New Bioactive Clerodane Diterpenoids from the Roots of Casearia membranacea

New Bioactive Clerodane Diterpenoids from the Roots of Casearia

membranacea

by Ching-Yu Chena_{), Yuan-Bin Cheng}b_{), Shun-Ying Chen}c_{), Ching-Te Chien}c_{), Yao-Haur Kuo}d_),

Jih-Hwa Guhe_{), Ashraf Taha Khalil}e_{), and Ya-Ching Shen*}e₎

a_{) Center of General Education, Tzu-Hui Institute of Technology, Kaohsiung, Taiwan, Republic of China} b_{) Department of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung}

804, Taiwan, Republic of China

c_{) Division of Silviculture, Taiwan Forestry Research Institute, Taipei, Taiwan, Republic of China} d_{) National Research Institute of Chinese Medicine, Taipei, Taiwan, Republic of China} e_{) School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan 100,}

Republic of China

(phone:þ 886-2-23123456 ext. 2226; fax: þ 886-2-23919098; e-mail: [email protected])

Bioassay-guided fractionation of the acetone extract of the roots of Casearia membranacea furnished three new clerodane diterpenes, caseamembrins S – U (1 – 3) and the known caseamembrin Q (4). Their structures were established by extensive spectroscopic analyses, especially 2D-NMR. Compounds 1 – 4 were tested against human tumor cells, including HeLa (cervical epitheloid carcinoma), DLD-1 (colon carcinoma), Daoy (medulloblastoma), and KB (oral epidermoid carcinoma) cell lines. Caseamembrin T (2) exhibited the most potent activity against Daoy cells (ED50¼ 10 ng/ml), superior to that of the

standard drug mitomycin.

Introduction. – Plants of the genus Casearia are reported to be a rich source of

clerodane diterpenes with interesting biological functions such as antimycobacterial,

antimalarial, and potent cytotoxic activities [1 – 5]. The tropical tree Casearia

membranacea Hance (Flacourtiaceae) grows wildly in the northern part of Taiwan

[6]. Despite the numerous clerodane diterpenoids isolated from the leaves and stems of

different collections of this species [7 – 11], the constituents of its roots have been

scarcely investigated so far [11]. In a preliminary investigation, a fraction from the root

extract of this plant showed promising cytotoxicity against human medulloblastoma

cancer cells, with an ED

value of 0.06 mg/ml. Bioassay-guided fractionation of the

acetone extract of the roots now led to the isolation of three new clerodane

diterpenoids, caseamembrins S – U (1 – 3), along with the known compound

case-amembrin Q (4) [12]. Herein, we report the isolation, structural elucidation, and the

biological evaluation of the isolates 1 – 4, which were tested against a panel of human

cancer cell lines.

Results and Discussion. – 1. Structure Elucidation. Compound 1 was isolated as a

colorless, amorphous, optically active solid ([a]

D

¼ þ 30.9 (MeOH)). Its molecular

formula was established as C

H

O

by HR-ESI-MS, indicating nine degrees of

unsaturation. Characteristic UV (223 nm) and IR (1731 cm

_{) absorption bands}

pointed to the presence of a conjugated diene and ester groups, respectively. The

and

_{C-NMR data of 1 (Tables 1 and 2, resp.), revealed an esterified}

clerodane-diterpene skeleton [13].

Inspection of the NMR data of 1 indicated two Ac and one propanoyl groups. In the

olefinic region, three signals with characteristic cis/trans couplings were found at d(H)

5.20 (d, J¼ 18 Hz, H

C(15)), 5.03 (d, J ¼ 10.6 Hz, H

C(15)), and 6.44 (dd, J ¼ 18,

11 Hz, HC(14)), which indicated the presence of a vinyl group. Two singlets at d(H)

4.93 and 5.06 (CH

(16)) further pointed to a 1,1-disubstituted C¼C bond, making up

the clerodane side chain at C(9) [14]. Two acetal H-atoms at d(H) 6.51 (HC(18)) and

