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Nov 13, 2015 - alone and with fluconazole on the growth and biofilms of Candida albicans. ... and reverse the tolerance of C. albicans biofilms to flu...
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Synergistic Antifungal Meroterpenes and Dioxolanone Derivatives from the Endophytic Fungus Guignardia sp. Tian-Xiao Li, Ming-Hua Yang,* Xiao-Bing Wang, Ying Wang, and Ling-Yi Kong* State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new meroterpenes (1−9) and one new dioxolanone derivative (10), along with seven known compounds (11−17), were isolated from solid cultures of the endophytic fungus Guignardia sp., obtained from Euphorbia sieboldiana. Their structures were elucidated by analysis of UV, IR, 1D and 2D NMR, and HRESIMS data, and their absolute configurations were determined by a combination of single-crystal X-ray studies, modified Mosher methods, and Rh2(OCOCF3)4- and Mo2(OCOCH3)4-induced electronic circular dichroism experiments. All compounds were evaluated for their inhibitory effects alone and with fluconazole on the growth and biofilms of Candida albicans. At 6.3 μg/mL combined with 0.031 μg/mL of fluconazole, compounds 8 and 16 were found to have prominent inhibition on the growth of C. albicans with fractional inhibitory concentration index values of 0.23 and 0.19, respectively. Combined with fluconazole, both of them (40 μg/mL for 8 and 20 μg/ mL for 16) could also inhibit C. albicans biofilms and reverse the tolerance of C. albicans biofilms to fluconazole. of 252 μg/mL. Further systematic chemical evaluation of this extract led to the isolation of nine new meroterpenes, namely, guignardones J−L (1−3), 13-hydroxylated guignardone A (4), 12-hydroxylated guignardone A (5), 17-hydroxylated guignardone A (6), guignardones M−O (7−9), and a new dioxolanone derivative, 10-hydroxylated guignardianone C (10), together with seven known compounds, guignardones A−C (11−13),9 guignardones G and H (14 and 15),10 guignardic acid (16),11 and palmarumycin C11 (17).12 These meroterpenes are structurally similar to the tricycloalternarenes (TCAs)13 with mixed biosynthetic origin,14 and compounds 4−9, possessing an additional tetrahydrofuran ring, expanded the relatively uncommon tetracyclic subclass of TCAs. More importantly, tetracyclic meroterpenes 8 and 12 were found to have pronounced synergistic inhibition on C. albicans growth at 6.3 μg/mL when combined with 0.031 μg/mL of fluconazole. Meanwhile, when combined with fluconazole, both could inhibit C. albicans biofilms and reverse the tolerance of C. albicans biofilms to fluconazole. The dioxolanone derivative 16

Candida albicans, residing on mucosal surfaces, is a pathogen that causes serious infections in both immunocompromised and healthy people.1 Under certain circumstances, C. albicans bloodstream infections may be life threatening.2 Although many antifungal medicines have been developed, drug resistance of C. albicans is continuously arising, which imposes problems in clinical treatment.3 It has been found that drug tolerance of C. albicans is mainly associated with the overexpression of drug efflux pump genes,4 the change of membrane lipid fluidity,4 and the formation of biofilms.5 To overcome the tolerance, clinical treatment has resorted to drug combination, but it still requires powerful and multiple combination modes.6 Notably, screens of natural products have afforded an increasing number of potential antifungal synergists like beauvericin6 and C15-surfactin.7 Such chemical compounds are promising agents in the combination treatment of fungal infections, since they can synergistically lower dosages and reduce side effects of antifungal drugs and reduce the development of resistance.6 In our ongoing search for potential antifungal agents,8 the crude extract from an Euphorbia sieboldiana endophytic fungal strain, Guignardia sp., showed antifungal activity against C. albicans with a minimal inhibitory concentration (MIC) value © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 6, 2015

A

DOI: 10.1021/acs.jnatprod.5b00008 J. Nat. Prod. XXXX, XXX, XXX−XXX

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was the best synergist and inhibited C. albicans biofilms at 20 μg/mL when combined with 63 μg/mL of fluconazole. Herein, the isolation, structure elucidation, and bioactive studies are described.

structure could exhibit enough anomalous dispersion of Cu Kα radiation with the Flack parameter x = 0.04(9).15 Thus, the absolute configuration of 1 was unambiguously assigned as 4S, 6R, 9S, 10R, 14R. Guignardone K (2), a colorless oil, was a derivative of 1 with one more OH unit deduced from its molecular formula C17H24O6 (m/z 369.1554 [M + COOH]−) and NMR data (Table 1). The downfield shifts of H-12 (δH 4.10, dd) and C-12 (δC 78.4) denoted the hydroxy group at C-12, as evidenced by the HMBC correlations from CH3-11 to C-12 and from H-12 to C-9, C-10, and C-14. In the relative configuration analysis, CH3-11, H-4, H-9, and the isopropenyl group were α-oriented due to the same NOE correlations as those of 1, and the apparent ROESY correlation of H-12/H-14 as well as the lack of an NOE signal between H-12 and H-9 confirmed the βorientation of H-12. In its electronic circular dichroism (ECD) spectrum (Supporting Information S16), the observed Cotton effects were identical with those of 1, suggesting the 4S, 6R, 9S, 10S, 12S, 14R absolute configuration for 2. Guignardone L (3), C17H24O4, was another tricyclic meroterpene and structurally resembled guignardone H10 due to their similar NMR data (Table 1). However, the difference of coupling constants (J = 10.6, 4.5 Hz) between H-6 and H-5 in 3 from those in guignardone H (J = 6.8, 3.6 Hz),10 along with the weak ROESY correlation of H-6/H-4, established the α-orientation of H-6. To determine the absolute configuration of 3, a Rh2(OCOCF3)4-induced ECD method was performed.16 According to the bulkiness rule,17 the positive Cotton effect (Supporting Information S25) at around 350 nm indicated a “bS” configuration of 4-OH, correlating to a 4S absolute configuration. Combined with the ROESY analysis, C6 was determined as R and the absolute configuration of 3 was assigned as depicted. 13-Hydroxylated guignardone A (4) was obtained as a white, amorphous powder. Its molecular formula, C17H22O5, was determined from the HRESIMS ion at m/z 305.1396 [M − H]− (calcd for 305.1394, C 17 H 21 O 5 ). The 1D NMR spectroscopic data of 4 (Table 1) were similar to those of guignardone A (11)9 except for the downfield shifts of H-13 (δH 4.23, dt) and C-13 (δC 69.6), indicating the hydroxylation of C-13. The observed HMBC correlations (Figure 1) from H13 to C-9, C-10, C-12, and C-14 and from H-12 and H-14 to C-13 denoted this above-mentioned deduction, and the correlations from H-4 to C-7 and from H-7 to C-1, C-4, C-5, and C-6 verified the existence of a tetrahydrofuran ring D. Extensive analysis of its HMBC spectrum further confirmed the planar structure of 4. Moreover, judged from their similar NOE correlations (Figure 2), compound 4 had the same relative configuration as guignardone A, and the additional ROESY correlation of H-13/H-14 along with the lack of an NOE signal between H-13 and H-9 collectively designated the β-orientation of H-13. Cotton effects (λ294 −34.6, λ261 +39.7, and λ223 +22.3) in the ECD spectrum (Supporting Information S33) of 4 were in agreement with those in the experimental and calculated ECD curves for guignardone A (11),9 indicating the same absolute configuration of the core. To further confirm this deduction, a modified Mosher method18 was carried out to give (S)- and (R)-MTPA esters 4a and 4b (Figure 4). The positive ΔδH(S−R) values of CH3-11 and H-12 as well as the negative ΔδH(S−R) values of H-16 and CH3-17 unambiguously determined the 13R configuration, also confirming the 4S, 6R, 9S, 10R, 13R, 14R configuration of 4.



