Phytochemistry 71 (2010) 15731578

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Phytochemistry
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Labdane diterpenoids and highly methoxylated bibenzyls from the liverwort Frullania inouei
Dong-Xiao Guo a, Feng Xiang a, Xiao-Ning Wang a, Hui-Qing Yuan b, Guang-Min Xi b, Yan-Yan Wang a, Wen-Tao Yu c, Hong-Xiang Lou a,*
a b c

Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University, No. 44 West Wenhua Road, Jinan 250012, Peoples Republic of China Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, No. 44 West Wenhua Road, Jinan 250012, Peoples Republic of China State Key Laboratory of Crystal Materials, Shandong University, No. 27 Shanda Nanlu, Jinan 250100, Peoples Republic of China

a r t i c l e

i n f o

a b s t r a c t
Four undescribed labdane diterpenoids, 1,2-dehydro-3,7-dioxo-manoyl oxide (1), 1,2-dehydro-7bhydroxy-3-oxo-manoyl oxide (2), 3,7-dioxo-manoyl oxide (3), and 3b-hydroxy-7-oxo-manoyl oxide (4) together with three known diterpenoids (57) and four highly methoxylated bibenzyls (811) were isolated from the liverwort Frullania inouei. The absolute structures of 14 were established by combined analysis of NMR data, CD data coupled with TDDFT CD calculations, and single-crystal X-ray diffraction measurement. Cytotoxicity tests to human tumor KB, KB/VCR, K562 or K562/A02 cells showed bibenzyls 811 inhibited cell proliferation with ID50 values ranging from 11.3 to 49.6 lM and overcame the multidrug resistance (MDR) with the reversal fold (RF) values ranging from 3.19 to 10.91 (5 lM) for vincristine-resistant KB/VCR and RF values from 4.40 to 8.26 (5 lM) for adriamycin-resistant K562/A02 cells, respectively. However, none of the diterpenoids were found to be active (ID50 > 50 lM). 2010 Elsevier Ltd. All rights reserved.

Article history: Received 3 February 2010 Received in revised form 17 May 2010 Available online 17 June 2010 Keywords: Frullania inouei Liverworts Diterpenoids TDDFT CD calculations Cytotoxicity Multidrug resistance Chemosystematics

1. Introduction Liverworts (Hepaticae) are known to be rich sources of terpenoids and aromatic compounds with interesting biological activities, including antifungal, anti-HIV, antioxidative, insect antifeedant, neurotrophic, cytotoxic, and multidrug resistance (MDR) reversal activities (Asakawa, 2004, 2007; Shi et al., 2008). With over 1000 described taxa, Frullania is a large, complex genus whose (sub)generic boundaries remain unresolved (Asakawa et al., 2003). Dozens of Frullania species have been chemically investigated, and were divided into ten chemo-types according to chemotaxonomy (Asakawa, 2004). This genus contains a variety of sesquiterpene lactones, diterpenoids, and bibenzyl derivatives; which can cause potent allergenic contact dermatitis. The extract also possess piscicidal activity as well as cytotoxicity to tumor cells (Asakawa et al., 2003, 2009). As part of our ongoing research on bioactive substances from Chinese liverworts (Xie and Lou, 2009), the liverwort Frullania inouei Hatt., collected in the mountain area (3000 m) of Yunnan Province was phytochemically investigated. Seven labdane diterpenoids (17) and four highly methoxylated bibenyl (811) derivatives were isolated. The similar labdanes and the same bibenzyls

as those found in the present species have been isolated from F. hamatiloba (Toyota et al., 1988), F. serrata (Asakawa, 1995), and F. brittoniae ssp. truncatifolia (Asakawa et al., 1976). In this paper, the absolute structures of four undescribed diterpenoids (14) were determined by combined application of CD measurement, TDDFT CD calculations, as well as X-ray diffraction measurement. Cytotoxicities and MDR reversal activities of the isolated compounds were evaluated in vincristine-resistant KB/VCR, adriamycin-resistant K562/A02 and in their parental cells by MTT assays.

