Uncommon Polyoxygenated Sesquiterpenoids from South China Sea

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Uncommon Polyoxygenated Sesquiterpenoids from South China Sea Soft Coral Lemnalia flava Qihao Wu, Fei Ye, Xiao-Lu Li, Lin-Fu Liang, Jiadong Sun, Han Sun, Yue-Wei Guo, and Hong Wang J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02912 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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The Journal of Organic Chemistry

Uncommon Polyoxygenated Sesquiterpenoids from South China Sea Soft Coral Lemnalia flava Qihao Wu,†,‡,# Fei Ye,‡,# Xiao-Lu Li,§ Lin-Fu Liang,⊥ Jiadong Sun,║ Han Sun,§ Yue-Wei Guo,*,†,‡ and Hong Wang*,† College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, China State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China § Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, Berlin 13125, Germany. ⊥ College of Material Science and Engineering, Central South University of Forestry and Technology, Changsha 410004, China ║ Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, Maryland 20892, USA. † ‡

Supporting Information

ABSTRACT: A detailed chemical investigation of the Chinese soft coral Lemnalia flava yielded four new nardosinane-type sesquiterpenoids (1−4), one new neolemnane-type sesquiterpenoid (5), and one new sesquiterpenoid with an uncommon 6/9 fused bicyclic skeleton (6), together with two known related compounds (7 and 8). The structures and absolute configurations of 1−8 were determined on the basis of extensive spectroscopic data analyses, X-ray diffraction analysis, chemical reactions and computerassisted structural elucidation including 13C NMR data calculation, residual dipolar coupling (RDC) based NMR analysis, and TDDFT-ECD calculation. Plausible biogenetic pathways of two uncommon sesquiterpenoids (4 and 6) were proposed. ■ INTRODUCTION

Soft corals of the genus Lemnalia have been regarded as a rich source of sesquiterpenoids and diterpenoids with intriguing structural features and various carbon skeletons.1 Many of these secondary metabolites merit further investigations due to their potent bioactivities ranging from neuroprotective1d and cytotoxic2 to anti-inflammatory3 properties. The genus Lemnalia consists 33 species which are wildly inhabited in tropical waters, in particular, in South China Sea. However, only a few of them were chemically investigated.2a, 4 The title animal L. flava, is abundant in South China Sea and has been an intensive research subject for marine natural product chemists because of its structurally diversified secondary metabolites.1a In the course of our continuing effort to explore chemically fascinating and biologically active secondary metabolites of invertebrates from South China Sea,5 we have collected two samples (No. 13XS-52, and No. 13XS-28) of the soft coral L. flava, in the same area but different locations near the Xisha

Island. An exhaustive chemical investigation of these two samples has resulted in the isolation and identification of six nardosinane-type sesquiterpenoids (1−4, 7−8) including four new ones, one new neolemnane-type sesquiterpenoid (5), and one novel sesquiterpenoid lactone (6) featuring an uncommon 6/9 fused bicyclic skeleton. Herein, we report the isolation, stereostructural elucidation, and bioactivity evaluation of these isolates. Plausible biogenetic pathways of these sesquiterpenoids were also proposed. ■ RESULT AND DISCUSSION

The frozen bodies of two collections were cut into pieces and extracted with acetone for six times. The Et2O-soluble portion of the acetone extracts were chromatographed

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(δC 172.9, qC) in 1. Due to the oxidation at C-12, the 13C NMR chemical shifts of the carbons at C-10 (δC 82.2, qC), and C-11 (δC 34.7, CH) were apparently downfield-shifted (Δδ= +7.1 and +8.1 ppm, respectively) comparing to those of paralemnolin R (Table 2). Furthermore, analysis of NOE of 1 and comparison with those of 8 indicated that the relative configurations at C-1, C-4, C-5, C-6, C-10, C-11 of both compounds were identical (Figure 3). Thus, 1 was determined as 12-oxo derivative of paralemnolin R (8).

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Figure 1. Structures of compounds 1−10. sequentially over silica gel, Sephadex LH-20, and RP-HPLC to yield pure sesquiterpenoids 1 (4.7 mg), 2 (2.4 mg), 3 (2.1 mg), 4 (5.0 mg), 5 (5.6 mg), 6 (4.1 mg), 7 (20.0 mg), and 8 (4.8 mg) (Figure 1). The known compound 7 was identified as nardosinanol A, a nardosinane-type sesquiterpenoid that previously isolated from south Kenya soft coral Lemnalia sp., by comparing its NMR spectroscopic data and specific optical rotation with those reported in the literature.6 The structural identification of compound 8 was straightforward. The X-ray diffraction (XRD) analysis of the crystal form of 8 (by employing Ga Kα radiation; λ= 1.34139 Å) unambiguously recognized its structure to be the same as paralemnolin R7 (Figure 2) and its absolute configuration (AC) was defined as 1R,4R,5S,6R,10S,11R. It is notable that this is the first time that the AC of 8 was defined.

Figure 2. Perspective ORTEP drawing of X-ray structure of 8 (ellipsoids shown at the 50 % probability level). New compounds 1−4 showed similar NMR spectra as those of 7 and 8 indicating that they are also nardosinane-type sesquiterpenoids. In fact, they differ from each other only by the oxidative patterns within the nardosinane skeleton. Xishaflavalin A (1) was isolated as an optically active colorless oil {[α]20 D −28 (c 0.1, CHCl3)}. Its molecular formula, C15H20O4, was established by HREIMS at m/z 264.1355 ([M]+; calcd. 264.1356), 14 mass units more than that of the co-occurring sesquiterpenoid 8, indicating five degrees of unsaturation. The IR spectrum of 1, like 8,7 showed the absorptions of α,β-unsaturated ketone (νmax 1680 cm-1), and hydroxy (νmax 3417 cm-1) groups. In addition, the new compound 1 showed IR absorption indicative of the presence of ester carbonyl group (1720 cm-1). The 1H- and 13C-NMR data of 1 were very similar to those of 8 except for the signals assigned to C-12, where the oxygenated sp3 methylene carbon (δC 64.6, CH2) in 8 was replaced by an ester carbonyl carbon

