Xanthones from Hypericum chinense and their cytotoxicity evaluation

Phytochemistry 70 (2009) 1456–1461

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Xanthones from Hypericum chinense and their cytotoxicity evaluation Naonobu Tanaka a,1, Yoshiki Kashiwada a,*, Sang-Yong Kim a, Michiko Sekiya b, Yasumasa Ikeshiro b, Yoshihisa Takaishi a a b

Graduate School of Pharmaceutical Sciences, University of Tokushima, Shomachi 1-78, Tokushima 770-8505, Japan Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Higashijima 265-1, Niigata 956-8683, Japan

a r t i c l e

i n f o

Article history: Received 6 March 2009 Received in revised form 9 July 2009 Available online 18 September 2009 Keywords: Hypericum chinense Clusiaceae Xanthonolignoid Phenylxanthone Xanthone Cytotoxicity

a b s t r a c t A xanthonolignoid, 2-O-demethylkielcorin, and a phenylxanthone, chinexanthone A, were isolated from stems of Hypericum chinense together with four known xanthonolignoids and seven known xanthones. Their structures were established by spectroscopic analysis, as their optical properties and absolute stereochemistry determined. The cytotoxicities of the isolated xanthone derivatives as well as additional 32 xanthones against a panel of human cancer cell lines were also evaluated. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction The family Clusiaceae is a rich source of xanthones, which show various bioactivities (Sultanbawa, 1980; Bennett and Lee, 1989; Pinto et al., 2005). Natural xanthonolignoids are a class of compounds with a phenylpropane skeleton linked to an ortho-dihydroxyxanthone by a dioxane ring, formed by radical oxidative coupling. They are considered to be very promising compounds due to a variety of interesting biological activities associated with xanthones and the benzodioxane moiety (Pinto and Sousa, 2003). The biological activities of xanthonolignoids such as protein kinase C inhibition (Saraiva et al., 2003), inhibition of human lymphocytes proliferation (Sousa et al., 2002), hepatoprotective activity (Fernandes et al., 1995), and cytotoxicity (Pinto and Sousa, 2003; Sousa et al., 2002; Abou-Shoer et al., 1989) have been reported. Hypericum chinense L. (Clusiaceae) is an undershrub, which is used as a folk medicine for treatment of female disorders (Tanaka et al., 2005). In the course of our search for the constituents of Hypericum plants (Matsuhisa et al., 2002; Tanaka et al., 2004, 2005, 2008a,b, 2009; W. Hashida et al., 2007, 2008; C. Hashida et al., 2008; Tanaka and Takaishi, 2006, 2007), we reported several prenylated and simple (hydroxy and/or methoxy) xanthones from Hypericum scabrum (Tanaka et al., 2004), Hypericum ascyron (Hashida et al., 2007), and H. chinense (Tanaka and Takaishi, 2006, 2007). Further study on * Corresponding author. Tel./fax: +81 88 633 7276. E-mail address: [email protected] (Y. Kashiwada). 1 Present address: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060–0812, Japan. 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.08.015

constituents of H. chinense has resulted in the isolation of a new xanthonolignoid (1) and a new phenylxanthone, chinexanthone A (6) together with four known xanthonolignoids (2–5) and eight known xanthones. In this paper, we describe the structure elucidation and cytotoxicity of these compounds as well as additional 32 xanthones against human cancer cell lines including multidrugresistant cancer cells.

2. Results and discussion The MeOH extracts of H. chinense stems were partitioned successively with n-hexane, EtOAc, and H2O. From the EtOAc-soluble fraction, two new (1 and 6) and 12 known xanthone derivatives were isolated. The following known compounds were identified by comparison with the literature data: kielcorin (2) (Silva and Pint, 2005), subalatin (3) (Chen and Chen, 1985), 50 -demthoxycadensin G (4) (Sia et al., 1995), cadensin G (5) (Abou-Shoer et al., 1989), 2-hydroxy-5-methoxy-xanthone (Cardona et al., 1985), 1,2,5-trihydroxy-xanthone (9) (Wu et al., 1998), 1,3-dihydroxy-5-methoxy-xanthone (10) (Cassady et al., 1987), 3,5-dihydroxy-1-methoxy-xanthone (Ghosal et al., 1976), 4-hydroxy-2, 3-dimethoxy-xanthone (Gottlieb et al., 1966), 3,5,6-trihydroxy-1methoxy-xanthone (Jackson et al., 1966), and 3,6-dihydroxy-1, 7-dimethoxy-xanthone (Hu et al., 1999). The structure of 3,7-dihydroxy-1-methoxy-xanthone (Ellis et al., 1976) was assigned by 2DNMR analyses including HMBC, HSQC, and NOESY spectra, since the NMR spectroscopic data for this compound have not been reported previously.

