English

• 1.   Introduction

  Chrysin is a flavonoid compound extracted from the Senmen oroxyli of the genus Dictyophora. It exhibits a wide range of biological activities including antioxidant, ^[1] antiviral, ^[2] antiallergy, ^[3] anti-inflammatory, ^[4] antianxiety^[5] and antiproliferative^[6~9] effects. The antiproliferative activities of chrysin and its derivatives have attracted considerable attention in the recent years. Other biochemical properties of chrysin, such as low solubility in vitro and low bioavailability in vivo, restrict its clinical application.^{[10, 11]} Therefore, the discovery of highly active derivatives via the structural modification of chrysin during the development of antiproliferative agents is promising.

  Triazole fragments are important pharmacophores that display diverse biological functions, especially antiproliferative activities. The introduction of triazole fragments can improve the pharmacokinetic property, and biological activities of the parent compounds.^[12] Many reports have shown that introducing 1, 2, 3-triazole or 1, 2, 4-triazole fragments in different natural products can improve their antiproliferative activities. For example, compound A (Figure 1) exhibited a significantly improved antiproliferative activity when compared with the parent compound.^[13] Furthermore, Liu et al.^[14] reported a series of compounds linked to 1, 2, 4-triazole, and as the result, compound B showed promising antiproliferative activities against different types of cancer cell lines.

  Figure 1

  

  Figure 1.  Design of target compounds based on the combination principles

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  Previous studies showed that the structural modification in the 7 hydroxyl group can improve the bioactivity of chrysin.^[15] In this study, we synthesized two series of novel chrysin derivatives, linking 1, 2, 3-triazole and 1, 2, 4-tria- zole fragments in the 7-OH position. The antiproliferative activity of the target compounds was evaluated on human gastric cancer (MGC-803), human hepatocellular carcinoma (BEL-7402), human liver cancer (HepG2), human cervical cancer (HeLa), human lung cancer (A549), and human gastric cancer (SGC-7901).

  2.   Results and discussion

  2.1   Chemistry

  Intermediate Ⅰ was prepared by the nucleophilic substitution reaction of chrysin with propargyl bromide, and compounds 1a~1h were obtained by a click reaction of intermediate Ⅰ with different substitutions of 1-(azidome- thyl)benzene (yield: 31.3%~41.2%). Intermediate Ⅱ was prepared by the nucleophilic substitution reaction of chrysin with ethyl bromoacetate, and intermediate Ⅲ was generated from intermediate Ⅱ and hydrazine hydrate. Compounds 2a~2k were products of the reaction of intermediate Ⅲ with different substitutions of aniline at 82 ℃. The yields of 2a~2k were in the range of 10.3%~25.5%.

  2.2   Evaluation of the bioactivity

  As summarized in Table 1, compounds 1a, 1b, 1c, and 1g exhibited stronger antiproliferative activities against the MGC-803 cell line than the lead compound chrysin at 100 μmol/L, while compounds 1e, 1h, and 2b showed a consi- derably higher antiproliferative activity against the BEL-7402 cell line. However, most compounds lost their antiproliferative activity at lower concentrations and only four compounds had an IC₅₀ value (< 100 μmol/L) lower than that of chrysin against the MGC-803 cell line.

  Table 1

  

  Table 1.  Antiproliferative activity of compounds^a

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  Compd.	Growth inhibition/% at 100 μmol/L
  	MGC-803	BEL-7402	HepG-2	HeLa	A549	SGC-7901
  1a	66.83±1.46	33.18±0.81	14.20±0.22	31.90±0.99	44.67±0.72	19.31±0.65
  1b	52.71±0.71	44.01±0.77	11.89±0.86	24.42±1.01	41.20±1.55	47.68±1.36
  1c	52.82±2.25	3.31±0.07	1.02±0.02	16.81±0.17	27.89±0.56	7.30±0.14
  1d	41.52±1.12	31.44±0.88	4.91±0.05	24.40±0.26	27.89±0.19	12.78±0.18
  1e	33.81±0.25	98.18±1.35	6.81±0.07	6.32±0.02	33.91±0.67	12.64±0.13
  1f	10.59±0.08	12.80±0.14	7.43±0.25	16.70±0.09	43.46±0.22	20.98±0.15
  1g	74.28±1.68	17.44±0.12	33.02±0.26	35.07±0.18	50.71±0.87	45.64±0.88
  1h	78.33±0.36	97.60±0.98	12.92±0.11	4.11±0.17	29.13±0.14	6.44±0.12
  2a	27.33±0.11	57.24±0.76	34.01±0.33	27.30±0.05	39.49±0.17	51.71±1.00
  2b	44.51±0.57	78.26±1.77	5.11±0.02	26.81±0.18	9.01±0.07	6.81±0.05
  2c	10.72±0.12	70.70±1.02	4.52±0.12	26.42±0.26	15.01±0.07	8.22±0.12
  2d	34.62±0.23	15.49±0.11	88.40±1.66	29.33±0.20	44.11±0.31	49.22±0.32
  2e	25.21±0.21	32.30±0.33	7.50±0.08	35.41±0.37	31.11±0.33	19.70±0.26
  2f	22.62±0.24	44.33±0.08	15.62±0.12	40.92±0.40	37.80±0.08	37.71±0.37
  2g	31.71±0.15	19.01±0.12	31.02±0.24	20.10±0.20	27.90±0.13	18.41±0.10
  2h	16.80±0.10	35.71±0.11	38.79±0.31	46.50±0.40	43.01±0.23	47.83±0.32
  2i	42.11±0.34	49.81±0.37	38.82±0.21	35.51±0.13	30.11±0.13	51.09±0.05
  2j	36.89±0.33	57.64±0.32	51.60±0.22	30.49±0.61	46.02±0.64	47.11±0.69
  2k	21.73±0.27	29.44±0.04	58.11±0.97	38.22±0.22	44.52±0.12	36.54±0.20
  Chrysin	50.89±0.50	73.02±0.23	39.33±0.22	62.89±0.33	8.80±0.04	16.30±0.13
  a Growth inhibitions are presented as the mean±SD (standard error of the mean) from three separated experiment.

