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

Abstract

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.

1. Introduction

Agarwood, known as “Chenxiang” in Chinese, is the resinous heartwood of Aquilaria or Gyrinops genus (Thymelaeaceae) formed after various forms of natural or artificial injury [1]. As a rare traditional herbal medicine and natural spice, it possesses a panoply of effects such as aphrodisiac, sedative, cardiatonic and carminative activity, and it is used to relieve gastric problems, coughs, rheumatism and high fever [2]. Until now, the chemical constituents of agarwood harvested from A. malassensis, A. sinensis, A. crassna, A. agallocha, and G. salicifolia have been well or partly investigated. 2-(2-Phenylethyl)chromone derivatives and sesquiterpenes were reported as the main chemical constituents of agarwood and exhibited various of biological activities, including cytotoxicity, antibacterial, acetylcholinesterase (AChE) inhibitory, α-glucosidase inhibitory, antineuroinflammatory, neuroprotective and antidepressant activities [3,4,5,6,7,8].

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.

2. 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

Table 1. ¹H-NMR data for compounds 1–3 and unit as of (−)-6″-hydroxyaquisinenone B (4) and (−)-aquisinenone D (5) (δ in ppm, J in Hz).

Position	1 a	Unit A of 4 b	2 c	Unit A of 5 d	3 e
3	6.07, s	6.42, s	6.05, s	6.02, s	6.22, s
5	4.31, t (2.1)	4.29, t (2.5)	4.53, dd (3.0, 1.9)	4.49, br s
6	4.23, dd (3.4, 2.1)	4.40, m	4.41, dd (4.9, 3.0)	4.32, dd (4.5, 3.0)	6.79, d (8.3)
7	4.61, m	4.87, br s	4.74, dt (4.9, 1.9)	4.73, m	7.52, t (8.3)
8	4.33, br s	4.55, d (7.0)	4.47, d (1.9)	4.46, d (2.0)	6.96, d (8.3)
2′	7.18, d (7.2)	7.27, m	7.07, d (8.6)	7.06, d (8.5)	7.20, d (2.1)
3′	7.22, t (7.2)	7.27, m	6.76, d (8.6)	6.75, d (8.5)
4′	7.14, t (7.2)	7.19, m
5′	7.22, t (7.2)	7.27, m	6.76, d (8.6)	6.75, d (8.5)	6.89, d (8.3)
6′	7.18, d (7.2)	7.27, m	7.07, d (8.6)	7.06, d (8.5)	7.10, dd (8.3, 2.1)
7′	2.81, 2.89, m	2.99, m	2.84, 2.93, m	2.90, m	7.54, d (15.9)
8′	2.81, m	2.92, m	2.93, m	2.82, m	6.61, d (15.9)
5′′	6.70, d (8.3)		6.76, d (8.3)
6′′	6.56, d (8.3)		6.66, d (8.3)
7′′	2.76, 3.48, m		2.93, 3.64, m
8′′	2.48, 2.61, m		2.61, 2.71, m
10′′	2.06, s		2.18, s
4′-OCH3			3.73, s		3.95, s
4″-OCH3	3.64, s		3.77, s
6-OH		5.87, d (3.0)
8-OH		6.11, d (8.0)

^a Recorded at 600 MHz in DMSO-d₆, ^b Recorded at 500 MHz in DMSO-d₆, ^c Recorded at 600 MHz in CD₃OD, ^d Recorded at 500 MHz in CD₃OD, ^e Recorded at 500 MHz in CDCl₃.

and

Table 2. ¹³C-NMR data for compounds 1–3 and unit as of (−)-6″-hydroxyaquisinenone B (4) and (−)-aquisinenone D (5) (δ in ppm).

