Cytotoxic Cleistanthane and Cassane Diterpenoids from the

Jun 2, 2014 - infected Diaspidiotus sp. collected from Cang Mountain, Dali, Yunnan Province, People's Republic of China, in August 2011. The isolate w...
10 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/jnp

Cytotoxic Cleistanthane and Cassane Diterpenoids from the Entomogenous Fungus Paraconiothyrium hawaiiense Shenxi Chen,†,§ Yang Zhang,‡ Shubin Niu,†,§ Xingzhong Liu,† and Yongsheng Che*,‡ †

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ‡ State Key Laboratory of Antitoxic Drugs & Toxicology, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China S Supporting Information *

ABSTRACT: Hawaiinolides A−D (1−4), four new secondary metabolites including three cleistanthane (1, 3, and 4) and one cassane (2) type of diterpene lactones, were isolated from the crude extract of Paraconiothyrium hawaiiense, a fungus entomogenous to the Septobasidium-infected insect Diaspidiotus sp. The structures of 1−4 were elucidated by nuclear magnetic resonance experiments, and 1 and 3 were further confirmed by X-ray crystallography. The absolute configuration of 1 was assigned via single-crystal X-ray diffraction analysis using Cu Kα radiation, whereas that of 2−4 was deduced via the circular dichroism data. Compound 1 showed significant cytotoxicity against a small panel of five human tumor cell lines, A549, T24, HeLa, HCT116, and MCF-7.

T

prepared from solid-substrate fermentation products of the fungus showed cytotoxicity against five human tumor cell lines, A549 (lung carcinoma epithelial), T24 (bladder carcinoma), HeLa (cervical epithelium), HCT116 (colon carcinoma), and MCF-7 (breast cancer). Bioassay-directed fractionation of the active extract led to the isolation of hawaiinolides A−D (1−4, respectively), four new diterpene lactones including three cleistanthanes (1, 3, and 4) and one cassane (2). Details of the isolation, structure characterization, and cytotoxicity of 1−4 are reported herein.

erpenoids are the largest group of natural products, showing diverse structural features and interesting biological activities.1−4 Although most of the terpenoids were isolated from plant sources, members of this class of compounds are also frequently encountered as fungal secondary metabolites, with notable examples including the thiersinines, 5 asperolides A−C,6 and the guanacastane diterpenes.7 Diterpenoids are generally classified on the basis of biogenetic cyclization and/or rearrangement reactions.8 The cleistanthane and cassane diterpenoids are tricyclic compounds derived from the pimaranes, in which migration of either the C13 vinyl or methyl group to C-14 forms the cleistanthane or cassane diterpenes.8 Diaspidiotus sp. is a Diaspididae insect that feeds on the phloem or parenchyma of woody plants and grasses, showing putative mutualism with phytopathogenic fungi Septobasidium spp.9,10 Since the insects obtain nutrition from the host plants, they cause a huge loss in agriculture.11 Recently, dasipidids have received much attention as promising subjects for the evolutionary study of physiology, genetics, and coevolution due to their close relationships with the host plants, parasitoids, predators, fungi, and bacteria.9 Our prior chemical investigation of an entomogenous fungus Hypocrea sp., isolated from a Septobasidium-infected insect, Serrataspis sp., has afforded a botryane metabolite with a new mixed-biogenetic skeleton.12 Encouraged by this finding, and as part of our ongoing search for new cytotoxic natural products from fungi of unique niches,13 we selected a strain of Paraconiothyrium hawaiiense, a fungus entomogenous to the Septobasidium-infected insect Diaspidiotus sp., which was collected at Cang Mountain, Dali, Yunnan Province, People’s Republic of China, for chemical study. An EtOAc extract © 2014 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Hawaiinolide A (1) was assigned a molecular formula of C20H26O4 (eight degrees of unsaturation) on the basis of highresolution electrospray ionization mass spectrometry (HRESIMS). Analysis of its 1H and 13C nuclear magnetic resonance (NMR) data (Table 1) revealed the presence of one exchangeable proton (δH 5.12), two methyl groups, five methylenes, four methines (including one O-methine), three Received: April 5, 2014 Published: June 2, 2014 1513

dx.doi.org/10.1021/np500302e | J. Nat. Prod. 2014, 77, 1513−1518

Journal of Natural Products

Article

Table 1. NMR Data for 1 and 2 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OH-5

δC,a mult. 28.7, 24.0, 78.2, 59.1, 81.7, 207.7, 44.4, 43.5, 46.1, 44.7, 27.6, 36.3, 151.0, 55.6, 139.7, 117.6, 107.9, 19.1, 172.1, 16.0,

CH2 CH2 CH qC qC qC CH2 CH CH qC CH2 CH2 qC CH CH CH2 CH2 CH3 qC CH3

δHb (J in Hz)

2 δC,c mult.

