Synthesis and biological evaluation of novel pyranochalcone derivatives as a new class of microtubule stabilizing agents

Article abstract of DOI:10.1016/j.ejmech.2013.01.007

Twenty-five novel pyranochalcone derivatives were synthesized and evaluated for their in vitro and in vivo antiproliferative activities. Among them, compound 10i exhibited superior potent activity against 21 tumor cell lines including multidrug resistant phenotype with the IC₅₀ values ranged from 0.09 to 1.30 μM. In addition, 10i significantly induced cell cycle arrest in G2/M phase, promoted tubulin polymerization into microtubules and caused microtubule stabilization. Further studies confirmed that 10i significantly suppressed the growth of tumor volume in HepG2 xenograft tumor model. Our study demonstrated that 10i could have beneficial antitumor activity as a novel microtubule stabilizing agent.

Full text of DOI:10.1016/j.ejmech.2013.01.007

European Journal of Medicinal Chemistry 62 (2013) 579e589
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel pyranochalcone
derivatives as a new class of microtubule stabilizing agents
,
,
Dong Cao a 1, Xiaolei Han a 1, Guangcheng Wang a, Zhuang Yang b, Fei Peng a, Liang Ma a,
Ronghong Zhang a, Haoyu Ye a, Minghai Tang a, Wenshuang Wu a, Kai Lei a, Jiaolin Wen a,
Jinying Chen a, Jingxiang Qiu a, Xiaolin Liang a, Yan Ran a, Yun Sang a, Mingli Xiang a,
Aihua Peng 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 Chemistry College, Sichuan University, Chengdu, Sichuan 610064, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-five novel pyranochalcone derivatives were synthesized and evaluated for their in vitro and
in vivo antiproliferative activities. Among them, compound 10i exhibited superior potent activity against
21 tumor cell lines including multidrug resistant phenotype with the IC50 values ranged from 0.09 to
Received 18 October 2012
Received in revised form
2 January 2013
Accepted 7 January 2013
Available online 16 January 2013
1.30 mM. In addition, 10i significantly induced cell cycle arrest in G2/M phase, promoted tubulin poly-
merization into microtubules and caused microtubule stabilization. Further studies confirmed that 10i
significantly suppressed the growth of tumor volume in HepG2 xenograft tumor model. Our study
demonstrated that 10i could have beneficial antitumor activity as a novel microtubule stabilizing agent.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Pyranochalcone
Tubulin
Anticancer activity
Microtubule stabilizing agents
1. Introduction
mitosis and apoptosis. However, these compounds have some
limitations such as the drug resistance, high toxicity, difficulty of
Tubulin which plays a crucial role in cell growth involved in the
formation of the mitotic spindle [1,2] has been recognized as an
attractive molecular target for anticancer agents recently [3]. Drugs
attack microtubules by interfering with the dynamics of tubulin
polymerization or dissociation, resulting in the mitotic arrest. Two
major classes of anticancer agents exert their effectiveness involved
in this mechanism [4], defined as microtubule destabilizing (Vinca
alkaloids [5], Colchicine and Combretastatin [6,7]) and microtubule
stabilizing agents (Taxol, Epothilone and Discodermolide).
synthesis and complex isolation procedure. Recently, several small
synthetic molecules that stimulate tubulin polymerization and
induce microtubule stabilization have been reported as potential
microtubule stabilizing agents (Compounds 1, 2, 3 in Fig.1) [14e16].
The success of previous research encourages scientists to search for
simple small molecules for microtubule stabilizing agents.
Millepachine, a natural pyranochalcone, was for the first time
isolated from Millettia pachycarpa in our lab [17]. It exhibited a potent
cytotoxic activity and induced apoptosis effects in several human
Taxol (Paclitaxel), generally regarded as the first microtubule
stabilizing agent [8,9], is one of the strongest chemotherapeutic
agents currently used in clinic [10,11]. Three non-taxoid compounds
have also been shown to stabilize microtubules: Epothilone A,
Epothilone B [12] and Discodermolide [13]. These compounds share
with Taxol the ability to promote tubulin polymerization into mi-
crotubules and block microtubule dynamics, resulting in abnormal
cancer cell lines with an IC50 values ranging from 1.51 to 4.0
mM [18].
Similar to other reported chalcones [19,20], the cytotoxic activity of
millepachineis associated with tubulin [17,18]. The easilymodification
and structural similarity to Combretastatin A4 (CA4) of millepachine
hint that millepachine might be a potential lead compound for anti-
tumor drug via targeting tubulin and encourage us to establish more
advanced structureeactivity relationships(SAR)around millepachine.
It is well known that CA4 has three important pharmacophone
components: two hydrophobic rings (rings A and B), a linking bridge
between the two rings (Fig. 1). Similarly to the structure of CA4,
millepachine has two aromatic rings and an unsaturated chain.
Hence, weassumed thatitisfeasibletointroducea30-hydroxyl group
* Corresponding author. Tel.: þ86 28 85164063; fax: þ86 28 85164060.
E-mail address: lijuan17@hotmail.com (L. Chen).
1
These authors contributed equally and should be considered as co-first authors.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.007

580
D. Cao et al. / European Journal of Medicinal Chemistry 62 (2013) 579e589
Fig. 1. Some tubulin interacting agents and lead structures.
on B ring and further modified on hydroxyl group of millepachine to
obtain diverse derivatives. Herein, a series of millepachine-based
pyranochalcones were synthesized and evaluated for their cytotox-
icity in several human tumor cell lines including multidrug resistant
phenotype. The effects on tubulin polymerization and the tumor cell
cycle distribution of selected compounds were investigated to
determine their exact mechanism. The most promising compound
was further tested for anti-tumor activity and toxicity in vivo.
The key intermediate 6 was prepared by condensation of
paeonol 4 and 3-chloro-3-methyl-1-butyne, and followed cycliza-
tion upon heating in pyridine at 120 ꢀC overnight. Subsequently,
the chalcone analog 8 was obtained by ClaiseneSchmidt con-
densation of 6 and aldehyde 7. Although the synthetic procedure
was suitable for a series of appropriate aldehydes, it had a low
yielding or even unsuccessfully accomplished for the hydroxyl-
substituent aldehydes because of the special chemical environ-
ment. To solve the problem, the hydroxyl-substituent benzalde-
hydes were initially protected by methoxy-methylene chloride
(MOM-Cl), which was stable under the base-promoted condition.
Compound 9 was readily obtained by the deprotection with dilute
acid treatment, for the further generation of 10aex.
2. Results and discussion
2.1. Chemistry
Although several synthetic approaches to pyranochalcones have
been established [21e23], we reported an efficient and simple
methodology for preparing naturally occurring pyranochalcones in
previous work [24,25]. The ClaiseneSchmidt condensation was used
as a key procedure to synthesize the chalcone analogs, which proved
to be satisfactory for the formation of E isomer [26,27] (Scheme 1).
2.2. Biological activities
2.2.1. Biological activities in vitro cell growth inhibitory activity
The synthesized derivatives 9, 10ae10x were evaluated for
antiproliferative activities in vitro by MTT assay against HepG2 cells
Scheme 1. Reagents and conditions: (a) 3-chloro-3-methylbut-1-yne, CuCl2$2H2O, DBU, CH3CN, 0 ꢀC, 5 h; (b) Pyridine, 120 ꢀC, 12 h; (c) NaOH (50% w/v aqueous solution), CH3OH, rt,
12 h; (d) HCl/EA, rt, 2h; (e) Method A for 10aed: K2CO3, RCl,CH3CN, reflux, 6 h; method B for 10eeh: NaH, DMF, RCl, 12 h; method C for 10iek: (RCO)2O, pyridine, rt, 6 h; method D
for 10len: RCOCl, CH2Cl2, rt, 6 h; method E for 10oex: RCOOH, EDCI, DMAP, CH2Cl2, rt, 12 h.

D. Cao et al. / European Journal of Medicinal Chemistry 62 (2013) 579e589
581
Table 1
In vitro cytotoxicity of 9, 10aex.
