Neolignans from the Arils of Myristica fragrans as Potent Antagonists

Jul 15, 2016 - CC chemokine receptor 3 (CCR3) is expressed selectively in eosinophils, basophils, and some Th2 cells and plays a major role in allergi...
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Neolignans from the Arils of Myristica f ragrans as Potent Antagonists of CC Chemokine Receptor 3 Toshio Morikawa,*,†,‡ Ikuko Hachiman,† Kazuhiko Matsuo,§ Eriko Nishida,† Kiyofumi Ninomiya,†,‡ Takao Hayakawa,† Osamu Yoshie,⊥ Osamu Muraoka,†,‡ and Takashi Nakayama*,§ †

Pharmaceutical Research and Technology Institute, ‡Antiaging Center, and §Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan ⊥ Faculty of Medicine, Kindai University, 377-2 Ohno-higashi, Osaka-sayama, Osaka 589-8511, Japan S Supporting Information *

ABSTRACT: CC chemokine receptor 3 (CCR3) is expressed selectively in eosinophils, basophils, and some Th2 cells and plays a major role in allergic diseases. A methanol extract from the arils of Myristica f ragrans inhibited CC chemokine ligand 11-induced chemotaxis in CCR3-expressing L1.2 cells at 100 μg/mL. From this extract, eight new neolignans, maceneolignans A−H (1−8), were isolated, and their stereostructures were elucidated from their spectroscopic values and chemical properties. Of those constituents, compounds 1, 4, 6, and 8 and (+)-erythro-(7S,8R)-Δ8′-7hydroxy-3,4-methylenedioxy-3′,5′-dimethoxy-8-O-4′-neolignan (11), (−)-(8R)-Δ8′-3,4-methylenedioxy-3′,5′-dimethoxy-8-O-4′neolignan (17), (+)-licarin A (20), nectandrin B (25), verrucosin (26), and myristicin (27) inhibited CCR3-mediated chemotaxis at a concentration of 1 μM. Among them, 1 (EC50 1.6 μM), 6 (1.5 μM), and 8 (1.4 μM) showed relatively strong activities, which were comparable to that of a synthetic CCR3 selective antagonist, SB328437 (0.78 μM).

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that a methanol extract of the arils of Myristica f ragrans Houtt. (Myristicaceae) showed an inhibitory effect on CCL11-induced chemotaxis in L1.2 cells expressing CCR3. From this extract, eight new neolignans, maceneolignans A−H (1−8), were isolated together with 28 known compounds (9−31). Also obtained were quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-O[α-L-rhamnopyranosyl-(1→2)]-O-β-D-galactopyranoside, palmitic acid, oleic acid, oleic acid methyl ester, and linoleic acid. In this work, the absolute stereostructures of the new neolignans (1−8) were elucidated. The isolated compounds were evaluated as CCR3 antagonists in a chemotaxis assay.

hemokines are a family of chemotactic cytokines that regulate migration and tissue localization of various kinds of cells in the body.1−3 Specifically, they participate in inflammatory leukocyte recruitment, lymphocyte recirculation and homing,1−4 and cancer metastasis.1,5 Chemokines are grouped into four subfamilies, CXC, CC, C, and CX3C, based on the arrangement of the two N-terminal cysteine residues.1 CC chemokine receptor 3 (CCR3) has been shown to belong to a family of seven transmembrane-spanning G proteincoupled receptors and to be the principal receptor for CC chemokine ligand (CCL) 11/eotaxin-1, CCL24/eotaxin-2, and CCL26/eotaxin-3.6−9 In the early stage of inflammation, it has been reported that CCR3 is expressed selectively in eosinophils and is involved in eosinophil infiltration.6,10,11 It has also been reported that CCR3 is expressed in a subset of Th2 cells,12 mast cells,13 airway smooth muscle cells,14 and airway epithelial cells.15 The association of CCR3, its ligands, and eosinophils with allergic diseases, such as allergic asthma, allergic rhinitis, and atopic dermatitis, has been studied widely.16−19 For example, studies have shown that eotaxin and CCR3 mRNA are expressed and co-localized in the bronchial mucosa of asthmatics. Moreover, the intensity of their expression correlates with the increase in airway responsiveness in patients with atopic asthma.16,20,21 Thus, CCR3 antagonists may have potential as therapeutic agents for eosinophil-related allergic diseases.22 During the course of characterization studies on antiallergic principles from natural resources,23−29 it was found © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Dried arils of M. f ragrans cultivated in Indonesia were extracted using methanol to give a MeOH extract (33.4% from the dried material). The inhibitory effects of this extract (100 μg/mL) were examined in a chemotaxis assay in a panel of mouse L1.2 cell lines stably expressing CCR1−7 to their specific ligands. As shown in Figure S1, Supporting Information, the MeOH extract was found to suppress significantly CCL11-induced chemotaxis in CCR3-expressing cells (65.2% migration versus a corresponding control), whereas the other expressing cells were not affected. Thus, the MeOH extract was found to show selective CCR3 antagonistic activity against CCR1−7. Received: March 23, 2016

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DOI: 10.1021/acs.jnatprod.6b00262 J. Nat. Prod. XXXX, XXX, XXX−XXX

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aromatic ring. The UV spectrum exhibited absorption maxima at 223 (log ε 4.40) and 270 nm (4.18), which were suggestive of a conjugated aromatic moiety. The EIMS of 1 showed a molecular ion peak at m/z 356 (M+), and the molecular formula was determined as C21H24O5 by high-resolution EIMS measurement. The 1H and 13C NMR spectra of 1 (CDCl3, Table 1), which were assigned with the aid of DEPT, 1H−1H COSY, HMQC, and HMBC experiments, showed signals assignable to two methyls [δ 1.39 (3H, d, J = 7.2 Hz, H3-9), 1.87 (3H, dd, J = 1.6, 6.6 Hz, H3-9′)], two methines [δ 3.46 (1H, dq, J = 9.6, 7.2 Hz, H-8), 5.08 (1H, d, J = 9.6 Hz, H-7)], three methoxy groups [δ 3.88 (6H, s, CH3O-3,5), 3.90 (3H, s, CH3O-3′)], a disubstituted trans-olefin [δ 6.11 (1H, dq, J = 16.0, 6.6 Hz, H-8′), 6.36 (1H, dq, J = 16.0, 1.6 Hz, H-7′)], and two tetrasubstituted benzene rings [δ 6.66 (2H, s, H-2,6), 6.77 (1H, br s, H-6′), 6.79 (1H, br s, H-2′)]. The 1H−1H COSY experiment on 1 indicated the presence of partial structures shown in bold lines in Figure 1. In the HMBC experiment, long-range correlations were observed between the following proton and carbon pairs: H-2,6 and C-4, 7; H-8 and C-1, 5′; H3-9 and C-5′; H-2′ and C-4′, 6′, 7′; H-6′ and C-8, 2′, 4′, 7′; H-7′ and C-2′, 6′; H-8′ and C-1′; CH3O-3,5 and C-3,5; CH3O3′ and C-3′. Thus, the linkage positions of the quaternary carbons were clarified unambiguously, and the planar structure of 1 was elucidated. Next, the relative stereostructure of 1 was investigated using a nuclear Overhauser effect spectroscopy (NOESY) experiment, in which NOE correlations were observed between the following proton pairs: H-2,6 and CH3O-3,5; H-7 and H3-9; H-2′ and CH3O-3′, as shown in Figure S2, Supporting Information. The absolute stereostructure of 1 was elucidated using circular dichroism (ECD) spectroscopic analysis. The ECD spectrum (in MeOH) of 1 showed positive Cotton effects [242 nm (Δε −3.13), 268 nm (+4.00) in MeOH], which indicated that the absolute configurations of the C-7 and C-8 positions are 7R and 8R.37 Otsuka et al. have reported the isolation and absolute stereostructure determination of the enantiomer of 1, odoratisol A, where the opposite specific rotation was observed ([α]25D −35.1 in CHCl3) and Cotton effects in the ECD spectrum were observed at 242 nm (+4.35) and 269 nm (−5.93) in MeOH.51 The above-mentioned evidence enabled the absolute stereostructure of 1 to be determined as (7R,8R)7,8-dihydro-7-(4-hydroy-3,5-dimethoxyphenyl)-3′-methoxy-8methyl-1′-trans-propenylbenzofuran. Maceneolignan B (2) was also isolated as a colorless oil with positive rotation ([α]26D +42 in CHCl3). The EIMS of 2 showed a molecular ion peak at m/z 354 [M+], and the molecular formula was determined as C21H22O5, using HREIMS. The 1H and 13C NMR spectra of 2 (CDCl3, Table 1) showed signals assignable to two methyls [δ 1.39 (3H, d, J = 6.8 Hz, H3-9), 1.86 (3H, dd, J = 1.7, 6.6 Hz, H3-9′)], two methines [δ 3.41 (1H, dq, J = 9.0, 6.8 Hz, H-8), 5.07 (1H, d, J = 9.0 Hz, H-7)], two methoxy groups [δ 3.893 (3H, s, CH3O3′), 3.894 (3H, s, CH3O-5)], a methylenedioxy [δ 5.96 (2H, br s, −OCH2O−)], a disubstituted trans-olefin [δ 6.10 (1H, dq, J = 15.7, 6.6 Hz, H-8′), 6.35 (1H, dq, J = 15.7, 1.7 Hz, H-7′)], and two tetrasubstituted benzene rings [δ 6.61 (1H, d, J = 1.5 Hz, H-2), 6.66 (1H, d, J = 1.5 Hz, H-6), 6.75 (1H, br s, H-6′), 6.78 (1H, br s, H-2′)]. The 1H and 13C NMR spectroscopic properties of 2 were generally superimposable on those of fragrasol-D,52 but a difference was due to the hydroxymethyl group at C-9′ in 2. As shown in Figure 2, the connectivity of the quaternary carbons in 2 was determined using 1H−1H

