Design, synthesis and biological evaluation of millepachine derivatives as a new class of tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.02.002

A series of novel tubulin polymerization inhibitors (9a-9p) have been synthesized and evaluated for their in vitro and in vivo biological activities. Among these compounds, 9e displayed strong antiproliferative activity against several tumor cell lines (I

Full text of DOI:10.1016/j.bmc.2013.02.002

Bioorganic & Medicinal Chemistry 21 (2013) 6844–6854
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of millepachine
derivatives as a new class of tubulin polymerization inhibitors
Guangcheng Wang a, , Fei Peng a, , Dong Cao a, Zhuang Yang a,b, Xiaolei Han a, Juan Liu a
Wenshuang Wu a, Lin He a, Liang Ma a, Jinying Chen a, Yun Sang a,b, Mingli Xiang a, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel tubulin polymerization inhibitors (9a–9p) have been synthesized and evaluated for their
in vitro and in vivo biological activities. Among these compounds, 9e displayed strong antiproliferative
activity against several tumor cell lines (IC50 = 0.15–0.62 lM). Compound 9e was also shown to arrest
cells in the G2/M phase of the cell cycle and inhibit the polymerization of tubulin. Molecular docking
studies suggested that 9e binds into the colchicine binding site of tubulin. In xenograft experiments,
9e exerted more potent anticancer effect than anticancer drug taxol against the H460 Human lung car-
cinoma in BALB/c nude mice. In summary, these findings suggest that 9e is a promising new antimitotic
compound for the potential treatment of cancer.
Received 15 December 2012
Revised 4 February 2013
Accepted 4 February 2013
Available online 13 February 2013
Keywords:
Millepachine
Chalcone
Ó 2013 Elsevier Ltd. All rights reserved.
Tubulin
Anticancer
1. Introduction
Nevertheless, clinical use of these compounds is facing severe
disadvantages, including high toxicity, marginal oral bioavailabil-
Microtubules, composed of
a
/b-tubulin heterodimers, are major
ity, poor solubility, complex synthesis, and drug resistance.18,19
Therefore, synthetic low molecular weight compounds with high
oral bioavailability and potent antitumor activity for first and
second line therapy are urgently needed.
components of the cytoskeleton of eukaryotic cells and play an
important role in a wild number of fundamental cellar functions,
such cell division, formation, maintenance of cell shape, regulation
of motility, cell signaling, secretion, and intracellular transport.1–5
The formation of microtubules is a dynamic process that involves
Chalcones are naturally occurring compounds acting as precur-
sors in the biosynthesis of flavonoids and isoflavonoids, which are
an important pharmacophore of various natural products.
Chalcones also display a variety of biological activities, such
as antimitotic,20 antiproliferative,21 anti-inflammatory,22 anti-
malarial,23 anti-bacterial,24 and anti-fungal activities.25 Over the
last few years, the development of chalcones as tubulin-binding
agents has been drawn widely attention. Recent studies have
shown that some compounds containing the chalcone skeleton
act as cytotoxic agents or as microtubule destabilizing agents,
targeting the colchicine binding site.20,26–28
Millepachine, one of naturally occurring pyranochalcones, is
isolated from Millettia pachycarpa for the first time in our group,
exhibited a potent apoptosis inducing effects, being a promised
candidate for the treatment of cancer.29,30 As previously re-
ported,31 we designed and synthesized a series of millepachine
derivatives and evaluated for their in vitro and in vivo antiprolifer-
ative activity as well as tubulin polymerization inhibitory activity.
Among these novel derivatives, compound 1 was identified as a po-
tent anti-tumor agent which is a potential tubulin polymerization
inhibitor. Unfortunately, the poor solubility and low bioavailability
the polymerization and depolymerization of
a and b tubulin
heterodimers.1 Disruption of the dynamic equilibrium blocks the
cell division machinery at mitosis and leads to an increase in the
number of cells in metaphase arrest, resulting in cell death.6,7
Given their significant role in the growth and function of cells,
the microtubule has become an important target for the design
and development of new antimitotic anticancer agents.8,9
Antimitotic agents are generally derived from natural products
and fall into two classes: (1) microtubule stabilizers including tax-
oids (e.g., paclitaxel and docetaxel)10 along with the more recently
discovered epothilones,11 discodermolide,12 and eleutherobin,13
which prevent the depolymerization of tubulin; and (2) microtu-
bule disrupters, such as Vinca alkaloids (e.g., vincristine,
vinblastine),14 colchicines,15 podophyllotoxin,16 and combretasta-
tin A-4,17 which inhibit the polymerization of tubulin (Fig. 1).
⇑
Corresponding author. Tel.: +86 28 85164063; fax: +86 28 85164060.
E-mail address: Lijuan17@hotmail.com (L. Chen).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.002

G. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6844–6854
6845
Figure 1. Examples of antimitotic natural products.
(7.08%) of compound 1 possibly limited the druggability. Our goal
was to discover an orally available, low toxicity, tubulin inhibitor.
Therefore, as a consequence of the structural optimization, com-
pound 1 was taken for further modification in order to get better
antiproliferative activity and oral bioavailability.
Herein, we report the synthesis and bioactivities of a series of
substituted pyranochalcone compounds based on lead compound
1. The synthesized compounds were evaluated for their cytotoxic
activity toward five different human cancer cell lines and one nor-
mal human cell line. Among these derivatives, compound 9e exhib-
ited most potent antiproliferative activity against HepG2, A549,
A375, SMMC-7221 and K562 cells (IC50 = 0.15, 0.36, 0.62, 0.61
at room temperature to provide 3. Treatment of 3 with 3-chloro-
3-methyl-1-butyne and NaH in anhydrous DMF to afford
compound 4 which heating in pyridine at 120 °C for overnight,
underwent Claisen rearrangement, leading to compound 5.
Condensation of 5 and 4-(diethylamino)benzaldehyde 6 under
Claisen–Schmidt conditions using 50% KOH in methanol provided
the desired compound 7. Deprotection of the MOM groups using
HCl in ethyl acetate at room temperature yielded key intermediate
8, followed by an esterification or etherification of the liberated
phenol with diethyl sulfate, alkyl halide, acid anhydride, sulfonyl
chloride and acid to afford the final desired products 9a–9p.
and 0.52 lM, respectively). Compound 9e was also investigated
3. Biological results and discussion
3.1. In vitro antiproliferative activities
for its inhibition of tubulin polymerization activity. In addition,
computer-based docking studies have been performed to under-
stand the interaction between tubulin and small molecule
inhibitors.
