Design, synthesis, and structure-activity relationship studies of novel millepachine derivatives as potent antiproliferative agents

Article abstract of DOI:10.1016/j.ejmech.2012.06.034

In this paper, 38 millepachine derivatives have been designed, synthesized and evaluated for their in vitro and in vivo antiproliferative activity. Among these novel derivatives, 15 displayed more potent antiproliferative activity than millepachine agains

Full text of DOI:10.1016/j.ejmech.2012.06.034

European Journal of Medicinal Chemistry 54 (2012) 793e803
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, and structureeactivity relationship studies of novel
millepachine derivatives as potent antiproliferative agents
,
,
,
Guangcheng Wang a 1, Wenshuang Wu a 1, Fei Peng a, Dong Cao a, Zhuang Yang a b, Liang Ma a, Neng Qiu a,
Haoyu Ye a, Xiaolei Han a, Jinying Chen a, Jingxiang Qiu a, Yun Sang a b, Xiaolin Liang a, Yan Ran a
,
,
,c
Aihua Peng a, Yuquan Wei a, Lijuan Chen a
,
*
a State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, Gaopeng Street, Chengdu 610041, China
b College of Chemistry of Sichuan University, Chengdu, Sichuan 610064, China
c Pharmacy College of West China of Sichuan University, Chengdu, Sichuan 610065, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper, 38 millepachine derivatives have been designed, synthesized and evaluated for their in vitro
and in vivo antiproliferative activity. Among these novel derivatives, 15 displayed more potent
antiproliferative activity than millepachine against HepG2, K562, SK-OV-3, HCT116, HT29, and SW620
Received 21 March 2012
Received in revised form
13 June 2012
Accepted 16 June 2012
Available online 23 June 2012
tumor cells (mean IC50 ¼ 0.64 vs. 2.86
mM, respectively). Furthermore, 15 could effectively inhibit tubulin
polymerization in HepG2 cells, and induce the HepG2 cell cycle arrest at the G2/M phase in a concen-
tration-dependant manner. Further studies confirmed that 15 significantly suppressed the growth of
tumor volume and exerted more potent anticancer potency than millepachine and anticancer drug
cisplatin in A549 lung xenograft tumor model.
Keywords:
Millepachine
Chalcone
Ó 2012 Elsevier Masson SAS. All rights reserved.
Tubulin
Antiproliferative activity
Cell cycle arrest
Anticancer activity
1. Introduction
important in the treatment of leukemias, lymphomas and several
types of solid tumors [10e14]. The clinical success of paclitaxel has
Microtubules, the key components of the mitotic spindle of
eukaryotic cells, are involved in a variety of fundamental cellar
processes, including regulation of motility, cell signaling formation,
maintenance of cell shape and vesicle transport [1e4]. Microtubules
are in dynamic equilibrium with tubulin dimmers. Disruption of the
dynamic equilibrium blocks thecelldivision machineryatmitosis and
leads to an increase in the number of cells in metaphase arrest [5,6].
For this reason, tubulin is one of the most common and attractive
pharmacological targets in anticancer drug development [7,8].
Natural products are very important source of antimicrotubule
agents: (i) The vinca alkaloids, vinblastine and vincristine, were
isolated from the Madagascar periwinkle Catharanthus roseus. As
the first natural products be used in clinical, vincristine is used in
the treatment of acute lymphoblastic leukemias and lymphomas,
while vinblastine is used in the treatment of bladder and breast
cancers [9]. (ii) The taxanes, such as paclitaxel and docetaxel are
spurred enormous interest in finding additional microtubule-
targeting drugs. (iii) Combretastatin A-4 (CA-4) was originally
isolated by Pettit et al. from the root bark of the South African tree
Combretum caffrum [15], has been displayed potent anti-
proliferative activity against several human cancer cell lines as well
as multiple drug-resistant cancer cell lines [16] (Fig. 1). However,
vinca alkaloids, taxanes have been used in clinical for the treatment
of cancer, their adverse effects, the complex synthesis, difficult
formulation, lack of oral availability and high cost limited these
drugs suboptimum for clinical treatment of cancer [17,18]. Hence,
the search of novel molecules that inhibit tubulin polymerization
with better activity is still in progress.
Our previous studies have shown that millepachine, which
isolated from Millettia pachycarpa for the first time in our group,
exhibited a potent apoptosis induces effects, being promising
candidates for the treatment of cancer [19]. As a part of continuing
search for novel anticancer agents [20e22], a series of novel mil-
lepachine derivatives have been designed, synthesized and evalu-
ated for antitumor activity by MTT assay in six human cancer cell
lines. Among these derivatives, compound 15 exhibited more
* Corresponding author. Tel.: þ86 28 85164063; fax: þ86 28 85164060.
E-mail address: Lijuan170@hotmail.com (L. Chen).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.034

794
G. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 793e803
Fig. 1. Structure of some antimitiotic drugs and millepahcine.
potent antiproliferative activity than millepachine against HepG2,
condensation of 3 with 37be42b was performed in the presence of
methanolic KOH solution at room temperature for 48 h and led to
the appropriate intermediates. deprotection of which through
a treatment with 2 M HCl (aq.) in methanol at 60 ꢀC for 2 h, gave the
title products (37e42), in 10e15% overall yield for the two steps.
K562, SK-OV-3, HCT116, HT29, and SW620 cells (mean IC50 ¼ 0.64
vs. 2.86
mM, respectively). Furthermore, 15 effectively inhibited
cellular tubulin polymerization, and induce the cell cycle arrest at
the G2/M phase in HepG2 cells. The results of in vivo study showed
that 15 exerted more potent inhibitory potency on the growth of
tumor volume than millepachine and cisplatin in A549 lung
xenograft tumor model. Herein, we report the antiproliferative
activity and mechanism of action of these derivatives.
3. Results and discussion
3.1. Antiproliferative effects of millepachine derivatives
2. Chemistry
The cytotoxicity of compounds 4 e 42 was evaluated against six
human tumor cell lines by MTT assay. The results were summarized
in Table 1. Among the newly derivatives, 15 displayed more potent
antiproliferative activity than millepachine against HepG2, K562,
SK-OV-3, HCT116, HT29, and SW620 tumor cells (mean IC50 ¼ 0.64
A general synthesis of millepachine derivatives is exhibited in
Scheme 1. Treatment of 1 with 3-chloro-3-methyl-1-butyne in the
presence of DBU and catalytic amounts of CuCl2$2H2O proceeded
smoothly to afforded 1,1-dimethylpropargyl ether 2 in good yield,
which, upon heating in pyridine at 120 ꢀC for overnight, underwent
Claisen rearrangement, leading to key intermediate 3.
The millepachine and its derivatives (5e36) were prepared by
ClaiseneSchimidt condensation of 3 with various commercially
available aromatic aldehydes. The reaction was carried out at room
temperature for 48 h, and the corresponding products were isolated
by crystallization or silica gel chromatography (Scheme 1, Route 1).
However, as for the hydroxyl benzaldehydes (37ae42a), the
above-mentioned KOH catalyzed ClaiseneSchimidt condensation
was not successfully accomplished because of the special chemical
environment of the phenolic hydroxyl group [23,24]. Therefore, as
shown in Scheme 1 (Route 2), the hydroxyl benzaldehydes
(37ae42a) were initially protected using methoxy-methylene
chloride (MOMeCl) and NaH as base in dry DMF to afford the
corresponding products (37be42b) in 40e95% yield. The
vs. 2.86 mM, respectively). In addition, compounds (12 and 13) also
exhibited stronger cytotoxicity than millepachine.
With respect to the study of structureeactivity relationship
(SAR), the importance of the substitution of the right phenyl ring
was preferentially discussed in this study. The introduction of
electron-withdrawing groups (F, Cl, Br, NO2, CF3, Ph) to the right
phenyl ring has been proven to be detrimental to anti-tumor
activity. It is interesting to point out that 12 and 15 containing
the dialkylamine group (dimethyl in 12, and diethyl in 15) at para-
position of the right phenyl ring significantly improved the anti-
proliferative activity, especially with the IC50 value ranging from
0.55 to 0.79 mM in 15 (Table 1). Additionally, the introduction of
electro-donating dimethylamino (12), diethylamino (15) and pyr-
rolydinyl group (17) was more favorable to the inhibitory activity
against the six tumor cells than the electro-withdrawing diphe-
nylamine group (16) at para-position.

G. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 793e803
795
Scheme 1. Reagents and conditions: (a) DBU, CuCl2$2H2O, 3-chloro-3-methyl-1-butyne, CH3CN, 0 ꢀC, 5 h; (b) pyridine 120 ꢀC, 12 h; (c) KOH, methanol, 48 h, room temperature; (d)
NaH, MOMCl, DMF, 2 h, room temperature; (e) (i) KOH, methanol, 48 h, room temperature; (ii) 2 M HCl (aq.), methanol, 60 ꢀC, 2 h.
Although the methoxyl and hydroxyl groups were very prevalent
in the natural products, the presence of methoxyl group (5, 6, 8, 25,
and 36) and hydroxyl group (37e42) substituted in the right phenyl
ring failed to effectively improve the anti-tumor activity compared
to millepachine in our study. The unsubstituted phenyl ring of 23 is
immunofluorescence staining of tubulin using monoclonal antibody
to -tubulin. As a result, the microtubule polymerization and spindle
formation were significantly suppressed, suggesting that tubulin
a
might be an effective target for its anti-tumor activity (Fig. 2).
also showed inactive (IC50 > 10.0 mM was defined as inactive).