The MeOH extract was subjected to normal- and reversedphase column chromatography and finally HPLC to give maceneolignans A (1, 0.061%), B (2, 0.0010%), C (3, 0.0007%), D (4, 0.0089%), E (5, 0.0084%), F (6, 0.015%), G (7, 0.0045%), and H (8, 0.83%), (+)-erythro-(7S,8R)-Δ8′-7hydroxy-3,4,5,3′,5′-pentamethoxy-8-O-4′-neolignan 30 (9, 0.35%), (+)-erythro-(7S,8R)-Δ8′-4,7-dihydroxy-3,3′,5′-trimethoxy-8-O-4′-neolignan31 (10, 0.54%), (+)-erythro-(7S,8R)Δ8′-7-hydroxy-3,4-methylenedioxy-3′,5′-dimethoxy-8-O-4′-neolignan32 (11, 0.018%), (−)-erythro-(7R,8S)-Δ8′-4,7-dihydroxy3,3′,5′-trimethoxy-8-O-4′-neolignan31 (12, 0.60%), myrislignanometin E33 (13, 0.0030%), (−)-erythro-(7R,8S)-Δ8′-7-hydroxy-3,4,3′,5′-tetramethoxy-8-O-4′-neolignan31 (14, 0.056%), (−)-erythro-(7R,8S)-Δ8′-7-acetoxy-3,4-methylenedioxy-3′,5′-dimethoxy-8-O-4′-neolignan32 (15, 0.032%), myrifralignan C34 (16, 0.0053%), (−)-(8R)-Δ8′-3,4-methylenedioxy-3′,5′-dimethoxy-8-O-4′-neolignan35 (17, 0.0039%), (−)-(8R)-Δ8′-4-hydroxy-3,3′,5′-trimethoxy-8-O-4′-neolignan31 (18, 0.31%), (−)-(8R)-Δ 8 ′ -3,4,5,3′,5′-pentamethoxy-8-O-4′-neolignan 36 (19, 0.12%), (+)-licarin A37−39 (20, 0.59%), licarin B39,40 (21, 0.28%), (7S,8S)-7-(4-hydroxy-3-methoxyphenyl)-1′-formyl-3′methoxy-8-methyldihydrobenzofuran41 (22, 0.0043%), dihydrocarinatidin42,43 (23, 0.0028%), threo-austrobailignan-544,45 (24, 0.0026%), nectandrin B46 (25, 0.019%), verrucosin46 (26, 0.0093%), myristicin (27, 2.91%), anthriscinol47 (28, 0.027%), 4-allyl-2,6-dimethoxyphenol48 (29, 0.11%), malabaricone C49 (30, 0.0090%), and vanillin (31, 0.017%), together with quercetin 3-O-α-L-rhamnopyranosyl-(1→6)-O-[α-L-rhamnopyranosyl-(1 → 2)]-O-β-D-galactopyranoside50 (0.0028%), palmitic acid (0.065%), oleic acid (0.027%), oleic acid methyl ester (0.41%), and linoleic acid (0.092%). Compounds 27, 31, palmitic acid, oleic acid, and oleic acid methyl ester were identified by comparing their NMR and MS data with those of commercially available samples.

Maceneolignan A (1) was obtained as a colorless oil with positive specific rotation ([α]26D +34 in CHCl3). Its IR spectrum showed absorption bands at 3578, 1617, 1497, 1456, and 1070 cm−1 ascribable to hydroxy and ether groups and an B

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Table 1. 13C NMR Spectroscopic Data (200a and 150b MHz, CDCl3) for Maceneolignans A−H (1−8) position

1a

2a

3b

4a

5a

6a

7a

8a

1 2 3 4 5 6 7 8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ CH3O-3 CH3O-4 CH3O-5 O−CH2−O CH3COO CH3COO CH3O-3′ CH3O-5′

133.2 103.5 147.0 134.8 147.0 103.5 94.0 45.7 17.6 132.3 109.3 144.2 146.5 131.2 113.3 130.9 123.5 18.3 56.4

135.0 100.7 149.0 135.2 143.6 106.2 93.5 45.8 17.8 132.3 109.4 144.1 146.5 133.0 113.3 130.9 123.5 18.3

134.8 100.7 135.2 149.0 143.6 106.2 93.7 45.7 17.9 130.4 110.1 144.3 147.6 133.2 114.5 134.6 120.9 65.3

131.3 108.0 146.8 146.1 114.3 120.0 94.5 45.1 17.7 128.1 111.9 144.7 150.7 134.1 117.4 153.2 126.4 193.5 56.1

129.8 109.2 146.3 145.0 114.0 119.6 88.5 41.6 17.1 133.5 116.5 144.0 145.5 134.2 112.0 40.2 137.9 115.5 56.1

131.2 102.7 146.9 133.6 146.9 102.7 73.0 82.4 12.8 136.2 105.5 153.5 133.1 153.5 105.5 40.6 137.0 116.2 56.3

131.6 108.6 146.5 144.7 114.0 118.8 73.2 83.1 12.7 129.9 105.5 153.9 137.7 153.9 105.5 152.3 128.2 193.4 55.9

130.4 110.2 148.6 148.4 110.7 119.2 76.6 80.8 14.4 135.6 105.4 153.3 133.7 153.3 105.4 40.4 137.1 115.8 55.8 55.7

56.4

56.7 101.5

56.7 101.6 170.9 21.1 56.0

55.9

56.0

56.3

56.0 56.0

56.1 56.1

56.3 56.3

170.0 21.1 55.9 55.9

Figure 1. 1H−1H COSY and HMBC correlations of 1−8.

