Sesquiterpenoids from the Mangrove-Derived Endophytic Fungus

Sep 24, 2012 - Department of Chemistry, University of Malaya, Kuala Lumpur 50603, Malaysia ... known compounds 10 and 11 were isolated from a culture ...
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Sesquiterpenoids from the Mangrove-Derived Endophytic Fungus Diaporthe sp. Le Yun Zang,† Wei Wei,† Ye Guo,† Ting Wang,† Rui Hua Jiao,† Seik Weng Ng,‡,§ Ren Xiang Tan,† and Hui Ming Ge*,† †

Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, School of Lifescience, Nanjing University, Nanjing 210093, People's Republic of China ‡ Department of Chemistry, University of Malaya, Kuala Lumpur 50603, Malaysia § Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah 80203, Saudi Arabia S Supporting Information *

ABSTRACT: A new sesquiterpenoid, diaporol A (1), possessing a unique tricyclic lactone framework, eight new drimane sesquiterpenoids, diaporols B−I (2−9), and the known compounds 10 and 11 were isolated from a culture of the mangrove-derived endophyte Diaporthe sp. The absolute configurations of 1−5 were determined by low-temperature (100 K) single-crystal X-ray diffraction with Cu Kα radiation. The compounds were evaluated for cytotoxic activity; however, no compound showed significant cytotoxicity against the tested cell lines at a concentration of 20 μM.

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angroves, a consortium of higher plants that inhabit the intertidal zones in subtropical and tropical climates, are important ecologically as fish nurseries, pollution sinks, shoreline barriers, bird sanctuaries, and wildlife refuges.1−3 Mangrove-derived fungi have attracted much attention due to their unique habitat in tidal mudflats, where they are exposed to wave energy, high moisture levels, high salt concentration, and lack of oxygen.2 They are also considered a rich source of evolutionary preselected lead compounds that are indispensable for drug development.4−6 As part of our ongoing search for novel bioactive compounds from microorganisms isolated from specialized niches,7−9 we focused on a strain of fungus that was isolated from the healthy leaves of Rhizophora stylosa, one of the most easily recognized mangroves because of its distinctive root system and widespread distribution in Hainan Island (China). The fungus was subsequently identified as Diaporthe sp. by analysis of its morphological characteristics and 18S rDNA sequence. The fungus was grown in a solid-substrate fermentation culture. Fractionation of the ethanol extract afforded nine new sesquiterpenoids, namely, the diaporols (1− 9), and the known compounds 3β-hydroxyconfertifolin (10)10 and diplodiatoxin (11).11 Compound 1 featured a new tricyclic framework, which could be derived from the known compound 3β-hydroxyconfertifolin (10) via a tandem oxidation and esterification rearrangement. Herein, we report on the isolation, structure elucidation, and biological activity screening of these sesquiterpenoids.

HRESIMS spectrum, indicating six indices of hydrogen deficiency. The 13C NMR spectrum (Table 1) disclosed two ester carbonyl (δ 172.9, C-2 and δ 170.4, C-7) and two vinyl carbon signals (δ 166.2, C-11 and δ 125.0, C-3), accounting for three of the indices of hydrogen deficiency. The three remaining indices were attributed to three ring systems. The HMBC correlations of H-1/C-2, C-3, and C-11 suggested the presence of a γ-lactone moiety. The 1H−1H COSY data indicated the isolated H2-4/H2-5 and H2-8/H2-9 proton spin systems. The HMBC correlations of H-5/C-3, C-10 and H-12/ C-6, C-10, C-11 indicated the presence of a cyclohexene ring

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RESULTS AND DISCUSSION Diaporol A (1), obtained as colorless crystals, was assigned the molecular formula C15H20O5 according to an [M + Na]+ ion at m/z 303.1204 (calcd for C15H20O5Na, 303.1203) in its © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 13, 2012

A

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Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data for 1−4 1a no.

