Synthesis and Anti-proliferative in-vitro Activity of Two Natural Dihydrostilbenes and their Analogues.
код для вставкиСкачать244 Arch. Pharm. Chem. Life Sci. 2007, 340, 244 – 250 Full Paper Synthesis and Anti-proliferative in-vitro Activity of Two Natural Dihydrostilbenes and their Analogues Wei-Ge Zhang1,3, Rui Zhao1, Jian Ren1, Li-Xiang Ren2, Jin-Guang Lin1, Dai-Lin Liu3, Ying-Liang Wu2, and Xin-Sheng Yao3 1 School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, P. R. China School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, P. R. China 3 Shenzhen Keylab for Research and Development of New Drugs, Shenzhen, P. R. China 2 A total synthetic route for two natural dihydrostilbenes with significant cytotoxicity toward human cancer cell lines, (3-(2-(7-methoxybenzo[d][1,3]dioxol-5-yl)ethyl)phenol 1a and 6-(3-hydroxyphenethyl)benzo[d][1,3]dioxol-4-ol 1b), which were isolated from Bulbophyllum odoratissimum Lindl, was developed via Wittig–Horner reaction. The natural products 1a and 1b were obtained in 28% and 20% overall yield, respectively. Additionally, nine analogues, 1c-1k, of the two natural dihydrostilbenes were synthesized and evaluated for their anti-proliferative activity against human SGC-7901, KB and HT-1080 cell lines by MTT assay. The activities of 1c and 1d were in the same range as those of the natural products 1a and 1b. Keywords: Anti-proliferative activity / Natural dihydrostilbene / Total synthesis / Received: September 12, 2006; accepted: November 7, 2006 DOI 10.1002/ardp.200600146 Introduction Stilbenoids, such as dihydrostilbenes, stilbenes, phenanthrenes, and their oligomers, constitute an important class of natural products, which attract increasing interest chiefly due to their various pharmacological effects, for instance, anti-oxidant activity [1], cancer chemopreventive effect [2], anti-tumor activity [3], inhibition of cyclooxygenase [4], inhibition of platelet aggregation [5]. In addition, some synthetic dihydrostilbenes, known as the analogues of combretastatin A-4, were found to be tubulin polymerization inhibitors and display strong anti-mitotic activity to a broad spectrum of human cancer lines [6]. In our effort to search for naturally-occurring cytotoxic agents present in traditional Chinese medicines using the Pyricularia oryzae bioassay [7], a set of dihydrostilbenes, including 3-(2-(7-methoxybenzo[d][1,3]dioxol-5-yl)- Correspondence: Wei-Ge Zhang, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District Shenyang 110016, P. R. China E-mail: zhangweige2000@sina.com Fax: +86 24 23986393 i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 1. Structures of two natural dihydrostilbenes and their analogues. ethyl)phenol 1a and 6-(3-hydroxyphenethyl)benzo[d][1,3]dioxol-4-ol 1b (Fig. 1), were obtained from Bulbophyllum odoratissimum Lindl, a folk herb for the treatment of phthisis and rheumatism in the southern part of China Arch. Pharm. Chem. Life Sci. 2007, 340, 244 – 250 Natural Dihydrostilbenes and Their Analogues 245 Reagents and conditions: a) CH3OH, H2SO4, reflux, 12 h; b) PhCH2Cl, K2CO3, DMF, 608C, 16 h; c) NaBH4, I2, THF, 08C, 2 h, then 708C, 6 h; d) KMnO4, ZrOCl268 H2O, THF, rt, ultrasound irradiation, 10 h; e) (CH3O)2SO2 or PhCH2Cl, Na2B4O7610 H2O, rt, overnight; f) CH2Cl2, K2CO3, DMF, reflux, 4 h; g) NaBH4, I2, THF, 08C, 2 h, then 708C, 8 h; h) SOCl2, N(C2H5)3, ClCH2CH2Cl, rt, 6 h; i) P(OC2H5)3, 1408C, 8 h; j) aldehyde 5, NaH, DMF, 08C, 1 h, rt, 6 h; k) H2, Pd-C, EtOAc, rt, 48 h. Scheme 1. Synthesis route of natural