Compds
R
IC50 (mM)
HepG2
A375
K562
LO2
Millepachine
9
NAa
H
1.51
0.45
3.58
0.80
4.56
0.63
>20.0
>10.0
10a
10b
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
10c
10d
>10.0
>10.0
>10.0
>10.0
>20.0
>10.0
9.60
8.75
10e
10f
10g
>10.0
>10.0
4.40
8.20
3.81
3.00
8.35
3.05
2.08
>10.0
9.50
9.15
10h
10i
5.55
0.09
4.10
0.24
2.92
0.16
8.60
>10.0
10j
0.10
0.25
0.22
0.31
0.24
0.57
>10.0
>10.0
10k
10l
0.16
0.12
0.39
0.45
0.30
0.40
>10.0
>10.0
10m
10n
10o
0.22
0.87
0.38
0.80
0.41
0.79
>10.0
>10.0
10p
10q
10r
0.68
0.57
0.60
0.42
0.48
0.70
1.11
0.29
0.43
8.40
>10.0
>10.0
10s
10t
0.43
0.70
2.63
9.20
0.38
0.47
18.0
>10.0
10u
0.37
0.68
0.45
>10.0
(continued on next page)

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D. Cao et al. / European Journal of Medicinal Chemistry 62 (2013) 579e589
Table 1 (continued )
Compds
R
IC50 (mM)
HepG2
A375
K562
9.24
LO2
10v
10w
10x
9.18
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
3.60
2.40
7.85
a
NA, not applicable.
(human hepatocellular carcinoma), A375 cells (human melanoma),
K562 cells (human leukemia) and LO2 cells (normal liver cell line)
(Table 1). Generally, the antiproliferative activity of the most potent
compounds was visibly greater against human liver HepG2 tumor
cell lines as compared with the other cell lines.
The products by esterification were distinctly more potent than
ether derivatives. The aliphatic products 10ie10n displayed potent
antiproliferative activities against tumor cell lines, particularly 10i
which showed the strongest activity against HepG2 cells and K562
Although many anticancer drugs in clinical use are effective, their
potentials are limited by the development of drug resistance [28]. Thus,
the antiproliferative effects of 10i and 10j were evaluatedinMCF-7/ADR
(multidrug-resistant human breast cancer cells), HCT-8/T (Tax-
ol-resistant colon cancer cells), A2780/T (Taxol-resistant human ovar-
ian cancer cells), compared with the tubulin interacting agents: Taxol
and Colchicines (Table 2). These cell lines have been reported to express
high levels of P-glycoprotein and were resistant toTaxol and Colchicine
[28]. Compounds 10i and 10j were found to be sensitive both in sen-
sitive and resistant cancer cells with similar IC50 values ranged from
cells (IC50 0.09
mM and 0.16
mM), and 10j, the most active molecule
against A375 cells (IC50 0.22
m
M). However, the activities of prod-
0.16 to 1.30
respective 0.19
1.33 M in A2780 and A2780/T in contrast to Taxol, which was very
sensitive to parental cell lines, was less active in the MCF-7/MDR cell
line, with a shift in IC50 values from 0.66 to 6.9 M; and the Pgp-1
overexpressing cell line A2780/T, shifting its IC50 values from 0.02 to
8.25 M, showing 400-fold decrease in activity. The same results were
obtained in Colchicine. These results show that the activities of 10i and
10j are not affected by Pgp-1 overexpressing, suggesting that 10i and
10j might be useful in the treatment of drug refractory tumors, in
particular those with resistance to other antitubulin drugs.
m
M. For example, the IC50 values of compound 10i were
ucts 10oe10t which contained benzene ring in the substitutional
group were sharply decreased. The ether derivatives 10ae10h were
the least active compounds in the series, with IC50 values over
m
Mand0.16 M in MCF-7 and MCF-7/MDR, 0.22 Mand
m
m
m
10
mM against almost all cell lines.
m
Then we tested the activity of the two compounds, 10i and 10j,
against various human tumor cell lines, with Taxol and Colchicine
as the positive controls. The tumor cells we evaluated included
cancer cell lines coming from colon, lung, liver, breast, ovarian and
prostate (Table 2). Similar to Taxol and Colchicine, the two com-
pounds showed potent inhibitory activity with IC50 values ranged
m
from 0.1 to 0.5
H460 lung cancer cell lines. It is worth notice that the IC50 values of
10i and 10j (0.31 M and 0.30 M) showed around 3e5-fold
increase than those of Taxol (1.05 M) and Colchicine (1.72 M)
against C26 cells (murine colon adenocarcinoma). In general, the
wide range of cell killing across multiple tumor types may suggest
that this kind of compounds induce tumor cell killing by inhibiting
an intrinsically important process of tumor cell division.
mM against almost all cell lines except SPC-A1 and
2.2.2. Analysis of immunofluorescence staining
m
m
The biological activity of chalcones has been reported to be
bound up with tubulin [19]. To investigate whether the anti-
proliferative activities of 10i and 10j were derived from an inter-
action with tubulin, we examined the effect on the cellular
microtubule network treated with the selected compounds for 24 h
m
m
and stained for DNA (blue) and
a-tubulin (green). As shown in
Fig. 2, in contrast with the microtubule network in HepG2 cells
absence of drug exhibited normal arrangement and organization,
after exposure the cells to 1 mM 10i or 10j for 24 h, the microtubule
Table 2
Activity of selected compound against various human tumor cell lines.
polymerization and spindle formation showed distinct abnormal.
HepG2 cells became arrested in mitosis and the mitotic cells had
abnormal mitotic spindles. The mitotic cells acquired multiple
tubulin bundles (multipolar) without chromosomes aligned at the
metaphase plate with more dispersion of the chromosomes. From
the results obtained so far, it seems that tubulin might be an
effective target for 10i and 10j (Fig. 2).
Cell lines
Colon
IC50 of compds (
m
M)
Taxol
10i
10j
Colchicine
C26
HCT-15
HT29
NCI-H358
SPC-A1
H460
HepG2
BEL-7402
SK-HEP-1
MCF-7
SK-OV-3
ES-2
A2780S
PC-3
DU145
MCF-7/ADRa
HCT-8/T
A2780/T
0.31
0.10
0.20
0.15
22.3
4.05
0.09
0.40
0.32
0.19
0.35
0.10
0.22
0.09
0.19
0.16
1.69
1.33
0.30
0.10
0.19
0.09
18.1
5.45
0.10
>2.0
0.60
0.25
0.27
0.10
0.33
0.12
0.21
0.17
2.30
5.92
1.05
0.12
0.45
0.01
5.3
1.72
0.08
0.02
0.02
80
Lung
Liver
24
27
0.19
>2.0
0.24
0.66
0.10
0.10
0.02
0.14
0.03
6.90
13.3
8.25
0.21
1.31
0.17
0.29
0.12
0.09
0.02
0.04
0.30
3.12
11.8
>20.0
2.2.3. Analysis of cell cycle
Due to the anti-mitotic drugs induced cell cycle arrest at G2/M
phase in various cancer cell lines and lead to an increment of the
relative peak in the DNA histogram [29], the effects of compounds
10i, 10j at different concentration on cell cycle progression were
examined in HepG2 cells. After treated 24 h, both 10i and 10j
caused a remarkable G2/M arrest pattern in a concentration-
dependent manner. When cells were treated with selected com-
pound, an accumulation of tumor cells occurred the G2/M phase of
the cell cycle, the G2/M peak raised from 17.07% (Control) to 67.76%
Breast
Ovarian
Prostate
Drug resistant

D. Cao et al. / European Journal of Medicinal Chemistry 62 (2013) 579e589
583
Fig. 2. Effects of 10i and 10j on the microtubule network of HepG2 cancer cells. Untreated (Control) and cells treated with 10i, 10j at concentration of 1.0
mM for 24 h, were fixed and
stained with anti-
a-tubulin-FITC specific antibodies followed by propidium iodide. Microtubules and unassembled tubulin are shown in green, and DNA, stained with propidium
iodide 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.)
