Chinese Journal of Natural Medicines 2015, 13(4): 03110315
Chinese Journal of Natural Medicines
Synthesis and cytotoxicity of longistylin C derivatives SHAN Yan1, HONG Ting1, WANG Yan-Fei1, ZHANG Nen-Ling2, YU Bo1, LU Yu1*, QIU Sheng-Xiang2* 1
Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China;
2
South China Botanical Garden, the Chinese Academy of Science, Guangzhou 510560, China
Available online 20 Apr. 2015 [ABSTRACT] The present study was designed to identify potent anti-tumor compounds from a series of new longistylin C derivatives. Ten longistylin C derivatives were synthesized and their structures were confirmed by 1H NMR, MS, and elemental analyses. Their cytotoxicity in vitro against three human cancer cell lines (A549, HepG2, and MCF-7) were evaluated by the MTT assay. Among these compounds, DT-6 and DT-9 displayed much better cytotoxicity against A549, HepG2, and MCF-7 cells, DT-1 exhibited selective cytotoxicity against HepG2, and the structure–activity relationships were investigated. In conclusion, Compounds DT-6 and DT-9 may serve as potential lead compounds for the discovery of new anti-cancer drugs. [KEY WORDS] Synthesis; LongistylinC derivatives; Cytotoxicity
[CLC Number] R284.3; R965
[Document code] A
[Article ID] 2095-6975(2015)04-0311-05
Introduction Longistylin C (3-methoxy-4-(3-methylbut-2-enyl)-5styrylphenol) is a natural product belonging to stilbene compounds containing a prenyl. Longistylin C is rich in pigeonpea roots, stems, leaves and seeds. It was first extracted and isolated from Lonchocarpus violaceus by Delle et al. in 1977 [1] and was isolated from the pigeon-pea in 1985 [2]. It is a naturally phytoalexin which can protect plants from environmental stresses such as fungal infections and ultraviolet radiation [3-5]. In recently years, Longistylin C has attracted a great deal of attentions for its wide physiological properties and potential therapeutic values, such as anticancer, antioxidant, anti-free radical, estrogenic, and heart protecting activities, effects on alzheimer disease, and bone metabo[Received on] 06-Apr.-2014 [Research Funding] This work was supported by the ‘115’ ‘New Drugs Invention’ the national science and technology major projects fund (2009zx09103-436), Ministry of Science and Technology of China and Cross-fund of Nanchang University. [*Corresponding author] Tel: 86-791-8333529, Fax: 86-79183337081, E-mail: [email protected] (LU Yu); Tel: 86-20137081190, Fax: 86-20-37081190, E-mail: [email protected] (QIU Sheng-Xiang) All the authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
lism as well as acting as an endothelin antagonist [6-17]. Despite its good medicinal properties, there are some limitations such as structure instability and low activity; there is an urgent need for further search for new effective Longistylin C derivatives. To our best knowledge, there have been few reports about the cytotoxic activity of Longistylin C and its derivatives in cancer cells. In the present study, a series of new Longistylin C derivatives DT-1-10 (Fig. 1) were designed and synthesized to discover more potent antitumor compounds based on Longistylin C. We also determined if Phenolic-OH was important for a functional molecule and synthesized the derivatives DT-1-DT-8. Additionally, to investigate the effects of double bond on the biological activity of Longistylin C, the derivatives DT-9 and DT-10 were synthesized. The details for chemical structures and synthetic pathways are shown in Fig. 1.
Results and Discussion Chemistry A series of resveratrol derivatives DT-1-DT-10 were synthesized and their structures were illustrated in Fig1. The phenolic-OH groups of longistylin C were etherified with various groups. The synthesis of compound DT-1 began with the reaction of Longistylin C and dimethyl sulphate in the presence of K2CO3 in acetone, with stirring at 30 °C for 6 h under a flow of nitrogen. The solvent was filtered, and the filtrate was
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Fig. 1 Structures of Longistylin C and its derivatives
diluted with ethyl acetate and washed with water, and the obtained organic phase was dried with Na2SO4, and then concentrated under reduced pressure. The resulting mixture was purified by silicagel chromatography to give derivative DT-1. Compounds DT-2-DT-7 were synthesized by reacting Longistylin C with 1-bromo-3-chloropropane, benzenesulfonyl chloride, benzoyl chloride, ethyl bromoacetate, acetic anhydride, and 1-chloro-3-methyl-2-butene, respectively. All the reactions were conducted with a similar set of procedures and reaction conditions. There were two steps required to give derivative DT-8, adding Longistylin C and 1-chloro-3-methyl-2-butene in anhydrous DMF and the solution refluxed in DMSO, through the Claisen rearrangement and cyclization reaction. To study the effect of double bonds in Longistylin C, through catalytic reduction reaction, its double bonds was reduced to give DT-9. The synthesis of DT-10 required two steps, first removing the methyl group in Longistylin C by reacting it with bromine hydride, followed by cyclization reaction. The structures of the synthesized compounds were confirmed by MS, 1H NMR, and elemental analyses. Biological evaluation The biological activities of the Longistylin C derivatives DT-1-DT-10 were evaluated by the MTT assay carried out with three human tumor cell lines (lung adenocarcinoma cell A549, hepatoma carcinoma cell HepG2, and breast cancer cell MCF-7) using the previously reported method [16]. The naturally occurring Longistylin C and Doxorubicin were used as the positive controls. The IC50 values of these compounds are shown in Table 1.
Table 1 Cytotoxicity of Longistylin C and synthesized derivatives against A549, HepG2, and MCF-7 cells Compounds
Cytotoxicity ( IC50, µmol·L−1 ) A549
HepG2
MCF-7
108.8
3.7
1378.4
DT-2
9.6
25.3
42.5
DT-3
21.3
58.0
25.5
DT-4
196.2
98.7
1832.8
DT-5
11.5
24.6
21.3
DT-6
7.6
6.2
13.9
DT-7
129.2
96.0
1072.4
DT-8
65.8
97.2
171.8
DT-1
DT-9
5.6
6.9
7.3
DT-10
64.4
55.4
165.3
Longistylin C
7.8
11.8
12.7
Doxorubicin
3.7
4.4
5.7
As shown in Table 1, the derivatives DT-6 and DT-9 displayed distinct cytotoxicities which were better than that of natural Longistylin C, and similar to that of Doxorubicin, while the other derivatives showed poorer cytotoxic activities against A549, HepG2 and MCF-7 cell lines. Compounds DT-1-DT-8 with a modified phenolic-OH showed less cytotoxicity, which indicated that the phenolic-OH of Longistylin C is very significant to preserve the cytotoxic activity of Longistylin C. Interestingly, DT-1 and DT-6 with modified phenolic-OH showed potent cytotoxicity, and DT-1 exhibited
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selective cytotoxicity against HepG2 cell line (IC50 = 3.7). DT-6 displayed a board cytotoxicity against A549, HepG2 and MCF-7 cell lines (IC50 = 7.7, 6.2, and 13.9, respectively); the reason might be that acetyl group was hydrolyzed to give the free phenolic-OH in tumor cells. The antitumor mechanism needs further investigation. Compound DT-9 exhibited higher cytotoxicity than Longistylin C, and the IC50 values were 5.6, 6.9, and 7.3, respectively. The obtained results indicated that the presence of double bond conferred a negative effect on the cytotoxicity against A549, HepG2, and MCF-7 cells. However, since DT-9 lost two double bonds, further study are needed to confirm which one plays a critical role in cytotoxic activity.