  Scheme 1

  

  Scheme 1.  Synthetic routes of the target compounds

  1a: R＝H, 1b: R＝4-F, 1c: R＝3-F, 1d: R＝2-F, 1e: R＝4-Cl, 1f: R＝3-Cl, 1g: R＝2-Cl, 1h: R＝4-CH₃; 2a: R＝H, 2b: R＝4-F, 2c: R＝3-F, 2d: R＝2-F, 2e: R＝4-Cl, 2f: R＝3-Cl, 2g: R＝2-Cl, 2h: R＝3-Br, 2i: R＝4-CH₃, 2j: R＝3-CH₃, 2j: R＝4-OCH₃
  Reagents and conditions: (a) propargyl bromide, K₂CO₃, CH₃COCH₃, 57 ℃; (b) different substitutions of 1-(azidomethyl)benzene, CuSO₄• 5H₂O, sodium ascorbate, t-BuOH/H₂O (V:V＝1:1), 30 ℃; (c) ethyl bromoacetate, K₂CO₃, CH₃COCH₃, 57 ℃; (d) N₂H₄•H₂O, CH₃CH₂OH, 78 ℃; (e) DMFDMA, CH₃CN, different substitutions of aniline, CH₃COOH, 82 ℃

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  As listed in Table 2, only compounds 1a, 1b, 1c and 1g exhibited higher antiproliferative activities, indicating that the antiproliferative activity induced by introducing 1, 2, 3- triazole in the 7-hydroxyl group of chrysin was better than that induced by the introduction of 1, 2, 4-triazole. Among them, compounds 1c and 1g exhibited significant antiproliferative potency against the MGC-803 cell line with IC₅₀ values of (18.4±0.14) and (5.92±0.02) μmol/L, respectively, which were even better than that of 5-fluorouracil (5-FU). Compound 1g, with a chlorine atom introduced at the ortho position of the benzene ring, had the strongest antiproliferative activity among all target compounds, and improved potency up to two digits μmol/L compared with that of chrysin. It is possible that the chlorine atom acts as a hydrogen bond acceptor and binds to some key proteins in- volved in cancer cell metabolism, inhibiting their ex- pression. Compound 1g was 5.2-folds more active than 5-FU, which demonstrated that it may be a valuable candidate for further studies.

  Table 2

  

  Table 2.  IC₅₀ values (μmol/L) of several active compounds

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  Compd.	IC50 against MGC-803 cell lines/(μmol•L－1)
  1a	26.79±0.03
  1b	95.58±0.07
  1c	18.40±0.14
  1g	5.92±0.02
  Chrysin	> 100
  5-FU	30.52±0.36
  IC50: concentration that inhibits 50% of cell growth. The values are presented as the mean±SD (standard error of the mean) from three separated experiments.

  3.   Conclusions

  In summary, 19 novel chrysin derivatives were synthesized and their chemical structures were characterized via ¹H NMR, ¹³C NMR, IR and mass spectra. Biological assays revealed that the introduction of 1, 2, 3-triazole fragments in the 7-hydroxyl group of chrysin could improve its antiproliferative activity against MGC-803 cell lines. These results are useful for research on more potent novel antiproliferative compounds and further studies are ongoing.

  4.   Experimental section

  All reactions were monitored by thin-layer chromatography (TLC) performed on silica gel plates. IR spectra were recorded (in KBr) on an IR Prestige-21. ¹H NMR and ¹³C NMR spectra were recorded on an AV-300 spectrometers (Bruker BioSpin, Switzerland) at room temperature, tetramethylsilane (TMS) was used as the internal standard. Mass spectra were measured using a matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)/TOF mass spectrometer (Bruker Daltonik, Germany). The major chemicals were purchased from Aldrich Chemical Corporation (Milwaukee, WI). High-resolution mass spectra were recorded using a Thermo Scientific LTQ Orbitrap XL in the electrospray ionisation (ESI) mode. All other chemicals were analytical grade.

  3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO). All cells were initially purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 media, and foetal bovine serum (FBS) were provided from Gibco Company (Grand Island, NY, USA). The cells were maintained in RPMI-1640 or DMEM, supplemented with 10% FBS, 100 mg/mL streptomycin and 100 IU/mL penicillin (Grand Island, NY, USA), and at 37 ℃ in a humidified atmosphere containing 5% CO₂.

  4.1   General procedure for the syntheses of intermediates Ⅰ, Ⅱ, and Ⅲ

  5-Hydroxy-2-phenyl-7-(prop-2-ynyloxy)-4H-chromen-4-one (Ⅰ) and 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen- 7-yloxy)acetohydrazide (Ⅲ) were synthesized as per the protocol described in a previous study.^{[16, 17]}

  Chrysin (1.27 g, 5.0 mmol), K₂CO₃ (1.00 g, 7.5 mmol) and ethyl bromoacetate (1.00 g, 6.0 mmol) were added to CH₃COCH₃ (50 mL). The mixture was stirred at 57 ℃ for 2 h. Then, the solvent was evaporated in vacuo, water was added (20 mL), the mixture was filtered, and the residue was washed with water to obtain ethyl 2-(5-hydroxy-4- oxo-2-phenyl-4H-chromen-7-yloxy)acetate (Ⅱ):^{[18] 1}H NMR (CDCl₃, 300 MHz) δ: 12.83 (s, 1H), 8.11 (dd, J＝7.9, 1.5 Hz, 2H), 7.68~7.53 (m, 3H), 7.08 (s, 1H), 6.87 (d, J＝2.2 Hz, 1H), 6.44 (d, J＝2.2 Hz, 1H), 4.97 (s, 2H), 4.19 (q, J＝7.1 Hz, 2H), 1.23 (t, J＝7.1 Hz, 3H).

