Article pubs.acs.org/jnp

ent-Abietane-Type and Related Seco-/Nor-diterpenoids from the Rare Chloranthaceae Plant Chloranthus sessilifolius and Their Antineuroinflammatory Activities Li-Jun Wang,† Juan Xiong,*,† Shu-Ting Liu,† Li-Long Pan,*,‡ Guo-Xun Yang,† and Jin-Feng Hu*,† †

Department of Natural Products Chemistry and ‡Department of Pharmacology, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Fourteen new ent-abietane-type diterpenoids, sessilifols A−N (1−14), and three related new norditerpenoids (15−17) were isolated from Chloranthus sessilifolius. The absolute configurations were determined by single-crystal Xray diffraction analysis, the modified Mosher’s method, and/or the observed Cotton effects in their electronic circular dichroism spectra. Sessilifols A (1) and B (2) possess an uncommon five-membered C-ring rearranged by oxidative cleavage of the C-13/C-14 bond in abieta-7,13-diene followed by the formation of a new C−C bond between C-12 and C-14. Sessilifol C (3) is a rare 7,8-seco-9-spiro-fused ent-abietane, whereas sessilifol O (15) represents the first example of a naturally occurring 14-norabietane-type diterpenoid. Compounds 6 and 9 were found to have moderate antineuroinflammatory activities by inhibiting the nitric oxide production in lipopolysaccharide-stimulated murine BV-2 microglial cells, with IC50 values of 8.3 and 7.4 μM, respectively.

N

C. angustifolius,8 C. anhuiensis,9 C. elatior,10 C. fortunei,9,11 C. henryi,6c,9,12 C. holostegius,9 C. japonicus,9,13 C. multistachys,9,14 C. serratus,9,15 C. spicatus,9,16 and C. tianmushanensis.9 Previous studies revealed that this genus is a rich source of sesquiterpenoids, but only ent-kaurane and labdane-type diterpenoids with interesting anti-inflammatory activity have been obtained from this genus.9,10a,b,15 C. sessilifolius K. F. Wu, a rare Chloranthus species endemic in China, is a perennial herb generally growing in damp areas in forests at an altitude of about 1000−1200 m in the Fengqi Mountain areas in the Sichuan Province of China.7,17 During continuing research toward the discovery of novel antineuroinflammatory natural products from Chloranthus plants,6c,18 a number of entabietane-type and related seco-/nor-diterpenoids (1−17, Figure 1) were isolated from C. sessilifolius. We herein describe their isolation, structural elucidation, and antineuroinflammatory activities.

eurodegenerative disorders are characterized by progressive degeneration and loss of neurons in the brain, although the pathological mechanisms remain elusive.1 Neuroinflammation has been implicated in the pathogenesis of neurological disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD).2 Microglia are the resident immune cells in the central nervous system (CNS) and the activated microglia play a key role in inflammatory responses in the brain, suggesting that the activation of microglial cells is, at least in part, responsible for neurodegenerative diseases.3 Lipopolysaccharides (LPS), the major constituent of the Gram-negative bacterial cell wall, play a pivotal role in the initiation of inflammation mediated by releasing inflammatory mediators, such as nitric oxide (NO) and tumor necrosis factor-α (TNFα), which are thought to be responsible for neuroglia-mediated neuroinflammation.4 Thus, inhibiting the synthesis or release of these inflammatory mediators may represent a potential therapeutic approach for neurodegenerative diseases.5 In fact, finding novel bioactive compounds with diverse structures from natural sources has been a major concern for pharmaceuticals research. In our previous reports,6 naturally occurring phenolic compounds from Cratoxylum formosum ssp. pruniflorum, lignans from Clematis armandii, and sesquiterpenoids from Chloranthus henryi showed remarkable antineuroinflammatory effects by suppressing the NO production and/or the TNF-α release in LPS-stimulated BV-2 microglial cells. Chloranthus (family: Chloranthaceae) is a small genus with 13 species and five variations occurring in China.7 Among them, 11 species have been chemically investigated, including © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The MeOH extract of the whole plant of C. sessilifolius was suspended in H2O and then partitioned successively with petroleum ether, EtOAc, and n-BuOH. The entire EtOAc fraction was subjected repeatedly to column chromatography over silica gel, MCI gel, and semipreparative HPLC to afford compounds 1−17 (Figure 1). Received: March 3, 2015

A

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three quaternary carbons (one olefinic at δ 142.4). In the 1H NMR spectrum (Table 2), three tertiary [δ 1.00 (s, Me-18), 0.88 (s, Me-19), and 0.74 (s, Me-20)] and two secondary [δ 1.10 (6H, d, J = 7.0 Hz, Me-16 and Me-17)] methyls, two oxymethines [δ 3.26 (dd, J = 11.6, 3.8 Hz, H-3), 4.62 (br d, J = 8.2 Hz, H-14)], and one olefinic proton [δ 5.64 (br s, H-7)] were observed. Detailed 2D NMR (COSY, HSQC, and HMBC) spectroscopic analyses suggested that 1 is a rearranged 14(13→12)-abeo-abietane diterpenoid similar to the semisynthetic methyl 7α,8α-epoxy-14α-hydroxy-13-oxo-14(13→ 12)-abeo-abieta-18-oate.19 Unlike this known structure, compound 1 has an additional hydroxy group (δH 3.26, δC 79.2) at C-3, a methyl group (δH 1.00, δC 27.6) instead of a methoxycarbonyl group at C-18, and a Δ7,8 double bond (δH 5.64, δC 116.9, 142.4) replacing the 7α,8α-epoxy ring, which were confirmed by the key correlations between Me-18/Me19/H-5 and C-3, between H-6 and C-7/C-8, and between H11/H-14 and C-8 in its HMBC spectrum (Figure 2). The relative configuration of 1 was established by analyses of the proton−proton coupling constants (Table 2) and the NOESY NMR spectrum. The large values observed for JH‑1β,2α (12.8 Hz), JH‑2α,3 (11.6 Hz), and JH‑5,6α (11.7 Hz) implied that H-1β, H-2α, H-3, and H-5 were axially oriented. NOE correlations of Me-18/H-3, Me-18/H-5, H-5/H-3, H-3/H-1β, H-1β/H-9, and H-9/H-14 (Figure 3) indicated these protons are all β-oriented, while the NOE correlaltions of Me-19/H-6α, H-6α/Me-20, and Me-20/H-12 indicated that Me-19, Me-20, and H-12 were α-oriented. The absolute configuration of 1 was defined by X-ray crystallography based on an anomalous dispersion of Cu Kα radiation (Figure 3). On the basis of the above findings, the structure of compound 1 was characterized as (3R,5S,9S,10R,12R,14S)-3,14-dihydroxy-ent-14(13→12)abeo-abieta-7-en-13-one (sessilifol A). Compound 1 represents the first example of a naturally occurring 14(13→12)-abeoabietane.

Figure 1. Chemical structures of compounds 1−17.

Compound 1 was obtained as colorless crystals (from MeOH), and its molecular formula was determined to be C20H32O3 based on the 13C NMR data and a sodium adduct ion at m/z 343.2259 ([M + Na]+) in its positive mode HRESIMS, implying five indices of hydrogen deficiency. The IR spectrum of 1 showed absorption bands (νmax) attributable to hydroxy (3405 cm−1), carbonyl (1699 cm−1), and olefinic (1650, 1465 cm−1) groups. The 13C and DEPT NMR data of 1 (Table 1) showed 20 carbon resonances, consisting of one carbonyl at δ 216.2, five methyls, four methylenes, seven methines (two oxygenated at δ 79.2 and 75.2 and one olefinic at δ 116.9), and

Table 1. 13C NMR Data (δ in ppm) for Compounds 1−8 and 10−17a no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe

1b 37.3 27.2 79.2 38.8 49.6 23.5 116.9 142.4 49.9 34.5 25.1 56.7 216.2 75.2 40.6 17.9 18.1 27.6 14.8 12.9

2b 38.0 27.4 79.2 38.7 49.6 24.1 122.1 144.4 54.7 34.3 25.1 54.3 215.7 73.0 40.5 17.8 18.5 28.0 15.0 13.6

3b 30.7 26.7 77.4 38.6 42.0 28.5 171.9 196.7 88.9 39.7 30.1 26.7 171.7 124.3 35.4 20.9 20.6 27.5 15.3 14.9

4b 36.3 27.5 78.5 38.9 50.0 36.2 200.5 136.5 50.4 36.1 20.8 32.8 72.5 141.1 37.2 16.7 17.0 27.2 14.6 13.8

5b 35.0 26.8 75.9 42.7 178.8 125.7 187.6 136.7 48.0 39.0 18.1 28.9 71.4 138.3 37.9 16.4 17.4 24.4 23.1 26.1

6b 36.8 27.3 78.6 38.9 49.5 37.1 200.0 140.1 50.1 36.0 18.6 26.9 75.5 139.3 32.6 15.8 17.5 27.4 14.7 14.0 51.2

7b d

37.0 27.3 78.7 39.0 49.3 37.0d 200.7 138.8 51.2 35.8 19.1 30.0 73.8 137.6 74.8 24.0 24.8 27.5 14.8 14.2

8b

10b

11b

12b

13b

14b

15b

16b

17c

37.0 27.3 78.6 39.0 49.6 36.9 199.7 139.4 50.4 35.9 28.2 69.0 73.3 136.3 33.5 16.6 17.8 27.5 14.8 14.2