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Figure 3. 1H- 1H COSY, key HMBC and NOESY correlations of compound 1. Xishaflavalin B (2) was isolated as an optically active colorless oil {[α]20 D −95 (c 0.1, CHCl3)}. The molecular formula of 2 was determined to be C15H22O3 by HREIMS (m/z 250.1559 [M]+, calcd. 250.1563), indicating five degrees of unsaturation. The 1H NMR data of 2 showed typical signals (δH 5.24, dt, J= 9.6, 2.4 Hz; δH 5.97, dt, J= 9.6, 3.6 Hz) assignable to a cis 1,2-disubstituted double bond. Besides, the signals of two secondary methyls (δH 1.06, d, J= 6.6 Hz; δH 1.24, d, J= 6.6 Hz) and a tertiary methyl (δH 0.99, s) were observed, indicating that compound 2 is a nardosinane-type sesquiterpenoid. The 13C NMR data of 2 showed the presence of 15 carbons, which were assigned to three sp3 methyls, four sp3 methylenes (including a mono-oxygenated carbon resonating at δC 77.9), three sp3 methines, three sp3 quaternary carbons (including a mono-oxygenated carbon resonating at δC 79.2 and a ketal carbon resonating at δC 109.8), and two sp2 methines of an olefin. The data above accounted for one of the five degrees of unsaturation, indicating a tetracyclic structure for 2. Detailed analysis of the 1H- 1H COSY spectrum allowed to establish three spin systems from H-1 to H3-14, H-6 (δH 1.63, d, J= 10.2 Hz) to H3-13 and H2-12 (δH 4.30, t, J= 8.4 Hz; δH 3.54, t, J= 7.8 Hz) through H-11 (δH 2.74, m), and H2-8 (δH 2.23, m, δH 1.82, td, J= 12.0, 3.6 Hz) to H2-9 (δH 2.28, m; δH 2.10, m). The HMBC experiment showed the following correlations: H-2 to C-10 (δC 79.2, qC); H-4 (δH 2.78, m) to C5 (δC 42.6, qC), C-15 (δC 20.8, CH3); H-6 to C-5, C-15, C-4 (δC 29.1, CH); H2-8 to C-6 (δC 60.6, CH), C-7 (δC 109.8, qC), C-10; H3-15 to C-5, C-4, C-6, C-10 allowing to construct rings A−C with a C-15 methyl bearing at C-5 (Figure 4). Subtraction of above elaborated rings A−C from the molecular formula of 2, two oxygen atoms remain to be assigned. Bearing in mind that the remaining one degree of unsaturation, along with the existence of C-10 mono-oxygenated carbon and the C-7 ketal carbon, a peroxide bridge should be lying between C-7 and C-10 to form the planar structure of compound 2. The relative configuration of 2 was determined on the basis of NOESY correlations, as shown in Figure 4. The remarkable NOESY correlations from H-6 to H3-15, H3-13, and H3-14 placed three methyls (H3-13, H3-14 and H3-15) and H-6 on the


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same face (β-orientation). Additionally, the obvious correlations from H3-15 to H-9a (δH 2.28, m) and H-6 to H-8b (δH 1.82, td, J= 12.0, 3.6 Hz) placed the peroxide group as αorientation.

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Figure 4. 1H- 1H COSY, key HMBC and NOESY correlations of compound 2. In order to corroborate the proposed relative configuration of 2, the theoretical calculation of 13C NMR chemical shifts of epimers 2a (α-orientation for peroxide group) and 2b (βorientation for peroxide group) were carried out by geometry optimization at the DFT(B3LYP)/6-31G(d) level followed by computation of the NMR shielding with the standard gaugeincluding atomic orbital (GIAO) approach at the lager basis functions of 6-311+G(d,p), using the Gaussian 09 program8. The calculated shifts for two conformers, 2a and 2b, were corrected by the slope and intercept to get the corrected 13C NMR chemical shifts, and the differences between the corrected and experimental 13C NMR chemical shifts were analyzed (Figure 5). The result showed that the correlation coefficient R2 of 2a (0.9856) was higher than that of 2b (0.9756). Meanwhile, the MAE (mean absolute error) and MD (maximum deviation) of 2a (MAE= 4.06, MD= 8.7) were lower than that of 2b (MAE= 5.06, MD= 10.3), indicating that the calculated shifts of 2a was more consistent with the experimental values. Thus, the peroxide group was unequivocally assigned to be α-orientation as depicted.

Xishaflavalin C (3) was obtained as an optically active colorless oil {[α]20 D +328 (c 0.1, CHCl3)}. The HRESIMS of 3 exhibited an ion peak at m/z 265.1444 [M−H]-, consistent with the molecular formula C15H22O4 which required five degrees of unsaturation. The IR spectrum of 3 showed absorptions at νmax 1722 cm-1, and νmax 3385 cm-1, indicating the presence of the ester and hydroxy groups, respectively. The NMR spectroscopic data exhibited the presence of one aldehyde group (δH 9.66, s, δC 199.4, CH), one trisubstituted double bond (δH 5.48, dd, J= 5.4, 2.4 Hz, δC 125.6, CH; δC 135.0, qC). The above functionalities account for two of the five degrees of unsaturation, suggesting a tricyclic structure in 3. The above structural features were reminiscent of previously reported paralemnolin O (9), which was isolated from the Formosan soft coral Paralemnalia thyrsoide.9 The comparison of NMR data revealed the considerable similarity between compounds 3 and 9, possessing the same nardosinane ring system. The only significant differences of these two compounds were the oxygenated sp3 methine at C-7 (δC 71.9, CH) in 99 was replaced by a hemiketal group (δC 107.2, qC) in 3, the chemical shifts of carbons at C-11 and C-6 (δC 92.3, qC; δC 71.6, CH) were apparently downfield-shifted compared with the model compound (δC 80.5, qC, Δδ= +11.8 ppm; δC 50.2, CH, Δδ= +21.4 ppm). By considering the molecular formula, the 13C NMR chemical shifts (Table 2), and the HMBC correlations (Figure 6), the planar structure of 3 possessing a peroxide group between C-7 and C-11, was fully established. The relative configuration of 3 was also determined by careful interpretation of its NOESY spectrum with the correlations of H3-14 (δH 0.90, d, J= 6.6 Hz)/H3-15 (δH 1.11, s), H-6 (δH 2.97, s)/H3-15, H3-13 (δH 1.43, s)/H3-14, and H-6/H313 (Figure 6), except for the chiral center at C-7, as it is a hemiketal group and no NOE could be detected. To tackle this problem, we decided to perform RDC-based NMR analysis10 to assign the relative configuration of 3. 9

