2-(6-Aryl-3(Z)-hexen-1,5-diynyl)anilines as a new class of potent antitubulin agents

Article abstract of DOI:10.1021/jm070820n

Compounds 2a-h and 6 displayed significant GI₅₀ values of 10^-7-10^-6 M against various cancer cell lines. Of these compounds, 2-(6-(2-trifluoromethylphenyl))-3(Z)-hexen-1,5-diynyl)aniline (2c) showed the most potent growth

Full text of DOI:10.1021/jm070820n

2682
J. Med. Chem. 2008, 51, 2682–2688
2-(6-Aryl-3(Z)-hexen-1,5-diynyl)anilines as a New Class of Potent Antitubulin Agents
Yu-Hsiang Lo,†,‡ Ching-Chih Lin,‡,# Chi-Fong Lin,| Ying-Ting Lin, Tsai-Hui Duh, Yi-Ren Hong,# Sheng-Huei Yang,
,X
Shinne-Ren Lin, Shyh-Chyun Yang,† Long-Sen Chang,∆,X and Ming-Jung Wu*,
Graduate Institute of Pharmaceutical Sciences, Graduate Institute of Biochemistry, Faculty of Biotechnology, Faculty of Medicinal and Applied
Chemistry, Kaohsiung Medical UniVersity, Kaohsiung, Taiwan, Department of Biological Science and Technology, Chung Hwa College of
Medical Technology, Tainan, Taiwan, Institute of Biomedical Science, National Sun Yat-Sen UniVersity, Kaohsiung, Taiwan, and National Sun
Yat-Sen UniVersity-Kaohsiung Medical UniVersity Joint Research Center, Kaohsiung, Taiwan
ReceiVed July 9, 2007
Compounds 2a-h and 6 displayed significant GI50 values of 10-7-10-6 M against various cancer cell
lines. Of these compounds, 2-(6-(2-trifluoromethylphenyl))-3(Z)-hexen-1,5-diynyl)aniline (2c) showed the
most potent growth inhibition activity. Compound 2c also arrested cancer cells in the G2/M phase and in
low concentration reduced a significant percentage of MDA-MB-231/ATCC breast cancer tetraploid cells.
In addition to the G2/M block, compound 2c caused microtubule depolymerization and induced apoptosis
via activation of the caspase family.
Introduction
compounds were evaluated in the in vitro antitumor protocol
of the NCI. They were also evaluated in the cell cycle analysis,
the caspase-3 colorimetric assay, and microtubule depolymer-
ization assay.
Microtubules are cytoskeletal protein polymers formed by
highly dynamic assemblies of tubulin heterodimers, including
R-tubulin and ꢀ-tubulin. Microtubules and their dynamics play
a crucial role in many biological processes, including mitosis,
intracellular transport, exocytosis, and cell growth.1a Since
microtubules are important in mitosis and cell division, they
have been a target for the development of a number of new
anticancer drugs.1b There are two major groups of these
antitumor agents: microtubule stabilizers such as paclitaxel2 and
microtubule destabilizers (colchicine,3 combretastatin A-4,4 and
vinca alkaloids,5 Chart 1) One of these compounds, combret-
astatin A-4 (CA-4), shows potent inhibition of microtubule
polymerization by blocking colchicine’s binding site and rapidly
shutting down existing tumor vasculature.6 Most of these
compounds accumulate cells in the G2/M phase of cell cycle
and induce apoptosis,7 a cell suicide mechanism.
Chemistry
The syntheses of the 2-(6-aryl-3(Z)-hexen-1, 5-diynyl)anilines
2a-h were carried out using vinyl chloride 39 and 2-ethynyl-
aniline 410 as starting materials. The palladium-catalyzed
Sonogashira coupling reaction11 of 3 and 4 gave 2-(6-trimeth-
ylsilyl-3(Z)-hexen-1,5-diynyl)aniline (5) in 75% yield. Treatment
of compound 5 with CuI in the presence of K2CO3 in methanol
gave 6 in 55% yield. Desilylation and palladium-catalyzed
coupling reaction of 5 in the presence of K2CO3 and MeOH
with various aryl iodides (7a-h) gave 2a-h in 34-78% yield.
The results are summarized in Scheme 1.