Compounds 9a–9p were evaluated for in vitro cytotoxic activity
against five human cancer cell lines, including human hepatocellu-
lar carcinoma (HepG2), human lung adenocarcinoma (A549), hu-
man malignant melanoma (A375), human hepatocellular
carcinoma (SMMC-7221), human chronic myelogenous leukemia
(K562) and one normal human cell line (LO2). The results ex-
pressed as IC50 values, are reported in Table 1. It was notable that
compound 9e, 9l, and 9n exhibited stronger cytotoxicity activity
2. Chemistry
The synthetic pathway to achieve title compounds 9a–9p is
shown in Scheme 1. 2,4-Dihydroxyacetophenone 2 was regioselec-
tively monoprotected on the nonhydrogen-bonded phenol using
K2CO3 in dry acetone and methoxymethylene chloride (MOMCl)

6846
G. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6844–6854
Scheme 1. Reagents and conditions: (a) K2CO3, MOMCl, acetone, rt; (b) NaH, 3-chloro-3-methyl-1-butyne, DMF, rt; (c) pyridine, 120 °C; (d) HCl-EA, EA, rt; (e) 50% KOH,
methanol, rt; (f) compound 9a: K2CO3, (EtO)2SO2, acetone, rt; compounds 9b–9d: K2CO3, RBr, acetone, rt; compounds 9e, 9h, 9k, 9l: (RCO)2O, pyridine, rt; compounds 9f, 9g,
9m: RSO2Cl, Et3N, CH2Cl2, rt; compounds 9i, 9j, 9n, 9o, 9p: RCOOH, EDCI, DMAP, CH2Cl2, rt.
than compound 1. Compound 9e, the most potent one, which con-
tains a propionyloxy group at the 4-position of the left phenyl ring,
exhibited enhanced inhibitory activity against HepG2, A549, A375,
SMMC-7221 and K562 cancer cell lines, with IC50 values of cells
0.15, 0.36, 0.62, 0.61 and 0.52 lM, respectively. Thus, it showed
greater than twice the inhibitory activity of 1 against cancer cell
microtubules at 37 °C. The assembly is accompanied by an increase
in turbidity which can be measured spectrophotometrically at k
340 nm. As shown in Figure 2, compound 9e proved to be a strong
inhibitor of tubulin polymerization, showed a trend similar to
colchicine. The results demonstrated that 9e growth inhibition is
correlated with inhibition of tubulin polymerization.
lines and had low cytoxicity on normal human cell line (LO2).
3.3. Immunofluorenscence staining
3.2. Tubulin polymerization assay
In order to confirm the anti-microtubule effect in cells, we
investigated microtubule structure and distribution in cultured
HepG2 cells. Microtubules were labelled by the indirect immuno-
fluorescence method and analysed by fluorescence microscopy.
To investigate whether the antiproliferative activity of
compound 9e was related to the interaction with tubulin, it was
evaluated for its inhibition of tubulin polymerization in a cell-free
in vitro assay. The assay was using a polymerisation enhancer
(paclitaxel) (data not shown) and polymerisation suppressor
(colchicine) as a positive control. In the presence of GTP and
Cells treated with DMSO (control cells) stained with
a-tubulin
monoclonal anti-body demonstrated a well-organized microtubule
network throughout the cells (Fig. 3). In contrast, cells treated with
9e (0.1 or 0.5 lM) showed a disruption of the tubulin network and
Mg2+
,
a/b-tubulin is known to be able to assemble in vitro into

G. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6844–6854
6847
Table 1
Cytotoxic activity (IC50, lM) of test compounds against tumor cell and normal cell lines
Compounds
IC50 (lM)
HepG2
A549
A375
SMMC-7221
K562
LO2
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
1
3.01 0.18
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
2.95 0.21
>10.0
2.22 0.29
>10.0
>10.0
>10.0
0.61 0.04
>10.0
0.23 0.02
>10.0
8.60 0.45
9.70 0.49
>10.0
0.78 0.09
3.38 0.20
0.77 0.02
1.62 0.21
0.59 0.05
0.80 0.04
8.60 0.27
>10.0
0.95 0.09
>10.0
>10.0
>10.0
>10.0
>10.0
>10.0
8.20 0.11
>10.0
>10.0
8.60 0.56
>10.0
>10.0
>10.0
>10.0
>10.0
0.15 0.01
9.70 0.52
0.19 0.00
>10.0
5.55 0.17
0.70 0.03
>10.0
0.59 0.02
8.20 0.09
0.53 0.03
1.07 0.13
9.45 0.47
0.59 0.04
0.36 0.03
9.70 0.57
8.45 0.27
>10.0
6.90 0.75
1.61 0.08
>10.0
0.94 0.06
>10.0
0.41 0.05
0.50 0.02
9.10 0.37
1.80 0.16
0.62 0.06
8.40 0.08
0.45 0.02
9.60 0.56
4.10 0.57
9.20 0.31
>10.0
0.61 0.09
3.75 0.36
0.26 0.02
0.48 0.06
>10.0
0.52 0.03
4.42 0.23
0.53 0.02
8.75 0.48
2.92 0.02
0.59 0.02
>10.0
0.32 0.01
4.25 0.23
0.51 0.02
1.05 0.24
6.57 0.56
0.79 0.23
>10.0
>10.0
>10.0
>10.0
0.85 0.41
a The cytotoxic activities of the compounds against all the tested cell lines were determined using the MTT assay. The results were expressed as the IC50, date are the mean of
three independent experiments.
5.22% (control) to 15.95% (60 nM), 38.59% (125 nM), 50.86%
(250 nM), 75.77% (500 nM) and 94.84% (1000 nM), respectively
(Fig. 4). Our results shown that 9e arrests HepG2 cells at the
G2/M phase in a dose-dependent manner. In summary, the effect
of compound 9e on cell cycle progression is correlated well with
its strong antiproliferative and antitubulin activity. Compound 9e
has been identified as candidate antimitotic agents.
3.5. In vivo antitumor activity
To evaluate in vivo antitumor activity of compound 9e, we
tested the antitumor activity of compound 9e on a human tumor
xenograft model in mice. Eighteen female BALB/c nude mice were
randomly divided into three groups: a vehicle control group, a
taxol (5 mg/kg) treatment group, and a 9e (20 mg/kg) treatment
group. Human lung carcinoma xenografts were established by sub-
cutaneous injection of H460 cells in right gluteal region of nude
mice. Ten days after, tumor volume reached 100 mm3, treatment
started with 9e injected intraperitoneally twice daily at doses of
20 mg/kg or with taxol injected intraperitoneally at doses of
5 mg/kg every 4 days for 14 days.
Figure 2. Effects of 9e on in vitro tubulin polymerization assay. Purified bovine
The results are shown in the Figure 5. Compound 9e caused a
remarkable reduction in tumor growth (52.06%) as compared with
administration of vehicle only and better than positive control
anticancer drug taxol, which caused a 43.50% of reduction. Other-
wise, there were no significant changes in body weights for the 9e
and taxol treated groups compared to the vehicle treated group.
tubulin and GTP were mixed in a 96-well plate. The reaction was started by
warming the solution from 4 to 37 °C. Colchicine (2 M) was used as a reference,
while ethanol (1% v/v) was used as a vehicle control. The effect on tubulin assembly
was monitored in Spectramax 340PC spectrophotometer at 340 nm at 60 s
l
a
intervals for 60 min at 37 °C.
the appearance of a bundle at a pole of the cell compared with con-
trol cells (Fig. 3). Taken together, these results confirm tubulin is
the molecular target of compound 9e.
3.6. Oral efficacy
The original lead compound 1 was only 7.08% bioavailable in
rats when dosed orally at a dose of 12 mg/kg. However, compound
9e, which replaced the methoxyl group at the 4-position of the left
phenyl ring with propionyloxy group, was improved oral bioavail-
ability to 26.25% at a dose of 12 mg/kg. Our results show that 9e
displays modest to excellent oral bioavailability (Table 2).
3.4. Flow cytometric assay
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 rel-
ative peak in the DNA histogram.32,33 To gain further insight into
the mode of action, the effect of compound 9e on the cell cycle
was measured by flow cytometry against HepG2 human cancer
cells. HepG2 cells were treated with compound 9e at 60, 125,
250, 500 and 1000 nM concentration and the majority of control
cells treated with DMSO showed 5.22% of cells in G2/M phase.
