Phenylethylchromones with In Vitro Antitumor Promoting Activity from Aquilaria filaria

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Antiproliferative activity assay
• In vitro Epstein-Barr virus early antigen activation experiments
• Supporting Information
• Acknowledgements
• References

Abstract

A new chromone, 2-(2-hydroxy-2-phenylethyl)chromone (1), was isolated together with ten known phenylethyl chromones from MeOH extracts of agarwood (Aquilaria filaria). The selected compounds were evaluated in an antiproliferative assay against five human tumor cell lines, including a multidrug-resistant cell line. They were also tested for antitumor promoting activity, as mediated by 12-O-tetradecanoylphorbol-13-acetate-induced activation of the Epstein-Barr virus early antigen in Raji cells. Among all compounds, 4′,7-dimethyoxy-6-hydroxychromone (2) displayed broad spectrum antiproliferative activity against all tumor cell lines tested with IC₅₀ values of 25–38 µM, while 8 was selectively inhibitory against multidrug-resistant cells. All tested compounds suppressed tumor promotion at noncytotoxic concentrations. 4′,6-Dihydroxyphenylethylchromone (7) exhibited the most potent effect with an IC₅₀ value of 319 mol ratio relative to 12-O-tetradecanoylphorbol-13-acetate. This study is the first to report the antitumor promoting activity of 2-(2-phenylethyl)chromone derivatives, as well as the selective antiproliferative activity of 8 against a multidrug-resistant tumor cell line.

Introduction

2-(2-Phenylethyl)chromone is a member of an unusual class of chromones connected at the C-2 position with a phenyl group through an ethyl linker. Compounds of this type have been isolated from only a few plant species, such as Imperata cylindrica [1], Bothriochloa ischaemum [2], and Cucumis melo var. reticulatus [3]. Agarwood, which is also called eaglewood as well as other vernacular names by different cultures, is the best source of 2-(2-phenylethyl)chromones [4], [5], [6], [7], [8], [9], [10]. Agarwood is produced from some Aquilaria and Gyrinops species by natural or artificial damage, such as fungal infection and cutting. It has been used for centuries as incense and in traditional medicine [11]. More than sixty 2-(2-phenylethyl)chromone derivatives with various substitution patterns of hydroxyl and/or methoxy groups have been isolated from agarwood [5], [12], [13], [14], [15], [16], [17], [18].

In the course of our phytochemical research on rainforest plants, a methanolic extract of Indonesian agarwood showed potent antiproliferative activity against human tumor cell lines with IC₅₀ values ranging from 1.0 to 7.1 µg/mL against lung (A549) and breast (MDA-MB-231) cancer cells, respectively. In addition, the antiproliferative potency was greater against the multidrug-resistant (MDR) cell line KB-VIN than the parent chemosensitive KB cell line. These preliminary results suggest that the crude extract contains multiple bioactive compounds. We report herein the isolation and structure determination of a new phenylethylchromone (1) and ten known chromones from Indonesian agarwood. The isolated phenylethylchromones were tested for antiproliferative activity against five human tumor cell lines, including an MDR cell line, and for antitumor promoting effects, which have not been reported previously.

Results and Discussion

A crude methanolic extract of agarwood was partitioned between EtOAc and water. The EtOAc extract was fractionated by column chromatography to provide 11 phenylethylchromones, including a new compound (1) ([Fig. 1]). Ten known compounds were identified as 6-hydroxy-7-methoxy-2-[2-(4-methoxyphenyl)ethyl]chromone (2) [15], 8-chloro-6-hydroxy-2-(2-phenylethyl)chromone (3) [5], 6-hydroxy-7-methoxy-2-(2-phenylethyl)chromone (4) [16], 6,7-dimethoxy-2-(2-phenylethyl)chromone (5) [8], 6-hydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone (6) [17], 6-hydroxy-2-[2-(4-hydroxyphenyl)ethyl]chromone (7) [16], 6-hydroxy-2-(2-phenylethyl)chromone (8) [8], 8-hydroxy-2-(2-phenylethyl)chromone (9) [18], 6,8-dihydroxy-2-(2-phenylethyl)chromone (10) [16], and 2-(2-phenylethyl)chromone (11) [7] by comparison with their previously reported spectroscopic data.

