Design, synthesis, biological evaluation and molecular modeling studies of 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diynes as a new class of potent antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2013.01.011

A series of novel enediyne-containing molecules, 1-aryl-6-(3,4,5- trimethoxyphenyl)-3(Z)-hexen-1,5-diynes, were synthesized and displayed significant IC₅₀ values of 10^-7 to 10^-6 M against various cancer cell lines. Of these compounds, 1-(2-pyridinyl)-6-(3,4,5- trimethoxyphenyl)-3(Z)-hexen-1,5-diyne (8) demonstrated the greatest growth inhibition activity. Compound 8 also arrested cancer cells in the G2/M phase and induced apoptosis via activation of Caspase-3. In addition to the G2/M block, compound 8 caused microtubule depolymerization at low concentrations and markedly decreased tumor size in xenographic studies.

Full text of DOI:10.1016/j.ejmech.2013.01.011

European Journal of Medicinal Chemistry 62 (2013) 526e533
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, biological evaluation and molecular modeling
studies of 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diynes as
a new class of potent antitumor agents
,
,
,
b,e,
*
Yu-Hsiang Lo a 1, Ying-Ting Lin c 1, Yu-Peng Liu d, Tsai-Hui Duh e, Pei-Jung Lu d , Ming-Jung Wu
**
a Graduate Institute of Pharmaceutical Science, Kaohsiung Medical University, Kaohsiung, Taiwan
b Department of Chemistry, National Sun Yat-sen University, Kaohsiung, Taiwan
c Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan
d Institute of Clinical Medicine, National Cheng Kung University, Tainan, Taiwan
e Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan
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 enediyne-containing molecules, 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-
diynes, were synthesized and displayed significant IC50 values of 10ꢀ7 to 10ꢀ6 M against various can-
cer cell lines. Of these compounds, 1-(2-pyridinyl)-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diyne (8)
demonstrated the greatest growth inhibition activity. Compound 8 also arrested cancer cells in the G2/M
phase and induced apoptosis via activation of Caspase-3. In addition to the G2/M block, compound 8
caused microtubule depolymerization at low concentrations and markedly decreased tumor size in
xenographic studies.
Received 29 November 2012
Received in revised form
1 January 2013
Accepted 8 January 2013
Available online 21 January 2013
Keywords:
Enediyne
Ó 2013 Elsevier Masson SAS. All rights reserved.
G2/M arrest
Apoptosis
Microtubule depolymerization
1. Introduction
(CA-4) shows potent inhibition of microtubule polymerization by
blocking the colchicine binding site and rapidly shutting down
Microtubules are a component of the cytoskeleton formed by
highly dynamic assemblies of tubulin heterodimers, including
and -tubulins. Microtubules and their dynamics play a crucial role
existing tumor vasculature at doses below the maximum tolerated
dose [8]. CA-4 may block cells in the G2/M phase of the cell cycle
and induce apoptosis [9].
a
-
b
in many biological processes, including mitosis, intracellular
transport, exocytosis and cell growth [1]. Because microtubules are
important in mitosis and cell division, they have been targets for
the development of a number of new anticancer drugs [2]. These
drugs are often classified into two major groups: the microtubule-
stabilizing agents (inhibit microtubule depolymerization) and the
microtubule-destabilizing agents (enhance microtubule depoly-
merization) [3]. The microtubule-stabilizing agents bind in the
taxoid binding site, such as taxol and docetaxel [4]. Most
microtubule-destabilizing agents bind in the “vinca” binding site or
the “colchicine” binding site, such as vinca alkaloids [5], colchicine
[6], and combretastatin A-4 [7]. For instance, combretastatin A-4
In our earlier publications [10], we reported a series of 1,6-
diaryl-3(Z)-1,5-diynes as new antitumor agents. Of those com-
pounds, 2-(6-(2-trifluoromethylphenyl))-3(Z)-(hexen-1,5-diynyl)
aniline (1) demonstrated the most potent cytotoxic activity against
tumor cells at a concentration of 10ꢀ7 M, inducing a massive
accumulation of cells in G2/M phase and apoptosis via activation of
the Caspase family [10c]. A brief exposure of MDA-MB-231/ATCC
cells to compound 1 was sufficient to produce sustained depoly-
merization of the microtubules in a concentration-dependent
manner. According to our ligand-docking experiment [11], com-
pound 1 binds to
cine. The amino group of compound 1 forms hydrogen bonds with
the amino acids 179-Thr and 181-Val of -tubulin, as would the
hydroxyl group of CA-4 and the carbonyl group of colchicine. The
trifluoromethylphenyl group of compound 1 sits in the pocket of
a- and b-tubulin in the same manner as colchi-
a
* Corresponding author. Department of Chemistry, National Sun Yat-sen Uni-
versity, Kaohsiung, Taiwan. Fax: þ886 7 5253909.
** Corresponding author.
b
-
tubulin, similar to the trimethoxylphenyl subunits of colchicine and
CA-4. Although several 1,6-diaryl-3(Z)-1,5-diynes with trimethox-
ylphenyl subunits have been reported by Provot [12], such as
E-mail address: mijuwu@faculty.nsysu.edu.tw (M.-J. Wu).
Equal contribution.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.011

Y.-H. Lo et al. / European Journal of Medicinal Chemistry 62 (2013) 526e533
527
compounds 2a and 2b [12], most of the compounds demonstrated
a poor ability to inhibit growth in tumor cell lines and did not
interfere with microtubule polymerization. To analyze and com-
pare the structureeactivity relationship of compounds 2a and 2b
with compound 1, we hypothesized that an ortho substituent, such
as an amino group, on the phenyl ring is essential for the formation
of hydrogen bonding with the amino acids 179-Thr and 181-Val of
and the structure of each compound are summarized in Table 2.
