Anti-inflammatory Meroterpenoids from Baeckea frutescens - Journal

Article pubs.acs.org/jnp

Anti-inflammatory Meroterpenoids from Baeckea f rutescens Ji-Qin Hou,†,‡ Cui Guo,† Jian-Juan Zhao,† Yang-Yang Dong,† Xiao-Long Hu,† Qi-Wei He,† Bao-Bao Zhang,† Ming Yan,*,‡ and Hao Wang*,†,‡ †

State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, and ‡Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: Frutescones H−R (1−11), new sesqui- or monoterpene-based meroterpenoids, were isolated from the aerial parts of Baeckea f rutescens. Their structures and absolute configurations were established by means of spectroscopic analyses (HRESIMS, 1D and 2D NMR, and ECD), as well as single-crystal X-ray crystallography of 1, (−)-7, and 9. The anti-inflammatory activities of all isolates were evaluated by measuring their inhibitory effects on NO production in LPSstimulated RAW 264.7 macrophages, and the structure− activity relationships of 1−11 are also discussed. Compound 8 exhibited anti-inflammatory activity with an IC50 value of 0.36 μM, which might be related to the regulation of the NF-κB signaling pathway via the suppression of p65 nuclear translocation and the consequent decrease of IL-6 and TNF-α.

M

indicating eight indices of hydrogen deficiency. The IR spectrum displayed absorption bands for carbonyl (1665 cm−1) and olefinic (1616 cm−1) functionalities. The 1H NMR data of 1 (Table 1) revealed an olefinic proton [δH 5.37 (1H, brs)], seven methyl groups [δH 1.35, 1.28 (each 3H, s); 0.94 (3H, d, J = 6.5 Hz), 0.92 (3H, d, J = 7.0 Hz), 0.90 (3H, d, J = 7.3 Hz), 0.74 (3H, d, J = 6.5 Hz), 0.58 (3H, d, J = 7.0 Hz)], a vinylic methyl group [δH 1.87 (3H, s)], and an O-methyl group [δH 3.83 (3H, s)]. The 13C NMR data (Table 3) displayed 29 carbon resonances categorized into nine methyl, five methylene, seven methine, four quaternary, three oxygenated tertiary, and one carbonyl carbon. The partial structure of an isobutyrylphloroglucinol enone-type moiety (1a) was established by the HMBC correlations (Figure 1). The remaining 15 carbons implied a bicyclosesquiphellandrene unit (1b),9 which was confirmed by the 1H−1H COSY correlations (Figure 1) and the HMBC correlations from Me-15′ to C-1′ and C-9′, from Me-12′ and Me-13′ to C-7′, from H-5′ to C-1′, C-3′, and C-7′, and from H2-2′ to C-4′. Additionally, the HMBC correlations from H-5′ to C-4′ and C-14′ revealed the linkage between C-5′ and C-14′ through the deshielded C-4′ resonance (δC 76.1). Combined with the 1H−1H COSY correlations of H2-14′/H-10/H-11/Me-12 and Me-13 and the HMBC correlations from H2-14′ to C-1 and from H-10 to C-6, it revealed an oxa-spiro [5.5] ring system connecting the phloroglucinol (1a) and sesquiterpene (1b) moieties. The 2D structure of 1 was thus defined. The relative configuration of 1 was established by a ROESY experiment. As shown in Figure 2, the ROESY correlations of H-10 with H-3′a and H-3′a with H-14′a indicated that these

eroterpenoids, a class of hybrid natural products, are constructed from phloroglucinol-terpene adducts and have attracted attention due to their interesting structural skeletons and biological activities.1 Baeckea f rutescens L. (Myrtaceae) has long been used as a folk herbal medicine for treating inflammatory diseases, including rheumatism, snake bites, dermitis, and cold.2 Previous phytochemical studies focused on the leaves and roots of B. f rutescens, resulting in the isolation of phloroglucinols,3 sesquiterpenoids,4 chromones and chromanones,5 flavanones,6 and biflavonoids.7 As part of a continuing search for bioactive novel natural products,8 the petroleum ether extract of B. f rutescens was found to possess potent anti-inflammatory activity with an IC50 value of 4.00 μg/ mL against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. Further bioactivity-guided fractionation methods led to the isolation of 11 new sesqui- or monoterpene-based meroterpenoids, frutescones H−R (1−11), from the aerial parts of B. f rutescens. Their structures and absolute configurations were determined by a combination of 1D and 2D NMR spectroscopy, electronic circular dichroism (ECD) data, and X-ray diffraction analysis. All isolates were evaluated for their inhibitory activities on NO levels in LPS-stimulated RAW 264.7 macrophages. Notably, compounds 7 and 8 exhibited anti-inflammatory activities, with IC50 values of 1.80 and 0.36 μM, respectively. Herein, the isolation, structural elucidation, plausible biosynthesis, and biological activity of these compounds are discussed.

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RESULTS AND DISCUSSION Frutescone H (1) was obtained as colorless crystals from a MeOH solution. Its molecular formula of C29H44O3 was established by the HRESIMS protonated molecular ion [M + H]+ at m/z 441.3369 (calcd for C29H45O3, 441.3363), © 2017 American Chemical Society and American Society of Pharmacognosy

Received: January 15, 2017 Published: July 28, 2017 2204

DOI: 10.1021/acs.jnatprod.7b00042 J. Nat. Prod. 2017, 80, 2204−2214

Journal of Natural Products

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Chart 1

were cofacial and assigned as α-oriented, and H-7′ and Me-15′ were β-oriented. The absolute configuration of 2 was assigned by the ECD data, because the pattern of the Cotton effects of this class of compounds is mainly determined by the chirality at the C-10 nearest to the chromophore.10,11 Hence, the (10R) absolute configuration was defined via comparison of the ECD spectrum with that of 1 (Figure 4). The absolute configuration of 2 was thus assigned as (10R, 1′R, 4′R, 7′S, 10′R), which was confirmed by the calculated ECD spectrum using the timedependent density functional theory (TDDFT) method at the B3LYP/6-31G(d) level (Figure 4). Frutescone J (3), with a molecular formula of C29H44O3, the same as those of 1 and 2, was obtained as a light yellow oil. Its 1 H and 13C NMR data (Tables 1 and 3) were analogous to those of 1 and 2, suggesting that they shared the same 2D structure. This structural assignment was confirmed by analysis of the 2D NMR data, especially the 1H−1H COSY and HMBC correlations. The ROESY correlations of H-10 with H-3′a and H-3′a with H-14′b/H-1′ indicated that these protons were cofacial and assigned as α-oriented. The relative configuration

