Weakly Anti-inflammatory Limonoids from the Seeds of Xylocarpus

John W. Blunt , Brent R. Copp , Robert A. Keyzers , Murray H. G. Munro ... Souvik Kusari , Dennis Eckelmann , Abraham Yeboah Mensah , Ferdinand M. Tal...
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Weakly Anti-inflammatory Limonoids from the Seeds of Xylocarpus rumphii Chanin Sarigaputi,† Damrong Sommit,‡ Thapong Teerawatananond,§ and Khanitha Pudhom*,§ †

Program in Biotechnology and §Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand ‡ Department of Chemistry, Faculty of Science, Mahanakorn University, Bangkok 10530, Thailand S Supporting Information *

ABSTRACT: Seven new limonoids, namely, xylorumphiins E−J (1−2 and 4−7) and 2-hydroxyxylorumphiin F (3), along with three known derivatives (8−10), were isolated from the seeds of Xylocarpus rumphii. 2-Hydroxyxylorumphiin F (3) and xylorumphiin I (6) displayed moderate inhibitory activity against nitric oxide production from lipopolysaccharide-activated macrophages with IC50 values of 24.5 and 31.3 μM, respectively.

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3), it was clear that six of the 12 indices of hydrogen deficiency came from two carbon−carbon double bonds (furan ring) and four ester carbonyl carbons. Therefore, it required six additional rings in the structure of 1. The NMR data of 1 and its 2D correlations (Figure 1) indicated the presence of four tertiary methyls [δH 0.75 s, 1.04 s (×2), and 1.19 s; δC 21.0, 22.1, 22.2, and 24.6], a typical β-substituted furan moiety [δH 6.39, 7.39, and 7.54 s; δ C 110.0, 121.0, 141.5, and 142.9], a methoxycarbonyl (δH 3.70 s; δC 51.9 and 173.9), and two isobutyryl groups [δH 1.04 br s (6H), 1.10 d (J = 6.8 Hz), 1.21 d (J = 6.8 Hz), 2.53 m, and 2.66 m; δC 17.5, 18.3, 19.1, 20.0, 33.3, 33.9, 174.9, and 177.0]. The aforementioned data strongly suggested that 1 was a mexicanolide-type limonoid. The NMR data of 1 were similar to those of xylorumphiin A,11 except for the presence of a methine (δH 2.61 m; δC 57.1) in place of an oxygenated carbon in xylorumphiin A. The methine functionality was assigned to C-2 by the 1H−1H COSY correlations from its proton at δH 2.61 to H-3 and H-30 and by its HMBC correlations to C-1, C-3, and C-30 (Figure 1a). The quaternary carbon resonance at δC 106.7 was assigned to C-1, a hemiacetal carbon, by its HMBC correlation with a proton resonance at δH 3.57 (1-OH) that did not show correlation with any carbon in the HSQC spectrum. The HMBC correlations from H-3 [δH 5.09 d (J = 9.2 Hz)] and H-30 (δH 6.17 br s) to the carbonyl

esearch on limonoids is of growing interest due to the range of biological activities, such as insect antifeedant, growth regulation, antibacterial, antifungal, antimalarial, anticancer, and antiviral activities.1−3 The mangroves of the genus Xylocarpus are known to produce a large number of limonoids, particularly mexicanolides and phragmalins.4−8 In our continuing search for new biologically active limonoids from the plants in this genus, we have reported the isolation of a number of limonoids from the seed kernels of all three species X. granatum, X. moluccensis, and X. rumphii, collected from several areas of the mangrove forests in Thailand.9−13 In this study, we report the isolation and structural determination of six new mexicanolides (1−6) and a new phragmalin (7), along with three known limonoids, xyloccensins X, E, and K (8−10), from the seeds of X. rumphii collected from Kudee Island, Thailand. The structures of these compounds were established via spectroscopic data or by comparison with literature data.9−14 Their anti-inflammatory activities were also evaluated via suppression of nitric oxide (NO) production in activated macrophages.

