Chinese Journal of Natural Medicines 2015, 13(5): 0375−0382
Chinese Journal of Natural Medicines
Synthesis and cytotoxic activities of E-resveratrol derivatives HONG Ting1, 2, JIANG Wei1, DONG Huai-Ming1, QIU Sheng-Xiang3, LU Yu1* 1
Sino-German Joint Research Institute of Nanchang University, Nanchang 330047, China; Jiangxi Provincial Institute for Food and Drug Control, Jiangxi Provincial Engineering Research Center for Drug and Medical Device Quality, Nanchang 330029, China; 3 Chinese Academy of Sciences, South China Botanical Garden, Guangzhou 510650, China 2
Available online 20 May 2015
[ABSTRACT] The present study was designed to synthesize derivatives of E-resveratrol and evaluate their cytotoxic activity in vitro. Different functional groups were conjugated with the phenolic hydroxyl group of E-resveratrol, and the double bond of E-resveratrol was reduced. The in vitro cytotoxicity of the synthetic derivatives was evaluated against three tumor cell lines (A549, LAC, and HeLa) using the MTT assay. Twenty-six E-resveratrol derivatives were synthesized and their structures were confirmed by 1 H NMR, MS, IR, and elemental analyses. Compounds 1−6, 12, 15−21, and 23−26 were reported for the first time. Among them, Compounds 1, 2, 4, 5, and 9−11, showed significant cytotoxicity against tumor cells; especially, Compound 1 showed an IC50 value of 4.38 µmol·L−1 in the A549 cells which was 15-fold more active than E-resveratrol; Compound 9 showed an IC50 value of 1.41 µmol·L−1 in the HeLa cell line which was 90-fold more active than E-resveratrol, and close to adriamycin. The structure–activity relationships were also investigated. Compounds 1, 2 and 9−11 may serve as potential lead compounds for the discovery of new anticancer drugs. [KEY WORDS] E-resveratrol derivatives; Synthesis; Cytotoxic activity; Structure–activity relationships
[CLC Number] R284.3, R965
[Document code] A
[Article ID] 2095-6975(2015)05-0375-08
Introduction The naturally occurring E-resveratrol and its various derivatives have attracted a great deal of attention due to their wide range of biological properties. E-resveratrol (3, 4', 5-trihydroxystilbene) is a stilbene-type polyphenolic natural product present in grapes and a variety of medicinal plants [1-7] . It is a naturally occurring phytoalexin which can be activated by adverse conditions of plants, protecting against fungal infections [8]. E-resveratrol possesses multiple biological activities that are beneficial to human health, including anticancer [9], anti-inflammatory [10], antibacterial antioxidant [11] , anti-free radica [12], heart protecting [13], liver protecting [14] , fatty acid synthase-inhibitory [15], nerve protecting [16], estrogenic [17], bone metabolism and endothelin antagonist [18], among others. Jang M et al reported the antitumor function of [Received on] 03-June-2014 [Research funding] This work was supported by the Cross-fund of Nanchang University. [*Corresponding author] Tel: 86-13870886467, Fax: 86-791-8333 7081, E-mail: [email protected] These authors have no conflict of interest to declare. Copyright © 2015, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
E-resveratrol in 1997 [19]. The usefulness of E-resveratrol, however, is limited by its instability upon exposure to light and oxygen. These stimuli may cause trans-cis transformation or oxidation that leads to a reduction in bioavailability and bioactivity. Recently, considerable attention has been focused on E-resveratrol derivatives. It has been reported that methylated E-resveratrol derivative R3 is an effective neuroprotective agent against free radicalmediated oxidative stress triggered by 6-OHDA in SH-SY5Y cells [20]. Yang LM et al have synthesized a series of E-resveratrol derivatives with sulfur thalidomide substitutes that showed inhibitory activities against breast cancer and colon cancer [21]. It is reported that methylated E-resveratrol derivatives are more effective than E-resveratrol in the prevention and treatment of cancer [22]. Cardile V and others have synthesized methoxy derivatives of E-resveratrol and E-resveratrol esters, tested their cytotoxicity in human prostate cancer cells, and found that the activity of these derivatives was stronger than E-resveratrol [23-25]. It has been reported that polymethoxy-stilbene analogs, especially (Z)-3, 5, 4-trimethoxystilbene, exhibit strong anti-proliferative activity [26-27]. Stivala LA et al have reported a number of E-resveratrol derivatives which showed good anti-proliferative activity, suggesting that the 7-methoxycoumarin nucleus, together with the 3, 5-disubstitution pattern of the trans-
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vinylbenzene moiety, are promising structural features to obtain excellent anticancer compounds endowed with a apoptosis-inducing capability [28]. Encouraged by the promising cytotoxic activity of the reported E-resveratrol derivatives, we designed and synthesized a series of E-resveratrol derivatives and evaluated their biological activities structure-activity relationships (SAR) in the present study. Our results indicated that some of the analogs can be developed as lead compounds as novel anticancer agents.
Materials and Methods Materials and instrumentation 1 H NMR spectra were recorded with a Bruker Avance 600 FT-NMR spectrometer (Bruker Biospin Ltd., Bern, Switzerland) in the indicated solvents (TMS as internal standard). Mass spectra were obtained using Waters 2695-4000 spectrometer (Waters Ltd., Milford, Massachusetts, USA). IR spectra were obtained on a Nicolet 380 spectrometer (Thermo Nicole Ltd, Madison, Wisconsin, USA). All melting points are measured using an Electrothermal engineering 9200 apparatus (Electrothermal Engineering Ltd, Stone Staffordshire, UK). Elemental analyses were performed using a TQ-3A elemental analysis instrument (Elementar Ltd, Hanau, Germany). All of the compounds synthesized were purified by column chromatography (CC) on silica gel 60 (200−300 mesh) and thin-layer chromatography (TLC) on silica gel 60 F254 plates (250 μm; Qingdao Marine Chemical Company, Qingdao, China). Most chemicals and solvents were of analytical grade. E-resveratrol (98%) was purchased from Shanxi Sciphar Hi-tech Industry Co., Ltd. (Shanxi, China). General procedures for the synthesis of compounds 1−25 E-resveratrol (1.14 g, 5.00 mmol) was dissolved in acetone (100 mL), and K2CO3 (1.04 g, 7.54 mmol) was added. Then, PhSO2Cl (0.6 mL, 4.70 mmol) were slowly added and the reaction mixture was stirred at reflux temperature for 9 h. The resultant clear solution was filtered and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; 9 : 1 chloroform/ethyl acetate elution) to give compound 1 (0.39 g, 22.5% yield) as white crystals: mp 152−153 ºC; IR (KBr) ν 3 413, 3 059, 3 017, 1 600, 1 515, 1 449, 1 353, 1 298, 1 171, 1 090, 986, 959, 847, 808, 570 cm–1; 1H NMR (600 MHz, CDCl3) δ: 6.66 (1H, s, C5-H), 6.73 (1H, s, C2-H), 6.74 (1H, d, J = 16.4 Hz, CH=), 6.82 (1H, s, C6-H) 6.83-6.84 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 6.88 (1H, d, J = 16.4 Hz, CH=), 7.35 (2H, d, J = 8.4 Hz, C2′-H and C6′-H), 7.54 (2H, dd, J = 7.6 Hz, Ar-H), 7.69 (1H, t, J = 7.6 Hz, Ar-H), 7.88 (2H, d, J = 7.6 Hz, Ar-H); ESI-MS m/z 369 [M + H]+; Anal. C20H16O5S, C 65.12, H 4.49 (Req C 65.20, H 4.38). Following the general procedure, compound 2 was prepared from E-resveratrol as white crystals (33.1% yield): m p 94−95 ºC; IR (KBr) ν 3 460, 1 615, 1 569, 1 501, 1 449, 1