3.3. Flow cytometric assay
The replacement of the right phenyl ring in millepachine with
various heterocycles (quinolin-2-yl in 19, 6-methoxylpyridin-3-yl in
24, thiophen-3-yl in 27, furan-2-yl in 28, naphthalen-1-yl in 29,
pyridin-2-yl in 30, and thiophen-2-yl in 33) resulted in a remarkable
decrease of cytotoxicity, whereas the substitution of naphthalen-2-
yl group (13) enhanced the anti-proliferative activity. These results
indicates the patterns of substitution in the right aryl system is
closely related to the biological activity of this class of compounds. In
summary, the information of SAR provided us a guideline to improve
the inhibitory activity in the future structural modification.
Because the anti-mitotic drugs induced cell cycle arrest at G2/M
phase in various cancer cell lines and led to an increment of the
relative peak in the DNA histogram [25,26], the inhibitory effect on
the cell cycle of HepG2 cells were performed. Millepachine and 15
were tested at 1.25, 2.50, and 5.0 mM, respectively, and the results
were illustrated in Fig. 3. They induced the arrest of cell cycle at G2/M
phase, raising the G2/M peak from 32.43% (control) to 79.65% (mil-
lepachine), and 78.35% (15) at a concentration of 2.5 mM, which were
inaccordancewith theobserved inhibition of tubulinpolymerization.
3.2. Immunofluorenscence staining
3.4. In vivo antitumor activity
To get an insight into the mechanism of action of 15, we inves-
tigated the potency to inhibit tubulin polymerization by immuno-
fluorescence staining. We observed the morphology of microtubules
and spindles in HepG2 cells treated with millepachine and 15 at
To evaluate in vivo antitumor activity of 15, human lung
adenocarcinoma xenografts were established by subcutaneous
injection of A549 cells in right gluteal region of nude mice. Twenty-
four mice were randomly assigned to four groups when the tumor
volume of A549 xenograft reached a size of 100 mm3. The mice in
concentration of 1.0
mM. After incubation for 24 h, the

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G. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 793e803
Table 1
development of novel antitumor agents. In the future research we
will be exploring for a clear structureeactivity relationship of this
type of compound and discovering more potent anti-tumor agents.
The antiproliferative activities of test compounds against six cancer cell lines.
Compds
IC50 (m
M)a
HepG2
K562
SK-OV-3
HCT116
HT29
SW620
5. Experimental section
Millepachine
1.51
4.56
2.36
4.66
3.29
0.76
5
6
7
8
>10.0
>10.0
7.56
>10.0
>10.0
>10.0
>10.0
0.73
0.21
>10.0
0.59
>10.0
4.74
>10.0
4.28
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
3.50
3.67
>10.0
0.79
>10.0
6.18
>10.0
3.02
9.36
9.70
4.88
>10.0
7.65
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
1.87
4.48
>10.0
0.55
>10.0
8.36
6.6.0
>10.0
9.15
6.57
>10.0
>10.0
>10.0
>10.0
0.57
2.14
>10.0
0.67
>10.0
7.15
>10.0
2.25
5.1. Chemistry
>10.0
>10.0
>10.0
>10.0
>10.0
1.92
5.82
>10.0
0.62
>10.0
8.59
>10.0
4.53
8.88
8.05
>10.0
>10.0
>10.0
>10.0
9.71
>10.0
>10.0
>10.0
6.63
>10.0
>10.0
>10.0
9.44
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
5.24
>10.0
>10.0
>10.0
>10.0
2.75
7.65
>10.0
0.61
>10.0
7.37
7.20
2.35
3.42
All starting materials and reagents were purchased from
commercial suppliers. TLC was performed on 0.20 mm Silica Gel 60
F254 plates (Qingdao Ocean Chemical Factory, Shandong, China).
Nuclear magnetic resonance spectra (NMR) were recorded at
400 MHz on a Varian spectrometer (Varian, Palo Alto, CA) and
reported in parts per million. Mass spectra (MS) were measured by
Q-TOF Premier mass spectrometer (Micromass, Manchester, UK).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
5.1.1. 1-(4-Methoxy-2-((2-methylbut-3-yn-2-yl)oxy)phenyl)
ethanone (2)
3.79
3.73
4.26
2.61
2.83
7.77
5.87
3.52
4.09
To a solution of 1 (1.66 g, 10 mmol) in CH3CN (100 mL) at 0 ꢀC
was added DBU (1.5 mL, 15 mmol), CuCl2$2H2O (5 mg, 0.3% mol),
and 3-chloro-3-methyl-1-butyne (1.53 g, 15 mmol). The mixture
was stirred at 0 ꢀC for 5 h and monitored by TLC. After completed,
1 N HCl (aq.) was added to adjust pH ¼ 2. After removing the
solvent under reduced pressure, the residue was poured into water
(50 mL) and the white precipitate was collected by filtration to
9.72
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
5.76
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
6.64
>10.0
>10.0
>10.0
>10.0
7.85
>10.0
>10.0
>10.0
5.40
>10.0
>10.0
>10.0
9.03
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
5.51
>10.0
>10.0
>10.0
>10.0
9.33
>10.0
>10.0
>10.0
8.39
>10.0
>10.0
>10.0
9.94
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
6.27
>10.0
>10.0
>10.0
>10.0
9.05
>10.0
>10.0
>10.0
2.12
>10.0
>10.0
>10.0
7.58
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
4.97
>10.0
>10.0
>10.0
>10.0
7.87
>10.0
>10.0
>10.0
5.00
>10.0
>10.0
>10.0
7.70
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
4.23
obtain 2 (1.97 g, 85%). 1H NMR (CDCl3, 400 MHz)
d: 1.75 (s, 6H), 2.58
(s, 3H), 2.69 (s, 1H), 3.84 (s, 3H), 6.58 (dd, 1H, J ¼ 8.8 Hz, 2.0 Hz), 7.19
(d,1H, J ¼ 2.0 Hz), 7.75 (d,1H, J ¼ 8.8 Hz); 13C NMR (CDCl3,100 MHz)
d
: 29.6, 31.9, 31.9, 55.5, 73.1, 75.1, 85.3, 104.9, 107.4, 124.6, 132.1,
156.9, 163.3, 198.5; MS (ESI, m/z): 255.05 [M þ Na]þ.
5.1.2. 1-(5-Methoxy-2,2-dimethyl-2H-chromen-8-yl)ethanone (3)
A suspension of 2 (2.32 g, 10 mmol) in pyridine (50 mL) was
stirred at 120 ꢀC for 12 h. After cooling to room temperature, the
mixture concentrated under reduced pressure to give 3 as black oil,
which was used in the next step without further purification. 1H
NMR (CDCl3, 400 MHz) d: 1.49 (s, 6H), 2.62 (s, 3H), 3.87 (s, 3H), 5.61
a
The cytotoxic activities of the compounds against six cancer cell lines were
determined using the MTT assay. The results were expressed as the IC50, date are the
mean of three independent experiments.
(d, 1H, J ¼ 10.0 Hz), 6.47 (d, 1H, J ¼ 8.8 Hz), 6.66 (d, 1H, J ¼ 10.0 Hz),
7.73 (d, 1H, J ¼ 8.8 Hz); MS (ESI, m/z): 255.14 [M þ Na]þ.
5.1.3. General procedure A for the synthesis of 4e36
tested group received tail intravenously 2 day intervals for 18 days
at a dosage of 20 mg/kg of millepachine, or 15 dissolved in 0.2 mL of
physiological saline. The positive group was injected with cisplatin
per a week at a dose of 5 mg/kg, and the control group received
injection of physiological saline alone following the time schedule
for the test groups.
As shown in Fig. 4, compound 15 caused a remarkable reduction
in tumor growth (84.1%) as compared with administration of
vehicle only and better than parent compound millepachine and
positive control anticancer drug cisplatin, which caused a 69.4% and
62.3% of reduction respectively.
A solution of 50% KOH (3 mL, aq.) was added dropwise to
a stirred solution of 3 (232 mg, 1.0 mmol) and substituted aromatic
aldehydes (1.0 mmol) in methanol (10 mL) at 0 ꢀC. The resulting
mixture was stirred at room temperature for 48 h under N2
atmosphere. After the end of reaction, the mixture was poured into
ice water and neutralized with 2 N HCl. The precipitated was
filtered, washed with water, dried, and crystallized from ethanol to
afford a solid. If no solid formed, the mixture was extracted with
ethyl acetate and purified by silica gel column chromatography.
5.1.3.1. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(4-met
During the whole therapeutic period, no significant weight
changes were observed in the millepachine and 15 treated groups,
however, in the cisplatin group the weight of animal decrease signif-
icantly (Fig. 4), stating that 15 is potentially less toxic than cisplatin.
hoxyphenyl)-pr-op-2-en-1-one (4). The title compound
prepared according to the general procedure A. Recrystallization of
4 was
the crude compound from EtOH afforded 4 (34.4%) as a yellow solid.