Compound 9e caused significant arrest of the cells at the G2/M
phase relative to the untreated control, raising the G2/M peak from
3.7. Molecular docking
To gain better understanding on the potency of the studied
compounds and guide further SAR studies, we proceeded to exam-
ine the interaction of compound 9e with tubulin which 3D struc-
ture was gained from Protein Data Bank (PDB ID: 3E22).34 The

6848
G. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6844–6854
Figure 3. Effects of 9e on the microtubule network of HepG2 cancer cells. Untreated (control) and cells treated with compound 9e at concentration of 0.5 and 0.1
lM for 24 h,
were fixed in methanol and stained with -tubulin and counterstained with 4,6-diamidino-2-phenylindole (DAPI). Microtubules and unassembled tubulin are shown in
a
green. DNA, stained with DAPI, is shown in blue.
molecular docking was performed by simulation of compound 9e
into the colchicine binding site of tubulin.
suggested that compound 9e binds into the colchicine binding site
of tubulin. In summary, these findings suggest that 9e is a promis-
ing new antimitotic compound for the potential treatment of
cancer.
Molecule 9e was built with ChemBio3D and optimized at
molecular mechanical and semi-empirical level by using Hyper-
chem software.35 Conformers of the compound 9e were created
by the aid of Omega36 and the up limit of conformer number was
set to 2000. Then they were docked to the binding site of tubulin
by employing a protein-ligand docking program FRED.37 Scoring
function chemgauss338 was used for exhaustive searching, solid
body optimizing and interaction scoring. The pose with the most
favorable score was remained.
The binding site of tubulin is a semi-enclosed pocket. As shown
in Figure 6, the N,N-diethylaniline moiety of the compounds was
surrounded by residues Asn258, Met259, Thr314, Asn350,
Asn349, Val315, Lys352 and Ala316 which are consist of a hydro-
phobic domain. Residues Leu255, Leu252, Ala250, Leu242,
Val238, Cys241, Val318, Ala354 and Leu248 form a polar domain,
and the other part of the compound 9e is located there.
Overall, the result of the molecular docking study showed that
compound 9e could act synergistically to interact with the colchi-
cine binding site of tubulin, suggested that compound 9e was a po-
tential inhibitor of tubulin.
5. Experimental section
5.1. Chemistry
All starting materials and reagents were purchased from com-
mercial 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 re-
ported in parts per million. Mass spectra (MS) were measured by
Q-TOF Priemier mass spectrometer (Micromass, Manchester, UK).
5.1.1. 1-(2-Hydroxy-4-(methoxymethoxy)phenyl)ethanone (3)
Chloromethyl methyl ether (9.8 mL, 130 mmol) was added
dropwise into
a mixture of 2,4-dihydroxy acetophenone 2
(15.2 g, 100 mmol) and potassium carbonate (18.0 g, 130 mmol)
in anhydrous acetone (150 mL). The reaction mixture was stirred
at room temperature for 2.5 h. After filtration, the filtrate was
solved in water and extracted with CH2Cl2 (3 Â 100 mL). The or-
ganic layer was dried over Na2SO4 and evaporated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy to afford 3 (75%) as a yellow oil. 1H NMR (400 MHz, CDCl3) d:
2.56 (s, 3H), 3.47 (s, 3H), 5.20 (s, 2H), 6.53–6.58 (m, 2H), 7.63 (d,
1H, J = 8.8 Hz), 12.62 (s, 1H).
4. Conclusions
In conclusion, a series of novel tubulin polymerization inhibi-
tors (9a–9p) have been synthesized and evaluated for their biolog-
ical activities. These compounds exhibited potent tubulin
polymerization inhibitory activities and antiproliferative activities.
Among all these compounds, compound 9e with a propionyloxy
group at the 4-position of the left phenyl ring showed the most po-
tent inhibition activity which inhibited the growth of HepG2, A549,
A375, SMMC-7221 and K562 cancer cell lines with IC50 values of
5.1.2. 1-(4-(Methoxymethoxy)-2-((2-methylbut-3-yn-2-
yl)oxy)phenyl)ethanone (4)
To a solution of 3 (1.96 g, 10 mmol) in anhydrous DMF (80 mL)
was added NaH (0.8 g, 35 mmol). After stirred for 10 min,
3-chloro-3-methylbut-1-yne (3.59 g, 35 mmol) was added drop-
wise into the mixture and stirred for 12 h at room temperature.
0.15, 0.36, 0.62, 0.61 and 0.52 lM, respectively. Compound 9e
was also shown to arrest cells in the G2/M phase of the cell cycle
and inhibit the polymerization of tubulin. Molecular docking study

G. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6844–6854
6849
Figure 4. Effects of compound 9e on HepG2 cell cycle progression. The HepG2 cancer cells were cultured without any compounds (control) or with 9e at 60, 125, 250, 500 or
1000 nM concentration, respectively. The cells were collected and the DNA content was analyzed by flow cytometer (TASC240, USA).
The reaction mixture was poured into ice-cold water and
extracted with ethyl acetate (3 Â 200 mL). The organic layer
was dried over Na2SO4 and evaporated under reduced pressure.
The residue was purified by silica gel column chromatography
to give 4 as a red oil (40%). 1H NMR (400 MHz, CDCl3) d: 1.74
(s, 6H), 2.57 (s, 3H), 2.67 (s, 1H), 3.48 (s, 3H), 5.19 (s, 2H), 6.70
(dd, 1H, J = 8.8 Hz, 2.4 Hz), 7.31 (d, 1H, J = 2.4 Hz), 7.71 (d, 1H,
J = 8.8 Hz).

6850
G. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6844–6854
Figure 5. The antitumor effects of 9e in a human lung carcinoma H460 mouse model. (A) The curves of inhibition of tumors. H460 tumor-bearing nude mice were
administered vehicle alone or 20 mg/kg of 9e twice daily for 14 days, the positive group received injections of taxol at a dose of 5 mg/kg every four days. Each group contained
six mice; bars, SD. (B) Effects of 9e and taxol on mice body weight, bars, SD. (C) The picture of the stripping tumor from mice. (D) Effects of 9e and taxol on tumor weight. ⁄
<0.05, ⁄⁄P <0.01, and ⁄⁄⁄P <0.001, significantly different compared with control by t test.
P
Table 2
Pharmacokinetic parameters of 1 and 9e after iv and po administration
6.64 (d, 2H, J = 8.8 Hz), 6.68 (d, 1H, J = 8.8 Hz), 6.71 (d, 1H,
J = 10.0 Hz), 7.43 (d, 1H, J = 15.2 Hz), 7.46 (d, 2H, J = 8.4 Hz), 7.61–
7.67 (m, 2H); MS (ESI, m/z): 422.25 [M+H]+.
Parameter
1
9e
iv
po
iv
po
Dose (mg/kg)
4
12
3
12
5.1.5. (E)-3-(4-(Diethylamino)phenyl)-1-(5-hydroxy-2,2-
dimethyl-2H-chromen-8-yl)prop-2-en-1-one (8)
AUC (
Cz g/L)
Tmax (h)
Cmax g/L)
lg/L h)
532.468
59.864
0.083
62.916
126.858
1.256
113.075
184.688
6.25
11.742
3.192
44.818
212.444
7.08%
1028.599
1.951
0.083
1500.143
14.068
2.902
1080.168
0.543
4.6
340.608
1.995
19.009
62.268
26.25%
(l
To a solution of 7 (2.1 g, 5 mmol) in ethyl acetate (50 mL) was
added 20 mL HCl–EtOAc solution (1 M), and the mixture was stir-
red at room temperature for 2 h. The reaction mixture was washed
with water, brine and dried over Na2SO4, and then concentrated in
vacuo. The residue was purified by silica gel column chromatogra-
phy to obtain 8 as a yellow solid (85%). 1H NMR (400 MHz, CDCl3)
d: 1.21 (t, 6H, J = 6.8 Hz), 1.52 (s, 6H), 3.39 (q, 4H, J = 6.8 Hz), 5.63
(d, 1H, J = 10.0 Hz), 6.41 (d, 1H, J = 8.4 Hz), 6.65 (d, 2H, J = 8.4 Hz),
6.69 (d, 1H, J = 10.0 Hz), 7.47 (d, 2H, J = 8.8 Hz), 7.50 (d, 1H,
J = 16.0 Hz), 7.57 (d, 1H, J = 8.8 Hz), 7.67 (d, 1H, J = 16.0 Hz). MS
(ESI, m/z): 378.22 [M+H]+.