36.9 28.0 78.7 39.0 49.8 18.9 30.9 134.8 147.8 37.4 124.4 122.0 146.1 124.8 72.3 31.6 31.6 28.2 15.4 24.9

34.2 27.8 78.7 38.3 50.4 129.6 128.5 132.6 146.7 37.5 121.8 123.7 145.9 122.6 72.3 31.6 31.6 27.6 16.4 20.3

34.4 27.4 76.4 43.4 172.1 125.3 185.9 129.5d 151.6 40.7 125.7 129.5d 147.5 121.6 72.4 31.7 31.7 27.2 22.3 29.7

37.1 27.9 78.5 38.7 48.4 30.0 71.4 137.6 146.6 38.0 124.5 125.9 146.6 125.1 33.7 23.9 24.1 28.1 15.5 25.3

36.4 26.6 78.5 39.0 56.2 68.5 153.5 143.3 49.0 39.9 21.0 42.3 215.3 194.9 40.7 18.2 18.3 30.8 15.6 15.0

36.7 27.2 78.4 39.2 53.1 38.8 211.2 42.8 49.6 36.2 23.0 38.0 214.3

37.0 27.2 78.7 38.6 45.9 29.1 71.5 163.5 46.8 39.3 20.3 36.6 200.7 127.5

37.6 27.6 78.7 39.5 51.6 32.4 72.0 169.1 49.8 40.0 21.1 36.6 202.2 122.3

28.2 15.6 14.7

28.5 16.0 15.2

41.0 18.3 18.3 27.7 14.9 12.8

a

Assignments were made by a combination of 1D and 2D NMR experiments. bRecorded in CDCl3. cRecorded in CD3OD. dOverlapped with the same superscript in the same column. B

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Table 2. 1H NMR Data (CDCl3, δ in ppm, J in Hz) of Compounds 1−3, 14, and 15a no. 1α 1β 2 3 5 6α 6β 7 8α 8β 9 11a 11b 12a 12b 14 15 16 17 18 19 20 a

1 1.65, 1.15, 1.65, 3.26, 1.31, 2.01, 2.21, 5.64,

m ddd (12.8, 12.8, 4.5) m, 2H dd (11.6, 3.8) dd (11.7, 5.2) m br dd, overlapped br s

2 1.72, 1.26, 1.65, 3.26, 1.26, 2.03, 2.21, 5.87,

m ddd (11.6, 11.6, 4.5) m, 2H dd (10.2, 5.2) dd (11.0, 4.5) m m br s

2.20, 1.81, 1.69, 2.73,

dd, overlapped ddd (13.8, 9.2, 5.2) ddd, overlapped ddd (10.0, 9.2, 8.2)

2.05, 1.76, 1.73, 2.99,

dd, overlapped m, α-H m, β-H ddd (11.6, 5.2, 5.2)

4.62, 2.72, 1.10, 1.10, 1.00, 0.88, 0.74,

br d (8.2) m d (7.0) d (7.0) s s s

4.71, 2.81, 1.12, 1.12, 1.01, 0.89, 0.77,

br d (5.2) m d (7.0) d (7.0) s s s

3 1.81, 1.57, 1.64, 3.17, 1.89, 2.38, 2.70,

ddd (12.5, 3.2, 2.9) ddd, overlappedb m, 2H dd (10.0, 5.5) dd (12.3, 6.2) dd (18.5,12.3) dd (18.5, 6.2)

14 1.90, 1.20, 1.61, 3.25, 1.20, 4.64,

ddd (13.2, 4.0, 4.0) ddd (13.5, 13.5, 4.0) m; 1.68, m dd (11.8, 4.3) d (10.0) ddd (10.0, 2.4, 2.4)

15 1.95, 1.17, 1.67, 3.27, 1.27, 2.36, 2.41,

ddd (13.6, 3.6, 3.6) ddd (13.5, 13.5, 3.4) m; 1.73, m dd (11.8, 4.5) dd (12.9, 6.8) m ddd (15.6, 3.9, 2.0)

2.05, 2.33, 1.25, 1.92, 1.20, 2.47, 2.36,

dd (15.0, 12.9) dd (15.0, 6.8) m m m m m

2.57, 1.08, 1.08, 0.96, 0.85, 1.00,

m d (7.0) d (7.0) s s s

6.60, t (2.4)

2.59, 2.14, 2.51, 2.47, 6.03, 2.45, 1.12, 1.12, 0.95, 0.89, 1.18,

ddd (14.6, 5.2, 2.9) ddd (14.6, 10.5, 6.5) m m br s m d (7.0) d (7.0) s s s

1.96, 1.47, 1.81, 3.09, 2.43, 9.45, 2.61, 1.09, 1.09, 1.28, 1.06, 0.85,

m m m ddd (17.4, 10.9, 4.5) ddd (17.4, 11.2, 5.0) s m d (7.0) d (7.0) s s s

Assignments were made by a combination of 1D and 2D NMR experiments. bOverlapped with a broad water peak in CDCl3.

Figure 2. HMBC correlations of the indicated compounds.

Figure 3. NOE correlations and the ORTEP drawing of 1.

The HRESIMS [m/z 343.2231 ([M + Na]+)] and the 13C NMR data of sessilifol B (2) indicated it possesses the same molecular formula (C20H32O3) as that of 1. These compounds have similar 1H and 13C NMR spectroscopic data, with differences evident for resonances of the C-ring (Tables 1 and 2). The planar structure of 2 is identical to that of 1 by 2D NMR experiments. Differing from those of 1, NOE correlations of H-9/H-12 and H-9/H-14 were observed for compound 2, but lack such a correlation between Me-20 and H-12 (see Supporting Information), suggesting H-9, H-12, and H-14 were cofacial. Thus, the structure of compound 2 was elucidated as the C-12 epimer of 1.

Sessilifol C (3), obtained as colorless crystals (from CHCl3/ n-hexane, 1:2, v/v), has a molecular formula of C20H30O4 as deduced by its positive mode HRESIMS (Experimental Section) and 13C NMR data. The IR absorption bands (νmax) of 3 indicated the presence of hydroxy (3417 cm−1), lactone (1716 cm−1), and α,β-unsaturated carbonyl (1663, 1463 cm−1) functionalities. Similar to compounds 1 and 2, the 1H NMR spectrum (Table 2) of 3 also displayed signals of three tertiary [δ 0.95 (s, Me-18), 0.89 (s, Me-19), and 1.18 (s, Me-20)] and two secondary [δ 1.12 (6H, d, J = 7.0 Hz, Me-16 and Me-17)] methyl groups and an olefinic proton (δ 6.03, br s, H-14), but only one oxymethine at δ 3.17 (dd, J = 10.0, 5.5 Hz, H-3). The C

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Sessilifol D (4) was obtained as colorless crystals (from MeOH) and was assigned the molecular formula C20H32O3 by its HRESIMS (Experimental Section) and 13C NMR data. A strong absorption (νmax) at 1680 cm−1 in its IR spectrum together with the absorption (λmax) at 248 nm in its UV (MeOH) spectrum is indicative of a conjugated carbonyl group in the structure of 4. Similar to compound 3, the 1H NMR spectrum of 4 (Table 3) also showed signals of three methyl singlets [δ 0.99 (s, Me-18), 0.87 (s, Me-19), and 0.83 (s, Me20)], two methyl doublets [δ 0.94 (d, J = 7.0 Hz, Me-16); 0.96 (d, J = 7.0 Hz, Me-17)], a characteristic C-3 oxymethine (δ 3.30, dd, J = 11.4, 4.0 Hz, H-3), and an olefinic proton at δ 6.74 (br s, H-14). The 13C NMR data (Table 1) of 4, showing 20 carbon signals, indicated the presence of an α,β-unsaturated carbonyl group (δC 200.5, 141.1, and 136.5), an oxymethine (δC 78.5), and an oxygenated tertiary carbon (δC 73.5). These data suggested that 4 is also a diterpenoid, carrying an enone group and a secondary and a tertiary hydroxy group. The planar structure of 4 as a common abietane-type skeleton was then verified by 2D (COSY, HSQC, and HMBC) NMR experiments, from which the hydroxy groups at C-3 and C-13, a Δ8(14) double bond, and a C-7 carbonyl were evident (Figure 2). The relative configuration of 4 was determined by analyses of the proton−proton coupling constants (Table 3) and the NOE correlations (see Supporting Information). In particular, the large values of J2α,H‑3 (11.4 Hz) and JH‑5,6α (13.5 Hz) unambiguously indicated that H-3 and H-5 were in axial positions. The NOE correlations of H-3 with Me-18 and H-5 revealed that OH-3 was α-orientated, whereas those of H-12α with Me-16 and Me-17 showed that OH-13 adopted the βorientation. The absolute configuration of 4 was unequivocally established by a single-crystal X-ray diffraction experiment using Cu Kα radiation (Figure 5). Thus, the structure of compound 4 was defined as (3R,5S,9S,10R,13S)-3,13-dihydroxy-ent-abieta-8(14)-en-7-one. The molecular formula (C20H30O3) of sessilifol E (5) was established by its HRESIMS (Experimental Section) and 13C

C and DEPT NMR data of 3 (Table 1) exhibited 20 carbon signals classified as five methyls, five methylenes, four methines (one oxygenated at δ 77.4 and one olefinic at δ 124.3), one oxygenated tertiary carbon (δ 88.9), three quaternary carbons (one olefinic at δ 171.7), and two carbonyl groups (δ 171.9, 196.7). The above spectroscopic data were similar to those of decandrinin,20 the first 7,8-seco-9-spiro-fused ent-abietane recently isolated from the Godavari mangrove (Ceriops decandra). The only difference is that the C-3 carbonyl group in decandrinin is replaced by a hydroxymethine group in 3, which was confirmed by the correlations from Me-18, Me-19, and H-5 (δ 1.89, dd, J = 12.3, 6.2 Hz) to C-3 (δC 77.4) in the HMBC spectrum of 3 (Figure 2). Moreover, the large values observed for JH‑2α,3 (10.0 Hz) and JH‑5,6α (12.3 Hz) were indicative of the axial orientations of H-3 and H-5. The relative configuration of 3 was determined by the NOESY NMR spectrum (Figure 4). Like compound 1, the absolute configuration (3R,5R,9S,10R) of 3 was confirmed by singlecrystal X-ray (Cu Kα) diffraction analysis (Figure 4).