Conclusion In the present study, a series of new Longistylin C derivatives were synthesized and evaluated for their cytotoxic activity in three human tumor cell lines. Among them, DT-1, DT-6, and DT-9 were found potent cytotoxic activity. The structure–activity relationship study revealed that the phenolic-OH and the double bond both had an impact on their cytotoxic activity, but the mechanism needs further investigations. The further structural modification and activity study in vivo are needed.
Experimental Biological materials 1 H NMR spectra was obtained on a Bruker Avance 600 FT-NMR spectrometer (Bruker, Switzerland) operating at 600 MHz and 1H NMR chemical shifts (δ ) was relative to tetramethylsilane. Mass spectra were obtained on ESI-MS (Waters 2695-ZQ4000 spectrometer, Waters Corporation, Milford, America). All melting points were measured using Electrothermal engineering 9200 apparatus (Syngene Corporation, Cambridge shire, England). Element analyses were conducted using TQ-3A element analysis instrument (Henan MingShengKeji Company, China). All chemicals and reagents were purchased from Shanghai Kefeng Chemical Reagents Company (Shanghai, China), and used without further purification. Solvents used for the chemical synthesis were of analytical grade. All solvents were purified and dried when necessary. Concentration of solutions after reactions involved the use of a rotatory evaporator (Buchi, Switzerland) operating at reduced pressure. Yields were reported after crystallization and chromatographic purification. Following up of the reactions, the purity determination of the compounds was made by TLC on silica gel-precoated aluminum sheets (Type 60 F254, Qingdao Haiyang Chemical Factory, Qingdao, China). Preparation of the compounds 3, 5-dimethoxy-2-isopentenyl-stilbene (DT-1) Longistylin C (0.5 g, 1.7 mmol) was dissolved in acetone (50 mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, Me2SO4 (0.2 mL,1.7 mmol) was slowly added and the solvent
was stirred at 30 °C for 6 h. The solvent was filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate=19:1 as eluent), to give a white crystal (DT-1) 0.48 g, yield 91.6%. mp 60–61 °C; 1H NMR (CDCl3) δ: 1.67 (s, 3H), 1.8 0 ( s , 3 H ) , 3.43 (d, J = 6.6Hz, 2H), 3.78 (s, 3H), 3.82 (s, 3H), 5.13(t, J = 6.6Hz, 1H), 6.41(s, 1H), 6.74 (s, 1H), 6.96 (d, J = 16.2Hz, 1H), 7.24 (t, J = 7.8Hz,1H), 7.34 (t, J = 7.8Hz, 2H), 7.36 (d, J = 16.2Hz, 1H), 7.48 (d, J = 7.8Hz, 2H). ESI-MS m/z 309 ([M + H]+, 100%). Anal. Calcd. for C21H24O2: C, 81.81; H, 7.76. Found: C, 81.80; H, 7.79. 5-chlorpromazineoxygen-3-methoxy-2-isopentenyl-stilbene (DT-2) Longistylin C (0.25 g, 0.85 mmol) was dissolved in dried DMF (10 mL), and K2CO3 (0.1 g, 0.85 mmol) was added. Then, BrCH2CH2CH2Cl (0.084 mL, 5 mmol) was slowly added under N2 atmosphere, and the solvent was stirred at 30 °C for 6 h. The solvent was filtered and the filtrate was diluted with ethyl acetate (25 mL) and extracted with water (3 × 10 mL). The combined organic extracts were dried (Na2SO4), and the solvent was filtered and concentrated. The crude product was purified by silica gel chromatography (petroleum ether: ethyl acetate = 19 : 1 as eluent), to give a white crystal (DT-2) 0.28g, yield 88.8%, mp 62–63 °C; 1HNMR(CDCl3)δ: 1.67(s, 3H), 1.80(s, 3H), 2.25(m, 2H), 3.43(d, J = 7.2Hz, 2H), 3.76(t, J = 6.3 Hz, 2H), 3.81 (s, 3H), 4.16 (t, J = 5.7 Hz, 2H), 5.12 (t, J = 7.2 Hz, 1H), 6.42 (s, 1H), 6.76 (s, 1H), 6.96 (d, J = 16.2 Hz, 1H), 7.25 (t, J= 7.8 Hz, 1H), 7.35(t, J = 7.8 Hz, 2H), 7.36 (d, J = 16.2 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H). ESI-MS m/z 371 ([M + H]+, 5%). Anal. Calcd. for C23H27O2Cl: C, 74.48; H, 7.34. Found: C, 74.49; H, 7.35. 3-methoxy-2-isopentenyl-5-benzenesulfonyloxygen-stilbene (DT-3) Longistylin C (0.5 g, 1.7 mmol) was dissolved in acetone (10 mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, PhSO2Cl (0.22mL, 1.7mmol) was slowly added and the solvent was stirred at reflux for 8 h. The solvent was filtered and concentrated. The residue was recrystallized with acetone to give (DT-3) 0.7 g, yield 95%, mp 109–111 °C. 1H NMR (DMSO) δ: 1.60 (s, 3H), 1.73 (s, 3H), 3.40 (d, J = 7.2 Hz, 2H), 3.65 (s, 3H), 4.97 (t, J = 7.2 Hz, 1H), 6.50 (s, 1H), 6.82 (s, 1H), 6.84 (d, J =1 6.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.33 (d, J = 16.2 Hz, 1H),7.40 (t, J = 7.8 Hz, 2H),7.56 (d, J = 7.2 Hz, 2H),7.73 (d, J = 7.8 Hz, 2H), 7.87 (t, J = 7.8 Hz, 1H), 7.94 (d, J = 7.8 Hz, 2H). ESI-MS m/z 473 ([M + K]+ , 100%). Anal. Calcd. for C26H26O4S: C, 71.86; H, 6.03. Found: C, 71.88; H, 6.01. 3-methoxy-2-isopentenyl -5-benzyloxy -stilbene (DT-4) Longistylin C (0.5 g, 1.7 mmol) was dissolved in dry DMSO (15 mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, PhCH2Cl (0.195 mL, 1.7 mmol) was slowly added under N2 atmosphere, and the solvent was stirred at 85 °C f or 12 h. The solvent was filtered and the filtrate was diluted with ethyl acetate (15 mL) and extracted with water (3 × 25mL). The combined organic extracts were dried (Na2SO4), and the solvent