Compounds 5, 6, and 8 displayed a broad spectrum of growth in-
hibition against most of the cancer cell lines. Compounds 7, 11 and
12 were inactive against the tested cell lines. Comparing the IC50 of
the novel compounds 3e12 with the previously designed com-
pound 1, compounds 4, 5, 6, 8 and 9 demonstrated higher potency
than compound 1. The results support our hypothesis that two vital
factors, the presence of the trimethoxyphenyl ring and an aryl
group bearing an only ortho substituent, are essential for the bio-
logical activity of these compounds. Although the cytotoxicity of
several compounds was not as potent as colchicine, compounds 5
and 8 demonstrated stronger cytotoxic activity than colchicine in
BT483, HeLa and SK Hep-1 cell lines. Comparing the structuree
activity relationship of compounds 5 and 12, compound 12 had
an extra methoxyl group at the para position of the phenyl ring,
which did not contribute to its cytotoxic activity. A similar phe-
nomenon was also observed in compounds 6 and 7. Replacing an
oxygen atom of the methoxy group of compound 5 to produce 11
decreased the IC50 activity.
a
-tubulin and the biological activity based on our ligand-docking
experiments. In this study, we designed a series of 1-aryl-6-
(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diynes (3e12) that con-
tain two vital components: a trimethoxyphenyl ring that could sit
in the pocket of
substituent that could strengthen the molecular interactions with
the amino acids of -tubulin. The biological activity of these com-
b-tubulin and the other aryl group bearing an ortho
a
pounds was explored both in vitro, and the most active compound
was tested in an animal model (Fig. 1).
2. Results and discussion
Because compound 8 demonstrated the highest level of activity
among these synthetic enediynes, its detailed mechanism of action
was investigated in the BT483 cancer cell line. BT483 cells were
2.1. Chemistry
The synthesis of compounds 3e12 is shown in Scheme 1 and the
yields are summarized in Table 1. 1-Chloro-4-trimethylsilyl-1(Z)-
buten-3-yne (13) [10] was coupled with 3,4,5-trimethoxy-
phenylethyne (14) under Sonogashira coupling reaction condi-
tions to give 15 with a yield of 85%. Compound 15 was then coupled
with various aryl iodides 16aei using Pd(PPh3)4 as the catalyst in
the presence of K2CO3 in MeOH, giving 3e9 and 11,12 with yields of
48e79%. The nitro group of 9 was reduced to an amino group using
SnCl2 to give 10 with a yield of 36%.
treated with 0.2 mM of compound 8 for 12 h and 24 h, and the cell
cycle distribution was analyzed by flow cytometry. As shown in
Fig. 2A, compound 8 significantly arrested the cell cycle in the G2/M
phase at 12 h post-treatment. A significant accumulation of sub-G1
cells was observed when the cells were treated with compound 8
for 24 h, suggesting that apoptosis may occur after treatment.
To confirm the occurrence of apoptosis induced by compound 8,
BT483 cells were treated with compound 8 at different concen-
trations for 24 h, and the cell lysate was subjected to Western blot
analysis. Treatment with irinotecan was used as a positive control
for apoptosis and DNA damage events. As shown in Fig. 2B, treat-
2.2. Biological assay results
ment with compound 8 at a 1 mM concentration for 24 h was suf-
2.2.1. Cytotoxicity activities, cell cycle analysis and electrophoresis
Compounds 3e12 were tested on a panel of human cancer cell
lines, including non-small-cell lung adenocarcinoma (A549),
stomach adenocarcinoma (AGS), prostate cancer (PC-3), breast
carcinoma (BT483), cervical epithelioid carcinoma (Hela), oral
squamous cell carcinoma (SAS), hepatoma (SK Hep-1), and esoph-
ageal squamous cell carcinoma (CE81T). The IC50 in each cell line
ficient to induce Caspase-3 and poly (ADP-ribose) polymerase
(PARP) cleavage, the early stages of apoptosis. In contrast, we found
that the histone H2A variant H2AX was phosphorylated at Ser139 in
response to DNA double-strand breaks after compound 8 treat-
ment. These results indicated that compound 8 triggered a DNA
damage response, consequently leading to cell apoptosis.
OCH3
H3CO
OCH3
O
H3CO
H3CO
H3CO
H3CO
NH
A
B
H3CO
A
H3CO
H3CO
H3CO
C
C
OCH3
O
OH
OCH3
OCH3
Colchicine
H3CO
H3C
Combretastatin A-4 (CA-4)
NH2
2a
2b
Y
F3C
NH2
X = N
X = C, Y = O, N, S
X
H3CO
H3CO
OCH3
Ortho substitution
interacts with microtubulin
1
trimethoxy benzene ring
Fig. 1. Comparison and design of new antitubulin agents.

528
Y.-H. Lo et al. / European Journal of Medicinal Chemistry 62 (2013) 526e533
OCH3
OCH3
OCH3
OCH3
OCH3
SiMe3
+
Pd(PPh3)4
CuI
OCH3
ArI, Pd(PPh3)4
OCH3
OCH3
n-BuNH2
ether, 85%
CuI, K2CO3
MeOH
OCH3
Cl
SiMe3
Ar
13
14
15
3-9, 11, 12
Scheme 1.
