Dioxatricyclic and Oxabicyclic Polyketides from Trichocladium opacum

Article pubs.acs.org/jnp

Dioxatricyclic and Oxabicyclic Polyketides from Trichocladium opacum Shenxi Chen,†,§ Fengxia Ren,‡ 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 ‡ 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: Five new polyketides, trichocladinols D−H (1−5) with dioxatricyclic (1−3) and oxabicyclic (4 and 5) skeletons, and the known massarilactone C (6) were isolated from the solid-substrate fermentation cultures of the ascomycete fungus Trichocladium opacum. The structures of 1−5 were determined mainly by NMR experiments, and 1, 3, and 4 were confirmed by X-ray crystallography. The absolute configurations of 1 and 3 were assigned by Xray crystallography using Cu Kα radiation, whereas that of C-5 in 2 and 4 was deduced via the circular dichroism (CD) data. Compounds 2−4 showed weak cytotoxicity against the human tumor cell lines A549, HCT116, and SW480.

F

ungi inhabiting special and competitive environments are more likely to produce secondary metabolites with diverse structural features and interesting biological activities, presumably due to their highly evolved metabolic systems adapted during the natural selection process.1−3 Known as the “Roof of the World”, the Qinghai-Tibetan plateau, with an average elevation exceeding 4000 m,4 is such an environment that harbors unique organisms including Ophiocordyceps sinensis and related fungi.5 Recently, we have chemically investigated the fungal species isolated from either O. sinensis or the soil samples collected in alpine regions of the Qinghai-Tibetan plateau and discovered a variety of bioactive secondary metabolites.6,7 Trichocladium opacum is a common soil fungus frequently isolated from various soil samples on the Qinghai-Tibetan plateau. However, the chemistry of this fungal species remained largely unexplored. The only documented example is our prior chemical study on a Cordyceps-colonizing strain of T. opacum, which afforded trichocladinols A and B, two metabolites featuring an undescribed 2,9-dioxatricyclo[5.2.1.03,8]dec-4-ene skeleton.8 During an ongoing search for new cytotoxic natural products from rarely studied fungi from unique niches, a strain of T. opacum isolated from a soil sample that was also collected in Linzhi, Tibet, People’s Republic of China, was subjected to chemical investigation. Fractionation of an EtOAc extract prepared from a solid-substrate fermentation culture led to the isolation of three new dioxatricyclic (1−3) and two new oxabicyclic (4 and 5) polyketides, which we named trichocladinols D−H (1−5), and the known compound massarilactone C (6).9 Details of the isolation, structure elucidation, and cytotoxicity of the new metabolites are reported herein. © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Trichocladinol D (1) was assigned the molecular formula C11H14O5 by HRESIMS (five degrees of unsaturation). Analysis of its 1H and 13C NMR data (Table 1) revealed the presence of two exchangeable protons (δH 5.41 and 6.37, respectively), two methyl groups, four methines including three O-methines, two sp3 quaternary carbons with one doubly oxygenated (δC 111.2), two protonated olefinic carbons, and one carboxylic carbon (δC 173.1). These data accounted for all the NMR resonances for 1 and suggested that it was a tricyclic compound. The 1H−1H COSY NMR data of 1 showed the two isolated proton spinsystems of C-4−OH-4 and C-6−C-10 (including OH-7 and C12). HMBC correlations from H-6 and OH-4 to C-4 and C-5 and from H-10 and H3-12 to C-5 indicated that C-4, C6, and C-10 were all attached to C-5, connecting the above-mentioned Received: June 14, 2013 Published: December 19, 2013 9

dx.doi.org/10.1021/np4004799 | J. Nat. Prod. 2014, 77, 9−14

Journal of Natural Products

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Table 1. NMR Data for 1−3 1 pos. 1 3 4 5 6 7 8 8a 9 9a 10 11 12 OH-4 OH-7

δC,a mult. 173.1, 111.2, 77.0, 56.5, 75.9, 69.5, 127.9,

qC qC CH qC CH CH CH

132.4, CH 30.2, CH 14.4, CH3 15.6, CH3

2

δHb (J in Hz)

δC,a mult.