(
l
Half-life (h)
CLz ((L/h)/kg)
Vz (L/kg)
128.042
60.441
Oral F (%)
5.1.3. 1-(5-(Methoxymethoxy)-2,2-dimethyl-2H-chromen-8-
yl)ethanone (5)
A mixture of 4 (17.5 g) and pyridine (75 mL) was stirred at
120 °C for 12 h. After cooling to room temperature, the mixture
concentrated under reduced pressure to give 5 as black oil, which
was used in the next step without further purification.
5.1.6. (E)-3-(4-(Diethylamino)phenyl)-1-(5-ethoxy-2,2-
dimethyl-2H-chromen-8-yl)prop-2-en-1-one (9a)
Ethyl sulfate (154 mg, 1 mmol) were added to a solution of 7
(188.7 mg; 0.5 mmol) in acetone (5 mL) containing also K2CO3
(172.6 mg, 1.25 mmol). The reaction mixture was stirred for 12 h
at room temperature. After filtration, the filtrate was concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography to give 9a as a yellow solid (39%); mp 99–
100 °C; 1H NMR (400 MHz, CDCl3) d: 1.20 (t, 6H, J = 7.2 Hz), 1.44
(t, 3H, J = 7.2 Hz), 1.51 (s, 6H), 3.38 (q, 4H, J = 7.2 Hz), 4.07 (q, 2H,
J = 7.2 Hz), 5.61 (d, 1H, J = 10.0 Hz), 6.47 (d, 1H, J = 8.4 Hz), 6.65
(d, 2H, J = 8.4 Hz), 6.72 (d, 1H, J = 10.0 Hz), 7.47–7.53 (m, 3H),
7.65 (d, 1H, J = 15.6 Hz), 7.66 (d, 1H, J = 8.8 Hz); 13C NMR (CDCl3,
100 MHz) d: 11.6, 11.6, 13.7, 27.0, 27.0, 43.4, 43.4, 63.0, 75.6,
103.1, 109.4, 110.3, 110.3, 116.0, 120.9, 121.2, 121.5, 127.4,
129.3, 129.3, 130.5, 141.8, 148.1, 152.3, 156.3, 189.2; MS (ESI, m/
z): 406.27 [M+H]+.
5.1.4. (E)-3-(4-(Diethylamino)phenyl)-1-(5-(methoxymethoxy)-
2,2-dimethyl-2H-chromen-8-yl)prop-2-en-1-one (7)
A solution of 50% KOH (30 mL, aq) was added dropwise to a
stirred solution of 5 (2.62 g, 10 mmol) and 4-(diethylamino)benz-
aldehyde 6 (1.77 g, 10 mmol) in methanol (100 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 pH was brought back to 7.0 by the careful addi-
tion of 1 N HCl solution. The aqueous layer was extracted with
ethyl acetate, washed with brine, dried (Na2SO4), and then concen-
trated in vacuo. The residue was purified by silica gel column
chromatography to provide 7 as a yellow solid (73%). 1H NMR
(400 MHz, CDCl3) d: 1.20 (t, 6H, J = 7.2 Hz), 1.51 (s, 6H), 3.38 (q,
4H, J = 7.2 Hz), 3.50 (s, 3H), 5.23 (s, 2H), 5.64 (d, 1H, J = 10.0 Hz),

G. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6844–6854
6851
Figure 6. The interaction mode of compound 9e within the binding site of tubulin (PDB ID: 3E22). Ligand is represented with thick stick and carbon atoms are depicted with
green color. Residues within the binding site were expressed with thin stick and colored by their types.
5.1.7. (E)-3-(4-(Diethylamino)phenyl)-1-(2,2-dimethyl-5-((3-
methylbut-2-en-1-yl)oxy)-2H-chromen-8-yl)prop-2-en-1-one
(9b)
(d, 2H, J = 8.4 Hz), 7.49 (d, 1H, J = 15.6 Hz), 7.65 (d, 1H,
J = 15.6 Hz), 7.66 (d, 1H, J = 8.8 Hz); 13C NMR (CDCl3, 100 MHz) d:
12.6, 12.6, 16.8, 17.7, 25.7, 28.0, 28.0, 39.6, 44.5, 44.5, 59.4, 65.5,
76.6, 104.5, 110.6, 111.3, 111.3, 117.1, 119.3, 120.6, 121.9, 122.2,
123.8, 128.4, 130.3, 130.3, 131.4, 131.9, 141.7, 142.7, 149.2,
153.4, 157.4, 190.2; MS (ESI, m/z): 514.32 [M+H]+.
The compound 9b was prepared in a similar manner to the pro-
cedure described for the preparation of 9a to yield a yellow solid
(37%); mp 92–95 °C; 1H NMR (400 MHz, CDCl3) d: 1.20 (t, 6H,
J = 7.2 Hz), 1.51 (s, 6H), 1.74 (s, 3H), 1.80 (s, 3H), 3.38 (q, 4H,
J = 7.2 Hz), 4.57 (d, 2H, J = 6.8 Hz), 5.48 (t, 1H, J = 6.8 Hz), 5.60 (d,
1H, J = 10.0 Hz), 6.49 (d, 1H, J = 8.8 Hz), 6.64 (d, 2H, J = 8.0 Hz),
6.71 (d, 1H, J = 10.0 Hz), 7.47 (d, 2H, J = 8.0 Hz), 7.49 (d, 1H,
J = 15.2 Hz), 7.65 (d, 1H, J = 15.2 Hz), 7.66 (d, 1H, J = 8.8 Hz); 13C
NMR (CDCl3, 100 MHz) d: 11.6, 11.6, 17.3, 24.8, 27.0, 27.0, 43.4,
43.4, 64.4, 75.6, 103.4, 109.6, 110.3, 110.3, 116.1, 118.4, 120.9,
121.2, 121.6, 127.4, 129.3, 129.3, 130.4, 137.0, 141.7, 148.1,
152.3, 156.3, 189.2; MS (ESI, m/z): 446.26 [M+H]+.
5.1.10. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl propionate (9e)
Propionic anhydride (650 mg, 5 mmol) was added to a solution
of 8 (188.7 mg, 0.5 mmol) in pyridine. The reaction mixture was
stirred at room temperature for 24 h. The solvent was removed un-
der reduced pressure and the residue was purified by silica gel
chromatography to give a yellow solid (68%); mp 81–83 °C; 1H
NMR (400 MHz, CDCl3) d: 1.20 (t, 6H, J = 7.2 Hz), 1.30 (t, 3H,
J = 7.6 Hz), 1.50 (s, 6H), 2.61 (q, 2H, J = 7.6 Hz), 3.38 (q, 4H,
J = 7.2 Hz), 5.70 (d, 1H, J = 10.0 Hz), 6.36 (d, 1H, J = 10.0 Hz), 6.64
(d, 2H, J = 8.0 Hz), 6.69 (d, 1H, J = 8.8 Hz), 7.28 (d, 1H, J = 15.6 Hz),
7.46 (d, 2H, J = 8.0 Hz), 7.55 (d, 1H, J = 8.8 Hz), 7.61 (d, 1H,
J = 15.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 9.2, 12.6, 12.6, 27.6,
28.1, 28.1, 44.5, 44.5, 77.1, 111.3, 111.3, 114.4, 114.6, 116.2,
121.4, 122.1, 126.7, 130.1, 130.6, 130.6, 131.3, 144.3, 148.6,
149.5, 152.7, 172.4, 191.2; MS (ESI, m/z): 434.22 [M+H]+.
