Secondary Metabolites from the Chinese Liverwort Cephaloziella

Δ Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of ... Publication Date (Web): September 13, 2013 ... (17 and 1...
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Secondary Metabolites from the Chinese Liverwort Cephaloziella kiaeri Rui-Juan Li,† Rong-Xiu Zhu,‡ Yao-Yao Li,† Jin-Chuan Zhou,† Jiao-Zhen Zhang,† Song Wang,† Jian-Ping Ye,§ Yue-Hu Wang,Δ Susan L. Morris-Natschke,Δ Kuo-Hsiung Lee,Δ and Hong-Xiang Lou*,† †

Department of Natural Products Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Science, Shandong University, Jinan 250012, People’s Republic of China ‡ School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, People’s Republic of China § Guangxi Mount Maoer National Nature Reserve, Guilin 541001, People’s Republic of China Δ Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7568, United States S Supporting Information *

ABSTRACT: Sixteen new clerodane diterpenoids, cephaloziellins A−P (1−16), and two known analogues (17 and 18) were isolated from an EtOH extract of the Chinese liverwort Cephaloziella kiaeri. The structures of the new compounds were elucidated from extensive spectroscopic data (IR, UV, HRESIMS, 1D NMR, and 2D NMR), and the structures of 5, 9, and 15 were confirmed by single-crystal X-ray diffraction analyses. The absolute configurations of all new compounds were established by comparing experimental and calculated electronic circular dichroism spectra.

L

compared with that calculated by means of the time-dependent density functional theory (TDDFT) method.

iverworts (Hepaticae) are a rich source of terpenoids and aromatic compounds, which are characterized by high structural diversity and a broad spectrum of biological activities, including antifungal, antioxidative, cytotoxic, allelopathic, insect antifeedant, and multidrug resistance (MDR) reversal activities. 1−5 Numerous terpenoids have been isolated and characterized from various families/species in Jungermanniales, the largest order of liverworts.2,6−10 However, few phytochemical investigations on the family Cephaloziellaceae have been reported so far, and to our best knowledge, there is only one report on the isolation of several sesquiterpenoids from Cephaloziella recurvifolia.11 Thus, this is the first chemical study on the liverwort Cephaloziella kiaeri (Austin) S. W. Arnell (Cephaloziellaceae), which is distributed predominantly in south and southeastern Asia. This article reports 16 new clerodane diterpenoids, cephaloziellins A−P (1−16), along with two known analogues (17 and 18) isolated from the liverwort C. kiaeri, collected from Mount Maoer in Guangxi Zhuang Autonomous Region, South China. Structure elucidation of the compounds was achieved by spectroscopic methods and by comparison with closely related known compounds. For compounds 5, 9, and 15, relative configurations were confirmed by single-crystal X-ray diffraction analyses. In order to determine the absolute configurations of all new compounds, their ECD spectra were measured and © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION An EtOH−H2O (95:5) extract of C. kiaeri was suspended in H2O and partitioned successively with Et2O and n-BuOH. The Et2O fraction was separated by chromatography over MCI gel, silica gel, Sephadex LH-20, and further semipreparative HPLC to yield 16 new clerodane diterpenoids (1−16) and two known analogues (17 and 18). Compound 1 was isolated as a white, amorphous powder. Its molecular formula, C20H24O6, was established from a quasimolecular ion peak at m/z 378.1914 [M + NH4]+ (calcd 378.1911) in its HRESIMS, which suggested nine degrees of unsaturation in a clerodane framework. The IR spectrum showed absorption bands due to OH (3444 cm−1) and lactone (1770 cm−1) groups. Analysis of 1D NMR data (Tables 1 and 4) of 1 revealed a typical β-monosubstituted furan ring [δH 6.28 (br s, H-14), 7.38 (br s, H-15), and 7.35 (br s, H-16); δC 128.9 (C-13), 108.2 (C-14), 144.0 (C-15), and 138.5 (C-16)], an ester carbonyl [δC 177.8 (C-18)], an acetal [δC 105.8 (C-20)], three oxygenated methines [δC 72.1 (C-3), 84.6 (C-6), and 70.6 (C-12)], an oxygenated quaternary carbon [δC 73.2 (CReceived: May 30, 2013

A

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8)], and two methyl groups [δH 1.21 (3H, s, H3-17) and 1.37 (3H, s, H3-19)]. According to its molecular formula and by comparing its NMR data with those of known 3-hydroxyteubutilin A,12 compound 1 was elucidated as a highly oxygenated clerodane diterpenoid. The planar structure of 1 was determined by extensive analyses of its 2D NMR spectra. A 1H−1H COSY experiment established the connectivity from C-1 to C-4, C-6 to C-7, C-11 to C-12, and C-14 to C-15 (Figure 1). On the basis of the HMBC correlations (Figure 1) from H3-19 to C-4, C-5, C-6, and C-10 and from H3-17 to C-7, C-8, and C-9, the 6/6 dicyclic core of the diterpene with 5,8-dimethyl and 3,6,8-trioxygenated substitutions was established. A γ-lactone ring was deduced by HMBC correlations of H-6/C-18 and H-3/C-18. Also, an octahydrofuro[2,3-b]oxepine system was constructed based on HMBC correlations from H-3, H2-11, and H-12 to C-20. The furan ring was located at C-12 by the HMBC correlations from H-12 to C-14 and C-16. Thus, the planar structure of 1 with a six-ring system was elucidated as shown. The relative configuration of 1 was established from its NOE spectrum. Correlations of H3-19 with H-4, H-6, and H-10 showed that all were cofacial and were arbitrarily assigned to be α-oriented. H-1α and H-1β were determined by NOE correlations with H3-19 and H-12, respectively. NOEs observed between H-16 and H3-17 established the β-orientation of CH317. H-20 was assigned to be β-oriented by NOE correlation of H3-17 with H-20. H-11β and H-7β were determined by correlations with H3-17 and H-20, respectively (Figure 2). Table 1. 1H NMR Spectroscopic Data for Compounds 1−7a position

1.95 m (α-H) 2.07 m (β-H)

2a 2b 3 4 6a

1.63 2.86 4.63 1.98 4.61

6b 7a 7b 8 9 10 11a 11b 12a 12b 14 15 16 17 19 20 OMe a

1

1a 1b

2

3

4

4.34 dd (10.8, 7.2)

7

4.34 dd (10.8, 7.2)

4.69 d (9.6)

1.87 m

6.95 d (16.2)

2.30 dd (12.5, 7.8) (α- 1.90 m (α-H) H) 1.78 t like (12.0) (β1.29 m (β-H) H) 1.46 m