2. Results and discussion 2.1. Structure elucidation The HRESIMS spectrum of compound 1 showed the [M+Na]+ ion peak at m/z 339.1936 (calcd. 339.1931) ascribable to the molecular formula C20H28O3, which indicated seven degrees of unsaturation. The IR absorptions at 1732 and 1661 cm1 were ascribable to a carbonyl and a conjungated carbonyl, respectively. The 1H NMR spectrum of 1 (Table 1) exhibited the presence of a monosubstituted vinyl group (CH = CH2) at dH 5.92 (H-14, dd, J = 17.4, 10.8 Hz), 5.23 (H-15a, dd, J = 17.4, 1.2 Hz) and 4.99 (H-15b, dd, J = 10.8, 1.2 Hz), and ve tertiary methyls at dH 1.57 (H3-17, s), 1.36 (H3-16, s), 1.29 (H3-20, s), 1.15 (H3-18, s) and 1.11 (H3-19, s). Addi-

* Corresponding author. Tel.: +86 531 88382012; fax: +86 531 88382019. E-mail address: louhongxiang@sdu.edu.cn (H.-X. Lou). 0031-9422/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.05.023

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16 13 14 15

D.-X. Guo et al. / Phytochemistry 71 (2010) 15731578

1 2 3

20

11 9

12 17 8 7

O
18

10

O R

O H R H O R H H

O H H COOH 7

H
6

19

1R =O 2 R = -H, -OH H 3CO H 3CO R1

3R =O 4 R = -H, -OH

5R=O 6 R = -H, -OAc H 3 CO

R2 OCH 3

H 3CO H 3 CO 11

O O OCH 3

OCH 3 8 R1 = R2 = H 9 R1 = OCH3 , R 2 = H 10 R 1 = R 2 = OCH 3

tionally, resonances for a pair of olenic hydrogens at dH 7.06 (H-1, d, J = 10.8 Hz) and 5.93 (H-2, d, J = 10.8 Hz) were observed. The 13C NMR (Table 2) and HSQC spectra conrmed the presence of a monosubstituted double bond (CH = CH2) and a disubstituted one (CH = CH), two carbonyls, ve methyls, three methylenes, two methines, as well as four quaternary carbons (including two

Table 1 1 H NMR spectroscopic data for compounds 14.a Position 1 1 7.06 d (10.8) 2 7.06 d (10.2) 3 4

a 1.43 ddd (13.2,

10.8, 6.6) b 1.94 ddd (13.2, 7.2, 3.6) a 2.44 ddd (16.2, 6.6, 3.6) b 2.62 ddd (16.2, 10.8, 7.2)

a 1.01 m
b 1.68 m

5.93 d (10.8)

5.86 d (10.2)

a 1.72 m
b 1.66 m 3.24 dd (11.4, 4.2) 1.28 br d (14.4) 2.42 br d (14.4) 2.63 t (14.4)

3 5 6a 6b 7 9 11 12a 12b 14 15a 15b 16 17 18 19 20 2.02 dd (11.4, 4.8) 1.91 m (2H) 1.84 m 1.91 m 5.92 dd (17.4, 10.8) 5.23 dd (17.4, 1.2) 4.99 dd (10.8, 1.2) 1.36 s (3H) 1.57 s (3H) 1.15 s (3H) 1.11 s (3H) 1.29 s (3H) 2.12 dd (14.4, 2.4) 2.44 dd (14.4, 2.4) 2.80 t (14.4) 1.85 m 1.87 m 1.54 dt (13.8, 12.0) 3.69 dd (12.0, 4.8) 1.51 m 1.85 dd (14.4, 3.0) 2.35 dd (14.4, 3.0) 2.74 t (14.4)