  4.2   General procedure for the syntheses of compounds 1a~1h

  A mixture of compound Ⅰ (146 mg, 0.5 mmol), different substitutions of 1-(azidomethyl)benzene (0.5 mmol), CuSO₄•5H₂O (10 mg, 0.05 mmol) and sodium ascorbate (17 mg, 0.1 mmol) were added to t-BuOH/H₂O (V:V＝1:1, 30 mL) and stirred at 30 ℃ for 12 h. After confirming the reaction progress by TLC, t-BuOH was evaporated in vacuo. Then, the mixture was extracted with dichloromethane (10 mL×3) and washed with saline (10 mL). Finally, the mixture was purified using silica gel column chromatography and eluted with dichloromethane/methanol (V:V＝100:1) to obtain the target compounds 1a~1h.

  5-Hydroxy-2-phenyl-7-((1-phenyl-1H-1, 2, 3-triazol-4-'yl)methoxy)-4H-chromen-4-one (1a): Pale yellow solid, yield 41.2%. m.p. 186~187 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.72 (s, 1H, OH), 7.88 (d, J＝5.9 Hz, 2H, ArH), 7.57~7.52 (m, 4H, ArH), 7.40~7.37 (m, 3H, ArH), 7.31~7.26 (m, 2H, ArH), 6.67 (s, 1H, ArH), 6.61 (d, J＝2.0 Hz, 1H, ArH), 6.41 (d, J＝2.0 Hz, 1H, ArH), 5.55 (s, 2H, CH₂), 5.26 (s, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.50, 164.13, 164.03, 162.15, 157.77, 134.28, 133.18, 131.92, 131.21, 129.22 (2C), 129.11 (2C), 128.95, 128.20 (2C), 126.34 (2C), 122.88, 106.08, 105.92, 99.06, 93.27, 62.42, 54.39; IR (KBr) ν: 3138, 1660, 1612, 1498, 1446, 1350, 1165 cm^－1; MS-MALDI m/z: 426 [M+H⁺]; HRMS (ESI) calcd for C₂₅H₂₀N₃O₄ [M+H⁺] 426.1448, found 426.1443.

  7-((1-(4-Fluorophenyl)-1H-1, 2, 3-triazol-4-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (1b): Pale yellow solid, yield 39.5%. m.p. 171~173 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.75 (s, 1H, OH), 7.91 (dd, J＝7.7, 1.9 Hz, 2H, ArH), 7.59~7.52 (m, 4H, ArH), 7.34~7.29 (m, 2H, ArH), 7.13~7.07 (m, 2H, ArH), 6.70 (s, 1H, ArH), 6.63 (d, J＝2.2 Hz, 1H, ArH), 6.44 (d, J＝2.2 Hz, 1H, ArH), 5.55 (s, 2H, CH₂), 5.30 (s, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.50, 164.16, 163.99, 162.90 (d, J＝151.5 Hz), 162.16, 157.77, 143.60, 137.79, 131.94, 131.19, 130.10 (d, J＝151.5 Hz, 2C), 129.12 (2C), 126.34 (2C), 122.74, 116.26 (d, J＝21.8 Hz, 2C), 106.09, 105.92, 99.02, 93.27, 62.38, 53.62; IR (KBr) ν: 3136, 1662, 1614, 1502, 1452, 1348, 1165 cm^－1; MS-MALDI m/z: 444 [M+H⁺]; HRMS (ESI) calcd for C₂₅H₂₀N₃O₄ [M+H⁺] 444.1354, found 444.1354.

  7-((1-(3-Fluorophenyl)-1H-1, 2, 3-triazol-4-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (1c): Pale yellow solid, yield 33.5%. m.p. 175~177 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.75 (s, 1H, OH), 7.91 (dd, J＝7.7, 1.8 Hz, 2H, ArH), 7.63~7.51 (m, 4H, ArH), 7.38 (dd, J＝13.8, 7.9 Hz, 1H, ArH), 7.11~7.06 (m, 2H, ArH), 7.00 (d, J＝9.2 Hz, 1H, ArH), 6.69 (s, 1H, ArH), 6.64 (d, J＝2.2 Hz, 1H, ArH), 6.44 (d, J＝2.2 Hz, 1H, ArH), 5.57 (s, 2H, CH₂), 5.31 (s, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.51, 168.44 (d, J＝132.0 Hz), 164.16, 163.97, 162.15, 157.76, 143.69, 136.64 (d, J＝7.5 Hz), 131.95, 131.18, 130.91 (d, J＝8.3 Hz), 129.13 (2C), 126.35 (2C), 123.66 (d, J＝3.0 Hz), 122.96, 116.00 (d, J＝21.0 Hz), 115.13 (d, J＝21.8 Hz), 114.98, 105.91, 99.03, 93.26, 62.37, 53.71; IR (KBr) ν: 3165, 1660, 1612, 1502, 1450, 1359, 1168 cm^－1. MS-MALDI m/z: 444 [M+H⁺]; HRMS (ESI) calcd for C₂₅H₁₉FN₃O₄ [M+H⁺] 444.1354, found 444.1354.

