Highly Oxygenated ent-Pimarane-Type Diterpenoids from the Chinese

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Highly Oxygenated ent-Pimarane-Type Diterpenoids from the Chinese Liverwort Pedinophyllum interruptum and Their Allelopathic Activities Na Liu,† Rui-Juan Li,† Xiao-Ning Wang,† Rong-Xiu Zhu,‡ Lei Wang,† Zhao-Min Lin,† Yu Zhao,† and Hong-Xiang Lou*,† †

Department of Natural Products Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, No. 44 West Wenhua Road, Jinan 250012, People’s Republic of China ‡ School of Chemistry and Chemical Engineering, Shandong University, No. 27 Shanda Nanlu, Jinan 250100, People’s Republic of China S Supporting Information *

ABSTRACT: Ten highly oxygenated ent-pimarane-type diterpenoids, pedinophyllols A−J (1−10), were isolated from the Chinese liverwort Pedinophyllum interruptum. Their structures were determined by comprehensive analysis of spectroscopic data together with single-crystal X-ray diffraction analysis. The absolute configurations were elucidated by comparison of experimental and theoretically calculated electronic circular dichroism spectra. Allelopathic testing showed that several new diterpenoids inhibited germination of Arabidopsis thaliana seeds.

iverworts, which are likely the first plant type to successfully transition from water to land, have yielded various secondary metabolites with interesting chemical structures and significant biological activities.1 Unlike vascular plants, liverworts possess fewer protective structural features against environmental stress, such as phytotoxins, pathogen attack, UV injury, and insect/animal predation.2,3 They must develop biochemical capabilities and active molecular architectures as protectants, some of which also act as chemical communication between liverworts and other plants, microorganisms, animals, or insects. Allelopathic interactions play a crucial role in natural ecosystems of liverworts.4,5 However, few research studies have been carried out so far to explore such interactions.6 Over the past decades, chemical research on the family Plagiochilaceae focused mainly on the genus Plagiochila,7,8,4 while investigations of the Pedinophyllum species lagged far behind due to difficulty in collection and taxonomy.9 Our ongoing research on bioactive compounds from Chinese liverworts has led to the identification of 10 highly oxygenated ent-pimarane diterpenoids, pedinophyllols A−J (1−10), from Pedinophyllum interruptum (Nee.) Lindb. (Figure 1), a liverwort collected in the Xishui Nature Reserve, Guizhou Province, P. R. China.10,11 This study marks the first isolation of ent-pimarane derivatives from the family Plagiochilaceae.9 Their chemical structures were elucidated by extensive spectroscopic analysis, and the absolute structures of compounds 1 and 4 were

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© 2013 American Chemical Society and American Society of Pharmacognosy

established by comparing experimental ECD spectra with TDDFT-calculated ECD spectra. In addition, based on ECD spectra as well as biosynthetic considerations, the absolute configurations of other co-occurring diterpenoids were also assigned. Allelopathic testing showed that several new isolates retarded Arabidopsis thaliana seed germination.

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RESULTS AND DISCUSSION Compound 1 was assigned the molecular formula C20H30O3, as determined by HRESIMS (m/z 336.2546 [M + NH4]+), requiring six indices of hydrogen deficiency. IR absorptions at 3217 and 1685 cm−1 provided evidence for hydroxy and conjugated carbonyl groups, respectively. The 13C NMR spectrum (Table 1) revealed signals for four methyl groups, five sp3 methylenes, two sp3 methines (one oxygenated at δC 72.7), one carbonyl (δC 201.6), two double bonds (δC 115.4, 142.1, 138.4, and 141.1), and four quaternary sp3 carbons (one oxygenated at δC 79.0). The 1H NMR spectrum (Table 2) displayed signals characteristic for a vinyl group at δH 5.70 (dd, J = 17.6, 10.7 Hz, H-15), 4.79 (d, J = 17.6 Hz, H-16a), and 5.07 (d, J = 10.7 Hz, H-16b), an olefinic proton at δH 6.96 (m, H-7), and four tertiary methyls at δH 1.21 (s, H3-17), 0.98 (s, H3-18 and H3-19), and 0.80 (s, H3-20). The aforementioned spectroscopic data were consistent with a pimarane-type Received: April 22, 2013 Published: September 4, 2013 1647