370, 1 292, 1 192, 1 148, 1 091, 984, 972, 874, 750, 581 cm–1; H NMR(600 MHz, CDCl3) δ: 6.42 (1H, s, C5-H), 6.66 (1H, s, C2-H), 6.78 (1H, d, J = 16.4 Hz, CH=), 6.82 (1H, s, C6-H), 6.84 (1H, d, J = 16.4 Hz, CH=), 6.94 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 7.32 (2H, d, J = 8.4 Hz, C2′-H and C6′-H), 7.54 (4H, t, J = 12.0 Hz, Ar-H), 7.66-7.69 (2H, m, Ar-H), 7.83−7.88 (4H, m, Ar-H); ESI-MS m/z 531 [M + Na]+; Anal. C26H20O7S2, C 61.58; H 3.89 (Req C 61.40, H 3.96). Following the general procedure, compound 3 was prepared from E-resveratrol as white crystals (96.3% yield) as a white crystal: mp 1 4 4 −145 ºC; IR (KBr) ν 3 079, 3 029, 1 611, 1 578, 1 500, 1 449, 1 383, 1 350, 1 304, 1 191, 1 089, 968, 866, 843, 771, 682, 633, 563 cm−1; 1H NMR (600 MHz, CDCl3) δ: 6.49 (1H, s, C4-H), 6.79 (1H, d, J = 16.2 Hz, CH=), 6.84 (1H, d, J = 16.2 Hz, CH=), 6.98 (2H, d, J = 9.0 Hz, C3′-H and C5′-H), 7.04 (2H, s, C2-H and C6-H), 7.35 (2H, d, J = 9.0 Hz, C2′-H and C6′-H), 7.53−7.57 (6H, m, Ar-H), 7.69−7.71 (3H, m, Ar-H), 7.80 (4H, t, J = 8.4 Hz, Ar-H), 7.82 (2H, d, J = 8.4 Hz, Ar-H); ESI-MS m/z 687 [M + K]+; Anal. C32H24O9S3, C 59.06, H 3.69 (Req C 59, H 3.73). Following the general procedure, compound 4 was prepared from E-resveratrol as white crystals (30.9% yield): mp 152−154 ºC; IR (KBr) ν 3 395, 3 249, 2 959, 2 929, 2 872, 1 605, 1 511, 1 459, 1 350, 1 306, 1 258, 1 168, 1 148, 1 038, 962, 831, 682 cm−1; 1H NMR(600 MHz, CDCl3) δ: 2.26 (2H, m, CH2), 3.75 (2H, d, J = 6.3 Hz, CH2Cl), 4.14 (2H, d, J = 6.3 Hz, OCH2), 6.25 (1H, s, C4-H), 6.55 (2H, s, C2-H and C6-H), 6.83 (1H, d, J = 16.2 Hz, CH=), 6.89 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 6.99 (1H, d, J = 16.2Hz, CH=), 7.42 (2H, d, J = 8.4 Hz, C2′-H and C6′-H); ESI-MS m/z 305 [M + H]+; Anal. C17H17ClO3, C 68.84, H 5.81 (Req C 67.00, H 5.62). Following the general procedure, compound 5 was prepared from E-resveratrol as white crystals (47.9% yield): m p 9 4 − 9 5 ºC; IR (KBr) ν 3 339, 3 015, 2 964, 2 934, 2 860, 1 600, 1 511, 1 441, 1 341, 1 251, 1 230, 1 164, 1 064, 957, 832, 676, 656 cm−1; 1H NMR(600 MHz, CDCl3) δ: 2.23−2.26 (4H, m, 2 × CH2), 3.74−3.77 (4H, m, 2 × CH2Cl), 4.12−4.15 (4H, m, 2 × OCH2), 6.31 (1H, s, C4-H), 6.57 (1H, s, C2-H), 6.63 (1H, s, C6-H), 6.86 (1H, d, J = 16.2 Hz, CH=), 6.89 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 7.01 (1H, d, J = 16.2 Hz, CH=), 7.42−7.43 (2H, d, J = 8.4 Hz, C2′-H and C6′-H); ESI-MS m/z 381 [M + H]+; Anal. C20H22Cl2O3, C 63.13, H 5.77 (Req C 63.00, H 5.82). Following the general procedure, compound 6 was prepared from E-resveratrol as white crystals (53.1% yield): m p 88−89 ºC; IR (KBr) ν 2 955, 2 930, 2 876, 1 587, 1 512, 1 439, 1 257, 1 165, 1 062, 955, 833, 677 cm−1; 1H NMR(600 MHz, CDCl3) δ: 2.23−2.27 (6H, m, 3 × CH2), 3.76 (6H, t, J = 8.0 Hz, 3 × CH2Cl), 4.14 (6H, t, J = 8.0 Hz, 3 × OCH2), 6.38 (1H, s, C4-H), 6.66 (2H, s, C2-H and C6-H), 6.88 (1H, d, J = 16.2 Hz, CH=), 6.90 (2H, d, J = 8.0 Hz, C3′-H and C5′-H), 7.03 (1H, d, J = 16.2 Hz, CH=), 7.43 (2H, d, J = 8.0 Hz, C2′-H and C6′-H); ESI-MS m/z 457 [M + H]+; Anal. C23H27Cl3O3, C 60.51, H 6.01 (Req C 60.34, H 5.94). 1
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Following the general procedure, compound 7 was prepared from E-resveratrol as white crystals (93.4% yield): m p 138−140 ºC; IR (KBr) ν 3 054, 3 030, 2 921, 2 859, 1 597, 1 512, 1 443, 1 371, 1 276, 1 155, 1 062, 1 014, 956, 833, 736, 684 cm−1; 1H NMR (600 MHz, CDCl3) δ: 5.06 (4H, s, 2 × OCH2), 5.08 (2H, s, OCH2), 6.53 (1H, s, C4-H), 6.74 (2H, s, C2-H and C6-H), 6.89 (1H, d, J = 16.2 Hz, CH=), 6.96 (2H, d, J = 7.8 Hz, C3′-H and C5′-H), 7.01 (1H, d, J = 16.2 Hz, CH=), 7.32 (3H, t, J = 7.8 Hz, Ar-H), 7.38 (6H, t, J = 7.8 Hz, Ar-H), 7.39-7.40 (6H, J = 7.8 Hz, Ar-H), 7.42−7.45 (2H, d, J = 7.8 Hz, C2′-H and C6′-H); ESI-MS m/z 499 [M + H]+; Anal. C35H30O3, C 84.17, H 5.97 (Req C 84.31, H 6.06). Following the general procedure, compound 8 was prepared from E-resveratrol as white crystals (43.9% yield): mp 142−143 ºC; IR(KBr) ν 3 419, 3 083, 3 029, 2 868, 1 600, 1 512, 1 453, 1 383, 1 350, 1 383, 1 350, 1 265, 1 175, 1 147, 1 016, 961, 842, 745, 699 cm−1; 1H NMR (600 MHz, CDCl3) δ: 5.07 (2H, s, OCH2), 5.09 (2H, s, OCH2), 6.38 (1H, s, C4-H), 6.59 (1H, s, C2-H), 6.71(1H, s, C6-H), 6.89 (1H, d, J = 16.0 Hz, CH=), 6.96−6.97 (2H, m, C3′-H and C5′-H), 7.01 (1H, d, J = 16.0 Hz, CH=), 7.32−7.35 (2H, m, C2′-H and C6′-H), 7.38−7.45 (10H, m, 2 × Ar-H); ESI-MS m/z 409 [M + H]+; Anal. C28H24O3, C 82.12, H 5.89 (Req C 82.33, H 5.92). Following the general procedure, compound 9 was prepared from E-resveratrol as white crystals (30.2% yield): mp 143−144 ºC; IR (KBr) ν 3 563, 3 386, 3 029, 2 950, 1 632, 1 603, 1 509, 1 445, 1 334, 1 283, 1 253, 1 134, 1 050, 970, 847, 749, 735, 696, 632 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 5.07 (2H, s, OCH2), 6.28 (1H, s, C4-H), 6.52 (1H, s, C2-H), 6.67 (1H, s, C6-H), 6.76 (2H, d, J = 6.0 Hz, C3′-H and C5′-H), 6.86 (1H, d, J = 16.0 Hz, CH=), 7.03 (1H, d, J = 16.0 Hz, CH=), 7.34 (1H, t, J = 6.0 Hz, Ar-H), 7.39−7.41 (4H, m, Ar-H), 7.46 (2H, d, J = 6.0 Hz, C2′-H and C6′-H), 9.40 (1H, s, OH), 9.56 (1H, s, OH); ESI-MS m/z 319 [M + H]+; Anal. C21H18O3, C 79.19, H 5.83 (Req C 79.22, H 5.70). Following the general procedure, compound 10 was prepared from E-resveratrol as a white crystals (14.5% yield): mp 193−194 ºC; IR (KBr) ν 3 393, 3 278, 3 029, 2 897, 2 886, 1 603, 1 511, 1 458, 1 382, 1 330, 1 263, 1 148, 1 038, 1 025, 963, 830, 694, 731, 607 cm–1; 1H NMR (600 MHz, DMSO-d6) δ: 5.13 (2H, s, OCH2), 6.12 (1H, t, C4-H), 6.40 (2H, s, C2-H and C6-H), 6.90 (1H, d, J = 16.2 Hz, CH=), 6.98 (1H, d, J = 16.2 Hz, CH=), 7.05 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 7.33 (1H, t, J = 4.8 Hz, Ar-H), 7.40 (2H, dd, J = 4.8 Hz, Ar-H), 7.46 (2H, d, J = 4.8 Hz, Ar-H), 7.51 (2H, d, J = 8.4 Hz, C2′-H and C6′-H), 9.20 (2H, s, 2 × OH); ESI-MS m/z 319 [M + H]+; Anal. C21H18O3, C 79.15, H 5.88 (Req C 79.22, H 5.70). Following the general procedure, compound 11 was prepared from E-resveratrol as white crystals (72.6% yield): mp 113−114 ºC; IR (KBr) ν 3 448, 3 075, 1 758, 1 612, 1 589, 1 507, 1 370, 1 214, 1 127, 1 018, 912 cm−1; 1H NMR (600 MHz, CDCl3) δ: 2.30 (9H, s, 3 × OCH3), 6.82 (1H, s, C4-H), 6.97 (1H, d, J = 16.0 Hz, CH=), 7.06 (1H, d, J = 16.0 Hz, CH=), 7.09 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.11 (2H, s,
C2-H and C6-H), 7.48 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 355 [M + H]+; Anal. C20H18O6, C 67.83, H 5.01 (Req C 67.79, H 5.12). Following the general procedure, compound 12 was prepared from compound 6 as white crystals (95.2% yield): mp 41−43 ºC; IR (KBr) ν 3 037, 2 933, 2 104, 1 598, 1 510, 1 452, 1 350, 1 291, 1 252, 1 169, 1 060, 963, 833, 680 cm−1; 1 H NMR (600 MHz, CDCl3) δ: 2.03−2.09 (6H, m, 3 × CH2N3), 3.53 (6H, t, J = 6.0 Hz, 3 × CH2), 4.07 (6H, t, J = 6.0 Hz, 3 × OCH2), 6.37 (1H, s, C4-H), 6.65 (2H, s, C2-H and C6-H), 6.89 (1H, d, J = 16 Hz, CH=), 6.87 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 7.03 (1H, d, J = 16 Hz, CH=), 7.43 (2H, d, J = 8.4 Hz, C2′-H and C6′-H); ESI-MS m/z 478 [M + H]+; Anal. C23H27N9O3, C 57.74, H 5.82 (Req C 57.85, H 5.70). Following the general procedure, compound 13 was prepared from E-resveratrol as white crystals (52.7% yield): mp 83−84 ºC; IR (KBr) ν 3 429, 2 988, 2 909, 1 759, 1 723, 1 595, 1 169, 834 cm−1; 1H NMR (600 MHz, CDCl3) δ: 1.25−1.33 (9H, m, 3 × CH3), 4.26−4.31 (6H, m, 3 × COCH2), 4.62−4.64 (6H, m, 3 × OCH2CO), 6.40 (1H, s, C4-H), 6.67 (2H, s, C2-H and C6-H), 6.90 (1H, d, J = 16.0 Hz, CH=), 6.91 (2H, t, J = 8.5 Hz, C3′-H and C5′-H), 6.97 (1H, d, J = 16.0 Hz, CH=), 7.43 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 485 [M − H]−; Anal. C26H30O9, C 64.38, H 6.12 (Req C 64.19, H 6.22). Following the general procedure, compound 14 was prepared from compound 13 as white crystals (82.0% yield): mp 240−242 ºC; IR (KBr) ν 3 530, 3 408, 3 170, 2 362, 1 736, 1 595, 1 248, 1 178, 1 090, 835, 673 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 4.68 (6H, s, 3 × OCH2), 6.36 (1H, s, C4-H), 6.74 (2H, s, C2-H and C6-H), 6.92 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.01 (1H, d, J = 16.0 Hz, CH=), 7.20 (1H, d, J =16.0 Hz, CH=), 7.52 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 401 [M − H]−; Anal. C20H18O9, C 59.58, H 4.57 (Req C 59.70, H 4.51). Following the general procedure, compound 15 was prepared from E-resveratrol as beige crystals (73.1% yield): mp 183−184 ºC; IR (KBr) ν 3 386, 3 236, 2 943, 2 886, 1 674, 1 526, 1 250, 1 059, 962, 838, 677, 581 cm−1; 1H NMR (600 MHz, CD3OD) δ: 3.43−3.46 (6H, m, 3 × NCH2), 3.64−3.68 (6H, m, 3 × CH2), 4.57 (6H, s, 3 × OCH2CO), 6.59 (1H, s, C4-H), 6.87 (2H, d, J = 8.5 Hz, C2-H and C6-H), 6.94 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.02 (1H, d, J = 16.0 Hz, CH=), 7.18 (1H, d, J = 16.0 Hz, CH=), 7.55 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 532 [M + H]+; Anal. C26H33N3O9, C 58.79, H 6.32, N 8.09 (Req C 58.75, H 6.26, N 7.91). Following the general procedure, compound 16 was prepared from compound 14 and (R)-Amino alcohol as light brown crystals (76.2% yield): mp 192−194 ºC; [α]20D +129 (c 1.0, EtOH); IR (KBr) ν 3 422, 3 316, 2 959, 2 871, 1 652, 1 551, 1 176, 1 078, 980, 836, 595 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 1.05−1.08 (9H, m, 3 × CH3), 3.85−3.92 (3H, m, 3 × NCH), 4.43 (6H, s, 3 × OCH2CO), 4.79−4.83 (3H, m, 3 × CH2), 6.49 (1H, s, C4-H), 6.80 (1H, s, C2-H and C6-H), 6.99