  7-((1-(2-Fluorophenyl)-1H-1, 2, 3-triazol-4-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (1d): Pale yellow solid, yield 31.3%. m.p. 201~203 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.75 (s, 1H, OH), 7.91 (dd, J＝7.6, 1.9 Hz, 2H, ArH), 7.70 (s, 1H, ArH), 7.57~7.54 (m, 3H, ArH), 7.32~7.34 (m, 2H, ArH), 7.21~7.12 (m, 2H, ArH), 6.70 (s, 1H, ArH), 6.65 (d, J＝2.2 Hz, 1H, ArH), 6.45 (d, J＝2.2 Hz, 1H, ArH), 5.64 (s, 2H, CH₂), 5.28 (s, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.53, 164.15, 164.03, 162.14, 158.86 (d, J＝165.0 Hz), 143.44, 131.94, 131.21 (d, J＝0.8 Hz), 131.10, 130.75 (d, J＝3.0 Hz), 129.13 (2C), 126.35 (2C), 124.97, 124.92, 123.14, 123.12, 118.83 (d, J＝19.5 Hz), 115.95 (d, J＝21.0 Hz), 106.80, 99.05, 93.27, 62.36, 50.92; IR (KBr) ν: 3134, 1670, 1614, 1496, 1452, 1350, 1161 cm^－1; MS-MALDI m/z: 444 [M+H⁺]; HRMS (ESI) calcd for C₂₅H₂₀N₃O₄ [M+H⁺] 444.1354, found 444.1353.

  7-((1-(4-Chlorophenyl)-1H-1, 2, 3-triazol-4-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (1e): Pale yellow solid, yield 37.2%. m.p. 172~173 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.74 (s, 1H, OH), 7.92~7.89 (m, 2H, ArH), 7.60~7.54 (m, 4H, ArH), 7.40~7.37 (m, 2H, ArH), 7.28~7.24 (m, 2H, ArH), 6.69 (s, 1H, ArH), 6.63 (d, J＝2.2 Hz, 1H, ArH), 6.43 (d, J＝2.2 Hz, 1H, ArH), 5.54 (s, 2H, CH₂), 5.30 (s, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.51, 164.17, 163.98, 162.17, 157.77, 143.69, 135.04, 132.77, 131.95, 131.20, 129.51 (2C), 129.45 (2C), 129.13 (2C), 126.35 (2C), 122.80, 106.10, 105.93, 99.02, 93.28, 62.37, 53.62; IR (KBr) ν: 3128, 1664, 1612, 1496, 1450, 1346, 1161 cm^－1; MS-MALDI m/z: 460 [M+H⁺]; HRMS (ESI) calcd for C₂₅H₁₉ClN₃O₄ [M+H⁺] 460.1058, found 460.1056.

  7-((1-(3-Chlorophenyl)-1H-1, 2, 3-triazol-4-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (1f): Pale yellow solid, yield 35.1%. m.p. 152~153 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.74 (s, 1H, OH), 7.91~7.88 (m, 2H, ArH), 7.63~7.54 (m, 4H, ArH), 7.45~7.29 (m, 3H, ArH), 7.19 (d, J＝4.7 Hz, 1H, ArH), 6.70 (s, 1H, ArH), 6.63 (d, J＝2.2 Hz, 1H, ArH), 6.44 (d, J＝2.1 Hz, 1H, ArH), 5.55 (s, 2H, CH₂), 5.29 (s, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.52, 164.17, 163.96, 162.14, 157.77, 143.73, 136.20, 135.11, 131.96, 131.16, 130.54, 129.18, 129.13 (2C), 128.20, 126.35 (2C), 126.20, 122.96, 106.09, 105.90, 99.05, 93.26, 62.35, 53.65; IR (KBr) ν: 3072, 1666, 1612, 1498, 1452, 1354, 1165 cm^－1; MS-MALDI m/z: 460 [M+H⁺]; HRMS (ESI) calcd for C₂₅H₁₉ClN₃O₄ [M+H⁺] 460.1059, found 460.1060.

  7-((1-(2-Chlorophenyl)-1H-1, 2, 3-triazol-4-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (1g): Pale yellow solid, yield 31.7%. m.p. 146~147 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.75 (s, 1H, OH), 7.92~7.89 (m, 2H, ArH), 7.71 (s, 1H, ArH), 7.56~7.45 (m, 5H, ArH), 7.37~7.30 (m, 2H, ArH), 6.70~6.64 (m, 2H, ArH), 6.45 (d, J＝2.1 Hz, 1H, ArH), 5.71 (s, 2H, CH₂), 5.29 (s, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.52, 164.15, 164.02, 162.13, 157.76, 143.32, 133.61, 132.13, 131.94, 131.19, 130.57, 130.48, 130.04, 129.13 (2C), 127.70, 126.35 (2C), 123.28, 106.08, 105.91, 99.06, 93.30, 62.38, 51.63; IR (KBr) ν: 3143, 1660, 1614, 1498, 1444, 1377, 1159 cm^－1; MS- MALDI m/z: 460 [M+H⁺]; HRMS (ESI) calcd for C₂₅H₁₉- ClN₃O₄ [M+H⁺] 460.1059, found 460.1055.

