Article pubs.acs.org/jnp
Guanacastane Diterpenoids from the Plant Endophytic Fungus Cercospora sp. Yu Feng,†,⊥ Fengxia Ren,‡ Shubin Niu,†,⊥ Lin Wang,†,⊥ Li Li,§ 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 and Toxicology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, People’s Republic of China § Institute of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College, Beijing 100050, People’s Republic of China ⊥ University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China S Supporting Information *
ABSTRACT: Cercosporenes A−F (1−6, respectively), six new guanacastane diterpenes, including a homodimer (5) and a heterodimer (6), were isolated from the crude extract of the fungus Cercospora sp., endophytic to the medicinal plant Fallopia japonica. The structures of 1−6 were elucidated by nuclear magnetic resonance experiments, and 4 and 5 were further confirmed by X-ray crystallography. The absolute configuration of 1 and 3 was assigned by electronic circular dichroism calculations, whereas that of 6 was deduced by the application of the circular dichroism exciton chirality method. In addition to its cytotoxicity against a panel of five human tumor cell lines, HeLa, A549, MCF-7, HCT116, and T24, heterodimer 6 also induced autophagy in HCT116 cells.
against five human tumor cell lines, HeLa (cervical epithelium), A549 (lung carcinoma epithelial), MCF-7 (breast cancer), HCT116 (colon carcinoma), and T24 (bladder carcinoma). Fractionation of the crude extract afforded cercosporenes A−F (1−6, respectively), six new guanacastane diterpenoids, including a homodimer (5) and a heterodimer (6). Details of the isolation, structure elucidation, and biological activity of 1− 6 are reported herein.
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lant endophytic fungi are microorganisms that inhabit the internal tissues of the hosts for all or part of their life cycles without causing apparent pathogenic symptoms.1 This class of fungi has been demonstrated to be a valuable source of bioactive natural products.2,3 A notable example is the anticancer drug paclitaxel, a highly functionalized diterpene that was initially isolated from the Pacific yew tree, Taxus brevifolia,4 and later found in low yields from several species of the plant endophytic fungi in the genus Pestalotiopsis.5−8 Terpenoids are the largest group of natural products, with more than 25000 structures being reported to date, most of which were isolated from plants.9−12 Terpenoids not only possess highly diverse chemical structures but also show significant biological effects.12−14 In nature, they are known to function as metabolic controls, mediating inter- and intraspecies antagonistic and beneficial interactions among organisms.15−17 Members of this class of compounds are also frequently encountered as fungal secondary metabolites.18 Examples include libertellenones A−D,19 asperolides A−C,20 and thiersinines A and B.21 In an effort to search for new cytotoxic natural products from fungi of unique niches,22 a strain of Cercospora sp., which was isolated from leaves of the medicinal plant Fallopia japonica collected at Mingyue Mountain, Jiangxi Province, People’s Republic of China, was chemically investigated. An EtOAc extract prepared from solid substrate fermentation products of the fungus showed cytotoxicity © 2014 American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Cercosporene A (1) was assigned a molecular formula of C20H26O5 (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 three exchangeable protons (δH 3.77, 4.11, and 7.92), four methyl groups, four methylenes with one oxygenated, two methines (including one O-methine), two sp3 quaternary carbons, six olefinic carbons (one of which was protonated), and two ketone carbons (δC 187.4 and 199.3). These data accounted for all the NMR resonances of 1 and five of the eight unsaturations, suggesting that 1 was a tricyclic compound. Interpretation of the 1H−1H COSY NMR data of 1 revealed the presence of Received: November 20, 2013 Published: February 27, 2014 873
dx.doi.org/10.1021/np4009688 | J. Nat. Prod. 2014, 77, 873−881
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Table 1. NMR Data for 1 and 2 1 position
δC,a multiplet
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OH-3 OH-10 OH-15
133.2, qC 199.3, qC 68.4, CH 46.8, CH2 38.0, qC 34.4, CH2 30.3, CH2 43.8, qC 155.0, qC 150.5, qC 187.4, qC 142.0, qC 126.2, CH 160.6, qC 55.4, CH2 26.1, CH3 19.7, CH3 25.7, CH 19.8, CH3 19.9, CH3
a
2
δHb [J (Hz)]
HMBCa
4.60, ddd (14.0, 5.5, 2.0) 2.14, m; 1.86, m
2, 4 2, 3, 5, 6, 14
2.65, m; 1.78, m 1.98, m; 1.76, m
5, 7, 8, 14, 16 5, 8, 9
7.14, s
1, 2, 5, 8, 11, 12, 14
4.19, 1.13, 1.18, 2.56, 1.33, 1.31, 4.11, 7.92, 3.77,
1, 4, 7, 8, 9, 9, 2, 9, 1,
m s s m d (7.0) d (7.0) m s m
δC,a multiplet 130.5, qC 200.1, qC 69.2, CH 47.6, CH2 38.4, qC 35.3, CH2 31.3, CH2 44.6, qC 152.7, qC 151.7, qC 188.1, qC 142.5, qC 127.6, CH 158.2, qC 12.1, CH3 27.1, CH3 26.1, CH3 34.8, CH 15.5, CH3 65.8, CH2
2, 14 5, 6, 14 8, 9, 12 9, 10, 19, 20 18, 20 18, 19 3, 4 10, 11 15
δHb [J (Hz)]
HMBCa
4.45, dd (14.0, 5.5) 2.02, dd (13.0, 5.5); 1.92, dd (14.0, 13.0)
2, 4, 5 2, 3, 5, 6, 14, 16
2.53, m; 1.75, m 1.94, m; 1.78, m
5, 7, 8, 14, 16 5, 8, 12
6.86, s
1, 5, 8, 11, 12
1.62, 1.00, 1.13, 2.45, 1.27, 3.80, 4.09,
1, 4, 7, 8, 9, 9,
s s s m d (7.0) dd (10.0, 6.0); 3.69, dd (10.0,6.0) brs
2, 14 5, 6, 14 8, 9, 12 9, 19, 20 18, 20 18, 19
Recorded at 125 MHz in acetone-d6. bRecorded at 500 MHz in acetone-d6.