RESULTS AND DISCUSSION The fungal strain was cultivated on rice for 30 days, and then the EtOAc extract of the rice cultures was purified by silica gel column chromatography (CC) and preparative HPLC to yield 10 new compounds (1−10), as well as seven known compounds (11−17). Guignardone J (1) was isolated as colorless crystals that had the molecular formula C17H24O5 as established by the negative HRESIMS ion at m/z 353.1608 [M + COOH]− (calcd for 353.1606, C18H25O7) and showing six degrees of unsaturation. The IR absorption bands at 3446 and 1634 cm−1 indicated the presence of hydroxy and carbonyl groups. Its 1H NMR spectrum (Table 1) apparently displayed one terminal olefinic bond (δH 4.72 and 4.63, each 1H, s), one hydroxymethyl (δH 3.71 and 3.64, each 1H, d), and two methyls (δH 1.65, 1.33, each 3H, s). The 13C NMR data (Table 1) combined with HSQC spectra demonstrated the presence of two methyls (δC 23.6, 19.3), six methylenes (δC 111.7, 69.5, 38.2, 37.9, 27.2, 16.5), three methines (δC 65.4, 48.9, 43.8), and five quaternary carbons (δC 199.7, 168.2, 106.4, 88.7). Besides the three degrees of unsaturation taken off by one carbonyl and two olefinic bonds, the remaining three degrees revealed the tricyclic scaffold of 1, which, combined with the analysis of NMR data, suggested that 1 was the TCA class of compounds. A comparison of its 1D NMR data with those of guignardone G10 denoted 1 to be an analogue of guignardone G with an additional hydroxymethyl. The same skeleton was confirmed by the observed HMBC correlations (Figure 1) from H-13 (δH 1.93, m, and δH 1.55, m) to C-9, C-10, and C-14, from H-8 (δH 2.31, d, and δH 2.16, dd) to C-1, C-3, and C-14, and from H-5 (δH 2.23, m) to C-1 and C-3, and the aforementioned hydroxymethyl was located at C-7 by the HMBC correlations from itself to C-1, C-5, and C-6. The relative configuration of 1 was established on the basis of ROESY analysis. The ROESY cross-peaks (Figure 2) of CH311/H-4, CH3-11/H-9, and H-9/H-16b indicated the same orientation of CH3-11, H-4, H-9, and the isopropenyl group, whereas 7-hydroxymethyl might be oriented differently due to the absence of an NOE signal between H-4 and 7hydroxymethyl. This assumption was confirmed by singlecrystal X-ray analysis using colorless needle-like crystals of 1 (Figure 3). Meanwhile, the presence of five oxygen atoms in the B

DOI: 10.1021/acs.jnatprod.5b00008 J. Nat. Prod. XXXX, XXX, XXX−XXX

a

1 2 3 4 5a 5b 6 7a 7b 8a 8b 9 10 11 12a 12b 13a 13b 14 15 16a 16b 17

C

s m ddd (14.0, 10.9, 7.1) m m m

1.33, 2.14, 1.82, 1.93, 1.55, 2.26,

4.72, s 4.63, s 1.65, s

d (11.0) d (11.0) d (17.4) dd (17.7, 6.1) m

3.71, 3.64, 2.31, 2.16, 1.93,

4.41, br s 2.23, m

δH (J in Hz)

Measured in CDCl3. bMeasured in methanol-d4.