  5-Hydroxy-2-phenyl-7-((1-(p-tolyl)-1H-1, 2, 3-triazol-4-yl)methoxy)-4H-chromen-4-one (1h): Pale yellow solid, yield 37.8%. m.p. 166~168 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.82 (s, 1H, OH), 8.32 (s, 1H, ArH), 8.10~8.08 (m, 2H, ArH), 7.64~7.56 (m, 3H, ArH), 7.21 (dd, J＝20.2, 8.0 Hz, 4H, ArH), 7.05 (s, 1H, ArH), 6.94 (d, J＝2.1 Hz, 1H, ArH), 6.49 (d, J＝2.1 Hz, 1H, ArH), 5.57 (s, 2H, ArH), 5.28 (s, 2H, ArH), 2.27 (s, 3H, CH₃); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.31, 163.93, 161.97, 157.59, 143.30, 138.77, 131.79, 131.16, 131.03, 129.74 (2C), 128.97 (2C), 128.72, 128.13 (2C), 126.18 (2C), 122.76, 105.90, 105.72, 98.92, 93.11, 62.29, 54.04, 21.07; IR (KBr) ν: 3120, 1656, 1620, 1496, 1446, 1377, 1157 cm^－1; MS-MALDI m/z: 440 [M+H⁺]; HRMS (ESI) calcd for C₂₆H₂₂N₃O₄ [M+H⁺] 440.1605, found 440.1602.

  4.3   General procedure for the syntheses of compounds 2a~2k

  Compound Ⅲ (652.0 mg, 2.0 mmol) and N, N-dimethyl- formamide dimethyl acetal (DMFDMA, 260.0 mg, 2.2 mmol) were added to CH₃CN (30 mL). The mixture was stirred at 50 ℃ for 2 h. Then, different substitutions of aniline (3.0 mmol) and CH₃COOH (0.72 g, 12 mmol) were added, and the mixture was stirred at 82 ℃ for 12 h. After confirming the reaction progress by TLC, the solvent was evaporated in vacuo. The mixture was then purified using silica gel column chromatography and eluted using a gradient of dichloromethane/methanol (V:V＝120:1) to obtain the target compounds 2a~2k.

  5-Hydroxy-2-phenyl-7-((4-phenyl-4H-1, 2, 4-triazol-3-yl)methoxy)-4H-chromen-4-one (2a): Pale yellow solid, Yield 23.5%. m.p. 188~189 ℃; ¹H NMR (DMSO-d₆, 300 MHz) δ: 12.80 (s, 1H, OH), 8.92 (s, 1H, Triazole-H), 8.07 (d, J＝7.0 Hz, 2H, ArH), 7.78~7.35 (m, 8H, ArH), 7.03 (s, 1H, ArH), 6.85 (d, J＝1.5 Hz, 1H, ArH), 6.40 (d, J＝1.5 Hz, 1H, ArH), 5.38 (s, 2H, CH₂); ¹³C NMR (DMSO-d₆, 75 MHz) δ: 187.89, 182.58, 164.07, 163.62, 161.60, 157.58, 149.17, 145.88, 134.02, 132.67, 130.98, 130.27, 129.85, 129.63, 126.92, 125.77, 105.91, 105.85, 99.17, 94.17, 60.48; IR (KBr) ν: 3089, 1666, 1622, 1502, 1450, 1550, 1338, 1153 cm^－1; MS-MALDI m/z: 412 [M+H⁺]; HRMS (ESI) calcd for C₂₆H₂₂N₃O₄ [M+H⁺] 412.1292, found 412.1291.

  7-((4-(4-Fluorophenyl)-4H-1, 2, 4-triazol-3-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (2b): Pale yellow solid, yield 24.5%. m.p. 177~179 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.76 (s, 1H, OH), 8.35 (s, 1H, Triazole-H), 7.91 (d, J＝6.0 Hz, 2H, ArH), 7.57~7.42 (m, 5H, ArH), 7.27 (d, J＝6.0 Hz, 2H, ArH), 6.70 (s, 2H, ArH), 6.35 (s, 1H, ArH), 5.29 (d, J＝15.5 Hz, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.49, 164.32, 162.92, 162.25, 159.84 (d, J＝135.8 Hz), 157.76, 149.40, 144.83, 132.04, 131.06, 130.75, 129.29 (d, J＝3.0 Hz), 129.14 (2C), 127.56 (d, J＝9.0 Hz, 2C), 126.38 (2C), 117.20 (d, J＝23.3 Hz, 2C), 106.51, 106.00, 99.04, 93.11, 59.63; IR (KBr) ν: 3068, 1654, 1614, 1512, 1496, 1450, 1338, 1155 cm^－1; MS-MALDI m/z: 430 [M+H⁺]; HRMS (ESI) calcd for C₂₄H₁₇FN₃O₄ [M+H⁺] 430.1198, found 430.1197.

  7-((4-(3-Fluorophenyl)-4H-1, 2, 4-triazol-3-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (2c): Pale yellow solid, yield 21.5%. m.p. 174~176 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.76 (s, 1H, OH), 8.39 (s, 1H, Triazole-H), 7.93~7.90 (m, 2H, ArH), 7.55~7.53 (m, 3H, ArH), 7.28~7.25 (m, 4H, ArH), 6.76~6.68 (m, 2H, ArH), 6.37 (d, J＝2.3 Hz, 1H, ArH), 5.31 (d, J＝4.6 Hz, 2H, CH₂); ¹³C NMR (DMSO-d₆, 75 MHz) δ: 182.57, 164.10, 163.58, 162.54 (d, J＝244.5 Hz), 161.62, 157.58, 149.11, 145.82, 135.38 (d, J＝10.5 Hz), 132.67, 132.02 (d, J＝9.0 Hz), 130.98, 129.62 (2C), 126.92 (2C), 122.03 (d, J＝3.0 Hz), 116.80 (d, J＝21.0 Hz), 113.51 (d, J＝24.8 Hz), 105.94 (2C), 99.16, 94.20, 60.56; IR (KBr) ν: 3066, 1656, 1614, 1512, 1496, 1448, 1340, 1155 cm^－1; MS-MALDI 430 [M+H⁺]; HRMS (ESI) calcd for C₂₄H₁₇FN₃O₄ [M+H⁺] 430.1198, found 430.1192.