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RESULTS AND DISCUSSION Xylorumphiin E (1), a white, amorphous powder, had the molecular formula C35H48O11, as established by 13C NMR data and an HRESIMS m/z 667.2996 [M + Na]+ ion (calcd 667.3089), corresponding to 12 indices of hydrogen deficiency. From the 1H and 13C NMR spectroscopic data (Tables 1 and © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 29, 2014

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dx.doi.org/10.1021/np5003687 | J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Spectroscopic Data of 1−4a

position

3

4

(mult., J in Hz)

(mult., J in Hz)

2.61, 5.09, 2.59, 2.32,

9

2.28, br s 1.44, d (12.4)

2.25, d (16.4) 1.42, m

11

1.92, d (14.0)

1.92, dd (3.6, 13.2) 1.66, m 1.83, d (16.4) 1.31, m 2.19, d (9.6)

14 15

17 18 19 21 22 23 28 29 30 7-OMe 1-OH 2-OH 3-Acyl 2′ 3′ 4′ 5′ 30-Acyl 2″ 3″ 4″ 5″ a

2 (mult., J in Hz)

2 3 5 6

12

carbons (C-1′ and C-1″) of the isobutyryl groups confirmed their location at C-3 and C-30, respectively. The relative configuration of 1 was established by NOE interactions (Figure 1b). The NOESY spectrum showed close similarity to reported NOE data of xylorumphiin A.11 The NOE cross-peaks observed from H-3 to H-2 and H3-29 indicated an α-orientation of these protons and hence a 3β-isobutyryl group, whereas the lack of an interaction from H-3 to H-5 indicated a β-orientation of H-5. The α-orientation of the 30-isobutyryl group was deduced by the NOE cross-peak between H-5 and H-30, without the interactions between H-3/H-30 and/or H-2/H-30. On the basis of the above results, the structure of 1 was elucidated as shown. Xylorumphiin F (2), a white, amorphous powder, gave a molecular formula of C36H50O11 as determined by 13C NMR data and an HRESIMS ion at m/z 657.3270 [M − H]− (calcd 657.3269). The MS and NMR data suggested the presence of an additional −CH2− unit in 2 compared to xyrolumphiin E (1).11 The NMR spectroscopic data of 2 (Tables 1 and 3) were similar to those of 1 except for the replacement of an isobutyryl group in 1 by a 2-methylbutyryl group at C-30. The existence of the 2-methylbutyryl group was confirmed by 1H−1H COSY correlations between H3-5″/H-2″, H-2″/H2-3″, and H2-3″/H34″. The HMBC correlation from H-30 (δH 6.17 br s) to the carbonyl carbon (δC 174.9) of the 2-methylbutyryl group defined its location at C-30. The absolute configuration at C-2″ in the 2-methylbutyryl group could be determined according to the specific rotation of the corresponding acid derived from the alkaline hydrolysis of 2 ([α]D −14.3 for (R)-2-methylbutyric acid and [α]D +19.2 for (S)-2-methylbutyric acid).15,16 Although a 1:1 mixture with isobutanoic acid was obtained from the hydrolysis, isobutanoic acid is optically active. Therefore, the absolute configuration at C-2″ in the 2methylbutyryl group was assigned as S from the [α]20D value of +10 (c 0.05, MeOH) of this mixture. Both compounds 1 and 2 shared the same relative configuration, as confirmed by similar NOE correlations. 2-Hydroxyxylorumphiin F (3), a white, amorphous powder, was assigned a molecular formula of C36H50O12 by 13C NMR

1 (mult., J in Hz)

1.67, 1.83, 1.33, 2.19,

m d (9.2) m d (10.0)

m d (16.4) m d (10.0)

2.59, 5.11, 2.61, 2.33,

m d (9.6) m d (9.6)

