Cytotoxic and novel skeleton compounds from the heartwood of Chamaecyparis obtusa var. formosana

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Cytotoxic and novel skeleton compounds from the heartwood

of Chamaecyparis obtusa var. formosana

Shih-Chang Chien,

a

Jang-Yang Chang,

b,c

Ching-Chuan Kuo,

b

Cheng-Chih Hsieh,

d

Ning-Sun Yang

e

and Yueh-Hsiung Kuo

a,e,f,g,*

a

Department of Chemistry, National Taiwan University, Taipei 106, Taiwan

b

Division of Cancer Research, National Health Research Institutes, Taipei 114, Taiwan

c

Division of Hematology/Oncology, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan

d_{Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei 114, Taiwan} e

Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan

f_{College of Pharmacy, China Medical University, Taichung 404, Taiwan} g

Research Center of Food and Biomolecules, College of Bioresources and Agriculture, National Taiwan University, Taipei 106, Taiwan

Received 25 September 2006; revised 28 December 2006; accepted 4 January 2007 Available online 7 January 2007

Abstract—The novel skeleton compounds, chamaecypanone C (3) and obtunorlignan A (4) were isolated from the heartwood of Chamaecyparis obtusa var. formosana. The structure of 3 was elucidated as a dimeric of monoterpene and norlignan with tricyclo-[5.2.2.02.6]undecane and the structure of 4 was elucidated as a norlignan skeleton by spectroscopic methods. Compound 3 exhibits potent cytotoxic activity against several human cancer cells with IC50 values ranging from 0.19 to 0.52 lM, whereas 4 has no

activity.

2007 Published by Elsevier Ltd.

The trunk of Chamaecyparis obtusa var. formosana Rehd. (Taiwan hinoki; Cupressaceae) is an important building material in Taiwan due to its decay-resistant characteristics. We have previously investigated the chemical components of the heartwood of this plant,

and found various monoterpenes, sesquiterpenes,

diterpenes and lignans.1–8 _{Two interesting compounds,}

bicyclo[2.2.2]octane skeleton diterpenes, obtunone (1)1

and 8,12-dihydroxydielmentha-5,9-diene-7,11-dione (2)1

were observed. The biosyntheses of 1 and 2 were proposed as the adducts from 1-hydroxymetha-3,5-dien-2-one with myrcene and itself, respectively, via bio-Diels–Alder reaction. Further detailed investigation of the same extraction from the heartwood has fur-nished two novel skeleton compounds, chamaecypanone C (3) and obtunorlignan A (4). The structural elucida-tion of these compounds are reported here.

The air-dried slices of heartwood of C. obtusa var.

for-mosana were extracted with Me2CO at room

tempera-ture. After evaporation of Me2CO, the extract was

partitioned with an EtOAc–water mixture to give an EtOAc-soluble fraction and an aqueous phase. The EtOAc-soluble fraction (680 g) was repeatedly

chro-matographed on SiO2 column and HPLC [Merck

Lichrosorb Si 60, 250· 10 mm i.d., EtOAc–CH2Cl2

(3:2)] to give chamaecypanone C (3) and obtunorlignan A (4).

Chamaecypanone C (3) was isolated as an amorphous

solid with a positive optical rotation ½½a23_D +175.7 (c

0.85, MeOH)] and UV kmax at 227, 282 and 302 nm.

The positive-ion fast atom bombardment (FAB-MS) of 3 showed a quasi-molecular ion peak at m/z 431

(M+H)+, and the molecular formula C27H26O5 of 3

was resolved using high-resolution MS measurement.9

OH O H OH O H H OH O 1 2

0040-4039/$ - see front matter 2007 Published by Elsevier Ltd. doi:10.1016/j.tetlet.2007.01.011

Keywords: Chamaecyparis obtusa var. formosana; Dimer of monoter-pene and norlignan; Norlignan.

* Corresponding author. Tel.: +886 2 33661671; fax: +886 2 23636359; e-mail:yhkuo@ntu.edu.tw

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The IR (KBr) spectrum of 3 showed absorption bands at

3379, 1740, 1701, 1616 and 1517 cm1 ascribable to

hydroxyl, carbonyl and aromatic groups. In the UV spectrum of 3, absorption maxima were observed at 227, 282 and 302 nm revealing the presence of the conjugated

system. The 1H NMR (acetone-d6) spectrum9,10 of 3

showed an isopropyl group attached to a double bond

[1380 and 1368 cm1, d 0.91 and 0.94 (3H each, d,

J = 6.8 Hz), 2.29 (1H, sep, J = 6.8 Hz)], one trisubsti-tuted double bond [d 5.83 (1H, dd, J = 6.5, 1.2 Hz, H-5)], a methyl group attached to a quaternary carbon bearing a hydroxyl group [d 1.26 (3H, s, H-15)], a methine proton located between the carbonyl and ole-ﬁnic groups (d 3.71, d, J = 1.2 Hz, H-1) and a methine proton considered to be linked between an oleﬁnic and methine group (d 3.18, 1H, dd, J = 6.5, 3.5 Hz, H-4).