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Compound 1 was obtained as yellow powder and the molecular formula was determined as C23H18O8 by the HRESIMS. The IR spectrum showed absorption bands of a hydroxy (3367 cm1) and a conjugated carbonyl (1657 cm1) groups. The 1H NMR spectrum (Table 1) indicated the presence of one 1,2-disubstituted aromatic ring, one 1,2,4-trisubstituted aromatic ring, one penta-substituted aromatic ring, two oxygenated methines, one methoxy group, and one hydroxy methyl group. The 13C NMR spectrum (Table 1) showed 23 signals including one conjugated carbonyl and 18 aromatic carbons. These data suggested that 1 is a xanthonolignoid with one methoxy and two phenolic hydroxy groups. The 13C NMR spectroscopic data of 1 was similar to that of kielcorin (2) (Silva and Pint, 2005), except for the lack of one of methoxy signals as well as the chemical shifts of C-1 and C-2 (dC 100.2 and 143.8 in 1; dC 96.4 and 145.6 in 2), suggesting 1 to be a demethy derivative of kielcorin. This was further supported by the fact that methylation of 1 yielded 30 -O-methyl kielcorin. The position of the methoxy group at C-30 was confirmed by the NOE correlation between methoxy protons and H-20 . The trans configurations of H-70 and H-80 were concluded from the J-value (8 Hz) of H-70 . Compounds 1 was optically active [1: [a]D +15.4 (c 0.5 MeOH)], in spite of many xanthonolignoids being reported as a racemic mixture (Silva and Pint, 2005). The presence of enantiomers was shown by HPLC analysis using a chiral resolution column (Chiral RU-2), which showed two peaks in a ratio of about 85:15. In order to elucidate the optical nature for 1, (S)-a-methoxy-a-(trifluoromethyl)phenylacetic acid (MTPA) ester derivative of 1 was prepared. 2,40 ,90 -Tri-MTPA ester derivative obtained from 1 showed a major and minor sets of 1H NMR signals in a ratio of 88:12, indicating that 1 is in 76% ee. 1H NMR chemical shifts and coupling patterns of H-70 , H-80 , and H2-90 in major ester (1a) and minor ester (1b) were similar to those in 40 ,90 -di-(S)-MTPA esters of (70 R,80 R) and (70 S,80 S)-venkatasin (Sajeli et al., 2006), respectively (Tables 2 and 3), indicating the major enantiomer of 1 to be 70 R,80 R as shown in Fig. 1. Thus, the structure of 1 was elucidated. Known xanthonolignoids, kielcorin (2), subalatin (3), 50 demethoxycadesin G (4), and cadensin G (5) were also optically active [2: [a]D +5.7 (c 0.09 MeOH); 3: +28.3 (c 0.1 MeOH); 4: [a]D +10.9 (c 0.14 MeOH); 5: +10.3 (c 0.08 MeOH)], and therefore their

Table 1 1 H and 13C NMR spectroscopic data for 1 in DMSO-d6.

a b

Position

13

1 2 3 4 4a 10a 5 6 7 8 8a 9 9a 10 20 30 40 50 60 70 80 90 30 -OMe

100.2 143.8 139.3 132.7 140.1 155.4 118.1 134.7 124.1 125.9 120.7 174.9 114.3 126.9 112.1 147.7 147.3 115.4 120.8 76.3 77.8 60.0 55.8

C

1

Ha

7.16 – – – – – 7.63 7.80 7.43 8.15 – – – – 7.08 – – 6.83 6.92 5.09 4.38 3.74 3.79

(1H, s)

(1H, (1H, (1H, (1H,

d, 8.0) dt, 1.2, 8.0) t, 8.0) dd, 8.0, 1.2)

(1H, d, 1.6)

(1H, (1H, (1H, (1H, (1H, (3H,

Coupling constants given (J, Hz) in parentheses. Overlapped with the signal of DOH.

d, 8.0) dd, 8.0, 1.6) d, 8.0) m) brd, 14.0), 3.48 (1H, m)b s)

Table 2 1 H NMR spectroscopic data for (70 R,80 R)-venkatasin 40 ,90 -di-(S)-MTPA ester (Sajeli et al., 2006) and 1a–3a in CDCl3. Position

(70 R,80 R)-venkatasin40 ,90 -di-(S)-MTPA ester

1a

2a

3a

70

4.97 (d, 7.6)

80

4.41 (ddd, 7.6, 4.5, 2.4)

90

4.75 (dd, 12.5, 2.4)

4.93 (d, 8.4) 4.44 (ddd, 8.4, 4.3, 2.2) 4.96 (dd, 12.8, 2.2) 4.24 (dd, 12.8, 4.3)

4.90 (d, 7.8) 4.45 (ddd, 7.8, 4.5, 2.4) 4.95 (dd, 12.5, 2.4) 4.25 (dd, 12.5, 4.5)

4.92 (d, 8.6) 4.42 (ddd, 8.6, 4.3, 2.6) 5.03 (dd, 12.8, 2.6) 4.25 (dd, 12.8, 4.3)

4.24 (dd, 12.5, 4.5)

Coupling constants given (J, Hz) in parentheses.