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(2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.00 (1H, d, J = 16.0 Hz, CH=), 7.18 (1H, d, J = 16.0 Hz, CH=), 7.55 (2H, d, J = 8.5 Hz, C2′-H and C6′-H), 7.55-7.79 (3H, m, 3 × CH2); ESI-MS m/z 596 [M + Na]+; Anal. C29H39N3O9, C 60.81, H 6.87, N 7.28 (Req C 60.72, H 6.85, N 7.33). Following the general procedure, compound 17 was prepared from compound 14 and (S)-Amino alcohol as light brown crystals (76.2% yield): mp 192−194 ºC; [α]20D −129 (c 1.0, EtOH); IR (KBr) ν 3 422, 3 316, 2 959, 2 871, 1 652, 1 551, 1 176, 1 078, 980, 836, 595 cm–1; 1H NMR (600 MHz, DMSO-d6) δ: 1.05−1.08 (9H, m, 3 × CH3), 3.85−3.92 (3H, m, 3 × NCH), 4.43 (6H, s, 3 × OCH2CO), 4.79−4.83 (3H, m, 3 × CH2), 6.49 (1H, s, C4-H), 6.80 (1H, s, C2-H and C6-H), 6.99 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.00 (1H, d, J = 16.0 Hz, CH=), 7.18 (1H, d, J = 16.0 Hz, CH=), 7.55 (2H, d, J = 8.5 Hz, C2′-H and C6′-H), 7.55-7.79 (3H, m, 3 × CH2); ESI-MS m/z 596 [M + Na]+; Anal. C29H39N3O9, C 60.81, H 6.87, N 7.28 (Req C 60.72, H 6.85, N 7.33). Following the general procedure, compound 18 was prepared from compound 14 and (R)-Amino alcohol as white crystals (63.2% yield): mp 207−209 ºC; [α]20D +64 (c 1.0, EtOH); IR (KBr) ν 3 416, 3 270, 2 963, 2 880, 1 654, 1 597, 1 546, 1 170, 1 066, 971, 836 cm–1; 1H NMR (600 MHz, DMSO-d6) δ: 0.80−0.87 (18H, m, 6 × CH3), 1.83 (3H, m, 3 × CH), 3.63 (3H, m, 3 × NCH), 4.54 (6H, s, 3 × OCH2CO), 4.64−4.67 (3H, m, 3 × CH2), 6.49 (2H, s, C2-H and C6-H), 6.79 (1H, s, C4-H) , 7.00 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.03 (1H, d, J = 16.0 Hz, CH=), 7.17 (1H, d, J =16.0 Hz, CH=), 7.50 (2H, d, J = 8.5 Hz, C2′-H and C6′-H), 7.58 (3H, m, 3 × CH2); ESI-MS m/z 680 [M + Na]+; Anal. C35H51N3O9, C 64.02, H 7.73, N 6.44 (Req C 63.91, H 7.81, N 6.39). Following the general procedure, compound 19 was prepared from compound 14 and (S)-Amino alcohol as white crystals (63.7% yield): mp 207−209 ºC; [α]20D −64 (c 1.0, EtOH); IR (KBr) ν 3 416, 3 270, 2 963, 2 880, 1 654, 1 597, 1 546, 1 170, 1 066, 971, 836 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 0.80−0.87 (18H, m, 6 × CH3), 1.83 (3H, m, 3 × CH), 3.63 (3H, m, 3 × NCH), 4.54 (6H, s, 3 × OCH2CO), 4.64−4.67 (3H, m, 3 × CH2), 6.49 (2H, s, C2-H and C6-H), 6.79 (1H, s, C4-H), 7.00 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.03 (1H, d, J = 16.0 Hz, CH=), 7.17 (1H, d, J = 16.0 Hz, CH=), 7.50 (2H, d, J = 8.5 Hz, C2′-H and C6′-H), 7.58 (3H, m, 3 × CH2); ESI-MS m/z 680 [M + Na]+; Anal. C35H51N3O9, C 64.02, H 7.73, N 6.44 (Req C 63.91, H 7.81, N 6.39). Following the general procedure, compound 20 was prepared from compound 14 and (R)-Amino alcohol as white crystals (68.9% yield): mp 224−227 ºC; [α]20D +11 (c 1.0, EtOH); IR (KBr) ν 3 392, 3 277, 1 662, 1 540, 1 167, 1067, 955, 835, 698 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 2.76−2.81 (3H, m, 3 × CH2Ph), 2.91−2.96 (3H, m, 3 × CH2Ph), 4.08−4.09 (3H, m, 3 × NCH), 4.51 (6H, s, 3 × OCH2CO), 4.96−4.99 (3H, m, 3 × CH2), 6.51 (1H, s, C4-H), 6.85 (2H, s, C2-H and C6-H), 6.96 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.09 (1H, d, J = 16.0 Hz, CH=), 7.19 (1H, d, J =
16.0 Hz, CH=), 7.21−7.34 (15H, m, Ar-H), 7.58 (2H, d, J = 8.5Hz, C2′-H and C6′-H), 7.88−7.93 (3H, m, 3 × CH2); ESI-MS m/z 802 [M + H]+; Anal. C47H51N3O9, C 70.31, H 6.38, N 5.27 (Req C 70.39, H, 6.41, N 5.24). Following the general procedure, compound 21 was prepared from compound 14 and (S)-Amino alcohol as white crystals (68.1% yield): mp 224−227 ºC; [α]20 D −118(c 1.0, EtOH); IR (KBr) ν 3 392, 3 277, 1 662, 1 540, 1 167, 1 067, 955, 835, 698 cm–1; 1H NMR (600 MHz, DMSO-d6) δ: 2.76−2.81 (3H, m, 3 × CH2Ph), 2.91−2.96 (3H, m, 3 × CH2Ph), 4.08−4.09 (3H, m, 3 × NCH), 4.51 (6H, s, 3 × OCH2CO), 4.96−4.99 (3H, m, 3 × CH2), 6.51 (1H, s, C4-H), 6.85 (2H, s, C2-H and C6-H), 6.96 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.09 (1H, d, J = 16.0 Hz, CH=), 7.19 (1H, d, J = 16.0 Hz, CH=), 7.21-7.34 (15H, m, Ar-H), 7.58 (2H, d, J = 8.5 Hz, C2′-H and C6′-H), 7.88-7.93 (3H, m, 3 × CH2); ESI-MS m/z 802 [M + H]+; Anal. C47H51N3O9, C 70.31, H 6.38, N 5.27 (Req C 70.39, H 6.41, N 5.24). Following the general procedure, compound 22 was prepared from E-resveratrol as white crystals (43.0% yield): mp 47−48 ºC; IR (KBr) ν 3 070, 2 990, 2 934, 2 833, 1 593, 1 509, 1 458, 1 278, 1 249, 1 155, 1 067, 1 031, 959, 828, 682 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 3.83 (9H, s, 3 × OCH3), 6.37 (1H, s, C4-H), 6.49 (2H, s, C2-H and C6-H), 6.63 (1H, d, J = 16.0 Hz, CH2=), 6.95 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.04 (1H, d, J = 16.0 Hz, CH2=), 7.45 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 271 [M + H]+; Anal. C17H18O3, C 75.65, H 6.79 (Req C 75.53, H 6.79). Following the general procedure, compound 23 was prepared from E-resveratrol as white crystals (22.8% yield): mp 88−89 ºC; IR (KBr) ν 3 547, 3 444, 3 236, 1 608, 1 586, 1 512, 1 453, 1 296, 1 233, 1 148, 1 052, 959, 830, 673 cm−1; 1 H NMR (600 MHz, CDCl3) δ: 3.83 (6H, s, 2 × OCH3), 6.38 (1H, s, C4-H), 6.64 (2H, s, C2-H and C6-H) ,6.82 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 6.89 (1H, d, J = 16.4 Hz, CH2=), 7.03 (1H, d, J = 16.4 Hz, CH2=), 7.40 (2H, d, J = 8.4 Hz, C2′-H and C6′-H); ESI-MS m/z 256 [M + H]+; Anal. C16H16O3, C 75.02, H 6.23 (Req C 74.98, H 6.29). Following the general procedure, compound 24 was prepared from E-resveratrol as white crystals (14.0% yield): mp 115−117 ºC; IR (KBr) ν 3 547, 3 446, 3 237, 1 590, 1 509, 1 454, 1 150, 1 051, 958, 833, 680 cm−1; 1H NMR (600 MHz, CDCl3) δ: 3.83 (3H, s, OCH3), 4.87 (1H, s, C4-H), 6.31 ( 1H, s, C2-H), 6.60 (2H, d, J = 16.0 Hz, CH2=), 6.84 (1H, s, C6-H), 6.89 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.02 (1H, d, J = 16.0 Hz, CH2=), 7.44 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 241 [M − H]−; Anal. C15H14O3, C 74.47, H 5.90 (Req C 74.36, H 5.82). Following the general procedure, compound 25 was prepared from E-resveratrol as white crystals (10.7% yield): mp 168−172 ºC; IR (KBr) ν 3 360, 1 601, 1 511, 1 349, 1 301, 1 261, 1 170, 1 007, 962, 832, 677 cm−1; 1H NMR (600 MHz, CDCl3) δ: 3.82 (3H, s, OCH3), 4.84 (1H, s, C4-H), 6.25 (1H, s, C2-H), 6.55 (1H, s, C6-H), 6.83 (1H, d, J = 16.0 Hz, CH2=),
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6.90 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.00 (1H, d, J = 16.0 Hz, CH2=), 7.43 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 241 [M − H]−; Anal. C15H14O3, C 74.45, H 5.86 (Req C 74.36, H 5.82). General procedures for the synthesis of compound 26 Compound 10 (2.40 g, 7.55 mmol) was dissolved in acetone (100 mL), and K2CO3 (2.59 g, 18.8 mmol) was added. Then, Me2SO4 (1.8 mL, 19.0 mmol) was slowly added and the mixture was stirred at reflux for 5 h. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified on silica gel column using petroleum ether:ethyl acetate (4 : 1) as an eluent to give a white solid [3, 5-dimethoxy-4'-O-benzylresveratrol (1.96 g, 75.1% yield). The white solid (1.96 g, 5.66 mmol) was dissolved in ethyl acetate and was stirred at room temperature under 2 atm H2 pressure in the presence of Pd/C (13 mg) for 24 h. The reaction mixture was filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel; 3 : 2 petroleum ether/ethyl acetate elution) to give compound 23 (1.79 g，90.9% yield) as white crystals: mp 69−70 ºC ; IR (KBr) ν 3 017, 2 950, 1 601, 1 511, 1 349, 1 261, 1 237, 1 179, 1 170, 1 009, 962, 830 cm−1; 1H NMR (600 MHz, CDCl3) δ: 2.81−2.86 (4H, m, 2 × CH2), 3.76 (6H, s, 2 × OCH3), 5.05 (2H, s, OCH2), 6.31 (1H, s, C4-H), 6.33 (2H, s, C2-H and C6-H), 6.90 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 7.10 (2H, d, J = 8.4 Hz, C2′-H and C6′-H), 7.32 (1H, t, J = 7.6 Hz, Ar-H), 7.38 (2H, dd, J = 7.6 Hz, Ar-H), 7.43 (2H, d, J = 7.6 Hz, Ar-H); ESI-MS m/z 349 [M + H]+; Anal C23H24O3, C 79.32, H 7.01 (Req C 79.28, H 6.94).