dihydrostilbene compounds 1a, b. [8]. Compound 1a is a new natural product firstly found in our laboratory, which demonstrates significant cytotoxic activity [9]. Although compound 1b had also been isolated from the orchid Cirrhopetalum andersonii [10], the total synthesis and bioactivity of 1b, to our knowledge, have not been reported yet. For the purpose of searching for new antitumor agents and understanding their structure-activity relationships, we developed a general synthetic route to the two natural dihydrostilbenes 1a, 1b and their analogues 1c – 1k (Fig. 1) and preliminarily evaluated in vitro the anti-proliferative activity of all synthesized compounds. Results and discussion The first aim of our present work was the total synthesis of the natural dihydrostilbenes 1a and 1b. Retro-synthetic analysis of 1a and 1b led to 3-hydroxybenzoic acid and methyl 3,4,5-trihydroxybenzoate, while our general approach to the total synthesis of these compounds is described in Scheme 1. Aldehyde 5 was viewed as the requisite intermediate for the total synthesis of the natural dihydrostilbenes 1a and 1b. The commercially available and cheap 3-hydroxy- i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim benzoic acid was used as starting material, which was treated with CH3OH/H2SO4 to give benzoic ester 2. The mOH group of 2 was protected with benzyl chloride to yield 3. Employing NaBH4/I2 as a safe and inexpensive reducing agent [11] to replace LiAlH4, we got 4 in 88.9% yield. Since KMnO4/ZrOCl268 H2O had been proven to be efficient for the highly selective oxidation of primary benzylic alcohols to the corresponding aldehyde [12], we obtained aldehyde 5 in 96.3% yield by treatment of 4 with this new oxidant in diethyl ether at room temperature for 36 h. As this oxidation is a liquid-solid biphasic process, we considered that ultrasound should promote this reaction, and this assumption was readily proven valid by experiment. Thus, when we carried out this reaction for 8 h under ultrasound irradiation, the desired aldehyde 5 was obtained in 98.5% yield. Following the reported methodologies [13] with slight modifications, compound 7a and 7b were prepared in 73.3 and 49.1%, respectively, via mono-alkylation of mOH of methyl 3,4,5-trihydroxybenzoate and cyclization by treatment with CH2Cl2 and K2CO3 in DMF. The resultant esters were subjected to reduction with NaBH4/I2 to provide benzyl alcohol 8a and 8b in 89.3 and 86.7% yield, which were treated with SOCl2 and Et3N to form benzyl chloride 9a and 9b in 96.7 and 95.5% yield, respectively. www.archpharm.com 246 W.-G. Zhang et al. Arch. Pharm. Chem. Life Sci. 2007, 340, 244 – 250 Reagents and conditions: l) NaH, DMF, 08C, 1 h, rt, 6 h; m) H2, Pd-C, EtOAc, rt, 48 h. Scheme 2. Synthesis of dihydrostilbene analogues 1c – 1k. Further, Michaelis-Arbuzov reaction of 9a and 9b with P(OEt)3 provided 10a and 10b, both of which could be used directly in the next step. Phosphite 10a and 10b were treated with NaH in DMF, followed by addition of aldehyde 5 to afford the trans-stilbenes 10a and 10b by Wittig-Horner reaction in 47.3 and 51.1% yield, respectively. Catalytic hydrogenation of 10a and 10b over palladium on carbon provided the natural dihydrostilbenes 1a and 1b in 94.5 and 98.3% yield, resulting from reduction of the double bond and removal of the benzyl protecting group. The synthetic materials 1a and 1b were identical in 1H-NMR and MS with the samples of the natural products. Having accomplished the total synthesis of the natural