Structural modification of isoalantolactone and biological activity against the hepatoma cell lines

Rui-Xia Guo 1 , Li-Geng Li 1 , Man-Li Zhang 1 , Françoise Sauriol 2 , Qing-Wen Shi 1 , Yu-Cheng Gu 3 , and Hiromasa Kiyota 4
  • 1 School of Pharmaceutical Sciences, Hebei Key Laboratory of Forensic Medicine, Hebei Medical University, Shijiazhuang, Hebei Province, 050017, P.R. China
  • 2 Department of Chemistry, Queen’s University, Kingston, Ontario, K7L 3N6, Canada
  • 3 Syngenta Jealott’s Hill International Research Centre, Berkshire, RG42 6EY, UK
  • 4 Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan
Rui-Xia Guo, Li-Geng Li, Man-Li Zhang, Françoise Sauriol, Qing-Wen Shi, Yu-Cheng Gu and Hiromasa Kiyota

Abstract

Structural modifications were performed on isoalantolactone in an effort to find compounds with potential anticancer activity. Seven previously unknown adducts of active methylene compounds with isoalantolactone were synthesized by the Michael reaction. Moreover, four derivatives of aryl-substituted isoalantolactone were prepared by the Heck reaction. All synthetic products were evaluated for toxic activities against three different hepatoma cell lines, Bel-7402, SMMC-7721, and Hep G2. Products prepared through the Heck reaction and 3a,b show potential antiproliferative activity against the Hep G2 cell.

Introduction

As part of a search for anticancer agents from medicinal herbs, we focused our attention on one such species, Inula helenium, a traditional Chinese medicinal herb [1]. It grows wild in Europe, North America, and the eastern part of Asia. In addition, it is distributed in northeast, northwest, and central regions of China [2]. Sesquiterpene lactones are components of I. helenium [3]. Among them, isoalantolactone (1) is the major and important bioactive constituent. It possesses toxicity for leukocytes in vitro cultures, significant anti-inflammatory and hepatoprotective activity similar to that of silymarin, anticancer, and antifungal activities [4, 5]. Spiridonov et al. [6] found that 1 possesses marked cytotoxicity, suppressing the growth of cultured human lymphoblastoid Raji cells. The sesquiterpene lactone 1 exhibits significant antiproliferative activity to human tumor cells cultivated in vitro, the U251SP, HLE, and MM1-CB cells [7, 8].

In this paper, we describe a modification of 1 by the Heck reaction and the Michael reaction. Active methylene compounds react well with 1, adding to the α-methylene lactone subunit in a conjugative manner. This reaction therefore presents a potentially efficient method for the preparation of a range of isoalantolactone derivatives for screening. The goal of such modifications is to enhance the biological activity of the natural compound or to impart it to new types of activity. This article also discusses the relationship between the structure and activity of these derivatives. The results provide some useful information for further research on isoalantolactone 1.

Results and discussion

It is widely believed that α,β-unsaturated carbonyl compounds, and particularly α-methylene lactones, exert their biological effects by acting as alkylating agents. The lactones can form covalent adducts in vivo with proteins and other nucleophilic biomolecules, via a Michael-type addition of a free sulfhydryl or amino group. The retention of activity upon addition of the amine can be explained by the reversible nature of the Michael-type reaction.

Michael addition reactions using the active methylene compounds and proper base were performed on isoalantolactone 1 (Scheme 1). Compound 1 was allowed to react with active methylene compounds 2a–g under mild conditions, that is, stirring the reagents in absolute alcohol at room temperature for 0.5–3 h. Some products, 3a and 3b, crystallized directly from the reaction mixture. The reaction rates and product yields depend on the nature of the starting compounds. The methylene compounds 2a and 2b are the most reactive. In the presence of potassium hydroxide, additions of 1 to both 2a and 2b produced the desired products 3a and 3b in yields of 95% and 93%, respectively. However, the yields of the diethyl malonate adduct 3d and ethyl acetoacetate adduct 3g were low. The explanation could be that reaction yields are reduced with the increase of steric hindrance. Besides, as electron-withdrawing ability of compounds becomes strong, the reactivity becomes higher.

Scheme 1
Scheme 1

Synthesis of compounds 3a–g through the Michael reaction.