three isolated spin systems, which were C-3−C-4 (including OH-3), C-6−C-7, and C-19−C-20 (via C-18). In the HMBC spectrum of 1, correlations from H3-16 to C-4, C-5, C-6, and C14 led to the connections of C-4, C-6, and C-14 to C-5. HMBC correlations from H-3 and H2-4 to C-2 and from H2-15 to C-1, C-2, and C-14 established a cyclohexenone unit with an oxymethylene and a free hydroxy group attached to C-1 and C3, respectively. HMBC cross-peaks from H-13 to C-11 (δC 187.4), C-12 (δC 142.0), and C-14 (δC 160.6) established an α,β-unsaturated ketone moiety (C-11−C-13) located at C-14 of the cyclohexenone ring. In turn, correlations from H3-17 to C7, C-8, C-9, and C-12 indicated that C-7, C-9, and C-17 are all connected to C-8, leading to completion of a cycloheptene ring fused to the cyclohexenone at C-5/C-14. Additional crosspeaks from OH-10 to C-9, C-10, and C-11 established a cyclopentenone ring fused to the cycloheptene at C-8/C-12, completing the same 7,8,8a,9,10,10a-hexahydrobenzo[f ]azulene-3,6-dione core that is seen in guanacastepenes A− C.23−25 Finally, HMBC correlations from OH-15 to C-1 and C15 and from H3-19 and H3-20 to C-9 located the carbinol unit and the isopropyl group at C-1 and C-9, respectively, permitting assignment of the planar structure for cercosporene A. The relative configuration of 1 was proposed on the basis of NOESY data (Figure 1A). NOESY correlations of H-6a (δH 2.65) with H-3 and H3-17 revealed their proximity in space, whereas those of H-6b (δH 1.78) with H3-16 placed these protons on the opposite face of the ring system. The absolute configuration of 1 was deduced by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by time-dependent density functional theory (TDDFT)26 for enantiomers (3R,5R,8R)-1 (1a) and (3S,5S,8S)-1 (1b). The MMFF94 conformational search followed by reoptimization using TDDFT at the B3LYP/6-
Figure 1. Key NOESY correlations for compounds 1−6 (A−F, respectively) in three-dimensional models.
31G(d) basis set level afforded four lowest-energy conformers for 1a and 1b (Figure S45 of the Supporting Information). The overall calculated ECD spectra of 1a and 1b were then generated by Boltzmann weighting of the four conformers with 75.01, 18.75, 2.93, and 3.31% populations by their relative free energies (Figure 2). The absolute configuration of 1 was then extrapolated by comparison of the experimental and calculated ECD spectra of 1a and 1b (Figure S46 of the Supporting 874
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C NMR data (Table 1) revealed the same 7,8,8a,9,10,10ahexahydrobenzo[f ]azulene-3,6-dione core structure that is found in 1, except that the C-20 methyl group (δH 1.31 and δC 19.9) and the C-15 oxymethylene (δH 4.19 and δC 55.4) in 1 were replaced by a carbinol moiety (δH 3.80 and 3.69 and δC 65.8) and a methyl group (δH 1.62 and δC 12.1), respectively, which were supported by relevant HMBC correlations. The relative configuration of the core structure in 2 was determined to be the same as that of 1 by analysis of NOESY data (Figure 1B). A NOESY correlation of H3-17 with H-18 indicated that these protons adopt the same orientation. Because the CD spectrum of 2 (Figure S42 of the Supporting Information) was nearly identical to that of 1, the absolute configuration of 2 was therefore deduced to be 3R,5R,8R,18S. The molecular formula of cercosporene C (3) was established as C20H26O4 (eight degrees of unsaturation) on the basis of HRESIMS data. Although the 1H and 13C NMR spectra of 3 showed structural features similar to the features of those of 1, the resonances corresponding with the cyclohexenone and cyclopentenone units are significantly different from those of 1 (Table 2). Specifically, the C-9 and C-10 enol carbons of the cyclopentenone ring in 1 were reduced to a methine and a methylene, respectively, as evidenced by the 1 H−1H COSY correlations of H-9 with H2-10 and H-18, whereas the C-3/C-4 fragment of the cyclohexenone in 1 was oxidized to an enol moiety, which was supported by the HMBC correlations from the newly observed olefinic proton at 6.05 ppm (H-4) to C-2, C-5, and C-14, and chemical shifts for the newly observed olefinic carbons at 127.1 (C-4) and 161.4 (C-3) ppm. Therefore, the planar structure of cercosporene C was proposed as shown in 3. Analysis of the NOESY data (Figure 1C) for relevant protons permitted assignment of the relative configuration of 3. Correlations of H-7a with H3-16 and H-9 and of H-7b with
Figure 2. Experimental CD spectrum of 1 in MeOH and the calculated ECD spectra of (3R,5R,8R)-1 (1a) and (3S,5S,8S)-1 (1b).
