Phytochemistry 71 (2010) 1573–1578

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Phytochemistry

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Labdane diterpenoids and highly methoxylated bibenzyls fromthe 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 Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University, No. 44 West Wenhua Road, Jinan 250012, People’s Republic of Chinab Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, No. 44 West Wenhua Road, Jinan 250012, People’s Republic of Chinac State Key Laboratory of Crystal Materials, Shandong University, No. 27 Shanda Nanlu, Jinan 250100, People’s Republic of China

a r t i c l e i n f o

Article history:Received 3 February 2010Received in revised form 17 May 2010Available online 17 June 2010

Keywords:Frullania inoueiLiverwortsDiterpenoidsTDDFT CD calculationsCytotoxicityMultidrug resistanceChemosystematics

0031-9422/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.phytochem.2010.05.023

* Corresponding author. Tel.: +86 531 88382012; faE-mail address: louhongxiang@sdu.edu.cn (H.-X. L

a b s t r a c t

Four undescribed labdane diterpenoids, 1,2-dehydro-3,7-dioxo-manoyl oxide (1), 1,2-dehydro-7b-hydroxy-3-oxo-manoyl oxide (2), 3,7-dioxo-manoyl oxide (3), and 3b-hydroxy-7-oxo-manoyl oxide (4)together with three known diterpenoids (5–7) and four highly methoxylated bibenzyls (8–11) were iso-lated from the liverwort Frullania inouei. The absolute structures of 1–4 were established by combinedanalysis of NMR data, CD data coupled with TDDFT CD calculations, and single-crystal X-ray diffractionmeasurement. Cytotoxicity tests to human tumor KB, KB/VCR, K562 or K562/A02 cells showed bibenzyls8–11 inhibited cell proliferation with ID50 values ranging from 11.3 to 49.6 lM and overcame the mul-tidrug resistance (MDR) with the reversal fold (RF) values ranging from 3.19 to 10.91 (5 lM) for vincris-tine-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.

1. Introduction

Liverworts (Hepaticae) are known to be rich sources ofterpenoids and aromatic compounds with interesting biologicalactivities, including antifungal, anti-HIV, antioxidative, insect anti-feedant, neurotrophic, cytotoxic, and multidrug resistance (MDR)reversal activities (Asakawa, 2004, 2007; Shi et al., 2008).

With over 1000 described taxa, Frullania is a large, complexgenus whose (sub)generic boundaries remain unresolved (Asaka-wa et al., 2003). Dozens of Frullania species have been chemicallyinvestigated, and were divided into ten chemo-types according tochemotaxonomy (Asakawa, 2004). This genus contains a varietyof sesquiterpene lactones, diterpenoids, and bibenzyl derivatives;which can cause potent allergenic contact dermatitis. The extractalso possess piscicidal activity as well as cytotoxicity to tumor cells(Asakawa et al., 2003, 2009).

As part of our ongoing research on bioactive substances fromChinese liverworts (Xie and Lou, 2009), the liverwort Frullania in-ouei Hatt., collected in the mountain area (3000 m) of YunnanProvince was phytochemically investigated. Seven labdane diter-penoids (1–7) and four highly methoxylated bibenyl (8–11) deriv-atives were isolated. The similar labdanes and the same bibenzyls

ll rights reserved.

x: +86 531 88382019.ou).

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 diter-penoids (1–4) were determined by combined application of CDmeasurement, TDDFT CD calculations, as well as X-ray diffractionmeasurement. Cytotoxicities and MDR reversal activities of the iso-lated compounds were evaluated in vincristine-resistant KB/VCR,adriamycin-resistant K562/A02 and in their parental cells by MTTassays.