Compound 1 was obtained as a colorless solid. High-resolution fast atom bombardment MS (HR-FAB-MS) analysis indicated a molecular ion at m/z 267.1020 [M + H]⁺ corresponding to the molecular formula C₁₇H₁₄O₃ (calcd. for C₁₇H₁₅O₃, 267.1021). The ¹H- and ¹³C-NMR spectra were very similar to those of 2-phenethyl-4H-chromen-4-one (11) except the signals of the methylene groups H₂-7′ and H₂-8′, which were replaced in the ¹H NMR spectrum of 1 by an ABX pattern observed at δ _H 3.08 (1H, dd, J = 14.4, 8.4 Hz), 3.01 (1H, dd, J = 14.4, 4.8 Hz), and 5.12 (1H, m) in CDCl₃ ([Table 1]). In addition, a signal for an oxymethine group was present at 72.0 ppm in the ¹³C-NMR spectrum. These data suggested the presence of a hydroxyl group at C-7′ or C-8′. Based on an HMBC correlation between H-3 and C-8′ (δ _c 44.2), compound 1 was determined as 2-(2-hydroxy-2-phenylethyl)-4H-chromen-4-one ([Fig. 2]). A similar 2-phenylethylchromenone with a hydroxyl group at the C7′ position has been identified by Yaguraʼs group [19]. Comparison of the spectroscopic data also supported the structure of compound 1. The configuration of C-7′ was established by the Mosher ester method [20]. Compound 1 was treated with (S)-(+)- and (R)-(−)-α-methoxy-α-trifluoromethyl-phenylacetyl chloride (MTPA-Cl), triethylamine (TEA), and N,N-dimethyl-4-aminopyridine (DMAP) in CH₂Cl₂ to obtain, in each case, a mixture of diastereomeric esters 1a and 1b ([Fig. 3]). From the ¹H-NMR data of the esters, Δδ ^SR (= δ_S – δ_R ) values of relevant protons were calculated (Table 3S, Supporting information). The proton integrals of the peaks corresponding to H-3, H-5, and H-7′ in each ester obtained with (S)-(+)-MTPA-Cl indicated a 4 : 1 mixture of 1a to 1b, respectively. The same ratio was observed for the esters obtained with (R)-(−)-MTPA-Cl, but the intensities of the signals of both diastereomers in the ¹H NMR spectrum were reversed. Thus, 1a obtained with (S)-(+)-MTPA-Cl and 1b obtained with (R)-(−)-MTPA-Cl are enantiomers, and not distinguishable by NMR. These results showed that compound 1 was a 4 : 1 mixture of C-7′ enantiomers, the majority with an (R)-configuration ([Fig. 4]).

Selected compounds 2 and 4–11 were evaluated for antiproliferative activity against five human tumor cell lines, KB (originally isolated from epidermoid carcinoma of the nasopharynx), KB-subline KB-VIN showing an MDR phenotype with overexpression of P-glycoprotein (P-gp), A549 (lung carcinoma), MCF-7 (estrogen receptor-positive and HER2-negative breast cancer), and MDA-MD-231 (triple-negative breast cancer) cell lines ([Table 2]). The antiproliferative effects were assessed by the sulforhodamine B (SRB) assay, and IC₅₀ values were calculated from at least three independent experiments, each performed in duplicate. Most of the 2-(2-phenylethyl)chromones did not exhibit significant antiproliferative activity; however, 4′,7-dimethoxy-6-hydroxy chromone 2 showed moderate antiproliferative activity against all tested tumor cell lines with IC₅₀ values of 25 to 38 µM. Interestingly, chromone 8 displayed selective antiproliferative activity against the P-gp overexpressing MDR tumor cell line KB-VIN with an IC₅₀ value of 19.2 µM, although no activity was observed against the parent KB cell line at 40 µM.

In vitro antitumor promoting activity measured as the inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells generally correlates well with cancer chemopreventive effects in vivo [21]. This correlation has been verified by many of our previous studies using various natural products derivatives, such as betulinic acid [22], [23], dimethyl dicarboxylate biphenyl [24], and coumarins [25], [26]. Because the antitumor promoting effects of phenylethylchromones have not been reported previously, selected compounds 2–4 and 7–11 were examined with the EBV-EA activation assay in Raji cells ([Table 3]). All tested compounds showed low cytotoxicity against Raji cells, as exhibited by less than 40 % growth inhibition (60 % viability of Raji cells), even at the highest concentration (32 µM) of the compound (a compound/TPA molar ratio of 1000 : 1) (data not shown). Among them, chromone 7 displayed the most significant inhibitory activity with an IC₅₀ value of 319 mol ratio/32 pmol TPA. The percentages of EBV-EA positive cells within the growing cells were 5.3, 33.4, 76.2, and 100 %, meaning 94.7, 66.6, 23.8, and 0 % inhibition of activation at concentrations of 1000, 500, 100, and 10 mol ratio/TPA, respectively. From our previous observations, the presence of a hydroxyl group tends to increase the inhibitory potency. This study also suggests that the hydroxyl group is important for antitumor promoting activity, since unsubstituted chromone 11 exhibited lower activity than compounds containing a hydroxyl group. Furthermore, a compound with a chlorine atom at the 8-position in addition to a hydroxyl group at the 6-position was less potent than its analogous non-chlorinated compound (compare 3 vs. 8). Comparison of 3 and 10 also suggested that the hydroxyl is favored, while the halogen function is disfavored. This result supported our previous studies showing that a halogen is not preferred for antitumor promoting activity. The presence of a hydroxyl group(s) could improve the compoundsʼ solubility in the medium, thus positively affecting the activity. In fact, good correlations of CLogP values and activities were observed ([Table 3]). Finally, a hydroxyl group at the 4′-position is important for antitumor promoting activity, since chromone 6 with a methoxy rather than a hydroxyl group at this position exhibited lower activity (IC₅₀ value of 486 mol ratio/32 pmol TPA) than 7.