a

δC

δH (J in Hz) 4.96, dt (17.3, 2.6) 4.86, dd (17.3, 2.4)

2a δC

3a

δH (J in Hz)

1

68.7

2

172.9

33.4

3

125.0

215.9

35.3

4

16.8

52.6

38.2

5 6

26.6 89.7

7

170.4

8 9

26.8 29.1

10 11

39.5 166.2

12 13 14

25.3 77.9 29.1

1.44, s 1.29, s

24.1 18.8 23.0

15 5-OH 11-OH 12-OH 13-OH 14-OH

27.7

1.31, s

22.0

2.02, m 2.16, br d (15.5) 1.90, m

33.2

77.8 24.9 36.5

2.36, dd (5.0, 9.5) 1.56, dt (13.5, 5.0) 2.96, dt (13.5, 9.5)

71.8 52.5 40.6 59.3

1.58, 1.98, 2.34, 2.45,

δC

1.70, 1.43, 1.37, 1.94,

ddd (3.5, 8.0, 13.0) mc ddd (3.5, 8.0, 13.0) m

td dt dt td

(13.0, (13.0, (13.0, (13.0,

17.9

56.3 19.9

3.5) 3.5) 3.5) 3.5)c

44.2 72.2 60.8

2.17, dd (4.0, 8.0)

3.57, 3.77, 1.15, 0.92, 1.04,

40.0

36.9 58.9

dd (4.0, 11.0) dd (8.0, 11.0) s s s

24.2 16.1 62.7

0.98, s 4.35, s

27.5

1.01, 1.71, 1.31, 1.48, 0.81, 1.73,

4b

δH (J in Hz)

δC

δH (J in Hz)

td (13.0, 3.5) mc mc m td (13.5, 4.0) mc

55.5

2.48, dd (2.0, 12.5) 2.25, d (12.5)

210.7 56.0

2.18, dd (2.0, 13.5) 2.32, d (13.5)

38.4 0.98, 1.24, 1.59, 1.35, 1.67,

dd (1.5, 12.0)c m m td (12.5, 3.5)c dt (12.5, 3.0)

43.7 74.5 59.7

1.31, mc

3.61, 3.72, 1.09, 0.73, 3.15, 3.43, 0.86,

55.2 20.3

m ddd (3.5, 7.5, 10.0) s s dd (5.0, 10.5) dd (5.0, 10.5) s

42.4 60.7

1.58, 1.38, 1.80, 1.63, 1.96,

dd (2.5, 10.0) dt (2.5, 13.5) mc dt (4.0, 13.5) td (3.5, 13.5)

1.77, dd (3.5, 10.0)

24.1 17.2 33.6

3.79, 3.95, 1.35, 0.81, 1.08,

dd (3.5, 11.0) dd (10.0, 11.0) s s s

23.2

0.87, s

4.59, dd (3.5, 5.0) 4.73, s 5.01, s 4.11, t (5.0) b

c

Recorded in DMSO-d6. Recorded in CDCl3. Overlapped signals.