Table 3 1 H NMR spectroscopic data for (70 S,80 S)-venkatasin 40 ,90 -di-(S)-MTPA ester (Sajeli et al., 2006) and 1b–3b in CDCl3. Position

(70 S,80 S)-venkatasin40 ,90 -di-(S)-MTPA ester

1b

2b

3b

70

5.00 (d, 8.0)

80

4.38 (dt, 8.0, 2.5)

90

4.99 (dd, 12.6, 2.5)

5.09 (d, 8.5) 4.35 (dt, 8.5, 2.5) 5.12 (dd, 12.5, 2.5) 4.02 (dd, 12.5, 2.5)

5.03 (d, 8.0) 4.42 (dt, 8.0, 2.4) 5.13 (dd, 12.8, 2.4) 4.05 (dd, 12.8, 2.4)

5.07 (d, 8.2) 4.34 (dt, 8.2, 2.3) 5.25 (dd, 12.8, 2.3) 4.04 (dd, 12.8, 2.3)

3.98 (dd, 12.6, 2.5)

Coupling constants given (J, Hz) in parentheses.

(S)-MTPA esters were also prepared. The ratio of enantiomers in 2 and 3 were estimated to be 52:48 and 58:42, respectively, from 1H NMR signals for 40 ,90 -di-(S)-MTPA esters of 2 and 3. Both major enantiomers were concluded to be 70 R,80 R in each case by the same manner as for 1 (Tables 2 and 3). In contrast, 4 and 5 yielded a mixture of 3,40 ,90 -tri-(S)- and 1,3,40 ,90 -tetra-(S)-MTPA esters, and therefore the optical nature for these compounds failed to analyze. Chinexanthone A (6) was obtained as yellow powder and its molecular formula was assigned as C29H24O11 by HRESIMS. The 1 H NMR spectrum (Table 4) showed four aromatic proton signals [dH 7.98 (1H, d, J = 7.6 Hz), 7.70 (1H, t, J = 7.6 Hz), 7.57 (1H, d, J = 7.6 Hz), and 7.31 (1H, t, J = 7.6 Hz)], ascribable to a 1,2-disubstituted aromatic ring, two aromatic singlets [dH 6.71 and 6.66 (each 1H, s)], and two meta-coupled aromatic protons [dH 6.07 and 6.00 (each 1H, d, J = 2.4 Hz)], together with four methoxy resonances [dH 3.85, 3.64, 3.60, and 3.49 (each 3H, s)]. The 13C NMR spectrum (Table 4) showed 29 signals including one conjugated carbonyl, 24 aromatic, and four methoxy carbons. Analysis of HMBC spectrum established the presence of a 1,2,3,5- and a 1,2,4,5-tetra-substituted aromatic rings, whose structures were elucidated as 2,5-dimethoxy-3-hydroxy-phenol and 2-methoxy-4,5-dihydroxyphenyl moieties by detailed HMBC and NOESY analyses as shown in Fig. 2. In addition, NOESY correlations between H-6” and H-60 as well as 5”-OMe indicated that these moieties were connected at C-50 and C-1” through an ether linkage. In contrast, the remaining 13 carbon resonances, including the conjugated carbonyl resonance, together with one methoxyl carbon resonance, suggested the existence of a 1,2,3,4-tetra-substituted xanthone moiety. These resonances were similar to those of 1,2-dihydroxy-3-methoxyxanthone, except for the observation an sp2 quaternary carbon resonance instead of C-1 methine carbon resonance. The HMBC correlation from H-60 to the sp2 quaternary carbon at dC 123.1 as well as the NOESY correlation between 2-OMe and H-60 indicated that the 40 -hydroxy-20 -methoxy-50 -(3”-hydroxy-2”,5”-dimethoxy)phenoxyphenyl moiety was bound to the xanthone moiety through

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N. Tanaka et al. / Phytochemistry 70 (2009) 1456–1461

O

O

8

1 8a

7

9

9a

HO

OH 3'

10a 5

O

4a

O

4

O 2'

O

7'

8'

R2O

5'

2'

3

6

3'

OMe MeO

4'

OR4

O

OH

O

MeO O

6' 5'

HO

6'

1 ; R1,R2,R3,R4=H 1a; R1,R2,R4=(S)-MTPA, R3=H 2 ; R1=Me, R2,R3,R4=H 2a; R1=Me, R2,R4=(S)-MTPA, R3=H 3 ; R, R2,R4=H, R3=OMe 3a; R1,R2,R4=(S)-MTPA, R3=OMe

3"

6

OH

OH

4"

6"

5"

OMe

OH

O

4 ; R=H 5 ; R=OMe O

2"

1"

OMe

OH R

R3

O

1'

1' 9'

OMe

4'

OR1

2

OH O

OH

HO

OMe OH O

O

7

O 8

Fig. 1. Structures of xanthonolignoids (1–5), chinexanthone A (6), and cytotoxic xanthones (7, 8).