Materials and Methods

General experimental procedures

All solvents were used as purchased. NMR spectra were recorded on a JNM-ECS400 or JNM-ECA600 spectrometer with TMS as the internal standard. Chemical shifts are reported in ppm and apparent scalar coupling constants J in Hz. High-resolution mass spectroscopic data were obtained on a JMS-700 Mstation (FAB) or JMS-T100TD (DART) mass spectrometer. Infrared spectra were measured with a SHIMADZU FTIR-8700 instrument in CHCl₃. Optical rotations were recorded on a JASCO P-2200 digital polarimeter. Analytical TLC was carried out on Merck precoated glass silica gel sheets (TLC Silica gel 60 RP-18F-254S). Column chromatography was performed with silica gel 60 N (spherical, 63–210 µm, neutral, Kanto chemical). Silica gel 60 RP-18 F254S (0.25 mm) for reverse-phased and 60 F254 (1 mm) for normal-phased preparative TLCs (Merck) were used.

Plant material

Agarwoods were collected from Fak-Fak, West Papua Province, Indonesia, in September 2012, and were identified as Aquilaria filaria (Oken) Merr. (Thymelaeaceae) by one of the authors (Y. S). A voucher of specimen (KNG-FD-04) has been deposited in the Laboratory of Molecular Pharmacognosy, School of Pharmacy, School of Pharmaceutical Science, Kanazawa University, Japan.

Extraction and isolation

Air-dried and powdered agarwoods (390 g) were extracted with 95 % MeOH (5.0 L) at room temperature for 72 h. The extract was concentrated in vacuo to yield a semisolid (33.8 g), from which 6.2 g was further partitioned between EtOAc and water. The EtOAc-soluble fraction (4.0 g) was subjected to column chromatography (CC) on silica gel eluted with n-hexane–EtOAc–MeOH mixtures (n-hexane; n-hexane–EtOAc, 4 : 1 to 1 : 2; EtOAc; EtOAc–MeOH, 19 : 1 and 9 : 1; MeOH) to afford 35 fractions of 45 mL each. The fractions with a similar TLC profile were combined and resulted in 11 fractions (F1–11). Fraction 5 (F5, 140.3 mg) was further fractionated by CC on silica gel using n-hexane–EtOAc (from 9 : 1 to 0 : 1, v/v) as the eluting solvent to yield 11 (4.2 mg). Fraction 6 (F6, 41.5 mg) was subjected to CC on silica gel by gradient elution using CH₂Cl₂-EtOAc (from 19 : 1 to 0 : 1, v/v) to provide five fractions (F6-1 to F6-5). F6-3 (6.7 mg) and F6-4 (15.7 mg) were further purified on preparative reversed-phase TLC with MeOH-H₂O (2 : 1 and 9 : 11, v/v) to yield 9 (2.6 mg) and 3 (1.2 mg), respectively. Fraction 7 (F7, 165.0 mg) was also subjected to CC on silica gel by gradient elution using n-hexane–acetone (from 5 : 1 to 0 : 1, v/v) to provide seven fractions (F7-1 to F7-7). F7-3 (39.1 mg) was further fractionated by CC on silica gel using CH₂Cl₂-EtOAc (from 1 : 0 to 0 : 1, v/v) as the eluting solvent to give eight fractions (F7-3–1 to F7-3–8). Compound 5 (1.1 mg) was isolated from F7-3–4 (4.7 mg) by preparative reversed-phase TLC with MeOH-H₂O (6 : 5 and 2 : 1, v/v). F7-3–6 (3.3 mg) was further purified over preparative reversed-phase TLC with MeOH-H₂O (2 : 1 and 1 : 1, v/v) to yield 1 (0.6 mg). Compound 8 (15.0 mg) was isolated from F7-4 (35.8 mg) by recrystallization with EtOAc. Compound 6 (1.6 mg) was obtained from F7-5 (27.1 mg) by repeated CC on silica gel using n-hexane–acetone (from 3 : 1 to 0 : 1, v/v) and then preparative TLC on silica gel with CH₂Cl₂-EtOAc (from 5 : 1 to 10 : 3, v/v). Fraction 8 (F8, 387.5 mg) was separated into six fractions (F8-1 to F8-6) using CC on silica gel eluted with an n-hexane–EtOAc mixture (from 1 : 0 to 0 : 1, v/v). F8-2 (132.1 mg) was further purified by CC on silica gel using n-hexane–acetone (5 : 1, v/v) as the eluting solvent to provide 10 fractions (F8-2–1 to F8-2–10). Compound 4 (2.3 mg) was isolated from F8-2–6 (24.8 mg) by recrystallization (EtOAc). F8-2–8 (25.9 mg) was subjected to CC on silica gel by gradient elution using n-hexane–acetone (from 4 : 1 to 0 : 1, v/v) repeatedly to give five fractions (F8-2–9–1 to − 5). F8-2 − 9 −3 (5.2 mg) was purified using preparative reversed-phase TLC with MeOH-H₂O (3 : 2, v/v) to yield 2 (1.8 mg), 10 (3.2 mg), and 7 (0.8 mg).