HMBCa

4.08, d (5.8)

1, 3, 5, 6, 10

4.31, d (8.8) 3.74, m 5.30, dt (1.5, 10.1)

1, 3, 4, 5, 9, 10 6, 8, 9 6, 9, 10, 12

174.8, 111.3, 79.1, 57.8, 75.0, 69.1, 128.4,

5.57, ddd (2.2, 4.1, 10.1)

5, 6, 7, 10, 12

131.9, CH

2.78, 1.53, 1.08, 6.37, 5.41,

1, 3, 5, 3, 6,

m s d (7.4) d (5.8) d (6.0)

5, 4 9, 4, 7,

6, 8, 9, 12

qC qC CH qC CH CH CH

27.9, CH 14.9, CH3 15.0, CH3

10 5 8, 9

3 δHb (J in Hz)

4.19, d (4.0) 4.05, d (8.7) 3.69, m 5.31, dt (1.5, 10.1)

δC,c mult. 173.7, 110.4, 78.8, 59.2, 78.3, 71.5, 29.6,

qC qC CH qC CH CH CH2

5.62, ddd (2.2, 4.2, 10.1)

28.6, CH2

2.60, 1.52, 1.03, 6.31, 5.44,

29.2, CH 14.0, CH3 13.7, CH3

m s d (7.4) d (4.0) d (5.7)

δHd (J in Hz)

4.12, d (5.5) 4.43, 3.43, 1.75, 1.43, 2.36, 1.34, 2.44, 1.52, 1.15, 5.43, 4.18,

d (10.3) m m m m m m s d (7.4) d (5.5) d (4.4)

a

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

two partial structures at C-5 to complete the cyclohexene ring in 1. Correlations from OH-4 to C-3 and from H3-11 to C-3 and C-4 led to the connection of C-3 and C-4 and located the second methyl group in 1 at C-3. A key HMBC cross-peak from the oxymethine proton H-6 to the doubly oxygenated sp3 quaternary carbon C-3 established a tetrahydrofuran (THF) ring fused to the cyclohexene moiety via C-5−C-6. Additional HMBC correlations from H-6 and H-10 to the carboxylic carbon C-1 led to the connection of C-1 to C-5. Considering the unsaturation requirement of 1, C-1 and C-3 should be attached to the remaining oxygen atom to form a hydroxylated γ-lactone ring joined to the THF unit at C-3 and C-5, completing the gross structure of 1 as shown. The proposed structure of 1 was confirmed by X-ray crystallographic analysis, and a perspective ORTEP plot is shown in Figure 1. The X-ray data also allowed assignment of the relative configuration of 1. In addition, due to the presence of a relatively high percentage of oxygen, 1 exhibited enough anomalous dispersion of Cu Kα radiation to allow assignment of its absolute configuration.10 Therefore, the absolute configuration of 1 was deduced to be 3S, 4R, 5R, 6S, 7S, 10R based on the value of the Flack parameter, −0.1(2).11