2.2.2. Microtubule depolymerization assay
colchicine, which fits better than compound 1. On the other side,
the amino group of compound 6 and the nitrogen atom of com-
The previously reported lead compound 2-(6-(2-trifluoromethyl
phenyl))-3(Z)-(hexen-1,5-diynyl)aniline (1) is known to destabilize
microtubules. It displays significant microtubule-depolymerizing
pound 8 hydrogen bond with Thr179 of
a-tubulin, as shown in
Fig. 4D and E. In short, these finding could explain why the tri-
methoxyphenyl ring and ortho substitution of enediyne serve as
vital factors for the observed bioactivity (For interpretation of the
references to color in this paragraph, the reader is referred to the
web version of this article.).
activity at a concentration of 3.6 mM. To investigate the effects of
compound 8 on microtubule polymerization, a microtubule depo-
lymerization assay was carried out. Our data show that a brief
exposure of BT483 cells to compound 8 was sufficient to produce
sustained depolymerization of the microtubules at a low concen-
tration of 1
mM (Fig. 2C). These results suggest that the loss of
3. Conclusion
microtubule function in compound 8-treated cells at interphase
may block these cells from progressing to mitosis (Fig. 2A).
We have successfully designed a series of novel enediyne-
containing compounds, 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-
hexen-1,5-diynes 3e12, based on our ligand-docking experiment
results and found that these compounds show significant growth
inhibition against various human cancer cell lines. After a detailed
investigation of the biological activities and bio-mechanisms of
these novel synthetic enediynes, we have reached several conclu-
sions: (1) as demonstrated in the structureeactivity relationship
study, the cytotoxicity of compound 6 is better than compound 1,
suggesting the significance of the trimethoxyphenyl ring; (2) the
addition of any substituent to the phenyl ring and substitution at
the ortho position would reduce the potency of cytotoxicity, as
evidenced by the activities of compounds 7, 10 and 12; (3) atoms at
the ortho position that can form strong hydrogen bonds with the
2.2.3. Xenographic studies
The inhibitory function of compound 8 on tumor growth was
evaluated in a nude mouse xenograft model. We first injected
5 ꢁ 106 BT483 cells into nude mice subcutaneously. After the tumor
grew to an appropriate size, 20 mg/kg of compound 8 or vehicle
only was intraperitoneally (i.p.) injected daily for 2 weeks, and the
tumor size was measured every 3 days. As shown in Fig. 3, 20 mg/kg
of compound 8 dramatically diminished tumor growth compared
to the tumor volume of vehicle-treated mice. These results indi-
cated that compound 8 exhibits anti-tumor activity and may be
used as an anti-breast cancer agent in future pre-clinical trials.
2.2.4. Molecular modeling
amino acids in a-tubulin are vital for biological activity, as dem-
We performed docking experiments using the LigandFit pro-
gram to investigate the binding of these synthetic compounds. A
molecular model of colchicines (gray pattern) was fit to an X-ray
structure of colchicines (gold) as a positive control, as shown in
onstrated by the activities of compounds 6 and 8; (4) according to
the results from the cell cycle distribution and Western blot ana-
lyses, compound 8 leads to a massive accumulation of cancer cells
in the G2/M phase after 12 h of treatment and induces apoptosis via
the activation of Caspase-3 after 24 h of treatment; (5) as demon-
strated by the microtubule depolymerization assay, compound 8
Fig. 4A. We previously hypothesized that compound 1 binds to
and -tubulin in the same manner as colchicine [11]. The tri-
fluoromethylphenyl group of 1 sits in the pocket of -tubulin in
a-
b
b
can interfere with the polymerization of microtubules at 1 mM; (6)
a similar manner as the trimethoxyphenyl subunit of colchicine and
the amino group of compound 1, forming hydrogen bonds with
Thr179 of a-tubulin, as shown in Fig. 4B. With regard to compounds
6 and 8, the trimethoxyphenyl rings of compounds 6 and 8 are in
xenographic studies indicated that compound 8 efficiently reduced
tumor sizes in BT483 tumor-bearing mice; and (7) molecular
modeling studies further implicated the significance of a trime-
thoxyphenyl ring and ortho substitution at the other aryl group of
the enediynes.
the same orientation as the trimethoxyphenyl subunit of
This study reveals a new potent antitumor agent, compound 8,
for further development as a new anticancer drug. We also believe
that the information disclosed in this paper will significantly help
in the development of new anticancer drugs.
Table 1
Chemical yields of compounds 3e9, 11 and 12.
Entry
ArI
Products/yields (%)
1
2
3
4
5
6
7
8
9
16a, Ar ¼ 2-nitrophenyl
3/58
4/77
5/72
6/69
7/78
8/79
9/48
11/65
12/49
4. Experimental section
16b, Ar ¼ 2-cyanophenyl
16c, Ar ¼ 2-anisyl
4.1. Cell lines and culture conditions
16d, Ar ¼ 2-anilinyl
16e, Ar ¼ 2-amino-5-trifluoromethylphenyl
16f, Ar ¼ 2-pyridinyl
All cell lines were purchased from the Bioresource Collection
and Research Center (Hsinchu, Taiwan). Each cell line was main-
tained in standard medium and grown as a monolayer in Dulbec-
co’s modified Eagle’s medium (DMEM) containing 10% fetal bovine
16g, Ar ¼ 2-nitro-4-methoxyphenyl
16h, Ar ¼ 2-thioanisyl
16i, Ar ¼ 2,4-dimethoxyphenyl

Y.-H. Lo et al. / European Journal of Medicinal Chemistry 62 (2013) 526e533
529
Table 2
Growth inhibition (IC50, mM) of human cancer cell lines by compounds 1, 3e12.