HMBCa

1.95, m; 1.67, m 2.31, m; 2.03, m 4.32, dd (2.5, 6.3)

2, 3, 5, 9, 10, 20 1, 3, 4, 10 1, 2, 18, 19

2.68, dd (13.4, 12.8); 2.19, dd (4.8, 13.4) 1.51, m 2.34, m

6, 8, 9, 14 5, 6, 8, 9, 14 9, 11, 14, 15 7, 11, 12, 14, 20

1.86, m; 1.16, m 2.47, m; 2.15, m

8, 9, 12, 13 9, 11, 13, 14, 17

2.51, 5.67, 5.15, 4.72, 1.49,

8, 15, 16 8, 13, 14 14, 15 12, 13, 14 3, 4, 5, 19

m m dd (2.1, 10.2); 5.02, dd (2.1, 17.2) d (1.9); 4.58, d (1.6) s

0.89, s 5.12, s

1, 5, 9, 10 4, 5, 6, 10

27.9, 24.2, 78.1, 59.3, 81.9, 208.5, 41.5, 38.2, 35.4, 44.4, 26.6, 128.9, 142.1, 32.6, 14.7, 139.3, 110.4, 19.2, 172.2, 15.8,

CH2 CH2 CH qC qC qC CH2 CH CH qC CH2 CH qC CH CH3 CH CH2 CH3 qC CH3

δHd (J in Hz)

HMBCc

1.97, m; 1.60, m 2.33, m; 2.00, m 4.33, dd (2.6, 6.1)

2, 3, 5, 9, 10, 20 1, 3, 4, 10 1, 2, 4, 5, 18, 19

3.10, dd (12.8, 13.1); 2.05, m 1.93, m 2.72, m

6, 8, 9, 14 7, 9, 11, 14 1, 5, 7, 8, 10, 11, 14

2.23, m; 2.03, m 5.69, t (4.0)

8, 9, 12, 13, 16 9, 11, 13, 14, 15, 16

2.48, 1.04, 6.26, 5.10, 1.53,

7, 8, 9, 12, 13, 16 8, 13, 14 12, 13, 14, 17 12, 13, 16 3, 4, 5, 19

m d (7.0) dd (10.9, 17.7) d (17.7); 4.91, d (10.9) s

0.91, s 5.15, s

1, 5, 9, 10 4, 5, 6, 10

a

Recorded at 100 MHz in acetone-d6. bRecorded at 400 MHz in acetone-d6. cRecorded at 150 MHz in acetone-d6. dRecorded at 600 MHz in acetone-d6.

sp3 quaternary carbons with one oxygenated, four olefinic carbons (three of which were protonated) accounting for an exocyclic olefin and a vinyl group, one carboxylic carbon (δC 172.1), and one ketone carbon (δC 207.7). These data accounted for all the NMR resonances of 1 and four of the eight unsaturations, suggesting that 1 was a tetracyclic compound. Interpretation of the 1H−1H COSY NMR data of 1 revealed the presence of two isolated spin-systems, which were C-1−C-3 and C-7−C-12, with the C-14−C-16 fragment attached to C-8. In the HMBC spectrum of 1, correlations from the olefinic proton signal H2-17 to C-12, C-13, and C-14 indicated that C-13 is connected to both C-12 and C-14, establishing a cyclohexane ring with an exocyclic olefin and a vinyl group attached to C-13 and C-14, respectively. HMBC cross-peaks from H3-20 to C-1, C-5, C-9, and C-10 led to the connections of C-1, C-5, and C-9 to C-10. In turn, correlations from the exchangeable proton OH-5 to C-4, C-5, C-6, and C10 and from H2-7 to C-5 and C-6 located the only hydroxy group in 1 and established a cyclohexanone moiety fused to the cyclohexane unit at C-8/C-9. Further HMBC correlations from H3-18 to C-3, C-4, C-5, and C-19 indicated that C-3, C-18, and C-19 are all connected to C-4, completing the second cyclohexane ring fused to the cyclohexanone moiety at C-5/ C-10. Although the three-bond HMBC correlation from H-3 to C-19 could not be used to connect C-19 and C-3, the unsaturation requirement of 1 indicated that the C-19 carboxylic carbon acylated the C-3 oxygen to form a β-lactone, which was consistent with an IR absorption at 1808 cm−1. Therefore, the planar structure of hawaiinolide A was proposed to possess the same tetradecahydro-2H-phenanthro[2,1-b]oxet2-one core as that in sarcolide A,14 a cassane diterpene isolated from the cultured mycelium of Sarcodon scabrosus. Although comparison of the NMR spectroscopic data of 1 (Table 1) with those for sarcolide A suggested their structural similarity, it is not feasible to simply deduce the configuration for 1 via the

