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Spongian Diterpenes Including One with a Rearranged Skeleton from the Marine Sponge Spongia of ficinalis Qian Chen,†,⊥ Qiqi Mao,†,⊥ Miao Bao,† Yongxiao Mou,† Chengyan Fang,† Min Zhao,† Wei Jiang,‡ Xia Yu,§ Chaojie Wang,† Lishang Dai,† Wenfei He,† Jianyong Dong,† Jianzhang Wu,*,† and Pengcheng Yan*,† †

School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, People’s Republic of China School of Environmental Science and Engineering, Yangzhou University, Yangzhou, Jiangsu 225127, People’s Republic of China § Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, Hunan 410013, People’s Republic of China

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‡

S Supporting Information *

ABSTRACT: Five new diterpenes, including an unprecedented 5,5,6,6,5-pentacyclic diterpene, sponalactone (1), two new spongian diterpenes, 17-O-acetylepispongiatriol (2) and 17-O-acetylspongiatriol (3), and two new spongian diterpene artifacts, 15α,16α-dimethoxy-15,16-dihydroepispongiatriol (4) and 15α-ethoxyepispongiatriol-16(15H)-one (5), were isolated from a South China Sea collection of the marine sponge Spongia of f icinalis, together with three known analogues (6−8). The structures of the new diterpenes were elucidated by extensive spectroscopic analysis. The absolute configurations were established on the basis of ECD data. Compounds 1−5 and 7 exhibited moderate inhibition against LPS-induced NO production in RAW264.7 macrophages with IC50 values of 12−32 μM.

S

report the details of isolation, structure characterization, and biological evaluation of these spongian diterpenes. Compound 1 had a molecular formula of C20H26O6 as determined by HRESIMS data, requiring eight degrees of unsaturation. IR absorptions at 3413 and 1763 cm−1 indicated the presence of hydroxy and carbonyl functionalities. The 13C NMR spectrum showed 20 carbon signals including a carbonyl (δC 179.7, C-3) and four olefinic carbons at δC 138.6 (CH, C15), 138.1 (CH, C-16), 131.2 (C, C-14), and 120.9 (C, C-13) (Table 2), which accounted for three of the eight degrees of unsaturation. Thus, 1 was probably a pentacyclic diterpene. The 1H NMR signals at δH 7.18 (s, H-15) and 7.17 (s, H-16) (Table 1) and the four olefinic carbon signals mentioned above were suggestive of a 3,4-disubstituted furan moiety, which is a typical functionality of spongian diterpene derivatives from sponges of the genus Spongia.6 In addition, an oxygenated nonprotonated carbon (δC 83.6, C, C-2), an oxygenated methine (δC 82.7, CH, C-1), and two oxygenated methylenes (δC 74.1, CH2, C-19; 63.3, CH2, C-17) were recognized with the aid of an HMQC spectrum. An α-hydroxy-β-methyl-γlactone moiety was established by HMBC correlations from the methyl singlet H3-18 (δH 1.11, s) to the oxygenated nonprotonated carbon C-2, oxygenated methylene carbon C19, and aliphatic nonprotonated carbon C-4 (δC 48.0, C) and from the oxygenated methylene protons H2-19 (δH 4.39, d, J = 9.0 Hz; 3.86, d, J = 9.0 Hz) to the carbonyl carbons C-3 and C-

pongian diterpenes are a family of natural products containing a 6,6,6,5-tetracyclic ring system and mainly have been isolated from marine sponges of the orders Dictyoceratid and Dendroceratid, often from the genus Spongia.1 To date, about 200 spongian diterpenes and rearranged derivatives have been reported2 since the first spongian diterpene, named isoagatholactone, was isolated from the sponge Spongia of f icinalis (family Spongiidae) collected in the Mediterranean.3 Some of these compounds were reported to possess a variety of pharmaceutical bioactivities, such as cytotoxic, anti-inflammatory, and antiviral properties.4−9 Spongia species of the South China Sea have been rarely chemically investigated, with only three studies reporting three new spongian diterpenes10 and two novel trinorsesquiterpenoids11 from the title species and nine sesquiterpene quinones/hydroquinones from S. pertusa,12 making them an understudied resource of chemical diversity for potential drug discovery efforts. In the course of our continuing research on bioactive molecules from marine invertebrates of the South China Sea,13−15 a specimen of the previously investigated sponge, S. off icinalis, was re-collected. Chemical examination of the EtOAc extract led to the isolation of eight spongian diterpene derivatives, including a new rearranged diterpene bearing a 5,5,6,6,5-pentacyclic ring system (1), four previously undescribed diterpenes (2−5), and three known analogues (6−8). These compounds were evaluated for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 murine macrophages. Herein we © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 25, 2019