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(Figure S6) of H-1/H3-20, H-6α/H3-20, H-5/H-6β, H-11α/ H-15, and H-12α/H-15 in the NOESY spectrum, which was unambiguously confirmed by the single-crystal X-ray diffraction analysis (Figure 3). From the above evidence, 1 was determined to be 1β,9β-dihydroxy-ent-pimara-7,15-dien-14-one and named pedinophyllol A. Compound 2 displayed an [M + NH4]+ ion at m/z 334.2390 in the HRESIMS, consistent with a molecular formula of C20H28O3. The 13C and 1H NMR spectroscopic data of 2 (Tables 1 and 2) resembled those of 1, except for the resonance of a ketocarbonyl (δC 218.5) in 2 instead of an oxymethine group (δH 4.03, δC 72.7) in 1 at C-1. This assignment was further confirmed by an HMBC correlation between H3-20 (δH 1.19) and C-1. The relative configuration of 2, furnished by a NOESY experiment, also resembled that of 1. Accordingly, 2 was determined as 9β-hydroxy-ent-pimara-7,15-dien-1,14-dione and named pedinophyllol B. The molecular formula of 3, C20H30O3, was determined by HRESIMS (m/z 336.2545 [M + NH4]+). The 13C and 1H NMR spectra of 3 (Tables 1 and 2) were similar to those of 1, except for the position of the α,β-unsaturated carbonyl group. The 2D NMR data of 3 revealed that a Δ8(14) double bond and 7-carbonyl group were present based on HMBC correlations of H-5 to C-7, H-6 to C-7 and C-8, H-14 to C-7 and C-9, and H317 to C-14. Compounds 3 and 1 have the same stereostructure for 3 based on similar NOESY correlations. Thus, the structure of compound 3 was established as 1β,9β-dihydroxy-ent-pimara8(14),15-dien-7-one and named pedinophyllol C. Compound 4, colorless needles, was assigned the molecular formula C20H30O3 from its positive HRESIMS (m/z 341.2085 [M + Na]+). The 1D NMR spectra of 4 (Tables 1 and 2), in comparison with those of 1, revealed the absence of an olefinic proton and the presence of an oxymethine signal at δH 4.53 (d, J = 2.4 Hz, H-7) in 4. A spin system of CH(5)−CH2(6)− CH(7) was deduced from the 1H−1H COSY spectrum together with the HMBC correlations of H-7/C-8, H-7/C-9, H-7/C-14,

Figure 1. Structures of compounds 1−10.

diterpenoid skeleton,12 which was confirmed by HMBC (Figure 2). Specifically, HMBC correlations from H3-17 to C12, C-13, C-14, and C-15, from H-7 to C-14, and from H-16 to C-13 indicated that a methyl and a vinyl were connected to C13 and a carbonyl was present at C-14. HMBC correlations from H3-20 to C-9, from H-7 to C-5, C-8, C-9, and C-14, and from H-11 to C-8 suggested that a hydroxy group was positioned at C-9 and a double bond was located between C-7 and C-8. Another hydroxy group was confirmed at C-1 by HMBC correlations of H-3 (δH 1.19 and 1.84), H-5 (δH 2.26), and H3-20 to C-1 (δC 72.7) (Figure 2). The relative configuration of 1 was determined by NOE correlations

Table 1. 13C NMR Data of Compounds 1−10 (150 MHz) in CDCl3 (δ in ppm) position

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OAc-7 OMe-7

72.7 26.3 33.7 32.7 36.0 25.0 141.1 138.4 79.0 41.8 24.7 31.3 50.4 201.6 142.1 115.4 24.6 33.1 22.2 17.4

218.5 37.1 41.5 32.7 44.2 24.6 137.8 138.4 76.8 54.1 26.8 31.1 50.8 203.1 142.5 115.1 24.8 32.0 22.4 15.4

73.5 26.9 33.7 33.0 36.3 37.2 201.4 138.1 76.1 42.5 26.0 30.5 39.4 144.0 144.1 113.7 28.1 32.7 21.4 17.3

71.0 25.8 34.0 32.8 37.9 25.7 62.9 132.4 166.5 45.0 20.8 33.6 46.9 204.4 141.6 113.7 22.2 32.7 21.5 18.9

70.9 25.9 33.8 32.6 38.3 24.8 64.7 128.7 168.0 44.7 21.1 33.6 46.7 200.3 141.7 113.6 21.9 32.6 21.4 19.1 170.4

70.3 25.7 33.6 32.7 42.1 34.7 201.3 133.5 167.8 44.5 22.1 30.7 38.6 70.6 146.0 111.8 18.2 32.3 21.2 18.0

71.3 25.4 34.3 32.5 37.9 27.0 69.8 130.9 143.4 43.6 21.1 29.4 39.7 76.7 144.5 112.5 20.1 32.9 21.8 18.8

70.8 25.2 34.1 32.7 41.6 23.7 77.5 119.9 141.7 43.4 20.6 31.6 39.2 72.4 146.8 111.8 17.3 32.9 21.6 20.3

70.0 25.8 33.6 32.9 43.5 35.1 199.1 129.3 168.4 44.7 21.6 22.3 40.9 76.7 79.9 70.9 18.4 32.4 21.2 18.2