Citation: Heterocyclic Communications 20, 2; 10.1515/hc-2013-0220

Reaction of isoalantolactone 1 with aryl iodides 4a–d is catalyzed by the system Pd(OAc)2-(o-Tol)3P in DMF in the presence of triethylamine (Scheme 2). The main reaction products were isolated in 60–91% yield by column chromatography on silica gel. In agreement with the general rule of metal-catalyzed coupling reactions, the coupling of the aromatic iodide with an electron-donating substituent was efficient.

Scheme 2
Scheme 2

Synthesis of compounds 5a–d through the Heck reaction.

Citation: Heterocyclic Communications 20, 2; 10.1515/hc-2013-0220

The synthesized compounds were screened in vitro for their anticancer potential against three different hepatoma cell lines Bel-7402, SMMC-7721, and Hep G2, with three anticancer agents, cisplatin, jifitinib, and taxol, used as controls. The concentration of these compounds was 100 mm. The results, shown in Figure 1, indicate that none of these compounds exhibits any significant or selective hepatoma cell growth inhibitory properties. However, it was encouraging to see that the compounds are able to inhibit Hep G2 cell growth in the submicromolar concentration range. Most derivatives show more potent inhibitory activity than cisplatin to the Hep G2 cell but weaker than taxol. The compounds obtained by Heck reaction inhibit hepatoma cell growth, especially the growth of Hep G2. In addition, the Michael reaction products 3a and 3b can significantly inhibit the proliferation of the Hep G2 cell.

Figure 1
Figure 1

Cell growth inhibition properties of isoalantolactone derivatives against hepatoma cells.

Citation: Heterocyclic Communications 20, 2; 10.1515/hc-2013-0220

Conclusion

In total, 14 isoalantolactone derivatives were synthesized by using the Michael reaction or the Heck reaction, of which seven are new compounds. The reactions proceed in high yields. All products were tested in vitro for anticancer activity on three different hepatoma cell lines: Bel-7402, SMMC-7721, and Hep G2. Several compounds show potential antiproliferative activity against the Hep G2 cell.

Experimental

General

Melting points were obtained on a Laboratory Devices SGW X-4B melting apparatus and are uncorrected. NMR spectra were recorded on a Bruker AV-500 instrument at 500 MHz (1H) and 125 MHz (13C) in CDCl3. HR mass spectra (MS) were acquired with an Agilent Accurate-Mass-Q-TOF MS 6520 system equipped with an electrospray ionization (ESI) source. All MS experiments were conducted in the positive ionization mode. Isoalantolactone (1) was isolated from I. helenium by our laboratory and its purity was determined by analytical HPLC (purity > 95%).

Extraction and isolation of isoalantolactone (1)

Dried crushed roots of I. helenium (3 kg) were extracted three times with 99% ethanol at refluxing temperature for 2 h. The residue was scattered in saturated brine (1 g/mL) and then extracted with petroleum ether, dichloromethane, and ethyl acetate. The dichloromethane extract (58 g) was subjected to purification on a silica gel column, eluting with petroleum ether and ethyl acetate (9:1–1:1) gradient. Compound 1 was visualized using HPLC detection. Further separation of the mixture was achieved by column chromatography using an SiO2-AgNO3 (90:10) adsorbent and a benzene-petroleum ether-ethyl acetate (3:15:3) as an eluent. White needles of isoalantolactone (1) were obtained. EI-MS (acetone): m/z 232 [M]+; 1H NMR: δ 6.13 (1H, br, s, H-13a), 5.59 (1H, br, s, H-13b), 4.77 (1H, br, s, H-15a), 4.45 (1H, br, s, H-15b), 4.51 (1H, br, s, H-8), 2.99 (1H, m, H-7), 2.34 (1H, d, J = 12.0 Hz, H-3b), 2.21 (1H, d, J = 16.0 Hz, H-9a), 2.01 (1H, m, H-3a), 1.85 (1H, J = 12.5 Hz, H-5), 1.75 (1H, m, H-6a), 1.71 (1H, m, H-9b), 1.60 (2H, m, H-2), 1.54 (1H, m, H-1a), 1.40 (1H, m, H-6b), 1.25 (1H, m, H-1b), 0.83 (3H, s, 14-CH3); 13C NMR: δ 41.3 (C-1), 22.7 (C-2), 36.8 (C-3), 148.9 (C-4), 46.2 (C-5), 27. 5 (C-6), 40.5 (C-7), 76.8 (C-8), 42.2 (C-9), 34.3 (C-10), 142.2 (C-11), 170.7 (C-12), 120.2 (C-13), 17.7 (C-14), 106.6 (C-15). The data above are virtually identical to literature data [9].