These data together with13C NMR data [dC59.8 (CH,

C-1), 47.8 (CH, C-4), 124.5 (CH, C-5), 147.9 (C, C-6), 209.8 (C, C-7), 71.0 (C, C-8), 33.9 (CH, C-12), 20.7

(CH3, C-13), 21.2 (CH3, C-14) and 26.7 (CH3, C-15)]

are also similar to p-methenone moiety in compounds 1 and 2. The partial structure was further proved by

1

H–1H COSY, HMBC (seeFig. 1) and NOESY spectra

(see Fig. 2). The 1H–1H COSY experiment on 3 indi-cated the presence of partial structure in bold lines as in Figure 1. The pronounced NOESY correlation

be-tween H-5 and H3-15 established two protons are in

syn face. The diﬀerences between 3 and 2 are that H-2 is not observed, and H-1 only couples with H-5 via allylic coupling. Two p-hydroxyphenyl groups were

obviously revealed from the1H NMR data [d 6.73 and

7.35 (2H each, d, J = 8.7 Hz, H2-30, 50 and H2-20, 60,

respectively), 6.80 and 7.65 (2H each, d, J = 8.7 Hz,

H2-300, 500 and H2-200, 600, respectively) and 8.23, 8.50

(1H each, exchangeable)]. H-1 exhibited NOESY

corre-lations with H-12, 13, 14, and H-20_{, 6}0_{, as well as HMBC}

correlation with C-10_{. This evidence suggests that the}

p-hydroxyphenyl group linked at C-2 with b-orientation.

The remaining sp3 methine proton at d 3.58 (d,

J = 3.5 Hz) was assigned as H-3 due to coupling with H-4 (d 3.18, dd, J = 6.5, 3.5 Hz) and HMBC correlation

with C-8, C-9 (dC71.0, 160.1) and C-10 (dC133.1). The

pronounced NOESY correlation between 3 and

H-20_{, 6}0 _{conﬁrmed the H-3 and one of p-hydroxyphenyl}

group are in syn face. The UV, IR (1740 cm1) and1H

and 13C NMR [dC 209.0 (C-11), dH 7.58 (H-9, dC

160.1), dC 142.8 (C-10)] signals indicated the presence

of cyclopentenone with a p-hydroxyphenyl substituent at the a-position. Fifteen indices of hydrogen deﬁciency

(IHD) were determined from the13C NMR, DEPT and

HR-FAB-MS experiments. On the basis of the above evidence, the structure of 3 was elucidated as shown in the formula, a dimeric of monoterpene and norlignan

with tricyclo[5.2.2.02.6]undecane skeleton.

The absolute conﬁguration of 2 was obtained from CD measurements and determined to be 8R. Compound 2, isolated from this plant, expressed the same speciﬁc

rotation value as isolated from Callitric macleayana.11,12

Based on the same biological pathway, C-8 in chamae-cypanone C was assigned as R-conﬁguration. The biosynthesis of this novel skeleton may occur from 1-hydroxymentha-3,5-dien-2-one (5) and 1,3-bis(4-hydr-oxyphenyl)cyclopenta-1,3-diene (6, a norlignan) via endo addition of bio-Diels–Alder reaction, and then was oxidized to produce compound 3.

HO O

OH

5 6

Obtunorlignan A (4) was isolated as an amorphous solid

with negative optical rotation ½½a20_D 10.3 (c 0.35,

MeOH)] and UVmax at 225 and 255 nm. The IR

spec-trum showed aromatic (1616 and 1516 cm1) and

hydr-oxy (3387 cm1) groups. The 1H NMR spectrum13,14

revealed two p-hydroxyphenyls [d 6.75, 7.30 (2H each, d, J = 8.8 Hz) and 6.77, 7.26 (2H each, d, J = 8.8 Hz)].