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was removed under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether: ethyl acetate = 18 : 2 as eluent), to give a white crystal (DT-4) 0.62 g, yield 96%. mp 71–72 °C. 1H NMR (DMSO) δ: 1.60 (s, 3H), 1.75 (s, 3H), 3.40 (d, J = 7.2 Hz, 2H), 3.77 (s, 3H), 5.01 (t, J = 7.2Hz, 1H), 5.16 (s, 2H), 6.59 (s, 1H), 6.94 (s, 1H), 7.14 (d, J = 16.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.33 (d, J = 16.2 Hz, 1H), 7.40 (t, J = 7.8 Hz, 2H), 7.56 (d, J = 7.2 Hz, 2H), 7.73 (d, J = 7.8Hz, 2H), 7.87 (t, J = 7.8Hz, 1H), 7.94 (d, J = 7.8 Hz, 2H). ESI-MS m/z 385 ([M + H]+, 5%). Anal. Calcd. for C27H28O2: C, 84.34; H, 7.34. Found: C, 84.36; H, 7.33. 5-O-acetic ether-3-methoxy -2-isopentenyl-stilbene(DT-5) Longistylin C (0.5 g, 1.7 mmol) was dissolved in dry DMF (15mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, BrCH2COOC2H5 (0.22 mL, 2.0 mmol) was slowly added under N2 atmosphere, and the solvent was stirred at 70 °C for 20 h. The solvent was filtered and the filtrate was diluted with water (15 mL) and extracted with CH2Cl2 (5 × 10 mL). The combined organic extracts were dried (Na2SO4), and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether: ethyl acetate = 17 : 3 as eluent), to give a white crystal (DT-5) 0.6 g, yield 92.3%, mp 61–62 °C.1HNMR(CDCl3)δ:1.32 (t, J = 7.2 Hz, 3H), 1.68 (s, 3H), 1.80 (s, 3H), 3.43 (d, J = 7.2 Hz, 2H), 3.81 (s, 3H), 4.30 (m, 2H), 4.66 (s, 2H), 5.11 (t, J = 7.2 Hz, 1H), 6.50 (s, 1H), 6.70 (s, 1H), 6.94 (d, J = 16.2 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.35 (d, J = 16.2 Hz, 1H), 7.38 (t, J = 7.8 Hz, 2H), 7.50 (d, J = 7.8 Hz, 2H). ESI-MS m/z 381 ([M + H]+, 100%). Anal. Calcd for C24H28O4: C, 75.76; H, 7.42. Found: C, 75.75; H, 7.43. 5-acetoxy-3-methox-2-isopentenyl-stilbene (DT-6) Longistylin C (0.5 g, 1.7 mmol) was dissolved in p yridine (12mL), Ac2O (0.2 mL, 2.1 mmol) was slowly a dded, and the solvent was stirred at 30 °C for 6 h. The n, the solvent was acidified with 1.0N HCl to PH 2, an d the residue was extracted with ethyl acetate (3 × 15 mL), the combined organic extracts were dried (Na2SO4), and the solvent was removed under reduced pressure. T he crude product was purified by silica gel chromatograp hy (petroleum ether–ethyl acetate = 18 : 2 as eluent), to give (DT-6) 0.51 g, yield 89.5%. mp 62–63 °C. 1H NMR (CDCl3) δ: 1.62 (s, 3H), 1.77 (s, 3H), 2.28 (s, 3H), 3.47 (d, J = 6.6Hz, 2H), 3.78 (s, 3H), 5.03 (t, J = 6.6Hz, 1 H), 6.72 (s, 1H), 7.05 (s, 1H), 7.12(d, J = 16.2Hz, 1H), 7.2 9 ( J = 7.8 Hz, 1H), 7.37 (d, J = 16.2 Hz, 1H), 7.40 (t, J =7.8 Hz, 2H), 7.59 (d, J = 7.8 Hz, 2H). ESI-MS m/z 337 ([M + H]+, 100%). Anal.Calcd for C22H24O3:C, 78.5 4; H, 7.19. Found: C, 78.53; H, 7.20. 5-O-isopentenyl-3-methoxy-2-isopentenyl-stilbene (DT-7) Longistylin C (0.4 g, 1.36 mmol) was dissolved in dry DMF (15 mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, ClCH2CH=C(CH3)2 (0.16 mL, 1.4 mmol) was slowly added under N2 atmosphere, and the solvent was stirred at 30 °C for 6 h. The solvent was filtered. The filtrate was diluted with
ethyl acetate(25 mL) and extracted with water (3 × 15 mL).The combined organic extracts were dried (Na2SO4), Then, The solvent was filtered and concentrated. The crude product was purified by silica-gel chromatography (petroleum ether: ethyl acetate = 19 : 1 as eluent), to give a white crystal (DT-7) 0.47 g, yield 95.9%, mp 111–112 °C. 1H NMR (DMSO) δ: 1.62 (s, 3H), 1.73 (s, 3H), 1.75 (s, 3H), 1.76 (s, 3H), 3.42 (d, J = 6.9 Hz, 2H), 3.76 (s, 3H), 4.59 (d, J = 6.6 Hz, 2H), 5.07 (t, J = 6.9 Hz, 1H), 5.47 (t, J = 6.6 Hz, 1H), 6.51 (s, 1H), 6.87 (s, 1H), 7.14 (d, J = 16.2Hz, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.38 (t, J = 7.2 Hz, 2H), 7.40 (d, J = 16.2 Hz, 1H), 7.57 (d, J = 7.2 Hz, 2H). ESI-MS m/z 363 ([M + H]+, 10%). Anal. Calcd. for C25H30O2: C, 82.83; H, 8.34. Found: C, 82.82; H, 8.35. 2,3,3-trimethyl-4-methoxy-5-prenyl-6-styryl-benzo tetrahydrofuran (DT-8) Longistylin C (0.4 g, 1.36 mmol) was dissolved in dry DMF(15 mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, ClCH2CH=C(CH3)2 (0.16 mL, 1.4 mmol) was slowly added under N2 atmosphere, and the solvent was stirred at 30 °C for 6 h. The solvent was filtered. The filtrate was diluted with ethyl acetate (25 mL) and extracted with water (3 × 15 mL). The combined organic extracts were dried (Na2SO4), the solvent was filtered and concentrated. Then, the remainder was dissolved in dry DMSO (15 mL) and the solvent was stirred at reflux for 10 h, after reaction is completed, cooling to room temperature, the solvent was diluted with ethyl acetate (25 mL) and extracted with water (5 × 20 mL). The combined organic extracts were dried (Na2SO4), the solvent was filtered and concentrated. The crude product was purified by silica gel chromatography (petroleum ether: ethyl acetate = 200 : 5 as eluent), to give a white crystal (DT-8) 0.38g, yield 77.6%. mp 81–82 °C. 1H NMR (DMSO) δ: 1.01 (s, 3H), 1.23 (s, 3H), 1.26 (d, J = 6.6 Hz, 3H), 1.52 (s, 3H), 1.62 (s, 3H), 3.22 (d, J = 6.6 Hz, 2H), 3.73 (s, 3H), 4.22 (q, J = 6.6 Hz, 1H), 5.06 (t, J = 6.6 Hz, 1H), 6.43 (s, 1H), 6.51 (d, J =16.2 Hz, 1H), 7.22 (d, J = 16.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H),7.40 (t, J =7.2 Hz, 2H), 7.55 (d, J =7.2 Hz, 2H). ESI-MS m/z 362 ([M – H]– , 10%). Anal. Calcd. for C25H30O2: C, 82.83; H, 8.34. Found: C, 82.84; H, 8.35. 2-isoamyl-3-methoxy-1-phenylethyl-5-phenol (DT-9) Longistylin C (0.5 g, 1.7 mmol) was dissolved in dry DMF (15 mL), and 0.02 g of 10% Pd/C was added. Then, H2 was slowly filled, and the solvent was stirred at 30 °C for 6 h. The solvent was filtered. The filtrate was diluted with ethyl acetate (25 mL) and extracted with water (3 × 25 mL). The combined organic extracts were dried (Na2SO4), and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether: ethyl acetate = 18 : 2 as eluent), to give a white crystal (DT-9) 0.5g, yield 98%. mp 70–71 °C. 1HNMR (DMSO) δ: 0.90 (s, 3H), 0.91 (s, 3H), 1.23 (m, 2H), 1.50 (t, J = 6.6 Hz, 1H), 2.45 (s , J = 8.1 Hz, 2H), 2.72 (d, J = 7.5 Hz, 2H), 2.76 (q, J = 7.5 Hz, 2H), 3.69 (s, 3H), 6.24 (s, 1H), 6.25 (s, 1H), 7.20 (t, J = 7.2 Hz, 1H),