General procedure for the synthesis of 3a,b

Methyl cyanoacetate or ethyl cyanoacetate (0.2 mmol) and potassium hydroxide (11 mg, 0.2 mmol) were added to a solution of 1 (46.4 mg, 0.200 mmol) in 5 mL of 100% anhydrous EtOH. The mixture was stirred at room temperature for 0.5 h and the resulting white precipitate was crystallized from acetone.

Methyl (3aR,4aS,8aR,9aR)-2-cyano-3-[(8a-methyl-5-methylene-2-oxododecahydronaphtho[2,3-b](3-furyl)]propanoate (3a)

White solid; yield 94%; mp 194–195°C; 1H NMR: δ 0.79 (3H, s, H-14), 1.23 (1H, m, H-1b), 1.34 (2H, m, H-13), 1.47 (1H, m, H-6b), 1.54–1.59 (4H, m, H-1a, 2, 2, 9b), 1.77 (1H, d, J = 12.0 Hz, H-6a), 1.85 (1H, m, H-5), 1.99 (1H, m, H-3b), 2.18 (1H, dd, J = 15.5, 4 Hz, H-9a), 2.32 (1H, m, H-3a), 2.65 (1H, m, H-11), 2.75 (1H, m, H-7), 2.95 (1H, br, t, H-16), 4.29 (3H, m, H-19), 4.47–4.51 (2H, m, H-8, 15b), 4.80 (1H, d, J = 8.5 Hz, H-15a); 13C NMR: δ 17.7 (C-14), 21.6 (C-13), 22.6 (C-2), 28.6 (C-6), 32.1 (C-16), 34.8 (C-10), 36.7 (C-3), 39.4 (C-7), 41.4 (C-1), 42.1 (C-9), 46.5 (C-5), 48.6 (C-11), 63.7 (C-19), 78.2 (C-8), 106.7 (C-15), 118.2 (C-17), 148.8 (C-4), 168.0 (C-18), 176.5 (C-12); HR-ESI-MS. Calcd for C19H25NNaO4 ([M+Na]+): m/z 354.1675, found: m/z 354.1676.

Ethyl (3aR,4aS,8aR,9aR)-2-cyano-3-[(8a-methyl-5-methylene-2-oxododecahydronaphtho[2,3-b](3-furyl)]propanoate (3b)

White solid; yield 93%; mp 193–194°C; 1H NMR: δ 0.79 (3H, s, H-14), 1.09 (3H, m, H-20), 1.23 (1H, m, H-1b), 1.34 (2H, m, H-13), 1.48 (1H, m, H-6b), 1.53–1.62 (4H, m, H-1a, 2, 2, 9b), 1.77 (1H, m, H-6a), 1.85 (1H, d, J = 12.0 Hz, H-5), 1.99 (1H, m, H-3b), 2.15 (1H, m, H-9a), 2.31 (1H, m, H-3a), 2.66 (1H, m, H-11), 2.75 (1H, m, H-7), 2.95 (1H, br, t, H-16), 4.28 (2H, m, H-19), 4.47–4.51 (2H, m, H-8, 15b), 4.80 (1H, d, J = 2.0 Hz, H-15a); 13C NMR: δ 13.89 (C-20), 17.73 (C-14), 21.63 (C-13), 22.63 (C-2), 28.56 (C-6), 32.04 (C-16), 34.76 (C-10), 36.67 (C-3), 39.44 (C-7), 41.35 (C-1), 42.12 (C-9), 46.45 (C-5), 48.58 (C-11), 63.68 (C-19), 78.17 (C-8), 106.70 (C-15), 118.19 (C-17), 148.84 (C-4), 167.94 (C-18), 176.54 (C-12); HR-ESI-MS. Calcd for C20H27NNaO4 ([M+Na]+): m/z 368.1832, found: m/z 368.1834.

