Labdane Diterpenes from the Fruits of Sinopodophyllum emodi

Two new labdane diterpenes, sinoditerpene A (1) and B (2), were isolated from the fruits of Sinopodophyllum emodi, along with two known analogues 3 and 4. Their structures were established on the basis of extensive spectroscopic analysis. The isolation of compounds 1–4 represents the first report of diterpenes from the genus Sinopodophyllum. The cytotoxic activities of all isolated compounds were evaluated in comparison with 5-fluorouracil against the MCF-7 and HepG2 cell lines, towards which 3 showed more potent cytotoxicity.


Introduction
Sinopodophyllum emodi is an important medicinal plant that has been described in the Chinese Pharmacopoeia and Tibetan medicine [1]. Its dried roots and rhizomes (called Taoerqi in Chinese) are frequently used for the treatment of certain cancers, various verrucosis [2], constipation, verminosis [3], rheumatoid pain [4], and pyogenic skin tissue infections [5]. The dried ripe fruits (called "Xiaoyelian" in Chinese) are clinically applied to the treatment of amenorrhea, dead fetus, and placental retention [1]. Previous phytochemical and pharmacological investigations revealed that S. emodi is particularly rich in aryltetralin lactone lignans and prenylated flavonoids, and has attracted wide attention due to their cytotoxic properties [1][2][3][6][7][8][9]. In our search for cytotoxic natural products, we previously reported the isolation, identification and cytotoxic activity of aryltetralin lactone and tetrahydrofuranoid lignans, and prenylated flavonoids from S. emodi [10][11][12]. In a further examination of the fruits of this plant, two new labdane diterpenes, sinoditerpene A (1) and B (2), were obtained together with two known analogues 3 and 4 ( Figure 1). For the first time, the NMR signals for compound 4 were completely assigned by 2D NMR spectra ( 1 H-1 H COSY, HSQC, HMBC, NOESY). Details of the isolation, structure elucidation, and cytotoxicity of all isolated compounds against MCF-7 and HepG2 cell lines are described here.
The relative configuration of the ring substituents of compound 1 was determined by analyzing the NOESY spectrum ( Figure 3). The NOESY correlations from H-3 to Me-18, H-5, and H-1α indicated that they were cofacial and were assigned α-orientation. The NOESY cross peak of Me-19/Me-20 and Me-20/H-11 showed that these protons were β-oriented. Additionally, the stereochemistry of the side-chain double bond was established as E by virtue of the Me-16 chemical shifts (δH 1.70; δC 16.6)
Chinese herbal medicines produce a wide variety of secondary metabolites, which can be exploited as potential anticancer agents. Previous chemical investigations on S. emodi revealed the presence of aryltetralin lactone and tetrahydrofuranoid lignans [2,3,7,8,10], flavonoids [1,6,9,11,20], steroids [21], and phenolics [22]. Cytotoxic activities of some isolated S. emodi constituents have been shown in various cancer cell lines. In the HeLa and KB cell lines, deoxypodphyllotoxin was about 579 and 1123 times more toxic than etoposide, respectively [10]. Sinolignan C displayed cytotoxicity  Compound 4 was obtained as a sticky oil and possessed a molecular formula C 18 H 30 O 2 with four degrees of unsaturation, as revealed from its HR-ESI-MS analysis (m/z 301.2148 [M + Na] + , calcd 301.2143). The IR spectrum displayed the presence of carbonyl (1714 cm´1) and hydroxy (3415 cm´1) groups. Its 1 H-and 13 C-NMR (Tables 1 and 2) were similar to those of compound 3, except that a carbonyl group δ C 208.7 were observed instead of two olefinic carbons δ C 140.0, 123.5, and one oxygenated methylene δ C 59.4 in 1. Due to apparent differences of carbon signal number in 4 (20 carbons) and 3 (18 carbons), it was reasonably assumed that 4 was a dinor-derivative of 3. The HMBC correlation between the carbonyl group δ C 208.7 (C-13) and the methyl δ H 2.11 (3H, s, H-16), the methylene δ H 2.40 (1H, m, H-12), 2.63 (1H, m, H-12), in combination with 1 H-1 H COSY cross peak of H-11 with H-12 and H-9, indicated that 3-oxobutyl was linked to C-9.
Chinese herbal medicines produce a wide variety of secondary metabolites, which can be exploited as potential anticancer agents. Previous chemical investigations on S. emodi revealed the presence of aryltetralin lactone and tetrahydrofuranoid lignans [2,3,7,8,10], flavonoids [1,6,9,11,20], steroids [21], and phenolics [22]. Cytotoxic activities of some isolated S. emodi constituents have been shown in various cancer cell lines. In the HeLa and KB cell lines, deoxypodphyllotoxin was about 579 and 1123 times more toxic than etoposide, respectively [10]. Sinolignan C displayed cytotoxicity against the KB cell line and was more cytotoxic than etoposide [12]. 3-Methoxyquercetin showed cytotoxicity against the MCF-7 and HepG2 cell lines, with IC 50 values of 3.14 and 2.08 µM, respectively [11].
All of the isolated labdane diterpenoids were evaluated for cytotoxic activities against the MCF-7 and HepG2 cell lines (Table 3). Compound 3 was more cytotoxic than 5-fluorouracil, whereas compounds 1, 2 and 4 displayed no cytotoxicity against MCF-7 and HepG2 cell lines. Compounds 1-4 have the same structural skeleton, so the variation in cytotoxicity between them indicates a free hydroxyl group at C-15 was structurally required for the cytotoxity against the MCF-7 and HepG2 cells lines. Esterification at C-15 drastically reduced the cytotoxic activity of the parent compound 3. These results revealed the potential of compound 3 as an ideal antitumor lead compound.