4.89, s 2.64, m 2.37, dd (9.6, 16.4) 2.27, br s 1.51, m 1.89, m 1.69, m 1.83, m 1.35, m 2.75, dd (9.2, 19.6) 2.22, d (9.2) 3.13, d (19.6)

3.25, d (20.0) 2.73, dd (10.0, 20.0) 5.26, s 1.04, br s 1.04, br s 7.54, s 6.39, s 7.39, s 0.75, s 1.19, s 6.17, br s 3.70, s 3.57, s

3.26, d (20.0) 2.73, dd (9.6, 20.0) 5.25, s 1.05, s 1.04, s 7.53, s 6.39, s 7.39, s 0.75, s 1.19, s 6.17, br s 3.70, s

2.53, m 1.10, d (6.8)

2.65, m 1.11, d (6.8)

2.61, m 1.09, d (6.8)

1.21, d (6.8)

1.21 d (6.8)

1.22, d (6.8)

2.66, m 1.04, br s

2.41, 1.66, 1.42, 0.90, 1.20,

2.43, 1.67, 1.28, 0.91, 1.08,

1.04, br s

m m m t (7.6) d (7.2)

5.18, 1.03, 1.12, 7.55, 6.40, 7.40, 0.73, 1.25, 6.23, 3.21, 4.21, 3.21,

s s s s s s s s s s s s

m m m t (7.6) d (6.4)

4.89, s 2.64, m 2.35, m 2.24, m 1.48, dd (2.8, 12.8) 1.89, m 1.69, 1.85, 1.33, 2.22,

m m m d (9.6)

3.15, d (20.0) 2.74, dd (9.6, 20.0) 5.18, s 1.05, s 1.12, s 7.53, s 6.39, s 7.40, s 0.73, s 1.25, s 6.24, d (5.2) 3.69, s 4.25, s

2.35, 1.41, 1.65, 0.90, 1.21,

m m m t (7.6) d (7.2)

2.45, 1.28, 1.67, 0.91, 1.08,

m m m t (7.6) d (7.2)

Data were measured in CDCl3 at 400 MHz.

data and an HRESIMS m/z 697.3169 [M + Na]+ ion (calcd 697.3194). Its 1H and 13C NMR data (Tables 1 and 3) closely resembled those of 2, except for the presence of an oxygenated tertiary carbon (δC 82.1) instead of a methine group in 2. This carbon was assigned to C-2 due to its HMBC correlation with H-3 (δH 4.89 s). A proton resonance at δH 3.21 that did not show correlation with any carbon in the HSQC spectrum was assigned to 2-OH by its HMBC correlations to C-1, C-2, C-3, and C-30 (Figure S1a). Similar NOE correlations suggested that compound 3 possessed the same relative configuration as those of 1 and 2. The key NOE cross-peak observed in 3 from 2-OH to H-3, along with the lack of correlation from 2-OH to H-30, confirmed the α-orientation of 2-OH (Figure S1b). The absolute configuration at C-2″ in the 2-methylbutyryl group B

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Table 2. 1H NMR Spectroscopic Data of 5−8a 5

6

position

(mult., J in Hz)

(mult., J in Hz)

2 3 5

4.85, s 2.61, m

6

9 11

12 14 15

17 18 19 21 22 23 28 29 30 32 7-OMe 1-OH 2-OH 15-OH 3-Acyl 2′ 3′

2.36, dd (9.2, 16.4) 2.25, d (16.4)

4″ 5″ 2-Acyl 2‴ a

2.35, m

1.48, dd (2.4, 12.8) 1.90, dd (3.2, 13.2) 1.69, m 1.83, m 1.33, m 2.21, d (9.2) 3.11, d (19.6) 2.74, d (9.2, 19.6) 5.21, s 1.05, s 1.12, s 7.55, s 6.39, s 7.40, s 0.72, s 1.24, s

2.20, m

6.19, s

5.61, s

3.70, s 4.25, s 3.27, s

3.69, s 4.52, s 3.90, s

8

(mult., J in Hz)