Information). The experimental CD spectrum of 1 was nearly identical to the calculated ECD curve of (3R,5R,8R)-1 (1a), both showing one positive Cotton effect (CE) in the range of 230−250 nm and two negative CEs in the ranges of 280−320 and 330−350 nm (Figure 2). Therefore, the absolute configuration of 1 was deduced to be 3R,5R,8R. Cercosporene B (2) was assigned the same molecular formula, C20H26O5, as 1 by HRESIMS. Analysis of its 1H and Table 2. NMR Data for 3 and 4 3
4
position
δ C, a multiplet
1 2 3 4 5 6 7 8 9 10
135.4, qC 180.9, qC 161.4, qC 127.1, CH 40.5, qC 35.9, CH2 36.3, CH2 46.1, qC 51.2, CH 40.5, CH2
11 12 13 14 15
203.4, qC 150.3, qC 127.1, CH 159.4, qC 54.5, CH2
7.28, s
1, 5, 8, 11, 12
4.47, d (12.0); 4.34, d (12.0)
1, 2, 14
212.6, qC 66.5, CH 82.0, CH 167.3, qC 72.0, CH2
16 17 18 19 20
23.7, 18.0, 28.3, 21.7, 23.4,
1.31, 1.10, 1.85, 0.99, 1.09,
4, 7, 8, 9, 9,
22.6, 21.2, 38.7, 18.2, 67.3,
a
CH3 CH3 CH CH3 CH3
δHb [J (Hz)]
HMBCa
6.05, s
2, 5, 6, 14, 16
1.99, m; 1.87, m 2.20, m; 1.86, m
5, 7, 8, 16 5, 8
1.79, m 2.50, dd (18.0, 7.5); 2.28, dd (18.0, 13.0)
8, 10, 17, 18, 19, 20 8, 9, 11, 12, 18
s s m d (6.5) d (6.5)
5, 6, 14 8, 9, 12 9, 10, 19, 20 18, 20 18, 19
δ C, a multiplet 131.9, qC 190.5, qC 35.2, CH2 39.7, CH2 35.8, qC 36.1, CH2 34.2, CH2 44.6, qC 48.9, CH 41.6, CH2
CH3 CH3 CH CH3 CH2
δHb [J (Hz)]
HMBCa
2.70, m; 2.33, m 1.99, m; 1.91, m
2, 4, 5 2, 3, 16
1.67, m; 1.63, m 2.01, m
4, 5, 7, 8, 16 5, 8
2.39, m 2.62, m; 2.06, m
7, 8, 10, 17, 18, 19, 20 8, 11
2.03, d (10.5) 5.28, ddd (10.5, 5.0, 1.5)
7, 11, 13, 17 1, 8, 12, 14, 15
4.59, dd (12.0, 5.0); 4.52, dd (12.0, 1.5) 1.35, s 1.00, s 1.72, m 1.16, d (7.0) 3.58, m; 3.42, m
1, 13, 14 4, 7, 8, 9, 9,
5, 6, 14 8, 9, 12 9, 10, 19, 20 18, 20 18, 19
Recorded at 125 MHz in acetone-d6. bRecorded at 500 MHz in acetone-d6. 875
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established a 2,5-dihydrofuran unit fused to the cycloheptane ring. The ketone C-2 atom is now required to be attached to C1 to satisfy the unsaturation requirement of 4, even though no additional evidence of this linkage was provided by the HMBC data. Collectively, these data permitted the tentative assignment of the planar structure of 4. The proposed structure for cercosporene D (4) was confirmed by single-crystal X-ray crystallographic analysis. The perspective ORTEP plot is shown in Figure 4. The Xray data also allowed assignment of its relative configuration.
H3-17 indicated that H-9 adopts the same orientation as H3-16. The absolute configuration of 3 was also deduced by comparison of the experimental and calculated ECD spectra generated for enantiomers (5R,8R,9R)-3 (3a) and (5S,8S,9S)-3 (3b) (Figure 3). The MMFF94 conformational search followed
Figure 4. Thermal ellipsoid representation of 4. Note that a different numbering system is used for the structural data deposited with the CCDC.