  7-((4-(2-Fluorophenyl)-4H-1, 2, 4-triazol-3-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (2d): Pale yellow solid, yield 14.5%. m.p. 175~177 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.71 (s, 1H, OH), 8.35 (s, 1H, Triazole-H), 7.90 (dd, J＝7.8, 1.7 Hz, 2H, ArH), 7.54 (dd, J＝7.2, 3.4 Hz, 4H, ArH), 7.43~7.33 (m, 3H, ArH), 6.69 (s, 1H, ArH), 6.61 (d, J＝2.2 Hz, 1H), 6.20 (d, J＝2.2 Hz, 1H, ArH), 5.33 (d, J＝7.1 Hz, 2H, CH₂); ¹³C NMR (CDCl_3, 75 MHz) δ: 182.48, 164.28, 162.95, 162.54 (d, J＝133.5 Hz), 162.09, 157.69, 149.73, 145.04, 141.64, 132.12 (d, J＝8.3 Hz), 132.03, 131.04, 129.13 (2C), 127.94, 126.37 (2C), 125.32 (d, J＝3.8 Hz), 117.18 (d, J＝19.5 Hz), 106.45, 105.95, 99.07, 92.91, 60.27; IR (KBr) ν: 3066, 1654, 1612, 1514, 1496, 1448, 1338, 1157 cm^－1; MS-MALDI m/z: 430 [M+H⁺]; HRMS (ESI) calcd for C₂₄H₁₇FN₃O₄ [M+H⁺] 430.1198, found 430.1199.

  7-((4-(4-Chlorophenyl)-4H-1, 2, 4-triazol-3-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (2e): Pale yellow solid, yield 25.5%. m.p. 166~168 ℃; ¹H NMR (DMSO- d₆, 300 MHz) δ: 12.81 (s, 1H, OH), 8.92 (s, 1H, Triazole-H), 8.08 (d, J＝8.0 Hz, 2H, ArH), 7.64~7.29 (m, 7H, ArH), 7.04 (s, 1H, ArH), 6.84 (d, J ＝2.1 Hz, 1H, ArH), 6.41 (d, J＝2.1 Hz, 1H, ArH), 5.41 (s, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.43, 164.26, 162.84, 162.20, 157.69, 149.18, 144.58, 136.30, 131.99, 131.71, 131.00, 130.30 (2C), 129.09 (2C), 126.72 (2C), 126.34 (2C), 106.48, 105.94, 98.99, 93.10, 59.58; IR (KBr) ν: 3107, 1656, 1614, 1504, 1452, 1350, 1159 cm^－1. MS-MALDI m/z: 446 [M+H⁺]; HRMS (ESI) calcd for C₂₄H₁₇ClN₃O₄ [M+H⁺] 446.0902, found 446.0907.

  7-((2-(3-Chlorophenyl)-2H-1, 2, 4-triazol-3-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (2f): Pale yellow solid, yield 22.1%. m.p. 159~160 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.75 (s, 1H, OH), 8.36 (s, 1H, Triazole-H), 7.90 (d, J＝8.1 Hz, 2H, ArH), 7.56~7.46 (m, 6H, ArH), 7.34 (s, 1H, ArH), 6.70 (d, J＝2.6 Hz, 2H, ArH), 6.35 (d, J＝2.2 Hz, 1H, ArH), 5.28 (s, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.45, 164.31, 162.34, 144.44, 135.87, 134.39, 131.96, 131.10, 130.33, 129.11 (2C), 126.37 (2C), 125.90, 123.63, 113.03, 106.58, 106.04, 99.10, 93.24, 77.41, 76.99, 76.57, 59.80; IR (KBr) ν: 3111, 1660, 1618, 1502, 1450, 1350, 1157 cm^－1; MS-MALDI m/z: 446 [M+H⁺]; HRMS (ESI) calcd for C₂₄H₁₇ClN₃O₄ [M+H⁺] 446.0902, found 446.0905.

  7-((4-(2-Chlorophenyl)-4H-1, 2, 4-triazol-3-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (2g): Pale yellow solid, yield 10.3%. m.p. 157~158 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.67 (s, 1H, OH), 8.31 (s, 1H, Triazole-H), 7.87 (dd, J＝7.8, 1.5 Hz, 2H, ArH), 7.63~7.60 (m, 1H, ArH), 7.54~7.48 (m, 4H, ArH), 7.46~7.40 (m, 2H, ArH), 6.66 (s, 1H, ArH), 6.57 (d, J＝2.2 Hz, 1H, ArH), 6.17 (d, J＝2.2 Hz, 1H, ArH), 5.30 (d, J＝5.3 Hz, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.44, 164.25, 162.99, 162.03, 157.66, 149.67, 144.91, 132.01, 131.84, 131.50, 131.06, 131.00, 130.78, 129.11 (2C), 128.83, 128.14, 126.35 (2C), 106.38, 105.89, 99.06, 92.86, 60.21; IR (KBr) ν: 3103, 1654, 1618, 1504, 1454, 1346, 1157 cm^－1; MS-MALDI m/z: 446 [M+H⁺]; HRMS (ESI) calcd for C₂₄H₁₇ClN₃O₄ [M+H⁺] 446.0902, found 446.0901.