Cytotoxicity studies in vitro Biological materials A549 (lung adenocarcinoma), LAC (lung adenocarcinoma), and HeLa cell lines (cervical cancer) cell lines were obtained from Gentest, BD Biosciences (Woburn, MA, USA). Dimethyl sulfoxide (DMSO) and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) were purchased from Sigma Company (St. Louis, Missouri, USA). RPMI-1640 and DMEM Media were purchased from Gibco Company (California, USA). FBS was obtained from Jiangxi Medical College (Nanchang, China). All other chemicals and reagents were of analytical grade. Cell culture A549 and LAC cells were cultured in RPMI-1640, and the HeLa cells were cultured in DMEM, bothsupplemented with 5% FBS, 100 units·mL−1 of penicillin, 100 μg·mL−1 of streptomycin; the cells were maintained in culture flasks in a 5% CO2−95% air atmosphere at 37 °C. The media were changed twice per week, and when the cells became confluent, they were trypsinized, passed, and reseeded for specific studies. MTT assay The MTT assay was employed for the in vitro cytotoxicity assay and performed in 96-well plates. A549 cells at the log phase of their growth cycle (5 × 104 cell·mL−1) were added to each well (10 μL/well), in six replicates,
treated with test compounds at various concentrations (3.125 - 100 μmol·mL −1), and then incubated for 72 h. The MTT solution (0.5 mg·mL−1, 20 μL per well,) was added, the cells were incubated for an additional 4 h at 37 °C. DMSO was then added to each well (200 μL/well), and after 10 min at room temperature, the OD of each well was measured on a Microplate Reader at the wavelength of 570 nm. In these experiments, the negative reference agent was 0.1% DMSO, and adriamycin at various concentrations (3.125− 100 μmol·mL−1) was used as the positive reference substance. The same method was used for the cytotoxic testing against the LAC and HeLa cells. All assays were performed in triplicate. The data were expressed as the percentage of relative viability compared with the untreated cells. IC50 calculations were performed using Microsoft Excel software for PC.
Results and Discussion Chemistry The synthetic routes are outlined in Scheme 1. A series of E-resveratrol derivatives 1−26 were synthesized. The E-resveratrol phenolic hydroxy groups were etherified with various groups. Treatment of E-resveratrol with benzene sulfonyl chloride and anhydrous potassium carbonate in acetone afforded compounds 1−3. Compounds 4−6 were prepared by the treatment of E-resveratrol with Br(CH2)3Cl and anhydrous potassium carbonate in DMF . Treatment of the E-resveratrol with benzyl chloride and anhydrous potassium carbonate in DMSO afforded compounds 7−10. Treatment of the E-resveratrol with acetic anhydride in pyridine afforded compound 11. Compound 12 was synthesized by the treatment of compound 6 with NaN3. Treatment of the E-resveratrol with ethyl chloroacetate and anhydrous potassium carbonate in DMF afforded Compound 13. Compound 14 was obtained by hydrolysis of Compound 13. Compounds 15−21 were prepared by the treatment of compound 14 with amino alcoholsin solvent-less conditions. While the amino alcohol has two configuration isomer (R , S), so corresponding compounds 16−21 have (R)- and (S)configurations. The treatment of the E-resveratrol with dimethyl sulfate and anhydrous potassium carbonate in acetone afforded compounds 22−25. Compound 26 was obtained by the treatment of compound 10 with dimethyl sulfate and then dissolving in ethyl acetate at room temperature under 2 atm H2 pressure in the presence of Pd/C. In vitro cytotoxicity The 26 derivatives of E-resveratrol were evaluated for their cytotoxicity against three cancer cell lines (A549, LAC, and HeLa) by the MTT assay in vitro. The activity of the derivatives was compared with the parent compound E-resveratrol. As shown in Table 1, the results revealed that eleven of the E-resveratrol derivatives were more cytotoxic than E-resveratrol in vitro.
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Scheme 1
Structure of E-resveratrol and synthetic derivatives 1−26
Compounds 1 and 2 were the most active compounds among the synthetic derivatives. Especially, Compound 1 showed an IC 50 value of 4.38 µmol·L −1 in A549 cells which was 15-fold more active than E-resveratrol. The dose response curve for compound 1 is shown in Fig. 1. Compounds 4, 5, 9, and 10 showed remarkable cytotoxic activity, and Compound 9, with an IC 50 value of 1.41 μmol·L −1 in the HeLa cells, was 90-fold more active than E-resveratrol and similar to adriamycin. Compound 20 showed improved cytotoxic activity against the LAC cells. Preliminary analysis of the SAR suggested that most derivatives with one or two phenolic hydroxy groups
etherified showed greater cytotoxic activity than those with three phenolic hydroxyl groups etherified and E-resveratrol. For example, compounds 1 and 2 exhibited stronger cytotoxicity than compound 3, and compounds 4, 5, 9, 10, and 23−25 exhibited stronger cytotoxicity than Compounds 6, 7, and 22. Compounds 1, 2, and 9 etherified with groups bearing an aromatic nucleus exhibited stronger cytotoxicity than any of the other derivatives. However, compound 11 with three phenolic hydroxy groups etherified exhibited stronger cytotoxicity than the other compounds with three phenolic hydroxyl groups etherified. The mechanisms of action of these derivatives are unknown and await further investigation.
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Moreover, recent results from Hai SL et al [29-30] are in agreement with our findings; they have reported that the pharmacokinetics of (E)-3, 5, 4'-trimethoxystilbene (10) in rat plasma are significantly different for that of E-resveratrol, with greater plasma exposure, longer elimination half-lives, and lower clearance being observed with the derivative compound.
Conclusions In the present study, a series of E-resveratrol derivativeswere synthesized and tested for their cytotoxic activity in A549, LAC, and HeLa cells. Compounds 1−6, 12, 15−21, and 23−26 were synthesized for the first time. Most derivatives with one or two phenolic hydroxy groups etherified showed greater cytotoxic activity than those with three phenolic hydroxylgroups etherified and E-resveratrol, and the derivatives
Fig. 1 Dose-response curve of compound 1
Table 1 Cytotoxic activity of synthesized compounds 1–26 in vitro from different tumor types ( A549, LAC, and HeLa cells) Compounds
a) b)
IC50/(μmol·L−1)
Compounds
IC50/(μmol·L−1)
A549
LAC
HeLa
A549
LAC
HeLa
1
4.38 ± 0.71
10.19 ± 0.37
8.16 ± 0.95
16
≥100 ± 1.35
≥100 ± 1.27
≥100 ± 1.03
2
4.90 ± 1.18
5.55 ± 1.07
6.48 ± 1.56
17
≥100 ± 1.46
≥100 ± 0.23
≥100 ± 1.06
3
≥100 ± 1.05
≥100 ± 1.46
≥100 ± 1.82
18
≥100 ± 0.89
≥100 ± 0.52
≥100 ± 1.13
4
14.17 ± 1.28
27.71 ± 2.01
17.08 ± 0.79
19
≥100 ± 1.05
≥100 ± 0.83
≥100 ± 2.04
5
10.15 ± 2.07
16.35 ± 0.31
8.89 ± 1.63
20
≥100 ± 1.17
≥100 ± 0.84
≥100 ± 0.52
6
49.85 ± 2.55
≥100 ± 2.74
≥100 ± 1.04
21
≥100 ± 1.20
≥100 ± 2.33
≥100 ± 1.67
7
≥100 ± 1.56
≥100 ± 2.02
≥100 ± 3.12
22
≥100 ± 0.20
21.06 ± 0.35
≥100 ± 3.12
8
≥100 ± 2.34
≥100 ± 0.79
≥100 ± 1.40
23
60.05 ± 0.78
8.50 ± 1.87
84.37 ± 3.41
9
11.80 ± 0.75
7.96 ± 2.87
1.41 ± 3.21
24
≥100 ± 0.69
9.80 ± 1.07
53.06 ± 3.24
10
26.29 ± 0.20
9.43 ± 1.03
8.25 ± 2.06
25
59.51 ± 1.60
7.58 ± 1.02
52.21 ± 0.93
11
9.43 ± 3.41
30.70 ± 2.57
20.14 ± 1.46
26
≥100 ± 1.05
≥100 ± 2.32
≥100 ± 1.74
12
≥100 ± 2.35
≥100 ± 0.94
≥100 ± 0.86
E-resveratrol
66.25 ± 2.07
24.89 ± 1.40
98.17 ± 0.54
13
≥100 ± 2.31
≥100 ± 3.35
≥100 ± 0.24
2.36 ± 1.01
1.18 ± 1.78
0.48 ± 0.96
14
≥100 ± 1.42
≥100 ± 1.82
≥100 ± 2.12
15
≥100 ± 3.14
≥100 ± 2.71
≥100 ± 0.98
a
Adriamycin
Used as a positive control. The IC50 value is the concentration at which 50% survival of cells was observed The results are reported as mean ± SE for at least three experiments
etherified with groups bearing an aromatic nucleus were more active than those bearing aliphatic groups. Compounds 1, 2, and 9−11 were identified to be the most promising candidates with improved cytotoxic activity, making them potential candidates for cancer drug discovery.
[3]
[4]
Acknowledgments The authors are thankful to the Analysis and Testing Center of Nanchang University for facilities and support.
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pinostilbene, a resveratrol methylated derivative, against 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells [J]. J Nutrit Biochem, 2010, 21: 482-489. Yang LM, Lin SJ, Hsu FL, et al. Antitumor agents. Part 3: synthesis and cytotoxicity of new trans-stilbene benzenesulfonamide derivatives [J]. Bioorg Med Chem Lett, 2002, 12: 1013-1022. Gosslau A, Chen M, Ho CT, et al. Amethoxy derivative of resveratrol analogue selectively induced activation of the mitochondrial apoptotic pathway in transformed fibroblasts [J]. BJ Cancer, 2005, 92(3): 513-521. Cardile V, Lombardo L, Spatafora C, et al. Chemoenzymatic synthesis and cell-growth inhibition activity of resveratrol analogues [J]. Bioorg Chem, 2005, 33(1): 22-33. Burkhardt S, Reiter RJ, Tan DX, et al. DNA oxidatively damaged by chromium(III) and H2O2 is protected by the antioxidants melatonin, N-acetyl-N-formyl-5-methoxykynu ramine, resveratrol and uric acid [J]. Int J Biochem Cell Biol, 2001, 33: 775-781. Bhat KP, Pezzuto JM. Cancer chemopreventive activity of resveratrol [J]. Ann NY Acad Sci, 2002, 957: 210-229. Schneider Y, Chabert P, Stutzmann J, et al. Resveratrol analog (Z)-3, 5, 4’-trimethoxystilbene is a potent anti-mitotic drug inhibiting tubulin poly-merization [J]. Int J Cancer, 2003, 107 (2): 189-196. Chillemi R, Sciuto S, Spatafora C, et al. Anti-tumor properties of stilbene-based resveratrol analogues: recent results [J]. Nat Prod Commun, 2007, 2(4): 499-513. Stivala LA, Savio M, Carafoli F, et al. Specific structural determinants are responsible for the, antioxidant activity and the cell cycle effects of resveratrol [J]. J Biol Chem, 2001, 276(25): 22586-22594. Hai SL, Paul CH. A rapid HPLC method for the quantification of 3, 5, 4′-trimethoxy-trans-stilbene (TMS) in rat plasma and its application in pharmacokinetic study [J]. J Pharm Biomed Anal. 2009, 49(2): 387-392. Hai SL, Corrado T, Carmela S, et al. A simple and sensitive HPLC-UV method for the quantification of piceatannol analog trans-3, 5, 3′, 4′-tetramethoxystilbene in rat plasma and its application for a pre-clinical pharmacokinetic study [J]. J Pharm Biomed Anal, 2010, 51(3): 679-684.