2. Results and discussion

2.1. Structure elucidation

The HRESIMS spectrum of compound 1 showed the [M+Na]+ ionpeak at m/z 339.1936 (calcd. 339.1931) ascribable to the molecularformula C20H28O3, which indicated seven degrees of unsaturation.The IR absorptions at 1732 and 1661 cm�1 were ascribable to a car-bonyl and a conjungated carbonyl, respectively. The 1H NMR spec-trum of 1 (Table 1) exhibited the presence of a monosubstitutedvinyl 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 five 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-

OH

HO R

12

34 5

6 7

8

9

10

1112

13 14

15

16

17

18 19

20

1 R = O2 R = α-H, β-OH

OH

HR O

3 R = O4 R = α-H, β-OH

OH

HR

5 R = O6 R = α-H, β-OAc

OH

COOHH

7

H3CO

H3CO

R1OCH3

OCH3

R2H3CO

H3CO

H3COOCH3

O

O

8 R1 = R2 = H9 R1 = OCH3, R2 = H10 R1 = R2 = OCH3

11

1574 D.-X. Guo et al. / Phytochemistry 71 (2010) 1573–1578

tionally, resonances for a pair of olefinic 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 13CNMR (Table 2) and HSQC spectra confirmed the presence of amonosubstituted double bond (–CH = CH2) and a disubstitutedone (–CH = CH–), two carbonyls, five methyls, three methylenes,two methines, as well as four quaternary carbons (including two

Table 11H NMR spectroscopic data for compounds 1–4.a

Position 1 2 3 4

1 7.06 d (10.8) 7.06 d (10.2) a 1.43 ddd (13.2,10.8, 6.6)

a 1.01 m

b 1.94 ddd (13.2,7.2, 3.6)

b 1.68 m

2 5.93 d (10.8) 5.86 d (10.2) a 2.44 ddd (16.2,6.6, 3.6)

a 1.72 m

b 2.62 ddd (16.2,10.8, 7.2)

b 1.66 m

3 3.24 dd(11.4, 4.2)

5 2.12 dd(14.4, 2.4)

1.85 m 1.85 dd (14.4, 3.0) 1.28 br d(14.4)

6a 2.44 dd(14.4, 2.4)

1.87 m 2.35 dd (14.4, 3.0) 2.42 br d(14.4)

6b 2.80 t (14.4) 1.54 dt (13.8,12.0)

2.74 t (14.4) 2.63 t (14.4)

7 3.69 dd(12.0, 4.8)

9 2.02 dd(11.4, 4.8)

1.51 m 1.76 m 1.65 m

11 1.91 m (2H) a 1.83 m a 1.69 m 1.64 m (2H)b 1.77 m b 1.76 m

12a 1.84 m 1.69 m 1.76 m 1.72 m12b 1.91 m 1.86 m 1.83 m 1.80 m14 5.92 dd

(17.4, 10.8)5.85 dd(17.4, 10.8)

5.91 dd (16.8,10.2)

5.91 dd(17.4, 10.8)

15a 5.23 dd(17.4, 1.2)

5.14 dd(17.4, 1.2)

5.21 br d (17.4) 5.20 br d(17.4)

15b 4.99 dd(10.8, 1.2)

4.94 dd(10.8, 1.2)

4.96 br d (10.8) 4.95 br d(10.8)

16 1.36 s (3H) 1.30 s (3H) 1.33 s (3H) 1.31 s (3H)17 1.57 s (3H) 1.35 s (3H) 1.54 s (3H) 1.48 s (3H)18 1.15 s (3H) 1.17 s (3H) 1.07 s (3H) 0.96 s (3H)19 1.11 s (3H) 1.10 s (3H) 1.05 s (3H) 0.81 s (3H)20 1.29 s (3H) 1.05 s (3H) 1.17 s (3H) 1.02 s (3H)

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

oxygenated). It is suggested that this compound was a manoyloxide-type diterpenoid (Toyota et al., 1988) bearing an a,b-unsat-urated ketone and another carbonyl. The HMBC correlations(Fig. 1) of H3-18 and H3-19 to the carbon signal at dC 203.5 indi-cated that the carbonyl was at C-3. Another carbonyl at C-7 wasconfirmed 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 olefinic carbon (C-1) at dC

155.4, and H-1 with C-3 and C-5 (dC 52.8) assigned the locationof a double bound D1,2, which was supported by the correlationsof an olefinic 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 configu-ration of 1. To determine the absolute configuration of 1, the CDexciton chirality method was applied (Berova and Nakanishi,2000; Harada et al., 1981). The CD of 1 (Fig. 3) exhibited a positivesplit 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 213C NMR spectroscopic data for compounds 1–4.a