Figure 4. NOE correlations and the ORTEP drawing of 3.

Table 3. 1H NMR Data (400 MHz, CDCl3, δ in ppm, J in Hz) of Compounds 4−8a no. 1α 1β 2 3 5 6α 6β 9 11α 11β 12α 12β 14 15 16 17 18 19 20 OMe a

4 1.75, 1.26, 1.64, 3.30, 1.51, 2.37, 2.59, 2.07, 1.41, 1.74, 2.14, 1.41, 6.74, 1.78, 0.94, 0.98, 0.99, 0.87, 0.83,

m ddd (13.6, 13.6, 3.4) m; 1.70, m dd (11.4, 4.0) dd (13.5, 5.0) dd (18.6, 13.5) dd (18.6, 5.1) m m m ddd (10.5, 4.7, 2.0) m br s m d (7.0) d (7.0) s s s

5 1.89, 1.44, 1.82, 3.48,

ddd (13.9, 4.0, 3.7) m m, 2H dd (8.9, 6.5)

6.24, s 2.38, m 1.74, m, 2H 1.77, 1.46, 6.90, 1.82, 0.89, 0.98, 1.29, 1.19, 1.12,

m m br s m d (7.0) d (7.0) s s s

6 1.84, 1.27, 1.65, 3.31, 1.53, 2.41, 2.60, 1.92, 1.51, 1.66, 1.72, 1.38, 6.79, 2.08, 0.77, 0.99, 0.99, 0.88, 0.86, 3.19,

ddd (13.5, 3.5, 3.5) ddd (13.1, 13.1, 5.3) m; 1.73, m dd (11.4, 4.2) dd (13.5, 4.9) dd (18.9, 13.5) dd (18.9, 4.9) m m m m m br s m d (7.0) d (7.0) s s s s

7 1.84, 1.27, 1.65, 3.30, 1.52, 2.38, 2.58, 1.94, 1.50, 1.71, 1.83, 1.46, 7.01,

ddd (13.4, 3.5, 3.5) ddd (13.3, 13.3, 4.3) m; 1.72, m dd (11.2, 4.2) dd (13.5, 5.2) dd (18.6, 13.2) dd (18.6, 5.2) m m m m ddd (13.8, 13.6, 2.8) br s

1.13, 1.22, 0.98, 0.87, 0.83,

s s s s s

8 1.82, 1.27, 1.69, 3.31, 1.53, 2.38, 2.60, 2.11, 1.52, 1.82,

m ddd (13.5, 13.5, 3.3) m; 1.72, m dd (11.6, 4.1) dd (13.3, 5.4) dd (18.8, 13.3) dd (18.8, 5.4) m m m

3.76, 6.78, 2.13, 0.87, 1.04, 0.99, 0.88, 0.85,

dd (12.0, 3.8) br s m d (7.0) d (7.0) s s s

Assignments were made by a combination of 1D and 2D NMR experiments. D

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data (Experimental Section). The 1H and 13C NMR data (Tables 1 and 3) of 7 and 8 showed similarities to those of 4, except for an additional tertiary (for 7, δC 74.8) or secondary (for 8, δH 3.76; δC 69.0) hydroxy group. Their planar structures were confirmed by inspection of their 2D NMR spectra. For compound 7, the key HMBC correlations from H-14 (δ 7.01), Me-16 (δ 1.13), and Me-17 (δ 1.22) to the oxygenated tertiary carbons at δC 73.8 and 74.8 revealed that both C-13 and C-15 are hydroxylated (see Supporting Information). The relative configuration of 7 was found to be identical to that of 6 according to its NOESY NMR data (see Supporting Information). As for compound 8, the hydroxy groups at C3, C-12, and C-13 were umambiguously assigned based on the long-range correlations in its HMBC NMR spectrum. As shown in Figure 2, HMBC correlations were found between H-5 (δ 1.53)/Me-18 (δ 0.99)/Me-19 (0.88) and C-3 (δ 78.6), between H-14 (δ 6.78)/H-15 (δ 2.13) and C-12 (δ 69.0), and between H-15/Me-16 (δ 0.87)/Me-17 (δ 1.04) and C-13 (δ 73.3). The large value (12.0 Hz) of JH‑11β,12, together with the observed NOE correlation of H-12/Me-20, indicated that H-12 is axial and α-oriented. To determine the absolute configurations of compounds 7 and 8, each with a vic-diol segment, the Snatzke’s method22 was applied, involving the induced electronic circular dichroism (IECD) spectrum due to the in situ complexation of a 1,2-diol with dimolybdenum tetraacetate [Mo2(OAc)4] in DMSO solution. As shown in Figure 7, the complex of 7 (with a

Figure 5. ORTEP drawing of 4.

NMR data, indicating it has one more index of hydrogen deficiency than that of compound 4. Comparing the NMR data (Tables 1 and 3) of 5 with those of 4 revealed their structural similarities. Compound 5 possesses an additional trisubstituted double band (δH 6.24, s; δC 178.8, 125.7), which was assigned to be at C-5−C-6 by the HMBC correlations from H-3 (δ 3.48), Me-18 (δ 1.29), Me-19 (δ 1.19), and Me-20 (δ 1.12) to C-5 (δ 178.8) and from H-6 (δ 6.24) to C-5, C-8 (δ 136.7), and C-10 (δ 39.0). The relative configuration of 5 was determined via NOE correlations (see Supporting Information). Consequently, the structure of compound 5 was defined as 3α,13β-dihydroxy-ent-abieta-5,8(14)-dien-7-one. Sessilifol F (6) has the molecular formula (C21H34O3) established by its HRESIMS (m/z 357.2400 [M + Na]+, calcd 357.2400) and the 13C NMR data. Its 1H and 13C NMR data (Tables 1 and 3) were similar to those of 4, except for a methoxy group (δH 3.19, s; δC 51.2) instead of the tertiary hydroxy group at C-13 in 4, which was secured by the key HMBC correlations from OMe to C-13 (δC 75.5) (see Supporting Information). Differing from the β-oriented OH13 in 4, the methoxy group at C-13 in 6 was found to be αoriented based on a key NOE correlation between Me-20 and OMe (see Supporting Information). Since compound 6 (20.8 mg) is a relatively major component, the modified Mosher’s method21 was successfully utilized to determine its absolute configuration. As shown in Figure 6, the ΔδH (ΔδH = δS − δR)

Figure 7. IECD spectra for Mo2(OAc)4 complexes of 7 and 8.

negative sign of the O−C−C−O torsion angle of the diol unit) and Mo2(OAc)4 in anhydrous DMSO gave a significant IECD spectrum, in which the Cotton effect at 315 nm is visible as a positive minimum only. Nevertheless, the tendency to form a minimum by this band is clearly visible, which permitted the assignment of a 13S configuration in 7.22a Likewise, the Cotton effect at 297 nm shown as a positive minimum in the IECD spectrum of the metal complex of 8 established the 12R,13R configurations (Figure 7). Therefore, the structures of compounds 7 and 8 were defined as (3R,5S,9S,10R,13S)3,13,15-trihydroxy-ent-abieta-8(14)-en-7-one and (3R,5S,9S,10R,12R,13R)-3,12,13-trihydroxy-ent-abieta-8(14)en-7-one, respectively. Sessilifol I (9) has the same molecular formula (C20H32O3) and the same NMR data (Experimental Section) as those of 13β,18-dihydroxyabieta-8(14)-en-7-one,23 a known abietenetype diterpenoid previously isolated from the cones of Larix

Figure 6. Results with the modified Mosher’s method (ΔδH = δS − δR).

values for H2-1 and H2-2 were positive, while negative values were observed for H-5, Me-18, and Me-19. The results allowed the assignment of a 3R configuration. Thus, the structure of compound 6 was deduced as (3R,5S,9S,10R,13R)-3-hydroxy13-methoxy-ent-abieta-8(14)-en-7-one. Sessilifols G (7) and H (8) were assigned the same molecular formula of C20H32O4 based on their HRESIMS and 13C NMR E

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Table 4. 1H NMR Data (CDCl3, 400 MHz, δ in ppm, J in Hz) of Compounds 10−13 and 16a no.

a

10 ddd (13.0, 3.2, 3.2) ddd (13.0, 13.0, 4.4) m, 2H dd (11.0, 5.0) dd (12.4, 2.3) m m br dd (17.2, 7.2) ddd (17.2, 11.0, 7.4)

11 ddd (12.2, 2.8, 2.8) m m, 2H dd (11.0, 4.0) br d (3.7) dd (9.6, 3.7)