Three lower shift oxygenated sp3 carbons at dC 66.2

(CH2, C-9), 69.0 (CH, C-80) and 82.6 (CH, C-70)

indi-cated the remaining 2O-atoms which existed as one ether and one alcohol group. One trisubstitutes oleﬁnic

proton at the lower ﬁeld dH 6.04 (H-8, resonanted at

dC 124.5) exhibited coupling with H2-9 with dd,

J = 2.4, 2.0 Hz and conjugating with p-hydroxyphenyl

(UVmax255 nm). H-80 expressed signal at dH 4.64 with

diaxial coupling to H-70 _(d

H 4.41, J = 6.8 Hz) and

homoallylic compiling to H-9b (dH 4.22, J = 3.6 Hz)

and to H-9a (dH4.36, J = 2.4 Hz). Except with geminal

coupling (J = 16.8 Hz), H-9b and H-9a exhibited vicinal coupling with H-8 with J = 2.0 and 2.4 Hz, respectively. From the above evidence, the gross structure of 4 can be elucidated as 2,4-bis-(4-hydroxyphenyl)-3-hydroxy-4,5-dehydrotetrahydropyranane. The further proof was conﬁrmed from its HMBC correlation. The HMBC

H-H COSY HMBC O O OH OH OH _4' 1 2 3 4 6 7 8 9 10 11 12 13 15 1' 1" 4"

Figure 1. 1H–1_{H COSY and key HMBC correlations of 3.}

O O OH OH OH 4' 1 2 3 4 6 7 8 9 10 11 12 13 15 1' 1" 4"

Figure 2. Key NOESY correlations of 3.

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spectrum (Fig. 3) indicated that the two

p-hydroxy-phenyl groups were located at C-7 and C-70_{. The}1

H–1H

COSY experiment on 4 indicated the presence of partial

structure in bold lines as inFigure 3. H-80_{and H-2}0_,

H-60_{have NOESY (}_{Fig. 4}_{) correlation to give the same side}

evidence. The coupling constant (J = 6.8 Hz) between

H-70 _{and H-8}0_{determined the quasi-diaxial correlation.}

On the basis of the above evidence, the structure of (4) was elucidated.

Chamaecypanone C (3) and obtunorlignan A (4) were evaluated for their cytotoxicity against KB (human oral epidermoid carcinoma), HONE-1 (human nasopharyn-geal carcinoma) and TSGH (human gastric carcinoma) cells. The cell viability were assessed through a

methyl-ene blue dye assay,15 _{and the results are shown in}

Table 1.

Compound 3 exhibited the higher susceptibility with

IC50ranges from 0.19 to 0.52 lM than that of the

clin-ically used anticancer drug etoposide (VP-16). However, 4 displayed no cytotoxic activity. Further studies, aim-ing to investigate a possible mechanism responsible for 3-mediated cytotoxic eﬀect among human cancer cells, are actually in progress.

Acknowledgments

The authors thank the National Science Council of the Republic of China, for ﬁnancial support.