1.89 m (α-H)

2.90 dd (17.4, 9.6)

2.20 m 2.93 m

5.96 d (16.2)

1.26 m (β-H)

2.72 d (17.4)

2.28 m

2.24 m 2.20 dd (14.4, 8.4) (αH) 2.33 dd (14.4, 6.0) (βH) 5.29 dd (8.4, 6.0)

2.00 m 1.74 m 3.00 dd (13.2, 7.8) (α- 1.54 m H) 1.98 m (β-H) 1.76 m

1.79 d (4.8) 1.48 m

2.74 m 2.40 m 2.44 m (2H)

3.03 t (9.6) 1.62 m 2.44 m (2H)

2.85 t (9.6) 1.79 d (12.0) 2.44 dd (9.6, 6.0) (2H)

1.71 m

5.32 t like (8.4)

2.20 m (2H)

5.55 t (6.0)

5.49 br d (6.6)

5.46 t (6.0)

6.28 7.38 7.35 1.21 1.37 5.25

6.50 7.37 7.44 1.18 1.34 5.49

6.86 br s 6.13 br s

6.37 7.44 7.44 2.26 1.21

6.38 7.45 7.44 2.12 1.20

6.34 7.44 7.41 2.33 1.43

br s br s br s s s s

4.60 dd (11.4, 7.8)

6

1.65 m (α-H) 1.96 m (β-H)

2.26 dd (14.4, 8.4) (αH) 1.80 dd (14.4, 9.6) (βH)

1.92 m (2H)

5

2.12 m (α-H) 1.58 m (α-H) 1.92 dd (14.4, 6.0) (β- 1.96 m (β-H) H) 2.49 m (α-H) 2.37 m (α-H) 2.38 m (β-H) 2.18 m (β-H) 6.90 t (3.0) 6.93 br s

m (α-H) m (β-H) m br s t like (8.90)

2.11 m (α-H) 1.95 m (α-H) 1.86 dd (15.6, 7.2) (β- 1.89 m (β-H) H) 2.40 m (α-H) 2.39 m (2H) 2.62 m (β-H) 6.95 br s 6.87 t (3.6)

br s br s br s s s s

2.24 m 2.39 m 5.89 br s 6.03 d (4.8) 0.85 dd (6.6, 2.4) 1.27 s 0.77 s

2.36 m (2H) 6.85 m

2.40 br s (α-H) 2.24 m (β-H) 7.10 br s

1.49 m

0.85 d (6.6) 1.26 s 0.73 s

br s br s br s s s

br s br s br s s s

3.70 s

br s br s br s s s

3.67 s

Recorded at 600 MHz in CDCl3. Chemical shifts (δ) are expressed in ppm, and J values are presented in Hz. B

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Table 2. 1H NMR Spectroscopic Data for Compounds 8− 11a position

8

9

1a

1.89 m (α-H)

1.90 m (α-H)

1b

1.74 m (β-H)

1.78 dd (13.8, 6.6) (β-H)

2a

2.32 m (2H)

2.37 dt (19.8, 5.4) 2.22 m

6.82 dd (3.6, 3.0)

6.79 br s

4.70 dd (10.2, 6.6) 2.01 dd (13.8, 6.6) (α-H) 2.16 dd (13.8, 10.2) (β-H) 2.57 dd (9.0, 7.2) 2.63 dt (13.0, 7.2) (α-H) 2.27 ddd (13.0, 9.0, 6.0) (βH) 5.45 dd (7.2, 6.0) 6.36 br s 7.43 br s 7.42 br s 1.51 s 1.35 s

4.71 dd (9.4, 6.3) 2.31 dd (13.8, 6.3) (α-H) 1.42 dd (13.8, 9.46) (β-H) 2.70 dd (12.0, 8.4) 2.08 q (12.0) (α-H) 2.47 ddd (12.0, 8.4, 6.3) (βH) 5.23 dd (11.5, 6.3) 6.36 br s 7.44 br s 7.45 br s 1.68 s 1.35 s

2b 3a 3b 4 6 7a 7b 9 11a 11b 12 14 15 16 17 19

10

11

1.69 td (13.8, 5.4) (α-H) 1.79 m (βH)

2.75 m (αH) 2.28 dd (19.2, 9.6) (β-H) 2.77 m (2H)

1.55 m (αH) 1.80 m (βH) 1.46 qd (6.6, 3.0) 1.92 m 2.81 dd (12.6, 3.0) 4.56 t (8.8)

Thus, the structure of 1, named cephaloziellin A, was elucidated as shown. Cephaloziellin B (2) was assigned the molecular formula C20H22O5 by HRESIMS, corresponding to one more degree of unsaturation than that of 1. The 1D NMR data (Tables 1 and 4) showed that the structure of compound 2 was similar to that of 1, except for the presence of a trisubstituted double bond [δH 6.95 (br s, H-3); δC 138.1 (C-3) and 132.7 (C-4)]. Further analysis of the NMR data gave evidence that the acetal methine [δC 100.7 (C-20)] was located between C-8 (δC 72.8) and C-12 (δC 74.5), rather than between C-3 and C-12 as found in 1. Furthermore, formation of this four-membered oxygen ring satisfied the molecular formula, and the other oxygenated carbon [δC 84.6 (C-6)] was unequivocally located in the 18,6lactone ring by a key HMBC correlation of H-6/C-18. NOE correlations between H3-19/H-6, H-6/H3-17, H3-17/H-10, and H-10/H3-19 established that all were α-oriented. H-1α and H1β were determined by correlations with H3-19 and H-12, respectively. Likewise, H-7α and H-7β were determined by correlations with H3-17 and H-20, respectively (Figure S12). Compound 3 was assigned the molecular formula C20H26O5 on the basis of the HRESIMS peak at m/z 347.1854 [M + H]+. The 1D NMR spectrum of 3 resembled that of amphiacrolide F (17),13 indicating that compound 3 was a clerodane diterpenoid. Careful comparison of the 1H NMR data of 3 and 17 (Table S190) revealed a replacement of the oxygenated methylene in 17 [H2-16 at δH 4.76 (d, J = 2.4 Hz)] by a hemiacetal methine in 3 [H-16 at δH 6.03 (d, J = 4.8 Hz)]. In addition, in the 13C NMR spectrum of 3, differences from that of 17, such as downfield shifts of 2.2 ppm for C-14 and 25.6 ppm for C-16, as well as an upfield shift of 3.2 ppm for C-15, were observed (Table S190). The above data suggested that the 13-buten-15,16-olide moiety attached to C-12 in 17 was replaced by a 16-hydroxy-13-buten-15,16-olide moiety in 3. NOE correlations of H3-19 with H-6 and H-10, as well as H-6 with H-8, determined that all were α-oriented. H-1α and H-1β were determined by NOE correlations with H3-19 and H3-20,

7.19 t (4.2)

4.62 d (12.6)

2.27 dd (15.0, 8.8) 1.90 dd (15.0, 8.8) 2.36 d (4.0)

2.65 d (12.6)

4.36 d (4.0)

2.70 m

1.93 t like (12.6)

3.12 dd (15.6, 8.0) 5.36 s 6.33 7.48 7.44 1.74 1.19

br s br s br s s s

5.41 t like (7.8) 6.36 br s 7.40 br s 7.44 br s 2.33 br s 1.46 s

a Recorded at 600 MHz in CDCl3. Chemical shifts (δ) are expressed in ppm, and J values are presented in Hz.

