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

Article abstract of DOI:10.1515/hc-2013-0220

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. M

Full text of DOI:10.1515/hc-2013-0220

ꢁꢁꢁꢂ
ꢀHeterocycl. Commun. 2014; 20(2): 117–121
DOI 10.1515/hc-2013-0220ꢀ
Rui-Xia Guo, Li-Geng Li, Man-Li Zhang, Françoise Sauriol, Qing-Wen Shi*, Yu-Cheng Gu
and Hiromasa Kiyota*
Structural modification of isoalantolactone and
biological activity against the hepatoma cell lines
Abstract: Structural modifications were performed on isoa- activity similar to that of silymarin, anticancer, and anti-
lantolactone in an effort to find compounds with potential fungal activities [4, 5]. Spiridonov et al. [6] found that 1
anticancer activity. Seven previously unknown adducts of possesses marked cytotoxicity, suppressing the growth of
active methylene compounds with isoalantolactone were cultured human lymphoblastoid Raji cells. The sesquiter-
synthesized by the Michael reaction. Moreover, four deriv- pene lactone 1 exhibits significant antiproliferative activ-
atives of aryl-substituted isoalantolactone were prepared ity to human tumor cells cultivated in vitro, the U251SP,
by the Heck reaction. All synthetic products were evalu- HLE, and MM1-CB cells [7, 8].
ated for toxic activities against three different hepatoma
In this paper, we describe a modification of 1 by the
cell lines, Bel-7402, SMMC-7721, and Hep G2. Products pre- Heck reaction and the Michael reaction. Active methylene
pared through the Heck reaction and 3a,b show potential compounds react well with 1, adding to the α-methylene
antiproliferative activity against the Hep G2 cell.
lactone subunit in a conjugative manner. This reaction
therefore presents a potentially efficient method for the
Keywords: antiproliferative activity; Heck reaction; isoa- preparation of a range of isoalantolactone derivatives for
lantolactone; Michael reaction; structural modification.
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.
*Corresponding authors: Qing-Wen Shi, School of Pharmaceutical
Sciences, Hebei Key Laboratory of Forensic Medicine, Hebei Medical
University, Shijiazhuang, Hebei Province, 050017, P.R. China,
e-mail: shiqingwen@hebmu.edu.cn; and Hiromasa Kiyota,
Graduate School of Environmental and Life Science, Okayama
University, 1-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan,
e-mail: kiyota@okayama-u.ac.jp
Rui-Xia Guo, Li-Geng Li and Man-Li Zhang: School of Pharmaceutical
Sciences, Hebei Key Laboratory of Forensic Medicine, Hebei Medical
University, Shijiazhuang, Hebei Province, 050017, P.R. China
Françoise Sauriol: Department of Chemistry, Queen’s University,
Kingston, Ontario, K7L 3N6, Canada
Yu-Cheng Gu: Syngenta Jealott’s Hill International Research Centre,
Berkshire, RG42 6EY, UK
Results and discussion
It is widely believed that α,β-unsaturated carbonyl com-
pounds, 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.
Introduction
Michael addition reactions using the active methylene
As part of a search for anticancer agents from medicinal compounds and proper base were performed on isoalan-
herbs, we focused our attention on one such species, tolactone 1 (Scheme 1). Compound 1 was allowed to react
Inula helenium, a traditional Chinese medicinal herb [1]. with active methylene compounds 2a–g under mild con-
It grows wild in Europe, North America, and the eastern ditions, that is, stirring the reagents in absolute alcohol
part of Asia. In addition, it is distributed in northeast, at room temperature for 0.5–3 h. Some products, 3a and
northwest, and central regions of China [2]. Sesquiterpene 3b, crystallized directly from the reaction mixture. The
lactones are components of I. helenium [3]. Among them, reaction rates and product yields depend on the nature
isoalantolactone (1) is the major and important bioactive of the starting compounds. The methylene compounds
constituent. It possesses toxicity for leukocytes in vitro cul- 2a and 2b are the most reactive. In the presence of potas-
tures, significant anti-inflammatory and hepatoprotective sium hydroxide, additions of 1 to both 2a and 2b produced
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118ꢀ ꢀR.-X. Guo et al.: Structural modification of isoalantolactone
R
2a-2g
H
explanation could be that reaction yields are reduced with
the increase of steric hindrance. Besides, as electron-with-
drawing ability of compounds becomes strong, the reac-
tivity becomes higher.