Position 1 2 3 4

1 155.4 157.2 36.9 36.92 126.5 126.3 33.6 27.03 203.5 204.9 214.9 78.44 44.9 44.7 47.5 39.35 52.8 51.0 54.8 55.16 35.8 27.0 36.1 35.77 207.4 80.3 208.3 209.48 80.5 78.5 79.9 80.39 49.4 48.4 53.6 54.410 39.4 39.7 36.7 37.011 15.5 15.3 15.6 15.312 33.9 35.6 34.1 34.313 75.0 73.8 74.7 74.714 146.3 147.3 146.4 146.715 112.0 110.8 111.6 111.516 29.8 28.6 29.3 29.517 24.7 20.6 24.1 24.318 27.0 27.9 25.7 27.619 21.1 21.5 20.9 15.020 18.0 19.1 14.3 15.0

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

O

O O3

1

6

89

1112

13

14

15

16

17

1918

20

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

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

D.-X. Guo et al. / Phytochemistry 71 (2010) 1573–1578 1575

D14,15 double bond (209 nm, De �1.00, p ? p* transition) (Haradaet al., 1981), indicating that the transition dipole moments of thetwo chromophores were oriented in a clockwise manner (Fig. 3).Thus the absolute configuration of the five chiral centers in 1was deduced as 5R, 8S, 9R, 10S, and 13R, which was further sup-ported by the result of the TDDFT CD calculation (Fig. 3). Accord-ingly, 1 was a manoyl oxide derivative, and elucidated definitelyas 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 1Hand 13C NMR spectroscopic data of 2 (Tables 1 and 2) resembledthose of 1, except for the resonance of an oxymethine group (dH

-6

0

6

12

18

CD

(Δε )

exp.boltz.

200 250 300 350 400

0.0

0.2

0.4

0.6

0.8

UV

(A)

Wavelength (nm)

1

Fig. 3. Experimental CD/UV and simulated CD spectra of 1 and 2. The red lines denottransition dipole of the chromophores for 1 and 2.

3.69, dC 80.3) in 2 instead of the ketone carbonyl (dC 207.4) in 1at C-7. This assignment was confirmed by the HMBC correlationfrom H3-17 (dH 1.35) to C-7 and the 1H–1H COSY correlation ofH-7 with H-6 (dH 1.87 and 1.54). The NOESY correlations of H-7to H-5 (dH 1.85) and H-9 (dH 1.51) indicated H-7 should be in anaxial position (a-oriented) and therefore the hydroxy group shouldbe equatorial (b-oriented), and this was supported by couplingconstants of J7,6b = 12.0 Hz and J7,6a = 4.8 Hz (Stavri et al., 2009).The relative configuration of 2, furnished by the NOESY experi-ment, resembled that of 1. The CD spectrum of 2 (Fig. 3) showeda 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 oxidederivative. The TDDFT CD calculation (Fig. 3) confirmed the abso-lute configuration as depicted. Therefore, 2 was determined as1,2-dehydro-7b-hydroxy-3-oxo-manoyl oxide.

The HRESIMS of compound 3 gave a [M+Na]+ ion peak at m/z341.2092 (calcd. 341.2087), corresponding to a molecular formulaof C20H30O3, which suggested one less degree of unsaturation than1. 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 configuration of 3was deduced by the NOESY experiment.

Compound 4 was assigned the molecular formula C20H32O3 forits HRESIMS ([M+Na]+ at m/z 343.2249, calcd. 343.2244). Analysisof 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 ke-tone carbonyl (dC 214.9) in 3 at C-3, which was confirmed by thelong-range correlations of H3-18 (dH 0.96) and H3-19 (dH 0.81) tothe oxymethine carbon signal in the HMBC spectrum. The b-orien-tation 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 NOESYcorrelations of H-3 to H-5 (dH 1.28) and H3-18.