carbonyl group, the remaining indices required 2 to contain two saturated rings. The 1H and 13C NMR data of 2 (Table 1) implied that 2 was a drimane sesquiterpenoid.8,9 The H2-1/H22, H2-6/H2-7, and H-9/H2-11 isolated proton spin systems were deduced from the 1H−1H COSY spectrum. The HMBC correlations between H-1/C-10; H-2/C-3; H-7/C-5; and H-9/ C-8, C-10, together with the chemical shift values of C-3 (δ 215.9), C-5 (δ 77.8), and C-8 (δ 71.8), suggested the presence of an oxygenated bicyclic ring. In addition, the HMBC correlations of H-14/C-3, C-4, C-5; H-15/C-3, C-4, C-5; H12/C-7, C-8, C-9; H-11/C-8, C-9, C-10; and H-13/C-1, C-9, C-10 enabled elucidation of the overall substitution pattern. Xray diffraction analysis established the absolute configuration of 2 as 5S, 8R, 9S, 10R (Figure 1). Diaporol C (3) was isolated as colorless crystals. Its molecular formula was established as C15H28O3 by the HRESIMS ion at m/z 279.1934 (calcd for C15H28O3Na, 279.1931). The 1H and 13C NMR data suggested that compound 3 was also a drimane sesquiterpenoid. The four proton spin systems of H2-1/H2-2/H2-3, H-5/H2-6/H2-7, OH11/H2-11/H-9, and OH-14/H2-14 were established by the 1 H−1H COSY spectrum. 3J HMBC correlations of H-14/C-3, C-5; H-15/C-4, C-5; H-11/C-8, C-10; and H-12/C-7, C-8, C-9 facilitated the assignment of structure to this compound. The absolute configuration of 3 was assigned as 4S, 5R, 8R, 9S, 10S by low-temperature X-ray analysis with Cu Kα radiation (Figure 1). Diaporol D (4) had the molecular formula C15H26O3 on the basis of the HRESIMS ion at m/z 277.1776 (calcd for C15H26O3Na, 277.1780), indicating three indices of hydrogen

with a methyl group (C-12) anchored to C-10. The HMBC correlations of H-9/C-6, C-7 and H-8/C-7, C-10, together with the chemical shift values of C-6 (δC 89.7) and C-7 (δC 170.4), suggested a δ-lactone system. Thus, the gross structure of 1 was established. The absolute configuration was assigned as 6S, 10R on the basis of low-temperature (100 K) Cu Kα X-ray diffraction data (Figure 1). Diaporol B (2), isolated as colorless crystals, was assigned the molecular formula C15H26O4 (three indices of hydrogen deficiency) based on an HRESIMS ion at m/z 293.1715 [M + Na]+ (calcd for C15H26O4Na, 293.1723). The 13C NMR spectrum (DMSO-d6) showed one carbonyl signal (δ 215.9, C3). Apart from one index of hydrogen deficiency of the

Figure 1. X-ray crystallographic analysis of 1−5. B

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Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Data for 5−9 5a

6b

no.

δC

1

35.6

2

18.4

3

41.3

4 5

32.4 44.8

1.39, mc

34.5 49.6

6

28.6

1.55, mc

35.0

7

67.8

3.73, t (5.0)

8 9 10 11

131.3 142.7 38.1 56.1

12 13 14

16.8 18.7 21.4

3.87, dd (5.0, 11.5) 3.93, dd (5.0, 11.5) 1.70, s 0.87, s 0.80, s

15

32.8

0.85, s

a

δH (J in Hz)

δC,

δH (J in Hz)

7b δC

δH (J in Hz)

8b δC

1.13, m 1.77, m

17.9

1.69, mc 1.76, mc

18.4

1.44, mc 1.64, m

34.5

1.32, d (13.5) 1.56, td (13.5, 4.0)c

41.8

1.17, m 1.41, mc

28.4

64.7

4.01, m

25.2

1.13, td (4.0, 13.0) 1.38, mc

50.4

1.23, t (12.0) 1.87, mc

74.9

1.75, m 2.00, td (13.5, 4.0)c 1.75, m 2.05, dt (13.5, 4.0)c 3.51, t (2.5)

37.3 43.3

2.24, t (9.0)

37.5 43.3

34.8

2.43, m

35.0

2.12, dd (14.0, 4.0) 2.39, dd (17.5, 14.0)c 2.43, dd (17.5, 4.0)c

33.2 55.5 20.3

200.6

200.8

201.0

44.0

132.4 161.8 41.2 58.4

132.5 162.6 39.6 58.6

132.5 163.1 39.7 58.5

72.9 66.2 38.1 175.4

4.36, d (11.5) 4.38, d (11.5)

4.34, d (11.5) 4.39, d (11.5)

δH (J in Hz)