19.3

48.9 145.6 111.7

27.2

43.8 88.7 23.6 37.9

16.5

73.5 69.5

δC

199.7 106.4 168.2 65.4 38.2

position

1a δC

19.3

48.6 147.1 112.2

39.1

41.8 90.5 19.2 78.4

17.3

75.1 68.8

200.2 108.5 170.2 66.0 40.4

d (10.9) dd (10.9, 4.4) dd (17.0, 10.3) ddd (17.0, 6.1, 1.7) m

4.73, br s 4.67, br s 1.70, s

2.40, ddd (14.0, 9.4, 6.9) 1.47, ddd (14.0, 8.2, 2.7) 2.25, m

1.30, s 4.10, dd (6.7, 2.6)

3.76, 3.55, 2.29, 2.10, 2.17,

4.39, dt (1.4, 5.5) 2.26, m 2.17, m

δH (J in Hz)

2b

Table 1. 1H and 13C NMR Spectroscopic Data for 1−5 (500, 125 MHz, J in Hz) δC

19.2

50.6 147.5 111.8

22.7

44.4 89.1 28.0 38.6

17.1

78.0 58.7

198.9 107.1 169.5 66.3 36.7 t (3.6) m m dd (10.6, 4.5) s

s m m m m m

4.74, br s 4.65, br s 1.69, s

1.33, 2.11, 1.88, 1.89, 1.60, 2.24,

2.32, d (17.2) 2.07, m 2.01, dd (11.4, 6.7)

4.35, 2.27, 2.10, 4.06, 3.48,

δH (J in Hz)

3b δC

24.2

53.9 141.2 114.6

69.6

39.5 88.2 24.1 48.1

15.4

82.0 70.8

198.9 103.1 172.7 78.6 44.2

s dd (15.6, 6.7) dd (15.6, 1.8) dt (1.5, 5.1)

5.17, s 4.81, s 1.73, s

2.18, dd (13.1, 5.2)

1.38, 2.62, 1.90, 4.23,

3.80, d (7.9) 3.45,d (7.9) 2.42, d (17.6) 2.27, dd (17.1, 6.6) 2.36, dd (13.1, 6.8)

4.53, d (5.5) 2.45, dd (10.7, 5.5) 2.02, d (10.7)

δH (J in Hz)

4a δC

19.2

47.3 144.9 112.4

38.4

40.2 90.5 19.2 77.6

15.6

82.1 70.8

199.0 104.2 172.8 78.5 44.3

d (7.8) d (7.9) t (16.2) m m

4.75, s 4.63, s 1.68, s

2.45, m 1.47, dd (14.2, 9.6) 2.14, m

1.32, s 4.15, br s

3.82, 3.48, 2.33, 2.16, 2.19,

4.54, d (5.2) 2.45, m 2.04, d (10.7)

δH (J in Hz)

5a

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DOI: 10.1021/acs.jnatprod.5b00008 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Selected HMBC correlations (H → C) of 1 and 4.

12-Hydroxylated guignardone A (5) was isolated as an isomer of 4 with the same molecular formula of C17H22O5 (at m/z 305.1395 [M − H]−). The slightly different chemical shifts of the hydroxy methine (δH 4.15, br s, and δC 77.6) from the comparison of their NMR data (Table 1) denoted a different hydroxylated position in 5, which was further demonstrated at C-12 by HMBC correlations from CH3-11 to C-12 and from H-12 to C-9, C-10, and C-14. The absolute configuration of 5 was determined in the same way as for 4. Both the Cotton effects in its ECD spectrum (Supporting Information S43) and the modified Mosher experiment (Figure 4) revealed the 12S configuration. 17-Hydroxylated guignardone A (6) had the same molecular formula as 4 (C17H22O5), suggesting 6 was another isomer of 4. The apparently different proton signal of H-17 (δH 4.07 and 4.00, each 1H, d) and the corresponding downfield carbon signal (δC 65.3) ascertained the hydroxylation at C-17, which was also verified by the HMBC correlations from CH2OH-17 to C-14, C-15, and C-16. Guignardone M (7) was a derivative of 6 with the addition of an acetyl group, as demonstrated by its molecular formula of C19H24O6 (at m/z 349.1643 [M + H]+) and the corresponding NMR signals (δH 2.06, 3H, s, and δC 170.8, 21.1). The key HMBC correlation from δH 2.06 to C-17 located this acetyl group at C-17. In addition, the Cotton effects in the ECD spectra of 6 and 7 were similar to those of guignardone A (11),9 thus establishing their identical absolute configuration. Guignardone N (8) and guignardone O (9) had the same molecular formula of C17H24O6 as determined by their HRESIMS data (m/z 323.1499 [M − H]− for 8 and 369.1559 [M + COOH]− for 9, respectively). Analysis of the NMR spectroscopic data (Table 2) revealed the similar structures of 8, 9, and 4. However, the terminal olefinic bond (Δ15(16)) in 4 was oxidized to 15,16-vicinal diols in both 8 and 9, as evidenced by the HMBC correlations seen from H-17 to C-14, C-15, and C-16 and from H-16 to C-14, C-15, and C-17. Additional observation of the different hydroxymethyl proton

Figure 3. ORTEP drawing of 1.

Figure 4. ΔδH(S−R) values of MTPA esters for 4 and 5 (measured in CDCl3).

signals at C-16 (δH 3.39, 3.34, each 1H, d, J = 11.0 Hz for 8 and δH 3.37, 2H, s for 9) indicated different configurations at C-15. A Mo2(OCOCH3)4-induced ECD method was performed to settle the absolute configurations of these acyclic diols in 8 and 9.19,20 According to Snatzke’s rule,21 the 15S configuration in 8 was determined by the induced positive Cotton effect (Figure 5) at around 310 nm, while the negative Cotton effect of 9 (Supporting Information S78) suggested the 15R configuration. Further analysis of the identical Cotton effects in their ECD spectra with those of 4 demonstrated their absolute configurations (4S, 6R, 9S, 10R, 14R, 15S for 8 and 4S, 6R, 9S, 10R, 14R, 15R for 9). 10-Hydroxylated guignardianone C (10) was obtained as an orange oil with the molecular formula C15H16O6 determined from the negative HRESIMS ion at m/z 291.0873 [M − H]− (calcd for 291.0874, C15H15O6). Its structure was elucidated on

Figure 2. Key ROESY correlations (H ↔ H) of 1 and 4. D

DOI: 10.1021/acs.jnatprod.5b00008 J. Nat. Prod. XXXX, XXX, XXX−XXX

a

1 2 3 4 5a 5b 6 7a 7b 8a 8b 9 10 11 12a 12b 13a 13b 14 15 16a 16b 17a 17b 18 19 10-OH

E

d (7.9) d (7.9) dd (17.2, 1.0) m m

s m ddd (14.2, 11.6, 6.3) m m m

d (1.0) br s d (13.9) d (13.9)

3.80, 3.47, 2.37, 2.19, 2.06,

1.33, 2.14, 1.86, 2.07, 1.56, 2.22,

5.10, 4.85, 4.07, 4.00,

4.54, d (5.5) 2.45, dd (10.7, 5.5) 2.03, d (10.7)

δH (J in Hz)

Measured in CDCl3. bMeasured in methanol-d4.

