The Diterpenes Ovoideal A–G from Tirpitzia ovoidea

Seven new diterpenes, named ovoideal A (1), B (2), C (3), D (4), E (5), F (6) and G (7), have been isolated along with eleven known diterpenes 8–18 from the petroleum ether soluble fraction of an ethanol extract of the aerial parts of Tirpitzia ovoidea. The structures of the new compounds were elucidated primarily by 1D and 2D NMR spectroscopy, as well as by the HR-ESI-MS spectrometry. All compounds were isolated from the Linaceae family for the first time. The in vitro cytotoxic activity of compounds 1, 3–5, 8–18 was evaluated against the Hela, HepG2 and K562 cell lines. Among them, compounds 3, 9, 11, 12, 13, 14, 15, 17, 18 showed moderate inhibitory activities.


Introduction
Tirpitzia ovoidea Chun et How ex Sha (Linaceae family) is distributed in Chongzuo, Jingxi, Longzhou, Mashan in Guangxi of China, while Vietnam has some distribution too [1]. The dried aerial parts, containing stems, branches and leaves of this species, is known in China as Baihuachai and has been used as a folk medicine in Guangxi. Baihuachai was used to promoting blood circulation for removing blood stasis, relieving muscle rigidity and activating collaterals and in the treatment of rheumatism, OPEN ACCESS traumatic hemorrhage, injuries from falls, fractures, contusions and strains [1]. The Mulao people use it to treat chronic hepatitis, traumatic infections, fractures, hepatalgia and anepithymia caused by liver cancer [2]. As a folk medicine it has precise therapeutic effects, while there has been no research on its chemical constituents and pharmacological activities. In particular, diterpenoids are thought by many to be the largest pool of chemically diverse and physiologically interesting metabolites [3,4], displaying properties such as anti-leishmania activity [5], cytotocity [6], anti-microbial activity [7], anti-HIV activity [8] and neuro-protection activity [9]. In our research eighteen diterpenes, including seven new compounds (ovoideal A, B, C, D, E, F and G, Figures 1 and 2) were isolated from the petroleum ether soluble fraction of an ethanol extract of the stems, branches and leaves of Tirpitzia ovoidea and their structures were elucidated. All of them were isolated from Linaceae family for the first time. Fourteen of the isolated compounds were evaluated for their cytotoxic activity.
Compound 3 (Figure 1) was obtained as a light yellow gum and its molecular formula was determined to be C20H30O2 by HRESIMS, which displayed a quasi-molecular ion peak at m/z 301.2190 ([M−H] − ). Comparing the 1 H-NMR and the 13 C-NMR spectra (Tables 1 and 2) of 3 with those of the known compound 11, we could note that their A and B rings were the same and they just differed in C ring. It was clear that the carbonyl group at C ring in 3 was reduced into a hydroxyl group. The signal at δC 72.4 belonged to the carbon connected with the hydroxyl group which had long-range HMBC couplings ( Figure 2 Compound 4 ( Figure 1) was obtained as colorless crystals and its molecular formula was determined to be C20H26O3 by HRESIMS, which displayed a quasi-molecular ion peak at m/z 315.1959 ([M+H] + ). 1 H-NMR spectrum (Table 1) showed signals at δ 1.94 (3H, s) for an olefinic methyl group, δ 1.09 (3H, s), 1.16 (3H, s), 1.57 (3H, s) for three tertiary methyl protons, δ 6.06 (1H, s) for an olefinic proton. It also gave signals for an ABX system at δ 6.64 (1H, dd, J = 17.9, 11.8 Hz, H-15), 5.57 (1H, dd, J = 17.9, 1.76 Hz, H-16a) and 5.67 (1H, dd, J = 11.9, 1.8 Hz, H-16b), corresponding to a monosubstituted vinyl group attached to an olefinic carbon nucleus. The combination of these groups in a single nucleus suggested this compound was a member of the cleistanthane series [22]. The 13 C-NMR spectrum ( Table 2) revealed all 20 carbons, which in combination with HSQC measurements were found to be four methyl, five methylene (one olefinic), three methine (two olefinic), and eight quaternary carbons (three olefinic, three oxygenated). 1 (1.16), thus confirmed the location of this carbonyl group was at C-3. Also, another carbonyl group was located at C-12 from the cross-peak at 189.0/1.94 with H-17. The carbon at δC 71.5, which was connected with a hydroxyl group, was assigned to C-8 because of the cross-peaks with H-6, H-7, H-11, H-15 and H-17. The coupling constant values of H-5 was 2.6 and 12.6, demonstrating that H-5 was axial-oriented [23]. Also cross-peaks in the NOESY (Figure 3) spectrum at 1.16/1.57 (H-19/H-20) revealed that C-20 methyl group was axial-oriented. Therefore we propose that the 8β-hydroxycleistanth-9(11),13,15-triene-3,12-dione structure for compound 4, which was named ovoideal D.   Compound 5 (Figure 1) was obtained as a white amorphous powder and its molecular formula was determined to be C20H28O3 by HRESIMS, which displayed a quasi-molecular ion peak at m/z 317.2115 ([M+H] + ). Comparing the 1 H-NMR (Table 1) and 13 C-NMR (Table 2) spectra of 5 with those of compound 4, we found that 5 just differed from 4 in the presence of a hydroxyl group instead of a carbonyl group. The carbon connected with the hydroxyl group at δC 78.3, has the long-range HMBC ( Figure 2) correlations with H-1, H-2, H-18 and H-19, indicated the location of this hydroxyl group at C-3. In NOESY spectrum there were correlations between H-3 and H-5, so we suggest the structure of compound 5 to be 3β,8β-dihydroxycleistanth-9(11),13,15-triene-12-one, named ovoideal E.
Compound 6 ( Figure 1) was obtained as colorless crystals and its molecular formula was determined to be C20H26O2 by HRESIMS, which displayed a quasi-molecular ion peak at m/z 297.1862 ([M−H] − ). Comparing the 1 H-NMR (Table 1) and 13 C-NMR (Table 2) spectra of 6 with those of the known compound 14, we noted that 6 differed from 14 only in the location of the hydroxyl group, as the carbon connected with the hydroxyl group at δC 64.9, has long-range HMBC ( Figure 2) correlations with H-5 and H-6; also the proton of this carbon correlated with C-8 and C-9, so we propose the peri location of this hydroxyl group at C-7. In the NOESY spectrum, there were correlations between H-7 and H-15, suggesting that the hydroxyl group was β-oriented. Considering the stereochemistry of compound 14, we propose that the structure of compound 6 is 7βhydroxycleistanth-8,11,13,15-tetraene-3-one, named ovoideal F. Compound 7 (Figure 1) was obtained as colorless crystals and its molecular formula was determined to be C18H24O2 by HRESIMS, which displayed a quasi-molecular ion peak at m/z 273.1855 ([M+H] + ). The molecular formula indicated the presence of seven double-bond equivalents in the molecule. The 13 C-NMR spectrum ( Table 2) further showed that 7 had one ketonic carbonyl carbon conjugated with the aromatic ring (δ 199.1), three tertiary and three quaternary olefinic (of the aromatic ring), four methyl, three methylene, two methane, and two saturated quaternary carbons. The 1 H-NMR spectrum (Table 1)  , and -CH=CH-(C13-C14), so the aromatic methyl substituent was at C-12. The assignment of the location of the carbonyl group was at C-7 by virtue of the HMBC correlations with C-7 and H-14. We also observed cross-peaks at 75.2/1.84, 75.2/1.02, confirming that the hydroxyl group was located at C-3. The H-3 peak was a broad singlet, revealing that the hydroxyl group was axial. On the basis of the general stereochemistry of the compounds isolated from this plant, we propose that the absolute configuration of 7 is 3S,5S,10R, so we suggest the structure of compound 7 to be 3α-hydroxy-12-methylpodocarpa-8,11,13-triene-7-one, named ovoideal G.