1H NMR (CDCl3, 400 MHz)
d: 1.51 (s, 6H), 3.85 (s, 3H), 3.88 (s, 3H), 5.62
(d,1H, J ¼ 10.0 Hz), 6.51 (d,1H, J ¼ 8.8 Hz), 6.69(d,1H, J ¼ 10.0 Hz), 6.91
(d, 2H, J ¼ 8.8 Hz), 7.54 (d, 2H, J ¼ 8.8 Hz), 7.58 (d,1H, J ¼ 15.6 Hz), 7.65
(d,1H, J ¼ 15.6 Hz), 7.70 (d,1H, J ¼ 8.8 Hz); 13C NMR (CDCl3,100 MHz)
4. Conclusions
This paper described the synthesis of a series of novel mil-
lepachine derivatives and the evaluation of their cytotoxic activity
and tubulin polymerization inhibitor activity. Among the tests
derivatives, compound 15 showed a potent anti-tumor activity
which as a potential tubulin polymerization inhibitor. This study
d: 28.0, 28.0, 55.4, 55.8, 77.3, 103.4, 110.4, 114.4, 114.4, 116.8, 121.9,
125.3, 128.4, 128.7, 129.8, 129.8, 131.7, 141.3, 153.6, 158.3, 161.1, 190.1;
MS (ESI, m/z): 351.17 [M þ H]þ; HPLC purity ¼ 99.4%.
5.1.3.2. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(3-met
presented here provide
a
new class structure type for the
hoxyphenyl)prop-2-en-1-one (5). The title compound 5 was

G. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 793e803
797
Fig. 2. Effect of compounds millepachine and 15 on the microtubule network of HepG2 cancer cells. Untreated (control) and cells treated with compound millepachine and 15 at
concentration of 1.0 M for 24 h, were fixed in methanol and stained with -tubulin and counterstained with 4,6-diamidino-2-phenylindole (DAPI). Microtubules and unassembled
m
a
tubulin are shown in green. DNA, stained with DAPI, is shown in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)
prepared according to the general procedure A. Purification of the
crude product by silica gel chromatography afforded 5 (50.7%) as
of the crude compound from EtOH afforded 7 (41.9%) as a yellow
solid. 1H NMR (CDCl3, 400 MHz)
: 1.51 (s, 6H), 3.84 (s, 3H), 3.89 (s,
d
a yellow oil. 1H NMR (CDCl3, 400 MHz)
d
: 1.51 (s, 6H), 3.86 (s, 3H),
3H), 5.62 (d, 1H, J ¼ 10.0 Hz), 6.51 (d, 1H, J ¼ 8.8 Hz), 6.64e6.70 (m,
3.88 (s, 3H), 5.62 (d, 1H, J ¼ 10.0 Hz), 6.51 (d, 1H, J ¼ 8.8 Hz), 6.89 (d,
1H, J ¼ 10.0 Hz), 6.92 (dd, 1H, J ¼ 8.0 Hz, 2.0 Hz), 7.13 (s, 1H), 7.19 (d,
1H, J ¼ 8.0 Hz), 7.31 (t, 1H, J ¼ 8.0 Hz), 7.63 (d, 1H, J ¼ 15.6 Hz), 7.71
(d, 1H, J ¼ 15.6 Hz), 7.72 (d, 1H, J ¼ 8.0 Hz); 13C NMR (CDCl3,
2H), 6.72 (dd,1H, J ¼ 8.8 Hz, 2.4 Hz), 7.50 (t,1H, J ¼ 8.8 Hz), 7.71e7.75
(m, 2H), 7.77 (d, 1H, J ¼ 15.6 Hz); 13C NMR (CDCl3, 100 MHz)
d: 27.9,
27.9, 55.7, 55.8, 77.4,102.0,102.2,103.5,110.5,110.7,116.7,121.6,127.5,
128.8, 130.9,131.8, 134.5, 153.7, 158.4,162.0, 164.0, 189.9; MS (ESI, m/
z): 369.12 [M þ H]þ; HPLC purity ¼ 98.5%.
100 MHz) d: 28.0, 28.0, 55.2, 55.8, 77.4, 103.6, 110.5, 112.8, 115.9,
116.8, 120.9, 121.6, 127.7, 128.6, 129.9, 131.8, 137.1, 141.2, 153.7, 158.6,
160.0, 189.9; MS (ESI, m/z): 351.19 [M þ H]þ; HPLC purity ¼ 99.2%.
5.1.3.5. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (8). The title compound 8 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 8 (41.9%) as a yellow solid.
5.1.3.3. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (6). The title compound 6 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 6 (47.4%) as a yellow solid.
1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.89 (s, 9H), 3.94 (s, 3H),
5.61 (d, 1H, J ¼ 10.0 Hz), 6.50 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H,
1H NMR (CDCl3, 400 MHz)
d: 1.52 (s, 6H), 3.90 (s, 12H), 5.63 (d, 1H,
J ¼ 10.0 Hz), 7.14 (s, 1H), 7.59 (d, 1H, J ¼ 16.0 Hz), 7.69 (d, 1H,
J ¼ 10.0 Hz), 6.52 (d, 1H, J ¼ 8.8 Hz), 6.70 (d, 1H, J ¼ 10.0 Hz), 6.85 (s,
J ¼ 8.8 Hz), 8.03 (d, 1H, J ¼ 16.0 Hz); 13C NMR (CDCl3, 100 MHz)
d:
2H), 7.59 (d, 1H, J ¼ 15.6 Hz), 7.66 (d, 1H, J ¼ 15.6 Hz), 7.73 (d, 1H,
27.9, 27.9, 55.8, 56.1, 56.3, 56.5, 77.4, 97.1, 103.4, 110.2, 110.4, 116.2,
116.9,122.2,125.2, 128.6,131.7,136.5,143.2,151.9,153.3,154.2, 158.1,
190.2; MS (ESI, m/z): 411.24 [M þ H]þ; HPLC purity ¼ 98.7%.
J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
d: 28.0, 28.0, 55.8, 56.1, 56.1,
61.0, 77.4, 103.6, 105.2, 105.2, 110.4, 116.9, 121.6, 126.8, 128.5, 131.2,
131.8, 139.8, 141.3, 153.4, 153.4, 153.6, 158.5, 189.6; MS (ESI, m/z):
411.25 [M þ H]þ; HPLC purity ¼ 97.3%.
5.1.3.6. (E)-3-(4-Fluorophenyl)-1-(5-methoxy-2,2-dimethyl-2H-chro
men-8-yl)prop-2-en-1-one (9). The title compound 9 was prepared
according to the general procedure A. Recrystallization of the crude
compound from EtOH afforded 9 (44.4%) as a yellow solid. 1H NMR
5.1.3.4. (E)-3-(2-fluoro-4-methoxyphenyl)-1-(5-methoxy-2,2-dimet
hyl-2H-chromen-8-yl)prop-2-en-1-one (7). The title compound 7
was prepared according to the general procedure A. Recrystallization
(CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.89 (s, 3H), 5.63 (d, 1H,

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G. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 793e803
Fig. 3. Millepachine and 15 affected the cell cycle distribution in HepG2 cancer cells. HepG2 cancer cells were treatment with compounds millepachine and 15 at 1.25
mM, 2.50 mM
or 5.00 M concentration for 24 h, respectively and control group indicates the untreated cells. After treatment, the cells were collected and the DNA content was analyzed by flow
m
cytometer (TASC240, USA).
J ¼ 10.0 Hz), 6.51 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H, J ¼ 10.0 Hz), 7.09 (t,
2H, J ¼ 8.8 Hz), 7.56e7.60 (m, 2H), 7.61 (s, 2H), 7.71 (d, 1H,
7.06e7.09 (m, 1H), 7.26e7.30 (m, 1H), 7.35e7.38 (m, 2H), 7.60 (d, 1H,
J ¼ 16.0 Hz), 7.71 (d, 1H, J ¼ 16.0 Hz), 7.72 (d, 1H, J ¼ 8.8 Hz); 13C
J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
d
: 28.1, 28.1, 55.8, 77.4, 103.6,
NMR (CDCl3, 100 MHz) d: 28.1, 28.1, 55.8, 77.4, 103.6, 110.5, 114.0,
110.5, 115.9, 116.1, 116.8, 121.6, 127.2, 128.6, 129.9, 129.9, 131.8, 140.0,
153.7, 158.5, 162.4, 164.9, 189.7; MS (ESI, m/z): 339.16 [M þ H]þ;
HPLC purity ¼ 99.0%.
116.5, 116.7, 121.3, 124.2, 128.7, 130.4, 131.8, 138.0, 139.6, 153.9, 158.7,
161.8, 164.3, 189.5; MS (ESI, m/z): 339.14 [M þ H]þ; HPLC
purity ¼ 98.9%.
