Kaohsiung Medical University Institutional Repository:Item 310902000/6503

We have continued to study vibsane-type diterpenes

occur-ring in Viburnum awabuki

1)

and V. odoratissimum

2)

from a

phytochemical point of view. More than 30 kinds of

vibsane-type diterpenes have been found so far, and they can be

suc-cessfully categorized into three sub-classes.

3,4)

As part of our

studies on biologically active compounds in Viburnum

species, we have examined chemical components in the

leaves of V. luzonicum, an evergreen shrub widely distributed

in Taiwan. Since one paper reported that a 70% acetone

ex-tract of this plant inhibited KB cell growth,

5)

our attention

was directed to exploration of the cytotoxic principles,

result-ing in the isolation of four new iridoid aldehydes 1—4

named luzonials A and B, luzonidials A and B. In this paper,

we report the structure elucidation of these new compounds

and their inhibitory activity against HeLa S3 cancer cells.

The methanol extract of the leaves of V. luzonicum was

fractionated on a silica gel column into fractions 1—17.

Fractions 3 and 10 were purified by a combination of silica

gel chromatography and preparative HPLC, which yielded

the four new iridoids 1—4.

Luzonial A (1) had a [M]

ion peak at m/z 360.1204 in

high-resolution (HR)-EI-MS, corresponding to the molecular

formula C

19

H

20

O

7

and its IR spectrum displayed absorptions

due to the presence of hydroxyl (3338 cm

1

) and carbonyl

(1683 cm

1

) groups, and an aromatic (1587, 1515 cm

1

)

moiety. The UV (313 nm) and NMR data of 1 (Table 1)

showed the presence of an (E)-p-coumaroyl group [d 6.80

(2H, d, J

8.8 Hz), 7.47 (2H, d, J8.8 Hz), 6.37 (1H, d,

J

15.9 Hz), 7.64 (1H, d, J15.9 Hz); d 168.7], which was

supported by the observation of prominent fragment peaks at

m/z 147 and 164 in the EI-MS. The remaining NMR spectral

data (Table 1) of 1 indicated the presence of an aldehyde [d

9.58 (s); d 196.0] and an exo-methylene [d 6.24 (s), 6.55 (d,

J

1.1 Hz); d 134.8, 153.4], which formed a conjugated

sys-tem on the basis of the correlation of the exo-methylene

sig-nals with the aldehyde carbonyl in the

1

_H-detetced

heteronu-clear multi-bond correlation (HMBC), as well as of an acetal

moiety [d 5.34 (d, J

0.8 Hz); d 104.6] and an isolated

oxymethylene [d 3.93 (d, J

9.5 Hz), 4.22 (d, J9.5 Hz); d

73.5]. The routine analyses of

1

_H–

1

_{H shift correlation}

spec-troscopy (COSY) and

1

H-detected heteronuclear correlation

through multiple quantum coherence (HMQC) indicated the

presence of a partial structure (bold line), as shown in Fig. 2.