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Figure 6. 1H- 1H COSY, key HMBC and NOESY correlations of compound 3. There are 5 stereocenters in compound 3 resulting in 16 possible relative configurations. Strong NOEs described above indicated that H-6, H3-14 and H3-15 shared the same orientation, reducing the possible relative configuration to be 4S*5S*6S*7S*11S*, 4S*5S*6S*7S*11R*, 4S*5S*6S*7R*11S*, and 4S*5S*6S*7R*11R* respectively. For the RDC analysis, compound 3 was aligned in a selfassembled oligopeptide (AAKLVFF) phase, which has been recently proposed as a liquid crystalline based alignment medium that is compatible with MeOH.11 The concentration of AAKLVFF is about 36 mg/mL, leading to a corresponding quadrupolar splitting of 26.0 Hz (Figure S1, Supporting Information (SI)). 1DCH RDCs were measured as the


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difference (D = T − J) between the couplings measured in the anisotropic (T) and isotropic (J) samples using the [1H-13C]CLIP-HSQC experiment (Figure S2).12 Altogether we were able to extract 9 1DCH values (Table S2) ranging from −43.2 to 45.5 Hz with high accuracy. The conformational space of the compound 3 was explored by conformational search at the OPLS3 level implemented in the program Schrödinger.13 Below an energy threshold of 21 kJ/mol 8 structures were generated. Superimposing all structures revealed two main conformations of the backbone ring system (Figure 7). Strong NOE of H-4 and H-12 while very weak NOE of H-4 and H3-13 suggested that conformers 4 , 5, and 6 as the main conformation. Experimental RDCs of C1-H1, C2-H2a, C2-H2b, C4-H4, C6-H6, C9-H9a, C13-H13, C14-H14 and C15-H15 were used to fit to the DFT-optimized structure of four possible configuration. DFT optimizations were performed at the B3LYP/6-31G(d) level of theory using Gaussian09.14 Alignment tensor of each structure was calculated by singular value decomposition (SVD) method as implemented in the RDC module of the MSpin program.15 Theoretically predicted RDCs were determined from the computed alignment tensor and further compared with the experimentally calculated ones. Q-factor was used to evaluate the results as it can be considered as a quantitative assessment of the quality of the fit between experimental and backcalculated RDC values. Among all possible configurations, configuration 1 (4S*5S*6S*7S*11S*) has lowest Q factor with an excellent fit between experimental and back-calculated RDCs. Comparison of two configurations with lowest Q factors (configurations 1 and 2) indicated that only configuration 1 (4S*5S*6S*7S*11S*) has a full agreement with the J-coupling and NOE data (Figure 8 and Table S3). Combining all results, the relative configuration of compound 3 was determined as 4S*,5S*,6S*,7S*,11S*.

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Xishaflavalin D (4) was obtained as a yellow oil {[α]20 D 21 (c 0.1, CHCl3)}. The molecular formula was determined as C15H19NO by HRESIMS (m/z 230.1539 [M+H]+, calcd. 230.1539) indicating seven degrees of unsaturation. The IR spectrum of 4 displayed strong absorption at νmax 1640 cm-1, bearing in mind the odd molecular weight, suggesting the presence of lactam carbonyl group, which was verified by the UV absorptions at λmax 273 nm (log ε= 3.7) (Figure S47). The NMR spectroscopic data displayed the presence of two trisubstituted double bonds (δH 5.54, m, δC 122.1, CH; δC 138.5, qC and δH 5.60, dd, J= 6.0, 3.0 Hz, δC 109.3, CH; δC 137.2, qC), one tetrasubstituted double bond (δC 151.0, qC; δC 121.8, qC). The above functionalities account for four out of the total seven degrees of unsaturation, suggesting a tricyclic structure in 4. In addition, the COSY spectrum revealed two proton sequences from H-1 to H3-14 (δH 1.05, d, J= 6.6 Hz), H-8 to H2-9 (δH 3.18, d, J= 18.6 Hz; δH 2.67, dd, J= 18.6, 6.0 Hz) indicated the presence of the same ring A as the cooccurring 3 (Figure 9). In addition, the HMBC cross-peaks between H3-13 (δH 1.99, s) and C-11, C-6, C-12, as well as amide proton (δH 7.52 s) and C-12, C-11, C-6, C-7 revealed the presence of an α,β-unsaturated γ-lactam at C-11 (α), C-6 (β), C-7 (γ), and C-12 (C=O). The NOESY correlations of H314 with H3-15 (δH 1.20, s) reflected the same β-orientation of two methyls at C-4 and C-5 (Figure 9). On the basis of the above findings, the planar structure of 4 was fully established. 1 2 3

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Figure 9. 1H- 1H COSY, key HMBC and NOESY correlations of compound 4.

Figure 7. The 8 conformers of compound 3 showed by state (a) and by object (b). The conformational research used the program Schrödinger below an energy threshold of 21 kJ/mol. means the position of H-12 in conformers 1−3 and 7−8; means the position of H-12 in conformers 4−6.