In our earlier publications8 that described new enediyne
antitumor agents, we found that 2-(6-(2-anilinyl)-3(Z)-hexen-
1,5-diynyl)benzonitrile (1), which is structurally similar to CA-
4, showed potent cytotoxic activity against all 60 tumor cell
lines at a concentration of 10-7 M. This compound is particu-
larly potent against the MDA-MB-435 cell line of human breast
cancer (0.11 µM) and significantly arrested G2/M in the cell
cycle and induced apoptosis. For further investigation of the
role of G2/M phase blockage or to obtain lead compounds that
would arrest the G2/M state more potently and with enhanced
apoptotic effects, a series of 2-(6-aryl-3(Z)-hexen-1,5-diynyl)-
anilines 2a-h and 6 were designed and synthesized. These
Biological Assay Results
(A) Cytotoxicity Activities, Cell Cycle Analysis, and
Caspase-3, -8, and -9 Activities. Compounds 2a-h and 6 were
submitted to the National Cancer Institute for testing against a
panel of approximately 60 tumor cell lines. Details of this test
system have been published by others.12 Compounds 2b and
2d are found to be inactive in the NCI screen, and the log10
GI50 values of the active compounds are shown in Table 1. The
GI50 values of these compounds are in the range 10-7-10-6
M. Compound 2c showed the most potent growth inhibitiory
activity against all tumor cell lines. In particular, compound 2c
displayed a two-digit nanomolar value of GI50 against the SNB-
75 of CNS cancer cell line. To investigate the mechanism of
the inhibition activities of synthetic enediynes 2a-h and 6, we
examined their cell cycle distribution by treating K-562 cells
with these synthetic compounds for 24 h, and the cell cycle
profile was analyzed by flow cytometry. The results are
summarized in Table 2. Compounds 2c, 2d, and 2e significantly
blocked the K-562 cell cycle in the G2/M phase. The results
indicated a significant increase of the G2/M phase percentage
from 14.4% (control) to 73.9%, 81.4%, and 71.4% by com-
pounds 2c, 2d, and 2e, respectively. With the exception of potent
G2/M cell cycle arrest, some of the enediyne derivatives also
induced apoptosis in K-562 cells. Compared to the sub-G1 area
(3.86%) of control, all compounds showed significant apoptotic
* To whom correspondence should be addressed. Tel: 886-7-3121101
ext 2220. Fax: 886-7-3125339. E-mail: mijuwu@kmu.edu.tw.
†
Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical
University.
‡
Contribution was equal to that of the first author.
Department of Biological Sciences, National Sun Yat-Sen University.
Department of Biological Science and Technology, Chung Hwa College
§
|
of Medical Technology.
Faculty of Biotechnology, Kaohsiung Medical University.
Faculty of Medicinal and Applied Chemistry, Kaohsiung Medical
University.
#
Graduate Institute of Biochemistry, Kaohsiung Medical University.
Institute of Biomedical Science, National Sun Yat-Sen University.
National Sun Yat-Sen University-Kaohsiung Medical University Joint
∆
X
Research Center.
10.1021/jm070820n CCC: $40.75
2008 American Chemical Society
Published on Web 04/03/2008

Potent Antitubulin Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2683
Chart 1
Scheme 1a
studied (Figure 1B). The detailed percentages are included in
the Supporting Information. Moreover, the greatest accumulation
of cells arrested in the G2/M stage occurred at 9 µM, and a
relatively high concentration of 2c (36 µM) led to the greatest
apoptosis. However, all of these treatments were also correlated
with the capacity to induce tetraploid (8N) cells (Figure 1A).
The appearance of tetraploidy (8N) cells indicates that the cells
went through an extra DNA replication without completing
mitosis. Moreover, the phenomenon of generated tetraploidy
can be induced by disrupting chromosome segregation during
mitosis, disrupting the cell cleavage, or failed checkpoint control
during mitosis.13
A major part of the phenomenon could be mediated by
deregulation in cell cycle progression governed by families of
caspases. Caspase-3, -8, and -9 in particular are the principal
promoters of apoptosis. Colorimetric assays of capase-3, -8, and
-9 provided evidence of the relationship between compound 2c
and the programmed cell death. As shown in Figure 1C,
apoptosis was shown by inducing caspase-3, -8, and -9 activities
with 36 µM treatment for 72 h. It was demonstrated that the
activity of caspase-3, -8, and -9 enhanced by 3.4, 4.0, and 3.9,
respectively, relative to the control (Figure 1C). Taken together,
our results suggested that the effects of compound 2c on cell-
cycle progression correlates well with its strong ability to induce
a massive accumulation of cells in the G2/M phase and also to
induce apoptosis via activation of caspase-3, -8, and -9.
a Reagents and conditions: (a) Pd(PPh3)4, CuI, nBuNH2, ether, rt; (b)
CuI, K2CO3, methanol, rt; (c) Pd(PPh3)4, CuI, K2CO3, methanol, rt.
activity (9.17% to 30.55%). More noteworthy is that compounds
2d (28.68%), 2e (30.55%), and 2g (27.34%) have a greater
ability to induce apoptosis.
