chromones from the Resinous Wood of - ACS Publications - American

Article Cite This: J. Nat. Prod. 2018, 81, 543−553

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Anti-inflammatory Dimeric 2‑(2-Phenylethyl)chromones from the Resinous Wood of Aquilaria sinensis Hui-Xia Huo,† Zhi-Xiang Zhu,† Yue-Lin Song,† She-Po Shi,† Jing Sun,† Hui Sun,† Yun-Fang Zhao,† Jiao Zheng,† Daneel Ferreira,‡ Jordan K. Zjawiony,‡ Peng-Fei Tu,† and Jun Li*,† †

Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China ‡ Department of BioMolecular Sciences, Division of Pharmacognosy, and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677-1848, United States S Supporting Information *

ABSTRACT: Sixteen new 2-(2-phenylethyl)chromone dimers, including four pairs of enantiomers (1a/1b, 3a/3b, 6a/6b, and 8a/8b), along with eight optically pure analogues (2, 4, 5, 7, and 9−12) were isolated from the resinous wood of Aquilaria sinensis. Their structures were determined by extensive spectroscopic analysis (1D and 2D NMR, UV, IR, and HRMS) and experimental and computed ECD data. Compounds 1−10 feature an unusual 3,4-dihydro-2H-pyran ring linkage connecting two 2-(2-phenylethyl)chromone monomeric units, while compounds 11 and 12 possess an unprecedented 6,7-dihydro-5H-1,4dioxepine moiety in their structures. A putative biosynthetic pathway of the representative structures via a diepoxy derivative of a chromone with a nonoxygenated A-ring is also proposed. Compounds 1a/1b, 2, 3a/3b, 5, 7, 8a/8b, and 10−12 exhibited significant inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells with IC50 values in the range 7.0−12.0 μM.

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constituents of Chinese agarwood.8,9 In a continuing search for anti-inflammatory agents from Aquilaria plants,3−6,10,11 the EtOAc-soluble fraction from a 95% EtOH extract of the resinous wood of A. sinensis was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells (95% inhibition at 20 μg/mL). Bioassayguided separation of this active fraction led to the identification of an active subfraction, containing a variety of 2-(2phenylethyl)chromone derivatives in trace amounts as revealed by LCMS-IT-TOF analysis. Subsequent LC-MS-guided separation and purification afforded 16 new 2-(2-phenylethyl)chromone dimers, including four pairs of scalemic mixtures (1a/1b, 3a/3b, 6a/6b, and 8a/8b), and eight optically pure 2(2-phenylethyl)chromone dimers (2, 4, 5, 7, and 9−12). Herein, the isolation and structure elucidation of the new compounds as well as their inhibitory effects on NO production

atural products continue to play a highly significant role in the identification of lead compounds for the development of anti-inflammatory drugs.1 2-(2-Phenylethyl)chromones are characterized as a class of uncommon chromones possessing a phenylethyl substituent at C-2. This type of natural product has been found from a limited number of plant species such as Aquilaria (Thymelaeaceae), Bothriochloa ischaemum (Gramineae), Cucumis melo (Cucurbitaceae), Eremophila georgei (Myoporaceae), Flindersia laevicarpa (Rutaceae), and Imperata cylindrica (Gramineae).2,3 Recently, 2-(2phenylethyl)chromone derivatives have been reported to possess pronounced anti-inflammatory activities via multiple underlying mechanisms and have the potential to be developed into therapeutic drugs for inflammation-related diseases.3−6 Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae) is mainly distributed in southern China. Its resinous woods have been used as Chinese agarwood (“Chenxiang” in Chinese) in Traditional Chinese Medicine for centuries to treat inflammation-related disorders such as rheumatism, arthritis, body pain, asthma, and gout.7 2-(2-Phenylethyl)chromone derivatives and sesquiterpenoids have been reported to be the main active © 2017 American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of Susan Horwitz Received: November 1, 2017 Published: December 11, 2017 543

DOI: 10.1021/acs.jnatprod.7b00919 J. Nat. Prod. 2018, 81, 543−553

Journal of Natural Products

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Chart 1

five sp2 oxygenated tertiary (δC 167.3, 165.9, 161.7, 150.9, 148.9), 14 sp2 methine, four sp3 methine (three oxygenated at δC 73.7, 68.3, 63.1), and four sp3 methylene carbons. The aforementioned spectroscopic data suggested that 1 is a 2-(2phenylethyl)chromone dimer comprising a 5,6,7,8-tetrahydro2-(2-phenylethyl)chromone (unit A) and a 2-(2-phenylethyl)chromone (unit B).12−17 The structure of unit A was found to be similar to isoagarotetrol18 except for the replacement of the C-5 oxymethine function in isoagarotetrol by a methine in 1, while unit B was similar to 6-hydroxy-2-(2-phenylethyl)chromone19 except for the absence of H-5″ in 1. These deductions were supported by the HMBC correlations between H-3 and C-4/C-10/C-8′, H-5 and C-4/C-9/C-10, H-6 and C10, H-7 and C-5/C-9, H-8 and C-6/C-10, H-7′ and C-2′/C-6′, H-8′ and C-1′/C-7′, H-3″ and C-4″/C-10″/C-8‴, H-7″ and C5″/C-6″/C-9″, H-8″ and C-6″/C-10″, and H-7‴ and C-2″/C1‴/C-2‴/C-6‴/C-8‴ (Figure 1), as well as the 1H−1H COSY correlations of H-5/H-6, H-6/H-7, and H-7/H-8 (Figure 1). Taking the indices of hydrogen deficiency into account, the monomeric A and B units accounted for 20 indices of hydrogen deficiency and, thus, required the presence of an additional ring in 1. The HMBC correlations from H-5 to C-5″/C-6″/C-10″, H-7 to C-6″, H-7″ to C-5″/C-6″/C-8″/C-9″, and H-8″ to C6″/C-10″ (Figure 1) indicated the linkages of units A and B via a (5,5″)-carbon−carbon bond and a (7,O,6″)-ether bond to

in LPS-stimulated RAW264.7 cells are described. Putative biosynthetic pathways toward the formation of representative structures via a diepoxy derivative of a chromone with a nonoxygenated A-ring are also proposed.

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RESULTS AND DISCUSSION A scalemic mixture of aquisinenone A (1) was obtained as a white, amorphous powder, [α]25 D +70 (c 0.1, MeOH). Its molecular formula was established as C34H28O7 on the basis of the protonated molecular ion at m/z 549.1912 [M + H]+ (calcd for C34H29O7, 549.1908) in positive-ion HRESIMS and from its 13 C NMR spectroscopic data, requiring 21 indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3423 cm−1), α,β-unsaturated carbonyl (1656 cm−1), and phenyl (1603, 1496 cm−1) groups. The 1H NMR data of 1 (Table 1) exhibited resonances characteristic for two sets of monosubstituted phenyl groups [δH 7.26 (2H, m), 7.24 (4H, m), 7.20 (2H, m), and 7.19 (2H, m)], a 1,2,3,4-tetrasubstituted phenyl group [δH 7.28 (1H, overlapped), 7.08 (1H, d, J = 9.0 Hz)], four consecutive methines [δH 6.20 (1H, br s), 4.71 (1H, br s), 4.47 (1H, br s), 4.39 (1H, d, J = 8.5 Hz)], four methylenes [δH 2.97 (2H, m), 2.88 (4H, m), 2.79 (2H, m)], two hydroxy groups [δH 6.28 (1H, s), 5.27 (1H, d, J = 9.0 Hz)], and two olefinic protons [δH 6.14 (1H, s), 5.94 (1H, s)]. The 13 C NMR data of 1 (Table 1) showed 34 carbon resonances comprising two carbonyl (δC 178.1, 177.1), five sp2 quaternary, 544