5.1.3.7. (E)-3-(3-Fluorophenyl)-1-(5-methoxy-2,2-dimethyl-2H-chro
men-8-yl)prop-2-en-1-one (10). The title compound 10 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 10 (41.0%) as a yellow
5.1.3.8. (E)-3-(4-Bromophenyl)-1-(5-methoxy-2,2-dimethyl-2H-chro
men-8-yl)prop-2-en-1-one (11). The title compound 11 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 11 (46.9%) as a yellow
solid. 1H NMR (CDCl3, 400 MHz)
d: 1.51 (s, 6H), 3.89 (s, 3H), 5.63 (d,
solid. 1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.89 (s, 3H), 5.63 (d,
1H, J ¼ 10.0 Hz), 6.52 (d, 1H, J ¼ 8.8 Hz), 6.68 (d, 1H, J ¼ 10.0 Hz),
1H, J ¼ 10.0 Hz), 6.52 (d,1H, J ¼ 8.8 Hz), 6.68 (d,1H, J ¼ 10.0 Hz), 7.44

G. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 793e803
799
Fig. 4. Inhibition of human xenograft growth in vivo by compound millepachine and 15. (A) the curves of inhibition of tumors. A549 tumor-bearing nude mice were administered
vehicle alone or 20 mg/kg of millepachine, or 15 intravenous 2 day intervals for 18 days, the positive group received injections every week of cisplatin at a dose of 5 mg/kg. Each
group contained 6 mice; bars, ꢁ SD. (B) Effects of 15 and millepachine on mice body weight, bars, ꢁ SD. (C) the picture of the stripping tumor from mice. (D) Effects of 15 and
millepachine on tumor weight. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, significantly different compared with control by t test.
(d, 2H, J ¼ 8.8 Hz), 7.52 (d, 2H, J ¼ 8.8 Hz), 7.58 (d, 1H, J ¼ 16.0 Hz),
7.70e7.74 (m, 2H); 13C NMR (CDCl3, 100 MHz)
: 28.1, 28.1, 55.8,
the crude compound from EtOH afforded 14 (21.5%) as a yellow
solid. 1H NMR (CDCl3, 400 MHz)
: 1.53 (s, 6H), 3.90 (s, 3H), 5.64 (d,
d
d
77.4, 103.6, 110.5, 116.8, 121.4, 123.9, 128.0, 128.6, 129.5, 129.5, 131.8,
131.8, 132.1, 134.7, 139.7, 153.8, 158.6, 189.6; MS (ESI, m/z): 401.05
[M þ H]þ; HPLC purity ¼ 98.7%.
1H, J ¼ 10.0 Hz), 6.53 (d,1H, J ¼ 8.8 Hz), 6.70 (d,1H, J ¼ 10.0 Hz), 7.38
(d, 1H, J ¼ 7.6 Hz), 7.47 (t, 2H, J ¼ 7.6 Hz), 7.63e7.80 (m, 9H); 13C
NMR (CDCl3, 100 MHz) d: 28.1, 28.1, 55.8, 77.3, 103.3, 103.6, 110.5,
116.8, 121.7, 127.0, 127.0, 127.3, 127.6, 127.6, 127.8, 128.7, 128.7, 128.9,
128.9, 131.2, 131.8, 134.7, 140.3, 140.9, 153.7, 158.5, 189.9; MS (ESI, m/
z): 397.20 [M þ H]þ; HPLC purity ¼ 99.0%.
5.1.3.9. (E)-3-(4-(Dimethylamino)phenyl)-1-(5-methoxy-2,2-dimethyl-
2H-chromen-8-yl)prop-2-en-1-one (12). The title compound 12 was
prepared according to the general procedure A. Purification of the
crude product by silica gel chromatography afforded 12 (14.3%) as an
5.1.3.12. (E)-3-(4-(Diethylamino)phenyl)-1-(5-methoxy-2,2-dimethyl-
2H-chromen-8-yl)prop-2-en-1-one (15). The title compound 15 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 15 (49.0%) as a yellow
orange solid. 1H NMR (CDCl3, 400 MHz)
d: 1.51 (s, 6H), 3.03 (s, 6H),
3.89 (s, 3H), 5.62 (d, 1H, J ¼ 10.0 Hz), 6.50 (d, 1H, J ¼ 8.8 Hz),
6.68e6.71 (m, 3H), 7.50e7.55 (m, 3H), 7.66 (d,1H, J ¼ 15.6 Hz), 7.68 (d,
1H, J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
d
: 28.0, 28.0, 40.2, 40.2,
solid.1H NMR (CDCl3, 400 MHz)
d
: 1.20 (t, 6H, J ¼ 6.8 Hz),1.51 (s, 6H),
55.8, 77.3, 103.3, 110.4, 112.0, 112.0, 116.8, 122.3, 122.6, 123.5, 128.7,
130.0, 130.0, 131.5, 142.7, 151.6, 153.3, 158.0, 190.2; MS (ESI, m/z):
364.25 [M þ H]þ; HPLC purity ¼ 99.5%.
3.40 (d, 4H, J ¼ 6.8 Hz), 3.88 (s, 3H), 5.62 (d, 1H, J ¼ 10.0 Hz), 6.50 (d,
1H, J ¼ 8.8 Hz), 6.65 (d, 2H, J ¼ 6.8 Hz), 6.68 (d, 1H, J ¼ 10.0 Hz),
7.48e7.52 (m, 3H), 7.65e7.70 (m, 2H); 13C NMR (CDCl3, 100 MHz)
d:
12.6, 12.6, 28.0, 28.0, 44.5, 44.5, 55.7, 77.2, 103.3, 103.3, 110.4, 111.3,
116.8, 121.9, 122.4, 122.5, 128.6, 130.3, 130.3, 131.5, 142.9,
149.2, 153.2, 157.9, 190.2; MS (ESI, m/z): 392.28 [M þ H]þ; HPLC
purity ¼ 96.9%.
5.1.3.10. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(naph-
thalen-2-yl)prop-2-en-1-one (13). The title compound 13 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 13 (48.3%) as yellow
crystals. 1H NMR (CDCl3, 400 MHz)
d: 1.54 (s, 6H), 3.90 (s, 3H), 5.64
5.1.3.13. (E)-3-(4-(Diphenylamino)phenyl)-1-(5-methoxy-2,2-dimethyl-
2H-chromen-8-yl)prop-2-en-1-one (16). The title compound 16 was
prepared according to the general procedure A. Purification of the
crude product by silica gel chromatography afforded 16 (13.6%) as
(d, 1H, J ¼ 10.0 Hz), 6.53 (d, 1H, J ¼ 8.8 Hz), 6.70 (d, 1H, J ¼ 10.0 Hz),
7.50e7.52 (m, 2H), 7.75 (d, 2H, J ¼ 8.8 Hz), 7.83e7.88 (m, 5H), 8.00 (s,
1H); 13C NMR (CDCl3, 100 MHz)
d: 28.1, 28.1, 55.8, 77.2, 103.5, 110.5,
116.8, 121.7, 123.6, 126.6, 127.0, 127.8, 127.8, 128.5, 128.6, 128.7, 130.1,
131.8, 133.2, 133.5, 134.1, 141.4, 153.8, 158.5, 189.9; MS (ESI, m/z):
371.19 [M þ H]þ; HPLC purity ¼ 99.1%.
a yellow solid. 1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.88 (s, 3H),
5.61 (d, 1H, J ¼ 10.0 Hz), 6.53 (d, 1H, J ¼ 8.8 Hz), 6.68 (d, 1H,
J ¼ 10.0 Hz), 7.01 (d, 2H, J ¼ 8.8 Hz), 7.07e7.11 (m, 2H), 7.13e7.15 (m,
4H), 7.28e7.32 (m, 4H), 7.44 (d, 2H, J ¼ 8.8 Hz), 7.59 (d,1H, J ¼ 15.2 Hz),
7.65 (d, 1H, J ¼ 15.2 Hz), 7.71 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3,
5.1.3.11. (E)-3-([1,10-Biphenyl]-4-yl)-1-(5-methoxy-2,2-dimethyl-2H-
chromen-8-yl)prop-2-en-1-one (14). The title compound 14 was
prepared according to the general procedure A. Recrystallization of
100 MHz) d: 28.1, 28.1, 55.8, 77.3, 103.5, 110.4, 116.8, 121.9, 121.9, 123.9,
123.9, 124.9, 125.4, 125.4, 125.4, 125.4, 126.3, 128.6, 128.8, 129.4, 129.5,

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G. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 793e803
129.5, 129.5, 129.5, 129.7, 131.7, 141.4, 147.0, 147.0, 149.6, 153.5, 158.3,
5.1.3.19. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(4-
(trifluoromethyl)phenyl)prop-2-en-1-one (22). The title compound
22 was prepared according to the general procedure A. Recrystal-
lization of the crude compound from EtOH afforded 22 (51.8%) as
189.9; MS (ESI, m/z): 488.26 [M þ H]þ; HPLC purity ¼ 99.1%.
5.1.3.14. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(4-(pyr-
rolidin-1-yl)phenyl)prop-2-en-1-one (17). The title compound 17 was
prepared according to the general procedure A. Purification of the
crude product by silica gel chromatography afforded 17 (8.4%) as an
a yellow solid. 1H NMR (CDCl3, 400 MHz)
d: 1.51 (s, 6H), 3.90 (s, 3H),
5.64 (d, 1H, J ¼ 10.0 Hz), 6.53 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H,
J ¼ 10.0 Hz), 7.65e7.71 (m, 5H), 7.74 (d, 1H, J ¼ 8.8 Hz), 7.78 (d, 1H,
orange solid. 1H NMR (CDCl3, 400 MHz)
d: 1.51 (s, 6H), 2.18 (s, 2H),
J ¼ 15.6 Hz); 13C NMR (CDCl3, 100 MHz)
d: 28.1, 28.1, 55.8, 77.4,
3.40 (s, 2H), 3.88 (s, 3H), 5.62 (d, 1H, J ¼ 10.0 Hz), 6.50 (d, 1H,
103.7, 110.5, 116.7, 121.2, 125.8, 125.8, 125.9, 125.9, 128.1, 128.1, 128.6,
129.7, 131.9, 139.0, 139.2, 153.9, 158.8, 189.4; MS (ESI, m/z): 389.30
[M þ H]þ; HPLC purity ¼ 99.3%.