2-(2-Hydroxy-2-phenylethyl)-4H-chromen-4-one (1)

Colorless solid; [α]_D ²⁵ − 8.8 (c 0.025, MeOH); IR: 3630, 3013, 2943, 1711, 1647, 1219, 1016, 745, 731, 669 cm⁻¹; ¹H NMR (CDCl₃ and CD₃OD, 600 MHz) and ¹³C NMR (CDCl₃, 150 MHz), see [Table 1]; HR-FAB-MS m/z 267.1020 [M + H]⁺ (calcd for C₁₇H₁₅O₃ 267.1021).

2-(2-Phenylethyl)chromones 2–11

Spectroscopic data are provided as Supporting Information.

Reaction of compound 1 with (S)-MTPA-Cl: Compound 1 (0.6 mg), (S)-MTPA-Cl (0.9 µl, 2.0 eq.), TEA (0.9 µl, 2.6 eq.), and DMAP (0.73 mg, 2.6 eq.) in CH₂Cl₂ (150 µl) were stirred at room temperature for 1.5 h. The mixture was concentrated under reduced pressure and purified by preparative TLC on silica gel n-hexane-EtOAc (2 : 1, v/v) to obtain compounds 1a and 1b as a 4 : 1 mixture (0.7 mg, 60 %) along with starting material (0.2 mg, 33 %).

Mixture of compounds 1a and 1b: ¹H-NMR (600 MHz, acetone-d ₆) δ _H: 8.05 and 8.08 (1H, dd, J = 7.8, 1.8 Hz, H-5 for 1a and 1b, 4 : 1), 7.78 and 7.79 (1H, m, H-7 for 1a and 1b, 4 : 1), 7.61 and 7.42 (2H, m, H-2′, 6′ for 1a and 1b, 4 : 1), 7.52 and 7.56 (1H, d, J = 8.1 Hz, H-8 for 1a and 1b, 4 : 1), 6.58 and 6.53 (1H, dd, J = 9.6, 4.8 Hz, H-7′ for 1a and 1b, 4 : 1), and 6.14 and 6.29 (1H, s, H-3 for 1a and 1b, 4 : 1).

Reaction of compound 1 with (R)-MTPA-Cl: Compound 1 (0.2 mg), which was recovered from the above reaction, (R)-MTPA-Cl (0.9 µl, 6.0 eq.), TEA (0.9 µl, 7.8 eq.), and DMAP (0.73 mg, 7.8 eq.) in CH₂Cl₂ (150 µl) were treated under the same conditions as described above to obtain a 4 : 1 mixture of compounds 1a and 1b (0.3 mg, 77 %). Chemical shifts were identical but the intensities of the signals were reversed compared to 1a and 1b obtained with (S)-MTPA-Cl.

Antiproliferative activity assay

Antiproliferative activity assay was performed as described previously [27]. In brief, freshly trypsinized cell suspensions were seeded in 96-well microtiter plates at densities of 4000–12 000 cells per well with compounds. The highest concentration of DMSO in the cultures (0.1 % v/v) used for the antiproliferative activity assay was without effect on cell growth under the culture conditions used. Paclitaxel (Sigma-Aldrich, purity > 95 %) was used as a reference compound. After 72 h in culture with test compounds, attached cells were fixed with 10 % trichloroacetic acid and then stained with 0.04 % sulforhodamine B. After solubilizing the protein-bound dye with 10 mM Tris base, absorbance at 515 nm was measured using a microplate reader (ELx800, BioTek) with Gen5 software (BioTek). The mean IC₅₀ is the concentration of agent that reduced cell growth by 50 % compared with vehicle (DMSO) control under the experimental conditions used and is the average from at least three independent experiments with duplicate samples (N = 6). All values presented in [Table 2] were calculated by Excel (Microsoft). The following human tumor cell lines were used in the assay: A549 (lung carcinoma), KB (originally isolated from epidermoid carcinoma of the nasopharynx), KB-VIN [vincristine (VIN)-resistant KB subline showing MDR phenotype by overexpressing P-gp], MCF-7 (estrogen receptor-positive, HER2-negative breast cancer), and MDA-MB-231 (estrogen receptor-negative, progesterone receptor-negative, HER2-negative breast cancer). All cell lines were obtained from the Lineberger Comprehensive Cancer Center (UNC-CH) or from ATCC, except KB-VIN, which was a gift of Professor Y.-C. Cheng (Yale University). Cells were cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine and 25 mM HEPES (CORNING), supplemented with 10 % heat-inactivated FBS (HyClone), 100 µg/mL streptomycin, 100 IU/mL penicillin, and 0.25 µg/mL amphotericin B (CORNING). MDR stock cells (KB-VIN) were maintained in the presence of 100 nM VIN.