Trichocladinol E (2) was determined to have the same molecular formula, C11H14O5, as 1 by HRESIMS (five degrees of unsaturation). Analysis of its 1H and 13C NMR data (Table 1) revealed nearly identical structural features to those found in 1, except that the chemical shift values for the C-4 and C-6 oxymethines in 2 (δH/δC 4.19/79.1 and 4.05/75.0) were different from those in 1 (δH/δC 4.08/77.0 and 4.31/75.9). Interpretation of the 1H and 13C NMR data established the same planar structure as 1, which was supported by relevant 1 H−1H COSY and HMBC data, suggesting that 2 is a stereoisomer of 1. The relative configuration of 2 was proposed by analysis of 1H−1H coupling constants and NOESY data. A coupling constant of 8.7 Hz observed for H-6 in 2, compared to that of 8.8 Hz for the same proton in 1, suggested a trans relationship for H-6 and H-7. NOESY correlations of H-6 with H-4 and H3-12 placed these protons on the same face of the cyclohexene ring, whereas that of H3-11 with H-4 revealed their proximity in space, thereby allowing deduction of the relative configuration for 2. The absolute configuration of C-5 in 2 was deduced by application of the CD exciton chirality method. 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 moiety.12 In this case, the CD spectrum of 2 (Figure S12) showed a negative Cotton effect at 214−219 nm, correlating with a 5R absolute configuration. Combining the relative configuration established the 3S, 4S, 5R, 6S, 7S, 10R absolute configuration was deduced for 2. Trichocladinol F (3) gave a pseudomolecular ion [M + Na]+ peak by HRESIMS, indicating a molecular formula of C11H16O5 (four degrees of unsaturation). The 1H and 13C NMR spectra of 3 showed resonances similar to those of 1 and 2, except that the C-8−C-9 olefin was replaced by two methylene units in 3, which was confirmed by relevant 1H−1H COSY and HMBC data, indicating that 3 is a hydrogenated analogue of 1. The relative configuration of 3 was deduced to be the same as that of 1 by analysis of 1H−1H coupling constants and NOESY data for relevant protons, which was further confirmed by singlecrystal X-ray diffraction analysis (Figure 2). The CD spectrum of 3 (Figure S13) is nearly identical to that of 1, indicating that 3 possesses the same absolute configuration as 1.

Figure 1. Thermal ellipsoid representation of 1. (Note: A different numbering system is used for the structural data deposited with the CCDC.) 10



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proposed structure of 4 was again confirmed by X-ray crystallographic analysis (Figure 3), which also allowed

Figure 2. Thermal ellipsoid representation of 3.

Figure 3. Thermal ellipsoid representation of 4. (Note: A different numbering system is used for the structural data deposited with the CCDC.)

The molecular formula of trichocladinol G (4) was determined to be C11H16O5 (four degrees of unsaturation) on the basis of its HRESIMS data. Its 1H and 13C NMR data (Table 2) showed three exchangeable protons (δH 3.99, 4.99,

assignment of the relative configuration of 4. The absolute configuration of C-5 in 4 was also deduced via the CD data. The CD spectrum of 4 (Figure S14) showed a negative Cotton effect at 214−219 nm, correlating with a 5R absolute configuration.12 Therefore, the 3R, 4S, 5R, 6S, 7R, 10S absolute configuration was proposed for 4. The elemental composition of trichocladinol H (5) was established as C12H18O6 (four degrees of unsaturation) by HRESIMS, which is the same as the co-isolated known compound massarilactone C (6).9 Interpretation of the 1H and 13 C NMR (Table 2) as well as 2D NMR data of 5 established the same gross structure as 6, indicating their isomeric relationship. The relative configuration of 5 was proposed by analysis of its 1H−1H coupling constants and NOESY data. A coupling constant of 7.9 Hz observed for H-6 and H-7 in 5 indicated that both protons are pseudoaxially oriented with respect to the cyclohexane-1,2-diol moiety, whereas that of 4.9 Hz observed for H-9 indicated that H-9 and H-10 are pseudoequatorially oriented.9 NOESY correlations of H-4 with H-7 and of H3-12 with H-6 and H-9 supported the above assignments. A NOESY correlation of H-10 with H3-11 placed these protons on the same face of the ring system, permitting assignment of the relative configuration of 5. The absolute configuration of the 6,7-diol moiety in 5 was assigned using the in situ dimolybdenum CD method developed by Snatzke and Frelek.13,14 Upon addition of dimolybdenum tetraacteate [Mo2(OAc)4] to 5 in DMSO solution, a metal complex was generated. After subtraction of the inherent CD spectrum, the observed sign of the Cotton effect in the induced spectrum of 5 originates solely from the chirality of the vic-diol moiety expressed by the sign of the O− C−C−O torsion angle. The positive Cotton effect observed at around 310 and 400 nm, respectively, in the induced CD spectrum (Figure 4) permitted assignment of the 6S and 7S absolute configuration on the basis of the empirical rule proposed by Snatzke. Therefore, 5 was deduced to have the 4R, 5R, 6S, 7S, 9S, 10S absolute configuration. The remaining known compound 6 was identified as massarilactone C by comparison of its NMR and MS data with those reported.9 Compounds 1−5 were tested for cytotoxicity against the following four human tumor cell lines: HeLa (cervical epithelial cells), A549 (lung carcinoma epithelial cells), SW480 (human

Table 2. NMR Data for 4 and 5 4 pos.