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7.23 (d, J = 7.2 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 9.06 (s, 1H). ESI-MS m/z 297([M – H]–, 100%). Anal. Calcd. for C20H26O2: C, 80.50; H, 8.78. Found: C, 80.51; H, 879. 2,2-dimethyl-5-styryl-7-hydroxy-benzotetrahydro-pyran(DT-10) Longistylin C (0.5 g, 1.7 mmol) was dissolved in 20 mL CH3COOH and 33%HBr, and the solvent was stirred at 70 °C for 8 h. Then, the solvent was diluted with ethyl acetate (15 mL) and extracted with water (5 × 15 mL). The combined organic extracts were dried (Na2SO4), and the solvent was removed under reduced pressure. The crude product was purified by silica-gel chromatography (petroleum ether: ethyl acetate = 200 : 5 as eluent), to give a white crystal (DT-10) 0.21 g, yield 44.1%. mp 78–79 °C. 1H NMR (CDCl3) δ: 1.33 (s, 6H), 1.83 (t, J = 6.6 Hz, 2H), 2.77 (t, J = 6.6 Hz, 2H), 4.82 (s, 1H), 6.27 (s, 1H), 6.69 (s, 1H), 6.98 (d, J = 16.2 Hz, 1H), 7.25 (t, J = 16.2 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.37 (t, J = 7.8 Hz, 2H), 7.50 (d, J = 7.8 Hz, 2H). ESI-MS m/z 281([M + H]+, 100%). Anal. Calcd. for C19H20O2: C, 81.40; H, 7.19. Found: C, 81.41; H, 7.18. Biological activity assays The cancer cells (200 L per well) were cultured in 96-well plates (Costar Electronic Material Co., Ltd., America)1640 with 10% fetal calf serum under 5% CO2 at 37 °C. After 24 h, the appropriate test compound was added with different indicated concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 μmol·L−1, respectively, for another 72 h incubation. Then, 100 μL of 0.5 g·L−1 MTT was added to each well after discarding the culture medium, and an additional
4 h incubation was allowed. The resulting formazan was dissolved in 200 μL of dimethyl sulfoxide (DMSO) after aspiration of the culture medium. After shaking for 30 min at a plate shaker, the plate was read immediately at 570 nm using a microplate reader (Bio-Rad Model 550, Bio-Rad Laboratories, America,). The specific cytotoxicity of each compound was determined based on (1 – ODexperiments/ODpositive controls) × 100%.
[2] [3]
[4]
[5]
[6] [7]
[8]
[9]
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[11] [12]
[13]
[14]
Acknowledgments
[15]
Authors are thankful to Analysis and Testing Center of Nanchang University for facilities and support.
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References [1]
Delle-Monache F, Cuca-Suarez LE, Marini-Bettolo GB. Flavonoids from the seeds of six Lonchocarpus species [J]. Phytochemistry, 1978, 17(10): 1812-1813.
[17]
Chen DH, Li HY, Lin H. Chemical constituents of Cajanus cajan [J]. Tradit Chin Med Res (in Chin),1985, 16(10): 2-7. Duke-Eshun G, Jaroszewski JW, Asomaning WA. Antiplasmodial constituents of Cajanus cajan [J]. Phytother Res, 2004, 18(2): 128-130. Monache FD, Marletti F, Marini-Bettolo GB, et al. Isolation and structure of longistylins A, B, C, D, new prenylated stilbenes from Lonchocarpus violaceus [J]. Lloydia, 1977, 40(2): 201-208. Soleas GJ, Diamandis EP, Goldberg DM. Resveratrol: a molecule whose time has come? and gone ? [J]. Clin Biochem, 1997, 30(2): 91-113. Lu Y, Qiu SX, et al. Application of Longistylin C and its derivative for treating cancer. CN 102276433 [P]. 2011-12-14. Wang DW, Xiao MY. Determination of longistylin A and longistylin C in Cajanus cajan [J]. Chin J Chin Mater Med, 2011, 36(19): 2680-2683. Luo QF, Sun L, Si JY, et al. Hypocholesterolemic effect of stilbenes containing extract-fraction from Cajanus cajan on diet-induced hypercholesterolemia in mice [J]. Phytomedicine, 2008, 15(11): 932-939. Ruana CJ, Si JY, Zhang L, et al. Protective effect of stilbenes containing extract-fraction from Cajanus cajan L. on Aβ 25-35-induced congnitive deficits in mice [J]. Neurosci Lett, 2009, 467(2): 159-163. Grover JK, Yadav S, Vats V. Medicinal plants of India with anti-diabetic potential [J]. J Ethnopharmacol, 2002, 81(1): 81-100. Amazonian Ethnobotanical Dictionary [M]. CRC Press: Boca Raton, FL, 1994. Al-Saeedi AH, Hossain MA. Phenols, total flavoniods contents and free radical scavenging activity of seeds crude extracts of pigeon pea traditionally used in Oman for the treatment of several chronic diseases [J]. Asian Pac J Trop Dis, 2015, 5(4): 316-321. Balasubramanian A, Durairajpandian V, et al. Structural and functional studies on urease from pigeon pea (Cajanus cajan) [J]. Biomacromolecules, 2013, 58: 301-309. Cheng GT. An introduction to diabetes and its treatment [J]. Tradit Chin Med Res (in Chin), 2000, 13: 54. Kuete V, Viertel K, Efferth T. 18-Antiproliferative potential of African medicinal plants. Med Plant Res in Africa [M]. 2013: 711-724. Dzoyem JP, Kuete V. Review of the antifungal potential of African medicinal plants. Antifungal Metabolites from Plants [M]. 2013: 79-153. Zhang ZW, Zhang JQ, Hui L, et al. First synthesis and biological evaluation of novel spin-labeled derivatives of deoxypodophyllotoxin [J]. Eur J Med Chem, 2010, 45(4): 1673-1677.