39.9

1.46, t (12.0) 2.38, d (12.0)c

b

δC

1.49, td (13.5, 4.0)c 2.00, d (13.5)

44.6

2.39, dd (17.5, 14.5)c 2.54, dd (3.5, 17.5)

δH (J in Hz)

35.1

1.21, td (4.0, 13.5) 1.81, m 1.43, mc 1.57, mc

1.73, dd (3.5, 14.5)

9b

11.3 19.6 22.1

1.87, sc 1.16, s 0.97, s

11.3 18.4 27.4

1.87, s 1.15, s 0.97, s

11.3 18.9 70.6

32.5

0.96, s

21.6

0.96, s

17.2

4.31, d (11.6) 4.37, d (11.6)

1.85, 1.16, 3.10, 3.40, 0.86,

s s d (11.0) d (11.0) s

0.92, dd (2.0, 12.5) 1.35, mc 1.70, m 1.50, td (13.0, 3.5) 1.93, dt (12.5, 3.0) 2.34, s

24.7 15.1 21.4

1.45, s 1.14, s 0.82, s

33.4

0.88, s

c

Recorded in DMSO-d6. Recorded in CDCl3. Overlapped signals.

Figure 2. Key 1H−1H COSY, HMBC, and ROESY correlations of 6−9.

complete NMR and crystallographic analysis,15 and was confirmed by a single-crystal X-ray diffraction experiment using Cu Kα radiation to establish its absolute configuration as 5S, 7R, 10S (Figure 1). The molecular formula of 6, C15H24O3, was evident from the molecular ion peak at m/z 251.1654 [M − H]− (calcd for C15H23O3, 251.1647) in the HRESIMS spectrum, indicating four indices of hydrogen deficiency. The 13C NMR spectrum displayed one carbonyl (δ 200.6, C-7) and two vinylic carbons (δ 132.4, C-8 and δ 161.8, C-9), accounting for two indices of hydrogen deficiency. Thus, the remaining indices were attributed to two saturated rings. The 1H and 13C NMR and HSQC spectroscopic data (Table 2) additionally revealed four methyl, four methylene, two methine, and two sp3 quaternary carbons and two exchangeable protons. Analysis of the 1H−1H

deficiency. The similarity of the NMR data of 4 and 2 (Table 1) indicated their structural resemblance and showed one fewer oxygenated C atoms in 4. Further analysis of 2D NMR spectra showed a carbonyl group at C-2 instead of C-3 and a C-5 methine group rather than an oxygenated quaternary carbon as in 2. The absolute configuration of 4 was unambiguously determined by single-crystal X-ray diffraction analysis as 5S, 8R, 9S, 10S (Figure 1). The structure of 4 matched that of a synthesis intermediate without any data reported.14 The molecular formula of compound 5 was established as C15H26O2 by HRESIMS analysis (m/z 261.1823, calcd for C15H26O2Na, 261.1830) with three indices of hydrogen deficiency. Its 1H and 13C NMR and HSQC data (Table 2) showed that it was a drimane sesquiterpenoid. The structure matched that of a synthesis intermediate reported without C