2.51, 1.67, 2.05, 3.44,

13

2.31, 1.53, 1.81, 3.29, 1.32, 1.76, 1.91, 2.97, 2.86,

7.20, d (8.3) 7.23, dd (8.3, 2.0)

7.12, d (8.2) 7.31, dd (8.2, 2.0)

7.50, d (8.3) 7.78, dd (8.3, 2.0)

7.14, d (8.3) 7.10, dd (8.3, 2.0)

14 15 16 17 18 19 20

7.17, d (2.0)

7.19, d (2.0)

8.17, d (2.0)

1.56, 1.56, 1.07, 0.90, 1.19,

1.57, 1.57, 1.05, 1.03, 1.09,

1.61, 1.63, 1.38, 1.32, 1.54,

7.39, 2.88, 1.24, 1.24, 1.08, 0.90, 1.26,

s s s s s

2.22, 1.81, 1.84, 3.37, 2.13, 6.02,

12

1α 1β 2 3 5 6α 6β 7α 7β 9 11 12

ddd (13.5, 3.5, 3.5) ddd (13.5, 13.5, 3.2) m, 2H dd (11.6, 4.0)

6.59, s

6.60, dd (9.6, 1.7)

s s s s s

s s s s s

2.29, 1.50, 1.81, 3.29, 1.34, 1.71, 2.29, 4.81,

m ddd (13.0, 13.0, 5.2) m, 2H dd (11.0, 4.9) dd (12.6, 1.0) ddd (12.3, 12.3, 10.2) m dd (10.2, 7.2)

d (2.0) m d (7.0) d (7.0) s s s

16 1.78, 1.31, 1.73, 3.35, 1.73, 1.77, 1.93, 4.41,

ddd, overlapped ddd (13.4, 13.4, 3.0) m; 1.58, m dd (11.8, 3.9) dd (11.7, 5.2) ddd, overlapped br d (13.2) br s

2.48, 2.03, 2.44, 2.28, 6.00,

dd (7.5, 7.5) m, α-H; 1.79, m, β-H ddd (16.0, 4.9, 4.9), α-H ddd (16.3, 13.2, 5.0), β-H br s

1.06, s 0.85, s 0.81, s

Assignments were made by a combination of 1D and 2D NMR experiments.

kaempferi. However, in contrast with the positive specific rotation {[α]23D +8.9 (c 0.64, CHCl3)} observed for the known compound,23 compound 9 has a negative specific rotation {[α]25D −7.5 (c 0.08, CHCl3)}. Thus, these two compounds are regarded as a pair of enantiomers. Moreover, similar Cotton effects observed for 9 (Δε245 −2.3, Δε341 +0.6) and 7 (Δε245 −3.1, Δε342 +0.8) in their ECD spectra (Experimental Section) indicated that they shared the same absolute configurations at C-9 (S) and C-13 (R). Thus, the structure of compound 9 was defined as (4S,5S,9S,10R,13R)-13,18-dihydroxy-ent-abieta8(14)-en-7-one. Sessilifol J (10) was obtained as colorless crystals (from CHCl3). A sodium adduct ion at m/z 325.2132 ([M + Na]+, calcd 325.2144) in its HRESIMS and the 13C NMR data indicated that compound 10 possesses the molecular formula C20H30O2 with six indices of hydrogen deficiency. Like compounds 1−9, the 13C and DEPT NMR spectra of 10 (Table 1) also exhibited 20 carbon resonances, consisting of five methyls, four methylenes, five methines (one oxygenated at δ 78.7 and three olefinic at δ 122.0, 124.4, 124.8), one oxygenated tertiary carbon at δ 72.3, and five quaternary carbons (three olefinic at δ 134.8, 146.1, 147.8). Similar to compound 7, the signals of five tertiary methyls at δ 0.90 (3H, Me-19), 1.07 (3H, Me-18), 1.19 (3H, Me-20), and 1.56 (6H, Me-16/Me-17) and one oxymethine double doublet at δ 3.29 (dd, J = 11.0, 5.0 Hz, H-3) were evident in the 1H NMR spectrum of 10 (Table 4). However, a deshielded ABX spin system reminiscent of a 1,2,4-trisubstituted benzene ring, as demonstrated by signals at δ 7.17 (d, J = 2.0 Hz), 7.20 (d, J = 8.3 Hz), and 7.23 (dd, J = 8.3, 2.0 Hz), was evident. These revealed that 10 is a 3,15-dihydroxyabieta-8,11,13-triene derivative,24 which was further confirmed by its 2D NMR data, especially the HMBC correlations. Accordingly, H-3 and H-5 were axially oriented based on their large coupling constants (Table 4). NOE correlations of Me-18/H-3, Me-18/H-5, H-5/H-3, and Me-19/Me-20 indicated that Me-18, H-3, and H-5 were cofacial, whereas Me-19 and Me-20 were located on the opposite side. The absolute

configuration of 10 was defined by a single-crystal X-ray diffraction analysis using the anomalous scattering of Cu Kα radiation (Figure 8). Therefore, the structure of compound 10 was assigned as (3R,5S,10R)-ent-abieta-3,15-dihydroxy-8,11,13triene.

Figure 8. ORTEP drawing of 10.

The positive mode HRESIMS (m/z 323.1994 [M + Na]+) and the 13C NMR data of sessilifol K (11) indicated the molecular formula of C20H28O2 with one more index of hydrogen deficiency than that of compound 10. Its 1H and 13C NMR data (Tables 1 and 4) resembled those of 10, except for the presence of an additional double bond at C-6 [δH 6.02 (dd, J = 9.6, 3.7 Hz, H-6), δC 129.6; δH 6.60 (dd, J = 9.6, 1.7 Hz, H7), δC 128.5] in 11. Accordingly, the structure of 11 was defined as 3α,15-dihydroxy-ent-abieta-6,8,11,13-tetraene. Sessilifol L (12) was assigned the molecular formula C20H26O3 based on its HRESIMS (Experimental Section) and 13C NMR data. Comparison of its NMR data (Tables 1 and 4) with those of 10 and 11 indicated the presence of a conjugated Δ5,6-7-one unit [δH 6.59, s (H-6); δC 172.1 (C-5), F

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125.3 (C-6), 185.9 (C-7)] in 12. This was verified by the key HMBC correlations from H-6 to C-4 (δ 43.4), C-8 (δ 129.5), and C-10 (δ 40.7), from H-14 (δ 8.17) to C-7, and from Me-18 (δ 1.38), Me-19 (δ 1.32), and Me-20 (δ 1.54) to C-5. The orientaions of H-3 and Me-20 in 12 were identical to those in compounds 10 and 11 according to the analysis of the proton− proton coupling constants (Table 4) and the observed NOE correlations (see Supporting Information). Thus, the structure of compound 12 was determined to be 3α,15-dihydroxy-entabieta-5,8,11,13-tetraen-7-one. Sessilifol M (13) possesses the same molecular formula (C20H30O2) as compound 10 by its HRESIMS (Experimental Section) and 13C NMR data. Likewise, the 1H and 13C NMR data of 13 were similar to those of 10 (Tables 1 and 4), except that the signals for a tertiary hydroxy group at C-15 in 10 were replaced by a secondary hydroxy group (δH 4.81, dd, J = 10.2, 7.2 Hz; δC 71.4) in 13. The latter was concluded to be at C-7 based on the HMBC correlations from H-7 (δ 4.81) to C-6 (δ 30.0)/C-8 (δ 137.6) and from H-5 (δ 1.34)/H-14 (δ 7.39) to C-7 (δ 71.4) (Figure 2). Furthermore, the magnitude (10.2 Hz) of JH‑6α,H‑7 and the NOE correlations of Me-18/H-5 and H-5/ H-7 indicated that H-7 was in the β-orientation. Thus, compound 13 was identified as 3α,7α-dihydroxy-ent-abieta8,11,13-triene. The molecular formula of sessilifol N (14) was determined to be C20H32O4 based on a sodium adduct ion at m/z 359.2203 [M + Na]+ in its positive HRESIMS and the 13C NMR data, implying five indices of hydrogen deficiency. The 1H NMR spectrum of 14 (Table 2) showed the presence of three tertiary methyl singlets at δ 1.28 (s, Me-18), 1.06 (s, Me-19), and 0.85 (s, Me-20), two equivalent secondary methyl doublets at δ 1.12 (6 H, d, J = 7.0 Hz, Me-16 and Me-17), two oxymethines at δ 3.25 (dd, J = 11.8, 4.3 Hz, H-3) and 4.64 (dt, J = 10.0, 2.4, 2.4 Hz, H-6), an olefinic proton at δ 6.60 (t, J = 2.4 Hz, H-7), and a typical formyl proton at δ 9.45 (s, H-14). The 13C NMR data of 14 (Table 1) suggested that it is also a diterpenoid with the 20 carbons classified as five methyls, four methylenes, six methines (two oxygenated at δ 78.5 and 68.5, one olefinic at δ 153.5, and a formyl at δ 194.9), four quaternary carbons (one olefinic at δ 143.3), and one carbonyl group at δ 215.3. As the ketocarbonyl and the α,β-unsaturated formyl groups accounted for three out of the five indices of hydrogen deficiency, the remaining two indicated that 14 is a bicyclic compound. These structural features were similar to those of 13,14-seco-13,14-dioxoabieta7-en-18-oic acid, a known 13,14-seco-abietene-type diterpenoid isolated from the cones of Larix kaempferi.25 More detailed information about the planar structure of 14 came from inspection of the HMBC spectrum (Figure 2), from which the hydroxy groups at C-3 and C-6, a Δ7,8-14-al α,β-unsaturated formyl unit, and the carbonyl group at C-13 were all verified. The relative configuration of 14, depicted in Figure 1, was determined by analyses of the proton−proton coupling constants (Table 2) and ROE correlations. The magnitudes of JH‑2α,3 (11.8 Hz) and JH‑5,6 (10.0 Hz) indicated that H-2α, H3, H-5, and H-6 were all in axial positions. ROE correlations of Me-18/H-3, Me-18/H-5, H-3/H-5, H-5/H-9, Me-19/Me-20, Me-19/H-6, and Me-20/H-6 were observed. Thus, the structure of compound 14 was established as 13,14-seco3α,6β-dihydroxy-13-oxo-ent-abieta-7-en-14-al. The 13,14-secoabietanes are quite rare from natural sources, and only two examples have been previously reported.25,26 Sessilifol O (15) has the molecular formula C19H32O3 as determined by the HRESIMS (m/z 331.2242 ([M + Na]+) and

