Glycolipids from the Formosan Soft Coral Lobophytum crassum

During the course of our investigation on the bioactive

chemical constituents from marine invertebrates,

_{we have}

investigated a soft coral Lobophytum crassum collected from

Taiwanese waters. Earlier studies of the genus Lobophytum

have led to the isolation of terpenoids, of which some have

shown cytotoxic,

_anti-HIV,

_{and antibacterial}

activi-ties. In this paper we report the isolation and structural

eluci-dation of three new glycolipids 1—3 (Fig. 1) from this

organ-ism. The absolute configurations of 1—3 were determined by

the comparison of specific optical rotations of sugar and

aglycon moieties with known compounds, chemical

conver-sions, and the application of Mosher’s method. Cytotoxicity

of metabolites 1—3 against the growth of a limited panel of

cancer cells of HepG2, Hep3B (human liver carcinoma),

MDA-MB-231 (human breast carcinoma), and Ca9-22

(human gingival carcinoma) is also discussed.

Results and Discussion

(2R)-1-Hydroxy-3-hexadecyloxy-propyl-b

-D-arabinopyra-noside (1) was isolated as amorphous white solid. Its

HR-ESI-MS exhibited a pseudomolecular ion peak at m/z

471.3296 [M

Na]

(Calcd for C

H

O

Na, 471.3298),

cor-responding to the molecular formula C

H

O

, indicating

one degree of unsaturation. The 1 : 2 ratio of carbon and

pro-ton atoms and high oxygen content in its molecular formula

and strong absorption bands at n

3391, 1142, 1075, and

1007 cm

in IR spectrum suggested that 1 might be a

gly-colipid. An anomeric glycoside proton resonance was

ob-served at d 5.67 (1H, d, J

3.3 Hz), corresponding to a

car-bon resonance at d 100.4 (d) in the HMQC spectrum. Except

for the above carbon (C-1

), the

_{C-NMR spectrum of 1}

ex-hibited eight oxygenated carbons, including four

oxymethyl-enes at d

71.7 (t), 71.1 (t), 64.5 (t), and 63.0 (t), and four

oxymethines at d

78.5 (d), 71.0 (d), 70.8 (d), and 70.1 (d)

(Table 1). Above data together with the methyl group

reso-nanting at d

0.85 (3H, t, J

6.6 Hz) and methylenes at d

1.26 (br s) revealed the structure of 1 to be a glycoside with a

long aliphatic chain.

The gross structure of metabolite 1 was further

establish-ed by the 2D-NMR studies, particularly in

H–

H COSY,

HMQC and HMBC experiments. The correlations of

H–

H

COSY revealed proton–proton sequences, from H-1

 to H-5,

H

-1 to H

-3, H

-1

 to H

-2

, and H

-15

 to H

-16

, as shown

in Fig. 2. The HMBC correlations from H-1

 to 5 and

C-2, as well as other correlations illustrated in Fig. C-2,

estab-lished a pyranose and a glycerol ether moiety. The pyranose

was identified as b-arabinopyranose by analysis of coupling

constants of the related protons (Table 1). HR-ESI mass

spectrum and the above 2D-NMR spectroscopic analysis led

to the establishment of the aglycon to be chimyl alcohol.

The absolute configurations on the sugar portion and lipid

aglycon were determined on the basis of methanolysis,

chemical transformation and the application of the modified

Mosher’s method. As shown in Chart 1, methanolysis of 1

with 1

HCl

–MeOH (1 : 1) yielded the methyl

arabinopy-ranoside and the chimyl alcohol. The glycerol-based portion

was found to be 2R-chimyl alcohol, [a]

_2°.