5.1.8. (E)-3-(4-(Diethylamino)phenyl)-1-(2,2-dimethyl-5-(prop-
2-yn-1-yloxy)-2H-chromen-8-yl)prop-2-en-1-one (9c)
The compound 9c was prepared in a similar manner to the pro-
cedure described for the preparation of 9a to yield a yellow solid
(45%); mp 140–142 °C; 1H NMR (400 MHz, CDCl3) d: 1.22 (t, 6H,
J = 7.2 Hz), 1.51 (s, 6H), 2.55 (t, 1H, J = 2.0 Hz), 3.40 (q, 4H,
J = 7.2 Hz), 4.76 (d, 2H, J = 2.0 Hz), 5.63 (d, 1H, J = 10.0 Hz), 6.59
(d, 1H, J = 8.8 Hz), 6.65 (d, 2H, J = 8.0 Hz), 6.70 (d, 1H, J = 10.0 Hz),
7.42–7.51 (m, 3H), 7.64 (d, 1H, J = 15.2 Hz), 7.65 (d, 1H,
J = 8.8 Hz); 13C NMR (CDCl3, 100 MHz) d: 11.6, 11.6, 27.0, 27.0,
43.4, 43.4, 55.2, 74.9, 75.7, 77.1, 103.4, 109.9, 110.3, 110.3, 115.7,
120.7, 121.4, 122.1, 127.9, 129.3, 129.3, 130.1, 142.1, 148.2,
152.2, 154.7, 189.3; MS (ESI, m/z): 416.21 [M+H]+.
5.1.11. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl 4-methylbenzenesulfonate (9f)
To a solution of 8 (188.7 mg, 0.5 mmol) in CH2Cl2 (5 mL) was
added 4-toluene sulfonyl chloride (190 mg, 1 mmol) and Et3N
(101 mg, 1 mmol) at room temperature. The mixture stirred at
room temperature for 12 h, and washed with water, brine, dried
over Na2SO4, and concentrated under reduce pressure. The residue
was purified by silica gel column chromatography to provide a yel-
low solid 9f (46%); mp 115–117 °C; 1H NMR (400 MHz, CDCl3) d:
1.20 (t, 6H, J = 7.2 Hz), 1.39 (s, 6H), 2.45 (s, 3H), 3.39 (q, 4H,
J = 7.2 Hz), 5.58 (d, 1H, J = 10.0 Hz), 6.40 (d, 1H, J = 10.4 Hz), 6.60
(d, 1H, J = 8.8 Hz), 6.64 (d, 2H, J = 8.0 Hz), 7.21 (d, 1H, J = 8.8 Hz),
7.31 (d, 2H, J = 8.4 Hz), 7.42 (d, 1H, J = 15.6 Hz), 7.44 (d, 2H,
J = 8.0 Hz), 7.57 (d, 1H, J = 15.6 Hz), 7.73 (d, 2H, J = 8.4 Hz); 13C
NMR (CDCl3, 100 MHz) d: 12.6, 12.6, 21.7, 27.9, 27.9, 44.5, 44.5,
77.2, 111.3, 111.3, 114.5, 115.9, 116.2, 121.1, 121.9, 127.8, 128.6,
5.1.9. (E)-3-(4-(Diethylamino)phenyl)-1-(5-(((E)-3,7-
dimethylocta-2,6-dien-1-yl)oxy)-2,2-dimethyl-2H-chromen-8-
yl)prop-2-en-1-one (9d)
The compound 9d was prepared in a similar manner to the pro-
cedure described for the preparation of 9a to yield a yellow solid
(57%); mp 55–57 °C; 1H NMR (400 MHz, CDCl3) d: 1.20 (t, 6H,
J = 7.2 Hz), 1.25 (s, 6H), 1.51 (s, 6H), 1.68 (s, 3H), 2.08–2.03 (m,
4H), 3.38 (q, 4H, J = 7.2 Hz), 4.56–4.61 (m, 2H), 5.08–5.11 (m,
1H), 5.46–5.49 (m, 1H), 5.60 (d, 1H, J = 10.0 Hz), 6.49 (d, 1H,
J = 8.8 Hz), 6.64 (d, 2H, J = 8.4 Hz), 6.71 (d, 1H, J = 10.0 Hz), 7.47

6852
G. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6844–6854
128.6, 129.9, 129.9, 129.9, 130.6, 130.6, 131.4, 132.2, 144.6, 145.7,
5.1.16. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl pentanoate (9k)
147.1, 149.6, 152.5, 190.9; MS (ESI, m/z): 532.23 [M+H]+.
The compound 9k was prepared in a similar manner to the pro-
cedure described for the preparation of 9e to yield a yellow solid
(76%); mp 53–55 °C; 1H NMR (400 MHz, CDCl3) d: 0.92 (t, 3H,
J = 7.6 Hz), 1.25 (t, 6H, J = 7.2 Hz), 1.50 (s, 6H), 1.60 (m, 2H), 1.80
(m, 2H), 2.35 (t, 2H, J =7.6 Hz), 3.47 (q, 4H, J = 7.2 Hz), 5.70 (d,
1H, J = 10.0 Hz), 6.35 (d, 1H, J = 10.0 Hz), 6.64 (d, 2H, J = 8.0 Hz),
6.68 (d, 1H, J = 8.8 Hz), 7.28 (d, 1H, J = 15.6 Hz), 7.45 (d, 2H,
J = 8.0 Hz), 7.61 (d, 1H, J = 15.6 Hz) 7.65 (d, 1H, J = 8.8 Hz); 13C
NMR (CDCl3, 100 MHz) d: 12.6, 12.6, 13.7, 22.2, 27.0, 28.1, 28.1,
34.0, 44.5, 44.5, 77.1, 111.3, 111.3, 114.4, 114.6, 116.2, 121.4,
122.1, 126.7, 130.1, 130.5, 130.5, 131.2, 144.4, 148.6, 149.5,
152.7, 171.6, 191.3; MS (ESI, m/z): 462.22 [M+H]+.
5.1.12. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl methanesulfonate (9g)
The compound 9g was prepared in a similar manner to the pro-
cedure described for the preparation of 9f to yield a yellow solid
(49%); mp 120–122 °C; 1H NMR (400 MHz, CDCl3) d: 1.22 (t, 6H,
J = 7.2 Hz), 1.50 (s, 6H), 3.20 (s, 3H), 3.39 (q, 4H, J = 7.2 Hz), 5.78
(d, 1H, J = 10.0 Hz), 6.63 (d, 1H, J = 10.0 Hz), 6.66 (d, 2H,
J = 8.4 Hz), 6.91 (d, 1H, J = 8.4 Hz), 7.23 (d, 1H, J = 15.6 Hz), 7.46
(d, 2H, J = 8.4 Hz), 7.53 (d, 1H, J = 8.4 Hz), 7.59 (d, 1H, J = 15.6 Hz);
13C NMR (CDCl3, 100 MHz) d: 12.6, 12.6, 28.1, 28.1, 38.0, 44.5,
44.5, 77.5, 111.3, 111.3, 114.1, 115.7, 116.2, 121.1, 121.9, 128.2,
130.2, 130.7, 130.7, 132.2, 144.9, 146.6, 149.6, 152.8, 191.0; MS
(ESI, m/z): 456.17 [M+H]+.