Compounds
Cell linea
A549
AGS
PC-3
BT483
HeLa
SAS
SK Hep-1
CE81T
Colchinice
1
0.07
6.57
5.71
0.79
0.52
0.57
12.41
3.99
5.95
6.42
11.66
NA
0.05
8.24
4.66
4.56
0.78
0.98
NA
0.13
2.78
5.75
9.53
NA
0.06
8.26
4.56
8.68
0.62
0.73
NA
0.51
3.79
11.67
17.39
22.93
1.45
8.56
4.24
10.52
1.23
1.98
21.23
0.86
4.53
9.56
15.22
NA
0.66
5.49
1.89
1.07
0.57
0.64
24.76
0.39
2.26
5.00
15.87
12.77
3.50
5.83
1.37
10.60
9.88
4.53
15.77
1.16
0.65
12.28
12.81
NA
0.78
6.68
1.34
1.20
0.34
0.20
28.26
0.43
4.28
5.23
13.27
14.39
1.78
2.91
6.91
6.39
6.95
8.98
21.00
6.87
OCH3
OCH3
3 (R1 ¼ NO2, R2 ¼ H, R3 ¼ H, X ¼ C)
4 (R1 ¼ CN, R2 ¼ H, R3 ¼ H, X ¼ C)
5 (R1 ¼ OCH3, R2 ¼ H, R3 ¼ H, X ¼ C)
6 (R1 ¼ NH2, R2 ¼ H, R3 ¼ H, X ¼ C)
7 (R1 ¼ NH2, R2 ¼ H, R3 ¼ CF3, X ¼ C)
8 (R1 ¼ none, R2 ¼ H, R3 ¼ H, X ¼ N)
9 (R1 ¼ NO2, R2 ¼ OCH3, R3 ¼ H, X ¼ C)
10 (R1 ¼ NH2, R2 ¼ OCH3, R3 ¼ H, X ¼ C)
11 (R1 ¼ SCH3, R2 ¼ H, R3 ¼ H, X ¼ C)
12 (R1 ¼ OCH3, R2 ¼ OCH3, R3 ¼ H, X ¼ C)
OCH3
R3
R2
6.32
29.97
13.91
17.43
X
R1
NA ¼ not available.
a
A549 (non-small-cell lung cancer); AGS (human stomach adenocarcinoma); PC-3 (prostate cancer); BT483 (breast carcinomas); HeLa (human cervical epithelioid car-
cinoma); SAS (oral squamous cell carcinoma); SK Hep (hepatocellular carcinoma); CE81T (esophageal carcinoma).
serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml
streptomycin. Cultures were maintained at 37 ꢂC with 5% CO2 in
a humidified atmosphere.
4.5. Xenograft tumor growth
The nude mice (6e8 weeks old, male, 20e25 g body weight)
were obtained from the National Laboratory Animal Center. The
mice were maintained in a specific pathogen-free (SPF) environ-
ment at the Laboratory Animal Center of National Cheng-kung
University. Animal care was provided in accordance with the Lab-
oratory Animal Welfare Act, Guide for the Care and Use of Labo-
ratory Animals, approved by the Institutional Animal Care and Use
Committee of National Cheng Kung University. For the animal ex-
periments, each mouse was subcutaneously inoculated with
1 ꢁ 106 BT483 cells in the left flank of legs using 50% matrigel. The
tumor size was measured every 3 days with a caliper, and tumor
volume was calculated as width (mm) ꢁ length (mm) ꢁ height
(mm). Tumor nodules were allowed to grow to a volume of
w20 mm3 before initiating treatment. Tumor-bearing mice were
randomly assigned to two groups (n ¼ 5e6 in each group). Test
animals received intraperitoneal (i.p.) injections of DMSO (n ¼ 5) or
compound 8 (20 mg/kg/day) (n ¼ 6) every day for 2 weeks. The
tumor size was measured every 3 days beginning from the first day
of compound 8 treatment.
4.2. Flow cytometric analysis
Apoptosis and cell cycle profiles were assessed using DNA flu-
orescence flow cytometry. BT-483 cells treated with DMSO or
0.2 mM compound 8 for 12 and 24 h were harvested, rinsed in PBS,
resuspended, fixed in 80% ethanol, and stored at ꢀ20 ꢂC in fixation
buffer until they were ready for analysis. The pellets were sus-
pended in 1 ml of fluorochromic solution (0.08 mg/ml PI), 0.1%
Triton X-100, and 0.2 mg/ml RNase A in 1ꢁ PBS at room tempera-
ture in the dark for 30 min. The DNA content was analyzed using
a flow cytometer (FACScan; Becton Dickinson, Mountain View, CA)
and CellQuest software (Becton Dickinson).
4.3. Microtubule depolymerization assay
BT483 cells were maintained at 37 ꢂC in a humidified atmo-
sphere containing 5% CO2. To depolymerize microtubules, cells
were incubated at 4 ꢂC for 4 h. To study the effect of compound 8 on
microtubule polymerization, the cells were pre-treated in the cold
as described above and incubated at 37 ꢂC for 6 h in culture medium
4.6. Molecular modeling
supplemented with DMSO or 1
mM compound 8. After fixation,
All molecular modeling studies were performed on an Asus
personal computer with an Intel Core i7 2.67 GHz processor run-
ning Windows 7 using ChemBioOffice 2010 [13] and Discovery
Studio 2.1 (DS) [14]. The receptor structure two tubulin dimers co-
crystallized with a stathmin-like domain and N-deacetyl-N-(2-
mercaptoacetyl)-colchicine (DAMA-colchicine) was downloaded
from the PDB data bank (http://www.rcsb.org/pdb/index.html; PDB
code: 1SA0) [15]. Compound structures were built with ChemBio3D
immunofluorescence assays were performed with anti-tubulin
antibody to examine the sub-cellular distribution of microtubules.