  7-((4-(3-Bromophenyl)-4H-1, 2, 4-triazol-3-yl)methoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (2h): Pale yellow solid, yield 20.8%. m.p. 179~181 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.77 (s, 1H, OH), 8.37 (s, 1H, Triazole-H), 7.92 (d, J＝8.2 Hz, 2H, ArH), 7.70 (d, J ＝8.3 Hz, 1H, ArH), 7.63~7.55 (m, 4H, ArH), 7.48~7.38 (m, 2H, ArH), 6.71 (s, 2H, ArH), 6.38 (d, J ＝2.2 Hz, 1H, ArH), 5.31 (s, 2H, CH₂); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.46, 164.32, 162.91, 162.35, 157.77, 149.18, 144.44, 134.47, 133.27, 131.96, 131.23, 131.15, 129.11 (2C), 128.80, 126.37 (2C), 124.10, 123.49, 106.59, 106.04, 99.11, 93.25, 59.80; IR (KBr) ν: 3107, 1660, 1618, 1500, 1452, 1350, 1157 cm^－1; MS-MALDI m/z: 490 [M+H⁺]; HRMS (ESI) calcd for C₂₄H₁₇BrN₃O₄ [M+H⁺] 490.0397, found 490.0393.

  5-Hydroxy-2-phenyl-7-((4-p-tolyl-4H-1, 2, 4-triazol-3-yl)methoxy)-4H-chromen-4-one (2i): Pale yellow solid, yield 25.4%. m.p. 165~166 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.81 (s, 1H, OH), 8.88 (s, 1H, Triazole-H), 8.09~8.06 (m, 2H, ArH), 7.64~7.57 (m, 3H, ArH), 7.41 (dd, J＝28.2, 8.2 Hz, 4H, ArH), 7.05 (s, 1H, ArH), 6.87 (d, J＝2.1 Hz, 1H, ArH), 6.43 (d, J＝2.1 Hz, 1H, ArH), 5.36 (s, 2H, CH₂), 2.35 (s, 3H, CH₃); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.32, 164.08, 163.02, 161.99, 157.56, 149.23, 144.73, 140.27, 131.88, 130.90, 130.48 (2C), 128.99 (2C), 126.21 (2C), 125.09 (2C), 106.25, 105.76, 98.99, 93.08, 90.29, 59.53, 21.09; IR (KBr) ν: 3115, 1664, 1612, 1517, 1448, 1336, 1155 cm^－1; MS-MALDI m/z: 426 [M+H⁺]; HRMS (ESI) calcd for C₂₅H₂₀N₃O₅ [M+H⁺] 426.1448, found 426.1446.

  5-Hydroxy-2-phenyl-7-((4-m-tolyl-4H-1, 2, 4-triazol-3-yl)methoxy)-4H-chromen-4-one (2j): Pale yellow solid, yield 15.4%. m.p. 175~177 ℃; ¹H NMR (CDCl₃, 300 MHz) δ: 12.75 (s, 1H, OH), 8.37 (s, 1H, TriazoleH), 7.93~7.90 (m, 2H, ArH), 7.56 (d, J ＝7.3 Hz, 3H, ArH), 7.46~7.34 (m, 2H, ArH), 7.23 (s, 2H, ArH), 6.72~6.71 (m, 2H, ArH), 6.35 (d, J＝2.2 Hz, 1H, ArH), 5.28 (s, 2H, CH₂), 2.44 (s, 3H, CH₃); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.51, 164.29, 163.14, 162.18, 157.75, 153.43, 144.73, 142.39, 140.45, 132.01, 131.09, 130.80, 129.82, 129.13 (2C), 126.38 (2C), 126.01, 122.38, 106.44, 105.97, 99.15, 93.21, 59.69, 21.32; IR (KBr) ν: 3072, 1658, 1618, 1502, 1450, 1350, 1159 cm^－1; MS-MALDI m/z: 426 [M+H⁺]; HRMS (ESI) calcd for C₂₅H₂₀N₃O₅ [M+H⁺] 426.1448, found 426.1446.

  5-Hydroxy-7-((4-(4-methoxyphenyl)-4H-1, 2, 4-triazol-3-yl)methoxy)-2-phenyl-4H-chromen-4-one (2k): Pale yellow solid, yield 24.9%. m.p. 163~165 ℃; ¹H NMR (DMSO-d₆, 300 MHz) δ: 12.82 (s, 1H, OH), 8.85 (s, 1H, Triazole-H), 8.10~8.07 (m, 2H, ArH), 7.62~7.59 (m, 3H, ArH), 7.50 (d, J＝8.9 Hz, 2H, ArH), 7.11~7.06 (m, 3H, ArH), 6.87 (d, J＝2.1 Hz, 1H, ArH), 6.44 (d, J＝2.1 Hz, 1H, ArH), 5.34 (s, 2H, CH₂), 3.79 (s, 3H, CH₃); ¹³C NMR (CDCl₃, 75 MHz) δ: 182.31, 164.08, 163.03, 161.99, 160.50, 157.57, 149.43, 144.92, 131.86, 130.90, 128.98 (2C), 126.73 (2C), 126.21 (2C), 125.70, 114.95 (2C), 106.25, 105.77, 98.99, 93.06, 59.52, 55.57; IR (KBr) ν: 3078, 1658, 1616, 1517, 1452, 1348, 1157 cm^－1; MS-MALDI 442 [M+H⁺]; HRMS (ESI) calcd for C₂₅H₂₀N₃O₅ [M+H⁺] 442.1398, found 442.1396.