Cite this article as: HONG Ting, JIANG Wei, DONG Huai-Ming, QIU Sheng-Xiang, LU Yu. Synthesis and cytotoxic activities of E-resveratrol derivatives [J]. Chinese Journal of Natural Medicines, 2015, 13 (5): 375-382.
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Chinese Journal of Natural Medicines
Synthesis and cytotoxic activities of E-resveratrol derivatives HONG Ting1, 2, JIANG Wei1, DONG Huai-Ming1, QIU Sheng-Xiang3, LU Yu1* 1
Sino-German Joint Research Institute of Nanchang University, Nanchang 330047, China; Jiangxi Provincial Institute for Food and Drug Control, Jiangxi Provincial Engineering Research Center for Drug and Medical Device Quality, Nanchang 330029, China; 3 Chinese Academy of Sciences, South China Botanical Garden, Guangzhou 510650, China 2
Available online 20 May 2015
[ABSTRACT] The present study was designed to synthesize derivatives of E-resveratrol and evaluate their cytotoxic activity in vitro. Different functional groups were conjugated with the phenolic hydroxyl group of E-resveratrol, and the double bond of E-resveratrol was reduced. The in vitro cytotoxicity of the synthetic derivatives was evaluated against three tumor cell lines (A549, LAC, and HeLa) using the MTT assay. Twenty-six E-resveratrol derivatives were synthesized and their structures were confirmed by 1 H NMR, MS, IR, and elemental analyses. Compounds 1−6, 12, 15−21, and 23−26 were reported for the first time. Among them, Compounds 1, 2, 4, 5, and 9−11, showed significant cytotoxicity against tumor cells; especially, Compound 1 showed an IC50 value of 4.38 µmol·L−1 in the A549 cells which was 15-fold more active than E-resveratrol; Compound 9 showed an IC50 value of 1.41 µmol·L−1 in the HeLa cell line which was 90-fold more active than E-resveratrol, and close to adriamycin. The structure–activity relationships were also investigated. Compounds 1, 2 and 9−11 may serve as potential lead compounds for the discovery of new anticancer drugs. [KEY WORDS] E-resveratrol derivatives; Synthesis; Cytotoxic activity; Structure–activity relationships
[CLC Number] R284.3, R965
[Document code] A
[Article ID] 2095-6975(2015)05-0375-08
Introduction The naturally occurring E-resveratrol and its various derivatives have attracted a great deal of attention due to their wide range of biological properties. E-resveratrol (3, 4', 5-trihydroxystilbene) is a stilbene-type polyphenolic natural product present in grapes and a variety of medicinal plants [1-7] . It is a naturally occurring phytoalexin which can be activated by adverse conditions of plants, protecting against fungal infections [8]. E-resveratrol possesses multiple biological activities that are beneficial to human health, including anticancer [9], anti-inflammatory [10], antibacterial antioxidant [11] , anti-free radica [12], heart protecting [13], liver protecting [14] , fatty acid synthase-inhibitory [15], nerve protecting [16], estrogenic [17], bone metabolism and endothelin antagonist [18], among others. Jang M et al reported the antitumor function of [Received on] 03-June-2014 [Research funding] This work was supported by the Cross-fund of Nanchang University. [*Corresponding author] Tel: 86-13870886467, Fax: 86-791-8333 7081, E-mail: [email protected] These authors have no conflict of interest to declare. Copyright © 2015, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
E-resveratrol in 1997 [19]. The usefulness of E-resveratrol, however, is limited by its instability upon exposure to light and oxygen. These stimuli may cause trans-cis transformation or oxidation that leads to a reduction in bioavailability and bioactivity. Recently, considerable attention has been focused on E-resveratrol derivatives. It has been reported that methylated E-resveratrol derivative R3 is an effective neuroprotective agent against free radicalmediated oxidative stress triggered by 6-OHDA in SH-SY5Y cells [20]. Yang LM et al have synthesized a series of E-resveratrol derivatives with sulfur thalidomide substitutes that showed inhibitory activities against breast cancer and colon cancer [21]. It is reported that methylated E-resveratrol derivatives are more effective than E-resveratrol in the prevention and treatment of cancer [22]. Cardile V and others have synthesized methoxy derivatives of E-resveratrol and E-resveratrol esters, tested their cytotoxicity in human prostate cancer cells, and found that the activity of these derivatives was stronger than E-resveratrol [23-25]. It has been reported that polymethoxy-stilbene analogs, especially (Z)-3, 5, 4-trimethoxystilbene, exhibit strong anti-proliferative activity [26-27]. Stivala LA et al have reported a number of E-resveratrol derivatives which showed good anti-proliferative activity, suggesting that the 7-methoxycoumarin nucleus, together with the 3, 5-disubstitution pattern of the trans-
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vinylbenzene moiety, are promising structural features to obtain excellent anticancer compounds endowed with a apoptosis-inducing capability [28]. Encouraged by the promising cytotoxic activity of the reported E-resveratrol derivatives, we designed and synthesized a series of E-resveratrol derivatives and evaluated their biological activities structure-activity relationships (SAR) in the present study. Our results indicated that some of the analogs can be developed as lead compounds as novel anticancer agents.
Materials and Methods Materials and instrumentation 1 H NMR spectra were recorded with a Bruker Avance 600 FT-NMR spectrometer (Bruker Biospin Ltd., Bern, Switzerland) in the indicated solvents (TMS as internal standard). Mass spectra were obtained using Waters 2695-4000 spectrometer (Waters Ltd., Milford, Massachusetts, USA). IR spectra were obtained on a Nicolet 380 spectrometer (Thermo Nicole Ltd, Madison, Wisconsin, USA). All melting points are measured using an Electrothermal engineering 9200 apparatus (Electrothermal Engineering Ltd, Stone Staffordshire, UK). Elemental analyses were performed using a TQ-3A elemental analysis instrument (Elementar Ltd, Hanau, Germany). All of the compounds synthesized were purified by column chromatography (CC) on silica gel 60 (200−300 mesh) and thin-layer chromatography (TLC) on silica gel 60 F254 plates (250 μm; Qingdao Marine Chemical Company, Qingdao, China). Most chemicals and solvents were of analytical grade. E-resveratrol (98%) was purchased from Shanxi Sciphar Hi-tech Industry Co., Ltd. (Shanxi, China). General procedures for the synthesis of compounds 1−25 E-resveratrol (1.14 g, 5.00 mmol) was dissolved in acetone (100 mL), and K2CO3 (1.04 g, 7.54 mmol) was added. Then, PhSO2Cl (0.6 mL, 4.70 mmol) were slowly added and the reaction mixture was stirred at reflux temperature for 9 h. The resultant clear solution was filtered and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; 9 : 1 chloroform/ethyl acetate elution) to give compound 1 (0.39 g, 22.5% yield) as white crystals: mp 152−153 ºC; IR (KBr) ν 3 413, 3 059, 3 017, 1 600, 1 515, 1 449, 1 353, 1 298, 1 171, 1 090, 986, 959, 847, 808, 570 cm–1; 1H NMR (600 MHz, CDCl3) δ: 6.66 (1H, s, C5-H), 6.73 (1H, s, C2-H), 6.74 (1H, d, J = 16.4 Hz, CH=), 6.82 (1H, s, C6-H) 6.83-6.84 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 6.88 (1H, d, J = 16.4 Hz, CH=), 7.35 (2H, d, J = 8.4 Hz, C2′-H and C6′-H), 7.54 (2H, dd, J = 7.6 Hz, Ar-H), 7.69 (1H, t, J = 7.6 Hz, Ar-H), 7.88 (2H, d, J = 7.6 Hz, Ar-H); ESI-MS m/z 369 [M + H]+; Anal. C20H16O5S, C 65.12, H 4.49 (Req C 65.20, H 4.38). Following the general procedure, compound 2 was prepared from E-resveratrol as white crystals (33.1% yield): m p 94−95 ºC; IR (KBr) ν 3 460, 1 615, 1 569, 1 501, 1 449, 1
