English

• Cancer is a large family of diseases resulting from uncontrolled cell growth, which remains one of the most potentially life threatening diseases worldwide.^[1] Therefore, the development of new anticancer agents and more effective treatment strategies for cancer has attracted more and more attention.^[2]

  Resveratrol (trans-3, 4', 5-trihydroxystilibene, 1), a natural phytoalexin presenting in grape skin and peanuts, displays a broad range of biological activities, including anti-tumor, antioxidant, cardiovascular protection, anti-inflammatory, antitubulin and so on.^[3~5] Especially, resveratrol has been considered as a potential cancer chemopreventive agent. In addition, the anti-tumor effects are mediated through multiple mechanisms, which include cell cycle disruption, apoptosis induction and blockade of nuclear factor-kappa B (NF-κB), and cyclooxygenase (COX).^{[6, 7]} Recently, resveratrol derivatives have attracted considerable interests for their versatile properties in chemistry and pharmacology as chemotherapeutic agents. For example, resveratrol derivatives A possessing chalcone moiety had good antiproliferative activity, and resveratrol amide derivatives B showed good cytotoxic activities against a human nasopharyngeal epidermoid tumor cell line κB (Figure 1).^{[8, 9]}

  Figure 1

  

  Figure 1.  Structure of resveratrol and derivatives

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  Natural and synthetic chalcones (1, 3-diaryl-2-propen-1-ones) exhibit a broad range of biological activities, which are widely used as antioxidant, anti-inflammatory, anti-bacterial, anti-tumor and so on.^[10~12] Due to their α, β-unsaturated carbonyl system, chalcones are playing the vital role in drug research. In former work, we have reported that N-heterocyclic substituted chalcone compounds showed potent anti-inflammatory and cytotoxic activities.^[13] Especially, the hybrid compounds between chalcone and N-aryl piperazine displayed potential anti-tumor activities.^[14~16]

  Herein we report the synthesis and biological evaluation of novel hybrids towards the combination of resveratrol and chalcone possessing piperazine moiety as potential anti-inflammatory and anti-tumor agents on the basis of previous research. In order to study the structure-activity relationship (SAR) of hybrid compounds, various R—X were selected, including acyl chloride and sulfonyl chloride (Scheme 1). The biological results demonstrated that the combination of resveratrol and chalcone may contribute to the potential anti-inflammatory and anti-tumor activities.

  Scheme 1

  

  Scheme 1.  Designed strategy of resveratrol-chalcone hybrids

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  1.   Results and discussion

  1.1   Chemistry

  The synthetic route of the hybrid compounds is outlined in Scheme 2. The intermediate (E)-2, 4-dimethoxy-6-(4-methoxystyryl)benzaldehyde (3) was prepared according to literature procedure.^[17] Subsequently, treatment of 4-fluoro-acetophenone with compound 3 gave the stilibene-chalcone 4 in the presence of KOH resulted by the Aldol condensation. The key intermediate 5 was prepared by substitution of compound 4 with piperazine in the presence of K₂CO₃ at 110 ℃ in N, N-dimethylformamide (DMF). In order to characterize the configuration of two double bonds of compound 5, the nuclear overhauser effect (NOE) experiment was performed. From the data, it could be seen that two double bonds were trans-configuration. With the desired intermediate 5 in hand, the title amide derivatives 6~18 were prepared by reaction of 5 with various acyl chloride in good yields (60%~90%), respectively.

  Scheme 2

  

  Scheme 2.  Synthetic routes of hybrids

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  To explored the structure-activity relationships (SAR), various substituents (F, Cl, Br and CF₃, etc) were introduced into the phenyl rings of the title derivatives according to their electronic characteristics. The quantitative and positional effects of the substituents were also explored by examining biological activity of the hybrids with substituents at various positions on the phenyl ring.

  Comparative data for novel derivatives with respective to structures, melting point and yield are provided in Table 1.

  Table 1

  

  Table 1.  Structures and yields of compounds

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  Compound	R	m.p.a/℃	Yieldb/%
  6	CH3—	137~139	87
  7		141~143	90
  8		170~172	83
  9		176~178	80
  10		179~181	84
  11		186~188	85
  12		170~172	79
  13		188~190	80
  14		182~184	62
  15		134~136	86
  16		169~171	71
  17		185~187	60
  18		176~178	84
  a Melting point was uncorrected. b Yields represent isolated yields.

  All of the synthetic compounds were characterized by ¹H NMR and ¹³C NMR, and some active compounds were characterized by HRMS analysis as well.

  1.2   Biological evaluation

  1.2.1   Cytotoxic activity

  In vitro cytotoxic activities of novel synthesized compounds were tested against human lung cancer cell lines (A549), human cervical carcinoma (Hela) and human gastric carcinoma (SGC7901) by MTT assay, using cisplatin (DDP) as the reference drug. The cell inhibition data of compounds are summarized in Table 2.