dihydrostilbenes 1a and 1b, our attention was focused on the synthesis of their analogues 1c – 1k. This task was achieved in a two-step reaction sequence similar to the method described above, which is outlined in Scheme 2. Wittig-Horner reaction of phosphite 10a or 10b with diverse aldehydes 12a – 12f provided corresponding transstilbenes 13a – 13i in 46.5 to 55.8% yield, which were converted into dihydrostilbenes 1c – 1k by catalytic hydrogenation in 94.7 to 99.1% yield. The structures of those dihydrostilbenes 1c – 1k were characterized by the application of MS and 1H-NMR. The anti-proliferative activity of all obtained compounds was assessed in vitro against three human cancer cell lines, SGC-7901 gastric carcinoma, KB nasopharyngeal carcinoma, and HT-1080 fibrosarcoma, by colorimetric MTT assay using cisplatin as positive control and expressed as IC50 values. IC50 is the concentration (lM) required to inhibit tumor cell proliferation by 50% after i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Table 1. Anti-proliferative activity in vitro of the natural dihydrostilbenes 1a, 1b, and their analogs 1c – 1k against three human cancer cell lines. Compounds 1a 1b 1c 1d 1e 1f 1g 1h 1i 1j 1k Cisplatin IC50 (lM) SGC-7901 KB HT-1080 8.30 9.20 7.30 9.90 A 100 60.5 A 100 A 100 A 100 A 100 A 100 9.48 5.50 8.70 4.20 8.50 A 100 A 100 A 100 >100 87.5 A 100 49.4 1.46 25.5 40.0 37.9 43.3 89.3 87.7 A 100 A 100 A 100 A 100 54.7 21.3 24 h of exposure of the cells to a tested compound. The measured IC50 values for the natural dihydrostilbenes 1a, 1b, and their analogues 1c – 1k are summarized in Table 1. As can be seen from the presented data, natural dihydrostilbenes 1a, 1b and their analogues 1c, 1d were found to have significant anti-proliferative activity, which IC50 values against SGC-7901 and KB cell lines were smaller than 10.0 lM. Conversion of the m-OMe (1a and 1c) to a hydrophilic m-OH group on the ring A (1b and 1d) caused a 1.6 to 2.0-fold decrease in inhibition of the growth of KB cell, and there was only a slight reduction in inhibitory activity against SGC-7901 and HT-1080 cell lines. Removal of the hydroxyl group at the m-position on the ring A of www.archpharm.com Arch. Pharm. Chem. Life Sci. 2007, 340, 244 – 250 1a or 1b and replacement with an amino group, to afford its derivative 1c or 1d, resulted in a slight increase in the activity against all three cell lines. The introduction of –OH, –F, –OCH3, or –N(CH3)2 at the p-position on the ring B, along with the removal of the hydroxyl or amino group at the m-position on the ring B, almost eliminated growth inhibition activity. Furthermore, it is clear that activity of the natural dihydrostilbenes and their analogues against SGC-7901 and KB cell lines is generally higher than that against the HT-1080 cell line. In summary, the first total synthesis of two natural dihydrostilbenes, 3-(2-(7-methoxybenzo[d][1,3]dioxol-5-yl)ethyl)phenol 1a and 6-(3-hydroxyphenethyl)benzo[d][1,3]dioxol-4-ol 1b, had thus been achieved in 28.3 and 20.4% overall yield, respectively. In addition, nine analogues of 1a and 1b were prepared and tested for their anti-proliferative activity against SGC-7901, KB, and HT-1080 cell lines using MTT assay. Among the analogues synthesized, 1c and 1d bearing an amino moiety in place of the phenolic OH of the natural products 1a and 1b showed significant anti-proliferative activity against SGC-7901 and KB cell lines (IC50 a 10.0 lM), while the cytotoxicity of other analogues was markedly reduced with respect to