5.1.17. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl isobutyrate (9l)
5.1.13. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl acetate (9h)
The compound 9l was prepared in a similar manner to the pro-
cedure described for the preparation of 9e to yield a yellow solid
(69%); mp 81–82 °C; 1H NMR (400 MHz, CDCl3) d: 1.21 (t, 6H,
J = 7.2 Hz), 1.34 (d, 6H, J = 15.2 Hz), 1.50 (s, 6H), 2.79–2.89 (m,
1H), 3.41 (q, 4H, J = 7.2 Hz), 5.69 (d, 1H, J = 10.0 Hz), 6.35 (d, 1H,
J = 10.0 Hz), 6.64 (d, 2H, J = 8.0 Hz), 6.67 (d, 1H, J = 8.8 Hz), 7.29
(d, 1H, J = 15.6 Hz), 7.42 (d, 2H, J = 8.0 Hz), 7.55 (d, 1H, J = 8.8 Hz),
7.61 (d, 1H, J = 15.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 12.6, 12.6,
19.0, 19.0, 28.1, 28.1, 34.2, 44.5, 44.5, 77.3, 111.3, 111.3, 114.4,
114.6, 116.1, 121.5, 122.2, 126.7, 130.1, 130.5, 130.5, 131.2,
144.2, 146.9, 148.7, 152.7, 174.9, 191.1; MS (ESI, m/z): 448.20
[M+H]+.
The compound 9h was prepared in a similar manner to the pro-
cedure described for the preparation of 9e to yield a yellow solid
(54%); mp 109–110 °C; 1H NMR (400 MHz, CDCl3) d: 1.20 (t, 6H,
J = 7.2 Hz), 1.50 (s, 6H), 2.35 (s, 3H), 3.38 (q, 4H, J = 7.2 Hz), 5.71
(d, 1H, J = 10.0 Hz), 6.38 (d, 1H, J = 10.0 Hz), 6.64 (d, 2H,
J = 8.0 Hz), 6.69 (d, 1H, J = 8.4 Hz), 7.28 (d, 1H, J = 15.6 Hz), 7.46
(d, 2H, J = 8.0 Hz), 7.55 (d, 1H, J = 8.4 Hz), 7.61 (d, 1H, J = 15.6 Hz);
13C NMR (CDCl3, 100 MHz) d: 12.6, 12.6, 20.9, 28.1, 28.1, 44.5,
44.5, 77.1, 111.3, 111.3, 114.4, 114.6, 116.2, 121.5, 122.2, 126.9,
130.1, 130.5, 130.5, 131.3, 144.2, 148.5, 149.5, 152.7, 168.8,
191.1; MS (ESI, m/z): 420.22 [M+H]+.
5.1.18. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl cyclopropanesulfonate (9m)
The compound 9m was prepared in a similar manner to the pro-
cedure described for the preparation of 9f to yield a yellow solid
(54%); mp 143–145 °C; 1H NMR (400 MHz, CDCl3) d: 0.83–0.89
(m, 2H), 1.22 (t, 6H, J = 7.2 Hz), 1.31–1.35 (m, 2H), 1.50 (s, 6H),
2.63–2.67 (m, 1H), 3.40 (q, 4H, J = 7.2 Hz), 5.77 (d, 1H,
J = 10.0 Hz), 6.66 (d, 2H, J = 8.0 Hz), 6.69 (d, 1H, J = 10.0 Hz), 6.95
(d, 1H, J = 8.8 Hz), 7.23 (d, 1H, J = 15.6 Hz), 7.47 (d, 2H, J = 8.0 Hz),
7.53 (d, 1H, J = 8.8 Hz), 7.60 (d, 1H, J = 15.6 Hz); 13C NMR (CDCl3,
100 MHz) d: 6.5, 6.5, 12.6, 12.6, 28.1, 28.1, 28.3, 44.5, 44.5, 77.2,
111.3, 111.3, 114.5, 115.8, 116.4, 121.2, 122.0, 128.0, 130.1,
130.6, 130.6, 131.8, 144.7, 147.1, 149.6, 152.7, 191.0; MS (ESI,
m/z): 482.13 [M+H]+.
5.1.14. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl acrylate (9i)
A mixture of 8 (188.7 mg, 0.5 mmol), acrylic acid (72 mg,
1 mmol), EDCI (155 mg, 1 mmol) and DMAP (30 mg, 0.25 mmol)
in CH2Cl2 (10 mL) was stirred at room temperature for overnight
and washed with water, brine, dried over Na2SO4, and concen-
trated under reduce pressure. The residue was purified by silica
gel column chromatography to provide a yellow solid 9i (67%);
mp 111–112 °C; 1H NMR (400 MHz, CDCl3) d: 1.20 (t, 6H,
J = 7.2 Hz), 1.50 (s, 6H), 3.39 (q, 4H, J = 7.2 Hz), 5.70 (d, 1H,
J = 10.0 Hz), 6.06 (dd, 1H, J = 10.4 Hz, J = 1.2 Hz), 6.32 (dd,
1H, J = 17.2 Hz, J = 10.4 Hz), 6.37 (d, 1H, J = 10.0 Hz), 6.64 (dd, 1H,
J = 17.2 Hz, J = 1.2 Hz), 6.64 (d, 2H, J = 8.4 Hz), 6.73 (d, 1H,
J = 8.8 Hz), 7.29 (d, 1H, J = 15.6 Hz), 7.46 (d, 2H, J = 8.4 Hz), 7. 56
(d, 1H, J = 8.8 Hz), 7.62 (d, 1H, J = 15.6 Hz); 13C NMR (CDCl3,
100 MHz) d: 12.6, 12.6, 28.1, 28.1, 44.5, 44.5, 77.1, 111.3, 111.3,
114.3, 114.6, 116.2, 121.5, 122.1, 126.9, 127.4, 130.1, 130.5,
130.5, 131.3, 133.3, 144.2, 148.4, 149.4, 152.7, 163.9, 191.1; MS
(ESI, m/z): 432.19 [M+H]+.
5.1.19. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl cyclopropanecarboxylate (9n)
The compound 9n was prepared in a similar manner to the pro-
cedure described for the preparation of 9i to yield a yellow solid
(67%); mp 101–102 °C; 1H NMR (400 MHz, CDCl3) d: 0.83–0.86
(m, 2H), 1.03–1.09 (m, 2H), 1.21 (t, 6H, J = 7.2 Hz), 1.49 (s, 6H),
1.86–1.90 (m, 1H), 3.37 (q, 4H, J = 7.2 Hz), 5.69 (d, 1H,
J = 10.0 Hz), 6.39 (d, 1H, J = 10.0 Hz), 6.63 (d, 2H, J = 8.0 Hz), 6.68
(d, 1H, J = 8.4 Hz), 7.28 (d, 1H, J = 15.6 Hz), 7.45 (d, 2H, J = 8.0 Hz),
7.53 (d, 1H, J = 8.4 Hz), 7.60 (d, 1H, J = 15.6 Hz); 13C NMR (CDCl3,
100 MHz) d: 9.4, 9.4, 12.6, 12.6, 12.9, 28.1, 28.1, 44.5, 44.5, 77.2,
111.3, 111.3, 114.4, 114.6, 116.2, 121.5, 122.2, 126.7, 130.1,
130.5, 130.5, 131.1, 144.2, 148.6, 149.4, 152.6, 172.8, 191.1; MS
(ESI, m/z): 446.21 [M+H]+.