  4.4   Biological testing

  The antiproliferative activities of the target compounds against MGC-803, BEL-7402, HepG2, HeLa, A549, and SGC-7901 cell lines were evaluated by MTT assay in vitro, with 5-FU as the positive control. All cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Cells were detached by trypsin, seeded at 9×10³ cells in a 96-well plate and incubated in 5% CO₂ at 37 ℃ overnight. Then the cells are treated with the test compounds at different concentrations (1, 10, 50, and 100 μmol/L) and incubated for 48 h. Fresh MTT solutions were added to each well and incubated at 37 ℃ for 4 h. The MTT-formazan formed by metabolically viable cells was dissolved by dimethyl sulfoxide (DMSO, 150 μL) in each well and monitored by a microplate reader at a wavelength of 492 nm. IC₅₀ was defined as the drug concentrations that inhibited the cell number to 50% after 48 h.

  Supporting Information NMR spectra of target compounds. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn/.

  
  
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• 

  Figure 1  Design of target compounds based on the combination principles

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  Scheme 1  Synthetic routes of the target compounds

  1a: R＝H, 1b: R＝4-F, 1c: R＝3-F, 1d: R＝2-F, 1e: R＝4-Cl, 1f: R＝3-Cl, 1g: R＝2-Cl, 1h: R＝4-CH₃; 2a: R＝H, 2b: R＝4-F, 2c: R＝3-F, 2d: R＝2-F, 2e: R＝4-Cl, 2f: R＝3-Cl, 2g: R＝2-Cl, 2h: R＝3-Br, 2i: R＝4-CH₃, 2j: R＝3-CH₃, 2j: R＝4-OCH₃
  Reagents and conditions: (a) propargyl bromide, K₂CO₃, CH₃COCH₃, 57 ℃; (b) different substitutions of 1-(azidomethyl)benzene, CuSO₄• 5H₂O, sodium ascorbate, t-BuOH/H₂O (V:V＝1:1), 30 ℃; (c) ethyl bromoacetate, K₂CO₃, CH₃COCH₃, 57 ℃; (d) N₂H₄•H₂O, CH₃CH₂OH, 78 ℃; (e) DMFDMA, CH₃CN, different substitutions of aniline, CH₃COOH, 82 ℃

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  Table 1.  Antiproliferative activity of compounds^a

  Compd.	Growth inhibition/% at 100 μmol/L
  	MGC-803	BEL-7402	HepG-2	HeLa	A549	SGC-7901
  1a	66.83±1.46	33.18±0.81	14.20±0.22	31.90±0.99	44.67±0.72	19.31±0.65
  1b	52.71±0.71	44.01±0.77	11.89±0.86	24.42±1.01	41.20±1.55	47.68±1.36
  1c	52.82±2.25	3.31±0.07	1.02±0.02	16.81±0.17	27.89±0.56	7.30±0.14
  1d	41.52±1.12	31.44±0.88	4.91±0.05	24.40±0.26	27.89±0.19	12.78±0.18
  1e	33.81±0.25	98.18±1.35	6.81±0.07	6.32±0.02	33.91±0.67	12.64±0.13
  1f	10.59±0.08	12.80±0.14	7.43±0.25	16.70±0.09	43.46±0.22	20.98±0.15
  1g	74.28±1.68	17.44±0.12	33.02±0.26	35.07±0.18	50.71±0.87	45.64±0.88
  1h	78.33±0.36	97.60±0.98	12.92±0.11	4.11±0.17	29.13±0.14	6.44±0.12
  2a	27.33±0.11	57.24±0.76	34.01±0.33	27.30±0.05	39.49±0.17	51.71±1.00
  2b	44.51±0.57	78.26±1.77	5.11±0.02	26.81±0.18	9.01±0.07	6.81±0.05
  2c	10.72±0.12	70.70±1.02	4.52±0.12	26.42±0.26	15.01±0.07	8.22±0.12
  2d	34.62±0.23	15.49±0.11	88.40±1.66	29.33±0.20	44.11±0.31	49.22±0.32
  2e	25.21±0.21	32.30±0.33	7.50±0.08	35.41±0.37	31.11±0.33	19.70±0.26
  2f	22.62±0.24	44.33±0.08	15.62±0.12	40.92±0.40	37.80±0.08	37.71±0.37
  2g	31.71±0.15	19.01±0.12	31.02±0.24	20.10±0.20	27.90±0.13	18.41±0.10
  2h	16.80±0.10	35.71±0.11	38.79±0.31	46.50±0.40	43.01±0.23	47.83±0.32
  2i	42.11±0.34	49.81±0.37	38.82±0.21	35.51±0.13	30.11±0.13	51.09±0.05
  2j	36.89±0.33	57.64±0.32	51.60±0.22	30.49±0.61	46.02±0.64	47.11±0.69
  2k	21.73±0.27	29.44±0.04	58.11±0.97	38.22±0.22	44.52±0.12	36.54±0.20
  Chrysin	50.89±0.50	73.02±0.23	39.33±0.22	62.89±0.33	8.80±0.04	16.30±0.13
  a Growth inhibitions are presented as the mean±SD (standard error of the mean) from three separated experiment.

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  Table 2.  IC₅₀ values (μmol/L) of several active compounds

  Compd.	IC50 against MGC-803 cell lines/(μmol•L－1)
  1a	26.79±0.03
  1b	95.58±0.07
  1c	18.40±0.14
  1g	5.92±0.02
  Chrysin	> 100
  5-FU	30.52±0.36
  IC50: concentration that inhibits 50% of cell growth. The values are presented as the mean±SD (standard error of the mean) from three separated experiments.

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