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Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data for Compounds 1−6 (δ in ppm, J in Hz) 1a δH

position 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ OH-6 OH-8 OH-5″ OH-6″ OCH3-4′ OCH3-4‴ a

5.94, s 6.20, 4.47, 4.71, 4.39,

br s br s br s d (8.5)

7.20, 7.24, 7.19, 7.24, 7.20, 2.88, 2.79,

mc mc mc mc mc mc m

6.14, s

7.08, d (9.0) 7.28, mc

7.24, 7.26, 7.19, 7.26, 7.24, 2.97, 2.88, 6.28, 5.27,

mc mc mc mc mc m mc s d (9.0)

2a δC 167.3 112.7 177.1 29.5 63.1 73.7 68.3 161.7 121.1 140.0 128.2 128.3 126.2 128.3 128.2 31.9 33.9 165.9 110.2 178.1 121.0 148.9 121.4 118.1 150.9 120.6 140.2 128.3 128.4 126.2 128.4 128.3 31.9 34.3

δH

δC

5.92, s 6.19, 4.47, 4.71, 4.39,

br br br br

s s s s

7.10, mc 6.79, d (8.5) 6.83, 7.17, 2.80, 2.75,

d (8.5) mc m m

6.12, s

7.08, mc 7.28, mc

7.27, 7.27, 7.19, 7.27, 7.27, 2.97, 2.87, 6.30, 5.30,

mc mc mc mc mc m m s s

3b 167.4 112.7 177.0 29.5 63.1 73.7 68.3 161.7 121.1 131.8 129.1 113.7 157.6 113.7 129.1 31.0 34.2 165.8 110.2 178.0 121.0 148.9 121.4 118.0 150.8 120.6 140.1 128.2 128.3 126.1 128.3 128.2 31.9 34.2

4a

δH 6.12, s 4.44, 4.56, 4.97, 4.58,

br s t (3.5) br s br s

7.16, 7.24, 7.12, 7.24, 7.16, 2.96, 2.87,

mc mc mc mc mc mc m

6.08, s 7.45, d (8.0) 7.33, d (8.0)

7.22, 7.18, 7.12, 7.18, 7.22, 3.06, 3.00, 5.86, 5.95,

mc mc mc mc mc m mc sa sa

δC 171.0 113.7 180.3 36.0 63.6 78.2 70.1 163.9 122.7 141.2 129.6 129.4 127.4 129.4 129.6 33.9 36.3 171.1 110.6 180.0 116.6 126.0 130.3 141.8 147.1 124.6 141.2 129.5 129.5 127.4 129.5 129.5 34.0 36.9

δH 6.42, s 4.29, 4.40, 4.87, 4.55,

t (2.5) m br s d (7.0)

7.27, 7.27, 7.19, 7.27, 7.27, 2.99, 2.92,

mc mc mc mc mc mc mc

6.10, s 6.78, s

7.27, 7.27, 7.19, 7.27, 7.27, 2.96, 2.97, 5.87, 6.11,

mc mc mc mc mc mc mc d (3.0) d (8.0)

9.84, s 3.70, s

54.9

5a δC 170.1 111.8 180.0 29.3 61.3 77.2 68.1 164.1 121.1 140.0 128.3 128.4 126.2 128.4 128.3 32.1 34.6 167.9 108.8 176.0 100.7 150.5 117.8 140.9 139.6 123.4 139.8 128.2 128.3 126.1 128.3 128.2 31.8 34.2

δH 6.41, s 4.28, 4.40, 4.87, 4.56,

t (3.0) t (3.5) br s br s

7.14, mc 6.81, mc 6.81, 7.14, 2.89, 2.89,

mc mc mc mc

6.08, s 6.77, s

7.16, mc 6.83, mc 6.83, 7.16, 2.91, 2.91, 5.89, 6.12,

mc mc mc mc d (2.5) d (7.5)

9.86, s 3.69, s 3.70, s

6a δC 170.2 111.8 180.0 29.4 61.3 77.3 68.1 164.1 121.1 131.7 129.2 113.7 157.7 113.7 129.2 31.0 34.6 168.1 108.8 176.1 100.7 150.6 117.8 140.9 139.6 123.5 131.8 129.3 113.8 157.7 113.8 129.3 31.2 35.0

δH 6.17, s 4.26, 4.38, 4.74, 4.38,

br s m br s m

7.23, 7.27, 7.18, 7.27, 7.23, 2.92, 2.84,

mc mc mc mc mc m m

6.18, s

6.87, s

7.26, mc 7.26, mc 7.18, mc 7.26, mc 7.26, mc 2.97, mc 2.98, mc 5.80, m 5.80, m 12.62, s

δC 168.0 112.4 176.8 34.6 61.5 76.5 68.3 161.7 120.6 140.0 128.2 128.3 126.1 128.3 128.2 31.9 34.0 170.9 107.3 182.9 146.5 134.4 132.1 105.1 148.7 109.3 139.9 128.3 128.3 126.2 128.3 128.3 31.9 34.9

54.9 54.9

Recorded in DMSO-d6. bRecorded in methanol-d4. cOverlapped signals without designating multiplicity.

of enantiomers 1a and 1b (Figure S118, Supporting Information) in a 7:2 ratio and favoring the (+)-enantiomer. These compounds showed opposite electronic circular dichroism (ECD) curves and specific rotations of opposite signs. The absolute configurations of 1a and 1b were established by comparison of their experimental and calculated ECD spectra. The computed ECD spectrum (Figure 4) of (5S,6R,7S,8R)-1a agreed well with the experimental curve for 1a. Thus, the absolute configurations of (+)- and (−)-aquisinenone (1a and 1b) were defined as (5S,6R,7S,8R) and (5R,6S,7R,8S), respectively. Compound 2 was obtained as a white, amorphous powder, [α]25 D −102 (c 0.1, MeOH). Its molecular formula, C35H30O8, was deduced from the HRESIMS (m/z 579.2029 [M + H]+, calcd for C35H31O8, 579.2013) and 13C NMR data, indicating 21 indices of hydrogen deficiency. Analysis of the 1H and 13C NMR spectroscopic data (Table 1) of 2 showed a close

form a 3,4-dihydro-2H-pyran ring. Therefore, the 2D structure of 1 was established as depicted. The relative configuration of 1 was established as shown on the basis of the ROESY data (Figure 2). The NOE correlation between OH-6 and OH-8 indicated that these two hydroxy groups occupy axial positions in the cyclohexene ring that adopted a half-chair conformation. Moreover, NOEs of H-5/ OH-6, OH-6/H-7, and H-7/OH-8 suggested that H-5 and H-7 are cofacial. Such a constitution was confirmed by single-crystal X-ray crystallographic analysis (Figure 3). Based on its positive specific rotation, [α]25 D +70 (c 0.1, MeOH), compound 1 was initially assumed to be an optically pure compound; thus a single-crystal X-ray diffraction experiment was used to determine the C-5, C-6, C-7, and C-8 absolute configurations (Figure 3). However, the crystal structure data of 1 were reminiscent of a scalemic mixture. Subsequent chiral-phase HPLC of 1 on a CHIRALPAK ID column led to the isolation 545



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Figure 1. Selected HMBC (arrows point from protons to carbons) and 1H−1H COSY correlations of compounds 1, 6, 7, and 11.

Figure 2. Selected ROESY correlations of compounds 1, 6, 7, and 11.