protons were cofacial and arbitrarily designated as β-oriented. The correlations of H-1′ with H-3′a/H-11′/Me-13′ indicated that H-1′ and the C-7′ isopropyl group were cofacial and assigned as β-oriented, and H-7′ was α-oriented. The αorientation of Me-15′ was deduced from the ROESY correlation of Me-15′ with H-7′. The (10R, 1′R, 4′S, 7′S, 10′R) absolute configuration of 1 was defined by an X-ray diffraction analysis using Cu Kα radiation with the Flack parameter [0.07(6)] (Figure 3). Frutescone I (2) possessed the same molecular formula of C29H45O3 as 1 based on the HRESIMS ion at m/z 441.3353 [M + H]+ (calcd for C29H45O3, 441.3363). The 1H and 13C NMR data (Tables 1 and 3) of 2 were similar to those of 1, except for the deshielded C-3′ (ΔδC +6.1) and the shielded C5′ (ΔδC −4.5), indicating that 2 might be the C-4′ epimer of 1. This deduction was supported by the diagnostic ROESY correlation between H-10 and H-5′. The relative configuration of the sesquiterpene unit in 2 was established by the ROESY correlations of H-1′ with H-3′b/H-11′/Me-13′ and Me-15′ with H-7′, indicating that H-1′ and the C-7′ isopropyl group 2205



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Table 1. 1H NMR Data of Compounds 1−6 in CDCl3 1a

position 7 8 9 10 11 12 13 1′ 2′a 2′b 3′a 3′b 5′ 6′a 6′b 7′a 7′b 8′a 8′b 9′a 9′b 10′ 11′ 12′ 13′ 14′a 14′b 15′a 15′b OMe a

1.87, 1.35, 1.28, 2.72 2.91, 0.92, 0.58, 2.23, 1.68, 1.45, 1.87, 1.20, 5.37,

2a

s s s

3b

1.88, s 1.31, s 1.19, s 2.82, ddd (11.5, 7.3, 4.0) 2.94, dtd (13.7, 6.9, 4.1) 0.93, d (7.0) 0.56, d (7.0) 2.35−2.26, m 1.90, mc 1.51, mc 1.77, mc 1.28, mc 5.48, d (1.6)

1.87, 1.31, 1.23, 2.69, 2.87, 0.93, 0.59, 2.33, 1.72, 1.37, 1.59, 1.32, 5.41,

1.68, mc

1.52, mc

1.68, mc

1.45, mc

1.60, mc

1.66, mc

1.87, mc 1.37, mc 1.96, mc 1.82, mc 0.94 (d, 6.6) 0.74 (d, 6.6) 1.89, mc 1.60, mc 0.90, d (7.3)

1.80, mc 1.30, mc 1.92, mc 1.79, mc 0.86 (d, 6.6) 0.64 (d, 6.6) 1.80, mc 1.53, mc 0.85, d (6.9)

1.82, 1.35, 1.91, 1.87, 0.94, 0.83, 1.68, 1.62, 0.87,

dt (11.3, 4.0) d (7.0) d (7.0) m mc mc mc mc brs

3.83, s

3.83, s 1

4b

s s s dt (3.7, 7.3) m d (7.0) d (7.0) m mc mc mc mc brs

1.92, s 1.32, s 1.31, s 2.82, t (4.5) 1.97, mc 1.18, d (6.9) 0.70, d (6.9 1.53, m 1.41, mc 1.33−1.20, mc 1.93, mc 1.73, m 2.05, m 1.89, m 1.40, mc 2.40, dd (13.3, 8.4) 2.14, mc

mc mc mc mc d (6.6) d (6.6) mc mc d (7.0)

1

s s s mc mc d (6.9) d (6.9)

1.87, 1.34, 1.24, 2.62, 2.79, 0.92, 0.62,

1.87, 1.55, 1.72, 1.05, 0.92, 1.01,

mc mc mc mc mc mc

2.00, m 1.47, mc 1.60, mc 1.10, mc 1.04, mc 0.5, d (2.85)

1.45, 0.85, 1.66, 0.54, 1.70, 1.60, 1.04, 0.97, 1.71, 1.62, 0.96,

1.67, t (8.9) 0.97, s 0.88, s 1.17, s 4.83, s 4.76, s 3.83, s

6b

1.86, 1.33, 1.26, 2.73, 2.82, 0.95, 0.59,

1.00, mc

2.50, q (9.1)

3.83, s

b

5a

mc m mc mc mc mc d (6.7) d (6.7) mc mc d (6.3)

3.83, s

s s s ddd (11.2, 7.2, 4.5) dq (12.4, 6.6) d (6.9) d (6.9)

1.05, mc 1.43, 0.86, 1.65, 0.60, 1.80, 1.58, 0.95, 0.91, 1.82, 1.67, 1.05,

mc mc mc mc mc mc d (6.6) d (6.6) mc mc d (6.6)

3.83, s

c

Measured at 300 MHz ( H). Measured at 500 MHz ( H). Overlapped signals without designating multiplicity.

Table 2. 1H NMR Data of Compounds 7−11 in CDCl3 7b

position 7 8 9 10 11 12 13 1′a 1′b 2′ 3′a 3′b 4′a 4′b 5′a 5′b 6′a 6′b 7′a 7′b 8′ 9′ 10′ OMe a

1.24, 1.21, 1.84, 2.73, 2.85, 0.94, 0.62,

s s s ddd (11.8, 7.8, 4.0) ddd (14.0, 6.8, 4.4) d (7.0) d (7.0)

1.16, dd (8.0, 3.6) 0.85, m 0.52, dd (8.0, 5.2)

1.67, m 1.80, 1.12, 1.75, 1.69, 1.41, 0.95, 0.90, 3.76,

mc m mc mc p (6.7) d (6.8) d (6.8) s

8b

9a

1.20, s 1.16, s 1.77, s 2.56−2.50, m 2.77, pd (7.0, 4.4) 0.84, d (7.0) 0.57, d (7.0)

1.85, 1.33, 1.21, 2.61, 2.89, 0.93, 0.56,

s s s ddd (11.3, 7.0, 4.4) dtd (13.9, 6.9, 4.6) d (6.9) d (6.8)

1.17, mc 0.42, dd (8.1, 5.9) 0.29, dd (5.5, 3.6)