The

sugar-containing portion was further treated with 1

HCl

and

the sugar thus obtained was shown to be (

)-

1720 Vol. 55, No. 12

Glycolipids from the Formosan Soft Coral Lobophytum crassum

Chih-Hua C

,

Ho-Cheng H

,

Yang-Chang W

,

Chung-Kuang L

,

Chang-Feng D

,

and

Jyh-Horng S

*

a_{Department of Marine Biotechnology and Resources, National Sun Yat-sen University; Kaohsiung 804, Taiwan, R.O.C.:} b_{Department of Chemical and Materials Engineering, Cheng Shiu University; Kaohsiung 833, Taiwan, R.O.C.:} c_Graduate

Institute of Natural Products, Kaohsiung Medical University; Kaohsiung 807, Taiwan, R.O.C.: d_{National Museum of}

Marine Biology and Aquarium; e_{Institute of Marine Biotechnology, National Dong Hwa University; Checheng, Pingtung}

944, Taiwan, R.O.C.: and f_{Institute of Oceanography, National Taiwan University; Taipei 106, Taiwan, R.O.C.: and} g_Asian

Pacific Ocean Research Center, National Sun Yat-sen University; Kaohsiung 804, Taiwan, R.O.C.

Received August 6, 2007; accepted September 14, 2007

Three glycolipids (1—3), possessing a sugar moiety at C-2 of glycerol ether, have been isolated from the For-mosan soft coral Lobophytum crassum. Their structures were elucidated by spectroscopic methods, particularly in 1D- and 2D-NMR experiments. The absolute configurations on the sugar portion and lipid aglycon of 1—3 were determined by methanolysis, chemical transformation and the application of Mosher’s method on 1 and 3. Compounds 1—3 exhibited weak cytotoxic activities.

Key words Lobophytum crassum; glycolipid; coral; R-batyl alcohol; R-chimyl alcohol; arabinopyranoside Chem. Pharm. Bull. 55(12) 1720—1723 (2007)

© 2007 Pharmaceutical Society of Japan ∗ To whom correspondence should be addressed. e-mail: [email protected]

[a]

110°,

by comparison of specific optical rotation

and TLC analysis with the authentic sample. The absolute

configuration of chimyl alcohol (1a) was further confirmed

by the calculation of the chemical shift difference of two

di-astereotopic protons of H

-1 in bis-(R)-MTPA ester (1b),

which was prepared from 1a with (S)-MTPA chloride.

Previ-ous study has shown that the chemical shift difference of two

diastereotopic protons of H

-1, resonating as two doublets of

doublets, in 2S-glycerol ether was 0.26 ppm, while that in

2R-glycerol ether was 0.31 ppm.

_{In the case of 1b, the}

above two protons were found to resonate at d 4.73 and 4.42

(D

0.31 ppm), indicating R configuration at C-2, the same

as that deduced by specific optical rotation. The absolute

configuration of the sugar moiety was also determined by the

application of Mosher’s method

on the acetonide

deriva-tive 1e. The chemical shift differences of (S)-MTPA ester

(1c) and (R)-MTPA ester (1d) were summarized in Chart 1

and indicated the S-configuration at C-2

; hence, the sugar

moiety of 1 was deduced as D-arabinose.

(2R)-1-Hydroxy-3-octadecyloxy-propyl-b

-D-arabinopyra-noside (2) had the molecular formula of C

H

O

, 28 mass

units higher than that of 1, as determined by HR-ESI-MS.

The

H- and

C-NMR spectral data of 2 were found to be

very similar with those of 1. Compound 2 is more strongly

retained by the reverse stationary phase in ODS column than

1, implying that 2 might possess longer aliphatic chain.