oxygenated). It is suggested that this compound was a manoyl oxide-type diterpenoid (Toyota et al., 1988) bearing an a,b-unsaturated ketone and another carbonyl. The HMBC correlations (Fig. 1) of H3-18 and H3-19 to the carbon signal at dC 203.5 indicated that the carbonyl was at C-3. Another carbonyl at C-7 was conrmed by the HMBC correlations of H-5 (dH 2.12), H-6 (dH 2.80 and 2.44) and H3-17 to the carbon (C-7) signal at dC 207.4. The cross-peaks of H3-20 with the olenic carbon (C-1) at dC 155.4, and H-1 with C-3 and C-5 (dC 52.8) assigned the location of a double bound D1,2, which was supported by the correlations of an olenic hydrogen (H-2) with C-4 (dC 44.9) and C-10 (dC 39.4), respectively, in the HMBC spectrum. The NOEs between the protons in NOESY and the single-crystal X-ray diffraction analysis (Fig. 2) determined the relative conguration of 1. To determine the absolute conguration of 1, the CD exciton chirality method was applied (Berova and Nakanishi, 2000; Harada et al., 1981). The CD of 1 (Fig. 3) exhibited a positive split between the two chromophores of the a,b-unsaturated ketone (232 nm, De +9.87, p ? p* transition) (Koreeda et al., 1973) and the

Table 2 13 C NMR spectroscopic data for compounds 14.a Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

a

1 155.4 126.5 203.5 44.9 52.8 35.8 207.4 80.5 49.4 39.4 15.5 33.9 75.0 146.3 112.0 29.8 24.7 27.0 21.1 18.0

2 157.2 126.3 204.9 44.7 51.0 27.0 80.3 78.5 48.4 39.7 15.3 35.6 73.8 147.3 110.8 28.6 20.6 27.9 21.5 19.1

3 36.9 33.6 214.9 47.5 54.8 36.1 208.3 79.9 53.6 36.7 15.6 34.1 74.7 146.4 111.6 29.3 24.1 25.7 20.9 14.3

4 36.9 27.0 78.4 39.3 55.1 35.7 209.4 80.3 54.4 37.0 15.3 34.3 74.7 146.7 111.5 29.5 24.3 27.6 15.0 15.0

1.76 m

1.65 m 1.64 m (2H) 1.72 m 1.80 m 5.91 dd (17.4, 10.8) 5.20 br d (17.4) 4.95 br d (10.8) 1.31 s (3H) 1.48 s (3H) 0.96 s (3H) 0.81 s (3H) 1.02 s (3H)

a 1.83 m
b 1.77 m 1.69 m 1.86 m 5.85 dd (17.4, 10.8) 5.14 dd (17.4, 1.2) 4.94 dd (10.8, 1.2) 1.30 s (3H) 1.35 s (3H) 1.17 s (3H) 1.10 s (3H) 1.05 s (3H)

a 1.69 m
b 1.76 m 1.76 m 1.83 m 5.91 dd (16.8, 10.2) 5.21 br d (17.4) 4.96 br d (10.8) 1.33 1.54 1.07 1.05 1.17 s s s s s (3H) (3H) (3H) (3H) (3H)

a Recorded in CDCl3 at 600 MHz. Chemical shifts are given in ppm. Figures in parentheses are coupling constants (J) in Hz.

Recorded in CDCl3 at 150 MHz. Chemical shifts are given in ppm.

D.-X. Guo et al. / Phytochemistry 71 (2010) 15731578

1575

16 11 20 12 1 713 14 15

1 3

9 8

O O

O
18 19

Fig. 1. Key HMBC (H ? C) correlations of 1.

Fig. 2. Single crystal X-ray structure of 1.

D14,15 double bond (209 nm, De 1.00, p ? p* transition) (Harada et al., 1981), indicating that the transition dipole moments of the two chromophores were oriented in a clockwise manner (Fig. 3). Thus the absolute conguration of the ve chiral centers in 1 was deduced as 5R, 8S, 9R, 10S, and 13R, which was further supported by the result of the TDDFT CD calculation (Fig. 3). Accordingly, 1 was a manoyl oxide derivative, and elucidated denitely as 1,2-dehydro-3,7-dioxo-manoyl oxide. Compound 2 was assigned the molecular formular C20H30O3 from its HREIMS ([M]+ at m/z 318.2193, calcd. 318.2195). The 1H and 13C NMR spectroscopic data of 2 (Tables 1 and 2) resembled those of 1, except for the resonance of an oxymethine group (dH
18 12 exp. boltz.