Cytotoxity Activity
Many diterpenes have strong anti-tumor activities on human cancer cell lines [25,26], so the in vitro cytotoxity activities for all these compounds, except for 2, 6 and 7, against Hela, HepG2 and K562 were evaluated in our study. Clonogenic-type assays for comparing the growth of treated cells showed some inhibitory activity for compounds 3, 9, 11-15, 17, 18 in a range of tumor cell lines ( Table 3). The anti-cancer activity of compounds 3, 11, 12, 15 and 18 are reported for the first time in this study.

Plant Material
The aerial parts of Tirpitzia ovoidea were collected from Wuming, Guangxi, China in April 2012 and identified by Associate Professor Ming-Ying Shang. A voucher specimen (No. 7349) was deposited in the Herbarium of Pharmacognosy, School of Pharmaceutical Sciences, Peking University Health Science Center.

Cytotoxicity Activity
The cytotoxic effects of compounds were estimated against the Hela, HepG2 and K562 cancer cell lines by using the MTT assay method. The cell suspensions were distributed into 96-well cell culture plates and cultured at 37 °C under a 5% CO2 atmosphere in an incubator for 24 h. The compounds were dissolved with limited DMSO and diluted to five different concentrations (0.1, 1, 10, 100, 200 µM) with RPMI 1640 before adding to the corresponding well. After 48 h cultivation, MTT was added to each well for 4 h cultivation. Finally, the supernatant was discarded and limited DMSO was added to the well to dissolve the blue-violet crystal, then the optical density (OD) values were read on the microplate reader at 492 nm. All tests and analyses were carried out in triplicate. DMSO, 5-FU and taxol were applied as the blank control and positive control, respectively.