Table 3. 1H NMR Spectroscopic Data for Compounds 12−16a position 1a 1b 2a 2b 3a 3b 4 6a 6b 7 9 10 11 12 14 15 16 17 19 OMe a

12

13

1.52 m (α-H) 1.68 m (β-H) 2.26 m (2H)

1.61 m (α-H) 1.81 m (β-H) 2.28 m (2H)

6.95 t (4.2)

2.73 1.35 2.74 2.62 2.16 2.92 5.04 6.51 7.43 7.43 2.25 1.30 3.71

m (α-H) t (14.4) (β-H) dd (10.2, 3.0) dd (8.4, 2.4) dt (12.0, 2.4) ddd (10.2, 8.4, 1.2) br s br s br s br s s s s

14

6.92 dd (4.8, 3.0)

1.48 1.82 1.40 1.83 1.70

m m m m m

2.45 1.17 2.64 2.77 2.25 3.39 5.44 6.20 7.40 7.44 1.67 1.23 3.68

2.40 1.58 1.85 2.75 2.81 1.89 2.65 4.97 6.41 7.42 7.43 1.90 1.17 3.65

dd (9.6, 6.6) dd (14.4, 4.2) (α-H) m (β-H) td (11.0, 4.2) dd (13.8, 3.6) m dt (13.8, 11.0) d (11.0) br s br s br s s s s

dd (13.0, 3.6) (α-H) t (13.0) (β-H) td (13.0, 3.6) dd (8.4, 1.2) m ddd (13.0, 8.4, 5.5) d (5.5) br s t (1.8) br s s s s

(α-H) (β-H) (α-H) (β-H) (2H)

15 1.46 m (2H) 1.36 1.78 1.58 1.63 2.40 2.00 1.65 2.72 2.47 2.02 2.92 5.00 6.51 7.42 7.41 2.26 1.09 3.68

m (α-H) m (9.0, 3.0) (β-H) m (α-H) m (β-H) dd (12.0, 4.2) dd (13.8, 3.6) (α-H) m (β-H) td (12.0, 3.6) d (8.4) t (8.4) dd (12.0, 8.4) br s br s br s br s s s s

16 1.56 1.61 1.38 1.81 1.59

m (α-H) m (β-H) m (α-H) br d (13.8, 3.0) (β-H) m (2H)

2.37 1.67 1.49 2.64 2.65 2.09 3.40 5.41 6.19 7.39 7.43 1.62 1.03 3.63

dd (11.4, 5.4) m (α-H) t (13.8) (β-H) td (12.0, 4.2) d (8.4) dd (12.6, 4.2) ddd (12.0, 8.4, 5.4) dd (5.4) br s br s br s s s s

Recorded at 600 MHz in CDCl3. Chemical shifts (δ) are expressed in ppm, and J values are presented in Hz. C

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Table 4. 13C NMR Spectroscopic Data (δ) for Compounds 1−7a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe a

1 18.2 25.0 72.1 54.9 39.1 84.6 41.8 73.2 56.0 42.7 40.0 70.6 128.9 108.2 144.0 138.5 26.9 177.8 29.6 105.8

2 t t d d s d t s s d t d s d d d q s q d

19.4 23.8 138.1 132.7 39.5 84.9 42.1 72.8 60.3 46.2 37.8 74.5 128.7 109.5 143.8 140.1 24.4 169.5 30.4 100.7

3 t t d s s d t s s d t d s d d d q s q d

17.9 24.8 136.3 133.6 39.7 85.2 34.9 32.3 39.3 41.6 34.8 21.3 169.0 117.8 170.6 98.7 15.4 169.9 30.8 16.5

4 t t d s s d t d s d t t s d s d q s q q

17.8 24.8 136.5 133.6 39.6 85.4 34.9 32.1 39.2 41.4 35.4 18.9 138.6 143.0 96.9 171.6 15.4 170.3 30.7 16.5

5 t t d s s d t d s d t t s d d s q s q q

23.5 23.1 136.4 133.2 43.4 78.7 43.0 204.5 41.6 38.1 34.2 72.6 124.2 108.4 144.4 139.8 30.9 168.8 23.1 177.3

6 t t d s s d t s d d t d s d d d q s q s

21.2 26.2 139.6 136.6 39.4 30.0 41.8 209.4 38.3 48.4 37.4 72.4 124.8 108.5 144.4 139.5 30.1 167.8 27.4 176.6 51.7

7 t t d s s s t s d d t d s d d d q s q s q

20.7 26.4 141.3 134.9 42.8 151.1 131.3 200.1 38.5 47.5 36.7 72.3 124.7 108.4 144.4 139.5 26.2 166.6 23.3 176.0 51.7

t t d s s d d s d d t d s d d d q s q s q

Data were measured in CDCl3 at 150 MHz. Chemical shifts (δ) are expressed in ppm.