Figure 8. The DFT-optimized structures of configuration 4S*5S*6S*7S*11S* (pink) and 4S*5S*6S*7S*11R* (yellow) at the B3LYP/6-31G(d) level.

Based on the assignment of relative configurations of compounds 1−4, we further decided to perform the timedependent density functional theory electronic circular dichroism (TDDFT-ECD) calculation to assign the ACs of 1−4, since TDDFT-ECD calculation is a powerful and reliable tool for the ACs determination of the natural products with chiral centers near the chromophore groups.16 However, the CD spectrum showed no obvious cotton effect (CE) for compound 2 due to lack of strong chromophore near the chiral centers. Thus, only compounds 1, 3 and 4 were carried out for the TDDFT-ECD calculation. As shown in Figures 10−12, multiple CEs occurred for these three compounds, the ECD spectra (MeCN) of compound 1 displayed a negative CE at 230 nm, compound 3 showed positive CEs at 224 nm and 312 nm, compound 4 displayed a negative CE at 260 nm and positive CE at 280 nm. The initial torsional sampling (MCMM) and OPLS_2005 force field conformational searches of (1R,4S,5S,6R,10R,11S)-1, (4S,5S,6S,7S,11S)-3 and (4S,5R)-4 were performed and afforded 7, 4 and 3 conformers, respectively, within the 21 kJ/mol energy window. The Boltzmann populations for all the conformers were calculated based on the potential energy from the OPLS_2005 force field, which afforded 3 conformers for 1, 4 conformers for 3


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and 2 conformers for 4 above 1% population for further reoptimization. To verify that the re-optimized geometries were at the energy minima, the above geometries were reoptimized (B3LYP/6-311G (d,p) level with IEFPCM solvent model for CH3CN), and frequency analysis was also performed. Detailed comparison of the Boltzmann-averaged ECD spectra with those of the experimental ones disclosed that the ACs of 1, 3 and 4 are 1R,4S,5S,6R,10R,11S, 4S,5S,6S,7S,11S, and 4S,5R, respectively, which could be further confirmed by completely opposite curves of those of their enantiomers to the calculated (Figures 10−12). Since the AC of several nardosinanes isolated from the soft coral of the genera Lemnalia1a including co-occurring compounds presented herewith, had been determined, it was therefore, on the basis of biogenetic consideration, to assign the ACs at C-4 and C-5 of 2 being identical as illustrated in compounds 1, 3, 4 and 8. As a consequence, the absolute stereochemistry of 2 was tentatively assigned as 4S,5S,6R,7S,10S,11S. Calculated ECD of (1R,4S,5S,6R,10R, 11S)-1 Mirrored spectrum of calculated ECD Experimental ECD of xishaflavalin A (1)

Figure 10. Experimental ECD spectrum of xishaflavalin A (1) (black), the calculated ECD spectra of 1 (red) and their enantiomers (blue), respectively. Calculated ECD of (4S,5R)-2

Mirrored spectrum of calculated ECD Mirrored spectrum of calculated Experimental ECD of ECD lemnalianoid B (2)

Calculated ECD of (4S,5S,6S,7S,11S)-3

Experimental ECD of xishaflavalin C (3)

Figure 11. Experimental ECD spectrum of xishaflavalin C (3) (black), the calculated ECD spectra of 3 (red) and their enantiomers (blue), respectively.

Calculated ECD of (4S,5R)-4

Calculated ECD of (4S,5R)-2

Mirrored spectrum of calculated Mirrored spectrum of calculated ECD ECD Experimental ECD of ECD of Experimental xishaflavalin D (4) lemnalianoid B (2)

Figure 12. Experimental ECD spectrum of xishaflavalin D (4) (black), the calculated ECD spectra of 4 (red) and their enantiomers (blue), respectively. Xishaflavalin E (5) was obtained as a colorless oil {[α]20 D +110 (c 0.1, CHCl3)}. The molecular formula of 5 was determined to be C15H24O2 by HREIMS (m/z 236.1779 [M]+, calcd. 236.1771), indicating four degrees of unsaturation. The IR spectrum of 5 showed the presence of hydroxyl groups (νmax 3300−3500 cm-1). In addition, the 13C NMR and DEPT spectra of 5 showed signals of 15 carbons (Table 2), including two trisubstituted double bonds (δC 123.7, CH, δC 144.8, qC; δC 136.9, CH, δC 133.4, qC). The above functionalities account for two out of the four degrees of unsaturation, suggesting a bicyclic structure in 5. Analysis of COSY and HMBC spectra, aided by the comparison with co-occurring molecules 3, 4 and model compound 10, paralemnolin D, a neolemnane-type sesquiterpenoid previously isolated from P. thyrsoides,17 revealed that compound 5 possessed the common ring A part as 3 and 4. The only significant differences of the NMR spectroscopic data between 5 and 10 were confined to the remarkable upfield-shifted of H-8 (from δH 6.49 to δH 5.14) and C-8 (from δC 76.3 to δC 71.6) and the relatively small upfield-shifted of H-9 (from δH 3.93 to δH 3.62) and small downfield-shifted C-9 (from δC 72.1 to δC 73.4) (Table 2).17 All these data were easily rationalized by the deacetylation at C-9. Finally, the location of the hydroxyl groups at C-8 and C9 in 5 was further confirmed by the clear HMBC correlations from H-8 to C-7, C-6, C-9, C-14 and C-10, from H-9 to C-11, C-8 and C-7 (Figure 13). Thus, the planar structure of 5 was established. The relative configuration of 5 was elucidated from the correlations observed in NOESY experiment (Figure 13). The Z geometry of the 6,7-trisubstituted double bond was established by the correlation between H-6 and H3-14. Also, the correlations of H3-15 (δH 0.97, s)/H3-14 (δH 0.89, d, J= 7.0 Hz), H3-15/H-11β (δH 2.16, m), H-11β/H-9 and H-4 (δH 1.65, m)/H-8 were found, while no obvious correlation could be observed between H-8 and H-9, suggesting that the two methyls (H3-14 and H3-15) and H-9 should be on the same face (β-orientation) and both H-4 and H-8 should be on the αorientation.