The nuclei were counterstained by DAPI.
4.4. Western blot
After the cells were treated with vehicle (1% DMSO) or com-
pound 8 at various concentrations for 24 h, they were washed twice
in ChemBioOffice and minimized using the MMFF94 force field
ꢀꢀ1
with PBS, and the reaction was terminated by adding 100
mL of lysis
until an RMSD gradient of 0.1 kcal molꢀ1
A
was reached. The
buffer. For Western blot analysis, 50 g of protein was separated
m
partial charges were automatically calculated, and the structure
was saved as a mol file. Docking simulations were carried out using
the DS LigandFit docking module. The DAMA-colchicine present
between chain A and chain B of the structure was used to define the
using electrophoresis in 15% SDS-PAGE and then transferred to
a polyvinylidene fluoride membrane. After it had been incubated
overnight at 4 ꢂC in 5% nonfat milk, the membrane was washed and
incubated with monoclonal primary antibodies for 2 h at room
temperature. The antibodies used in this study were anti-Caspase-
ꢀ
binding site, which extended 3.0 A from the boundary of the
crystallized ligand. The GTP molecule situated at the edge of the
binding site was included as a part of the receptor. DAMA-
colchicine was manually removed from the active site before the
docking experiment. The Jain scoring function [16] was used to
measure the interaction energy between various ligand confor-
mations and the receptor. The output of LigandFit docking was
3, anti-PARP, anti-phospho-g-H2AX, and anti-b-actin (Cell Signal-
ing Technology). After the membrane was washed three more
times, anti-mouse or anti-rabbit IgG (dilution 1:5000) was applied
to the membranes, and the signals were detected using enhanced
chemiluminescence reagents.

530
Y.-H. Lo et al. / European Journal of Medicinal Chemistry 62 (2013) 526e533
Fig. 2. (A) Analysis of the cancer cell cycle. (B) Expression of apoptosis-associated proteins. (C) The effect of compound 8 on microtubule depolymerization.
visualized in the DS graphics environment, and the distances of
hydrogen bonds were measured between the related heavy atoms
of the docked ligand and the receptor residues.
a degassed solution of compound 13 (12 mmol) containing
Pd(PPh3)4 (0.8 mmol) in ether (25 ml). The resulting reaction
mixture was stirred at room temperature for 4 h and then
quenched with saturated aqueous NH4Cl. The aqueous layer was
extracted with EtOAc (30 ml ꢁ 3), and the combined organic ex-
tracts were washed with saturated aqueous Na2CO3 (40 ml) and
dried over anhydrous MgSO4. After filtration and removal of the
solvent in vacuo, the residue was purified by column chromatog-
raphy on a silica gel (hexane/EA ¼ 10:1 as the eluent) to give 15
with a yield of 85% as a brown solid. Mp ¼ 49e50 ꢂC. 1H NMR
4.7. Chemistry
4.7.1. Synthesis of 2-(6-trimethylsilyl-3(Z)-hexen-1,5-diynyl)-1,2,3-
trimethoxybenzene (15)
To
a degassed solution of 14 (12 mmol) containing CuI
(3.2 mmol) and n-BuNH2 (30 mmol) in ether (20 ml) was added

Y.-H. Lo et al. / European Journal of Medicinal Chemistry 62 (2013) 526e533
531
Fig. 2. (continued).
(CDCl3, 400 MHz)
d
6.71 (s, 2H), 6.05 (d, 1H, J ¼ 10.8 Hz), 5.87 (d, 1H,
combined organic extracts were washed with saturated aqueous
Na2CO3 (40 ml) and dried over anhydrous MgSO4. After filtration
and removal of the solvent in vacuo, the residue was purified by
column chromatography on a silica gel to produce the desired
products.
J ¼ 10.8 Hz), 3.86 (s, 3H), 3.84 (s, 6H), 0.24 (s, 9H). 13C NMR (CDCl3,
100 MHz)
d 150.3 (2C), 139.2, 120.6, 119.1, 118.0, 108.9 (2C), 103.2,
102.3, 97.6, 86.2, 60.9, 56.1 (2C), ꢀ0.1 (3C). HRMS calcd for
C
C
18H22O3Si, Mr
18H22O3Si: C, 68.75; H, 7.05. Found: C, 68.50; H, 7.06.