Since compound 2c is the most active compound among these
synthetic enediynes, it was chosen to investigate the detailed
mechanism of its action against the MDA-MB-231/ATCC breast
cancer cell line. Parts A and B of Figure 1 display the cell cycle
patterns of MDA-MB-231/ATCC treated with compound 2c for
24 and 72 h, respectively. The concentration of 2c chosen in
this study is based on its LC50 value against this tumor cell line
(Supporting Information) and graduate diluted to 1/10th of that
concentration. Thus, treatment of MDA-MB-231/ATCC cells
with compound 2c at different concentrations (3.6, 9.0, 18, 27,
and 36 µM) for 24 h led to profound perturbations of the cell
cycle profile with colchicine chosen as the control (Figure 1A).
Our results show that cells treated with compound 2c at a
concentration of 3.6 µM for 24 h were sufficient to induce a
massive accumulation of cells in the G2/M phase, and this
phenomenon was also in a dose-dependent manner. In parallel
to the G2/M block, a characteristic hypodiploid DNA content
area (sub-G1) increased after 72 h of drug treatment at all doses
(B) Microtubule Depolymerization Assay. The structure of
synthetic enediyne 2c is very similar to the skeleton of CA-4
or colchicines. For this reason, we anticipated that there is a
relationship between the G2/M phase arrest caused by 2c and
microtubule function. To investigate the effect of 2c upon
microtubules, the microtubule depolymerization assay was
carried out. Our data show that a brief exposure of the MDA-
MB-231/ATCC cells to compound 2c was sufficient to produce
sustained depolymerization of the microtubules in a concentra-
tion-dependent manner (Figure 2). As shown in Figure 2, cells
were treated with a low concentration (3.6 µM and 9 µM) of
compound 2c for 2 h. Microtubules were still seen in interphase
cells, but their number was decreased and the microtubule
cytoskeleton network was disorganized (Figure 2B,C), compared
to the control cells (Figure 2A). At a higher concentration (18
and 27 µM), compound 2c caused significant depolymerization

2684 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Lo et al.
Table 1. In Vitro Screening of Enediynes, Colchicine, and CA-4 against the NCI’s Cancer Cell Linesa
b
log10GI50
leukemia
(K-562)
colon cancer
(HCT-15)
CNS cancer
(SNB-75)
melanoma
(UACC-62)
ovarian cancer
(OVCAR-3)
breast cancer
(MDA-MB-231)
breast cancer
(MDA-MB-435)
compd
colchinicec
-7.9
-7.5
-6.3
-6.0
-5.6
-5.3
-4.8
-5.5
-6.1
-6.9
-7.5
-6.1
-5.9
-5.4
-4.9
-4.8
-5.3
-5.4
-7.6
-6.1
-4.9
-7.1
-5.6
NA
-4.4
NA
-5.5
-7.6
-8.0
-5.7
-6.6
-5.2
-4.8
-4.9
-5.1
-5.7
-7.8
-7.4
-5.4
-6.2
-5.4
-5.2
-4.8
-5.5
-5.6
-6.5
-8.0
-6.4
-6.4
-5.3
-4.8
-5.6
-5.0
-5.8
-8.0
-7.5
-6.4
-6.4
-5.9
-5.6
-4.8
-5.7
-5.5
combretastatin A-4c
2a
2c
2e
2f
2g
2h
6
a Data obtained from the NCI’s in vitro human tumor cell screen. NA ) not available. b The concentration produces 50% reduction in cell growth. c The
log10GI50 of colchinice (NS C757) and combretastatin A-4(NSC613729) were obtained from NCI’s web site (http://dtp.nci.nih.gov/dtpstandard/dwindex/
index.jsp).
Table 2. Cell Cycle Distribution of K-562 Cell Line Treated with Synthetic Enediynes
cell cycle percentagea
2a
2b
2c
2d
2e
2f
2g
2h
6
G0/G1%
S%
G2/M%
Sub-G1 (apoptosis area)
42.7
41.0
16.3
11.13
35.0
46.2
18.8
15.78
6.7
19.3
73.9
19.40
5.9
12.7
81.4
28.68
4.5
24.1
71.4
30.55
32.5
33.1
34.4
10.04
43.6
38.5
17.9
27.34
22.1
40.9
37.0
22.8
25.0
52.2
11.52
9.19
a The percentages of the cells in each phase were calculated by using the WinMDI software for the flow cytometry treated with sample for 24 h used at
a concentration of 50 µm by flow cytometry analysis. Control percentage: G0/G1 (26.7%), S (58.9%), G2/M (14.4%), and Sub-G1(3.86%).
