Two New Diterpenoids from Thuja orientalis and Their Cytotoxicity

Experimental Section

General. Optical rotations were obtained on a JASCO P-1020 Polarimeter. IR spectra were recorded on a Bruker Vector 22 IR spectrophotometer. NMR spectra, including 1H-1H COSY, HMQC, HMBC and NOESY experiments, were recorded on a Varian UNITY INOVA 500 NMR spectrometer operating at 500 MHz (1H) and 125 MHz (13C). HRFAB and HREI mass spectra were obtained on a JEOL JMS700 mass spectrometer. Preparative HPLC was performed using a Gilson 306 pump with a Shodex refractive index detector. Silica gel 60 (230-400 mesh) and RP-C18 silica gel (230-400 mesh) were used for the column chromatography. TLC was performed using the precoated Silica gel F254 plates and RP-18 F254s plates (Merck). Spots were detected on TLC by heating after spraying with 10% H2SO4 in EtOH (v/v).

Plant Material. The leaves of T. orientalis were collected in Yeongcheon City, Korea during May 2009. The plant was identified by one of the authors (K. R. Lee). A voucher specimen (SKKU-NPL 0819) of the plant was deposited at the herbarium of the School of Pharmacy at the Sungkyunkwan University in Suwon, Korea.

Extraction and Isolation. An amount of 4 kg leaves of T. orientalis was extracted at room temperature with 80% MeOH and evaporated under reduced pressure with 405 g residue. The residue was dissolved in water (800 mL × 2) and solvent-partitioned to n-hexane (73 g), CHCl3 (41 g), EtOAc (42 g) and n-BuOH (104 g) layers. The n-hexane-soluble layer (36 g) was chromatographed on a silica gel column (230-400 mesh, 600 g, 5 × 60 cm) eluted with n-hexane:EtOAc (5:1 ~ 1:1, gradient system) to yield four fractions (H1-H4). Fraction H1 (3.6 g) was separated over a RP-C18 silica gel column (230-400 mesh, 150 g, 3 × 30 cm) with 90% MeOH to five subfractions (H11-H15). Fraction H13 (620 mg) was separated over a silica gel column (230-400 mesh, 50 g, 2 × 25 cm) with CHCl3:MeOH (150:1) and purified with a RP-C18 prep. HPLC with 75% CH3CN at a flow rate of 2.0 mL/min (Econosil RP-18 10 μm column; 250 × 10 mm; 10 μ particle size; Shodex refractive index detector) to yield 1 (4 mg, tR = 17.1 min) and 2 (3 mg, tR = 18.1 min). Fraction H13 (620 mg) was separated over a silica gel column (230-400 mesh, 50 g, 2 × 25 cm) with CHCl3:MeOH (150:1) and purified with a RP-C18 prep. HPLC with 75% CH3CN to yield 3 (4 mg, tR = 19.8 min). Fraction H14 (440 mg) was further separated on a silica gel (230-440 mesh, 50 g, 2 × 25 cm) eluted with n-hexane:EtOAc (20:1) to yield four subfractions (H141-H144). Fraction H141 (50 mg) was purified with a RP-C18 prep. HPLC with 85% CH3CN to yield 5 (4 mg, tR = 20.2 min), 6 (8 mg, tR = 23.1 min) and 7 (4 mg tR = 29.8 min). Fraction H142 (80 mg) was purified with a RP-C18 prep. HPLC with 85 % CH3CN to yield 9 (32 mg, tR = 27.2 min). Fraction H144 (90 mg) was purified with a RP-C18 prep. HPLC with 90% CH3CN to yield 8 (27 mg, tR =20.2 min). All fractions were purified as described above. Fraction H2 (3.6 g) was separated over a RP-C18 silica gel column (230-400 mesh, 150 g, 3 × 30 cm) with 90% MeOH to give two subfractions (H21-H22). Fraction H21 (150 mg) was purified with a silica gel prep. HPLC with n-hexane: EtOAc (4:1) at a flow rate of 2.0 mL/min (Apollo Silica 5 μm column; 250 × 10 mm; 5 μ particle size; Shodex refractive index detector) to yield compound 4 (15 mg, tR = 15.8 min).

Thujuoric Acid A (1): Colorless gum; +11.0° (c 0.4, MeOH); IR (KBr) νmax 3078, 2932, 2845, 1691, 1641, 1263, 1164, 1099, 1029 cm−1; 1H NMR see Table 1; 13C NMR see Table 2; HRFABMS m/z 401.2300 [M + Na]+ (calcd for C22H34NaO5, 401.2304).

Thujuoric Acid B (2): Colorless gum; +5.0° (c 0.4, MeOH); IR (KBr) νmax: 3079, 2928, 2844, 1693, 1645, 1266, 1163, 1100, 1027 cm−1; 1H NMR see Table 1; 13C NMR see Table 2; HRFABMS m/z 401.2302 [M + Na]+ (calcd for C22H34NaO5, 401.2304).

Thujuoric Acid C (3): Colorless gum; +36.6° (c 0.2, CHCl3); IR (KBr) νmax: 3078, 2932, 2845, 1691, 1641, 1263, 1164, 1099, 1029 cm−1; 1H NMR see Table 1; 13C NMR see Table 2; HRFABMS m/z 401.2304 [M + Na]+ (calcd for C22H34NaO5, 401.2304).

8β,18-Dihydroxysandaracopimar-15-ene (4): Colorless oil; −2.0° (c 0.35, CHCl3); IR (KBr) νmax 3424, 2922, 1635, 1444, 1385, 1034, 909, 757 cm−1; 1H NMR see Table 1; 13C NMR see Table 2; HREIMS m/z 306.2557 [M]+ (calcd for C20H34O2, 306.2559).

Cytotoxicity Assay. A SRB bioassay was used to determine the cytotoxicity of each compound isolated against four cultured human tumor cell lines.16 The assays were performed at the Korea Research Institute of Chemical Technology. The cell lines which were used were A549, SK-OV-3, A498 and HCT-15. Doxorubicin was used as a positive control. The cytotoxicities of doxorubicin against the A549, SK-OV-3, A498, and HCT-15 cell lines were IC50 0.0007, 0.1274, 0.0094, and 0.2149 μM, respectively.