70.7 25.7 33.9 32.7 37.7 25.9 62.4 132.0 168.8 45.0 20.8 30.9 46.4 207.9 75.8 61.9 16.1 32.6 21.5 18.5

55.3 1648

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Table 2. 1H NMR Data of Compounds 1−5 (600 MHz) in CDCl3 (δ in ppm and J in Hz) position 1 2α 2β 3α 3β 5 6α 6β 7 11α 11β 12α 12β 14 15 16a 16b 17 18 19 20 7-OAc a

1 4.03 1.84 1.57 1.84 1.19 2.26 2.15 2.40 6.96 2.08 1.87 1.78 2.23

br s m m m m m m m m m m m m

5.70 5.07 4.79 1.21 0.98 0.98 0.80

dd (10.7, 17.6) d (10.7) d (17.6) s s s s

2 2.82 dt (4.9, 13.3) 2.23 m 1.79−1.83 ma 2.39 dd (3.8, 12.6) 2.28 m 6.80 1.96 2.36 1.73 2.24

d (4.8) dt (17.9, 2.5) m m m

5.70 5.07 4.83 1.23 1.02 1.19 1.19

dd (10.7, 17.6) d (10.7) d (17.6) s s s s

3 4.08 1.98 1.55 1.84 1.28 2.81 2.66 2.32

br s m m m m dd (5.8, 12.6) dd (5.8, 18.6) dd (12.6, 18.6)

1.95 1.83 1.63 1.83 6.63 5.69 4.99 4.79 1.20 0.96 0.98 0.88

m m m m s dd (10.5, 17.5) d (10.5) d (17.5) s s s s

4

5

3.99 1.96 1.65 1.64 1.29 1.93 1.98 1.89 4.53 2.40 2.75 1.96 1.87

br s m m m m m m m t (2.4) dt (4.8, 18.3) dt (4.8, 18.3) m m

4.07 2.04 1.67 1.67 1.31 1.93 1.94 1.69 5.77 2.44 2.79 1.98 1.90

br s m m m m m m m t (2.7) dt (4.6, 18.2) m m m,

6.00 5.11 4.99 1.23 0.89 1.01 1.03

dd (10.7, 17.6) d (10.7) d (17.6) s s s s

5.99 5.10 4.98 1.23 0.93 0.89 1.04 2.01

dd (10.7, 17.6) d (10.7) d (17.6) s s s s s

Signals were overlapped.

Figure 2. Key 1H−1H COSY (−) and HMBC (H→C) correlations of 1 and 4.

Figure 4. Single-crystal X-ray structures of 4.

Compound 5 was determined to be an acetylated derivative of 4. Its molecular weight was 42 amu higher than that of 4, as revealed by HRESIMS (m/z 383.2205 [M + Na]+), and its NMR data (Tables 1 and 2) showed resonances for an acetyl group (δH 2.01, s; δC 21.4 and 170.4). The acetoxy group at C7 was verified by the downfield-shifted H-7 signal at δH 5.77 (Δδ 1.24 ppm) and the HMBC correlations of H-7 (δH 5.77, br s) to the acetyl carbonyl (δC 170.4). The configurations of 5 were the same as that of 4 based on similar NOE relationships. Therefore, 5 was elucidated as 1β-hydroxy-7β-acetoxy-entpimara-8,15-dien-14-one and named 7-O-acetylpedinophyllol E. Compound 6 had the same molecular formula as that of 4, as determined by HRESIMS (m/z 341.2093 [M + Na]+). The 1H NMR data of 6 (Table 3) were similar to those of 4, except for the splitting pattern of one oxymethine proton (a triplet at δH 4.53 for 4; a singlet at δH 4.43 for 6). The key HMBC correlations between the signal at δH 4.43 (H-14) and carbon signals at δC 167.8 (C-9), 146.0 (C-15), and 18.2 (C-17)

Figure 3. Single-crystal X-ray structure of 1.

and H3-20/C-9 (Figure 2) and, furthermore, confirmed the positions of the hydroxy group and olefinic bond. The βorientation of OH-7 was determined from the rather small vicinal coupling constant (J = 2.4 Hz) detected from the H-7 signal at δH 4.53.13 Proof for the proposed structure and evidence for the stereochemistry of 4 were again confirmed by X-ray crystallography (Figure 4). Thus, 4 was determined as 1β,7β-dihydroxy-ent-pimara-8,15-dien-14-one and named pedinophyllol D. 1649

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Table 3. 1H NMR Data of Compounds 6−10 (600 MHz) in CDCl3 (δ in ppm and J in Hz) position 1 2α 2β 3 5 6 7 11α 11β 12α 12β 14 15 16α 16β 17 18 19 20 7-OMe a