Figure 1. (A) Cell cycle distribution of MDA-MB-231/ATCC after treatment with compound 2c at different concentrations for 24 h (A) and 72 h
(B) and analyzed by flow cytometry G0/G1, S, and G2/M indicated the phase and sub-G1 area refers to the portion of apoptotic cells. (C) The
caspase-3, -8, and -9 colorimetric assay of compound 2c that absorption fold of control: caspase-3 (3.4), -8 (4.0), and -9(3.9), which can be quantified
spectrophotometrically at a wavelength of 405 nm in 36 µM treatment for 72 h.
of microtubules in interphase cells (Figure 2D,E), and 36 µM
compound 2c induced extensive depolymerization of interphase
microtubules (Figure 2F). In summary, these results suggest that
the loss of function of microtubules in interphase cells treated
with 2c may prevent these cells from progressing into mitosis
(Figures 1 and 2).
(C) Microtubule Regrowth Assay. To further examine
whether the effect of compound 2c on microtubule disassembly
is reversible, the microtubule regrowth assay was carried out.
MDA-MB-231/ATCC cells were treated with 36 µM of
compound 2c for 2 h, the drug was removed, and the cells were
incubated in fresh culture medium for microtubule regrowth at
the indicated time points as shown in Figure 3A-D. The well-
known microtubule-depolymerizing agents, nocodazole and
colchicine, were used as control. Nocodazole (Figure 3E-H)
is a reversible antitubulin agent that was used as a positive
control, and colchicine (Figure 3I-L) is an irreversible antitu-
bulin agent that was employed as a negative control. As shown
in Figure 3, the disruption of microtubule assembly is reversible
in a time-dependent manner when compound 2c was removed
(Figure 3A-D). Taken together, our data suggest that the effect
of compound 2c on microtubule depolymerization should be
reversible.
(D) Compound 2c Inhibits Tubulin Polymerization In
Vitro. To further explore whether the growth-inhibitory effect
of these novel compounds was related to an interaction with
the tubulin system, an in vitro tubulin polymerization assay was
performed. The effects of 2c (3.6, 9, 18, 27, and 36 µM) on
microtubule formation were monitored by the increase in
fluorescent intensity of the reaction mixture. As expected,
colchicine (the positive control) potently inhibited tubulin
polymerization and paclitaxel (the negative control) promoted
the polymerization of tubulin. According to Figure 4, the IC50
value of inhibition of tubulin polymerization for compound 2c
is about 9–10 µM and that for colchicine is much less than 3

Potent Antitubulin Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2685
Figure 2. Dose effect of compound 2c on microtubule depolymerization. MDA-MB-231/ATCC cells were treated with compound 2c at 37 °C in
0 (A), 3.6 (B), 9 (C), 18 (D), 27 (E), and 36 µM (F) for 2 h, respectively. Then cells were fixed and immunostained with antininein (green in
merged images) antibody and anti-R-tubulin (red in merged images) antibody and counterstained with DAPI for nucleus staining. Scale bars represent
10 µM.
Figure 3. Effect of compound 2c on depolymerization of microtubules is reversible. MDA-MB-231/ATCC cells were treated with compound 2c
in 36 µM (A–D) and 20 µM nocodazole (E, F) and 5 µM colchicine (I-L) for 2 h at 37 °C. The drugs were then removed and the cells incubated
in prewarmed medium at 37 °C for microtubule regrowth. Cells were fixed at the indicated time points and immunostained with antininein (green
in merged images) antibody as a centrosome marker and anti-R-tubulin (red in merged images) antibody and counterstained with DAPI for nucleus
staining. Scale bars represent 10 µM.
µM. Compound 2c inhibited tubulin polymerization in a
concentration-dependent manner (Figure 4).
by fluoro atom as compound 2h, the inhibition activity dropped
dramatically. (ii) After a 24 h treatment with compound 2c, a
strong ability to induce a massive accumulation of cells in the
G2/M phase was observed (Figure 1A). After 72 h treatment
with compound 2c, some of the cells undergo apoptosis via
activation of caspase-3, -8, and -9 (Figure 1B,C). (iii) A brief
exposure of the MDA-MB-231/ATCC cells to compound 2c is
sufficient to produce sustained depolymerization of the micro-
tubules in a concentration-dependent manner (Figure 2). (iv)
The disruption of microtubule is reversible when the drug is
removed, which indicates a lower toxicity of this compound
(Figure 3).
Conclusion
In this work, we have synthesized a series of 2-(6-aryl-3(Z)-
hexen-1, 5-diynyl)anilines and found that these compounds show
good growth inhibition activity against various human cancer
cell lines. After a detailed investigation of the biological
activities and mechanisms of activity of these synthetic ene-
diynes, several conclusions can be reached: (i) The structure–
activity relationship study shows that compound 2c bearing a
trifluoromethyl group at the 2-position on one of the phenyl
rings, shows the most potent inhibitiory activity. Introducing
an oxygen atom into the triflouoromethylphenyl ring of 2c to
afford 2e decreased the activity by 10-fold relative to the average
GI50 values. When the trifluoromethyl group of 2c was replaced
In conclusion, this study provides a new lead, such as
compound 2, for the development of antitumor agent and also
provides a direction for further structure modification to obtain
a more potent antitumor drug. We believe that the information

2686 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9
Lo et al.