65.3

44.9 150.0 110.3

28.5

44.3 89.4 23.5 37.5

15.9

82.0 70.9

δC

199.0 103.2 173.0 78.7 44.2

position

6a δC

170.8 21.1

65.9

45.3 144.8 113.5

28.2

44.2 89.3 23.4 37.5

15.8

82.0 70.9

199.0 103.1 172.9 78.7 44.1

s m ddd (14.1, 11.6, 6.4) m m m

d (7.9) d (7.9) dd (17.2, 0.8) m m

2.06, s

5.11, d (0.9) 4.92, s 4.48, s

1.33, 2.15, 1.85, 2.06, 1.54, 2.23,

3.81, 3.47, 2.38, 2.19, 2.04,

4.54, d (5.5) 2.46, dd (10.7, 5.5) 2.03, d (10.7)

δH (J in Hz)

7a

Table 2. 1H and 13C NMR Spectroscopic Data for 6−10 (500, 125 MHz, J in Hz) δC

22.6

48.1 75.6 69.6

24.6

42.1 92.0 23.2 39.7

19.1

83.5 72.5

200.6 104.8 174.8 80.2 45.4

s dt (12.0, 2.5) m m

d (7.8) d (7.8) d (16.8) dd (16.8, 6.1) m

3.39, d (11.0) 3.34, d (11.0) 1.15, s

1.75, m

1.34, 1.95, 1.70, 1.75,

3.72, 3.53, 2.56, 2.23, 2.17,

4.55, d (5.5) 2.31, dd (10.7, 5.5) 2.09, d (10.7)

δH (J in Hz)

8b δC

21.2

47.8 76.0 69.9

25.2

42.0 91.7 23.1 39.4

18.9

83.5 72.3

200.7 105.2 174.6 80.2 45.5

s m m m m m

d (7.8) d (7.8) dt (15.5, 3.5) m m

1.11, s

3.37, s

1.33, 1.96, 1.69, 1.78, 1.54, 1.79,

3.71, 3.56, 2.71, 2.21, 2.19,

4.54, d (5.6) 2.31, dd (10.7, 5.5) 2.09, d (10.7)

δH (J in Hz)

9b

14.8

53.7 33.3 15.5

166.6

116.1 157.0 116.1 132.1

132.1

110.3 125.1

134.2 163.7

108.7

δC

5.72, br s

1.06, d (6.9)

3.85, s 2.66, m 1.06, d (6.9)

6.88, d (8.4) 7.57, d (8.4)

6.88, d (8.4)

7.57, d (8.4)

6.46, s

δH (J in Hz)

10a

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DOI: 10.1021/acs.jnatprod.5b00008 J. Nat. Prod. XXXX, XXX, XXX−XXX

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meroterpenes 4, 5, 8, 9, and 12, fluconazole significantly reduced the biofilms at the concentration of 63 μg/mL (Table 4). Compounds 4 and 8 were observed to have equivalent synergistic activities (FICI < 0.31) to beauvericin, which made biofilms sensitive to fluconazole and enhanced the potency of fluconazole more than 16-fold. In addition, guignardic acid (16) was the best synergist among all tested compounds. Observed under scanning electron microscopy (SEM),28 the typical biofilms were formed with compact mutilayered hyphae and Candida cells at 1000 μg/mL of fluconazole (Figure 6). However, when treated simultaneously with 40 μg/mL of 8 and 63 μg/mL of fluconazole, the biofilms were obviously reduced and damaged. In the treatment with 80 μg/mL of 8 and 63 μg/ mL of fluconazole, the biofilms were significantly inhibited, leaving a few hyphae and cells. TCAs have been reported to be antibacterial,10 and all of the isolated compounds were evaluated for their activities against Gram-positive bacteria (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633) and Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 9027). As shown in Table 5, compounds 1, 3, 4, 8, 11, 12, and 17 exhibited some antibacterial activity, and 11 was the most potent against P. aeruginosa, with an MIC value of 25.5 μg/mL. Compounds 1−17 were inactive against three human tumor cell lines, U2OS, MCF-7, and HepG2, as evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazalium bromide] method with cis-platin29 as the positive control.

Figure 5. Mo2(OCOCH3)4-induced ECD spectrum of 8 (in DMSO).

the basis of NMR data (Table 2) and was similar to guignardianone C.22 The broad phenolic hydroxy signal (δH 5.72) as well as the downfield carbon signal (δC 157.0) revealed the presence of a phenolic hydroxy group shown to be at C-10 from HMBC correlations seen from H-8 and H-9 to C-10. In addition, the negative optical rotation and the negative Cotton effect (Supporting Information S86) at 315 nm resembled those of guignardianone C, suggesting the 2S configuration. Compounds 1−17 were evaluated for their inhibition of C. albicans growth. Four compounds (8, 12, 16, and 17, Table 3) were found to be inhibitory, and the known compound guignardone B (12) was the most potent, with an MIC value of 15.5 μg/mL (positive control fluconazole, 0.25 μg/mL). In contrast, the less active guignardone N (8) and other inactive tested compounds demonstrated that the oxidized isopropenyl group may be important for the antifungal activities of these tetracyclic meroterpenes. Although most meroterpenes did not show inhibitory activities when used alone, the tetracyclic compounds 4, 8, 9, and 12 tested were synergistic (fractional inhibitory concentration index, FICI < 0.5, Table 3)23,24 when combined with fluconazole, whereas the tricyclic compounds evaluated were not. Guignardic acid (16) exhibited the most promising synergistic activity (FICI = 0.19). Although biofilms formed in vivo and in vitro are quite different, the in vitro XTT colorimetric method was widely used to evaluate the inhibition on biofilms.25−27 In this assay, fluconazole was inactive even at the concentration of 1000 μg/ mL, as were the compounds 1−16 when tested alone (MIC > 200 μg/mL). However, when combined with tetracyclic