This partial structure was found to contain the conjugated

exo-methylene aldehyde unit at the C-5 position and the

p-coumaroyl group at the C-7 position according to the HMBC

correlations of H-5 and H-7 resonating at d 2.99 and 5.22

with the exo-methylene carbons C-4 and C-1 and the ester

carbonyl of a p-coumaroyl group, respectively. In the HMBC

as summarized in Fig. 2, the H-10 oxy-methylene signals

showed correlations with the acetal carbon C-1, whereas the

H-1 acetal and H-9 signals had cross peaks to the C-8

quater-nary oxygen-bearing carbon at d 90.7, which further

corre-lated with H-7. These spectral data indicated that C-8 was

connected to the p-coumaroyl-bearing oxycarbon C-7 and the

methine 9 was involved in the partial unit, and also that

C-10 was bonded to C-8 as well as to C-1 through an ether

bond, resulting in the formation of an iridoid structure fused

with two five-membered rings, as shown in Fig. 1. In

addi-tion, stirring a methanol solution of 1 in the presence of

Am-berlyst 15E gave 1a, which confirmed the presence of a

cyclic acetal ring in 1. The relative stereochemistry of 1 was

elucidated on the basis of the following nuclear Overhauser

and exchange spectroscopy (NOESY) correlations as shown

in Fig. 2: H-5/H-9, H-5/H-6b, H-7/H-10a, and H-10b/H-9,

which can account for a p-coumaroyl group at C-7 and a

con-jugated aldehyde moiety at C-5 taking a b- and an

a-config-uration, respectively. However, the configuration on the C-1

acetal carbon was unable to be determined by the NOESY

because H-5 observes NOE for H-1a or H-1b. The most

sta-ble conformation for 1 obtained by a MM2 calculation using

Marcromodel

®

_{indicates that a dihedral angle between H-5}

and H-1a, and H-5 and H-1b is 82° and 39°, respectively.

Considering the small J value (0.8 Hz) observed for H-1, the

OH located at C-1 should take a b configuration.

Accord-ingly, luzonial A (1) was represented as 1.

Luzonial B (2) was assigned the same molecular formula

C

19

H

20

O

7

as 1, obtained from HR-EI-MS at m/z 360 [M]

,

and exhibited physical and NMR data (Table 1) very similar

to those of compound 1 except for the NMR data assignable

to a p-coumaroyl group. Namely, a small J value (12.6 Hz)

was observed for two vicinal olefinic protons, indicating the

presence of a (Z)-p-coumaroyl group. It was also evident

from the HMBC correlation of the H-7 signal at d 5.19 with

the ester carbonyl at d

_C

167.8 that this p-coumaroyl group

was located at the C-7 position. Other NMR data including

HMBC and NOESY were consistent with those of 1. Thus

the structure of luzonial B (2) was determined to be

7-O-(Z)-January 2005 Notes Chem. Pharm. Bull. 53(1) 125—127 (2005) 125

∗ To whom correspondence should be addressed. e-mail: [email protected] © 2005 Pharmaceutical Society of Japan

Cytotoxic Iridoid Aldehydes from Taiwanese Viburnum luzonicum

Yoshiyasu F

UKUYAMA

,*

, a

Yuka M

INOSHIMA

,

a

Yoshiko K

ISHIMOTO

,

a

Ih-Sheng C

HEN

,

b

Hironobu T

AKAHASHI

,

a

and Tomoyuki E

SUMIa

a_{Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University; Yamashiro-cho,}

Tokushima 770–8514, Japan: and b_{Department of Pharmacy, Kaohsiung Medical University; Kaohsiung, 807, Taiwan.} Received August 20, 2004; accepted October 4, 2004

Four new iridoid aldehydes bearing (E)- or (Z)-p-coumaroyl group, luzonial A (1), luzonial B (2), luzonidial A (3), and luzonidial B (4), were isolated from a methanol extract of the dried leaves of Viburnum luzonicum col-lected in Kaoshiung, Taiwan and their structures were elucidated by analysis of spectroscopic data. Compounds 1—3 exhibited moderate inhibitory activity against HeLa S3 cancer cells.

p-coumaroyl derivative of 1.

Luzonidial A (3) had a molecular formula C

₁₉

H

₁₈

O

₆

,

de-termined by HR-EI-MS at m/z 342 [M]