C NMR data, implying four indices of hydrogen deficiency. In accordance with the molecular formula, only 19 carbon signals were detected in the 13C NMR spectrum of 15 (Table 1). By comparing the 1H and 13C NMR spectroscopic data of 15 with those of compound 14 (Tables 1 and 2), a bicyclic norabietane scaffold was determined for 15. Differing from 14 with a Δ7,814-al unit, an additional saturated carbonyl (δC 211.2) presented at C-7 in 15, which was established by 2D NMR experiments. Particularly, two spin systems of CH(5)−CH2(6) and CH2(8)−CH(9)−CH2(10)−CH2(11) in the COSY spectrum and the HMBC correlations from H2-6 (δ 2.36/ 2.41), H2-8 (δ 2.05/2.33), and H-9 (δ 1.25) to C-7 were evident. The large values of JH‑2α,3 (11.8 Hz) and JH‑8α,9 (12.9 Hz) indicated that both H-3 and H-9 were in axial orientations. The relative configuration of 15 was determined as depicted in Figure 1 from the NOE correlations (see Supporting Information). On the basis of the above evidence, 15 was identified as 13,14-seco-3α-hydroxy-ent-14-norabieta-7,13dione. Compound 15 represents the first example of a naturally occurring 14-norabietane-type diterpenoid. Only one analogue (1-naphthalenecarboxylic acid = 13,14-seco-8,13-dioxo-14norabieta-18-oic acid) with such a scaffold has been reported, as a semisynthetic product in 1941.27 The molecular formula of sessilifol P (16) was determined as C17H26O3 from the 13C data and an [M + H]+ peak at m/z 279.1961 in the HRESIMS. The absorption bands (1698, 1667, 1455 cm−1) in its IR spectrum and the absorption maximum at 237 nm in its UV spectrum indicated an α,β-unsaturated carbonyl unit in 16. The 1H NMR data of 16 (Table 4) displayed signals for three tertiary methyls ((δ 1.06 (s, Me-18), 0.85 (s, Me-19), and 0.81 (s, Me-20)), two oxymethine protons (δ 3.35 (dd, J = 11.8, 3.9 Hz, H-3) and 4.41 (br s, H-7)), and one vinylic proton (δ 6.00 (br s, H-14)). The 13C NMR spectrum (Table 1) exhibited only 17 carbon resonances of three methyls, five methylenes, five methines (two oxygenated at δ 78.7 and 71.5, one olefinic at δ 127.5), three quaternary carbons (one olefinic at δ 163.5), and a carbonyl group at δ 200.7. These data showed general features similar to those of 7α-dihydroxypodocarp-8(14)-en-13-one, a known podocarpane-type C17 norditerpene isolated from Abies georgei.28 The only difference was that an additional secondary hydroxy group was found in 16, which is a typical 3α-OH resembling the above ent-abietanes 1−15. The relative configuration of 16 was defined by the NOE correlations of Me-18/H-3, H-3/H-5, H5/H-9, and Me-20/H-7. Moreover, the ECD spectrum of 16 showed a negative Cotton effect at 234 nm but a positive one at 326 nm, which were opposite of those of (4R,5R,7R,9R,10S)7,18-dihydroxypodocarp-8(14)-en-13-one,23 allowing the definition of absolute configurations of C-7 (S) and C-9 (S) in 16. Thus, compound 16 was characterized as (3R,5S,7S,9S,10R)3,7-dihydroxy-ent-podocarpa-8(14)-en-13-one. Sessilifol Q (17) was found to have the same molecular formula (C17H26O3) as 16 from the 13C NMR data and an [M + Na]+ ion at m/z 301.1789 in its HRESIMS. Comparison of the NMR data of these two compounds (Table 1 and Experimental Section) suggested that 17 shares the same planar structure (Figure 2) as 16, but requires a different configuration at C-7. This inference was thereafter confirmed by the observation of a large value (11.5 Hz) of JH‑6α,H‑7 and the NOE correlations of H-7 with H-5 and H-9 (see Supporting Information). Thus, the structure of 17 was defined as the C-7 epimer of compound 16. G

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Among the 17 new isolates, sessilifols A (1) and B (2) are the first two abietane-type representatives possessing an unusual five-membered C-ring. Sessilifol C (3) is the second 7,8-seco-9-spiro-fused ent-abietane, whereas sessilifol O (15) is the first example of naturally occurring 14-norabietane-type diterpenoids. As for 3, it might be formed via a biosynthetic pathway previously proposed for decandrinin,20 the first 7,8seco-9-spiro-fused ent-abietane. It seems more reasonable that the 3-OH group in 3 should be introduced by oxidation at the final but not the initial step as described by Wang et al.20 We herein only briefly discuss putative biogenetic pathways toward compounds 1, 2, and 15 (Scheme 1). As shown in Scheme 1, a

Table 5. Inhibitory Effects on LPS-Induced NO Production in BV-2 Cells compound 6 8 9 10 13 d L-NMMA

IC50a,b (μM) 8.3 37.7 7.4 17.8 43.9 14.4

± ± ± ± ± ±

1.3 4.5 0.8 1.8 3.3 1.2

cell viabilityc (%) 96.7 98.4 96.8 95.4 94.7 98.5

± ± ± ± ± ±

4.3 2.9 5.5 4.0 5.5 3.4

a IC50 value of each compound was defined as the concentration (μM) of indicated compound that caused 50% inhibition of NO production in LPS-stimulated BV-2 cells. bThe results are averages of three independent experiments, and the data were expressed as mean ± SD. c Cell viability after treatment with 50 μM of each tested compound was expressed as a percentage (%) of untreated control cells. dLNMMA: positive control.

Scheme 1. Putative Biogenetic Pathways of Compounds 1, 2, and 15

that are rich in sesquiterpenoids, the current research shows that ent-abietane-type diterpenoids are the major secondary metabolites of C. sessilifolius. A literature survey showed that ent-abietanoids have been previously obtained only from the following genera: Ceriops (family: Rhizophoraceae),20 Suregada (family: Euphorbiaceae),30 Euphorbia (family: Euphorbiaceae),31 Isodon (family: Labiatae),32 Goldf ussia (family: Acanthaceae),33 Doellingeria,34 and Solidago (family: Compositae).35 Our findings add to the diversity and complexity of entabietanes and their natural origins. Moreover, compounds 6 and 9 showed inhibitory effects on NO production in LPS-stimulated BV-2 microglial cells. This is the first report on the antineuroinflammatory activity of naturally occurring (ent-)abietane-type diterpenoids. This study may contribute to further exploration of the therapeutic potential of the ent-abietenes in neurodegenerative and other aging-associated diseases.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on an Autopol IV automatic polarimeter. UV and IR spectra were recorded on a Hitachi U-2900E double-beam spectrophotometer and a Thermo Scientific Nicolet Is5 FTIR spectrometer, respectively. ECD spectra were recorded on a JASCO810 spectropolarimeter. NMR spectra were recorded on a Bruker Avance III 600 MHz and/or a Bruker Avance III 400 MHz spectrometer. Chemical shifts are expressed in δ (ppm) and referenced to the residual solvent signals. ESIMS were measured on a Waters UPLC H-Class SQD or on an Agilent 1100 series mass spectrometer; HRESIMS were measured on an Aglient Technologies 6224 TOF LC/ MS or on an LCT Premier XE (Waters) mass spectrometer or on an Agilent Technologies 1290 Infinity LC System. X-ray data were collected on a Bruker Apex Duo diffractometer using graphitemonochromated Cu Kα radiation. Semipreparative HPLC were performed on a Waters e2695 system with a Waters 2998 PAD, a Waters 2424 ELSD, and SunFire ODS columns (5 μm, 250 × 10 mm; flow rate: 3.0 mL/min). Column chromatography (CC) was performed using silica gel (Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, China) and MCI gel CHP20P (75−150 μm, Mitsubishi Chemical Industries, Tokyo, Japan). Gel-precoated plates (GF254, 0.25 mm, Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, China) were used for TLC detection. Spots were visualized under UV light (254 nm) and by spraying with 15% (v/v) H2SO4/EtOH followed by heating to 120 °C. Plant Material. Whole plants of C. sessilifolius were collected by one of the authors (S.-T.L.) in August 2012 from the Fengqi Mountains in the Sichuan Province of China. The plant was identified by Prof. Baokang Huang (Department of Pharmacognosy, Second Military Medical University of China). A voucher specimen (No.