Cite this article as: SHAN Yan, HONG Ting, WANG Yan-Fei, ZHANG Nen-Ling, YU Bo, LU Yu, QIU Sheng-Xiang. Synthesis and cytotoxicity of longistylin C derivatives [J]. Chinese Journal of Natural Medicines, 2015, 13(4): 311-315
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Chinese Journal of Natural Medicines
Synthesis and cytotoxicity of longistylin C derivatives SHAN Yan1, HONG Ting1, WANG Yan-Fei1, ZHANG Nen-Ling2, YU Bo1, LU Yu1*, QIU Sheng-Xiang2* 1
Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China;
2
South China Botanical Garden, the Chinese Academy of Science, Guangzhou 510560, China
Available online 20 Apr. 2015 [ABSTRACT] The present study was designed to identify potent anti-tumor compounds from a series of new longistylin C derivatives. Ten longistylin C derivatives were synthesized and their structures were confirmed by 1H NMR, MS, and elemental analyses. Their cytotoxicity in vitro against three human cancer cell lines (A549, HepG2, and MCF-7) were evaluated by the MTT assay. Among these compounds, DT-6 and DT-9 displayed much better cytotoxicity against A549, HepG2, and MCF-7 cells, DT-1 exhibited selective cytotoxicity against HepG2, and the structure–activity relationships were investigated. In conclusion, Compounds DT-6 and DT-9 may serve as potential lead compounds for the discovery of new anti-cancer drugs. [KEY WORDS] Synthesis; LongistylinC derivatives; Cytotoxicity
[CLC Number] R284.3; R965
[Document code] A
[Article ID] 2095-6975(2015)04-0311-05
Introduction Longistylin C (3-methoxy-4-(3-methylbut-2-enyl)-5styrylphenol) is a natural product belonging to stilbene compounds containing a prenyl. Longistylin C is rich in pigeonpea roots, stems, leaves and seeds. It was first extracted and isolated from Lonchocarpus violaceus by Delle et al. in 1977 [1] and was isolated from the pigeon-pea in 1985 [2]. It is a naturally phytoalexin which can protect plants from environmental stresses such as fungal infections and ultraviolet radiation [3-5]. In recently years, Longistylin C has attracted a great deal of attentions for its wide physiological properties and potential therapeutic values, such as anticancer, antioxidant, anti-free radical, estrogenic, and heart protecting activities, effects on alzheimer disease, and bone metabo[Received on] 06-Apr.-2014 [Research Funding] This work was supported by the ‘115’ ‘New Drugs Invention’ the national science and technology major projects fund (2009zx09103-436), Ministry of Science and Technology of China and Cross-fund of Nanchang University. [*Corresponding author] Tel: 86-791-8333529, Fax: 86-79183337081, E-mail: [email protected] (LU Yu); Tel: 86-20137081190, Fax: 86-20-37081190, E-mail: [email protected] (QIU Sheng-Xiang) All the authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
lism as well as acting as an endothelin antagonist [6-17]. Despite its good medicinal properties, there are some limitations such as structure instability and low activity; there is an urgent need for further search for new effective Longistylin C derivatives. To our best knowledge, there have been few reports about the cytotoxic activity of Longistylin C and its derivatives in cancer cells. In the present study, a series of new Longistylin C derivatives DT-1-10 (Fig. 1) were designed and synthesized to discover more potent antitumor compounds based on Longistylin C. We also determined if Phenolic-OH was important for a functional molecule and synthesized the derivatives DT-1-DT-8. Additionally, to investigate the effects of double bond on the biological activity of Longistylin C, the derivatives DT-9 and DT-10 were synthesized. The details for chemical structures and synthetic pathways are shown in Fig. 1.
Results and Discussion Chemistry A series of resveratrol derivatives DT-1-DT-10 were synthesized and their structures were illustrated in Fig1. The phenolic-OH groups of longistylin C were etherified with various groups. The synthesis of compound DT-1 began with the reaction of Longistylin C and dimethyl sulphate in the presence of K2CO3 in acetone, with stirring at 30 °C for 6 h under a flow of nitrogen. The solvent was filtered, and the filtrate was
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Fig. 1 Structures of Longistylin C and its derivatives
diluted with ethyl acetate and washed with water, and the obtained organic phase was dried with Na2SO4, and then concentrated under reduced pressure. The resulting mixture was purified by silicagel chromatography to give derivative DT-1. Compounds DT-2-DT-7 were synthesized by reacting Longistylin C with 1-bromo-3-chloropropane, benzenesulfonyl chloride, benzoyl chloride, ethyl bromoacetate, acetic anhydride, and 1-chloro-3-methyl-2-butene, respectively. All the reactions were conducted with a similar set of procedures and reaction conditions. There were two steps required to give derivative DT-8, adding Longistylin C and 1-chloro-3-methyl-2-butene in anhydrous DMF and the solution refluxed in DMSO, through the Claisen rearrangement and cyclization reaction. To study the effect of double bonds in Longistylin C, through catalytic reduction reaction, its double bonds was reduced to give DT-9. The synthesis of DT-10 required two steps, first removing the methyl group in Longistylin C by reacting it with bromine hydride, followed by cyclization reaction. The structures of the synthesized compounds were confirmed by MS, 1H NMR, and elemental analyses. Biological evaluation The biological activities of the Longistylin C derivatives DT-1-DT-10 were evaluated by the MTT assay carried out with three human tumor cell lines (lung adenocarcinoma cell A549, hepatoma carcinoma cell HepG2, and breast cancer cell MCF-7) using the previously reported method [16]. The naturally occurring Longistylin C and Doxorubicin were used as the positive controls. The IC50 values of these compounds are shown in Table 1.
Table 1 Cytotoxicity of Longistylin C and synthesized derivatives against A549, HepG2, and MCF-7 cells Compounds
Cytotoxicity ( IC50, µmol·L−1 ) A549
HepG2
MCF-7
108.8
3.7
1378.4
DT-2
9.6
25.3
42.5
DT-3
21.3
58.0
25.5
DT-4
196.2
98.7
1832.8
DT-5
11.5
24.6
21.3
DT-6
7.6
6.2
13.9
DT-7
129.2
96.0
1072.4
DT-8
65.8
97.2
171.8
DT-1
DT-9
5.6
6.9
7.3
DT-10
64.4
55.4
165.3
Longistylin C
7.8
11.8
12.7
Doxorubicin
3.7
4.4
5.7
As shown in Table 1, the derivatives DT-6 and DT-9 displayed distinct cytotoxicities which were better than that of natural Longistylin C, and similar to that of Doxorubicin, while the other derivatives showed poorer cytotoxic activities against A549, HepG2 and MCF-7 cell lines. Compounds DT-1-DT-8 with a modified phenolic-OH showed less cytotoxicity, which indicated that the phenolic-OH of Longistylin C is very significant to preserve the cytotoxic activity of Longistylin C. Interestingly, DT-1 and DT-6 with modified phenolic-OH showed potent cytotoxicity, and DT-1 exhibited
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selective cytotoxicity against HepG2 cell line (IC50 = 3.7). DT-6 displayed a board cytotoxicity against A549, HepG2 and MCF-7 cell lines (IC50 = 7.7, 6.2, and 13.9, respectively); the reason might be that acetyl group was hydrolyzed to give the free phenolic-OH in tumor cells. The antitumor mechanism needs further investigation. Compound DT-9 exhibited higher cytotoxicity than Longistylin C, and the IC50 values were 5.6, 6.9, and 7.3, respectively. The obtained results indicated that the presence of double bond conferred a negative effect on the cytotoxicity against A549, HepG2, and MCF-7 cells. However, since DT-9 lost two double bonds, further study are needed to confirm which one plays a critical role in cytotoxic activity.