References and notes

1. Kuo, Y. H.; Chen, C. H.; Huang, S. L. Chem. Pharm. Bull. 1998, 46, 181–183.

2. Kuo, Y. H.; Chen, C. H.; Huang, S. L. J. Nat. Prod. 1998, 61, 829–831.

3. Kuo, Y. H.; Chen, C. H. Tetrahedron Lett. 2001, 42, 2985– 2986.

4. Kuo, Y. H.; Chen, C. H.; Chiang, Y. M. Tetrahedron Lett. 2001, 42, 6731–6735.

5. Kuo, Y. H.; Chen, C. H.; Chien, S. C.; Lin, Y. L. J. Nat. Prod. 2002, 65, 25–28.

6. Kuo, Y. H.; Chen, C. H.; Lin, Y. L. Chem. Pharm. Bull. 2002, 50, 978–980.

7. Kuo, Y. H.; Chen, C. H.; Wein, Y. S. Helv. Chem. Acta 2002, 85, 2657–2663.

8. Kuo, Y. H.; Chen, C. H.; Chien, S. C.; Lin, H. C. Chem. Pharm. Bull. 2004, 52, 764–766.

9. Compound 3: amorphous solid; ½a23_D +175.7 (c 0.85, MeOH); high-resolution positive-ion FAB-MS calcd for C27H27O5 (M+H) + 431.1935, found 431.1930; UV (MeOH, log e) 227 (4.32), 282 (3.93), 302 (3.86, sh) nm; IR (KBr) 3379, 1740, 1701, 1616, 1517, 1474, 1380, 1368, 1271, 1225, 1174, 837, 757 cm1; positive-ion FAB-MS m/z 431 (M+H)+;1H NMR (acetone-d6) d 0.91, 0.94 (3H each, d, J = 6.8 Hz, H3-13, 14), 1.26 (3H, s, H3-15), 2.29 (1H, sep, J = 6.8 Hz, H-12), 3.18 (1H, dd, J = 6.5, 3.5 Hz, H-4), 3.58 (1H, d, J = 3.5 Hz, H-3), 3.71 (1H, d, J = 1.2 Hz, H-1), 4.41, 8.23, 8.50 (1H each, br s, OH-8, 40, 400), 5.83 (1H, dd, J = 6.5, 1.2 Hz, H-5), 6.73 (2H, d, J = 8.7 Hz, H-30, 50), 6.80 (2H, d, J = 8.7 Hz, H-300, 500), 7.35 (2H, d, J = 8.7 Hz, H-20_{, 6}0_{), 7.58 (1H, s, H-9), 7.65} (2H, d, J = 8.7 Hz, H-200_{, 6}00_); 13_{C NMR (acetone-d} 6) dC 20.7 13), 21.2 14), 26.7 15), 33.9 12), 47.8 4), 53.5 2), 53.6 3), 59.8 1), 71.0 8), 116.1 (C-30_{, 5}0_{, 3}00_{, 5}00_{), 123.7 (C-1}00_{), 124.5 (C-5), 129.4 (C-2}00_{, 6}00_), 129.5 (C-20_{, 6}0_{), 133.1 (C-1}0_{), 142.8 (C-10), 147.9 (C-6),} 157.0 (C-40_{), 158.8 (C-4}00_{), 160.1 (C-9), 209.0 (C-11), 209.8} (C-7).

10. The1H and13C NMR spectra of 3 were assigned with the aid of NOESY,1H–1H COSY, DEPT, HSQC and HMBC experiments.

11. Carmam, R. M.; Lambert, L. K.; Robinson, W. T.; Van Dongen, J. M. A. Aust. J. Chem. 1986, 39, 1843–1850. 12. Carmam, R. M.; Owsia, S.; Van Dongen, J. M. A. Aust. J.

Chem. 1987, 40, 333–340.

13. Compound 4: amorphous solid; ½a20_D 10.3 (c 0.35, MeOH); high-resolution EI–MS calcd for C17H16O4–

H2O (MH2O)+266.0939, found 266.0941; UV (MeOH,

log e) 225 (4.02, sh), 255 (4.01) nm; IR (KBr) 3387, 1616, 1516, 1233, 833 cm1; EI–MS (rel. int. %) m/z 266 (MH2O)+ (3), 250 (4), 163 (17), 162 (100), 133 (68), 121 (17); 1_{H NMR (methanol-d} 4) d 4.22 (1H, ddd, J = 16.8, 3.6, 2.0 Hz, H-9), 4.36 (1H, dt, J = 16.8, 2.4 Hz, H-9), 4.41 (1H, d, J = 6.8 Hz, H-70_{), 4.64 (1H,} ddd, J = 6.8, 3.6, 2.4 Hz, H-80_{), 6.04 (1H, dd, J = 2.4,} 2.0 Hz, H-8), 6.75 (2H, d, J = 8.8 Hz, H-3, 5), 6.77 (2H, d, J = 8.8 Hz, H-30_{, 5}0_{), 7.26 (2H, d, J = 8.8 Hz, H-2}0_{, 6}0_), 7.30 (2H, d, J = 8.8 Hz, H-2, 6);13C NMR (methanol-d4) dC66.2 (C-9), 69.0 (C-80), 82.6 (C-70), 116.1 (C-3, 5), 116.1 (C-30_{, 5}0_{), 124.5 (C-8), 128.7 (C-2, 6), 130.2 (C-2}0_{, 6}0_), 131.5 (C-1), 132.1 (C-10_{), 139.8 (C-7), 158.0 (C-4), 158.5} (C-40).

14. The1H and13C NMR spectra of 4 were assigned with the aid of NOESY,1H–1H COSY, DEPT, HSQC and HMBC experiments.

15. Finlay, G. J.; Baguley, B. C.; Wilson, W. R. Anal. Biochem. 1984, 139, 272–277. H-H COSY HMBC O OH HO OH 1 4 1' 4' 7 8 9 7' 8'

Figure 3. 1H–1_{H COSY and key HMBC correlations of 4.}

O OH HO OH 1 4 1' 4' 7 8 9 7' 8'

Figure 4. Key NOESY correlations of 4.

Table 1. Cytotoxicity of 3 and 4

Cell line IC50 (lM)

3 4 VP-16a

KB 0.19 ± 0.08 >50 1.10 ± 0.12 HONE-1 0.24 ± 0.09 >50 0.51 ± 0.35 TSGH 0.52 ± 0.11 >50 2.74 ± 0.94

a

Positive control substance.