NMR data since the configuration of sarcolide A was not reported.14 The proposed structure of 1 was confirmed by single-crystal X-ray diffraction analysis using Cu Kα radiation, and a perspective ORTEP plot is shown in Figure 1. The X-ray

Figure 1. Thermal ellipsoid representation of 1.

data also allowed assignment of the relative configuration of 1. In addition, the presence of a relatively high percentage of oxygen in 1 and the value of the Flack parameter, 0.00(16),15,16 determined by X-ray analysis also permitted assignment of the 3S,4S,5S,8R,9S,10R,14S absolute configuration. Hawaiinolide B (2) was determined to have the same molecular formula C20H26O4 as 1 by HRESIMS. Analysis of its 1 H and 13C NMR data (Table 1) revealed the presence of the same octahydro-1H-naphtho[2,1-b]oxete-1,8(2aH)-dione partial structure as found in 1, but the signals corresponding to the 1514

dx.doi.org/10.1021/np500302e | J. Nat. Prod. 2014, 77, 1513−1518

Journal of Natural Products

Article

The elemental composition of hawaiinolide D (4) was determined to be C22H30O5 (eight degrees of unsaturation) by HRESIMS. The 1H and 13C NMR spectra of 4 showed resonances similar to those of 3, except that the H-3 oxygenated methine proton (δH 4.45) was shifted downfield to 5.64 ppm. In addition, NMR resonances corresponding to an acetyl group (δH/δC 2.02/21.0, 169.9) were observed, indicating that the C-3 oxygen of 4 was acylated, which was supported by HMBC correlations from H-3 to the carboxylic carbon at 169.9 ppm. On the basis of these data, 4 was determined as the C-3 monoacetate of 3. The relative configuration of 4 was deduced to be the same as that of 3 on the basis of NOESY data. Since the CD spectrum of 4 (Figure 4) was nearly identical to that of 3, the 3R,4S,5S,6R,8R,9S,10R,14S absolute configuration was proposed for 4. Compounds 1−4 were tested for cytotoxicity against a panel of five human tumor cell lines, A549, T24, HeLa, HCT116, and MCF-7 (Table 3). Compound 1 showed significant cytotoxic effects against all the cell lines tested, showing IC50 values of 7.48, 4.26, 2.84, 6.23, and 7.43 μM, respectively (the positive control cisplatin showed IC50 values ranging from 7.78 to 16.4 μM) (Table 3). Compound 2 did not show detectable cytotoxicity at 50 μM. Hawaiinolide A (1) represents the first example of a cleistanthane diterpenoid possessing the tetradecahydro-2Hphenanthro[2,1-b]oxet-2-one skeleton. The unique skeleton was previously found only in a cassane diterpenoid, sarcolide A,14 which was isolated from the cultured mycelium of Sarcodon scabrosus with no configuration or biological activity being reported.14 Although compound 1 is structurally related to sarcolide A, they belong to different subclasses (cleistanthane and cassane) of diterpenoids, in addition to the differences at C-1 and C-6. Compound 2 is a new member of the cassane subclass closely related to sarcolide A,14 but shows structural variations at C-1, C-6, C-12/C-13, and C-14/C-15. Diterpenoids incorporating a β-lactone moiety are rare, with the plant metabolites rubesanolides A and B as the other documented precedents,22 in which the β-lactone unit fused to the middle six-membered ring at C-9/C-10.22 Compounds 3 and 4 are new members of the cleistanthane diterpenes closely related to fungal secondary metabolites sonomolide A19 and zythiostromolide,20 but show different configurations at C-3 and C-14, respectively. To our knowledge, compounds 1−4 are the first secondary metabolites to be reported from the entomogenous strain of P. hawaiiense.