A scalemic mixture of aquisinenone B (3) was obtained as a 13 white, amorphous powder, [α]25 D −50 (c 0.1, MeOH). Its C NMR and positive-ion HRESIMS data, exhibiting a protonated molecular ion at m/z 549.1897 [M + H]+, corresponded to a molecular formula of C 34 H 28 O 7 (calcd for C 34 H 29 O 7 , 549.1908), the same as that of scalemic aquisinenone A (1). Analysis of the 1H and 13C NMR spectroscopic data (Table 1) of 3 showed a close structural resemblance to 1. The major difference found was that the units A and B are linked via (5,7″)-carbon−carbon and (7,O,8″)-ether bonds in 3. This assignment was verified by the HMBC correlations from H-5 to C-4/C-7/C-9/C-10/C-6″/C-7″/C-8″, H-7 to C-9/C-8″, H-6″ to C-5/C-8″/C-10″, and H-5″ to C-4″/C-7″/C-9″ (Figure S25, Supporting Information). The C-5, C-6, C-7, and C-8 relative configurations of 3 were the same as assigned for 1 on the basis of ROESY data (Figure S28, Supporting Information). Although 3 showed low-amplitude Cotton effects in the ECD spectrum (Figure S131, Supporting Information) and a

structural resemblance to 1, except for the presence of a methoxy group [δH 3.70 (3H, s); δC 54.9] in 2. The deshielded C-4′ (δC 157.6; ΔδC +31.4) resonances in 2 compared to 1 indicated that the methoxy group is located at C-4′, which was confirmed by the HMBC correlations between OCH3-4′ and C-4′, H-3′ and C-1′/C-4′, and H-2′ and C-4′/C-6′/C-7′, as well as NOE correlations between OCH3-4′ and H-3′/5′ (Figures S16 and S18, Supporting Information). NOE correlations of OH-6/OH-8, H-5/OH-6, OH-6/H-7, and H7/OH-8 in the ROESY spectrum revealed that the relative configuration of 2 was the same as assigned for 1. The similar value and sign of the specific rotation of 2 and 1b, i.e., [α]25 D −102 (c 0.1, MeOH) for 2 and [α]25 D −159 (c 0.1, MeOH) for 1b, as well as their similar experimental ECD spectra (Figure S131, Supporting Information), indicated the absolute configuration of 2 to be (5R,6S,7R,8S). Accordingly, the structure of (−)-4′-methoxyaquisinenone A (2) was established as shown. 546



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565.1857), the same as that of 4. Analysis of the 1H and 13C NMR spectroscopic data (Table 1) of 6 showed a close structural resemblance to 4. The major differences were that the hydroxy group in unit B is located at C-5″ and that units A and B are linked via (5,7″)-carbon−carbon and (7,O,6″)-ether bonds in 6. This assignment was verified by the HMBC correlations between H-5 and C-4/C-7/C-9/C-10/C-6″/C-7″, H-7 and C-9/C-6″, and H-8″ and C-5/C-6″/C-9″/C-10″ (Figure 1). The relative configuration of 6 was established on the basis of the ROESY spectrum, which showed NOE correlations of OH-6/OH-8, H-5/OH-6, OH-6/H-7, and H-7/ OH-8 (Figure 2). The chiral-phase resolution of 6 (Figure S120, Supporting Information) also led to a pair of enantiomers, (+)-6a and (−)-6b (1:1.36 ratio), and their absolute configurations were established as (5R,6S,7R,8S) and (5S,6R,7S,8R), respectively, via comparison of the experimental and calculated ECD spectra (Figure 4). Compound 7 was obtained as a white, amorphous powder, [α]25 D −20 (c 0.1, MeOH). Its molecular formula was deduced as C37H34O10 from the 13C NMR and positive-ion HRESIMS data (m/z 639.2199 [M + H]+, calcd for C37H35O10, 639.2225), indicating 21 indices of hydrogen deficiency. The IR spectrum exhibited the presence of hydroxy (3306 cm−1), carbonyl (1711, 1654 cm−1), and phenyl (1614, 1513 cm−1) groups. The 1 H NMR spectrum of 7 showed resonances of a 1,2,4trisubstituted phenyl group [δH 7.52 (1H, d, J = 9.5 Hz), 7.49 (1H, d, J = 3.0 Hz), 7.35 (1H, dd, J = 9.5, 3.0 Hz)], a 1,2,3,4tetrasubstituted phenyl group [δH 6.73 (1H, d, J = 8.0 Hz), 6.68 (1H, d, J = 8.0 Hz)], a 1,4-disubstituted phenyl group [δH 7.06 (2H, d, J = 8.5 Hz), 6.75 (2H, d, J = 8.5 Hz)], four consecutive methines [δH 4.73 (1H, m), 4.49 (1H, br s), 4.46 (1H, d, J = 2.0 Hz), 4.32 (1H, dd, J = 4.5, 3.0 Hz)], four methylenes [δH 4.02, 3.07 (each 1H, m); 2.90 (4H, m); 2.82 (2H, m)], two olefinic protons [δH 6.17 (1H, s), 6.02 (1H, s)], and three methoxy groups [δH 3.88, 3.75, 3.72, (each 3H, s)]. The 13C NMR data (Table 2), in conjunction with the HSQC experiment, showed 37 carbon resonances comprising two carbonyl (δC 180.9, 180.4), five sp2 quaternary, eight sp2 oxygenated tertiary (δC 171.8, 170.6, 164.3, 159.7, 158.6, 152.9, 148.0, 142.8), 11 sp2 methine, four sp3 methine (three oxygenated at δC 75.8, 70.3, 65.5), four sp3 methylene, and three O-methyl carbons. The aforementioned data revealed that 7 is also a 2-(2-phenylethyl)chromone dimer comprising a 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone (unit A) and a 2-(2-phenylethyl)chromone (unit B). The structure of unit A was similar to that of aquilarone C4 except for the replacement of the C-5 oxymethine function in aquilarone C by a methine in 7, while unit B was similar to 6-methoxy-2-[2-(3-hydroxy-4methoxyphenyl)ethyl]chromone20 except for the absence of H2‴ in 7. These deductions were supported by the 1H−1H COSY correlations of H-5/H-6, H-6/H-7, and H-7/H-8, as well as the HMBC correlations from H-3 to C-2/C-4/C-10/C8′, H-5 to C-4/C-9/C-10, H-7 to C-5/C-9, H-8 to C-6/C-10, H-2′ to C-4′/C-7′, H-3′ to C-1′/C-4′, OCH3-4′ to C-4′, H-7′ to C-2/C-1′/C-2′/C-6′/C-8′, H-3″ to C-2″/C-4″/C-10″/C8‴, H-5″ to C-4″/C-6″, OCH3-6″ to C-6″, H-7″ to C-5″/C-9″, H-8″ to C-6″/C-10″, OCH3-4‴ to C-4‴, H-5‴ to C-1‴/C-3‴, H-6‴ to C-2‴/C-4‴/C-7‴, and H-7‴ to C-2″/C-2‴/C-6‴/C8‴ (Figure 1). The presence of (5,2‴)-carbon−carbon and (7,O,3‴)-ether linkages connecting units A and B was supported by the HMBC correlation from H-5 to C-1‴/C2‴/C-3‴ and H-7 to C-3‴. The relative configuration of 7 was established as shown by the ROESY spectrum recorded in

Figure 3. X-ray ORTEP drawing of compound 1.