Table 4 1 H and 13C NMR spectroscopic data for 6 in CD3OD. Position 1 2 3 4 4a 10a 5 6 7 8 8a 9 9a 2-OMe

a

13

C

123.1 145.1 146.1 134.3 145.8 156.7 118.6 135.3 124.8 127.1 123.4 178.2 113.8 61.1

1

Ha

– – – – – – 7.57 7.70 7.31 7.98 – – – 3.49

Position

(1H, (1H, (1H, (1H,

d, 7.6) t, 7.6) t, 7.6) d, 7.6)

1 20 30 40 50 60 1” 2” 3” 4” 5” 6” 0

2 -OMe 2”-OMe 5”-OMe

(3H, s)

13

1

Ha

119.0 156.3 101.5 150.0 137.0 124.7 154.0 132.6 152.4 97.4 157.7 95.0

– – 6.66 – – 6.71 – – – 6.07 – 6.00

56.2 61.6 55.9

3.64 (3H, s) 3.85 (3H, s) 3.60 (3H, s)

C

0

(s)

(s)

(1H, d, 2.4) (1H, d, 2.4)

Coupling constants given (J, Hz) in parentheses.

C–C linkage. The position of this phenyl moiety was concluded to be C-1 from the NOESY correlation between OMe-5” and H-8. The biphenyl moiety of 6 was considered to be racemic from its

small optical rotation value and CD spectrum, in which obvious cotton effect was not observed. Compounds 1–6, and 39 known xanthones, including 32 xanthones previously isolated from H. chinense (Tanaka and Takaishi, 2006, 2007), were examined for their cytotoxic activity against a panel of human cancer cell lines, namely, the KB (human epidermoid carcinoma of nasopharynx), K562 (leukemia), MCF7 (breast carcinoma), and COLO205 (colon carcinoma) cell lines, as well as multidrug-resistant (MDR) human cancer cell lines including KBC2 (colchicine-resistant KB) and K562/Adr (doxorubicin-resistant K562). The IC50 values are shown in Table 5. The xanthones evaluated classified into three groups, including xanthonolignoids (1–5), a phenyl xanthone (6), prenylated xanthones [7 and 1,3,7-trihydroxy-2-(2-hydroxy-3-methylbut-3-enyl)-xanthone], and simple xanthones (8–20 as well as those indicated in the footnote for Table 5). Among the evaluated xanthonolignoids, kielcorin (2) demonstrated relatively potent cytotoxicity against KB cells with an IC50 value of 8.1 lg/mL. It also showed about threefold reversal effect of colchicine resistance against colchicine-resistant KB (KB-C2) cells (6.5 lg/mL), as compared with its cytotoxicity (IC50 18.4 lg/ mL) against KB-C2 cells. Replacement of the methoxy group at C2 to a hydroxy group in 2 yielded a slightly less cytotoxic compound as 1. However, 1 displayed relatively potent cytotoxicity

OH

MeO HO

OMe O

MeO O OMe OH

O OH

HMBC

Fig. 2. Key HMBC and NOESY correlations in 6.

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N. Tanaka et al. / Phytochemistry 70 (2009) 1456–1461 Table 5 Cytotoxicity of xanthonolignoids and selected xanthone derivativesa isolated from H. chinense against human cancer cell lines (IC50 in lg/mL). Cell lines KBb

(Substitution pattern for simple xanthones) C-1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Doxorubicin

OMe OH OH H OH H H H OH OH OH H OH

C-2

C-3

OH H OH H H OH OH H H H -O-CH2-OOMe OH OH H H OMe H OH H OH OH OMe H OH

C-4

H H H H H H H H H H H OMe H

C-5

H OH OMe H H H H OH OH OH H H OMe

C-6

H H H H H H H H H OH OH OH OMe

C-7

H H H H OH H H H H H OH H OH

KB-C2c

C-8

H H H H H H H H H H H H H

KB-C2 (+Col.)