and its IR spectrum

displayed absorptions due to a hydroxyl group at 3368 cm

1

,

a conjugated carbonyl group at 1680 cm

1

, and an aromatic

moiety at 1587 and 1515 cm

1

. The NMR spectral data of 3

showed the presence of two aldehyde groups [d 9.85 (s, H-3),

193.5 (C-3); d 10.1 (s, H-1), 188.3 (C-1)], an exo-methylene

[d 6.03 (s, H-11), 6.15 (d, J

0.8 Hz, H-11); d 134.0 (C-11),

150.3 (C-4)], an isolated oxymethylene [d

4.38 (d,

J

15.1 Hz) and 4.85 (d, J15.1 Hz); d 56.3 (C-10)], and a

p-coumaroyl group with an E-geometry (J

15.9 Hz). In

ad-dition to these structural fragments, one partial structure,

–C

₍₅₎

H–C

₍₆₎

H

₂

–C

₍₇₎

H–O–, was obtained from analysis of

1

H–

1

H COSY and HMQC. The H-5 signal at d 4.26 showed

not only a long-range coupling (0.8 Hz) to one of the H-11

exo-methylene protons but also HMBC correlations with

C-3, C-4, C-11, and two quaternary sp

2

_{carbons C-8 (d 158.2)}

and C-9 (d 141.5), indicating that C-5 connects to both C-9

and the exo-methylene C-4 bearing an aldehyde group.

Ac-cording to the additional HMBC, the H-7 signal correlated

with the C-8 and C-9 resonances, which further had

cross-peaks with the H-10 oxy-methylene and the remaining

alde-hyde H-1 signal, indicating that both 7 and 10 link to

C-8 and the other aldehyde function bonds to C-9. The

p-coumaroyl group was confirmed to be located at the C-7

po-sition on the basis of a HMBC correlation between the H-7

signal resonated at d 6.20 and its ester carbonyl at d 167.8.

Thus, 3 is comprised of a basic iridoid skeleton as shown in

Fig. 1. A trans relationship between H-5 and H-7 was

eluci-dated on the basis of the following NOESY: H-5/H-6b and

H-6a/H-7. The structure of luzonidial A was therefore

repre-sented as 3.

Luzonidial B (4) was found to have the same molecular

formula C

19

H

18

O

6

as that of 3, determined by HR-EI-MS at

m/z 342 [M]

. The spectral data of 4 were similar to those of

3 except for a big difference in the chemical shifts and J

val-ues corresponding to the coumaroyl H-1

 and H-2. A small

J

1,2

value (12.6 Hz) indicated the double bond geometry for

this p-coumaroyl to be Z. The NOESY experiments indicated

that the relative stereochemistry of 4 was the same as that of

3. Thus the structure of 4 was assigned as a Z variant of a

p-coumaroyl group located at the C-7 position in luzonidial A.

All the new iridoids isolated in this study are structurally

analogous to those found in many other species of

Viburnum.

6)

They are characterized by the presence of (E)- or

(Z)-p-coumaroyl group at position 7. Although these iridioids

bearing (E)- or (Z)-p-coumaroyl group are readily isolated in

a pure form, they gradually interconvert into a mixture of E

and Z at room temperature. In a cytotoxicity assay with the

HeLa S3 (human epithelial cancer) cell line, iridoid

gluco-sides (1) and (2) exhibited moderate inhibitory activity at

IC

₅₀

values of 1.9—3.5

m

M

as summarized in Table 2.

Experimental

General Procedure Optical rotations were measured on a JASCO DIP-1000 digital polarimeter. IR spectra were measured on a JASCO FT-IR 5300

126 Vol. 53, No. 1

Fig. 1. New Iridoids Isolated from V. luzonicum

Fig. 2. Representative HMBC and NOESY for 1 Table 1. 13_{C-NMR (150 MHz) and} 1_{H-NMR (600 MHz) Spectral Data of}