COSY and HMBC experiments. NOE correlations in the NOESY experiment were observed between H-6 and CH3O-5, between H-7 and H3-9, and between H-2′ and CH3O-3′ (Figure S2, Supporting Information), and on this basis, the relative configuration in the 7,8-dihydrobenzofuran moiety was concluded as trans. The ECD spectrum was used to determine the absolute configuration of 2 as 7R and 8R conformations from positive Cotton effects [244 nm (Δε − 3.24), 268 nm (+5.05)], which was noted to be the same as in (+)-licarin A [231 nm (θ −1200), 268 nm (+520) in MeOH].37 This configuration agreed well with the sign of the optical rotation of (+)-licarin A ([α]25D +43.5 in CHCl3).37 Consequently, the absolute stereostructure of 2 was elucidated as (7R,8R)-7,8-

dihydro-7-(5-methoxy-3,4-methylenedioxyphenyl)-3′-methoxy8-methyl-1′-trans-propenylbenzofuran. Maceneolignan C (3) was obtained as a colorless oil with positive rotation ([α]25D +25 in CHCl3), and its molecular formula was determined as C23H24O7 using HREIMS. The 1H and 13C NMR spectroscopic properties (Table 1) of 3 were quite similar to those of (−)-licarin A;37 however, an additional acetyl group was evident in 3 [δ 2.10 (3H, s)]. Treatment of 3 with 0.5% sodium methoxide (NaOMe)−MeOH produced fragransol-D52 (Figure S2, Supporting Information). According to the observed HMBC correlation between H2-9′ [δ 4.72 (2H, d, J = 6.7 Hz)] and the acetyl ester carbonyl carbon (δC 170.9), C

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Figure 2. Effects of the isolates (1, 2, 4, 6, 8−12, 14, 15, 17−22, and 24−31) on CCL11-induced chemotaxis in CCR3-expressing L1.2 cells. Each experiment was performed using a concentration of 1 μM. Migration of CCR3-expressing L1.2 cells was induced by 10 nM CCL11. Results from three experiments are shown as mean ± SEM. Significant difference was analyzed by Dunnett’s test, *p < 0.05, **p < 0.01 (vs control).

d, J = 2.0 Hz, H-2), 6.89 (1H, d, J = 8.0 Hz, H-5)], and a monosubstituted olefin {δ [5.07 (1H, dd, J = 1.9, 17.0 Hz), 5.10 (1H, dd, J = 1.9, 10.0 Hz), H2-9′], 5.98 (1H, ddt, J = 10.0, 17.0, 6.7 Hz, H-8′)}. A 1H−1H COSY experiment on 5 indicated the presence of the partial structures shown in bold lines in Figure 1. In the HMBC experiment, long-range correlations were observed between the following proton and carbon pairs: H-2 and C-4, 6, 7; H-5 and C-1, 3; H-6 and C-2, 4, 7; H-7 and C-2, 6, 5′; H-8 and C-1, 5′; H3-9 and C-5′; H-2′ and C-4′, 6′, 7′; H6′ and C-8, 2′, 4′, 7′; H2-7′ and C-2′, 6′; H-8′ and C-1′; CH3O3 and C-3; CH3O-3′ and C-3′. Thus, the linkage positions of the quaternary carbons in 5 were determined unambiguously. The configuration of 5 was characterized using the NOESY experiment, which showed the NOE correlations between H-7 and H-8 as shown in Figure S2, Supporting Information. The cis-7-aryl-8-methyl dihydrobenzofuran moiety in 5 was also supported by comparison of the proton signals in the 1H NMR spectrum with those of isolicarin A [δ 0.83 (3H, d, J = 7.0 Hz, H3-9), 3.59 (1H, dq, J = 8.5, 7.0 Hz, H-8), 5.77 (1H, d, J = 8.5 Hz, H-7) in CDCl3].54 The absolute configuration of 5 was established as 7S and 8R based on its optical rotation and the ECD spectroscopic analysis, which were similar to those exhibited by isolicarin A.54 On the basis of the abovementioned evidence, the absolute stereostructure of 5 was determined as (7S,8R)-7,8-dihydro-7-(4-hydroxy-3-methoxyphenyl)-3′-methoxy-8-methyl-1′-allylbenzofuran. Maceneolignan F (6) was obtained as a colorless oil with positive specific rotation ([α]27D +14 in CHCl3). The molecular formula of 6 was determined by EIMS and HREIMS measurements to be C22H28O7. The 1H and 13C NMR spectra (Table 1, CDCl3) of 6 showed signals assignable to a methyl [δ 1.11 (3H, d, J = 6.4 Hz, H3-9)], a methylene [δ 3.38 (2H, d, J = 6.9 Hz, H2-7′)], four methoxy groups [δ 3.87 (6H, s, CH3O3,5), 3.88 (6H, s, CH3O-3′,5′)], two methines [δ 4.31 (1H, dq, J = 3.0, 6.4 Hz, H-8), 4.78 (1H, d, J = 3.0 Hz, H-7)], two tetrasubstituted benzene rings [δ 6.47 (2H, s, H-2′,6′), 6.55 (2H, s, H-2,6)], and a vinyl group {δ [5.12 (1H, dd, J = 1.6, 10.0 Hz), 5.14 (1H, dd, J = 1.6, 17.0 Hz), H2-9′], 5.99 (1H, ddt, J = 10.0, 17.0, 6.9 Hz, H-8′)}. These data were quite similar to those of (+)-erythro-(7S,8R)-Δ8′-7-hydroxy-3,4,5,3′,5′-pentamethoxy-8-O-4′-neolignan30 (9), but signals were lacking for a CH3O-4 group. Treatment of 6 with trimethylsilyldiazomethane (TMSCHN2) yielded 9 (Figure S2, Supporting Information), which indicated that the structure of compound 6 including its absolute configuration is (+)-erythro-(7S,8R)Δ8′-4,7-dihydroxy-3,5,3′,5′-tetramethoxy-8-O-4′-neolignan.