exocyclic olefin and vinyl group attached to the cyclohexane portion of 1 were significantly different. Analysis of the 1H−1H COSY NMR data of 2 identified the C-16−C-17 vinyl group and the C-7−C-12 unit with the C-14−C-15 fragment attached to C-8. HMBC cross-peaks from the olefinic proton H-16 to C12, C-13, and C-14 established the C-8/C-9 fused cyclohexene ring with a vinyl and a methyl group attached to C-13 and C14, respectively. On the basis of these data, the planar structure of hawaiinolide B was established as shown in 2. The relative configuration of 2 was proposed on the basis of NOESY data and by comparison to that of 1. NOESY correlations of H-7a (δH 3.10) with H-9, H3-15, and H3-18 indicated that these protons adopt the same orientation, whereas those of H3-20 with H-7b (δH 2.05) and H-8 placed these protons on the opposite face of the ring system. Further correlations of H3-18 with H-3 and OH-5 revealed their proximity in space, thereby allowing deduction of the relative configuration for 2. The absolute configuration of 2 was deduced using the octant rule for the Cotton effects (CEs) arising from the n → π* transition of the cyclohexanones.17,18 The negative CE observed at 317 nm (Δε of −0.10) of the CD spectrum (Figure 2) is associated with the n → π* transition of the

Figure 2. CD spectrum of 2 in MeOH.

cyclohexanone chromophore.17,18 On the basis of the octant rule for cyclohexanones, and combining the relative configuration established by NOESY data, the absolute configuration of 2 was deduced to be 3S,4S,5S,8S,9S,10R,14R. The molecular formula of hawaiinolide C (3) was established as C20H28O4 (seven degrees of unsaturation) on the basis of HRESIMS data. Interpretation of the 1H and 13C NMR data of 3 (Table 2) revealed the same planar structure as found in the known compounds sonomolide A19 and zythiostromolide,20 suggesting that 3 is a stereoisomer of both compounds. The proposed structure for 3 was again confirmed by singlecrystal X-ray crystallography. The perspective ORTEP plot is shown in Figure 3. The X-ray data also allowed assignment of its relative configuration. The absolute configuration of the C-4 sp3 quaternary carbon in 3 was deduced from the CD data. It has been demonstrated that the sign of the n → π* band (214− 219 nm) can be used to correlate with the absolute configuration for Cα in a γ-lactone ring.21 In this case, the CD spectrum of 3 (Figure 4) showed a positive CE at 214−219 nm, correlating with the 4S absolute configuration. 21 Considering the relative configuration established by X-ray data, the 3R,4S,5S,6R,8R,9S,10R,14S absolute configuration was deduced for 3. Therefore, 3 was determined as the C-3 and C-4 isomer of sonomolide A and zythiostromolide, respectively.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rodolph Research analytical automatic polarimeter, and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. 1H and 13C NMR data were acquired with Varian Mercury-400 and NMR system-600 spectrometers using solvent signals (acetone-d6: δH 2.05/δC 29.8, 206.1) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. ESIMS and HRESIMS data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. The fragmentor and capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 300 °C. The flow rate of the drying gas and the pressure of the nebulizer were 10 L/min and 10 psi, respectively. All 1515

dx.doi.org/10.1021/np500302e | J. Nat. Prod. 2014, 77, 1513−1518

Journal of Natural Products

Article

Table 2. NMR Data for 3 and 4 3 position

a

δC,a mult.

δHb (J in Hz)

4 δC,a mult.