δC , mult. a

δH (J in Hz)

δC , mult. c

175.7, 68.8, 75.1, 57.9, 79.3,

7 8 8a

78.6, CH 126.5, CH

4.44, d (5.7) 6.08, m

70.1, CH 34.7, CH2

9

137.9, CH

5.68, ddd (1.1, 2.8, 9.3) 3.19, m 1.33, d (6.1) 1.02, d (7.2)

78.4, CH

3.86, dd (5.2, 7.9) 3.43, m 1.95, m 1.44, dd (10.2, 13.7) 4.21, t (4.9)

40.7, 29.0, 9.3, 51.7,

2.65, m 2.1, s 1.02, d (7.2) 3.58, s

37.1, CH 20.1, CH3 16.0, CH3

4.12, m 4.18, m 4.57, t (3.2)

3.99, d (5.7) 4.99, d (6.7) 5.78, t (3.2)

171.1, 207.0, 81.2, 61.7, 71.8,

qC qC CH qC CH

δHd (J in Hz)

1 3 4 5 6

10 11 12 13 OH-3 OH-4 OH-6 OH-7

qC CH CH qC CH

5 b

CH CH3 CH3 CH3

4.45, s

5.25, d (5.2) 4.86, d (6.6)

a Recorded at 100 MHz in acetone-d6. bRecorded at 400 MHz in acetone-d6. cRecorded at 100 MHz in DMSO-d6. dRecorded at 400 MHz in DMSO-d6

and 5.78, respectively), two methyl groups, five methines including four oxymethines, one sp3 quaternary carbon, one disubstituted olefin, and one carboxylic carbon (δC 175.7). Analysis of the 1H and 13C NMR data of 4 revealed the presence of the same cyclohexene ring as found in 1−3, with the ester carbonyl group carbon and a methyl group similarly attached to C-5 and C-10, respectively. The isolated proton spin-system C-11−C-3−C-4 (including OH-3 and OH-4) was also connected to C-5 based on the HMBC correlations from H-3 and H-4 to C-5. An HMBC cross-peak from the H-7 oxymethine proton to the only ester carbonyl carbon in 4 established the γ-lactone moiety, completing the 6oxabicyclo[3.2.1]oct-3-en-7-one gross structure of 4. The 11