(mult., J in Hz)

5.11, s 2.99, d (10.0) 2.46, dd (10.0, 16.4) 2.24 m

4.80, s 2.66, br d (9.2) 2.36, m 2.16, d (15.2) 2.21, d (10.0) 2.34, m

2.35, m

2.06, m

1.81, m 2.04, m 1.40, m

1.67, 1.51, 1.20, 2.09, 5.06,

m m m d (2.4) br s

1.80, m 2.05, m 1.39, m

5.58, 1.05, 1.16, 7.56, 6.48, 7.42, 0.89, 1.98, 1.68, 6.32, 1.73, 3.68,

s s s s s s s d (11.2) d (11.2) s s s

4.91, 1.21, 1.14, 7.48, 6.41, 7.41, 0.79, 1.31,

6.01, s

4.92, 1.22, 1.14, 7.48, 6.41, 7.41, 0.78, 1.30,

s s s s s s s s

6.07, s

s s s s s s s s

5.58, s 3.68, s 4.66, s 4.13, s

3.12, br s 2.07, 5

2.30, m 1.66, m 1.40, m 0.90, t (7.2) 1.15, d (7.2)

2.26, s

2.29, 1.65, 1.40, 0.90, 1.14,

2.65, m 1.09, d (6.4)

2.30, m 1.66, m 1.40, m 0.92, t (7.6) 1.10, d (7.2)

1.94, s

2.54, m 1.12, d (7.2)

4′ 5′ 30-Acyl 2″ 3″

4.83, s 2.66, d (8.8) 2.18, m

7

and H-30 to the respective carbonyl carbon of the 2methylbutyryl groups placed these acyl groups at C-3 and C30. A proton at δH 4.25, which did not display correlation with a carbon in the HSQC spectrum, was assigned to 1-OH; the second 2-methylbutyryl group was thus assumed to be attached at C-3. In order to clarify the full structure and establish the relative configuration of 4, single-crystal X-ray diffraction analysis was utilized. Thus, the 2-methylbutyryl groups at C-3 and C-30 were α-oriented (Figure 2). The absolute configurations at C-2′ and C-2″ in the respective 2methylbutyryl groups were determined as S due to the [α]20D value of +15 (c 0.03, MeOH) of the corresponding acid obtained from alkaline hydrolysis. Xylorumphiin H (5), a white, amorphous powder, had a molecular formula of C33H44O12 as determined by 13C NMR data and HRESIMS based on the molecular ion m/z 631.2781 [M − H]− (calcd 631.2749). The NMR spectroscopic data of 5 (Tables 2 and 3) were similar to those of xylorumphiin A,11 with the only difference being the presence of an acetyl rather than an isobutyryl group at C-3. This deduction was validated by the HMBC correlation from H-3 to the acetyl carbonyl (δC 171.3). The relative configuration of 5 was identical to that of xylorumphiin A based on analysis of the NOESY data. Xylorumphiin I (6) was obtained as a white, amorphous powder. It gave a molecular formula of C37H50O12 as established by 13C NMR and an HRESIMS ion at m/z 685.3266 [M − H]− (calcd 685.3219). The NMR spectroscopic data of 6 (Tables 2 and 3) were similar to those of 4, except for the presence of a Δ14,15 double bond (δH 6.01 s; δC 118.3 and 158.6). The location of the Δ14,15 double bond was corroborated by HMBC correlations from H-15 to C-8 and C16 (Figure 3). The presence of two 2-methylbutyryl groups was confirmed by 1H−1H COSY correlations. Their positions at C3 and C-30 were validated by the HMBC correlations from H-3 and H-30 to the respective 2-methylbutyryl carbonyls, C-1′ and C-1″. The absolute configurations at C-2′ and C-2″ in the respective 2-methylbutyryl groups were assigned as S from the [α]20D value of +18 (c 0.05, MeOH) of the corresponding acid. The similar NOE correlations suggested that compound 6 possessed the same relative configuration as those of 3−5. Xylorumphiin J (7), a white solid, had the molecular formula C35H42O15 as established by 13C NMR and HRESIMS (m/z 701.2471 [M − H]−, calcd 701.2440) data. Analysis of 1D and 2D NMR spectroscopic data revealed that 7 was a phragmalin ortho ester. The existence of an ortho acetate group was characterized by a methyl singlet at δH 1.73, showing an HMBC correlation with a quaternary carbon at δC 119.4. This was further supported by the presence of three oxygenated tertiary carbons at δC 85.4, 85.1 and 86.8, assigned as C-1, C-8, and C9, respectively, by HMBC correlations of H-14/C-8, H-30/C-8, H3-19/C-1, H3-19/C-9, and H2-29/C-1 (Figure 4a). These data suggested that 7 was a phragmalin 1,8,9-ortho acetate. The NMR data of 7 (Tables 2 and 3) were similar to those of xyloccensin E,13 except for the presence of an oxygenated methine (δH 5.06 br s; δC 64.2) instead of the C-15 methylene group in xyloccensin E. HMBC cross-peaks from a hydroxy proton at δH 3.12, which was not correlated with any carbon in the HSQC spectrum, to C-14, C-15, and C-16 confirmed the location of this hydroxy group at C-15. Similar NOE correlations suggested that compound 7 possessed the same relative configuration as xyloccensin E.13 The α-orientation of H-15 was established by its NOE correlation with H-14 (Figure 4b).