6.53 (HC(19)) exhibited HMBC correlations to the C¼O resonances at d(C) 170.1

and 169.9 of the two AcO groups (Fig. 1). The oxymethine signal at d(H) 3.78

(HC(6)) was correlated to a quaternary C-atom at d(C) 53.6 (C(5)), and a second

oxymethine at d(H) 5.44 – 5.50 was assigned to HC(2), based on HMBC correlations

of HC(10) (d(H) 2.23 – 2.29) with C(2), C(6), and C(19). The propanoyloxy group

was positioned at C(2) due to a correlation of HC(2) to the carboxy group at d(C)

173.9. These assignments were supported by HMQC, HMBC, and COSY correlations;

the latter indicated connectivities of HC(10)/CH

(1)/HC(2)/HC(3), of HC(6)/

CH

(7)/HC(8)/Me(20), and of CH

(11)/CH

(12) (Fig. 1).

The relative configuration of 1 was determined on the basis of NOESY correlations

(Fig. 2). NOEs of HC(2)/HC(3), of HC(18)/HC(3), of HC(18)/HC(19),

and of HC(19)/HC(6) indicated b-orientation of the AcO groups at C(2), C(18),

and C(19), as well as of 6-OH. In addition, the NOE interactions between Me(17)/

HC(8) and HC(10)/Me(20) were in agreement with b-orientated Me(20) and

CH

(11) groups. Thus, from the above data, the structure of compound 1 was

established as

(2S*,5R*,6R*,8S*,18R*,19S*)-18,19-diacetoxy-18,19-epoxy-6-hydroxy-cleroda-3,13(16),14-trien-2-yl propanoate, and named caseamembrin S.

Compound 2 was isolated as a colorless, amorphous, optically active solid ([a]

¼

þ 32.6 (MeOH)). The molecular formula of 2 was determined by HR-ESI-MS as

C

H

O

, which is 14 mass units higher than in the case of 1. The

H- and

C-NMR

data of 2 (Tables 1 and 2, resp.) were similar to those of 1, indicating that 2 was a

clerodane-diterpene analogue of 1 with essentially the same substitution pattern [15].

Compound 2 had two AcO groups (d(H) 1.88, 2.04 (2s)) and a butanoyloxy group

[d(H) 2.35 (m); 1.70 (m); 0.98 (t, J¼ 6.9 Hz)], as inferred from correlations observed in

the

_H,

_{H-COSY, HMQC, and HMBC spectra. The oxymethine signal for HC(2)}

(d(H) 5.45) exhibited an HMBC correlation to the carboxy group at d(C) 173.1, and

the acetal signals HC(18) (d(H) 6.71) and HC(19) (d(H) 6.52) showed correlations

with the carboxy C-atoms at d(C) 170.1 and 169.8 ppm, respectively. Comparison of the

chemical shifts and coupling constants of 1 and 2 allowed us to identify the latter as the

butanoyl analogue of 1, and was named caseamembrin T.

Caseamembrin U (3) was isolated as a colorless, amorphous, optically active solid

([a]

D

¼ þ 19.5 (MeOH)). ItLs molecular formula was determined as C

H

O

by

HR-ESI-MS and NMR analyses (Tables 1 and 2). The

_{H- and}

_{C-NMR data of 3 were very}

similar to those of 2, differing only in the signals of one ester substituent. An AcO, a

butanoyloxy, and a MeO group were identified, with typical correlations in the

_H,

H-COSY, HMQC, and HMBC spectra. The MeO group at d(H) 3.43 showed an HMBC

correlation with the acetal resonance at d(C) 104.6. The signals of HC(2) [d(H) 5.45 –

5.50; d(C) 66.2] and HC(6) [d(H) 3.75; d(C) 73.3] of 3 were superimposable to those

of 2, indicating the same substitution pattern and configuration at these positions, which

3; d in ppm J in Hz. Assignments were confirmed by

COSY and HMBC techniques. Arbitrary atom numbering.