2.20, 2.04, 1.45, 1.92,

m mc d (9.9) mc

1.78, 1.49, 1.60, 0.99, 1.72, 1.60, 1.35, 0.96, 0.91, 3.71,

mc dd (12.3, 8.0) mc mc dd (13.6, 11.0) mc p (6.9) d (6.8) d (6.8) s

2.04−1.82, mc 2.15, 1.87, 2.04, 1.60,

mc mc mc t (6.1)

1.19, s 0.94, s 3.82, s

10b 1.94, 1.37, 1.35, 2.34, 1.54, 0.77, 0.74, 2.44, 2.24, 5.18,

s s s mc mc d (6.8) d (6.8) d (17.4) dd (17.0, 4.8) m

11a 1.90, 1.40, 1.33, 2.36, 1.49, 0.79, 0.73, 2.14, 2.00, 5.29,

s s s m ddt (13.8, 6.9, 3.5) d (6.8) d (6.8) mc m s

2.32, mc 2.10, mc 2.03, m

2.43, m 2.23, mc 2.01, m

2.12, mc

2.12, mc

5.12, t (6.8)

5.12, t (6.8)

1.69, s 1.62, s 3.92, s

1.69, s 1.61, s 3.90, s

Measured at 300 MHz (1H). bMeasured at 500 MHz (1H). cOverlapped signals without designating multiplicity. 2206



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Table 3. 13C NMR Data of Compounds 1−11 in CDCl3

a

position

1a

2a

3b

4b

5a

6b

7b

8b

9a

10b

11a

1 2 3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ OMe

111.2 188.2 117.8 171.9 43.0 170.7 9.9 24.4 24.3 32.7 26.2 20.7 15.5 37.8 22.5 29.1 76.1 127.9 145.7 50.9 22.4 29.2 33.5 27.2 21.5 21.9 33.1 14.5 61.8

111.7 188.2 117.8 171.9 42.9 170.3 9.9 24.0 24.2 32.7 26.2 20.6 15.5 36.9 23.2 35.2 76.5 123.4 144.1 51.3 22.9 29.4 34.8 26.9 21.4 21.8 32.9 14.6 61.8

111.0 188.2 117.9 171.8 42.9 170.6 9.9 24.2 24.4 32.7 26.4 20.7 15.8 36.6 23.3 28.9 76.6 127.8 144.2 51.3 22.9 29.4 35.1 26.8 21.5 21.4 31.9 14.6 61.8

111.4 188.9 117.3 172.0 42.8 168.9 10.2 24.5 23.6 35.3 25.7 26.6 19.6 58.1 23.4 41.0 83.5 39.3 27.6 36.8 155.4 42.7 38.0 33.5 29.7 22.3 23.0 109.8 61.9

111.9 188.2 117.9 171.8 42.8 171.7 9.9 23.7 24.2 33.3 26.5 20.6 15.6 33.2 29.5 30.0 87.9 37.4 23.3 44.8 27.8 32.1 30.8 34.1 19.9 20.6 30.6 19.0 61.8

111.9 188.1 117.7 171.6 42.6 170.2 9.7 23.8 24.2 33.9 26.6 20.5 15.9 34.3 29.6 31.1 89.3 37.2 24.1 44.3 26.8 31.7 30.2 33.7 19.8 19.9 29.7 18.9 61.6

109.2 200.2 49.4 167.4 112.0 168.1 22.5 25.8 9.8 33.4 26.2 20.6 15.8 87.6 31.9 11.7 33.0 25.6 30.5 30.2 32.6 19.7 19.7

109.4 200.3 49.4 167.7 112.0 166.5 22.4 25.9 10.0 34.2 26.4 20.7 16.1 89.8 32.2 13.0 34.4 25.1 31.5 29.1 32.6 19.7 20.3

111.0 188.1 117.8 171.8 43.0 170.8 9.8 24.3 24.3 33.7 26.1 20.7 15.7 84.0 45.3 26.4 40.8 24.9 31.0 32.9 38.2 27.7 23.2

201.7 117.9 175.2 50.2 210.9 59.9 11.1 27.6 23.7 44.8 30.6 19.5 23.2 36.8 114.3 139.5 29.8 37.5 26.6 124.6 131.4 25.9 17.9

199.8 118.4 174.4 49.9 214.2 61.3 10.9 23.6 27.3 45.0 30.2 24.1 18.9 27.9 116.1 137.3 34.9 37.5 26.6 124.6 131.4 25.9 17.9

61.8

61.8

61.8

62.0

62.0

Measured at 75 MHz (13C). bMeasured at 125 MHz (13C).

Figure 1. 1H−1H COSY and key HMBC correlations of compounds 1, 4, 5, 7, 9, and 10.

of the sesquiterpene unit in 3 was identical to that in 2 based on the ROESY correlations of H-1′ with H-11′/Me-13′ and Me-

15′ with H-7′. The experimental ECD curves of 3 and 1 were mirror-image-like (Figure 4), indicating that their configu2207



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Figure 2. Key ROESY correlations of compounds 1, 4, 5, 7, 8, 10, and 11.

Figure 3. X-ray ORTEP drawings of 1, (−)-7, and 9.

structure as frutescone E,8 a phloroglucinol-caryophyllene derivative, on the basis of analysis of the 2D NMR data, especially the 1H−1H COSY and HMBC correlations (Figure 1). In the ROESY spectrum of 4 (Figure 2), the cross-peaks of Me-14′ with H-11/Me-13 indicated that Me-14′ and the C-10 isopropyl group were cofacial and arbitrarily assigned to be α-

rations at C-10 were opposite. Thus, the (10S) absolute configuration was assigned, and the absolute configuration of 3 was defined as (10S, 1′R, 4′R, 7′S, 10′R). Compound 4 gave the molecular formula C29H44O3, as deduced by the HRESIMS ion at [M + H]+ m/z 441.3367 (calcd for C29H45O3, 441.3363). It possessed the same 2D 2208



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Figure 4. Calculated and experimental ECD spectra of compounds 2−6, 8, 10, and 11.

