against human osteosarcoma MG-63 cells and A-549 lung cancer cells using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] colorimetric method. Adriamycin was used as the positive control with IC50 values of 2.2 and 2.9 μM for MG-63 and A549 cell lines, respectively. The cytotoxicities of the compounds against the human hepatic cancer stem cells Huh7 were evaluated using the CCK-8 [Cell Counting Kit-8] colorimetric method. Adriamycin was used as the positive control with an IC50 value of 14.8 μM.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00453. Supplementary schemes, tables, and figures giving NMRcalculated values, HRESIMS and NMR spectra for 1 and 2, the low-energy conformers for 1 and 2, atom coordinates and absolute energies of the computed structures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax (W. Zhang): 86 21 81871257. E-mail: [email protected]. Author Contributions ‡

P. Sun and Q. Yu contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research work was financially supported by NSFC (81573342, U1405227, 41576157), the International S&T Cooperation Program between China and Italy (CN code 2014DFG32640; IT code CN13MO2), the Youth Program of SMMU (2014QN08), the Program of Shanghai Subject Chief Scientist (15XD1504600), the Hundred Talents Program of SMCH (XBR2013111), the key project of STCSM (14431902900), and the Hungarian National Research Foundation (OTKA K105871).

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REFERENCES

(1) Li, Y.; Liang, L.; Xiao, W.; Liang, J.; Guo, Y. Youji Huaxue 2013, 33, 1157−1166. (2) Jia, R.; Kurtán, T.; Mándi, A.; Yan, X.-H.; Zhang, W.; Guo, Y.-W. J. Org. Chem. 2013, 78, 3113−3119. (3) Chen, W.-T.; Li, J.; Wang, J.-R.; Li, X.-W.; Guo, Y.-W. RSC Adv. 2015, 5, 23973−23980. (4) Huang, C.-Y.; Sung, P.-J.; Uvarani, C.; Su, J.-H.; Lu, M.-C.; Hwang, T.-L.; Dai, C.-F.; Wu, S.-L.; Sheu, J.-H. Sci. Rep. 2015, 5, 15624. (5) Xi, Z.; Bie, W.; Chen, W.; Liu, D.; van Ofwegen, L.; Proksch, P.; Lin, W. Helv. Chim. Acta 2013, 96, 2218−2227. (6) Nguyen, X. N.; Quang, N. V.; Minh, C. V.; Hang, D. T. T.; Anh, H. L. T.; Tai, B. H.; Yen, P. H.; Hoai, N. T.; Thung, D. C.; Kiem, P. V. Nat. Prod. Commun. 2013, 8, 1209−1212. (7) Su, J.; Long, K.; Pang, T.; He, C. H.; Clardy, J. J. Am. Chem. Soc. 1986, 108, 177−178. (8) Iwagawa, T.; Hashimoto, K.; Yokogawa, Y.; Okamura, H.; Nakatani, M.; Doe, M.; Morimoto, Y.; Takemura, K. J. Nat. Prod. 2009, 72, 946−949. (9) Yan, X.-H.; Gavagnin, M.; Cimino, G.; Guo, Y.-W. Tetrahedron Lett. 2007, 48, 5313−5316. (10) Ichige, T.; Okano, Y.; Kanoh, N.; Nakata, M. J. Am. Chem. Soc. 2007, 129, 9862−9863. (11) Gong, J.; Sun, P.; Jiang, N.; Riccio, R.; Lauro, G.; Bifulco, G.; Li, T.-J.; Gerwick, W. H.; Zhang, W. Org. Lett. 2014, 16, 2224−2227. (12) Li, C.; La, M.-P.; Li, L.; Li, X.-B.; Tang, H.; Liu, B.-S.; Krohn, K.; Sun, P.; Yi, Y.-H.; Zhang, W. J. Nat. Prod. 2011, 74, 1658−1662. (13) Raju, B. L.; Subbaraju, G. V.; Reddy, M. C.; Rao, D. V.; Rao, C. B.; Raju, V. S. J. Nat. Prod. 1992, 55, 904−911. (14) Liang, L.-F.; Kurtán, T.; Mándi, A.; Gao, L.-X.; Li, J.; Zhang, W.; Guo, Y.-W. Eur. J. Org. Chem. 2014, 2014, 1841−1847. 2557