  Table 2

  

  Table 2.  In vitro cytotoxic activities [IC₅₀/(μmol•L^－1)] of title compounds^a

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  Compound	A549	Hela	SGC7901
  1	> 40	> 40	> 40
  2	> 40	> 40	> 40
  3	> 40	24.16	> 40
  4	> 40	> 40	19.42
  5	24.20	> 40	16.72
  6	25.15	> 40	32.24
  7	28.23	> 40	> 40
  8	> 40	> 40	> 40
  9	0.26	7.35	29.68
  10	> 40	37.50	12.82
  11	> 40	8.54	12.17
  12	21.20	> 40	38.71
  13	2.14	9.83	3.87
  14	> 40	15.24	18.45
  15	> 40	> 40	34.41
  16	1.22	22.51	> 40
  17	> 40	> 40	28.16
  18	11.52	31.56	6.18
  DDP	11.54	20.52	12.44
  a Cytotoxicity as IC50 values for each cell line, the concentration of compound that inhibit 50% of the cell growth measured by MTT assay.

  From the activity data, it could be observed that the structures of compounds have an obvious influence on anticancer activities. Among all compounds, amide derivatives containing electron withdrawing groups and halide of benzene ring such as electron withdrawing trifluoromethyl (CF₃), and chloro (Cl) substituents exhibited more sensitive to anticancer activities on three tested cancer cell lines. For example, compounds 11, 13, 14 and 16 were more potent than compounds with other substituted groups. In addition, α, β-unsaturated amides showed good cytotoxic activity as well, such as compound 9 (IC₅₀＝0.26 μmol•L^－1 against A549, 7.35 μmol•L^－1 against Hela) and 18 (IC₅₀＝11.52 μmol•L^－1 against A549, 7.18 μmol•L^－1 against SGC7901). Among all derivatives, acrylamide 9 exhibited the best growth inhibitory activities against two human cancer cell lines (A549, Hela), and hybrid 13 showed satisfied anti-tumor activities against 3 cancer cell lines. From the comparative analysis data, it could be seen that lack of electronic of benzene ring led to positive activities. Although the biological study was preliminary and the further research was undergoing, the SAR analysis was summarized. The anticancer activity was related to the number and category of substituents on the benzene ring, but had nothing to do with position of substituents.

  1.2.2   FACs analysis

  Impaired apoptosis is one of the hallmarks of cancer and contributes to tumor progression and resistance to conventional cancer therapy. In order to further characterize the potential mechanism of anticancer activities of title derivatives, an apoptotic assay by flow cytometry with Annexin V and PI staining was carried out on compound 9. The cells were treated with 9 for 48 h, and apoptosis was determined by Annexin V/PI double-staining assay. As shown in Figure 2, following the treatment with 5 and 10 μmol•L^－1 of compound 9, the percentage of early apoptotic cells were decreased, and the percentage of late apoptotic and necrotic cells were increased. These results indicated that compound 9 suppressed cell proliferation in A549 cell caused by inducing apoptosis.

  Figure 2

  

  Figure 2.  Compound 9 induced apoptosis in A549 cell

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  2.   Conclusions

  In summary, a series of novel hybrid compounds between resveratrol and chalcone possessing piperazine moiety have been synthesized, and screened in vitro cytotoxic activity. The results showed that the amide compounds contributed good cytotoxic activity. Especially, 9 showed the best cytotoxic activity against A549 and Hela (IC₅₀＝0.26 and 7.35 μmol•L^－1, respectively), which was identified as the most potent anti-tumor agent. Annexin V/ PI-staining result indicated that compound 9 significantly induced apoptosis. Further research is currently undergoing and the results will be reported in due course.

  3.   Experimental section

  3.1   General considerations

  Starting materials were analytically pure. Melting points were measured on a YANACO microscopic melting point meter and were uncorrected. 1D and 2D NMR spectra were recorded on a Bruker AV 400 spectrometer, using TMS as internal standard and CDCl₃ as solvent, respectively. High-resolution mass spectra were performed on an Agilent CL/MSD spectrometer. FT-IR spectra were performed on a Perkin-Elmer FT-IR Fronter spectrometer. Thin layer chromatographic (TLC) analysis was carried out on silica gel plates GF₂₅₄.

  3.2   Synthesis of (E)-2, 4-dihydroxy-6-(4-hydro-xystyryl)benzaldehyde (2)

  To a stirred solution of resveratrol (4.56 g, 20 mmol) and DMF (1.61 g, 22 mmol) in anhydrous CH₃CN (100 mL), POCl₃ (4.60 g, 30 mmol) was added at 0 ℃ for 0.5 h. Then the reaction mixture was stirred for another 3 h at room temperature. Following, the mixture was poured into cold water (300 mL) and the solution was stirred under 60 ℃ for 1 h. After cooling to ambient temperature, the solution was filtered, and the filtrate was washed with water (30 mL×3) and dried to afford yellow powder, m.p. 211~213 ℃. The characterization data was the same to that in reference 9.