13,14-seco-abietane structure i, generated from abieta-7,13diene via an oxidative cleavage at Δ13,14,29 is probably the common precursor to compounds 1, 2, and 15. An unusual [6,6,5]-tricyclic abietane scaffold could then be constructed by an intramolecular aldol reaction19 of i, resulting in the formation of a C-12−C-14 bond in 1 and 2. On the other hand, the 14-norabietane 15 may be generated from the key intermediate i through a sequence of oxidation reactions followed by decarboxylization. The above isolates (except 11) were evaluated for their inhibitory effects against the NO production in LPS-stimulated BV-2 cells. As shown in Table 5, compounds 6 and 9 showed the most significant activities (IC50 values of 8.3 and 7.4 μM, respectively), whereas compounds 8, 10, and 13 also showed moderate inhibitory effects. The rest were inactive (IC50 values >100 μM). None of the tested compounds (each at a concentration up to 50 μM) was found to exhibit cytotoxicity against the BV-2 cells. From the above results, the regular C20 ent-abietane-type diterpenoids generally showed better activities than the seco- and nor-abietanes. The position, configuration, and substitution of the hydroxy groups also seem to be crucial for the potency of these ent-abietane-type diterpenoids. Thus, 14 new C20 ent-abietane-type diterpenoids (sessilifols A−N, 1−14) and three related new norditerpenoids (sessilifols O−Q, 15−17) were isolated from C. sessilifolius, a rare Chloranthaceae plant. Unlike other studied Chloranthus species H

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2961, 2932, 2873, 1716, 1663, 1463, 1255, 1054 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 335.2 [M + H]+, 691.4 [2 M + Na]+; HRESIMS m/z 691.4189 [2 M + Na]+ (calcd for C40H60O8Na, 691.4186, Δ = +0.4 ppm). X-ray crystal data of 3. C20H30O4; colorless crystals were obtained from a solution of CHCl3/n-hexane (1:2, v/v). M = 334.44, orthorhombic, space group P212121, a = 7.03520(10) Å, b = 9.560180(10) Å, c = 26.5701(3) Å, α = 90°, β = 90°, γ = 90°, V = 1794.83(4) Å3, T = 140(2) K, Z = 4, μ(Cu Kα) = 0.676 mm−1, Dcalc = 1.238 mg/m3, F(000) = 728, 8925 reflections collected, 3237 independent reflections (Rint = 0.0394). The final R1 = 0.0412, wR(2) = 0.1077 (I > 2σ(I)), Flack parameter = 0.04(7). Sessilifol D [(3R,5S,9S,10R,13S)-3,13-Dihydroxy-ent-abieta-8(14)en-7-one, 4]: colorless crystals; [α]25D +50 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 248 (3.62) nm; ECD (c 1.56 × 10−3 M, MeOH) λmax (Δε) 254 (+5.9), 330 (+1.8) nm; IR (film) νmax 3417 (br), 2960, 2936, 2871, 1680, 1557, 1457, 1307, 1033 cm−1; for 1H and 13C NMR data, see Tables 1 and 3; ESIMS m/z 343.2 [M + Na]+; 663.4 [2 M + Na]+; HRESIMS m/z 663.4617 [2 M + Na] + (calcd for C40H64O6Na, 663.4601, Δ = +2.4 ppm). X-ray crystal data of 4: C20H32O3; colorless crystals were obtained from MeOH. M = 320.45, monoclinic, space group P21, a = 6.1311(2) Å, b = 17.3576(7) Å, c = 8.5062(4) Å, α = 90°, β = 98.395(2)°, γ = 90°, V = 895.54(6) Å3, T = 140(2) K, Z = 2, μ(Cu Kα) = 0.610 mm−1, Dcalc = 1.188 mg/m3, F(000) = 352, 6754 reflections collected, 2979 independent reflections (Rint = 0.0290). The final R1 = 0.0382, wR(2) = 0.1009 (I > 2σ(I)), Flack parameter = 0.01(10). Sessilifol E [3α,13β-Dihydroxy-ent-abieta-5,8(14)-dien-7-one, 5]: colorless, amorphous powder; [α]25D +51 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 267 (3.46) nm; ECD (c 1.10 × 10−3 M, MeOH) λmax (Δε) 221 (−0.7), 247 (+1.3), 281 (−0.6), 361 (+1.3) nm; IR (film) νmax 3401 (br), 2941, 2930, 1650, 1614, 1455, 1383, 1038 cm−1; for 1H and 13C NMR data, see Tables 1 and 3; ESIMS m/z 319.2 [M + H]+, 659.4 [2 M + Na]+; HRESIMS m/z 659.4312 [2 M + Na]+ (calcd for C40H60O6Na, 659.4288, Δ = +3.6 ppm). Sessilifol F [(3R,5S,9S,10R,13R)-13-Methoxy-3-hydroxy-ent-abieta-8(14)-en-7-one, 6]: colorless, amorphous powder; [α]25D −7 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 242.5 (3.69) nm; ECD (c 8.68 × 10−4 M, MeOH) λmax (Δε) 245 (−1.8), 337 (+0.6) nm; IR (film) νmax 3421 (br), 2945, 2932, 1704, 1614, 1458, 1387, 1073 cm−1; for 1H and 13 C NMR data, see Tables 1 and 3; ESIMS m/z 357.2 [M + Na]+; HRESIMS m/z 357.2400 [M + Na]+ (calcd for C21H34O3Na, 357.2400, Δ = 0.0 ppm). Preparation of (R)- and (S)-MTPA Esters of 6. Similar to previous reports,21 compound 6 (5.5 mg) was dissolved in CH2Cl2 (2 mL), to which (S)-MTPA (46.4 mg), 4-DMAP (47.6 mg), and EDC·HCl (38.2 mg) were added successively. The reaction mixture was stirred at room temperature for 12 h and then evaporated to give a yellow residue, which was applied to a silica gel column (CH2Cl2/EtOAc, 10:1, v/v) and further purified by semipreparative HPLC (MeOH, 90:10, v/v, 3 mL/min) to yield the (S)-MTPA ester of 6 (6a, 1.4 mg, 16.5%). The (R)-MTPA ester of 6 (6b, 4.3 mg, 30.7%) was obtained by using the same procedure as described above but using (R)-MTPA. (S)-MTPA ester of 6 (6a): colorless gum; 1H NMR (CDCl3, 600 MHz) δ 7.54−7.57 (2H, m), 7.39−7.43 (3H, m), 3.58 (OMe, s), 1.42 (ddd, J = 13.5, 13.5, 2.5 Hz, H-1α), 1.90 (1H, ddd, J = 13.5, 2.9, 2.9 Hz, H-1β), 1.73 (m, H-2a), 1.66 (m, H-2b), 4.76 (dd, J = 11.3, 4.0 Hz, H-3), 1.64 (dd, J = 13.2, 5.2 Hz, H-5), 2.40 (dd, J = 18.7, 13.2 Hz, H6α), 2.60 (dd, J = 18.7, 5.2 Hz, H-6β), 1.96 (m, H-9), 1.52 (m, H11α), 1.66 (m, H-11β), 1.86 (m, H-12α), 1.37 (m, H-12β), 6.82 (br s, H-14), 2.09 (m, H-15), 0.78 (d, J = 6.9 Hz, Me-16), and 0.90 (d, J = 6.9 Hz, Me-17), 0.82 (s, Me-18), 0.89 (s, Me-19), 0.90 (3H, s, Me-20) ppm; ESIMS m/z 573.2 [M + Na]+. (R)-MTPA ester of 6 (6b): colorless gum; 1H NMR (CDCl3, 400 MHz) δ 7.51−7.54 (2H, m), 7.39−7.43 (3H, m), 3.53 (OMe, s), 1.41 (ddd, J = 13.2, 13.2, 3.6 Hz, H-1α), 1.87 (overlapped, H-1β), 1.72 (m, H-2a), 1.65 (m, H-2b), 4.79 (dd, J = 11.5, 4.0 Hz, H-3), 1.65 (dd, J = 12.4, 5.0 Hz, H-5), 2.40 (dd, J = 18.8, 12.2 Hz, H-6α), 2.60 (dd, J = 18.7, 5.0 Hz, H-6β), 1.96 (m, H-9), 1.52 (m, H-11α), 1.67 (m, H11β), 1.86 (m, H-12α), 1.37 (m, H-12β), 6.82 (br s, H-14), 2.09 (m,