3.69, dC 80.3) in 2 instead of the ketone carbonyl (dC 207.4) in 1 at C-7. This assignment was conrmed by the HMBC correlation from H3-17 (dH 1.35) to C-7 and the 1H1H COSY correlation of H-7 with H-6 (dH 1.87 and 1.54). The NOESY correlations of H-7 to H-5 (dH 1.85) and H-9 (dH 1.51) indicated H-7 should be in an axial position (a-oriented) and therefore the hydroxy group should be equatorial (b-oriented), and this was supported by coupling constants of J7,6b = 12.0 Hz and J7,6a = 4.8 Hz (Stavri et al., 2009). The relative conguration of 2, furnished by the NOESY experiment, resembled that of 1. The CD spectrum of 2 (Fig. 3) showed a positive split between the a,b-unsaturated ketone (237 nm, De +8.20, p ? p* transition) and D14,15 double bond (210 nm, De 3.05, p ? p* transition), indicating 2 was also a manoyl oxide derivative. The TDDFT CD calculation (Fig. 3) conrmed the absolute conguration as depicted. Therefore, 2 was determined as 1,2-dehydro-7b-hydroxy-3-oxo-manoyl oxide. The HRESIMS of compound 3 gave a [M+Na]+ ion peak at m/z 341.2092 (calcd. 341.2087), corresponding to a molecular formula of C20H30O3, which suggested one less degree of unsaturation than 1. The 1H and 13C NMR spectroscopic data of 3 (Tables 1 and 2) were similar to those of 1 except for the difference that the D1,2 double bond in 1 was absent in 3. The relative conguration of 3 was deduced by the NOESY experiment. Compound 4 was assigned the molecular formula C20H32O3 for its HRESIMS ([M+Na]+ at m/z 343.2249, calcd. 343.2244). Analysis of the 1H and 13C NMR spectroscopic data of 4 (Tables 1 and 2) found that an oxymethine (dH 3.24, dC 78.4) in 4 replaced the ketone carbonyl (dC 214.9) in 3 at C-3, which was conrmed by the long-range correlations of H3-18 (dH 0.96) and H3-19 (dH 0.81) to the oxymethine carbon signal in the HMBC spectrum. The b-orientation of OH-3 was suggested from the coupling constants of H-3 (J3,2b = 11.4 Hz, J3,2a = 4.2 Hz) (Stavri et al., 2009) and the NOESY correlations of H-3 to H-5 (dH 1.28) and H3-18. As manoyl oxide derivatives, 3 and 4 were established as 3,7-dioxo-manoyl oxide and 3b-hydroxy-7-oxo-manoyl oxide, respectively. Their absolute structures were determined by comparing their negative Cotton effects at ca. 293 nm in CD (Fig. S1, Supplementary material) corresponding to the n ? p* transition of the carbonyl at C-7 (Toyota et al., 1988) with that of 1 and this was further supported by the TDDFT CD calculations (Fig. S1, Supplementary material). The known compounds were elucidated as 3-oxo-manoyl oxide (5) (Chaichantipyuth et al., 2005), 3b-acetoxy-manoyl oxide (6) (Dominguez et al., 1975), 13-epi-manoyl oxide-19-oic acid (7)
18 12

exp. boltz.

CD ()

6 0 -6 0.8 0.6

CD ()
UV (A)
200 250 300 350 400

6 0 -6 0.8 0.6 0.4 0.2 0.0 200 250 300 Wavelength (nm) 350 400

UV (A)

0.4 0.2 0.0

Wavelength (nm)

Fig. 3. Experimental CD/UV and simulated CD spectra of 1 and 2. The red lines denote the simulated Boltzmann-averaged CD spectrum. Bold lines denote the electric transition dipole of the chromophores for 1 and 2.