In-spection of 2D-NMR spectral data of 2 allowed the

establish-ment of the same planar structure as that of 1, with the slight

difference of aliphatic chain length at 3-O position. Hence,

the glycerol moiety of 2 was derived from batyl alcohol. The

absolute stereochemistry of 2 was suggested to be the same

as that of 1 due to the biogenetic consideration as well as the

December 2007 1721

Table 1. 1_{H- and} 13_{C-NMR Spectral Data of Compounds 1—3}

C # 1 2 3 1_Ha) 13_Cb) 1_Ha) 13_Cb) 1_Ha) 13_Cb) 1 4.14 br s 63.0 (t)d) _{4.14 br s 63.0 (t) 4.55 dd (11.4, 3.9) 65.1 (t)} 4.46 dd (11.4, 6.6) 2 4.37 m 78.5 (d) 4.37 m 78.5 (d) 4.36 m 74.9 (d) 3 3.89 dd (10.2, 4.2)c) _{71.1 (t) 3.89 dd (10.2, 4.2) 71.1 (t) 3.75 dd (10.2, 5.4) 70.2 (t)} 3.85 dd (10.2, 5.4) 3.85 dd (10.2, 5.4) 3.67 dd (10.2, 5.4) 1 5.67 d (3.3) 100.4 (d) 5.67 d (3.3) 100.4 (d) 5.55 d (3.6) 100.7 (d) 2 4.62 dd (9.0. 3.3) 70.8 (d) 4.62 dd (9.0. 3.3) 70.8 (d) 4.61 dd (9.0. 3.6) 70.5 (d) 3 4.50 dd (9.0, 3.3) 71.0 (d) 4.50 dd (9.0, 3.3) 71.0 (d) 4.47 m 70.9 (d) 4 4.36 br s 70.1 (d) 4.36 br s 70.1 (d) 4.40 br s 70.2 (d) 5 4.47 dd (12.0, 1.5) 64.5 (t) 4.47 dd (12.0, 1.5) 64.5 (t) 4.36 dd (12.0, 1.8) 64.5 (t) 4.07 dd (12.0, 2.7) 4.07 dd (12.0, 2.7) 4.09 dd (12.0, 2.4) 1 3.46 t (6.3) 71.7 (t) 3.46 t (6.3) 71.7 (t) 3.43 t (6.3) 71.7 (t) 2 1.54 m 30.1 (t) 1.54 m 30.1 (t) 1.55 m 30.0 (t) 3 1.26 m 26.4 (t) 1.26 m 26.4 (t) 1.26 m 26.4 (t) 4—13 1.26 br s 29.6—30.0 (t) 1.26 br s 29.6—30.0 (t) 1.26 br s 29.6—30.0 (t) 14 1.26 br s 32.1 (t) 1.26 br s 29.6—30.0 (t) 1.26 br s 32.1 (t) 15 1.26 br s 22.9 (t) 1.26 br s 29.6—30.0 (t) 1.26 br s 22.9 (t) 16 0.85 t (6.6) 14.2 (q) 1.26 br s 32.1 (t) 0.86 t (6.6) 14.3 (q) 17 1.26 br s 22.9 (t) 18 0.85 t (6.6) 14.2 (q) OAc 2.02 s 20.7 (q) 170.7 (s)

a) Spectra recorded at 300 MHz in pyridine-d5. b) Spectra recorded at 75 MHz in pyridine-d5. c) J values (in Hz) in parentheses. d) Multiplicity deduced by DEPT and

indicated by usual symbols.

same sign of specific optical rotations, and similarity of

NMR spectral data.

(2R)-1-Acetoxy-3-hexadecyloxy-propyl-b

-D-arabinopyra-noside (3) was obtained as a white powder. Its HR-ESI-MS

exhibited a molecular ion peak at m/z 513.3404 [M

Na]

and established a molecular formula of C

H

O

,

corre-sponding to two degrees of unsaturation. The IR spectrum of

3 revealed the presence of hydroxyl (

n

3418 cm

) and

ester (n

1741 cm

) moieties. The ester was identified

as an acetoxyl from the

H-NMR data at d 2.02 (3H, s)

and the

_{C-NMR data at d 20.7 (q) and 170.7 (s) (Table 1).}

The attaching of acetoxy group at C-1 was confirmed

by the HMBC correlation from H

-1 to the acetate carbonyl

carbon. Therefore, 3 was identified as a C-1 acetoxy

deriva-tive of 1.

To establish the absolute configuration on C-2

 of

the sugar moiety of 3, the modified Mosher’s method

was applied on the acetonide derivative of 3 (Chart 2). As

shown in Chart 2, the chemical conversion from 3 to

1d through methanolysis of 3b at 50 °C followed by the

treatment with 2,2-dimethoxypropane and then (S)-MTPA

chloride in pyridine to obtain 1d demonstrated that both 1

and 3 possess the same absolute configurations at all chiral

centers. The chemical shift differences of (S)-MTPA ester

(3a) and (R)-MTPA ester (3b) were summarized in Chart 2

and also suggested the S-configuration at C-2

, the same as

that of 1.

The cytotoxicity of compounds 1—3 against HepG2,

Hep3B, MDA-MB-231, and Ca9-22 cancer cells was shown

in Table 2. It was found that all of 1—3 showed cytotoxic

ac-tivities toward the above cancer cell lines with IC

’s ranged

from 9.2 to 15.0

mg/ml.