(10i), 53.47% (10j) at a concentration of 0.2
m
M, and 76.02% (10i),
2.2.6. In vivo anticancer activity
69.18% (10j) at a concentration of 1 M, respectively. These trends
were in accordance with the anti-mitotic drugs, further indicating
that tubulin probably be an effective target for 10i and 10j (Fig. 3).
m
The in vivo efficacy of 10i was studied in s.c. xenograft models
using human liver HepG2 tumor cell lines. HepG2 cells (5 ꢁ 106)
were inoculated in each flank of null mice. Once tumor volume
reached a size of around 100 mm3, 10i was administered at doses of
2.5, 5, 10 mg/kg every two days. As the positive control, Taxol was
injected at 5 mg/kg every four days. As shown in Fig. 6 and Table 3,
during 20 days therapeutic treatment, 10i was administered at 2.5,
5, 10 mg/kg dose-dependently inhibited tumor growth on day 20
(T/C ¼ 34.9%, 32.4% and 33.9%, respectively), while the T/C of Taxol
at 5 mg/kg was 32.4%. Tumor growth inhibition was extremely
distinct in mice treated with 10i at 10 mg/kg/2days. Compared with
positive control, treatment with 10i at 10 mg/kg resulted in the
inhibitory rate of 69.7% and compared with mice treated by Taxol
with an inhibitory rate of 59.1%.
During the therapeutic treatment, no distinct changes in gross
measures were observed, including weight loss, feeding, and
behavior. It was demonstrated that 10i did not cause serious tox-
icity in mice at doses that associated with remarkable anti-tumor
effects. All these results showed that 10i could inhibit tumor
growth significantly and seemed more safety than Taxol when
possessing a similar tumor suppression. The exact mechanism still
needs further research.
2.2.4. Tubulin polymerization assay
Since the structural similarity, we selected 10i to further verify
whether the activity of 10i was related to the direct interaction with
microtubules, the effects on the assembly of purified tubulin were
evaluated by measuring an increase in absorbance at 340 nm at
37 ꢀC [30]. In this experiment, Taxol, a microtubule stabilizer to
enhance the rate of tubulin polymerization, was assessed as a pos-
itive control. Similar to Taxol, we found that 10i did not depoly-
merize tubulin but stabilized tubulin polymerization, which was
different from previous reports about chalcones [19,20]. As shown
in Fig. 4, 10i significantly and concentration-dependently stabilized
tubulin polymerization. Treatment at respective 5, 10 and 20
10i induced 10.0%, 16.3% and 32.0% increases of tubulin polymer-
ization. At the same concentration, 5 M and 20 M of 10i treat-
mM of
m
m
ment showed stronger ability to promote the assembly of tubulin
than Taxol (6.3% and 23.8%). This trend for accelerating tubulin
polymerization was in good correlation with their cytotoxic activ-
ity, further ensuring that 10i represents one novel microtubule
stabilizing agent.
3. Conclusion
2.2.5. Western blot analysis
Given that tubulin binding molecules attack microtubules by
interfering with the dynamics of tubulin polymerization or dis-
sociation, we next studied the alterations in expression of tubulin
that regulated cell division. Comparing to the control group,10i was
characterized by increasing the level of polymerization-tubulin
while decreasing the level of dissociation-tubulin. The exper-
imental results further confirm that 10i promoted tubulin poly-
merization into microtubules and induced microtubule
stabilization (Fig. 5).
In our research, we described the synthesis of a series of mil-
lepachine derivatives, which induced apoptotic death of a wide
variety of tumor cell lines in the double-digit nanomolar range by
attacking microtubules. Compound 10i exerted the most potent
activity in several human tumor cell lines including multidrug
resistant human tumor cell lines. A flow cytometric study showed
that compound 10i significantly induced cell cycle arrest in G2/M
phase at 0.2
mM. Furthermore, in vitro immunofluorescence stain-
ing and tubulin polymerization assay displayed that 10i, similar to

584
D. Cao et al. / European Journal of Medicinal Chemistry 62 (2013) 579e589
Fig. 3. 10i and 10j affected the cell cycle distribution in HepG2 cancer cells. Untreated (Control) and cells treated with 10i and 10j at 0.2 mM, 1.0 mM concentration for 24 h.
Taxol, promoted tubulin polymerization into microtubules and
caused microtubule stabilization, which suggesting that compound
10i is a microtubule stabilizer via enhancing the rate of tubulin
polymerization. This result was also evidenced by western blot
analysis of the level of polymerization-tubulin. Comparing to the
control group, 10i, characterized by increasing the level of
polymerization-tubulin while decreasing the level of dissociation-
tubulin, was suggested to promote tubulin polymerization into
microtubules and induced microtubules stabilization. Then in vivo
study, compound 10i exhibited potent inhibitory activity in human
HepG2 xenograft tumor models. This study demonstrated that 10i
was supposed to be a new microtubule stabilizing agent and pro-
vided a new molecular scaffold for the further development of anti-
Fig. 4. Effect of 10i on tubulin polymerization. Tubulin had been pre-incubated for 5 min
Fig. 5. 10i affected the proportion of assembled tubulin. HepG2 cancer cells were
with 10i at 5
mM, 10
mM and 20
mM, Taxol at 5
m
M and 20
m
M or vehicle DMSO at room
treatment with compound 10i at 0.1 mM or 0.5 mM concentration. Comparing to the
temperature before GTP was added to start the tubulin polymerization reactions. The
reaction was monitored at OD340 nm at 37 ꢀC. Taxol was included as positive control.
control, the proportion of assembled tubulin was increased, and the level of unas-
sembled tubulin was decreased.

D. Cao et al. / European Journal of Medicinal Chemistry 62 (2013) 579e589
585
measured by Q-TOF Priemier mass spectrometer (Micromass,
Manchester, UK).
4.1.1. General preparation of 10aed (method A)
To a solution of compound 9 (0.365 g, 1 mmol) in anhydrous
CH3CN (10 mL) was added anhydrous K2CO3 (0.345 g, 2.5 mmol,
2.5 equiv). After stirred over 15 min at room temperature, requisite
halogenated compound (2 mmol, 2 equiv) was added into the
slurry. The reaction mixture was stirred at room temperature for
6 h. The reaction mixture was filtered, diluted with EtOAc and
subsequently washed with water, then brine. The organic layer was
dried over sodium sulfate and concentrated in vacuo. The resulting
residue was purified by flash chromatography using petroleum
ether/ethyl acetate (4:1) as eluent to yield light yellow solids.
4.1.1.1. (E)-1-(5-Methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(4-
methoxy-3-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
(10a). Yield: 60%; light yellow liquid; 1H NMR (CDCl3, 400 MHz)
d:
1.50 (s, 3H), 1.61 (s, 3H), 1.72e1.77 (m, 6H), 3.87 (s, 3H), 3.89 (s, 3H),
4.59 (d, 2H, J ¼ 6.4 Hz), 5.53 (m, 1H), 5.62 (d, 1H, J ¼ 10.0 Hz), 6.50
(d, 1H, J ¼ 8.8 Hz), 6.68 (d, 1H, J ¼ 9.6 Hz), 6.85e6.88 (m, 1H), 7.14e
7.17 (m, 2H), 7.56 (d, 1H, J ¼ 16.0 Hz), 7.62 (d, 1H, J ¼ 16.0 Hz), 7.70
(d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
d: 18.1, 25.9, 28.0,
28.0, 55.8, 56.0, 65.6, 76.8, 103.5, 108.6, 112.4, 116.9, 119.5, 121.9,
122.2, 126.2, 127.4, 128.7, 128.8, 131.6, 138.1, 140.1, 140.3, 148.4, 151.3,
158.3, 189.9. MS found (M þ H)þ (m/z): 435.27. HPLC purity ¼ 98.9%.
4.1.1.2. (E)-1-(5-Methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(4-meth-
oxy-3-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one (10b). Yield: 47%;
light yellow liquid; 1H NMR (CDCl3, 400 MHz)
d: 1.52 (s, 6H), 2.52 (s,
1H), 3.89 (s, 3H), 3.92 (s, 3H), 4.80 (s, 2H), 5.63 (d, 1H, J ¼ 10.0 Hz),
6.51 (d, 1H, J ¼ 8.4 Hz), 6.69 (d, 1H, J ¼ 10.0 Hz), 6.91 (d, 1H,
J ¼ 8.0 Hz), 7.23e7.31 (m, 2H), 7.58 (d, 1H, J ¼ 15.6 Hz), 7.65 (d, 1H,
Fig. 6. 10i inhibits the growth of HepG2 xenograft tumor. (A) The curves of inhibition
of tumors. The three test groups were administered with 2.5, 5, 10 mg/kg of 10i every
two days. The positive group received Taxol at a dosage of 5 mg/kg every four days, and
the control group received injection of physiological saline alone. Each group con-
tained 6 mice. (B) The bar charts of tumor weight and the inhibitory rate of 10i. *,
P < 0.05, **, P < 0.01, and ***, P < 0.001, significantly different compared with control
by t test.