oriented. The 13C NMR data of 4 [C-4′ (δC 83.5) and C-5′ (δC 39.3)] were similar to those of tomentodione A [(4′S) (δC 83.2) and (5′R) (δC 40.6)],11 but different from those of tomentodione C [(4′R) (δC 85.5) and (5′S) (δC 38.7)],11 permitting assignment of the (4′S, 5′R) absolute configuration for 4, consistent with the ROESY correlations. The (10S) absolute configuration of 4 was identical to those of frutescones D and E,8 by comparison of their similar ECD Cotton effects. Consequently, the well-matched calculated and experimental ECD spectra of 4 (Figure 4) permitted assignment of a (10S, 1′R, 4′S, 5′R, 9′S) absolute configuration for 4. The molecular formula of frutescone L (5) was assigned as C29H44O3 by its HRESIMS ion [M + H]+ at m/z 441.3373 (calcd for C29H45O3, 441.3363). Comparison of the NMR data of 5 (Tables 1 and 3) with those of 1−4 suggested 5 also possessed an isobutyrylphloroglucinol enone-type unit (5a). The presence of a β-cubebene moiety (5b)12 was deduced from the 1H−1H COSY correlations (Figure 1) and the HMBC correlations from H-10′ to C-2′ and C-6′, from H2-2′ and H-6′ to C-4′, and from H2-3′ to C-5′ (Figure 1). Additionally, the HMBC correlations from H-10 to C-6 and from H2-14′ to C-3′ and C-4′, as well as the 1H−1H COSY correlations of H2-14′/ H-10/Me-12 and Me-13, established an oxa-spiro [5.5] ring system. The relative configuration of 5 was determined by a ROESY experiment (Figure 2). The ROESY correlations of H10 with H-3′a and H-3′a with H-14′a indicated that these protons were cofacial and assigned to be α-oriented. The correlations of H-5′ with H-7′/H-14′b, H-6′ with H-11′/Me13′, H-6′/H-10′ with H-2′a, and Me-15′ with H-2′b permitted assignment of β-orientations for H-5′, H-7′, H-14′b, and Me15′ and α-orientations for H-6′, H-10′, and the C-7′ isopropyl group. The (10S) absolute configuration was defined based on the similarity of the ECD spectra of 5 and 3. Hence, the absolute configuration of 5 was defined as (10S, 1′R, 4′R, 5′R, 6′R, 7′S, 10′R), which was confirmed by the calculated ECD spectrum (Figure 4). Frutescone M (6) possessed the molecular formula C29H44O3 based on the HRESIMS ion at m/z 441.3365 [M

+ H]+ (calcd for C29H45O3, 441.3363). The NMR data of 6 (Tables 1 and 3) resembled those of 5, suggesting that they shared the same 2D structure. This deduction was confirmed by analysis of the 2D NMR data, especially the 1H−1H COSY and HMBC correlations. In the ROESY spectrum of 6 (Figure 2), the correlations of H-10 with H-3′a and H-3′a with H-14′a indicated that these protons were cofacial and designated as βoriented. The correlations of H-5′ with H-7′/H-14′b, H-6′ with H-11′/Me-13′, H-6′/H-10′ with H-2′a, and Me-15′ with H-2′b permitted assignment of α-orientations for H-5′, H-7′, H-14′b, and Me-15′ and β-orientations for H-6′, H-10′, and the C-7′ isopropyl group. Compound 6 exhibited a mirror-image-like ECD spectrum in comparison with that of 5 (Figure 4), indicating that their configurations at C-10 were opposite. Consequently, the absolute configuration of 6 was defined as (10R, 1′R, 4′S, 5′R, 6′R, 7′S, 10′R). (±)-Frutescone N (7) was obtained as colorless crystals. Its molecular formula was determined as C29H44O3 by the HRESIMS ion [M + H]+ at m/z 373.2741 (calcd for C24H37O3, 373.2737). The 1H NMR data of 7 (Table 2) showed six methyl groups [δH 1.24, 1.21 (each 3H, s); 0.95 (3H, d, J = 6.8 Hz), 0.94 (3H, d, J = 7.0 Hz), 0.90 (3H, d, J = 6.8 Hz), 0.62 (3H, d, J = 7.0 Hz)], a vinylic methyl group [δH 1.84 (3H, s)], and an O-methyl group [δH 3.76 (3H, s)]. The 13 C NMR data (Table 3) displayed 24 carbon resonances categorized into eight methyl, four methylene, four methine, four quaternary, three oxygenated tertiary, and one carbonyl carbon. The UV absorption maxima at 222 and 340 nm revealed an isobutyrylphloroglucinol enone-type moiety (7a), different from those of 1−6. This deduction was confirmed by the HMBC correlations from Me-7 and Me-8 to C-2 and C-4, MeO-4 to C-4, Me-9 to C-4 and C-6, Me-12 and Me-13 to C10, H-10 to C-6, and H-11 to C-1 (Figure 1). The presence of a sabinene unit (7b)9c was deduced from the 1H−1H COSY correlations (Figure 1) and the HMBC correlations from Me-9′ and Me-10′ to C-4′, H-8′ to C-3′ and C-5′, H-2′ to C-5′ and C6′, and H2-3′ and H2-5′ to C-1′. Additionally, the HMBC 2209



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Scheme 1. Plausible Biogenetic Pathway of Compounds 1−11

with the protonated HRESIMS ion at m/z 373.2739 (calcd for C24H37O3, 373.2737). Using the 1H−1H COSY, HSQC, and HMBC data, the 1H and 13C NMR signals of 9 were assigned as shown in Tables 2 and 3. The NMR data of 9 were similar to those of the phloroglucinol derivative BF-2,3a except for C-10 (ΔδC +1.5), C-11 (ΔδC −6.1), C-2′ (ΔδC −7.8), and C-6′ (ΔδC +4.6), indicating that 9 might be the C-10 epimer of BF2. This deduction was verified by an X-ray diffraction analysis of 9 [Flack parameter: −0.04(7)] (Figure 3), which unambiguously permitted assignment of a (10R, 1′R, 2′R, 4′R) absolute configuration for 9. The HRESIMS data of compound 10 showed a protonated molecular ion at m/z 373.2742 (calcd for C24H37O3, 373.2737), consistent with the molecular formula C24H36O3. The IR spectrum showed absorption bands for carbonyl (1712 and 1655 cm−1) and olefinic (1633 cm−1) functionalities. The NMR data of 10 (Tables 2 and 3) were analogous to those of (±)-calliviminone A,13 indicating that 10 was a phloroglucinolmyrcene adduct featuring an all-carbon spiro [5.5] ring system. The main difference involved the partial structure of the isobutyrylphloroglucinol unit (10a). Analysis of the 1H−1H COSY and HMBC correlations (Figure 1) supported the above structural elucidation. The ROESY correlations of Me-9 with H-11/Me-13, Me-8 with H-1′a, and H-10 with H-1′b indicated that Me-9 and the C-10 isopropyl group were cofacial and arbitrarily assigned to be α-oriented, and Me-8 and H-10 were β-oriented (Figure 2). Compound 10 was demonstrated to be racemic with an enantiomeric ratio of 1:1 (Figure S112, Supporting Information), and the enantiomers displayed typical