Position	1 a	Unit A of 4 b	2 c	Unit A of 5 d	3 e
2	168.0, C	170.1, C	170.7, C	170.6, C	163.3, C
3	112.7, CH	111.8, CH	113.8, CH	113.7, CH	108.5, CH
4	178.0, C	180.0, C	181.0, C	180.9, C	183.6, C
5	31.7, CH	29.3, CH	33.4, CH	33.4, CH	160.9, C
6	63.6, CH	61.3, CH	65.6, CH	65.5, CH	111.3, CH
7	74.7, CH	77.2, CH	75.6, CH	75.8, CH	135.3, CH
8	68.8, CH	68.1, CH	70.4, CH	70.3, CH	106.9, CH
9	162.5, C	164.1, C	164.3, C	164.3, C	156.3, C
10	121.7, C	121.1, C	122.9, C	122.8, C	111.0, C
1′	140.3, C	140.0, C	133.0, C	133.1, C	128.6, C
2′	128.6, CH	128.3, CH	130.4, CH	130.4, CH	112.8, CH
3′	128.7, CH	128.4, CH	114.9, CH	114.9, CH	146.1, C
4′	126.5, CH	126.2, CH	159.7, C	159.7, C	148.5, C
5′	128.7, CH	128.4, CH	114.9, CH	114.9, CH	110.8, CH
6′	128.6, CH	128.3, CH	130.4, CH	130.4, CH	121.7, CH
7′	32.2, CH2	32.1, CH2	33.1, CH2	33.1, CH2	138.0, CH
8′	34.3, CH2	34.6, CH2	36.5, CH2	36.5, CH2	117.9, CH
1′′	131.6, C		133.2, C
2′′	123.0, C		123.9, C
3′′	141.3, C		142.8, C
4′′	146.1, C		147.6, C
5′′	111.1, CH		112.4, CH
6′′	120.9, CH		122.3, CH
7′′	25.6, CH2		26.9, CH2
8′′	45.4, CH2		46.6, CH2
9′′	208.7, C		211.6, C
10′′	30.0, CH3		30.0, CH3
4′-OCH3			55.6, CH3		56.1, CH3
4″-OCH3	55.6, CH3		56.5, CH3

^a Recorded at 150 MHz in DMSO-d₆, ^b Recorded at 125 MHz in DMSO-d₆, ^c Recorded at 150 MHz in CD₃OD, ^d Recorded at 125 MHz in CD₃OD, ^e Recorded at 125 MHz in CDCl₃.

. HRESIMS and NMR spectra for compounds 1–3 are shown in the Supplementary Materials.

Compound 1 was obtained as a yellow powder. Its molecular formula was deduced to be C₂₈H₂₈O₇ with 15 degrees of unsaturation on the basis of the HRESIMS data. The ¹H-NMR displayed a monosubstituted benzene ring at δ_H 7.10–7.25 (H-2′–6′), two doublet aromatic protons (δ_H 6.56, H-6″; 6.70, H-5″), the characteristic olefinic proton singlet of 2-(2-phenylethyl)chromone at δ_H 6.07 (H-3), four methines (δ_H 4.61, H-7; 4.33, H-8; 4.31, H-5; and 4.23, H-6), one methoxy group singlet at δ_H 3.64 (OCH₃-4″), one methyl group singlet at δ_H 2.06 (H₃-10″), and four methylene groups ranging from δ_H 2.40 to 3.50. The DEPTQ spectrum showed the presence of two carbonyls at δ_C 208.7 (C-9″), and 178.0 (C-4), two benzene rings, and four olefinic carbons, accounting for 12 degrees of unsaturation. Apart from these signals, four methines at δ_C 74.7 (C-7), 68.8 (C-8), 63.6 (C-6), and 31.7 (C-5), four methylene groups at δ_C 45.4 (C-8″), 34.3 (C-8′), 32.2 (C-7′), and 25.6 (C-7″), one methoxy group at δ_C 55.6 (OCH₃-4″) and one methyl group at δ_C 30.0 (CH₃-10″) were observed in DEPTQ spectrum of 1. Detailed analysis of its 1D and 2D NMR data revealed that compound 1 was a dimer comprising a 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone unit and a benzylacetone unit. The 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone unit was identical to that of monomeric A of (−)-6″-hydroxyaquisinenone B by comparison of NMR data and key HMBC correlations from H-2′ and 6′ (δ_H 7.18) to C-7′, from H-3 to C-8′ and C-5, from H-5 to C-4, C-6 and C-7, from H-6 to C-10 (δ_C 121.7), from H-7 to C-9 (δ_C 162.5), from H-8 to C-9, C-10, C-6, and C-7, and ¹H-¹H COSY of H-6/H-7, H-2′/H-3′/H-4′/H-5′/H-6′, H-7′/H-8′ [11]. The structure of the benzylacetone unit was elucidated by ¹H-¹H COSY of H-5″/H-6″, H₂-7″ (δ_H 2.76 and 3.48)/H₂-8″ (δ_H 2.48 and 2.61), and HMBC correlations from H₃-10″ to C-8″ and C-9″, from H₂-7″ to C-8″, C-9″, C-1″ (δ_C 131.6), C-2″ (δ_C 123.0) and C-6″ (δ_C 120.9), and from H-6″ to C-2″, C-4″ (δ_C 146.1), and C-7″. The position of the methoxy group at C-4″ was elucidated by HMBC correlations from 4″-OCH₃ to C-4″, and by NOE correlation between 4″-OCH₃ and H-5″. The linkage between two units by C-5/C-2″ and C-7/O/C-3″ (δ_C 141.3) was determined by HMBC correlations from H-7 to C-3″, and from H-5 to C-1″, C-2″ and C-3″ as shown. Due to the formation of a 3,4-dihydro-2H-pyran ring between two units, H-5 and H-7, as equatorial hydrogens, were oriented towards the same face of the cyclohexene ring in a half-chair conformation, which was confirmed by “W” coupling between H-5 and H-7 (⁴J_5,7 = 2.1 Hz). The trans-type relationships of H-7/H-8 was deduced by their small coupling constant (~90° dihedral angle of H₇–C₇–C₈–H₈). The remaining relative configuration of H-6 was elucidated as opposite to H-5 by its close ³J_5,6 (2.1 Hz) to that of (−)-6″-hydroxyaquisinenone B (³J_5,6 = 2.5 Hz) and ³J_6,7 (3.4 Hz) to that of (+)-6″-hydroxy-4′,4′′′-dimethoxyaquisinenone B (³J_6,7 = 3.5 Hz) [11]. The relative configuration of 1 was identical to that of (−)-6″-hydroxyaquisinenone B and (+)-6″-hydroxy-4′,4′′′-dimethoxyaquisinenone B by further detailed comparison of their coupling constants of H-5–8. Thus, the structure of 1 was established as depicted and named gyrinone A.