6 4.02 2.04 1.65 1.72 1.31 2.24 2.41 2.55

br s m m m m dd (3.4, 14.7) dd (3.4, 17.6) t (14.7, 17.6)

2.30 2.79 1.69 1.47 4.43 5.92 5.04 5.06 1.09 0.97 0.97 1.11

dt (4.8, 18.7) m m m s dd (10.7, 17.6) d (10.7) d (17.6) s s s s

7 3.89 1.89 1.59 1.71 1.25 1.94 1.82 1.65 4.14 2.31 2.06 1.76 1.49 3.83 5.72 5.01

t (2.1) m m m m d (13.3) m m br s m m m m s dd (10.7, 17.6) d (10.7)a

1.07 1.00 0.89 0.95

s s s s

8

9

3.84 1.92 1.54 1.64 1.22 1.55 2.18 1.46 4.17 2.28 2.05 1.48

t (2.1) dt (4.3, 13.2) m m m m m m br s m dt (4.5, 13.2) ma

4.07 2.08 1.64 1.69 1.31 2.19 2.57 2.48

4.19 5.92 5.07 5.02 1.02 0.95 0.90 1.05 3.40

s dd (10.7, 17.6) d (10.7) d (17.6) s s s s s

4.31 4.29 4.19 3.64 0.99 0.96 0.95 1.20

br s m m m m dd (3.1, 14.6) dd (3.1, 14.6) m

2.44 m 2.73 m 1.55−1.61 ma s t (7.8) t (7.8) t (7.8) s s s s.

10 3.99 2.03 1.64 1.64 1.31 1.94 1.94 1.64 4.58 2.49 2.83 1.87 1.74

br s m m m m t (12.4) t (12.4) m d (3.2) br d (12.8) dt (12.8, 4.6) dt (4.6, 12.8) br d (12.8)

3.73 m 3.64−3.72 ma 1.19 1.03 0.92 1.07

s s s s

Signals were overlapped.

Figure 5. Experimental ECD (red) and simulated Boltzmann-averaged ECD (black) spectra of 3 and 4.

indicated a hydroxy group at C-14. The carbonyl carbon at δC 201.3 was assigned at C-7 from the HMBC correlations of H-5 and H-14 to C-7. Thus, the carbonyl and hydroxy groups were positioned at C-7 and C-14, respectively, in 6 rather than at C14 and C-7, respectively, in 4. The proton at C-14 was assigned a β-orientation on the basis of NOESY correlations between H14 and Me-17. Compound 6 was thus established as 1β,14αdihydroxy-ent-pimara-8,15-dien-7-one and named pedinophyllol F. The HRESIMS of compound 7 exhibited an [M + NH4]+ ion peak at m/z 338.2699, corresponding to a molecular formula of C20H32O3. The NMR data indicated that the C-14 ketocarbonyl group in 4 was reduced to an oxymethine group (δH 3.83, s; δC 76.7) in 7, which was supported by HMBC correlations of H14 with δC 130.9 (C-8), 143.4 (C-9), and 69.8 (C-7), and H317 with δC 76.7 (C-14). The methoxy derivative (8) of 7, with the molecular formula C21H34O3 from its HRESIMS (m/z 352.2851 [M + Na]+; calcd 352.2847), was also obtained and identified by 2D NMR experiments. This compound might be

an artifact from compound 7 with the allylic alcohol being converted to a methyl ether during isolation in the presence of MeOH.14 Analysis of NOESY correlations determined the relative configurations of these two compounds. Accordingly, 7 and 8 were established as 1β,7β,14α-trihydroxy-ent-pimara8,15-diene and 7β-methoxy-1β,14α-dihydroxy-ent-pimara-8,15diene and named pedinophyllol G and 7-O-methylpedinophyllol H, respectively. Compound 9 was obtained as a colorless oil. A molecular formula of C20H32O5 was assigned for 9 based on the [M + Na]+ ion peak at m/z 357.2045 in the HRESIMS. The 13C and 1 H NMR data (Tables 1 and 3) of 9 resembled those of 6 except for the absence of the vinyl group and the presence of an oxymethine (δC 79.9, C-15) and an oxymethylene group (δC 70.9, C-16). The remaining unsaturation and downfield shift of the C-14 signal (δC 76.7) suggested an ether linkage between C-14 and C-16, which was verified by HMBC correlations between H-14/C-16, H-14/C-15, and H-16/C-13. NOE relationships between H-14/H3-17, H-15/H-12α, and H-15/ 1650