correlations from H-10 to C-1′ and C-6 and from H-11 and H26′ to C-7′, as well as the 1H−1H COSY correlations of H2-7′/ H-10/Me-12 and Me-13, established an oxa-spiro [5.4] ring system. The 2D structure of 7 was thus assigned as depicted. The relative configuration of 7 was established by a ROESY experiment (Figure 2). The ROESY cross-peaks of H-10 with H-6′a, H-6′a with H-7′a, and H-2′ with H-7′b/H-8′/Me-10′ indicated that H-10, H-6′a, and H-7′a were α-oriented and H2′, H-7′b, and the C-4′ isopropyl group were β-oriented. Chiral HPLC separation of 7 afforded two enantiomers with a ratio of approximately 1:1 (Figure S111, Supporting Information). The absolute configurations of (+)-7 and (−)-7 were assigned as (10R, 1′S, 2′S, 4′R) and (10S, 1′R, 2′R, 4′S), respectively, by an X-ray diffraction analysis of (−)-7 [Flack parameter: −0.08(5)] (Figure 3). Frutescone O (8), with a molecular formula of C24H36O3, possessed the same 2D structure as 7 by analysis of their similar NMR data (Tables 2 and 3), as well as the 1H−1H COSY and HMBC correlations (Figure 1). The ROESY correlations of H10 with H-6′b, H-6′b with H-7′b/H-3′b, and H-2′ with H-7′a/ H-8′/Me-9′ indicated that H-10, H-3′b, H-6′b, and H-7′b were β-oriented and H-2′, H-7′a, and the C-4′ isopropyl group were α-oriented. The (10R) absolute configuration was assigned via comparison of the ECD spectrum with that of (+)-7. The absolute configuration of 8 was thus assigned as (10R, 1′S, 2′R, 4′S), which was supported by the calculated ECD spectrum (Figure 4). Compound 9 was obtained as colorless crystals. Its HRESIMS data provided a molecular formula of C24H36O3 2210



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activities with IC50 values ranging from 0.36 to 6.50 μM, comparable to that of the positive control L-NMMA (IC50: 30.92 μM), while compounds 1−6 showed moderate or weak activities. The pro-inflammatory cytokines TNF-α and IL-6 are important inflammatory mediators that contribute to inflammation and various related diseases.15 Compound 8 was evaluated for its inhibitory effects against LPS-induced upregulation of TNF-α and IL-6. As shown in Figure 5, the level of TNF-α was dramatically decreased by pretreatment with compound 8 (0.2, 0.4, and 0.8 μM) in a dose-dependent manner, while only a weak inhibitory effect was observed for the level of IL-6. For exploration of the anti-inflammatory mechanism of compound 8, Western blotting was performed to determine the translocation of NF-κB p65 in the nucleus. As seen in Figure 6, compound 8 significantly inhibited nuclear translocation of NFκB p65 in a dose-dependent manner. These findings suggested that the anti-inflammatory activity of compound 8 was partly attributed to the suppression of the NF-κB p65 nuclear translocation and the consequent decrease of IL-6 and TNF-α.

antipodal ECD curves (Figure 4). Four possible configurations existed due to the C-6 and C-10 stereogenic centers. The (6S, 10S) absolute configuration of (+)-10 was defined by comparison of the experimental ECD spectrum of (+)-10 and the calculated ECD spectrum of (6S,10S)-10 (Figure 4). Thus, the structure of 10, (±)-frutescone Q, was elucidated as shown. (±)-Frutescone R (11), a constitutional isomer of 10, possessed the same molecular formula of C24H36O3 (m/z 373.2741 [M + H]+) as 10. The main differences involved the deshielded C-5 (ΔδC +3.3), C-9 (ΔδC +3.6), and C-12 (ΔδC +4.6) and the shielded C-8 (ΔδC −4.0), C-13 (ΔδC −4.3), and C-1′ (ΔδC −8.9), suggesting that their configurations at C-6 were different. The HMBC correlations from H-10 to C-2′, from H2-4′ to C-1, and from H2-5′ to C-2′ and C-4′ and the 1 H−1H COSY correlations of H-2′/H2-1′/H-10/H-11/Me-12 and Me-13, as well as comparison of the similar NMR data with those of (±)-calliviminone B,13 indicated that the phloroglucinol unit (11a) was conjugated with the myrcene moiety (11b) in different ways from that of 10. Thus, the 2D structure of 11 was assigned as depicted. The ROESY correlations of Me8 with H-11/Me-12, Me-9 with H-4′b, and H-4′a with H-11 indicated that Me-8 and the C-10 isopropyl group were cofacial and arbitrarily assigned to be β-oriented, and Me-9 was αoriented (Figure 2). Chiral HPLC analysis indicated that 11 was a racemic mixture. As shown in Figure 4, the calculated ECD spectrum for (6R,10S)-11 agreed with the experimental ECD curve of (+)-11, permitting assignment of (6R, 10S) and (6S, 10R) absolute configurations for (+)-11 and (−)-11, respectively. Biosynthetically, compounds 1−11 are presumably derived from a common precursor, tasmanone,14 by divergent heteroDiels−Alder (HDA) reactions. As illustrated in Scheme 1, reduction of the putative precursor tasmanone and subsequent dehydration would generate intermediate A1, which could undergo HDA reactions with various sesqui- or monoterpenoids, including bicyclosequiphellandrene, β-caryophyllene, βcubebene, β-pinene, (−)-sabinene, and myrcene, the constituents of B. f rutescens essential oil,4 to obtain compounds 1−11 via regio- and stereoselective [4 + 2] cycloaddition reaction. Compounds 1−11 were evaluated for their inhibitory activities on NO production in LPS-induced RAW 264.7 macrophages. As shown in Table 4, monoterpene-based phloroglucinols (7−11) exhibited significant anti-inflammatory

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Table 4. Inhibitory Effects of Compounds 1−11 on LPSActivated NO Production in RAW 264.7 Cells compound 1 2 3 4 5 6 7 8 9 10 11 a L-NMMA a