J ¼ 15.6 Hz), 7.71 (d, 1H, J ¼ 8.8 Hz); 13C NMR (DMSO, 100 MHz)
d:
27.6, 27.6, 55.6, 55.8, 55.9, 76.8, 78.5, 79.0, 104.1, 109.8, 112.1, 112.3,
115.9, 121.2, 123.7, 124.9, 127.3, 129.3, 131.0, 141.5, 146.6, 151.3, 152.6,
157.7, 188.8. MS found (M þ H)þ (m/z): 405.17. HPLC purity ¼ 98.1%.
4.1.1.3. (E)-3-(3-(Allyloxy)-4-methoxyphenyl)-1-(5-methoxy-2,2-
tumor agents. In the future research we will be exploring for a clear
structure activity relationship of this type of compound and dis-
covering more potent anti-tumor agents.
dimethyl-2H-chromen-8-yl)prop-2-en-1-one (10c). Yield: 61%; light
yellow liquid; 1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 3.88 (s, 3H),
3.91 (s, 3H), 4.63 (d, 2H, J ¼ 4.0 Hz), 5.28 (dd, 1H, J ¼ 9.2 Hz, 1.6 Hz),
5.38 (dd, 1H, J ¼ 17.2 Hz, 1.6 Hz), 5.61 (d, 1H, J ¼ 10.0 Hz), 6.04e6.12
(m, 1H), 6.50 (d, 1H, J ¼ 8.8 Hz), 6.68 (d, 1H, J ¼ 10.0 Hz), 6.89 (d, 1H,
J ¼ 8.4 Hz), 7.14 (d, 1H, J ¼ 2.0 Hz), 7.19 (dd, 1H, J ¼ 8.4 Hz, 1.6 Hz),
7.54 (d, 1H, J ¼ 15.6 Hz), 7.60 (d, 1H, J ¼ 15.6 Hz), 7.71 (d, 1H,
4. Experimental
4.1. Chemistry
J ¼ 8.4 Hz); 13C NMR (DMSO, 100 MHz)
d: 27.5, 27.5, 55.6, 55.9, 68.8,
Chemistry reagents of analytical grade were purchased from
Changzheng Chemical Factory, Chengdu, Sichuan, PR China. TLC
was performed on 0.20 mm Silica Gel 60 F254 plates (Qingdao Ocean
Chemical Factory, Shandong, China). Proton (1H) and carbon (13C)
NMR spectra were recorded at 400 MHz and 100 MHz, respectively,
on a Varian spectrometer (Varian, Palo Alto, CA) model Gemini 400
and reported in parts per million. Mass spectra (MS) were
76.7, 104.1, 109.8, 111.8, 112.0, 116.0, 117.5, 121.2, 122.8, 124.8, 127.5,
129.3, 131.0, 133.5, 141.5, 151.1, 152.5, 157.7, 163.4, 188.8. MS found
(M þ H)þ (m/z): m/z 407.21. HPLC purity ¼ 97.9%.
4.1.1.4. (E)-3-(3-(2-Hydroxyethoxy)-4-methoxyphenyl)-1-(5-
methoxy-2,2-dimethyl -2H-chromen-8-yl)prop-2-en-1-one (10d).
Yield: 70%; light yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 1.51 (s,
6H), 3.89 (s, 6H), 3.98 (t, 2H, J ¼ 4.4 Hz), 4.16 (t, 2H, J ¼ 4.4 Hz), 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),
6.90 (d, 1H, J ¼ 8.4 Hz), 7.20 (d, 1H, J ¼ 1.6 Hz), 7.57 (d, 1H,
Table 3
Summary of tumor growth inhibition.
J ¼ 16.0 Hz), 7.62 (d, 1H, J ¼ 16.0 Hz); 13C NMR (DMSO, 100 MHz)
d:
Tumor
HepG2
Compds
Dose
(mg/kg/injection)
Inhibition rate
(100%)
T/C valuea
(100%)
27.5, 27.5, 55.5, 55.9, 59.5, 70.1, 76.7, 104.1, 109.8, 111.3, 111.9, 116.0,
121.2, 122.6, 124.8, 127.6, 129.3, 131.0, 141.6, 148.3, 151.0, 152.6, 157.7,
188.8. MS found (M þ H)þ (m/z): 411.27. HPLC purity ¼ 98.8%.
10i
10i
10i
Taxol
2.5
5
10
5
54.3
57.6
69.7
59.1
34.9
32.4
33.9
32.4
4.1.2. General preparation of 10eeh (method B)
To a solution of compound 9 (0.59 g,1.6 mmol) in anhydrous DMF
(10 mL) under nitrogen was added NaH (0.38 g of a 60% dispersion,
a
T/C value < 42% is considered as significant antitumor activity by the Drug
Evaluation Branch of the Division of Cancer Treatment (NCI).

586
D. Cao et al. / European Journal of Medicinal Chemistry 62 (2013) 579e589
9.5 mmol, 6 equiv). The mixture was stirred at room temperature
over 30 min, by which point H2 evolution had ceased. The reaction
mixture was heated to 90 ꢀC, and the halogenated compound
(3.6 mmol, 2.25 equiv) was added in portions over 30 min. The re-
action mixture was stirred at 90 ꢀC for another 12 h. The completion
of the reaction was monitored by TLC. On completion, the mixture
was cooled to ambient temperature, and isopropyl alcohol (3 mL)
was added to destroy excess NaH. The slurry was partitioned be-
tween water (50 mL) and ethyl acetate (50 mL), and the water was
extracted with ethyl acetate (3 ꢁ 20 mL). The combined ethereal
extract was dried (Na2SO4), filtered, and concentrated in vacuo to
yield a brown oil. Chromatographic separation (hexaneseethyl
acetateetriethylamine,10:5:1) gave the product as a pale yellow oil.
4.1.3. General preparation of 10iek (method C)
To the solution of Compound 9 (184 mg, 0.5 mmol) in 5 mL of
pyridine was added the requisite anhydride (1 mL). The reaction
mixture was stirred at room temperature for 6 h. After removal of
the solvent, the compound was purified by flash chromatography
using petroleum ether/ethyl acetate (10:1) as eluent and yielded
a pale yellow solid.
4.1.3.1. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
8-yl)-3-oxoprop-1-en-1-yl)phenyl propionate (10i). Yield: 72%; light
yellow solid; 1H NMR (CDCl3, 400 MHz)
d
: 1.29 (t, 3H, J ¼ 6.4 Hz),
1.49 (s, 6H), 2.64 (q, 2H, J ¼ 7.6 Hz), 3.87 (s, 3H), 3.89 (s, 3H), 5.62 (d,
1H, J ¼ 10.0 Hz), 6.51 (d,1H, J ¼ 8.4 Hz), 6.68 (d,1H, J ¼ 10.0 Hz), 6.97
(d, 1H, J ¼ 8.4 Hz), 7.29 (d, 1H, J ¼ 2.0 Hz), 7.43 (dd, 1H, J ¼ 8.4 Hz,
2.0 Hz), 7.55 (d, 1H, J ¼ 15.6 Hz), 7.60 (d, 1H, J ¼ 15.6 Hz), 7.70 (d, 1H,
4.1.2.1. (E)-1-(5-Methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(4-metho-
xy-3-(2-morpholinoethoxy)phenyl)prop-2-en-1-one (10e). Yield: 23%;
J ¼ 8.4 Hz). 13C NMR (CDCl3, 100 MHz)
d: 9.2, 27.4, 28.0, 28.0, 55.8,
yellow liquid; 1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H), 2.63 (t, 4H,
56.0, 76.9, 103.4, 110.4, 112.4, 116.8, 121.7, 122.2, 126.2, 127.4, 128.7,
128.8, 131.6, 140.1, 140.3, 152.7, 153.6, 158.3, 172.3, 189.8. MS found
(M þ H)þ (m/z): 423.18. HPLC purity ¼ 99.6%.