Article

(15) Demarco, P. V.; Farkas, E.; Doddrell, D.; Mylari, B. L.; Wenkert, E. J. Am. Chem. Soc. 1968, 90, 5480−5486. (16) Kusumi, T.; Igari, M.; Ishitsuka, M. O.; Ichikawa, A.; Itezono, Y.; Nakayama, N.; Kakisawa, H. J. Org. Chem. 1990, 55, 6286−6289. (17) Bifulco, G.; Dambruoso, P.; Gomez-Paloma, L.; Riccio, R. Chem. Rev. 2007, 107, 3744−79. (18) Di Micco, S.; Chini, M. G.; Riccio, R.; Bifulco, G. Eur. J. Org. Chem. 2010, 2010, 1411−1434. (19) Grimblat, N.; Zanardi, M. M.; Sarotti, A. M. J. Org. Chem. 2015, 80, 12526−12534. (20) Nadmid, S.; Plaza, A.; Lauro, G.; Garcia, R.; Bifulco, G.; Müller, R. Org. Lett. 2014, 16, 4130−4133. (21) Masullo, M.; Bassarello, C.; Bifulco, G.; Piacente, S. Tetrahedron 2010, 66, 139−145. (22) Seco, J. M.; Quiñoá, E.; Riguera, R. Chem. Rev. 2004, 104, 17− 118. (23) Bowden, B.; Coll, J.; Gulbis, J.; Mackay, M.; Willis, R. Aust. J. Chem. 1986, 39, 803−812. (24) D’Ambrosio, M.; Guerriero, A.; Pietra, F. Helv. Chim. Acta 1989, 72, 1590−1596. (25) Venkateswarlu, Y.; Chavakula, R.; Farooq Biabani, M. A.; Rao, T. P. Nat. Prod. Lett. 1994, 5, 131−134. (26) Liu, Z.; Cheng, W.; Liu, D.; van Ofwegen, L.; Proksch, P.; Lin, W. Tetrahedron 2014, 70, 8703−8713. (27) Xi, Z.; Bie, W.; Chen, W.; Liu, D.; van Ofwegen, L.; Proksch, P.; Lin, W. Mar. Drugs 2013, 11, 3186−3196. (28) Zhang, M.; Long, K.; Huang, S.; Shi, K.; Mak, T. C. W. J. Nat. Prod. 1992, 55, 1672−1675. (29) Leone, P. A.; Bowden, B. F.; Carroll, A. R.; Coll, J. C.; Meehan, G. V. J. Nat. Prod. 1993, 56, 521−526. (30) Shen, S.; Zhu, H.; Chen, D.; Liu, D.; van Ofwegen, L.; Proksch, P.; Lin, W. Tetrahedron Lett. 2012, 53, 5759−5762. (31) Maestro 10.2; Schrödinger, LLC: New York, NY, 2015. (32) MacroModel, 10.8; Schrödinger, LLC: New York, NY, 2015. (33) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision B.01; Gaussian, Inc.: Wallingford, CT, 2010. (34) Bruhn, T.; Schaumloffel, A.; Hemberger, Y.; Bringmann, G. Chirality 2013, 25, 243−249. (35) MacroModel; Schrödinger, LLC, 2012, http://www.schrodinger. com/MacroModel. (36) Grimme, S. J. Comput. Chem. 2006, 27, 1787−1799. (37) Sun, P.; Xu, D.-X.; Mándi, A.; Kurtán, T.; Li, T.-J.; Schulz, B.; Zhang, W. J. Org. Chem. 2013, 78, 7030−7047. (38) Stephens, P. J.; Harada, N. Chirality 2010, 22, 229−233. (39) Varetto, U. MOLEKEL 5.4; Swiss National Supercomputing Centre: Manno, Switzerland, 2009.

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