The absolute configuration of the C-5 sp3 quaternary carbon in 4 was deduced using the helicity rule for the CEs arising from the π → π* transition of the α,β-unsaturated ketones.28 The negative CE observed in the range of 240−260 nm (Kband) of the CD spectrum (Figure S43 of the Supporting Information) suggested the 5R absolute configuration. Considering the relative configuration established by X-ray data, the absolute configuration of 4 was deduced to be 5R,8R,9R,12S,13R,18S. The elemental composition of cercosporene E (5) was determined to be C40H50O8 (16 degrees of unsaturation) by HRESIMS. Even though the 1H and 13C NMR data of 5 (Table 3) were nearly identical to those of 1, the NMR spectra of 5 showed only half of the resonances required by the elemental composition, indicating that 5 is a homodimer of 1 and is structurally related to radianspene M.27 In addition, resonances for the C-15 methylene were shifted upfield to 2.05 and 2.18 ppm because of the absence of the hydroxy group, indicating that 5 originates from two units of 1 via a dimethylene linkage. The relative configuration of 5 was deduced by analogy to 1 (Figure 1E) and confirmed by X-ray crystallography using Cu Kα radiation (Figure 5). In addition, the presence of a relatively high percentage of oxygen in 5 and the value of the Flack parameter, 0.07(19),29 determined by X-ray analysis also permitted assignment of the 3R,5R,8R,3′R,5′R,8′R absolute configuration. The molecular formula of cercosporene F (6) was determined to be C40H52O8 on the basis of HRESIMS data. The NMR data of 6 (Table 3) showed structural characteristics of 5, possessing one unit of the same monomer that is found in 5. However, those for other portions of 6 were different, indicating that 6 is a heterodimer related to 5. Interpretation of corresponding 1H−1H COSY and HMBC data established a 3hydroxy-4,5,6,7,8,8a-hexahydroazulen-2(1H)-one substructure fused to the cyclohexenone unit at C- 5′/C-14′ in 6, compared
Figure 3. Experimental CD spectrum of 3 in MeOH and the calculated ECD spectra of (5R,8R,9R)-3 (3a) and (5S,8S,9S)-3 (3b) after a UV correction of 30 nm.
by B3LYP/6-31G(d) DFT reoptimization afforded 11 lowestenergy conformers for two enantiomers (Figure S47 of the Supporting Information). The calculated ECD spectra of enantiomers 3a and 3b were then generated by Boltzmann weighting of the conformers (Figure S48 of the Supporting Information). The experimental CD spectrum of 3 was nearly identical to the calculated ECD curve of 3a, suggesting the 5R,8R,9R absolute configuration. Cercosporene D (4) gave a pseudomolecular ion [M + H]+ peak by HRESIMS, consistent with the molecular formula C20H28O4 (seven degrees of unsaturation). Its 1H and 13C NMR spectra showed resonances for three methyl groups, seven methylenes (including two oxygenated), three methines with one O-methine, two sp3 quaternary carbons, two olefinic carbons, and two ketone carbons (δC 190.5 and 212.6). These data accounted for all the NMR resonances of 4 except an exchangeable proton, revealing similar structural features for guanacastepene E25 and radianspene G.27 Analysis of the 1H and 13C NMR data of 4 (Table 2) revealed the same cyclopentanone moiety that is found in 3, but with a hydroxypropan-2-yl group attached to C-9 instead of an isopropyl unit, which was verified by relevant 1H−1H COSY and HMBC correlations. Further analysis of the two-dimensional (2D) NMR data indicated that the cyclopentanone in 4 is fused to a cycloheptane ring at C-8/C-12, rather than a cycloheptene in 3. HMBC cross-peaks from H2-4 to C-2 and C16 revealed the connections of the C-3−C-4 fragment to both C-2 and C-15. In turn, correlations from the oxymethine proton H-13 to C-1, C-14, and C-15 and from the oxymethylene protons H2-15 to C-1, C-13, and C-14 876
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Table 3. NMR Data for 5 and 6 5
a
position
δC,a multiplet
1/1′ 2/2′ 3/3′ 4/4′ 5/5′ 6/6′ 7/7′ 8/8′ 9/9′ 10/10′ 11/11′ 12/12′ 13/13′ 14/14′ 15/15′ 16/16′ 17/17′ 18/18′ 19/19′ 20/20′ OH-3/3′ OH-10/10′
134.3, qC 200.1, qC 68.4, CH 47.4, CH2 38.2, qC 34.5, CH2 30.4, CH2 43.7, qC 156.1, qC 150.9, qC 188.0, qC 142.0, qC 126.9, CH 156.8, qC 25.7, CH2 26.8, CH3 20.2, CH3 25.7, CH 20.4, CH3 20.3, CH3
6 δHb [J (Hz)]
4.38, m 1.94, m; 1.82, m 2.49, m; 1.61, m 1.98, m; 1.54, m
6.91, s 2.18, 0.97, 1.05, 2.45, 1.20, 1.18, 5.13, 9.17,
m; 2.05, m s s m d (7.0) d (7.0) d (4.5) s
position
δC,a multiplet
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OH-3 OH-10
134.0, qC 200.0, qC 68.3, CH 47.4, CH2 38.3, qC 34.4, CH2 30.4, CH2 43.8, qC 156.2, qC 151.0, qC 188.0, qC 141.9, qC 126.9, CH 157.3, qC 24.8, CH2 26.8, CH3 20.8, CH3 25.7, CH 20.7, CH3 20.7, CH3
δHb [J (Hz)]
4.46, m 1.97, m; 1.80, m 2.49, m; 1.63, m 1.85, m; 1.56, m
6.96, s 2.11, 0.97, 1.06, 2.49, 1.19, 1.17, 5.20, 9.23,
m; 1.85, m s s m d (5.0) d (5.0) d (4.5) s
position
δC,a multiplet
1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 16′ 17′ 18′ 19′ 20′ OH-3′ OH-11′
133.0, qC 199.3, qC 68.3, CH 49.4, CH2 40.8, qC 35.2, CH2 41.8, CH2 44.0, qC 61.1, CH 203.3, qC 148.2, qC 148.7, qC 29.5, CH2 161.2, qC 26.4, CH2 24.8, CH3 17.0, CH3 27.7, CH 19.2, CH3 23.4, CH3
δHb [J (Hz)]
4.24, m 1.83, m; 1.72, m 2.09, m; 1.44, m 1.74, m; 1.39, m 1.89, m
3.62; 3.56, d (19.0) 2.26, 0.99, 0.97, 1.96, 0.75, 1.09, 5.50, 9.11,
m; 1.95, m s s m d (5.0) d (5.0) d (4.5) s
Recorded at 125 MHz in DMSO-d6. bRecorded at 500 MHz in DMSO-d6.