Figure 1. 1H−1H COSY (bold lines) and key HMBC (arrows) correlations for compounds 1 and 14.

respectively (Figure S24). Thus, compound 3 was elucidated as an analogue of amphiacrolide F (17) and named cephaloziellin C. The same molecular formula as that of 3, C20H26O5, was assigned to cephaloziellin D (4) by HRESIMS. The 1D NMR data (Tables 1 and 4) of 4 and 3 were closely comparable, with the only differences evident in signals for the side chain at C-12. In the 1H NMR spectrum of 4, the proton signal of an oxygenated CH, assigned as H-16 in compound 3, was absent, and the corresponding oxygenated carbon was substituted by a carbonyl group at δC 171.6 (C-16) in 4. Moreover, signals of an oxygenated CH at δH 6.13 (br s, H-15) and the corresponding oxygenated carbon at δC 96.9 (C-15) in 4 were present rather than a carbonyl group assigned as C-15 in 3. Thus, a 15hydroxy-13-buten-16,15-olide moiety was assigned for the C-12 side chain in compound 4. The NOE spectrum of 4 displayed a correlative pattern similar to that of 3 (Figure S36), indicating the same relative orientations of H-6α, H-10α, H3-17β, H3-19α, and H3-20β. Cephaloziellin E (5) was obtained as colorless crystals. Its molecular formula, C20H22O6, with 10 degrees of unsaturation, was established on the basis of HRESIMS. Its 1D NMR spectra showed the presence of signals due to two methyl groups [δH

Figure 2. Key NOESY correlations (dashed arrows) for compounds 1 and 14.

2.26 (s, H3-17) and 1.21 (s, H3-19)], two oxygenated methines [δH 4.69 (d, J = 9.6 Hz, H-6) and 5.55 (t, J = 6.0 Hz, H-12); δC 78.7 (C-6) and 72.6 (C-12)], a trisubstituted double bond [δH 6.90 (t, J = 3.0 Hz, H-3); δC 136.4 (C-3) and 133.2 (C-4)], a typical β-monosubstituted furan ring [δC 124.2 (C-13), 108.4 (C-14), 144.4 (C-15), and 139.8 (C-16)], a ketone carbonyl group [δC 204.5 (C-8)], a conjugated ester group [δC 168.8 (C18)], and a lactone carbonyl group [δC 177.3 (C-20)]. The planar structure of 5 was achieved by analyses of its 1H−1H COSY and HMBC spectra (Figures S53 and S52). The D

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Figure 3. X-ray crystal structures of compounds 5, 9, and 15.

Table 5. 13C NMR Spectroscopic Data (δ) for Compounds 8−16a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe a

8 33.7 25.1 135.4 132.3 43.4 79.0 34.9 74.9 50.0 97.6 31.3 72.7 125.0 108.4 144.3 139.6 25.6 169.2 22.6 174.8

9 t t d s s d t s d s t d s d d d q s q s

33.1 25.1 135.2 133.0 43.2 79.1 37.7 73.9 51.8 97.3 32.2 71.7 124.0 108.2 144.3 140.0 23.9 169.3 22.7 174.6

10 t t d s s d t s d s t d s d d d q s q s

32.4 22.1 18.1 45.4 43.3 79.4 40.8 70.5 42.8 100.0 75.2 77.7 121.2 107.9 144.8 139.7 26.2 174.5 16.7 171.8

11 t t t d s d t s d s d d s d d d q s q s

36.4 23.8 136.4 132.5 52.5 83.6 40.4 147.6 122.7 209.6 35.2 70.4 125.4 108.2 144.1 139.9 18.1 167.3 24.4 169.3

12 t t d s s d t s s s t d s d d d q s q s

25.8 25.6 139.8 137.3 36.8 33.1 48.0 209.9 42.5 38.0 39.8 78.9 123.9 108.7 144.1 139.2 28.9 167.3 23.5 178.0 51.6

13 t t d s s t d s d d d d s d d d q s q s q

26.1 25.8 139.6 137.4 36.4 33.4 43.5 210.1 46.6 38.3 38.6 77.1 121.2 109.8 143.4 140.4 28.7 167.2 23.0 177.7 51.5

14 t t d s s t d s d d d d s d d d q s q s q

22.5 25.2 24.8 54.6 36.9 30.0 49.7 208.8 45.0 40.2 43.9 77.4 121.4 108.8 144.3 141.8 29.3 174.5 25.6 174.7 51.6

15 t t t d s t d s d d d d s d d d q s q s q

30.0 25.3 24.6 54.3 36.0 26.9 48.5 210.1 42.7 39.0 38.6 79.5 124.2 108.6 144.0 138.8 28.8 174.8 25.7 178.0 51.5

16 t t t d s t d s d d d d s d d d d s q s q

29.8 25.3 24.5 54.3 35.5 27.6 43.5 210.6 47.0 39.4 37.7 77.2 121.0 109.8 143.4 140.4 28.8 174.7 25.7 177.6 51.5

t t t d s t d s d d d d s d d d q s q s q

Data were measured in CDCl3 at 150 MHz. Chemical shifts (δ) are expressed in ppm.

attached to C-4 in 6, which was confirmed by HMBC correlations (Figure S59) from the protons at δH 3.70 to C-4 (δC 136.6) and C-18 (δC 167.8). The 1D NMR data (Tables 1 and 4) of cephaloziellin G (7) were similar to those of 6, with a noticeable difference being the presence of signals for a transdouble bond [δH 6.95 (d, J = 16.2 Hz, H-6) and 5.96 (d, J = 16.2 Hz, H-7); δC 151.1 (C-6) and 131.3 (C-7)], which was consistent with its molecular formula C21H24O6. The relative configurations of 6 and 7 were partially elucidated from NOE spectra. However, NOE correlations were not able to determine the orientations of H-9 and H-12 since the C(10)−C(9) single bond is free to rotate. Thus, the absolute configurations of 6 and 7 were elucidated to be the same as that of 5 based on the biogenic pathway and ECD calculations. Cephaloziellins H (8) and I (9) had the same molecular formula, C20H22O7, determined by HRESIMS. IR spectra showed absorption bands of OH, lactone, and conjugated ester groups at 3439, 1765, and 1684 cm−1 in 8 and 3378, 1755, and 1692 cm−1 in 9, respectively. The 1D NMR spectra of 8 and 9 were similar, with only small differences in the chemical shifts of H-9, H2-7, H2-11, and H3-17 (Tables 2 and 5). Further analyses of the 2D NMR spectra showed that the planar structures of 8 and 9 were similar to that of jamesoniellide E,14

presence of C-3(H)−C-2(H2)−C-1(H2)−C-10(H)−C-9(H)− C-11(H2)−C-12(H), C-6(H)−C-7(H2), and C-14(H)−C15(H) moieties was established by 1H−1H COSY correlations (Figure S47). The 18,6-lactone ring formation was supported by HMBC correlations from H-3 and H-6 to C-18. Similarly, the other γ-lactone ring attached to C-10 was established from correlations of H-10, H2-11, and H-12 (assigned by HMBC correlations to C-13, C-14, and C-16) with C-20. HMBC correlations of H3-17 with C-8 and C-7 confirmed the location of a propan-2-one group at C-6. These data suggested that compound 5 was a seco-clerodane diterpenoid cleaved between C8 and C9. The relative configuration of 5 was partially established by the application of NOE experiment (Figure S48) and confirmed by single-crystal X-ray diffraction measurement (Figure 3). The molecular formula of cephaloziellin F (6) was determined as C21H26O6 by HRESIMS, indicating one more carbon atom than 5, which was consistent with the presence of an additional oxygenated methyl in 6. Accordingly, the presence of signals of the oxygenated methyl [δH 3.70 (s); δC 51.7] and the methylene [δH 1.87 and 2.20 (m, H2-6); δC 30.0 (C-6)] in the 1D NMR spectra of 6 provided evidence that the 18,6-lactone ring in 5 was replaced by a carbomethoxy group E