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(Zinkel and Clarke, 1985), 3,30 ,4,40 -tetramethoxybibenzyl (8) (Pincock and Wedge, 1994), chrysotobibenzyl (9) (Pettit et al., 1988), brittonin A (10) (Asakawa et al., 1976) and brittonin B (11) (Asakawa et al., 1976) by comparison of their NMR, optical rotation and MS data with those reported in the literatures. Interestingly, compound 7 was deduced to be a 13-epi manoyl oxide based on the downeld shift of dC16 at 32.8 which differed from the congurations at C-13 of compounds 16 (dC ca. 28.5) (da Silva et al., 2008). This conclusion was further conrmed by the NOESY correlation. 2.2. Cytotoxicity and multidrug resistance reversal activity Cytotoxicities of compounds 111 were evaluated in vincristine-resistant KB/VCR, adriamycin-resistant K562/A02 and in their parental cells by MTT assays. The bibenzyls 811 exhibited moderate cytotoxicity against KB, KB/VCR, K562 or K562/A02 cells with

ID50 values ranging from 11.3 to 49.6 lM (Table 3). Among the tested compounds, 9 showed the strongest cytotoxicity to all cancer cells. However, none of the manoyl oxide derivatives (17) were active against those cancer cells (ID50 > 50 lM). As shown in Tables 4 and 5, we also tested the MDR reversal activities of 811 towards KB/VCR and K562/A02 cells. Compounds 811 showed moderate MDR reversal activities. They improved vincristine cytotoxicity in KB/VCR cells with the reversal fold (RF) values ranging from 3.19 to 10.91 (5 lM), and increased adriamycin cytotoxicity in K562/A02 cells with the RF values ranging from 4.40 to 8.26 (5 lM). 9 showed the most potent MDR reversal activity toward both KB/VCR and K562/A02 cells. 3. Concluding remarks Liverworts often elaborate sesqui- and diterpenoids enantiomeric to those found in higher plants (Asakawa, 1982, 1995), while the manoyl oxides isolated from the title liverwort of this paper and the highly oxidized manoyl oxides isolated from the species of the same genus, F. hamatiloba (Toyota et al., 1988), have the same absolute conguration as those reported in higher plants. In addition, F. inouei is chemically specic, since it produces both labdane diterpenoids and highly methoxylated bibenzyls as the major components, which is chemotaxomically different from the Frullania species reported before (Asakawa, 2004). 4. Experimental 4.1. General experimental procedures Melting points were measured with an X-6 micro-melting point apparatus and were uncorrected. Whereas optical rotations were obtained using a GYROMAT-HP polarimeter. UV spectra were acquired with an Agilent 8453E UVVisible spectroscopy system, with CD spectra being obtained on a Chirascan spectropolarimeter. IR spectra were recorded on a Thermo Nicolet NEXUS 470 FT-IR spectrometer in KBr discs. NMR spectra were measured on a Bruker Avance DRX-600 spectrometer operating at 600 (1H) and 150 (13C) MHz with TMS as internal standard. HREIMS spectra were obtained on a Waters GCT system mass spectrometer. HRESIMS were carried out on a LTQ-Orbitrap XL. HPLC was performed on an Agilent 1100 G1310A isopump equipped with an Agilent 1100 G1322A degasser, an Agilent 1100 G1314A VWD detector (210 nm) and a Phenomenex Luna 5 lm C18(2) column (250 4.60 mm). All solvents used were of analytical grade. Silica gel (200300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao, Peoples Republic of China) and Sephadex LH-20 (25100 lm; Pharmacia Biotek, Denmark) were used for column chromatography (CC). TLC was carried out with high-performance TLC plates precoated with silica gel GF254 (Qingdao Haiyang Chemical Co. Ltd.). Prep. TLC was performed on glass plates (20 10 cm) precoated with silica gel GF254 (Qingdao Haiyang Chemical Co. Ltd.). Layer thickness of the prep. TLC was ca. 1.5 mm, and amount of sample applied to one layer was ca. 5 mg. Spots of TLC were visualized within iodine vapor or by spraying with H2SO4EtOH (1:9) followed by heating. 4.2. Plant material F. inouei Hatt. was collected in July 2006, from Jiaozixueshan of Yunnan Province, PR China, and was authenticated by Prof. RuiLiang Zhu (School of Life Science, East China Normal University, PR China). A voucher specimen (No. 20060719-1) has been deposited in the Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University, PR China.