Figure 4. Pattern of various concentrations of compound 2c on in vitro tubulin polymerization assay.
a BCA protein assay kit (Pierce, Rockford, IL), and clear lysates
containing 50 µg of protein were incubated with 100 µM of enzyme-
specific colorigenic substrates (Biovision) and 2× reaction buffer
(Biovision) containing 10 mM DTT at 37 °C for 1 h. The activity
of caspases was described as the cleavage of colorimetric substrate
by measuring the absorbance at 405 nm.14
disclosed in this paper will have a significant impact upon the
development of new anticancer drugs.
Experimental Section
Cell Culture. (1) Human leukemia K-562 cells were obtained
from the American Type Culture Collection (ATCC, Manassas,
VA), and human purified lymphocyte preparation was obtained from
blood as described previously. Cells were maintained in RPMI 1640
medium supplemented with 10% fetal calf serum, 2 µM glutamine,
and antibiotics (100 units/mL penicillin and 100 µg/mL strepto-
mycin) at 37 °C in a humidified atmosphere of 5% CO2.
(2) Human leukemia cells and MDA-MB-231/ATCC breast
carcinoma cells were obtained from the American Type Culture
Collection (ATCC, Manassas, VA), and human purified lymphocyte
preparation was obtained from blood as described previously. K562
cells were maintained in RPMI 1640 medium supplemented with
10% fetal calf serum, 2 µM glutamine, and antibiotics (100 units/
mL penicillin and 100 µg/mL streptomycin), and MDA-MB-231
cells were cultured in DMEM medium supplemented with 10%
fetal bovine serum, 2 µM glutamine, 1% nonessential amino acids,
100 units/mL penicillin, and 100 µg/mL streptomycin (Gibco) at
37 °C in a humidified atmosphere of 5% CO2. Cell cultures were
treated with compound 2c (3.6, 9, 18, 27, or 36 µM) and 20 µM
nocodazole (Sigma) in DMEM, respectively, at the indicated time
periods before the cells were fixed.
Cell Cycle Analysis. Flow cytometry was used to measure cell
cycle profile and apoptosis. For cell cycle analysis, K-562 cells
treated with compounds 2a-h and 6 (50 µM) for 24 h were
harvested by centrifugation. After being washed with PBS, the cells
were fixed with ice-cold 70 °C ethanol for 30 min, washed with
PBS, and then treated with 1 mL of 1 mg/mL of RNase A solution
at 37 °C for 30 min. Cells were harvested by centrifugation at 1000
rpm for 5 min and further stained with 250 µL of DNA staining
solution (10 mg of propidium iodide [PI], 0.1 mg of trisodium
citrate, and 0.03 mL of Triton X-100 were dissolved in 100 mL
H2O) at room temperature for 30 min in the dark. After loading
500 µL of PBS, the DNA contents of 10000 events were measured
by FACScan (Elite ESP, Beckman Coulter, Brea, CA) and the cell
cycle profile was analyzed from the DNA content histograms with
WinCycle software. When cells were apoptosis, those containing
DNA were digested by endonuclease and then the sub G1 pick
appeared. The percentage in sub G1 were analyzed by gating on
cell cycle dot blots using Windows Multiple Document Interface
software (WinMDI).
Immunocytochemistry and Microscopy. These procedures
were modified from previously described protocols.15 In brief,
MDA-MB-231/ATCC cells were grown on glass coverslips at a
density of 1 × 104 cells for 24 h. Cell cultures were rinsed several
times with PBS, fixed in 4% paraformaldehyde for 5 min, and
permeabilized with 0.5% Triton X-100 in PBS for 5 min. Fixed
cells were rinsed in PBS, and nonspecific binding was blocked with
5% normal goat serum (NGS)/1% bovine serum albumin (BSA)
in PBS pH 7.4 for at least 30 min at 37 °C. After a brief wash, the
cells were incubated for 45 min at 37 °C with the primary antibodies
diluted in the same blocking solution. After extensive washes with
PBS, the cultures were then incubated with the appropriate
secondary antibody conjugated to either Alexa 488 or Alexa 568
for 45 min at 37 °C. Finally, the cells were incubated for 5 min
with DAPI (Roche) prior to mounting (Molecular Probes). Confocal
images were obtained using an OLYMPUS IX71 microscope (100
× UPlanFl objective 1.3 NA) at 0.2 µm z-steps, controlled by
FLUOVIEW software (Universal Imaging). All images were
imported into Adobe Photoshop v7.0 for contrast manipulation.