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a known seco-clerodane diterpenoid isolated from in vitro cultures of the liverwort Jamesoniella autumnalis, except for the presence of an additional OH at C-10 (δC 97.6 in 8 and 97.3 in 9, respectively), which was consistent with the molecular formula C20H22O7. The relative configuration of 9 was partially established by the NOE correlations (Figure S96) and confirmed by single-crystal X-ray diffraction measurement (Figure 3), indicating orientations of H-6α, H-9β, OH-10α, H-12β, H3-17α, and H3-19α. In compound 8, strong NOE correlations (Figure S84) of H3-19/H-6, H-6/H3-17, and H317/H-9 determined that these groups were all α-oriented. H1α, H-7α, and H-11β were determined by NOE correlations with H3-19, H-9, and H3-17, respectively. The α-orientation of the β-monosubstituted furan ring was determined by the NOE correlation of H-14 with H-11α. Thus, the β-orientation of H-9 in 9 and α-orientation of H-9 in 8 showed that compounds 9 and 8 were C-9 epimers. Cephaloziellin J (10) was assigned the molecular formula C20H22O7, indicating 10 degrees of unsaturation. The 1D NMR data (Tables 2 and 5) of 10 were close to those of 8, except for the presence of another oxygenated methine [δC 75.2 (C-11)] and the absence of a double bond. 1H−1H COSY correlations (Figure S107) showed a C-1(H2)−C-2(H2)−C-3(H2)−C4(H) segment, which confirmed the absence of Δ3,4. The oxygenation of C-11 was supported by the presence of the CH−CH segment (at δH 4.36, d, J = 4.0 Hz, H-11; 5.36, s, H12, respectively). The one remaining degree of unsaturation was satisfied by the presence of an ether bridge between the ketal carbon C-10 (δC 100.0) and the oxygenated methine carbon C-11 (δC 75.2). Key NOE correlations of H-4/H3-19, H3-19/H-6, H3-19/H-11, and H-6/H-9 established that all were α-oriented. The α-orientation of the β-monosubstituted furan ring was determined by the correlation of H-16 with H-11 (Figure S108). The molecular formula of cephaloziellin K (11) was C20H20O6 by HRESIMS. Its 1H−1H COSY spectrum identified four spin systems (Figure S119): C-1(H2)−C-2(H2)−C-3(H), C-6(H)−C-7(H2), C-11(H2)−C-12(H), and C-14(H)−C15(H) segments. The assignments of C-4, C-5, C-6, and C10 were established by HMBC correlations (Figure S119) with H3-19 (δH 1.46, s), while C-7, C-8, and C-9 were correlated with H3-17 (δH 1.74, s). The presence of a γ-lactone attached to C-8 through the Δ8,9 double bond was confirmed by HMBC correlations of H2-11 with C-8, C-9, C-20, and C-12 (assigned by correlations to C-13, C-14, and C-16). NOE correlation of H3-9 and H-6 showed that they were cofacial and assigned as αoriented. However, the relative configuration of H-12 was not elucidated due to the rotation of the C(6)−C(7) bond. Cephaloziellins L−P (12−16) had quite similar NMR data. Compounds 12 and 13 were assigned by HRESIMS and elemental analysis to the same molecular formula (C21H24O6), which was 2 amu lower than that obtained for compounds 14− 16 (C21H26O6). The one added degree of unsaturation in 12 and 13 resulted from a double bond conjugated to the ester group, which was confirmed by 13C NMR data [δC 139.8 (C-3) and 137.3 (C-4) in 12 and δC 139.6 (C-3) and 137.4 (C-4) in 13] and HMBC correlations of H-3 with C-4 and C-18. The 1D NMR spectra of compounds 12−16 showed almost identical shifts for a β-monosubstituted furan ring [δC 121.0− 123.9 (C-13), 108.6−109.8 (C-14), 143.4−144.3 (C-15), 138.8−141.8 (C-16)], a carbomethoxy group [δH 3.63−3.71 (s, OCH3); δC 51.5−51.6 (OCH3) and 167.2−167.8 (C-18)], an acetyl group [δH 1.62−2.26 (s, H3-17); δC 28.7−29.3 (C-17)

and 208.8−210.6 (C-8)], and a lactone ring [δC 77.1−79.5 (C12) and 174.7−178.0 (C-20)]. These signals were correlated with their carbons by HSQC and long-range correlations to neighboring carbon atom (HMBC and 1H−1H COSY) experiments. The spectroscopic data were very similar to those published for 18, a clerodane diterpenoid isolated from the liverwort Jamesoniella colorata,8 except that the conjugated double bond was absent in 14−16. In compound 12, NOE correlations of H3-19/H-10, H3-19/ H-7, and H-7/H-12 determined that all were α-oriented. H-6α and H-6β were assigned by correlations with H3-19 and H-11, respectively, while H-1α and H-1β were determined by correlations with H-9 and H-11, respectively. NOE correlations of compounds 13 and 12 were quite similar except for the presence of the correlation of H-9 with H-12, which determined the β-orientation of H-12 in 13. Thus, compound 12 is the C-9 epimer of 18, while compound 13 is the C-12 epimer of 12. In contrast to 12, 13, and 18, the Δ3,4 double bond was absent in compounds 14−16 and H-4 was assigned to be α-oriented by NOE correlations with H3-19 and H-10. Apart from this difference, the structures of compounds 14, 15, and 16 were identical to those of 18, 12, and 13, respectively. The structure of compound 15 was confirmed by single-crystal X-ray diffraction study (Figure 3). The absolute configurations of all new compounds were determined by comparing experimental and calculated ECD spectra predicted by TDDFT (Experimental Section; Figure 4 for compound 5 and Figure 5A for compound 15). The results showed that the experimental and calculated ECD spectra of all structures were in good agreement.

Figure 4. Experimental and calculated ECD spectra of compound 5.