Figure 6. CD spectra of 1 and 6 in MeOH.
proximity in space, whereas those of H-7′a (δH 1.74) with H-9′ and H3-16′ placed them on the opposite face of the molecule. The absolute configuration of the C-3′, C-5′, C-8′, and C-9′ stereogenic atoms in 6 was deduced via the CD data (Figure 7).30 Because of the transition reaction of the two α,βunsaturated ketone chromophores, the subtracted CD curve showed the negative first CE at 304 nm (Δε of −5.12) and positive second CE at 269 nm (Δε of 2.57) around the UV maximum (λmax of 277 nm), suggesting the 3′R,5′R,8′R,9′R absolute configuration for the other portion of 6. Therefore, 6 was deduced to have the 3/3′R,5/5′R,8/8′R,9′R absolute configuration. Compounds 1−6 were tested for cytotoxicity against a panel of five human tumor cell lines, HeLa, A549, MCF-7, HCT116, and T24 (Table 4). Heterodimer 6 was cytotoxic to all the cell lines tested, showing IC50 values of 19.3, 29.7, 46.1, 21.3, and 8.16 μM, respectively (the positive control cisplatin showed IC50 values of 18.2, 21.1, 10.2, 19.8, and 11.6 μM, respectively), whereas 1−5 did not show detectable activity at 50 μM.
Figure 5. Thermal ellipsoid representation of 5. Note that a different numbering system is used for the structural data deposited with the CCDC.
to a 2-hydroxy-4,5,6,7-tetrahydroazulen-1(3aH)-one moiety found in 5, thereby completing the planar sturcture of 6 as shown. Because 6 incorporated the same monomer as 5, the relative and absolute configurations of this portion of the compound were proposed by comparison of the 1H and 13C NMR data with those of 5 and verified via the NOESY (Figure 1F) and CD (Figure 6) data. The relative configuration of the other portion of 6 was proposed on the basis of the following NOESY data. Correlations of H-6′a (δH 2.09) with H-3′ and H3-17′ and of H3-17′ with H-7′b (δH 1.39) revealed their 877
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Figure 8. Compound 6 induces accumulation of LC3-II and enhances autophagic flux. (A) Electron microscopy of HCT116 cells treated with compound 6 (10 μM, 2 h). (B) HCT116 cells were transfected with a plasmid expressing GFP-LC3 for 12 h, and the cells were incubated at 37 °C in RPMI-1640 medium with 1/1000 DMSO (top) and 10 μM compound 6 (bottom). Following fixation, cells were stained with DAPi and immediately visualized by fluorescence microscopy. (C) HCT116 cells were treated with 2.5−10 μM compound 6 for 1 h. Densitometry was performed for quantification, and the ratios (representative of three experiments) of LC3-II to actin are presented in the graphs below the blots.
Figure 7. Subtracted CD (top) and UV (bottom) spectra of 6 in MeOH. Arrows denote the electric transition dipole of two α,βunsaturated ketone chromophores.
cyclopentenone units. Compound 4 is structurally similar to guanacastepene E25 and radianspene G;27 the latter was isolated from a fungal strain of Coprinus radian M65. Compounds 5 and 6 are closely related to radianspene M,27 a homodimer isolated from the fungus C. radian M65, and represent the second example of the dimeric guanacastepene.