H
H
H
O
O
O
O
base
r.t.
H
H
H
R
isoalantholactone (1)
3a-3g
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 chro-
matography on silica gel. In agreement with the general
rule of metal-catalyzed coupling reactions, the coupling
of the aromatic iodide with an electron-donating substitu-
ent was efficient.
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 anti-
cancer agents, cisplatin, jifitinib, and taxol, used as con-
trols. 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 encour-
aging 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.
CO2Me
CO2Et
2a, 3a: R =
2c, 3c: R =
2e, 3e: R =
2g, 3g: R =
2b, 3b: R =
2d, 3d: R =
2f, 3f: R =
CN
CN
CO2Me
CO2Et
CO2Me
CO2Et
CH2NO2
CH2NO2
Me
CO2Et
COMe
Scheme 1ꢀSynthesis of compounds 3a–g through the Michael
reaction.
I
H
O
+
O
H
H
R
4a-4d
1
Pd(OAc)2, (o-Tol)3P
Et3N, DMF, N2
110–120°C,10–15h
H
H
O
4a, 5a: R = Cl (60%)
4b, 5b: R = H (65%)
4c, 5c: R = Me (91%)
4d, 5d: R = OMe (84%)
O
H
5a-5d
R
Conclusion
Scheme 2ꢀSynthesis of compounds 5a–d through the Heck reaction.
In total, 14 isoalantolactone derivatives were synthesized
the desired products 3a and 3b in yields of 95% and 93%, by using the Michael reaction or the Heck reaction, of
respectively. However, the yields of the diethyl malonate which seven are new compounds. The reactions proceed in
adduct 3d and ethyl acetoacetate adduct 3g were low. The high yields. All products were tested in vitro for anticancer
Bel-7402
SMMC-7721
Hep G2
100
80
60
40
20
cisplapin↵Taxol↵Jifitini
0
3a
3b
3c
3d
3e
3f
3g
5c
5d
5a
5b
Figure 1ꢀCell growth inhibition properties of isoalantolactone derivatives against hepatoma cells.
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R.-X. Guo et al.: Structural modification of isoalantolactoneꢀ
ꢀ119
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.
activity on three different hepatoma cell lines: Bel-7402,
SMMC-7721, and Hep G2. Several compounds show poten-
tial antiproliferative activity against the Hep G2 cell.
Experimental
General
Ethylꢀ(3aR,4aS,8aR,9aR)-2-cyano-3-[(8a-methyl-5-methylene-
2-oxododecahydronaphtho[2,3-b](3-furyl)]propanoate
Melting points were obtained on a Laboratory Devices SGW X-4B melt-
ing 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 labora-
tory and its purity was determined by analytical HPLC (purity ꢀ> ꢀ 95%).
1
(3b)ꢀWhite solid; yield 93%; mp 193–194°C; H 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.
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 petro-
leum ether, dichloromethane, and ethyl acetate. The dichlorometh-
ane 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 separa-
tion 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 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 tem-
perature for 2 h and then quenched with deionized H2O. Afer extrac-
tion 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. Afer concentra-
tion, the residue was purified by silica gel column chromatography
eluting with petroleum ether-ethyl acetate.
Dimethylꢀ(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.
General procedure for the synthesis of 3a,b
Methyl cyanoacetate or ethyl cyanoacetate (0.2 mmol) and potas-
sium 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 precipi-
tate was crystallized from acetone.
Diethylꢀ(3aR,4aS,8aR,9aR)-2-[(8a-methyl-5-methylene-2-
oxododecahydronaphtho[2,3-b](3-furylmethyl)]malonate
1
Methylꢀ(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),
(3d)ꢀColorless liquid; yield 76%; H 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),
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120ꢀ ꢀR.-X. Guo et al.: Structural modification of isoalantolactone
2.32 (2H, m, H-13), 2.45 (1H, m, H-11), 2.75 (1H, m, H-7), 3.76 (1H, m, sieves was sealed under argon. The ampule was heated for 10–15 h at
H-16), 4.22 (4H, m, H-18, 18′), 4.45 (1H, m, H-8), 4.48 (1H, d, J ꢀ= ꢀ 1.0 120°C, then cooled and opened. The mixture was poured into water
Hz, H-15b), 4.78 (1H, d, J ꢀ= ꢀ 1.0 Hz, H-15a); 13C NMR: δ 14.0 (C-19), 14.1 and extracted with ethyl acetate. Afer concentration, the residue was
(C-19′), 17.7 (C-14), 21.1 (C-13), 22.6 (C-2), 24.0 (C-6), 34.8 (C-10), 36.7 purified by silica gel column chromatography eluting with petroleum
(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), ether-ethyl acetate.