The structures of the known compounds 17 (amphiacrolide F) and 18 were elucidated based on comparison of their NMR and MS data with those reported in the literature.8,13 Compound 18 is a known modified clerodane diterpenoid reported by M. Toyota from the New Zealand liverwort Jamesoniella colorata.8 However, its absolute configuration and name were not clarified in that study. From a biogenetic point of view, the stereochemistry of 18 was consistent with those of compounds 12−16. Accordingly, compound 18 was named cephaloziellin Q.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured with an X-6 micromelting point apparatus. Optical rotations were obtained using a GYROMAT-HP polarimeter. UV data were F

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acetone, 10:1 to 0:1] to give subfractions 2BA−2BC. Subfraction 2BA (50 mg) was further separated by HPLC (MeOH−H2O, 50:40, 1.5 mL/min) to afford 1 (1.6 mg), 2 (1.6 mg), and 9 (3.9 mg). Subfraction 2BB (167 mg) was separated by RP C18 Lobar CC (MeOH−H2O, 50:50) to give five subfractions (2BBA−2BBE). Subfraction 2BBB (53 mg) was separated by HPLC (MeCN−H2O, 40:60, 1.5 mL/min) to give 8 (37.0 mg) and 5 (3.0 mg). Subfractions 2BBC (12 mg) and 2BBD (17 mg) were purified by HPLC (MeOH− H2O, 45:55, 1.5 mL/min) and HPLC [MeCN−H2O (0.1% HOAc), 45:55, 1.8 mL/min] to afford 11 (1.7 mg) and 4 (7.0 mg), respectively. Fraction 3 (655 mg) was subjected to silica gel CC [200−300 mesh, petroleum ether−EtOAc, 30:1 to 0:1] to give 11 subfractions (3A−3K). Subfraction 3H (58 mg) was separated by HPLC (MeCN−H2O, 55:45, 1.8 mL/min) to yield 10 (7.0 mg), 18 (1.6 mg), and 6 (6.1 mg). Subfraction 3I (50 mg) was separated by HPLC (MeOH−H2O, 56:44, 1.5 mL/min) to give 14 (3.7 mg) and 7 (7.8 mg). Subfraction 3K (48 mg) was also separated by HPLC [MeOH−H2O (0.1% HOAc), 55:45, 1.5 mL/min] to yield 3 (3.2 mg) and 17 (2.1 mg). Fraction 4 (1.0 g) was subjected to silica gel CC [200−300 mesh, petroleum ether−acetone, 10:1 to 0:1] to give subfractions 4A−4C. Subfraction 4B was further purified by HPLC (MeOH−H2O, 58:42, 1.8 mL/min) to yield 16 (15.0 mg), 13 (26.0 mg), 12 (6.0 mg), and 15 (27.0 mg). Compound 1: white, amorphous powder (MeOH); [α]25D −17.8 (c 0.169, MeOH); UV (MeOH) λmax (log ε) 209 (3.78) nm; CD (c 4.7 × 10−3 M, MeOH) λmax Δε 238 (+0.12), 219 (−0.08) nm; IR νmax 3444, 2962, 1922, 1770, 1599, 1503, 1367, 1233, 1175, 1056, 866 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 4; HRESIMS m/z 378.1914 [M + NH4]+ (calcd for C20H28O6N, 378.1911). Compound 2: white crystals (MeOH); mp 156−158 °C; [α]25D 0.0 (c 0.166, MeOH); UV (MeOH) λmax (log ε) 214 (3.82) nm; CD (c 4.8 × 10−3 M, MeOH) λmax Δε 256 (+1.11) nm; IR νmax 3477, 2958, 2920, 1748, 1679, 1596, 1507, 1408, 1147, 1021, 971 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 4; HRESIMS m/z 343.1542 [M + H]+ (calcd for C20H23O5, 343.1540). Compound 3: white, amorphous powder (MeOH); [α]25D −27.6 (c 0.326, MeOH); UV (MeOH) λmax (log ε) 209 (3.89) nm; CD (c 9.4 × 10−3 M, MeOH) λmax Δε 262 (+0.15) nm; IR νmax 3347, 2953, 1755, 1689, 1647, 1454, 1265, 1164, 952 cm−1; 1H NMR (600 MHz) and 13 C NMR (150 MHz) data (CDCl3), see Tables 1 and 4; HRESIMS m/z 347.1854 [M + H]+ (calcd for C20H27O5, 347.1853). Compound 4: white, amorphous powder (MeOH); [α]25D −28.6 (c 0.350, MeOH); UV (MeOH) λmax (log ε) 211 (4.00) nm; CD (c 10.1 × 10−3 M, MeOH) λmax Δε 267 (+0.33) nm; IR νmax 3349, 2956, 2873, 1747, 1685, 1610, 1451, 1266, 1075, 1012, 919 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 4; HRESIMS m/z 347.1855 [M + H]+ (calcd for C20H27O5, 347.1853). Compound 5: colorless crystals (MeOH); mp 187−190 °C; [α]25D −22.8 (c 0.307, MeOH); UV (MeOH) λmax (log ε) 212 (3.97) nm; CD (c 8.6 × 10−3 M, MeOH) λmax Δε 317 (−0.10), 261 (+0.46) nm; IR νmax 2953, 1756, 1718, 1681, 1599, 1504, 1254, 1161, 1025, 947 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 4; HRESIMS m/z 376.1756 [M + NH4]+ (calcd for C20H26O6N, 376.1755). Compound 6: colorless oil (MeOH); [α]25D +32.6 (c 0.613, MeOH); UV (MeOH) λmax (log ε) 210 (3.92) nm; CD (c 16.4 × 10−3 M, MeOH) λmax Δε 264 (+0.08) nm; IR νmax 2951, 2876, 1767, 1711, 1634, 1503, 1433, 1262, 1162, 1061 cm−1; 1H NMR (600 MHz) and 13 C NMR (150 MHz) data (CDCl3), see Tables 1 and 4; HRESIMS m/z 375.1804 [M + H]+ (calcd for C21H27O6, 375.1802). Compound 7: colorless oil (MeOH); [α]25D +29.2 (c 0.787, MeOH); UV (MeOH) λmax (log ε) 217 (3.82) nm; CD (c 21.2 × 10−3 M, MeOH) λmax Δε 322 (−0.12) nm; IR νmax 2949, 1766, 1714, 1672, 1640, 1434, 1265, 1058 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 1 and 4; HRESIMS m/z 373.1649 [M + H]+ (calcd for C21H25O6, 373.1646).