5.1.15. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl furan-2-carboxylate (9j)
The compound 9j was prepared in a similar manner to the pro-
cedure described for the preparation of 9i to yield a yellow solid
(73%); mp 57–58 °C; 1H NMR (400 MHz, CDCl3) d: 1.22 (t, 6H,
J = 7.2 Hz), 1.51 (s, 6H), 3.44 (q, 4H, J = 7.2 Hz), 5.70 (d, 1H,
J = 10.0 Hz), 6.45 (d, 1H, J = 10.0 Hz), 6.62 (dd, 1H, J = 1.2 Hz,
J = 3.2 Hz), 6.65 (d, 2H, J = 8.4 Hz), 6.82 (d, 1H, J = 8.4 Hz), 7.29 (d,
1H, J = 15.6 Hz), 7.43 (d, 1H, J = 3.2 Hz), 7.47 (d, 2H, J = 8.4 Hz),
7.58 (d, 1H, J = 8.4 Hz), 7.62 (d, 1H, J = 15.6 Hz), 7.71 (d, 1H,
J = 1.2 Hz); 13C NMR (CDCl3, 100 MHz) d: 12.6, 12.6, 28.1, 28.1,
44.5, 44.5, 77.2, 111.3, 111.3, 112.4, 114.4, 114.8, 116.2, 119.9,
121.4, 122.1, 127.1, 130.1, 130.5, 130.5, 131.4, 143.5, 144.3,
147.5, 147.9, 149.4, 152.7, 156.2, 191.1; MS (ESI, m/z): 472.19
[M+H]+.
5.1.20. (E)-8-(3-(4-(Diethylamino)phenyl)acryloyl)-2,2-
dimethyl-2H-chromen-5-yl 2-(3-
(trifluoromethyl)phenyl)acetate (9o)
The compound 9o was prepared in a similar manner to the pro-
cedure described for the preparation of 9i to yield a yellow solid
(77%); mp 52–54 °C; 1H NMR (400 MHz, CDCl3) d: 1.20 (t, 6H,

G. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6844–6854
6853
J = 7.2 Hz), 1.47 (s, 6H), 3.38 (q, 4H, J = 6.8 Hz), 3.97 (s, 2H), 5.63 (d,
1H, J = 10.0 Hz), 6.11 (d, 1H, J = 10.40 Hz), 6.64 (d, 2H, J = 8.0 Hz),
6.68 (d, 1H, J = 8.8 Hz), 7.26 (d, 1H, J = 15.6 Hz), 7.45–7.50 (m,
3H), 7.52–7.62 (m, 3H), 7.59 (d, 1H, J = 8.8 Hz), 7.63 (d, 1H,
J = 15.6 Hz); 13C NMR (CDCl3, 100 MHz) d: 12.6, 12.6, 28.1, 28.1,
41.0, 44.5, 44.5, 77.2, 111.3, 111.3, 113.9, 114.2, 114.5, 115.7,
121.4, 122.0, 124.5, 126.2, 127.0, 129.4, 130.1, 130.6, 130.6,
130.7, 131.5, 132.9, 134.1, 144.4, 148.2, 149.5, 152.7, 168.6,
191.1; MS (ESI, m/z): 564.19 [M+H]+.
concentrations of 9e. After treatment, the cells were stained with
propidium iodide (PI) and the cells Cycle were analyzed by flow
cytometer (TASC240, USA). The date was analyzed using Modfit
2.8 software.
5.2.5. Antitumor activity in vivo
Four weeks old, female BALB/c nude mice (15–18 g) were
purchased from the animal breeding laboratories of Sichuan
University Animal Center (Sichuan, Chengdu, China). Eighteen fe-
male BALB/c nude mice were randomly divided into three groups:
a vehicle control group, a taxol (5 mg/kg) treatment group, and a
9e (20 mg/kg) treatment group. Human lung carcinoma xeno-
grafts were established by subcutaneous injection of H460 cells
in right gluteal region of nude mice. Ten days after tumor volume
reached 100 mm3, treatment started with 9e injected intraperito-
neally twice daily at doses of 20 mg/kg or with taxol injected
intraperitoneally at doses of 5 mg/kg every four days for 14 days.
5.1.21. (1r,3r,5r,7r)-8-((E)-3-(4-(Diethylamino)phenyl)acryloyl)-
2,2-dimethyl-2H-chromen-5-yl adamantane-2-carboxylate (9p)
The compound 9p was prepared in a similar manner to the pro-
cedure described for the preparation of 9i to yield a yellow solid
(62%); mp 133–135 °C; 1H NMR (400 MHz, CDCl3) d: 1.21 (t, 6H,
J = 7.2 Hz), 1.49 (s, 6H), 1.58–1.67 (m, 2H), 1.73–1.85 (m, 10H),
1.92–1.98 (m, 2H), 2.05–2.10 (m, 1H), 3.41 (q, 4H, J = 7.2 Hz),
5.68 (d, 1H, J = 10.0 Hz), 6.33 (d, 1H, J = 10.0 Hz), 6.21–6.64 (m,
3H), 7.28 (d, 1H, J = 15.6 Hz), 7.46 (d, 2H, J = 8.0 Hz), 7.54 (d, 1H,
J = 8.4 Hz), 7.60 (d, 1H, J = 15.2 Hz); 13C NMR (CDCl3, 100 MHz) d:
12.6, 12.6, 27.9, 27.9, 28.1, 28.1, 29.7, 36.4, 36.4, 36.4, 36.4, 38.9,
38.9, 41.3, 44.5, 44.5, 77.3, 111.3, 111.3, 114.4, 114.7, 116.2,
121.6, 122.2, 126.6, 130.1, 130.5, 130.5, 131.1, 144.1, 148.9,
149.4, 152.7, 175.4, 191.1; MS (ESI, m/z): 540.29 [M+H]+.
Tumor burden was measured every 2 days with
a caliper
(calculated volume (mm3) =
p
/6  length  width  width). After
completing the treatment schedule, tumor-bearing mice were
euthanized.
5.2.6. Statistical analysis
Unless indicated differently, the results are presented as
mean SEM. The differences between different treatments were
analyzed using the two-sided Student’s t test. P values lower than
0.05 were considered significant.
5.2. Biological assay methods
5.2.1. Cell proliferation inhibition assay (MTT assay)
MTT assay: MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide] was used to identify viable cells which reduce
it to a violet formazan.39 Cells were seeded in 96-well culture plates
(1 Â 104 cells per well) and cultured for 24 h. Incubation (5% CO2,
95% humidity, 37 °C) of the cells following treatment with the test
compounds was continued for 24, 48, or 72 h. MTT in phosphate
Acknowledgement
This study was supported by National Key Technologies R&D
Program of China (2012ZX09103101-009).
References and notes
buffered saline (20
bated at 37 °C for 4 h. The formazan precipitate was dissolved in
150 L of DMSO, after the medium was carefully removed, shaken
lL of 5 mg/mL) was added to each well and incu-
1. Amos, L. A. Org. Biomol. Chem. 2004, 2, 2153.
2. Downing, K. H.; Nogales, E. Curr. Opin. Struct. Biol. 1998, 8, 785.
3. Downing, K. H. Annu. Rev. Cell Dev. Biol. 2000, 16, 89.
4. Sorger, P. K.; Dobles, M.; Tournebize, R.; Hyman, A. A. Curr. Opin. Cell Biol. 1997,
9, 807.
l
mechanically for 30 min, and then absorbance values at a wave-
length of 570 nm were determined with the Spectramax M5 Micro-
titer Plate Luminometer (Molecular Devices, USA). Values were
calculated using percentage of growth versus untreated control.