significant specific rotation, its scalemic nature was demonstrated by chiral-phase HPLC that afforded a pair of enantiomers 3a and 3b (1:1.66 ratio) (Figure S119, Supporting Information), which had mirror-like ECD spectra (Figure S131, Supporting Information). Specific rotation values of opposite signs also supported their enantiomeric relationship. The wellmatched calculated and experimental ECD spectra of 3a (Figure S131, Supporting Information) indicated that the absolute configurations of (+)- and (−)-aquisinenones (3a and 3b) were (5R,6S,7R,8S) and (5S,6R,7S,8R), respectively. Compounds 4 and 5 were assigned molecular formulas of C34H28O8 and C36H32O10, respectively, from their 13C NMR and HRESIMS data. The 1H and 13C NMR data (Table 1) exhibited characteristic resonances of a dimeric 2-(2phenylethyl)chromone skeleton similar to 3. The main differences found were that a C-6″ hydroxy group is present in both 4 and 5, and methoxy groups are located at C-4′ and C4‴ in 5. These assignments were corroborated by their HMBC and 1H−1H COSY spectra (Figures S35, S36, S44, and S45, respectively, Supporting Information). The relative configurations of 4 and 5 were the same as assigned for 3 on the basis of their ROESY data (Figures S37 and S46, Supporting Information). Furthermore, a comparison of the experimental and calculated ECD spectra (Figure S131, Supporting Information) facilitated assignment of the (5S,6R,7S,8R) absolute configuration of 4. The opposite signs of the specific rotations of 4 and 5, i.e., [α]25 D −20 (c 0.1, MeOH) for 4 and [α]25 D +30 (c 0.1, MeOH) for 5, as well as the mirror-like Cotton effects in their experimental ECD spectra (Figure S131, Supporting Information) indicated the absolute configuration of 4 and 5 as (5S,6R,7S,8R) and (5R,6S,7R,8S), respectively. Therefore, the structures of (−)-6″-hydroxyaquisinenone B (4) and (+)-6″-hydroxy-4′,4‴-dimethoxyaquisinenone B (5) were defined as shown. A scalemic mixture of aquisinenone C (6) was obtained as a 13 white, amorphous powder, [α]25 D −20 (c 0.1, MeOH). Its C NMR and positive-ion HRESIMS data, exhibiting a protonated molecular ion at m/z 565.1837 [M + H]+, corresponded to a molecular formula of C 34 H 28 O 8 (calcd for C34 H 29 O 8 , 547



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Figure 4. Experimental and calculated ECD spectra of compounds 1, 6, 7, and 11 (in MeOH).

S72, Supporting Information). The relative configuration of 8 was the same as that of 7 on the basis of ROESY data (Figure S75, Supporting Information). Compound 8 was also found to be a scalemic mixture by chiral-phase HPLC analysis (Figure S121, Supporting Information). Subsequently, the enantiomers 8a and 8b were obtained in a 1.91:1 ratio and showed opposite ECD Cotton effects and specific rotations of opposite signs (Figure S132, Supporting Information). Compound 8b had the same absolute configuration as (−)-aquisinenone D (7) based on their similar 2D structure, relative configuration, specific rotations, and ECD spectra (Figure S132, Supporting Information). Thus, the (5R,6S,7R,8S) and (5S,6R,7S,8R) absolute configurations of 8a and 8b were defined unambiguously. (+)-Aquisinenone E (9) was obtained as a colorless gum, 13 [α]25 C NMR and positive-ion D +10 (c 0.1, MeOH). Its HRESIMS data, exhibiting a protonated ion at m/z 609.2091,

DMSO-d6, which showed NOE correlations of OH-6/OH-8, OH-6/H-5, OH-6/H-7, and OH-8/H-7 (Figure 2). The (5S,6R,7S,8R) absolute configuration of 7 was also deduced by comparison of the experimental and calculated ECD spectra (Figure 4). Therefore, the structure of (−)-aquisinenone D (7) was established as depicted. A scalemic mixture of 4′-demethoxyaquisinenone D (8) gave a molecular formula of C36H32O9 on the basis of the 13C NMR data and a protonated ion at m/z 609.2091 [M + H]+ (calcd for C36H33O9, 609.2119) in the positive-ion HRESIMS, which was 30 mass units less than that of 7, suggesting it to be a de-Omethyl derivative of (−)-aquisinenone D (7). The 1H and 13C NMR spectroscopic data (Table 2) of 8 were similar to those of 7, with the difference being the replacement of the 1,4disubstituted phenyl group in 7 by a monosubstituted phenyl group in 8, as well as the absence of the resonances for OCH34′ in 8. This was confirmed by the HMBC spectrum (Figure 548



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Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Data for Compounds 7−12 (δ in ppm, J in Hz) 7a position 2 3 4 5 6 7 8 9 10 1′ 2′ 3′

6.02, s 4.49, br s 4.32, dd (4.5, 3.0) 4.73, m 4.46, d (2.0)

δC 170.6 113.7 180.9 33.4 65.5

7.06, d (8.5)

75.8 70.3 164.3 122.8 133.1 130.4

6.75, d (8.5)

δH

9a δC 170.5 113.7 180.9 33.4 65.5

6.05, s 4.49, m 4.32, m 4.73, m 4.46, d (1.5)

7.19, md

75.8 70.3 164.3 122.8 141.2 129.4

114.9

7.19, md

129.5

d

10a

δH

δC 170.9 113.8 180.5 35.9 64.5

6.02, s 4.20, br s 4.42, t (3.5) 4.68, br s 4.39, br s

7.06, d (8.0)

77.5 70.2 163.4 123.5 133.2 130.4

6.75, d (8.0)

δH 6.09, s 4.24, br s 4.42, t (3.5) 4.69, m 4.39, br s

11b δC 170.8 113.7 180.6 35.7 64.5

δH

12c δC

6.02, s 5.71, s 4.82, br s 4.90, br s 4.52, s

169.7 113.9 178.0 71.0 68.9

7.16, md

77.5 70.2 163.4 123.6 141.2 129.4

6.99, md

75.9 66.9 164.0 116.2 139.2 128.3

114.9

7.16, md

129.5

7.17, md

128.7

d

d

4′ 5′

6.75, d (8.5)

159.7 114.9

7.14, m 7.19, md

127.4 129.5

6.75, d (8.0)

159.7 114.9

7.13, m 7.16, md

127.4 129.5

7.17, m 7.17, md

126.8 128.7

6′

7.06, d (8.5)

130.4

7.19, md

129.4

7.06, d (7.5)

130.4

7.16, md

129.4

6.99, md

128.3

7′ 8′ 2″ 3″ 4″ 5″ 6″ 7″

2.90, md 2.82, m

33.1 36.5 171.8 110.0 180.4 105.6 158.6 124.9

2.97, md 2.85, md

33.9 36.2 171.8 110.0 180.4 105.6 158.6 124.9

2.89, m 2.82, m

33.2 36.7 171.3 110.1 180.3 105.7 158.6 124.8

2.97, md 2.84, md

33.9 36.4 171.3 109.9 180.3 105.6 158.6 124.9

2.86, md 2.75, m

32.7 35.4 169.2 109.5 177.8 118.2 145.0 152.8

6.93, s

7.18, md 6.66, md 2.94, md

120.7 152.8 124.9 142.3 116.8 152.7 123.1 129.9 121.7 33.6

7.17, 7.26, 7.17, 7.26, 7.17, 2.99,

2.94, md

36.7

2.86, md

3.88, s

56.3

8″ 9″ 10″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ OCH3-4′ OCH3-6″ OCH3-7″ OCH3-4‴ a

δH

8a

6.17, s 7.49, d (3.0) 7.35, dd (9.5, 3.0) 7.52, d (9.5)

6.73, d (8.0) 6.68, d (8.0) 4.02, m; 3.07, m 2.90, md 3.72, s 3.88, s 3.75, s

120.8 152.9 124.9 131.8 123.9 142.8 148.0 112.4 122.6 29.9 37.5 55.6 56.3 56.4

6.17, s 7.49, d (2.5) 7.35, dd (9.0, 2.5) 7.52, d (9.0)

6.74, d (8.0) 6.68, d (8.5) 4.02, m; 3.07, m 2.91, md

3.88, s 3.75, s

120.8 152.9 124.9 131.8 123.9 142.8 148.0 112.3 122.6 29.9 37.5

6.07, s 7.47, d (3.0) 7.35, dd (9.0, 3.0) 7.49, d (9.0)

6.66, d (8.5) 6.95, d (8.5) 2.94, md

120.7 152.9 124.8 133.2 129.6 125.0 151.2 117.0 129.7 33.0

2.94, md 3.73, s 3.87, s

37.0 55.6 56.3

7.13, br s

6.14, s 7.45, d (3.0) 7.31, dd (9.0, 3.0) 7.43, d (9.0)

