2-(2-Phenylethyl)chromone Derivatives of Agarwood Originating from Gyrinops salicifolia

Three new 2-(2-phenylethyl)chromone derivatives (1–3) and a new 2-(2-phenylethenyl)chromone derivative (4), together with two known 2-(2-phenylethyl)chromone derivatives (5–6), were isolated from agarwood originating from Gyrinops salicifolia Ridl. The structures of compounds 1–4 were elucidated by comprehensive spectroscopic techniques (UV, IR, 1D and 2D-NMR) and MS analysis, as well as by comparison with the literature. Compounds 1, 2, and 5 showed moderate cytotoxicity against human tumor K562, BEL-7402, and SGC-7901 cell lines with IC50 values of 5.76 to 20.1 µM.


Introduction
Agarwood (Chen-Xiang in Chinese) is the resinous heartwood of the plants from the Aquilaria or Gyrinops genus that belong to the family of Thymelaeaceae [1]. It is well known as a traditional medicinal and natural perfume material, and has become more and more prevalent in international trade [2,3]. As traditional medicine, agarwood can alleviate stomachaches and ease symptoms of cough, rheumatism and high fever. Furthermore, its special fragrance is able to calm people down and relieve fatigue [4,5]. Agarwood formation occurs slowly and infrequently in nature and the supply of wild agarwood cannot meet the market demand, so the studies on fragrant constituents and related biosynthetic genes [5][6][7][8][9], as well as the search for wild agarwood-producing species [10], become critical.
Gyrinops salicifolia, currently listed in CITES Appendix II [11], is one of the agarwood-producing endemic species in Papua New Guinea. Previous studies on agarwood mainly focused on the resins obtained from Aquilaria species [5,6], although Gyrinops species also produce agarwood. As far as we know, the only report on chemical constituents of Gyrinops species is on the leaves and stems of Gyrinops walla [12]. It is worth noting that the chemical constituents of resins from Gyrinops walla identified by GC-MS were chemically similar to those from Aquilaria sp. [13]. So far, it has been reported that 2-(2-phenylethyl)chromone derivatives and sesquiterpenes were the main chemical constituents of agarwood [5,6,14,15]. Our study on chemical constituents of the agarwood from Gyrinops salicifolia led to isolation of three new 2-(2-phenylethyl)chromone derivatives (1-3) and a new 2-(2-phenylethenyl)chromone derivative (4), together with two known chromone derivatives (5,6) ( Figure 1). In this paper, the isolation and structure elucidation of compounds 1-6 as well as cytotoxic activity against human tumor K562, BEL-7402, SGC-7901 cell lines is described.
Compound 4 was obtained as a yellow powder, and its molecular formula was deduced to be C 18  According to the results, we found that compounds 1, 2, 5 and 6 showed more effective cytotoxic activity to K-562 cells than to BEL-7402 and SGC-7901 cell lines. In addition, a comprehensive comparison of the cytotoxic activity of compounds 1-6 suggested that hydroxyls substituted at C-6 and C-7, respectively, as in 1 and 5, led to more potent activity, while 5 with methoxy at C-4 showed increased activity. Furthermore, the hydroxy group at C-6 was crucial for the cytotoxic activities among this type of compound.

Plant Material
The

Extraction and Isolation
The material of agarwood (491.1 g, dry weight) was extracted with 95% EtOH (2 L × 3) for three times at heating reflux and filtered. Then the EtOH was removed under vacuum, and get crude extract 177.4 g. After that scatter it completely in H 2 O (2 L), subsequently extracted with EtOAc (2 L × 3) followed by n-BuOH (2 L × 3). The EtOAc extract (141.2 g) was subjected to vacuum liquid chromatography with silica gel using a step gradient of CHCl 3

Bioassay of Cytotoxic Activity
Human cancer cell lines, gastric carcinoma (SGC-7901), myeloid leukemia (K562), and hepatocellular carcinoma (BEL-7402), were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology. MTT assay [20,21] was used to determine the growth inhibition of the tested cell lines. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin at 37 • C and 5% CO 2 with 90% humidity. The logarithmic phase cells (90 µL) were seeded onto 96-well plates at the concentration of 5 × 10 4 cell/mL. The following specific experimental procedures were the same as those described previously [22]. The results of the cytotoxic activity experiment are shown in Table 2.