¼
314.1338, found 314.1338. Anal. Calcd for
4.7.2.1. 2-(6-(2-Nitrophenyl)-3(Z)-hexen-1,5-diynyl)-1,2,3-
trimethoxybenzene (3). This compound was obtained at a 58% yield
as a brown solid using hexane/EA (3:1) as an eluent using the
general procedure. Mp ¼ 95e96 ꢂC. 1H NMR (CDCl3, 400 MHz)
4.7.2. General procedure for the synthesis of compounds 3e9, 11
and 12
To a degassed solution of 2-(6-trimethylsilyl-3(Z)-hexen-1,5-
diynyl)-1,2,3-trimethoxybenzene (15) (12 mmol) containing CuI
(3.2 mmol) and K2CO3 (30 mmol) in MeOH (15 ml) was added
a degassed solution of aryl iodides (16aei) (12 mmol) containing
Pd(PPh3)4 (0.8 mmol) in MeOH (20 ml). The resulting reaction
mixture was stirred at room temperature for 4 h. The solvent was
then removed in vacuo. The residue was quenched with saturated
aqueous NH4Cl and extracted with EtOAc (20 ꢁ 3 ml). The
d
8.09 (dd, 1H, J ¼ 8.4, 1.6 Hz), 7.69 (dd, 1H, J ¼ 7.6, 1.6 Hz), 7.57 (td,
1H, J ¼ 7.6, 1.2 Hz), 7.47 (td, 1H, J ¼ 7.6, 1.6 Hz), 6.90 (s, 2H), 6.21 (d,
1H, J ¼ 10.8 Hz), 6.12 (d,1H, J ¼ 10.8 Hz), 3.88 (s, 3H), 3.87 (s, 6H). 13
C
NMR (CDCl3, 100 MHz) d 153.0 (2C), 149.1, 139.3, 135.0, 132.8, 128.8,
124.7, 121.7, 118.7, 117.8, 117.7, 109.3 (2C), 99.2, 94.7, 92.2, 86.1, 60.9,
56.2 (2C). HRMS calcd for C21H17NO5, Mr ¼ 363.1107, found
363.1111. Anal. Calcd for C21H17NO5: C, 69.41; H, 4.72; N, 3.85.
Found: C, 68.99; H, 4.78; N, 3.35.
4.7.2.2. 2-(6-(2-Cyanophenyl)-3(Z)-hexen-1,5-diynyl)-1,2,3-
trimethoxybenzene (4). This compound was obtained at 77% yield
as a brown solid using hexane/EA (3:1) as the eluent using the
general procedure. Mp ¼ 64e65 ꢂC. 1H NMR (CDCl3, 400 MHz)
d
7.67 (dd, 1H, J ¼ 7.2, 0.8 Hz), 7.62 (dd, 1H, J ¼ 8.0, 0.8 Hz), 7.54 (td,
1H, J ¼ 7.6, 1.6 Hz), 7.42 (td, 1H, J ¼ 7.6, 1.2 Hz), 6.83 (s, 2H), 6.21 (d,
1H, J ¼ 11.2 Hz), 6.13 (d,1H, J ¼ 10.8 Hz), 3.87 (s, 3H), 3.86 (s, 6H). 13
C
NMR (CDCl3, 100 MHz) d 153.0 (2C), 132.7, 132.6, 132.2, 128.6, 127.0,
121.5, 121.5, 117.8, 117.5, 117.3, 114.9, 109.3 (2C), 99.1, 93.2, 92.7, 86.0,
60.9, 56.2 (2C). HRMS calcd for C22H17NO3, Mr ¼ 343.1208, found
343.1200. HPLC analysis: 96.48%.
4.7.2.3. 2-(6-(2-Anisyl)-3(Z)-hexen-1,5-diynyl)-1,2,3-
trimethoxybenzene (5). This compound was obtained at 72% yield
as a brown solid using hexane/EA (3:1) as the eluent using the
general procedure. Mp ¼ 66e67 ꢂC. 1H NMR (CDCl3, 400 MHz)
Fig. 3. Inhibitory effect of compound 8 against BT483 cell-derived tumors in nude
mice. BT-483 cells were subcutaneously injected into nude mice. The tumor-bearing
mice were intraperitoneally injected with either the vehicle (n ¼ 5) or 20 mg/kg/day
of compound 8 (n ¼ 6) for 2 weeks. The tumor volume was measured and calculated.
The data were presented as the mean ꢃ SEM for each group.
d
7.51 (dd, 1H, J ¼ 7.6, 1.6 Hz), 7.31 (td, 1H, J ¼ 7.6, 1.6 Hz), 6.92-6.88

532
Y.-H. Lo et al. / European Journal of Medicinal Chemistry 62 (2013) 526e533
Fig. 4. Molecular modeling: (A) X-ray structure of colchicines (gold) and molecular model of colchicines (gray pattern). (B) X-ray structure of colchicines (gold) and compound 2
(gray pattern). (C) X-ray structure of colchicines (gold) and compound 5 (gray pattern). (D) X-ray structure of colchicines (gold) and compound 6 (gray pattern). (E) X-ray structure of
colchicines (gold) and compound 8 (gray pattern) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
(m, 2H), 6.75 (s, 2H), 6.16 (d, 1H, J ¼ 10.4 Hz), 6.06 (d, 1H,
J ¼ 10.8 Hz), 3.86 (s, 3H), 3.84 (s, 6H), 3.82 (s, 3H). 13C NMR (CDCl3,
4.7.2.7. 2-(6-(4-Methoxy-2-nitrophenyl)-3(Z)-hexen-1,5-diynyl)-
1,2,3-trimethoxybenzene (9). This compound was obtained at 48%
yield as a brown solid using hexane/EA (5:1) as the eluent using the
general procedure. Mp ¼ 97e98 ꢂC. 1H NMR (CDCl3, 400 MHz)
100 MHz)
d 160.0, 153.0 (2C), 139.0, 133.6, 130.2, 120.4, 119.8, 118.9,
118.2, 112.4, 110.9, 108.9 (2C), 97.5, 94.0, 91.5, 86.7, 60.9, 56.1 (2C),
55.9. HRMS calcd for C22H20O4, Mr ¼ 348.1362, found 348.1362.
Anal. Calcd for C22H20O4: C, 75.84; H, 5.79. Found: C, 75.43; H, 6.04.