6.65, md

6.08, s 7.76, s

md md md md md m

111.3 153.8 120.2 139.6 128.4 128.8 126.8 128.8 128.4 33.0 36.1

δH 6.02, s 5.40, s 4.44, br s 4.68, br s 4.38, s

6.87, d (8.5) 6.66, d (8.5) 6.66, d (8.5) 6.87, d (8.5) 2.73, m 2.67, m 6.14, s 7.55, s

6.83, s

7.22, 7.25, 7.17, 7.25, 7.22, 2.97,

md md md md md m

2.92, m 3.68, s

δC 168.4 113.1 176.1 70.9 68.0 78.2 64.7 163.8 115.6 131.5 129.1 113.6 157.6 113.6 129.1 30.9 34.7 168.8 109.0 175.8 117.6 144.9 152.2 111.0 152.6 119.5 140.0 128.3 128.3 126.2 128.3 128.3 31.8 34.5 54.9

56.3 56.5

Recorded in methanol-d4. bRecorded in CDCl3. cRecorded in DMSO-d6. dOverlapped signals without designating multiplicity.

those of 7 on the basis of the ROESY data (Figure S84, Supporting Information). The (5S,6R,7S,8R) absolute configuration of 9 was defined by comparison of the experimental and calculated ECD spectra (Figure S132, Supporting Information). Compound 10 was obtained as a white, amorphous powder, 1 13 [α]25 C NMR D −50 (c 0.1, MeOH). Analysis of the H and spectroscopic data (Table 2) indicated that compound 10 is also a 2-(2-phenylethyl)chromone dimer and possesses similar tetrahydro-2-(2-phenylethyl)chromone and 2-(2-phenylethyl)chromone units as 9. The main differences were that the monomeric units were connected via (5,4‴)-carbon−carbon and (7,O,3‴)-ether bonds, and the proton and carbon resonances for OCH3-4′ were absent in 10. These assignments were supported by the 1H−1H COSY correlations of H-5/H-6,

corresponded to a molecular formula of C36H32O9 (calcd for C36H33O9, 609.2119), which was also 30 mass units less than that of (−)-aquisinenone D (7). The 1H and 13C NMR data (Table 2) of 9 were comparable to those of 7, with the difference being the replacement of the 1,2,3,4-tetrasubstituted phenyl group in 7 by a 1,2,4-trisubstituted phenyl group in 9, as well as the absence of the resonance for OCH3-4‴ in 9. The two monomeric units A and B of 9 were connected via (5,3‴)carbon−carbon and (7,O,4‴)-ether bonds. These assignments were verified by the HMBC correlations from H-2‴ to C-4‴/ C-6‴/C-7‴, H-5‴ to C-1‴/C-3‴, H-6‴ to C-4‴, H-7‴ to C2″/C-1‴/C-2‴/C-8‴, H-5 to C-2‴/C-3‴/C-4‴, and H-7 to C4‴ (Figure S82, Supporting Information). The relative configurations at C-5, -6, -7, and -8 of 9 were the same as 549



Article

11 on the basis of ROESY data (Figure S112, Supporting Information). The (5R,6S,7R,8S) absolute configuration of 12 was defined based on the opposite sign of their specific rotations and the mirror-like ECD spectra as compared with those of 11 (Figure S133, Supporting Information). Accordingly, the structure of (+)-4′-methoxyaquisinenone G (12) was established as depicted. Compounds 1−10 are novel 2-(2-phenylethyl)chromone dimers featuring an unusual 3,4-dihydro-2H-pyran ring connecting two 2-(2-phenylethyl)chromone monomeric moieties, while compounds 11 and 12 feature an unusual 6,7dihydro-5H-1,4-dioxepine structural moiety. Putative biosynthetic pathways toward compounds 1a and 11 are proposed as shown in Scheme 1. The biosynthetic precursors of 1a are

H-6/H-7, and H-7/H-8, as well as the HMBC correlations from H-5 to C-4/C-7/C-9/C-10/C-3‴/C-4‴/C-5‴, H-7 to C-9/C3‴, H-2‴ to C-3‴/C-6‴/C-7‴, and H-5‴ to C-5/C-1‴/C-3‴ (Figure S91, Supporting Information). The relative configurations at C-5, -6, -7, and -8 of 10 were the same as those of 9 on the basis of the ROESY data (Figure S93, Supporting Information). A comparison of the experimental and calculated ECD spectra (Figure S132, Supporting Information) facilitated assignment of the (5S,6R,7S,8R) absolute configuration of 10. Thus, the structure of (−)-aquisinenone F (10) was established as shown. (−)-Aquisinenone G (11) was obtained as a white, amorphous powder, [α]25 D −80 (c 0.1, MeOH). Its molecular formula, C34H28O8, was deduced from the HRESIMS (m/z 565.1831 [M + H]+, calcd for C34H29O8, 565.1857) and 13C NMR data, indicating 21 indices of hydrogen deficiency. The IR spectrum showed the presence of hydroxy (3376 cm−1) and α,β-unsaturated carbonyl (1662 cm−1) groups. The 1H NMR data of 11 (Table 2) suggested the presence of two olefinic protons [δH 6.08 (1H, s), 6.02 (1H, s)], two sets of monosubstituted phenyl groups [δH 7.26 (2H, m), 7.17 (6H, m), and 6.99 (2H, m)], a 1,2,4,5-tetrasubstituted phenyl group [δH 7.76 (1H, s), 6.93 (1H, s)], four consecutive methines [δH 5.71 (1H, s), 4.90 (1H, br s), 4.82 (1H, br s), 4.52 (1H, s)], and four methylenes [δH 2.99 (2H, m), 2.86 (4H, m), and 2.75 (2H, m)]. The 13C NMR spectrum showed 34 carbon resonances including two carbonyl (δC 178.0, 177.8), four sp2 quaternary, six sp2 oxygenated tertiary (δC 169.7, 169.2, 164.0, 153.8, 152.8, 145.0), 14 sp2 methine, four oxygenated sp3 tertiary (δC 75.9, 71.0, 68.9, 66.9), and four sp3 methylene carbons. The aforementioned data indicated 11 to be a dimeric 2-(2-phenylethyl)chromone derivative comprising 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone (unit A) and 2-(2phenylethyl)chromone (unit B) constituent units.16 Analysis of the 1D and 2D NMR data revealed that the 2D structure of unit A is the same as isoagarotetrol,18 and unit B was the same as 6,7-dihydroxy-2-(2-phenylethyl)chromone21 (Figure 1). HMBC correlations from H-5 to C-7″ and from H-7 to C-6″ indicated that two monomeric units of 11 were linked via (5,O,7″)- and (7,O,6″)-ether bonds to furnish an unusual 6,7dihydro-5H-1,4-dioxepine moiety. Interestingly, a NOE correlation between H-8 (δH 4.52) and H-5″ (δH 7.76) was observed clearly, suggesting a stacked arrangement of the 2-(2phenylethyl)chromone monomeric moieties, as shown in Figure 2. The relative configuration of 11 was established via the NOE correlations of OH-6/OH-8, OH-6/H-5, OH-6/H-7, and OH-8/H-7 (Figure 2). Furthermore, the (5S,6R,7S,8R) absolute configuration was established via comparison of the experimental and calculated ECD spectra (Figure 4). The molecular formula of compound 12 was determined as C35H30O9 by its 13C NMR (Table 2) and HRESIMS data (m/z 595.1941 [M + H]+), which is 30 mass units more than that of 11, suggesting it is an O-methyl derivative of 11. A comparison of the 1D and 2D NMR spectroscopic data (Table 2) of 12 showed a close structural resemblance to 11. The major difference involved the presence of a 4′-O-methyl group in 12. This assignment was supported by the significantly deshielded C-4′ resonance (δC 157.6; ΔδC +30.8) and shielded resonances of C-3′, 5′ (δC 113.6; ΔδC −15.1). This was further verified by the HMBC correlation from OCH3-4′ to C-4′ (Figure S110, Supporting Information) and by the NOE correlations of OCH3-4′ and H-3′/5′ (Figure S112, Supporting Information). The orientations of H-5, -6, -7, and -8 were the same as those in