HMBCa

1 2 3 4 5 6 7

30.8, 27.2, 66.8, 51.4, 79.7, 79.1, 30.9,

CH2 CH2 CH qC qC CH CH2

1.72, m; 1.34, m 1.83, m; 1.70, m 4.45, m

2, 3, 5, 10, 20 1, 3, 4 1, 2, 4, 5, 18, 19

4.47, m 2.13, dd (5.4, 16.2); 1.57, m

8 9 10 11 12 13 14

38.6, 43.8, 38.2, 26.8, 36.5, 151.6, 56.1,

CH CH qC CH2 CH2 qC CH

1.42, m 1.90, td (11.9, 3.2)

4, 7, 8, 10, 19 5, 6, 8, 9 5, 6, 8, 9, 14 7, 10, 11, 13, 14, 15 1, 7, 8, 10, 11, 12, 20

1.69, m; 1.15, m 2.43, m; 2.07, m

8, 9, 12, 13 9, 11, 13, 14, 15, 17

2.29, t (9.8)

15 16

140.2, CH 117.2, CH2

17 18 19 20 21 22 OH-3 OH-5

107.5, 12.1, 181.7, 20.2,

5.70, m 5.15, dd (2.2, 10.2); 5.02, dd (2.2, 17.2) 4.69, d (1.8); 4.56, d (1.4) 1.23, s 0.93, s

1, 5, 9, 10

CH2 CH3 qC CH3

30.5, 23.7, 69.7, 49.9, 79.7, 79.1, 30.6,

CH2 CH2 CH qC qC CH CH2

7, 8, 9, 13, 15, 16, 17

38.5, 43.7, 38.3, 26.7, 36.4, 151.4, 56.0,

CH CH qC CH2 CH2 qC CH

8, 13, 14 13, 14, 15

140.1, CH 117.3, CH2

12, 13, 14 3, 4, 5, 19

107.6, 12.8, 180.4, 20.2, 169.9, 21.0,

3.88, d (3.6) 4.16, s

CH2 CH3 qC CH3 qC CH3

δHb (J in Hz)

HMBCa

1.77, m; 1.40, m 1.87, m; 1.74, m 5.64, m

2, 3, 5, 10, 20 1, 3, 4 1, 2, 4, 5, 18, 19, 21

4.54, d (6.2) 2.16, dd (5.6, 16.5); 1.60, m

4, 5, 7, 8, 10 5, 6, 8, 9 6, 8, 9, 14

1.46, m 1.92, m

7, 10, 11, 13, 14, 15 1, 5, 7, 8, 10, 11, 12, 20

1.71, m; 1.18, m 2.44, m; 2.07, m

8, 9, 12, 13 9, 11, 13, 14, 15, 17

2.30, t (9.7)

7, 8, 9, 12, 13, 15, 16, 17 8, 13, 14 13, 14, 15

5.72, m 5.16, dd (2.2, 10.2); 5.02, dd (2.2, 17.2) 4.70, d (1.9); 4.56, d (1.5) 1.29, s

12, 13, 14 3, 4, 5, 19

0.99, s

1, 5, 9, 10

2.02, s

21

4.47, s b

Recorded at 150 MHz in acetone-d6. Recorded at 600 MHz in acetone-d6. MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100−1000 at 1.03 spectra/s. HPLC separations were performed on an Agilent 1260 instrument (Agilent, USA) equipped with a variable-wavelength UV detector. Fungal Material. The culture of P. hawaiiense was isolated from a Septobasidium sp.-infected Diaspidiotus sp. collected from Cang Mountain, Dali, Yunnan Province, People’s Republic of China, in August 2011. The isolate was identified by one of the authors (X.L.) based on morphology and sequence (GenBank accession no. KJ737370) analysis of the ITS region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar at 25 °C for 10 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm3) under aseptic conditions; 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract; the final pH of the media was adjusted to 6.5 and sterilized by autoclave). Three flasks of the inoculated media were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days to prepare the seed culture. Fermentation was carried out in 12 Fernbach flasks (500 mL), each containing 80 g of rice. Distilled H2O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 40 days. Extraction and Isolation. The fermented material was extracted repeatedly with EtOAc (4 × 1.0 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (9.5 g), which was fractionated by silica gel vacuum liquid chromatography using petroleum ether−EtOAc gradient elution. The fraction (900 mg) eluted with 35% EtOAc was separated by Sephadex LH-20 column chromatography (CC) eluting with MeOH. The resulting subfractions were purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 50% MeCN in H2O for 10 min, followed by 50−58% over 55 min; 2 mL/min) to afford 1 (9.3

Figure 3. Thermal ellipsoid representation of 3.

Figure 4. CD spectra of 3 and 4 in MeOH.