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Fungal Material. The strain IFB-3lp-10 was isolated from the healthy leaves of Rhizophora stylosa collected in August 2010 from the mangrove forest of Hainan Province of China. The strain was identified as Diaporthe sp. by comparing the morphological character and 18S rDNA sequence with that of a standard. The 18S rDNA sequence of the strain has been deposited at GenBank as JX536253. White-colored colonies on potato dextrose agar at 28 °C grew fast, reaching 20−25 mm in diameter in three days. Perithecia are globular and gregarious with a long neck at the top, while the innermost cells are hyaline and thin-walled. Asci are globular with a thin membrane and furnished an apical ring at the thickened tip. A voucher specimen has been deposited in our laboratory. After growing on PDA medium at 28 °C for 5 d, the fungus Diaporthe sp. IFB-3lp-10 was inoculated into Erlenmeyer flasks (1 L) containing 400 mL of ME liquid medium. After incubation for 4 d at 28 °C on a rotary shaker at 150 rpm, 20 mL of culture liquid was transferred as the seed into 250 mL flasks, each preloaded with the evenly mixed medium (7.5 g of grain, 7.5 g of bran, 0.5 g of yeast extract, 0.1 g of sodium tartrate, 0.01 g of FeSO4·7H2O, 0.1 g of sodium glutamate, and 30 mL of H2O). The fungus was allowed to grow for 30 d at 28 °C with humidity in the range 60−70%. Extraction and Isolation. The air-dried samples (30 kg) were extracted with 95% EtOH, and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (1.2 kg), which gave six fractions (Fr.1, 21 g; Fr.2, 75 g; Fr.3, 56 g; Fr.4, 25 g; Fr.5, 36 g; Fr.6, 158 g) upon column chromatography (10 × 120 cm) on silica gel (6000 g, 200−300 mesh) eluted with a gradient of CH2Cl2/MeOH (v/v 100:0, 100:1, 100:2, 100:4, 100:8, 100:20, 0:100, each 20 L) based on TLC monitoring. The fourth fraction was separated on a reversed-phase ODS column (4 cm × 40 cm) with a gradient of MeOH/H2O (v/v 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, each 3 L) to give subfractions 4.1−4.9. Purification of subfraction 4.3 by using CC (petroleum ether/EtOAc, 1:1) and Sephadex LH-20 (100% MeOH) followed by crystallization from a MeOH/CH2Cl2 (1:1) solution gave orthorhombic colorless crystals of 1 (10.8 mg). Purification of subfraction 4.4 by Si gel CC (petroleum ether/EtOAc, 2:1) and Sephadex LH-20 (100% MeOH) followed by crystallization from a MeOH/CH2Cl2 (1:1) solution gave orthorhombic colorless crystals of 4 (15.6 mg) and 5 (2.6 mg). Purification of subfraction 4.5 was the same as for subfraction 4.4 and gave 9 (3.2 mg), 10 (5.2 mg), and 11 (7.8 mg). The fifth fraction was also subjected to a reversed-phase ODS column (4 cm × 40 cm) with a gradient of MeOH/H2O (v/v 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, each 3 L) to give subfractions 5.1−5.9. Purification of subfraction 5.4 by using Sephadex LH-20 (100% MeOH) followed by crystallization from a MeOH/CH2Cl2 (1:1) solution gave orthorhombic colorless crystals of 2 (8.6 mg) and monoclinic colorless crystals of 3 (3.2 mg). Purification of subfraction 5.5 by Si gel CC (petroleum ether/EtOAc, 1:2) followed by HPLC (53% MeOH) gave 6 (6.1 mg), 7 (9.8 mg), and 8 (4.1 mg). Diaporol A (1): colorless crystals; mp 243 °C; [α]28 D +145.1 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 238 (3.3) nm; IR (KBr) νmax 3487.0, 2961.7, 1742.5, 1676.3, 1431.7, 1350.4, 1260.9, 1039.7, 802.6 cm−1; HRESIMS m/z 303.1204 ([M + Na]+, calcd for C15H20O5Na, 303.1203); for 1H and 13C NMR data, see Table 1. Diaporol B (2): colorless crystals; mp 175−178 °C; [α]28 D +78.5 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 248 (3.5) nm; IR (KBr) νmax 3346.1, 2960.3, 2923.3, 1669.9, 1609.1, 1454.4, 1417.1, 1033.1, 802.4 cm−1; HRESIMS m/z 293.1715 ([M + Na]+, calcd for C15H26O4Na, 293.1723); for 1H and 13C NMR data, see Table 1. Diaporol C (3): colorless crystals; mp 196 °C; [α]28 D +31.6 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 200 (2.6) nm; IR (KBr) νmax 3254.9, 2964.4, 1639.4, 1460.6, 1261.7, 1028.0, 800.9 cm−1; HRESIMS m/z 279.1934 ([M + Na]+, calcd for C15H28O3Na, 279.1931); for 1H and 13C NMR data, see Table 1. Diaporol D (4): colorless crystals; mp 90−95 °C; [α]28 D +81.3 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 244 (3.2) nm; IR (KBr) νmax 3477.7, 3357.5, 2969.5, 2179.3, 1687.2, 1271.4, 1017.1 cm−1; HRESIMS m/z 277.1776 ([M + Na]+, calcd for C15H26O3Na, 277.1780); for 1H and 13C NMR data, see Table 1.