Microtubule Regrowth Experiment. MDA-MB-231/ATCC
cells (1 × 104) were seeded on coverslips and grown for 24 h.
Microtubules were depolymerized by treating with 3.6, 9, 18, 27,
and 36 µM 2c in DMEM, respectively, for 2 h at 37 °C and 20 µM
nocodazole (Sigma) as control. For regrowth, cells were washed
with PBS three times and incubated in prewarmed DMEM at 37
°C to allow regrowth. Cells were fixed at indicated time points
and immunostained for ninein proteins and microtubules as
described above.
In Vitro Tubulin Polymerization Assay. In vitro tubulin
polymerization assays were conducted with reagents as described
by the manufacturer (Cytoskeleton, Inc.). In brief, compound 2c
was incubated with purified bovine tubulin and buffer containing
20% glycerol and 1 mM GTP at 37 °C, and the effect of compound
2c on tubulin polymerization was monitored kinetically using a
fluorescent plate reader (FLUOstar galaxy, BMG).16
2-(6-Trimethylsilyl-3(Z)-hexen-1,5-diynyl)aniline (5). To a
degassed solution of 4 (12 mmol) containing CuI (3.2 mmol) and
n-BuNH2 (30 mmol) in ether (20 mL) was added a degassed
solution of compound 3 (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 solution. The aqueous layer was extracted with EtOAc (30
mL × 3), and the combined organic extracts were washed with
saturated aqueous Na2CO3 solution (40 mL) and dried over
anhydrous MgSO4. After filtration and removal of solvent in vacuo,
Caspases Colorimetric Assay. After different treatments, K-562
cells (1 × 106 cells/mL) were collected and washed three times
with PBS. Cells were resuspended in 200 µL of cell lysis buffer
(Biovision) and incubated on ice for 10 min. Cell lysates were
clarified by centrifugation at 18000g for 3 min. The supernatant
(cytosolic extract) was transferred to a fresh tube and kept on ice.
The protein concentration in the supernatant was determined with

Potent Antitubulin Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 9 2687
the residue was purified by column chromatography on silica gel
(hexane/EA ) 10:1 as the eluent) to give 5 in 75% yield as brown
oil: 1H NMR (CDCl3, 200 MHz) δ 7.33–7.28 (m, 1H), 7.15 (ddd,
1H, J ) 8.2, 7.2, 1.6 Hz), 6.73–6.65 (m, 2H), 6.13 (d, 1H, J )
11.0 Hz), 5.88 (d, 1H, J ) 11.0 Hz), 4.32 (bs, 2H), 0.25 (s, 9H);
13C NMR (CDCl3, 50 MHz) δ 148.1, 132.1, 130.3, 120.5, 117.8,
117.7, 114.1, 107.2, 102.9, 102.8, 94.4, 92.8, 0.0; HRMS calcd for
C15H17NSi, Mr ) 239.1131, found 239.1127.
J ) 10.2, 3.6 Hz), 7.52–7.21 (m, 4H), 7.11 (td, 1H, J ) 7.4, 2.8
Hz), 6.22 (d, 2H, J ) 4.4 Hz), 4.38 (q, 2H, J ) 4.4 Hz), 4.08 (bs,
1H), 1.40 (t, 3H, J ) 3.4 Hz); 13C NMR (CDCl3, 50 MHz) δ166.0,
148.2, 134.4, 132.1, 131.9, 131.4, 130.3, 130.1, 128.1, 123.1, 119.8,
118.0, 117.5, 114.0, 107.2, 95.6, 94.5, 93.0, 92.3, 61.2, 64.1; HRMS
calcd for C21H17NO2, Mr ) 315.1259, found 315.1253.
2-(6-(2-(Trifluoromethoxy)phenyl)-3(Z)-hexen-1,5-diynyl)ani-
line (2e). This compound was obtained in 78% yield as a brown
1
6,6-Bis(3(Z)-hexen-1,5-diynyl)aniline (6). To a degassed solu-
tion of 2-(6-trimethylsilyl-3(Z)-hexen-1,5-diynyl)aniline (5) (0.1 g,
0.42 mmol) containing K2CO3 (0.56 g, 4.2 mmol) in MeOH (20
mL) was added CuI (80 mg, 0.42 mmol). The resulting reaction
mixture was stirred at room temperature for 3 h. Solvent was then
removed in vacuo. The residue was quenched with saturated
aqueous NH4Cl solution and extracted with EtOAc (20 mL × 3).