  3.3   Synthesis of (E)-2, 4-dimethoxy-6-(4-methoxy-styryl)benzaldehyde (3)

  To a stirred solution of compound 2 (2.56 g, 10 mmol) in acetone (50 mL), K₂CO₃ (6.21 g, 45 mmol) and CH₃I (8.52 g, 60 mmol) were added and reaction mixture was refluxed for 24 h. Then the reaction mixture was concentrated in vacuo and water (50 mL) was added. The solution was filtered, and the filtrate was washed with water (30 mL×3) and dried to afford pale yellow solid, m.p. 107~109 ℃. The characterization data was the same to that in Reference [9].

  3.4   Synthesis of (E)-3-(2, 4-dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-flurophenyl)prop-2-en-1-one (4)

  To a solution of compound 3 (2.98 g, 10 mmol) and 4-fluoroacetophenone (1.38 g, 10 mmol) in EtOH (40 mL), was added 20% KOH (15 mL) and left to react for 12 h at room temperature. After completion of the reaction as indicated by thin layer chromatography (TLC), the reaction was quenched by the addition of water (30 mL) and the mixture was filtered. Then the filtrate was washed with water (30 mL×3) and dried to afford pale yellow solid, m.p. 182~184 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.20 (d, J＝15.6 Hz, 1H), 7.97~8.01 (m, 2H), 7.55 (d, J＝15.6 Hz, 1H), 7.46 (d, J＝8.7 Hz, 2H), 7.33 (d, J＝16.0 Hz, 1H), 7.09 (d, J＝8.5 Hz, 1H), 7.07 (d, J＝8.8 Hz, 1H), 6.94 (d, J＝17.8 Hz, 1H), 6.90 (d, J＝8.8 Hz, 2H), 6.71 (d, J＝2.3 Hz, 1H), 6.42 (d, J＝2.2 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.82 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 189.44, 166.70 (C-F), 164.18 (C-F), 161.81, 160.94, 159.78, 141.90, 139.09, 135.12, 131.88, 131.10, 131.01, 128.11, 125.53, 124.72, 115.70, 115.48, 114.32, 103.88, 97.73, 55.80, 55.53, 55.41; IR (KBr) ν: 2363, 1668, 1593, 1508, 1418, 1302, 1153, 1084, 833 cm^－1.

  3.5   Synthesis of (E)-3-(2, 4-dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-piperazin-1-yl)phenyl-prop-2-en-1-one (5)

  To a stirred solution of compound 4 (4.18 g, 10 mmol) and K₂CO₃ (2.76 g, 20 mmol) in dried DMF (40 mL), piperazine (1.73 g, 20 mmol) was added and the mixture was stirred for 12 h at 120 ℃. After completion of the reaction as indicated by TLC, the reaction mixture was cooled to room temperature, and was extracted with the dichloromethane (DCM) (100 mL). Then organic layer was washed with water (50 mL×3). The organic layer was dried by anhydrous sodium sulfate, concentrated in vacuo and purified by column chromatography (2% MeOH/ DCM) to afford pale brown solid. m.p. 202~204 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (d, J＝15.7 Hz, 1H), 7.94 (d, J＝9.0 Hz, 2H), 7.57 (d, J＝15.7 Hz, 1H), 7.47 (d, J＝8.7 Hz, 2H), 7.37 (d, J＝16.1 Hz, 1H), 6.96 (d, J＝16.1 Hz, 1H), 6.89 (d, J＝8.8 Hz, 2H), 6.82 (d, J＝9.0 Hz, 2H), 6.72 (d, J＝2.3 Hz, 1H), 6.42 (d, J＝2.4 Hz, 1H), 3.87 (s, 6H), 3.81 (s, 3H), 3.25~3.28 (m, 4H), 2.97~2.99 (m, 4H), 1.83 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ: 188.69, 161.31, 160.61, 159.60, 154.32, 141.21, 137.11, 131.29, 130.68, 130.57, 130.07, 128.82, 128.08, 127.92, 125.91, 125.76, 116.66, 114.26, 113.56, 103.58, 97.71, 55.48, 55.37, 48.55, 45.91; IR (KBr) ν: 3466, 2936, 2833, 1641, 1597, 1510, 1454, 1308, 1248, 1186, 1151, 1082, 1030, 826 cm^－1; HRMS (ESI-TOF) calcd for C₃₀H₃₃N₂O₄ (M+H)⁺ 485.2435, found 485.2434.