1516

dx.doi.org/10.1021/np500302e | J. Nat. Prod. 2014, 77, 1513−1518

Journal of Natural Products

Article

Table 3. Cytotoxicity of Compounds 1, 3, and 4a IC50 (μM)

a

compound

A549

T24

1 3 4 cisplatin

7.48 ± 1.2

4.26 ± 0.83

43.6 ± 3.7 7.98 ± 1.1

20.7 ± 3.2 8.40 ± 1.5

HeLa

HCT116

MCF-7

± ± ± ±

6.23 ± 0.46

7.43 ± 1.6

37.3 ± 2.5 16.4 ± 0.64

23.4 ± 3.3 7.78 ± 0.62

2.84 50.3 43.7 8.85

0.35 6.2 3.3 0.91

Compound 2 was inactive at 50 μM. graphite-monochromated Cu Kα radiation, λ = 1.54184 Å at 97(2) K. Crystal data: C20H30O5, M = 350.44, space group orthorhombic, P2(1)2(1)2(1); unit cell dimensions a = 7.2054(3) Å, b = 8.0628(3) Å, c = 31.8423(11) Å, V = 1849.90(12) Å3, Z = 4, Dcalcd = 1.258 mg/ m3, μ = 0.722 mm−1, F(000) = 760. The structure was solved by direct methods using SHELXL-9724 and refined by using full-matrix leastsquares difference Fourier techniques. All non-hydrogen atoms were refined with anisotropic displacement parameters, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Absorption corrections were performed using SADABS.25 The 8427 measurements yielded 3528 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0369 and wR2 = 0.0945 [I > 2σ(I)]. Hawaiinolide D (4): colorless oil; [α]25D +83.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (3.02), 275 (2.52) nm; CD (c 1.3 × 10−3 M, MeOH) λmax (Δε) 224 (+1.22), 277 (−0.01) nm; IR (neat) νmax 3519 (br), 2993, 2951, 2879, 1748, 1643, 1456, 1384, 1269, 1227, 1068, 992 cm−1; 1H, 13C, and HMBC NMR data see Table 2; NOESY correlations (acetone-d6, 600 MHz) H-3 ↔ H3-20; H-6 ↔ H3-18; H7a ↔ H-8, 15; H-8 ↔ H-15, 20; H-12a ↔ H-17; H-14 ↔ H-7b, 9, 16b; H-15 ↔ H-17b; HRESIMS m/z 375.2171 (calcd for C20H31O5, 375.2166). MTS Assay (ref 27). In a 96-well plate, each well was plated with (2−5) × 103 cells (depending on the cell multiplication rate). After cell attachment overnight, the medium was removed, and each well was treated with 100 μL of medium containing 0.1% DMSO or appropriate concentrations of the test compounds and the positive control cisplatin (100 mM as stock solution of a compound in DMSO and serial dilutions; the test compounds showed good solubility in DMSO and did not precipitate when added to the cells). The plate was incubated for 48 h at 37 °C in a humidified, 5% CO2 atmosphere. Proliferation was assessed by adding 20 μL of MTS (Promega) to each well in the dark, followed by a 90 min incubation at 37 °C. The assay plate was read at 490 nm using a microplate reader. The assay was run in triplicate.