The combined organic extracts were washed with saturated aqueous
Na2CO3 solution (40 mL) and dried over anhydrous MgSO4. After
filtration and removal of solvent in vacuo, the residue was purified
by column chromatography using hexane/EA (1:1) as eluent on
silica gel to give the desired product 6 (76 mg, 55%) as a brown
oil: 1H NMR (CDCl3, 200 MHz) δ 7.28 (dd, 1H, J ) 7.8, 1.2 Hz),
7.04 (td, 1H, J ) 7.4, 1.6 Hz), 6.62 (td, 1H, J ) 7.6, 1.2 Hz), 6.46
(d, 1H, J ) 8.8 Hz), 6.31 (d, 1H, J ) 10.2 Hz), 5.96 (d, 1H, J )
10.6 Hz), 4.44 (bs, 2H); 13C NMR (CDCl3, 50 MHz) δ148.5, 131.9,
130.6, 123.6, 117.5, 116.0, 114.3, 106.4, 96.6, 93.1, 83.1, 80.7;
HRMS calcd for C24H16N2, Mr ) 332.1313, found 332.1322.
General Procedure for the Synthesis of Compounds 2a-h.
To a degassed solution of 2-(6-trimethylsilyl-3(Z)hexen-1,5-diy-
nyl)aniline (5) (12 mmol) containing CuI (3.2 mmol) and K2CO3
(30 mmol) in MeOH (15 mL) was added a degassed solution of
aryl iodides (7a–h) (12 mmol) containing Pd(PPh3)4 (0.8 mmol) in
MeOH (20 mL). The resulting reaction mixture was stirred at room
temperature for 6 h. Solvent was then removed in vacuo. The
residue was quenched with saturated aqueous NH4Cl solution and
extracted with EtOAc (20 × 3 mL). The combined organic extracts
were washed with saturated aqueous Na2CO3 solution (40 mL) and
dried over anhydrous MgSO4. After filtration and removal of solvent
in vacuo, the residue was purified by column chromatography on
silica gel to give the desired products.
2-(6-(2-Nitrophenyl)-3(Z)-hexen-1,5-diynyl)aniline (2a). This
compound was obtained in 75% yield as a brown oil using hexane/
EA (3:1) as eluent by the general procedure:1H NMR (CDCl3, 200
MHz) δ 8.06 (d, 1H, J ) 8.0 Hz), 7.70–7.38 (m, 3H), 7.11 (td,
2H, J ) 4.8, 1.8 Hz), 6.73–6.66 (m, 2H), 6.25 (dd, 2H, J ) 11, 2.6
Hz), 4.16 (bs, 2H); 13C NMR (CDCl3, 50 MHz) δ 148.2, 135.1,
132.7, 132.2, 130.3, 128.7, 124.5, 121.3, 118.3, 117.6, 116.9, 114.1,
107.0, 95.5, 94.8, 92.6, 91.8; HRMS calcd for C18H12N2O2, Mr )
288.0899, found 288.0890.
2-(6-(4-Nitrophenyl)-3(Z)-hexen-1,5-diynyl)aniline(2b). This
compound was obtained in 64% yield as a brown oil using hexane/
EA (3:1) as eluent by the general procedure: 1H NMR (CDCl3,
200 MHz) δ 8.20 (d, 2H, J ) 8.2 Hz), 7.63 (d, 3H, J ) 8.8 Hz),
7.41–7.20 (m, 3H), 6.88–6.67 (m, 2H), 6.28 (d, 1H, J ) 10.6 Hz),
6.09 (d, 1H, J ) 11 Hz), 4.11 (bs, 2H); 13C NMR (CDCl3, 50
MHz) δ148.1, 132.4, 132.0, 130.5, 123.8, 123.5, 121.7, 121.2,
117.9, 116.8, 114.2, 110.8, 102.6, 95.7, 94.6, 92.7, 92.6; HRMS
calcd for C18H12N2O2, Mr ) 288.0899, found 288.0876.
oil using hexane/EA (4:1) as eluent by the general procedure: H
NMR (CDCl3, 400 MHz) δ 7.56 (dd, 1H, J ) 8.0, 1.6 Hz),
7.38–7.27 (m, 3H), 7.14 (td, 1H, J ) 7.6, 1.2 Hz), 6.72–6.68 (m,
2H), 6.22 (d, 1H, J ) 10.8 Hz), 6.11 (d, 1H, J ) 10.8 Hz) 4.20
(bs, 2H); 13C NMR (CDCl3, 100 MHz) δ 147.8, 134.8, 134.7, 134.1,
132.1, 130.3, 127.8, 127.7, 121.1, 120.4, 118.0, 117.5, 114.4, 94.7,
92.9, 92.5, 90.8; HRMS calcd for C19H12F3NO, Mr ) 327.0871,
found 327.0877.
2-(6-(2,4-Difluorophenyl)-3(Z)-hexen-1,5-diynyl)aniline (2f).