IC50 ± SD (μM) >50 18.75 >50 >50 30.54 15.17 1.80 0.36 3.70 2.07 6.50 30.92

± 3.84

± ± ± ± ± ± ± ±

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Autopol IV polarimeter at room temperature. The UV and IR spectra were recorded on a Shimadzu UV-2450 UV/ vis spectrophotometer and Nicolet IS10 FT-IR spectrometer with KBr disks, respectively. The ECD spectra were obtained on a JASCO J-810 spectropolarimeter. HRESIMS data were acquired on an Agilent 6520 Q-TOF mass spectrometer (Agilent Technologies, USA). The 1D and 2D NMR spectra were recorded in CDCl3 on Bruker AV-500 or AV300 spectrometers with tetramethylsilane as internal standard. All NMR assignments were based on the 1H−1H COSY, HSQC, and HMBC spectroscopic data. Diffraction data were collected on a Gemini Ultra CCD diffractometer using Cu Kα graphite-monochromated radiation (λ = 154 184 Å). Column chromatography was performed with silica gel (100−200 and 200−300 mesh, Qingdao Marine Chemical Co., Ltd., China), Sephadex LH-20 (Pharmacia, Sweden), MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.), and YMC ODS-AQ (S-50 μm, 12 nm, YMC Co., Ltd., Japan). Thin-layer chromatography was performed on a precoated GF254 plate (Qingdao Marine Chemical Co., Ltd., China). Spots were detected under UV light followed by heating after spraying with 1% vanillin/H2SO4 solution. Preparative HPLC was carried out on a Shimadzu LC-8A instrument equipped with an SPD-20A detector and a YMC-pack C8 column (20 × 250 mm, 5 μm). Chiral HPLC separation was conducted on a semipreparative chiral OD-RH column (10 × 250 mm, 5 μm) (Daicel Chiral Technologies Co., Ltd., China). Plant Material. The aerial parts of B. f rutescens were collected from Nanning, Guangxi Province, China, in October 2014, and authenticated by Prof. Min-Jian Qin (Department of Medicinal Plants, China Pharmaceutical University). A voucher specimen (No. BF201410) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. The aerial parts of B. f rutescens (18.0 kg) were extracted with 95% EtOH (3 × 100 L, 4 h each time) at 83 °C, to afford a dark green residue (2.0 kg) after removal of the solvent in vacuo. The crude extract was suspended in H2O (5 L) and successively partitioned with petroleum ether, CHCl3, and n-BuOH. The petroleum ether extract (500 g) was subjected to a silica gel column using petroleum ether−EtOAc (100:0 → 0:100, v/v) as eluent to afford eight fractions (Fr. A−H). Fraction B (104 g) was further separated into five subfractions (Fr. B.1−B.5) on a silica gel column (petroleum ether−EtOAc, 100:0 → 30:70, v/v). Fraction B.2 (10.5 g) was subjected to an MCI gel column with a gradient of MeOH−H2O (60:40 → 100:0, v/v) as eluent, to give six subfractions (Fr. B.2.1− B.2.6). Fraction B.2.4 (310 mg) was loaded on an ODS column with

2.30 1.45 0.29 0.16 1.25 0.84 0.96 4.12

N-Monomethyl- L-arginine was used as a positive control. 2211



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Figure 5. Effects of compound 8 on LPS-induced TNF-α (A) and IL-6 (B) expression in RAW 264.7 macrophages. RAW 264.7 cells were treated with different concentrations of compound 8 (0.2, 0.4, and 0.8 μM) for 2 h followed by LPS stimulation for another 18 h. The levels of TNF-α and IL-6 in the culture medium were determined by ELISA. Data are presented as mean ± SD of three independent experiments: **p < 0.01 and ****p < 0.0001 for the LPS group versus the control group; #p < 0.05, ###p < 0.001, and ####p < 0.0001 versus the LPS group. [(+)-10 (3.8 mg) and (−)-10 (3.9 mg)], and [(+)-11 (3.5 mg) and (−)-11 (3.7 mg)], respectively, on a semipreparative chiral column using CH3CN−H2O (85:15, 90:10, 90:10, v/v) at a flow rate of 4 mL/ min with UV detection at 254 nm. Frutescone H (1): colorless needles (MeOH); mp 327−329 °C; [α]20 D +224 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.24), 249 (4.08), 296 (3.74) nm; ECD (MeOH) λmax (Δε) 209 (−17.3), 251 (+18.0), 292 (+13.9) nm; IR (KBr) νmax 2960, 2930, 2866, 1665, 1616, 1468, 1379, 1120, 982 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 441.3369 [M + H]+ (calcd for C29H45O3, 441.3363). Frutescone I (2): light yellowish oil; [α]20 D +127 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.39), 249 (4.16), 297 (3.80) nm; ECD (MeOH) λmax (Δε) 216 (−42.0), 251 (+11.4), 292 (+14.3) nm; IR (KBr) νmax 2926, 2959, 2856, 1655, 1624, 1466, 1383, 1122, 1032 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 441.3353 [M + H]+ (calcd for C29H45O3, 441.3363). Frutescone J (3): light yellowish oil; [α]20 D −123 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.33), 249 (4.05), 295 (3.74) nm; ECD (MeOH) λmax (Δε) 203 (+17.7), 250 (−11.1), 292 (−9.0) nm; IR (KBr) νmax 2957, 2931, 2868, 1663, 1618, 1467, 1383, 1123 cm−1; 1 H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 441.3367 [M + H]+ (calcd for C29H45O3, 441.3363). Frutescone K (4): light yellowish oil; [α]20 D −7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 245 (4.00), 305 (3.50) nm; ECD (MeOH) λmax (Δε) 248 (+8.8), 289 (+2.5), 324 (−1.7) nm; IR (KBr) νmax 2958, 2928, 1664, 1624, 1458, 1384, 1121 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 441.3367 [M + H]+ (calcd for C29H45O3, 441.3363). Frutescone L (5): light yellowish crystals; mp 150−152 °C; [α]20 D −223 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (4.11), 249 (4.08), 296 (3.74) nm; ECD (MeOH) λmax (Δε) 250 (−19.1), 291 (−12.2) nm; IR (KBr) νmax 2956, 2928, 2873, 1655, 1609, 1466, 1459, 1383, 1369, 1128 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 441.3373 [M + H]+ (calcd for C29H45O3, 441.3363). Frutescone M (6): light yellowish crystals; mp 132−136 °C; [α]20 D +45 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.23), 248 (3.98), 293 (3.62) nm; ECD (MeOH) λmax (Δε) 251 (+5.9), 285 (+4.7) nm; IR (KBr) νmax 2956, 2929, 2870, 1666, 1618, 1466, 1385, 1116 cm−1; 1 H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 441.3366 [M + H]+ (calcd for C29H45O3, 441.3363). (±)-Frutescone N (7): colorless needles (MeOH); mp 98−100 °C; 20 [α]20 D +54 (c 0.1, MeOH) for (+)-7; [α]D −60 (c 0.1, MeOH) for (−)-7; UV (MeOH) λmax (log ε) 222 (4.51), 340 (3.93) nm; ECD (MeOH) λmax (Δε) 227 (+30.5), 303 (+19.2), 347 (−14.9) nm for (+)-7; 227 (−31.5), 303 (−19.6), 347 (+17.2) nm for (−)-7; IR (KBr) νmax 2962, 2870, 1662, 1636, 1570, 1459, 1384, 1128 cm−1; 1H