Position 1 2 3 1 1.88 – 1.94 (m, Hb), 1.80 – 1.86 (m, Ha) 2.02 – 2.07 (m, Hb), 1.95 – 1.99 (m, Ha) 1.90 – 1.95 (m, Hb), 1.84 – 1.88 (m, Ha) 2 5.44 – 5.50 (m) 5.45 (br. s) 5.45 – 5.50 (m) 3 5.98 (d, J¼ 3.5) 5.99 (br. d, J¼ 3.2) 6.05 (d, J¼ 3.0) 6 3.78 (dd, J¼ 11.5, 4.0) 3.67 (d, J¼ 10.2) 3.75 (dd, J¼ 11, 3.5) 7 1.69 – 1.75 (m) 1.68 – 1.75 (m, 2 H ) 1.68 – 1.73 (m, 2 H ) 8 1.67 – 1.73 (m) 1.82 – 1.87 (m) 1.66 – 1.74 (m) 10 2.23 – 2.29 (m) 2.32 – 2.38 (m) 2.27 – 2.33 (m) 11 1.50 – 1.55 (m, Hb), 1.20 – 1.23 (m, Ha) 1.23 – 1.28 (m, Hb), ,1.21 – 1.27 (m, Ha) 1.21 – 1.25 (m, Hb), 1.21 – 1.26 (m, Ha) 12 1.15 – 1.20 (m, Hb), 2.06 – 2.12 (m, Ha) 2.10 – 2.15 (m, 2 H ) 2.05 – 2.10 (m, 2 H ) 14 6.44 (dd, J¼ 11, 18) 6.43 (dd, J¼ 10.8, 17.6) 6.40 – 6.47 (m) 15 5.20 (d, J¼ 18), 5.03 (d, J¼ 10.6) 5.17 (d, J¼ 17.6), 5.04 (d, J¼ 10.8) 5.18 (d, J¼ 18), 5.03 (d, J¼ 10.5) 16 5.06, 4.93 (2s) 5.02, 4.98 (2s) 5.05 (s, 2 H ) 17 0.96 (s) 0.99 (s) 0.97 (s) 18 6.51 (s) 6.71 (s) 5.48 (s) 19 6.53 (s) 6.52 (s) 6.44 (s) 20 0.92 (d, J¼ 6.7) 0.90 (d, J¼ 6.6) 0.91 (d, J¼ 6.5) 2’ 2.43 – 2.49 (m) 2.31 – 2.38 (m) 2.30 – 2.35 (m) 3’ 1.66 – 1.74 (m) 1.67 – 1.74 (m) 1.60 – 1.68 (m) 4’ – 0.95 – 1.00 (m) 0.94 – 1.01 (m) AcO 1.89 (s) 1.88 (s) 1.90 (s) AcO 2.06 (s) 2.04 (s) – MeO – – 3.43 (s)

Table 2.13_{C-NMR ( DEPT ) Data of 1 – 3. Recorded at 125 MHz in CDCl} 3; d in ppm. Arbitrary atom numbering. Position 1 2 3 Position 1 2 3 1 26.9 (t) 26.9 (t) 27.0 (t) 16 115.5 (t) 115.4 (t) 115.3 (t) 2 66.1 (d) 66.2 (d) 66.2 (d) 17 25.4 (q) 25.3 (q) 25.4 (q) 3 121.8 (d) 121.8 (d) 121.7 (d) 18 95.6 (d) 95.6 (d) 104.6 (d) 4 145.2 (s) 145.1 (s) 146.3 (s) 19 97.8 (d) 97.9 (d) 95.4 (d) 5 53.6 (s) 53.7 (s) 53.6 (s) 20 15.7 (q) 15.7 (q) 15.7 (q) 6 73.1 (d) 73.1 (d) 73.3 (d) 1’ 173.9 (s) 173.1 (s) 173.2 (s) 7 37.1 (t) 36.5 (t) 36.5 (t) 2’ 27.9 (t) 36.5 (t) 36.2 (t) 8 36.5 (d) 37.1 (d) 37.4 (d) 3’ 9.2 (q) 18.7 (t) 18.6 (t) 9 36.2 (s) 36.5 (s) 37.8 (s) 4’ – 13.6 (q) 13.7 (q) 10 37.3 (d) 37.4 (d) 37.6 (d) AcO 21.5 (q) 169.9 (s) 21.2 (q) 169.8 (s) 21.6 (q) 169.4 (s) 11 28.0 (t) 28.1 (t) 29.7 (t) AcO 21.2 (q) 170.1 (s) 21.4 (q) 170.1 (s) – 12 23.7 (t) 23.8 (t) 23.8 (t) 18-MeO – – 56.2 (q) 13 145.1 (s) 145.3 (s) 145.3 (s) 14 140.3 (d) 140.4 (d) 140.4 (d) 15 112.4 (t) 112.3 (t) 112.3 (t)