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(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 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 T. opacum was isolated from a soil sample collected in Linzhi, Tibet, People’s Republic of China, in July 2007. The strain was isolated from the soil suspension in distilled water by the plating technique on a PDA plate containing streptomycin. The isolate was identified by one of the authors (X.L.) based on morphology and sequence (Genbank Accession No. JQ 179993) analysis of the ITS region of the rDNA and assigned the accession number XZ07102-6 in X.L.’s culture collection at the Institute of Microbiology, Chinese Academy of Sciences, Beijing. 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, and 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 five days to prepare the seed culture. Spore inoculum was prepared by suspension in sterile, distilled H2O to give a final spore/cell suspension of 1 × 106/mL. 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 (6.2 g), which was fractionated by silica gel VLC using petroleum ether− CH2Cl2−MeOH gradient elution. The fraction (400 mg) eluted with 100:1 CH2Cl2−MeOH was separated by Sephadex LH-20 column chromatography (CC) eluting with MeOH, and the resulting fractions were combined and purified by RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 16% MeOH in H2O for 40 min; 2 mL/ min) to afford 1 (8.0 mg, tR 21.32 min), 2 (6.0 mg, tR 14.51 min), and 3 (7.0 mg, tR 34.45 min). The fraction (300 mg) eluted with 100:1.5 CH2Cl2−MeOH was fractionated by Sephadex LH-20 CC eluting with MeOH. The resulting fractions were further purified by RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 20% MeOH in H2O for 3 min, followed by 20−100% over 50 min; 2 mL/min) to afford 4 (3.0 mg, tR 13.71 min). The fraction (400 mg) eluted with 100:2 CH2Cl2−MeOH was purified by RP HPLC (Agilent Zorbax SBC18 column; 5 μm; 9.4 × 250 mm; 25% MeOH in H2O for 35 min; 2 mL/min) to afford 5 (5 mg, tR 22.40 min) and 6 (8.9 mg, tR 33.50 min). Trichocladinol D (1): white powder; mp 177−180 °C; [α]25D −9.2 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 208 (0.75), 210 (0.73), 215 (0.63) nm; CD (c 1.0 × 10−3 M, MeOH) λmax (Δε) 218 (−0.70) nm; IR (neat) νmax 3388 (br), 2992, 1785, 1186, 1062, 850 cm−1; 1H NMR, 13C NMR, and HMBC data see Table 1; HRESIMS m/z 249.0740 (calcd for C11H14O5Na, 249.0733). X-ray Crystallographic Analysis of 1 (ref 19). Upon crystallization from MeOH−H2O (20:1) using the vapor diffusion method, colorless crystals were obtained for 1. A crystal (0.80 × 0.50 × 0.50 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 Ǻ at 101(2) K. Crystal data: C11H14O5, M = 226.22, space group orthorhombic, P2(1); unit cell dimensions a = 9.9909(3) Ǻ , b = 7.57278(19) Ǻ , c = 13.7572(4) Ǻ , V = 1040.82(5) Ǻ 3, Z = 4, Dcalcd = 1.444 mg/m3, μ = 0.968 mm−1, F(000) = 480. The structure was solved by direct methods using SHELXL-9720 and refined by using

Figure 4. CD spectrum of 5 in DMSO containing Mo2(OAc)4 with the inherent CD spectrum subtracted.

colon adenocarcinoma cell line), and HCT116 (colon cancer cells) (Table 3). Compounds 2−4 showed weak cytotoxic Table 3. Cytotoxicity of Compounds 2−4a IC50 (μM) compound 2 3 4 cisplatin a

A549 83.6 61.0 52.3 4.3

± ± ± ±

13.9 6.5 3.5 0.17

SW480

HeLa

± ± ± ±

−a −a 44.3 ± 0.66 10.7 ± 0.29

54.9 51.9 43.6 11.0

4.6 7.8 1.0 1.3

HCT116 48.8 56.6 41.7 14.1

± ± ± ±

0.71 2.3 1.4 0.85

1 and 5 were inactive at 20 μg/mL.

effects against A549, HCT116, and SW480 cells, while 4 was also cytotoxic to HeLa cells, with an IC50 value of 44.3 μM (the positive control cisplatin showed an IC50 value of 10.7 μM). Compounds 1 and 5 did not show detectable cytotoxicity against the four cell lines at 20 μg/mL. Natural products incorporating a 7,9-dioxatricyclo[6.2.1.01,6]undec-3-en-10-one skeleton are rare, with fungal metabolite spiroleptosphol B (7) as the only documented one prior to this study.15 Trichocladinols D−F (1−3) are structurally related to 7, but differ from the known analogue by having a methyl group attached to C-10 instead of an (E)-3,5-dimethyl-1-heptenyl chain, in addition to different relative configurations at C-10 (the absolute configuration of 7 was not assigned). Trichocladinols G (4) and H (5) possess the 6oxabicyclo[3.2.1]octane skeleton, in which 4 is closely related to monoterpene lactones filifolides A and B,16 but has a 1,2diolpropyl unit at C-5, whereas 5 is a stereoisomer of massarilactone C (6)9 at C-4, C-5, C-6, and C-9, with massarilactones A17 and E−G18 as the other examples of related natural products. Biogenetically, trichocladinols D−H (1−5) may share the same biosynthetic precursors as the known natural products including trichocladinols A−C,8 the massarilactones,17,18 and spiroleptosphol B (7).15 To our knowledge, compounds 1−5 are the second examples of natural products to be reported from T. opacum.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 241 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 13 C NMR data were acquired with a Varian Mercury-400 spectrometer using solvent signals (acetone-d6: δH 2.05/δC 29.8, 206.1; DMSO-d6: δH 2.50/δC 39.5) 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 12



