Three New 2-(2-Phenylethyl)chromone Derivatives of Agarwood Originated from Gyrinops salicifolia

Two new 2-(2-phenylethyl)chromone derivatives (1–2), comprising 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone and benzylacetone moieties, together with one new 2-(2-phenylethenyl)chromone (3) were isolated from the ethyl acetate extraction of agarwood originated from Gyrinops salicifolia Ridl. All structures were unambiguously elucidated on the basis of 1D and 2D NMR spectra as well as by HRESIMS data. All isolated compounds were tested for acetylcholinesterase (AChE) inhibitory activity and cytotoxic activity against human myeloid leukemia cell line (K562). However, none of the compounds displayed AChE inhibitory activity at a concentration of 50 µg mL−1 or cytotoxic activity against K562 cell line.

Gyrinops salicifolia Ridl. is one of agarwood-producing endemic species in Papua New Guinea.In our previous studies on new bioactive chemical constituents from G. salicifolia, several 2-(2-phenylethyl)chromones and sesquiterpenes were identified and showed cytotoxicity and AChE inhibitory activities [9,10].In order to further explore the feature and active constituents of agarwood originating from G. salicifolia, contributing to the deeper understanding of the similarities and differences among agarwood, the investigation of ethyl acetate extraction of agarwood originated from G. salicifolia was continued and led to the identification of two new 2-(2-phenylethyl)chromone derivatives (1-2), comprising 2-(2-phenylethyl)chromone and benzylacetone moieties, and one new 2-(2-phenylethenyl)chromone (3) (Figure 1).Herein, this paper describes the isolation and elucidation of new compounds.

Results and Discussion
Chromatographic separation of ethyl acetate extraction of agarwood originated from G. salicifolia led to the isolation of three 2-(2-phenylethyl)chromone derivatives (1-3).Their structures were elucidated by HRESIMS and NMR spectroscopic analyses, the data as shown in Tables 1 and 2. HRESIMS and NMR spectra for compounds 1-3 are shown in the Supplementary Materials.

Results and Discussion
Chromatographic separation of ethyl acetate extraction of agarwood originated from G. salicifolia led to the isolation of three 2-(2-phenylethyl)chromone derivatives (1-3).Their structures were elucidated by HRESIMS and NMR spectroscopic analyses, the data as shown in Tables 1 and 2. HRESIMS and NMR spectra for compounds 1-3 are shown in the Supplementary Materials.
Compound 2 was isolated as a yellow amorphous solid.It had the molecular formula C 29 H 30 O 8 as established by HRESIMS, indicating the addition of a methoxy group compared to 1.The 1 H-and 13 C-NMR spectra were similar to those of 1, except for the presence of one more methoxy group.The 1 H-NMR spectra of 1 revealed the presence of a para-disubstituted benzene ring (δ H 6.76, H-3 /5 ; and 7.07, H-2 /6 ), suggested a methoxy group attached to C-4 (δ C 159.7).The deduction was confirmed by HMBC correlation from 4 -OCH 3 (δ H 3.73) to C-4 , and by NOE correlation from 4 -OCH 3 to H-3 and H-5 (Figure 2).The remaining substructures of 2 were identical to those of 1 based on detailed analysis of 1D-and 2D-NMR spectra.In the same way to 1, the relative configuration of 2 was identical to that of (−)-aquisinenone D ( 5) and 1 by analysis of their configuration and for their close chemical shifts of unit A and coupling constants of H-6 and H-8 (Tables 1 and 2) [11].Therefore, the structure of 2 was elucidated as shown (Figure 1) and named gyrinone B.
Compounds 1-3 were tested for AChE inhibitory activity in vitro and cytotoxicity against K562 human myeloid leukemia cell line.Unfortunately, none of the compounds displayed AChE inhibitory activity or cytotoxicity against K562 cell line.All compounds were tested for AChE inhibitory activity by Ellman's colorimetric method in vitro at a concentration of 50 µg mL −1 as described previously [12].Tacrine hydrochloride hydrate was used as positive control with an IC 50 value of 64.8 nM, and DMSO was served as negative control.

Bioassay for Cytotoxic Activity
The MTT assay was used to evaluate cytotoxicity of all compounds against human myeloid leukemia cell line (K562) as described previously [9].K562 cell line was obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology.Paclitaxel was performed as positive control with an IC 50 value of 0.89 µM.

Supplementary Materials :
HRESIMS and NMR spectra for compounds 1-3 are available online.Author Contributions: The list authors contributed to this work as follows: W.-H.D., H.W. And F.-J.G. performed the process of data, collection of the agarwood samples, and preparation of the manuscript.F.-D.K. and W.L. partially contributed the structure elucidation.W.-L.M. and H.-Q.C. contributed to the revision of this manuscript.The whole research was performed based on the planning of H.-F.D. and K.-B.Z.All authors approved the final version of the manuscript.