General procedure for the synthesis of 3c–g

Active methylene compound 2c–g (0.24 mmol) and triethylamine (0.24 mmol) were added to a solution of 1 (46.4 mg, 0.002 mmol) in 5 mL of 100% anhydrous EtOH. The mixture was stirred at room temperature for 2 h and then quenched with deionized H2O. After extraction with ethyl acetate (3 × 15 mL), the combined organic phases were washed successively with 0.1% hydrochloric acid (10 mL), a saturated NaCl solution (2 × 20 mL), and dried over Na2SO4. After concentration, the residue was purified by silica gel column chromatography eluting with petroleum ether-ethyl acetate.

Dimethyl (3aR,4aS,8aR,9aR)-2-[(8a-methyl-5-methylene-2-oxododecahydronaphtho[2,3-b](3-furylmethyl)]malonate (3c)

White needles; yield 83%; mp 105–107°C; 1H NMR: δ 0.80 (3H, s, H-14), 1.23 (1H, m, H-1b), 1.45 (1H, dd, J = 15.5, 4.5 Hz, H-6b), 1.52–1.61 (4H, H-1a, 2, 2, 9b), 1.79 (1H, d, J = 15.5 Hz, H-6a), 2.00 (1H, td, J = 12.5, 6.5 Hz, H-3b), 2.11–2.17 (3H, m, H-3a, 5, 9a), 2.34 (2H, m, H-13), 2.45 (1H, m, H-11), 2.74 (1H, m, H-7), 3.76 (6H, s, H-18, 18′), 3.86 (1H, dd, J = 8.5, 6.5 Hz, H-16), 4.45 (1H, td, J = 4.0, 2.0 Hz, H-8), 4.48 (1H, d, J = 1.0 Hz, H-15b), 4.78 (1H, d, J = 1.0 Hz, H-15a); 13C NMR: δ 17.8 (C-14), 21.1 (C-13), 22.7 (C-2), 24.2 (C-6), 34.8 (C-10), 36.7 (C-3), 39.7 (C-7), 41.5 (C-1), 42.2 (C-9), 44.5 (C-11), 46.5 (C-5), 49.3 (C-16), 52.7 (C-18), 52.7 (C-18′), 77.9 (C-8), 106.6 (C-15), 149.1 (C-4), 169.5 (C-17), 169.4 (C-17′), 177.7 (C-12); HR-ESI-MS. Calcd for C20H29O6 ([M+H]+): m/z 365.1959, found: m/z 365.1954.

Diethyl (3aR,4aS,8aR,9aR)-2-[(8a-methyl-5-methylene-2-oxododecahydronaphtho[2,3-b](3-furylmethyl)]malonate (3d)

Colorless liquid; yield 76%; 1H NMR: δ 0.80 (3H, s, H-14), 1.19 (1H, m, H-1b), 1.27 (6H, td, J = 7.5, 3.5 Hz, H-19, 19′), 1.45 (1H, dd, J = 15.5 Hz, 4.0 Hz, H-6b), 1.52–1.63 (4H, m, H-1a, 2, 2, 9b), 1.78 (1H, d, J = 12 Hz, H-6a), 1.98 (1H, m, H-3b), 2.10–2.17 (3H, m, H-3a, 5, 9a), 2.32 (2H, m, H-13), 2.45 (1H, m, H-11), 2.75 (1H, m, H-7), 3.76 (1H, m, H-16), 4.22 (4H, m, H-18, 18′), 4.45 (1H, m, H-8), 4.48 (1H, d, J = 1.0 Hz, H-15b), 4.78 (1H, d, J = 1.0 Hz, H-15a); 13C NMR: δ 14.0 (C-19), 14.1 (C-19′), 17.7 (C-14), 21.1 (C-13), 22.6 (C-2), 24.0 (C-6), 34.8 (C-10), 36.7 (C-3), 39.6 (C-7), 41.5 (C-1), 42.2 (C-9), 44.5 (C-11), 46.5 (C-5), 49.6 (C-16), 61.6 (C-18), 61.6 (C-18′), 77.9 (C-8), 106.5 (C-15), 149.1 (C-4), 168.9 (C-17), 169.1 (C-17′), 177.7 (C-12); HR-ESI-MS. Calcd for C22H32NaO6 ([M+Na]+): m/z 415.2091, found: m/z 415.2094.