1.11, d (6.4)

m m m t (7.2) d (6.8)

1.15, d (6.8)

2.16, s

Data were measured in CDCl3 at 400 MHz.

was determined as S, the same as that in 2, from the [α]20D value of +12 (c 0.05, MeOH) of the corresponding acid. Xylorumphiin G (4) was obtained as colorless crystals. The molecular formula C37H52O12 was assigned by 13C NMR and an HRESIMS ion at m/z 687.3390 [M − H]− (calcd 687.3381). The NMR data (Tables 1 and 3) indicated that 4 was similar to 3, except for the presence of a 2-methylbutyryl group in place of the isobutyryl group in 3. The HMBC correlations from H-3 C

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Table 3. 13C NMR Spectroscopic Data of 1−8a

a

position

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 31 32 7-OMe 3-Acyl 1′ 2′ 3′ 4′ 5′ 30-Acyl 1″ 2″ 3″ 4″ 5″ 2-Acyl 1‴ 2‴

106.7 57.1 73.4 38.1 40.5 32.5 173.9 82.4 64.4 43.7 19.6 35.9 36.3 46.6 29.0 169.9 77.1 21.0 22.2 121.0 141.5 110.0 142.9 24.6 22.1 75.9

106.6 57.1 73.5 38.0 40.4 32.5 173.9 82.3 63.4 43.7 19.6 35.9 36.3 46.6 29.0 170.0 77.1 22.2 21.0 121.0 141.5 110.0 143.0 22.1 24.6 75.8

107.2 82.1 80.3 38.9 40.3 32.3 173.9 81.1 63.3 42.6 19.7 35.8 36.3 46.5 29.0 169.6 77.2 22.2 21.0 120.8 141.6 109.9 143.1 24.2 22.0 76.7

107.2 82.1 80.3 38.9 40.4 32.3 173.8 81.1 63.3 42.6 19.7 35.9 36.3 46.5 29.1 169.5 77.0 22.1 21.0 120.9 141.5 109.3 143.1 24.2 22.0 75.6

107.1 82.2 80.5 38.7 40.3 32.3 173.9 81.0 63.3 42.6 19.7 35.9 36.2 46.4 29.0 169.5 77.1 22.2 21.0 120.8 141.6 110.0 143.0 23.8 21.9 75.6