d
7.59e7.57 (m, 2H), 7.10 (dd,1H, J ¼ 8.8, 2.8 Hz), 6.89 (s, 2H), 6.15 (d,
1H, J ¼ 10.8 Hz), 6.09 (d, 1H, J ¼ 10.8 Hz), 3.89 (s, 3H), 3.87 (s, 6H),
3.86 (s, 3H). 13C NMR (CDCl3, 100 MHz)
d
159.7, 153.0 (2C), 149.9,
4.7.2.4. 2-(6-(2-Anilinyl)-3(Z)-hexen-1,5-diynyl)-1,2,3-
trimethoxybenzene (6). This compound was obtained at 69% yield
as a brown solid using hexane/EA (5:1) as the eluent using the
general procedure. Mp ¼ 132e133 ꢂC. 1H NMR (CDCl3, 400 MHz)
139.1, 135.9, 120.6, 119.9, 118.1, 117.8, 110.7, 109.3, 109.2 (2C), 98.7,
93.0, 92.5, 86.2, 60.9, 56.1 (2C), 55.9. HRMS calcd for C22H19NO6,
Mr ¼ 393.1212, found 393.1210. HPLC analysis: 99.87%.
d
7.33 (dd, 1H, J ¼ 8.0, 1.6 Hz), 7.13 (td, 1H, J ¼ 8.0, 1.6 Hz), 6.75 (s,
4.7.2.8. 2-(6-(2-Amino-4-methoxyphenyl)-3(Z)-hexen-1,5-diynyl)-
1,2,3-trimethoxybenzene (10). This compound was obtained at 36%
yield as a brown oil using hexane/EA (1:1) as the eluent via the
reduction of compound 9 in a mixture of SnCl2 (5 eq) and MeOH
2H), 6.70e6.65 (m, 2H), 6.17 (d, 1H, J ¼ 10.8 Hz), 6.08 (d, 1H,
J ¼ 10.8 Hz), 4.36 (bs, 2H), 3.87 (s, 3H), 3.84 (s, 6H). 13C NMR (CDCl3,
100 MHz)
d 153.0 (2C), 148.2, 139.1, 131.9, 130.2, 119.6, 118.1, 117.8,
117.7, 114.1, 109.2 (2C), 107.3, 97.2, 94.4, 93.2, 87.0, 60.9, 56.1 (2C).
Anal. Calcd for C21H19NO3: C, 75.66; H, 5.74; N, 4.2. Found: C, 75.66;
H, 5.43; N, 3.9.
(10 ml). 1H NMR (CDCl3, 400 MHz)
d
7.24 (d, 1H, J ¼ 8.8 Hz), 6.74 (s,
2H), 6.27 (dd, 1H, J ¼ 8.4, 2.4 Hz), 6.16 (d, 1H, J ¼ 10.8 Hz), 6.02 (d,
1H, J ¼ 10.8 Hz), 3.87 (s, 3H), 3.84 (s, 6H), 3.76 (s, 3H). 13C NMR
(CDCl3, 100 MHz) d 161.3, 153.0 (2C), 149.8, 139.0, 133.1, 119.9, 117.9,
4.7.2.5. 2-(6-(2-Amino-5-trifluoromethylphenyl)-3(Z)-hexen-1,5-
diynyl)-1,2,3-trimethoxybenzene (7). This compound was obtained
at 78% yield as a brown solid using hexane/EA (5:1) as the eluent
using the general procedure. Mp ¼ 131e132 ꢂC. 1H NMR (CDCl3,
116.8, 109.1 (2C), 104.6, 100.3, 98.9, 96.7, 94.9, 92.5, 87.2, 60.9, 56.1
(2C), 55.1. HRMS calcd for C22H21NO4, Mr ¼ 363.1471, found
363.1472. HPLC analysis: 98.3%.
400 MHz)
d
7.57 (d, 1H, J ¼ 2.0 Hz), 7.34 (dd, 1H, J ¼ 8.8, 2.4 Hz), 6.73
4.7.2.9. 2-(6-(2-Thioanisyl)-3(Z)-hexen-1,5-diynyl)-1,2,3-
trimethoxybenzene (11). This compound was obtained at 65% yield
as a brown oil using hexane/EA (3:1) as the eluent using the general
(s, 2H), 6.72e6.70 (m, 1H), 6.17 (d, 1H, J ¼ 10.8 Hz), 6.13 (d, 1H,
J ¼ 10.8 Hz), 4.68 (br s, 2H), 3.88 (s, 3H), 3.83 (s, 6H). 13C NMR
(CDCl3, 100 MHz)
d
153.1, 150.4 (2C), 139.3, 127.0, 125.6, 122.9, 119.8,
procedure. 1H NMR (CDCl3, 400 MHz)
d
7.48 (dd, 1H, J ¼ 7.6, 1.2 Hz),
119.2, 119.0, 117.5, 113.5, 109.2 (2C), 106.9, 97.6, 93.9, 92.6, 86.7, 60.9,
56.1 (2C). HRMS calcd for C22H18F3NO3, Mr ¼ 401.1239, found
401.1245. Anal. Calcd for C22H18F3NO3: C, 65.83; H, 4.52; N, 3.49.
Found: C, 65.2; H, 4.35; N, 3.08.