EXPERIMENTAL SECTION

General Experimental Procedures. Melting point measurement was carried out on an X-4 instrument, and optical rotations were measured on a Jasco P-1020 polarimeter (Jasco, Tokyo, Japan). A UV2450 spectrophotometer (Shimadzu, Tokyo, Japan) was applied to UV spectra measurement. ECD spectra were recorded on a Jasco J-810 spectrometer, and IR spectra (KBr disks, in cm−1) were obtained on a Bruker Tensor 27 spectrometer (Bruker, Karlsruhe, Germany). A Bruker AVIII-500 NMR instrument was used to acquire NMR data (1H NMR, 500 MHz; 13C NMR, 125 MHz) with TMS as internal standard. HRESIMS data were acquired using an Agilent 6520B QTOF mass instrument and an Agilent 1100 series LC/MSD-Trap-SL mass analyzer, respectively (Agilent Technologies, Santa Clara, CA, USA). Preparative HPLC was performed on a Shimadzu LC-8A system with a binary channel UV detector at 210 and 265 nm, using a Shim-pack RP-C18 column (10 μm, 200 mm × 20 mm i.d., Shimadzu, Tokyo, Japan). CC was carried out using Sephadex LH-20 (Pharmacia, Stockholm, Sweden) and silica gel (Qingdao Marine Chemical Co. Ltd., Qingdao, China). After spraying with vanillin−sulfuric acid, spots were visualized by heating silica gel GF254 plates. X-ray diffraction

Table 3. Inhibition and Synergistic Inhibition on C. albicans Growtha compound (A, μg/mL) compound 4 8 9 12 16 17 beauvericine

MICsingle >100 57.3 >100 15.5 87.5 24.3 >100

± 2.6 ± 1.1 ± 3.4 ± 1.7

fluconazole (B, μg/mL)

MICcombination

FICAb

MICsingle

MICcombination

FICBc

FICId

25.0 6.3 25.0 6.3 6.3 6.3 1.3

200 >200 >200 >200 145 ± 11 >200

80 40 80 40 80 80 20 40 40

1000 >1000 >1000

63 63 63 63 63 63 63 63 63

0.063 0.063 0.063 0.063 0.063 0.063 0.063 0.063 0.063

100.0 67.7 59.5 1.0

± 5.8 ± 3.2 ± 3.1

± 2.3 ± 2.1 ± 0.1

E. coli >100.0 >100.0 88.3 ± 5.4 78.0 ± 3.5 >100.0 35.9 ± 2.1 >100.0 2.4 ± 0.4

P. aeruginosa >100.0 >100.0 53.2 >100.0 25.5 52.1 45.5

± 3.1 ± 2.0 ± 2.9 ± 2.2

1.7 ± 0.2

Compounds were inactive when MICs > 100 μg/mL, ±SD values were calculated based on three individual experiments, and the inactive compounds were not shown. bPositive control. a

sterilized rice and 10 mL of seed culture, and the flaks were cultivated at 28 °C for 30 days. Extraction and Isolation. The solid cultures were extracted with EtOAc three times at room temperature. After removing the solvent under vacuum, 7.9 g of the crude extract was fractionated by silica gel CC with a gradient elution (petroleum ether−EtOAc, 20:1 to 1:7), giving fractions A−G. Fraction E (0.8 g) was submitted to Sephadex LH-20 CC with CH2Cl2−MeOH (1:1) as eluent. After collection and combination, the second subfraction was further fractionated by Shimadzu LC-8A preparative HPLC with MeOH−H2O (50:50), to give 4 (3.2 mg, tR 10.5 min), 5 (10.5 mg, tR 15.4 min), 8 (9.0 mg, tR 17.2 min), 3 (1.0 mg, tR 26.2 min), 9 (3.0 mg, tR 28.4 min), and 10 (35.0 mg, tR 36.6 min), respectively. Fraction F (1.0 g) was further fractionated by Sephadex LH-20 CC using MeOH, and the third subfraction was also fractionated via preparative HPLC using MeOH− H2O (35:65) to acquire 2 (2.5 mg, tR 18.2 min), 6 (7.0 mg, tR 24.3 min), 7 (6.0 mg, tR 33.2 min), and 1 (42.0 mg, tR 56.5 min). Guignardone J (1): colorless crystals (MeOH); mp 150−152 °C; [α]25D +56.5 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 191 (3.54),

data were collected at room temperature (291 K) on an Oxford Diffraction Gemini-S Ultra CCD diffractometer with Cu Kα radiation (Oxford Instruments, Oxford, England). Optical density (OD) values were determined by a microplate reader (Tecan, Mannedorf, Switzerland), and SEM pictures were taken on an EVO-LS10 SEM (Zeiss, Jena, Germany). Fungal Material. The fungus was obtained from the fresh leaves of Euphorbia sieboldiana, which were collected from the campus of China Pharmaceutical University, Nanjing, Jiangsu, People’s Republic of China, in April 2013. The isolated strain was identified as Guignardia sp. on the basis of the morphological method and reinforced by 18S rDNA and internal transcribed spacer (ITS) sequences with 99% identity to the known Guignardia sp. (GenBank accession no. JN165708.1). After cultivation on potato dextrose agar (PDA) at 28 °C for 7 days, two pieces of the agar (about 1.0 cm3) were added to an Erlenmeyer flask (500 mL) with 150 mL of potato dextrose liquid medium, and the flask was incubated on a rotary shaker at 28 °C and 150 rpm for 5 days to prepare seed culture. Solid fermentation was carried out in 12 Erlenmeyer flasks (500 mL) each containing 120 g of G