As manoyl oxide derivatives, 3 and 4 were established as 3,7-di-oxo-manoyl oxide and 3b-hydroxy-7-oxo-manoyl oxide, respec-tively. Their absolute structures were determined by comparingtheir negative Cotton effects at ca. 293 nm in CD (Fig. S1, Supple-mentary material) corresponding to the n ? p* transition of thecarbonyl at C-7 (Toyota et al., 1988) with that of 1 and this was fur-ther supported by the TDDFT CD calculations (Fig. S1, Supplemen-tary 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)

exp.boltz.

CD

(Δε )

UV

(A)

-6

0

6

12

18

200 250 300 350 400

0.0

0.2

0.4

0.6

0.8

Wavelength (nm)

2

e the simulated Boltzmann-averaged CD spectrum. Bold lines denote the electric

1576 D.-X. Guo et al. / Phytochemistry 71 (2010) 1573–1578

(Zinkel and Clarke, 1985), 3,30,4,40-tetramethoxybibenzyl (8) (Pin-cock and Wedge, 1994), chrysotobibenzyl (9) (Pettit et al., 1988),brittonin A (10) (Asakawa et al., 1976) and brittonin B (11) (Asak-awa et al., 1976) by comparison of their NMR, optical rotation andMS data with those reported in the literatures.

Interestingly, compound 7 was deduced to be a 13-epi manoyloxide based on the downfield shift of dC

�16 at 32.8 which differedfrom the configurations at C-13 of compounds 1–6 (dC ca. 28.5) (daSilva et al., 2008). This conclusion was further confirmed by theNOESY correlation.

2.2. Cytotoxicity and multidrug resistance reversal activity

Cytotoxicities of compounds 1–11 were evaluated in vincris-tine-resistant KB/VCR, adriamycin-resistant K562/A02 and in theirparental cells by MTT assays. The bibenzyls 8–11 exhibited moder-ate cytotoxicity against KB, KB/VCR, K562 or K562/A02 cells with

Table 3Cytotoxicities of compounds 8–11 against cancer cells.

Compound ID50 (lM)a

KB KB/VCR K562 K562/A02

8 30.8 ± 1.74 >50 >50 39.2 ± 1.839 11.3 ± 1.43 12.8 ± 1.53 14.5 ± 1.94 12.0 ± 2.4510 42.1 ± 2.51 33.7 ± 1.28 49.6 ± 3.13 30.9 ± 2.5911 24.4 ± 1.11 26.3 ± 1.77 42.8 ± 3.11 21.0 ± 1.91

a Data are expressed as means ± standard deviation from three independentexperiments. *P < 0.05.

Table 4Inhibitory effect of compounds 8–11 on cancer cells.a

Compound % of inhibition

KB KB/VCR K562 K562/A02

8 9.44 7.33 1.09 1.449 29.39 23.83 15.44 11.4310 3.55 5.26 1.54 4.6511 2.02 1.96 5.44 5.95

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

Table 5Multidrug resistance (MDR) reversal activity of compounds 8–11 against vincristine-resistant KB/VCR and adriamycin-resistant K562/A02 cell lines.a

Treatment ID50b (lM)

(KB/VCR)RFc

(KB/VCR)

Treatment ID50b (mM)

(K562/A02)RFc

(K562/A02)

VCR 1.200 ± 0.133 ADR 0.722 ± 0.016VCR + 8

(5 lM)0.376 ± 0.077 3.19 ADR + 8

(5 lM)0.164 ± 0.006 4.40

VCR + 9(5 lM)

0.110 ± 0.016 10.91 ADR + 9(5 lM)

0.087 ± 0.008 8.26

VCR + 10(5 lM)

0.319 ± 0.038 3.76 ADR + 10(5 lM)

0.123 ± 0.011 5.85

VCR + 11(5 lM)

0.295 ± 0.014 4.07 ADR + 11(5 lM)

0.109 ± 0.010 6.60

a KB/VCR and K562/A02 cells were seeded at the density of 6 � 104/ml in 96-wellplates and cotreated with various concentrations of vincristine (VCR) and adria-mycin (ADR), respectively, in the presence of 8, 9, 10, or 11 at the concentration of5 lM. Cell viability was determined using the MTT assay.

b Data are expressed as means ± standard deviation from three independentexperiments. *P < 0.05.

c The reversal fold (RF) values, as potency of reversal, were obtained from fittingthe data to RF = ID50 of cytotoxic drug alone/ID50 of cytotoxic drug in the presenceof the test drugs.