mg, tR 33.20 min), 2 (5.1 mg, tR 31.34 min), and 4 (2.0 mg, tR 43.75 min). The fractions (700 mg) eluted with 45% and 50% EtOAc were combined and separated again by Sephadex LH-20 CC using MeOH as eluent, and the resulting subfractions were further purified by RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm) to afford 3 (8.0 mg, tR 27.42 min; 45% CH3CN in H2O over 40 min; 2 mL/min). Hawaiinolide A (1): colorless needles; mp 150−152 °C; [α]25D +45.7 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 214 (2.55), 302 (1.57) nm; CD (c 1.5 × 10−3 M, MeOH) λmax (Δε) 253 (−0.09), 309 (−0.40) nm; IR (neat) νmax 3478 (br), 2969, 2935, 2869, 1808, 1719, 1459, 1422, 1345, 1112, 913 cm−1; 1H, 13C, and HMBC NMR data see Table 1; NOESY correlations (acetone-d6, 400 MHz) H-3 ↔ H3-18; H-8 ↔ H-15, 16, 20; H-9 ↔ H-14; HRESIMS m/z 331.1909 (calcd for C20H27O4, 331.1904). X-ray Crystallographic Analysis of 1 (ref 23). Upon crystallization from MeOH−H2O (20:1) using the vapor diffusion method, colorless crystals were obtained for 1. A crystal (0.60 × 0.60 × 0.20 mm) was separated from the sample and mounted on a glass fiber, and data were collected using an Oxford Diffraction Gemini E diffractometer with graphite-monochromated Cu Kα radiation, λ = 1.54184 Å at 99(8) K. Crystal data: C20H26O4, M = 330.41, space group orthorhombic, P2(1)2(1)2(1); unit cell dimensions a = 7.3028(3) Å, b = 13.9746(13) Å, c = 16.4665(12) Å, V = 1680.5(2) Å3, Z = 4, Dcalcd = 1.306 mg/m3, μ = 0.721 mm−1, F(000) = 712. The structure was solved by direct methods using SHELXL-9724 and refined by using full-matrix least-squares difference Fourier techniques. All nonhydrogen atoms were refined with anisotropic displacement parameters, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with the relative isotropic parameters. Absorption corrections were performed using the Siemens Area Detector Absorption Program (SADABS).25 The 5578 measurements yielded 3215 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0344 and wR2 = 0.0882 [I > 2σ(I)]. Hawaiinolide B (2): colorless oil; [α]25D +20.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 209 (2.71), 215 (2.74), 249 (2.83) nm; CD (c 1.5 × 10−3 M, MeOH) λmax (Δε) 243 (−0.17), 317 (−0.10) nm; IR (neat) νmax 3484 (br), 2965, 2937, 2880, 1813, 1721, 1456, 1381, 1274, 1103, 1052 cm−1; 1H, 13C, and HMBC NMR data see Table 1; NOESY correlations (acetone-d6, 600 MHz) H-3 ↔ H-18; H-7a ↔ H9, 15, 18; H-9 ↔ H3-15; H-12 ↔ H-16; H3-15 ↔ H-17 and OH-5; H320 ↔ H-7b, 8; OH-5 ↔ H3-18; HRESIMS m/z 331.1910 (calcd for C20H27O4, 331.1904). Hawaiinolide C (3): colorless needles; mp 145−148 °C; [α]25D +123.9 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 201 (2.44), 219 (2.67), 231 (2.75) nm; CD (c 7.5 × 10−4 M, MeOH) λmax (Δε) 228 (+1.69), 280 (−0.02) nm; IR (neat) νmax 3445 (br), 2940, 2869, 2844, 1747, 1643, 1462, 1392, 1346, 1245, 1136, 1066, 1016 cm−1; 1H, 13C, and HMBC NMR data see Table 2; NOESY correlations (acetone-d6, 600 MHz) H-3 ↔ H3-20; H-6 ↔ H3-18; H-7a ↔ H-8, 15, 16a; H-8 ↔ H-15, 20; H-12a ↔ H-17; H-14 ↔ H-7b, 9, 16b; H-15 ↔ H-17b; H318 ↔ OH-3, OH-5; HRESIMS m/z 333.2064 (calcd for C20H29O4, 333.2060). X-ray Crystallographic Analysis of 3 (ref 26). Upon crystallization from MeOH−H2O (30:1) using the vapor diffusion method, colorless crystals were obtained for 1. A crystal (0.40 × 0.15 × 0.03 mm) was separated from the sample and mounted on a glass fiber, and data were collected using an Oxford Diffraction Gemini E diffractometer with

■

ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of 1−4 and CD spectrum of 1. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86 10 66932679. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Program of Drug Research and Development (2012ZX09301003) and Beijing Natural Science Foundation (5111003).

■

REFERENCES

(1) Fraga, B. M. Nat. Prod. Rep. 2011, 28, 1580−1610.