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202 (3.84), 265 (4.40) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 214 (−3.76), 262 (+45.17), 299 (−5.57) nm; IR (KBr) νmax 3743, 3446, 1867, 1634, 1454, 1396, 1167, 894 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 309.20 [M + H]+; HRESIMS m/z 353.1608 [M + COOH]− (calcd for 353.1606, C18H25O7). Guignardone K (2): colorless oil; [α]25D +38.7 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 195 (3.45), 202 (3.70), 264 (4.08) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 200 (−8.96), 262 (+18.57) nm; IR (KBr) νmax 3743, 3458, 1631, 1385, 1053, 891 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 359.03 [M + Cl]−; HRESIMS m/ z 369.1554 [M + COOH]− (calcd for 369.1555, C18H25O8). Guignardone L (3): amorphous powder; [α]25D +7.0 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 193 (3.26), 203 (3.57), 264 (3.70) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 204 (−2.59), 261 (+13.38) nm; IR (KBr) νmax 3744, 3461, 1622, 1455, 1386, 1079, 892 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 293.09 [M + H]+; HRESIMS m/z 607.3239 [2 M + Na]+ (calcd for 607.3241, C34H48NaO8). 13-Hydroxylated guignardone A (4): amorphous powder; [α]25D +2.9 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 193 (3.61), 202 (3.83), 263 (4.20) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 223 (+22.29), 261 (+39.68), 294 (−34.59) nm; IR (KBr) νmax 3732, 3445, 1622, 1455, 1386, 1249, 1023, 883 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 340.99 [M + Cl]−; HRESIMS m/z 305.1396 [M − H]− (calcd for 305.1394, C17H21O5). 12-Hydroxylated guignardone A (5): amorphous powder; [α]25D +47.1 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 191 (3.65), 202 (3.91), 262 (4.32) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 226 (+13.96), 260 (+35.48), 295 (−21.88) nm; IR (KBr) νmax 3457, 2973, 2916, 1662, 1620, 1451, 1404, 1361, 1246, 1045, 900 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 307.50 [M + H]+; HRESIMS m/z 305.1395 [M − H]− (calcd for 305.1394, C17H21O5). 17-Hydroxylated guignardone A (6): colorless oil; [α]25D +37.2 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 202 (3.84), 263 (4.26) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 223 (+25.71), 261 (+59.22), 294 (−42.67) nm; IR (KBr) νmax 3450, 2967, 1616, 1461, 1402, 1244, 1171, 1023 cm−1; 1H NMR and 13C NMR data, see Table 2; ESIMS m/z 307.11 [M + H]+; HRESIMS m/z 305.1397 [M − H]− (calcd for 305.1394, C17H21O5). Guignardone M (7): colorless oil; [α]25D +7.0 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 192 (3.62), 202 (3.84), 263 (4.29) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 223 (+20.77), 261 (+45.69), 294 (−32.79) nm; IR (KBr) νmax 3455, 1740, 1619, 1384, 1243, 1028, 903 cm−1; 1H NMR and 13C NMR data, see Table 2; ESIMS m/z 349.08 [M + H]+; HRESIMS m/z 349.1643 [M + H]+ (calcd for 349.1646, C19H25O6). Guignardone N (8): amorphous powder; [α]25D +41.8 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 192 (3.62), 203 (3.74), 264 (4.23) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 224 (+15.29), 262 (+28.51), 293 (−25.03) nm; IR (KBr) νmax 3743, 3461, 2963, 1651, 1617, 1453, 1405, 1017 cm−1; 1H NMR and 13C NMR data, see Table 2; ESIMS m/z 359.20 [M + Cl]−; HRESIMS m/z 323.1499 [M − H]− (calcd for 323.1500, C17H23O6). Guignardone O (9): amorphous powder; [α]25D +47.6 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 203 (3.77), 264 (4.25) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 223 (+20.77), 262 (+44.76), 293 (−36.79) nm; IR (KBr) νmax 3445, 2973, 1614, 1403, 1251, 1026 cm−1; 1H NMR and 13C NMR data, see Table 2; ESIMS m/z 325.20 [M + H]+; HRESIMS m/z 369.1559 [M + COOH]− (calcd for 369.1555, C18H25O8). 10-Hydroxylated guignardianone C (10): orange oil; [α]25D −172.3 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 202 (3.82), 228 (3.84), 320 (4.26) nm; ECD (0.2 mg/mL, MeOH) λ (Δε) 315 (−6.48) nm; IR (KBr) νmax 3459, 1784, 1639, 1515, 1396, 1268, 1172, 1049 cm−1; 1H NMR and 13C NMR data, see Table 2; ESIMS m/z 293.10 [M + H]+; HRESIMS m/z 291.0873 [M − H]− (calcd for 291.0874, C15H15O6). X-ray Crystallographic Analysis of Guignardone J (1). Colorless crystals were obtained from a MeOH solution. A needlelike crystal (0.39 × 0.31 × 0.24 mm3) was separated and submitted for the