all three cell lines. Natural Dihydrostilbenes and Their Analogues 247 was poured into ice water (500 mL). After filtration and washing with water, the crude product was purified by column chromatography (n-hexane : EtOAc = 3 : 1) to give 3 (39.1 g, 89.6% from mhydroxybenzoic acid) as a white solid: mp 74 – 768C; 1H-NMR (400 MHz, CDCl3) d: 3.95 (3H, s), 5.12 (2H, s), 7.19 (1H, m), 7.34 – 7.48 (6H, m), 7.67 (2H, m) [14]. (3-(Benzyloxy)phenyl)methanol 4 To a suspension of compound 3 (24.2 g, 0.100 mol) and NaBH4 (9.0 g, 0.235 mol) in dry THF (300 mL) was added a solution of I2 (25.4 g, 0.100 mol) in dry THF (200 mL) over 2 h at 08C under nitrogen atmosphere. The mixture was stirred at 708C for 6 h, and then concentrated in vacuo. The residue was dissolved in EtOAc (500 mL), and the resulting solution was washed with water, dried over anhydrous Na2SO4, and evaporated. The crude product was purified by column chromatography (n-hexane : EtOAc = 3 : 1) to give 4 (19.0 g, 88.9%) as a white solid: mp 46 – 488C; 1H-NMR (300 MHz, CDCl3) d: 4.65 (2H, s), 5.06 (2H, s), 6.89 – 7.01 (3H, m), 7.24 – 7.41 (6H, m) [15]. 3-(Benzyloxy)benzaldehyde 5 A mixture of 4 (15.2 g, 0.071 mol), KMnO4 (33.7 g, 0.213 mol) and ZrOCl268 H2O (6.75 g, 0.021 mol) in THF (300 mL) was treated under ultrasound irradiation for 8 h at room temperature. After filtration, the filter cake was washed with EtOAc. The filtrate was concentrated in vacuo and purified by column chromatography (n-hexane : EtOAc = 3 : 1) to give 5 (14.8 g, 98.5%) as a white solid: mp 57 – 588C (56 – 588C) [16]. Methyl-3,4-dihydroxy-5-methoxybenzoate 6a Experimental Melting points for the compounds were determined using a hotstage microscope and are uncorrected. 1H- and 13C-NMR spectra were taken in CDCl3 or DMSO-d6 solution on Bruker AVANCE400, Bruker ARX-300 or Bruker ARX-600 spectrometers with TMS as the internal reference (Bruker BioSciences, USA). MS spectra were obtained using Bruker Esqure 2000 or Shimadzu GCMSQP5050A spectrometers (Shimadzu, Japan). Elemental analyses (C, H, and N) were performed by Jilin University (Changchun, China). Column chromatography was run on silica gel (200 – 300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). Unless otherwise noted, all the materials were obtained from commercially available sources and were used without further purification. Chemistry Methyl-3-hydroxybenzoate 2 and Methyl-3(benzyloxy)benzoate 3 To a solution of 3-hydroxybenzoic acid (25.0 g, 0.180 mol) in CH3OH (250 mL) conc. H2SO4 (37.5 mL) was added dropwise. The resulting mixture was refluxed for 12 h. After cooling at room temperature, the mixture was diluted with water, extracted with EtOAc, and the organic layer was washed with saturated brine, dried over Na2SO4, filtered, and concentrated to give a crude product 2 which was used directly for the next step. Benzyl chloride (23.0 mL, 0.20 mol) was added to the suspension of 2 and K2CO3 (37.3 g, 0.27 mol) in DMF (150 mL), then the reaction mixture was stirred at 608C for 16 h. After cooling, the mixture i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim A solution of NaOH (19.5 g, 0.488 mol) in H2O (75 mL) and (CH3O)2SO2 (45 mL) were added to a stirred solution of methyl gallate (15 g, 0.083 mol) and Na2B4O7610 H2O (37.5 g, 0.098 mol) in H2O (750 mL), and the mixture was stirred overnight at room temperature. The resulting mixture was then acidified to pH 3 – 4 with 10% H2SO4. The solid was filtered, and the filtrate was extract with EtOAc. The extract was washed with