Compound 6 was also investigated for its autophagyinducing effect in HCT116 cells. After incubation with 6 in RPMI-1640 medium for 2 h, the obvious accrual of membrane vacuoles was observed in the cells, and autophagosome-like vacuoles with double-membrane structures were also observed (Figure 8A). To further measure autophagosome formation, cells treated with 6 were transfected with a fusion protein between green fluorescent protein (GFP) and LC3 (a specific marker of autophagosomes) and visualized by fluorescence microscopy. The treated cells showed increased levels of punctuate staining of GFP-LC3 compared to those of untreated ones (Figure 8B). Because the ratio of LC3-II to actin is an accurate indicator of autophagy, the expression of LC3- II in HCT116 cells was detected by Western blotting (Figure 8C). The ratio of LC3- II to actin in the cells treated with 6 for 2 h was increased compared to the control, indicating that 6 induces the autophagic process in HCT116 cells. Cercosporenes A−F (1−6, respectively) are new members of the guanacastane diterpene class of natural products.31 Compounds 1−3 are closely related to guanacastepenes A− C,23−25 respectively, which are the notable representatives of the guanacastepenes isolated from an endophytic fungus CR115 as antibacterial agents against the drug resistant strains of Staphylococcus aureus and Enterococcus faecalis.24,25 However, compounds 1−3 differ from the precedents by having different substituents and substitution patterns at the cyclohexenone and
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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 Inova-500 and NMR system-600 spectrometers using solvent signals (acetone-d6, δH 2.05 and δC 29.8 and 206.1; DMSO-d6, δH 2.50 and 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 (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 to 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 a rate of 1.03 spectra/s. HPLC separations were performed on an Agilent 1200 instrument equipped with a variablewavelength UV detector. Systematic conformational analysis for 1 and
Table 4. Cytotoxicity of Compound 6a IC50 (μM)
a
compound
HeLa
A549
MCF-7
HCT116
T24
6 cisplatin
19.3 ± 0.67 18.2 ± 1.2
29.7 ± 1.1 21.1 ± 1.0
46.1 ± 0.10 10.2 ± 1.4
21.3 ± 0.76 19.8 ± 1.8
8.16 ± 1.2 11.6 ± 0.46
Compounds 1−5 were inactive at 50 μM. 878
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(−26.6) nm; IR (neat) νmax 3369 (br), 2968, 2935, 2875, 1680, 1625, 1456, 1408, 1307, 1149, 1005 cm−1; 1H, 13C, and HMBC NMR data in Table 1; NOESY correlations (acetone-d6, 500 MHz) H-3 ↔ H-6a, H6a ↔ H3-17, H-6b ↔ H3-16, H-7a ↔ H3-16, H-7b ↔ H3-17, H-7b ↔ H-18, H3-17 ↔ H-18; HRESIMS m/z 347.1855 (calcd for C20H27O5, 347.1853). Cercosporene C (3): colorless oil; [α]25D −116.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 200 (3.53), 233 (3.39), 286 (3.59) nm; CD (c 3.0 × 10−4 M, MeOH) λmax (Δε) 220 (+11.7), 283 (−15.6), 355 (+1.27) nm; IR (neat) νmax 3412 (br), 2961, 2936, 2873, 1720, 1633, 1462, 1388, 1329, 1226, 1120, 1039, 1001 cm−1; 1H, 13C, and HMBC NMR data in Table 2; NOESY correlations (acetone-d6, 500 MHz) H6a ↔ H3-17, H-6b ↔ H3-16, H-7a ↔ H3-16, H-7a ↔ H-9, H-7b ↔ H3-17; HRESIMS m/z 331.1904 (calcd for C20H27O4, 331.1904). Cercosporene D (4): pale yellow powder; mp 115−118 °C; [α]25D +62.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 225 (3.25), 262 (3.44) nm; CD (c 3.0 × 10−4 M, MeOH) λmax (Δε) 209 (+7.53), 277 (−7.70) nm; IR (neat) νmax 3452 (br), 2959, 2932, 2877, 1731, 1670, 1454, 1398, 1283, 1205, 1057, 1033 cm−1; 1H, 13C, and HMBC NMR data in Table 2; NOESY correlations (acetone-d6, 500 MHz) H-6a ↔ H3-17, H-6b ↔ H3-16, H-12 ↔ H3-17, H-13 ↔ H3-16, H3-17 ↔ H18; HRESIMS m/z 333.2058 (calcd for C20H29O4, 333.2060). X-ray Crystallographic Analysis of 4.32 Upon crystallization from acetone using the vapor diffusion method, pale yellow crystals of 4 were obtained. A crystal (0.80 mm × 0.20 mm × 0.05 mm) was separated from the sample and mounted on a glass fiber, and data were collected using a Rigaku RAXIS RAPID IP diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.7107 Å) at 101(8) K. Crystal data: C20H28O4; M = 332.42; space group, orthorhombic P2(1)2(1)2(1); unit cell dimensions a = 8.2270(7) Å, b = 11.0537(9) Å, c = 18.858(13) Å; V = 1714.9(12) Å3; Z = 4; Dcalcd = 1.288 mg/m3; μ = 0.088 mm−1; F(000) = 720. The structure was determined by direct methods using SHELXL-9733 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 the Siemens Area Detector Absorption Program (SADABS).34 The 5848 measurements yielded 3358 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave the following values: R1 = 0.0527, and wR2 = 0.0870 [I > 2σ(I)]. Cercosporene E (5): colorless powder; mp 112−115 °C; [α]25D −77.