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H3-17 led to the assignments of H-14 and H-15 as β-oriented. Consequently, 9 was elucidated as 14α,16-epoxy-1β,15αdihydroxy-ent-pimara-8-en-7-one and named pedinophyllol I. Compound 10 possessed the molecular formula C20H30O4, as confirmed by the HRESIMS (m/z 375.2152 [M + Na]+), and was isolated as an oil. The 13C and 1H NMR spectroscopic data of 10 (Tables 1 and 3) indicated that an oxymethine and an oxymethylene group were present in 10 rather than the vinyl group in 4, which indicated two hydroxy groups at C-15 and C16. This assignment was confirmed by HMBC correlations of H-15 (δH 3.73, br s) to C-12 (δC 30.9), C-16 (δC 61.9), and C17 (δC 16.1) as well as H-16 (δH 3.64−3.72, m) to C-13 (δC 46.4). The 13C NMR spectrum of 10 showed a resonance at δC 75.8 (C-15) close to the value of 15S but different from that reported for the 15R epimer, which provided proof for the C-15 configuration.15 The conclusion was in accordance with a previous study of the C-15 configuration of naturally occurring pimarene-15,16-diols.16 The relative configurations at other stereocenters were deduced by NOESY experiments. The structure of compound 10 was thus defined as 1β,7β,15α,16tetrahydroxy-ent-pimara-8-en-14-one, and 10 has been named pedinophyllol J. Chiroptical properties of skewed α,β-unsaturated ketones are often used for determining the absolute configuration of organic compounds on the basis of empirical helicity rules.17 The ECD spectrum of compound 1, which is a cisoid enone, showed characteristic Cotton effects at 256 (Δε +0.82), 299 (Δε +0.11), and 345 (Δε −0.48) nm reflecting negative enone helicity.18 Enone 3 exhibited a positive n→π* band (Figure 5) on the basis of the positive enone torsion angle. Thus, the absolute configurations of C-1 and C-9 in 1 and 3 are deduced as R as shown.19 TDDFT ECD calculations (Figure 5) confirmed the absolute configuration of 3 as depicted. The ECD spectrum of 2 displayed a positive Cotton effect at 260 nm, identical with that of 1, indicating 2 was also an entpimarane-type diterpenoid. The difference between the ECD spectra of 1 and 2 at about 300 nm reflects the presence of the 1-oxo function in 2 (Δε = +1.62, in MeOH) and its absence in 1 (Δε = +0.11, in MeOH).20 The absolute configuration of 4 was suggested as shown in Figure 1 by the calculation of the ECD spectrum (Figure 5). The ECD spectra of compounds 5−10 (Supporting Information) resembled that of 4 at ca. 218 and 201 nm, and their absolute configurations were thus defined as shown. Additionally, from a biosynthetic standpoint, their absolute configurations should be the same. Pimarane-type diterpenoids functioning as allelochemicals have already been reported in moss.21 The allelopathic potential of the aforementioned diterpenoids from P. interruptum was tested with a model of A. thaliana germination. The root growth of A. thaliana was inhibited in a dosedependent manner after treatment with compound 1 (Figure 6A). The inhibitory effects of compounds 1−6, 9, and 10 at different concentrations as shown in Figure 6B were calculated by the equation described by Fan.22 However, compounds 7 and 8 were not available in sufficient quantity for this analysis. Results showed that compound 1 significantly retarded Arabidopsis seed germination with an IC50 (the effective concentration to afford 50% inhibition) value of 7.8 μg/mL. Lunularic acid, a reported allelochemical,23 was used as a positive control (IC50 14.2 μg/mL). Compounds 2−5 were found to display moderate activity, with IC50 values ranging

Figure 6. Effects of compounds on seedling growth of A. thaliana. (A) Growth of A. thaliana on Petri dishes treated with different concentrations of compound 1: 0, 8, 16, and 32 μg/mL. (B) Inhibition of different concentrations of compounds on A. thaliana root growth. Columns with different asterisks (*, **) show statistically significant difference, with (**) denoting more significant value (p < 0.01, 0.05). Lunularic acid was used as the positive control.

from 22 to 35 μg/mL, while the three remaining tested compounds exhibited weak inhibitory activity. In conclusion, 10 new highly oxygenated ent-pimarane-type diterpenoids (1−10) were isolated from the liverwort P. interruptum. To date, few chemical studies on the genus Pedinophyllum have been conducted. The only previous publication on the chemistry of P. interruptum, collected in Scotland, reported the presence of chromanes and prenyl benzoate derivatives.24 Our results showed that Chinese P. interruptum produces ent-pimaranes as main components, which is chemotaxomically different from the Scottish species.4 At a glance, there might be two chemo- or geographical types of P. interruptum. An allelopathitic activity test showed that the new entpimarane diterpenoids from P. interruptum could play a role as 1651