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structure was solved by direct methods using SHELXL-9720 and refined by using full-matrix least-squares 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.21 The 4604 measurements yielded 2341 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.1718 and wR2 = 0.4033 [I > 2σ(I)]. Trichocladinol H (5): white powder; [α]25D −60.0 (c 1, MeOH); UV (MeOH) λmax (log ε) 207 (0.58), 208 (0.65), 210 (0.60) nm; CD (c 3.4 × 10−4 M, DMSO) λmax (Δε) 297 (+6.14), 375 (+1.05) nm; IR (neat) νmax 3435 (br), 2953, 1728, 1437, 1355, 1254, 1073, 1048, 1022 cm−1; 1H and 13C NMR data see Table 2; HMBC correlations (DMSO-d6, 400 MHz) H-4 → C-1, 3, 5, 6, 9, 10, 11; H-6 → C-1, 3, 4, 5, 7, 10; H-7 → C-6, 8; H-8 → C-6, 7, 9, 10; H-9 → C-4, 5, 7, 10; H10 → C-1, 5, 6, 8, 9, 12; H3-11 → C-3, 4; H3-12 → C-5, 9, 10; HRESIMS m/z 281.1003 (calcd for C11H16O5Na, 281.0996). Absolute Configuration of the 6,7-Diol Moiety in 5 (refs (13 and 14). HPLC grade DMSO was dried with 4 Å molecular sieves. According to a published procedure,24 a mixture of 1:1.3 diolMo2(OAc)4 for 5 was subjected to CD measurements at a concentration of 0.5 mg/mL. The first CD spectrum was recorded immediately after mixing, and its time evolution was monitored until stationary (about 10 min after mixing). The inherent CD was subtracted. The observed signs of the diagnostic bands at around 310 and 400 nm in the induced CD spectrum were correlated to the absolute configuration of the 6,7-diol moiety. Massarilactone C (6): colorless oil; [α]25D +40.1 (c 0.7, MeOH); 1 H NMR, 13C NMR, and the MS data were consistent with literature values.9 MTS Assay (ref 25). The assay was run in triplicate. 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.