370, 1 292, 1 192, 1 148, 1 091, 984, 972, 874, 750, 581 cm–1; H NMR(600 MHz, CDCl3) δ: 6.42 (1H, s, C5-H), 6.66 (1H, s, C2-H), 6.78 (1H, d, J = 16.4 Hz, CH=), 6.82 (1H, s, C6-H), 6.84 (1H, d, J = 16.4 Hz, CH=), 6.94 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 7.32 (2H, d, J = 8.4 Hz, C2′-H and C6′-H), 7.54 (4H, t, J = 12.0 Hz, Ar-H), 7.66-7.69 (2H, m, Ar-H), 7.83−7.88 (4H, m, Ar-H); ESI-MS m/z 531 [M + Na]+; Anal. C26H20O7S2, C 61.58; H 3.89 (Req C 61.40, H 3.96). Following the general procedure, compound 3 was prepared from E-resveratrol as white crystals (96.3% yield) as a white crystal: mp 1 4 4 −145 ºC; IR (KBr) ν 3 079, 3 029, 1 611, 1 578, 1 500, 1 449, 1 383, 1 350, 1 304, 1 191, 1 089, 968, 866, 843, 771, 682, 633, 563 cm−1; 1H NMR (600 MHz, CDCl3) δ: 6.49 (1H, s, C4-H), 6.79 (1H, d, J = 16.2 Hz, CH=), 6.84 (1H, d, J = 16.2 Hz, CH=), 6.98 (2H, d, J = 9.0 Hz, C3′-H and C5′-H), 7.04 (2H, s, C2-H and C6-H), 7.35 (2H, d, J = 9.0 Hz, C2′-H and C6′-H), 7.53−7.57 (6H, m, Ar-H), 7.69−7.71 (3H, m, Ar-H), 7.80 (4H, t, J = 8.4 Hz, Ar-H), 7.82 (2H, d, J = 8.4 Hz, Ar-H); ESI-MS m/z 687 [M + K]+; Anal. C32H24O9S3, C 59.06, H 3.69 (Req C 59, H 3.73). Following the general procedure, compound 4 was prepared from E-resveratrol as white crystals (30.9% yield): mp 152−154 ºC; IR (KBr) ν 3 395, 3 249, 2 959, 2 929, 2 872, 1 605, 1 511, 1 459, 1 350, 1 306, 1 258, 1 168, 1 148, 1 038, 962, 831, 682 cm−1; 1H NMR(600 MHz, CDCl3) δ: 2.26 (2H, m, CH2), 3.75 (2H, d, J = 6.3 Hz, CH2Cl), 4.14 (2H, d, J = 6.3 Hz, OCH2), 6.25 (1H, s, C4-H), 6.55 (2H, s, C2-H and C6-H), 6.83 (1H, d, J = 16.2 Hz, CH=), 6.89 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 6.99 (1H, d, J = 16.2Hz, CH=), 7.42 (2H, d, J = 8.4 Hz, C2′-H and C6′-H); ESI-MS m/z 305 [M + H]+; Anal. C17H17ClO3, C 68.84, H 5.81 (Req C 67.00, H 5.62). Following the general procedure, compound 5 was prepared from E-resveratrol as white crystals (47.9% yield): m p 9 4 − 9 5 ºC; IR (KBr) ν 3 339, 3 015, 2 964, 2 934, 2 860, 1 600, 1 511, 1 441, 1 341, 1 251, 1 230, 1 164, 1 064, 957, 832, 676, 656 cm−1; 1H NMR(600 MHz, CDCl3) δ: 2.23−2.26 (4H, m, 2 × CH2), 3.74−3.77 (4H, m, 2 × CH2Cl), 4.12−4.15 (4H, m, 2 × OCH2), 6.31 (1H, s, C4-H), 6.57 (1H, s, C2-H), 6.63 (1H, s, C6-H), 6.86 (1H, d, J = 16.2 Hz, CH=), 6.89 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 7.01 (1H, d, J = 16.2 Hz, CH=), 7.42−7.43 (2H, d, J = 8.4 Hz, C2′-H and C6′-H); ESI-MS m/z 381 [M + H]+; Anal. C20H22Cl2O3, C 63.13, H 5.77 (Req C 63.00, H 5.82). Following the general procedure, compound 6 was prepared from E-resveratrol as white crystals (53.1% yield): m p 88−89 ºC; IR (KBr) ν 2 955, 2 930, 2 876, 1 587, 1 512, 1 439, 1 257, 1 165, 1 062, 955, 833, 677 cm−1; 1H NMR(600 MHz, CDCl3) δ: 2.23−2.27 (6H, m, 3 × CH2), 3.76 (6H, t, J = 8.0 Hz, 3 × CH2Cl), 4.14 (6H, t, J = 8.0 Hz, 3 × OCH2), 6.38 (1H, s, C4-H), 6.66 (2H, s, C2-H and C6-H), 6.88 (1H, d, J = 16.2 Hz, CH=), 6.90 (2H, d, J = 8.0 Hz, C3′-H and C5′-H), 7.03 (1H, d, J = 16.2 Hz, CH=), 7.43 (2H, d, J = 8.0 Hz, C2′-H and C6′-H); ESI-MS m/z 457 [M + H]+; Anal. C23H27Cl3O3, C 60.51, H 6.01 (Req C 60.34, H 5.94). 1
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Following the general procedure, compound 7 was prepared from E-resveratrol as white crystals (93.4% yield): m p 138−140 ºC; IR (KBr) ν 3 054, 3 030, 2 921, 2 859, 1 597, 1 512, 1 443, 1 371, 1 276, 1 155, 1 062, 1 014, 956, 833, 736, 684 cm−1; 1H NMR (600 MHz, CDCl3) δ: 5.06 (4H, s, 2 × OCH2), 5.08 (2H, s, OCH2), 6.53 (1H, s, C4-H), 6.74 (2H, s, C2-H and C6-H), 6.89 (1H, d, J = 16.2 Hz, CH=), 6.96 (2H, d, J = 7.8 Hz, C3′-H and C5′-H), 7.01 (1H, d, J = 16.2 Hz, CH=), 7.32 (3H, t, J = 7.8 Hz, Ar-H), 7.38 (6H, t, J = 7.8 Hz, Ar-H), 7.39-7.40 (6H, J = 7.8 Hz, Ar-H), 7.42−7.45 (2H, d, J = 7.8 Hz, C2′-H and C6′-H); ESI-MS m/z 499 [M + H]+; Anal. C35H30O3, C 84.17, H 5.97 (Req C 84.31, H 6.06). Following the general procedure, compound 8 was prepared from E-resveratrol as white crystals (43.9% yield): mp 142−143 ºC; IR(KBr) ν 3 419, 3 083, 3 029, 2 868, 1 600, 1 512, 1 453, 1 383, 1 350, 1 383, 1 350, 1 265, 1 175, 1 147, 1 016, 961, 842, 745, 699 cm−1; 1H NMR (600 MHz, CDCl3) δ: 5.07 (2H, s, OCH2), 5.09 (2H, s, OCH2), 6.38 (1H, s, C4-H), 6.59 (1H, s, C2-H), 6.71(1H, s, C6-H), 6.89 (1H, d, J = 16.0 Hz, CH=), 6.96−6.97 (2H, m, C3′-H and C5′-H), 7.01 (1H, d, J = 16.0 Hz, CH=), 7.32−7.35 (2H, m, C2′-H and C6′-H), 7.38−7.45 (10H, m, 2 × Ar-H); ESI-MS m/z 409 [M + H]+; Anal. C28H24O3, C 82.12, H 5.89 (Req C 82.33, H 5.92). Following the general procedure, compound 9 was prepared from E-resveratrol as white crystals (30.2% yield): mp 143−144 ºC; IR (KBr) ν 3 563, 3 386, 3 029, 2 950, 1 632, 1 603, 1 509, 1 445, 1 334, 1 283, 1 253, 1 134, 1 050, 970, 847, 749, 735, 696, 632 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 5.07 (2H, s, OCH2), 6.28 (1H, s, C4-H), 6.52 (1H, s, C2-H), 6.67 (1H, s, C6-H), 6.76 (2H, d, J = 6.0 Hz, C3′-H and C5′-H), 6.86 (1H, d, J = 16.0 Hz, CH=), 7.03 (1H, d, J = 16.0 Hz, CH=), 7.34 (1H, t, J = 6.0 Hz, Ar-H), 7.39−7.41 (4H, m, Ar-H), 7.46 (2H, d, J = 6.0 Hz, C2′-H and C6′-H), 9.40 (1H, s, OH), 9.56 (1H, s, OH); ESI-MS m/z 319 [M + H]+; Anal. C21H18O3, C 79.19, H 5.83 (Req C 79.22, H 5.70). Following the general procedure, compound 10 was prepared from E-resveratrol as a white crystals (14.5% yield): mp 193−194 ºC; IR (KBr) ν 3 393, 3 278, 3 029, 2 897, 2 886, 1 603, 1 511, 1 458, 1 382, 1 330, 1 263, 1 148, 1 038, 1 025, 963, 830, 694, 731, 607 cm–1; 1H NMR (600 MHz, DMSO-d6) δ: 5.13 (2H, s, OCH2), 6.12 (1H, t, C4-H), 6.40 (2H, s, C2-H and C6-H), 6.90 (1H, d, J = 16.2 Hz, CH=), 6.98 (1H, d, J = 16.2 Hz, CH=), 7.05 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 7.33 (1H, t, J = 4.8 Hz, Ar-H), 7.40 (2H, dd, J = 4.8 Hz, Ar-H), 7.46 (2H, d, J = 4.8 Hz, Ar-H), 7.51 (2H, d, J = 8.4 Hz, C2′-H and C6′-H), 9.20 (2H, s, 2 × OH); ESI-MS m/z 319 [M + H]+; Anal. C21H18O3, C 79.15, H 5.88 (Req C 79.22, H 5.70). Following the general procedure, compound 11 was prepared from E-resveratrol as white crystals (72.6% yield): mp 113−114 ºC; IR (KBr) ν 3 448, 3 075, 1 758, 1 612, 1 589, 1 507, 1 370, 1 214, 1 127, 1 018, 912 cm−1; 1H NMR (600 MHz, CDCl3) δ: 2.30 (9H, s, 3 × OCH3), 6.82 (1H, s, C4-H), 6.97 (1H, d, J = 16.0 Hz, CH=), 7.06 (1H, d, J = 16.0 Hz, CH=), 7.09 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.11 (2H, s,
C2-H and C6-H), 7.48 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 355 [M + H]+; Anal. C20H18O6, C 67.83, H 5.01 (Req C 67.79, H 5.12). Following the general procedure, compound 12 was prepared from compound 6 as white crystals (95.2% yield): mp 41−43 ºC; IR (KBr) ν 3 037, 2 933, 2 104, 1 598, 1 510, 1 452, 1 350, 1 291, 1 252, 1 169, 1 060, 963, 833, 680 cm−1; 1 H NMR (600 MHz, CDCl3) δ: 2.03−2.09 (6H, m, 3 × CH2N3), 3.53 (6H, t, J = 6.0 Hz, 3 × CH2), 4.07 (6H, t, J = 6.0 Hz, 3 × OCH2), 6.37 (1H, s, C4-H), 6.65 (2H, s, C2-H and C6-H), 6.89 (1H, d, J = 16 Hz, CH=), 6.87 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 7.03 (1H, d, J = 16 Hz, CH=), 7.43 (2H, d, J = 8.4 Hz, C2′-H and C6′-H); ESI-MS m/z 478 [M + H]+; Anal. C23H27N9O3, C 57.74, H 5.82 (Req C 57.85, H 5.70). Following the general procedure, compound 13 was prepared from E-resveratrol as white crystals (52.7% yield): mp 83−84 ºC; IR (KBr) ν 3 429, 2 988, 2 909, 1 759, 1 723, 1 595, 1 169, 834 cm−1; 1H NMR (600 MHz, CDCl3) δ: 1.25−1.33 (9H, m, 3 × CH3), 4.26−4.31 (6H, m, 3 × COCH2), 4.62−4.64 (6H, m, 3 × OCH2CO), 6.40 (1H, s, C4-H), 6.67 (2H, s, C2-H and C6-H), 6.90 (1H, d, J = 16.0 Hz, CH=), 6.91 (2H, t, J = 8.5 Hz, C3′-H and C5′-H), 6.97 (1H, d, J = 16.0 Hz, CH=), 7.43 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 485 [M − H]−; Anal. C26H30O9, C 64.38, H 6.12 (Req C 64.19, H 6.22). Following the general procedure, compound 14 was prepared from compound 13 as white crystals (82.0% yield): mp 240−242 ºC; IR (KBr) ν 3 530, 3 408, 3 170, 2 362, 1 736, 1 595, 1 248, 1 178, 1 090, 835, 673 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 4.68 (6H, s, 3 × OCH2), 6.36 (1H, s, C4-H), 6.74 (2H, s, C2-H and C6-H), 6.92 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.01 (1H, d, J = 16.0 Hz, CH=), 7.20 (1H, d, J =16.0 Hz, CH=), 7.52 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 401 [M − H]−; Anal. C20H18O9, C 59.58, H 4.57 (Req C 59.70, H 4.51). Following the general procedure, compound 15 was prepared from E-resveratrol as beige crystals (73.1% yield): mp 183−184 ºC; IR (KBr) ν 3 386, 3 236, 2 943, 2 886, 1 674, 1 526, 1 250, 1 059, 962, 838, 677, 581 cm−1; 1H NMR (600 MHz, CD3OD) δ: 3.43−3.46 (6H, m, 3 × NCH2), 3.64−3.68 (6H, m, 3 × CH2), 4.57 (6H, s, 3 × OCH2CO), 6.59 (1H, s, C4-H), 6.87 (2H, d, J = 8.5 Hz, C2-H and C6-H), 6.94 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.02 (1H, d, J = 16.0 Hz, CH=), 7.18 (1H, d, J = 16.0 Hz, CH=), 7.55 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 532 [M + H]+; Anal. C26H33N3O9, C 58.79, H 6.32, N 8.09 (Req C 58.75, H 6.26, N 7.91). Following the general procedure, compound 16 was prepared from compound 14 and (R)-Amino alcohol as light brown crystals (76.2% yield): mp 192−194 ºC; [α]20D +129 (c 1.0, EtOH); IR (KBr) ν 3 422, 3 316, 2 959, 2 871, 1 652, 1 551, 1 176, 1 078, 980, 836, 595 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 1.05−1.08 (9H, m, 3 × CH3), 3.85−3.92 (3H, m, 3 × NCH), 4.43 (6H, s, 3 × OCH2CO), 4.79−4.83 (3H, m, 3 × CH2), 6.49 (1H, s, C4-H), 6.80 (1H, s, C2-H and C6-H), 6.99