J ¼ 8.8 Hz), 6.68e6.71 (m, 3H), 7.51e7.55 (m, 3H), 7.65e7.71 (m, 2H);
13C NMR (CDCl3, 100 MHz)
d: 25.5, 25.5, 28.0, 28.0, 47.5, 47.5, 55.7,
77.4, 103.3, 110.4, 111.7, 111.7, 116.8, 121.9, 122.5, 122.8, 128.7, 130.2,
130.2, 131.5, 143.2, 149.2, 153.2, 157.9, 190.3; MS (ESI, m/z): 390.24
[M þ H]þ; HPLC purity ¼ 99.3%.
5.1.3.20. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-pheny
lprop-2-en-1-one (23). The title compound 23 was prepared
according to the general procedure A. Recrystallization of the crude
compound from EtOH afforded 23 (38.1%) as a yellow solid. 1H NMR
5.1.3.15. (E)-3-(4-(1H-imidazol-1-yl)phenyl)-1-(5-methoxy-2,2-dimethyl-
2H-chromen-8-yl)prop-2-en-1-one (18). The title compound 18 was
prepared according to the general procedure A. Recrystallization of the
crude compound from EtOH afforded 18 (43.4%) as a yellow solid. 1H
(CDCl3, 400 MHz)
d: 1.51 (s, 6H), 3.89 (s, 3H), 5.63 (d, 1H,
J ¼ 10.0 Hz), 6.52 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H, J ¼ 10.0 Hz),
7.37e7.42 (m, 3H), 7.60e7.67 (m, 2H), 7.71e7.76 (m, 3H); 13C NMR
NMR (CDCl3, 400 MHz)
d: 1.51 (s, 6H), 3.90 (s, 3H), 5.64 (d, 1H,
(CDCl3, 100 MHz) d: 28.0, 28.0, 55.7, 77.4, 103.5, 110.4, 116.7, 121.5,
J ¼ 10.0 Hz), 6.53 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H, J ¼ 10.0 Hz), 7.46e7.50
127.3, 128.1, 128.1, 128.6, 128.8, 128.8, 129.8, 131.7, 135.6, 141.2, 153.7,
(m, 2H), 7.59 (d, 2H, J ¼ 8.0 Hz), 7.65 (d, 1H, J ¼ 16.0 Hz), 7.34e7.81 (m,
158.4, 189.8; MS (ESI, m/z): 321.11 [M þ H]þ; HPLC purity ¼ 98.7%.
4H), 9.07 (s, 1H); 13C NMR (d6-DMSO, 100 MHz)
d: 28.1, 28.1, 56.5, 77.4,
104.7, 110.3, 116.4, 119.6, 121.3, 121.9, 121.9, 126.0, 128.5, 129.8, 130.0,
130.0, 131.6, 135.1, 135.5, 137.3, 139.8, 153.3, 158.5, 189.3; MS (ESI, m/z):
388.66 [M þ H]þ; HPLC purity ¼ 98.6%.
5.1.3.21. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(6-
methoxypyridin-3-yl)prop-2-en-1-one (24). The title compound 24
was prepared according to the general procedure A. Recrystalliza-
tion of the crude compound from EtOH afforded 24 (36.8%) as
5.1.3.16. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(qui-
nolin-2-yl)prop-2-en-1-one (19). The title compound 19 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 19 (41.3%) as a yellow
a yellow solid. 1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.89 (s, 3H),
3. 98 (s, 3H), 5.63 (d, 1H, J ¼ 10.0 Hz), 6.52 (d, 1H, J ¼ 8.8 Hz), 6.69 (d,
1H, J ¼ 10.0 Hz), 6.78 (d, 1H, J ¼ 8.8 Hz), 7.64 (s, 2H), 7.71 (d, 1H,
J ¼ 8.8 Hz), 7.81e7.84 (m, 1H), 8.37 (s, 1H); 13C NMR (CDCl3,
solid. 1H NMR (CDCl3, 400 MHz)
d
: 1.57 (s, 6H), 3.90 (s, 3H), 5.64 (d,
100 MHz) d: 28.1, 28.1, 53.8, 55.8, 77.4, 103.5, 110.4, 111.5, 116.8,
1H, J ¼ 10.0 Hz), 6.53 (d,1H, J ¼ 8.8 Hz), 6.69 (d,1H, J ¼ 10.0 Hz), 7.56
(t, 1H, J ¼ 7.6 Hz), 7.66 (d, 1H, J ¼ 8.8 Hz), 7.73e7.86 (m, 4H), 8.14 (s,
1H), 8.20 (d, 1H, J ¼ 8.0 Hz), 8.33 (d, 1H, J ¼ 15.6 Hz); 13C NMR
121.5, 125.0, 126.4, 128.6, 131.8, 136.5, 137.7, 148.3, 153.6, 163.5,
165.0, 189.5; MS (ESI, m/z): 352.19 [M þ H]þ; HPLC purity ¼ 98.6%.
(CDCl3, 100 MHz)
d
: 28.0, 28.0, 55.8, 77.3, 103.6, 110.6, 116.7, 120.9,
5.1.3.22. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(2,3,
4-trimethoxyphenyl)prop-2-en-1-one (25). The title compound 25
was prepared according to the general procedure A. Recrystalliza-
tion of the crude compound from EtOH afforded 25 (46.0%) as
121.4, 127.0, 127.6, 128.0, 128.9, 129.8, 129.9, 131.7, 132.8, 136.6,
139.9, 148.4, 154.2, 154.4, 158.7, 189.9; MS (ESI, m/z): 372.19
[M þ H]þ; HPLC purity ¼ 98.9%.
a yellow solid. 1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.88 (s, 3H),
5.1.3.17. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(4-
nitrophenyl)prop-2-en-1-one (20). The title compound 20 was
prepared according to the general procedure A. Purification of the
crude product by silica gel chromatography afforded 20 (11.0%) as
3.89 (s, 3H), 3.92 (s, 3H), 3.94 (s, 3H), 5.62 (d, 1H, J ¼ 10.0 Hz), 6.51
(d, 1H, J ¼ 8.8 Hz), 6.68 (d, 1H, J ¼ 10.0 Hz), 6.70 (d, 1H, J ¼ 8.4 Hz),
7.36 (d, 1H, J ¼ 8.8 Hz), 7.66 (d, 1H, J ¼ 15.6 Hz), 7.70 (d, 1H,
J ¼ 8.4 Hz), 7.91 (d, 1H, J ¼ 15.6 Hz); 13C NMR (CDCl3, 100 MHz)
d:
a yellow solid. 1H NMR (CDCl3, 400 MHz)
d
: 1.51 (s, 6H), 3.91 (s, 3H),
28.0, 28.0, 55.8, 56.0, 60.9, 61.5, 77.4, 103.4, 107.6, 110.5, 116.8, 122.0,
122.8, 122.8, 126.5, 128.7, 131.7, 136.4, 142.5, 153.5, 153.7, 155.2,
158.2, 190.2; MS (ESI, m/z): 411.26 [M þ H]þ; HPLC purity ¼ 97.7%.
5.64 (d, 1H, J ¼ 10.0 Hz), 6.54 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H,
J ¼ 10.0 Hz), 7.65 (d, 1H, J ¼ 15.6 Hz), 7.72 (d, 2H, J ¼ 8.8 Hz), 7.75 (d,
1H, J ¼ 8.8 Hz), 7.83 (d, 1H, J ¼ 15.6 Hz), 8.26 (d, 2H, J ¼ 8.8 Hz); 13C
NMR (CDCl3, 100 MHz)
d: 28.1, 28.1, 55.9, 77.3, 103.8, 110.5, 116.7,
5.1.3.23. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(3-
(trifluoromethyl)phenyl)prop-2-en-1-one (26). The title compound
26 was prepared according to the general procedure A. Recrystal-
lization of the crude compound from EtOH afforded 26 (38.8%) as
121.0, 124.2, 124.2, 128.5, 128.5, 128.6, 131.3, 131.9, 137.7, 142.1, 148.1,
154.0, 159.0, 189.0; MS (ESI, m/z): 366.18 [M þ H]þ; HPLC
purity ¼ 99.2%.
a yellow solid. 1H NMR (CDCl3, 400 MHz)
d: 1.52 (s, 6H), 3.90 (s, 3H),
5.1.3.18. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(3-
nitrophenyl)prop-2-en-1-one (21). The title compound 21 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 21 (40.0%) as a yellow
5.64 (d, 1H, J ¼ 10.0 Hz), 6.53 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H,
J ¼ 10.0 Hz), 7.53 (t, 1H, J ¼ 7.6 Hz), 7.61 (d, 1H, J ¼ 7.6 Hz), 7.66 (d,
1H, J ¼ 15.6 Hz), 7.72 (d, 1H, J ¼ 7.6 Hz), 7.76 (d, 1H, J ¼ 8.8 Hz), 7.82
(d, 1H, J ¼ 15.6 Hz), 7.89 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d: 28.0,
solid. 1H NMR (CDCl3, 400 MHz)
d: 1.55 (s, 6H), 3.91 (s, 3H), 5.65 (d,
28.0, 55.8, 77.3, 103.7, 110.5, 116.7, 121.2, 124.0, 124.1, 126.1, 126.1,
128.6, 129.2, 129.4, 131.5, 131.9, 136.6, 139.0, 154.0, 158.8, 189.2; MS
(ESI, m/z): 389.31 [M þ H]þ; HPLC purity ¼ 99.3%.