Scheme 1. Putative Biosynthetic Pathways toward the Formation of 1a and 11

proposed to be the co-occurring oxidoagarochromone A (A1) and 6-hydroxy-2-(2-phenylethyl)chromone (B1).19,22 First, diepoxide A1 would be transformed into a cationic intermediate via protonation, followed by an aromatic electrophilic substitution reaction with B1 to produce intermediate i, which would be susceptible to an intramolecular nucleophilic substitution reaction to afford 1a. Compound 11 is considered to be derived from oxidoagarochromone A (A1) and 6,7dihydroxy-2-(2-phenylethyl)chromone (B2).21,22 Substitution via the phenolic groups of B2 at the epoxy ring carbon atoms generates 11, in a similar fashion to the reaction between benzo[a]pyrene-7,8-diol-9,10-epoxide and ellagic acid.23 Compounds 1a/1b, 2, 3a/3b, 5, 7, 8a/8b, and 10−12 were also evaluated for their inhibitory effects on NO production in LPS-stimulated RAW264.7 cells. All these test compounds showed inhibition against NO production with IC50 values in the range 7.0−12.0 μM (Table 3). GYF-17 was used as a positive control (IC50 4.4 μM).5 In order to investigate whether the inhibitory activities of these compounds are due to the decrease of cell numbers (cytotoxicity), their effects on cell proliferation/viability were measured using the MTT method. None of the compounds (up to 80 μM) showed cytotoxicity with LPS treatment for 24 h. The bioassay data of 4, 6a/6b, and 9 could not be assessed due to quantity limitations. 550



Article

MS-guided separation and purification targeted the subfractions containing compounds with m/z > 500. Fraction H (1.16 g) was fractionated by silica gel column chromatography (CC) eluting with a stepwise gradient of CH2Cl2−MeOH (20:1 → 1:1) to yield four subfractions (H1−H4). Subfraction H1 (0.45 g) containing the targeted compounds was subjected to RP-C18 CC eluting with a stepwise gradient of aqueous MeOH (40% to 100% MeOH) to afford five fractions (H1a−H1e). H1a (37 mg) was purified by semipreparative RP-HPLC eluted with isocratic 42% aqueous CH3CN to produce 4 (1.1 mg, tR 69.0 min) and 6 (2.5 mg, tR 105.9 min). Subsequent resolution of 6 by chiral-phase HPLC using a CHIRALPAK ID column eluting with EtOH−n-hexane (50:50) afforded 6a (0.6 mg, tR 10.5 min) and 6b (0.8 mg, tR 19.1 min). H1c (140 mg) was purified by semipreparative RP-HPLC with isocratic 42% aqueous CH3CN to afford 5 (2.0 mg, tR 58.1 min) and three subfractions (H1c1−H1c3). Compounds 11 (4.0 mg, tR 63.2 min) and 12 (4.0 mg, tR 67.5 min) were purified from H1c2 (20 mg) by semipreparative RP-HPLC with isocratic 38% aqueous CH3CN as the mobile phase. Separation of H1d (40.3 mg) on semipreparative RP-HPLC (isocratic 78% aqueous MeOH) afforded 1 (10.0 mg, tR 25.4 min). The enantiomers 1a (4.5 mg, tR 19.1 min) and 1b (4.0 mg, tR 20.9 min) were resolved by chiral-phase HPLC using a CHIRALPAK ID column eluting with EtOH−n-hexane (28:72). Fraction H1e (120 mg) was repeatedly chromatographed on silica gel and RP-C18 CC and purified by semipreparative RP-HPLC (isocratic 70% aqueous MeOH) to afford 2 (4.5 mg, tR 73.5 min). Subfraction H4 (300 mg) was separated into three fractions (H4a−H4c) by semipreparative RP-HPLC eluted with isocratic 70% aqueous CH3CN. Compounds 3 (3.8 mg, tR 76.5 min), 7 (2.0 mg, tR 88.6 min), and 10 (2.4 mg, tR 92.7 min) were obtained from H4b (38 mg) by semipreparative RP-HPLC eluting with isocratic 63% aqueous MeOH. Compound 3 was further separated using a chiral-phase HPLC (CHIRALPAK IC column) eluting with EtOH−n-hexane (50:50) to give 3a (1.2 mg, tR 9.1 min) and 3b (1.8 mg, tR 11.1 min). Fraction H4c (12 mg) was purified by semipreparative RP-HPLC (isocratic 67% aqueous MeOH) to afford 8 (4.5 mg, tR 60.2 min) and 9 (1.3 mg, tR 68.5 min). Subsequently, chiral-phase resolution of 8 on a CHIRALPAK IA column (EtOH−n-hexane, 40:60) afforded 8b (1.6 mg, tR 15.7 min) and 8a (2.1 mg, tR 18.7 min). Aquisinenone A Scalemic Mixture (1): white, amorphous powder; [α]25 D +70 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 331 (3.62), 235 (4.38), 207 (4.44) nm; ECD (MeOH) λmax (Δε) 220 (−25.88), 246 (+41.36), 340 (−10.96) nm; IR (KBr) νmax 3423, 1656, 1603, 1583, 1496, 1462, 1427, 1377, 1197, 1175, 1041, 1004 cm−1; 1H and 13C NMR data see Table 1; positive-ion HRESIMS m/z 549.1912 [M + H]+ (calcd for C34H29O7, 549.1908). (+)-Aquisinenone A (1a): [α]25 D +169 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 222 (−48.80), 244 (+70.97), 338 (−17.66) nm. (−)-Aquisinenone A (1b): [α]25 D −159 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 222 (+49.25), 244 (−75.29), 338 (+19.22) nm. (−)-4′-Methoxyaquisinenone A (2): white, amorphous powder; [α]25 D −102 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 329 (3.74), 230 (4.53), 206 (4.52) nm; ECD (MeOH) λmax (Δε) 220 (+27.51), 248 (−50.03), 336 (+12.40) nm; IR (KBr) νmax 3422, 1654, 1583, 1512, 1462, 1377, 1244, 1177, 1037, 821, 700 cm−1; 1H and 13C NMR data see Table 1; positive-ion HRESIMS m/z 579.2029 [M + H]+ (calcd for C35H31O8, 579.2013). Aquisinenone B Scalemic Mixture (3): white, amorphous powder; [α]25 D −50 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 306 (3.56), 237 (4.40), 206 (4.45) nm; ECD (MeOH) λmax (Δε) 204 (+16.07), 240 (−21.36), 308 (−2.83) nm; IR (KBr) νmax 3305, 1711, 1655, 1570, 1494, 1451, 1388, 1362, 1218, 1052, 859, 700 cm−1; 1H and 13C NMR data see Table 1; positive-ion HRESIMS m/z 549.1897 [M + H]+ (calcd for C34H29O7, 549.1908). (+)-Aquisinenone B (3a): [α]25 D +124 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 218 (−50.18), 238 (+108.58), 266 (−13.69), 310 (+13.30) nm. (−)-Aquisinenone B (3b): [α]25 D −120 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 208 (+50.03), 238 (−99.60), 266 (+10.75), 308 (−10.81) nm.