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(2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.00 (1H, d, J = 16.0 Hz, CH=), 7.18 (1H, d, J = 16.0 Hz, CH=), 7.55 (2H, d, J = 8.5 Hz, C2′-H and C6′-H), 7.55-7.79 (3H, m, 3 × CH2); ESI-MS m/z 596 [M + Na]+; Anal. C29H39N3O9, C 60.81, H 6.87, N 7.28 (Req C 60.72, H 6.85, N 7.33). Following the general procedure, compound 17 was prepared from compound 14 and (S)-Amino alcohol as light brown crystals (76.2% yield): mp 192−194 ºC; [α]20D −129 (c 1.0, EtOH); IR (KBr) ν 3 422, 3 316, 2 959, 2 871, 1 652, 1 551, 1 176, 1 078, 980, 836, 595 cm–1; 1H NMR (600 MHz, DMSO-d6) δ: 1.05−1.08 (9H, m, 3 × CH3), 3.85−3.92 (3H, m, 3 × NCH), 4.43 (6H, s, 3 × OCH2CO), 4.79−4.83 (3H, m, 3 × CH2), 6.49 (1H, s, C4-H), 6.80 (1H, s, C2-H and C6-H), 6.99 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.00 (1H, d, J = 16.0 Hz, CH=), 7.18 (1H, d, J = 16.0 Hz, CH=), 7.55 (2H, d, J = 8.5 Hz, C2′-H and C6′-H), 7.55-7.79 (3H, m, 3 × CH2); ESI-MS m/z 596 [M + Na]+; Anal. C29H39N3O9, C 60.81, H 6.87, N 7.28 (Req C 60.72, H 6.85, N 7.33). Following the general procedure, compound 18 was prepared from compound 14 and (R)-Amino alcohol as white crystals (63.2% yield): mp 207−209 ºC; [α]20D +64 (c 1.0, EtOH); IR (KBr) ν 3 416, 3 270, 2 963, 2 880, 1 654, 1 597, 1 546, 1 170, 1 066, 971, 836 cm–1; 1H NMR (600 MHz, DMSO-d6) δ: 0.80−0.87 (18H, m, 6 × CH3), 1.83 (3H, m, 3 × CH), 3.63 (3H, m, 3 × NCH), 4.54 (6H, s, 3 × OCH2CO), 4.64−4.67 (3H, m, 3 × CH2), 6.49 (2H, s, C2-H and C6-H), 6.79 (1H, s, C4-H) , 7.00 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.03 (1H, d, J = 16.0 Hz, CH=), 7.17 (1H, d, J =16.0 Hz, CH=), 7.50 (2H, d, J = 8.5 Hz, C2′-H and C6′-H), 7.58 (3H, m, 3 × CH2); ESI-MS m/z 680 [M + Na]+; Anal. C35H51N3O9, C 64.02, H 7.73, N 6.44 (Req C 63.91, H 7.81, N 6.39). Following the general procedure, compound 19 was prepared from compound 14 and (S)-Amino alcohol as white crystals (63.7% yield): mp 207−209 ºC; [α]20D −64 (c 1.0, EtOH); IR (KBr) ν 3 416, 3 270, 2 963, 2 880, 1 654, 1 597, 1 546, 1 170, 1 066, 971, 836 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 0.80−0.87 (18H, m, 6 × CH3), 1.83 (3H, m, 3 × CH), 3.63 (3H, m, 3 × NCH), 4.54 (6H, s, 3 × OCH2CO), 4.64−4.67 (3H, m, 3 × CH2), 6.49 (2H, s, C2-H and C6-H), 6.79 (1H, s, C4-H), 7.00 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.03 (1H, d, J = 16.0 Hz, CH=), 7.17 (1H, d, J = 16.0 Hz, CH=), 7.50 (2H, d, J = 8.5 Hz, C2′-H and C6′-H), 7.58 (3H, m, 3 × CH2); ESI-MS m/z 680 [M + Na]+; Anal. C35H51N3O9, C 64.02, H 7.73, N 6.44 (Req C 63.91, H 7.81, N 6.39). Following the general procedure, compound 20 was prepared from compound 14 and (R)-Amino alcohol as white crystals (68.9% yield): mp 224−227 ºC; [α]20D +11 (c 1.0, EtOH); IR (KBr) ν 3 392, 3 277, 1 662, 1 540, 1 167, 1067, 955, 835, 698 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 2.76−2.81 (3H, m, 3 × CH2Ph), 2.91−2.96 (3H, m, 3 × CH2Ph), 4.08−4.09 (3H, m, 3 × NCH), 4.51 (6H, s, 3 × OCH2CO), 4.96−4.99 (3H, m, 3 × CH2), 6.51 (1H, s, C4-H), 6.85 (2H, s, C2-H and C6-H), 6.96 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.09 (1H, d, J = 16.0 Hz, CH=), 7.19 (1H, d, J =
16.0 Hz, CH=), 7.21−7.34 (15H, m, Ar-H), 7.58 (2H, d, J = 8.5Hz, C2′-H and C6′-H), 7.88−7.93 (3H, m, 3 × CH2); ESI-MS m/z 802 [M + H]+; Anal. C47H51N3O9, C 70.31, H 6.38, N 5.27 (Req C 70.39, H, 6.41, N 5.24). Following the general procedure, compound 21 was prepared from compound 14 and (S)-Amino alcohol as white crystals (68.1% yield): mp 224−227 ºC; [α]20 D −118(c 1.0, EtOH); IR (KBr) ν 3 392, 3 277, 1 662, 1 540, 1 167, 1 067, 955, 835, 698 cm–1; 1H NMR (600 MHz, DMSO-d6) δ: 2.76−2.81 (3H, m, 3 × CH2Ph), 2.91−2.96 (3H, m, 3 × CH2Ph), 4.08−4.09 (3H, m, 3 × NCH), 4.51 (6H, s, 3 × OCH2CO), 4.96−4.99 (3H, m, 3 × CH2), 6.51 (1H, s, C4-H), 6.85 (2H, s, C2-H and C6-H), 6.96 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.09 (1H, d, J = 16.0 Hz, CH=), 7.19 (1H, d, J = 16.0 Hz, CH=), 7.21-7.34 (15H, m, Ar-H), 7.58 (2H, d, J = 8.5 Hz, C2′-H and C6′-H), 7.88-7.93 (3H, m, 3 × CH2); ESI-MS m/z 802 [M + H]+; Anal. C47H51N3O9, C 70.31, H 6.38, N 5.27 (Req C 70.39, H 6.41, N 5.24). Following the general procedure, compound 22 was prepared from E-resveratrol as white crystals (43.0% yield): mp 47−48 ºC; IR (KBr) ν 3 070, 2 990, 2 934, 2 833, 1 593, 1 509, 1 458, 1 278, 1 249, 1 155, 1 067, 1 031, 959, 828, 682 cm−1; 1H NMR (600 MHz, DMSO-d6) δ: 3.83 (9H, s, 3 × OCH3), 6.37 (1H, s, C4-H), 6.49 (2H, s, C2-H and C6-H), 6.63 (1H, d, J = 16.0 Hz, CH2=), 6.95 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.04 (1H, d, J = 16.0 Hz, CH2=), 7.45 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 271 [M + H]+; Anal. C17H18O3, C 75.65, H 6.79 (Req C 75.53, H 6.79). Following the general procedure, compound 23 was prepared from E-resveratrol as white crystals (22.8% yield): mp 88−89 ºC; IR (KBr) ν 3 547, 3 444, 3 236, 1 608, 1 586, 1 512, 1 453, 1 296, 1 233, 1 148, 1 052, 959, 830, 673 cm−1; 1 H NMR (600 MHz, CDCl3) δ: 3.83 (6H, s, 2 × OCH3), 6.38 (1H, s, C4-H), 6.64 (2H, s, C2-H and C6-H) ,6.82 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 6.89 (1H, d, J = 16.4 Hz, CH2=), 7.03 (1H, d, J = 16.4 Hz, CH2=), 7.40 (2H, d, J = 8.4 Hz, C2′-H and C6′-H); ESI-MS m/z 256 [M + H]+; Anal. C16H16O3, C 75.02, H 6.23 (Req C 74.98, H 6.29). Following the general procedure, compound 24 was prepared from E-resveratrol as white crystals (14.0% yield): mp 115−117 ºC; IR (KBr) ν 3 547, 3 446, 3 237, 1 590, 1 509, 1 454, 1 150, 1 051, 958, 833, 680 cm−1; 1H NMR (600 MHz, CDCl3) δ: 3.83 (3H, s, OCH3), 4.87 (1H, s, C4-H), 6.31 ( 1H, s, C2-H), 6.60 (2H, d, J = 16.0 Hz, CH2=), 6.84 (1H, s, C6-H), 6.89 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.02 (1H, d, J = 16.0 Hz, CH2=), 7.44 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 241 [M − H]−; Anal. C15H14O3, C 74.47, H 5.90 (Req C 74.36, H 5.82). Following the general procedure, compound 25 was prepared from E-resveratrol as white crystals (10.7% yield): mp 168−172 ºC; IR (KBr) ν 3 360, 1 601, 1 511, 1 349, 1 301, 1 261, 1 170, 1 007, 962, 832, 677 cm−1; 1H NMR (600 MHz, CDCl3) δ: 3.82 (3H, s, OCH3), 4.84 (1H, s, C4-H), 6.25 (1H, s, C2-H), 6.55 (1H, s, C6-H), 6.83 (1H, d, J = 16.0 Hz, CH2=),
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6.90 (2H, d, J = 8.5 Hz, C3′-H and C5′-H), 7.00 (1H, d, J = 16.0 Hz, CH2=), 7.43 (2H, d, J = 8.5 Hz, C2′-H and C6′-H); ESI-MS m/z 241 [M − H]−; Anal. C15H14O3, C 74.45, H 5.86 (Req C 74.36, H 5.82). General procedures for the synthesis of compound 26 Compound 10 (2.40 g, 7.55 mmol) was dissolved in acetone (100 mL), and K2CO3 (2.59 g, 18.8 mmol) was added. Then, Me2SO4 (1.8 mL, 19.0 mmol) was slowly added and the mixture was stirred at reflux for 5 h. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified on silica gel column using petroleum ether:ethyl acetate (4 : 1) as an eluent to give a white solid [3, 5-dimethoxy-4'-O-benzylresveratrol (1.96 g, 75.1% yield). The white solid (1.96 g, 5.66 mmol) was dissolved in ethyl acetate and was stirred at room temperature under 2 atm H2 pressure in the presence of Pd/C (13 mg) for 24 h. The reaction mixture was filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography (silica gel; 3 : 2 petroleum ether/ethyl acetate elution) to give compound 23 (1.79 g，90.9% yield) as white crystals: mp 69−70 ºC ; IR (KBr) ν 3 017, 2 950, 1 601, 1 511, 1 349, 1 261, 1 237, 1 179, 1 170, 1 009, 962, 830 cm−1; 1H NMR (600 MHz, CDCl3) δ: 2.81−2.86 (4H, m, 2 × CH2), 3.76 (6H, s, 2 × OCH3), 5.05 (2H, s, OCH2), 6.31 (1H, s, C4-H), 6.33 (2H, s, C2-H and C6-H), 6.90 (2H, d, J = 8.4 Hz, C3′-H and C5′-H), 7.10 (2H, d, J = 8.4 Hz, C2′-H and C6′-H), 7.32 (1H, t, J = 7.6 Hz, Ar-H), 7.38 (2H, dd, J = 7.6 Hz, Ar-H), 7.43 (2H, d, J = 7.6 Hz, Ar-H); ESI-MS m/z 349 [M + H]+; Anal C23H24O3, C 79.32, H 7.01 (Req C 79.28, H 6.94).