Table 3 Cytotoxicities of compounds 811 against cancer cells. Compound ID50 (lM)a KB 8 9 10 11 30.8 1.74 11.3 1.43 42.1 2.51 24.4 1.11 KB/VCR >50 12.8 1.53 33.7 1.28 26.3 1.77 K562 >50 14.5 1.94 49.6 3.13 42.8 3.11 K562/A02 39.2 1.83 12.0 2.45 30.9 2.59 21.0 1.91

a Data are expressed as means standard deviation from three independent experiments. *P < 0.05.

Table 4 Inhibitory effect of compounds 811 on cancer cells.a Compound % of inhibition KB 8 9 10 11 9.44 29.39 3.55 2.02 KB/VCR 7.33 23.83 5.26 1.96 K562 1.09 15.44 1.54 5.44 K562/A02 1.44 11.43 4.65 5.95

a After treatment of cancer cells with 5 lM of each compound for 48 h, the inhibitory rate (%) was calculated using the MTT assay.

Table 5 Multidrug resistance (MDR) reversal activity of compounds 811 against vincristineresistant KB/VCR and adriamycin-resistant K562/A02 cell lines.a Treatment ID50b (lM) (KB/VCR) 1.200 0.133 0.376 0.077 0.110 0.016 0.319 0.038 0.295 0.014 RFc (KB/ VCR) 3.19 10.91 3.76 4.07 Treatment ID50b (mM) (K562/A02) 0.722 0.016 0.164 0.006 0.087 0.008 0.123 0.011 0.109 0.010 RFc (K562/ A02) 4.40 8.26 5.85 6.60

VCR VCR + 8 (5 lM) VCR + 9 (5 lM) VCR + 10 (5 lM) VCR + 11 (5 lM)

ADR ADR + 8 (5 lM) ADR + 9 (5 lM) ADR + 10 (5 lM) ADR + 11 (5 lM)

a KB/VCR and K562/A02 cells were seeded at the density of 6 104/ml in 96-well plates and cotreated with various concentrations of vincristine (VCR) and adriamycin (ADR), respectively, in the presence of 8, 9, 10, or 11 at the concentration of 5 lM. Cell viability was determined using the MTT assay. b Data are expressed as means standard deviation from three independent experiments. *P < 0.05. c The reversal fold (RF) values, as potency of reversal, were obtained from tting the data to RF = ID50 of cytotoxic drug alone/ID50 of cytotoxic drug in the presence of the test drugs.