Table 3. Inhibitory Activity against LPS-Induced NO Production in RAW264.7 Cells compound

IC50 (μM)a

compound

IC50 (μM)a

1a 1b 2 3a 3b 5 GYF-17b

11.5 ± 0.6 7.6 ± 0.1 9.3 ± 0.3 8.8 ± 0.2 8.6 ± 0.2 10.5 ± 0.2 4.4 ± 0.4

7 8a 8b 10 11 12

7.0 ± 0.1 8.5 ± 0.1 8.5 ± 0.4 12.0 ± 0.5 11.4 ± 0.3 8.0 ± 0.4

Values are presented as means ± SD based on three independent experiments. bPositive control.

a

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Autopol IV automatic polarimeter (NJ, USA). UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). ECD spectra were recorded on a J-810 CD spectrophotometer (JASCO, Japan). IR spectra were obtained using a Thermo Nicolet Nexus 470 FT-IR spectrophotometer (MA, USA) with KBr pellets. The NMR spectra were measured with a Varian INOVA-500 spectrometer (CA, USA) operating at 500 MHz for 1H NMR and 125 MHz for 13C NMR, respectively. HRESIMS were acquired using an LCMS-IT-TOF system fitted with a Prominence UFLC system and an ESI interface (Shimadzu, Kyoto, Japan). X-ray crystallographic data were collected using an Agilent Gemini E X-ray single-crystal diffractometer with Cu Kα radiation (Agilent Technologies, Yarnton, Oxfordshire, UK). Column chromatography was performed using silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), Sephadex LH-20 (Pharmacia), and LiChroprep RP-C18 gel (40−63 μm, Merck, Germany). Semipreparative HPLC was performed on a Shimadzu LC-20AT pump system (Shimadzu Corporation, Tokyo, Japan), equipped with an SPD-M20A photodiode array detector monitoring at 254 nm, and a Waters 2535 pump system (Waters Corporation, Milford, MA, USA), equipped with a 2998 photodiode array detector monitoring at 254 nm. A semipreparative reversed-phase (RP) C18 column (YMC-Pack ODS-A, 250 × 10 mm, 5 μm), chiral CHIRALPAK IA column (4.6 mm × 250 mm, 5 μm), CHIRALPAK IC column (4.6 mm × 250 mm, 5 μm), and CHIRALPAK ID column (4.6 mm × 250 mm, 5 μm) were employed for the isolation. The LC-MS-guided separation and purification procedure was performed on an Agilent ZORBAX SB-C18 column (250 × 4.6 mm, 5 μm) eluting with isocratic 0.1% aqueous formic acid−CH3CN (62:38) monitored by IT-TOFMS. TLC was carried out using precoated silica gel GF254 plates and visualized under a UV lamp at 254 nm. All purified compounds submitted for bioassay were at least 95% pure as judged by HPLC and supported by 1H NMR analysis. Plant Material. The resinous wood of Aquilaria sinensis was collected in Zhongshan, Guangdong Province, People’s Republic of China, in November 2012, and was authenticated by one of the authors (P.-F.T.). A voucher specimen (CX2012029) was deposited at the Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine. Extraction and Isolation. The dried Chinese agarwood sample (6.9 kg) was extracted with 95% EtOH (3 × 120 L, each 2.5 h) under reflux. After removing the solvent under reduced pressure, the extract was suspended in 80% aqueous MeOH (1 L) and successively partitioned with petroleum ether and EtOAc, to afford petroleum ether (36.4 g)- and EtOAc (682.0 g)-soluble extracts, respectively. The EtOAc extract (602.0 g) was subjected to vacuum-liquid chromatography on silica gel (200−300 mesh) eluted with a gradient of petroleum ether−EtOAc (8:1 → 0:1) and MeOH to provide eight fractions (A−H). Fraction H (1.16 g) inhibited NO production in LPS-stimulated RAW264.7 cells by 90% at 10 μg/mL. Subsequent LC551



Article

(−)-6″-Hydroxyaquisinenone B (4): white, amorphous powder; [α]25 D −20 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 333 (3.52), 245 (4.28), 206 (4.44) nm; ECD (MeOH) λmax (Δε) 206 (−11.32), 222 (+29.43), 246 (−33.57), 268 (+10.47), 288 (−8.15), 339 (+1.97) nm; IR (KBr) νmax 3306, 1652, 1597, 1456, 1393, 1245, 1178, 1055, 1026, 855, 750, 700 cm−1; 1H and 13C NMR data see Table 1; positive-ion HRESIMS m/z 565.1838 [M + H]+ (calcd for C34H29O8, 565.1857). (+)-6″-Hydroxy-4′,4‴-dimethoxyaquisinenone B (5): white, amorphous powder; [α]25 D +30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 333 (3.52), 243 (4.28), 224 (4.40), 204 (4.40) nm; ECD (MeOH) λmax (Δε) 222 (−37.47), 246 (+62.52), 269 (−16.42), 288 (+17.09), 338 (−3.03) nm; IR (KBr) νmax 3249, 1711, 1653, 1597, 1512, 1454, 1391, 1246, 1178, 1081, 1032, 850, 824 cm−1; 1H and 13C NMR data see Table 1; positive-ion HRESIMS m/z 625.2041 [M + H]+ (calcd for C36H33O10, 625.2068). Aquisinenone C Scalemic Mixture (6): white, amorphous powder; [α]25 D −20 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 350 (3.38), 250 (4.34), 207 (4.45) nm; ECD (MeOH) λmax (Δε) 222 (−22.08), 242 (+9.52), 264 (−10.79), 288 (+3.92), 344 (−1.17) nm; IR (KBr) νmax 3292, 1711, 1658, 1621, 1454, 1412, 1176, 1071, 1028, 699 cm−1; 1H and 13C NMR data see Table 1; positive-ion HRESIMS m/z 565.1837 [M + H]+ (calcd for C34H29O8, 565.1857). (+)-Aquisinenone C (6a): [α]25 D +59 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 206 (−33.97), 222 (+88.28), 242 (−32.70), 264 (+36.64), 290 (−11.73), 348 (+3.80) nm. (−)-Aquisinenone C (6b): [α]25 D −59 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 206 (+44.07), 222 (−109.61), 240 (+41.80), 264 (−45.78), 290 (+15.76), 350 (−5.02) nm. (−)-Aquisinenone D (7): white, amorphous powder; [α]25 D −20 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 324 (3.64), 225 (4.59), 207 (4.58) nm; ECD (MeOH) λmax (Δε) 208 (−67.67), 226 (+45.20), 252 (+21.92), 310 (−2.95) nm; IR (KBr) νmax 3306, 1711, 1654, 1614, 1513, 1485, 1436, 1362, 1274, 1245, 1205, 1177, 1085, 1033, 999, 825 cm−1; 1H and 13C NMR data see Table 2; positive-ion HRESIMS m/z 639.2199 [M + H]+ (calcd for C37H35O10, 639.2225). 4′-Demethoxyaquisinenone D Scalemic Mixture (8): white, amorphous powder; [α]25 D +10 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 324 (3.64), 225 (4.53), 207 (4.58) nm; ECD (MeOH) λmax (Δε) 206 (+21.94), 248 (−9.94), 306 (+0.79) nm; IR (KBr) νmax 3270, 1713, 1689, 1653, 1485, 1436, 1274, 1205, 1084, 1048 cm−1; 1H and 13C NMR data see Table 2; positive-ion HRESIMS m/z 609.2091 [M + H]+ (calcd for C36H33O9, 609.2119). (+)-4′-Demethoxyaquisinenone D (8a): [α]25 D +20 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 210 (+58.18), 226 (−40.30), 252 (−27.65), 304 (+3.58) nm. (−)-4′-Demethoxyaquisinenone D (8b): [α]D25 −20 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 210 (−57.19), 226 (+38.95), 252 (+23.33), 306 (−3.52) nm. (+)-Aquisinenone E (9): colorless gum; [α]25 D +10 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 321 (3.58), 224 (4.50), 205 (4.49) nm; ECD (MeOH) λmax (Δε) 208 (−37.65), 232 (+37.31), 274 (+11.76) nm; IR (KBr) νmax 3341, 1712, 1654, 1611, 1512, 1485, 1437, 1362, 1079, 1028, 823 cm−1; 1H and 13C NMR data see Table 2; positive-ion HRESIMS m/z 609.2091 [M + H]+ (calcd for C36H33O9, 609.2119). (−)-Aquisinenone F (10): white, amorphous powder; [α]25 D −50 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 322 (3.66), 224 (4.51), 207 (4.55) nm; ECD (MeOH) λmax (Δε) 204 (+21.22), 238 (−15.94) nm; IR (KBr) νmax 3306, 1711, 1654, 1575, 1484, 1434, 1362, 1245, 1204, 1080, 1030 cm−1; 1H and 13C NMR data see Table 2; positive-ion HRESIMS m/z 579.1997 [M + H]+ (calcd for C35H31O8, 579.2013). (−)-Aquisinenone G (11): white, amorphous powder; [α]25 D −80 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 312 (3.78), 234 (4.38), 206 (4.50) nm; ECD (MeOH) λmax (Δε) 206 (+35.30), 220 (−36.98), 250 (−30.50), 276 (+21.33), 314 (−5.87) nm; IR (KBr) νmax 3376, 1662, 1483, 1450, 1377, 1267, 1173, 1079, 1028, 1003, 848, 750, 699 cm−1; 1H and 13C NMR data see Table 2; positive-ion HRESIMS m/z 565.1831 [M + H]+ (calcd for C34H29O8, 565.1857). (+)-4′-Methoxyaquisinenone G (12): white, amorphous powder; [α]25 D +80 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 314 (3.73), 225 (4.40), 205 (4.43) nm; ECD (MeOH) λmax (Δε) 206 (−37.17), 222