Conclusion In the present study, a series of new Longistylin C derivatives were synthesized and evaluated for their cytotoxic activity in three human tumor cell lines. Among them, DT-1, DT-6, and DT-9 were found potent cytotoxic activity. The structure–activity relationship study revealed that the phenolic-OH and the double bond both had an impact on their cytotoxic activity, but the mechanism needs further investigations. The further structural modification and activity study in vivo are needed.
Experimental Biological materials 1 H NMR spectra was obtained on a Bruker Avance 600 FT-NMR spectrometer (Bruker, Switzerland) operating at 600 MHz and 1H NMR chemical shifts (δ ) was relative to tetramethylsilane. Mass spectra were obtained on ESI-MS (Waters 2695-ZQ4000 spectrometer, Waters Corporation, Milford, America). All melting points were measured using Electrothermal engineering 9200 apparatus (Syngene Corporation, Cambridge shire, England). Element analyses were conducted using TQ-3A element analysis instrument (Henan MingShengKeji Company, China). All chemicals and reagents were purchased from Shanghai Kefeng Chemical Reagents Company (Shanghai, China), and used without further purification. Solvents used for the chemical synthesis were of analytical grade. All solvents were purified and dried when necessary. Concentration of solutions after reactions involved the use of a rotatory evaporator (Buchi, Switzerland) operating at reduced pressure. Yields were reported after crystallization and chromatographic purification. Following up of the reactions, the purity determination of the compounds was made by TLC on silica gel-precoated aluminum sheets (Type 60 F254, Qingdao Haiyang Chemical Factory, Qingdao, China). Preparation of the compounds 3, 5-dimethoxy-2-isopentenyl-stilbene (DT-1) Longistylin C (0.5 g, 1.7 mmol) was dissolved in acetone (50 mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, Me2SO4 (0.2 mL,1.7 mmol) was slowly added and the solvent
was stirred at 30 °C for 6 h. The solvent was filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate=19:1 as eluent), to give a white crystal (DT-1) 0.48 g, yield 91.6%. mp 60–61 °C; 1H NMR (CDCl3) δ: 1.67 (s, 3H), 1.8 0 ( s , 3 H ) , 3.43 (d, J = 6.6Hz, 2H), 3.78 (s, 3H), 3.82 (s, 3H), 5.13(t, J = 6.6Hz, 1H), 6.41(s, 1H), 6.74 (s, 1H), 6.96 (d, J = 16.2Hz, 1H), 7.24 (t, J = 7.8Hz,1H), 7.34 (t, J = 7.8Hz, 2H), 7.36 (d, J = 16.2Hz, 1H), 7.48 (d, J = 7.8Hz, 2H). ESI-MS m/z 309 ([M + H]+, 100%). Anal. Calcd. for C21H24O2: C, 81.81; H, 7.76. Found: C, 81.80; H, 7.79. 5-chlorpromazineoxygen-3-methoxy-2-isopentenyl-stilbene (DT-2) Longistylin C (0.25 g, 0.85 mmol) was dissolved in dried DMF (10 mL), and K2CO3 (0.1 g, 0.85 mmol) was added. Then, BrCH2CH2CH2Cl (0.084 mL, 5 mmol) was slowly added under N2 atmosphere, and the solvent was stirred at 30 °C for 6 h. The solvent was filtered and the filtrate was diluted with ethyl acetate (25 mL) and extracted with water (3 × 10 mL). The combined organic extracts were dried (Na2SO4), and the solvent was filtered and concentrated. The crude product was purified by silica gel chromatography (petroleum ether: ethyl acetate = 19 : 1 as eluent), to give a white crystal (DT-2) 0.28g, yield 88.8%, mp 62–63 °C; 1HNMR(CDCl3)δ: 1.67(s, 3H), 1.80(s, 3H), 2.25(m, 2H), 3.43(d, J = 7.2Hz, 2H), 3.76(t, J = 6.3 Hz, 2H), 3.81 (s, 3H), 4.16 (t, J = 5.7 Hz, 2H), 5.12 (t, J = 7.2 Hz, 1H), 6.42 (s, 1H), 6.76 (s, 1H), 6.96 (d, J = 16.2 Hz, 1H), 7.25 (t, J= 7.8 Hz, 1H), 7.35(t, J = 7.8 Hz, 2H), 7.36 (d, J = 16.2 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H). ESI-MS m/z 371 ([M + H]+, 5%). Anal. Calcd. for C23H27O2Cl: C, 74.48; H, 7.34. Found: C, 74.49; H, 7.35. 3-methoxy-2-isopentenyl-5-benzenesulfonyloxygen-stilbene (DT-3) Longistylin C (0.5 g, 1.7 mmol) was dissolved in acetone (10 mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, PhSO2Cl (0.22mL, 1.7mmol) was slowly added and the solvent was stirred at reflux for 8 h. The solvent was filtered and concentrated. The residue was recrystallized with acetone to give (DT-3) 0.7 g, yield 95%, mp 109–111 °C. 1H NMR (DMSO) δ: 1.60 (s, 3H), 1.73 (s, 3H), 3.40 (d, J = 7.2 Hz, 2H), 3.65 (s, 3H), 4.97 (t, J = 7.2 Hz, 1H), 6.50 (s, 1H), 6.82 (s, 1H), 6.84 (d, J =1 6.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.33 (d, J = 16.2 Hz, 1H),7.40 (t, J = 7.8 Hz, 2H),7.56 (d, J = 7.2 Hz, 2H),7.73 (d, J = 7.8 Hz, 2H), 7.87 (t, J = 7.8 Hz, 1H), 7.94 (d, J = 7.8 Hz, 2H). ESI-MS m/z 473 ([M + K]+ , 100%). Anal. Calcd. for C26H26O4S: C, 71.86; H, 6.03. Found: C, 71.88; H, 6.01. 3-methoxy-2-isopentenyl -5-benzyloxy -stilbene (DT-4) Longistylin C (0.5 g, 1.7 mmol) was dissolved in dry DMSO (15 mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, PhCH2Cl (0.195 mL, 1.7 mmol) was slowly added under N2 atmosphere, and the solvent was stirred at 85 °C f or 12 h. The solvent was filtered and the filtrate was diluted with ethyl acetate (15 mL) and extracted with water (3 × 25mL). The combined organic extracts were dried (Na2SO4), and the solvent