18.6 8.1 43.6 31.7 25.2 48.5 9.1 14.3 16.9 33.3 19.1 15.1 28.1 20.3 26.1 28.1 25.6 33.3 34.2 20.9 0.22

32.0 18.4 >100 36.0 54.2 >100 6.2 14.6 61.8 24.6 14.1 12.3 20.6 19.6 25.2 22.0 >100 93.5 31.3 43.2 >100

MCF-7e

K562f

K562/Adrg

COLO205h

17.1 17.5 14.1 18.2 16.3 28.7 9.8 4.0 16.4 29.5 40.2 42.7 >100 43.5 26.6 32.2 18.9 18.6 35.7 23.7 0.33

>100 >100 >100 33.2 28.8 23.9 >100 21.6 63.9 >100 >100 >100 >100 >100 >100 >100 25.4 22.5 44.8 36.5 0.45

>100 >100 >100 26.0 33.7 >100 17.5 17.9 40.1 37.0 13.9 42.1 46.1 57.5 41.4 15.7 24.7 34.5 57.1 37.7 15.2

15.8 >100 >100 23.9 15.8 35.6 12.0 14.0 32.7 54.7 54.5 42.0 >100 87.3 30.2 44.2 26.5 28.5 36.2 23.3 0.50

d

22.9 6.5 >100 34.9 42.9 >100 4.8 15.1 >100 26.0 19.9 14.5 19.2 23.6 26.6 25.9 >100 >100 38.9 38.2

a Cytotoxicities for xanthones, including 1,3,7-trihydroxy-2-(2-hydroxy-3-methylbut-3-enyl)-xanthone, 2-hydroxy-5-methoxyxanthone, 3,5-dihydroxy-1-methoxyxanthone, 4-hydroxy-2,3-dimethoxyxanthone, 3,5,6-trihydroxy-1-methoxyxanthone, 3,6-dihydroxy-1,7-dimethoxyxanthone, 3,7-dihydroxy-1-methoxyxanthone, 1-hydroxyxanthone, 2,7-dihydroxy-xanthone, 1,3-dihydroxy-2-methoxy-xanthone, 3-hydroxy-,2,4-dimethoxy-xanthone, 2,5-dihydroxy-1-methoxy-xanthone, 3,4-dihydroxy-2methoxy-xanthone, 1,3-dihydroxy-2,4-dimethoxy-xanthone, 1,3,7-trihydroxy-5-methoxy-xanthone, 1,7-dihydroxy-5,6-dimethoxy-xanthone, 1,5-dihydroxy-6,7-dimethoxy-xanthone, 1,6-dihydroxy-7,8-dimethoxy-xanthone, 1,3,6-trihydroxy-5-methoxy-xanthone, 4,5-dihydroxy-2,3-dimethoxy-xanthone, 3,5-dihydroxy-1,2-dimethoxyxanthone, 1,3,5-trihydroxy-6-methoxy-xanthone, 4,6-dihydroxy-2,3-dimethoxy-xanthone, 3,6-dihydroxy-1,2-dimethoxy-xanthone, 4,7-dihydroxy-2,3-dimethoxy-xanthone, 3,7-dihydroxy-2,4-dimethoxy-xanthone, 3,5,6-trihydroxy-1-methoxy-xanthone, 3,6-dihydroxy-1,7-dimethoxy-xanthone, 3,5,6-trihydroxy-1-methoxy-xanthone, 3,6dihydroxy-1,7-dimethoxy-xanthone, and 1,3,5-trihydroxy-6,7-dimethoxy-xanthone, are not shown, since they only displayed little or no cytotoxicity. b Human epidermoid carcinoma. c Multidrug-resistant KB cells. d 2.5 lM colchicine. e Breast carcinoma. f Leukemia. g Doxorubicin-resistant K562 cells. h Colon carcinoma.

(15.8 lg/mL) against COLO205 cells, although 2 had no cellular toxicity against this cell line. Introduction of a methoxy group at C-50 in 2 also decreased its cytotoxicity against KB and KB-C2 cell lines as seen in 3. In contrast, xanthonolignoids 4 and 5 displayed moderate cytotoxicities against all the tested cell lines, including MDR cancer cell lines. Considering the cytotoxicity (IC50 > 100 lg/ mL) against KB-C2 cells for the simple xanthenes, 3,4-dihydroxy-2methoxy-xanthone and 1,3,5,6-tetrahydroxy-xanthone, corresponding to xanthonolignoids (2 and 4–5, respectively), introduction of a C6–C3 unit might enhance their cytotoxicity against KB-C2 cells. Xanthonolignoids also showed moderate cytotoxicities against MCF-7 cells. Prenylated xanthone possessing a dihydrofuran ring (7) showed relatively potent cytotoxicity against KB and MCF-7 cell lines (IC50 9.1 and 9.8 lg/mL, respectively). Cytotoxicities of 7 against MDR cancer cell lines were slightly more potent (KB-C2: 6.2 lg/mL; K562/Adr: 17.5 lg/mL) than those against sensitive cell lines. In addition, it showed significant cellular toxicity against KBC2 cells in the presence of colchicine. Among the evaluated simple xanthones, 2-hydroxy-1-methoxyxanthone (8) showed significant cytotoxicity against MCF-7 cells with an IC50 value of 4.0 lg/mL. Removal of the methoxy group at C-1 in 8 gave a less cytotoxic compound (IC50 40.2 lg/lL; MCF-7). Furthermore, compound 8 showed relatively potent cytotoxicity against KB, K562, and COLO205 cell lines with IC50 values ranging from 14.0 to 21.6 lg/ mL. In addition, its cytotoxicity against MDR cancer cell lines (KB-C2 and K562/Adr) was similar to those against sensitive cell lines. Its cytotoxicity against KB-C2 were not changed in the presence of 2.5 lM of colchicine, suggesting that compound 8 has no