J ¼ 4.8 Hz), 2.90 (t, 2H, J ¼ 5.6 Hz), 3.75 (t, 4H, J ¼ 4.8 Hz), 3.90 (s, 6H),
4.19 (t, 2H, J ¼ 5.6 Hz), 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), 6.88 (d, 1H, J ¼ 8.4 Hz), 7.16 (d, 1H, J ¼ 2.0 Hz),
7.19 (dd, 1H, J ¼ 8.4 Hz, 2.0 Hz), 7.52 (d, 1H, J ¼ 15.6 Hz), 7.60 (d, 1H,
4.1.3.2. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
J ¼ 16.0 Hz), 7.69 (d, 1H, J ¼ 9.2 Hz); 13C NMR (DMSO, 100 MHz)
d
:
8-yl)-3-oxoprop-1-en-1-yl)phenyl pentanoate (10j). Yield: 65%;
28.0, 28.0, 54.1, 55.8, 56.0, 56.1, 57.3, 57.4, 66.8, 66.9, 76.7, 103.6, 110.7,
111.3, 112.0, 116.8, 121.8, 122.6, 125.5, 127.6, 128.5, 130.1, 141.5, 148.4,
151.1, 153.5, 158.4, 190.8. MS found (M þ H)þ (m/z): 480.31. HPLC
purity ¼ 96.8%.
light yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 0.97 (t, 3H,
J ¼ 7.2 Hz), 1.43e1.47 (m, 2H), 1.48 (s, 6H), 1.76 (quint, 2H,
J ¼ 7.6 Hz), 2.59 (t, 2H, J ¼ 7.2 Hz), 3.86 (s, 3H), 3.88 (s, 3H), 5.61 (d,
1H, J ¼ 10.0 Hz), 6.50 (d,1H, J ¼ 8.8 Hz), 6.67 (d,1H, J ¼ 10.0 Hz), 6.96
(d, 1H, J ¼ 8.4 Hz), 7.25 (d, 1H, J ¼ 2.0 Hz), 7.42 (dd, 1H, J ¼ 8.4 Hz,
2.0 Hz), 7.58e7.60 (m, 2H), 7.69 (d, 1H, J ¼ 8.8 Hz). 13C NMR (CDCl3,
4.1.2.2. (E)-1-(5-Methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(4-
methoxy-3-(2-(pyrrolidin-1-yl)ethoxy)phenyl)prop-2-en-1-one (10f).
100 MHz) d: 13.8, 22.2, 27.1, 28.0, 28.0, 33.7, 55.8, 56.0, 76.9, 103.5,
Yield: 65%; yellow liquid; 1H NMR (CDCl3, 400 MHz)
d
: 1.51 (s, 6H),
110.4, 112.4, 116.8, 121.7, 122.1, 126.2, 127.5, 128.7, 128.8, 131.7, 140.1,
140.4, 152.6, 153.6, 158.4, 171.7, 189.8. MS found (M þ H)þ (m/z):
451.24. HPLC purity ¼ 99.5%.
1.81e1.82 (m, 4H), 2.65e2.66 (m, 4H), 2.98 (t, 2H, J ¼ 6.4 Hz), 3.84
(s, 6H), 4.20 (t, 2H, J ¼ 6.4 Hz), 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), 6.90 (d, 1H, J ¼ 8.4 Hz), 7.20 (d,
1H, J ¼ 1.6 Hz), 7.21 (dd, J ¼ 8.8 Hz, 1.6 Hz), 7.57 (d, 1H, J ¼ 16.0 Hz),
7.62 (d, 1H, J ¼ 16.0 Hz), 7.70 (d, 1H, J ¼ 8.4 Hz); 13C NMR (CDCl3,
4.1.3.3. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
8-yl)-3-oxoprop- 1-en-1-yl)phenyl isobutyrate (10k). Yield: 70%;
100 MHz)
d: 23.5, 23.5, 28.0, 28.0, 54.5, 54.5, 54.8, 55.7, 55.9, 67.7,
light yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 1.32 (s, 3H), 1.34 (s,
76.7, 103.5, 110.4, 111.5, 111.8, 116.8, 121.9, 122.8, 125.4, 128.5, 128.6,
131.6, 141.5, 148.4, 151.3, 153.5, 158.3, 189.8. MS found (M þ H)þ
(m/z): 464.31. HPLC purity ¼ 97.8%.
3H), 1.49 (s, 6H), 2.85 (m, 1H, J ¼ 7.6 Hz), 3.86 (s, 3H), 3.88 (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), 6.96 (d, 1H, J ¼ 8.4 Hz), 7.27e7.29 (m, 1H), 7.41 (d, 1H,
J ¼ 8.80 Hz), 7.59e7.61 (m, 2H), 7.70 (d, 1H, J ¼ 8.8 Hz); 13C NMR
4.1.2.3. (E)-1-(5-Methoxy-2,2-dimethyl-2H-chromen-8-yl)-3-(4-
(CDCl3, 100 MHz) d: 19.0, 19.0, 28.0, 28.0, 34.0, 55.8, 56.0, 76.9,
methoxy-3-(2-(piperidin-1-yl)ethoxy)phenyl)prop-2-en-1-one (10g).
103.5, 110.4, 112.3, 116.8, 121.7, 122.0, 126.2, 127.5, 128.7, 128.8, 131.7,
140.2, 140.4, 152.7, 153.6, 158.4, 175.0, 189.7. MS found (M þ H)þ
(m/z): 437.22. HPLC purity ¼ 99.2%.
Yield: 53%; yellow liquid; 1H NMR (CDCl3, 400 MHz)
d: 1.24e1.28
(m, 2H), 1.51 (s, 6H), 1.62e1.64 (m, 4H), 2.56e2.57 (m, 4H), 2.89 (t,
2H, J ¼ 6.4 Hz), 3.84 (s, 6H), 4.20 (t, 2H, J ¼ 6.4 Hz), 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), 6.88 (d,
1H, J ¼ 8.0 Hz), 7.17e7.20 (m, 2H), 7.56 (d, 1H, J ¼ 15.6 Hz), 7.62 (d,
1H, J ¼ 15.6 Hz), 7.70 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
4.1.4. General preparation of 10len (method D)
Compound 9 (147.3 mg, 0.4 mmol) was dissolved in dry CH2Cl2
(10 mL). Then, requisite acyl chloride was added (0.6 mmol),
followed by triethylamine (0.6 mmol, dropwise), and the reaction
mixture was stirred at room temperature for 6 h. Reaction
progress was followed by TLC. The reaction mixture was washed
with: 5 mL, 0.1 M HCl and 2 ꢁ 5 mL NaCl brine (pH w 6). The
organic phase was dried with Na2SO4, filtered, the solvent was
evaporated and the product was dried under vacuum (a pale
yellow solid).
d: 23.1, 24.7, 24.7, 27.0, 27.0, 53.9, 54.8, 54.9, 56.6, 64.8, 67.7, 76.7,
102.4, 109.4, 110.5, 110.8, 116.0, 120.7, 122.0, 124.4, 128.5, 128.6,
130.6, 141.5, 147.4, 151.3, 153.5, 157.4, 189.0. MS found (M þ H)þ
(m/z): 478.36. HPLC purity ¼ 97.3%.
4.1.2.4. (E)-3-(3-(3-(Dimethylamino)propoxy)-4-methoxyphenyl)-1-
(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)prop-2-en-1-one (10h).