In vitro Epstein-Barr virus early antigen activation experiments

EBV-EA positive serum from a patient with nasopharyngeal carcinoma (NPC) was a gift from Professor H. Hattori, Department of Otorhinolaryngology, Kobe University. The EBV genome carrying lymphoblastoid cells (Raji cells derived from Burkittʼs lymphoma) were cultured in 10 % FBS in RPMI-1640 medium (Sigma R8758). Spontaneous activation of EBV-EA in our subline of Raji cells was less than 0.1 %. The inhibition of EBV-EA activation was assayed using Raji cells (virus non-producer type) as described below. The cells were incubated at 37 °C for 48 h in 1 mL of medium containing n-butyric acid (4 mM), TPA (32 pM = 20 ng in 1 µL DMSO), and various amounts of the test compounds dissolved in 2 µL of DMSO. mears were made from the cell suspension. The EBV-EA inducing cells were stained by means of an indirect immunofluorescence technique. In each assay, at least 500 cells were counted, and the number of stained cells (positive cells) was recorded. Triplicate assays were performed for each compound. Under this condition, EBV-EV induction was ordinarily around 35 % of viable cells. The average EBV-EA induction of the test compound is expressed as a ratio relative to the control experiment, which was carried out with 32 pM TPA in the presence of the vehicle (DMSO). The viability of treated Raji cells was evaluated by a trypan blue assay.

Supporting Information

NMR and IR spectra of compound 1, NMR data of compounds 2–11, and the Δδ (= δ_S – δ_R ) values for Mosher esters of 1 are available as Supporting Information.

Acknowledgements

This study was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT KAKENHI, Japan) awarded to K. N. G. (Grant Number 25 293 024 & 25 670 054) and by a grant from the IBM Junior Faculty Development Awards as well as the University Research Council (UNC) awarded to M. G.

Conflict of Interest

The authors declare no conflict of interest.