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HMBC

NOESY

1H-1H

Figure 13. COSY, key HMBC, and NOESY correlations of compound 5. The determination of the absolute stereochemistry of 5 is worth discussing. Bearing in mind the ACs of co-occurring nardosinane-type sesquiterpenoids (1−4, 8), the AC at C-4 and C-5 of 5 could be determined. Consequently, the AC of 5 was assigned to be the same as compound 10 based on biogenetic consideration.17 However, to confirm this hypothesis, the following chemical reactions were employed. Compound 5 was firstly reacted with PPTS and 2,2-DMP in acetone18 to give dioxolane 5a (Figure S6). The remarkable NOE correlation between H-8 and H3-17, H-9 and H3-18 in 5a confirmed that the two hydroxyl groups are of the opposite orientation (Figure 14, Figure S8). NOESY

O O

18

9 8

5a

17

Figure 14. NOESY correlations of dioxolane 5a. Then, the AC of the 8,9-diol unit in 5 was assigned by Snatzke's method by measuring the Mo2(OAc)4 induced circular dichroism (ICD) of Mo-complex of 5.19 The ICD spectrum of the Mo-complex of 5 showed a positive CE at 310 nm (Figure 15), which could be attributed to the chirality of the vic-diol group at C-8 and C-9. With the aid of the relative configuration established by the NOESY data, the AC (4S,5S,8S,9S) of 5 was unambiguously assigned as depicted in Figure 1.

Page 6 of 11

and 13C NMR analysis established the molecular formula of 6 as C15H22O3 (m/z 250.1582 [M]+, calcd. 250.1563), requiring five degrees of unsaturation. The IR spectrum showed the presence of ketone carbonyl (νmax 1710 cm-1) and ester carbonyl (1740 cm-1) functionalities, and absence of hydroxyl group. The 13C NMR and DEPT spectra of 6 displayed 15 carbon signals: three sp3 methyls, five sp3 methylenes, two sp3 methines, one sp3 quaternary carbons, one sp2 methines, three sp2 quaternary carbons. From the more detailed analysis of the 13C NMR spectrum of 6, the typical downfield resonances at δC 216.2 and δC 174.0 further verified the presence of ketone and ester functionalities, respectively. Furthermore, one trisubstituted carbon-carbon double bond (δC 129.9, CH; δC 135.8, qC) was also recognized in the 13C NMR spectrum. The above functionalities account for three of the five degrees of unsaturation, suggesting a bicyclic structure of 6. Comparison of the NMR spectral data of 6 with those of 5 disclosed that they should possess the similar carbon skeleton with the variation at the ring B. The analysis of the 2D NMR data allowed to assign the planar structure of 6. As depicted in Figure 16, the 1H- 1H COSY spectrum revealed the presence of three structural fragments a−c by clear correlations of H-1 (δH 5.94, dt, J= 6.0, 2.0 Hz)/H2-2 (δH 2.18, m; 2.13, m)/H2-3 (δH 1.56, m; 1.26, qd, J= 12.0, 5.5 Hz)/ H-4 (δH 1.97, m)/H3-14 (δH 0.74, d, J= 7.0 Hz) (a); H3-13 (δH 0.92, d, J= 7.0 Hz)/H-7 (δH 3.42, m)/H2-8 (δH 4.53, dd, J= 10.0, 5.0 Hz; δH 3.81, dd, J= 11.5, 10.0 Hz) (b); H2-10 (δH 2.54, m; δH 2.40, m)/H2-11 (δH 2.24, m; δH 2.04, m) (c), respectively. Bearing in mind the presence of the ketone and ester functionalities, careful interpretation of the well resolved HMBC correlations connected the three spin systems (a−c), enabling the construction of the planar structure of 6. Fortunately, the single crystals of 6 were obtained in petroleum ether and Et2O (4: 1) and subjected to an X-ray diffraction experiment with Ga Kα radiation. The detailed structure and AC of 6 could be established unambiguously from XRD analysis (Figure 17).

c 10

O

11

1 12

2

9

O

5 3

a

6

4 15 14

O 1H- 1H

7

b

8

14

15

13

13

COSY

HMBC

NOESY

Figure 16. 1H-1H COSY, key HMBC, and NOESY correlations of compound 6.

Figure 15. CD spectrum of Mo-complex of 5 measured in DMSO (the Newman projection describes the conformation of the vic-diol at C-8 and C-9 in the Mo-complex of 5). Xishaflavalin F (6) was obtained as an optically active white powder {[α]20 D +58 (c 0.1, CHCl3)}. The HREIMS

Structurally, compound 6 presented an uncommon sesquiterpenoid skeleton which was constructed by a sixmembered ring (ring A, Figure 1) conjugated with a ninemembered ring macrolide (ring B, Figure 1). It was recognized that 6 possessed an unexpected chemical interaction with the co-occurring 7. It was proposed that compound 6 was transformed from 7 by Δ6/7 double bond oxidation. In order to determine the biosynthetic origin of the uncommon sesquiterpenoid 6, the chemical transformation between compound 6 and its proposed precursor 7 was carried out. The Δ6/7 double bond of 7 was oxidated with the existence of metachloroperoxybenzoic acid (m-CPBA) in dichloromethane (DCM) for 2h in room temperature.20 The NMR and optical


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rotation data of the product was consistent with those of 6 (Figures S11 and S12). In addition, compound 7 was previously isolated and identified from the same genus of the title animals by Kashman’s group in 2008,6 however, the AC of 7 in the literature was tentatively proposed on the basis of biogenetic consideration. In our case, for the first time, the AC of 7 was unambiguously elucidated as 4S,5R,11S by using chemical reaction.