  3.6   General procedure for the preparation of amine derivatives 6~18

  To a stirred solution of compound 5 (0.2 mmol) and Et₃N (0.3 mL) in dried DCM (10 mL), acyl chloride (0.3 mmol) was added and reaction mixture was stirred for 12 h at room temperature. After completion of the reaction as indicated by TLC, the reaction was quenched by the addition of saturated Na₂CO₃ (20 mL) and the mixture was extracted with DCM (10 mL×3). The organic layer was dried using anhydrous sodium sulfate, concentrated in vacuo and purified by column chromatography (1% MeOH/DCM) to afford products.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-acetyl)piperazin-1-yl)phenylprop-2-en-1-one (6): Yellow solid, m.p. 137~139 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (d, J＝15.6 Hz, 1H), 7.94 (d, J＝8.9 Hz, 2H), 7.57 (d, J＝15.7 Hz, 1H), 7.47 (d, J＝8.7 Hz, 2H), 7.36 (d, J＝16.1 Hz, 1H), 6.96 (d, J＝16.1 Hz, 1H), 6.89 (d, J＝8.7 Hz, 2H), 6.81 (d, J＝9.0 Hz, 2H), 6.72 (d, J＝2.2 Hz, 1H), 6.43 (d, J＝2.2 Hz, 1H), 3.88 (s, 6H), 3.81 (s, 3H), 3.55~3.60 (m, 4H), 3.27~3.34 (m, 4H), 2.12 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 188.78, 169.19, 161.40, 160.64, 159.59, 153.38, 141.32, 137.44, 131.39, 130.68, 130.57, 129.99, 129.47, 128.06, 127.88, 125.77, 125.41, 116.43, 114.24, 113.94, 113.58, 103.64, 97.68, 55.75, 55.36, 55.18, 47.54, 47.29, 45.75, 40.95, 21.29; IR (KBr) ν: 3429, 2934, 1645, 1597, 1512, 1456, 1429, 1186, 1151, 1080, 1030, 827 cm^－1; HRMS (ESI-TOF) calcd for C₃₂H₃₄N₂O₅Na (M+Na)⁺ 549.2360, found 549.2360.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-N-(ethyl carboxylate))piperazin-1-yl)phenylprop-2-en-1-one (7): Yellow solid, m.p. 141~143 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (d, J＝15.7 Hz, 1H), 7.94 (d, J＝9.0 Hz, 2H), 7.56 (d, J＝15.6 Hz, 1H), 7.47 (d, J＝8.7 Hz, 2H), 7.36 (d, J＝16.1 Hz, 1H), 6.95 (d, J＝16.1 Hz, 1H), 6.89 (d, J＝8.8 Hz, 2H), 6.82 (d, J＝9.0 Hz, 2H), 6.72 (d, J＝2.3 Hz, 1H), 6.42 (d, J＝2.3 Hz, 1H), 4.20 (q, J＝7.1 Hz, 2H), 3.87 (s, 6H), 3.81 (s, 3H), 3.57~3.65 (m, 4H), 3.29~3.34 (m, 4H), 1.30 (t, J＝7.1 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 188.77, 161.37, 160.63, 159.60, 155.49, 153.64, 141.29, 137.38, 131.35, 130.69, 130.59, 130.03, 129.39, 128.07, 127.90, 125.86, 125.55, 116.53, 114.24, 114.01, 113.98, 113.67, 103.62, 97.69, 61.63, 55.75, 55.48, 55.36, 47.41, 43.25, 14.72; IR (KBr) ν: 2835, 1699, 1597, 1512, 1462, 1425, 1248, 1221, 1186, 1153, 1080, 1030, 826; HRMS (ESI-TOF) calcd for C₃₃H₃₇N₂O₆ (M+H)⁺ 557.2646, found 557.2647.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-benzoyl)piperazin-1-yl)phenylprop-2-en-1-one (8): Yellow solid, m.p. 170~172 ℃; ¹H NMR(400 MHz, CDCl₃) δ: 8.17 (d, J＝15.6 Hz, 1H), 7.95 (d, J＝9.0 Hz, 2H), 7.57 (d, J＝15.6 Hz, 1H), 7.42~7.47 (m, 7H), 7.36 (d, J＝16.1 Hz, 1H), 6.96 (d, J＝16.0 Hz, 1H), 6.89 (d, J＝8.8 Hz, 2H), 6.83 (d, J＝9.1 Hz, 2H), 6.72 (d, J＝2.3 Hz, 1H), 6.43 (d, J＝2.3 Hz, 1H), 3.88 (s, 6H), 3.80 (s, 3H), 3.60~3.68 (m, 2H), 3.28~3.39 (m, 6H); ¹³C NMR(100 MHz, CDCl₃) δ: 188.91, 170.63, 161.44, 160.69, 159.63, 153.50, 141.37, 137.60, 135.39, 131.43, 130.74, 130.64, 130.10, 130.06, 129.73, 128.67, 128.43, 128.10, 127.93, 127.21, 125.86, 125.52, 116.53, 114.28, 114.21, 114.02, 113.85, 103.67, 97.74, 55.80, 55.52, 55.40, 47.79; IR (KBr) ν: 2835, 1636, 1597, 1508, 1458, 1420, 1288, 1186, 1153, 1082, 1028, 1011, 824, 712 cm^－1; HRMS (ESI-TOF) calcd for C₃₇H₃₇N₂O₅ (M+H)⁺ 589.2697, found 589.2697.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-acryloyl)piperazin-1-yl)phenylprop-2-en-1-one (9): Yellow solid, m.p. 176~178 ℃; ¹H NMR(400 MHz, CDCl₃) δ: 8.16 (d, J＝15.6 Hz, 1H), 7.94 (d, J＝8.7 Hz, 2H), 7.56 (d, J＝15.6 Hz, 1H), 7.47 (d, J＝8.6 Hz, 2H), 7.36 (d, J＝16.0 Hz, 1H), 6.96 (d, J＝9.0 Hz, 2H), 6.89 (d, J＝8.5 Hz, 2H), 6.82 (d, J＝16.0 Hz, 1H), 6.80 (d, J＝8.6 Hz, 1H), 6.73 (d, J＝1.7 Hz, 1H), 6.43 (d, J＝10.6 Hz, 1H), 6.33 (dd, J＝10.6 Hz, 1.8 Hz, 1H), 5.75 (d, J＝10.6 Hz, 1H), 3.88 (s, 6H), 3.82 (s, 3H), 3.73 (t, J＝6.1 Hz, 4H), 3.36 (t, J＝5.2 Hz, 4H); ¹³C NMR (100 MHz, CDCl₃) δ: 188.85, 165.57, 161.47, 160.71, 159.69, 153.40, 141.38, 137.51, 131.46, 130.74, 130.64, 130.10, 129.67, 129.03, 128.42, 128.11, 127.94, 127.27, 125.90, 125.61, 116.60, 114.32, 114.05, 114.01, 113.65, 103.76, 97.80, 55.82, 55.42, 55.24, 47.56, 45.30; IR (KBr) ν: 2932, 2835, 1645, 1597, 1512, 1456, 1441, 1186, 1151, 1082, 1028, 968, 827 cm^－1; HRMS (ESI-TOF) calcd for C₃₃H₃₅N₂O₅ (M+H)⁺ 539.2540, found 539.2539.