• References

• 1 Wang T, Li LF, Zhang K, Zhang WY, Pei YH. New 2-(2-phenylethyl)chromones from Bothriochloa ischaemum . J Asian Nat Prod Res 2001; 3: 145-149
  CrossrefPubMedSearch in Google Scholar
• 2 Yoon JS, Lee MK, Sung SH, Kim YC. Neuroprotective 2-(2-phenylethyl)chromones of Imperata cylindrica . J Nat Prod 2006; 69: 290-291
  CrossrefPubMedSearch in Google Scholar
• 3 Ibrahim SR. New 2-(2-phenylethyl)chromone derivatives from the seeds of Cucumis melo L var. reticulatus . Nat Prod Commun 2010; 5: 403-406
  PubMedSearch in Google Scholar
• 4 Dai HF, Liu J, Han Z, Zeng YB, Wang H, Mei WL. Two new 2-(2-phenylethyl)chromones from Chinese eaglewood. J Asian Nat Prod Res 2010; 12: 134-137
  CrossrefPubMedSearch in Google Scholar
• 5 Gao YH, Liu JM, Lu HX, Wei ZX. Two new 2-(2-phenylethyl)chromen-4-ones from Aquilaria sinensis (Lour.) Gilg. Helv Chim Acta 2012; 95: 951-954
  CrossrefPubMedSearch in Google Scholar
• 6 Liu J, Wu J, Zhao YX, Deng YY, Mei WL, Dai HF. A new cytotoxic 2-(2-phenylethyl)chromone from Chinese eaglewood. Chin Chem Lett 2008; 19: 934-936
  CrossrefPubMedSearch in Google Scholar
• 7 Hashimoto K, Nakahara S, Inoue T, Sumida Y, Takahashi M, Masada Y. A new chromone from agarwood and pyrolysis products of chromone derivatives. Chem Pharm Bull 1985; 33: 5088-5091
  CrossrefPubMedSearch in Google Scholar
• 8 Shimada Y, Tominaga T, Konishi T, Kiyosawa S. Studies on the agarwood (Jinko). I. Structures of 2-(2-phenylethyl)chromone derivatives. Chem Pharm Bull 1982; 30: 3791-3795
  CrossrefPubMedSearch in Google Scholar
• 9 Yagura T, Shibayama N, Ito M, Kiuchi F, Honda G. Three novel diepoxy tetrahydrochromones from agarwood artificially produced by intentional wounding. Tetrahedron Lett 2005; 46: 4395-4398
  CrossrefPubMedSearch in Google Scholar
• 10 Yang L, Qiao L, Xie D, Yuan Y, Chen N, Dai J, Guo SX. 2-(2-Phenylethyl)chromones from Chinese eaglewood. Phytochemistry 2012; 76: 92-97
  CrossrefPubMedSearch in Google Scholar
• 11 Okudera Y, Ito M. Production of agarwood fragrant constituents in Aquilaria calli and cell suspension cultures. Plant Biotechnol 2009; 26: 307-315
  CrossrefPubMedSearch in Google Scholar
• 12 Chen D, Xu Z, Chai X, Zeng K, Jia Y, Bi D, Ma Z, Tu P. Nine 2-(2-phenylethyl)chromone derivatives from the resinous wood of Aquilaria sinensis and their inhibition of LPS-induced NO production in RAW 264.7 cells. Eur J Org Chem 2012; 2012: 5389-5397
  CrossrefPubMedSearch in Google Scholar
• 13 Wu B, Kwon SW, Hwang GS, Park JH. Eight new 2-(2-phenylethyl)chromone (= 2-(2-phenylethyl)-4H-1-benzopyran-4-one) derivatives from Aquilaria malaccensis agarwood. Helv Chim Acta 2012; 95: 1657-1665
  CrossrefPubMedSearch in Google Scholar
• 14 Li W, Cai CH, Dong WH, Guo ZK, Wang H, Mei WL, Dai HF. 2-(2-Phenylethyl)chromone derivatives from Chinese agarwood induced by artificial holing. Fitoterapia 2014; 98: 117-123
  CrossrefPubMedSearch in Google Scholar
• 15 Liao G, Mei WL, Dong W, Li W, Wang P, Kong FD, Gai CJ, Song XQ, Dai HF. 2-(2-Phenylethyl)chromone derivatives in artificial agarwood from Aquilaria sinensis . Fitoterapia 2016; 110: 38-43
  CrossrefPubMedSearch in Google Scholar
• 16 Konishi T, Konoshima T, Shimada Y, Kiyosawa S. Six new 2-(2-phenylethyl)chromones from agarwood. Chem Pharm Bull (Tokyo) 2002; 50: 419-422
  CrossrefPubMedSearch in Google Scholar
• 17 Yang DL, Mei WL, Zeng YB, Guo ZK, Zhao YX, Wang H, Zuo WJ, Dong WH, Wang QH, Dai HF. 2-(2-Phenylethyl)chromone derivatives in Chinese agarwood “Qi-Nan” from Aquilaria sinensis . Planta Med 2013; 79: 1329-1334
  Thieme ConnectPubMedSearch in Google Scholar
• 18 Yang DL, Wang H, Guo ZK, Dong WH, Mei WL, Dai HF. A new 2-(2-phenylethyl)chromone derivative in Chinese agarwood ʼQi-Nanʼ from Aquilaria sinensis . J Asian Nat Prod Res 2014; 16: 770-776
  CrossrefPubMedSearch in Google Scholar
• 19 Yagura T, Ito M, Kiuchi F, Honda G, Shimada Y. Four new 2-(2-phenylethyl)chromone derivatives from withered wood of Aquilaria sinensis . Chem Pharm Bull (Tokyo) 2003; 51: 560-564
  CrossrefPubMedSearch in Google Scholar
• 20 Hoye TR, Jeffrey CS, Shao F. Mosher ester analysis for the determination of absolute configuration of stereogenic (chiral) carbinol carbons. Nat Protoc 2007; 2: 2451-2458
  CrossrefPubMedSearch in Google Scholar
• 21 Takasaki M, Konoshima T, Kozuka M, Tokuda H. Anti-tumor promoting activities of euglobals from Eucalyptus plants. Biol Pharm Bull 1995; 18: 435-438
  CrossrefPubMedSearch in Google Scholar
• 22 Nakagawa-Goto K, Yamada K, Taniguchi M, Tokuda H, Lee KH. Cancer preventive agents 9. Betulinic acid derivatives as potent cancer chemopreventive agents. Bioorg Med Chem Lett 2009; 19: 3378-3381
  CrossrefPubMedSearch in Google Scholar
• 23 Hung HY, Nakagawa-Goto K, Tokuda H, Iida A, Suzuki N, Qian K, Lee KH. A-ring modified betulinic acid derivatives as potent cancer preventive agents. Bioorg Med Chem Lett 2014; 24: 1005-1008
  CrossrefPubMedSearch in Google Scholar
• 24 Hung HY, Nakagawa-Goto K, Tokuda H, Iida A, Suzuki N, Morris-Natschke SL, Lee KH. Cancer preventive agents 11. Novel analogs of dimethyl dicarboxylate biphenyl as potent cancer chemopreventive agents. Pharm Biol 2012; 50: 18-24
  CrossrefPubMedSearch in Google Scholar
• 25 Suzuki M, Nakagawa-Goto K, Nakamura S, Tokuda H, Morris-Natschke SL, Kozuka M, Nishino H, Lee KH. Cancer preventive agents. Antitumor-promoting effects of coumarins and related compounds on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis. Pharm Biol 2006; 44: 178-182
  CrossrefPubMedSearch in Google Scholar
• 26 Wang X, Nakagawa-Goto K, Kozuka M, Tokuda H, Nishino H, Lee KH. Cancer preventive agents. Part 6: Chemopreventive potential of furanocoumarins and related compounds. Pharm Biol 2006; 44: 116-120
  CrossrefPubMedSearch in Google Scholar
• 27 Nakagawa-Goto K, Oda A, Hamel E, Ohkoshi E, Lee KH, Goto M. Development of a novel class of tubulin inhibitor from desmosdumotin B with a hydroxylated bicyclic B-ring. J Med Chem 2015; 58: 2378-2389
  CrossrefPubMedSearch in Google Scholar