The structural features of xishaflavalins D and F (4 and 6) are very different from the co-occurring nardosinane-type and neolemnane-type sesquiterpenoids. But interestingly, 4 and 6 share the same ring A of aforementioned sesquiterpenoids, inspiring us to explore the biogenetic origin of 4 and 6. Further analysis of the structures of 4 and 6 allowed us to raise a plausible biogenetic connection starting from the possible precursor 1(10)-aristolene. As outlined in the Scheme 1, the possible intermediate ii was generated by double bond migration and oxidation of i. The resulting carboxylic acid moiety was attacked by the lone pair electrons of nitrogen atom followed by the amidation and dehydration to give the final product 4. The generation of 7 involves the hydration of intermediate i followed by a cyclization and dehydration. Then, as described in the chemical reaction section, further oxidation of 7 could generate 6. All the isolated compounds were evaluated for cytotoxic, and anti-inflammatory effects. However, none of them showed positive effects in the aforementioned assays (Table S7 and S8).

Figure 17. Perspective ORTEP drawing of X-ray structure of 6 (ellipsoids shown at the 50 % probability level). Table 1. 1H NMR Data (δ in ppm, J in Hz) for compounds 1−6 in CDCl3. No. 1a 2a 3a 4a 5b 1 4.03 brt 5.24 dt (9.6, 2.4) 5.48 dd (5.4, 2.4) 5.54 m 5.48 t (4.0) 2a 2.14 m 5.97 dt (9.6, 3.6) 1.97 m 2.10 m 2.01 m 2b 1.76 m 1.86 m 1.85 m 3a 1.74 m 2.30 m 1.40 m 1.62 m 1.55 m 3b 1.46 m 1.72 m 1.36 m 1.53 m 1.41 m 4 2.10 m 2.78 m 1.80 m 2.71 m 1.65 m 5 6 2.68 d (6.6) 1.63 d (10.2) 2.97 s 5.22 s 7 8a 6.13 d (9.6) 2.23 m 2.18 m 5.60 dd (6.0, 3.0) 5.14 d (9.0) 8b 1.82 td (12.0, 3.6) 2.05 m 9a 6.95 d (9.6) 2.28 m 2.52 m 3.18 d (18.6) 3.62 m 9b 2.10 m 2.37 m 2.67 dd (18.6, 6.0) 10a 2.18 m 10b 1.71 m 11a 2.94, m 2.74 m 2.32 m 11b 2.16 m 12a 4.30 t (8.4) 9.66 s 12b 3.54 t (7.8) 13 1.14, d (7.2) 1.24 d (6.6) 1.43 s 1.99 s 1.71 d (1.5) 14 0.87, d (6.6) 1.06 d (6.6) 0.90 d (6.6) 1.05 d (6.6) 0.89 d (7.0) 15 1.18, s 0.99 s 1.11 s 1.20 s 0.97 s NH 7.52 s a Recorded at 600 MHz in CDCl , chemical shifts (ppm) referred to CHCl (δ 7.26). b Recorded at 500 MHz in CDCl , chemical 3 3 H 3 CHCl3 (δH 7.26). Assignments were deduced by analysis of 1D and 2D NMR spectra.

6b 5.94 dt (6.0, 2.0) 2.18 m 2.13 m 1.56 m 1.26 qd (12.0, 5.5) 1.97 m 3.42 m 4.53 dd (10.0, 5.0) 3.81 dd (11.5, 10.0) 2.54 m 2.40 m 2.24 m 2.04 m 0.92 d (7.0) 0.74 d (7.0) 1.00 s shifts (ppm) referred to

Table 2. 13C NMR Data (δ in ppm) for compounds 1−10 in CDCl3. No. 1a 2a 3a 4a 5a 1 71.5 CH 123.5 CH 125.6 CH 122.1 CH 123.7 CH 2 29.2 CH2 137.6 CH 25.5 CH2 20.9 CH2 24.4 CH2 3 23.7 CH2 35.3 CH2 26.5 CH2 27.5 CH2 26.9 CH2 4 31.0 CH 29.1 CH 33.0 CH 31.8 CH 39.4 CH 5 41.3 qC 42.6 qC 38.6 qC 42.4 qC 43.1 qC 6 57.1 CH 60.6 CH 71.6 CH 151.0 qC 136.9 CH 7 200.1 qC 109.8 qC 107.2 qC 137.2 qC 133.4 qC 8 129.9 CH 29.1 CH2 28.7 CH2 109.3 CH 71.6 CH 9 149.6 CH 28.6 CH2 25.4 CH2 32.6 CH2 73.4 CH 10 82.2 qC 79.2 qC 135.0 qC 138.5 qC 34.7 CH2 11 34.7 CH 32.3 CH 92.3 qC 121.8 qC 29.2 CH2 12 172.9 qC 77.9 CH2 199.4 CH 172.9 qC 144.8 qC 13 14.5 CH3 20.0 CH3 18.8 CH3 9.6 CH3 19.5 CH3 14 14.0 CH3 18.5 CH3 16.4 CH3 15.1 CH3 16.1 CH3 15 16.7 CH3 20.8 CH3 22.0 CH3 23.0 CH3 23.0 CH3 a Recorded at 150 MHz in CDCl , chemical shifts (ppm) referred to CHCl (δ 77.2). b Recorded at 125 MHz in CDCl , chemical 3 3 C 3 CHCl3 (δC 77.2). Assignments were deduced by analysis of 1D and 2D NMR spectra.