A

DOI: 10.1021/acs.jnatprod.9b00270 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

Table 1. 1H NMR Data for Compounds 1−5 (Acetone-d6)a 1b

no. 1

2c

3.80, s

3 5 6

1.92, 1.77, 1.56, 2.55, 1.25, 2.08, 1.90, 1.70, 2.70, 2.59, 7.18, 7.17, 3.80, 3.51, 1.11, 4.39, 3.86, 0.83,

7 9 11 12 15 16 17 18 19 20 OAc 15-OMe 16-OMe OEt

m m br d (13.5) m t (12.5) m m m dd (15.5, 4.5) m s s d (10.0) d (10.0) s d (9.0) d (9.0) s

3b

2.61, 2.37, 4.10, 1.90, 1.82,

d (12.6) d (12.6) s m m

2.45, 1.43, 1.72, 1.75,

dt (13.2, 3.0) m m m

2.81, 2.53, 7.28, 7.17, 4.37, 4.13, 1.33, 3.62, 3.39, 0.98, 1.97,

m m s s d (10.8) d (10.8) s d (10.8) dd (10.8, 7.2) s s

2.56, 2.30, 4.54, 1.60, 1.87, 1.75, 2.43, 1.38, 1.54, 1.76,

d (16.5) d (16.5) br s d (12.0) m m d (13.0) t (13.0) dd (8.0, 5.0) m

2.79, 2.56, 7.28, 7.17, 4.42, 4.14, 0.84, 3.54,

m m s s d (10.5) d (10.5) s s

1.25, s 1.98, s

4b 2.52, 2.32, 4.10, 1.80, 1.77, 1.72, 2.25, 1.47, 1.62, 1.79, 1.65, 2.20, 2.06, 5.67, 5.27, 3.91, 3.59. 1.33, 3.58, 3.32, 0.88,

d (12.0) d (12.0) br s m m m m td (13.0, 3.0) d (8.0) m m dd (18.0, 6.0) m s s br d (9.5) br d (9.5) s m m s

5c 2.56, 2.35, 4.10, 1.82, 1.82,

d (12.0) d (12.0) s dd (10.2, 1.8) m

2.41, 1.54, 1.67, 1.77,

dt (13.8, 3.0) td (12.6, 4.2) dd (11.4, 2.4) m

2.36, m 2.17, m 6.13, s 4.04, 3.66, 1.33, 3.59, 3.35, 0.91,

d d s d d s

(10.2) (10.2) (10.2) (10.2)

3.31, s 3.27, s 3.84, dq (9.6, 7.2) 3.74, dq (9.6, 7.2) 1.24, t (7.2)

a

The coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm. b500 MHz. c600 MHz.

Table 2. 13C NMR Data for Compounds 1−5 (Acetone-d6)a no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OAc 15-OMe 16-OMe OEt

1

b

82.7, CH 83.6, C 179.7, C 48.0, C 57.1, CH 18.5, CH2 35.3, CH2 41.3, C 49.0, CH 46.9, C 20.2, CH2 20.2, CH2 120.9, C 131.2, C 138.6, CH 138.1, CH 63.3, CH2 23.2, CH3 74.1, CH2 14.9, CH3

c

b

b

2 (Figure 1). HMBC correlations from the second methyl singlet H3-20 (δH 0.83, s) to the oxygenated methine carbon

c

2

3

4

5

53.7, CH2 210.4, C 84.1, CH 49.6, C 55.7, CH 19.7, CH2 36.6, CH2 39.3, C 57.1, CH 43.3, C 18.9, CH2 21.0, CH2 120.4, C 130.7, C 139.0, CH 137.9, CH 64.6, CH2 23.9, CH3 64.4, CH2 17.9, CH3 170.8, C 20.9, CH3

53.7, CH2 213.0, C 76.5, CH 47.1, C 54.3, CH 20.2, CH2 36.4, CH2 39.1, C 57.8, CH 39.1, C 19.4, CH2 21.2, CH2 120.5, C 130.5, C 139.1, CH 137.9, CH 64.7, CH2 20.9, CH3 65.0, CH2 20.1, CH3 170.8, C 20.9, CH3