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 227 (3.27), 282 (3.36) nm; CD (c 3.0 × 10−4 M, MeOH) λmax (Δε) 249 (+16.1), 313 (−13.6) nm; IR (neat) νmax 3370 (br), 2963, 2932, 2873, 1681, 1627, 1408, 1380, 1202, 1150, 1089, 1013 cm−1; 1H and 13C NMR data in Table 3; HMBC data (DMSO-d6, 500 MHz) H-3/3′ → 2/2′, 4/4′; H2-4/4′ → 2/2′, 3/3′, 5/5′, 6/6′, 14/14′; H2-6/6′ → 5/5′, 7/7′, 8/8′, 14/14′, 16/16′; H2-7/7′ → 5/5′, 6/6′, 8/8′, 12/12′, 17/17′; H-13/13′ → 1/1′, 2/2′, 5/5′, 8/8′, 11/11′, 12/12′, 14/14′; H2-15/15′ → 1/1′, 2/2′, 14/14′; H3-16/16′ → 4/4′, 5/5′, 6/6′, 14/14′; H3-17/17′ → 7/ 7′, 8/8′, 9/9′, 12/12′; H-18/18′ → 8/8′, 9/9′, 10/10′, 19/19′, 20/20′; H3-19/19′ → 9/9′, 18/18′, 20/20′; H3-20/20′ → 9/9′, 18/18′, 19/ 19′; OH-3/3′ → 2/2′, 3/3′, 4/4′; OH-10/10′ → 9/9′, 10/10′, 11/11′; HRESIMS m/z 659.3578 (calcd for C40H51O8, 659.3578). X-ray Crystallographic Analysis of 5.35 Upon crystallization from acetone using the vapor diffusion method, colorless crystals of 5 were obtained. A crystal (0.30 mm × 0.10 mm × 0.08 mm) was separated from the sample and mounted on a glass fiber, and data were collected using a Rigaku RAXIS RAPID IP diffractometer with graphite-monochromated Cu Kα radiation (λ = 1.54184 Å) at 104(2) K. Crystal data: C40H50O8; M = 658.80; space group, monoclinic C2; unit cell dimensions a = 12.8255(6) Å, b = 11.1968(4) Å, c = 12.6545(6) Å; V = 1736.14(13) Å3; Z = 2; Dcalcd = 1.260 mg/m3; μ = 0.698 mm−1; F(000) = 708. The structure was determined by direct methods using SHELXL-9733 and refined by using full-matrix leastsquares difference Fourier techniques. All non-hydrogen atoms were refined with anisotropic displacement parameters, and all hydrogen
3 was performed via Molecular Operating Environment (MOE) version 2009.10 (Chemical Computing Group, Montreal, QC) using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational analysis was further optimized using TDDFT at the B3LYP/6-31G(d) basis set level. The stationary points have been checked as the true minima of the potential energy surface by verifying they do not exhibit vibrational imaginary frequencies. The 30 lowest electronic transitions were calculated, and the rotational strengths of each electronic excitation were given using both dipole length and dipole velocity representations. ECD spectra were stimulated using a Gaussian function with halfbandwidths of 0.35 and 0.4 eV. Equilibrium populations of conformers at 298.15 K were calculated from their relative free energies (ΔG) using Boltzmann statistics. The overall ECD spectra were then generated according to Boltzmann weighting of each conformer. The systematic errors in the prediction of the wavelength and excited state energies are compensated for by employing UV correlation. All quantum computations were performed using the Gaussian03 package,36 on an IBM cluster machine located at the High Performance Computing Center of Peking Union Medical College. Fungal Material. The culture of Cercospora sp. was isolated from the leaves of F. japonica collected at Mingyue Mountain, Jiangxi Province, People’s Republic of China, in October 2010. The isolate was identified on the basis of morphology and sequence (GenBank entry KF577929) analysis of the ITS region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C for 10 days. Agar plugs were cut into small pieces (∼0.5 cm × ∼0.5 cm × ∼0.5 cm) under aseptic conditions; 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of medium (0.4% glucose, 1% malt extract, and 0.4% yeast extract; the final pH of the medium was adjusted to 6.5 and the mixture sterilized with an autoclave). Three flasks of the inoculated medium were incubated at 25 °C on a rotary shaker at 170 rpm for 5 days to prepare the seed culture. Fermentation was conducted 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 being autoclaved 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 (10.3 g), which was fractionated by silica gel VLC using petroleum ether/ EtOAc gradient elution. The fraction (420 mg) eluted with 30% EtOAc was separated by Sephadex LH-20 column chromatography (CC) eluting with MeOH. The resulting subfractions were purified using reversed-phase (RP) HPLC to afford 3 (4.3 mg; tR = 25.08 min; 55% MeOH in H2O for 30 min; 2 mL/min) and 4 (2.3 mg; tR = 25.08 min; 25−35% CH3CN in H2O over 30 min; 2 mL/min). The fractions (700 mg) eluted with 50 and 55% EtOAc were combined and separated again by Sephadex LH-20 CC using MeOH as eluents, and the resulting subfractions were further purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column, 5 μm, 9.4 mm × 250 mm) to afford 1 (25.0 mg; tR = 37.68 min; 20−30% CH3CN in H2O over 40 min; 2 mL/min), 2 (23.7 mg; tR = 22.63 min; the same gradient as in purification of 1), 5 (7.8 mg; tR = 23.93 min; 50−65% CH3CN in H2O over 30 min; 2 mL/min), and 6 (11.2 mg; tR = 24.92 min; the same gradient as in purification of 5). Cercosporene A (1): colorless needles; mp 113−115 °C; [α]25D −218.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 206 (3.11), 282 (3.17) nm; CD (c 2.9 × 10−4 M, MeOH) λmax (Δε) 246 (+22.2), 282 (−17.9), 341 (−9.76) nm; IR (neat) νmax 3359 (br), 2961, 2930, 2873, 1679, 1625, 1456, 1409, 1379, 1152, 1039 cm−1; 1H, 13C, and HMBC NMR data in Table 1; NOESY correlations (acetone-d6, 500 MHz) H3 ↔ H-6a, H-6a ↔ H3-17, H-6b ↔ H3-16, H3-17 ↔ H-18, H-7a ↔ H3-16, H-7b ↔ H3-17; HRESIMS m/z 347.1857 (calcd for C20H27O5, 347.1853). Cercosporene B (2): colorless oil; [α]25D −194.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 204 (3.09), 239 (2.98), 287 (3.25) nm; CD (c 2.9 × 10−4 M, MeOH) λmax (Δε) 242 (+37.3), 289 (−28.9), 329 879