20120801) was deposited at the Herbarium of the Department of Natural Products Chemistry, School of Pharmacy at Fudan University. Extraction and Isolation. The dried whole plant of C. sessilifolius (6.8 kg) was extracted with MeOH (3 × 20 L) at room temperature. After filtration, the solvents were removed under vacuum to give a crude extract (463 g), which was suspended in H2O (2 L) and partitioned successively with petroleum ether (PE, 3 × 1.5 L), EtOAc (3 × 1.5 L), and n-BuOH (3 × 1.5 L). The EtOAc extract (248.0 g) was subjected to silica gel (100−200 mesh) CC with PE/acetone gradients (from 9:1 to 0:1, v/v) to afford six fractions (A−F) monitored by TLC. Fr. A was separated by repeated CC over silica gel and purified using semipreparative HPLC (MeCN/H2O, 65:35, v/v, 3.0 mL/min) to yield compounds 6 (20.8 mg, tR = 19.1 min), 9 (1.7 mg, tR = 22.8 min), and 15 (3.4 mg, tR = 20.7 min). Fr. B was subjected to an MCI gel column (6 × 30 cm) eluted with gradients of MeOH/H2O (from 50% to 100%, v/v). The 70−80% MeOH fraction (Fr. BB, 15.0 g) was subjected to a silica gel column (200−300 mesh) with a PE/EtOAc gradient (from 4:1 to 2:1, v/v) to afford five subfractions (Fr. BB1−BB5) monitored by TLC. Fr. BB3 was subjected to a silica gel column (200−300 mesh) followed by semipreparative HPLC to afford compounds 1 (2.2 mg), 2 (0.7 mg), 10 (1.3 mg), and 13 (1.7 mg). The method employed consisted of an isocratic gradient of 60% MeOH in H2O for 15 min, followed by a linear gradient of MeOH from 60% to 70% over 20 min, and finally by 100% MeOH for 5 min [1: tR = 21.5 min; 2: tR = 20.6 min; 10: tR = 17.5 min; and 13: tR = 13.4 min]. Fr. BB5 was subjected to CC over silica gel (200−300 mesh) with CH2Cl2/EtOAc (2:1, v/v) and further purified by semipreparative HPLC with MeOH/H2O (50:55, v/v) to yield compounds 3 (1.9 mg, tR = 15.7 min), 11 (0.9 mg, tR = 18.2 min), and 12 (0.9 mg, tR = 19.2 min). Fr. D was subjected to an MCI gel column eluted with a gradient of MeOH/H2O (from 50% to 100%, v/v). The 70−80% MeOH fraction (Fr. DB, 10.5 g) was subjected to a silica gel column (100−200 mesh) with PE/EtOAc gradients (from 2:1 to 1:2, v/v) to afford six subfractions (DB1−DB6) monitored by TLC. From Fr. DB1, compounds 4 (6.3 mg, tR = 18.6 min) and 5 (1.1 mg, tR = 15.5 min) were purified by semipreparative HPLC (MeOH/ H2O, 65:35, v/v). Compounds 16 (7.6 mg, tR = 20.5 min) and 17 (15.8 mg, tR = 22.5 min) were isolated from Fr. DB4 by semipreparative HPLC with MeOH/H2O from a linear gradient from 50:50 to 60:40 (v/v) for 20 min, then followed by an isocratic gradient of 60% MeOH for 10 min and finally by 100% MeOH for 5 min. Fr. DB5 was subjected to CC over silica gel (200−300 mesh) with CH2Cl2/EtOAc (1:1, v/v) and further purified by semipreparative HPLC with MeOH/H2O from a linear gradient from 50:50 to 60:40 (v/v) for 20 min, to furnish 7 (14.1 mg, tR = 12.9 min), 8 (3.4 mg tR = 15.6 min), and 14 (14.9 mg, tR = 13.5 min). Sessilifol A [(3R,5S,9S,10R,12R,14S)-3,14-Dihydroxy-ent-14(13→ 12)-abeo- abieta-7-en-13-one, 1]: colorless crystals; [α]25D −26 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 236 (3.40) nm; IR (film) νmax 3405 (br), 2963, 2928, 2851, 1699, 1650, 1465, 1383, 1037 cm−1; for 1 H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 343.2 [M + Na]+; HRESIMS m/z 343.2259 [M + Na]+ (calcd for C20H32O3Na, 343.2249, Δ = +2.9 ppm). X-ray crystal data of 1: C20H32O3·1/2 H2O; colorless crystals were obtained from hydrous MeOH. M = 329.46, monoclinic, space group P21, a = 8.1519(11) Å, b = 20.549(3) Å, c = 10.9532(11) Å, α = 90°, β = 94.218(10)°, γ = 90°, V = 1829.8(4) Å3, T = 140(2) K, Z = 4, μ(Cu Kα) = 0.630 mm−1, Dcalc = 1.196 mg/m3, F(000) = 724, 13 244 reflections collected, 5568 independent reflections (Rint = 0.0606). The final R1 = 0.0644, wR(2) = 0.1726 (I > 2σ(I)), Flack parameter = −0.04(19). Sessilifol B (2): colorless, amorphous powder; [α]25D −23 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 237 (3.10) nm; IR (film) νmax 3420 (br), 2960, 2922, 2850, 1704, 1649, 1457, 1384, 1038 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 343.2 [M + Na]+; HRESIMS m/z 343.2231 [M + Na]+ (calcd for C20H32O3Na, 343.2249, Δ = −5.2 ppm). Sessilifol C (3): colorless crystals; [α]25D +81 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 244 (3.40) nm; ECD (c 8.98 × 10−4 M, MeOH) λmax (Δε) 245 (+17.8), 323 (+2.5) nm; IR (film) ν max 3417 (br.), I

DOI: 10.1021/acs.jnatprod.5b00195 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Sessilifol K [3α,15-Dihydroxy-ent-abieta-6,8,11,13-tetraene, 11]: colorless, amorphous powder; [α]25D +27 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 220 (3.51) nm; IR (film) νmax 3421 (br), 2940, 2925, 1649, 1384, 1260, 1030 cm−1; for 1H and 13C NMR data, see Tables 1 and 4; ESIMS m/z 323.2 [M + Na]+; HRESIMS m/z 323.1994 [M + Na]+ (calcd for C20H28O2Na, 323.1987, Δ = +2.2 ppm). Sessilifol L [3α,15-Dihydroxy-ent-abieta-5,8,11,13-tetraen-7-one, 12]: colorless, amorphous powder; [α]25D −14 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 257 nm (3.52); ECD (c 9.95 × 10−4 M, MeOH) λmax (Δε) 250 (−6.2), 279 (+2.4), 349 (+0.4) nm; IR (film) νmax 3419 (br.), 2959, 2917, 2849, 1737, 1649, 1602, 1462, 1366, 1260, 1039 cm−1; for 1H and 13C NMR data, see Tables 1 and 4; ESIMS m/z 315.2 [M + H]+, 651.4 [2 M + Na]+; HRESIMS m/z 651.3668 [2 M + Na]+ (calcd for C40H52O6Na, 651.3662, Δ = 0.9 ppm). Sessilifol M [3α,7α-Dihydroxy-ent-abieta-8,11,13-triene, 13]: colorless, amorphous powder; [α]25D −24 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 219 (3.83) nm; ECD (c 8.29 × 10−4 M, MeOH) λmax (Δε) 219 (−3.1), 268 (+0.4) nm; IR (film) νmax 3363 (br), 2957, 2928, 2867, 1459, 1383, 1067, 1023 cm−1; for 1H and 13C NMR data, see Tables 1 and 4; ESIMS m/z 325.2 [M + Na]+; HRESIMS m/z 325.2133 [M + Na]+ (calcd for C20H30O2Na, 325.2144, Δ = −3.4 ppm). Sessilifol N [13,14-Seco-3α,6β-dihydroxy-13-oxo-ent-abieta-7-en14-al, 14]: colorless, amorphous powder; [α]25D −19 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 234 (3.67) nm; ECD (c 8.69 × 10−4 M, MeOH) λmax (Δε) 215 (+20.0), 322 (−8.4) nm; IR (film) νmax 3407 (br), 2969, 2934, 2873, 2720, 1689 (br), 1639, 1463, 1384, 1039 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 359.2 [M + Na]+; HRESIMS m/z 359.2203 [M + Na]+ (calcd for C20H32O4Na, 359.2198, Δ = +1.4 ppm). Sessilifol O [13,14-Seco-3α-hydroxy-14-nor-ent-abieta-7,13dione, 15]: colorless, amorphous powder; [α]25D −6 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 247 (3.12) nm; IR (film) νmax 3440 (br), 2967, 2934, 2872, 1708, 1466, 1384, 1037 cm−1; for 1H and 13 C NMR data, see Tables 1 and 2; ESIMS m/z 331.2 [M + Na]+; HRESIMS m/z 331.2242 [M + Na]+ (calcd for C19H32O3Na, 331.2249, Δ = −2.1 ppm). Sessilifol P [(3R,5S,7S,9S,10R)-3,7-Dihydroxy-ent-podocarpa8(14)-en-13-one, 16]: colorless gum; [α]25D +44 (c 0.8, acetone); UV (MeOH) λmax (log ε) 237 (3.68) nm; ECD (c 1.08 × 10−3 M, MeOH) λmax (Δε) 234 (−4.7), 263 (+1.3), 326 (+3.7) nm; IR (film) νmax 3419 (br.), 2936, 2873, 1698, 1667, 1455, 1385, 1028 cm−1; for 1 H and 13C NMR data, see Tables 1 and 4; ESIMS m/z 279.2 [M + H]+; HRESIMS m/z 279.1961 [M + H]+ (calcd for C17H27O3, 279.1960, Δ = +0.4 ppm). Sessilifol Q [3α,7α-Dihydroxy-ent-podocarpa-8(14)-en-13-one, 17]: colorless gum; [α]25D +11 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 242 (3.69) nm; ECD (c 1.80 × 10−3 M, EtOH) λmax (Δε) 240 (−10.1), 322 (+1.8) nm; IR (film) νmax 3397 (br), 2928, 2870, 1654, 1455, 1384, 1026 cm−1; 1H NMR (400 MHz, CD3OD) δ 1.82 (ddd, J = 13.2, 3.5, 3.5, H-1α), 1.30 (overlapped, H-1β), 1.65 (m, H2-2), 3.26 (dd, J = 10.5, 5.2, H-3), 1.29 (overlapped, H-5), 1.56 (ddd, J = 12.4, 12.4, 11.5, H-6α), 2.11 (ddd, J = 12.4, 6.4, 1.9, H-6β), 4.15 (br dd, J = 11.5, 6.4, H-7β), 2.16 (dd, J = 7.5, 7.5, H-9), 2.05 (m, H-11α), 1.85 (m, H-11β), 2.37 (dt, J = 16.0, 5.2, H-12α), 2.27 (ddd, J = 16.0, 12.0, 5.0, H-12β), 6.27 (br s, H-14), 1.06 (s, Me-18). 0.86 (s, Me-19), 0.86 (s, Me-20); for 13C NMR data, see Table 1; ESIMS m/z 301.2 [M + Na]+; HRESIMS m/z 301.1789 [M + Na]+ (calcd for C17H26O3Na, 301.1774, Δ = +4.9 ppm). X-ray Crystallographic Experiment Details. The crystal structures of compounds 1, 3, 4, and 10 were solved by direct methods using SHELXS-97.36 Refinements were performed with SHELXL-2013 using full-matrix least-squares calculations on F2,37 with anisotropic displacement parameters for all the non-hydrogen atoms. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. CCDC-1038022 (1), CCDC1038120 (3), CCDC-1038021 (4), and CCDC-1038121 (10) contain the supplementary crystallographic data, which can be obtained free of