Compound 2 was isolated as a yellow amorphous solid. It had the molecular formula C₂₉H₃₀O₈ as established by HRESIMS, indicating the addition of a methoxy group compared to 1. The ¹H- and ¹³C-NMR spectra were similar to those of 1, except for the presence of one more methoxy group. The ¹H-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′-OCH₃ (δ_H 3.73) to C-4′, and by NOE correlation from 4′-OCH₃ 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 (Table 1 and Table 2) [11]. Therefore, the structure of 2 was elucidated as shown (Figure 1) and named gyrinone B.

Compound 3 was obtained as yellow powder, and its molecular formula was deduced to be C₁₈H₁₄O₅ on the basis of HRESIMS. Its ¹H-NMR spectroscopic data showed two trans-olefinic protons at δ_H 7.54 (H-7′) and δ_H 6.61 (H-8′), a 1,2,3-trisubstituted benzene ring (δ_H 6.79, H-6; 7.52, H-7; and 6.96, H-8), a set of ABX coupling aromatic system at δ_H 7.10 (H-6′), 6.89 (H-5′) and 7.20 (H-2′), and a methoxy group at δ_H 3.95 (4′-OCH₃). The ¹H and ¹³C-NMR spectroscopic data of 3 were similar to those of 5-hydroxy-2-[2-(4-methoxybenzene)ethenyl]chromone [9], except for containing an additional hydroxy group in 3. The HMBC correlations from 4′-OCH₃, H-2′, and H-6′ to C-4′ (δ_C 148.5), together with NOE of 4′-OCH₃ and H-5′, indicated that the methoxy group was attached to C-4′. Thus, C-5 (δ_C 160.9) and C-3′ (δ_C 146.1) were substituted by hydroxy groups based on their much further downfield chemical shifts. Finally, compound 3 was established to be 5-hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethenyl]chromone by a comprehensive analysis of its ²D-NMR data (Figure 2).

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.

3. Materials and Methods

3.1. General Procedures

¹D-and ²D-NMR experiments were recorded on Bruker AVANCE IIITM 600 MHz or Bruker AVIII 500 MHz spectrometers (Bruker, Bremen, Germany). Chemical shifts were referenced to the solvent residual peaks. The HRESIMS were acquired using an API Qstar Pulsar mass spectrometer (Bruker, Bremen, Germany). Optical rotations were recorded on an MCP 5100 polorimeter (Anton Paar, Graz, Austria). IR spectra were measured on a Nicolet 380 FT-IR spectrometer (Thermo, Pittsburgh, PA, America). HPLC analysis was performed on Agilent Technologies 1260 Infinity II (Agilent, Palo Alto, CA, USA) with a reversed-phased column (YMC-packed C18, 5 μm, 250 mm × 10 mm) using a Dionex P680 pump and detected with a Dionex UVD 170 U detector (λ = 254 nm). Silica gel (60–80, 200–300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, China), ODS gel (20–45 μm, Fujian Silysia Chemical Co. Ltd., Fuzhou, China) and Sephadex LH-20 (40–70 μm, Merck, Darmstadt, Germany) were used for column chromatography. TLC was carried out on silica gel G precoated plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, China), and the peaks on TLC were detected by a UV lamp at 254 nm and then sprayed with 5% H₂SO₄ in EtOH. The methanol used for HPLC analysis was of chromatographic grade (Concord Technology Co. Ltd., Tianjin, China). Tacrine hydrochloride hydrate (99%) and paclitaxel (99%) were purchased from Sigma Chemical.