(+56.00), 248 (+61.73), 276 (−42.50), 314 (+10.96) nm; IR (KBr) νmax 3270, 1711, 1662, 1631, 1512, 1449, 1375, 1247, 1176, 1028, 1004, 823, 700 cm−1; 1H and 13C NMR data see Table 2; positive-ion HRESIMS m/z 595.1941 [M + H]+ (calcd for C35H31O9, 595.1963). Cell Culture, Measurement of NO Production, and Cell Viability. RAW264.7 cells were purchased from Peking Union Medical College Cell Bank (Beijing, People’s Republic of China). Cell maintenance, experimental procedures, and data presentation for the inhibition of NO production and viability assay were the same as previously described.24−26 GYF-17 (IC50 4.4 μM) was used as a positive control.5 All the compounds were prepared as stock solutions in DMSO (final solvent concentration less than 0.2% in all assays). ECD Calculations. The relative configurations of compounds 1, 3, 4, 6, 7, and 9−11 were established initially on the basis of their ROESY spectra and subjected to random conformational analysis with the MMFF94s force field and using the SYBYL-X 2.0 software package. The conformers were further optimized by using the timedependent density functional theory (TDDFT) method at the B3LYP/6-31G(d) level, and the frequency was calculated at the same level of theory. The stable conformers without imaginary frequencies were subjected to ECD calculation by the TDDFT method at the B3LYP/6-31+G(d) level with the CPCM model in MeOH. The ECD spectra of different conformers were simulated using SpecDis v1.51 with a half-bandwidth of 0.16−0.40 eV, and the final ECD spectra were obtained according to the Boltzmanncalculated contribution of each conformer after UV correction. The calculated ECD spectra were compared with the experimental data. All calculations were performed with the Gaussian 09 program package.27−29 X-ray Crystallographic Analysis. Colorless plates of 1 were obtained from MeOH−H2O (95:5). Crystallographic data were collected at T = 107.2 K, with Cu Kα radiation (λ = 1.54178 Å) on an Agilent Gemini E X-ray single-crystal diffractometer, equipped with an Oxford Cryostream cooler. The structures were solved by direct methods using SHELXS-97 and refined anisotropically by full-matrix least-squares on F2 using SHELXL-97. Crystallographic data for 1: colorless, plates, C35H32O8, M = 580.61,30 crystal size 0.20 × 0.17 × 0.07 mm3, triclinic, a = 9.6731(4) Å, b = 11.8776(6) Å, c = 13.3871(6) Å, α = 110.146(4)°, β = 98.926(4)°, γ = 99.616(4)°, V = 1385.60(11) Å3, T = 107.2 K, space group P1̅ (no. 2), Z = 2, μ(Cu Kα) = 0.808 mm−1, Dcalcd = 1.392 mg/m3, F(000) = 612, 17 394 reflections measured, 5259 unique (Rint = 0.0324), which were used in all calculations. The final wR(F2) was 0.1064 (all data). The goodness-offit on F2 was 1.047. Crystallographic data for 1 reported in this paper have been deposited with the Cambridge Crystallographic Data Centre (deposition number: CCDC 1582684). Copies of these data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: + 44-(0)1223-336033 or e-mail: [email protected]).

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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.7b00919. Detailed HRESIMS, UV, IR, and NMR spectra of 1−12; chiral-phase HPLC chromatograms of 1, 3, 6, and 8; ECD spectra calculation details of compounds 1a, 3a, 4, 6a, 7, and 9−11; and experimental and calculated ECD spectra of 2−5, 8−10, and 12 (PDF) X-ray crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J. Li). ORCID

She-Po Shi: 0000-0003-3252-3108 552



Article

(24) Li, M. M.; Su, X. Q.; Sun, J.; Gu, Y. F.; Huang, Z.; Zeng, K. W.; Zhang, Q.; Zhao, Y. F.; Ferreira, D.; Zjawiony, J. K.; Li, J.; Tu, P. F. J. Nat. Prod. 2014, 77, 2248−2254. (25) Li, J.; Zeng, K. W.; Shi, S. P.; Jiang, Y.; Tu, P. F. Fitoterapia 2012, 83, 896−900. (26) Huang, Z.; Zhu, Z. X.; Li, Y. T.; Pang, D. R.; Zheng, J.; Zhang, Q.; Zhao, Y. F.; Ferreira, D.; Zjawiony, J. K.; Tu, P. F.; Li, J. J. Nat. Prod. 2015, 78, 2276−2285. (27) Zhang, C.; Wang, S.; Zeng, K. W.; Li, J.; Ferreira, D.; Zjawiony, J. K.; Liu, B. Y.; Guo, X. Y.; Jin, H. W.; Jiang, Y.; Tu, P. F. J. Nat. Prod. 2016, 79, 213−223. (28) Pang, D. R.; Su, X. Q.; Zhu, Z. X.; Sun, J.; Li, Y. T.; Song, Y. L.; Zhao, Y. F.; Tu, P. F.; Zheng, J.; Li, J. Fitoterapia 2016, 115, 135−141. (29) Sun, J.; Zhu, Z. X.; Song, Y. L.; Dong, D.; Zheng, J.; Liu, T.; Zhao, Y. F.; Ferreira, D.; Zjawiony, J. K.; Tu, P. F.; Li, J. J. Nat. Prod. 2016, 79, 1415−1422. (30) Owing to the presence of MeOH in the X-ray crystallographic structure of 1, the actual formula of 1 was C34H28O7, with a formula weight of 548.

Daneel Ferreira: 0000-0002-9375-7920 Jun Li: 0000-0001-8243-5267 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project was financially supported by the National Natural Science Foundation of China (Nos. 81503227, 81573572, 81530097), Project for Standardization of Chinese Materia Medica (ZYBZH-Y-GD-13-ZYY-2017-076), and Graduate Students Independent Subject of Beijing University of Chinese Medicine (No. 2017-JYB-XS-058).

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DEDICATION Dedicated to Dr. Susan Band Horwitz, of Albert Einstein College of Medicine, Bronx, NY, for her pioneering work on bioactive natural products.

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REFERENCES

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