53.8, CH2 210.6, C 84.2, CH 49.6, C 55.8, CH 19.5, CH2 32.8, CH2 42.3, C 56.8, CH 43.4, C 18.1, CH2 23.2, CH2 135.9, C 143.8, C 109.4, CH 107.6, CH 65.8, CH2 23.9, CH3 64.4, CH2 17.5, CH3

53.7, CH2 210.4, C 84.1, CH 49.5, C 55.5, CH 19.4, CH2 32.0, CH2 44.2, C 55.9, CH 43.4, C 17.6, CH2 21.9, CH2 129.4, C 164.6, C 105.5, CH 171.6, C 65.0, CH2 23.8, CH3 64.3, CH2 17.5, CH3

Figure 1. Key COSY and HMBC correlations for 1, 2, 4, and 5.

C-1, aliphatic methine carbon C-5 (δC 57.1, CH), and aliphatic nonprotonated carbon C-10 (δC 46.9, C), from H3-18 to C-5, and from the oxygenated methine proton H-1 (δH 3.80, s) to carbonyl carbon C-3 revealed that the α-hydroxy-β-methyl-γlactone moiety was fused with a cyclopentane ring B, in which C-1 and C-10 were substituted by hydroxy and methyl groups, respectively. Furthermore, COSY correlations of H-5 (δH 1.92, m)/H2-6 (δH 1.77, m; 1.56, br d, J = 13.5 Hz) and H2-6/H2-7 (δH 2.55, m; 1.25, t, J = 12.5 Hz), in combination with HMBC correlations from H3-20 to aliphatic methine carbon C-9 (δC

54.1, CH3 52.9, CH3 66.3, CH2 15.5, CH3

a

The assignments were based on HMQC, HMBC, and COSY spectra. b125 MHz. c150 MHz. B

DOI: 10.1021/acs.jnatprod.9b00270 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 2. Key NOE correlations and computer-generated models using MM2 force field calculations for 1, 4, and 5.

(Tables 1 and 2), indicating that 2 was an acetylated derivative of 7. The acetoxy group was attached to C-17, as evidenced by HMBC correlations from the oxygenated methylene protons H2-17 (δH 4.37, d, J = 10.8 Hz; 4.13, d, J = 10.8 Hz) to the ester carbonyl carbon (δC 170.8) and from H2-17 to C-7, C-8, C-9, and C-14 (Figure 1). The relative configuration of 2 was determined to be identical to that of 7 based on the similar NMR data and NOE correlations (Figure S37). The ECD spectrum showed a positive Cotton effect at 290 nm for the n−π* transition of the carbonyl (Figure S38), suggesting that the absolute configuration of 2 was consistent with that of 7 by using the octant rule.18 Thus, compound 2 was elucidated as 17-O-acetylepispongiatriol. Compound 3 was determined to be an isomer of 2 according to its HRESIMS and 1D NMR data (Tables 1 and 2) and detailed analysis of HMBC and COSY correlations (Figure 1). The significantly shielded chemical shift of C-3 (δC 76.5, CH) in comparison with that of 2 (δC 84.1, CH) suggested that 3 was the C-3 epimer of 2 and was closely related to the cooccurring analogue spongiatriol (8).17 This assignment was further supported by the NOE correlations (Figure S37). The absolute configuration of 3 was determined to be the same as that of 8 following the same protocol as depicted in 2 (Figure S38). Therefore, compound 3 was established as 17-Oacetylspongiatriol. Compounds 4 and 5 were established as two spongian artifacts derived from epispongiatriol (7)17 by oxidation of furan ring D. In 4, ring D was a 2,5-dimethoxy-2,5dihydrofuran, as indicated by NMR signals for a tetrasubstituted double bond (δC 143.8, C, C-14; 135.9, C, C-13), two acetals [δC 109.4 (CH, C-15), 107.6 (CH, C-16); δH 5.67 (s, H-15), 5.27 (s, H-16)], and two methoxy groups [δC 54.1 (CH3), 52.9 (CH3); δH 3.31 (s), 3.27 (s)], while in 5, the counterpart was a 5-ethoxyfuran-2(5H)-one, as evidenced by NMR data for an ester carbonyl (δC 171.6, C-16), a tetrasubstituted double bond (δC 164.6, C, C-14; 129.4, C, C-13), an acetal [δC 105.5 (CH, C-15); δH 6.13 (s, H-15)], and an ethoxy group [δC 66.3 (CH2), 15.5 (CH3); δH 3.84 (dq,