the structure of 3 including the absolute configuration was elucidated as 9′-O-acetyl fragransol-D. Maceneolignan D (4) was obtained as a colorless oil with negative optical rotation ([α]22D −51). In the EIMS, a molecular ion peak was observed at m/z 340, and HREIMS analysis revealed its molecular formula as C20H20O5. The 1H and 13C NMR spectra of 4 (CDCl3, Table 1) showed signals assignable to a methyl [δ 1.42 (3H, d, J = 6.9 Hz, H3-9)], two methines [δ 3.52 (1H, dq, J = 9.2, 6.9 Hz, H-8), 5.20 (1H, d, J = 9.2 Hz, H-7)], two methoxy groups [δ 3.89 (3H, s, CH3O-3), 3.93 (3H, s, CH3O-3′)], a tri- and a tetrasubstituted benzene ring [δ 6.90 (2H, br s, H-5, 6), 6.94 (1H, br s, H-2), 7.02 (1H, br s, H-2′), 7.04 (1H, br s, H-6′)], a disubstituted trans-olefin [δ 6.62 (1H, dd, J = 7.7, 15.8 Hz, H-8′), 7.43 (1H, d, J = 15.8 Hz, H-7′)], and an aldehyde [δ 9.66 (1H, d, J = 7.7 Hz, H-9′)]. These data were quite similar to those of (−)-licarin A;37 however, the signals were different due to the presence of an aldehyde group in 4. As shown in Figure 1, the connectivity of the aldehyde group in 4 was established using the results of the 1 H−1H COSY experiment. Using these results, the planar structure of 4 could be elucidated. This compound was reported previously as a derivative of lignin from acid degradation,53 although its stereostructure has not been reported. The relative stereostructure at C-7 and C-8 in the 7,8-dihydrobenzofuran moiety in 4 was deduced using a NOESY experiment, in which a NOE correlation was observed between H-7 and H3-9 (Figure S2, Supporting Information). In addition, the absolute stereostructure of 4 was determined using ECD spectroscopic analysis [235 nm (+1.10), 269 nm (−0.25) in MeOH], where the same Cotton effects were observed as in (−)-licarin A [231 nm (θ +1735), 270 nm (−1900) in MeOH].37 Consequently, the absolute configuration of 4 was determined to be 7S and 8S. Maceneolignan E (5) was obtained as a colorless oil with negative specific rotation ([α]25D −81 in CHCl3). In the positive-ion ESIMS, a quasimolecular ion peak was observed at m/z 349 [M + Na]+, and HRESIMS analysis revealed that its molecular formula is C20H22O4. The 1H and 13C NMR spectroscopic properties of 5 were superimposable on those of dihydrocarinatidin42,43 (23) and indicated the presence of the same functional groups. These were a methyl [δ 0.82 (3H, d, J = 7.3 Hz, H3-9)], a methylene [δ 3.35 (2H, d, J = 6.7 Hz, H2-7′)], two methines [δ 3.57 (1H, dq, J = 8.6, 7.3 Hz, H-8), 5.77 (1H, d, J = 8.6 Hz, H-7)], two methoxy groups [δ 3.87 (3H, s, CH3O-3), 3.90 (3H, s, CH3O-3′)], a tri- and a tetrasubstituted benzene ring [δ 6.62 (1H, br s, H-6′), 6.63 (1H, br s, H-2′), 6.80 (1H, dd, J = 2.0, 8.0 Hz, H-6), 6.88 (1H, D

DOI: 10.1021/acs.jnatprod.6b00262 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Maceneolignan G (7) was obtained as a colorless oil with positive specific rotation ([α]25D +22 in CHCl3), and its molecular formula, C21H24O7, was determined using positiveion EISMS and HRESIMS measurements. The 1H and 13C NMR spectra (Table 1, CDCl3) showed signals assignable to a methyl [δ 1.15 (3H, d, J = 6.4 Hz, H3-9)], three methoxy groups [δ 3.90 (3H, s, CH3O-3), 3.94 (6H, s, CH3O-3′,5′)], two methines [δ 4.44 (1H, dq, J = 2.9, 6.4 Hz, H-8), 4.79 (1H, d, J = 2.9 Hz, H-7)], a tri- and a tetrasubstituted aromatic ring [δ 6.68 (1H, dd, J = 1.7, 8.1 Hz, H-6), 6.84 (1H, d, J = 8.1 Hz, H-5), 6.85 (2H, s, H-2′,6′), 6.97 (1H, d, J = 1.7 Hz, H-2)], a trans-disubstituted olefin [δ 6.66 (1H, dd, J = 7.6, 15.8 Hz, H8′), 7.42 (1H, d, J = 15.8 Hz, H-7′)], and an aldehyde [δ 9.70 (1H, d, J = 7.6 Hz, H-9′)]. As shown in Figure 2, the 1H−1H COSY experiment indicated the presence of partial structures written in bold lines. In the HMBC experiment, long-range correlations were observed between the following protons and carbons: H-2 and C-4, 6, 7; H-5 and C-1, 3; H-6 and C-2, 4, 7; H-2′,6′ and C-4′, 7′; H-8′ and C-1′; CH3O-3 and C-3; CH3O3′,5′ and C-3′,5′. The positions of the methoxy groups in 7 were characterized using the NOESY experiment, which showed the NOE correlations between CH3O-3 and H-2 and between CH3O-3′,5′ and H-2′,6′ (Figure S2, Supporting Information). A comparison of the optical rotation value between 7 and those of similar neolignans, (+)-erythro-(7S,8R)and (−)-erythro-(7R,8S)-Δ8′-4,7-dihydroxy-3,3′,5′-trimethoxy8-O-4′-neolignan (10: [α]20D +25.28 and 12: [α]20D −25.53 both in CHCl3),31 led us to conclude that the absolute configurations of the 7- and 8-positions in 7 were in the S and R forms, respectively. Maceneolignan H (8) was obtained as a colorless oil with negative specific rotation ([α]24D −34 in CHCl3). In the EIMS, a molecular ion peak was observed at m/z 430 [M]+, and HREIMS analysis revealed the molecular formula to be C24H30O7. The 1H and 13C NMR spectroscopic properties (Table 1) of 8 were quite similar to those of (−)-erythro(7R,8S)-Δ 8′ -7-hydroxy-3,4,3′,5′-tetramethoxy-8-O-4′-neolignan31 (14), but an additional acetyl group was apparent in 8 [δ 2.17 (3H, s)]. Treatment of 8 with 0.5% NaOMe−MeOH produced 14 (Figure S2, Supporting Information). According to the observed HMBC correlation between H-7 [δ 5.85 (1H, d, J = 3.2 Hz)] and the acetyl ester carbonyl carbon (δC 170.0), the structure of 8, including its absolute stereochemistry, was elucidated as (−)-erythro-(7R,8S)-Δ8′-7-acetoxy-3,4,3′,5′-tetramethoxy-8-O-4′-neolignan. The effects of the isolates (1, 2, 4, 6, 8−12, 14, 15, 17−22, and 24−31) from the arils of M. f ragrans were evaluated against CCL-11-induced chemotaxis in CCR3-expressing L1.2 cells, and the results are summarized in Figure 2. The results showed that 1, 4, 6, 8, 11, 17, 20, and 25−27 inhibited chemotaxis at a concentration of 1 μM. Furthermore, three of the new neolignans, 1 (EC50 1.6 μM), 6 (1.5 μM), and 8 (1.4 μM), showed relatively potent activities, as shown in Figure 3. These constituents showed equivalent activity to that of a synthetic CCR3 selective antagonist, SB328437 (EC50 0.78 μM), without notable cytotoxic effects at the effective concentrations (data not shown). In light of its pivotal role in eosinophil migration, CCR3 is a promising therapeutic target of interest for the development of antiallergic drugs in eosinophil-associated inflammation. However, although many pharmaceutical companies are trying to develop new drugs targeting CCR3,55−57 none of these inhibitors have been approved for clinical use yet. The present results indicate that the neolignan constituents, 1,

Figure 3. Concentration dependence of 1 (A), 6 (B), 8 (C), and SB328437 (D) on CCL11-induced chemotaxis in CCR3-expressing L1.2 cells. Migration of CCR3-expressing L1.2 cells was induced by 10 nM CCL11. Results from three experiments are shown as mean ± SEM. Significant difference was analyzed by Dunnett’s test, **p < 0.01 (vs control).

6, and 8 are potential candidates for a new type of CCR3 antagonist derived from naturally occurring products and may be a useful tool in the treatment of allergic diseases. Further studies on the mechanism of action of these highly active neolignans as well as their structural requirements are in progress.