Yield: 59%; yellow liquid; 1H NMR (CDCl3, 400 MHz)
d: 1.50 (s, 6H),
2.04e2.07 (m, 2H), 2.28 (s, 6H), 2.50 (t, 2H, J ¼ 6.4 Hz), 3.85 (s, 6H),
4.09 (t, 2H, J ¼ 6.4 Hz), 5.61 (d, 1H, J ¼ 10.0 Hz), 6.50 (d, 1H,
J ¼ 9.2 Hz), 6.68 (d, 1H, J ¼ 10.0 Hz), 6.86 (d, 1H, J ¼ 8.0 Hz), 7.15e
7.17 (m, 2H), 7.56 (d, 1H, J ¼ 16.0 Hz), 7.61 (d, 1H, J ¼ 16.0 Hz), 7.69
4.1.4.1. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
8-yl)-3-oxoprop-1-en-1-yl)phenyl acrylate (10l). Yield: 43%; light
yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 1.49 (s, 6H), 3.88 (s, 6H),
5.62 (d, 1H, J ¼ 10.0 Hz), 6.02 (d, 1H, J ¼ 10.4 Hz), 6.36 (dd, 1H,
J ¼ 17.2 Hz, 10.4 Hz), 6.50 (d, 1H, J ¼ 8.4 Hz), 6.62 (d, 1H, J ¼ 17.2 Hz),
6.68 (d, 1H, J ¼ 10.0 Hz), 6.98 (d, 1H, J ¼ 8.8 Hz), 7.32 (d, 1H,
J ¼ 2.0 Hz), 7.46 (dd, 1H, J ¼ 8.4 Hz, 2.0 Hz), 7.54 (d, 1H, J ¼ 15.6 Hz),
7.59 (d, 1H, J ¼ 15.6 Hz), 7.70 (d, 1H, J ¼ 8.8 Hz); 13C NMR (DMSO,
(d, 1H, J ¼ 8.8 Hz); 13C NMR (DMSO, 100 MHz)
d: 26.6, 27.5, 27.5,
44.9, 44.9, 55.4, 55.6, 55.9, 66.5, 76.7, 104.1, 109.8, 111.0, 111.9, 116.0,
121.2, 122.8, 124.8, 127.6, 129.2, 131.1, 141.4, 148.3, 151.1, 152.6,
157.8, 188.6. MS found (M
purity ¼ 97.6%.
þ
H)þ (m/z): 452.30. HPLC
100 MHz) d: 27.5, 27.5, 55.9, 56.0, 76.7, 104.1, 109.8, 113.2, 116.0,

D. Cao et al. / European Journal of Medicinal Chemistry 62 (2013) 579e589
587
121.1, 122.4, 125.8, 127.2, 127.4, 127.9, 129.3, 131.0, 133.9, 139.2, 140.1,
152.5, 152.6, 157.8, 163.4, 188.7. MS found (M þ H)þ (m/z): 421.17.
HPLC purity ¼ 99.1%.
d: 1.44 (s, 6H), 3.80 (s, 3H), 3.87 (s, 3H), 3.96 (s, 2H), 5.60 (d, 1H,
J ¼ 10.0 Hz), 6.49 (d, 1H, J ¼ 9.2 Hz), 6.67 (d, 1H, J ¼ 10.0 Hz), 6.94 (d,
1H, J ¼ 8.4 Hz), 7.25e7.27 (m, 2H), 7.42 (d, 1H, J ¼ 8.8 Hz), 7.49 (d,
1H, J ¼ 8.0 Hz), 7.56e7.60 (m, 3H), 7.68e7.98 (m, 2H); 13C NMR
4.1.4.2. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
(CDCl3, 100 MHz) d: 27.9, 27.9, 40.7, 55.8, 55.9, 76.9, 103.5, 110.5,
8-yl)-3-oxoprop-1-en-1-yl)phenyl 2-chloroacetate (10m). Yield:
112.4,116.8,121.7, 121.9, 124.2, 126.2, 126.2, 126.4, 127.7,128.7, 128.9,
129.1, 131.7, 132.9, 134.5, 140.0, 140.1, 152.4, 153.6, 158.4, 168.7, 189.7.
MS found (M þ H)þ (m/z): 553.27. HPLC purity ¼ 98.2%.
73%; light yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 1.49 (s, 6H),
3.88 (s, 6H), 4.35 (s, 2H), 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), 6.99 (d, 1H, J ¼ 8.4 Hz), 7.33 (d,
1H, J ¼ 2.0 Hz), 7.46 (dd, 1H, J ¼ 8.4 Hz, 1.6 Hz), 7.54 (d, 1H,
J ¼ 15.6 Hz), 7.59 (d, 1H, J ¼ 15.6 Hz), 7.70 (d, 1H, J ¼ 8.8 Hz); 13C
4.1.5.4. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
8-yl)-3-oxoprop-1-en-1-yl)phenyl 2-(3,4-dichlorophenyl)acetate
NMR (DMSO, 100 MHz)
d
: 27.5, 27.5, 40.8, 55.9, 56.1, 76.7, 104.1,
(10r). Yield: 70%; light yellow solid; 1H NMR (CDCl3, 400 MHz)
d:
109.8, 113.4, 116.0, 121.0, 122.0, 125.9, 127.8, 127.9, 131.0, 139.1, 140.0,
152.2, 152.7, 157.8, 165.7, 188.7. MS found (M þ H)þ (m/z): 443.08.
HPLC purity ¼ 98.8%.
1.45 (s, 6H), 3.83 (s, 3H), 3.86 (s, 2H), 3.88 (s, 3H), 5.62 (d, 1H,
J ¼ 10.0 Hz), 6.50 (d, 1H, J ¼ 8.4 Hz), 6.68 (d, 1H, J ¼ 10.0 Hz), 6.96 (d,
1H, J ¼ 8.4 Hz), 7.23e7.26 (m, 2H) 7.43 (d, 2H, J ¼ 8.0 Hz), 7.54e7.58
(m, 3H), 7.70 (d, 1H, J ¼ 8.8 Hz); 13C NMR (DMSO, 100 MHz)
d: 27.4,
4.1.4.3. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
27.4, 38.6, 56.0, 56.1, 76.7, 104.2, 109.8, 113.3, 116.0, 121.0, 122.0,
125.8, 127.7, 127.9, 129.3, 129.8, 129.9, 130.5, 130.8, 131.1, 131.5,
135.0, 139.4, 140.1, 152.4, 152.6, 157.8, 168.8, 188.7. MS found
(M þ H)þ (m/z): 553.06. HPLC purity ¼ 98.4%.
8-yl)-3-oxoprop-1-en-1-yl)phenyl cyclopropanecarboxylate (10n).
Yield: 67%; light yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 1.02e
1.06 (m, 2H), 1.17e1.20 (m, 2H), 1.49 (s, 6H), 1.88e1.89 (m, 1H),
3.87 (s, 6H), 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), 6.96 (d, 1H, J ¼ 8.4 Hz), 7.31 (d, 1H, J ¼ 2.0 Hz), 7.42
(dd, 1H, J ¼ 8.4 Hz, 2.0 Hz), 7.54 (d, 1H, J ¼ 15.6 Hz), 7.59 (d, 1H,
4.1.5.5. (E)-2-Methoxy-5-((E)-3-(5-methoxy-2,2-dimethyl-2H-chro-
men-8-yl)-3-oxoprop-1-en-1-yl)phenyl 3-(4-methoxyphenyl)acrylate
J ¼ 15.6 Hz), 7.69 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
d
:
(10s). Yield: 72%; light yellow solid; 1H NMR (CDCl3, 400 MHz)
d:
9.3, 9.3, 12.8, 28.0, 28.0, 55.8, 56.1, 76.9, 103.4, 110.4, 112.4, 116.8,
121.7,122.3,126.2,127.4,128.7,128.8,131.7,140.0, 140.4,152.7,153.6,
158.3, 172.9, 189.9. MS found (M þ H)þ (m/z): 435.14. HPLC
purity ¼ 98.9%.
1.49 (s, 6H), 3.86 (s, 9H), 5.61 (d, 1H, J ¼ 10.0 Hz), 6.51 (d, 1H,
J ¼ 8.4 Hz), 6.53 (d, 1H, J ¼ 15.6 Hz), 6.67 (d, 1H, J ¼ 10.0 Hz), 6.93 (d,
2H, J ¼ 8.4 Hz), 7.00 (d, 1H, J ¼ 8.0 Hz), 7.38 (m, 1H), 7.46 (d, 1H,
J ¼ 7.6 Hz), 7.55 (d, 2H, J ¼ 7.6 Hz), 7.57 (d, 1H, J ¼ 14.8 Hz), 7.62 (d,
1H, J ¼ 14.8 Hz), 7.70 (d, 1H, J ¼ 7.6 Hz), 7.83 (d, 1H, J ¼ 15.6 Hz); 13C
4.1.5. General preparation of 10oex (method E)
NMR (CDCl3, 100 MHz) d: 28.0, 28.0, 55.4, 55.8, 56.1, 76.9, 103.5,
Requisite acid (0.45 mmol) was added to a stirred mixture of
compound 9 (147.3 mg, 0.4 mmol), EDCI (115.0 mg, 0.6 mmol),
DMAP (24.4 mg, 0.2 mmol) in anhydrous CH2Cl2 (5 mL). The mix-
ture was stirred at room temperature for 12 h. On completion, the
slurry was partitioned between water (20 mL) and CH2Cl2 (20 mL),
and the water was extracted with CH2Cl2 (3 ꢁ 10 mL). The com-
bined extract of CH2Cl2 and the solvent was removed under
reduced pressure to yield a yellow solid. Chromatographic sepa-
ration (petroleum ethereethyl acetate, 10:1) gave the product as
a pale yellow solid.