Figure 6. Effects of compound 8 on nuclear translocation of NF-κB p65 in LPS-stimulated RAW 264.7 macrophages. Cultured RAW 264.7 cells were incubated with different concentrations of compound 8 (0.2, 0.4, and 0.8 μM) for 2 h and then treated with 1 μg/mL LPS or left untreated for 16 h. Cell lysates were subjected to Western blotting analysis with NF-κB p65 and lamin B (internal control) antibodies. Data are presented as mean ± SD of three independent experiments: ***p < 0.001 for the LPS group compared with the control group; ##p < 0.01, ###p < 0.001, and ####p < 0.0001 compared with the LPS group. CH3CN−H2O (60:40 → 100:0, v/v) as eluent, to afford five subfractions (Fr. B.2.4.1−B.2.4.5). Fraction B.2.4.4 (183 mg) was applied to a Sephadex LH-20 column (CHCl3−MeOH, 50:50, v/v), followed by preparative HPLC using CH3CN−H2O (75:25, v/v) as the mobile phase, to afford 7 (47.9 mg), 8 (15.1 mg), and 9 (34.7 mg). Fraction B.3 (9.3 g) was chromatographed on an ODS column using a gradient of MeOH−H2O (65:35 → 100:0, v/v) to yield six subfractions (Fr. B.3.1−B.3.6). Fraction B.3.2 was purified on a Sephadex LH-20 column (CHCl 3 −MeOH, 50:50, v/v) and preparative HPLC (CH3CN−H2O, 75:25, v/v), to yield 2 (5.3 mg), 3 (6.8 mg), and 6 (17.2 mg). Compounds 10 (12.1 mg) and 11 (9.7 mg) were obtained from fraction B.3.3 by preparative HPLC (CH3CN−H2O, 75:25, v/v). Fraction B.3.6 was separated on a silica gel column eluting with petroleum ether−EtOAc (100:0 → 90:10, v/ v) and further purified by preparative HPLC (CH3CN−H2O, 80:20, v/v), to yield 1 (9.1 mg), 5 (5.0 mg), and 4 (6.3 mg). Compounds 7, 10, and 11 were resolved into [(+)-7 (5.2 mg) and (−)-7 (5.0 mg)], 2212



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and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 373.2741 [M + H]+ (calcd for C24H37O3, 373.2737). Frutescone O (8): light yellowish oil; [α]20 D −21 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 221 (4.24), 337 (3.64) nm; ECD (MeOH) λmax (Δε) 225 (+12.3), 303 (+10.7), 345 (−8.6) nm; IR (KBr) νmax 2959, 2869, 1661, 1632, 1562, 1463, 1373, 1177, 1123 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 373.2740 [M + H]+ (calcd for C24H37O3, 373.2737). Frutescone P (9): colorless needles (MeOH); mp 105−107 °C; [α]20 D +272 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (4.13), 250 (4.13), 297 (3.78) nm; ECD (MeOH) λmax (Δε) 249 (+25.7), 294 (+19.8) nm; IR (KBr) νmax 2957, 2932, 2869, 1661,1620, 1609, 1466, 1384, 1120 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 373.2739 [M + H]+ (calcd for C24H37O3, 373.2737). (±)-Frutescone Q (10): colorless oil; [α]20 D +133 (c 0.1, MeOH) for (+)-10; [α]20 D −147 (c 0.1, MeOH) for (−)-10; UV (MeOH) λmax (log ε) 203 (4.07), 256 (3.91) nm; ECD (MeOH) λmax (Δε) 210 (−3.2), 260 (+14.1), 315 (+2.5) nm for (+)-10; ECD (MeOH) λmax (Δε) 210 (+1.2), 260 (−15.4), 315 (−2.9) nm for (−)-10; IR (KBr) νmax 2961, 2929, 1712, 1655, 1633, 1467, 1375, 1324, 1201, 1118, 981 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 373.2742 [M + H]+ (calcd for C24H37O3, 373.2737). (±)-Frutescone R (11): colorless oil; [α]20 D +85 (c 0.1, MeOH) for (+)-11; [α]20 D −90 (c 0.1, MeOH) for (−)-11; UV (MeOH) λmax (log ε) 202 (4.17), 258 (3.89) nm; ECD (MeOH) λmax (Δε) 221 (+6.5), 256 (−1.8), 329 (+4.3) nm for (+)-11; 221 (−6.6), 256 (+3.6), 329 (−4.6) nm for (−)-11; IR (KBr) νmax 2961, 2928, 1712, 1657, 1634, 1467, 1374, 1322, 1202, 1119, 981 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 373.2741 [M + H]+ (calcd for C24H37O3, 373.2737). X-ray Crystallographic Data for Frutescone H (1). C29H44O3, M = 440.64, monoclinic crystal (0.220 × 0.200 × 0.170 mm), space group P21 (no. 4), a = 14.1074(2) Å, b = 5.82670(10) Å, c = 17.4282(2) Å, α = 90°, β = 112.703(2)°, γ = 90°, V = 1321.59(4) Å3, Z = 2, T = 291(2) K, μ(Cu Kα) = 0.537 mm−1, Dcalc = 1.107 g/cm3, 10 953 reflections measured (6.792° ≤ 2θ ≤ 142.604°), 4734 unique (Rint = 0.0158, Rsigma = 0.0162), which were used in all calculations. The final R1 was 0.0312 (I > 2σ(I)) and wR2 was 0.0899 (all data). The goodness-of-fit on F2 was 1.040. Flack parameter = 0.07(6) (CCDC 1519758). X-ray Crystallographic Data for (−)-Frutescone N (7). C29H36O3, M = 372.53, orthorhombic crystal (0.360 × 0.320 × 0.290 mm), space group P212121 (no. 19), a = 6.22190(10) Å, b = 12.66200(10) Å, c = 28.7249(3) Å, α = 90°, β = 90°, γ = 90°, V = 2263.00(5) Å3, Z = 4, T = 289(2) K, μ(Cu Kα) = 0.547 mm−1, Dcalc = 1.093 g/cm3, 24 103 reflections measured (7.63° ≤ 2θ ≤ 140.052°), 4256 unique (Rint = 0.0230, Rsigma = 0.0144), which were used in all calculations. The final R1 was 0.0425 (I > 2σ(I)) and wR2 was 0.1359 (all data). The goodness-of-fit on F2 was 1.077. Flack parameter = −0.08(5) (CCDC 1519763). X-ray Crystallographic Data for Frutescone P (9). C29H36O3, M = 372.53, hexagonal crystal (0.320 × 0.300 × 0.270 mm), space group P65 (no. 170), a = 10.12080(10) Å, b = 10.12080(10) Å, c = 37.1340(8) Å, α = 90°, β = 90°, γ = 120°, V = 3294.06(10) Å3, Z = 6, T = 289(2) K, μ(Cu Kα) = 0.563 mm−1, Dcalc = 1.127 g/cm3, 21 240 reflections measured (11.164° ≤ 2θ ≤ 140.166°), 3708 unique (Rint = 0.0226, Rsigma = 0.0177), which were used in all calculations. The final R1 was 0.0323 (I > 2σ(I)) and wR2 was 0.0883 (all data). The goodness-of-fit on F2 was 1.074. Flack parameter = −0.04(7) (CCDC 1519765). Crystallographic data for the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained free of charge on application to www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB21EZ, UK (fax: +44-(0)1223-336033 or e-mail: [email protected]. ac.uk). ECD Calculations of 2−6, 8, 10, and 11. The calculations of 2− 6, 8, 10, and 11 were performed using Gaussian 09.16 Briefly, the 3D structures of these compounds were first established according to the ROESY spectra. Conformational analysis was performed in the SYBYL