1517

dx.doi.org/10.1021/np500302e | J. Nat. Prod. 2014, 77, 1513−1518

Journal of Natural Products

Article

(2) Xiao, W. L.; Li, R. T.; Huang, S. X.; Pu, J. X.; Sun, H. D. Nat. Prod. Rep. 2008, 25, 871−891. (3) Sun, H. D.; Huang, S. X.; Han, Q. B. Nat. Prod. Rep. 2006, 23, 673−698. (4) Geris, R.; Simpson, T. J. Nat. Prod. Rep. 2009, 26, 1063−1094. (5) Li, C.; Gloer, J. B.; Wicklow, D. T.; Dowd, P. F. Org. Lett. 2002, 4, 3095−3098. (6) Sun, H. F.; Li, X. M.; Meng, L.; Cui, C. M.; Gao, S. S.; Li, C. S.; Huang, C. G.; Wang, B. G. J. Nat. Prod. 2012, 75, 148−152. (7) (a) Brady, S. F.; Singh, M. P.; Janso, J. E.; Clardy, J. J. Am. Chem. Soc. 2000, 122, 2116−2117. (b) Hughes, C. C.; Kennedy-Smith, J. J.; Trauner, D. Org. Lett. 2003, 5, 4113−4115. (c) Ou, Y. X.; Li, Y. Y.; Qian, X. M.; Shen, Y. M. Phytochemistry 2012, 78, 190−196. (d) Feng, Y.; Ren, F.; Niu, S.; Wang, L.; Li, L.; Liu, X.; Che, Y. J. Nat. Prod. 2014, 77, 873−881. (8) Peters, R. J. Nat. Prod. Rep. 2010, 27, 1521−1530. (9) Morse, G. E.; Normark, B. B. Syst. Entomol. 2006, 31, 338−349. (10) Couch, J. N. The Genus Septobasidium; University of North Carolina Press: Chapel Hill, NC, 1938; pp 53−54. (11) Alexopoulos, C.; Mims, C. W. Introductory Mycology; John Wiley and Sons., Co.: New York, 1979; pp 113−114. (12) Yuan, Y.; Feng, Y.; Ren, F.; Niu, S.; Liu, X.; Che, Y. Org. Lett. 2013, 15, 6050−6053. (13) Che, Y. Int. J. Pharm. Res. 2011, 38, 12−27. (14) Shibata, H.; Hayasaki, F.; Hirota, M. Shinshu Daigaku Nogakubu Kiyo 2002, 38, 55−58. (15) Luo, J.; Liu, X.; Li, E.; Guo, L.; Che, Y. J. Nat. Prod. 2013, 76, 107−112. (16) Flack, H. D. Acta Crystallogr., Sect. A 1983, 39, 876−881. (17) Ye, X. L. Stereochemistry; Beijing University Express: Beijing, 1999; pp 236−259. (18) Tian, Y.; Xu, W.; Zhu, C.; Lin, S.; Guo, Y.; Shi, J. J. Nat. Prod. 2013, 76, 1039−1046. (19) Morris, S. A.; Curotto, J. E.; Zink, D. L.; Dreikom, S.; Jenkins, R.; Bills, G. F.; Thompson, J. R.; Vicente, F.; Basilio, A.; Liesch, J. M.; Schwartz, R. E. Tetrahedron Lett. 1995, 36, 9101−9104. (20) Ayer, Y. A.; Khan, A. Q. Phytochemistry 1996, 42, 1647−1652. (21) Beecham, A. F. Tetrahedron Lett. 1968, 9, 2355−2360. (22) Zou, J.; Pan, L.; Li, Q.; Zhao, J.; Pu, J.; Yao, P.; Gong, N.; Lu, Y.; Kondratyuk, T. P.; Pezzuto, J. M.; Fong, H. H. S.; Zhang, H.; Sun, H. Org. Lett. 2011, 13, 1406−1409. (23) Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 991596). Copies of the data can be obtained, free of charge, on application to the director, CCDC 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223 336033 or e-mail: [email protected]). (24) Sheldrick, G. M. SHELXL-97, Program for X-ray Crystal Structure Solution and Refinement; University of Gö ttingen: Gö ttingen: Germany, 1997. (25) Sheldrick, G. M. SADABS, Program for Empirical Absorption Correction of Area Detector Data; University of Göttingen: Göttingen: Germany, 1999. (26) Crystallographic data for 3 have been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 991613). Copies of the data can be obtained, free of charge, on application to the director, CCDC 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223 336033 or e-mail: [email protected]). (27) Zhang, N.; Chen, Y.; Jiang, R.; Li, E.; Chen, X.; Xi, Z.; Guo, Y.; Liu, X.; Zhou, Y.; Che, Y.; Jiang, X. Autophagy 2011, 7, 598−612.

1518

dx.doi.org/10.1021/np500302e | J. Nat. Prod. 2014, 77, 1513−1518