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EXPERIMENTAL SECTION

General Experimental Procedures. The following instruments were used to obtain physical data: specific rotations, SEPA-300 digital polarimeter (Horiba Ltd., Kyoto, Japan, l = 5 cm); UV spectra, UV1600 spectrometer (Shimadzu Co., Kyoto, Japan); ECD spectra, J720WI spectrometer (JASCO Co., Tokyo, Japan); IR spectra, FTIR8100 spectrometer (Shimadzu Co.); 1H NMR spectra, JNM-ECA800 (800 MHz), JNM-ECA600 (600 MHz), and JNM-ECS400 (400 MHz) spectrometers (JEOL Ltd., Tokyo, Japan); 13C NMR spectra, JNM-ECA800 (200 MHz), JNM-ECA600 (150 MHz), and JNMECS400 (100 MHz) spectrometers (JEOL Ltd.) with tetramethylsilane as an internal standard; EIMS and HREIMS, JMS-GCMate mass spectrometer (JEOL Ltd.); ESIMS and HRESIMS, Exactive Plus mass spectrometer (Thermo Fisher Scientific Inc., MA, USA); HPLC detector, SPD-10Avp UV−vis detector (Shimadzu Co.); HPLC column, Cosmosil 5C18-MS-II and Cosmosil Cholestor (Nacalai Tesque, Inc., Kyoto, Japan), and Ceramospher Chiral RU-2 (Shiseido Co., Ltd., Tokyo, Japan) 4.6 mm i.d. × 250 mm and 20 mm i.d. × 250 mm for analytical and preparative purposes, respectively. The following experimental conditions were used for column chromatography (CC): normal-phase silica gel CC, silica gel 60N (Kanto Chemical Co., Ltd., Tokyo, Japan; 63-210 mesh, spherical, neutral); reversed-phase ODS CC, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., Aichi, Japan; 100−200 mesh); TLC, precoated TLC plates with silica gel 60F254 (Merck, Darmstadt, Germany, 0.25 mm, normal-phase) and silica gel RP-18 WF254S (Merck, Darmstadt, Germany, 0.25 mm, reversed-phase); reversedphase HPTLC, precoated TLC plates with silica gel RP-18 WF254S (Merck, 0.25 mm); detection was carried out by spraying 1% Ce(SO4)2−10% aqueous H2SO4, followed by heating. Plant Material. The arils of M. f ragrans, which were cultivated in Indonesia, were purchased in November 2011 from a wholesale firm in Tochimoto Tenkaido Co., Ltd., Osaka, Japan. Identification of the plant material was confirmed by the Garden of Medicinal Plants, Kindai University, and by one of the authors (T.M.). A voucher specimen (lot. no. 110997) of this plant material is on file in our laboratory. E

DOI: 10.1021/acs.jnatprod.6b00262 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

57.0 mg, 0.056%), and (−)-(8R)-Δ8′-4-hydroxy-3,3′,5′-trimethoxy-8O-4′-neolignan (18, 241.9 mg, 0.24%). Fraction 8-6 (141.7 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (70:30, v/v)] to give (−)-(8R)-Δ8′-3,4,5,3′,5′pentamethoxy-8-O-4′-neolignan (19, 59.2 mg, 0.014%). Fraction 8-7 (498.9 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (65:35, v/v)] to give 19 (245.2 mg, 0.11%). Fraction 8-8 (509.1 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (70:30, v/v)] to give maceneolignan A (1, 21.0 mg, 0.020%), (−)-erythro-(7R,8S)Δ8′-7-acetoxy-3,4-methylenedioxy-3′,5′-dimethoxy-8-O-4′-neolignan (15, 33.0 mg, 0.032%), and 20 (187.1 mg, 0.18%). Fraction 8-9 (125.1 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (70:30, v/v)] to give maceneolignan C (3, 3.0 mg, 0.0007%) and 20 (12.0 mg, 0.0028%). Fraction 9 (20.7 g) was subjected to reversed-phase ODS CC [660 g, MeOH−H2O (60:40 → 70:30, v/v) → MeOH → acetone] to afford 11 fractions {Fr. 9-1 (88.8 mg), Fr. 9-2 [= anthriscinol (28, 111.3 mg, 0.027%)], Fr. 9-3 (70.6 mg), Fr. 9-4 (663.2 mg), Fr. 9-5 (246.5 mg), Fr. 9-6 (7.00 g), Fr. 9-7 (1.30 g), Fr. 9-8 (5.60 g), Fr. 9-9 (794.5 mg), Fr. 9-10 (416.3 mg), and Fr. 9-11 (2.90 g)}. Fraction 9-4 (663.2 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (65:35, v/v)] to give maceneolignan D (4, 7.9 mg, 0.0019%), (7R,8S)-1′-formyl-7-(4-hydroxy-3-methoxyphenyl)-3′-methoxy-8-methyldihydrobenzofuran (22, 18.0 mg, 0.0043%), and nectandrin B (25, 44.4 mg, 0.011%). Fraction 9-5 (246.5 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (65:35, v/v)] to give 4 (7.5 mg, 0.0018%), maceneolignan F (6, 61.0 mg, 0.015%), 25 (34.8 mg, 0.0083%), and verrucosin (26, 38.8 mg, 0.0093%). Fraction 9-6 (102.2 mg) was purified by HPLC [Ceramospher Chiral RU-2, UV (254 nm), MeOH] to give (+)-erythro-(7S,8R)-Δ8′-4,7-dihydroxy-3,3′,5′-trimethoxy-8-O4′-neolignan (10, 33.2 mg, 0.54%) and (−)-erythro-(7R,8S)-Δ8′-4,7dihydroxy-3,3′,5′-trimethoxy-8-O-4′-neolignan (12, 36.6 mg, 0.60%). Fraction 9-7 (398.3 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (80:20, v/v)] to give (+)-erythro-(7S,8R)-Δ8′-7-hydroxy-3,4,5,3′,5′-pentamethoxy-8-O-4′neolignan (9, 259.0 mg, 0.20%). Fraction 9-8 (108.4 mg) was purified by HPLC [Cosmosil Cholestor, UV (254 nm), MeOH−1% aqueous AcOH (70:30, v/v)] to give maceneolignan H (8, 66.4 mg, 0.82%) and 9 (12.3 mg, 0.15%). Fraction 9-9 (402.2 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (80:20, v/v)] to give maceneolignan A (1, 86.0 mg, 0.041%). Fraction 9-10 (416.3 mg) was purified by HPLC [Cosmosil 5C18-MSII, UV (254 nm), CH3CN−1% aqueous AcOH (80:20, v/v)] to give malabaricone C (30, 37,6 mg, 0.0090%). Fraction 11 (3.64 g) was subjected to reversed-phase ODS CC [115 g, MeOH−H2O (75:25 → 90:10, v/v) → MeOH → acetone] to afford five fractions [Fr. 11-1 (695.3 mg), Fr. 11-2 (520.3 mg), Fr. 11-3 (118.9 mg), Fr. 11-4 (630.8 mg), and Fr. 11-5 (1.69 g)]. Fraction 11-1 (695.3 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (50:50, v/v)] to give 4 (8.3 mg, 0.0020%), maceneolignan G (7, 18.9 mg, 0.0045%), and (−)-erythro-(7R,8S)-Δ7′-4,7,9′-trihydroxy3,3′,5′-trimethoxy-8-O-4′-neolignan (13, 12.4 mg, 0.0030%). Fraction 11-2 (520.3 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (60:40, v/v)] to give 4 (13.6 mg, 0.0032%) and (−)-erythro-(7R,8S)-Δ7′-4,7-dihydroxy-3,5,3′-trimethoxy-8-O-4′-neolignan (16, 22.1 mg, 0.0053%). The known compounds isolated were unambiguously identified by comparison of their physical and spectroscopic data with those of commercially available samples. Maceneolignan A (1): colorless oil; [α]26D +34 (c 1.6, CHCl3); UV (MeOH) λmax (log ε) 217 (sh) (4.38), 223 (4.40), 270 (4.18) nm; ECD (MeOH) λ (Δε) 218 (+0.70), 242 (−3.13), 268 (+4.00) nm; IR (film) νmax 3578, 1617, 1552, 1497, 1456, 1374, 1070 cm−1; 1H NMR (CDCl3, 800 MHz) δ 1.39 (3H, d, J = 7.2 Hz, H3-9), 1.87 (3H, dd, J = 1.6, 6.6 Hz, H3-9′), 3.46 (1H, dq, J = 9.6, 7.2 Hz, H-8), 3.88 (6H, s, CH3O-3,5), 3.90 (3H, s, CH3O-3′), 5.08 (1H, d, J = 9.6 Hz, H-7), 6.11 (1H, dq, J = 16.0, 6.6 Hz, H-8′), 6.36 (1H, dq, J = 16.0, 1.6 Hz, H-7′), 6.66 (2H, s, H-2,6), 6.77 (1H, br s, H-6′), 6.79 (1H, br s, H-2′); 13C