Fig. 1. Key COSY (——) and HMBC (H! C) correlations of 1

was further confirmed by NOESY correlations. The above findings, thus, helped us to

elucidate the structure of 3 as

(2S*,5R*,6R*,8S*,18R*,19S*)-19-acetoxy-18,19-epoxy-6-hydroxy-18-methoxycleroda-3,13(16),14-trien-2-yl butanoate, which was named

case-amembrin U.

2. Biological Studies. Compounds 1 – 4 were tested for their cytotoxic activities

against human cervical epitheloid (HeLa), colon (DLD-1), medullablastoma (Daoy),

and oral epidermoid (KB) cancer cell lines. As indicated in Table 3, compounds 1 – 3

exhibited significant activity against the four tumor cell lines, whereas 4 was nearly

inactive. Among them, compound 2 showed the highest activity against all four tested

cell lines, its potency being superior to that of the standard compound minomycin,

when tested against Daoy cells (ED

¼ 10 ng/ml).

Financial support by the National Science Council of the Republic of China (94-2320-B-110-001) is gratefully acknowledged.

Experimental Part

General. Column Chromatography (CC): silica gel 60 (200 – 300 mesh; Merck). Thin-layer chromatography (TLC): silica gel GF254 (Merck). UV Spectra: Hitachi U-3210 spectrophotometer;

lmax(log e) in nm. Optical rotations: Jasco DIP-1000 spectropolarimeter. IR Spectra: Hitachi T-2001

spectrophotometer, in CH2Cl2. NMR Spectra: Varian Unity-INOVA-500 FT-NMR spectrometer; d in

ppm rel. to Me4Si, J in Hz. HR-ESI-MS: JEOL JMS-HX-110 mass spectrometer; in m/z.

Plant Material. The roots of Caseria membranacea Hance were collected in May 2003 from Taipei County, Taiwan. Identification was carried out by one of the authors (C.-T. C.). A voucher specimen (TP207 – 2) was deposited at the School of Pharmacy, National Taiwan University, Taipei, Taiwan.

Extraction and Isolation. The dried roots of C. membranacea (1.9 kg) were extracted with acetone (3 10 l). After filtration and solvent removal, the resulting residue was extracted with AcOEt/H2O 1 : 1.

The AcOEt-soluble part (23 g) was purified by CC (SiO2; hexane/AcOEt 100 : 1! 1 : 10, then AcOEt/

MeOH 50 : 1! 3 : 1) to afford 45 fractions. Fr. 23 (0.32 g) was separated by RP-HPLC (MeOH/MeCN/ H2O 75 : 5 : 20) to yield 1 (5 mg), 2 (12 mg), 3 (5 mg), and 4 (5 mg).