110.5, 112.5, 114.1, 114.4, 114.4, 116.7, 121.7, 122.4, 126.2, 126.9, 127.4,
128.7, 128.9, 130.1, 130.1, 131.6, 140.2, 140.5, 146.6, 152.8, 153.6,
158.4, 161.8, 165.1, 189.9. MS found (M þ H)þ (m/z): 527.26. HPLC
purity ¼ 97.5%.
4.1.5.6. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
8-yl)-3-oxoprop-1-en-1-yl)phenyl 4-(4-(bis(2-chloroethyl)amino)
phenyl)butanoate (10t). Yield: 39%; light yellow solid; 1H NMR
(CDCl3, 400 MHz) d: 1.48 (s, 6H), 2.03e2.07 (m, 2H), 2.61 (t, 2H,
J ¼ 7.2 Hz), 2.68 (t, 2H, J ¼ 7.2 Hz), 3.64 (t, 4H, J ¼ 6.8 Hz), 3.72 (t,
4H, J ¼ 6.8 Hz), 3.87 (s, 3H), 3.89 (s, 3H), 5.61 (d, 1H, J ¼ 10.0 Hz),
6.51 (d, 1H, J ¼ 8.8 Hz), 6.66 (d, 2H, J ¼ 8.0 Hz), 6.68 (d, 1H,
J ¼ 10.0 Hz), 6.98 (d, 1H, J ¼ 8.4 Hz), 7.12 (d, 2H, J ¼ 8.0 Hz), 7.27
(m, 1H), 7.44 (d, 1H, J ¼ 8.4 Hz), 7.56 (d, 1H, J ¼ 14.8 Hz), 7.59 (d,
1H, J ¼ 14.8 Hz), 7.70 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3,
4.1.5.1. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-8-
yl)-3-oxoprop-1-en-1-yl)phenyl 3-methoxybenzoate (10o). Yield: 82%;
light yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 1.49 (s, 6H), 3.86 (s,
9H), 5.61 (d, 1H, J ¼ 10.0 Hz), 6.50 (d, 1H, J ¼ 8.8 Hz), 6.67 (d, 1H,
J ¼ 10.0 Hz), 7.02 (d, 1H, J ¼ 8.80 Hz), 7.18 (dd, 1H, J ¼ 8.0 Hz, 2.4 Hz),
7.40e7.43 (m, 2H) 7.49 (d, 1H, J ¼ 8.4 Hz), 7.57 (d,1H, J ¼ 16.0 Hz), 7.62
(d, 1H, J ¼ 16.0 Hz), 7.69e7.72 (m, 2H), 7.82 (d, 1H, J ¼ 7.6 Hz); 13C
100 MHz) d: 26.9, 28.0, 28.0, 33.3, 33.8, 40.5, 40.5, 53.6, 53.6,
55.8, 55.9, 76.9, 103.5, 110.4, 110.4, 112.2, 112.4, 116.7, 121.7, 122.2,
126.2, 127.3, 128.7, 128.8, 129.8, 129.8, 130.5, 131.6, 140.0, 144.4,
152.6, 153.6, 158.4, 171.4, 189.8. MS found (M þ H)þ (m/z): 652.33.
HPLC purity ¼ 96.8%.
NMR (CDCl3, 100 MHz) d: 28.0, 28.0, 55.5, 55.8, 56.1, 76.9, 103.5,
110.5, 112.5, 114.6, 116.7, 120.2, 121.7, 122.4, 122.8, 126.3, 127.5, 128.7,
128.9, 129.6, 130.5, 131.6, 140.3, 140.4, 152.8, 153.6, 158.4, 159.7,
164.4, 189.9. MS found (M þ H)þ (m/z): 501.24. HPLC purity ¼ 98.6%.
4.1.5.7. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-8-
yl)-3-oxoprop-1-en-1-yl)phenyl furan-2-carboxylate(10u). Yield: 88%;
4.1.5.2. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
light yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 1.49 (s, 6H), 3.86 (s,
8-yl)-3-oxoprop-1-en-1-yl)phenyl 4-bromobenzoate(10p). Yield: 64%;
6H), 5.61 (d, 1H, J ¼ 10.0 Hz), 6.50 (d, 1H, J ¼ 8.4 Hz), 6.60e6.64 (m,
1H), 6.67 (d,1H, J ¼ 10.0 Hz), 7.01 (d, 2H, J ¼ 8.4 Hz), 7.41 (m, 2H), 7.48
(d, 1H, J ¼ 8.4 Hz), 7.56 (d, 1H, J ¼ 15.6 Hz), 7.61 (d, 1H, J ¼ 15.6 Hz),
light yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 1.49 (s, 6H), 3.86 (s,
3H), 3.91 (s, 3H), 5.61 (d,1H, J ¼ 10.0 Hz), 6.51 (d, 1H, J ¼ 8.4 Hz), 6.67
(d, 1H, J ¼ 10.0 Hz), 7.02 (d, 1H, J ¼ 8.4 Hz), 7.41 (m, 1H), 7.49 (d, 1H,
J ¼ 8.0 Hz), 7.59e7.63 (m, 2H), 7.66 (d, 2H, J ¼ 8.4 Hz), 7.70 (d, 1H,
J ¼ 8.4 Hz), 8.07 (d, 2H, J ¼ 8.4 Hz). MS found (M þ H)þ (m/z):
549.05. HPLC purity ¼ 98.9%.
7.68 (m, 1H), 7.70 (d, 1H, J ¼ 8.8 Hz); 13C NMR (DMSO, 100 MHz)
d:
27.5, 27.5, 55.9, 56.1, 76.7, 104.1, 109.8, 112.8, 113.3, 115.9, 120.4, 121.1,
122.7, 125.9, 127.5, 127.9, 129.3, 131.0, 138.7, 140.1, 142.6, 148.7, 152.5,
152.6, 155.7, 157.8, 188.9. MS found (M þ H)þ (m/z): 461.13. HPLC
purity ¼ 98.6%.
4.1.5.3. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
8-yl)-3-oxoprop-1-en-1-yl)phenyl 2-(3-(trifluoromethyl)phenyl)ace-
tate (10q). Yield: 69%; light yellow solid; 1H NMR (CDCl3, 400 MHz)
4.1.5.8. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
8-yl)-3-oxoprop-1-en-1-yl)phenylmethanesulfonate(10v). Yield: 78%;

588
D. Cao et al. / European Journal of Medicinal Chemistry 62 (2013) 579e589
light yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 1.52 (s, 6H), 3.89 (s,
4.2.4. Tubulin staining
3H), 3.94 (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), 7.01 (d, 2H, J ¼ 8.8 Hz), 7.46 (dd, 1H, J ¼ 8.4 Hz,
2.0 Hz), 7.59 (d, 1H, J ¼ 15.6 Hz), 7.61 (d, 1H, J ¼ 2.0 Hz), 7.65 (d, 1H,
HepG2 cells were grown on glass covers lip sin 6-well plates and
treated with 1 mM selected compound for 24 h. Then cells were
washed with 1 ꢁ PBS once, fixed in cold 4% PFA for 10 min. After 1 h
J ¼ 15.6 Hz), 7.72 (d, 1H, J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz)
d:
blocking with 5% goat serum albumin/0.3%Triton/PBS, the cells
28.0, 28.0, 38.3, 55.8, 56.2, 77.1, 103.5, 110.5, 112.9, 116.6, 121.4, 123.0,
126.9, 128.8, 129.2, 129.3, 131.7, 138.5, 139.4, 152.6, 153.8, 158.5, 189.4.