49.0, CH) and from the second oxygenated methylene H2-17 (δH 3.80, d, J = 10.0 Hz; 3.51, d, J = 10.0 Hz) to the aliphatic methylene carbon C-7 (δC 35.3, CH2), nonprotonated carbon C-8 (δC 41.3, C), and C-9, disclosed that ring B was fused with a cyclohexane ring C and C-8 was substituted by a hydroxymethyl group. Finally, COSY correlations of H-9 (δH 2.08, m)/H2-11 (δH 1.90, m; 1.70, m) and H2-11/H2-12 (δH 2.70, dd, J = 15.5, 4.5 Hz; 2.59, m) and HMBC correlations from H2-17 to the olefinic nonprotonated carbon C-14 and from H2-12 to three olefinic carbons (C-13, C-14, and C-16) of the furan moiety completed the establishment of the planar structure of 1. The relative configurations of stereogenic centers in 1 were determined by NOESY analysis. Significant NOE correlations of H3-20/H-19a (δH 4.39, d, J = 9.0 Hz) and H3-18/H-5 suggested the cis and trans fusions of rings A/B and B/C, respectively, and the same facial orientation of H3-18 and H-5, while NOE correlations of H3-20/H2-17 and H2-17/H-11a (δH 1.90, m) indicated the trans fusion of rings C/D and the same orientation of H2-17 and H3-20 (Figure 2). In addition, NOE correlation between H-1 and H-11b (δH 1.70, m) and lack of correlations of H-1/H-5 and H-1/H-9 suggested the βorientation of H-1. The absolute configurations at C-5, C-8, C-9, and C-10 were suggested to be in agreement with those of the coisolated spongian diterpenes isospongiatriol (6),16 epispongiatriol (7),17 and spongiatriol (8)17 due to a shared biogenesis. Accordingly, C-1, C-2, and C-4 were assigned as R, S, and S, respectively. In order to support this assignment, the electronic circular dichroism (ECD) calculation was performed for 1. However, the overall match of the experimental and calculated ECD curves was only partial (Figure S36). Interestingly, compound 1 represents a new pentacyclic skeleton that could be derived from 6−8 via a series of oxidation, nucleophilic addition, and rearrangement reactions,10 and it was named sponalactone. The NMR data of 2 were comparable to those of epispongiatriol (7)17 with the exception of additional signals for an acetyl group [δC 170.8 (C), 20.9 (CH3); δH 1.97 (s)] C

DOI: 10.1021/acs.jnatprod.9b00270 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Note

J = 9.6, 7.2 Hz), 3.74 (dq, J = 9.6, 7.2 Hz), 1.24 (t, J = 7.2 Hz)] (Tables 1 and 2). These assumptions were confirmed by detailed analysis of HMBC and COSY correlations as shown in Figure 1. In 4, two methoxy groups were suggested to be αoriented by NOE correlations of H-15/H2-17 (δH 3.91, br d, J = 9.5 Hz; 3.59, br d, J = 9.5 Hz), H-9 (δH 1.62, d, J = 8.0 Hz)/ H-12b (δH 2.06, m), and H-16/H-12a (δH 2.20, dd, J = 18.0, 6.0 Hz); in 5, the ethoxy group was also α-oriented according to an NOE correlation between H-15 and H2-17 (δH 4.04, d, J = 10.2 Hz; 3.66, d, J = 10.2 Hz); in addition, the relative configurations of the other stereogenic centers in 4 and 5 were determined to be identical to those of 7 by the similar NOE correlations (Figure 2). The absolute configuration of 4 was assigned as shown in Chart 1 following the same method as for