General Procedures
The IR spectra were measured on a Tensor 27 Fourier transform infrared (FTIR) spectrometer (Bruker Optics, Ettlingen, Germany) as KBr discs. The 1D and 2D NMR spectra were recorded on an AC (E)-500 spectrometer(Bruker Biospin, Fallanden, Switzerland) using TMS as an internal standard.

Plant Material
The plant material was collected from Deqin, Yunnan Province, China, in September 2013, and identified by Prof. Cheng-Ming Dong as the fruits of S. emodi, according to the Chinese Traditional Medicine Dictionary [23]. A voucher specimen (SE 20130929) was deposited at the School of Pharmacy, Henan University of Traditional Chinese Medicine.

Alkali Hydrolysis
Compound 1 (6 mg) was refluxed with 4N alcoholic KOH (5 mL) for 1 h. The resulting liquid was neutralized by 1N HCI until pH 7, and concentrated under reduced pressure, and then extracted with CH 2 Cl 2 (10 mLˆ3) after 10 mL water was added. The organic phase was washed with water, dried over Na 2 SO 4 and concentrated in vacuo. The residue was chromatographed on silica gel (PE-acetone 100:7-100:20) to obtain the hydrolyzed target compound.

Cytotoxicity Asssay
Tumor cells were maintained in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, penicillin (100 units/mL), streptomycin (100 µg/mL) under humidified air with 5% CO 2 at 37˝C. Exponentially growing cells were seeded into 96-well tissue culture-treated plates and precultured for 1 day. The cytotoxic activities of isolated compounds were tested against MCF-7 and HepG2 cell lines, using an established MTT assay protocol [10]. 5-fluorouracil was used as the positive control.

Conclusions
Further phytochemical studies on S. emodi resulted in the isolation of two new labdane diterpenes 1 and 2 and two known analogues 3 and 4. All the labdane diterpenoids contained 7(8) and 13 (14) double bonds. Isolation of diterpenoids from S. emodi is reported here for the first time, which also enriches our knowledge about the chemical diversity of this plant. Compound 1 is the first reported example of a labdane diterpene with 7(8) and 13 (14) double bonds exhibiting cytotoxicity in the MCF-7 and HepG2 cell lines. As an antitumor lead compound, further investigations are necessary to explore the structure-activity relationship and lead optimization. Our research demonstrated that the fruits of S. emodi have chemopreventive potential as a herbal medicine and its labdane diterpenes are partly responsible for the observed potency. In addition, these results will broaden the application field of S. emodi.