Cytotoxicity studies in vitro Biological materials A549 (lung adenocarcinoma), LAC (lung adenocarcinoma), and HeLa cell lines (cervical cancer) cell lines were obtained from Gentest, BD Biosciences (Woburn, MA, USA). Dimethyl sulfoxide (DMSO) and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) were purchased from Sigma Company (St. Louis, Missouri, USA). RPMI-1640 and DMEM Media were purchased from Gibco Company (California, USA). FBS was obtained from Jiangxi Medical College (Nanchang, China). All other chemicals and reagents were of analytical grade. Cell culture A549 and LAC cells were cultured in RPMI-1640, and the HeLa cells were cultured in DMEM, bothsupplemented with 5% FBS, 100 units·mL−1 of penicillin, 100 μg·mL−1 of streptomycin; the cells were maintained in culture flasks in a 5% CO2−95% air atmosphere at 37 °C. The media were changed twice per week, and when the cells became confluent, they were trypsinized, passed, and reseeded for specific studies. MTT assay The MTT assay was employed for the in vitro cytotoxicity assay and performed in 96-well plates. A549 cells at the log phase of their growth cycle (5 × 104 cell·mL−1) were added to each well (10 μL/well), in six replicates,
treated with test compounds at various concentrations (3.125 - 100 μmol·mL −1), and then incubated for 72 h. The MTT solution (0.5 mg·mL−1, 20 μL per well,) was added, the cells were incubated for an additional 4 h at 37 °C. DMSO was then added to each well (200 μL/well), and after 10 min at room temperature, the OD of each well was measured on a Microplate Reader at the wavelength of 570 nm. In these experiments, the negative reference agent was 0.1% DMSO, and adriamycin at various concentrations (3.125− 100 μmol·mL−1) was used as the positive reference substance. The same method was used for the cytotoxic testing against the LAC and HeLa cells. All assays were performed in triplicate. The data were expressed as the percentage of relative viability compared with the untreated cells. IC50 calculations were performed using Microsoft Excel software for PC.
Results and Discussion Chemistry The synthetic routes are outlined in Scheme 1. A series of E-resveratrol derivatives 1−26 were synthesized. The E-resveratrol phenolic hydroxy groups were etherified with various groups. Treatment of E-resveratrol with benzene sulfonyl chloride and anhydrous potassium carbonate in acetone afforded compounds 1−3. Compounds 4−6 were prepared by the treatment of E-resveratrol with Br(CH2)3Cl and anhydrous potassium carbonate in DMF . Treatment of the E-resveratrol with benzyl chloride and anhydrous potassium carbonate in DMSO afforded compounds 7−10. Treatment of the E-resveratrol with acetic anhydride in pyridine afforded compound 11. Compound 12 was synthesized by the treatment of compound 6 with NaN3. Treatment of the E-resveratrol with ethyl chloroacetate and anhydrous potassium carbonate in DMF afforded Compound 13. Compound 14 was obtained by hydrolysis of Compound 13. Compounds 15−21 were prepared by the treatment of compound 14 with amino alcoholsin solvent-less conditions. While the amino alcohol has two configuration isomer (R , S), so corresponding compounds 16−21 have (R)- and (S)configurations. The treatment of the E-resveratrol with dimethyl sulfate and anhydrous potassium carbonate in acetone afforded compounds 22−25. Compound 26 was obtained by the treatment of compound 10 with dimethyl sulfate and then dissolving in ethyl acetate at room temperature under 2 atm H2 pressure in the presence of Pd/C. In vitro cytotoxicity The 26 derivatives of E-resveratrol were evaluated for their cytotoxicity against three cancer cell lines (A549, LAC, and HeLa) by the MTT assay in vitro. The activity of the derivatives was compared with the parent compound E-resveratrol. As shown in Table 1, the results revealed that eleven of the E-resveratrol derivatives were more cytotoxic than E-resveratrol in vitro.
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Scheme 1
Structure of E-resveratrol and synthetic derivatives 1−26
Compounds 1 and 2 were the most active compounds among the synthetic derivatives. Especially, Compound 1 showed an IC 50 value of 4.38 µmol·L −1 in A549 cells which was 15-fold more active than E-resveratrol. The dose response curve for compound 1 is shown in Fig. 1. Compounds 4, 5, 9, and 10 showed remarkable cytotoxic activity, and Compound 9, with an IC 50 value of 1.41 μmol·L −1 in the HeLa cells, was 90-fold more active than E-resveratrol and similar to adriamycin. Compound 20 showed improved cytotoxic activity against the LAC cells. Preliminary analysis of the SAR suggested that most derivatives with one or two phenolic hydroxy groups
etherified showed greater cytotoxic activity than those with three phenolic hydroxyl groups etherified and E-resveratrol. For example, compounds 1 and 2 exhibited stronger cytotoxicity than compound 3, and compounds 4, 5, 9, 10, and 23−25 exhibited stronger cytotoxicity than Compounds 6, 7, and 22. Compounds 1, 2, and 9 etherified with groups bearing an aromatic nucleus exhibited stronger cytotoxicity than any of the other derivatives. However, compound 11 with three phenolic hydroxy groups etherified exhibited stronger cytotoxicity than the other compounds with three phenolic hydroxyl groups etherified. The mechanisms of action of these derivatives are unknown and await further investigation.
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Moreover, recent results from Hai SL et al [29-30] are in agreement with our findings; they have reported that the pharmacokinetics of (E)-3, 5, 4'-trimethoxystilbene (10) in rat plasma are significantly different for that of E-resveratrol, with greater plasma exposure, longer elimination half-lives, and lower clearance being observed with the derivative compound.
Conclusions In the present study, a series of E-resveratrol derivativeswere synthesized and tested for their cytotoxic activity in A549, LAC, and HeLa cells. Compounds 1−6, 12, 15−21, and 23−26 were synthesized for the first time. Most derivatives with one or two phenolic hydroxy groups etherified showed greater cytotoxic activity than those with three phenolic hydroxylgroups etherified and E-resveratrol, and the derivatives
Fig. 1 Dose-response curve of compound 1
Table 1 Cytotoxic activity of synthesized compounds 1–26 in vitro from different tumor types ( A549, LAC, and HeLa cells) Compounds
a) b)
IC50/(μmol·L−1)
Compounds
IC50/(μmol·L−1)
A549
LAC
HeLa
A549
LAC
HeLa
1
4.38 ± 0.71
10.19 ± 0.37
8.16 ± 0.95
16
≥100 ± 1.35
≥100 ± 1.27
≥100 ± 1.03
2
4.90 ± 1.18
5.55 ± 1.07
6.48 ± 1.56
17
≥100 ± 1.46
≥100 ± 0.23
≥100 ± 1.06
3
≥100 ± 1.05
≥100 ± 1.46
≥100 ± 1.82
18
≥100 ± 0.89
≥100 ± 0.52
≥100 ± 1.13
4
14.17 ± 1.28
27.71 ± 2.01
17.08 ± 0.79
19
≥100 ± 1.05
≥100 ± 0.83
≥100 ± 2.04
5
10.15 ± 2.07
16.35 ± 0.31
8.89 ± 1.63
20
≥100 ± 1.17
≥100 ± 0.84
≥100 ± 0.52
6
49.85 ± 2.55
≥100 ± 2.74
≥100 ± 1.04
21
≥100 ± 1.20
≥100 ± 2.33
≥100 ± 1.67
7
≥100 ± 1.56
≥100 ± 2.02
≥100 ± 3.12
22
≥100 ± 0.20
21.06 ± 0.35
≥100 ± 3.12
8
≥100 ± 2.34
≥100 ± 0.79
≥100 ± 1.40
23
60.05 ± 0.78
8.50 ± 1.87
84.37 ± 3.41
9
11.80 ± 0.75
7.96 ± 2.87
1.41 ± 3.21
24
≥100 ± 0.69
9.80 ± 1.07
53.06 ± 3.24
10
26.29 ± 0.20
9.43 ± 1.03
8.25 ± 2.06
25
59.51 ± 1.60
7.58 ± 1.02
52.21 ± 0.93
11
9.43 ± 3.41
30.70 ± 2.57
20.14 ± 1.46
26
≥100 ± 1.05
≥100 ± 2.32
≥100 ± 1.74
12
≥100 ± 2.35
≥100 ± 0.94
≥100 ± 0.86
E-resveratrol
66.25 ± 2.07
24.89 ± 1.40
98.17 ± 0.54
13
≥100 ± 2.31
≥100 ± 3.35
≥100 ± 0.24
2.36 ± 1.01
1.18 ± 1.78
0.48 ± 0.96
14
≥100 ± 1.42
≥100 ± 1.82
≥100 ± 2.12
15
≥100 ± 3.14
≥100 ± 2.71
≥100 ± 0.98
a
Adriamycin
Used as a positive control. The IC50 value is the concentration at which 50% survival of cells was observed The results are reported as mean ± SE for at least three experiments
etherified with groups bearing an aromatic nucleus were more active than those bearing aliphatic groups. Compounds 1, 2, and 9−11 were identified to be the most promising candidates with improved cytotoxic activity, making them potential candidates for cancer drug discovery.
[3]
[4]
Acknowledgments The authors are thankful to the Analysis and Testing Center of Nanchang University for facilities and support.
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Cite this article as: HONG Ting, JIANG Wei, DONG Huai-Ming, QIU Sheng-Xiang, LU Yu. Synthesis and cytotoxic activities of E-resveratrol derivatives [J]. Chinese Journal of Natural Medicines, 2015, 13 (5): 375-382.
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