Extraction and Isolation. Dried arils of M. f ragrans (500 g) were extracted three times with MeOH under reflux for 3 h. Evaporation of the combined extracts under reduced pressure yielded the MeOH extract (167 g, 33.4%). An aliquot (140.0 g) of the MeOH extract was subjected to normal-phase silica gel CC [3.3 kg, n-hexane → nhexane−EtOAc (50:1 → 10:1 → 5:1 → 3:1 → 1:1, v/v) → EtOAc → MeOH] to give 12 fractions [Fr. 1 (304.0 mg), Fr. 2 (18.7 g), Fr. 3 (16.4 g), Fr. 4 (8.0 g), Fr. 5 (8.4 g), Fr. 6 (3.9 g), Fr. 7 (4.4 g), Fr. 8 (8.8 g), Fr. 9 (20.7 g), Fr. 10 (1.4 g), Fr. 11 (3.6 g), and Fr. 12 (11.4 g)]. Fraction 2 (18.7 g) was subjected to reversed-phase ODS CC [660 g, MeOH−H2O (70:30 → 80:20 → 90:10, v/v) → MeOH] to afford seven fractions {Fr. 2-1 (683.8 mg), Fr. 2-2 [= myristicin (27, 12.20 g, 2.91%)], Fr. 2-3 (196.6 mg), Fr. 2-4 (215.8 mg), Fr. 2-5 (650.6 mg), Fr. 2-6 [= oleic acid methyl ester (1.70 g, 0.41%)], and Fr. 2-7 (1087.6 mg)}. Fraction 2-5 (510.6 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (230 nm), MeOH−H2O (90:10, v/v)] to give threo-austrobailignan-5 (24, 8.6 mg, 0.0026%). Fraction 3 (16.4 g) was subjected to reversed-phase ODS CC [560 g, MeOH−H2O (60:40 → 70:30 → 80:20 → 90:10, v/v) → MeOH → acetone] to afford six fractions {Fr. 3-1 (189.1 mg), Fr. 3-2 [= (7R,8R)-7-(3,4methylenedioxyphenyl)-3′-methoxy-8-methyl-1′-propenyldihydrobenofuran (21, 1.10 g, 0.26%)], Fr. 3-3 (196.5 mg), Fr. 3-4 (2.90 g), Fr. 3-5 (1.15 g), and Fr. 6-6 (8.40 g)}. Fraction 3-4 (500.0 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (230 nm), MeOH−1% aqueous AcOH (90:10, v/v)] to give palmitic acid (46.8 mg, 0.065%). Fraction 4 (7.96 g) was subjected to reversed-phase ODS CC [264 g, MeOH−H2O (65:35 → 90:10, v/v) → MeOH → acetone] to afford seven fractions [Fr. 4-1 (338.4 mg), Fr. 4-2 [= (−)-(8R)-Δ8′-3,4methylenedioxy-3′,5′-dimethoxy-8-O-4′-neolignan (17, 16.4 mg, 0.0039%)], Fr. 4-3 (25.9 mg), Fr. 4-4 [= 21 (62.8 mg, 0.015%)], Fr. 4-5 (5.52 g), Fr. 4-6 [= oleic acid (803.7 mg, 0.19%)], and Fr. 4-7 (599.4 mg). Fraction 6 (3.91 g) was subjected to reversed-phase ODS CC [500 g, MeOH−H2O (50:50 → 70:30 → 80:20 → 90:10, v/v) → MeOH → acetone] to afford nine fractions {Fr. 6-1 [= quercetin 3-Oα-L-rhamnopyranosyl-(1→6)-O-[α-L-rhamnopyranosyl-(1→2)]-O-βD-galactopyranoside (11.8 mg, 0.0028%)], Fr. 6-2 [= 4-allyl-2,6dimethoxyphenol (29, 186.7 mg, 0.044%)], Fr. 6-3 (13.7 mg), Fr. 6-4 (133.7 mg), Fr. 6-5 (35.1 mg), Fr. 6-6 (64.9 mg), Fr. 6-7 (765.5 mg), Fr. 6-8 (383.0 mg), and Fr. 6-9 (2.14 g)}. Fraction 6-4 (133.7 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (70:30, v/v)] to give (+)-erythro-(7S,8R)-Δ8′-7hydroxy-3,4-methylenedioxy-3′,5′-dimethoxy-8-O-4′-neolignan (11, 53.9 mg, 0.013%), (+)-licarin A (20, 4.5 mg, 0.0011%), and dihydrocarinatidin (23, 19.6 mg, 0.0047%). Fraction 6-6 (64.9 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH− 1% aqueous AcOH (80:20, v/v)] to give maceneolignan B (2, 4.3 mg, 0.0010%). Fraction 6-7 (516.7 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (90:10, v/v)] to give linoleic acid (260.8 mg, 0.092%) and oleic acid (31.9 mg, 0.011%). Fraction 6-8 (383.0 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (230 nm), MeOH−1% aqueous AcOH (80:20, v/v)] to give oleic acid (68.5 mg, 0.018%). Fraction 7 (4.45 g) was subjected to reversed-phase ODS CC [147 g, H2O → MeOH−H2O (50:50 → 60:40 → 70:30 → 90:10, v/v) → MeOH → acetone] to afford six fractions {Fr. 7-1 [= 29 (273.0 mg, 0.065%), Fr. 7-2 (21.8 mg), Fr. 7-3 [= (−)-(8R)-Δ8′-4-hydroxy-3,3′,5′,-trimethoxy-8-O-4′-neolignan (18, 39.4 mg, 0.0094%)], Fr. 7-4 (518.4 mg), Fr. 7-5 [= 20 (1.74 g, 0.41%)], and Fr. 7-6 (1.58 g)}. Fraction 7-4 (518.4 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−1% aqueous AcOH (70:30, v/v)] to give maceneolignan E (5, 35.0 mg, 0.0084%), 11 (20.2 mg, 0.0048%), 18 (241.6 mg, 0.057%), and 23 (94.6 mg, 0.023%). Fraction 8 (8.8 g) was subjected to reversed-phase ODS CC [330 g, MeOH−H2O (60:40, v/v) → MeOH → acetone] to afford 10 fractions {Fr. 8-1 [= vanillin (70.4 mg, 0.017%)], Fr. 8-2 (231.7 mg), Fr. 8-3 (779.6 mg), Fr. 8-4 (859.7 mg), Fr. 8-5 (2.10 mg), Fr. 8-6 (141.7 mg), Fr. 8-7 (979.3 mg), Fr. 8−-8 (2.10 g), Fr. 8-9 (125.1 mg), and Fr. 8-10 (1902.3 mg)}. Fraction 8-5 (502.8 mg) was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH−H2O (65:35, v/ v)] to give maceneolignan H (8, 8.3 mg, 0.0082%), (−)-erythro(7R,8S)-Δ8′-7-hydroxy-3,4,3′,5′-tetramethoxy-8-O-4′-neolignan (14, F