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).21 The 3860 measurements yielded 2612 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0432 and wR2 = 0.1132 [I > 2σ(I)]. Trichocladinol E (2): white powder; [α]25D −13.7 (c 0.33, MeOH); UV (MeOH) λmax (log ε) 210 (0.28), 214 (0.35), 230 (0.19) nm; CD (c 1.0 × 10−4 M, MeOH) λmax (Δε) 228 (−0.64) nm; IR (neat) νmax 3385 (br), 2970, 1778, 1701, 1640, 1401, 1141, 1078, 854 cm−1; 1H and 13C NMR data see Table 1; HMBC correlations (DMSO-d6, 400 MHz) H-4 → C-1, 3, 6, 11; H-6 → C-1, 3, 4, 5, 7, 8, 10; H-7 → C-6, 8, 9; H-8 → C-6, 10, 12; H-9 → C-5, 6, 7, 10, 12; H-10 → C-1, 5, 6, 8, 9, 12; H3-11 → C-3, 4; H3-12 → C-5, 9, 10; HRESIMS m/z 227.0918 (calcd for C11H15O5, 227.0914). Trichocladinol F (3): white powder; mp 158−161 °C; [α]25D −1.5 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 210 (0.57), 221 (0.64), 230 (0.48) nm; CD (c 0.5 × 10−4 M, MeOH) λmax (Δε) 220 (−0.69), 230 (−0.69) nm; IR (neat) νmax 3389 (br), 3242, 2963, 2950, 1775, 1444, 1348, 1203, 1107, 1057, 844 cm−1; 1H and 13C NMR data see Table 1; HMBC data (acetone-d6, 400 MHz) H-4 → C-3, 4, 6; H-6 → C-1, 4, 5, 7, 8; H-7 → C-6; H-8 → C-6, 7, 9; H-9 → C-5, 7, 8, 12; H10→ C-1, 4, 5, 6, 8, 9, 12; H3-11 → C-3, 4; H3-12 → C-5, 9, 10 NOESY correlations (acetone-d6, 400 MHz) H-4 ↔ H3-11, H3-12; H6 ↔ H3-12; HRESIMS m/z 251.0910 (calcd for C11H16O5Na, 251.0890). X-ray Crystallographic Analysis of 3 (ref 22). Upon crystallization from MeOH−H2O (20:1) using the vapor diffusion method, colorless crystals were obtained for 3. A crystal (0.50 × 0.30 × 0.30 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 Ǻ at 102(7) K. Crystal data: C11H16O5, M = 228.24, space group orthorhombic, P2(1); unit cell dimensions a = 9.9972(8) Ǻ , b = 7.3788(3) Ǻ , c = 15.5846(10) Ǻ , V = 1117.33(13) Ǻ 3, Z = 4, Dcalcd = 1.357 mg/m3, μ = 0.902 mm−1, F(000) = 488. The structure was solved by direct methods using SHELXL-9720 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 SADABS.21 The 3839 measurements yielded 3839 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0331 and wR2 = 0.0866 [I > 2σ(I)]. Trichocladinol G (4): white powder; mp 135−138 °C; [α]25D −2.2 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 206 (0.78), 207 (0.90), 210 (0.73) nm; CD (c 1.0 × 10−4 M, MeOH) λmax (Δε) 224 (−1.40) nm; IR (neat) νmax 3253 (br), 3095, 2986, 1766, 1484, 1336, 1156, 1083, 990 cm−1; 1H and 13C NMR data see Table 2; HMBC correlations (acetone-d6, 400 MHz) H-3 → C-4, 5, 11; H-4 → C-1, 3, 5, 6, 11; H-6 → C-1, 7, 10; H-7 → C-1, 5, 6, 8, 9; H-8 → C-7, 10; H-9 → C-5, 7, 10, 12; H-10 → C-1, 4, 5, 8, 9, 12; H3-11 → C-3, 4; H3-12 → C-5, 9, 10; NOESY correlations (acetone-d6, 400 MHz) H-4 ↔ H3-12; H-6 ↔ H3-11; H-10 ↔ H-3, 6, 11; H3-12↔ H-3, 4, 9; HRESIMS m/z 251.0892 (calcd for C11H16O5Na, 251.0890). X-ray Crystallographic Analysis of 4 (ref 23). Upon crystallization from MeOH−H2O (30:1) using the vapor diffusion method, colorless crystals were obtained for 4. A crystal (0.50 × 0.40 × 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 Mo Kα radiation, λ = 0.7107 Ǻ at 103(1) K. Crystal data: C11H18O6, M = 246.25, space group orthorhombic, P2(1)2(1)2(1); unit cell dimensions a = 6.6087(5) Ǻ , b = 6.6339(4) Ǻ , c = 27.605(2) Å, V = 10.23(15) Ǻ 3, Z = 4, Dcalcd = 1.352 mg/m3, μ = 0.110 mm−1, F(000) = 528. The

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ASSOCIATED CONTENT

* Supporting Information S

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H and 13C NMR spectra of 1−5 and CD spectra of 1−4. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation of China (30925039), Beijing Natural Science Foundation (5111003), the Ministry of Science and Technology of China (2012ZX09301-003), and the Chinese Academy of Sciences (KSCX2-EW-G-6).

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REFERENCES

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Dioxatricyclic and Oxabicyclic Polyketides from Trichocladium opacum

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