saturated brine, dried over anhydrous Na2SO4, and concentrated to give crude 6a (14.4 g). Methyl-3-(benzyloxy)-4,5-dihydroxybenzoate 6b A solution of Na2B4O7610 H2O (160 g, 0.42 mol) in H2O (480 mL) was added to a stirred suspension of methyl gallate (40 g, 0.22 mol) and K2CO3 (160 g, 1.16 mol) in DMF (320 mL), and the mixture was stirred for 30 min at room temperature. Then PhCH2Cl (27.2 mL, 0.22 mol) was added, and the mixture was stirred overnight at the same temperature. The reaction mixture was then acidified to pH 3 – 4 with 10% H2SO4, the resulting solid was filtered, and the filtrate was extracted with EtOAc. The extract was washed with saturated brine, dried over anhydrous Na2SO4, and concentrated to give crude 6b (42.3 g). Methyl-7-methoxybenzo[d][1,3]dioxole-5-carboxylate 7a To a suspension of CH2Cl2 (6.95 mL, 0.108 mol) and K2CO3 (19.9 g, 0.144 mol) in DMF (300 mL) was added dropwise a solution of 6a (14.4 g, 0.0727 mol) in DMF (250 mL) and the mixture was stirred and heated to reflux for 4 h. The mixture was cooled, then filtered and the filter cake was washed with EtOAc. The filtrate was washed with water, dried over anhydrous Na2SO4, and evaporated. The crude product was purified by column chromatograwww.archpharm.com 248 W.-G. Zhang et al. phy (n-hexane : EtOAc = 3 : 1) to give 7a (10.4 g, 59.8% from methyl gallate) as a white solid: mp 89 – 908C; 1H-NMR (400 MHz, CDCl3) d: 3.86 (3H, s), 3.88 (3H, s), 6.06 (2H, s), 7.21 (1H, d, J = 1.5Hz), 7.33 (1H, d, J = 1.5 Hz) [17]. Methyl 7-(benzyloxy)benzo[d][1,3]dioxole-5-carboxylate 7b Compound 7b was prepared from 6b following the procedure described for 7a and obtained in 49.1% yield (from methyl gallate) as a white solid: mp 78 – 798C; 1H-NMR (400 MHz, CDCl3) d: 3.87 (3H, s), 5.19 (2H, s), 6.04 (2H, s), 7.20 – 7.45 (7H, m); 13C-NMR (100 MHz, CDCl3) d: 52.1, 71.5, 102.2, 104.3, 112.2, 124.4, 127.7, 128.2, 128.6, 136.2, 139.9, 142.2, 148.9,166.3; MS (ESI) m/z 594.6 [2M + Na+]+, 303.8 [M + NH4+]+, 287.0 [M]+ [17]. (7-Methoxybenzo[d][1,3]dioxol-5-yl)methanol 8a To a suspension of compound 7a (5.00 g, 23.8 mmol) and NaBH4 (2.16 g, 57.1 mmol) in dry THF (150 mL) was added a solution of I2 (6.04 g, 23.8 mmol) in dry THF (100 mL) dropwise over 2 h at 08C under nitrogen atmosphere. The mixture was refluxed for 8 h, and then concentrated in vacuo to give the residue which was dissolved in EtOAc (300 mL), then washed with water, dried over anhydrous Na2SO4, and evaporated. The crude product was purified by column chromatography (n-hexane : EtOAc = 3 : 1) to give 8a (3.87 g, 89.3%) as a white solid: mp 66 – 678C; 1H-NMR (400 MHz, CDCl3) d: 3.87 (3H, s), 4.52 (2H, brs), 5.93 (2H, s), 6.50 (1H, s), 6.51 (1H, s) [18]. (7-(Benzyloxy)benzo[d][1,3]dioxol-5-yl)methanol 8b Compound 8b was prepared from 7b as described above for the preparation of 8a and obtained in 86.7% yield as a white solid: mp 62 – 648C; 1H-NMR (400 MHz, CDCl3) d: 1.83 (1H, s), 4.51 (2H, s), 5.16 (2H, s), 5.93 (2H, s), 6.53 (1H, s), 6.58 (1H, s), 7.28 – 7.43 (5H, m) [19]. 6-(Chloromethyl)-4-methoxybenzo[d][1,3]dioxole 9a To a solution of compound 8a (3.00 g, 16.5 mmol) and Et3N (2.43 mL, 17.5 mmol) in 1,2-dichloroethane (80 mL) was added dropwise SOCl2 (1.80 mL, 24.7 mmol) and the mixture was stirred for 6 h at room temperature. The reaction mixture was washed with 5% Na2CO3, dried over anhydrous Na2SO4, and evaporated. The crude product was purified by column chromatography (n-hexane : EtOAc = 3 : 1) to give 9a (3.21 g, 96.7%) as a white powder: mp 94 – 968C; 1H-NMR (400 MHz, CDCl3) d: 3.90 (3H, s), 4.43 (2H, s), 5.97 (2H, s), 6.56 (1H, s), 6.58 (1H, s) [20]. 