8.1 program by using the Monte Carlo protocol at the MMFF94s level with an energy cutoff of 10 kcal/mol to the global minima. The conformers were optimized using DFT at the B3LYP/6-31+G(d) level in the gas phase, and the most stable conformers were selected. The optimized stable conformers were used for TDDFT computation of the excited status at the same levels and considering the first 50 excitations. The overall ECD curve was weighted by Boltzmann distribution of each conformer (with a half-bandwidth of 0.3 eV) derived from their relative free energy values. The ECD spectra were produced by SpecDis 1.6 software.17 The calculated ECD spectra of 2−6, 8, 10, and 11 were subsequently compared with the experimental spectra. The ECD spectra were simulated by overlapping Gaussian functions for each transition according to

Δε =

1 × 2.297 × 10−39

1 2Πσ

A

2

∑ ΔΕiR ie−[(Ε−Εi)/(2σ)] j

where σ is the width of the band at 1/e height (fixed at 0.30 eV) and ΔEi and Ri are the excitation energies and rotatory strengths for transition i, respectively. Cell Culture. The murine RAW 264.7 macrophage cells (American Type Culture Collection, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heatinactivated fetal bovine serum (FBS) and 1% penicillin−streptomycin (100 U/mL, Gibco) at 37 °C and maintained in 5% CO2 humidified air. Measurement of Nitric Oxide Content and Cell Viability. RAW 264.7 macrophage cells in 10% FBS DMEM were cultured in 96well plates (1 × 105 cells/well) for 12 h, pretreated with the test compounds for 2 h, and incubated with LPS (1 μg/mL) for 20 h. NMonomethyl-L-arginine (L-NMMA, Sigma) was used as the positive control. NO production was determined by measuring the nitrite concentration in the culture supernatant using Griess reagent. Briefly, 100 μL of the supernatant from incubates was mixed with an equal volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid; 0.1% naphthylethylenediamine dihydrochloride in distilled water). After incubation for 10 min at room temperature, the absorbance was measured on a Safire2 fluorescence microplate reader at a test wavelength of 540 nm. All experiments were performed in three independent replicates. Cytotoxicity was determined using the Alamar Blue assay, after 20 h of incubation with the test compounds. Evaluation of Cytokine Secretion. RAW 264.7 cells (1 × 106 cells/well) were cultured in six-well plates with serum-free DMEM for 12 h and then pretreated with compound 8 (0.2, 0.4, 0.8 μM) for 2 h prior to stimulating with LPS (1 μg/mL) for 18 h. The supernatant was harvested, and the concentration of TNF-α and IL-6 in the culture medium was determined using commercial ELISA kits according to the instructions. Western Blot Analysis. RAW264.7 cells were lysed by RIPA lysis buffer (Vazyme, China), and the proteins were quantified using the BCA protein assay kit (Beyotime, China). The proteins were electrophoresed on 10% SDS−PAGE and then transferred onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were washed with Tris-buffered saline Tween-20 (TBST) buffer and blocked in 5% skimmed milk for 1 h at room temperature. The membranes were incubated with the primary antibodies at 4 °C overnight. After washing three times with TBST buffer, the membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Finally, the protein blots were detected using a ChemiDoc XRS imaging system (Bio-Rad Laboratories). βTubulin protein were used as the loading controls. Data Analysis. The data obtained are presented as the means ± SD of three independent experiments. A one-way analysis of variance (ANOVA) test was used for statistical analysis, followed by a Dunnett’s post hoc test for multiple comparisons. GraphPad Prism 5.02 (GraphPad Software Inc., San Diego, CA, USA) was used to perform the analyses. 2213



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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.7b00042. 1D and 2D NMR, HRESIMS, IR, UV, and ECD of 1−11 and computational details of 2−6, 8, 10, and 11 (PDF) Crystallographic data for 1 (CIF) Crystallographic data for (−)-7 (CIF) Crystallographic data for 9 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (M. Yan): [email protected]. *E-mail (H. Wang): [email protected]. Tel: 86-2583271328. Fax: 86-25-85301528. ORCID

Hao Wang: 0000-0003-3994-9806 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 81573309), the Open Project Program of Jiangsu Key Laboratory of Drug Screening (JKLD2015KF-01), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Research and Innovation Project for College Graduates of Jiangsu Province 2015 (No. KYLX15_0661).

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REFERENCES

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