Table 3. Inhibition of LPS-Induced NO Production

Chart 1

was performed using Sephadex LH-20 (GE Healthcare Biosciences AB), ODS (50 μm, YMC), and silica gel (200−300 mesh, Qingdao Marine Chemistry Co. Ltd.). Preparative reversed-phase HPLC was run on an Agilent 1100 series instrument using a YMC-Pack C18 column (10 μm, 250 × 10 mm). Animal Material. Specimens of the marine sponge Spongia of f icinalis were collected from the inner coral reef of Ximao Island, Hainan Province, China, in June 2016, at a depth of 10 m, and frozen immediately after collection. The DNA identification was carried out by one of the authors (L.D.). A voucher specimen (HS201601) was deposited at the Laboratory of Marine Natural Products Chemistry, Wenzhou Medical University, China. Extraction and Isolation. The frozen sample of S. of f icinalis (wet weight: 1.05 kg) was homogenized and extracted with 95% EtOH at room temperature. The concentrated extract was partitioned between H2O and EtOAc. Evaporation of EtOAc in vacuo yielded a dark residue of 15.9 g. The EtOAc fraction (15.0 g) was separated by silica gel vacuum column chromatography, eluting with a gradient of CH2Cl2/petroleum ether (PE) (1:9), EtOAc/PE (1:9), acetone/PE (1:9), EtOAc/CH2Cl2 (1:9), acetone/CH2Cl2 (1:9), MeOH/CH2Cl2 (1:9), and MeOH, to obtain seven fractions (A−G). Fraction F (3.6 g) was subjected to a Sephadex LH-20 column, eluting with PE/ CH2Cl2/MeOH (5:5:1), to afford four fractions (F1−F4). Fraction F2 (383.7 mg) was further separated on a silica gel column, eluting with a gradient of EtOAc/PE (1:2, 1:1, 2:1, and 4:1), to afford five fractions (F2a−F2e). Fractions F2b (37.3 mg) and F 2d (25.2 mg) were purified by semipreparative HPLC, using MeOH/H2O (70:30) as eluent, to yield 2 (7.0 mg) and 3 (5.1 mg), respectively. Fraction F3 (520.0 mg) was subjected to a silica gel column, eluting with a gradient of acetone/PE (1:3, 1:1, and 3:1), to afford three fractions (F3a−F3c). Fraction F3b (47.5 mg) was purified by HPLC (MeOH/ H2O, 65:35) to obtain 7 (5.2 mg). Fraction F4 (746.3 mg) was separated on a silica gel column, eluting with a gradient of EtOAc/PE (1:1, 3:1, 6:1, and 8:1), to yield six fractions (F4a−F4f). Fraction F4b (14.2 mg) was purified by HPLC (MeOH/H2O, 63:37) to afford 1 (3.2 mg). Fraction F4d (116.1 mg) was separated on an ODS column, eluting with MeOH/H2O (60:40, 70:30, and 80:20), to obtain three fractions (F4d1−F4d3). Fraction F4d2 (40.7 mg) was purified by HPLC (MeOH/H2O, 63:37) to yield 8 (11.8 mg). Fraction F4e (145.5 mg) was subjected to an ODS column, eluting with MeOH/ H2O (50:50, 60:40, 70:30, 80:20, and 90:10), to afford six fractions (F4e1−F4e6). Fractions F4e2 (13.8 mg), F4e3 (7.9 mg), and F4e5 (34.6 mg) were purified by HPLC (MeOH/H2O, 55:45, 50:50, and 63:37, respectively), to obtain 4 (3.4 mg), 5 (1.7 mg), and 6 (17.5 mg). Sponalactone (1): colorless oil; [α]25D +18 (c 0.05, MeOH); ECD (MeOH) λmax (Δε) 240 (−2.55), 220 (+3.02) nm; IR (KBr) νmax 3413, 2927, 2861, 1763, 1469, 1385, 1153, 1039, 737 cm−1; 1H and 13 C NMR data, Tables 1 and 2; HRESIMS m/z 385.1641 [M + Na]+ (calcd for C20H26O6Na, 385.1627). 17-O-Acetylepispongiatriol (2): colorless oil; [α]25D +14 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 290 (+1.62), 224 (+3.37) nm; IR (KBr) νmax 3485, 2951, 1734, 1457, 1384, 1238, 1036, 729 cm−1; 1H

compound 1 2 3 4 5 6 7 b L-NMMA a

32 15 12 22 12 >60 20 17

± ± ± ± ±

CC50a (μM)

4 3 2 3 2

>60 >60 >60 >60 >60 >60 >60 UDc

±2

CC50: cytotoxicity against RAW264.7 macrophages. NG-methyl-L-arginine acetate salt. cUD: undetected.

2 and 3 (Figure S38). The absolute configuration at C-15 in 5 was assigned as S by the negative Cotton effect at 225 nm (π−π* transition) in the ECD spectrum and application of the ECD helicity rule for α,β-unsaturated-γ-lactones (Figure S38).19 The absolute configurations of the other stereogenic centers in 5 were accordingly assigned to be the same as those of 7. Three known compounds were also isolated from the S. of f icinalis extract and identified as isospongiatriol (6),16 epispongiatriol (7),17 and spongiatriol (8)17 by comparison of their 1H and 13C NMR and MS spectroscopic data, as well as specific rotations with those reported in the literature. All compounds except 8, a known cytotoxic diterpene,7 were tested for their in vitro anti-inflammatory activities. In the primary assay, compounds 1−5 and 7 showed inhibition against lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages with IC50 values of 12−32 μM, whereas no inhibitory effect was observed for 6 (IC50 > 60 μM) (Table 3). The weaker activities of 1 and 6 in comparison with 2−5 and 7 suggested that the typical 2-oxo-3hydroxycyclohexane ring A was essential for the inhibitory activity.