108.4 80.9 82.4 38.7 40.3 31.9 173.7 80.4 51.5 42.2 15.1 25.1 38.9 158.6 118.3 163.1 81.2 19.6 20.6 119.9 141.3 109.9 142.9 24.5 21.7 75.4

108.5 81.0 82.6 38.7 40.3 31.9 173.7 80.4 51.5 42.3 15.1 25.0 38.9 158.5 118.4 163.1 81.3 19.6 42.3 120.0 141.3 109.9 142.9 24.5 21.7 75.4

51.9

51.9

52.0

51.9

51.9

51.9

85.4 86.0 81.1 46.2 35.5 33.3 172.6 85.1 86.8 45.8 25.5 28.8 36.2 53.0 64.2 174.2 78.8 19.2 16.7 120.5 141.0 109.6 143.2 14.6 40.1 69.8 119.4 20.9 52.0

177.0 33.3 18.3 20.0

177.2 33.9 18.3 20.0

177.6 33.9 18.2 20.2

177.1 40.5 16.8 25.3 11.2

171.3 21.3

176.7 40.9 26.1 11.4 16.2

170.0 21.0

176.8 41.0 26.1 11.4 16.1

174.9 33.9 19.1 17.5

174.9 40.6 25.3 11.2 16.4

174.3 40.7 27.2 12.0 14.9

174.1 40.6 27.0 11.9 14.8

174.5 34.0 17.9 19.5

174.7 40.9 26.4 11.6 15.7

168.2 21.0

175.2 34.3 18.8 19.0

51.9

170.4 21.7

Data were measured in CDCl3 at 100 MHz.

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Xyloccensin X (8) was obtained as colorless crystals, mp 214−216 °C. Compound 8 was first reported in 2006 as a mixture with xyloccensin Y.17 In the current study, the 1H and 13 C NMR data for compound 8 (Tables 2 and 3) were assigned unambiguously. All isolated compounds were evaluated for their antiinflammatory activities by monitoring the inhibition of LPSinduced NO production in J774.A1 macrophages. Only 2hydroxyxylorumphiin F (3) and xylorumphiin J (6) exhibited moderate anti-inflammatory activities, with IC50 values of 24.5 and 31.3 μM, respectively, while the remaining compounds did not show any significant effects at a dose of 50 μM.

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Stuart Scientific melting point apparatus and are uncorrected. Optical rotations were measured on a PerkinElmer 341 polarimeter at 20 °C. UV spectra were recorded on a Shimadzu UV160 UV−visible spectrometer. IR spectra were measured on a Nicolet 6700 FT-IR spectrophotometer. NMR spectra were acquired on a Varian Mercury-400 Plus NMR spectrometer with TMS as internal standard. HRESIMS was carried out on a micrOTOF-Q II ESI mass spectrometer. Single-crystal X-ray diffraction analysis was performed on an Oxford Gemini S Ultra diffractometer. Plant Material. The fruits of X. rumphii were collected in February 2011 from Kudee Island, Thailand, and authenticated by the Royal Forest Department, Bangkok, Thailand. A voucher specimen (BKF D