COSY and HMBC spectra (Figure 2) indicated the bicyclic structure of a drimane sesquiterpenoid. The H2-1/H-2/H2-3 and H-5/H2-6 isolated proton spin systems were deduced from the 1H−1H COSY spectrum. HMBC correlations from H-1 to C-3, C-9, C-10; from H-3 to C-4; from H-5 to C-1, C-4, C-7, C-9; and from H-6 to C-8, C-10 showed the presence of an A/ B ring substructure characteristic of drimane-type sesquiterpenoids. Additionally, HMBC correlations from H-11 to C-8, C-9, C-10; from H-12 to C-7, C-8, C-9; from H-13 to C-1, C-9, C10; and from H-14 and H-15 to C-3 and C-5 suggested the planar construction of 6. The trans A/B ring junction was inferred from ROESY interactions between H-5/H3-15, H3-13/ H3-14, and H3-13/H-6β. The ROESY correlations of H-2 with H3-13, H3-14, H-1β, and H-3β suggested an α-orientation of the hydroxy group. Accordingly, the structure of 6 was depicted as shown in Figure 2. Compounds 7 and 8 had the same the molecular formula as compound 6 and displayed similar 1H and 13C NMR spectra (Table 2) to those of compound 6. The 2D NMR data further suggested that compound 7 had a similar structure to 6 except for 2-OH located at C-3 in 7 (Figure 2). The ROESY correlations between H3-13/H3-14, H3-14/H-3, and H-5/H3-15 indicated the trans A/B ring junction and 3β-OH orientation. Compound 7 had four tertiary methyl groups based on the 1H NMR data, while compound 8 only had three tertiary methyl groups, which implied that one methyl group might be oxygenated. The planar structure shown in Figure 2 was confirmed by HSQC, 1H−1H COSY, and HMBC correlations. The ROESY correlations of H3-13/H3-15 and H-14/H-5 and the J value (dd, 14.0, 4.0 Hz) of H-5 suggested a 14αhydroxymethyl orientation and a trans-junction of the A/B ring system. Compound 9 had the molecular formula C15H26O3, established by an HRESIMS ion at m/z 277.1788 (calcd for C15H26O3Na, 277.1780). It was identified as 8-hydroxydriman11-oic acid, a synthetic intermediate, by analysis of its 1D (Table 2) and 2D NMR spectra (Figure 2) as well as comparison of its 1H NMR with literature data.16 However, it is described here as a natural product for the first time. All compounds were evaluated for their cytotoxicity against human gastric cancer SGC-7901, breast adenocarcinoma MCF7, lung adenocarcinoma epithelial A549, and hepatocellular carcinoma QGY-7701 cell lines using MTT methods. However, none of the compounds showed cytotoxicity against these cell lines at a concentration of 20 μM.