Experimental

General Procedure Optical rotations were measured on a Jasco P-1020 polarimeter. IR spectra were recorded on Jasco FT/IR-4100 fourier trans-form infrared spectrophotometer. The NMR spectra were recorded on Varian Mercury-Plus 300 FT-NMR instruments at 300 MHz for 1_{H, and 75 MHz for} 13_{C in pyridine-d}

5, and chemical shifts are referenced to dC135.5 ppm and

dH8.71 ppm. Nuclear Overhauser and exchange spectroscopy (NOESY), 1_H–1_{H correlation spectroscopy (COSY), heteronuclear multiple quantum}

coherence (HMQC), and 1_{H-detected multiple-bond heteronuclear multiple}

quantum coherence (HMBC) experiments were performed by using standard Varian pulse sequences. LR-MS and HR-MS were obtained by ESI on a Bruker APEX II mass spectrometer. Silica gel 60 (Merck, 230—400 mesh) was used for column chromatography. Precoated Silica gel plates (Merck Kieselgel 60 F2540.2 mm) were used for analytical TLC. High-performance

liquid chromatography (HPLC) was performed on a Shimadzu LC-10ATVP apparatus equipped with a Shimadzu SPD-10AVPUV detector and with a Purospher®_{STAR RP-18e column (25010 mm, 5 mm).}

()-D-Arabinose

was purchased from Aldrich.

Animal Material The soft coral L. crassum was collected by hand using scuba at the coast of Kenting, in January, 2004, at a depth of 10 m, and was stored in a freezer until extraction. A voucher specimen was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University (specimen no. 200401-9).

Extraction and Isolation The soft coral L. crassum (1.1 kg fresh wt.) was collected and freeze-dried. The freeze-dried material was minced and extracted exhaustively with EtOH (32 l). The organic extract was concen-trated to an aqueous suspension and was further partitioned between EtOAc and water. The EtOAc extract (23 g) was fractionated by open column chro-matography on silica gel using n-hexane–EtOAc and EtOAc–MeOH mix-tures of increasing polarity. A fraction eluted with EtOAc–MeOH (4 : 1) was subjected to separation by a Sephadex LH-20 column (290 cm) using ace-tone and followed by reverse phase HPLC (aceace-tone–H2O, 1 : 4) to afford

compounds 1—3 (24.6, 14.0, 13.2 mg, respectively).

(2R)-1-Hydroxy-3-hexadecyloxy-propyl-b -D-arabinopyranoside (1):

Amorphous white solid; [a]D

22_{26° (c2.46, MeOH); IR (KBr) n} max3391,

2919, 2851, 1142, 1075, 1007 cm1; for 1_{H- and} 13_{C-NMR data, see Tables 1}

and 2; ESI-MS m/z 471 [M
Na]
, HR-ESI-MS m/z 471.3296 [M
Na]
(Calcd for C24H48O7Na, 471.3298).

(2R)-1-Hydroxy-3-octadecyloxy-propyl-b -D-arabinopyranoside (2): Amorphous white solid; [a]D

22_{25° (c1.40, MeOH); IR (KBr) n} max3391,

2921, 2853, 1142, 1075, 1007 cm1; for 1_{H- and} 13_{C-NMR data, see Tables 1}

and 2; ESI-MS m/z 499 [M
Na]
, HR-ESI-MS m/z 499.3609 [M
Na]
(Calcd for C26H52O7Na, 499.3611).

(2R)-1-Acetoxy-3-hexadecyloxy-propyl-b -D-arabinopyranoside (3):

1722 Vol. 55, No. 12

Chart 2. Chemical Conversions from 3 to 1d and Chemical Shift Differences of MTPA Esters 3a and 3b Table 2. Cytotoxicity Data of Compounds 1—3

Compound

Cell lines IC50(mg/ml)

Hep G2 Hep 3B MDA-MB-231 Ca9-22

1 10.8 12.7 14.5 12.0

2 9.2 9.7 11.1 15.0

3 11.3 9.7 12.4 9.5

Amorphous white solid; [a]D

22_{20° (c1.32, MeOH); IR (KBr) n} max3418,

1741, 1238, 1142, 1079, 1007 cm1; for 1_{H- and} 13_{C-NMR data, see Tables 1}

and 2; ESI-MS m/z 513 [M
Na]
, HR-ESI-MS m/z 513.3404 [M
Na]
(Calcd for C26H50O8Na, 513.3403).

