Two New Diterpenoids from Thuja orientalis and Their Cytotoxicity

Thuja orientalis L. [= Biota orientalis (L.) Endl., Platycladus orientalis (L.) Franco] (Cupressaceae) is an evergreen arbor widely distributed throughout Korea and China. This plant has been used in the traditional medicine for the treatment of hemorrhoids and hypertension. Previous chemical investigations on this plant reported the isolation of terpenoids, flavonoids and lignans. The extract and some constituents of this plant were reported to exhibit anti-inflammatory, cytotoxic, and neuroprotective activities. We investigated the constituents of the leaves of T. orientalis as part of our continuing search for cytotoxic secondary metabolites from Korean medicinal plants and reported about cytotoxic diterpenoids. Further we isolated two new stereoisomers (12) and seven known compounds (3-9) (Figure 1). The chemical structures of 1-2 were determined by spectroscopic methods, including 1D and 2D NMR (H and C NMR, DEPT, H-H COSY, HMQC, HMBC, and NOESY). Previously, 3 was reported with incomplete stereochemistry, and now we reported the complete stereostructure of 3. Compounds 1, 2 and 4-9 were evaluated for their cytotoxicity against A549 (non small cell lung adenocarcinoma), SK-OV-3 (ovarian cancer cells), A498 (renal cell carcinoma) and HCT-15 (colon cancer cells) human tumor cell lines using an in vitro sulforhodamine B (SRB) assay. Compound 1 was obtained as a colorless gum. The molecular formula C22H34O5 was determined from the ion peak [M + Na] at m/z 401.2300 (calcd for C22H34NaO5, 401.2304) in positive ion HRFABMS. The H NMR spectrum showed the presence of signals due to three olefinic [δ 5.65, 4.86, and 4.50 (each brs)], two acetalic [δ 5.54 and 5.42 (each brs)], two methoxyl [δ 3.42 and 3.38 (each s)], and two tertiary methyl [δ 1.23 and 0.61 (each s)] protons. The C NMR spectrum showed 22 carbon signals which were attributed to four methyl, eight methylene, and five methine groups, as well as five quaternary carbons, including one carbonyl carbon (δ 182.7), four olefinic carbons (δ 147.8, 146.3, 123.7 and 106.7), and two acetalic carbons (δ 107.9 and 107.1), determined by the analysis of the DEPT and HMQC spectra. These spectroscopic data of 1 were closely resembled to those of 3 with the exception that H12a and H-12b signals of 1 (δ 2.25 and 2.02, respectively) were slightly shifted (δ 2.35 and 1.88, respectively, 3) in the H NMR spectrum, indicating that 1 may be a stereoisomer of 3. The planar structure of 1 was confirmed by the DEPT, H-H COSY, HMQC and HMBC spectra (Figure 2). A trans-fused A/B ring junction was inferred from the absence of NOESY correlation of H-5/H-20 and the NOESY crosspeaks of H-5/H-9 and H-18 and H-11/H-20 confirmed the relative stereochemistry at A and B rings (Figure 3). The H14, H-15, and H-16 signals in H NMR (δ 5.65, 5.54, and 5.42, respectively) were very similar to those of the cis-form of the 1,4-dihydro-1,4-dimethoxy furan ring (δ 5.64, 5.50 and 5.35), not to those of the trans-form (δ 5.80-5.70 and 5.68-5.60), indicating the H-15 and H-16 of 1 were in the