This compound was obtained in 48% yield as a yellow oil using
hexane/EA (3:1) as eluent by the general procedure: 1H NMR
(CDCl3, 400 MHz) δ 7.50–7.44 (m, 1H), 7.32 (dd, 1H, J ) 7.6,
1.2 Hz), 7.15 (td, 1H, J ) 6.8, 1.6 Hz), 6.91–6.85 (m, 2H),
6.73–6.69 (m, 2H), 6.20 (d, 1H, J ) 10.8 Hz), 6.07 (d, 1H, J )
11.2 Hz), 4.00 (bs, 2H); 13C NMR (CDCl3, 100 MHz) δ 134.7,
134.6, 132.1, 130.4, 120.2, 117.9, 117.3, 114.4, 111.8, 111.6, 107.4,
104.6, 104.3, 104.1, 94.8, 92.9, 92.3, 89.1; HRMS calcd for
C18H11F2N, Mr ) 279.0860, found 279.0866.
2-(6-(2,3,4,5,6-Pentafluorophenyl)-3(Z)-hexen-1,5-diynyl)ani-
line (2g). This compound was obtained in 35% yield as a yellow
1
oil using hexane/EA (3:1) as eluent by the general procedure: H
NMR (CDCl3, 400 MHz) δ 7.31–7.26 (m, 1H), 7.04 (t, 1H, J )
7.2, 1.2 Hz), 6.73–6.60 (m, 2H), 6.29 (d, 1H, J ) 5.2 Hz), 5.97 (d,
1H, J ) 10.4 Hz), 4.12 (bs, 2H); 13C NMR (CDCl3, 100 MHz)
δ148.5, 132.1, 130.6, 130.5, 123.8, 123.7, 118.0, 117.7, 117.5,
116.2, 116.0, 114.3, 114.2, 106.8, 96.6, 94.1, 83.1, 82.4; HRMS
calcd for C18H8F5N, Mr ) 333.0577, found 333.0580.
2-(6-(2-Fluorophenyl)-3(Z)-hexen-1,5-diynyl)aniline (2h). This
compound was obtained in 55% yield as a yellow oil using hexane/
EA (3:1) as eluent by the general procedure: 1H NMR (CDCl3,
200 MHz) δ 7.49 (td, 1H, J ) 9.8, 1.6 Hz), 7.39–7.28 (m, 2H),
7.18–7.05 (m, 3H), 6.73–6.65 (m, 2H), 6.21 (d, 1H, J ) 10.8 Hz),
6.10 (d, 1H, J ) 10.8 Hz), 4.41 (bs, 2H); 13C NMR (CDCl3, 50
MHz) δ148.3, 133.8, 132.1, 130.5, 130.3, 124.1, 124.0, 120.1,
117.6, 117.4, 115.7, 115.3, 114.1, 107.2, 94.9, 92.9, 92.5, 90.1;
HRMS calcd for C18H12FN, Mr ) 261.0954, found 261.0949.
Acknowledgment. We thank the National Science Council
of the Republic of China and National Sun Yat-Sen University-
Kaohsiung Medical University Joint Research Center for
financial support and the National Institutes of Health (National
Cancer Institute) for the cytotoxicity sceening.
Supporting Information Available: Data for the cell cycle
percentage for 24 and 72 h, HPLC purity data, and full panel
screening data of 2c. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
2-(6-(2-Trifluoromethylphenyl)-3(Z)-hexen-1,5-diynyl)aniline
(2c). This compound was obtained in 51% yield as a brown oil
using hexane/EA (3:1) as eluent by the general procedure: 1H NMR
(CDCl3, 200 MHz) δ 7.65 (t, 2H, J ) 5.4 Hz), 7.55–7.25 (m, 3H),
7.14 (td, 1H, J ) 7.6, 1.4 Hz), 6.69 (d, 2H, J ) 4.8 Hz), 6.25 (d,
1H, J ) 1.4 Hz), 6.20 (d, 1H, J ) 1.8 Hz), 4.8–4.2 (bs, 2H); 13C
NMR (CDCl3, 50 MHz) δ 147.0, 134.4, 132.1, 131.2, 130.3, 128.3,
127.3, 126.0, 125.9, 125.8, 120.5, 118.5, 117.6, 114.8, 108.0, 94.6,
92.9, 92.5; HRMS calcd for C19H12F3N, Mr ) 311.0922, found
311.0896.
2-(6-(2-Ethoxycarbonylphenyl)-3(Z)-hexen-1,5-diynyl)aniline
(2d). This compound was obtained in 34% yield as a brown oil
using hexane/EA (4:1) as eluent by the general procedure: 1H NMR
(CDCl3, 200 MHz) δ 7.89 (dd, 1H, J ) 7.4, 2.4 Hz), 7.66 (dd, 1H,
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