(3aR,4aS,8aR,9aR)-8a-Methyl-5-methylene-3-(2-nitroethyl)decahydronaphtho[2,3-b]furan-2-one (3e)

White solid; yield 80%; mp 113–114°C; 1H NMR: δ 0.81 (3H, s, H-14), 1.21 (1H, m, H-1b), 1.48 (1H, dd, J = 15.5, 4 Hz, 6b), 1.54–1.61 (4H, m, H-1a, 2, 2, 9b), 1.74 (1H, m, H-6a), 1.81 (1H, m, H-5), 2.00 (1H, m, H-3b), 2.18 (1H, dd, J = 15.5, 2.0 Hz, H-9a), 2.27 (1H, m, H-11), 2.35 (1H, m, H-3a), 2.41 (1H, m, H-13), 2.49 (1H, m, H-13), 2.78 (1H, m, H-7), 4.46 (1H, d, J = 1.5 Hz, H-15b), 4.51 (1H, td, J = 4.5, 2.0 Hz, H-8), 4.69 (2H, m, H-16), 4.79 (1H, d, J = 1.5 Hz, H-15a); 13C NMR: δ 17.8 (C-14), 21.3 (C-13), 22.6 (C-2), 23.1 (C-6), 34.8 (C-10), 36.7 (C-3), 39.6 (C-7), 41.4 (C-1), 42.2 (C-9), 44.0 (C-11), 46.4 (C-5), 73.3 (C-16), 78.1 (C-8), 106.6 (C-15), 149.0 (C-4), 177.3 (C-12); HR-ESI-MS. Calcd for C16H23NNaO4 ([M+Na]+): m/z 316.1519, found: m/z 316.1520.

(3aR,4aS,8aR,9aR)-8a-Methyl-5-methylene-3-(2-nitropropyl)decahydronaphtho[2,3-b]furan-2-one (3f)

White needles; yield 80%; mp 134–136°C; 1H NMR: δ 0.80 (3H, s, H-14), 1.25 (1H, m, H-1b), 1.47 (1H, m, H-6b), 1.53–1.59 (4H, m, H-1a, 2, 2, 9b), 1.62 (3H, dd, J = 6.5, 1.5 Hz, H-17), 1.81 (1H, m, H-5), 1.97 (1H, m, H-3b), 2.06–2.27 (2H, m, H-6a, 9a), 2.34 (1H, m, H-3a), 2.68 (1H, m, H-7), 2.53 (2H, m, H-13), 2.68 (1H, m, H-11), 4.44 (1H, d, J = 1.0 Hz, H-15b), 4.47 (1H, m, H-8), 4.77 (1H, s, H-15a), 5.05 (1H, m, H-16); 13C NMR: δ 17.7 (C-14), 19.2 (C-17), 22.6 (C-2), 23.0 (C-6), 30.6 (C-13), 34.7 (C-10), 36.7 (C-3), 39.8 (C-7), 41.4 (C-1), 42.1 (C-9), 44.2 (C-11), 46.4 (C-5), 78.1 (C-8), 82.1 (C-16), 106.5 (C-15), 149.0 (C-4), 177.6 (C-12); HR-ESI-MS. Calcd for C17H25NNaO4 ([M+Na]+): m/z 330.1676, found: m/z 330.1677.

Ethyl(3aR,4aS,8aR,9aR)-2-[(8a-methyl-5-methylene-2-oxododecahydronaphtho[2,3-b](3-furylmethyl)]-3-oxobutanoate (3g)

Colorless liquid; yield 71%. 1H NMR: δ 0.79 (3H, s, H-14), 1.19 (1H, m, H-1b), 1.29 (3H, m, H-19), 1.45 (1H, dd, J = 15.5, 4.0 Hz, H-6b), 1.52–1.61 (4H, m, H-1a, 2, 2, 9b), 1.78 (1H, d, J = 12.5 Hz, H-6a), 1.98 (1H, m, H-3b), 2.11–2.19 (4H, m, H-5, 9a, 13), 2.31 (4H, m, H-21, 3a), 2.44 (1H, m, H-11), 2.67 (1H, m, H-7), 4.05 (1H, m, H-16), 4.22 (2H, m, H-18), 4.44 (1H, m, H-15b), 4.48 (1H, dd, J = 4.0, 1.5 Hz, H-8), 4.78 (1H, s, H-15a); 13C NMR: δ 14.0 (C-21), 16.8 (C-18), 17.8 (C-14), 21.2 (C-13), 22.6 (C-2), 29.2 (C-6), 34.8 (C-10), 36.7 (C-3), 39.8 (C-7), 41.5 (C-1), 42.2 (C-9), 44.5 (C-11), 46.5 (C-5), 56.5 (C-16), 61.6 (C-20), 78.0 (C-8), 106.6 (C-15), 149.1 (C-4), 169.1 (C-19), 178.2 (C-12), 202.7 (C-17); HR-ESI-MS. Calcd for C21H31O5 ([M+H]+): m/z 363.2166, found: m/z 363.2163.