Caseamembrin S (¼ (2S*,5R*,6R*,8S*,18R*,19S*)-18,19-Diacetoxy-18,19-epoxy-6-hydroxycleroda-3,13(16),14-trien-2-yl Propanoate; ¼ (1R*,3S*,5S*,6aR*,7S*,8S*,10R*,10aR*)-1,3-Diacetoxy-3,5,6,6a, 7,8,9,10-octahydro-10-hydroxy-7,8-dimethyl-7-( 3-methylidenepent-4-en-1-yl)naphtho[1,8a-c]furan-5-yl Propanoate; 1). Colorless, amorphous powder. [a]25

D¼ þ 30.9 (c ¼ 0.2, MeOH). UV (MeOH): 223 (4.05).

IR (neat): 3441, 2926, 1731, 1455, 1231, 736.1_{H- and}13_{C-NMR: see Tables 1 and 2, resp. ESI-MS: 513}

([Mþ Na]þ_{). HR-ESI-MS: 513.2466 ([Mþ Na]}þ_{, C}

27H38NaOþ8; calc. 513.2464).

Caseamembrin T (¼ (2S*,5R*,6R*,8S*,18R*,19S*)-18,19-Diacetoxy-18,19-epoxy-6-hydroxycleroda-3,13(16),14-trien-2-yl Butanoate; ¼ (1R*,3S*,5S*,6aR*,7S*,8S*,10R*,10aR*)-1,3-Diacetoxy-3,5,6,6a, Table 3. Cytotoxicities of Clerodane Diterpenes against Different Human Tumor Cells. For details and

abbreviations, see Exper. Part.

Compound ED50[mg/ml]a) HeLa DLD-1 Daoy KB 1 6.74 3.34 3.50 10.41 2 0.62 0.52 0.01 2.50 3 7.92 3.28 4.72 9.39 4 14.4 19.1 5.44 21.84 Mitomycinb_{) 0.10 0.23 0.13 0.17}

7,8,9,10-octahydro-10-hydroxy-7,8-dimethyl-7-( 3-methylidenepent-4-en-1-yl)naphtho[1,8a-c]furan-5-yl Butanoate; 2). Colorless, amorphous powder. [a]25

D¼ þ 32.6 (c ¼ 0.2, MeOH). UV (MeOH): 221 (4.12).

IR (KBr): 3456, 2965, 1730, 1454, 1373, 1231, 736.1_{H- and}13_{C-NMR: see Tables 1 and 2, resp. ESI-MS:}

527 ([Mþ Na]þ_{). HR-ESI-MS: 527.2619 ([Mþ Na]}þ_{, C}

28H40NaOþ8; calc. 527.2621).

Caseamembrin U (¼ (2S*,5R*,6R*,8S*,18R*,19S*)-19-Acetoxy-18,19-epoxy-6-hydroxy-18-methoxy-cleroda-3,13(16) ,14-trien-2-yl Butanoate; ¼ (1R*,3S*,5S*,6aR*,7S*,8S*,10R*,10aR*)-1-Acetoxy-3,5, 6,6a,7,8,9,10-octahydro-10-hydroxy-3-methoxy-7,8-dimethyl-7-( 3-methylidenepent-4-en-1-yl)naphtho-[1,8a-c]furan-5-yl Butanoate; 3). Colorless, amorphous powder. [a]25

D¼ þ 19.5 (c ¼ 0.2, MeOH). UV

(MeOH): 224 (4.02). IR (KBr): 3453, 2962, 1730, 1596, 1454, 1373, 1227, 736.1_{H- and}13_{C-NMR: see}

Tables 1 and 2, resp. ESI-MS: 499 ([Mþ Na]þ_{). HR-ESI-MS: 499.2673 ([Mþ Na]}þ_{, C}

27H40NaOþ7; calc.

499.2672).