Figure 5. (A) Experimental and calculated ECD spectra of compound 15. (B) Experimental ECD spectra of compounds 12−16. recorded on a UV-2450 spectrophotometer (Shimadzu, Japan). CD spectra were obtained on a Chirascan spectropolarimeter. IR spectra were recorded using a Nicolet iN 10 Micro FTIR spectrometer. NMR spectra were recorded on a Bruker Avance DRX-600 spectrometer operating at 600 (1H) and 150 (13C) MHz with TMS as internal standard. HRESIMS were carried out on an LTQ-Orbitrap XL. HPLC separations were performed on an Agilent 1200 G1311A quat pump equipped with an Agilent 1200 G1322A degasser, an Agilent 1200 G1329B 1260ALS, an Agilent 1200 G1315D DAD detector, and a ZORBAX SB-C18 5 μm column (9.4 × 250 mm). All solvents used were of analytical grade. Silica gel (200−300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao, P. R. China), RP C18 silica gel (40−63 μm, FuJi), Sephadex LH-20 (25−100 μm; Pharmacia Biotek, Denmark), and MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.) were used for column chromatography (CC). Thinlayer chromatography (TLC) was carried out with glass precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.). Compounds were visualized under UV light and by spraying with H2SO4−EtOH (1:9, v/v) followed by heating. Plant Material. C. kiaeri was collected in April 2011 from Mount Maoer, Guangxi Zhuang Autonomous Region, P. R. China, and authenticated by Prof. Yuan-Xin Xiong, College of Life Sciences, Guizhou University, P. R. China. A voucher specimen (No. 2011041429) has been deposited at the Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University, P. R. China. Extraction and Isolation. The air-dried powder of the plant material of C. kiaeri (700 g) was extracted with 95% EtOH at room temperature (1.5 L × 4, each for one week). The crude extract (29.3 g) was suspended in H2O (200 mL) and partitioned successively with Et2O (4 × 200 mL) and n-BuOH (3 × 100 mL). After removal of organic solvent, the Et2O residue (10.0 g) was separated by MCI gel CC (CHP20P, 70−150 μm, MeOH−H2O, 3:7 to 9:1) to give fractions 1−6. Fraction 2 (1.2 g) was separated on a Sephadex LH-20 CC (MeOH) to give subfractions 2A and 2B. Subfraction 2B (745 mg) was subjected to silica gel CC [200−300 mesh, petroleum ether− G

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Compound 8: colorless crystals (MeOH); mp 150−153 °C; [α]25D −82.9 (c 0.350, MeOH); UV (MeOH) λmax (log ε) 211 (4.05) nm; CD (c 9.4 × 10−3 M, MeOH) λmax Δε 255 (+0.82) nm; IR νmax 3439, 2926, 2852, 1765, 1684, 1600, 1504, 1455, 1193, 966 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 2 and 5; HRESIMS m/z 392.1703 [M + NH4]+ (calcd for C20H26O7N, 392.1704). Compound 9: colorless needles (MeOH); mp 153−157 °C; [α]25D −70.4 (c 0.398, MeOH); UV (MeOH) λmax (log ε) 211 (3.98) nm; CD (c 10.6 × 10−3 M, MeOH) λmax Δε 257 (+0.75) nm; IR νmax 3378, 3107, 2946, 1755, 1692, 1634, 1509, 1454, 1077, 972 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 2 and 5; HRESIMS m/z 392.1709 [M + NH4]+ (calcd for C20H26O7N, 392.1704). Compound 10: white, amorphous powder (MeOH); [α]25D −75.0 (c 0.120, MeOH); UV (MeOH) λmax (log ε) 208 (3.79) nm; CD (c 3.2 × 10−3 M, MeOH) λmax Δε 228 (−0.36) nm; IR νmax 2956, 2928, 1686, 1581, 1494, 1396, 1367, 1195, 1105, 1069, 989 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 2 and 5; HRESIMS m/z 397.1619 [M + Na]+ (calcd for C20H22O7Na, 397.1622). Compound 11: white, amorphous powder (MeOH); [α]25D +64.7 (c 0.170, MeOH); UV (MeOH) λmax (log ε) 217 (4.26) nm; CD (c 4.8 × 10−3 M, MeOH) λmax Δε 316 (+0.14), 268 (+0.37), 247 (−0.22) nm; IR νmax 2917, 2850, 1747, 1712, 1662, 1576, 1539, 1196, 1024 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 2 and 5; HRESIMS m/z 357.1335 [M + H]+ (calcd for C20H21O6, 357.1333). Compound 12: white, amorphous powder (MeOH); [α]25D −9.1 (c 0.660, MeOH); UV (MeOH) λmax (log ε) 212 (4.09) nm; CD (c 17.7 × 10−3 M, MeOH) λmax Δε 291 (−1.03), 253 (+0.11) nm; IR νmax 2927, 1773, 1706, 1634, 1503, 1433, 1359, 1265, 1055, 773 cm−1; 1 H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 3 and 5; HRESIMS m/z 373.1648 [M + H]+ (calcd for C21H25O6, 373.1646). Compound 13: colorless crystals (MeOH); mp 167−170 °C; [α]25D +73.8 (c 0.420, MeOH); UV (MeOH) λmax (log ε) 212 (4.03) nm; CD (c 11.3 × 10−3 M, MeOH) λmax Δε 288 (−1.00), 250 (+0.48) nm; IR νmax 2920, 1774, 1705, 1630, 1503, 1444, 1358, 1054, 766 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 3 and 5; HRESIMS m/z 390.1915 [M + NH4]+ (calcd for C21H28O6N, 390.1911). Compound 14: white, amorphous powder (MeOH); [α]25D −31.9 (c 0.376, MeOH); UV (MeOH) λmax (log ε) 208 (3.76) nm; CD (c 11.3 × 10−3 M, MeOH) λmax Δε 289 (−0.23), 249 (−0.03) nm; IR νmax 2946, 2868, 1772, 1729, 1705, 1597, 1506, 1153, 1023 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 3 and 5; HRESIMS m/z 375.1804 [M + H]+ (calcd for C21H27O6, 375.1802). Compound 15: colorless crystals (MeOH); mp 154−157 °C; [α]25D −24.6 (c 0.528, MeOH); UV (MeOH) λmax (log ε) 210 (3.70) nm; CD (c 14.1 × 10−3 M, MeOH) λmax Δε 288 (−1.30) nm; IR νmax 2931, 1774, 1726, 1705, 1502, 1449, 1434, 1196, 1143, 1026, 948 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 3 and 5; HRESIMS m/z 375.1804 [M + H]+ (calcd for C21H27O6, 375.1802). Compound 16: colorless crystals (MeOH); mp 170−173 °C; [α]25D +11.5 (c 0.435, MeOH); UV (MeOH) λmax (log ε) 209 (3.59) nm; CD (c 11.6 × 10−3 M, MeOH) λmax Δε 286 (−1.12) nm; IR νmax 2952, 2863, 1782, 1727, 1705, 1643, 1498, 1193, 1026, 822 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (CDCl3), see Tables 3 and 5; HRESIMS m/z 392.2069 [M + NH4]+ (calcd for C21H30O6N, 392.2068). X-ray Crystallographic Analysis of Compound 5. C20H22O6, M = 358.38, monoclinic system, space group P21, a = 8.783(2) Å, b = 11.166(3) Å, c = 9.402(2) Å, V = 892.7(4) Å3, Z = 2, Dcalcd = 1.333 Mg/m3, μ(Mo Kα) = 0.098 mm−1, F(000) = 380, and T = 293(2) K. A crystal of dimensions 0.48 × 0.14 × 0.04 mm3 was selected for measurements on a Bruker APEX2 CCD area-detector diffractometer with a graphite monochromator (φ−ω scans), Mo Kα radiation (λ =