effect on P-glycoprotein function of KB-C2 cells. Compounds 10– 16 also showed relatively potent to moderate cytotoxicity against both KB and its MDR cancer cells (KB-C2) with IC50 values ranging from 12.3 to 25.4 lg/mL similar to 8. Although 10–16 were not cytotoxic (>100 lg/mL) against K562 cells, they displayed relatively potent to moderate cytotoxicity against K562/Adr cells. In contrast, compounds 9, 17–20 displayed moderate to weak cytotoxicity against both K562 and K562/Adr cell lines. Overall, these simple xanthones were sensitive against MDR cancer cell lines. Therefore, they might be potential leads for the chemotherapeutic agents against MDR in cancer, although the structure–activity relationships are not clear with these limited numbers of evaluated compounds.

3. Concluding remarks The new xanthonolignoid, 2-demthylkielcorin (1) and phenylxanthone, chinexanthone A (6) were isolated from the stems of H. chinense together with four known xanthonolignoids and seven known xanthones. Their structures were established by spectroscopic analysis. The new xanthonolignoid as well as known xanthonolignoids from H. chinense were shown to be optically active, though many xanthonolignoids reported so far were racemic mixtures. Assignments of the stereochemistry for the major enantiomer as well as the ratio of enantiomers were elucidated by analyzing their MTPA ester derivatives. In contrast, chinexanthone A, possessing a phenyl substituent in xanthone skeleton, appears to

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be a new class of xanthone as phenylxanthone. We also evaluated cytotoxicities for 45 xanthenes, including previously reported 32 xanthones, against a panel of six human cancer cell lines including two multidrug-resistant (MDR) human cancer cell lines. Though almost xanthones were non-cytotoxic, some xanthones were more sensitive against MDR cancer cells. In addition, some xanthones displayed enhanced cytotoxicities against MDR cancer cells in the presence of colchicine. Since MDR is one of major obstacles to cancer chemotherapy, overcoming MDR is most important to succeed in cancer chemotherapy. These xanthones are considered to be leads for the chemotherapeutic agents against MDR in cancer, and would provide more potent derivative with a suitable modification. 4. Experimental 4.1. General experimental procedures NMR experiments were run on a Bruker AVANCE instrument, 1H NMR: 400 MHz, 13C NMR 100 MHz, using TMS as int. stand. MS was obtained on a Waters LCT Premier. Chromatography column: silica gel 60 N (Merck, 63–210 lm), Sephadex LH-20 (Pharmacia), and Toyopearl HW-40 (TOSOH); HPLC: GPC (Shodex H-2001, 2002, CHCl3; Asahipak, GS-310 2G, MeOH), silica gel (YMC-Pack SIL-06 SH-043-5-06, 250  20 mm), ODS (Mightysil RP-18, 250  20 mm, Kanto Kagaku). Chiral RU-2 (Shiseido) column (4.6 mm i.d.  150 mm) [1% AcOH/MeOH] was used for optical resolution of xanthonolignoids. IR spectra were recorded on a 1720 Infrared Fourier Transform spectrometer (Perkin–Elmer). Optical rotations were measured with a JASCO DIP-370 digital polarimeter. 4.2. Plant material The aerial parts of H. chinense were collected in October 2002 in Tokushima Prefecture, Japan, and identified by Dr. Kotaro Murakami (Sojo University). Herbarium specimens were deposited in the botanical garden of the University of Tokushima (specimen number: UTP98008). 4.3. Extraction and isolation The stems of H. chinense (4.54 kg, dried) were crushed and extracted (3  18 L) with MeOH at 60 °C for 4 h. The MeOH extracts were concentrated in vacuo to give a residue (548 g), which was partitioned successively with n-hexane, EtOAc, and H2O. The EtOAc-soluble fraction (96.8 g) was subjected to silica gel CC eluted with solvents of increasing polarity n-hexane–EtOAc–MeOH to give 13 fractions (fr. 1–13). Fr. 7 (4.54 g) was applied to a Sephadex LH-20 column with MeOH to give four fractions (fr. 7.1–4). Fr. 7.4 was separated by silica gel CC (CHCl3–MeOH, 98:2, 96:4, 9:1, and 0:10) to give four fractions (fr. 7.4.1–4). Fr. 7.4.1 was loaded on GPC on HPLC (CHCl3) to give four fractions (fr. 7.4.1.1–4). Fr. 7.4.1.3 was purified by ODS-HPLC (MeOH/H2O, 8:2) and prep. TLC (CHCl3–MeOH–H2O, 9:1:0.1) to afford 4-hydroxy-2,3-dimethoxyxanthone (14 mg). Fr. 7.4.1.4 was subjected to GPC on HPLC (MeOH) to give 2-hydroxy-5-methoxyxanthone (1 mg) and 1,3dihydroxy-5-methoxy-xanthone (5 mg). Fr. 9 (8.85 g) was dissolved in MeOH to give a residue and a MeOH-soluble layer. The residue was purified by silica gel CC (CHCl3–MeOH, 9:1) to afford 2 (42 mg). The BuOH-soluble fraction was loaded to a Sephadex LH-20 column (MeOH) to give four fractions (fr. 9.1–4). Fr. 9.4 was subjected to silica gel CC (CHCl3–MeOH, 95:5, 9:1, 8:2, 6:4, and 0:10) to give nine fractions (fr. 9.4.1–9). Fr. 9.4.4 was purified by GPC on HPLC (MeOH) to give 1 (45 mg) and 4 (15 mg). Fr. 9.4.6 was purified fur-