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-(2-chlorobenzoyl))piperazin-1-yl)phenylprop-2-en-1-one (10): Pale yellow solid, m.p. 179~181 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.17 (d, J＝15.6 Hz, 1H), 7.94 (d, J＝8.9 Hz, 2H), 7.56 (d, J＝15.6 Hz, 1H), 7.46 (d, J＝8.8 Hz, 2H), 7.31~7.35 (m, 5H), 6.95 (d, J＝16.0 Hz, 1H), 6.88 (d, J＝8.7 Hz, 2H), 6.82 (d, J＝8.9 Hz, 2H), 6.72 (d, J＝2.3 Hz, 1H), 6.42 (d, J＝2.3 Hz, 1H), 3.87 (s, 6H), 3.79 (s, 3H), 3.32~3.43 (m, 8H); ¹³C NMR (100 MHz, CDCl₃) δ: 188.79, 166.97, 161.40, 160.64, 159.57, 153.36, 141.31, 137.50, 135.41, 131.38, 130.56, 130.48, 130.32, 129.97, 129.74, 129.67, 128.04, 127.87, 127.35, 125.76, 125.39, 116.40, 114.22, 113.95, 103.65, 97.65, 55.73, 55.46, 55.30, 47.95, 47.43, 46.16, 41.29; IR (KBr) ν: 2934, 1643, 1560, 1510, 1456, 1431, 1290, 1246, 1186, 1082, 1030, 1009, 827, 770, 745, 521 cm^－1; HRMS (ESI-TOF) calcd for C₃₇H₃₅ClN₂O₅Na (M+Na)⁺ 645.2127, found 645.2122.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-(3, 5-dichlorobenzoyl))piperazin-1-yl)phenylprop-2-en-1-one (11): Yellow solid, m.p. 186~188 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.17 (d, J＝15.6 Hz, 1H), 7.95 (d, J＝8.9 Hz, 2H), 7.76 (d, J＝9.0 Hz, 1H), 7.58 (d, J＝15.6 Hz, 1H), 7.43~7.47 (m, 3H), 7.32 (s, 2H), 6.95 (d, J＝16.0 Hz, 1H), 6.88 (d, J＝8.7 Hz, 2H), 6.83 (d, J＝9.2 Hz, 2H), 6.72 (d, J＝2.3 Hz, 1H), 6.43 (d, J＝2.3 Hz, 1H), 3.88 (s, 6H), 3.81 (s, 3H), 3.33~3.39 (m, 8H); ¹³C NMR (100 MHz, CDCl₃) δ: 189.10, 167.57, 161.50, 160.73, 159.63, 153.32, 141.48, 138.12, 137.85, 135.61, 135.23, 131.52, 130.80, 130.69, 130.17, 130.03, 128.45, 128.10, 127.92, 125.78, 125.68, 125.35, 114.33, 114.27, 114.02, 113.95, 103.72, 97.74, 55.80, 55.52, 55.36, 47.71; IR (KBr) ν: 2934, 1645, 1597, 1562, 1512, 1456, 1184, 1151, 1024, 806 cm^－1; HRMS (ESI-TOF) calcd for C₃₇H₃₅Cl₂N₂O₅ (M+H)⁺ 657.1918, found 657.1919.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-(4-flurobenzoyl))piperazin-1-yl)phenylprop-2-en-1-one (12): Pale brown solid, m.p. 170~172 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.17 (d, J＝15.6 Hz, 1H), 7.95 (d, J＝8.9 Hz, 2H), 7.56 (d, J＝15.7 Hz, 1H), 7.44~7.47 (m, 3H), 7.36 (d, J＝16.1 Hz, 1H), 7.14 (t, J＝8.6 Hz, 3H), 6.96 (d, J＝16.0 Hz, 1H), 6.89 (d, J＝8.8 Hz, 2H), 6.84 (d, J＝8.9 Hz, 2H), 6.73 (d, J＝2.3 Hz, 1H), 6.43 (d, J＝2.3 Hz, 1H), 3.88 (s, 6H), 3.81 (s, 3H), 3.65 (s, 2H), 3.35 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 188.83, 169.64, 161.45, 160.69, 159.64, 153.45, 141.37, 137.58, 131.44, 130.62, 130.06, 129.83, 129.64, 129.56, 128.11, 125.85, 125.50, 115.89, 115.67, 114.28, 114.24, 103.67, 97.74, 55.80, 55.53, 55.41, 47.83; IR (KBr) ν: 2835, 1638, 1599, 1510, 1423, 1221, 1186, 1153, 1009, 824, 760 cm^－1.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-(3-trifluromethylbenzoyl))piperazin-1-yl)phenyl-prop-2-en-1-one (13): Pale yellow solid, m.p. 188~190 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.35 (s, 1H), 8.27 (d, J＝7.8 Hz, 1H), 8.19 (d, J＝15.6 Hz, 1H), 7.96 (d, J＝8.9 Hz, 1H), 7.84 (d, J＝7.8 Hz, 1H), 7.73 (d, J＝9.1 Hz, 2H), 7.65 (d, J＝7.7 Hz, 1H), 7.57~7.59 (m, 2H), 7.47 (d, J＝8.7 Hz, 1H), 7.37 (d, J＝16.0 Hz, 1H), 6.96 (d, J＝16.0 Hz, 1H), 6.89 (d, J＝8.7 Hz, 2H), 6.85 (d, J＝9.0 Hz, 1H), 6.73 (d, J＝2.3 Hz, 1H), 6.44 (d, J＝2.3 Hz, 1H), 3.89 (s, 6H), 3.81 (s, 3H), 3.39 (t, J＝8.2 Hz, 8H); ¹³C NMR (100 MHz, CDCl₃) δ: 189.20, 169.16, 161.56, 160.78, 159.69 (C-F), 153.43, 141.52, 137.92, 136.14, 133.36, 131.55, 131.00, 130.87, 130.75, 130.59, 129.96, 129.92, 129.88, 129.40, 129.20, 128.15, 127.97, 127.11, 127.08, 126.96, 125.87, 125.47, 124.35, 124.31, 116.54, 114.37, 114.32, 114.07, 113.99, 103.76, 97.80, 55.84, 55.42, 55.24, 47.76; IR (KBr) ν: 2934, 1597, 1512, 1427, 1331, 1171, 1124, 1072, 1018, 822 cm^－1; HRMS (ESI-TOF) calcd for C₃₈H₃₆F₃N₂O₅ (M+H)⁺ 657.2571, found 657.2568.