4-(Benzyloxy)-6-(chloromethyl)benzo[d][1,3]dioxole 9b Compound 9b was prepared from 8b in a manner similar to that described above for 9a and obtained in 95.5% yield as a white powder: mp 85 – 878C; 1H-NMR (400 MHz, CDCl3) d: 4.44 (2H, s), 5.16 (2H, s), 5.97 (2H, s), 6.57 (1H, s), 6.61 (1H, s), 7.32 – 7.45 (5H, m). Diethyl(7-methoxybenzo[d][1,3]dioxol-5yl)methylphosphonate 10a and 6-(3-(Benzyloxy) styryl)-4methoxybenzo[d][1,3]dioxole 11a A mixture of 9a (0.300 g, 1.50 mmol) and triethyl phosphite (0.499 g, 3.00 mmol) was refluxed at 1408C for 8 h, then residual triethyl phosphite was removed in vacuo to give compound 10a. i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Arch. Pharm. Chem. Life Sci. 2007, 340, 244 – 250 To a suspension of NaH (0.240 g, 6.00 mmol) in dry DMF (5 mL) was added 10a in dry DMF (10 mL) dropwise at 08C under nitrogen atmosphere. The resulting solution was stirred at room temperature for 1 h, to which aldehyde 5 (0.318 g, 1.50 mmol) in dry DMF (5 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 6 h, and then was poured into ice water (100 mL). After filtration and washing with water, the crude product was purified by column chromatography (n-hexane : EtOAc = 3 : 1) to give 11a (0.256 g, 47.3%) as a viscous oil: 1H-NMR (300 MHz, CDCl3) d: 3.97 (3H, s), 5.12 (2H, brs), 6.01 (2H, s), 6.67 (1H, d, J = 1.2Hz), 6.77 (1H, d, J = 1.2Hz), 6.88 – 7.49 (11H, m); MS (EI) m/z 360 [M]+ (34), 91 (100). Compounds 13a – 13i were prepared as described as described above for 10a and 11a. Yields and spectroscopic date of compounds 10b, 11b, and 13a – 13i Diethyl(7-(benzyloxy)benzo[d][1,3]dioxol-5yl)methylphosphonate 10b and 4-(Benzyloxy)-6-3(benzyloxy)styryl)benzo[d][1,3]dioxole 11b Yield 51.1%; mp 105 – 1078C; 1H-NMR (300 MHz, CDCl3) d: 5.10 (2H, s), 5.21 (2H, s), 5.99 (2H, s), 6.69 (1H, d, J = 1.2Hz), 6.75 (1H, d, J = 1.2Hz), 6.89 – 7.47 (16H, m); MS (EI) m/z 436 [M]+ (25), 91 (100). 4-Methoxy-6-(3-nitrostyryl)benzo[d][1,3]dioxole 13a Yield 48.5%, oil; 1H-NMR (300 MHz, CDCl3) d: 3.88 (3H, s), 6.01 (2H, s), 6.74 (1H, d, J = 1.2 Hz), 6.77 (1H, d, J = 1.2Hz), 6.94 (1H, d, J = 16.2 Hz), 7.10 (1H, d, J = 16.2 Hz), 7.51 (1H, d, J = 7.8 Hz), 7.73 (1H, d, J = 7.8 Hz), 8.11 (1H, s), 8.35 (1H, s); MS (EI) m/z 299 [M]+. 4-(Benzyloxy)-6-(3-nitrostyryl)benzo[d][1,3]dioxole 13b Yield 50.2%; mp 109 – 1118C; 1H-NMR (300 MHz, CDCl3) d: 5.23 (2H, s),6.01 (2H, s), 6.73 (1H, d, J = 1.2 Hz), 6.75 (1H, d, J = 1.2 Hz), 6.93 (1H, d, J = 16.2 Hz), 7.08 (1H, d, J = 16.2 Hz), 7.51 (1H, d, J = 7.8Hz), 7.74 (1H, d, J = 7.8 Hz), 7.31 – 7.47 (5H, m), 8.07 (1H, s), 8.31 (1H, s); MS (EI) m/z 375 [M]+ (15), 91 (100). 6-(4-(Benzyloxy)styryl)-4-methoxybenzo[d][1,3]dioxole 3c Yield 46.5%, oil; 1H-NMR (300 MHz, CDCl3) d: 3.82 (3H, s), 5.12 (2H, s), 5.98 (2H, s), 6.72 (1H, d, J = 1.2 Hz), 6.74 (2H, d, J = 8.7 Hz), 6.77 (1H, d, J = 1.2 Hz), 6.90 (2H, s), 7.32 – 7.47 (5H, m), 7.80 (2H, d, J = 8.7 Hz). MS (EI) m/z: 360 [M]+ (12), 91 (100). 4-(Benzyloxy)-6-(4(benzyloxy)styryl)benzo[d][1,3]dioxole 13d Yield 50.2%; mp 127 – 1298C; 1H-NMR (300 MHz, CDCl3) d: 5.08 (2H, s), 5.21 (2H, s), 5.98 (2H, s), 6.67 (1H, d, J = 1.2 Hz), 6.73 (1H, d, J = 1.2 Hz), 6.85 (2H, s), 6.96 (2H, d, J = 8.7 Hz), 7.32 – 7.47 (10H, m), 7.84 (2H, d, J = 8.7 Hz). MS (EI) m/z: 436 [M]+ (21), 91 (100). 