ID50 values ranging from 11.3 to 49.6 lM (Table 3). Among thetested compounds, 9 showed the strongest cytotoxicity to all can-cer cells. However, none of the manoyl oxide derivatives (1–7)were active against those cancer cells (ID50 > 50 lM).

As shown in Tables 4 and 5, we also tested the MDR reversalactivities of 8–11 towards KB/VCR and K562/A02 cells. Compounds8–11 showed moderate MDR reversal activities. They improvedvincristine cytotoxicity in KB/VCR cells with the reversal fold (RF)values ranging from 3.19 to 10.91 (5 lM), and increased adriamy-cin cytotoxicity in K562/A02 cells with the RF values ranging from4.40 to 8.26 (5 lM). 9 showed the most potent MDR reversal activ-ity toward both KB/VCR and K562/A02 cells.

3. Concluding remarks

Liverworts often elaborate sesqui- and diterpenoids enantio-meric to those found in higher plants (Asakawa, 1982, 1995), whilethe manoyl oxides isolated from the title liverwort of this paperand the highly oxidized manoyl oxides isolated from the speciesof the same genus, F. hamatiloba (Toyota et al., 1988), have thesame absolute configuration as those reported in higher plants.In addition, F. inouei is chemically specific, since it produces bothlabdane diterpenoids and highly methoxylated bibenzyls as themajor components, which is chemotaxomically different from theFrullania species reported before (Asakawa, 2004).

4. Experimental

4.1. General experimental procedures

Melting points were measured with an X-6 micro-melting pointapparatus and were uncorrected. Whereas optical rotations wereobtained using a GYROMAT-HP polarimeter. UV spectra were ac-quired with an Agilent 8453E UV–Visible spectroscopy system,with CD spectra being obtained on a Chirascan spectropolarimeter.IR spectra were recorded on a Thermo Nicolet NEXUS 470 FT-IRspectrometer in KBr discs. NMR spectra were measured on a Bru-ker Avance DRX-600 spectrometer operating at 600 (1H) and 150(13C) MHz with TMS as internal standard. HREIMS spectra were ob-tained on a Waters GCT system mass spectrometer. HRESIMS werecarried out on a LTQ-Orbitrap XL. HPLC was performed on an Agi-lent 1100 G1310A isopump equipped with an Agilent 1100G1322A 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. Silicagel (200–300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao,People’s Republic of China) and Sephadex LH-20 (25–100 lm;Pharmacia Biotek, Denmark) were used for column chromatogra-phy (CC). TLC was carried out with high-performance TLC platesprecoated with silica gel GF254 (Qingdao Haiyang Chemical Co.Ltd.). Prep. TLC was performed on glass plates (20 � 10 cm) pre-coated with silica gel GF254 (Qingdao Haiyang Chemical Co. Ltd.).Layer thickness of the prep. TLC was ca. 1.5 mm, and amount ofsample applied to one layer was ca. 5 mg. Spots of TLC were visu-alized within iodine vapor or by spraying with H2SO4–EtOH (1:9)followed by heating.

4.2. Plant material

F. inouei Hatt. was collected in July 2006, from Jiaozixueshan ofYunnan Province, PR China, and was authenticated by Prof. Rui-Liang Zhu (School of Life Science, East China Normal University,PR China). A voucher specimen (No. 20060719-1) has beendeposited in the Department of Natural Products Chemistry, Schoolof Pharmaceutical Sciences, Shandong University, PR China.