Cytotoxicity Assay. The cells (HeLa, DLD-1, Daoy, or KB) were cultured in RPMI-1640 medium under a 5% CO2atmosphere in an incubator at 378. The cytotoxicity assay was based on the binding of

Methylene Blue to fixed cell monolayers at pH 8.5, washing, and releasing the dye by lowering the pH. Samples and positive controls were prepared at concentrations of 1, 10, 40, and 100 mg/ml. After seeding 2,880 cells/well in a 96-well microplate for 3 h, 20 ml of sample or standard agent was placed in each well, which was incubated at 378 for 3 d. After removing the medium from the microplates, the cells were fixed with 10% formaldehyde in 0.9% saline for 30 min, and dyed with 1% (w/v) Methylene Blue in 0.01m borate buffer (100 ml/well) for 30 min. The 96-well plate was dipped into a 0.01m borate-buffer soln. (4 ) to remove excess dye. Then, 100 ml/well of EtOH/0.1m HCl 1 : 1 was added as a dye-eluting solvent, and the VIS absorbance was measured with a microtiter plate reader (Dynatech MR-7000) at 650 nm. The ED50value was determined, by comparison with the untreated cells, as the concentration of test sample

resulting in 50% reduction of absorbance.

REFERENCES

[1] J. A. Beutler, K. L. McCall, K. Herbert, D. L. Herald, G. R. Petit, T. Johnson, R. H. Shoemaker, M. R. Boyd, J. Nat. Prod. 2000, 63, 657.

[2] N. H. Oberlies, J. P. Burgess, H. A. Navarro, R. E. Pinos, C. R. Fairchild, R. W. Peterson, D. D. Soejarto, N. R. Farnsworth, A. D. Kinghorn, M. C. Wani, M. E. Wall, J. Nat. Prod. 2002, 65, 95. [3] C. V. S. Prakash, J. M. Hoch, D. G. I. Kingston, J. Nat. Prod. 2002, 65, 100.

[4] L. S. Espindola, J. R. Vasconcelos Jr., M. L. de Mesquita, P. Marquie´, J. E. de Paula, L. Mambu, J. M. Santana, Planta Med. 2004, 70, 1095.

[5] S. Kanokmedhakul, K. Kanokmedhakul, T. Kanarsa, M. Buayairakes, J. Nat. Prod. 2005, 68, 183. [6] H. L. Li, H. C. Lo, in RFlora of TaiwanL, Editorial Committee of the Flora of Taiwan, 2nd edn.,

Taipei, Vol. III, 1993, p. 794.

[7] Y. C. Shen, C. H. Wang, Y. B. Cheng, L. T. Wang, J. H. Guh, C. T. Chien, A. T. Khalil, J. Nat. Prod. 2004, 67, 316.

[8] Y. C. Shen, L. T. Wang, C. H. Wang, A. T. Khalil, J. H. Guh, Chem. Pharm. Bull. 2004, 52, 108. [9] Y. C. Shen, C. L. Lee, A. T. Khalil, Y. B. Cheng, C. T. Chien, Y. H. Kuo, Helv. Chim. Acta 2005, 88, 68. [10] Y. C. Shen, Y. B. Cheng, A. F. Ahmed, C. L. Lee, S. Y. Chen, C. T. Chien, Y. H. Kuo, G. L. Tzeng, J.

Nat. Prod. 2005, 68, 1665.

[11] J. A. Beutler, K. L. McCall, K. Herbert, D. L. Herald, G. R. Petit, T. Johnson, R. H. Shoemaker, M. R. Boyd, J. Nat. Prod. 2000, 63, 657.

[12] Y. C. Shen, Y. B. Cheng, Y. H. Chen, A. T. Khalil, C. L. Ko, J. Chin. Chem. Soc. 2005, 52, 1263. [13] H. Itokawa, N. Totsuka, H. Morita, K. Takeya, Y. Iitaka, E. P. Schenkel, M. Motidome, Chem.

Pharm. Bull. 1990, 38, 3384.

[14] H. Morita, M. Nakayama, H. Kojima, K. Takeya, H. Itokawa, E. P. Schenkel, M. Motidome, Chem. Pharm. Bull. 1991, 39, 693.

[15] P. R. F. De Carvalho, M. Furlan, M. C. M. Young, D. G. I. Kingston, V. Bolzani, V. Das, Phytochemistry 1998, 49, 1659.