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]+): (3aR,4aS,8aR,9aR,E)-3-(4-Chlorobenzyl)-8a-methyl-5-
m/z 415.2091, found: m/z 415.2094.
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
(3aR,4aS,8aR,9aR)-8a-Methyl-5-methylene-3-(2-nitroethyl) 0.88 (3H, s, H-14), 1.27 (1H, m, H-1b), 1.43 (1H, m, H-6b), 1.54–1.63 (4H,
decahydronaphtho[2,3-b]furan-2-one (3e)ꢀWhite solid; yield 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
1
80%; mp 113–114°C; H NMR: δ 0.81 (3H, s, H-14), 1.21 (1H, m, H-1b), Hz, H-9a), 2.36 (1H, br, d, J ꢀ= ꢀ 13.5 Hz, H-3a), 3.40 (1H, m, H-7), 4.42 (1H,
1.48 (1H, dd, J ꢀ= ꢀ 15.5, 4 Hz, 6b), 1.54–1.61 (4H, m, H-1a, 2, 2, 9b), 1.74 d, J ꢀ= ꢀ 1.0 Hz, H-15b), 4.51 (1H, td, J ꢀ= ꢀ 4.5, 1.5 Hz, H-8), 4.78 (1H, d, J ꢀ= ꢀ
(1H, m, H-6a), 1.81 (1H, m, H-5), 2.00 (1H, m, H-3b), 2.18 (1H, dd, J ꢀ= ꢀ 1.5 Hz, H-15a), 7.39 (3H, m, H-3′, 5′, 13), 7.46 (2H, d, J ꢀ= ꢀ 8.5, H-2′, 6′).
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, (3aR,4aS,8aR,9aR,E)-3-Benzyl-8a-methyl-5-methylenede
H-15b), 4.51 (1H, td, J ꢀ= ꢀ 4.5, 2.0 Hz, H-8), 4.69 (2H, m, H-16), 4.79 (1H, cahydronaphtho[2,3-b]furan-2-one (5b)ꢀWhite needles; yield
1
d, J ꢀ= ꢀ 1.5 Hz, H-15a); 13C NMR: δ 17.8 (C-14), 21.3 (C-13), 22.6 (C-2), 23.1 65%; mp 220–222°C, lit. [10] mp 202–204°C; H NMR: δ 0.89 (3H, s,
(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), H-14), 1.28 (1H, m, H-1b), 1.44 (1H, m, H-6b), 1.52–1.63 (4H, m, H-1, 2,
46.4 (C-5), 73.3 (C-16), 78.1 (C-8), 106.6 (C-15), 149.0 (C-4), 177.3 (C-12); 2, 9), 1.95 (1H, d, J ꢀ= ꢀ 12.5 Hz, H-6a), 2.03 (2H, m, H-3b, 5), 2.27 (1H,
HR-ESI-MS. Calcd for C16H23NNaO4 ([M+Na]+): m/z 316.1519, found: dd, J ꢀ= ꢀ 15.5, 1.5 Hz, H-9a), 2.36 (1H, m, H-3a), 3.44 (1H, m, H-7), 4.43
m/z 316.1520.
(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,
(3aR,4aS,8aR,9aR)-8a-Methyl-5-methylene-3-(2-nitropropyl)- J ꢀ= ꢀ 8.5, H-2′, 6′).