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-(2-fluro-4-bromobenzoyl))piperazin-1-yl)phenyl-prop-2-en-1-one (14): Yellow solid, m.p. 182~184 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.17 (d, J＝15.6 Hz, 1H), 7.95 (d, J＝8.8 Hz, 2H), 7.57 (d, J＝15.6 Hz, 1H), 7.48 (d, J＝8.6 Hz, 2H), 7.38 (s, 1H), 7.33 (d, J＝7.2 Hz, 3H), 6.97 (d, J＝16.0 Hz, 1H), 6.90 (d, J＝8.7 Hz, 2H), 6.84 (d, J＝9.0 Hz, 2H), 6.73 (d, J＝2.2 Hz, 1H), 6.44 (d, J＝2.2 Hz, 1H), 3.89 (s, 6H), 3.82 (s, 3H), 3.40~3.47 (m, 6H), 3.30~3.34 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ: 188.88, 164.42, 161.47, 160.71 (C-F), 159.66 (C-F), 153.40, 141.40, 137.65, 131.46, 130.75, 130.71, 130.65, 130.58, 130.08, 129.94, 129.00, 128.46, 128.42, 127.96, 126.11, 125.89, 125.54, 119.79, 119.54, 116.56, 114.38, 114.31, 114.04, 114.00, 103.68, 97.77, 55.83, 55.56, 55.44, 48.16, 47.58, 46.61, 41.87; IR (KBr) ν: 2930, 1645, 1597, 1512, 1456, 1453, 1219, 1186, 1153, 1011, 889, 826 cm^－1.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-phenylacetyl)piperazin-1-yl)phenylprop-2-en-1-one (15):Yellow solid, m.p. 134~136 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (d, J＝15.6 Hz, 1H), 7.91 (d, J＝9.0 Hz, 2H), 7.55 (d, J＝15.6 Hz, 1H), 7.46 (d, J＝8.7 Hz, 2H), 7.23~7.35 (m, 7H), 6.95 (d, J＝16.0 Hz, 1H), 6.88 (d, J＝8.8 Hz, 2H), 6.75 (s, 1H), 6.72 (d, J＝2.3 Hz, 1H), 6.42 (d, J＝2.3 Hz, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.79 (s, 3H), 3.74 (s, 2H), 3.56 (t, J＝4.7 Hz, 2H), 3.27 (t, J＝5.6 Hz, 3H), 3.11 (t, J＝5.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 188.74, 169.68, 161.41, 160.64, 159.59, 153.31, 141.30, 137.46, 134.79, 131.38, 130.55, 129.98, 129.83, 129.41, 129.19, 128.94, 128.86, 128.68, 128.59, 128.44, 128.05, 127.88, 126.98, 125.78, 125.42, 116.44, 114.24, 113.97, 113.88, 103.65, 97.68, 55.74, 55.47, 55.35, 47.37, 47.18, 45.52, 41.30, 40.98; IR (KBr) ν: 2833, 1638, 1595, 1560, 1508, 1456, 1186, 1151, 1030, 964, 826, 698 cm^－1; HRMS (ESI-TOF) calcd for C₃₈H₃₉N₂O₅ (M+H)⁺603.2853, found 603.2853.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-(2, 4-dichlorophenoxyacetyl))piperazin-1-yl)phenyl-prop-2-en-1-one (16):Pale yellow solid, m.p. 169~171 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.17 (d, J＝15.6 Hz, 1H), 7.94 (d, J＝8.9 Hz, 2H), 7.56 (d, J＝15.6 Hz, 1H), 7.47 (d, J＝8.7 Hz, 2H), 7.38 (d, J＝2.5 Hz, 2H), 7.18 (dd, J＝2.5 Hz, 2.5 Hz, 1H), 6.96 (d, J＝9.0 Hz, 2H), 6.90 (d, J＝8.8 Hz, 2H), 6.82 (d, J＝9.0 Hz, 2H), 6.73 (d, J＝2.3 Hz, 1H), 6.44 (d, J＝2.3 Hz, 1H), 4.79 (s, 2H), 3.89 (s, 6H), 3.82 (s, 3H), 3.46~3.52 (m, 2H), 3.29~3.36 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 188.85, 165.78, 161.48, 160.72, 159.68, 153.34, 152.10, 141.41, 137.61, 131.48, 130.76, 130.65, 130.38, 130.08, 129.83, 128.13, 127.96, 127.11, 125.87, 125.52, 123.67, 116.55, 114.47, 114.32, 114.21, 114.06, 113.83, 103.69, 97.78, 68.86, 55.83, 55.56, 55.45, 48.07, 47.44, 45.11, 41.90; IR (KBr) ν: 2932, 1653, 1597, 1512, 1479, 1217, 1186, 1026, 827 cm^－1.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-(2-naphthoy))piperazin-1-yl)phenylprop-2-en-1-one (17): Yellow power, m.p. 185~187 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.18 (d, J＝15.7 Hz, 1H), 7.94 (t, J＝4.0 Hz, 2H), 7.83~7.88 (m, 4H), 7.49~7.57 (m, 4H), 7.44 (d, J＝8.7 Hz, 2H), 7.35 (d, J＝15.7 Hz, 1H), 6.94 (d, J＝16.0 Hz, 1H), 6.86 (d, J＝8.7 Hz, 2H), 6.80 (d, J＝9.0 Hz, 2H), 6.71 (d, J＝2.2 Hz, 1H), 6.41 (d, J＝2.2 Hz, 1H), 3.85 (s, 36H), 3.84 (s, 3H), 3.76 (s, 3H), 3.58~3.68 (m, 2H), 3.30~3.35 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 188.68, 170.45, 161.35, 160.59, 159.53, 153.38, 141.26, 137.39, 133.76, 132.66, 132.62, 131.33, 130.50, 129.93, 129.56, 128.44, 128.42, 128.01, 127.81, 127.25, 127.09, 126.82, 125.73, 125.36, 124.24, 116.37, 114.18, 114.07, 103.63, 97.61, 55.67, 55.41, 55.27, 47.65; IR (KBr) ν: 2833, 1638, 1597, 1510, 1423, 1186, 1151, 1082, 1024, 822, 758 cm^－1; HRMS (ESI-TOF) calcd for C₄₁H₃₉N₂O₅ (M+H)⁺ 639.2853, found 639.2851.