ther over Sephadex LH-20 to give three fractions (fr. 9.4.6.1–3). Fr. 9.4.6.3 was purified by GPC on HPLC (MeOH), and an ODS-HPLC (MeOH–H2O, 7:3) to furnish 6 (2.6 mg). Fr. 10 (30.9 g) was applied to a silica gel column eluted with solvents of increasing polarity (CHCl3–MeOH) to give eight fractions (fr. 10.1–8). Fr. 10.2 was separated by Toyopearl HW-40 CC (CHCl3–MeOH, 1:1) to give four fractions (fr. 10.2.1–4). Fr. 10.2.3 was purified by GPC on HPLC (CHCl3) and silica gel CC (CHCl3–MeOH, 96:4) to afford 3 (22 mg). Fr. 10.2.4 was separated by GPC on HPLC (MeOH) and purified by ODS-HPLC (MeOH–H2O, 7:3) to give 1,2,5-trihydroxyxanthone (9 mg). Fr. 10.3 was chromatographed on a Sephadex LH-20 column (MeOH) to give eight fractions (fr. 10.3.1–8). Fr. 10.3.5 was applied to a silica gel column eluting with the solvents increasing polarity (CHCl3–MeOH) to give eight fractions (fr. 10.3.5.1–8). Fr. 10.3.5.3 was separated by GPC on HPLC (MeOH) to give 5 (25 mg) and 4-hydroxy-2,3-dimethoxyxanthone (14 mg). Fr. 10.3.5.4 was purified by GPC on HPLC (MeOH) and ODS-HPLC (MeOH–H2O, 1:1) to give 3,5-dihydroxy-1-methoxyxanthone (45 mg), 3,7-dihydroxy-1-methoxyxanthone (27 mg), and 3,5,6trihydroxy-1-methoxyxanthone (10 mg). 4.4. 2-Demethylkielcorin (1) Yellow powder. [a]D +15.4 (c 0.5 MeOH); IR (KBr): mMAX cm1 3367, 1657, 1620, 1520, 1485, 1466, 1286, 1225, and 1136; HRESIMS: m/z 445.0905, [M+Na]+ (calcd for 445.0899, C23H18O8Na); for 1H and 13C NMR (DMSO-d6) spectroscopic data, see Table 1. 4.5. Methylation of 1 A solution of 1 (7 mg) in MeOH (2 mL) was treated with trimethylsilyldiazomethane (TMSCHN2, 0.5 ml, 10% in hexane) at room temperature for 3 h. After removal of the solvent by evaporation, the residue was purified by silica gel CC (benzene–acetone, 4:1) to afford 40 -O-methylkiekcorin (5 mg) (Nielsen and Arends, 1978). 4.6. Conversion of xanthonolignoids (1–3) to bis or tri-(S)-MTPA ester (1a,b–3a,b) Compounds 1–3 (each 0.5 mg) in pyridine (100 lL) were treated with (R)-MTPACl (5 lL) for 1 h. MeOH (30 lL) was added to the reaction mixture to destroy the acid chloride. Solvents were removed in vacuo to afford bis or tri-(S)-MTPA ester (1a,b–3a,b). 4.7. Chinexanthone A (6) Yellow powder. [a]D +1.2 (c 0.58 MeOH); IR (KBr): mMAX cm1 3410, 1651 (sh), 1620 (sh), 1599, 1500, 1465, 1295, 1200, and 1043; HRESIMS: m/z 571.1199, [M+Na]+ (calcd for 571.1216, C29H24O11Na); for 1H and 13C NMR (DMSO-d6) spectroscopic data, see Table 4. 4.8. 3,7-Dihydroxy-1-methoxyxanthone 1