H-15), 0.78 (d, J = 6.9 Hz, Me-16), and 0.90 (d, J = 6.9 Hz, Me-17), 0.90 (s, Me-18), 0.90 (s, Me-19), 0.86 (s, Me-20) ppm; ESIMS m/z 573.2 [M + Na]+. Sessilifol G [(3R,5S,9S,10R,13S)-3,13,15-Trihydroxy-ent-abieta8(14)-en-7-one, 7]: colorless, amorphous powder; [α]25D +3 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 244 (3.41) nm; ECD (c 8.92 × 10−4 M, MeOH) λmax (Δε) 243 (−3.1), 342 (+0.8) nm; IR (film) νmax 3425 (br.), 2946, 2937, 2872, 1681, 1614, 1461, 1385, 1033 cm−1; for 1 H and 13C NMR data, see Tables 1 and 3; ESIMS m/z 359.2 [M + Na]+; HRESIMS m/z 359.2208 [M + Na]+ (calcd for C20H32O4Na, 359.2198, Δ = +2.8 ppm). Induced Electronic Circular Dichroism of 7. Using a published method,22a 1.0 mg of compound 7 was dissolved in 1.5 mL of anhydrous DMSO, of which 0.75 mL was used for the measurement of the ECD spectrum of 7. Mo2(OAc)4 (0.8 mg) was added into the remaining 0.75 mL solution. The ECD spectrum of the complex was recorded 20 min later, and the inherent ECD spectrum was subtracted. ECD (c 8.53 × 10−4 M, DMSO) λmax (Δε) 286 (−0.6), 315 (0.07), 335 (+0.40), 395 (−0.08). Sessilifol H [(3R,5S,9S,10R,12R,13R)-3,12,13-Trihydroxy-ent-abieta-8(14)-en-7-one, 8]: colorless, amorphous powder; [α]25D +6 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 241 (3.45) nm; ECD (c 8.63 × 10−4 M, MeOH) λmax (Δε) 239 (−1.4), 345 (+0.3) nm; IR (film) νmax 3397 (br), 2940, 2926, 2871, 1682, 1614, 1455, 1385, 1042 cm−1; for 1 H and 13C NMR data, see Tables 1 and 3; ESIMS m/z 359.2 [M + Na]+, 695.4 [2 M + Na]+; HRESIMS m/z 695.4501 [2 M + Na]+ (calcd for C40H64O8Na, 695.4499, Δ = +0.3 ppm). Induced Electronic Circular Dichroism of 8. Similar to that of 7, 0.8 mg of compound 7 was dissolved in 1.5 mL of anhydrous DMSO, of which 0.75 mL was used for the measurement of the ECD of 8. Mo2(OAc)4 (0.75 mg) was added into the remaining 0.75 mL solution. The ECD spectrum of the complex was recorded 20 min later, and the inherent ECD spectrum was subtracted. ECD (c 1.59 × 10−3 M, DMSO) λmax (Δε) 256 (+1.58), 297 (+0.1), 352 (+0.61). Sessilifol I [(4S,5S,9S,10R,13R)-13,18-Dihydroxy-ent-abieta-8(14)en-7-one, 9]: colorless, amorphous powder; [α]25D −8 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 244 (3.67) nm; ECD (c 9.69 × 10−4 M, MeOH) λmax (Δε) 245 (−2.3), 341 (+0.6) nm; IR (film) νmax 3416 (br), 2940, 2928, 2871, 1781, 1614, 1454, 1384, 1042 cm−1; 1H NMR (400 MHz, CDCl3) δ 1.84 (ddd, J = 13.0, 3.0, 3.0 Hz, H-1α), 1.12 (ddd, J = 13.0, 13.0, 3.5 Hz, H-1β), 1.62 (m, H2-2), 1.36 and 1.51 (m, H2-3), 1.90 (dd, J = 13.5, 4.9 Hz, H-5), 2.31 (dd, J = 18.8, 4.9 Hz, H6α), 2.52 (dd, J = 18.8, 13.5 Hz, H-6β), 2.03 (m, H-9), 1.46 (m, H11α), 1.75 (m, H-11β), 1.73 (m, H-12α), 1.46 (m, H-12β), 6.73 (br s, H-14), 1.86 (m, H-15), 0.87 (d, J = 7.0 Hz, Me-16), 0.96 (d, J = 7.0 Hz, Me-17), 3.31 and 3.37 (ABq, J = 10.9 Hz, H2-18). 0.85 (s, Me-19), 0.87 (s, Me-20); 13C NMR (100 MHz, CDCl3) δ 38.5 (C-1), 18.0 (C2), 35.2 (C-3), 37.9 (C-4), 42.7 (C-5), 37.2 (C-6), 200.4 (C-7), 138.7 (C-8), 51.7 (C-9), 35.7 (C-10), 18.5 (C-11), 29.7 (C-12), 71.9 (C-13), 139.8 (C-14), 37.9 (C-15), 16.3 (C-16), 17.5 (C-17), 71.0 (C-18), 17.2 (C-19), 14.8 (C-20); ESIMS m/z 343.2 [M + Na]+; HRESIMS m/z 343.2251 [M + Na]+ (calcd for C20H32O3Na, 343.2244, Δ = +2.1 ppm). Sessilifol J [(3R,5S,10R)-3,15-Dihydroxy-ent-abieta-8,11,13-triene, 10]: colorless crystals; [α]25D −32 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 219 (3.54) nm; ECD (c 9.93 × 10−4 M, MeOH) λmax (Δε) 222 (−2.5), 244 (+0.2) nm; IR (film) νmax 3356 (br), 2940, 2924, 2871, 1593, 1454, 1383, 1233, 1092, 1038 cm−1; for 1H and 13C NMR data, see Tables 1 and 4; ESIMS m/z 325.2 [M + Na]+; HRESIMS m/z 325.2132 [M + Na]+ (calcd for C20H30O2Na, 325.2144, Δ = −3.4 ppm). X-ray crystal data of 10: C20H30O2·CHCl3; colorless crystals were obtained from a solution of CHCl3. M = 421.81, monoclinic, space group C2, a = 22.6971(4) Å, b = 11.6034(2) Å, c = 19.6097(6) Å, α = 90°, β = 124.0207(10)°, γ = 90°, V = 4280.18(17) Å3, T = 140(2) K, Z = 8, μ(Cu Kα) = 3.969 mm−1, Dcalc = 1.309 mg/m3, F(000) = 1792, 15 632 reflections collected, 6424 independent reflections (Rint = 0.0377). The final R1 = 0.0417, wR(2) = 0.1124 (I > 2σ(I)), Flack parameter = 0.041(8). J

DOI: 10.1021/acs.jnatprod.5b00195 J. Nat. Prod. XXXX, XXX, XXX−XXX

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charge from the Cambrige Crystallographic Data Centre via www.ccdc. cam.ac.uk/data_request/cif. Measurement of NO Production and Cell Viability in LPSActivated BV-2 Cells. Briefly, the mouse microglia cell line BV-2 was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium containing 1800 mg/L NaHCO3, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. The antineuroinflammatory activity in BV-2 cells was evaluated according to the reported protocol.6 The NO production was quantified by nitrite accumulation in the culture medium using the Griess reaction kit (Beyotime Biotechnology, China) according to the manufacturer’s instructions. BV-2 cells were pretreated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 μM) of indicated compounds for 4 h and stimulated with or without LPS (1 μg/mL, Sigma-Aldrich) for 24 h. The isolated supernatants were mixed with an equal volume of Griess reagent. NaNO2 was used to generate a standard curve, and NO production was determined by measuring the optical density at 540 nm by a microplate reader (M200, TECAN, Austria GmbH, Austria). Cell viability was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) colorimetric assay.6 NGmonomethyl-L-arginine (L-NMMA, Beyotime, purity ≥99%), a wellknown NO synthase inhibitor, served as a positive control. The IC50 values were determined by GraphPad Prism 5.

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ASSOCIATED CONTENT

S Supporting Information *

The NMR, ECD, and HRESIMS spectra of compounds 1−17. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00195.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Tel/Fax: +86 21 51980172. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSFC grants (Nos. 21472021, 81273401, 81202420), grants from the Ph.D. Programs Foundation of Ministry of Education (MOE) of China (Nos. 20120071110049, 20120071120049), and the National Basic Research Program of China (973 Program, Grant No. 2013CB530700).

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