General procedure for synthesis of 5a–d by the Heck reaction

Compounds 5ad were synthesized by modification of the literature methods [10, 11]. Briefly, a three-necked glass ampule, filled with isoalantolactone (1, 70 mg, 0.3 mmol), aromatic iodide (0.33 mmol), Pd(OAc)2 (0.015 mmol, 5 mol%), tris(o-tolyl)phosphine (0.051 mmol, 17 mol%), DMF (10 mL) and Et3N (61 mg, 0.6 mmol), and 3A molecular sieves was sealed under argon. The ampule was heated for 10–15 h at 120°C, then cooled and opened. The mixture was poured into water and extracted with ethyl acetate. After concentration, the residue was purified by silica gel column chromatography eluting with petroleum ether-ethyl acetate.

(3aR,4aS,8aR,9aR,E)-3-(4-Chlorobenzyl)-8a-methyl-5-methylenedecahydronaphtho[2,3-b]furan-2-one (5a)

White needles; yield 60%; mp 219–220°C, lit. [10] mp 207–209°C; 1H NMR: δ 0.88 (3H, s, H-14), 1.27 (1H, m, H-1b), 1.43 (1H, m, H-6b), 1.54–1.63 (4H, m, H-1, 2, 2, 9), 1.93–2.04 (3H, m, H-3b, 5, 6a), 2.27 (1H, dd, J = 15.5, 1.5 Hz, H-9a), 2.36 (1H, br, d, J = 13.5 Hz, H-3a), 3.40 (1H, m, H-7), 4.42 (1H, d, J = 1.0 Hz, H-15b), 4.51 (1H, td, J = 4.5, 1.5 Hz, H-8), 4.78 (1H, d, J = 1.5 Hz, H-15a), 7.39 (3H, m, H-3′, 5′, 13), 7.46 (2H, d, J = 8.5, H-2′, 6′).

(3aR,4aS,8aR,9aR,E)-3-Benzyl-8a-methyl-5-methylenede cahydronaphtho[2,3-b]furan-2-one (5b)

White needles; yield 65%; mp 220–222°C, lit. [10] mp 202–204°C; 1H NMR: δ 0.89 (3H, s, H-14), 1.28 (1H, m, H-1b), 1.44 (1H, m, H-6b), 1.52–1.63 (4H, m, H-1, 2, 2, 9), 1.95 (1H, d, J = 12.5 Hz, H-6a), 2.03 (2H, m, H-3b, 5), 2.27 (1H, dd, J = 15.5, 1.5 Hz, H-9a), 2.36 (1H, m, H-3a), 3.44 (1H, m, H-7), 4.43 (1H, d, J = 1.0 Hz, H-15b), 4.50 (1H, td, J = 5.0, 1.5 Hz, H-8), 4.78 (1H, d, J = 1.0 Hz, H-15a), 7.40–7.44 (4H, m, H-3′, 4′, 5′, 13), 7.53 (2H, d, J = 8.5, H-2′, 6′).

(3aR,4aS,8aR,9aR,E)-8a-Methyl-3-(4-methylbenzyl)-5-methylenedecahydronaphtho[2,3-b]furan-2-one (5c)

White needles; yield 91%; mp 239–240°C, lit. [10] mp 220–222°C; 1H NMR: δ 0.88 (3H, s, H-14), 1.28 (1H, m, H-1b), 1.43 (1H, m, H-6b), 1.52–1.63 (4H, m, H-1, 2, 2, 9), 1.95 (1H, d, J = 12.5 Hz, H-6a), 2.03 (2H, m, H-3b, 5), 2.27 (1H, dd, J = 15.5, 1.5 Hz, H-9a), 2.35 (1H, m, H-3a), 2.38 (3H, s, H-CH3), 3.43 (1H, m, H-7), 4.43 (1H, d, J = 1.5 Hz, H-15b), 4.49 (1H, td, J = 4.5, 1.5 Hz, H-8), 4.77 (1H, d, J = 1.5 Hz, H-15a), 7.22 (2H, d, J = 8.0 Hz, H-3′, 5′), 7.42 (3H, m, H-2′, 6′, 13).