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Journal of Natural Products

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Pedinophyllol A (1): colorless needles (MeOH); mp 161−163 °C; [α]20D +55 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 241 (3.92) nm; ECD (MeOH) λmax (Δε) 256 (+0.82), 299 (+0.11), 345 (−0.48) nm; IR (KBr) νmax 3217, 2924, 1685, 1622, 1413, 1366, 1196 cm−1; 13C NMR and 1H NMR data, see Tables 1 and 2; positive ESIMS m/z 336.5 [M + NH4]+; positive HRESIMS m/z 336.2538 [M + NH4]+ (calcd for C20H30O3, 336.2533). Pedinophyllol B (2): colorless needles (MeOH); mp 145−147 °C; [α]20D +200 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 234 (3.87) nm; ECD (MeOH) λmax (Δε) 234 (+2.53), 298 (+1.62) nm; IR (KBr) νmax 3417, 2953, 1710, 1621, 1461, 1370, 1192 cm−1; 13C NMR and 1H NMR data, see Tables 1 and 2; positive ESIMS m/z 334.5 [M + NH4]+; positive HRESIMS m/z 334.2390 [M + NH4]+ (calcd for C20H28O3, 334.2377). Pedinophyllol C (3): colorless needles (MeOH); mp 151−153 °C; [α]20D −59 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 236 (3.99) nm; ECD (MeOH) λmax (Δε) 216 (−2.59), 270 (+0.15), 331 (+0.71) nm; IR (KBr) νmax 3371, 2942, 1670, 1601, 1419, 1302, 1041 cm−1; 13C NMR and 1H NMR data, see Tables 1 and 2; positive ESIMS m/z 336.5 [M + NH4]+; positive HRESIMS m/z 336.2545 [M + NH4]+ (calcd for C20H30O3, 336.2533). Pedinophyllol D (4): colorless needles (MeOH); mp 154−156 °C; [α]20D −52 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 243 (3.93) nm; ECD (MeOH) λmax (Δε) 213 (−2.11), 242 (+0.12), 346 (−0.45) nm; IR (KBr) νmax 3442, 2934, 1736, 1647, 1456, 1374, 1234, 1050 cm−1; 13 C NMR and 1H NMR data, see Tables 1 and 2; positive ESIMS m/z 341.4 [M + Na]+; positive HRESIMS m/z 341.2085 [M + Na]+ (calcd for C20H30O3, 341.2087). Pedinophyllol E (5): colorless needles (MeOH); mp 147−149 °C; [α]20D −66 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 239 (3.70) nm; ECD (MeOH) λmax (Δε) 214 (−3.90), 260 (+0.09), 341 (−0.47) nm; IR (KBr) νmax 3500, 2935, 1733, 1666, 1458, 1373, 1244 cm−1; 13C NMR and 1H NMR data, see Tables 1 and 2; positive ESIMS m/z 361.4 [M + H]+; positive HRESIMS m/z 383.2205 [M + Na]+ (calcd for C22H32O4, 383.2217). Pedinophyllol F (6): white, amorphous powder (MeOH); [α]20D −18 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 236 (3.82) nm; ECD (MeOH) λmax (Δε) 218 (−1.87), 249 (+1.05), 342 (−0.41) nm; IR (KBr) νmax 3403, 2934, 1655, 1459, 1375, 1219, 1008 cm−1; 13C NMR and 1H NMR data, see Tables 1 and 3; positive ESIMS m/z 341.4 [M + Na]+; positive HRESIMS m/z 341.2093 [M + Na]+ (calcd for C20H30O3, 341.2087). Pedinophyllol G (7): colorless oil (MeOH); [α]20D +35 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (1.67) nm; ECD (MeOH) λmax (Δε) 211 (−1.07), 248 (+0.98) nm; IR (KBr) νmax 3434, 2929, 1732, 1629, 1429, 1369, 1241 cm−1; 13C NMR and 1H NMR data, see Tables 1 and 3; positive ESIMS m/z 338.5 [M + NH4]+; positive HRESIMS m/z 338.2699 [M + NH4]+ (calcd for C20H32O3, 338.2690). Pedinophyllol H (8): white, amorphous powder; [α]20D +21 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (1.42) nm; ECD (MeOH) λmax (Δε) 209 (−1.22), 243 (+1.02) nm; IR (KBr) νmax 3358, 2947, 1710, 1641, 1460, 1370, 1230 cm−1; 13C NMR and 1H NMR data, see Tables 1 and 3; positive ESIMS m/z 352.3 [M + NH4]+; positive HRESIMS m/z 352.2851 [M + NH4]+ (calcd for C21H34O3, 352.2847). Pedinophyllol I (9): colorless oil; [α]20D −56 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 240 (3.91) nm; ECD (MeOH) λmax (Δε) 211 (−4.67), 247 (+1.20), 333 (−0.89) nm; IR (KBr) νmax 3419, 2938, 1655, 1458, 1393, 1298, 1023 cm−1; 13C NMR and 1H NMR data, see Tables 1 and 3; positive ESIMS m/z 335.2 [M + H]+; positive HRESIMS m/z 357.2045 [M + Na]+ (calcd for C20H30O4, 357.2036). Pedinophyllol J (10): colorless oil; [α]20D −45 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 236 (3.91) nm; ECD (MeOH) λmax (Δε) 223 (−0.64), 250 (+0.32), 344 (−0.53) nm; IR (KBr) νmax 3416, 2940, 1644, 1458, 1379, 1233, 1053 cm−1; 13C NMR and 1H NMR data, see Tables 1 and 3; positive ESIMS m/z 375.4 [M + Na]+; positive HRESIMS m/z 375.2152 [M + Na]+ (calcd for C20H32O5, 375.2142). X-ray Crystallographic Analysis of 1 and 4. X-ray data for compounds 1 and 4 were collected on a Bruker APEX2 CCD area-