6a 129.9 CH 26.3 CH2 26.1 CH2 33.3 CH 61.3 qC 216.2 qC 39.6 CH 68.0 CH2 174.0 qC 35.8 CH2 28.4 CH2 135.8 qC 14.5 CH3 17.3 CH3 14.7 CH3 shifts (ppm) referred to


O

O

COOH

C OH O

singlet O2 ene cyclopropane ring open H oxidation

oxidation

ii

NH3

O

H

Page 8 of 11 Enamine formation O

O H 2N O -

C NH2 O

OH2

hydration

1(10)-aristolene

O

O

CH2OH

CH2OH

■ CONCLUSION

The chemical investigation of soft coral L. flava collected off Xisha Island, South China Sea, China, led to the isolation and identification of six new sesquiterpenoids and two related known ones. Among them, xishaflavalin F (6) represents an uncommon sesquiterpenoid skeleton containing a rare ninemembered ring lactone and the AC of compound 6 was determined by XRD analysis unambiguously. Besides, four new (1−4) and two known (7−8) nardosinane-type sesquiterpenoids were identified. In fact, nitrogen-containing sesquiterpenoid are rarely found in nature, xishaflavalin D (4) is a rare nitrogenous secondary metabolite from Lemnalia and the first example for nitrogen-containing nardosinane-type sesquiterpenoid. Besides, xishaflavalins B and C (2−3) represent the only peroxide bridge containing secondary metabolites from Lemnalia. The discovery of xishaflavalins (1−6) has expanded the chemical diversity and complexity of sesquiterpenoids from marine origin and may inspire further synthetic studies due to their intriguing structural features. The biogenetic origins and ecological roles of xishaflavalins in the life cycle of the soft coral warrant further investigation. ■ EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an SGW X-4 melting point instrument and were uncorrected. IR spectrum was recorded on a Nicolet iS50 spectrometer (Thermo Fisher Scientific, Madison, U.S.A.). Optical rotations were measured on a PerkinElmer 241MC polarimeter. 1H and 13C NMR spectra were acquired on a Bruker AVANCE III 500 and 600 spectrometer. Chemical shifts are reported with the residual CHCl3 (δH 7.26 ppm) as the internal standard for 1H NMR spectrometry, and CDCl3 (δC 77.0 ppm) for 13C NMR spectrometry. The LREIMS and HREIMS data were recorded on a Finnigan-MAT-95 mass spectrometer (Finnigan-MAT, San Jose, CA, U.S.A.). HRESIMS spectra were recorded on Agilent G6250 Q-TOF (Agilent, Santa Clara, CA, U.S.A.). Commercial silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 200˗300 mesh, 300˗400 mesh) was used for column chromatography, and precoated silica gel GF254 plates (Sinopharm Chemical Reagent Co., Shanghai, China) were used for analytical TLC. Sephadex LH-20 (Pharmacia, USA) was also used for column chromatography. Reversed-phase (RP) HPLC was performed on an Agilent 1260 series liquid chromatography equipped with a DAD G1315D detector at 210 nm (Agilent, Santa Clara, CA, U.S.A.). An Agilent semipreparative XDB-C18 column (5 μm, 250×9.4 mm) was employed for the purification. All solvents used for column chromatography and HPLC were of analytical grade

O O

O

-H2O

O 7

Scheme 1. Plausible biosynthetic pathway of 4 and 6.

O 4

double bond oxidation cleavage

Enol ether formation

i

NH

-H2O

6

(Shanghai Chemical Reagents Co., Ltd.) and chromatographic grade (Dikma Technologies Inc.), respectively. Collection of Biological Materials. The soft coral L. flava, a voucher specimen No. 13XS-52 was collected by scuba at a depth of –15 m from Xisha Island, South China Sea, China, in 2013. The other collection (No. 13XS-28) was collected by scuba at a depth of -15 m from different location but same sea area and same time. The voucher specimen with two different collections (No. 13XS-52 and No. 13XS-28) are available for inspection at the Shanghai Institute of Materia Medica, CAS. Extraction and Isolation. Extraction of L. flava 13XS-52 and Isolation of Xishaflavalins B–F (2–6), Nardosinanol A (7), and Paralemnolin R (8) The frozen animals (350 g, dry weight) were cut into pieces and extracted exhaustively with acetone at room temperature (6 × 2.0 L). The organic extract was evaporated to give a brown residue, which was then partitioned between H2O and Et2O. The upper layer was concentrated under reduced pressure to give a brown residue 8.0 g. The resulted residue was separated into seven fractions (A–G) by gradient Silica-gel column chromatography. The resulting fractions were then fractionated into sub-fractions by Sephadex LH-20. The sub-fraction E7 was purified by Semipreparative HPLC (80 % MeCN to 100 % MeCN in 20 min), yielding compound 3 (2.1 mg). The sub-fraction F4 of fraction F gave compound 6 (4.1 mg) and compound 2 (2.4 mg) while sub-fraction F5 gave compound 8 (20.0 mg). Compound 5 (1.7 mg) and compound 7 (4.8 mg) were obtained from subfraction F7. Finally, the sub-fraction G7 of fraction G gave compound 4 (3.0 mg). Extraction of L. flava 13XS-28 and Isolation of Xishaflavalins A (1), D (4), E (5). The frozen animals (144 g, dry weight) were cut into pieces and extracted exhaustively with acetone at room temperature (6 × 2.0 L). The organic extract was evaporated to give a brown residue, which was then partitioned between H2O and Et2O. The upper layer was concentrated under reduced pressure to give a brown residue 3.5 g. The isolation procedure described above was applied and resulted three purified compounds including xishaflavalin D (4, 2.0 mg), xishaflavalin A (1, 4.7 mg), and xishaflavalin E (5, 3.9 mg). Xishaflavalin A (1). Colorless oil; {[α]20 D−28 (c 0.1, CHCl3)}; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HREIMS: m/z calcd for C15H20O4 [M]+: 264.1355; found: 264.1356. Xishaflavalin B (2). Colorless oil; {[α]20 D −95 (c 0.1, CHCl3)}; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HREIMS: m/z calcd for C15H22O3 [M]+: 250.1563; found: 250.1559. Xishaflavalin C (3). Colorless oil; {[α]20 D+328 (c 0.1, CHCl3)}; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS: m/z calcd for C15H21O4 [M-H]-: 265.1445; found: 265.1444. Xishaflavalin D (4). Yellow oil; {[α]20 D −21 (c 0.1, CHCl3)}; for 1H and 13C NMR spectroscopic data, see Tables