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EXPERIMENTAL SECTION

General Experimental Procedures. HRESIMS spectra were recorded on an Agilent 6210 TOF LC/MS. IR spectra were obtained on a Nexus 870 FT-IR spectrometer. NMR spectra were recorded on a Bruker AV-500 NMR spectrometer. Melting points were determined on a Boetius micro-melting-point apparatus and are uncorrected. Single-crystal X-ray diffraction data were collected on an Agilent Technologies SuperNova Dual diffractometer with an Atlas detector (Cu Kα radiation, λ = 1.54184 Å). Optical rotations were recorded on a Rudolph Autopol III automatic polarimeter. UV spectra were recorded on a Hitachi U-3000 spectrophotometer. Silica gel (200−300 mesh; Qingdao Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 gel (Pharmacia Biotech, Sweden) were used for column chromatography (CC). HPLC was performed with a Hitachi L-7110 pump and L-7400 UV detector equipped with an Apollo C18 column (5 μm, 250 mm × 4.6 mm; Alltech Associates, Inc. Chicago, IL, USA). D

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Diaporol E (5): colorless crystals; mp 159 °C; [α]28 D +107.5 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 211 (3.4) nm; IR (KBr) νmax 3359.6, 3288.3, 2938.7, 2930.4, 1406.0, 1083.6, 1033.8, 993.6 cm−1; HRESIMS m/z 261.1823 ([M + Na]+, calcd for C15H26O2Na, 261.1830); for 1H and 13C NMR data, see Table 2. Diaporol F (6): colorless oil; [α]28 D +59.3 (c 0.8, MeOH); UV (MeOH) λmax (log ε) 250 (3.0) nm; IR (KBr) νmax 3379.9, 2959.1, 2931.7, 2873.4, 1654.2, 1459.1, 1380.5, 1327.4, 1036.6, 802.7 cm−1; HRESIMS m/z 251.1654 ([M − H]−, calcd for C15H23O3, 251.1647); for 1H and 13C NMR data, see Table 2. Diaporol G (7): colorless oil; [α]28 D +38.2 (c 0.6, MeOH); UV (MeOH) λmax (log ε) 252 (3.4) nm; IR (KBr) νmax 3429.7, 2952.2, 1654.8, 1390.9, 1333.7, 1071.8, 1014.8, 942.2, 579.6 cm−1; HRESIMS m/z 275.1601 ([M + Na]+, calcd for C15H24O3Na, 275.1618); for 1H and 13C NMR data, see Table 2. Diaporol H (8): colorless oil; [α]28 D +31.3 (c 0.7, MeOH); UV (MeOH) λmax (log ε) 258 (3.1) nm; IR (KBr) νmax 3403.2, 2932.5, 2871.8, 1651.0, 1455.1, 1328.8, 1048.9, 1002.8, 622.5 cm−1; HRESIMS m/z 275.1600 ([M + Na]+, calcd for C15H24O3Na, 275.1618); for 1H and 13C NMR data, see Table 2. Diaporol I (9): white powder; mp 140−145 °C; [α]28 D −29.2 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 200 (2.8) nm; IR (KBr) νmax 3425.4, 2922.5, 1720.8, 1461.6, 1388.1, 1229.8, 1084.5, 938.9, 692.1, 629.4 cm−1; HRESIMS m/z 277.1788 ([M + Na]+, calcd for C15H26O3Na, 277.1780); for 1H and 13C NMR data, see Table 2. X-ray Crystallographic Studies of 1−5. The absolute structure parameter y was calculated from Bayesian statistics by using PLATON.17,18 This method for determining the absolute structure does not, in principle, require the use of copper radiation, and it should work with molybdenum radiation for natural products. For compound 2, the Friedel coverage was 95%; the resulting Hooft value was −0.01 with an uncertainty of 0.05. This indicates that the absolute structure had been determined correctly. The other compounds had similar although slightly larger Hooft values. Colorless crystals of 1−5 were obtained by crystallization from a 1:1 solution of MeOH and CH2Cl2 (1:1). All single-crystal X-ray diffraction data were collected at 100 K on an Agilent Technologies SuperNova Dual diffractometer with an Atlas detector (Cu Kα radiation, λ = 1.54184 Å). The structure was solved and refined by SHELXS-97. CCDC-862584−862588 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www. ccdc.cam.ac.uk/data_request/cif. Crystal data of 1: C15H20O5, Mr = 280.31, prism, space group P212121, a = 8.0388(2) Å, b = 11.6016(2) Å, c = 14.5562(3) Å, α = β = γ = 90.00°, V = 1357.55(5) Å3, Z = 4, Dx = 1.371 g/cm3, μ(Cu Kα) = 0.849 mm−1, F(000) = 600. Crystal dimensions: 0.30 × 0.25 × 0.20 mm3. Independent reflections: 2605 (Rint = 0.0122). The final R1 values were 0.0306, wR2 = 0.0819 [I > 2σ(I)]. Flack parameter: 0.0(2). CCDC number: 862584. Crystal data of 2: C15H26O4, Mr = 270.36, block, space group P212121, a = 6.1878(1) Å, b = 12.2291(2) Å, c = 18.9781(2) Å, α = β = γ = 90.00°, V = 1436.10(4) Å3, Z = 4, Dx = 1.250 g/cm3, μ(Cu Kα) = 0.719 mm−1, F(000) = 592. Crystal dimensions: 0.30 × 0.20 × 0.10 mm3. Independent reflections: 2843 (Rint = 0.0150). The final R1 values were 0.0285, wR2 = 0.0783 [I > 2σ(I)]. Flack parameter: 0.0(1). CCDC number: 862585. Crystal data of 3: C15H28O3, Mr = 256.37, plate, space group P21, a = 5.9952(2) Å, b = 17.2742(3) Å, c = 7.1081(2) Å, α = γ = 90.00°, β = 112.765(3)°, V = 678.79(3) Å3, Z = 2, Dx = 1.254 g/cm3, μ(Cu Kα) = 0.671 mm−1, F(000) = 284. Crystal dimensions: 0.30 × 0.30 × 0.03 mm3. Independent reflections: 2830 (Rint = 0.0290). The final R1 values were 0.0626, wR2 = 0.1686 [I > 2σ(I)]. Flack parameter: 0.2(2). CCDC number: 862586. Crystal data of 4: C15H26O3·H2O, Mr = 272.37, prism, space group P212121, a = 7.9289(2) Å, b = 9.7894(2) Å, c = 19.0738(4) Å, α = β = γ = 90.00°, V = 1480.49(6) Å3, Z = 4, Dx = 1.222 g/cm3, μ(Cu Kα) = 0.698 mm−1, F(000) = 600. Crystal dimensions: 0.20 × 0.15 × 0.05 mm3. Independent reflections: 2851 (Rint = 0.0230). The final R1