Correspondence

• References

• 1 Wang T, Li LF, Zhang K, Zhang WY, Pei YH. New 2-(2-phenylethyl)chromones from Bothriochloa ischaemum . J Asian Nat Prod Res 2001; 3: 145-149
  CrossrefPubMedSearch in Google Scholar
• 2 Yoon JS, Lee MK, Sung SH, Kim YC. Neuroprotective 2-(2-phenylethyl)chromones of Imperata cylindrica . J Nat Prod 2006; 69: 290-291
  CrossrefPubMedSearch in Google Scholar
• 3 Ibrahim SR. New 2-(2-phenylethyl)chromone derivatives from the seeds of Cucumis melo L var. reticulatus . Nat Prod Commun 2010; 5: 403-406
  PubMedSearch in Google Scholar
• 4 Dai HF, Liu J, Han Z, Zeng YB, Wang H, Mei WL. Two new 2-(2-phenylethyl)chromones from Chinese eaglewood. J Asian Nat Prod Res 2010; 12: 134-137
  CrossrefPubMedSearch in Google Scholar
• 5 Gao YH, Liu JM, Lu HX, Wei ZX. Two new 2-(2-phenylethyl)chromen-4-ones from Aquilaria sinensis (Lour.) Gilg. Helv Chim Acta 2012; 95: 951-954
  CrossrefPubMedSearch in Google Scholar
• 6 Liu J, Wu J, Zhao YX, Deng YY, Mei WL, Dai HF. A new cytotoxic 2-(2-phenylethyl)chromone from Chinese eaglewood. Chin Chem Lett 2008; 19: 934-936
  CrossrefPubMedSearch in Google Scholar
• 7 Hashimoto K, Nakahara S, Inoue T, Sumida Y, Takahashi M, Masada Y. A new chromone from agarwood and pyrolysis products of chromone derivatives. Chem Pharm Bull 1985; 33: 5088-5091
  CrossrefPubMedSearch in Google Scholar
• 8 Shimada Y, Tominaga T, Konishi T, Kiyosawa S. Studies on the agarwood (Jinko). I. Structures of 2-(2-phenylethyl)chromone derivatives. Chem Pharm Bull 1982; 30: 3791-3795
  CrossrefPubMedSearch in Google Scholar
• 9 Yagura T, Shibayama N, Ito M, Kiuchi F, Honda G. Three novel diepoxy tetrahydrochromones from agarwood artificially produced by intentional wounding. Tetrahedron Lett 2005; 46: 4395-4398
  CrossrefPubMedSearch in Google Scholar
• 10 Yang L, Qiao L, Xie D, Yuan Y, Chen N, Dai J, Guo SX. 2-(2-Phenylethyl)chromones from Chinese eaglewood. Phytochemistry 2012; 76: 92-97
  CrossrefPubMedSearch in Google Scholar
• 11 Okudera Y, Ito M. Production of agarwood fragrant constituents in Aquilaria calli and cell suspension cultures. Plant Biotechnol 2009; 26: 307-315
  CrossrefPubMedSearch in Google Scholar
• 12 Chen D, Xu Z, Chai X, Zeng K, Jia Y, Bi D, Ma Z, Tu P. Nine 2-(2-phenylethyl)chromone derivatives from the resinous wood of Aquilaria sinensis and their inhibition of LPS-induced NO production in RAW 264.7 cells. Eur J Org Chem 2012; 2012: 5389-5397
  CrossrefPubMedSearch in Google Scholar
• 13 Wu B, Kwon SW, Hwang GS, Park JH. Eight new 2-(2-phenylethyl)chromone (= 2-(2-phenylethyl)-4H-1-benzopyran-4-one) derivatives from Aquilaria malaccensis agarwood. Helv Chim Acta 2012; 95: 1657-1665
  CrossrefPubMedSearch in Google Scholar
• 14 Li W, Cai CH, Dong WH, Guo ZK, Wang H, Mei WL, Dai HF. 2-(2-Phenylethyl)chromone derivatives from Chinese agarwood induced by artificial holing. Fitoterapia 2014; 98: 117-123
  CrossrefPubMedSearch in Google Scholar
• 15 Liao G, Mei WL, Dong W, Li W, Wang P, Kong FD, Gai CJ, Song XQ, Dai HF. 2-(2-Phenylethyl)chromone derivatives in artificial agarwood from Aquilaria sinensis . Fitoterapia 2016; 110: 38-43