  (E)-3-(2, 4-Dimethoxy-6-((E)-4-methoxystyryl)phenyl)-1-(4-(N-cinnamyl)piperazin-1-yl)phenylprop-2-en-1-one (18): Pale red solid, m.p. 176~178 ℃; ¹H NMR (400 MHz, CDCl₃) δ: 8.19 (d, J＝15.6 Hz, 1H), 7.96 (d, J＝9.0 Hz, 2H), 7.78 (s, 1H), 7.76 (d, J＝15.3 Hz, 1H), 7.74 (s, 1H), 7.59 (d, J＝15.8 Hz, 1H), 7.50~7.52 (m, 5H), 7.47 (d, J＝8.7 Hz, 2H), 6.95 (d, J＝15.1 Hz, 1H), 6.88 (d, J＝8.8 Hz, 2H), 6.81 (d, J＝9.0 Hz, 2H), 6.72 (d, J＝2.3 Hz, 1H), 6.47 (s, 1H), 6.43 (s, 1H), 3.86 (s, 6H), 3.78 (s, 3H), 3.39 (t, J＝4.9 Hz, 8H); ¹³C NMR (100 MHz, CDCl₃) δ: 189.01, 171.45, 165.91, 161.44, 160.69, 159.61, 153.34, 146.55, 143.69, 141.40, 137.64, 135.10, 134.20, 131.45, 130.69, 130.61, 130.02, 129.90, 129.42, 128.89, 128.32, 128.09, 127.93, 125.78, 125.41, 117.69, 116.54, 116.47, 114.26, 113.85, 103.70, 97.71, 55.74, 55.76, 55.34, 47.44; IR (KBr) ν: 2835, 1699, 1636, 1597, 1512, 1454, 1425, 1312, 1184, 1030, 980, 764 cm^－1; HRMS (ESI-TOF) calcd for C₃₉H₃₉N₂O₅ (M+H)⁺ 615.2853, found 615.2854.