allelochemicals for defense of the liverwort against other plants. Thus, the allelopathic potential of liverworts should be regarded as an important biochemical capability for their survival in the terrestrial ecosystem. However, evidence of allelopathic activity is not sufficient to explain how other plants are affected during the growth of liverworts.25 Therefore, further studies must to be carried out to better understand how allelopathic chemicals play their role, taking into account the possible incorporation of the powdered exudate, also in pellet formulation, into the soil.26

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a GYROMAT-HP polarimeter. UV spectra were acquired on a Shimadzu UV-9 spectrophotometer, and IR spectra on a Thermo Nicolet NEXUS 470 FT-IR spectrometer with KBr discs. ECD spectra were obtained on a Chirascan spectropolarimeter. NMR spectra were measured on a Bruker Avance DRX-600 spectrometer operating at 600 MHz for 1H NMR and 150 MHz for 13C NMR in CDCl3 with TMS as an internal standard. HRESIMS and ESIMS were determined using a LTQ-Orbitrap XL and an API 4000 triple-stage quadrupole instrument, respectively. Column chromatography (CC) was performed on silica gel (200−300 mesh, Haiyang Chemical Co. Ltd., Qingdao, P. R. China), Sephadex LH-20 (25−100 μm; Pharmacia Biotek, Denmark), or MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.). TLC was carried out on precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.). TLC spots were visualized under UV (254 nm) light and by spraying with 10% H2SO4/EtOH followed by heating. Semipreparative HPLC was performed on an Agilent 1100 G1310A isocratic pump equipped with a G1322A degasser, a G1314A VWD detector (210 nm), and a Phenomenex Luna C18(2) column (250 × 4.60 mm i.d., 5 μm), using an eluent of MeOH/H2O with a flow rate of 0.8 mL/min. Column loading was performed with a manual sample injector (Rheodyne Valve model 7725) equipped with a 200 μL injection loop. Plant Material. The liverwort P. interruptum (Nee.) Lindb. (Plagiochilaceae) was collected in July 2010, from Xishui Nature Reserve in Guizhou Province, P. R. China, and was authenticated by Prof. Yuan-Xin Xiong (College of Life Sciences, Guizhou University, P. R. China). A voucher specimen (no. 201007-31) 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 and powdered liverwort P. interruptum (29 g) was extracted exhaustively with 90% EtOH at room temperature (3 × 0.2 L, each for one week). The crude extract (1.82 g) was suspended in H2O (1000 mL), then partitioned successively with Et2O (3 × 150 mL) and n-BuOH (3 × 150 mL). The Et2O extract (0.79 g) was applied to MCI gel column chromatography (MeOH/H2O, 4:6 to 9:1) to give fractions A−F. Fraction D (67 mg) was subjected to silica gel CC, eluted with petroleum ether/Me2CO, 15:1 to 1:1, to give two subfractions (D1, D2). Fractions D1 (13 mg) and D2 (11 mg) were purified by semipreparative HPLC on a Phenomenex Luna C18 column (MeOH/H2O, 60:40, 0.8 mL/min, injection volume 20 μL) to give 10 (6.7 mg, tR = 16.3 min) and 9 (5.1 mg, tR = 21.1 min), respectively. Fraction E (40 mg) was subjected to Si gel column chromatography using petroleum ether/acetone (20:1) as the eluent to yield mixtures E1−E3. Further separation of fraction E2 (38 mg) by semipreparative HPLC (MeOH/H2O, 77:23, 0.8 mL/ min, injection volume 25 μL) gave 2 (25.3 mg, tR = 12.0 min). Fraction F (110 mg) was subjected to silica gel chromatography eluted with petroleum ether/Me2CO (30:1 to 0:1) in gradient to obtain 5 (4.3 mg) and four subfractions, F1−F4. Fraction F1 (29 mg) was further chromatographed on Sephadex LH-20 using CHCl3/MeOH (1:1) as eluent and then purified by semipreparative HPLC (MeOH/ H2O, 69:31, 0.8 mL/min, injection volume 25 μL) to yield 1 (2.8 mg tR = 13.2 min), 3 (2.9 mg tR = 15.9 min), and 4 (3.7 mg tR = 16.8 min). Separation of fraction F2 (30 mg) following a similar procedure to that for fraction F1 yielded 6 (2.5 mg, tR = 10.3 min), 7 (1.1 mg tR = 11.3 min), and 8 (1.3 mg tR = 18.8 min). 1652