0.71069 Å). APEX2 Software Suite (Bruker, 2005) was used for cell refinement and data reduction. The structure was refined with fullmatrix least-squares calculations on F2 using SHELXL-97 (Sheldrick, 1997). A total of 10 496 reflections, collected in the θ range 2.24° to 27.49°, yielded 4066 unique reflections (Rint = 0.0358). All nonhydrogen atoms were given anisotropic thermal parameters. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. The final stage converged to R1 = 0.0424 (wR2 = 0.0806) for 4066 observed reflections [with I > 2σ(I)] and 238 variable parameters, R1 = 0.0769 (wR2 = 0.0913) for all unique reflections, and goodness-of-fit = 0.986. X-ray Crystallographic Analysis of Compound 9. C20H22O7, M = 374.38, monoclinic system, space group P212121, a = 8.237(7) Å, b = 23.983(19) Å, c = 9.258(7) Å, V = 1829(3) Å3, Z = 4, Dcalcd = 1.360 Mg/m3, μ(Mo Kα) = 0.103 mm−1, F(000) = 792, and T = 293(2) K. A crystal of dimensions 0.35 × 0.32 × 0.03 mm3 was selected for measurements on a Bruker APEX2 CCD area-detector diffractometer with a graphite monochromator (φ−ω scans), Mo Kα radiation (λ = 0.71073 Å). APEX2 Software Suite (Bruker, 2005) was used for cell refinement and data reduction. The structure was refined with fullmatrix least-squares calculations on F2 using SHELXL-97 (Sheldrick, 1997). A total of 10 553 reflections, collected in the θ range 2.36° to 26.97°, yielded 3904 unique reflections (Rint = 0.1215). The final stage converged to R1 = 0.1052 (wR2 = 0.2603) for 3904 observed reflections [with I > 2σ(I)] and 248 variable parameters, R1 = 0.2108 (wR2 = 0.3173) for all unique reflections, and goodness-of-fit = 0.979. X-ray Crystallographic Analysis of Compound 15. C21H26O6, M = 374.42, monoclinic system, space group P21, a = 9.098(2) Å, b = 10.562(2) Å, c = 10.531(2) Å, V = 978.6(4) Å3, Z = 2, Dcalcd = 1.271 Mg/m3, μ(Mo Kα) = 0.093 mm−1, F(000) = 400, and T = 293(2) K. A crystal of dimensions 0.48 × 0.15 × 0.13 mm3 was selected for measurements on a Bruker APEX2 CCD area-detector diffractometer with a graphite monochromator (φ−ω scans), Mo Kα radiation (λ = 0.71073 Å). APEX2 Software Suite (Bruker, 2005) was used for cell refinement and data reduction. The structure was refined with fullmatrix least-squares calculations on F2 using SHELXL-97 (Sheldrick, 1997). A total of 6054 reflections, collected in the θ range 2.00° to 27.42°, yielded 4231 unique reflections (Rint = 0.0142). The final stage converged to R1 = 0.0381 (wR2 = 0.0906) for 4231 observed reflections [with I > 2σ(I)] and 248 variable parameters, R1 = 0.0452 (wR2 = 0.0956) for all unique reflections, and goodness-of-fit = 1.053. Crystallographic data for these structures have been deposited with the Cambridge Crystallographic Data Centre as CCDC 932263 for 5, CCDC 932261 for 9, and CCDC 932264 for 15. Copies of the data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/ retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; e-mail: [email protected]). Theory and Calculation Details. The calculations were performed by the Materials Studio software and the Gaussian 09 program package. Conformational search was performed by MD simulations based on the COMPASS force field15 and by scanning the potential energy surface on the main chain dihedral angles using the semiempirical AM1. The starting conformer of compound 5 came from the corresponding X-ray structure (Figure 3). All ground-state geometries were optimized at the B3LYP/6-31G* level at 298 K, and harmonic frequency analysis was computed to confirm the minima and, hence, calculation of room-temperature free energy. Multiple lowenergy conformations of compound 5 were found, and four lower energy conformations were selected to predict ECD spectra for compound 5. Electronic excitation energies and rotational strengths in the gas phase and in methanol were calculated using TDDFT16,17 at the same level in velocity formalism for the first 60 states. The solvent effect of methanol has been modeled by a conductor-like screening model for real solvents (COSMO).18,19 The ECD curves were simulated by using the Gaussian function:20

Δε(E) = H

1 1 × 2.296 × 10−39 σ π

2

∑ ΔEiR ie−[(E −ΔEi)/ σ ] i

dx.doi.org/10.1021/np400426a | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

where σ is half the bandwidth at 1/e height and ΔEi and Ri are the excitation energies and rotatory strengths for transition i, respectively. Here σ = 0.4 eV.

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ASSOCIATED CONTENT

S Supporting Information *

1D NMR, 2D NMR, ESIMS, HRESIMS, IR, and UV spectra of the new compounds 1−16; experimental and calculated ECD spectra of compounds 1−4, 6−14, and 16; and X-ray data of compounds 5, 9, and 15. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-531-8838-2012. Fax: +86-531-8838-2019. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 30925038 and 30730109) and Shandong Provincial Fund (Nos. Z2006C03 and JQ200806).

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REFERENCES

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dx.doi.org/10.1021/np400426a | J. Nat. Prod. XXXX, XXX, XXX−XXX