5. Honore, S.; Pasquier, E.; Braguer, D. Cell. Mol. Life Sci. 2005, 62, 3039.
6. Pasquier, E.; Andre, N.; Braguer, D. Curr. Cancer Drug Targets 2007, 7, 566.
7. Odlo, K.; Hentzen, J.; dit Chabert, J. F.; Ducki, S.; Gani, O. A. B. S. M.; Sylte, I.;
Skrede, M.; Florenes, V. A.; Hansen, T. V. Bioorg. Med. Chem. 2008, 16, 4829.
8. Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T. Med. Res. Rev. 1998, 18,
259.
9. Jordan, M. A.; Wilson, L. Nat. Rev. Cancer 2004, 4, 253.
10. Verweij, J.; Clavel, M.; Chevalier, B. Ann. Oncol. 1994, 5, 495.
11. Kowalski, R. J.; Giannakakou, P.; Hamel, E. J. Biol. Chem. 1997, 272, 2534.
12. Ter Haar, E.; Kowalski, R. J.; Hamel, E.; Lin, C. M.; Gunasekera, S. P.; Rosenkranz,
H. S.; Day, B. W. Biochemistry 1996, 35, 243.
13. Long, B. H.; Carboni, J. M.; Wasserman, A. J.; Cornell, L. A.; Casazza, A. M.;
Jensen, P. R. Cancer Res. 1998, 58, 1111.
14. Rowinsky, E. K.; Tolcher, A. W. In Cancer: Principles and Practice of Oncology;
DeVita, V. T., Hellman, S., Rosenberg, S. A., Eds., 6th ed.; Lippincott-Raven:
Philadelphia, 2001; pp 431–452.
5.2.2. Tubulin polymerization in vitro assay
Turbidimetric assays of microtubules were performed as de-
scribed by Lopes et al.40 MAP-rich tubulin (2 mg/mL) was preincu-
bated in polymerization buffer (0.1 M PIPES, pH 6.9, 1 mM MgCl2)
with various compound at 4 °C for 2 min before the addition of
1 mM GTP. The samples were then rapidly warmed to 37 °C in a
96-well plate thermostatically controlled spectrophotometer, and
the change in absorbance at 340 nm was periodically measured.
Colchicine was included as positive control.
15. Hastie, S. B. Pharmacol. Ther. 1991, 51, 377.
5.2.3. Immunofluorescence staining of tubulin
16. Cortese, F.; Bhattacharyya, B.; Wolff, J. J. Biol. Chem. 1977, 521, 1134.
17. Cushman, M.; He, H.; Lin, C. M.; Hamel, E. J. Med. Chem. 1993, 36, 2817.
18. Attard, G.; Greystoke, A.; Kaye, S.; De Bono, J. Pathol. Biol. 2006, 54, 72.
19. Kavallaris, M.; Verrills, N. M.; Hill, B. T. Drug Resist. Updat. 2001, 4, 392.
20. Edwards, M. L.; Stemerick, D. M.; Sunkara, P. S. J. Med. Chem. 1948, 1990, 33.
21. Loa, J.; Chow, P.; Zhang, K. Cancer Chemother. Pharmacol. 2009, 63, 1007.
22. Lee, S. H.; Sohn, D. H.; Jin, X. Y.; Kim, S. W.; Choi, S. C.; Seo, G. S. Biochem.
Pharmacol. 2007, 74, 870.
23. Li, R. S.; Kenyon, G. L.; Cohen, F. E.; Chen, X. W.; Gong, B. Q.; Dominguez, J. N.;
Davidson, E.; Kurzban, G.; Miller, R. E.; Nuzum, E. O.; Rosenthal, P. J.;
McKerrow, J. H. J. Med. Chem. 1995, 38, 5031.
24. Nielsen, S. F.; Boesen, T.; Larsen, M.; Schonning, K.; Kromann, H. Bioorg. Med.
Chem. 2004, 12, 3047.
HepG2 tumor cells (1 Â 104) were seeded in 96-well culture plate
and incubated for 12 h at 37 °C and 5% CO2. Cells were treatment
with DMSO, 9e (0.5 lM) or 9e (1.0 lM) for 24 h. The cells were fixed
with methanol and then permeabilized with 0.5% Triton X-100/PBS.
After blocking for 45 min in 5% BSA/PBS, cells were washed with PBS
and incubated with
a-tubulinfor 2 h, and then tubulin was immuno-
stained with monoclonal antibody to
a
-tubulin followed by fluores-
cence antibody. Nuclei were labeled with Hoechst 33258. Cells were
visualized using a fluorescence microscope.
25. Lahtchev, K. L.; Batovska, D. I.; Parushev, S. P.; Ubiyvovk, V. M.; Sibirny, A. A.
Eur. J. Med. Chem. 2008, 43, 2220.
26. Ducki, S. Anticancer Agents Med. Chem. 2009, 9, 336.
27. Peyrot, V.; Leynadier, D.; Sarrazin, M.; Briand, C.; Rodriquez, A.; Nieto, J. M.;
Andreu, J. M. J. Biol. Chem. 1989, 264, 21296.
5.2.4. Cell cycle analysis by flow cytometry
HepG2 tumor cells (2 Â 105) were cultured in 6-well cell culture
plates and treated with vehicle alone (0.1% DMSO) or various

6854
G. Wang et al. / Bioorg. Med. Chem. 21 (2013) 6844–6854
28. Peyrot, V.; Leynadier, D.; Sarrazin, M.; Briand, C.; Menendez, M.; Laynez, J.;
Andreu, J. M. Biochemistry 1992, 31, 11125.
33. Hadfield, J. A.; Ducki, S.; Hirst, N.; McGown, A. T. Prog. Cell Cycle Res. 2003, 5,
309.
29. Ye, H.; Fu, A.; Wu, W.; Li, Y.; Wang, G.; Tang, M.; Li, S.; He, S.; Zhong, S.; Lai, H.;
Yang, J.; Xiang, M.; Peng, A.; Chen, L. Fitoterapia 2012, 83, 1402.
30. Wu, W.; Ye, H.; Wang, G.; Han, X.; Hu, J.; Tang, M.; Duan, X.; Fan, Y.; He, S.;
Huang, L.; Pei, H.; Wang, X.; Li, X.; Xie, C.; Zhang, R.; Yuan, Z.; Mao, Y.; Chen, L.;
Wei, Y. Carcinogenesis, accepted for publication.
31. Wang, G.; Wu, W.; Peng, F.; Cao, D.; Yang, Z.; Ma, L.; Qiu, N.; Ye, H.; Han, X.;
Chen, J.; Qiu, J.; Sang, Y.; Liang, X.; Ran, Y.; Peng, A.; Wei, Y.; Chen, L. Eur. J. Med.
Chem. 2012, 54, 793.
34. Cormier, A.; Marchand, M.; Ravelli, R. B. G.; Knossow, M.; Gigant, B. EMBO Rep.
2008, 9, 1101.
35. Menikarachchi, L. C.; Gascón, J. A. Curr. Top. Med. Chem. 2010, 10, 46.
36. Hynes, R. O. Annu. Rev. Cell Dev. Biol. 2002, 110, 673.
37. Gao, J.; Amara, P.; Alhambra, C.; Field, M. J. J. Phys. Chem. A 1998, 102, 4714.
38. Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee, R. P. J. Comput.
Aided Mol. Des. 1997, 11, 425.
39. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
40. Lopes, N. M.; Miller, H. P.; Young, N. D.; Bhuyan, B. K. Cancer Chemother.
Pharmacol. 1997, 41, 37.
32. Lawrence, N. J.; McGown, A. T.; Ducki, S.; Hadfield, J. A. Anti-Cancer Drug Des.
2000, 15, 135.