DOI: 10.1021/acs.jnatprod.6b00262 J. Nat. Prod. XXXX, XXX, XXX−XXX

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NMR data, see Table 1; EIMS m/z 356 [M+] (100), 341 (16), 202 (8), 167 (15); HREIMS m/z 356.1617 (calcd for C21H24O5, 356.1624). Maceneolignan B (2): colorless oil; [α]26D +42 (c 1.1, CHCl3); UV (MeOH) λmax (log ε) 214 (4.58), 270 (4.11), 306 (sh) (3.38) nm; ECD (MeOH) λ (Δε) 217 (+1.57), 244 (−3.24), 268 (+5.05) nm; IR (film) νmax 1636, 1509, 1497, 1456, 1339, 1208, 1142, 1046 cm−1; 1H NMR (CDCl3, 800 MHz) δ 1.39 (3H, d, J = 6.8 Hz, H3-9), 1.86 (3H, dd, J = 1.7, 6.6 Hz, H3-9′), 3.41 (1H, dq, J = 9.0, 6.8 Hz, H-8), 3.893 (3H, s, CH3O-3′), 3.894 (3H, s, CH3O-5), 5.07 (1H, d, J = 9.0 Hz, H7), 5.96 (2H, br s, OCH2O), 6.10 (1H, dq, J = 15.7, 6.6 Hz, H-8′), 6.35 (1H, dq, J = 15.7, 1.7 Hz, H-7′), 6.61 (1H, d, J = 1.5 Hz, H-2), 6.66 (1H, d, J = 1.5 Hz, H-6), 6.75 (1H, br s, H-6′), 6.78 (1H, br s, H2′); 13C NMR data, see Table 1; EIMS m/z 354 [M+] (100), 339 (9), 202 (4), 165 (14); HREIMS m/z 354.11473 (calcd for C21H22O5, 354.1467). Maceneolignan C (3): colorless oil; [α]25D +25 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 213 (4.45), 278 (3.89), 306 (sh) (3.48) nm; ECD (MeOH) λ (Δε) 217 (+1.12), 244 (−1.61), 272 (+1.69) nm; IR (film) νmax 1734, 1576, 1509, 1456, 1339, 1227, 1104, 1046 cm−1; 1H NMR (CDCl3, 600 MHz) δ 1.40 (3H, d, J = 6.9 Hz, H3-9), 2.10 (3H, s, CH3COO), 3.43 (1H, dq, J = 9.1, 6.9 Hz, H-8), 3.899 (3H, s, CH3O-3′), 3.902 (3H, s, CH3O-5), 4.72 (2H, d, J = 6.7 Hz, H3-9′), 5.09 (1H, d, J = 9.1 Hz, H-7), 5.97 (2H, br s, OCH2O), 6.17 (1H, dt, J = 15.8, 6.7 Hz, H-8′), 6.60 (1H, d, J = 1.6 Hz, H-2), 6.61 (1H, d, J = 1.6 Hz, H-6), 6.61 (1H, d, J = 15.8 Hz, H-7′), 6.84 (1H, br s, H-6′), 6.84 (1H, br s, H-2′); 13C NMR data, see Table 1; EIMS m/z 412 [M+] (100), 354 (14); HREIMS m/z 412.1514 (calcd for C23H24O7, 412.1522). Maceneolignan D (4): colorless oil; [α]22D −51 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 230 (4.21), 289 (3.80), 342 (4.18) nm; ECD (MeOH) λ (Δε) 235 (+1.10), 269 (−0.25), 331 (−1.37) nm; IR (film) νmax 3415, 1671, 1595, 1519, 1456, 1331, 1271, 1208, 1129, 1034 cm−1; 1H NMR (CDCl3, 800 MHz) δ 1.42 (3H, d, J = 6.9 Hz, H3-9), 3.52 (1H, dq, J = 9.2, 6.9 Hz, H-8), 3.89 (3H, s, CH3O-3), 3.93 (3H, s, CH3O-3′), 5.20 (1H, d, J = 9.2 Hz, H-7), 6.62 (1H, dd, J = 7.7, 15.8 Hz, H-8′), 6.90 (2H, br s, H-5 and H-6), 6.94 (1H, br s, H-2), 7.02 (1H, br s, H-2′), 7.04 (1H, br s, H-6′), 7.43 (1H, d, J = 15.8 Hz, H-7′), 9.66 (1H, d, J = 7.7 Hz, H-9′); 13C NMR data, see Table 1; EIMS m/z 340 [M+] (100), 203 (11), 137 (24); HREIMS m/z 340.1319 (calcd for C20H20O5, 340.1311). Maceneolignan E (5): colorless oil; [α]25D −81 (c 0.8, CHCl3); UV (MeOH) λmax (log ε) 227 (4.38), 282 (sh) (3.93) nm; ECD (MeOH) λ (Δε) 242 (+3.01), 289 (−1.55) nm; IR (film) νmax 3456, 1607, 1516, 1456, 1333, 1213, 1140, 1034 cm−1; 1H NMR (CDCl3, 800 MHz) δ 0.82 (3H, d, J = 7.3 Hz, H3-9), 3.35 (2H, d, J = 6.7 Hz, H2-7′), 3.57 (1H, dq, J = 8.6, 7.3 Hz, H-8), 3.87 (3H, s, CH3O-3), 3.90 (3H, s, CH3O-3′), [5.07 (1H, dd, J = 1.9, 17.0 Hz), 5.10 (1H, dd, J = 1.9, 10.0 Hz), H2-9′], 5.77 (1H, d, J = 8.6 Hz, H-7), 5.98 (1H, ddt, J = 10.0, 17.0, 6.7 Hz, H-8′), 6.62 (1H, br s, H-6′), 6.63 (1H, br s, H-2′), 6.80 (1H, dd, J = 2.0, 8.0 Hz, H-6), 6.88 (1H, d, J = 2.0 Hz, H-2), 6.89 (1H, d, J = 8.0 Hz, H-5); 13C NMR data, see Table 1; positive-ion ESIMS m/z 349 [M + Na]+; HRESIMS m/z 349.1399 (calcd for C20H22O4Na, 349.1410). Maceneolignan F (6): colorless oil; [α]27D +14 (c 1.1, CHCl3); UV (MeOH) λmax (log ε) 236 (sh) (4.16), 273 (3.49), 317 (3.50) nm; IR (film) νmax 3506, 1590, 1518, 1460,1424, 1329, 1219, 1123 cm−1; 1H NMR (CDCl3, 800 MHz) δ 1.11 (3H, d, J = 6.4 Hz, H3-9), 3.38 (2H, d, J = 6.9 Hz, H2-7′), 3.87 (6H, s, CH3O-3,5), 3.88 (6H, s, CH3O3′,5′), 4.31 (1H, dq, J = 3.0, 6.4 Hz, H-8), 4.78 (1H, d, J = 3.0 Hz, H7), [5.12 (1H, dd, J = 1.6, 10.0 Hz), 5.14 (1H, dd, J = 1.6, 17.0 Hz), H2-9′], 5.99 (1H, ddt, J = 10.0, 17.0, 6.9 Hz, H-8′), 6.47 (2H, s, H2′,6′), 6.55 (2H, s, H-2,6); 13C NMR data, see Table 1; EIMS m/z 404 [M+] (2), 210 (10), 194 (100); HREIMS m/z 404.1838 (calcd for C22H28O7, 404.1835). Maceneolignan G (7): colorless oil; [α]25D +22 (c 0.6, CHCl3); UV (MeOH) λmax (log ε) 230 (4.05), 281 (3.68), 318 (3.75) nm; IR (film) νmax 3441, 1682, 1670, 1582, 1520, 1460, 1427, 1334, 1223, 1134, 1038 cm−1; 1H NMR (CDCl3, 800 MHz) δ 1.15 (3H, d, J = 6.4 Hz, H3-9), 3.90 (3H, s, CH3O-3), 3.94 (6H, s, CH3O-3′,5′), 4.44 (1H,