Acid Hydrolysis of 1 To a mixture of 1 (5.0 mg) and MeOH (0.3 ml) was added 0.3 ml of 1NHCl(aq). The reaction was carried out under refluxing

for 16 h. The reaction mixture was then cooled to room temperature and ex-tracted with n-hexane. Hexane layer was concentrated and chromatographed over silica gel using n-hexane–CH2Cl2(1 : 2) as eluent to obtain chimyl

alco-hol (2.5 mg, 68%), [a]D

22_{2° (c0.25, CHCl}

3). The MeOH–water layer,

containing methyl arabinoside (mixture of a- and b-anomers), was evapo-rated to dryness and followed by treated with 1N HCl at 190 °C for 1.5 h. Then, the aqueous layer was concentrated under reduce pressure and the residue was chromatographied over silica gel using CHCl3

–ace-tone–MeOH–H2O (8 : 2 : 3 : 1) as eluent to yield ()-D-arabinose (0.4 mg,

23%), [a]D

22 _{110° (c0.040, H}

2O), which was confirmed by

co-TLC analysis with authentic sample (CHCl3–acetone–MeOH–H2O,

6 : 2 : 4 : 1, Rf 0.47).

Preparation of Bis-(R)-MTPA Ester (1b) To a solution of chimyl alco-hol (1.0 mg) in dry pyridine (0.4 ml) was added (S)-MTPA chloride (20ml), and the solution was allowed to stand overnight at room temperature for 16 h. The reaction was quenched by the addition of 1.0 ml of water, followed by extraction with CH2Cl2(31 ml). The CH2Cl2-soluble layers were

com-bined, dried over anhydrous MgSO4and evaporated. The residue was

sub-jected to short silica-gel column using n-hexane–CH2Cl2(1 : 1) to yield

bis-(R)-MTPA ester (1b) (0.2 mg, 8%) in trace amount and the C-1 substituted mono-(R)-MTPA ester was the major product (1.5 mg, 89%). 1_H-NMR

(CDCl3) of 1b: dH7.33—7.49 (10H, m, 2Ph), 5.43 (1H, m, H-2), 4.73 (1H, dd, J12.3, 2.8 Hz, H-1a), 4.42 (1H, dd, J12.3, 6.3 Hz, H-1b), 3.49 (2H, m, H-3), 3.48 (3H, s, OCH3), 3.40 (3H, s, OCH3), 3.30 (2H, t, J6.6 Hz, H2-1), 1.26 (28H, br s, H2-2—H2-15), 0.88 t (3H, t, J6.6 Hz, H-16); 1_{H-NMR (CDCl} 3) of mono-(R)-MTPA ester: dH7.40—7.55 (5 H, m, Ph), 4.38 (2H, d, J5.0 Hz, H2-1), 4.03 (1H, m, H-2), 3.57 (3H, s, OCH3), 3.36—3.49 (4H, m, H2-3, H2-1), 1.26 (28H, br s, H2-2—H2-15), 0.88 t (3H, t, J6.6 Hz, H-16).

Preparation of MTPA Esters 1c, 1d, 3a and 3b To a solution of 1 (2 mg) in dry acetone (0.3 ml) was added 1,2-dimethoxypropane (0.3 ml) and catalytic amount of CF3COOH, and the reaction mixture was stirred at room

temperature for 16 h. The mixture was concentrated and divided into two equal portions. One portion was converted to (S)-MTPA ester (1c) (1.5 mg, 73%) with (R)-MTPA chloride (20ml) and the other was converted to (R)-MTPA ester (1d) (2.0 mg, 97%) with (S)-(R)-MTPA chloride (20ml) according to the procedure of the preparation of bis-(R)-MTPA ester (1b). 1_H-NMR