D.-X. Guo et al. / Phytochemistry 71 (2010) 1573–1578 1577

4.3. Extraction and isolation

The air-dried powder of F. inouei (360 g) was extracted withEtOH–H2O (95:5, v/v) at room temperature (3 l � 3, each for2 weeks). Evaporation of the solvent in vacuo provided a viscous,dark green residue (28 g). Subsequently, to the residue was addedH2O (250 ml) with the resulting suspension partitioned succes-sively with Et2O (250 ml � 3) and n-BuOH (250 ml � 3). TheEt2O-soluble fraction (9 g) was separated by silica gel CC elutedwith a gradient of petroleum ether (60–90 �C)–Me2CO (100:1 to0:1) to obtain eight fractions (A–H). Fraction A (0.6 g) was thensubjected to silica gel CC (petroleum ether–CHCl3, 1:1) to providefive subfractions (A1–A5). Further separation of fraction A3(30 mg) by prep. TLC (petroleum ether–Me2CO, 9:1) gave 5(5.1 mg, Rf 0.55) and 6 (1.1 mg, Rf 0.62). Fraction C (0.8 g) was sub-jected to silica gel CC (petroleum ether–Me2CO, 100:1 to 20:1) togive six subfractions (C1–C6). Fraction C2 (25 mg) was separatedby HPLC (MeOH–H2O, 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 sil-ica gel CC (petroleum ether–Me2CO, 60:1). Fraction D (0.3 g) wasapplied to a Sephadex LH-20 column (CHCl3–MeOH, 1:1), thenfractioned by silica gel CC (petroleum ether–Me2CO, 80:1) to afforda subfraction D1 (45 mg), which was purified by HPLC (MeOH–H2O, 69:31, 0.6 ml/min) to give 9 (9.2 mg, tR 16.1 min). Separationof fraction E (1.1 g) following the similar procedure as fraction Dyielded 10 (32.5 mg) and a subfraction E1 (50 mg). E1 was furtherfractioned by HPLC (MeOH–H2O, 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 purified by repeated silica gel CC (petroleum ether–Me2CO, 18:1) to give a major component, which was subjected toprep. TLC (petroleum ether–Me2CO, 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. 209–210 �C; ½a�20D þ 29:3 (c

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 cm�1: 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+H�H2O]+ (8); HRESIMS (positive mode) m/z 339.1936[M+Na]+ (calcd. for C20H28O3Na, 339.1931); for 1H and 13C NMRspectroscopic data, see Tables 1 and 2.

4.5. 1,2-Dehydro-7b-hydroxy-3-oxo-manoyl oxide (2)

White amorphous powder; ½a�20D þ 45:6 (c 0.112, MeOH); UV

(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 cm�1: 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+H�H2O]+ (73),283.6 [M+H�2H2O]+ (15); HREIMS m/z 318.2193 [M]+ (calcd. forC20H30O3, 318.2195); for 1H and 13C NMR spectroscopic data, seeTables 1 and 2.

4.6. 3,7-Dioxo-manoyl oxide (3)

White amorphous powder; ½a�20D � 27:3 (c 0.097, MeOH); UV

(MeOH) kmax (log e) nm: 202 (3.23); CD (MeOH) kmax (De) nm:293 (�2.90); IR (KBr) mmax cm�1: 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+H�H2O]+

(8); HRESIMS (positive mode) m/z 341.2092 [M+Na]+ (calcd. for

C20H30O3Na, 341.2087); for 1H and 13C NMR spectroscopic data,see Tables 1 and 2.

4.7. 3b-Hydroxy-7-oxo-manoyl oxide (4)