6-(4-Fluorostyryl)-4-methoxybenzo[d][1,3]dioxole 13e Yield 48.5%; mp 80 – 828C; 1H-NMR (300 MHz, CDCl3) d: 3.94 (3H, s), 5.99 (2H, s), 6.64 (1H, d, J = 1.2 Hz), 6.74 (1H, d, J = 1.2 Hz), 6.90 (2H, s), 7.03 (2H, m), 7.43 (2H, m); MS (EI) m/z 272 [M]+. www.archpharm.com Arch. Pharm. Chem. Life Sci. 2007, 340, 244 – 250 4-(Benzyloxy)-6-(4-fluorostyryl)benzo[d][1,3]dioxole 13f Yield 48.9%; mp 97 – 998C; 1H-NMR (300 MHz, CDCl3) d: 5.21 (2H, s), 5.99 (2H, s), 6.68 (1H, d, J = 1.2 Hz), 6.74 (1H, d, J = 1. 2Hz), 6.86 (2H, s), 7.03 (2H, t), 7.35 (2H, m), 7.39 – 7.46 (5H, m); MS (EI) m/z 348 [M+] (32), 91 (100). Natural Dihydrostilbenes and Their Analogues 249 (1H, s), 6.53 (1H, d, J = 7.8 Hz), 6.58 (1H, d, J = 7.8 Hz), 7.00 (1H, t, J = 7.8 Hz); MS (EI) m/z 271 [M]+ (35), 165 (100), 106 (24), 77 (16); Anal. Calcd. for C16H17NO3: C, 70.83; H, 6.32; N, 5.16. Found: C, 70.96; H, 6.53; N, 5.01. 6-(3-Aminophenethyl)benzo[d][1,3]dioxol-4-ol 1d 4-(Benzyloxy)-6-(4-methoxystyryl)benzo[d][1,3]dioxole 13g Yield 55.8%; mp 98 – 1008C; 1H-NMR (600 MHz, CDCl3) d: 3.82 (3H, s), 5.21 (2H, s), 5.98 (2H, s), 6.68 (1H, d, J = 1.2 Hz), 6.74 (1H, d, J = 1.2 Hz), 6.82 (1H, d, J = 16.2 Hz), 6.86 (1H, d, J = 16.2 Hz), 6.88 (2H, d, J = 8.4 Hz), 7.31 – 7.40 (5H, m), 7.46 (2H, d, J = 8.4 Hz); MS (EI) m/z 360 [M]+ (9), 91 (100). 4-(2-(7-(Benzyloxy)benzo[d][1,3]dioxol-5-yl)vinyl)-N,Ndimethylbenzenamine 13h Yield 52.9%; mp 154 – 1568C; 1H-NMR (300 MHz, CDCl3) d: 2.99 (6H, s), 5.21 (2H, s), 5.97 (2H, s), 6.70 (1H, d, J = 16.2 Hz), 6.80 (1H, d, J = 1.2Hz), 6.85 (1H, d, J = 16.2 Hz), 6.85 (1H, s), 6.86 (1H, d, J = 1.2 Hz), 6.89 (1H, s), 7.32-7.47 (7H, m); MS (EI) m/z 373 [M]+ (13), 91 (100). 6-(4-(Benzyloxy)-3-methoxystyryl)-4methoxybenzo[d][1,3]dioxole 13i Yield 50.3%; mp 108 – 1108C; 1H-NMR (600 MHz, CDCl3) d: 3.94 (3H, s), 3.95 (3H, s), 5.18 (2H, s), 5.98 (2H, s), 6.63 (1H, s), 6.73 (1H, s), 6.85 (1H, d, J = 16.2 Hz), 6.86 (1H, d, J = 16.2 Hz), 6.87 (1H, d, J = 7.8 Hz), 6.95 (1H, d, J = 7.8 Hz), 7.05 (1H, s), 7.29 – 7.45 (5H, m); MS (EI) m/z 390 [M]+ (20), 91 (100). Yield 97.3%; mp 135 – 1388C; 1H-NMR (300 MHz, DMSO) d: 2.64 (4H, s), 4.90 (2H, brs), 5.88 (2H, s), 6.26 (1H, s), 6.30 (1H, s), 6.36 (1H, d, J = 7.8 Hz), 6.38 (1H, d, J = 7.8 Hz), 6.41 (1H, s), 6.90 (1H, t, J = 7.8 Hz), 9.49 (1H, s); MS (EI) m/z 257 [M]+ (40), 151 (100), 106 (11), 77 (8); Anal. Calcd. for C15H15NO3: C, 70.02; H, 5.88; N, 5.44. Found: C, 70.36; H, 5.91; N, 5.23. 4-(2-(7-Methoxybenzo[d][1,3]dioxol-5-yl)ethyl)phenol 1e Yield 96.4%, oil; 1H-NMR (300 MHz, CDCl3) d: 2.79 (4H, brs), 3.85 (3H, s), 5.92 (2H, s), 6.27 (1H, d, J = 1.2 Hz), 6.36 (1H, d, J = 1.2 Hz), 6.74 (2H, d, J = 7.8 Hz), 7.02 (2H, d, J = 7.8 Hz); MS (EI) m/z 272 [M]+ (16), 165 (100), 107 (42), 77 (22); Anal. Calcd. for C16H16O4: C, 70.57; H, 5.92. Found: C, 70.85; H, 6.09. 6-(4-Hydroxyphenethyl)benzo[d][1,3]dioxol-4-ol 1f Yield 97.4%; mp 129 – 1308C; 1H-NMR (300 MHz, CDCl3) d: 2.77 (4H, brs), 5.92 (2H, s), 6.29 (1H, s), 6.31 (1H, s), 6.74 (2H, d, J = 7.8 Hz), 7.02 (2H, d, J = 7.8 Hz); MS (EI) m/z 258 [M]+ (12), 151 (86), 107 (100), 77 (37); Anal. Calcd. for C15H14O4: C, 69.76; H, 5.46. Found: C, 69.62; H, 5.32. 6-(4-Fluorophenethyl)-4-methoxybenzo[d][1,3]dioxole 1g Synthesis, yields and spectroscopic date of compounds 1a – 1k Yield 95.5%; mp 39 – 418C; 1H-NMR (300 MHz, CDCl3) d: 2.81 (4H, m), 3.84 (3H, s), 5.93 (2H, s), 6.25 (1H, d, J = 1.0 Hz), 6.35 (1H, d, J = 1.0 Hz), 6.95 (2H, m), 7.08 (2H, m); MS (EI) m/z 274 [M]+ (11), 165 (100), 109 (22), 77 (22); Anal. Calcd. for C16H15FO3: C, 70.06; H, 5.51. Found: C, 70.22; H, 5.37. 