dx.doi.org/10.1021/np4003178 | J. Nat. Prod. 2013, 76, 1647−1653

Journal of Natural Products

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detector diffractometer with a graphite monochromator (ϕ−ω scans), Mo Kα radiation (λ = 0.71069 Å). APEX2 Software Suite27 was used for cell refinement and data reduction. Crystal data of 1: C20H30O3, M = 318.44, orthorhombic, space group P212121, a = 7.9551(2) Å, b = 11.0331(3) Å, c = 21.0769(8) Å, V = 1849.91(10) Å3, Z = 4, Dcalcd = 1.143 g/cm3, T = 293 K, F(000) = 696, and μ(Mo Kα) = 0.075 mm−1. A total of 9553 reflections (4187 unique, Rint = 0.0357) were collected from 3.16° to 27.40° in θ and index ranges 10 ≥ h ≥ −10, 14 ≥ k ≥ −14, 27 ≥ l ≥ −22. The final stage converged to R1 = 0.0544 (wR2 = 0.1350) for 4187 observed reflections [with I > 2σ(I)] and 214 variable parameters and R1 = 0.1006 (wR2 = 0.1625) for all unique reflections and GoF = 1.002. Crystal data of 4: C20H30O3, M = 318.44, orthorhombic, space group P212121, a = 8.9471(5) Å, b = 19.6621(8) Å, c = 20.8630(8) Å, V = 3670.2(3) Å3, Z = 8, Dcalcd = 1.153 g/cm3, T = 293 K, F(000) = 1392, and μ(Mo Kα) = 0.075 mm−1. A total of 18 250 reflections (8053 unique, Rint = 0.0565) were collected from 1.95° to 27.49° in θ and index ranges 11 ≥ h ≥ −9, 19 ≥ k ≥ −25, 27 ≥ l ≥ −25. The final stage converged to R1 = 0.0769 (wR2 = 0.1920) for 8053 observed reflections [with I > 2σ(I)] and 416 variable parameters and R1 = 0.1714 (wR2 = 0.2431) for all unique reflections and GoF = 1.012. The structures were refined with full-matrix least squares calculations on F2 using SHELXL-97.28 All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in geometrically calculated positions and refined as riding atoms with the relative isotropic parameters. The refined fractional atomic coordinates, bond lengths, bond angles, and thermal parameters have been deposited at the Cambridge Crystallographic Data Centre (CCDC). CCDC 844915 and 844916 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/deposit or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: C44 1223 336 033; e-mail: [email protected]). Seedling Growth Test. Seeds of A. thaliana were surface sterilized using 5% sodium hypochlorite for 5 min, followed by five washes with sterile distilled water. Compounds were dissolved with DMSO and added to 25 mL of 1/2 MS medium supplemented with 0.8% (w/v) agar to produce plates with different concentrations of compounds (0, 8, 16, 32, 64 μg mL−1). Lunularic acid and the same volume of DMSO were used as positive and negative controls, respectively. Fifteen seeds were distributed on each Petri dish. Three replicates were performed for each concentration. The Petri dishes were placed in a growth chamber at 23 ± 1 °C under 18 h light and 6 h dark.22 Lengths of seedling roots were measured after seven days.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Selected NOEs of compound 1; 1H NMR and 13C NMR, 1 H−1H COSY, HSQC, HMBC, NOESY, CD, and UV spectra of 1−10. CIF files and X-ray crystallographic data of 1 and 4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 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 Financial support from the National Natural Science Foundation of China (nos. 30730109 and 30925038) is gratefully acknowledged. We kindly acknowledge D.-X. Guo and L.-N. Wang for collection of plant material, Mrs. J. Ren and Mr. B. Ma for NMR measurements, and Mrs. Y.-H. Gao for HRESIMS determination. 1653

dx.doi.org/10.1021/np4003178 | J. Nat. Prod. 2013, 76, 1647−1653