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was removed under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether: ethyl acetate = 18 : 2 as eluent), to give a white crystal (DT-4) 0.62 g, yield 96%. mp 71–72 °C. 1H NMR (DMSO) δ: 1.60 (s, 3H), 1.75 (s, 3H), 3.40 (d, J = 7.2 Hz, 2H), 3.77 (s, 3H), 5.01 (t, J = 7.2Hz, 1H), 5.16 (s, 2H), 6.59 (s, 1H), 6.94 (s, 1H), 7.14 (d, J = 16.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.33 (d, J = 16.2 Hz, 1H), 7.40 (t, J = 7.8 Hz, 2H), 7.56 (d, J = 7.2 Hz, 2H), 7.73 (d, J = 7.8Hz, 2H), 7.87 (t, J = 7.8Hz, 1H), 7.94 (d, J = 7.8 Hz, 2H). ESI-MS m/z 385 ([M + H]+, 5%). Anal. Calcd. for C27H28O2: C, 84.34; H, 7.34. Found: C, 84.36; H, 7.33. 5-O-acetic ether-3-methoxy -2-isopentenyl-stilbene(DT-5) Longistylin C (0.5 g, 1.7 mmol) was dissolved in dry DMF (15mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, BrCH2COOC2H5 (0.22 mL, 2.0 mmol) was slowly added under N2 atmosphere, and the solvent was stirred at 70 °C for 20 h. The solvent was filtered and the filtrate was diluted with water (15 mL) and extracted with CH2Cl2 (5 × 10 mL). The combined organic extracts were dried (Na2SO4), and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether: ethyl acetate = 17 : 3 as eluent), to give a white crystal (DT-5) 0.6 g, yield 92.3%, mp 61–62 °C.1HNMR(CDCl3)δ:1.32 (t, J = 7.2 Hz, 3H), 1.68 (s, 3H), 1.80 (s, 3H), 3.43 (d, J = 7.2 Hz, 2H), 3.81 (s, 3H), 4.30 (m, 2H), 4.66 (s, 2H), 5.11 (t, J = 7.2 Hz, 1H), 6.50 (s, 1H), 6.70 (s, 1H), 6.94 (d, J = 16.2 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.35 (d, J = 16.2 Hz, 1H), 7.38 (t, J = 7.8 Hz, 2H), 7.50 (d, J = 7.8 Hz, 2H). ESI-MS m/z 381 ([M + H]+, 100%). Anal. Calcd for C24H28O4: C, 75.76; H, 7.42. Found: C, 75.75; H, 7.43. 5-acetoxy-3-methox-2-isopentenyl-stilbene (DT-6) Longistylin C (0.5 g, 1.7 mmol) was dissolved in p yridine (12mL), Ac2O (0.2 mL, 2.1 mmol) was slowly a dded, and the solvent was stirred at 30 °C for 6 h. The n, the solvent was acidified with 1.0N HCl to PH 2, an d the residue was extracted with ethyl acetate (3 × 15 mL), the combined organic extracts were dried (Na2SO4), and the solvent was removed under reduced pressure. T he crude product was purified by silica gel chromatograp hy (petroleum ether–ethyl acetate = 18 : 2 as eluent), to give (DT-6) 0.51 g, yield 89.5%. mp 62–63 °C. 1H NMR (CDCl3) δ: 1.62 (s, 3H), 1.77 (s, 3H), 2.28 (s, 3H), 3.47 (d, J = 6.6Hz, 2H), 3.78 (s, 3H), 5.03 (t, J = 6.6Hz, 1 H), 6.72 (s, 1H), 7.05 (s, 1H), 7.12(d, J = 16.2Hz, 1H), 7.2 9 ( J = 7.8 Hz, 1H), 7.37 (d, J = 16.2 Hz, 1H), 7.40 (t, J =7.8 Hz, 2H), 7.59 (d, J = 7.8 Hz, 2H). ESI-MS m/z 337 ([M + H]+, 100%). Anal.Calcd for C22H24O3:C, 78.5 4; H, 7.19. Found: C, 78.53; H, 7.20. 5-O-isopentenyl-3-methoxy-2-isopentenyl-stilbene (DT-7) Longistylin C (0.4 g, 1.36 mmol) was dissolved in dry DMF (15 mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, ClCH2CH=C(CH3)2 (0.16 mL, 1.4 mmol) was slowly added under N2 atmosphere, and the solvent was stirred at 30 °C for 6 h. The solvent was filtered. The filtrate was diluted with
ethyl acetate(25 mL) and extracted with water (3 × 15 mL).The combined organic extracts were dried (Na2SO4), Then, The solvent was filtered and concentrated. The crude product was purified by silica-gel chromatography (petroleum ether: ethyl acetate = 19 : 1 as eluent), to give a white crystal (DT-7) 0.47 g, yield 95.9%, mp 111–112 °C. 1H NMR (DMSO) δ: 1.62 (s, 3H), 1.73 (s, 3H), 1.75 (s, 3H), 1.76 (s, 3H), 3.42 (d, J = 6.9 Hz, 2H), 3.76 (s, 3H), 4.59 (d, J = 6.6 Hz, 2H), 5.07 (t, J = 6.9 Hz, 1H), 5.47 (t, J = 6.6 Hz, 1H), 6.51 (s, 1H), 6.87 (s, 1H), 7.14 (d, J = 16.2Hz, 1H), 7.27 (t, J = 7.2 Hz, 1H), 7.38 (t, J = 7.2 Hz, 2H), 7.40 (d, J = 16.2 Hz, 1H), 7.57 (d, J = 7.2 Hz, 2H). ESI-MS m/z 363 ([M + H]+, 10%). Anal. Calcd. for C25H30O2: C, 82.83; H, 8.34. Found: C, 82.82; H, 8.35. 2,3,3-trimethyl-4-methoxy-5-prenyl-6-styryl-benzo tetrahydrofuran (DT-8) Longistylin C (0.4 g, 1.36 mmol) was dissolved in dry DMF(15 mL), and K2CO3 (0.23 g, 1.7 mmol) was added. Then, ClCH2CH=C(CH3)2 (0.16 mL, 1.4 mmol) was slowly added under N2 atmosphere, and the solvent was stirred at 30 °C for 6 h. The solvent was filtered. The filtrate was diluted with ethyl acetate (25 mL) and extracted with water (3 × 15 mL). The combined organic extracts were dried (Na2SO4), the solvent was filtered and concentrated. Then, the remainder was dissolved in dry DMSO (15 mL) and the solvent was stirred at reflux for 10 h, after reaction is completed, cooling to room temperature, the solvent was diluted with ethyl acetate (25 mL) and extracted with water (5 × 20 mL). The combined organic extracts were dried (Na2SO4), the solvent was filtered and concentrated. The crude product was purified by silica gel chromatography (petroleum ether: ethyl acetate = 200 : 5 as eluent), to give a white crystal (DT-8) 0.38g, yield 77.6%. mp 81–82 °C. 1H NMR (DMSO) δ: 1.01 (s, 3H), 1.23 (s, 3H), 1.26 (d, J = 6.6 Hz, 3H), 1.52 (s, 3H), 1.62 (s, 3H), 3.22 (d, J = 6.6 Hz, 2H), 3.73 (s, 3H), 4.22 (q, J = 6.6 Hz, 1H), 5.06 (t, J = 6.6 Hz, 1H), 6.43 (s, 1H), 6.51 (d, J =16.2 Hz, 1H), 7.22 (d, J = 16.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H),7.40 (t, J =7.2 Hz, 2H), 7.55 (d, J =7.2 Hz, 2H). ESI-MS m/z 362 ([M – H]– , 10%). Anal. Calcd. for C25H30O2: C, 82.83; H, 8.34. Found: C, 82.84; H, 8.35. 2-isoamyl-3-methoxy-1-phenylethyl-5-phenol (DT-9) Longistylin C (0.5 g, 1.7 mmol) was dissolved in dry DMF (15 mL), and 0.02 g of 10% Pd/C was added. Then, H2 was slowly filled, and the solvent was stirred at 30 °C for 6 h. The solvent was filtered. The filtrate was diluted with ethyl acetate (25 mL) and extracted with water (3 × 25 mL). The combined organic extracts were dried (Na2SO4), and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether: ethyl acetate = 18 : 2 as eluent), to give a white crystal (DT-9) 0.5g, yield 98%. mp 70–71 °C. 1HNMR (DMSO) δ: 0.90 (s, 3H), 0.91 (s, 3H), 1.23 (m, 2H), 1.50 (t, J = 6.6 Hz, 1H), 2.45 (s , J = 8.1 Hz, 2H), 2.72 (d, J = 7.5 Hz, 2H), 2.76 (q, J = 7.5 Hz, 2H), 3.69 (s, 3H), 6.24 (s, 1H), 6.25 (s, 1H), 7.20 (t, J = 7.2 Hz, 1H),