3-(2-(7-Methoxybenzo[d][1,3]dioxol-5-yl)ethyl)phenol 1a 6-(4-Fluorophenethyl)benzo[d][1,3]dioxol-4-ol 1h A solution of 11a (0.120 g, 0.333 mmol) in EtOAc (10.0 mL) was stirred in the presence of 10% Pd-C (0.006 g) under a hydrogen atmosphere at room temperature for 48 h. The catalyst was removed by filtration and washed with EtOAc. The filtrate was concentrated in vacuo and purified by column chromatography (n-hexane : EtOAc = 3 : 1) to give dihydrostilbene 1a (0.0857 g, 94.5%) as a white powder: mp 55 – 578C; 1H-NMR (300 MHz, CDCl3) d: 2.81 (4H, brs), 3.85 (3H, brs), 4.82 (1H, brs), 5.93 (2H, s), 6.29 (1H, d, J = 1.5 Hz), 6.38 (1H, d, J = 1.5Hz), 6.65 (1H, s), 6.67 (1H, d, J = 7.5 Hz), 6.74 (1H, d, J = 7.5Hz), 7.14 (1H, t, J = 7.5 Hz); MS (EI) m/z 272 [M]+ (13), 165 (100), 77 (36); Anal. Calcd. for C16H16O4: C, 70.57; H, 5.92. Found: C, 70.80; H, 5.69. Compounds 1b – k were prepared using the same method. Yield 94.7%; mp 91 – 938C; 1H-NMR (300 MHz, CDCl3) d: 2.78 (4H, m), 4.70 (1H, brs), 5.93 (2H, s), 6.28 (1H, s), 6.29 (1H, s), 6.95 (2H, m), 7.09 (2H, m); MS (EI) m/z 260 [M]+ (12), 151 (100), 109 (69), 95 (5); Anal. Calcd. for C15H13FO3: C, 69.22; H, 5.03. Found: C, 69.46; H, 5.09. 6-(3-Hydroxyphenethyl)benzo[d][1,3]dioxol-4-ol 1b Yield 98.3%; mp 125 – 1288C; 1H-NMR (300 MHz, DMSO) d: 2.50 (2H, brs), 2.68 (2H, brs), 5.88 (2H, s), 6.25 (1H, s), 6.31 (1H, s), 6.56 (1H, s), 6.57 (1H, d, J = 7.5 Hz), 6.59 (1H, d, J = 7.5 Hz), 7.04 (1H, t, J = 7.5 Hz), 9.24 (1H, s), 9.53 (1H, s); MS (EI) m/z 258 [M]+ (19), 151 (100), 77 (13); Anal. Calcd. for C15H14O4: C, 69.76; H, 5.46. Found: C, 69.99; H, 5.24. 3-(2-(7-Methoxybenzo[d][1,3]dioxol-5-yl)ethyl)benzenamine 1c 1 Yield 97.8%, oil; H-NMR (300 MHz, CDCl3) d 2.78 (4H, s), 3.85 (3H, s), 5.91 (2H, s), 6.30 (1H, d, J = 1.2 Hz), 6.38 (1H, d, J = 1.2 Hz), 6.50 i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 3.3.9 6-(4-Methoxyphenethyl)benzo[d][1,3]dioxol-4-ol 1i Yield 96.8%; mp 96 – 978C; 1H-NMR (300 MHz, CDCl3) d: 2.78 (4H, brs), 3.78 (3H, s), 5.90 (2H, s), 6.29 (1H, s), 6.31 (1H, s), 6.80 (2H, d, J = 7.8 Hz), 7.05 (2H, d, J = 7.8 Hz); MS (EI) m/z 272 [M]+ (7), 151 (21), 121 (100), 77 (11); Anal. Calcd. for C16H16O4: C, 70.57; H, 5.92. Found: C, 70.64; H, 5.88. 6-(4-(Dimethylamino)phenethyl)benzo[d][1,3]-dioxol-4-ol 1j Yield 99.1%; mp 189 – 1918C; 1H-NMR (300 MHz, CDCl3) d: 2.64 (4H, brs), 2.82 (6H, s), 5.86 (2H, s), 6.24 (1H, d, J = 1.2Hz), 6.29 (1H, d, J = 1.2Hz), 6.62 (2H, d, J = 7.8 Hz), 7.00 (2H, d, J = 7.8 Hz); MS (EI) m/z 285 [M]+ (5), 134 (100); Anal. Calcd. for C17H19NO3: C, 71.56; H, 6.71; N, 4.91. Found: C, 71.20; H, 6.53. 2-Methoxy-4-(2-(7-methoxybenzo[d][1,3]dioxol-5yl)ethyl)phenol 1k Yield 97.2%; mp 59 – 618C; 1H-NMR (300 MHz, CDCl3) d: 2.79 (4H, s), 3.85 (6H, s), 5.45 (1H, brs), 5.93 (2H, s), 6.28 (1H, d, J = 1.2 Hz), 6.37 (1H, d, J = 1.2 Hz), 6.64 (1H, dd, J = 7.9 Hz, J = 1.2 Hz), 6.68 (1H, www.archpharm.com 250 W.-G. Zhang et al. d, J = 1.2 Hz), 6.83 (1H, d, J = 7.9 Hz); MS (EI) m/z 302 [M]+ (11), 165 (100), 137 (98), 77 (32); Anal. Calcd. for C17H18O5: C, 67.54; H, 6.00. Found: C, 67.79; H, 6.16. Cytotoxicity assays An MTT colorimetric assay was employed according to the established procedure [21]. Since compounds 1a – 1k had very low aqueous solubility, all dilutions involving these compounds were performed in DMSO prior to the addition of 0.5 lL aliquots to each well. After the addition of the samples to the cell cultures, the cells were incubated for 24 h before the MTT reagent was added. The assays were performed in the Cell Culture Laboratory. 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