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Figure 1. (a) Selected HMBC and COSY correlations of 1. (b) Diagnostic NOE correlations of 1. No. 1638) was deposited at the Forest Herbarium, Royal Forest Department, Bangkok, Thailand. Extraction and Isolation. The air-dried, powdered seeds of X. rumphii (5 kg) were extracted three times with MeOH (10 L, each for 3 days) at room temperature. The extract was concentrated under reduced pressure. The combined MeOH extract was partitioned between EtOAc and H2O to obtain the EtOAc crude extract (107 g). The EtOAc extract was fractionated over a column of silica gel with a gradient of EtOAc−n-hexane (from 1:9 to 1:0) to give 14 fractions, A−N. Fraction G (7.84 g) was subjected to passage over a column of Sephadex LH20 (MeOH) to afford four fractions (G1−G4), and fraction G2 (376.31 mg) was rechromatographed over silica gel eluting with an EtOAc−n-hexane gradient (from 3:7 to 1:1) to yield seven subfractions (G2a−G2g). Fraction G2a (47.9 mg) was purified by silica gel CC (EtOAc−n-hexane, from 2:3 to 1:1) to give 4 (4.3 mg). Fraction G2e (74.6 mg) was chromatographed over a silica gel column with MeOH−CH2Cl2 (2:98), and the major fraction (G2e.4) was rechromatographed with EtOAc−n-hexane (from 3:7 to 1:1) to obtain compounds 6 (7.7 mg) and 8 (19.3 mg). Fraction G2f (22.0 mg) was purified by CC over silica gel with EtOAc−n-hexane (2:3) to give compounds 1 (2.9 mg) and 3 (11.3 mg). Using the same procedure, fraction G2g (19.7 mg) yielded compound 2 (11.2 mg). Fraction J was recrystallized from MeOH to give xyloccensin E (9, 178.8 mg). Fraction K (3.44 g) was subjected to a column of Sephadex LH-20 with MeOH to afford six fractions, K1−K6. Fraction K2 (115.6 mg) was further purified by silica gel CC eluted with MeOH−CH2Cl2 (4:96) to yield compounds 5 (7.6 mg) and 7 (9.9 mg). Fraction K4 was recrystallized from MeOH to afford xyloccensin K (10, 85.3 mg). Xylorumphiin E (1): white, amorphous powder; [α]20D −13 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 209 (3.24) nm; IR (neat) νmax 3370 (br), 2974, 2933, 2878, 1715, 1453, 1377, 1295, 1264, 1195,

Figure 2. ORTEP drawing of 4.

Figure 3. Selected HMBC and COSY correlations of 6.

Figure 4. (a) Selected HMBC and COSY correlations of 7. (b) Diagnostic NOE correlations of 7. E