(3aR,4aS,8aR,9aR,E)-3-(4-Methoxybenzyl)-8a-methyl-5-methylenedecahydronaphtho[2,3-b]furan-2-one (5d)

White solid; yield 84%; mp 202–204°C, lit. [11] mp 199–201°C; 1H NMR: δ 0.88 (3H, s, H-14), 1.27 (1H, m, H-1b), 1.41 (1H, m, H-6b), 1.53–1.63 (4H, m, H-1, 2, 2, 9), 1.94 (1H, d, J = 12.5 Hz, H-6a), 2.01–2.06 (2H, m, H-3b, 5), 2.26 (1H, dd, J = 15.5, 1.5 Hz, H-9a), 2.35 (1H, m, H-3a), 3.41 (1H, m, H-7), 3.85 (3H, s, OCH3), 4.43 (1H, d, J = 1.5 Hz, H-15b), 4.49 (1H, m, H-8), 4.77 (1H, d, J = 1.0 Hz, H-15a), 6.94 (2H, d, J = 9.0 Hz, H-3′, 5′), 7.39 (1H, s, H-13), 7.49 (2H, d, J = 9.0 Hz, H-2′, 6′).

In vitro anticancer activity

For comparison of cell viability in human hepatoma carcinoma, cells were treated with compounds, cisplatin, taxol, and jifitinib (reference agents), for 2 days, followed by estimation of cell viability by the MTT assay, as described below. Data are presented as means ± SD of three independent experiments. The compounds were dissolved in DMSO (SERVA, Germany) and immediately after dissolving used for the test. Anticancer drugs, cisplatin, taxol, jifitinib (Wako Pure Chemicals Industries, Ltd.), and Methotrexate (F6627, Sigma, St. Louis, MO, USA), were also dissolved in DMSO. The following human hepatoma carcinoma cells were used: Bel-7402, SMMC-7721, and Hep G2. Cells were purchased from ShangHai MEIXUAN Biological Science and Technology Ltd. (China). Hep G2 cells was cultured in DMEM (GIBCO, USA), containing 10% (v/v) calf serum (Biological Industries, BioInd) and antibiotics, 100 μg/mL of streptomycin (Nalgene, China) and 100 units/mL of penicillin G (Nalgene, China), at 37°C in a humidified atmosphere containing 5% CO2. Instead, RPMI 1640 (GIBCO, USA) and fetal calf serum (Biological Industries, BioInd), respectively, were used for culture of Bel-7402, SMMC-7721 cells.Cell viability was estimated by the MTT assay as described elsewhere [12]. Briefly, logarithmically proliferating cells were plated onto 96-well plates (JETBIOFIL, China) (1 × 104 cells/well) with the medium containing test compounds at 100 μmol/L doses, followed by culture for 2 days. After the culture, the activity of mitochondrial succinic dehydrogenase was measured by further incubation of the cells with 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (TOKYO KASEI KOGYO, Japan) for 4 h, followed by measurement of absorbance at 570 nm with a reference wavelength at 655 nm. Cell survival was calculated from absorbance and presented as a percentage of the surviving cells.

Acknowledgments

We are grateful for financial support from the National Natural Science Foundation of China (grant numbers 81302664, 81072551, 81241101). We also wish to extend our sincere thanks for financial support from Syngenta Ltd. (2013-Hebei Medical University-Syngenta-04), and JSPS KAKENHI (grant numbers 19580120, 22560112, 25450144).

References

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    Qiu, C. C.; Dai, B. Isolation and identification of alantolactone and isoalantolactone from Inulahelenium. J. Chin. Med. Mater.1995, 18, 30–31.

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    Belovodskii, A. V.; Shul’ts, E. E.; Shakirov, M. M.; Bagryanskaya, I. Y.; Gatilov, Y. V.; Tolstikov, G. A. Synthetic transformations of methylenelactones of eudesmanic type. Behavior of isoalantolactone under the conitions of heck reaction. Russ. J. Org. Chem.2010, 46, 1719–1734.