values were 0.0419, wR2 = 0.1106 [I > 2σ(I)]. Flack parameter: 0.1(2). CCDC number: 862587. Crystal data of 5: C15H26O2, Mr = 238.36, prism, space group P212121, a = 7.2909(2) Å, b = 7.7433(1) Å, c = 24.0515(4) Å, α = β = γ = 90.00°, V = 1357.84(5) Å3, Z = 4, Dx = 1.166 g/cm3, μ(Cu Kα) = 0.581 mm−1, F(000) = 528. Crystal dimensions: 0.40 × 0.30 × 0.20 mm3. Independent reflections: 2782 (Rint = 0.0154). The final R1 values were 0.0355, wR2 = 0.0955 [I > 2σ(I)]. Flack parameter: −0.2(2). CCDC number: 862588.

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ASSOCIATED CONTENT

S Supporting Information *

Copies of 1D and 2D NMR for 1−9, cif data for 1−5. This can be accessed free of charge via the Internet at http://pubs.acs. org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was cofinanced by the National Natural Science Foundation (81121062, 81172948, 21132004, 30901846, 90813036, and 21072092), New Century Excellent Talents in University (NCET-10-0477), Jiangsu Provincial Government (BK2009010), Ministry of Science and Technology of China (2011AA09070204), and University of Malaya (UM.C/625/1/ HIR/033/10).

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