dq, J = 2.9, 6.4 Hz, H-8), 4.79 (1H, d, J = 2.9 Hz, H-7), 6.66 (1H, dd, J = 7.6, 15.8 Hz, H-8′), 6.68 (1H, dd, J = 1.7, 8.1 Hz, H-6), 6.84 (1H, d, J = 8.1 Hz, H-5), 6.85 (2H, s, H-2′,6′), 6.97 (1H, d, J = 1.7 Hz, H-2), 7.42 (1H, d, J = 15.8 Hz, H-7′), 9.70 (1H, d, J = 7.6 Hz, H-9′); 13C NMR data, see Table 1; positive-ion EISMS m/z 411 [M + Na]+; HREIMS m/z 411.1408 (calcd for C21H24O7Na, 411.1414). Maceneolignan H (8): colorless oil; [α]24D −34 (c 1.5, CHCl3); UV (MeOH) λmax (log ε) 230 (sh) (4.17), 287 (3.50) nm; IR (film) νmax 1744, 1590, 1464, 1422, 1331, 1240, 1129, 1028 cm−1; 1H NMR (CDCl3, 800 MHz) δ 1.28 (3H, d, J = 6.4 Hz, H3-9), 2.17 (3H, s, CH3COO), 3.33 (2H, d, J = 7.2 Hz, H2-7′), 3.845 (3H, s, CH3O-4), 3.853 (3H, s, CH3O-3), 3.77 (6H, s, CH3O-3′,5′), 4.43 (1H, dq, J = 6.4, 3.2 Hz, H-8), [5.08 (1H, dd, J = 1.6, 13.6 Hz), 5.10 (1H, dd, J = 1.6, 16.8 Hz), H2-9′], 5.85 (1H, d, J = 3.2 Hz, H-7), 5.96 (1H, ddt, J = 13.6, 16.8, 7.2 Hz, H-8′), 6.39 (2H, s, H-2′,6′), 6.80 (1H, d, J = 8.8 Hz, H-5), 6.84 (1H, dd, J = 1.6, 8.8 Hz, H-6), 6.88 (1H, d, J = 1.6 Hz, H2); 13C NMR data, see Table 1; EIMS m/z 430 [M+] (3), 237 (66), 194 (100); HREIMS m/z 430.1982 (calcd for C24H30O7, 430.1991). Deacetylation of Maceneolignans C (3) and H (8). A solution of 3 (5.0 mg) in 0.5% NaOMe−MeOH (1.0 mL) was stirred at room temperature for 2 h. The reaction mixture was neutralized with Dowex HCR-W2 (H+ form), and the resins were removed by filtration. Evaporation of the solvent from the filtrate under reduced pressure gave fragransol-D52 (4.5 mg, quant.). Using a similar procedure, 1431 (2.8 mg, quant.) was obtained from 8 (3.1 mg). Methylation of Maceneolignan F (6). A solution of 6 (7.7 mg) and trimethylsilyldiazomethane (TMSCHN2, 10% in hexane, ca. 0.5 mL) in MeOH (1.0 mL) was stirred at room temperature for 1 h. Removal of the solvent under reduced pressure gave a residue, which was purified by HPLC [Cosmosil 5C18-MS-II, UV (254 nm), MeOH− 1% aqueous AcOH (65:35, v/v)] to give 930 (7.9 mg, 99%). Bioassays. Reagents. RPMI 1640 medium was purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA); bovine serum albumin (BSA) was from Seikagaku Co. (Tokyo, Japan); recombinant murine (rm) CCL2, CCL 3, CCL 11, CCL 20, CCL 21, and CCL 22 were from R&D Systems (Minneapolis, MN, USA); and other chemicals were from Wako Pure Chemical Industries, Co., Ltd. (Osaka, Japan) and Nacalai Tesque Inc. (Kyoto, Japan). Establishments of Chemokine Receptors Expressing L1.2 Cell Lines. The mouse pre-B cell line L1.2 was a kind gift provided by E. Butcher (Stanford University School of Medicine, Stanford, CA, USA). The panels of L1.2 transfectants stably expressing human chemokine receptors (CCR1−7) were generated using retroviral vector pMX-IRES-EGFP as described previously.58,59 L1.2 cells were infected with the recombinant viruses, and cells expressing the EGFP marker were sorted using FACSVantage SE (BD Biosciences, Mountain View, CA, USA). This study was approved by the ethical committee of Kindai University (Osaka, Japan, protocol number: KDPS-27-0001). Chemotaxis Assay. The chemotaxis assay was performed using a 96-well chemoTx chamber (Neuroprobe, Inc., Gaithersburg, MD, USA). Each corresponding chemoattractant [CCL3 for CCR1, CCL2 for CCR2, CCL11 for CCR3, CCL22 for CCR4, CCL3 for CCR5, CCL20 for CCR6, and CCL21 for CCR7 (final concentration, each 10 nM)] with or without a test sample was diluted in phenol red-free RPMI 1640 medium supplemented with 1 mg/mL BSA and placed in the lower wells (28 μL/well). Cells were suspended in phenol red-free RPMI 1640 medium containing 1% BSA at 8 × 106 cells/mL and added to the upper cells (25 μL/well), which were separated from the lower wells using a polyvinylpyrrolidone-free polycarbonate filter with 5 μm pores. The chamber was incubated for 1.5 h at 37 °C. The cells that migrated into the lower wells were lysed with 0.1% Triton X-100 (Wako, Osaka, Japan) and quantified using PicoGreen dsDNA reagent (Molecular Probes, Eugene, OR, USA). Each test sample was dissolved in DMSO, and the solution was added to the medium (final DMSO concentration, 0.5%). Commercially available CCR3 selective antagonist SB328437 was used as a reference compound. Statistical Analysis. Values are expressed as means ± SEM. Significant differences were calculated using a Student’s t-test or a G

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Dunnett’s test. Probability (p) values of less than 0.05 are considered significant.

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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.6b00262. Effects of the methanol extract of M. f ragrans arils on chemokine-induced chemotaxis in L1.2 cells expressing CCR1−7; stereostructures of 1−8 (PDF) NMR spectra of maceneolignans A−H (1−8) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +81 6 4307 4306. Fax: +81 6 6729 3577. E-mail: [email protected] (T. Morikawa). *Tel: +81 6 4307 4025. Fax: +81 6 6730 1394. E-mail: [email protected] (T. Nakayama). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2014−2018 (S1411037, T.M. and T.N.), and by a Grant-in-Aid for Scientific Research [KAKENHI, Grant Numbers 15K08008 (T.M.), 15K08009 (K.N.), and 16K08313 (O.M.)]. Thanks are also due to Kobayashi International Scholarship Foundation for the financial support (T.M.).

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