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13

4.3. Extraction and isolation The air-dried powder of F. inouei (360 g) was extracted with EtOHH2O (95:5, v/v) at room temperature (3 l 3, each for 2 weeks). Evaporation of the solvent in vacuo provided a viscous, dark green residue (28 g). Subsequently, to the residue was added H2O (250 ml) with the resulting suspension partitioned successively with Et2O (250 ml 3) and n-BuOH (250 ml 3). The Et2O-soluble fraction (9 g) was separated by silica gel CC eluted with a gradient of petroleum ether (6090 C)Me2CO (100:1 to 0:1) to obtain eight fractions (AH). Fraction A (0.6 g) was then subjected to silica gel CC (petroleum etherCHCl3, 1:1) to provide ve subfractions (A1A5). Further separation of fraction A3 (30 mg) by prep. TLC (petroleum etherMe2CO, 9:1) gave 5 (5.1 mg, Rf 0.55) and 6 (1.1 mg, Rf 0.62). Fraction C (0.8 g) was subjected to silica gel CC (petroleum etherMe2CO, 100:1 to 20:1) to give six subfractions (C1C6). Fraction C2 (25 mg) was separated by HPLC (MeOHH2O, 71:29, 0.8 ml/min) to give 2 (1.6 mg, tR 18.7 min), 8 (1.2 mg, tR 12.6 min), and 11 (3.0 mg, tR 14.4 min), respectively, while fraction C3 (30 mg) afforded 7 (1.5 mg) after silica gel CC (petroleum etherMe2CO, 60:1). Fraction D (0.3 g) was applied to a Sephadex LH-20 column (CHCl3MeOH, 1:1), then fractioned by silica gel CC (petroleum etherMe2CO, 80:1) to afford a subfraction D1 (45 mg), which was puried by HPLC (MeOH H2O, 69:31, 0.6 ml/min) to give 9 (9.2 mg, tR 16.1 min). Separation of fraction E (1.1 g) following the similar procedure as fraction D yielded 10 (32.5 mg) and a subfraction E1 (50 mg). E1 was further fractioned by HPLC (MeOHH2O, 67:33, 0.8 ml/min) to afford 1 (8.0 mg, tR 12.4 min) and 3 (10.2 mg, tR 14.9 min). Fraction G (0.5 g) was puried by repeated silica gel CC (petroleum ether Me2CO, 18:1) to give a major component, which was subjected to prep. TLC (petroleum etherMe2CO, 3:1) to give 4 (2.1 mg, Rf 0.43). 4.4. 1,2-Dehydro-3,7-dioxo-manoyl oxide (1) Colorless platelets (MeOH), m.p. 209210 C; a20 29:3 (c D 0.105, MeOH); UV (MeOH) kmax (log e) nm: 227 (4.03); CD (MeOH) kmax (De) nm: 209 (1.00), 232 (+9.87), 293 (2.79), 342 (1.51); IR (KBr) mmax cm1: 2972, 2926, 1732, 1661, 1380, 1123, 1066, 826; ESIMS (positive mode) m/z (rel. int.): 651.0 [2 M+NH4]+ (14), 339.6 [M+Na]+ (18), 334.8 [M+NH4]+ (100), 317.7 [M+H]+ (76), 299.7 [M+HH2O]+ (8); HRESIMS (positive mode) m/z 339.1936 [M+Na]+ (calcd. for C20H28O3Na, 339.1931); for 1H and 13C NMR spectroscopic data, see Tables 1 and 2. 4.5. 1,2-Dehydro-7b-hydroxy-3-oxo-manoyl oxide (2) White amorphous powder; a20 45:6 (c 0.112, MeOH); UV D (MeOH) kmax (log e) nm: 228 (3.80); CD (MeOH) kmax (De) nm: 210 (3.05), 237 (+8.20), 344 (1.56); IR (KBr) mmax cm1: 3473, 2946, 1669, 1385, 1116, 1080, 994, 826; ESIMS (positive mode) m/z (rel. int.): 660.0 [2 M+Na]+ (10), 341.6 [M+Na]+ (51), 336.9 [M+NH4]+ (100), 319.6 [M+H]+ (47), 301.8 [M+HH2O]+ (73), 283.6 [M+H2H2O]+ (15); HREIMS m/z 318.2193 [M]+ (calcd. for C20H30O3, 318.2195); for 1H and 13C NMR spectroscopic data, see Tables 1 and 2. 4.6. 3,7-Dioxo-manoyl oxide (3) White amorphous powder; a20 27:3 (c 0.097, MeOH); UV D (MeOH) kmax (log e) nm: 202 (3.23); CD (MeOH) kmax (De) nm: 293 (2.90); IR (KBr) mmax cm1: 2993, 2944, 1731, 1697, 1457, 1111, 1071, 1042, 995, 919; ESIMS (positive mode) m/z (rel. int.): 660.0 [2 M+Na]+ (9), 654.9 [2 M+NH4]+ (25), 341.6 [M+Na]+ (28), 336.8 [M+NH4]+ (98), 319.5 [M+H]+ (100), 301.7 [M+HH2O]+ (8); HRESIMS (positive mode) m/z 341.2092 [M+Na]+ (calcd. for

C20H30O3Na, 341.2087); for 1H and see Tables 1 and 2.