7.29 (td, 1H, J ¼ 7.6, 1.2 Hz), 7.16 (d, 1H, J ¼ 8.0 Hz), 7.07 (td, 1H,
J ¼ 7.6, 1.2 Hz), 6.78 (s, 2H), 6.17 (d, 1H, J ¼ 10.4 Hz), 6.11 (d, 1H,
J ¼ 10.8 Hz), 3.86 (s, 3H), 3.82 (s, 6H), 2.40 (s, 3H). 13C NMR (CDCl3,
100 MHz)
d 152.9 (2C), 141.8, 139.0, 132.6, 129.0, 124.2, 124.1, 121.2,
119.4, 119.1, 118.2, 109.2 (2C), 97.8, 94.6, 93.6, 86.7, 60.9, 56.1 (2C),
15.1. HRMS calcd for C22H20O3S, Mr ¼ 346.1133, found 346.1136.
HPLC analysis: 99.68%.
4.7.2.6. 2-(6-(2-Pyridinyl)-3(Z)-hexen-1,5-diynyl)-1,2,3-trimethoxy-
benzene (8). This compound was obtained at 79% yield as a brown
oil using hexane/EA (3:1) as the eluent using the general procedure.
1H NMR (CDCl3, 400 MHz)
d
8.58 (ddd, 1H, J ¼ 4.8, 1.6, 0.8 Hz), 7.64
4.7.2.10. 2-(6-(2,4-Dimethoxyphenyl)-3(Z)-hexen-1,5-diynyl)-1,2,3-
trimethoxybenzene (12). This compound was obtained at 49% yield
as a brown solid using hexane/EA (5:1) as the eluent using the
general procedure. Mp ¼ 77e78 ꢂC. 1H NMR (CDCl3, 400 MHz)
(td, 1H, J ¼ 8.0, 2.0 Hz), 7.50 (dt, 1H, J ¼ 8.0, 0.8 Hz), 7.23 (ddd, 1H,
J ¼ 7.6, 4.8, 1.2 Hz), 6.78 (s, 2H), 6.17 (d, 1H, J ¼ 10.8 Hz), 6.09 (d, 1H,
J ¼ 10.8 Hz), 3.85 (s, 3H), 3.83 (s, 6H). 13C NMR (CDCl3, 100 MHz)
d
153.0, 150.1 (2C), 143.3, 135.9,127.3, 127.3, 122.9, 121.4, 118.5, 117.9,
d
7.41 (d, 1H, J ¼ 9.2 Hz), 6.75 (s, 2H), 6.44e6.41 (m, 2H), 6.14 (d, 1H,
109.0 (2C), 98.6, 96.1, 86.8, 86.4, 60.9, 56.0 (2C). HRMS calcd for
20H17NO3, Mr ¼ 319.1208, found 319.1212. HPLC analysis: 99.90%.
J ¼ 10.4 Hz), 6.01 (d, 1H, J ¼ 10.8 Hz), 3.86 (s, 3H), 3.82 (s, 6H), 3.81
C
(s, 3H), 3.80 (s, 3H). 13C NMR (CDCl3, 100 MHz)
d
161.6, 161.3, 153.0

Y.-H. Lo et al. / European Journal of Medicinal Chemistry 62 (2013) 526e533
533
(b) P.E. Thorpe, Vascular targeting agents as cancer therapeutics, Clin. Cancer
Res. 10 (2004) 415e427.
[8] (a) G.M. Tozer, C. Kanthou, C.S. Parkins, S.A. Hill, The biology of the com-
bretastatins as tumour vascular targeting agents, Int. J. Exp. Pathol. 83 (2002)
21e38;
(2C), 134.5, 120.2, 120.2, 118.4, 117.8, 108.9 (2C), 105.1, 104.9, 98.5,
97.1, 94.4, 90.4, 86.9, 60.9, 56.1 (2C), 55.9, 55.5. Anal. Calcd for
C
23H22O5: C, 73.00; H, 5.86. Found: C, 72.99; H, 6.00.
(b) V.E. Prise, D.J. Honess, M.R. Stratford, J. Wilson, G.M. Tozer, The vascular
response of tumor and normal tissues in the rat to the vascular targeting
agent, combretastatin A-4-phosphate, at clinically relevant doses, Int. J.
Oncol. 21 (2003) 717e726;
(c) G.G. Dark, S.A. Hill, V.E. Prise, G.M. Tozer, G.R. Pettit, D.J. Chaplin, Com-
bretastatin A-4, an agent that displays potent and selective toxicity toward
tumor vasculature, Cancer Res. 57 (1997) 1829e1834.
Acknowledgments
We would like to thank the National Science Council of the
Republic of China for the financial support.
Appendix A. Supplementary data
[9] I. Vitale, A. Antoccia, C. Cenciarelli, P. Crateri, S. Meschini, G. Arancia,
C. Pisano, C. Tanzarella, Combretastatin (CA-4) and combretastatin deriva-
tive induce mitotic catastrophe dependent on spindle checkpoint and
Caspase-3 activation in non-small cell lung cancer cells, Apoptosis 12 (2006)
155e166.
[10] (a) Y.H. Lo, C.F. Lin, M.C. Hsieh, M.J. Wu, Remarkable G2/M phase arrest and
apoptotic effect performed by 2-(6-aryl-3-hexen-1,5-diynyl) benzonitrile
antitumor agents, Bioorg. Med. Chem. 12 (2004) 1047e1053;
(b) C.F. Lin, Y.H. Lo, M.C. Hsieh, Y.H. Chen, J.J. Wang, M.J. Wu, Cytotoxicities,
cell cycle and caspase evaluations of 1,6-diaryl-3(Z)-hexen-1,5-diynes, 2-(6-
aryl-3(Z)-hexen-1,5-diynyl)anilines and their derivatives, Bioorg. Med.
Chem. 13 (2005) 3565e3575;
Supplementary data associated with this article can be found
in the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.
01.011.
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