1H, J ¼ 10.0 Hz), 6.54 (d,1H, J ¼ 8.8 Hz), 6.70 (d,1H, J ¼ 10.0 Hz), 7.59
(t, 1H, J ¼ 8.0 Hz), 7.68 (d, 1H, J ¼ 15.6 Hz), 7.77 (d, 1H, J ¼ 8.8 Hz),
7.84 (d, 1H, J ¼ 8.0 Hz), 7.90 (d, 1H, J ¼ 15.6 Hz), 8.21 (dd, 1H,
J ¼ 8.0 Hz, 1.2 Hz), 8.52 (s, 1H); 13C NMR (CDCl3, 100 MHz)
d
: 28.1,
5.1.3.24. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(thio-
phen-3-yl)prop-2-en-1-one (27). The title compound 27 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 27 (46.0%) as a yellow
28.1, 55.9, 77.3, 103.8, 110.5, 116.7, 120.9, 121.7, 123.9, 128.7, 130.0,
130.2, 132.0, 134.2, 137.6, 137.8, 148.7, 154.1, 159.0, 188.9; MS (ESI, m/
z): 366.18 [M þ H]þ; HPLC purity ¼ 99.5%.

G. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 793e803
801
solid. 1H NMR (CDCl3, 400 MHz)
d
: 1.51 (s, 6H), 3.89 (s, 3H), 5.62 (d,
127.8, 128.7, 128.7, 129.6, 130.1, 131.8, 134.9, 137.6, 139.4, 153.9, 158.7,
1H, J ¼ 10.0 Hz), 6.51 (d, 1H, J ¼ 8.8 Hz), 6.68 (d, 1H, J ¼ 10.0 Hz),
189.5; MS (ESI, m/z): 355.28 [M þ H]þ; HPLC purity ¼ 98.4%.
7.35e7.36 (m, 2H), 7.53 (s, 1H), 7.54 (d, 1H, J ¼ 15.6 Hz), 7.67 (d, 1H,
J ¼ 15.6 Hz), 7.71 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
d:
5.1.3.30. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(thio-
phen-2-yl)prop-2-en-1-one (33). The title compound 33 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 33 (42.6%) as a yellow
28.0, 28.0, 55.8, 77.3, 103.5, 110.5, 116.8, 121.6, 125.1, 126.8, 127.3,
127.8, 128.6, 131.7, 134.9, 138.9, 153.7, 158.4, 190.0; MS (ESI, m/z):
327.24 [M þ H]þ; HPLC purity ¼ 98.8%.
solid. 1H NMR (CDCl3, 400 MHz)
d: 1.55 (s, 6H), 3.89 (s, 3H), 5.63 (d,
5.1.3.25. (E)-3-(Furan-2-yl)-1-(5-methoxy-2,2-dimethyl-2H-chro-
men-8-yl)prop-2-en-1-one (28). The title compound 28 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 28 (30.7%) as a yellow
1H, J ¼ 10.0 Hz), 6.51 (d,1H, J ¼ 8.8 Hz), 6.68 (d,1H, J ¼ 10.0 Hz), 7.06
(dd, 1H, J ¼ 4.8 Hz, 3.6 Hz), 7.28 (d, 1H, J ¼ 3.6 Hz), 7.34 (d, 1H,
J ¼ 4.8 Hz), 7.60 (d, 1H, J ¼ 15.6 Hz), 7.74 (d, 1H, J ¼ 8.8 Hz), 7.82 (d,
1H, J ¼ 15.6 Hz); 13C NMR (CDCl3, 100 MHz)
d: 28.0, 28.0, 55.8, 77.4,
solid. 1H NMR (CDCl3, 400 MHz)
d
: 1.52 (s, 6H), 3.88 (s, 3H), 5.62 (d,
103.6, 110.5, 116.7, 121.4, 126.7, 127.6, 128.2, 128.7, 131.2, 131.8, 133.8,
141.5, 153.9, 158.6, 189.0; MS (ESI, m/z): 327.25 [M þ H]þ; HPLC
purity ¼ 99.2%.
1H, J ¼ 10.0 Hz), 6.48e6.49 (m, 1H), 6.50 (d, 1H, J ¼ 8.8 Hz), 6.63 (d,
1H, J ¼ 3.2 Hz), 6.71 (d, 1H, J ¼ 10.0 Hz), 7.45e7.49 (m, 2H), 7.64 (d,
1H, J ¼ 15.6 Hz), 7.71 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
d: 27.9, 27.9, 55.8, 77.4, 103.5, 110.5, 112.3, 114.6, 116.7, 121.6, 125.2,
5.1.3.31. (E)-3-(3,4-Dichlorophenyl)-1-(5-methoxy-2,2-dimethyl-2H-
chromen-8-yl)prop-2-en-1-one (34). The title compound 34 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 34 (48.7%) as a yellow
127.8, 128.8, 131.7, 144.3, 152.4, 153.8, 158.4, 189.4; MS (ESI, m/z):
311.12 [M þ H]þ; HPLC purity ¼ 98.3%.
5.1.3.26. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(naph
thalen-1-yl)prop-2-en-1-one (29). The title compound 29 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 29 (43.7%) as a yellow
solid. 1H NMR (CDCl3, 400 MHz)
d: 1.51 (s, 6H), 3.90 (s, 3H), 5.64 (d,
1H, J ¼ 10.0 Hz), 6.52 (d,1H, J ¼ 8.8 Hz), 6.69 (d,1H, J ¼ 10.0 Hz), 7.39
(dd, 1H, J ¼ 8.4 Hz, 2.0 Hz), 7.46 (d, 1H, J ¼ 8.4 Hz), 7.53 (d, 1H,
J ¼ 15.6 Hz), 7.67 (d, 1H, J ¼ 2.0 Hz), 7.70 (d, 1H, J ¼ 15.6 Hz), 7.73 (d,
solid. 1H NMR (CDCl3, 400 MHz)
d
: 1.51 (s, 6H), 3.90 (s, 3H), 5.63 (d,
1H, J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
d: 28.1, 28.1, 55.8, 77.4,
1H, J ¼ 10.0 Hz), 6.54 (d, 1H, J ¼ 8.8 Hz), 6.70 (d, 1H, J ¼ 10.0 Hz),
103.7, 110.5, 116.7, 121.1, 127.0, 128.6, 129.1, 129.5, 130.9, 131.8, 133.1,
133.5, 135.9, 138.1, 153.9, 158.7, 189.1; MS (ESI, m/z): 391.14
[M þ H]þ; HPLC purity ¼ 99.1%.
7.49e7.61 (m, 3H), 7.79e7.91 (m, 5H), 8.32 (d, 1H, J ¼ 8.0 Hz), 8.53
(d, 1H, J ¼ 15.6 Hz); 13C NMR (CDCl3, 100 MHz)
d: 28.0, 28.0, 55.8,
77.4, 103.6, 110.5, 116.8, 121.7, 123.8, 124.7, 125.5, 126.2, 126.7, 128.7,
128.8, 129.9, 130.1, 131.9, 131.9, 133.1, 133.8, 138.3, 153.8, 158.5,
189.8; MS (ESI, m/z): 371.22 [M þ H]þ; HPLC purity ¼ 97.6%.
5.1.3.32. (E)-3-(2-Chlorophenyl)-1-(5-methoxy-2,2-dimethyl-2H-
chromen-8-yl)prop-2-en-1-one (35). The title compound 35 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 35 (44.3%) as a yellow
5.1.3.27. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(pyr-
idin-2-yl)prop-2-en-1-one (30). The title compound 30 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 30 (49.2%) as a yellow
solid. 1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.89 (s, 3H), 5.62 (d,
1H, J ¼ 10.0 Hz), 6.52 (d, 1H, J ¼ 8.8 Hz), 6.68 (d, 1H, J ¼ 10.0 Hz),
7.28e7.31 (m, 2H), 7.42e7.44 (m, 1H), 7.70e7.75 (m, 3H), 8.07 (d,
solid. 1H NMR (CDCl3, 400 MHz)
d
: 1.52 (s, 6H), 3.89 (s, 3H), 5.62 (d,
1H, J ¼ 15.6 Hz); 13C NMR (CDCl3, 100 MHz)
d: 28.0, 28.0, 55.8, 77.4,
1H, J ¼ 10.0 Hz), 6.51 (d, 1H, J ¼ 8.8 Hz), 6.70 (d, 1H, J ¼ 10.0 Hz),
7.25e7.28 (m, 1H), 7.48 (d, 1H, J ¼ 8.0 Hz), 7.62 (d, 1H, J ¼ 15.6 Hz),
7.71e7.75 (m, 2H), 8.18 (d, 1H, J ¼ 15.6 Hz), 8.66 (d, 1H, J ¼ 4.4 Hz);
103.6, 110.4, 116.7, 121.4, 127.0, 127.3, 128.6, 129.7, 130.2, 130.5, 131.8,
133.8, 135.3, 136.8, 153.8, 158.6, 189.5; MS (ESI, m/z): 355.29
[M þ H]þ; HPLC purity ¼ 99.3%.