H NMR (pyridine-d5): dH 8.26 (1H, d, J = 3.2 Hz, H-8), 7.46 (1H, dd, J = 8.8, 3.2 Hz, H-6), 7.38 (1H, d, J = 8.8 Hz, H-5), 6.79 (1H, d, J = 2.0 Hz, H-2), 6.67 (1H, d, J = 2.0 Hz, H-4), and 3.85 (3H, s, OMe-1); 13C NMR (pyridine- d5): dC 175.1 (C-9), 165.4 (C-3), 163.2 (C-1), 160.6 (C-4a), 155.4 (C-7), 149.2 (C-10a), 124.7 (C-8a), 123.8 (C-6), 118.9 (C-5), 110.7 (C-8), 106.5 (C-9a), 96.6 (C-2), 96.2 (C-4), and 56.3 (OMe-1). 4.9. Cell lines and cell culture Human epidermoid carcinoma cells (KB) were cultured in Dulbecco’s modified Eagles medium (DMEM) with 10% fetal bovine

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serum (FBS). Multidrug-resistant KB cells (KB-C2) were maintained in DMEM medium in the presence of 10% FBS and 5 lM colchicine. MCF-7 (breast carcinoma), COLO205 (colon carcinoma), K-562 (leukemia) cells were cultured in RPMI1640 supplemented with 10% FBS. K-562/Adr (doxorubicin-resistant K562 cell line) cells were cultured in RPMI1640 medium containing 10% FBS and 0.5 lM doxorubicin. All cells were incubated at 37 °C in a humidified atmosphere with 5% CO2–95% air. 4.10. In vitro cytotoxicity assay Cells were seeded at each density (1  105 cells/well for K562 and K562/Adr, 5  104 cells /well for KB and KB-C2, or 5  104 cells/well for MCF7 and COLO205) in 96-well plate and pre-incubated for 24 h. Test samples were dissolved in small amounts of DMSO and diluted in the appropriate culture medium (final concentration of DMSO < 0.5%). After removal of pre-incubated culture medium, 100 ll of medium containing various concentration of test compound were added and further incubated for 48 h. Cell viability was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Mosmann, 1983). IC50 values (concentration in lg/ml required to inhibit cell viability by 50%) were calculated using the concentration–inhibition curve. References Abou-Shoer, M., Habib, A.A., Chang, C.J., Cassady, J.M., 1989. Seven xanthonolignoids from Psorospermum febrifugum. Phytochemistry 28, 2483–2487. Bennett, G.J., Lee, H.-K., 1989. Xanthones from Guttiferae. Phytochemistry 28, 967– 998. Cardona, M.L., Pedro, J.R., Seoane, E., Vidal, R., 1985. Xanthone constituents of Hypericum canarienis. J. Nat. Prod. 48, 467–469. Cassady, J.M., Byrn, S.R., MacKenzie, A.T., Ho, D.K., 1987. O5-methyl-(±)-(20 R, 30 S)psorospermin. J. Org. Chem. 52, 342–347. Chen, M.-T., Chen, C.-M., 1985. Xanthonolignoids from Hypericum subalatum. Heterocycles 27, 2589–2594. Ellis, R.C., Whalley, W.B., Ball, K., 1976. The chemistry of fungi. Part LXX. Synthesis of some xanthones. J. Chem. Soc. Perkin Trans. 1, 1377–1382. Fernandes, E.R., Carvalho, F.D., Remiao, F.G., Bastos, M.L., Pinto, M.M., Gottlieb, O.R., 1995. Hepatoprotective activity of xanthones and xanthonolignoids against tert-butylhydroperoxyde-induced toxicity in isolated rat hepatocytescomparison with silybin. Pharm. Res. 12, 1756–1760. Ghosal, S., Chauhan, R.B.P.S., Biswas, K., Chaudhuri, R.K., 1976. New 1,3, 5trioxygenated xanthones in Canscora decussata. Phytochemistry 15, 1041–1043. Gottlieb, O.R., Taveira Magalháes, M., Camey, M., Lins Mesquita, A.A., de Barros Corréa, D., 1966. The chemistry of brazilian Guttiferae-V: 2,3,4- and 1,3,5trioxygenatd xanthones from Kielmeyer species. Tetrahedron 22, 1777–1784. Hashida, C., Tanaka, N., Kashiwada, Y., Ogawa, M., Takaishi, Y., 2008. Prenylated phloroglucinol derivatives from Hypericum perforatum var. angustifolium. Chem. Pharm. Bull. 56, 1164–1167. Hashida, W., Tanaka, N., Takaishi, Y., 2007. Prenylated xanthones from Hypericum ascyron. J. Nat. Med. 61, 371–374.

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