White amorphous powder; ½a�20D � 49:8 (c 0.110, MeOH); UV

(MeOH) kmax (log e) nm: 202 (3.36); CD (MeOH) kmax (De) nm:292 (�3.01); IR (KBr) mmax cm�1: 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+H�H2O]+ (7); HRESIMS (positivemode) 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 groupP212121, a = 6.3107(6), b = 12.2516(12), c = 22.849(2) Å, V =1766.6(3) Å3, Z = 4, Dcalcd = 1.190 Mg/m3, l(MoKa) = 0.078 mm�1,F(000) = 688, and T = 293(2) K. A crystal of dimensions0.34 � 0.25 � 0.19 mm3 was selected for measurements on a Bru-ker APEX2 CCD area-detector diffractometer with a graphite mono-chromator (/–x scans), MoKa radiation (k = 0.71073 Å). APEX2Software Suite (Bruker, 2005) was used for cell refinement anddata reduction. The structure was refined with full-matrix least-squares calculations on F2 using SHELXL-97 (Sheldrick, 1997). A to-tal of 20234 reflections, collected in the h range 1.78 to 27.58�,yielded 2357 unique reflections (Rint = 0.0326). All non-hydrogenatoms were given anisotropic thermal parameters. The hydrogenatom positions were geometrically idealized and allowed to rideon their parent atoms. The final stage converged to R1 = 0.0400(wR2 = 0.0855) for 2357 observed reflections [with I > 2r(I)] and214 variable parameters, R1 = 0.0508 (wR2 = 0.0915) for all uniquereflections and goodness-of-fit = 1.019.

Crystallographic data for 1 have been deposited in the Cam-bridge Crystallographic Data Centre (deposition No. CCDC-757310).Copies of the data can be obtained free of charge via www.ccdc.ca-m.ac.uk/conts/retrieving.html or from the Cambridge Crystallo-graphic 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 soft-ware (version 4.0; Accerlys Software Inc.) and the Gaussian 03 pro-gram package (Frisch et al., 2004). MD simulations based on theCOMPASS force field (Sun, 1998) at respective 300, 400, and500 K were employed to search the possible conformations. Thestarting conformer of 1 comes from the corresponding X-ray struc-ture, which is also regarded as the template for compounds 2–4. Allground-state geometries were optimized at the B3LYP/6-31+G*

level at 298 K, and harmonic frequency analysis was computed toconfirm the minima and thence calculation of room-temperaturefree energy. TDDFT (Casida, 1995; Gross et al., 1996; Gross andKohn, 1990; Runge and Gross, 1984) at B3LYP/TZVP level (Schaferet al., 1994; Schwabe and Grimme, 2007; Zhao and Truhlar, 2008)in the gas phase was employed to calculate the excitation energyand rotatory strength R for the first 40 states. The ECD spectra werethen simulated by overlapping Gaussian functions for each transi-tion according to:

DeðEÞ ¼ 22:296� 10�39 �

1ffiffiffiffipp

w

X

i

DE0iR0ie�½2ðE�DE0iÞ=w�2

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

1578 D.-X. Guo et al. / Phytochemistry 71 (2010) 1573–1578

strengths for the transition from 0 to i, respectively (Stephens andHarada, 2010).

4.10. Biological evaluation

4.10.1. Cell cultureKB, KB/VCR, K562, and K562/A02 cells were cultured in RPMI

1640 medium supplemented with 10% fetal bovine serum. Thecells were maintained in 5% CO2 at 37 �C until reaching approxi-mately 50–70% confluence, and then treated with differentamounts of chemicals. DMSO was used as a control vehicle.

4.10.2. Cytotoxicity and multidrug resistance reversal assayMTT assay was used to measure the cytotoxicity of tested com-

pounds in 96-well plates (Mosmann, 1983). The cells were treatedwith vehicle and tested compounds alone for 48 h. After addition ofMTT (10 ll/well, 5 mg/ml in phosphated-buffered saline), theplates were incubated for 4 h under 5% CO2 at 37 �C. Then, theabsorbance was determined at 570 nm.

The MDR reversal ability of tested compounds to potentiate vin-cristine and adriamycin cytotoxicity was evaluated in KB/VCR cellsand in K562/A02 cells, respectively, by the MTT assay describedabove. The cells were treated with vehicle and tested compoundcombined with desired concentrations of chemicals. ID50 valuesfor all tested compounds were calculated from plotted resultsusing untreated cells as 100%.

Acknowledgements

Financial supports from the National Natural Science Founda-tion of China (Nos. 30730109 and 30925038) and ShandongProvincial Fund (Nos. Z2006C03 and JQ200806) are gratefullyacknowledged.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.phytochem.2010.05.023.

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