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    Belovodskii, A. V.; Shul’ts, E. E.; Shakirov, M. M.; Tolstikov, G. A. Sesquiterpene metylenelactones in a palladium-catalyzed cross-coupling reaction. Dokl. Chem.2009, 426, 138–142.

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    Wu, Y. B.; Ni, Z. Y.; Huo, C. H.; Su, J.; Dong, M.; Sauriol, F.; Shi, Q. W.; Gu, Y. C.; Kiyota, H. Xylomexicanins C and D, new mexicanolide-type limonoids from Xylocarpus granatum.Biosci. Biotechnol. Biochem.2013, 77, 736–740.

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  • [1]

    Ducki, S.; Hadfield, J. A.; Hepworth, L. A.; Lawrence, N. J.; Liu, C.; McGown, A. T. Synthesis and cell growth inhibitory properties of substituted (E)-1-phenylbut-1-en-3-ones. Bioorg. Med. Chem. Lett.1997, 7, 3091–3094.

  • [2]

    Institute of Botany, The Chinese Academy of Sciences. Flora of China; Science Press: Beijing, 1979; Vol. 75, pp. 248–254.

  • [3]

    Zhao, Y. M.; Zhang, M. L.; Shi, Q. W.; Kiyota, H. Chemical constituents of plants from the genus Inula. Chem. Biodivers.2006, 3, 371–384.

    • PubMed
  • [4]

    Huo, Y.; Shi, H. M.; Wang, M. Y.; Li, X. B. Chemical constituents and pharmacological properties of Radix Inulae. Pharmazie2008, 63, 699–703.

    • PubMed
  • [5]

    Zhang, S.; Won, Y. K.; Ong, C. N.; Shen, H. M. Anti-cancer potential of sesquiterpene lactones: bioactivity and molecular mechanisms. Curr. Med. Chem.2005, 5, 239–249.

  • [6]

    Spiridonov, N. A.; Konovalov, D. A.; Arkhipov, V. V. Cytotoxicity of some Russian ethnomedicinal plants and plant compounds. Phytother. Res. 2005, 19, 428–432.

  • [7]

    Li, Y.; Li, T. K.; Shi, S. W.; Zhang, Y. F.; Tian, Z. Q.; Shi, Q. W.; Suzuki, S. In vivo and in vitro anti-tumor activity studies of five sesquiterpenoids from Inula helenium. Chin. Pharm. Bull.2010, 26, 112–115.

  • [8]

    Yu, F.; Wang, S.; Dong, M.; Ni, Z. Y.; Dong, F. J.; Shi, Q. W.; Suzuki, N.; Cong, B. Studies on the anti-growth activity of three sesquiterpene compounds to human tumor cell lines. Nat. Prod. Res. Dev.2010, 22, 506–509.

  • [9]

    Qiu, C. C.; Dai, B. Isolation and identification of alantolactone and isoalantolactone from Inulahelenium. J. Chin. Med. Mater.1995, 18, 30–31.

  • [10]

    Belovodskii, A. V.; Shul’ts, E. E.; Shakirov, M. M.; Bagryanskaya, I. Y.; Gatilov, Y. V.; Tolstikov, G. A. Synthetic transformations of methylenelactones of eudesmanic type. Behavior of isoalantolactone under the conitions of heck reaction. Russ. J. Org. Chem.2010, 46, 1719–1734.

  • [11]

    Belovodskii, A. V.; Shul’ts, E. E.; Shakirov, M. M.; Tolstikov, G. A. Sesquiterpene metylenelactones in a palladium-catalyzed cross-coupling reaction. Dokl. Chem.2009, 426, 138–142.

  • [12]

    Wu, Y. B.; Ni, Z. Y.; Huo, C. H.; Su, J.; Dong, M.; Sauriol, F.; Shi, Q. W.; Gu, Y. C.; Kiyota, H. Xylomexicanins C and D, new mexicanolide-type limonoids from Xylocarpus granatum.Biosci. Biotechnol. Biochem.2013, 77, 736–740.

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    Synthesis of compounds 3a–g through the Michael reaction.

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    Synthesis of compounds 5a–d through the Heck reaction.

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    Cell growth inhibition properties of isoalantolactone derivatives against hepatoma cells.