Compounds 1 and 2 in CD3OD 1 2 Position dC dH dC dH 1 104.6 5.34 d (0.8) 104.7 5.27 d (0.8) 3 196.0 9.58 s 196.0 9.57 s 4 153.4 153.5 5 39.5 2.99 dddd 39.3 2.89 dddd (7.5, 7.1, 6.9, 1.1) (7.1, 7.1, 6.6, 1.1) 6 36.6 1.99 ddd 35.9 1.92 ddd (13.3, 7.1, 5.9) (13.5, 7.1, 6.4) 2.18 ddd 2.15 ddd (13.3, 7.5, 5.8) (13.5, 7.1, 6.1) 7 82.0 5.22 dd 81.7 5.19 dd (5.9, 5.8) (6.4, 6.1) 8 90.7 90.3 9 64.4 2.31 dd 64.0 2.27 dd (6.9, 0.8) (6.6, 0.8) 10 73.5 3.93 d (9.5) 73.6 3.84 d (9.8) 4.22 d (9.5) 4.07 d (9.8) 11 134.8 6.24 s 134.7 6.23 s 6.55 d (1.1) 6.54 d (1.1) Coumaroyl 1 168.7 167.8 2 114.9 6.37 d (15.9) 116.5 5.81 d (12.6) 3 147.1 7.64 d (15.9) 145.8 6.91 d (12.6) 4 127.1 127.7 5, 9 131.3 7.47 d (8.8) 133.6 7.61 d (8.4) 6, 8 116.9 6.80 d (8.8) 115.9 6.75 d (8.4) 7 161.5 160.2

All assignments were made by extensive analyses of 1D and 2D NMR (COSY, DEPT, HMQC, and HMBC).

Table 2. Cytotoxic Activities of Compounds 1—3 against HeLa S3a) Compound IC50(mM) 1 3.50 2 1.93 3 24.5 4 NDb) Fluorouracil 5.40 Cisplatin 2.46

infrared spectrophotometer, and 1D- and 2D-NMR spectra were recorded on a Varian Unity 600 instrument. Chemical shifts are given as d (ppm) with TMS as an internal standard. MS were recorded on a JEOL AX-500 instru-ment. Column chromatography was carried out on Kieselgel 60 (70—230 mesh) or Wako gel C-300.

Plant Material The leaves of Viburnum lozonicum were collected in Kaoshiung, Taiwan in May 2002. Prof. Ih-Sheng Chen identified the plant and a voucher specimen (1714LF) has been deposited at the Institute of Pharmacognosy, TBU.

Extraction and Isolation The dried leaves of V. luzonicum (7.0 kg) were extracted with MeOH to yield 800 g of the MeOH extract. The extract (150 g) was chromatographed on a silica gel (Kieselgel 60) column eluting with a step gradient of CH2Cl2(100%), CH2Cl2–EtOAc (9 : 1), CH2Cl2–

EtOAc (3 : 2), CH2Cl2–EtOAc (2 : 3), EtOAc (100%), EtOAc–MeOH (9 : 1),

and EtOAc–MeOH (4 : 1) to give seventeen fractions (fr. 1—17).

Fraction 10 (7.7 g) was first subjected to silica gel (Wako C-300) chro-matography eluting with CHCl3–EtOAc (1 : 1) to give fractions 18—27.

Fraction 25 (195 mg) was separated by silica gel chromatography with CHCl3–MeOH–H2O (8 : 2 : 0.2), followed by reversed-phase HPLC

[Cos-mosil 5C18-AR-II, f 10250 mm; H2O–MeOH (11 : 9), 2.0 ml/min] to give

compounds 1 (6.7 mg) and 2 (3.1 mg). MeOH soluble portion (1.6 g) of frac-tion 3 (3.7 g) was separated by column chromatography over silica gel elut-ing with CHCl3–EtOAc (1 : 1) to nine fractions. The eighth fraction (71 mg)

was purified by HPLC (Cosmosil 5C18-AR-II, f 10250 mm) using MeOH–H2O (11 : 9, 2.5 ml/min) to give compounds 3 (5.4 mg) and 4

(2.2 mg).

Luzonial A (1): Yellow oil; [a]D217.1° (c1.04, MeOH); IR nmax(film)

cm1: 3338 (OH), 1683 (conj. CO), 1587, 1515 (aroma.); UV lmax(EtOH)

nm: 313 (e 11100), 297 (e 9000), 210 (9300); EI-MS m/z (rel. int.): 360 [M]
(2), 164 (100), 147 (89); HR-EI-MS m/z: 360.1204 (M
Calcd for C19H20O7: 360.1209); 1H- and 13C-NMR, see Table 1.