13C NMR (CDCl3,100 MHz)
d: 27.9, 27.9, 55.8, 77.4,103.5,110.6,116.6,
121.5, 123.7, 124.1, 128.9, 131.5, 131.6, 136.6, 139.5, 150.1, 154.1, 154.2,
5.1.3.33. (E)-1-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(2-meth
oxyphenyl)prop-2-en-1-one (36). The title compound 36 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 36 (17.8%) as a yellow solid.
158.6, 190.2; MS (ESI, m/z): 322.14 [M þ H]þ; HPLC purity ¼ 98.5%.
5.1.3.28. (E)-3-(4-Chlorophenyl)-1-(5-methoxy-2,2-dimethyl-2H-chro
men-8-yl)prop-2-en-1-one (31). The title compound 31 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 31 (50.7%) as a yellow
1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.89 (s, 3H), 3.89 (s, 3H), 5.62
(d, 1H, J ¼ 10.0 Hz), 6.50 (d, 1H, J ¼ 8.8 Hz), 6.68 (d, 1H, J ¼ 10.0 Hz),
6.92 (d, 1H, J ¼ 8.0 Hz), 6.98 (t, 1H, J ¼ 7.6 Hz), 7.35 (t, 1H, J ¼ 7.6 Hz),
7.61 (dd, 1H, J ¼ 8.0 Hz, 1.6 Hz), 7.69 (d, 1H, J ¼ 8.8 Hz), 7.72 (d, 1H,
solid. 1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.89 (s, 3H), 5.63 (d,
1H, J ¼ 10.0 Hz), 6.52 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H, J ¼ 10.0 Hz), 7.36
(d, 2H, J ¼ 8.8 Hz), 7.51 (d, 2H, J ¼ 8.8 Hz), 7.60 (d, 1H, J ¼ 15.6 Hz),
7.68 (d, 1H, J ¼ 15.6 Hz), 7.72 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3,
J ¼ 15.6 Hz), 8.03 (d, 1H, J ¼ 15.6 Hz); 13C NMR (CDCl3, 100 MHz)
d:
27.9, 27.9, 55.5, 55.8, 77.4, 103.4, 110.4, 111.2, 116.7, 120.7, 122.0, 124.6,
127.8, 128.4, 128.7, 131.1, 131.7, 136.7, 153.5, 158.2, 158.6, 190.5; MS (ESI,
m/z): 351.28 [M þ H]þ; HPLC purity ¼ 99.1%.
100 MHz) d: 28.1, 28.1, 55.8, 77.4, 103.6, 110.5, 116.8, 121.4, 127.9,
128.6,129.2,129.2,129.3,129.3,131.8,134.2,135.6,139.7,153.8,158.6,
189.6; MS (ESI, m/z): 355.45 [M þ H]þ; HPLC purity ¼ 99.3%.
5.1.4. General procedure B for the synthesis of 37be42b
To a solution of hydroxyl substituted aromatic aldehydes
(1.0 mmol) in anhydrous DMF (10 mL) at 0 ꢀC was added NaH
(2.0 mmol, 60%). Then methyl chloromethyl ether (2.0 mmol) was
added dropwise to the mixture. The ice bath was removed and
stirred at room temperature for 2 h. The reaction mixture was
quenched with water, extracted with ethyl acetate and purified by
silica gel column chromatography.
5.1.3.29. (E)-3-(3-Chlorophenyl)-1-(5-methoxy-2,2-dimethyl-2H-chro
men-8-yl)prop-2-en-1-one (32). The title compound 32 was
prepared according to the general procedure A. Recrystallization of
the crude compound from EtOH afforded 32 (43.5%) as a yellow
solid. 1H NMR (CDCl3, 400 MHz)
d: 1.52 (s, 6H), 3.90 (s, 3H), 5.64 (d,
1H, J ¼ 10.0 Hz), 6.52 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H, J ¼ 10.0 Hz),
7.33e7.35 (m, 2H), 7.45e7.47 (m,1H), 7.58 (d,1H, J ¼ 15.6 Hz), 7.59 (s,
1H), 7.72 (d, 1H, J ¼ 15.6 Hz), 7.73 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3,
5.1.4.1. 4-(Methoxymethoxy)benzaldehyde (37b). The compound
37b was preparation according to the general procedure B to yield
100 MHz) d: 28.1, 28.1, 55.8, 77.4, 103.6, 110.5, 116.8, 121.3, 126.3,

802
G. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 793e803
a yellow oil (91.2%). 1H NMR (CDCl3, 400 MHz)
(s, 2H), 7.14 (d, 2H, J ¼ 8.8 Hz), 7.83 (d, 2H, J ¼ 8.8 Hz), 9.90 (s, 1H).
d: 3.50 (s, 3H), 5.26
1H), 5.63 (d, 1H, J ¼ 10.0 Hz), 6.52 (d, 1H, J ¼ 8.8 Hz), 6.68 (d, 1H,
J ¼ 10.0 Hz), 6.86 (dd, 1H, J ¼ 8.0 Hz, 1.6 Hz), 7.10 (s, 1H), 7.19 (d, 1H,
J ¼ 8.0 Hz), 7.28 (d, 1H, J ¼ 8.0 Hz), 7.62 (d,1H, J ¼ 15.6 Hz), 7.69 (d,1H,
5.1.4.2. 3-(Methoxymethoxy)benzaldehyde (38b). The compound
38b was preparation according to the general procedure B to yield
J ¼ 15.6 Hz), 7.72 (d, 1H, J ¼ 8.8 Hz); 13C NMR (d6-DMSO, 100 MHz)
d:
28.0, 28.0, 56.5, 77.3, 104.8, 110.3, 114.5, 116.4, 118.0, 119.9, 121.4, 127.2,
129.9, 130.6, 131.7, 136.6, 141.7, 153.3, 158.3, 158.5, 189.1; MS (ESI, m/z):
337.28 [M þ H]þ; HPLC purity ¼ 97.1%.
a yellow oil (91.2%). 1H NMR (CDCl3, 400 MHz)
d: 3.47 (s, 3H), 5.21
(s, 2H), 7.26e7.29 (m, 1H), 7.43 (t, 1H, J ¼ 8.0 Hz), 7.49e7.52 (m, 2H),
9.95 (s, 1H).
5.1.5.3. (E)-3-(3-ethoxy-4-hydroxyphenyl)-1-(5-methoxy-2,2-dimethyl-
2H-chromen-8-yl)prop-2-en-1-one (39). The title compound 39 was
preparation according to the general procedure C to yield a yellow
5.1.4.3. 3-Ethoxy-4-(methoxymethoxy)benzaldehyde
compound 39b was preparation according to the general procedure
B to yield a yellow oil (83.6%). 1H NMR (CDCl3, 400 MHz)
: 1.46 (t,
(39b). The
d
solid (11.5%). 1H NMR (CDCl3, 400 MHz)
d: 1.46e1.50 (m, 9H), 3.89 (s,
3H, J ¼ 6.8 Hz), 3.51 (s, 3H), 4.15 (q, 2H, J ¼ 6.8 Hz, 13.6 Hz), 5.29 (s,
3H), 4.15 (q, 2H, J ¼ 7.6 Hz, 13.6 Hz), 5.62 (d, 1H, J ¼ 10.0 Hz), 5.89 (s,
1H), 6.51 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H, J ¼ 10.0 Hz), 6.93 (d, 1H,
J ¼ 8.4 Hz), 7.10 (s, 1H), 7.14 (d, 1H, J ¼ 8.4 Hz), 7.55 (d, 1H, J ¼ 15.6 Hz),
7.61 (d, 1H, J ¼ 15.6 Hz), 7.70 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3,
2H), 7.23 (d, 1H, J ¼ 8.0 Hz), 7.39e7.41 (m, 2H), 9.84 (s, 1H).
5.1.4.4. 3-Bromo-4-(methoxymethoxy)benzaldehyde
compound 41b was preparation according to the general procedure
B to yield a yellow oil (41.7%). 1H NMR (CDCl3, 400 MHz)
: 3.50 (s,
(40b). The
100 MHz) d: 14.9, 28.0, 28.0, 55.8, 64.5, 77.3, 103.5, 110.4, 110.6, 114.8,
d
116.9, 121.9, 122.8, 125.1, 128.2, 128.6, 131.7, 141.9, 146.0, 147.8, 153.5,
3H), 5.32 (s, 2H), 7.23e7.27 (m, 1H), 7.75e7.78 (m, 1H), 8.05e8.07
(m, 1H), 9.84 (s, 1H).
158.3, 190.0; MS (ESI, m/z): 381.24 [M þ H]þ; HPLC purity ¼ 98.4%.