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7.23 (d, J = 7.2 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 9.06 (s, 1H). ESI-MS m/z 297([M – H]–, 100%). Anal. Calcd. for C20H26O2: C, 80.50; H, 8.78. Found: C, 80.51; H, 879. 2,2-dimethyl-5-styryl-7-hydroxy-benzotetrahydro-pyran(DT-10) Longistylin C (0.5 g, 1.7 mmol) was dissolved in 20 mL CH3COOH and 33%HBr, and the solvent was stirred at 70 °C for 8 h. Then, the solvent was diluted with ethyl acetate (15 mL) and extracted with water (5 × 15 mL). The combined organic extracts were dried (Na2SO4), and the solvent was removed under reduced pressure. The crude product was purified by silica-gel chromatography (petroleum ether: ethyl acetate = 200 : 5 as eluent), to give a white crystal (DT-10) 0.21 g, yield 44.1%. mp 78–79 °C. 1H NMR (CDCl3) δ: 1.33 (s, 6H), 1.83 (t, J = 6.6 Hz, 2H), 2.77 (t, J = 6.6 Hz, 2H), 4.82 (s, 1H), 6.27 (s, 1H), 6.69 (s, 1H), 6.98 (d, J = 16.2 Hz, 1H), 7.25 (t, J = 16.2 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.37 (t, J = 7.8 Hz, 2H), 7.50 (d, J = 7.8 Hz, 2H). ESI-MS m/z 281([M + H]+, 100%). Anal. Calcd. for C19H20O2: C, 81.40; H, 7.19. Found: C, 81.41; H, 7.18. Biological activity assays The cancer cells (200 L per well) were cultured in 96-well plates (Costar Electronic Material Co., Ltd., America)1640 with 10% fetal calf serum under 5% CO2 at 37 °C. After 24 h, the appropriate test compound was added with different indicated concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 μmol·L−1, respectively, for another 72 h incubation. Then, 100 μL of 0.5 g·L−1 MTT was added to each well after discarding the culture medium, and an additional
4 h incubation was allowed. The resulting formazan was dissolved in 200 μL of dimethyl sulfoxide (DMSO) after aspiration of the culture medium. After shaking for 30 min at a plate shaker, the plate was read immediately at 570 nm using a microplate reader (Bio-Rad Model 550, Bio-Rad Laboratories, America,). The specific cytotoxicity of each compound was determined based on (1 – ODexperiments/ODpositive controls) × 100%.
[2] [3]
[4]
[5]
[6] [7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
Acknowledgments
[15]
Authors are thankful to Analysis and Testing Center of Nanchang University for facilities and support.
[16]
References [1]
Delle-Monache F, Cuca-Suarez LE, Marini-Bettolo GB. Flavonoids from the seeds of six Lonchocarpus species [J]. Phytochemistry, 1978, 17(10): 1812-1813.
[17]
Chen DH, Li HY, Lin H. Chemical constituents of Cajanus cajan [J]. Tradit Chin Med Res (in Chin),1985, 16(10): 2-7. Duke-Eshun G, Jaroszewski JW, Asomaning WA. Antiplasmodial constituents of Cajanus cajan [J]. Phytother Res, 2004, 18(2): 128-130. Monache FD, Marletti F, Marini-Bettolo GB, et al. Isolation and structure of longistylins A, B, C, D, new prenylated stilbenes from Lonchocarpus violaceus [J]. Lloydia, 1977, 40(2): 201-208. Soleas GJ, Diamandis EP, Goldberg DM. Resveratrol: a molecule whose time has come? and gone ? [J]. Clin Biochem, 1997, 30(2): 91-113. Lu Y, Qiu SX, et al. Application of Longistylin C and its derivative for treating cancer. CN 102276433 [P]. 2011-12-14. Wang DW, Xiao MY. Determination of longistylin A and longistylin C in Cajanus cajan [J]. Chin J Chin Mater Med, 2011, 36(19): 2680-2683. Luo QF, Sun L, Si JY, et al. Hypocholesterolemic effect of stilbenes containing extract-fraction from Cajanus cajan on diet-induced hypercholesterolemia in mice [J]. Phytomedicine, 2008, 15(11): 932-939. Ruana CJ, Si JY, Zhang L, et al. Protective effect of stilbenes containing extract-fraction from Cajanus cajan L. on Aβ 25-35-induced congnitive deficits in mice [J]. Neurosci Lett, 2009, 467(2): 159-163. Grover JK, Yadav S, Vats V. Medicinal plants of India with anti-diabetic potential [J]. J Ethnopharmacol, 2002, 81(1): 81-100. Amazonian Ethnobotanical Dictionary [M]. CRC Press: Boca Raton, FL, 1994. Al-Saeedi AH, Hossain MA. Phenols, total flavoniods contents and free radical scavenging activity of seeds crude extracts of pigeon pea traditionally used in Oman for the treatment of several chronic diseases [J]. Asian Pac J Trop Dis, 2015, 5(4): 316-321. Balasubramanian A, Durairajpandian V, et al. Structural and functional studies on urease from pigeon pea (Cajanus cajan) [J]. Biomacromolecules, 2013, 58: 301-309. Cheng GT. An introduction to diabetes and its treatment [J]. Tradit Chin Med Res (in Chin), 2000, 13: 54. Kuete V, Viertel K, Efferth T. 18-Antiproliferative potential of African medicinal plants. Med Plant Res in Africa [M]. 2013: 711-724. Dzoyem JP, Kuete V. Review of the antifungal potential of African medicinal plants. Antifungal Metabolites from Plants [M]. 2013: 79-153. Zhang ZW, Zhang JQ, Hui L, et al. First synthesis and biological evaluation of novel spin-labeled derivatives of deoxypodophyllotoxin [J]. Eur J Med Chem, 2010, 45(4): 1673-1677.
Cite this article as: SHAN Yan, HONG Ting, WANG Yan-Fei, ZHANG Nen-Ling, YU Bo, LU Yu, QIU Sheng-Xiang. Synthesis and cytotoxicity of longistylin C derivatives [J]. Chinese Journal of Natural Medicines, 2015, 13(4): 311-315
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