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IC50 (μM)

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were acquired on a PoLAAR 3005 digital polarimeter. UV and IR spectra were measured with a TU 1901 spectrometer and a Bruker Equinox 55 spectrometer, respectively. ECD spectra were recorded on a Chirascan circular dichroism spectrometer. NMR spectra were obtained with Bruker Avance III NMR spectrometers operating at 500 or 600 MHz for 1H and 125 or 150 MHz for 13C. Tetramethylsilane was used as an internal standard. (+) HRESIMS data were obtained with a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer. Column chromatography D

b

L-NMMA:

DOI: 10.1021/acs.jnatprod.9b00270 J. Nat. Prod. XXXX, XXX, XXX−XXX

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and 13C NMR data, Tables 1 and 2; HRESIMS m/z 413.1953 [M + Na]+ (calcd for C22H30O6Na, 413.1940). 17-O-Acetylspongiatriol (3): colorless oil; [α]25D +12 (c 0.10, MeOH); ECD (MeOH) λmax (Δε) 290 (+0.72), 223 (+3.61) nm; IR (KBr) νmax 3446, 2949, 1734, 1385, 1238, 1038, 735 cm−1; 1H and 13 C NMR data, Tables 1 and 2; HRESIMS m/z 413.1931 [M + Na]+ (calcd for C22H30O6Na, 413.1940). 15α,16α-Dimethoxy-15,16-dihydroepispongiatriol (4): colorless oil; [α]25D −20 (c 0.05, MeOH); ECD (MeOH) λmax (Δε) 290 (+2.27) nm; IR (KBr) νmax 3423, 2951, 2836, 1714, 1451, 1375, 1093, 1011, 737 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 433.2213 [M + Na]+ (calcd for C22H34O7Na, 433.2202). 15α-Ethoxyepispongiatriol-16(15H)-one (5): colorless oil; [α]25D +28 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 220 (3.77) nm; ECD (MeOH) λmax (Δε) 250 (+4.09), 225 (−12.39) nm; IR (KBr) νmax 3444, 2956, 2894, 1747, 1330, 1084, 735 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 431.2058 [M + Na]+ (calcd for C22H32O7Na, 431.2046). Isospongiatriol (6): [α]25D −28 (c 0.05, CHCl3), no literature value. Epispongiatriol (7): [α]25D +22 (c 0.05, CHCl3); lit. value [α]D +26 (c 0.50, CHCl3).17 Spongiatriol (8): [α]25D +30 (c 0.05, CHCl3); lit. value [α]D +142 (c 0.50, CHCl3).17 ECD Calculations. The structure for 1 was optimized at the CAM-B3LYP/6-311G(2d,p) level. Then the ECD calculation was performed at the B3LYP/Def2-TZVP level. The solvent effect was taken into account in the calculation by using the polarizable continuum model (PCM, MeOH as the solvent). The quantum mechanical calculation was carried out using Gaussian’16 and Multiwfn program packages. Assay for Inhibitory Activity against Nitric Oxide Production. A previously reported protocol20 was followed except that NGmethyl-L-arginine acetate salt in DMSO was used as the positive control, and each test compound (30 mM) in DMSO was diluted to 1−60 μM at room temperature before the experiment.

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University (No. 604091809), and the Opening Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences (No. 201707).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00270. 1D and 2D NMR and HRESIMS spectra of the new compounds 1−5, ECD spectra of 1−5, calculated ECD spectrum for 1, octant rules of 2−4, helicity rule of 5, and key NOE correlations for 2 and 3 (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (J. Wu): [email protected]. *Tel (P. Yan): +86-577-86699572. E-mail: [email protected]. cn. ORCID

Pengcheng Yan: 0000-0002-9114-2244 Author Contributions ⊥

Q. Chen and Q. Mao made equal contributions to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of Zhejiang Province, China (No. LQ19H300003), Public Project of Zhejiang Province, China (2017C37042), National Nature Science Foundation of China (No. 81803580), Outstanding Youth Foundation from Wenzhou Medical E

DOI: 10.1021/acs.jnatprod.9b00270 J. Nat. Prod. XXXX, XXX, XXX−XXX