Luzonial B (2): Yellow oil; [a]D

21_{1.9° (c1.17, MeOH). IR n} max(film)

cm1: 3364 (OH), 1688 (CO), 1586, 1514 (aroma.); UV lmax(EtOH) nm:

315 (e 10300), 300 (e 8600), 225 (e 9200), 212 (e 10900); EI-MS m/z (rel. int.): 360 [M]
(1), 164 (100), 147 (92); HR-EI-MS m/z: 360.1207 (M
Calcd for C19H20O7: 360.1209);

1_{H- and} 13_{C-NMR, see Table 1.}

Luzonidial A (3): Yellow oil; [a]D

21_{183.4° (c0.94, CHCl}

3); IR nmax

(film) cm1: 3368 (OH), 1680 (conj. CO), 1587, 1515 (aroma.); UV lmax

(EtOH) nm: 316 (e 16000), 228 (e 14300); 1_{H-NMR (600 MHz, CD} 3OD) d: 2.18 (1H, ddd, J14.1, 7.7, 3.8 Hz, H-6), 2.36 (1H, ddd, J14.1, 9.1, 5.1 Hz, H-6), 4.26 (1H, ddd, J9.1, 3.8, 0.8 Hz, H-5), 4.38 (1H, d, J15.1 Hz, H-10), 4.85 (1H, d, J15.1 Hz, H-10), 6.03 (1H, s, H-11), 6.15 (1H, d, J0.8 Hz, H-11), 6.20 (1H, dd, J7.7, 5.1 Hz, H-7), 6.30 (1H, d, J15.9 Hz, 2), 6.86 (2H, d, J8.5 Hz, 6, 8), 7.47 (2H, J8.5 Hz, H-5, 9), 7.69 (1H, J15.9 Hz, H-3), 9.85 (1H, s, H-3), 10.1 (1H, s, H-1); 13_{C-NMR (150 MHz, CD} 3OD) d: 36.6 (C-6), 40.9 (C-2), 56.3 (C-10), 78.6 (C-7), 114.1 (C-2), 116.0 (C-6, 8), 126.8 (C-4), 130.3 (C-5, 9), 134.0 (C-11), 141.5 (C-9), 146.3 (C-3), 150.3 ( C-4), 158.1 (C-7), 158.2 (C-8), 167.8 (C-1), 188.3 (C-1), 193.5 (C-3); EI-MS m/z (rel. int): 342 [M]
, 147 (100); HR-EI-MS m/z: 342.1118 (M
Calcd for C19H18O6: 342.1103).

Luzonidial B (4): Yellow oil; [a]D2132.4° (c0.54, CHCl3); IR (film)

nmax (film) cm1: 3400 (OH), 1682 (conj. CO), 1604, 1514; 1_H-NMR (600 MHz, CD3OD) d: 2.14 (1H, ddd, J14.1, 7.6, 4.3 Hz, H-6), 2.24 (1H, ddd, J14.1, 9.1, 4.9 Hz, H-6), 4.17 (1H, ddd, J9.1, 4.3, 0.8 Hz, H-5), 4.36 (1H, d, J15.1 Hz, H-10), 4.78 (1H, d, J15.1 Hz, H-10), 5.84 (1H, d, J12.6 Hz, H-2), 6.03 (1H, s, H-11), 6.09 (1H, dd, J7.6, 4.9 Hz, H-7), 6.13 (1H, d, J0.8 Hz, H-11), 6.83 (2H, d, J8.5 Hz, H-6, 8), 6.96 (1H, d, J12.6 Hz, H-3), 7.62 (2H, d, J8.5 Hz, H-5, 9), 9.55 (1H, s, H-3), 10.1 (1H, s, H-1): 13_{C-NMR (150 MHz, CD} 3OD) d: 36.7 (C-6), 41.0 (C-5), 56.6 (C-10), 78.9 (C-7), 115.1 (C-6, 8), 116.0 2), 127.2 4), 131.1 (C-11), 132.5 (C-5, 9), 141.5 9, C-1), 145.7 3), 150.2 4), 157.0 (C-7), 158.0 (C-8), 188.4 (C-1), 193.5 (C-3); EI-MS m/z (rel. int.): 342 [M]
, 147 (100); HR-EI-MS m/z: 342.1118 (M
Calcd for C19H18O6: 342.1103).