MS found (M þ H)þ (m/z): 445.10. HPLC purity ¼ 98.2%.
were incubated with monoclonal anti-a tubulin (Santa Cruz)
overnight at 4 ꢀC, washed in PBS and incubated with Alexa Fluor
488 goat anti-mouse IgG (H þ L; Invitrogen) for 30 min at room
temperature. Cells were washed in PBS and microtubule distribu-
tion imaged on either an Olympus IX81 fluorescence microscope or
Bio-Rad MRC1024 confocal microscope. Propidium iodide was
4.1.5.9. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chromen-
8-yl)-3-oxoprop-1-en-1-yl)phenyl phenylmethanesulfonate (10w).
Yield: 81%; light yellow solid; 1H NMR (CDCl3, 400 MHz)
d: 1.51 (s,
added at 5 mg/mL to stain for nuclei.
6H), 3.88 (s, 3H), 3.93 (s, 3H), 4.59 (s, 2H), 5.61 (d, 1H, J ¼ 10.0 Hz),
6.51 (d, 1H, J ¼ 8.8 Hz), 6.67 (d, 1H, J ¼ 10.0 Hz), 6.99 (d, 1H,
J ¼ 8.4 Hz), 7.26e7.29 (m, 1H), 7.36e7.49 (m, 6H), 7.52 (d, 1H,
J ¼ 15.6 Hz), 7.57 (d, 1H, J ¼ 15.6 Hz), 7.70 (d, 1H, J ¼ 8.8 Hz); 13C
4.2.5. In vitro tubulin polymerization assay
2 mg/mL Tubulin form pig brain (Cytoskeleton) was resus-
pended in PEM buffer [80 mM PIPES, (pH 6.9), 0.5 mM EGTA,
2 mM MgCl2and 15% glycerol], and then was pre-incubated with
compounds or its vehicle DMSO on ice. PEG containing GTP was
added to the final concentration of 1 mM before detecting the
tubulin polymerization reaction. The reaction was monitored by
a spectrophoto-meter in absorbance at 340 nm at 37 ꢀC every
30 s. The final concentrations of the compound were list as
NMR (CDCl3, 100 MHz) d: 28.0, 28.0, 55.8, 56.2, 57.6, 77.2, 103.5,
110.5, 112.8, 116.6, 121.5, 122.9, 126.9, 127.4, 128.8, 128.8, 128.9,
128.9, 129.2, 129.2, 130.9, 130.9, 131.7, 138.7, 139.5, 152.8, 153.8,
158.5, 189.6. MS found (M þ H)þ (m/z): 521.14. HPLC purity ¼ 98.0%.
4.1.5.10. (E)-2-Methoxy-5-(3-(5-methoxy-2,2-dimethyl-2H-chro-
men-8-yl)-3-oxoprop-1-en-1-yl)phenyl 4-methylbenzenesulfonate
follow: 10i (5, 10, 20
mM), Taxol (5, 20 mM).
(10x). Yield: 79%; light yellow solid; 1H NMR (CDCl3, 400 MHz)
d:
1.52 (s, 6H), 2.35 (s, 3H), 3.60 (s, 3H), 3.89 (s, 3H), 5.63 (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), 6.83 (d,
1H, J ¼ 8.4 Hz), 7.29 (d, 1H, J ¼ 8.0 Hz), 7.38 (dd, 1H, J ¼ 8.8 Hz,
2.0 Hz), 7.44 (d, 1H, J ¼ 2.0 Hz), 7.53 (d, 1H, J ¼ 16.0 Hz), 7.58 (d, 1H,
J ¼ 15.6 Hz), 7.71 (d, 1H, J ¼ 8.8 Hz), 7.74 (d, 1H, J ¼ 8.4 Hz); 13C NMR
4.2.6. Western blot analysis
Cells were treated with indicated concentrations of the selected
test agents for treatment duration. After 24 h treatment, cells were
washed with PBS three times. Then added lysis buffer (20 mM Trise
HCl, pH 6.8, 1 mM MgCl2, 2 mM EGTA, 1 mM PMSF, 1 mM ortho-
vanadate, 0.5% NP-40and 20 mg/mL proteinase inhibitor) on ice for
30 min. Supernatants were collected after centrifugation at
15,000 g for 10 min at 4 ꢀC. This yielded soluble tubulin dimers in
the supernatant and polymerized microtubules in the pellet. Equal
amounts of the two fractions were analyzed by western blot probed
(CDCl3, 100 MHz) d: 21.7, 28.0, 28.0, 55.8, 55.9, 77.2, 103.5, 110.5,
112.7, 116.5, 121.5, 122.9, 126.9, 128.6, 128.6, 128.7, 128.8, 128.8,
129.2, 129.2, 131.7, 132.9, 138.7, 139.5, 145.2, 153.1, 153.8, 158.5,
189.6. MS found (M þ H)þ (m/z): 521.19. HPLC purity ¼ 98.2%.
with a-tubulin antibody and secondary HRP-conjugated antibody.
4.2. Biological assay methods
The blots were developed using an Enhanced Chemiluminescence
reagent kit, followed by development on Kodak Bio-MAX MR film
(Rochester, NY).
4.2.1. Tissue culture and reagents
Taxol was purchased from Sigma. Cell lines were purchased from
American Type Culture Collection (ATCC) and maintained following
ATCCrecommendations. Cell lines were routinelygrown in DMEMor
RPM1 (CellGro) supplemented with 10% fetal bovine serum (Cell-
Generation, CO) and 1 unit/mL penicillinestreptomycin (Gibco).
4.2.7. In vivo tumor models
Thirty mice were randomly assigned to five groups. For
HepG2 xenografts (5 ꢁ 106 per mouse), we used 5e6-week-old
female Balb/C and athymic nude mice respectively, implanted the
indicated number of cells suspended in 100
mE HBSS in the
4.2.2. Cell proliferation assay
right flank of mice. Length and width of tumors were measured,
Antiproliferative activities were maintained in the HepG2 cells,
A375 cells, K562 cells, normal cell line L02 cells. Cells in logarithmic
phase were diluted to a density of 40,000e50,000 cells/mL in cul-
ture medium based on growth characteristics, supplemented with
10% fetal bovine serum, 2 mM glutamine and antibiotics, at 37 ꢀC in
humid atmosphere of 5% CO2. Cells were seeded on 96-well plates
and selected compound were added at various doses for indicated
durations. MTT was then added for 4-h incubation. The MTT for-
mazan precipitate was then dissolved in DMSO, and the absorbance
was measured at a wave length of 590 nm.
and the tumor volume (mm3) was calculated by the formula,
p
/6 ꢁ length ꢁ width2, after tumor cells inoculated 10 days. Six
mice per group were randomly assigned. When tumor volumes
reached 100 mm3, the animals were treated for three weeks at the
doses of 10i (2.5 mg/kg),10i (5 mg/kg) and 10i (10 mg/kg) every two
days. And the positive control was Taxol (5 mg/kg) every four days.
Signs of toxicity and mortality were observed daily. Tumor volumes
and body weights were measured every two days when adminis-
trated with a caliper (calculated volume (mm3) ¼
p/6 ꢁ length-
ꢁ width ꢁ width). The anti-tumor activity of compound was
evaluated by two parameters: T/C value (%) ¼ (tumor volume of
4.2.3. Flow cytometry
treated group)/(
Dtumor volume of control group) ꢁ 100%, and tu-
HepG2 cells were incubated with various concentrations (0.2,
mor inhibitor ¼ (1 ꢂ tumor weight of treat group/tumor weight of
1 m
M) of selected compound or DMSO vehicle for 24 h at 37 ꢀC in an
control group) ꢁ 100%.
incubator supplemented with 5% CO2. Then cells were fixed in ice-
cold 70% ethanol overnight at 4 ꢀC. The cell DNA was stained with
Acknowledgments
5
mg/mL propidium iodide containing 10 mg/mL of DNase-free
RNase at 4 ꢀC for a minimum of 10 min. After treatment, the cells
were collected and the DNA content was analyzed by flowcy-
tometer (TASC240, USA).
The authors greatly appreciate the financial support from
National Key Programs of China during the 12th Five-Year Plan
Period (2012ZX09103101-009).

D. Cao et al. / European Journal of Medicinal Chemistry 62 (2013) 579e589
589
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Appendix A. Supplementary material
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.01.007.
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