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1143, 1061 cm−1; 1H and 13C NMR spectroscopic data (see Tables 1 and 3); HRESIMS m/z 667.2996 [M + Na]+ (calcd for C35H48O11Na, 667.3089). Xylorumphiin F (2): white, amorphous powder; [α]20D +18 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.50) nm; IR (neat) νmax 3424 (br), 2970, 2928, 2863, 1726, 1461, 1383, 1302, 1189, 1140, 1059 cm−1; 1H and 13C NMR spectroscopic data (see Tables 1 and 3); HRESIMS m/z 657.3270 [M − H]− (calcd for C36H49O11, 657.3269). 2-Hydroxyxylorumphiin F (3): white, amorphous powder; [α]20D −19 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (3.61) nm; IR (neat) νmax 3404, 2970, 2934, 2876, 1720, 1454, 1379, 1392, 1227, 1143, 1062 cm−1; 1H and 13C NMR spectroscopic data (see Tables 1 and 3); HRESIMS m/z 697.3169 [M + Na]+ (calcd for C36H50O12Na, 697.3194). Xylorumphiin G (4): colorless crystals (MeOH); mp 162−164 °C; [α]20D −68 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.51) nm; IR (neat) νmax 3410 (br), 2967, 2924, 2883, 1720, 1457, 1377, 1292, 1189, 1147, 1066 cm−1; 1H and 13C NMR spectroscopic data (see Tables 1 and 3); HRESIMS m/z 687.3390 [M − H]− (calcd for C37H51O12, 687.3381). Xylorumphiin H (5): white, amorphous powder; [α]20D +12 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (3.64) nm; IR (neat) νmax 3440 (br), 2967, 2931, 2883, 1717, 1461, 1370, 1260, 1224, 1137, 1014 cm−1; 1H and 13C NMR spectroscopic data (see Tables 2 and 3); HRESIMS m/z 631.2781 [M − H]− (calcd for C33H43O12, 631.2749). Xylorumphiin I (6): white, amorphous powder; [α]20D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (4.16) nm; IR (neat) νmax 3375 (br), 2973, 2934, 2876, 1720, 1461, 1377, 1289, 1260, 1143, 1062 cm−1; 1H and 13C NMR spectroscopic data (see Tables 2 and 3); HRESIMS m/z 685.3266 [M − H]− (calcd for C37H49O12, 685.3319). Xylorumphiin J (7): white solid; decomposed at 238 °C; [α]20D −7 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (3.64) nm; IR (neat) νmax 3492, 2957, 2924, 2850, 1730, 1460, 1428, 1370, 1311, 1240, 1088 cm−1; 1H and 13C NMR spectroscopic data (see Tables 2 and 3); HRESIMS m/z 701.2471 [M − H]− (calcd for C35H41O15, 701.2440). X-ray Crystallographic Analysis of 4. The single-crystal X-ray diffraction data were collected at 296 K on a Bruker APEX-II CCD diffractometer with Mo Kα radiation (λ = 0.710 73 Å). The crystal structure was solved by direct methods using SHELXS-9718 and refined with full-matrix least-squares on all F2 data using SHELXS-97 to final R values.19 All non-hydrogen atoms were anisotropically refined, except for the oxygen C-3A atom, which was refined isotropically. The C-3A atom is disordered over two positions with site occupancies of 0.52 and 0.48. All hydrogen atoms were added at calculated positions and refined using a rigid model, with C−H = 0.93 Å (aromatic), 0.97 Å (CH2), and 0.98 Å (CH3) and O−H = 0.82 Å. Crystallographic data for 4 have been deposited with the Cambridge Crystallographic Data Centre under the deposition number CCDC 998571. Crystal data of 4: colorless crystal; C37H50O12, Mr = 688.80, prism, a = 12.1699(10) Å, b = 12.3165(16) Å, c = 24.075(3) Å, space group P212121, Z = 4, V = 3608.6(7) Å3, μ(Mo Kα) = 0.07 mm−1, and F(000) = 1200. Crystal dimensions: 0.38 × 0.22 × 0.20 mm3. Independent reflections: 3394 (Rint = 0.035). Absolute Configuration of C-2 of the 2-Methylbutyryl Group of Xylorumphiins F, G, and I (2, 4, and 6) and 2Hydroxyxylorumphiin F (3). Each compound (3 mg) was dissolved in EtOH (0.5 mL) and treated with 10% aqueous KOH solution (1 mL). After stirring overnight, the mixture was concentrated and washed with EtOAc (×3), and the aqueous layer was acidified with HCl to pH 3.0 and extracted with CH2Cl2 (×3). The combined organic layer was subjected to Sephadex LH-20 CC (CH2Cl2−MeOH, 1:1) to yield 2S-methylbutanoic acid, [α]20D +10, +12, +15, and +18, for compounds 2−4 and 6, respectively, lit. [α]25D +19.2.16 Anti-inflammatory Bioassay. The inhibitory effects of samples on NO production were evaluated in LPS-activated murine macrophage J774.A1 cells according to a reported method.20 The cell lines were seeded in 24-well plates with 1 × 105 cells/well and allowed to adhere for 24 h at 37 °C in 5% CO2. The cells were pretreated with various concentrations of test compounds or vehicle (DMSO) for 2 h

and then activated with 1 μg/mL of LPS for 20 h. Indomethacin (IC50 = 28.56 μM) was used as a positive control. The culture supernatant (50 μL) of each well was collected, and the concentration of NO was further measured by using Griess reagent. The absorbance was measured at 540 nm with a microplate reader. The nitrite level in the samples was calculated from the standard curve created from known concentrations of sodium nitrite.

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ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra of compounds 1−7 and CIF file for compound 4. These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +66-2-2187641. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support from Thailand Research Fund and Chulalongkorn University through the Royal Golden Jubilee Ph.D. Program (PHD/0009/2553) and the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endownment Fund). This research has also been partially supported by the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (RES560530208-AS).

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