(CDCl3) of 1c: dH7.36—7.56 (10H, m, 2Ph), 5.24 (1H, d, J3.3 Hz, H-1), 5.09 (1H, dd, J7.9, 3.3 Hz, H-2), 4.59 (1H, dd, J12.0, 3.3 Hz, H-1a), 4.25 (1H, dd, J12.0, 6.3 Hz, H-1b), 4.07 (1H, m, H-2), 3.99 (1H, dd, J7.9, 5.4 Hz, H-3), 3.91 (1H, dd, J13.7, 2.5 Hz, H-5a), 3.82 (1H, d, J13.7 Hz, H-5b), 3.81 (1H, m, H-4), 3.55 (3H, s, OCH3), 3.51 (3H, s, OCH3), 3.38 (2H, m H-3), 3.30 (2H, t, J6.6 Hz, H-1), 1.52 (3H, s, CH3), 1.47 (1H, m, H-2a) 1.30 (3H, s, CH3), 1.26 (1H, br s, H-2b), 1.26 (26H, br s, H2-3—H2-15), 0.88 t (3H, t, J6.6 Hz, H-16); 1H-NMR (CDCl3) of 1d: dH7.39—7.58 (10H, m, 2Ph), 5.09 (1H, dd, J7.9, 3.3 Hz, H-2), 5.07 (1H, br s, H-1), 4.54 (1H, dd, J11.7, 2.9 Hz, H-1a), 4.22 (1H, dd, J11.7, 5.9 Hz, H-1b), 4.19 (1H, dd, J7.9, 5.4 Hz, H-3), 4.01 (1H, dd, J5.4, 2.5 Hz, H-4), 3.89 (1H, dd, J13.4, 2.5 Hz, H-5a), 3.85 (1H, m, H-2), 3.75 (1H, d, J13.4 Hz, H-5b), 3.58 (3H, s, OCH3), 3.47 (3H, s, OCH3), 3.25 (2H, br t, J6.3 Hz, H-1), 3.13 (2H, br d, J6.0 Hz, H-3), 1.54 (3H, s, CH3), 1.47 (1H, m, H-2a) 1.34 (3H, s, CH3), 1.26 (1H, br s, H-2b), 1.26

(26H, br s, H2-3—H2-15), 0.88 t (3H, t, J6.6 Hz, H-16). The same

pro-cedure was treated with 3 (2.5 mg) to obtain the (S)-MTPA ester (3a) (1.6 mg, 84%) and (R)-MTPA ester (3b) (1.9 mg, 100%). Selective 1_H-NMR

(CDCl3) of 3a: dH7.40—7.59 (5H, m, Ph), 5.28 (1H, d, J3.4 Hz, H-1),

5.18 (1H, dd, J7.5, 3.4 Hz, H-2), 4.30 (1H, m, H-3), 4.00 (1H, m, H-2), 3.55 (3H, s, OCH3), 3.42 (2H, m, H-3), 3.34 (2H, t, J6.6 Hz, H-1), 1.55—

1.58 (3H, overlapped with H2O, CH3), 1.47 (1H, m, H-2a), 1.34 (3H, s,

CH3), 1.26 (1H, br s, H-2b), 1.26 (26H, br s, H2-3—H2-15), 0.88 t (3H, t, J6.6 Hz, H-16); Selective 1_{H-NMR (CDCl}

3) of 3b: dH7.39—7.60 (5H,

m, Ph), 5.15 (1H, dd, J7.5, 2.2 Hz, H-2), 5.13 (1H, br s, H-1), 4.33 (1H, m, H-3), 3.85 (1H, m, H-2), 3.59 (3H, s, OCH3), 3.17 (2H, m, H-3), 3.27

(2H, t, J6.6 Hz, H-1), 2.03 (3H, s, OAc), 1.55—1.58 (3H, overlapped with H2O, CH3), 1.47 (1H, m, H-2a), 1.37 (3H, s, CH3), 1.26 (1H, br s, H-2b),

1.26 (26H, br s, H2-3—H2-15), 0.88 t (3H, t, J6.6 Hz, H-16).