  3.3   Biological activity

  3.3.1   Cytotoxic activity

  The assay was carried out using the method previously described.^[16] About 1×10⁴ cell/well were seeded into 96-well microtiter plates. The test compounds were dissolved in dimethylsulfoxide (DMSO). After 24 h post-seeding, cells were treated with vehicle control or various concentrations of samples for 48 h. 20 μL of MTT solution (5 mg/mL) was added to each well and the tumor cells were incubated at 37 ℃ in a humidified atmosphere of 5% CO₂ air for 4 h. Upon removal of MTT/medium, 150 μL of DMSO was added to each well and the plate was agitated at oscillator for 5 min to dissolve the MTT-formazan. The assay plate was read at a wavelength of 570 nm using a microplate reader.

  3.3.2   Assessment of apoptosis

  Apoptosis was measured by Annexin V/PI-staining by flow cytometry, A549 cell were harvested after exposure to compound 9 for 48 h, the cells were washed twice with cold phosphate-buffered saline (PBS) and then resuspended in binding buffer, the cells were stained with 5 μg/mL FITC-conjugated Annexin V and PE-conjugated PI for 15 min in the dark at the room temperature and analyzed with a FACS can flow cytometer (Becton-Dickinson). The FL-1 and FL-2 channels were used simultaneously to gate annexin V-positive cells and PI-positive cells, respectively.

  Supporting Information The ¹H NMR, ¹³C NMR and HRMS spectra of compounds 4~18. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn.

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• 

  Figure 1  Structure of resveratrol and derivatives

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  Scheme 1  Designed strategy of resveratrol-chalcone hybrids

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  Scheme 2  Synthetic routes of hybrids

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  Figure 2  Compound 9 induced apoptosis in A549 cell

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  Table 1.  Structures and yields of compounds

  Compound	R	m.p.a/℃	Yieldb/%
  6	CH3—	137~139	87
  7		141~143	90
  8		170~172	83
  9		176~178	80
  10		179~181	84
  11		186~188	85
  12		170~172	79
  13		188~190	80
  14		182~184	62
  15		134~136	86
  16		169~171	71
  17		185~187	60
  18		176~178	84
  a Melting point was uncorrected. b Yields represent isolated yields.

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  Table 2.  In vitro cytotoxic activities [IC₅₀/(μmol•L^－1)] of title compounds^a

  Compound	A549	Hela	SGC7901
  1	> 40	> 40	> 40
  2	> 40	> 40	> 40
  3	> 40	24.16	> 40
  4	> 40	> 40	19.42
  5	24.20	> 40	16.72
  6	25.15	> 40	32.24
  7	28.23	> 40	> 40
  8	> 40	> 40	> 40
  9	0.26	7.35	29.68
  10	> 40	37.50	12.82
  11	> 40	8.54	12.17
  12	21.20	> 40	38.71
  13	2.14	9.83	3.87
  14	> 40	15.24	18.45
  15	> 40	> 40	34.41
  16	1.22	22.51	> 40
  17	> 40	> 40	28.16
  18	11.52	31.56	6.18
  DDP	11.54	20.52	12.44
  a Cytotoxicity as IC50 values for each cell line, the concentration of compound that inhibit 50% of the cell growth measured by MTT assay.

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•