Methylation of 1 A mixture of 1 (2 mg) and Amberlyst 15E (2 pieces) in MeOH (1 ml) was strirred for 12 h at room temperature. After the catalyst was filtered, the filtrate was condensed in vacuo to give 1a (2.1 mg) as yel-low oil. [a]D

21_{23.3° (c0.62, MeOH); IR n}

max(film) cm1: 3350 (OH),

1689 (CO), 1586, 1515 (aroma.); UV lmax nm (e): 315 (14800), 298

(11800), 215 (14400); 1_{H-NMR (600 MHz, CDCl} 3) d: 2.12 (1H, ddd, J13.5, 7.1, 4.4 Hz, H-6), 2.16 (1H, ddd, J13.5, 9.1, 8.7 Hz, H-6), 2.50 (1H, d, J4.7 Hz, H-9), 3.06 (1H, dddd, J8.7, 4.7, 4.4, 1.1 Hz, H-5), 3.37 (3H, s, OMe), 3.70 (1H, s, 8-OH), 3.84 (1H, d, J9.9 Hz, H-10), 4.22 (1H, d, J9.9 Hz, H-10), 4.92 (1H, s, H-1), 5.20 (1H, dd, J9.1, 7.1 Hz, H-7), 6.17 (1H, s, H-11), 6.33 (1H, d, J15.9 Hz, H-2), 6.59 (1H, d, J1.1 Hz, H-11), 6.85 (2H, d, J8.5 Hz, H-6, 8), 7.45 (2H, J8.5 Hz, H-5, 9), 7.66 (1H, J15.9 Hz, H-3), 9.58 (1H, s, H-3); 13_{C-NMR (150 MHz, CDCl} 3) d: 34.7 (C-6), 36.7 (C-5), 54.6 (OCH3), 60.9 (C-9), 73.4 (C-10), 82.1 (C-7), 88.7 (C-8), 110.4 (C-1), 114.4 (C-2), 115.9 (C-6, 8), 127.0 (C-4), 130.3 (C-5, 9), 134.2 11), 146.0 3), 151.8 4), 157.9 7), 168.2 (C-1), 194.0 (C-3); EI-MS m/z (rel. int.): 374 [M]
(4), 343 (2), 147 (100); HR-EI-MS m/z: 374.1357 (M
Calcd for C20H22O7: 374.1366).

Cell Proliferation Assay Cell proliferation assay was carried out using a Cell Counting Kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan). In brief, HeLa S3 cells were plated in 384-well plates at a density of 500 cells/well in minimum essential medium. Following overnight culture, drugs were added to final concentrations of 0.1, 1, 10, and 100mM, and the cells were incubated for 72 h. After 72 h, WST-1 was added according to the manufacturer's protocol and the cells were further incubated for 2 h. The plates were read at a wavelength of 450 nm using a Microplate Reader Wal-lac 1420 ARVOsx (Perkin Elmer Life and Analytical Sciences, Inc., Boston, MA, U.S.A.). The assay results are summarized in Table 2.

Acknowledgements We would like to thank Dr. Masami Tanaka and Miss Yasuko Okamoto for measuring NMR and mass spectra, and Dr. Hi-roaki Tsujimoto, Taiho Pharmaceutical Co., Ltd., for conducting a cytotoxic-ity assay. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (16510172), and the Open Research Center Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan.

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