  CrossrefPubMedSearch in Google Scholar
• 16 Konishi T, Konoshima T, Shimada Y, Kiyosawa S. Six new 2-(2-phenylethyl)chromones from agarwood. Chem Pharm Bull (Tokyo) 2002; 50: 419-422
  CrossrefPubMedSearch in Google Scholar
• 17 Yang DL, Mei WL, Zeng YB, Guo ZK, Zhao YX, Wang H, Zuo WJ, Dong WH, Wang QH, Dai HF. 2-(2-Phenylethyl)chromone derivatives in Chinese agarwood “Qi-Nan” from Aquilaria sinensis . Planta Med 2013; 79: 1329-1334
  Thieme ConnectPubMedSearch in Google Scholar
• 18 Yang DL, Wang H, Guo ZK, Dong WH, Mei WL, Dai HF. A new 2-(2-phenylethyl)chromone derivative in Chinese agarwood ʼQi-Nanʼ from Aquilaria sinensis . J Asian Nat Prod Res 2014; 16: 770-776
  CrossrefPubMedSearch in Google Scholar
• 19 Yagura T, Ito M, Kiuchi F, Honda G, Shimada Y. Four new 2-(2-phenylethyl)chromone derivatives from withered wood of Aquilaria sinensis . Chem Pharm Bull (Tokyo) 2003; 51: 560-564
  CrossrefPubMedSearch in Google Scholar
• 20 Hoye TR, Jeffrey CS, Shao F. Mosher ester analysis for the determination of absolute configuration of stereogenic (chiral) carbinol carbons. Nat Protoc 2007; 2: 2451-2458
  CrossrefPubMedSearch in Google Scholar
• 21 Takasaki M, Konoshima T, Kozuka M, Tokuda H. Anti-tumor promoting activities of euglobals from Eucalyptus plants. Biol Pharm Bull 1995; 18: 435-438
  CrossrefPubMedSearch in Google Scholar
• 22 Nakagawa-Goto K, Yamada K, Taniguchi M, Tokuda H, Lee KH. Cancer preventive agents 9. Betulinic acid derivatives as potent cancer chemopreventive agents. Bioorg Med Chem Lett 2009; 19: 3378-3381
  CrossrefPubMedSearch in Google Scholar
• 23 Hung HY, Nakagawa-Goto K, Tokuda H, Iida A, Suzuki N, Qian K, Lee KH. A-ring modified betulinic acid derivatives as potent cancer preventive agents. Bioorg Med Chem Lett 2014; 24: 1005-1008
  CrossrefPubMedSearch in Google Scholar
• 24 Hung HY, Nakagawa-Goto K, Tokuda H, Iida A, Suzuki N, Morris-Natschke SL, Lee KH. Cancer preventive agents 11. Novel analogs of dimethyl dicarboxylate biphenyl as potent cancer chemopreventive agents. Pharm Biol 2012; 50: 18-24
  CrossrefPubMedSearch in Google Scholar
• 25 Suzuki M, Nakagawa-Goto K, Nakamura S, Tokuda H, Morris-Natschke SL, Kozuka M, Nishino H, Lee KH. Cancer preventive agents. Antitumor-promoting effects of coumarins and related compounds on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis. Pharm Biol 2006; 44: 178-182
  CrossrefPubMedSearch in Google Scholar
• 26 Wang X, Nakagawa-Goto K, Kozuka M, Tokuda H, Nishino H, Lee KH. Cancer preventive agents. Part 6: Chemopreventive potential of furanocoumarins and related compounds. Pharm Biol 2006; 44: 116-120
  CrossrefPubMedSearch in Google Scholar
• 27 Nakagawa-Goto K, Oda A, Hamel E, Ohkoshi E, Lee KH, Goto M. Development of a novel class of tubulin inhibitor from desmosdumotin B with a hydroxylated bicyclic B-ring. J Med Chem 2015; 58: 2378-2389
  CrossrefPubMedSearch in Google Scholar

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