crystallographic analysis. The structure was solved with the ShelXS structure solution program using direct methods and refined with the ShelXL refinement package using least-squares minimization. After being placed in calculated positions, the H atoms were refined using a riding model. Molecular graphics were computed with Ortep-3. Crystallographic data of compound 1 have been deposited at the Cambridge Crystallographic Data Centre with the deposition number CCDC 1042185. Copies of the crystallographic data can be acquired, free of charge, from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44-(0)1223-336033; email: [email protected]] or through www.ccdc.cam.ac.uk/ products/csd/request. Crystal data of guignardone J (1): C17H24O5, M = 308.36 g/mol, orthorhombic, space group P212121, a = 7.23410(10) Å, b = 12.0603(2) Å, c = 19.2403(3) Å; α = β = γ = 90°, V = 1678.63(4) Å3, Z = 4, T = 291(2) K, μ(Cu Kα) = 0.730 mm−1, Dcalc = 1.220 g/ cm3, 3115 unique (Rint = 0.0210, Rsigma = 0.0170) reflections were used in all calculations from 12 311 measurements (8.654° ≤ 2θ ≤ 139.24°). The final R1 was 0.0358 and wR2 was 0.0978 (I > 2σ(I)). The absolute configuration was determined correctly with the Flack parameter value x = 0.04(9). Absolute Configuration of 4-OH in 3. A sample of 3 (0.4 mg) was dissolved in CH2Cl2 (800 μL). After mixing with 0.8 mg of Rh2(OCOCF3)4, the first induced ECD spectrum was record at once, and the following spectra were measured every 5 min three times. The observed Cotton effect at the E bond (around 350 nm) in the induced ECD spectrum was correlated to the absolute configuration of the secondary alcohol at C-4. Preparation of (S)- and (R)-MTPA Esters of 4 and 5. After drying under vacuum overnight, 0.8 mg of 4 together with several crystals of dimethylaminopyridine (DMAP) was dissolved in 150 μL of freshly distilled dry pyridine. Treatment with (R)-(−)- and (S)-(+)-αmethoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl, 8 μL) at room temperature overnight yielded the (S)- and (R)-MTPA esters, respectively. The reactions were quenched with 200 μL of methanol, and the residues were purified through Shimadzu LC-8A preparative HPLC using 75% MeOH, to give the (S)-MTPA ester (tR 28.4 min) and the (R)-MTPA ester (tR 30.4 min). The (S)- and (R)-MTPA esters of 5 were prepared in the same way at 50 °C overnight and obtained at 28.6 and 32.0 min (87% MeOH), respectively. 13-Hydroxylated guignardone A-(S)-MTPA ester (4a): 1H NMR data (500 MHz, CDCl3) δH 7.482 (2H, d, J = 7.3 Hz, aromatic), 7.390 (3H, m, aromatic), 5.538 (1H, dt, J = 2.0, 6.9 Hz, H-13), 4.763 (1H, s, H-16a), 4.613 (1H, s, H-16b), 4.537 (1H, d, J = 5.5 Hz, H-4), 3.797 (1H, d, J = 8.0 Hz, H-7a), 3.489 (3H, s, OCH3), 3.446 (1H, d, J = 8.0 Hz, H-7b), 2.837 (1H, dd, J = 15.9, 7.2 Hz, H-12a), 2.456 (1H, dd, J = 10.8, 5.5 Hz, H-5a), 2.425 (1H, d, J = 17.3 Hz, H-8a), 2.358 (1H, dd, J = 13.9, 6.5 Hz, H-9), 2.298 (1H, dd, J = 12.2, 5.8 Hz, H-8b), 2.268 (1H, dd, J = 13.0, 5.8 Hz, H-14), 2.019 (1H, d, J = 10.8 Hz, H-5b), 1.902 (1H, dd, J = 15.9, 2.2 Hz, H-12b), 1.525 (3H, s, 17-CH3), 1.378 (3H, s, 11-CH3); ESIMS m/z 523.11 [M + H]+. 13-Hydroxylated guignardone A-(R)-MTPA ester (4b): 1H NMR data (500 MHz, CDCl3) δH 7.363−7.485 (5H, m, aromatic), 5.481 (1H, dt, J = 2.0, 7.0 Hz, H-13), 4.926 (1H, s, H-16a), 4.703 (1H, s, H16b), 4.530 (1H, d, J = 5.4 Hz, H-4), 4.149 (1H, s, 6-OH), 3.797 (1H, d, J = 8.0 Hz, H-7a), 3.472 (3H, s, OCH3), 3.446 (1H, d, J = 8.0 Hz, H-7b), 2.804 (1H, dd, J = 16.3, 7.2 Hz, H-12a), 2.450 (1H, dd, J = 10.7, 5.2 Hz, H-5a), 2.417 (1H, d, J = 16.2 Hz, H-8a), 2.299 (1H, dd, J = 10.0, 6.0 Hz, H-9), 2.274 (1H, d, J = 10.6 Hz, H-8b), 2.245 (1H, dd, J = 15.2, 6.8 Hz, H-14), 2.012 (1H, d, J = 10.7 Hz, H-5b), 1.763 (1H, dd, J = 15.9, 2.4 Hz, H-12b), 1.621 (3H, s, 17-CH3), 1.286 (3H, s, 11CH3); ESIMS m/z 523.12 [M + H]+. 12-Hydroxylated guignardone A-(S)-MTPA ester (5a): 1H NMR data (500 MHz, CDCl3) δH 7.693 (2H, m, aromatic), 7.505 (2H, m, aromatic), 7.421 (6H, m, aromatic), 5.262 (1H, dd, J = 6.8, 2.0 Hz, H12), 4.699 (1H, s, H-16a), 4.625 (1H, d, J = 5.5 Hz, H-4), 4.556 (1H, s, H-16b), 4.101 (1H, d, J = 9.0 Hz, H-7a), 4.061 (1H, d, J = 9.0 Hz, H-7b), 3.636 (3H, s, OCH3), 3.536 (3H, s, OCH3), 2.832 (1H, d, J = 10.7 Hz, H-5a), 2.585 (1H, ddd, J = 15.2, 9.8, 7.2 Hz, H-13a), 2.352 (1H, dd, J = 10.7, 5.8 Hz, H-5b), 2.327 (1H, d, J = 17.3 Hz, H-8a), H