5.1.5.4. (E)-3-(3-bromo-4-hydroxyphenyl)-1-(5-methoxy-2,2-dimethyl-
2H-chromen-8-yl)prop-2-en-1-one (40). The title compound 40 was
preparation according to the general procedure C to yield a yellow
5.1.4.5. 3,5-Dimethoxy-4-(methoxymethoxy)benzaldehyde (41b). The
compound 42b was preparation according to the general procedure
B to yield a yellow oil (93.2%). 1H NMR (CDCl3, 400 MHz)
3H), 3.92 (s, 6H), 5.22 (s, 2H), 7.13 (s, 2H), 9.86 (s, 1H).
d: 3.59 (s,
solid (14.9%). 1H NMR (CDCl3, 400 MHz)
d: 1.51 (s, 6H), 3.89 (s, 3H),
5.63 (d, 1H, J ¼ 10.0 Hz), 5.77 (s, 1H), 6.51 (d, 1H, J ¼ 8.8 Hz), 6.68 (d, 1H,
J ¼ 10.0 Hz), 7.03 (d, 1H, J ¼ 8.4 Hz), 7.46 (dd, 1H, J ¼ 8.4 Hz, 2.0 Hz),
7.54 (d, 1H, J ¼ 15.6 Hz), 7.60 (d, 1H, J ¼ 15.6 Hz), 7.71e7.73 (m, 2H); 13C
5.1.4.6. 5-Chloro-2-(methoxymethoxy)benzaldehyde
compound 43b was preparation according to the general procedure
B to yield a yellow oil (95.7%). 1H NMR (CDCl3, 400 MHz)
: 3.49 (s,
(42b). The
NMR (CDCl3,100 MHz) d: 28.0, 28.0, 55.8, 77.4, 103.6, 110.5, 110.8, 116.6,
d
116.8, 121.5, 126.4, 128.7, 129.1, 129.9, 131.8, 132.0, 139.7, 153.8, 153.9,
3H), 5.26 (s, 2H), 7.16 (d, 1H, J ¼ 8.8 Hz), 7.41 (dd, 1H, J ¼ 9.2, 2.8 Hz),
158.6, 189.8; MS (ESI, m/z): 417.12 [M þ H]þ; HPLC purity ¼ 98.3%.
7.73 (d, 1H, J ¼ 2.4 Hz), 10.39 (s, 1H).
5.1.5.5. (E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-1-(5-methoxy-2,2-
5.1.5. General procedure C for the synthesis of compounds 37e42
A solution of 50% KOH (3 mL, aq.) was added dropwise to
a stirred solution of 3 (1.0 mmol) and MOM-protected aromatic
aldehydes (1.0 mmol) in methanol (10 mL) at 0 ꢀC. The resulting
mixture was stirred at room temperature for 48 h under N2
atmosphere. After the end of reaction, the mixture was poured into
ice water and neutralized with 2 N HCl. The resulting mixture was
extracted with ethyl acetated (3 ꢂ 20 mL), the combined organic
layer was separated, dried over anhydrous Na2SO4, and concen-
trated under reduced pressure. The residue was dissolved in
methanol (10 mL), then 2 M HCl (aq.) was added. The mixture was
reflux at 60 ꢀC for 2 h. After cooling to room temperature, the
mixture was poured into 20 mL water, and extracted with ethyl
acetated (3 ꢂ 20 mL). The combined organic layer was separated,
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The residue was purified by silica gel chromatography to
obtain title products.
dimethyl-2H-chromen-8-yl)prop-2-en-1-one
compound 41 was preparation according to the general procedure
C to yield a yellow solid (12.3%). 1H NMR (CDCl3, 400 MHz)
: 1.52 (s,
(41). The
title
d
6H), 3.90 (s, 3H), 3.93 (s, 6H), 5.63 (d, 1H, J ¼ 10.0 Hz), 6.52 (d, 1H,
J ¼ 8.8 Hz), 6.70 (d, 1H, J ¼ 10.0 Hz), 6.87 (s, 2H), 7.63 (s, 2H), 7.72 (d,
1H, J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
d: 28.0, 28.0, 55.8, 56.2,
56.2, 77.4, 103.5, 105.1, 105.1, 110.4, 116.9, 121.7, 125.5, 127.2, 128.5,
131.7, 136.9, 141.9, 147.2, 147.2, 153.6, 158.4, 189.7; MS (ESI, m/z):
397.36 [M þ H]þ; HPLC purity ¼ 96.7%.
5.1.5.6. (E)-3-(5-chloro-2-hydroxyphenyl)-1-(5-methoxy-2,2-dimethyl-
2H-chromen-8-yl)prop-2-en-1-one (42). The title compound 42 was
preparation according to the general procedure C to yield a yellow
solid (13.5%). 1H NMR (CDCl3, 400 MHz)
d: 1.52 (s, 6H), 3.90 (s, 3H),
5.63 (d, 1H, J ¼ 10.0 Hz), 6.50 (s, 1H), 6.52 (d, 1H, J ¼ 8.8 Hz), 6.68 (d,
1H, J ¼ 10.0 Hz), 6.85 (d, 1H, J ¼ 8.8 Hz), 7.18 (d,1H, J ¼ 8.8 Hz), 7.52 (d,
1H, J ¼ 2.0 Hz), 7.74 (d,1H, J ¼ 8.8 Hz), 7.78 (d, 1H, J ¼ 15.6 Hz), 7.97 (d,
1H, J ¼ 15.6 Hz); 13C NMR (d6-DMSO, 100 MHz)
d: 27.3, 27.3, 55.9, 76.8,
5.1.5.1. (E)-3-(4-Hydroxyphenyl)-1-(5-methoxy-2,2-dimethyl-2H-chro-
men-8-yl)prop-2-en-1-one (37). The title compound 37 was prepara-
tion according to the general procedure C to yield a yellow solid
104.2, 109.8, 116.0, 117.9, 121.0, 122.9, 123.4, 128.2, 128.5, 129.4, 130.6,
131.2, 135.8, 152.8, 155.9, 157.9, 188.9; MS (ESI, m/z): 371.28 [M þ H]þ;
HPLC purity ¼ 98.2%.
(10.4%). 1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.89 (s, 3H), 5.22 (s,
1H), 5.63 (d, 1H, J ¼ 10.0 Hz), 6.51 (d, 1H, J ¼ 8.8 Hz), 6.69 (d, 1H,
J ¼ 10.0 Hz), 6.86 (d, 2H, J ¼ 8.8 Hz), 7.49 (d, 2H, J ¼ 8.8 Hz), 7.57 (d,1H,
J ¼ 15.6 Hz), 7.65 (d, 1H, J ¼ 15.6 Hz), 7.70 (d, 1H, J ¼ 8.8 Hz); 13C NMR
5.2. Biological assay methods
5.2.1. Cell proliferation inhibition assay (MTT assay)
(d6-DMSO, 100 MHz)
d
: 27.5, 27.5, 55.9, 76.7, 104.1, 109.8, 116.0, 116.0,
1 ꢂ 104 cells per well were plated in 96-well culture plates and
incubated for 24 h. After incubation, Tetrazolium dye [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide, MTT]
116.0, 121.3, 123.6, 125.9, 129.3, 130.1, 130.1, 131.0, 141.7, 152.5, 157.6,
159.8, 188.8; MS (ESI, m/z): 337.28 [M þ H]þ; HPLC purity ¼ 96.3%.
solution (20
incubated until a purple precipitate was visible. Then, the precipi-
tates were dissolved in 150 L of DMSO, after the medium was
mL of 5 mg/mL) in PBS was added to each well. This was
5.1.5.2. (E)-3-(3-Hydroxyphenyl)-1-(5-methoxy-2,2-dimethyl-2H-chro-
men-8-yl)prop-2-en-1-one (38). The title compound 38 was prepa-
ration according to the general procedure C to yield a yellow solid
m
carefully removed, shaken mechanically for 30 min, and then
absorbance values at a wavelength of 570 nm were determined
(15.9%). 1H NMR (CDCl3, 400 MHz)
d: 1.51 (s, 6H), 3. 89 (s, 3H), 5.21 (s,

G. Wang et al. / European Journal of Medicinal Chemistry 54 (2012) 793e803
803
with the Spectramax M5 Microtiter Plate Luminometer (Molecular
Devices, USA). Values were calculated using percentage of growth
versus untreated control.
analyzed using the two-sided Student’s t test. P values lower than
0.05 were considered significant.
Acknowledgments
5.2.2. Immunofluorescence staining of tubulin
HepG2 tumor cells (1 ꢂ 104) were maintained in 6-well culture
plate, After incubation for 12 h, cells prior to treatment with DMSO,
This study was supported by National Key Technologies R&D
Program of China (2012ZX09103101-009).
millepachine (1.0 mM) or 15 (1.0 m
M) for 24 h at 37 ꢀC. Cells were
fixed with blocking buffer (PBS containing 3% BSA, 2% goat serum
and 0.2% triton ꢂ 100) for 1 h after being washed with PBS. And
Appendix A. Supplementary material
then cells would be incubated with
a-tubulin for 2 h, and then
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2012.06.034.
tubulin was immunostained with monoclonal antibody to
a-
tubulin followed by fluorescence antibody. Nuclei were labeled
with Hoechst 33258. Fluorescence labeled tubulin and nuclei were
observed using fluorescence microscopy.
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5.2.5. Statistical analysis
Unless indicated differently, the results are presented as
mean ꢁ S.E.M. The differences between different treatments were