New Dihydro-β-agarofuran Sesquiterpenes from Parnassia wightiana Wall: Isolation, Identification and Cytotoxicity against Cancer Cells

Five new (4–8) and three known (1–3) dihydro-β-agarofuran sesquiterpene polyesters were isolated from the whole plants of Parnassia wightiana. The structures of all compounds were elucidated through spectroscopic analysis including 2D-NMR and HR-MS. The absolute configuration of these compounds was established by X-ray diffraction analysis, comparison of NOESY spectra and biogenetic means. The cytotoxities of compounds 2–8 were evaluated in vitro against HL-60, SMMC-7721, A549, MCF-7 and SW480 cell lines. Compounds 5–7 exhibited the highest activities with IC50 values of 11.8–30.1 μM in most cases. The SAR revealed that the introduction of hydroxyl group was able to significantly improve the activities of the compounds for most of the cell lines.


Introduction
The sesquiterpene polyesters with a dihydroagarofuran skeleton have attracted considerable attention from synthetic organic chemists and pharmacologists due to their complex and diverse chemical structures and wide range of biological activities [1], including insect antifeedant and/or insecticidal activity [2,3], cytotoxic activity [4], antitumor [5], antitumor promoting activity [6], antitubercular [7], immunosuppressive [8], anti-HIV [9], anti-inflammatory activity [10] and reversal of the multidrug resistance (MDR) phenotype [11]. Until now, hundreds of dihydro-β-agarofuran compounds have been isolated from dozens of species of plants. It is noteworthy that most of the natural dihydro-β-agarofurans originated mainly from plants of the Celastraceae family. Only a minority were found in other family plants such as Hippocrateaceae and Lamiaceae [1]. Therefore, dihydro-β-agarofurans have been considered as chemotaxonomic indicators of the Celastraceae family.
Parnassia wightiana Wall (Saxifragaceae), commonly known as Ji-mei-hua-cao, Cang-er-qi, Qiao-mai-ye or Ding-chuang-cao, is a perennial herb and is distributed in the Qinling Mountains in China. Dried whole plants have been used as Chinese folk medicine for the treatment of leukorrhea, cough, haematemesis, carbuncle, irregular menstruation, hypertension, malaria, kidney stones, gall stones, and so on. Recently, Wang and coworkers isolated one dihydro-β-agarofuran compound from this plant, which exhibited cytotoxicity against HepG2 and MDA-10 cells and antifeedant activity against Mythimna separata larvae [12,13]. Besides that, up to now, no systematic phytochemical investigation of this plant has been reported.
In this context, this study aimed at investigating in more detail the composition, structural characteristics and bioactivity of dihydro-β-agarofuran compounds in P. wightiana. Herein, we described the isolation and structure elucidation of eight dihydro-β-agarofuran sesquiterpenes ( Figure 1) including five new compounds (4)(5)(6)(7)(8) as well as their cytotoxic activity against five cancer cell lines. The structures of all compounds were elucidated by means of HR-MS, 1D-and 2D-NMR spectroscopic analysis including DEPT, 1 H-1 H COSY, NOESY, HSQC and HMBC experiments. Their absolute configurations were established by X-ray crystallographic analysis, biogenetic means and comparison of NOESY spectra. The isolated compounds were evaluated for cytotoxic properties against five cancer cell lines.