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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).34 The 5373 measurements yielded 3311 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave the following values: R1 = 0.0366, and wR2 = 0.0976 [I > 2σ(I)]. Cercosporene F (6): colorless oil; [α]25D −90.5 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 230 (3.13), 277 (3.50) nm; CD (c 3.0 × 10−4 M, MeOH) λmax (Δε) 233 (+23.5), 287 (−19.3), 340 (−9.44) nm; IR (neat) νmax 3366 (br), 2964, 2936, 2874, 1678, 1626, 1458, 1409, 1381, 1221, 1177, 1150, 1089 cm−1; 1H and 13C NMR data in Table 3; HMBC data (DMSO-d6, 500 MHz) H-3 → 2, 4; H2-4 → 2, 3, 5, 6, 16; H2-6 → 5, 7, 8, 14, 16; H2-7 → 6, 8, 12, 17; H-13 → 1, 2, 5, 8, 11, 12; H2-15 → 1, 2, 14, 15′; H3-16 → 4, 5, 6, 14; H3-17 → 7, 8, 9, 12; H-18 → 8, 9, 10, 19, 20; H3-19 → 9, 18, 20; H3-20 → 9, 18, 19; H3′ → 4′; H2-4′ → 2′, 3′, 5′, 6′, 16′; H2-6′ → 5′, 8′, 14′; H2-7′ → 5′, 8′, 12′, 14′; H-9′ → 7′, 8′, 10′, 12′, 18′, 19′, 20′; H2-13′ → 1′, 5′, 8′, 11′, 12′, 14′; H2-15′ → 15, 1′, 2′, 14′; H3-16′ → 4′, 5′, 6′, 14′; H3-17′ → 7′, 8′, 9′, 12′; H-18′ → 8′, 9′, 10′, 19′, 20′; H3-19′ → 9′, 18′, 20′; H320′ → 9′, 18′, 19′; OH-3 → 2, 3, 4; OH-10 → 9; OH-3′ → 2′, 3′, 4′; OH-11′ → 11′, 12′; NOESY correlations (acetone-d6, 500 MHz) H-3 ↔ H-6a, H-6a ↔ H3-17, H-6b ↔ H3-16, H-7a ↔ H3-16, H-7b ↔ H317, H-3′ ↔ H-6a′, H-6a′ ↔ H3-17′, H-6b′ ↔ H3-16′, H-7a′ ↔ H3-16′, H-7b′ ↔ H3-17′, H-7a′ ↔ H-9′; HRESIMS m/z 661.3735 (calcd for C40H53O8, 661.3735). (MTS) Assay.37 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 a 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. Cell Culture and Western Blotting. Polyclonal antibodies against LC3 (L7543) were purchased from Sigma-Aldrich. Antibodies to actin (sc-1616) were purchased from Santa Cruz Biotechnology. HCT116 human colon carcinoma cells were grown in RPMI-1640 medium (HyClone, catalog no. SH30809.01B) supplemented with fetal bovine serum and antibiotics. Cells were split overnight and grown to 50% confluence before the addition of compound 6. Whole cell lysates were prepared by lysis using Triton X-100/glycerol buffer, which contained 50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, and 1 mM dithiothreitol, supplemented with 1% Triton X-100 and protease inhibitors, and then separated on a sodium dodecyl sulfate− polyacrylamide gel electrophoresis gel (15 or 8% according to the molecular weights of the proteins of interest) and transferred to a polyvinylidene fluoride membrane. Western blotting was performed using appropriate primary antibodies and the horseradish peroxidaseconjugated suitable secondary antibodies, followed by detection with enhanced chemiluminescence (Pierce Chemical, catalog no. 34080). Electron Microscopy. Electron microscopy was performed as described previously.38 Briefly, HCT116 cell samples were washed three times with PBS, trypsinized, and collected by centrifugation. The cell pellets were fixed with 4% paraformaldehyde overnight at 4 °C, postfixed with 1% OsO4 in cacodylate buffer at room temperature for 1 h, and dehydrated stepwise with ethanol. The dehydrated pellets were rinsed with propylene oxide for 30 min and then embedded in Spurr resin for sectioning. Images of the thin sections were observed under a transmission electron microscope (model JEM1230) Fluorescence Microscopy. The GFP-LC3 plasmid was a kind gift of T. Yoshimori (Osaka University, Osaka, Japan). HCT116 cells were transfected with the GFP-LC3-expressing plasmid, and after 24 h, cells were treated with compound 6, the fluorescence of GFP-LC3 was
viewed, and the images were acquired via confocal microscopy (Leica, TCS SP5).
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ASSOCIATED CONTENT
* Supporting Information S
1
H, 13C, 2D NMR, and HRESIMS spectra of 1−6; CD spectra of 2, 4, and 5; and UV and CD calculations for 1 and 3. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Telephone and fax: +86 10 66932679. E-mail:
[email protected]. cn. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the Beijing Natural Science Foundation (5111003), the National Program of Drug Research and Development (2012ZX09301-003), and the Chinese Academy of Sciences (KSCX2-EW-G-6).
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REFERENCES
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