3.2. Plant Material

The agarwood sample was collected in Papua New Guinea, then traded in Macao, one of China′s special administrative regions, in Dec. 2014, and identified as agarwood originating from Gyrinops salicifolia Ridl. by Prof. Dr. Hao-Fu Dai and Dr. Jun Wang (Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences & Hainan engineering research center of agarwood). A voucher specimen (CX 20141222) has been deposited at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

3.3. Extraction and Isolation

The dried agarwood sample (491.1 g) was extracted with 95% EtOH (2 L) for three times at heating reflux and filtered. After removing EtOH under reduced pressure, the crude extract (177.4 g) was obtained and then suspended in H₂O (2 L), subsequently extracted with EtOAc (2 L), followed by n-BuOH (2 L). The EtOAc extract (141.2 g) was subjected to vacuum liquid chromatography with silica gel (10 × 55 cm) using a step gradient of CHCl₃-MeOH (v/v, 1:0, 50:1, 25:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1, 6 L of each) to yield 10 fractions (Fr.1~10). Fr.6 (27.2 g) was applied to ODS column (3 × 40 cm) chromatography with MeOH-H₂O (v/v, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 1:0, 4 L of each) divided to 14 fractions (Fr.6-1~14). Fr.6-3 (85.2 mg) was submitted to Sephadex LH-20 (column: 1.2 × 50 cm) in MeOH to get Fr.6-3-1 (36.0 mg), then purified through silica gel column (1.2 × 40 cm) chromatography with CHCl₃-MeOH (v/v = 60:1) to obtain compound 3 (3.5 mg). Fr.6-7 (182.9 mg) was separated on Sephadex LH-20 (petroleum ether:CHCl₃:MeOH = 1:1:1), and then chromatographed on silica gel with CHCl₃-MeOH (v/v, 100:1, 50:1) to afford compounds 1 (1.1 mg) and 2 (1.1 mg).

Gyrinone A (1): yellow powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ = −8.4 (c 0.05, MeOH); UV (MeOH) 298, 224 nm; IR (KBr) ν_max 3434, 2977, 2922, 1672, 1635, 1400, 1384, 1048 cm⁻¹; ¹H- and ¹³C-NMR data, see Table 1 and Table 2; HRESIMS m/z 499.1730 [M + Na]⁺ (calcd. C₂₈H₂₈NaO₇ for 499.1727).

Gyrinone B (2): yellow amorphous solid; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{25}$ = −11.2 (c 0.04, MeOH); UV (MeOH) 297, 222 nm; IR (KBr) ν_max 3434, 2977, 2921, 1673, 1403, 1387, 1140, 1029 cm⁻¹; ¹H- and ¹³C-NMR data, see Table 1 and Table 2; HRESIMS m/z 529.1839 [M + Na]⁺ (calcd. C₂₉H₃₀NaO₈ for 529.1833).

5-Hydroxy-2-[2-(3-hydroxy-4-methoxyphenyl)ethenyl]chromone (3): yellow powder; UV (MeOH) 383, 225 nm; IR (KBr) ν_max 3440, 2968, 1649, 1602, 1512, 1475, 1411, 1260, 1155, 1131, 1029, 961 cm⁻¹; ¹H- and ¹³C-NMR data, see Table 1 and Table 2; HRESIMS m/z 311.0923 [M + H]⁺ (calcd. C₁₈H₁₅O₅ for 311.0914).

3.4. Bioassays

3.4.1. Bioassay for AChE Inhibitory Activity In Vitro

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 IC₅₀ value of 64.8 nM, and DMSO was served as negative control.

3.4.2. 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 IC₅₀ value of 0.89 µM.

4. Conclusions

Three 2-(2-phenylethyl)chromone derivatives (1–3) were isolated from agarwood originating from Gyrinops salicifolia Ridl. Gyrinones A and B, comprising 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone and benzylacetone units, were elucidated as novel structures. However, bioassay tests of all compounds showed that there was no inhibition effect on AChE inhibitory activity or cytotoxicity against K562 cell line.