Conversion of 3b to 1d To a mixture of 3b (1.0 mg) and MeOH (0.3 ml) was added 0.3 ml of 1NHCl(aq). The reaction was stirred at 50 °C for

4 h. Water (1 ml) was added to the mixture and then extracted with n-hexane (32 ml). Hexane layer was concentrated and then dried in vacuum. The residue was redissolved in acetone (0.3 ml), and then subsequently added 1,2-dimethoxypropane (0.3 ml) and catalytic amount of CF3COOH. The

re-action mixture was stirred at room temperature for 16 h and then concen-trated to dryness. The residue was redissolved in dry pyridine (0.4 ml) and added (S)-MTPA chloride (20ml). The reaction was stirred for another 16 h. The reaction mixture was concentrated and chromatographed over silica gel using n-hexane–EtOAc (8 : 1) as eluent to obtain a diester (0.9 mg, 73%), of which the 1_{H-NMR data was consistent with that of 1d.}

Cytotoxicity Testing Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicity assays were performed using the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide] colorimetric method.18,19)

Acknowledgments Financial support was provided by Ministry of Edu-cation (C030313) and National Science Council of Taiwan (NSC 95-2323-B-110-002) awarded to J.-H. S.

References

1) Wu S.-L., Sung P.-J., Su J.-H., Sheu J.-H., J. Nat. Prod., 66, 1252— 1256 (2003).

2) Sheu J.-H., Huang L.-F., Chen S.-P., Yang Y.-L., Sung P.-J., Wang G.-H., Su J.-G.-H., Chao C.-G.-H., Hu W.-P., Wang J.-J., J. Nat. Prod., 66, 917— 921 (2003).

3) Sheu J.-H., Wang G.-H., Duh C.-Y., Soong K., J. Nat. Prod., 66, 662— 666 (2003).

4) Ahmed A. F., Su J.-H., Shiue R.-T., Pan X.-J., Dai C.-F., Kuo Y.-H., Sheu J.-H., J. Nat. Prod., 67, 592—597 (2004).

5) Ahmed A. F., Su J.-H., Kuo Y.-H., Sheu J.-H., J. Nat. Prod., 67, 2079—2082 (2004).

6) Chao C.-H., Huang L.-F., Yang Y.-L., Su J.-H., Wang G.-H., Chiang M. Y., Wu Y.-C., Dai C.-F., Sheu J.-H., J. Nat. Prod., 68, 880—885 (2005).

7) Wang S. K., Duh C. Y., Wang Y., Cheng M. C., Soong K., Fang L. S.,

J. Nat. Prod., 55, 1430—1435 (1992).

8) Morris L, A., Christie E. M., Jaspars M., van Ofwegen L. P., J. Nat.

Prod., 61, 538—541 (1998).

9) Duh C. Y., Wang S. K., Huang B. T., Dai C. F., J. Nat. Prod., 63, 884—885 (2000).

10) Wang L. T., Wang S. K., Soong K., Duh C. Y., Chem. Pharm. Bull.,

55, 766—770 (2007).

11) Rashid M. A., Gustafson K. R., Boyd M. R., J. Nat. Prod., 63, 531— 533 (2000).

12) Vanisree M., Subbaraju G. V., Asian J. Chem., 14, 957—960 (2002). 13) Myers B. L., Crews P., J. Org. Chem., 48, 3583—3585 (1983). 14) Koren-Goldshlager G., Klein P., Rudi A., Benayahu Y., Schleyer M.,

Kashman Y., J. Nat. Prod., 59, 262—266 (1996).

15) Do M. N., Erickson K. L., Tetrahedron Lett., 24, 5699—5702 (1983). 16) Ohtani I., Kusumi T., Kashman Y., Kakisawa H., J. Am. Chem. Soc.,

113, 4092—4096 (1991).

17) Randazzo A., Bifulco G., Giannini C., Bucci M., Debitus C., Cirino G., Gomez-Paloma L., J. Am. Chem. Soc., 123, 10870—10876 (2001). 18) Alley M. C., Scudiero D. A., Monks A., Hursey M. L., Czerwinski M.

J., Fine D. L., Abbott B. J., Mayo J. G., Shoemaker R. H., Boyd M. R.,

Cancer Res., 48, 589—601 (1988).

19) Scudiero D. A., Shoemaker R. H., Paull K. D., Monks A., Tierney S., Nofziger T. H., Currens M. J., Seniff D., Boyd M. R., Cancer Res., 48, 4827—4833 (1988).