Results and Discussion
Three known compounds were identified as ejap-4 (1) [14], celahin B (2) [15,16] and 1α-acetoxy-8α-hydroxy-6β,9β-dibenzyloxy-2-oxodihydro-β-agarofuran (3) [12,13] by spectroscopic methods, but the previous reports did not confirm their absolute configurations. For this reason, a single-crystal X-ray diffraction experiment of 1 was performed in the present research. Its crystallographic data revealed that the torsion angle obtained corresponded to a decalin system with a trans union between rings A and B, of which A adopted a chair conformation and B, a half-chair conformation, whereas the furan ring presented an envelope conformation ( Figure 2). Thus, 1 was elucidated as (1R,2S,4R,5S,6R,7R,8R,9R,10S)- 1,2,6,8,15-pentaacetoxy-9-benzoyloxydihydro-β-agarofuran. Taking into account biosynthetic consideration and the same NOE effects of 2 as that of 1, the absolute configuration of 2 can be proposed as 1R,2S,4R,5S,6R,7R,9S,10R because the only difference of 2 from 1 is the absence of acetoxyl group at C-8. Compound 3 as a new compound from the same plant was reported by Wang et al. [12,13], but the authors proposed an inconsistent stereochemistry for 3, due to the absence of the corresponding 2D-NMR analysis. In addition, they did not also provide the complete 1D-NMR data and other spectrometric data as well as physical properties. Therefore, the more detailed structure characterization of 3 is herein necessary.
The 13 C NMR and DEPT spectra (Tables 1 and 2) indicated that 4 had one additional methylene group and one less oxygenated methine group than 3. The 1 H and 13 C NMR, EI-MS, IR and UV spectra displayed the presence of one acetate ester, two benzoate esters and one ketone carbonyl group. The 1 H and 13 C NMR spectra of 4 were similar to those of 3 except that the methylene group at C-8 of 4 replaced the oxygenated methine at C-8 of 3. Thus, 4 was identified as a 1,6,9-trisubstituted dihydroagarofuran-2-one, which was further supported by the 1 H-1 H COSY and HMBC spectra. The complete assignments of the protonated carbons were made from the HSQC spectrum.
In the NOESY spectrum, the NOEs of 8 were observed to be the same as those of 7 ( Figure 2). As the only difference of 8 from 7 was the transfer of the acetoxy group at the 2 site in 7 to the 1 site in 8, this compound lastly was identified as having the same stereochemistry as 7. Accordingly, the structure of 8 was deduced as (1R,2S,4R,5S,6R,7R,8R,9R,10S)-1-acetoxy- 6,9-dibenzoyloxy-2,8-dihydroxydihydroβ-agarofuran. To explore the potential bioactivities of the isolates from P. wightiana Wall, the compounds 2-8 were evaluated for in vitro cytotoxic properties against HL-60, SMMC-7721, A549, MCF-7 and SW480 cell lines by MTT assays. Because of poor solubility, compound 1 was not tested for cytotoxicity. The cytotoxicity data are shown in Table 3. The anticancer agent cis-diaminodichloroplatinum was used as a positive control.
All the test compounds showed the definite activities in varying degrees against all tested cell lines at the concentration of 40 μM. For most of the cell lines, compounds 3, 5-8 exhibited moderate activities with IC 50 values ranging from 11.8 to 30.6 μM, while 2 and 4 displayed poor activities with IC 50 values >40 μM in most cases. It was worth noting that 4 showed higher activity against A-549 (IC 50 = 17.4 μM). For each of the tested cell lines, 5, 6 or 7 were the most cytotoxic.  Preliminary analysis of the structure-activity relationship revealed that the compounds with hydroxyl groups (3,(5)(6)(7)(8) had greater cytotoxicity than the compounds without hydroxyl groups (2,4). The reduction of ketone carbonyl group in 4 to hydroxyl group such as 5 or the introduction of 8-OH into 4 such as 3 led to a significant improvement of activity, except against A-549 cells, showing that 2-OH and 8-OH were beneficial for the improvement of activity against most of the cell lines, and the presence of 2-carbonyl group was able to increase activity against A-549 cells. However, the 2,8-dihydroxy compound 8 did not demonstrate higher activity than 2-hydroxy compound 5 or 8-hydroxy compound 3. The higher activity of 7 than 8 showed that the combination of 1,8-dihydroxy groups led to higher activity than that of 2,8-dihydroxy groups. In addition, the 1,8-dihydroxy compound 7 demonstrated higher activity than the corresponding 1-acetoxy-8-hydroxy compound 6 against HL-60, MCF-7 and SW480 but lower activity against SMMC-7721 and A-549.

Plant Material
The entire plants of P. wightiana were collected from Qinling Mountains, Shaanxi province, China, in June 2013, and identified by Professor Fang Miao, a co-author of this paper. A voucher specimen (No. 20061202) is deposited in botanic specimen center of Northwest A&F University, Yangling, China.

Conclusions
In conclusion, we isolated five new and three known dihydro-β-agarofuran sesquiterpene polyesters from Parnassia wightiana and evaluated their cytotoxicities in vitro against five cancer cell lines. The structures of all the compounds were established through spectroscopic analysis, and their absolute configurations were established by X-ray diffraction analysis and biogenetic means. For most of tested cell lines, 3, 5, 6 and 7 exhibited moderate activity with IC 50 values of 11.8-30.1 μM. In addition, the structure-activity relationship was discussed as well.