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2.153 (1H, m, H-9), 2.131 (1H, dd, J = 13.1, 5.8 Hz, H-8b), 2.058 (1H, dd, J = 11.6, 6.3 Hz, H-14), 1.461 (1H, dd, J = 8.2, 2.2 Hz, H13b), 1.432 (3H, s, 17-CH3), 1.243 (3H, s, 11-CH3); ESIMS m/z 739.16 [M + H]+. 12-Hydroxylated guignardone A-(R)-MTPA ester (5b): 1H NMR data (500 MHz, CDCl3) δH 7.711 (2H, m, aromatic), 7.517 (2H, m, aromatic), 7.421 (6H, m, aromatic), 5.339 (1H, dd, J = 6.8, 1.8 Hz, H12), 4.764 (1H, s, H-16a), 4.641 (1H, d, J = 5.6 Hz, H-4), 4.626 (1H, s, H-16b), 4.018 (1H, d, J = 8.7 Hz, H-7a), 3.892 (1H, d, J = 8.7 Hz, H-7b), 3.662 (3H, s, OCH3), 3.534 (3H, s, OCH3), 2.998 (1H, d, J = 10.7 Hz, H-5a), 2.601 (1H, ddd, J = 15.3, 9.0, 7.3 Hz, H-13a), 2.370 (1H, d, J = 16.8 Hz, H-8a), 2.304 (1H, dd, J = 10.8, 5.8 Hz, H-5b), 2.161 (1H, m, H-9), 2.130 (1H, m, H-8b), 2.118 (1H, m, H-14), 1.526 (1H, m, H-13b), 1.568 (3H, s, 17-CH3), 1.144 (3H, s, 11-CH3); ESIMS m/z 739.15 [M + H]+. Absolute Configurations of the 15-Tertiary Alcohols in 8 and 9. A 0.8 mg amount of samples and 1.3 mg of Mo2(OCOCH3)4 were dissolved in dry DMSO and were recorded immediately for the first induced ECD spectra. The additional induced ECD spectra were recorded every 10 min until reaching the stationary state. The absolute configurations of the 15-tertiary alcohols were demonstrated from the observed Cotton effects at around 310 nm in their induced ECD spectra. Inhibition on C. albicans Growth. The activity tests of compounds on C. albicans ATCC 24433 growth were performed using a broth microdilution method30 in 96-well microtiter plates (Thermo, USA). Briefly, 24-h-old colonies were suspended in 5 mL of 0.85% saline and were diluted with RPMI-1640 (Sigma) plus MOPS [3-(N-morpholino)propanesulfonic acid, Sigma] medium to a final concentration of 1.0 × 104 cfu/mL. Then 100 μL of sample solutions together with 100 μL of Candida suspensions was added to each well. After incubation at 35 °C for 24 h, OD values were determined by a microplate reader at 595 nm. MIC values were defined as the lowest drug concentration that produced 80% reduction of the growth. Synergistic Inhibition on C. albicans Growth. A checkerboard assay was used to evaluate the synergistic antifungal effect.23,24 In the 96-well microtiter plates, serial dilutions of the compound solutions were used in the horizontal orientation from 25 to 0.8 μg/mL, and gradient concentrations of fluconazole solutions from 4.0 to 0.0078 μg/mL were added in the vertical orientation. The MIC values were defined as the final concentrations of fluconazole and compounds in selected wells that had at least 80% reduction of the growth. Most of the minimum FICIs for different compounds were found at the fluconazole concentration of 0.031 μg/mL, and the data under this concentration were used for a direct comparison of the synergistic activities. FICI values were used to evaluate the synergistic activities, which were defined as FICI = FICA + FICB = MICA combination/ MICA single + MICB combination/MICB single [MICA combination (MIC of compound in combination), MICA single (MIC of compound alone), MICB combination (MIC of fluconazole in combination), MICB single (MIC of fluconazole alone)]. FICI ≥ 4 means antagonism between fluconazole and the tested compound, 0.5 < FICI < 4 means no interaction between them, and FICI ≤ 0.5 means synergy of the combination; the synergistic effect is stronger when the FICI value is lower.24 Inhibition and Synergistic Inhibition on C. albicans Biofilms. For biofilm assays, the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, Sigma] colorimetric method25,26 was employed. Candida cells were cultivated in YPD medium (1% yeast extract, 2% peptone, and 2% dextrose) overnight. After centrifugation, cells were washed with sterile PBS (phosphate-buffered saline) and suspended in RPMI-1640 plus MOPS medium to 1.0 × 106 cfu/mL. A 100 μL amount of suspensions was further added into each well and cultivated at 37 °C for 24 h to form the biofilms. After washing the biofilms with PBS three times, prepared solutions (100 μL) were added to the selected wells and were incubated at 37 °C for another 24 h, followed by treatment with 0.5 mg/mL of XTT solution for 2 h. MIC values were defined as the lowest concentrations that caused ≥80% reduction in XTT reduction evaluation, which was measured at 490 nm using a microplate reader. The synergistic

inhibition test was carried out by the checkerboard assay using gradient concentrations of compounds from 160 to 5 μg/mL and gradient concentrations of fluconazole from 500 to 8 μg/mL and was also evaluated by FICI values. SEM for the Inhibition on Biofilms. In the 24-well microtiter plates (Thermo, USA), 300 μL of the standard cell suspensions (1.0 × 106 cfu/mL) was added to each well, which contained glass coverslips treated with poly-L-lysine hydrobromidein (Sigma), and was incubated at 37 °C for 24 h to form biofilms.28 The biofilms were washed with PBS three times and were cultivated for another 24 h after 150 μL of fluconazole solution and 150 μL of compound solution were added. Then the biofilms were washed and treated with 2.5% glutaraldehyde in 0.2 M PBS (pH 7.2) overnight and 1% Zetterquist’s osmium for 2 h, followed by dehydrating in increasing ethanol solutions and drying at the critical point. Finally, the samples were coated with gold and observed through an EVO-LS10 SEM in the high-vacuum mode at 10 kV. The photos were processed for display by Photoshop software (Adobe, Mountain View, USA). Antibacterial Assays. The antibacterial activities against S. aureus, B. subtilis, E. coli, and P. aeruginosa were measured in sterile 96-well plates using the broth microdilution method.8 MICs were determined by comparing OD values with the growth control using amoxicillin and streptomycin as the positive controls.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00008. HRESIMS, IR, 1H NMR, 13C NMR, HSQC, HMBC, ROESY, and ECD spectra of 1−10 (PDF) Crystallographic data of 1 (CIF)



AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86-25-8618-5039. E-mail: [email protected] (M.-H. Yang). *Tel/Fax: +86-25-8327-1405. E-mail: [email protected] (L.-Y. Kong). Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research work was funded by the National Natural Science Foundation of China (81503218), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Youth Fund Project of Basic Research Program of Jiangsu Province (Natural Science Foundation, BK20130651), and the Fundamental Research Funds for the Central Universities (JKQZ2013016).



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DOI: 10.1021/acs.jnatprod.5b00008 J. Nat. Prod. XXXX, XXX, XXX−XXX