C NMR spectroscopic data,

4.7. 3b-Hydroxy-7-oxo-manoyl oxide (4) White amorphous powder; a20 49:8 (c 0.110, MeOH); UV D (MeOH) kmax (log e) nm: 202 (3.36); CD (MeOH) kmax (De) nm: 292 (3.01); IR (KBr) mmax cm1: 3436, 2938, 2852, 1708, 1468, 1375, 1114, 1049, 922; ESIMS (positive mode) m/z (rel. int.): 659.0 [2 M+NH4]+ (17), 343.6 [M+Na]+ (8), 338.7 [M+NH4]+ (76), 321.5 [M+H]+ (100), 303.6 [M+HH2O]+ (7); HRESIMS (positive mode) m/z 343.2249 [M+Na]+ (calcd. for C20H32O3Na, 343.2244); for 1H and 13C NMR spectroscopic data, see Tables 1 and 2. 4.8. X-ray crystallographic analysis of 1 C20H28O3, M = 316.42, orthorhombic system, space group P212121, a = 6.3107(6), b = 12.2516(12), c = 22.849(2) , V = 1766.6(3) 3, Z = 4, Dcalcd = 1.190 Mg/m3, l(MoKa) = 0.078 mm1, F(000) = 688, and T = 293(2) K. A crystal of dimensions 0.34 0.25 0.19 mm3 was selected for measurements on a Bruker APEX2 CCD area-detector diffractometer with a graphite monochromator (/x scans), MoKa radiation (k = 0.71073 ). APEX2 Software Suite (Bruker, 2005) was used for cell renement and data reduction. The structure was rened with full-matrix leastsquares calculations on F2 using SHELXL-97 (Sheldrick, 1997). A total of 20234 reections, collected in the h range 1.78 to 27.58, yielded 2357 unique reections (Rint = 0.0326). All non-hydrogen atoms were given anisotropic thermal parameters. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. The nal stage converged to R1 = 0.0400 (wR2 = 0.0855) for 2357 observed reections [with I > 2r(I)] and 214 variable parameters, R1 = 0.0508 (wR2 = 0.0915) for all unique reections and goodness-of-t = 1.019. Crystallographic data for 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition No. CCDC-757310). Copies of the data can be obtained free of charge via 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 336033; e-mail: deposit@ccdc.cam.ac.uk). 4.9. Theory and calculation details The calculations were performed by the Materials Studio software (version 4.0; Accerlys Software Inc.) and the Gaussian 03 program package (Frisch et al., 2004). MD simulations based on the COMPASS force eld (Sun, 1998) at respective 300, 400, and 500 K were employed to search the possible conformations. The starting conformer of 1 comes from the corresponding X-ray structure, which is also regarded as the template for compounds 24. All ground-state geometries were optimized at the B3LYP/6-31+G* level at 298 K, and harmonic frequency analysis was computed to conrm the minima and thence calculation of room-temperature free energy. TDDFT (Casida, 1995; Gross et al., 1996; Gross and Kohn, 1990; Runge and Gross, 1984) at B3LYP/TZVP level (Schafer et al., 1994; Schwabe and Grimme, 2007; Zhao and Truhlar, 2008) in the gas phase was employed to calculate the excitation energy and rotatory strength R for the rst 40 states. The ECD spectra were then simulated by overlapping Gaussian functions for each transition according to:

DeE

2 2:296 10
39

2 1 X p DE0i R0i e2EDE0i =w pw i

where w is the bandwidth at 1/e peak height and expressed in energy units. DE0i and R0i are the excitation energies and rotatory

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strengths for the transition from 0 to i, respectively (Stephens and Harada, 2010). 4.10. Biological evaluation 4.10.1. Cell culture KB, KB/VCR, K562, and K562/A02 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cells were maintained in 5% CO2 at 37 C until reaching approximately 5070% conuence, and then treated with different amounts of chemicals. DMSO was used as a control vehicle. 4.10.2. Cytotoxicity and multidrug resistance reversal assay MTT assay was used to measure the cytotoxicity of tested compounds in 96-well plates (Mosmann, 1983). The cells were treated with vehicle and tested compounds alone for 48 h. After addition of MTT (10 ll/well, 5 mg/ml in phosphated-buffered saline), the plates were incubated for 4 h under 5% CO2 at 37 C. Then, the absorbance was determined at 570 nm. The MDR reversal ability of tested compounds to potentiate vincristine and adriamycin cytotoxicity was evaluated in KB/VCR cells and in K562/A02 cells, respectively, by the MTT assay described above. The cells were treated with vehicle and tested compound combined with desired concentrations of chemicals. ID50 values for all tested compounds were calculated from plotted results using untreated cells as 100%. Acknowledgements Financial supports from the National Natural Science Foundation of China (Nos. 30730109 and 30925038) and Shandong Provincial Fund (Nos. Z2006C03 and JQ200806) are gratefully acknowledged. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2010.05.023. References
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