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), (3aR,4aS,8aR,9aR,E)-8a-Methyl-3-(4-methylbenzyl)-5-
1.47 (1H, m, H-6b), 1.53–1.59 (4H, m, H-1a, 2, 2, 9b), 1.62 (3H, dd, J ꢀ= ꢀ methylenedecahydronaphtho[2,3-b]furan-2-one
(5c)ꢀWhite
6.5, 1.5 Hz, H-17), 1.81 (1H, m, H-5), 1.97 (1H, m, H-3b), 2.06–2.27 (2H, needles; yield 91%; mp 239–240°C, lit. [10] mp 220–222°C; 1H NMR: δ
m, H-6a, 9a), 2.34 (1H, m, H-3a), 2.68 (1H, m, H-7), 2.53 (2H, m, H-13), 0.88 (3H, s, H-14), 1.28 (1H, m, H-1b), 1.43 (1H, m, H-6b), 1.52–1.63 (4H,
2.68 (1H, m, H-11), 4.44 (1H, d, J ꢀ= ꢀ 1.0 Hz, H-15b), 4.47 (1H, m, H-8), 4.77 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, s, H-15a), 5.05 (1H, m, H-16); 13C NMR: δ 17.7 (C-14), 19.2 (C-17), 22.6 (1H, dd, J ꢀ= ꢀ 15.5, 1.5 Hz, H-9a), 2.35 (1H, m, H-3a), 2.38 (3H, s, H-CH3),
(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), 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
42.1 (C-9), 44.2 (C-11), 46.4 (C-5), 78.1 (C-8), 82.1 (C-16), 106.5 (C-15), Hz, H-8), 4.77 (1H, d, J ꢀ= ꢀ 1.5 Hz, H-15a), 7.22 (2H, d, J ꢀ= ꢀ 8.0 Hz, H-3′, 5′),
149.0 (C-4), 177.6 (C-12); HR-ESI-MS. Calcd for C17H25NNaO4 ([M+Na]+): 7.42 (3H, m, H-2′, 6′, 13).
m/z 330.1676, found: m/z 330.1677.
(3aR,4aS,8aR,9aR,E)-3-(4-Methoxybenzyl)-8a-methyl-5-
methylenedecahydronaphtho[2,3-b]furan-2-one
(5d)ꢀWhite
Ethyl(3aR,4aS,8aR,9aR)-2-[(8a-methyl-5-methylene-2-
oxododecahydronaphtho[2,3-b](3-furylmethyl)]-3-oxobu-
tanoate (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.
1
solid; yield 84%; mp 202–204°C, lit. [11] mp 199–201°C; H 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 afer dissolving used for the
test. Anticancer drugs, cisplatin, taxol, jifitinib (Wako Pure Chemi-
General procedure for synthesis of 5a–d by
the Heck reaction
Compounds 5a–d were synthesized by modification of the literature cals Industries, Ltd.), and Methotrexate (F6627, Sigma, St. Louis, MO,
methods [10, 11]. Briefly, a three-necked glass ampule, filled with USA), were also dissolved in DMSO. The following human hepatoma
isoalantolactone (1, 70 mg, 0.3 mmol), aromatic iodide (0.33 mmol), carcinoma cells were used: Bel-7402, SMMC-7721, and Hep G2. Cells
Pd(OAc)2 (0.015 mmol, 5 mol%), tris(o-tolyl)phosphine (0.051 mmol, were purchased from ShangHai MEIXUAN Biological Science and
17 mol%), DMF (10 mL) and Et3N (61 mg, 0.6 mmol), and 3A molecular Technology Ltd. (China). Hep G2 cells was cultured in DMEM (GIBCO,
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ꢂꢁꢁꢁ
R.-X. Guo et al.: Structural modification of isoalantolactoneꢀ
ꢀ121
USA), containing 10% (v/v) calf serum (Biological Industries, BioInd) survival was calculated from absorbance and presented as a percent-
and antibiotics, 100 μg/mL of streptomycin (Nalgene, China) and 100 age of the surviving cells.
units/mL of penicillin G (Nalgene, China), at 37°C in a humidified
atmosphere containing 5% CO2. Instead, RPMI 1640 (GIBCO, USA)
Acknowledgments: We are grateful for financial support
and fetal calf serum (Biological Industries, BioInd), respectively,
from the National Natural Science Foundation of China
were used for culture of Bel-7402, SMMC-7721 cells.Cell viability was
(grant numbers 81302664, 81072551, 81241101). We also
wish to extend our sincere thanks for financial support
from Syngenta Ltd. (2013-Hebei Medical University-Syn-
genta-04), and JSPS KAKENHI (grant numbers 19580120,
22560112, 25450144).
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.
Afer the culture, the activity of mitochondrial succinic dehydroge-
nase 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
Received December 13, 2013; accepted January 15, 2014
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