Page 9 of 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 and 2; HRESIMS: m/z calcd for C15H19NO [M+H]+: 230.1539; found: 230.1539. Xishaflavalin E (5). Colorless oil; {[α]20 D+110 (c 0.1, CHCl3)}; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HREIMS: m/z calcd for C15H24O2 [M]+: 236.1771; found: 236.1779. Xishaflavalin F (6). Colorless crystal, mp 99−100 °C; {[α]20 D+58 (c 0.1, CHCl3)}; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HREIMS: m/z calcd for C15H22O3 [M]+: 250.1563; found: 250.1582. X-ray Crystallographic Analysis for Compounds 6 and 8. The crystals of 6 and 8 were recrystallized from (Petroleum ether: Et2O= 4: 1) and dichloromethane, respectively. X-ray analyses were carried out on a Bruker D8 Venture diffractometer with Ga Kα radiation (λ = 1.34139 Å). The acquisition parameters for 6 and 8 were provided in supporting information and crystallographic data for compounds 6 (deposition no. CCDC1864976) and 8 (deposition no. CCDC 1864975) have been deposited at the Cambridge Crystallographic Data Center. Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html. 13C NMR Data Calculation for Xishaflavalin B (2). The theoretical calculation of 13C NMR chemical shifts of epimers 2a and 2b were carried out by geometry optimization at the DFT(B3LYP)/6-31G(d) level followed by computation of the NMR shielding with the standard gauge-including atomic orbital (GIAO) approach at the lager basis functions of 6311+G(d,p), using the Gaussian 09 program. The experimental shifts were plotted against the calculated ones (Table S1), and least-squares fit lines was confirmed. The calculated shifts for 2a and 2b were corrected by the slope and intercept to get the corrected 13C shifts, and the differences between the corrected and experimental 13C NMR chemical shifts were analyzed. TDDFT-ECD Calculation of Compounds 1, 3 and 4. The torsional sampling (MCMM) method and OPLS_2005 force field were carried out to perform the conformational searches. Re-optimization for conformers above 1% population was conducted at the B3LYP/6-311G (d, p) level with IEFPCM (Polarizable Continuum Model using the Integral Equation Formalism variant) solvent model for acetonitrile. ECD spectra were obtained by TDDFT calculations for the geometries with the same functional, basis set and solvent model as the energy optimization. The Boltzmann-averaged ECD spectra of the three compounds were obtained with SpecDis1.62.21 Torsional sampling (MCMM) conformational searches using OPLS_2005 force field were carried out by means of the conformational search module in the Macromodel 9.9.223 software applying an energy window of 21 kJ/mol. The Boltzmann populations of the conformers were obtained based on the potential energy provided by the OPLS_2005 force field, which afforded conformers for 1, 3 and 4 above 1% population for re-optimization. The reoptimization and the following TDDFT calculations of the reoptimized geometries were all performed with Gaussian 0922 at the B3LYP/6-311G(d,p) level with IEFPCM solvent model for acetonitrile. Frequency analysis was performed as well to confirm that the re-optimized geometries were at the energy minima. Finally, the SpecDis1.62 software was applied to obtain the Boltzmann-averaged ECD spectra of the two compounds and visualize the results. Mo2(OAc)4 Induced Circular Dichroism (ICD) Experiment of 5. The diol functionality in compound 5 was protected as its acetonide by treatment with 2,2-DMP and

PPTS (catalytic amount) to furnish compound 5a in 94% yield (Figure S6 and S7). The NOE experiment of 5a determined the relative configuration of the vicinal diol (Figure S8), allowing the application of Snatzke's method to confirm its AC by measuring the Mo2(OAc)4 ICD of Mo-complex of 5. A total of 0.3 mg compound 5 was added into dry 0.5 mL DMSO (c= 0.6 mg/mL). Then, 0.5 mg Mo2(AcO)4 was added into the solvent. The absolute configuration of the 8, 9-diol moiety in 5 was verified by Mo2(OAc)4 ICD experiment developed by Snatzke and Frelek19. Chemical Transformation between Xishaflavalin F (6) and Nardosinanol A (7). A total of 5.0 mg nardosinanol A (7) was dissolved in 2.0 mL DCM and added a balanced amount of m-CPBA, then stirred for 2 h at room temperature. The obtained mixture was subjected to the 300−400 mesh silica gel and eluted by Et2O. The eluent was evaporated to dry and analyzed by both TLC and HPLC (Figure S9 and S10). The mixture showed two main chromatographic peaks and one of them was speculated as compound 1 (tR= 8.9 min). This was then verified by the comparison of HPLC profiles between standard xishaflavalin F (6) and obtained mixture. Then, semiprep HPLC was applied for isolation, and the mixture gave enough amount to determine the chemical structure of transformation product by the comparison of 1H NMR (Figure S11), 13C NMR (Figure S12), and specific optical rotation. The results indicated the transformation products was xishaflavalin F (6). ■ ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The 13C NMR calculation of 2; the RDC based NMR analysis of 3; the ECD calculations of 1, 3 and 4; the Mo2(OAc)4 induced circular dichroism experiment of 5; the chemical transformation between 6 and 7; the crystallographic data of 6 and 8; and the 1D and 2D NMR, MS and IR spectra of 1–6 (PDF) Crystal data for 6 in CIF format (CIF) Crystal data for 8 in CIF format (CIF) ■ AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]

Author Contributions #Q.W.

and Y.F. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest. ■ ACKNOWLEDGEMENTS This research work was financially supported by the National Key Research and Development Program of China (No. 2018YFC0310903), the Drug Innovation Major Project (No. 2018ZX09711-001-001-009), the National Natural Science Foundation of China (NSFC) (Nos. 81520108028, 21672230, 81773628), NSFC-Shandong Joint Fund for Marine Science Research Centers (No. U1606403).



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Uncommon Polyoxygenated Sesquiterpenoids from South China Sea

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